[Show abstract][Hide abstract] ABSTRACT: The global population remains vulnerable in the face of the next pandemic influenza virus outbreak, and reformulated vaccinations are administered annually to manage seasonal epidemics. Therefore, development of a new generation of vaccines is needed to generate broad and persistent immunity to influenza viruses. Here, we describe three adjuvants that enhance the induction of stalk-directed antibodies against heterologous and heterosubtypic influenza viruses when administered with chimeric HA proteins. Addavax, an MF59-like nanoemulsion, poly(I:C), and an RNA hairpin derived from Sendai virus (SeV) Cantell were efficacious intramuscularly. The SeV RNA and poly(I:C) also proved to be effective respiratory mucosal adjuvants. Although the quantity and quality of antibodies induced by the adjuvants varied, immunized mice demonstrated comparable levels of protection against challenge with influenza A viruses on the basis of HA stalk reactivity. Finally, we present that intranasally, but not intramuscularly, administered chimeric HA proteins induce mucosal IgA antibodies directed at the HA stalk.
[Show abstract][Hide abstract] ABSTRACT: Severe human disease caused by the emerging H7N9 influenza virus in China warrants a rapid response. Here, we present a recombinant
Newcastle disease virus expressing a North American lineage H7 influenza virus hemagglutinin. Sera from immunized mice were
cross-reactive to a broad range of H7 subtype viruses and inhibited hemagglutination by the novel H7 hemagglutinin. Immunized
mice were protected against a heterologous H7 subtype challenge, and genetic analysis suggested that cross-protective antibodies
recognize conserved antigenic sites.
Full-text · Article · May 2013 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: A serum hemagglutination inhibition (HAI) titer of 40 or greater is thought to be associated with reduced influenza virus pathogenesis in humans and is often used as a correlate of protection in influenza vaccine studies. We have previously demonstrated that intramuscular vaccination of guinea pigs with inactivated influenza virus generates HAI titers greater than 300 but does not protect vaccinated animals from becoming infected with influenza virus by transmission from an infected cage mate. Only guinea pigs intranasally inoculated with a live influenza virus or a live attenuated virus vaccine, prior to challenge, were protected from transmission (1). Because serum HAI titer is mostly determined by IgG content, these results led us to speculate that prevention of viral transmission may require IgA antibodies or cellular immune responses. To evaluate this hypothesis, guinea pigs and ferrets were administered a potent, neutralizing mouse IgG monoclonal antibody 30D1 (Ms30D1 IgG) against the A/California/04/2009 (H1N1) virus hemagglutinin and exposed to respiratory droplets from animals infected with this virus. Even though HAI titers were greater than 160 one day post-administration, Ms30D1 IgG did not prevent airborne transmission to passively immunized recipient animals. In contrast, intramuscular administration of recombinant 30D1 IgA (Ms30D1 IgA) prevented transmission to 88% of recipient guinea pigs, and Ms30D1 IgA was detected in animal nasal washes. Ms30D1 IgG administered intranasally also prevented transmission, suggesting the importance of mucosal immunity in preventing influenza virus transmission. Collectively, our data indicate that IgG antibodies may prevent pathogenesis associated with influenza virus infection but do not protect from virus infection by airborne transmission, while IgA antibodies are more important for preventing transmission of influenza viruses.
Full-text · Article · May 2013 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: The influenza virus nonstructural protein 1 (NS1) inhibits innate immunity by multiple mechanisms. We previously reported
that NS1 is able to inhibit the production of type I interferon (IFN) and proinflammatory cytokines in human primary dendritic
cells (DCs). Here, we used recombinant viruses expressing mutant NS1 from the A/Texas/36/91 and A/Puerto Rico/08/34 strains
in order to analyze the contribution of different NS1 domains to its antagonist functions. We show that the polyadenylation
stimulating factor 30 (CPSF30) binding function of the NS1 protein from A/Texas/36/91 influenza virus, which is absent in
the A/Puerto Rico/08/34 strain, is essential for counteracting these innate immune events in DCs. However, the double-stranded
RNA (dsRNA) binding domain, present in both strains, specifically inhibits the induction of type I IFN genes in infected DCs,
while it is essential only for inhibition of type I IFN proteins and proinflammatory cytokine production in cells infected
with influenza viruses lacking a functional CPSF30 binding domain, such as A/Puerto Rico/08/34.
