VACCINE IMMUNOGENS

Abstract

An immunogenic composition comprising: a) one or more Plasmodium-derived ribosomal or ribosomal associated protein or immunogenic fragment thereof which has a sequence which is at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a ribosomal or ribosomal associated protein or an immunogenic fragment of a ribosomal or ribosomal associated protein recited in FIG. 1; or a ribosomal or ribosomal associated protein or peptide or immunogenic fragment thereof as recited in FIG. 2 or FIG. 3; and/or b) a polynucleotide encoding one or more protein, peptide or immunogenic fragment of a); wherein the immunogenic composition is for use in eliciting an immune response in a subject to treat or prevent malaria. Also provided are Plasmodium-derived ETRAMPs and/or histones, or immunogenic fragments thereof, for use in eliciting an immune response in a subject, preferably to treat or prevent malaria.

Claims

1. An immunogenic composition comprising: a) one or more Plasmodium-derived ribosomal or ribosomal associated protein, peptide or immunogenic fragment thereof which has: i) a sequence which is at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a ribosomal or ribosomal associated protein, or an immunogenic fragment of a ribosomal or ribosomal associated protein, recited in FIG. 1, or ii) a sequence which is at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a ribosomal or ribosomal associated protein or peptide, or immunogenic fragment of a ribosomal or ribosomal associated protein or peptide, recited in FIG. 2 or FIG. 3; and/or b) a polynucleotide encoding one or more protein, peptide or immunogenic fragment of a); wherein the immunogenic composition is for use in eliciting an immune response in a subject to treat or prevent malaria.

2. The immunogenic composition of claim 1, wherein the one or more Plasmodium-derived ribosomal or ribosomal associated protein, peptide or immunogenic fragment thereof is derived from Plasmodium falciparum and/or from Plasmodium vivax.

3. The immunogenic composition of claim 1, wherein the immune response elicited is a protective immune response.

4. The immunogenic composition of claim 1, wherein the immune response elicited is a CD8+ T-cell response.

5. The immunogenic composition of claim 1, wherein the Plasmodium-derived ribosomal or ribosomal associated protein, peptide or immunogenic fragment thereof has a sequence which is at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the 40S ribosomal protein S20e or a fragment of 40S ribosomal protein S20e.

6. The immunogenic composition of claim 5 wherein the 40S ribosomal protein S20e is derived from P. falciparum (Accession Q8IK02) or P. vivax (Accession A5K757), preferably from P. falciparum (Accession Q8IK02).

7. The immunogenic composition of claim 1, wherein the Plasmodium-derived ribosomal or ribosomal associated protein, peptide or immunogenic fragment thereof has a sequence which is at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any of the ribosomal or ribosomal associated peptides recited in FIG. 9.

8. The immunogenic composition of claim 1, wherein the Plasmodium-derived ribosomal or ribosomal associated protein or immunogenic fragment thereof has a sequence which is at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a protein or a fragment of a protein selected from: 60S ribosomal subunit protein L4/L1, putative (Accession A5KAZ9); 40S ribosomal protein S25, putative (Accession A5K124); 40S ribosomal protein S2, putative (Accession A5K3U1); 60S ribosomal protein L9, putative (Accession A5K306); 60S ribosomal protein L30, putative (Accession A5KCD7); 40S ribosomal protein S23, putative (Accession A5KB86); 40S ribosomal protein S30 (Accession A5KBT5); 60S ribosomal protein L29 (Accession A5K3F2); 40S ribosomal protein S6, putative (Accession A5K858); 40S ribosomal protein S8 (Accession A5K1E3); 60S ribosomal protein L13, putative (Accession A5JZN9); 60S ribosomal protein L23a, putative (Accession A5K303); Ribosomal protein L37 (Accession A5KA70); and 40S ribosomal claims 1, 16, 18 and 21 protein SI 1, putative (Accession A5K7Q0).

9. The immunogenic composition of claim 1, further comprising a pharmaceutically acceptable excipient or carrier.

10. The immunogenic composition of claim 1, wherein the polynucleotide is provided in a vector.

11. The immunogenic composition of claim 10, wherein the vector is a viral vector, DNA vector or RNA vector.

12. The immunogenic composition of claim 11, wherein the viral vector comprises a Modified Vaccinia Ankara (MVA) virus or an adenovirus.

13. The immunogenic composition of claim 10, wherein the vector comprises a trypanosomatid vector.

14. The immunogenic composition of claim 1, wherein the composition comprises two or more different vectors and/or two or more ribosomal or ribosomal associated proteins, peptides or immunogenic fragments thereof.

15. A method of treating or preventing malaria in a subject comprising administering to a subject the immunogenic composition of claim 1.

16. One or more Plasmodium-derived protein, peptide or immunogenic fragment thereof which has a sequence which is at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a protein or a fragment of a protein recited in FIG. 1, or a protein or a peptide or a fragment of a protein or peptide recited in FIG. 2 or FIG. 3, for use in eliciting an immune response in a subject.

17. The one or more Plasmodium-derived protein, peptide or immunogenic fragment thereof for the use of claim 16 wherein immune response in a subject is to treat or prevent malaria.

