Interferons (IFNs) induce antiviral activity in many cell types. The ability of IFN-γ to inhibit replication of ectromelia, vaccinia, and herpes simplex-1 viruses in mouse macrophages correlated with the cells' production of nitric oxide (NO). Viral replication was restored in IFN-γ-treated macrophages exposed to inhibitors of NO synthase. Conversely, epithelial cells with no detectable NO synthesis restricted viral replication when transfected with a complementary DNA encoding inducible NO synthase or treated with organic compounds that generate NO. In mice, an inhibitor of NO synthase converted resolving ectromelia virus infection into fulminant mousepox. Thus, induction of NO synthase can be necessary and sufficient for a substantial antiviral effect of IFN-γ.

The large size of poxvirus genomes has stymied attempts to identify determinants recognized by CD8⁺ T cells and greatly impeded development of mouse smallpox vaccination models. Here, we use a vaccinia virus (VACV) expression library containing each of the predicted 258 open reading frames to identify five peptide determinants that account for approximately half of the VACV-specific CD8⁺ T cell response in C57BL/6 mice. We show that the primary immunodominance hierarchy is greatly affected by the route of VACV infection and the poxvirus strain used. Modified vaccinia virus ankara (MVA), a candidate replacement smallpox vaccine, failed to induce responses to two of the defined determinants. This could not be predicted by genomic comparison of viruses and is not due strictly to limited MVA replication in mice. Several determinants are immunogenic in cowpox and ectromelia (mousepox) virus infections, and immunization with the immunodominant determinant provided significant protection against lethal mousepox. These findings have important implications for understanding poxvirus immunity in animal models and bench-marking immune responses to poxvirus vaccines in humans.

Parasite burden predicts disease severity in malaria and risk of death in cerebral malaria patients. In murine experimental cerebral malaria (ECM), parasite burden and CD8⁺ T cells promote disease by mechanisms that are not fully understood. We found that the majority of brain-recruited CD8⁺ T cells expressed granzyme B (GzmB). Furthermore, gzmB^−/− mice harbored reduced parasite numbers in the brain as a consequence of enhanced antiparasitic CD4⁺ T cell responses and were protected from ECM. We showed in these ECM-resistant mice that adoptively transferred, Ag-specific CD8⁺ T cells migrated to the brain, but did not induce ECM until a critical Ag threshold was reached. ECM induction was exquisitely dependent on Ag-specific CD8⁺ T cell-derived perforin and GzmB, but not IFN-γ. In wild-type mice, full activation of brain-recruited CD8⁺ T cells also depended on a critical number of parasites in this tissue, which in turn, was sustained by these tissue-recruited cells. Thus, an interdependent relationship between parasite burden and CD8⁺ T cells dictates the onset of perforin/GzmB-mediated ECM.

Viral infection often activates the interferon (IFN)-γ–inducible gene, nitric oxide synthase 2 (NOS2). Expression of NOS2 can limit viral growth but may also suppress the immune system and damage tissue. This study assessed each of these effects in genetically deficient NOS2^−/− mice after infection with influenza A, a virus against which IFN-γ has no known activity. At inocula sufficient to cause consolidating pneumonitis and death in wild-type control mice, NOS2^−/− hosts survived with little histopathologic evidence of pneumonitis. Moreover, they cleared influenza A virus from their lungs by an IFN-γ–dependent mechanism that was not evident in wild-type mice. Even when the IFN-γ–mediated antiviral activity was blocked in NOS2^−/− mice with anti–IFN-γ mAb, such mice failed to succumb to disease. Further evidence that this protection was independent of viral load was provided by treating NOS2^+/+ mice with the NOS inhibitor, N^ω-methyl-l-arginine (l-NMA). l-NMA prevented mortality without affecting viral growth. Thus, host NOS2 seems to contribute more significantly to the development of influenza pneumonitis in mice than the cytopathic effects of viral replication. Although NOS2 mediates some antiviral effects of IFN-γ, during influenza infection it can suppress another IFN-γ–dependent antiviral mechanism. This mechanism was observed only in the complete absence of NOS2 activity and appeared sufficient to control influenza A virus growth in the absence of changes in cytotoxic T lymphocyte activity.