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.

Keywords: H9N2; avian influenza virus; genetic evolution; mammalian non-pathogenicity; recombinant vaccine strain.

Figure 1

E. coli toxic ORF in the HA2 subunit gene and hotspots of deletion mutations. MF434468.1 was identical to the A/chicken/Egypt/ME543V/2016 (H9N2) (ME543), and synthetic H9 genes had a single nucleotide deletion (~) in four different positions of the hot spot (A at 1336, C at 1350, C at 1359 or A at 1365), forming a stop codon and terminating HA protein synthesis early. They encode the leucine-rich sequence (ELLVLL) and are mutated into the same nucleotide sequence of 01310 without amino acid change. Additionally, the Shine–Dalgarno sequence (GAGG) prior to the toxic ORF was eliminated by mutation into GAAG to reduce the expression. * means the stop codon and red box was site described with the text above it.

Figure 2

The six internal genes of 01310 induce reverse (L226Q) and additional (P221S) mutations in the HA of recombinant Egyptian H9N2 strains during ECEs passages. Recombinant Egypt H9N2 viruses with internal genes of 01310 were passaged to increase antigen yields in ECEs. All viruses showed increased replication efficiency in ECEs after the third passage by acquisition of the Q226L mutation in the HA protein, and rEgH9N2(310)-PB21U4 only had the additional P221S mutation in the HA protein after the fourth passage.

Figure 3

Comparison of the 2SBS amino acid sequences of H9N2 AIVs and mutated genes in this study. The 2SBS sequence of A/turkey/Minnesota/511/78(H9N2) was compared with others as the prototype. Three kinds of mutated NA genes were generated by replacing mutations of ME543V in the 370-loop (N2-av370L), the 400-loop (N2-av400L), or both (N2-av370L-av400L) with prototype sequences.

Figure 4

Most mutations decreasing avian receptor affinity have no effect on the ratio of avian to mammalian receptor affinities. Each virus was diluted to the same concentration (2⁶ HAU) with neuraminidase inhibitor, blocking buffer was added, and the virus was adsorbed on a 96-well immunoplate. (A) Avian receptor analog (3′SLN-PAA) and (B) human receptor analog (6′SLN-PAA) three-fold diluents were added and reacted with HRP-conjugated streptavidin after 1 h. HRP was developed by TMB, and the absorbance at 450 nm was measured after the addition of 0.1 M sulfuric acid. (C) The relative binding affinity was calculated by dividing the 3′SLN binding affinity by the 6′ SLN binding affinity, *: rEgH9N2(P)-L226Q had a significantly higher binding affinity to 3′SLN than other viruses, **: rEgH9N2(P)-P221S-L226Q showed significantly lower affinity to 3′SLN than other viruses and to 6′SLN than rEgH9N2(P)-P221S and rEgH9N2(P)-L226Q (p < 0.05), ***: rEgH9N2(P)-P221S was significantly different from other viruses (p < 0.05).

Figure 5

Reverse mutations in the 370-loop and 400-loop of NA increase neuraminidase activity via increased affinity to substrate. The 128(2⁷) HAU of each virus was diluted five-fold, and neuraminidase activity was measured in triplicate using the NA-star™ Influenza Neuraminidase Inhibitor Resistance Detection Kit. *, rEgH9N2(P)-av370L was significantly different from rEgH9N2(P); **, rEgH9N2(P)-av370L was significantly different from the others (p < 0.05).

Figure 6

Relatively high heat stability and low pH susceptibility of recombinant Egyptian H9N2 strains. (A) Each recombinant virus was diluted to 32 (2⁵) HAU and incubated at 56 °C for 0.5, 1, 1.5, 2, 3, and 4 h. After heat treatment, the HA titer of each aliquot was measured and recorded. (B) A total of 10⁷ EID₅₀ of viruses was mixed with pH buffer (5.0–5.6) at a 1:100 ratio and incubated at 37 °C for 1 h. Mixtures were diluted with PBS (pH 7.4) at a 1:100 ratio and inoculated into three 10 day old ECEs. The HA titers of viral replicates in harvested allantoic fluid after 72 h were compared.

Figure 7

The E627V mutation of PB2 is a key MPM of Egyptian H9N2 AIVs. Based on the plasmid encoding PB2 of 01310 with I66M, I109V, and I133V mutations (MVV), single (E627V) and multiple (I292T, K526R, G590C, E627V, and S714G) mutations acquired by Egypt H9N2 viruses were introduced. Each PB2 plasmid was cotransfected with plasmids for PB1, PA, NP of PR8, firefly luciferase, and pRL-TK plasmid into 293T cells. Firefly luciferase activity was normalized to Renilla luciferase activity and compared with the PB2 of PR8. The average and standard deviation of three independent replicates are shown. * Significantly different from the PB2 gene of 01310, ** Significantly different with others (p < 0.05).

Figure 8

More efficient replication of PR8-derived recombinant Egyptian H9N2 strains than rPR8 and their attenuation by 01310 PB2 in mammalian cells. A total of 5 × 10⁵ EID₅₀ (1 MOI) of each virus was inoculated into (A) MDCK and (B) A549 cells prepared in 12-well plates. After 1 h of incubation at 37 °C, the cells were washed with PBS, and 1 mL of fresh medium was added to each well. During 72 h of incubation, supernatants were obtained at 24, 48, and 72 h, and viral titers in supernatants were measured in TCID₅₀. * The amount of rPR8 was significantly lower than that of other recombinant H9N2 viruses (p < 0.05).

Figure 9

Different amounts of viral proteins and interactions with sialoproteins in allantoic fluids. The harvested allantoic fluid of SPF ECEs infected with vaccine candidate viruses was mixed with OptiPrep density gradient medium and ultracentrifuged at 45,000 rpm for 1 h. (A) Total viral protein purified and deglycosylated by PNGase F of vaccine candidate viruses was separated by SDS-PAGE and stained with Coomassie blue. (B) Separated protein-transferred membranes were blocked with 5% skim milk and treated with HRP-conjugated goat anti-chicken IgG (IgY) antibody for 1 h. HRP was developed with TMB solution, and chicken IgY was confirmed.