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

Abstract

H9N2 avian influenza A viruses (AIVs) cause economic losses in the poultry industry and provide internal genomic segments for the evolution of H5N1 and H7N9 AIVs into more detrimental strains for poultry and humans. In addition to the endemic Y439/Korea-lineage H9N2 viruses, the Y280-lineage spread to Korea since 2020. Conventional recombinant H9N2 vaccine strains, which bear mammalian pathogenic internal genomes of the PR8 strain, are pathogenic in BALB/c mice. To reduce the mammalian pathogenicity of the vaccine strains, the PR8 PB2 was replaced with the non-pathogenic and highly productive PB2 of the H9N2 vaccine strain 01310CE20. However, the 01310CE20 PB2 did not coordinate well with the hemagglutinin (HA) and neuraminidase (NA) of the Korean Y280-lineage strain, resulting in a 10-fold lower virus titer compared to the PR8 PB2. To increase the virus titer, the 01310CE20 PB2 was mutated (I66M-I109V-I133V) to enhance the polymerase trimer integrity with PB1 and PA, which restored the decreased virus titer without causing mouse pathogenicity. The reverse mutation (L226Q) of HA, which was believed to decrease mammalian pathogenicity by reducing mammalian receptor affinity, was verified to increase mouse pathogenicity and change antigenicity. The monovalent Y280-lineage oil emulsion vaccine produced high antibody titers for homologous antigens but undetectable titers for heterologous (Y439/Korea-lineage) antigens. However, this defect was corrected by the bivalent vaccine. Therefore, the balance of polymerase and HA/NA activities can be achieved by fine-tuning PB2 activity, and a bivalent vaccine may be more effective in controlling concurrent H9N2 viruses with different antigenicities.

Keywords: H9N2; avian influenza A virus; bivalent oil emulsion vaccine; mammalian pathogenicity; recombinant vaccine strain.

Figure 1

Growth kinetics of recombinant and wild-type Korean Y280-lineage H9N2 viruses. Recombinant SL20 viruses and wild-type SL20 virus (SL20wt) (0.1 MOI) were infected into (A) MDCK cells and (B) A549 cells. After 1h incubation, inoculum was replaced with fresh media and supernatant was obtained at each time point (0, 24, 48, and 72 h). The viral titer was measured as TCID₅₀/mL in MDCK cells, and the result was the average of three independent repeated experiments. Statistical significance was analyzed by one-way ANOVA and significant difference with other groups was marked in panels (A) *, rSL20(P) and rSL20(P)-L226Q; **, SL20wt and rSL20-MVV310PB2, and (B) #; rSL20(P); ##; rSL20(P)-L226Q; ###; rPR8 (p < 0.05).

Figure 2

Weight loss and survival rate of recombinant Korean Y280-lineage H9N2 virus-infected mouse. Five six-week-old female BALB/c mice in each group were infected with 10⁶ EID₅₀/50 μL via intranasal route and weighed over the course of 1 week. Mice with a weight loss of 20% or more were considered dead and euthanized, and average weight loss (A) and survival rate of each group (B) are shown.

Figure 3

Hemagglutination inhibition test of SPF chicken and mouse serum samples against recombinant SL20 viruses. Serum samples from SPF chickens and BALB/c mice were treated with two methods: RDE− with heat-treated serum only (56 °C, 30 min) and RDE+ with RDE and heat-treated serum for inactivation of non-specific inhibitors. Each serum sample was serially two-fold diluted with PBS and reacted with the same volume of virus (4 HAU). After 40 min incubation at 4 °C, 1% chicken RBC was added, and the results were recorded after 40 min at 4 °C.

Figure 4

Neuraminidase-inhibiting activity of serum samples from vaccinated chickens. Neuraminidase activities of (A) H9N2 vaccine strains, rSL20(P)-MVV310PB2, and r310-NS28; (B) differently treated SPF chicken serum, RDE+ heat treatment (56 °C, 30 min), and heat treatment. Neuraminidase-inhibiting activity of heat-treated serum samples from vaccinated and negative control groups of Exp. 2. Serum samples collected at 3 wpv were reacted with 8 HAU of (C) rSL20-MVV310PB2 and (D) r310-NS28. The data are shown as the relative neuraminidase activity (%) of the average of the five chicken serums in each vaccine group to that of the negative control group. *, rSL20(P)-MVV310PB2 and bivalent vaccine groups were significantly different from V-r310-NS28 and negative control; **, V-r310-NS28 group was significantly different from the bivalent vaccine and negative control groups.