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Transmission-blocking vaccines interrupting malaria transmission within mosquitoes represent an ideal public health tool to eliminate malaria at the population level. Plasmodium falciparum and P. vivax account for more than 90% of the global malaria burden, co-endemic in many regions of the world. P25 and P48/45 are two leading candidates for both species and have shown promising transmission-blocking activity in preclinical and clinical studies. However, neither of these target antigens as individual vaccines has induced complete transmission inhibition in mosquitoes. In this study, we assessed immunogenicity of combination vaccines based on P25 and P48/45 using a DNA vaccine platform to broaden vaccine specificity against P. falciparum and P. vivax. Individual DNA vaccines encoding Pvs25, Pfs25, Pvs48/45 and Pfs48/45, as well as various combinations including (Pvs25 + Pvs48/45), (Pfs25 + Pfs48/45), (Pvs25 + Pfs25), and (Pvs48/45 + Pfs48/45), were evaluated in mice using in vivo electroporation. Potent antibody responses were induced in mice immunized with individual and combination DNA vaccines, and specific antibody responses were not compromised when combinations of DNA vaccines were evaluated against individual DNA vaccines. The anti-Pvs25 IgG from individual and combination groups revealed concentration-dependent transmission-reducing activity (TRA) in direct membrane feeding assays (DMFA) using blood from P. vivax-infected donors in Brazil and independently in ex vivo MFA using Pvs25-transgenic P. berghei. Similarly, anti-Pfs25 and anti-Pfs48/45 IgGs from mice immunized with Pfs25 and Pfs48/45 DNA vaccines individually and in various combinations revealed antibody dose-dependent TRA in standard membrane feeding assays (SMFA) using culture-derived P. falciparum gametocytes. However, antibodies induced by immunization with Pvs48/45 DNA vaccines were ineffective in DMFA and require further vaccine construct optimization, considering the possibility of induction of both transmission-blocking and transmission-enhancing antibodies revealed by competition ELISA. These studies provide a rationale for combining multiple antigens to simultaneously target transmission of malaria caused by P. falciparum and P. vivax., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Apical membrane antigen-1 (AMA1) is a conserved malarial vaccine candidate essential for the formation of tight junctions with the rhoptry neck protein (RON) complex, enabling Plasmodium parasites to invade human erythrocytes, hepatocytes, and mosquito salivary glands. Despite its critical role, extensive surface polymorphisms in AMA1 have led to strain-specific protection, limiting the success of AMA1-based interventions beyond initial clinical trials. Here, we identify an i-body, a humanised single-domain antibody-like molecule that recognises a conserved pan-species conformational epitope in AMA1 with low nanomolar affinity and inhibits the binding of the RON2 ligand to AMA1. Structural characterisation indicates that the WD34 i-body epitope spans the centre of the conserved hydrophobic cleft in AMA1, where interacting residues are highly conserved among all Plasmodium species. Furthermore, we show that WD34 inhibits merozoite invasion of erythrocytes by multiple Plasmodium species and hepatocyte invasion by P. falciparum sporozoites. Despite a short half-life in mouse serum, we demonstrate that WD34 transiently suppressed P. berghei infections in female BALB/c mice. Our work describes the first pan-species AMA1 biologic with inhibitory activity against multiple life-cycle stages of Plasmodium. With improved pharmacokinetic characteristics, WD34 could be a potential immunotherapy against multiple species of Plasmodium., (© 2024. The Author(s).)

Background: A highly effective vaccine for malaria remains an elusive target, at least in part due to the under-appreciated natural parasite variation. This study aimed to investigate genetic and structural variation, and immune selection of leading malaria vaccine candidates across the Plasmodium falciparum's life cycle., Methods: We analysed 325 P. falciparum whole genome sequences from Zambia, in addition to 791 genomes from five other African countries available in the MalariaGEN Pf3k Database. Ten vaccine antigens spanning three life-history stages were examined for genetic and structural variations, using population genetics measures, haplotype network analysis, and 3D structure selection analysis., Findings: Among the ten antigens analysed, only three in the transmission-blocking vaccine category display P. falciparum 3D7 as the dominant haplotype. The antigens AMA1, CSP, MSP1 19 and CelTOS, are much more diverse than the other antigens, and their epitope regions are under moderate to strong balancing selection. In contrast, Rh5, a blood stage antigen, displays low diversity yet slightly stronger immune selection in the merozoite-blocking epitope region. Except for CelTOS, the transmission-blocking antigens Pfs25, Pfs48/45, Pfs230, Pfs47, and Pfs28 exhibit minimal diversity and no immune selection in epitopes that induce strain-transcending antibodies, suggesting potential effectiveness of 3D7-based vaccines in blocking transmission., Interpretation: These findings offer valuable insights into the selection of optimal vaccine candidates against P. falciparum. Based on our results, we recommend prioritising conserved merozoite antigens and transmission-blocking antigens. Combining these antigens in multi-stage approaches may be particularly promising for malaria vaccine development initiatives., Funding: Purdue Department of Biological Sciences; Puskas Memorial Fellowship; National Institute of Allergy and Infectious Diseases (U19AI089680)., Competing Interests: Declaration of interests All authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

The Duffy-binding protein (DBP) is a promising antigen for a malaria vaccine that would protect against clinical symptoms caused by Plasmodium vivax infection. Region II of DBP (DBP-II) contains the receptor-binding domain that engages host red blood cells, but DBP-II vaccines elicit many non-neutralizing antibodies that bind distal to the receptor-binding surface. Here, we engineered a truncated DBP-II immunogen that focuses the immune response to the receptor-binding surface. This immunogen contains the receptor-binding subdomain S1S2 and lacks the immunodominant subdomain S3. Structure-based computational design of S1S2 identified combinatorial amino acid changes that stabilized the isolated S1S2 without perturbing neutralizing epitopes. This immunogen elicited DBP-II-specific antibodies in immunized mice that were significantly enriched for blocking activity compared to the native DBP-II antigen. This generalizable design process successfully stabilized an integral core fragment of a protein and focused the immune response to desired epitopes to create a promising new antigen for malaria vaccine development., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Cryptocaryoniasis, caused by Cryptocaryon irritans, is a significant threat to marine fish cultures in tropical and subtropical waters. However, controlling this disease remains a challenge. Fish infected with C. irritans acquires immunity; however, C. irritans is difficult to culture in large quantities, obstructing vaccine development using parasite cells. In this study, we established a method for expressing an arbitrary protein on the surface of Tetrahymena thermophila, a culturable ciliate, to develop a mimetic C. irritans. Fusing the signal peptide (SP) and glycosylphosphatidylinositol (GPI) anchor sequences of the immobilization antigen, a surface protein of C. irritans, to the fluorescent protein, monomeric Azami-green 1 (mAG1) of the stony coral Galaxea fascicularis, allowed protein expression on the surface and cilia of transgenic Tetrahymena cells. This technique may help develop transgenic Tetrahymena displaying parasite antigens on their cell surface, potentially contributing to the development of vaccines using "mimetic parasites"., (© 2023. The Author(s).)

The sequestration of Plasmodium falciparum-infected erythrocytes to the host endothelium is central to the pathogenesis of malaria. The sequestration is mediated by the parasite´s diverse Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variants, which bind select human receptors on the endothelium. Severe malaria is associated with PfEMP1 binding human endothelial protein C receptor (EPCR) via their CIDRα1 domains. Antibodies binding and inhibiting across the sequence diverse CIDRα1 domains are likely important in acquired immunity against severe malaria. In this study, we explored if immunization with AP205 bacteriophage capsid-virus-like particles (cVLPs) presenting a mosaic of diverse CIDRα1 protein variants would stimulate broadly reactive and inhibitory antibody responses in mice. Three different mosaic cVLP vaccines each composed of five CIDRα1 protein variants with varying degrees of sequence conservation of residues at and near the EPCR binding site, were tested. All mosaic cVLP vaccines induced functional antibodies comparable to those induced by matched cocktails of cVLPs decorated with the single CIDRα1 variant. No broadly reactive responses were observed. However, the vaccines did induce some cross-reactivity and inhibition within the CIDRα1 subclasses included in the vaccines, demonstrating potential use of the cVLP vaccine platform for the design of multivalent vaccines., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: C. F. and A. F. S. are employees of AdaptVac, a company commercializing virus-like particle display technology and vaccine, including several patents. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Riedmiller et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis, with clinical outcomes ranging from asymptomatic infections to severe invasive diseases. The innate immune system, particularly macrophages, is of paramount importance in resisting the invasion of host tissues and organs by the trophozoites of E. histolytica . Parasite-derived pathogenic factors, such as lectins, play a pivotal role in the promotion of macrophage polarization phenotypes that have undergone alteration. Nevertheless, the precise mechanisms by which E. histolytica modulates immune polarization remain largely unknown. The current study focused on the immunomodulatory effects of the Igl-C fragment of E. histolytica Gal/GalNAc lectin on macrophage polarization. These results demonstrated that Igl-C could induce the secretion of IL-1β, IL-6, and other cytokines, activating a mixed M1/M2 polarization state. M1 polarization of macrophages occurs in the early stages and gradually transitions to M2 polarization in the later stages, which may contribute to the persistence of the infection. Igl-C induces the macrophage M1 phenotype and causes the release of immune effector molecules, including iNOS and cytokines, by activating the NF-κB p65 and JAK-STAT1 transcription factor signaling pathways. Furthermore, Igl-C supports the macrophage M2 phenotype via JAK-STAT3 and IL-4-STAT6 pathways, which activate arginase expression in later stages, contributing to the tissue regeneration and persistence of the parasite. The involvement of distinct signaling pathways in mediating this response highlights the complex interplay between the parasite and the host immune system. These findings enhance our understanding of the Igl-C-mediated pathogenic mechanisms during E. histolytica infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dong, Zhang, Li, Zhang, Cheng and Feng.)

Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is a leading blood-stage malaria vaccine antigen target, currently in a phase 2b clinical trial as a full-length soluble protein/adjuvant vaccine candidate called RH5.1/Matrix-M. We identify that disordered regions of the full-length RH5 molecule induce non-growth inhibitory antibodies in human vaccinees and that a re-engineered and stabilized immunogen (including just the alpha-helical core of RH5) induces a qualitatively superior growth inhibitory antibody response in rats vaccinated with this protein formulated in Matrix-M adjuvant. In parallel, bioconjugation of this immunogen, termed "RH5.2," to hepatitis B surface antigen virus-like particles (VLPs) using the "plug-and-display" SpyTag-SpyCatcher platform technology also enables superior quantitative antibody immunogenicity over soluble protein/adjuvant in vaccinated mice and rats. These studies identify a blood-stage malaria vaccine candidate that may improve upon the current leading soluble protein vaccine candidate RH5.1/Matrix-M. The RH5.2-VLP/Matrix-M vaccine candidate is now under evaluation in phase 1a/b clinical trials., Competing Interests: Declaration of interests S.J.D. is an inventor on patent applications relating to RH5 malaria vaccines and antibodies, is a co-founder of and shareholder in SpyBiotech, and has been a consultant to GSK on malaria vaccines. A.M.M. has been a consultant to GSK on malaria vaccines, has an immediate family member who is an inventor on patent applications relating to RH5 malaria vaccines and antibodies, and is a co-founder of and shareholder in SpyBiotech. M.R.H. is an inventor on patents relating to peptide targeting via spontaneous amide bond formation and is a co-founder of and shareholder in SpyBiotech. S.B. is an inventor on patent applications relating to vaccines made using spontaneous amide bond formation and is a co-founder of, shareholder in, and employee of SpyBiotech. J.J. is an inventor on patent applications relating to vaccines made using spontaneous amide bond formation and is a co-founder of and shareholder in SpyBiotech. R.A.D. is an inventor on patent applications relating to vaccines made using spontaneous amide bond formation and shareholder in SpyBiotech. L.D.W.K., J.R.B., D.Q., A.M.L., S.E.S., B.G.W., K. McHugh, I.C., S.J.F., and D.P. are inventors on patent applications relating to RH5 malaria vaccines and/or antibodies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Strain-transcending antibodies against virulence-associated subsets of P. falciparum -infected erythrocyte surface antigens could protect children from severe malaria. However, the evidence supporting the existence of such antibodies is incomplete and inconsistent. One subset of surface antigens associated with severe malaria, rosette-mediating Plasmodium falciparum Erythrocyte Membrane Protein one (PfEMP1) variants, cause infected erythrocytes to bind to uninfected erythrocytes to form clusters of cells (rosettes) that contribute to microvascular obstruction and pathology. Here, we tested plasma from 80 individuals living in malaria-endemic regions for IgG recognition of the surface of four P. falciparum rosetting strains using flow cytometry. Broadly reactive plasma samples were then used in antibody elution experiments in which intact IgG was eluted from the surface of infected erythrocytes and transferred to heterologous rosetting strains to look for strain-transcending antibodies. We found that seroprevalence (percentage of positive plasma samples) against allopatric rosetting strains was high in adults (63%-93%) but lower in children (13%-48%). Strain-transcending antibodies were present in nine out of eleven eluted antibody experiments, with six of these recognizing multiple heterologous rosetting parasite strains. One eluate had rosette-disrupting activity against heterologous strains, suggesting PfEMP1 as the likely target of the strain-transcending antibodies. Naturally acquired strain-transcending antibodies to rosetting P. falciparum strains in humans have not been directly demonstrated previously. Their existence suggests that such antibodies could play a role in clinical protection and raises the possibility that conserved epitopes recognized by strain-transcending antibodies could be targeted therapeutically by monoclonal antibodies or vaccines., Competing Interests: The authors declare no conflict of interest.

Trypanosoma brucei is the causal agent of African Trypanosomiasis in humans and other animals. It maintains a long-term infection through an antigenic variation based population survival strategy. To proliferate in a mammal, T. brucei acquires iron and haem through the receptor mediated uptake of host transferrin and haptoglobin-hemoglobin respectively. The receptors are exposed to host antibodies but this does not lead to clearance of the infection. Here we discuss how the trypanosome avoids this fate in the context of recent findings on the structure and cell biology of the receptors., (© 2024 The Authors. BioEssays published by Wiley Periodicals LLC.)

Our developed cell division-specific 'centrin' gene deleted Leishmania donovani (LdCen1 -/- ) the causative parasite of the fatal visceral-leishmaniasis (VL), exhibits a selective growth arrest at the intracellular stage and is anticipated as a live attenuated vaccine candidate against VL. LdCen1 -/- immunization in animals has shown increased IFN-γ secreting CD4+ and CD8+ T cells along with protection conferred by a protective proinflammatory immune response. A label-free proteomics approach has been employed to understand the physiology of infection and predict disease interceptors during Leishmania-host interactions. Proteomic modulation after infection of human macrophage cell lines suggested elevated annexin A6, implying involvement in various biological processes such as membrane repair, transport, actin dynamics, cell proliferation, survival, differentiation, and inflammation, thereby potentiating its immunological protective capacity. Additionally, S100A8 and S100A9 proteins, known for maintaining homeostatic balance in regulating the inflammatory response, have been upregulated after infection. The inhibitory clade of serpins, known to inhibit cysteine proteases (CPs), was upregulated in host cells after 48 h of infection. This is reflected in the diminished expression of CPs in the parasites during infection. Such proteome analysis confirms LdCen1 -/- efficacy as a vaccine candidate and predicts potential markers in future vaccine development strategies against infectious diseases., Competing Interests: Declaration of competing interest None of the authors has any conflict of interest to disclose., (Copyright © 2024 Institut Pasteur. All rights reserved.)

Malaria remains a public health challenge. Since many control strategies have proven ineffective in eradicating this disease, new strategies are required, among which the design of a multivalent vaccine stands out. However, the effectiveness of this strategy has been hindered, among other reasons, by the genetic diversity observed in parasite antigens. In Plasmodium vivax, the Erythrocyte Binding Protein (PvEBP, also known as DBP2) is an alternate ligand to Duffy Binding Protein (DBP); given its structural resemblance to DBP, EBP/DBP2 is proposed as a promising antigen for inclusion in vaccine design. However, the extent of genetic diversity within the locus encoding this protein has not been comprehensively assessed. Thus, this study aimed to characterize the genetic diversity of the locus encoding the P. vivax EBP/DBP2 protein and to determine the evolutionary mechanisms modulating this diversity. Several intrapopulation genetic variation parameters were estimated using 36 gene sequences of PvEBP/DBP2 from Colombian P. vivax clinical isolates and 186 sequences available in databases. The study then evaluated the worldwide genetic structure and the evolutionary forces that may influence the observed patterns of genetic variation. It was found that the PvEBP/DBP2 gene exhibits one of the lowest levels of genetic diversity compared to other vaccine-candidate antigens. Four major haplotypes were shared worldwide. Analysis of the protein's 3D structure and epitope prediction identified five regions with potential antigenic properties. The results suggest that the PvEBP/DBP2 protein possesses ideal characteristics to be considered when designing a multivalent effective antimalarial vaccine against P. vivax., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Chicken coccidiosis is a protozoan disease that leads to considerable economic losses in the poultry industry. Live oocyst vaccination is currently the most effective measure for the prevention of coccidiosis. However, it provides limited protection with several drawbacks, such as poor immunological protection and potential reversion to virulence. Therefore, the development of effective and safe vaccines against chicken coccidiosis is still urgently needed., Methods: In this study, a novel oral vaccine against Eimeria tenella was developed by constructing a recombinant Lactobacillus plantarum (NC8) strain expressing the E. tenella RON2 protein. We administered recombinant L. plantarum orally at 3, 4 and 5 days of age and again at 17, 18 and 19 days of age. Meanwhile, each chick in the commercial vaccine group was immunized with 3 × 10 2 live oocysts of coccidia. A total of 5 × 10 4 sporulated oocysts of E. tenella were inoculated in each chicken at 30 days. Then, the immunoprotection effect was evaluated after E. tenella infection., Results: The results showed that the proportion of CD4 + and CD8 + T cells, the proliferative ability of spleen lymphocytes, inflammatory cytokine levels and specific antibody titers of chicks immunized with recombinant L. plantarum were significantly increased (P < 0.05). The relative body weight gains were increased and the number of oocysts per gram (OPG) was decreased after E. tenella challenge. Moreover, the lesion scores and histopathological cecum sections showed that recombinant L. plantarum can significantly relieve pathological damage in the cecum. The ACI was 170.89 in the recombinant L. plantarum group, which was higher than the 150.14 in the commercial vaccine group., Conclusions: These above results indicate that L. plantarum expressing RON2 improved humoral and cellular immunity and enhanced immunoprotection against E. tenella. The protective efficacy was superior to that of vaccination with the commercial live oocyst vaccine. This study suggests that recombinant L. plantarum expressing the RON2 protein provides a promising strategy for vaccine development against coccidiosis., (© 2024. The Author(s).)

