Thomas Muster

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

Background 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. Methodology/Principal Findings We applied this technology to the development of a pandemic H5N1 vaccine candidate. The vaccine virus was constructed by reverse genetics in Vero cells, as a 5∶3 reassortant, encoding four proteins HA, NA, M1, and M2 of the A/Vietnam/1203/04 virus while the remaining genes were derived from IVR-116. The HA cleavage site was modified in a trypsin dependent manner, serving as the second attenuation factor in addition to the deleted NS1 gene. The vaccine candidate was able to grow in the Vero cells that were cultivated in a serum free medium to titers exceeding 8 log10 TCID50/ml. The vaccine virus was replication deficient in interferon competent cells and did not lead to viral shedding in the vaccinated animals. The studies performed in three animal models confirmed the safety and immunogenicity of the vaccine. Intranasal immunization protected ferrets and mice from being infected with influenza H5 viruses of different clades. In a primate model (Macaca mulatta), 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. Conclusion/Significance 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.

Development of a live-attenuated influenza B ΔNS1 intranasal vaccine candidate

We discovered a unique, single amino acid mutation in the influenza B M1 protein promoting viral growth of NS1 truncation mutants in Vero cells. Due to this mutation, we were able to generate an influenza B virus lacking the complete NS1 open reading frame (DeltaNS1-B virus) by reverse genetics, which was growing to titers of 8log(10)TCID(50)/ml in a Vero cell culture-based micro-carrier fermenter. The DeltaNS1-B vaccine candidate was attenuated in IFN-competent hosts such as human alveolar epithelial cells (A549) similar to influenza A DeltaNS1 viruses. In ferrets, the DeltaNS1-B virus was replication-deficient and did not provoke any clinical symptoms. Importantly, a single intranasal immunization of ferrets at a dose as low as 6 log(10)TCID(50)/animal induced a significant HAI response and provided protection against challenge with wild-type influenza B virus. So far, the lack of a DeltaNS1-B virus component growing to high titers in cell culture has been limiting the possibility to formulate a trivalent vaccine based on deletion of the NS1 gene. Our study closes this gap and paves the way for the clinical evaluation of a seasonal, trivalent, live replication-deficient DeltaNS1 intranasal influenza vaccine.

Influenza B mutant viruses with truncated NS1 proteins grow efficiently in Vero cells and are immunogenic in mice.

Contemporary influenza B virus strains were generated encoding C-terminally truncated NS1 proteins. Viable viruses containing the N-terminal 14, 38, 57 or 80 aa of the NS1 protein were rescued in Vero cells. The influenza B virus NS1-truncated mutants were impaired in their ability to counteract interferon (IFN) production, induce antiviral pro-inflammatory cytokines early after infection and show attenuated or restricted growth in IFN-competent hosts. In Vero cells, all of the mutant viruses replicated to high titres comparable to the wild-type influenza B virus. Mice that received a single, intranasal immunization of the NS1-truncated mutants elicited an antibody response and protection against wild-type virus challenge. Therefore, these NS1-truncated mutants should prove useful as potential candidates for live-attenuated influenza virus vaccines.