Expanding Mouse-Adapted Yamagata-like Influenza B Viruses in Eggs Enhances In Vivo Lethality in BALB/c Mice

Despite the yearly global impact of influenza B viruses (IBVs), limited host range has been a hurdle to developing a readily accessible small animal disease model for vaccine studies. Mouseadapting IBV can produce highly pathogenic viruses through serial lung passaging in mice. Previous studies have highlighted amino acid changes throughout the viral genome correlating with increased pathogenicity, but no consensus mutations have been determined. We aimed to show that growth system can play a role in mouse-adapted IBV lethality. Two Yamagata-lineage IBVs were serially passaged 10 times in mouse lungs before expansion in embryonated eggs or Madin–Darby canine kidney cells (London line) for use in challenge studies. We observed that virus grown in embryonated eggs was significantly more lethal in mice than the same virus grown in cell culture. Ten additional serial lung passages of one strain again showed virus grown in eggs was more lethal than virus grown in cells. Additionally, no mutations in the surface glycoprotein amino acid sequences correlated to differences in lethality. Our results suggest growth system can influence lethality of mouse-adapted IBVs after serial lung passaging. Further research can highlight improved mechanisms for developing animal disease models for IBV vaccine research

Protection against Divergent Influenza H1N1 Virus by a Centralized Influenza Hemagglutinin

Influenza poses a persistent worldwide threat to the human population. As evidenced by the 2009 H1N1 pandemic, current vaccine technologies are unable to respond rapidly to this constantly diverging pathogen. We tested the utility of adenovirus (Ad) vaccines expressing centralized consensus influenza antigens. Ad vaccines were produced within 2 months and protected against influenza in mice within 3 days of vaccination. Ad vaccines were able to protect at doses as low as 107 virus particles/kg indicating that approximately 1,000 human doses could be rapidly generated from standard Ad preparations. To generate broadly cross-reactive immune responses, centralized consensus antigens were constructed against H1 influenza and against H1 through H5 influenza. Twenty full-length H1 HA sequences representing the main branches of the H1 HA phylogenetic tree were used to create a synthetic centralized gene, HA1-con. HA1-con minimizes the degree of sequence dissimilarity between the vaccine and existing circulating viruses. The centralized H1 gene, HA1-con, induced stronger immune responses and better protection against mismatched virus challenges as compared to two wildtype H1 genes. HA1-con protected against three genetically diverse lethal influenza challenges. When mice were challenged with 1934 influenza A/PR/8/34, HA1-con protected 100% of mice while vaccine generated from 2009 A/TX/05/09 only protected 40%. Vaccination with 1934 A/PR/8/34 and 2009 A/TX/05/09 protected 60% and 20% against 1947 influenza A/FM/1/47, respectively, whereas 80% of mice vaccinated with HA1-con were protected. Notably, 80% of mice challenged with 2009 swine flu isolate A/California/4/09 were protected by HA1-con vaccination. These data show that HA1-con in Ad has potential as a rapid and universal vaccine for H1N1 influenza viruses

Protection from the 2009 H1N1 Pandemic Influenza by an Antibody from Combinatorial Survivor-Based Libraries

Influenza viruses elude immune responses and antiviral chemotherapeutics through genetic drift and reassortment. As a result, the development of new strategies that attack a highly conserved viral function to prevent and/or treat influenza infection is being pursued. Such novel broadly acting antiviral therapies would be less susceptible to virus escape and provide a long lasting solution to the evolving virus challenge. Here we report the in vitro and in vivo activity of a human monoclonal antibody (A06) against two isolates of the 2009 H1N1 pandemic influenza virus. This antibody, which was obtained from a combinatorial library derived from a survivor of highly pathogenic H5N1 infection, neutralizes H5N1, seasonal H1N1 and 2009 “Swine” H1N1 pandemic influenza in vitro with similar potency and is capable of preventing and treating 2009 H1N1 influenza infection in murine models of disease. These results demonstrate broad activity of the A06 antibody and its utility as an anti-influenza treatment option, even against newly evolved influenza strains to which there is limited immunity in the general population

A Contributing Role for Anti-Neuraminidase Antibodies on Immunity to Pandemic H1N1 2009 Influenza A Virus

Exposure to contemporary seasonal influenza A viruses affords partial immunity to pandemic H1N1 2009 influenza A virus (pH1N1) infection. The impact of antibodies to the neuraminidase (NA) of seasonal influenza A viruses to cross-immunity against pH1N1 infection is unknown.Antibodies to the NA of different seasonal H1N1 influenza strains were tested for cross-reactivity against A/California/04/09 (pH1N1). A panel of reverse genetic (rg) recombinant viruses was generated containing 7 genes of the H1N1 influenza strain A/Puerto Rico/08/34 (PR8) and the NA gene of either the pandemic H1N1 2009 strain (pH1N1) or one of the following contemporary seasonal H1N1 strains: A/Solomon/03/06 (rg Solomon) or A/Brisbane/59/07 (rg Brisbane). Convalescent sera collected from mice infected with recombinant viruses were measured for cross-reactive antibodies to pH1N1 via Hemagglutinin Inhibition (HI) or Enzyme-Linked Immunosorbent Assay (ELISA). The ectodomain of a recombinant NA protein from the pH1N1 strain (pNA-ecto) was expressed, purified and used in ELISA to measure cross-reactive antibodies. Analysis of sera from elderly humans immunized with trivalent split-inactivated influenza (TIV) seasonal vaccines prior to 2009 revealed considerable cross-reactivity to pNA-ecto. High titers of cross-reactive antibodies were detected in mice inoculated with either rg Solomon or rg Brisbane. Convalescent sera from mice inoculated with recombinant viruses were used to immunize naïve recipient Balb/c mice by passive transfer prior to challenge with pH1N1. Mice receiving rg California sera were better protected than animals receiving rg Solomon or rg Brisbane sera.The NA of contemporary seasonal H1N1 influenza strains induces a cross-reactive antibody response to pH1N1 that correlates with reduced lethality from pH1N1 challenge, albeit less efficiently than anti-pH1N1 NA antibodies. These findings demonstrate that seasonal NA antibodies contribute to but are not sufficient for cross-reactive immunity to pH1N1

