[PubMed] [Google Scholar] 25. immune response. Though no obvious differences were detected in immune responses to challenge in mice. is usually a facultative, intracellular, gram-negative bacterial pathogen that can cause abortion in pregnant cattle and undulant fever in humans (6). Upon contamination, enters macrophage-monocyte lineage cells and multiplies within the phagosomes by inhibiting phagosome-lysosome fusion (7, 22). strains exhibiting a easy phenotype contain a surface-exposed O-polysaccharide chain (O antigen) as part of their lipopolysaccharide (LPS) structure (5). Truly rough strains do not contain O antigen in their LPS. Clean LPS is usually a virulence factor of strains; infected animals develop antibodies to this antigen. However, as in most intracellular bacterial infections, cell-mediated immunity (CMI) plays a major role in acquired resistance to infection. Hence, vaccination of cattle with live rough strains, such as RB51, results in effective protection against brucellosis. strain RB51 is an attenuated stable rough mutant derived from the virulent strain 2308 (25). Strain RB51 is currently used as the live vaccine of choice against bovine brucellosis in the United States and many other countries. Being a rough mutant, strain RB51 does not induce antibodies to the O antigen, thereby facilitating obvious serological differentiation of vaccinated from infected animals. Vaccination with live strain RB51 results in a biased T-helper 1 (Th1)-specific immune response characterized by the induction of specific CD8+ cytotoxic T cells, CD4+ helper T cells that secrete gamma interferon (IFN-), and predominance of immunoglobulin G2a (IgG2a) isotype antigen-specific antibodies (11, 27, 28, 29, 30). is usually a potent stimulator of CMI. Heat-killed promotes a strong Th1-type immune response and simultaneously inhibits primary as well as secondary Th2 immune responses (1, 26, 32). Studies performed with heat-killed made up of a chemically conjugated human immunodeficiency computer virus (HIV) peptide indicated that can enhance cell-mediated immune responses, including cytotoxic CD8+ T lymphocytes, even in major histocompatibility complex class II-knockout mice, although these mice have low CD4+ T-cell figures and Rabbit Polyclonal to PAR4 defective CD4-dependent T-helper responses (9, 16). Interleukin-12 (IL-12) plays an important role in the induction of a Th1 immune response (21). In vivo studies showed that heat-killed induces the secretion of IL-12 and tumor necrosis factor alpha (TNF-) and recognized dendritic cells (DCs) as a major source of IL-12 (13, 14). The LPS of is usually 1,000-fold less toxic than the LPS of (10). Taken together, these features form a strong basis for using as a vaccine carrier to induce a Th1-biased immune response against specific antigens. We are exploring the development of strain RB51 as a vaccine expression and delivery platform for the purpose of inducing a Th1-biased immune response against specific protein antigens. Previously, using a plasmid-based system, we have shown that strain RB51 can Befetupitant be engineered to express heterologous proteins, Befetupitant and mice vaccinated with such recombinant RB51 strains develop a strong Th1-type immune response to the foreign proteins (29). It is apparent that the use of live, recombinant RB51 strains for vaccination of a variety of animal species would be limited by security concerns. Therefore, we are researching strategies to inactivate recombinant RB51 strains without interfering with the induction of Th1-type immune responses. In this study we examined the effect of two different modes of inactivation, heat treatment and gamma irradiation, around the induction of a heterologous antigen-specific immune response by a recombinant RB51 strain. Befetupitant Our studies exhibited that strain RB51G/LacZ (a recombinant RB51 strain expressing -galactosidase of strain 2308 challenge. MATERIALS AND METHODS Bacterial strains. strains RB51 and 2308 were from our culture collection. Generation of strain RB51G/LacZ was explained previously (29). This strain harbors plasmid pBBgroE/lacZ, which contains the gene of expressed from your promoter. The bacteria were produced in tryptic soy broth (TSB) or tryptic soy agar (TSA) at 37C as previously explained (25). Strain RB51G/LacZ was produced in the presence of 30 g/ml of chloramphenicol. Vaccine preparation. Strain RB51G/LacZ was produced in TSB with chloramphenicol to mid-log phase, and aliquots of 1 1 1011 CFU/ml were stored at ?80C until use. On the day of immunization, an aliquot of the vaccine was subjected to gamma irradiation using a 60Co source irradiator (Gammacell 220 irradiator). Another aliquot was subjected to heat killing by.