RECOMBINANT MEASLES VIRUS EXPRESSING PROTEINS OF A PLASMODIUM PARASITE AND THEIR APPLICATIONS

Abstract

The present invention relates to recombinant measles virus expressing proteins of a Plasmodium parasite and their applications, in particular in inducing preventive protection against a Plasmodium infection. The present invention is directed to recombinant measles virus (MV) expressing (i) at least the circumsporozoite (CS) protein of a Plasmodium parasite or an antigenic fragment thereof, and at least a chimeric antigen of a Plasmodium parasite as defined below, or (ii) at least the CS protein of a Plasmodium parasite or an antigenic fragment thereof, at least a chimeric antigen of a Plasmodium parasite as defined below and at least the reticulocyte-binding protein homologue 5 (RH5) of a Plasmodium parasite or an antigenic fragment thereof, and concerns recombinant infectious virus partides of said MV-malaria able to replicate in a host after an administration. The present invention provides means, in particular nucleic acids, vectors, cells and rescue systems to produce these recombinant infectious virus particles. The present invention also relates to the use of these recombinant infectious virus particles, in particular under the form of a composition, more particularly in a vaccine composition, for the prevention of a Plasmodium infection or for the preventive protection against clinical outcomes of infection by a Plasmodium parasite.

Claims

1. A chimeric measles virus (MV)-based nucleic acid construct suitable for the expression of heterologous polypeptides, which comprises: a cDNA molecule encoding a full-length, infectious antigenomic (+) RNA strand of a MV; and (1) a first heterologous polynucleotide encoding at least the circumsporozoite (CS) protein of a Plasmodium parasite or an antigenic fragment thereof; and (2) a second heterologous polynucleotide encoding at least a chimeric antigen of a Plasmodium parasite; and wherein said chimeric antigen as defined in (2) comprises or consists of the following fragments of (a), (b), (c) and (d) assembled in a fusion polypeptide, wherein the fragments of (a), (b), (c) and (d) elicit a human leukocyte antigen (HLA)-restricted CD8.sup.+ and/or CD4.sup.+ T cell response against a Plasmodium parasite, and are directly or indirectly fused in this order: (a) a fragment of the inhibitor of cysteine protease (ICP) (18-10) of a Plasmodium parasite, (b) a fragment of the protein Ag45 (11-10) of a Plasmodium parasite, (c) a fragment of the thrombospondin related anonymous protein (TRAP) of a Plasmodium parasite, and (d) the protein Ag40 (11-09) of a Plasmodium parasite or a fragment thereof, or a chimeric antigen variant thereof, which consists of a chimeric antigen having an amino acid sequence which has at least 90% sequence identity or more than 95% sequence identity or 99% sequence identity with the sequence of the fusion polypeptide consisting of fused fragments of (a), (b), (c) and (d), from which it derives by point mutation of one or more amino acid residues, over its whole length; wherein the first heterologous polynucleotide is operatively linked, in particular cloned within an additional transcription unit (ATU) inserted within the cDNA molecule; and wherein the second heterologous polynucleotide is operatively linked, in particular cloned within an ATU inserted within the cDNA molecule at a location distinct from the location of the first linked, in particular cloned heterologous polynucleotide.

2. The nucleic acid construct according to claim 1, further comprising a third heterologous polynucleotide encoding at least the reticulocyte-binding protein homologue 5 (RH5) of a Plasmodium parasite or an antigenic fragment thereof, wherein said third heterologous polynucleotide is directly fused or indirectly fused to the first heterologous polynucleotide.

3. The nucleic acid construct according to claim 1, wherein said nucleic acid construct complies with the rule of six of the MV genome.

4. The nucleic acid construct according to claim 1, comprising the following polynucleotides encoding polypeptides from 5′ to 3′: (a) a polynucleotide encoding the N protein of the MV; (b) a polynucleotide encoding the P protein of the MV; (c) the first heterologous polynucleotide encoding at least the CS protein of the Plasmodium parasite or the antigenic fragment thereof; (d) a polynucleotide encoding the M protein of the MV; (e) a polynucleotide encoding the F protein of the MV; (f) a polynucleotide encoding the H protein of the MV; (g) the second heterologous polynucleotide encoding the at least a chimeric antigen of the Plasmodium parasite; and (h) a polynucleotide encoding the L protein of the MV; wherein said polynucleotides are operatively linked in the nucleic acid construct and under the control of viral replication and transcription regulatory sequences such as MV leader and trailer sequences.

5. The nucleic acid construct according to claim 2, comprising the following polynucleotides encoding polypeptides from 5′ to 3′: (a) a polynucleotide encoding the N protein of the MV; (b) a polynucleotide encoding the P protein of the MV; (c) the first heterologous polynucleotide encoding at least the CS protein of the Plasmodium parasite or the antigenic fragment thereof; (d) the third heterologous polynucleotide encoding at least the RH5 of the Plasmodium parasite or the antigenic fragment thereof, which is directly fused or indirectly fused to the first heterologous polynucleotide of (c); (e) a polynucleotide encoding the M protein of the MV; (f) a polynucleotide encoding the F protein of the MV; (g) a polynucleotide encoding the H protein of the MV; (h) the second heterologous polynucleotide encoding the at least a chimeric antigen of the Plasmodium parasite; and (i) a polynucleotide encoding the L protein of the MV; wherein said polynucleotides are operatively linked in the nucleic acid construct and under the control of viral replication and transcription regulatory sequences such as MV leader and trailer sequences.

6. The nucleic acid construct according to claim 1, wherein said measles virus is an attenuated virus strain selected from the group consisting of the Schwarz strain, the Zagreb strain, the AIK-C strain and the Moraten strain.

7. The nucleic acid construct according to claim 1, wherein the Plasmodium parasite is Plasmodium falciparum or Plasmodium berghei.

8. The nucleic acid construct according to claim 7, wherein the Plasmodium parasite is Plasmodium falciparum and wherein said first heterologous polynucleotide encoding at least the CS protein of Plasmodium falciparum or the antigenic fragment thereof further encodes (i) the signal peptide from the F protein of the MV or (ii) the signal peptide from the F protein of the MV and the intracytoplasmic and transmembrane domains of the F protein of the MV.

9. The nucleic acid construct according to claim 1, wherein the second heterologous polynucleotide encoding the at least a chimeric antigen of the Plasmodium parasite further encodes (i) the signal peptide from the F protein of the MV.

