Exploring the genetic diversity pattern of PvEBP/DBP2: A promising candidate for an effective Plasmodium vivax vaccine.

Malaria remains a public health challenge. Since many control strategies have proven ineffective in eradicating this disease, new strategies are required, among which the design of a multivalent vaccine stands out. However, the effectiveness of this strategy has been hindered, among other reasons, by the genetic diversity observed in parasite antigens. In Plasmodium vivax, the Erythrocyte Binding Protein (PvEBP, also known as DBP2) is an alternate ligand to Duffy Binding Protein (DBP); given its structural resemblance to DBP, EBP/DBP2 is proposed as a promising antigen for inclusion in vaccine design. However, the extent of genetic diversity within the locus encoding this protein has not been comprehensively assessed. Thus, this study aimed to characterize the genetic diversity of the locus encoding the P. vivax EBP/DBP2 protein and to determine the evolutionary mechanisms modulating this diversity. Several intrapopulation genetic variation parameters were estimated using 36 gene sequences of PvEBP/DBP2 from Colombian P. vivax clinical isolates and 186 sequences available in databases. The study then evaluated the worldwide genetic structure and the evolutionary forces that may influence the observed patterns of genetic variation. It was found that the PvEBP/DBP2 gene exhibits one of the lowest levels of genetic diversity compared to other vaccine-candidate antigens. Four major haplotypes were shared worldwide. Analysis of the protein's 3D structure and epitope prediction identified five regions with potential antigenic properties. The results suggest that the PvEBP/DBP2 protein possesses ideal characteristics to be considered when designing a multivalent effective antimalarial vaccine against P. vivax.

Evaluating the genetic diversity of the Plasmodium vivax siap2 locus: A promising candidate for an effective malaria vaccine?

Malaria is the deadliest parasitic disease in the world. Traditional control measures have become less effective; hence, there is a need to explore alternative strategies, such as antimalarial vaccines. However, designing an anti-Plasmodium vivax vaccine is considered a challenge due to the complex parasite biology and the antigens' high genetic diversity. Recently, the sporozoite invasion-associated protein 2 (SIAP2) has been suggested as a potential antigen to be considered in vaccine design due to its significance during hepatocyte invasion. However, its use may be limited by the incomplete understanding of gene/protein diversity. Here, the genetic diversity of pvsiap2 using P. vivax DNA samples from Colombia was assessed. Through PCR amplification and sequencing, we compared the Colombian sequences with available worldwide sequences, revealing that pvsiap2 displays low genetic diversity. Molecular evolutionary analyses showed that pvsiap2 appears to be influenced by directional selection. Moreover, the haplotypes found differ by a few mutational steps and several of them were shared between different geographical areas. On the other hand, several conserved regions within PvSIAP2 were predicted as potential B-cell or T-cell epitopes. Considering these characteristics and its role in hepatocyte invasion, the PvSIAP2 protein emerges as a promising antigen to be considered in a multi-antigen-multi-stage (multivalent) fully effective vaccine against P. vivax malaria.

Plasmodium vivax ookinete surface protein (Pvs25) is highly conserved among field isolates from five different regions of the Brazilian Amazon.

The Plasmodium vivax Ookinete Surface Protein (Pvs25) is one of the leading malaria Transmission-Blocking Vaccine candidates based on its high immunogenicity in animal models, transmission-blocking activity of antibodies elicited in clinical trials and high conservation among P. vivax isolates from endemic areas. However, the polymorphism in gene encoding Pvs25 in endemic areas from South America has been poorly studied so far. Here, we investigated the genetic polymorphism of pvs25 in P. vivax isolates from five different regions of the Brazilian Amazon (Cruzeiro do Sul, Mâncio Lima, Guajará, Manaus and Oiapoque) and its impact on antigenicity of predicted B-cell epitopes using gene sequencing and epitope prediction tools. Firstly, only a non-synonymous substitution was found in the 657 bp amplified fragment in all sequenced samples, which represented an exchange of Gln by Lys at position 87 (Q87K) of protein amino acid sequence (domain II EGF-like). Q87K substitution was also present in all studied sites with a total frequency of 37.8%. Cruzeiro do Sul presented Q87K substitution in almost half of the isolates (48.4%), and an expressive frequency (40.5%) was also found in Manaus, while in Mâncio Lima, Guajará and Oiapoque, the frequencies were low (23.5%, 25% and 22.2% respectively). We also observed the Q87K mutation in a predicted B-cell epitope of pvs25, with no significant changes on its putative antigenicity. Our data suggest that the pvs25 gene is conserved among isolates from different Brazilian Amazon geographic regions, an important observation considering the antigen potentiality as a vaccine candidate to cover distinct P. vivax endemic areas worldwide.

