Critical Role of Perforin-dependent CD8+ T Cell Immunity for Rapid Protective Vaccination in a Murine Model for Human Smallpox

Kremer, Melanie; Suezer, Yasemin; Volz, Asisa; Frenz, Theresa; Majzoub, Monir; Hanschmann, Kay-Martin; Lehmann, Michael H.; Kalinke, Ulrich; Sutter, Gerd

Previous immunization experiments in T and B cell deficient RAG-1 2/2 mice had already indicated that adaptive responses are indispensible to achieving rapidly protective immunity [41, 42] . We also suspected a key role of humoral immunity in rapidly protective immunization, since MVA can induce antibody responses much faster than conventional VACV [39, 41] . Moreover vaccinia immune globulin is effective in post-exposure treatment of lethal orthopoxvirus infections [28, 53, 79] . However surprisingly, we found that vaccinated B cell-deficient mice were still fully protected. MVA immunization prevented the onset of any detectable disease in B cell deficient animals for at least four weeks following respiratory challenge infection. This is remarkable because in this intranasal infection model (at low dosage of 200 PFU ECTV) normal C57BL/6 mice (not vaccinated) suffer from severe systemic mousepox and succumb within 10 to 14 days after challenge [80] . It is worth noting, however, that ECTV can persist for several months without any signs of disease in naïve C57BL/6 mice following footpad inoculation and, in infected B cell deficient animals, the onset of symptoms must not occur until very late in infection [72, 73] . Yet, on the contrary, depletion of CD4+ or/and CD8+ T cells in C57BL/6 mice completely abrogated the protective capacity of immunization against the respiratory ECTV challenge. Moreover, the need for T cellmediated immunity was underlined by the transfer of naïve CD3+ T cells into RAG-1 2/2 mice, which supported protective vaccination of these immunocompromised animals against lethal ECTV challenge. Furthermore, depletion of CD4+ T cells was sufficient to inhibit the protective effect of MVA immunization, although we observed clearly delayed onset of morbidity. This observation may be best explained by an essential role of T helper cells in mediating efficient clearance of virus by CD8+ T cell activity. Importantly, this possibility is clearly supported by our demonstration that depletion of CD4+ T cells significantly reduced the in vivo expansion of endogenous VACV-specific CD8+ T cells. Similarly, CD4+ T cells have been found essential for maintaining a robust or protective cytotoxic T cell memory response upon vaccination with recombinant VACV expressing lymphocytic choriomeningitis virus glycoprotein, or upon infection of mice with Listeria monocytogenes bacteria [81, 82, 83] The possibility that clearance of ECTV cannot be accomplished because of the absence of T helper cell dependent antibody responses appears unlikely in the view of the fact that CD4depleted animals still mounted substantial levels of VACV-specific antibodies. Moreover, we clearly demonstrated the essential need for the direct cytotoxic effector function of CD8+ T cells to mediate rapid protection as the absence of perforin completely abrogated the protective capacity of immunization. Nonetheless, we observed reduced levels of ECTV also in the livers of vaccinated Prf2/2 mice indicating that MVA induced innate responses might have modulated the course of infection. This hypothesis is in agreement with previous findings of an early enhanced production of chemokines and cytokines after in vivo inoculation of MVA but not other strains of VACV [46, 84] .