Search Results (12)

Background/Objectives: Malaria, caused by infection with Plasmodium parasites, exacts a heavy toll worldwide. There are two licensed vaccines for malaria as well as two monoclonal antibodies that have shown promising efficacy in field trials. The vaccines and monoclonal antibodies target the major surface protein (circumsporozoite protein, CSP) of Plasmodium falciparum. Yet P. falciparum is only one of the four major species of Plasmodium that infect humans. Plasmodium vivax is the second leading cause of malaria, but the P. vivax vaccine and monoclonal development lags far behind that for P. falciparum owing to the lack of basic preclinical tools such as in vitro culture or mouse models that replicate the key biological features of P. vivax. Notably among these features is the ability to form dormant liver stages (hypnozoites) that reactivate and drive the majority of the P. vivax malaria burden. Plasmodium cynomolgi is a simian parasite which is genotypically very close and phenotypically similar to P. vivax; it can infect non-human primates commonly used in research and replicates many features of P. vivax, including relapsing hypnozoites. Methods: Recently, a strain of P. cynomolgi has been adapted to in vitro cultures allowing parasite transgenesis. Here, we created a transgenic P. cynomolgi parasite in which the endogenous P. cynomolgi CSP has been replaced with P. vivax CSP, with the goal of enabling the preclinical study of anti-P. vivax CSP interventions to protect against primary and relapse infections. Results: We show that the in vitro-generated transgenic Pcy[PvCSP] parasite expresses both serotypes of P. vivax CSP and retains full functionality in vivo, including the ability to transmit to laboratory-reared Anopheles mosquitoes and cause relapsing infections in rhesus macaques. To our knowledge, this is the first gene replacement in a relapsing Plasmodium species. Conclusions: This work can directly enable the in vivo development of anti-P. vivax CSP interventions and provide a blueprint for the study of relapsing malaria through reverse genetics. Full article

Background: Noroviruses, which cause epidemic acute gastroenteritis, and Plasmodium parasites, which lead to malaria, are two infectious pathogens that pose threats to public health. The protruding (P) domain of norovirus VP1 and the αTSR domain of the circumsporozoite protein (CSP) of Plasmodium sporozoite are the glycan receptor-binding domains of the two pathogens for host cell attachment, making them excellent targets for vaccine development. Modified norovirus P domains self-assemble into a 24-meric octahedral P nanoparticle (P₂₄ NP). Methods: We generated a unique P₂₄-αTSR NP by inserting the αTSR domain into a surface loop of the P domain. The P-αTSR fusion proteins were produced in the Escherichia coli expression system and the fusion protein self-assembled into the P₂₄-αTSR NP. Results: The formation of the P₂₄-αTSR NP was demonstrated through gel filtration, electron microscopy, and dynamic light scattering. A 3D structural model of the P₂₄-αTSR NP was constructed, using the known cryo-EM structure of the previously developed P₂₄ NP and P₂₄-VP8* NP as templates. Each P₂₄-αTSR NP consists of a P₂₄ NP core, with 24 surface-exposed αTSR domains that have retained their general conformations and binding function to heparan sulfate proteoglycans. The P₂₄-αTSR NP is immunogenic, eliciting strong antibody responses in mice toward both the norovirus P domain and the αTSR domain of Plasmodium CSP. Notably, sera from mice immunized with the P₂₄-αTSR NP bound strongly to Plasmodium sporozoites and blocked norovirus VLP attachment to their glycan receptors. Conclusion: These data suggest that the P₂₄-αTSR NP may serve as a combination vaccine against both norovirus and Plasmodium parasites. Full article

The genetic diversity within the circumsporozoite surface protein (PvCSP) of Plasmodium vivax, the predominant malaria species in Thailand, is primarily observed in the northwestern region along the Thailand–Myanmar border. However, as P. vivax cases shift to southern provinces, particularly Yala Province near the Thailand–Malaysia border, PvCSP diversity remains understudied. Between 2018 and 2020, 89 P. vivax isolates were collected in Yala Province, a significant malaria hotspot. Employing polymerase chain reaction amplification, restriction fragment length polymorphism (PCR-RFLP), and DNA sequencing, the gene encoding PvCSP (Pvcsp) was analyzed. All Yala P. vivax isolates belonged to the VK210 type, distinct from strains in the western region near the Myanmar border. The central repeat region of Pvcsp revealed two common peptide repeat motifs—GDRADGQPA and GDRAAGQPA—across all southern isolates. Sequence analysis identified two subtypes, with S1 more prevalent (92%) than S2 (8%). This study underscores the limited diversity of VK210 variants of P. vivax populations in southern Thailand. These baseline findings facilitate monitoring for potential new parasite variants, aiding in the future control and management of P. vivax in the region. Full article

