Comparison of multiple vaccine vectors in a single heterologous prime-boost trial

The prevention of infectious disease via prophylactic immunization is a mainstay of global public health efforts. Vaccine design would be facilitated by a better understanding of the type and durability of immune responses generated by different vaccine vectors. We report here the results of a comparative immunogenicity trial of six different vaccine vectors expressing the same insert antigen, cowpox virus B5 (CPXV-B5). Of those vectors tested, recombinant adenovirus (rAd5) was the most immunogenic, inducing the highest titer anti-B5 antibodies and conferring protection from sublethal vaccinia virus challenge in mice after a single immunization. We tested select heterologous prime-boost combinations and identified recombinant vesicular stomatitis virus (rVSV) and recombinant Venezuelan equine encephalitis virus replicons (VRP) as the most synergistic regimen. Comparative data such as those presented here are critical to efforts to generate protective vaccines for emerging infectious diseases as well as for biothreat agents

NLRX1 Sequesters STING to Negatively Regulate the Interferon Response, Thereby Facilitating the Replication of HIV-1 and DNA Viruses

SummaryUnderstanding the negative regulators of antiviral immune responses will be critical for advancing immune-modulated antiviral strategies. NLRX1, an NLR protein that negatively regulates innate immunity, was previously identified in an unbiased siRNA screen as required for HIV infection. We find that NLRX1 depletion results in impaired nuclear import of HIV-1 DNA in human monocytic cells. Additionally, NLRX1 was observed to reduce type-I interferon (IFN-I) and cytokines in response to HIV-1 reverse-transcribed DNA. NLRX1 sequesters the DNA-sensing adaptor STING from interaction with TANK-binding kinase 1 (TBK1), which is a requisite for IFN-1 induction in response to DNA. NLRX1-deficient cells generate an amplified STING-dependent host response to cytosolic DNA, c-di-GMP, cGAMP, HIV-1, and DNA viruses. Accordingly, Nlrx1−/− mice infected with DNA viruses exhibit enhanced innate immunity and reduced viral load. Thus, NLRX1 is a negative regulator of the host innate immune response to HIV-1 and DNA viruses

IL-1R−/− mice control VSV replication, make strong humoral and cellular immune responses, and are immune to rechallenge.

<p>Adult female wild type or IL-1R−/− (n = 3 per group) mice were immunized intramuscularly with a single injection of 5×10⁸ PFU rVSV in the rear quadriceps. Mice were sacrificed at 24 hours after infection and viral titers determined in the quadriceps muscle (Panel A). There was no significant difference in viral loads between the two groups. In Panels B–D, adult female wild type (n = 5) or IL-1R−/− (n = 4) mice were immunized intramuscularly with a single injection of 5×10⁸ 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, Two-tailed T test) VSV N specific CD8 T cells than IL-1R−/− 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 (1×10⁸ 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.</p

Mice deficient in caspase-1 are partially protected from acute weight loss after intramuscular immunization.

<p>Groups of adult C57BL/6 wild type or caspase 1−/− mice were immunized with two injections (5×10⁸ 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-1β in the draining popliteal LN (Panel A left) and quadriceps muscle (Panel A right) was quantitated via ELISA. The amount of IL-1β produced by wild type and caspase 1−/− mice was not significantly different in either organ. The comparison of IL-1β production by wild type and caspase 1−/− mice has been performed twice with consistent results. Panel B shows average percent initial weight for wild type and caspase 1−/− mice (n = 5 per group) after intramuscular challenge with 5×10⁸ PFU of rVSV. The comparison of WT and caspase 1−/− mice has been performed twice with consistent results. Caspase 1−/− 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 1−/− mice were not significantly different. Panel D shows average serum neutralizing antibody titers for wild type and caspase 1−/− 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 ± 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 1−/− mice has been performed twice with consistent results.</p

Wild type mice challenged with rVSV produce IL-1β locally and systemically.

<p>Groups of adult female C57BL/6 mice were immunized with two injections (5×10⁸ 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 = 4 per timepoint), were sacrificed and IL-1β in the blood (Panel A), draining popliteal LN (Panel B), and quadriceps muscle (Panel C) was quantitated via ELISA. Open and filled bars represent the 12 and 24 hour timepoints respectively. Dotted line on graphs shows the average amount of IL-1β detected in the blood and tissues of sham-inoculated mice (n = 4). This experiment has been performed twice with consistent results.</p

Mice deficient in the inflammasome adaptor ASC (ASC−/−) are partially protected from acute weight loss after intramuscular immunization with rVSV.

<p>Groups of adult female C57BL/6 wild type or ASC−/− mice were immunized with two injections (5×10⁸ 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-1β 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-1β produced by wild type and ASC−/− mice was not significantly different at any time or in any organ. The comparison of IL-1β production by wild type and ASC−/− mice has been performed twice with consistent results. Panel B shows average percent initial weight for wild type (n = 6) and ASC−/− (n = 5) mice after intramuscular challenge with 5×10⁸ PFU of rVSV. The comparison of WT and ASC−/− mice has been performed four times with consistent results. Panel C shows average percent initial weight for wild type (n = 5), ASC−/− (n = 5), and IL-1R−/− (n = 4) mice infected with rVSV. IL-1R−/− mice lost significantly less weight than wild type (days 2–4) or ASC−/− mice (days 1–2) (P<0.05 via one way ANOVA with Bonferroni test). The comparison of WT, IL-1R−/−, and ASC−/− mice has been performed twice with consistent results. Panel D shows average serum neutralizing titers for WT (n = ) and ASC−/− (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 ASC−/− mice (n = 9), but by day 28 the difference was no longer significant (P = 0.07). At eight weeks after the primary infection pre-immune WT (n = 10) and ASC−/− (n = 6) mice were challenged intranasally with a semi-lethal dose of rVSV (1×10⁸ 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.</p