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

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

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

IL-17A production in response to PPD and increasing concentrations (1 and 10 µg/ml) of ADP-ribosylating toxins, detoxified mutants or subunits.

a<p>IL-17A concentration (pg/ml) in supernatants from cells isolated from BCG vaccinated volunteers (n = 6) expressed as medians (range). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> shown are from 3 independent experiments.</p>b<p>Fold rises in IL-17A concentration as compared to stimulation with PPD alone.</p>*<p>P<0.05 and **P<0.01; compared to cells stimulated with PPD alone.</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

dmLT enhances IL-17A and IL-13 responses to PHA stimulation.

<p>PBMCs from volunteers (n = 9) were stimulated with PHA, in combination with increasing concentrations of dmLT (0, 1 and 10 µg/ml) and production of IL-17A (A) and IL-13 (B) were determined. Responses to medium alone and dmLT alone (10 µg/ml) are shown by the leftmost and rightmost bars in each graph. Bars represent mean + SEM. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> shown are from 6 independent experiments. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison post test. * P<0.05, ** P<0.01 and *** P<0.001; compared to cells stimulated with PHA alone.</p

dmLT enhances IL-17A responses to components of novel ETEC and pneumococcal vaccines.

<p>(A and B) PBMCs from 20 volunteers collected pre and post-vaccination with oral inactivated whole cell vaccines against ETEC containing CTB or LCTB<i>A</i>, were stimulated with 10 µg/ml LTB with and without 1 µg/ml dmLT, and the resulting IL-17A (A) and IFN-γ (B) production was determined. Statistical analysis was performed using the Wilcoxon signed rank test. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> shown are from 6 independent experiments. (C and D) PBMCs from 8 volunteers were stimulated with WCA and increasing concentrations (0, 1 and 10 µg/ml) of dmLT, and the resulting IL-17A (C) and IFN-γ (D) production was determined. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison post test. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> shown are from 6 independent experiments. (A–D) Bars represent mean + SEM. * P<0.05, ** P<0.01; compared to cells stimulated with LTB (A and B) or WCA (C and D) alone. In A and B, separate comparisons of post- versus pre-vaccination responses are also indicated.</p

dmLT enhances IL-17A and IL-13 responses in CD4+ T cells.

<p>PBMCs and PBMCs depleted of CD4+ T cells isolated from BCG vaccinated volunteers were stimulated with PPD (A, n = 4), or PHA (B–C, n = 3), in combination with 10 µg/ml dmLT, and the IL-17A production was determined. (D–E) CD4+ T cells from another set of volunteers (n = 6) were stimulated with beads coated with anti-CD3/CD28 antibodies and increasing concentrations (0, 1 and 10 µg/ml) of dmLT, and the IL-17A (D) and IL-13 (E) concentration in culture supernatants were determined. Bars represent mean + SEM. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> shown are from 2 (A–C) or 3 (D–E) independent experiments. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison post test. * P<0.05 and ** P<0.01; compared to cells stimulated with anti-CD3/CD28 beads alone.</p

dmLT enhances IL-17A production from T cells via soluble factors and monocytes.

<p>(A) PBMCs from BCG vaccinated volunteers (n = 7) were stimulated with PHA in the presence of supernatants (Sup) derived from PBMCs stimulated with PPD, dmLT or PPD + dmLT, and the IL-17A production was determined. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> shown are from 3 independent experiments. (B) PBMCs from BCG vaccinated volunteers (n = 6) were stimulated with PPD and dmLT in the presence and absence of neutralising antibodies (Abs) against IL-1β, IL-6 and IL-23, and the IL-17A production was determined. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> shown are from 3 independent experiments. (A and B) Bars represent mean + SEM. Statistical analysis was performed using the Friedman test with Dunn's multiple comparison post test. * P<0.05, ** P<0.01 and *** P<0.001; compared to cells stimulated with PHA alone (A) or PPD plus dmLT (B). In (B), indicated differences were also significant (P<0.05) compared to treatments with isotype control antibodies. (C) CD14+ monocytes isolated from BCG vaccinated volunteers (n = 4) were pulsed with PPD alone or together with 10 µg/ml dmLT, and then washed. CD4+ T cells were then added to the monocytes, and the resulting IL-17A production was determined. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051718#s3" target="_blank">Results</a> shown are from 2 independent experiments.</p

Survival likelihood of neonatal CD-1 mice following oral challenge with wild-type O1 Ogawa <i>V. cholerae</i> O395.

<p>Three- to five-day-old pups (cohort size 20) were orally gavaged with 50 µl of a preparation containing 2.3×10⁹ CFU of wild type <i>V. cholerae</i> O395 mixed with a 1∶250 dilution of pooled day 56 serum from mice intramuscularly immunized with conjugate vaccine (OSP:TThc) and immunoadjuvantative dmLT, or dmLT alone. Survival curves were compared by log rank testing.</p