Centrin1 gene deleted Leishmania donovani parasite (LdCen1 -/- ) was developed and extensively tested experimentally as an intracellular stage-specific attenuated and immunoprotective live parasite vaccine candidate ex vivo using human PBMCs and in vivo in animals. Here we report manufacturing and pre-clinical evaluation of current Good-Laboratory Practice (cGLP) grade LdCen1 -/- parasites, as a prerequisite before proceeding with clinical trials. We screened three batches of LdCen1 -/- parasites manufactured in bioreactors under cGLP conditions, for their consistency in genetic stability, attenuation, and safety. One such batch was preclinically tested using human PBMCs and animals (hamsters and dogs) for its safety and protective immunogenicity. The immunogenicity of the CGLP grade LdCen1 -/- parasites was similar to one grown under laboratory conditions. The cGLP grade LdCen1 -/- parasites were found to be safe and non-toxic in hamsters and dogs even at 3 times the anticipated vaccine dose. When PBMCs from healed visceral leishmaniasis (VL) cases were infected with cGLP LdCen1 -/- , there was a significant increase in the stimulation of cytokines that contribute to protective responses against VL. This effect, measured by multiplex ELISA, was greater than that observed in PBMCs from healthy individuals. These results suggest that cGLP grade LdCen1 -/- manufactured under cGMP complaint conditions can be suitable for future clinical trials., (© 2024. The Author(s).)

In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice's splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Matos, Soares, Rodrigues-da-Silva, Rodolphi, Albrecht, Donassolo, Lopez-Camacho, Ano Bom, Neves, Conte, Pratt-Riccio, Daniel-Ribeiro, Totino and Lima-Junior.)

Resistance to clinical malaria takes years to develop even in hyperendemic regions and sterilizing immunity has rarely been observed. To evaluate the maturation of the host response against controlled repeat exposures to P. falciparum (Pf) NF54 strain-infected mosquitoes, we systematically monitored malaria-naïve participants through an initial exposure to uninfected mosquitoes and 4 subsequent homologous exposures to Pf-infected mosquitoes over 21 months (n = 8 males) (ClinicalTrials.gov# NCT03014258). The primary outcome was to determine whether protective immunity against parasite infection develops following repeat CHMI and the secondary outcomes were to track the clinical signs and symptoms of malaria and anti-Pf antibody development following repeat CHMI. After two exposures, time to blood stage patency increases significantly and the number of reported symptoms decreases indicating the development of clinical tolerance. The time to patency correlates positively with both anti-Pf circumsporozoite protein (CSP) IgG and CD8 + CD69+ effector memory T cell levels consistent with partial pre-erythrocytic immunity. IFNγ levels decrease significantly during the participants' second exposure to high blood stage parasitemia and could contribute to the decrease in symptoms. In contrast, CD4-CD8 + T cells expressing CXCR5 and the inhibitory receptor, PD-1, increase significantly after subsequent Pf exposures, possibly dampening the memory response and interfering with the generation of robust sterilizing immunity., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Malaria is preventable yet causes >600 000 deaths annually. RTS,S, the first marketed malaria vaccine, has modest efficacy, but improvements are needed for eradication., Methods: We conducted an open-label, dose escalation phase 1 study of a full-length recombinant circumsporozoite protein vaccine (rCSP) administered with adjuvant glucopyranosyl lipid A-liposome Quillaja saponaria 21 formulation (GLA-LSQ) on days 1, 29, and 85 or 1 and 490 to healthy, malaria-naive adults. The primary end points were safety and reactogenicity. The secondary end points were antibody responses and Plasmodium falciparum parasitemia after homologous controlled human malaria infection., Results: Participants were enrolled into 4 groups receiving rCSP/GLA-LSQ: 10 µg × 3 (n = 20), 30 µg × 3 (n = 10), 60 µg × 3 (n = 10), or 60 µg × 2 (n = 9); 10 participants received 30 µg rCSP alone × 3, and there were 6 infectivity controls. Participants experienced no serious adverse events. Rates of solicited and unsolicited adverse events were similar among groups. All 26 participants who underwent controlled human malaria infection 28 days after final vaccinations developed malaria. Increasing vaccine doses induced higher immunoglobulin G titers but did not achieve previously established RTS,S benchmarks., Conclusions: rCSP/GLA-LSQ had favorable safety results. However, tested regimens did not induce protective immunity. Further investigation could assess whether adjuvant or schedule adjustments improve efficacy., Clinical Trials Registration: NCT03589794., Competing Interests: Potential conflicts of interest . All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: To prepare and characterize the mouse polyclonal antibody against the dense granule protein 24 (GRA24) of Toxoplasma gondii , and explore its preliminary applications., Methods: The GRA24 coding sequences of different T. gondii strains were aligned using the MEGA-X software, and the dominant peptide of the GRA24 protein was analyzed with the Protean software. The base sequence encoding this peptide was amplified using PCR assay and ligated into the pET-28a vector, and the generated GRA24 truncated protein was transformed into Escherichia coli BL21. After induction by isopropyl-beta-D-thiogalactopyranoside (IPTG), the expression and purification of the recombinant GRA24 protein was analyzed using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). BALB/c mice were immunized by subcutaneous injection with the purified recombinant GRA24 truncated protein to generate the polyclonal antibody, and the titer of the polyclonal antibody was measured using enzyme linked immunosorbent assay (ELISA). The specificity of the polyclonal antibody was tested using Western blotting, and the intracellular localization of the polyclonal antibody was investigated using immunofluorescence assay (IFA)., Results: SDS-PAGE showed successful construction of the recombinant expression plasmid, and Coomassie brilliant blue staining showed the generation of the high-purity recombinant GRA24 truncated protein. ELISA measured that the titer of the polyclonal antibody against the GRA24 truncated protein was higher than 1:208 400, and Western blotting showed that the polyclonal antibody was effective to recognize the endogenous GRA24 proteins of different T. gondii strains and specifically recognize the recombinant GRA24 truncated protein. Indirect IFA showed that the GRA24 protein secreted 16 hour following T. gondii invasion in host cells., Conclusions: The polyclonal antibody against the T. gondii GRA24 protein has been successfully prepared, which has a widespread applicability, high titers and a high specificity. This polyclonal antibody is available for Western blotting and IFA, which provides the basis for investigating the function of the GRA24 protein.

SUMMARYMalaria remains one of the biggest health problems in the world. While significant reductions in malaria morbidity and mortality had been achieved from 2000 to 2015, the favorable trend has stalled, rather significant increases in malaria cases are seen in multiple areas. In 2022, there were 249 million estimated cases, and 608,000 malaria-related deaths, mostly in infants and children aged under 5 years, globally. Therefore, in addition to the expansion of existing anti-malarial control measures, it is critical to develop new tools, such as vaccines and monoclonal antibodies (mAbs), to fight malaria. In the last 2 years, the first and second malaria vaccines, both targeting Plasmodium falciparum circumsporozoite proteins (PfCSP), have been recommended by the World Health Organization to prevent P. falciparum malaria in children living in moderate to high transmission areas. While the approval of the two malaria vaccines is a considerable milestone in vaccine development, they have much room for improvement in efficacy and durability. In addition to the two approved vaccines, recent clinical trials with mAbs against PfCSP, blood-stage vaccines against P. falciparum or P. vivax , and transmission-blocking vaccine or mAb against P. falciparum have shown promising results. This review summarizes the development of the anti-PfCSP vaccines and mAbs, and recent topics in the blood- and transmission-blocking-stage vaccine candidates and mAbs. We further discuss issues of the current vaccines and the directions for the development of next-generation vaccines., Competing Interests: The authors declare no conflict of interest.

Background: Malaria transmission-blocking vaccines (TBVs) aim to inhibit malaria parasite development in mosquitoes and prevent further transmission to the human host. The putative-secreted ookinete protein 25 (PSOP25), highly conserved in Plasmodium spp., is a promising TBV target. Here, we investigated PvPSOP25 from P. vivax as a TBV candidate using transgenic murine parasite P. berghei and clinical P. vivax isolates., Methods and Findings: A transgenic P. berghei line expressing PvPSOP25 (TrPvPSOP25Pb) was generated. Full-length PvPSOP25 was expressed in the yeast Pichia pastoris and used to immunize mice to obtain anti-rPvPSOP25 sera. The transmission-blocking activity of the anti-rPvPSOP25 sera was evaluated through in vitro assays and mosquito-feeding experiments. The antisera generated by immunization with rPvPSOP25 specifically recognized the native PvPSOP25 antigen expressed in TrPvPSOP25Pb ookinetes. In vitro assays showed that the immune sera significantly inhibited exflagellation and ookinete formation of the TrPvPSOP25Pb parasite. Mosquitoes feeding on mice infected with the transgenic parasite and passively transferred with the anti-rPvPSOP25 sera showed a 70.7% reduction in oocyst density compared to the control group. In a direct membrane feeding assay conducted with five clinical P. vivax isolates, the mouse anti-rPvPSOP25 antibodies significantly reduced the oocyst density while showing a negligible influence on mosquito infection prevalence., Conclusions: This study supported the feasibility of transgenic murine malaria parasites expressing P. vivax antigens as a useful tool for evaluating P. vivax TBV candidates. Meanwhile, the moderate transmission-reducing activity of the generated anti-rPvPSOP25 sera necessitates further research to optimize its efficacy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Recent data indicate increasing disease burden and importance of Plasmodium vivax (Pv) malaria. A robust assay will be essential for blood-stage Pv vaccine development. Results of the in vitro growth inhibition assay (GIA) with transgenic P. knowlesi (Pk) parasites expressing the Pv Duffy-binding protein region II (PvDBPII) correlate with in vivo protection in the first PvDBPII controlled human malaria infection (CHMI) trials, making the PkGIA an ideal selection tool once the precision of the assay is defined. To determine the precision in percentage of inhibition in GIA (%GIA) and in GIA 50 (antibody concentration that gave 50 %GIA), ten GIAs with transgenic Pk parasites were conducted with four different anti-PvDBPII human monoclonal antibodies (mAbs) at concentrations of 0.016 to 2 mg/mL, and three GIAs with eighty anti-PvDBPII human polyclonal antibodies (pAbs) at 10 mg/mL. A significant assay-to-assay variation was observed, and the analysis revealed a standard deviation (SD) of 13.1 in the mAb and 5.94 in the pAb dataset for %GIA, with a LogGIA 50 SD of 0.299 (for mAbs). Moreover, the ninety-five percent confidence interval (95 %CI) for %GIA or GIA 50 in repeat assays was calculated in this investigation. The error range determined in this study will help researchers to compare PkGIA results from different assays and studies appropriately, thus supporting the development of future blood-stage malaria vaccine candidates, specifically second-generation PvDBPII-based formulations., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Chetan E. Chitnis is an inventor on patents that relate to binding domains of erythrocyte-binding proteins of Plasmodium parasites including PvDBP (patent no. 6962987; binding domains from Plasmodium vivax and Plasmodium falciparum erythrocyte binding proteins)., (Copyright © 2024. Published by Elsevier Ltd.)