Immunogenicity of synthetic peptide constructs based on PvMSP9E795-A808, a linear B-cell epitope of the P. vivax Merozoite Surface Protein-9.

Plasmodium vivax Merozoite Surface Protein-9 (PvMSP-9) is a malaria vaccine candidate naturally immunogenic in humans and able to induce high antibody titers in animals when delivered as a recombinant protein. Recently, we identified the sequence EAAPENAEPVHENA (PvMSP9E795-A808) as the main linear B-cell epitope in naturally exposed individuals. However, the potential of PvMSP9E795-A808 as an immunogen in experimental animal models remained unexplored. Here we assess the immunogenicity of PvMSP9E795-A808 using synthetic peptides. The peptides tested in BALB/c mice include two repeats of the sequence EAAPENAEPVHENA tested alone (peptide RII), or linked to an autologous (PvMSP9 peptide pL; pLRII) or heterologous (p2 tetanus toxin universal T cell epitope; TTRII) T cell epitope. Immune responses were evaluated by ELISA, FLUOROSPOT, and indirect immunofluorescence. We show that all of the peptide constructs tested were immunogenic eliciting specific IgG antibodies at different levels, with a prevalence of IgG1 and IgG2. Animals immunized with synthetic peptides containing T cell epitopes (pLRII or TTRII) had more efficient antibody responses that resulted in higher antibody titers able to recognize the native protein by immunofluorescence. Relevantly, the frequency of IFN-γ secreting SFC elicited by immunization with TTRII synthetic peptide was comparable to that reported to the PvMSP9-Nt recombinant protein. Taken together, our study indicates that PvMSP9E795-A808 is highly immunogenic in mice and further studies to evaluate its value as promising vaccine target are warranted. Moreover, our study supports the critical role of CD4 T cell epitopes to enhance humoral responses induced by subunit based vaccines.

Prime-boost vaccination with recombinant protein and adenovirus-vector expressing Plasmodium vivax circumsporozoite protein (CSP) partially protects mice against Pb/Pv sporozoite challenge.

Vaccine development against Plasmodium vivax malaria lags behind that for Plasmodium falciparum. To narrow this gap, we administered recombinant antigens based on P. vivax circumsporozoite protein (CSP) to mice. We expressed in Pichia pastoris two chimeric proteins by merging the three central repeat regions of different CSP alleles (VK210, VK247, and P. vivax-like). The first construct (yPvCSP-AllFL) contained the fused repeat regions flanked by N- and C-terminal regions. The second construct (yPvCSP-AllCT) contained the fused repeat regions and the C-terminal domain, plus RI region. Mice were vaccinated with three doses of yPvCSP in adjuvants Poly (I:C) or Montanide ISA720. We also used replication-defective adenovirus vectors expressing CSP of human serotype 5 (AdHu5) and chimpanzee serotype 68 (AdC68) for priming mice which were subsequently boosted twice with yPvCSP proteins in Poly (I:C) adjuvant. Regardless of the regime used, immunized mice generated high IgG titres specific to all CSP alleles. After challenge with P. berghei ANKA transgenic parasites expressing Pb/PvVK210 or Pb/PvVK247 sporozoites, significant time delays for parasitemia were observed in all vaccinated mice. These vaccine formulations should be clinically tried for their potential as protective universal vaccine against P. vivax malaria.

Natural immune response to Plasmodium vivax alpha-helical coiled coil protein motifs and its association with the risk of P. vivax malaria.