18. An immunogenic composition comprising: a) one or more Plasmodium-derived protein, peptide or immunogenic fragment thereof which has a sequence which is at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a protein or a fragment of a protein recited in FIG. 1, or a protein or a peptide or a fragment of a protein or a peptide recited in FIG. 2 or FIG. 3; and/or b) a polynucleotide encoding one or more Plasmodium-derived protein, peptide or immunogenic fragment thereof which has a sequence which is at least about 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a protein or a fragment of a protein recited in FIG. 1, or a protein or peptide, or a fragment of a protein or peptide recited in FIG. 2 or FIG. 3.

19. The one or more Plasmodium-derived protein, peptide or immunogenic fragment of claim 17, or the immunogenic composition of claim 18, wherein the protein, peptide or fragment is an ETRAMP or is derived from an ETRAMP.

20. The one or more Plasmodium-derived protein or immunogenic fragment of claim 17, or the immunogenic composition of claim 18, wherein the protein, peptide or fragment is a histone or is derived from a histone.

21. A vector comprising a polynucleotide encoding one or more Plasmodium-derived protein, peptide or immunogenic fragment thereof which has a sequence which is 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a protein or a fragment of a protein recited in FIG. 1, or a protein or a peptide or a fragment thereof recited in FIG. 2 or FIG. 3.

22. The vector of claim 21, wherein the vector is a viral or trypanosomatid vector.

Description

[0116] There now follows by way of example only a detailed description of the present invention with reference to the accompanying drawings, in which;

[0117] FIG. 1—is a table of Plasmodium-derived proteins, including ribosomal proteins or ribosomal associated proteins, and ETRAMPs, and histones, and other non-ribosomal associated proteins which are marked with an asterix (*) derived from P. falciparum and P. vivax.

[0118] peptides identified the majority come from plasmodial ribosomal proteins. Non-ribosomal associated proteins are marked with an asterix (*)

[0119] FIG. 3—is a table of all the fragments of Plasmodium-derived identified after elution from HLA class I molecules of P. vivax-infected reticulocytes and immunopeptidomic mass spectrometry sequence analysis. Of all the peptides identified the majority come from plasmodial ribosomal proteins.

[0120] FIG. 4—demonstrates the protective efficacy of Ad-MVA encoding PfS20e ribosomal protein in immunized mice after challenge with PfS20e-expressing sporozoites transgenic for this gene, expressed using the Pbuis4 promoter. Kaplan-Meier analysis of the survival curves showed a highly significant protective effect with 2/8 mice sterilely protected with a P value of P=0.0001.

[0121] FIG. 5—demonstrates the protective efficacy of the ChAdOx1-MVA P. falciparum S20e vaccine in CD-1 outbred mice (n=10 vaccinated and 10 naive). The mice were vaccinated i.m. with 108 ifu of PfS20e-ChAdOx1 as a prime vaccination, followed eight weeks later by 107 pfu of PfS20e-MVA as a boost vaccination (by i.m. route). Mice were challenged with 1000 chimeric sporozoites i.v. The Kaplan-Meier curves illustrate the time to 1% parasitaemia, whilst statistical significance between the survival curves was assessed using the Log-Rank (Mantel-Cox) Test: p<0.0001.

[0122] FIG. 6—shows the protective efficacy of the ChAdOx1-MVA P. falciparum S20e vaccine in CD-1 inbred mice (n=10 vaccinated and 10 naive). The mice were vaccinated i.m. with 10.sup.8 ifu of PfS20e-ChAdOx1 as prime followed three weeks later by 10.sup.7 pfu of PfS20e-MVA as a target-boost vaccination (by i.v. route). Mice were challenged with 1000 chimeric sporozoites i.v. The Kaplan-Meier curves illustrate the time to 1% parasitaemia, whilst statistical significance between the survival curves was assessed using the Log-Rank (Mantel-Cox) Test: p<0.0001.

[0123] FIG. 7—demonstrates relative expression of the 60S ribosomal protein L30 P. berghei homologue compared to CSP in P. berghei-infected hepatocytes at 12, 24, 36 and 48 hours post infection by RT-qPCR normalised to levels of 18SrRNA. Average 2-ACT was calculated from two biological replicates, which had three technical replicates each. Error bars show the standard deviation for each sample. Kruskal Wallis Multiple Comparison Dunn's test compared to the relative expression of CSP. *P<0.05, **P<0.01.

[0124] FIG. 8—demonstrates peptide validation by ex vivo ELIspot assay. Selected peptides (see FIG. 9) were tested using PBMC isolated from patients infected with P. vivax (n=24), P. falciparum (n=7) and healthy donors from endemic (n=15) and non-endemic (n=6) regions for malaria. Cells were stimulated with 40S ribosomal peptides (a), 60S ribosomal peptides (b), ETRAMP peptides (c), histone peptides (d) and other peptides (e). Each symbol represents one individual. The circles in the top graphs in FIGS. 7a, b, c, d and e represent P. vivax patients, the circles in the middle graphs in FIGS. 7a, b, c, d and e represent P. falciparum patients, the triangles in the bottom graphs in FIGS. 7a, b, c, d and e represent endemic healthy donors and the squares in the bottom graph in FIGS. 7a, b, c, d and e represent non-endemic healthy donors. IFN-γ production was measured by spot counting and the results are expressed as spot-forming cells (SFC) by 1×10.sup.6 PBMCs. Positive peptides were considered as responses that induced ≥30 spots in each patient—above the dashed horizontal line. Percentage of positive individuals for each tested peptide in each infected or healthy group are depicted by colour intensity with the legend indicated at the right of the figure (f). Red, yellow, and green represent the high, middle, or poor levels of responders in the ELIspot assay.

