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

Development of high yield reassortants for influenza type B viruses and analysis of their gene compositions

AbstractA critical step in producing the annual inactivated influenza vaccine is the development of high yield (hy) seed viruses by reassortment for improved growth in ovo. Although hy reassortants for type A influenza viruses have been developed for many years, hy B influenza reassortant virus development for vaccine production has proven difficult. In this study, we have developed fourteen hy influenza type B reassortants as vaccine candidate strains with B/Lee/40 as the donor virus. Upon characterization by the Influenza Division at the Centers for Disease Control and Prevention (CDC) and the verification of HA by sequencing, all B reassortants were found to be antigenically indistinguishable from the wild type (wt) parents and suitable for vaccine production. However, only one hy reassortant seed virus from this group was used by a manufacturer for vaccine production. In general, hy reassortants showed an increase in hemagglutination (HA) titers over their wt parents by approximately 8 fold (range 1–32 fold). Gene compositions of the hy B reassortants were analyzed by restriction fragment length polymorphism (RFLP) and the wt origin of the HA and neuraminidase (NA) were confirmed. However, in contrast to hy A reassortants which require the M gene (hy donor A/PR/8/34) for high yield, all fourteen hy B reassortants obtained the NP gene from the hy donor strain (B/Lee/40). The parental source for the remaining genes varied among the hy B reassortants. The results indicate that the B/Lee/40 NP and PB1 gene segments are important contributors to high yield growth in influenza B reassortant viruses for both Yamagata and Victoria lineages. The B/Lee/40 PB2 gene along with wt NS gene also contributed to the improved growth for hy reassortants of Yamagata lineage

Frequency of Origin of Internal Genes in High Yield Reassortants.

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

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

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

Structural model of H3N2 HA (monomer) with coding changes found in the H3N2 HYRs in this study.

<p>The structure was generated by PyMOL Molecular Graphics System (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>). Single letter amino acid code is used. The numbers in the parentheses indicate recurrent changes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone-0065955-t009" target="_blank">Table 9</a>); underlined changes were reported earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone.0065955-Robertson1" target="_blank">[25]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone.0065955-Kumari1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone.0065955-Stevens1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone.0065955-Kilbourne3" target="_blank">[43]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone.0065955-Widjaja1" target="_blank">[45]</a> and also found in our study. ¹indicates unique changes found in the HA1 in this study and ² indicates unique changes found in the HA2 region.</p