Malaria pre-erythrocytic antigens as a fusion polypeptide and their use in the elicitation of a protective immune response in a host
20210187089 · 2021-06-24
Inventors
- Rogerio AMINO (BAGNEUX, FR)
- Pierre Charneau (Paris, FR)
- Catherine Blanc (Chantilly, FR)
- Vanessa LAGAL (FONTENAY SOUS BOIS, FR)
Cpc classification
C12N2740/15043
CHEMISTRY; METALLURGY
C12N2740/16043
CHEMISTRY; METALLURGY
C12N2760/20243
CHEMISTRY; METALLURGY
C12N2740/15034
CHEMISTRY; METALLURGY
A61K39/015
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C12N15/86
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to chimeric Plasmodium antigenic polypeptides derived from pre-erythrocytic (PE) antigens and associated in a fusion polypeptide. In particular, the invention relates to antigenic fusion polypeptides of malaria parasites wherein said antigenic polypeptides exhibit a protective effect, especially that of eliciting a protective immune response in a host against challenge by Plasmodium sporozoites or a sterile response. Such identified antigenic fusion polypeptides may thus constitute active ingredients suitable for the design of a vaccine candidate, in particular a vaccine suitable for a human host.
Claims
1. A chimeric antigenic polypeptide which comprises or consists of a fusion of at least 2, in particular of 2, 3, or 4 antigens of a Plasmodium parasite infecting human or antigenic fragments: (a) wherein said antigens are antigens of a Plasmodium parasite, and are selected from the group of antigens designated as TRAP, 18-10 (ICP), 11-10 (Ag45) and 11-09 (Ag40) and, (b) when the chimeric antigenic polypeptide comprises antigenic fragments of antigens instead of such antigens as defined in (a), such antigenic fragments comprise T cell epitopes in particular CD8+ T cell epitopes and are either truncated antigens or antigenic fragments with amino acid deletion(s) in particular said antigenic fragments contain respectively: v. the 18-10 antigen devoid of its signal peptide, and/or or devoid of 1 to 15, in particular 1 to 10 or 1 to 3 amino acid residues, vi. the C-terminal fragment of the 11-10 antigen, and/or a fragment thereof deleted from 1 to 3 amino acid residues in its N-terminal end and/or its C-terminal end, vii. the N-terminal fragment of the TRAP antigen, and/or a fragment thereof deleted from 1 to 3 amino acid residues in its N-terminal end, viii. a fragment of the 11-09 antigen deleted from 1 to 6 amino acid residues, in particular at its N-terminal end.
2. The chimeric antigenic polypeptide according to claim 1 which comprises or consists of a fusion of antigens or antigenic fragments thereof wherein the antigens and the antigens suitable to provide such fragments are as follows: a. at least 3, in particular 3 antigens of Plasmodium parasite wherein at least 2 of such antigens are selected from the group of antigens designated as TRAP, 18-10 (ICP), 11-10 (Ag45) and 11-09 (Ag40) and the antigens further include CSP, in particular wherein the antigens include CSP and TRAP or b. at least 4, in particular 4 antigens of Plasmodium parasite wherein at least 3 of such antigens are selected from the group of antigens designated as TRAP, 18-10 (ICP), 11-10 (Ag45) and 11-09 (Ag40) and the antigens further include CSP, in particular wherein the antigens include CSP and TRAP or c. at least 5, in particular of 5 antigens of Plasmodium parasite which are TRAP, 18-10 (ICP), 11-10 (Ag45), 11-09 (Ag40) and CSP.
3. The chimeric antigenic polypeptide according to claim 1 or 2, whose primary structure results from a junction, in particular a fusion of the following antigens and/or fragments thereof: i. the TRAP antigen, the 18-10 antigen devoid of its signal peptide, the C-terminal fragment of the 11-10 antigen, and antigen 11-09 (Ag40) in any order of fusion or ii. the CSP antigen, the TRAP antigen, the 18-10 antigen devoid of its signal peptide, the C-terminal fragment of the 11-10 antigen, and antigen 11-09 (Ag40) in any order of fusion.
4. The chimeric antigenic polypeptide according to any one of claims 1 to 3 wherein the amino acid sequence of the antigens of Plasmodium parasite and/or antigenic fragments as defined in claim 1(a) and (b) consists of: 1. the amino acid sequence of SEQ ID No. 96 or SEQ ID No. 98 or SEQ ID No. 100, or SEQ ID No. 114 or the fusion of the amino acid sequences SEQ ID No. 96 and 98 for the 18-10 antigenic fragment fused to, 2. the amino acid sequence of SEQ ID No. 102 or SEQ ID No. 104 for the 11-10 antigenic fragment fused to, 3. the amino acid sequence of SEQ ID No. 106 or SEQ ID No. 108 for the TRAP antigenic fragment fused to, 4. the amino acid sequence of SEQ ID No. 110 or SEQ ID No. 112 for the 11-09 antigen, wherein said fragments are devoid or an initial Methionine residue when they feature an internal fusion partner and optionally are devoid of the 1 to 6, or 1 to 5, in particular the 1 to 3 N-terminal residues of the original antigen and further optionally wherein said fragments contain 1 to 3 additional amino acid residues and/or deleted amino acid residues in their N- and/or C-terminal end to enable the junction with the contiguous antigen or antigenic fragment.
5. The chimeric antigenic polypeptide according to any one of claims 1 to 4 wherein the amino acid sequence: i. contains 1 to 3 additional junctional amino acid residues at the junction of SEQ ID No. 104 for the 11-10 antigenic fragment and SEQ ID No. 108 for the TRAP antigen, in particular contains one amino acid residue such as the Glutamic acid residue at said junction or, ii. contains a deletion of 1 to 6 or 1 to 5 amino acid residues present in the N-terminal end of the original antigen or antigenic fragment thereof present in the fusion such as the deletion of MA contiguous amino acid residues or MANG contiguous amino acid residues.
6. The chimeric antigenic polypeptide according to any one of claims 1 to 5 wherein all the antigens or antigenic fragments thereof are from Plasmodium falciparum or from Plasmodium vivax.
7. The chimeric antigenic polypeptide according to any one of claims 1 to 6 whose amino acid sequence is SEQ ID No. 116 or SEQ ID No. 120, or SEQ ID No. 122 (for Plasmodium berghei) or SEQ ID No. 118 (for Plasmodium falciparum).
8. A nucleic acid molecule encoding the chimeric antigenic polypeptide according to any one of claims 1 to 7, in particular a DNA or a RNA.
9. A nucleic acid molecule according to claim 8, which is a mammalian codon-optimized, in particular a human codon-optimized sequence for expression in mammalian, respectively human cells.
10. A nucleic acid molecule according to claim 9 whose nucleotide sequence is SEQ ID No. 115 or SEQ ID No. 119 or SEQ ID No. 121 (for Plasmodium berghei) or SEQ ID No. 117 (for Plasmodium falciparum).
11. A vector, in particular a viral vector expressing the chimeric antigenic polypeptide according to any one of claims 1 to 7, in particular an Herpes virus-based vector such as a CMV-based vector, a pox virus-based vector such as a canarypox-based vector, a vaccinia-based vector, flavivirus-based vector, adenovirus-based vector, a MVA-based vector, a Measles virus-based vector, Yellow fever virus-based vector or a VSV-based vector.
12. A vector according to claim 11 which is a lentiviral vector, in particular a HIV-based vector, more particularly a HIV1-based vector.
