Immunogenic compositions and vaccines against Plasmodial infection
comprising an Rh polypeptide or a fragment or variant thereof are
disclosed. Also disclosed are Rh5 polypeptides or fragments or variants
thereof capable of binding CD147 and conferring protection against
infection and/or disease caused by multiple Plasmodial strains or
Plasmodial species, inhibitors of the interaction between Rh5 and CD147
and methods for producing polypeptides in a mammalian expression system.
| Inventors: |
WRIGHT; Gavin J.; (London, GB)
; RAYNER; Julian C.; (London, GB)
; CROSNIER; Cecile; (London, GB)
; BUSTAMANTE; Leyla Y.; (London, GB)
; BARTHOLDSON; S. Josefin; (London, GB)
|
| Applicant: | | Name | City | State | Country | Type |
GENOME RESEARCH LIMITED |
London | |
GB
| | |
|---|
| Family ID:
|
43304257
|
| Appl. No.:
|
15/911816
|
| Filed:
|
March 5, 2018 |
Related U.S. Patent Documents
| | | | |
|---|
|
| Application Number | Filing Date | Patent Number |
|---|
| | 15204155 | Jul 7, 2016 | |
| | 15911816 | | |
| | 13878149 | Nov 22, 2013 | |
| | PCT/GB2011/051936 | Oct 7, 2011 | |
| | 15204155 | | |
|
|
| Current U.S. Class: |
1/1 |
| Current CPC Class: |
A61K 39/015 20130101; C07K 2/00 20130101; C07K 16/205 20130101; A61K 2039/70 20130101; C12N 15/115 20130101; Y02A 50/30 20180101; G01N 33/566 20130101 |
| International Class: |
A61K 39/015 20060101 A61K039/015 |
Foreign Application Data
| Date | Code | Application Number |
|---|
| Oct 8, 2010 | GB | 1016969.6 |
Claims
1. An immunogenic composition or vaccine against Plasmodial infection
comprising an Rh5 polypeptide or a fragment; or variant thereof.
2. An immunogenic composition or vaccine comprising an Rh5 polypeptide or
a fragment or variant thereof, wherein the Rh5 polypeptide or fragment or
variant thereof is capable of binding CD147.
3. An immunogenic composition or vaccine comprising an Rh5 polypeptide or
a fragment or variant thereof, wherein the vaccine is capable of
conferring protection against infection and tor disease caused by
multiple Plasmodial strains or Plasmodial species.
4. An immunogenic composition or vaccine as claimed in any one of claims
1 to 3, wherein the immunogenic composition or vaccine comprises
additional antigens, such as Plasmodial antigens.
5. An Rh5 polypeptide or fragment or variant thereof as claimed in any
one of claims 1 to 4 for conferring protection against infection or
disease by multiple Plasmodial strains or species.
6. A Rh5 polypeptide or a fragment or variant thereof as claimed in any
one of claims 1 to 4 for the prevention and/or treatment of Plasmodial
infection and/or disease.
7. An inhibitor of the interaction between Rh5 and CD147.
8. An inhibitor of the interacted of Rh5 and CD147 for the prevention and
or treatment of Plasmodium infection and or disease.
9. Use of the inhibitor of the interaction of Rh5 and CD147 in the
preparation of a medicament for prevention and or treatment of Plasmodium
infection and/or malarial disease.
10. An inhibitor or use according to any one of claims 7 to 9 which is an
antibody or fragment or derivative thereof which binds to CD147,
preferably wherein the antibody is Metuximab.
11. An inhibitor or use according to according to any one of claims 7 to
9 which is a soluble fragment of Rh5.
12. An inhibitor or use according to any one of claims 7 to 9 which is an
antibody or fragment or derivative thereof which binds to Rh5.
13. An inhibitor according to any one of claims 7 to 12 which is a
nucleic acid aptamer or peptide aptamer capable of binding to CD147 or
Rh5.
14. A method for producing a polypeptide, the method comprising
expression of nucleic acid encoding the polypeptide in a eukaryotic cell,
and optionally purification of the polypeptide so expressed, wherein: (i)
optionally the expressed polypeptide is not N-gylcosylated in the cell
(ii) the nucleic acid encodes an exogenous eukaryotic signal sequence
effective to deliver the polypeptide into the secretory pathway of the
eukaryotic cell; and (iii) the nucleic acid has been codon optimised for
express ion of the polypeptide in the eukaryotic cell.
15. A polypeptide produced according to claim 14.
16. A polypeptide produced according to claim 14 for the prevention or
treatment of Plasmodium infection or malarial disease.
17. A method or polypeptide according to any preceding claim wherein the
polypeptide is an ectodomain of a secreted Plasmodium polypeptide.
18. A method or polypeptide according to claim 17 wherein the polypeptide
is selected from the ectodomain of MSP1, MSP2, MSP4, MSP5, MSP10, Pf12,
Pf38, Pf92, Pf113, ASP, RAMA, EBA140, EBA175, EBA181, EBL1, AMA1, MTRAP,
MSP3, MSP6, H101, H103, MSP7, Pf41, RhopH3: Rh5, SPATR, TLP, Pf34,
PF14_0344, PF10_0323, PFF0335c, AARP, MSP3.4, MSP3.8, MSRP1, MSRP2,
MSRP3, RON6, Pf12p, MSP9, GAMA, PF11_0373, Rh1, Rh2b and Rh4.
19. A nucleic acid encoding a polypeptide as disclosed in claims 14 to 18
which contains no N-glycosylation sites operably linked to art exogenous
eukaryotic signal sequence effective to deliver the polypeptide into the
secretory pathway of a eukaryotic cell, which nucleic acid has been codon
optimised for expression of the polypeptide in the eukaryotic cell.
20. A vector comprising the nucleic acid of claim 19.
21. A cell comprising the vector of claim 19.
22. A vaccine comprising a polypeptides or polynucleotides as disclosed
in any of claims 14 to 19, or combination thereof.
23. Use of a Plasmodium polypeptide expressed according to claim 14 in
the identification of a red blood cell (RBC) receptor, the method
comprising screening red blood cells and/or RBC proteins with the
polypeptide to identify RBC components that bind to the polypeptide.
24. An antibody raised to, or specifically reactive with, a polypeptide
claimed in any one of the preceding claims, or functionally equivalent
fragment thereof.
25. A nucleic acid or peptide aptamer capable of binding to a polypeptide
claimed in any one of the preceding claims.
26. A polypeptide expressed by the method of claim 14 for use in
prevention or treatment of disease.
27. A polypeptide from a merozoite expressed by the method of claim 14
for use in prevention or treatment of disease.
Description
SUMMARY
[0001] The present invention relates to polypeptides, inhibitors of
polypeptide interactions, methods for expressing polypeptides and
polypeptides so expressed.
BACKGROUND
[0002] Parasites of the Plasmodium genus are the etiological agents
responsible for malaria, a disease mostly occurring in sub-tropical areas
and affecting potentially up to 40% of the world population. Amongst the
various species that can affect humans, Plasmodium falciparum is by far
the most virulent, causing over a million deaths annually, mostly in
children under the age of five. Despite intensive efforts from the
research community, an effective vaccine has yet to be produced. There is
currently no approved vaccine for malaria. One vaccine currently in Phase
III trails in Africa is RTS,S (produced by GSK). This vaccine targets the
liver stages of the parasite life-cycle. Phase II trials with RTS,S have
shown between 39% and 59% efficacy, depending on the adjuvant dose, and
clinical end-point used (eg. Asante et al., Lancet Infect Dis. 2011, PMID
21782519; Olutu et al., Lancet Infect Dis, 2011, PMID 21237715). There
are ongoing efforts to produce a more effective vaccine for subsequent
release.
[0003] All of the clinical symptoms of malaria occur during the asexual
erythrocytic stage of the parasite life cycle, when the parasite's
merozoites invade human red blood cells, replicate and release up to 32
additional merozoites [ref1]. For this reason, and because merozoites are
exposed to the host immune system, erythrocytic invasion has been the
main centre of attention. Several proteins displayed at the merozoite
surface are believed to be critical for invasion and are therefore good
vaccine candidates, yet their precise function remains poorly understood.
This is largely due to the difficulty in producing large amounts of
functional recombinant parasite proteins [ref 2]. The high AT content of
Plasmodium genomes, the high prevalence of low complexity regions in the
parasite's proteins, and the difficulty at identifying clear structural
domains within these proteins using standard prediction programs are all
contributing factors. Production of extracellular proteins, which often
contain structurally critical disulfide bonds, adds another level of
complexity as correct folding in a heterologous system will only occur in
an oxidising environment (typically the secretory pathway of the organism
in which the recombinant parasitic proteins am expressed).
Membrane-tethered proteins are also difficult to solubilise as they
contain hydrophilic ectodomains in close apposition to hydrophobic
transmembrane domains [ref 3].
[0004] Invasion of the red blood cell requires interaction between
proteins displayed on the surface of the merozoite and the red blood
cell. As a result almost all merozoite proteins have been previously
suggested to be vaccine candidates, based primarily on their location and
supposed function. (Baum et al., Int J Parasitol 2009, PMID 19000690).
[0005] Of all the potential blood stage candidate antigens, two have
reached Phase II vaccine trials, MSP1 and AMA1. In both cases, no
protection was observed (AMA1: Sagara et al., Vaccine 2009, PMID 1974925;
Thera et al, N Engl J Med 2011, PMID 21916638; MSP1: Ogutu et al., PLoS
One 2009, PMID 19262754). In both cases, the central problem appears to
be the inability of the vaccine to protect across multiple strains. AMA1
in particular is known to induce antibody responses that primarily target
the immunizing sequence, and do not cross-protect against other
sequences. Like other blood stage proteins, the Rh family (six related
proteins in P. falciparum) have been suggested for vaccine development,
with attempts being made to combine multiple Rh antigens into a single
multi-component vaccine (Lopaticki et al., Infect Immun 2011, PMID
21149582).
[0006] Cross-protection is widely viewed as the central problem facing
blood stage vaccines (Takala and Plowe, Parasite Immunol 2009, PMID
19691559).
[0007] The present invention relates to polypeptide expression systems,
polypeptides and inhibitors for the prevention and/or treatment of
Plasmodium infection and/or disease, and vaccines comprising such
polypeptides, or inhibitors.
STATEMENT OF INVENTION
[0008] The present invention relates to a method for producing a
polypeptide, the method comprising expression of nucleic acid encoding
the polypeptide in a eukaryotic cell, and optionally purification of the
polypeptide, so expressed, wherein: [0009] (i) optionally the expressed
polypeptide is not N-glycosylated in the cell [0010] (ii) the nucleic
acid encodes an exogenous eukaryotic signal sequence effective to deliver
the polypeptide into the secretory pathway of the eukaryotic cell; and
[0011] (iii) the nucleic acid has been codon optimised for expression of
the polypeptide in the eukaryotic cell.
[0012] The invention also relates to a polypeptide expressed by the method
of the invention.