10. The nucleic acid construct according to claim 2, wherein the third heterologous polynucleotide encoding at least the RH5 of the Plasmodium parasite or the antigenic fragment thereof further encodes (i) the signal peptide from the F protein of the MV or (ii) the signal peptide from the F protein of the MV and the signal peptide from the F protein of the MV and the intracytoplasmic and transmembrane domains of the F protein of the MV.

11. The nucleic acid construct according to claim 1, wherein the fragment of the ICP (18-10) of the Plasmodium parasite of (a) has the amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18, the fragment of the protein Ag45 (11-10) of the Plasmodium parasite of (b) has the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 22, the fragment of the TRAP of the Plasmodium parasite of (c) has the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 26, and the protein Ag40 (11-09) of the Plasmodium parasite or the fragment thereof of (d) has the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30.

12. The nucleic acid construct according to claim 1, wherein the first heterologous polynucleotide encoding at least the CS protein of the Plasmodium parasite or the antigenic fragment thereof has a sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, and wherein the second heterologous polynucleotide encoding the at least a chimeric antigen of the Plasmodium parasite has a sequence selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46.

13. The nucleic acid construct according to claim 2, wherein the third heterologous polynucleotide encoding at least the RH5 of the Plasmodium parasite or the antigenic fragment thereof has the sequence of SEQ ID NO: 32 or the sequence of SEQ ID NO: 34 or the sequence of SEQ ID NO: 56 or the sequence of SEQ ID NO: 58.

14. The nucleic acid construct according to claim 1, wherein the first heterologous polynucleotide encodes the CS protein of the Plasmodium parasite or the antigenic fragment thereof whose sequence is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13, and the second heterologous polynucleotide encodes the chimeric antigen of the Plasmodium parasite whose sequence is selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45 and SEQ ID NO: 47.

15. The nucleic acid construct according to claim 2, wherein the third heterologous polynucleotide encodes the RH5 of the Plasmodium parasite or the antigenic fragment thereof whose sequence is SEQ ID NO: 33 or SEQ ID NO: 35 or SEQ ID NO: 57 or SEQ ID NO: 59.

16. The nucleic acid construct according to claim 1, wherein said nucleic acid construct comprises a first polynucleotide whose sequence is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, and a second polynucleotide whose sequence is selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46.

17. The nucleic acid construct according to claim 2, wherein said nucleic acid construct comprises a first polynucleotide whose sequence is SEQ ID NO: 36 or SEQ ID NO: 38, and a second polynucleotide whose sequence is selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46.

18. A transfer vector plasmid, comprising the nucleic acid construct according to claim 1.

19. The transfer vector plasmid according to claim 18, whose sequence is SEQ ID NO: 54 or SEQ ID NO: 55.

20. Transformed cells comprising inserted in their genome the nucleic acid construct according to claim 1.

21. Recombinant infectious replicating measles virus (MV)-malaria virus particles, which comprise as their genome a nucleic acid construct according to claim 1.

22. Recombinant infectious replicating MV-malaria virus particles according to claim 21, which are rescued from a helper cell line expressing an RNA polymerase recognized by said cell line, for example a T7 RNA polymerase, a nucleoprotein (N) of a MV, a phosphoprotein (P) of a MV, and optionally an RNA polymerase large protein (L) of a MV.

23. The recombinant infectious replicating MV-malaria virus particles according to claim 21, wherein said virus particles comprise in their genome a polynucleotide sequence comprising (i) a first polynucleotide whose sequence is selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12, and a second polynucleotide whose sequence is selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46, or (ii) a first polynucleotide whose sequence is SEQ ID NO: 36 or SEQ ID NO: 38, and a second polynucleotide whose sequence is selected from the group consisting of SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 and SEQ ID NO: 46.

24. A composition or an assembly of immunologically active ingredients comprising the recombinant infectious replicating MV-malaria virus particles according to claim 21 and a pharmaceutically acceptable vehicle.

25. A method for eliciting elicitation of antibodies directed against said proteins of a Plasmodium parasite, and/or of a cellular immune response, in a host, comprising administering the composition or the assembly of immunologically active ingredients according to claim 24 to the host.

26. A method for the prevention of a Plasmodium infection in a subject or in the prevention of clinical outcomes of infection by a Plasmodium parasite in a subject, in particular in a human comprising administering the recombinant infectious replicating MV-malaria virus particles according to claim 21 to the subject.

27. A process to rescue recombinant infectious replicating measles virus (MV)-malaria virus particles expressing (i) at least the circumsporozoite (CS) protein of a Plasmodium parasite or an antigenic fragment thereof, and at least a chimeric antigen of a Plasmodium parasite, or (ii) at least the CS protein of a Plasmodium parasite or an antigenic fragment thereof, at least a chimeric antigen of a Plasmodium parasite and at least the reticulocyte-binding protein homologue 5 (RH5) of a Plasmodium parasite or an antigenic fragment thereof, wherein said chimeric antigen comprises or consists of the following fragments of (a), (b), (c) and (d) assembled in a fusion polypeptide, wherein the fragments of (a), (b), (c) and (d) elicit a human leukocyte antigen (HLA)-restricted CD8.sup.+ and/or CD4.sup.+ T cell response against a Plasmodium parasite, and are directly or indirectly fused in this order: (a) a fragment of the inhibitor of cysteine protease (ICP) (18-10) of a Plasmodium parasite, (b) a fragment of the protein Ag45 (11-10) of a Plasmodium parasite, (c) a fragment of the thrombospondin related anonymous protein (TRAP) of a Plasmodium parasite, and (d) the protein Ag40 (11-09) of a Plasmodium parasite or a fragment thereof, or a chimeric antigen variant thereof, which consists of a chimeric antigen having an amino acid sequence which has at least 90% sequence identity or more than 95% sequence identity or 99% sequence identity with the sequence of the fusion polypeptide consisting of fused fragments of (a), (b), (c) and (d), from which it derives by point mutation of one or more amino acid residues, over its whole length, and wherein said process comprises: 1) co-transfecting helper cells, in particular HEK293 helper cells, that stably express T7 RNA polymerase, and measles N and P proteins with (i) the transfer vector plasmid according to claim 18 and with (ii) a vector, especially a plasmid, encoding the MV L polymerase; 2) cultivating said co-transfected helper cells in conditions enabling the production of recombinant MV-malaria virus particles; 3) propagating the thus produced recombinant MV-malaria virus particles by co-cultivating said helper cells of step 2) with cells enabling said propagation such as Vero cells; 4) recovering recombinant infectious replicating MV-malaria virus particles expressing (i) at least the CS protein of the Plasmodium parasite or the antigenic fragment thereof, and said chimeric antigen of the Plasmodium parasite, or (ii) at least the CS protein of the Plasmodium parasite or the antigenic fragment thereof, said chimeric antigen of the Plasmodium parasite and the RH5 of the Plasmodium parasite.