Efficacy data on two malaria vaccines, RTS,S and R21, targeting Plasmodium falciparum circumsporozoite protein (PfCSP), are encouraging. Efficacy may be improved by induction of additional antibodies to neutralizing epitopes outside of the central immunodominant repeat domain of PfCSP. We designed four rPfCSP-based vaccines in an effort to improve the diversity of the antibody response. We also evaluated P. falciparum merozoite surface protein 8 (PfMSP8) as a malaria-specific carrier protein as an alternative to hepatitis B surface antigen. We measured the magnitude, specificity, subclass, avidity, durability, and efficacy of vaccine-induced antibodies in outbred CD1 mice. In comparison to N-terminal- or C-terminal-focused constructs, immunization with near full-length vaccines, rPfCSP (#1) or the chimeric rPfCSP/8 (#2), markedly increased the breadth of B cell epitopes recognized covering the N-terminal domain, junctional region, and central repeat. Both rPfCSP (#1) and rPfCSP/8 (#2) also elicited a high proportion of antibodies to conformation-dependent epitopes in the C-terminus of PfCSP. Fusion of PfCSP to PfMSP8 shifted the specificity of the T cell response away from PfCSP toward PfMSP8 epitopes. Challenge studies with transgenic Plasmodium yoelii sporozoites expressing PfCSP demonstrated high and consistent sterile protection following rPfCSP/8 (#2) immunization. Of note, antibodies to conformational C-terminal epitopes were not required for protection. These results indicate that inclusion of the N-terminal domain of PfCSP can drive responses to protective, repeat, and non-repeat B cell epitopes and that PfMSP8 is an effective carrier for induction of high-titer, durable anti-PfCSP antibodies. Full article

Background: malaria caused by Plasmodium parasites remains a public health threat. The circumsporozoite proteins (CSPs) of Plasmodium sporozoite play a key role in Plasmodium infection, serving as an excellent vaccine target. Methods: using a self-assembled S₆₀ nanoparticle platform, we generated pseudovirus nanoparticles (PVNPs) displaying CSPs, named S-CSPs, for enhanced immunogenicity. Results: purified Hisx6-tagged or tag-free S-CSPs self-assembled into PVNPs that consist of a norovirus S₆₀ inner shell and multiple surface-displayed CSPs. The majority of the PVNPs measured ~27 nm with some size variations, and their three-dimensional structure was modeled. The PVNP-displayed CSPs retained their glycan receptor-binding function. A mouse immunization study showed that PVNPs induced a high antibody response against CSP antigens and the PVNP-immunized mouse sera stained the CSPs of Plasmodium sporozoites at high titer. Conclusions and discussion: the PVNP-displayed CSPs retain their authentic antigenic feature and receptor-binding function. The CSP-specific antibody elicited by the S-CSP PVNPs binds original CSPs and potentially inhibits the attachment of Plasmodium sporozoites to their host cells, a key step for liver invasion by the sporozoites. Thus, S-CSP PVNPs may be an excellent vaccine candidate against malaria caused by Plasmodium parasites. Full article

High levels of genetic variants of Plasmodium vivax have previously been reported in Thailand. Circumsporozoite surface protein (CSP), merozoite surface protein (MSP), and microsatellite markers were used to determine the genetic polymorphisms of P. vivax. This study aimed to investigate the molecular epidemiology of P. vivax populations at the Thai–Myanmar border by genotyping the PvCSP, PvMSP-3α, and PvMSP-3β genes. Four hundred and forty P. vivax clinical isolates were collected from the Mae Sot and Sai Yok districts from 2006–2007 and 2014–2016. Polymerase chain reaction with restriction fragment length polymorphism (RFLP) was used to investigate the genetic polymorphisms of the target genes. Based on PCR band size variations, 14 different PvCSP alleles were identified: eight for VK210 and six for VK247. The VK210 genotype was the dominant variant during both sample collection periods. Based on PCR genotyping, three distinct types (A, B, and C) for both PvMSP-3α and PvMSP-3β were observed. Following RFLP, 28 and 14 allelic variants of PvMSP-3α and 36 and 20 allelic variants of PvMSP-3β with varying frequencies were identified during the first and second periods, respectively. High genetic variants of PvMSP-3 and PvCSP were found in the study area. PvMSP-3β exhibited a higher level of genetic diversity and multiple-genotype infection versus PvMSP-3α. Full article

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. Full article

The circumsporozoite surface protein of Plasmodium vivax (PvCSP) plays a critical role in parasite biology. It has been extensively studied as a leading vivax-malaria-vaccine candidate. In this study, the genetic polymorphism and natural selection of pvcsp in P. vivax isolates collected from the Central Highlands, Vietnam were analyzed to understand the genetic structure of the parasite circulating in the endemic area and to provide baseline information for effective vaccine development based on the protein. Only two major alleles, VK210 and VK247, were detected in Vietnamese pvcsp, with VK247 being the predominant one. The N-terminal and C-terminal regions of Vietnamese VK210 and VK247 variants showed a low genetic diversity. Amino acid substitutions, insertions of a single amino acid or octapeptide (ANKKAEDA in VK210 and ANKKAGDA in VK247), and tetrapeptide repeat motifs (GGNA) were the main factors generating genetic diversity in the two regions of the Vietnamese VK210 and VK247 variants. Interestingly, these two regions of Vietnamese pvcsp displayed a unique natural selection pressure distinct from global pvcsp, particularly with the neighboring Southeast Asian pvcsp population. Meanwhile, the central repeat region (CRR) in both the VK210 and VK247 variants showed a high degree of polymorphic characters, caused by varying numbers, types, and combinations of peptide repeat motifs (PRMs) in Vietnamese pvcsp. Highly complicated polymorphic patterns of the CRR were also detected in global pvcsp. These results expand our understanding of the genetic structure of Vietnamese pvcsp and the population dynamics of P. vivax in the Central Highlands, Vietnam. Full article

