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.
Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Florian Krammer reports a relationship with Castlevax, Dynavax, Third Rock Ventures, Avimex, VIR, Gritstone Bio that includes: board membership, consulting or advisory, equity or stocks, and funding grants. Florian Krammer has patent #Influenza virus vaccines and therapeutics, SARS-CoV-2 vaccines, SARS-CoV-2 diagnostic tests with royalties paid to Castlevax, Avimex, Leiden Labs. The Icahn School of Medicine at Mount Sinai has filed patent applications relating to SARS-CoV-2 serological assays, NDV-based SARS-CoV-2 vaccines influenza virus vaccines and influenza virus therapeutics which list Florian Krammer as co-inventor. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, CastleVax, to develop SARS-CoV-2 vaccines. Florian Krammer is co-founder and scientific advisory board member of CastleVax. Florian Krammer has consulted for Merck, Curevac, GSK, Seqirus and Pfizer and is currently consulting for 3rd Rock Ventures, Gritstone and Avimex. The Krammer laboratory is collaborating with Dynavax on influenza vaccine development and with VIR on influenza virus therapeutics. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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