Abstract Rickettsiae are a group of arthropod-associated, obligately intracellular Gram-negative bacteria that are closely related but potentially cause life-threatening infection in humans. Tick-borne rickettsioses (TBRs) are increasingly posing serious threat to the public health in the US due to increased incidence and lack of commercially available vaccine for prevention. Therefore, a safe and effective vaccine against TBRs will be an important resource that will ensure the Nation’s capability to prevent this group of diseases. Our long- term goal is to develop novel approaches for safe and effective immunoprophylaxis of TBRs. Our recently published and pilot studies demonstrated that live-attenuated R. parkeri mutant 3A2 conferred full protection against lethal challenge of two highly virulent rickettsiae in mouse models, accompanied by generating high titers of IgG antibodies reactive against several pathogenic rickettsial species and significantly elevated type 1 T cell memory immunity. These findings support the proof-of-concept that live-attenuated vaccine candidate serves a feasible and effective approach to prevent TBRs and study vaccine-induced memory immunity against TBRs. Thus, the objectives of this R01 application are to generate a safe and efficacious live-attenuated vaccine (LAV) against TBRs, define the protective efficacy of LAV against homologous and heterologous rickettsial strains, and mechanistically determine the immune correlates of vaccine-elicited protection against tick transmission of TBRs. To achieve these objectives, we propose three specific aims to test the central hypothesis that the LAV will elicit high quality CD8+ T memory cells and rickettsiae-specific neutralizing antibodies to confer complete protection against natural acquisition of TBRs as a safe and efficacious polyvalent vaccine candidate. Aim 1 will optimize R. parkeri mutants genetically to reduce virulence, enhance safety while maintaining immunogenicity. Aim 2 will define the efficacy of genetically optimized R. parkeri mutants in protecting against TBRs in both needle-challenge and tick transmission animal models. Aim 3 will identify the immunological correlates of vaccine-induced protection against TBRs. Upon the completion of the proposed research, we expect to define the first multivalent vaccine candidate for TBR and reveal the novel elements of host immunity responsible for prevention from natural transmission of TBRs. This will have significant positive effects on human health because it will provide the basic information required to ultimately develop a safe, effective, and tractable vaccine against TBRs.