Christian Kittel

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.

Optimization of baculovirus transduction on freestyleTM293 cells for the generation of influenza B/Lee/40

Recombinant baculovirus expression vectors derived from the Autographa californica nuclear polyhedrosis virus can serve as efficient gene-transfer vehicles for transient expression of recombinant proteins in a wide range of mammalian cell types and are able to produce multisubunit particles such as viruses or virus like particles.In this study, we constructed eight recombinant baculoviruses each containing one of the influenza B/Lee/40 virus genes in a bidirectional expression cassette for simultaneous mRNA and viral RNA transcription. Baculoviruses were transduced into FreeStyleTM293 in combination with the specific histone deacetylase inhibitor trichostatin A (TSA). Contransduction conditions were optimized with a set of five baculoviruses (influenza B/Lee/40 PB1, PB2, PA, and NP and the control construct NCR-NS-minus-sense orientated encoding green fluorescent protein [rGFP]), which led to GFP expression in each host cell transduced with all five constructs.Based on the optimization with five constructs, transduction with eight baculoviruses was performed at MOI 50 and 100 with high yield stocks and 1 μM TSA and led to successful rescue of infectious influenza B/Lee/40 viruses.

A genetically adjuvanted influenza B virus vector increases immunogenicity and protective efficacy in mice

The existence of multiple antigenically distinct types and subtypes of influenza viruses allows the construction of a multivalent vector system for the mucosal delivery of foreign sequences. Influenza A viruses have been exploited successfully for the expression of extraneous antigens as well as immunostimulatory molecules. In this study, we describe the development of an influenza B virus vector whose functional part of the interferon antagonist NS1 was replaced by human interleukin 2 (IL2) as a genetic adjuvant. We demonstrate that IL2 expressed by this viral vector displays immune adjuvant activity in immunized mice. Animals vaccinated with the IL2 viral vector showed an increased hemagglutination inhibition antibody response and higher protective efficacy after challenge with a wild-type influenza B virus when compared to mice vaccinated with a control virus. Our results demonstrate that it is feasible to construct influenza B vaccine strains expressing immune-potentiating foreign sequences from the NS genomic segment. Based on these data, it is now hypothetically possible to create a trivalent (or quadrivalent) live attenuated influenza vaccine in which each component expresses a selected genetic adjuvant with tailored expression levels.