A replication competent Plasmodium falciparum parasite completely attenuated by dual gene deletion.

Vaccination with infectious Plasmodium falciparum (Pf) sporozoites (SPZ) administered with antimalarial drugs (PfSPZ-CVac), confers superior sterilizing protection against infection when compared to vaccination with replication-deficient, radiation-attenuated PfSPZ. However, the requirement for drug administration constitutes a major limitation for PfSPZ-CVac. To obviate this limitation, we generated late liver stage-arresting replication competent (LARC) parasites by deletion of the Mei2 and LINUP genes (mei2-/linup- or LARC2). We show that Plasmodium yoelii (Py) LARC2 sporozoites did not cause breakthrough blood stage infections and engendered durable sterilizing immunity against various infectious sporozoite challenges in diverse strains of mice. We next genetically engineered a PfLARC2 parasite strain that was devoid of extraneous DNA and produced cryopreserved PfSPZ-LARC2. PfSPZ-LARC2 liver stages replicated robustly in liver-humanized mice but displayed severe defects in late liver stage differentiation and did not form liver stage merozoites. This resulted in complete abrogation of parasite transition to viable blood stage infection. Therefore, PfSPZ-LARC2 is the next-generation vaccine strain expected to unite the safety profile of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac.

Genetic diversity and natural selection of apical membrane antigen-1 (ama-1) in Cameroonian Plasmodium falciparum isolates.

Antigenic variation associated with genetic diversity in global Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a major impediment to designing an effective malaria vaccine. Here, we report the first study on genetic diversity and natural selection of the Pfama-1 gene in P. falciparum isolates from Cameroon. A total of 328 P. falciparum positive samples collected during 2016 and 2019 from five localities of Cameroon were analysed. The ectodomain coding fragment of Pfama-1 gene was amplified for polymorphism profiling and natural selection analysis. A total of 108 distinct haplotypes were found in 203 P. falciparum isolates with considerable nucleotide diversity (π = 0.016) and haplotype diversity (Hd = 0.976). Most amino acid substitutions detected were scattered in ectodomain-I and few specific mutations viz P145L, K148Q, K462I, L463F, N471K, S482L, E537G, K546R and I547F were seen only in Cameroonian isolates. A tendency of natural selection towards positive diversifying selection was observed (Taj-D = 2.058). Five positively selected codon sites (P145L, S283L, Q308E/K, P330S and I547F) were identified, which overlapped with predicted B-cell epitopes and red blood cell (RBC) binding sites, suggesting their potential implication in host immune pressure and parasite-RBC binding complex modulation. The Cameroonian P. falciparum populations indicated a moderate level of genetic differentiation when compared with global sequences, with few exceptions from Vietnam and Venezuela. Our findings provide baseline data on existing Pfama-1 gene polymorphisms in Cameroonian field isolates, which will be useful information for malaria vaccine design.

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

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

High genetic and haplotype diversity in vaccine candidate Pfceltos but not Pfrh5 among malaria-infected children in Ibadan, Nigeria.

