We undertook this study with the goal of determining which cytokines induced by rVSV contribute to acute pathology after intramuscular immunization. This is important because while VSV is a highly promising vaccine vector and oncolytic agent, its clinical development has lagged behind that of other live viruses because of concerns about vector-associated pathology. Fever, myalgia, and the ''sickness response'' are induced by many live viral or bacterial vaccines [32, 33, 34, 35, 36] , and these vaccineinduced side effects are among the leading reasons why some individuals elect not to receive protective vaccines [37, 38, 39] . Consistent with our results obtained in the mouse model and presented here, several recent studies have correlated increased levels of IL-1 or other pro-inflammatory cytokines with the induction of high fevers or other adverse events in response to live There was no significant difference in viral loads between the two groups. In Panels B-D, adult female wild type (n = 5) or IL-1R2/2 (n = 4) mice were immunized intramuscularly with a single injection of 5610 8 PFU rVSV in the rear quadriceps. At the indicated timepoints after infection mice were bled and humoral and cellular immune responses were assayed. Panel B shows average anti-VSV neutralizing antibody responses by group as measured by microneutralization assay. Error bars represent the upper and lower limits of the 95% confidence interval. Panel C shows average percent CD8 T cells specific for the VSV N1 epitope as measured by MHC Class I tetramer. At 14 days after immunization, WT mice had significantly more (P = 0.03, Twotailed T test) VSV N specific CD8 T cells than IL-1R2/2 mice, but by day 28 the difference was no longer significant (P = 0.13). At eight weeks after the primary infection all mice were challenged intranasally with a semi-lethal dose of rVSV (1610 8 PFU). A cohort of naïve wild type mice (n = 7) was challenged at the same time. All pre-immune mice had robust immunity to rechallenge (Panel D) and did not lose weight or exhibit other signs of pathology. Two of the naïve mice succumbed to infection. Days on which naïve animals succumbed are indicated with an asterisk on the graph. doi:10.1371/journal.pone.0046516.g003 Figure 4 . Mice deficient in the inflammasome adaptor ASC (ASC2/2) are partially protected from acute weight loss after intramuscular immunization with rVSV. Groups of adult female C57BL/6 wild type or ASC2/2 mice were immunized with two injections (5610 8 PFU per injection) of rVSV in each rear quadriceps, or were sham inoculated with sterile PBS. At 12 and 24 hours after infection mice (n = at least 4 per timepoint), were sacrificed and IL-1b in the blood (Panel A left), draining popliteal LN (Panel A middle), and quadriceps muscle (Panel A right) was quantitated via ELISA. The amount of IL-1b produced by wild type and ASC2/2 mice was not significantly different at any time or in any organ. The comparison of IL-1b production by wild type and ASC2/2 mice has been performed twice with consistent results. Panel B shows average percent initial weight for wild type (n = 6) and ASC2/2 (n = 5) mice after intramuscular challenge with 5610 8 PFU of rVSV. The comparison of WT and ASC2/2 mice has been performed four times with consistent results. Panel C shows average percent initial weight for wild type (n = 5), ASC2/2 (n = 5), and IL-1R2/2 (n = 4) mice infected with rVSV. IL-1R2/2 mice lost significantly less weight than wild type (days 2-4) or ASC2/2 mice (days 1-2) (P,0.05 via one way ANOVA with Bonferroni test). The comparison of WT, IL-1R2/2, and ASC2/2 mice has been performed twice with consistent results. Panel D shows average serum neutralizing titers for WT (n = ) and ASC2/2 (n = ) mice after primary immunization with VSV. There were no significant differences in neutralizing titer between the two groups. Panel E shows average percent CD8 T cells specific for the VSV N1 epitope as measured by MHC Class I tetramer. At 14 days after immunization, WT mice (n = 9) had significantly more (P = 0.001, Two-tailed T test) VSV N specific CD8 T cells than ASC2/2 mice (n = 9), but by day 28 the difference was no longer significant (P = 0.07). At eight weeks after the primary infection preimmune WT (n = 10) and ASC2/2 (n = 6) mice were challenged intranasally with a semi-lethal dose of rVSV (1610 8 PFU). A cohort of naïve wild type mice (n = 5) was challenged at the same time. All pre-immune mice had robust immunity to rechallenge (Panel F) and did not lose weight or exhibit other signs of pathology. One of the naïve mice succumbed to infection. doi:10.1371/journal.pone.0046516.g004 Figure 5 . Mice deficient in caspase-1 are partially protected from acute weight loss after intramuscular immunization. Groups of adult C57BL/6 wild type or caspase 12/2 mice were immunized with two injections (5610 8 PFU per injection) of rVSV in each rear quadriceps, or were sham inoculated with sterile PBS. At 24 hours after infection mice (n = 2-3 mice per timepoint), were sacrificed and IL-1b in the draining popliteal LN (Panel A left) and quadriceps muscle (Panel A right) was quantitated via ELISA. The amount of IL-1b produced by wild type and caspase 12/2 mice was not significantly different in either organ. The comparison of IL-1b production by wild type and caspase 12/2 mice has been performed twice with consistent results. Panel B shows average percent initial weight for wild type and caspase 12/2 mice (n = 5 per group) after intramuscular challenge with 5610 8 PFU of rVSV. The comparison of WT and caspase 12/2 mice has been performed twice with consistent results. Caspase 12/2 mice lost significantly less weight than wild type controls on days 2 and 3 after challenge (P,0.05 via Mann Whitney test). Panel C shows average viral loads in the quadriceps muscle of infected mice (n = 6 per group). Data is compiled from two identically performed experiments. Viral loads in WT and caspase 12/2 mice were not significantly different. Panel D shows average serum neutralizing antibody titers for wild type and caspase 12/2 mice immunized with rVSV (n = 5 per group per timepoint). Error bars represent the upper and lower limits of the 95% confidence interval. Panel E shows average percent 6 SEM of CD8 T cells in the blood binding to an MHC Class I tetramer recognizing an immunodominant epitope within VSV N. There were no significant differences in the antibody or CD8 T cell responses between the two groups at any time. The comparison of humoral and cellular immune responses in WT and caspase 12/2 mice has been performed twice with consistent results. doi:10.1371/journal.pone.0046516.g005 virus vaccination in humans [36, 40] , or have identified genetic polymorphisms in the IL-1 gene which predispose individuals to severe adverse events after receiving live virus vaccines [41] . For these reasons it is important to determine the ways in which proinflammatory cytokines contribute to reactogenicity of VSV vectors in particular, not only because those findings could advance development of VSV-based therapeutics, but also because our findings might help to inform the development of other live virus vaccines and oncotherapies.