Vaccines
11197920 · 2021-12-14
Assignee
Inventors
Cpc classification
A61K39/39
HUMAN NECESSITIES
A61K39/015
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
A61K39/015
HUMAN NECESSITIES
Abstract
The present invention relates to particles, particularly virus-like particles (VLPs), comprising fusion polypeptides comprising selected repeat units derived from the repeating regions of Type I and Type II circumsporozoite proteins (CSP) of Plasmodium vivax (Pv), together with an amino acid sequence derived from the C-terminal PvCSP sequence. In some embodiments, the fusion polypeptide additionally comprises an amino acid sequence derived from the N-terminal PvCSP sequence and/or a surface antigen polypeptide derived from Hepatitis B virus (HBV-S). The invention also relates to nucleotide sequences coding for such fusion polypeptides, vectors and plasmids comprising such nucleotide sequences, and host cells comprising such vectors and plasmids. The invention additionally relates to compositions, particularly vaccine compositions, comprising the fusion polypeptides or VLPs for use as vaccines for the prevention of malaria.
Claims
1. A fusion polypeptide or a particle comprising the fusion polypeptide, wherein the fusion polypeptide comprises: (i) the Plasmodium vivax Type I circumsporozoite protein (CSP) repeat units: TABLE-US-00010 (SEQ ID NO: 3) (GDRAAGQPA)*5 (SEQ ID NO: 4) (GDRADGQPA)*4 (SEQ ID NO: 5) (GNGAGGQAA)*1 (SEQ ID NO: 11) (ANGAGNQPG-ANGAGGQAA)*2 (SEQ ID NO: 12) (ANGAGDQPG-ANGAGDQPG)*1 (SEQ ID NO: 13) (ANGADDQPG-ANGAGDQPG)*1 and (SEQ ID NO: 14) (EDGAGNQPG-ANGAGDQPG)*1 (ii) the Plasmodium vivax Type II circumsporozoite (CSP) repeat units: TABLE-US-00011 (ANGAGNQPG-ANGAGGQAA)*2 (SEQ ID NO: 11) (ANGAGDQPG-ANGAGDQPG)*1 (SEQ ID NO: 12) (ANGADDQPG-ANGAGDQPG)*1 (SEQ ID NO: 13) and (EDGAGNQPG-ANGAGDQPG)*1 (SEQ ID NO: 14) wherein the above Type I and Type II repeat units are joined contiguously in the above order in a N-C orientation, and *5 represents multiplied by 5, *4 represents multiplied by 4, *2 represents multiplied by 2, and *1 represents multiplied by 1; and (iii) (a) an amino acid sequence having at least 80% sequence identity to one or more of SEQ ID NOs: 29-32; or (b) a fragment of SEQ ID NOs: 29-32 which is at least 50% of the length of SEQ ID NOs: 29-32.
2. The fusion polypeptide or particle as claimed in claim 1, wherein the fusion polypeptide additionally comprises: (iv) at least one repeat unit of a circumsporozoite protein (CSP) of a Plasmodium vivax-like malaria parasite, wherein the amino acid sequence of the at least one repeat unit is APGANQ(E/G)GGAA (SEQ ID NO: 19).
3. The fusion polypeptide or particle as claimed in claim 1, wherein the fusion polypeptide additionally comprises: (v) (a) an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 38; or (b) a fragment of SEQ ID NO: 38 which is at least 80% of the length of SEQ ID NO: 38.
4. A pharmaceutical composition comprising the fusion polypeptide or particle as claimed in claim 1, optionally together with one or more pharmaceutically-acceptable carriers, excipients or diluents.
5. A vaccine composition comprising the fusion polypeptide or particle as claimed in claim 1, together with a pharmaceutically-acceptable adjuvant.
Description
BRIEF DESCRIPTION OF THE FIGURES
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(8) Right panel: D. PbANKA-PvCSP(r).sub.PbCSP: 2196cl1 and 2199cl1. the correct integration of the PvCSP expression construct into the GIMO locus was confirm by showing the removal of the hdhfr::yfcu SM cassette in the cloned chimeric parasite line (2196cl1 and 2199cl1). The southern blot is hybridized with a mixture of two probes: one recognizing hdhfr and a control probe recognizing chr-5. As an additional control (ctrl), parasite line 2117cl1 is used with the hdhfr::yfcu SM integrated into chr-3.
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(13) Groups of mice (n=6) were immunized with vv expressing PvCSP (N210/N247C, N210C, N247C, NC; see
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EXAMPLES
(20) The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
(21) The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.
Example 1: Materials and Methods
(22) Materials and Methods
(23) Animals
(24) Female inbred BALB/c (H-2.sup.d), C57BL/6 (H-2.sup.b) and outbred CD1 (ICR) mice were used for the assessment of immunogenicity and protection after challenge. Tuck-ordinary (TO) outbred mice were used for parasite production and transmission. Mice were purchased from Harlan (UK). Transgenic parasites were developed in Leiden University Medical Centre (LUMC) using 6-week old Swiss mice (Charles River).
(25) Parasite Production
(26) Wild type and transgenic parasites used to challenge mice were produced at the insectary of the Jenner Institute. Female Anopheles stephensi mosquitoes were fed on infected TO mice. Briefly, exflagellation was first confirmed and mosquitoes were exposed to anaesthetized infected mice for 10 minutes. Mosquitoes were then maintained for 21 days in a humidified incubator at a temperature of 19-21° C. on a 12 hour day-night cycle and fed with a fructose/PABA solution.
(27) Parasites
(28) The wild type (WT) reference line cl15cy1 of P. berghei ANKA [48] and the reporter PbANKA parasite line PbGFP-Luc.sub.con (676m1cl1). PbGFP-Luc.sub.con parasite expresses a fusion protein of GFP (mutant3) and firefly luciferase (LUC-IAV) under the constitutive eefla promoter and is SM free [48]. The reporter-cassette is integrated into the neutral 230p locus (PBANKA_030600). For details of PbGFP-Luc.sub.con, see RMgmDB entry #29 (www.pberghei.eu/index.php?rmgm=29).
(29) Laboratory Animals and Ethics Statement
(30) For the generation of the chimeric parasites Swiss mice (OF1 ico, Construct 242; 6 weeks old; 25-26 g; Charles River) were used. All animal experiments performed at the LUMC were approved by the Animal Experiments Committee of the Leiden University Medical Center (DEC 12042). The Dutch Experiments on Animals Act was established under European guidelines (EU directive no. 86/609/EEC regarding the Protection of Animals used for Experimental and Other Scientific Purposes).
(31) Generation of DNA Constructs and Genotyping of the Chimeric Parasites
(32) To generate the chimeric parasites where the P. berghei csp gene (PBANKA_040320) coding sequence (CDS) has been replaced by the CDS of P. vivax csp (PVX_119355), we used a 2-step GIMO transfection protocol [31, 49]. In the first step, we deleted the P. berghei csp CDS and replaced it with the positive-negative selectable marker, to create a P. berghei csp deletion GIMO line (PbANKA-CS GIMO). In order to do this, we generated pL1929 construct that is based on the standard GIMO DNA construct pL0034 [49]. This construct contains the positive-negative (hdhfr.:yfcu) selection marker (SM) cassette, and was used to insert both the Pbcsp 5′ and 3′ gene targeting regions (TR), encompassing the full length promoter and transcription terminators sequences respectively. The linear pL1929 DNA construct was introduced into PbGFP-Luc.sub.con parasites using standard methods transfection [48]. Transfected parasites were selected in mice by applying positive selection by providing pyrimethamine in the drinking water [48]. Transfected parasites were cloned by limiting dilution [50], resulting in the PbANKA-CS GIMO line (2151cl1). Correct deletion of the P. berghei csp CDS was confirmed by diagnostic PCR-analysis on gDNA and Southern analysis of pulsed field gel (PFG) separated chromosomes as described [48]. Primers used for PCR genotyping are listed in Table S1 below.
