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Differences in innate immune ‘imprinting’ between vaccine adjuvants may mediate dissimilar effects on the quantity/quality of persisting adaptive responses. We compared antibody avidity maturation, antibody/memory B cell/CD4+ T cell response durability, and recall responses to non-adjuvanted fractional-dose antigen administered 1-year post-immunization (Day [D]360), between hepatitis B vaccines containing Adjuvant System (AS)01B, AS01E, AS03, AS04, or Alum (NCT00805389). Both the antibody and B cell levels ranked similarly (AS01B/E/AS03 > AS04 > Alum) at peak response, at D360, and following their increases post-antigen recall (D390). Proportions of high-avidity antibodies increased post-dose 2 across all groups and persisted at D360, but avidity maturation appeared to be more strongly promoted by AS vs. Alum. Post-antigen recall, frequencies of subjects with high-avidity antibodies increased only markedly in the AS groups. Among the AS, total antibody responses were lowest for AS04. However, proportions of high-avidity antibodies were similar between groups, suggesting that MPL in AS04 contributes to avidity maturation. Specific combinations of immunoenhancers in the AS, regardless of their individual nature, increase antibody persistence and avidity maturation.

Protection against influenza is mediated by neutralizing antibodies, and their induction at high and sustained titers is key for successful vaccination. Optimal B cells activation requires delivery of help from CD4 ⁺ T lymphocytes. In lymph nodes and tonsils, T-follicular helper cells have been identified as the T cells subset specialized in helping B lymphocytes, with interleukin-21 (IL-21) and inducible costimulatory molecule (ICOS1) playing a central role for this function. We followed the expansion of antigen-specific IL-21 ⁺ CD4 ⁺ T cells upon influenza vaccination in adults. We show that, after an overnight in vitro stimulation, influenza-specific IL-21 ⁺ CD4 ⁺ T cells can be measured in human blood, accumulate in the CXCR5 ⁻ICOS1 ⁺ population, and increase in frequency after vaccination. The expansion of influenza-specific ICOS1 ⁺IL-21 ⁺ CD4 ⁺ T cells associates with and predicts the rise of functionally active antibodies to avian H5N1. We also show that blood-derived CXCR5 ⁻ICOS1 ⁺ CD4 ⁺ T cells exert helper function in vitro and support the differentiation of influenza specific B cells in an ICOS1- and IL-21–dependent manner. We propose that the expansion of antigen-specific ICOS1 ⁺IL-21 ⁺ CD4 ⁺ T cells in blood is an early marker of vaccine immunogenicity and an important immune parameter for the evaluation of novel vaccination strategies.

Respiratory syncytial virus (RSV) is the leading cause of death from lower respiratory tract infection in infants and children, and is responsible for considerable morbidity and mortality in older adults. Vaccines for pregnant women and elderly which are in phase III clinical studies target people with pre‐existing natural immunity against RSV. To investigate the background immunity which will be impacted by vaccination, we single cell‐sorted human memory B cells and dissected functional and genetic features of neutralizing antibodies (nAbs) induced by natural infection. Most nAbs recognized both the prefusion and postfusion conformations of the RSV F‐protein (cross‐binders) while a smaller fraction bound exclusively to the prefusion conformation. Cross‐binder nAbs used a wide array of gene rearrangements, while preF‐binder nAbs derived mostly from the expansion of B‐cell clonotypes from the IGHV1 germline. This latter class of nAbs recognizes an epitope located between Site Ø, Site II, and Site V on the F‐protein, identifying an important site of pathogen vulnerability. Synopsis Several RSV vaccines are currently under clinical development and these will impact the pre‐existing immunity of pre‐infected individuals. We performed in depth analyses of the protective antibody response following natural infection to understand the B cells that will be impacted by vaccination. The majority of nAbs elicited following natural infection recognize epitopes shared between the preF and postF form of the RSV F‐protein. The repertoire analyses of neutralizing antibodies reveal that preF specific nAbs derive mostly from the expansion of B cells from the IGHV1 germline. IGHV1‐derived nAbs recognize an important site of pathogen vulnerability on the preF form of the RSV F‐protein. Graphical Abstract Several RSV vaccines are currently under clinical development and these will impact the pre‐existing immunity of pre‐infected individuals. We performed in depth analyses of the protective antibody response following natural infection to understand the B cells that will be impacted by vaccination.