28. The process according to claim 27, wherein the transfer vector plasmid has the sequence of SEQ ID NO: 54 or SEQ ID NO: 55.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0181] FIG. 1. rMV-CSPb and rMV CSPf. (A) Schematic representation of measles vector expressing CS protein from Plasmodium berghei ANKA (CSPb) or Plasmodium falciparum (CSPf). The synthetic sequence was mammalian codon-optimized and cloned into the Additional Transcription Unit (ATU) position 2 of pTM-Schwarz. The MV genes are indicated as follows: nucleoprotein (N), phosphoprotein (P), V and C accessory proteins, matrix (M), fusion (F), hemagglutinin (H) and polymerase (L). T7 RNA polymerase promoter (T7), T7 RNA polymerase terminator (T7t), hepatitis delta virus ribozyme (∂), hammerhead ribozyme (hh) are requested for viral rescue. (B) Growth curves of MV-Schwarz, rMV-CSPb and rMV-CSPf in Vero cells infected at an MOI of 0.1. Cell-associated virus titers are indicated in TCID50/ml. (C) Detection by western-blot of CSPb or CSPf in cell lysates (L) or supernatant (SN) of Vero cells infected by rMV-CSPb and rMV CSPf. (D) Immunofluorescence detection of CSPb and CSPf in Vero cells infected for 24 hours with rMV-CSPb and rMV-CSPf at an MOI of 0.1. Infected cells formed syncytia where are localized CS proteins.

[0182] FIG. 2. Blood parasitemia of C57BL/6 and hCD46IFNAR mice after skin microinjection of 5,000 sporozoites of Plasmodium berghei ANKA. Percentage of infected red blood cells (iRBCs) at day 4, 5 and 6 post-infection (p.i.) was log transformed for parasitemia normalization before statistical analysis. No statistically significant difference was observed between both groups. N.I. threshold of parasitemia detection.

[0183] FIG. 3. Immunogenicity and protective efficacy of rMV-CSPb. Antibody response induced in hCD46IFNAR mice immunized with rMV-CSPb at day 0 and 4 weeks later. The data show the reciprocal endpoint dilution titers of specific antibodies to MV (A) and CSPb (B). Percentage of asymptomatic (C) and non-infected (D) hCD46IFNAR mice (6 mice per group) immunized twice at one month of interval and challenged 3 weeks after with 5,000 Pb ANKA sporozoïtes intradermally. (E) Log of parasitemia at day 4, 5 and 6 post-infection (p.i.) (means+/−SD). Asterisks (*) indicate significant mean differences (** for p<0.01; *** for p<0,001). L.D. level of detection. N.I. threshold of parasitemia detection.

[0184] FIG. 4. Immunogenicity and protective efficacy of rMV-CSPf. (A-C) Antibody response induced in hCD46IFNAR mice immunized with rMV-CSPf at day 0 and 4 weeks later. Long-term memory was assessed at week 22 post-priming. The data show the reciprocal endpoint dilution titers of specific antibodies to MV (A) and CSPf (B). (C) IgG subtypes of CSPf antibodies elicited by rMV-CSPf 4 weeks after the second immunization. (D-E) Infectious challenge with 5,000 sporozoïtes of a recombinant PbA expressing CSPf repeat sequence 3 weeks (D) or 16 weeks (E) after the second immunization. Log of parasitemia at days 4, 5 and 6 post-infection (p.i.) (means+/−SD). Asterisks (*) indicate significant mean differences (* for p<0.05; ** for p<0,01). L.D. level of detection. N.I. threshold of parasitemia detection.

[0185] FIG. 5. Cellular response to rMV-CSPf. (A) IFNγ Elispot assay and (B) intracellular cytokine staining assay were done on freshly extracted splenocytes 7 days after one immunization i.p. with 1.105 TCID50 of MV-Schwarz or rMV-CSPf. Splenocytes were restimulated with inactivated MV-Schwarz at an MOI of 1 or CSPf recombinant LPS-free protein at 50 μg/ml. CD4+ and CD8+ T-cells were stained against IFNγ and TNFα. Negative controls, cultured with media alone, showed less than 0.05% of positive cells (data not shown). Percentile IFNγ and TNFα cytokine distribution for CD4+ and CD8+ T-cells reactive against MV-Schwarz (B1) and upon CSPf restimulation (B2). Asterisks (*) indicate significant mean differences (** for p<0.01).

[0186] FIG. 6. Scheme of a chimeric antigen of Plasmodium berghei (P. berghei Fusion 4cPEAg). 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 fusion 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).

[0187] FIG. 7. P. falciparum 4cPEAg fusion. 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 FIG. 6. SP, signal peptide. Antigens are (a) Pf18-10, (b) Pf11-10, (c) PfTRAP, (d) Pf11-09. (e) Selected regions of the Pf 4cPEAgs (white bars) were chimerized generating the Pf 4cPEAg Fusion. The dotted lines represent the junction between two adjacent antigens/protective domains and show the absence of formation of neo-epitopes.

[0188] FIG. 8. Plasmodium berghei infectious challenge in hCD46IFNAR mice immunized with MV Schwarz (G1, control group), MV-CSPb (G2), MV-Pb Fusion (G3), MV-CSPb-Pb Fusion (G4), and MV-CSPb+MV-Pb Fusion (G5). (A) Parasitemia at day 3, 4, 5, and 6 post-challenge. (B) Percentage of non-infected mice after challenge for the five groups (sterile protection). Groups of 6 mice were immunized i.p. (100 μl, 10.sup.5 TCID.sub.50 of each virus, two viruses in two injections for G5) two times at one month interval. The infectious challenge was done with Plasmodium berghei ANKA strain, a model of cerebral malaria, four weeks after the second immunization, with 5,000 sporozoites injected i.d. in footpads. Blood parasitemia was assessed by flow cytometry on a drop of blood from day 3 to 6 post-challenge, until day 10 for sterilely protected mice.

