Anti-Mouse IgG2b polyclonal antibody [AP]

Staphylococcal enterotoxin B (SEB) is a potent toxin that is produced by Staphylococcus aureus strains and is classified as a category B select agent. We have previously shown that monoclonal antibody (MAb) 20B1, a murine anti-SEB IgG1, successfully treats SEB-induced lethal shock (SEBILS) and bacteremia that is caused by SEB-producing S. aureus. In this study, we have generated two isotype switch variants of the original IgG1 MAb 20B1, an IgG2a and IgG2b, both bearing the same variable region sequence, and compared their neutralizing and protective activity in in vitro and in vivo assays, respectively. All 3 isotypes demonstrated comparable affinity to SEB and comparable 50% inhibitory concentrations (IC(50)s) in T cell proliferation assays. In vivo, however, the IgG2a isotype variant of 20B1 exhibited significantly greater protection than IgG1 or IgG2b in murine SEB intoxication and S. aureus sepsis models. Protection was associated with downmodulation of inflammatory host response. Our data demonstrate that changing the isotype of already protective MAbs, without affecting their antigen specificity or sensitivity, can result in an enhancement of their protective ability. Isotype selection, therefore, should be carefully considered in the development of toxin-neutralizing MAbs and the design of antibody therapeutics. IMPORTANCE The purpose of this study was to enhance the protective efficacy of an existing, protective monoclonal antibody against staphylococcal enterotoxin B. Using two in vivo mouse models, our study demonstrates that the protective efficacy of a monoclonal antibody may be improved by inducing an isotype switch at the Fc region of an antibody, without altering the antigen specificity or sensitivity of the antibody. The development of therapeutic MAbs with higher efficacy may allow for the achievement of equal therapeutic benefit with a lower dosage. In turn, the use of lower doses may reduce the cost of these therapies, while reducing the potential for adverse side effects.

Novel adjuvants are in demand for improving the efficacy of human vaccines. The immunomodulatory properties of Mycobacterium tuberculosis cell wall components have been highlighted in the formulation of complete Freund's adjuvant (CFA). We have explored the adjuvant potential of poly-alpha-L-glutamine (PLG), a lesser-known constituent of the pathogenic mycobacterial cell wall. Immune parameters indicated that the adjuvant potency of PLG was statistically comparable to that of CFA and better than that of alum in the context of H1 antigen (Ag85B and ESAT-6 fusion). At 1 mg/dose, PLG augmented the immune response of Ag85B, BP26, and protective antigen (PA) by increasing serum antibodies and cytokines in the culture supernatant of antigen-stimulated splenocytes. PLG modulated the humoral response of vaccine candidate ESAT-6, eliciting significantly higher levels of total IgG and isotypes (IgG1, IgG2a, and IgG2b). Additionally, the splenocytes from PLG-adjuvanted mice displayed a robust increase in the Th1-specific gamma interferon, tumor necrosis factor alpha, interleukin-2 (IL-2), Th2-specific IL-6 and IL-10, and Th17-specific IL-17A cytokines upon antigenic stimulation. PLG improved the protective efficacy of ESAT-6 by reducing bacillary load in the lung and spleen as well as granuloma formation, and it helped in maintaining vital health parameters of mice challenged with M. tuberculosis. The median survival time of PLG-adjuvanted mice was 205 days, compared to 146 days for dimethyl-dioctadecyl ammonium bromide-monophosphoryl lipid A (DDA-MPL)-vaccinated groups and 224 days for Mycobacterium bovis BCG-vaccinated groups. PLG enhanced the efficiency of the ESAT-6 vaccine to the level of BCG and better than that of DDA-MPL (P < 0.05), with no ill effect in C57BL/6J mice. Our results propose that PLG is a promising adjuvant candidate for advanced experimentation.