Novel production of attenuated influenza B viruses

This project application represents an idea of rescuing attenuated influenza B viruses in mammalian Vero cells (green monkey kidney cells) using baculovirus as a carrier of its genome. The development of modern live influenza vaccines requires continuous and laborious genetic studies on the establishment of attenuated laboratory mutants which can serve as master strains for attenuation. For example establishment of influenza A gene engineering methods permitted obtaining of genetically stable attenuated strains. No such strategy has yet been successful for the construction of attenuated influenza B viruses, due to a lack of appropriate temperature sensitive mutants and molecular biology tools. The genome of influenza virus is single stranded RNA, of negative polarity and comprises approximately 13500 nucleotides. The genome of both influenza A and B viruses is distributed into eight different segments, coding for nine structural proteins and one non- structural protein. Our goal is to rescue infectious influenza B virus particles and to provide vectors that can be used for attenuation and genetic engineering. Baculoviruses have been shown to efficiently transduce various mammalian cell lines, they are easy to generate and allow the insertion of multiple foreign genes, thus, they seem to be the ideal gene delivery vehicle for the production of multi-subunit particles such as viruses or virus like particles. This strategy will serve to express all influenza B viral genes in green monkey kidney cells (Vero), which have previously been shown to be capable of producing infectious influenza virus. Thus, by creating appropriate baculovirus vectors, we are hoping to rescue infectious influenza B virus particles, that can be genetically modified as desired, e.g. for attenuation and designed antigenic properties. Based on our work with baculovirus cloning systems and insect cell culture during the past years and our development of influenza A attenuated mutants we have all tools and expertise at our hands that are needed. We believe, that this approach could create a new generation of more safe live attenuated influenza vaccines and viral vectors.

Influenza is of major public health importance worldwide. Epidemics of influenza disrupt economic and social activities and kill thousands of people every year. The highest attack rates for influenza occur in children and elderly. A lot of efforts have been made in order to develop efficient influenza vaccines. Although, inactivated influenza vaccines are available, their efficacy is suboptimal. Live attenuated influenza virus vaccines present new possibilities for the prevention and control of influenza. Additionally, due to the high mutation rates within the influenza virus genomes and the fact that gene segments can be exchanged between different strains, every year a new vaccine has to be generated. Efficient methods are therefore required. For fast and safe generation of live vaccine a gene transfer system is needed, which is flexible and efficient. Necessary influenza virus genes can be transferred into mammalian cells for the generation of intact influenza virus progeny, which due to targeted deletions have been attenuated in infectivity, and can thus, be used as live vaccine. A novel gene transfer system has been used, which is based on insect viruses, in particular baculoviruses. These viruses enter mammalian cells, but cannot replicate in this foreign host and are therefore regarded as mere gene delivery tool. This system is faster, more efficient and safer than other methods used. Genetic modification of baculoviruses is fairly easy and we constructed eight baculoviruses, each containing one of the eight influenza virus genes. One additional baculovirus was generated, containing a reporter gene, which proved the functionality of the four polymerase genes of influenza virus. Simultaneous delivery of eight different baculovirus clones was successful and led to the production of infectious influenza virus progeny. Thus, "proof of concept" of suggested strategy was successfully demonstrated, thereby making a novel and safe method for the production of live influenza virus available.