Regulation of antinucleoprotein IgG by systemic vaccination and its effect on influenza virus clearance

Abstract

Seasonal influenza epidemics recur due to antigenic drift of envelope glycoprotein antigens and immune evasion of circulating viruses. Additionally, antigenic shift can lead to influenza pandemics. Thus, a universal vaccine that protects against multiple influenza virus strains could alleviate the continuing impact of this virus on human health. In mice, accelerated clearance of a new viral strain (cross-protection) can be elicited by prior infection (heterosubtypic immunity) or by immunization with the highly conserved internal nucleoprotein (NP). Both heterosubtypic immunity and NP-immune protection require antibody production. Here, we show that systemic immunization with NP readily accelerated clearance of a 2009 pandemic H1N1 influenza virus isolate in an antibody-dependent manner. However, human immunization with trivalent inactivated influenza virus vaccine (TIV) only rarely and modestly boosted existing levels of anti-NP IgG. Similar results were observed in mice, although the reaction could be enhanced with adjuvants, by adjusting the stoichiometry among NP and other vaccine components, and by increasing the interval between TIV prime and boost. Importantly, mouse heterosubtypic immunity that had waned over several months could be enhanced by injecting purified anti-NP IgG or by boosting with NP protein, correlating with a long-lived increase in anti-NP antibody titers. Thus, current immunization strategies poorly induce NP-immune antibody that is nonetheless capable of contributing to long-lived cross-protection. The high conservation of NP antigen and the known longevity of antibody responses suggest that the antiviral activity of anti-NP IgG may provide a critically needed component of a universal influenza vaccine.

Fig. 1.

NP-immune antibody promotes pandemic H1N1 influenza virus clearance. The indicated mice were immunized i.p. with 30 μg LPS with or without 50 μg rNP (PR8 sequence) on days 0, 10, 20, and 30. (A) NP-reactive serum IgG on day 123 postprime. Mean ± standard deviation of 5 mice/group. (B) On day 124, the mice were challenged i.n. with 2.5 LD₅₀ of pandemic H1N1 influenza virus A/CA/04/2009. Shown are lung viral titers at day 8 postinfection. (C) Body weights on the indicated days postinfection. Mean ± standard deviation of 5 mice/group.

Fig. 2.

NP content of 2008/09 TIV. (A) Capture ELISA to measure NP in TIV as described in Materials and Methods. (B) Western blot analysis of the indicated μg rNP compared with the indicated μl TIV as described in Materials and Methods.

Fig. 3.

TIV rarely changes anti-NP IgG titers in humans. Samples were collected from healthy human subjects on the indicated days relative to i.m. vaccination with TIV. NP-reactive IgG was detected by endpoint ELISA. (A and B) Study 1. Plasma from 64 subjects pre- and post-2008/09 TIV. (C and D) Study 2. Serum from 43 subjects pre- and post-2003/04 TIV. (A and C) Anti-NP IgG titers. (B and D) Fold change of anti-NP IgG titer from that at day 0 (prevaccination).

Fig. 4.

Anti-NP IgG response in humans does not consistently correlate with starting titer or with HAI response. (A) Regression analysis derived from human ELISA data in Fig. 3. (B) Fold change in anti-NP IgG in study 2 serum according to HAI response (left; individuals with ≥4-fold change in HAI titer) or no response (right), as well as age (in years). Bars show median fold changes for each group.

Fig. 5.

Poor induction of anti-NP IgG in TIV-immunized mice. Influenza virus-naive C57BL/6 mice were injected i.p. as indicated on days 0 and 10. Sera were collected at day 26 and analyzed by endpoint ELISA. rNP/LPS, 50 μg rNP plus 20 μg LPS. TIV was unadjuvanted and dosed as μg HA equivalent (HA eq). (A) IgG reactive with total TIV protein. (B) NP-reactive IgG. Results representative of at least 3 similar experiments. Mean ± standard deviation of 5 mice/group.

Fig. 6.

