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The malaria parasite Plasmodium falciparum proliferates in the blood stream where the host immune system is most active. To escape from host immunity, P. falciparum has developed a number of evasion mechanisms. Serine repeat antigen 5 (SERA5) is a blood stage antigen highly expressed at late trophozoite and schizont stages. The P47 N-terminal domain of SERA5, the basis of SE36 antigen of the blood stage vaccine candidate under clinical trials, covers the merozoite surface. Exploring the role of the P47 domain, screening of serum proteins showed that vitronectin (VTN) directly binds to 20 residues in the C-terminal region of SE36. VTN co-localized with P47 domain in the schizont and merozoite stages. Phagocytosis assay using THP-1 cells demonstrated that VTN bound to SE36 prevented engulfment of SE36-beads. In addition, several serum proteins localized on the merozoite surface, suggesting that host proteins camouflage merozoites against host immunity via binding to VTN. [ABSTRACT FROM AUTHOR]

The study of antigenic targets of naturally-acquired immunity is essential to identify and prioritize antigens for further functional characterization. We measured total IgG antibodies to 38 P. vivax antigens, investigating their relationship with prospective risk of malaria in a cohort of 1-3 years old Papua New Guinean children. Using simulated annealing algorithms, the potential protective efficacy of antibodies to multiple antigen-combinations, and the antibody thresholds associated with protection were investigated for the first time. High antibody levels to multiple known and newly identified proteins were strongly associated with protection (IRR 0.44-0.74, p<0.001-0.041). Among five-antigen combinations with the strongest protective effect (>90%), EBP, DBPII, RBP1a, CyRPA, and PVX_081550 were most frequently identified; several of them requiring very low antibody levels to show a protective association. These data identify individual antigens that should be prioritized for further functional testing and establish a clear path to testing a multicomponent P. vivax vaccine. [ABSTRACT FROM AUTHOR]

Background: Thirty-one glycosylphosphatidylinositol (GPI)-anchored proteins of Plasmodium vivax, merozoite surface protein 1 (MSP1), MSP1 paralogue, MSP4, MSP5, MSP8, and MSP10 have been reported from homologs of Plasmodium falciparum by gene annotation with bioinformatics tools. These GPI-anchored proteins contain two epidermal growth factor (EGF)-like domains at its C-terminus. Here, P. vivax merozoite surface protein 8 (PvMSP8) are considered as potential targets of protective immunity. Methods: Recombinant PvMSP8 (rPvMSP8) was expressed, purified, and used for the assessment of humoral and cellular immune responses in P. vivax-infected patients and immune mice. Moreover, the target epitope of ant-PvMSP8 antibodies and subcellular localization of PvMSP8 was also determined. Results: The rPvMSP8 was successfully expressed and purified as soluble form as ~55 kDa. PvMSP8 was localized to the outer circle of pigments associated with the food vacuole. The rPvMSP8 protein had a high antigenicity (73.2% in sensitivity and 96.2% in specificity) in patients infected with P. vivax. IgG2 antibody subtype was the predominantly responses to this antigen. Antibody response to PvMSP8 increased up to day 7 and after that slightly decreased within a month. The longevity of anti-PvMSP8 antibody was stably sustained up to 12-year recovery patient samples. Most anti-PvMSP8 antibodies recognized two epitopes that were located outside the C-terminal EGF-like domain. The cellular immune response in P. vivax-exposed individuals produced high levels of IFN-γ and IL-10 upon PvMSP8 antigen stimulation in vitro. Conclusions: All data in this study suggest that PvMSP8 antigen has a potential to induce both humoral and cellular immune responses in patients with P. vivax infection. The subcellular localization of PvMSP8 confirmed that it was associated with the parasite food vacuole in blood-stage parasites. A further characterization of this protein will be useful for blood stage P. vivax vaccine development. [ABSTRACT FROM AUTHOR]

