Rishi Pathirana

T-Helper 1 Cells Elicited by H5N1 Vaccination Predict Seroprotection

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 influenza-specific 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.

Longevity of B-Cell and T-Cell Responses After Live Attenuated Influenza Vaccination in Children

Background. The live attenuated influenza vaccine (LAIV) is the preferred vaccine for children, but the mechanisms behind protective immune responses are unclear, and the duration of immunity remains to be elucidated. This study reports on the longevity of B-cell and T-cell responses elicited by the LAIV. Methods. Thirty-eight children (3–17 years old) were administered seasonal LAIV. Blood samples were collected before vaccination with sequential sampling up to 1 year after vaccination. Humoral responses were evaluated by a hemagglutination inhibition assay, and memory B-cell responses were evaluated by an enzyme-linked immunosorbent spot assay (ELISpot). T-cell responses were evaluated by interferon γ (IFN-γ) ELISpot analysis, and intracellular cytokine staining of CD4+ T cells for detection of IFN-γ, interleukin 2, and tumor necrosis factor α was performed using flow cytometry. Results. LAIV induced significant increases in B-cell and T-cell responses, which were sustained at least 1 year after vaccination. Strain variations were observed, in which the B strain elicited stronger responses. IFN-γ–expressing T cell counts increased significantly, and remained higher than prevaccination levels 1 year later. Expression of T-helper type 1 intracellular cytokines (interleukin 2, IFN-γ, and tumor necrosis factor α) increased after 1 dose and were boosted after the second dose. Hemagglutination inhibition titers were sustained for 1 year. Vaccine-induced memory B cell counts were significantly increased, and the response persisted for one year. Conclusions. LAIV elicited B-cell and T-cell responses that persisted for at least 1 year in children. This is a novel finding that will aid future vaccine policy.

Serum IgG titres, but not avidity, correlates with neutralizing antibody response after H5N1 vaccination

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<0.0001). By elution ELISA, serum antibody avidity was highest at days 180 and 360 post vaccination and did not correlate with virus neutralization. Long-lasting H5 HA1-specific memory B cells produced high IgG antibody avidity similar to serum IgG. CONCLUSIONS Maturation of serum antibody avidity continued up to day 360 after influenza H5N1 vaccination. Virus neutralization correlated with serum H5 HA1-specific IgG antibody concentrations and not antibody avidity.

The mucosal and systemic immune responses elicited by a chitosan-adjuvanted intranasal influenza H5N1 vaccine

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.

A study of Chitosan and c-di-GMP as mucosal adjuvants for intranasal influenza H5N1 vaccine

Please cite this paper as: Svindland et al. (2012) A study of Chitosan and c‐di‐GMP as mucosal adjuvants for intranasal influenza H5N1 vaccine. Influenza and Other Respiratory Viruses 10.1111/irv.12056000(000), 000–000.

Live Attenuated Influenza Vaccine in Children Induces B-Cell Responses in Tonsils

Background. Tonsils play a key role in eliciting immune responses against respiratory pathogens. Little is known about how tonsils contribute to the local immune response after intranasal vaccination. Here, we uniquely report the mucosal humoral responses in tonsils and saliva after intranasal live attenuated influenza vaccine (LAIV) vaccination in children. Methods. Blood, saliva, and tonsils samples were collected from 39 children before and after LAIV vaccination and from 16 age-matched, nonvaccinated controls. Serum antibody responses were determined by a hemagglutination inhibition (HI) assay. The salivary immunoglobulin A (IgA) level was measured by an enzyme-linked immunosorbent assay. Antibody-secreting cell (ASC) and memory B-cell (MBC) responses were enumerated in tonsils and blood. Results. Significant increases were observed in levels of serum antibodies and salivary IgA to influenza A(H3N2) and influenza B virus strains as early as 14 days after vaccination but not to influenza A(H1N1). Influenza virus–specific salivary IgA levels correlated with serum HI responses, making this a new possible indicator of vaccine immunogenicity in children. LAIV augmented influenza virus–specific B-cell responses in tonsils and blood. Tonsillar MBC responses correlated with systemic MBC and serological responses. Naive children showed significant increases in MBC counts after LAIV vaccination. Conclusions. This is the first study to demonstrate that LAIV elicits humoral B-cell responses in tonsils of young children. Furthermore, salivary IgA analysis represents an easy method for measuring immunogenicity after vaccination.