[0013] The invention also relates to a nucleic acid encoding a polypeptide
as disclosed herein, operably linked to an exogenous eukaryotic signal
sequence effective to deliver the polypeptide into the secretory pathway
of a eukaryotic cell and which nucleic acid has been codon optimised for
expression of the polypeptide in the eukaryotic cell. Optionally the
nucleic acid encodes a polypeptide which contains no N-glycosylation
sites.
[0014] The invention further relates to a vector comprising the nucleic
acid, and cell comprising such a vector.
[0015] In a further aspect the invention relates to an immunogenic
composition or vaccine comprising one or more polypeptides or
polynucleotides of the invention, or combination of polypeptides and
polynucleotides of the invention.
[0016] The invention also relates to an immunogenic composition or vaccine
comprising an Rh5 polypeptide or a fragment or variant thereof for the
prevention and/or treatment of Plasmodial infection and/or disease.
[0017] The invention also relates to an Rh5 polypeptide or a fragment or
variant thereof for the prevention and/or treatment of Plasmodial
infection and/or disease and also relates to an Rh5 polypeptide or a
fragment or variant thereof conferring protection across multiple
Plasmodial strains. In one aspect the Rh5 polypeptide or fragment or
variant thereof may elicit an immune response which is capable of
recognizing the same Rh5 polypeptide or Rh5 polypeptides having different
sequences, which may be natural variant sequences.
[0018] The invention also relates to an anti-Rh5 antibody, or fragment or
derivative thereof, for use in the prevention or treatment of malarial
disease and/or Plasmodial infection, and to an anti-Rh5 antibody, or
fragment or derivative thereof capable of preventing red blood cell
infection by Plasmodial species, for example species which have a
different Rh5 sequence to those against which the antibody was raised or
otherwise generated.
[0019] The invention also relates to an inhibitor of the interaction
between Rh5 and CD147, and use of that inhibitor in the prevention or
treatment of malaria or malarial infection.
[0020] The invention also relates to use of a Plasmodial polypeptide of
the invention in the identification of a red blood cell (RBC) receptor,
the method comprising screening red blood cells and/or RBC proteins with
the polypeptide to identity RBC components that bind to the malaria
polypeptide.
[0021] The invention also relates to an antibody specifically raised to,
or reactive with, polypeptide produced according to the invention. Where
the polypeptide of the invention is a fragment of a full length protein
(Such as an ectodomain) then in one aspect the antibody shows greater
specificity to binding of the fragment when compared to the full length
protein.
[0022] The invention further relates to a polypeptide expressed by the
method of the invention for use in prevention or treatment of disease,
such as treatment or prevention of malaria where the polypeptide is a
Plasmodium polypeptide.
FIGURES
[0023] FIG. 1. Expression of recombinant secreted and cell surface
merozoite proteins from P. falciparum.
[0024] FIG. 2. Functional activity and immunogenicity of recombinant P.
falciparum merozoite proteins.
[0025] FIG. 3. Demonstration that MSP1 and MSP7 are correctly folded and
functional.
[0026] FIG. 4. Immunogenicity of the recombinant P. falciparum merozoite
surface antigens.
[0027] FIG. 5. AVEXIS identifies two splice variants of the erythrocyte
surface protein BASIGIN as a receptor for P. falciparum Rh5.
[0028] FIG. 6. Biophysical characterisation of the Rh5-BSG interaction
using surface plasmon resonance.
[0029] FIG. 7a Soluble recombinant ectodomains of BSG-S and BSG-L potently
reduce the efficiency of P. falciparum erythrocyte invasion.
[0030] FIG. 7b. Soluble BSG potently block erythrocyte invasion across
multiple strains.
[0031] FIG. 8a. Mouse monoclonal antibodies to human BSG block the
invasion of P. falciparum into human erythrocytes. Purified monoclonal
antibodies MEM-M6/1 (circles) and MEM-M6/6 (squares) were added at the
indicated concentrations to an in vitro P. falciparum invasion assay
using the 3D7 strain. The proteins reduced invasion efficiency relative
to an isotype-matched negative control (diamonds)
[0032] FIG. 8b. Anti-BSG antibodies potently block erythrocyte invasion.
TABLE-US-00001
[0033] Sequences
amino acid sequence for Rh5 ectodomain
SEQ ID No. 1
FENAIKKTKNQENNLALLPIKSTEEEKDDIKNGKDIKKEIDNDKENIKTN
NAKDHSTYIKSYLNTNVNDGLKYLFIPSHNSFIKKYSVFNQINDGMLLNE
KNDVKNNEDYKNVDYKVNNFLQYHFKELSNYNIANSIDILQEKEGHLDFV
IIPHYTFLDYYKHLSYNSIYHKSSTYGKCIAVDAFIKKINEAYDKVKSKC
NDIKNDLIATIKKLEHPYDINNKNDDSYRYDISEEIDDKSEETDDETEEV
EDSIQDTDSNHAPSNKKKNDLMNRAFKKMMDEYNTKKKKLIKCIKNHEND
FNKICMDMKNYGTNLFEQLSCYNNNFCNTNGIRYHYDEYIHKLILSVKSK
NLNKDLSDMTNILQQSELLLTNLNKKMGSYIYIDTIKFIHKEMKHIFNRI
EYHTKIINDKTKIIQDKIKLNIWRTFQKDELLKRILDMSNEYSLFITSDH
LRQMLYNTFYSKEKHLNNIFHHLIYVLQMKFNDVPIKMEYFQTYKKNKPL
TQ
DETAILED DESCRIPTION
[0034] The present invention relates to an expression system for
expression of polypeptides. In one aspect the invention relates to the
expression of polypeptides produced in the malaria, parasite, which are
generally not glycosylated.
[0035] Plasmodial proteins are difficult to express [Birkholtz and Blatch
"Heterologous expression of Plasmodial proteins for structural studies
and functional annotation." Malaria Journal v7 p197 (2008)].
[0036] In the studies described, we have attempted to express the 50
entire ectodomain fragments and 3 partial extracellular regions of
cell-surface merozoite proteins from P. falciparum, using human embryonic
kidney (HEK) 293E cells. Using the polypeptide expression system as
described in the Example herein we were able to detect expression of 40
proteins by ELISA and 44 by western blot with 2 showing a lower molecular
weight than expected. Thus the invention provides a standardised
expression system platform suitable for effective expression of a wide
range of different polypeptides.
[0037] The present invention relates to a method for producing a
polypeptide, the method comprising expression of nucleic acid encoding
the polypeptide in a eukaryotic cell, wherein: [0038] (i) optionally
the expressed polypeptide is not N-glycosylated in the cell; [0039] (ii)
the nucleic acid encodes an exogenous eukaryotic signal sequence
effective to deliver the polypeptide into the secretory pathway of the
eukaryotic cell; and [0040] (iii) the nucleic acid is codon optimised for
expression of the polypeptide in the eukaryotic cell.
[0041] The method may further comprise the steps of purifying the
expressed polypeptide, and optionally formulation of the resulting
polypeptide with excipients or carriers or with adjuvants as disclosed
herein.
[0042] The eukaryotic cell may be a mammalian cell. The signal sequence
may be a mammalian signal sequence.
[0043] In one aspect the polypeptide is a eukaryotic polypeptide, and in
one aspect a Plasmodial polypeptide, such as a merozoite polypeptide,
such as merozoite surface polypeptide or part thereof. In one aspect the
polypeptide is an ectodomain which is generally a region of a protein
that is located outside of the cell. In one aspect the polypeptide is a
secreted polypeptide. In one aspect the polypeptide is a cell surface
polypeptide. In one aspect the polypeptide is exposed on the surface of a
merozoite from Plasmodium falciparum strain, such as 3D7, or on the
surface of a merozoite from Plasmodium vivax.
[0044] Polypeptides suitable for expression, in whole or in part, using
the method of the invention include Plasmodium proteins: MSP1, MSP2,
MSP4, MSP5, MSP10, Pf12, Pf38, Pf92, Pf113, ASP, RAMA, EBA140, EBA175,
EBA181, EBL1, AMA1, MTRAP, MSP3, MSP6, H101, H103, MSP7, Pf41, RhopH3
Rh5, SPATR, TLP, Pf34, PF14_0344, PF10_0323, PFF0335c, AARP, MSP3.4,
MSP3.8, MSRP1, MSRP2, MSRP3, RON6, Pf12p, MSP9, GAMA, PF11_0373. In one
aspect, the polypeptide is Rh5 or an ectodomain of Rh5 or a fragment or
variant thereof. The Rh5 or fragment or variant thereof suitably binds to
BASIGIN (CD147), which may be assessed by methods disclosed herein.
[0045] Table I lists the accession number for each protein, along with the
first and last amino aside of a suitable ectodomain region that may be
expressed.
[0046] It will be appreciated that in one aspect a polypeptide to be
produced in the invention will not naturally contain any N-glycosylation
sequences. In that case the sequence or the wild type polypeptide, or
part of it may be expressed. However, in another aspect polypeptide
sequences will naturally contain N-glycosylation sequences, and in that
case the nucleic acid encoding the wild type polypeptide sequence is
modified to remove that N-glycosylation sequence from the polypeptide, or
the system is arranged in another way to prevent N-glycosylation. These
polypeptides are therefore variants of the original (naturally occurring)
polypeptide sequence. Therefore, reference herein to polypeptides for
expression in the invention encompasses variants of those polypeptides in
which the polypeptide has been modified when compared to the wild type
polypeptide so as to lack any or all N-glycosylation sites, by
modification of the nucleic acid encoding the wild type polypeptide. Any
reference to polypeptides suitable for use herein includes reference to
such variants, where the wild type polypeptide has glycosylation
sequences, unless otherwise apparent from the context.
[0047] Any suitable polypeptide may be expressed in the present invention,
which may be a naturally occurring polypeptide or a part or mutant
thereof, Mutants include polypeptides which differ in sequence from the
naturally occurring sequence by the presence of addition, substitution or
deletions. A polypeptide may differ from a naturally occurring
polypeptide sequences at 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, or more ammo
acids. In one aspect the mutant sequence retains substantially the same
function and/or immunogenicity as the naturally occurring wild type
sequence. The difference in sequence may be assessed across only the
secreted or ectodomain portion of a polypeptide.
[0048] Polypeptides suitable for use in the invention, such as suitable
for expression or other uses as disclosed herein, may be exogenous
polypeptides which are not encoded by the genome of a mammalian cell but
which are found in mammalian cells in nature, suitably in a non
glycosylated form. Such polypeptides may be introduced into mammalian
cells cell by infection, for example, by a virus, bacteria or other
parasite, preferably a eukaryotic parasite. The invention is thus not
restricted to expression of Plasmodial merozoite polypeptides, although
these are in one aspect preferred.
[0049] In one aspect N-glycosylation within the cell is prevented by
provision of nucleic acid encoding a polypeptide which contains no
N-glycosylation sites. In this aspect the method comprises modification
of the nucleic acid encoding the polypeptide to remove any
N-glycosylation sites present in the polypeptide, encoded by Asn-X-Ser or
Asn-X-Thr motifs, where X is any amino acid. Methods for the modification
of nucleic acid sequences such as site directed mutagenesis are well
known in the art. In one aspect the encoded serine or threonine residue
in the motif is replaced with a different amino acid residue, such as an
alanine residue.
