Development of Opsonic Mouse Monoclonal Antibodies against Multidrug-Resistant Enterococci.

Multidrug-resistant enterococci are major causes of hospital-acquired infections. Immunotherapy with monoclonal antibodies (MAbs) targeting bacterial antigens would be a valuable treatment option in this setting. Here, we describe the development of two MAbs through hybridoma technology that target antigens from the most clinically relevant enterococcal species. Diheteroglycan (DHG), a well-characterized capsular polysaccharide of Enterococcus faecalis, and the secreted antigen A (SagA), an immunogenic protein from Enterococcus faecium, are both immunogens that have been proven to raise opsonic and cross-reactive antibodies against enterococcal strains. For this purpose, a conjugated form of the native DHG with SagA was used to raise the antibodies in mice, while enzyme-linked immunosorbent assay and opsonophagocytic assay were combined in the selection process of hybridoma cells producing immunoreactive and opsonic antibodies targeting the selected antigens. From this process, two highly specific IgG1(κ) MAbs were obtained, one against the polysaccharide (DHG.01) and one against the protein (SagA.01). Both MAbs exhibited good opsonic killing against the target bacterial strains: DHG.01 showed 90% killing against E. faecalis type 2, and SagA.01 showed 40% killing against E. faecium 11231/6. In addition, both MAbs showed cross-reactivity toward other E. faecalis and E. faecium strains. The sequences from the variable regions of the heavy and light chains were reconstructed in expression vectors, and the activity of the MAbs upon expression in eukaryotic cells was confirmed with the same immunological assays. In summary, we identified two opsonic MAbs against enterococci which could be used for therapeutic or prophylactic approaches against enterococcal infections.

Generation of glucosylated sn-1-glycerolphosphate teichoic acids: glycerol stereochemistry affects synthesis and antibody interaction.

Lipoteichoic acids (LTAs) have been addressed as possible antigen candidates for vaccine development against several opportunistic Gram-positive pathogens. The study of structure-immunogenicity relationship represents a challenge due to the heterogenicity of LTA extracted from native sources. LTAs are built up from glycerol phosphate (GroP) repeating units and they can be substituted at the C-2-OH with carbohydrate appendages or d-alanine residues. The substitution pattern, but also the absolute chirality of the GroP residues can impact the interaction with chiral biomolecules including antibodies and biosynthesis enzymes. We have generated a set of diastereomeric GroP hexamers bearing a glucosyl modification at one of the residues. The chirality of the glycerol building block had an important impact on the stereoselectivity of the glycosylation reaction between the glycosyl donor and the glycerol C-2-OH acceptor. The GroP C-2-chirality also played an important role in the interaction with TA recognizing antibodies. These findings have important implications for the design and synthesis of synthetic TA fragments for diagnostic and therapeutic applications.

Epitope Recognition of a Monoclonal Antibody Raised against a Synthetic Glycerol Phosphate Based Teichoic Acid.

Glycerol phosphate (GroP)-based teichoic acids (TAs) are antigenic cell-wall components found in both enterococcus and staphylococcus species. Their immunogenicity has been explored using both native and synthetic structures, but no details have yet been reported on the structural basis of their interaction with antibodies. This work represents the first case study in which a monoclonal antibody, generated against a synthetic TA, was developed and employed for molecular-level binding analysis using TA microarrays, ELISA, SPR-analyses, and STD-NMR spectroscopy. Our findings show that the number and the chirality of the GroP residues are crucial for interaction and that the sugar appendage contributes to the presentation of the backbone to the binding site of the antibody.

Advances and Prospects in Vaccine Development against Enterococci.

Enterococci are the second most common Gram-positive pathogen responsible for nosocomial infections. Due to the limited number of new antibiotics that reach the medical practice and the resistance of enterococci to the current antibiotic options, passive and active immunotherapies have emerged as a potential prevention and/or treatment strategy against this opportunistic pathogen. In this review, we explore the pathogenicity of these bacteria and their interaction with the host immune response. We provide an overview of the capsular polysaccharides and surface-associated proteins that have been described as potential antigens in anti-enterococcal vaccine formulations. In addition, we describe the current status in vaccine development against enterococci and address the importance and the current advances toward the development of well-defined vaccines with broad coverage against enterococci.