514G3 antibody binds to WT SpA with specificity and picomolar affinity.

<p>A. Antigen binding profiles of 514G3 captured on an active flow cell on a Biacore 3000. The colored lines on the sensogram represent the recorded binding response signals at different antigen concentrations from 2-0nM, and the overlaid black lines represent the fitted curves. Binding of SpA to 514G3 was monitored in real time to obtain on (ka) and off (kd) rates. The equilibrium constant (K_D) was calculated from the observed Ka and Kd and found to be 46.7pM. B. A graph showing the relative binding of 514G3 to SpA, MabSelect and MabSelect SuRe ligands. The three different SpA domain variants were coated on the wells of an ELISA plate, and then probed with biotinylated 514G3 or VH3/IgG3-k isotype control at 4μg/ml, and detected with streptavidin-HRP. The graph shows OD at 450nm on the y-axis and the different SpA domain variants on the X-axis. The error bars show the standard deviation from three replicates.</p

514G3 mediates opsonophagocytic killing of <i>S</i>. <i>aureus</i>.

<p>A. Opsonophagocytic killing of MRSA by human blood cells. Opsonophagocytic killing assay was done using healthy human whole blood. <i>S</i>. <i>aureus</i> was pre-incubated with either 514G3 or VH3/IgG3-k isotype control, followed by addition to anticoagulated human blood. After 20 min of incubation, the white blood cells were spun down, treated with gentamicin to kill <i>S</i>. <i>aureus</i> bound on the surface of phagocytes, lysed with saponin and plated at different time points to measure the number of surviving bacteria within the phagocytes. The y-axis shows CFUs in 100μl of lysed sample. The graph shows that >99% of the opsonized bacteria were killed within 2.5 hrs of phagocytosis and the numbers in 514G3 sample, declined over time at a higher rate when compared to either VH3/IgG3-k isotype control or PBS. The error bars show the standard deviation from three replicates. B. 514G3 mediated opsonophagocytosis of MRSA resulted in a reduction of total bacterial load in whole human blood. Anticoagulated human blood was incubated with <i>S</i>. <i>aureus</i> (NR-46223) in the presence of 514G3 or VH3/IgG3-k isotype control (10μg/ml) for 30 min, 1.5 hr, and 2.5 hr; and survival in the whole blood was measured. Relative survival was calculated at 30 min, 1.5 and 2.5 hrs as the percentage difference in CFU in the 514G3 samples compared with the VH3/IgG3-k isotype control samples. The error bars show the standard deviation from three replicates. The statistical significance was measured using a two-tailed unpaired t-test.</p

514G3 protects mice from <i>S</i>. <i>aureus</i> mediated bacteremia.

<p>A. Prophylactic administration of 514G3 protects mice against lethal challenge with MRSA in a bacteremia model: BALB/c mice (N = 10) were injected intravenously with 10 mg of 514G3 or VH3/IgG3-k isotype control 3 hours prior to intravenous injection of MRSA (3 × 10⁷ CFUs). The challenged mice were monitored for 14 days, at which point all the remaining mice were sacrificed. The vehicle control mice were injected with vehicle only. At day 14, sixty percent (6/10) from 514G3 group survived, and 0% of mice that received either VH3/IgG3-k isotype control or vehicle only survived the bacterial challenge. The p-values are 514G3 vs. vehicle control p = 0.0006, and 514G3 vs. VH3/IgG3-k isotype control p = 0.0013. B. Effect of 514G3 in combination with vancomycin: Female Balb/c mice (10 per group) from Charles River were injected with one of the following: (i) 0.5 mg of vancomycin via intraperitoneal route, (ii) sub-efficacious dose of 514G3 (2.5 mg) via intravenous route or (iii) 0.5 mg of vancomycin and 2.5mg of 514G3 two hours prior to infection with MRSA (NR-46223 at 2 X 10⁷ CFU i.v.). The control mice received only vehicle. The mice were observed for 14 days at which point all the remaining mice were sacrificed. At day 14, 10% of mice in the no treatment group and sub-efficacious dose of 514G3 survived (1/10), 30% of mice in vancomycin only survived, but 60% of mice in the vancomycin plus 514G3 survived indicating an additive effect due to co-treatment of 514G3 and vancomycin (p = 0.021 compared to antibody only group).</p

514G3 mediated antibody dependent <i>S</i>. <i>aureus</i> phagocytosis.

<p>A. Visualization of opsonophagocytic uptake of <i>S aureus</i> by RAW264.7 cells. <i>S</i>. <i>aureus</i> were labeled with CFSE (Green), 514G3 (top panel) or VH3/IgG3-k isotype control (bottom panel) was conjugated with APC (Red) and nuclei was stained with DAPI (Blue). The stained cells were imaged on a confocal microscope by focusing on the center of the macrophage to visualize only the bacteria that have been internalized. The top panel shows co-localization of the bacteria and 514G3 as yellow-orange dots inside the cytoplasm of the RAW 264.7 cells. The bottom panel shows that the isotype control does not promote significant opsonophagocytosis as seen by the absence of yellow-orange dots inside the cells.</p

<i>In vitro</i> characterization of 514G3.

