Search

BackgroundTransmission-blocking vaccines (TBVs) can effectively prevent the community’s spread of malaria by targeting the antigens of mosquito sexual stage parasites. At present, only a few candidate antigens have demonstrated transmission-blocking activity (TBA) potential in P. vivax. Quiescin-sulfhydryl oxidase (QSOX) is a sexual stage protein in the rodent malaria parasite Plasmodium berghei and is associated with a critical role in protein folding by introducing disulfides into unfolded reduced proteins. Here, we reported the immunogenicity and transmission-blocking potency of the PvQSOX in P. vivax.Methods and findingsThe full-length recombinant PvQSOX protein (rPvQSOX) was expressed in the Escherichia coli expression system. The anti-rPvQSOX antibodies were generated following immunization with the rPvQSOX in rabbits. A parasite integration of the pvqsox gene into the P. berghei pbqsox gene knockout genome was developed to express full-length PvQSOX protein in P. berghei (Pv-Tr-PbQSOX). In western blot, the anti-rPvQSOX antibodies recognized the native PvQSOX protein expressed in transgenic P. berghei gametocyte and ookinete. In indirect immunofluorescence assays, the fluorescence signal was detected in the sexual stages, including gametocyte, gamete, zygote, and ookinete. Anti-rPvQSOX IgGs obviously inhibited the ookinetes and oocysts development both in vivo and in vitro using transgenic parasites. Direct membrane feeding assays of anti-rPvQSOX antibodies were conducted using four field P. vivax isolates (named isolates #1–4) in Thailand. Oocyst density in mosquitoes was significantly reduced by 32.00, 85.96, 43.52, and 66.03% with rabbit anti-rPvQSOX antibodies, respectively. The anti-rPvQSOX antibodies also showed a modest reduction of infection prevalence by 15, 15, 20, and 22.22%, respectively, as compared to the control, while the effect was insignificant. The variation in the DMFA results may be unrelated to the genetic polymorphisms. Compared to the P.vivax Salvador (Sal) I strain sequences, the pvqsox in isolate #1 showed no amino acid substitution, whereas isolates #2, #3, and #4 all had the M361I substitution.ConclusionsOur results suggest that PvQSOX could serve as a potential P. vivax TBVs candidate, which warrants further evaluation and optimization.

Background: With the emergence of resistance to front-line antimalarials, there is an urgent need to develop new medicines, including those targeting sexual development. This study aimed to assess the activity of a panel of phosphatase inhibitors against the sexual development of Plasmodium berghei and evaluate their potential as transmission-blocking agents. Methods: Twenty-five compounds were screened for transmission-blocking activity in vitro using the P. berghei ookinete culture assay. The inhibitory effects on male gametogenesis, gamete-ookinete, and zygote-ookinete formation were evaluated. The transmission-blocking activity of two compounds was evaluated using an in vivo mosquito feeding assay. Their cytotoxic effects were assessed on the human cell line HepG2. Results: Twelve compounds inhibited P. berghei ookinete formation with an IC50

Abstract Background Plasmodium vivax transmission-blocking vaccines (TBVs) are receiving increasing attention. Based on excellent transmission-blocking activities of the PbPH (PBANKA_0417200) and PbSOP26 (PBANKA_1457700) antigens in Plasmodium berghei, their orthologs in P. vivax, PVX_098655 (PvPH) and PVX_101120 (PvSOP26), were selected for the evaluation of their potential as TBVs. Methods Fragments of PvPH (amino acids 22–304) and PvSOP26 (amino acids 30–272) were expressed in the yeast expression system. The recombinant proteins were used to immunize mice to obtain antisera. The transmission-reducing activities of these antisera were evaluated using the direct membrane feeding assay (DMFA) using Anopheles dirus mosquitoes and P. vivax clinical isolates. Results The recombinant proteins PvPH and PvSOP26 induced robust antibody responses in mice. The DMFA showed that the anti-PvSOP26 sera significantly reduced oocyst densities by 92.0 and 84.1% in two parasite isolates, respectively, whereas the anti-PvPH sera did not show evident transmission-reducing activity. The variation in the DMFA results was unlikely due to the genetic polymorphisms of the two genes since their respective sequences were identical in the clinical P. vivax isolates. Conclusion PvSOP26 could be a promising TBV candidate for P. vivax, which warrants further evaluation. Graphical Abstract

