PfSPZ Vaccine induces focused humoral immune response in HIV positive and negative Tanzanian adults.

BACKGROUND: PfSPZ Vaccine, a promising pre-erythrocytic stage malaria vaccine candidate based on whole, radiation-attenuated Plasmodium falciparum (Pf) sporozoites (SPZ), has proven safe and effective in mediating sterile protection from malaria in malaria-naïve and exposed healthy adults. Vaccine-induced protection presumably depends on cellular responses to early parasite liver stages, but humoral immunity contributes. METHODS: On custom-made Pf protein microarrays, we profiled IgG and IgM responses to PfSPZ Vaccine and subsequent homologous controlled human malaria infection (CHMI) in 21 Tanzanian adults with (n = 12) or without (n = 9) HIV infection. Expression of the main identified immunogens in the pre-erythrocytic parasite stage was verified by immunofluorescence detection using freshly purified PfSPZ and an in vitro model of primary human hepatocytes. FINDINGS: Independent of HIV infection status, immunisation induced focused IgG and IgM responses to circumsporozoite surface protein (PfCSP) and merozoite surface protein 5 (PfMSP5). We show that PfMSP5 is detectable on the surface and in the apical complex of PfSPZ. INTERPRETATION: Our data demonstrate that HIV infection does not affect the quantity of the total IgG and IgM antibody responses to PfCSP and PfMSP5 after immunization with PfSPZ Vaccine. PfMSP5 represents a highly immunogenic, so far underexplored, target for vaccine-induced antibodies in malaria pre-exposed volunteers. FUNDING: This work was supported by the Equatorial Guinea Malaria Vaccine Initiative (EGMVI), the Clinical Trial Platform of the German Center for Infection Research (TTU 03.702), the Swiss Government Excellence Scholarships for Foreign Scholars and Artists (grant 2016.0056) and the Interdisciplinary Center for Clinical Research doctoral program of the Tübingen University Hospital. The funders had no role in design, analysis, or reporting of this study.

Identification of RTS,S/AS01 vaccine-induced humoral biomarkers predictive of protection against controlled human malaria infection.

BACKGROUNDThe mechanism(s) responsible for the efficacy of WHO-recommended malaria vaccine RTS,S/AS01 are not completely understood. We previously identified RTS,S vaccine-induced Plasmodium falciparum circumsporozoite protein-specific (PfCSP-specific) antibody measures associated with protection from controlled human malaria infection (CHMI). Here, we tested the protection-predicting capability of these measures in independent CHMI studies.METHODSVaccine-induced total serum antibody (immunoglobulins, Igs) and subclass antibody (IgG1 and IgG3) responses were measured by biolayer interferometry and the binding antibody multiplex assay, respectively. Immune responses were compared between protected and nonprotected vaccinees using univariate and multivariate logistic regression.RESULTSBlinded prediction analysis showed that 5 antibody binding measures, including magnitude-avidity composite of serum Ig specific for PfCSP, major NANP repeats and N-terminal junction, and PfCSP- and NANP-specific IgG1 subclass magnitude, had good prediction accuracy (area under the receiver operating characteristic curves [ROC AUC] > 0.7) in at least 1 trial. Furthermore, univariate analysis showed a significant association between these antibody measures and protection (odds ratios 2.6-3.1). Multivariate modeling of combined data from 3 RTS,S CHMI trials identified the combination of IgG1 NANP binding magnitude plus serum NANP and N-junction Ig binding magnitude-avidity composite as the best predictor of protection (95% confidence interval for ROC AUC 0.693-0.834).CONCLUSIONThese results reinforce our previous findings and provide a tool for predicting protection in future trials.TRIAL REGISTRATIONClinicalTrials.gov NCT03162614, NCT03824236, NCT01366534, and NCT01857869.FUNDINGThis study was supported by Bill & Melinda Gates Foundation's Global Health-Discovery Collaboratory grants (INV-008612 and INV-043419) to GDT.

Polarization toward Tfh2 cell involved in development of MBC and antibody responses against Plasmodium vivax infection.