[0050] Alternatively the glycosylation of a polypeptide being expressed in
the cell is prevented by use of cells in which the N-glycosylation
pathway has been inactivated fey mutation of one of more of the elements
of the glycosylation pathway, and/or the cells are treated with
inhibitors of N-glycosylation.
[0051] The nucleic acids of the invention are codon optimised for
expression in the cell type selected for expression. Suitably the codon
optimisation is such that expression is optimised for a cell in which the
signal sequence is effective and/or is naturally found associated with
polypeptides. Codon optimisation may be full or partial optimisation.
[0052] The nucleic acids of the invention may encode all or part of a
naturally occurring polypeptide, or encode a mutant thereof, as above.
[0053] The nucleic acid encodes an exogenous eukaryotic signal sequence
affective to deliver the expressed polypeptide into the secretory pathway
of the cell. In one aspect this signal sequence is from a secreted
protein such as an antibody, such as leader sequence of the mouse
variable .kappa. light chain 7-33. However, the invention is not
restricted by this signal sequence and any other suitable leader sequence
which directs polypeptides into the cellular secretory pathway may be
used.
[0054] The signal sequence is operably linked with the nucleic acid
encoding the polypeptide to allow the secretion of the expressed
polypeptide from the cell.
[0055] The signal sequence its exogenous, not being naturally found linked
with the polypeptide to be produced. The term `exogenous` thus refers to
the origin of the signal sequence with respect to the polypeptide to be
expressed.
[0056] In one aspect the polypeptide produced is immunogenic, for example
as assessed by the ability to raise an immune response in a human or
other mammal.
[0057] In one aspect the expressed polypeptide has one, or more, or all,
activities of the naturally occurring polypeptide, for example is capable
of reacting with antibodies generated by individuals exposed to the
naturally occurring polypeptide, or binding to known binding partners,
for example as assessed by using methods disclosed herein. In one aspect
the binding is of the same affinity as the wild type polypeptide or
equivalent fragment of the wild type polypeptide, but may be higher or
lower as long as some degree of binding affinity and specificity is
observed. Similarly the expressed polypeptide suitably is substantially
as immunogenic as the naturally occurring polypeptide, although the
immunogenicity may be higher or lower, as long as some degree of
immunogenicity is observed.
[0058] in one aspect the nucleic acid encodes a polypeptide sequence that
acts as a tag, to allow the polypeptide to easily purified and/or
identified. For example, the tag may be a biotinylation sequence, to
allow for biotinylated recombinant proteins to be produced in cell
culture, and isolated by streptavidin affinity chromatography, or capture
of biotinylated proteins on streptavidin-coated solid phases. Other tags
and identification/purification systems are well known. The tag is not
considered to be a part of the polypeptide of the invention and may
comprise a sequence that is glycosylated if cellular conditions allow.
[0059] The invention also relates to any polypeptide expressed according
to Use present invention, as described above. Suitably the polypeptide is
soluble, in particular the polypeptide is an ectodomain of any one of
MSP1, MSP2, MSP4, MSP5, MSP10, Pf12, Pf38, Pf92, Pf113, ASP, RAMA,
EBA140, EBA175, EBA1801, EBL1, AMA1, MTRAP, MSP3, MSP6, H101, H103, MSP7,
Pf41, RhopH3, Rh5, SPATR, TLP, Pf34, PF14_0344, PF10 _0323, PFF0335c,
AARP, MSP3.4, MSP38.8, MSRP1, MSRP2, MSRP3, RON6, Pf12p, MSP9, GAMA,
PF11_0373. In one aspect the polypeptide is not secreted from the cell as
part of an organelle. In one aspect, the polypeptide is an ectodomain of
Rh5, or a fragment or variant thereof.
[0060] The invention further relates to an isolated nucleic acid encoding
a polypeptide which contains no N-glycosylation sites, operably linked to
an exogenous eukaryotic signal sequence effective to deliver the
polypeptide into the-secretory pathway of the cell, which has been codon
optimised, suitably for expression in a cell in which the signal sequence
is effective.
[0061] The invention also relates to vectors, such as expression vectors
comprising the nucleic acid and cells comprising the expression vector.
Suitable vectors include plasmid vectors and viral vectors, as well as
transposons. Cells include both bacterial cells which may be used in
standard cloning methodologies, and eukaryotic, such as mammalian cells,
in which the nucleic acid is be expressed.
[0062] Previous work on vaccine development evolving the Rh family (six
related proteins in P. falciparum, of which Rh5 is one) made attempts to
combine multiple Rh antigens into a single multi-component vaccine
(Lopaticki et al., Infect Immun 2011, PMID 21149582). Rh5 has been
included as a minor component of these studies, but only by expressing
small fragments in a bacterial expression system. Importantly, there is
no evidence that expressing Rh5 in a bacterial expression system leads to
correctly-folded protein.
[0063] RH5 may also be referred to as PfRh5 herein.
[0064] The inventors have now shown that red blood cell invasion
critically depends on a single receptor-ligand pair between a parasite
protein called PfRh5 and a host receptor called BASIGIN (which is also
referred to herein as BSG or CD147). BSG has not been previously
identified as a receptor used for red blood cell invasion.
[0065] The inventors have made the following observations: [0066] 1.
Blocking the Rh5-BSG interaction with antibodies completely blocks
invasion. This puts Rh5 in a different class to many other invasion
ligands, including the other Rhs, which are largely redundant, catalysing
overlapping pathways. Antibodies against these other proteins will only
ever therefore partially block invasion, hence the focus on
multi-component vaccine strategies for these proteins. [0067] 2. Blocking
the Rh5-BSG interaction blocks invasion in all P. falciparum strains that
we have tested to date rely on the Rh5-BSG interaction, including strains
recently isolated from P. falciparum infected individuate. We have tested
9 laboratory adapted strains, representing 7 different PfRh5 sequences
(see FIG. 8b (C)) and 6 field isolates (FIG. 8b (D)). This suggests that
the Rh5-BSG interaction is universal, and may provide the critical
cross-strain protection that has been impossible to generate with other
blood stage targets such as MSP1 and AMA1.
[0068] The identification of the Rh5-BSG interaction as possibly universal
and essential for invasion was unexpected. In one aspect Rh5 may be
developed as a single universal target that could allow cross-protection
across strains. The advantage of a single component over multi-component
vaccine is obviously the lower cost of production--an important
consideration for diseases that affect less developed countries.
[0069] In particular it has been demonstrated herein that CD147 interacts
with PfRh5, and that this interaction is involved in invasion. Blocking
the interaction using antibodies prevents invasion.
[0070] Thus in one aspect the invention relates to an inhibitor of the
interaction of Rh5 end CD147. In another aspect the invention relates to
an inhibitor of the interaction of Rh5 and CD147 for use in the
prevention and of treatment of Plasmodium infection and or disease, and
in a yet further aspect to the use of the inhibitor in the preparation of
a medicament for prevention and or treatment of Plasmodium infection
and/or malarial disease.
[0071] The invention also relates to a method of prevention or treatment
of Plasmodium infection or malarial disease, the method comprising
delivery of an inhibitor of the interaction of Rh5 and CD147 to an
individual in need thereof.
[0072] The inhibitor in one aspect is an antibody, which may be a
polyclonal or monoclonal antibody, or an antigen-binding derivative or
fragments thereof. Welt known antigen binding fragments include, for
example, single domain antibodies (dAbs; which consist essentially of
single VL or VH antibody domains), Fv fragment including single chain Fv
fragment (scFv), Fab fragment, and F(ab')2 fragment. Methods for the
construction of such antibody molecules are well known in the art. In one
aspect the antibody is humanised.
[0073] Modified antibody formats have been developed which retain binding
specificity, but have other characteristics that may be desirable,
including for example, bispecificity, multivalence (more than two binding
sites), and compact size (e.g., binding domains alone). Single chain
antibodies lack some of all of the constant domains of the whole
antibodies from which they are derived. Therefore, they can overcome some
of the problems associated with the use of whole antibodies. For example,
single-chain antibodies tend to be free of certain undesired interactions
between heavy-chain constant regions and other biological molecules.
Additionally, single-chain antibodies are considerably smaller than whole
antibodies and can have greater permeability than whole antibodies,
allowing single-chain antibodies to localize and bind to target
antigen-binding sites more efficiently. Furthermore, the relatively small
size of single-chain antibodies makes them less likely to provoke an
unwanted immune response in a recipient than whole antibodies. Multiple
single chain antibodies, each single chain having one VH and one VL
domain covalently linked by a first peptide linker, can be covalently
linked by at least one or more peptide linker to form multivalent single
chain antibodies, which can be monospecific or multispecific. Each chain
of a multivalent single chain antibody includes a variable light chain
fragment and a variable heavy chain fragment, and is linked by a peptide
linker to at least one other chain. The peptide linker is composed of at
least fifteen amino acid residues. The maximum number of linker amino
acid residues is approximately one hundred. Two single chain antinomies
can be combined to form a diabody, also known as a bivalent dimer.
Diabodies have two chains and two binding sites, and can be monospecific
or bispecific. Each chain of the diabody includes a VH domain connected
to a VL domain. The domains are connected with linkers that are short
enough to prevent pairing between domains on the same chain, thus driving
the pairing between complementary domains on different chains to recreate
the two antigen-binding sites. Three single chain antibodies can be
combined to form triabodies, also known as trivalent trimers. Triabodies
are constructed with the amino acid terminus of a VL or VH domain
directly fused to the carboxyl terminus of a VL or VH domain, i.e.,
without any linker sequence. The triabody has three Fv heads with the
polypeptides arranged in a cyclic, head-to-tail fashion. A possible
conformation of the triabody is planar with the three bonding sites
located in a plane at an angle of 120 degrees from one another.
Triabodies can be monospecific, bispecific or trispecific. Thus,
antibodies useful in the methods described herein include, but are not
limited to, naturally occurring antibodies bivalent fragments such as
(Fab')2, monovalent fragments such as Fab, single chain antibodies,
single chain Fv (scFv), single domain antibodies multivalent single chain
antibodies, diabodies, triabodies, and the like that bind specifically
with an antigen.
[0074] Antibodies can also be raised against a polypeptide or portion of a
polypeptide by methods known to those skilled in the art. Antibodies are
readily raised in animals such as rabbits or mice by immunization with
the gene product, or a fragment thereof. Immunized mice are particularly
useful for providing sources of B cells for the manufacture of
hybridomas, which in turn are cultured to produce large quantities of
monoclonal antibodies. While both polyclonal and monoclonal antibodies
can be used in the methods described herein. It is preferred that a
monoclonal antibody is used where conditions require increased
specificity for a particular protein.
[0075] In another aspect the inhibitor is an oligonucleotide (eg DNA or
RNA) or peptide aptamer which can bind either a polypeptide made
according to the present invention, or the binding target of a
polypeptide made according to the present invention, and/or which can
prevent interaction of the wild type equivalent of a polypeptide of the
invention with its target red blood cell receptor.
