A Tetraspecific VHH-Based Neutralizing Antibody Modifies Disease Outcome in Three Animal Models of Clostridium difficile Infection

Abstract

Clostridium difficile infection (CDI), a leading cause of nosocomial infection, is a serious disease in North America, Europe, and Asia. CDI varies greatly from asymptomatic carriage to life-threatening diarrhea, toxic megacolon, and toxemia. The incidence of community-acquired infection has increased due to the emergence of hypervirulent antibiotic-resistant strains. These new strains contribute to the frequent occurrence of disease relapse, complicating treatment, increasing hospital stays, and increasing morbidity and mortality among patients. Therefore, it is critical to develop new therapeutic approaches that bypass the development of antimicrobial resistance and avoid disruption of gut microflora. Here, we describe the construction of a single heteromultimeric VHH-based neutralizing agent (VNA) that targets the two primary virulence factors of Clostridium difficile, toxins A (TcdA) and B (TcdB). Designated VNA2-Tcd, this agent has subnanomolar toxin neutralization potencies for both C. difficile toxins in cell assays. When given systemically by parenteral administration, VNA2-Tcd protected against CDI in gnotobiotic piglets and mice and to a lesser extent in hamsters. Protection from CDI was also observed in gnotobiotic piglets treated by gene therapy with an adenovirus that promoted the expression of VNA2-Tcd.

Structural Insights into Rational Design of Single-Domain Antibody-Based Antitoxins against Botulinum Neurotoxins

“…In earlier studies, we found that VHH-based neutralizing agents (VNAs) consisting of VHH heterodimers joined by a flexible peptide linker possessed significantly improved antitoxin potencies (Mukherjee et al, 2012). Similar results have been obtained when developing VNAs for other toxins, such as ricin, anthrax, Shiga toxins, and Clostridium difficile toxins (Herrera et al, 2015;Schmidt et al, 2016;Sheoran et al, 2015;Vance et al, 2013;Vrentas et al, 2016;Yang et al, 2014). We hypothesized that novel heterodimeric VNAs, which are composed of two VHHs connected by a spacer that permits simultaneous binding, could be rationally designed based on structural and mechanistic knowledge of individual VHHs.…”

“…Small proteins such as heavy-chain-only camelid antibodies (called VHHs, nanobodies, or single-domain antibodies) and de novo designed mini-proteins against the toxins are currently being developed as alternatives (Chevalier et al, 2017;Conway et al, 2010;Godakova et al, 2019;Mukherjee et al, 2012;Thanongsaksrikul et al, 2010). These small proteins have high stability, can be economically produced, display high binding affinity, and have been shown to function effectively as antitoxins in animal models (Dong et al, 2010;Herrera et al, 2015;Schmidt et al, 2016;Sheoran et al, 2015;Vance et al, 2013;Vrentas et al, 2016). However, the therapeutic applications of these antitoxins have been limited by a lack of understanding of the molecular mechanisms underlying BoNT neutralization, the extreme potency of BoNTs (in vivo lethal blood concentrations at sub-pM), as well as the diverse sequences among different BoNT serotypes and subtypes.…”

Structure of the full-length Clostridium difficile toxin B

“…Taken together, these findings suggest that 5D neutralizes TcdB 31 , 46 by preventing the pore-forming region from completing the necessary pH-induced conformational change. Notably, the pore-forming region recognized by 5D is highly conserved among a family of large clostridial glucosylating toxins (LCGTs), which include TcdA and TcdB, C. novyi α-toxin (Tcnα), C. sordellii lethal and hemorrhagic toxins (TcsL and TcsH), and C. perfringens toxin (TpeL) ( Fig.…”

Genetically engineered red cells expressing single domain camelid antibodies confer long-term protection against botulinum neurotoxin

“…We showed that a single VHH (GPA-VHH7) is also able to neutralize BoNT/A. Since each VHH comprises a single immunoglobulin domain stabilized by one or two intramolecular disulfide bonds that fold independently 36 , 37 , it is likely possible to engineer GPA or Kell chimeras that contain three or more VHH domains and express these on the same RBCs. In this way, one could engineer RBCs that bind multiple foreign toxins or viruses and thus offer long-term prophylactic protection against multiple pathogens.…”