1. A vaccine combination comprising: a) a first vaccine comprising an
immunologically effective amount of one or more recombinant adenovirus
vectors together comprising a first nucleic acid encoding a first
polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and a
second nucleic acid encoding a second polypeptide comprising the amino
acid sequence of SEQ ID NO: 20, together with a pharmaceutically
acceptable carrier; and b) a second vaccine comprising an immunologically
effective amount of a recombinant Modified Vaccinia Ankara (MVA) vector
comprising a third nucleic acid encoding a third polypeptide comprising
the amino acid sequence of SEQ ID NO: 1 and a fourth nucleic acid
encoding a fourth polypeptide comprising SEQ ID NO: 20, together with a
pharmaceutically acceptable carrier; wherein the MVA vector comprises
MVA-BN or derivatives thereof.
2. The vaccine combination according to claim 1, wherein the first
vaccine and the second vaccine each further comprise a nucleic acid
encoding a fifth polypeptide comprising the amino acid sequence of SEQ ID
NO: 28 and a nucleic acid encoding a sixth polypeptide comprising the
amino acid sequence of SEQ ID NO: 31.
3. The vaccine combination according to claim 1, wherein the first
polypeptide and the third polypeptide each further comprise the amino
acid sequence of SEQ ID NO:28 and wherein the second polypeptide and the
fourth polypeptide each further comprise the amino acid sequence of SEQ
ID NO: 31.
4. A vaccine combination according to claim 1, wherein the first nucleic
acid and the third nucleic acid each encode a polypeptide comprising the
amino acid sequence of SEQ ID NO: 3, and wherein the second nucleic acid
and the fourth nucleic acid each encode a polypeptide comprising the
amino acid sequence of SEQ ID NO: 22.
5. The vaccine combination according to claim 1, wherein the first
nucleic acid and the third nucleic acid each have at least 90% sequence
identity to the polynucleotide sequence of SEQ ID NO: 2, and the second
nucleic acid and the fourth nucleic acid each have at least 90% sequence
identity to the polynucleotide sequence of SEQ ID NO: 21.
6. The vaccine combination according to claim 4, wherein the first
nucleic acid and the third nucleic acid each have at least 90% sequence
identity to the polynucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 24
and the second nucleic acid and the fourth nucleic acid each have at
least 90% sequence identity to the polynucleotide sequence of SEQ ID NO:
23 or SEQ ID NO: 25.
7. The vaccine combination according to claim 1, wherein the recombinant
adenovirus vector is rAd26.
8. The vaccine combination according to claim 1, wherein the first
vaccine comprises a first recombinant adenovirus vector comprising the
first nucleic acid and a second recombinant adenovirus comprising the
second nucleic acid.
9. A recombinant Modified Vaccinia Ankara (MVA) vector comprising: (a) a
first nucleic acid encoding at least one of a polypeptide comprising the
amino acid sequence of SEQ ID NO: 1 and a polypeptide comprising the
amino acid sequence of SEQ ID NO: 3, and (b) a second nucleic acid
encoding at least one of a polypeptide comprising the amino acid sequence
of SEQ ID NO: 20 and a polypeptide comprising the amino acid sequence of
SEQ ID NO: 22; wherein the MVA vector is MVA-BN or derivatives thereof.
10. The recombinant Modified Vaccinia Ankara (MVA) vector according to
claim 9, wherein the first nucleic acid encodes the polypeptide
comprising the amino acid sequence of SEQ ID NO: 1, and the second
nucleic acid encodes the polypeptide comprising the amino acid sequence
of SEQ ID NO: 20.
11. The recombinant MVA vector according to claim 9, wherein the first
nucleic acid encodes the polypeptide comprising the amino acid sequence
of SEQ ID NO: 3, and the second nucleic acid encodes the polypeptide
comprising the amino acid sequence of SEQ ID NO: 22.
12. The recombinant MVA vector according to claim 10, wherein the first
nucleic acid has at least 90% sequence identity to the polynucleotide
sequence of SEQ ID NO: 2, and the second nucleic acid has at least 90%
sequence identity to the polynucleotide sequence of SEQ ID NO: 21.
13. The recombinant MVA vector according to claim 11, wherein the first
nucleic acid has at least 90% sequence identity to the polynucleotide
sequence of SEQ ID NO: 4 or SEQ ID NO: 24, and the second nucleic acid
has at least 90% sequence identity to the polynucleotide sequence of SEQ
ID NO: 23 or SEQ ID NO: 25.
14. A recombinant MVA vector comprising at least one nucleic acid
encoding at least one polypeptide selected from the group consisting of
polypeptides comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID
NO: 3, SEQ ID NO: 20, and SEQ ID NO:22, wherein the at least one nucleic
acid is operably linked to a promoter comprising at least one of the
polynucleotide sequences of SEQ ID NO: 26 and SEQ ID NO: 27.
15. A vaccine comprising a recombinant MVA vector according to claim 9
and a pharmaceutically acceptable carrier.
16. A method for treating a persistent Human Papilloma Virus (HPV)
infection, vulvar intraepithelial neoplasia (VIN), cervical
intraepithelial neoplasia (CIN), vaginal intraepithelial neoplasia
(VaIN), anal intraepithelial neoplasia (AIN), cervical cancer (such as
cervical squamous cell carcinoma (SCC)), oropharyngeal cancer, penile
cancer, vaginal cancer or anal cancer in a subject in need thereof, the
method comprising administering to the subject a vector, vaccine, or
vaccine combination according to claim 1.
17. A method for inducing an immune response against Human Papilloma
Virus (HPV) in a subject in need thereof, the method comprising: (a)
administering to the subject a first vaccine comprising an
immunologically effective amount of either (i) a recombinant adenovirus
vector comprising a first nucleic acid encoding a first polypeptide
comprising the amino acid sequence of SEQ ID NO:1 and a second nucleic
acid encoding a second polypeptide comprising SEQ ID NO: 20, or (ii) a
first recombinant adenovirus vector comprising a first nucleic acid
encoding a first polypeptide comprising the amino acid sequence of SEQ ID
NO:1 and a second recombinant adenovirus vector comprising a second
nucleic acid encoding a second polypeptide comprising the amino acid
sequence of SEQ ID NO: 20, together with a pharmaceutically acceptable
carrier; and (b) administering to the subject a second vaccine comprising
an immunologically effective amount of a recombinant Modified Vaccinia
Ankara (MVA) vector comprising a third nucleic acid encoding a third
polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a
fourth nucleic acid encoding a fourth polypeptide comprising the amino
acid sequence of SEQ ID NO: 20, together with a pharmaceutically
acceptable carrier; wherein the first vaccine is administered to the
subject as a priming vaccine and the second vaccine is administered to
the subject as a boosting vaccine.
18. The method according to claim 17, wherein the first polypeptide and
the third polypeptide each further comprise the amino acid sequence of
SEQ ID NO: 28 and wherein the second polypeptide and the fourth
polypeptide each further comprise the amino acid sequence of SEQ ID NO:
31.
19. The method according to claim 17, wherein the first vaccine comprises
the first recombinant adenovirus vector comprising the first nucleic acid
encoding the first polypeptide comprising the amino acid sequence of SEQ
ID NO: 1 or SEQ ID NO: 3 and the second recombinant adenovirus vector
comprising the second nucleic acid encoding the second polypeptide
comprising the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 22.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is entitled to priority pursuant to 35 U.S.C.
.sctn. 119(e) to U.S. Provisional Patent Application No. 62/209,091 filed
Aug. 24, 2015, and to U.S. Provisional Patent Application No. 62/210,655,
filed Aug. 27, 2015, the disclosures of which are herein incorporated by
reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to the field of medicine and more in
particular to nucleic acid constructs, polypeptides, vectors, vaccines,
vaccine combinations that can be used as therapeutics against human
papillomavirus type 18, and/or type 16.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0003] This application contains a sequence listing, which is submitted
electronically via EFS-Web as an ASCII formatted sequence listing with a
file name "0269 US_P00_PRO_ST25", creation date of Apr. 25, 2016, and
having a size of about 64,700 bytes. The sequence listing submitted via
EFS-Web is part of the specification and is herein incorporated by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0004] The family of human papillomaviruses (HPVs) consist of more than
100 types (also referred to as subtypes) that are capable of infecting
keratinocytes of the skin or mucosal membranes. Over 40 types of HPV are
typically transmitted through sexual contact and HPV infections of the
anogenital region are very common in both men and women. Some sexually
transmitted HPV types may cause genital warts. Persistent infections with
"high-risk" HPV types (e.g. types 16, 18, 31, 45)--different from the
ones that cause skin warts--may progress to precancerous lesions and
invasive cancer, e.g. of the cervix, vulva, vagina, penis, oropharynx and
anus. The majority of HPV infections are spontaneously cleared within one
to two years after infection. In healthy individuals circulating Th1- and
Th2-type CD4+ T-cells specific for the viral early proteins E2, E6 and E7
of HPV-16 as well as E6-specific CD8+ T-cells, migrate into the skin upon
antigenic challenge, indicating that successful defense against HPV-16
infection is commonly associated with a systemic effector T-cell response
against these viral early antigens. In a minority (.about.1%) of infected
individuals, HPV infection persists, ultimately resulting in genital
neoplastic lesions. Among the high-risk HPVs, HPV16 and HPV18 are the
main causes of cervical cancer, together causing about 70% of the cases,
and these two types also play a major role in other HPV-induced cancers
such as anal and oropharyngeal cancer. Worldwide, HPV is one of the most
important infectious agents causing cancer.
[0005] Vaccination against HPV is deemed a feasible strategy to reduce the
incidence or effects of infection by HPV (van der Burg and Melief, 2011,
Curr Opinion Immunol 23: 252-257).
[0006] Prophylactic HPV vaccines based on virus like particles (VLPs)
formed by the (envelope) protein L1 of the HPV types 16 and 18, are very
efficient in the prevention of persistent infection and the associated
disease by HPV16 and HPV18. These vaccines are believed to provide
sterile immunity via the induction of neutralizing antibodies against the
L1 proteins. Addition of L1-based VLPs from additional high-risk HPV
types may further increase the breadth of protection conferred by such
vaccines.
[0007] However, while such vaccines can prevent initial infection (i.e.,
they result in prophylaxis), there is no evidence of a beneficial effect
on established genital lesions caused by HPV16 and HPV18, so they are not
considered therapeutic vaccines against HPV (Hildesheim et al., 2007,
JAMA 298: 743-53).
[0008] Despite the introduction of these prophylactic vaccines, large
numbers of people have already obtained or are still at risk of obtaining
persistent high-risk HPV infections and, therefore, are at risk of
getting cancer. Therapeutic vaccines for the eradication of established
HPV infections and associated diseases are an urgent unmet medical need.
[0009] Some attempts to address this need have been described. For
example, clinical trials have been carried out with a variety of
different vaccination strategies, such as a fusion protein consisting of
a heat shock protein (Hsp) from Mycobacterium bovis and HPV-16 E7 or a
fusion protein of E6, E7 and L2 from HPV-16 and HPV-18, chimeric L1-E7
VLPs, recombinant vaccinia viruses expressing either E6 and E7 of HPV-16
and HPV-18 or bovine papilloma virus E2, DNA vaccines expressing CTL
epitopes of E6 and E7 of HPV-16 and HPV-18, a live-attenuated Listeria
monocytogenes (Lm) that secretes the HPV-16 E7 antigen, and synthetic
long-peptides (SLPs) comprising HPV-16 E6 and E7 peptides. While some of
these approaches show some, but limited, clinical efficacy, most have
failed, demonstrating that improvement of the current strategies is
needed.
[0010] Integration of the genes encoding the early HPV proteins E6 and E7
is a necessary step in the process from infection to cancer and
continuous expression of E6 and E7 is required for the maintenance of the
neoplastic phenotype of cervical cancer cells. E6 and E7 are therefore
considered good targets for therapeutic vaccination. As mentioned some
studies have shown that therapeutic vaccination of women infected with
high-risk HPV can induce regression of existing lesions. Kenter et al
showed a durable and complete regression in 47% of patients having Vulvar
Intraepithelial Neoplasia (VIN) using SLPs derived from the HPV16 E6 and
E7 proteins and an adjuvant as a therapeutic vaccine (Kenter et al.,
2009, N Engl J Med 361: 1838-47). Similarly, a study in which a
protein-based vaccine (TA-CIN, consisting of a fusion protein of HPV16
E6, E7 and L2) was combined with local immune modulation in VIN 2/3
patients, showed complete regression in 63% of patients (Daayana et al.,
2010, Br J Cancer 102: 1129-36). Possible drawbacks of the synthetic long
peptides as a vaccine include manufacturability at large scale and costs
associated therewith, the need for potentially reactogenic adjuvant and
the associated adverse effects associated with immunization (especially
pain and swelling). Due to the high level of discomfort it is not likely
that SLPs will be used in early stage disease when the spontaneous
clearance rate is still high. Similarly, due to the need for local
imiquimod treatment in the case of TA-CIN treatment, tolerability is a
significant issue as the majority of women experience local and systemic
side effects lasting for the duration of imiquimod treatment, which may
affect daily activities.
[0011] A possible alternative is to use nucleic acid based vaccination
such as DNA vaccines or viral vectored vaccines encoding the HPV E6
and/or E7 protein for vaccination.
[0012] However, the HPV E6 and E7 proteins have oncogenic potential and
thus vaccination with vaccines that comprise nucleic acids encoding these
proteins poses a risk of inducing cellular transformation due to the
possibility of prolonged expression of the antigens.
[0013] Therefore, in case of genetic vaccination, non-oncogenic/detoxified
versions of E6 and/or E7 can be used in order to exclude any risk of
cellular transformation due to the vaccination. Loss of oncogenic
potential of wild-type E6 and E7 is commonly achieved by deletion and/or
substitution of residues known to be important for the function of these
proteins (e.g., Smahel et al., 2001, Virology 281:231-38; Yan et al.,
2009, Vaccine 27: 431-40; Wieking et al., 2012, Cancer Gene Ther 19:
667-74). However, a disadvantage of these approaches is that they carry
the risk of removing important T-cell epitopes from and/or introducing
new undesired T-cell epitopes into the proteins, and may thus not lead to
the desired immune response.
[0014] In an alternative strategy to remove the oncogenic potential of
HPV16 E6 and E7, shuffled versions (i.e. polypeptides wherein fragments
of the wild-type protein are re-ordered) of the E6 and E7 proteins have
been constructed (e.g. Ohlschlager et al., 2006, Vaccine 24: 2880-93;
Oosterhuis et al., 2011, Int J Cancer 129: 397-406; Oosterhuis et al.,
2012, Hum Gen Ther 23: 1301-12). However, these approaches would still
require manufacturing, formulation and administration of multiple
molecules to ensure inclusion of all possible epitopes of both the E6 and
E7 proteins, resulting in sub-optimal logistics and relatively high
costs, and moreover the strategies described introduce potentially strong
non-natural epitopes that are not present in E6 and E7 and since immune
responses could be diverted from relevant E6/E7 epitopes towards such
non-natural epitopes, the described constructs may not have the optimal
immunological characteristics. A therapeutic DNA vaccine expressing an
intracellularly targeted fusion protein with built-in genetic adjuvant
and shuffled fragments of E6 and E7 of both HPV16 and HPV18 has also been
described, and electroporation-enhanced immunization therewith elicited a
significant E6/E7-specific T-cell response in CIN3 patients (Kim et al.,
2014).
[0015] There remains a need in the art for therapeutic vaccines against
HPV, preferably having less of the drawbacks of the approaches described
before.
SUMMARY OF THE INVENTION
[0016] The present invention provides one or more vectors, vaccines, and
vaccine combinations that can be used for generating an immune response
against HPV infections. In various embodiments, the present invention
comprises nucleic acid molecules that encode polypeptides, or fusion
proteins, that comprise essentially all possible T-cell epitopes of HPV16
or HPV18 oncoproteins E6 and E7, but nevertheless have a strongly reduced
(as compared to wt E6 and E7), up to non-detectable, transforming
activity, by comprising fragments of the E6 and E7 proteins that have
been re-ordered, while at the same time containing a minimized number of
undesired strong neo-epitopes. This is in contrast to molecules
previously reported by others. The invention provides molecules that can
be used in therapeutic vaccines against either HPV16 or HPV18.
[0017] In various additional embodiments, the vectors, vaccines, and
vaccine combinations comprise nucleic acid molecules that encode
polypeptides, or fusion proteins, that comprise essentially all possible
T-cell epitopes of HPV16 or HPV18 oncoproteins E6 and E7, but
nevertheless have a strongly reduced (as compared to wt E6 and E7), up to
non-detectable transforming activity. At least in one aspect this is
accomplished as the vectors, vaccines, and vaccine combinations comprise
fragments of the E6 and E7 proteins that have been re-ordered, while at
the same time containing a minimized number of undesired strong
neo-epitopes. This is in contrast to molecules previously reported by
others. In preferred embodiments, the polypeptides or fusion proteins
encoded by the vectors, vaccines or vaccine combinations further comprise
E2 protein or fragments thereof of HPV16 or HPV18. The invention provides
molecules that can be used in therapeutic vaccines against either HPV16
or HPV18. Such molecules can also be combined in therapeutic vaccines
against both HPV16 and HPV18.
[0018] In certain embodiments, the invention for HPV16 provides vectors,
vaccines, and vaccine combinations comprising a nucleic acid molecule
encoding a polypeptide comprising the amino acid sequence as set forth in
SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO: 28, or combinations
thereof. In certain preferred embodiments, the invention for HPV16
provides vectors, vaccines, and vaccine combinations comprising a nucleic
acid molecule encoding a polypeptide comprising the amino acid sequence
as set forth in SEQ ID NO: 3. In other embodiments, for HPV18, the
invention provides vectors, vaccines, and vaccine combinations comprising
a nucleic acid molecule encoding a polypeptide comprising the amino acid
sequence as set forth in SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 31, or
combinations thereof. In certain preferred embodiments, for HPV18, the
invention provides vectors, vaccines, and vaccine combinations comprising
a nucleic acid molecule encoding a polypeptide comprising the amino acid
sequence as set forth in SEQ ID NO: 22.
[0019] In some aspects, the encoded polypeptide of the invention can
further comprise a leader sequence.
[0020] In certain embodiments, the encoded polypeptide comprises at least
one epitope of an HPV16 E2 protein or an HPV18 E2 protein. The E2 protein
can be inactivated in for instance its transactivation and/or DNA binding
domain, e.g. by deletion, mutation or by structural rearrangement of
different parts of the protein. In certain embodiments for HPV16, the
encoded polypeptide comprises the amino acid sequence as set forth in SEQ
ID NO: 28. In certain embodiments for HPV18, the encoded polypeptide
comprises the amino acid sequence as set forth in SEQ ID NO: 31.
[0021] In certain embodiments, for HPV16, the nucleic acid sequence
comprises the polynucleotide sequence as set forth in SEQ ID NO: 2, SEQ
ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 24, SEQ ID NO: 29, SEQ ID NO: 30, or
combinations thereof. In certain preferred embodiments, for HPV16, the
nucleic acid sequence comprises the polynucleotide sequence as set forth
in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 24.
[0022] In certain other embodiments, for HPV18, the nucleic acid sequence
comprises the polynucleotide sequence as set forth in SEQ ID NO: 21, SEQ
ID NO: 23, SEQ ID NO:25, SEQ ID NO: 32, SEQ ID NO: 33, or combinations
thereof. In certain preferred embodiments, for HPV18, the nucleic acid
sequence comprises the polynucleotide sequence as set forth in SEQ ID NO:
21, SEQ ID NO: 23, or SEQ ID NO: 25.
[0023] The invention also provides a vaccines, and vaccine combinations
comprising a recombinant virus vector according to the invention, and a
pharmaceutically acceptable excipient. The recombinant virus vector
comprises one or more nucleic acid molecules according to the invention,
wherein the sequence encoding the polypeptide is operably linked to a
promoter.
[0024] In certain embodiments the vector is a viral vector, such as an
recombinant poxviral vector or a recombinant adenoviral vector. In
certain preferred embodiments, the vector is a recombinant adenovirus or
a recombinant MVA virus. In additional preferred embodiments, the MVA
virus vector is MVA-BN or derivatives thereof. In still additional
preferred embodiments, the adenoviral vector is selected from rAd26 and
rAd35, and most preferably it is rAd26.
[0025] In certain preferred embodiments, there is a vaccine combination
comprising:
[0026] a) a first vaccine comprising an immunologically effective amount
of one or more recombinant adenovirus vectors together comprising a first
nucleic acid encoding a first polypeptide comprising the amino acid
sequence of SEQ ID NO: 1 and a second nucleic acid encoding a second
polypeptide comprising the amino acid sequence of SEQ ID NO: 20, together
with a pharmaceutically acceptable carrier; and
[0027] b) a second vaccine comprising an immunologically effective amount
of a recombinant Modified Vaccinia Ankara (MVA) vector comprising a third
nucleic acid encoding a third polypeptide comprising the amino acid
sequence of SEQ ID NO: 1 and a fourth nucleic acid encoding a fourth
polypeptide comprising the amino acid sequence of SEQ ID NO: 20, together
with a pharmaceutically acceptable carrier;
[0028] wherein the MVA vector comprises MVA-BN or derivatives thereof.
[0029] According to embodiments of the invention, the first polypeptide
and the third polypeptide can be identical or different. For example, one
of the first and third polypeptides can contain an additional amino acid
sequence that is absent from the other polypeptide, or the first and
third polypeptides can contain additional amino acid sequences that are
different from each other. Similarly, the second and the fourth
polypeptides can be identical or different. The first nucleic acid and
the third nucleic acid can be identical or different. For example, the
first and third nucleic acids can be different because they encode
different first and third polypeptides, and/or use different codons for
the same amino acids. Similarly, the second and fourth nucleic acids can
be identical or different.
[0030] In certain additional preferred embodiments, the first vaccine and
the second vaccine of both a) and b) each further comprise a nucleic acid
encoding a fifth polypeptide comprising the amino acid sequence of SEQ ID
NO: 28 and a nucleic acid encoding a sixth polypeptide comprising the
amino acid sequence of SEQ ID NO: 31. The fifth and the sixth
polypeptides can each be expressed independently or preferably as a part
of a fusion protein that contains an E6 and E7 polypeptide of the
invention.
[0031] In other additional preferred embodiments, the nucleic acid
encoding the polypeptide comprising the amino acid sequence of SEQ ID
NO:1 of each of the first vaccine and second vaccine further encodes the
fifth polypeptide. Preferably, the polypeptide comprising the amino acid
sequence of SEQ ID NO:1 and the fifth polypeptide are expressed together
in a fusion protein, such as a polypeptide comprising the amino acid
sequence of SEQ ID NO:3 or SEQ ID NO:5. The nucleic acid encoding the
polypeptide comprising the amino acid sequence of SEQ ID NO: 20 of each
of the first and second vaccines preferably further encodes the sixth
polypeptide. Preferably, the polypeptide comprising the amino acid
sequence of SEQ ID NO:20 and the sixth polypeptide are expressed together
in a fusion protein, such as a polypeptide comprising the amino acid
sequence of SEQ ID NO:22.
[0032] In still other preferred embodiments, the nucleic acid encoding the
polypeptide comprising SEQ ID NO:1 of one or both the first vaccine and
second vaccine is part of a nucleic acid encoding a polypeptide
comprising SEQ ID NO: 3. In still additional preferred embodiments, the
nucleic acid encoding the polypeptide comprising SEQ ID NO: 20 of one or
both the first vaccine and second vaccine is part of a nucleic acid
encoding a polypeptide comprising SEQ ID NO: 22.
[0033] In various embodiments, the nucleic acid encoding a polypeptide
comprising the amino acid sequence of SEQ ID NO: 1 has at least 90%
sequence identity to the polynucleotide sequence of SEQ ID NO: 2 and the
nucleic acid encoding a polypeptide comprising the amino acid sequence of
SEQ ID NO: 20 has at least 90% sequence identity to the polynucleotide
sequence of SEQ ID NO: 21. In other embodiments, the nucleic acid
encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3
has at least 90% sequence identity to the polynucleotide sequence of SEQ
ID NO: 4 or SEQ ID NO: 24 and the nucleic acid encoding a polypeptide
comprising the amino acid sequence of SEQ ID NO: 22 has at least 90%
sequence identity to the polynucleotide sequence of SEQ ID NO: 23 or SEQ
ID NO: 25.
[0034] The invention also provides a method of inducing an immune response
against HPV, in particular HPV16 or HPV18, or HPV16 and HPV18 in a
subject in need thereof, the method comprises administering to the
subject a vector, vaccine, or vaccine combination according to the
invention. The invention also provides a vector, vaccine, or vaccine
combination according to the invention for use in inducing an immune
response against HPV, in particular HPV16 or HPV18, or both HPV16 and
HPV18 in a subject in need thereof.
[0035] In certain embodiments, the vectors or vaccines of the present
invention are administered to the subject more than once.
[0036] In certain embodiments, the vector, vaccine, or vaccine combination
according to the invention are administered to a subject in need thereof,
preferably a human subject, as a prime-boost regimen. In a preferred
embodiment, the prime-boost regimen comprises a priming vaccine
comprising an immunologically effective amount of either (i) a
recombinant adenovirus vector comprising a nucleic acid encoding a
polypeptide according to the invention, together with a pharmaceutically
acceptable carrier; or (ii) a first recombinant adenovirus vector
comprising a nucleic acid encoding a polypeptide according to the
invention and a second recombinant adenovirus vector comprising a nucleic
acid encoding a different polypeptide according to the invention,
together with a pharmaceutically acceptable carrier. There is also a
boosting vaccine comprising an immunologically effective amount of a
recombinant Modified Vaccinia Ankara (MVA) vector comprising a nucleic
acid encoding a polypeptide according to the invention, preferably
encoding two different polypeptides according to the invention, together
with a pharmaceutically acceptable carrier.
[0037] In other various embodiments, the prime-boost regimen comprises a
priming vaccine comprising an immunologically effective amount of a
recombinant Modified Vaccinia Ankara (MVA) vector comprising a nucleic
acid encoding a polypeptide according to the invention, together with a
pharmaceutically acceptable carrier. There is also a boosting vaccine
comprising an immunologically effective amount of either (i) a
recombinant adenovirus vector comprising a nucleic acid encoding a
polypeptide according to the invention, together with a pharmaceutically
acceptable carrier; or (ii) a first recombinant adenovirus vector
comprising a nucleic acid encoding a polypeptide according to the
invention and a second recombinant adenovirus vector comprising a nucleic
acid encoding a different polypeptide according to the invention,
together with a pharmaceutically acceptable carrier.
[0038] The invention also provides a method for treating any of:
persistent HPV infection (in particular persistent HPV16 or HPV18
infection), vulvar intraepithelial neoplasia (VIN), cervical
intraepithelial neoplasia (CIN), vaginal intraepithelial neoplasia
(VaIN), anal intraepithelial neoplasia (AIN), cervical cancer (such as
cervical squamous cell carcinoma (SCC)), oropharyngeal cancer, penile
cancer, vaginal cancer or anal cancer in a subject in need thereof, the
method comprises administering to the subject a vector, vaccine, or
vaccine combination according to the invention. The invention also
provides a vector, vaccine, or vaccine combination according to the
invention for use in treatment of any of: persistent HPV infection (in
particular persistent HPV16 or HPV18 infection), vulvar intraepithelial
neoplasia (VIN), cervical intraepithelial neoplasia (CIN), vaginal
intraepithelial neoplasia (VaIN), anal intraepithelial neoplasia (AIN),
cervical cancer (such as cervical squamous cell carcinoma (SCC)),
oropharyngeal cancer, penile cancer, vaginal cancer or anal cancer in a
subject in need thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0039] The foregoing summary, as well as the following detailed
description of the invention, will be better understood when read in
conjunction with the appended drawings. It should be understood that the
invention is not limited to the precise embodiments shown in the
drawings.
[0040] FIG. 1. Expression of fusion proteins of HPV16 E6 and E7. HEK-293T
cells were transiently transfected with DNA vectors expressing the
transgenes indicated above the figure. 24 hr after transfection the cells
were harvested and cell extracts were analyzed by SDS-PAGE and western
blotting with an antibody against HPV16 E7 (upper panel). A loading
control showing NF-kB (lower panel) confirms similar loading of cell
lysates in all lanes. A molecular weight marker is indicated on the left.
Expected sizes of the fusion proteins: E6E7SH approx. 38 kDa; E2E6E7SH
and E6E7E2SH approx. 75 kDa, LSE2E6E7SH approx. 78 kDa.
[0041] FIG. 2. Colony formation in soft agar. A) Schematic representation
of the setup of the soft-agar assay. B) Representative microscopic images
at 40.times. magnification of the cells in agar six weeks post seeding.
The white arrows highlight colonies observed in the E7 wt transfected
cells. C) Colony quantification six weeks post seeding in agar using the
Gelcount.TM. and associated software. *: p<0.05 (Poisson regression
model); **: non-inferior (generalized linear model with non-inferiority
margin of 5%).
[0042] FIG. 3. HPV16 E6E7SH has lost E6 and E7 activities. A)
Representative western blot demonstrating absence of p53 degradation by
E6E7SH. Human p53 null NCI-H1299 cells were co-transfected with a plasmid
expressing p53 in combination with a plasmid expressing HPV16 E6
wild-type, HPV16 E6E7SH or the empty vector. Non-TF indicates
non-transfected cells. 24 hours after transfection cell lysates were
prepared and 30 .mu.g of total protein was loaded on gel. Upper
panel--p53 staining, middle panel--E6 staining, lower panel--NF-kB
staining (loading control). (B) Quantification of p53 levels in four
independent assays. The p53 signal was normalized to the NF-.kappa.B
signal. C) Western blot demonstrating lack of pRb degradation by E6E7SH.
pRb null Saos-2 cells were transfected with a plasmid expressing pRb in
combination with a plasmid expressing HPV16 E7 wild-type, HPV16 E6E7SH or
the empty vector. Non-TF indicates non-transfected cells. 24 hours after
transfection cell lysates were prepared and 10 .mu.g of total protein was
loaded on gel. Upper panel--pRb staining, middle panel--E7 staining,
lower panel--NF-.kappa.B staining (loading control). D) Quantification of
pRb levels in four independent assays. The pRb signal was normalized to
the NF-.kappa.B signal. *: p<0.05 (ANOVA models); **: non-inferior
(testing was based on 95% CI's derived from ANOVA models. Non-inferiority
margin was set at 75%).
[0043] FIG. 4. HPV16 E6E7SH does not immortalize primary human epidermal
keratinocytes. Primary human epidermal keratinocytes were transduced with
lentiviruses encoding either the wild-type E6- and E7-encoding open
reading frame of HPV16 (E6E7 wt), the HPV16 E6E7SH sequence or eGFP.
Non-transduced donor cells were used as a control. Only expression of
E6E7 wt induces immortalization of primary keratinocytes as indicated by
the extended lifespan and hTERT activation around day 200 (not shown).
The cross symbol indicates that the cells died in senescence and could
not be further cultured. For details see example 2. Similar results were
obtained in two additional donors (not shown).
[0044] FIG. 5. Immune response induced by HPV16 E6E7SH after DNA
immunization--IFN.gamma. ELISPOT analysis. A. Immunization scheme. CB6F1
mice were immunized with DNA plasmids expressing HPV16 E6E7SH or a
plasmid expressing no transgene (control). Two weeks after immunization
the mice were sacrificed and isolated splenocytes were stimulated
overnight with 15mer peptide pools corresponding to E7. B. HPV16
E7-specific immune responses in individual mice as measured by IFN.gamma.
ELISPOT assays are given as spot forming units (SFU) per 10.sup.6
splenocytes.
[0045] FIG. 6. Immunogenicity of HPV16 E6E7SH--IFN.gamma. ELISPOT
analysis. (A). Immunization scheme. Mice were immunized with adenovectors
with inserts as indicated. E7-specific responses at two weeks (B) and at
eight weeks (C) were analyzed by IFN.gamma. ELISPOT (represented as
spot-forming units (SFU) per 10.sup.6 splenocytes). The closed circles
represent mice immunized with a dosage of 1*10.sup.10 vp, and open
circles represent mice immunized with 5*10.sup.9 vp. The black bar
represents the geometric mean of the responses. The dotted line indicates
the lower detection limit in the ELISPOT assay. ANOVA Post-hoc Bonferroni
statistical analysis was performed on log transformed data. *: p<0.05.
For details see example 3.
[0046] FIG. 7. Immunogenicity of HPV16 E2E6E7SH-E7-tetramer staining. (A).
Immunization scheme. CB6F1 mice were immunized with 1*10.sup.10 vp of
adenovectors expressing the transgenes as indicated. Two weeks after
immunization the mice were sacrificed and isolated splenocytes analyzed
for the presence of CD8+ cells capable of interacting with
E7.sub.49-57-H2-Db tetramers (B). The percentage of E7-tetramer positive
CD8+ T-cells is indicated on the y-axis. ANOVA Post-hoc Bonferroni
statistical analysis was performed on log transformed data, the
differences between the different E6E7SH variants were not statistically
significant.
[0047] FIG. 8. Immunogenicity of HPV16 E2E6E7SH--IFN.gamma. ELISPOT
analysis. (A). Immunization scheme. CB6F1 mice were immunized with
adenovectors expressing the transgenes indicated below panels B and C.
Two weeks after immunization the mice were sacrificed and isolated
splenocytes were stimulated overnight with 15mer peptide pools
corresponding to E2 (B), E6 (not shown) or E7 (C). Responses are given as
SFU per 10.sup.6 splenocytes. ANOVA Post-hoc Bonferroni statistical
analysis was performed on log transformed data. The E2 response induced
by Adenovectors encoding E2 alone is higher than the response induced by
the polypeptides of the invention that include the E6 and E7 fragments.
The difference is significant for E2 vs E2E6E7SH and E2 vs E6E7E2SH (*:
p<0.05). ANOVA Post-hoc Bonferroni statistical analysis was performed
on log transformed data.
