In Silico Analysis Reveals High Levels of Genetic Diversity of Plasmodium knowlesi Cell Traversal Protein for Ookinetes and Sporozoites (PkCelTOS) in Clinical Samples.

The cell-traversal protein for ookinetes and sporozoites (CelTOS), expressed on the surface of ookinetes and sporozoitesin Plasmodium species, is a promising malaria vaccine candidate. CelTOS is essential for parasite invasion into mosquito midgut and human hepatocytes, thereby contributing to malaria transmission and disease pathogenesis. This study explores the genetic diversity, polymorphisms, haplotypes, natural selection, phylogenetic analysis, and epitope prediction in the full-length Plasmodium knowlesi CelTOS gene in clinical samples from Sarawak, Malaysian Borneo, and long-term laboratory strains from Peninsular Malaysia and the Philippines. Our analysis revealed a high level of genetic variation in the PkCelTOS gene, with a nucleotide diversity of π ~ 0.021, which was skewed towards the 3' end of the gene. This level of diversity is double that observed in PfCelTOS and 20 times that observed in PvCelTOS from worldwide clinical samples. Tests of natural selection revealed evidence for positive selection within clinical samples. Phylogenetic analysis of the amino acid sequence of PkCelTOS revealed the presence of two distinct groups, although no geographical clustering was observed. Epitope prediction analysis identified two potential epitopes (96AQLKATA102 and 124TIKPPRIKED133) using the IEDB server and one epitope (125IKPPRIKED133) by Bcepred server on the C' terminal region of PkCelTOS protein. Both the servers predicted a common epitope region of nine amino acid length (IKPPRIKED) peptide, which can be studied in the future as a potential candidate for vaccine development. These findings shed light on the genetic diversity, polymorphism, haplotypes, and natural selection within PkCelTOS in clinical samples and provide insights about its future prospects as a potential candidate for P. knowlesi malaria vaccine development.

Population genetic analysis of Plasmodium falciparum cell-traversal protein for ookinetes and sporozoite among malaria patients from southern Nigeria.

Plasmodium falciparum immune escape mechanisms affect antigens being prioritized for vaccine design. As a result of the multiple surface antigens the parasite exhibits at different life cycle stages, designing a vaccine that would efficiently boost the immune system in clearing infections has been challenging. The P. falciparum cell-traversal protein for ookinetes and sporozoite (Pfceltos) is instrumental for ookinete traversal of the mosquito midgut and sporozoites invasion of the human liver cells. Pfceltos elicits both humoral and cellular immune response but has been reported with multiple single nucleotide polymorphisms in global isolates. A cross-sectional survey, conducted in southern Nigeria, between January-March 2021 recruited 283 individuals. Of this, 166 demonstrated P. falciparum infections (86 from Cross River and 80 from Edo), 48 (55.8%) while only 36 (45%) were amplified for Pfceltos gene from both sites respectively. Fifty amplified samples were sequenced and analysed for their diversity, polymorphisms and population structure of the gene. The number of segregating sites in Edo State was higher (34) than that of Cross River State. Though nucleotide diversity was higher for Edo compared to Cross River State (θw = 0.02505; π = 0.03993 versus θw = 0.00930; π = 0.01033 respectively), the reverse was the case for haplotype diversity (0.757 versus 0.890 for Edo and Cross River respectively). Of the twelve haplotypes observed from both states, only two (KASLPVEK and NAFLSFEK) were shared, with haplotype prevalence higher in Edo (16% and 36%) than Cross River (8% and 4%). The Tajima's D test was positive for both states, with Fst value showing a strong genetic differentiation (Fst = 0.25599), indicating the occurrence of balancing selection favoring haplotype circulation at a low frequency. The shared haplotypes, low Hst and Fst values presents a challenge to predict the extent of gene flow. High LD values present a grim public health consequence should a Pfceltos-conjugated vaccine be considered for prophylaxis in Nigeria.

Implications of conformational flexibility, lipid binding, and regulatory domains in cell-traversal protein CelTOS for apicomplexan migration.

