The presence of circulating antibody secreting cells and long-lived memory B cell responses to reticulocyte binding protein 1a in Plasmodium vivax patients.

BACKGROUND: Development of an effective vaccine against blood-stage malaria requires the induction of long-term immune responses. Plasmodium vivax Reticulocyte Binding Protein 1a (PvRBP1a) is a blood-stage parasite antigen which is associated with invasion of red blood cells and induces antibody responses. Thus, PvRBP1a is considered as a target for design of a blood-stage vaccine against vivax malaria. METHODS: Both cross-sectional and cohort studies were used to explore the development and persistence of long-lived antibody and memory B cell responses to PvRBP1a in individuals who lived in an area of low malaria endemicity. Antibody titers and frequency of memory B cells specific to PvRBP1a were measured during infection and following recovery for up to 12 months. RESULTS: IgG antibody responses against PvRBP1a were prevalent during acute vivax malaria, predominantly IgG1 subclass responses. High responders to PvRBP1a had persistent antibody responses for at least 12-month post-infection. Further analysis of high responder found a direct relation between antibody titers and frequency of activated and atypical memory B cells. Furthermore, circulating antibody secreting cells and memory B cells specific to PvRBP1a were generated during infection. The PvRBP1a-specific memory B cells were maintained for up to 3-year post-infection, indicating the ability of PvRBP1a to induce long-term humoral immunity. CONCLUSION: The study revealed an ability of PvRBP1a protein to induce the generation and maintenance of antibody and memory B cell responses. Therefore, PvRBP1a could be considered as a vaccine candidate against the blood-stage of P. vivax.

Longitudinal analysis of antibody responses to Plasmodium vivax sporozoite antigens following natural infection.

BACKGROUND: P. vivax malaria is a major global health burden hindering social and economic development throughout many tropical and sub-tropical countries. Pre-erythrocytic (PE) vaccines emerge as an attractive approach for the control and elimination of malaria infection. Therefore, evaluating the magnitude, longevity and prevalence of naturally acquired IgG antibody responses against PE candidate antigens is useful for vaccine design. METHODOLOGY/PRINCIPAL FINDINGS: The antigenicity of five recombinant PE antigens (PvCSP-VK210, PvSSP3, PvM2-MAEBL, PvCelTOS and PvSPECT1) was evaluated in plasma samples from individuals residing in low transmission areas in Thailand (Ranong and Chumphon Provinces). The samples were collected at the time of acute vivax malaria and 90, 270 and 360 days later. The prevalence, magnitude and longevity of total IgG and IgG subclasses were determined for each antigen using the longitudinal data. Our results showed that seropositivity of all tested PE antigens was detected during infection in at least some subjects; anti-PvCSP-VK210 and anti-PvCelTOS antibodies were the most frequent. Titers of these antibodies declined during the year of follow up, but notably seropositivity persisted. Among seropositive subjects at post-infection, high number of subjects possessed antibodies against PvCSP-VK210. Anti-PvSSP3 antibody responses had the longest half-life. IgG subclass profiling showed that the predominant subclasses were IgG1 and IgG3 (cytophilic antibodies), tending to remain detectable for at least 360 days after infection. CONCLUSIONS/SIGNIFICANCE: The present study demonstrated the magnitude and longevity of serological responses to multiple PE antigens of P. vivax after natural infection. This knowledge could contribute to the design of an effective P. vivax vaccine.

Naturally acquired antibodies against Plasmodium vivax pre-erythrocytic stage vaccine antigens inhibit sporozoite invasion of human hepatocytes in vitro.