Protein-polysaccharide conjugate vaccines rely on the induction of T-cell-dependent responses that support germinal center (GC) reactions to potentiate the expansion of antigen-specific memory B-cell (MBC) populations and high-avidity antibody responses. The effects of adjuvants on B-cell and antibody responses are well described for protein antigens but remain largely unexplored for conjugated polysaccharidic antigens. We assessed the effects of five adjuvants present in licensed vaccines (AS01, AS03, AS04, and aluminum hydroxide [Alum]) or under clinical evaluation (AS37) on the magnitude and quality of antigen-specific antibody responses and local/systemic B-cell responses. Naive mice received three immunizations of adjuvanted or non-adjuvanted model glycoconjugate vaccine containing (SA) capsular polysaccharide serotypes 5/8 (CP5/8) conjugated to a tetanus toxoid carrier and inactivated SA HlaH35L toxin. All AS-containing vaccines increased CP5/8-specific antibody titers and B-cell immunity relative to Alum- or non-adjuvanted formulations. After two immunizations, AS03 (α-tocopherol-containing oil-in-water emulsion) most robustly enhanced CP5/8-specific immunity relative to the other adjuvants or no adjuvant. AS03 induced higher responses of high-avidity antibodies persisting for at least 25 weeks post-immunization and greater expansions of populations of splenic GC B cells, mature MBCs in the lymph node or spleen, and long-lived plasma cells in the bone marrow. These effects increased with each immunization, suggesting the presence of avidity maturation and highlighting the role of the carrier in improving the quality of GC reactions. While HlaH35L-specific responses were augmented by each adjuvant, they lacked significant inter-group differences, pointing to profound differences in the adjuvants' effects on polysaccharide vs. protein antigens in the mice of the present study. Investigating the antibody quantity and quality and local and systemic B-cell population expansions in a naive model supports our understanding of how different adjuvants shape the response to the tested polysaccharidic antigens.

Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.

Generalized Modules for Membrane Antigens (GMMA) are outer membrane vesicles derived from Gram-negative bacteria that can be used to design affordable subunit vaccines. GMMA have been observed to induce a potent humoral immune response in preclinical and clinical studies. In addition, in preclinical studies, it has been found that GMMA can be exploited as optimal antigen carriers for both protein and saccharide antigens, as they are able to promote the enhancement of the antigen-specific humoral immune response when the antigen is overexpressed or chemically conjugated to GMMA. Here we investigated the mechanism of this GMMA carrier effect by immunizing mice and using factor H binding protein and GMMA of Neisseria meningitidis B as an antigen–GMMA model. We confirmed that the antigen displayed on the GMMA surface increased the antigen-specific IgG production and, above all, the antibody functionality measured by the serum bactericidal activity. We found that the enhancement of the bactericidal capacity induced by GMMA carrying the antigen on the surface was associated with the increase in antibody affinity to the antigen, and with the switching toward IgG subclasses with more bactericidal potential. Thus, we conclude that the potent carrier effect of GMMA is due to their ability to promote a better quality of humoral immunity.

Natural immunity against obligate and/or facultative intracellular pathogens is usually mediated by both humoral and cellular immunity. The identification of those antigens stimulating both arms of the immune system is instrumental for vaccine discovery. Although high-throughput technologies have been applied for the discovery of antibody-inducing antigens, few examples of their application for T-cell antigens have been reported. We describe how the compilation of the immunome, here defined as the pool of immunogenic antigens inducing T- and B-cell responses in vivo, can lead to vaccine candidates against Chlamydia trachomatis. We selected 120 C. trachomatis proteins and assessed their immunogenicity using two parallel high-throughput approaches. Protein arrays were generated and screened with sera from C. trachomatis-infected patients to identify antibody-inducing antigens. Splenocytes from C. trachomatis-infected mice were stimulated with 79 proteins, and the frequency of antigen-specific CD4⁺/IFN-γ⁺ T cells was analyzed by flow cytometry. We identified 21 antibody-inducing antigens, 16 CD4⁺/IFN-γ⁺-inducing antigens, and five antigens eliciting both types of responses. Assessment of their protective activity in a mouse model of Chlamydia muridarum lung infection led to the identification of seven antigens conferring partial protection when administered with LTK63/CpG adjuvant. Protection was largely the result of cellular immunity as assessed by CD4⁺ T-cell depletion. The seven antigens provided robust additive protection when combined in four-antigen combinations. This study paves the way for the development of an effective anti-Chlamydia vaccine and provides a general approach for the discovery of vaccines against other intracellular pathogens.

Understanding the impact that human memory B-cells (MBC), primed by previous infections or vaccination, exert on neutralizing antibody responses against drifted influenza hemagglutinin (HA) is key to design best protective vaccines. A major obstacle to these studies is the lack of practical tools to analyze HA-specific MBCs in human PBMCs ex vivo. We report here an efficient method to identify MBCs carrying HA-specific BCR in frozen PBMC samples. By using fluorochrome-tagged recombinant HA baits, and vaccine antigens from mismatched influenza strains to block BCR-independent binding, we developed a protocol suitable for quantitative, functional and molecular analysis of single MBCs specific for HA from up to two different influenza strains in the same tube. This approach will permit to identify the naive and MBC precursors of plasmablasts and novel MBCs appearing in the blood following infection or vaccination, thus clarifying the actual contribution of pre-existing MBCs in antibody responses against novel influenza viruses. Finally, this protocol can allow applying high throughput deep sequencing to analyze changes in the repertoire of HA⁺ B-cells in longitudinal samples from large cohorts of vaccinees and infected subjects with the ultimate goal of understanding the in vivo B-cell dynamics driving the evolution of broadly cross-protective antibody responses.

