Intramuscular immunization of mice with live influenza virus is more immunogenic and offers greater protection than immunization with inactivated virus

<p>Abstract</p> <p>Background</p> <p>Influenza virus continues to cause significant hospitalization rates in infants and young children. A 2-dose regime of trivalent inactivated vaccine is required to generate protective levels of hemagglutination inhibiting (HAI) antibodies. A vaccine preparation with enhanced immunogenicity is therefore desirable.</p> <p>Methods</p> <p>Mice were inoculated intramuscularly (IM) with live and inactivated preparations of A/Wisconsin/67/2005 (H3N2). Serum cytokine levels, hemagglutinin (HA)-specific antibody responses and nucleoprotein (NP)-specific CD8+ T cell responses were compared between vaccinated groups, as well as to responses measured after intranasal infection. The protective efficacy of each vaccine type was compared by measuring virus titers in the lungs and weight loss of mice challenged intranasally with a heterosubtypic virus, A/PR/8/34 (H1N1).</p> <p>Results</p> <p>Intramuscular administration of live virus resulted in greater amounts of IFN-α, IL-12 and IFN-γ, HA-specific antibodies, and virus-specific CD8+ T cells, than IM immunization with inactivated virus. These increases corresponded with the live virus vaccinated group having significantly less weight loss and less virus in the lungs on day 7 following challenge with a sublethal dose of a heterosubtypic virus.</p> <p>Conclusions</p> <p>Inflammatory cytokines, antibody titers to HA and CD8+ T cell responses were greater to live than inactivated virus delivered IM. These increased responses correlated with greater protection against heterosubtypic virus challenge, suggesting that intramuscular immunization with live influenza virus may be a practical means to increase vaccine immunogenicity and to broaden protection in pediatric populations.</p

Antibody contributes to heterosubtypic protection against influenza A-induced tachypnea in cotton rats

<p>Abstract</p> <p>Background</p> <p>Influenza virus infection or vaccination evokes an antibody response to viral hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins, which results in immunity against influenza A viruses of the same HA and NA subtype. A heterosubtypic immune response that offers some protection against different influenza A subtypes has been suggested from epidemiologic studies in human influenza outbreaks, and has been induced in experimental animal models. Original studies of such cross-protection showed that cytotoxic T lymphocytes (CTL) protect H3N2-immune mice from a lethal H1N1 infection. More recent studies in mice demonstrate that antibodies also contribute to heterosubtypic immunity (HSI). We previously demonstrated that HSI in cotton rats (<it>Sigmodon hispidus</it>) is characterized by protection of H3N2-immune animals from influenza H1N1-induced increase in respiratory rate (tachypnea). Alternatively, H1N1-immune animals are protected from H3N2-induced tachypnea. The experiments described in this report were designed to elucidate the immune mechanism that prevents this very early sign of disease.</p> <p>Results</p> <p>Our results show that cotton rats provided with H1N1-immune serum prior to challenge with an H3N2 virus were protected from influenza-associated tachypnea, with the degree of protection correlating with the antibody titer transferred. Immunization with an inactivated preparation of virus delivered intramuscularly also provided some protection suggesting that CTL and/or mucosal antibody responses are not required for protection. Antibodies specific for conserved epitopes present on the virus exterior are likely to facilitate this protection since prophylactic treatment of cotton rats with anti-M2e (the extracellular domain of M2) but not anti-nucleoprotein (NP) reduced virus-induced tachypnea.</p> <p>Conclusion</p> <p>In the cotton rat model of heterosubtypic immunity, humoral immunity plays a role in protecting animals from influenza-induced tachypea. Partial protection against respiratory disease caused by different influenza A subtypes can be attained with either live virus administered intranasally or inactivated virus delivered intramuscularly suggesting that either vaccine regimen may provide some protection against potential pandemic outbreaks in humans.</p

Comparability of neuraminidase inhibition antibody titers measured by enzyme-linked lectin assay (ELLA) for the analysis of influenza vaccine immunogenicity