In Plasmodium vivax, the most studied vaccine antigens are aimed at blocking merozoite invasion of erythrocytes and disease development. Very few studies have evaluated pre-erythrocytic (PE) stage antigens. The P. vivax circumsporozoite protein (CSP), is considered the leading PE vaccine candidate, but immunity to CSP is short-lived and variant specific. Thus, there is a need to identify other potential candidates to partner with CSP in a multivalent vaccine to protect against infection and disease. We hypothesize that sporozoite antigens important for host cell infection are considered potential targets. In this study, we evaluated the magnitude and quality of naturally acquired antibody responses to four P. vivax PE antigens: sporozoite surface protein 3 (SSP3), sporozoite protein essential for traversal 1 (SPECT1), cell traversal protein of ookinetes and sporozoites (CelTOS) and CSP in plasma of P. vivax infected patients from Thailand. Naturally acquired antibodies to these antigens were prevalent in the study subjects, but with significant differences in magnitude of IgG antibody responses. About 80% of study participants had antibodies to all four antigens and only 2% did not have antibodies to any of the antigens. Most importantly, these antibodies inhibited sporozoite infection of hepatocytes in vitro. Significant variations in magnitude of antigen-specific inhibitory antibody responses were observed with individual samples. The highest inhibitory responses were observed with anti-CelTOS antibodies, followed by anti-SPECT1, SSP3 and CSP antibodies respectively. These data highlight the vaccine potential of these antigens in protecting against hepatocyte infection and the need for a multi-valent pre-erythrocytic vaccine to prevent liver stage development of P. vivax sporozoites.

Characterization of Duffy Binding Protein II-specific CD4+T cell responses in Plasmodium vivax patients.

Plasmodium vivax Duffy Binding Protein region II (PvDBPII) is a leading vaccine candidate against blood-stage vivax malaria. Anti-PvDBPII antibodies potentially block parasite invasion by inhibition of erythrocyte binding. However, knowledge of PvDBPII-specific T cell responses is limited. Here, to assess the responses of PvDBPII-specific CD4+T cells in natural P. vivax infection, three cross-sectional studies were conducted in recovered subjects. In silico analysis was used for potential T cell epitope prediction and selection. PBMCs from P. vivax subjects were stimulated with selected peptides and examined for cytokine production by ELISPOT or intracellular cytokine staining. Six dominant T cell epitopes were identified. Peptide-driven T cell responses showed effector memory CD4+T cell phenotype, secreting both IFN-γ and TNF-α cytokines. Single amino acid substitutions in three T cell epitopes altered levels of IFN-γ memory T cell responses. Seropositivity of anti-PvDBPII antibodies were detected during acute malaria (62%) and persisted up to 12 months (11%) following P. vivax infection. Further correlation analysis showed four out of eighteen subjects had positive antibody and CD4+T cell responses to PvDBPII. Altogether, PvDBPII-specific CD4+T cells were developed in natural P. vivax infections. Data on their antigenicity could facilitate development of an efficacious vivax malaria vaccine.

Preliminary characterization of Plasmodium vivax sporozoite antigens as pre-erythrocytic vaccine candidates.

Plasmodium vivax pre-erythrocytic (PE) vaccine research has lagged far behind efforts to develop Plasmodium falciparum vaccines. There is a critical gap in our knowledge of PE antigen targets that can induce functionally inhibitory neutralizing antibody responses. To overcome this gap and guide the selection of potential PE vaccine candidates, we considered key characteristics such as surface exposure, essentiality to infectivity and liver stage development, expression as recombinant proteins, and functional immunogenicity. Selected P. vivax sporozoite antigens were surface sporozoite protein 3 (SSP3), sporozoite microneme protein essential for cell traversal (SPECT1), sporozoite surface protein essential for liver-stage development (SPELD), and M2 domain of MAEBL. Sequence analysis revealed little variation occurred in putative B-cell and T-cell epitopes of the PE candidates. Each antigen was tested for expression as refolded recombinant proteins using an established bacterial expression platform and only SPELD failed. The successfully expressed antigens were immunogenic in vaccinated laboratory mice and were positively reactive with serum antibodies of P. vivax-exposed residents living in an endemic region in Thailand. Vaccine immune antisera were tested for reactivity to native sporozoite proteins and for their potential vaccine efficacy using an in vitro inhibition of liver stage development assay in primary human hepatocytes quantified on day 6 post-infection by high content imaging analysis. The anti-PE sera produced significant inhibition of P. vivax sporozoite invasion and liver stage development. This report provides an initial characterization of potential new PE candidates for a future P. vivax vaccine.

