Engineering Protein Nanoparticles Functionalized with an Immunodominant Coxiella burnetii Antigen to Generate a Q Fever Vaccine

Coxiella burnetii is the causative agent of Q fever, for which there is yet to be an FDA-approved vaccine. This bacterial pathogen has both extra- and intracellular stages in its life cycle, and therefore both a cell-mediated (i.e., T lymphocyte) and humoral (i.e., antibody) immune response are necessary for effective eradication of this pathogen. However, most proposed vaccines elicit strong responses to only one mechanism of adaptive immunity, and some can either cause reactogenicity or lack sufficient immunogenicity. In this work, we aim to apply a nanoparticle-based platform toward producing both antibody and T cell immune responses against C. burnetii. We investigated three approaches for conjugation of the immunodominant outer membrane protein antigen (CBU1910) to the E2 nanoparticle to obtain a consistent antigen orientation: direct genetic fusion, high affinity tris-NTA-Ni conjugation to polyhistidine-tagged CBU1910, and the SpyTag/SpyCatcher (ST/SC) system. Overall, we found that the ST/SC approach yielded nanoparticles loaded with the highest number of antigens while maintaining stability, enabling formulations that could simultaneously co-deliver the protein antigen (CBU1910) and adjuvant (CpG1826) on one nanoparticle (CBU1910-CpG-E2). Using protein microarray analyses, we found that after immunization, antigen-bound nanoparticle formulations elicited significantly higher antigen-specific IgG responses than soluble CBU1910 alone and produced more balanced IgG1/IgG2c ratios. Although T cell recall assays from these protein antigen formulations did not show significant increases in antigen-specific IFN-γ production compared to soluble CBU1910 alone, nanoparticles conjugated with a CD4 peptide epitope from CBU1910 generated elevated T cell responses in mice to both the CBU1910 peptide epitope and whole CBU1910 protein. These investigations highlight the feasibility of conjugating antigens to nanoparticles for tuning and improving both humoral- and cell-mediated adaptive immunity against C. burnetii.


Figure SI- 2 .
Figure SI-2.tNTA-Ni + CBU1910 conjugation conditions optimization.Table of excipients tested to stabilize CBU1910-E2 constructs during tNTA-Ni/HisTag conjugation.Aggregation/precipitation and intact nanoparticle structure were determined based on DLS analysis.+++: high percentage of monodispersed particles; ++: mixture of monodispersed and aggregated particles; +: extreme aggregation of particles.A high NaCl concentration of 360 mM in a HEPES buffer system using low CBU1910:E2 molar ratios demonstrated the most consistent ability to alleviate aggregation during conjugation.

Figure SI- 4 .
Figure SI-4.Alphafold2 Colab to predict the folded structure of CBU1910.The amino acid sequence of the transmembrane truncated form of CBU1910 was input into Alphafold2 and its output .pdbfile was viewed on ChimeraX.Orange highlighted region = CBU1910p sequence.

Figure SI- 5 .
Figure SI-5.ST/SC reaction conditions optimization.Table of excipients tested to stabilize CBU1910-E2 constructs during ST/SC reaction.Aggregation/precipitation and intact nanoparticle structure were determined based on SDS-PAGE and DLS analysis.+++X: highly soluble but disassembled particles; +++: high percentage of monodispersed particles; ++: mixture of monodispersed and aggregated particles; +: extreme aggregation of particles.A 0.08-0.0875%(w/v) SLS condition demonstrated consistent ability to alleviate aggregation while keeping nanoparticles intact.

Table SI - 1 .
Abbreviations and description of vaccine components.N/A denotes "not applicable."

Table SI - 2 .
DNA and protein sequences of E2 nanoparticle mutants and antigen mutants.