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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.

Proactive priming before the next pandemic could induce immune memory responses to novel influenza antigens. In an open-label study, we analyzed B cell memory and antibody responses of 54 adults who received 2 7.5-μg doses of MF59-adjuvanted A/Vietnam/1194/2004 clade 1 (H5N1) vaccine. Twenty-four subjects had been previously primed with MF59-adjuvanted or plain clade 0-like A/duck/Singapore/1997 (H5N3) vaccine during 1999-2001. The prevaccination frequency of circulating memory B cells reactive to A/Vietnam/1194/2004 was low in both primed and unprimed individuals. However, at day 21 after boosting, MF59-adjuvanted primed subjects displayed a higher frequency of H5N1-specific memory B cells than plain-primed or unprimed subjects. The immune memory was rapidly mobilized by a single vaccine administration and resulted in high titers of neutralizing antibodies to antigenically diverse clade 0, 1, and 2 H5N1 viruses already at day 7. In general, postvaccination antibody titers were significantly higher in primed subjects than in unprimed subjects. Subjects primed with MF59-adjuvanted vaccine responded significantly better than those primed with plain vaccine, most notably in early induction and duration of cross-reacting antibody responses. After 6 months, high titers of cross-reactive antibody remained detectable among MF59-primed subjects. We conclude that distant priming with clade 0-like H5N3 induces a pool of cross-reactive memory B cells that can be boosted rapidly years afterward by a mismatched MF59-adjuvanted vaccine to generate high titers of crossreactive neutralizing antibodies rapidly. These results suggest that pre-pandemic vaccination strategies should be considered.

Immune responses to vaccination are tested in clinical trials. This process usually requires years especially when immune memory and persistence are analyzed. Markers able to quickly predict the immune response would be very useful, particularly when dealing with emerging diseases that require a rapid response, such as avian influenza. To address this question we vaccinated healthy adults at days 1, 22, and 202 with plain or MF59-adjuvanted H5N1 subunit vaccines and tested both cell-mediated and antibody responses up to day 382. Only the MF59-H5N1 vaccine induced high titers of neutralizing antibodies, a large pool of memory H5N1-specific B lymphocytes, and H5-CD4⁺ T cells broadly reactive with drifted H5. The CD4⁺ response was dominated by IL-2⁺ IFN-γ⁻ IL-13⁻ T cells. Remarkably, a 3-fold increase in the frequency of virus-specific total CD4⁺ T cells, measurable after 1 dose, accurately predicted the rise of neutralizing antibodies after booster immunization and their maintenance 6 months later. We suggest that CD4⁺ T cell priming might be used as an early predictor of the immunogenicity of prepandemic vaccines.

Invariant natural killer T (iNKT) cells are innate-like lymphocytes recognizing CD1d-restricted glycolipid antigens, such as α-galactosylceramide (αGC). We assessed whether iNKT cells help B lymphocyte responses and found that mice immunized with proteins and αGC develop antibody titers 1-2 logs higher than those induced by proteins alone. Activation of iNKT cells enhances protection against infections such as influenza and elicits higher frequencies of memory B cells and higher antibody responses to booster immunizations. Protein vaccination with αGC, but not with conventional adjuvants, elicits IgG responses in mice lacking MHC class II molecules, demonstrating that iNKT cells can substitute for CD4⁺ T cell help to B cells. Interestingly, the decay of circulating antibodies is faster in mice lacking iNKT cells. These findings point to a homeostatic role for iNKT cells on critical features of the antibody response such as immunity and B cell memory.

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.

The standard serological methods present limitations for the measurement of immunity against H5N1 influenza strains. The hemagglutination inhibition (HI) assay lacks sensitivity and requires standardization, while the viral micro-neutralization (MN) assay needs handling of live virus. We produced pseudoparticles expressing hemagglutinin from clades 1 or 2 H5N1 in order to measure neutralizing antibodies in human sera after prime-boost vaccination with plain or MF59-adjuvanted H5N1 clade 1 subunit vaccines. Titers measured by pseudoparticle neutralization (PPN) assay significantly correlated with those measured by HI, single radial haemolysis or MN, with a PPN titer of 1:357 corresponding to an MN titer of 1:80. Notably, results from the PPN assay, confirm that MF59-H5N1 vaccine induces potent and long-lasting neutralizing antibody responses not only against the vaccine strain, but also against several heterologous clade 2 strains. Overall, the PPN assay represents a valid alternative to conventional serological methods for the evaluation of H5N1 vaccine immunogenicity.

