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.

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.

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.

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.

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.

The expansion of activated naive DNA autoreactive B cells and its association with disease activity in systemic lupus erythematosus patients.

BACKGROUND: Autoreactive B cells are well recognized as key participants in the pathogenesis of systemic lupus erythematosus (SLE). However, elucidating the particular subset of B cells in producing anti-dsDNA antibodies is limited due to their B cell heterogeneity. This study aimed to identify peripheral B cell subpopulations that display autoreactivity to DNA and contribute to lupus pathogenesis. METHODS: Flow cytometry was used to detect total B cell subsets (n = 20) and DNA autoreactive B cells (n = 15) in SLE patients' peripheral blood. Clinical disease activities were assessed in SLE patients using modified SLEDAI-2 K and used for correlation analyses with expanded B cell subsets and DNA autoreactive B cells. RESULTS: The increases of circulating double negative 2 (DN2) and activated naïve (aNAV) B cells were significantly observed in SLE patients. Expanded B cell subsets and DNA autoreactive B cells represented a high proportion of aNAV B cells with overexpression of CD69 and CD86. The frequencies of aNAV B cells in total B cell populations were significantly correlated with modified SLEDAI-2 K scores. Further analysis showed that expansion of aNAV DNA autoreactive B cells was more related to disease activity and serum anti-dsDNA antibody levels than to total aNAV B cells. CONCLUSION: Our study demonstrated an expansion of aNAV B cells in SLE patients. The association between the frequency of aNAV B cells and disease activity patients suggested that these expanded B cells may play a role in SLE pathogenesis.

The persistence of naturally acquired antibodies and memory B cells specific to rhoptry proteins of Plasmodium vivax in patients from areas of low malaria transmission.

BACKGROUND: Rhoptries are the large, paired, secretory organelles located at the apical tip of the malaria merozoite that are considered important for parasite invasion processes. Plasmodium vivax rhoptry proteins have been shown to induce humoral immunity during natural infections. Therefore, these proteins may be potential novel vaccine candidates. However, there is a lack of data on the duration of antibody and memory B cell (MBC) responses. Here, the longitudinal analysis of antibody and MBC responses to the P. vivax rhoptry proteins PvRALP1-Ecto and PvRhopH2 were monitored and analysed in individuals to determine their persistence. METHODS: Thirty-nine samples from P. vivax-infected subjects (age 18-60 years) were recruited to explore the frequency and persistence of antibody and MBC responses against rhoptry proteins (PvRALP1-Ecto and PvRhopH2) using both cross-sectional and longitudinal cohort study designs. Antibody levels were determined by ELISA during clinical malaria, and at 3, 9 and 12 months post-infection. The frequency of MBC sub-sets and presence of rhoptry-specific MBCs in subjects 18 months after treatment were detected by flow cytometry and ELISPOT assay. RESULTS: The seroprevalence of antibodies against PvRALP1-Ecto and PvRhopH2 proteins was found to be high during acute infection, with IgG1, IgG2 and IgG3 sub-classes predominant. However, these anti-rhoptry responses were short-lived and significantly decreased at 9 months post-infection. To relate the durability of these antibody responses to MBC persistence at post-infection, 18-month post-infection peripheral blood mononuclear cells (PBMCs) samples were taken to detect rhoptry-specific MBCs and frequency of MBC sub-sets, and correlate with antibody responses. These late post-infection samples revealed that rhoptry-specific MBCs were present in about 70% of total subjects. However, the persistence of specific MBCs was not correlated with antibody responses as the majority of malaria subjects who were positive for PvRALP1-Ecto- or PvRhopH2-specific MBCs were seronegative for the rhoptry antigens. The frequencies of classical MBCs were increased after infection, whereas those of activated and atypical MBCs were decreased, indicating that MBC responses could switch from activated or atypical MBCs to classical MBCs after parasite clearance, and were maintained in blood circulating at post-infection. CONCLUSION: The study showed that rhoptry antigens induced the development and persistence of MBC responses in P. vivax-infected subjects who lived in a region of low malaria transmission, which were not related to the longevity of antibody responses.

The acquisition of long-lived memory B cell responses to merozoite surface protein-8 in individuals with Plasmodium vivax infection.

BACKGROUND: The ability of a malaria antigen to induce effective, long-lasting immune responses is important for the development of a protective malaria vaccine. Plasmodium vivax merozoite surface protein-8 (PvMSP8) has been shown to be immunogenic in natural P. vivax infections and produces both cell-mediated and antibody-mediated immunity. Thus, PvMSP8 has been proposed as a vaccine candidate following fusion with other merozoite antigens in blood stage vaccine design. Here, the long-term responses of antibodies and memory B cells (MBCs) specific to PvMSP8 in individuals were monitored in a longitudinal cohort study. METHODS: Both cross-sectional surveys and cohort studies were utilized to explore the persistence of antibody and MBC responses to PvMSP8. Antibody titers were detected in individuals with acute disease and those who recovered from an infection for 4 years. The dominant peptide epitope of PvMSP8 recognized by naturally acquired antibodies was examined to observe the durability of the post-infection antibody response. PvMSP8-specific MBCs were also in subjects 4 years post-infection using an enzyme-linked immunospot assay. RESULTS: The prevalence of antibodies to PvMSP8 was high during and after infection. The antibody levels in individual responders were monitored for up to 12 months post-infection and showed that most patients maintained their seropositive response. Interestingly, the anti-PvMSP8 antibody responses stably persisted in some patients who had recovered from an infection for 4 years. Positive PvMSP8-specific MBCs were also detected at 4 years post-infection. However, analysis in these individuals showed no correlation with the presence or titer of circulating antibody. CONCLUSION: PvMSP8 had the ability to induce a long-term humoral immune response. The antibodies and MBCs specific for this antigen developed and persisted in subjects who acquired a natural P. vivax infection. Inclusion of the PvMSP8 antigen in blood stage vaccine design should be considered.