Abstract

BackgroundInfluenza A virus vaccines undergo yearly reformulations due to the antigenic variability of the virus caused by antigenic drift and shift. It is critical to the vaccine manufacturing process to obtain influenza A seed virus that is antigenically identical to circulating wild type (wt) virus and grows to high titers in embryonated chicken eggs. Inactivated influenza A seasonal vaccines are generated by classical reassortment. The classical method takes advantage of the ability of the influenza virus to reassort based on the segmented nature of its genome. In ovo co-inoculation of a high growth or yield (hy) donor virus and a low yield wt virus with antibody selection against the donor surface antigens results in progeny viruses that grow to high titers in ovo with wt origin hemagglutinin (HA) and neuraminidase (NA) glycoproteins. In this report we determined the parental origin of the remaining six genes encoding the internal proteins that contribute to the hy phenotype in ovo.MethodologyThe genetic analysis was conducted using reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). The characterization was conducted to determine the parental origin of the gene segments (hy donor virus or wt virus), gene segment ratios and constellations. Fold increase in growth of reassortant viruses compared to respective parent wt viruses was determined by hemagglutination assay titers.SignificanceIn this study fifty-seven influenza A vaccine candidate reassortants were analyzed for the presence or absence of correlations between specific gene segment ratios, gene constellations and hy reassortant phenotype. We found two gene ratios, 6∶2 and 5∶3, to be the most prevalent among the hy reassortants analyzed, although other gene ratios also conferred hy in certain reassortants.

Highlights

  • Due to the genetic instability of influenza viruses, caused by antigenic drift and periodically antigenic shift, the influenza vaccine needs to undergo yearly reformulations

  • Inactivated hy reassortants generated in embryonated chicken eggs are used to produce 400 million yearly doses of influenza vaccine [23]

  • Hy reassortants allow manufacturers to produce the necessary amounts of virus for the seasonal vaccines in a shorter time frame with smaller quantities of embryonated chicken eggs

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Summary

Introduction

Due to the genetic instability of influenza viruses, caused by antigenic drift and periodically antigenic shift, the influenza vaccine needs to undergo yearly reformulations. Its genome is composed of eight individual RNA segments that encode eleven proteins. The remaining six gene segments encode internal and non-structural viral proteins. The segmented structure of the genome allows for the exchange of the individual gene segments between influenza viruses, a process defined as reassortment. It is critical to the vaccine manufacturing process to obtain influenza A seed virus that is antigenically identical to circulating wild type (wt) virus and grows to high titers in embryonated chicken eggs. In ovo co-inoculation of a high growth or yield (hy) donor virus and a low yield wt virus with antibody selection against the donor surface antigens results in progeny viruses that grow to high titers in ovo with wt origin hemagglutinin (HA) and neuraminidase (NA) glycoproteins. In this report we determined the parental origin of the remaining six genes encoding the internal proteins that contribute to the hy phenotype in ovo

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