Quiescin sulfhydryl oxidase (QSOX), present in a wide variety of eukaryotic species, catalyzes the insertion of disulfide bonds into unfolded, reduced proteins. Here we characterized the QSOX protein from the rodent malaria parasite Plasmodium berghei (PbQSOX), which is conserved in all sequenced malaria parasite species. The PbQSOX protein was not expressed in asexual erythrocytic stages, but was most abundantly expressed in ookinetes. Indirect immunofluorescence assays revealed PbQSOX was not only localized in cytoplasm of gametocytes, gametes and ookinetes, but also expressed on the surface of gametes and ookinetes. Western blot identified extracellular presence of PbQSOX in the culture medium of ookinetes suggestive of secretion. Pbqsox deletion (Δpbqsox) did not affect asexual intraerythrocytic development, but reduced exflagellation of male gametocytes as well as formation and maturation of ookinetes. Pbqsox deletion also led to a significant increase in the reduced thiol groups of ookinete surface proteins, suggesting that it may play a role in maintaining the integrity of disulfide bonds of surface proteins, which might be needed for ookinete development. Mosquitoes that fed on Δpbqsox-infected mice showed a significant reduction in ookinete and oocyst numbers compared to those fed on wild-type parasite-infected mice. Further, both polyclonal mouse antisera and a monoclonal antibody against the recombinant PbQSOX exhibited substantial transmission-blocking activities in in vitro and mosquito feeding assays, suggesting QSOX is a potential target for blocking parasite transmission.

Abstract Background A vaccine that targets multiple developmental stages of malaria parasites would be an effective tool for malaria control and elimination. Methods A conserved gene in Plasmodium, the Plasmodium berghei gene (PBANKA_020570) encoding a 51 kDa protein (pb51 gene), was identified through search of the PlasmoDB database using a combination of expression and protein localization criteria. A partial domain of the Pb51 protein was expressed in a prokaryotic expression system (rPb51) and used for immunization in mice. The protein expression profile and localization were studied by Western blot and indirect immunofluorescence assay (IFA), respectively. The inhibitory effect of the anti-rPb51 antibodies on parasite proliferation was evaluated in erythrocytes in vivo. The transmission-blocking activity of the immune sera was determined by in vitro ookinete conversion assay and by direct mosquito feeding assay (DFA). Results The rPb51 elicited specific antibodies in mice. Western blot confirmed Pb51 expression in schizonts, gametocytes and ookinetes. IFA showed localization of Pb51 on the outer membranes of schizonts, gametocytes, zygotes, retorts, ookinetes and sporozoites of P. berghei. Mice immunized with the rPb51 protein significantly reduced parasite proliferation and gametocyte conversion in vivo. Moreover, the rPb51 antisera also significantly reduced the in vitro ookinete conversion when added into the ookinete culture medium. In DFA, mice immunized with the rPb51 reduced the prevalence of mosquito infection by 21.3% and oocyst density by 54.8%. Conclusions In P. berghei, P51 was expressed in both asexual erythrocytic and sexual stages and localized on the surface of these stages with the exception of the ring stage. The anti-rPb51 antibodies inhibited both P. berghei proliferation in mice and transmission of the parasite to mosquitoes.

Abstract 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.

The pathogenesis of cerebral malaria is biologically complex and involves multi-factorial mechanisms such as microvascular congestion, immunopathology by the pro-inflammatory cytokine and endothelial dysfunction. Recent data have suggested that a pleiotropic T-cell immunomodulatory protein (TIP) could effectively mediate inflammatory cytokines of mammalian immune response against acute graft-versus-host disease in animal models. In this study, we identified a conserved homologue of TIP in Plasmodium berghei (PbTIP) as a membrane protein in Plasmodium asexual stage. Compared with PBS control group, the pathology of experimental cerebral malaria (ECM) in rPbTIP intravenous injection (i.v.) group was alleviated by the downregulation of pro-inflammatory responses, and rPbTIP i.v. group elicited an expansion of regulatory T-cell response. Therefore, rPbTIP i.v. group displayed less severe brain pathology and feverish mice in rPbTIP i.v. group died from ECM. This study suggested that PbTIP may be a novel promising target to alleviate the severity of ECM. [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: Artemisinin resistance in Plasmodium falciparum has emerged as a major threat for malaria control and elimination worldwide. Mutations in the Kelch propeller domain of PfK13 are the only known molecular markers for artemisinin resistance in this parasite. Over 100 non-synonymous mutations have been identified in PfK13 from various malaria endemic regions. This study aimed to investigate the genetic diversity of PvK12, the Plasmodium vivax ortholog of PfK13, in parasite populations from Southeast Asia, where artemisinin resistance in P. falciparum has emerged. Methods: The PvK12 sequences in 120 P. vivax isolates collected from Thailand (22), Myanmar (32) and China (66) between 2004 and 2008 were obtained and 353 PvK12 sequences from worldwide populations were retrieved for further analysis. Results: These PvK12 sequences revealed a very low level of genetic diversity (π = 0.00003) with only three single nucleotide polymorphisms (SNPs). Of these three SNPs, only G581R is nonsynonymous. The synonymous mutation S88S is present in 3% (1/32) of the Myanmar samples, while G704G and G581R are present in 1.5% (1/66) and 3% (2/66) of the samples from China, respectively. None of the mutations observed in the P. vivax samples were associated with artemisinin resistance in P. falciparum. Furthermore, analysis of 473 PvK12 sequences from twelve worldwide P. vivax populations confirmed the very limited polymorphism in this gene and detected only five distinct haplotypes. Conclusions: The PvK12 sequences from global P. vivax populations displayed very limited genetic diversity indicating low levels of baseline polymorphisms of PvK12 in these areas. [ABSTRACT FROM AUTHOR]