[0125] FIG. 9—this table details the peptides used in the ELIspot assays in FIG. 8. Non-ribosomal associated proteins are marked with an asterix (*)

MATERIALS AND METHODS

[0126] 1. Humanised Mice Infected with P. falciparum

[0127] TK-NOG mice were transplanted/engrafted with human primary hepatocytes, which repopulate the damaged liver (repopulation (60-80%) as described in Soulard, V. et al. Nat Commun 6 (2015). This model allows the complete hepatic development of P. falciparum and the transition to erythrocytic stages, including the appearance of mature gametocytes. This mouse model closely mimics the physiological complexity and specificity of an in vivo infection in the human environment and is also a source of fresh human hepatocytes. Every mouse received hepatocytes from the same donor. The donor HLA alleles were HLA-A*03:01, HLA-A*11:01, HLA-B*40:01, HLA-B*50:01, HLA-C*03:02 and HLA-C*06:02. Thirteen TK-NOG mice were used in three independent experiments and infected with P. falciparum sporozoites in the tail vein as described in the Table 1.

[0128] Mice were infected sporozoites from P. falciparum, NF54 or NF135 strains, in the tail vein, and livers were harvested at discreet time points post-infection as described in Table 1 below.

TABLE-US-00001 TABLE 1 Experimental details of humanised mice infected with P. falciparum Number of Liver P. P. falciparum Harvest Sample Experiment falciparum sporozoites (days post- number number strain (10{circumflex over ( )}6) infection) 1 1 NF54 None Non-Infected 2 3 3 3 3 5 4 2 NF54 None Non-Infected 5 3 3 6 3 4 7 3 5 8, 9, 10, 11 3 NF135 None Non-Infected 12 10  3 13 10  5

[0129] 50 μm thick liver sections were obtained and histology was performed to determine the rate of infection. The rate of infection was determined by staining the parasites with an anti-HSP70 antibody and by calculating the average of schizonts counted, divided by the total number of human hepatocytes in each liver. The total number of human hepatocytes infected (rate of infection multiplied by the total number of human hepatocytes) was also calculated. The rate of infection in these mice varied from 30,000 to 60,000 per liver.

[0130] Starting from 200 mg of liver material, cells were lysed and total proteins were collected for mass spectrometry (MS) analysis. See section 3 for details.

[0131] 2. Human Reticulocytes Infected with P. vivax

[0132] Human reticulocytes from patients infected with P. vivax were purified as described in Junqueira, C. et al. Nature medicine 24, 1330-1336 (2018). Seven samples containing >99% P. vivax-infected reticulocytes varying from 4.3×10.sup.7 to 7×10.sup.8 cells (Table 2), were lysed and total proteins were collected for mass spectrometry analysis. See section 3 for details.

TABLE-US-00002 Sample ID No retics Parasitaemia 24 .sup. 5 × 10.sup.7 Pv +++ 26 1.1 × 10.sup.8 Pv +++ 28 .sup. 7 × 10.sup.8 Pv +++ 30 4.3 × 10.sup.7 Pv ++ 31 7.2 × 10.sup.7 Pv ++ 34 6.2 × 10.sup.7 Pv +++ 35 6.2 × 10.sup.7 Pv ++

[0133] Parasitemia was determined at the site of sample collection following local clinical protocols.

TABLE-US-00003 Sample HLA class I Allele ID A A B B C C 24 11:01 23:01 35:01 81:01 04:01 18:01 26 01:04 02:01 51:04 57:01 07:01 15:02 28 02:01 02:22 15:20 27:02 02:02 04:01 30 02:01 24:02 18:01 35:04 04:01 07:01 31 03:01 33:03 35:01 51:01 07:05 15:02 34 24:02 24:02 08:01 51:01 07:01 15:02 35 02:01 02:01 35:01 40:02 03:04 04:01

[0134] 3. Peptide Identification by Immunopeptidomics

[0135] 3.1 Mass Spectrometry

[0136] Cells were lysed in 1 ml lysis buffer (0.5% Igepal, 150 mM NaCl, 50 mM Tris, pH 8.0, supplemented with Complete™ protease inhibitor cocktail (Roche)). HLA complexes were immunoprecipitated using 1 mg monoclonal antibody W6/32 against HLA-ABC complexes (GE healthcare) cross-linked to Protein A Sepharose beads using dimethyl pimelimidate (DMP, Sigma). Lysates were incubated overnight. Beads were subsequently washed with 10 column volumes of 2×150 mM NaCl in 50 mM Tris, 1×450 mM NaCl in 50 mM Tris and 50 mM Tris buffer without salt. Peptides bound to the HLA groove are released after mild acid elution with 5 mL 10% acetic acid to denaturate of the alpha-chains and beta-2-microglobulin. The HLA-bound peptides were further purified from beta-2-microglobulin and alpha-chains by HPLC (Ultimate 3000) on a ProSwift RP-IS 4.6×50 mm column (Thermo Scientific) by applying a linear gradient of 2-35% (v/v) acetonitrile in 0.1% (v/v) formic acid in water over 10 min. Alternating fractions that did not contain beta-2-microglobulin or alpha-chains were pooled into two final fractions, concentrated and kept at −80° C. prior to MS analysis.

