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Recurrence of coronavirus disease 19 (COVID-19) symptoms and SARS-CoV-2 viral load relapse have been reported in people treated with nirmatrelvir/ritonavir (NM/r). However, little is understood about the etiology of this phenomenon. Our aim was to investigate the relation between the host's immune response and viral rebound. We described three cases of COVID-19 rebound that occurred after treatment with nirmatrelvir/ritonavir (group A). In addition, we compared spike-specific antibody response and plasma cytokine/chemokine patterns of the rebound cases with those of (i) control patients treated with nirmatrelvir/ritonavir who did not show rebound (group B), and (ii) subjects not treated with any anti-SARS-CoV-2 drug (group C). The anti-spike antibodies and plasma cytokines/chemokines were similar in groups A and B. However, we observed a higher anti-BA.2 spike IgG response in patients without antiviral treatment (group C) [geometric mean titer 210,807, 5.1- and 8.2-fold higher compared to group A (p = 0.039) and group B (p = 0.032)]. Moreover, the patients receiving antiviral treatment (groups A-B) showed higher circulating levels of platelet-derived growth factor subunit B (PDGF-BB) and vascular endothelial growth Factors (VEGF) and lower levels of interleukin-9 (IL-9), interleukine-1 receptor antagonist (IL-1 RA), and regulated upon activation normal T cell expressed and presumably secreted chemokine (RANTES) when compared to group C. In conclusion, we observed lower anti-spike IgG levels and different cytokine patterns in nirmatrelvir/ritonavir-treated patients compared to those not treated with anti-SARS-CoV-2 drugs. This suggests that early antiviral treatment, by reducing viral load and antigen presentation, could mitigate the immune response against SARS-CoV-2. The clinical relevance of such observation should be further investigated in larger populations. [ABSTRACT FROM AUTHOR]

The mRNA vaccines for SARS-CoV-2 have demonstrated efficacy and immunogenicity in the real-world setting. However, most of the research on vaccine immunogenicity has been centered on characterizing the antibody response, with limited exploration into the persistence of spike-specific memory B cells. Here we monitored the durability of the memory B cell response up to 9 months post-vaccination, and characterized the trajectory of spike-specific B cell phenotypes in healthy individuals who received two doses of the BNT162b2 vaccine. To profile the spike-specific B cell response, we applied the tSNE and Cytotree automated approaches. Spike-specific IgA+ and IgG+ plasmablasts and IgA+ activated cells were observed 7 days after the second dose and disappeared 3 months later, while subsets of spike-specific IgG+ resting memory B cells became predominant 9 months after vaccination, and they were capable of differentiating into spike-specific IgG secreting cells when restimulated in vitro. Other subsets of spike-specific B cells, such as IgM+ or unswitched IgM+IgD+ or IgG+ double negative/atypical cells, were also elicited by the BNT162b2 vaccine and persisted up to month 9. The analysis of circulating spike-specific IgG, IgA, and IgM was in line with the plasmablasts observed. The longitudinal analysis of the antigen-specific B cell response elicited by mRNA-based vaccines provides valuable insights into our understanding of the immunogenicity of this novel vaccine platform destined for future widespread use, and it can help in guiding future decisions and vaccination schedules. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 inhouse 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 interferongamma (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 (LLOQULOQ), 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. [ABSTRACT FROM AUTHOR]

The rVSV-ZEBOV Ebolavirus vaccine confers protection within days after immunization, suggesting the contribution of innate immune responses. We report modulation of rVSV-ZEBOV vaccinee blood CD56+ NK cell numbers, NKG2D or NKp30 surface receptor expression, Killer Immunoglobulin-like Receptor (KIR)+ cell percentages and NK-cell-related genes on day 1 post immunization. Inverse correlations existed between the concentration of several plasma cytokines and inhibitory KIR+ CD56dim or cytokine-responsive CD56bright NK cells. Thus, NK cells may contribute to the early protective efficacy of rVSV-ZEBOV in humans. [ABSTRACT FROM AUTHOR]