(33) TABLE-US-00006 TABLE S1 Expected PCR PCR size Primer name Primer sequence.sup.1 product (bp) PvCSP-trans (F6) TGACATGCATATGTGTTGGTTG PvCSP 5′ 744 PvCSP-trans (R5) (SEQ ID NO: 48)/ int. GCTGATTGTCCAACATGTGC (SEQ ID NO: 49) 1054 CCAAAGGAACTTAAACGAGCTATG PbCSP 744 1055 (SEQ ID NO: 50)/ CTTATACCAGAACCACATGTTACG (SEQ ID NO: 51) 7393 ATCGACTAGTAAAGCCTCGCTACG PbTRAP 1052 7394 (SEQ ID NO: 52)/ Control GTGAGTCAGATGGACTTTCTGGTAG (SEQ ID NO: 53) .sup.1The primer sequences (i.e. Forward/Reverse) surrounding the regulatory region are shown with the restriction site used (bold and underlined) to introduce the region into the transfection construct.
(34) In the second step, we replaced the positive-negative SM in the PbANKA-CS GIMO genome with the CDS of either P. vivax VK210 or VK247 csp by GIMO transfection to create the two P. berghei transgenic CSP replacement lines. This was achieved by modifying the construct used in the first step (pL1929); specifically, the hdfhr::yfcu SM cassette was removed and replaced with P. vivax csp CDS sequence. The P. vivax csp CDS was ordered from GeneArt (Regensburg, Germany) (i.e. VK210) or cDNA (VK247) Both the P. vivax VK210 and VK247 CSP constructs (pL1942 and pL1943, respectively) were sequenced to ensure there were no mutations in the P. vivax csp CDS. These constructs were linearized using SacI and PacI restriction enzymes outside of the 5′ and 3′ TRs before transfection. These constructs were used to transfect parasites of the PbANKA-CS GIMO line (2151cl1) using standard methods of GIMO-transfection [30]. Transfected parasites were selected in mice by applying negative selection by providing 5-fluorocytosine (5-FC) in the drinking water of mice [51]. Negative selection results in selection of chimeric parasites where the hdhfr.:yfcu SM in the csp locus of PbANKA-CS GIMO line is replaced by the CDS of P. vivax CSP Selected chimeric parasites were cloned by the method of limiting dilution [50]. Correct integration of the constructs into the genome of chimeric parasites was analyzed by diagnostic PCR-analysis on gDNA and Southern analysis of pulsed field gel (PFG) separated chromosomes as described [48]. Primers used for PCR genotyping are listed in Table S1. This method creates chimeric ‘gene replacement’ P. berghei parasites that do not contain P. berghei csp gene CDS but express either P. vivax VK210 (PbANKA-PvCS VK210(r).sub.PbCS; 2196cl1) or VK247 csp (PbANKA-PvCS VK247(r).sub.PbCS; 2199cl1) under the control of the P. berghei csp regulatory sequences.
(35) Phenotyping of Reporter and Chimeric Parasites
(36) Growth of blood stages of the reporter and chimeric P. berghei parasites was determined during the cloning period as described [30, 48]. Feeding of A. stephensi mosquitoes, determination of oocyst production, sporozoite collection were performed as described [30]. Expression of PvCSP-VK210 and PvCSP-VK247 antigens in sporozoites of the chimeric parasites was analysed by immunofluorescence-staining assay (IFA), using anti-P. vivax antigen monoclonal antibodies (anti-PvCSP-VK210 (MR4) or anti-PvCSP-247 (MR4) antibodies; diluted 200 times) or anti-PbCSP 3D11 antibodies as a control; diluted 1000 times. Purified sporozoites were fixed with 4% paraformaldehyde in PBS for 20 min on ice, then washed three times with PBS and blocked with 20 ul 10% FCS+1% BSA in PBS for 30 min at room temperature. The excess blocking medium was removed, followed by the addition of 20-25 uL primary monoclonal antibody in 10% FCS+1% BSA in PBS (blocking medium) for 1-2 hours at room temperature or overnight at 4° C. After incubation the primary antibody was removed and the slides washed three times with PBS, followed by the staining with the secondary antibody (Alexa Fluor® 488 Goat Anti-Mouse IgG from life technologies, Cat #A-11001) diluted 800 times in 10% FCS+1% BSA in PBS (blocking medium) for 1 hour at room temperature. After washing three times with PBS, nuclei were stained with 2% Hoechst-33342 (Cell Signaling Technology #4082S) in PBS for 10 minutes at room temperature, washed twice with PBS and left to air-dry, this followed by adding Fluorescence Mounting Medium (Dako, code S3023) before complete dry out. Cover slips were mounted onto the slides, and the slides were sealed with nail polish and left to dry overnight in dark. The parasites in both blue and green channels were analyzed using a DMI-300B Leica fluorescence microscope and images processed using ImageJ software.
(37) Antibody recognition to native, wild type P. vivax isolates from Mexico was assessed by IFA using a technique described earlier [52]. Briefly, sporozoites were produced by infection of laboratory-reared An. albimanus mosquitoes that fed on blood from patients infected with P. vivax. Mosquitoes were maintained for 15-18 days and sporozoites were collected by dissection of the salivary glands and deposited in multiwell IFAT slides at a concentration of 2,000 sporozoites/well. Slides were kept frozen at −70° C. until used. The assay was performed using sera from C57Bl/6 and CD-1 mice, which was incubated with three different batches each of sporozoites, yielding similar results between both strains. Air-dried sporozoites were also incubated with anti-VK210 or anti-VK247 monoclonal antibodies as positive controls [29]. Slides were analyzed using a confocal microscope and titers were calculated using the highest dilution that gave positive fluorescence.
(38) Efficacy Studies: Determination of Liver Parasite Liver Load by Real Time Imaging and Determination Prepatent Period (after Challenge of Immunized Mice with Chimeric Sporozoites)
(39) To determine the efficacy of the liver-stage vaccines, chimeric P. berghei infected A. stephensi mosquitoes were dissected 21 days post-feed and salivary gland sporozoites resuspended in RPMI-1640 media (Sigma Aldrich). 2000 sporozoites were injected i.v. into the tail vain per mouse, into both vaccinated and naïve controls.
(40) In Vivo Imaging Using the IVIS System
(41) All the transgenic parasite lines were generated to express the fusion protein GFP-Luciferase under the control of the constitutive eeflα promoter. The gfp-luc expression cassette is stably integrated into the Pb230p locus without introduction of a drug-selectable marker {Spaccapelo, 2010 #257; Janse, 2006 #181}. In vivo imaging of mice was performed using the IVIS 200 imaging system to determined parasite loads in livers of infected mice 44 hours post-infection {Ploemen, 2009 #258}. Mice were firstly shaved over the area of the liver, then anaesthetized and subcutaneously (s.c.) injected with 50 μl of 50 mg/ml D-luciferin substrate. Eight minutes after the injection of luciferin, mice were imaged for two minutes. Quantification of the bioluminescence signal was performed using the Living Image 4.2 image analysis software program. The readings were expressed as the total flux of photons emitted per second of exposure time.
(42) Transgenic Parasite Fitness
(43) Parasite fitness studies indicated similar infectivity, growth rates, gametocyte production and production of oocysts and sporozoites for the chimeric parasites expressing the P. vivax CSP genes compared to those of the WT P. berghei line. The blood stage growth and prepatent time to reach 1% parasitaemia in naïve mice for chimeric parasites were identical to the WT P. berghei line.
(44) P. vivax CSP DNA Sequences
(45) Four versions of ChAd63 and four of MVA were designed and produced to express various versions of the P. vivax CSP protein. One consisted of only the N- and C-terminal regions (NC); a second viral vector expressed VK210 repeats inserted in between the N- and C-terminal sequences (N210C). A third vector expressed VK247 inserted between the N- and C-terminal sequences (N247C) and a final design consisted on a chimeric VK210/247 flanked by similar N- and C-terminal repeats (sequences detailed below).