[0137] Peptides were suspended in 20 μL buffer A (1% acetonitrile, 0.1% TFA in water) and analyzed by nUPLC-MS/MS using an Ultimate 3000 RSLCnano System coupled with an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific). 9 μl of each sampled was injected and trapped onto a 3 μm particle size 0.075 mm×150 mm Acclaim PepMap RSLC column at 8 μl/min flowrate. Peptide separation was performed at 40° C. by applying a 1 h linear gradient of 3-25% (v/v) acetonitrile in 0.1% (v/v) formic acid, 5% DMSO in water at a flow rate of 250 μl/min on a 2 μm particle size, 75 μm×50 cm Acclaim PepMap RSLC column. For HLA class II samples, a linear gradient of 5-30% (v/v) acetonitrile was applied. Peptides were introduced to a Fusion Lumos mass spectrometer (Thermo Scientific) via an Easy-Spray source at 2000 V. The ion transfer tube temperature was set to 305° C. Measurement of precursor peptides was performed with a resolution of 120,000 for full MS (300-1500 m/z scan range) at an AGC target of 400,000. Precursor ion selection and fragmentation by high-energy collisional dissociation (HCD at 28% collision energy for charge state 2-4, 35% for charge state 1) was performed in TopSpeed mode at an isolation window of 1.2 Da for singly to quarterly charged ions at a resolution of 30,000 and an AGC target of 300,000 in the Orbitrap for a cycle duration of 2 s. Singly charged ions were acquired with lower priority.

[0138] 3.2 Peptide Identification

[0139] MS data was analyzed with Peaks 8 (Bioinformatics Solutions and Tran, N. H. et al. Nature methods 16, 63-66 (2019)) for identification of peptide sequences. Spectra were matched to all reviewed human proteins combined with Plasmodium falciparum (isolate 3D7) or Plasmodium vivax (Salvador I), produced based on UniProt proteomes (UniProt, C. UniProt: a worldwide hub of protein knowledge. Nucleic acids research 47, (2019)). The results were filtered using a score cut-off of −1g10P=15. The searches were performed with the following parameters: no enzyme specificity, no static and variable modifications, peptide tolerance: ±5 ppm and fragment tolerance: ±0.03 Da.

[0140] Human sequences were disregarded from the analysis. The peptide spectrum matches (PSMs) of plasmodial origin obtained were analysed following a pipeline consisting of: 1) Size exclusion: MHC-I peptides longer than 15 amino were excluded. Peptides smaller than 8 amino acid long were excluded; 2) A Peaks score cut-off of 15 was applied to all samples; 3) False positive peptides, that is, peptides with incorrect identification were removed from the samples; 4) A stringent blast analysis was performed in every peptides sequence using the DeBosT script (see section 3.3). Peptides with two or more amino acid different from human sequences were considered non-human and therefore identified as plasmodial peptides. Peptides with higher netMHC rank were prioritized. The amino acids leucine (L) and isoleucine (I) are isomers, which are indistinguishable from each other through the mass spectrometry protocol. All peptides were blasted for all possible combinations of I and L, and when a combination matched a human peptide, the sequence was excluded. Applying these four-step data analysis criteria, a list of Plasmodium peptides were identified, see FIG. 2 and FIG. 3.

[0141] 3.3 DeBosT Script

[0142] Blast searches (National Library of Medicine, https.//blast.ncbi.nlm.nih.gov) of putative plasmodial sequences were performed using a batch script. Sequences that have less than two amino acid differences compared to human sequences were excluded from downstream analysis (Bettencourt, P. et al. Identification of antigens presented by MHC for vaccines against tuberculosis. npj Vaccines 5, 2 (2020)).

[0143] 3.4 Peptide Validation

[0144] Malaria infected patients and healthy donors. P. vivax infected patients were recruited in the Tropical Medicine Research Center (Porto Velho-Brazil) along with healthy donors from the same endemic region and from a non-endemic region (Belo Horizonte-Brazil). All participants provided written informed consent for participation in the study, which has a protocol approved by the Institutional Review Boards of the Oswaldo Cruz Foundation and National Ethical Council (CAAE: 59902816.7.0000.5091). Samples were collected at three different times. Firstly, 7 samples from P. vivax infected patients were obtained for the mass spec experiments. Twenty two samples from P. vivax infected patients, eighteen healthy donors from the endemic region and 6 from a non-endemic region posteriorly were collected for the Elispot assay.

[0145] PBMC and P. vivax infected reticulocytes obtention. 100 mL of blood was collected from infected individuals and controls. First, mononuclear cells were separated from peripheral blood (PBMCs). For this, the blood was diluted in a 1:1 ratio, was gently added to a tube containing 15 mL of Ficoll (GE Healthcare, USA). Red blood cell pellet resulting from PBMC purification was resuspended in RPMI culture medium in a 1:4 ratio. Diluted blood was added carefully into a 50 mL tube containing Percoll 45% (GE Healthcare, USA), 5× the volume of the red blood cell pellet. Samples were centrifuged for 15 minutes at 2000 rpm. After centrifugation, the reticulocyte interface was collected. Reticulocyte purified samples had 99% purity.