AbstractNeuraminidase-inhibition (NI) antibody titers can be used to evaluate the immunogenicity of inactivated influenza vaccines and have provided evidence of serologic cross-reactivity between seasonal and pandemic H1N1 viruses. The traditional thiobarbituric acid assay is impractical for large serologic analyses, and therefore many laboratories use an enzyme-linked lectin assay (ELLA) to determine serum NI antibody titers. The comparability of ELLA NI antibody titers when measured in different laboratories was unknown. Here we report a study conducted through the Consortium for the Standardisation of Influenza SeroEpidemiology (CONSISE) to evaluate the variability of the ELLA. NI antibody titers of a set of 12 samples were measured against both N1 and N2 neuraminidase antigens in 3 independent assays by each of 23 laboratories. For a sample repeated in the same assay, ≥96% of N1 and N2 assays had less than a 4-fold difference in titer. Comparison of the titers measured in assays conducted on 3 different days in the same laboratory showed that a four-fold difference in titer was uncommon. Titers of the same sera measured in different laboratories spanned 3 to 6 two-fold dilutions (i.e., 8–64 fold difference in titer), with an average percent geometric coefficient of variation (%GCV) of 112 and 82% against N1 and N2 antigens, respectively. The difference in titer as indicated by fold range and %GCV was improved by normalizing the NI titers to a standard that was included in each assay. This study identified background signal and the amount of antigen in the assay as critical factors that influence titer, providing important information toward development of a consensus ELLA protocol

Comparison of airway measurements during influenza-induced tachypnea in infant and adult cotton rats

<p>Abstract</p> <p>Background</p> <p>Increased respiratory rate (tachypnea) is frequently observed as a clinical sign of influenza pneumonia in pediatric patients admitted to the hospital. We previously demonstrated that influenza infection of adult cotton rats (<it>Sigmodon hispidus</it>) also results in tachypnea and wanted to establish whether this clinical sign was observed in infected infant cotton rats. We hypothesized that age-dependent differences in lung mechanics result in differences in ventilatory characteristics following influenza infection.</p> <p>Methods</p> <p>Lung tidal volume, dynamic elastance, resistance, and pleural pressure were measured in a resistance and compliance system on mechanically-ventilated anesthestized young (14–28 day old) and adult (6–12 week old) cotton rats. Animals at the same age were infected with influenza virus, and breathing rates and other respiratory measurements were recorded using a whole body flow plethysmograph.</p> <p>Results</p> <p>Adult cotton rats had significantly greater tidal volume (TV), and lower resistance and elastance than young animals. To evaluate the impact of this increased lung capacity and stiffening on respiratory disease, young and adult animals were infected intra-nasally with influenza A/Wuhan/359/95. Both age groups had increased respiratory rate and enhanced pause (<it>Penh</it>) during infection, suggesting lower airway obstruction. However, in spite of significant tachypnea, the infant (unlike the adult) cotton rats maintained the same tidal volume, resulting in an increased minute volume. In addition, the parameters that contribute to <it>Penh </it>were different: while relaxation time between breaths and time of expiration was decreased in both age groups, a disproportionate increase in peak inspiratory and expiratory flow contributed to the increase in <it>Penh </it>in infant animals.</p> <p>Conclusion</p> <p>While respiratory rate is increased in both adult and infant influenza-infected cotton rats, the volume of air exchanged per minute (minute volume) is increased in the infant animals only. This is likely to be a consequence of greater lung elastance in the very young animals. This model replicates many respiratory features of humans and consequently may be a useful tool to investigate new strategies to treat respiratory disease in influenza-infected infants.</p

Neuraminidase activity provides a practical read-out for a high throughput influenza antiviral screening assay

Abstract Background The emergence of influenza strains that are resistant to commonly used antivirals has highlighted the need to develop new compounds that target viral gene products or host mechanisms that are essential for effective virus replication. Existing assays to identify potential antiviral compounds often use high throughput screening assays that target specific viral replication steps. To broaden the search for antivirals, cell-based replication assays can be performed, but these are often labor intensive and have limited throughput. Results We have adapted a traditional virus neutralization assay to develop a practical, cell-based, high throughput screening assay. This assay uses viral neuraminidase (NA) as a read-out to quantify influenza replication, thereby offering an assay that is both rapid and sensitive. In addition to identification of inhibitors that target either viral or host factors, the assay allows simultaneous evaluation of drug toxicity. Antiviral activity was demonstrated for a number of known influenza inhibitors including amantadine that targets the M2 ion channel, zanamivir that targets NA, ribavirin that targets IMP dehydrogenase, and bis-indolyl maleimide that targets protein kinase A/C. Amantadine-resistant strains were identified by comparing IC50 with that of the wild-type virus. Conclusion Antivirals with specificity for a broad range of targets are easily identified in an accelerated viral inhibition assay that uses NA as a read-out of replication. This assay is suitable for high throughput screening to identify potential antivirals or can be used to identify drug-resistant influenza strains.</p