Evaluation of naturally acquired immune responses against novel pre-erythrocytic Plasmodium vivax proteins in a low endemic malaria population located in the Peruvian Amazon Basin.

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.

Immune responses to malaria pre-erythrocytic stages: Implications for vaccine development.

Radiation-attenuated sporozoites induce sterilizing immunity and remain the 'gold standard' for malaria vaccine development. Despite practical challenges in translating these whole sporozoite vaccines to large-scale intervention programmes, they have provided an excellent platform to dissect the immune responses to malaria pre-erythrocytic (PE) stages, comprising both sporozoites and exoerythrocytic forms. Investigations in rodent models have provided insights that led to the clinical translation of various vaccine candidates-including RTS,S/AS01, the most advanced candidate currently in a trial implementation programme in three African countries. With advances in immunology, transcriptomics and proteomics, and application of lessons from past failures, an effective, long-lasting and wide-scale malaria PE vaccine remains feasible. This review underscores the progress in PE vaccine development, focusing on our understanding of host-parasite immunological crosstalk in the tissue environments of the skin and the liver. We highlight possible gaps in the current knowledge of PE immunity that can impact future malaria vaccine development efforts.

Repurposing Drugs to Fight Hepatic Malaria Parasites.

Malaria remains one of the most prevalent infectious diseases worldwide, primarily affecting some of the most vulnerable populations around the globe. Despite achievements in the treatment of this devastating disease, there is still an urgent need for the discovery of new drugs that tackle infection by Plasmodium parasites. However, de novo drug development is a costly and time-consuming process. An alternative strategy is to evaluate the anti-plasmodial activity of compounds that are already approved for other purposes, an approach known as drug repurposing. Here, we will review efforts to assess the anti-plasmodial activity of existing drugs, with an emphasis on the obligatory and clinically silent liver stage of infection. We will also review the current knowledge on the classes of compounds that might be therapeutically relevant against Plasmodium in the context of other communicable diseases that are prevalent in regions where malaria is endemic. Repositioning existing compounds may constitute a faster solution to the current gap of prophylactic and therapeutic drugs that act on Plasmodium parasites, overall contributing to the global effort of malaria eradication.

[Development of malaria vaccines-state of the art]. / Entwicklung von Impfstoffen gegen Malaria ­ aktueller Stand.

Globally, 3.1 billion people live in areas endemic for malaria (the tropics and subtropics). Annually, around 200 million fall ill, and around 500,000 persons die as a result of this infection. Mainly children are the victims. In order to control and eventually prevent any new infection, the development of effective vaccines is pivotal. In this review, background information about the history of vaccine development and malaria disease as well as possibilities for therapy and control is given. In the main part of the article, an update on the development of vaccines against Plasmodium falciparum is provided followed by an extensive discussion.Malaria is a parasitic infectious disease caused by the single cell organism Plasmodium. Five different Plasmodium species can induce disease in humans with P. falciparum being the origin for more than 99% of infections in Africa. The vector is the Anopheles mosquito. The life cycle of Plasmodium offers several approaches for vaccines to have an impact. Out of around 70 candidates, pre-erythrocytic vaccine candidates interfering with the liver phase of the parasite are the most developed. However, a vaccine with more than 75% efficacy, as required by the World Health Organization (WHO), is not yet in sight.Currently, for the first time, a moderately efficacious vaccine (RTS,S/AS01) is being applied in large-scale operations. But it is obvious that malaria can only be controlled in combination with concurring measures. For example, the use of impregnated mosquito nets, indoor residual spraying, elimination of vector breeding sites, rapid diagnosis, and therapy of the infection as well as a functioning health system are important elements, which can hardly be guaranteed in areas characterized by poverty.

RTS,S/AS01E immunization increases antibody responses to vaccine-unrelated Plasmodium falciparum antigens associated with protection against clinical malaria in African children: a case-control study.