[0146] ELISPOT assay. The ex vivo IFN-γ ELISpot assays were performed using 5×10.sup.5 fresh PBMCs from P. vivax infected patients, endemic and non-endemic healthy donors. Cells were plated in duplicates into 96-well ELISpot plates (Merck Millipore) precoated with 4 μg/ml anti-human-IFN-γ (clone 1-D1K; Mabtech). Peptides of Table 3 (FIG. 9) were tested with stimulation of 10 μg/ml and stimulated for 18-20 hour at 37° C. under 5% CO.sub.2. Anti-CD3/anti-CD28 antibodies were used as positive control and medium alone as negative control (subtracted from all conditions). After cell removal, plates were developed for 2 hours in the same temperature condition of the stimulus in the presence of 0.2 μg/ml IFN-γ, 7-B6-Biotin (Mabtech). Spot detection was performed following incubation for 30 min in the dark with BCIP/NBT Alkaline Phosphatase Substrate (Sigma). Spot-forming cells (SFC) were counted using the ImmunoSpot automated Elispot counter.

TABLE-US-00004 TABLE 3 peptides used for ELISpot assay. Accession number ELISPOT ID Protein Peptide Sequence A5K3U1 40S S2 40S ribosomal protein S2 LETYQNMKIQKQTP A5K858 40S S6-1 40S ribosomal protein S6 SKNGKNRFIKPKIQ A5K858 40S S6-2 40S ribosomal protein S6 GVKKDVAK A5K858 40S S6- 3 40S ribosomal protein S6 GPKRATKIRK A5K1E3 40S S8- 1 40S ribosomal protein S8 RLTGGKKKIHKKK A5K1E3 40S S8- 2 40S ribosomal protein S8 GSKQVHV A5K7Q0 40S S11 40S ribosomal protein S11 SFFNSKKIKKGSKS A5KB86 40S S23 40S ribosomal protein S23 SSHAKGIVVEKV A5K124 40S S25-1 40S ribosomal protein S25 GKGKNKEKL A5K124 40S S25-2 40S ribosomal protein S25 GKGKNKEKLNHAVF A5KBT5 40S S30-1 40S ribosomal protein S30 SDGTGRKKGPNSKL A5KBT5 40S S30-2 40S ribosomal protein S30 GTGRKKGPNSKL A5KBT5 40S S30-3 40S ribosomal protein S30 TGRKKGPNSKL A5K3E2 50S S28e 50S ribosomal protein S28e GDTELSGRFL A5KAZ9 60S L4/L1-1 60S ribosomal subunit protein L4/L1 ANKALLPTAGDD A5KAZ9 60S L4/L1-2 60S ribosomal subunit protein L4/L1 NKALLPTAGDD A5KAZ9 60S L4/L1-3 60S ribosomal subunit protein L4/L1 YGRIFKKKITKK A5K306 60S L9 60S ribosomal protein L9 VSEVTTVEKDE A5K186 60S L10 60S ribosomal protein L10 GAFGKPNGV A5K762 60S 13a 60S ribosomal protein L13a MYKKVYVID A5JZN9 60S L13-1 60S ribosomal protein L13 YESIEVSKID A5JZN9 60S L13-2 60S ribosomal protein L13 GTPIEKLHPI A5JZN9 60S L13-3 60S ribosomal protein L13 KNIKSKNGIGGIPAD A5K3F2 60S L29 60S ribosomal protein L29 PKFFKNQRY A5KCD7 60S L30 60S ribosomal protein L30 VITDVGDSDIIKTNE A5KAW8 60S L31 60S ribosomal protein L31 AKAVKKQKKTLKPV A5K6B0 60S L32-1 60S ribosomal protein L32 AVKKVGKIVKKRT A5K6B0 60S L32-2 60S ribosomal protein L32 AVKKVGKIVK A5K4R0 60S L35 60S ribosomal protein L35 KKYKNKKFKPY A5KBH5 ETRAMP-1 Early transcribed membrane protein (ETRAMP) KKVAAGYKKLTD A5KBH5 ETRAMP-2 Early transcribed membrane protein (ETRAMP) GLNQKQPTKGSNIQ A5KBH5 ETRAMP-3 Early transcribed membrane protein (ETRAMP) LNQKQPTKGSNIQ A5KBH5 ETRAMP-4 Early transcribed membrane protein (ETRAMP) LGGLNQKQPT A5K676 ETRAMP-5 Early transcribed membrane protein (ETRAMP) KSAGADSKSLKKLD A5K676 ETRAMP-6 Early transcribed membrane protein (ETRAMP) TPIITNKPFG A5K214 Hist. H2A-1 Histone H2A GRIGRYLKKGKYAK A5K214 Hist. H2A-2 Histone H2A ASGGVLPNIHNV A5K214 Hist. H2A-3 Histone H2A SGGVLPNIHNV A5K214 Hist. H2A-4 Histone H2A GRIGRYLKKGKYA A5K7L8 Hist. H2A-5 Histone H2A KVPVPPTQAKKPKKN A5K1U7 Hist. H3 Histone H3 APISAGIKKPHR A5K8J8 Unch A5K8J8 Uncharacterized protein LILRAAIKTK A5JZN7 Unch A5JZN7.1 Uncharacterized protein DNNEHVVQEKTVSF A5JZN7 Unch A5JZN7.2 Uncharacterized protein DNNEHVVQEKTV A5K8G9 Unch A5K8G9 Uncharacterized protein EDYSPRKV A5K2R4 Ubqt/ribos-1 Ubiquitin/ribosomal AIEPSLAQLAQK A5K2R4 Ubqt/ribos-2 Ubiquitin/ribosomal NQLRPKKKLK A5K197 Don Juan Sperm-specific protein Don juan AQKIKKKKKLTPA

[0147] 3.5 Spectral Match Validation

[0148] To further confirm the identity of the peptide sequences identified, a spectral match validation experiment will be performed. Synthetic peptides will be produced and compared to a selection of PSMs obtained from the experiments (the biological peptides). Synthetic peptides will be run in the same experimental conditions as biological peptides. The mass over charge [m/z] for each peptide, the charge state, the intensity and distribution of each peak within each peptide sequences, as well as the peptide specific retention time (RT) on the Liquid Chromatography will be compared between synthetic and biological peptides.

