Efficacy of Intracellular Activated Promoters for Generation ofSalmonella-Based Vaccines

Evaluation of Salmonella enterica Type III Secretion System Effector Proteins as Carriers for Heterologous Vaccine Antigens

“…The immune responses to vaccination with live Salmonella carriers translocating fusion proteins of listeriolysin (Llo) to various effectors were analyzed as previously described (25). Briefly, BALB/c female mice 6 weeks of age (Jackson Laboratory) were maintained under standard conditions at the Baylor College of Medicine animal facility and were treated according to an IACUC-approved protocol.…”

“…Stimulation of T cell proliferation was analyzed as described before (25). Briefly, BM-DCs from C57BL/6 mice were seeded at 10 5 per well in a 96-well plate.…”

“…B3Z is a T cell hybridoma that recognizes the OVA-derived SIIN FEKL epitope in the context of H2K b and expresses the lacZ reporter gene under the control of the NFAT enhancer. The ␤-galactosidase activity thus is a measure of the antigendependent stimulation (25). Very low stimulation was observed with the vector controls without expression cassettes, while addi- Expression cassettes with fusions to sseF were described previously (11).…”

“…As a second approach, we used the OVA-specific T cells isolated from OT-I or OT-II transgenic mice (25). OT-I mice are transgenic for CD8 ϩ T cells that recognize the OVA epitope SIIN FEKL (OVA 257-276 ) in the context of MHC class I molecules, while OT-II mice are transgenic for CD4 ϩ cells that respond to the OVA 323-339 peptide ISQAVHAAHAEINEAGR in the context of the MHC class II I-A 2 molecule.…”

“…In order to quantify the immune response to vaccination, blood samples were analyzed on days 10, 21, and 35 after the initial immunization. The elicitation of Listeria monocytogenes antigen-specific cytotoxic T lymphocytes (CTL) is a reliable correlate of protective immunity against L. monocytogenes infections in murine models (11,14,18,25). The proportion of Llo-specific CTL was determined using specific pentamers (Fig.…”