Background: Transmission-blocking vaccine (TBV) is a promising strategy for interrupting the malaria transmission cycle. Current TBV candidates include both pre- and post-fertilization antigens expressed during sexual development of the malaria parasites. Methods: We tested whether a TBV design combining two sexual-stage antigens has better transmission-blocking activity. Using the rodent malaria model Plasmodium yoelii, we pursued a DNA vaccination strategy with genes encoding the gametocyte antigen Pys48/45 and the major ookinete surface protein Pys25. Results: Immunization of mice with DNA constructs expression either Pys48/45 or Pys25 elicited strong antibody responses, which specifically recognized a ~45 and ~25 kDa protein from gametocyte and ookinete lysates, respectively. Immune sera from mice immunized with DNA constructs expressing Pys48/45 and Pys25 individually and in combination displayed evident transmission-blocking activity in in vitro ookinete culture and direct mosquito feeding experiments. With both assays, the Pys25 sera had higher transmission-blocking activity than the Pys48/45 sera. Intriguingly, compared with the immunization with the individual DNA vaccines, immunization with both DNA constructs produced lower antibody responses against individual antigens. The resultant immune sera from the composite vaccination had significantly lower transmission-blocking activity than those from Pys25 DNA immunization group, albeit the activity was substantially higher than that from the Pys48 DNA vaccination group. Conclusions: This result suggested that vaccination with the two DNA constructs did not achieve a synergistic effect, but rather caused interference in inducing antigen-specific antibody responses. This result has important implications for future design of composite vaccines targeting different sexual antigens. [ABSTRACT FROM AUTHOR]

Background: Transmission-blocking vaccines (TBVs) are a promising strategy for malaria control and elimination. However, candidate TBV antigens are currently limited, highlighting the urgency of identifying new antigens for TBV development. Methods: Using a combination of bioinformatic analysis and functional studies in the rodent malaria model Plasmodium berghei, we identified a conserved Plasmodium protein PbPH (PBANKA_041720) containing a pleckstrin homology (PH) domain. The expression of PbPH was detected by Western blot and indirect immunofluorescence assay (IFA). The function of PbPH was tested by genetic knockout. The TB activity was confirmed by in vitro ookinete conversion assay and mosquito feeding. Results: PbPH was detected in Western blot as highly expressed in sexual stages (gametocytes and ookinetes). IFA revealed localizations of PbPH on the surface of gametes, zygotes, and ookinetes. Deletion of the pbph gene did not affect asexual growth, but significantly reduced the formation of gametocytes, ookinetes, and oocysts, indicating that PbPH protein is required for parasite sexual development. Recombinant PbPH expressed and purified from bacteria elicited strong antibody responses in mice and the antibodies significantly inhibited exflagellation of male gametocytes and formation of ookinetes in a concentration-dependent manner. Mosquito feeding experiments confirmed that mosquitoes fed on mice immunized with PbPH had 13 % reduction in the prevalence of infection and almost 48 % reduction in oocyst density. Conclusions: Pbph is a highly conserved Plasmodium gene and is required for parasite sexual development. PbPH protein is expressed on the surface of gametes and ookinetes. Immunization of mice against the recombinant PbPH protein induced strong antibody responses that effectively reduced the formation of male gametes and ookinetes in vitro and blocked transmission of the parasites to mosquitoes. These results highlight PbPH as a potential TBV candidate that is worth future investigations in human malaria parasites. [ABSTRACT FROM AUTHOR]

In animal models of experimental cerebral malaria (ECM), neuropathology is associated with an overwhelming inflammatory response and sequestration of leukocytes and parasite-infected RBCs in the brain. In this study, we explored the effect of vitamin D (VD; cholecalciferol) treatment on host immunity and outcome of ECM in C57BL/6 mice during Plasmodium berghei ANKA (PbA) infection. We observed that oral administration of VD both before and after PbA infection completely protected mice from ECM. VD administration significantly dampened the inducible systemic inflammatory responses with reduced circulating cytokines IFN-γ and TNF and decreased expression of these cytokines by the spleen cells. Meanwhile, VD also resulted in decreased expression of the chemokines CXCL9 and CXCL10 and cytoadhesion molecules (ICAM-1, VCAM-1, and CD36) in the brain, leading to reduced accumulation of pathogenic T cells in the brain and ultimately substantial improvement of the blood-brain barriers of PbA-infected mice. In addition, VD inhibited the differentiation, activation, and maturation of splenic dendritic cells. Meanwhile, regulatory T cells and IL-10 expression levels were upregulated upon VD treatment. These data collectively demonstrated the suppressive function of VD on host inflammatory responses, which provides significant survival benefits in the murine ECM model. [ABSTRACT FROM AUTHOR]