[0149] 3.6 CD8+ T-Cell Response

[0150] The CD8+ T-cell responses from healthy patients from endemic and non-endemic areas for malaria, and those of P. falciparum and P. vivax-infected patients were analysed against peptides identified by immunopeptidomics (Table 3), using IFN-gamma ELISpot. Several peptides, including ribosomal protein peptides were shown to be recognised in ex vivo ELISPOT assays. This demonstrated that peptides displayed on MHC-1 molecules on reticulocytes, including those derived from ribosomal proteins, are naturally immunogenic in human infections and thus are good immunogens for vaccination.

[0151] 3.7 Efficacy and Immunogenicity of New Vaccine Candidates

[0152] The immunogens described here may be cloned into viral vectors for use as vaccines. The platform of subunit vaccines has proved to be very safe and is very powerful in inducing CD8+ T-cell responses against the antigen that is being expressed. Generation of ChAdOx1, AdHu5 and/or MVA expressing each antigen may be cloned using GeneArt Technology (ThermoFisher Scientific, UK). Subunit vaccines expressing liver stage antigens may be produced. Immunogens may be mammalian codon-optimized flanked by a Kozak consensus sequence, a tPA leader sequence and a GS linker at the 5′-end and at the 3′ end, cloned into a GeneArt entry vector and then recombined into an ChAdOx1, AdHu5 and/or MVA destination plasmid as previously described in Dicks, M. D. et al. PloS one 7 (2012) and McConkey, S. J. et al. Nature medicine 9, 729-735 (2003). Efficacy and immunogenicity of the viral vectors will be evaluated in a malaria challenge mouse model.

EXAMPLES

Example 1

[0153] Plasmodium vivax is the most widespread cause of human malaria and the second most lethal after P. falciparum. Unusually, P. vivax preferentially infects reticulocytes and recently it has been demonstrated that P. vivax-infected patients have circulating CD8+ T cells that recognize and form immunological synapses with P. vivax-infected reticulocytes in an HLA-dependent manner, releasing their cytotoxic granules to kill both host cell and intracellular parasite, preventing reinvasion. 50 and 700 million reticulocytes per subject were obtained from seven Brazilian subjects infected with P. vivax, as described in section of the materials and methods.

[0154] 453 unique peptides were identified by tandem mass spectrometry sequencing and these were from 176 distinct P. vivax antigens. There was significant overlap in immunogens identified in the six subjects, with peptides from 29 antigens found in at least 50% of the subjects and peptides from two antigens found in all six subjects, with high quality data. A most striking and unexpected finding was that over half of the peptides (57%) came from a single class of proteins, plasmodial ribosomal or ribosome associated proteins. A list of peptides identified and the protein they are derived from is provided in FIG. 2 and FIG. 3.

[0155] Ribosomal proteins are species-specific and between humans and Plasmodia, most ribosomal proteins share approximately 60% sequence identity on average. This divergence provides adequate differences for regions on non-identify between human/mammalian ribosomal and parasite ribosomal sequences to avoid self-tolerance and be suitably immunogenic. However, proteins with less homology to humans are preferred so as to potentially maximise immunogenicity. Furthermore proteins with identify or greater similarity between P. falciparum and P. vivax are preferred because they are more likely to provide a cross-species protective effect.

[0156] Further, ribosomes are required for protein production and their structure and mechanism of polypeptide generation are well understood. Cells and microbes that are rapidly dividing or very metabolically active may need a lot of ribosomes and ribosomal proteins to engage in the required protein synthesis. This is likely to be true of parasitized reticulocytes in which P. vivax grows very rapidly. Similarly, within hepatocytes, malaria parasites generally grow very rapidly. For example, in the case of P. falciparum, one sporozoite infects a liver cell and seven days later 20,000 parasites with a largely different antigenic composition, malaria merozoites, emerge from the same liver cell. Therefore, the findings of the mass spectrometry analysis support the fact the parasite will need to generate a lot of ribosomal proteins intracellularly, which are capable of ending up on the HLA class I molecules in parasitized cells.

