Cross-reactive, cell-mediated immunity and protection of chickens from lethal H5N1 influenza virus infection in Hong Kong poultry markets

Abstract

In 1997, avian H5N1 influenza virus transmitted from chickens to humans resulted in 18 confirmed infections. Despite harboring lethal H5N1 influenza viruses, most chickens in the Hong Kong poultry markets showed no disease signs. At this time, H9N2 influenza viruses were cocirculating in the markets. We investigated the role of H9N2 influenza viruses in protecting chickens from lethal H5N1 influenza virus infections. Sera from chickens infected with an H9N2 influenza virus did not cross-react with an H5N1 influenza virus in neutralization or hemagglutination inhibition assays. Most chickens primed with an H9N2 influenza virus 3 to 70 days earlier survived the lethal challenge of an H5N1 influenza virus, but infected birds shed H5N1 influenza virus in their feces. Adoptive transfer of T lymphocytes or CD8(+) T cells from inbred chickens (B(2)/B(2)) infected with an H9N2 influenza virus to naive inbred chickens (B(2)/B(2)) protected them from lethal H5N1 influenza virus. In vitro cytotoxicity assays showed that T lymphocytes or CD8(+) T cells from chickens infected with an H9N2 influenza virus recognized target cells infected with either an H5N1 or H9N2 influenza virus in a dose-dependent manner. Our findings indicate that cross-reactive cellular immunity induced by H9N2 influenza viruses protected chickens from lethal infection with H5N1 influenza viruses in the Hong Kong markets in 1997 but permitted virus shedding in the feces. Our findings are the first to suggest that cross-reactive cellular immunity can change the outcome of avian influenza virus infection in birds in live markets and create a situation for the perpetuation of H5N1 influenza viruses.

FIG. 1

Dose-response challenge of chickens. Ten chickens per group primed 30 days earlier by infection with 10³ CID₅₀ of A/Chicken/HK/G9/97(H9N2) influenza virus were challenged with various doses of an H5N1 influenza virus. Chickens were monitored to determine how many died each day until 15 days after the challenge. Chickens immunized 30 days earlier with P/Chicken/HK/QB4/99 (Newcastle disease virus) were challenged with 10 CLD₅₀ of H5N1 influenza virus.

FIG. 2

Adoptive transfer of immune splenocytes and subtypes of T lymphocytes. (A) Splenic lymphocytes (4 × 10⁷) collected 10 days postinfection from four inbred chickens (B²/B²) immunized by infection with 10³ CID₅₀ of A/Chicken/HK/G9/97(H9N2) influenza virus were adoptively transferred through the wing veins to four naive inbred chickens (B²/B²), and 1 day later chickens were challenged with 10 LD₅₀ of an H5N1 influenza virus. As a control, whole splenic cells from unimmunized chickens were transferred to naive chickens prior to challenge. Results were evaluated for deaths of chickens 15 days postchallenge. (B) Splenic T cells were collected from four inbred chickens (B²/B²) immunized 7 days earlier with 10³ CID₅₀ of A/Chicken/HK/G9/97(H9N2) influenza virus. T lymphocytes were depleted of CD4⁺ or CD8⁺ subtype of T cells using pan-mouse IgG-coated Dynabeads. Subtypes of T cells 2 × 10⁷ were transferred to four naive inbred chickens (B²/B²) through the wing veins, and 1 day later chickens were challenged with 10 LD₅₀ of an H5N1 influenza virus. As a control, CD4⁺ or CD8⁺ T cells from unimmunized chickens were transferred to naive chickens prior to challenge. Results were evaluated for deaths of chickens 15 days postchallenge.

FIG. 2

Adoptive transfer of immune splenocytes and subtypes of T lymphocytes. (A) Splenic lymphocytes (4 × 10⁷) collected 10 days postinfection from four inbred chickens (B²/B²) immunized by infection with 10³ CID₅₀ of A/Chicken/HK/G9/97(H9N2) influenza virus were adoptively transferred through the wing veins to four naive inbred chickens (B²/B²), and 1 day later chickens were challenged with 10 LD₅₀ of an H5N1 influenza virus. As a control, whole splenic cells from unimmunized chickens were transferred to naive chickens prior to challenge. Results were evaluated for deaths of chickens 15 days postchallenge. (B) Splenic T cells were collected from four inbred chickens (B²/B²) immunized 7 days earlier with 10³ CID₅₀ of A/Chicken/HK/G9/97(H9N2) influenza virus. T lymphocytes were depleted of CD4⁺ or CD8⁺ subtype of T cells using pan-mouse IgG-coated Dynabeads. Subtypes of T cells 2 × 10⁷ were transferred to four naive inbred chickens (B²/B²) through the wing veins, and 1 day later chickens were challenged with 10 LD₅₀ of an H5N1 influenza virus. As a control, CD4⁺ or CD8⁺ T cells from unimmunized chickens were transferred to naive chickens prior to challenge. Results were evaluated for deaths of chickens 15 days postchallenge.

FIG. 3

Identification of the T-lymphocyte phenotypes of splenic T cells depleted of CD4⁺ or CD8⁺ T cells by flow cytometry. Splenic T cells depleted of CD4⁺ or CD8⁺ T cells were stained with mouse anti-chicken CD4 or CD8 monoclonal antibody and FITC-labeled goat anti-mouse IgG. Control T cells were stained with FITC-labeled goat anti-mouse IgG (A), splenic T cells depleted of CD4⁺ T cells were stained with mouse anti-chicken CD8 antibody (B), and splenic T cells depleted of CD8⁺ T cells were stained with mouse anti-chicken CD4 antibody (C).

FIG. 4

Cytotoxicity of splenic T cells from chickens previously infected with an H9N2 virus. (A) The effector cells were splenocytes collected from four inbred chickens (B²/B²) infected with A/Chicken/HK/G9/97 (H9N2) influenza virus 7 days earlier. Lung target cells infected with either an H9N2 or H5N1 influenza virus served as the target cells. CTL activity was measured with the nonradioactive CTL assay that detects LDH release. (B) Purified T cells were isolated from inbred chickens (B²/B²) that had been infected 7 days earlier with A/Chicken/HK/G9/97 (H9N2) influenza virus. Before the CTL assays were performed, the populations were depleted of CD4⁺ or CD8⁺ T cells using pan anti-mouse IgG-coated Dynabeads and mouse anti-chicken CD4 or CD8 monoclonal antibodies. Inbred lung cells (B²/B²) infected with either an H9N2 or H5N1 influenza virus were used as target cells. (C) Splenic T cells were isolated from inbred chickens (B²/B²) that had been infected 60 days earlier with A/Chicken/HK/G9/97 (H9N2) influenza virus (MOI of 2). Before CTL assays were performed, the splenic T cells were stimulated with inbred splenic cells infected with A/Chicken/HK/G9/97 (H9N2) for 7 days. Inbred chicken lung cells (B²/B²) infected with an H9N2 or H5N1 influenza virus were used as target cells.