Strain-specific immunity may drive adaptive polymorphism in the merozoite surface protein 1 of the rodent malaria parasite Plasmodium chabaudi

Clinical immunity against malaria is slow to develop, poorly understood and strongly strain-specific. Understanding how strain-specific immunity develops and identifying the parasite antigens involved is crucial to developing effective vaccines against the disease. In previous experiments we have shown that strain-specific protective immunity (SSPI) exists between genetically distinct strains (cloned lines) of the rodent malaria parasite Plasmodium chabaudi chabaudi in mice [Cheesman, S., Raza, A., Carter, R., 2006. Mixed strain infections and strain-specific protective immunity in the rodent malaria parasite P. chabaudi chabaudi in mice. Infect. Immun. 74, 2996-3001]. In two subsequent studies, we identified the highly polymorphic Merozoite Surface Protein 1 (MSP-1) as being the principal candidate molecule for the control of SSPI against P. c. chabaudi malaria [Martinelli et al., 2005; Pattaradilokrat, S., Cheesman, S.J., Carter R., 2007. Linkage group selection: towards identifying genes controlling strain-specific protective immunity in malaria. PLoS ONE 2(9):e857]. In the present study, we sequenced the whole msp1 gene of several genetically distinct strains of P. chabaudi and found high levels of genetic diversity. Protein sequence alignments reveal extensive allelic polymorphism between the P. chabaudi strains, concentrated primarily within five regions of the protein. The 3′-end sequence region, encoding the C-terminal 21 kDa region (MSP-1₂₁), which is analogous and homologous to MSP-1₁₉ of Plasmodium falciparum, appears to have been subject to balancing selection. We have found that the strains with the lowest sequence identity at MSP-1₂₁ (i.e. AS/CB and AJ/CB) induce robust and reciprocal SSPI in experimental mice. In contrast, two strains that do not induce reciprocal SSPI are identical at the 21 kDa region. Final identification of the region(s) controlling SSPI will provide important information to help guide decisions about MSP-1 based vaccines.     

Antibody responses to a panel of Plasmodium falciparum malaria blood-stage antigens in relation to clinical disease outcome in Sudan

Despite many intervention programmes aimed at curtailing the scourge, malaria remains a formidable problem of human health. Immunity to asexual blood-stage of Plasmodium falciparum malaria is thought to be associated with protective antibodies of certain immunoglobulin classes and subclasses. We have analysed immunoglobulin G profiles to six leading blood-stage antigens in relation to clinical malaria outcome in a hospital-based study in Sudan. Our results revealed a linear association with anti-AMA-1-IgG1 antibodies in children <5 years and reduced risk of severe malaria, while the responses of the IgG3 antibodies against MSP-2, MSP-3, GLURP in individuals above 5 years were bi-modal. A dominance of IgG3 antibodies in >5 years was also observed. In the final combined model, the highest levels of IgG1 antibodies to AMA-1, GLURP-R0, and the highest levels of IgG3 antibodies to 3D7 MSP-2 were independently associated with protection from clinical malaria. The study provides further support for the potential importance of the studied merozoite vaccine candidate antigens as targets for parasite neutralizing antibody responses of the IgG1 and IgG3 subclasses.     

Antibody response to allelic variants of 19kDa fragment of MSP-1: recognition of a variant and protection associated with ethnicity in Assam, India

Evidence suggests association of anti MSP-1₁₉ antibodies with protection from clinical malaria. However, the target epitope was reported to vary with respect to response to conserved or variant epitopes in different studies. We have investigated here humoral response of naturally exposed individuals of Tibeto-Burman and Austro-Asiatic ethnic groups to E-TSR and Q-KNG variants of MSP-1₁₉ in comparison to whole merozoite extract (WME) of local strain of Plasmodium falciparum in a longitudinal prospective cohort study. The association of antibodies in relation to risk of infection and disease severity was determined.A relatively lower seropositivity to MSP1₁₉ peptides derived from 3D7 and FVO strains in comparison to whole merozoite extract of local P. falciparum strain was observed. Recognition of Q-KNG variant was markedly lower in TB (p < 0.0001) indicating a role of ethnicity. The Tea tribes of Austro-Asiatic affinity had higher antibody response (E-TSR; p = 0.038 and Q-KNG; p = 0.004) and equally recognized the two variants. A reduced risk of clinical infection in high transmission summer season was seen in presence of anti MSP-1₁₉ antibodies (p = 0.013) and antibody level was predictive of risk of clinical malaria (ROC = 0.729). Anti E-TSR antibodies were inversely associated to disease severity at KTE (λ²p = 0.013; t-test p = 0.032).The present study demonstrated antibody response to MSP-1₁₉ was associated with protection from frequent episodes of malaria and disease severity and that the host genetic background was important factor in response to MSP-1₁₉ allelic variant.     

