Protection against a lethal avian influenza A virus in a mammalian system

Abstract

The question of how best to protect the human population against a potential influenza pandemic has been raised by the recent outbreak caused by an avian H5N1 virus in Hong Kong. The likely strategy would be to vaccinate with a less virulent, laboratory-adapted H5N1 strain isolated previously from birds. Little attention has been given, however, to dissecting the consequences of sequential exposure to serologically related influenza A viruses using contemporary immunology techniques. Such experiments with the H5N1 viruses are limited by the potential risk to humans. An extremely virulent H3N8 avian influenza A virus has been used to infect both immunoglobulin-expressing (Ig+/+) and Ig-/- mice primed previously with a laboratory-adapted H3N2 virus. The cross-reactive antibody response was very protective, while the recall of CD8(+) T-cell memory in the Ig-/- mice provided some small measure of resistance to a low-dose H3N8 challenge. The H3N8 virus also replicated in the respiratory tracts of the H3N2-primed Ig+/+ mice, generating secondary CD8(+) and CD4(+) T-cell responses that may contribute to recovery. The results indicate that the various components of immune memory operate together to provide optimal protection, and they support the idea that related viruses of nonhuman origin can be used as vaccines.

FIG. 1

(A) Naive B6 mice were infected i.n. with 10-fold dilutions of the H3N8 virus and monitored daily for survival. Four mice per group were infected as follows: diagonal stripes, 10⁵ EID₅₀; dotted, 10⁴ EID₅₀; horizontal stripes, 10³ EID₅₀; open, 10² EID₅₀; filled, 10¹ EID₅₀. (B to F) Naive B6 mice were infected i.n. with 10⁴ EID₅₀ of H3N8 virus, and virus titers were determined for the lungs, brain, spleen, blood, and liver in embryonated chicken eggs.

FIG. 2

Naive B6 mice were primed i.n. with 10^6.8 EID₅₀ of the H3N2 virus and rested for 1 month prior to challenge i.n. with 10⁴ EID₅₀ of the H3N8 virus. Virus titers in the lung were determined on days 1 to 3 after infection.

FIG. 3

Naive B6 mice were infected i.n. with 10^6.8 EID₅₀ of the H3N2 virus and rested for 4 months. Rat Ig-treated control mice (A) or mice depleted of both CD4⁺ and CD8⁺ T cells (B) were infected i.n. with 10⁴ EID₅₀ of the H3N8 virus. BAL was done on days 0, 7, and 10 after secondary infection. The BAL cells were removed by centrifugation, and H3N2-specific IgG titers were determined by ELISA. Each curve represents a single animal, and three animals are shown per time point. O.D. 405, optical density at 405 nm.

FIG. 4

(A and B) μMT mice were primed i.n. with 10^6.8 EID₅₀ of the H3N2 virus (A) or i.p. with 10^5.2 EID₅₀ of the H3N8 virus (B) and rested for 6 weeks. They were then either mock (rat Ig)-treated (•) or depleted of CD4⁺ T cells (□), CD8⁺ T cells (▴), or both (▵) as described in Materials and Methods and then challenged i.n. with 10^4.0 EID₅₀ of the H3N8 virus. Virus titers in the lungs were determined. (C) μMT mice were infected i.n. with 10^6.8 EID₅₀ of H3N2 virus, rested for 10 weeks, and then challenged i.n. with 10³ EID₅₀ of the H3N8 virus. Virus titers in the lungs were determined.

FIG. 5

(A to C) Staining profiles for CD8⁺ NPP⁺ lymphocytes obtained for BAL cells harvested from naive (A), H3N2-immune (B), or H3N8-immune (C) μMT mice on day 6 after i.n. challenge with 10^4.0 EID₅₀ of the H3N8 virus. (D to F) Prevalence of virus-specific CD8⁺ T cells for BAL (D), MLN (E), and spleen (F) cells harvested from μMT mice on day 12 after i.n. challenge with 250 EID₅₀ of the H3N8 virus. Staining with the control SEV9 tetramer was always <0.1%. Percentages in parentheses denote levels of specific ⁵¹Cr release (see footnotes to Table 4) after direct assay of freshly isolated lymphocyte populations; numbers in brackets are reciprocal Thp frequencies for IFN-γ-producing CD4⁺ T cells (see footnotes to Table 3).