A phase IIa, randomized, double-blind, safety, immunogenicity and efficacy trial of Plasmodium falciparum vaccine antigens merozoite surface protein 1 and RTS,S formulated with AS02 adjuvant in healthy, malaria-naïve adults.

BACKGROUND: To improve the efficacy of Plasmodium falciparum malaria vaccine RTS,S/AS02, we conducted a study in 2001 in healthy, malaria-naïve adults administered RTS,S/AS02 in combination with FMP1, a recombinant merozoite surface-protein-1, C-terminal 42kD fragment. METHODS: A double-blind Phase I/IIa study randomized N = 60 subjects 1:1:1:1 to one of four groups, N = 15/group, to evaluate safety, immunogenicity, and efficacy of intra-deltoid half-doses of RTS,S/AS02 and FMP1/AS02 administered in the contralateral (RTS,S + FMP1-separate) or same (RTS,S + FMP1-same) sites, or FMP1/AS02 alone (FMP1-alone), or RTS,S/AS02 alone (RTS,S-alone) on a 0-, 1-, 3-month schedule. Subjects receiving three doses of vaccine and non-immunized controls (N = 11) were infected with homologous P. falciparum 3D7 sporozoites by Controlled Human Malaria Infection (CHMI). RESULTS: Subjects in all vaccination groups experienced mostly mild or moderate local and general adverse events that resolved within eight days. Anti-circumsporozoite antibody levels were lower when FMP1 and RTS,S were co-administered at the same site (35.0 µg/mL: 95 % CI 20.3-63), versus separate arms (57.4 µg/mL: 95 % CI 32.3-102) or RTS,S alone (62.0 µg/mL: 95 % CI: 37.8-101.8). RTS,S-specific lymphoproliferative responses and ex vivo ELISpot CSP-specific interferon-gamma (IFN-γ) responses were indistinguishable among groups receiving RTS,S/AS02. There was no difference in antibody to FMP1 among groups receiving FMP1/AS02. After CHMI, groups immunized with a RTS,S-containing regimen had âˆ¼ 30 % sterile protection against parasitemia, and equivalent delays in time-to-parasitemia. The FMP1/AS02 alone group showed no sterile immunity or delay in parasitemia. CONCLUSION: Co-administration of RTS,S and FMP1/AS02 reduced anti-RTS,S antibody, but did not affect tolerability, cellular immunity, or efficacy in a stringent CHMI model. Absence of efficacy or delay of patency in the sporozoite challenge model in the FMP1/AS02 group did not rule out efficacy of FMP1/AS02 in an endemic population. However, a Phase IIb trial of FMP1/AS02 in children in malaria-endemic Kenya did not demonstrate efficacy against natural infection. CLINICALTRIALS: gov identifier: NCT01556945.

Genetic polymorphism of merozoite surface protein 1 and antifolate-resistant genes in Plasmodium falciparum from Mali and Niger.

Since 2015, countries in the Sahel region have implemented large-scale seasonal malaria chemoprevention (SMC). However, the mass use of sulfadoxine-pyrimethamine (SP) plus amodiaquine impacts the genetic diversity of malaria parasites and their sensitivity to antimalarials. This study aimed to describe and compare the genetic diversity and SP resistance of Plasmodium falciparum strains in Mali and Niger. We collected 400 blood samples in Mali and Niger from children aged 3-59 months suspected of malaria. Of them, 201 tested positive (Niger, 111, 55.2%; Mali, 90, 44.8%). Polymorphism of merozoite surface protein 1 (msp1) genetic marker showed 201 allotypes. The frequency of the RO33 allotype was significantly higher in Niger (63.6%) than in Mali (39.3%). There was no significant difference in the frequency of the K1 and MAD20 allotypes between the 2 countries. The multiplicity of infection was 2 allotypes per patient in Mali and one allotype per patient in Niger. The prevalence of strains with the triple mutants Pfdhfr51I/Pfdhfr59R/Pfdhps436A/F/H and Pfdhfr51I/Pfdhfr59R/Pfdhps437G was 18.1% and 30.2%, respectively, and 7.7% carried the quadruple mutant Pfdhfr51I/Pfdhfr59R/Pfdhps436A/F/H/Pfdhps437G. Despite the significant genetic diversity of parasite populations, the level of SP resistance was comparable between Mali and Niger. The frequency of mutations conferring resistance to SP still allows its effective use in intermittent preventive treatment in pregnant women and in SMC.