Introduction: Trypanosoma cruzi is a protozoan parasite that causes the tropical ailment known as Chagas disease, which has its origins in South America. Globally, it has a major impact on health and is transported by insect vector that serves as a parasite. Given the scarcity of vaccines and the limited treatment choices, we conducted a comprehensive investigation of core proteomics to explore a potential reverse vaccine candidate with high antigenicity., Methods: To identify the immunodominant epitopes, T. cruzi core proteomics was initially explored. Consequently, the vaccine sequence was engineered to possess characteristics of non-allergenicity, antigenicity, immunogenicity, and enhanced solubility. After modeling the tertiary structure of the human TLR4 receptor, the binding affinities were assessed employing molecular docking and molecular dynamics simulations (MDS)., Results: Docking of the final vaccine design with TLR4 receptors revealed substantial hydrogen bond interactions. A server-based methodology for immunological simulation was developed to forecast the effectiveness against antibodies (IgM + IgG) and interferons (IFN-g). The MDS analysis revealed notable levels of structural compactness and binding stability with average RMSD of 5.03 Aring;, beta-factor 1.09e+5 Å, Rg is 44.7 Aring; and RMSF of 49.50 Aring;. This is followed by binding free energies calculation. The system stability was compromised by the complexes, as evidenced by their corresponding Gibbs free energies of -54.6 kcal/mol., Discussion: Subtractive proteomics approach was applied to determine the antigenic regions of the T cruzi. Our study utilized computational techniques to identify B- and T-cell epitopes in the T. cruzi core proteome. In current study the developed vaccine candidate exhibits immunodominant features. Our findings suggest that formulating a vaccine targeting the causative agent of Chagas disease should be the initial step in its development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Albaqami, Altharawi, Althurwi and Alharthy.)

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric "RCR-complex". We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called "R78C", combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial., (© 2024. The Author(s).)

Introduction: Toxoplasma gondii is an intracellular parasite of importance to human and veterinary health. The structure and diversity of the genotype population of T. gondii varies considerably with respect to geography, but three lineages, type I, II and III, are distributed globally. Lineage III genotypes are the least well characterized in terms of biology, host immunity and virulence. Once a host is infected with T.gondii , innate immune mechanisms are engaged to reduce the parasite burden in tissues and create a pro-inflammatory environment in which the T H 1 response develops to ensure survival. This study investigated the early cellular immune response of Swiss-Webster mice post intraperitoneal infection with 10 tachyzoites of four distinct non-clonal genotypes of lineage III and a local isolate of ToxoDB#1. The virulence phenotype, cumulative mortality (CM) and allele profiles of ROP5, ROP16, ROP18 and GRA15 were published previously., Methods: Parasite dissemination in different tissues was analyzed by real-time PCR and relative expression levels of IFNγ, IL12-p40, IL-10 and TBX21 in the cervical lymph nodes (CLN), brain and spleen were calculated using the ΔΔCt method. Stage conversion was determined by detection of the BAG1 transcript in the brain., Results: Tissue dissemination depends on the virulence phenotype but not CM, while the TBX21 and cytokine levels and kinetics correlate better with CM than virulence phenotype. The earliest detection of BAG1 was seven days post infection. Only infection with the genotype of high CM (69.4%) was associated with high T-bet levels in the CLN 24 h and high systemic IFNγ expression which was sustained over the first week, while infection with genotypes of lower CM (38.8%, 10.7% and 6.8%) is characterized by down-regulation and/or low systemic levels of IFNγ. The response intensity, as assessed by cytokine levels, to the genotype of high CM wanes over time, while it increases gradually to genotypes of lower CM., Discussion: The results point to the conclusion that the immune response is not correlated with the virulence phenotype and/or allele profile, but an early onset, intense pro-inflammatory response is characteristic of genotypes with high CM. Additionally, high IFNγ level in the brain may hamper stage conversion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Uzelac, Klun and Djurković-Djaković.)

The Plasmodium secreted protein with an altered thrombospondin repeat (SPATR) has been known to play an important role in the malaria parasite's invasion into host erythrocytes. This protein is immunogenic and has been considered as one of the potential vaccine candidates against malaria parasite infection. Thus far, only a handful immunological studies have been carried out on P. knowlesi SPATR (PkSPATR), and none of these studies investigated the immunoprotective properties of the protein. In the present study, the ability of anti-PkSPATR antibodies to inhibit invasion of human erythrocytes was assessed in an in vitro merozoite invasion inhibition assay. The antibodies were harvested from the serum of a rabbit which was immunised with recombinat PkSPATR. Results from the merozoite invasion inhibition assay revealed significant antibody invasion inhibitory activity in a concentration dependent manner (concentration range: 0.375 - 3.00 mg/ml) with inhibition rate ranging from 20% to 32%. Future studies, such as anti-PkSPATR antibodies inhibitory effect on sporozoite invasion of human liver cells, need to be carried out to assess the potential of PkSPATR as a knowlesi malaria vaccine candidate.

