Coxiella burnetii is an obligate intracellular bacterium and the causative agent of Q fever. C. burnetii is considered a potential bioterrorism agent because of its low infectious dose; resistance to heat, drying, and common disinfectants; and lack of prophylactic therapies. Q-Vax, a formalin-inactivated whole-bacteria vaccine, is currently the only prophylactic measure that is protective against C. burnetii infections but is not U.S. Food and Drug Administration approved. To overcome the safety concerns associated with the whole-bacteria vaccine, we sought to generate and evaluate recombinant protein subunit vaccines against C. burnetii. To accomplish this, we formulated C. burnetii Ags with a novel TLR triagonist adjuvant platform, which used combinatorial chemistry to link three different TLR agonists together to form one adjuvanting complex. We evaluated the immunomodulatory activity of a panel of TLR triagonist adjuvants and found that they elicited unique Ag-specific immune responses both in vitro and in vivo. We evaluated our top candidates in a live C. burnetii aerosol challenge model in C56BL/6 mice and found that several of our novel vaccine formulations conferred varying levels of protection to the challenged animals compared with sham immunized mice, although none of our candidates were as protective as the commercial vaccine across all protection criteria that were analyzed. Our findings characterize a novel adjuvant platform and offer an alternative approach to generating protective and effective vaccines against C. burnetii.

The vast majority of seasonal influenza vaccines administered each year are derived from virus propagated in eggs using technology that has changed little since the 1930s. The immunogenicity, durability, and breadth of response would likely benefit from a recombinant nanoparticle-based approach. Although the E2 protein nanoparticle (NP) platform has been previously shown to promote effective cell-mediated responses to peptide epitopes, it has not yet been reported to deliver whole protein antigens. In this study, we synthesized a novel maleimido tris-nitrilotriacetic acid (NTA) linker to couple protein hemagglutinin (HA) from H1N1 influenza virus to the E2 NP, and we evaluated the HA-specific antibody responses using protein microarrays. We found that recombinant H1 protein alone is immunogenic in mice but requires two boosts for IgG to be detected and is strongly IgG1 (Th2) polarized. When conjugated to E2 NPs, IgG2c is produced leading to a more balanced Th1/Th2 response. Inclusion of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) significantly enhances the immunogenicity of H1−E2 NPs while retaining the Th1/Th2 balance. Interestingly, broader homo-and heterosubtypic cross-reactivity is also observed for conjugated H1−E2 with MPLA, compared to unconjugated H1 with or without MPLA. These results highlight the potential of an NP-based delivery of HA for tuning the immunogenicity, breadth, and Th1/Th2 balance generated by recombinant HA-based vaccination. Furthermore, the modularity of this protein−protein conjugation strategy may have utility for future vaccine development against other human pathogens.

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An important function of antibodies is their ability to mark cells for phagocytosis and subsequent degradation in a process known as opsonization. When developing new vaccine candidates, it is critical to determine whether the antigen specific antibodies elicited in response to immunization have this function. Due to the high containment working constraints of several important human pathogens, it is not always possible to directly study this property using the pathogen of interest. Therefore, we sought to develop a pathogen-free, flow cytometry based assay to quantify the opsonizing activity of antibodies elicited in response to immunization. We studied this using vaccine candidates against Coxiella burnetii, the causative agent of Q-fever that is a potential bioterrorism agent. In our assay, fluorescent polystyrene beads were conjugated to C. burnetii proteins then incubated with the antibody-containing sera. The antibody-coated beads were incubated with macrophages and phagocytosis of the fluorescent beads was assayed by flow cytometry. This assay allows for rapid quantification of the opsonizing capacity of antigen specific antibodies elicited in response to immunization.