Induction of Antigen-Specific Th1-Type Immune Responses by Gamma-Irradiated RecombinantBrucella abortusRB51

Abstract

Brucella abortus strain RB51 is an attenuated rough mutant used as the live vaccine against bovine brucellosis in the United States and other countries. We previously reported the development of strain RB51 as a bacterial vaccine vector for inducing Th1-type immune responses against heterologous proteins. Because safety concerns may preclude the use of strain RB51-based recombinant live vaccines, we explored the ability of a gamma-irradiated recombinant RB51 strain to induce heterologous antigen-specific immune responses in BALB/c mice. Exposure of strain RB51G/LacZ expressing Escherichia coli ␤-galactosidase to a minimum of 300 kilorads of gamma radiation resulted in complete loss of replicative ability. These bacteria, however, remained metabolically active and continued to synthesize ␤-galactosidase. A single intraperitoneal inoculation of mice with 10 9 CFU equivalents of gamma-irradiated, but not heat-killed, RB51G/LacZ induced a ␤-galactosidasespecific Th1-type immune response. Though no obvious differences were detected in immune responses to B. abortus-specific antigens, mice vaccinated with gamma-irradiated, but not heat-killed, RB51G/LacZ developed significant protection against challenge with virulent B. abortus. In vitro experiments indicated that gammairradiated and heat-killed RB51G/LacZ induced maturation of dendritic cells; however, stimulation with gamma-irradiated bacteria resulted in more interleukin-12 secretion. These results suggest that recombinant RB51 strains exposed to an appropriate minimum dose of gamma radiation are unable to replicate but retain their ability to stimulate Th1 immune responses against the heterologous antigens and confer protection against B. abortus challenge in mice.

Nondividing but Metabolically Active Gamma-IrradiatedBrucella melitensisIs Protective against VirulentB. melitensisChallenge in Mice

“…It is noteworthy that irradiated Brucella demonstrated enhanced protection compared to metabolically inactive heat-treated bacteria. Our results are consistent with previous reports using gamma-irradiated B. abortus strain RB51 and gamma-irradiated Listeria monocytogenes (13,38) Importantly, different approaches generating nonreplicating metabolically active organisms result in effective protection against intracellular pathogens (8,13,23,38). Together, these findings suggest that despite the inactivation method, metabolic activity is the key component mediating effective immunity.…”

“…Irradiation has long been used as a method to prevent mammalian cell proliferation without inhibition of cellular activity (39). Irradiated microorganisms also retain metabolic activity, as suggested by researchers utilizing the irradiated bacteria B. abortus strain RB51 (38). Furthermore, other researchers demonstrated that the gamma-irradiated protozoan Toxoplasma gondii retains "morphology, metabolism, and cell invasion properties," suggesting that the cellular functions were not abrogated by irradiation (19).…”

Immunization of mice with gamma-irradiated Brucella neotomae and its recombinant strains induces protection against virulent B. abortus, B. melitensis, and B. suis challenge

“…Therefore, we used exposure to a minimum dose of gamma radiation as a means to abolish the ability of B. neotomae to replicate. The use of ionizing radiation has been used in the development of vaccines for preventing infectious diseases of animals and humans that are caused by different viruses, bacteria, and parasites [13, 25-29]. However, a recently developed strategy specifically uses exposure to a minimum dose of radiation, which is sufficient to abolish replication of the organism, for developing safer vaccines for diseases caused by intracellular pathogens [13, 28-29].…”

“…The use of ionizing radiation has been used in the development of vaccines for preventing infectious diseases of animals and humans that are caused by different viruses, bacteria, and parasites [13, 25-29]. However, a recently developed strategy specifically uses exposure to a minimum dose of radiation, which is sufficient to abolish replication of the organism, for developing safer vaccines for diseases caused by intracellular pathogens [13, 28-29]. Similar to the previously reported findings for B. abortus RB51 and B. melitensis Rev1 [13, 29], B. neotomae exposed to a minimum of 300-350 krads of radiation lost its ability to replicate on nutrient rich TSA medium.…”

“…For the Alamar blue assay, irradiated samples were washed in saline solution and resuspended in TSB to the original concentration. Ninety microliters of the irradiated suspension were mixed with 10 μl of Alamar blue and incubated at 37°C for 1 hour, and the change in color from blue to pink was monitored as described previously [13]. The manufacturer’s instructions were followed while using the LIVE/DEAD Baclight kit which utilizes a mixture of the green-fluorescent SYTO® 9 and the red-fluorescent propidium iodide to stain the bacterial nucleic acids.…”