Inhibition of toxic shock by human monoclonal antibodies against staphylococcal enterotoxin B

Abstract

Background: Staphylococcus aureus is implicated in many opportunistic bacterial infections around the world. Rising antibiotic resistance and few alternative methods of treatment are just two looming problems associated with clinical management of S. aureus. Among numerous virulence factors produced by S. aureus, staphylococcal enterotoxin (SE) B is a secreted protein that binds T-cell receptor and major histocompatibility complex class II, potentially causing toxic shock mediated by pathological activation of T cells. Recombinant monoclonal antibodies that target SEB and block receptor interactions can be of therapeutic value.

Methodology/principal findings: The inhibitory and biophysical properties of ten human monoclonal antibodies, isolated from a recombinant library by panning against SEB vaccine (STEBVax), were examined as bivalent Fabs and native full-length IgG (Mab). The best performing Fabs had binding affinities equal to polyclonal IgG, low nanomolar IC(50)s against SEB in cell culture assays, and protected mice from SEB-induced toxic shock. The orthologous staphylococcal proteins, SEC1 and SEC2, as well as streptococcal pyrogenic exotoxin C were recognized by several Fabs. Four Fabs against SEB, with the lowest IC(50)s, were converted into native full-length Mabs. Although SEB-binding kinetics were identical between each Fab and respective Mab, a 250-fold greater inhibition of SEB-induced T-cell activation was observed with two Mabs.

Conclusions/significance: Results suggest that these human monoclonal antibodies possess high affinity, target specificity, and toxin neutralization qualities essential for any therapeutic agent.

Figure 1. Antibody detection of SEB in a complex antigen mixture by Western blot.

A 10–20% gradient SDS-PAGE separated crude S. aureus antigen preparations subsequently transferred onto nitrocellulose. Each Fab (1 µg/ml) was used to probe the nitrocellulose for 1 h. Lanes include: A- 50 ng purified SEB; B – 20 µg cell lysate; and C – 20 µg culture supernatant.

Figure 2. Therapeutic capabilities of antibodies added after SEB in human PBMC cultures.

Fab or native full-length (FL) Mabs were used at a final concentration of 1000 nM and added at designated times after SEB (0.35 nM). Culture fluids were collected 16 h after SEB addition and IFNγ concentrations ascertained as described in the Materials and Methods. Panels A and C respectively show Fab and FL-Mab results. Antibody controls consisted of a SEA-specific Fab and SEB affinity-purified IgG from human sera (Panel B). Results are representative of two separate experiments.