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 in vitro 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. [ABSTRACT FROM AUTHOR]

The human immune system and the microbiota co-evolve, and their balanced relationship is based on crosstalk between the two systems through the course of life. This tight association and the overall composition and richness of the microbiota play an important role in the modulation of host immunity and may impact the immune response to vaccination. The availability of innovative technologies, such as next-generation sequencing (NGS) and correlated bioinformatics tools, allows a deeper investigation of the crosstalk between the microbiota and human immune responses. This review discusses the current knowledge on the influence of the microbiota on the immune response to vaccination and novel tools to deeply analyze the impact of the microbiome on vaccine responses. [ABSTRACT FROM AUTHOR]

Transcriptomic profiling of the immune response induced by vaccine adjuvants is of critical importance for the rational design of vaccination strategies. In this study, transcriptomics was employed to profile the effect of the vaccine adjuvant used for priming on the immune response following re-exposure to the vaccine antigen alone. Mice were primed with the chimeric vaccine antigen H56 of Mycobacterium tuberculosis administered alone or with the CAF01 adjuvant and boosted with the antigen alone. mRNA sequencing was performed on blood samples collected 1, 2, and 7 days after priming and after boosting. Gene expression analysis at day 2 after priming showed that the CAF01 adjuvanted vaccine induced a stronger upregulation of the innate immunity modules compared with the unadjuvanted formulation. The immunostimulant effect of the CAF01 adjuvant, used in the primary immunization, was clearly seen after a booster immunization with a low dose of antigen alone. One day after boost, we observed a strong upregulation of multiple genes in blood of mice primed with H56 + CAF01 compared with mice primed with the H56 alone. In particular, blood transcription modules related to innate immune response, such as monocyte and neutrophil recruitment, activation of antigen-presenting cells, and interferon response were activated. Seven days after boost, differential expression of innate response genes faded while a moderate differential expression of T cell activation modules was appreciable. Indeed, immunological analysis showed a higher frequency of H56-specific CD4+ T cells and germinal center B cells in draining lymph nodes, a strong H56-specific humoral response and a higher frequency of antibody-secreting cells in spleen of mice primed with H56 + CAF01. Taken together, these data indicate that the adjuvant used for priming strongly reprograms the immune response that, upon boosting, results in a stronger recall innate response essential for shaping the downstream adaptive response. [ABSTRACT FROM AUTHOR]

The induction and modulation of the immune response to vaccination can be rationally designed by combining different vaccine formulations for priming and boosting. Here, we investigated the impact of heterologous prime-boost approaches on the vaccine-specific cellular and humoral responses specific for a mycobacterial vaccine antigen. C57BL/6 mice were primed with the chimeric vaccine antigen H56 administered alone or with the CAF01 adjuvant, and boosted with H56 alone, or combined with CAF01 or with the squalene-based oil-in-water emulsion adjuvant (o/w squalene). A strong secondary H56-specific CD4+ T cell response was recalled by all the booster vaccine formulations when mice had been primed with H56 and CAF01, but not with H56 alone. The polyfunctional nature of T helper cells was analyzed and visualized with the multidimensional flow cytometry FlowSOM software, implemented as a package of the R environment. A similar cytokine profile was detected in groups primed with H56 + CAF01 and boosted with or without adjuvant, except for some clusters of cells expressing high level of IL-17 together with TNF-a, IL-2, and IFN-γ, that were significantly upregulated only in groups boosted with the adjuvants. On the contrary, the comparison between groups primed with or without the adjuvant showed a completely different clusterization of cells, strengthening the impact of the formulation used for primary immunization on the profiling of responding cells. The presence of the CAF01 adjuvant in the priming formulation deeply affected also the secondary humoral response, especially in groups boosted with H56 alone or o/w squalene. In conclusion, the presence of CAF01 adjuvant in the primary immunization is crucial for promoting primary T and B cell responses that can be efficiently reactivated by booster immunization also performed with antigen alone. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The design of heterologous prime-boost vaccine combinations that optimally shape the immune response is of critical importance for the development of next generation vaccines. Here, we tested different prime-boost combinations using the tuberculosis vaccine antigen H56 with CAF01 or CpG ODN 1826 adjuvants, administered by the parenteral and nasal routes. Using peptide-MHC class II tetramers, antigen-specific CD4+ T cells were tracked following primary and booster immunizations. Both parenteral priming with H56 plus CAF01 and nasal priming with H56 plus CpG elicited significant expansion of CD4+ tetramer-positive T cells in the spleen; however, only parenterally primed cells responded to booster immunization. Subcutaneous (SC) priming with H56 and CAF01 followed by nasal boosting with H56 and CpG showed the greater expansion of CD4+ tetramer-positive T cells in the spleen and lungs compared to all the other homologous and heterologous prime-boost combinations. Nasal boosting exerted a recruitment of primed CD4+ T cells into lungs that was stronger in subcutaneously than nasally primed mice, in accordance with different chemokine receptor expression induced by primary immunization. These data demonstrate that SC priming is fundamental for eliciting CD4+ T cells that can be efficiently boosted by the nasal route and results in the recruitment of antigen-experienced cells into the lungs. Combination of different vaccine formulations and routes of delivery for priming and boosting is a strategic approach for improving and directing vaccine-induced immune responses. [ABSTRACT FROM AUTHOR]

