Functional activities against HIV-1 of immunoglobulins purified from pooled stool samples of children breastfed by HIV-1-infected milk.

<p>Immunoglobulins (Ig) purified by immunoaffinity from pooled stool samples from 8 HIV-1-infected mothers and their breast milk exposed non HIV-infected babies (group I) and 6 couples of HIV-1-infected mothers and their breastfed HIV-1-infected babies (group II), and containing F(ab’)2 to gp160 with similar specific activities, were constituted. <b>A and B.</b> Inhibition of the attachment of HIV-1NDKon HT29 cells and of HIV-1NDKand HIV-1JRCSF on monocyte-derived macrophages (MDM) by Ig purified from pooled stools of children breastfed by HIV-1-infected milk.HT29 intestinal cell lines and MDM were incubated with HIV-1NDKor HIV-1JRCSFin the presence of 30 (A) or 50 (B) µg/ml of pooled stools purified Igfor 1 hour at 37°C. Cells were then washed, lysed, and quantities of attached virus were evaluated by HIV p24 antigen measurement in the culture lysate using capture ELISA. Lactoferrin and the monoclonal antibody IgG1B12 were used as positive controls for inhibition; IVIg and total Ig purified from pooled stools of HIV non exposed, HIV-seronegative babies were used as negative controls. The experiments were carried out in triplicate with cells from three different donors. The HIV-1NDK or HIV-1JRCSF attachment inhibitions are expressed as percentage ± standard error of three independent experiments; <b>C.</b> Inhibition of the HIV-1JRCSF infection of MDM by Ig (50 µg/ml) purified from pooled stools of children breastfed by HIV-1-infected milk.The monoclonal antibody IgG2G12 was used as positive control for inhibition; IVIg and total Ig purified from pooled stools of HIV non exposed, HIV-seronegative babies were used as negative controls. The levels of viral production at day 6 postinfection were assessed by HIV p24 antigen measurement in the culture supernatants using capture ELISA. The experiments were carried out in triplicate with cells from three different donors. The HIV-1JRCSF infection inhibition is expressed as percentage ± standard error of three independent experiments. <i>Nota bene</i>. HT-29 epithelial cells were stained with mouse phycoerythrin (PE)-conjugated monoclonal antibodies anti-CD4 (Leu3a) (Becton Dickinson Biosciences, Mountain View, CA) and CXCR4 (12G5) (BD PharMingen, Le Pont de Claix, France), and with fluorescein isothiocyanate (FITC)-conjugated anti-human monoclonal antibodies DC-SIGN (DCN46) and CCR5 (2D7) from BD Biosciences (San Diego, CA) and GalCer (MAB342) (Chemicon International, Paris, France). Analysis was assessed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences). Results are presented as the percentage of receptor-positive cells. Forty-six percent of HT-29 cells expressed GalCer, 29% CXCR4 and 10% CCR5, whereas very low (<0.1%) expression of CD4 and DC-SIGN was detected.</p

Main characteristics of study mother-child couples, including 8 couples of HIV-1-infected mothers and their breastfed non HIV-infected babies (group I), and 6 couples of HIV-1-infected mothers and their breastfed HIV-1-infected babies (group II).

£<p>World Health Organization (WHO) clinical staging of HIV/AIDS for adults and adolescent with established HIV infection according to the 2010-revised WHO recommendations for HIV care in in babies and children for resource-limited settings <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063408#pone.0063408-WHO2" target="_blank">[31]</a>;</p>$<p>WHO clinical staging of HIV/AIDS for children with established HIV infection according to the 2010-revised WHO recommendations for HIV care in babies and children for resource-limited settings <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063408#pone.0063408-WHO1" target="_blank">[30]</a>; CD4 T cell count in infants were not available because the national AIDS programme has focused on universal treatment for infants less than 12 months, independently of their clinical or immunological status;</p>*<p>Plasma HIV-1 RNA load was measuredby the Generic HIV-1 RNA quantification assay (Biocentric, Bandol, France), which threshold of detectability is 300 copies/ml <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063408#pone.0063408-Rouet2" target="_blank">[35]</a>.The molecular diagnosis of HIV infection was carried out at time of sampling; the timing of HIV infection in study children could not be assessed.</p><p>NA: Not applicable.</p

Relative ratios of excretion (RRE) by reference to lactoferrin of total IgA, IgG and IgM (A) and anti-gp160 antibodies of the IgA, IgG and IgM classes (B) in stool samples from8 HIV-1-infected mothers and their breast milk exposed non HIV-infected babies (group I) (grey bars) and from 6 couples of HIV-1-infected mothers and their breastfed HIV-1-infected babies (group II) (hatched grey bars).

<p>RRE is expressed as mean±standard error. The stars indicate the significant differences between the mean RRE of IgA, IgG and IgM, and those of HIV-specific IgA, IgG and IgM in the whole 14 study babies breastfed by their HIV-infected mothers (groups I and II) (* P<0.01).</p

Levels of total IgA, IgG and IgM in breast milk (white bars in left) and children’s stools (black bars in right) samples from 8 HIV-1-infected mothers and their breast milk exposed non HIV-infected babies (group I), 6 couples of HIV-1-infected mothers and their breastfed HIV-infected babies (group II), and 10 healthy HIV-negative breast-feeding women and their breastfed non HIV-infected babies as negative controls.

<p>The mean concentrations of milk IgA were higher than those of IgG or IgM in HIV-infected mothers as in HIV-negative mothers. The levels of milk IgG was 5.5 higher in HIV-infected mothers than in HIV-negative mothers. The levels of stool IgA and IgG in babies born from HIV-infected mothers were higher than those of HIV-negative control babies. Immunoglobulins levels are expressed in µg/ml ± standard error. The stars indicate the significant differences by comparison to HIV-negative control samples (* P<0.01).</p