[0048] FIG. 9. Sustained HPV16 immune responses in immunized mice. In
particular, (A) Immunization scheme. CB6F1 mice were immunized with
1*10.sup.10 vp of Ad35 vectors expressing variants HPV16 LSE2E6E7SH,
HPV16 E2E6E7SH, HPV16 E6E7SH, or with an adenovector not expressing a
transgene (Emtpy). Blood samples were taken every two weeks to determine
the percentage E7-specific CD8+ T-cells by tetramer staining. (B) Immune
responses two weeks after immunization. The vector including a leader
sequence induced a higher response than vectors without the leader
sequence; LSE2E6E7SH vs E2E6E7SH (*: p<0.05). (C) Kinetics of the
responses. ANOVA Post-hoc Bonferroni statistical analysis was performed
on log transformed data of the week 2 data set. The E7 response induced
by molecules including E2 tend to be higher compared to the molecule
without E2, though the results were not statistically significant.
[0049] FIG. 10. Use of different Adenoviral vectors to boost immune
responses. (A). Immunization scheme. CB6F1 mice were immunized with an
Ad26 vector expressing HPV16 E2E6E7SH (HPV16-Tx) or with an Ad26 vector
expressing no transgene (empty). Two weeks later the immunizations were
repeated with Ad35-based vectors as indicated below the figure. Four
weeks after the second immunization the mice were sacrificed and blood
samples were used to determine the percentage of E7-specific CD8+ T-cells
by tetramer staining (B). * indicates the comparison of
Ad26.HPV16-Tx/Ad35.HPV16-Tx versus Ad26.HPV16-Tx/Ad35.Empty, p<0.05
(student t-test on log transformed data, with alpha=0.01 for multiple
comparisons).
[0050] FIG. 11. Cellular immunogenicity of HPV16 E2E6E7SH in Rhesus
macaques. (A) Immunization scheme. Rhesus macaques were immunized at day
0: Eight animals received Ad26.HPV16-E2E6E7SH and two control animals
received Ad26.Empty by intramuscular immunization (i.m). A boost
immunization was given (Ad26.HPV16-E2E6E7SH or Ad26.Empty) at 8 weeks. At
16 weeks, animals received a second boost immunization with Ad35 vectors
expressing the same HPV16 E2E6E7SH, while control animals received
Ad35.Empty. The dose of adenovectors was 1*10.sup.11vp per immunization.
Blood drawings were performed at several time points. (B) Cellular immune
responses in PBMCs were measured by IFN.gamma. ELISPOT. PBMCs were
stimulated with peptide pools corresponding to HPV16 E2, E6 or E7 and the
number of spot-forming units (SFU) in 1*10.sup.6PBMCs are depicted. The
empty control animal (n=2) showed no detectable response. For details see
example 4.
[0051] FIG. 12. Therapeutic effect of Adenovectors expressing
HPV16-E2E6E7SH. (A) TC-1 injection and immunization scheme. CB6F1 mice
were injected sub-cutaneously with 1*10.sup.5 TC-1 cells at day 0. After
six days, when tumors were palpable, mice were immunized with two SLPs
covering HPV16 E6 and E7 immunodominant epitopes (i.e., HPV16 E6, aa41-65
(KQQLLRREVYDFAFRDLCIVYRDGN; SEQ ID NO: 18) and HPV16 E7 aa 43-77
(GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR; SEQ ID NO: 19)) at 150 .mu.g in a
final volume of 200 .mu.l 0.9% saline supplemented with 5 nmol
ODN1826-CpG (B) or Ad26.HPV16-E2E6E7SH (C). Control mice received either
CpG alone (D) or Ad26.Empty (E). All mice received a boost immunization
at day 20. Mice that received Ad26 vectors in the prime immunization were
subsequently immunized with the corresponding Ad35 vectors. The other
mice received, SLP adjuvanted with CpG or CpG alone as in the prime
immunizations. (B-E) Tumor measurement in TC-1 injected mice. Tumor
volume was calculated as (width.sup.2*length)/2. Mice were sacrificed
when tumor volumes surpassed 1000 mm.sup.3. Two mice had to be sacrificed
due to weight loss of more than 20% (indicated with asterisks). (F-G)
Close up of panels B and C for first 35 days. (H) Survival after TC-1
injection. The survival of mice treated with Ad.HPV16-E2E6E7SH was
significantly increased compared with mice immunized with SLP and CpG
(Log-rank test p<0.05). Three mice immunized with the
Ad.HPV16-E2E6E7SH were tumor free at the end of the experiment (at day
92).
[0052] FIG. 13. Adenoviral vectors carrying transgenes encoding either
HPVAg or LSE2E6E7SH show increased viral yields on cells capable of
repressing transgene expression. A) Viral yield assay for Ad35 vectors.
PER.C6, PER.C6/CymR, and PER.C6/TetR cells were infected by Ad35 vectors
carrying GFP-Luc- or HPVAg-encoding transgenes. These transgenes were
driven by either CuO- or TetO-containing CMV promoters. Viral yields were
determined four days after infection by an Ad35 hexon-specific qPCR-based
method. B) Viral yield assay for Ad26 vectors. PER.C6 and PER.C6/TetR
cells were infected by Ad26 vectors carrying GFP-Luc, HPVAg, or
LSE2E6E7SH-encoding transgenes, which were all driven by a
TetO-containing CMV promoter. Viral yields were determined three days
after infection by an Ad26 hexon-specific qPCR-based method. For details
see Example 6.
[0053] FIG. 14. Employment of a repressor system to repress transgene
expression during vector production prevents transgene cassette
instability in an adenoviral vector carrying an HPVAg-encoding transgene.
An Ad35 vector expressing HPVAg under the control of CMVCuO was rescued
by DNA transfection on either PER.C6 or PER.C6/CymR cell lines. Resultant
viral plaques were picked--five per cell line--and used for consecutive
infection rounds on the respective cell lines. A) Analysis of the
integrity of the vector transgene cassette region by PCR after 10 viral
passages. PCR products obtained from viral isolates passaged on PER.C6
and PER.C6/CymR are shown in the middle and right panels, respectively.
The full-length-appearing PCR products obtained for PER.C6-passaged viral
isolates 1, 2, 4, and 5, and those seen for PER.C6/CymR-passaged isolates
1 to 5 were analyzed by Sanger DNA sequencing. Analysis of the
chromatogram traces (not shown) revealed that all isolates grown on
PER.C6, but not those grown on PER.C6/CymR, contained either
frameshifting small deletions or premature stop mutations within the
coding sequence for HPVAg. B) Analysis of the ability of the vectors to
express HPVAg after seven viral passages. A549 cells were transduced by
the PER.C6- and PER.C6/CymR-grown viral isolates and HPVAg expression was
analyzed by Western Blot using an HPV16 E7-specific antibody. The
predicted size for HPVAg is 83 kDa. For details see Example 6.
[0054] FIG. 15. Expression of fusion proteins of HPV18 E6 and E7. HEK-293T
cells were transiently transfected with DNA vectors expressing the
transgenes indicated above the figure. 24 hr after transfection the cells
were harvested and cell extracts were analyzed by SDS-PAGE and western
blotting with an antibody against HPV18 E6 (upper panel). A loading
control showing NF-kB (lower panel) confirms similar loading of cell
lysates in both lanes. A molecular weight marker is indicated on the left
and arrows indicate the fusion proteins. Expected sizes: E6E7SH approx.
38 kDa; E2E6E7SH approx. 75 kDa.
[0055] FIG. 16. No colony formation in soft agar by the HPV18 E6E7SH
designer construct. A) Representative microscopic images at 40.times.
magnification of the cells in agar six weeks post seeding. Large colonies
are observed in the E7 wt transfected cells. B) Colony quantification six
weeks post seeding in agar using the Gelcount.TM. and associated
software. *: p<0.05 (Poisson regression model); **: non-inferior
(generalized linear model with non-inferiority margin of 5%).
[0056] FIG. 17. HPV18 E6E7SH has lost the ability to degrade p53 and pRb.
(A) Representative western blot demonstrating absence of p53 degradation
by HPV18 E6E7SH. Human p53 null NCI-H1299 cells were co-transfected with
a plasmid expressing p53 in combination with a plasmid expressing HPV18
E6 wild-type, E6E7SH or the empty vector. Non-TF indicates
non-transfected cells. 24 hours after transfection cell lysates were
prepared and 30 .mu.g of total protein was loaded on gel. Upper
panel--p53 staining, middle panel--E6 staining, lower panel--NF-kB
staining (loading control). (B) Quantification of p53 levels in four
independent assays. The p53 signal was normalized to the NF-.kappa.B
signal. C) Western blot demonstrating lack of pRb degradation by HPV18
E6E7SH. pRb null Saos-2 cells were transfected with a plasmid expressing
pRb in combination with a plasmid expressing HPV18 E7 wild-type, E6E7SH
or the empty vector. Non-TF indicates non-transfected cells. 24 hours
after transfection cell lysates were prepared and 10 .mu.g of total
protein was loaded on gel. Upper panel--pRb staining, middle panel--E7
staining, lower panel--NF-.kappa.B staining (loading control). D)
Quantification of pRb levels in four independent assays. The pRb signal
was normalized to the NF-.kappa.B signal. *: p<0.05 (ANOVA models);
**: non-inferior (testing was based on 95% CI's derived from ANOVA
models. Non-inferiority margin was set at 75%).
[0057] FIG. 18. HPV18 E6E7SH does not immortalize primary human genital
keratinocytes. Primary human genital keratinocytes were transduced with
lentiviruses encoding either the wild-type E6- and E7-encoding open
reading frame of HPV18 (E6E7 wt), the E6E7SH sequence or eGFP.
Non-transduced donor cells were used as a control. Only expression of
HPV18 E6E7 wt induces immortalization of primary keratinocytes as
indicated by the extended lifespan (and hTERT activation around day 200,
data not shown). The cross symbol indicates that the cells died in
senescence and could not be further cultured. For details see example 8.
Similar results were obtained in two additional donors (data not shown).
[0058] FIG. 19. Immunogenicity of HPV18 E6E7SH variants--Intracellular
Cytokine staining. CB6F1 mice were immunized with adenovectors expressing
the transgenes indicated below the panels. Two weeks after immunization
the mice were sacrificed and isolated splenocytes were stimulated
overnight with 15mer peptide pools corresponding to HPV18 E6. Responses
are given as percentage of IFN.gamma.-positive CD8+ T-cells.
[0059] FIG. 20. Immunogenicity of combined HPV16 and HPV18
vectors--IFN.gamma. ELISPOT analysis. CB6F1 mice were immunized with
adenovectors (type 26) expressing the E2E6E7SH transgenes from both HPV16
(encoding SEQ ID NO: 3) and HPV18 (encoding SEQ ID NO: 22). Four weeks
after prime immunization the mice received an heterologous boost
immunization with adenoviral vectors of type 35 with the same E2E6E7SH
transgenes. Two weeks after the boost immunization the mice were
sacrificed and isolated splenocytes were stimulated overnight with 15mer
peptide pools corresponding to HPV16 E7 (A) or HPV18 E6 (B). Responses
are given as SFU per 10.sup.6 splenocytes.
[0060] FIG. 21. Cellular immunogenicity of combined HPV16 and HPV18
vaccine in Rhesus macaques. Rhesus macaques were immunized according to
the scheme as presented in FIG. 11, with a combination of HPV16 and HPV18
designer constructs. At day 0: Eight animals received a mixture of
Ad26.HPV16-E2E6E7SH and Ad26.HPV18-E2E6E7SH by intramuscular immunization
(i.m). A boost immunization with the same vectors was given at 8 weeks.
At 16 weeks, animals received a second boost immunization with a mixture
of two Ad35 vectors expressing the same HPV16 and HPV18 E2E6E7SH fusion
proteins. The dose of adenovectors was 1*10.sup.11 vp per vector per
immunization. Blood drawings were performed at several time points.
Cellular immune responses in PBMCs were measured by IFN.gamma. ELISPOT.
PBMCs were stimulated with peptide pools corresponding to E2, E6 or E7 of
HPV16 and HPV18 and the number of spot-forming units (SFU) in 1*10.sup.6
PBMCs were determined. The figure shows cumulative responses for all six
tested peptide pools at 2 weeks after each immunization. For details see
example 11.
[0061] FIG. 22. Therapeutic effect of combined adenovectors expressing
HPV16 and HPV18 E2E6E7SH. C57BL/6 mice were injected sub-cutaneously with
5*10.sup.4 TC-1 cells at day 0. After six days, when tumors were
palpable, mice were immunized with Ad26.HPV16-E2E6E7SH or a mixture of
Ad26.HPV16-E2E6E7SH and Ad26.HPV18-E2E6E7SH. Control mice received
Ad26.Empty. All mice received a boost immunization at day 20 with the
corresponding Ad35 vectors. Tumor volume was calculated as
(width.sup.2*length)/2. Mice were sacrificed when tumor volumes surpassed
1000 mm.sup.3. The graphs show survival after TC-1 injection. Three mice
immunized with the combined HPV16+HPV18 vaccine were tumor free at the
end of the experiment. The median survival time of mice treated with
Ad.HPV16-E2E6E7SH was not significantly different compared with mice
immunized with Ad.HPV16/18-E2E6E7SH.
[0062] FIG. 23. Use of Modified Vaccinia Ankara (MVA) vectors to boost
immune responses. (A). Immunization scheme. CB6F1 mice were immunized
with a mixture of an Ad26 vector expressing HPV16 E2E6E7SH (HPV16) and
Ad26 vector expressing HPV18 E2E6E7SH, or with an Ad26 vector expressing
no transgene (empty). Eight weeks later the immunizations were repeated
with an MVA-BN or with an Ad35 vector expressing the same antigen as the
Ad26 vectors. Control animals were boost-immunized with an MVA-BN vector
expressing no transgene (control). Two weeks after the second
immunization the mice were sacrificed and isolated splenocytes were
stimulated overnight with 15mer peptide pools corresponding to E2, E6 or
E7 of HPV16 and HPV18 E6. (B) shows the total IFN.gamma. response per
group as SFU per 10.sup.6 splenocytes. (student t-test on log transformed
data, with alpha=0.05, excluding negative control).
[0063] FIG. 24. Cellular immunogenicity induced by Ad26 prime and MVA
boost in Rhesus macaques. Rhesus macaques were immunized according to the
scheme as presented in FIG. 24A, with a combination of HPV16 and HPV18
designer constructs. At day 0: Several animals received a mixture of
Ad26.HPV16-E2E6E7SH and Ad26.HPV18-E2E6E7SH by intramuscular immunization
(i.m). A boost immunization MVA-BN encoding HPV16 E2E6E7SH and HPV18
E2E6E7SH vectors was given at 8 weeks. The dose of adenovectors was
1*10.sup.11 vp per vector. The dose of MVA was 1.8.times.10.sup.8
TCID.sup.50. Blood drawings were performed at several time points.
Cellular immune responses in PBMCs were measured by IFN.gamma. ELISPOT.
PBMCs were stimulated with peptide pools corresponding to HPV16 E2 (B),
HPV16 E6 (C), HPV16 E7 (D), HPV18 E2 (E), HPV18 E6 (F), HPV18 E7 (G) and
the number of spot-forming units (SFU) in 1*10.sup.6 PBMCs were
determined and shown in FIG. 24B-24G, respectively. FIG. 24H shows the
number of antigens per animal per time point. The cut-off was set to 50
SFU per 1*10.sup.6 PBMCs. FIG. 24I shows the cumulative responses for all
six tested peptide pools at different time points. Statistical analysis
for panel B-H: Wilcoxon Signed Rank test comparing week 13 with week 2,
and week 24 with week 13. A Bonferroni correction for 2 comparisons was
applied (adjusted p-values).
[0064] FIG. 25. Therapeutic effect of Ad26 priming and boost with either
Ad35 or MVA vectors expressing HPV16 and HPV18 E2E6E7SH. C57BL/6 mice
were injected sub-cutaneously with 5*10.sup.4 TC-1 cells at day 0. After
six days, when tumors were palpable, mice were immunized with a mixture
of Ad26.HPV16-E2E6E7SH and Ad26.HPV18-E2E6E7SH. Control mice received
Ad26.Empty. Mice received a boost immunization at day 20 with Ad35
vectors or MVA encoding HPV16/18 E2E6E7SH. Control animals were immunized
with a MVA not encoding a transgene. Tumor volume was calculated as
(width.sup.2*length)/2. Mice were sacrificed when tumor volumes surpassed
1000 mm.sup.3. The graphs show survival after TC-1 injection. One mouse
boost immunized with Ad35.HPV16 E2E6E7SH and Ad35.HPV18 E2E6E7SH and one
mouse boost immunized with MVA-BN HPV16/18 E2E6E7SH were tumor free at
the end of the experiment. The median survival time of mice boosted with
Ad35.HPV16/18-E2E6E7SH was not significantly different compared with mice
boost immunized with MVA-BN HPV16/18-E2E6E7SH.
DETAILED DESCRIPTION OF THE INVENTION
[0065] Various publications, articles and patents are cited or described
in the background and throughout the specification; each of these
references is herein incorporated by reference in its entirety.
Discussion of documents, acts, materials, devices, articles or the like
which has been included in the present specification is for the purpose
of providing context for the invention. Such discussion is not an
admission that any or all of these matters form part of the prior art
with respect to any inventions disclosed or claimed.
[0066] Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood to one of ordinary
skill in the art to which this invention pertains. Otherwise, certain
terms used herein have the meanings as set forth in the specification.
All patents, published patent applications and publications cited herein
are incorporated by reference as if set forth fully herein. It must be
noted that as used herein and in the appended claims, the singular forms
"a", "an", and "the" include plural reference unless the context clearly
dictates otherwise.
[0067] Unless otherwise stated, any numerical value, such as a
concentration or a concentration range described herein, are to be
understood as being modified in all instances by the term "about." Thus,
a numerical value typically includes .+-.10% of the recited value. For
example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL.
Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to
11% (w/v). As used herein, the use of a numerical range expressly
includes all possible subranges, all individual numerical values within
that range, including integers within such ranges and fractions of the
values unless the context clearly indicates otherwise.
[0068] The invention provides one or more vectors, vaccines, and vaccine
combinations that can be used for generating an immune response against
HPV infections and diseases associated therewith.
[0069] The vectors, vaccines, and vaccine combinations of embodiments of
the present invention comprise a nucleic acid molecule encoding one or
more polypeptides that are carefully designed molecules containing
virtually the complete E6 and E7 amino acid sequences, and in some
embodiments E2 as well, of HPV16 in the form of fragments that are
re-ordered and partly overlapping such that (essentially) all T-cell
epitopes of the HPV16 E6 and E7 protein are present. The vectors,
vaccines, and vaccine combinations of embodiments of the present
invention additionally comprise a nucleic acid molecule encoding one or
more polypeptides that are carefully designed molecules containing
virtually the complete E6 and E7 amino acid sequences, and in some
embodiments E2 as well, of HPV18 in the form of fragments that are
re-ordered and partly overlapping such that (essentially) all T-cell
epitopes of the HPV18 E6 and E7 protein are present. Earlier molecules
with some potential as HPV vaccines have been described by others (e.g.
Kenter et al., 2009, N Engl J Med 361: 1838-47; Daayana et al., 2010, Br
J Cancer 102: 1129-36; Smahel et al., 2001, Virology 281: 231-38; Yan et
al., 2009, Vaccine 27: 431-40; Ohlschlager et al., 2006, Vaccine 24:
2880-93; Oosterhuis et al., 2011, Int J Cancer 129: 397-406; EP1183368,
WO 2013/083287), but each of these molecules has one or more drawbacks.
The vectors, vaccines, and vaccine combinations of the present invention
are advantageous in at least one and typically several aspects with
respect to the approaches described earlier. In particular, advantages of
the present invention include, but are not limited to: (i) they have a
desired safety profile, as the nucleic acid molecules have a strongly
reduced (as compared to native E6 and E7 proteins), down to
non-detectable, transforming activity; (ii) they are single nucleic acid
molecules, which are easy to manufacture at industrial scale in an
economically feasible manner, and do not pose logistic challenges unlike
multiple molecule approaches; (iii) the encoded polypeptides of the
vaccines, and vaccine combinations comprise essentially all T-cell
epitopes of the native HPV16 and HPV 18 E6 and E7 proteins; (iv) the
design of the encoded polypeptides has minimized the introduction of
undesired potential strong neo-epitopes (i.e. epitopes not present in the
native E6 and E7 proteins); (v) in certain embodiments, they are not
dependent on highly reactogenic adjuvants to raise a desired immune
response; and (vi) in certain embodiments, as shown herein, the combined
administration (e.g. in prime-boost schedule) of the adenoviral vaccine
and the MVA vaccine provide an enhanced immune response, as compared to
administrations of the vaccines alone.
[0070] Thus, the vectors, vaccines, and vaccine combinations of
embodiments of the invention represent a major step forward by combining
various advantageous characteristics in a single design, and are
excellent candidates primarily for therapeutic vaccination against HPV16
and HPV18. These vectors, vaccines, and vaccine combinations could also
be used as prophylactic vaccines against HPV16 and HPV18, meaning that
they are likely to prevent persistent infection with HPV16, HPV18, or
both HPV16 and HPV18 of vaccinated subjects.
[0071] In developing certain embodiments of the invention, we used the
IEDB-AR to determine the possible formation of non-natural strong
epitopes that could be introduced at the newly created junctions between
the different E6 and E7 fragments. In certain embodiments for the HPV16
designer molecule, by careful design, the number of neo-epitopes with a
length of nine amino acids with a predicted binding affinity <50 nM
for the 20 most common HLA-A, 20 most common HLA-B and 20 most common
HLA-C alleles in the re-ordered HPV16 E6 and E7 sequences was minimized
to only 1. This is a significant improvement over constructs described by
others, which for a single shuffled HPV16 E6 protein already contained
more than 30 of such neo-epitopes, and which constructs will highly
likely comprise even several more neo-epitopes in sequences that were
appended to these constructs to prevent loss of epitopes (Ohlschlager et
al., 2006, Vaccine 24: 2880-93). Hence the constructs of the invention
have a significantly improved immunologic profile since chances of an
altered immune response as compared to native E6 and E7 have been
minimized in the molecules of the invention, as compared to approaches
described by others.
[0072] Skilled persons can, using routine techniques, make nucleotide
substitutions that do not affect the polypeptide sequence encoded by the
polynucleotides described to reflect the codon usage of any particular
host organism in which the polypeptides are to be expressed. Therefore,
unless otherwise specified, a "nucleotide sequence encoding an amino acid
sequence" includes all nucleotide sequences that are degenerate versions
of each other and that encode the same amino acid sequence. Nucleotide
sequences that encode proteins and RNA can include introns.
[0073] In a preferred embodiment, nucleic acid molecules encoding the
polypeptides, or HPV fusion proteins, according to the invention have
been configured for optimal expression and efficacy in one or more
vectors of the present invention. For example, a nucleic acid molecule
encoding an HPV 16 polypeptide comprising the amino acid sequence of SEQ
ID NO:1 in certain embodiments comprises the polynucleotide sequence of
SEQ ID NO: 2, a nucleic acid molecule encoding an HPV16 polypeptide
comprising the amino acid sequence of SEQ ID NO:3 in certain embodiments
comprises the polynucleotide sequence of SEQ ID NO:4 or SEQ ID NO:24, and
a nucleic acid molecule encoding an HPV16 polypeptide comprising the
amino acid sequence of SEQ ID NO:5 in certain embodiments comprises the
polynucleotide sequence of SEQ ID NO:6. Additionally, a nucleic acid
molecules encoding an HPV 18 polypeptide comprising the amino acid
sequence of SEQ ID NO:20 in certain embodiments comprises the
polynucleotide sequence of SEQ ID NO: 21 and a nucleic acid molecule
encoding an HPV18 polypeptide comprising the amino acid sequence of SEQ
ID NO:22 in certain embodiments comprises the polynucleotide sequence of
SEQ ID NO:23 or SEQ ID NO:25.
[0074] In certain embodiments, the nucleic acids according to the
invention encompass homologous or variant sequences. These sequences are
defined as having a percentage of sequence identity to the nucleic acids
of the invention.
[0075] Preferably, such homologues or variants have at least about 50%, at
least about 60% or 65%, at least about 70% or 75%, at least about 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically, at least
about 90%, 91%, 92%, 93%, or 94% and even more typically at least about
95%, 96%, 97%, 98% or 99%, most typically, at least about 99% sequence
identity to the referenced nucleic acid sequence. The term homologue or
variant also encompasses truncated, deleted or otherwise modified
nucleotide sequences.
[0076] Techniques for determining sequence identity between nucleic acids
are known in the art. Two or more sequences can be compared by
determining their "percent identity." The percent identity of two
sequences is the number of exact matches between two aligned sequences
divided by the length of the shorter sequences and multiplied by 100.
[0077] "Percent (%) sequence identity" with respect to nucleotide sequence
or nucleic acids is defined as the percentage of nucleotide residues in a
candidate sequence that are identical with the nucleotide residues in the
reference sequence (i.e., the nucleic acid sequence from which it is
derived), after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and not
considering any conservative substitutions as part of the sequence
identity. Alignment for purposes of determining percent amino acid
sequence identity can be achieved in various ways that are within the
skill in the art, for example, using publically available computer
software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those
skilled in the art can determine appropriate parameters for measuring
alignment, including any algorithms needed to achieve maximum alignment
over the full length of the sequences being compared.
[0078] For example, an appropriate alignment for nucleic acid sequences is
provided by the local homology algorithm of Smith and Waterman, (1981),
Advances in Applied Mathematics 2:482-489. This algorithm can be applied
to amino acid sequences by using the scoring matrix developed by Dayhoff,
Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.
3:353-358, National Biomedical Research Foundation, Washington, D.C.,
USA, and normalized by Gribskov (1986), Nucl. Acids Res. 14(6):6745-6763.
An exemplary implementation of this algorithm to determine percent
identity of a sequence is provided by the Genetics Computer Group
(Madison, Wis.) in the "BestFit" utility application. The default
parameters for this method are described in the Wisconsin Sequence
Analysis Package Program Manual, Version 8 (1995) (available from
Genetics Computer Group, Madison, Wis.). A preferred method of
establishing percent identity in the context of the present invention is
to use the MPSRCH package of programs copyrighted by the University of
Edinburgh, developed by John F. Collins and Shane S. Sturrok, and
distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this
suite of packages the Smith-Waterman algorithm can be employed where
default parameters are used for the scoring table (for example, gap open
penalty of 12, gap extension penalty of one, and a gap of six). From the
data generated the "Match" value reflects "sequence identity." Other
suitable programs for calculating the percent identity or similarity
between sequences are generally known in the art, for example, another
alignment program is BLAST, used with default parameters. For example,
BLASTN and BLASTP can be used using the following default parameters:
genetic code=standard; filter=none; strand=both; cutoff=60; expect=10;
Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;
Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+Swiss protein+Spupdate+PIR. Details of these programs can be
found at the following internet address:
http://http://blast.ncbi.nlm.nih.gov/.
[0079] Nucleic acid sequences can be cloned using routine molecular
biology techniques, or generated de novo by DNA synthesis, which can be
performed using routine procedures by service companies having business
in the field of DNA synthesis and/or molecular cloning (e.g. GeneArt,
GenScripts, Invitrogen, Eurofins).
[0080] It will be appreciated by a skilled person that changes can be made
to a protein, e.g. by amino acid substitutions, deletions, additions,
etc, e.g. using routine molecular biology procedures. Generally,
conservative amino acid substitutions may be applied without loss of
function or immunogenicity of a polypeptide. This can be checked
according to routine procedures well known to the skilled person.
[0081] In certain embodiments, the encoded polypeptides according to at
least one aspect of the invention further comprise a leader sequence,
also referred to as signal sequence or signal peptide. This is a short
(typically 5-30 amino acids long) peptide present at the N-terminus of
the majority of newly synthesized proteins that are destined towards the
secretory pathway. The presence of such a sequence may lead to increased
expression and immunogenicity. Non-limiting examples that can be used are
an IgE leader peptide (see e.g. U.S. Pat. No. 6,733,994; e.g. having
sequence MDWTWILFLVAAATRVHS (SEQ ID NO: 7)) or a HAVT20 leader peptide
(e.g. having sequence MACPGFLWALVISTCLEFSMA (SEQ ID NO: 9)). One of these
can optionally be added to the N-terminus of a polypeptide of the
invention. In other embodiments, a polypeptide according to the invention
does not comprise a leader sequence.
[0082] Diverse types of HPV exist (over 120 types have been identified and
are referred to by number), and generally for each type that needs to be
covered by a vaccine, type-specific antigens may need to be incorporated
in the vaccine, although for certain antigens some cross-reactivity might
exist. Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and
82 are carcinogenic "high-risk" sexually transmitted HPVs and may lead to
the development of cervical intraepithelial neoplasia (CIN), vulvar
intraepithelial neoplasia (VIN), vaginal intraepithelial neoplasia
(VaIN), penile intraepithelial neoplasia (PIN), and/or anal
intraepithelial neoplasia (AIN). The HPV according to the invention (i.e.
the HPV from which the E6 and E7 fragments in the encoded polypeptide are
derived) is HPV16 (for SEQ ID NOs: 1-6), or HPV18 (for SEQ ID NOs:
20-23). It can be used for subjects that are infected with HPV16 or
HPV18, respectively. It can in certain embodiments also suitably be
combined with vaccines against other HPV types. In certain embodiments,
this combination is with a vaccine against HPV of a high risk type as
identified above, e.g. a vaccine against HPV16 with a vaccine against
HPV18. In other embodiments, the vaccine of the invention is combined
with a vaccine against one or more of HPV-16, -18, -31, -33, -35, -39,
-45, -51, -52, -56, -58, -59, -68, -73, or -82. Such combinations could
for instance be used if the exact type of HPV infection is not yet
certain, or if an immune response with a prophylactic effect is desired
against more than one HPV type. Also combinations of the vaccines of the
invention with vaccines against HPV types that cause genital warts, such
as HPV6 and/or HPV11, are envisaged. Sequences of these HPV types and the
proteins encoded thereby (e.g. E6, E7, E2) are available to the skilled
person in public databases, such as the GenBank sequence database
provided by the National Center for of technology Information (NCBI).
[0083] A polypeptide according to one aspect of the invention for HPV16
comprises the amino acid sequence of SEQ ID NO: 1, and in one embodiment
a nucleic acid molecule according to the invention comprises the
polynucleotide sequence of SEQ ID NO: 2. A polypeptide according to the
invention for HPV18 comprises the amino acid sequence of SEQ ID NO: 20,
and in one embodiment a nucleic acid molecule according to the invention
comprises the polynucleotide sequence of SEQ ID NO: 21.
[0084] Sequences herein are provided from 5' to 3' direction or from N- to
C-terminus, as custom in the art.
[0085] The encoded polypeptides according to the invention comprise the
epitopes of HPV16 E6 and E7 proteins, or alternatively the epitopes of
HPV18 E6 and E7 proteins. In certain embodiments, the polypeptide
according to the invention further comprises (and hence the nucleic acid
encoding the polypeptide further encodes) at least one further antigen or
epitope(s) of such further antigen. Such a further antigen preferably is
an HPV antigen, preferably of the same HPV type as the E6 and E7 proteins
in the polypeptide, i.e. HPV16 or HPV18 respectively. Such a further
antigen can thus be an HPV protein or an immunogenic fragment thereof,
and in certain embodiments comprises an E2 protein or a fragment thereof
comprising at least one epitope of E2 of HPV, preferably from HPV16 or
HPV18. Such further antigens or epitopes can be expressed independently
of an E6 and E7 polypeptide according to the invention. Such further
antigens or epitopes can also be expressed as a part of a fusion protein,
for example, being placed internally between two fragments of an E6
and/or E7 in a polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 or SEQ ID NO: 20, but preferably being fused N-terminally or
C-terminally to an E6/E7 in a polypeptide comprising the amino acid
sequence of SEQ ID NO: 1 or SEQ ID NO: 20. Alternatively or in addition,
amino acid sequences can be present that stimulate the immune response.
Thus, in certain embodiments the invention provides nucleic acid
molecules according to the invention, encoding a polypeptide comprising
the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 20, and wherein the
polypeptide further comprises at least one other antigen, e.g. HPV E2
protein or at least one epitope, but preferably more epitopes, thereof.
One advantage of the addition of E2 antigen for the instant invention is
that E2 is known to be expressed early during infection/in low grade
lesions where E6 and E7 expression is still very low.
[0086] During the development towards cervical cancer E2 expression is
lost and as a result E6 and E7 levels are increased (Yugawa and Kiyono,
2009, Rev Med Virol 19: 97-113). Combining epitopes from E2, E6 and E7 in
one vaccine allows for treatment in a broad target group of patients,
ranging from having persistent infection to invasive cervical cancer (or
other HPV16-caused cancers). In certain embodiments, the E2 protein is a
wild-type E2 protein. In certain other embodiments, the E2 protein has a
deletion or one or more mutations in its DNA binding domain (as compared
to a wild type E2 protein). The sequence of the HPV16 and HPV18 E2
proteins can be found in the NCBI protein database
(www.ncbi.nlm.nih.gov/protein) under numbers NP_041328.1 and AAP20597.1,
respectively. Several single amino acid changes in HPV16 E2 such as
G293V, K299M, or C300R in the C-terminal part of this protein are known
to abrogate DNA binding. For HPV18 E2, the corresponding amino acid
changes are G294V, K300M, C301R.
[0087] An advantage of using a variant or fragment of E2 that lacks DNA
binding capacity is that it could prevent unpredictable transcriptional
changes via direct binding to host cell DNA in the cells where it is
expressed. In addition to or as an alternative to mutations in the DNA
binding domain described above, further approaches to prevent E2 activity
are to introduce mutations that abrogate activity of the more
N-terminally located E2 transactivation domain, and/or that are reported
to affect the structure of the E2 polypeptide. For HPV16 E2, non-limiting
examples of amino acid changes at positions that have previously been
described (e.g. Brokaw et al, 1996; Sakai et al, 1996) are R37A, I73A,
W92A, E39A, W33A, P106A and G156A, and HPV16 E2 according to the
invention could optionally comprise one or more of these mutations in the
transactivation domain. In one preferred embodiment, the HPV16 E2
fragment comprises a nucleic acid encoding a polypeptide comprising the
amino acid sequence of SEQ ID NO: 28. In a more particular embodiment,
the nucleic acid sequence encoding for SEQ ID NO: 28 comprises the
polynucleotide sequence of SEQ ID NO: 29 or SEQ ID NO: 30. For HPV18 E2,
the corresponding amino acid changes are R41A, I77A, W96A, E43A, W37A,
P110A and G161A, and HPV18 E2 according to the invention could thus
optionally comprise one or more of these mutations in the transactivation
domain. In one preferred embodiment, the HPV18 E2 fragment comprises a
nucleic acid encoding a polypeptide comprising the amino acid sequence of
SEQ ID NO: 31. In a more particular embodiment, the nucleic acid sequence
encoding for SEQ ID NO: 31 comprises the polynucleotide sequence of SEQ
ID NO: 32 or SEQ ID NO: 33.
