Monoclonal antibodies protect from Staphylococcal Enterotoxin K (SEK) induced toxic shock and sepsis by USA300 Staphylococcus aureus

Figures & data

Figure 1. MAbs inhibit SEK-induced proliferation of human immune cells. PBMCs from human whole blood were plated at 1 × 10⁵ cells per well and incubated in the presence of SEK for 96 hours at 37°C and 10% CO₂. (A) SEK induces maximal proliferation of immune cells (up to 6-fold greater than controls) at 10ng/ml. Immune cell proliferation after treatment with 10ng/ml of SEK is inhibited in a dose-dependent manner by concurrent incubation with 1, 10, and 100ug/ml of (B) mAb-4G3, (C) mAb-5G2, and (D) mAb-9H2. Relative luminescence units (RLU); Treatment with media alone (RPMI); Murine antibody isotype control (IC); Student's t-test: * = p < 0.05; ** = p < 0.01.

Figure 2. MAbs inhibit SEK-induced cytokine secretion by human immune cells. T cells from human whole blood were plated at 5 × 10⁴ cells per well and incubated in the presence of SEK and 1ug/ml of mAb(s) for 8 hours at 37°C and 10% CO₂. Supernatants were subsequently collected and pro-inflammatory cytokines were measured by ELISA for human (A) IL-2 or (B) INF-γ. Treatment with media alone (RPMI); Murine antibody isotype control (IC). Student's t-test: * = p < 0.05; ** = p < 0.01; *** < 0.001.

Figure 3. SEK binding competition between mAb combinations. A 96-well ELISA plate was coated with 25ng of SEK per well, blocked with 1% BSA, and then simultaneously treated with (A) the combination of mAb-4G3 (IgG2b) and mAb-5G2 (IgG1) or (B) the combination of mAb-9H2 (IgG1). MAb-4G3 and mAb-5G2 do no exhibit competition for epitope(s) on SEK. mAb-4G3 and mAb-9H2 exhibit competition for epitope(s) on SEK. All experiments were performed in duplicate. GAM = Goat anti-mouse, AP = Alkaline phosphatase.

Table 1. Characterization of SEK monoclonal antibodies.

mAb	Isotype	KD (M)	IC50 (M)
4G3	IgG2b	9.4 × 10^−10	1.04 × 10^−7
5G2	IgG1	4.3 × 10^−9	2.58 × 10^−7
9H2	IgG1	2.4 × 10^−9	1.49 × 10^−6

Note. Relative binding affinities to SEK (K_D) were determined by ELISA. The 50% inhibitory concentration (IC₅₀) were determined by analysis of the PBMC proliferation data from Fig. 3-1 (b-d). M = molar concentration.

Table 2. The combination of non-competing mAbs protects mice from SEK-induced lethal shock.

Treatment	Total	Survived	Died	p-value
SEK + PBS	10	1	9
SEK + mAb-4G3	10	1	9	ns
SEK + mAb-5G2	10	2	8	ns
SEK + mAb-4G3 + mAb-5G2	10	8	2	0.006

Note. Six to eight week-old female BALB/c mice were treated with a series of 3 intraperitoneal injections: first with 25mg of D-galactosamine, followed within 10 minutes by either antibody treatment or an equivalent volume of PBS, and then immediately followed by 50ug of SEK. All deaths occurred within 24 hours after SEK administration, and all surviving mice remained without symptoms for up to 30 days of monitoring. Data analyzed by Fisher's Exact Test (GraphPad Prism 6 software). NS = no significant difference.

Table 3. Competitive mAb combination does not protect from SEK lethal shock.

Treatment	Total	Survived	Died	p-value
SEK + PBS	5	0	5
SEK + mAb-4G3	10	2	8	NS
SEK + mAb-9H2	10	1	9	NS
SEK + mAb-4G3 + mAb-9H2	10	3	7	NS
SEK + mAb-4G3 + mAb-9H2 (intravenous)	5	0	5	NS

Note. Six to eight week-old female BALB/c mice were treated with a series of 3 intraperitoneal injections, first with 25mg of D-galactosamine, followed 10 minutes later by either antibody treatment or an equivalent volume of PBS, and then immediately followed by 100ug of SEK. All deaths occurred within 24 hours after SEK administration, and all surviving mice remained without symptoms for up to 30 days of monitoring. Data analyzed by Fisher's Exact Test (GraphPad Prism 6 software). NS = no significant difference.

Figure 4. MAb combination is protective against sepsis by S. aureus USA300. (A) 6-8 week old mice female BALB/c mice were intravenously administered 1mg of mAb-4G3 or mAb-5G2 or both (1:1: ratio) or an equivalent volume of PBS control, followed within 1 hr by intravenous administration of 5.1 × 10⁶ CFUs of USA300 strain, B-155. Where indicated, vancomycin was administered once daily for 3 days starting 24 hrs before infection. Survival curves were analyzed by Log-rank (Mantel-Cox) test (GraphPad Prism 6 software). (B) Body weight was monitored daily during the course of 21-day experiment. Change in body weight is relative to weight of same group of mice on Day 0, prior to bacterial challenge. Mann-Whitney test (GraphPad Prism 6 software): * P ≤ 0.05, ** P ≤ 0.01.

Figure 5. mAb booster is mildly protective from sepsis with SEB+ s. aureus. (A) Six to 8 week-old female BALB/c mice were intravenously administered 1mg of mAb-4G3 (IgG2b) or mAb-5G2 (IgG1) or both (1:1 ratio) or an equivalent volume of PBS control, followed within 1 hr by intravenous administration of 1.1 × 10⁸ CFUs of MRSA strain W-132. (B) Similarly to panel A, mice were intravenously administered 1.2 × 10⁸ CFUs of W-132, and received an additional intravenous administration of antibody or PBS control at 5 days post-infection. Survival curves were analyzed by Log-rank (Mantel-Cox) test (GraphPad Prism 6 software).