Full-text · Article · Dec 2012 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: The limited availability of approved influenza virus antivirals highlights the importance of studying the fitness and transmissibility
of drug-resistant viruses. S247N is a novel, naturally occurring N1 neuraminidase mutation that reduces oseltamivir sensitivity
and greatly potentiates oseltamivir resistance in the context of the H275Y mutation. Here we show that highly oseltamivir-resistant
viruses containing both the S247N and H275Y mutations transmit efficiently in the guinea pig transmission model.
Full-text · Article · Feb 2012 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Oseltamivir is routinely used worldwide for the treatment of severe influenza A virus infection, and should drug-resistant pandemic 2009 H1N1 viruses become widespread, this potent defense strategy might fail. Oseltamivir-resistant variants of the pandemic 2009 H1N1 influenza A virus have been detected in a substantial number of patients, but to date, the mutant viruses have not moved into circulation in the general population. It is not known whether the resistance mutations in viral neuraminidase (NA) reduce viral fitness. We addressed this question by studying transmission of oseltamivir-resistant mutants derived from two different isolates of the pandemic H1N1 virus in both the guinea pig and ferret transmission models. In vitro, the virus readily acquired a single histidine-to-tyrosine mutation at position 275 (H275Y) in viral neuraminidase when serially passaged in cell culture with increasing concentrations of oseltamivir. This mutation conferred a high degree of resistance to oseltamivir but not zanamivir. Unexpectedly, in guinea pigs and ferrets, the fitness of viruses with the H275Y point mutation was not detectably impaired, and both wild-type and mutant viruses were transmitted equally well from animals that were initially inoculated with 1:1 virus mixtures to naïve contacts. In contrast, a reassortant virus containing an oseltamivir-resistant seasonal NA in the pandemic H1N1 background showed decreased transmission efficiency and fitness in the guinea pig model. Our data suggest that the currently circulating pandemic 2009 H1N1 virus has a high potential to acquire drug resistance without losing fitness.
Full-text · Article · Nov 2010 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Several members of the human APOBEC3 family of cytidine deaminases can potently restrict retroviruses such as HIV-1. The single-domain APOBEC3H (A3H) is encoded by four haplotypes, of which only A3H haplotype II-RDD (hapII-RDD) restricts HIV-1 efficiently. The goal of this study was to elucidate the mechanisms underlying the differences in antiviral activity among A3H haplotypes. The naturally occurring A3H hapI-GKE and hapII-RDD variants differ at three amino acid positions. A panel of six site-directed mutants containing combinations of the three variable residues was used to determine A3H protein expression, requirements of A3H virion incorporation, and A3H-Gag interactions. The catalytic activity of each A3H protein was assessed directly by using an Escherichia coli mutator assay. We found that the incorporation efficiencies of A3H variants into HIV-1 virions were comparable despite major differences in cellular expression. An assessment of the enzymes' catalytic activities showed that the deaminase activity of each A3H variant correlated with protein expression, suggesting similar enzymatic efficiencies. Surprisingly, virion incorporation experiments using Gag deletion mutants demonstrated that A3H haplotypes interacted with different Gag regions. A3H hapII-RDD associated with nucleocapsid in an RNA-dependent manner, whereas A3H hapI-GKE associated with the C-terminal part of matrix and the N-terminal capsid domain. Our results show that the A3H hapII-RDD interaction with nucleocapsid is critical for its antiviral activity and that the inability of A3H hapI-GKE to interact with nucleocapsid underlies its limited antiviral potential. Thus, the antiviral activity of A3H haplotypes is determined by its incorporation into the viral core, in proximity to the reverse transcription complex.
Full-text · Article · Aug 2010 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: The 1918 influenza A virus caused the most devastating pandemic, killing approximately 50 million people worldwide. Immunization with 1918-like and classical swine H1N1 virus vaccines results in cross-protective antibodies against the 2009 H1N1 pandemic influenza, indicating antigenic similarities among these viruses. In this study, we demonstrate that vaccination with the 2009 pandemic H1N1 vaccine elicits 1918 virus cross-protective antibodies in mice and humans, and that vaccination or passive transfer of human-positive sera reduced morbidity and conferred full protection from lethal challenge with the 1918 virus in mice. The spread of the 2009 H1N1 influenza virus in the population worldwide, in addition to the large number of individuals already vaccinated, suggests that a large proportion of the population now have cross-protective antibodies against the 1918 virus, greatly alleviating concerns and fears regarding the accidental exposure/release of the 1918 virus from the laboratory and the use of the virus as a bioterrorist agent.
Full-text · Article · Jun 2010 · Nature Communications