Analysis of post-translational modification dynamics unveiled novel insights into Rice responses to MSP1.

Magnaporthe oryzae snodprot1 homologous protein (MSP1) is known to function as a pathogen-associated molecular pattern (PAMP) and trigger PAMP-triggered immunity (PTI) in rice including induction of programmed cell death and expression of defense-related genes. The involvement of several post-translational modifications (PTMs) in the regulation of plant immune response, especially PTI, is well established, however, the information on the regulatory roles of these PTMs in response to MSP1-induced signaling is currently elusive. Here, we report the phosphoproteome, ubiquitinome, and acetylproteome to investigate the MSP1-induced PTMs alterations in MSP1 overexpressed and wild-type rice. Our analysis identified a total of 4666 PTMs-modified sites in rice leaves including 4292 phosphosites, 189 ubiquitin sites, and 185 acetylation sites. Among these, the PTM status of 437 phosphorylated, 53 ubiquitinated, and 68 acetylated peptides was significantly changed by MSP1. Functional annotation of MSP1 modulated peptides by MapMan analysis revealed that these were majorly associated with cellular immune responses including signaling, transcription factors, DNA and RNA regulation, and protein metabolism, among others. Taken together, our study provides novel insights into post-translational mediated regulation of rice proteins in response to M. oryzae secreted PAMP which help in understanding the molecular mechanism of MSP1-induced signaling in rice in greater detail. SIGNIFICANCE: The research investigates the effect of overexpression of MSP1 protein in rice leaves on the phosphoproteome, acetylome, and ubiquitinome. The study found that MSP1 is involved in rice protein phosphorylation, particularly in signaling pathways, and identified a key component, PTAC16, in MSP1-induced signaling. The analysis also revealed MSP1's role in protein degradation and modification by inducing ubiquitination of the target rice proteins. The research identified potential kinases involved in the phosphorylation of rice proteins, including casein kinase II, 14-3-3 domain binding motif, ß-adrenergic receptor kinase, ERK1,2 kinase substrate motif, and casein kinase I motifs. Overall, the findings provide insights into the molecular mechanisms underlying of MSP1 induced signaling in rice which may have implications for improving crop yield and quality.

Natural Plasmodium falciparum Infection Stimulates Human Antibodies to MSP1 Epitopes Identified in Mice Infection Models upon Non-Natural Modified Peptidomimetic Vaccination.

(1) Background: Malaria, a vector-borne infectious disease, is caused by parasites of the Plasmodium genus, responsible for increased extreme morbidity and mortality rates. Despite advances in approved vaccines, full protection has not yet been achieved upon vaccination, thus the development of more potent and safe immuno-stimulating agents for malaria prevention is a goal to be urgently accomplished. We have focused our research on a strategy to identify Plasmodium spp. epitopes by naturally acquired human antibodies and rodent malaria infection models immunized with site-directed non-natural antigens. (2) Methods: Some predictive algorithms and bioinformatics tools resembling different biological environments, such as phagosome-lysosome proteolytic degradation, affinity, and the high frequency of malaria-resistant and -sensitive HLA-II alleles were regarded for the proper selection of epitopes and potential testing. Each epitope's binding profile to both host cells and HLA-II molecules was considered for such initial screening. (3) Results: Once selected, we define each epitope-peptide to be synthesized in terms of size and hydrophobicity, and introduced peptide-bond surrogates and non-natural amino acids in a site-directed fashion, and then they were produced by solid-phase peptide synthesis. Molecules were then tested by their antigenic and immunogenic properties compared to human sera from Colombian malaria-endemic areas. The antigenicity and protective capacity of each epitope-peptide in a rodent infection model were examined. The ability of vaccinated mice after being challenged with P. berghei ANKA and P. yoelii 17XL to control malaria led to the determination of an immune stimulation involving Th1 and Th1/Th2 mechanisms. In silico molecular dynamics and modeling provided some interactions insights, leading to possible explanations for protection due to immunization. (4) Conclusions: We have found evidence for proposing MSP1-modified epitopes to be considered as neutralizing antibody stimulators that are useful as probes for the detection of Plasmodium parasites, as well as for sub-unit components of a site-directed designed malaria vaccine candidate.

