We next determined the mechanism(s) responsible for effective vaccination. We vaccinated mice lacking both major histocompatibility complex (MHC) Class I and b2 microglobulin (b2M) [43] (CD8 T cells deficiency [44] ), MHC Class II (CD4 T cells deficiency [45] ), or B cell deficient mice [46] ( Figure 3A) . These experiments were conducted concurrently with the experiments in Figure 2 above, as such the data from WT mice are repeated in the figure for comparison. Live MNV vaccination induced significant protection against MNV challenge in both the distal ileum and the MLN of B cell-/-, MHC Class II-/-and MHC Class I6b2M-/-mice (p,0.05 in all cases, Figure 3B and 3C). However, there was considerable variation in the efficacy of vaccination in distal ileum and MLN between different immunodeficient strains. In B cell-/-mice, after vaccination with live virus, only 2 out of 15 mice had any titer (and those two mice had less than 100 PFU of MNV) and in MHC Class I6b2M-/-mice, similar vaccination led to undetectable viral titers in the distal ileum ( Figure 3B ) but detectable titers in the MLN ( Figure 3C ). In MHC Class II-/-mice, there were detectable titers in both tissues ( Figure 3B and 3C). Results for ORF2 vaccination showed that protection required the activity of all major aspects of the adaptive immune response ( Figure 3B and 3C). Moreover, there was no protection elicited by ORF2 vaccination in either intestine or MLN tissue after vaccination of MHC Class I6b2M-/-mice with ORF2 VRPs (Figure 3B and 3C) indicating that protection by VRPs critically depends on CD8 T cells. These data demonstrated that complete protection in all tissues after vaccination with live virus required the concerted actions of B cells, MHC Class II, MHC Class I and b2M. Further, the results were consistent with tissue specific roles for B cells, CD4 T cells and CD8 T cells in the development of complete protection against MNV infection.