BACKGROUND: Plasmodium vivax is the dominant Plasmodium spp. causing malaria throughout tropical and sub-tropical countries. Humoral immunity is induced during P. vivax infection. However, data on longevity of antibody and memory B cell (MBC) responses is lacking. Follicular helper T cells (Tfh) are drivers of high-affinity and long-lived antibody responses. Understanding of Tfh-mediated immunity against malaria is valuable for vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: We enrolled 31 acutely infected P. vivax patients in low malaria transmission areas of Thailand to detect frequencies, phenotypes and kinetics of different subsets of circulating Tfh (cTfh) and MBCs, and to evaluate their association with humoral immunity following natural P. vivax infection. Expansion of cTfh2 cells, activated and atypical MBCs were shown during acute malaria. To relate increased cTfh2 cells to humoral immunity, P. vivax-specific MBCs and antibodies were assessed. High anti-PvCSP and -PvDBPII seropositivity was detected and most subjects produced MBCs specific to these antigens. The increased cTfh2 cells were positively related to atypical MBCs, plasmablasts/plasma cells, and anti-PvDBPII IgM and IgG levels. Distributions of memory cTfh cell subsets were altered from central memory (CM) to effector memory (EM) during infection. The highest ratios of cTfh-EM/cTfh-CM were represented in cTfh2 cells. Positive correlation of cTfh17-EM with activated and atypical MBCs was observed, while cTfh2-CM and cTfh17-CM cells were positively related to PvDBPII-specific MBCs and IgM levels. CONCLUSIONS/SIGNIFICANCE: Present study demonstrated that P. vivax infection induced cTfh polarization into cTfh2 subset, and alteration of memory cTfh2 phenotype from CM to EM phase. These P. vivax-induced cTfh responses significantly associated with generation of MBCs and antibody responses. Therefore, cTfh2 cells might possibly influence humoral immunity by inducing expansion of activated and atypical MBCs, and by generating P. vivax-specific MBCs and antibody responses following natural infection.

Exploring the naturally acquired response to Pvs47 gametocyte antigen.

Malaria represents a challenging global public health task, with Plasmodium vivax being the predominant parasite in Brazil and the most widely distributed species throughout the world. Developing a vaccine against P. vivax malaria demands innovative strategies, and targeting gametocyte antigens shows promise for blocking transmission prevention. Among these antigens, Pvs47, expressed in gametocytes, has shown remarkable efficacy in transmission blocking. However, remains underexplored in vaccine formulations. This study employed in silico methods to comprehensively characterize the physicochemical properties, structural attributes, epitope presence, and conservation profile of Pvs47. Additionally, we assessed its antigenicity in individuals exposed to malaria in endemic Brazilian regions. Recombinant protein expression occurred in a eukaryotic system, and antigenicity was evaluated using immunoenzymatic assays. The responses of naturally acquired IgM, total IgG, and IgG subclasses were analyzed in three groups of samples from Amazon region. Notably, all samples exhibited anti-Pvs47 IgM and IgG antibodies, with IgG3 predominating. Asymptomatic patients demonstrated stronger IgG responses and more diverse subclass responses. Anti-Pvs47 IgM and IgG responses in symptomatic individuals decrease over time. Furthermore, we observed a negative correlation between anti-Pvs47 IgM response and gametocytemia in samples of symptomatic patients, indicating a gametocyte-specific response. Additionally, negative correlation was observed among anti-Pvs47 antibody response and hematocrit levels. Furthermore, comparative analysis with widely characterized blood antigens, PvAMA1 and PvMSP119, revealed that Pvs47 was equally or more recognized than both proteins. In addition, there is positive correlation between P. vivax blood asexual and sexual stage immune responses. In summary, our study unveils a significant prevalence of anti-Pvs47 antibodies in diverse Amazonian samples and the importance of IgM response for gametocytes depuration. These findings regarding the in silico characterization and antigenicity of Pvs47 provide crucial insights for potential integration into P. vivax vaccine formulations.

Increased pathogen exposure of a marine apex predator over three decades.

