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

Abstract

For the development of an optimized Egyptian H9N2 vaccine candidate virus for poultry, various recombinant Egyptian H9N2 viruses generated by a PR8-based reverse genetics system were compared in terms of their productivity and biosafety since Egyptian H9N2 avian influenza viruses already possess mammalian pathogenicity-related mutations in the hemagglutinin (HA), neuraminidase (NA), and PB2 genes. The Egyptian HA and NA genes were more compatible with PR8 than with H9N2 AIV (01310) internal genes, and the 01310-derived recombinant H9N2 strains acquired the L226Q reverse mutation in HA after passages in eggs. Additionally, the introduction of a strong promoter at the 3′-ends of PB2 and PB1 genes induced an additional mutation of P221S. When recombinant Egyptian H9N2 viruses with intact or reverse mutated HA (L226Q and P221S) and NA (prototypic 2SBS) were compared, the virus with HA and NA mutations had high productivity in ECES but was lower in antigenicity when used as an inactivated vaccine due to its high binding affinity into non-specific inhibitors in eggs. Finally, we substituted the PB2 gene of PR8 with 01310 to remove the replication ability in mammalian hosts and successfully generated the best recombinant vaccine candidate in terms of immunogenicity, antigenicity, and biosafety.

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

“…The conflicting results may be partially explained by the increased resistance of rSL20(P)-L226Q to non-specific inhibitors in SPF chicken and mouse serum. A similar result was also reported in the G1-lineage Egyptian H9N2 strains (Figure 3) [35]. Serum contains heat-stable α and γ inhibitors, as well as heat-labile β inhibitors.…”

“…In a previous study, we observed lower immunogenicity in the G1-lineage Egyptian H9N2 vaccine strain with the L226Q mutation. This was hypothesized to be due to the masking of HA epitopes by formalin-mediated cross-linking of bound sialoproteins [35]. In this study, we did not find an effect of L226Q on immunogenicity, but the HI titer to rSL20(P)-L226Q was significantly lower than that of rSL20(P) (Table 4).…”

“…However, the higher virus titers of rSL20(P) and rSL20(P)-L226Q than rPR8 in both cells raises strong public health concerns about the conventional reverse genetics system using the highly pathogenic PR8 PB2 gene. Our experiences in testing the growth kinetics of various PR8derived clade 2.3.2.1c H5N1, clade 2.3.4.4 H5N6 and H5N8, Y439/Korea-lineage, and G1-lineage Egyptian H9N2 recombinant strains in MDCK and/or A549 cells showed that rSL20(P) and rSL20(P)-L226Q outgrow rPR8, similar to the G1-lineage Egyptian H9N2 strain [25,30,35,36]. However, the similar growth kinetics of rSL20-MVV310PB2 to rPR8 in MDCK cells was unexpected, as MVV310PB2 did not have a dominant-negative effect.…”

“…The sialic acid in α and γ inhibitors interacts with the receptor binding site of HA, while the mannose-binding lectin activity in β inhibitors interacts with mannose-rich glycans on the HA and NA [58][59][60]. RDE and heat treatment remove the inhibitory activity of serum, which may contain sialic acid [35,61,62]. The increased affinity of L226Q to the avian receptor may increase its susceptibility to non-specific inhibitors in serum.…”

“…The Y280 and G1-lineage H9N2 viruses have been reported to have mammalian pathogenicity, although fatal infections in humans have never been reported [12,31]. As a result, it is important to prevent H9N2 infections in poultry farms, and various vaccine strains have been developed to achieve this goal [4,[32][33][34][35].…”