B cells play a fundamental role in host defenses against viral infections. Profiling the B cell response elicited by SARS-CoV-2 vaccination, including the generation and persistence of ...antigen-specific memory B cells, is essential for improving the knowledge of vaccine immune responsiveness, beyond the antibody response. mRNA-based vaccines have shown to induce a robust class-switched memory B cell response that persists overtime and is boosted by further vaccine administration, suggesting that memory B cells are critical in driving a recall response upon re-exposure to SARS-CoV-2 antigens. Here, we focus on the role of the B cell response in the context of SARS-CoV-2 vaccination, offering an overview of the different technologies that can be used to identify spike-specific B cells, characterize their phenotype using machine learning approaches, measure their capacity to reactivate following antigen encounter, and tracking the maturation of the B cell receptor antigenic affinity.
SARS-CoV-2 mRNA vaccines have demonstrated high efficacy and immunogenicity, but limited information is currently available on memory B cell generation and long-term persistence. Here, we ...investigated spike-specific memory B cells and humoral responses in 145 subjects, up to 6 months after the BNT162b2 vaccine (Comirnaty) administration. Spike-specific antibodies peaked 7 days after the second dose and significant antibody titers and ACE2/RBD binding inhibiting activity were still observed after 6 months, despite a progressive decline over time. Concomitant to antibody reduction, spike-specific memory B cells, mostly IgG class-switched, increased in the blood of vaccinees and persisted 6 months after vaccination. Following the
restimulation, circulating memory B cells reactivated and produced spike-specific antibodies. A high frequency of spike-specific IgG
plasmablasts, identified by computational analysis 7 days after boost, positively correlated with the generation of IgG
memory B cells at 6 months. These data demonstrate that mRNA BNT162b2 vaccine elicits strong B cell immunity with spike-specific memory B cells that still persist 6 months after vaccination, playing a crucial role for a rapid response to SARS-CoV-2 virus encounter.
Adjuvants contribute to enhancing and shaping the vaccine immune response through different modes of action. Here early biomarkers of adjuvanticity after primary immunization were investigated using ...four different adjuvants combined with the chimeric tuberculosis vaccine antigen H56. C57BL/6 mice were immunized by the subcutaneous route with different vaccine formulations, and the modulation of primary CD4
T cell and B cell responses was assessed within draining lymph nodes, blood, and spleen, 7 and 12 days after priming. Vaccine formulations containing the liposome system CAF01 or a squalene-based oil-in-water emulsion (o/w squalene), but not aluminum hydroxide (alum) or CpG ODN 1826, elicited a significant primary antigen-specific CD4
T cell response compared to antigen alone, 7 days after immunization. The effector function of activated CD4
T cells was skewed toward a Th1/Th17 response by CAF01, while a Th1/Th2 response was elicited by o/w squalene. Differentiation of B cells in short-lived plasma cells, and subsequent early H56-specific IgG secretion, was observed in mice immunized with o/w squalene or CpG adjuvants. Tested adjuvants promoted the germinal center reaction with different magnitude. These results show that the immunological activity of different adjuvants can be characterized by profiling early immunization biomarkers after primary immunization. These data and this approach could give an important contribution to the rational development of heterologous prime-boost vaccine immunization protocols.
Analysis of multifunctional CD4
T cells is fundamental for characterizing the immune responses to vaccination or infection. Major histocompatibility complex (MHC)/peptide tetramers represent a ...powerful technology for the detection of antigen-specific T cells by specific binding to their T-cell receptor, and their combination with functional assays is fundamental for characterizing the antigen-specific immune response. Here we optimized a protocol for the detection of multiple intracellular cytokines within epitope-specific CD4
T cells identified by the MHC class II tetramer technology. The optimal procedure for assessing the functional activity of tetramer-binding CD4
T cells was based on the simultaneous intracellular staining with both MHC tetramers and cytokine-specific antibodies upon
restimulation of cells with the vaccine antigen. The protocol was selected among procedures that differently combine the steps of cellular restimulation and tetramer staining with intracellular cytokine labeling. This method can be applied to better understand the complex functional profile of CD4
T-cell responses upon vaccination or infection.
