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

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

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

Matrix-M Adjuvated Seasonal Virosomal Influenza Vaccine Induces Partial Protection in Mice and Ferrets against Avian H5 and H7 Challenge

<div><p>There is a constant threat of zoonotic influenza viruses causing a pandemic outbreak in humans. It is virtually impossible to predict which virus strain will cause the next pandemic and it takes a considerable amount of time before a safe and effective vaccine will be available once a pandemic occurs. In addition, development of pandemic vaccines is hampered by the generally poor immunogenicity of avian influenza viruses in humans. An effective pre-pandemic vaccine is therefore required as a first line of defense. Broadening of the protective efficacy of current seasonal vaccines by adding an adjuvant may be a way to provide such first line of defense. Here we evaluate whether a seasonal trivalent virosomal vaccine (TVV) adjuvated with the saponin-based adjuvant Matrix-M (MM) can confer protection against avian influenza H5 and H7 virus strains in mice and ferrets. We demonstrate that mice were protected from death against challenges with H5N1 and H7N7, but that the protection was not complete as evidenced by severe clinical signs. In ferrets, protection against H7N9 was not observed. In contrast, reduced upper and lower respiratory tract viral loads and reduced lung pathology, was achieved in H5N1 challenged ferrets. Together these results suggest that, at least to some extent, Matrix-M adjuvated seasonal virosomal influenza vaccine can serve as an interim measure to decrease morbidity and mortality associated with a pandemic outbreak.</p></div

Ferrets are not protected against highly pathogenic H7N9 after TVV+MM vaccination.

<p>Groups of 7–8 ferrets received two intramuscular injections with TVV, TVV+MM, PBS, PBS+MM or inactivated H7N9 virus as positive control (Control). 4 weeks later the animals were challenged with a sub-lethal dose of 10^5.5 TCID₅₀ of influenza A H7N9 A/Anhui/1/2013. Ferrets were monitored for 4 consecutive days and sacrificed at day 4 post challenge. (A) Infectious viral load in lung tissue (B) infectious throat viral load (day 1 to 4), (C) percentage of body weight change during the observation period (D) lung weight as determined after sacrifice. Dots indicate individual animals and horizontal lines represent group means (A and D). Lines represent group mean with 95% confidence interval (B) or the interquartile range (C). Asterisks indicate statistically significant differences compared to PBS injected animals (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, according to the materials and methods section).</p

Ferrets are partially protected against highly pathogenic H5N1 after TVV+MM vaccination.

<p>Groups of 7–8 ferrets received two intramuscular injections with TVV, TVV+MM, PBS, PBS+MM or inactivated H5H1 virus as positive control (Control). 4 weeks later the animals were challenged with a sub-lethal dose of 10⁴ TCID₅₀ of influenza A H5N1 A/Indonesia/05/2005. Ferrets were monitored for 4 consecutive days and sacrificed at day 4 post challenge. (A) Infectious viral load in lung tissue (B) infectious throat viral load (day 1 to 4), (C) percentage of body weight change during the observation period and (D) lung weight as determined after sacrifice. Dots indicate individual animals and horizontal lines represent group means (A and D). Lines represent group mean with 95% confidence interval (B) or the interquartile range (C). Asterisks indicate statistically significant differences compared to PBS injected animals (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, according to the materials and methods section).</p

TVV+MM protects mice against avian H5N1.

<p>Mice (n = 9-10/group) were immunized 1x or 2x with TVV with or without MM. Four weeks later, mice were challenged with 25xLD₅₀ wild-type A/Hong Kong /156/97 (H5N1) and monitored for 21 days for survival, body weight loss and clinical symptoms. Graphs represent the Kaplan-Meier survival curve (A and D) or mean bodyweight change with 95% confidence interval (B and E) or mean clinical scores with interquartile range (C and F). Asterisks indicate statistically significant differences compared to the vehicle control group (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, according to the materials and methods section).</p

TVV+MM induces cross-reactive H5 and H7 antibody responses.

<p>Mice (n = 9-10/group) were immunized 1x or 2x with TVV with or without MM. 27 days later (1 day before challenge) individual serum samples were obtained and tested for (A) vaccine homologous recH1 of A/California/07/07, (B) vaccine homologous recH3 of the A/Victoria/210/09-like A/Perth/16/09 (98.8% homologous), (C) recH5 of A/Hong Kong/156/97, and (D) recH7 of A/Netherlands/219/03 (99.6% homologous to the challenge strain A/chicken/Netherlands/621557/03) antibody responses. Serum pools of mice (n = 50/group) that received 1x or 2x TVV+MM or no immunization (-) were tested for (E) vaccine homologous recN1 A/California/04/09 and (F) recN1 of A/Hong Kong/156/97 reactive antibody responses. Black bars indicate medians of log-10 transformed ELISA titers (EU). Asterisks indicate statistically significant differences compared to the vehicle control group (*p<0.05, **p<0.01, ***p<0.001, according to the materials and methods section).</p

Additional file 1: Table S1. of Matrix-M™ adjuvation broadens protection induced by seasonal trivalent virosomal influenza vaccine

Summary statistical analysis HAI assays. (DOCX 17 kb