Malaria remains a global public health challenge. The disease has a great impact in sub-Saharan Africa among children under five years of age and pregnant women. Malaria control programs targeting the parasite and mosquitoes vectors with combinational therapy and insecticide-treated bednets are becoming obsolete due to the phenomenon of resistance, which is a challenge for reducing morbidity and mortality. Malaria vaccines would be effective alternative to the problem of parasite and insecticide resistance, but focal reports of polymorphisms in malaria candidate antigens have made it difficult to design an effective malaria vaccine. Therefore, studies geared towards elucidating the polymorphic pattern and how genes targeted for vaccine design evolve are imperative. We have carried out molecular and genetic analysis of two genes encoding vaccine candidates-the Plasmodium falciparum cell traversal ookinetes and sporozoites (Pfceltos) and P. falciparum reticulocyte binding protein 5 (Pfrh5) in parasite isolates from malaria-infected children in Ibadan, Nigeria to evaluate their genetic diversity, relatedness and pattern of molecular evolution. Pfceltos and Pfrh5 genes were amplified from P. falciparum positive samples. Amplified fragments were purified and sequenced using the chain termination method. Post-sequence edit of fragments and application of various population genetic analyses was done. We observed a higher number of segregating sites and haplotypes in the Pfceltos than in Pfrh5 gene, the former also presenting higher haplotype (0.942) and nucleotide diversity (θ = 0.01219 and π = 0.01148). In contrast, a lower haplotype (0.426) and nucleotide diversity (θ = 0.00125; π = 0.00095) was observed in the Pfrh5 gene. Neutrality tests do not show deviation from neutral expectations for Pfceltos, with the circulation of multiple low frequency haplotypes (Tajima's D = -0.21637; Fu and Li's D = -0.08164; Fu and Li's F = -0.14051). Strong linkage disequilibrium was observed between variable sites, in each of the genes studied. We postulate that the high diversity and circulation of multiple haplotypes has the potential of making a Pfceltos-subunit vaccine ineffective, while the low genetic diversity of Pfrh5 gene substantiates its evolutionary conservation and potential as a malaria vaccine candidate.

A dual-antigen malaria vaccine targeting Pb22 and Pbg37 was able to induce robust transmission-blocking activity.

BACKGROUND: Despite years of effort to develop an effective vaccine against malaria infection, a vaccine that provides individuals with sufficient protection against malaria illness and death in endemic areas is not yet available. The development of transmission-blocking vaccines (TBVs) is a promising strategy for malaria control. A dual-antigen malaria vaccine targeting both pre- and post-fertilization antigens could effectively improve the transmission-blocking activity of vaccines against the sexual stages of the parasite. METHODS: A chimeric recombinant protein Pb22-Pbg37 (Plasmodium berghei 22-P. berghei G37) composed of 19-218 amino acids (aa) of Pb22 and the N-terminal 26-88 aa of Pbg37 was designed and expressed in the Escherichia coli expression system. The antibody titers of the fusion (Pb22-Pbg37) and mixed (Pb22+Pbg37) antigens, as well as those of Pb22 and Pbg37 single antigens were evaluated by enzyme-linked immunosorbent assay. Immunofluorescence and western blot assays were performed to test the reactivity of the antisera with the native proteins in the parasite. The induction of transmission-blocking activity (TBA) by Pb22-Pbg37 and Pb22+Pbg37 were evaluated by in vitro gametocyte activation, gamete and exflagellation center formation, ookinete conversion, and in the direct mosquito feeding assay. RESULTS: The Pb22-Pbg37 fusion protein was successfully expressed in vitro. Co-administration of Pb22 and Pbg37 as a fusion or mixed protein elicited comparable antibody responses in mice and resulted in responses to both antigens. Most importantly, both the mixed and fusion antigens induced antibodies with significantly higher levels of TBA than did each of the individual antigens when administered alone. In addition, the efficacy of vaccination with the Pb22-Pbg37 fusion protein was equivalent to that of vaccination with the mixed single antigens. CONCLUSIONS: Dual-antigen vaccines, which expand/lengthen the period during which the transmission-blocking antibodies can act during sexual-stage development, can provide a promising higher transmission-reducing activity compared to single antigens.

Evaluating the stability of host-reference gene expression and simultaneously quantifying parasite burden and host immune responses in murine malaria.

