ABSTRACT
The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.
Subject(s)
Antibodies, Monoclonal/metabolism , Antitoxins/metabolism , Clostridioides difficile/physiology , Enterocolitis, Pseudomembranous/drug therapy , Enterocolitis, Pseudomembranous/immunology , Animals , Antibodies, Monoclonal/immunology , Antitoxins/immunology , Bacterial Proteins/adverse effects , Bacterial Proteins/immunology , Bacterial Toxins/adverse effects , Bacterial Toxins/immunology , Binding Sites, Antibody/immunology , CHO Cells , Clostridioides difficile/pathogenicity , Cricetinae , Cricetulus , Diarrhea/etiology , Diarrhea/prevention & control , Drug Combinations , Enterocolitis, Pseudomembranous/microbiology , Enterocolitis, Pseudomembranous/physiopathology , Enterotoxins/adverse effects , Enterotoxins/immunology , Epitope Mapping , Humans , Mice , Protein BindingABSTRACT
Cytotoxic T-lymphocytes (CTLs) are critical for the defense against herpesvirus infections, in which cell-to-cell spread occurs earlier than the hematogenous spread. The ability of bovine herpesvirus-1 (BHV-1) to undergo latency, to induce apoptosis of CD4(+) T-lymphocytes, and to down-regulate the expression of major histocompatibility complex (MHC) class I molecules, necessitates the development of immunization strategies that do not involve the live virus. The objective of this study was to evaluate the feasibility of DNA immunization as a means of induction of CTLs against BHV-1. Mice were injected either by intramuscular (IM) or intradermal (ID) route with a Sindbis virus-based plasmid carrying the gene encoding the glycoprotein D (gD) of BHV-1. Splenocytes from the immunized mice were re-stimulated in vitro with gD-transduced syngeneic fibroblasts. The CTLs generated specifically lysed syngeneic targets, either transduced with gD or infected with BHV-1. IM route of inoculation induced a better CTL response when compared to ID route with respect to onset, magnitude and duration of immunity. These results indicate the feasibility of using a plasmid carrying the gene encoding BHV-1 gD as an immunogen to induce CTLs against BHV-1.
