Critical Role of Perforin-dependent CD8+ T Cell Immunity for Rapid Protective Vaccination in a Murine Model for Human Smallpox

Kremer, Melanie; Suezer, Yasemin; Volz, Asisa; Frenz, Theresa; Majzoub, Monir; Hanschmann, Kay-Martin; Lehmann, Michael H.; Kalinke, Ulrich; Sutter, Gerd

Statistical comparison of different groups of mice was performed as means of the area under the weight curve (AUC) in percent of individual weight at baseline. The AUC was additionally weighted with the length of the observation period (usually day of challenge (day 0) until day 22, or the day the animal died). The differences between vaccination groups were analyzed with a one-factorial analysis of variance model. For multiple comparisons p-values were adjusted with the Bonferroni method. The statistical evaluation was performed with SAS/STAT software, version 9.2, SAS System for Windows. For statistical significant results the following convention was used: * -p-value,0.05, ** -pvalue,0.01 and *** -p-value,0.001. In all experiments weight loss of individual mice was monitored daily (n = 2 to 3 per group). +indicate the individual time of death. Error bars indicate SEMs, and the numbers of surviving/total animals are given in parentheses. (TIF) Figure S8 C57BL/6 mice and B cell-deficient JHT mice mount comparable VACV specific T cell responses. At 7 days after MVA immunization spleen cells from individual C57BL/6 (wt) (n = 6) mice or B-cell deficient JHT mice (n = 5) were stimulated with VACV specific peptide B8R 20-27 and subsequently CD8+ CD62L low T cells were analyzed by intracellular cytokine staining and FACS for gamma interferon expression (IFN-y+). (TIF) Figure S9 Heat-inactivated MVA vaccine does not protect from morbidity and mortality following ECTV challenge. (A) C57BL/6 mice were i.m. immunized with MVA (10 8 PFU) (n = 5) or heatinactivated MVA (corresponding to 10 8 PFU) (n = 5) two days before 36LD 50 ECTV challenge. Mock-challenged (&) (n = 3) and mock-vaccinated (m) (n = 5) mice served as controls. In all experiments weight loss of individual mice was monitored daily (n = 3 to 5 per group). The data shown are representative for two similar experiments. +indicate the individual time of death. Error bars indicate SEMs, and the numbers of surviving/total animals are given in parentheses. Statistical significance of differences between groups is indicated by * for p-value,0.05, ** for pvalue,0.01 and *** for p-value,0.001. (B) Confirmation of MVA inactivation. Heat-treatment (60uC for 4 hours) of MVA vaccine preparation prevents activation of viral early gene transcription. Human THP-1 cells were infected with MVA or heat-treated MVA (corresponding to an MOI of 4) and incubated for 6 h at 37uC. Total RNA was isolated from infected and mock-infected cells, and analyzed by RT-PCR using specific oligonucleotide primers for the products of VACV early gene E3L and human GAPDH.