The efficacy of pre-erythrocytic stage malaria antigens or vaccine platforms is routinely assessed in murine models challenged with Plasmodium sporozoites. Relative liver-stage parasite burden is quantified using reverse transcription quantitative PCR (RTqPCR), which relies on constitutively expressed endogenous control reference genes. However, the stability of host-reference gene expression for RTqPCR analysis following Plasmodium challenge and immunization has not been systematically evaluated. Herein, we evaluated the stability of expression of twelve common RTqPCR reference genes in a murine model of Plasmodium yoelii sporozoite challenge and DNA-adenovirus IV 'Prime-Target' immunization. Significant changes in expression for six of twelve reference genes were shown by one-way ANOVA, when comparing gene expression levels among challenge, immunized, and naïve mice groups. These changes were attributed to parasite challenge or immunization when comparing group means using post-hoc Bonferroni corrected multiple comparison testing. Succinate dehydrogenase (SDHA) and TATA-binding protein (TBP) were identified as stable host-reference genes suitable for relative RTqPCR data normalisation, using the RefFinder package. We defined a robust threshold of 'partial-protection' with these genes and developed a strategy to simultaneously quantify matched host parasite burden and cytokine responses following immunisation or challenge. This is the first report systematically identifying reliable host reference genes for RTqPCR analysis following Plasmodium sporozoite challenge. A robust RTqPCR protocol incorporating reliable reference genes which enables simultaneous analysis of host whole-liver cytokine responses and parasite burden will significantly standardise and enhance results between international malaria vaccine efficacy studies.

Plasmodium knowlesi circumsporozoite protein: genetic characterisation and predicted antigenicity of the central repeat region.

Circumsporozoite protein (CSP) central repeat region is one of the main target regions of the RTS,S/AS01 vaccine for falciparum infection as it consists of immunodominant B cell epitopes. However, there is a lack of study for P. knowlesi CSP central repeat region. This study aims to characterise the CSP repeat motifs of P. knowlesi isolates in Peninsular Malaysia. CSP repeat motifs of 64 P. knowlesi isolates were identified using Rapid Automatic Detection and Alignment of Repeats (RADAR). Antigenicity of the repeat motifs and linear B cell epitopes were predicted using VaxiJen 2.0, BepiPred-2.0 and BCPred, respectively. A total of 35 dominant repeat motifs were identified. The repeat motif "AGQPQAQGDGANAGQPQAQGDGAN" has the highest repeat frequency (n=15) and antigenicity index of 1.7986. All the repeat regions were predicted as B cell epitopes. In silico approaches revealed that all repeat motifs were antigenic and consisted of B cell epitopes which could be designed as knowlesi malaria vaccine.

Molecular and functional properties of human Plasmodium falciparum CSP C-terminus antibodies.

Human monoclonal antibodies (mAbs) against the central repeat and junction domain of Plasmodium falciparum circumsporozoite protein (PfCSP) have been studied extensively to guide malaria vaccine design compared to antibodies against the PfCSP C terminus. Here, we describe the molecular characteristics and protective potential of 73 germline and mutated human mAbs against the highly immunogenic PfCSP C-terminal domain. Two mAbs recognized linear epitopes in the C-terminal linker with sequence similarity to repeat and junction motifs, whereas all others targeted conformational epitopes in the α-thrombospondin repeat (α-TSR) domain. Specificity for the polymorphic Th2R/Th3R but not the conserved RII+/CS.T3 region in the α-TSR was associated with IGHV3-21/IGVL3-21 or IGLV3-1 gene usage. Although the C terminus specific mAbs showed signs of more efficient affinity maturation and class-switching compared to anti-repeat mAbs, live sporozoite binding and inhibitory activity was limited to a single C-linker reactive mAb with cross-reactivity to the central repeat and junction. The data provide novel insights in the human anti-C-linker and anti-α-TSR antibody response that support exclusion of the PfCSP C terminus from malaria vaccine designs.

Designing a multi-epitope chimeric protein from different potential targets: A potential vaccine candidate against Plasmodium.

Malaria is an infectious disease that has been a continuous threat to mankind since the time immemorial. Owing to the complex multi-staged life cycle of the plasmodium parasite, an effective malaria vaccine which is fully protective against the parasite infection is urgently needed to deal with the challenges. In the present study, essential parasite proteins were identified and a chimeric protein with multivalent epitopes was generated. The designed chimeric protein consists of best potential B and T cell epitopes from five different essential parasite proteins. Physiochemical studies of the chimeric protein showed that the modeled vaccine construct was thermo-stable, hydrophilic and antigenic in nature. And the binding of the vaccine construct with Toll-like receptor-4 (TLR-4) as revealed by the molecular docking suggests the possible interaction and role of the vaccine construct in activating the innate immune response. The constructed vaccine being a chimeric protein containing epitopes from different potential candidates could target different stages or pathways of the parasite. Moreover, the approach used in this study is time and cost effective, and can be applied in the discoveries of new potential vaccine targets for other pathogens.