[0088] In certain embodiments, E2 has mutations in the transactivation
domain, in other embodiments E2 has mutations in the DNA binding domain,
and in further embodiments E2 has mutations in both the transactivation
domain and in the DNA binding domain. In yet another alternative
embodiment, the E2 polypeptide according to the invention is divided in
fragments which are reordered (shuffled), to abrogate E2 activity while
maintaining the E2 epitopes for immunogenicity. Such embodiment could
optionally be combined with one or more of the mutations described above,
e.g. in the DNA binding domain and/or in the transactivation domain.
Besides wild-type HPV E2 polypeptides, all such E2 mutants can be used as
the E2 protein or part or variant thereof according to the invention.
[0089] The E2 protein or part or variant thereof, such as, but not limited
to those described herein, can be added internally, but preferably is
fused to the N-terminus or to the C-terminus of a polypeptide of the
invention having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:
20. In one embodiment for HPV16, a nucleic acid molecule of the invention
encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 3.
In one embodiment thereof, the nucleic acid molecule of the invention
comprises the polynucleotide sequence of SEQ ID NO: 4 or SEQ ID NO:24. In
another embodiment for HPV16, a nucleic acid molecule of the invention
encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 5.
In one embodiment thereof, a nucleic acid molecule of the invention
comprises the polynucleotide sequence of SEQ ID NO: 6. In one embodiment
for HPV18, a nucleic acid molecule of the invention encodes a polypeptide
comprising the amino acid sequence of SEQ ID NO: 22. In one embodiment
thereof, a nucleic acid molecule of the invention comprises the
polynucleotide sequence of SEQ ID NO: 23 or SEQ ID NO: 25.
[0090] It is also possible to make further fusions of the designer
polypeptides of the invention with further proteins, e.g. so called
carrier proteins, such as Calreticulin, Mycobacterium Tubercelosis heat
shock protein-70, IP10, or Tetanus toxin fragment C (see Oosterhuis et
al., Human Gene Ther, 2012, supra, for more examples), which could
further enhance the immune response to the HPV E6 and E7 (and optionally
E2) epitopes. The invention thus also provides such further fusion
proteins, and nucleic acids encoding such.
[0091] In certain embodiments, one or more of the nucleic acid molecules
according to the invention are incorporated into a vector. A "vector" as
used herein, is typically a vehicle to artificially carry foreign genetic
material into another cell, where it can be replicated and/or expressed,
and according to the invention can be any nucleic acid molecule that
incorporates a nucleic acid molecule according to the invention. These
can be prepared according to routine molecular biology techniques such as
cloning. Typically such vectors can be propagated in at least one type of
suitable hosts such as bacteria, yeast, insect cells, mammalian cells,
and the like. Four major types of vectors are plasmids, viral vectors,
cosmids, and artificial chromosomes. The vector itself is generally a DNA
sequence that consists of an insert (transgene; in the present invention
the nucleic acid encoding the fusion polypeptide of the invention) and a
sequence that serves as the "backbone" of the vector. The purpose of a
vector which transfers genetic information to another cell is typically
to isolate, multiply, or express the insert in the target cell.
[0092] Preferably, the sequence encoding the polypeptide is operably
linked to a promoter in the vector. The term "operably linked" is
intended to mean that the nucleotide sequence of interest is linked to
the promoter in a manner that allows for expression of the nucleotide
sequence (e.g., in a host cell when the vector is introduced into the
host cell). Expression regulatory sequences can be operably linked to a
transgene. In certain embodiments, vectors are designed for the
expression of the transgene in the target cell, and generally have a
promoter sequence that drives expression of the transgene. In certain
embodiments, one or more of routinely used vector elements such as
transcription terminator sequences, polyadenylation tail sequences, Kozak
sequences, UTRs, origin of replication, multiple cloning sites, genetic
markers, antibiotic resistance, and further sequences may be present, and
the skilled person can design a vector such that it has the desired
properties, e.g. for replication in certain cells for propagation and
multiplication of the vector, and for expression of the transgene of the
vector in target cells into which the vector is introduced. Vectors
comprising the nucleic acid encoding the polypeptide according to the
invention, preferably designed for expression in mammalian cells, are
suitable as vaccines according to the invention. In certain embodiments,
a vector according to the invention is a plasmid, a cosmid, a yeast
artificial chromosome, a bacterial artificial chromosome, a viral vector,
or the like. The person skilled in the art is aware that various
promoters can be used to obtain expression of a gene in host cells. Some
well-known and much used promoters for expression in eukaryotic cells
comprise promoters derived from viruses, such as adenovirus, e.g. the E1A
promoter, promoters derived from cytomegalovirus (CMV), such as the CMV
immediate early (IE) promoter (referred to herein as the CMV promoter)
(obtainable for instance from pcDNA, Invitrogen), promoters derived from
Simian Virus 40 (SV40) (e.g. obtainable from pIRES, cat. no. 631605, BD
Sciences), and the like. Suitable promoters can also be derived from
eukaryotic cells, such as methallothionein (MT) promoters, elongation
factor 1.alpha. (EF-1.alpha.) promoter, ubiquitin C or UB6 promoter,
actin promoter, an immunoglobulin promoter, heat shock promoters, and the
like (see e.g. WO 2006/048459). A non-limiting example of a suitable
promoter for obtaining expression in eukaryotic cells is a CMV-promoter
(U.S. Pat. No. 5,385,839), e.g. the CMV immediate early promoter, for
instance comprising nt. -735 to +95 from the CMV immediate early gene
enhancer/promoter, e.g. a CMV promoter as provided herein with a sequence
as set forth in SEQ ID NO: 13. A polyadenylation signal, for example the
bovine growth hormone polyA signal (U.S. Pat. No. 5,122,458), may be
present behind the transgene(s). In another, non-limiting example, the
promoter can be a PrMVA13.5long promoter (WO 2014/063832) or a PrHyb
promoter (U.S. Pat. Nos. 8,394,385, 8,613,936), which comprise the
polynucleotide sequence of SEQ ID NO: 26 and SEQ ID NO: 27, respectively,
and which are particularly useful for driving expression of transgenes in
MVA vectors.
[0093] Further regulatory sequences may also be added. The term
"regulatory sequence" is used interchangeably with "regulatory element"
herein and refers to a segment of nucleic acid, typically but not limited
to DNA, that modulate the transcription of the nucleic acid sequence to
which it is operatively linked, and thus acts as a transcriptional
modulator. A regulatory sequence often comprises nucleic acid sequences
that are transcription binding domains that are recognized by the nucleic
acid-binding domains of transcriptional proteins and/or transcription
factors, enhancers or repressors etc. For example, it is possible to
operably couple a repressor sequence to the promoter, which repressor
sequence can be bound by a repressor protein that can decrease or prevent
the expression of the transgene in a production cell line that expresses
this repressor protein. This may improve genetic stability and/or
expression levels of the nucleic acid molecule upon passaging and/or when
this is produced at high quantities in the production cell line. Such
systems have been described in the art. For example, a regulatory
sequence could include one or more tetracycline operon operator sequences
(tetO), such that expression is inhibited in the presence of the
tetracycline operon repressor protein (tetR). In the absence of
tetracycline, the tetR protein is able to bind to the tetO sites and
repress transcription of a gene operably linked to the tetO sites. In the
presence of tetracycline, however, a conformational change in the tetR
protein prevents it from binding to the operator sequences, allowing
transcription of operably linked genes to occur. In certain embodiments,
a nucleic acid molecule, e.g. when present in a recombinant adenovirus
vector, of the present invention can optionally include tetO operatively
linked to a promoter, such that expression of one or more transgenes is
inhibited in recombinant adenoviruses that are produced in the producer
cell line in which tetR protein is expressed. Subsequently, expression
would not be inhibited if the recombinant adenovirus is introduced into a
subject or into cells that do not express the tetR protein (e.g.,
international patent application WO 07/073513). In certain other
embodiments, a nucleic acid molecule of the present invention, e.g. when
present in a recombinant adenovirus, can optionally include a cumate
gene-switch system, in which regulation of expression is mediated by the
binding of the repressor (CymR) to the operator site (CuO), placed
downstream of the promoter (e.g., Mullick et al. BMC Biotechnol. 2006
6:43). As used herein, the term "repressor," refers to entities (e.g.,
proteins or other molecules) having the capacity to inhibit, interfere,
retard and/or repress the production of heterologous protein product of a
recombinant expression vector. For example, by interfering with a binding
site at an appropriate location along the expression vector, such as in
an expression cassette. Examples of repressors include tetR, CymR, the
lac repressor, the trp repressor, the gal repressor, the lambda
repressor, and other appropriate repressors known in the art. Examples of
the use of the tetO/tetR operator/repressor system and of the CuO/CymR
operator/repressor system are provided herein. Repression of vector
transgene expression during vector propagation can prevent transgene
instability, and may increase yields of vectors having a transgene of the
invention during production. Hence, in some embodiments, the vectors of
the invention have a promoter that can be repressed by binding of a
repressor protein, e.g. by having a promoter that is operably coupled to
a repressor operator sequence (e.g. in non-limiting embodiments, a
TetO-containing sequence, e.g. the one set forth in the polynucleotide
sequence of SEQ ID NO: 11, or a CuO-containing sequence, e.g. the one set
forth in the polynucleotide sequence of SEQ ID NO: 12), to which a
repressor protein (e.g. the TetR protein, e.g. having the amino acid
sequence as set forth in SEQ ID NO: 15, or the CymR protein, e.g. having
the amino acid sequence as set forth in SEQ ID NO: 17) can bind.
[0094] In preferred embodiments, the vector is a recombinant viral vector,
which can be replication competent or replication deficient or defective.
In certain embodiments, a viral vector comprises a recombinant DNA
genome. In certain embodiments, a vector according to the invention is,
for instance, a recombinant adenovirus, a recombinant pox virus such as
an orthopoxvirus (e.g., a vaccinia virus, Modified Vaccinia Ankara
(MVA)).
[0095] In one or more preferred embodiments, the vector according to the
invention is recombinant poxvirus such as, but not limited to an
orthopoxvirus. The recombinant orthopoxvirus can be a vaccinia virus
(VV), a Wyeth strain, ACAM 1000, ACAM 2000, MVA, or MVA-BN.
[0096] In a more preferred embodiment, the recombinant poxvirus is MVA. In
certain preferred embodiments, the MVA is MVA-BN. MVA-BN is replication
incompetent, which is a significant advantage over other types of MVA.
MVA-BN was deposited on Aug. 30, 2000, at the European Collection of Cell
Cultures (ECACC) under number V00083008, and is described in
International PCT publication WO2002042480 (see also e.g. EP Patent No.
1335987, U.S. Pat. Nos. 6,761,893, 6,913,752, 7,335,364, 7,459,270,
7,939,086, and 8,268,325). As described in those patent publications,
MVA-BN does not reproductively replicate in cell lines 293, 143B, HeLa
and HaCat.
[0097] In certain embodiments, a recombinant MVA is a derivative of
MVA-BN. Such "derivatives" include viruses exhibiting essentially the
same replication characteristics as the deposited strain (ECACC No.
V00083008), but exhibiting differences in one or more parts of its
genome. MVA-BN derivatives, as used herein, are characterized: i) in
being capable of reproductive replication in chicken embryo fibroblasts
(CEF) cells and the Baby Hamster Kidney cell line BHK but not capable of
reproductive replication in the human cell lines HaCat, HeLa, and 143B;
and ii) by a failure to replicate in a mouse strain that is incapable of
producing mature B and T cells and as such is severely immune compromised
and highly susceptible to a replicating virus. These characteristics and
tests therefor have been well defined in the art (e.g. WO2002042480, U.S.
Pat. Nos. 6,761,893 and 6,913,752).
[0098] In certain embodiments, the nucleic acid molecules described herein
are incorporated in a variety of insertion sites, or intergenic regions
in the MVA genome, or in the MVA-BN genome. The nucleic acid molecules
can be inserted into the recombinant MVA, or MVA-BN as separate
transcriptional units or as fusion genes, as described herein. In certain
embodiments, the nucleic acid molecules are inserted into one or more
intergenic regions (IGR) of the MVA, or MVA-BN. The IGR may be selected
from IGR07/08, IGR 44/45, IGR 64/65, IGR 88/89, IGR 136/137, and IGR
148/149, preferably from IGR64/65, IGR88/89, and/or IGR 148/149. These
IGRs are further characterized in WO 03/097845 (see also, e.g., EP Patent
No. 1407033 and U.S. Pat. Nos. 7,550,147, 7,964,374, 8,034,354, and
8,741,308). The nucleic acid molecules may, additionally or
alternatively, be inserted into one or more of the naturally occurring
deletion sites I, II, II, IV, V, or VI of the MVA, or MVA-BN. In certain
embodiments, less than 5, 4, 3, or 2 of the integration sites comprise
the nucleic acid molecules of the present disclosure.
[0099] The number of insertion sites of MVA, or MVA-BN comprising the
nucleic acid molecules can be 1, 2, 3, 4, 5, 6, 7, or more. The
recombinant MVA, or MVA-BN can comprise the nucleic acid molecules
inserted into 4, 3, 2, or 1 insertion sites.
[0100] In certain preferred embodiments, the nucleic acid molecules of the
present disclosure are inserted into a single insertion site. In a
preferred embodiment, nucleic acid molecules inserted in the single
insertion site is a nucleic acid molecule encoding a polypeptide
comprising an amino acid sequence selected from the group consisting of:
SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 20, SEQ ID NO: 22,
and combinations thereof. In other preferred embodiments, nucleic acid
molecules inserted in the single insertion site encode the amino acid
sequences of SEQ ID NO: 3 and SEQ ID NO: 22. In still other preferred
embodiments, a nucleic acid molecule inserted in the single insertion
site is one or more nucleic acid molecules having at least 90% sequence
identity to a polynucleotide sequence selected from the group consisting
of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 21, SEQ ID NO:
23, SEQ ID NO: 24, and SEQ ID NO: 25, preferably comprising a
polynucleotide sequence selected from the group consisting of SEQ ID NO:
2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:
24, and SEQ ID NO: 25. In still more preferred embodiments, two or more
nucleic acid molecules are inserted in the single insertion site, the
nucleic acid molecules have at least 90% sequence identity to the
polynucleotide sequences of SEQ ID NO: 24 and ID NO: 25, respectively,
preferably, comprise the polynucleotide sequences of SEQ ID NO: 24 and ID
NO: 25, respectively. In another preferred embodiment the single
insertion site is IGR88/89.
[0101] The recombinant MVA viruses provided herein can be generated by
routine methods known in the art in view of the present disclosure.
Methods to obtain recombinant poxviruses or to insert heterologous
nucleotide sequences into a poxviral genome are well known to the person
skilled in the art. For example, methods for standard molecular biology
techniques such as cloning of DNA, DNA and RNA isolation, Western blot
analysis, RT-PCR and PCR amplification techniques are described in
Molecular Cloning, A laboratory Manual (2nd Ed.) [J. Sambrook et al.,
Cold Spring Harbor Laboratory Press (1989)], and techniques for the
handling and manipulation of viruses are described in Virology Methods
Manual [B. W. J. Mahy et al. (eds.), Academic Press (1996)]. Similarly,
techniques and know-how for the handling, manipulation and genetic
engineering of MVA are described in Molecular Virology: A Practical
Approach [A. J. Davison & R. M. Elliott (Eds.), The Practical Approach
Series, IRL Press at Oxford University Press, Oxford, UK (1993)(see,
e.g., Chapter 9: Expression of genes by Vaccinia virus vectors)] and
Current Protocols in Molecular Biology [John Wiley & Son, Inc.
(1998)(see, e.g., Chapter 16, Section IV: Expression of proteins in
mammalian cells using vaccinia viral vector)].
[0102] For the generation of the various recombinant MVAs disclosed
herein, different methods can be applicable. The nucleotide sequences to
be inserted into the virus can be placed into an E. coli plasmid
construct into which DNA homologous to a section of DNA of the MVA has
been inserted. Separately, the DNA sequence to be inserted can be ligated
to a promoter. The promoter-gene linkage can be positioned in the plasmid
construct so that the promoter-gene linkage is flanked on both ends by
DNA homologous to a DNA sequence flanking a region of MVA DNA containing
a non-essential locus. The resulting plasmid construct can be amplified
by propagation within E. coli bacteria and isolated. The isolated plasmid
containing the DNA gene sequence to be inserted can be transfected into a
cell culture, e.g., of chicken embryo fibroblasts (CEFs), at the same
time the culture is infected with MVA. Recombination between homologous
MVA DNA in the plasmid and the viral genome, respectively, can generate
an MVA modified by the presence of foreign DNA sequences.
[0103] According to a preferred embodiment, a cell of a suitable cell
culture such as, e.g., CEF cells, can be infected with a poxvirus. The
infected cell can be, subsequently, transfected with a first plasmid
vector comprising a foreign gene or genes, preferably under the
transcriptional control of a poxvirus expression control element. As
explained above, the plasmid vector also comprises sequences capable of
directing the insertion of the exogenous sequence into a selected part of
the poxviral genome. Optionally, the plasmid vector also contains a
cassette comprising a marker and/or selection gene operably linked to a
poxviral promoter. Suitable marker or selection genes are, e.g., the
genes encoding the green or red fluorescent protein, beta-galactosidase,
neomycin-phosphoribosyltransferase, ecogpt, or other markers. The use of
selection or marker cassettes simplifies the identification and isolation
of the generated recombinant poxvirus. However, a recombinant poxvirus
can also be identified by PCR technology. Subsequently, a further cell
can be infected with the recombinant poxvirus obtained as described above
and transfected with a second vector comprising a second foreign gene or
genes. In case, this gene can be introduced into a different insertion
site of the poxviral genome, the second vector also differs in the
poxvirus-homologous sequences directing the integration of the second
foreign gene or genes into the genome of the poxvirus. After homologous
recombination has occurred, the recombinant virus comprising two or more
foreign genes can be isolated. For introducing additional foreign genes
into the recombinant virus, the steps of infection and transfection can
be repeated by using the recombinant virus isolated in previous steps for
infection and by using a further vector comprising a further foreign gene
or genes for transfection.
[0104] Alternatively, the steps of infection and transfection as described
above are interchangeable, i.e., a suitable cell can at first be
transfected by the plasmid vector comprising the foreign gene and, then,
infected with the poxvirus. As a further alternative, it is also possible
to introduce each foreign gene into different viruses, coinfect a cell
with all the obtained recombinant viruses and screen for a recombinant
including all foreign genes. A third alternative is ligation of DNA
genome and foreign sequences in vitro and reconstitution of the
recombined vaccinia virus DNA genome using a helper virus. A fourth
alternative is homologous recombination in E. coli or another bacterial
species between a vaccinia virus genome cloned as a bacterial artificial
chromosome (BAC) and a linear foreign sequence flanked with DNA sequences
homologous to sequences flanking the desired site of integration in the
vaccinia virus genome.
[0105] In other preferred embodiments, a vector according to the invention
is a recombinant adenovirus. Advantages of adenoviruses for use as
vaccines include ease of manipulation, good manufacturability at large
scale, and an excellent safety record based on many years of experience
in research, development, manufacturing and clinical trials with numerous
adenoviral vectors that have been reported. Adenoviral vectors that are
used as vaccines generally provide a good immune response to the
transgene-encoded protein, including a cellular immune response. An
adenoviral vector according to the invention can be based on any type of
adenovirus, and in certain embodiments is a human adenovirus, which can
be of any serotype. In other embodiments, it is a simian adenovirus, such
as chimpanzee or gorilla adenovirus, which can be of any serotype. In
certain embodiments, a vector according to the invention is of a human
adenovirus serotype 26 or 35. The preparation of recombinant adenoviral
vectors is well known in the art. In certain embodiments, an adenoviral
vector according to the invention is deficient in at least one essential
gene function of the E1 region, e.g. the E1a region and/or the E1b
region, of the adenoviral genome that is required for viral replication.
In certain embodiments, an adenoviral vector according to the invention
is deficient in at least part of the non-essential E3 region. In certain
embodiments, the vector is deficient in at least one essential gene
function of the E1 region and at least part of the non-essential E3
region.
[0106] Adenoviral vectors, methods for construction thereof and methods
for propagating thereof, are well known in the art and are described in,
for example, U.S. Pat. Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806,
5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, and
6,113,913, and Thomas Shenk, "Adenoviridae and their Replication", M. S.
Horwitz, "Adenoviruses", Chapters 67 and 68, respectively, in Virology,
B. N. Fields et al., eds., 3d ed., Raven Press, Ltd., New York (1996),
and other references mentioned herein. Typically, construction of
adenoviral vectors involves the use of standard molecular biological
techniques, such as those described in, for example, Sambrook et al.,
Molecular Cloning, a Laboratory Manual, 2d ed., Cold Spring Harbor Press,
Cold Spring Harbor, N.Y. (1989), Watson et al., Recombinant DNA, 2d ed.,
Scientific American Books (1992), and Ausubel et al., Current Protocols
in Molecular Biology, Wiley Interscience Publishers, NY (1995), and other
references mentioned herein.
[0107] Particularly preferred serotypes for the recombinant adenovirus are
human serotype 35 or human serotype 26, most preferably human serotype 26
(rAd26). Preparation of rAd26 vectors is described, for example, in WO
2007/104792 and in Abbink et al., 2007 Virology 81: 4654-63. Exemplary
genome sequences of Ad26 are found in GenBank Accession EF 153474 and in
SEQ ID NO:1 of WO 2007/104792. Preparation of rAd35 vectors is described,
for example, in U.S. Pat. No. 7,270,811, in WO 00/70071, and in Vogels et
al., 2003, J Virol 77: 8263-71. Exemplary genome sequences of Ad35 are
found in GenBank Accession AC_000019 and in FIG. 6 of WO 00/70071.
[0108] In certain embodiments, the adenovirus is replication deficient,
e.g. because it contains a deletion in the E1 region of the genome. As
known to the skilled person, in case of deletions of essential regions
from the adenovirus genome, the functions encoded by these regions have
to be provided in trans, preferably by the producer cell, i.e. when parts
or whole of E1, E2 and/or E4 regions are deleted from the adenovirus,
these have to be present in the producer cell, for instance integrated in
the genome thereof, or in the form of so-called helper adenovirus or
helper plasmids. The adenovirus may also have a deletion in the E3
region, which is dispensable for replication, and hence such a deletion
does not have to be complemented.
[0109] A producer cell (sometimes also referred to in the art and herein
as `packaging cell` or `complementing cell`) that can be used can be any
producer cell wherein a desired adenovirus can be propagated. For
example, the propagation of recombinant adenovirus vectors is done in
producer cells that complement deficiencies in the adenovirus. Such
producer cells preferably have in their genome at least an adenovirus E1
sequence, and thereby are capable of complementing recombinant
adenoviruses with a deletion in the E1 region. Any E1-complementing
producer cell can be used, such as human retina cells immortalized by E1,
e.g. 911 or PER.C6 cells (see U.S. Pat. No. 5,994,128), E1-transformed
amniocytes (See EP patent 1230354), E1-transformed A549 cells (see e.g.
WO 98/39411, U.S. Pat. No. 5,891,690), GH329:HeLa (Gao et al., 2000, Hum
Gene Ther 11: 213-19), 293, and the like. In certain embodiments, the
producer cells are for instance HEK293 cells, or PER.C6 cells, or 911
cells, or IT293 SF cells, and the like. Production of adenoviral vectors
in producer cells is reviewed in (Kovesdi et al., 2010, Viruses 2:
1681-703).
[0110] In certain embodiments, an E1-deficient adenovirus comprises the
E4-orf6 coding sequence of an adenovirus of subgroup C such as Ad5. This
allows propagation of such adenoviruses in well known complementing cell
lines that express the E1 genes of AdS, such as for example 293 cells or
PER.C6 cells (see, e.g. Havenga et al., 2006, J Gen Virol 87: 2135-43; WO
03/104467, incorporated in its entirety by reference herein).
[0111] "Heterologous nucleic acid" (also referred to herein as
`transgene`) in vectors of the invention is nucleic acid that is not
naturally present in the vector, and according to the present invention
the nucleic acid encoding the fusion polypeptide of the invention is
considered heterologous nucleic acid when present in a vector. It is
introduced into the vector for instance by standard molecular biology
techniques. It can for instance be cloned into a deleted E1 or E3 region
of an adenoviral vector, or in the region between the E4 region and the
rITR. A transgene is generally operably linked to expression control
sequences. In preferred embodiments, the transgene is cloned into the
E1-region of an adenoviral vector.
[0112] Production of vectors such as MVA vectors, or recombinant
adenovirus vectors, can be performed according to various methods well
known to the person skilled in the art in view of the present disclosure.
Generally, the production entails propagation in cultured cells to
generate a substantial amount of vector material, followed by harvest of
the vector from the cell culture, and typically followed by further
purification of the vector to remove other substances and obtain purified
vectors that can be formulated into pharmaceutical compositions (e.g.,
Hoganson et al., 2002, BioProcessing J 1: 43-8; Evans et al., 2004, J
Pharm Sci 93:2458-75). For example, methods for harvesting adenovirus
from cultures of producer cells have for instance been extensively
described in WO 2005/080556. For example WO 2010/060719, and WO
2011/098592, both incorporated by reference herein, describe suitable
methods for obtaining and purifying large amounts of recombinant
adenoviruses.
[0113] In additional aspects, the invention further provides vaccines, and
vaccine combinations comprising nucleic acid molecules, vectors, or
recombinant viruses according to the invention, wherein embodiments for
each of these aspects can include those as described herein. In certain
embodiments, a vaccine according to the invention comprises a nucleic
acid molecule described herein. In preferred embodiments, the vaccine
comprises a vector according to the invention, preferably a recombinant
poxvirus vector such as an MVA vector, preferably MVA-BN vector or
derivatives thereof, and/or a recombinant adenovirus vector, such as a
rAd26 vector.
[0114] In certain embodiments, a vaccine according to the invention that
encodes the HPV16 designer polypeptide comprises further active
ingredients, e.g. nucleic acid encoding at least one epitope of E6 and/or
E7 protein of at least one HPV type different from HPV16, e.g. a high
risk HPV type such as HPV18, -31, -33, -35, -39, -45, -51, -52, -56, -58,
-59, -68, -73, or -82. In certain embodiments, a vaccine according to the
invention that encodes the HPV18 designer polypeptide comprises further
active ingredients, e.g. nucleic acid encoding at least one epitope of E6
and/or E7 protein of at least one HPV type different from HPV18, e.g. a
high risk HPV type such as HPV16, -31, -33, -35, -39, -45, -51, -52, -56,
-58, -59, -68, -73, or -82.
[0115] Particularly preferred are vectors, vaccines, or vaccine
combinations comprising nucleic acids encoding both HPV16 and HPV18
designer polypeptides of the invention, e.g., encoding a polypeptide
comprising the amino acid sequence of SEQ ID NO: 1 as well as a
polypeptide comprising the amino acid sequence of SEQ ID NO: 20; e.g.,
encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 3
as well as a polypeptide comprising the amino acid sequence of SEQ ID NO:
22. In such vectors, vaccines, vaccine compositions, or vaccine
combinations, the HPV16 and HPV18 components can be in the same
composition as separate molecules, or they can be in the same molecule
e.g. encoded on the same vector. One advantage of such combinations is
that such vaccines can work therapeutically in subjects that are infected
with either HPV16 or with HPV18 (the two most prevailing high risk HPV
types that together account for the majority of HPV-induced cancers), so
that such vaccines have increased applicability over the monotype
vaccines that have either HPV16 or HPV18 designer molecules.
[0116] In other embodiments, the HPV16 and HPV18 components could be
provided as a kit or composition of parts with a separate HPV16 component
and a separate HPV18 component for combined use in vaccination, e.g. for
reconstitution prior to administration, or for separate but essentially
simultaneous administration. One advantage of such combinations is that
such vaccines can work therapeutically in subjects that are infected with
either HPV16 or with HPV18. The term "vaccine" refers to an agent or
composition containing an active component effective to induce a
prophylactic and/or therapeutic degree of immunity in a subject against a
certain pathogen or disease, in this case therapeutically against HPV.
The vaccine typically comprises the nucleic acid molecule, or vector, or
recombinant virus according to the invention, and a pharmaceutically
acceptable excipient. Upon administration to a subject, the polypeptide
encoded by the nucleic acid molecule according to the invention will be
expressed in the subject, which will lead to an immune response towards
antigenic fragments that are present in the encoded polypeptide. The
advantage of a vaccine of the present invention is that essentially all
T-cell epitopes of E6 and E7 of HPV16 (e.g., SEQ ID NOs: 1-6, and 24) or
HPV18 (e.g., SEQ ID NOs: 20-23, and 25), and optionally epitopes of E2 of
HPV16 or HPV18 are present and thus a T-cell response to any epitope
present in wild-type E6, or E7, or optionally E2, can be mounted in the
vaccinee. Further, the vaccine has all the safety and efficacy advantages
as outlined above for the nucleic acid molecules according to the
invention.
[0117] For administering to humans, the invention may employ
pharmaceutical compositions comprising the vector and a pharmaceutically
acceptable carrier or excipient. In the present context, the term
"Pharmaceutically acceptable" means that the carrier or excipient, at the
dosages and concentrations employed, will not cause any unwanted or
harmful effects in the subjects to which they are administered. Such
pharmaceutically acceptable excipients are well known in the art (see
Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed.,
Mack Publishing Company [1990]; Pharmaceutical Formulation Development of
Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor &
Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition,
A. Kibbe, Ed., Pharmaceutical Press [2000]). An excipient is generally a
pharmacologically inactive substance formulated with the active
ingredient of a medication. Excipients are commonly used to bulk up
formulations that contain potent active ingredients (thus often referred
to as "bulking agents," "fillers," or "diluents"), to allow convenient
and accurate dispensation of a drug substance when producing a dosage
form. They also can serve various therapeutic-enhancing purposes, such as
facilitating drug absorption or solubility, or other pharmacokinetic
considerations. Excipients can also be useful in the manufacturing
process, to aid in the handling of the active substance concerned such as
by facilitating powder flowability or non-stick properties, in addition
to aiding in vitro stability such as prevention of denaturation over the
expected shelf life. The selection of appropriate excipients also depends
upon the route of administration and the dosage form, as well as the
active ingredient and other factors.
[0118] The purified nucleic acid molecule, vector or polypeptide
preferably is formulated and administered as a sterile solution although
it is also possible to utilize lyophilized preparations. Sterile
solutions are prepared by sterile filtration or by other methods known
per se in the art. The solutions are then lyophilized or filled into
pharmaceutical dosage containers. The pH of the solution generally is in
the range of pH 3.0 to 9.5, e.g., pH 5.0 to 7.5. The nucleic acid
molecule or vector or polypeptide typically is in a solution having a
suitable buffer, and the solution of vector may also contain a salt.
Optionally stabilizing agent may be present, such as albumin. In certain
embodiments, detergent is added. In certain embodiments, vaccine may be
formulated into an injectable preparation. These formulations contain
effective amounts of nucleic acid molecule, vector or polypeptide are
either sterile liquid solutions, liquid suspensions or lyophilized
versions and optionally contain stabilizers or excipients.
[0119] For instance recombinant adenovirus vector may be stored in the
buffer that is also used for the Adenovirus World Standard (Hoganson et
al., 2002, Bioprocessing J 1: 43-8): 20 mM Tris pH 8, 25 mM NaCl, 2.5%
glycerol. Another useful formulation buffer suitable for administration
to humans is 20 mM Tris, 2 mM MgCl.sub.2, 25 mM NaCl, sucrose 10% w/v,
polysorbate-80 0.02% w/v. Another formulation buffer that is suitable for
recombinant adenovirus comprises 10-25 mM citrate buffer pH 5.9-6.2, 4-6%
(w/w) hydroxypropyl-beta-cyclodextrin (HBCD), 70-100 mM NaCl,
0.018-0.035% (w/w) polysorbate-80, and optionally 0.3-0.45% (w/w)
ethanol. An exemplary formulation buffer suitable for MVA vectors can be
10 mM Tris, 140 mM NaCl, pH 7.7 (or 7.4). Obviously, many other buffers
can be used, and several examples of suitable formulations for the
storage and for pharmaceutical administration of purified vectors are
known.
[0120] In certain embodiments a composition comprising the vector further
comprises one or more adjuvants. Adjuvants are known in the art to
further increase the immune response to an applied antigenic determinant.
The terms "adjuvant" and "immune stimulant" are used interchangeably
herein, and are defined as one or more substances that cause stimulation
of the immune system. In this context, an adjuvant is used to enhance an
immune response to the polypeptides encoded by the nucleic acid molecules
in the vectors of the invention. Examples of suitable adjuvants include
aluminium salts such as aluminium hydroxide and/or aluminium phosphate
and/or aluminium potassium phosphate; oil-emulsion compositions (or
oil-in-water compositions), including squalene-water emulsions, such as
MF59 (see e.g. WO 90/14837); saponin formulations, such as for example
QS21 and Immunostimulating Complexes (ISCOMS) (see e.g. U.S. Pat. No.
5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620);
bacterial or microbial derivatives, examples of which are monophosphoryl
lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motif containing
oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof,
such as E. coli heat labile enterotoxin LT, cholera toxin CT, and the
like. It is also possible to use vector-encoded adjuvant, e.g. by using
heterologous nucleic acid that encodes a fusion of the oligomerization
domain of C4-binding protein (C4 bp) to the antigen of interest (e.g.
Solabomi et al., 2008, Infect Immun 76: 3817-23), or by using a vector
encoding both the transgene of interest and a TLR-3 agonist such as
heterologous dsRNA (e.g. WO 2007/100908), or the like.
[0121] In other embodiments, the compositions of the invention do not
comprise adjuvants.