Adjuvanting TIV can enhance anti-NP IgG responses in mice. (A) Influenza virus-naive C57BL/6 mice were immunized i.p. with 100 μg rNP and 20 μg LPS or with 5 μg HA eq of 2008/09 TIV with or without 20 μg LPS on days 0, 10, and 20. Serum was collected for analysis on day 27. (B) C57BL/6 mice were infected i.n. with 0.25 LD₅₀ of influenza virus X31. On day 30 postinfection, the mice were immunized i.p. with 20 μg LPS, 1 μg HA eq of TIV, or both. Similar results were observed when 10 μg HA eq of TIV was used. Mean ± standard deviation of 3 to 5 mice/group.

Fig. 7.

The stoichiometry of NP and other vaccine components influences anti-NP IgG responses to TIV. Influenza virus-naive C57BL/6 mice were injected i.p. as indicated on days 0 and 10. Sera were collected at day 34 and analyzed by endpoint ELISA. (A) Mice injected with 30 μg rNP plus 20 μg LPS with or without the indicated HA eq doses of 2008/09 TIV. (B) Mice injected with 100 μg rNP plus 20 μg LPS with or without the indicated HA eq doses of 2008/09 TIV. Results representative of 2 similar experiments.

Fig. 8.

A longer prime-boost interval enhances anti-NP IgG response to TIV in mice. Influenza virus-naive C57BL/6 mice were immunized i.p. with PBS alone or with 10 μg HA eq of TIV on day 0. On the indicated days, different groups of TIV-primed mice were boosted with TIV. Sera were collected on day 50 for ELISA. Results representative of 2 similar experiments.

Fig. 9.

Protein boosting increases anti-NP IgG and enhances long-term Het-I. (A) Experimental design. C57BL/6 mice were infected i.n. with 0.25 LD₅₀ of influenza virus X31 or PBS alone on day 0. On day 90, half of the X31-immune mice were boosted with 100 μg rNP i.p. On day 200, all mice were challenged with 2.5 LD₅₀ of influenza virus PR8 i.n. (Not shown is that sera were collected at regular intervals prior to PR8 challenge.) (B) NP-reactive IgG in mouse sera prior to PR8 challenge. Mean and standard deviation of 5 mice/group. (C) Lung viral load at the indicated day post-PR8 challenge. *, P = 0.01; ***, P < 0.0001. Results representative of 3 similar experiments.

Fig. 10.

Anti-NP IgG enhances long-term Het-I. C57BL/6 mice were infected with 0.25 LD₅₀ of influenza virus H3N2 X31 as in Fig. 9. On days 239 to 242, the indicated mice were injected i.p. with PBS, with the indicated serum (A and B), or with the indicated MAb (C and D). All mice were infected i.n. on day 241 with 2.5 LD₅₀ of influenza virus H1N1 PR8 and then bled on day 243 (1 day after last injection). (A and B) Mice injected with 400 μl of the indicated immune sera. (A) NP-reactive IgG in recipients' sera. (B) Lung viral titer on day 247 (6 days post-PR8 challenge). (C and D) Mice injected with PBS or a mixture of 300 μg anti-NP IgG1, IgG2a, and IgG2b (striped bar in panel C and filled squares in panel D) during PR8 challenge. The black bar in panel C and filled circles in panel D indicate an additional group of mice that were instead previously boosted on day 90 with 100 μg purified rNP. (C) Serum anti-NP IgG on day 243. (D) Lung viral titer on day 247 (6 days post-PR8 challenge).

Fig. 11.

NP-specific IgG protects long-term heterosubtypic immune mice. C57BL/6 mice were infected with 0.25 LD₅₀ of H1N1 influenza virus PR8 i.n. on day 0. On days 631 to 634, the mice were injected with control (ctrl) or anti-NP IgG (300-μg mixtures of IgG1, IgG2a, and IgG2b) i.p. On day 633, the mice were challenged i.n. with 2.5 LD₅₀ of H3N2 influenza virus X31 i.n. (A) Serum NP-reactive IgG on day 635 (1 day after last injection). Mean ± standard deviation of 5 mice/group. (B) Relative body weights on indicated day postchallenge. Mean ± standard deviation of 5 mice/group. (C) Survival on indicated day postchallenge. Mice losing 30% of body weight were humanely euthanized and scored as nonsurvivors.