► Antibody CD4 T and B cells response to 2009 A/H1N1 pandemic vaccination in adults. ► Comparable day 0 frequency of CD4 T and memory B cells reactive to pandemic/seasonal H1N1. ► Vaccination expand pandemic- and seasonal-specific CD4+ T cells and memory B cells. ► Antibodies and plasmablasts accumulating by day 8 are IgG-switched. ► By day 8 in antibodies inhibiting hemagglutination and infection rose up to 8-fold. Protective antibody responses to a single dose of 2009 pandemic vaccines have been observed in the majority of healthy subjects aged more than 3 years. These findings suggest that immune memory lymphocytes primed by previous exposure to seasonal influenza antigens are recruited in the response to A/H1N1 pandemic vaccines and allow rapid seroconversion. However, a clear dissection of the immune memory components favoring a fast response to pandemic vaccination is still lacking. Here we report the results from a clinical study where antibody, CD4+ T cell, plasmablast and memory B cell responses to one dose of an MF59-adjuvanted A/H1N1 pandemic vaccine were analyzed in healthy adults. While confirming the rapid appearance of antibodies neutralizing the A/H1N1 pandemic virus, we show here that the response is dominated by IgG-switched antibodies already in the first week after vaccination. In addition, we found that vaccination induces the rapid expansion of pre-existing CD4+ T cells and IgG-memory B lymphocytes cross-reactive to seasonal and pandemic A/H1N1 antigens. These data shed light on the different components of the immune response to the 2009 H1N1 pandemic influenza vaccination and may have implications in the design of vaccination strategies against future influenza pandemics.

Invariant NKT cells are a peculiar subset of T lymphocytes whose features, highly conserved both in the mouse and the human system, strongly recall those of other “innate lymphocytes”. Following recognition of CD1d-presented glycosphingolipid antigens invariant NKT promptly release high amount of diverse cytokines concurring to the activation of the actors of both innate and acquired immune responses. For this reason, in recent years NKT cells have been the object of intensive study, aimed to understand their role in diverse patho-physiological conditions and to exploit the possibility to take advantage of their “adjuvant-like” activity in the formulation of new vaccines. As antibodies are an essential part of many immune responses, we focused our attention on invariant NKT–B cell interactions analyzing their influences on B cell activation and effector functions. The results of this study demonstrate that human invariant NKT cells can provide direct help for B cell proliferation and antibody production through CD1d-restricted mechanisms. Remarkably, help to B lymphocytes by invariant NKT cells is delivered also in the absence of exogenous antigen, suggesting the existence of an endogenous ligand presented by CD1d on B cells.

Immune responses to vaccination are tested in clinical trials. This process usually requires years especially when immune memory and persistence are analyzed. Markers able to quickly predict the immune response would be very useful, particularly when dealing with emerging diseases that require a rapid response, such as avian influenza. To address this question we vaccinated healthy adults at days 1, 22, and 202 with plain or MF59-adjuvanted H5N1 subunit vaccines and tested both cell-mediated and antibody responses up to day 382. Only the MF59-H5N1 vaccine induced high titers of neutralizing antibodies, a large pool of memory H5N1-specific B lymphocytes, and H5-CD4+ T cells broadly reactive with drifted H5. The CD4+ response was dominated by IL-2+ IFN-γ− IL-13− T cells. Remarkably, a 3-fold increase in the frequency of virus-specific total CD4+ T cells, measurable after 1 dose, accurately predicted the rise of neutralizing antibodies after booster immunization and their maintenance 6 months later. We suggest that CD4+ T cell priming might be used as an early predictor of the immunogenicity of prepandemic vaccines.

Immune responses to vaccination are tested in clinical trials. This process usually requires years especially when immune memory and persistence are analyzed. Markers able to quickly predict the immune response would be very useful, particularly when dealing with emerging diseases that require a rapid response, such as avian influenza. To address this question we vaccinated healthy adults at days 1, 22, and 202 with plain or MF59-adjuvanted H5N1 subunit vaccines and tested both cell-mediated and antibody responses up to day 382. Only the MF59-H5N1 vaccine induced high titers of neutralizing antibodies, a large pool of memory H5N1-specific B lymphocytes, and H5-CD4 super(+) T cells broadly reactive with drifted H5. The CD4 super(+) response was dominated by IL-2 super(+) IFN- gamma super(-) IL-13 super(-) T cells. Remarkably, a 3-fold increase in the frequency of virus-specific total CD4 super(+) T cells, measurable after 1 dose, accurately predicted the rise of neutralizing antibodies after booster immunization and their maintenance 6 months later. We suggest that CD4 super(+) T cell priming might be used as an early predictor of the immunogenicity of prepandemic vaccines.