[0157] In addition to the vast majority of ribosomal proteins identified in the mass spectrometry analysis of peptides eluted from HLA-I expressed on infected reticulocytes, two Pv ETRAMPs peptides that are expressed both in hepatocyte and reticulocyte stages were also identified. The ETRAMPs compose a family of polymorphic, small, highly-charged transmembrane proteins unique to malaria parasites, they localize in the parasitophorous vacuole membrane (PVM) with the C-terminal region exposed to the RBC cytosol and are also exported to the host cell cytoplasm. Therefore, the ETRAMPs are accessible to the protein machinery that processes and presents endogenous antigens. Furthermore, they are expressed in the first hours of invasion and, thus, the infected reticulocytes may become targets to CTLs at the early stages of infection. In addition, the ETRAMPs are recognized by antibodies from Plasmodium falciparum and Plasmodium vivax malaria patients and CD4+ T cells from P. berghei-infected mice. In conclusion, the HLA-I binding and biology of ETRAMPs suggest that they could be key targets for protective CD8+ T cell-mediated immunity against malaria. Like ribosomal proteins, ETRAMPs have not been employed or evaluated for CD8+ T cell mediated immunity.

Example 2

[0158] As a proof of concept, one plasmodial ribosomal protein gene was selected and expressed to test the concept that plasmodial ribosomal proteins could be suitable immunogens for developing immunogenic, protective malaria vaccines.

[0159] The P. falciparum 40S ribosomal protein S20e was selected, which is 118 amino acids in length (sequence: MSKLMKGAIDNEKYRLRRIRIALTSKNLRAIEKVCSDIMKG AKEKNLNVSGPVRLPVKTLRITTRKSPCGEGTNTWDRFELRIYKRLIDLYSQCE VVTQMTSINIDPVVEVEVIITDS, Uniprot accession: PF3D7_1003500.1). The protein was expressed in both a simian adenoviral vector ChAdOx1 and in MVA. BALB/c Mice were immunised with a single shot of 1×10.sup.8 infectious units of the ChAd recombinant and boosted with the MVA three weeks later with a dose of 1×10.sup.8 pfu and challenged intravenously three weeks later with 1000 P. berghei sporozoites transgenic for P. falciparum 40S ribosomal protein S20e (expressed under a Pbuis4 promoter). There was highly significant protective efficacy with 2 of 8 mice sterilely protected and the remaining six delayed in time to parasitaemia reflecting a substantial reduction of liver parasite load, P=0.0001 (FIG. 5).

[0160] CD-1 mice were immunised with a single shot of 1×10.sup.8 infectious units of the ChAd recombinant and boosted with the MVA eight weeks later with a dose of 1×10.sup.8 pfu and challenged intravenously three weeks later with 1000 P. berghei sporozoites transgenic for P. falciparum 40S ribosomal protein S20e (expressed under a Pbuis4 promoter). There was highly significant protective efficacy with 3 of 10 mice sterilely protected and the remaining six delayed in time to parasitaemia reflecting a substantial reduction of liver parasite load, P=0.0001 (FIG. 6).

[0161] Using prime-target vaccination regime in CD-1 mice, there was highly significant protective efficacy with 5 of 10 mice sterilely protected and the remaining 5 delayed in time to parasitaemia reflecting a substantial reduction of liver parasite load, P=0.0001 (FIG. 7).

[0162] This efficacy with a Plasmodium falciparum ribosomal protein immunogen demonstrates that ribosomal protein immunogens are a new class of antigens for malaria vaccination, especially to target the liver-stage of infection.

Example 3

[0163] To allow analysis of larger numbers of P. falciparum-infected human hepatocytes, a recently described mouse strain (TK-NOG) in which most of the mouse liver has been replaced by human hepatocytes was utilised (which unlike mouse hepatocytes will support invasion and growth of P. falciparum).

[0164] The TK-NOG mice (Soulard et al Nature Communications 2015) express the HSVtk transgene under the albumin promoter onto the NOD SCID IL2Rg/background. In this mouse strain, the loss of endogenous hepatocytes is inducible by a brief exposure to a non-toxic dose of ganciclovir, a method that is rapid and temporally restricted, and routinely leads to substantial repopulation (60-80%) with human hepatocytes. The human herpes simplex virus thymidine kinase type 1 gene (HSVtk) acts as a conditional lethal marker in mammalian cells. The HSVtk-encoded enzyme is able to phosphorylate certain nucleoside analogs (e.g. ganciclovir, an antiherpetic drug), thus converting them to toxic DNA replication inhibitors. The utility of HSVtk is a conditional negative-selection marker.

[0165] These TK-NOG mice were infected with 1×10.sup.7 P. falciparum sporozoites of a rapidly growing strain (e.g. P. falciparum NF135) and livers were removed at 3-5 days post-infection. After applying the immunopeptidomics pipeline to identify peptides bound to MHC molecules, Table 4 was obtained. Remarkably, the peptide sequence VITDVGDSDIIKTNE that is part of the protein W4IGC0 form P. falciparum NF135 (Ribosomal_L7Ae domain-containing protein) (FIGS. 2 and 3), was also found amongst the peptides eluted from P. vivax infected reticulocytes, here designated as protein A5KCD7 (60S ribosomal protein L30) (Table 4). Moreover, another peptide from the protein W41D94 form P. falciparum NF135 (Ribosomal_L23eN domain-containing protein) was identified in this example (Table 4). Peptides from the corresponding homologous protein in P. vivax were also identified amongst the peptides eluted from P. vivax infected reticulocytes, here designated as protein A5K303 (60S ribosomal protein L23a) (FIGS. 2 and 3).

[0166] Finding two antigens in a short list of few confirmed eluted peptides from this type of experiment using human hepatocytes, provides clear evidence that ribosomal protein peptides can be presented on the HLA class I molecules of P. falciparum-infected liver cells. This further supports the concept that vaccines based on ribosomal proteins would be effective malaria vaccines.