(46) DNA transgenes were synthesized by GeneArt (Regensburg, Germany) and constructs were previously modified to improve antigen expression within the hosts cells, modifications included codon optimization for mammalian use and replacement of the endogenous PvCSP leading sequence for tPA (human plasminogen activator) (GenBank Accession no. K03021). In addition, the transmembrane GPI-anchor domain was removed from the Plasmodium vivax circumsporozoite protein (PvCSP) genes to allow protein secretion from any virus-transduced cell. The chimeric PvCSP vaccine insert consisted of the N- and C-terminal region from the Salvador I strain (NCBI Reference Sequence XP_001613068.1) flanking the central repeat regions of the circumsporozoite (CSP) VK210 of the Belem strain (GenBank accession number P08677) or VK247 of the Papua New Guinea (PNG) (GenBank accession number M69059.1). The central repeat region was designed using segments of 9 amino acid repeats as follows:
(47) TABLE-US-00007 VK210 (SEQ ID NO: 3) 5x(GDRAAGQPA), (SEQ ID NO: 4) 4x(GDRADGQPA), (SEQ ID NO: 5) 1x(GNGAGGQAA)) VK247 (SEQ ID NO: 11) 2x (ANGAGNQPG/ANGAGGQAA), (SEQ ID NO: 12) 1x (ANGAGDQPG/ANGAGDQPG), (SEQ ID NO: 13) 1x (ANGADDQPG/ANGAGDQPG), (SEQ ID NO: 14) 1x (EDGAGNQPG/ANGAGDQPG)).
(48) A region II-plus was included in the C-terminal sequence of the vaccine (EWTPCSVTCG) (SEQ ID NO: 31) [53], as well as an insertion region in the C-terminal sequence of PvCSP (GAGGQAAGGNA) (SEQ ID NO: 33) [54]. The VK210 vaccine insert consisted of the Salvador I strain sequence, while the VK247 consisted of the PNG sequence with the accession numbers mentioned above. A control viral vector lacking the expression of any transgene was used as a control in mock-vaccinated mice.
(49) Viral Vector Construction
(50) All of DNA constructs required for ChAd63 were cloned in two steps. In the first step, unique Acc65I and NotI sites were used to insert the synthetic transgenes into an adenovirus entry plasmid. The transgene was placed upstream of BGH poly(A) transcription termination sequence and under the control of the long cytomegalovirus (CMV) promoter (containing a regulatory element, an enhancer and an intron A). The entry plasmid also contained attachment L (attL) sequences, which were required for site-specific recombination with attachment R (attR) sites located on the destination vector.
(51) In the second step of cloning, an in vitro Gateway reaction was performed mediated by LR Clonase II system (Invitrogen), whereby the transgene of the entry vector was integrated into the destination plasmid by site-specific recombinase through an attL-attR interaction. The diagnostic PCR was performed to confirm the desired integration before completing the production of the recombinant ChAd63.
(52) Similarly, all of the malarial genes were inserted into an MVA shuttle vector using a similar cloning strategy. Unique Acc65I and XhoI sites were used for transgene restriction and ligation and the transgenes were inserted under the control of an endogenous P7.5 promoter. PCR and RFLP were used to verify the correct insertion before linearization of the MVA plasmid.
(53) Rv21 HepB Surface Antigen VLP Design and Development
(54) A gene containing a chimeric CSP sequence comprising the repeat regions VK210 and VK247, followed by the C-terminal sequence (210/247C) was designed to be fused to the Hepatitis B surface antigen (HepBsAg). The sequence was codon-optimised for optimal expression in yeast and purchased in GeneArt® (InVitrogen). DNA sequences were similar to those expressed by recombinant ChAd and MVA viruses, but without the N-terminal CSP region. The construct was cloned into the pPink-HC intracellular Pichia plasmid and amplified in E. coli. Upon plasmid linearization, four strains of Pichia pastoris (knock out for ade2, ade2-pep4, ade2-prb1, ade2-pep4-prb1) were electroporated and white colonies were selected using PAD selection plates. Protein expression was induced by addition of methanol and a kinetics analysis was used to select the highest protein production and an optimal time point. Purification of the protein/VLP was made in an affinity column in presence of imidazol, followed by gel filtration. Fractions from gel filtration and affinity column were analyzed by SDS-PAGE and subsequently transferred to nitrocellulose membranes, blocked in 5% skimmed milk/PBS followed by addition of primary and secondary antibodies. VLP-containing fractions from the gel filtration column were confirmed with anti-HepBsAg, anti-PvCSP VK210 and anti-PvCSP VK247 primary mouse antibodies, diluted in 3% BSA/PBS to 1:200, 1:20,000 and 1:20,000 respectively (MR4). A secondary donkey anti-mouse-AP conjugate in 3% BSA/PBS and Sigmafast™ BCIP/NBT tablets (Sigma-Aldrich) were used for development. Silver staining was used to analyse purity of the fractions from the FPLC column and of progressive stages of the VLP purification process. The samples were run on Mini-PROTEAN 12% gels (BioRad) with the Pierce unstained protein ladder (Thermo Scientific) run for reference. The gels were subjected to silver staining using a Pierce silver stain kit (Thermo Scientific). Direct staining of gels was carried out according to manufacturer's instructions. The particle-containing fraction from the sephacryl gel filtration column was negatively stained with 2% uranyl acetate. The sample was then imaged using a FEI Tecnai 12 Transmission Electron Microscope (TEM).
(55) Immunization of Mice
(56) Mice were primed with simian adenoviral vector 63 (ChAd63) encoding PvCSP at a dose of 1×10.sup.8 infectious units (iu) and 8 weeks later boosted with vaccinia-modified virus strain Ankara (MVA) encoding the same transgene at a concentration of 1×10.sup.6 plaque forming unit (pfu) per mouse. All viral vector vaccines were administered intramuscularly (i.m.) in endotoxin-free PBS. The Rv21 VLP vaccine was administered i.m. at a dose of 0.5 μg/mouse in endotoxin-free PBS.
(57) Whole IgG Enzyme-Linked Immunosorbent Assay (ELISA)
(58) ELISAs measuring total IgG were carried out as described previously [24]. Serum antibody endpoint titers were taken as the x-axis intercept of the dilution curve at an absorbance value three standard deviations greater than the OD405 for serum from a naïve mouse. Results were also calculated using a standardized ELISA by including a high-titre reference serum from hyper-immune mice to each ELISA plate to produce a standard curve, which in turn was used to quantify and assign ELISA units to each sample [55]. Briefly, Nunc Maxisorp Immuno ELISA plates were coated with the antigens diluted in PBS to a final concentration of 2 μg/mL. Each plate contained a standard curve, negative and positive controls, as well as serum samples of mice pre-boost (1:300) or post-boost (1:3000). An anti-mouse IgG coupled to alkaline phosphatase was used as a secondary antibody. Development was made with 4-nitrophenylphosphate diluted in diethanolamine buffer. Data was fitted to a four parameter hyperbolic curve [56].
(59) Expression and Purification of PvCSP VK210 and VK247 Proteins
(60) The codon optimized genes of P. vivax CSP containing VK210 or VK247 repeats were cloned into the pHLsec vector [57] with a C-terminal hexahistidine tag. Protein was expressed by transient transfection in HEK-293T cells and purified from dialyzed (against PBS buffer) conditioned medium by immobilized Co.sup.2+-affinity chromatography followed by size-exclusion chromatography in 20 mM Tris-HCl pH 8.0, 300 mM NaCl [40].
(61) Peptides
(62) Crude 20-mer peptides overlapping by 10 amino acids and representing full-lengths of P. vivax CSP VK210 and VK247 were synthesized by Mimotopes (Victoria, Australia). Individual peptide pools were used at a final concentration of 5 μg/mL.
