Mucosal delivery of inactivated influenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal influenza A H5N1 virus infection

Abstract

Influenza vaccines that induce greater cross-reactive or heterosubtypic immunity (Het-I) may overcome limitations in vaccine efficacy imposed by the antigenic variability of influenza A viruses. We have compared mucosal versus traditional parenteral administration of inactivated influenza vaccine for the ability to induce Het-I in BALB/c mice and evaluated a modified Escherichia coli heat-labile enterotoxin adjuvant, LT(R192G), for augmentation of Het-I. Mice that received three intranasal (i.n.) immunizations of H3N2 vaccine in the presence of LT(R192G) were completely protected against lethal challenge with a highly pathogenic human H5N1 virus and had nasal and lung viral titers that were at least 2,500-fold lower than those of control mice receiving LT(R192G) alone. In contrast, mice that received three vaccinations of H3N2 vaccine subcutaneously in the presence or absence of LT(R192G) or incomplete Freund's adjuvant were not protected against lethal challenge and had no significant reductions in tissue virus titers observed on day 5 post-H5N1 virus challenge. Mice that were i.n. administered H3N2 vaccine alone, without LT(R192G), displayed partial protection against heterosubtypic challenge. The immune mediators of Het-I were investigated. The functional role of B and CD8+ T cells in Het-I were evaluated by using gene-targeted B-cell (IgH-6(-/-))- or beta2-microglobulin (beta2m(-/-))-deficient mice, respectively. beta2m(-/-) but not IgH-6(-/-) vaccinated mice were protected by Het-I and survived a lethal infection with H5N1, suggesting that B cells, but not CD8+ T cells, were vital for protection of mice against heterosubtypic challenge. Nevertheless, CD8+ T cells contributed to viral clearance in the lungs and brain tissues of heterotypically immune mice. Mucosal but not parenteral vaccination induced subtype cross-reactive lung immunoglobulin G (IgG), IgA, and serum IgG anti-hemagglutinin antibodies, suggesting the presence of a common cross-reactive epitope in the hemagglutinins of H3 and H5. These results suggest a strategy of mucosal vaccination that stimulates cross-protection against multiple influenza virus subtypes, including viruses with pandemic potential.

FIG. 1

Mucosal but not parenteral influenza virus vaccination induces Het-I that is augmented by adjuvant. Groups of BALB/c mice received three i.n. or s.c. inoculations at weekly intervals of 20 μg of formalin-fixed H3N2 virus in the presence or absence of the indicated adjuvant. One additional group was vaccinated i.n. with live H3N2 virus. Control mice received adjuvant only. Four weeks after live virus vaccine boost and 2 weeks after the final fixed vaccine boost, mice were challenged i.n. with lethal H5N1 (A/Hong Kong/483/97) (A and B) or nonlethal H1N1 virus (A/Taiwan/1/86) (C) and monitored for survival (A) or euthanatized 5 days later for collection of lung, nose, and brain tissue. Individual tissues were homogenized in 1 ml of PBS and titrated for virus infectivity in eggs. Virus endpoint titers are expressed as mean log₁₀ EID₅₀/ml (B and C). An asterisk indicates the H3N2/LT(R192G)-vaccinated group was significantly (P < 0.05) different from the adjuvant-only control group by analysis of variance.

FIG. 2

Mucosal vaccination with influenza B virus vaccine fails to protect against lethal influenza A virus challenge. Groups of BALB/c mice received three i.n. inoculations of 20 μg of formalin-fixed H3N2 or B/Harbin/7/94 virus in the presence or absence of 2 μg of LT(R192G). An additional group of mice received three s.c. inoculations of 20 μg of fixed H3N2 virus together with 2 μg of LT(R192G). Two weeks after the final vaccine boost, mice were challenged i.n. with a lethal dose of H5N1 virus and monitored for survival (A) or euthanatized 5 days later for collection of lung and nose tissue (B). An asterisk indicates the vaccinated group was significantly (P < 0.05) different from the adjuvant-only control group by analysis of variance.

FIG. 3

Effect of T-cell depletion on survival of H3N2-immune mice. Groups of mice were vaccinated with H3N2 in the presence of LT(R192G) as described in Materials and Methods. The adjuvant control mice received LT(R192G) only (●). Mice received 1 mg of the anti-CD4 (○), anti-CD8 (◊), a combination of both MAbs (anti-CD4/CD8) (□), or rat IgG (▵) control antibody on days −2, +2, +6, and +10 relative to the time of challenge. Mice were challenged i.n. with a lethal dose of H5N1 and were monitored for survival (A) or euthanatized for collection of tissues on day 5 (B). Individual lung and brain tissues were titrated for virus infectivity as described in the legend to Fig. 1. An asterisk indicates the T cell-depleted group was significantly (P < 0.05) different from the rat IgG control group by analysis of variance.

FIG. 4

Susceptibility of B-cell-deficient mice to heterosubtypic challenge. Four groups (eight per group) of mice were vaccinated i.n. three times with H3N2/LT(R192G) vaccine at weekly intervals. The immunized groups of IgH-6^−/− (■) and wt (□) control mice were treated with 1 mg of anti-CD8 (clone 2.43) ascites on days −2, +2, and +6 relative to the time of virus challenge. In addition, immunized groups of IgH-6^−/− (●) and wt (○) mice received control ascites (clone SFR3-DR5) in place of anti-CD8 MAb. Control wt (▵) and IgH-6^−/− (▴) mice received LT(R192G) adjuvant only. Two weeks after the final vaccine boost, mice received a lethal heterosubtypic challenge with 100 MID₅₀ of H5N1 virus. Blood samples were collected from the orbital plexus on day 3 p.c. from all mice and were tested for the presence of serum IgG and IgA antiviral titers by ELISA and HAI as described in Materials and Methods. No IgG, IgA, or HAI antibodies could be detected in IgH-6^−/− H3N2-immunized mice, whereas wt-immunized mice had mean HAI titers of 2,560 and IgA or IgG serum titers of ≥64,000 to X-31 virus. In addition, two mice from each group were euthanatized on day 3 p.c. to confirm depletion of CD8⁺ T cells and/or CD45R/B220⁺ B cells by flow cytometry. Cells from the spleen were analyzed for CD3⁺, CD4⁺, CD8⁺, and CD45R/B220⁺ B cells as described in Materials and Methods. Anti-CD8 treatment resulted in more than 95% reduction of the T-cell subpopulation in IgH-6^−/− (■) and wt (□) mice. Analysis of CD45R/B220⁺ B cells in the wt mice revealed a normal range (55 to 65%) of spleen cells in comparison to 1.9 to 2.2% detected in IgH-6^−/− mice.