[0076] In one aspect the inhibitor, such as an aptamer or an antibody or
derivative or fragment thereof, binds to CD147.
[0077] In one aspect the inhibitor, such as an aptamer or an antibody or
derivative or fragment thereof binds to a merozoite target from
Plasmodium falciparum such as Rh5, or equivalent protein in other
Plasmodium species.
[0078] Thus the invention in particular relates to any anti-CD147
antibody, such as Metuximab, or any anti-Rh5 antibody, for use in the
prevention and/or treatment of Plasmodial infection and/or malarial
disease.
[0079] In one aspect the antibody is an anti-CD147 (also called HAb18G)
antibody which has been licensed for use to treat hepatocellular
carcinoma by Chinese State Food and Drug Administration (No. S20050039).
It is a radiolabeled F(ab)'2 called Licartin (or metuximab).
[0080] In one aspect the antibody is an antibody that is capable of
competing with MEM-M6/6 or TRA-1-85 for binding to CD147.
[0081] In one aspect the antibody is a humanised version of either of the
antibodies MEM-M6/6 or MEM-M6/1. (MEM-M6/1) was purchased from
AbD-Serotec and purified using protein G columns (GE Healthcare) using
standard procedures.
[0082] In one aspect, the invention relates to a humanized anti-CD147
antibody.
[0083] In one aspect the invention relates to antibodies against
Plasmodium falciparum Rh5.
[0084] In one aspect the antibody is a humanised version of any anti-Rh5
polypeptide or fragment or variant thereof.
[0085] In one aspect the inhibitor of binding is a small molecule, which
binds to either the red blood cell (e.g. to CD147) or to a Plasmodial
target (eg Rh5) to prevent interaction, for example to prevent Rh5and
CD147 interaction.
[0086] In a further aspect the inhibitor may be a soluble fragment of one
of Rh5 or CD147, which has been shown to help prevent invasion of P.
falciparum in vitro herein, such as a fragment generated by the methods
disclosed herein.
[0087] For the avoidance of doubt the inhibitor need not be restricted to
P. falciparum Rh5, but may be an inhibitor from any Plasmodium species.
[0088] Furthermore, for the avoidance of doubt, Rh5 may be expressed in
any suitable form, by any method, not limited to that disclosed
specifically herein, and may be expressed without any protein sequence
modification (e.g. with the native signal sequence and glycosylation
sites). In one aspect the Rh5 polypeptide or sequence of variant thereof
is expressed with a native signal sequence. In one aspect the Rh5
polypeptide is expressed in a mammalian expression system.
[0089] The invention also relates to an Rh5 polypeptide or a fragment or
variant thereof for the prevention and/or treatment of Plasmodial
infection and/or disease. In one aspect, the invention relates to an Rh5
polypeptide or a fragment or variant thereof for conferring protection
across multiple Plasmodial strains.
[0090] In a yet further aspect the invention relates to the use of an Rh5
polypeptide or a fragment or variant thereof in the preparation of a
medicament for prevention and or treatment of Plasmodium infection and/or
malarial disease.
[0091] In a yet further aspect the invention relates to the use of the Rh5
polypeptide or a fragment or variant thereof in the preparation of a
medicament for conferring protection across multiple Plasmodial strains
or species.
[0092] Thus in one aspect the invention relates to the use of an Rh5
polypeptide or a fragment or variant thereof from a first Plasmodium
strain in the of an immunogenic composition capable of preventing
Plasmodium infection or related disease by a different Plasmodium strain
or species, such as one having a different Rh5 polypeptide sequence.
[0093] The invention also relates to a method of prevention or treatment
of Plasmodium infection or malarial disease, the method comprising
delivery of Rh5 polypeptide or a fragment or variant thereof to an
individual in need thereof.
[0094] The invention also relates to a method of conferring protection
across multiple Plasmodial strains, or species, the method comprising
delivery of Rh5 polypeptide or a fragment or variant thereof to an
individual in need thereof.
[0095] The invention also relates to an immunogenic composition or a
vaccine comprising the Rh5 polypeptide or a fragment of variant thereof.
The immunogenic composition or vaccine may comprise additional antigens,
such as antigens CSP, MSP-1, AMA1, or part thereof. In one aspect, the
immunogenic composition or vaccine does not comprise additional antigens.
In one aspect, the immunogenic composition or vaccine does not comprise
other members of PfRh and/or EBL families.
[0096] In one aspect, the Rh5 or a fragment or variant thereof is
expressed using an expression system and method as described herein,
preferably using a mammalian expression system, but is not limited to
being expressed in this way. In one aspect the Rh5 polypeptide is
expressed in a mammalian expression system.
[0097] In one aspect the term "Rh5 polypeptide" refers to a polypeptide
comprising, consisting essentially of, or consisting of the amino acid
sequence as shown in SEQ ID No. 1. Reference to Rh5 polypeptide includes
a polypeptide that comprises SEQ ID No. 1 with an N-terminal signal
peptide and a C-terminal rat Cd4 domains 3 and 4 tag.
[0098] It will be appreciated that fragments or variants of Rh5, such as
additions, substitutions or deletions, which may be naturally occurring,
may be used in as immunogenic or vaccine compositions. For the avoidance
of doubt, the polypeptide variants of Rh5 are not limited to variants
that affect glycosylation.
[0099] The Rh5 or fragment or variant thereof used in the invention
suitably has the ability to bind BASIGIN (CD147). The ability to bind
BASIGIN is indicative of correctly-folded protein. Expressing Rh5 using a
mammalian expression system as shown herein can demonstrably produce
correctly-folded protein as assessed by its ability to bind to BASIGIN.
The ability to bind BASIGIN can be assessed using techniques such as
surface plasmon resonance. In one aspect, the binding affinity of Rh5 or
Rh5 fragment to BASIGIN is similar to or stronger than a KD of 1 .mu.M.
[0100] In one aspect of the invention, treatment or prevention of
Plasmodium infection or malarial disease refers to the complete blocking
of invasion of human red blood cells by the Plasmodium, for example as
assessed by methods disclosed herein. In one aspect, treatment or
prevention of Plasmodium infection or malarial disease refers to the
substantial or significant blocking of invasion of human red blood cells
by the Plasmodium, for example as assessed by methods disclosed herein.
The methods disclosed herein are as described in example 2 and shown in
FIGS. 7a and 7b showing inhibition of Plasmodium falciparum invasion in
vitro by blocking the Rh5-CD147 interaction.
[0101] In a further aspect the invention relates to an immunogenic
composition comprising a polypeptide or polynucleotide of the invention.
The invention also relates to vaccines comprising a polypeptide or
polynucleotide of the invention. In particular malaria vaccines using
Plasmodium antigens expressed using methods described herein.
[0102] The immunogenic composition or vaccine may comprise one or more
polypeptides or polynucleotides of the invention, or a combination of
polypeptides or polynucleotides, preferably a polynucleotide in
combination with all or a part of the polypeptide encoded by it,
expressed by the methods of the invention.
[0103] Compositions and vaccines may comprise pharmaceutically acceptable
excipients. Suitable excipients are well known in the art and proteins,
saccharides, polylactic acids, polyglycolic acids, polymeric amino acids,
amino acid copolymers, sucrose (Paoletti et al., 2001, Vaccine, 19:2118),
trehalose (WO 00/56365), lactose and lipid aggregates (such as oil
droplets or liposomes), diluents, such as water, saline, glycerol etc.
Additionally, auxiliary substances, such as wetting or emulsifying
agents, pH buffering substances, and the like, may be present. Sterile
pyrogen-free, phosphate buffered physiologic saline is a typical
excipient. A thorough discussion of pharmaceutical acceptable excipients
is available in reference Gennaro, 2000, Remington: The Science and
Practice of Pharmacy, 20.sup.th edition, ISBN:0683306472.
[0104] The vaccine of the present disclosure may be used to protect or
treat a mammal susceptible to infection, by means of administering said
vaccine via systemic or mucosal route. These administrations may include
injection via the intramuscular, intraperitoneal, intradermal or
subcutaneous routes; or via mucosal administration to the
oral/alimentary, respiratory, genitourinary tracts. Thus one aspect of
the present disclosure is a method of immunizing a human host against a
disease, which method comprises administering to the host an
immunoprotective dose of the vaccine or composition of the present
disclosure.
[0105] The amount of antigen in a vaccine dose is selected as an amount
which induces an immunoprotective response without significant, adverse
side effects in typical vaccines. Such amount will vary depending upon
which specific immunogen is employed and how it is presented. Generally,
it is expected that each dose will comprise 10 pg-1 mg, such as 1-100 ug
of protein antigen, suitably 5-50 ug, and most typically in the range
5-25 ug.
[0106] An optimal amount for a particular vaccine can be ascertained by
standard studies involving observation of appropriate immune responses in
subjects.
[0107] Following an initial vaccination, subjects may receive one or
several booster immunisations adequately spaced.
[0108] Following an initial vaccination, subjects may also receive further
vaccinations with antigens which are different from the initial vaccine,
for example containing a polypeptide which is naturally occurring but
with a different sequence, or is a mutant or variant of a wild type
sequence.
[0109] The immunogenic composition or vaccine may comprise additional
antigens, such as Plasmodium antigens, for example those, comprising CSP,
for example RTSS or AMA1.
[0110] The immunogenic composition or vaccine of the invention may also
comprise suitable adjuvants to increase the immune response to any
vaccine. Suitable adjuvants include those inducing either a Th1 or Th2
response, or both, and adjuvants may comprise an aluminium salt, oil in
water emulsion, a saponin such as QS21 or an lipid A derivative such as
3D-MPL, or combinations thereof, such as the GSK AS01, AS02, AS03, AS04
adjuvant, or the Novartis MF59 adjuvant.
[0111] In a further aspect the invention relates to the use of polypeptide
made using the present invention in screening for interactions with a
receptor or other binding partner, suitably by exposing the polypeptide
to various targets and detecting binding. For example, the invention
relates to the use of a Plasmodium polypeptide of the invention in the
identification of a RBC receptor, the method comprising screening red
blood cells or RBC proteins with the polypeptide to identity RBC
components that bind to the soluble polypeptide. The methodology
disclosed in Kauth, V. W. et al. [interactions between merozoite surface
proteins 1, 6, and 7 of the malaria parasite Plasmodium falciparum. The
Journal of biological chemistry 281, 31517-31527 (2006).] can be used to
screen polypeptides against a red blood cells, or RBC extracts, or red
blood cell polypeptides. In one aspect the interaction assay heroin
termed AVEXIS is used, as described in Bushell, Genome Research v18 p 622
(2008).
[0112] The same interaction assay approach could be used to introduce
specific mutations within proteins and observe the effects of naturally
occurring variations on protein function.
[0113] Thus the invention relates to mutants of the polypeptides of the
invention, comprising additions or substitutions or deletions, and
polynucleotides encoding the same, and to use of such mutants in
screening for the effects of variations on polypeptide binding and/or
function.
