(C) Flow cytometry of lung cells was performed on day 8 post-infection
(C) Flow cytometry of lung cells was performed on day 8 post-infection. a soluble protein. This approach induced high titers of NP-specific serum antibody, but only poorly detectable NP-specific T cell responses. Nevertheless, rNP immunization was effective at reducing morbidity and reducing viral BKI-1369 titers after challenge with influenza virus. Importantly, antibody-deficient mice were not protected by this vaccination strategy. Furthermore, rNP-immune serum could transfer these protective effects to BKI-1369 na?ve hosts in an antibody-dependent manner. Therefore, antibody is essential for rNP-immune protection, strongly suggesting that NP-specific antibody can convey immunity to influenza virus. Thus, antibody to conserved, internal viral proteins, such as NP can provide an important mechanism of protection that may be utilized together with cytoxic T cells to elicit heterosubtypic immunity by future vaccines. INTRODUCTION Influenza virus causes acute respiratory illness that leads to ~94,000 hospitalizations (1) and 36,000 deaths annually in the United States (2). Vaccines against influenza have been available for many years, and are often highly effective at preventing infection as well as reducing morbidity and mortality associated with seasonal influenza outbreaks. Current vaccines are designed to elicit antibodies directed against the external glycoproteins of influenza: hemagglutinin (HA) and neuraminidase (NA). Neutralizing anti-HA antibodies prevent influenza virus infection of cultured epithelial cells (neutralization) and can passively protect mice from infection (3, 4). In fact, neutralizing antibody titers are considered to be the gold-standard correlate of vaccine-induced immunity, and are presumed to provide the mechanism for vaccine-induced protection (5C7). Despite the efficacy of neutralizing antibodies, their utility is limited, as they only protect against viral serotypes that express the same HA and NA proteins contained in the vaccine. Because mutations rapidly accumulate in the HA and NA proteins of influenza virus, particularly in the epitopes recognized by neutralizing antibodies, influenza vaccines must be reformulated each year to include the HA and NA proteins predicted to dominate in the following influenza season. Consequently, generating annual vaccines is cumbersome and costly, and if serotypes are not accurately predicted, the resulting immunity may not be very effective. By contrast, vaccines that elicit immunity to conserved, often internal viral proteins, such as nucleoprotein (NP), provide some protection from multiple strains and subtypes of influenza virus. For example, mice vaccinated with influenza NP (as purified protein or using DNA expression vectors) have higher frequencies of NP-specific CD8 T cells before infection, as well as lower viral titers after challenge with H3N2 and H1N1 strains of influenza. This vaccination also protects from virus-induced lethality (8C13), including lethality induced by highly pathogenic H5N1 human isolates (14). T cell responses to conserved epitopes in these proteins are thought to be the main mechanism of protection, because restimulated T cells can transfer protection to na?ve mice (15, 16), and because T cell BKI-1369 depletion in the vaccinated mice can abrogate protection (14, 15). As a result, many investigations have focused on targeting antigens to the MHC class I pathway (e.g., using DNA-based vectors) to elicit CD8 T cell responses. Although ETV4 CD4 and CD8 T cells can each contribute to protection elicited by vaccination with BKI-1369 NP, T cells appear to be dispensable in some situations (13, 17), suggesting that other mechanisms, such as antibody production, may also contribute. Both natural infection with influenza virus and vaccination with recombinant NP elicit NP-specific antibodies (18, 19). However, anti-NP antibodies were considered to be ineffective because they do not neutralize virus, and because passive transfer of such antibodies do not protect na?ve immunodeficient recipient mice (4). However, it has recently been shown that immune complexes formed with anti-NP monoclonal antibodies can promote dendritic cell maturation, Th1 cytokine production, and anti-influenza CD8+ CTL responses in na?ve immunocompetent recipients (20). Additionally, anti-NP IgG can stimulate complement-mediated lysis of infected P815 mastocytoma cells ?/?) 102:553 with mice lacking the secretory form of IgM (?/? mice) JI 160:4776. Because ?/? mice cannot isotype switch their antibody genes, and mice have B cells, but cannot secrete antibody of any isotype. We vaccinated C57BL/6 mice and mice with rNP/LPS or with LPS alone, challenged them with influenza virus on day 40. Figure 4A shows that, even after vaccination and influenza infection, mice do not generate any NP-specific antibodies compared to vaccinated and infected C57BL/6 mice. As observed earlier, rNP-immune C57BL/6 mice had BKI-1369 significantly lower viral titers than LPS-vaccinated controls on day 8 post-infection (Fig. 4B). However, rNP-immune, antibody-deficient mice had viral titers that were as high as those in LPS-vaccinated control mice. Importantly, rNP-immune mice still had an enhanced NP-specific CD8 T cell response that was still detectable.