Malaria and other apicomplexan-caused diseases affect millions of humans, agricultural animals, and pets. Cell traversal is a common feature used by multiple apicomplexan parasites to migrate through host cells and can be exploited to develop therapeutics against these deadly parasites. Here, we provide insights into the mechanism of the Cell-traversal protein for ookinetes and sporozoites (CelTOS), a conserved cell-traversal protein in apicomplexan parasites and malaria vaccine candidate. CelTOS has previously been shown to form pores in cell membranes to enable traversal of parasites through cells. We establish roles for the distinct protein regions of Plasmodium vivax CelTOS and examine the mechanism of pore formation. We further demonstrate that CelTOS dimer dissociation is required for pore formation, as disulfide bridging between monomers inhibits pore formation, and this inhibition is rescued by disulfide-bridge reduction. We also show that a helix-destabilizing amino acid, Pro127, allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C terminus of CelTOS is a negative regulator that limits pore formation. Finally, we highlight that lipid binding is a prerequisite for pore assembly as mutation of a phospholipids-binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. These findings identify critical regions in CelTOS and will aid in understanding the egress mechanism of malaria and other apicomplexan parasites as well as have implications for studying the function of other essential pore-forming proteins.

Exploring in vitro expression and immune potency in mice using mRNA encoding the Plasmodium falciparum malaria antigen, CelTOS.

The secreted malarial protein, Cell-Traversal protein for Ookinetes and Sporozoites (CelTOS), is highly conserved among Plasmodium species, and plays a role in the invasion of mosquito midgut cells and hepatocytes in the vertebrate host. CelTOS was identified as a potential protective antigen based on a proteomic analysis, which showed that CelTOS stimulated significant effector T cells producing IFN-γ in peripheral blood mononuclear cells (PBMCs) from radiation attenuated sporozoite-immunized, malaria-naïve human subjects. In a rodent malaria model, recombinant full-length CelTOS protein/adjuvant combinations induced sterile protection, and in several studies, functional antibodies were produced that had hepatocyte invasion inhibition and transmission-blocking activities. Despite some encouraging results, vaccine approaches using CelTOS will require improvement before it can be considered as an effective vaccine candidate. Here, we report on the use of mRNA vaccine technology to induce humoral and cell-mediated immune responses using this antigen. Several pfceltos encoding mRNA transcripts were assessed for the impact on protein translation levels in vitro. Protein coding sequences included those to evaluate the effects of signal sequence, N-glycosylation on translation, and of nucleoside substitutions. Using in vitro transfection experiments as a pre-screen, we assessed the quality of the expressed CelTOS target relative to the homogeneity, cellular localization, and durability of expression levels. Optimized mRNA transcripts, which demonstrated highest protein expression levels in vitro were selected for encapsulation in lipid nanoparticles (LNP) and used to immunize mice to assess for both humoral and cellular cytokine responses. Our findings indicate that mRNA transcripts encoding pfceltos while potent for inducing antigen-specific cellular cytokine responses in mice, were less able to mount PfCelTOS-specific antibody responses using a two-dose regimen. An additional booster dose was needed to overcome low seroconversion rates in mice. With respect to antibody fine specificities, N-glycosylation site mutated immunogens yielded lower immune responses, particularly to the N-terminus of the molecule. While it remains unclear the impact on CelTOS antigen as immunogen, this study highlights the need to optimize antigen design for vaccine development.

Plasmodium vivax Cell Traversal Protein for Ookinetes and Sporozoites (CelTOS) Functionally Restricted Regions Are Involved in Specific Host-Pathogen Interactions.