13. A combination or a composition of compounds, comprising at least one active ingredient that consists of a chimeric antigenic polypeptide of a Plasmodium parasite that is a fusion of at least 2, in particular 3 or 4 antigens of Plasmodium parasite or antigenic fragments as defined in any one of claims 1 or 3 to 7, a polynucleotide encoding such chimeric antigenic polypeptide according to any one of claims 8 to 10, or a vector according to claim 11 or 12, in particular a viral vector, especially a lentiviral vector, expressing such chimeric antigenic polypeptide of a Plasmodium parasite in association with respectively at least one further antigen of a Plasmodium parasite, a nucleic acid encoding such further antigen of a Plasmodium parasite or a vector encoding such further antigen of a Plasmodium parasite and optionally a vehicle for administration to a mammalian host, especially a human host.
14. A combination or a composition of compounds according to claim 13, wherein the chimeric antigenic polypeptide is as defined in any one of claims 1 or 2 to 7 or the nucleic acid is according to any one of claims 8 to 10 or a vector according to claim 11 or 12 and the further antigenic polypeptide or respectively nucleic acid or vector encoding same is the circumsporozoite protein (CSP) or a polypeptidic derivative thereof provided the polypeptidic derivative keeps essentially the protective properties of the CSP antigen from which it derives.
15. A combination or a composition of compounds according to any one of claims 13 to 14, wherein the distinct active ingredients of Plasmodium are provided as separate active ingredients or as separate compositions of multiple active ingredients in the combination of compounds, in particular as separate doses of active ingredients for administration to the host.
16. A combination of compounds according to any one of claims 13 to 15, wherein the distinct active ingredients of Plasmodium are provided as a single composition.
17. A combination or a composition of compounds according to any one of claims 13 to 16, wherein the active ingredients consist of antigens and/or chimeric antigenic polypeptides of antigens of a human-infecting Plasmodium parasite or consist of nucleic acids, in particular DNA or RNA, encoding such antigens or chimeric antigenic polypeptides or consist of lentiviral vector(s) expressing antigens and/or chimeric antigenic polypeptides of a human-infecting Plasmodium parasite, or consist in a mixture or an association of such antigens and/or chimeric antigenic polypeptides and viral vectors, in particular wherein the Plasmodium parasite is Plasmodium falciparum or Plasmodium vivax, more preferably Plasmodium falciparum.
18. A combination or a composition of compounds according to any one of claims 13 to 17, wherein the active ingredients comprise circumsporozoite protein (CSP) characterized by the sequence of SEQ ID No. 11, 12, 14, 15, 17 or 18. or a variant thereof or a polypeptidic derivative thereof which consists of a sequence with at least 70% of amino acid identity, preferably at least 95% of amino acid identity with the CSP antigen from which it derives and which keeps the protective properties of the polypeptide from which it derives.
19. A composition suitable for administration to a host, which comprises: i. a vector, in particular a lentiviral vector expressing a chimeric polypeptide as defined in any one of claims 1 to 7 or which comprises in its genome a nucleic acid of any one of claims 8 to 10 or, ii. a nucleic acid encoding a chimeric polypeptide as defined in any one of claims 1 to 7 such as a nucleic acid which is a DNA or a RNA according to any one of claims 8 to 10.
20. A combination or a composition of compounds according to claim 17 which is a combination or a composition of viral vectors, in particular lentiviral vectors, wherein one vector expresses the chimeric antigenic polypeptide as defined in any one of claims 1 to 7, in particular is a vector comprising in its genome the nucleic acid sequence of claim 8, 9 or 10, and a distinct vector expresses the CSP antigen, in particular the CSP antigen of SEQ ID No. 11, 12, 14, 15, 17, 18 or 124.
21. A combination or a composition of compounds according to any one of claim 13 to 18 or 20, or a composition according to claim 18 wherein the active ingredients comprise or consist of human lentiviral vector(s) expressing the antigens, chimeric antigenic polypeptides, in particular HIV-1 lentiviral vector(s), wherein the antigens and chimeric antigenic polypeptides are expressed either individually from separate vectors and/or from a single vector.
22. A combination or a composition of compounds or a composition according to claim 21 wherein each lentiviral vector is a replication-incompetent pseudotyped lentiviral vector, in particular a replication-incompetent pseudotyped HIV-1 lentiviral vector, wherein said vector contains a genome comprising a mammal codon-optimized synthetic nucleic acid, in particular a human-codon optimized synthetic nucleic acid, wherein said synthetic nucleic acid encodes the antigen(s) and/or the chimeric antigenic polypeptide(s) of a Plasmodium parasite infecting a mammal, in particular a human host.
23. A combination or a composition of compounds or a composition according to claim 21 or 22 wherein each lentiviral vector is an integrative and/or a non-integrative pseudotyped lentiviral vector, in particular an integrative HIV-1 vector, wherein said vector contains a genome comprising a mammal-codon optimized synthetic nucleic acid, in particular a human-codon optimized synthetic nucleic acid, wherein said synthetic nucleic acid encodes the antigens and/or the chimeric antigenic polypeptide(s) of a Plasmodium parasite infecting a mammal, in particular a human host.
24. A combination or a composition of compounds or a composition according to any one of claims 20 to 23 wherein the genome of the lentiviral vector genome is obtained from the pTRIP vector plasmid wherein the Plasmodium synthetic nucleic acid encoding the antigenic polypeptide or polypeptidic derivative thereof has been cloned under control of a promoter functional in mammalian cells, in particular a promoter of the MHC class 1 group in particular the human beta-2 microglobulin promoter, and optionally wherein the vector comprises the post-transcriptional regulatory element of the woodchuck hepatitis virus (WPRE).
25. A combination or a composition of compounds or a composition according to any one of claims 20 to 24 wherein the lentiviral vector is pseudotyped with the glycoprotein G from a Vesicular Stomatitis Virus (VSV-G) such as a VSV-G of a VSV chosen among the group of Carajas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahan virus (ISFV), Maraba virus (MARAV), Piry virus (PIRYV), Vesicular stomatitis Alagoas virus (VSAV), Vesicular stomatitis Indiana virus (VSIV) and Vesicular stomatitis New Jersey virus (VSNJV) and/or stains provisionally classified in the vesiculovirus genus as Grass carp rhabdovirus, BeAn 157575 virus (BeAn 157575), Boteke virus (BTKV), Calchaqui virus (CQIV), Eel virus American (EVA), Gray Lodge virus (GLOV), Jurona virus (JURV), Klamath virus (KLAV), Kwatta virus (KWAV), La Joya virus (LJV), Malpais Spring virus (MSPV), Mount Elgon bat virus (MEBV), Perinet virus (PERV), Pike fry rhabdovirus (PFRV), Porton virus (PORV), Radi virus (RADIV), Spring viremia of carp virus (SVCV), Tupaia virus (TUPV), Ulcerative disease rhabdovirus (UDRV) and Yug Bogdanovac virus (YBV), in particular with the glycoprotein G from a Vesicular Stomatitis Virus (VSV-G) of Indiana or of New-Jersey serotype.
26. A combination of compounds or a composition according to any one of claims 20 to 25, wherein the nucleic acid molecules which encodes the CSP antigen is a mammalian codon-optimized synthetic nucleic acids characterized by the following sequences: SEQ ID No. 10 or SEQ ID No. 124 for CSP of P. berghei, or, SEQ ID No. 13 for CSP of P. falciparum or, SEQ ID No. 16 for CSP of P. vivax.
27. A formulation suitable for administration to a mammalian host comprising a combination of compounds or a composition according to any one of claims 20 to 26 as active ingredient for protection against a Plasmodium infection or against the parasite-induced condition or disease, together with excipient(s) suitable for administration to a host in need thereof, in particular a human host.