Protein α-helical coiled coil structures are known to induce antibodies able to block critical functions in different pathogens. In a previous study, a total of 50 proteins of Plasmodium vivax erythrocytic asexual stages containing α-helical coiled coil structural motifs were identified in silico, and the corresponding peptides were chemically synthesized. A total of 43 peptides were recognized by naturally acquired antibodies in plasma samples from both Papua New Guinea (PNG) and Colombian adult donors. In this study, the association between IgG antibodies to these peptides and clinical immunity was further explored by measuring total IgG antibody levels to 24 peptides in baseline samples from a longitudinal study of children aged 1-3 years (n = 164) followed for 16 months. Samples were reactive to all peptides tested. Eight peptides were recognized by >50% of individuals, whereas only one peptide had < 20% reactivity. Children infected at baseline were seropositive to 23/24 peptides. No significant association was observed between antibody titers and age or molecular force of infection, suggesting that antibody levels had already reached an equilibrium. There was a strong association between antibody levels to all peptides and protection against P. vivax clinical episodes during the 16 months follow-up. These results suggest that the selected coiled coil antigens might be good markers of both exposure and acquired immunity to P. vivax malaria, and further preclinical investigation should be performed to determine their potential as P. vivax vaccine antigens.

Generation, characterization and immunogenicity of a novel chimeric recombinant protein based on Plasmodium vivax AMA-1 and MSP119.

Plasmodium vivax is the most widely distributed malaria species and the most prevalent species of malaria in America and Asia. Vaccine development against P. vivax is considered a priority in the global program for the eradication of malaria. Earlier studies have characterized the Apical Membrane Antigen 1 (AMA-1) ectodomain and the C-terminal region (19kDa) of the Merozoite Surface Protein 1 (MSP-1) of P. vivax as immunodominant antigens. Based on this characterization, we designed a chimeric recombinant protein containing both merozoite immunodominant domains (PvAMA166-MSP119). The recombinant PvAMA166-MSP119 was successfully expressed in Pichia pastoris and used to immunize two different mouse strains (BALB/c and C57BL/6) in the presence of the Poly (I:C) as an adjuvant. Immunization with the chimeric protein induced high antibody titers against both proteins in both strains of mice as detected by ELISA. Antisera also recognized the native proteins expressed on the merozoites of mature P. vivax schizonts. Moreover, this antigen was able to induce IFN-gamma-secreting cells in C57BL/6 mice. These findings indicate that this novel yeast recombinant protein containing PvAMA166 and PvMSP119 is advantageous, because of improved antibody titers and cellular immune response. Therefore, this formulation should be further developed for pre-clinical trials in non-human primates as a potential candidate for a P. vivax vaccine.

Global genetic diversity of the Plasmodium vivax transmission-blocking vaccine candidate Pvs48/45.

BACKGROUND: Plasmodium vivax 48/45 protein is expressed on the surface of gametocytes/gametes and plays a key role in gamete fusion during fertilization. This protein was recently expressed in Escherichia coli host as a recombinant product that was highly immunogenic in mice and monkeys and induced antibodies with high transmission-blocking activity, suggesting its potential as a P. vivax transmission-blocking vaccine candidate. To determine sequence polymorphism of natural parasite isolates and its potential influence on the protein structure, all pvs48/45 sequences reported in databases from around the world as well as those from low-transmission settings of Latin America were compared. METHODS: Plasmodium vivax parasite isolates from malaria-endemic regions of Colombia, Brazil and Honduras (n = 60) were used to sequence the Pvs48/45 gene, and compared to those previously reported to GenBank and PlasmoDB (n = 222). Pvs48/45 gene haplotypes were analysed to determine the functional significance of genetic variation in protein structure and vaccine potential. RESULTS: Nine non-synonymous substitutions (E35K, Y196H, H211N, K250N, D335Y, E353Q, A376T, K390T, K418R) and three synonymous substitutions (I73, T149, C156) that define seven different haplotypes were found among the 282 isolates from nine countries when compared with the Sal I reference sequence. Nucleotide diversity (π) was 0.00173 for worldwide samples (range 0.00033-0.00216), resulting in relatively high diversity in Myanmar and Colombia, and low diversity in Mexico, Peru and South Korea. The two most frequent substitutions (E353Q: 41.9 %, K250N: 39.5 %) were predicted to be located in antigenic regions without affecting putative B cell epitopes or the tertiary protein structure. CONCLUSIONS: There is limited sequence polymorphism in pvs48/45 with noted geographical clustering among Asian and American isolates. The low genetic diversity of the protein does not influence the predicted antigenicity or protein structure and, therefore, supports its further development as transmission-blocking vaccine candidate.

