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. 2004 Mar;78(6):2770-9.
doi: 10.1128/jvi.78.6.2770-2779.2004.

Induction of potent humoral and cell-mediated immune responses by attenuated vaccinia virus vectors with deleted serpin genes

Fatema A Legrand  1 , Paulo H Verardi, Leslie A Jones, Kenneth S Chan, Yue Peng, Tilahun D Yilma
Affiliations

Induction of potent humoral and cell-mediated immune responses by attenuated vaccinia virus vectors with deleted serpin genes

Fatema A Legrand et al. J Virol. 2004 Mar.

Abstract

Vaccinia virus (VV) has been effectively utilized as a live vaccine against smallpox as well as a vector for vaccine development and immunotherapy. Increasingly there is a need for a new generation of highly attenuated and efficacious VV vaccines, especially in light of the AIDS pandemic and the threat of global bioterrorism. We therefore developed recombinant VV (rVV) vaccines that are significantly attenuated and yet elicit potent humoral and cell-mediated immune responses. B13R (SPI-2) and B22R (SPI-1) are two VV immunomodulating genes with sequence homology to serine protease inhibitors (serpins) that possess antiapoptotic and anti-inflammatory properties. We constructed and characterized rVVs that have the B13R or B22R gene insertionally inactivated (vDeltaB13R and vDeltaB22R) and coexpress the vesicular stomatitis virus glycoprotein (v50DeltaB13R and v50DeltaB22R). Virulence studies with immunocompromised BALB/cBy nude mice indicated that B13R or B22R gene deletion decreases viral replication and significantly extends time of survival. Viral pathogenesis studies in immunocompetent CB6F(1) mice further demonstrated that B13R or B22R gene inactivation diminishes VV virulence, as measured by decreased levels of weight loss and limited viral spread. Finally, rVVs with B13R and B22R deleted elicited potent humoral, T-helper, and cytotoxic T-cell immune responses, revealing that the observed attenuation did not reduce immunogenicity. Therefore, inactivation of immunomodulating genes such as B13R or B22R represents a general method for enhancing the safety of rVV vaccines while maintaining a high level of immunogenicity. Such rVVs could serve as effective vectors for vaccine development and immunotherapy.