Proofreading of protein localization mediated by a mitochondrial AAA-ATPase Msp1.

Normal cellular functions rely on correct protein localization within cells. Protein targeting had been thought to be a precise process, and even if it fails, the mistargeted proteins were supposed to be quickly degraded. However, this view is rapidly changing. Tail-anchored (TA) proteins are a class of membrane proteins that possess a single transmembrane domain (TMD) near the C-terminus and are posttranslationally targeted to the endoplasmic reticulum (ER) membrane, mitochondrial outer membrane (OM), and peroxisomal membrane, yet they can be mistargeted to the mitochondrial OM. The mistargeted TA proteins can be extracted from the OM by a mitochondrial AAA-ATPase Msp1/ATAD1 and transferred to the ER. If they are regarded as aberrant by the ER protein quality control system, they are extracted from the ER membrane for proteasomal degradation in the cytosol. If they are not regarded as aberrant, they are further transported to downstream organelles or original destinations along the secretory pathway. Thus, Msp1 contributes to not only degradation but also "proofreading" of the targeting of mislocalized TA proteins.

[Humoral response to Plasmodium falciparum antigenic peptides (MSP1, MSP2, and SR-11.1) in subjects living in endemic areas]. / La réponse humorale contre les peptides antigéniques de Plasmodium falciparum (MSP1, MSP2 et SR-11.1) chez des sujets vivant en zone endémique.

Introduction: vaccines are the key tools for controlling and eradicating malaria worldwide. Currently, research and development are focused on three types of vaccine candidates that target different life stages of Plasmodium falciparum in humans and the vector. The purpose of this study is to evaluate the humoral response to Plasmodium falciparum antigenic peptides (MSP1, MSP2 and SR-11.1) in subjects living in endemic areas. Methods: we conducted a cross-sectional study of serum samples collected from 182 Vietnamese subjects living in endemic areas over a period of 5 months. Whole blood was centrifuged and the serum was aliquoted into cryovials and preserved at -20°C until IgG testing. ELISA was used for total immunoglobulin G (IgG) antibodies testing after peptide coupling with glutaraldehyde. Results: a total of 182 sera samples from Vietnamese subjects living in endemic areas were included. In the different study age groups, total antigen-specific IgG antibodies against Plasmodium falciparum antigenic peptides (MSP-1; MSP-2 and SR-11.1) showed age-dependent distribution. In subjects aged 3-19 years, the level of total antigen-specific IgG antibodies against Plasmodium falciparum were lower than those of the age groups over 20 years of age (p=0.07). Comparison of specific antigen-specific IgG antibody levels with Plasmodium falciparum antigenic peptides MSP-1, MSP-2, and SR-11.1 in the age groups showed a significantly higher mean in MSP-1 and MSP-2 compared to anti-SR-11.1 (p=0.04). Conclusion: this study highlights that the level of specific antibodies against Plasmodium falciparum antigenic peptides (MSP-1; MSP-2 and SR-11.1) is age dependent. Of the three antigenic peptides, MSP-1 and MSP-2 appear to be more immunogenic than SR.11.1. Therefore, SR.11.1 is a macromolecule to the immune system and, from this point of view, appears to be significantly less immunogenic than other peptides.

Cyclic constrained immunoreactive peptides from crucial P. falciparum proteins: potential implications in malaria diagnostics.

