Generation of a Genetically Stable High-Fidelity Influenza Vaccine Strain

Abstract

Vaccination is considered the most effective preventive means for influenza control. The development of a master virus with high growth and genetic stability, which may be used for the preparation of vaccine viruses by gene reassortment, is crucial for the enhancement of vaccine performance and efficiency of production. Here, we describe the generation of a high-fidelity and high-growth influenza vaccine master virus strain with a single V43I amino acid change in the PB1 polymerase of the high-growth A/Puerto Rico/8/1934 (PR8) master virus. The PB1-V43I mutation was introduced to increase replication fidelity in order to design an H1N1 vaccine strain with a low error rate. The PR8-PB1-V43I virus exhibited good replication compared with that of the parent PR8 virus. In order to compare the efficiency of egg adaptation and the occurrence of gene mutations leading to antigenic alterations, we constructed 6:2 genetic reassortant viruses between the A(H1N1)pdm09 and the PR8-PB1-V43I viruses; hemagglutinin (HA) and neuraminidase (NA) were from the A(H1N1)pdm09 virus, and the other genes were from the PR8 virus. Mutations responsible for egg adaptation mutations occurred in the HA of the PB1-V43I reassortant virus during serial egg passages; however, in contrast, antigenic mutations were introduced into the HA gene of the 6:2 reassortant virus possessing the wild-type PB1. This study shows that the mutant PR8 virus possessing the PB1 polymerase with the V43I substitution may be utilized as a master virus for the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production.IMPORTANCE Vaccination represents the most effective prophylactic option against influenza. The threat of emergence of influenza pandemics necessitates the ability to generate vaccine viruses rapidly. However, as the influenza virus exhibits a high mutation rate, vaccines must be updated to ensure a good match of the HA and NA antigens between the vaccine and the circulating strain. Here, we generated a genetically stable master virus of the A/Puerto Rico/8/1934 (H1N1) backbone encoding an engineered high-fidelity viral polymerase. Importantly, following the application of the high-fidelity PR8 backbone, no mutation resulting in antigenic change was introduced into the HA gene during propagation of the A(H1N1)pdm09 candidate vaccine virus. The low error rate of the present vaccine virus should decrease the risk of generating mutant viruses with increased virulence. Therefore, our findings are expected to be useful for the development of prepandemic vaccines and live attenuated vaccines with higher safety than that of the present candidate vaccines.

Epistatic interactions within the influenza virus polymerase complex mediate mutagen resistance and replication fidelity

“…We determined the baseline mutation rate of our three 5FU resistant variants using a Luria-Delbrück fluctuation test that can measure the rates of all 12 mutational classes ((11) and Figure 3). We also evaluated PB1 V43I, which has been reported to be a fidelity variant (20, 43). We used this novel fluctuation test to interrogate four of the most common mutational classes (A to G, C to U, G to A, and U to C), for which the assay has the greatest discriminatory power.…”

“…This mutation, which mediates ribavirin resistance in the A/Wuhan/359/95 H3N2 and A/Vietnam/1203/04 H5N1 strains, is sensitive to drug in the PR8 genetic background (20). The PB1 V43I mutant, which has been suggested to be a fidelity variant even in the PR8 background (43), shows no difference in the rate of transition mutations in PR8. These findings suggest that there are likely to be epistatic interactions governing polymerase activity and fidelity in influenza virus, and that the ability of one virus to evolve resistance to a mutagen may not be reflective of how another strain evolves in the face of the same selective pressure.…”

A comprehensive influenza reporter virus panel for high-throughput deep profiling of neutralizing antibodies

“…However, it is possible to prepare virus stocks relatively quickly with five or less passages, and we confirm that these stocks retain an active reporter gene expression. Additionally, genomic stability of R3 influenza viruses can be improved by utilizing the variant PB1 polymerase with lower error-rates as reported recently( 70 ) and designing R3 viruses capable of replicating only when the engineered genomic segment is functional, such as inducible gene-expression systems. Alternatively, plaque purification or rederiving virus strains may be necessary periodically.…”

Influenza vaccine response: future perspectives

“…Therefore, the development of a master virus with high genetic stability may be crucial for the preparation of vaccines [14]. In a recent work, Naito et al described the generation of a high-fidelity and high-growth influenza vaccine MSV with a single V43I amino acid change in the polymerase basic 1 (PB1) of the high-growth A/Puerto Rico/8/1934 (PR8) master virus [15]. This can lead to the generation of high-growth vaccine viruses with high polymerase fidelity, low error rates of gene replication, and reduced antigenic diversity during virus propagation in eggs for vaccine production.…”