[0114] The invention also relates to an antibody raised to, or
specifically reactive with, a polypeptide produced according to the
invention. Where the polypeptide of the invention is a fragment of a full
length protein (such an an ectodomain) then in one aspect the antibody
shows greater specificity to binding or the fragment when compared to the
full length protein. Antibodies may be whole antibodies, antibody
fragments or subfragments. Antibodies can be whole immunoglobulins of any
class e.g., IgG, IgM, IgA, IgD or IgE, chimeric antibodies or hybrid
antibodies with dual specificity to two or more antigens of the
disclosure. They may also be fragments e. g. F(ab')2. Fab', Fab. Fv and
the like including hybrid fragments. An immunoglobulin also includes
natural, synthetic or genetically engineered proteins that act like an
antibody by binding to specific antigens to form a complex.
[0115] The invention also relates to peptide or nucleic acid (e.g. DNA or
RNA) aptamers which bind polypeptides according to the invention.
[0116] In another aspect the invention rotates to use of Rh5 or a fragment
or variant thereof in identification of an Rh5 ligand suitable for the
prevention or treatment of Plasmodial infection or related disease. In
another aspect the invention relates to use of CD147 or a fragment or
variant thereof in identification of an CD147 ligand suitable for the
prevention or treatment of Plasmodial infection or related disease.
[0117] In one aspect prevention of treatment of red blood infection by
Plasmodia is considered as prevention of treatment of Plasmodial
infection or related disease.
[0118] In one aspect the invention relates to a cell line expressing
PfRh5, suitably a stable cell line in a preferred aspect the invention
relates to a method for producing a merozoite polypeptide ectodomain from
Plasmodium falciparum, the method comprising expression of nucleic acid
encoding the polypeptide in a mammalian human embryonic kidney (HEK)
cell, and optionally purification of the polypeptide so expressed,
wherein:
(i) (optionally) the expressed polypeptide is not N-glycosylated in the
cell; (ii) the nucleic acid encodes an exogenous mammalian signal
sequence from the mouse variable .kappa. light chain 7-33 effective to
deliver the polypeptide into the secretory pathway of the mammalian cell;
and (iii) the nucleic acid has been colon optimised for expression of the
polypeptide in the HEK cell.
[0119] It will be understood that particular embodiments described herein
are shown by way of illustration and not as limitations of the invention.
The principal features of this invention can be employed in various
embodiments without departing from the scope of the invention. Those
skilled in the art will recognize, or be able to ascertain using no more
than routine study, numerous equivalents to the specific procedures
described herein. Such equivalents are considered to be within the scope
of this invention and are covered by the claims. All publications and
patent applications mentioned in the specification are indicative of the
level of skill of those skilled in the art to which this invention
pertains. All publications and patent applications are herein
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
indicated to be incorporated by reference. The use of the word "a", or
"an" when used in conjunction with the term "comprising" in the claims
and/or the specification may mean "one," but it is also consistent with
the meaning of "one or more," "at least one," and "one or more than one."
The use of the term "or" in the claims issued to mean "and/or" unless
explicity indicated to refer to alternatives only or the alternatives are
mutually exclusive, although the disclosure supports a definition that
refers to only alternatives and "and/or." Throughout this application,
the term "about" is used to indicate that a value includes the inherent
variation of error for the device, the method being employed to determine
the value, or the variation that exists among the study subjects.
[0120] As used in this specification and claim(s), the word "comprising"
(and any form of comprising, such as "comprise" and "comprises"),
"having" (and any form of having, such as "have" and "has"), "including"
(and any form of including, such as "includes" and "include") or
"containing" (and any form of containing, such as "contains" and
"contain") are inclusive or open-ended and do not exclude additional,
unrecited elements or method steps. In one aspect such open ended terms
also comprise within their scope a restricted or closed definition, for
example such as "consisting essentially of", or "consisting of".
[0121] The term "or combinations thereof" as used herein refers to all
permutations and combinations of the listed items preceding the term. For
example, "A, B, C, or combinations thereof is intended to include at
least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a
particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
Continuing with this example, expressly included are combinations that
contain repeats of one or more item or term, such as BB, AAA, AB, BBC,
AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will
understand that typically there is no limit on the number of items or
forms in any combination, unless otherwise apparent from the context.
[0122] All or the compositions and/or methods disclosed and claimed herein
can be made and executed without undue experimentation in light of the
present disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to these of skill in the art that variations may be applied to
the compositions and/or methods and in the steps or in the sequence of
steps of the method described herein without departing from the concept,
spirit and scope of the invention. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by the
appended claims.
[0123] All documents referred to herein are incorporated by reference to
the fullest extent permissible.
[0124] Any element of a disclosure is explicitly contemplated in
combination with any other element of a disclosure, unless otherwise
apparent from the context of the application.
[0125] The present invention is further described by reference fa the
following examples, not limiting upon the present invention.
Example 1
A Library of Functional Recombinant Plasmodium falciparum Merozoite
Surface Proteins
[0126] In this study, we have attempted to express the extracellular
domain of 53 secreted or cell-surface merozoite proteins from P.
falciparum, using human embryonic kidney (HEK) 293E cells. To improve the
production of functional recombinant proteins, all coding sequences were
codon-optimised for expression in human cells and any potential N-linked
glycosylation site modified so as to more closely mimic the shape of the
native protein. Endogenous signal sequences were removed and replaced by
an exogenous mammalian signal sequence to promote correct addressing of
the recombinant proteins to the secretory pathway.
[0127] Using this approach, we were able to detect expression of 40
proteins by ELISA and 44 by Western blot. The recombinant proteins were
shown to be correctly folded and functional by demonstrating their
ability to interact with known binding partners, and by showing their
immunogenicity against human sera from malaria-infected patients.
Material and Methods
Generation of a Recombinant Merozoite Protein Library
[0128] Sequences encoding the extracellular domains of 53 merozoite
cell-surface proteins, with the exception of their signal peptide, were
made by gene synthesis (Geneart) and are presented in Table 1. All
sequences were codon-optimised for expression into human cells and all
potential N-linked glycosylation sites identified by the canonical
sequence NXS/T ware modified by replacing all serine or threonine
residues within the canonical motifs by an alanine residue.
[0129] The coding sequences, flanked by unique Notl and Ascl sites, were
cloned into a derivative of the pTT3 expression vector 4 between the
leader sequence of the mouse variable .kappa. light chain 7-33, and a rat
CD4 domains 3 and 4 tag followed by an enzymatic biotinylation sequence
as previously described 5. All expression constructs were cotransfected
with the BirA biotinylation enzyme into HEK293E cells. The soluble
biotinylated recombinant proteins were collected from the cell culture
supernatant 6 days post transfection, and dialysed into HBS before
analysis.
ELISA Test
[0130] The biotinylated ectodomains of the P. falciparum library were
serially diluted 1:2 op to a final dilution of 1:128 and all dilutions
were immobilized on streptavidin-coated plates (NUNC) before being
mounted for one hour with 10 .mu.g/ml OX68 antibody, which binds the CD4
tag. The plates were washed in PBS/0.1% Tween20 (PBST) before incubation
with an anti-mouse immunoglobulin antibody coupled to alkaline
phosphatase (Sigma) for one hour at room temperature. After washes in
PBST and PBS, wells were incubated with p-nitrophenyl phosphate at 1
mg/ml and optical density measurements (OD) taken at 405 nm.
Western Blot
[0131] Between 5 and 30 .mu.L of dialysed transfection medium containing
the recombinant proteins was resolved by SDS-PAGE under reducing
conditions (with the exception of EBA181 and EBL1 which were run in
non-reducing condition) before blotting onto Hybond-P PVDF membrane (GE
Healthcare) overnight at 30 V. Membranes were blocked with 2% BSA in PBST
and incubated with 0.02 .mu.g/ml of streptavidin-HRP (Jackson
Immunoresearch) diluted in 0.2% BSA and detected with the Supersignal
West pico chemiluminescent substrate (Pierce).
AVEXIS Screen
[0132] Interaction between MSP1 and MSP7 proteins was identified using the
AVEXIS method as previously described 5. Briefly, the codon-optimized
sequence for MSP1 and MSP7 was cloned into a prey construct between the
leader sequence of the mouse variable .kappa. light chain 7-33, and a rat
CD4 domains 3 and 4 tag followed by the pentamerisation domain of rat
cartilaginous oligomeric matrix protein and the betalactamase coding
sequence, as previously described 5. MSP1 and MSP7 prey pentamers were
screened against the whole biotinylated merozoite library, and positive
interactions identified using nitrocefin (Calbiochem). OD measurements
were taken at 485 nm.
Flow Cytometry
[0133] Biotinylated EBA 175-CD4d3+4 ectodomains or CD4 domains 3+4 alone
(negative control) were immobilized on streptavidin-coated Nile Red
fluorescent 0.4-0.6 .mu.m microbeads (Spherotech Inc.) by incubation for
45 min al 4.degree. C. and then presented to human erythrocytes. After
incubating for 1 hour at 4.degree. C., cells were washed three times in
PBS-BSA-NaN3 to remove non-bound beads, re-suspended in 1% formalin and
analyzed by flow cytometry using an LSR II machine (BD Biosciences). To
test for binding specificity, purified human erythrocytes were either
treated with 5 mU of Vibrio cholera neuraminidase (Sigma) for 1 hour at
37.degree. C. and washed twice, or preincubated with the anti-GYPA BRIC
256 monoclonal antibody at a concentration of 0.5 .mu.g/106 cells, prior
to incubation with EBA-175-coated microbeads.
Results
[0134] The codon-optimized ectodomain sequences of 53 cell-surface and
secreted merozoite proteins from P. falciparum were co-transfected with
the biotinylation enzyme BirA into HEK293E cells (Table 1). Soluble
biotinylated recombinant proteins were harvested six days
post-transfection and dialysed into HBS. Expression levels of all
proteins ere first assessed by ELISA: of the 53 proteins tested, all but
12 (RAP1, RAP2, RAP3, RhopH1, RhopH2, RON3, Rh1, Rh2b, Rh4, PF14_0293,
EBL1 and PTRAMP) showed clear signals (data not shown).
[0135] All proteins were than tested by western blot (FIG. 1). As expected
from the ELISA test, no expression was detected for RAP1, RAP2, RAP3,
RhopH1, RhopH2, RON3, EBL1, PF14.sub.13 0293 and PTRAMP. Most proteins
showed expression of one major form.
[0136] To show that those recombinant proteins were functional, we next
assessed their ability to interact with known binding partners. As a
first example, we focused on the micronemal protein EBA175, which is
known to interact, through its region II, to GLYCOPHORIN A (GYPA)
expressed at the surface of human red blood cells 6. This interaction
requires sialylation of GYPA as pre-treatment of erythrocytes with
neuraminidase which cleaves off sialic acid residues, is sufficient to
abolish binding. To test whether we could recapitulate these observations
using our recombinant EBA175 ectodomain, human erythrocytes were
presented with Nile Red microbeads coated either with the biotinylated
EBA175 extracellular domain, or the biotinylated CD4 domains 3+4 as a
negative control (FIG. 2). EBA175-coated but not CD4-coated microbeads
bound robustly to human red blood cells. This binding could be
specifically blocked by either pre-treatment of erythrocytes with
neuraminidase (FIG. 2A), or pre-incubation of the red blood cells with an
anti-GYPA monoclonal antibody (FIG. 2B) demonstrating that the
full-length recombinant EBA175 ectodomain could specifically internet
with the native GYPA present on the surface of erythrocytes.