Analysis of sequence diversity in Plasmodium falciparum glutamic acid-rich protein (PfGARP), an asexual blood stage vaccine candidate.

Glutamic acid-rich protein of Plasmodium falciparum (PfGARP) binds to erythrocyte band 3 and may enhance cytoadherence of infected erythrocytes. Naturally acquired anti-PfGARP antibodies could confer protection against high parasitemia and severe symptoms. While whole genome sequencing analysis has suggested high conservation in this locus, little is known about repeat polymorphism in this vaccine candidate antigen. Direct sequencing was performed from the PCR-amplified complete PfGARP gene of 80 clinical isolates from four malaria endemic provinces in Thailand and an isolate from a Guinean patient. Publicly available complete coding sequences of this locus were included for comparative analysis. Six complex repeat (RI-RVI) and two homopolymeric glutamic acid repeat (E1 and E2) domains were identified in PfGARP. The erythrocyte band 3-binding ligand in domain RIV and the epitope for mAB7899 antibody eliciting in vitro parasite killing property were perfectly conserved across isolates. Repeat lengths in domains RIII and E1-RVI-E2 seemed to be correlated with parasite density of the patients. Sequence variation in PfGARP exhibited genetic differentiation across most endemic areas of Thailand. Phylogenetic tree inferred from this locus has shown that most Thai isolates formed closely related lineages, suggesting local expansion/contractions of repeat-encoding regions. Positive selection was observed in non-repeat region preceding domain RII which corresponded to a helper T cell epitope predicted to be recognized by a common HLA class II among Thai population. Predicted linear B cell epitopes were identified in both repeat and non-repeat domains. Besides length variation in some repeat domains, sequence conservation in non-repeat regions and almost all predicted immunogenic epitopes have suggested that PfGARP-derived vaccine may largely elicit strain-transcending immunity.

Genetic variations of Plasmodium falciparum circumsporozoite protein and the impact on interactions with human immunoproteins and malaria vaccine efficacy.

In October 2021, the world's first malaria vaccine RTS,S was endorsed by WHO for broad use in children, despite its low efficacy. This study examined polyclonal infections and the associations of parasite genetic variations with binding affinity to human leukocyte antigen (HLA). Multiplicity of infection was determined by amplicon deep sequencing of PfMSP1. Genetic variations in PfCSP were examined across 88 samples from Ghana and analyzed together with 1655 PfCSP sequences from other African and non-African isolates. Binding interactions of PfCSP peptide variants and HLA were predicted using NetChop and HADDOCK. High polyclonality was detected among infections, with each infection harboring multiple non-3D7 PfCSP variants. Twenty-seven PfCSP haplotypes were detected in the Ghanaian samples, and they broadly represented PfCSP diversity across Africa. The number of genetic differences between 3D7 and non-3D7 PfCSP variants does not influence binding to HLA. However, CSP peptide length after proteolytic degradation significantly affects its molecular weight and binding affinity to HLA. Despite the high diversity of HLA, the majority of the HLAI and II alleles interacted/bound with all Ghana CSP peptides. Multiple non-3D7 strains among P. falciparum infections could impact the effectiveness of RTS,S. Longer peptides of the Th2R/Th3R CSP regions should be considered in future versions of RTS,S.

Immunogenicity of Wild Type and Mutant Hepatitis B Surface Antigen Virus-like Particles (VLPs) in Mice with Pre-Existing Immunity against the Wild Type Vector.