28. A combination or a composition of compounds or a composition according to any one of claims 20 to 27, for use in the protective immunisation against malaria parasite infection or against parasite-induced condition or disease, in a mammalian host, especially a human host, optionally in association with an appropriate delivery vehicle and optionally with an adjuvant component and/or with an immunostimulant component.
29. A combination of compounds according to claim 28 for use in the protective immunisation against malaria parasite infection or against parasite-induced condition or disease, in a mammalian host, especially a human host, optionally in association with an appropriate delivery vehicle and optionally with an adjuvant component and/or with an immunostimulant component, wherein the compounds are suitable for a heterologous prime boost regimen wherein the compounds for the prime administration are selected in order to elicit at least a humoral response and the compounds for the boost administration are selected to elicit at least a cellular immune response or vice versa.
30. A combination of compounds according to claim 29 wherein the compounds for the prime and boost administration are such that: a. all the compounds are lentiviral particles wherein the lentiviral particles express different antigens or antigenic fragments thereof as defined in any one of claims 1 to 7 b. the compounds for prime administration are lentiviral particles expressing antigens or antigenic fragments thereof as defined in any one of claims 1 to 7 and the compounds for the boost are not lentiviral particles and are selected among viral vectors or nucleic acids expressing antigens or antigenic fragments thereof as defined in any one of claims 1 to 7 which are identical or different from the antigens or antigenic fragments of the prime or c. the compounds for prime administration are not lentiviral particles and are selected among viral vectors or nucleic acids expressing antigens or antigenic fragments thereof as defined in any one of claims 1 to 7 and the compounds for the boost are lentiviral particles that express antigens or antigenic fragments thereof as defined in any one of claims 1 to 7 which are identical or different from the antigens or antigenic fragments of the prime or d. the compounds for prime administration are not lentiviral particles and are selected among viral vectors or nucleic acids expressing antigens or antigenic fragments thereof as defined in any one of claims 1 to 7 and the compounds for the boost are not lentiviral particles and are selected among viral vectors and nucleic acids expressing antigens or antigenic fragments thereof as defined in any one of claims 1 to 7 wherein said vector or nucleic acid in prime and boost are identical or different and the antigens or antigenic fragments of the prime and boost are different.
31. A combination or a composition of compounds or a composition according to any one of claims 13 to 30, for use in the protective immunisation against malaria parasite infection or against parasite-induced condition or disease, in a mammalian host, especially in a human host, said use involving an immunisation pattern comprising administering an effective amount of the compounds, in particular of the lentiviral vector(s), to prime the cellular immune response of the host and later in time administering an effective amount of the compounds, in particular of the lentiviral vector(s), to boost the cellular immune response of the host, and optionally repeating said administration step for boosting, wherein the compounds, in particular the lentiviral vector(s), administered in each of the priming or boosting steps are pseudotyped with distinct envelope protein(s) which do not cross-seroneutralise, and wherein said priming and boosting steps are separated in time by at least 6 weeks, in particular by at least 8 weeks.
32. A combination or a composition of compounds or a composition according to any one of claims 27 to 31, for use in the protective immunisation against malaria parasite infection or against parasite-induced condition or disease in a mammalian host, especially in a human host, in a dosage regimen comprising separately provided doses of the compounds, in particular of the lentiviral vector(s), wherein the dose intended for priming the cellular immune response is a moderate dose and the dose intended for boosting the cellular immune response is higher than the dose for priming.
33. A combination or a composition of compounds or a composition according to any one of claim 27 or 32, for use in the protective immunisation against malaria parasite infection or against parasite-induced condition or disease in a mammalian host, especially in a human host, in a dosage regimen comprising separately provided doses of the lentiviral vector(s) wherein the dose intended for priming and the dose intended for boosting the cellular immune response each comprises from 10.sup.5 to 10.sup.9 TU (Transcription Unit), in particular from 10.sup.5 to 10.sup.8 TU when integrative-competent vector particles are used and the dose intended for priming and for boosting the cellular immune response comprises from 10.sup.7 to 10.sup.10 TU when integrative-incompetent vector particles are used.
34. Use of a combination or a composition of compounds or a composition as defined in any one of claims 12 to 31, in the manufacture of an administration form for injection to a mammalian host wherein the administration form comprises a dose of said compounds enabling protective immunisation against malaria parasite infection or against parasite-induced condition or disease in a mammalian host, especially in a human host.
Description
LEGENDS OF THE FIGURES
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[0245] Amino acid sequences of protective orthologous antigens from rodent-infecting P. berghei, macaque-infecting P. cynomolgi, and human-infecting P. falciparum and P. vivax parasites were aligned by MUltiple Sequence Comparison by Log-Expectation (MUSCLE). Vertical black bars represent identical amino acids conserved in the four plasmodial species, short dark gray bars represent repetitive regions and short light gray bars, insertional gaps used for the alignment.
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[0248] TU of non-concentrated VSV.sup.NJ B2M LPs in the first immunization/1×10.sup.7 TU of non-concentrated VSV.sup.IND B2M LPs in the second immunization) of the control antigen AL11-luciferase (Luc, white triangles), with the individual dose of CSP plus 6×Luc (gray triangles), or with the individual doses of CSP and of 6 conserved PE antigens (11-05, 11-06, 11-07, 11-09, 11-10 and 18-10; black triangles, 7cPEAg). 4 weeks after the second immunization dose, mice were challenged with 5,000 GFP SPZs. Both graphs show the individual log of parasitemia at day 5 post-challenge. (a) The graph shows the pooled results of three independent experiments. Number of sterile protected/challenged mice: 7×Luc (0/21), 1×CSP 6×Luc (1/20) and 1×7cPEAg (18/21). (b) Three and one day before sporozoite challenge, 1×7cPEAg immunized mice were injected with 400 pg of control (Ctr), CD4-depleting (a-CD4+, clone GK1.5) and CD8-depleting (a-CD8+, clone 2.43) monoclonal antibodies. GFP data comes from experiment showed in
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[0253] The sequence of the plasmid is constituted by the following functional regions wherein the cis-active lentiviral regions are derived from the HIV genome, and the promoter driving the expression of the protein (GFP) is CMV:
[0254] The insert in the plasmid that provides the vector genome is composed as follows: LTR-ψ-RRE-cPPT/CTS-CMV-GFP-WPRE-ΔU3LTR, wherein
LTR is Long Terminal Repeat
[0255] Psi (ψ) is Packaging signal
RRE is Rev Responsive Element
[0256] CMV is Immediate early CytoMegaloVirus promoter
cPPT is central PolyPurine Tract, and wherein the nucleotide segment from
cPPT to CTS forms the flap sequence
CTS is Central Termination Sequence
[0257] WPRE is Woodchuck hepatitis virus Post Regulatory Element
[0258] The nucleotide sequence is provided as SEQ ID No. 1
[0259]
[0260] The insert in the plasmid that provides the vector genome is composed as follows:
[0261] LTR-ψ-RRE-cPPT/CTS-B2M-GFP-WPRE-ΔU3LTR.
[0262] The nucleotide sequence is provided as SEQ ID No. 2.
[0263]
[0264] The plasmid provides the required GAG and POL coding sequences of the HIV-1 lentivirus under the control of the CMV promoter.
[0265] The nucleotide sequence is provided as SEQ ID No. 3.
[0266]
[0267] The envelope protein is the VSV-G of the Indiana strain and the coding sequence has been mouse-codon optimized.
[0268] The nucleotide sequence is provided as SEQ ID No. 4.
[0269]
[0270] The envelope protein is the VSV-G of the New-Jersey strain and the coding sequence has been mouse-codon optimized.
[0271] The nucleotide sequence is provided as SEQ ID No. 5.
