Animal Models for Influenza Viruses: Implications for Universal Vaccine Development

Influenza virus infections are a significant cause of morbidity and mortality in the human population. Depending on the virulence of the influenza virus strain, as well as the immunological status of the infected individual, the severity of the respiratory disease may range from sub-clinical or mild symptoms to severe pneumonia that can sometimes lead to death. Vaccines remain the primary public health measure in reducing the influenza burden. Though the first influenza vaccine preparation was licensed more than 60 years ago, current research efforts seek to develop novel vaccination strategies with improved immunogenicity, effectiveness, and breadth of protection. Animal models of influenza have been essential in facilitating studies aimed at understanding viral factors that affect pathogenesis and contribute to disease or transmission. Among others, mice, ferrets, pigs, and nonhuman primates have been used to study influenza virus infection in vivo, as well as to do pre-clinical testing of novel vaccine approaches. Here we discuss and compare the unique advantages and limitations of each model

Residual Baculovirus in Insect Cell-Derived Influenza Virus-Like Particle Preparations Enhances Immunogenicity

<div><p>Influenza virus-like particles are currently evaluated in clinical trials as vaccine candidates for influenza viruses. Most commonly they are produced in baculovirus- or mammalian- expression systems. Here we used different vaccination schemes in order to systematically compare virus-like particle preparations generated in the two systems. Our work shows significant differences in immunogenicity between the two, and indicates superior and broader immune responses induced by the baculovirus-derived constructs. We demonstrate that these differences critically influence protection and survival in a mouse model of influenza virus infection. Finally, we show that the enhanced immunogenicity of the baculovirus-derived virus-like particles is caused by contamination with residual baculovirus which activates the innate immune response at the site of inoculation.</p> </div

Seroconversion in mice immunized with different vaccine preparations.

<p>Pooled sera (<b>A</b>) or sera from individual mice (<b>B</b>) vaccinated by different immunization schemes with either mammalian cell- or baculovirus-derived VLPs, were tested for binding to purified PR8 virus or recombinant PR8 HA, respectively.</p

<i>In vivo</i> protection by vaccination with VLP preparations generated in either mammalian or insect cells.

<p>Mice were vaccinated once with mammalian cell-, or baculovirus- derived VLPs containing 50 ng of HA, and were intranasally infected with 100 mLD50 of PR8 virus three weeks post vaccination. (<b>A</b>) Body weight loss was monitored daily for 14 days and (<b>B</b>) animals that lost more than 25% of their initial body weight were scored dead (Kaplan-Meier curve).</p

Geometric means of hemagglutination-inhibition titers against homologous virus (PR8) induced by vaccination with mammalian- or baculovirus- derived VLP preparations.

1<p>numbers in brackets represent the range of hemagglutination-inhibition values detected in sera of individual animals in each group.</p

Assessment of innate response activation by different VLPs or purified baculovirus.

<p>Mice were immunized (intramuscularly or intranasally) with one dose of vaccine or the corresponding dose of wild type baculovirus (in PFU/dose) and 6 hours post-vaccination lung or muscle tissue was harvested and the level of induction of gene specific mRNA for IFN β, and Mx1 was analyzed as fold induction over tissue samples from animals mock-immunized with PBS.</p

Antibody isotype profile in serum and mucosal samples from vaccinated animals.

<p>(<b>A</b>) Serum antibody isotype profile induced by different VLPs and immunization regimens. Serum was collected 3 weeks after the second immunization and analyzed for relative distribution of immunoglobulin subclasses by ELISA assay, using a recombinant PR8 HA protein as substrate and different secondary antibodies recognizing the different Ig isotypes. Signal was detected using a tertiary antibody coupled to an alkaline phosphatase (AP) enzyme. (<b>B</b>) Mucosal IgA antibody levels induced by the different vaccination regimens as analyzed by testing binding activity in nasal washes obtained from animals three weeks after the second immunization. Samples were pooled and binding was detected using an AP-coupled secondary antibody specific for the IgA subtype. Baselines represent background levels detected in this assay, when testing sera from animals vaccinated with HA negative, eGFP-Gag containing, mammalian cell- (orange) or baculovirus-derived (yellow) VLPs.</p

Assessment of cross-reactive activity in sera of mice immunized with different VLP preparations.

<p>Pooled sera from animals vaccinated with either mammalian cell- or baculovirus-derived VLPs were tested in ELISA for the ability to bind recombinant chimeric H6/1 (cH6/1 HA), H1 (A/California/04/2009 HA) or H5 (A/Viet Nam/1203/2004 HA) hemagglutinin. Data shown are from serum samples diluted 1∶300. Baselines represent background levels detected in this assay, when testing sera from animals vaccinated with HA negative, eGFP-Gag containing, mammalian cell- (red) or baculovirus-derived (black) VLPs.</p