[0137] As a second example, we tested interactions between merozoite
proteins. The merozoite surface proteins 1, 5 and 7 have previously been
shown to form a noncovalent complex at the surface of merozoites, which
is subsequently cleaved off upon erythrocyte invasion. All three proteins
are believed to undergo proteolytic maturation before forming the
complex. Using full-length recombinant MSP7 produced in E. coli, Kauth
and coworkers (ref 2) demonstrated association with the aminoterminal
p83, p30 and p38 subfragments of MSP1, but not with the carboxyterminal
p42 region. The p38 fragment of MSP1 was also able to bind the processed
carboxyterminal MSP636 form of MSP6. To reproduce binding between these
different merozoite surface proteins we used the AVEXIS method. The
codon-optimized sequence of MSP1 and MSP7 ectodomains were cloned into a
prey construct and expressed as pentameric proteins fused to
beta-lactamase by transfection into HEK293E cells. These 2 preys were
then normalised before screening against the whole recombinant merozoite
library. Using this approach, we were able to detect the MSP1-MSP7
interaction in both orientations: the MSP1 prey was only captured by the
MSP7 bait, and similarly the MSP7 prey was only captured by the MSP1 bait
(FIG. 3) demonstrating that both proteins were functional interestingly
this binding was detected using the whole unprocessed ectodomains of MSP1
and MSP7. No interaction was detected between MSP6 and MSP7.
Interestingly, we did not observe any binding between MSP1 and MSP6,
confirming the previous observation that only the processed form MSP636
can bind MSP1 (ref 2).
Example 2: Immunogenicity Testing
[0138] We tested whether any of the recombinant ectodomains were
immunogenic by testing them against sera from patients exposed to
Plasmodium falciparum compared to patients that were malaria naive. This
approach has been previously used to provide an indication that P.
falciparum recombinant proteins are folded correctly. To extend this
rationale, we compared the responses of the sera to untreated proteins
and those that had been heated for 10 minutes at 80.degree. C. with the
thinking that many conformational-dependent epitopes would be heat
labile. All recombinant proteins gave a higher response to pooled serum
from malaria-exposed patients relative to non-exposed controls (FIG. 4).
Heat-treatment decreased the reading for all but 9 proteins (Pf12p, MSP3,
MSP6, H103, MSRP3, ASP, RON6, RAMA, TLP, PF10_0323) demonstrating that
most proteins contain heat-labile epitopes implying that they are
correctly folded.
AVEXIS Identifies CD147 as a Receptor for PfRh5
[0139] One anticipated use of the recombinant P. falciparum merozoite
surface protein library is to identity erythrocyte receptors for the
parasite ligands. Here we show how this has been performed for the P.
falciparum merozoite protein, Rh5.
[0140] To identify an erythrocyte receptor for the P. falciparum invasion
ligand Rh5, we used a recombinant Rh5 protein taken from the merozoite
surface protein protein library. Rh5 was expressed either as a monomeric
enzymatically biotinylated "bait" or a beta-lactamase-tagged pentamerised
"prey" and their expression activities normalised to a stringent
threshold suitable for screening by AVEXIS (7). A protein library
produced in an identical fashion but containing the ectodomain regions of
human erythrocyte receptors contained 41 baits and 37 preys. The Rh5 prey
was screened against the erythrocyte bait library and an interaction with
BASIGIN (BSG-L) was detected (FIG. 5A, left panel). The next best
interaction was with a shorted splice variant of the same proteins,
BSG-S. To verify these interactions, the screen was performed in the
reciprocal orientation with the erythrocyte prey library screened against
the Rh5 bait. We found that both isoforms of the BASIGIN protein
interacted with Rh5 (FIG. 5A, right panel). No other interactions with
recombinant erythrocyte receptors were detected in our screen.
[0141] BASIGIN (also known as CD147, EMMPRIN and M6) is a widely expressed
member of the immunoglobulin superfamily (IgSF) (FIG. 5B) that has been
implicated in many biological functions ranging from embryo implantation
and spermatogenesis (8) to retinal development (9). It has also been
implicated in disease processes including tumour metastasis (10,
rheumatoid arthritis and human immunodeficiency virus infection. The two
IgSF domains belong to the C and V sets but are unusual in that the C-set
IgSF domain is located N-terminal to the V-set domain (11). Rh5
interacted with both isoforms of the protein suggesting that the Rh5
binding site resided with the two membrane-proximal IgSF domains.
Rh5 Directly Interacts with BSG: a Quantitative Analysis
[0142] To demonstrate that Rh5 and BSG directly interact and to quantify
the bending parameters of the interaction, we used surface plasmon
resonance as implemented in a BiAcore machine which is able to detect
even very transient interactions. The ectodomain fragment of Rh5 was
purified and separated by gel titration before injecting increasing
concentrations over the biotinylated BSG-SCd4d3+4bio immobilised on a
streptavidin-coated sensor chip with CD4d3+4bio used as a reference. The
binding once equilibrium had been reached (FIG. 6A--inset) is represented
by the difference in response units observed in the BSG-S and control
flow cell and is plotted as a binding curve (FIG. 6A). Saturable binding
was observed and an equilibrium dissociation constant (K.sub.D) of
1.14.+-.0.03 .mu.M was calculated from a non-linear curve fit to the
data.
[0143] To determine the kinetic parameters of the interaction, serial
dilutions of purified PfRh5Cd4d3+4-6H were injected over immobilised
BSG-S and a reference flow cell at high flow rates (100 .mu.l/min) to
minimise rebinding effects. A global fit of a simple 1:1 binding model
fitted the curves well (FIG. 6B) and yielded a dissociation rate constant
(k.sub.d) of 0.240.+-.0.001 s.sup.-1 (corresponding to an interaction
half-life of 2.9 seconds) and an association rate constant (k.sub.a) of
2.2.+-.0.01.times.10.sup.5 M.sup.-1 s.sup.-1. The same analysis was
performed on the three domain isoform of BSG which showed a slightly
shopper interaction strength (K.sub.D=0.71.+-.0.02 .mu.M;
k.sub.d0.1436.+-.0.0003 S.sup.-1) suggesting that the additional IgSf
domain of the long BSG isoform marginally increased Rh5 binding affinity.
Soluble BSG-S and BSG-L Inhibit P. falciparum Invasion In Vitro
[0144] To determine whether the interaction between the erythrocyte BSG
receptor and P. falciparum invasion ligand Rh5 is necessary for invasion,
we attempted to specifically block the interaction by adding purified
pentamerised recombinant soluble ectodomain fragments of both the long
and short terms of human BSG to invasion cultures. We found that BSG-S
inhibited invasion of the neuraminidase-sensitive Dd2 P. falciparum
strain in a dose-dependent manner which had the shape of a typical
dose-response curve with an IC.sub.50 of .about.1 .mu.M (FIG. 7a(A)).
This protein also inhibited the invasion of a neuraminidase-independent
strain, 3D7, although with reduced efficacy (FIG. 7a(B)). In addition,
BSG-L was able to inhibit invasion in both strains (FIG. 7).
[0145] FIG. 7b shows that soluble BSG potently block erythrocyte invasion
across multiple stains.
Monoclonal Antibodies Against BSG Inhibit P. falciparum Invasion In Vitro
[0146] Soluble forms of BSG consisting of the extracellular regions are
known to have biological effects such as up regulation of matrix
metalloproteases which might indirectly affect erythrocyte invasion
efficiency. To rule out this possibility, we added purified monoclonal
antibodies which are known to bind human BSG to the in vitro invasion
assay. Two independent mouse monoclonal antibodies that recognise human
BSG (MEM-M6/6 and MEM-M6/1) both potently blocked invasion (FIG. 8). To
our knowledge, this is the first identification of a human--P. falciparum
receptor ligand pair that is essential for invasion.
Materials and Methods
Recombinant Protein Production
[0147] Proteins for inclusion within the human erythrocyte protein library
were selected from a comprehensive proteomics analysis of human
erythrocyte ghost preparations and included all type I, GPI and type II
receptors and secreted proteins. Bait and prey constructs were produced
essentially as described (7). Briefly, each construct contained the
entire extracellular region (including the native signal peptide) flanked
by unique Notl and Ascl sites to facilitate cloning into a vector that
added a C-terminal rat CD4d3+4-tag and either a enzymatically
biotinylatable peptide (baits) or the pentamerising peptide from the rat
cartilage oligomeric matrix protein (COMP) followed by the enzymes
beta-lactamase (preys). Bait proteins were enzymatically biotinylated
during expression by cotransfection of a secreted term of the E. coli
BirA protein biotin ligase (7). The Rh5 bait and prey constructs differed
in that the low-scoring endogenous signal peptide (17) was replaced by a
high-scoring signal peptide from the mouse immunoglobulin kappa light
chain and potential N-linked glycan sites were mutated. All constructs
were codon optimised for mammalian expression and chemically synthesized
(Geneart AG, Regensburg, Germany) and subcloned into both bait and prey
expression vectors. Monomeric proteins were purified by subcloning the
Notl-/Ascl flanked extracellular regions into a similar vector encasing a
CD4d3+4 tag followed be a hexa-His tag and purified using 1 ml HiTrap
Ni.sup.2+ IMAC columns (GE Healthcare; as described (7). Purified
pentameric proteins used in invasion assays were made by replacing the
beta-lactamase reporter in the pray plasmid with a hexa-his tag and the
supernatants from transient transfections purified on HiTrap columns as
described above. Purified proteins were then dialysed against PBS and
1.times. against RPMI prior to use. Individual domains at human BSG were
produced by identifying domain boundaries using the structure of the BSG
extracellular region (11, 18) and amplifying these domains using primers
with suitable restriction cloning sites.
Interacation Screening by AVEXIS
Interaction Screening was Carried out as Described (7).
Antibodies
[0148] Antibodies were purchased from AbD-Serotec (MEM-M6/1) or were a
kind gift from Vaclav Horeijsi (Institute of Molecular Genetics, Czech
Republic) (MEM-M6/6) and purified using protein G columns (GE Healthcare)
using standard procedures.
Surface Plasmon Resonance
[0149] Surface plasmon resonance studies were performed essentially as
described (7, 20) using a BIAcore T100 instrument. Briefly, biotinylated
bait proteins were captured on a streptavidin-coated sensor chip
(BIAcore, GE Healthcare) using molar equivalents of rat CD4 domains 3 and
4 as a reference. Purified analyte proteins wore separated by gel
filtration just prior to use in SPR experiments to remove small amounts
of protein aggregates which are known to influence binding kinetic
binding measurements (21) increasing connect rations of purified proteins
were injected at 10 .mu.l/min for equilibrium studies or 100 .mu.l/min
for kinetic analyses to minimise rebinding effects. Binding data were
analysed in BIAevaluation software (BiAcore) using a global fits to the
entire sensorgrams (both association and dissociation phases) to a
dilution series of ligand. All experiments were performed at 37.degree.
