Brucella are Gram-negative intracellular bacteria that cause abortion and infertility in livestock and chronic disease in humans. The Centers for Disease Control and Prevention (CDC) categorizes them as class B pathogens due to their zoonotic potential. Currently, there are no efficacious Brucella vaccines for humans available. Very few studies have focused on identifying protective vaccines against respiratory exposure. Protection by B. abortus rough vaccine strains RB51 and RB51SOD is through strong CD4⁺ Th₁ and CD8⁺ Tc₁ adaptive immunity. However, limited information is available on how they stimulate innate immunity. This knowledge is critical for improving these vaccines for their potential use in humans. Dendritic cells (DCs) play a crucial role bridging innate and adaptive immunity. Therefore, enhancing the ability of rough vaccine strains to induce DC maturation and function could be critical for upregulating protective T-cell responses. Herein, we demonstrated that live vaccine strain RB51 induced significantly better (p≤0.05) DC maturation and function in vitro and upon intranasal inoculation in vivo compared to strain RB51SOD or strain 2308. Due to safety concerns of live vaccines, irradiated and heat killed vaccines were also tested; only live strain RB51 infected DCs induced significant (p≤0.05) DC function based on TNF-α and IL-12 secretion. DC activation occurs through Toll-like receptors (TLRs) 2, 4 and 9. Our study reported that strain RB51 induced significant (p≤0.05) DC activation compared to strain 2308, which was not dependent on a specific TLR. However, strain RB51 induced TNF – α production was TLR2 and TLR9 dependent and IL-12 production was TLR2 and TLR4 dependent. TLR4 KO mice had significantly (p≤0.05) higher number of strain RB51 colonies present at day 14 post infection. By unraveling the innate immune responses to Brucella, the ultimate goal of these studies is to develop a protective vaccine for animals and people against respiratory challenge. As such, we tested several vaccination strategies. Despite enhanced DC activation and function achieved by vaccine strains, they failed to protect mice against intranasal challenge with strain 2308. Future experiments will address host-pathogen interaction at the lung microenvironment and elucidate immune mechanisms that will enhance protection against aerosol exposure. / Ph. D.

Brucella abortus is amongst the top 5 zoonotic diseases worldwide. The overall goal of this research is to generate a safe and effective vaccine for humans. Brucella abortus strain RB51, approved for use in cattle, provides protection by initiating a strong T-helper 1 (Th1) type response is a candidate vaccine. Based on a model for aerosol exposure mice were vaccinated intranasally (IN) with strain RB51 and challenged IN with B. abortus strain 2308, strain RB51 did not protect. Protection against Brucella is mediated through TLRs 2, 4 and 9. The addition of TLR 2 or TLR 4 and a trend with TLR9 agonists with intranasal RB51 vaccination significantly increased bacterial clearance in the lung after strain 2308 challenge. Therefore, we hypothesized that combining TLR agonists 2, 4, and 9 with strain RB51 IN would upregulate protection and clearance in the lung against strain 2308 challenge (IN), by upregulating the DC1 and CD4 Th1 and CD8 immune response. This study showed that protection is not upregulated by combining all TLR agonists. Overall the addition of TLR 2 and 4 vs. TLR 2, 4 and 9 agonists affects the immune response and impacts the level of clearance. Our data support the development of a DC1 Th1 CD8 response, based on serology, and both DC and T-cell activation and function by the group which received the TLR 2 and 4 agonists and to a lesser degree the group receiving TLR 2, 4, and 9 agonists. Additional studies are warranted to further define the differential mechanisms and endpoints of protection. / Master of Science