Environmental changes associated with global warming create new opportunities for pathogen and parasite transmission in Arctic wildlife. As an apex predator ranging over large, remote areas, changes in pathogens and parasites in polar bears are a useful indicator of changing transmission dynamics in Arctic ecosystems. We examined prevalence and risk factors associated with exposure to parasites and viral and bacterial pathogens in Chukchi Sea polar bears. Serum antibodies to six pathogens were detected and prevalence increased between 1987-1994 and 2008-2017 for five: Toxoplasma gondii, Neospora caninum, Francisella tularensis, Brucella abortus/suis, and canine distemper virus. Although bears have increased summer land use, this behavior was not associated with increased exposure. Higher prevalence of F. tularensis, Coxiella burnetii, and B. abortus/suis antibodies in females compared to males, however, could be associated with terrestrial denning. Exposure was related to diet for several pathogens indicating increased exposure in the food web. Elevated white blood cell counts suggest a possible immune response to some pathogens. Given that polar bears face multiple stressors in association with climate change and are a subsistence food, further work is warranted to screen for signs of disease.

Nanospheres as the delivery vehicle: novel application of Toxoplasma gondii ribosomal protein S2 in PLGA and chitosan nanospheres against acute toxoplasmosis.

Toxoplasma gondii (T. gondii) is a zoonotic disease that poses great harm to humans and animals. So far, no effective T. gondii vaccine has been developed to provide fully protection against such parasites. Recently, numerous researches have focused on the use of poly-lactic-co-glycolic acid (PLGA) and chitosan (CS) for the vaccines against T. gondii infections. In this study, we employed PLGA and CS as the vehicles for T. gondii ribosome protein (TgRPS2) delivery. TgRPS2-PLGA and TgRPS2-CS nanospheres were synthesized by double emulsion solvent evaporation and ionic gelation technique as the nano vaccines. Before immunization in animals, the release efficacy and toxicity of the synthesized nanospheres were evaluated in vitro. Then, ICR mice were immunized intramuscularly, and immune protections of the synthesized nanospheres were assessed. The results showed that TgRPS2-PLGA and TgRPS2-CS nanospheres could induce higher levels of IgG and cytokines, activate dendritic cells, and promote the expression of histocompatibility complexes. The splenic lymphocyte proliferation and the enhancement in the proportion of CD4+ and CD8+ T lymphocytes were also observed in immunized animals. In addition, two types of nanospheres could significantly inhabit the replications of T. gondii in cardiac muscles and spleen tissues. All these obtained results in this study demonstrated that the TgRPS2 protein delivered by PLGA or CS nanospheres provided satisfactory immunoprotective effects in resisting T. gondii, and such formulations illustrated potential as prospective preventive agents for toxoplasmosis.

Development and longevity of naturally acquired antibody and memory B cell responses against Plasmodium vivax infection.

Plasmodium vivax malaria causes significant public health problems in endemic regions. Considering the rapid spread of drug-resistant parasite strains and the development of hypnozoites in the liver with potential for relapse, development of a safe and effective vaccine for preventing, controlling, and eliminating the infection is critical. Immunity to malaria is mediated by antibodies that inhibit sporozoite or merozoite invasion into host cells and protect against clinical disease. Epidemiologic data from malaria endemic regions show the presence of naturally acquired antibodies to P. vivax antigens during and following infection. But data on the persistence of these antibodies, development of P. vivax-specific memory B cells (MBCs), and their relation to reduction of malaria severity and risk is limited. This review provides an overview of the acquisition and persistence of naturally acquired humoral immunity to P. vivax infection. Also, we summarize and discuss current progress in assessment of immune responses to candidate vaccine antigens in P. vivax patients from different transmission settings. Longitudinal studies of MBC and antibody responses to these antigens will open new avenues for developing vaccines against malaria infection and its transmission.

Saurian-associated Leishmania tarentolae in dogs: Infectivity and immunogenicity evaluation in the canine model.