(63) Ex-Vivo IFN-γ EL/SPOT Assay
(64) Ex vivo IFN-gamma (IFN-γ) ELISPOTs were carried out using PBMCs isolated from the blood as previously described [58, 59]. MAIP ELISPOT plates (Millipore) were used to plate cells. Anti-mouse IFN-γ mAb and development reagents were used according to the manufacturer specifications (Mabtech).
(65) Intracellular Cytokine Staining (ICS)
(66) Peripheral mononuclear cells (PBMCs) were stimulated for 5 hours in the presence of TRAP peptide pools described above. Hepatic cellular responses were assessed from perfused livers that were digested with collagenase and treated with ACK buffer to lyse red blood cells. Phenotypic and functional analysis of CD8.sup.+ and CD4.sup.+ T cells were performed using the following antibody clones: anti-CD8 PerCP-Cy5.5 (clone 53-6.7), and eFluor650 coupled anti-CD4 (GK1.5), anti-IFN-γ APC (XMG1.2), anti-TNF-α eFluor 450 (MP6-XT22), anti-IL-2 PE-Cy 7 (JES6-5H4). Flow cytometric analyses were performed using an LSRII instrument. Frequencies of cells producing cytokines in the graphs represent data where background from non-stimulated cells was subtracted. Data were analyzed with either FACSDiva or FlowJo software.
(67) Infection of Mice
(68) Mice were challenged with 2,000 wild type or transgenic P. berghei sporozoites. Infection was monitored from day 5 to 20 by Giemsa staining of blood smears.
(69) Statistical Model for Parasitaemia Prediction
(70) Percent parasitaemia was used to calculate the time required to reach a blood-stage infection of 1%, or time to 1% parasitaemia. This was predicted using a linear regression model as described previously [25]. Briefly, blood parasite counts were obtained for 3-5 consecutive days starting on day 5 after the challenge. Blood smears were stained with Giemsa, and percentages of parasitaemia calculated in all animals. The logarithm to base 10 of the calculated percentage of parasitaemia was plotted against the time after challenge and Prism 5 for Mac OS X (GraphPad software) statistical analysis package used for generating a linear regression model on the linear part of the blood-stage growth curve.
(71) Statistical Analysis
(72) For all statistical analyses, GraphPad Prism version 5.0 for Max OS was used unless indicated otherwise. Prior to statistical analysis to compare two or more populations, the Kolmogorov-Smirnov test for normality was used to determine whether the values followed a Gaussian distribution. An unpaired t-test was employed to compare two normally distributed groups, whereas Mann-Whitney rank test was used for comparing two non-parametric groups. If more than two groups were present non-parametric data was compared using Kruskal-Wallis test with Dunn's multiple comparison post-test, whereas normally distributed data were analyzed by one-way ANOVA with Bonferroni's multiple comparison post-test. The effect of two variables was explored using two-way ANOVA with Bonferroni's multiple comparison post-test. Correlation strength was tested using either Pearson's or Spearman's tests as indicated in the results chapters. Kaplan-Meier survival curves were used to represent protective efficacy to a challenge with any P. berghei parasite lines. All ELISA titres were also log 10 transformed before analysis. The value of p<0.05 was considered statistically significant (*p<0.05, **p<0.01, **p<0.001, and ***p<0.001).
Example 2: Cellular Immune Responses are not Induced in Mice Immunized with P. vivax Circumsporozoite Viral-Vectored Vaccines (PvCSP vv)
(73) We first assessed whether viral-vectored vaccines (vv) expressing the P. vivax circumsporozoite protein (PvCSP) could induce cellular immune responses in inbred or outbred mouse strains. We generated four recombinant adenoviruses from chimpanzee origin (ChAd63) and four Modified Vaccinia Ankara (MVA) vv expressing various versions of PvCSP (PvCSP vv). The resulting recombinant viruses contained a series of PvCSP expression cassettes encoding the PvCSP N- and C-terminal sequences of the Salvador I strain, including or excluding certain selected central repeat sequences which are present in the two major PvCSP alleles VK210 and VK247 (
(74) First, a set of ChAd63 and MVA vv expressing only the N- and C-terminal sequences and no central repeats were generated (NC); a second set of vv contained a series of selected VK210 repeats [8] flanked by the same NC sequences (N210C); a third set expressed VK247 repeats flanked by NC (N247C). Finally we generated a ChAd63/MVA set expressing a chimeric CSP containing both type of repeats (VK210/247) flanked by NC (N210/247C). Groups of mice (n=6) received a homologous ChAd63-MVA prime-boost at an interval of eight weeks between immunizations (
Example 3: Humoral Immune Responses are Induced in Mice Immunized with the PvCSP vv
(75) We next assessed antibody responses induced by the ChAd63 and MVA vaccination protocols described above, using C57BL/6 mice (
Example 4: P. vivax Sporozoites Isolated from Chiapas, Mexico are Recognized by Antibodies Induced in Mice after PvCSP vv Vaccination
(76) We addressed the question whether mouse antibodies raised by immunisation with various PvCSP antigens encoded by the vv could recognize CSP on the surface of P. vivax sporozoites collected from patients in the endemic region of Tapachula, Chiapas [29]. We performed immunofluorescence assays (IFAs) using two types of sporozoites, VK210 and VK247 belonging to three different sporozoite batches, all yielding similar results. Results shown in
Example 5: Development of a Rodent Challenge Model for Assessing Protective Efficacy of the PvCSP Vaccination
(77) In order to assess the in vivo protective efficacy of the P. vivax vaccines, we developed rodent challenge models, which involved creating transgenic P. berghei sporozoites where Pbcsp was replaced with either full length VK210 Pvcsp or VK247 Pvcsp. These transgenic parasites were generated using the gene insertion/marker out (GIMO) based transfection technology [30, 31]. Using this technology, we generated two Double-step Replacement (DsR) mutants [32] that resulted in the coding sequence (CDS) of P. berghei csp being replaced with the CDS of the Pvcsp in a two-step GIMO-transfection procedure (
Example 6: Protective Efficacy of PvCSP vv Vaccines Against a Sporozoite Challenge with P. berghei PvCSP Transgenic Parasites
(78) We next determined vaccination efficacy by challenging vv-immunized mice with the transgenic P. berghei PvCSP transgenic parasites. These mice were immunised with the 4 different (ChAd63-MVA) PvCSP vv, as described above (
(79) Our results indicated that the PvCSP central repeat sequences are necessary to generate protective immunity, as immunisations with NC only (i.e. lacking either VK210 or 247 repeat sequences) failed to induce protective immunity, demonstrated by the prepatent period being similar to mock immunized control mice (
(80) While significant protective immunity was maintained 12 weeks after immunisation with the N210/247C vector as determined by a prolonged prepatent period after challenge with either VK210 (p=0.005) or VK247 (p=0.016) transgenic sporozoites, sterile protection was not induced as all mice eventually developed a blood stage infection (
Example 7: Vaccination with the Rv21 VLP, Presenting the Chimeric PvCSP VK210/247 Antigen Fused to the Hepatitis B Surface (HepB S), Antigen Induces High Levels of Protective Immunity in Mice
(81) The ability to induce antibody-mediated protective immunity by the PvCSP vv in the absence of detectable cellular responses led us to develop a VLP vaccine platform with a greater potential to enhance humoral responses against PvCSP. VLPs are known for their ability to induce high antibody titres, and are a leading vaccine platform not only for malaria [5] but also for HPV [35]. RTS,S/AS is based on the hepatitis B surface antigen virus-like particle (VLP) platform, genetically-engineered to include the central repeats and carboxy (C-) terminus (amino acids 207-395) of the P. falciparum CSP antigen [36]. We developed a VLP consisting of the chimeric PvCSP VK210/VK247 central repeats and the CSP C-terminal sequence fused to the Hepatitis B Surface Antigen (HepB-S) gene. Codon usage of the fusion gene was optimized for expression in Pichia pastoris and production of the fusion protein (PvCSP-HepB-S) was assessed in four yeast strains using a kinetics analysis.
