Protective efficacy of i.m. (A) or i.n. (B) immunization with MVA, or i.m. immunization with NYVAC (C) in BALB/c mice. Groups of mice (five per group) were immunized i.m. or i.n. with 0, 10³,10⁴,10⁵,10⁶,or10⁷ pfu of MVA or NYVAC, or with 5 × 10⁵ pfu of Wyeth strain. One month later, mice were challenged i.n. with 10⁶ pfu of WR vaccinia. Individual weight loss measured daily is presented as means for each group. These experiments were performed twice with comparable results.

Vaccinia virus-specific antibody is necessary and sufficient for protection against WR. (A) B cell-deficient mice (on the BALB/c background) were immunized with 10⁶ pfu of MVA. One month later, mice were challenged with 10⁶ pfu of WR, and weight loss was monitored daily. Three days before challenge, group 1 of B cell-deficient mice (n = 3) was untreated with antibody, group 2 (n = 3) was treated with anti-CD4 and anti-CD8 antibodies, and group 3 (n = 3) unimmunized BALB/c mice were left untreated with antibody as a control. Weight loss monitored daily after i.n. challenge with WR is presented as means for each group. (B) Protection against WR challenge after syngeneic transfer of immune serum. Immune serum collected 1 month after tail scratch immunization with 5 × 10⁵ pfu of Wyeth was transferred into three syngeneic BALB/c mice per group starting 2 days before challenge (200 μl of serum i.p daily for 7 days). Experiments were performed twice with comparable results.

Mechanisms of protection. Induction of virus-specific antibody (A) and IFN-γ-producing cells by ELISPOT (B) after i.m. immunization of BALB/c mice with MVA. Groups of mice (five per group) were immunized i.m. with 0, 10³,10⁴,10⁵,10⁶,or10⁷ pfu of MVA or with 5 × 10⁵ pfu of Wyeth. One month after immunization, mice were bled and then killed, and spleens were harvested. Virus-specific antibody response (end-point titer) was measured by ELISA. The vaccinia virus-specific T cell response was studied by the ELISPOT assay for IFN-γ-producing class I MHC-restricted T cells. (C) BALB/c or CD1^–/– mice were immunized with 10⁶ pfu of MVA i.m. One month later, mice were challenged with 10⁶ pfu of WR. Three days before challenge, mice in group 1 (n = 5) were treated i.p. with anti-CD8 antibody (clone 2.43; 0.5 mg per mouse per day) daily for 4 days (▪); group 2 was treated with anti-CD4 antibody (GK 1.5 ascites) (1 mg per mouse per day) daily for 4 days (♦); group 3 was untreated before challenge (▴); group 4 was unimmunized and untreated (▵); and group 5 was CD1^–/– mice (•). Individual weight loss measured daily after challenge is presented as means for each group (C). Flow cytometric analysis showed that >98% of CD4 and CD8 T cells were depleted by the treatment with the respective Abs. All experiments were performed at least twice with comparable results.

(A) Natural resistance to WR virus depends on CD4 and CD8 cells. Naïve BALB/c mice (n = 5) were challenged i.n. with 10⁴ pfu of WR. Three days before challenge, BALB/c mice were treated with anti-CD4 antibody (group 1), anti-CD8 antibody (group 2), or anti-CD4 and anti-CD8 antibodies (group 3), or untreated (group 4). After i.n. challenge with 10⁴ pfu of WR, weight loss was monitored. (B) CD8 CTL and CD4 T cell responses are not required for protection induced by immunization with Wyeth. BALB/c mice were immunized with 5 × 10⁵ pfu of Wyeth by tail scratch. One month after immunization, mice were challenged with 10⁶ pfu of WR. Three days before challenge, group 1 (n = 5) mice were treated i.p. with anti-CD8 antibody (0.5 mg per mouse per day) daily for 4 days, group 2 was treated with anti-CD4 antibody (ascites) (1 mg per mouse per day) daily for 4 days, and group 3 was untreated before challenge. A fourth group of BALB/c mice was left unimmunized. These were compared with a group of B cell-deficient BALB/c mice that were immunized with Wyeth. Experiments were performed twice with comparable results.