The identification of immune-reactive proteins recognised in response to Coxiella burnetii infection
Student thesis: Doctoral Thesis
Infection with Coxiella burnetii in humans is associated with severe disease, known as Q fever. Acute infection, that can be sub-clinical in nature, can lead to the development of a chronic life-threatening cardiac condition. The largest reported human outbreak of Q fever recently occurred in the Netherlands between 2007 and 2010. This outbreak gave rise to over 4,000 reported human cases and efforts such as the culling of 50,000 pregnant goats in an attempt to control the outbreak. While an effective vaccine exists for protection against infection, it has an unsatisfactory safety profile that has led to it being licenced in only a single country and in a defined group at risk of occupational exposure to infection. In addition, this vaccine involves culturing the organisms at high levels of biological containment; this makes the vaccine expensive to produce and presents a challenge in attaining reproducible batch-to-batch variation.
In this work, two species of laboratory animals were infected by the aerosol route to closely mimic the likely natural route of infection in humans. The species investigated were guinea pigs (Dunkin Hartley strain) and mice (BALB/c and A/J strains). The infection in guinea pigs more closely mimicked human acute disease than that in the two different strains of mice: In terms of a more acute febrile response and greater clearance of the organism from the tissues. After clinical recovery from disease, the antibodies from the guinea pigs’ sera were used to isolate immune-reactive proteins found in axenically-grown C. burnetii organisms. Isolation of protein was achieved by picking spots from 2D-PAGE gels corresponding to reactive spots on parallel Western blots as well as immunoprecipitation (pull down) of bacterial protein extracts, a method not previously applied to this type of analysis. These isolated proteins were then identified using nanoLC-electrospray ionisation tandem mass-spectrometry (Q Exactive™ instrument).
More than 100 proteins were identified, 71 of which had not been previously described in the literature. The identified proteins were considered for their suitability for inclusion in a future subunit vaccine to protect against Q fever. The most promising candidates were an OmpA-like surface protein similar to a molecule that has already been described as an invasin, two surface-exposed components of the bacterial type IV secretion system (DotA and IcmX), and two hypothetical proteins that warrant further study.
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