ELISA analysis of sera reactivity against GMMA.

<p>Groups 1–6 received 2 µg of GMMA with or without Freund’s adjuvant (FA), group 1) GMMA from <i>S. sonnei</i> Δ<i>tolR</i> Δ<i>galU</i> (grown in HTMC, 37°C), 2) GMMA of group 1 plus FA, 3) GMMA <i>S. sonnei</i> –pSS Δ<i>tolR</i> (defined medium, 37°C), 4) GMMA of group 3 plus FA, 5) GMMA from <i>S. sonnei</i> –pSS Δ<i>tolR</i> Δ<i>msbB</i> (defined medium, 30°C), 6) GMMA of group 5 plus FA. Group 7 received 0.2 µg of GMMA from <i>S. sonnei</i> –pSS Δ<i>tolR</i> Δ<i>msbB</i>. Control groups were immunized with PBS alone (group 8) or FA alone (group 9). Sera from individual mice obtained 14 days after the third immunization and pooled preimmune sera from each group respectively were assayed in dilutions of 1∶1000, 1∶10,000, and 1∶100,000 on GMMA from <i>S. sonnei</i> 53G –pSS Δ<i>tolR</i> as coating and arbitrary units were calculated. Data are presented as scatter plots of ELISA units determined in individual mice (groups 1–9) or of the pooled preimmune sera (pre). The horizontal lines represent the geometric mean. ELISA units of groups 1–6 receiving 2 µg of GMMA were analyzed using the non-parametric Kruskal-Wallis test to compare the immunogenicity of the different GMMA to each other and with and without FA. No statistically significant differences were found (n.s.). Reduction of the immunization dosage of <i>S. sonnei</i> –pSS Δ<i>tolR</i> Δ<i>msbB</i> GMMA to 0.2 µg (group 7) resulted in statistically significant reduction of ELISA units in the sera of the immunized animals compared to sera of mice immunized with 2 µg of the same GMMA (group 5) as determined by Mann-Whitney test (p = 0.0047). All groups receiving GMMA showed higher <i>S. sonnei</i> –pSS Δ<i>tolR</i>-specific antibody responses than groups immunized with PBS or FA alone (Mann-Whitney, p≤0.003). For all comparisons a p value smaller than 0.05 was considered to be significant.