In canine leishmaniosis endemic areas, Leishmania infantum may occur in sympatry with the non-pathogenic Leishmania tarentolae, which is associated to reptiles. The potential infectivity of L. tarentolae for mammals raises questions about the interactions between the two Leishmania species, and the potential cross-immune protection in dogs. This study aimed to assess the outcome of experimental L. tarentolae infection in dogs, determining: i) the anti-L. tarentolae antibody production, ii) the duration of the immunity and cytokine expression, and iii) the possible pathogenic effect in the canine host. Twelve purpose-bred beagle dogs were randomly allocated to three groups (intravenous inoculation, G1; intradermal inoculation, G2; negative control, G3). G1 and G2 dogs were inoculated twice (day 0, day 28) with 108 promastigotes of L. tarentolae strain (RTAR/IT/21/RI-325) isolated from a Tarentola mauritanica gecko. The animals were followed until day 206. Blood, serum, conjunctival swabs and lymph node aspirate samples were collected monthly and bone marrow, liver and spleen biopsies on day 91. Hematological and biochemical parameters were assessed monthly, as well as serology (IFAT and ELISA) and molecular identification of L. tarentolae. Mononuclear cells (PBMC) were obtained to assess the cytokine expression through in vitro stimulation or (re-) infection. Data from this study demonstrated that DNA from L. tarentolae is detectable up to 3 months post-infection, with seroconversion after day 28. Moreover, the non-pathogenic nature of L. tarentolae was confirmed, with a neutral Th1/Th2 polarization, and a possible shift to Th1 phenotype after derived macrophages (re-) infection, as demonstrated by the expression of IFN-gamma. Therefore, L. tarentolae demonstrated a great potential as a surrogate pathogen and/or immune-prophylaxis/immune-therapy against Leishmania infections in dogs and humans.

Interleukin-27 Regulates Adaptative Immune Responses Associated With Control of Parasite Replication in Canine Leishmaniasis.

Interleukin 27 (IL-27) is a cytokine that regulates susceptibility to Leishmania infantum infection in humans and experimental models. This cytokine has not yet been described in canine leishmaniasis (CanL). Therefore, we investigated whether IL-27 has a regulatory role in CanL. The EBI3 and p28 subunits of IL-27 were measured in splenic leukocytes culture supernatant from dogs with CanL and compared to control dogs. We also correlated EBI3 and p28 levels with IL-21, anti-L. infantum antibodies and parasite loads. We performed functional assays followed by IL-27 blockade and measured parasite loads, production of cytokines in splenic leukocytes culture supernatant, and the expression of PD-1, CTLA-4, phospho-Stat-1/3, T-bet, GATA3 and nitric oxide production (NO). Both IL-27 subunits increased in the supernatant of dogs with CanL compared to control dogs. EBI3 and p28 levels showed a moderate positive correlation with IL-21 (r = 0.67, p < 0.0001 and r = 0.45, p < 0.012, respectively), and the EBI3 subunit was positively associated with anti-L. infantum IgG antibodies (r = 0.38, p < 0.040) and parasite load (r = 0.47, p < 0.009). IL-27 and IL-21 participate of immune responses in CanL. IL-27 may be associated with the failure of immunity to control parasite replication via upregulation of the expression of PD-1, CTLA-4, T-bet and NO in splenic leukocytes from dogs with CanL. These findings suggest that the pathways regulated by IL-27 are involved in CanL pathogenesis in the host, and may be targets for new therapies.

Humoral and cellular immunity in response to an in silico-designed multi-epitope recombinant protein of Theileria annulata.

Tropical theileriosis is a lymphoproliferative disease caused by Theileria annulata and is transmitted by Ixodid ticks of the genus Hyalomma. It causes significant losses in livestock, especially in exotic cattle. The existing methods for controlling it, chemotherapeutic agents and a vaccine based on an attenuated schizont stage parasite, have several limitations. A promising solution to control this disease is the use of molecular vaccines based on potential immunogenic proteins of T. annulata. For this purpose, we selected five antigenic sequences of T. annulata, i.e. SPAG-1, Tams, TaSP, spm2, and Ta9. These were subjected to epitope prediction for cytotoxic T lymphocytes, B-cells, and helper T lymphocytes. CTL and B-cell epitopes with a higher score whereas those of HTL with a lower score, were selected for the construct. A single protein was constructed using specific linkers and evaluated for high antigenicity and low allergenicity. The construct was acidic, hydrophobic, and thermostable in nature. Secondary and tertiary structures of this construct were drawn using the PSIPRED and RaptorX servers, respectively. A Ramachandran plot showed a high percentage of residues in this construct in favorable, allowed, and general regions. Molecular docking studies suggested that the complex was stable and our construct could potentially be a good candidate for immunization trials. Furthermore, we successfully cloned it into the pET-28a plasmid and transformed it into the BL21 strain. A restriction analysis was performed to confirm the transformation of our plasmid. After expression and purification, recombinant protein of 49 kDa was confirmed by western blotting. An ELISA detected increased specific antibody levels in the sera of the immunized animals compared with the control group, and flow cytometric analysis showed a stronger cell-mediated immune response. We believe our multi-epitope recombinant protein has the potential for the large-scale application for disease prevention globally in the bovine population. This study will act as a model for similar parasitic challenges.