Malaria is still a global challenge with significant morbidity and mortality, especially in the African, South-East Asian, and Latin American regions. Malaria diagnosis is a crucial pillar in the control and elimination efforts, often accomplished by the administration of mass-scale Rapid diagnostic tests (RDTs). The inherent limitations of RDTs- insensitivity in scenarios of low transmission settings and deletion of one of the target proteins- Histidine rich protein 2/3 (HRP-2/3) are evident from multiple reports, thus necessitating the need to explore novel diagnostic tools/targets. The present study used peptide microarray to screen potential epitopes from 13 antigenic proteins (CSP, EXP1, LSA1, TRAP, AARP, AMA1, GLURP, MSP1, MSP2, MSP3, MSP4, P48/45, HAP2) of P. falciparum. Three cyclic constrained immunoreactive peptides- C6 (EXP1), A8 (MSP2), B7 (GLURP) were identified from 5458 cyclic constrained peptides (in duplicate) against P. falciparum-infected sera. Peptides (C6, A8, B7- cyclic constrained) and (G11, DSQ, NQN- corresponding linear peptides) were fairly immunoreactive towards P. falciparum-infected sera in dot-blot assay. Using direct ELISA, cyclic constrained peptides (C6 and B7) were found to be specific to P. falciparum-infected sera. A substantial number of samples were tested and the peptides successfully differentiated the P. falciparum positive and negative samples with high confidence. In conclusion, the study identified 3 cyclic constrained immunoreactive peptides (C6, B7, and A8) from P. falciparum secretory/surface proteins and further validated for diagnostic potential of 2 peptides (C6 and B7) with field-collected P. falciparum-infected sera samples.

Conserved structural elements specialize ATAD1 as a membrane protein extraction machine.

The mitochondrial AAA (ATPase Associated with diverse cellular Activities) protein ATAD1 (in humans; Msp1 in yeast) removes mislocalized membrane proteins, as well as stuck import substrates from the mitochondrial outer membrane, facilitating their re-insertion into their cognate organelles and maintaining mitochondria's protein import capacity. In doing so, it helps to maintain proteostasis in mitochondria. How ATAD1 tackles the energetic challenge to extract hydrophobic membrane proteins from the lipid bilayer and what structural features adapt ATAD1 for its particular function has remained a mystery. Previously, we determined the structure of Msp1 in complex with a peptide substrate (Wang et al., 2020). The structure showed that Msp1's mechanism follows the general principle established for AAA proteins while adopting several structural features that specialize it for its function. Among these features in Msp1 was the utilization of multiple aromatic amino acids to firmly grip the substrate in the central pore. However, it was not clear whether the aromatic nature of these amino acids were required, or if they could be functionally replaced by aliphatic amino acids. In this work, we determined the cryo-EM structures of the human ATAD1 in complex with a peptide substrate at near atomic resolution. The structures show that phylogenetically conserved structural elements adapt ATAD1 for its function while generally adopting a conserved mechanism shared by many AAA proteins. We developed a microscopy-based assay reporting on protein mislocalization, with which we directly assessed ATAD1's activity in live cells and showed that both aromatic amino acids in pore-loop 1 are required for ATAD1's function and cannot be substituted by aliphatic amino acids. A short α-helix at the C-terminus strongly facilitates ATAD1's oligomerization, a structural feature that distinguishes ATAD1 from its closely related proteins.

Investigation of liposomal self-adjuvanting peptide epitopes derived from conserved blood-stage Plasmodium antigens.

Malaria is a vector born parasitic disease causing millions of deaths every year. Despite the high mortality rate, an effective vaccine against this mosquito-borne infectious disease is yet to be developed. Up to date, RTS,S/AS01 is the only vaccine available for malaria prevention; however, its efficacy is low. Among a variety of malaria antigens, merozoite surface protein-1(MSP-1) and ring-infected erythrocyte surface antigen (RESA) have been proposed as promising candidates for malaria vaccine development. We developed peptide-based Plasmodium falciparum vaccine candidates that incorporated three previously reported conserved epitopes from MSP-1 and RESA into highly effective liposomal polyleucine delivery system. Indeed, MSP-1 and RESA-derived epitopes conjugated to polyleucine and formulated into liposomes induced higher epitope specific antibody titres. However, immunized mice failed to demonstrate protection in a rodent malaria challenge study with Plasmodium yoelii. In addition, we found that the three reported P. falciparum epitopes did not to share conformational properties and high sequence similarity with P. yoelii MSP-1 and RESA proteins, despite the epitopes were reported to protect mice against P. yoelii challenge.

Reconstitution of Msp1 Extraction Activity with Fully Purified Components.

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Polymorphisms in Plasmodium falciparum dihydropteroate synthetase and dihydrofolate reductase genes in Nigerian children with uncomplicated malaria using high-resolution melting technique.

