Development of Opsonic Mouse Monoclonal Antibodies against Multidrug-Resistant Enterococci

Abstract

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.

Advances and Prospects in Vaccine Development against Enterococci

“…Interestingly, the mAb against DHG exhibited lower killing compared to the polyclonal serum raised against the same antigen, revealing a variability in the presence and exposure of the recognizable epitope between the strains [126]. A combination therapy using antibodies that target a variety of antigenic epitopes would provide broad coverage and overcome the lack of diagnostic methods [126]. Similar strategies are being explored against other types of diseases, e.g., cancer, and could also be implemented in infectious diseases [133,134].…”

“…Passive immunotherapy using mAbs is an emerging field with many promising candidates to fight these health threats, either by replacing the common antibiotic therapy or by co-administration with antibiotics [125,126]. mAbs are also of great importance for the development of chemically defined vaccines by solving the bottleneck of epitope identification [127].…”

“…Both antibodies exhibited high specificity and opsonic killing against several enterococcal strains. Interestingly, the mAb against DHG exhibited lower killing compared to the polyclonal serum raised against the same antigen, revealing a variability in the presence and exposure of the recognizable epitope between the strains [126]. A combination therapy using antibodies that target a variety of antigenic epitopes would provide broad coverage and overcome the lack of diagnostic methods [126].…”

Carriers and Antigens: New Developments in Glycoconjugate Vaccines

“…Kalfopoulou et al provided data on two highly specific antibodies directed against the polysaccharide and carrier protein. Both antibodies showed good opsonic killing against target bacterial strains in upwards of 40% for E. faecium and 90% for E. faecalis respectively [ 140 ]. Romero-Saavedra et al showed similar results with antibodies against the corresponding conjugate, as well as against the respective protein and carbohydrate antigens.…”

Enterococcal Membrane Vesicles as Vaccine Candidates

“…Given the prevalence of enterococcal infections and the treatment challenges, there is an urgent need to develop new approaches to treat or prevent these infections. Immunotherapies, among other options, represent a promising avenue of research [5].…”