Enhancing NA immunogenicity through novel VLP designs.

Current influenza virus vaccines poorly display key neuraminidase (NA) epitopes and do not robustly induce NA-reactive antibodies; instead, they focus on the induction of hemagglutinin (HA)-reactive antibodies. Next-generation influenza vaccines should be optimized in order to activate NA-reactive B cells and to induce a broadly cross-reactive and protective antibody response. We aimed at enhancing the immunogenicity of the NA on vaccines by two strategies: (i) modifying the HA:NA ratio of the vaccine preparation and (ii) exposing epitopes on the lateral surface or beneath the head of the NA by extending the NA stalk. The H1N1 glycoproteins from the influenza virus A/California/04/2009 strain were displayed on human immunodeficiency virus 1 (HIV-1) gag-based virus-like particles (VLP). Using the baculovirus insect cell expression system, we biased the quantity of surface glycoproteins employing two different promoters, the very late baculovirus p10 promoter and the early and late gp64 promoter. This led to a 1:1 to 2:1 HA:NA ratio, which was approximately double or triple the amount of NA as present on the wild-type influenza A virus (HA:NA ratio 3:1 to 5:1). Furthermore, by insertion of 15 amino acids from the A-New York/61/2012 strain (NY12) which prolongates the NA stalk (NA long stalk; NA-LS), we intended to improve the accessibility of the NA. Six different types of VLPs were produced and purified using a platform downstream process based on Capto-Core 700™ followed by Capto-Heparin™ affinity chromatography combined with ultracentrifugation. These VLPs were then tested in a mouse model. Robust titers of antibodies that inhibit the neuraminidase activity were elicited even after vaccination with two low doses (0.3 µg) of the H1N1 VLPs without compromising the anti-HA responses. In conclusion, our results demonstrate the feasibility of the two developed strategies to retain HA immunogenicity and improve NA immunogenicity as a future influenza vaccine candidate.

Increased efficacy of influenza virus vaccine candidate through display of recombinant neuraminidase on virus like particles.

Vaccination against influenza virus can reduce the risk of influenza by 40% to 60%, they rely on the production of neutralizing antibodies specific to influenza hemagglutinin (HA) ignoring the neuraminidase (NA) as an important surface target. Vaccination with standardized NA concentration may offer broader and longer-lasting protection against influenza infection. In this regard, we aimed to compare the potency of a NA displayed on the surface of a VLP with a soluble NA. The baculovirus expression system (BEVS) and the novel virus-free Tnms42 insect cell line were used to express N2 NA on gag-based VLPs. To produce VLP immunogens with high levels of purity and concentration, a two-step chromatography purification process combined with ultracentrifugation was used. In a prime/boost vaccination scheme, mice vaccinated with 1 µg of the N2-VLPs were protected from mortality, while mice receiving the same dose of unadjuvanted NA in soluble form succumbed to the lethal infection. Moreover, NA inhibition assays and NA-ELISAs of pre-boost and pre-challenge sera confirm that the VLP preparation induced higher levels of NA-specific antibodies outperforming the soluble unadjuvanted NA.