Serum samples from an ETEC-endemic area contain antibodies to both A- and B-subunits of LT.

<p>Human patient serum from Sierra Leone were evaluated for antibodies to LT, A-subunit, and B-subunit compared to commercially purchased control serum using endpoint titer analysis. (A) Anti-LT, anti-A, and anti-B ELISA antibody titers for all tested patient samples with geometric means indicated (lines). (B) Correlations between anti-LT and anti-A (open squares) or anti-B titers (gray diamonds) by Spearman’s Rank Order Correlation Coefficient (r) analysis. Antibody responses to LT holotoxin significantly correlated with both anti-A (<i>P</i><0.0001) and anti-B (<i>P</i><0.0001).</p

Anti-A or anti-B subunit antibodies neutralize LT toxicity, with maximal responses when both are present.

<p>(A) Neutralization of toxin-mediated intestinal fluid secretion in the patent mouse assay after intragastric feeding with buffer (1) or 25 μg LT combined with anti-A (2), anti-B (3), negative control sera (4), or toxin alone (5). (B,E) Neutralization of epithelial cell cAMP intoxication in Caco-2 cells after 3 h treatment with 0.1 μg LT pre-incubated with positive control GM1 monosialoganglioside (GM1+LT), various dilutions and combinations of anti-serum, or toxin alone. (C) Blocking of toxin GM1 binding by ELISA detection using 0.1 μg LT toxin pre-incubated with anti-sera or alone. (D) Western blot of Caco-2 cells lysates after 3 h treatment with 0.1 μg LT toxin pre-incubated with anti-sera. Blots were probed for ADP-ribosylated proteins using rAF1521 macrodomains (bottom blot) then re-probed for presence of the B-subunit using anti-B rabbit serum (top blot). Boxes indicate proteins with altered ADP-ribosylation, potentially Gsα. Significance testing for secretion and cAMP panels was done using one-way ANOVA with Tukey’s Multiple Comparison post-test. <i>P</i>-values were coded as follows: *<0.05, **<0.01, ***<0.001.</p

ETEC-challenged human serum pool contains antibodies to both A- and B-subunits of LT.

<p>(A) ETEC challenge serum (pooled 10 days after oral H10407 challenge) anti-LT, anti-A, and anti-B antibody responses detected by ELISA (gray line, circles) compared to commercially purchased control sera (black lines, open circles) using dilutions of each sample. (B) ETEC-challenge serum (1) or control serum (2) immunoblot testing for anti-LT antibodies using unboiled LT-loaded lanes or boiled LT-loaded lanes. In unboiled SDS-PAGE gels, LT runs as an 84 kD polymeric protein, pentameric B-subunit (56 kD), and LT-A (28 kD). When boiled and subjected to SDS-PAGE, LT separates into LT-A (28 kD) and monomeric LT-B (11.5 kD). (C) ETEC-challenge serum or control serum anti-LT, anti-A, or anti-B responses detected with a modified Immunoblot using a slot blot apparatus to load 0.1 μg protein (LT, A, A1, or B) with raw images (top) and quantified band density of these images for unboiled, loaded proteins graphed (bottom).</p

Both A- and B-subunits are immunogenic and induce anti-LT antibodies following mucosal immunization.

<p>For these studies, we employed an <i>in vitro</i> ELISA assay following immunization by two different mucosal routes (intranasal, sublingual). For intranasal immunizations, BALB/c mice were immunized weekly for three weeks with 10 μl saline containing 5 μg of protein antigens A1, A, B, A+B, or dmLT. For sublingual immunizations, BALB/c mice were sublingually immunized once with 10 μl saline containing 5 μg of protein antigens A, B, A+B, dmLT, or LT. All mice were sacrificed 21 days after the primary immunization. The integrity of the antigens was demonstrated by SDS-PAGE (A). Anti-LT serum IgG (B,C) or fecal IgA (C) responses were detected by ELISA. Significance testing for ELISA panels was done using one-way ANOVA with Tukey’s Multiple Comparison post-test. <i>P</i>-values were coded as follows: *<0.05, **<0.01, ***<0.001.</p

Age distribution of cases presenting to the KGH Lassa Ward, 2008–12.

<p>Panel A: Age distributions of patients presenting while antigenemic (Ag+/IgM±). Panel B: Age distributions of nonantigenemic patients presenting with serum anti-LASV IgM (Ag−/IgM+). Panel C: Age distributions of nonantigenemic patients presenting without anti-LASV IgM seropositivity (Ag−/IgM−). In Panels A–C yellow portion of bars represent patients who were discharged and black portion of bars represent patients who died. Panel D: Age demographic for the population of Sierra Leone (2010 estimate). Among patients who died, the age distributions differed significantly between the Ag+/IgM± and Ag−/IgM− groups (p = .005). Distributional comparisons were carried out using the Kolmogorov-Smirnov technique (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002748#pntd.0002748.s006" target="_blank">Table S5</a>).</p

Case fatality rates for suspected LF cases by ribavirin treatment status and serostatus.

<p>The presence of LASV Ag in serum of patients with observed survival outcomes and verified treatment status was assessed by recombinant Ag− and IgM-capture ELISA. Statistical significance for within and between group comparisons was determined using a multivariate logistic regression model (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002748#pntd.0002748.s010" target="_blank">Table S9</a>). NS = not significant.</p

Suspected cases of LF evaluated at the KGH Lassa Laboratory and numbers of patients admitted to the KGH Lassa Ward, 2008–12.

<p>Non-admitted patients include those where only blood samples were submitted for screening from referral health-posts, patients dying en route to the hospital (DOA = dead on arrival), and patients not meeting the LF suspected case criteria (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002748#pntd-0002748-t001" target="_blank">Table 1</a>). Characteristics of study patients are compiled in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002748#pntd.0002748.s002" target="_blank">Table S1</a>.</p

CFRs in suspected LF cases presenting to the KGH Lassa Ward by serostatus, 2008–12.

<p>Panel A: CFR by serostatus. The presence of LASV Ag and anti-LASV IgM in serum of patients with verifiable outcomes was assessed by recombinant Ag− and IgM− capture ELISA, respectively. Panel B: Alternative calculation of CFRs. Ag+/IgM± plus Ag−/IgM+ compared to Ag−/IgM−. Statistical significance was determined using a logistic regression model predicting CFR (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002748#pntd.0002748.s004" target="_blank">Table S3</a>). NS = not significant.</p

Geographic distribution of patients presenting to the KGH with LASV antigenemia and anti-LASV IgM serpositivity, 2008–12.

<p>Confirmed cases of LF as assessed by LASV Ag in serum or cases anti-LASV IgM are shown by year of presentation, district of residence and frequency of cases. Panel A: Patients presenting in 2008–9. Panel B: Patients presenting in 2010. Panel C: Patients presenting in 2011. Panel D: Patients presenting in 2012.</p

Monthly distribution of suspected LF cases presenting to the KGH Lassa Ward by serostatus, 2008–2012.

<p>Panel A: antigenemic Lassa fever cases (Ag+/IgM±). Panel B: Patients with serum anti-LASV IgM (Ag−/IgM+). Panel C: Patients with no Lassa virus seropositivity (Ag−/IgM−). The monthly frequency distributions differed between each of the serostatus group comparisons as assessed using a Poisson regression model (p<.001 for all serostatus comparisons; data not shown).</p