In silico Identification and Validation of a Linear and Naturally Immunogenic B-Cell Epitope of the Plasmodium vivax Malaria Vaccine Candidate Merozoite Surface Protein-9.

Synthetic peptide vaccines provide the advantages of safety, stability and low cost. The success of this approach is highly dependent on efficient epitope identification and synthetic strategies for efficacious delivery. In malaria, the Merozoite Surface Protein-9 of Plasmodium vivax (PvMSP9) has been considered a vaccine candidate based on the evidence that specific antibodies were able to inhibit merozoite invasion and recombinant proteins were highly immunogenic in mice and humans. However the identities of linear B-cell epitopes within PvMSP9 as targets of functional antibodies remain undefined. We used several publicly-available algorithms for in silico analyses and prediction of relevant B cell epitopes within PMSP9. We show that the tandem repeat sequence EAAPENAEPVHENA (PvMSP9E795-A808) present at the C-terminal region is a promising target for antibodies, given its high combined score to be a linear epitope and located in a putative intrinsically unstructured region of the native protein. To confirm the predictive value of the computational approach, plasma samples from 545 naturally exposed individuals were screened for IgG reactivity against the recombinant PvMSP9-RIRII729-972 and a synthetic peptide representing the predicted B cell epitope PvMSP9E795-A808. 316 individuals (58%) were responders to the full repetitive region PvMSP9-RIRII, of which 177 (56%) also presented total IgG reactivity against the synthetic peptide, confirming it validity as a B cell epitope. The reactivity indexes of anti-PvMSP9-RIRII and anti-PvMSP9E795-A808 antibodies were correlated. Interestingly, a potential role in the acquisition of protective immunity was associated with the linear epitope, since the IgG1 subclass against PvMSP9E795-A808 was the prevalent subclass and this directly correlated with time elapsed since the last malaria episode; however this was not observed in the antibody responses against the full PvMSP9-RIRII. In conclusion, our findings identified and experimentally confirmed the potential of PvMSP9E795-A808 as an immunogenic linear B cell epitope within the P. vivax malaria vaccine candidate PvMSP9 and support its inclusion in future subunit vaccines.

Immunization with the MAEBL M2 Domain Protects against Lethal Plasmodium yoelii Infection.

Malaria remains a world-threatening disease largely because of the lack of a long-lasting and fully effective vaccine. MAEBL is a type 1 transmembrane molecule with a chimeric cysteine-rich ectodomain homologous to regions of the Duffy binding-like erythrocyte binding protein and apical membrane antigen 1 (AMA1) antigens. Although MAEBL does not appear to be essential for the survival of blood-stage forms, ectodomains M1 and M2, homologous to AMA1, seem to be involved in parasite attachment to erythrocytes, especially M2. MAEBL is necessary for sporozoite infection of mosquito salivary glands and is expressed in liver stages. Here, the Plasmodium yoelii MAEBL-M2 domain was expressed in a prokaryotic vector. C57BL/6J mice were immunized with doses of P. yoelii recombinant protein rPyM2-MAEBL. High levels of antibodies, with balanced IgG1 and IgG2c subclasses, were achieved. rPyM2-MAEBL antisera were capable of recognizing the native antigen. Anti-MAEBL antibodies recognized different MAEBL fragments expressed in CHO cells, showing stronger IgM and IgG responses to the M2 domain and repeat region, respectively. After a challenge with P. yoelii YM (lethal strain)-infected erythrocytes (IE), up to 90% of the immunized animals survived and a reduction of parasitemia was observed. Moreover, splenocytes harvested from immunized animals proliferated in a dose-dependent manner in the presence of rPyM2-MAEBL. Protection was highly dependent on CD4(+), but not CD8(+), T cells toward Th1. rPyM2-MAEBL antisera were also able to significantly inhibit parasite development, as observed in ex vivo P. yoelii erythrocyte invasion assays. Collectively, these findings support the use of MAEBL as a vaccine candidate and open perspectives to understand the mechanisms involved in protection.

