Preclinical Evaluation of a Replication-Deficient Intranasal ΔNS1 H5N1 Influenza Vaccine

We developed a novel intranasal influenza vaccine approach that is based on the construction of replication-deficient vaccine viruses that lack the entire NS1 gene (ΔNS1 virus). We previously showed that these viruses undergo abortive replication in the respiratory tract of animals. The local release of type I interferons and other cytokines and chemokines in the upper respiratory tract may have a “self-adjuvant effect”, in turn increasing vaccine immunogenicity. As a result, ΔNS1 viruses elicit strong B- and T- cell mediated immune responses.), one dose of vaccine delivered intranasally was sufficient for the induction of antibodies against homologous A/Vietnam/1203/04 and heterologous A/Indonesia/5/05 H5N1 strains.Our findings show that intranasal immunization with the replication deficient H5N1 ΔNS1 vaccine candidate is sufficient to induce a protective immune response against H5N1 viruses. This approach might be attractive as an alternative to conventional influenza vaccines. Clinical evaluation of ΔNS1 pandemic and seasonal influenza vaccine candidates are currently in progress

Single HA2 Mutation Increases the Infectivity and Immunogenicity of a Live Attenuated H5N1 Intranasal Influenza Vaccine Candidate Lacking NS1

Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals

Egg- or cell culture-derived hemagglutinin mutations impair virus stability and antigen content of inactivated influenza vaccines

Egg-derived viruses are the only available seed material for influenza vaccine production. Vaccine manufacturing is done in embryonated chicken eggs, MDCK or Vero cells. In order to contribute to efficient production of influenza vaccines, we investigate whether the quality of inactivated vaccines is influenced by the propagation substrate. We demonstrate that H3N2 egg-derived seed viruses (A/Brisbane/10/07, IVR147, and A/Uruguay/716/07) triggered the hemagglutinin (HA) conformational change under less acidic conditions (0.2-0.6 pH units) than antigenically similar primary isolates. This phenotype was associated with HA1 (A138S, L194P) and HA2 (D160N) substitutions, and strongly related to decreased virus stability towards acidic pH and elevated temperature. The subsequent propagation of H3N2 and H1N1 egg-derived seed viruses in MDCK and Vero cells induced HA2 N50K (H1N1) and D160E (H3N2) mutations, improving virus growth in cell culture but further impairing virus stability. The prevention of the loss or recovery of stability was possible by cultivation at acidified conditions. Viruses carrying less stable HAs are more sensitive for HA conformational change during concentration, purification and storage. This results in decreased detectable HA antigen content - the main potency marker for inactivated influenza vaccines. Thus, virus stability can be a useful marker for predicting the manufacturing scope of seed viruses

The Intranasal Application of Zanamivir and Carrageenan Is Synergistically Active against Influenza A Virus in the Murine Model

<div><p>Background</p><p>Carrageenan is a clinically proven and marketed compound for the treatment of viral upper respiratory tract infections. As infections caused by influenza virus are often accompanied by infections with other respiratory viruses the combination of a specific anti-influenza compound with the broadly active antiviral polymer has huge potential for the treatment of respiratory infections. Thus, the combination of the specific anti-influenza drug Zanamivir together with carrageenan in a formulation suitable for intranasal application was evaluated <i>in-vitro</i> and <i>in-vivo</i>.</p><p>Principal Findings</p><p>We show <i>in-vitro</i> that carrageenan and Zanamivir act synergistically against several influenza A virus strains (H1N1(09)pdm, H3N2, H5N1, H7N7). Moreover, we demonstrate in a lethal influenza model with a low pathogenic H7N7 virus (HA closely related to the avian influenza A(H7N9) virus) and a H1N1(09)pdm influenza virus in C57BL/6 mice that the combined use of both compounds significantly increases survival of infected animals in comparison with both mono-therapies or placebo. Remarkably, this benefit is maintained even when the treatment starts up to 72 hours post infection.</p><p>Conclusion</p><p>A nasal spray containing carrageenan and Zanamivir should therefore be tested for prevention and treatment of uncomplicated influenza in clinical trials.</p></div

Therapeutic efficacy in influenza H1N1(09)pdm lethally infected mice.

<p>Mice (n = 20 per group) were lethally intranasally infected without anesthesia on day 0 and intranasally treated twice per day either with placebo or a combination of carrageenan and Zanamivir (1 mg/kg BW/day). Treatment started either 48 hpi or 72 hpi and continued for 5 days. On the y-axis the survival of mice [%] and on the x-axis the time post infection [days] is given. Placebo treated uninfected control mice showed 100% survival (data not shown). Statistical analyses were conducted using log rank test and are shown beneath the graphs. Values of p<0.05 were considered statistically significant.</p

IC₅₀ values of carrageenan and Zanamivir for influenza A viruses of human and animal origin.

<p>^a IC₅₀ values were calculated in comparison to untreated infected cells. Each value represents the mean IC₅₀ of 6 replicates/assay and their standard deviation.</p><p>IC₅₀ values of carrageenan and Zanamivir for influenza A viruses of human and animal origin.</p

Isobologram of compound interaction.

<p>Comparison of the combination of different doses of both compounds necessary to reach 50% replication inhibition of (A) H7N7 and (B) H1N1(09)pdm (◆) to a model of dose additivity that would represent a curve progression of 1 (□). Dose response was tested with an adapted plaque reduction assay with semi-liquid overlay in MDCK cells. On the x-axis the concentration of Zanamivir and on the y-axis the concentration of carrageenan is presented. The concentrations (determined as mean of 3 replicates) are given as the fraction of the respective IC₅₀ values of the different viruses with the particular compound (IC₅₀ = 1).</p