In 2005, the Nigerian Federal Ministry of Health revised the treatment policy for uncomplicated malaria with the introduction of artemisinin-based combination therapies (ACTs). This policy change discouraged the use of Sulphadoxine-pyrimethamine (SP) as the second-line treatment of uncomplicated falciparum malaria. However, SP is used as an intermittent preventive treatment of malaria in pregnancy (IPTp) and seasonal malaria chemoprevention (SMC) in children aged 3-59 months. There have been increasing reports of SP resistance especially in the non-pregnant population in Nigeria, thus, the need to continually monitor the efficacy of SP as IPTp and SMC by estimating polymorphisms in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes associated with SP resistance. The high resolution-melting (HRM) assay was used to investigate polymorphisms in codons 51, 59, 108 and 164 of the dhfr gene and codons 437, 540, 581 and 613 of the dhps gene. DNA was extracted from 271 dried bloodspot filter paper samples obtained from children (< 5 years old) with uncomplicated malaria. The dhfr triple mutant I51R59N108, dhps double mutant G437G581 and quadruple dhfr I51R59N108 + dhps G437 mutant haplotypes were observed in 80.8%, 13.7% and 52.8% parasites, respectively. Although the quintuple dhfr I51R59N108 + dhps G437E540 and sextuple dhfr I51R59N108 + dhps G437E540G581 mutant haplotypes linked with in-vivo and in-vitro SP resistance were not detected, constant surveillance of these haplotypes should be done in the country to detect any change in prevalence.

Recombinant proteins of Plasmodium malariae merozoite surface protein 1 (PmMSP1): Testing immunogenicity in the BALB/c model and potential use as diagnostic tool.

BACKGROUND: Plasmodium malariae is the third most prevalent human malaria-causing species and has a patchy, but ample distribution in the world. Humans can host the parasite for years without presenting significant symptoms, turning its diagnosis and control into a difficult task. Here, we investigated the immunogenicity of recombinant proteins of P. malariae MSP1. METHODS: Five regions of PmMSP1 were expressed in Escherichia coli as GST-fusion proteins and immunized in BALB/c mice. The specificity, subtyping, and affinity of raised antibodies were evaluated by enzyme-linked immunosorbent assays. Cellular immune responses were analyzed by lymphoproliferation assays and cytokine levels produced by splenocytes were detected by cytometry. RESULTS: We found that N-terminal, central regions, and PmMSP119 are strongly immunogenic in mice. After three doses, the induced immune responses remained high for 70 days. While antibodies induced after immunization with N-terminal and central regions showed similar affinities to the target antigens, affinities of IgG against PmMSP119 were higher. All proteins induced similar antibody subclass patterns (predominantly IgG1, IgG2a, and IgG2b), characterizing a mixed Th1/Th2 response. Further, autologous stimulation of splenocytes from immunized mice led to the secretion of IL2 and IL4, independently of the antigen used. Importantly, IgG from P. malariae-exposed individuals reacted against PmMSP1 recombinant proteins with a high specificity. On the other hand, sera from P. vivax or P. falciparum-infected individuals did not react at all against recombinant PmMSP1 proteins. CONCLUSION: Recombinant PmMSP1 proteins are very useful diagnostic markers of P. malariae in epidemiological studies or in the differential diagnosis of malaria caused by this species. Immunization with recombinant PmMSP1 proteins resulted in a significant humoral immune response, which may turn them potential component candidates for a vaccine against P. malariae.

Lack of quadruple and quintuple mutant alleles associated with sulfadoxine-pyrimethamine resistance in Plasmodium vivax isolates from Brazilian endemic areas