Virus-like particles (VLPs), composed of the small hepatitis B virus surface antigen (HBsAgS), are the antigenic components of the hepatitis B virus (HBV) vaccine and represent the backbones for a chimeric anti-malaria vaccine and various vaccine candidates. Biological vectors have to face pre-existing anti-vector immune responses due to previous immune exposure. Vector recognition after natural infections or vaccinations can result in unwarranted outcomes, with compromising effects on clinical outcomes. In order to evaluate the impact of a pre-existing anti-HBsAgS immune response, we developed mutant VLPs composed of subunits with reduced HBsAgS-specific antigenicity. The insertion of a Plasmodium falciparum circumsporozoite protein (CSP)-derived epitope as a read-out allowed the assessment of wild type (wt) and mutant VLPs in the context of a pre-existing immune response. Mutant and wt VLP platforms with a CSP-epitope insert are immunogenic and have the ability to generate anti-CSP antibody responses in both naïve BALB/c mice and mice with a pre-existing anti-HBsAgS immune response, but with superior anti-CSP responses in mice with a pre-existing immunity. The data indicate that previous HBsAgS exposure facilitates enhanced antibody responses against foreign epitopes delivered by the HBsAgS platform, and, in this context, the state of immune sensitization alters the outcome of subsequent vaccinations.

Evaluation of transmission-blocking potential of Pv22 using clinical Plasmodium vivax infections and transgenic Plasmodium berghei.

Antigens expressed during the sexual development of malaria parasites are transmission-blocking vaccine (TBV) targets. Pb22, a protein expressed and localized to the plasma membrane of gametes and ookinetes in Plasmodium berghei, is an excellent TBV candidate. Here, we evaluated the TB potential of the Plasmodium vivax ortholog Pv22 using a transgenic P. berghei parasite line and P. vivax clinical isolates. The full-length recombinant Pv22 (rPv22) protein was produced and used to immunize mice and rabbits to obtain antibodies. We generated a transgenic P. berghei line (TrPv22Pb) by inserting the pv22 gene into the pb22 locus and showed that Pv22 expression completely rescued the defects in male gametogenesis of the pb22 deletion parasite. Since Pv22 in the transgenic parasite showed similar expression and localization patterns to Pb22, we used the TrPv22Pb parasite as a surrogate to evaluate the TB potential of Pv22. In mosquito feeding assays, mosquitoes feeding on rPv22-immunized mice infected with TrPv22Pb parasites showed a 49.3-53.3 % reduction in the oocyst density compared to the control group. In vitro assays showed that the rPv22 immune sera significantly inhibited exflagellation and ookinete formation of the TrPv22Pb parasites. In a direct membrane feeding assay using three clinical P. vivax isolates, the rabbit anti-rPv22 antibodies also significantly decreased the oocyst density by 53.7, 30.2, and 26.2 %, respectively. This study demonstrated the feasibility of using transgenic P. berghei parasites expressing P. vivax antigens as a potential tool to evaluate TBV candidates. However, the much weaker TB activity of Pv22 obtained from two complementary assays suggest that Pv22 may not be a promising TBV candidate for P. vivax.

Engineering of a plant-produced virus-like particle to improve the display of the Plasmodium falciparum Pfs25 antigen and transmission-blocking activity of the vaccine candidate.

Malaria kills around 409,000 people a year, mostly children under the age of five. Malaria transmission-blocking vaccines work to reduce malaria prevalence in a community and have the potential to be part of a multifaceted approach required to eliminate the parasites causing the disease. Pfs25 is a leading malaria transmission-blocking antigen and has been successfully produced in a plant expression system as both a subunit vaccine and as a virus-like particle. This study demonstrates an improved version of the virus-like particle antigen display molecule by eliminating known protease sites from the prior A85 variant. This re-engineered molecule, termed B29, displays three times the number of Pfs25 antigens per virus-like particle compared to the original Pfs25 virus-like particle. An improved purification scheme was also developed, resulting in a substantially higher yield and improved purity. The molecule was evaluated in a mouse model and found to induce improved transmission-blocking activity at lower doses and longer durations than the original molecule.

Genetic diversity of transmission-blocking vaccine candidate antigens Pvs25 and Pvs28 in Plasmodium vivax isolates from China.