Interferon-γ signal drives differentiation of T-bethi atypical memory B cells into plasma cells following Plasmodium vivax infection.

For development of a long-lasting protective malaria vaccine, it is crucial to understand whether Plasmodium-induced memory B cells (MBCs) or plasma cells develop and stably contribute to protective immunity, or on the contrary the parasite suppresses antibody responses by inducing MBC dysfunction. The expansion of T-bethi atypical MBCs is described in chronic Plasmodium falciparum-exposed individuals. However, it remains unclear whether accumulation of T-bethi atypical MBCs is indicative of a protective role or rather an impaired function of the immune system in malaria. Here, the phenotypic and functional features of T-bethi atypical MBCs were studied in P. vivax patients living in an area of low malaria transmission. During P. vivax infection, the patients produced a twofold higher frequency of T-bethi atypical MBCs compared to malaria non-exposed individuals. This distinct atypical MBC subset had a switched IgG phenotype with overexpression of activation markers and FcRL5, and decreased Syk phosphorylation upon BCR stimulation. Post-infection, expansion of T-bethi IgG+ atypical MBCs was maintained for at least 3 months. Further studies of the contribution of T-bethi atypical MBC function to humoral immunity showed that synergizing IFN-γ with TLR7/8 and IL-21 signals was required for their differentiation into plasma cells and antibody secretion.

Cross-reactive inhibitory antibody and memory B cell responses to variant strains of Duffy binding protein II at post-Plasmodium vivax infection.

Duffy binding protein region II (DBPII) is considered a strong potential vaccine candidate of blood-stage P. vivax. However, the highly polymorphic nature of this protein often misdirects immune responses, leading them to be strain-specific. Details of cross-reactive humoral immunity to DBPII variants have therefore become an important focus for the development of broadly protective vaccines. Here, cross-reactive humoral immunity against a panel of Thai DBPII variants (DBL-THs) was demonstrated in immunized BALB/c mice and P. vivax patients, by in vitro erythrocyte-binding inhibition assay. Sera from immunized animals showed both strain-transcending (anti-DBL-TH2 and -TH4) and strain-specific (anti-DBL-TH5, -TH6 and -TH9) binding to DBL-TH variants. Using anti-DBL-TH sera at 50% inhibitory concentration (IC50) of the homologous strain, anti-DBL-TH2 sera showed cross inhibition to heterologous DBL-TH strains, whereas anti-DBL-TH5 sera exhibited only strain-specific inhibition. In P. vivax patients, 6 of 15 subjects produced and maintained cross-reactive anti-DBL-TH inhibitory antibodies through the 1-year post-infection timepoint. Cross-reactive memory B cell (MBC) responses to DBL-TH variants were analyzed in subjects recovered from P. vivax infection (RC). The plasma samples from 5 RC subjects showed broad inhibition. However, MBC-derived antibodies of these patients did not reveal cross-inhibition. Altogether, broadly anti-DBP variant inhibitory antibodies developed and persisted in P. vivax infections. However, the presence of cross-reactive anti-DBL-TH inhibitory function post-infection was not related with MBC responses to these variants. More detailed investigation of long-lasting, broadly protective antibodies to DBPII will guide the design of vivax malaria vaccines.

CD4+ T-cell cooperation promoted pathogenic function of activated naïve B cells of patients with SLE.