BACKGROUND: Vaccination and naturally acquired immunity against microbial pathogens may have complex interactions that influence disease outcomes. To date, only vaccine-specific immune responses have routinely been investigated in malaria vaccine trials conducted in endemic areas. We hypothesized that RTS,S/A01E immunization affects acquisition of antibodies to Plasmodium falciparum antigens not included in the vaccine and that such responses have an impact on overall malaria protective immunity. METHODS: We evaluated IgM and IgG responses to 38 P. falciparum proteins putatively involved in naturally acquired immunity to malaria in 195 young children participating in a case-control study nested within the African phase 3 clinical trial of RTS,S/AS01E (MAL055 NCT00866619) in two sites of different transmission intensity (Kintampo high and Manhiça moderate/low). We measured antibody levels by quantitative suspension array technology and applied regression models, multimarker analysis, and machine learning techniques to analyze factors affecting their levels and correlates of protection. RESULTS: RTS,S/AS01E immunization decreased antibody responses to parasite antigens considered as markers of exposure (MSP142, AMA1) and levels correlated with risk of clinical malaria over 1-year follow-up. In addition, we show for the first time that RTS,S vaccination increased IgG levels to a specific group of pre-erythrocytic and blood-stage antigens (MSP5, MSP1 block 2, RH4.2, EBA140, and SSP2/TRAP) which levels correlated with protection against clinical malaria (odds ratio [95% confidence interval] 0.53 [0.3-0.93], p = 0.03, for MSP1; 0.52 [0.26-0.98], p = 0.05, for SSP2) in multivariable logistic regression analyses. CONCLUSIONS: Increased antibody responses to specific P. falciparum antigens in subjects immunized with this partially efficacious vaccine upon natural infection may contribute to overall protective immunity against malaria. Inclusion of such antigens in multivalent constructs could result in more efficacious second-generation multistage vaccines.

Plasmodium yoelii S4/CelTOS is important for sporozoite gliding motility and cell traversal.

Gliding motility and cell traversal by the Plasmodium ookinete and sporozoite invasive stages allow penetration of cellular barriers to establish infection of the mosquito vector and mammalian host, respectively. Motility and traversal are not observed in red cell infectious merozoites, and we have previously classified genes that are expressed in sporozoites but not merozoites (S genes) in order to identify proteins involved in these processes. The S4 gene has been described as criticaly involved in Cell Traversal for Ookinetes and Sporozoites (CelTOS), yet knockout parasites (s4/celtos¯) do not generate robust salivary gland sporozoite numbers, precluding a thorough analysis of S4/CelTOS function during host infection. We show here that a failure of oocysts to develop or survive in the midgut contributes to the poor mosquito infection by Plasmodium yoelii (Py) s4/celtos¯ rodent malaria parasites. We rescued this phenotype by expressing S4/CelTOS under the ookinete-specific circumsporozoite protein and thrombospondin-related anonymous protein-related protein (CTRP) promoter (S4/CelTOSCTRP ), generating robust numbers of salivary gland sporozoites lacking S4/CelTOS that were suitable for phenotypic analysis. Py S4/CelTOSCTRP sporozoites showed reduced infectivity in BALB/c mice when compared to wild-type sporozoites, although they appeared more infectious than sporozoites deficient in the related traversal protein PLP1/SPECT2 (Py plp1/spect2¯). Using in vitro assays, we substantiate the role of S4/CelTOS in sporozoite cell traversal, but also uncover a previously unappreciated role for this protein for sporozoite gliding motility.

Evidence of cross-stage CD8+ T cell epitopes in malaria pre-erythrocytic and blood stage infections.

Malaria parasites have a complex, multistage life cycle and there is a widely held view that each stage displays a distinct set of antigens presented to the immune system. Yet, molecular analysis of malaria parasites suggests that many putative antigenic targets are shared amongst the different stages. The specificities of these cross-stage antigens and the functions of the immune responses they elicit are poorly characterized. It is well-known that CD8+ T cells play opposing immune functions following Plasmodium berghei (Pb) infection of C57BL/6 mice. Whilst these cells play a crucial role in protective immunity against pre-erythrocytic stages, they are implicated in the development of severe disease during blood stages. Recently, CD8+ T cell epitopes derived from proteins supposedly specific for either pre-erythrocytic or blood stages have been described. In this brief report, we have compiled and confirmed data that the majority of the mRNAs and/or proteins from which these epitopes are derived display expression across pre-erythrocytic and blood stages. Importantly, we provide evidence of cross-stage immune recognition of the majority of these CD8+ T cell epitopes. Hence, our findings provide a resource to further examine the relevance of antigen-specific cross-stage responses during malaria infections.