(82) We selected a P. pastoris colony that had optimal expression levels, which peaked after 108 hours (
(83) The protocol for purification of the fusion protein involved two steps. The first consisted of an affinity purification using a capture select C-tag molecule (Thermofisher) bound to the fusion protein. In addition to collecting the expected protein band corresponding to the PvCSP-HepB S protein, the sample also contained additional proteins. VLP particle formation was confirmed using transmission electron microscopy (TEM;
(84) The protective efficacy of each prime-boost regimen was assessed by challenging the Rv21-immunized CD-1 mice with a dose of 2000 PbANKA-PvCS VK210(r).sub.PbCS or PbANKA-PvCS VK247(r).sub.PbCS transgenic sporozoites (
(85) These results indicated the importance of both the VLP and the adjuvant in inducing protective immune responses of this vaccination regime. In addition, our results showed that PvCSP VLP-based vaccination induced higher protective efficacy than immunisation with the viral-vectored vaccine candidates. ELISA analyses of antibody responses to vaccination supported the importance of Matrix-M adjuvant to elicit high antibody titres to PvCSP, even after a single (priming) immunisation with Rv21 (
(86) We tested Rv21 efficacy in C57BL/6, a mouse strain that has previously shown to be highly sensitive to a P. berghei sporozoite infection and for which we have previously failed to induce sterile immunity using a P. vivax TRAP vaccine candidate [40]. Immunisation of mice with an Rv21 dose as described above (0.5 μg) followed by a challenge with 2,000 transgenic PbANKA-PvCS VK210(r).sub.PbCS sporozoites failed to show protective efficacy (
(87) Our results indicate that immunization with a chimeric VK210/247 vaccine can induce immunity to the two major strains of P. vivax, based on the type of PvCSP central repeats. Moreover, high levels of protective immunity levels can be achieved using a VLP platform in presence of the Matrix-M adjuvant, even in a mouse strain that is difficult to protect against a sporozoite infection.
Example 8: The Effect of Adjuvants and a Combination of Viral Vectors on Rv21 Anti-Vivax CSP Immunogenicity
(88) BALB/c mice were immunized using two consecutive doses (prime-boost) of Rv21-VLP presenting vivax CSP on the surface. The results are shown in
Example 9: The Effect of Adjuvants and a Combination of Viral Vectors on Rv21 Protective Efficacy Against a Sporozoite Challenge
(89) BALB/c mice were immunized using two consecutive doses (prime-boost) of Rv21-VLP presenting vivax CSP on the surface, or a combination with Rv21+Adjuvant+vvTRAP (viral vectors expressing vivax TRAP). The results are shown in
Example 10: A Combination of Rv21+Viral Vectors (vv) Require Both Antigens to be from Vivax to Induce Complete Protection Against a Sporozoite Challenge
(90) The results of this example are shown in
(91) TABLE-US-00008 Ad MVA Ad MVA Rv21 TRAP TRAP R21 Tc24 Tc24 Group μg iu pfu μg iu pfu Rv21/ 1 μg — — — 1 × 10.sup.8 1 × 10.sup.7 vvControl R21control/ — 1 × 10.sup.8 1 × 10.sup.7 1 μg vvTRAP Rv21/ 1 μg 1 × 10.sup.8 1 × 10.sup.7 — — — vvTRAP naïve — — — — — —
(92) In
Example 11: Effect of the Presence of the N-Terminus Region of P. vivax CSP on the Protective Efficacy Against a Malaria Sporozoite Challenge
(93) Two forms of P. vivax CSP were produced: a full version containing the N-terminus, repeats of VK210 and VK247 and C-terminus region (
Example 12: Additional Characterization of Rv21 by Western Blot and ELISA
(94) The Rv21 VLP particle (
REFERENCES
(95) 1. Guerra, C. A., et al., The international limits and population at risk of Plasmodium vivax transmission in 2009. PLoS Negl Trop Dis, 2010. 4(8): p. e774. 2. White, N. J., Determinants of relapse periodicity in Plasmodium vivax malaria. Malar J, 2011. 10: p. 297. 3. Markus, M. B., Malaria: origin of the term “hypnozoite”. J Hist Biol, 2011. 44(4): p. 781-6. 4. Duffy, P. E., et al., Pre-erythrocytic malaria vaccines: identifying the targets. Expert Rev Vaccines, 2012. 11(10): p. 1261-80. 5. Rts, S. C. T. P., Efficacy and safety of the RTS,S/AS01 malaria vaccine during 18 months after vaccination: a phase 3 randomized, controlled trial in children and young infants at 11 African sites. PLoS Med, 2014. 11(7): p. e1001685. 6. Almeida, A. P., et al., Long-lasting humoral and cellular immune responses elicited by immunization with recombinant chimeras of the Plasmodium vivax circumsporozoite protein. Vaccine, 2014. 32(19): p. 2181-7. 7. Bennett, J. W., et al., Phase 1/2a Trial of Plasmodium vivax Malaria Vaccine Candidate VMP001/AS01B in Malaria-Naive Adults: Safety, Immunogenicity, and Efficacy. PLoS Negl Trop Dis, 2016. 10(2): p. e0004423. 8. Lim, C. S., L. Tazi, and F. J. Ayala, Plasmodium vivax: recent world expansion and genetic identity to Plasmodium simium. Proc Natl Acad Sci USA, 2005. 102(43): p. 15523-8. 9. Teixeira, L. H., et al., Immunogenicity of a prime-boost vaccine containing the circumsporozoite proteins of Plasmodium vivax in rodents. Infect Immun, 2014. 82(2): p. 793-807. 10. Vanloubbeeck, Y., et al., Comparison of the immune responses induced by soluble and particulate Plasmodium vivax circumsporozoite vaccine candidates formulated in AS01 in rhesus macaques. Vaccine, 2013. 31(52): p. 6216-24. 11. Yadava, A., et al., A novel chimeric Plasmodium vivax circumsporozoite protein induces biologically functional antibodies that recognize both VK210 and VK247 sporozoites. Infect Immun, 2007. 75(3): p. 1177-85. 12. Yadava, A., et al., Protective efficacy of a Plasmodium vivax circumsporozoite protein-based vaccine in Aotus nancymaae is associated with antibodies to the repeat region. PLoS Negl Trop Dis, 2014. 