[0272]
[0274] 4cPEAg refers to the combination of PbTRAP, antigen Pb18-10, antigen Pb11-10 and antigen Pb11-09. Fusion 4cPEAg refers to the chimeric antigen formed by the fus of the antigen Pb18-10 without its signal peptide (SP), followed by the protective domains 11-10CT and TRAPNT, and the antigen Pb11-09. GPI (glycosylphosphatidylinositol), TSR (thrombospondin type I repeat). [0275] (b) Four weeks-old C57BL/6 mice (n=6-7 per group) were acclimated for 3 weeks and immunized intraperitoneally with 5×10.sup.5 TU/antigen using non-concentrated VSV.sup.NJ B2M LPs. Four weeks after the first immunization, all groups received intraperitoneally 3×10.sup.7 TU per respective antigen of concentrated VSV.sup.IND B2M LPs. Four weeks after the second immunization, mice were challenged with the microinjection of 5,000 GFP PbSPZs in the footpad. The graph shows the log of parasitemia of individual mouse at day 5 post-challenge. Traces represent the mean of the log parasitemia. The superior and inferior dotted lines delineate the 95% tolerance interval (grey box) of the control group established in the experiment of
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[0281] Epitopes from the Pf 4cPEAgs predicted to bind to the Human Leukocyte Antigen (HLA) were identified using the immune epitope database (iedb; www.iedb.org). (a-c) Bars represent epitopes predicted to bind on the HLA-DRB1*01:01, *03:01, *04:01, *04:05, *07:01, *08:02, *09:01, *11:01, *12:01, *13:02 and *15:01. Triangles represent epitopes predicted to bind to the HLA A*01:01, *02:01, *02:03, *02:06, *03:01, *11:01, *23:01,*24:02, *26:01, *30:01, *30:02, *31:01, *32:01, *33:01, *68:01 and *68:02. Inverted triangles represent epitopes predicted to bind to the HLA-B*07:02, *08:01, *15:01, *35:01, *40:01, *44:02, *44:03, *51:01, *53:01, *57:01 and *58:01. White horizontal bars represent the regions used to design the Pf 4cPEAg fusion based on the content of class I and II predicted epitopes and structural/sequence similarity with the protective domains tested using P. berghei. Gray shadows represent conserved structural domains depicted in the
[0282]
[0283] The following table provides the list and identification of the sequences contained in the sequence listing.
TABLE-US-00001 SEQ ID No. Sequence designation Origin Type 1 pTRIP CMV GFP DNA 2 pTRIP B2M GFP DNA 3 PACKAGING 8.74 PLASMID DNA 4 pCMV-VSV-INDco DNA 5 pCMV-VSV-Njco DNA 6 eGFP DNA 7 eGFP protein protein 8 AL11-Luciferase protein 9 AL11-Luciferase protein 10 circumsporozoite (CS) protein P. berghei DNA (CSP) mouseCO + Kozak ANKA strain 11 PbCSP (mouseCO + Kozak) P. berghei protein ANKA strain 12 PbCSP P. berghei protein ANKA strain 13 PfCSP humanCO + Kozak P falciparum DNA 3D7 strain 14 PfCSP (humanC0 + Kozak) P falciparum protein 3D7 strain 15 PfCSP P falciparum protein 16 PvCSP humanCO + Kozak P vivax Sal-1 DNA strain 17 PvCSP (humanCO + Kozak) P vivax Sal-1 protein strain 18 PvCSP P vivax Sal-1 protein strain 19 thrombospondin-related P. berghei DNA anonymous protein ANKA strain (PbTRAP) mouseCO + Kozak 20 PbTRAP (mouseCO + Kozak) P. berghei protein ANKA strain 21 PbTRAP P. berghei protein ANKA strain 22 PfTRAP humanCO + Kozak P falciparum DNA 3D7 strain 23 PfTRAP (humanCO + Kozak) P falciparum protein 3D7 strain 24 PfTRAP P falciparum protein 25 PvTRAPhumanCO P vivax Sal-1 DNA strain 26 PvTRAP P vivax Sal-1 protein strain 27 PvTRAP P vivax protein 28 inhibitor of cysteine proteases (ICP) P. berghei DNA mouseCO + Kozak ANKA strain 29 PbICP (mouseCO + Kozak) P. berghei protein ANKA strain 30 PbICP P. berghei protein ANKA strain 31 PfICP humanCO P falciparum DNA 3D7 strain 32 PfICP P falciparum protein 3D7 strain 33 PfICP P falciparum protein 34 PvICP humanCO + Kozac P vivax Sal-1 DNA strain 35 PvICP (humanCO + Kozac) P vivax Sal-1 protein strain 36 PvICP P vivax protein 37 Bergheilysin-A-mouseCO + P. berghei DNA Kozak ANKA strain 38 Bergheilysin-A (1-777, P. berghei protein mouse CO + Kozak) ANKA strain 39 Bergheilysin entire ORF (1-1149) P. berghei protein ANKA strain 40 Falcilysin human CO + Kozak P falciparum DNA 3D7 strain 41 Falcilysin (human CO + Kozak) P falciparum protein 3D7 strain 42 Falcilysin P falciparum protein 3D7 strain 43 PvFalcilysin human CO + Kozak P vivax Sal-1 DNA strain 44 PvFalcilysin (humanCO + Kozak) P vivax Sal-1 protein strain 45 PvFalcilysin P vivax Sal-1 protein strain 46 Bergheilysin-B- mouseCO + P. berghei DNA Kozak + signal peptide (SP) ANKA strain 47 Bergheilysin-B (SP + 778-1149, P. berghei protein mouse CO + Kozak) ANKA strain 48 perforin like protein 1 (SPECT2) P. berghei DNA mouseCO + Kozak ANKA strain 49 PbSPECT2 (mouseCO + Kozak) P. berghei protein ANKA strain 50 PbSPECT2 P. berghei protein ANKA strain 51 PfSPECT2 human CO + Kozak P falciparum DNA 3D7 strain 52 PfSPECT2 (humanCO + Kozak) P falciparum protein 3D7 strain 53 PfSPECT2 P falciparum protein 54 PvSPECT2 human CO + Kozak P vivax Sal-1 DNA strain 55 PvSPECT2 (human CO + Kozak) P vivax Sal-1 protein strain 56 PvSPECT2 P vivax protein 57 GPI_P113 mouseCO + Kozak P. berghei DNA ANKA strain 58 Pb GPI_P113 (mouseCO + Kozak) P. berghei protein ANKA strain 59 Pb GPI_P113 P. berghei protein ANKA strain 60 PfP113 human CO + Kozak P falciparum DNA 3D7 strain 61 PfP113 (human CO + Kozak) P falciparum protein 3D7 strain 62 P113 P falciparum protein 63 PvP113 human CO + Kozak P vivax Sal-1 DNA strain 64 PvP113 (human CO + Kozak) P vivax Sal-1 protein strain 65 P113 P vivax protein 66 PbAg40 mouse CO + Kozak P. berghei DNA ANKA strain 67 PbAg40 (mouse CO + Kozak) P. berghei protein ANKA strain 68 PbAg40 P. berghei protein ANKA strain 69 PfAg40 human CO + Kozak P falciparum DNA 3D7 strain 70 PfAg40 (human CO + Kozak) P falciparum protein 3D7 strain 71 Ag40 P falciparum protein 72 PvAg40 human CO + Kozak P vivax Sal-1 DNA strain 73 PvAg40 (human CO + Kozak) P vivax Sal-1 protein strain 74 PvAg40 P vivax Sal-1 protein strain 75 PbAg45 mouse CO + Kozak P. berghei DNA ANKA strain 76 PbAg45 (mouse CO + Kozak) P. berghei protein ANKA strain 77 PbAg45 P. berghei protein ANKA strain 78 PfAg45 human CO + Kozak P falciparum DNA 3D7 strain 79 PfAg45 (human CO + Kozak) P falciparum protein 3D7 strain 80 PfAg45 P falciparum protein 81 PvAg45 human CO + Kozak P vivax Sal-1 DNA strain 82 PvAg45 (human CO + Kozak) P vivax Sal-1 protein strain 83 PvAg45 P vivax protein 84 Kozak consensus sequence DNA 85 Kozak consensus sequence DNA 86 BamHI site DNA 87 Xhol site DNA 88-94 CD8 T cell epitopes protein
DNA and Amino Acid Sequences Used in the Chimeric Fusion Antigenic Polypeptides for P. berghei and P. falciparum