TABLE-US-00005 TABLE 4 Peptides identified by immunopeptidomics in humanized mice infected with P. falciparum NF135. Peptide Identifier AAEEGAKAGAL tr|W4I970|W4I970_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_05454 PE = 4 SV = 1 DNNNYDDDEII tr|W4IEZ4|W4IEZ4_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_03293 PE = 4 SV = 1 E(-18.01) tr|W4IC91|W4IC91_PLAFA Actin-1 OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 EYDESGPSIVHR GN = PFNF135_04291 PE = 3 SV = 1 GGATGAGLAL tr|W4IN72|W4IN72_PLAFA Glycerol-3-phosphate dehydrogenase OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_00472 PE = 3 SV = 1 GK(+42.05) tr|W4IAJ3|W4IAJ3_PLAFA Elongation factor 1-alpha OS = Plasmodium falciparum NF135/5.C10 EKTHINLVVIGHVD OX = 1036726 GN = PFNF135_05027 PE = 3 SV = 1 HGNNMNTCLM tr|W4IGK7|W4IGK7_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_03672 PE = 4 SV = 1 KIYEKKILK tr|W4IH49|W4IH49_PLAFADNA polymerase OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_03009 PE = 3 SV = 1 KTATLGVI tr|W4IGU9|W4IGU9_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_02418 PE = 4 SV = 1:tr|W4IGJ3|W4IGJ3_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_02842 PE = 4 SV = 1 LEGKELPG tr|W4IHK5|W4IHK5_PLAFA Phosphoglycerate kinase OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_02647 PE = 3 SV = 1 LVTDVGDSDIIKTNE tr|W4IGC0|W4IGC0_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_03035 PE = 4 SV = 1 NEAEEFEDY tr|W4IKH7|W4IKH7_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_01561 PE = 4 SV = 1 PEEVAEELV tr|W4IFR1|W4IFR1_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_03212 PE = 4 SV = 1 PQNINEYF tr|W4ICP6| W4ICP6_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX-1036726 GN = PFNF135_05101 PE = 4 SV = 1 QGGGSPLLGT tr|W4IM59|W4IM59_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_00968 PE = 4 SV = 1 QGISDDSSIHH tr|W4IBP5|W4IBP5_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_05175 PE = 4 SV = 1 SLGSSILTK tr|W4IB92|W4IB92_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_05237 PE = 4 SV = 1 SLNDALIVSI tr|W4IAD7|W4IAD7_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_06050 PE = 4 SV = 1 VANKIGIL tr|W4ID94|W4ID94_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_04649 PE = 3 SV = 1 VITDVGDSDIIKTNE tr|W4IGC0|W4IGC0_PLAFA Uncharacterized protein OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_03035 PE = 4 SV = 1 VLPELNGK tr|W4I9B4|W4I9B4_PLAFA Glyceraldehyde-3-phospliate dehydrogenase OS = Plasmodium falciparum NF135/5.C10 OX = 1036726 GN = PFNF135_05644 PE = 3 SV = 1

Example 4

[0167] Peptide Validation by ELIspot Assay

[0168] 48 peptides were tested by ex vivo IFN-γ ELIspot assay. The peptides are detailed in FIG. 9. All peptides were tested in P. vivax, P. falciparum and Endemic and Non-endemic healthy donors. The peptides were divided into ribosomal peptides, ETRAMP peptides, hi stone peptides and other peptides.

[0169] All ribosomal peptides were immunogenic in the P. vivax tested samples and twelve were positive in at least 70% of the patients (FIG. 8a). Peptide 40S S11 for example, was immunogenic in 86% of P. vivax samples, and 60S L4/L1-2, 60S L9, 60S L13-a, 60S L13-3 and 60S L29 were positive in 72%. P. falciparum samples were positive for nine ribosomal peptides. Among these peptides, three were immunogenic in more than half of P. falciparum samples, the 40S S8, 40S S11, 60S L13-1 and the 60S L32-2. Only one healthy donor individual from a non-endemic area showed a positive response to seven ribosomal peptides, whereas in healthy donors from an endemic malaria region, only 26% of the individuals showed a positive response after stimulation with ribosomal peptides.

[0170] All the ETRAMP peptides tested were immunogenic in the P. vivax samples, ranging from 47.8 to 82.6% positivity in the tested patients (FIG. 8c). Regarding P. falciparum, from six ETRAMP peptides tested, three showed a positive response for at least half of the patients. The higher positivity in healthy donors from an endemic area was 26%, while healthy donors from a non-endemic area did not show a positive response to any peptide.

[0171] All the histone peptides tested were immunogenic in all P. vivax and all P. falciparum tested samples. In P. vivax the rate of responder patients ranged between 40% and 71.4% (FIG. 8d) and P. falciparum at least two in seven patients obtained a positive response to these peptides. No healthy individual from a non-endemic area showed a positive response, whereas the rate of positivity in healthy individuals from an endemic area was only 13.33%.

[0172] The Uncharacterized proteins or Don Juan peptides showed a rate of patients with positive responses between 45 and 63.6% (FIG. 8e). From the groups of patients infected with P. falciparum or from healthy donors in an endemic malaria region, a positive response was observed in one individual for all the peptides tested. No individual from a non-endemic area showed a positive response to these peptides.