Protection against H5N1 Influenza Virus Induced by Matrix-M Adjuvanted Seasonal Virosomal Vaccine in Mice Requires Both Antibodies and T Cells.

BACKGROUND:It remains important to develop the next generation of influenza vaccines that can provide protection against vaccine mismatched strains and to be prepared for potential pandemic outbreaks. To achieve this, the understanding of the immunological parameters that mediate such broad protection is crucial. METHOD:In the current study we assessed the contribution of humoral and cellular immune responses to heterosubtypic protection against H5N1 induced by a Matrix-M (MM) adjuvanted seasonal influenza vaccine by serum transfer and T-cell depletion studies. RESULTS:We demonstrate that the heterosubtypic protection against H5N1 induced by MM adjuvanted vaccine is partially mediated by antibodies. The serum contained both H5N1 cross-reactive hemagglutinin (HA)- and neuraminidase (NA)-specific antibodies but with limited virus neutralizing and no hemagglutination inhibiting activity. The cross-reactive antibodies induced antibody-dependent cellular cytotoxicity (ADCC) in vitro, suggesting a role for the Fc part of the antibodies in protection against H5N1. Besides H5N1 specific antibody responses, cross-reactive HA- and NA-specific T-cell responses were induced by the adjuvanted vaccine. T-cell depletion experiments demonstrated that both CD4+ and CD8+ T cells contribute to protection. CONCLUSION:Our study demonstrates that cross-protection against H5N1 induced by MM adjuvanted seasonal virosomal influenza vaccine requires both the humoral and cellular arm of the immune system

CD4⁺ and CD8⁺ T cells both contribute to H5N1 protection.

<p>Mice (n = 8-10/group) were immunized once with TVV+MM or PBS as negative control 4 weeks before challenge and (A) CD8⁺ or the combination of CD4⁺ and CD8⁺ T cells were depleted or (B) CD4⁺ T cells only were depleted with antibodies injected 4 days and 1 day before challenge. Mice were challenged with 12.5xLD₅₀ of wild type H5N1 A/Hong Kong/156/97 and monitored for 21 days for (A and B) survival and (C and D) bodyweight-loss. Graphs A and B represent the Kaplan-Meier survival curves and graphs C and D represent mean bodyweight change with 95% confidence interval.</p

H5 and N1 cross-reactive T cells are induced by TVV+MM.

<p>Mice (n = 8/group) were immunized once or twice with TVV or TVV+MM. Three weeks later, spleens were harvested. The number of IFN-γ producing T cells was determined by ex vivo stimulation of splenocytes with peptide pools consisting of 15-mer peptides that cover the total (A) HA or (B) NA sequence of H5/HK with an 11-mer overlap. Black bars indicate medians of IFN-γ⁺ T cells per 10⁶ splenocytes. SFU = Spot forming units.</p

Cross-reactive HA and NA-specific ADCC responses are induced by TVV+MM.

<p>Mice (n = 8/group) were immunized once or twice with TVV or TVV+MM. Three weeks later, serum samples were obtained and tested for H5N1 A/Hong Kong/156/97 cross-reactive HAI responses (A), neutralizing antibody responses (B), HA-specific antibodies (C) and NA-specific antibodies (D) or ADCC responses against H5 expressing cells (E) or N1 expressing cells (F). Black bars indicate medians of log-2 transformed HAI and neutralizing titers (NT) or log-10 transformed ELISA titers (EU). Error bars in ADCC assays indicate the standard error of the duplicate means. Control in HAI assay = H5/HK specific sheep serum.</p

Serum antibodies confer partial protection against H5N1.

<p>(A) Mice (n = 8/group) were immunized once with TVV+MM or PBS as negative control 4 weeks before challenge. (B) Recipient mice (n = 11-12/group) received 400μl immune sera of 1-time or 2-times TVV+MM immunized donors or naïve serum intraperitoneally one day before challenge. Mice were challenged with 12.5xLD₅₀ of wild type H5N1 A/Hong Kong/156/97 and monitored for 21 days for survival and weight-loss. Graphs A and B represent the Kaplan-Meier survival curves and graphs C and D represent mean bodyweight change with 95% confidence interval. ST = serum transfer.</p

Mini-hemagglutinin vaccination induces cross-reactive antibodies in pre-exposed NHP that protect mice against lethal influenza challenge

Seasonal vaccines are currently the most effective countermeasure against influenza. However, seasonal vaccines are only effective against strains closely related to the influenza strains contained in the vaccine. Recently a new hemagglutinin (HA) stem-based antigen, the so-called "mini-HA", has been shown to induce a cross-protective immune response in influenza-naive mice and non-human primates (NHP). However, prior exposure to influenza can have a profound effect on the immune response to subsequent influenza infection and the protective efficacy of vaccination. Here we show that mini-HA, compared to a trivalent influenza vaccine (TIV), elicits a broadened influenza-specific humoral immune response in NHP previously exposed to influenza. Serum transfer experiments showed that antibodies induced by both mini-HA and seasonal vaccine protected mice against lethal challenge with a H1N1 influenza strain heterologous to the H1 HA included in the TIV. However, antibodies elicited by mini-HA showed an additional benefit of protecting mice against lethal heterosubtypic H5N1 influenza challenge, associated with H5 HA-specific functional antibodies

A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen

The identification of human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem revitalized hopes of developing a universal influenza vaccine. Using a rational design and library approach, we engineered stable HA stem antigens ("mini-HAs") based on an H1 subtype sequence. Our most advanced candidate exhibits structural and bnAb binding properties comparable to those of full-length HA, completely protects mice in lethal heterologous and heterosubtypic challenge models, and reduces fever after sublethal challenge in cynomolgus monkeys. Antibodies elicited by this mini-HA in mice and nonhuman primates bound a wide range of HAs, competed with human bnAbs for HA stem binding, neutralized H5N1 viruses, and mediated antibody-dependent effector activity. These results represent a proof of concept for the design of HA stem mimics that elicit bnAbs against influenza A group 1 viruse