Chagas disease-caused by the parasite Trypanosoma cruzi-is a neglected tropical disease for which available drugs are not fully effective in the chronic stage and a vaccine is not available yet. Microalgae represent a promising platform for the production and oral delivery of low-cost vaccines. Herein, we report a vaccine prototype against T. cruzi produced in a microalgae platform, based on the candidate antigen Tc24 with a C terminus fusion with the Co1 peptide (Tc24:Co1 vaccine prototype). After modeling the tertiary structure, in silico studies suggested that the chimeric protein is antigenic, not allergenic, and molecular docking indicated binding with Toll-like receptors 2 and 4. Thus, Tc24:Co1 was expressed in the marine microalga Schizochytrium sp., and Western blot confirmed the expression at 48 h after induction, with a yield of 632 µg/L of algal culture (300 μg/g of lyophilized algal cells) as measured by the enzyme-linked immunosorbent assay (ELISA). Upon oral administration of whole-cell Schizochytrium sp. expressing Tc24:Co1 (7.5 µg or 15 µg of Tc24:Co1 doses) in mice, specific serum IgG and intestinal mucosa IgA responses were detected in addition to an increase in serum Th1/Th2 cytokines. In conclusion, Schizochytrium sp.-expressing Tc24:Co1 is a promising oral vaccine prototype to be evaluated in an animal model of Trypanosoma cruzi infection., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cryptosporidium causes debilitating diarrheal disease in patients with primary and acquired defects in T cell function. However, it has been a challenge to understand how this infection generates T cell responses and how they mediate parasite control. Here, Cryptosporidium was engineered to express a parasite effector protein (MEDLE-2) that contains the major histocompatibility complex-I restricted SIINFEKL epitope which is recognized by T cell receptor transgenic OT-I(OVA-TCR-I) clusters of differentiation (CD)8 + T cells. These modified parasites induced expansion of endogenous SIINFEKL-specific and OT-I CD8 + T cells that were a source of interferon-gamma (IFN-γ) that could restrict growth of Cryptosporidium. This T cell response was dependent on the translocation of the effector and similar results were observed with another secreted parasite effector (rhoptry protein 1). Although infection and these translocated effector proteins are restricted to intestinal epithelial cells, type 1 conventional dendritic cells were required to generate CD8 + T cell responses to these model antigens. These data sets highlight Cryptosporidium effectors as potential targets of the immune system and suggest that crosstalk between enterocytes and type 1 conventional dendritic cells is crucial for CD8 + T cell responses to Cryptosporidium., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Visceral Leishmaniasis, caused by Leishmania infantum, is a tropical neglected disease and the most dangerous form of Leishmaniasis. It occurs zoonotically, with domestic transmission posing risks to humans as dogs have high susceptibility and are natural reservoirs of the parasite. Given their epidemiological role, improvements are needed in diagnosing Canine Visceral Leishmaniasis (CVL). Thus, we mapped linear epitopes from the rLiNTPDase2 antigen through peptide microarray and identified six positive epitopes. Validation through peptide ELISA revealed three promising peptides with accuracies of 78.6%, 85.92%, and 79.59%. Their combination yielded 97.58% accuracy. Negative epitopes were also found, which interacted with CVL-negative and Chagas Disease positive samples. Their removal from the rLiNTPDase2 sequence resulted in the rNT2.neg, which obtained enhanced specificity over rLiNTPDase2. The rNT2.neg validation achieved 87.50% sensitivity, 90.55% specificity, and 93.5% accuracy within 127 CVL-positive and 96 CVL-negative samples. Therefore, three peptides and rNT2.neg show significant promise for CVL diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Toxoplasma gondii is an important protozoan pathogen, which can cause severe diseases in the newborns and immunocompromised individuals. Developing an effective vaccine against Toxoplasma infection is a critically important global health priority. Immunofluorescence staining analysis revealed that TgSAG2 and TgSRS2 are membrane associated and displayed on the surface of the parasite. Immunizations with pBud-SAG2, pBud-SRS2 and pBud-SAG2-SRS2 DNA vaccines significantly increased the production of specific IgG antibodies. Immunization with pBud-SAG2-SRS2 elicited cellular immune response with higher concentrations of IFN-γ and IL-4 compared to the control group. Antigen-specific lymphocyte proliferations in the pBud-SRS2 and pBud-SAG2-SRS2 groups were significantly higher compared to that in the control group. Furthermore, 30 % of mice immunized with pBud-SAG2-SRS2 survived after the challenge infection with virulent T. gondii RH tachyzoites. This study revealed that immunization with pBud-SAG2-SRS2 induced potent immune responses, and has the potential as a promising vaccine candidate for the control of T. gondii infection., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Neosporosis is the major infectious cause of abortion and reproductive losses in cattle worldwide; however, there are no available vaccines or drugs to control this disease. Recently, a dual (positive and negative) DIVA-like (Differentiation of Infected from Vaccinated Animals) vaccine was evaluated in a pregnant mouse model of neosporosis, showing promising immunogenic and protective results. The current report aimed to study the safety, the dose-dependent immunogenicity and the dual DIVA-like character of a recombinant subunit vaccine composed of the major surface antigen from Neospora caninum (rNcSAG1) and the carrier/adjuvant Heat shock protein 81.2 from Arabidopsis thaliana (rAtHsp81.2) in cattle. Healthy heifers were separated and assigned to experimental groups A-F and subcutaneously immunized with 2 doses of vaccine formulations 30 days apart as follows: A (n = 4): 50 μg rNcSAG1 + 150 μg rAtHsp81.2; B (n = 4): 200 μg rNcSAG1 + 600 μg rAtHsp81.2; C (n = 4): 500 μg rNcSAG1 + 1,500 μg rAtHsp81.2; D (n = 3): 150 μg rAtHsp81.2; E (n = 3):1,500 μg rAtHsp81.2, and F (n = 3) 2 ml of sterile PBS. The immunization of heifers with the different vaccine or adjuvant doses (groups A-E) was demonstrated to be safe and did not modify the mean value of the evaluated serum biomarkers of metabolic function (GOT/ASP, GPT/ALT, UREA, Glucose and total proteins). The kinetics and magnitude of the immune responses were dose-dependent. The higher dose of the vaccine formulation (group C) stimulated a broad and potent humoral and cellular immune response, characterized by an IgG1/IgG2 isotype profile and IFN-γ secretion. In addition, this was the first time that dual DIVA-like character of a vaccine against neosporosis was demonstrated, allowing us to differentiate vaccinated from infected heifers by two different DIVA compliant test approaches. These results encourage us to evaluate its protective efficacy in infected pregnant cattle in the future., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Toxoplasma gondii is an intracellular protozoan parasite that infects all nucleated cells except the red blood cells. Currently, nucleic acid vaccines are being widely investigated in Toxoplasma gondii control, and several nucleic acid vaccine candidate antigens have shown good protection in various studies. The aim of this study was to construct a nucleic acid vaccine with Toxoplasma gondii SRS29C as the target gene. We explored the nucleic acid vaccine with Toxoplasma surface protein SRS29C and the combined gene of SRS29C and SAG1 and evaluated its immunoprotective effect against Toxoplasma gondii. To amplify the gene fragment and clone it to the expression vector, the recombinant plasmid pEGFP-SRS29C was constructed by PCR. Eukaryotic cells were transfected with the plasmid, and the expression of the target protein was assessed using the Western blot method. The level of serum IgG was determined via ELISA, and the splenic lymphocyte proliferation ability was detected using the CCK-8 method. The percentages of CD4 + and CD8 + T cells were measured by flow cytometry. Mice were immunised three times with single-gene nucleic acid vaccine and combination vaccine. Splenic lymphocytokine expression was determined using ELISA kits. The mice's survival time was monitored and recorded during an in vivo insect assault experiment, and the vaccine's protective power was assessed. The outcomes showed that PCR-amplification of an SRS29C gene fragment was successful. The 4,733-bp vector fragment and the 1,119-bp target segment were both recognised by double digestion. Additionally, after transfection of the recombinant plasmid pEGFP-SRS29C, Western blot examination of the extracted protein revealed the presence of a target protein strip at 66 kDa. The test results demonstrated that the IgG content in the serum of the pEGFP-SRS29C group and the co-immunization group was significantly higher than that of the PBS group and the empty vector group. The IgG potency induced by the co-immunization group was higher than that of the pEGFP-SRS29C group and the pEGFP-SAG1 group, the number of splenic lymphocyte proliferation number was higher than that of the PBS group and the empty vector group. The CD4 + /CD8 + T ratio was higher than that of the PBS group and the empty vector group. The expression of IFN-γ and TNF-α in the splenocytes of the pEGFP-SRS29C group and the combined immunisation group was significantly higher following antigen stimulation. In the worm attack experiments, mice in the PBS and empty vector groups perished within 9 days of the worm attack, whereas mice in the pEGFP-SRS29C group survived for 18 days, mice in the pEGFP-SAG1 group survived for 21 days, and mice in the co-immunization group survived for 24 days. This demonstrates that the constructed Toxoplasma gondii nucleic acid vaccine pEGFP-SRS29C and the combined gene vaccine can induce mice to develop certain humoral and cellular immune responses, and enhance their ability to resist Toxoplasma gondii infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Conflicts of interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Despite decades of effort, Plasmodium falciparum malaria remains a leading killer of children. The absence of a highly effective vaccine and the emergence of parasites resistant to both diagnosis as well as treatment hamper effective public health interventions., Methods and Results: To discover new vaccine candidates, we used our whole proteome differential screening method and identified PfGBP130 as a parasite protein uniquely recognized by antibodies from children who had developed resistance to P. falciparum infection but not from those who remained susceptible. We formulated PfGBP130 as lipid encapsulated mRNA, DNA plasmid, and recombinant protein-based immunogens and evaluated the efficacy of murine polyclonal anti-PfGBP130 antisera to inhibit parasite growth in vitro. Immunization of mice with PfGBP130-A (aa 111-374), the region identified in our differential screen, formulated as a DNA plasmid or lipid encapsulated mRNA, but not as a recombinant protein, induced antibodies that inhibited RBC invasion in vitro . mRNA encoding the full ectodomain of PfGBP130 (aa 89-824) also generated parasite growth-inhibitory antibodies., Conclusion: We are currently advancing PfGBP130-A formulated as a lipid-encapsulated mRNA for efficacy evaluation in non-human primates., Competing Interests: Author JK is a scientific co-founder of Ocean Biomedical which seeks to develop malaria vaccines. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Johnson, Shakri, Pond-Tor, Jnawali, Najrana, Wu, Badhai, Alameh, Weissman, Kabyemela, Duffy, Fried, Kurtis and Raj.)

Background: The spleen plays a critical role in the immune response against malaria parasite infection, where splenic fibroblasts (SFs) are abundantly present and contribute to immune function by secreting type I collagen (collagen I). The protein family is characterized by Plasmodium vivax tryptophan-rich antigens (PvTRAgs), comprising 40 members. PvTRAg23 has been reported to bind to human SFs (HSFs) and affect collagen I levels. Given the role of type I collagen in splenic immune function, it is important to investigate the functions of the other members within the PvTRAg protein family., Methods: Protein structural prediction was conducted utilizing bioinformatics analysis tools and software. A total of 23 PvTRAgs were successfully expressed and purified using an Escherichia coli prokaryotic expression system, and the purified proteins were used for co-culture with HSFs. The collagen I levels and collagen-related signaling pathway protein levels were detected by immunoblotting, and the relative expression levels of inflammatory factors were determined by quantitative real-time PCR., Results: In silico analysis showed that P. vivax has 40 genes encoding the TRAg family. The C-terminal region of all PvTRAgs is characterized by the presence of a domain rich in tryptophan residues. A total of 23 recombinant PvTRAgs were successfully expressed and purified. Only five PvTRAgs (PvTRAg5, PvTRAg16, PvTRAg23, PvTRAg30, and PvTRAg32) mediated the activation of the NF-κBp65 signaling pathway, which resulted in the production of inflammatory molecules and ultimately a significant reduction in collagen I levels in HSFs., Conclusions: Our research contributes to the expansion of knowledge regarding the functional role of PvTRAgs, while it also enhances our understanding of the immune evasion mechanisms utilized by parasites., (© 2024. The Author(s).)

