Therapeutic efficacy of a potent anti-Venezuelan equine encephalitis virus antibody is contingent on Fc effector function.

The development of specific, safe, and potent monoclonal antibodies (Abs) has led to novel therapeutic options for infectious disease. In addition to preventing viral infection through neutralization, Abs can clear infected cells and induce immunomodulatory functions through engagement of their crystallizable fragment (Fc) with complement proteins and Fc receptors on immune cells. Little is known about the role of Fc effector functions of neutralizing Abs in the context of encephalitic alphavirus infection. To determine the role of Fc effector function in therapeutic efficacy against Venezuelan equine encephalitis virus (VEEV), we compared the potently neutralizing anti-VEEV human IgG F5 (hF5) Ab with intact Fc function (hF5-WT) or containing the loss of function Fc mutations L234A and L235A (hF5-LALA) in the context of VEEV infection. We observed significantly reduced binding to complement and Fc receptors, as well as differential in vitro kinetics of Fc-mediated cytotoxicity for hF5-LALA compared to hF5-WT. The in vivo efficacy of hF5-LALA was comparable to hF5-WT at -24 and + 24 h post infection, with both Abs providing high levels of protection. However, when hF5-WT and hF5-LALA were administered + 48 h post infection, there was a significant decrease in the therapeutic efficacy of hF5-LALA. Together these results demonstrate that optimal therapeutic Ab treatment of VEEV, and possibly other encephalitic alphaviruses, requires neutralization paired with engagement of immune effectors via the Fc region.

Development of potent and effective synthetic SARS-CoV-2 neutralizing nanobodies.

The respiratory virus responsible for coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected nearly every aspect of life worldwide, claiming the lives of over 3.9 million people globally, at the time of this publication. Neutralizing humanized nanobody (VHH)-based antibodies (VHH-huFc) represent a promising therapeutic intervention strategy to address the current SARS-CoV-2 pandemic and provide a powerful toolkit to address future virus outbreaks. Using a synthetic, high-diversity VHH bacteriophage library, several potent neutralizing VHH-huFc antibodies were identified and evaluated for their capacity to tightly bind to the SARS-CoV-2 receptor-binding domain, to prevent binding of SARS-CoV-2 spike (S) to the cellular receptor angiotensin-converting enzyme 2, and to neutralize viral infection. Preliminary preclinical evaluation of multiple VHH-huFc antibody candidates demonstrate that they are prophylactically and therapeutically effective in vivo against wildtype SARS-CoV-2. The identified and characterized VHH-huFc antibodies described herein represent viable candidates for further preclinical evaluation and another tool to add to our therapeutic arsenal to address the COVID-19 pandemic.

Discovery of Small-Molecule Inhibitors of SARS-CoV-2 Proteins Using a Computational and Experimental Pipeline.

A rapid response is necessary to contain emergent biological outbreaks before they can become pandemics. The novel coronavirus (SARS-CoV-2) that causes COVID-19 was first reported in December of 2019 in Wuhan, China and reached most corners of the globe in less than two months. In just over a year since the initial infections, COVID-19 infected almost 100 million people worldwide. Although similar to SARS-CoV and MERS-CoV, SARS-CoV-2 has resisted treatments that are effective against other coronaviruses. Crystal structures of two SARS-CoV-2 proteins, spike protein and main protease, have been reported and can serve as targets for studies in neutralizing this threat. We have employed molecular docking, molecular dynamics simulations, and machine learning to identify from a library of 26 million molecules possible candidate compounds that may attenuate or neutralize the effects of this virus. The viability of selected candidate compounds against SARS-CoV-2 was determined experimentally by biolayer interferometry and FRET-based activity protein assays along with virus-based assays. In the pseudovirus assay, imatinib and lapatinib had IC50 values below 10 µM, while candesartan cilexetil had an IC50 value of approximately 67 µM against Mpro in a FRET-based activity assay. Comparatively, candesartan cilexetil had the highest selectivity index of all compounds tested as its half-maximal cytotoxicity concentration 50 (CC50) value was the only one greater than the limit of the assay (>100 µM).