TABLE-US-00002 SEQ ID N. Description of sequence 95 DNA sequence of PD Plasmodium berghei ANKA 18-10NT— mouse codon optimized, with adaptors and Kozak sequence (372 bp) 96 Amino acid sequence of PD Plasmodium berghei ANKA 18-10NT (117 aa) 97 DNA sequence of PD Plasmodium berghei ANKA 18-10CT— mouse codon optimized, with adaptors and Kozak sequence (528 bp). 98 Amino acid sequence of PD Plasmodium berghei ANKA 18-10CT (169 aa) 99 DNA sequence of PD Plasmodium falciparum 3D7 18-10 minus Signal Peptide (SP.sup.−), human codon optimized, with adaptors and Kozak sequence (1197 bp). 100 Amino acid sequence of PD Plasmodium falciparum 3D7 18-10-SP (392 aa) 101 DNA sequence of PD Plasmodium berghei ANKA 11-10CT— mouse codon optimized, with adaptors and Kozak sequence (528 bp). 102 Amino acid sequence of PD Plasmodium berghei ANKA 11-10CT (169 aa) 103 DNA sequence of PD Plasmodium falciparum 3D7 11-10CT— human codon optimized, with adaptors and Kozak sequence (561 bp). 104 Amino acid sequence of PD Plasmodium falciparum 3D7 11-10CT (180 aa) 105 DNA sequence of PD Plasmodium berghei ANKA TRAP NT— mouse codon optimized, with adaptors and Kozak sequence (747 bp). 106 Amino acid sequence of PD Plasmodium berghei ANKA TRAP NT (242 aa) 107 DNA sequence of PD Plasmodium falciparum 3D7 TRAP NT—human codon optimized, with adaptors and Kozak sequence (903 bp). 108 Amino acid sequence of PD Plasmodium falciparum 3D7 TRAP NT (294 aa) 109 DNA sequence of PD Plasmodium berghei ANKA 11-09— mouse codon optimized, with adaptors and Kozak sequence (654 bp). 110 Amino acid sequence of PD Plasmodium berghei ANKA 11-09 (211 aa) 111 DNA sequence of PD Plasmodium falciparum 3D7 11-09— human codon optimized, with adaptors and Kozak sequence (642 bp). 112 Amino acid sequence of PD Plasmodium falciparum 3D7 11-09 (207 aa) 113 DNA sequence of Plasmodium berghei ANKA Fusion of PDPb18-10NT and PD Pb18-10CT—mouse codon optimized + ATG (852pb) 114 Amino acid sequence of Plasmodium berghei ANKA Fusion of PD Pb18-10NT and PD Pb18-10CT (284aa) 115 DNA sequence of Plasmodium berghei ANKA Fusion 4cPEAg—mouse codon optimized, with adaptors and Kozak sequence (2715 bp). 116 Amino acid sequence of Plasmodium berghei ANKA Fusion 4cPEAg (898 aa). 117 DNA sequence of Plasmodium falciparum 3D7 Fusion 4cPEAg—human codon optimized, with adaptors and Kozak sequence (3234 bp). 118 Amino acid sequence of Plasmodium falciparum 3D7 Fusion 4cPEAg (1070 aa) 119 DNA sequence of Plasmodium berghei Fusion 5cPEAg— mouse codon optimized, with adaptors and Kozak sequence (3597 bp). 120 Amino acid sequence of Plasmodium berghei Fusion 5cPEAg SP.sup.− (1192 aa) 121 DNA sequence of Plasmodium berghei Fusion 5cPEAg SP.sup.+—mouse codon optimized, with adaptors and Kozak sequence (3663 bp). 122 Amino acid sequence of Plasmodium berghei Fusion 5cPEAg SP.sup.+ (1214 aa) 123 DNA sequence of Plasmodium berghei ANKA CSP— mouse codon optimized, with adaptors and Kozak sequence (1044 bp). 124 Amino acid sequence of Plasmodium berghei ANKA CSP (341 aa)
[0284] Additional information relating to some of the sequences disclosed in the above table are provided in the table below.
TABLE-US-00003 SEQ ID GenBank strain pubmed 15 BAM84930.1 Plasmodium falciparum isolate 23295064 Pal97-042 origin: Philippines ACO49323 Plasmodium falciparum″ isolate 19460323 A5 origin: Thailand 18 AAA29535.1 P. vivax (strain Thai; isolate 2290443 NYU Thai) origin: Thailand 24.sup.(1) EWC74605.1 Plasmodium falciparum UGT5.1 strain origin: Uganda 27 AIU97014.1 Plasmodium vivax isolate = “TMS38” origin: Thailand 36.sup.(3) KMZ87332.1 Plasmodium vivax Brazil I strain 56 KMZ82648.1 Plasmodium vivax India VII 65 KMZ78214.1 Plasmodium vivax India VII 83 KMZ90984.1 Plasmodium vivax Mauritania I .sup.(1)https://www.ncbi.nlm.nih.govhiosample/SAMN01737342 .sup.(2)https://www.ncbi.nlm.nih.gov/biosample/SAMEA2394724 .sup.(3)https://www.ncbi.nlm.nih.gov/biosample/SAMN00710434
EXAMPLES
[0285] To approach the complex problem of identifying protective antigens, the inventors devised a functional screening to identify and combine novel PE protective antigens using a rodent malaria model where mice (C57BL/6) are extremely susceptible to Plasmodium berghei (Pb) sporozoite infection. In this model, sterilizing protection induced by live irradiated sporozoites is mediated by antibodies and mainly by CD8 T cell responses against sporozoites and liver stages, respectively. The inventors' screening strategy was designed based on four main features: i) parameterized selection of 55 PE antigens based on abundance, orthology, predicted topology and function, ii) synthesis of codon-optimized antigens to avoid AT-rich plasmodial sequences and maximize the expression in mammalian cells, iii) immunization using HIV-based lentiviral vector that elicits strong humoral and cellular responses.sup.11,12, and iv) measurement of protection after a stringent challenge of sporozoites inoculated sub-cutaneously in the immunized mice.
1. Setting Up the Parameters of the Screening.
[0286] In a proof-of-concept experiment aimed at validating the viability of the strategy to screen antigens at a medium-throughput, the inventors ordered mouse-codon optimized synthetic genes of Pb CSP (SEQ ID No. 11), a known protective antigen, and of more 4 other sporozoite antigens (Celtos, SPECT, HSP20 and Ag13), which were previously correlated with protection.sup.13. The synthetic plasmodial genes were cloned in to the pTRIP vector plasmid, which drives their expression in mammalian cells via the immediate-early cytomegalovirus promoter (CMV) and the post-transcriptional regulatory element of woodchuck hepatitis virus (WPRE) (
[0287] Groups of five mice were immunized with a single intra-muscular dose of 5e7 TU of ultracentrifugation-concentrated vsv.sup.IND pseudotyped lentiviral particles (LPs). Thirty days after immunization, mice were challenged with 10,000 bioluminescent sporozoites inoculated sub-cutaneously in the footpad. Two days later, the parasite load in the liver was measured by bioluminescence. Surprisingly, CSP-immunization decreased 15×-fold the parasite load in the liver after a challenge using 10,000 bioluminescent sporozoites, versus a 5×-fold decrease in animals immunized intravenously with 50,000 irradiated sporozoites, our golden standard of protection (
[0288] The inventors next aimed at the transposition of these optimal experimental conditions to those of a larger screening. This transposition included the validation of the use of non-concentrated LPs, the choice of the best promoter driving the expression of the plasmodial antigens, and the dose of immunization.