Background: Plasmodium vivax represents the most geographically widespread human malaria parasite affecting civilian and military populations in endemic areas. Targeting the pre-erythrocytic (PE) stage of the parasite life cycle is especially appealing for developing P. vivax vaccines as it would prevent disease and transmission. Here, naturally acquired immunity to a panel of P. vivax PE antigens was explored, which may facilitate vaccine development and lead to a better understanding of naturally acquired PE immunity., Methods: Twelve P. vivax PE antigens orthologous to a panel of P. falciparum antigens previously identified as highly immunogenic in protected subjects after immunization with radiation attenuated sporozoites (RAS) were used for evaluation of humoral and cellular immunity by ELISA and IFN-γ ELISpot. Samples from P. vivax infected individuals (n = 76) from a low endemic malaria region in the Peruvian Amazon Basin were used., Results: In those clinical samples, all PE antigens evaluated showed positive IgG antibody reactivity with a variable prevalence of 58-99% in recently P. vivax diagnosed patients. The magnitude of the IgG antibody response against PE antigens was lower compared with blood stage antigens MSP1 and DBP-II, although antibody levels persisted better for PE antigens (average decrease of 6% for PE antigens and 43% for MSP1, p < 0.05). Higher IgG antibodies was associated with one or more previous malaria episodes only for blood stage antigens (p < 0.001). High IgG responders across PE and blood stage antigens showed significantly lower parasitaemia compared to low IgG responders (median 1,921 vs 4,663 par/µl, p < 0.05). In a subgroup of volunteers (n = 17),positive IFN-γ T cell response by ELISPOT was observed in 35% vs 9-35% against blood stage MSP1 and PE antigens, respectively, but no correlation with IgG responses., Conclusions: These results demonstrate clear humoral and T cell responses against P. vivax PE antigens in individuals naturally infected with P. vivax. These data identify novel attractive PE antigens suitable for use in the potential development and selection of new malaria vaccine candidates which can be used as a part of malaria prevention strategies in civilian and military populations living in P. vivax endemic areas., (© 2024. The Author(s).)

Plasmodium falciparum glutamic acid-rich protein (PfGARP) is a recently characterized cell surface antigen encoded by Plasmodium falciparum, the causative agent of severe human malaria pathophysiology. Previously, we reported that the human erythrocyte band 3 (SLC4A1) serves as a host receptor for PfGARP. Antibodies against PfGARP did not affect parasite invasion and growth. We surmised that PfGARP may play a role in the rosetting and adhesion of malaria. Another study reported that antibodies targeting PfGARP exhibit potent inhibition of parasite growth. This inhibition occurred without the presence of any immune or complement components, suggesting the activation of an inherent density-dependent regulatory system. Here, we used polyclonal antibodies against PfGARP and a monoclonal antibody mAb7899 to demonstrate that anti-PfGARP polyclonal antibodies, but not mAb7899, exerted potent inhibition of parasite growth in infected erythrocytes independent of PfGARP. These findings suggest that an unknown malaria protein(s) is the target of growth arrest by polyclonal antibodies raised against PfGARP., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Diversity in malarial antigens is an immune evasion mechanism that gives malaria parasites an edge over the host. Immune responses against one variant of a polymorphic antigen are usually not fully effective against other variants due to altered epitopes. This study aimed to evaluate diversity in the Plasmodium falciparum antigens apical membrane antigen 1 (PfAMA1) and circumsporozoite protein (PfCSP) from circulating parasites in a malaria-endemic community in southern Ghana and to determine the effects of polymorphisms on antibody response specificity., Methods: The study involved 300 subjects, whose P. falciparum infection status was determined by microscopy and PCR. Diversity within the two antigens was evaluated by msp2 gene typing and molecular gene sequencing, while the host plasma levels of antibodies against PfAMA1, PfCSP, and two synthetic 24mer peptides from the conserved central repeat region of PfCSP, were measured by ELISA., Results: Of the 300 subjects, 171 (57%) had P. falciparum infection, with 165 of the 171 (96.5%) being positive for either or both of the msp2 allelic families. Gene sequencing of DNA from 55 clonally infected samples identified a total of 56 non-synonymous single nucleotide polymorphisms (SNPs) for the Pfama1 gene and these resulted in 44 polymorphic positions, including two novel positions (363 and 365). Sequencing of the Pfcsp gene from 69 clonal DNA samples identified 50 non-synonymous SNPs that resulted in 42 polymorphic positions, with half (21) of these polymorphic positions being novel. Of the measured antibodies, only anti-PfCSP antibodies varied considerably between PCR parasite-positive and parasite-negative persons., Discussion: These data confirm the presence of a considerable amount of unique, previously unreported amino acid changes, especially within PfCSP. Drivers for this diversity in the Pfcsp gene do not immediately seem apparent, as immune pressure will be expected to drive a similar level of diversity in the Pfama1 gene., Competing Interests: MB, HG and JH was employed by Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Kusi, Amoah, Acquah, Ennuson, Frempong, Ofori, Akyea-Mensah, Kyei-Baafour, Osei, Frimpong, Singh, Theisen, Remarque, Faber, Belmonte, Ganeshan, Huang, Villasante and Sedegah.)

Visceral leishmaniasis is a deadly infectious disease and is one of the world's major neglected health problems. Because the symptoms of infection are similar to other endemic diseases, accurate diagnosis is crucial for appropriate treatment. Definitive diagnosis using splenic or bone marrow aspirates is highly invasive, and so, serological assays are preferred, including the direct agglutination test (DAT) or rK39 strip test. These tests, however, are either difficult to perform in the field (DAT) or lack specificity in some endemic regions (rK39), making the development of new tests a research priority. The availability of Leishmania spp. genomes presents an opportunity to identify new diagnostic targets. Here, we use genome data and a mammalian protein expression system to create a panel of 93 proteins consisting of the extracellular ectodomains of the Leishmania donovani cell surface and secreted proteins. We use these panel and sera from murine experimental infection models and natural human and canine infections to identify new candidates for serological diagnosis. We observed a concordance between the most immunoreactive antigens in different host species and transmission settings. The antigen encoded by the LdBPK&#95;323600.1 gene can diagnose Leishmania infections with high sensitivity and specificity in patient cohorts from different endemic regions including Bangladesh and Ethiopia. In longitudinal sampling of treated patients, we observed reductions in immunoreactivity to LdBPK&#95;323600.1 suggesting it could be used to diagnose treatment success. In summary, we have identified new antigens that could contribute to improved serological diagnostic tests to help control the impact of this deadly tropical infectious disease., Importance: Visceral leishmaniasis is fatal if left untreated with patients often displaying mild and non-specific symptoms during the early stages of infection making accurate diagnosis important. Current methods for diagnosis require highly trained medical staff to perform highly invasive biopsies of the liver or bone marrow which pose risks to the patient. Less invasive molecular tests are available but can suffer from regional variations in their ability to accurately diagnose an infection. To identify new diagnostic markers of visceral leishmaniasis, we produced and tested a panel of 93 proteins identified from the genome of the parasite responsible for this disease. We found that the pattern of host antibody reactivity to these proteins was broadly consistent across naturally acquired infections in both human patients and dogs, as well as experimental rodent infections. We identified a new protein called LdBPK&#95;323600.1 that could accurately diagnose visceral leishmaniasis infections in humans., Competing Interests: The authors declare no conflict of interest.

The Plasmodium falciparum merozoite surface protein MSPDBL2 is a polymorphic antigen targeted by acquired immune responses, and normally expressed in only a minority of mature schizonts. The potential relationship of MSPDBL2 to sexual commitment is examined, as variable mspdbl2 transcript levels and proportions of MSPDBL2-positive mature schizonts in clinical isolates have previously correlated with levels of many sexual stage parasite gene transcripts, although not with the master regulator ap2-g . It is demonstrated that conditional overexpression of the gametocyte development protein GDV1, which promotes sexual commitment, also substantially increases the proportion of MSPDBL2-positive schizonts in culture. Conversely, truncation of the gdv1 gene is shown to prevent any expression of MSPDBL2. However, across diverse P. falciparum cultured lines, the variable proportions of MSPDBL2 positivity in schizonts do not correlate significantly with variable gametocyte conversion rates, indicating it is not involved in sexual commitment. Confirming this, examining a line with endogenous hemagglutinin-tagged AP2-G showed that the individual schizonts expressing MSPDBL2 are mostly different from those expressing AP2-G. Using a selection-linked integration system, modified P. falciparum lines were engineered to express an intact or disrupted version of MSPDBL2, showing the protein is not required for sexual commitment or early gametocyte development. Asexual parasite multiplication rates were also not affected by expression of either intact or disrupted MSPDBL2 in a majority of schizonts. Occurring alongside sexual commitment, the role of the discrete MSPDBL2-positive schizont subpopulation requires further investigation in natural infections where it is under immune selection., Importance: Malaria parasites in the blood are remarkably variable, able to switch antigenic targets so they may survive within humans who have already developed specific immune responses. This is one of the challenges in developing vaccines against malaria. MSPDBL2 is a target of naturally acquired immunity expressed in minority proportions of schizonts, the end stages of each 2-day replication cycle in red blood cells which contain merozoites prepared to invade new red blood cells. Results show that the proportion of schizonts expressing MSPDBL2 is positively controlled by the expression of the regulatory gametocyte development protein GDV1. It was previously known that expression of GDV1 leads to increased expression of AP2-G which causes parasites to switch to sexual development, so a surprising finding here is that MSPDBL2-positive parasites are mostly distinct from those that express AP2-G. This discrete antigenic subpopulation of mostly asexual parasites is regulated alongside sexually committed parasites, potentially enabling survival under stress conditions., Competing Interests: The authors declare no conflict of interest.