BACKGROUND: Transmission-blocking vaccines (TBVs) target the sexual stages of malaria parasites to reduce or interrupt the transmission cycle in human and mosquito populations. The genetic diversity of TBVs candidate antigens, Pvs25 and Pvs28, in Plasmodium vivax could provide evidence for the development of TBVs. METHODS: Dry blood spots from P. vivax patients were collected from Dandong, Suining, Hainan, Nyingchi, Tengchong, and Yingjiang in China. The pvs25 and pvs28 genes were amplified and sequenced. The genetic diversity of pvs25 and pvs28 were analyzed using DNASTAR, MEGA6, and DnaSP 5.0 programs. RESULTS: A total of 377 samples were collected, among which 324 and 272 samples were successfully amplified in the pvs25 and pvs28 genes, respectively. Eight haplotypes were identified in Pvs25, for which the predominant mutation was I130T with 100% prevalence. A variety of 22 haplotypes in Pvs28 were identified. The number of GSGGE/D repeats of Pvs28 was a range of 4-8, among which, high (7-8) and low (4-5) copy numbers of tandem repeats were found in haplotypes H2 and H17, respectively. The nucleotide diversity of pvs28 (π = 0.00305 ± 0.00061) was slightly higher than that of pvs25 (π = 0.00146 ± 0.00007), thus they were not significantly different (P > 0.05). The Tajima's D value of pvs25 was positive whereas pvs28 was negative, which indicated that both genes were affected by natural selection. CONCLUSION: The genetic diversity of pvs25 and pvs28 genes in China was relatively limited, which provided valuable information for TBVs design and optimization.

Structure-guided insights into potential function of novel genetic variants in the malaria vaccine candidate PfRh5.

The recent stall in the global reduction of malaria deaths has made the development of a highly effective vaccine essential. A major challenge to developing an efficacious vaccine is the extensive diversity of Plasmodium falciparum antigens. While genetic diversity plays a major role in immune evasion and is a barrier to the development of both natural and vaccine-induced protective immunity, it has been under-prioritized in the evaluation of malaria vaccine candidates. This study uses genomic approaches to evaluate genetic diversity in next generation malaria vaccine candidate PfRh5. We used targeted deep amplicon sequencing to identify non-synonymous Single Nucleotide Polymorphisms (SNPs) in PfRh5 (Reticulocyte-Binding Protein Homologue 5) in 189 P. falciparum positive samples from Southern Senegal and identified 74 novel SNPs. We evaluated the population prevalence of these SNPs as well as the frequency in individual samples and found that only a single SNP, C203Y, was present at every site. Many SNPs were unique to the individual sampled, with over 90% of SNPs being found in just one infected individual. In addition to population prevalence, we assessed individual level SNP frequencies which revealed that some SNPs were dominant (frequency of greater than 25% in a polygenomic sample) whereas most were rare, present at 2% or less of total reads mapped to the reference at the given position. Structural modeling uncovered 3 novel SNPs occurring under epitopes bound by inhibitory monoclonal antibodies, potentially impacting immune evasion, while other SNPs were predicted to impact PfRh5 structure or interactions with the receptor or binding partners. Our data demonstrate that PfRh5 exhibits greater genetic diversity than previously described, with the caveat that most of the uncovered SNPs are at a low overall frequency in the individual and prevalence in the population. The structural studies reveal that novel SNPs could have functional implications on PfRh5 receptor binding, complex formation, or immune evasion, supporting continued efforts to validate PfRh5 as an effective malaria vaccine target and development of a PfRh5 vaccine.

Structural organization and sequence diversity of the complete nucleotide sequence encoding the Plasmodium malariae merozoite surface protein-1.

