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BackgroundUnderstanding the characteristics of the humoral immune responses following COVID-19 vaccinations is crucial for refining vaccination strategies and predicting immune responses to emerging SARS-CoV-2 variants.MethodsA longitudinal analysis of SARS-CoV-2 spike receptor binding domain (RBD) specific IgG antibody responses, encompassing IgG subclasses IgG1, IgG2, IgG3, and IgG4 was performed. Participants received four mRNA vaccine doses (group 1; n=10) or two ChAdOx1 nCoV-19 and two mRNA booster doses (group 2; n=19) in Bangladesh over two years.ResultsFindings demonstrate robust IgG responses after primary Covishield or mRNA doses; declining to baseline within six months. First mRNA booster restored and surpassed primary IgG responses but waned after six months. Surprisingly, a second mRNA booster did not increase IgG levels further. Comprehensive IgG subclass analysis showed primary Covishield/mRNA vaccination generated predominantly IgG1 responses with limited IgG2/IgG3, Remarkably, IgG4 responses exhibited a distinct pattern. IgG4 remained undetectable initially but increased extensively six months after the second mRNA dose, eventually replacing IgG1 after the 3rd/4th mRNA doses. Conversely, initial Covishield recipients lack IgG4, surged post-second mRNA booster. Notably, mRNA-vaccinated individuals displayed earlier, robust IgG4 levels post first mRNA booster versus Covishield counterparts. IgG1 to IgG4 ratios decreased with increasing doses, most pronounced with four mRNA doses. This study highlights IgG response kinetics, influenced by vaccine type and doses, impacting immunological tolerance and IgG4 induction, shaping future vaccination strategies.ConclusionsThis study highlights the dynamics of IgG responses dependent on vaccine type and number of doses, leading to immunological tolerance and IgG4 induction, and shaping future vaccination strategies.

BackgroundInfection with enterotoxigenic Escherichia coli (ETEC) gives rise to IgA antibodies against both the heat labile toxin (LT) and colonization factors (CFs), which are considered to synergistically protect against ETEC diarrhea. Since the development of ETEC-specific long lived plasma cells and memory B cells is likely to be dependent on T helper (Th) cells, we investigated if natural ETEC diarrhea elicits ETEC-specific Th cells and their relation to IgA responses.MethodsTh cell subsets were analyzed in adult Bangladeshi patients hospitalized due to ETEC diarrhea by flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) isolated from blood collected day 2, 7, 30 and 90 after hospitalization as well as in healthy controls. The LT- and CF-specific Th responses were determined by analysis of IL-17A and IFN-γ in antigen stimulated PBMC cultures using ELISA. ETEC-specific IgA secreted by circulating antibody secreting cells (plasmablasts) were analyzed by using the antibodies in lymphocyte supernatants (ALS) ELISA-based method and plasma IgA was also measured by ELISA.ResultsETEC patients mounted significant ALS and plasma IgA responses against LTB and CFs on day 7 after hospitalization. ETEC patients had significantly elevated proportions of memory Th cells with a Th17 phenotype (CCR6+CXCR3-) in blood compared to controls, while frequencies of Th1 (CCR6-CXCR3+) or Th2 (CCR6-CXCR3-) cells were not increased. Antigen stimulation of PBMCs revealed IL-17A responses to LT, most clearly observed after stimulation with double mutant heat labile toxin (dmLT), but also with LT B subunit (LTB), and to CS6 in samples from patients with LT+ or CS6+ ETEC bacteria. Some individuals also mounted IFN-γ responses to dmLT and LTB. Levels of LTB specific IgA antibodies in ALS, but not plasma samples correlated with both IL-17A (r=0.5, p=0.02) and IFN-γ (r=0.6, p=0.01) responses to dmLT.ConclusionsOur results show that ETEC diarrhea induces T cell responses, which are predominantly of the Th17 type. The correlations between IL-17A and IFN-g and intestine-derived plasmablast responses support that Th responses may contribute to the development of protective IgA responses against ETEC infection. These observations provide important insights into T cell responses that need to be considered in the evaluation of advanced ETEC vaccine candidates.

BackgroundImmune responses that target sialidase occur following natural cholera and have been associated with protection against cholera. Sialidase is a neuraminidase that facilitates the binding of cholera toxin (CT) to intestinal epithelial cells. Despite this, little is known about age-related sialidase-specific immune responses and the impact of nutritional status and co-infection on sialidase-specific immunity.MethodsWe enrolled 50 culture-confirmed Vibrio cholerae O1 cholera cases presenting to the icddr,b Dhaka hospital with moderate to severe dehydration. We evaluated antibody responses out to 18 months (day 540) following cholera. We assessed immune responses targeting sialidase, lipopolysaccharide (LPS), cholera toxin B subunit (CtxB), and vibriocidal responses. We also explored the association of sialidase-specific immune responses to nutritional parameters and parasitic co-infection of cases.ResultsThis longitudinal cohort study showed age-dependent differences in anti-sialidase immune response after natural cholera infection. Adult patients developed plasma anti-sialidase IgA and IgG responses after acute infection (P

