Interlaboratory comparison of a multiplex immunoassay that measures human serum IgG antibodies against six-group B streptococcus polysaccharides.

Measurement of IgG antibodies against group B streptococcus (GBS) capsular polysaccharide (CPS) by use of a standardized and internationally accepted multiplex immunoassay is important for the evaluation of candidate maternal GBS vaccines in order to compare results across studies. A standardized assay is also required if serocorrelates of protection against invasive GBS disease are to be established in infant sera for the six predominant GBS serotypes since it would permit the comparison of results across the six serotypes. We undertook an interlaboratory study across five laboratories that used standardized assay reagents and protocols with a panel of 44 human sera to measure IgG antibodies against GBS CPS serotypes Ia, Ib, II, III, IV, and V. The within-laboratory intermediate precision, which included factors like the lot of coated beads, laboratory analyst, and day, was generally below 20% relative standard deviation (RSD) for all six serotypes, across all five laboratories. The cross-laboratory reproducibility was < 25% RSD for all six serotypes, which demonstrated the consistency of results across the different laboratories. Additionally, anti-CPS IgG concentrations for the 44-member human serum panel were established. The results of this study showed assay robustness and that the resultant anti-CPS IgG concentrations were reproducible across laboratories for the six GBS CPS serotypes when the standardized assay was used.

Sublingual immunisation with GBS serotype III capsular polysaccharide-tetanus toxoid conjugate vaccine induces systemic and mucosal antibody responses which are opsonophagocytic and inhibit GBS colonisation of vaginal epithelial cells.

No vaccines are currently licensed against Group B streptococcus (GBS), an important cause of morbidity and mortality in babies and adults. Using a mouse model, and in vitro opsonophagocytosis and colonisation assays, we evaluated the potential of a sublingually-administered polysaccharide-conjugate vaccine against GBS serotype III. Sublingual immunisation of mice with 10 µg of GBS conjugate vaccine once a week for 5 weeks induced a substantial systemic IgG anti-polysaccharide response which was similar to the level induced by subcutaneous immunsation. In addition, sublingual immunisation also induced mucosal (IgA) antibody responses in the mouth, intestines and vagina. Immune sera and intestinal washes were functionally active at mediating killing of the homologous GBS serotype III in an opsonophagocytosis assay. In addition, intestinal and vaginal washes inhibited the colonisation of mouse vaginal epithelial cells by the vaccine homologous strain. These results suggest that, in addition to the induction of high levels of IgG antibodies that could be transduced from the immunised mother to the foetus to protect the newborn against GBS infection, sublingual immunisation can elicit a substantial mucosal antibody response which might play an important role in the prevention of GBS colonisation in immunised women, thereby eliminating the risk of GBS transmission from the mother to the baby during pregnancy or at birth.

Purification and characterization of nisin P produced by a strain of Streptococcus gallolyticus.

Introduction. Against the backdrop of increasing resistance to conventional antibiotics, bacteriocins represent an attractive alternative, given their potent activity, novel modes of action and perceived lack of issues with resistance.Aim. In this study, the nature of the antibacterial activity of a clinical isolate of Streptococcus gallolyticus was investigated.Methods. Optimization of the production of an inhibitor from strain AB39 was performed using different broth media and supplements. Purification was carried out using size exclusion, ion exchange and HPLC. Gel diffusion agar overlay, MS/MS, de novo peptide sequencing and genome mining were used in a proteogenomics approach to facilitate identification of the genetic basis for production of the inhibitor.Results. Strain AB39 was identified as representing Streptococcus gallolyticus subsp. pasteurianus and the successful production and purification of the AB39 peptide, named nisin P, with a mass of 3133.78 Da, was achieved using BHI broth with 10 % serum. Nisin P showed antibacterial activity towards clinical isolates of drug-resistant bacteria, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and penicillin-resistant Streptococcus pneumoniae. In addition, the peptide exhibited significant stability towards high temperature, wide pH and certain proteolytic enzymes and displayed very low toxicity towards sheep red blood cells and Vero cells.Conclusion. To the best of our knowledge, this study represents the first production, purification and characterization of nisin P. Further study of nisin P may reveal its potential for treating or preventing infections caused by antibiotic-resistant Gram-positive bacteria, or those evading vaccination regimens.