Background: The R21/Matrix-M vaccine has demonstrated high efficacy against Plasmodium falciparum clinical malaria in children in sub-Saharan Africa. Using trial data, we aimed to estimate the public health impact and cost-effectiveness of vaccine introduction across sub-Saharan Africa., Methods: We fitted a semi-mechanistic model of the relationship between anti-circumsporozoite protein antibody titres and vaccine efficacy to data from 3 years of follow-up in the phase 2b trial of R21/Matrix-M in Nanoro, Burkina Faso. We validated the model by comparing predicted vaccine efficacy to that observed over 12-18 months in the phase 3 trial. Integrating this framework within a mathematical transmission model, we estimated the cases, malaria deaths, and disability-adjusted life-years (DALYs) averted and cost-effectiveness over a 15-year time horizon across a range of transmission settings in sub-Saharan Africa. Cost-effectiveness was estimated incorporating the cost of vaccine introduction (dose, consumables, and delivery) relative to existing interventions at baseline. We report estimates at a median of 20% parasite prevalence in children aged 2-10 years (PfPR 2-10 ) and ranges from 3% to 65% PfPR 2-10 ., Findings: Anti-circumsporozoite protein antibody titres were found to satisfy the criteria for a surrogate of protection for vaccine efficacy against clinical malaria. Age-based implementation of a four-dose regimen of R21/Matrix-M vaccine was estimated to avert 181 825 (range 38 815-333 491) clinical cases per 100 000 fully vaccinated children in perennial settings and 202 017 (29 868-405 702) clinical cases per 100 000 fully vaccinated children in seasonal settings. Similar estimates were obtained for seasonal or hybrid implementation. Under an assumed vaccine dose price of US$3, the incremental cost per clinical case averted was $7 (range 4-48) in perennial settings and $6 (3-63) in seasonal settings and the incremental cost per DALY averted was $34 (29-139) in perennial settings and $30 (22-172) in seasonal settings, with lower cost-effectiveness ratios in settings with higher PfPR 2-10 ., Interpretation: Introduction of the R21/Matrix-M malaria vaccine could have a substantial public health benefit across sub-Saharan Africa., Funding: The Wellcome Trust, the Bill & Melinda Gates Foundation, the UK Medical Research Council, the European and Developing Countries Clinical Trials Partnership 2 and 3, the NIHR Oxford Biomedical Research Centre, and the Serum Institute of India, Open Philanthropy., Competing Interests: Declaration of interests AVSH and KJE are named as co-inventors on patent applications related to R21 and are entitled to a royalty share on any future income in conformity with the University of Oxford's policy. KJE was an employee of the University of Oxford at the time of the work and is now an employee of GSK. KJE holds restricted shares in the GSK group of companies. The University of Oxford has received funding from the Serum Institute of India to support funding of several African trials including the ongoing phase 3 trial of R21/Matrix-M. AVSH is chief investigator of these trials. PW has received funding from Gavi and the Wellcome Trust in the past 36 months. ACG has received funding from Gavi and NIH and consulting fees from the Global Fund in the past 36 months and is a trustee at Malaria No More UK. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Malaria is a global disease affecting a large portion of the world's population. Although vaccines have recently become available, their efficacies are suboptimal. We generated virus-like particles (VLPs) that expressed either apical membrane antigen 1 (AMA1) or microneme-associated antigen (MIC) of Plasmodium berghei and compared their efficacy in BALB/c mice. We found that immune sera acquired from AMA1 VLP- or MIC VLP-immunized mice specifically interacted with the antigen of choice and the whole P. berghei lysate antigen, indicating that the antibodies were highly parasite-specific. Both VLP vaccines significantly enhanced germinal center B cell frequencies in the inguinal lymph nodes of mice compared with the control, but only the mice that received MIC VLPs showed significantly enhanced CD4+ T cell responses in the blood following P. berghei challenge infection. AMA1 and MIC VLPs significantly suppressed TNF-α and interleukin-10 production but had a negligible effect on interferon-γ. Both VLPs prevented excessive parasitemia buildup in immunized mice, although parasite burden reduction induced by MIC VLPs was slightly more effective than that induced by AMA1. Both VLPs were equally effective at preventing body weight loss. Our findings demonstrated that the MIC VLP was an effective inducer of protection against murine experimental malaria and should be the focus of further development.

A systems analysis was conducted to determine the potential molecular mechanisms underlying differential immunogenicity and protective efficacy results of a clinical trial of the radiation-attenuated whole-sporozoite PfSPZ vaccine in African infants. Innate immune activation and myeloid signatures at prevaccination baseline correlated with protection from P. falciparum parasitemia in placebo controls. These same signatures were associated with susceptibility to parasitemia among infants who received the highest and most protective PfSPZ vaccine dose. Machine learning identified spliceosome, proteosome, and resting DC signatures as prevaccination features predictive of protection after highest-dose PfSPZ vaccination, whereas baseline circumsporozoite protein-specific (CSP-specific) IgG predicted nonprotection. Prevaccination innate inflammatory and myeloid signatures were associated with higher sporozoite-specific IgG Ab response but undetectable PfSPZ-specific CD8+ T cell responses after vaccination. Consistent with these human data, innate stimulation in vivo conferred protection against infection by sporozoite injection in malaria-naive mice while diminishing the CD8+ T cell response to radiation-attenuated sporozoites. These data suggest a dichotomous role of innate stimulation for malaria protection and induction of protective immunity by whole-sporozoite malaria vaccines. The uncoupling of vaccine-induced protective immunity achieved by Abs from more protective CD8+ T cell responses suggests that PfSPZ vaccine efficacy in malaria-endemic settings may be constrained by opposing antigen presentation pathways.

Among Plasmodium spp. responsible for human malaria, Plasmodium vivax ranks as the second most prevalent and has the widest geographical range; however, vaccine development has lagged behind that of Plasmodium falciparum , the deadliest Plasmodium species. Recently, we developed a multistage vaccine for P. falciparum based on a heterologous prime-boost immunization regimen utilizing the attenuated vaccinia virus strain LC16m8Δ (m8Δ)-prime and adeno-associated virus type 1 (AAV1)-boost, and demonstrated 100% protection and more than 95% transmission-blocking (TB) activity in the mouse model. In this study, we report the feasibility and versatility of this vaccine platform as a P. vivax multistage vaccine, which can provide 100% sterile protection against sporozoite challenge and >95% TB efficacy in the mouse model. Our vaccine comprises m8Δ and AAV1 viral vectors, both harboring the gene encoding two P. vivax circumsporozoite (PvCSP) protein alleles (VK210; PvCSP-Sal and VK247; -PNG) and P25 (Pvs25) expressed as a Pvs25-PvCSP fusion protein. For protective efficacy, the heterologous m8Δ-prime/AAV1-boost immunization regimen showed 100% (short-term; Day 28) and 60% (long-term; Day 242) protection against PvCSP VK210 transgenic Plasmodium berghei sporozoites. For TB efficacy, mouse sera immunized with the vaccine formulation showed >75% TB activity and >95% transmission reduction activity by a direct membrane feeding assay using P. vivax isolates in blood from an infected patient from the Brazilian Amazon region. These findings provide proof-of-concept that the m8Δ/AAV1 vaccine platform is sufficiently versatile for P. vivax vaccine development. Future studies are needed to evaluate the safety, immunogenicity, vaccine efficacy, and synergistic effects on protection and transmission blockade in a non-human primate model for Phase I trials., Competing Interests: Authors SY, HS, HM, and MI are credited as inventors of patents concerning viral-vectored malaria vaccines 2022-24221. HS is also credited as an inventor on a pending patent related to LC16m8Δ WO 2005/054451 A1. However, neither of these products has been brought to market. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yamamoto, Fabbri, Okuhara, Takagi, Kawabata, Katayama, Iyori, Hasyim, Sakamoto, Mizukami, Shida, Lopes and Yoshida.)

Malaria vaccine development is hampered by extensive antigenic variation and complex life stages of Plasmodium species. Vaccine development has focused on a small number of antigens, many of which were identified without utilizing systematic genome-level approaches. In this study, we implement a machine learning-based reverse vaccinology approach to predict potential new malaria vaccine candidate antigens. We assemble and analyze P. falciparum proteomic, structural, functional, immunological, genomic, and transcriptomic data, and use positive-unlabeled learning to predict potential antigens based on the properties of known antigens and remaining proteins. We prioritize candidate antigens based on model performance on reference antigens with different genetic diversity and quantify the protein properties that contribute most to identifying top candidates. Candidate antigens are characterized by gene essentiality, gene ontology, and gene expression in different life stages to inform future vaccine development. This approach provides a framework for identifying and prioritizing candidate vaccine antigens for a broad range of pathogens., (© 2024. The Author(s).)

Merozoite surface protein 3 of Plasmodium vivax (PvMSP3) contains a repertoire of protein members with unique sequence organization. While the biological functions of these proteins await elucidation, PvMSP3 has been suggested to be potential vaccine targets. To date, studies on natural immune responses to this protein family have been confined to two members, PvMSP3α and PvMSP3β. This study analyzed natural IgG antibody responses to PvMSP3γ recombinant proteins derived from two variants: one containing insert blocks (CT1230nF) and the other without insert domain (NR25nF). The former variant was also expressed as two subfragment proteins: one encompassing variable domain I and insert block A (CT1230N) and the other spanning from insert block B to conserved block III (CT1230C). Serum samples were obtained from 246 symptomatic vivax malaria patients in Tak (n = 50) and Ubon Ratchathani (n = 196) Provinces. In total, 176 (71.5%) patients could mount antibodies to at least one recombinant PvMSP3γ antigen. IgG antibodies directed against antigens CT1230nF, CT1230N, CT1230C and NR25nF occurred in 96.6%, 61.4%, 71.6% and 68.2% of samples, respectively, suggesting the widespread occurrence of B-cell epitopes across PvMSP3γ. The rates of seropositivity seemed to correlate with the number of previous malaria episodes. Isotype analysis of anti-PvMSP3γ antibodies has shown predominant cytophilic subclass responses, accounting for 75.4-81.7% for IgG1 and 63.6-77.5% for IgG3. Comparing with previous studies in the same cohort, the numbers of serum samples reactive to antigens derived from P. vivax merozoite surface protein 9 (PvMSP9) and thrombospondin-related anonymous protein (PvTRAP) were higher than those to PvMSP3γ, being 92.7% and 87.0% versus 71.5%, respectively. Three (1.22%) serum samples were nonresponsive to all these malarial proteins. Nevertheless, the relevance of naturally acquired antibodies to PvMSP3γ in host protection requires further studies., (© 2024. The Author(s).)