Recombinant Pvs48/45 antigen expressed in E. coli generates antibodies that block malaria transmission in Anopheles albimanus mosquitoes.

Transmission of malaria parasites from humans to Anopheles mosquitoes can be inhibited by specific antibodies elicited during malaria infection, which target surface Plasmodium gametocyte/gamete proteins. Some of these proteins may have potential for vaccine development. Pvs48/45 is a P. vivax gametocyte surface antigen orthologous to Pfs48/45, which may play a role during parasite fertilization and thus has potential for transmission blocking (TB) activity. Here we describe the expression of a recombinant Pvs48/45 protein expressed in Escherichia coli as a ∼60kDa construct which we tested for antigenicity using human sera and for its immunogenicity and transmission blocking activity of specific anti-mouse and anti-monkey Pvs48/45 antibodies. The protein reacted with sera of individuals from malaria-endemic areas and in addition induced specific IgG antibody responses in BALB/c mice and Aotus l. griseimembra monkeys. Sera from both immunized animal species recognized native P. vivax protein in Western blot (WB) and immunofluorescence assays. Moreover, sera from immunized mice and monkeys produced significant inhibition of parasite transmission to An. Albimanus mosquitoes as shown by membrane feeding assays. Results indicate the presence of reactive epitopes in the Pvs48/45 recombinant product that induce antibodies with TB activity. Further testing of this protein is ongoing to determine its vaccine potential.

Evaluation of naturally acquired IgG antibodies to a chimeric and non-chimeric recombinant species of Plasmodium vivax reticulocyte binding protein-1: lack of association with HLA-DRB1*/DQB1* in malaria exposed individuals from the Brazilian Amazon.

The development of modular constructs that include antigenic regions targeted by protective immune responses is an attractive approach for subunit vaccine development. However, a main concern of using these vaccine platforms is how to preserve the antigenic identity of conformational B cell epitopes. In the present study we evaluated naturally acquired antibody responses to a chimeric protein engineered to contain a previously defined immunodominant domain of the Plasmodium vivax reticulocyte binding protein-1 located between amino acid positions K435-I777. The construct also includes three regions of the cognate protein (F571-D587, I1745-S1786 and L2235-E2263) predicted to contain MHC class II promiscuous T cell epitopes. Plasma samples from 253 naturally exposed individuals were tested against this chimeric protein named PvRMC-RBP1 and a control protein that includes the native sequence PvRBP123-751 in comparative experiments to study the frequency of total IgG and IgG subclass reactivity. HLA-DRB1 and HLA-DQB1 allelic groups were typed by PCR-SSO to evaluate the association between major HLA class II alleles and antibody responses. We found IgG antibodies that recognized the chimeric PvRMC-RBP1 and the PvRBP123-751 in 47.1% and 60% of the studied population, respectively. Moreover, the reactivity index against both proteins were comparable and associated with time of exposure (p<0.0001) and number of previous malaria episodes (p<0.005). IgG subclass profile showed a predominance of cytophilic IgG1 over other subclasses against both proteins tested. Collectively these studies suggest that the chimeric PvRMC-RBP1 protein retained antigenic determinants in the PvRBP1435-777 native sequence. Although 52.9% of the population did not present detectable titers of antibodies to PvRMC-RBP1, genetic restriction to this chimeric protein does not seem to occur, since no association was observed between the HLA-DRB1* or HLA-DQB1* alleles and the antibody responses. This experimental evidence strongly suggests that the identity of the conformational B cell epitopes is preserved in the chimeric protein.

An unstable Th epitope of P. falciparum fosters central memory T cells and anti-CS antibody responses.