BACKGROUND AND OBJECTIVE Brazil is responsible for a large number of Plasmodium vivax cases in America. Given the emergence of P. vivax parasites resistant to chloroquine and the effectiveness of antifolates in vivax malaria treatment together with a correlation between mutations in P. vivax dhfr and dhps genes and SP treatment failure, the point mutations in these genes were investigated. METHODS Blood samples from 54 patients experiencing vivax malaria symptomatic episodes in the Amazonian Region were investigated. Genomic DNA was extracted using a DNA extraction kit (QIAGENTM). Nested polymerase chain reaction (PCR) amplification was carried out followed by Sanger sequencing to detect single nucleotide polymorphisms (SNPs). FINDINGS All tested isolates showed non-synonymous mutations in pvdhfr gene: 117N (54/54, 100%) and 58R (25/54, 46%). Double mutant allele 58R/117N (FRTNI, 28%) was the most frequent followed by triple mutant alleles (58R/117N/173L, FRTNL, 11%; 58R/61M/117N, FRMNI, 5% 117N/173L, FSTNL, 4%) and quadruple mutant allele (58R/61M/117N/173L, FRMNL, 2%). A single mutation was observed at codon C383G in pvdhps gene (SGKAV, 48%). CONCLUSION No evidence of molecular signatures associated with P. vivax resistance to SP was observed in the Brazilian samples.

Plasmodium-specific atypical memory B cells are short-lived activated B cells.

A subset of atypical memory B cells accumulates in malaria and several infections, autoimmune disorders and aging in both humans and mice. It has been suggested these cells are exhausted long-lived memory B cells, and their accumulation may contribute to poor acquisition of long-lasting immunity to certain chronic infections, such as malaria and HIV. Here, we generated an immunoglobulin heavy chain knock-in mouse with a BCR that recognizes MSP1 of the rodent malaria parasite, Plasmodium chabaudi. In combination with a mosquito-initiated P. chabaudi infection, we show that Plasmodium-specific atypical memory B cells are short-lived and disappear upon natural resolution of chronic infection. These cells show features of activation, proliferation, DNA replication, and plasmablasts. Our data demonstrate that Plasmodium-specific atypical memory B cells are not a subset of long-lived memory B cells, but rather short-lived activated cells, and part of a physiologic ongoing B-cell response.

Molecular Surveillance of Plasmodium falciparum Drug Resistance Markers in Clinical Samples from Botswana.

Drug-resistant Plasmodium falciparum is a major threat to global malaria control and elimination efforts. In Botswana, a southern African country approaching malaria elimination, P. falciparum molecular data are not available. Parasites were assessed through pollymerase chain reaction (PCR) for confirmation of positive rapid diagnostic tests, multiplicity of infection (MOI), and drug resistance markers among isolates from clinical uncomplicated malaria cases collected at health facilities. Of 211 dried blood spot samples selected for the study, 186 (88.2%) were PCR positive for P. falciparum. The mean MOI based on MSP1 genotyping was 2.3 and was not associated with age. A high prevalence of wild-type parasites for pfcrt and pfmdr1 was found, with a haplotype frequency (K76/N86) of 88.8% and 17.7% of the isolates having two copies of the pfmdr1 gene. For pfATPase6, all the parasites carried the wild-type S769 allele. Sequencing showed no evidence of non-synonymous mutations associated with reduced artemisinin derivative sensitivity in the P. falciparum k13 gene. In conclusion, we found that P. falciparum parasites in Botswana were mostly wild type for the drug resistance markers evaluated. Yet, there was a high rate of a molecular marker associated to reduced sensitivity to lumefantrine. Our results indicate the need for systematic drug efficacy surveillance to complement malaria elimination efforts.

Genetic diversity and natural selection of Plasmodium knowlesi merozoite surface protein 1 paralog gene in Malaysia.