•An AS37-adjuvanted vaccine induced immune responses which maintained for 6 months.•Extensive immune profiling was conducted on a subset of participants.•AS37 increased expression of interferon-inducible genes and serum CXCL10 (IP-10).•AS37 upregulated specific innate immune cells and Ag-specific B and T lymphocytes.•The immune signature is consistent with toll-like receptor 7 engagement. The candidate Adjuvant System AS37 contains a synthetic toll-like receptor agonist (TLR7a) adsorbed to alum. In a phase I study (NCT02639351), healthy adults were randomised to receive one dose of licensed alum-adjuvanted meningococcal serogroup C (MenC-CRM197) conjugate vaccine (control) or MenC-CRM197 conjugate vaccine adjuvanted with AS37 (TLR7a dose 12.5, 25, 50 or 100 µg). A subset of 66 participants consented to characterisation of peripheral whole blood transcriptomic responses, systemic cytokine/chemokine responses and multiple myeloid and lymphoid cell responses as exploratory study endpoints. Blood samples were collected pre-vaccination, 6 and 24 h post-vaccination, and 3, 7, 28 and 180 days post-vaccination. The gene expression profile in whole blood showed an early, AS37-specific transcriptome response that peaked at 24 h, increased with TLR7a dose up to 50 µg and generally resolved within one week. Five clusters of differentially expressed genes were identified, including those involved in the interferon-mediated antiviral response. Evaluation of 30 cytokines/chemokines by multiplex assay showed an increased level of interferon-induced chemokine CXCL10 (IP-10) at 24 h and 3 days post-vaccination in the AS37-adjuvanted vaccine groups. Increases in activated plasmacytoid dendritic cells (pDC) and intermediate monocytes were detected 3 days post-vaccination in the AS37-adjuvanted vaccine groups. T follicular helper (Tfh) cells increased 7 days post-vaccination and were maintained at 28 days post-vaccination, particularly in the AS37-adjuvanted vaccine groups. Moreover, most of the subjects that received vaccine containing 25, 50 and 100 µg TLR7a showed an increased MenC-specific memory B cell responses versus baseline. These data show that the adsorption of TLR7a to alum promotes an immune signature consistent with TLR7 engagement, with up-regulation of interferon-inducible genes, cytokines and frequency of activated pDC, intermediate monocytes, MenC-specific memory B cells and Tfh cells. TLR7a 25–50 µg can be considered the optimal dose for AS37, particularly for the adjuvanted MenC-CRM197 conjugate vaccine.

[Display omitted] •B cell receptor repertoire analysis in RSV post-fusion subunit vaccinees.•Post-fusion subunit vaccine expands pre-/post-fusion cross-reactive memory B cells.•VH4-encoded B cell receptors preferentially selected for memory B cell expansion.•Clonotype analysis applied for detection of RSV-neutralizing antibodies. Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory illness in children of less than 5 years of age which usually results in hospitalization or even in death. Vaccine development is hampered in consequence of a failed vaccine trial with fatalities in the 1960s. Even though research has been more focused on the RSV fusion protein in its pre-fusion conformation, maternal vaccination with post-fusion protein (post F) was considered as a promising vaccine strategy for passive immunization of babies, because post F preserves very potent neutralizing epitopes. We extensively analyzed post F-binding B cell receptor (BCR) repertoires of three vaccinees who received a post F-subunit vaccine in the context of a first-in-human, Phase 1, randomized, observer-blind, placebo-controlled clinical trial (ClinicalTrials.gov Identifier: NCT02298179). In order to compare the vaccine-induced BCR repertoires with BCR repertoires induced by natural infection, we also analyzed pre F- and post F-binding BCRs isolated from a healthy blood donor with relatively high F-binding memory B cell (MBC) frequencies. Analysis of the vaccine-induced repertoires revealed that preferentially VH4-encoded BCRs were expanded in response to vaccination. Estimation of antigen-driven selection further demonstrated that expanded BCRs accumulated positively selected replacement mutations which substantiated the hypothesis that post F-vaccination induces diversification of VH4-encoded BCRs in germinal centers. Comparison of the vaccine-induced BCR repertoires with clonally related pre and post F-binding BCRs of the healthy blood donor suggested that the vaccine expanded pre/post F cross-reactive MBCs. Interestingly, several vaccine-induced BCRs shared stereotypic VDJ gene junctions with known neutralizing Abs. Once expressed for functional characterization, the selected monoclonal Abs demonstrated the predicted neutralization activities in plaque reduction neutralization assays indicating that the post F-vaccine induced expansion of neutralizing BCRs.