Malaria is transmitted by Plasmodium-infected anopheles mosquitoes. Widespread resistance of mosquitoes to insecticides and resistance of parasites to drugs highlight the urgent need for malaria vaccines. The most advanced malaria vaccines target sporozoites, the infective form of the parasite. A major target of the antibody response to sporozoites are the repeat epitopes of the circumsporozoite (CS) protein, which span almost one half of the protein. Antibodies to these repeats can neutralize sporozoite infectivity. Generation of protective antibody responses to the CS protein (anti-CS Ab) requires help by CD4 T cells. A CD4 T cell epitope from the CS protein designated T* was previously identified by screening T cells from volunteers immunized with irradiated P. falciparum sporozoites. The T* sequence spans twenty amino acids that contains multiple T cell epitopes restricted by various HLA alleles. Subunit malaria vaccines including T* are highly immunogenic in rodents, non-human primates and humans. In this study we characterized a highly conserved HLA-DRß1*04:01 (DR4) restricted T cell epitope (QNT-5) located at the C-terminus of T*. We found that a peptide containing QNT-5 was able to elicit long-term anti-CS Ab responses and prime CD4 T cells in HLA-DR4 transgenic mice despite forming relatively unstable MHC-peptide complexes highly susceptible to HLA-DM editing. We attempted to improve the immunogenicity of QNT-5 by replacing the P1 anchor position with an optimal tyrosine residue. The modified peptide QNT-Y formed stable MHC-peptide complexes highly resistant to HLA-DM editing. Contrary to expectations, a linear peptide containing QNT-Y elicited almost 10-fold lower long-term antibody and IFN-γ responses compared to the linear peptide containing the wild type QNT-5 sequence. Some possibilities regarding why QNT-5 is more effective than QNT-Y in inducing long-term T cell and anti-CS Ab when used as vaccine are discussed.

Invasion-inhibitory antibodies elicited by immunization with Plasmodium vivax apical membrane antigen-1 expressed in Pichia pastoris yeast.

In a recent vaccine trial performed with African children, immunization with a recombinant protein based on Plasmodium falciparum apical membrane antigen 1 (AMA-1) conferred a significant degree of strain-specific resistance against malaria. To contribute to the efforts of generating a vaccine against Plasmodium vivax malaria, we expressed the ectodomain of P. vivax AMA-1 (PvAMA-1) as a secreted soluble protein in the methylotrophic yeast Pichia pastoris. Recognized by a high percentage of sera from individuals infected by P. vivax, this recombinant protein was found to have maintained its antigenicity. The immunogenicity of this protein was evaluated in mice using immunization protocols that included homologous and heterologous prime-boost strategies with plasmid DNA and recombinant protein. We used the following formulations containing different adjuvants: aluminum salts (Alum), Bordetella pertussis monophosphoryl lipid A (MPLA), flagellin FliC from Salmonella enterica serovar Typhimurium, saponin Quil A, or incomplete Freund's adjuvant (IFA). The formulations containing the adjuvants Quil A or IFA elicited the highest IgG antibody titers. Significant antibody titers were also obtained using a formulation developed for human use containing MPLA or Alum plus MPLA. Recombinant PvAMA-1 produced under "conditions of good laboratory practice" provided a good yield, high purity, low endotoxin levels, and no microbial contaminants and reproduced the experimental immunizations. Most relevant for vaccine development was the fact that immunization with PvAMA-1 elicited invasion-inhibitory antibodies against different Asian isolates of P. vivax. Our results show that AMA-1 expressed in P. pastoris is a promising antigen for use in future preclinical and clinical studies.

Immunogenicity of a prime-boost vaccine containing the circumsporozoite proteins of Plasmodium vivax in rodents.

Plasmodium vivax is the most widespread and the second most prevalent malaria-causing species in the world. Current measures used to control the transmission of this disease would benefit from the development of an efficacious vaccine. In the case of the deadly parasite P. falciparum, the recombinant RTS,S vaccine containing the circumsporozoite antigen (CSP) consistently protects 30 to 50% of human volunteers against infection and is undergoing phase III clinical trials in Africa with similar efficacy. These findings encouraged us to develop a P. vivax vaccine containing the three circulating allelic forms of P. vivax CSP. Toward this goal, we generated three recombinant bacterial proteins representing the CSP alleles, as well as a hybrid polypeptide called PvCSP-All-CSP-epitopes. This hybrid contains the conserved N and C termini of P. vivax CSP and the three variant repeat domains in tandem. We also generated simian and human recombinant replication-defective adenovirus vectors expressing PvCSP-All-CSP-epitopes. Mice immunized with the mixture of recombinant proteins in a formulation containing the adjuvant poly(I·C) developed high and long-lasting serum IgG titers comparable to those elicited by proteins emulsified in complete Freund's adjuvant. Antibody titers were similar in mice immunized with homologous (protein-protein) and heterologous (adenovirus-protein) vaccine regimens. The antibodies recognized the three allelic forms of CSP, reacted to the repeated and nonrepeated regions of CSP, and recognized sporozoites expressing the alleles VK210 and VK247. The vaccine formulations described in this work should be useful for the further development of an anti-P. vivax vaccine.