[0289] The first parameter tested was the use of non-concentrated, instead of concentrated LPs, to avoid a costly and time consuming ultracentrifugation concentration step in the protocol of LP production, which requires large volumes of non-concentrated LP suspensions.
[0290] Next two promoters were tested to identify which one induced the best protection using the codon optimized Pb CSP. The inventors compared the use of the strong and constitutive cytomegalovirus (CMV) promoter versus a human beta-2 microglobulin (B2M) promoter, which direct gene expression in many cell types, particularly in dendritic cells.
[0291] During this period of optimization the inventors observed some variations in the CSP-induced protection using the same stock of LPs, as can be seen in the
[0292] Next, the best protective immunization dose was tested, ranging from 1×10.sup.8 to 1×10.sup.5 TU of B2M CSP non-concentrated LPs. As shown in
[0293] In summary, an immunization protocol was set up based on CSP that relied on a single intraperitoneal injection of 10.sup.7 TU of non-concentrated VSV.sup.IND B2M LP in C57BL/6 mice of 7 weeks-old, acclimated for 3 weeks in the animal facility. In the pooled data, this protocol leaded in average to a ˜5-fold decrease in the parasite liver load, as assessed by bioluminescence imaging, using a subcutaneous challenge of 5,000 luciferase-expressing SPZ.
[0294] However, this bioluminescent method of detection of parasites presents some disadvantages such as the use and associated costs of anesthesia and luminescent substrate, limited capacity of analysis of a few animals per acquisition, being time-consuming and not sensible enough to predict sterile protection. Therefore, the inventors decided to use fluorescent parasites to check protection by measuring parasitemia at day 4, 5, 6 and 10 post-inoculation by flow cytometry. The inventors analyze at least 100,000 red blood cells, which gives the sensibility to detect a parasitemia of 0.001%. At day 4 to 6, parasites grow exponentially in the blood therefore the log transform of parasitemia can be fitted using a linear regression where the slope represents the time of parasite replication per day. Consequently, the inventors use this parameter to determine if the immunization impacts the parasite growth in the blood. For quantifying protection, the inventors used the log of parasitemia at day 5 post inoculation. This represents an indirect measure of liver infection and it is more robust than the measure at day 4 because more events of infected blood cells are registered. Finally the inventors defined that immunized mice are sterile protected if infected red blood cells are not detected after 10 days post inoculation. After defining the protocol of immunization and the method for the quantification of parasite infection the inventors started to screen the protective activity of down-selected antigens.
2. Parameterized Selection of Antigens
[0295] By merging proteomic and transcriptomic data using PlasmoDB (www.plasmodb.org), the inventors identified ˜9000 genes expressed in plasmodial pre-erythrocytic stages—salivary gland sporozoites and liver-stages—of three different plasmodial species, with 3654 syntenic orthologs in Plasmodium falciparum (Pf), the most lethal human-infecting plasmodial species. By analyzing the repertoire of pathogen transcripts, as inferred by the amount of expressed sequence tags (ESTs) obtained in cDNA libraries of different stages and species of malaria parasites, the inventors have observed that ˜50% of the total amount of ESTs are coming from only ˜10% of genes represented in these libraries, corresponding to approximately 100 genes in these libraries (
3. First Screening of Protective Antigens Using a Single Dose of LPs
[0296] Usually, 6-10 plasmodial antigens were tested by experiment, with a negative (GFP) and positive (CSP) control of protection. After three weeks post-immunization, the immune-sera were tested on permeabilized and non-permeabilized sporozoites, allowing the determination of (i) the efficiency of the host humoral response and therefore the immunogenicity of the lentivirus-delivered antigen, and (ii) the localization of the parasite antigen (surface vs intracellular). As shown in the
[0297] Four weeks post-immunization the animals were challenged with 5,000 GFP-expressing sporozoites, microinjected in the footpad of immunized mice. Parasitemia was determined by flow cytometry. To define protection, parasitemia of all GFP groups (day 5 post-infection, n=35) was log transformed, pooled and the 95% tolerance interval was calculated (
[0298] In the first set of 43 antigens tested (
[0299] To verify the robustness of our screening, the inventors selected 4 protective antigens (CSP, 09-06, 10-05 and 07-03), 6 non-protective antigens (GFP, 09-07, 07-05, 07-06, 06-06 and 10-06), and instead of only one immunization dose, the inventors administered one dose of 5×10.sup.5 TU of non-concentrated VSV.sup.NJ B2M LPs and one month later, a second dose of 1×10.sup.7 TU of VSV.sup.IND B2M LPs. As shown in the
[0300] These results validated some of our protective antigens detected with a single immunization dose, but also showed that some good protective antigens were not detected in our first screen, leading to the decision of repeating the screening using two immunization doses.
4. Second Screening of Protective Antigens Using Two Doses of LP
[0301] By functionally screening the protective activity of 55 down-selected plasmodial PE antigens using the protocol of two immunization doses, the inventors identified 16 antigens that protected at least one immunized mice per group. Among these 16 antigens, 7 of them (black circles/bars in the
[0302] All of them presented a similar or an inferior protective activity when compared individually to our standard of protection, the CSP (
TABLE-US-00004 TABLE I Percent Identity Matrix created by CLUSTAL 2.1. Amino acid sequence of Pb antigen swere pBlasted against Pf and Pv taxids (organism) and the best matched sequence was used to align the orthologous proteins using MUSCLE(http://www.ebi.ac.uk/Tool/msa/muscle). The table shows the percentage of identical amino acids between species. Raw data is presented in the figures Amino acid identity (%) Antigen Pb/Pf Pb/Pv Pf/Pv 30-03 74.65 77.90 73 11-09 66.19 66.19 79.05 11-06 64.92 63.40 64.44 11-10 56.94 50.15 62.28 CSP 42.06 33.53 26.36 18-10 39.60 41.23 49.30 11-05 38.75 44.67 42.86 11-07 37.53 33.42 46.30
[0303] In a decreasing order of protection the first antigen identified is TRAP.sup.14 (thrombospondin related anonymous protein; 11-05) (SEQ ID No 20 and 21), which validated our method of screening since immunization with TRAP is known to induce protection both in rodents.sup.15 and humans.sup.16. TRAP is a type I transmembrane protein harbouring two extracellular adhesive domains, a von Willebrand factor type A domain and a thrombospondin type 1 domain, followed by a proline-rich repetitive region. TRAP is stored in micronemal secretory vesicles and following parasite activation, the protein is translocated to the surface of sporozoites where it serves as a linker between a solid substrate and the cytoplasmic motor of sporozoites. Intriguingly, anti-TRAP antibodies do not impair parasite motility and infectivity.sup.17 CD8+ T cells seem to mediate the protection mediated by TRAP immunization.sup.10,15,16,18.