Immunization against Plasmodium chabaudi malaria using combined formulations of apical membrane antigen-1 and merozoite surface protein-1

The control of Plasmodium falciparum malaria by vaccination will require immunization with multiple parasite antigens effectively formulated in combination. In this regard, proteins expressed on the surface of blood-stage merozoites are attractive as vaccine targets given their functional importance in the invasion of erythrocytes and accessibility to serum antibodies. We have utilized a Plasmodium chabaudi vaccine model to begin to evaluate the efficacy of immunization with combined formulations of apical membrane antigen-1 (AMA-1) and merozoite surface protein-1 (MSP-1). Using a pET/T7 RNA polymerase bacterial expression system, we have expressed, purified and refolded recombinant antigens representing the 54 kDa ectodomain of Pc AMA-1 and the 42 kDa C-terminus of Pc MSP-1. Immunization with recombinant Pc AMA-1+Pc MSP-1(42) induced a high level of protection against P. chabaudi malaria with protective efficacy varying with antigen dose, choice of adjuvant, and immunization protocol. Based on the reduction of P. chabaudi parasitemia, Alum proved effective for use with the combination of Pc AMA-1 and Pc MSP-1(42). The use of Quil A was similarly effective with single or combined antigen immunizations, particularly with low antigen dose. In general, serological analysis of prechallenge sera indicated a dominant IgG1 response. For a given formulation, immunization with the combination of Pc AMA-1 and Pc MSP-1(42) elicited IgG responses comparable to those observed following immunization with each antigen alone. However, prechallenge antibody titers alone were not predictive of protective efficacy. While Pc AMA-1 and Pc MSP-1(42) can be effectively formulated in combination, further study is needed to define measurable parameters of protective T cell and B cell responses induced by Pc AMA-1+Pc MSP-1(42) that are predictive of vaccine efficacy.     

Expression,purification and characterization of allelic variants of MSP-142 from Indian Plasmodium falciparum isolates

The C-terminal 19 and 42 kDa fragments of Plasmodium falciparum merozoite surface protein 1 (MSP-1) have shown to be protective in animals against lethal parasite challenge. The MSP-1₁₉ being highly conserved may lack sufficient number of T-cell epitopes in order to elicit a broader response in genetically diverse populations. The inclusion of additional epitopes from the N-terminal MSP-1₄₂ has shown to enhance the protective efficacy of MSP-1₁₉ vaccine. In an attempt to examine the strain specific immunogenicity to MSP-1, we have cloned and expressed three diverse allelic variants of MSP-1₄₂ from Indian P. falciparum isolates in bacteria. Among three alleles, one was extremely rare and not been found before. These purified and refolded recombinant products were recognized by conformation specific monoclonal antibodies and hyper-immune sera. Immunization of mice and rabbits with the purified proteins generated high titer biologically active polyclonal antibodies supporting further development of this vaccine candidate antigen.     

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 (19 kDa) 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.     

Genetic diversity in the C-terminus of merozoite surface protein 1 among Plasmodium knowlesi isolates from Selangor and Sabah Borneo,Malaysia

