Long-term egg-yolk adaptation of the Orientia tsutsugamushi for preparation of a formalinized immunogen

To assess the feasibility of preparing a killed rickettsial vaccine against the scrub typhus, the Karp and Gilliam strains of the Orientia tsutsugamushi were adapted through sequential passages in eggs for more than 100 times over 9 years to produce approximately five times more antigen than the unadapted rickettsia. The egg-grown rickettsia was purified by differential centrifugation and batch-type ion-exchange chromatography, and inactivated by formalin treatment. Strong protective immunity was acquired against lethal challenges of the homologous strains and lasted fully for longer than 8 months in the C3H/He mice immunized with either of the single-strain immunogen or in combination of the two strain immuinogens. However, neither immunogen protected animals from the challenges with the two heterologous strains or Boryong, the prevalent strain in Korea, despite that three antigenic proteins of 47, 56, and 110 kDa were eminent in both preparations. IgM, IgG, and neutralizing antibody were induced in the immunized mice in a level and pattern comparable to that in animals infected with live rickettsiae.