Background: Although a number of Plasmodium vivax proteins have been identified, few have been investigated as potential vaccine candidates. This study characterized the Plasmodium vivax merozoite surface antigen 180 (PvMSA180, PVX_094920), a novel P. vivax antigenic protein. Methods: The target gene was amplified as four overlapping domains (D1, D2, D3 and D4) to enable expression of the recombinant protein using cell-free and bacterial expression systems. The recombinant PvMSA180 proteins were used in protein microarrays to evaluate the humoral immune response of 72 vivax-infected patients and 24 vivax-naïve individuals. Antibodies produced in mice against the PvMSA180-D1 and -D4 domains were used to assess the subcellular localization of schizont-stage parasites with immunofluorescence assays. A total of 51 pvmsa180 sequences from 12 countries (41 sequences from PlasmoDB and 6 generated in this study) were used to determine the genetic diversity and genealogical relationships with DNAsp and NETWORK software packages, respectively. Results: PvMSA180 consists of 1603 amino acids with a predicted molecular mass of 182 kDa, and has a signal peptide at the amino-terminus. A total of 70.8% of patients (51/72) showed a specific antibody response to at least one of the PvMSA180 domains, and 20.8% (15/72) exhibited a robust antibody response to at least three of the domains. These findings suggest that PvMSA180 is targeted by the humoral immune response during natural infection with P. vivax. Immunofluorescence analysis demonstrated that PvMSA180 is localized on the merozoite surface of schizontstage parasites, and pvmsa180 sequences originating from various geographic regions worldwide showed low genetic diversity. Twenty-two haplotypes were found, and haplotype 6 (Hap_6, 77%) of pvmsa180 was detected in isolates from six countries. Conclusions: A novel P. vivax surface protein, PvMSA180, was characterized in this study. Most of P. vivax-infected patients had specific antibodies against particular antigenic domains, indicating that this protein is immunogenic in naturally exposed populations. Genetic analysis of worldwide isolates showed that pvmsa180 is less polymorphic than other well-known candidates and that some haplotypes are common to several countries. However, additional studies with a larger sample size are necessary to evaluate the antibody responses in geographically separated populations, and to identify the function of PvMSA180 during parasite invasion. [ABSTRACT FROM AUTHOR]

Background: Individual drug treatment may select resistant parasites in the human body, a process termed in vivo selection. Some single nucleotide polymorphisms in Plasmodium falciparum chloroquine-resistance transporter (pfcrt) and multidrug resistance gene 1 (pfmdr1) genes have been reportedly selected after artemether-lumefantrine treatment. However, there is a paucity of data regarding in vivo selection of P. falciparum Kelch propeller domain (pfkelch13) polymorphisms, responsible for artemisinin-resistance in Asia, and six putative background mutations for artemisinin resistance; D193Y in ferredoxin, T484I in multiple resistance protein 2, V127M in apicoplast ribosomal protein S10, I356T in pfcrt, V1157L in protein phosphatase and C1484F in phosphoinositide-binding protein. Methods: Artemether-lumefantrine efficacy study with a follow-up period of 28 days was conducted in northern Uganda in 2014. The above-mentioned genotypes were comparatively analysed before drug administration and on days; 3, 7, and 28 days after treatment. Results: In 61 individuals with successful follow-up, artemether-lumefantrine treatment regimen was very effective with PCR adjusted efficacy of 95.2%. Among 146 isolates obtained before treatment, wild-type alleles were observed in 98.6% of isolates in pfkelch13 and in all isolates in the six putative background genes except I356T in pfcrt, which had 2.4% of isolates as mixed infections. In vivo selection study revealed that all isolates detected in the follow-up period harboured wild type alleles in pfkelch13 and the six background genes. Conclusion: Mutations in pfkelch13 and the six background genes may not play an important role in the in vivo selection after artemether-lumefantrine treatment in Uganda. Different mechanisms might rather be associated with the existence of parasites after treatment. [ABSTRACT FROM AUTHOR]

