Avian influenza viral nucleocapsid and hemagglutinin proteins induce chicken CD8+ memory T lymphocytes

Abstract

The avian influenza viruses (AIVs) can be highly contagious to poultry and a zoonotic threat to humans. Since the memory CD8(+) T lymphocyte responses in chickens to AIV proteins have not been defined, these responses to H5N9 AIV hemagglutinin (HA) and nucleocapsid (NP) proteins were evaluated by ex vivo stimulation with virus infected non-professional antigen presenting cells. Secretion of IFNgamma by activated T lymphocytes was evaluated through macrophage induction of nitric oxide. AIV specific, MHC-I restricted memory CD8(+) T lymphocyte responses to NP and HA were observed 3 to 9 weeks post-inoculation (p.i.). The responses specific to NP were greater than those to HA with maximum responses being observed at 5 weeks p.i. followed by a decline to weakly detectable levels by 9 weeks p.i. The cross-reaction of T lymphocytes to a heterologous H7N2 AIV strain demonstrated their ability to respond to a broader range of AIV.

Fig. 1

In vitro expression of pcDNA3.1/V5-His-TOPO TA vectored AIV proteins in transfected CHO-K1 cells (magnification, 200 ×). Expression was detected by an IFA using AIV positive reference serum as the source of primary antibodies. Cells were transfected with plasmids expressing (A) LacZ, (B) HA, and (C) NP.

Fig. 2

Antibody titers induced in individual birds (n = 6) by NP and HA expressing plasmids at 3 weeks p.i. (A) Serum HI antibody titers from chickens inoculated with HA expressing plasmid against the homologous H5N9 AIV strain and heterologous H7N2 AIV strain. (B) Serum anti-NP antibody ELISA titers from chickens inoculated with the NP expressing plasmid. Neither HI nor anti-NP antibodies were detectable in the sera of 4 control birds inoculated with PBS. Each symbol represents the response of an individual chicken.

Fig. 3

Chicken memory T lymphocyte responses to AIV HA and NP proteins between 3 and 9 weeks p.i. with NP and/or HA expression plasmids. Chickens of the B19/B19 MHC haplotype were inoculated with DNA plasmids expressing AIV HA, NP or both HA and NP (HN). Memory T lymphocytes were stimulated ex vivo with H5N9 AIV infected MHC matched B19/B19 and mismatched B2/B2 APCs. Production of NO by HD11 macrophage cells induced by the secretion of IFNγ from stimulated T lymphocytes was used to quantify lymphocyte activation. Results represent the average (± S.E.) of two separate experiments. Each ex vivo stimulation assay is denoted by the source of T lymphocytes and MHC haplotype of the APCs infected with the virus. The differences in stimulation by matched and mismatched APCs were significant (p ≤ 0.003 to 0.02) for each inoculated antigen and time point. The responses to HN (p ≤ 0.03) at 3 weeks and NP (p ≤ 0.03) and HN (p ≤ 0.007) at 5 weeks p.i. were significantly greater than the responses to HA at the same time points. The p value for the difference between responses to NP and HA at 3 weeks p.i. was 0.07.

Fig. 4

T lymphocytes from B19 birds inoculated with either H5N9 derived HA or NP expression plasmids respond to a heterologous (H7N2) virus. At 8 weeks p.i., T lymphocytes from chickens receiving either HA or NP cloned from the H5N9 strain were co-cultured with APCs infected with H5N9 or H7N2 viruses. The T cell responses are expressed as the average (± S.E.) of NO production for each treatment group. T lymphocytes from plasmid-inoculated chickens had significantly greater responses to H7N2 AIV strain than those from the PBS control group (p ≤ 0.01). PBMC were prepared from three individual chickens for each stimulation assay.

Fig. 5

In vivo infection of B19 chickens with the low path H5N9/Tur/Wis/68 AIV generates AIV specific, MHC matched memory T lymphocytes. The potential for infectious AIV to also produce a memory T lymphocyte response was determined 5 weeks p.i. with H5N9. Mean (±S.E.) NO production by each treatment group is represented by the bars. APCs used for ex vivo stimulation were either uninfected (Uninf) or virus-infected (Inf). Significantly lower (p ≤ 0.01) NO production by AIV infected mismatched APCs derived from the CKC of homozygous B2 chicks compared to matched APCs derived from CKC of homozygous B19 chicks indicate MHC restriction. T lymphocytes from three individual birds were used for each ex vivo stimulation assay.