Generation of live attenuated influenza vaccine by codon usage bias

Abstract

Seasonal influenza epidemics and emerging avian influenza have posed a significant burden to public health. Vaccination is one of the most effective preventive measures, but current licensed vaccines have various limitations and require annual immunization against the most updated strains. New strategies of developing influenza vaccine that can safely elicit cross-subtype immune response are needed to get a step closer to universal influenza vaccine.

Previous studies have shown that influenza viruses circulating in different hosts had distinct codon usage patterns, and the pattern had a directional change over time. Therefore, it has been suggested that the viral codon usage bias may be related to host adaptation. Consequently, altering the codon usage of a human virus may lead to attenuation. In this study, the codon usage of a human seasonal influenza virus A/Brisbane/59/07 (H1N1) containing avian codon usage bias was generated by incorporating hundreds of synonymous mutations.

Results showed that the mutant virus with avian codon usage was attenuated in human cells and mice but could replicate well in embryonated eggs, and thus could be produced cost-effectively by existing egg-based vaccine production pipeline. A single dose of intranasal vaccination of the mutant virus could stimulate potent humoral, cellular and innate immune responses, and protected mice from homologous and heterologous viral challenges. In addition, the mutant virus could be used as a vaccine master donor strain by substituting hemagglutinin and neuraminidase of other influenza viruses. The strategy of virus attenuation was also validated in another virus strain A/PR/8/34 (H1N1), which had even better growth in eggs.

Studies on the attenuation mechanism of A/Brisbane/59/07 mutant virus showed that protein expression was upregulated in chicken cells but not in human cells. The packaging efficiency of infectious viral particles was slightly boosted in embryonated eggs but significantly reduced in mammalian cells. Reduction of M and NS segment spliced products was also observed and might contribute to delay in virus life cycle. These effects from multiple viral segments probably combined to result in the attenuation of mutant virus. Genetic stability study of this virus revealed a V97I mutation in the M1 protein that led to enhanced viral fitness. The mutation led to reduction of M gene products and affected the nuclear accumulation of M1 and NP proteins. The molecular mechanism behind will be further investigated.

Findings from this study have shed light on the biology of influenza virus, the causes of codon usage bias and the potential applications of codon usage alteration. More importantly, this study have demonstrated that human to avian codon usage alteration is a feasible virus attenuation strategy and the virus might be superior than licensed vaccines in terms of safety, immune response induction and crosssubtypic protection. This strategy allows rapid design and generation of attenuated viruses that present identical proteins of the target strains, together with the ability of the virus to serve as master donor strain, may contribute to better protection against seasonal and emerging influenza viruses.