Background: Plasmodium ookinete surface proteins as post-fertilization target antigens are potential malaria transmission-blocking vaccine (TBV) candidates. Putative secreted ookinete protein 25 (PSOP25) is a highly conserved ookinete surface protein, and has been shown to be a promising novel TBV target. Here, we further investigated the TBV activities of the full-length recombinant PSOP25 (rPSOP25) protein in Plasmodium berghei, and characterized the potential functions of PSOP25 during the P. berghei life-cycle. Methods: We expressed the full-length P. berghei PSOP25 protein in a prokaryotic expression system, and developed polyclonal mouse antisera and a monoclonal antibody (mAb) against the recombinant protein. Indirect immunofluorescence assay (IFA) and Western blot were used to test the specificity of antibodies. The transmission-blocking (TB) activities of antibodies were evaluated by the in vitro ookinete conversion assay and by direct mosquito feeding assay (DFA). Finally, the function of PSOP25 during Plasmodium development was studied by deleting the psop25 gene. Results: Both polyclonal mouse antisera and anti-rPSOP25 mAb recognized the PSOP25 proteins in the parasites, and IFA showed the preferential expression of PSOP25 on the surface of zygotes, retorts and mature ookinetes. In vitro, these antibodies significantly inhibited ookinetes formation in an antibody concentration-dependent manner. In DFA, mice immunized with the rPSOP25 and those receiving passive transfer of the anti-rPSOP25 mAb reduced the prevalence of mosquito infection by 31.2 and 26.1%, and oocyst density by 66.3 and 63.3%, respectively. Genetic knockout of the psop25 gene did not have a detectable impact on the asexual growth of P. berghei, but significantly affected the maturation of ookinetes and the formation of midgut oocysts. Conclusions: The full-length rPSOP25 could elicit strong antibody response in mice. Polyclonal and monoclonal antibodies against PSOP25 could effectively block the formation of ookinetes in vitro and transmission of the parasites to mosquitoes. Genetic manipulation study indicated that PSOP25 is required for ookinete maturation in P. berghei. These results support further testing of the PSOP25 orthologs in human malaria parasites as promising TBV candidates. [ABSTRACT FROM AUTHOR]

Background: Serological markers for exposure to different Plasmodium species have recently been used in multiplex immunoassays based on the Luminex technology. However, interpretation of the assay results requires consideration of the half-life of specific antibodies against these markers. Therefore, the aim of the present study was to document the half-life of malaria specific serological makers, as well as assessing the sensitivity of these markers to pick up recent changes in malaria exposure. Methods: A recently developed multiplex immunoassay was used to measure the intensity of antibody (Ab) responses against 19 different Plasmodium specific antigens, covering different human malaria parasites and two vector saliva antigens. Therefore, 8439 blood samples from five cross-sectional surveys in Ratanakiri, Cambodia, were analysed. These involve a random selection from two selected surveys, and an additional set of blood samples of individuals that were randomly re-sampled three, four or five times. A generalized estimating equation model and linear regression models were fitted on log transformed antibody intensity data. Results: Results showed that most (17/21) Ab-responses are higher in PCR positive than PCR negative individuals. Furthermore, these antibody-responses follow the same upward trend within each age group. Estimation of the half-lives showed differences between serological markers that reflect short-(seasonal) and long-term (year round) transmission trends. Ab levels declined significantly together with a decrease of PCR prevalence in a group of malaria endemic villages. Conclusion: For Plasmodium falciparum, antibodies against LSA3.RE, GLURP and Pf.GLURP.R2 are most likely to be a reflexion of recent (range from 6 to 8 months) exposure in the Mekong Subregion. PvEBP is the only Plasmodium vivax Ag responding reasonably well, in spite of an estimated Ab half-life of more than 1 year. The use of Ab intensity data rather dichotomizing the continuous Ab-titre data (positive vs negative) will lead to an improved approach for serological surveillance. [ABSTRACT FROM AUTHOR]

Successful invasion of human erythrocytes by P. falciparum merozoites is required for infection of the host and parasite survival. The early stages of invasion are mediated via merozoite surface proteins that interact with human erythrocytes. The nature of these interactions are currently not well understood, but it is known that Merozoite Surface Protein 1 (MSP1) is critical for successful erythrocyte invasion. Here, we show that the peripheral merozoite surface proteins MSP3, MSP6, MSPDBL1, MSPDBL2 and MSP7 bind directly to MSP1, but independently of each other, to form multiple forms of the MSP1 complex on the parasite surface. These complexes have overlapping functions that interact directly with human erythrocytes. We also show that targeting the p83 fragment of MSP1 using inhibitory antibodies inhibits all forms of MSP1 complexes and disrupts parasite growth in vitro. [ABSTRACT FROM AUTHOR]