The merozoite surface protein-1 (MSP1) is a prime candidate for an asexual blood stage vaccine against malaria. However, polymorphism in this antigen could compromise the vaccine's efficacy. Although the extent of sequence variation in MSP1 has been analyzed from various Plasmodium species, little is known about structural organization and diversity of this locus in Plasmodium malariae (PmMSP1). Herein, we have shown that PmMSP1 contained five conserved and four variable blocks based on analysis of the complete coding sequences. Variable blocks were characterized by short insertion and deletion variants (block II), polymorphic nonrepeat sequences (block IV), complex repeat structure with size variation (block VI) and degenerate octapeptide repeats (block VIII). Like other malarial MSP1s, evidences of intragenic recombination have been found in PmMSP1. The rate of nonsynonymous nucleotide substitutions significantly exceeded that of synonymous nucleotide substitutions in block IV, suggesting positive selection in this region. Codon-based analysis of deviation from neutrality has identified a codon under purifying selection located in close proximity to the homologous region of the 38 kDa/42 kDa cleavage site of P. falciparum MSP1. A number of predicted linear B-cell epitopes were identified across both conserved and variable blocks of the protein. However, polymorphism in repeat-containing blocks resulted in alteration of the predicted linear B-cell epitope scores across variants. Although a number of predicted HLA-class II-binding peptides were identified in PmMSP1, all variants of block IV seemed not to be recognized by common HLA-class II alleles among Thai population, suggesting that diversity in this positive selection region could probably affect host immune recognition. The data on structural diversity in PmMSP1 could be useful for further studies such as vaccine development and strain characterization of this neglected malaria parasite.

Engineered DNA-encoded monoclonal antibodies targeting Plasmodium falciparum circumsporozoite protein confer single dose protection in a murine malaria challenge model.

Novel approaches for malaria prophylaxis remain important. Synthetic DNA-encoded monoclonal antibodies (DMAbs) are a promising approach to generate rapid, direct in vivo host-generated mAbs with potential benefits in production simplicity and distribution coupled with genetic engineering. Here, we explore this approach in a malaria challenge model. We engineered germline-reverted DMAbs based on human mAb clones CIS43, 317, and L9 which target a junctional epitope, major repeat, and minor repeat of the Plasmodium falciparum circumsporozoite protein (CSP) respectively. DMAb variants were encoded into a plasmid vector backbone and their expression and binding profiles were characterized. We demonstrate long-term serological expression of DMAb constructs resulting in in vivo efficacy of CIS43 GL and 317 GL in a rigorous mosquito bite mouse challenge model. Additionally, we engineered an Fc modified variant of CIS43 and L9-based DMAbs to ablate binding to C1q to test the impact of complement-dependent Fc function on challenge outcomes. Complement knockout variant DMAbs demonstrated similar protection to that of WT Fc DMAbs supporting the notion that direct binding to the parasite is sufficient for the protection observed. Further investigation of DMAbs for malaria prophylaxis appears of importance.

Production of a high purity, C-tagged hepatitis B surface antigen fusion protein VLP vaccine for malaria expressed in Pichia pastoris under cGMP conditions.

Virus-like particles (VLPs) induce strong humoral and cellular responses and have formed the basis of some currently licensed vaccines. Here, we present the method used for the production of R21, a VLP-based anti-sporozoite malaria vaccine, under current Clinical Good Manufacturing Practice regulations (cGMP). Previous preclinical studies in BALB/c mice showed that R21 produced almost complete protection against sporozoite challenge with transgenic Plasmodium berghei parasites. Here, we have modified the preclinical production process to enable the production of sufficient quantities of highly pure, clinical-grade material for use in human clinical trials. The R21 construct was re-engineered to include a C-tag to allow affinity-based separation from the major contaminant alcohol oxidase 1 (AOX 1, ~74 kDa). To our knowledge, this is the first use of C-tag technology to purify a VLP vaccine candidate for use in human clinical trials. The R21 vaccine has shown high-level efficacy in an African Phase IIb trial, and multiple clinical trials are underway to assess the safety and efficacy of the vaccine. Our findings support the future use of C-tag platform technologies to enable cGMP-compliant biomanufacturing of high purity yeast-expressed VLP-based vaccines for early phase clinical trials when clinical grade material is required in smaller quantities in a quick time frame.

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.