Protective CD4+ and CD8+ T Cells against Influenza Virus Induced by Vaccination with Nucleoprotein DNA

DNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745–1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4⁺ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8⁺ T cells and cytokine-secreting CD4⁺ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.Cellular immune responses play an important role in protection from disease caused by infectious pathogens, such as viruses and certain bacteria (e.g., Mycobacterium tuberculosis). The specific T cells involved in conferring immunity can include both CD4⁺ and CD8⁺ T cells, often through the action of secreted cytokines and cytolytic activity, respectively. Certain types of vaccines, such as subunit proteins and whole or partially purified preparations of inactivated organisms, in general induce CD4⁺ T-cell responses but not CD8⁺ cytotoxic T lymphocytes (CTL). In contrast, live attenuated organisms and subunit proteins formulated with certain experimental adjuvants can induce both types of responses. Recently, a different approach consisting of direct immunization with plasmid DNA expression vectors (i.e., DNA vaccines) has shown promise as a viable means of inducing broad-spectrum T-cell responses. The effectiveness of DNA vaccines in animal models is likely due, at least in part, to expression of antigens in situ (35), leading to the induction of CTL (29), antibodies (3, 4, 10, 21, 22, 32), and cytokine-secreting lymphocyte responses (12, 36). During the past 5 years, many reports have been published on the immunogenicity of DNA vaccines encoding various antigens in several animal models, thereby illustrating the applicability of the technology to many pathogens (for a review, see reference 6). However, in only a few instances has the nature of the effector cells responsible for protective immunity been described (7, 16). In the present study, we have analyzed in detail the cellular immune responses induced by influenza virus nucleoprotein (NP) DNA and have established that both CD4⁺ T cells secreting Th1-type cytokines and CD8⁺ cytotoxic T cells play important effector roles in heterosubtypic protective immunity against lethal influenza virus challenge in mice.