[0304] The second protective antigen identified is an inhibitor of cysteine protease (ICP, 18-10).sup.19 (SEQ ID No 29, 30). ICP seems to be involved in the motility and infectivity capacity of sporozoites via the processing of CSP.sup.20,21,, as well as, in the parasite intra-hepatic development.sup.22. Although the protein does not present structural signatures of membrane localization, there is evidence that the protein is located on the surface of sporozoites.sup.19,20. Opposing results are published regarding the secretion of the protein following parasite activation.sup.21,22. Similarly, there are contradictory results regarding the inhibition of host cell invasion by sporozoites in vitro in the presence of anti-ICP immune sera.sup.20,23.
[0305] The third protective antigen identified is a metallopeptidase (Falcilysin/Bergheilysin, 30-03).sup.24 (SEQ ID No 38 for Bergheilysin A, No 47 for Bergheilysin B, and No 39 for the entire Bergheilysin ORF). This protease seems to be involved in the catabolism of hemoblobin in the parasite blood stages.sup.25. A H-2K.sup.b-restricted CD8 T cell epitope was recently described during the parasite blood infection.sup.25 suggesting that CD8 T cells could mediate the protection elicited by the antigen 30-03 during the hepatic infection.
[0306] The fourth protective antigen is a GPI-anchored protein (P113, 11-07) (SEQ ID No. 58 and 59) initially described in blood stages.sup.16 and also expressed in PE stages. P113 seems to be important for liver infection, dispensable for blood infection, but its precise function is unclear.sup.17.
[0307] The fifth antigen is the pore-forming like protein SPECT2 (11-06).sup.28 (SEQ ID No 49 and 50). This protein has a membrane attack complex/perforin (MACPF) domain and is involved in the sporozoite cell traversal activity, being important for the progression of sporozoites in the dermis.sup.29 and survival to phagocytosis in the liver.sup.30.
[0308] The sixth antigen identified is a hypothetical protein that the inventors called 11-09 or Ag40 (SEQ ID No 67 and 68). This protein has 4-5 annotated transmembrane domains. Deletion of the gene coding for the antigen 11-09 caused impairment of Pb parasite development in the liver.
[0309] The seventh antigen is also a hypothetical protein that the inventors called 11-10 or Ag45 (SEQ ID No 76 and 77). This protein doesn't have annotated domains, but possesses a central region with negatively charged amino acids. Recently the 11-10 ortholog of Plasmodium yoelii, another rodent-infecting plasmodial species, was also identified as a protective antigen.sup.21. The deletion of the gene coding for the antigen 11-10 blocked the Pb sporozoite invasion of salivary glands and completely abolished the capacity of sporozoites to infect the liver.
[0310] To determine if CSP based protection could be additively or synergistically improved by the combination of antigens, the inventors assessed the protection elicited by a sub-optimal dose of CSP (5×10.sup.5 TU of VSV.sup.NJ/5×10.sup.6 TU of VSV.sup.IND B2M LPs) in the absence or presence of a usual dose of protective antigens (5×10.sup.5 TU of VSV.sup.NJ/1×10.sup.7 TU of VSV.sup.IND B2M LPs). This protection induced by CSP+protective antigens was compared to the protection elicited by these antigens alone (data from
5. Identification of Multi-Antigenic Formulations Capable of Sterilizing Sporozoite Infection Via a CD8+ T Cell Response
[0311] Since testing all possible combinations of antigens was technically unfeasible, the inventors decided to evaluate the protection elicited by the combination of these multiple protective antigens. Remarkably, two immunizations of mice with the combination of CSP and 6 of these antigens (11-05, 11-06, 11-07, 11-09, 11-10, 18-10) elicited sterile protection in the vast majority of challenged animals (7PEAg, 86-100%,
[0312] The same protective efficacy was observed using only a single immunization for the 7PEAg or for the 7PEAg+30-03 (
6. Design of a Chimeric Antigen Containing the Protective Domains of Down-Selected PE Antigens
[0313] To determine a minimal antigenic composition capable of eliciting this additional protective CD8+ T cell response, the inventors first identified the antigens whose protective activity was dependent on these T cells. Protection induced by two immunizations using TRAP, 18-10 and 11-09 was significantly reduced after depletion of CD8+ cells, as shown in the
[0314] In order to combine the protective domains of each of these 5 antigens in a single chimeric molecule and thus avoid the costs associated with the production and delivery of five different antigens, the inventors mapped the protective regions of each antigen according to the localization of predicted epitopes binding to MHC class I molecules (
[0315] As shown in the
[0316] Analysis of the distribution of epitopes of Pb antigens predicted to bind to MHC class I molecules of C57BL/6 mice (H2-Kb, H2-Db) or of the Pf orthologues predicted to bind to HLA A02:01, a high prevalent human HLA allele, revealed that most of predicted good binders are clustering in the regions that are conserved among different plasmodial species (Pb, Pc, Pv and Pf,
[0317] In summary, using a parameterized selection of antigens, a screening based on lentiviral vaccination and a direct measurement of protection in vivo against a stringent sporozoite challenge, the inventors identified 8 protective antigens, including the vaccine candidates CSP and TRAP, out of 55 tested antigens. All these 8 antigens are conserved across several plasmodial species. Remarkably, immunization using a combination of seven or eight of these antigens elicited sterile protection in the vast majority of challenged mice, either using one or two immunizations. More importantly, this protection was far superior than the one elicited by CSP, so far the best protective PE antigen. Depletion of CD8+ T cells abolished sterilizing immunity, indicating that these cells are essential for this protective phenotype, similarly to the protection conferred by irradiated sporozoites. A minimal combination of 5 of these antigens was also capable of eliciting sterile protection in most of challenged animals. Mapping of the protective domains of these 5 antigens allowed the design of a chimeric antigen containing the fused protective domains of these 5 down-selected antigens. The human-infecting parasite orthologs of these protective antigens, or of their protective domains are potential candidates for being used in the development of a malaria vaccine formulation containing multiple protective antigens or multiple protective domains fused in a single molecule.
Chimeric Antigenic Polypeptide as a Fusion of Protective Domains of Plasmodium Antigens and Immunogenic Response
[0318] Immunization using a combination of CSP and 6 or 7 of screened conserved protective antigens delivered by lentiviral particles, were shown to confer sterile protection in ≈85% of mice challenged with 5,000 sporozoites (
[0319] The inventors have also shown that combination of four pre-erythrocytic conserved protective antigens (4cPEAg i.e., 4 separated antigens used in combination for administration) administered together with CSP elicited sterile protection at a similar level of that of the combination using 7 or 8 antigens (
[0320] This data confirmed the protective efficacy of the chimeric antigen prepared as a fusion of the protective domains, which satisfactorily substituted the 4cPEAg. Since predicted CD8+ T cell epitopes clustered in conserved regions of the antigens, independently of the plasmodial species and MHC class I restriction (
[0321] A further chimeric construct was designed in order to benefit from the immune response elicited by the CSP protein, taking into consideration that the lentiviral particles were shown to sustain the presence of large antigens. accordingly a fusion of 5 antigens or their protective domains was prepared, adding the CSP antigen to the fusion 4cPE Ag in a structure containing the CSP devoid, or not, of its signal peptide and devoid of its GPI followed by the PD 18-10NT and CT, the PD 11-10CT, followed by the PD TRAPNT and the antigen 11-09. The construct obtained for P. berghei has the amino acid sequence of SEQ ID No. 120 and the DNA of SEQ ID No. 121 or has the amino acid sequence of SEQ ID No. 122 and the DNA of SEQ ID No. 123. Specific amino acid residues were deleted from the original antigens where appropriate in order to preclude the formation of neo-epitopes.