Bacterial superglue generates a full-length circumsporozoite protein virus-like particle vaccine capable of inducing high and durable antibody responses.

BACKGROUND: Malaria, caused by Plasmodium falciparum, continues to have a devastating impact on global health, emphasizing the great need for a malaria vaccine. The circumsporozoite protein (CSP) is an attractive target for a malaria vaccine, and forms a major component of RTS,S, the most clinically advanced malaria vaccine. The clinical efficacy of RTS,S has been moderate, yet has demonstrated the viability of a CSP-based malaria vaccine. In this study, a vaccine comprised of the full-length CSP antigen presented on a virus-like particle (VLP) is produced using a split-intein conjugation system (SpyTag/SpyCatcher) and the immunogenicity is tested in mice. METHODS: Full-length 3d7 CSP protein was genetically fused at the C-terminus to SpyCatcher. The CSP-SpyCatcher antigen was then covalently attached (via the SpyTag/SpyCatcher interaction) to Acinetobacter phage AP205 VLPs which were modified to display one SpyTag per VLP subunit. To evaluate the VLP-display effect, the immunogenicity of the VLP vaccine was tested in mice and compared to a control vaccine containing AP205 VLPs plus unconjugated CSP. RESULTS: Full-length CSP was conjugated at high density (an average of 112 CSP molecules per VLP) to AP205 SpyTag-VLPs. Vaccination of mice with the CSP Spy-VLP vaccine resulted in significantly increased antibody titres over a course of 7 months as compared to the control group (2.6-fold higher at 7 months after immunization). Furthermore, the CSP Spy-VLP vaccine appears to stimulate production of IgG2a antibodies, which has been linked with a more efficient clearing of intracellular parasite infection. CONCLUSION: This study demonstrates that the high-density display of CSP on SpyTag-VLPs, significantly increases the level and quality of the vaccine-induced humoral response, compared to a control vaccine consisting of soluble CSP plus AP205 VLPs. The SpyTag-VLP platform utilized in this study constitutes a versatile and rapid method to develop highly immunogenic vaccines. It might serve as a generic tool for the cost-effective development of effective VLP-vaccines, e.g., against malaria.

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

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

IgM antibody responses against Plasmodium antigens in neotropical primates in the Brazilian Atlantic Forest.

Introduction: Zoonotic transmission is a challenge for the control and elimination of malaria. It has been recorded in the Atlantic Forest, outside the Amazon which is the endemic region in Brazil. However, only very few studies have assessed the antibody response, especially of IgM antibodies, in Neotropical primates (NP). Therefore, in order to contribute to a better understanding of the immune response in different hosts and facilitate the identification of potential reservoirs, in this study, naturally acquired IgM antibody responses against Plasmodium antigens were evaluated, for the first time, in NP from the Atlantic Forest. Methods: The study was carried out using 154 NP samples from three different areas of the Atlantic Forest. IgM antibodies against peptides of the circumsporozoite protein (CSP) from different Plasmodium species and different erythrocytic stage antigens were detected by ELISA. Results: Fifty-nine percent of NP had IgM antibodies against at least one CSP peptide and 87% against at least one Plasmodium vivax erythrocytic stage antigen. Levels of antibodies against PvAMA-1 were the highest compared to the other antigens. All families of NP showed IgM antibodies against CSP peptides, and, most strikingly, against erythrocytic stage antigens. Generalized linear models demonstrated that IgM positivity against PvCSP and PvAMA-1 was associated with PCR-detectable blood-stage malaria infection and the host being free-living. Interestingly, animals with IgM against both PvCSP and PvAMA-1 were 4.7 times more likely to be PCR positive than animals that did not have IgM for these two antigens simultaneously. Discussion: IgM antibodies against different Plasmodium spp. antigens are present in NP from the Atlantic Forest. High seroprevalence and antibody levels against blood-stage antigens were observed, which had a significant association with molecular evidence of infection. IgM antibodies against CSP and AMA-1 may be used as a potential marker for the identification of NP infected with Plasmodium, which are reservoirs of malaria in the Brazilian Atlantic Forest.

