Immunogenicity of PvCyRPA, PvCelTOS and Pvs25 chimeric recombinant protein of Plasmodium vivax in murine model.

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.

A Nanobody/Monoclonal Antibody "hybrid" sandwich technology offers an improved immunoassay strategy for detection of African trypanosome infections.

The scarcity of reliable devices for diagnosis of Animal African trypanosomiasis (AAT) presents a limitation to control of the disease. Existing high-sensitivity technologies such as PCR are costly, laborious, time-consuming, complex, and require skilled personnel. Hence, utilisation of most diagnostics for AAT is impracticable in rural areas, where the disease occurs. A more accessible point-of-care test (POCT) capable of detecting cryptic active infection, without relying on expensive equipment, would facilitate AAT detection. In turn, early management, would reduce disease incidence and severity. Today, several ongoing research projects aim at modifying complex immunoassays into POCTs. In this context, we report the development of an antigen (Ag) detection sandwich ELISA prototype for diagnosis of T. congolense infections, which is comprised of nanobody (Nb) and monoclonal antibody (mAb) reagents. The Nb474H used here, originated from a past study. Briefly, the Nb was engineered starting from mRNA of peripheral blood lymphocytes of an alpaca immunized with soluble lysate of Trypanosoma congolense (TC13). T. congolense glycosomal fructose-1,6-bisphosphate aldolase (TcoALD) was discovered as the cognate Ag of Nb474H. In this study, splenocytes were harvested from a mouse immunized with recombinant TcoALD and fused with NS01 cells to generate a hybridoma library. Random screening of the library on TcoALD retrieved a lone binder, designated IgM8A2. Using Nb474H as Ag-capture reagent in combination with the IgM8A2 monoclonal antibody Ag-detection reagent resulted in a tool that effectively detects native TcoALD released during infection by T. congolense parasites. Hitherto, development of POCT for detection of active trypanosome infection is elusive. The Nanobody/Monoclonal Antibody (Nb/mAb) "hybrid" sandwich technology offers prospects for exploration, using the unique specificity of Nb as a key determinant in Ag capturing, while using the versatility of monoclonal Ab to adapt to various detection conditions.

Structure-based design of a Plasmodium vivax Duffy-binding protein immunogen focuses the antibody response to functional epitopes.

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.

Preclinical development of a stabilized RH5 virus-like particle vaccine that induces improved antimalarial antibodies.

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.

Identification and serological responses to a novel Plasmodium vivax merozoite surface protein 1 (PvMSP-1) derived synthetic peptide: a putative biomarker for malaria exposure.

Background: The integration of diagnostic methods holds promise for advancing the surveillance of malaria transmission in both endemic and non-endemic regions. Serological assays emerge as valuable tools to identify and delimit malaria transmission, serving as a complementary method to rapid diagnostic tests (RDT) and thick smear microscopy. Here, we evaluate the potential of antibodies directed against peptides encompassing the entire amino acid sequence of the PvMSP-1 Sal-I strain as viable serological biomarkers for P. vivax exposure. Methods: We screened peptides encompassing the complete amino acid sequence of the Plasmodium vivax Merozoite Surface Protein 1 (PvMSP-1) Sal-I strain as potential biomarkers for P. vivax exposure. Here, immunodominant peptides specifically recognized by antibodies from individuals infected with P. vivax were identified using the SPOT-synthesis technique followed by immunoblotting. Two 15-mer peptides were selected based on their higher and specific reactivity in immunoblotting assays. Subsequently, peptides p70 and p314 were synthesized in soluble form using SPPS (Solid Phase Peptide Synthesis) and tested by ELISA (IgG, and subclasses). Results: This study unveils the presence of IgG antibodies against the peptide p314 in most P. vivax-infected individuals from the Brazilian Amazon region. In silico B-cell epitope prediction further supports the utilization of p314 as a potential biomarker for evaluating malaria transmission, strengthened by its amino acid sequence being part of a conserved block of PvMSP-1. Indeed, compared to patients infected with P. falciparum and uninfected individuals never exposed to malaria, P. vivax-infected patients have a notably higher recognition of p314 by IgG1 and IgG3.

[Preparation and preliminary application of the polyclonal antibody against Toxoplasma gondii dense granule protein 24].

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.

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex.

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.

Machine learning prediction of malaria vaccine efficacy based on antibody profiles.

