Gene Constellation of Influenza A Virus Reassortants with High Growth Phenotype Prepared as Seed Candidates for Vaccine Production

BACKGROUND: Influenza 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. METHODOLOGY: The 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. SIGNIFICANCE: In 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

HA1 (Hemagglutinin) Quantitation for Influenza A H1N1 and H3N2 High Yield Reassortant Vaccine Candidate Seed Viruses by RP-UPLC

The only effective measure to decrease morbidity and mortality caused by the influenza virus in the human population is worldwide vaccination. Vaccination produces neutralizing antibodies that target the HA1 subunit of the HA (hemagglutinin) protein and are strain specific. The effectiveness of new influenza vaccines are linked to two factors, the correct prediction of the circulating strains in the population in a particular season and the concentration of the HA1 protein in the vaccine formulation. With the advent of the licensing of quadrivalent vaccines, pharmaceutical manufacturers are under considerable pressure due to time constraints and dedicated resources to deliver 194-198 million doses (2020-2021 U.S. market) of vaccine. Considering the valuable resources needed to produce the influenza vaccine in a timely manner, the efficient quantitation of the HA1 protein (the main component in the influenza vaccine) is required. Currently the only method approved by regulatory agencies for quantitation of the HA antigen in vaccines is the single radial immunodiffusion assay (SRID), an antibody dependent assay that is not time efficient. Time efficient methods that are antibody independent e.g. reverse phase-high performance liquid chromatography (RP-HPLC) or size exclusion-HPLC (SE-HPLC) are available. An improved method implementing reverse phase-ultra performance liquid chromatography (RP-UPLC) has been developed to quantitate the HA1 protein antigen present in the high yield reassortant vaccine seed viruses from influenza A H1N1 and H3N2 subtypes harvested from inoculated embryonated chicken eggs. This method differentiates between high yield and lower yielding reassortants in order to select the best vaccine candidate seed virus with the highest growth \u27in ovo\u27. This direct capability to monitor the HA1 concentration of potential reassortant seed viruses and to choose the best yielding HA influenza reassortant when faced with multiple viral seed candidates provides a major advantage on the industrial scale to the influenza vaccine process

HY Reassortants: Gene Constellations and Fold Increase in HA Titer.

<p>*HA Titer is given as reciprocal of viral dilution at titration end point.</p><p>**[FI]: fold increase in HA titer over wt parent virus.</p>a<p>Used in seasonal influenza vaccine production.</p>b<p>Used in 2009 H1N1pdm vaccine production.</p><p>P: hy donor virus A/PR/8/1934 gene. <i>WT</i>: wild type virus gene.</p

Gene Constellations Present in Hy Reassortants.

<p>P: hy donor virus A/PR/8/1934 gene. <i>WT</i>: wild type virus gene.</p

Restriction Enzymes used for RFLP.

<p>Gene segments PB2, PB1 and M were digested by a single enzyme.</p><p>*<i>Sml</i>I was used to digest 2009 H1N1pdm M gene segment.</p

Frequency of Origin of Internal Genes in High Yield Reassortants.

<p>Frequency of Origin of Internal Genes in High Yield Reassortants.</p

Molecular Signature of High Yield (Growth) Influenza A Virus Reassortants Prepared as Candidate Vaccine Seeds

<div><p>Background</p><p>Human influenza virus isolates generally grow poorly in embryonated chicken eggs. Hence, gene reassortment of influenza A wild type (wt) viruses is performed with a highly egg adapted donor virus, A/Puerto Rico/8/1934 (PR8), to provide the high yield reassortant (HYR) viral ‘seeds’ for vaccine production. HYR must contain the hemagglutinin (HA) and neuraminidase (NA) genes of wt virus and one to six ‘internal’ genes from PR8. Most studies of influenza wt and HYRs have focused on the HA gene. The main objective of this study is the identification of the molecular signature in all eight gene segments of influenza A HYR candidate vaccine seeds associated with high growth <i>in ovo.</i></p><p>Methodology</p><p>The genomes of 14 wt parental viruses, 23 HYRs (5 H1N1; 2, 1976 H1N1-SOIV; 2, 2009 H1N1pdm; 2 H2N2 and 12 H3N2) and PR8 were sequenced using the high-throughput sequencing pipeline with big dye terminator chemistry.</p><p>Results</p><p>Silent and coding mutations were found in all internal genes derived from PR8 with the exception of the M gene. The M gene derived from PR8 was invariant in all 23 HYRs underlining the critical role of PR8 M in high yield phenotype. None of the wt virus derived internal genes had any silent change(s) except the PB1 gene in X-157. The highest number of recurrent silent and coding mutations was found in NS. With respect to the surface antigens, the majority of HYRs had coding mutations in HA; only 2 HYRs had coding mutations in NA.</p><p>Significance</p><p>In the era of application of reverse genetics to alter influenza A virus genomes, the mutations identified in the HYR gene segments associated with high growth <i>in ovo</i> may be of great practical benefit to modify PR8 and/or wt virus gene sequences for improved growth of vaccine ‘seed’ viruses.</p></div

Total number of nucleotide changes in HYRs.

<p>Total Changes: The majority of the nucleotide changes (Silent and Coding, black bars) in the internal genes was found in the NS gene followed by PB1, PB2, NP, and PA. The HA gene had a total of 33 nucleotide changes; in contrast NA had only 2 nucleotide changes, both coding changes. M gene had no nucleotide change in any of the 23 HYRs. Silent Changes: Silent nucleotide changes were found in the internal genes of the HYR derived from hy donor virus, PR8 (blue bars). No silent changes were found in the internal genes derived from wt viruses except in NYMC X-157 PB1 derived from wt virus NY/04 which had 13 silent changes. HA had 3 silent nucleotide changes (green bar). NA and M genes had no silent changes in any of the 23 HYRs. Coding Changes: Among the internal proteins, the majority of the coding changes (orange bars) were found in PR8 derived NS (14 total; 7 in NS1 and 7 in NS2), followed by PB1 (10 total; 7 in PB1 and 3 in PB1-F2), NP (9) and PA (8). PB2 had only 1 coding change in contrast with 20 silent changes. M had no coding changes in any of the 23 HYRs. HA had 30 coding changes; in contrast NA had only 2 coding changes (red bars).</p