Improvement of PR8-Derived Recombinant Clade 2.3.4.4c H5N6 Vaccine Strains by Optimization of Internal Genes and H103Y Mutation of Hemagglutinin

Abstract

Clade 2.3.4.4c H5N6 avian influenza A viruses (AIVs) may have originally adapted to infect chickens and have caused highly pathogenic avian influenza (HPAI) in poultry and human fatalities. Although A/Puerto Rico/8/1934 (H1N1) (PR8)-derived recombinant clade 2.3.4.4c H5N6 vaccine strains have been effective in embryonated chicken eggs-based vaccine production system, they need to be improved in terms of immunogenicity and potential mammalian pathogenicity. We replaced the PB2 gene alone or the PB2 (polymerase basic protein 2), NP (nucleoprotein), M (matrix protein) and NS (non-structural protein) genes together in the PR8 strain with corresponding genes from AIVs with low pathogenicity to remove mammalian pathogenicity and to match CD8+ T cell epitopes with contemporary HPAI viruses, respectively, without loss of viral fitness. Additionally, we tested the effect of the H103Y mutation of hemagglutinin (HA) on antigen productivity, mammalian pathogenicity and heat/acid stability. The replacement of PB2 genes and the H103Y mutation reduced the mammalian pathogenicity but increased the antigen productivity of the recombinant vaccine strains. The H103Y mutation increased heat stability but unexpectedly decreased acid stability, probably resulting in increased activation pH for HA. Interestingly, vaccination with inactivated recombinant virus with replaced NP, M and NS genes halted challenge virus shedding earlier than the recombinant vaccine without internal genes replacement. In conclusion, we successfully generated recombinant clade 2.3.4.4c H5N6 vaccine strains that were less pathogenic to mammals and more productive and heat stable than conventional PR8-derived recombinant strains by optimization of internal genes and the H103Y mutation of HA.

Engineering an Optimal Y280-Lineage H9N2 Vaccine Strain by Tuning PB2 Activity

“…One issue with PR8-derived recombinant vaccine strains is that they contain the PB2 of PR8, which includes multiple MPMs, including the highly potent E627K mutation, which increases the mammalian pathogenicity of the vaccine strain and poses a biosafety risk during vaccine production. To address this problem, the PR8 PB2 was replaced with a non-pathogenic PB2 (01310 PB2) to successfully develop safe and highly productive H5Nx and Egyptian G1-lineage vaccine strains [29,30,36]. In this study, we aimed to generate the optimal vaccine strain for the prevention of Korean Y280-lineage viruses, considering factors such as replication efficiency in ECEs, antigenicity, and mammalian non-pathogenicity.…”

Selection of an Optimal Recombinant Egyptian H9N2 Avian Influenza Vaccine Strain for Poultry with High Antigenicity and Safety

“…The P221S mutation has been reported in H5N1 AIVs to reduce HA trimer integrity to cause low heat and acid stabilities [49,50]. The P221S mutation among H9N2 AIVs is seemingly not natural; it may induce early viral envelope and endosome fusion due to low pH stability and speed up virus replication cycles [40,51]. Interestingly, the lower heat and acid stabilities and avian-to-mammalian-receptor-affinity ratio did not hinder virus replication in MDCK and A549 cells.…”

“…To date, the gain-of-function of viruses has been a concern for a long time due to the potential risks of laboratory and manufacturing leaks of viruses, but why the generation of PR8-derived vaccine strains against mammalian pathogenic AIVs has not been discussed in biosafety and biosecurity aspects in depth is strange [56]. In our previous studies, the simple exchange of PR8 PB2 with 01310 PB2 genes attenuated the mammalian pathogenicity of AIVs with maintenance of virus replication efficiency in ECEs and immunogenicity [36,40]. In this study, we verified the usefulness of 01310 PB2 again, but our additional intention to reduce mammalian pathogenicity and increase virus replication efficiency in ECEs by reversing mutations in HA and NA was proven to be ineffective.…”

“…Virus passages through ECEs were repeated 3 or 4 times, and allantoic fluid was harvested and stored at −80 • C until use. The presence of virus was detected with a hemagglutination assay using 1% (v/v) chicken red blood cells (RBCs), and the full genome sequences were confirmed by RT-PCR and sequencing as previously described [40].…”

“…To date, the 01310 PB2 gene has been successfully used for the development of AI vaccine strains in terms of high virus titers, strong immunogenicity, and mammalian nonpathogenicity [36,37,40]. Therefore, we generated recombinant strains with various combinations of mutations (Table 4).…”