Following the injection of Plasmodium sporozoites by a female Anopheles mosquito into the dermis, they become engaged on a long journey to hepatic tissue where they must migrate through different types of cell to become established in parasitophorous vacuoles in hepatocytes. Studies have shown that proteins such as cell traversal protein for Plasmodium ookinetes and sporozoites (CelTOS) play a crucial role in cell-traversal ability. Although CelTOS has been extensively studied in various species and included in pre-clinical assays it remains unknown which P. vivax CelTOS (PvCelTOS) regions are key in its interaction with traversed or target cells (Kupffer or hepatocytes) and what type of pressure, association and polymorphism these important regions could have to improve their candidacy as important vaccine antigens. This work has described producing a recombinant PvCelTOS which was recognized by ~30% P. vivax-infected individuals, thereby confirming its ability for inducing a natural immune response. PvCelTOS' genetic diversity in Colombia and its ability to interact with HeLa (traversal cell) and/or HepG2 cell (target cell) external membrane have been assessed. One region in the PvCelTOS amino-terminal region and another in its C-terminus were seen to be participating in host-pathogen interactions. These regions had important functional constraint signals (ω < 0.3 and several sites under negative selection) and were able to inhibit specific rPvCelTOS binding to HeLa cells. This led to suggesting that sequences between aa 41-60 (40833) and 141-160 (40838) represent promising candidates for an anti-P. vivax subunit-based vaccine.

Cell-traversal protein for ookinetes and sporozoites (CelTOS) formulated with potent TLR adjuvants induces high-affinity antibodies that inhibit Plasmodium falciparum infection in Anopheles stephensi.

BACKGROUND: Plasmodium falciparum parasite is the most deadly species of human malaria, and the development of an effective vaccine that prevents P. falciparum infection and transmission is a key target for malarial elimination and eradication programmes. P. falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an advanced vaccine candidate. A comparative study was performed to characterize the immune responses in BALB/c mouse immunized with Escherichia coli-expressed recombinant PfCelTOS (rPfCelTOS) in toll-like receptor (TLR)-based adjuvants, CpG and Poly I:C alone or in combination (CpG + Poly I:C), followed by the assessment of transmission-reducing activity (TRA) of anti-rPfCelTOS antibodies obtained from different vaccine groups in Anopheles stephensi. METHODS: The aim of the current work was achieved by head-to-head comparison of the vaccine groups using conventional and avidity enzyme-linked immunosorbent assay (ELISA), immunofluorescence test (IFAT), and standard membrane feeding assay (SMFA). RESULTS: Comparing to rPfCelTOS alone, administration of rPfCelTOS with two distinct TLR-based adjuvants in vaccine mouse groups showed a significant increase in responses (antibody level, IgG subclass analysis, avidity, and Th1 cytokines) and was able to induce reasonable transmission-reducing activity. Also, comparable functional activity of anti-rPfCelTOS antibodies was found in group that received antigen in either CpG or Poly I:C (69.9%/20% and 73.5%/24.4%, respectively, reductions in intensity/prevalence). However, the vaccine group receiving rPfCelTOS in combination with CpG + Poly I:C showed a significant induction in antibody titers and inhibitory antibodies in oocysts development (78.3%/19.6% reductions in intensity/prevalence) in An. stephensi. CONCLUSIONS: A key finding in this investigation is that rPfCelTOS administered alone in BALB/c mouse is poorly immunogenic, with relatively low IgG level, avidity, inhibitory antibodies, and mixed Th1/Th2 responses. However, immunological characteristic (IgG level, cytophilic IgG2a and IgG2b, avidity, and Th1 cytokines) and TRA of anti-rPfCelTOS significantly enhanced in the presence of co-administration of TLR-based adjuvants, confirming that targeting TLRs would be an effective means for the enhancement of inducing TRA against rPfCelTOS.

Heterogeneity in the acquisition of naturally acquired antibodies to cell-traversal protein for ookinetes and sporozoites (CelTOS) and thrombospondin-related adhesion protein (TRAP) of Plasmodium falciparum in naturally infected patients from unstable malaria areas in Iran.