EXAMPLES

Material and Methods

Cell Culture

[0189] Vero cells (African green monkey kidney cells) and HEK293-T7-MV (human embryonic kidney cells) helper cells were maintained in Dulbecco's modified Eagle medium (DMEM; Gibco) supplemented with 10% heat-inactivated fetal calf serum (GE Healthcare) and 10.000 U/ml of penicillin-streptomycin (Life technologies). HEK293-T7-MV helpers cells stably expressed T7 polymerase and MV-N and MV-P proteins and were used for measles viral rescue (WO2008/078198).

Construction of pTM2-MVSchw-CSPb and pTM2-MVSchw-CSPf Plasmids and Rescue of rMV-CSPb and rMV-CSPf Recombinant Viruses

[0190] The plasmid pTM2-Schw (Combredet, et al. J. Virol. 2003, 77(21):11546-54) encodes the cDNA of the anti-genome of the Schwarz MV vaccine strain with an additional transcription unit (ATU) between the phosphoprotein (MV-P) and the matrix (MV-M) genes, flanked by BsiWI/BssHII restriction sites. Two cDNAs encoding the circumsporozoite protein of Plasmodium berghei ANKA (CSPb ANKA full length sequence, mammalian codon optimized synthetic gene, Eurofins Genomics) and the circumsporozolte protein of Plasmodium falciparum 3D7 (CSPf, truncated form from 19 to 369 aa, without GPI anchored signal at C-terminus, signal sequence from MV Fusion protein at N-terminus, chemically synthesized; Genscript, USA) were inserted in ATU2, to produce respectively pTM2-MVSchw-CSPb and pTM2-MVSchw-CSPf plasmids. The sequences, which were codon optimized for expression in mammalian cells, respected the “rule of six”, which stipulates that the number of nucleotides in the MV genome must be a multiple of 6, and contain BsiWI/BssHII restriction sites at both ends. Rescue of both recombinant viruses (rMV-CSPb and rMV-CSPf) was performed as previously described (Combredet, et al. J. Virol. 2003, 77(21):11546-54) using the helper-cell-based rescue method described by Radecke et al. (Radecke, et al. EMBO J. 1995, 14(23):5773-84; Parks, et al., J. Virol. 1999, 73(5):3560-6) and modified by Parks et al. (Parks, et al., J. Virol. 1999, 73(5):3560-6). rMV-CSPb and rMV-CSPf were grown on Vero cells.

Virus Growth Curves

[0191] Monolayers of Vero cells grown in 24-mm-diameter dishes (6-well plates) were infected with rMV-CSPb and rMV-CSPf at an MOI of 1. At various times post-infection, cells were scraped into culture medium. After freeze thawing of cells and medium, and clarification of cell debris, virus titers were determined by endpoint dilution assay. For this purpose, Vero cells were seeded into 96-well plates (7,500 cells/well) and infected with serial 1:10 dilutions of virus sample in DMEM-5% FCS. After incubation for 7 days, cells were stained with crystal violet, and the TCID50 values were calculated by use of the Spaerman-Kärber method (Spaerman Br. J. Psychol. 1908(2):227-42).

Antigens Expression

[0192] Expression of CSPf and CSPb was assessed in Vero cells infected with rMV-CSPb and rMV-CSPf by IFA and Western blotting. IFA was performed on Vero cells at 36 hours post-infection with rMV-CSPb and rMV-CSPf at an MOI of 0.1. Cells were probed with 3D11 mouse anti-CSPb monoclonal antibody (1/1,000 dilution) (#MR4-100 hybridoma) or 2A10 mouse anti-CSPf monoclonal antibody (1/1,000 dilution) (#MR4-183 hybridoma). Cy3-conjugated goat anti-mouse IgG (Jackson immunoresearch laboratories) was used as secondary antibody (1/1,000 dilution). Western blotting was performed on infected Vero cell lysates fractionated by SDS-PAGE and transferred to cellulose membranes. 3D11 mouse anti-CSPb monoclonal antibody and 2A10 mouse anti-CSPf monoclonal antibody were used to detect CS proteins. A goat anti-mouse IgG-horseradish peroxidase (HRP) conjugate (#P0447, Dako) was used as secondary antibody.

Mice Immunizations and Challenge

[0193] Mice deficient for type-I IFN receptor (IFNAR) and expressing human CD46 (hCD46) (Combredet, et al. J. Virol. 2003, 77(21):11546-54) were housed under pathogen-free conditions at the Institut Pasteur animal facility. Group of 6 six-week-old hCD46IFNAR mice were inoculated with different doses of MV-Schwarz, rMV-CSPb and rMV-CSPf, via the intraperitoneal route (i.p.). To study cellular response, only one immunization was administered and spleens were extracted eight days later. For humoral response and infectious challenge, two immunizations were administered within a 4 weeks interval. Sera were collected before the first immunization (day 0, negative control) and 3 weeks after each immunization, and 4 months after the second immunization to study long-term memory responses. Immunized mice were challenged with P. berghei ANKA sporozoites expressing the green fluorescent protein (GFP) under the control of the hsp70 promoter (Ishino, et al. Mol. Microbiol; 2006, 59(4):1175-84). Alternatively, mice immunized with rMV-CSPf were challenged with P. berghei NK65 sporozoites expressing the GFP under the control of the hsp70 promoter (Demarta-Gatsi, et al., J. Exp. Med. 2016, 213(8): 1419-28) and the CSPb harboring the central repetitive region of CSPf (rGFP-Pb-CSPf repeat) (Persson, et al. J. Immunol. 2002, 169(12): 6681-5). rGFP-Pb-CSPf repeat parasites were generated by a genetic cross as described by Ishino et al. (Ishino, et al. Mol. Microbiol. 2006, 59(4):1175-84). Sporozoites were freshly collected from the salivary gland of infected Anopheles stephensi in D-PBS and filtered using a 35pm nylon mesh cell strainer snap cap (Corning Falcon). Infectious challenges were executed 4 weeks after the second immunization (early response), or 4 months after the second immunization (long-term memory response) by the microinjection of 5,000 sporozoites in one microliter of D-PBS in the posterior footpad using a 35G microsyringe (World Precision Instruments). After challenge, parasitemia was monitored from day 3 to day 10. Blood samples (2 μl) were diluted in 500 μl of PBS and analyzed by flow cytometry (MacsQuant, Miltenyi Biotec). Doublets and clusters of red blood cells (RBCs) were excluded from counts. Single GFP+RBCs (infected RBC, iRBCs) among total RBCs were estimated and data analyzed by the MACSQuantify™ Software. As no protection against blood stage parasites was expected, mice were sacrificed at day 10 post-challenge in the presence of iRBCs in order to avoid unnecessary suffering, or before in the presence of severe symptoms that were ethical endpoints (signs of cerebral malaria: motor troubles, ruffled fur and sometimes convulsions). Non-parasitemic mice at day 10 were considered sterile protected. Experiments were conducted following the guidelines of the Office of Laboratory Animal Care at Institut Pasteur.