BACKGROUND: Human infections due to the monkey malaria parasite Plasmodium knowlesi is on the rise in most Southeast Asian countries specifically Malaysia. The C-terminal 19 kDa domain of PvMSP1P is a potential vaccine candidate, however, no study has been conducted in the orthologous gene of P. knowlesi. This study investigates level of polymorphisms, haplotypes and natural selection of full-length pkmsp1p in clinical samples from Malaysia. METHODS: A total of 36 full-length pkmsp1p sequences along with the reference H-strain and 40 C-terminal pkmsp1p sequences from clinical isolates of Malaysia were downloaded from published genomes. Genetic diversity, polymorphism, haplotype and natural selection were determined using DnaSP 5.10 and MEGA 5.0 software. Genealogical relationships were determined using haplotype network tree in NETWORK software v5.0. Population genetic differentiation index (F ST ) and population structure of parasite was determined using Arlequin v3.5 and STRUCTURE v2.3.4 software. RESULTS: Comparison of 36 full-length pkmsp1p sequences along with the H-strain identified 339 SNPs (175 non-synonymous and 164 synonymous substitutions). The nucleotide diversity across the full-length gene was low compared to its ortholog pvmsp1p. The nucleotide diversity was higher toward the N-terminal domains (pkmsp1p-83 and 30) compared to the C-terminal domains (pkmsp1p-38, 33 and 19). Phylogenetic analysis of full-length genes identified 2 distinct clusters of P. knowlesi from Malaysian Borneo. The 40 pkmsp1p-19 sequences showed low polymorphisms with 16 polymorphisms leading to 18 haplotypes. In total there were 10 synonymous and 6 non-synonymous substitutions and 12 cysteine residues were intact within the two EGF domains. Evidence of strong purifying selection was observed within the full-length sequences as well in all the domains. Shared haplotypes of 40 pkmsp1p-19 were identified within Malaysian Borneo haplotypes. CONCLUSIONS: This study is the first to report on the genetic diversity and natural selection of pkmsp1p. A low level of genetic diversity and strong evidence of negative selection was detected and observed in all the domains of pkmsp1p of P. knowlesi indicating functional constrains. Shared haplotypes were identified within pkmsp1p-19 highlighting further evaluation using larger number of clinical samples from Malaysia.

Evaluation of a Plasmodium-Specific Carrier Protein To Enhance Production of Recombinant Pfs25, a Leading Transmission-Blocking Vaccine Candidate.

Challenges with the production and suboptimal immunogenicity of malaria vaccine candidates have slowed the development of a Plasmodium falciparum multiantigen vaccine. Attempting to resolve these issues, we focused on the use of highly immunogenic merozoite surface protein 8 (MSP8) as a vaccine carrier protein. Previously, we showed that a genetic fusion of the C-terminal 19-kDa fragment of merozoite surface protein 1 (MSP119) to P. falciparum MSP8 (PfMSP8) facilitated antigen production and folding and the induction of neutralizing antibodies to conformational B cell epitopes of MSP119 Here, using the PfMSP1/8 construct, we further optimized the recombinant PfMSP8 (rPfMSP8) carrier by the introduction of two cysteine-to-serine substitutions (CΔS) to improve the yield of the monomeric product. We then sought to test the broad applicability of this approach using the transmission-blocking vaccine candidate Pfs25. The production of rPfs25-based vaccines has presented challenges. Antibodies directed against the four highly constrained epidermal growth factor (EGF)-like domains of Pfs25 block sexual-stage development in mosquitoes. The sequence encoding mature Pfs25 was codon harmonized for expression in Escherichia coli We produced a rPfs25-PfMSP8 fusion protein [rPfs25/8(CΔS)] as well as unfused, mature rPfs25. rPfs25 was purified with a modest yield but required the incorporation of refolding protocols to obtain a proper conformation. In comparison, chimeric rPfs25/8(CΔS) was expressed and easily purified, with the Pfs25 domain bearing the proper conformation without renaturation. Both antigens were immunogenic in rabbits, inducing IgG that bound native Pfs25 and exhibited potent transmission-reducing activity. These data further demonstrate the utility of PfMSP8 as a parasite-specific carrier protein to enhance the production of complex malaria vaccine targets.

A Plasmodium vivax Plasmid DNA- and Adenovirus-Vectored Malaria Vaccine Encoding Blood-Stage Antigens AMA1 and MSP142 in a Prime/Boost Heterologous Immunization Regimen Partially Protects Aotus Monkeys against Blood-Stage Challenge.

