Isotype switching increases efficacy of antibody protection against staphylococcal enterotoxin B-induced lethal shock and Staphylococcus aureus sepsis in mice

Abstract

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 (IC50s) 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.

FIG 1

(A) Comparable binding of three IgG subclasses of SEB-specific MAb 20B1 isotype switch variants 20B1 IgG1, 20B1 IgG2a, and 20B1 IgG2b. Experiment was performed in triplicates. Each point represents the mean value of triplicates, and bars represent the standard error derived from measurements in the same experiment. (B, C, D) Dose response curves for binding of murine MAb 20B1 isotypes to biotinylated SEB were captured by streptavidin biosensors using BLItz. Analyses were performed using Forte Bio’s software to determine R equilibrium. Measured kinetic constants are reported.

FIG 2

IgG subclasses of MAb 20B1 showed comparable inhibition of SEB-induced proliferation in murine splenocytes. Calculated IC₅₀ of MAb 20B1 IgG1 was 0.28 nM (A), 20B1 IgG2a was 0.68 nM (B), and 20B1 IgG2b was 1.04 nM (C). Experiments performed in triplicates. The circles represent an average from triplicates of the relative luminescence units (RLU). Bars represent the standard deviations derived from measurements in triplicate wells in the same experiment. IC₅₀s were determined from concentration-response curve analysis (GraphPad Prism 6).

FIG 3

BALB/c mice (n = 10 per group) were treated with various IgG subclasses of MAb 20B1 at different doses or PBS intraperitoneally (i.p.) and challenged with 20 µg of SEB and sensitized with 25 mg of galactosamine (i.p.). Mice treated with 250-µg doses of 20B1 isotypes showed 100% survival (A), variable protection with the 100-µg dose (B), and IgG2a was 100% effective at the 50-µg dose, but IgG2b and IgG1 failed to protect (C). (D) Enhanced protection was observed when 20B1 isotypes (50 µg) were combined with 14G8 IgG1 (50 µg) compared to PBS treatment (20B1 IgG1 and 14G8, P = 0.003; 20B1 IgG2b and 14G8, P = 0.001; 20B1 IgG2a and 14G8, P = 0.0001). Analysis of survival data was performed using log rank (Mantel-Cox test).

FIG 4

Multiplex cytokine analysis of serum (after 2 h) from mice (n = 5) treated with MAb 20B1 isotypes (50 µg) or PBS and challenged with SEB in administration with galactosamine. Levels of proinflammatory cytokines (IL-6, IFN-γ, TNF-α, IL-10, IL-1β, and KC-GRO) were significantly lower in mice treated with 20B1 IgG2a than in mice treated with PBS or the IgG2b isotype of MAb 20B1 (P < 0.05). Cytokines IL-6, IFN-γ, and KC-GRO were significantly lower (P < 0.05) in 20B1 IgG2a-treated mice than in those treated with the parental 20B1 IgG1. All controls and samples were done in duplicates. Bars are represented as mean values + standard deviations. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc multiple-comparison test to compare the mean of each data set with the mean of every other datasets using GraphPad Prism 6 software, and data were considered significant as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. NS, not significant.

FIG 5

Eight different sets of mice (n = 5) were treated either with isotypes of MAb 20B1 (500 µg) or PBS and challenged with SEB. ELISAs were performed to measure the SEB levels from sera, kidney, liver, and spleen at 2 h and 6 h after SEB challenge. SEB level was higher in blood at the initial time point at 2 h, as well as at 6 h in mice treated with all isotypes of MAb 20B1. As expected, higher SEB clearance was observed from sera of mice treated with PBS only. Bars represent the standard error derived from the SEB measurement of a group of 5 mice in each data set.

FIG 6

Different IgG subclasses of MAb 20B1 (i.v.) (300 µg) were injected into mice and challenged with S. aureus (MRSA strain 38, i.v. 5 × 10⁷ CFU) in a sepsis model. Treatment with the IgG2a isotype resulted in significantly longer survival than that in mice treated with 20B1 IgG2b (P = 0.01), 20B1 IgG1 (P = 0.004), or PBS (P = 0.0001). Analyses of survival data were performed using log rank (Mantel-Cox test).