Immunization with DNA followed by modified vaccinia computer virus Ankara strain, both expressing the antigen 85A, induced both CD4+- and CD8+-T-cell responses in BALB/c mice. essential for immunity against (20). Classical class I-restricted CD8+ T cells may also be important (20), but their functional significance in vivo remains unclear. A encouraging candidate antigen for a new tuberculosis (TB) vaccine is usually antigen 85A, which is usually protective in mice and guinea pigs (10, 9). Antigen 85A contains several CD4+-T-cell epitopes and at least one CD8+-T-cell epitope in BALB/c mice (4), and CD4+- and CD8+-T-cell responses have been recognized in humans (12, 18). Heterologous prime-boost immunization strategies can induce high levels of CD8+ and CD4+ T cells in patients with malaria, human immunodeficiency computer virus, and TB (7, 14, 15). We used DNA and recombinant altered vaccinia computer virus Ankara strain (MVA) vaccines, each encoding antigen 85A, to identify two immunodominant epitopes within antigen 85A, a CD4+-restricted epitope and a CD8+-restricted epitope. DCs pulsed with both of these epitopes but not a mixture of DCs pulsed with each epitope separately conferred protection against challenge equivalent to the protection conferred by the prime-boost immunization regimen and by BCG. H37Rv stocks were prepared as previously explained (14). The antigen 85A gene and the tissue plasminogen activator leader sequence were ligated together as a single coding sequence in plasmid vector pSG2 and vaccinia computer virus shuttle vector pSC11. BHK cells were infected with nonrecombinant MVA at a multiplicity of contamination of 0.05 and then transfected with the recombinant shuttle vector. Recombinant computer virus was selected for by plaque purification. Autologous bone marrow cells were cultured in RPMI 1640 made up of 10% fetal calf serum (Labtech International, Uckfield, United Kingdom) and 1 ng of recombinant granulocyte-macrophage colony-stimulating factor (Peprotech, Rocky Hill, N.J.) per ml for 7 days. The medium and recombinant granulocyte-macrophage colony-stimulating factor (0.5 ng/ml) were replenished on days 3 and 6. DCs were pulsed Gossypol distributor with peptide (2 g/ml) overnight, washed twice, and counted before injection. Incubation of DCs with peptide did not induce DC maturation, as indicated by expression of surface molecules, such as major histocompatibility complex classes I and II, CD86, and CD40 (determined by fluorescence-activated cell sorter analysis) (data not shown). Female BALB/c mice (age, 4 to 6 6 weeks; Harlan Orlac, Shaws Farm, Blackthorn, United Kingdom) were injected intramuscularly with plasmid DNA (50 g/immunization). Recombinant MVA (106 PFU) and DCs (106 cells) were injected intravenously. BCG Glaxo (4 105 CFU) was injected intradermally at the time of the first immunization. Immunizations were given at 2-week intervals, and immunogenicity was evaluated 2 weeks after the last immunization. RGS9 Splenocytes were prepared as previously explained (14). The number of gamma interferon-secreting specific T cells was determined by using an ex vivo Elispot assay and 20-mer peptides (overlapping by 10 amino acids) spanning the length of antigen 85A (Research Genetics, Huntsville, Ala.) (14). The nonamer H-2Kd-restricted epitope within peptide p11 (WYDQSGLSV) was used for some DC experiments. The numbers of spot-forming cells (SFC) per 106 splenocytes were decided. Cell depletion was performed on cells restimulated in vitro for 7 days as previously explained (14). In the challenge experiments, mice were infected with 106 or 5 106 CFU of by intraperitoneal injection 2 weeks after Gossypol distributor the final immunization. Organs were homogenized 8 weeks later with a mini-bead beater (Biospec Products, Bartlesville, Okla.), and dilutions were plated onto Middlebrook plates. The plates were incubated for 21 days at 37C, and the numbers of colony counts per organ Gossypol distributor were calculated. Data were expressed as the log10 mean and standard error for each experimental group. Student’s test was used to determine statistical significance between groups. The values presented below are one-tailed values determined by comparing immunized groups with nonimmunized controls. BALB/c mice were immunized with Gossypol distributor DNA (D regimen), MVA (M regimen), or a combination of the two (DM or MD regimen). Using an ex lover vivo Elispot assay, we recognized responses to the following four peptides from your splenocytes of immunized mice: p11, p15, p24, and p27 (Table ?(Table1).1). The strongest responses were the responses to peptides p11 and p15. Magnetic bead depletion studies performed with restimulated splenocytes from mice immunized with DNA and then MVA showed that this response to p11 was eliminated by CD8+-T-cell depletion. The responses to p15, p24, and p27 were eliminated by CD4+-T-cell depletion. In this study we focused on the dominant epitopes contained in p11 and p15. Figure ?Figure11 shows that the responses generated by single or repeated immunizations with either construct were weak. Heterologous boosting of DNA with MVA resulted in higher frequencies of both the CD4+(p15) and CD8+(p11) T cells. Heterologous boosting of MVA with DNA increased only the.