ELISA

[0194] Measles virus antigen, Edmonston strain (#PR-BA102-S-L, Jena Bioscience) antigen at 1 μg/ml in PBS, and CSPb or CSPf recombinant proteins (produced at the Recombinant Protein and Antibodies Production Core Facility of the Institut Pasteur by J. Bellalou and V. Bondet, using the BioPod F800 microfermentor battery) at 1 pg/ml in carbonate buffer were coated overnight at 4° C. onto 96-well plates (#439454, Thermo Scientific) and then blocked for 1 h at 37° C. with a saturation buffer (PBS, 0.05% Tween, 3% BSA). Sera samples from immunized mice were serially diluted (PBS, 0.05% Tween, 1% BSA) and incubated on plates for 1 h at 37° C. After washing steps (0.05% Tween in PBS), a secondary horseradish peroxidase conjugated goat anti-mouse Ig antibody (#115-035-146, Jackson ImmunoResearch) was added at a dilution of 1/1,000 for 1h at 37° C. Antibody binding was revealed by addition of the TMB substrate (#5120-0047, Eurobio) and the reaction was stopped by addition of H.sub.2SO.sub.4 1M. The optical densities (O.D.) were recorded at 450 nm. The endpoint titers for each individual serum were calculated as the reciprocal of the last dilution giving twice the absorbance of negative control sera.

ELISPOT Assay

[0195] Freshly extracted splenocytes from immunized mice were tested for their capacity to secrete IFN-γ upon specific stimulation. Multiscreen-HA 96-well plates (#MSIP4510, Millipore) were coated overnight at 4° C. with 5 μg/ml of anti-mouse IFN-γ (#551216, BD Biosciences Pharmingen) in PBS and, after washing, were blocked for 2 h at 37° C. with complete MEM (MEM—10% FCS supplemented with non-essential amino-acids 1%, sodium pyruvate 1% and β-mercapto-ethanol). The medium was then replaced with 100 μl of cell suspension containing 2×10.sup.5 splenocytes in each well (triplicate) and 100 μl of stimulating agent in complete MEM. Plates were incubated for 40h at 37° C. Cells were stimulated with Concanavalin A (#C-5275, Sigma) as positive control, complete MEM as negative control, live attenuated MV-Schwarz virus at an MOI of 1, and CSPf recombinant protein at 50 μg/ml. After incubation and washing, biotinylated anti-mouse IFN-γ antibody (#554410, BD Biosciences Pharmingen) was added and plates were incubated for 90 minutes at room temperature. After extensive washing, streptavidin-alkaline phosphatase conjugate (#7100-05, Clinisciences) was added and plates were incubated 1 h at room temperature. Spots were developed with BCIP/NBT (#61911, Sigma) and counted in an ELISPOT reader (CTL ImmunoSpot®).

Intracellular Cytokine Staining

[0196] Freshly extracted splenocytes from immunized mice were analyzed by flow cytometry for their capacity to secrete IFN-γ and TNF-α upon specific stimulation. Spleen cells were cultured for 16 hours in U-bottom 96-well plates (1.0×10.sup.6 cells/well) in a volume of 0.2 ml complete medium (MEM—10% FCS supplemented with non-essential amino-acids 1%, sodium pyruvate 1% and β-mercapto-ethanol). Cells were stimulated with PMA/ionomycin (#00-4970, ebioscience) as positive control, complete MEM as negative control, live attenuated MV-Schwarz virus at an MOI of 1, and CSPf LPS-free recombinant protein at 50 μg/ml. Brefeldin A (#66542, Sigma) was then added at 10 μg/ml for 6 more hours of incubation. Stimulated cells were harvested, washed in phosphate-buffered saline containing 1% bovine serum albumin and 0.1% w/w sodium azide (FACS buffer), incubated 10 minutes with Fc blocking Ab (CD16/32 clone 2.4G2, PharMingen) and surface stained in FACS buffer with Live/Dead fixable dead cell violet kit (#L34955, invitrogen), anti-mouse CD4-PECy7 mAb (#552775, BD Biosciences) and anti-mouse CD8-APCH7 mAb (#560182, BD Biosciences) for 30 minutes at 4° C. in the dark. After washing, cells were fixed and permeabilised for intracellular cytokine staining using the Cytofix/Cytoperm kit (#554922, BD Bioscience). Cells were then incubated in a mix of anti-mouse IFNγ-APC mAb (#554413, BD Biosciences) and anti-mouse TNF-α-FITC mAb (#554418, BD Biosciences) diluted in permwash buffer (#557885, BD Bioscience) for 30 minutes in the dark. After washing with permwash buffer and FACS buffer, cells were fixed with 1% formaldehyde in PBS. Data were acquired using a MacsQuant® Analyzer (Miltenyi Biotec), and analysed using Flow Jo™ 9.3.2 software and are presented as % of CD4+ or CD8+ cells expressing TNF-α or IFNγ among total CD4 or CD8 populations.

Statistical Analysis

[0197] Parasitemia was Log transformed for normalization. Statistical analyses were done using the t-test. Differences were considered statistically significant when p<0.05.

EXAMPLE 1

Production of rMVs Expressing CSPb and CSPf Proteins

[0198] The inventors constructed an rMV expressing CSPb protein (rMV-CSPb) and an rMV expressing CSPf protein (rMV-CSPf) by inserting mammalian codon-optimized sequences of both proteins in additional transcription unit 2 (ATU2) of pTM-MVSchw plasmid, which encodes the antigenome of the Schwarz MV vaccine strain (Combredet, et al. J. Virol. 2003, 77(21):11546-54) (FIG. 1A). The ATU2 allowed high-level expression of the protein, as there was a decreasing gradient of gene expression generated by MV replication (from high nucleoprotein “N” expression to low polymerase “L” expression). Both plasmids were transfected into HEK293T-helper cells for rescue and co-cultured with Vero cells for virus spread. The rescued rMV-CSPb and rMV-CSPf had slightly delayed growth curves, as compared to empty MV (FIG. 1B), but still reached high titers on Vero cells. Viral stocks were made from unique syncytia after rescue and were therefore considered as clonal. The expression of CS was assessed by Western blot, and found in the lysate and in the supernatant of infected Vero cells (FIG. 1C). The CS expression in infected cells forming syncytia was also demonstrated by immunofluorescence (FIG. 1D). For rMV-CSPf, the stability of transgene expression was demonstrated after 10 passages of the recombinant virus on Vero cells by immunofluorescence, Western blot and sequencing (data not shown). The stability of rMV-CSPb was not tested as the mouse model was only used for proof of concept.