8(10): p. e3268. 13. Cespedes, N., et al., Antigenicity and immunogenicity of a novel Plasmodium vivax circumsporozoite derived synthetic vaccine construct. Vaccine, 2014. 32(26): p. 3179-86. 14. Cespedes, N., et al., Antigenicity and immunogenicity of a novel chimeric peptide antigen based on the P. vivax circumsporozoite protein. Vaccine, 2013. 31(42): p. 4923-30. 15. Udomsangpetch, R., et al., Short-term in vitro culture of field isolates of Plasmodium vivax using umbilical cord blood. Parasitol Int, 2007. 56(1): p. 65-9. 16. Targett, G. A., V. S. Moorthy, and G. V. Brown, Malaria vaccine research and development: the role of the WHO MALVAC committee. Malar J, 2013. 12: p. 362. 17. Golenda, C. F., J. Li, and R. Rosenberg, Continuous in vitro propagation of the malaria parasite Plasmodium vivax. Proc Natl Acad Sci USA, 1997. 94(13): p. 6786-91. 18. Galinski, M. R., et al., A reticulocyte-binding protein complex of Plasmodium vivax merozoites. Cell, 1992. 69(7): p. 1213-26. 19. Reyes-Sandoval, A. and M. F. Bachmann, Plasmodium vivax malaria vaccines: why are we where we are? Hum Vaccin Immunother, 2013. 9(12): p. 2558-65. 20. Galinski, M. R. and J. W. Barnwell, Plasmodium vivax: who cares? Malar J, 2008. 7 Suppl 1: p. S9. 21. Espinosa, D. A., et al., Development of a chimeric Plasmodium berghei strain expressing the repeat region of the P. vivax circumsporozoite protein for in vivo evaluation of vaccine efficacy. Infect Immun, 2013. 81(8): p. 2882-7. 22. Whitacre, D. C., et al., P. falciparum and P. vivax Epitope-Focused VLPs Elicit Sterile Immunity to Blood Stage Infections. PLoS One, 2015. 10(5): p. e0124856. 23. Ewer, K. J., et al., Protective CD8+T-cell immunity to human malaria induced by chimpanzee adenovirus-MVA immunisation. Nat Commun, 2013. 4: p. 2836. 24. Reyes-Sandoval, A., et al., Prime-boost immunization with adenoviral and modified vaccinia virus Ankara vectors enhances the durability and polyfunctionality of protective malaria CD8+T-cell responses. Infect Immun, 2010. 78(1): p. 145-53. 25. Reyes-Sandoval, A., et al., CD8+T effector memory cells protect against liver-stage malaria. J Immunol, 2011. 187(3): p. 1347-57. 26. Heppner, D. G., The malaria vaccine—status quo 2013. Travel Med Infect Dis, 2013. 11(1): p. 2-7. 27. Sun, P., et al., Protective immunity induced with malaria vaccine, RTS,S, is linked to Plasmodium falciparum circumsporozoite protein-specific CD4+ and CD8+ T cells producing IFN-gamma. J Immunol, 2003. 171(12): p. 6961-7. 28. Moorthy, V. S. and W. R. Ballou, Immunological mechanisms underlying protection mediated by RTS,S: a review of the available data. Malar J, 2009. 8: p. 312. 29. Gonzalez-Ceron, L., et al., Plasmodium vivax: a monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface. Exp Parasitol, 1998. 90(3): p. 203-11. 30. Lin, J. W., et al., A novel ‘gene insertion/marker out’ (GIMO) method for transgene expression and gene complementation in rodent malaria parasites. PLoS One, 2011. 6(12): p. e29289. 31. Longley, R. J., et al., Comparative assessment of vaccine vectors encoding ten malaria antigens identifies two protective liver-stage candidates. Sci Rep, 2015. 5: p. 11820. 32. Khan, S. M., et al., Standardization in generating and reporting genetically modified rodent malaria parasites: the RMgmDB database. Methods Mol Biol, 2013. 923: p. 139-50. 33. Rosenberg, R., et al., Circumsporozoite protein heterogeneity in the human malaria parasite Plasmodium vivax. Science, 1989. 245(4921): p. 973-6. 34. Yoshida, N., et al., Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science, 1980. 207(4426): p. 71-3. 35. Wang, J. W. and R. B. Roden, Virus-like particles for the prevention of human papillomavirus-associated malignancies. Expert Rev Vaccines, 2013. 12(2): p. 129-41. 36. Garcon, N., D. G. Heppner, and J. Cohen, Development of RTS,S/AS02: a purified subunit-based malaria vaccine candidate formulated with a novel adjuvant. Expert Rev Vaccines, 2003. 2(2): p. 231-8. 37. Magnusson, S. E., et al., Immune enhancing properties of the novel Matrix-M adjuvant leads to potentiated immune responses to an influenza vaccine in mice. Vaccine, 2013. 31(13): p. 1725-33. 38. Reimer, J. M., et al., Matrix-M adjuvant induces local recruitment, activation and maturation of central immune cells in absence of antigen. PLoS One, 2012. 7(7): p. e41451. 39. Bengtsson, K. L., et al., Matrix-M adjuvant: enhancing immune responses by ‘setting the stage’ for the antigen. Expert Rev Vaccines, 2013. 12(8): p. 821-3. 40. Bauza, K., et al., Efficacy of a Plasmodium vivax malaria vaccine using ChAd63 and modified vaccinia Ankara expressing thrombospondin-related anonymous protein as assessed with transgenic Plasmodium berghei parasites. Infect Immun, 2014. 82(3): p. 1277-86. 41. Herrera, S., G. Corradin, and M. Arevalo-Herrera, An update on the search for a Plasmodium vivax vaccine. Trends Parasitol, 2007. 23(3): p. 122-8. 42. Nahrendorf, W., et al., Memory B-cell and antibody responses induced by Plasmodium falciparum sporozoite immunization. J Infect Dis, 2014. 210(12): p. 1981-90. 43. Warimwe, G. M., et al., Peripheral blood monocyte-to-lymphocyte ratio at study enrollment predicts efficacy of the RTS, S malaria vaccine: analysis of pooled phase II clinical trial data. BMC Med, 2013. 11: p. 184. 44. Cabrera-Mora, M., et al., Induction of Multifunctional Broadly Reactive T Cell Responses by a Plasmodium vivax Circumsporozoite Protein Recombinant Chimera. Infect Immun, 2015. 83(9): p. 3749-61. 