Currently, there is no subunit malaria vaccine capable of providing long-lasting protection, and a vaccine based on a single-antigen has shown moderate to unsatisfactory efficacies in clinical trials. As in malaria elimination and eradication strategies, the primary objective is reduction in disease and death due to P. falciparum, in the present investigation, for the first time, we attempted to determine and compare the naturally acquired immune responses to two well-recognized sporozoite antigens, cell-traversal protein for ookinetes and sporozoites (CelTOS) and thrombospondin-related adhesion protein (TRAP), in P. falciparum-infected individuals (n = 204) in low malaria transmission settings of Iran using ELISA. Besides, the profile of IgG isotype responses, the avidity of IgG, IgG1, and IgG3, and the association of anti-PfCelTOS and -PfTRAP antibodies with host age were evaluated. Positive antibody responses to PfCelTOS and PfTRAP antigens were detected in 16.2% and 31.9% of Iranian P. falciparum-infected individuals, respectively, indicating significantly lower immune response to PfCelTOS than PfTRAP (P <0.0001, McNemar's test). Also, among the positive samples for anti-PfCelTOS (n = 33) and -PfTRAP (n = 65) total IgG, the cytophilic IgG1 and IgG3 antibodies were predominant. A significant proportion of the examined positive responders had high- and intermediate-avidity for IgG (93.9%, 87.7%), IgG1 (96.3%, 87.7%), and IgG3 (76%, 78.7%) antibodies to both PfCelTOS and PfTRAP antigens, respectively, with no correlation with age (P >0.05; Spearman's correlation test). In conclusion, the present data suggests the acquisition of heterogenic immune responses to both antigens in the same patients naturally infected with P. falciparum from settings of low malaria transmission intensity in Iran in which their role in protection to malaria needs further study.

Analysis of genetic diversity and population structure of gene encoding cell-traversal protein for ookinetes and sporozoites (CelTOS) vaccine candidate antigen in global Plasmodium falciparum populations.

Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) has been reported as one of the most attractive malaria vaccine candidate antigens. To design a broadly effective malaria vaccine based on this antigen, it is crucial to have adequate information on genetic diversity in global PfCelTOS. Therefore, the extent of sequence diversity at the full-length of the pfceltos was assessed among both natural P. falciparum isolates collected from Iran (n = 93) and from available global pfceltos sequence data retrieved from PlasmoDB database (n = 159). Also, recombination, natural selection, the degree of genetic differentiation as well as the predicted immunodominant regions in PfCelTOS were analyzed. In total, 40 SNPs (including 1 synonymous and 39 non-synonymous) were detected in 34 positions, as compared to 3D7 sequence, which led to 66 distinct haplotypes with different frequencies. Among those haplotypes, 34 (51.5%, excluded from further analysis) were singleton haplotype and mostly detected among Senegalese parasite isolates. PfCelt-1 was found as predominant haplotype (32.6% total frequency) that was only detected in Iranian P. falciparum isolates. Nucleotide diversity was low in French Guiana (0.00236 ±â€¯0.00203) and Iranian (0.00259 ±â€¯0.00048) P. falciparum isolates in comparison with African populations. Evidence for positive selection by host immunity and intragenic recombination were detected that are two key factors responsible for gene evolution and genetic diversity of pfceltos gene. The results of Fst analysis and haplotype network revealed that PfCelTOS antigen displayed evident genetic structure between geographical parasite populations. In conclusion, the present analysis demonstrates that there is a limited antigenic diversity and geographic variation in global PfCelTOS, and this finding may be associated with the critical function of this antigen in cell traversal of the parasite in sporozoite and ookinete. Besides, most of the predicted B- and T-cell epitopes were located in the conserved region of the gene, but most of the amino acid replacements were located at the C-terminal region of PfCelTOS. The obtained results in this investigation could provide knowledge for better design of PfCelTOS-based malaria vaccine.

Evaluation of Plasmodium vivax Cell-Traversal Protein for Ookinetes and Sporozoites as a Preerythrocytic P. vivax Vaccine.