EXAMPLE 2

Susceptibility of hCD46IFNAR Mice to Plasmodium berghei ANKA Challenge

[0199] Mice are naturally resistant to MV, which is restricted to human and non-human primates. The usual mouse model to test rMV vaccine candidates is deficient for type-I IFN receptor (IFNAR) and expresses human receptor CD46 (hCD46) (Combredet, et al. J. Virol. 2003, 77(21):11546-54). The genetic background of hCD46IFNAR mouse used in the present invention was Sv129, which had the same major histocompatibility complex haplotype as C57BL/6 mouse (H-2Db, H-2Kb, I-Ab). C57BL/6 mice infected with P. berghei ANKA (PbA) was a model for cerebral malaria, which lead to death. C57BL/6 mice were easily infected and highly susceptible, as compared to Balb/c mice (Jaffe, et al. Am. J. Trop. Med. Hyg. 1990, 42(4):309-13; Hafalla, et al. PLoS Pathog. 2013, 9(5):e1003303). In order to validate the model of infection in hCD46IFNAR mice, the inventors inoculated 5,000 GFP-expressing PbA (GFP PbA) sporozoites in the footpad of six C57BL/6 and six hCD46IFNAR mice. The inventors monitored the parasitemia from day 4 to day 6 post-inoculation. Mice were sacrificed at day 6 post-challenge in the presence of iRBCs in order to avoid unnecessary suffering (ethical endpoints). Although parasitemia was slightly higher in hCD46IFNAR group, there was no statistically significant difference between both groups of mice (FIG. 2). So, the inventors concluded that both mouse models were comparable for sporozoite challenge. These observations validated the use of hCD46IFNAR mouse for the rest of the study.

EXAMPLE 3

Immunogenicity and Protective Efficacy of rMV-CSPb as a Proof of Concept

[0200] Six-week-old hCD46IFNAR mice (6 mice per group) received 10.sup.5 TCID50 of rMV-CSPb, or MVSchw as negative control, by intraperitoneal (i.p.) route at day 0 and at day 28. Sera were collected before the first immunization (control) and 3 weeks after each immunization. Antibodies to MV were induced at similar levels in all immunized mice (FIG. 3A). Antibodies to CSPb were efficiently induced from the first immunization with limiting dilution titers of about 10.sup.4, then boosted after the second immunization to reach 10.sup.5 (FIG. 3B). Mice were challenged 3 weeks after the second immunization with 5,000 sporozoïtes of GFP-PbA injected in the footpad. In MVSchw immunized group (control), the inventors sacrificed mice at day 6 post-challenge (FIG. 3C), due to start of cerebral symptoms, which were ethical endpoints of the study. In rMV-CSPb immunized group, two mice (33%) achieved sterile protection (no detectable iRBC at day 10 post-challenge) (FIG. 3D). The other mice showed a significant delayed and decreased parasitemia (FIG. 3E), with no observed severe symptoms. Moreover, at day 10 post-challenge, the parasitemia in rMV-CSPb immunized mice was still less than 1%. So, immunization with rMV-CSPb achieved sterile protection in 33% of hCD46IFNAR mice and completely protected mice from severe and lethal PbA-induced cerebral malaria.

EXAMPLE 4

Immunogenicity of rMV-CSPf: Thi IgG Subtype Profile and Long-Term Memory

[0201] Six-week-old hCD46IFNAR mice (6 mice per group) received 10.sup.5 TCID50 of rMV-CSPf, or MVSchw as negative control, by intraperitoneal (i.p.) route at day 0 and at day 28. Sera were collected before the first immunization (control), 3 weeks after each immunization, and 22 weeks after the first immunization for a group of 6 mice dedicated to long-term memory study. As for rMV-CSPb, antibodies to MV were induced at similar levels in all immunized mice (FIG. 4A) and antibodies to CSPf were efficiently induced from the first immunization with limiting dilution titers of about 10.sup.4, then boosted after the second immunization to reach 10.sup.5 (FIG. 4B). Interestingly, this high antibody titer was maintained 22 weeks post-prime. The humoral response profile corresponded to Th1 polarization with high titers of IgG2a antibodies (FIG. 4C), as expected for a replicative viral vector. Mice were challenged 3 weeks after the second immunization (early challenge) or 22 weeks post-prime (late challenge) with 5,000 sporozoites of recombinant GFP-Pb expressing CSPb with CSPf repeat sequence (rGFP-Pb-CSPf repeat), microinjected in the mouse footpad. In MVSchw immunized group (control), all mice were sacrificed at day 6 post-challenge, due to start of cerebral symptoms, which were ethical endpoints of the study. In rMV-CSPf immunized group, there was no induction of sterile protection, but a decreased and delayed parasitemia, whether for early (FIG. 4D) or late challenge (FIG. 4E). Mice started to present symptoms of cerebral malaria at day 7 and were sacrificed to avoid unnecessary suffering. This decreased parasitemia was therefore less important than the one observed for rMV-CSPb. The inventors hypothesized that the observed difference was due to the challenge model with rGFP-PbA-CSPf repeat that allow only to study protection relying on neutralizing antibodies directed against the repeat sequence. The inventors therefore evaluated the cellular response in the Pf model.