A systems serology approach to identifying key antibody correlates of protection from cerebral malaria in Malawian children.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins are expressed on the surface of infected erythrocytes, mediating parasite sequestration in the vasculature. PfEMP1 is a major target of protective antibodies, but the features of the antibody response are poorly defined. METHODS: In Malawian children with cerebral or uncomplicated malaria, we characterized the antibody response to 39 recombinant PfEMP1 Duffy binding like (DBL) domains or cysteine-rich interdomain regions (CIDRs) in detail, including measures of antibody classes, subclasses, and engagement with Fcγ receptors and complement. Using elastic net regularized logistic regression, we identified a combination of seven antibody targets and Fc features that best distinguished between children with cerebral and uncomplicated malaria. To confirm the role of the selected targets and Fc features, we measured antibody-dependent neutrophil and THP-1 cell phagocytosis of intercellular adhesion molecule-1 (ICAM-1) and endothelial protein C (EPCR) co-binding infected erythrocytes. RESULTS: The selected features distinguished between children with cerebral and uncomplicated malaria with 87% accuracy (median, 80-96% interquartile range) and included antibody to well-characterized DBLß3 domains and a less well-characterized CIDRγ12 domain. The abilities of antibodies to engage C1q and FcγRIIIb, rather than levels of IgG, correlated with protection. In line with a role of FcγRIIIb binding antibodies to DBLß3 domains, antibody-dependent neutrophil phagocytosis of ICAM-1 and EPCR co-binding IE was higher in uncomplicated malaria (15% median, 8-38% interquartile range) compared to cerebral malaria (7%, 30-15%, p < 0.001). CONCLUSIONS: Antibodies associated with protection from cerebral malaria target a subset of PfEMP1 domains. The Fc features of protective antibody response include engagement of FcγRIIIb and C1q, and ability to induce antibody-dependent neutrophil phagocytosis of infected erythrocytes. Identifying the targets and Fc features of protective immunity could facilitate the development of PfEMP1-based therapeutics for cerebral malaria.

Rational structure-guided design of a blood stage malaria vaccine immunogen presenting a single epitope from PfRH5.

There is an urgent need for improved malaria vaccine immunogens. Invasion of erythrocytes by Plasmodium falciparum is essential for its life cycle, preceding symptoms of disease and parasite transmission. Antibodies which target PfRH5 are highly effective at preventing erythrocyte invasion and the most potent growth-inhibitory antibodies bind a single epitope. Here we use structure-guided approaches to design a small synthetic immunogen, RH5-34EM which recapitulates this epitope. Structural biology and biophysics demonstrate that RH5-34EM is correctly folded and binds neutralising monoclonal antibodies with nanomolar affinity. In immunised rats, RH5-34EM induces PfRH5-targeting antibodies that inhibit parasite growth. While PfRH5-specific antibodies were induced at a lower concentration by RH5-34EM than by PfRH5, RH5-34EM induced antibodies that were a thousand-fold more growth-inhibitory as a factor of PfRH5-specific antibody concentration. Finally, we show that priming with RH5-34EM and boosting with PfRH5 achieves the best balance between antibody quality and quantity and induces the most effective growth-inhibitory response. This rationally designed vaccine immunogen is now available for use as part of future malaria vaccines, alone or in combination with other immunogens.

Immunogenicity and transmission-blocking potential of quiescin sulfhydryl oxidase in Plasmodium vivax.

