Contributions of antinucleoprotein IgG to heterosubtypic immunity against influenza virus

Abstract

Influenza A virus causes recurring seasonal epidemics and occasional influenza pandemics. Because of changes in envelope glycoprotein Ags, neutralizing Abs induced by inactivated vaccines provide limited cross-protection against new viral serotypes. However, prior influenza infection induces heterosubtypic immunity that accelerates viral clearance of a second strain, even if the external proteins are distinct. In mice, cross-protection can also be elicited by systemic immunization with the highly conserved internal nucleoprotein (NP). Both T lymphocytes and Ab contribute to such cross-protection. In this paper, we demonstrate that anti-NP IgG specifically promoted influenza virus clearance in mice by using a mechanism involving both FcRs and CD8(+) cells. Furthermore, anti-NP IgG rescued poor heterosubtypic immunity in B cell-deficient mice, correlating with enhanced NP-specific CD8 T cell responses. Thus, Ab against this conserved Ag has potent antiviral activity both in naive and in influenza-immune subjects. Such antiviral activity was not seen when mice were vaccinated with another internal influenza protein, nonstructural 1. The high conservation of NP Ag and the known longevity of Ab responses suggest that anti-NP IgG may provide a critically needed component of a universal influenza vaccine.

FIGURE 1

Diversified Ab is required for Het-I. A, Experimental design. The indicated mice were infected i.n. with 0.25 LD₅₀ influenza X31 (H3N2) on day 0 and challenged on day 30 with 2.5 LD₅₀ influenza PR8 (H1N1). B, Lung viral load at the indicated day post-PR8 challenge. **p = 0.004 versus C57BL/6, ***p < 0.0001 versus C57BL/6 by Student t test; n = 5 mice/group/time point. C, NP-specific CD8 T cells in the lung on day 6 post-PR8 challenge. n.s., not significant by Mann–Whitney U test.

FIGURE 2

NP-reactive IgG response during X31 priming infection. C57BL/6 mice were treated i.n. with either sterile PBS or 0.25 LD₅₀ influenza X31 virus. At day 10, serum and spleen were collected for analysis. A–C, Serum IgG analysis by ELISA. A, Total serum IgG. B, NP-reactive serum IgG. Quantitative estimation in micrograms per milliliter was determined by comparing signal from diluted serum with that of a known monoclonal anti-NP IgG2a. C, Anti-NP IgG as a percentage of total serum IgG, based on values from A and B. D–F, Spleen ASC measured by ELISPOT analysis. D, Total IgG ASC in spleen. E, NP-reactive ASC in spleen. F, Anti-NP ASC as a percentage of total splenic IgG ASC.

FIGURE 3

Anti-NP IgG has antiviral activity. B cell-deficient μMT mice were injected i.p. with PBS alone, 400 μl LPS-immune serum or NP/LPS-immune serum (prepared as described in Materials and Methods), or with 300 μg each NP-specific IgG1, IgG2a, and IgG2b 1 d before i.n. infection with 0.13 LD₅₀ influenza PR8. Shown are lung viral titers on day 10 postinfection. The p values calculated by Student t test. Representative of at least three similar experiments.

FIGURE 4

Anti-NP IgG cooperates with existing influenza immunity. A, Experimental design. μMT mice were treated i.n. with either PBS (“naive”) or 0.25 LD₅₀ influenza X31 on day 0. Purified IgG was injected as in Fig. 2. Influenza PR8 was used at 2.5 LD₅₀ i.n. B, Lung viral titers of μMT recipient mice on the indicated day post-PR8 challenge. ***p < 0.0005 by Student t test. C, CD8⁺ T cells binding the indicated Db tetramer in the spleen of the indicated mice on day 5 (C) and day 6 (D) post-PR8 challenge. **p = 0.008 by Mann–Whitney U test. Representative of at least three similar experiments. BD, below the limit-of-detection.

FIGURE 5

CD8 depletion inhibits antiviral activity of NP-immune serum. μMT mice were treated with rat anti-KLH (“ctrl”) or anti-CD8 every other day beginning on day −1 as indicated in Materials and Methods. CD8 depletion was confirmed in representative lung samples, as well as in spleen and mediastinal lymph node. Serum (400 μl) from LPS- or from NP/LPS-immune C57BL/6 mice was transferred into μMT recipients 1 d prior to infection with 0.13 LD₅₀ PR8. Lung viral titers in μMT recipients on day 10 postinfection. The p values were calculated by Student t test.

FIGURE 6

NP exposure and involvement of FcR. A, C57BL/6 mice were infected with 0.25 LD₅₀ influenza PR8 or treated with PBS i.n. Bronchoalveolar lavage was collected at the indicated time points. ELISA plates coated with anti-NP IgG mAb were incubated with the indicated samples. NP was detected with biotinylated serum IgG purified from NP-immune mice. n = 2 mice per PBS and 3 PR8 mice per time point. Representative of three similar experiments. B, Purified mAb (300 μg each IgG1, IgG2a, and IgG2b) was transferred into the indicated recipients 1 d prior to infection with 0.13 LD₅₀ PR8. Lung viral titers on day 10 postinfection. Representative of two similar experiments. The p values calculated by Student t test. C, LPS- or NP-immune serum (400 μl) was injected i.p. into the indicated mice 1 d before i.n. infection with 0.13 LD₅₀ influenza PR8. D, Irradiated μMT and μMT/FcR^−/− mice were each reconstituted with μMT bone marrow (BM). After 8 wk, the chimeras were injected i.p. with a mixture of 300 μg of the indicated IgG1, IgG2a, and IgG2b. One day later, the mice were challenged i.n. with 0.13 LD₅₀ influenza PR8.

FIGURE 7

C57BL/6 mice were given 400-μl i.p. injections of LPS- or NP/LPS-immune serum according to the schedules indicated in A and D. B and E, Serum NP-reactive IgG in donors and recipients of immune and control serum. C and F, Day-8 lung viral titers in serum recipients. A–C, Mice injected beginning on day −1 relative to challenge with 0.25 LD₅₀ influenza PR8. D–F, Mice injected beginning on day −3 relative to challenge with 0.25 LD₅₀ influenza PR8.

FIGURE 8

Three hundred micrograms of mixed IgG1, IgG2a, and IgG2b control or anti-NP IgG mAb were injected i.p. into the indicated mice on days −3 to +1 relative to influenza infection. A, On day 0, recipient mice were challenged i.n. with 0.25 LD₅₀ influenza PR8, and lungs were assayed for virus titers on day 8. B, On day 0, mice were challenged i.n. with 2.5 LD₅₀ PR8. On day 7 postinfection, lungs were assayed for viral titers. Each experiment was performed twice with similar results.