Immunization through repeated direct venous inoculation of Plasmodium falciparum (Pf) sporozoites (PfSPZ) under chloroquine chemoprophylaxis, using the PfSPZ Chemoprophylaxis Vaccine (PfSPZ-CVac), induces high-level protection against controlled human malaria infection (CHMI). Humoral and cellular immunity contribute to vaccine efficacy but only limited information about the implicated Pf-specific antigens is available. Here, we examined Pf-specific antibody profiles, measured by protein arrays representing the full Pf proteome, of 40 placebo- and PfSPZ-immunized malaria-naïve volunteers from an earlier published PfSPZ-CVac dose-escalation trial. For this purpose, we both utilized and adapted supervised machine learning methods to identify predictive antibody profiles at two different time points: after immunization and before CHMI. We developed an adapted multitask support vector machine (SVM) approach and compared it to standard methods, i.e. single-task SVM, regularized logistic regression and random forests. Our results show, that the multitask SVM approach improved the classification performance to discriminate the protection status based on the underlying antibody-profiles while combining time- and dose-dependent data in the prediction model. Additionally, we developed the new fEature diStance exPlainabilitY (ESPY) method to quantify the impact of single antigens on the non-linear multitask SVM model and make it more interpretable. In conclusion, our multitask SVM model outperforms the studied standard approaches in regard of classification performance. Moreover, with our new explanation method ESPY, we were able to interpret the impact of Pf-specific antigen antibody responses that predict sterile protective immunity against CHMI after immunization. The identified Pf-specific antigens may contribute to a better understanding of immunity against human malaria and may foster vaccine development.

Repeat controlled human Plasmodium falciparum infections delay bloodstream patency and reduce symptoms.

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.

ELISA with recombinant antigen Lb6H validated for the diagnosis of American tegumentary leishmaniasis.

American tegumentary leishmaniasis (ATL) diagnosis is an open question, and the search for a solution is urgent. The available tests that detect the etiological agent of the infection are specific for ATL diagnosis. However, they present disadvantages, such as low sensitivity and the need for invasive procedures to obtain the samples. Immunological methods (leishmanin skin test and search for anti-Leishmania antibodies) are good alternatives to the etiological diagnosis of ATL. Presently, we face problems with disease confirmation due to the discontinuity in the production of leishmanin skin test antigen, particularly in resource-poor settings. Aiming to diagnose ATL, we validated rLb6H-ELISA for IgG antibodies using 1,091 samples from leishmaniasis patients and healthy controls, divided into four panels, living in 19 Brazilian endemic and non-endemic states. The rLb6H-ELISA showed a sensitivity of 98.6% and a specificity of 100.0%, with the reference panel comprising 70 ATL patient samples and 70 healthy controls. The reproducibility evaluation showed a coefficient of variation of positive samples ≤ 8.20% for repeatability, ≤ 17,97% for reproducibility, and ≤ 8.12% for homogeneity. The plates sensitized with rLb6H were stable at 4°C and -20°C for 180 days and 37°C for seven days, indicating 12 months of validity. In samples of ATL patients from five research and healthcare centers in endemic and non-endemic areas, rLb6H-ELISA showed a sensitivity of 84.0%; no significant statistical difference was observed among the five centers (chi-square test, p = 0.13). In samples of healthy controls from four areas with different endemicity, a specificity of 92.4% was obtained; lower specificity was obtained in a visceral leishmaniasis high endemicity locality (chi-square test, p<0.001). Cross-reactivity was assessed in 166 other disease samples with a positivity of 13.9%. Based on the good diagnostic performance and the reproducibility and stability of the antigen, we suggest using ELISA-rLb6H to diagnose ATL.

Immunization with PfGBP130 generates antibodies that inhibit RBC invasion by P. falciparum parasites.

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.

From proteome to candidate vaccines: target discovery and molecular dynamics-guided multi-epitope vaccine engineering against kissing bug.

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.

Plasmodium vivax serological exposure markers: PvMSP1-42-induced humoral and memory B-cell response generates long-lived antibodies.

Plasmodium vivax serological exposure markers (SEMs) have emerged as promising tools for the actionable surveillance and implementation of targeted interventions to accelerate malaria elimination. To determine the dynamic profiles of SEMs in current and past P. vivax infections, we screened and selected 11 P. vivax proteins from 210 putative proteins using protein arrays, with a set of serum samples obtained from patients with acute P. vivax and documented past P. vivax infections. Then we used a murine protein immune model to initially investigate the humoral and memory B cell response involved in the generation of long-lived antibodies. We show that of the 11 proteins, especially C-terminal 42-kDa region of P. vivax merozoite surface protein 1 (PvMSP1-42) induced longer-lasting long-lived antibodies, as these antibodies were detected in individuals infected with P. vivax in the 1960-1970s who were not re-infected until 2012. In addition, we provide a potential mechanism for the maintenance of long-lived antibodies after the induction of PvMSP1-42. The results indicate that PvMSP1-42 induces more CD73+CD80+ memory B cells (MBCs) compared to P. vivax GPI-anchored micronemal antigen (PvGAMA), allowing IgG anti-PvMSP1-42 antibodies to be maintained for a long time.