Background: Transmission-blocking vaccines (TBVs) can effectively prevent the community's spread of malaria by targeting the antigens of mosquito sexual stage parasites. At present, only a few candidate antigens have demonstrated transmission-blocking activity (TBA) potential in P. vivax. Quiescin-sulfhydryl oxidase (QSOX) is a sexual stage protein in the rodent malaria parasite Plasmodium berghei and is associated with a critical role in protein folding by introducing disulfides into unfolded reduced proteins. Here, we reported the immunogenicity and transmission-blocking potency of the PvQSOX in P. vivax. Methods and findings: The full-length recombinant PvQSOX protein (rPvQSOX) was expressed in the Escherichia coli expression system. The anti-rPvQSOX antibodies were generated following immunization with the rPvQSOX in rabbits. A parasite integration of the pvqsox gene into the P. berghei pbqsox gene knockout genome was developed to express full-length PvQSOX protein in P. berghei (Pv-Tr-PbQSOX). In western blot, the anti-rPvQSOX antibodies recognized the native PvQSOX protein expressed in transgenic P. berghei gametocyte and ookinete. In indirect immunofluorescence assays, the fluorescence signal was detected in the sexual stages, including gametocyte, gamete, zygote, and ookinete. Anti-rPvQSOX IgGs obviously inhibited the ookinetes and oocysts development both in vivo and in vitro using transgenic parasites. Direct membrane feeding assays of anti-rPvQSOX antibodies were conducted using four field P. vivax isolates (named isolates #1-4) in Thailand. Oocyst density in mosquitoes was significantly reduced by 32.00, 85.96, 43.52, and 66.03% with rabbit anti-rPvQSOX antibodies, respectively. The anti-rPvQSOX antibodies also showed a modest reduction of infection prevalence by 15, 15, 20, and 22.22%, respectively, as compared to the control, while the effect was insignificant. The variation in the DMFA results may be unrelated to the genetic polymorphisms. Compared to the P.vivax Salvador (Sal) I strain sequences, the pvqsox in isolate #1 showed no amino acid substitution, whereas isolates #2, #3, and #4 all had the M361I substitution. Conclusions: Our results suggest that PvQSOX could serve as a potential P. vivax TBVs candidate, which warrants further evaluation and optimization.

Therapeutic glycan-specific antibody binding mediates protection during primary amoebic meningoencephalitis.

Naegleria fowleri (N. fowleri) infection via the upper respiratory tract causes a fatal CNS disease known as primary amoebic meningoencephalitis (PAM). The robust in vivo immune response to N. fowleri infection underlies the immunopathology that characterizes the disease. However, little is known about why this pathogen evades immune control. Infections occur in seemingly healthy individuals and effective clinical options are lacking, thus a nearly 98% fatality rate. It is unclear how or if host factors may contribute to susceptibility or disease exacerbation, yet mechanistic studies of the in vivo immune response and disease progression are hampered by a lack of tools. In this study, we have generated monoclonal antibodies to N. fowleri surface antigens and shown them to be excellent tools for studying the in vivo immune response. We also identified one monoclonal, 2B6, with potent inherent anti-amoebastatic activity in vitro. This antibody is also able to therapeutically prolong host survival in vivo and furthermore, recombinant antibodies with an isotype more capable of directing immune effector activity further improved survival when given therapeutically. Thus, we report the generation of a novel monoclonal antibody to N. fowleri that can enhance beneficial immune functions, even when given therapeutically during disease. We believe this provides evidence for the potential of therapeutic antibody treatments in PAM.IMPORTANCENaegleria fowleri (N. fowleri) is a free-living amoeba that is found ubiquitously in warm freshwater. While human exposure is common, it rarely results in pathogenesis. However, when N. fowleri gains access to the upper airway, specifically the olfactory mucosa, infection leads to a lethal disease known as primary amoebic meningoencephalitis (PAM). As a free-living amoeba, N. fowleri does not need a mammalian host; indeed, it can be accurately described as an accidental opportunistic pathogen. While most opportunistic infections occur in humans who are immunocompromised, there are no reported immune dysfunctions associated with N. fowleri infection. Therefore, the basis for N. fowleri opportunism is not known, and the reasons why some humans develop PAM while others do not are simply not well understood. It is reasonable to speculate that local or acute immune failures, potentially even a lack of prior adaptive immunity, are related to disease susceptibility. Careful immune profiling and characterization of the in vivo immune response to N. fowleri in a mammalian host are desperately needed to understand which host factors are critical to defense, and how these responses might be compromised in a way that results in lethal infection. To identify genes and pathways that provide resistance against in vivo N. fowleri infection, we generated surface reactive monoclonal antibodies (Abs) that provide rapid amoeba detection and quantification in vivo. Interestingly, N. fowleri binding Abs have been readily detected in the serum and saliva of humans and animals suggesting that non-lethal exposure drives a humoral immune response against the amoeba. Yet, how Abs might interact with Naegleria in vivo or contribute to preventing lethal infection is not well understood. In this study, we have generated and characterized a monoclonal antibody (Ab), Clone 2B6, that recognizes a glycosylated surface antigen present in cultured in vitro N. fowleri as well as mouse passaged N. fowleri. When clone 2B6 binds to N. fowleri, it inhibits amoeba motility and feeding behavior, leading to strong growth inhibition. Mice treated systemically and intracerebrally with Ab displayed a delayed disease onset and prolonged survival. In addition, we found that enhancing immune-directed effector activity via antibody isotype could further enhance survival without obvious immunopathogenic side effects. These findings show the potential for antibody treatment as an additional therapeutic to those used currently in PAM.