T-cell priming is a critical event in the initiation of the immune response to vaccination since it deeply influences both the magnitude and the quality of the immune response induced. CD4+ T-cell priming, required for the induction of high-affinity antibodies and immune memory, represents a key target for improving and modulating vaccine immunogenicity. A major challenge in the study of in vivo T-cell priming is due to the low frequency of antigen-specific T cells. This review discusses the current knowledge on antigen-specific CD4+ T-cell priming in the context of vaccination, as well as the most advanced tools for the characterization of the in vivo T-cell priming and the opportunities offered by the application of systems biology. [ABSTRACT FROM AUTHOR]

The magnitude and quality of cell-mediated immune responses elicited by natural infection or vaccination are commonly measured by Interferon-ɣ (IFN-ɣ) Enzyme-Linked ImmunoSpot (ELISpot) and Intracellular Cytokine Staining (ICS). To date, laboratories apply a variety of in-house procedures which leads to diverging results, complicates interlaboratory comparisons and hampers vaccine evaluations. During the FLUCOP project, efforts have been made to develop harmonized Standard Operating Procedures (SOPs) for influenza-specific IFN-ɣ ELISpot and ICS assays. Exploratory pilot studies provided information about the interlaboratory variation before harmonization efforts were initiated. Here we report the results of two proficiency tests organized to evaluate the impact of the harmonization effort on assay results and the performance of participating FLUCOP partners. The introduction of the IFN-ɣ ELISpot SOP reduced variation of both background and stimulated responses. Post-harmonization background responses were all lower than an arbitrary threshold of 50 SFU/million cells. When stimulated with A/California and B/Phuket, a statistically significant reduction in variation (p < 0.0001) was observed and CV values were strongly reduced, from 148% to 77% for A/California and from 126% to 73% for B/Phuket. The harmonizing effect of applying an ICS SOP was also confirmed by an increased homogeneity of data obtained by the individual labs. The application of acceptance criteria on cell viability and background responses further enhanced the data homogeneity. Finally, as the same set of samples was analyzed by both the IFN-ɣ ELISpot and the ICS assays, a method comparison was performed. A clear correlation between the two methods was observed, but they cannot be considered interchangeable. In conclusion, proficiency tests show that a limited harmonization effort consisting of the introduction of SOPs and the use of the same in vitro stimulating antigens leads to a reduction of the interlaboratory variation of IFN-ɣ ELISpot data and demonstrate that substantial improvements for the ICS assay are achieved as comparable laboratory datasets could be generated. Additional steps to further reduce the interlaboratory variation of ICS data can consist of standardized gating templates and detailed data reporting instructions as well as further efforts to harmonize reagent and instrument use. [ABSTRACT FROM AUTHOR]

