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Background Influenza remains a major threat to public health. Live-attenuated influenza vaccines (LAIV) have been shown to be effective, particularly in children. Follicular T helper (TFH) cells provide B-cell help and are crucial for generating long-term humoral immunity. However the role of TFH cells in LAIV-induced immune responses is unknown. Methods We collected tonsils, plasma, and saliva samples from children and adults receiving LAIV prior to tonsillectomy. We measured influenza-specific TFH-cell responses after LAIV by flow cytometry and immunohistochemistry. Systemic and local antibody responses were analysed by hemagglutination inhibition assay and enzyme-linked immunosorbent assay. Results We report that LAIV induced early (3–7 days post-vaccination) activation of tonsillar follicles and influenza-specific TFH-cell (CXCR5+CD57+CD4+ T cell) responses in children, and to a lesser extent in adults. Serological analyses showed that LAIV elicited rapid (day 14) and long-term (up to 1 year post-vaccination) antibody responses (hemagglutination inhibition, influenza-specific IgG) in children, but not adults. There was an inverse correlation between pre-existing influenza-specific salivary IgA concentrations and tonsillar TFH-cell responses, and a positive correlation between tonsillar TFH-cell and systemic IgG induction after LAIV. Conclusions Our data, taken together, demonstrate an important role of tonsillar TFH cells in LAIV-induced immunity in humans., Live attenuated influenza vaccine induces rapid influenza-specific tonsillar TFH-cell and antibody responses in children, but is less potent in adults. The magnitude of influenza-specific TFH-cell responses inversely correlated with pre-existing salivary IgA, and directly associated with post-vaccination antibody fold-induction.

Influenza vaccination often results in a large percentage of low responders, especially in high-risk groups. As a first line of defense, natural killer (NK) cells play a crucial role in the fight against infections. However, their implication with regard to vaccine responsiveness is insufficiently assessed. Therefore, this study aimed at the validation of essential NK cell features potentially associated with differential vaccine responsiveness with a special focus on NKG2C- and/or CD57-expressing NK cells considered to harbor memory-like functions. To this end, 16 healthy volunteers were vaccinated with an adjuvanted pandemic influenza vaccine. Vaccine responders and low responders were classified according to their hemagglutination inhibition antibody titers. A majority of responders displayed enhanced frequencies of NKG2C-expressing NK cells 7- or 14-days post-vaccination as compared to low responders, whereas the expression of CD57 was not differentially modulated. The NK cell cytotoxic potential was found to be confined to CD56dimCD16+ NKG2C-expressing NK cells in the responders but not in the low responders, which was further confirmed by stochastic neighbor embedding analysis. The presented study is the first of its kind that ascribes CD56dimCD16+ NKG2C-expressing NK cells a crucial role in biasing adaptive immune responses upon influenza vaccination and suggests NKG2C as a potential biomarker in predicting pandemic influenza vaccine responsiveness.

Healthcare workers (HCW) were prioritized for vaccination during the 2009 influenza A(H1N1)pdm09 pandemic. We conducted a clinical trial in October 2009 where 237 HCWs were immunized with a AS03-adjuvanted A(H1N1)pdm09 monovalent vaccine. In the current study, we analyzed the homologous and cross-reactive H1N1 humoral responses using prototype vaccine strains dating back to 1977 by the haemagglutinin inhibition (HI), single radial hemolysis SRH), antibody secreting cell (ASC) and memory B cell (MBC) assays. The cellular responses were assessed by interferon-γ (IFN-γ) ELISPOT and by intracellular staining (ICS) for the Th1 cytokines IFN-γ, interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α). All assays were performed using blood samples obtained prior to (day 0) and 7, 14 and 21 d post-pandemic vaccination, except for ASC (day 7) and ICS (days 0 and 21). Vaccination elicited rapid HI, SRH and ASC responses against A(H1N1)pdm09 which cross reacted with seasonal H1N1 strains. MBC responses were detected against the homologous and seasonal H1N1 strains before vaccination and were boosted 2 weeks post-vaccination. An increase in cellular responses as determined by IFN-γ ELISPOT and ICS were observed 1–3 weeks after vaccination. Collectively, our data show that the AS03-adjuvanted A(H1N1)pdm09 vaccine induced rapid cellular and humoral responses against the vaccine strain and the response cross-reacted against prototype H1N1 strains dating back to 1977. publishedVersion

