One of the important lessons learned from the 2009 H1N1 pandemic is that a high yield influenza vaccine virus is essential for efficient and timely production of pandemic vaccines in eggs. The current seasonal and pre-pandemic vaccine viruses are generated either by classical reassortment or reverse genetics. Both approaches utilize a high growth virus, generally A/Puerto Rico/8/1934 (PR8), as the donor of all or most of the internal genes, and the wild type virus recommended for inclusion in the vaccine to contribute the hemagglutinin (HA) and neuraminidase (NA) genes encoding the surface glycoproteins. As a result of extensive adaptation through sequential egg passaging, PR8 viruses with different gene sequences and high growth properties have been selected at different laboratories in past decades. The effect of these related but distinct internal PR8 genes on the growth of vaccine viruses in eggs has not been examined previously. Here, we use reverse genetics to analyze systematically the growth and HA antigen yield of reassortant viruses with 3 different PR8 backbones. A panel of 9 different HA/NA gene pairs in combination with each of the 3 different lineages of PR8 internal genes (27 reassortant viruses) was generated to evaluate their performance. Virus and HA yield assays showed that the PR8 internal genes influence HA yields in most subtypes. Although no single PR8 internal gene set outperformed the others in all candidate vaccine viruses, a combination of specific PR8 backbone with individual HA/NA pairs demonstrated improved HA yield and consequently the speed of vaccine production. These findings may be important both for production of seasonal vaccines and for a rapid global vaccine response during a pandemic.

BACKGROUND: The emergence of avian influenza A(H7N9) virus in poultry causing zoonotic human infections was reported on March 31, 2013. Development of A(H7N9) candidate vaccine viruses (CVV) for pandemic preparedness purposes was initiated without delay. Candidate vaccine viruses were derived by reverse genetics using the internal genes of A/Puerto/Rico/8/34 (PR8). The resulting A(H7N9) CVVs needed improvement because they had titers and antigen yields that were suboptimal for vaccine manufacturing in eggs, especially in a pandemic situation. METHODS: Two CVVs derived by reverse genetics were serially passaged in embryonated eggs to improve the hemagglutinin (HA) antigen yield. The total viral protein and HA antigen yields of six egg-passaged CVVs were determined by the BCA assay and isotope dilution mass spectrometry (IDMS) analysis, respectively. CVVs were antigenically characterized by hemagglutination inhibition (HI) assays with ferret antisera. RESULTS: Improvement of total viral protein yield was observed for the six egg-passaged CVVs; HA quantification by IDMS indicated approximately a two-fold increase in yield of several egg-passaged viruses as compared to that of the parental CVV. Several different amino acid substitutions were identified in the HA of all viruses after serial passage; however HI tests indicated that the antigenic properties of two CVVs remained unchanged. CONCLUSIONS: If influenza A(H7N9) viruses were to acquire sustained human to human transmissibility, the improved HA yield of the egg-passaged CVVs generated in this study could expedite vaccine manufacturing for pandemic mitigation.

Vaccination is the most effective means to prevent influenza and its serious complications. Influenza viral strains undergo rapid mutations of the surface proteins hemagglutinin (HA) and neuraminidase (NA) requiring vaccines to be frequently updated to include current circulating strains. It is nearly impossible to predict which strains will be circulating in the next influenza season. It is, therefore, imperative that the process of producing a vaccine be streamlined and as swift as possible. We have developed an isotope dilution mass spectrometry (IDMS) method to quantify HA and NA in H7N7, H7N2, and H7N9 influenza. The IDMS method involves enzymatic digestion of viral proteins and the specific detection of evolutionarily conserved target peptides. The four target peptides that were initially chosen for analysis of the HA protein of H7N2 and H7N7 subtypes were conserved and available for analysis of the H7N9 subtype that circulated in China in the spring of 2013. Thus, rapid response to the potential pandemic was realized. Quantification of a protein is performed by employing multiple peptides to ensure that the enzymatic digestion of the protein is efficient in the region of the target peptides, verify the accuracy of the measurement, and provide flexibility in the case of amino acid changes among newly emerging strains. The IDMS method is an accurate, sensitive, and selective method to quantify the amount of HA and NA antigens in primary liquid standards, crude allantoic fluid, purified virus samples, and final vaccine presentations.