table_1_Elevated IgG Responses in Infants Are Associated With Reduced Prevalence of Mycobacterium tuberculosis Infection.PDF

Background<p>It is unclear whether antibodies can prevent Mycobacterium tuberculosis (Mtb) infection. In this study, we examined the relationship between total plasma IgG levels, IgG elicited by childhood vaccines and soil-transmitted helminths, and Mtb infection prevalence, defined by positive QuantiFERON (QFT) test.</p>Methods<p>We studied 100 Mtb uninfected infants, aged 4–6 months. Ten infants (10%) converted to positive QFT test (QFT+) within 2 years of follow-up for Mtb infection. Antibody responses in plasma samples acquired at baseline and tuberculosis investigation were analyzed by enzyme-linked immunosorbent assay and ImmunoCAP^® assay.</p>Results<p>QFT− infants displayed a significant increase in total IgG titers when re-tested, compared to IgG titers at baseline, which was not observed in QFT+ infants. Bacille Calmette-Guérin (BCG) vaccine-specific IgG2 and live-attenuated measles vaccine-specific IgG were raised in QFT− infants, and infants who acquired an Mtb infection did not appear to launch a BCG-specific IgG2 response. IgG titers against the endemic helminth Ascaris lumbricoides increased from baseline to QFT re-testing in all infants.</p>Conclusion<p>These data show raised IgG associates with a QFT-status. Importantly, this effect was also associated with a trend showing raised IgG titers to BCG and measles vaccine. Our data suggest a possible protective association between raised antibody titers and acquisition of Mtb infection, potentially mediated by exposure to antigens both related and unrelated to Mtb.</p

image_3_Elevated IgG Responses in Infants Are Associated With Reduced Prevalence of Mycobacterium tuberculosis Infection.tif

Background<p>It is unclear whether antibodies can prevent Mycobacterium tuberculosis (Mtb) infection. In this study, we examined the relationship between total plasma IgG levels, IgG elicited by childhood vaccines and soil-transmitted helminths, and Mtb infection prevalence, defined by positive QuantiFERON (QFT) test.</p>Methods<p>We studied 100 Mtb uninfected infants, aged 4–6 months. Ten infants (10%) converted to positive QFT test (QFT+) within 2 years of follow-up for Mtb infection. Antibody responses in plasma samples acquired at baseline and tuberculosis investigation were analyzed by enzyme-linked immunosorbent assay and ImmunoCAP^® assay.</p>Results<p>QFT− infants displayed a significant increase in total IgG titers when re-tested, compared to IgG titers at baseline, which was not observed in QFT+ infants. Bacille Calmette-Guérin (BCG) vaccine-specific IgG2 and live-attenuated measles vaccine-specific IgG were raised in QFT− infants, and infants who acquired an Mtb infection did not appear to launch a BCG-specific IgG2 response. IgG titers against the endemic helminth Ascaris lumbricoides increased from baseline to QFT re-testing in all infants.</p>Conclusion<p>These data show raised IgG associates with a QFT-status. Importantly, this effect was also associated with a trend showing raised IgG titers to BCG and measles vaccine. Our data suggest a possible protective association between raised antibody titers and acquisition of Mtb infection, potentially mediated by exposure to antigens both related and unrelated to Mtb.</p

Co-infection alters the frequency of T-zone localised FoxP3 cells.

<p>Spleen sections were generated for immunohistology at day 5 post-infection from mice which were infected as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003341#pntd-0003341-g001" target="_blank">Figure 1A</a>. Sections were double-stained for FoxP3 with CD3. These sections were then used to quantify FoxP3+CD3+ T cells in the T-zone per mm². Representative images show double-staining with FoxP3 (blue) and IgD (brown) with images acquired using a Leica microscope DM6000 using a 20× objective. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Data is representative of 4–6 mice per group with experiments performed twice for each time point. T = T zone; F = B cell follicle (^*P<0.05 and ^**P<0.005).</p

Prior Infection with Nb alters the control of STm and production of anti-STm IgG.

<p>WT mice were infected with 500 L3 Nb larvae and at day 16 mice were challenged with 5×10⁵ STm alongside naïve control mice. Splenic bacterial numbers were assessed at days 5 and 25 post-STm infection. Serum anti-STm IgM, IgG, IgG2a and IgG2b antibody titres were assessed by ELISA against a total outer membrane preparation of STm. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Groups contained 4–6 mice. (^*P<0.05 and ^**P<0.005).</p

Co-infection does not prevent Th1 and Th2 cell polarization.