Antigenicity and immunogenicity of a novel chimeric peptide antigen based on the P. vivax circumsporozoite protein.

BACKGROUND: Plasmodium vivax circumsporozoite (PvCS) protein is a major sporozoite surface antigen involved in parasite invasion of hepatocytes and is currently being considered as vaccine candidate. PvCS contains a dimorphic central repetitive fragment flanked by conserved regions that contain functional domains. METHODS: We have developed a chimeric 137-mer synthetic polypeptide (PvCS-NRC) that includes the conserved region I and region II-plus and the two natural repeat variants known as VK210 and VK247. The antigenicity of PvCS-NRC was tested using human sera from PNG and Colombia endemic areas and its immunogenicity was confirmed in mice with different genetic backgrounds, the polypeptide formulated either in Alum or GLA-SE adjuvants was assessed in inbred C3H, CB6F1 and outbred ICR mice, whereas a formulation in Montanide ISA51 was tested in C3H mice. RESULTS: Antigenicity studies indicated that the chimeric peptide is recognized by a high proportion (60-70%) of residents of malaria-endemic areas. Peptides formulated with either GLA-SE or Montanide ISA51 adjuvants induced stronger antibody responses as compared with the Alum formulation. Sera from immunized mice as well as antigen-specific affinity purified human IgG antibodies reacted with sporozoite preparations in immunofluorescence and Western blot assays, and displayed strong in vitro inhibition of sporozoite invasion (ISI) into hepatoma cells. CONCLUSIONS: The polypeptide was recognized at high prevalence when tested against naturally induced human antibodies and was able to induce significant immunogenicity in mice. Additionally, specific antibodies were able to recognize sporozoites and were able to block sporozoite invasion in vitro. Further evaluation of this chimeric protein construct in preclinical phase e.g. in Aotus monkeys in order to assess the humoral and cellular immune responses as well as protective efficacy against parasite challenge of the vaccine candidate must be conducted.

Immunogenicity of recombinant proteins consisting of Plasmodium vivax circumsporozoite protein allelic variant-derived epitopes fused with Salmonella enterica Serovar Typhimurium flagellin.

A Plasmodium falciparum circumsporozoite protein (CSP)-based recombinant fusion vaccine is the first malaria vaccine to reach phase III clinical trials. Resistance to infection correlated with the production of antibodies to the immunodominant central repeat region of the CSP. In contrast to P. falciparum, vaccine development against the CSP of Plasmodium vivax malaria is far behind. Based on this gap in our knowledge, we generated a recombinant chimeric protein containing the immunodominant central repeat regions of the P. vivax CSP fused to Salmonella enterica serovar Typhimurium-derived flagellin (FliC) to activate the innate immune system. The recombinant proteins that were generated contained repeat regions derived from each of the 3 different allelic variants of the P. vivax CSP or a fusion of regions derived from each of the 3 allelic forms. Mice were subcutaneously immunized with the fusion proteins alone or in combination with the Toll-like receptor 3 (TLR-3) agonist poly(I·C), and the anti-CSP serum IgG response was measured. Immunization with a mixture of the 3 recombinant proteins, each containing immunodominant epitopes derived from a single allelic variant, rather than a single recombinant protein carrying a fusion of regions derived from each of 3 allelic forms elicited a stronger immune response. This response was independent of TLR-4 but required TLR-5/MyD88 activation. Antibody titers significantly increased when poly(I·C) was used as an adjuvant with a mixture of the 3 recombinant proteins. These recombinant fusion proteins are novel candidates for the development of an effective malaria vaccine against P. vivax.

