BackgroundThe lack of a universal influenza vaccine is a global health problem. Interest is now focused on structurally conserved protein domains capable of eliciting protection against a broad range of influenza virus strains. The long alpha helix (LAH) is an attractive vaccine component since it is one of the most conserved influenza hemagglutinin (HA) stalk regions. For an improved immune response, the LAH domain from H3N2 strain has been incorporated into virus-like particles (VLPs) derived from hepatitis B virus core protein (HBc) using recently developed tandem core technology.ResultsFermentation conditions for recombinant HBc-LAH were established in yeast Pichia pastoris and a rapid and efficient purification method for chimeric VLPs was developed to match the requirements for industrial scale-up. Purified VLPs induced strong antibody responses against both group 1 and group 2 HA proteins in mice.ConclusionOur results indicate that the tandem core technology is a useful tool for incorporation of highly hydrophobic LAH domain into HBc VLPs. Chimeric VLPs can be successfully produced in bioreactor using yeast expression system. Immunologic data indicate that HBc VLPs carrying the LAH antigen represent a promising universal influenza vaccine component.Electronic supplementary materialThe online version of this article (10.1186/s12896-017-0396-8) contains supplementary material, which is available to authorized users.

Long alpha helix (LAH) from influenza virus hemagglutinin (HA) stem or stalk domain is one of the most conserved influenza virus antigens. Expression of N-terminally extended LAH in E. coli leads to assembly of α-h elical homotrimer which is structurally nearly identical to the corresponding region of post-fusion form of native HA. This novel tri-stalk protein was able to differentiate between group 1 and 2 influenza in ELISA with virus-infected mice sera. It was also successfully applied for enzyme-linked immunospot assay to estimate the number of HA stem-reactive antibody (Ab)-secreting cells in mice. An in-house indirect ELISA was developed using a HA tri-stalk protein as a coating antigen for evaluation of HA stem-specific Ab levels in human sera collected in Luxembourg from 211 persons with occupational exposure to swine before the pandemic H1N1/09 virus had spread to Western Europe. Our results show that 70% of these pre-pandemic sera are positive for HA stem-specific Abs. In addition, levels of HA stem-specific Abs have positive correlation with the corresponding IgG titers and neutralizing activities against pandemic H1N1/09 virus.

Structure determination of adjuvant‐coupled antigens is essential for rational vaccine development but has so far been hampered by the relatively low antigen content in vaccine formulations and by their heterogeneous composition. Here we show that magic‐angle spinning (MAS) solid‐state NMR can be used to assess the structure of the influenza virus hemagglutinin stalk long alpha helix antigen, both in its free, unformulated form and once chemically coupled to the surface of large virus‐like particles (VLPs). The sensitivity boost provided by high‐field dynamic nuclear polarization (DNP) and proton detection at fast MAS rates allows to overcome the penalty associated with the antigen dilution. Comparison of the MAS NMR fingerprints between the free and VLP‐coupled forms of the antigen provides structural evidence of the conservation of its native fold upon bioconjugation. This work demonstrates that high‐sensitivity MAS NMR is ripe to play a major role in vaccine design, formulation studies, and manufacturing process development.

Influenza, an acute, highly contagious respiratory disease, remains a significant threat to public health. More effective vaccination strategies aimed at inducing broad cross-protection not only against seasonal influenza variants, but also zoonotic and emerging pandemic influenza strains are urgently needed. A number of conserved protein targets to elicit such cross-protective immunity have been under investigation, with long alpha-helix (LAH) from hemagglutinin stalk and ectodomain of matrix protein 2 ion channel (M2e) being the most studied ones. Recently, we have reported the three-dimensional structure and some practical applications of LAH expressed in Escherichia coli system (referred to as tri-stalk protein). In the present study, we investigated the immunogenicity and efficacy of a panel of broadly protective influenza vaccine prototypes based on both influenza tri-stalk and triple M2e (3M2e) antigens integrated into phage AP205 virus-like particles (VLPs). While VLPs containing the 3M2e alone induced protection against standard homologous and heterologous virus challenge in mice, only the combination of both conserved influenza antigens into a single VLP fully protected mice from a high-dose homologous H1N1 influenza infection. We propose that a combination of genetic fusion and chemical coupling techniques to expose two different foreign influenza antigens on a single particle is a perspective approach for generation of a broadly-effective vaccine candidate that could protect against the constantly emerging influenza virus strains.

Die Strukturbestimmung Wirkverstärker‐gekoppelter Antigene ist für eine rationale Impfstoffentwicklung unerlässlich, wurde aber bisher durch den recht geringen Antigengehalt in Impfstoff‐Formulierungen und durch deren heterogene Zusammensetzung erschwert. Wir zeigen, dass die Festkörper‐NMR mit Magic Angle Spinning (MAS) zur Bestimmung der Struktur des Influenza‐Virus‐Hämagglutinin‐Stiel‐Antigens verwendet werden kann, und dies sowohl in seiner freien Form sowie nach chemischer Kopplung an die Oberfläche großer virusähnlicher Partikel (VLPs). Die Steigerung der Sensitivität durch dynamische nukleare Polarisation (DNP) und Protonen‐Detektion bei schnellen MAS‐Raten ermöglicht es, den mit der Antigenverdünnung verbundenen Nachteil auszugleichen. Der Vergleich der MAS‐NMR‐Fingerabdrücke zwischen freier und VLP‐gekoppelter Antigenform liefert strukturelle Beweise für die Erhaltung seiner nativen Faltung nach der Bio‐Konjugation. Die hochempfindliche MAS‐NMR ist reif, um eine wichtige Rolle bei Impfstoffdesign, Formulierungsstudien und der Entwicklung von Herstellungsprozessen zu spielen.