Global gene expression of human malaria parasite liver stages throughout intrahepatocytic development.

Plasmodium falciparum (Pf) is causing the greatest malaria burden, yet the liver stages (LS) of this most important parasite species have remained poorly studied. Here, we used a human liver-chimeric mouse model in combination with a novel fluorescent PfNF54 parasite line (PfNF54cspGFP) to isolate PfLS-infected hepatocytes and generate transcriptomes that cover the major LS developmental phases in human hepatocytes. RNA-seq analysis of early Pf LS trophozoites two days after infection, revealed a central role of translational regulation in the transformation of the extracellular invasive sporozoite into intracellular LS. The developmental time course gene expression analysis indicated that fatty acid biosynthesis, isoprenoid biosynthesis and iron metabolism are sustaining LS development along with amino acid metabolism and biosynthesis. Countering oxidative stress appears to play an important role during intrahepatic LS development. Furthermore, we observed expression of the variant PfEMP1 antigen-encoding var genes, and we confirmed expression of PfEMP1 protein during LS development. Transcriptome comparison of the late Pf liver stage schizonts with P. vivax (Pv) late liver stages revealed highly conserved gene expression profiles among orthologous genes. A notable difference however was the expression of genes regulating sexual stage commitment. While Pv schizonts expressed markers of sexual commitment, the Pf LS parasites were not sexually committed and showed expression of gametocytogenesis repression factors. Our results provide the first comprehensive gene expression profile of the human malaria parasite Pf LS isolated during in vivo intrahepatocytic development. This data will inform biological studies and the search for effective intervention strategies that can prevent infection.

Malaria vaccines: the 60-year journey of hope and final success-lessons learned and future prospects.

BACKGROUND: The world has made great strides towards beating malaria, although about half of the world population is still exposed to the risk of contracting malaria. Developing an effective malaria vaccine was a huge challenge for medical science. In 2021 the World Health Organization (WHO) approved the first malaria vaccine, RTS,S/AS01 vaccine (Mosquirix™), for widespread use. This review highlights the history of development, and the different approaches and types of malaria vaccines, and the literature to date. It covers the developmental stages of RTS,S/AS01 and recommends steps for its deployment. The review explores other potential vaccine candidates and their status, and suggests options for their further development. It also recommends future roles for vaccines in eradicating malaria. Questions remain on how RTS,S vaccine will work in widespread use and how it can best be utilized to benefit vulnerable communities. CONCLUSION: Malaria vaccines have been in development for almost 60 years. The RTS,S/AS01 vaccine has now been approved, but cannot be a stand-alone solution. Development should continue on promising candidates such as R21, PfSPZ and P. vivax vaccines. Multi-component vaccines may be a useful addition to other malaria control techniques in achieving eradication of malaria.

Effective Functional Immunogenicity of a DNA Vaccine Combination Delivered via In Vivo Electroporation Targeting Malaria Infection and Transmission.