Failure to account for genetic diversity of antigens during vaccine design may lead to vaccine escape. To evaluate the vaccine escape potential of antigens used in vaccines currently in development or clinical testing, we surveyed the genetic diversity, measured population differentiation, and performed in silico prediction and analysis of T-cell epitopes of ten such Plasmodium falciparum pre-erythrocytic-stage antigens using whole-genome sequence data from 1010 field isolates. Of these, 699 were collected in Africa (Burkina Faso, Cameroon, Guinea, Kenya, Malawi, Mali, and Tanzania), 69 in South America (Brazil, Colombia, French Guiana, and Peru), 59 in Oceania (Papua New Guinea), and 183 in Asia (Cambodia, Myanmar, and Thailand). Antigens surveyed include cell-traversal protein for ookinetes and sporozoites, circumsporozoite protein, liver-stage antigens 1 and 3, sporozoite surface proteins P36 and P52, sporozoite asparagine-rich protein-1, sporozoite microneme protein essential for cell traversal-2, and upregulated-in-infectious-sporozoite 3 and 4 proteins. The analyses showed that a limited number of these protein variants, when combined, would be representative of worldwide parasite populations. Moreover, predicted T-cell epitopes were identified that could be further explored for immunogenicity and protective efficacy. Findings can inform the rational design of a multivalent malaria vaccine. Full article

The sole currently approved malaria vaccine targets the circumsporozoite protein—the protein that densely coats the surface of sporozoites, the parasite stage deposited in the skin of the mammalian host by infected mosquitoes. However, this vaccine only confers moderate protection against clinical diseases in children, impelling a continuous search for novel candidates. In this work, we studied the importance of the membrane-associated erythrocyte binding-like protein (MAEBL) for infection by Plasmodium sporozoites. Using transgenic parasites and live imaging in mice, we show that the absence of MAEBL reduces Plasmodium berghei hemolymph sporozoite infectivity to mice. Moreover, we found that maebl knockout (maebl-) sporozoites display reduced adhesion, including to cultured hepatocytes, which could contribute to the defects in multiple biological processes, such as in gliding motility, hepatocyte wounding, and invasion. The maebl- defective phenotypes in mosquito salivary gland and liver infection were reverted by genetic complementation. Using a parasite line expressing a C-terminal myc-tagged MAEBL, we found that MAEBL levels peak in midgut and hemolymph parasites but drop after sporozoite entry into the salivary glands, where the labeling was found to be heterogeneous among sporozoites. MAEBL was found associated, not only with micronemes, but also with the surface of mature sporozoites. Overall, our data provide further insight into the role of MAEBL in sporozoite infectivity and may contribute to the design of future immune interventions. Full article

Recently, short synthetic peptides have gained interest as targeting agents in the design of site-specific nanomedicines. In this context, our work aimed at developing new tools for the diagnosis and/or therapy of hepatocellular carcinoma (HCC) by grafting the hepatotropic George Baker (GB) virus A (GBVA10-9) and Plasmodium circumsporozoite protein (CPB)-derived peptides to the biocompatible poly(benzyl malate), PMLABe. We successfully synthesized PMLABe derivatives end-functionalized with peptides GBVA10-9, CPB, and their corresponding scrambled peptides through a thiol/maleimide reaction. The corresponding nanoparticles (NPs), varying by the nature of the peptide (GBVA10-9, CPB, and their scrambled peptides) and the absence or presence of poly(ethylene glycol) were also successfully formulated using nanoprecipitation technique. NPs were further characterized by dynamic light scattering (DLS), electrophoretic light scattering (ELS) and transmission electron microscopy (TEM), highlighting a diameter lower than 150 nm, a negative surface charge, and a more or less spherical shape. Moreover, a fluorescent probe (DiD Oil) has been encapsulated during the nanoprecipitation process. Finally, preliminary in vitro internalisation assays using HepaRG hepatoma cells demonstrated that CPB peptide-functionalized PMLABe NPs were efficiently internalized by endocytosis, and that such nanoobjects may be promising drug delivery systems for the theranostics of HCC. Full article

Malaria is a mosquito-borne infectious disease caused by the parasite Plasmodium spp. Malaria continues to have a devastating impact on human health. Sporozoites are the infective forms of the parasite inside mosquito salivary glands. Circumsporozoite protein (CSP) is a major and immunodominant protective antigen on the surface of Plasmodium sporozoites. Here, we report a generation of specific monoclonal antibodies that recognize the central repeat and C-terminal regions of P. falciparum CSP. The monoclonal antibodies 3C1, 3C2, and 3D3—specific for the central repeat region—have higher titers and protective efficacies against challenge with sporozoites compared with 2A10, a gold standard monoclonal antibody that was generated in early 1980s. Full article