Protection in passively immunized mice following i.n. challenge with <i>B. pseudomallei</i> strain 1026b.

<p>BALB/c mice were administered 1 mg of either CPS IgG3 mAb 3C5 or LPS IgG3 mAb 4C7 alone or 1 mg of each mAb in combination by the i.p. route. Intranasal challenge was performed 18 h later with 15 LD₅₀ of <i>B. pseudomallei</i>. Mice were monitored for 21 days after which gross pathology and spleen cfu were determined on survivors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035386#pone-0035386-t001" target="_blank">Table 1</a>). Control mice were treated with 1 mg of an irrelevant IgG3 mAb. <i>p</i> values of survival vs. controls are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035386#pone-0035386-t001" target="_blank">Table 1</a>.</p

Survival and gross pathology of mice passively treated with mAbs.

a<p><i>p</i> value vs. controls determined from Kaplan-Meier survival plots by log-rank (Mantel-Cox) test, bold values are statistically significant (<i>p</i><0.05).</p>b<p>positive spleen cfu was determined on survivors and assumed to occur in mice that died before study endpoint.</p>c<p><i>p</i> values vs. controls determined by Fisher's exact test, bold values are statistically significant (<i>p</i><0.05).</p>d<p>spleen cfu was assessed on survivors only; values indicate cfu determined by plating 100 µl from a 1 ml spleen homogenate; T indicates too numerous to count.</p>e<p>determination of abscess formation on internal organs was performed on survivors only.</p

Detection of CPS within a splenic abscess by IHC.

<p>Organs were harvested from control BALB/c mice (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035386#pone-0035386-g003" target="_blank">Fig. 3</a>) that were infected with <i>B. pseudomallei</i> strain 1026b. A tissue section from a spleen that contained multiple large abscesses is shown (left panel). Location of CPS was identified by HRP-labeled mAb 3C5 (brown). Box within the panel on the left indicates the boundary of an abscess and surrounding normal splenic tissue (tissue within box is magnified in right panel). White scale bars indicate 50 µm.</p

Protection in passively immunized mice following i.n. challenge with <i>B. pseudomallei</i> strain K96243.

<p>mAbs were administered by the i.p. route at the doses (µg) listed. Intranasal challenge was performed 18 h later with 2 LD₅₀ of <i>B. pseudomallei</i>. Mice were monitored for 21 days. Control mice were treated with 1 mg of an irrelevant IgG3 mAb. <i>p</i> values of survival vs. controls are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035386#pone-0035386-t001" target="_blank">Table 1</a>.</p

Effect of mAb dose and combination therapy in mice challenged with <i>B. pseudomallei</i> strain 1026b.

<p>Mice were administered mAb(s) by the i.p. route followed 18 h later by i.n. challenge with 15 LD₅₀ of <i>B. pseudomallei</i>. (A) Dose-response experiment in which mice were treated with the doses (µg) listed of each mAb alone. (B) Multiple doses of mAbs 3C5 and 4C7 were administered in combination at the doses (µg) listed. Mice were monitored for 42 days after which gross pathology and spleen cfu were determined on survivors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035386#pone-0035386-t001" target="_blank">Table 1</a>). Control mice were not treated with mAb. <i>p</i> values of survival vs. controls are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035386#pone-0035386-t001" target="_blank">Table 1</a>.</p

Nomenclature and construction of heavy-chain hybrids of mAb F26G3.

<p>The CH1, CH2 and CH3 domains of IgG3 mAb were replaced with the respective domains of the IgG2b subclass switch variant of mAb F26G3.</p

Net charge of each murine IgG subclass and domain at pH 7.4^a.

a<p>Net charges were calculated assuming an environment of pH = 7.4 using amino acid sequences obtained from the ImMunoGeneTics online database (<a href="http://www.IMGT.org" target="_blank">www.IMGT.org</a>).</p

LPS binding affinity of each 1A4 subclass mAb analyzed by SPR using an antibody capture analysis approach.

<p>Anti-mouse antibody was covalently immobilized on a CM5 sensor chip. Each subclass of mAb 1A4 was injected individually over the chip surface, followed by injection of various concentrations of LPS (60–8,000 nM). Data shown is representative of three independent experiments with similar results. <b>Panel A</b> illustrates the complex formed on the chip surface. <b>Panel B</b> presents the sensorgrams (<b>top</b>) and steady-state binding analysis (<b>bottom</b>) of each 1A4 subclass variant.</p

Binding of subclass switch families of mAbs F24F2 and F26G3 to γdPGA.

<p>A – Scatchard plots for determination of functional affinities from equilibrium binding. Binding was assessed by SPR using intact mAb and sensor chips coated with 25-mers of γdPGA. B – K_D for binding of each mAb shown in Panel A. K_A was determined from the slope of the regression lines in Panel A. K_D was calculated as 1/K_A. C - Binding of each mAb as assessed by ELISA in which plates were coated with purified native γdPGA. Results are reported as the concentration of each mAb (ng/ml) that produced an OD₄₅₀ = 2.0 in a standard ELISA format.</p

Assessment of binding of subclass switch families of mAbs F24F2 and F26G3 by fluorescence perturbation.

<p>Changes in mAb fluorescence (excitation wavelength = 284 nm; emission wavelength = 341 nm) are reported for parental F24F2 or F26G3 IgG3 and subclass switch variants upon addition of increasing amounts of synthetic (γ-d-Glu)₅. The solid lines are hyperbolic fits of the data to the equation: y = (ax^b)/(c^b+x^b), where c is the apparent dissociation constant (K_D) in µM.</p