<p>A. 514G3 binds to <i>S</i>. <i>aureus</i> and leaves the Fc region exposed for FcγR1A receptor binding: 514G3 or VH3/IgG3-k isotype control was incubated with <i>S</i>. <i>aureus</i> cells for 15 min followed by the addition of biotin labeled FcγR1A. The figures show the overlay of median fluorescence intensities generated by the labeled antibodies or the Fcϒ receptor. There was a specific binding of 514G3 to the <i>S</i>. <i>aureus</i> cells within 15 min (left panel); and FcγR1A receptor could subsequently bind to the Fc region of 514G3 antibody bound to the surface of <i>S</i>. <i>aureus</i> (right panel). The right panel also shows two controls where <i>S</i>. <i>aureus</i> cells were incubated with biotinylated FcγR1A along with streptavidin-APC (light green trace) or streptavidin-APC only (dark green trace) to show that the high background is due to streptavidin-APC. The cells were analyzed on a BD Accuri and the data was analyzed using FlowJo 10.0.8. B. 514G3 is capable of binding to <i>S</i>. <i>aureus</i> in the presence of pooled human IgG. <i>S</i>. <i>aureus</i> was pre-incubated with 5mg of pooled human immunoglobulins at 37°C, before the addition of 100 μg/ml of 514G3 or the VH3/IgG3-k isotype control labeled with APC. There was an increase in the binding of 514G3 to <i>S</i>. <i>aureus</i> cell surface over the 2.5 hours tested. However, the signal with VH3/IgG3-k isotype remained the same over the time points tested. The graph plots the signal in the FL4 (APC) channel versus the total cell count. The median fluorescence intensity values are indicated in the column Median:FL4. The cells were analyzed on a BD Accuri and the data was analyzed using FlowJo 10.0.8, and the data is representative from multiple experimental repeats.</p

A natural human monoclonal antibody targeting Staphylococcus Protein A protects against <i>Staphylococcus aureus</i> bacteremia

<div><p><i>Staphylococcus aureus</i> can cause devastating and life-threatening infections. With the increase in multidrug resistant strains, novel therapies are needed. Limited success with active and passive immunization strategies have been attributed to <i>S</i>. <i>aureus</i> immune evasion. Here, we report on a monoclonal antibody, 514G3, that circumvents a key <i>S</i>. <i>aureus</i> evasion mechanism by targeting the cell wall moiety Protein A (SpA). SpA tightly binds most subclasses of immunoglobulins via their Fc region, neutralizing effector function. The organism can thus shield itself with a protective coat of serum antibodies and render humoral immunity ineffective. The present antibody reactivity was derived from an individual with natural anti-SpA antibody titers. The monoclonal antibody is of an IgG3 subclass, which differs critically from other immunoglobulin subclasses since its Fc is not bound by SpA. Moreover, it targets a unique epitope on SpA that allows it to bind in the presence of serum antibodies. Consequently, the antibody opsonizes <i>S</i>. <i>aureus</i> and maintains effector function to enable natural immune mediated clearance. The data presented here provide evidence that 514G3 antibody is able to successfully rescue mice from <i>S</i>. <i>aureus</i> mediated bacteremia.</p></div

Screening of anti-SpA antibodies using ELISA and Opsonophagocytosis assays.

<p>A and B: Phage ELISA to monitor reactivity of the clones after seven rounds of panning: Sixty-two high affinity clones were analyzed by Phage ELISA against SpA from <i>S</i>. <i>aureus</i>. The x-axis has the different phage clones and the y-axis has the absorbance at 450 nm. The empty vector has a 180 bp stuffer sequence between the two Sfi sites within the phagemid and phage generated with the empty vector is used as a negative control. The error bars show the standard deviation from three replicates. Top eight clones were further tested by opsonophagocytosis assay. C: Anti-SpA antibodies mediate opsonophagocytosis: Differentiated HL60 cells were incubated with opsonized fluorescently stained <i>S</i>. <i>aureus</i> cells. Increase in fluorescence intensities within cells correlates directly with the internalization of the <i>S</i>. <i>aureus</i> bacteria through opsonophagocytosis. Greater than 85% of the HL60 cells had phagocytosed CFSE labeled bacteria in the presence of all eight of the anti-SpA antibodies. The background opsonophagocytosis by the HL60 cells in the presence of VH3/IgG3-k isotype control was about 30%. The error bars show the standard deviation from three technical replicates.</p