Characterizing the impact of the vaccination schedule on the induction of B and T cell immune responses is critical for improving vaccine immunogenicity. Here we compare the effect of a short (4 ...weeks) or a long (18 weeks) interval between priming and boosting in mice, using a model vaccine formulation based on the chimeric tuberculosis vaccine antigen H56 combined with alum. While no significant difference was observed in serum antigen-specific IgG response and the induction of antigen-specific T follicular helper cells into draining lymph nodes after the two immunization schedules, a longer interval between priming and boosting elicited a higher number of germinal center-B cells and H56-specific antibody-secreting cells and modulated the effector function of reactivated CD4+ T cells. These data show that the scheduling of the booster immunization could affect the immune response elicited by vaccination modulating and improving the immunogenicity of the vaccine.
Influenza continues to be the most important cause of viral respiratory disease, despite the availability of vaccines. Today’s evaluation of influenza vaccines mainly focuses on the quantitative and ...functional analyses of antibodies to the surface proteins haemagglutinin (HA) and neuraminidase (NA). However, there is an increasing interest in measuring cellular immune responses targeting not only mutation-prone surface HA and NA but also conserved internal proteins as these are less explored yet potential correlates of protection. To date, laboratories that monitor cellular immune responses use a variety of in-house procedures. This generates diverging results, complicates interlaboratory comparisons, and hampers influenza vaccine evaluation. The European FLUCOP project aims to develop and standardize assays for the assessment of influenza vaccine correlates of protection. This report describes the harmonization and qualification of the influenza-specific interferon-gamma (IFN-γ) Enzyme-Linked ImmunoSpot (ELISpot) assay. Initially, two pilot studies were conducted to identify sources of variability during sample analysis and spot enumeration in order to develop a harmonized Standard Operating Procedure (SOP). Subsequently, an assay qualification study was performed to investigate the linearity, intermediate precision (reproducibility), repeatability, specificity, Lower and Upper Limits of Quantification (LLOQ-ULOQ), Limit of Detection (LOD) and the stability of signal over time. We were able to demonstrate that the FLUCOP harmonized IFN-γ ELISpot assay procedure can accurately enumerate IFN-γ secreting cells in the analytical range of 34.4 Spot Forming Units (SFU) per million cells up to the technical limit of the used reader and in the linear range from 120 000 to 360 000 cells per well, in plates stored up to 6 weeks after development. This IFN-γ ELISpot procedure will hopefully become a useful and reliable tool to investigate influenza-specific cellular immune responses induced by natural infection or vaccination and can be an additional instrument in the search for novel correlates of protection.
Despite the knowledge that cell-mediated immunity (CMI) contributes to the reduction of severe influenza infection, transmission, and disease outcome, the correlates of protection for cell-mediated ...immunity remain still unclear. Therefore, measuring the magnitude and quality of influenza-specific T cell responses in a harmonized way is of utmost importance to improve characterisation of vaccine-induced immunity across different clinical trials. The present study, conducted as part of the FLUCOP project, describes the development of a consensus protocol for the intracellular cytokine staining (ICS) assay, in order to reduce inter-laboratory variability, and its qualification. In order to develop a consensus protocol, the study was divided into different stages. Firstly, two pilot studies evaluated critical parameters in the analytical (read-outs) and post-analytical (gating strategies and data analysis) methods applied by eight different laboratories within the FLUCOP consortium. The methods were then harmonized by fixing the critical parameters and the subsequent consensus protocol was then qualified by one FLUCOP member. The antigen-specific cell population was defined as polypositive CD4
T cells (i.e. positive for at least two markers among CD40L/IFNγ/IL2/TNFα), which was shown to be the most sensitive and specific read-out. The qualification of this consensus protocol showed that the quantification of polypositive CD4
T cells was precise, linear and accurate, and sensitive with a lower limit of quantification of 0.0335% antigen-specific polypositive CD4
T cells. In conclusion, we provide the description of a harmonized ICS assay, which permits quantitative and qualitative evaluation of influenza vaccine-induced T cell responses. Application of this harmonized assay may allow for future comparisons of T cell responses to different influenza vaccines. It may facilitate future assessments of potential correlates of protection with the promise of application across other pathogens.