Plasmodium knowlesi, a malaria parasite of macaques, has emerged as an important parasite of humans. Despite the significance of P. knowlesi malaria in parts of Southeast Asia, very little is known about the genetic variation in this parasite. Our aim here was to explore sequence variation in a molecule called the 42 kDa merozoite surface protein-1 (MSP-1), which is found on the surface of blood stages of Plasmodium spp. and plays a key role in erythrocyte invasion. Several studies of P. falciparum have reported that the C-terminus (a 42 kDa fragment) of merozoite surface protein-1 (MSP-1₄₂; consisting of MSP-1₁₉ and MSP-1₃₃) is a potential candidate for a malaria vaccine. However, to date, no study has yet investigated the sequence diversity of the gene encoding P. knowlesi MSP-1₄₂ (comprising Pk-msp-1₁₉ and Pk-msp-1₃₃) among isolates in Malaysia. The present study explored this aspect. Twelve P. knowlesi isolates were collected from patients from hospitals in Selangor and Sabah Borneo, Malaysia, between 2012 and 2014. The Pk-msp-1₄₂ gene was amplified by PCR and directly sequenced. Haplotype diversity (Hd) and nucleotide diversity (л) were studied among the isolates. There was relatively high genetic variation among P. knowlesi isolates; overall Hd and л were 1 ± 0.034 and 0.01132 ± 0.00124, respectively. A total of nine different haplotypes related to amino acid alterations at 13 positions, and the Pk-MSP-1₁₉ sequence was found to be more conserved than Pk-msp-1₃₃. We have found evidence for negative selection in Pk-msp-₄₂ as well as the 33 kDa and 19 kDa fragments by comparing the rate of non-synonymous versus synonymous substitutions. Future investigations should study large numbers of samples from disparate geographical locations to critically assess whether this molecule might be a potential vaccine target for P. knowlesi.     

Induction of humoral immune response against PfMSP-1(19) and PvMSP-1(19) using gold nanoparticles along with alum

The C-terminal 19 kDa fragment of merozoite surface protein 1(MSP-1₁₉) is an important vaccine candidate but a poor immunogen with available human compatible adjuvants like alum. We have investigated if Gold Nanoparticles (GNPs) based delivery system would enhance immune response to recombinant PfMSP-1₁₉ and PvMSP-1₁₉. We found that PfMSP-1₁₉/PvMSP-1₁₉ coated on GNPs or in alum was poorly immunogenic in mice, a robust antibody response was observed when PfMSP-1₁₉/PvMSP-1₁₉ coated GNPs were formulated with alum. PfMSP-1₁₉/PvMSP-1₁₉ coated GNPs strongly recognized conformation specific monoclonal antibodies showing that capping on to GNPs did not compromise with conformational integrity of these antigens, which is critical for the generation of functional antibody response. Anti-PfMSP-1₁₉ antibodies raised with GNP-alum formulation recognized the native protein on parasite surface and showed invasion inhibition of Plasmodium falciparum parasite in an in vitro assay. Antigen coated GNPs with alum may be useful in the development of an alternate adjuvant formulation.     

Dynamic changes of Plasmodium vivax population structure in South Korea

The vivax malaria epidemic has persisted in South Korea since its reemergence in 1993. Although there has been a significant decrease in the number of malaria cases in recent years, vivax malaria is still a major public health concern. To gain in-depth insight into the genetic makeup of Korean Plasmodium vivax, we analyzed polymorphic patterns of two major antigens, merozoite surface protein-1 (MSP-1) and MSP-3α, in 255 Korean P. vivax isolates collected over an extended period from 1998 to 2013. Combinational genetic analysis of polymorphic patterns of MSP-1 and MSP-3α in the isolates suggests that the P. vivax population in South Korea has been diversifying rapidly, with the appearance of parasites with new genotypes, despite the recent reduction of disease incidence. These results highlight the importance of molecular epidemiological investigations to supervise the genetic variation of the parasite in South Korea.     

Mixed allele malaria vaccines: host protection and within-host selection

Malaria parasites are frequently polymorphic at the antigenic targets of many candidate vaccines, presumably as a consequence of selection pressure from protective immune responses. Conventional wisdom is therefore that vaccines directed against a single variant could select for non-target variants, rendering the vaccine useless. Many people have argued that a solution is to develop vaccines containing the products of more than one variant of the target. However, we are unaware of any evidence that multi-allele vaccines better protect hosts against parasites or morbidity. Moreover, selection of antigen-variants is not the only evolution that could occur in response to vaccination. Increased virulence could also be favored if more aggressive strains are less well controlled by vaccine-induced immunity. Virulence and antigenic identity have been confounded in all studies so far, and so we do not know formally from any animal or human studies whether vaccine failure has been due to evasion of protective responses by variants at target epitopes, or whether vaccines are just less good at protecting against more aggressive strains. Using the rodent malaria model Plasmodium chabaudi and recombinant apical membrane antigen-1 (AMA-1), we tested whether a bi-allelic vaccine afforded greater protection from parasite infection and morbidity than did vaccination with the component alleles alone. We also tested the effect of mono- and bi-allelic vaccination on within-host selection of mixed P. chabaudi infections, and whether parasite virulence mediates pathogen titres in immunized hosts. We found that vaccination with the bi-allelic AMA-1 formulation did not afford the host greater protection from parasite infection or morbidity than did mono-allelic AMA-1 immunization. Mono-allelic immunization increased the frequency of heterologous clones in mixed clone infections. There was no evidence that any type of immunization regime favored virulence. A single AMA-1 variant is a component of candidate malaria vaccines current in human trials; our results suggest that adding extra AMA-1 alleles to these vaccines would not confer clinical benefits, but that that mono-allelic vaccines could alter AMA-1 allele frequencies in natural populations.     