Background: The male gamete fertilization factor P48/45 in malaria parasites is a prime transmission-blocking vaccine (TBV) candidate. Efforts to develop antimalarial vaccines are often thwarted by genetic diversity of the target antigens. Here we evaluated the genetic diversity of Pvs48/45 gene in global Plasmodium vivax populations. Methods: We determined 200 Pvs48/45 sequences collected from temperate and subtropical parasite populations in China. Population genetic and evolutionary analyses were performed to determine the levels of genetic diversity, potential signature of selection, and population differentiation. Results: Analysis of the Pvs48/45 sequences from 200 P. vivax parasites collected in a temperate and a tropical region revealed a low level of genetic diversity (π = 0.0012) with 14 single nucleotide polymorphisms, of which 11 were nonsynonymous. Analysis of 344 Pvs48/45 sequences from nine worldwide P. vivax populations detected a total of 38 haplotypes, of which 13 haplotypes were present only once. Multiple tests for selection confirmed a signature of positive selection on Pvs48/45 with selection skewed to the second cysteine domain. Haplotype network analysis and Wright’s fixation index showed large geographical differentiation with the presence of continent-or region-specific mutations in this gene. Conclusions: Pvs48/45 displays low levels of genetic diversity with the presence of region-specific mutations. Some of the mutations may be potential epitope targets based on their positions in the predicted structure, highlighting the need for future evaluation of these mutations in designing Pvs48/45-based TBV. [ABSTRACT FROM AUTHOR]

Background: Proteins secreted from the rhoptry in Plasmodium merozoites are associated with the formation of tight junctions and parasitophorous vacuoles during invasion of erythrocytes and are sorted within the rhoptry neck or bulb. Very little information has been obtained to date about Plasmodium vivax rhoptry-associated leucine (Leu) zipper-like protein 1 (PvRALP1; PVX_096245), a putative rhoptry protein. PvRALP1 contains a signal peptide, a glycine (Gly)/glutamate (Glu)-rich domain, and a Leu-rich domain, all of which are conserved in other Plasmodium species. Methods: Recombinant PvRALP1s were expressed as full-length protein without the signal peptide (PvRALP1-Ecto) and as truncated protein consisting of the Gly/Glu- and Leu-rich domains (PvRALP1-Tr) using the wheat germ cell-free expression system. The immunoreactivity to these two fragments of recombinant PvRALP1 protein in serum samples from P. vivax-infected patients and immunized mice, including analysis of immunoglobulin G (IgG) subclasses, was evaluated by enzyme-linked immunosorbent assay or protein microarray technology. The subcellular localization of PvRALP1 in blood stage parasites was also determined. Results: Recombinant PvRALP1-Ecto and PvRALP1-Tr proteins were successfully expressed, and in serum samples from P. vivax patients from the Republic of Korea, the observed immunoreactivities to these proteins had 58.9% and 55.4% sensitivity and 95.0% and 92.5% specificity, respectively. The response to PvRALP1 in humans was predominantly cytophilic antibodies (IgG1 and IgG3), but a balanced Th1/Th2 response was observed in mice. Unexpectedly, there was no significant inverse correlation between levels of parasitaemia and levels of antibody against either PvRALP1-Ecto (R² = 0.11) or PvRALP1-Tr (R² = 0.14) antigens. PvRALP1 was localized in the rhoptry neck of merozoites, and this was the first demonstration of the localization of this protein in P. vivax. Conclusions: This study analysed the antigenicity and immunogenicity of PvRALP1 and suggested that PvRALP1 may be immunogenic in humans during parasite infection and might play an important role during invasion of P. vivax parasites. [ABSTRACT FROM AUTHOR]

Despite decades of effort battling against malaria, the disease is still a major cause of morbidity and mortality. Transmission-blocking vaccines (TBVs) that target sexual stage parasite development could be an integral part of measures for malaria elimination. In the 1950s, Huff et al. first demonstrated the induction of transmission-blocking immunity in chickens by repeated immunizations with Plasmodium gallinaceum-infected red blood cells. Since then, significant progress has been made in identification of parasite antigens responsible for transmission-blocking activity. Recombinant technologies accelerated evaluation of these antigens as vaccine candidates, and it is possible to induce effective transmission-blocking immunity in humans both by natural infection and now by immunization with recombinant vaccines. This chapter reviews the efforts to produce TBVs, summarizes the current status and advances and discusses the remaining challenges and approaches. [ABSTRACT FROM AUTHOR]