Four different vaccine platforms, each targeting the human malaria parasite Plasmodium vivax cell-traversal protein for ookinetes and sporozoites (PvCelTOS), were generated and assessed for protective efficacy. These platforms consisted of a recombinant chimpanzee adenoviral vector 63 (ChAd63) expressing PvCelTOS (Ad), a recombinant modified vaccinia virus Ankara expressing PvCelTOS (MVA), PvCelTOS conjugated to bacteriophage Qß virus-like particles (VLPs), and a recombinant PvCelTOS protein expressed in eukaryotic HEK293T cells (protein). Inbred BALB/c mice and outbred CD-1 mice were immunized using the following prime-boost regimens: Ad-MVA, Ad-VLPs, and Ad-protein. Protective efficacy against sporozoite challenge was assessed after immunization using a novel chimeric rodent Plasmodium berghei parasite (Pb-PvCelTOS). This chimeric parasite expresses P. vivax CelTOS in place of the endogenous P. berghei CelTOS and produces fully infectious sporozoites. A single Ad immunization in BALB/c and CD-1 mice induced anti-PvCelTOS antibodies which were boosted efficiently using MVA, VLP, or protein immunization. PvCelTOS-specific gamma interferon- and tumor necrosis factor alpha-producing CD8+ T cells were induced at high frequencies by all prime-boost regimens in BALB/c mice but not in CD-1 mice; in CD-1 mice, they were only marginally increased after boosting with MVA. Despite the induction of anti-PvCelTOS antibodies and PvCelTOS-specific CD8+ T-cell responses, only low levels of protective efficacy against challenge with Pb-PvCelTOS sporozoites were obtained using any immunization strategy. In BALB/c mice, no immunization regimens provided significant protection against a Pb-PvCelTOS chimeric sporozoite challenge. In CD-1 mice, modest protective efficacy against challenge with chimeric P. berghei sporozoites expressing either PvCelTOS or P. falciparum CelTOS was observed using the Ad-protein vaccination regimen.

The Plasmodium falciparum Cell-Traversal Protein for Ookinetes and Sporozoites as a Candidate for Preerythrocytic and Transmission-Blocking Vaccines.

Recent studies have shown that immune responses against the cell-traversal protein for Plasmodium ookinetes and sporozoites (CelTOS) can inhibit parasite infection. While these studies provide important evidence toward the development of vaccines targeting this protein, it remains unknown whether these responses could engage the Plasmodium falciparum CelTOS in vivo Using a newly developed rodent malaria chimeric parasite expressing the P. falciparum CelTOS (PfCelTOS), we evaluated the protective effect of in vivo immune responses elicited by vaccination and assessed the neutralizing capacity of monoclonal antibodies specific against PfCelTOS. Mice immunized with recombinant P. falciparum CelTOS in combination with the glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) or glucopyranosyl lipid adjuvant-liposome-QS21 (GLA-LSQ) adjuvant system significantly inhibited sporozoite hepatocyte infection. Notably, monoclonal antibodies against PfCelTOS strongly inhibited oocyst development of P. falciparum and Plasmodium berghei expressing PfCelTOS in Anopheles gambiae mosquitoes. Taken together, our results demonstrate that anti-CelTOS responses elicited by vaccination or passive immunization can inhibit sporozoite and ookinete infection and impair vector transmission.

Self-adjuvanting bacterial vectors expressing pre-erythrocytic antigens induce sterile protection against malaria.

Genetically inactivated, Gram-negative bacteria that express malaria vaccine candidates represent a promising novel self-adjuvanting vaccine approach. Antigens expressed on particulate bacterial carriers not only target directly to antigen-presenting cells but also provide a strong danger signal thus circumventing the requirement for potent extraneous adjuvants. E. coli expressing malarial antigens resulted in the induction of either Th1 or Th2 biased responses that were dependent on both antigen and sub-cellular localization. Some of these constructs induced higher quality humoral responses compared to recombinant protein and most importantly they were able to induce sterile protection against sporozoite challenge in a murine model of malaria. In light of these encouraging results, two major Plasmodium falciparum pre-erythrocytic malaria vaccine targets, the Cell-Traversal protein for Ookinetes and Sporozoites (CelTOS) fused to the Maltose-binding protein in the periplasmic space and the Circumsporozoite Protein (CSP) fused to the Outer membrane (OM) protein A in the OM were expressed in a clinically relevant, attenuated Shigella strain (Shigella flexneri 2a). This type of live-attenuated vector has previously undergone clinical investigations as a vaccine against shigellosis. Using this novel delivery platform for malaria, we find that vaccination with the whole-organism represents an effective vaccination alternative that induces protective efficacy against sporozoite challenge. Shigella GeMI-Vax expressing malaria targets warrant further evaluation to determine their full potential as a dual disease, multivalent, self-adjuvanting vaccine system, against both shigellosis, and malaria.