Highlights • Efficacy of vaccination in the elderly is strongly reduced compared to younger adults. • Need to design vaccines tailored for the elderly considering immunosenescence and inflammaging. • Adjuvants should be specifically designed for stimulating the ageing immune system. • Systems biology can contribute to identification of biomarkers and stratification of subpopulations for elderly vaccination. • Immunobiography could guide vaccination strategies for specific elderly subpopulations. Abstract The unprecedented increase of life expectancy challenges society to protect the elderly from morbidity and mortality making vaccination a crucial mean to safeguard this population. Indeed, infectious diseases, such as influenza and pneumonia, are among the top killers of elderly people in the world. Elderly individuals are more prone to severe infections and less responsive to vaccination prevention, due to immunosenescence combined with the progressive increase of a proinflammatory status characteristic of the aging process (inflammaging). These factors are responsible for most age-related diseases and correlate with poor response to vaccination. Therefore, it is of utmost interest to deepen the knowledge regarding the role of inflammaging in vaccination responsiveness to support the development of effective vaccination strategies designed for elderly. In this review we analyse the impact of age-associated factors such as inflammaging, immunosenescence and immunobiography on immune response to vaccination in the elderly, and we consider systems biology approaches as a mean for integrating a multitude of data in order to rationally design vaccination approaches specifically tailored for the elderly. [ABSTRACT FROM AUTHOR]

Abstract: Typhoid fever is a public health problem, especially among young children in developing countries. To address this need, a glycoconjugate vaccine Vi-CRM197, composed of the polysaccharide antigen Vi covalently conjugated to the non-toxic mutant of diphtheria toxin CRM197, is under development. Here, we assessed the antibody and cellular responses, both local and systemic, following subcutaneous injection of Vi-CRM197. The glycoconjugate elicited Vi-specific serum IgG titers significantly higher than unconjugated Vi, with prevalence of IgG1 that persisted for at least 60 days after immunization. Vi-specific IgG, but not IgA, were present in intestinal washes. Lymphocytes proliferation after restimulation with Vi-CRM197 was observed in spleen and mesenteric lymph nodes. These data confirm the immunogenicity of Vi-CRM197 and demonstrate that the vaccine-specific antibody and cellular immune responses are present also in the intestinal tract, thus strengthening the suitability of Vi-CRM197 as a promising candidate vaccine against Salmonella Typhi. [Copyright &y& Elsevier]

Abstract: NadA and NhhA, two surface proteins of serogroup B Neisseria meningitidis identified as candidate vaccine antigens, were expressed on the surface of the human oral commensal bacterium Streptococcus gordonii. Recombinant strains were used to immunize BALB/c mice by the intranasal route and the local and systemic immune response was assessed. Mice were inoculated with recombinant bacteria administered alone or with LTR72, a partially inactivated mutant of Escherichia coli heat-labile enterotoxin, as a mucosal adjuvant. Intranasal immunization with live bacteria expressing NadA induced a significant serum antibody response, with a prevalence of the IgG2a subclass, bactericidal activity in the sera of 71% of animals, and a NadA-specific IgA response in nasal and bronchoalveolar lavages. A formalin-inactivated recombinant strain of S. gordonii expressing NadA was also administered intranasally, inducing a systemic and mucosal humoral response comparable to that of live bacteria. The administration of recombinant bacteria with the mucosal adjuvant LTR72 stimulated a stronger systemic antibody response, protective in 85% of sera, while did not increase the local IgA response. Recombinant S. gordonii expressing NhhA induced a systemic but not mucosal antibody response. These data support the role of NadA as vaccine candidate against serogroup B meningococci, and the use of S. gordonii as vector for intranasal vaccination. [Copyright &y& Elsevier]