Background: Proteins secreted from the rhoptry in Plasmodium merozoites are associated with the formation of tight junctions and parasitophorous vacuoles during invasion of erythrocytes and are sorted within the rhoptry neck or bulb. Very little information has been obtained to date about Plasmodium vivax rhoptry-associated leucine (Leu) zipper-like protein 1 (PvRALP1; PVX_096245), a putative rhoptry protein. PvRALP1 contains a signal peptide, a glycine (Gly)/glutamate (Glu)-rich domain, and a Leu-rich domain, all of which are conserved in other Plasmodium species. Methods: Recombinant PvRALP1s were expressed as full-length protein without the signal peptide (PvRALP1-Ecto) and as truncated protein consisting of the Gly/Glu- and Leu-rich domains (PvRALP1-Tr) using the wheat germ cell-free expression system. The immunoreactivity to these two fragments of recombinant PvRALP1 protein in serum samples from P. vivax-infected patients and immunized mice, including analysis of immunoglobulin G (IgG) subclasses, was evaluated by enzyme-linked immunosorbent assay or protein microarray technology. The subcellular localization of PvRALP1 in blood stage parasites was also determined. Results: Recombinant PvRALP1-Ecto and PvRALP1-Tr proteins were successfully expressed, and in serum samples from P. vivax patients from the Republic of Korea, the observed immunoreactivities to these proteins had 58.9% and 55.4% sensitivity and 95.0% and 92.5% specificity, respectively. The response to PvRALP1 in humans was predominantly cytophilic antibodies (IgG1 and IgG3), but a balanced Th1/Th2 response was observed in mice. Unexpectedly, there was no significant inverse correlation between levels of parasitaemia and levels of antibody against either PvRALP1-Ecto (R2 = 0.11) or PvRALP1-Tr (R2 = 0.14) antigens. PvRALP1 was localized in the rhoptry neck of merozoites, and this was the first demonstration of the localization of this protein in P. vivax. Conclusions: This study analysed the antigenicity and immunogenicity of PvRALP1 and suggested that PvRALP1 may be immunogenic in humans during parasite infection and might play an important role during invasion of P. vivax parasites. [ABSTRACT FROM AUTHOR]

Background: Circumsporozoite protein (CSP) is essential for sporozoite formation and sporozoite invasion into human hepatocyte. Previously, a recombinant P. vivax CSP based on chimeric repeats (rPvCSP-c) representing two major alleles VK210 and VK247 within central region has been designed. Naturally acquired humoral immune responses study show that antigenicity of rPvCSP-c was much higher than that of native strain. However, the serologic reactivity of rPvCSP-c was still unclear in detail. Methods: In present study, recognition of rPvCSP-c in vivax malaria typed VK210 and VK247 alleles was assessed. VK210 typed and VK247 typed sera from adult residents reacted specifically with rPvCSP-c using protein array and immunoblot assay. Additionally, anti-rPvCSP-c serum recognized the fixed VK210 and VK247 sporozoites by immunofluorescence assay. Furthermore, statistic analysis was performed for correlational detection. Results: The rPvCSP-c reacted with both VK210 typed and VK247 typed P. vivax infected patient sera and antirPvCSP- c immune serum also reacted with VK210 and VK247 sporozoite parasites of P. vivax specifically. There was a positive correlation between increased antibody level, age of patients and also associated with pvcsp repeat number, although the level of responses did vary considerably in their reactivity to the rPvCSP-c from negative to very high level within each age group. Conclusions: These data confirmed the serologic reactivity of the novel rPvCSP-c in exposed both VK210 and VK247 populations. These results strongly suggested that this recombinant CSP was biologically active and potently immunogenic across major strains and raised the prospect that this protein could be used as serologic marker. [ABSTRACT FROM AUTHOR]