High-throughput screening to discover inhibitors of the CarD·RNA polymerase protein-protein interaction in Mycobacterium tuberculosis.

Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) accounts for 3.7% of new cases of TB annually worldwide and is a major threat to global public health. Due to the prevalence of the MDR-TB and extensively drug resistant tuberculosis (XDR-TB) cases, there is an urgent need for new drugs with novel mechanisms of action. CarD, a global transcription regulator in MTB, binds RNAP and activates transcription by stabilizing the transcription initiation open-promoter complex (RPo). CarD is required for MTB viability and it has highly conserved homologues in many eubacteria. A fluorescence polarization (FP) assay which monitors the association of MTB RNAP, native rRNA promoter DNA and CarD has been developed. Overall, our objective is to identify and characterize small molecule inhibitors which block the CarD/RNAP interaction and to understand the mechanisms by which CarD interacts with the molecules. We expect that the development of a new and improved anti-TB compound with a novel mechanism of action will relieve the burden of resistance. This CarD FP assay is amenable to HTS and is an enabling tool for future novel therapeutic discovery.

Source of the Fitness Defect in Rifamycin-Resistant Mycobacterium tuberculosis RNA Polymerase and the Mechanism of Compensation by Mutations in the ß' Subunit.

Mycobacterium tuberculosis is a critical threat to human health due to the increased prevalence of rifampin resistance (RMPr). Fitness defects have been observed in RMPr mutants with amino acid substitutions in the ß subunit of RNA polymerase (RNAP). In clinical isolates, this fitness defect can be ameliorated by the presence of secondary mutations in the double-psi ß-barrel (DPBB) domain of the ß' subunit of RNAP. To identify factors contributing to the fitness defects observed in vivo, several in vitro RNA transcription assays were utilized to probe initiation, elongation, termination, and 3'-RNA hydrolysis with the wild-type and RMPrM. tuberculosis RNAPs. We found that the less prevalent RMPr mutants exhibit significantly poorer termination efficiencies relative to the wild type, an important factor for proper gene expression. We also found that several mechanistic aspects of transcription of the RMPr mutant RNAPs are impacted relative to the wild type. For the clinically most prevalent mutant, the ßS450L mutant, these defects are mitigated by the presence of secondary/compensatory mutations in the DPBB domain of the ß' subunit.

Structural basis for rifamycin resistance of bacterial RNA polymerase by the three most clinically important RpoB mutations found in Mycobacterium tuberculosis.

Since 1967, Rifampin (RMP, a Rifamycin) has been used as a first line antibiotic treatment for tuberculosis (TB), and it remains the cornerstone of current short-term TB treatment. Increased occurrence of Rifamycin-resistant (RIFR ) TB, ∼41% of which results from the RpoB S531L mutation in RNA polymerase (RNAP), has become a growing problem worldwide. In this study, we determined the X-ray crystal structures of the Escherichia coli RNAPs containing the most clinically important S531L mutation and two other frequently observed RIFR mutants, RpoB D516V and RpoB H526Y. The structures reveal that the S531L mutation imparts subtle if any structural or functional impact on RNAP in the absence of RIF. However, upon RMP binding, the S531L mutant exhibits a disordering of the RIF binding interface, which effectively reduces the RMP affinity. In contrast, the H526Y mutation reshapes the RIF binding pocket, generating significant steric conflicts that essentially prevent any RIF binding. While the D516V mutant does not exhibit any such gross structural changes, certainly the electrostatic surface of the RIF binding pocket is dramatically changed, likely resulting in the decreased affinity for RIFs. Analysis of interactions of RMP with three common RIFR mutant RNAPs suggests that modifications to RMP may recover its efficacy against RIFR TB.