[0122] Pharmaceutical compositions can be administered to a subject, e.g.
a human subject. The total dose of the vaccine active component provided
to a subject during one administration can be varied as is known to the
skilled practitioner, and for adenovirus is generally from
1.times.10.sup.7 viral particles (vp) to 1.times.10.sup.12 vp, preferably
from 1.times.10.sup.8 vp to 1.times.10.sup.11 vp, for instance from
3.times.10.sup.8 to 5.times.10.sup.10 vp, for instance from 10.sup.9 to
3.times.10.sup.10 vp; for MVA virus a total dose of the vaccine is
generally from 1.times.10.sup.5 TCID.sub.50 (tissue culture infection
dose) to 1.times.10.sup.10 TCID.sub.50, preferably from 1.times.10.sup.7
TCID.sub.50 to 1.times.10.sup.10, and more preferably from
1.times.10.sup.8 TCID.sub.50 to 1.times.10.sup.9 TCID.sub.50.
Administration of pharmaceutical compositions can be performed using
standard routes of administration. Non-limiting embodiments include
parenteral administration, such as by injection, e.g. intradermal,
intramuscular, etc, or subcutaneous or transcutaneous, or mucosal
administration, e.g. intranasal, oral, intravaginal, rectal, and the
like. In one embodiment a composition is administered by intramuscular
injection, e.g. into the deltoid muscle of the arm, or vastus lateralis
muscle of the thigh. In certain embodiments the vaccine is a DNA vaccine,
and this can for instance be administered intradermally, e.g. by DNA
tattooing (see, e.g. Oosterhuis et al., 2012, Curr Top Microbiol Immunol
351: 221-50). This route is also feasible for adenoviral vectors and
poxviral vectors. In certain embodiments a composition according to the
invention comprises an adenoviral vector or a poxviral vector, or both an
adenoviral vector and a poxviral vector and is administered by
intramuscular injection. The skilled person knows the various
possibilities to administer a composition, such as a vaccine in order to
induce an immune response to the antigen(s) in the vaccine.
[0123] A subject as used herein preferably is a mammal, for instance a
rodent, e.g. a mouse, or a non-human-primate, or a human. Preferably, the
subject is a human subject.
[0124] The vaccines of the invention can be used to treat patients having
one of various stages of diseases caused by HPV (in particular type 16
for vaccines comprising or encoding any of SEQ ID NOs: 1-6, 24, and 28 or
type 18 for vaccines comprising or encoding any of SEQ ID NOs: 20-23, 25,
and 31 or both types for vaccines that comprise or encode both HPV16 and
HPV18 designer molecules described herein), from incident and persistent
HPV infection as such (e.g. as detected by HPV DNA testing), thus before
(pre-)cancerous lesions are formed, as well as cervical intraepithelial
neoplasia (CIN; also known as cervical dysplasia and cervical
interstitial neoplasia, which is the potentially premalignant
transformation and abnormal growth (dysplasia) of squamous cells on the
surface of the cervix) up to and including cervical cancer (such as
cervical squamous cell carcinoma (SCC)). In addition, other HPV-induced
neoplasias, such as vulvar intraepithelial neoplasia (VIN), vaginal
intraepithelial neoplasia (VaIN), penile intraepithelial neoplasia (PIN),
anal intraepithelial neoplasia (AIN) can be targeted as well as more
advanced stages of oropharyngeal cancer (also known as head- and neck
cancer), penile cancer, vaginal cancer, vulvar cancer and anal cancer.
The vaccines of the invention thus can target a wide range of HPV induced
lesions, and are likely most effective at the precancerous stages of
HPV-induced disease, e.g. at the (persistent) infection and/or the
neoplasia stages, where expression of E2, E6 and/or E7 is highest. It is
also possible to combine the treatment using a vaccine of the invention
with compounds that counteract or can overcome immune escape mechanisms
in advanced cancer cells e.g. anti-PD1/PD-L1 antibodies, anti CTLA-4
antibodies such as Ipilimumab, anti-LAG-3 antibodies, anti-CD25
antibodies, IDO-inhibitors, CD40 agonistic antibodies, CD137 agonistic
antibodies, etc (see, e.g. Hamid and Carvajal, 2013, Expert Opinion Biol
Ther 13: 847-861; Mellman et al., 2011, Nature Rev 480: 480-89).
[0125] As used herein, `treating` means administration of the vaccine to
induce a therapeutic immune response against cells that express (epitopes
of) HPV16 or 18 E6, and/or E7, and/or optionally E2, in the patient,
which leads to at least reduction of the level of and preferably complete
removal of HPV16 or 18 infection, which results in at least slowing and
preferably stopping the progress of HPV16- or HPV18-caused disease such
as neoplasias and/or symptoms thereof. Preferably treatment with the
vaccine results also in remission of more advanced stages of HPV-induced
cancers. It is preferred to administer the vaccine to patients that have
an established HPV infection that has been typed, so that the vaccine
that encodes the polypeptide of the corresponding HPV type can be
administered. In the absence of screening the vaccine can also be
administered in the part of the population that is likely to be HPV
infected, i.e. sexually active people. It is also possible to administer
a vaccine of the invention to subjects that have not been infected by
HPV16 or 18, e.g. for prophylactic use, possibly in combination with a
vaccine against another HPV type by which the patient has been infected,
or alternatively in non-infected subjects. A vaccine of the invention can
also be administered to a subject that is subject to further treatment by
other means, e.g. surgery (removal of a lesion caused by HPV16 or 18
infection), or treatment with imiquimod (comprising a TLR-7/8 agonist,
see e.g. Dayaana et al., 2010, Br J Cancer 102: 1129-36). The effect of
the treatment can be measured either by cytology or by HPV testing.
[0126] In certain embodiments, the vaccination and methods described
herein comprise administering the vaccine of the invention to a subject
or patient at least once. It is also possible to provide one or more
booster administrations of one or more further vaccines. If a boosting
vaccination is performed, typically, such a boosting vaccination will be
administered to the same subject at a moment between one week and one
year, preferably between two weeks and four months, after administering
an immunogenic composition with the same antigen to the subject for the
first time (which is in such cases referred to as `priming vaccination`).
In alternative boosting regimens, it is also possible to administer
different vectors, e.g. one or more adenoviruses of different serotype,
or other vectors such as MVA, or DNA, or protein, to the subject as a
priming or boosting vaccination. In certain embodiments, the same form of
a vaccine of the invention is administered at least twice to the same
patient in a prime-boost regimen, e.g. with the same recombinant
adenovirus (such as Ad26) according to the invention.
[0127] In certain preferred embodiments, a vaccine of the invention is
administered at least twice in a prime-boost regimen, but the vector of
the vaccine is different, e.g. two different viral vectors are used, e.g.
priming with recombinant Ad26 and boosting with a recombinant poxvirus,
or vice versa. Non-limiting exemplary embodiments include: a) priming
with a recombinant Ad26 vector and boosting with a recombinant MVA
vector; b) priming with a recombinant Ad26 vector and a recombinant Ad35
vector and boosting with a recombinant MVA vector; c) priming with a
recombinant poxviral vector (e.g., MVA) and boosting with a recombinant
Ad26 vector; d) priming with a recombinant poxviral vector (e.g., MVA)
and boosting with a recombinant Ad26 vector and a recombinant Ad35
vector; wherein in each case the priming and boosting vector each
comprise at least one nucleic acid encoding a designer polypeptide of the
invention. In certain preferred embodiments the priming and boosting
vector each encode the same designer polypeptide of the invention. Each
of the priming and/or boosting administrations can optionally be
administered more than once to the same subject.
[0128] In certain embodiments, a vaccine, or recombinant virus according
to the invention is administered at least three times in a
prime-boost-boost regimen, for example, first in a priming
administration, and second and third in two subsequent boosting
administrations. In additional embodiments, further booster
administrations might be added to the regimen. It is also possible to
simultaneously or substantially simultaneously (e.g. not more than 10
minutes apart) administer an adenoviral vector and an MVA vector (which
can either be in the same composition or in different compositions), to
induce an immune response (see e.g. WO 2010/073043).
[0129] It is also an aspect of the invention to induce a CTL response
against HPV16 or HPV18 in a subject, comprising administering a vector,
vaccine, or vaccine combination according to the invention to the
subject. The skilled person will understand that the vaccines that
include HPV16 sequences (e.g., encoding or comprising any of SEQ ID NOs:
1-6, 24, and 28) work best against and are intended for use against HPV16
infection, while the vaccines that include HPV18 sequences (e.g.,
encoding or comprising any of SEQ ID NOs: 20-23, 25, and 31) work best
against and are intended for use against HPV18 infection.
[0130] 1. The invention provides also the following non-limiting
embodiments: [0131] 1) A vaccine combination comprising: [0132] a) a
first vaccine comprising an immunologically effective amount of one or
more recombinant adenovirus vectors together comprising a first nucleic
acid encoding a first polypeptide comprising the amino acid sequence of
SEQ ID NO: 1 and a second nucleic acid encoding a second polypeptide
comprising the amino acid sequence of SEQ ID NO: 20, together with a
pharmaceutically acceptable carrier; and [0133] b) a second vaccine
comprising an immunologically effective amount of a recombinant Modified
Vaccinia Ankara (MVA) vector comprising a third nucleic acid encoding a
third polypeptide comprising the amino acid sequence of SEQ ID NO: 1 and
a fourth nucleic acid encoding a fourth polypeptide comprising the amino
acid sequence of SEQ ID NO: 20, together with a pharmaceutically
acceptable carrier;
[0134] 2. wherein the MVA vector comprises MVA-BN or derivatives thereof.
[0135] 2) The vaccine combination according to embodiment 1, wherein the
first vaccine and the second vaccine each further comprise a nucleic acid
encoding a fifth polypeptide comprising the amino acid sequence of SEQ ID
NO: 28 and a nucleic acid encoding a sixth polypeptide comprising the
amino acid sequence of SEQ ID NO: 31. [0136] 3) The vaccine combination
according to any one of embodiments 1-2, wherein the first and third
polypeptides each further comprise SEQ ID NO:28 and wherein the second
and fourth polypeptides each further comprise SEQ ID NO: 31. [0137] 4) A
vaccine combination according to embodiment 1, wherein the first nucleic
acid and the third nucleic acid each encode a polypeptide comprising the
amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:5, and wherein the
second nucleic acid and the fourth nucleic acid each encode a polypeptide
comprising the amino acid sequence of SEQ ID NO: 22. [0138] 5) The
vaccine combination according to any one of embodiments 1-4, wherein the
nucleic acid encoding a polypeptide comprising SEQ ID NO: 1 has at least
90% sequence identity to the polynucleotide sequence of SEQ ID NO: 2 and
the nucleic acid encoding a polypeptide comprising SEQ ID NO: 20 has at
least 90% sequence identity to the polynucleotide sequence of SEQ ID NO:
21. [0139] 6) The vaccine combination according to any one of embodiments
1-5, wherein the nucleic acid encoding a polypeptide comprising SEQ ID
NO: 1 has at least 95% sequence identity to SEQ ID NO: 2 and the nucleic
acid encoding a polypeptide comprising SEQ ID NO: 20 has at least 95%
sequence identity to SEQ ID NO: 21. [0140] 7) The vaccine combination
according to any one of embodiments 1-6, wherein the nucleic acid
encoding a polypeptide comprising SEQ ID NO: 1 comprises SEQ ID NO: 2 and
the nucleic acid encoding a polypeptide comprising SEQ ID NO: 20
comprises SEQ ID NO: 21. [0141] 8) The vaccine combination according to
embodiment 4, wherein the nucleic acid encoding a polypeptide comprising
SEQ ID NO: 3 has at least 90% sequence identity to the polynucleotide
sequence of SEQ ID NO: 4 or SEQ ID NO: 24, and the nucleic acid encoding
a polypeptide comprising SEQ ID NO: 22 has at least 90% sequence identity
to the polynucleotide sequence of SEQ ID NO: 23 or SEQ ID NO: 25. [0142]
9) The vaccine combination according to any one of embodiments 4 and 8,
wherein the nucleic acid encoding a polypeptide comprising SEQ ID NO: 3
has at least 95% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 24, and
the nucleic acid encoding a polypeptide comprising SEQ ID NO: 22 has at
least 95% sequence identity to SEQ ID NO: 23 or SEQ ID NO: 25. [0143] 10)
The vaccine combination according to any one of embodiments 4 and 8-9,
wherein the nucleic acid encoding a polypeptide comprising SEQ ID NO: 3
comprises SEQ ID NO: 4 or SEQ ID NO: 24 and the nucleic acid encoding a
polypeptide comprising SEQ ID NO: 22 comprises SEQ ID NO: 23 or SEQ ID
NO: 25. [0144] 11) The vaccine combination according to any one of
embodiments 4 and 8-10, wherein the nucleic acid encoding a polypeptide
comprising SEQ ID NO: 3 has at least 90% sequence identity to SEQ ID NO:
4 and the nucleic acid encoding a polypeptide comprising SEQ ID NO: 22
has at least 90% sequence identity to SEQ ID NO: 23. [0145] 12) The
vaccine combination according to any one of embodiments 4 and 8-11,
wherein the nucleic acid encoding a polypeptide comprising SEQ ID NO: 3
has at least 95% sequence identity to SEQ ID NO: 4 and the nucleic acid
encoding a polypeptide comprising SEQ ID NO: 22 has at least 95% sequence
identity to SEQ ID NO: 23. [0146] 13) The vaccine combination according
to any one of embodiments 4 and 8-12, wherein the nucleic acid encoding a
polypeptide comprising SEQ ID NO: 3 comprises SEQ ID NO: 4 and the
nucleic acid encoding a polypeptide comprising SEQ ID NO: 22 comprises
SEQ ID NO: 23. [0147] 14) The vaccine combination according to any one of
embodiments 4 and 8-10, wherein the nucleic acid encoding a polypeptide
comprising SEQ ID NO: 3 has at least 90% sequence identity to SEQ ID NO:
24 and the nucleic acid encoding a polypeptide comprising SEQ ID NO: 22
has at least 90% sequence identity to SEQ ID NO: 25. [0148] 15) The
vaccine combination according to any one of embodiments 4, 8-10 and 14,
wherein the nucleic acid encoding a polypeptide comprising SEQ ID NO: 3
has at least 95% sequence identity to SEQ ID NO: 24 and the nucleic acid
encoding a polypeptide comprising SEQ ID NO: 22 has at least 95% sequence
identity to SEQ ID NO: 25. [0149] 16) The vaccine combination according
to any one of embodiments 4, 8-10 and 14-15, wherein the nucleic acid
encoding a polypeptide comprising SEQ ID NO: 3 comprises SEQ ID NO: 24
and the nucleic acid encoding a polypeptide comprising SEQ ID NO: 22
comprises SEQ ID NO: 25. [0150] 17) The vaccine combination according to
any one of embodiments 2-3, wherein the nucleic acid encoding a
polypeptide comprising SEQ ID NO: 28 has at least 90% sequence identity
to the polynucleotide sequence of SEQ ID NO: 29 or SEQ ID NO: 30, and
wherein the nucleic acid encoding SEQ ID NO: 31 has at least 90% sequence
identity to the polynucleotide seuqnce of SEQ ID NO: 32 or SEQ ID NO: 33.
[0151] 18) The vaccine combination according to any one of embodiments
2-3 and 17, wherein the nucleic acid encoding a polypeptide comprising
SEQ ID NO: 28 has at least 95% sequence identity to SEQ ID NO: 29 or SEQ
ID NO: 30, and wherein the nucleic acid encoding SEQ ID NO: 31 has at
least 95% sequence identity to SEQ ID NO: 32 or SEQ ID NO: 33. [0152] 19)
The vaccine combination according to any one of embodiments 2-3 and
17-18, wherein the nucleic acid encoding a polypeptide comprising SEQ ID
NO: 28 comprises SEQ ID NO: 29 or SEQ ID NO: 30, and wherein the nucleic
acid encoding SEQ ID NO: 31 comprises SEQ ID NO: 32 or SEQ ID NO: 33.
[0153] 20) The vaccine combination according to embodiments 2-3 and
17-19, wherein the nucleic acid encoding a polypeptide comprising SEQ ID
NO: 28 has at least 90% sequence identity to SEQ ID NO: 29 and wherein
the nucleic acid encoding SEQ ID NO: 31 has at least 90% sequence
identity to SEQ ID NO: 32. [0154] 21) The vaccine combination according
to embodiments 2-3 and 17-20, wherein the nucleic acid encoding a
polypeptide comprising SEQ ID NO: 28 has at least 95% sequence identity
to SEQ ID NO: 29 and wherein the nucleic acid encoding SEQ ID NO: 31 has
at least 95% sequence identity to SEQ ID NO: 32. [0155] 22) The vaccine
combination according to embodiments 2-3 and 17-21, wherein the nucleic
acid encoding a polypeptide comprising SEQ ID NO: 28 comprises SEQ ID NO:
29 and wherein the nucleic acid encoding SEQ ID NO: 31 comprises SEQ ID
NO: 32. [0156] 23) The vaccine combination according to embodiments 2-3
and 17-19, wherein the nucleic acid encoding a polypeptide comprising SEQ
ID NO: 28 has at least 90% sequence identity to SEQ ID NO: 30 and wherein
the nucleic acid encoding SEQ ID NO: 31 has at least 90% sequence
identity to SEQ ID NO: 33. [0157] 24) The vaccine combination according
to embodiments 2-3, 17-19 and 23, wherein the nucleic acid encoding a
polypeptide comprising SEQ ID NO: 28 has at least 95% sequence identity
to SEQ ID NO: 30 and wherein the nucleic acid encoding SEQ ID NO: 31 has
at least 95% sequence identity to SEQ ID NO: 33. [0158] 25) The vaccine
combination according to embodiments 2-3, 17-19 and 23-24, wherein the
nucleic acid encoding a polypeptide comprising SEQ ID NO: 28 comprises
SEQ ID NO: 29 and wherein the nucleic acid encoding SEQ ID NO: 31
comprises SEQ ID NO: 32. [0159] 26) The vaccine combinations according to
any one of embodiments 1-25, wherein derivatives of MVA-BN are
characterized: i) in being capable of reproductive replication in chicken
embryo fibroblasts (CEF) cells and the Baby Hamster Kidney cell line BHK,
but not capable of reproductive replication in the human cell lines
HaCat, HeLa, and 143B; and ii) by a failure to replicate in a mouse
strain that is incapable of producing mature B and T cells and as such is
severely immune compromised and highly susceptible to a replicating
virus. [0160] 27) The vaccine combination according to any one of
embodiments 1-26, wherein the recombinant adenovirus vector is selected
from the group consisting of rAd26 and rAd35. [0161] 28) The vaccine
combination according to any one of embodiments 1-27, wherein the
recombinant adenovirus vector is rAd26. [0162] 29) The vaccine
combination according to any one of embodiments 1-27, wherein the
recombinant adenovirus vector is rAd35. [0163] 30) The vaccine
combination according to any one of embodiments 1-29, wherein the first
vaccine comprises a first recombinant adenovirus vector comprising the
first nucleic acid encoding the first polypeptide comprising SEQ ID NO: 1
and a second recombinant adenovirus vector comprising the second nucleic
acid encoding the second polypeptide comprising SEQ ID NO: 20. [0164] 31)
A recombinant Modified Vaccinia Ankara (MVA) vector comprising: (a) a
nucleic acid encoding at least one of a polypeptide comprising the amino
acid sequence of SEQ ID NO: 1 and a polypeptide comprising the amino acid
sequence of SEQ ID NO: 3, and (b) another nucleic acid encoding at least
one of a polypeptide comprising the amino acid sequence of SEQ ID NO: 20
and a polypeptide comprising the amino acid sequence of SEQ ID NO: 22;
[0165] wherein the MVA vector is MVA-BN or derivatives thereof [0166] 32)
The recombinant Modified Vaccinia Ankara (MVA) vector according to
embodiment 31, wherein the MVA vector comprises a nucleic acid encoding a
polypeptide comprising SEQ ID NO: 1 and a nucleic acid encoding a
polypeptide comprising SEQ ID NO: 20. [0167] 33) The recombinant MVA
vector according to any one of embodiments 31-32, wherein the MVA vector
further comprises at least one of a nucleic acid encoding a polypeptide
comprising the amino acid sequence of SEQ ID NO: 28 and a nucleic acid
encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:
31. [0168] 34) The recombinant MVA vector according to any one of
embodiments 31-33, wherein the polypeptide comprising SEQ ID NO:1 further
comprises SEQ ID NO: 28 and wherein the polypeptide comprising SEQ ID NO:
20 further comprises SEQ ID NO: 31. [0169] 35) The recombinant MVA vector
according to embodiment 31, wherein the MVA vector comprises a nucleic
acid encoding a polypeptide comprising SEQ ID NO: 3 and a nucleic acid
encoding a polypeptide comprising SEQ ID NO: 22. [0170] 36) The
recombinant MVA vector according to claim 31, wherein the nucleic acid
encoding a polypeptide comprising SEQ ID NO: 1 is part of a nucleic acid
encoding a polypeptide comprising SEQ ID NO: 3, and wherein the nucleic
acid encoding a polypeptide comprising SEQ ID NO: 20 is part of a nucleic
acid encoding a polypeptide encoding SEQ ID NO: 22. [0171] 37) The
recombinant MVA vector according to any one of embodiments 31-36, wherein
the nucleic acid encoding a polypeptide comprising SEQ ID NO: 1 has at
least 90% sequence identity to the polynucleotide sequence of SEQ ID NO:
2 and the nucleic acid encoding a polypeptide comprising SEQ ID NO: 20
has at least 90% sequence identity to the polynucleotide sequence of SEQ
ID NO: 21. [0172] 38) The recombinant MVA vector according to any one of
embodiments 31-37, wherein the nucleic acid encoding a polypeptide
comprising SEQ ID NO: 1 has at least 95% sequence identity to SEQ ID NO:
2 and the nucleic acid encoding a polypeptide comprising SEQ ID NO: 20
has at least 95% sequence identity to SEQ ID NO: 21. [0173] 39) The
recombinant MVA vector according to any one of embodiments 31-38, wherein
the nucleic acid encoding a polypeptide comprising SEQ ID NO: 1 comprises
SEQ ID NO: 2 and the nucleic acid encoding a polypeptide comprising SEQ
ID NO: 20 comprises SEQ ID NO: 21. [0174] 40) The recombinant MVA vector
according to any one of embodiments 31 and 35, wherein the nucleic acid
encoding SEQ ID NO: 3 has at least 90% sequence identity to the
polynucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 24 and the nucleic
acid encoding SEQ ID NO: 22 has at least 90% sequence identity to the
polynucleotide sequence of SEQ ID NO: 23 or SEQ ID NO: 25. [0175] 41) The
recombinant MVA vector according to any one of embodiments 31, 35 and 40,
wherein the nucleic acid encoding SEQ ID NO: 3 has at least 95% sequence
identity to SEQ ID NO: 4 or SEQ ID NO: 24 and the nucleic acid encoding
SEQ ID NO: 22 has at least 95% sequence identity to SEQ ID NO: 23 or SEQ
ID NO: 25. [0176] 42) The recombinant MVA vector according to any one of
embodiments 31, 35 and 40-41, wherein the nucleic acid encoding SEQ ID
NO: 3 comprises SEQ ID NO: 4 or SEQ ID NO: 24 and the nucleic acid
encoding SEQ ID NO: 22 comprises SEQ ID NO: 23 or SEQ ID NO: 25. [0177]
43) The recombinant MVA vector according to any one of embodiments 31, 35
and 40-42, wherein the nucleic acid encoding SEQ ID NO: 3 has at least
90% sequence identity to SEQ ID NO: 4 and the nucleic acid encoding SEQ
ID NO: 22 has at least 90% sequence identity to SEQ ID NO: 23. [0178] 44)
The recombinant MVA vector according to any one of embodiments 31, 35,
and 40-43, wherein the nucleic acid encoding SEQ ID NO: 3 has at least
95% sequence identity to SEQ ID NO: 4 and the nucleic acid encoding SEQ
ID NO: 22 has at least 95% sequence identity to SEQ ID NO: 23. [0179] 45)
The recombinant MVA vector according to any one of embodiments 31, 35 and
40-44, wherein the nucleic acid encoding SEQ ID NO: 3 comprises SEQ ID
NO: 4 and the nucleic acid encoding SEQ ID NO: 22 comprises SEQ ID NO:
23. [0180] 46) The recombinant MVA vector according to any one of
embodiments 31, 35 and 40-42, wherein the nucleic acid encoding SEQ ID
NO: 3 has at least 90% sequence identity to SEQ ID NO: 24 and the nucleic
acid encoding SEQ ID NO: 22 has at least 90% sequence identity to SEQ ID
NO: 25. [0181] 47) The recombinant MVA vector according to any one of
embodiments 31, 35, 40-42 and 46, wherein the nucleic acid encoding SEQ
ID NO: 3 has at least 95% sequence identity to SEQ ID NO: 24 and the
nucleic acid encoding SEQ ID NO: 22 has at least 95% sequence identity to
SEQ ID NO: 25. [0182] 48) The recombinant MVA vector according to any one
of embodiments 31, 35, 40-42 and 46-47, wherein the nucleic acid encoding
SEQ ID NO: 3 comprises SEQ ID NO: 24 and the nucleic acid encoding SEQ ID
NO: 22 comprises SEQ ID NO: 25. [0183] 49) A recombinant MVA vector
comprising at least one nucleic acid encoding a polypeptide comprising a
sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO:
3, SEQ ID NO: 20, and SEQ ID NO:22, wherein the at least one nucleic acid
is operably linked to a promoter comprising at least one of a nucleic
acid having at least 95% sequence identity to the polynucleotide sequence
of SEQ ID NO: 26 and a nucleic acid having at least 95% sequence identity
to the polynucleotide sequence of SEQ ID NO: 27.
[0184] 50) The recombinant MVA vector according to embodiment 49, wherein
the promoter comprises at least one of SEQ ID NO:26 and SEQ ID NO: 27.
[0185] 51) The recombinant MVA vector according to any one of embodiments
49-50, wherein the promoter is SEQ ID NO:26. [0186] 52) The recombinant
MVA vector according to any one of embodiments 49-50, wherein the
promoter is SEQ ID NO:27. [0187] 53) The recombinant MVA vector according
to any one of embodiments 49-51, wherein SEQ ID NO: 1 or SEQ ID NO: 3 is
operably linked to SEQ ID NO: 26. [0188] 54) The recombinant MVA vector
according to embodiment 53, wherein SEQ ID NO: 3 is operably linked to
SEQ ID NO: 26. [0189] 55) The recombinant MVA vector according to any one
of embodiments 49-50 or 52, wherein SEQ ID NO: 20 or SEQ ID NO: 22 is
operably linked to SEQ ID NO: 27. [0190] 56) The recombinant MVA vector
according to embodiment 55, wherein SEQ ID NO: 22 is operably linked to
SEQ ID NO: 27. [0191] 57) The recombinant MVA vector according to any one
of embodiments 49 and 53, wherein SEQ ID NO: 1 is encoded by a nucleic
acid in accordance with any one of embodiments 5-7 and 36-39. [0192] 58)
The recombinant MVA vector according to any one of embodiments 49 and
53-54, wherein SEQ ID NO: 3 is encoded by a nucleic acid in accordance
with any one of embodiments 4, 8-16, 35, 36, and 40-48. [0193] 59) The
recombinant MVA vector according to any one of embodiments 49 and 55,
wherein SEQ ID NO: 20 is encoded by a nucleic acid according to any one
of embodiments 3-7, 32, 34 and 36-39. [0194] 60) The recombinant MVA
vector according to any one of embodiments 49 and 55-56, wherein SEQ ID
NO: 22 is encoded by a nucleic acid according to any one of embodiments
4, 8-16, 35, 36, and 40-48. [0195] 61) A vaccine comprising a recombinant
MVA vector according to any one of embodiments 31-60 and a
pharmaceutically acceptable carrier. [0196] 62) A method for treating a
persistent HPV infection, vulvar intraepithelial neoplasia (VIN),
cervical intraepithelial neoplasia (CIN), vaginal intraepithelial
neoplasia (VaIN), anal intraepithelial neoplasia (AIN), cervical cancer
(such as cervical squamous cell carcinoma (SCC)), oropharyngeal cancer,
penile cancer, vaginal cancer or anal cancer in a subject, the method
comprising administering to the subject a vector, vaccine, or vaccine
combination according to any one of embodiments 1-61. [0197] 63) A method
for inducing an immune response against Human Papilloma Virus (HPV) in a
subject, the method comprising: (a) administering to the subject a first
vaccine comprising an immunologically effective amount of either [0198]
1. (i) a recombinant adenovirus vector comprising a first nucleic acid
encoding a first polypeptide comprising the amino acid sequence of SEQ ID
NO:1 and a second nucleic acid encoding a second polypeptide comprising
the amino acid sequence of SEQ ID NO: 20, or [0199] 2. (ii) a first
recombinant adenovirus vector comprising a first nucleic acid encoding a
first polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a
second recombinant adenovirus vector comprising a second nucleic acid
encoding a second polypeptide comprising the amino acid sequence of SEQ
ID NO: 20, together with a pharmaceutically acceptable carrier; [0200]
and (b) administering to the subject a second vaccine comprising an
immunologically effective amount of a recombinant Modified Vaccinia
Ankara (MVA) vector comprising a third nucleic acid encoding a third
polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a
fourth nucleic acid encoding a fourth polypeptide comprising the amino
acid sequence of SEQ ID NO: 20, together with a pharmaceutically
acceptable carrier; [0201] wherein the first vaccine is administered to
the subject as a priming vaccine and the second vaccine is administered
to the subject as a boosting vaccine. [0202] 64) A method for inducing an
immune response against Human Papilloma Virus (HPV) in a subject, the
method comprising: (a) administering to the subject a first vaccine
comprising an immunologically effective amount of either [0203] 3. (i) a
recombinant adenovirus vector comprising a first nucleic acid encoding a
first polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a
second nucleic acid encoding a second polypeptide comprising the amino
acid sequence of SEQ ID NO: 20, or [0204] (ii) a first recombinant
adenovirus vector comprising a first nucleic acid encoding a first
polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a
second recombinant adenovirus vector comprising a second nucleic acid
encoding a second polypeptide comprising the amino acid sequence of SEQ
ID NO: 20, together with a pharmaceutically acceptable carrier; [0205]
and (b) administering to the subject a second vaccine comprising an
immunologically effective amount of a recombinant Modified Vaccinia
Ankara (MVA) vector comprising a third nucleic acid encoding a third
polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a
fourth nucleic acid encoding a fourth polypeptide comprising the amino
acid sequence of SEQ ID NO: 20, together with a pharmaceutically
acceptable carrier; [0206] wherein either the first vaccine or the second
vaccine is administered to the subject as a priming vaccine and the other
vaccine is administered to the subject as a boosting vaccine. [0207] 65)
The method according to any one of embodiments 63-64, wherein the first
vaccine and the second vaccine each further comprise a nucleic acid
encoding a fifth polypeptide comprising the amino acid sequence of SEQ ID
NO: 28 and a nucleic acid encoding a sixth polypeptide comprising the
amino acid sequence of SEQ ID NO: 31. [0208] 66) The method according to
any one of embodiments 63-65, wherein each of the first and third
polypeptides further comprises SEQ ID NO: 28 and wherein each of the
second and third polypeptides further comprises SEQ ID NO: 31. [0209] 67)
The method according to any one of embodiments 63-66, wherein the first
vaccine comprises a first recombinant adenovirus vector comprising a
first nucleic acid encoding a first polypeptide comprising SEQ ID NO: 1
or SEQ ID NO: 3 and a second recombinant adenovirus vector comprising a
second nucleic acid encoding a second polypeptide comprising SEQ ID NO:
20 or SEQ ID NO: 22. [0210] 68) The method according to any one of
embodiments 63-67 wherein SEQ ID NO: 1 is encoded by a nucleic acid in
accordance with any one of embodiments 5-7 and 36-39. [0211] 69) The
method according to any one of embodiments 63-67, wherein SEQ ID NO: 3 is
encoded by a nucleic acid in accordance with any one of embodiments 4,
8-16, 35, 36 and 40-48. [0212] 70) The method according to any one of
embodiments 63-67, wherein SEQ ID NO: 20 is encoded by a nucleic acid
according to any one of embodiments 3-7, 32, 34 and 36-39. [0213] 71) The
method according to any one of embodiments 63-67, wherein SEQ ID NO: 22
is encoded by a nucleic acid according to any one of embodiments 4, 8-16,
35, 36 and 40-48. [0214] 72) The method according to any one of
embodiments 65-66, wherein SEQ ID NO: 28 is encoded by a nucleic acid
according to any one of embodiments 17-24. [0215] 73) The method
according to any one of embodiments 65-66, wherein SEQ ID NO: 31 is
encoded by a nucleic acid according to any one of embodiments 17-24.
[0216] 74) A nucleic acid molecule comprising a polynucleotide sequence
selected from the group consisting of SEQ ID NO:24, SEQ ID NO: 25, SEQ ID
NO: 30, and SEQ ID NO: 33. [0217] 75) A nucleic acid molecule comprising
SEQ ID NO:24. [0218] 76) A nucleic acid molecule comprising SEQ ID NO:25.
