Malone, K. | Search bioRxiv

bioRxiv2021

Deciphering Bedaquiline and Clofazimine Resistance in Tuberculosis: An Evolutionary Medicine Approach

Bedaquiline (BDQ) and clofazimine (CFZ) are core drugs for treatment of multidrug resistant tuberculosis (MDR-TB), however, our understanding of the resistance mechanisms for these drugs is sparse which is hampering rapid molecular diagnostics. To address this, we employed a unique approach using experimental evolution, protein modelling, genome sequencing, and minimum inhibitory concentration data combined with genomes from a global strain collection of over 14,151 Mycobacterium tuberculosis complex isolates and an extensive literature review. Overall, 230 genomic variants causing elevated BDQ and/or CFZ MICs could be discerned, with 201 (87.4%) variants affecting the transcriptional repressor (Rv0678) of an efflux system (mmpS5-mmpL5). Structural modelling of Rv0678 suggests four major mechanisms that confer resistance: impairment of DNA binding, reduction in protein stability, disruption of protein dimerization, and alteration in affinity for its fatty acid ligand. These modelling and experimental techniques will improve personalized medicine in an impending drug resistant era.

microbiology↗

bioRxiv2020

High-resolution mapping of the neutralizing and binding specificities of polyclonal rabbit serum elicited by HIV Env trimer immunization

Mapping the epitope specificities of polyclonal serum is critical to rational vaccine design. However, most high-resolution mapping approaches involve isolating and characterizing individual monoclonal antibodies, which incompletely defines the full polyclonal response. Here we use two complementary approaches to directly map the specificities of the neutralizing and binding antibodies of polyclonal anti-HIV-1 sera from rabbits immunized with BG505 Env SOSIP trimers. To map the neutralizing specificity, we used mutational antigenic profiling to determine how all amino-acid mutations in Env affected viral neutralization. To map the binding specificity, we used electron microscopy polyclonal epitope mapping (EMPEM) to directly visualize the Fabs in serum bound to Env trimers. Mutational antigenic profiling showed that the dominant neutralizing specificities were the C3/V5 and/or 241/289 glycan hole epitopes, which were generally only a subset of the more diverse binding specificities mapped with EMPEM. Additional differences between binding and neutralization reflected antigenicity differences between virus and soluble Env trimer. Further, mutational antigenic profiling was able to refine epitope specificity in residue-level detail directly from sera, revealing subtle differences across rabbits. Together, mutational antigenic profiling and EMPEM allow for a holistic view of the binding and neutralizing specificity of polyclonal sera and could be used to finely evaluate and guide vaccine design.

microbiology↗