A novel role for non-neutralizing antibodies against nucleoprotein in facilitating resistance to influenza virus

Abstract

Current influenza vaccines elicit Abs to the hemagglutinin and neuraminidase envelope proteins. Due to antigenic drift, these vaccines must be reformulated annually to include the envelope proteins predicted to dominate in the following season. By contrast, vaccination with the conserved nucleoprotein (NP) elicits immunity against multiple serotypes (heterosubtypic immunity). NP vaccination is generally thought to convey protection primarily via CD8 effector mechanisms. However, significant titers of anti-NP Abs are also induced, yet the involvement of Abs in protection has largely been disregarded. To investigate how Ab responses might contribute to heterosubtypic immunity, we vaccinated C57BL/6 mice with soluble rNP. This approach induced high titers of NP-specific serum Ab, but only poorly detectable NP-specific T cell responses. Nevertheless, rNP immunization significantly reduced morbidity and viral titers after influenza challenge. Importantly, Ab-deficient mice were not protected by this vaccination strategy. Furthermore, rNP-immune serum could transfer protection to naive hosts in an Ab-dependent manner. Therefore, Ab to conserved, internal viral proteins, such as NP, provides an unexpected, yet important mechanism of protection against influenza. These results suggest that vaccines designed to elicit optimal heterosubtypic immunity to influenza should promote both Ab and T cell responses to conserved internal proteins.

Figure 1. Immunization with purified rNP protects C57BL/6 mice from influenza-induced morbidity and reduces viral titers

(A) 500 ng of affinity-purified, sterile-filtered C-terminal 6Xhistidine-tagged rNP was fractionated using SDS-PAGE under reducing conditions, and stained with Coomassie Blue. (B) C57BL/6 mice were vaccinated i.p. with 30 μg rNP and 20 μg LPS (ν) or with LPS alone (λ) on days 0 and 10. T cells before challenge (C) Sera from mice vaccinated as in panel B were assayed for NP-specific antibody by ELISA. (D) Vaccinated mice were challenged i.n. on day 40 with 500 EIU PR8. Mice were weighed prior to infection, and the change in body weight was calculated as % of initial weight. Mean ± S.D., 5 mice/group. (E) Mice were immunized and infected as in panel D. Lung viral titers were assayed 8 days after influenza challenge.

Figure 2. rNP immunization alters the kinetics of the CD8 T cell response after viral challenge

C57BL/6 mice were vaccinated intraperitoneally with 30 μg rNP and 20 μg LPS (ν) or with LPS alone (λ) on days 0 and 10, and challenged with 500 EIU PR8 on day 40. NP-specific (A) and PA-specific (B) CD8 T cells in the lung were measured by flow cytometry on the indicated day subsequent to challenge infection. NP-specific (C) and PA-specific (D) CD8 T cells were measured in the spleen. Mean ± S.D., 5 mice/group.

Figure 3. rNP-elicited protection from influenza requires CD40

(A) C57BL/6 mice were vaccinated i.p. with 30 μg rNP and 20 μg LPS (ν) or with LPS alone (□) on days 0 and 10. CD40^−/− mice were also vaccinated i.p. with 30 μg rNP and 20 μg LPS (λ) on days 0 and 10. All groups were challenged i.n. with 500 EIU PR8 on day 40, and relative body weights were determined. Mean ± S.D., 5 mice/group. (B) Viral titers were assayed in the lung at day 8 post-infection in mice vaccinated with LPS ± rNP, as indicated. Mean ± S.D., 5 mice/group. (C) NP-specific CD8 T cells in the lung were determined by flow cytometry on day 7 post-infection. (D) Serum titers of NP-specific IgM and IgG were measured by ELISA in vaccinated mice on day 39 (one day prior to infection). Mean ± S.D., 5 mice/group.

Figure 4. rNP vaccination-mediated reduction in viral titers requires antibody

C57BL/6 and antibody-deficient AID/μS mice were i.p.-immunized with either 30 μg rNP and 20 μg LPS or with LPS alone on days 0 and 10. All groups were subsequently challenged i.n. with 500 EIU PR8 on day 40. (A) Titers of NP-specific IgM and IgG were measured by ELISA in serum from mice at day 8 post influenza infection. Mean ± S.D., 5 mice/group. The low titers of anti-NP IgM in C57BL/6 mice are not consistently detected among experiments performed at this timepoint. (B) Lung viral titers were measured on day 8 after infection. (C) NP-specific CD8 T cells in the lung were determined by flow cytometry on day 8 post-infection.

Figure 5. rNP-immune serum protects μMT mice against influenza-induced morbidity

Serum was obtained on day 40 from C57BL/6 mice vaccinated with either 30 μg rNP and 20 μg LPS (ν) or with LPS alone (λ) at days 0 and 10. 200 μl sera were injected i.p. into naïve μMT mice 1d prior to influenza PR8 infection (500 EIU). (A) Mice were weighed daily and relative body weights were determined. Mean ± S.D., 5 mice/group. (B) Lung viral titers were determined on day 10 post-infection. (C) Flow cytometry of lung cells was performed on day 8 post-infection. Values shown are percentage of the CD8⁺ gate. (D) Kinetics of the NP-specific CD8 T cell response in the spleen. Mean ± S.D., 5 mice/group. The total number of NP-specific CD8 T cells per spleen was calculated from flow cytometry data as in panel C.

Figure 6. Protection of μMT mice by transfer of rNP-immune serum requires antibody

(A) Serum was obtained from rNP/LPS-vaccinated C57BL/6 mice (ν), rNP/LPS-vaccinated AID/μS mice (σ), and LPS-vaccinated C57BL/6 mice (λ) 40 days post-vaccination. 200 μl of serum were transferred to μMT mice 1 day prior to i.n. challenge with 500 EIU influenza PR8. (A) Weight loss was measured daily and relative body weights were determined. Mean ± S.D., 5 mice/group. (B) Viral titers were measured in the lungs of the recipient mice on d10 post- infection.