Heterologous immunization targeting the CST1 antigen confers better protection than ROP18 in mice.

Aim: To evaluate the protective efficacy induced by heterologous immunization with recombinant baculoviruses or virus-like particles targeting the CST1 and ROP18 antigens of Toxoplasma gondii.Materials & methods: Recombinant baculovirus and virus-like particle vaccines expressing T. gondii CST1 or ROP18 antigens were developed to evaluate protective immunity in mice upon challenge infection with 450 Toxoplasma gondii (ME49).Results: Immunization with CST1 or ROP18 vaccines induced similar levels of T. gondii-specific IgG and IgA responses. Compared with ROP 18, CST1 vaccine showed better antibody-secreting cell response, germinal center B cell activation, and significantly reduced brain cyst burden and body weight loss.Conclusion: Our findings suggest that CST1 heterologous immunization elicited better protection than ROP18, providing important insight into improving the toxoplasmosis vaccine design strategy.

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A Multi-Epitope Protein for High-Performance Serodiagnosis of Chronic Chagas Disease in ELISA and Lateral Flow Platforms.

We developed a protein to rapidly and accurately diagnose Chagas disease, a life-threatening illness identified by the WHO as a critical worldwide public health risk. Limitations in present day serological tests are complicating the current health situation and contributing to most infected persons being unaware of their condition and therefore untreated. To improve diagnostic testing, we developed an immunological mimic of the etiological agent, Trypanosoma cruzi, by combining ten pathogen-specific epitopes within the beta-barrel protein structure of Thermal Green Protein. The resulting multi-epitope protein, DxCruziV3, displayed high specificity and sensitivity as the antibody capture reagent in an ELISA platform with an analytical sensitivity that exceeds WHO recommendations. Within an immunochromatographic platform, DxCruziV3 showed excellent performance for the point of application diagnosis in a region endemic for multiple diseases, the municipality of Barcelos in the state of Amazonas, Brazil. In total, 167 individuals were rapidly tested using whole blood from a finger stick. As recommended by the Brazilian Ministry of Health, venous blood samples were laboratory tested by conventional assays for comparison. Test results suggest utilizing DxCruziV3 in different assay platforms can confidently diagnose chronic infections by T. cruzi. Rapid and more accurate results will benefit everyone but will have the most noticeable impact in resource-limited rural areas where the disease is endemic.

Molecular Mimicry between Toxoplasma gondii B-Cell Epitopes and Neurodevelopmental Proteins: An Immunoinformatic Approach.

Epidemiological studies and meta-analyses have shown a strong association between high seroprevalence of Toxoplasma gondii (T. gondii) and schizophrenia. Schizophrenic patients showed higher levels of anti-Toxoplasma immunoglobulins M and G (IgM and IgG) when compared to healthy controls. Previously, in a rat model, we demonstrated that the progeny of mothers immunized with T. gondii lysates before gestation had behavioral and social impairments during adulthood. Therefore, we suggested that T. gondii infection can trigger autoreactivity by molecularly mimicking host brain proteins. Here, we aimed to identify the occurrence of antigenic mimicry between T. gondii epitopes and host brain proteins. Using a bioinformatic approach, we predicted T. gondii RH-88 B cell epitopes and compared them to human cell-surface proteins involved in brain development and differentiation (BrainS). Five different algorithms for B-cell-epitope prediction were used and compared, resulting in 8584 T. gondii epitopes. We then compared T. gondii predicted epitopes to BrainS proteins by local sequence alignments using BLASTP. T. gondii immunogenic epitopes significantly overlapped with 42 BrainS proteins. Among these overlapping proteins essential for brain development and differentiation, we identified HSP90 and NOTCH receptors as the proteins most likely to be targeted by the maternally generated pathogenic antibodies due to their topological overlap at the extracellular region of their sequence. This analysis highlights the relevance of pregestational clinical surveillance and screening for potential pathogenic anti-T. gondii antibodies. It also identifies potential targets for the design of vaccines that could prevent behavioral and cognitive impairments associated with pre-gestational T. gondii exposure.