Plasmodium falciparum circumsporozoite protein (PfCSP) and Pfs25 are leading candidates for the development of pre-erythrocytic and transmission-blocking vaccines (TBV), respectively. Although considerable progress has been made in developing PfCSP- and Pfs25-based vaccines, neither have elicited complete protection or transmission blocking in clinical trials. The combination of antigens targeting various life stages is an alternative strategy to develop a more efficacious malaria vaccine. In this study, female and male mice were immunized with DNA plasmids encoding PfCSP and Pfs25, administered alone or in combination via intramuscular in vivo electroporation (EP). Antigen-specific antibodies were analyzed for antibody titers, avidity and isotype by ELISA. Immune protection against sporozoite challenge, using transgenic P. berghei expressing PfCSP and a GFP-luciferase fusion protein (PbPfCSP-GFP/Luc), was assessed by in vivo bioluminescence imaging and blood-stage parasite growth. Transmission reducing activity (TRA) was evaluated in standard membrane feeding assays (SMFA). High levels of PfCSP- and Pfs25-specific antibodies were induced in mice immunized with either DNA vaccine alone or in combination. No difference in antibody titer and avidity was observed for both PfCSP and Pfs25 between the single DNA and combined DNA immunization groups. When challenged by PbPfCSP-GFP/Luc sporozoites, mice immunized with PfCSP alone or combined with Pfs25 revealed significantly reduced liver-stage parasite loads as compared to mice immunized with Pfs25, used as a control. Furthermore, parasite liver loads were negatively correlated with PfCSP-specific antibody levels. When evaluating TRA, we found that immunization with Pfs25 alone or in combination with PfCSP elicited comparable significant transmission reduction. Our studies reveal that the combination of PfCSP and Pfs25 DNAs into a vaccine delivered by in vivo EP in mice does not compromise immunogenicity, infection protection and transmission reduction when compared to each DNA vaccine individually, and provide support for further evaluation of this DNA combination vaccine approach in larger animals and clinical trials.

Norovirus-VLPs expressing pre-erythrocytic malaria antigens induce functional immunity against sporozoite infection.

Despite the development of prophylactic anti-malarial drugs and practices to prevent infection, malaria remains a health concern. Preclinical testing of novel malaria vaccine strategies achieved through rational antigen selection and novel particle-based delivery platforms is yielding encouraging results. One such platform, self-assembling virus-like particles (VLP) is safer than attenuated live viruses, and has been approved as a vaccination tool by the FDA. We explore the use of Norovirus sub-viral particles lacking the natural shell (S) domain forming the interior shell but that retain the protruding (P) structures of the native virus as a vaccine vector. Epitope selection and their surface display has the potential to focus antigen specific immune responses to crucial epitopes. Recombinant P-particles displaying epitopes from two malaria antigens, Plasmodium falciparum (Pf) CelTOS and Plasmodium falciparum (Pf) CSP, were evaluated for immunogenicity and their ability to confer protection in a murine challenge model. Immune responses induced in mice resulted either in sterile protection (displaying PfCelTOS epitopes) or in antibodies with functional activity against sporozoites (displaying PfCSP epitopes) in an in vitro liver-stage development assay (ILSDA). These results are encouraging and support further evaluation of this platform as a vaccine delivery system.

Low immunogenicity of malaria pre-erythrocytic stages can be overcome by vaccination.

Immunogenicity is considered one important criterion for progression of candidate vaccines to further clinical evaluation. We tested this assumption in an infection and vaccination model for malaria pre-erythrocytic stages. We engineered Plasmodium berghei parasites that harbour a well-characterised epitope for stimulation of CD8+ T cells, either as an antigen in the sporozoite surface-expressed circumsporozoite protein or the parasitophorous vacuole membrane associated protein upregulated in sporozoites 4 (UIS4) expressed in exo-erythrocytic forms (EEFs). We show that the antigen origin results in profound differences in immunogenicity with a sporozoite antigen eliciting robust, superior antigen-specific CD8+ T-cell responses, whilst an EEF antigen evokes poor responses. Despite their contrasting immunogenic properties, both sporozoite and EEF antigens gain access to antigen presentation pathways in hepatocytes, as recognition and targeting by vaccine-induced effector CD8+ T cells results in high levels of protection when targeting either antigen. Our study is the first demonstration that poorly immunogenic EEF antigens do not preclude their susceptibility to antigen-specific CD8+ T-cell killing, which has wide-ranging implications on antigen prioritisation for next-generation pre-erythrocytic malaria vaccines.