Annually, influenza virus infection has a large socioeconomic burden on society, with 500 000 fatal cases globally [1–3]. The World Health Organization estimates that 20% of children are infected with influenza virus each year, and they are the main source of spread of the virus and have a high burden of the disease [4]. Vaccination is the cornerstone of prophylaxis and is recommended for high-risk patients. The trivalent inactivated influenza vaccine (TIV) is safe and provides protection but may not be the optimal vaccine for young children, owing to their lack of previous infection. Since 2003, the cold-adapted live attenuated influenza vaccine (LAIV), administered as a nasal spray, has been approved in the United States for individuals aged 2–49 years. The vaccine was licensed in Europe in 2012 (for individuals aged 2–17 years) and was implemented in the British childhood vaccination campaign, beginning in 2013 [5]. In June 2014, the Advisory Committee on Immunization Practices preferentially recommended LAIV in healthy children 2–8 years old when it is immediately available [6]. Despite many years on the market, there are no established correlates of protection for LAIV. Efficacy studies and observational data suggest that the LAIV provides higher levels of protection than TIV in children [7–11]. The hemagglutination inhibition (HI) titer is widely used as a surrogate correlate of protection after TIV receipt; however, the cutoff titer of 1:40 is based on adult trials. There is debate on both the protective HI level in children and the fact that an HI titer underestimates the protection obtained by LAIV [12–14]. Cellular immunity may be a better measure of protective immunity after LAIV in children [15]. CD4+ T cells have the ability to act as effector cells and to direct and generate specific memory CD4+ and CD8+ T-cell subsets with diverse functions [16–18]. Animal and human studies have demonstrated protective cellular immunity after LAIV [10, 19–21]. Therefore, the duration and quality of the immune response in humans after LAIV needs to be studied. We conducted a pediatric clinical trial to elucidate the immunological mechanisms induced by LAIV, with emphasis on the long-term, strain-specific cellular immune responses. Our study is unique, as we obtained sequential blood samples from young children up to 1 year after vaccination, allowing us to compare and analyze serum and cellular responses in the same child. Our results indicate that LAIV induces long-term humoral and cellular immune responses in children and that priming is important in determining the magnitude of the response.

Background. We conducted a clinical trial in October 2009 to evaluate the immunogenicity of the AS03adjuvanted influenza vaccine (pH1N1 vaccine) in health care workers (HCWs) (www.clinicaltrials.gov, NCT01003288). By 2 weeks after vaccination, 97% had protective hemagglutinin inhibition (HI) titers (≥40) however, 16% were low responders (LR) and failed to maintain a protective response 90 days after vaccination. Methods. We analyzed the humoral responses (HI, antibody-secreting cell [ASC], and serum immunoglobulin G [IgG]) in 15 LRs and 25 control HCWs. Twelve LRs were revaccinated with the pH1N1 vaccine, and 7 were subsequently vaccinated with the 2010 seasonal trivalent influenza vaccine. We conducted a long-term analysis of the humoral and CD4 + T-helper (Th) 1 responses. Results. The LRs had a slower HI antibody response than the control HCWs, with protective antibody titers not reached until 2 weeks after vaccination in the majority of the participants. The LRs also had significantly lower IgG ASCs at day 7 and HA1-specific serum IgG responses at day 21, compared with the control HCWs. Revaccination with the pH1N1 vaccine elicited rapid HI antibody, ASC, memory B cell, and multifunctional CD4 + Th1 cell responses. Conclusion. This study shows that revaccination of low-responding HCWs with the pH1N1 vaccine is required for maintaining long-term protection.

Background. Vaccination is the best measure to protect the population against a potential influenza H5N1 pandemic, but 2 doses of vaccine are needed to elicit protective immune responses. An immunological marker for H5N1 vaccine effectiveness is needed for early identification of the best vaccine candidate. Methods. We conducted a phase I clinical trial of a virosomal H5N1 vaccine adjuvanted with Matrix M. Sixty adult volunteers were vaccinated intramuscularly with 2 doses of either 30 μg hemagglutinin (HA) alone or with 1.5, 7.5, or 30 μg HA and Matrix M adjuvant (50 μg). The humoral response was measured by the hemagglutination inhibition (HI), microneutralization (MN), and single radial hemolysis (SRH) assays, and the CD4 + T-helper 1 (Th1)–cell response was measured by intracellular staining for the cytokines interleukin 2, interferon γ, and tumor necrosis factor α. Results. The adjuvanted vaccine effectively induced CD4 + Th1-cell responses, and the frequency of influenzaspecific Th1 cells after the first vaccine dose predicted subsequent HI, MN, and SRH seroprotective responses after the second vaccination. Conclusions. These results support early identification of Th1-cell responses as a predictive biomarker for an efficient vaccine response, which could have great implications for early identification of persons with low or no response to vaccine when evaluating future pandemic influenza vaccines. Clinical Trials Registration. NCT00868218.