EXAMPLE 5

Induction of Specific Cellular Immune Response

[0202] Cell-mediating immune response (CMI) elicited by immunization with rMV-CSPf was assessed using IFNy Elispot assay and intracellular cytokine staining (IFNγ and TNFα) on freshly extracted splenocytes collected 7 days after a single immunization with 1.10.sup.5 TCID50 in 100 μl i.p. (FIG. 5). Splenocytes were stimulated ex vivo with inactivated MV-Schwarz at an MOI of 1 or CSPf recombinant LPS-free protein at 50 μg/ml. A moderate but significant (p<0.01) number of CSPf-specific cells (up to 100/10.sup.6 splenocytes) were detected by the ELISPOT assay (FIG. 5A), which corresponds to 5-10% of the number of MV-specific spots. The phenotype of MV- and CSPf-specific cells induced by rMV-CSPf was analyzed by flow cytometry (FIG. 5B). The mean frequency of MV-specific T cells secreting IFNγ and TNFα in CD4+ cells (B1 left panel) was respectively 1.5% and 0.2%. The mean frequency of MV-specific T cells secreting IFNγ and TNFα in CD8+ cells (B1 right panel) was respectively 2.6% and 0.2%. The mean frequency of CSPf-specific T cells secreting IFNγ and TNFα in CD4+ cells (B2 left panel) was respectively 0.16% and 0.14%. The mean frequency of MV-specific T cells secreting IFNγ and TNFα in CD8+ cells (B2 right panel) was respectively 0.3% and 0.18%. An induction of CD4+ cells secreting IFNγ and CD8+ cells secreting IFNγ or TNFα was observed, as compared to control group but statistically not significant (p=0.083, p=0.088 and p=0.057 respectively). Even if no CD8+ epitopes of CSPf were described in C57BL/6 mouse, the inventors showed the induction of a moderate but significant CMI as early as 7 days after a single immunization with rMV-CSPf, with CD4+ and CD8+ activated phenotype.

EXAMPLE 6

Construction of a Chimeric Antigen of a Plasmodium Parasite

[0203] The identification of the protective domains (PD) of four pre-erythrocytic conserved protective antigens allowed the construction of a chimeric antigen formed by the fusion in this order of a fragment of the ICP (18-10) of a Plasmodium parasite, a fragment of the protein Ag45 (11-10) of a Plasmodium parasite, a fragment of the TRAP of a Plasmodium parasite, and the protein Ag40 (11-09) of a Plasmodium parasite or a fragment thereof.

[0204] For example, a chimeric antigen of Plasmodium berghei ANKA have been constructed by the fusion in this order of the PD Pb18-10NT of SEQ ID NO: 15, the PD Pb18-10CT of SEQ ID NO: 17, the PD Pb11-10CT of SEQ ID NO: 21, the PD PbTRAPNT of SEQ ID NO: 25 and the antigen Pb11-09 of SEQ ID NO: 29. This chimeric antigen was called P. berghei Fusion 4cPEAg (SEQ ID NO: 41 or 43) and its structure is shown in FIG. 6.

[0205] As another example, a chimeric antigen of Plasmodium berghei ANKA has been constructed by the fusion in this order of the PD Pb18-10NT of SEQ ID NO: 15 and the PD Pb18-10CT if SEQ ID NO: 17.

[0206] As another example, a chimeric antigen of Plasmodium falciparum 3D7 have been constructed by the fusion in this order of the PD Pf18-10 of SEQ ID NO: 19, the PD Pf11-10CT of SEQ ID NO: 23, the PD PfTRAPNT of SEQ ID NO: 27 and the antigen Pf11-09 of SEQ ID NO: 31. This chimeric antigen was called P. falciparum Fusion 4cPEAg (SEQ ID NO: 45 or 47).

[0207] As another example, a chimeric antigen of Plasmodium falciparum 3D7 have been constructed by the insertion of the full antigen ICP 18-10 devoid of its signal peptide, in particular ICP 18-10 of SEQ ID NO: 19.

[0208] Since predicted CD8+T cell epitopes clustered in conserved regions of the antigens, independently of the plasmodial species and MHC class I restriction, this particularity was used to select the regions of P. falciparum 4cPEAg, corresponding to the protective domains of P. berghei 4cPEAg. More HLA class I and II allelles were analyzed, including the mapping of 9-mers peptides predicted to bind to 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, 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., and 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 (FIG. 7). This extended analysis corroborated the initial observation, using the HLA A*02:01 and the H2Db/Kb, that good binders tend to cluster in regions associated with structural/functional conserved domains, transmembrane domains, as well as in signal peptide and GPI-anchoring sequences. Based on this clustering of epitopes, the sequences/structures of P. berghei antigens were used to retrieve the cognate regions in P. falciparum antigens as shown in FIG. 7. These putative protective domains were fused as in P. berghei avoiding the creation of neo-epitopes in the junction of antigens/protective domains as shown in FIG. 7e. When inevitable, an amino acid residue was introduced in the fusion sequence to avoid the creation of neo-epitopes with high binding affinity to HLA. The only amino acid added in the Pf fusion 4cPE Ag was a glutamic acid (E) at the end of the Pf11-10CT.

EXAMPLE 7

Plasmodium Berghei Infectious Challenge in hCD46IFNAR Mice

[0209] As shown in FIG. 8(A), the expression of CSPb alone (G2) reduced the parasitemia, as compared to the control group (G1). The expression of Pb Fusion alone (G3) had no effect on parasitemia. However, when Pb Fusion was expressed simultaneously to CSPb (G4 and G5), the parasitemia was highly decreased. The effect was higher when both antigens were expressed in the same virus (G4) than in two viruses injected separately (G5) where a competitive effect could not be excluded.

[0210] As shown in FIG. 8(B), mice from G4 (33%) and G5 (17%) showed sterile protection, i.e. no blood infection at day 10 post-challenge. The expression of a single malaria antigen, either CSPb (G2) or Pb Fusion (G3) was not able to achieve sterile protection in this experiment.

[0211] This experiment clearly showed the synergistic effect of both antigens to achieve protection.

Discussion

[0212] Following the moderate protection and short memory response induced by RTS,S vaccine candidate in phase III clinical trial (Aaby, et al. Lancet 2015, 386(10005):1735-6), there is strong support for developing a second-generation malaria vaccine with higher efficacy and longer duration of protection. Because of its central place in infant vaccine schedules all over the world, measles provides a promising viral vector to deliver malaria antigens, either as a single delivery platform or in a prime boost strategy. The inventors have reported the use of measles-based vaccine platform to deliver CS malaria antigen as a proof of concept of the feasibility and advantages of this vector, in a murine model. Importantly, the inventors showed induction of cellular response and long-term memory with high antibody titers. These are the two main characteristics required for second-generation malaria vaccine candidates.