Introduction: Babesia bovis , a tick-borne apicomplexan parasite causing bovine babesiosis, remains a significant threat worldwide, and improved and practical vaccines are needed. Previous studies defined the members of the rhoptry associated protein-1 (RAP-1), and the neutralization-sensitive rhoptry associated protein-1 related antigen (RRA) superfamily in B. bovis , as strong candidates for the development of subunit vaccines. Both RAP-1 and RRA share conservation of a group of 4 cysteines and amino acids motifs at the amino terminal end (NT) of these proteins., Methods and Results: Sequence comparisons among the RRA sequences of several B. bovis strains and other Babesia spp parasites indicate a high level of conservation of a 15-amino acid (15-mer) motif located at the NT of the protein. BlastP searches indicate that the 15-mer motif is also present in adenylate cyclase, dynein, and other ATP binding proteins. AlphaFold2 structure predictions suggest partial exposure of the 15-mer on the surface of RRA of three distinct Babesia species. Antibodies in protected cattle recognize a synthetic peptide representing the 15-mer motif sequence in iELISA, and rabbit antibodies against the 15-mer react with the surface of free merozoites in immunofluorescence., Discussion and Conclusion: The presence of the 15-mer-like regions in dynein and ATP-binding proteins provides a rationale for investigating possible functional roles for RRA. The demonstrated presence of a surface exposed B-cell epitope in the 15-mer motif of the B. bovis RRA, which is recognized by sera from protected bovines, supports its inclusion in future subunit epitope-based vaccines against B. bovis ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Rojas, Bastos, Navas, Laughery, Lacy and Suarez.)

African trypanosomes replicate within infected mammals where they are exposed to the complement system. This system centres around complement C3, which is present in a soluble form in serum but becomes covalently deposited onto the surfaces of pathogens after proteolytic cleavage to C3b. Membrane-associated C3b triggers different complement-mediated effectors which promote pathogen clearance. To counter complement-mediated clearance, African trypanosomes have a cell surface receptor, ISG65, which binds to C3b and which decreases the rate of trypanosome clearance in an infection model. However, the mechanism by which ISG65 reduces C3b function has not been determined. We reveal through cryogenic electron microscopy that ISG65 has two distinct binding sites for C3b, only one of which is available in C3 and C3d. We show that ISG65 does not block the formation of C3b or the function of the C3 convertase which catalyses the surface deposition of C3b. However, we show that ISG65 forms a specific conjugate with C3b, perhaps acting as a decoy. ISG65 also occludes the binding sites for complement receptors 2 and 3, which may disrupt recruitment of immune cells, including B cells, phagocytes, and granulocytes. This suggests that ISG65 protects trypanosomes by combining multiple approaches to dampen the complement cascade., Competing Interests: AC, MC, MH No competing interests declared, (© 2023, Cook et al.)

Background: The stalling global progress in malaria control highlights the need for novel tools for malaria elimination, including transmission-blocking vaccines. Transmission-blocking vaccines aim to induce human antibodies that block parasite development in the mosquito and mosquitoes becoming infectious. The Pfs48/45 protein is a leading Plasmodium falciparum transmission-blocking vaccine candidate. The R0.6C fusion protein, consisting of Pfs48/45 domain 3 (6C) and the N-terminal region of P. falciparum glutamate-rich protein (R0), has previously been produced in Lactococcus lactis and elicited functional antibodies in rodents. Here, we assess the safety and transmission-reducing efficacy of R0.6C adsorbed to aluminium hydroxide with and without Matrix-M™ adjuvant in humans., Methods: In this first-in-human, open-label clinical trial, malaria-naïve adults, aged 18-55 years, were recruited at the Radboudumc in Nijmegen, the Netherlands. Participants received four intramuscular vaccinations on days 0, 28, 56 and 168 with either 30 µg or 100 µg of R0.6C and were randomised for the allocation of one of the two different adjuvant combinations: aluminium hydroxide alone, or aluminium hydroxide combined with Matrix-M1™ adjuvant. Adverse events were recorded from inclusion until 84 days after the fourth vaccination. Anti-R0.6C and anti-6C IgG titres were measured by enzyme-linked immunosorbent assay. Transmission-reducing activity of participants' serum and purified vaccine-specific immunoglobulin G was assessed by standard membrane feeding assays using laboratory-reared Anopheles stephensi mosquitoes and cultured P. falciparum gametocytes., Results: Thirty-one participants completed four vaccinations and were included in the analysis. Administration of all doses was safe and well-tolerated, with one related grade 3 adverse event (transient fever) and no serious adverse events occurring. Anti-R0.6C and anti-6C IgG titres were similar between the 30 and 100 µg R0.6C arms, but higher in Matrix-M1™ arms. Neat participant sera did not induce significant transmission-reducing activity in mosquito feeding experiments, but concentrated vaccine-specific IgGs purified from sera collected two weeks after the fourth vaccination achieved up to 99% transmission-reducing activity., Conclusions: R0.6C/aluminium hydroxide with or without Matrix-M1™ is safe, immunogenic and induces functional Pfs48/45-specific transmission-blocking antibodies, albeit at insufficient serum concentrations to result in transmission reduction by neat serum. Future work should focus on identifying alternative vaccine formulations or regimens that enhance functional antibody responses., Trial Registration: The trial is registered with ClinicalTrials.gov under identifier NCT04862416., (© 2024. The Author(s).)

This study presents an evaluation of seventeen newly produced recombinant trivalent chimeric proteins (containing the same immunodominant fragment of SAG1 and SAG2 of Toxoplasma gondii antigens, and an additional immunodominant fragment of one of the parasite antigens, such as AMA1, GRA1, GRA2, GRA5, GRA6, GRA7, GRA9, LDH2, MAG1, MIC1, MIC3, P35, and ROP1) as a potential alternative to the whole-cell tachyzoite lysate (TLA) used in the detection of infection in small ruminants. These recombinant proteins, obtained by genetic engineering and molecular biology methods, were tested for their reactivity with specific anti- Toxoplasma IgG antibodies contained in serum samples of small ruminants (192 samples of sheep serum and 95 samples of goat serum) using an enzyme-linked immunosorbent assay (ELISA). The reactivity of six recombinant trivalent chimeric proteins (SAG1-SAG2-GRA5, SAG1-SAG2-GRA9, SAG1-SAG2-MIC1, SAG1-SAG2-MIC3, SAG1-SAG2-P35, and SAG1-SAG2-ROP1) with IgG antibodies generated during T. gondii invasion was comparable to the sensitivity of TLA-based IgG ELISA (100%). The obtained results show a strong correlation with the results obtained for TLA. This suggests that these protein preparations may be a potential alternative to TLA used in commercial tests and could be used to develop a cheaper test for the detection of parasite infection in small ruminants.

Introduction: in areas with intense perennial malaria transmission, limited data is available on the impact of environmental conditions especially rainfall on naturally acquired immunity against promising malaria vaccine candidates. For this reason, we have compared IgG antibody responses specific to Plasmodium spp. derived MSP3 and UB05 vaccine candidates, in plasma of children living in two areas of Cameroon differing in rainfall conditions., Methods: data about children less than 5 years old was collected during the years 2017 and 2018. Next malaria asymptomatic P. falciparum (Pf) infected children were selected following malaria test confirmation. MSP3 and UB05 specific IgG antibody responses were measured in participant´s plasma using enzyme-linked immunosorbent assay (ELISA)., Results: interestingly, IgG antibody responses specific to UB05 were significantly higher (p<0.0001) in Pf-negative children when compared to their asymptomatic Pf-infected counterparts living in monomodal rainfall areas. In contrast, a significantly higher (p<0.0001) IgG response to MSP3 was observed instead in asymptomatic Pf-infected children in the same population. In addition, IgG responses specific to UB05 remained significantly higher in bimodal when compared to monomodal rainfall areas irrespective of children´s Pf infection status (p<0.0055 for Pf-positive and p<0.0001 for negative children). On the contrary, IgG antibody responses specific to MSP3 were significantly higher in bimodal relative to monomodal rainfall areas (P<0.0001) just for Pf-negative children., Conclusion: thus IgG antibody responses specific to UBO5 are a better correlate of naturally acquired immunity against malaria in Pf-negative Cameroonian children especially in monomodal rainfall areas., Competing Interests: The authors declare no competing interests., (Copyright: Loveline Ngu et al.)

Malaria remains a global health challenge, necessitating the development of effective vaccines. The RTS,S vaccination prevents Plasmodium falciparum (Pf) malaria but is ineffective against Plasmodium vivax (Pv) disease. Herein, we evaluated the murine immunogenicity of a recombinant PvCSP incorporating prevalent polymorphisms, adjuvanted with Alhydrogel or Poly I:C. Both formulations induced prolonged IgG responses, with IgG1 dominance by the Alhydrogel group and high titers of all IgG isotypes by the Poly I:C counterpart. Poly I:C-adjuvanted vaccination increased splenic plasma cells, terminally-differentiated memory cells (MBCs), and precursors relative to the Alhydrogel-combined immunization. Splenic B-cells from Poly I:C-vaccinated mice revealed an antibody-secreting cell- and MBC-differentiating gene expression profile. Biological processes such as antibody folding and secretion were highlighted by the Poly I:C-adjuvanted vaccination. These findings underscore the potential of Poly I:C to strengthen immune responses against Pv malaria., Competing Interests: RM, AG, and IS are co-inventors of the potential P. vivax vaccine evaluated in this study. The patent is under evaluation process, application number BR102022005915-2. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Costa-Gouvea, Françoso, Marques, Gimenez, Faria, Cariste, Dominguez, Vasconcelos, Nakaya, Silveira and Soares.)