Please cite this paper as: Svindland et al. The mucosal and systemic immune responses elicited by a chitosan-adjuvanted intranasal influenza H5N1 vaccine. Influenza and Other Respiratory Viruses DOI:10.1111/j.1750-2659.2011.00271.x. Background Development of influenza vaccines that induce mucosal immunity has been highlighted by the World Health Organisation as a priority (Vaccine 2005;23:1529). Dose-sparing strategies and an efficient mass-vaccination regime will be paramount to reduce the morbidity and mortality of a future H5N1 pandemic. Objectives This study has investigated the immune response and the dose-sparing potential of a chitosan-adjuvanted intranasal H5N1 (RG-14) subunit (SU) vaccine in a mouse model. Methods Groups of mice were intranasally immunised once or twice with a chitosan (5 mg/ml)-adjuvanted SU vaccine [7·5, 15 or 30 μg haemagglutinin (HA)] or with a non-adjuvanted SU vaccine (30 μg HA). For comparison, another group of mice were intranasally immunised with a whole H5N1 (RG-14) virus (WV) vaccine (15 μg HA), and the control group consisted of unimmunised mice. Results The chitosan-adjuvanted SU vaccine induced an immune response superior to that of the non-adjuvanted SU vaccine. Compared with the non-adjuvanted SU group, the chitosan-adjuvanted SU vaccine elicited higher numbers of influenza-specific antibody-secreting cells (ASCs), higher concentrations of local and systemic antibodies and correspondingly an improved haemagglutination inhibition (HI) and single radial haemolysis (SRH) response against both the homologous vaccine strain and drifted H5 strains. We measured a mixed T-helper 1/T-helper 2 cytokine response in the chitosan-adjuvanted SU groups, and these groups had an increased percentage of virus-specific CD4+ T cells producing two Thelper 1 (Th1) cytokines simultaneously compared with the non-adjuvanted SU group. Overall, the WV vaccine induced higher antibody concentrations in sera and an HI and SRH response similar to that of the chitosan-adjuvanted SU vaccine. Furthermore, the WV vaccine formulation showed a stronger bias towards a T-helper 1 profile than the SU vaccine and elicited the highest frequencies of CD4+ Th1 cells simultaneously secreting three different cytokines (INFγ+, IL2+ and INFα+). As expected, two immunisations gave a better immune response than one in all groups. The control group had very low or not detectable results in the performed immunoassays. Conclusion The cross-clade serum reactivity, improved B- and T-cell responses and dose-sparing potential of chitosan show that a chitosan-adjuvanted intranasal influenza vaccine is a promising candidate vaccine for further preclinical development.

Background Influenza H5N1 virus constitutes a pandemic threat and development of effective H5N1 vaccines is a global priority. Anti-influenza antibodies directed towards the haemagglutinin (HA) define a correlate of protection. Both antibody concentration and avidity may be important for virus neutralization and resolving influenza disease. Methods We conducted a phase I clinical trial of a virosomal H5N1 vaccine adjuvanted with the immunostimulating complex Matrix M™. Sixty adults were intramuscularly immunized with two vaccine doses (21 days apart) of 30 μg HA alone or 1.5, 7.5 or 30 μg HA adjuvanted with Matrix M™. Serum H5 HA1-specific antibodies and virus neutralization were determined at days 0, 21, 42, 180 and 360 and long-term memory B cells at day 360 post-vaccination. The binding of the HA specific antibodies was measured by avidity NaSCN-elution ELISA and surface plasmon resonance (SPR). Results The H5 HA1-specific IgG response peaked after the second dose (day 42), was dominated by IgG1 and IgG3 and was highest in the adjuvanted vaccine groups. IgG titres correlated significantly with virus neutralization at all time points (Spearman r ≥ 0.66, p