Children with hemoglobin C or S trait have low serologic responses to a subset of malaria variant surface antigens.

Children with hemoglobin AC or AS have decreased susceptibility to clinical malaria. Parasite variant surface antigen (VSA) presentation on the surface of infected erythrocytes is altered in erythrocytes with hemoglobin C (Hb AC) or sickle trait (Hb AS) mutations in vitro. The protective role of incomplete or altered VSA presentation against clinical malaria in individuals with Hb AC or AS is unclear. Using a high-throughput protein microarray, we sought to use serological responses to VSAs as a measure of host exposure to VSAs among Malian children with Hb AC, Hb AS, or wildtype hemoglobin (Hb AA). In uncomplicated malaria, when compared to Hb AA children, Hb AC children had significantly lower serological responses to extracellular Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) domains but did not differ in responses to intracellular PfEMP1 domains and other VSAs, including members of the repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR) family. Healthy children with Hb AC and Hb AS genotypes recognized fewer extracellular PfEMP1s compared to children with Hb AA, especially CD36-binding PfEMP1s. These reduced serologic responses may reflect reduced VSA presentation or lower parasite exposure in children with Hb AC or AS and provide insights into mechanisms of protection.

Inhibition of Plasmodium knowlesi merozoite invasion into human erythrocytes by antibodies raised against the parasite's secreted protein with altered thrombospondin repeat (SPATR).

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.

Anti-Toxoplasma gondii IgG seroprevalence in the general population in Iran: A systematic review and meta-analysis, 2000-2023.

Toxoplasmosis ranks among the most prevalent parasitic diseases globally. It seems that chronic toxoplasmosis is associated with several neuropsychiatric and other harmful effects in infected people, therefore, there is a need to investigate the prevalence of toxoplasmosis across various world regions. In this study, we conducted a meticulous meta-analysis to estimate the seroprevalence of anti-Toxoplasma gondii IgG antibodies within the general population in Iran (GPI). International and national scientific databases for studies published between January 1, 2000, and September 30, 2023, were searched. Observational studies reporting anti-T. gondii IgG seroprevalence in the GPI was selected/included. The data were synthesized using a random-effects model to calculate with a 95% confidence interval (95% CI) the national and regional anti-T. gondii IgG seroprevalence rates in Iran. Additionally, subgroup analyses were conducted to investigate the frequency of exposition to T. gondii in different socio-demographic, climatic, and geographical scenarios. From 18661 identified studies, 327 were included in the present meta-analysis, encompassing 122,882 individuals across the 31 Iranian provinces. The pooled nationwide anti-T. gondii IgG seroprevalence among the GPI was determined to be 32.9% (95% CI: 30.9-35.1%). The highest anti-T. gondii IgG seroprevalence was observed in Mazandaran province (North of Iran) (61%), whereas the lowest was in Semnan province (12.5%).Anti-T. gondii IgG seroprevalence demonstrated a higher occurrence in provinces characterized by moderate temperatures of 16-21°C, high relative humidity, and annual precipitation. Additionally, a higher anti-T. gondii IgG seroprevalence was identified among individuals with a habit of consumption of undercooked meat, raw fruits or vegetables, and untreated water. Moreover, those reporting direct contact with cats, possessing a lower level of education, residing in rural areas, being engaged in farming occupations, or playing the role of housewives exhibited higher anti-T. gondii IgG seroprevalence figures.The anti-T. gondii IgG seroprevalence within GPI closely aligns with the estimated worldwide average exposition rates. This underscores the imperative for public health policymakers to prioritize educational efforts regarding toxoplasmosis transmission pathways and its link to harmful effects.