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Figures

FIG. 1.
FIG. 1.
Construction of rVVs with a deletion in the B13R or B22R gene. (A) The VV transfer vector p2B13R was generated by joining pUC18, pB13R1167, and p2B8R PCR fragments. (B) The VV transfer vector p2B22R was derived by joining pUC18, pB22R1150, and p2B8R PCR fragments. For both vectors, the two back-to-back synthetic VV promoters (dsP) are active in both early and late stages of infection. The multiple cloning sites adjacent to each side of the dsP facilitate cloning of heterologous genes (only unique sites are shown). (C) The WR strain of VV was used for the generation of recombinants with a deletion in the TK and B13R or B22R gene. The gpt gene was subcloned into the p2B13R and p2B22R transfer vectors, which were then used for the insertional inactivation and partial deletion of either the VV B13R or the B22R gene by homologous recombination. The gpt gene, utilized for rVV selection, is under one of the two dsPs, and the lacZ gene for β-galactosidase screening of rVVs is under the control of the VV P11 late promoter. v50, used as a parental virus, expresses VSV-G under the VV P7.5 promoter and is inserted into the TK site. The symbol Δ denotes insertional inactivation/deletion.
FIG. 2.
FIG. 2.
B22R and B13R gene inactivation does not affect VV in vitro replication. Monolayers of BS-C-1 cells were infected at an MOI of 0.01, and at the indicated time points, intracellular (pelleted cells) (A) and extracellular (B) viral fractions (cell supernatants) were collected and their titers were determined. The data shown represent the mean values from triplicate samples assayed in duplicate; error bars indicate standard deviation.
FIG. 3.
FIG. 3.
B13R and B22R gene function is inactivated in rVVs with deleted serpin genes. (A) B13R gene inactivation restores caspase-1 activity. To assess caspase-1 activity, HeLa S3 cells were infected with the various rVVs to induce apoptosis. UV-exposed HeLa S3 cells served as a positive control, and mock-infected HeLa S3 cells were used as a negative control. (B) B22R gene inactivation reduces plaque numbers by approximately fourfold in A549 cells. Plaques of rVV-infected BS-C-1 and A549 cell monolayers (20 PFU per well) were photographed at 36 h postinfection. (C and D) B22R gene inactivation reduces viral replication in A549 cells. Monolayers of A549 cells were infected at an MOI of 0.01, and at the indicated time points, intracellular (pelleted cells) (C) and extracellular (cell supernatants) (D) viral fractions were collected and their titers were determined. The data shown represent the mean values from triplicate samples assayed in duplicate; error bars indicate standard deviation.
FIG. 4.
FIG. 4.
Serpin-inactivated rVVs are attenuated in immunodeficient mice. (A) Serpin gene deletion decreases viral replication in VV-infected immunodeficient mice. Female BALB/cBy athymic mice were inoculated i.p. with 107 PFU of each rVV. Ovaries and spleen were removed at 3, 6, and 12 days postinfection. Significant reduction in viral replication was observed in the ovaries on 3, 6, and 12 days postinfection for v50ΔB13R-inoculated mice and on 6 and 12 days postinfection for v50ΔB22R-inoculated mice (Mann-Whitney test, P < 0.05). Viral replication was significantly reduced in the spleen 6 days postinfection for both v50ΔB13R- and v50ΔB22R-inoculated mice (P < 0.05). (B) Serpin gene deletion increases survival of VV-infected nude mice. Male athymic mice inoculated i.p. with 107 PFU of v50 (n = 15), v50ΔB13R (n = 11), or v50ΔB22R (n = 11) had median survival rates of 16, 45, and 35 days, respectively. Statistical analysis of survival time (log-rank test) yielded P < 0.0005 for v50ΔB13R or v50ΔB22R versus v50.
FIG. 5.
FIG. 5.
Inactivation of B13R or B22R decreases rVV virulence in immunocompetent mice. (A) Female CB6F1 mice were inoculated i.p. with 107 PFU of WR, vΔB13R, or vΔB22R. Three days postinfection, the mice were sacrificed, ovaries were removed, and viral titers (PFU per organ) were determined. A bar indicates the mean titer per group. A significant reduction in viral titers was found in the ovaries of mice inoculated with vΔB13R or vΔB22R (Mann-Whitney test, P < 0.004) compared to those inoculated with WR parental virus. (B) Normal mice (CB6F1, n = 11) were inoculated intranasally with 105 PFU of WR, vΔB13R, or vΔB22R. Each animal was weighed and monitored daily for signs of disease. The mean group weight (± standard error) was expressed as the percentage of the mean weight of that group of animals immediately after infection. vΔB13R- or vΔB22R-inoculated mice did not display significant weight loss, whereas mice infected with wild-type WR virus exhibited marked weight loss. This difference was statistically significant (analysis of variance followed by Tukey's studentized range test) from days 5 through 21 postinfection (P < 0.05) and was highly significant at days 6 through 10 (P < 0.0005).
FIG. 6.
FIG. 6.
Serpin gene inactivation does not alter humoral immune responses to rVV. Groups of normal mice (11 animals per group) were inoculated i.m. with 105 PFU of the rVVs v50, v50ΔB13R, and v50ΔB22R. Animals were boosted with VSV i.p. at 4 weeks postvaccination. Antibody titers to VV (A) and VSV (B) were determined by ELISA from pooled samples assayed in duplicate.
FIG. 7.
FIG. 7.
VVs having an inactivated B13R or B22R gene elicit strong T-helper responses. Female CB6F1 mice were immunized i.p. with 107 PFU of each rVV. Splenocytes were harvested 10 days postvaccination and stimulated with 0, 0.25, and 0.5 μg of VSV-G per ml for 4 days and pulsed with [3H]thymidine. Data analysis was based on cpm in triplicates, and error bars represent the standard error of the mean.
FIG. 8.
FIG. 8.
rVVs with an inactivated serpin gene induce potent cytotoxic T-cell responses. CB6F1 mice were inoculated i.p. with 107 PFU of v50, v50ΔB13R, or v50ΔB22R. CTL immune responses were measured 10 days postvaccination. (A) Effector splenocytes were stimulated with haplotype-matched (H-2b/d) target splenocytes that were infected with UV-treated VSV. (B) Infected L929 cells (H-2k) served as negative target controls. The lysis values for negative control targets were <5%. Specific cytolysis values at the indicated effector/target cell ratios represent the means of triplicate experiments assayed in duplicate. Error bars represent standard deviation.
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