A(H1N1)pdm09 Vaccination of Health Care Workers: Improved Immune Responses in Low Responders Following Revaccination

BACKGROUND We conducted a clinical trial in October 2009 to evaluate the immunogenicity of the AS03-adjuvanted influenza vaccine (pH1N1 vaccine) in health care workers (HCWs). 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. CLINICAL TRIALS REGISTRATION NCT01003288.

Intranasal vaccination with a plant-derived H5 HA vaccine protects mice and ferrets against highly pathogenic avian influenza virus challenge

Highly pathogenic avian influenza H5N1 infection remains a public health threat and vaccination is the best measure of limiting the impact of a potential pandemic. Mucosal vaccines have the advantage of eliciting immune responses at the site of viral entry, thereby preventing infection as well as further viral transmission. In this study, we assessed the protective efficacy of hemagglutinin (HA) from the A/Indonesia/05/05 (H5N1) strain of influenza virus that was produced by transient expression in plants. The plant-derived vaccine, in combination with the mucosal adjuvant (3′,5′)-cyclic dimeric guanylic acid (c-di-GMP) was used for intranasal immunization of mice and ferrets, before challenge with a lethal dose of the A/Indonesia/05/05 (H5N1) virus. Mice vaccinated with 15 μg or 5 μg of adjuvanted HA survived the viral challenge, while all control mice died within 10 d of challenge. Vaccinated animals elicited serum hemagglutination inhibition, IgG and IgA antibody titers. In the ferret challenge study, all animals vaccinated with the adjuvanted plant vaccine survived the lethal viral challenge, while 50% of the control animals died. In both the mouse and ferret models, the vaccinated animals were better protected from weight loss and body temperature changes associated with H5N1 infection compared with the non-vaccinated controls. Furthermore, the systemic spread of the virus was lower in the vaccinated animals compared with the controls. Results presented here suggest that the plant-produced HA-based influenza vaccine adjuvanted with c-di-GMP is a promising vaccine/adjuvant combination for the development of new mucosal influenza vaccines.

Single dose vaccination of the ASO3-adjuvanted A(H1N1)pdm09 monovalent vaccine in health care workers elicits homologous and cross-reactive cellular and humoral responses to H1N1 strains

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.

Matrix M H5N1 Vaccine Induces Cross-H5 Clade Humoral Immune Responses in a Randomized Clinical Trial and Provides Protection from Highly Pathogenic Influenza Challenge in Ferrets

Background and Methods Highly pathogenic avian influenza (HPAI) viruses constitute a pandemic threat and the development of effective vaccines is a global priority. Sixty adults were recruited into a randomized clinical trial and were intramuscularly immunized with two virosomal vaccine H5N1 (NIBRG-14) doses (21 days apart) of 30μg HA alone or 1.5, 7.5 or 30μg HA adjuvanted with Matrix M. The kinetics and longevity of the serological responses against NIBRG-14 were determined by haemagglutination inhibition (HI), single radial haemolysis (SRH), microneutralization (MN) and ELISA assays. The cross-H5 clade responses in sera were determined by HI and the antibody-secreting (ASC) cell ELISPOT assays. The protective efficacy of the vaccine against homologous HPAI challenge was evaluated in ferrets. Results The serological responses against the homologous and cross-reactive strains generally peaked one week after the second dose, and formulation with Matrix M augmented the responses. The NIBRG-14-specific seroprotection rates fell significantly by six months and were low against cross-reactive strains although the adjuvant appeared to prolong the longevity of the protective responses in some subjects. By 12 months post-vaccination, nearly all vaccinees had NIBRG-14-specific antibody titres below the protective thresholds. The Matrix M adjuvant was shown to greatly improve ASC and serum IgG responses following vaccination. In a HPAI ferret challenge model, the vaccine protected the animals from febrile responses, severe weight loss and local and systemic spread of the virus. Conclusion Our findings show that the Matrix M-adjuvanted virosomal H5N1 vaccine is a promising pre-pandemic vaccine candidate. Trial Registration ClinicalTrials.gov NCT00868218