Malaria is caused by parasites of the genus Plasmodium, which are transmitted to humans by the bites of Anopheles mosquitoes. After the elimination of Plasmodium falciparum, it is predicted that Plasmodium vivax will remain an important cause of morbidity and mortality outside Africa, stressing the importance of developing a vaccine against P. vivax malaria. In this study, we assessed the immunogenicity and protective efficacy of two P. vivax antigens, apical membrane antigen 1 (AMA1) and the 42-kDa C-terminal fragment of merozoite surface protein 1 (MSP142) in a plasmid recombinant DNA prime/adenoviral (Ad) vector boost regimen in Aotus monkeys. Groups of 4 to 5 monkeys were immunized with plasmid DNA alone, Ad alone, prime/boost regimens with each antigen, prime/boost regimens with both antigens, and empty vector controls and then subjected to blood-stage challenge. The heterologous immunization regimen with the antigen pair was more protective than either antigen alone or both antigens delivered with a single vaccine platform, on the basis of their ability to induce the longest prepatent period and the longest time to the peak level of parasitemia, the lowest peak and mean levels of parasitemia, the smallest area under the parasitemia curve, and the highest self-cure rate. Overall, prechallenge MSP142 antibody titers strongly correlated with a decreased parasite burden. Nevertheless, a significant proportion of immunized animals developed anemia. In conclusion, the P. vivax plasmid DNA/Ad serotype 5 vaccine encoding blood-stage parasite antigens AMA1 and MSP142 in a heterologous prime/boost immunization regimen provided significant protection against blood-stage challenge in Aotus monkeys, indicating the suitability of these antigens and this regimen for further development.

The presence of T cell epitopes is important for induction of antibody responses against antigens directed to DEC205+ dendritic cells.

In vivo antigen targeting to dendritic cells (DCs) has been used as a way to improve immune responses. Targeting is accomplished with the use of monoclonal antibodies (mAbs) to receptors present on the DC surface fused with the antigen of interest. An anti-DEC205 mAb has been successfully used to target antigens to the DEC205+CD8α+ DC subset. The administration of low doses of the hybrid mAb together with DC maturation stimuli is able to activate specific T cells and induce production of high antibody titres for a number of different antigens. However, it is still not known if this approach would work with any fused protein. Here we genetically fused the αDEC205 mAb with two fragments (42-kDa and 19-kDa) derived from the ~200 kDa Plasmodium vivax merozoite surface protein 1 (MSP1), known as MSP142 and MSP119, respectively. The administration of two doses of αDEC-MSP142, but not of αDEC-MSP119 mAb, together with an adjuvant to two mouse strains induced high anti-MSP119 antibody titres that were dependent on CD4+ T cells elicited by peptides present in the MSP133 sequence, indicating that the presence of T cell epitopes in antigens targeted to DEC205+ DCs increases antibody responses.

Antibody levels against GLURP R2, MSP1 block 2 hybrid and AS202.11 and the risk of malaria in children living in hyperendemic (Burkina Faso) and hypo-endemic (Ghana) areas.

BACKGROUND: Differences in parasite transmission intensity influence the process of acquisition of host immunity to Plasmodium falciparum malaria and ultimately, the rate of malaria related morbidity and mortality. Potential vaccines being designed to complement current intervention efforts therefore need to be evaluated against different malaria endemicity backgrounds. METHODS: The associations between antibody responses to the chimeric merozoite surface protein 1 block 2 hybrid (MSP1 hybrid), glutamate-rich protein region 2 (GLURP R2) and the peptide AS202.11, and the risk of malaria were assessed in children living in malaria hyperendemic (Burkina Faso, n = 354) and hypo-endemic (Ghana, n = 209) areas. Using the same reagent lots and standardized protocols for both study sites, immunoglobulin (Ig) M, IgG and IgG sub-class levels to each antigen were measured by ELISA in plasma from the children (aged 6-72 months). Associations between antibody levels and risk of malaria were assessed using Cox regression models adjusting for covariates. RESULTS: There was a significant association between GLURP R2 IgG3 and reduced risk of malaria after adjusting age of children in both the Burkinabe (hazard ratio 0.82; 95 % CI 0.74-0.91, p < 0.0001) and the Ghanaian (HR 0.48; 95 % CI 0.25-0.91, p = 0.02) cohorts. MSP1 hybrid IgM was associated (HR 0.85; 95 % CI 0.73-0.98, p = 0.02) with reduced risk of malaria in Burkina Faso cohort while IgG against AS202.11 in the Ghanaian children was associated with increased risk of malaria (HR 1.29; 95 % CI 1.01-1.65, p = 0.04). CONCLUSION: These findings support further development of GLURP R2 and MSP1 block 2 hybrid, perhaps as a fusion vaccine antigen targeting malaria blood stage that can be deployed in areas of varying transmission intensity.