[0219] 77) A nucleic acid molecule comprising SEQ ID NO: 30. [0220] 78) A
nucleic acid molecule comprising SEQ ID NO:33. [0221] 79) A nucleic acid
molecule having at least 90% or 95% sequence identity to the nucleic acid
molecules of any one of embodiments 74-78. [0222] 80) An isolated nucleic
acid molecule comprising the nucleic acid molecule of any one of
embodiments 74-79. [0223] 81) A vector comprising the nucleic acid
molecule of any one of embodiments 74-79. [0224] 82) The vector according
to embodiment 81, wherein the vector is selected from a poxvirus and an
adenovirus. [0225] 83) The vector according to any one of embodiments
81-82, wherein the vector is a poxvirus. [0226] 84) The vector according
to embodiment 83, wherein the poxvirus is an orthopoxvirus or an
avipoxvirus. [0227] 85) The vector according to embodiment 84, wherein
the poxvirus is an orthopoxvirus. [0228] 86) The vector according to
embodiment 85, wherein the orthopoxvirus is a vaccinia virus. [0229] 87)
The vector according to embodiment 86, wherein the vaccinia virus is a
MVA virus. [0230] 88) The vector according to embodiment 87, wherein the
MVA virus is MVA-BN or a derivative thereof. [0231] 89) The vector
according to any one of embodiments 81-82, wherein the vector is an
adenovirus. [0232] 90) The vector according to embodiment 89, wherein the
adenovirus is selected from rAd26 and rAd35. [0233] 91) A vaccine
combination comprising: a) a first vaccine comprising an immunologically
effective amount of either [0234] (i) a recombinant adenovirus vector
comprising a first nucleic acid encoding a first polypeptide comprising
the amino acid sequence of SEQ ID NO: 3 and a second nucleic acid
encoding a second polypeptide comprising the amino acid sequence of SEQ
ID NO: 22, or [0235] (ii) a first recombinant adenovirus vector
comprising a first nucleic acid encoding a first polypeptide comprising
the amino acid sequence of SEQ ID NO: 3 and a second recombinant
adenovirus vector comprising a second nucleic acid encoding a second
polypeptide comprising the amino acid sequence of SEQ ID NO: 22, together
with a pharmaceutically acceptable carrier; [0236] and (b) a second
vaccine comprising an immunologically effective amount of a recombinant
Modified Vaccinia Ankara (MVA) vector comprising a third nucleic acid
encoding a third polypeptide comprising the amino acid sequence of SEQ ID
NO: 3 and a fourth nucleic acid encoding a fourth polypeptide comprising
the amino acid sequence of SEQ ID NO: 22, together with a
pharmaceutically acceptable carrier; [0237] wherein the either the first
vaccine or the second vaccine is administered to the subject as a priming
vaccine and the other vaccine is administered to the subject as a
boosting vaccine; and [0238] wherein the MVA vector comprises MVA-BN or
derivatives thereof [0239] 92) The vaccine combination according to
embodiment 91, wherein a polypeptide comprising SEQ ID NO: 3 is encoded
by a nucleic acid in accordance with any one of embodiments 4, 8-16, 35,
36 and 40-48. [0240] 93) The vaccine combination according to embodiment
91, wherein a polypeptide comprising SEQ ID NO: 22 is encoded by a
nucleic acid according to any one of embodiments 4, 8-16, 35, 36 and
40-48. [0241] 94) Use of any one of the nucleic acids, polypeptides,
vectors, vaccines, or vaccine combinations according to any one of
embodiments 1-61 and 74-93 in treating a persistent HPV infection, vulvar
intraepithelial neoplasia (VIN), cervical intraepithelial neoplasia
(CIN), vaginal intraepithelial neoplasia (VaIN), anal intraepithelial
neoplasia (AIN), cervical cancer (such as cervical squamous cell
carcinoma (SCC)), oropharyngeal cancer, penile cancer, vaginal cancer or
anal cancer in a subject in need thereof. [0242] 95) Use of any one of
the the nucleic acids, polypeptides, vectors, vaccines, or vaccine
combinations according to any one of embodiments 1-61 and 74-93 in the
preparation of a pharmaceutical composition or medicament for inducing an
immune response against Human Papilloma Virus (HPV) in a subject in need
thereof. [0243] 96) A kit comprising any one of the the nucleic acids,
polypeptides, vectors, vaccines, or vaccine combinations according to any
one of embodiments 1-61 and 74-93. [0244] 97) The vaccine combination
according to embodiment 30, wherein the first recombinant adenovirus
vector is rAd26 and the second recombinant adenovirus vector is rAd26.
[0245] 98) The vaccine combination according to embodiment 97, wherein
the first recombinant adenovirus vector comprises a first nucleic acid
encoding a first polypeptide comprising SEQ ID NO: 3 and the second
recombinant adenovirus vector comprises a second nucleic acid encoding a
second polypeptide comprising SEQ ID NO: 22. [0246] 99) The vaccine
combination according to embodiment 91, wherein the first vaccine
comprises an immunologically effective amount of a recombinant adenovirus
vector comprising a first nucleic acid encoding a first polypeptide
comprising SEQ ID NO: 3 and a second nucleic acid encoding a second
polypeptide comprising SEQ ID NO: 22, together with a pharmaceutically
acceptable carrier. [0247] 100) The vaccine combination according to
embodiment 99, wherein the recombinant adenovirus vector is rAd26. [0248]
101) The vaccine combination according to embodiment 91, wherein the
first vaccine comprises an immunologically effective amount of a first
recombinant adenovirus vector comprising a first nucleic acid encoding a
first polypeptide comprising SEQ ID NO: 3 and a second recombinant
adenovirus vector comprising a second nucleic acid encoding a second
polypeptide comprising SEQ ID NO: 22, together with a pharmaceutically
acceptable carrier. [0249] 102) The vaccine combination according to
embodiment 101, wherein the first recombinant adenovirus vector is rAd26
and the second recombinant adenovirus vector is rAd26. [0250] 103) The
vaccine combination according to any one of embodiments 91 and 99-102,
wherein the first vaccine is a priming vaccine and the second vaccine is
a boosting vaccine. [0251] 104) A method for treating persistent HPV
infection, vulvar intraepithelial neoplasia (VIN), cervical
intraepithelial neoplasia (CIN), vaginal intraepithelial neoplasia
(VaIN), anal intraepithelial neoplasia (AIN), cervical cancer (such as
cervical squamous cell carcinoma (SCC)), oropharyngeal cancer, penile
cancer, vaginal cancer or anal cancer in a subject in need thereof, the
method comprising: (a) administering to the subject a first vaccine
comprising an immunologically effective amount of either [0252] 4. (i) a
recombinant adenovirus vector comprising a first nucleic acid encoding a
first polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a
second nucleic acid encoding a second polypeptide comprising the amino
acid sequence of SEQ ID NO: 20, or [0253] 5. (ii) a first recombinant
adenovirus vector comprising a first nucleic acid encoding a first
polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a
second recombinant adenovirus vector comprising a second nucleic acid
encoding a second polypeptide comprising the amino acid sequence of SEQ
ID NO: 20, together with a pharmaceutically acceptable carrier; [0254]
and (b) administering to the subject a second vaccine comprising an
immunologically effective amount of a recombinant Modified Vaccinia
Ankara (MVA) vector comprising a third nucleic acid encoding a third
polypeptide comprising the amino acid sequence of SEQ ID NO:1 and a
fourth nucleic acid encoding a fourth polypeptide comprising the amino
acid sequence of SEQ ID NO: 20, together with a pharmaceutically
acceptable carrier;
wherein the first vaccine is administered to the subject as a priming
vaccine and the second vaccine is administered to the subject as a
boosting vaccine. [0255] 105) The method according to any one of
embodiments 63, 64 or 104, wherein the first vaccine comprises an
immunologically effective amount of a first recombinant adenovirus vector
comprising a first nucleic acid encoding a first polypeptide comprising
SEQ ID NO: 1 and a second recombinant adenovirus vector comprising a
second nucleic acid encoding a second polypeptide comprising SEQ ID NO:
20, together with a pharmaceutically acceptable carrier. [0256] 106) The
method according to embodiment 105, wherein the first recombinant
adenovirus vector is rAd26 and the second recombinant adenovirus vector
is rAd26. [0257] 107) THE METHOD ACCORDING TO ANY ONE OF EMBODIMENTS 63,
64 OR 104, WHEREIN THE FIRST VACCINE COMPRISES AN IMMUNOLOGICALLY
EFFECTIVE AMOUNT OF A RECOMBINANT ADENOVIRUS VECTOR COMPRISING A FIRST
NUCLEIC ACID ENCODING A FIRST POLYPEPTIDE COMPRISING SEQ ID NO: 1 AND A
SECOND NUCLEIC ACID ENCODING A SECOND POLYPEPTIDE COMPRISING SEQ ID NO:
20, TOGETHER WITH A PHARMACEUTICALLY ACCEPTABLE CARRIER [0258] 108) THE
METHOD ACCORDING TO EMBODIMENT 107, WHEREIN THE RECOMBINANT ADENOVIRUS
VECTOR IS RAD26. [0259] 109) THE METHOD ACCORDING TO ANY ONE OF
EMBODIMENTS 104-108, WHEREIN THE NUCLEIC ACID ENCODING A POLYPEPTIDE
COMPRISING SEQ ID NO: 1 IS PART OF A NUCLEIC ACID ENCODING A POLYPEPTIDE
COMPRISING SEQ ID NO: 3, AND WHEREIN THE NUCLEIC ACID ENCODING A
POLYPEPTIDE COMPRISING SEQ ID NO: 20 IS PART OF A NUCLEIC ACID ENCODING A
POLYPEPTIDE ENCODING SEQ ID NO: 22. [0260] 110) A recombinant Modified
Vaccinia Ankara (MVA) vector comprising: (a) a nucleic acid encoding at
least one of a polypeptide comprising the amino acid sequence of SEQ ID
NO: 1 and a polypeptide comprising the amino acid sequence of SEQ ID NO:
3, and (b) a nucleic acid encoding at least one of a polypeptide
comprising the amino acid sequence of SEQ ID NO: 20 and a polypeptide
comprising the amino acid sequence of SEQ ID NO: 22; [0261] 6. wherein at
least one of the nucleic acids from (a) and (b) is inserted in the MVA
intergenic region (IGR) 88/89. [0262] 111) The recombinant MVA vector
according to embodiment 110, wherein the nucleic acids from both (a) and
(b) are inserted into the MVA intergenic region (IGR) 88/89. [0263] 112)
The recombinant MVA vector according to any one of embodiments 110-111,
wherein the MVA is MVA-BN or derivatives thereof. [0264] 113) The
recombinant Modified Vaccinia Ankara (MVA) vector according to any one of
embodiments 110-112, wherein the MVA vector comprises a nucleic acid
encoding a polypeptide comprising SEQ ID NO: 1 and a nucleic acid
encoding a polypeptide comprising SEQ ID NO: 20. [0265] 114) The
recombinant MVA vector according to any one of embodiments 110-113,
wherein the MVA vector further comprises at least one of a nucleic acid
encoding a polypeptide comprising SEQ ID NO: 28 and a nucleic acid
encoding a polypeptide comprising SEQ ID NO: 31. [0266] 115) The
recombinant MVA vector according to any one of embodiments 110-114,
wherein the polypeptide comprising SEQ ID NO:1 further comprises SEQ ID
NO: 28 and wherein the polypeptide comprising SEQ ID NO: 20 further
comprises SEQ ID NO: 31. [0267] 116) The recombinant MVA vector according
to embodiment 110, wherein the MVA vector comprises a nucleic acid
encoding a polypeptide comprising SEQ ID NO: 3 and a nucleic acid
encoding a polypeptide comprising SEQ ID NO: 22.
[0268] The practice of this invention will employ, unless otherwise
indicated, conventional techniques of immunology, molecular biology,
microbiology, cell biology, and recombinant DNA, which are within the
skill of the art. See e.g. Sambrook, Fritsch and Maniatis, Molecular
Cloning: A Laboratory Manual, 2.sup.nd edition, 1989; Current Protocols
in Molecular Biology, Ausubel F M, et al., eds, 1987; the series Methods
in Enzymology (Academic Press, Inc.); PCR2: A Practical Approach,
MacPherson M J, Hams B D, Taylor G R, eds, 1995; Antibodies: A Laboratory
Manual, Harlow and Lane, eds, 1988.
[0269] The invention is further explained in the following examples. The
examples do not limit the invention in any way. They merely serve to
clarify the invention.
EXAMPLES
Example 1: Construction of a Designer Polypeptide Comprising Essentially
all HPV16 E6 and E7 CTL Epitopes
[0270] We designed a novel, non-tumorigenic polypeptide (and nucleic acid
encoding such) that contains essentially all CTL epitopes of HPV16 E6 and
E7 proteins, and has a minimum number of anticipated/predicted strong
neo-epitopes (neo-epitopes meaning epitopes not present in the wild type
HPV16 E6 and E7 proteins). A polypeptide of the invention (also sometimes
referred to as `E6E7SH` herein) for HPV16 comprises a sequence as
provided in SEQ ID NO: 1. A codon-optimized nucleic acid encoding this
polypeptide is provided in SEQ ID NO: 2.
[0271] The molecules of the invention are single molecules, which provides
manufacturing advantages over strategies where multiple molecules are
used. In addition, a polypeptide of the invention comprises essentially
all putative CTL epitopes that are present in wild-type E6 and E7 of
HPV16, and at the same time have a minimum number of
anticipated/predicted strong neo-epitopes that could potentially be
immunodominant and thus divert the immune response from relevant
wild-type CTL epitopes. Thus the constructs of the present invention are
immunologically more favourable than molecules described by others that
either lack possible CTL epitopes and/or that contain more or stronger
neo-epitopes.
[0272] For instance, the construct of SEQ ID NO: 1 contains only one
neo-epitope with a length of nine amino acids with a predicted binding
affinity <50 nM for the 20 most common HLA-A, 20 most common HLA-B and
20 most common HLA-C alleles (HLA-A*01:01, HLA-A*02:01, HLA-A*02:03,
HLA-A*02:06, HLA-A*02:07, HLA-A*03:01, HLA-A*11:01, HLA-A*23:01,
HLA-A*24:02, HLA-A*26:01, HLA-A*29:02, HLA-A*30:01, HLA-A*30:02,
HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*33:03, HLA-A*34:01,
HLA-A*68:01, HLA-A*68:02, HLA-B*07:02, HLA-B*07:04, HLA-B*08:01,
HLA-B*13:01, HLA-B*15:01, HLA-B*18:01, HLA-B*35:01, HLA-B*37:01,
HLA-B*39:01, HLA-B*40:01, HLA-B*40:02, HLA-B*40:06, HLA-B*44:02,
HLA-B*44:03, HL-B*46:01, HLA-B*48:01, HLA-B*51:01, HLA-B*52:01,
HLA-B*53:01, HLA-B*58:01, HLA-C*07:02, HLA-C*04:01, HLA-C*03:04,
HLA-C*01:02, HLA-C*07:01, HLA-C*06:02, HLA-C*03:03, HLA-C*08:01,
HLA-C*15:02, HLA-C*12:02, HLA-C*02:02, HLA-C*05:01, HLA-C*14:02,
HLA-C*03:02, HLA-C*16:01, HLA-C*08:02, HLA-C*12:03, HLA-C*04:03,
HLA-C*17:01, HLA-C*14:03), as determined using the ANN (Lundegaard et
al., 2008, Nucl Acids Res 36: W509-12) and SMM method (Peters et al.,
2003, Bioinformatics 19: 1765-72) for HLA-A and HLA-B and the NetMHCpan
method (Hoof et al., 2009, Immunogenetics 61: 1-13) for HLA-C of the
prediction tool for `Peptide binding to MHC class I molecules` at the
IEDB website
(http://tools.immuneepitope.org/analyze/html/mhc_binding.html, version
2009-09-01B).
[0273] As a non-limiting example, using the SMM prediction tool at the
IEDB website, the shuffled E6 and E7 sequences as described by Oosterhuis
et al., 2011, Int J Cancer 129: 397-406 and Ohlschlager et al., 2006,
Vaccine 24: 2880-93 contain each nine potential strong unique
neo-epitopes (ANN or SMM IC50<50 nM) for the 20 most HLA-A and --B, in
the core part. This even excludes the appendices used in that approach
(in which appendices will further contribute to additional neo-epitopes,
and may miss out on more native MHC II epitopes due to the limited length
of the `overlap`). Indeed, a reportedly improved molecule containing a
variant with shuffled E6 and E7 proteins that was described in WO
2013/083287, contains 22 unique neo-epitopes with a length of nine amino
acids with a predicted IC50<50 nM (ANN, SMM or NetMHCPan) for the 20
most common HLA-A, 20 most common HLA-B and 20 most common HLA-C alleles.
[0274] Hence, the designer molecules of the invention clearly are
favourable in having much lower number of predicted neo-epitopes compared
to other published approaches where E6 and E7 where shuffled to remove
functionality.
[0275] Nucleic acid encoding our thus designed HPV16 E6E7SH molecule (i.e.
a polypeptide having amino acid sequence as provided in SEQ ID NO:1) was
synthesized, the nucleic acid sequence comprising SEQ ID NO: 2, and
flanked by a HindIII site and a Kozak sequence on the 5'end and an XbaI
site on the 3' site (custom synthesis and standard molecular cloning at
Invitrogen Life technologies, Germany).
[0276] The synthezised fragments were cloned using HindIII and XbaI into a
standard expression vector, pCDNA2004.Neo, harbouring both a bacterial
resistance marker (Ampiciline) and a mammalian resistance marker
(Neomycine), to obtain plasmid vectors encoding a molecule of the
invention, e.g. for (transient) transfection based experiments.
[0277] These molecules could be used as such, but also as the basis for
further molecules that contain additional features. As non-limiting
examples, some further variants were prepared as described below.
[0278] The HPV16 E6E7SH fusion protein sequence can be combined with
sequences of other HPV16 early proteins to target individuals with
persistent infection and to broaden the immune repertoire in an immunized
individual. Immune responses against E2 have been suggested to play an
important role in the clearance of HPV16 infections (de Jong et al.,
2002, Cancer Res 62: 472-479). Fusion of E2 to E6E7SH will give a vaccine
component that harbours antigens against the stages of HPV-related cancer
from persistent infection to invasive cancer or recurrent/refractory
disease after LEEP surgery. Therefore, as a non-limiting example of such
embodiments, we prepared a sequence coding for a fusion protein of E6E7SH
with E2 at its N-terminus. In the E2 sequence modifications can be made
to abrogate DNA binding activity that might affect gene expression in
cells expressing the fusion protein. We mutated Glycine at position 293,
Lysine at position 299 and Cysteine at position 300 of the wt HPV16 E2
protein into respectively Valine, Methionine and Arginine. Each of these
mutations on its own already completely abrogates the binding of E2 to
DNA sequences that harbour E2 binding domains (Prakash et al., 1992,
Genes Dev 6: 105-16).
[0279] The resulting polypeptide is referred to as HPV16 E2E6E7SH and
comprises SEQ ID NO: 3. A codon-optimized sequence encoding this
polypeptide was prepared and is provided in SEQ ID NO: 4.
[0280] We also constructed a variant wherein the same E2 mutant protein
was fused to the C-terminus of the HPV16 E6E7SH fusion polypeptide,
giving rise to a polypeptide referred to as HPV16 E6E7E2SH, which
comprises SEQ ID NO: 5. The sequence encoding this construct is provided
as SEQ ID NO: 6.
[0281] For control purposes, we also constructed sequences encoding a
polypeptide that contains the wild-type sequences for full-length HPV16
E6 and E7 as a fusion protein (E6 from aa 1 to 158 directly fused to E7
from aa 1 to 98, named herein E6E7 wt).
[0282] We also tested the effect of adding leader sequences to the
polypeptide. As a non-limiting example, a sequence encoding an IgE leader
sequence (see e.g. U.S. Pat. No. 6,733,994) [the sequence of the leader
peptide is provided in SEQ ID NO: 7] was fused at the N-terminus of some
of the constructs, e.g. in the E6E7 wt construct, which rendered LSE6E7
wt, and in the E2E6E7SH construct, which rendered LSE2E6E7SH. The effect
thereof was significantly (p<0.05) enhanced immunogenicity in
comparison to the same antigen without the LS sequence as measured by
E7-tetramer analysis in immunized mice (as can for instance be seen in
FIG. 9).
[0283] The sequences that encode the E6E7SH polypeptides of the invention,
with or without E2, can for instance be expressed from DNA constructs,
from RNA or from viral vectors. FIG. 1 demonstrates expression in
HEK-293T cells upon transient transfection with DNA vectors expressing
the transgenes as described above. After transfection, cells were
harvested and cell extracts were analyzed by SDS-PAGE and western
blotting with an antibody against HPV16 E7. This experiment demonstrates
expression of the expected fusion proteins of appropriate size upon
transfection of the expression vectors.
[0284] Adenoviral vectors can be used to express the E6E7, either with or
without E2, and with or without additional sequences to augment the
immunogenicity of the encoded fusion protein.
[0285] The genes, coding for HPV16 E6E7 wt control or HPV16 designer
sequences described above were gene optimized for human expression and
synthesized, at Geneart. A Kozak sequence (5' GCCACC 3') was included
directly in front of the ATG start codon, and two stop codons (5' TGA TAA
3') were added at the end of the respective coding sequence. The genes
were inserted in the pAdApt35BSU plasmid and in the pAdApt26 plasmid
(Havenga et al., 2006, J Gen Virol 87, 2135-43) via HindIII and XbaI
sites.
[0286] All adenoviruses were generated in PER.C6 cells by single
homologous recombination and produced as previously described (for rAd35:
Havenga et al., 2006, J Gen Virol 87: 2135-43; for rAd26: Abbink et al.,
2007, J Virol 81: 4654-63). PER.C6 cells (Fallaux et al., 1998, Hum Gene
Ther 9: 1909-17) were maintained in Dulbecco's modified Eagle's medium
(DMEM) with 10% fetal bovine serum (FBS), supplemented with 10 mM MgCl2.
[0287] Briefly, PER.C6 cells were transfected with Ad vector plasmids,
using Lipofectamine according to the instructions provided by the
manufacturer (Life Technologies). Cells were harvested one day after full
cytopathic effect (CPE) was reached, freeze-thawed, centrifuged for 5 min
at 3,000 rpm, and stored at -20.degree. C. The viruses were plaque
purified and amplified in PER.C6 cells cultured in a single well of a
multiwell 24 tissue culture plate. Further amplification was carried out
in PER.C6 cells cultured in a T25 tissue culture flask and subsequently
in a T175 tissue culture flask. Of the crude lysate prepared from the
cells obtained after the T175 flask, 3 to 5 ml was used to inoculate
24.times.T1000 five-layer tissue culture flasks containing 70% confluent
layers of PER.C6 cells. The virus was purified using a two-step CsCl
purification method. Finally, the virus was stored in aliquots at
-85.degree. C.
[0288] Ad35.HPV16-E6E7 wt, and Ad35.HPV16-E6E7SH are recombinant
adenovirus serotype 35 (Ad35) vectors comprising the codon-optimized
nucleotide sequences for the expression of, respectively, a fusion
protein of the wild type HPV16 E6 and E7 proteins (E6E7 wt), and a
designer fusion protein variant as described above (E6E7SH, having the
amino acid sequence provided in SEQ ID NO: 1). The combined E6 and E7
sequences were placed under the control of a CMV promoter in the E1
region of the E1,E3 deleted adenovirus genome. Ad26.HPV16-E6E7 wt, and
Ad26.HPV16-E6E7SH are the equivalent vectors based on recombinant
adenovirus serotype 26.
[0289] Similarly, Ad26 and Ad35-based recombinant adenoviral vectors were
produced that encode the HPV16 E2E6E7SH (SEQ ID NO: 3) variant. Likewise,
Ad26 and Ad35 encoding the HPV16 E6E7E2SH (SEQ ID NO: 5) variant were
produced. Also, an Ad35 vector encoding the E2E6E7SH fusion protein with
an IgE leader sequence at the N-terminus was produced, named
Ad35.HPV16-LSE2E6E7SH. Also a control adenovirus with the E6E7 wt fused
to the IgE leader sequence at the N-terminus was produced.
[0290] The recombinant adenoviruses were produced on PER.C6 cells and
purified by centrifugation on cesium chloride gradients.
[0291] Further examples of constructs that were coupled to repressor
systems are provided in a later example below.
Example 2. Lack of Transforming Activity of the HPV16 Designer Constructs
[0292] Wild-type HPV16 E6 and E7 proteins have tumorigenic potential,
which is apparent as transforming activity in certain assays, such as
colony formation in a soft-agar assay (Massimi and Banks, 2005, Methods
Mol Med 119: 381-395). The E6E7SH polypeptide as described in example 1
comprises the fragments of the E6 and E7 proteins in a re-ordered
fashion. This is expected to remove the tumorigenic potential, as can be
measured for instance by a significantly reduced transforming activity as
compared to either of wt E6 and E7 proteins in such assays.
[0293] Others reported that gene-shuffled variants of HPV16 E6 and E7 have
indeed lost their oncogenic potential (Ohlschlager et al., 2006, Vaccine
24: 2880-93; Henken et al., 2012, Vaccine 30: 4259-66), demonstrating
that gene shuffling destroys the wild-type functions of E6 and E7
proteins.
[0294] To assess the loss of tumorigenic properties, we assessed the
ability of our E6E7SH constructs to confer the ability to grow in soft
agar upon NIH 3T3 cells (as described by e.g. Massimi and Banks, 2005,
Methods Mol Med 119: 381-395). Transfection of NIH3T3 cells with a
plasmid expressing the wild type HPV16 E7 resulted consistently in colony
formation. In these assays, expression of wild type HPV16 E6 alone did
not cause colony formation above background. This is in line with
published observations that E7 wt is much more efficient than E6 wt in
this assay (Sedman et al., 1991, J Virol 65: 4860-66). Transfection with
our E6E7SH construct did not lead to growth of colonies of cells in soft
agar (FIG. 2) in four independent experiments, demonstrating that nucleic
acids encoding a polypeptide of the invention, E6E7SH, have lost the
transforming capacity that is associated with E7.
[0295] The tumorigenic potential of E6 and E7 is associated with their
ability to reduce the levels of the cellular proteins p53 and pRb
respectively. p53 and pRb degradation assays were performed to
demonstrate that nucleic acid encoding a polypeptide of the invention,
E6E7SH, construct does not have the biological activity associated with
the wild-type E6 and E7 at the molecular level. In short, HPV16 E6 wt and
our E6E7SH construct were expressed in NCI-H1299 cells that lack
endogenous p53 for the p53 degradation assay. For the pRb degradation
assay HPV16 E7 wt and the E6E7SH construct were expressed in pRb null
Saos-2 cells. As can be seen in FIG. 3, co-expression of p53 with E6 wt,
but not with E6E7SH, leads to reduced p53 levels (panels A and B).
Likewise, panels 3C and 3D show that co-expression of pRb with E7 wt, but
not with E6E7SH, leads to reduced pRB levels. These data demonstrate that
nucleic acid encoding a polypeptide of the invention has no ability to
form colonies in soft agar and does not contain main biological
activities of the wild-type E6 and E7 polypeptides, namely the
inactivation of p53 and pRb respectively.
[0296] To further demonstrate the safety of nucleic acid constructs
encoding polypeptide of the invention, we made use of primary human
foreskin keratinocytes that are the natural target cells for HPV mediated
transformation. Immortalization of primary human keratinocytes requires
the action of both E6 and E7 wild-type (Munger et al., 1989, J Virol 63:
4417-21). This assay is probably the physiologically most relevant in
vitro assay to demonstrate the safety of our constructs (Massimi and
Banks, 2005, Methods Mol Med 119: 381-395). Cells transduced with
lentiviruses expressing wild type E6 and E7 from HPV16 (E6E7 wt) induce
immortalization in primary keratinocytes as indicated by the extension of
their lifespan as compared to non-transduced control cells (FIG. 4) and
activation of hTERT, the catalytic subunit of telomerase (data not
shown). Expression of a polypeptide of the invention (E6E7SH) is not able
to to extend the lifespan compared to GFP-transduced or non-transduced
keratinocytes. A similar result was obtained in two additional
independent donors (data not shown). Taken together these data
demonstrate that our constructs have lost the ability to induce
immortalization in primary human keratinocytes, that are considered a
highly physiological model.
[0297] Another construct wherein comparable fragments of HPV16 E6 and E7
were recombined in a different order was also incapable of
immortalization of primary human foreskin keratinocytes. However, an
expanded life span up to approximately 120-150 days was observed for that
construct. This indicates some unpredictability in this field, and
demonstrates the superiority of the selected designer molecules according
to the invention in this safety-related aspect.
[0298] All together the experiments in this example provide strong
evidence of the lack of transforming activity of nucleic acids encoding
HPV16 designer polypeptides according to the invention, and thus a
strongly improved safety over HPV16 E6 and E7 wt constructs.
Example 3. Immune Responses to the HPV16 E6E7SH Designer Constructs
[0299] We have prepared DNA vectors and adenoviral vectors, as described
in example 1.
[0300] We used the CB6F1 mouse strain for measuring immune responses,
based on initial experiments where mice where immunized with DNA plasmids
encoding wild type E2, or E6 or E7, and immunization with HPV16 E2, E6
and E7 antigens induced a broader cellular immune response in CB6F1 than
in C57BL/6 mice or Balb/c mice. In a separate experiment mice were
immunized with DNA vectors encoding molecules of the invention and
cellular immune responses were measured. HPV16 E7-specific immune
responses could be measured in mice immunized with DNA plasmids
expressing E6E7SH (FIG. 5).
[0301] The following data shown in this example are from mouse experiments
that were carried out with adenoviral vectors.
[0302] To evaluate the vaccine induced immunogenicity, CB6F1 mice were
immunized with adenovectors (Ad35) expressing HPV16 E6E7 wt, LSE6E7 wt,
E6E7SH or adenovectors not encoding a transgene (Empty). Two doses were
tested for administration to the mice: 5*10.sup.9 viral particles (vp)
and 1*10.sup.10 vp. Two and eight weeks after immunization the mice were
sacrificed and isolated splenocytes were stimulated overnight with an
HPV16 E7 15mer peptide pool. E7-specific responses at two weeks and at
eight weeks were analyzed by IFN.gamma. ELISPOT. The data are presented
in FIG. 6.
[0303] This shows that immunization of mice with Ad35.HPV16-E6E7SH induces
E7-specific immune responses as measured by ELISPOT analysis. In
addition, the results in FIG. 6 demonstrates the possibility to enhance
the immune response against an adenoviral expressed transgene by adding
an N-terminal leader sequence to the transgene.
[0304] Next the effect of adding HPV16 E2 to the HPV16 E6E7SH polypeptide
with respect to immunogenicity was tested. The Ad35 vectors encoded
polypeptides that had E2 either fused to the N-terminus (E2E6E7SH) or to
the C-terminus (E6E7E2SH). CB6F1 mice were immunized with a dose of
1.times.10.sup.10 vp. FIG. 7 (E7-tetramer staining) and FIG. 8 (Panel C,
IFN.gamma. ELISPOT) show the immune responses against E7, which for the
designer constructs including E2 tends to be higher in comparison to the
construct without E2, although the differences were not statistically
significant. The response against E2 was higher for adenoviral vectors
encoding only E2 compared to the response for adenoviral vectors that had
E2 fused to the E6E7SH designer polypeptide (FIG. 8B), with differences
being significant for both E2 vs E2E6E7SH and E2 vs E6E7E2SH (p-value:
<0.05).
[0305] It is concluded that the designer constructs that further include
E2 can still provide an immune response against E7, and in addition also
provide an immune response against E2, thus increasing the breadth of the
immune response over the constructs that do not include E2.
[0306] Addition of a leader sequence was shown to result in higher
E7-specific responses when fused to the N-terminus of the fusion protein
of wild type E6 and E7 (FIG. 6C). Similarly, the effect of the leader
sequence on immunogenicity of the E2E6E7SH fusion protein was determined.
Therefore, Ad35 vectors encoding the HPV16 designer polypeptide, with or
without N-terminal E2 and an Ad35 vector encoding LSE2E6E7SH were used
for immunization of mice and blood samples were taken at two-week
intervals to measure E7-specific immune responses (FIG. 9). As shown in
FIGS. 7 and 8 the presence of E2 at either N- or C-terminally fused to
E6E7SH tended to increase the immune responses. Addition of the IgE
leader sequence further increased the E7-specific response (FIG. 9B).
Over time sustained immune responses were observed for all three
adenoviral vectors that encoded designer molecules according to the
invention, and the highest response after the immunization corresponded
with the highest responses over the duration of the experiment.
[0307] It is concluded that the responses that are induced by the designer
construct that further includes N-terminal E2 can be increased by
addition of specific sequences, e.g., the IgE leader sequence, that
target the encoded protein to specific cellular compartments.
[0308] The cellular immune response against the peptide of the invention
can be induced with different types of adenoviral vectors. In the
previous experiment we used Ad35 vectors, while in the experiment of FIG.
10, mice were immunized with an Ad26 adenoviral vector expressing HPV16
E2E6E7SH. The data show that also immunization with an Ad26-based vaccine
induced E7-specific T-cells. In addition, the results demonstrate that a
second immunization with an Ad35 adenoviral vector expressing HPV16
E2E6E7SH further boosted the cellular immune responses (FIG. 10).
Example 4. Immunogenicity of HPV16 Designer Constructs in Rhesus Macaques
[0309] To evaluate the ability of the adenoviral vectors expressing the
designer sequence of the invention to induce immune responses in
non-human primates, rhesus macaques were immunized by intramuscular
injection with adenovectors (Ad26) expressing HPV16 E2E6E7SH or
adenovectors not encoding a transgene (Empty), with a dose of 1*10.sup.11
vp. Eight weeks after the immunization the immune responses were boosted
by immunization with Ad26 vectors expressing the same antigen. At week 16
the animals received one more injection with the Ad35 vectors expressing
the same antigen. Blood samples were taken at several time points and
isolated white blood cells were stimulated overnight with a peptide pools
corresponding to HPV16 E2, E6 or E7. Specific responses were measured by
IFN.gamma. ELISPOT. The data are presented in FIG. 11. In addition at
week 10 and week 18 post prime immunization, the cellular immune response
specific to peptides spanning the novel junctions in the invention was
evaluated. The induction of IFN.gamma. response was in all animals below
the limit of detection of <50 SFU per 1*10.sup.6 PBMC (data not
shown).
[0310] The data show that immunization of non-human primates with
Ad26.HPV16-E2E6E7SH resulted in cellular immune responses against all
three HPV16 proteins that are present in the encoded transgene, but not
against the novel junctions. Responses could be boosted by the additional
immunization with Ad26.HPV16-E2E6E7SH and additional boost at week 16
with the corresponding Ad35 vector further increased the HPV16 E2, E6 and
E7-specific immune responses.
[0311] In a separate experiment (not shown), Rhesus macaques were
immunized by intravaginal administration with a combination of two
adenoviral vectors, one expressing HPV16 E6E7SH and the other the HPV16
L1 protein. Low but measurable cellular responses were measured in
peripheral mononuclear blood cells against both E6 and E7. In these
experiments, strong cellular immune responses against L1 were detected.