The study of the initial phase of the adaptive immune response after first antigen encounter provides essential information on the magnitude and quality of the immune response. This phase is ...characterized by proliferation and dissemination of T cells in the lymphoid organs. Modeling and identifying the key features of this phenomenon may provide a useful tool for the analysis and prediction of the effects of immunization. This knowledge can be effectively exploited in vaccinology, where it is of interest to evaluate and compare the responses to different vaccine formulations. The objective of this paper is to construct a stochastic model based on branching process theory, for the dissemination network of antigen-specific CD4+ T cells. The devised model is validated on in vivo animal experimental data. The model presented has been applied to the vaccine immunization context making references to simple proliferation laws that take into account division, death and quiescence, but it can also be applied to any context where it is of interest to study the dynamic evolution of a population.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
BackgroundInvasive non-typhoidal Salmonella (iNTS) disease is a significant health concern in sub-Saharan Africa. While our knowledge of a larger-scale variation is growing, understanding of the ...subnational variation in iNTS disease occurrence is lacking, yet crucial for targeted intervention.MethodWe performed a systematic review of reported occurrences of iNTS disease in sub-Saharan Africa, consulting literature from PubMed, Embase and Web of Science published since 2000. Eligibility for inclusion was not limited by study type but required that studies reported original data on human iNTS diseases based on the culture of a normally sterile site, specifying subnational locations and the year, and were available as full-text articles. We excluded studies that diagnosed iNTS disease based on clinical indications, cultures from non-sterile sites or serological testing. We estimated the probability of occurrence of iNTS disease for sub-Saharan Africa on 20 km × 20 km grids by exploring the association with geospatial covariates such as malaria, HIV, childhood growth failure, access to improved water, and sanitation using a boosted regression tree.ResultsWe identified 130 unique references reporting human iNTS disease in 21 countries published from 2000 through 2020. The estimated probability of iNTS occurrence grids showed significant spatial heterogeneity at all levels (20 km × 20 km grids, subnational, country and subregional levels) and temporal heterogeneity by year. For 2020, the probability of occurrence was higher in Middle Africa (0.34, 95% CI: 0.25 to 0.46), followed by Western Africa (0.33, 95% CI: 0.23 to 0.44), Eastern Africa (0.24, 95% CI: 0.17 to 0.33) and Southern Africa (0.08, 95% CI: 0.03 to 0.11). Temporal heterogeneity indicated that the probability of occurrence increased between 2000 and 2020 in countries such as the Republic of the Congo (0.05 to 0.59) and Democratic Republic of the Congo (0.10 to 0.48) whereas it decreased in countries such as Uganda (0.65 to 0.23) or Zimbabwe (0.61 to 0.37).ConclusionThe iNTS disease occurrence varied greatly across sub-Saharan Africa, with certain regions being disproportionately affected. Exploring regions at high risk for iNTS disease, despite the limitations in our data, may inform focused resource allocation. This targeted approach may enhance efforts to combat iNTS disease in more affected areas.
Preventing SARS-CoV-2 infection is of utmost importance in allogeneic hematopoietic cell transplantation patients (allo-HCT), given their heightened susceptibility to adverse outcomes associated with ...SARS-CoV-2 infection. However, limited data are available regarding the immune response to COVID-19 vaccines in these subjects, particularly concerning the generation and persistence of spike-specific memory response. Here, we analyzed the spike-specific memory B cells in a cohort of allo-HCT recipients vaccinated with multiple doses of the mRNA-1273 vaccine and monitored the spike-specific antibody response from baseline up to one month after the fourth dose. After the primary vaccine series, the frequency of spike-specific B cells, detected within the pool of Ig-switched CD19+ cells, significantly increased. The booster dose further induced a significant expansion, reaching up to 0.28% of spike-specific B cells. The kinetics of this expansion were slower in the allo-HCT recipients compared to healthy controls. Spike-specific IgG and ACE2/RBD binding inhibition activity were observed in 80% of the allo-HCT recipients after the first two doses, with a significant increase after the third and fourth booster doses, including in the subjects who did not respond to the primary vaccine series. Additionally, 87% of the allo-HCT recipients exhibited positive cross-inhibition activity against the BA.1 variant. Our findings provide evidence that allo-HCT recipients need repeated doses of the mRNA-1273 vaccine to induceSARS-CoV-2 specific immune response similar to that observed in healthy individuals. This is particularly crucial for vulnerable individuals who may exhibit a limited response to the primary series of SARS-CoV-2 vaccination.
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