OBJECTIVE: To explore cooperation between activated naïve (aNAV) B cells and CD4+ T cells in the pathogenesis of SLE through autoantibody production, T-cell differentiation and inflammatory cytokine secretion. METHODS: Peripheral blood mononuclear cell samples were obtained from 31 patients with SLE and used to characterise phenotype of aNAV B cells (n=14) and measured the phosphorylation of B-cell receptor (BCR) signalling molecules (n=5). Upregulation of T-cell costimulatory molecules after BCR and toll-like receptor (TLR)-7/TLR-8 stimulation was detected in cells from four subjects. To explore the role of these cells in SLE pathogenesis via T cell-dependent mechanisms, four subjects were analysed to detect the promotion of CD4+ T-cell activation and antibody-secreting cell (ASC) differentiation after CD4+ T-cell-B-cell cocultures. The aNAV B cells from four patients were used to assess cytokine secretion. RESULTS: The aNAV B cells of patients with SLE had increased expression of surface CD40, HLA-DR and interleukin-21 receptor (IL-21R) and FCRL5 molecules. With BCR stimulation, these cells greatly increased PLCγ2 phosphorylation. Integrated BCR and TLR-7/TLR-8 signals induced overexpression of CD40, CD86, IL-21R and HLA-DR on lupus aNAV B cells. In T-cell-B-cell cocultures, lupus aNAV B cells (with upregulated costimulatory molecules) promoted CD4+ T-cell proliferation and polarisation toward effector Th2 and Th17 cells. Importantly, in this coculture system, CD4+ T-cell signals enhanced aNAV B-cell differentiation into auto-ASCs and produced anti-DNA antibodies. The interaction between CD4+ T cell and aNAV B cell increased production of inflammatory cytokines (IL-6, IL-8 and IL-23). CONCLUSION: Cooperation between aNAV B cells and CD4+ T cells contributed to SLE pathogenesis by promoting both differentiation of pathogenic T cells (Th2 and Th17) and autoantibody secretion.

Mechanistic investigation of phosphoprotein enrichment by fly ash-based chromatography.

In this work, the mechanistic details contributing to the binding of phosphoproteins on fly ash (FA) has been investigated. The effects of factors influencing adsorption of phosphoprotein, i.e., contact time, pH, ionic strength, initial concentration of proteins, and contribution of ligand exchange, were thoroughly examined. Results showed that the adsorption efficiency of phosphoproteins to FA was enhanced with increasing contact time. Intriguingly, the adsorption of phosphoproteins to FA was not profoundly affected by high ionic strength, suggesting that electrostatic interaction does not play a pivotal role in phosphoprotein binding on the surface of FA particles. The interaction between phosphoproteins and FA could be instead disturbed when NaF and phosphate ion were used as competing electrolytes/ions. Also, it was found that at a high pH condition has a substantial effect on the adsorption of phosphoproteins through ligand exchange mechanism. To this end, our results clearly indicated that ligand exchange mechanism exerted by F-, phosphate ion and hydroxide ion with the metal oxide surface of FA is the mechanism that majorly contributed to the phosphoprotein binding on the surface of FA particles.

Fly-ash as a low-cost material for isolation of phosphoproteins.

Metal oxide affinity chromatography (MOAC) is one of the most commonly used techniques for selective isolation phosphoproteins and phosphopeptides. This technique is capable of capturing the phosphorylated biomolecules through the affinity of the phosphoryl group for metal oxides/hydroxides. Fly-ash (FA), a by-product of coal-combustion power plants, is primarily composed of oxides of silicon and metals, among which iron and titanium. A number of studies have demonstrated the potential of these metal oxides for phosphoprotein and phosphopeptide enrichment. FA is annually produced over hundred million tons worldwide and generally considered as hazardous waste. It is thus of great importance to enhance its utilization. Here we present the first demonstration of the utility of FA as a low-cost MOAC material for the enrichment of phosphoproteins. With an FA-microcolumn, phosphoproteins can be successfully sequestered from other proteins. FA-microcolumns are shown to be simple, cheap and selective devices for phosphoprotein enrichment from a small volume of mixtures.