Example 5. Therapeutic Efficacy in a Mouse Tumor Model
[0312] A polypeptide of the invention for HPV16 (comprising SEQ ID NO: 1)
is capable of inducing HPV16-specific cellular immune response in
animals, which can exert a therapeutic effect on cells expressing HPV16
E6 and/or E7. Therapeutic immunization, i.e. immunization after tumor
growth has started, can be used to demonstrate efficacy of a therapeutic
HPV vaccine candidate. The therapeutic effect of Ad26 and Ad35 vectors
was tested in mice that were injected with TC-1 cells (mouse cells
expressing HPV16 E6 and E7) (Lin et al., 1996, Cancer Res 56: 21-6). TC-1
cells will form solid tumor within a few days to weeks after
sub-cutaneous injection in mice. Without vaccine the tumors grew rapidly
and reach a pre-determined size of 1000 mm.sup.3 within 30 days (panels D
and E). Upon reaching this size the mice are sacrificed for ethical
reasons.
[0313] With a prime-boost immunization scheme with SLPs (used as a
positive control; Kenter et al., 2009, N Engl J Med 361:1838-47;
Zwaveling et al., 2002, J Immunol 169:350-8) or adenoviral vectors
expressing HPV16-E2E6E7SH, a marked decrease of the growth of TC-1
induced tumors was observed (FIG. 12, panels B and C). Closer inspection
of the first 30 days after the prime immunizations (Panels F and G) shows
that the immunization with the adenovectors expressing E2E6E7SH have a
substantially larger impact on tumor growth than immunization with the
SLPs. The initial growth rate is much lower and in most cases the tumors
shrunk. In 3 out of 11 mice immunized with the adenoviral vectors, the
tumors were completely eradicated, which is reflected in the survival
plot (panel H).
[0314] In conclusion, immunization with adenoviral vectors expressing an
HPV16 designer polypeptide of the invention significantly reduced tumor
growth or completely eradicated established tumors in a well-established
challenge model for HPV16-induced cancer.
Example 6: Employment of Repressor Systems to Improve the Productivity and
Genetic Stability of Adenoviral Vectors Expressing HPV-Derived Antigens
[0315] It has previously been reported that transgenes inserted into
adenovirus vectors under the control of powerful constitutively active
promoters can, depending on the properties of the transgene product,
negatively impact vector production (Yoshida & Yamada, 1997, Biochem
Biophys Res Commun 230:426-30; Rubinchik et al., 2000, Gene Ther
7:875-85; Matthews et al., 1999, J Gen Virol 80:345-53; Edholm et al.,
2001, J Virol 75:9579-84; Gall et al., 2007, Mol Biotechnol 35:263-73).
Examples of transgene-dependent vector productivity issues include
inefficient vector rescue and growth, low final vector yields, and, in
severe cases, rapid outgrowth of viral mutants with defective transgene
cassettes. To solve these issues, multiple studies explored the
possibility to silence vector transgene expression during vector
replication in producer cells (Matthews et al., 1999, J Gen Virol
80:345-53; Edholm et al., 2001, J Virol 75:9579-84; Gall et al., 2007,
Mol Biotechnol 35:263-73; Cottingham et al., 2012, Biotechnol Bioeng
109:719-28; Gilbert et al., 2014, J Virol Methods 208:177-88). In this
regard, different repression systems have previously been implemented in
the context of Ad vectors and have indeed shown to improve vector
productivity and genetic stability for vectors encoding different types
of (inhibitory) transgenes.
[0316] It was observed that some of the adenovirus vectors described
herein, as well as some other adenoviral vectors encoding certain HPV
antigen variants, displayed some of the transgene-dependent vector
productivity issues described above, and therefore could possibly be
further improved in that respect. We therefore sought to investigate
whether usage of systems to repress vector transgene expression can
improve production characteristics of Ad vectors expressing HPV-derived
antigens as those described herein. For this purpose, we implemented two
existing repressor-operator systems, i.e. TetR/TetO (Yao & Eriksson,
1999, Hum Gene Ther 10:419-22, EP0990041B1) and CymR/CuO (Mullick et al.,
2006, BMC Biotechnol 6:43), into our adenovirus vector platform. Both the
TetR/TetO and the CymR/CuO system have previously been used by others to
improve adenovirus vector productivity through vector transgene silencing
during vector replication (Gall et al., 2007, Mol Biotechnol 35:263-73;
Cottingham et al., 2012, Biotechnol Bioeng 109:719-28; Gilbert et al.,
2014, J Virol Methods 208:177-88). Implementation of these two systems
involved the generation of adenoviral vectors expressing genes of
interest under the control of either a TetO or a CuO sequence-containing
CMV promoter. Furthermore, the implementation entailed the generation of
cell lines stably expressing the respective cognate repressors proteins
(i.e. TetR or CymR).
[0317] Several E1-deleted, Ad26- and Ad35-based vectors were generated in
which sequences encoding heterologous polypeptides were operably linked
to a CMV promoter containing either TetO or CuO operator sequences.
First, certain TetO- or CuO-containing sequences (SEQ ID NO: 11 and SEQ
ID NO: 12, respectively) were inserted near the transcription start site
(TSS) of the CMV promoter (SEQ ID NO: 13) of pAdapt26 and pAdapt35.Bsu
plasmids (Abbink et al., 2007, J Virol 81:4654-63; Havenga et al., 2006,
J Gen Virol 87:2135-43). The operator-containing sequences were inserted
at precisely the same positions of the CMV promoter as previously
described for the two systems (Yao & Eriksson, 1999, Human Gene Ther
10:419-22; EP0990041B1: Mullick et al., 2006, BMC Biotechnol 6:43;
EP1385946B1). Specifically, relative to the TSS (as originally assigned;
Stenberg et al. 1984, J Virol 49:190-9), the TetO- and CuO-containing
sequences were inserted directly downstream of positions -20 and +7,
respectively. In SEQ ID NO: 13, these two positions correspond to
positions 716 and 742, respectively. The resulting operator-containing
CMV promoters are termed, respectively, CMVTetO and CMVCuO. Next,
different transgenes were inserted downstream of the (modified) CMV
promoters of the resulting constructs using HindIII and XbaI restriction
sites. These transgenes included genes encoding a fusion protein of green
fluorescent protein and luciferase (GFP-Luc), HPV16 LSE2E6E7SH as
described above in example 1, and another polypeptide with some
similarity to HPV16 LSE2E6E7SH (a construct referred to in this example
as `HPVAg`). HPVAg comprises the same leader sequence as present in
LSE2E6E7SH, as well as E2, E6, and E7 sequences of HPV16. Using methods
as described herein, the resulting modified pAdapt26 and pAdapt35.Bsu
plasmids were used for the generation of adenoviral vectors expressing
the above mentioned reporter and HPV transgenes under the control of
either the CMVTetO or the CMVCuO promoter.
[0318] Cell lines expressing either TetR or CymR were generated by stable
transfection of PER.C6.RTM. cells using, respectively, plasmid
pcDNA.TM.6/TR (LifeTechnologies, V1025-20) and a derivative of
pcDNA.TM.6/TR in which the TetR-coding sequence (SEQ ID NO: 14, which
encodes polypeptide SEQ ID NO: 15) is replaced by a codon-optimized
CymR-coding sequence (SEQ ID NO: 16, which encodes polypeptide SEQ ID NO:
17). Stable cell line generation was performed largely as described by
the supplier of pcDNA.TM.6/TR using a transient transfection-based assay
to screen for cell clones capable of repressing expression of CMVTetO- or
CMVCuO-driven genes. The resulting PER.C6/TetR and PER.C6/CymR cell lines
were analyzed for their ability to repress transgene expression during
vector replication in these cells. Experiments conducted with vectors
expressing GFP-Luc under the control of operator-containing CMV-promoters
showed at least a 10-fold reduction of luciferase gene expression
throughout the complete virus replication cycle in the cell lines
expressing the repressor corresponding to the respective operator
sequences (data not shown). This confirmed that the PER.C6/TetR and
PER.C6/CymR cell lines were capable of repressing vector transgene
expression in the context of replicating adenovirus vectors.
[0319] The effect of TetR- and CymR-mediated repression of adenovector
transgene expression on vector yields was investigated for Ad35-based
vectors expressing HPVAg (FIG. 13A). To this end, PER.C6, PER.C6/TetR,
and PER.C6/CymR cell lines, seeded at 3*10.sup.5 cells per well in
24-well plate wells, were subjected to quadruplicate infections--at 1000
virus particles per cell and for a duration of three hours--by vectors
expressing HPVAg from either CMVTetO or CMVCuO promoters. As controls,
parallel infections were performed with corresponding vectors expressing
GFP-Luc instead of HPVAg. Four days after infection, crude viral lysates
were prepared by subjecting the contents of the wells (i.e. infected
cells and medium) to two freeze-thaw cycles. Adenovector titers were
subsequently determined by an Ad35 hexon sequence-specific quantitative
PCR-based protocol that uses a purified Ad35 vector with known virus
particle titer as a standard. The results show that both the TetO- and
the CuO-containing HPVAg-encoding Ad35 vectors, compared to the control
vectors expressing GFP-Luc, display decreased vector yields on normal
PER.C6 cells. By contrast, when produced on cells expressing their
cognate repressors (i.e. TetR and CymR, respectively), these same vectors
gave yields as high as those obtained with the control vectors. These
data indicate that repression of transgene expression during vector
production in producer cells can be beneficial for the productivity of
Ad35 vectors carrying HPVAg as a transgene.
[0320] The effect that repression of adenovector transgene expression may
have on vector yields was also investigated for vectors derived from
adenovirus serotype 26 (Ad26) (FIG. 13B). In an assay performed
essentially as described above for the Ad35 vectors, Ad26 vectors
carrying CMVTetO promoter-controlled transgenes encoding either GFP-Luc,
HPVAg, or LSE2E6E7SH were used to infect PER.C6 and PER.C6/TetR cells at
1500 virus particles per cell. Three days later the infections were
harvested and virus particle titers determined by an Ad26 hexon
sequence-specific quantitative PCR-based method. The results show that on
PER.C6 cells the yields for the vectors encoding HPVAg and LSE2E6E7SH are
lower than obtained with the control vector encoding GFP-Luc. In
contrast, on PER.C6/TetR cells, both these vectors showed titers that are
as high as that obtained for the control vector. Together with the
results above (for Ad35 vectors), these data indicate that repression of
transgene expression during adenovector production increases the yields
of vectors expressing HPVAg and LSE2E6E7SH.
[0321] We have observed major issues regarding the genetic stability of an
adenovirus vector that carried a CMV promoter-driven transgene for HPVAg.
For example, it was observed that after several passaging rounds of this
vector on PER.C6 the majority of the vector population consisted of a
mutant vector that carried a large deletion in the HPVAg coding sequence
(data not shown).
[0322] We reasoned that employment of a transgene expression repression
system, such as one of the two described above, could prevent genetic
stability issues associated with transgenes, such as HPVAg that are
inhibitory to vector growth. To test this, an Ad35-based vector with
CMVCuO promoter-driven HPVAg expression was assessed for transgene
cassette stability upon growth of the vector on either PER.C6 or
PER.C6/CymR cells (FIG. 14). In brief, vector DNA was transfected into
the two different cell lines and resultant viral plaques were allowed to
grow under an agarose layer. From each of the two transfections, five
viral plaques were isolated and separately passaged further on the same
cell line (i.e. as used for the transfection), for ten consecutive viral
passages. Transgene integrity was assessed by PCR amplification of the
transgene cassette at viral passage number ten (VPN10), and the
subsequent analysis of resultant PCR products by gel electrophoresis and
Sanger sequencing. In addition, at VPN7, the passaged viral clones were
assessed for their ability to express HPVAg. This was done by using the
passaged viral isolates to infect A549 cells at 1000 virus particles per
cell, lysing the cells at 48 hours post infection, and subsequently
analyzing the expression of HPVAg by western blotting using a monoclonal
antibody directed against HPV16 E7 (Santa-Cruz Biotechnology). The
results of the gel electrophoresis and sequencing analyses showed that
all five viral isolates that had been passaged on PER.C6 each carried
either small frameshifting deletions or premature stop mutations within
the transgene cassette. By contrast, such deletions or mutations could
not be detected in any of the vector isolates that had been passaged on
the cell line expressing CymR (PER.C6/CymR). In agreement with these
data, all PER.C6/CymR-propagated vector isolates were able to express
HPVAg, while all PER.C6-grown vectors completely lost this ability,
suggesting defective transgene cassettes for these vectors. In
conclusion, our data demonstrate that employment of a repressor system,
as for instance the CymR/CuO system, to repress vector transgene
expression during vector propagation is an effective means to prevent
severe transgene cassette instability, such as that seen for vectors
carrying a transgene expressing HPVAg.
Example 7: Construction of a Designer Polypeptide Comprising Essentially
all HPV18 E6 and E7 CTL Epitopes
[0323] Similar to our design for HPV16 E6 and E7, we designed a novel,
non-tumorigenic polypeptide (and nucleic acid encoding such) that
contains essentially all CTL epitopes of HPV18 E6 and E7 proteins, and
has a minimum number of anticipated/predicted strong neo-epitopes
(neo-epitopes meaning epitopes not present in the wild type HPV18 E6 and
E7 proteins). A polypeptide of the invention for HPV18 (also sometimes
referred to as HPV18 `E6E7SH` herein) comprises the amino acid sequence
as provided in SEQ ID NO: 20. A codon-optimized nucleic acid encoding
this polypeptide is provided in SEQ ID NO: 21.
[0324] The molecules of the invention for HPV18 have the same advantages
as described under example 1 for HPV16. They are single molecules, which
provides manufacturing advantages over strategies where multiple
molecules are used. In addition, a polypeptide of the invention comprises
essentially all putative CTL epitopes that are present in wild-type E6
and E7 of HPV18, and at the same time have a minimum number of
anticipated/predicted strong neo-epitopes that could potentially be
immunodominant and thus divert the immune response from relevant
wild-type CTL epitopes. Thus the constructs of the present invention are
immunologically more favourable than molecules described by others that
either lack possible CTL epitopes and/or that contain more or stronger
neo-epitopes.
[0325] For instance, the HPV18 designer construct of SEQ ID NO: 20
contains only five neo-epitopes with a length of nine amino acids with a
predicted binding affinity <50 nM for the 20 most common HLA-A, 20
most common HLA-B and 20 most common HLA-C alleles, as described in
example 1 for the HPV16 designer construct (having SEQ ID NO: 1).
[0326] Nucleic acid encoding our thus designed HPV18 E6E7SH molecule (i.e.
a polypeptide having amino acid sequence as provided in SEQ ID NO:20) was
synthesized, the nucleic acid sequence comprising SEQ ID NO: 21, and
flanked by a HindIII site and a Kozak sequence on the 5'end and an XbaI
site on the 3' site (custom synthesis and standard molecular cloning at
Invitrogen Life technologies, Germany).
[0327] The synthezised fragments were cloned using HindIII and XbaI into a
standard expression vector, pCDNA2004.Neo, harbouring both a bacterial
resistance marker (Ampiciline) and a mammalian resistance marker
(Neomycine), to obtain plasmid vectors encoding an HPV18 designer
molecule of the invention, e.g. for (transient) transfection based
experiments.
[0328] These molecules could be used as such, but also as the basis for
further molecules that contain additional features. As non-limiting
examples, some further variants were prepared as described below.
[0329] The HPV18 E6E7SH fusion protein sequence can be combined with
sequences of other HPV18 early proteins to target individuals with
persistent infection and to broaden the immune repertoire in an immunized
individual. As a non-limiting example of such embodiments, we prepared a
sequence coding for a fusion protein of E6E7SH with E2 at its N-terminus.
We mutated Glycine at position 294, Lysine at position 300 and Cysteine
at position 301 of the wt HPV18 E2 protein (Genbank: AAP20597.1) into
respectively Valine, Methionine and Arginine to abrogate DNA binding
activity. Each of these mutations on its own already completely abrogates
the binding of E2 to DNA sequences that harbour E2 binding domains
(Prakash et al., 1992, Genes Dev 6: 105-16).
[0330] The resulting polypeptide is referred to as HPV18 E2E6E7SH and
comprises SEQ ID NO: 22. A codon-optimized sequence encoding this
polypeptide was prepared and is provided in SEQ ID NO: 23.
[0331] The sequences that encode the HPV18 E6E7SH polypeptides of the
invention, with or without E2, can for instance be expressed from DNA
constructs, from RNA or from viral vectors. FIG. 15 demonstrates
expression in HEK-293T cells upon transient transfection with DNA vectors
expressing transgenes as described above. After transfection, cells were
harvested and cell extracts were analyzed by SDS-PAGE and western
blotting with an antibody that recognizes E6 of HPV18. This experiment
demonstrates expression of the expected fusion proteins of appropriate
size upon transfection of the expression vectors.
[0332] Adenoviral vectors can be used to express the E6E7, either with or
without E2, and with or without additional sequences to augment the
immunogenicity of the encoded fusion protein.
[0333] The genes, coding for HPV18 designer sequences described above were
gene optimized for human expression and synthesized, at Geneart. A Kozak
sequence (5' GCCACC 3') was included directly in front of the ATG start
codon, and two stop codons (5' TGA TAA 3') were added at the end of the
respective coding sequence. The genes were inserted in the pAdApt35BSU
plasmid and in the pAdApt26 plasmid (Havenga et al., 2006, J Gen Virol
87, 2135-43) via HindIII and XbaI sites.
[0334] Ad35.HPV18-E6E7SH is a recombinant adenovirus serotype 35 (Ad35)
vector comprising the codon-optimized nucleotide sequences for the
expression of the HPV18 designer fusion protein variant as described
above (HPV18 E6E7SH, having the amino acid sequence provided in SEQ ID
NO: 20). The combined E6 and E7 sequences were placed under the control
of a CMV promoter in the E1 region of the E1,E3 deleted adenovirus
genome. Ad26.HPV18-E6E7SH is the equivalent vector based on recombinant
adenovirus serotype 26.
[0335] Similarly, Ad26 and Ad35-based recombinant adenoviral vectors were
produced that encode the HPV18 E2E6E7SH (SEQ ID NO: 22) variant.
[0336] All adenoviruses were generated, prepared, purified and stored as
described in example 1 above.
Example 8. Lack of Transforming Activity of the HPV18 Designer Constructs
[0337] The E6 and E7 proteins of HPV18 have tumorigenic potential, which
is apparent as transforming activity in certain assays, such as colony
formation in a soft-agar assay (Massimi and Banks, 2005, Methods Mol Med
119: 381-395). The E6E7SH polypeptide as described in example 7 comprises
the fragments of the E6 and E7 proteins in a re-ordered fashion. This is
expected to remove the tumorigenic potential, as can be measured for
instance by lack of transforming activity as compared to either of wt E6
and E7 proteins in such assays.
[0338] Others reported that gene-shuffled variants of HPV16 E6 and E7 have
indeed lost their oncogenic potential (Ohlschlager et al., 2006, Vaccine
24: 2880-93; Henken et al., 2012, Vaccine 30: 4259-66), demonstrating
that gene shuffling destroys the wild-type functions of HPV16 E6 and E7
proteins. In example 2, we have shown that our designer construct for
HPV16 has lost its E6 and E7 activities.
[0339] To assess the loss of tumorigenic properties, we assessed the
ability of our HPV18 E6E7SH construct to confer the ability to grow in
soft agar upon NIH 3T3 cells (as described by e.g. Massimi and Banks,
2005, Methods Mol Med 119: 381-395). Transfection of NIH3T3 cells with a
plasmid expressing the wild type HPV18 E7 resulted consistently in colony
formation. Similar to the results obtained with HPV16 E6, expression of
wild type HPV18 E6 alone did not cause colony formation above background.
Transfection with our HPV18 E6E7SH construct did not lead to growth of
colonies of cells in soft agar (FIG. 16) in four independent experiments,
demonstrating that nucleic acids encoding a polypeptide of the invention,
HPV18 E6E7SH, have lost the transforming capacity that is associated with
E7.
[0340] The tumorigenic potential of E6 and E7 is associated with their
ability to reduce the levels of the cellular proteins p53 and pRb
respectively. p53 and pRb degradation assays were performed to
demonstrate that nucleic acid encoding a polypeptide of the invention,
HPV18 E6E7SH, does not have the biological activity associated with the
wild-type E6 and E7 at the molecular level. In short, HPV18 E6 wt and our
HPV18 E6E7SH construct were expressed in NCI-H1299 cells that lack
endogenous p53 for the p53 degradation assay. For the pRb degradation
assay HPV18 E7 wt and the HPV18 E6E7SH construct were expressed in pRb
null Saos-2 cells. As can be seen in FIG. 17, co-expression of p53 with
HPV18 E6 wt, but not with HPV18 E6E7SH, leads to reduced p53 levels
(panels A and B). Likewise, panels 17C,D show that co-expression of pRb
with HPV18 E7 wt, but not with HPV18 E6E7SH, leads to reduced pRB levels.
These data demonstrate that nucleic acid encoding an HPV18 designer
polypeptide of the invention has no ability to form colonies in soft agar
and does not contain main biological activities of the wild-type HPV18 E6
and E7 polypeptides, namely the inactivation of p53 and pRb respectively.
[0341] To further demonstrate the safety of nucleic acid constructs
encoding polypeptide of the invention, we made use of primary human
genital keratinocytes derived from neonatal foreskin (HEKn cells) that
closely resemble the natural target cells for HPV mediated
transformation. Immortalization of primary human keratinocytes requires
the action of both E6 and E7 wild-type (Munger et al., 1989, J Virol 63:
4417-21). This assay is probably the physiologically most relevant in
vitro assay to demonstrate the safety of our constructs (Massimi and
Banks, 2005, Methods Mol Med 119: 381-395). Cells transduced with
lentiviruses expressing wild type E6 and E7 from HPV18 (E6E7 wt) induce
immortalization in primary keratinocytes as indicated by the extension of
their lifespan as compared to non-transduced control cells (FIG. 18) and
activation of hTERT, the catalytic subunit of telomerase (data not
shown). Expression of the HPV18 designer polypeptide of the invention
(HPV18 E6E7SH) is not able to to extend the lifespan compared to
GFP-transduced or non-transduced keratinocytes. A similar result was
obtained in two additional independent donors (data not shown). Taken
together these data demonstrate that our constructs have lost the ability
to induce immortalization in primary human keratinocytes, that are
considered a highly physiological model.
[0342] Another construct wherein comparable fragments of HPV18 E6 and E7
were recombined in a different order was also incapable of
immortalization of primary human foreskin keratinocytes. However, and
similar to the results with an alternative E6E7 sequence for HPV16 (See
example 2), an expanded life span was observed for that alternative HPV18
construct. This indicates some unpredictability in this field, and
demonstrates the superiority of the selected designer molecules according
to the invention in this safety-related aspect.
[0343] All together the experiments in this example provide strong
evidence of the lack of transforming activity of nucleic acids encoding
polypeptides according to the invention, and thus a strongly improved
safety over HPV18 E6 and E7 wt constructs.
Example 9. Immune Responses to the HPV18 E6E7SH Designer Constructs
[0344] We have prepared DNA vectors and adenoviral vectors, as described
in example 7. To evaluate the vaccine induced immunogenicity, CB6F1 mice
were immunized with adenovectors (Ad35) expressing HPV18 E6E7SH or
E2E6E7SH, or with adenovectors not encoding a transgene (Empty) as
controls. Two weeks after the prime immunization the mice were sacrificed
and isolated splenocytes were stimulated overnight with an HPV18 E6 15mer
peptide pool. E6-specific immune responses were analyzed by intracellular
cytokine staining. In a separate experiment, CB6F1 mice were immunized
with adenovectors (Ad35 or Ad26) expressing HPV18 E2E6E7SH or with
adenovectors not encoding a transgene (Empty) as control.
[0345] FIG. 19A shows that immunization of mice with Ad35.HPV18-E6E7SH
induces E6-specific immune responses as measured by ICS analysis. In
addition, the results in FIG. 19A demonstrate that fusion of E2 to the
N-terminus of the designer construct does not decrease the
immunogenicity, despite the lower expression of this E2E6E7 variant that
was observed upon transfection (FIG. 15). FIG. 19B shows that
immunization of mice with Ad35.HPV18-E6E7SH or Ad26.HPV18-E2E6E7SH
induces comparable percentage of IFN.gamma.-producing HPV18-E6 specific
CD8 T-cells.
[0346] The cellular immune response against the peptide of the invention
can be induced with different types of adenoviral vectors. In the
experiment presented in FIG. 19B, mice were immunized with either Ad26 or
Ad35 adenoviral vectors expressing HPV18 E2E6E7SH. The data show that
these adenoviral vectors induced HPV18 E6-specific T-cells to similar
levels.
Example 10. Combining Adenoviral Vectors Expressing HPV16 and HPV18
Designer Constructs
[0347] Combining designer constructs for different HPV types offers the
possibility to make a treatment vaccine for different HPV types. To
evaluate the ability of the adenoviral vectors expressing different
designer sequences to induce immune responses, mice were immunized by
intramuscular injection with the adenovectors (Ad26) expressing HPV16
E2E6E7SH (encoding protein comprising amino acid sequence set forth in
SEQ ID NO: 3) and with Ad26 expressing HPV18 E2E6E7SH (encoding protein
comprising amino acid sequence set forth in SEQ ID NO: 22) with a dose of
1*10.sup.10 vp for each vector or adenovectors not encoding a transgene
(Empty). Four weeks after the immunization the immune responses were
boosted by immunization with Ad35 vectors expressing the same antigens.
Immune responses were measured two weeks after the boost immunization.
Cells were stimulated overnight with peptide pools corresponding to E6 of
HPV18 or E7 of HPV16 and responses were measured by IFN.gamma. ELISPOT.
The data are presented in FIG. 20.
[0348] The data show that immunization of mice with Ad26/35 vectors
expressing HPV16 E2E6E7SH and HPV18 E2E6E7SH resulted in cellular immune
responses against both (i.e. HPV16 and HPV18) designer proteins.
[0349] In an independent experiment with a similar immunization schedule
(Ad26 prime and Ad35 boost) we compared the immune response induced by Ad
expressing HPV16 E2E6E7SH and Ad expressing HPV18 E2E6E7SH together to
that induced in mice immunized with Ad expressing HPV16 E2E6E7SH alone or
Ad expressing HPV18 E2E6E7SH alone. Immune responses were measured two
weeks after boost immunization, and cells were stimulated overnight with
peptide pools corresponding to E2, E6 or E7 of HPV16 and HPV18 and the
responses were measured by IFN.gamma. ELISPOT as well as intracellular
cytokine staining. Although co-administration in a single composition of
Ad expressing HPV16 E2E6E7SH and Ad expressing HPV18 E2E6E7SH did result
in an overall lower magnitude of CD4 and CD8 responses as compared to
animals that were only immunized with the individual vaccine components,
the co-administration induced a similar breadth of the immune responses
(data not shown).
[0350] Co-administration of HPV16 E2E6E7SH and HPV18 E2E6E7SH expressing
constructs according to the invention is thus possible to induce cellular
immune responses to both HPV16 and HPV18.
Example 11. Immunogenicity of Combined Designer Constructs in Rhesus
Macaques
[0351] To evaluate the ability of the adenoviral vectors expressing the
designer sequences of the invention to induce immune responses in
non-human primates, rhesus macaques were immunized by intramuscular
injection with the mix of two separate adenovectors as in the previous
example, i.e. Ad26 vectors together expressing HPV16 and HPV18 E2E6E7SH,
at a dose of 1*10.sup.11 vp for each vector, or adenovectors not encoding
a transgene (Empty). Eight weeks after the immunization, animals received
a boost immunization with Ad26 vectors expressing the same antigens. At
week 16 the animals received one more injection with the Ad35 vectors
expressing the same antigens. Blood samples were taken at several time
points and isolated white blood cells were stimulated overnight with
peptide pools corresponding to E2, E6 or E7 for both HPV16 and HPV18.
Specific responses were measured by IFN.gamma. ELISPOT. The data are
presented in FIG. 21. In addition at week 10 and week 18 post prime
immunization, the cellular immune response specific to peptides spanning
the novel junctions in the HPV18 designer molecules of the invention was
evaluated. The induction of IFN.gamma. response against these junctional
peptides was in all animals below the limit of detection of <50 SFU
per 1*10.sup.6 PBMC (data not shown).
[0352] The data show that immunization of non-human primates with a
combination of Ad26 vectors together expressing HPV16 E2E6E7SH and HPV18
E2E6E7SH resulted in cellular immune responses against several of the HPV
proteins that are present in the encoded transgenes. Responses could be
boosted by the additional immunization with Ad26 vectors. The additional
boost immunization at week 16 with the corresponding Ad35 vector further
increased the immune responses.
Example 12. Therapeutic Efficacy of Combined Constructs in a Mouse Tumor
Model
[0353] A polypeptide of the invention corresponding to HPV16 E6 and E7 is
capable of inducing cellular immune responses in mice that will lead to a
therapeutic effect in the TC-1 model (as shown in example 5). The
therapeutic effect of a combination of adenoviral vectors together
expressing both HPV16 and HPV18 designer proteins was tested in this same
model. Without vaccine the tumors grew rapidly and reach a pre-determined
size of 1000 mm.sup.3 within 30 days at which point the mice were
sacrificed for ethical reasons.
[0354] In this experiment, C57BL/6 mice were injected sub-cutaneously with
5*10.sup.4 TC-1 cells at day 0. After six days, when tumors were
palpable, mice were immunized with Ad26.HPV16-E2E6E7SH or a mixture of
Ad26.HPV16-E2E6E7SH and Ad26.HPV18-E2E6E7SH. All mice also received a
boost immunization at day 20 with the corresponding Ad35 vectors. It was
observed that the prime-boost immunizations with adenoviral vectors
expressing HPV16 E2E6E7SH prolonged the survival of the mice
significantly (FIG. 22). With a combination of adenoviral vectors
together expressing both HPV16 E2E6E7SH and HPV18 E2E6E7SH, a similar
mean survival time was observed. In the group of mice that received the
combination vaccine, three animals were tumor free at the end of the
monitoring period of 90 days.
[0355] Results from other experiments showed that the prime-boost
immunizations with adenoviral vectors together expressing HPV16 E2E6E7SH
and HPV18 E2E6E7SH also prolonged the survival of the mice significantly
when the prime immunization was administered earlier, e.g., 4 days after
the mice were sub-cutaneously injected with the TC-1 cells (data not
shown).
[0356] In conclusion, immunization with a combination of adenoviral
vectors together expressing HPV16- and HPV18-specific designer
polypeptides of the invention significantly reduced tumor growth or
completely eradicated established tumors in a well-established challenge
model for HPV16-induced cancer.
Example 13: Construction of MVA Vector for HPV16E2E6E7 and HPV18E2E6E7
(MVA-BN mBN411)
[0357] In the instant example, we have generated an MVA-BN vector
including HPV16 E2E6E7 and HPV18 E2E6E7. It is understood that MVA-BN
vectors can be used to express the E6E7, either with or without E2, and
with or without additional sequences to augment the immunogenicity of the
encoded polypeptide.
[0358] We designed a novel nucleic acid (SEQ ID NO: 24) coding for the
polypeptide HPV16 E2E6E7 (SEQ ID NO: 3) and a novel nucleic acid (SEQ ID
NO: 25) coding for the polypeptide HPV18 E2E6E7 (SEQ ID NO: 22). The
novel nucleic acids were designed for human expression and minimal
homology among each other. The nucleic acid sequences were synthesized at
Geneart.
[0359] The PrMVA13.5long promoter (SEQ ID NO: 26) was included in front of
the ATG start codon of HPV16 E2E6E7 and two stop codons (5' TGA TGA 3')
were added at the end of the coding sequence. The PrHyb promoter (SEQ ID
NO: 27) was included in front of the ATG start codon of HPV18 E2E6E7 and
two stop codons (5' TGA TGA 3') were added at the end of the coding
sequence. Early termination signals (5' TTTTTAT 3') were inserted behind
the respective stop codons of both nucleic acid sequences.
[0360] The genes were inserted via SacII and NheI into pBNX202, a transfer
vector encoding IGR88/89 MVA-BN homologous regions and therefore allow
insertion into an insertion site of IGR88/89 of MVA-BN via homologous
recombination. Moreover pBNX202 encodes mRFP1 and ecogpt for positive
selection as well as a repetitive sequence of the IGR 88/89 MVA-BN
homologous region Flank 2 for later excision of the selection cassette
via homologous recombination in the absence of selective pressure.
[0361] The MVA based vectors were generated in primary chicken fibroblasts
(CEF) and produced as described herein.
[0362] The CEF cells were weekly isolated of chicken embryos and
maintained in VP-SFM medium without FBS.
[0363] Briefly, CEF cells were transfected with MVA vector plasmid, using
Fugene according to the instructions provided by the manufacturer
(Promega) and a coinfection with MVA-BN has been performed. Cells were
harvested after two days, sonified and further plaque purified. The virus
was plaque purified and amplified in CEF cells cultured in a single well
of a multiwell 24-tissue culture plate or a single well of a multiwell
96-tissue culture plate respectively. Further amplification was carried
out in CEF cells cultured in a single well of a multiwell 6-tissue plate
and subsequently in a T175 tissue culture flask.
[0364] To generate the virus mBN 411A, eleven passages were generated,
three passages of which were plaque purifications in VP-SFM medium
containing Mycophenolic acid/xanthine and hypoxanthine. To generate
mBN411B, seventeen passages were generated, six passage of which were
plaque purifications in VP-SFM medium without selective pressure to allow
excision of the selection cassette via homologous recombination.
[0365] The MVA mBN 411 virus is thus an MVA-BN comprising in its IGR88/89
region a nucleic acid encoding designer polypeptide HPV16 E2E6E7SH (SEQ
ID NO: 3) under control of a PrMVA13.5long promoter (SEQ ID NO: 26) and a
nucleic acid encoding designer polypeptide HPV18 E2E6E7SH (SEQ ID NO: 22)
under control of a a PrHyb promoter (SEQ ID NO: 27). The MVA mBN411 virus
was used in subsequent experiments in prime-boost regimens with
adenovirus vectors encoding designer polypeptides.
