Kinetics for clearance of CPS from serum.

<p>Mice were intravenously injected with 100 μg, 20 μg, or 4 μg of CPS. Blood samples were collected at the designated time points, and CPS concentrations in serum samples were determined using quantitative sandwich ELISA. The data were best described by a two-parameter monophasic exponential decay model (<i>y = ae</i>^<i>-bx</i><i>)</i>, where <i>a</i> is the <i>Y</i> intercept and <i>b</i> is the rate constant for clearance. Data shown are mean ± standard deviation for five mice per dose per time point. Half-life (t_1/2) values calculated from the elimination rate constant (<i>b</i>) derived from the model fitting were similar for all three doses of CPS. The results demonstrate that CPS is eliminated rapidly from serum with a half-life of 4 hours, 4.4 hours, or 2.9 hours for the doses of 100 μg, 20 μg, or 4 μg CPS, respectively.</p

<i>In vivo</i> Distribution and Clearance of Purified Capsular Polysaccharide from <i>Burkholderia pseudomallei</i> in a Murine Model

<div><p><i>Burkholderia pseudomallei</i> is the causative agent of melioidosis, a severe infection prominent in northern Australia and Southeast Asia. The “gold standard” for melioidosis diagnosis is bacterial isolation, which takes several days to complete. The resulting delay in diagnosis leads to delayed treatments, which could result in death. In an attempt to develop better methods for early diagnosis of melioidosis, <i>B</i>. <i>pseudomallei</i> capsular polysaccharide (CPS) was identified as an important diagnostic biomarker. A rapid lateral flow immunoassay utilizing CPS-specific monoclonal antibody was developed and tested in endemic regions worldwide. However, the <i>in vivo</i> fate and clearance of CPS has never been thoroughly investigated. Here, we injected mice with purified CPS intravenously and determined CPS concentrations in serum, urine, and major organs at various intervals. The results indicate that CPS is predominantly eliminated through urine and no CPS accumulation occurs in the major organs. Immunoblot analysis demonstrated that intact CPS was excreted through urine. To understand how a large molecule like CPS was eliminated without degradation, a 3-dimenational structure of CPS was modeled. The predicted CPS structure has a rod-like shape with a small diameter that could allow it to flow through the glomerulus of the kidney. CPS clearance was determined using exponential decay models and the corrected Akaike Information Criterion. The results show that CPS has a relatively short serum half-life of 2.9 to 4.4 hours. Therefore, the presence of CPS in the serum and/or urine suggests active melioidosis infection and provides a marker to monitor treatment of melioidosis.</p></div

Organ distribution of CPS.

<p>Mice were intravenously injected with 100 μg CPS per mouse. Internal organs (lungs, liver, spleen, and kidneys) were collected at various time points post-injection. The organs were homogenized in PBS. CPS amount per organ was determined by quantitative sandwich ELISA. The amount of CPS in blood samples was calculated from the CPS concentration in serum as shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005217#pntd.0005217.g001" target="_blank">Fig 1</a>. Data shown are mean ± standard deviation for five mice per time point. The negative values after subtraction of CPS amounts from serum found in each organ were adjusted to zero. The results showed that no significant amount of CPS accumulated in any of the colletced organs.</p

Western blot analysis of excreted CPS.

<p>Purified CPS (lane 1) and urine samples (lanes 2–7) were separated on 7.5% SDS-PAGE gels. All samples including purified CPS were incubated with proteinase K at 60°C for 1 hour, followed by boiling for 10 min before loading on the gels. Lanes 2, 3, and 4 were loaded with control urine spiked with CPS and incubated at 37°C for 30 min, 2 hours, and 8 hours, respectively. Lanes 5, 6, and 7 were urine from CPS-injected mice collected at 30 min, 2 hours, and 8 hours post-injection, respectively. The volume of sample loaded into each lane was adjusted to contain an equal amount of CPS, approximately 1 μg/lane. After blotting, membranes were probed with mAb 4C4 (1 μg/mL). Intact CPS was observed in urine samples from CPS-treated mice.</p

Excreted CPS detection by AMD^™ LFI.

<p>A urine sample from a CPS-treated mouse was serially diluted in mouse control urine to the indicated CPS concentrations. Each concentration of the urine sample then was tested with AMD^™ LFI. The tests were assessed by four examiners in a randomized, semi-blinded manner (panel A), and by using a lateral flow reader (panel B). The results demonstrated that AMD^™ LFI could detect excreted CPS as low as 0.2 ng/mL.</p

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

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

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

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