<p>Splenocytes from mice which were infected as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003341#pntd-0003341-g001" target="_blank">Figure 1A</a> were re-stimulated ex-vivo with anti-CD3 in the presence of anti-CD28: IFNγ and IL-13 induction in <b>A</b>) CD3⁺CD4⁺ T cells and <b>B</b>) CD3⁻CD4⁻ cells was measured 6 hours post-stimulation by intracellular FACS and is represented as a proportion and/or absolute numbers. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Data is representative of 4–6 mice per group with experiments performed twice for each time point. (NS = Non-significant, ^*P<0.05 and ^**P<0.005).</p

Prior Nb Infection impairs antibody titres and vaccine efficacy following porin-immunization.

<p><b>A</b>) WT mice were infected with 5×10⁵ STm and splenic bacterial numbers were examined at day 5. Prior to infection mice were given either: i) PBS (dashed), ii) infected with 500 L3 Nb (open bar), iii) immunized with 20 µg porins (black bar) or iv) infected with 500 L3 Nb and then immunized with 20 µg porins (grey bar). <b>B</b>) WT mice were infected with 5×10⁵ STm opsonised with complement-inactivated serum from mice that had either been infected with STm for 35 days or primed with porins for 18 days and then boosted for 7 days. Splenic bacterial numbers were assessed 5 days post-infection. Prior to STm infection mice were either immunized with PBS or infected with 500 L3 Nb larvae for 16 days. Naïve control mice were infected with non-opsonised STm (open bar). <b>C</b>) Serum anti-porin IgG, IgG1 and IgG2a antibody titres were assessed by ELISA on serum isolated from mice immunized as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003341#pntd-0003341-g007" target="_blank">Figure 7A</a>, but pre STm-infection. <b>D</b>) WT mice were either i) immunized with PBS (dashed bar) ii) infected with Nb for 18 days (open bar) before immunization with 20 µg porins for 18 days or iii) immunized with PBS (black bar) iv) infected with Nb for 18 days (grey bar) before immunization with 20 µg porins for 18 days followed by a second booster immunization for 7 days. Anti-porin IgG titres were then assessed by ELISA. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Data is representative of 4–6 mice per group and experiments were performed twice. POR = Porins. (^*P<0.05).</p

Immunoglobulin-switching patterns are maintained during co-infection but serum immunoglobulin responses to STm are reduced.

<p><b>A</b>) Spleen sections were generated for immunohistology from mice which were infected as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003341#pntd-0003341-g001" target="_blank">Figure 1A</a>. Sections were double-stained for IgD with IgG1, IgG2a, IgE or CD3. These sections were used to quantify extrafollicular plasma cells per mm² and determine the proportion of the spleen occupied by germinal centres. Germinal centres were identified as areas of the follicle which were IgD^lo. <b>B</b>) Serum anti-STm IgM, IgG, IgG2a and anti-Nb IgM, IgG and IgG1 antibody titres were quantified by ELISA against a total outer membrane preparation from STm and homogenized L3 larvae, respectively. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Data is representative of 4–6 mice per group with experiments performed twice for each time point. (NS = Non-significant, ^*P<0.05 and ^**P<0.005).</p

Co-infection with Nb and STm impairs control of each pathogen.

<p><b>A</b>) WT mice were infected with either 5×10⁵ STm, 500 L3 Nb larvae or both for 5, 10, 18 or 32 days. <b>B</b>) Splenic and liver bacterial numbers were quantified from STm-infected animals. Small intestines were isolated and total worm burdens were assessed from Nb-infected mice. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Groups contained 4–6 mice with experiments performed twice for each time point. (N.D = Not detected, ^*P<0.05, ^**P<0.005 and ^***P<0.0005).</p

Co-infection alters type-specific T-helper responses to Nb but not STm.

<p>Splenocytes from mice which were infected as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003341#pntd-0003341-g001" target="_blank">Figure 1A</a> were plated with anti-CD28 and re-stimulated ex-vivo with: <b>A</b>) anti-CD3 or <b>B</b>) 10 µg/ml heat-killed STm (HK_STm) and <b>C</b>) control wells were re-stimulated with PBS. IFNγ, IL-13, IL-4 and IL-10 secretion was measured by ELISA from supernatants 48–72 hours post-stimulation. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Data is representative of 4–6 mice per group with experiments performed twice for each time point. (NS = Non-significant, N.D = Not detected, ^*P<0.05 and ^**P<0.005).</p