The longevity of immune responses induced by different degrees of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection provides information important to understanding protection against coronavirus disease 2019 (COVID-19). Here, we report the persistence of SARS-CoV-2 spike receptor-binding domain (RBD) specific antibodies and memory B cells recognizing this antigen in sequential samples from patients in Bangladesh with asymptomatic, mild, moderate and severe COVID-19 out to six months following infection. Since the development of long-lived memory B cells, as well as antibody production, is likely to be dependent on T helper (Th) cells, we also investigated the phenotypic changes of Th cells in COVID-19 patients over time following infection. Our results show that patients with moderate to severe COVID-19 mounted significant levels of IgG antibodies out to six months following infection, while patients with asymptomatic or mild disease had significant levels of IgG antibodies out to 3 months following infection, but these then fell more rapidly at 6 months than in patients with higher disease severity. Patients from all severity groups developed circulating memory B cells (MBCs) specific to SARS-CoV-2 spike RBD by 3 months following infection, and these persisted until the last timepoint measured at 6 months. A T helper cell response with an effector memory phenotype was observed following infection in all symptomatic patients, while patients with asymptomatic infection had no significant increases in effector Th1, Th2 and Th17 effector memory cell responses. Our results suggest that the strength and magnitude of antibody and memory B cells induced following SARS-CoV-2 infection depend on the severity of the disease. Polarization of the Th cell response, with an increase in Th effector memory cells, occurs in symptomatic patients by day 7 following infection, with increases seen in Th1, Th2, Th17 and follicular helper T cell subsets.

Coronavirus disease 2019 (COVID-19) is a protean disease causing different degrees of clinical severity including fatality. In addition to humoral immunity, antigen-specific T cells may play a critical role in defining the protective immune response against SARS-CoV-2, the virus that causes this disease. As a part of a longitudinal cohort study in Bangladesh to investigate B and T cell-specific immune responses, we sought to evaluate the activation-induced marker (AIM) and the status of different immune cell subsets during a COVID-19 infection. We analyzed a total of 115 participants, which included participants with asymptomatic, mild, moderate, and severe clinical symptoms. We observed decreased mucosal-associated invariant T (MAIT) cell frequency on the initial days of the COVID-19 infection in symptomatic patients compared to asymptomatic patients. However, natural killer (NK) cells were found to be elevated in symptomatic patients just after the onset of the disease compared to both asymptomatic patients and healthy individuals. Moreover, we found a significant increase of AIM+ (both OX40+CD137+ and OX40+CD40L+) CD4+ T cells in moderate and severe COVID-19 patients in response to SARS-CoV-2 peptides (especially spike peptides) compared to pre-pandemic controls who are unexposed to SARS-CoV-2. Notably, we did not observe any significant difference in the CD8+ AIMs (CD137+CD69+), which indicates the exhaustion of CD8+ T cells during a COVID-19 infection. These findings suggest that patients who recovered from moderate and severe COVID-19 were able to mount a strong CD4+ T-cell response against shared viral determinants that ultimately induced T cells to mount further immune responses to SARS-CoV-2.

Enhancement of mucosal immune responses in children and infants using novel adjuvants such as double mutant heat labile toxin (dmLT) is an important goal in the enteric vaccine field. dmLT has been shown to enhance mucosal IgA responses to the oral inactivated enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX. dmLT can enhance IL-17A production from adult T cells, which may increase the production and secretion of mucosal IgA antibodies. However, the adjuvant mechanism remains to be fully elucidated and might differ between infants and adults due to age-related differences in the development of the immune system. The main objective of this study was to determine how dmLT influences antigen presenting cells and T cells from infants compared to adults, and the role of IL-1β for mediating the adjuvant activity. Peripheral blood mononuclear cells (PBMCs) from Bangladeshi infants (6-11 months) and adults (18-40 years) were stimulated with the mitogen phytohaemagglutinin (PHA), the superantigen Staphylococcal enterotoxin B (SEB), ETVAX whole cell component (WCC) or E. coli lipopolysaccharide (LPS) ± dmLT, and cytokine production was measured using ELISA and electrochemiluminescence assays. The adjuvant dmLT significantly enhanced SEB- and PHA-induced IL-17A, but not IFN-γ responses, in PBMCs from both infants and adults. Blocking experiments using an IL-1 receptor antagonist demonstrated the importance of IL-1 signaling for the adjuvant effect. dmLT, ETVAX WCC and LPS induced dose-dependent IL-1β responses of comparable magnitudes in infant and adult cells. Depletion experiments suggested that IL-1β was mainly produced by monocytes. dmLT enhanced IL-1β responses to low doses of WCC and LPS, and the adjuvant effect appeared over a wider dose-range of WCC in infants. dmLT and WCC also induced IL-6, IL-23 and IL-12p70 production in both age groups and dmLT tended to particularly enhance IL-23 responses to WCC. Our results show that dmLT can induce IL-1β as well as other cytokines, which in turn may enhance IL-17A and potentially modulate other immunological responses in both infants and adults. Thus, dmLT may have an important function in promoting immune responses to the ETVAX vaccine, as well as other whole cell- or LPS-based vaccines in infants in low- and middle-income countries.