A threshold concentration of anti-merozoite antibodies is required for protection from clinical episodes of malaria

Antibodies to selected Plasmodium falciparum merozoite antigens are often reported to be associated with protection from malaria in one epidemiological cohort, but not in another. Here, we sought to understand this paradox by exploring the hypothesis that a threshold concentration of antibodies is necessary for protection. We analyzed data from two independent cohorts along the Kenyan coast, one in which antibodies to AMA1, MSP-2 and MSP-3 were associated with protection from malaria (Chonyi) and another in which this association was not observed (Junju). We used a malaria reference reagent to standardize antibody measurements across both cohorts, and applied statistical methods to derive the threshold concentration of antibodies against each antigen that best correlated with a reduced risk of malaria (the protective threshold), in the Chonyi cohort. We then tested whether antibodies in Junju reached the protective threshold concentrations observed in the Chonyi cohort. Except for children under 3 years, the age-matched proportions of children achieving protective threshold concentrations of antibodies against AMA1 and MSP-2 were significantly lower in Junju compared to Chonyi (Fishers exact test, P < 0.01). For MSP-3, this difference was significant only among 4–5 year olds. We conclude that although antibodies are commonly detected in malaria endemic populations, they may be present in concentrations that are insufficient for protection. Our results have implications for the analysis and interpretation of similar data from immuno-epidemiological studies.     

Temporal variations in immune responses to conserved regions of Plasmodium falciparum merozoite surface proteins related to the severity of a prior malaria episode in Gabonese children

We measured cellular and humoral responses to conserved regions of Plasmodium falciparum merozoite surface proteins 1 and 2 (MSP-1 and MSP-2) at different times during and after acute infection in matched groups of Gabonese children who presented with either mild or severe malaria. We used an MSP-1(19) recombinant protein and peptides corresponding to conserved epitopes in MSP-1 and MSP-2 N- and C-terminal regions. The prevalences of peptide-induced cell-mediated responses were maximal in both groups when they were healthy, but were consistently higher in the mild malaria group, whereas peptide-specific antibody responses were consistently highest in the severe malaria group. Recombinant MSP-1(19) protein-specific antibody levels in the 2 groups were similar both prior to and 1 month post-treatment but declined later when the children were healthy and parasite-free, to a significantly lower level in those admitted with severe malaria, reflecting the profile of the predominant MSP-1(19)-specific immunoglobulin G1 isotype. This finding implies a defect in the ability of children with a history of severe malaria to maintain an antibody response putatively associated with immunity to P. falciparum malaria.     

A recombinant vaccine based on domain II of Plasmodium vivax Apical Membrane Antigen 1 induces high antibody titres in mice

The Apical Membrane Antigen 1 (AMA-1) is considered a promising candidate for development of a malaria vaccine against asexual stages of Plasmodium. We recently identified domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a highly immunogenic region recognised by IgG antibodies present in many individuals during patent infection with P. vivax. The present study was designed to evaluate the immunogenic properties of a bacterial recombinant protein containing PvAMA-1 DII. To accomplish this, the recombinant protein was administered to mice in the presence of each of the following six adjuvants: Complete/Incomplete Freund's Adjuvant (CFA/IFA), aluminium hydroxide (Alum), Quil A, QS21 saponin, CpG-ODN 1826 and TiterMax. We found that recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of any of the tested adjuvants. Importantly, we show that DII-specific antibodies recognised the native AMA-1 protein expressed on the surface of P. vivax merozoites isolated from the blood of infected patients. These results demonstrate that a recombinant protein containing PvAMA-1 DII is immunogenic when administered in different adjuvant formulations, and indicate that this region of the AMA-1 protein should continue to be evaluated as part of a subunit vaccine against vivax malaria.     

