C57BL/6 female mice were vaccinated 5 times with Tem1-TT or TT vaccine followed by mating 1 week later. (A) Tem1-TT did not alter reproduction. Vaccination had no effect on fertility, gestation time, number of pups, or weight of pups (n = 15 per group). Data are representative of 2 independent experiments. Statistical analyses were performed with Student’s t test. (B) Tem1-TT vaccine did not affect embryonic development. H&E staining of embryos of female mice vaccinated with TT and Tem1-TT showed no abnormalities detected for either group. (C) Tem1-TT vaccination did not induce tissue pathology. Histological analysis of lung, heart, liver, spleen, kidney, colon, ovary, uterus, and placenta from pregnant (postpartum) and nonpregnant mice (no placenta) revealed no pathological effects by either vaccine. Original magnification, ×10, except spleen and ovary (×4). (D) Tem1-TT vaccination did not affect the estrus cycle. In a separate independent experiment, sera from immunized mice had synchronized estrus cycles and were evaluated for reproductive hormones. No fluctuations were noted. (E) TEM1-specific T cell responses were unaltered by pregnancy. 1 × 10⁶ splenocytes of vaccinated pregnant and nonpregnant mice were tested against TEM1_696–710 peptide or control peptide (TEM1_691–705) using IFN-γ ELISpot. Mice were boosted with a single injection of Tem1-TT or TT 45 days after the last immunization, then sacrificed 2 weeks later. There was significant recognition of TEM1_696–710 peptide by Tem1-TT– versus TT-vaccinated splenocytes from both pregnant and nonpregnant mice splenocytes (n = 5).