Coxiella burnetii, which causes Q-fever, is a bacterial pathogen that infects and resides internally in host cells. For this reason, a T lymphocyte response, in addition to an adequate antibody response, is necessary for eradication of the pathogen. In our previous studies, utilization of the E2 protein nanocapsule (E2) in a viral-mimetic strategy for cancer vaccines resulted in formulations that elicited strong T cell responses against tumors. However, we have not yet applied this strategy towards protection against bacterial pathogens. The E2 nanoparticle is a 60-subunit, self-assembling 25-nm dodecahedral scaffold with high thermal stability that can be genetically engineered for the precise definition of chemical conjugation sites at the external surface and internal cavity. Our current studies aim to integrate full-length proteins from C. burnetii onto the surface of E2. Multiple approaches were explored, including utilization of high affinity Tris-NTA-Ni conjugation to polyhistidine-tagged (His-tag) antigens, direct genetic fusion of antigens to the E2 nanoparticle, and the SpyTag/SpyCatcher system. Using the Tris-NTA-Ni strategy, we identified experimental conditions that successfully conjugated C. burnetii protein antigen CBU 1910 to E2 nanoparticles. Direct fusion of the protein antigen CBU1910 onto the E2 nanoparticle using a recombinant system resulted in expression, but aggregation in inclusion bodies. Implementation of the SpyTag/SpyCatcher protein-protein conjugation system resulted in uniquely-engineered formulations that can co-deliver the antigen (CBU1910) and adjuvant (CpG1826) on an E2 nanoparticle. To examine the formulations’ potential as prophylactic vaccines, we have evaluated the humoral (e.g., IgG, IgG1, IgG2c antibodies) and cell-mediated (e.g., cytokine release from T cell assays) immune responses that were elicited after immunization.

Engineering protein nanoparticles conjugated with antigens towards a Q-fever vaccine

ACS Spring 2022

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