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1₁₉) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1₁₉ component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His₆FliC-MSP1₁₉ fusion protein was expressed from a recombinant Escherichia coli and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His₆MSP1₁₉, mice subcutaneously immunised with the recombinant His₆FliC-MSP1₁₉ developed strong MSP1₁₉-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund's adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-γ secretion by immune spleen cells. MSP1₁₉-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity.     

Antibody levels detected by the fluorescent antibody technique in mice infected with Plasmodium vinckei and P. chabaudi

The malaria parasites of mice are convenient models for immunological studies. Plasmodium vinckei and P. chabaudi are similar parasites which behave differently in mice, the former invariably being fatal whereas the latter seldom kills the host. The experiments described in this paper were performed in order to compare the antibody levels in the Ig, IgM and IgG serum fractions in mice cured of P. vinckei infections and naturally recovered from P. chabaudi. The technique involved using specific labelled antimouse-Ig, -IgM and -IgG sera and had not previously been applied to malaria infections. The results showed that the patterns of antibody production were similar in cured and naturally recovered mice. IgG antibodies were involved from an early stage of the infection and IgM antibodies persisted throughout the period of experiment and even after challenge. These results are significant in that they indicate a pattern of immune response different from the classically accepted one and draw attention to the fact that even in highly immune animals considerable amounts of IgM may be present.     

Do mixed infections matter? Assessing virulence of mixed-clone infections in experimental human and murine malaria

BackgroundMalaria parasites within an individual infection often consist of multiple strains (clonal populations) of a single species, which have the potential to interact both with one another, and with the host immune system. Several effects of these interactions have been measured in different parasite systems including competition and mutualism; however, direct observation of these effects in human malaria has been limited by sampling complexities and inherent ethical limitations.MethodsUsing multiple complementary epidemiological models, we propose a suite of analyses to more fully utilize data from challenge experiments, and re-examine historical human challenge studies with mixed-strain Plasmodium vivax inocula. We then compare these results with murine model systems using mixed-strain Plasmodium yoelii or Plasmodium chabaudi, to explore the utility of these methods in fully utilizing these data, including the first quantitative estimates of effect sizes for mixed-strain parasitemia. These models also provide a method to assess consistency within these animal model systems.ResultsWe find that amongst a limited set of P. vivax (incubation time) and P. yoelii infections (time-to-mortality), survival times at a study population-level are intermediate between each single-clone infection, and are not dominated by the more virulent clone; in P. vivax relapses, mixed clone infections also show intermediate survival curves. In these infections, the results strongly suggest that highly virulent clones have their virulence attenuated by the presence of less-virulent clones. The analysis of multiple experiments with P. chabaudi suggests greater nuances in the interactions between strains, and that mortality and time-to-event in mixed-strain infections are both indistinguishable from single infections with the more virulent strain.ConclusionsThese divergent dynamics support earlier work that suggested drivers of virulence may differ in fundamental ways between malaria species that are reticulocyte-specific and those that readily infect all red blood cell stages which should be studied in greater detail. The effect sizes (magnitude of biological effects) from these analyses are significant, and suggest the potential for important gains in malaria control by greater incorporation of evolutionary epidemiology theory. Moreover, we suggest that using these epidemiological models may generally allow fuller use of data from experimentally challenging animal model experiments.     

Direct effect of Plasmodium vivax recombinant vaccine candidates AMA-1 and MSP-119 on the innate immune response

The recombinant apical membrane antigen 1 (AMA-1) and 19-kDa fragment of merozoite surface protein (MSP-1(19)) are the lead candidates for inclusion in a vaccine against blood stages of malaria due to encouraging protective studies in humans and animals. Despite the importance of an efficacious malaria vaccine, vaccine-related research has focused on identifying antigens that result in protective immunity rather than determining the nature of anti-malarial immune effector mechanisms. Moreover, emphasis has been placed on adaptive rather than innate immune responses. In this study, we investigated the effect of Plasmodium vivax vaccine candidates Pv-AMA-1 and Pv-MSP-1(19) on the immune response of malaria-na?ve donors. Maturation of dendritic cells is altered by Pv-AMA-1 but not Pv-MSP-1(19), as observed by differential expression of cell surface markers. In addition, Pv-AMA-1 induced an increased production of MIP-1alpha/CCL3 and decreased production of TARC/CCL17 levels in both dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs). Finally, a significant pro-inflammatory response was elicited by Pv-AMA-1-stimulated PBMCs. These results suggest that the recombinant vaccine candidate Pv-AMA-1 may play a direct role on innate immune response and might be involved in parasite destruction.     