Towards Eradication of Malaria: Is the WHO's RTS,S/AS01 Vaccination Effective Enough?

BACKGROUND: Recent advances in mosquito eradication and antimalarial treatments have reduced the malaria burden only modestly. An effective malaria vaccine remains a high priority, but its development has several challenges. Among many potential candidates, the RTS,S/AS01 vaccine (MosquirixTM) remains the leading candidate. OBJECTIVE AND METHOD: This review aims to understand the advances in the RTS,S/AS01 vaccine, and future comments regarding the vaccine's effectiveness in malaria eradication. Literature review for the past five decades was performed searching PubMed, EMBASE Ovid, and Cochrane Library, with using the following search items: ("malaria" OR "WHO's malaria" OR "Plasmodium falciparum" OR "RTS,S" OR "RTS,S/AS01" OR "RTS,S/AS02" OR "pre-erythrocytic malaria" OR "circumsporozoite" OR "Mosquirix") AND ("vaccine" OR "vaccination"). RESULTS: RTS,S/AS01, a recombinant pre-erythrocytic vaccine containing Plasmodium falciparum surface-protein (circumsporozoite) antigen, is safe, well-tolerated, and immunogenic in children. Three doses, along with a booster, have a modest efficacy of about 36% in children (age 5-17 months) and about 26% in infants (age 6-12 weeks) against clinical malaria during a 48-month follow-up. However, the efficacy varies among population subgroups and with the parasite strain, it reduces without a booster and offers protection for a limited duration. Because of its potential cost-effectiveness and positive public health effect, the vaccine is being investigated in a pilot program for mortality benefits and broader deployment. CONCLUSION: The RTS,S/AS01 vaccine prevents malaria; however, it should be considered another addition to the malaria-control program and not as an eradication tool because of its relatively low to modest efficacy.

Conservation of S20 as an Ineffective and Disposable IFNγ-Inducing Determinant of Plasmodium Sporozoites Indicates Diversion of Cellular Immunity.

Despite many decades of research to develop a malaria vaccine, only one vaccine candidate has been explored in pivotal phase III clinical trials. This candidate subunit vaccine consists of a portion of a single Plasmodium antigen, circumsporozoite protein (CSP). This antigen was initially identified in the murine malaria model and shown to contain an immunodominant and protective CD8+ T cell epitope specific to the H-2K d (BALB/c)-restricted genetic background. A high-content screen for CD8+ epitopes in the H2K b /D b (C57BL/6)-restricted genetic background, identified two distinct dominant epitopes. In this study, we present a characterization of one corresponding antigen, the Plasmodium sporozoite-specific protein S20. Plasmodium berghei S20 knockout sporozoites and liver stages developed normally in vitro and in vivo. This potent infectivity of s20(-) sporozoites permitted comparative analysis of knockout and wild-type parasites in cell-based vaccination. Protective immunity of irradiation-arrested s20(-) sporozoites in single, double and triple immunizations was similar to irradiated unaltered sporozoites in homologous challenge experiments. These findings demonstrate the presence of an immunogenic Plasmodium pre-erythrocytic determinant, which is not essential for eliciting protection. Although S20 is not needed for colonization of the mammalian host and for initiation of a blood infection, it is conserved amongst Plasmodium species. Malarial parasites express conserved, immunogenic proteins that are not required to establish infection but might play potential roles in diverting cellular immune responses.

Absence of PEXEL-Dependent Protein Export in Plasmodium Liver Stages Cannot Be Restored by Gain of the HSP101 Protein Translocon ATPase.

