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To calculate LD₅₀, four female BALB/c or C57BL/6 mice (Charles River Laboratories) were infected i.v. with 5-fold dilutions starting at 1 ´10⁹ colony-forming units (cfu) of the indicated strain. The mice were monitored daily for 10 days, killed when they showed signs of distress, and the LD₅₀ was calculated.

Protective immunity was assessed by determining the cfu in spleen and liver of vaccinated mice challenged with wild-type Listeria at a dose equal to 2´LD₅₀ as described in ref. 1. Briefly, C57BL/6 mice were vaccinated with the indicated Listeria strains at a dose equal to 0.1 LD₅₀. Twenty-eight days later, mice were injected i.v. with wild-type Listeria (2´LD₅₀) diluted in 200 ml of Hanks’ balanced salt solution (HBSS). The level of infection in each mouse was determined 3 days after Listeria challenge by enumerating bacteria in liver and spleen organ homogenates.

For the s.c. tumor model, female BALB/c mice were implanted s.c. with 2 ´10⁵ CT26 cells on day 0. Groups of 10 mice were vaccinated i.v. with the indicated Listeria strains at different days after tumor implantation. Tumor size was measured twice a week throughout the experiment. Tumor volume in mm³ was calcutated by using the formula (length ´width ´ height)/2. Mice were killed when the tumor reached a size larger than 2,000 mm³.

1. Busch, D. H., Vijh, S. & Pamer, E. (2000) Current Protocols in Immunology (Wiley, New York), pp. 19.9.1–19.9.9.

Fig. 5. Listeria D actA/D inlB induces potent antigen-specific T cell responses at safe doses and with clinically relevant routes of administration. (A) C57BL/6 mice were vaccinated i.v. with the indicated strain and dose. Ovalbumin (OVA)-specific CD8⁺ T cell immunity was assessed on day 7 after vaccination by using intracellular cytokine staining. Representative results are shown of three independently performed experiments. (B) C57BL/6 mice were administered through different routes with 0.1 LD₅₀ of Listeria D actA/DinlB-OVA strain. The injection volume was adjusted depending on the route. OVA-specific immunity was assessed in spleen cells at day 7 after vaccination by using ICS. A representative of two independent experiments is shown. IV, i.v.; IP, i.p.; SC, s.c.; ID, intradermal; IM, i.m.

Fig. 6. (A) The in vivo growth kinetic in spleen was assessed for wild type, DactA, DinlB, and D actA/DinlB. C57BL/6 mice were injected i.v. with 0.1 LD₅₀ of the indicated strain, and bacteria per organ were determined from three mice at each time point. A representative of two experiments is shown. (B) Liver toxicity was determined after infection. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined at 24 h, when serum levels peaked. cfu, colony-forming units.

Fig. 7. Recombinant attenuated Listeria D actA/DinlB-AH1-A5 breaks tolerance to self that results in potent therapeutic antitumor activity and prolongation of life. (A) Female BALB/c mice were vaccinated once with 0.1 LD₅₀ of the indicated strain. Antigen-specific T cell responses were assessed at day 7 by using ICS. The percent of antigen-specific CD8⁺ T cells are shown. β-gal, β-galactosidase; LLO, listeriolysin O. (B) Female BALB/c mice were vaccinated either once or twice 28 days apart, and AH1- and AH1-A5-specific T cell responses were determined 7 days after the final immunization by using ICS. (C) BALB/c mice (n = 10 per group) were implanted s.c. with 2 ´10⁵ CT26 cells on day 0. Mice were vaccinated i.v. on day 7 or 10 after tumor implantation. Tumor growth was monitored twice per week throughout the experiment. D actA/DinlB-AH1-A5 vaccination resulted in statistically significant tumor growth delay (P < 0.004).