MATERIAL and METHODS
[0322] Parasite strains: Plasmodium berghei ANKA strain constitutively expressing the GFP under the control of the hsp70 promoter (Ishino et al, 2006) was used in the challenges using parasitemia, quantified by flow cytometry, as protective readout. Plasmodium berghei ANKA strain constitutively expressing a GFP-luciferase fusion under the control of the eef-1alfa promoter (Franke-Fayard et al, 2008) was used in the challenges using liver infection, assessed by bioluminescence, as protective readout. Of note, parasitemia quantified using hsp70-gfp parasites was at least 10 times more sensible than using eef-1a gfp:luc parasites due to more intense expression level of GFP. [0323] Ishino T, Orito Y, Chinzei Y, Yuda M (2006) A calcium-dependent protein kinase regulates Plasmodium ookinete access to the midgut epithelial cell. Mol Microbiol 59:1175-1184. [0324] Franke-Fayard B, Djokovic D, Dooren M W, Ramesar J, Waters A P, et al. (2008) Simple and sensitive antimalarial drug screening in vitro and in vivo using transgenic luciferase expressing Plasmodium berghei parasites. Int J Parasitol 38:1651-1662.
[0325] Mouse strains: C57BL/6 Rj and Swiss mice were purchased from Elevage Janvier (France). All experiments were approved by the Animal Care and Use Committee of Institut Pasteur (CETEA 2013-0093) and were performed in accordance with European guidelines and regulations (MESR-01324).
[0326] Production of Lentiviral Particles stock: Down-selected plasmodial antigens were synthesized by Eurofins MWG as mouse codon-optimized genes with the addition of a Kozak consensus sequence (GCCACCATGGCT(C) (SEQ ID No. 85 and 86), representing the first 12 nucleotides in the coding sequences of the antigenic polypeptides), encompassing the first translated ATG. This modification adds an extra alanine after the first methionine. A BamHI (GGATCC-SEQ ID No. 87) and Xho I (CTCGAG-SEQ ID No. 88) restriction sites were also inserted in the 5′ and 3′ extremities of the construct, respectively. These synthetic codon-optimized genes were then cloned into the BamHI and Xho I restriction sites of the pTRIP plasmid harboring either the CMV or B2M promoter (
[0327] Immunization protocol: For the screening using one single dose of LPs, 4 weeks-old C57BL/6 mice (n=5 per group per experiment) were acclimated for 3 weeks and intraperitoneally immunized with a single dose of 1×10.sup.7 TU of non-concentrated VSV.sup.IND B2M LPs. For the protocol using two immunization doses. 4 weeks-old C57BL/6 mice (n=5 per group per experiment) were acclimated for 3 weeks and intraperitoneally immunized with a first dose of 5×10.sup.5 TU of non-concentrated VSV.sup.NJ B2M LPs. Thirty days after the first immunization, the animals received a second dose of 1×10.sup.7 TU of non-concentrated VSV.sup.IND B2M LPs. For testing combinations of a sub-optimal dose of CSP+ an optimal dose of down-selected antigens, mice were immunized twice, four weeks apart, with a sub-optimal dose of CSP (5×10.sup.5 TU of non-concentrated VSV.sup.NJ B2M LP in the first immunization and 5×10.sup.6 TU of non-concentrated VSV.sup.IND B2M LP in the second immunization) and the usual dose of protective plasmodial antigens (5×10.sup.5 TU of non-concentrated VSV.sup.NJ B2M LP in the first immunization and 1×10.sup.7 TU of non-concentrated VSV.sup.IND B2M LP in the second immunization). For testing the combination of multiple antigens, mice were immunized twice, four weeks apart, with 7× the individual dose (1 dose=5×10.sup.5 TU of non-concentrated VSV.sup.NJ B2M LPs in the first immunization/1×10.sup.7 TU of non-concentrated VSV.sup.IND B2M LPs in the second immunization) of the control antigen AI11-luciferase (Luc), with the individual dose of CSP plus 6 doses of Luc or with the individual doses of CSP and of the 6 conserved PE antigens (11-05, 11-06, 11-07, 11-09, 11-10 and 18-10). For this experiment the inventors used ultrafiltration and lenti-X (Clontech) concentrated stocks. The average volume of injection was 500 uL of LPs diluted in DMEM.
[0328] In all cases, thirty days after last immunization, mice were challenged with 5,000 GFP-expressing sporozoites micro-injected subcutaneously in the mice footpad.
[0329] Sporozoite challenge: Anopheles stephensi (Sda500 strain) mosquitoes were reared using standard procedures. 3-5 days after emergence, mosquitoes were fed on infected Swiss mice with a parasitemia superior to 2%, and kept as described in Amino et al, 2007. Between 20 and 23 days post-feeding, the salivary glands of infected mosquitoes were dissected in PBS, collected in 20 uL of sterile PBS on ice and disrupted using an eppendorf pestle. The suspension of parasites was filtered through a nylon mesh of 40 um, counted using Kova glasstic slide (Hycor) and adjusted to a concentration of 5,000 or 10,000 sporozoites/uL with sterile PBS. This suspension was divided in individual tubes, one for each group of immunized mice (n=4-7 per group), and kept on ice until the challenge. One microliter of parasite suspension was injected in the right footpad of mice using a Nanofil syringe (World Precision Instruments) with a 35 GA bevelled needle (NF35BV). [0330] Amino R, Thiberge S, Blazquez S, Baldacci P, Renaud O, et al. (2007) Imaging malaria sporozoites in the dermis of the mammalian host. Nat Protoc 2:1705-1712.
[0331] Measurement of Parasite Infection: Hepatic parasite loads were quantified at ˜44h by bioluminescence in fur shaved mice infected with GFP LUC parasites. Infected mice were first anesthetized with isoflurane and injected subcutaneously with D-luciferin (150 mg/kg, Caliper LifeSciences). After a 5 minutes incubation allowing the distribution of the substrate in the body of the anesthetized animals, mice were transferred to the stage of an intensified charge-coupled device photon-counting video camera box where anesthesia was maintained with 2.5% isoflurane delivered via nose cones. After 5 minutes of signal acquisition controlled by the Living Image software (Xenogen Corporation), animals were returned to their cage. Automated detection of bioluminescence signals by the system resulted in the generation of bioluminescence signal maps superimposed to the gray-scale photograph of the experimental mice. These images were then quantified using the Living Image software. Briefly, regions of interest (ROI) encompassing the liver were manually defined, applied to all animals and the average radiance within these ROIs was automatically calculated. Background signal was measured in the lower region of the abdomen, and the average values of background signal obtained.
[0332] Alternatively, blood infection was assessed by flow cytometry using hsp70-GFP parasites. At day 4, 5, 6 and >10 post-challenge, a millimetric excision was performed in the tail of mice allowing the collection of a drop of blood that was readily diluted in 500 uL of PBS. This diluted blood was analyzed using a flow cytometer. 500,000 events were analyzed at day 4 post-challenge and 100,000 events in the subsequent days. Non-infected mice after day 10 were considered as sterile protected.
[0333] Statistical analysis: Parasitemia data from GFP immunized control were log transformed and pooled for the calculation of 95% tolerance of interval with 95% of certitude. For the immunization protocol of one dose this limit comprised the interval of the mean value±2.49 SD (mean=−0.3906, SD=0.3392, n=35). Similarly, for the immunization protocol of two doses this limit comprised the interval of the mean value±2.51 (mean=−0.3002, SD=0.3305, n=33). All mice with a log parasitemia inferior to the lower limit (mean—2.5 SD) were considered as significantly different from the control mice (P<0.05), and therefore considered as protected. In the protocol using two immunization doses, the difference in the numbers of protected mice, following the definition above, between the test group and the GFP control group was assessed using the Fisher's exact test. The average of the log parasitemia of the groups with significant differences in the Fisher's Test were compared to the GFP group using one-way ANOVA (Holm-Sidak's multiple comparison test).
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