[0213] The inventors first showed the possibility of stably expressing a malaria antigen using the measles virus as a delivery vector. CSPb and CSPf sequences were successfully inserted in MV-Schwarz genome and stably maintained after 10 passages in Vero cell culture, without any mutation. Nevertheless, the inventors were unable to rescue a virus with CS native sequence (data not shown) and therefore mammalian codon-optimized sequence is required. The P. falciparum genome is AT rich (Gardner, et al. Nature 2002, 419(6906):498-511) and polyA/polyU probably disturbed measles polymerase, either for replication or transcription. As MV-Schwarz vector is able to insert 6 kb in its additional transcription units, other antigens could be easily added to CS to improve vaccine efficacy.

[0214] Then the inventors showed in the hCD46IFNAR mouse model the induction of high antibody titers that were maintained at least until 22 weeks post-prime in a two-immunization schedule with one-month interval. This maintenance of high antibody level was longer than the one observed with CS administered in a three doses regimen at 50 μg with complete Freund's adjuvant in C57BL/6 mice (Wirtz, et al. Exp. Parasitol. 1987, 63(2):166-72), whereas rMV delivered only ng of heterologous antigens (Brandler, et al. PLoS Negl. Trop. Dis. 2007, 1(3):e96). R16HBsAg, a precursor of RTS,S, induced high level of antibodies in mice when administered with alum in a three dose regimen, but was not assessed more than 5 weeks after the last immunization (Rutgers, et al., Nat. Biotechnol. 1988, 6:1065-70). In monkeys, RTS,S/AS01B formulation has shown a rapid decrease of CS antibodies 8 weeks after each boost (Mettens, et al. Vaccine 2008, 26(8):1072-82). The long-term persistence of neutralizing antibodies against heterologous antigens vectored by rMV has already been described for an rMV expressing HIV antigens in both mouse (Guerbois, et al. Virol. 2009, 388(1):191-203) and non-human primate (NHP) models (Stebbings, et al. PLoS One, 2012, 7(11):e50397). Thus, the observed maintenance of high anti-CS antibody level is promising regarding MV efficiency to induce life-long memory. IgG sub-types were predominantly IgG2a, which was expected for a replicating viral vector. This subclass is cytophilic in mice (Waldmann, et al. Annu. Rev. Immunol. 1989, 7:407-44), with complement fixation and pathogen opsonization. Moreover, induction of cytophilic CS antibodies has been associated with protection from re-infection in the field (John, et al. Am. J. Trop. Med. Hyg. 2005, 73(1):222-8). Nevertheless, it is important to remember that the parasite itself escapes immunity by modulating immune responses (Wykes, et al. EMBO Rep. 2013, 14(8):661). Thus, further investigations of memory B cells' survival (Liu, et al. Eur. J. Immunol. 2012, 42(12):3291-301) and dendritic cells' functionality (Wykes, et al. Nat. Rev. Microbiol. 2008, 6(11):864-70) after infectious challenge would help identify predictive factors of long-term efficacy in human.

[0215] To evaluate protection, the inventors used C57BL/6 mice and PbA model, which was a relevant model of liver stage immunity that closely resembles the situation in humans. In this model, sterile protection was not as easy as for Balb/c mice, where CSPb was target of immuno-dominant and protective CD8+ T cell response (Romero, et al. Nature 1989, 341(6240):323-6). Indeed, CS seemed to contain no naturally processed and presented H-2b restricted epitopes (Hafalla, et al. PLoS Pathog. 2013, 9(5):e1003303). Sv129 hCD46IFNAR mice and C57BL/6 mice both expressed H-2b major histocompatibility complex. The inventors showed that they were similarly sensitive to PbA challenge, with similar clinical features and no statistical difference in parasitemia on days 3, 4, 5 and 6 post-infection. Palomo et al. showed a slightly delayed experimental cerebral malaria development and prolonged survival of C57BL/6 IFNAR mice, as compared to wild-type mice (Palomo, et al. Eur. J. Immunol. 2013, 43(10):2683-95). Nevertheless, the inventors defined ethical endpoints at the beginning of the study that had imposed an early sacrifice at day 6 or 7 post-infection, and the inventors did not wait for natural death to avoid unnecessary suffering. rMV-CSPb was able to elicit sterile protection in 33% of mice and to protect all of them from severe disease, with a reduced and delayed parasitemia, and no severe clinical symptoms. In the rGFP-PbA-CSPf repeat challenge model, there was no sterile protection and reduction in parasitemia was less compared to the PbA model. This suggested that sterile protection was not induced by neutralizing antibodies directed against the repeat sequence of CSPf, but might involve antibodies against C and N-terminal domains of CS and cell-mediated immune responses. In fact, phagocytic activity of antibodies induced by RTS,S/AS01 malaria vaccine had been correlated with full-length CS and C-terminal specific antibody titer, but not to repeat region antibody titer (Chaudhury, et al. Malar. J. 2016, 15:301). Accordingly, the inventors showed a moderate but significant induction of cell-mediated immune response that appeared as early as 7 days after a single immunization, with an increase in CD4+ and CD8+ specific T cells secreting IFNγ or TNFα. As there was no described CD8+ epitope for CSPf in H-2b mice, the increase observed, even if moderate, was of great interest. Indeed, protection against malaria had been correlated to CSPf CD8+ T cell response in human immune system (HIS) mice harboring functional human CD8+ T cells (Li, et al. Vaccine, 2016, 34(38):4501-6). This major role for CD8+ T cells to induce protection was already shown by in vivo depletion of CD8+ T cells that abrogated sporozoite-induced protective immunity in mice (Weiss, et al. PNAS 1988, 85(2):573-6). Thus, even if the protection resulting from rGFP-PbA-CSPf repeated challenge model was not indicative of real protection, it brought indications of efficient immune mechanisms involved in protection.

[0216] To conclude, the inventors demonstrated the promising potential of using measles vector to deliver malaria antigens by showing induction of cellular immune responses and long-term memory with high antibody titers in mice. These are two critical desired characteristics for second-generation malaria vaccines. As expected, expression of CS alone was not able to induce sterile protection in this mouse model and the inventors had used it only as a ‘gold standard’ to validate their viral vector. Further recombinant measles-vectored malaria vaccine candidates expressing additional pre-erythrocytic and/or blood-stage antigens in combination with CS is under evaluation. It remains to be seen if such combinations yield synergistic effects to provide protection with higher efficacy and for longer duration. rMV-vectored malaria vaccine candidates expressing additional pre-erythrocytic and/or blood-stage antigens in combination with rMV expressing PfCS may provide a path to development of next generation malaria vaccines with higher efficacy.