The evolution and diversity of a low complexity vaccine candidate,merozoite surface protein 9 (MSP-9), in Plasmodium vivax and closely related species

The merozoite surface protein-9 (MSP-9) has been considered a target for an anti-malarial vaccine since it is one of many proteins involved in the erythrocyte invasion, a critical step in the parasite life cycle. Orthologs encoding this antigen have been found in all known species of Plasmodium parasitic to primates. In order to characterize and investigate the extent and maintenance of MSP-9 genetic diversity, we analyzed DNA sequences of the following malaria parasite species: Plasmodium falciparum, Plasmodium reichenowi, Plasmodium chabaudi, Plasmodium yoelii, Plasmodium berghei, Plasmodium coatneyi, Plasmodium gonderi, Plasmodium knowlesi, Plasmodium inui, Plasmodium simiovale, Plasmodium fieldi, Plasmodium cynomolgi and Plasmodium vivax and evaluated the signature of natural selection in all MSP-9 orthologs. Our findings suggest that the gene encoding MSP-9 is under purifying selection in P. vivax and closely related species. We further explored how selection affected different regions of MSP-9 by comparing the polymorphisms in P. vivax and P. falciparum, and found contrasting patterns between these two species that suggest differences in functional constraints. This observation implies that the MSP-9 orthologs in human parasites may interact differently with the host immune response. Thus, studies carried out in one species cannot be directly translated into the other.     

Absence of Asymptomatic Malaria Infection in Endemic Area of Bashagard District,Hormozgan Province,Iran

Background

A successful malaria elimination program calls for enough attention to parasite carriers, especially asymptomatic malaria, as well as the diagnosis and treatment of clinical cases. Asymptomatic malaria is an infection that patients do not show any symptom; thus, these patients play critical role in the concept of an elimination program. The current investigation was conducted to evaluate the presence of these cases in Bashagard District, formerly a high malaria transmission area in Hormozgan Province, Iran.

Methods

Blood samples (n = 500) were collected from symptomless individuals residing in Bashagard to evaluate Plasmodium infection by using microscopic, serological and nested-PCR techniques.

Results

Regarding the microscopic and nested-PCR analysis, no asymptomatic infection was detected among studied individuals. Totally, 1% of the studied population (5 of 500) had anti PvMSP-1₁₉-specific IgG antibody; however, only 0.2% (1 of 500) of the individuals was seropositive to recombinant PfMSP-1₁₉, using ELISA.

Conclusion

This study showed no asymptomatic malaria infection in the studied population; hence malaria elimination is feasible and can be successfully carried out in this region.     

Protective immune responses elicited by immunization with a chimeric blood-stage malaria vaccine persist but are not boosted by Plasmodium yoelii challenge infection

An efficacious malaria vaccine remains elusive despite concerted efforts. Using the Plasmodium yoelii murine model, we previously reported that immunization with the C-terminal 19 kDa domain of merozoite surface protein 1 (MSP1₁₉) fused to full-length MSP8 protected against lethal P. yoelii 17XL, well beyond that achieved by single or combined immunizations with the component antigens. Here, we continue the evaluation of the chimeric PyMSP1/8 vaccine. We show that immunization with rPyMSP1/8 vaccine elicited an MSP8-restricted T cell response that was sufficient to provide help for both PyMSP1₁₉ and PyMSP8-specific B cells to produce high and sustained levels of protective antibodies. The enhanced efficacy of immunization with rPyMSP1/8, in comparison to a combined formulation of rPyMSP1₄₂ and rPyMSP8, was not due to improved conformation of protective B cell epitopes in the chimeric molecule. Unexpectedly, rPyMSP1/8 vaccine-induced antibody responses were not boosted by exposure to P. yoelii 17XL infected RBCs. However, rPyMSP1/8 immunized and infected mice mounted robust responses to a diverse set of blood-stage antigens. The data support the further development of an MSP1/8 chimeric vaccine but also suggest that vaccines that prime for responses to a diverse set of parasite proteins will be required to maximize vaccine efficacy.