Host cell remodeling is critical for successful Plasmodium replication inside erythrocytes and achieved by targeted export of parasite-encoded proteins. In contrast, during liver infection the malarial parasite appears to avoid protein export, perhaps to limit exposure of parasite antigens by infected liver cells. HSP101, the force-generating ATPase of the protein translocon of exported proteins (PTEX) is the only component that is switched off during early liver infection. Here, we generated transgenic Plasmodium berghei parasite lines that restore liver stage expression of HSP101. HSP101 expression in infected hepatocytes was achieved by swapping the endogenous promoter with the ptex150 promoter and by inserting an additional copy under the control of the elongation one alpha (ef1α) promoter. Both promoters drive constitutive and, hence, also pre-erythrocytic expression. Transgenic parasites were able to complete the life cycle, but failed to export PEXEL-proteins in early liver stages. Our results suggest that PTEX-dependent early liver stage export cannot be restored by addition of HSP101, indicative of alternative export complexes or other functions of the PTEX core complex during liver infection.

Profiling Humoral Immune Response Against Pre-Erythrocytic and Erythrocytic Antigens of Malaria Parasites Among Neotropical Primates in the Brazilian Atlantic Forest.

Human malaria due to zoonotic transmission has been recorded in the Atlantic Forest, an extra-Amazonian area in Brazil, which are a challenge for malaria control. Naturally acquired humoral immune response against pre-erythrocytic and erythrocytic antigens of Neotropical primates (NP) was evaluated here to improve the knowledge about the exposure of those animals to the malaria transmission and support the identification of the potential reservoirs of the disease in the Atlantic Forest. Blood samples of 154 monkeys from three areas of the Atlantic Forest were used to identify IgG antibodies against peptides of the repeat region of the major pre-erythrocytic antigen, the circumsporozoite protein (CSP), of Plasmodium vivax (PvCSP), Plasmodium brasilianum/Plasmodium malariae (Pb/PmCSP), and Plasmodium falciparum (PfCSP) by ELISA. Antibodies against erythrocytic recombinant antigens of P. vivax, Apical membrane antigen 1 (PvAMA-1), Erythrocyte binding protein 2 (PvEBP-2) and domain II of Duffy binding protein (PvDBPII) were also evaluated. Parameters, such as age, sex, PCR positivity, and captivity, potentially associated with humoral immune response were analyzed. Eighty-five percent of NP had antibodies against at least one CSP peptide, and 76% against at least one P. vivax erythrocytic antigen. A high percentage of adults compared to non-adults were seropositive and showed increased antibody levels. Neotropical primates with PCR positive for P. simium had a significantly higher frequency of positivity rate for immune response against PvEBP-2, PvDBPII and also higher antibody levels against PvDBPII, compared to PCR negative NPs for this species. Monkeys with PCR positive for P. brasilianum/P. malariae showed higher frequency of seropositivity and antibody levels against Pb/PmCSP. Levels of antibodies against Pb/PmCSP, PvEBP-2 and PvDBPII were higher in free-living than in captive monkeys from the same area. All Platyrrhine families showed antibodies against CSP peptides, however not all showed IgG against erythrocytic antigens. These findings showed a high prevalence of naturally acquired antibodies against CSP repeats in all studied areas, suggesting an intense exposure to infected-mosquitoes bites of NP from all families. However, mainly monkeys of Atelidae family showed antibodies against P. vivax erythrocytic antigens, suggesting blood infection, which might serve as potential reservoirs of malaria in the Atlantic Forest.

Partial protection against P. vivax infection diminishes hypnozoite burden and blood-stage relapses.

Latent forms of Plasmodium vivax, called hypnozoites, cause malaria relapses from the liver into the bloodstream and are a major obstacle to malaria eradication. To experimentally assess the impact of a partially protective pre-erythrocytic vaccine on reducing Plasmodium vivax relapses, we developed a liver-humanized mouse model that allows monitoring of relapses directly in the blood. We passively infused these mice with a suboptimal dose of an antibody that targets the circumsporozoite protein prior to challenge with P. vivax sporozoites. Although this regimen did not completely prevent primary infection, antibody-treated mice experienced 62% fewer relapses. The data constitute unprecedented direct experimental evidence that suboptimal efficacy of infection-blocking antibodies, while not completely preventing primary infection, has a pronounced benefit in reducing the number of relapses. These findings suggest that a partially efficacious pre-erythrocytic Plasmodium vivax vaccine can have a disproportionately high impact in positive public health outcomes.