C.
In Vitro Culture of P. falciparum Parasites
[0150] P. falciparum parasite strains 3D7, Dd2, and HB3 were routinely
cultured in human O+ erythrocytes (NHS Blood and Transplant, Cambridge,
UK) at 5% hematocrit in complete medium containing 10% human sear, under
an atmosphere of 1% O2, 3% CO2, and 96% NS (BOC, Guildford, UK). Parasite
cultures were synchronized on early stages with 5% D-sorbitol
(Sigma-Aldrich, Dorset, UK). Use of erythrocytes from human donors for P.
falciparum culture was approved by NHS Cambridgeshire 4 Research Ethics
Committee.
Parasite Labeling
[0151] Parasite cultures were stained with a DNA dye according to the
following protocol. The cells were washed with PBS before staining with 2
.mu.M. Hoechst 33342 (Invitrogen, Paisley, UK) in RPMI 1640. After
staining, the cells were washed with PBS, before being fixed with a 2%
paraformaldehyde (Sigma-Aldrich, Dorset, UK), 0.2% glutaraldehyde
(Sigma-Aldrich, Dorset, UK) solution in PBS for 1 h at 4.degree. C.
Finally, the suspension was washed with PBS before acquisition on a flow
cytometer. The cells were next washed with PBS before staining with the
DNA dyes as described earlier. Finally, the cells were washed with PBS
before acquisition on a flow cytometer.
Erythrocyte Labeling
[0152] Erythrocytes were labeled with amine-reactive fluorescent dyes. The
required volume of O+ erythrocytes at 2% haematocrit in RPMI 1640 was
centrifuged and the pellet resuspended to 2% hematocrit with either 20
.mu.M CFDA-SE (Invitrogen, Paisley, UK) or 10 .mu.M DDAO-SE (Invitrogen,
Paisley, UK) in RPMI 1640 and incubated for 2 h at 37.degree. C. The
suspension was washed with complete medium and the pellet resuspended to
2% hematocrit with complete medium and incubated for 30 min at 37.degree.
C. The suspension was then washed twice with incomplete medium (without
human sera) and finally resuspended to 2% hematocrit with incomplete
medium. The cells were stored until use at 4.degree. C. for up to 24 h.
Flow Cytometry and Data Analysis
[0153] Stained samples were examined with a 355 nm 20 mW UV laser, a 488
nm 20 mW blue laser, and a 633 nm 17 mW red laser on a BD LSRII flow
cytometer (BD Biosciences, Oxford, UK). Ethidium bromide (EB) was excited
by a blue laser and detected by a 610/20 filter. Hoechst 33342 was
excited by a UV laser and detected by a 450/50 niter SYBR Green I and
CFDA-SE were excited by a blue laser and detected by a 530/30 filter.
DDAO-SE was excited by a red laser and detected by a 660/20 filter. BO
FACS Diva (BD Biosciences, Oxford, UK) was used to collect 100,000 events
tor each sample. FSC and SSC voltages of 423 and 198, respectively, and a
threshold of 2,000 on FSC were applied to gate on the erythrocyte
copulation. The data collected was then further analysed with FlowJo
(Tree Star, Ashland, Oreg.). All experiments were carried out in
triplicate and the data is presented as the mean.+-.standard error of the
mean. GraphPad Prism (GraphPad Software, La Jolla, Calif.) was used to
plot parasitemia data generated and carry out statistical analysis.
P. falciparum Invasion Assays
[0154] Invasion assays were carried out in round-bottom 96-well plates,
with a culture volume of 100 .mu.L per well at a hematocrit of 2%. Plates
were incubated inside an incubator culture chamber (VWR, Lutterworth,
UK), gassed with 1% O.sub.2, 3% CO.sub.2, and 90% N.sub.2, and kept at
for 48 h. Erythrocytes labelled with either 20 .mu.M CFDA-SE (Invitrogen,
Paisley, UK) or 10 .mu.M DDAO-SE (Invitrogen, Paisley, UK) were pelleted
and washed with incomplete media. The pellet was resuspended to 2%
hematocrit with incomplete medium and aliquoted into individual microfuge
tubes. Neuraminidase from Vibrio cholerae (Sigma-Aldrich, Dorset, UK) was
added to the appropriate tubes to obtain a final concentration of 20
mU/mL, and all of the tubes were incubated under rotation at 37.degree.
C. for 1 h. The cell suspensions were pelleted and washed with incomplete
media. The pellets were then resuspended to 2% hematocrit with incomplete
medium. pRBC were then added to each well and the well suspension mixed
before incubation for 48 h. At the end of the incubation period. RBC were
harvested and pRBC were stained as described earlier. Data collection and
statistical analysis were carried out as described earlier. Detailed
Standard Operating Procedures for all invasion assays are available at
http://www.sanger.ac.uk/research/projects/malariaprogramme-rayner/(Resour-
ces section).
Immunogenicity to Malaria-Exposed Serum
[0155] The biotinylated ectodomains of the P. falciparum library were
immobilized on streptavidin-coated plates (NUNC) with or without prior
heat denaturation for 10 minutes at 80.degree.C. The concentration of
each ectodomain was adjusted so as to obtain saturation of the
streptavidin on the well. After a brief wash, the immobolised ectodomains
were incubated for 2 hours at room temperature with pooled sera from
malaria-exposed end malaria-naive individuals diluted 1:1000 in HBST/2%
BSA. The plates ware washed in HBS/0.1% Tween20 (HBST) before incubation
with an anti-human immunoglobulin antibody coupled to alkaline
phosphatase (Sigma) for one hour at room temperature. After washes in
HBST and HBS, wells were incubated with p-nitrophenyl phosphate at 1
mg/ml and optical dentisity measurements (OD) taken at 405 nm.
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DISCUSSION/CONCLUSION
[0176] In this study, we have used a simple systematic approach based on
codon-optimised constructs transiently expressed in mammalian cells to
create a resource of recombinant proteins of the P. falciparum merozoite
surface protein repertoire. With the exception of rhoptry proteins, which
proved difficult to express, most proteins were successfully produced.
This convenient high throughput system does net require complex refolding
procedures using a cost effective delivery reagent which results in
typical transfection efficiencies of 20%, we were aisle to obtain good
expression levels reacting up to 1.4 mg of purified protein from a 50 mL
transfection in some cases. This yield could probably be further
increased by creating high-secreting stable cell lines. All proteins were
expressed as soluble recombinant ectodomains and, where known, were shown
to be correctly folded and functional based on their ability to
recapitulate known binding events. The carboxy-terminal tag present on
the proteins includes a sequence that can be enzymatically
monobiotinylated during protein expression and enables the proteins to be
quantified and captured in an oriented fashion on streptavidin-coated
solid phases. In this study, we used it to create highly avid binding
reagents to identify host erythrocyte receptor interactions and we
believe that the proteins in this resource will facilitate the discovery
of novel erythrocyte receptors for merozoite surface proteins, many of
which are still unknown. The same approach could now be used to easily
introduce specific mutations within merozoite proteins and observe the
effects of naturally occurring variations on protein function. These
proteins could also be fed into protein structure initiatives which will
eventually aid the rational design of novel therapeutic drugs. Despite
decades of research, malaria continues to be a global health problem and
the emergence and rapid spread of drug-resistant strains makes the
development of novel therapeutics an urgent research priority. One
strategy has been to develop a vaccine based on targeting the merozoite
since this form of the parasite is exposed to the host humoral immune
system and passive transfer of immunoglobulins to patients with clinical
malaria can reduce parasitaemia and resolve symptoms. Many of the current
leading vaccine targets are proteins known to be located on the exposed
surface of the merozoite. However, the difficulty in producing these
proteins in a soluble recombinant form has often led to targets being
selected on criteria such as high-level expression in a convenient
expression system rather than producing correctly folded
antigenically-active proteins that make the most effective vaccines.
Because it is increasingly likely that an effective anti-malarial vaccine
will not consist of a single protein but will be a multi-component
vaccine, we believe that the resource described in this study will
represent a significant step towards this goal. Finally, thin
demonstration that the Rh5-BSG interaction is essential for erythrocyte
invasion now provides new opportunities for novel therapeutic
intervention strategies. This could include a modification of the AVEXIS
assay to identify small molecule inhibitors of the Rh5-BSG interaction or
the humanisation of the MEM-M6/1 and MEM-M6/6/antibodies.
FIGURE LEGENDS
[0177] FIG. 1. Expression of recombinant secreted and cell surface
merozoite proteins from P. falciparum. Expression of biotinylated
recombinant merozoite proteins was assessed by western blot. The expected
molecular weight for each recombinant protein is indicated in brackets
above each column.
[0178] FIG. 2. Functional activity and immunogenicity of recombinant P.
falciparum merozoite proteins, (A) Recombinant biotinylated PfEBA-175
(top panel) and PfEBA-140 (bottom panel) immobilized on fluorescent
streptavidin-coated beads bound to untreated erythrocytes (thick solid
grey line). Binding was blocked by pre-treating the erythrocytes with
neuraminidase (thin grey line) or (for PfEBA-175) pre-incubating
erythrocytes with an anti-GYPA monoclonal antibody (dotted line).
Negative controls were Cd4d3+4-coated beads (thin solid black line).
[0179] FIG. 3. Demonstration that MSP1 and MSP7 are correctly folded and
functional. The interaction between recombinant PfMSP-1 and PfMSP-7 was
detected i n both bait-prey orientations by screening the whole library
with PfMSP1 and PfMSP7 preys using the AVEXIS assay. Baits labeled with
an asterisk were below threshold levels required fro the assay.
[0180] FIG. 4. Immunogenicity of the recombinant P. falciparum merozoite
surface antigens. The immunogenicity of the recombinant proteins was
systematically compared using sera pooled from adult patients unexposed
to malaria (naive sera, open bars) or living within malaria-endemic
regions (immune sera, grey bar). Recombinant proteins largely contained
heat-labile (conformational); epitopes as seen by the reduced response of
immune sera to hoar denatured antigen (black bar).
[0181] FIG. 5. AVEXIS identifies two splice variants of the erythrocyte
surface protein BASIGIN as a receptor for P. falciparum Rh5. (A) Rh5
interacted with both long and short isoforms of BASIGIN but no other
erythrocyte receptor protein when screened as either a prey (left panel)
or a bait (right panel). Error bars indicate the standard deviation from
three replicates. (B) Schematic showing the domain architecture of Rh5
and the BSG isoforms. Rh5 is a secreted protein and contains a region of
sequence homology to other Rh-family members indicated by the red box.
The long and short BSG isoforms contain three and two IgSF domains
respectively, numbered 0 to 2 according to convention. Signal peptides
are indicated fry an untried rectangle and putative N-linked
glycosylation sites by lollipops.