The role of amino acid electron-donor/acceptor atoms in host-cell binding peptides is associated with their 3D structure and HLA-binding capacity in sterile malarial immunity induction.

Plasmodium falciparum malaria continues being one of the parasitic diseases causing the highest worldwide mortality due to the parasite's multiple evasion mechanisms, such as immunological silence. Membrane and organelle proteins are used during invasion for interactions mediated by high binding ability peptides (HABPs); these have amino acids which establish hydrogen bonds between them in some of their critical binding residues. Immunisation assays in the Aotus model using HABPs whose critical residues had been modified have revealed a conformational change thereby enabling a protection-inducing response. This has improved fitting within HLA-DRß1(∗) molecules where amino acid electron-donor atoms present in ß-turn, random or distorted α-helix structures preferentially bound to HLA-DR53 molecules, whilst HABPs having amino acid electron-acceptor atoms present in regular α-helix structure bound to HLA-DR52. This data has great implications for vaccine development.

Low genetic polymorphism of merozoite surface proteins 7 and 10 in Colombian Plasmodium vivax isolates.

The merozoite surface protein (MSP) family is involved in the initial interaction between merozoites and erythrocytes in Plasmodium species, its members are therefore becoming major targets for vaccine development. Considering that antigens included in a subunit malaria vaccine should be both accessible to the immune system and lack genetic diversity or have very limited polymorphism, we have analyzed the genetic diversity of three msp genes (msp-7A, msp-7K and msp-10) in different geographical regions of Colombia. The results showed that these genes follow the neutral model of evolution and also display low genetic diversity. The strong conservation found for msp-7 haplotypes in isolates from geographically different regions further suggests that these proteins could be good components of a vaccine against Plasmodium vivax malaria, thereby avoiding strain-specific immune responses.

Limited variation in vaccine candidate Plasmodium falciparum Merozoite Surface Protein-6 over multiple transmission seasons.

BACKGROUND: Plasmodium falciparum Merozoite Surface Protein-6 (PfMSP6) is a component of the complex proteinacious coat that surrounds P. falciparum merozoites. This location, and the presence of anti-PfMSP6 antibodies in P. falciparum-exposed individuals, makes PfMSP6 a potential blood stage vaccine target. However, genetic diversity has proven to be a major hurdle for vaccines targeting other blood stage P. falciparum antigens, and few endemic field studies assessing PfMSP6 gene diversity have been conducted. This study follows PfMSP6 diversity in the Peruvian Amazon from 2003 to 2006 and is the first longitudinal assessment of PfMSP6 sequence dynamics. METHODS: Parasite DNA was extracted from 506 distinct P. falciparum infections spanning the transmission seasons from 2003 to 2006 as part of the Malaria Immunology and Genetics in the Amazon (MIGIA) cohort study near Iquitos, Peru. PfMSP6 was amplified from each sample using a nested PCR protocol, genotyped for allele class by agarose gel electrophoresis, and sequenced to detect diversity. Allele frequencies were analysed using JMP v.8.0.1.0 and correlated with clinical and epidemiological data collected as part of the MIGIA project. RESULTS: Both PfMSP6 allele classes, K1-like and 3D7-like, were detected at the study site, confirming that both are globally distributed. Allele frequencies varied significantly between transmission seasons, with 3D7-class alleles dominating and K1-class alleles nearly disappearing in 2005 and 2006. There was a significant association between allele class and village location (p-value = 0.0008), but no statistically significant association between allele class and age, sex, or symptom status. No intra-allele class sequence diversity was detected. CONCLUSIONS: Both PfMSP6 allele classes are globally distributed, and this study shows that allele frequencies can fluctuate significantly between communities separated by only a few kilometres, and over time in the same community. By contrast, PfMSP6 was highly stable at the sequence level, with no SNPs detected in the 506 samples analysed. This limited diversity supports further investigation of PfMSP6 as a blood stage vaccine candidate, with the clear caveat that any such vaccine must either contain both alleles or generate cross-protective responses that react against both allele classes. Detailed immunoepidemiology studies are needed to establish the viability of these approaches before PfMSP6 advances further down the vaccine development pipeline.