One microgram protein per sample was prepared in Tris-glycine-SDS sample buffer (Invitrogen) and NuPAGE sample reducing agent (Invitrogen) and boiled at 100C for 5 min before being loaded onto 10% Mini-PROTEAN TGX precast gels (Bio-Rad) and run under denaturing conditions in the presence of sodium dodecyl sulfate (SDS)

One microgram protein per sample was prepared in Tris-glycine-SDS sample buffer (Invitrogen) and NuPAGE sample reducing agent (Invitrogen) and boiled at 100C for 5 min before being loaded onto 10% Mini-PROTEAN TGX precast gels (Bio-Rad) and run under denaturing conditions in the presence of sodium dodecyl sulfate (SDS). virus vaccines shows better protection against influenza virus challenge. Our results provide evidence that the immunodominance of STING agonist-1 HA stems in part from its abundance on the viral surface, and that rewiring viral packaging signalsthereby increasing the NA content on viral particlesis a viable strategy for improving the immunogenicity of NA in an influenza virus vaccine. IMPORTANCEInfluenza virus infections are a major source of morbidity and mortality worldwide. Increasing evidence highlights neuraminidase as a potential vaccination target. This report demonstrates the efficacy of rewiring influenza virus packaging signals for creating vaccines with more neuraminidase content which provide better neuraminidase (NA)-based protection. == INTRODUCTION == Influenza virus entry and egress are mediated predominantly by the two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). These two proteins function antagonisticallyHA is responsible for sialic acid binding while NA cleaves sialic acid (1). Current seasonal influenza virus vaccination strategies focus heavily on eliciting an immune response against the viral HA, as anti-HA antibodies are often neutralizing and hemagglutination inhibition is an established correlate of protection (2,3). Antigenic drift of the HA head domain necessitates constant reformulation of seasonal vaccines, and annual vaccine effectiveness is highly variable (4). Anti-NA antibody titers have been shown to correlate with reductions in both viral shedding and infection severity (3,5,6), and small molecules which inhibit NA currently serve as first-line therapeutics for active influenza virus infection (7). The amino acid drift rates for NA are lower than those for HA (8,9), and substantial evidence exists for the ability of humoral NA antibody responses to confer heterologous protection (1015). Despite strong evidence that NA-based immunity is protective, current Rabbit Polyclonal to GJC3 seasonal vaccines are only required to contain 15 g of HA, without standardization of NA content (16). Recent STING agonist-1 work has demonstrated that, in contrast to natural infection, seasonal vaccination STING agonist-1 fails to induce robust anti-NA immune responses (17). While a number of platforms such as recombinant NA protein (1315) or NA-only virus-like particles (VLPs) (18,19) have been put forth as vaccine candidates, few strategies exist for boosting the host immune response against NA in the context of influenza virus vaccines that also induce HA immunity. HA is the predominant glycoprotein on the virus surface, outnumbering NA at estimates ranging from 4:1 to 14:1 (20,21), and its immunodominance over NA has been well characterized during both vaccination and infection (22,23). In this study, we demonstrate that by rewiring the terminal 5 and 3 packaging signals of the HA and NA genomic segments, viruses can be rescued that express more NA and less HA. Vaccination with these viruses induces stronger anti-NA humoral responses that protect mice in passive transfer studies against influenza virus challenge. == RESULTS == == Design of rewired PR8 virus. == Prior studies have demonstrated that segments coding for foreign proteins such as green fluorescent protein (GFP) can be efficiently packaged with the influenza virus genome by flanking the open reading frames (ORFs) of these proteins with variable stretches of nucleotides taken from the 5 and 3 termini of influenza virus gene segments (2430). These terminal stretches, composed of both the untranslated regions (UTRs) and a portion of.