[0182] FIG. 6. Biophysical characterisation of the Rh5-BSG interaction
using surface plasmon resonance (A) Equilibrium binding analysis. Serial
dilutions of purified PfRh5Cd4d3+4-6H were injected (solid bar) through
flow cells with 325RU of BSG-SCd4d3+4bio or 150RU of Cd4d3+4 (control)
for 200 seconds until equilibrium was reached (inset).
Reference-subtracted binding data were plotted as a binding curve and a
KD of .about.1.1 .mu.M was calculated using non-linear regression fitting
of a simple Langmuir binding isotherm to the data (solid line) (B)
Kinetic binding analysis. The indicated concentrations of PfRh5Cd4d3+4-5H
ware injected over immobilized BSG-S surface at 100 .mu.l/min. The
binding curves were globally fitted to a 1:1 binding model (red line).
[0183] FIG. 7a. Soluble recombinant ectodomains BSG-S and BSG-L potently
reduce the efficiency of P. falciparum erythrocyte invasion. Purified
pentameric ectodomains of the short (squares) and long (circles) forms of
BSG were added at the indicated concentrations to an in vitro P.
falciparum invasion assay using the Dd2 (A) and 3D7 (B) strains. The
proteins reduced invasion efficiency relative to a control (CD4-COMP
(triangles)).
[0184] FIG. 7b. Soluble BSG potently block erythrocyte invasion across
multiple strains. (a) Erythrocyte invasion was inhibited by purified
pentamerised BSG-S-Cd4d3+4-COMP-His ectodomains but not by the two
non-binding BSG-S domains added individually or Cd4d3+4-COMP-His
(control); strain=Dd2. (b) Cross-strain inhibition of invasion using
pentamerised BSG-S.
[0185] FIG. 8a. Mouse monoclonal antibodies to human BSG block the
invasion of P. falciparum into human erythrocytes. Purified monoclonal
antibodies MEM-M6/1 (circles) and MEM-M6/6 (squares) were added at the
indicated concentrations to an in vitro P. falciparum invasion assay
using the 3D7 strain. The proteins reduced invasion efficiency relative
to an isotype-matched negative control (diamonds).
[0186] FIG. 8b. Anti-BSG antibodies potently block erythrocyte invasion.
(A) Anti-BSG monoclonal antibodies. TRA-1-85 and MEM-M6/6 potently
inhibited invasion of erythrocytes; strain=3D7. (B) MEM-M6/6
concentrations .gtoreq.10 .mu.g/ml prevented all detectable invasion by
microscopic observation of cultures, strain=3D7. (C, D) MEM-M6/6
inhibited invasion of synchronised P. falciparum culture-adapted lines
(C) and unsynchronised field isolates (D).
TABLE-US-00002
TABLE 1
Table 1. A list of recombinant merozoite proteins from P. falciparum.
The first and last amino-acid of the ectodomain region expressed
in HEK293E cells is shown for each protein, along with the number
of potential N-linked glycosylation sites that were modified and the
level of expression as assessed by ELISA.
Expres-
sion
Region N-Gly level
Protein name Accession # expressed Length sites (.mu.g/ml)
MSP1 PFI1475w V20-S1701 1682 13 1.25
MSP2 PFB0300c I20-N246 227 4 50
MSP4 PFB0310c Y29-S253 225 2 25
MSP5 PFB0305c N22-S251 230 4 5
MSP10 PFF0995c H27-S503 477 9 2.5
Pf12 PFF0615c H26-S323 298 7 0.62
Pf38 PFE0395c Q22-S328 307 4 1.25
Pf92 PF13_0338 A26-S770 745 15 0.03
Pf113 PF14_0201 Y23-K942 920 9 0.62
ASP PFD0295c A20-S708 689 10 0.39
RAMA AAQ89710 Y17-K838 821 10 0.15
EBA140 MAL13P1.60 I26-P1135 1110 11 0.004
EBA175 MAL7P1.176 A21-P1424 1404 18 0.015
EBA181 PFA0125c I27-S1488 1462 17 0.015
EBL1 PF13_0115 K22-N2584 2563 29 <0.002
AMA1 PF11_0344 Q25-T541 517 6 25
MTRAP PF10_0281 I23-K432 410 10 0.62
MSP3 PF10_0345 K26-H354 328 4 12.5
MSP6 PF10_0346 Y17-N371 355 3 0.156
H101 PF10_0347 Q23-N424 402 6 0.39
H103 PF10_0352 K27-Y405 379 4 1.56
MSP7 PF13_0197 T28-M351 324 2 6.24
Pf41 PFD0240c K21-S378 358 6 6.24
RAP1 PF14_0102 I23-D782 760 7 <0.002
RAP2 PFE0080c D22-L398 387 2 <0.002
RAP3 PFE0075c N23-K400 378 4 <0.002
RhopH1 PFC0110w K21-H1416 1396 10 <0.002
RhopH2 PFI1445w L20-S1378 1359 13 <0.002
RhopH3 PFI0265c K25-L897 873 4 0.312
Rh1 PFD0110w Q24-T666 643 7 <0.002
Rh2b* PF13_0198 + H25-Y75 + 1003 13 <0.002
MAL13P1.176 M1-S953
Rh4 PFD1150c I27-T1148 1122 20 <0.002
Rh5 PFD1145c F25-Q526 502 4 0.078
PTRAMP PFL0870w N25-S306 282 7 <0.002
SPATR PFB0570w E22-C250 229 2 12.5
TLP PFF0800w E24-P1306 1283 27 0.195
Pf34 PFD0955w N25-S306 282 2 6.24
PF14_0344 PF14_0344 A20-N993 974 13 0.39
PF10_0323 PF10_0323 R25-R52 28 0 1.56
RON3 PFL2505c N22-N249 228 1 <0.002
PFF0335c PFF0335c V23-K299 277 3 12.5
AARP PFD1105w K18-P191 174 5 0.312
MSP3.4 PF10_0348 N26-K697 672 11 0.2
MSP3.8 PF10_0355 Y23-N762 740 10 0.39
MSRP1 PF13_0196 Y22-T379 358 4 1.56
MSRP2 MAL13P1.174 K24-T280 257 5 0.312
MSRP3 PF13_0193 Q24-S298 275 3 1.56
RON6 PFB0680w F16-T949 934 15 0.01
Pf12p PFF0620c Y21-T349 329 3 0.15
MSP9 PFL1385c N24-S742 719 9 0.03
GAMA PF08_0008 L22-P710 689 9 0.78
PF11_0373 PF11_0373 L19-G656 638 20 0.78
PF14_0293 PF14_0293 N25-S968 944 23 0.01
*Because of the absence of clear signal peptide in the Rh2b protein
sequence (MAL13P1.176), the N-terminus of the Rh2a sequence (PF13_0198)
was added at the amino-terminus.
Sequence CWU
1
1
11502PRTPlasmodium falciparum 1Phe Glu Asn Ala Ile Lys Lys Thr Lys Asn Gln
Glu Asn Asn Leu Ala 1 5 10
15 Leu Leu Pro Ile Lys Ser Thr Glu Glu Glu Lys Asp Asp Ile Lys Asn
20 25 30 Gly Lys
Asp Ile Lys Lys Glu Ile Asp Asn Asp Lys Glu Asn Ile Lys 35
40 45 Thr Asn Asn Ala Lys Asp His
Ser Thr Tyr Ile Lys Ser Tyr Leu Asn 50 55
60 Thr Asn Val Asn Asp Gly Leu Lys Tyr Leu Phe Ile
Pro Ser His Asn 65 70 75
80 Ser Phe Ile Lys Lys Tyr Ser Val Phe Asn Gln Ile Asn Asp Gly Met
85 90 95 Leu Leu Asn
Glu Lys Asn Asp Val Lys Asn Asn Glu Asp Tyr Lys Asn 100
105 110 Val Asp Tyr Lys Asn Val Asn Phe
Leu Gln Tyr His Phe Lys Glu Leu 115 120
125 Ser Asn Tyr Asn Ile Ala Asn Ser Ile Asp Ile Leu Gln
Glu Lys Glu 130 135 140
Gly His Leu Asp Phe Val Ile Ile Pro His Tyr Thr Phe Leu Asp Tyr 145
150 155 160 Tyr Lys His Leu
Ser Tyr Asn Ser Ile Tyr His Lys Ser Ser Thr Tyr 165
170 175 Gly Lys Cys Ile Ala Val Asp Ala Phe
Ile Lys Lys Ile Asn Glu Ala 180 185
190 Tyr Asp Lys Val Lys Ser Lys Cys Asn Asp Ile Lys Asn Asp
Leu Ile 195 200 205
Ala Thr Ile Lys Lys Leu Glu His Pro Tyr Asp Ile Asn Asn Lys Asn 210
215 220 Asp Asp Ser Tyr Arg
Tyr Asp Ile Ser Glu Glu Ile Asp Asp Lys Ser 225 230
235 240 Glu Glu Thr Asp Asp Glu Thr Glu Glu Val
Glu Asp Ser Ile Gln Asp 245 250
255 Thr Asp Ser Asn His Ala Pro Ser Asn Lys Lys Lys Asn Asp Leu
Met 260 265 270 Asn
Arg Ala Phe Lys Lys Met Met Asp Glu Tyr Asn Thr Lys Lys Lys 275
280 285 Lys Leu Ile Lys Cys Ile
Lys Asn His Glu Asn Asp Phe Asn Lys Ile 290 295
300 Cys Met Asp Met Lys Asn Tyr Gly Thr Asn Leu
Phe Glu Gln Leu Ser 305 310 315
320 Cys Tyr Asn Asn Asn Phe Cys Asn Thr Asn Gly Ile Arg Tyr His Tyr
325 330 335 Asp Glu
Tyr Ile His Lys Leu Ile Leu Ser Val Lys Ser Lys Asn Leu 340
345 350 Asn Lys Asp Leu Ser Asp Met
Thr Asn Ile Leu Gln Gln Ser Glu Leu 355 360
365 Leu Leu Thr Asn Leu Asn Lys Lys Met Gly Ser Tyr
Ile Tyr Ile Asp 370 375 380
Thr Ile Lys Phe Ile His Lys Glu Met Lys His Ile Phe Asn Arg Ile 385
390 395 400 Glu Tyr His
Thr Lys Ile Ile Asn Asp Lys Thr Lys Ile Ile Gln Asp 405
410 415 Lys Ile Lys Leu Asn Ile Trp Arg
Thr Phe Gln Lys Asp Glu Leu Leu 420 425
430 Lys Arg Ile Leu Asp Met Ser Asn Glu Tyr Ser Leu Phe
Ile Thr Ser 435 440 445
Asp His Leu Arg Gln Met Leu Tyr Asn Thr Phe Tyr Ser Lys Glu Lys 450
455 460 His Leu Asn Asn
Ile Phe His His Leu Ile Tyr Val Leu Gln Met Lys 465 470
475 480 Phe Asn Asp Val Pro Ile Lys Met Glu
Tyr Phe Gln Thr Tyr Lys Lys 485 490
495 Asn Lys Pro Leu Thr Gln 500
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