Cross-Reactive Neutralizing Antibodies Directed against Pandemic H1N1 2009 Virus Are Protective in a Highly Sensitive DBA/2 Mouse Influenza Model▿

Our ability to rapidly respond to an emerging influenza pandemic is hampered somewhat by the lack of a susceptible small-animal model. To develop a more sensitive model, we pathotyped 18 low-pathogenic non-mouse-adapted influenza A viruses of human and avian origin in DBA/2 and C57BL/6 mice. The majority of the isolates (13/18) induced severe morbidity and mortality in DBA/2 mice upon intranasal challenge with 1 million infectious doses. Also, at a 100-fold-lower dose, more than 50% of the viruses induced severe weight loss, and mice succumbed to the infection. In contrast, only two virus strains were pathogenic for C57BL/6 mice upon high-dose inoculation. Therefore, DBA/2 mice are a suitable model to validate influenza A virus vaccines and antiviral therapies without the need for extensive viral adaptation. Correspondingly, we used the DBA/2 model to assess the level of protection afforded by preexisting pandemic H1N1 2009 virus (H1N1pdm) cross-reactive human antibodies detected by a hemagglutination inhibition assay. Passive transfer of these antibodies prior to infection protected mice from H1N1pdm-induced pathogenicity, demonstrating the effectiveness of these cross-reactive neutralizing antibodies in vivo

Multiple Introductions of Avian Influenza Viruses (H5N1), Laos, 2009–2010

Avian influenza viruses (H5N1) of clades 2.3.4.1, 2.3.4.2, and 2.3.2.1 were introduced into Laos in 2009–2010. To investigate these viruses, we conducted active surveillance of poultry during March 2010. We detected viruses throughout Laos, including several interclade reassortants and 2 subgroups of clade 2.3.4, one of which caused an outbreak in May 2010

Active Surveillance for Avian Influenza Virus, Egypt, 2010–2012

Continuous circulation of influenza A(H5N1) virus among poultry in Egypt has created an epicenter in which the viruses evolve into newer subclades and continue to cause disease in humans. To detect influenza viruses in Egypt, since 2009 we have actively surveyed various regions and poultry production sectors. From August 2010 through January 2013, >11,000 swab samples were collected; 10% were positive by matrix gene reverse transcription PCR. During this period, subtype H9N2 viruses emerged, cocirculated with subtype H5N1 viruses, and frequently co-infected the same avian host. Genetic and antigenic analyses of viruses revealed that influenza A(H5N1) clade 2.2.1 viruses are dominant and that all subtype H9N2 viruses are G1-like. Cocirculation of different subtypes poses concern for potential reassortment. Avian influenza continues to threaten public and animal health in Egypt, and continuous surveillance for avian influenza virus is needed

Multiple introductions of highly pathogenic avian influenza H5N1 viruses into Bangladesh

Highly pathogenic H5N1 and low pathogenic H9N2 influenza viruses are endemic to poultry markets in Bangladesh and have cocirculated since 2008. H9N2 influenza viruses circulated constantly in the poultry markets, whereas highly pathogenic H5N1 viruses occurred sporadically, with peaks of activity in cooler months. Thirty highly pathogenic H5N1 influenza viruses isolated from poultry were characterized by antigenic, molecular, and phylogenetic analyses. Highly pathogenic H5N1 influenza viruses from clades 2.2.2 and 2.3.2.1 were isolated from live bird markets only. Phylogenetic analysis of the 30 H5N1 isolates revealed multiple introductions of H5N1 influenza viruses in Bangladesh. There was no reassortment between the local H9N2 influenza viruses and H5N1 genotype, despite their prolonged cocirculation. However, we detected two reassortant H5N1 viruses, carrying the M gene from the Chinese H9N2 lineage, which briefly circulated in the Bangladesh poultry markets and then disappeared. On the other hand, interclade reassortment occurred within H5N1 lineages and played a role in the genesis of the currently dominant H5N1 viruses in Bangladesh. Few ‘human-like’ mutations in H5N1 may account for the limited number of human cases. Antigenically, clade 2.3.2.1 H5N1 viruses in Bangladesh have evolved since their introduction and are currently mainly homogenous, and show evidence of recent antigenic drift. Although reassortants containing H9N2 genes were detected in live poultry markets in Bangladesh, these reassortants failed to supplant the dominant H5N1 lineage