Detection of naturally acquired, strain-transcending antibodies against rosetting Plasmodium falciparum strains in humans.

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.

Virus-like particles expressing microneme-associated antigen of Plasmodium berghei confer better protection than those expressing apical membrane antigen 1.

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.

In silico study to explore the mechanism of Toxoplasma-induced inflammation and target therapy based on sero and salivary Toxoplasma.

We aimed to assess salivary and seroprevalence of Toxoplasma immunoglobulins in risky populations and evaluate drug docking targeting TgERP. A cross-sectional study was conducted in Alexandria University hospitals' outpatient clinics. 192 participants were enrolled from September 2022 to November 2023. Anti-Toxoplasma IgG and IgM were determined in serum and saliva by ELISA. An in-Silico study examined TgERP's protein-protein interactions (PPIs) with pro-inflammatory cytokine receptors, anti-inflammatory cytokine, cell cycle progression regulatory proteins, a proliferation marker, and nuclear envelope integrity-related protein Lamin B1. Our findings revealed that anti-T. gondii IgG were detected in serum (66.1%) and saliva (54.7%), with 2.1% of both samples were positive for IgM. Salivary IgG had 75.59% sensitivity, 86.15% specificity, 91.40% PPV, 64.40% NPP, 79.17% accuracy and fair agreement with serum IgG. On the other hand, the sensitivity, specificity, PPV, NPV, and accuracy in detecting salivary IgM were 75.0%, 99.47%, 75.0%, 99.47%, and 98.96%. AUC 0.859 indicates good discriminatory power. Examined synthetic drugs and natural products can target specific amino acids residues of TgERP that lie at the same binding interface with LB1 and Ki67, subsequently, hindering their interaction. Hence, salivary samples can be a promising diagnostic approach. The studied drugs can counteract the pro-inflammatory action of TgERP.

Vaccine efficacy induced by virus-like particles containing Leishmania donovani surface glycoprotein GP63.

Leishmania donovani surface glycoprotein 63 (GP63) is a major virulence factor involved in parasite escape and immune evasion. In this study, we generated virus-like particles (VLPs) expressing L. donovani GP63 using the baculovirus expression system. Mice were intramuscularly immunized with GP63-VLPs and challenged with L. donovani promastigotes. GP63-VLP immunization elicited higher levels of L. donovani antigen-specific serum antibodies and enhanced splenic B cell, germinal center B cell, CD4+, and CD8+ T cell responses compared to unimmunized controls. GP63-VLPs inhibited the influx of pro-inflammatory cytokines IFN-γ and IL-6 in the livers, as well as thwarting the development of splenomegaly in immunized mice. Upon L. donovani challenge infection, a drastic reduction in splenic parasite burden was observed in VLP-immunized mice. These results indicate that GP63-VLPs immunization conferred protection against L. donovani challenge infection by inducing humoral and cellular immunity in mice.

Evaluation of transmission-blocking potential of PvPSOP25 using transgenic murine malaria parasite and clinical isolates.

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.

Identification of Babesia microti immunoreactive antigens by phage display cDNA screen.

Human babesiosis is a malaria-like illness caused by protozoan parasites of the genus Babesia. Babesia microti is responsible for most cases of human babesiosis in the United States, particularly in the Northeast and the Upper Midwest. Babesia microti is primarily transmitted to humans through the bite of infected deer ticks but also through the transfusion of blood components, particularly red blood cells. There is a high risk of severe and even fatal disease in immunocompromised patients. To date, serology testing relies on an indirect immunofluorescence assay that uses the whole Babesia microti antigen. Here, we report the construction of phage display cDNA libraries from Babesia microti-infected erythrocytes as well as human reticulocytes obtained from donors with hereditary hemochromatosis. Plasma samples were obtained from patients who were or had been infected with Babesia microti. The non-specific antibody reactivity of these plasma samples was minimized by pre-exposure to the human reticulocyte library. Using this novel experimental strategy, immunoreactive segments were identified in three Babesia microti antigens termed BmSA1 (also called BMN1-9; BmGPI12), BMN1-20 (BMN1-17; Bm32), and BM4.12 (N1-15). Moreover, our findings indicate that the major immunoreactive segment of BmSA1 does not overlap with the segment that mediates BmSA1 binding to mature erythrocytes. When used in combination, the three immunoreactive segments form the basis of a sensitive and comprehensive diagnostic immunoassay for human babesiosis, with implications for vaccine development.

How are Trypanosoma brucei receptors protected from host antibody-mediated attack?

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.