Non-replicating adenovirus vectors expressing avian influenza virus hemagglutinin and nucleocapsid proteins induce chicken specific effector, memory and effector memory CD8(+) T lymphocytes

Abstract

Avian influenza virus (AIV) specific CD8(+) T lymphocyte responses stimulated by intramuscular administration of an adenovirus (Ad) vector expressing either HA or NP were evaluated in chickens following ex vivo stimulation by non-professional antigen presenting cells. The CD8(+) T lymphocyte responses were AIV specific, MHC-I restricted, and cross-reacted with heterologous H7N2 AIV strain. Specific effector responses, at 10 days post-inoculation (p.i.), were undetectable at 2 weeks p.i., and memory responses were detected from 3 to 8 weeks p.i. Effector memory responses, detected 1 week following a booster inoculation, were significantly greater than the primary responses and, within 7 days, declined to undetectable levels. Inoculation of an Ad-vector expressing human NP resulted in significantly greater MHC restricted, activation of CD8(+) T cell responses specific for AIV. Decreases in all responses with time were most dramatic with maximum activation of T cells as observed following effector and effector memory responses.

Fig. 1

Kinetics of the humoral immune response induced following i.m. inoculation with Ad-HA. Serum HI antibody titers of individual chickens were evaluated, expressed as log₂ of the reciprocal of the greatest dilution of serum inhibiting agglutination of 1% chicken RBCs by 4 HA units of the H5N9/Turkey/Wis/68 AIV. Results shown were derived from two separate experiments.

Fig. 2

Kinetics of the AIV specific T lymphocyte responses from B19/B19, MHC haplotype chickens following primary and booster inoculations with 1 × 10⁸ ifu of RCA-free human Ad5 adenovirus vector expressing AIV HA (Ad-HA). T lymphocytes isolated from birds inoculated with Ad-HA were ex vivo stimulated with H5N9 AIV infected MHC matched B19/B19 APCs before evaluating induction of the T lymphocyte response. Activation was quantified as IFNγ secretion from T cells determined through production of NO by an HD11 macrophage cell line. Results are expressed as the average (±S.E.) of two experiments (n=6 or 7 birds). Each ex vivo stimulation assay is denoted by the adenovirus vector inoculated animal source of T lymphocytes and virus infected MHC-I APCs. (A) The APCs were kidney cells derived from B19/B19 chicks. (B) The APCs were derived from mismatched B2/B2 chicks. The difference in the responses of T lymphocytes from Ad-HA inoculated birds and AdE inoculated control birds at 10 days and at 3 and 5 weeks p.i. were significant (p≤0.001).

Fig. 3

An IFNγ ELISA confirmed induction of specific T lymphocyte responses by primary and booster administration of Ad-HA. Concentration of IFNγ secreted in the supernatant of ex vivo APC-stimulated T lymphocytes was determined using a commercial ELISA (Invitrogen, La Jolla, CA). Results are expressed as the average (± S.E.) of 3 birds. Each ex vivo stimulation assay, using AIV infected B19/B19 derived APCs, is denoted by the adenovirus vector inoculated animal source of T lymphocyte and virus infected MHC-I APCs. Uninfected APCs did not elicit a response. The difference between IFNγ secretion by activation of T lymphocytes derived from Ad-HA and AdE inoculated chickens was significant (p≤0.001).

Fig. 4

T lymphocytes from Ad-HA inoculated chickens responsible for the AIV specific, MHC restricted responses were CD8⁺. At 6 weeks p.i., the T lymphocytes from the peripheral blood mononuclear cells were separated into CD4⁺ and CD8⁺ subpopulations prior to stimulation with AIV infected and uninfected B19 APCs. The activation of the T lymphocytes was measured by indirect stimulation of NO production. Results are expressed as average NO production (±S.E) of an n of 3 birds.

Fig. 5

CD8⁺ T lymphocytes from B19 birds inoculated with Ad-HA respond to APCs infected with the homologous (H5N9) or a heterologous (H7N2) virus. At 7 weeks p.i., purified CD4⁺ and CD8⁺ T lymphocytes from chickens (n = 3) inoculated with Ad-HA were stimulated by B19 derived APCs infected with either the H5N9 or H7N2 strain. Assays represent the mean (± S.E.) of T lymphocytes of 3 birds; (A) The T lymphocyte responses as determined by the indirect IFNγ assay, and (B) The T lymphocyte responses of the same birds as determined by the direct IFNγ ELISA. Responses of CD8⁺ T lymphocytes were significantly greater than those of the CD4⁺ T lymphocytes (p≤0.01 and p≤0.001, respectively, as determined by the two assays).

Fig. 6

AIV specific CD8⁺ T lymphocytes were detected from spleens of Ad-HA inoculated chickens 4 weeks p.b. Unseparated or the antibody enriched CD4⁺ and CD8⁺ splenic T lymphocytes isolated from chickens inoculated with adenovirus vector were stimulated ex vivo with MHC matched B19/B19 APCs infected with homologous H5N9 virus. Production of NO by macrophages induced by secretion of IFNγ from stimulated T lymphocytes was used to quantify the activation of the lymphocytes. Results are expressed as the average (± S.E.) of 4 birds. Each ex vivo stimulation assay is denoted by the adenovirus vector inoculated animal source of T lymphocyte and virus infected MHC-I APCs. AIV specific memory CD8⁺ T lymphocyte responses in the spleens were significantly greater than the CD4⁺ T lymphocyte responses (p≤0.003), but not significantly different from the response of the unseparated lymphocytes.

Fig. 7

CD8⁺ lymphocyte responses of birds receiving the Ad-NP vector were greater than those from birds receiving the Ad-HA vector. T lymphocytes, prepared from B19/B19 chickens inoculated with either vector (n = 3), were ex vivo stimulated with H5N9 AIV infected B19 APCs. The responses of the T lymphocytes were determined using an IFNγ ELISA. Results are expressed as the average (± S.E.) of 3 birds. Each ex vivo stimulation assay is denoted by the adenovirus vector inoculated animal source of T lymphocytes and virus infected MHC-I APCs.