Example 14. Immunogenicity of HPV16 and HPV18 Designer Constructs in a
Ad26 Prime and MVA Boost Immunization in Mice
[0366] HPV16 and HPV18-specific immune response induced by a prime
immunization with adenovectors (Ad26) and boost immunization with
Modified Vaccinia Ankara Virus (MVA) were evaluated. As a priming
immunization, mice were vaccinated by intramuscular injection with
adenovector (Ad26) expressing HPV16 E2E6E7SH (encoding protein comprising
amino acid sequence set forth in SEQ ID NO: 3) and with Ad26 expressing
HPV18 E2E6E7SH (encoding protein comprising amino acid sequence set forth
in SEQ ID NO: 22), using a dose of 1*10.sup.10 vp for each vector, or as
a control with adenovectors not encoding a transgene (Empty). Eight weeks
after the prime immunization animals were boost-immunized with MVA
expressing the same antigens as during prime immunization (MVA BN mBN
411A, at a dose of 8.9.times.10.sup.7 TCID50/mouse), while another group
of mice was boost-immunized with Ad35 vectors expressing the same
antigens as during prime immunization. Control animals were
boost-immunized with an MVA vector not encoding a transgene (Control).
[0367] Immune responses were measured two weeks after the boost
immunization. Cells were stimulated overnight with peptide pools
corresponding to E2, E6 or E7 of HPV16 or HPV18 and responses were
measured by IFN.gamma. ELISPOT. The data are presented in FIG. 23.
[0368] The data show that immunization of mice with either Ad26/Ad35 or
Ad26/MVA vectors expressing HPV16 E2E6E7SH and HPV18 E2E6E7SH resulted in
cellular immune responses against both (i.e. HPV16 and HPV18) designer
proteins. The overall response was highest in animals boost-immunized
with MVA expressing HPV16 E2E6E7SH and HPV18 E2E6E7SH.
Example 15. Immunogenicity of HPV16 and HPV18 Designer Constructs in a
Ad26 Prime and MVA Boost Immunization in Rhesus Macaques
[0369] We evaluated the ability of the adenoviral vectors and MVA vectors
expressing the designer sequences of the invention to induce immune
responses in non-human primates. Rhesus macaques (non-human primates,
NHP) were prime immunized by intramuscular injection with the mix of two
separate adenovectors as in example 11, i.e. Ad26 expressing HPV16
E2E6E7SH and Ad26 expressing HPV18 E2E6E7SH, at a dose of 1*10.sup.11 vp
for each adenoviral vector. Eight weeks after the prime immunization,
animals were boosted with MVA-BN (mBN 411A, a vector expressing these
same antigens, at a dose of about 1.80.times.10.sup.8 TCID50/NHP).
[0370] Blood samples were taken at several time points and isolated white
blood cells are stimulated overnight with peptide pools corresponding to
E2, E6 or E7 for both HPV16 and HPV18. Specific responses are measured by
IFN.gamma. ELISPOT. The data are presented in FIG. 24.
[0371] The data show that immunization of Rhesus macaques with Ad/MVA
expressing HPV16 E2E6E7SH and HPV18 E2E6E7SH resulted in cellular immune
responses against the designed antigens. Moreover the induced cellular
responses appear to be broadened by MVA boosting, with responses against
3-5 of the 6 different HPV16 and HPV18 antigens that are expressed by the
vaccine vectors.
Example 16. Therapeutic Efficacy of HPV16 and HPV18 Designer Constructs in
a Ad26 Prime and MVA Boost Immunization in Mice
[0372] The therapeutic effect of a prime boost immunization with
adenoviral vectors and MVA expressing HPV16 and HPV18 designer proteins
were tested in the same TC-1 model as described under example 12. The
tumor growth was followed over time, animals are sacrificed for ethical
reasons once the tumor volume reaches >1000 mm.sup.3.
[0373] The experimental design is as follows:
TABLE-US-00001
Group Tumor cell inoculation Prime day 6 Boost day 20 Sacrifice
(n = 36 total) (day 0) (dose) (dose) (day)
1. Pos control 50,000 TC-1 Ad26.HPV16-Tx + Ad35.HPV16-Tx + 90 or earlier
if tumor
(n = 12) cells Ad26.HPV18-Tx Ad35.HPV18-Tx volume >1000 mm.sup.3
(1 .times. 10.sup.10 vp/vector) (1 .times. 10.sup.10 vp/vector)
2. Test 50,000 TC-1 Ad26.HPV16-Tx + MVA-BN-HPV16/18-Tx 90 or earlier if
tumor
(n = 12) cells Ad26.HPV18-Tx (8.9 .times. 10.sup.7 TCID50) volume >1000
mm.sup.3
(1 .times. 10.sup.10 vp/vector)
3. Neg control 50,000 TC-1 Ad26.empty MVA-BN-control 90 or earlier if
tumor
(n = 12) cells (1 .times. 10.sup.10 vp/vector) (8.9 .times. 10.sup.7
TCID50) volume >1000 mm.sup.3
[0374] Treatment Groups.
[0375] At the day of priming tumors were palpable in minimum 50% of the
animals. (HPV16-Tx and HPV18-Tx are indications for the constructs of the
invention encoding polypeptides having SEQ ID NOs: 3 and 20,
respectively).
[0376] Blood was drawn before TC-1 tumor cell inoculation, at day 19 (i.e.
one day before boost administration) and at day 34 (i.e. two weeks after
boost administration), and in some mice blood was also drawn at day 90
after TC-1 tumor cell inoculation.
[0377] In this experiment, C57BL/6 mice were injected sub-cutaneously with
5*10.sup.4 TC-1 cells at day 0. After six days, when tumors were
palpable, mice were immunized with a mixture of Ad26.HPV16-E2E6E7SH and
Ad26.HPV18-E2E6E7SH. Mice received a boost immunization at day 20 with
either Ad26.HPV16-E2E6E7SH and Ad26.HPV18-E2E6E7SH or MVA-BN-HPV16/18-Tx.
Control mice were primed with an Ad26 not encoding a transgene and
boosted with a MVA not encoding a transgene. Animals immunized with
either Ad26/Ad35 encoding E2E6E7SH or MVA-BN encoding HPV16/18 E2E6E7SH
resulted in a comparable prolonged survival and median survival time; in
both groups one mouse was alive and tumor free at the end of the
monitoring period of 90 days. The data are represented in FIG. 25.
[0378] The examples in the specification are considered as exemplary only,
with a true scope and spirit of the invention being indicated by the
following claims.
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Sequence CWU
1
1
331328PRTArtificial SequenceHPV16-E6E7SH designer polypeptide 1Met His Gln
Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro1 5
10 15Arg Lys Leu Pro Gln Leu Cys Thr Glu
Leu Gln Thr Thr Ile His Asp 20 25
30Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Glu Asp Glu Ile
35 40 45Asp Gly Pro Ala Gly Gln Ala
Glu Pro Asp Arg Ala His Tyr Asn Ile 50 55
60Val Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln65
70 75 80Ser Thr His Val
Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr 85
90 95Leu Gly Ile Val Cys Pro Ile Cys Ser Gln
Lys Pro Gly Thr Thr Leu 100 105
110Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile
115 120 125Asn Cys Gln Lys Pro Leu Cys
Pro Glu Glu Lys Gln Arg His Leu Asp 130 135
140Lys Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg
Cys145 150 155 160Met Ser
Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Met His
165 170 175Gly Asp Thr Pro Thr Leu His
Glu Tyr Met Leu Asp Leu Gln Pro Glu 180 185
190Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser
Glu Glu 195 200 205Glu Asp Glu Ile
Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala 210
215 220His Tyr Asn Ile Val Thr Phe Cys Cys Gln Leu Cys
Thr Glu Leu Gln225 230 235
240Thr Thr Ile His Asp Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln
245 250 255Leu Leu Arg Arg Glu
Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile 260
265 270Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys Asp
Lys Cys Leu Lys 275 280 285Phe Tyr
Ser Lys Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr 290
295 300Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro
Leu Cys Asp Leu Leu305 310 315
320Ile Arg Cys Ile Asn Cys Gln Lys
3252990DNAArtificial Sequencent sequence encoding HPV16-E6E7SH designer
polypeptide 2atgcaccaga aacggaccgc catgttccag gacccccagg aacggcccag
aaagctgccc 60cagctgtgca ccgagctgca gaccaccatc cacgacatca tcctggaatg
cgtgtactgc 120aagcagcagc tggaagatga gatcgacggc cctgctggcc aggccgaacc
cgacagagcc 180cactacaata tcgtgacctt ctgctgcaag tgcgacagca ccctgcggct
gtgcgtgcag 240agcacccacg tggacatccg gaccctggaa gatctgctga tgggcaccct
gggcatcgtg 300tgccccatct gcagccagaa gcccggcacc accctggaac agcagtacaa
caagcccctg 360tgcgacctgc tgatccggtg catcaactgc cagaaacccc tgtgccccga
ggaaaagcag 420cggcacctgg acaagaagca gcggttccac aacatccggg gcagatggac
aggcagatgc 480atgagctgct gcagaagcag ccggaccaga cgggaaaccc agatgcacgg
cgacaccccc 540accctgcacg agtacatgct ggacctgcag cccgagacaa ccgacctgta
ctgctacgag 600cagctgaacg acagcagcga ggaagaggac gagattgacg gacccgctgg
acaggccgag 660cctgaccggg ctcactataa catcgtgaca ttttgctgtc agctctgtac
tgaactccag 720acaacaattc acgatattat tctcgaatgt gtgtattgta aacagcagct
cctgcggaga 780gaggtgtacg acttcgcctt ccgggacctc tgcatcgtgt atcgggacgg
caacccctac 840gccgtgtgcg acaagtgcct gaagttctac agcaagatca gcgagtaccg
gcactactgc 900tacagcctgt acggaacaac actcgaacag cagtataaca aaccactctg
tgatctgctg 960attcgctgta tcaattgtca gaagtgataa
9903693PRTArtificial SequenceHPV16 E2E6E7SH designer
polypeptide 3Met Glu Thr Leu Cys Gln Arg Leu Asn Val Cys Gln Asp Lys Ile
Leu1 5 10 15Thr His Tyr
Glu Asn Asp Ser Thr Asp Leu Arg Asp His Ile Asp Tyr 20
25 30Trp Lys His Met Arg Leu Glu Cys Ala Ile
Tyr Tyr Lys Ala Arg Glu 35 40
45Met Gly Phe Lys His Ile Asn His Gln Val Val Pro Thr Leu Ala Val 50
55 60Ser Lys Asn Lys Ala Leu Gln Ala Ile
Glu Leu Gln Leu Thr Leu Glu65 70 75
80Thr Ile Tyr Asn Ser Gln Tyr Ser Asn Glu Lys Trp Thr Leu
Gln Asp 85 90 95Val Ser
Leu Glu Val Tyr Leu Thr Ala Pro Thr Gly Cys Ile Lys Lys 100
105 110His Gly Tyr Thr Val Glu Val Gln Phe
Asp Gly Asp Ile Cys Asn Thr 115 120
125Met His Tyr Thr Asn Trp Thr His Ile Tyr Ile Cys Glu Glu Ala Ser
130 135 140Val Thr Val Val Glu Gly Gln
Val Asp Tyr Tyr Gly Leu Tyr Tyr Val145 150
155 160His Glu Gly Ile Arg Thr Tyr Phe Val Gln Phe Lys
Asp Asp Ala Glu 165 170
175Lys Tyr Ser Lys Asn Lys Val Trp Glu Val His Ala Gly Gly Gln Val
180 185 190Ile Leu Cys Pro Thr Ser
Val Phe Ser Ser Asn Glu Val Ser Ser Pro 195 200
205Glu Ile Ile Arg Gln His Leu Ala Asn His Pro Ala Ala Thr
His Thr 210 215 220Lys Ala Val Ala Leu
Gly Thr Glu Glu Thr Gln Thr Thr Ile Gln Arg225 230
235 240Pro Arg Ser Glu Pro Asp Thr Gly Asn Pro
Cys His Thr Thr Lys Leu 245 250
255Leu His Arg Asp Ser Val Asp Ser Ala Pro Ile Leu Thr Ala Phe Asn
260 265 270Ser Ser His Lys Gly
Arg Ile Asn Cys Asn Ser Asn Thr Thr Pro Ile 275
280 285Val His Leu Lys Val Asp Ala Asn Thr Leu Met Arg
Leu Arg Tyr Arg 290 295 300Phe Lys Lys
His Cys Thr Leu Tyr Thr Ala Val Ser Ser Thr Trp His305
310 315 320Trp Thr Gly His Asn Val Lys
His Lys Ser Ala Ile Val Thr Leu Thr 325
330 335Tyr Asp Ser Glu Trp Gln Arg Asp Gln Phe Leu Ser
Gln Val Lys Ile 340 345 350Pro
Lys Thr Ile Thr Val Ser Thr Gly Phe Met Ser Ile Met His Gln 355
360 365Lys Arg Thr Ala Met Phe Gln Asp Pro
Gln Glu Arg Pro Arg Lys Leu 370 375
380Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu385
390 395 400Glu Cys Val Tyr
Cys Lys Gln Gln Leu Glu Asp Glu Ile Asp Gly Pro 405
410 415Ala Gly Gln Ala Glu Pro Asp Arg Ala His
Tyr Asn Ile Val Thr Phe 420 425
430Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His
435 440 445Val Asp Ile Arg Thr Leu Glu
Asp Leu Leu Met Gly Thr Leu Gly Ile 450 455
460Val Cys Pro Ile Cys Ser Gln Lys Pro Gly Thr Thr Leu Glu Gln
Gln465 470 475 480Tyr Asn
Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn Cys Gln
485 490 495Lys Pro Leu Cys Pro Glu Glu
Lys Gln Arg His Leu Asp Lys Lys Gln 500 505
510Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys Met
Ser Cys 515 520 525Cys Arg Ser Ser
Arg Thr Arg Arg Glu Thr Gln Met His Gly Asp Thr 530
535 540Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln Pro
Glu Thr Thr Asp545 550 555
560Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu
565 570 575Ile Asp Gly Pro Ala
Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn 580
585 590Ile Val Thr Phe Cys Cys Gln Leu Cys Thr Glu Leu
Gln Thr Thr Ile 595 600 605His Asp
Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg 610
615 620Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp Leu
Cys Ile Val Tyr Arg625 630 635
640Asp Gly Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser
645 650 655Lys Ile Ser Glu
Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr 660
665 670Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp
Leu Leu Ile Arg Cys 675 680 685Ile
Asn Cys Gln Lys 69042085DNAArtificial Sequencent sequence encoding
HPV16 E2E6E7SH designer polypeptide 4atggaaaccc tgtgccagcg
gctgaacgtg tgccaggaca agatcctgac ccactacgag 60aacgacagca ccgacctgcg
ggaccacatc gactactgga agcacatgcg gctggaatgc 120gccatctact acaaggccag
agagatgggc ttcaagcaca tcaaccacca ggtggtgccc 180accctggccg tgtccaagaa
caaggccctg caggccatcg agctgcagct gaccctggaa 240accatctaca acagccagta
cagcaacgag aagtggaccc tgcaggacgt gtccctggaa 300gtgtacctga ccgctcccac
cggctgcatc aagaaacacg gctacaccgt ggaagtgcag 360ttcgacggcg acatctgcaa
caccatgcac tacaccaact ggacccacat ctacatctgc 420gaagaggcca gcgtgaccgt
ggtggaaggc caggtggact actacggcct gtactacgtg 480cacgagggca tccggaccta
cttcgtgcag ttcaaggacg acgccgagaa gtacagcaag 540aacaaagtgt gggaggtgca
cgctggcggc caggtcatcc tgtgccccac cagcgtgttc 600agcagcaacg aggtgtccag
ccccgagatc atccggcagc acctggccaa tcaccctgcc 660gccacccaca caaaggccgt
ggccctgggc accgaggaaa cccagaccac catccagcgg 720cccagaagcg agcccgacac
cggcaatccc tgccacacca ccaagctgct gcaccgggac 780agcgtggaca gcgcccctat
cctgaccgcc ttcaacagca gccacaaggg ccggatcaac 840tgcaacagca acaccacccc
catcgtgcac ctgaaggtgg acgccaacac cctgatgcgg 900ctgcggtaca gattcaagaa
gcactgcacc ctgtacaccg ccgtgtcctc cacctggcac 960tggaccggcc acaacgtgaa
gcacaagagc gccatcgtga ccctgaccta cgacagcgag 1020tggcagcggg accagttcct
gagccaggtc aaaatcccca agaccatcac cgtgtccacc 1080ggcttcatga gcatcatgca
ccagaaacgg accgccatgt tccaggaccc ccaggaacgg 1140cccagaaagc tgccccagct
gtgcaccgag ctgcagacca ccatccacga catcatcctg 1200gaatgcgtgt actgcaagca
gcagctggaa gatgagatcg acggccctgc tggccaggcc 1260gaacccgaca gagcccacta
caatatcgtg accttctgct gcaagtgcga cagcaccctg 1320cggctgtgcg tgcagagcac
ccacgtggac atccggaccc tggaagatct gctgatgggc 1380accctgggca tcgtgtgccc
catctgcagc cagaagcccg gcaccaccct ggaacagcag 1440tacaacaagc ccctgtgcga
cctgctgatc cggtgcatca actgccagaa acccctgtgc 1500cccgaggaaa agcagcggca
cctggacaag aagcagcggt tccacaacat ccggggcaga 1560tggacaggca gatgcatgag
ctgctgcaga agcagccgga ccagacggga aacccagatg 1620cacggcgaca cccccaccct
gcacgagtac atgctggacc tgcagcccga gacaaccgac 1680ctgtactgct acgagcagct
gaacgacagc agcgaggaag aggacgagat tgacggaccc 1740gctggacagg ccgagcctga
ccgggctcac tataacatcg tgacattttg ctgtcagctc 1800tgtactgaac tccagacaac
aattcacgat attattctcg aatgtgtgta ttgtaaacag 1860cagctcctgc ggagagaggt
gtacgacttc gccttccggg acctctgcat cgtgtatcgg 1920gacggcaacc cctacgccgt
gtgcgacaag tgcctgaagt tctacagcaa gatcagcgag 1980taccggcact actgctacag
cctgtacgga acaacactcg aacagcagta taacaaacca 2040ctctgtgatc tgctgattcg
ctgtatcaat tgtcagaagt gataa 20855693PRTArtificial
SequenceHPV16 E6E7E2SH designer polypeptide 5Met His Gln Lys Arg Thr Ala
Met Phe Gln Asp Pro Gln Glu Arg Pro1 5 10
15Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr
Ile His Asp 20 25 30Ile Ile
Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Glu Asp Glu Ile 35
40 45Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp
Arg Ala His Tyr Asn Ile 50 55 60Val
Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln65
70 75 80Ser Thr His Val Asp Ile
Arg Thr Leu Glu Asp Leu Leu Met Gly Thr 85
90 95Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Lys Pro
Gly Thr Thr Leu 100 105 110Glu
Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile 115
120 125Asn Cys Gln Lys Pro Leu Cys Pro Glu
Glu Lys Gln Arg His Leu Asp 130 135
140Lys Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys145
150 155 160Met Ser Cys Cys
Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Met His 165
170 175Gly Asp Thr Pro Thr Leu His Glu Tyr Met
Leu Asp Leu Gln Pro Glu 180 185
190Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser Glu Glu
195 200 205Glu Asp Glu Ile Asp Gly Pro
Ala Gly Gln Ala Glu Pro Asp Arg Ala 210 215
220His Tyr Asn Ile Val Thr Phe Cys Cys Gln Leu Cys Thr Glu Leu
Gln225 230 235 240Thr Thr
Ile His Asp Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln
245 250 255Leu Leu Arg Arg Glu Val Tyr
Asp Phe Ala Phe Arg Asp Leu Cys Ile 260 265
270Val Tyr Arg Asp Gly Asn Pro Tyr Ala Val Cys Asp Lys Cys
Leu Lys 275 280 285Phe Tyr Ser Lys
Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr 290
295 300Gly Thr Thr Leu Glu Gln Gln Tyr Asn Lys Pro Leu
Cys Asp Leu Leu305 310 315
320Ile Arg Cys Ile Asn Cys Gln Lys Met Glu Thr Leu Cys Gln Arg Leu
325 330 335Asn Val Cys Gln Asp
Lys Ile Leu Thr His Tyr Glu Asn Asp Ser Thr 340
345 350Asp Leu Arg Asp His Ile Asp Tyr Trp Lys His Met
Arg Leu Glu Cys 355 360 365Ala Ile
Tyr Tyr Lys Ala Arg Glu Met Gly Phe Lys His Ile Asn His 370
375 380Gln Val Val Pro Thr Leu Ala Val Ser Lys Asn
Lys Ala Leu Gln Ala385 390 395
400Ile Glu Leu Gln Leu Thr Leu Glu Thr Ile Tyr Asn Ser Gln Tyr Ser
405 410 415Asn Glu Lys Trp
Thr Leu Gln Asp Val Ser Leu Glu Val Tyr Leu Thr 420
425 430Ala Pro Thr Gly Cys Ile Lys Lys His Gly Tyr
Thr Val Glu Val Gln 435 440 445Phe
Asp Gly Asp Ile Cys Asn Thr Met His Tyr Thr Asn Trp Thr His 450
455 460Ile Tyr Ile Cys Glu Glu Ala Ser Val Thr
Val Val Glu Gly Gln Val465 470 475
480Asp Tyr Tyr Gly Leu Tyr Tyr Val His Glu Gly Ile Arg Thr Tyr
Phe 485 490 495Val Gln Phe
Lys Asp Asp Ala Glu Lys Tyr Ser Lys Asn Lys Val Trp 500
505 510Glu Val His Ala Gly Gly Gln Val Ile Leu
Cys Pro Thr Ser Val Phe 515 520
525Ser Ser Asn Glu Val Ser Ser Pro Glu Ile Ile Arg Gln His Leu Ala 530
535 540Asn His Pro Ala Ala Thr His Thr
Lys Ala Val Ala Leu Gly Thr Glu545 550
555 560Glu Thr Gln Thr Thr Ile Gln Arg Pro Arg Ser Glu
Pro Asp Thr Gly 565 570
575Asn Pro Cys His Thr Thr Lys Leu Leu His Arg Asp Ser Val Asp Ser
580 585 590Ala Pro Ile Leu Thr Ala
Phe Asn Ser Ser His Lys Gly Arg Ile Asn 595 600
605Cys Asn Ser Asn Thr Thr Pro Ile Val His Leu Lys Val Asp
Ala Asn 610 615 620Thr Leu Met Arg Leu
Arg Tyr Arg Phe Lys Lys His Cys Thr Leu Tyr625 630
635 640Thr Ala Val Ser Ser Thr Trp His Trp Thr
Gly His Asn Val Lys His 645 650
655Lys Ser Ala Ile Val Thr Leu Thr Tyr Asp Ser Glu Trp Gln Arg Asp
660 665 670Gln Phe Leu Ser Gln
Val Lys Ile Pro Lys Thr Ile Thr Val Ser Thr 675
680 685Gly Phe Met Ser Ile 69062085DNAArtificial
Sequencent sequence encoding HPV16 E6E7E2SH designer polypeptide
6atgcaccaga aacggaccgc catgttccag gacccccagg aacggcccag aaagctgccc
60cagctgtgca ccgagctgca gaccaccatc cacgacatca tcctggaatg cgtgtactgc
120aagcagcagc tggaagatga gatcgacggc cctgctggcc aggccgaacc cgacagagcc
180cactacaata tcgtgacctt ctgctgcaag tgcgacagca ccctgcggct gtgcgtgcag
240agcacccacg tggacatccg gaccctggaa gatctgctga tgggcaccct gggcatcgtg
300tgccccatct gcagccagaa gcccggcacc accctggaac agcagtacaa caagcccctg
360tgcgacctgc tgatccggtg catcaactgc cagaaacccc tgtgccccga ggaaaagcag
420cggcacctgg acaagaagca gcggttccac aacatccggg gcagatggac aggcagatgc
480atgagctgct gcagaagcag ccggaccaga cgggaaaccc agatgcacgg cgacaccccc
540accctgcacg agtacatgct ggacctgcag cccgagacaa ccgacctgta ctgctacgag
600cagctgaacg acagcagcga ggaagaggac gagattgacg gacccgctgg acaggccgag
660cctgaccggg ctcactataa catcgtgaca ttttgctgtc agctctgtac tgaactccag
720acaacaattc acgatattat tctcgaatgt gtgtattgta aacagcagct cctgcggaga
780gaggtgtacg acttcgcctt ccgggacctc tgcatcgtgt atcgggacgg caacccctac
840gccgtgtgcg acaagtgcct gaagttctac agcaagatca gcgagtaccg gcactactgc
900tacagcctgt acggaacaac actcgaacag cagtataaca aaccactctg tgatctgctg
960attcgctgta tcaattgtca gaagatggaa accctgtgcc agcggctgaa cgtgtgccag
1020gacaagatcc tgacccacta cgagaacgac agcaccgacc tgcgggacca catcgactac
1080tggaagcaca tgcggctgga atgcgccatc tactacaagg ccagagagat gggcttcaag
1140cacatcaacc accaggtggt gcccaccctg gccgtgtcca agaacaaggc cctgcaggcc
1200atcgagctgc agctgaccct ggaaaccatc tacaacagcc agtacagcaa cgagaagtgg
1260accctgcagg acgtgtccct ggaagtgtac ctgaccgctc ccaccggctg catcaagaaa
1320cacggctaca ccgtggaagt gcagttcgac ggcgacatct gcaacaccat gcactacacc
1380aactggaccc acatctacat ctgcgaagag gccagcgtga ccgtggtgga aggccaggtg
1440gactactacg gcctgtacta cgtgcacgag ggcatccgga cctacttcgt gcagttcaag
1500gacgacgccg agaagtacag caagaacaaa gtgtgggagg tgcacgctgg cggccaggtc
1560atcctgtgcc ccaccagcgt gttcagcagc aacgaggtgt ccagccccga gatcatccgg
1620cagcacctgg ccaatcaccc tgccgccacc cacacaaagg ccgtggccct gggcaccgag
1680gaaacccaga ccaccatcca gcggcccaga agcgagcccg acaccggcaa tccctgccac
1740accaccaagc tgctgcaccg ggacagcgtg gacagcgccc ctatcctgac cgccttcaac
1800agcagccaca agggccggat caactgcaac agcaacacca cccccatcgt gcacctgaag
1860gtggacgcca acaccctgat gcggctgcgg tacagattca agaagcactg caccctgtac
1920accgccgtgt cctccacctg gcactggacc ggccacaacg tgaagcacaa gagcgccatc
1980gtgaccctga cctacgacag cgagtggcag cgggaccagt tcctgagcca ggtcaaaatc
2040cccaagacca tcaccgtgtc caccggcttc atgagcatct gataa
2085718PRTArtificial SequenceIgE leader peptide 7Met Asp Trp Thr Trp Ile
Leu Phe Leu Val Ala Ala Ala Thr Arg Val1 5
10 15His Ser854DNAArtificial Sequencent sequence
encoding IgE leader peptide 8atggactgga cctggatcct gttcctggtg gctgccgcaa
cccgggtgca cagc 54921PRTArtificial SequenceHAVT20 leader
peptide 9Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser Thr Cys Leu1
5 10 15Glu Phe Ser Met
Ala 201063DNAArtificial Sequencent sequence encoding HAVT20
leader peptide 10atggcctgcc ccggctttct gtgggccctg gtcatcagca cctgtctgga
attcagcatg 60gcc
631154DNAArtificial Sequence2xTetO-containing sequence
11gagctctccc tatcagtgat agagatctcc ctatcagtga tagagatcgt cgac
541228DNAArtificial Sequence2xCuO-containing sequence 12aacaaacaga
caatctggtc tgtttgta
2813829DNAArtificial SequenceCMV promoter 13tcaatattgg ccattagcca
tattattcat tggttatata gcataaatca atattggcta 60ttggccattg catacgttgt
atccatatca taatatgtac atttatattg gctcatgtcc 120aacattaccg ccatgttgac
attgattatt gactagttat taatagtaat caattacggg 180gtcattagtt catagcccat
atatggagtt ccgcgttaca taacttacgg taaatggccc 240gcctggctga ccgcccaacg
acccccgccc attgacgtca ataatgacgt atgttcccat 300agtaacgcca atagggactt
tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360ccacttggca gtacatcaag
tgtatcatat gccaagtacg ccccctattg acgtcaatga 420cggtaaatgg cccgcctggc
attatgccca gtacatgacc ttatgggact ttcctacttg 480gcagtacatc tacgtattag
tcatcgctat taccatggtg atgcggtttt ggcagtacat 540caatgggcgt ggatagcggt
ttgactcacg gggatttcca agtctccacc ccattgacgt 600caatgggagt ttgttttggc
accaaaatca acgggacttt ccaaaatgtc gtaacaactc 660cgccccattg acgcaaatgg
gcggtaggcg tgtacggtgg gaggtctata taagcagagc 720tcgtttagtg aaccgtcaga
tcgcctggag acgccatcca cgctgttttg acctccatag 780aagacaccgg gaccgatcca
gcctccgcgg ccgggaacgg tgcattgga 82914657DNAArtificial
Sequencent sequence encoding TetR polypeptide 14atgtctagat tagataaaag
taaagtgatt aacagcgcat tagagctgct taatgaggtc 60ggaatcgaag gtttaacaac
ccgtaaactc gcccagaagc taggtgtaga gcagcctaca 120ttgtattggc atgtaaaaaa
taagcgggct ttgctcgacg ccttagccat tgagatgtta 180gataggcacc atactcactt
ttgcccttta gaaggggaaa gctggcaaga ttttttacgt 240aataacgcta aaagttttag
atgtgcttta ctaagtcatc gcgatggagc aaaagtacat 300ttaggtacac ggcctacaga
aaaacagtat gaaactctcg aaaatcaatt agccttttta 360tgccaacaag gtttttcact
agagaatgca ttatatgcac tcagcgctgt ggggcatttt 420actttaggtt gcgtattgga
agatcaagag catcaagtcg ctaaagaaga aagggaaaca 480cctactactg atagtatgcc
gccattatta cgacaagcta tcgaattatt tgatcaccaa 540ggtgcagagc cagccttctt
attcggcctt gaattgatca tatgcggatt agaaaaacaa 600cttaaatgtg aaagtgggtc
cgcgtacagc ggatcccggg aattcagatc ttattaa 65715218PRTArtificial
SequenceTetR polypeptide 15Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn
Ser Ala Leu Glu Leu1 5 10
15Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln
20 25 30Lys Leu Gly Val Glu Gln Pro
Thr Leu Tyr Trp His Val Lys Asn Lys 35 40
45Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His
His 50 55 60Thr His Phe Cys Pro Leu
Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg65 70
75 80Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu
Ser His Arg Asp Gly 85 90
95Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr
100 105 110Leu Glu Asn Gln Leu Ala
Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu 115 120
125Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu
Gly Cys 130 135 140Val Leu Glu Asp Gln
Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr145 150
155 160Pro Thr Thr Asp Ser Met Pro Pro Leu Leu
Arg Gln Ala Ile Glu Leu 165 170
175Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu
180 185 190Ile Ile Cys Gly Leu
Glu Lys Gln Leu Lys Cys Glu Ser Gly Ser Ala 195
200 205Tyr Ser Gly Ser Arg Glu Phe Arg Ser Tyr 210
21516612DNAArtificial Sequencent sequence encoding CymR