45. Mizutani, M., et al., Baculovirus-vectored multistage Plasmodium vivax vaccine induces both protective and transmission-blocking immunities against transgenic rodent malaria parasites. Infect Immun, 2014. 82(10): p. 4348-57. 46. Hodgson, S. H., et al., Evaluation of the efficacy of ChAd63-MVA vectored vaccines expressing circumsporozoite protein and ME-TRAP against controlled human malaria infection in malaria-naive individuals. J Infect Dis, 2015. 211(7): p. 1076-86. 47. Magnusson, S. E., et al., Matrix-M adjuvanted envelope protein vaccine protects against lethal lineage 1 and 2 West Nile virus infection in mice. Vaccine, 2014. 32(7): p. 800-8. 48. Janse, C. J., B. Franke-Fayard, and A. P. Waters, Selection by flow-sorting of genetically transformed, GFP-expressing blood stages of the rodent malaria parasite, Plasmodium berghei. Nat Protoc, 2006. 1(2): p. 614-23. 49. Cox, R. J., et al., Evaluation of a virosomal H5N1 vaccine formulated with Matrix M adjuvant in a phase I clinical trial. Vaccine, 2011. 29(45): p. 8049-59. 50. Menard, R. and C. Janse, Gene targeting in malaria parasites. Methods, 1997. 13(2): p. 148-57. 51. Orr, R. Y., N. Philip, and A. P. Waters, Improved negative selection protocol for Plasmodium berghei in the rodent malarial model. Malar J, 2012. 11: p. 103. 52. Gonzalez-Ceron, L., et al., Plasmodium vivax: impaired escape of Vk210 phenotype ookinetes from the midgut blood bolus of Anopheles pseudopunctipennis. Exp Parasitol, 2007. 115(1): p. 59-67. 53. Gantt, S. M., et al., Cell adhesion to a motif shared by the malaria circumsporozoite protein and thrombospondin is mediated by its glycosaminoglycan-binding region and not by CSVTCG. J Biol Chem, 1997. 272(31): p. 19205-13. 54. Hernandez-Martinez, M. A., et al., Antigenic diversity of the Plasmodium vivax circumsporozoite protein in parasite isolates of Western Colombia. Am J Trop Med Hyg, 2011. 84(2 Suppl): p. 51-7. 55. Miura, K., et al., Development and characterization of a standardized ELISA including a reference serum on each plate to detect antibodies induced by experimental malaria vaccines. Vaccine, 2008. 26(2): p. 193-200. 56. Studnicka, G. M., ELISA assay optimization using hyperbolic regression. Comput Appl Biosci, 1991. 7(2): p. 217-24. 57. Aricescu, A. R., W. Lu, and E. Y. Jones, A time-and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr D Biol Crystallogr, 2006. 62(Pt 10): p. 1243-50. 58. Reyes-Sandoval, A., et al., Potency assays for novel T-cell-inducing vaccines against malaria. Curr Opin Mol Ther, 2009. 11(1): p. 72-80. 59. Reyes-Sandoval, A., et al., Single-dose immunogenicity and protective efficacy of simian adenoviral vectors against Plasmodium berghei. Eur J Immunol, 2008. 38(3): p. 732-41. 60. Shoukat et al., Journal of Infectious Disease 1993, 168:1485-9
Additional Sequences
(96) TABLE-US-00009 Type I VK210 wild type sequence. P. vivax CSP Belem strain P08677 (ref. sequence from www.uniprot.org) SEQ ID NO: 1 MKNFILLAVSSILLVDLFPTHCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGE NPDDEEGDAKKKKDGKKAEPKNPRENKLKQPGDRADGQPAGDRADGQPAGDRADGQPAGDRAAG QPAGDRADGQPAGDRADGQPAGDRADGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRADGQ PAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRADGQPAGDRAAGQPAGDRAAGQPAGDRAAGQP AGDRAAGQPAGNGAGGQAAGGNAGGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTC GVGVRVRRRVNAANKKPEDLTLNDLETDVCTMDKCAGIFNVVSNSLGLVILLVLALFN. Type II VK247 wild type sequence. P. vivax CSP PND (Papua New Guinea) strain Q7M3X0 (ref. sequence from www.uniprot.org) SEQ ID NO: 2 MKNFILLAVSSILLVDLFPTHCGHNVDLSKAINLNGVGFNNVDASSLGAAHVGQSASRGRGLGE NPDDEEGDAKKKKDGKKAEPKNPRENKLKQPEDGAGNQPGANGAGNQPGANGAGNQPGANGAGD QPGANGAGNQPGANGAGDQPGANGAGNQPGANGAGNQPGANGAGNQPGANGADDQPGANGAGNQ PGANGAGNQPGANGAGNQPGANGAGDQPGANGAGNQPGANGAGDQPGANGAGNQPGANGAGNQP GANGAGNQPGANGAGNQPGANGAGGQAAGGNAANKKAGDAGAGQGQNNEGANATNEKSVKEYLD KVRATVGTEWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCTMDKCAGIFNVVSNSLG LVILLVLALFN Pv VK210 Repeats DNA Sequence (SEQ ID NO: 15) GGTGATAGAGCTGCTGGTCAACCTGCTGGTGACAGAGCTGCTGGACAGCCAGCTGGTGATAGAG CTGCTGGTCAGCCTGCTGGTGATAGAGCTGCTGGACAACCTGCTGGTGATAGAGCTGCTGGTCA ACCTGCTGGTGATAGAGCTGACGGTCAGCCAGCTGGTGATAGAGCTGACGGTCAACCTGCTGGT GACAGAGCTGACGGACAACCTGCTGGTGATAGAGCTGATGGACAACCAGCTGGAAATGGTGCTG GTGGTCAAGCTGCT Amino acid sequence (SEQ ID NO: 16) GDRAAGQPAGDRAAGQPAGDRAAGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDRADGQPAG DRADGQPAGDRADGQPAGNGAGGQAA Pv VK247 Repeats DNA Sequence (SEQ ID NO: 17) GCTAATGGTGCTGGAAATCAACCAGGTGCTAACGGTGCTGGTGGACAGGCTGCTGCTAACGGTG CTGGTAACCAGCCTGGTGCTAATGGTGCTGGTGGACAAGCTGCTGCTAACGGTGCTGGTGATCA ACCAGGTGCTAATGGTGCTGGTGATCAGCCTGGTGCTAACGGTGCTGATGACCAACCTGGTGCT AACGGTGCTGGTGACCAGCCAGGTGAGGACGGTGCTGGTAATCAACCTGGTGCTAACGGTGCTG GTGATCAACCTGGT Amino acid sequence (SEQ ID NO: 18) ANGAGNQPGANGAGGQAAANGAGNQPGANGAGGQAAANGAGDQPGANGAGDQPGANGADDQPGA NGAGDQPGEDGAGNQPGANGAGDQPG P vivax-like repeats Protein ID: UniProtKB-Q26124 (Q26124_9APIC) Repeat I DNA Sequence (SEQ ID NO: 22) gccccaggagcaaatcaggaaggtggagcagcagccccaggagcaaatcaggaaggtggagcag cagccccaggagcaaatcaggaaggtggagcagca Amino acid sequence (SEQ ID NO: 23) APGANQEGGAAAPGANQEGGAAAPGANQEGGAA P. vivax-like repeat II DNA Sequence (SEQ ID NO: 24) gcaccaggagcaaaccagggaggtggagcagcagcaccaggagcaaaccagggaggtggagcag cagcaccaggagcaaaccagggaggtggagcagca Amino acid sequence (SEQ ID NO: 25) APGANQGGGAAAPGANQGGGAAAPGANQGGGAA P. vivax-like repeat I + P. vivax-like repeat II Amino acid sequence (SEQ ID NO: 26) APGANQEGGAAAPGANQEGGAAAPGANQEGGAA APGANQGGGAAAPGANQGGGAAAPGANQGGGAA P. vivax-like repeat II + P. vivax-like repeat I Amino acid sequence (SEQ ID NO: 27) APGANQGGGAAAPGANQGGGAAAPGANQGGGAA APGANQEGGAAAPGANQEGGAAAPGANQEGGAA C-Terminus of PvCSP. Protein ID in UNIPROT: UniProtKB-P08677 (CSP_PLAVB) DNA Sequence (SEQ ID NO: 28) CAAGCTGCTGGTGGTAATGCTGGTGGTGGTCAGGGTCAAAACAACGAGGGTGCTAATGCTCCAA ACGAGAAGTCCGTTAAGGAATACTTGGACAAAGTTAGAGCTACTGTTGGTACTGAGTGGACTCC ATGTTCCGTTACTTGTGGTGTTGGTGTTAGAGTTAGAAGAAGAGTTAACGCTGCTAACAAGAAG CCAGAGGACTTGACTTTGAACGACTTGGAGACTGACGTTTGTACTATGGACAAG Protein Sequence (SEQ ID NO: 29) QAAGGNAGGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKK PEDLTLNDLETDVCTMDK C-Terminus fragment of PvCSP Amino acid sequence (SEQ ID NO: 30) EWTPCSVTCGVGVRVRRRVNAANKKPEDLTLNDLETDVCTMDK N-terminus sequence from PvCSP in Adenovirus. Gene ID PVX_187290. Plasmodium vivax (strain Salvador I), UNIPROT UniProtKB-A5KDP2 (A5KDP2_PLAVS) DNA Sequence (SEQ ID NO: 34) ACCCACTGCGGCCACAACGTGGACCTGAGCAAGGCCATCAACCTGAACGGCGTGAACTTCAACA ATGTGGACGCCTCTAGCCTGGGAGCTGCTCACGTGGGCCAGAGCGCCAGCAGAGGCAGAGGCCT GGGCGAGAACCCCGACGATGAGGAAGGCGACGCCAAGAAGAAGAAGGACGGCAAGAAGGCCGAG CCCAAGAACCCCAGAGAGAACAAGCTGAAGCAGCCC Amino acid sequence (SEQ ID NO: 35) THCGHNVDLSKAINLNGVNFNNVDASSLGAAHVGQSASRGRGLGENPDDEEGDAKKKKDGKKAE PKNPRENKLKQP Hepatitis B S Antigen DNA sequence (SEQ ID NO: 37) CCAGTTACTAATATGGAAAACATCACTTCCGGTTTCTTGGGTCCTTTGTTGGTTTTGCAGGCTG GATTCTTCTTGTTGACTAGAATCTTGACTATCCCACAGTCCTTGGACTCTTGGTGGACTTCCTT GAACTTCTTGGGTGGTTCCCCAGTTTGTTTGGGTCAGAACTCTCAATCCCCAACTTCTAACCAC TCCCCAACATCCTGTCCTCCAATTTGTCCAGGTTACAGATGGATGTGTTTGAGAAGATTCATCA TTTTCTTGTTCATCTTGTTGTTGTGTTTGATCTTCTTGTTGGTTTTGTTGGACTACCAGGGTAT GTTGCCAGTTTGTCCATTGATCCCAGGTTCCACTACTACAAACACTGGTCCATGTAAGACTTGT ACTACTCCAGCTCAGGGTAACTCTATGTTCCCTTCCTGTTGTTGTACTAAGCCAACTGACGGTA ACTGTACTTGTATCCCAATTCCATCCTCCTGGGCTTTCGCTAAGTACTTGTGGGAATGGGCTTC CGTTAGATTCTCTTGGTTGTCCTTGTTGGTTCCATTCGTTCAGTGGTTCGTTGGTTTGTCCCCA ACTGTTTGGTTGTCTGCTATCTGGATGATGTGGTACTGGGGTCCATCCTTGTACTCTATCGTTT CCCCATTCATCCCTTTGTTGCCAATCTTCTTCTGTTTGTGGGTTTACATC Amino acid sequence (SEQ ID NO: 38) PVTNMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNH SPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTNTGPCKTC TTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSP TVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI Preferred Rv21 sequence Amino acid sequence SEQ ID NO: 39 (SEQ ID NOs: 16 + 18 + 29 + 38) GDRAAGQPAGDRAAGQPAGDRAAGQPAGDRAAGQPAGDRAAGQPAGDRADGQPAGDRADGQPAG DRADGQPAGDRADGQPAGNGAGGQAA ANGAGNQPGANGAGGQAAANGAGNQPGANGAGGQAAANGAGDQPGANGAGDQPGANGADDQPGA NGAGDQPGEDGAGNQPGANGAGDQPG QAAGGNAGGGQGQNNEGANAPNEKSVKEYLDKVRATVGTEWTPCSVTCGVGVRVRRRVNAANKK PEDLTLNDLETDVCTMDK PVTNMENITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGSPVCLGQNSQSPTSNH SPTSCPPICPGYRWMCLRRFIIFLFILLLCLIFLLVLLDYQGMLPVCPLIPGSTTTNTGPCKTC TTPAQGNSMFPSCCCTKPTDGNCTCIPIPSSWAFAKYLWEWASVRFSWLSLLVPFVQWFVGLSP TVWLSAIWMMWYWGPSLYSIVSPFIPLLPIFFCLWVYI PvTRAP sequence from SalI strain of P. vivax (wild-type) Protein sequence (SEQ ID NO: 40) MKLLQNKSYLLVVFLLYVSIFARGDEKVVDEVKYSEEVCNESVD LYLLVDGSGSIGYPNWITKVIPMLNGLINSLSLSRDTINLYMNLFGNYTTELIRLGSG QSIDKRQALSKVTELRKTYTPYGTTNMTAALDEVQKHLNDRVNREKAIQLVILMTDGV PNSKYRALEVANKLKQRNVSLAVIGIGQGINHQFNRLIAGCRPREPNCKFYSYADWNE AVALIKPFIAKVCTEVERVANCGPWDPWTACSVTCGRGTHSRSRPSLHEKCTTHMVSE CEEGECPVEPEPLPVPAPLPTVPEDVNPRDTDDENENPNENKGLDVPDEDDDEVPPAN ERADGNPVEENVFPPADDSVPDESNVLPLPPAVPGGSSEEFPADVQNNPDSPEELPME QEVPQDNNVNEPERSDSKGYGVNEKVIPNPLDNERDMANKNKTVHPGRKDSARDRYAR PHGSTHVNNNRANENSDIPNNPVPSDYEQPEDKAKKSSNNGYKIAGGVIAGLALVGCV GFAYNFVAGGGAAGMAGEPAPFDEAMAEDEKDVAEADQFKLPEDNEWN PvTRAP variant sequence Protein sequence (SEQ ID NO: 41) DEKVVDEVKYSEEVCNESVDLYLLVDGSGSIGYPNWITKVIPMLNGLINSLSLSRDTINLYMNL FGNYTTELIRLGSGQSIDKRQALSKVTELRKTYTPYGTTNMTAALDEVQKHLNDRVNREKAIQL VILMTDGVPNSKYRALEVANKLKQRNVSLAVIGVGQGINHQFNRLIAGCRPREPNCKFYSYADW NEAVALIKPFIAKVCTEVERVANCGPWDPWTACSVTCGRGTHSRSRPSLHEKCTTHMVSECEEG ECPVEPEPLPVPAPLPTVPEDVNPRDTDDENENPNFNKGLDVPDEDDDEVPPANEGADGNPVEE NVFPPADDSVPDESNVLPLPPAVPGGSSEEFPADVQNNPDSPEELPMEQEVPQDNNVNEPERSD SNGYGVNEKVIPNPLDNERDMANKNKTVHPGRKDSARDRYARPHGSTHVNNNRANENSDIPNNP VPSDYEQPEDKAKKSSNNGYK C-tag DNA sequence (SEQ ID NO: 42) GAGCCCGAGGCC Amino acid sequence (SEQ ID NO: 43) EPEA Pvs25 DNA sequence (SEQ ID NO: 44) ATGgccgtcacggtagacaccatatgcaaaaatggacagctggttcaaatgagtaaccacttta agtgtatgtgtaacgaagggctggtgcacctttccgaaaatacatgtgaagaaaaaaatgaatg caagaaagaaaccctaggcaaagcatgcggggaatttggccagtgtatagaaaacccagaccca gcacaggtaaacatgtacaaatgtggttgcattgagggctacactttgaaggaagacacttgtg tgcttgatgtatgtcaatacaaaaattgtggagaaagtggcgaatgcattgttgagtacctctc ggaaatccaaagtgcaggttgctcatgtgctattggcaaagtccccaatccagaagatgagaaa aaatgtaccaaaacgggagaaactgcttgtcaattgaaatgtaacacagataatgaagtctgca aaaatgttgaaggagtttacaagtgccagtgtatggaaggctttacgttcgacaaagagaaaaa tgtatgcctttcctattctgtatttaacatcctaaactactccctcttctttatcatcctgctt gtcctttcgtacgtcatataa Pvs25 Amino acid sequence (SEQ ID NO: 45) MAVTVDTICKNGQLVQMSNHFKCMCNEGLVHLSENTCEEKNECKKETLGKACGEFGQCIENPDP AQVNMYKCGCIEGYTLKEDTCVLDVCQYKNCGESGECIVEYLSEIQSAGCSCAIGKVPNPEDEK KCTKTGETACQLKCNTDNEVCKNVEGVYKCQCMEGFTFDKEKNVCLSYSVFNILNYSLFFIILL VLSYVI Pvs28 Amino acid sequence (SEQ ID NO: 46) MKVTAETQC KNGYVVQMSN HFECKCNDGF 80 90 100 VMANENTCEE KRDCTNPQNV NKNCGDYAVC ANTRMNDEER ALRCGCILGY 110 120 130 140 150 TVMNEVCTPN KCNGVLCGKG KCILDPANVN STMCSCNIGT TLDESKKCGK 160 170 180 190 200 PGKTECTLKC KANEECKETQ NYYKCVAKGS GGEGSGGEGS GGEGSGGEGS 210 220 230 GGEGSGGDTG AAYSLMNGSA VISILLVFAF FMMSLV Pvs25 + Pvs28 fusion Amino acid sequence (SEQ ID NO: 47) MAVTVDTICKNGQLVQMSNHFKCMCNEGLVHLSENTCEEKNECKKETLGKACGEFGQCIENPDP AQVNMYKCGCIEGYTLKEDTCVLDVCQYKNCGESGECIVEYLSEIQSAGCSCAIGKVPNPEDEK KCTKTGETACQLKCNTDNEVCKNVEGVYKCQCMEGFTFDKEKNVCLSYSVFNILNYSLFFIILL VLSYVIKVTAETQCKNGYVVQMSNHFECKCNDGFVMANENTCEEKRDCTNPQNVNKNCGDYAVC ANTRMNDEERALRCGCILGYTVMNEVCTPNKCNGVLCGKGKCILDPANVNSTMCSCNIGTTLDE SKKCGKPGKTECTLKCKANEECKETQNYYKCVAKGSGGEGSGGEGSGGEGSGGEGSGGEGSGGD TGAAYSLMNGSAVISILLVFAFFMMSLV