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Abstract Antibodies generated against West Nile virus (WNV) during infection are essential for controlling dissemination. Recent studies have demonstrated that epitopes in all three domains of the flavivirus envelope protein (E) are targets for neutralizing antibodies, with determinants in domain III (DIII) eliciting antibodies with strong inhibitory properties. In order to increase the magnitude and quality of the antibody response against the WNV E protein, DNA vaccines with derivatives of the WNV E gene (full length E, truncated E, or DIII region, some in the context of the pre-membrane [prM] gene) were conjugated to the molecular adjuvant P28. The P28 region of the complement protein C3d is the minimum CR2-binding domain necessary for the adjuvant activity of C3d. Delivery of DNA-based vaccines by gene gun and intramuscular routes stimulated production of IgG antibodies against the WNV DIII region of the E protein. With the exception of the vaccine expressing prM/E given intramuscularly, only mice that received DNA vaccines by gene gun produced protective neutralizing antibody titers (FRNT80 titer >1/40). Correspondingly, mice vaccinated by the gene gun route were protected to a greater level from lethal WNV challenge. In general, mice vaccinated with P28-adjuvated vaccines produced higher IgG titers than mice vaccinated with non-adjuvanted vaccines.

Vaccination is the most effective way to prevent influenza virus infections. However, the diversity of antigenically distinct isolates is a challenge for vaccine development. In order to overcome the antigenic variability and improve the protective efficacy of influenza vaccines, our research group has pioneered the development of computationally optimized broadly reactive antigens (COBRA) for hemagglutinin (HA). Two candidate COBRA HA vaccines, P1 and X6, elicited antibodies with differential patterns of hemagglutination inhibition (HAI) activity against a panel of H1N1 influenza viruses. In order to better understand how these HA antigens elicit broadly reactive immune responses, epitopes in the Cb, Sa, or Sb antigenic sites of seasonal-like and pandemic-like wild-type or COBRA HA antigens were exchanged with homologous regions in the COBRA HA proteins to determine which regions and residues were responsible for the elicited antibody profile. Mice were vaccinated with virus-like particles (VLPs) expressing one of the 12 modified HA antigens (designated V1 to V12), COBRA HA antigens, or wild-type HA antigens. The elicited antisera was assessed for hemagglutination inhibition activity against a panel of historical seasonal-like and pandemic-like H1N1 influenza viruses. Primarily, the pattern of glycosylation sites and residues in the Sa antigenic region, around the receptor binding site (RBS), served as signatures for the elicitation of broadly reactive antibodies by these HA immunogens. Mice were vaccinated with VLPs expressing HA antigens that lacked a glycosylation site at residue 144 and a deleted lysine at position 147 residue were more effective at protecting against morbidity and mortality following infection with pandemic-like and seasonal-like H1N1 influenza viruses. IMPORTANCE There is a great need to develop broadly reactive or universal vaccines against influenza viruses. Advanced, next-generation hemagglutinin (HA) head-based vaccines that elicit protective antibodies against H1N1 influenza viruses have been developed. This study focused on understanding the specific amino acids around the receptor binding site (RBS) that were important in elicitation of these broadly reactive antibodies. Specific glycan sites and amino acids located at the tip of the HA molecule enhanced the elicitation of these broadly reactive antibodies. A better understanding of the HA structures around the RBS will lead to more effective HA immunogens. [ABSTRACT FROM AUTHOR]

Unlabelled: Broadly reactive antibodies targeting the conserved hemagglutinin (HA) stalk region are elicited following sequential infection or vaccination with influenza viruses belonging to divergent subtypes and/or expressing antigenically distinct HA globular head domains. Here, we demonstrate, through the use of novel chimeric HA proteins and competitive binding assays, that sequential infection of ferrets with antigenically distinct seasonal H1N1 (sH1N1) influenza virus isolates induced an HA stalk-specific antibody response. Additionally, stalk-specific antibody titers were boosted following sequential infection with antigenically distinct sH1N1 isolates in spite of preexisting, cross-reactive, HA-specific antibody titers. Despite a decline in stalk-specific serum antibody titers, sequential sH1N1 influenza virus-infected ferrets were protected from challenge with a novel H1N1 influenza virus (A/California/07/2009), and these ferrets poorly transmitted the virus to naive contacts. Collectively, these findings indicate that HA stalk-specific antibodies are commonly elicited in ferrets following sequential infection with antigenically distinct sH1N1 influenza virus isolates lacking HA receptor-binding site cross-reactivity and can protect ferrets against a pathogenic novel H1N1 virus., Importance: The influenza virus hemagglutinin (HA) is a major target of the humoral immune response following infection and/or seasonal vaccination. While antibodies targeting the receptor-binding pocket of HA possess strong neutralization capacities, these antibodies are largely strain specific and do not confer protection against antigenic drift variant or novel HA subtype-expressing viruses. In contrast, antibodies targeting the conserved stalk region of HA exhibit broader reactivity among viruses within and among influenza virus subtypes. Here, we show that sequential infection of ferrets with antigenically distinct seasonal H1N1 influenza viruses boosts the antibody responses directed at the HA stalk region. Moreover, ferrets possessing HA stalk-specific antibody were protected against novel H1N1 virus infection and did not transmit the virus to naive contacts., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: The influenza A virus genome is composed of eight single-stranded RNA segments of negative polarity. Although the hemagglutinin and neuraminidase genes are known to play a key role in host adaptation, the polymerase genes (which encode the polymerase segments PB2, PB1, PA) and the nucleoprotein gene are also important for the efficient propagation of the virus in the host and for its adaptation to new hosts. Current efforts to understand the host-specificity of the virus have largely focused on the amino acid differences between avian and human isolates., Results: Here we show that the folding free energy of the RNA segments may play an equally important role in the evolution and host adaptation of the influenza virus. Folding free energy may affect the stability of the viral RNA and influence the rate of viral protein translation. We found that there is a clear distinction between the avian and human folding free energy distributions for the polymerase and the nucleoprotein genes, with human viruses having substantially higher folding free energy values. This difference is independent of the amino acid composition and the codon bias. Furthermore, the folding free energy values of the commonly circulating human viruses tend to shift towards higher values over the years, after they entered the human population. Finally, our results indicate that the temperature in which the cells grow affects infection efficiency., Conclusions: Our data suggest for the first time that RNA structure stability may play an important role in the emergence and host shift of influenza A virus. The fact that cell temperature affects virus propagation in mammalian cells could help identify those avian strains that pose a higher threat to humans.