polypeptide 16atgtctccca aacgacggac tcaagcggaa agggcaatgg aaactcaggg
taagctgatt 60gccgcggctc tgggagtgct gcgagagaaa gggtatgccg ggtttcgcat
agccgacgtt 120cctggagctg caggcgtaag cagaggagcc caatctcatc actttccgac
caagctggag 180cttttgctgg ctaccttcga atggctgtac gagcagatca cggaaaggag
tcgtgctagg 240ctggccaagc tgaaacccga ggatgatgtc attcagcaga tgctggacga
tgcagccgag 300ttcttcctgg acgacgactt cagcatcagt ctcgacctca tcgtagccgc
agatcgcgat 360ccagctttgc gcgagggcat acagagaaca gtcgagcgga atcggtttgt
ggtggaggac 420atgtggcttg gtgttctggt gagcagaggc ctctcacggg atgatgccga
ggacatcctg 480tggctgatct ttaactccgt cagagggttg gcagtgaggt ccctttggca
gaaggacaaa 540gaacggtttg aacgtgtgcg aaactcaaca ctcgagattg ctagggaacg
ctacgccaag 600ttcaagagat ga
61217203PRTArtificial SequenceCymR polypeptide 17Met Ser Pro
Lys Arg Arg Thr Gln Ala Glu Arg Ala Met Glu Thr Gln1 5
10 15Gly Lys Leu Ile Ala Ala Ala Leu Gly
Val Leu Arg Glu Lys Gly Tyr 20 25
30Ala Gly Phe Arg Ile Ala Asp Val Pro Gly Ala Ala Gly Val Ser Arg
35 40 45Gly Ala Gln Ser His His Phe
Pro Thr Lys Leu Glu Leu Leu Leu Ala 50 55
60Thr Phe Glu Trp Leu Tyr Glu Gln Ile Thr Glu Arg Ser Arg Ala Arg65
70 75 80Leu Ala Lys Leu
Lys Pro Glu Asp Asp Val Ile Gln Gln Met Leu Asp 85
90 95Asp Ala Ala Glu Phe Phe Leu Asp Asp Asp
Phe Ser Ile Ser Leu Asp 100 105
110Leu Ile Val Ala Ala Asp Arg Asp Pro Ala Leu Arg Glu Gly Ile Gln
115 120 125Arg Thr Val Glu Arg Asn Arg
Phe Val Val Glu Asp Met Trp Leu Gly 130 135
140Val Leu Val Ser Arg Gly Leu Ser Arg Asp Asp Ala Glu Asp Ile
Leu145 150 155 160Trp Leu
Ile Phe Asn Ser Val Arg Gly Leu Ala Val Arg Ser Leu Trp
165 170 175Gln Lys Asp Lys Glu Arg Phe
Glu Arg Val Arg Asn Ser Thr Leu Glu 180 185
190Ile Ala Arg Glu Arg Tyr Ala Lys Phe Lys Arg 195
2001825PRTArtificial SequenceHPV16 E6 aa41-65 18Lys Gln Gln
Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp1 5
10 15Leu Cys Ile Val Tyr Arg Asp Gly Asn
20 251935PRTArtificial SequenceHPV16 E7 aa43-77
19Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys1
5 10 15Cys Lys Cys Asp Ser Thr
Leu Arg Leu Cys Val Gln Ser Thr His Val 20 25
30Asp Ile Arg 3520331PRTArtificial
SequenceHPV18-E6E7SH designer polypeptide 20Met Ala Arg Phe Glu Asp Pro
Thr Arg Arg Pro Tyr Lys Leu Pro Asp1 5 10
15Leu Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu
Ile Thr Cys 20 25 30Val Tyr
Cys Lys Thr Val Leu Asp Leu Leu Cys His Glu Gln Leu Ser 35
40 45Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp
Gly Val Asn His Gln His 50 55 60Leu
Pro Ala Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met65
70 75 80Cys Cys Lys Cys Glu Ala
Arg Ile Glu Leu Val Val Glu Ser Ser Ala 85
90 95Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe Leu Asn
Thr Leu Ser Phe 100 105 110Val
Cys Pro Trp Cys Ala Ser Gln His Tyr Ser Asp Ser Val Tyr Gly 115
120 125Asp Thr Leu Glu Lys Leu Thr Asn Thr
Gly Leu Tyr Asn Leu Leu Ile 130 135
140Arg Cys Leu Arg Cys Gln Lys Pro Leu Asn Pro Ala Glu Lys Leu Arg145
150 155 160His Leu Asn Glu
Lys Arg Arg Phe His Asn Ile Ala Gly His Tyr Arg 165
170 175Gly Gln Cys His Ser Cys Cys Asn Arg Ala
Arg Gln Glu Arg Leu Gln 180 185
190Arg Arg Arg Glu Thr Met His Gly Pro Lys Ala Thr Leu Gln Asp Ile
195 200 205Val Leu His Leu Glu Pro Gln
Asn Glu Ile Pro Val Asp Leu Leu Cys 210 215
220His Glu Gln Leu Ser Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp
Gly225 230 235 240Val Asn
Pro Asp Leu Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile
245 250 255Glu Ile Thr Cys Val Tyr Cys
Lys Thr Val Leu Glu Leu Thr Glu Val 260 265
270Phe Glu Phe Ala Phe Lys Asp Leu Phe Val Val Tyr Arg Asp
Ser Ile 275 280 285Pro His Ala Ala
Cys His Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg 290
295 300Glu Leu Arg His Tyr Ser Asp Ser Val Tyr Gly Asp
Thr Leu Glu Lys305 310 315
320Leu Thr Asn Thr Gly Leu Tyr Asn Leu Leu Ile 325
33021999DNAArtificial Sequencent sequence of HPV18-E6E7SH
designer sequence 21atggccagat tcgaggaccc caccagacgg ccctacaagc
tgcccgacct gtgcaccgag 60ctgaacacat ctctgcagga catcgagatc acatgcgtgt
actgcaagac cgtgctggac 120ctgctgtgcc acgagcagct gtccgactcc gaggaagaaa
acgacgagat cgacggcgtg 180aaccatcagc atctgcccgc cagacgggcc gagccccaga
gacacaccat gctgtgcatg 240tgctgcaagt gcgaggcccg gattgagctg gtggtggaaa
gcagcgccga cgacctgcgg 300gccttccagc agctctttct gaataccctg agcttcgtgt
gcccttggtg cgccagccag 360cactacagcg actccgtgta cggcgatacc ctggaaaagc
tgaccaatac cggcctgtat 420aacctgctga tccggtgcct gcggtgccag aagcccctga
atcccgccga gaaactgaga 480cacctgaacg agaagcggcg gttccacaat atcgccggcc
actacagagg ccagtgccac 540agctgctgca accgggccag acaggaacgg ctgcagcgga
ggcgggaaac catgcacgga 600cccaaggcca ccctccagga cattgtcctg cacctggaac
cccagaacga gatccccgtc 660gatctgctgt gtcatgaaca gctcagcgac agcgaagagg
aaaatgacga aattgacggg 720gtcaaccctg acctctgtac cgaactcaat accagtctcc
aggatatcga aattacctgt 780gtctactgta aaaccgtcct cgagctgacc gaggtgttcg
agttcgcctt caaggacctg 840tttgtggtgt acagagacag catcccccac gccgcctgcc
acaagtgcat cgacttctac 900agccggatca gagagctgcg gcactactcc gattctgtgt
atggcgacac actcgagaag 960ctcacaaaca caggactgta caatctgctc atctgataa
99922696PRTArtificial SequenceHPV18-E2E6E7SH
designer sequence 22Met Gln Thr Pro Lys Glu Thr Leu Ser Glu Arg Leu Ser
Ala Leu Gln1 5 10 15Asp
Lys Ile Ile Asp His Tyr Glu Asn Asp Ser Lys Asp Ile Asp Ser 20
25 30Gln Ile Gln Tyr Trp Gln Leu Ile
Arg Trp Glu Asn Ala Ile Phe Phe 35 40
45Ala Ala Arg Glu His Gly Ile Gln Thr Leu Asn His Gln Val Val Pro
50 55 60Ala Tyr Asn Ile Ser Lys Ser Lys
Ala His Lys Ala Ile Glu Leu Gln65 70 75
80Met Ala Leu Gln Gly Leu Ala Gln Ser Ala Tyr Lys Thr
Glu Asp Trp 85 90 95Thr
Leu Gln Asp Thr Cys Glu Glu Leu Trp Asn Thr Glu Pro Thr His
100 105 110Cys Phe Lys Lys Gly Gly Gln
Thr Val Gln Val Tyr Phe Asp Gly Asn 115 120
125Lys Asp Asn Cys Met Thr Tyr Val Ala Trp Asp Ser Val Tyr Tyr
Met 130 135 140Thr Asp Ala Gly Thr Trp
Asp Lys Thr Ala Thr Cys Val Ser His Arg145 150
155 160Gly Leu Tyr Tyr Val Lys Glu Gly Tyr Asn Thr
Phe Tyr Ile Glu Phe 165 170
175Lys Ser Glu Cys Glu Lys Tyr Gly Asn Thr Gly Thr Trp Glu Val His
180 185 190Phe Gly Asn Asn Val Ile
Asp Cys Asn Asp Ser Met Cys Ser Thr Ser 195 200
205Asp Asp Thr Val Ser Ala Thr Gln Leu Val Lys Gln Leu Gln
His Thr 210 215 220Pro Ser Pro Tyr Ser
Ser Thr Val Ser Val Gly Thr Ala Lys Thr Tyr225 230
235 240Gly Gln Thr Ser Ala Ala Thr Arg Pro Gly
His Cys Gly Leu Ala Glu 245 250
255Lys Gln His Cys Gly Pro Val Asn Pro Leu Leu Gly Ala Ala Thr Pro
260 265 270Thr Gly Asn Asn Lys
Arg Arg Lys Leu Cys Ser Gly Asn Thr Thr Pro 275
280 285Ile Ile His Leu Lys Val Asp Arg Asn Ser Leu Met
Arg Leu Arg Tyr 290 295 300Arg Leu Arg
Lys His Ser Asp His Tyr Arg Asp Ile Ser Ser Thr Trp305
310 315 320His Trp Thr Gly Ala Gly Asn
Glu Lys Thr Gly Ile Leu Thr Val Thr 325
330 335Tyr His Ser Glu Thr Gln Arg Thr Lys Phe Leu Asn
Thr Val Ala Ile 340 345 350Pro
Asp Ser Val Gln Ile Leu Val Gly Tyr Met Thr Met Met Ala Arg 355
360 365Phe Glu Asp Pro Thr Arg Arg Pro Tyr
Lys Leu Pro Asp Leu Cys Thr 370 375
380Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr Cys Val Tyr Cys385
390 395 400Lys Thr Val Leu
Asp Leu Leu Cys His Glu Gln Leu Ser Asp Ser Glu 405
410 415Glu Glu Asn Asp Glu Ile Asp Gly Val Asn
His Gln His Leu Pro Ala 420 425
430Arg Arg Ala Glu Pro Gln Arg His Thr Met Leu Cys Met Cys Cys Lys
435 440 445Cys Glu Ala Arg Ile Glu Leu
Val Val Glu Ser Ser Ala Asp Asp Leu 450 455
460Arg Ala Phe Gln Gln Leu Phe Leu Asn Thr Leu Ser Phe Val Cys
Pro465 470 475 480Trp Cys
Ala Ser Gln His Tyr Ser Asp Ser Val Tyr Gly Asp Thr Leu
485 490 495Glu Lys Leu Thr Asn Thr Gly
Leu Tyr Asn Leu Leu Ile Arg Cys Leu 500 505
510Arg Cys Gln Lys Pro Leu Asn Pro Ala Glu Lys Leu Arg His
Leu Asn 515 520 525Glu Lys Arg Arg
Phe His Asn Ile Ala Gly His Tyr Arg Gly Gln Cys 530
535 540His Ser Cys Cys Asn Arg Ala Arg Gln Glu Arg Leu
Gln Arg Arg Arg545 550 555
560Glu Thr Met His Gly Pro Lys Ala Thr Leu Gln Asp Ile Val Leu His
565 570 575Leu Glu Pro Gln Asn
Glu Ile Pro Val Asp Leu Leu Cys His Glu Gln 580
585 590Leu Ser Asp Ser Glu Glu Glu Asn Asp Glu Ile Asp
Gly Val Asn Pro 595 600 605Asp Leu
Cys Thr Glu Leu Asn Thr Ser Leu Gln Asp Ile Glu Ile Thr 610
615 620Cys Val Tyr Cys Lys Thr Val Leu Glu Leu Thr
Glu Val Phe Glu Phe625 630 635
640Ala Phe Lys Asp Leu Phe Val Val Tyr Arg Asp Ser Ile Pro His Ala
645 650 655Ala Cys His Lys
Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu Arg 660
665 670His Tyr Ser Asp Ser Val Tyr Gly Asp Thr Leu
Glu Lys Leu Thr Asn 675 680 685Thr
Gly Leu Tyr Asn Leu Leu Ile 690 695232094DNAArtificial
Sequencent sequence encoding HPV18-E2E6E7SH designer sequence
23atgcagaccc ccaaagagac actgagcgag cggctgagcg ccctgcagga caagatcatc
60gaccactacg agaacgacag caaggacatc gacagccaga tccagtactg gcagctgatc
120agatgggaga acgccatctt cttcgccgcc agagagcacg gcatccagac cctgaaccac
180caggtggtgc ccgcctacaa catcagcaag agcaaggccc acaaggctat cgagctgcag
240atggccctgc agggactggc ccagagcgcc tacaagaccg aggactggac cctgcaggat
300acctgcgagg aactgtggaa caccgagccc acccactgct tcaagaaagg cggccagacc
360gtgcaggtgt acttcgacgg caacaaggac aactgcatga cctacgtggc ctgggacagc
420gtgtactaca tgaccgacgc cggcacctgg gacaagaccg ccacctgtgt gtcccaccgg
480ggcctgtact acgtgaaaga gggctacaac accttctaca tcgagttcaa gagcgagtgc
540gagaagtacg gcaacaccgg cacatgggag gtgcacttcg gcaacaacgt gatcgactgc
600aacgacagca tgtgcagcac cagcgacgac accgtgtccg ccacccagct ggtgaaacag
660ctgcagcaca cccccagccc ctacagcagc accgtgtctg tgggcaccgc caagacctac
720ggccagacca gcgccgccac cagacctgga cactgtggcc tggccgagaa gcagcactgc
780ggccctgtga accctctgct gggagccgcc acccccaccg gcaacaacaa gcggagaaag
840ctgtgcagcg gcaacaccac ccccatcatc cacctgaagg tggaccggaa cagcctgatg
900cggctgcggt acagactgcg gaagcacagc gaccactacc gggacatcag cagcacctgg
960cactggaccg gcgctggcaa cgagaaaacc ggcatcctga ccgtgaccta ccacagcgaa
1020acccagcgga ccaagttcct gaacaccgtg gccatccccg acagcgtgca gatcctggtg
1080ggatatatga ccatgatggc cagattcgag gaccccacca gacggcccta caagctgccc
1140gacctgtgca ccgagctgaa cacatctctg caggacatcg agatcacatg cgtgtactgc
1200aagaccgtgc tggacctgct gtgccacgag cagctgtccg actccgagga agaaaacgac
1260gagatcgacg gcgtgaacca tcagcatctg cccgccagac gggccgagcc ccagagacac
1320accatgctgt gcatgtgctg caagtgcgag gcccggattg agctggtggt ggaaagcagc
1380gccgacgacc tgcgggcctt ccagcagctc tttctgaata ccctgagctt cgtgtgccct
1440tggtgcgcca gccagcacta cagcgactcc gtgtacggcg ataccctgga aaagctgacc
1500aataccggcc tgtataacct gctgatccgg tgcctgcggt gccagaagcc cctgaatccc
1560gccgagaaac tgagacacct gaacgagaag cggcggttcc acaatatcgc cggccactac
1620agaggccagt gccacagctg ctgcaaccgg gccagacagg aacggctgca gcggaggcgg
1680gaaaccatgc acggacccaa ggccaccctc caggacattg tcctgcacct ggaaccccag
1740aacgagatcc ccgtcgatct gctgtgtcat gaacagctca gcgacagcga agaggaaaat
1800gacgaaattg acggggtcaa ccctgacctc tgtaccgaac tcaataccag tctccaggat
1860atcgaaatta cctgtgtcta ctgtaaaacc gtcctcgagc tgaccgaggt gttcgagttc
1920gccttcaagg acctgtttgt ggtgtacaga gacagcatcc cccacgccgc ctgccacaag
1980tgcatcgact tctacagccg gatcagagag ctgcggcact actccgattc tgtgtatggc
2040gacacactcg agaagctcac aaacacagga ctgtacaatc tgctcatctg ataa
2094242085DNAArtificial Sequencent sequence encoding HPV16 E2E6E7SH
designer polypeptide 24atggaaacgc tctgccagag actcaatgtc tgccaggata
agattctcac gcattatgag 60aacgattcca cggacctgcg cgatcacatt gattattgga
aacacatgcg ccttgagtgt 120gctatctatt ataaggctcg cgaaatgggt ttcaaacata
tcaatcatca ggtcgtccct 180accctcgccg tcagcaaaaa caaagctctg caggctattg
aactccagct caccctcgag 240acaatctaca attcccagta ctccaatgag aaatggacgc
tccaggatgt gtctctcgag 300gtctacctga cagctcctac aggatgtatt aagaaacacg
ggtacacggt ggaagtccag 360tttgatggcg atatatgtaa taccatgcat tatacgaatt
ggacgcatat ctatatttgt 420gaagaggcct ctgtgacagt ggtcgaggga caggtcgact
attatgggct gtattatgtg 480cacgaaggga tcagaacata ctttgtccag tttaaggatg
atgctgagaa gtattctaag 540aacaaagttt gggaagtcca tgccggtgga caagtgattc
tgtgtcctac ctccgtgttc 600agctctaatg aggtgtcctc tccagagatc attagacagc
atctggccaa ccatcctgct 660gctacacata ccaaggctgt ggctctggga acagaagaga
cacagacaac aatccagagg 720cctcggagcg agcctgatac aggcaaccct tgtcacacaa
caaaactgct gcacagagac 780tccgtggact ccgctcctat tctgacagcc tttaactcct
cccacaaagg gagaatcaat 840tgcaattcca ataccacgcc gatcgtccac ctcaaagtgg
atgctaatac tctcatgcgg 900ctccgctacc gcttcaagaa acactgtaca ctgtatacag
ctgtgtccag cacatggcat 960tggacgggac acaatgtgaa acataagtcc gccatcgtca
cgctcacata cgattccgag 1020tggcagagag atcagtttct gtcccaagtc aagattccga
aaacgatcac cgtcagcacc 1080ggctttatgt ctattatgca ccagaaacgc acggctatgt
ttcaagatcc acaagagcga 1140cccagaaaac tgcctcagct gtgtacagaa ctgcaaacaa
ccatccatga catcattctt 1200gagtgtgttt attgcaagca acagctcgag gacgaaatcg
atggacctgc tggacaggcc 1260gaacccgata gggctcacta caacatcgtc acgttttgtt
gcaagtgtga ctccaccctg 1320agactgtgtg tgcagtctac ccatgtggat atcagaaccc
tcgaggacct gctcatggga 1380acactcggta ttgtgtgtcc tatctgctcc cagaagcctg
gaacaactct cgagcaacag 1440tacaacaagc cgctctgtga tctcctgatc agatgcatta
actgtcagaa gcctctctgc 1500cctgaagaga agcagagaca cctcgataag aaacagcgct
ttcacaatat cagaggccgg 1560tggaccggca gatgcatgtc ctgctgtcgg agcagcagaa
ccagacgcga gacacagatg 1620catggcgata cacctacact ccatgagtat atgctcgatc
tccagcctga aaccaccgat 1680ctctactgtt atgagcagct caatgacagc agcgaagagg
aagatgagat tgacggacca 1740gctgggcaag ccgagccaga tcgcgcacat tacaatatcg
ttaccttttg ttgtcagctc 1800tgcacagagt tgcaaacgac gattcacgac attatattgg
agtgcgtgta ctgtaaacaa 1860cagctgctgc ggagagaagt ctacgatttc gcctttagag
atctctgcat cgtctacaga 1920gatgggaatc cctatgccgt ctgtgataag tgtctcaagt
tttactccaa gatctccgag 1980tatcgccatt actgttactc cctgtacggg acaaccttgg
agcagcagta taacaagcca 2040ttgtgtgatc tgctcattcg gtgtattaac tgccaaaagt
gatga 2085252094DNAArtificial Sequencenucleotide
sequence encoding HPV16 E2E6E7SH designer polypeptide 25atgcagacgc
cgaaagaaac cctgtccgag agactgagcg ctctccaaga taagattatt 60gatcactatg
agaatgactc caaggacatt gattcccaga ttcagtattg gcaacttatt 120cgctgggaaa
atgccatctt ctttgccgct cgggaacacg ggattcagac actgaatcat 180caagtggtcc
cagcctacaa tatctccaag tccaaagccc acaaagctat cgaactccaa 240atggctctcc
agggactggc tcagtccgct tataagacag aagattggac actccaggac 300acgtgtgaag
aactctggaa taccgaaccg acgcactgtt ttaagaaggg tggacagaca 360gttcaagtct
actttgatgg gaacaaagat aattgcatga catatgtcgc ttgggattcc 420gtctactata
tgacagatgc tgggacgtgg gataagacag ccacatgcgt cagccacaga 480gggctgtact
atgtcaaaga agggtataat acgttctata tcgagtttaa gtctgaatgc 540gagaaatatg
ggaatacggg aacctgggaa gtgcattttg ggaacaatgt catcgattgc 600aatgactcca
tgtgctccac ctccgatgac acagtcagcg ccacacagct cgtgaaacag 660ctccagcata
caccatctcc ctactcctcc actgtgtccg tgggaacagc caaaacatat 720ggacagacct
ccgctgccac acggcctggc cattgcggac tggccgagaa acagcattgt 780ggaccagtca
accctctgct gggagctgct actccaacag ggaacaacaa gaggcggaaa 840ctgtgttccg
gcaatacgac acctatcatt cacctcaagg tcgacagaaa ctccctgatg 900agactgagat
accggctgag aaagcactcc gatcactaca gagatatcag ctctacttgg 960cattggacag
gtgctggaaa cgaaaagaca gggatcctga cagtgacgta tcactccgag 1020actcagcgca
ccaaatttct caatactgtg gccattcccg attccgtgca gattcttgtc 1080ggatatatga
cgatgatggc tcgctttgag gatcctacaa gaaggcctta taagctccct 1140gatctctgca
ctgagcttaa caccagcctg caagacattg aaatcacttg tgtctattgc 1200aaaacggtcc
tggacctcct ctgtcacgaa caactgtctg atagtgaaga ggagaatgat 1260gagatcgatg
gtgtcaacca ccagcacctc cctgctcgga gagccgagcc tcagcggcat 1320acaatgctct
gtatgtgttg taaatgcgag gccagaatcg aactcgtggt cgagtcctcc 1380gccgacgatc
tgagagcttt tcagcaactg tttctcaaca ccctgtcctt tgtctgtcct 1440tggtgtgcct
cccagcatta ctccgattct gtgtatggcg acactctcga gaagctcact 1500aacacgggac
tgtataatct gctcatccgc tgtctgagat gccagaaacc tctgaatcct 1560gccgagaaac
tgcgccacct caatgagaag agaagattcc acaacattgc cggacattat 1620cgaggccagt
gtcactcctg ctgtaacaga gctcggcaag agagactgca gagaaggcgc 1680gagacaatgc
acggacctaa agccaccctc caggacattg tgctgcatct cgagcctcag 1740aatgagattc
ctgtcgactt gctctgccat gagcaactgt ccgattctga ggaagaaaat 1800gacgaaatag
atggcgtgaa ccctgacttg tgcactgaac tcaatacttc cctgcaagat 1860atagagataa
cgtgcgttta ctgtaagacg gtcctcgaac tcaccgaagt ctttgagttt 1920gcctttaagg
atctctttgt cgtctaccgc gactccatcc ctcacgctgc ttgtcacaaa 1980tgtatcgatt
tttactctcg catcagagaa ctgcggcact actctgactc agtgtatggc 2040gatactttgg
agaagcttac caatacgggc ctctataact tgctgatctg atga
209426124DNAArtificial SequencePrMVA Promoter 26taaaaataga aactataatc
atataatagt gtaggttggt agtattgctc ttgtgactag 60agactttagt taaggtactg
taaaaataga aactataatc atataatagt gtaggttggt 120agta
12427227DNAArtificial
SequencePrHyb Promoter 27gttttgaaaa tttttttata ataaatatcc ggtaaaaatt
gaaaaactat tctaatttat 60tgcacggtcc ggtaaaaatt gaaaaactat tctaatttat
tgcacggtcc ggtaaaaatt 120gaaaaactat tctaatttat tgcacggtcc ggtaaaaatt
gaaaaactat tctaatttat 180tgcacggtcc ggtaaaaatt gaaaaactat tctaatttat
tgcacgg 22728365PRTArtificial SequenceHPV 16 E2 fragment
28Met Glu Thr Leu Cys Gln Arg Leu Asn Val Cys Gln Asp Lys Ile Leu1
5 10 15Thr His Tyr Glu Asn Asp
Ser Thr Asp Leu Arg Asp His Ile Asp Tyr 20 25
30Trp Lys His Met Arg Leu Glu Cys Ala Ile Tyr Tyr Lys
Ala Arg Glu 35 40 45Met Gly Phe
Lys His Ile Asn His Gln Val Val Pro Thr Leu Ala Val 50
55 60Ser Lys Asn Lys Ala Leu Gln Ala Ile Glu Leu Gln
Leu Thr Leu Glu65 70 75
80Thr Ile Tyr Asn Ser Gln Tyr Ser Asn Glu Lys Trp Thr Leu Gln Asp
85 90 95Val Ser Leu Glu Val Tyr
Leu Thr Ala Pro Thr Gly Cys Ile Lys Lys 100
105 110His Gly Tyr Thr Val Glu Val Gln Phe Asp Gly Asp
Ile Cys Asn Thr 115 120 125Met His
Tyr Thr Asn Trp Thr His Ile Tyr Ile Cys Glu Glu Ala Ser 130
135 140Val Thr Val Val Glu Gly Gln Val Asp Tyr Tyr
Gly Leu Tyr Tyr Val145 150 155
160His Glu Gly Ile Arg Thr Tyr Phe Val Gln Phe Lys Asp Asp Ala Glu
165 170 175Lys Tyr Ser Lys
Asn Lys Val Trp Glu Val His Ala Gly Gly Gln Val 180
185 190Ile Leu Cys Pro Thr Ser Val Phe Ser Ser Asn
Glu Val Ser Ser Pro 195 200 205Glu
Ile Ile Arg Gln His Leu Ala Asn His Pro Ala Ala Thr His Thr 210
215 220Lys Ala Val Ala Leu Gly Thr Glu Glu Thr
Gln Thr Thr Ile Gln Arg225 230 235
240Pro Arg Ser Glu Pro Asp Thr Gly Asn Pro Cys His Thr Thr Lys
Leu 245 250 255Leu His Arg
Asp Ser Val Asp Ser Ala Pro Ile Leu Thr Ala Phe Asn 260
265 270Ser Ser His Lys Gly Arg Ile Asn Cys Asn
Ser Asn Thr Thr Pro Ile 275 280
285Val His Leu Lys Val Asp Ala Asn Thr Leu Met Arg Leu Arg Tyr Arg 290
295 300Phe Lys Lys His Cys Thr Leu Tyr
Thr Ala Val Ser Ser Thr Trp His305 310
315 320Trp Thr Gly His Asn Val Lys His Lys Ser Ala Ile
Val Thr Leu Thr 325 330
335Tyr Asp Ser Glu Trp Gln Arg Asp Gln Phe Leu Ser Gln Val Lys Ile
340 345 350Pro Lys Thr Ile Thr Val
Ser Thr Gly Phe Met Ser Ile 355 360
365291095DNAArtificial Sequencent sequence encoding HPV 16 E2 fragment
29atggaaaccc tgtgccagcg gctgaacgtg tgccaggaca agatcctgac ccactacgag
60aacgacagca ccgacctgcg ggaccacatc gactactgga agcacatgcg gctggaatgc
120gccatctact acaaggccag agagatgggc ttcaagcaca tcaaccacca ggtggtgccc
180accctggccg tgtccaagaa caaggccctg caggccatcg agctgcagct gaccctggaa
240accatctaca acagccagta cagcaacgag aagtggaccc tgcaggacgt gtccctggaa
300gtgtacctga ccgctcccac cggctgcatc aagaaacacg gctacaccgt ggaagtgcag
360ttcgacggcg acatctgcaa caccatgcac tacaccaact ggacccacat ctacatctgc
420gaagaggcca gcgtgaccgt ggtggaaggc caggtggact actacggcct gtactacgtg
480cacgagggca tccggaccta cttcgtgcag ttcaaggacg acgccgagaa gtacagcaag
540aacaaagtgt gggaggtgca cgctggcggc caggtcatcc tgtgccccac cagcgtgttc
600agcagcaacg aggtgtccag ccccgagatc atccggcagc acctggccaa tcaccctgcc
660gccacccaca caaaggccgt ggccctgggc accgaggaaa cccagaccac catccagcgg
720cccagaagcg agcccgacac cggcaatccc tgccacacca ccaagctgct gcaccgggac
780agcgtggaca gcgcccctat cctgaccgcc ttcaacagca gccacaaggg ccggatcaac
840tgcaacagca acaccacccc catcgtgcac ctgaaggtgg acgccaacac cctgatgcgg
900ctgcggtaca gattcaagaa gcactgcacc ctgtacaccg ccgtgtcctc cacctggcac
960tggaccggcc acaacgtgaa gcacaagagc gccatcgtga ccctgaccta cgacagcgag
1020tggcagcggg accagttcct gagccaggtc aaaatcccca agaccatcac cgtgtccacc
1080ggcttcatga gcatc
1095301095DNAArtificial Sequencenucleotide sequence encoding HPV 16 E2
fragment 30atggaaacgc tctgccagag actcaatgtc tgccaggata agattctcac
gcattatgag 60aacgattcca cggacctgcg cgatcacatt gattattgga aacacatgcg
ccttgagtgt 120gctatctatt ataaggctcg cgaaatgggt ttcaaacata tcaatcatca
ggtcgtccct 180accctcgccg tcagcaaaaa caaagctctg caggctattg aactccagct
caccctcgag 240acaatctaca attcccagta ctccaatgag aaatggacgc tccaggatgt
gtctctcgag 300gtctacctga cagctcctac aggatgtatt aagaaacacg ggtacacggt
ggaagtccag 360tttgatggcg atatatgtaa taccatgcat tatacgaatt ggacgcatat
ctatatttgt 420gaagaggcct ctgtgacagt ggtcgaggga caggtcgact attatgggct
gtattatgtg 480cacgaaggga tcagaacata ctttgtccag tttaaggatg atgctgagaa
gtattctaag 540aacaaagttt gggaagtcca tgccggtgga caagtgattc tgtgtcctac
ctccgtgttc 600agctctaatg aggtgtcctc tccagagatc attagacagc atctggccaa
ccatcctgct 660gctacacata ccaaggctgt ggctctggga acagaagaga cacagacaac
aatccagagg 720cctcggagcg agcctgatac aggcaaccct tgtcacacaa caaaactgct
gcacagagac 780tccgtggact ccgctcctat tctgacagcc tttaactcct cccacaaagg
gagaatcaat 840tgcaattcca ataccacgcc gatcgtccac ctcaaagtgg atgctaatac
tctcatgcgg 900ctccgctacc gcttcaagaa acactgtaca ctgtatacag ctgtgtccag
cacatggcat 960tggacgggac acaatgtgaa acataagtcc gccatcgtca cgctcacata
cgattccgag 1020tggcagagag atcagtttct gtcccaagtc aagattccga aaacgatcac
cgtcagcacc 1080ggctttatgt ctatt
109531365PRTArtificial SequenceHPV 18 E2 fragment 31Met Gln
Thr Pro Lys Glu Thr Leu Ser Glu Arg Leu Ser Ala Leu Gln1 5
10 15Asp Lys Ile Ile Asp His Tyr Glu
Asn Asp Ser Lys Asp Ile Asp Ser 20 25
30Gln Ile Gln Tyr Trp Gln Leu Ile Arg Trp Glu Asn Ala Ile Phe
Phe 35 40 45Ala Ala Arg Glu His
Gly Ile Gln Thr Leu Asn His Gln Val Val Pro 50 55
60Ala Tyr Asn Ile Ser Lys Ser Lys Ala His Lys Ala Ile Glu
Leu Gln65 70 75 80Met
Ala Leu Gln Gly Leu Ala Gln Ser Ala Tyr Lys Thr Glu Asp Trp
85 90 95Thr Leu Gln Asp Thr Cys Glu
Glu Leu Trp Asn Thr Glu Pro Thr His 100 105
110Cys Phe Lys Lys Gly Gly Gln Thr Val Gln Val Tyr Phe Asp
Gly Asn 115 120 125Lys Asp Asn Cys
Met Thr Tyr Val Ala Trp Asp Ser Val Tyr Tyr Met 130
135 140Thr Asp Ala Gly Thr Trp Asp Lys Thr Ala Thr Cys
Val Ser His Arg145 150 155
160Gly Leu Tyr Tyr Val Lys Glu Gly Tyr Asn Thr Phe Tyr Ile Glu Phe
165 170 175Lys Ser Glu Cys Glu
Lys Tyr Gly Asn Thr Gly Thr Trp Glu Val His 180
185 190Phe Gly Asn Asn Val Ile Asp Cys Asn Asp Ser Met
Cys Ser Thr Ser 195 200 205Asp Asp
Thr Val Ser Ala Thr Gln Leu Val Lys Gln Leu Gln His Thr 210
215 220Pro Ser Pro Tyr Ser Ser Thr Val Ser Val Gly
Thr Ala Lys Thr Tyr225 230 235
240Gly Gln Thr Ser Ala Ala Thr Arg Pro Gly His Cys Gly Leu Ala Glu
245 250 255Lys Gln His Cys
Gly Pro Val Asn Pro Leu Leu Gly Ala Ala Thr Pro 260
265 270Thr Gly Asn Asn Lys Arg Arg Lys Leu Cys Ser
Gly Asn Thr Thr Pro 275 280 285Ile
Ile His Leu Lys Val Asp Arg Asn Ser Leu Met Arg Leu Arg Tyr 290
295 300Arg Leu Arg Lys His Ser Asp His Tyr Arg
Asp Ile Ser Ser Thr Trp305 310 315
320His Trp Thr Gly Ala Gly Asn Glu Lys Thr Gly Ile Leu Thr Val
Thr 325 330 335Tyr His Ser
Glu Thr Gln Arg Thr Lys Phe Leu Asn Thr Val Ala Ile 340
345 350Pro Asp Ser Val Gln Ile Leu Val Gly Tyr
Met Thr Met 355 360
365321095DNAArtificial Sequencenucleotide sequence encoding HPV 18 E2
fragment 32atgcagaccc ccaaagagac actgagcgag cggctgagcg ccctgcagga
caagatcatc 60gaccactacg agaacgacag caaggacatc gacagccaga tccagtactg
gcagctgatc 120agatgggaga acgccatctt cttcgccgcc agagagcacg gcatccagac
cctgaaccac 180caggtggtgc ccgcctacaa catcagcaag agcaaggccc acaaggctat
cgagctgcag 240atggccctgc agggactggc ccagagcgcc tacaagaccg aggactggac
cctgcaggat 300acctgcgagg aactgtggaa caccgagccc acccactgct tcaagaaagg
cggccagacc 360gtgcaggtgt acttcgacgg caacaaggac aactgcatga cctacgtggc
ctgggacagc 420gtgtactaca tgaccgacgc cggcacctgg gacaagaccg ccacctgtgt
gtcccaccgg 480ggcctgtact acgtgaaaga gggctacaac accttctaca tcgagttcaa
gagcgagtgc 540gagaagtacg gcaacaccgg cacatgggag gtgcacttcg gcaacaacgt
gatcgactgc 600aacgacagca tgtgcagcac cagcgacgac accgtgtccg ccacccagct
ggtgaaacag 660ctgcagcaca cccccagccc ctacagcagc accgtgtctg tgggcaccgc
caagacctac 720ggccagacca gcgccgccac cagacctgga cactgtggcc tggccgagaa
gcagcactgc 780ggccctgtga accctctgct gggagccgcc acccccaccg gcaacaacaa
gcggagaaag 840ctgtgcagcg gcaacaccac ccccatcatc cacctgaagg tggaccggaa
cagcctgatg 900cggctgcggt acagactgcg gaagcacagc gaccactacc gggacatcag
cagcacctgg 960cactggaccg gcgctggcaa cgagaaaacc ggcatcctga ccgtgaccta
ccacagcgaa 1020acccagcgga ccaagttcct gaacaccgtg gccatccccg acagcgtgca
gatcctggtg 1080ggatatatga ccatg
1095331095DNAArtificial Sequencenucleotide sequence encoding
HPV 18 E2 fragment 33atgcagacgc cgaaagaaac cctgtccgag agactgagcg
ctctccaaga taagattatt 60gatcactatg agaatgactc caaggacatt gattcccaga
ttcagtattg gcaacttatt 120cgctgggaaa atgccatctt ctttgccgct cgggaacacg
ggattcagac actgaatcat 180caagtggtcc cagcctacaa tatctccaag tccaaagccc
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