We extracted amplified fragments from a 1

We extracted amplified fragments from a 1.5% agarose gel BNC105 (MinElute Gel extraction kit, Qiagen) and sequenced using forward primer JA168. Mothers infected with subtype A more effectively neutralized subtype A than D. Conclusions: Breast milk from HIV-infected women showed homotypic and cross-subtype neutralization of HIV by IgG-dependent and -impartial mechanisms. These data direct further investigations into mechanisms of resistance against postnatal transmission of HIV to infants from their mothers. Keywords: Breast milk, HIV, Neutralization, IgG, IgA, Uganda, Subtype A, Subtype D, Mucosal immunity Introduction Breastfeeding accounts for 25C44% of the up to 330,000 cases of mother-to-child transmission (MTCT) of HIV contamination annually, particularly in sub-Saharan Africa.1,2 The risk of postnatal MTCT by breastfeeding is 8C12%,1,3 despite daily exposure to HIV in breast milk. Breast milk also lowers morbidity and mortality of children from malnutrition, respiratory and diarrheal diseases. 3C5 As a result, current WHO recommendations for infant feeding among HIV-infected mothers in resource-poor nations encourage continued unique breastfeeding for up to 6 months.6 The primary vehicles for MTCT of HIV infection are cell-free and cell-associated HIV virions found in maternal blood, vaginal fluids and breast milk.7 The amount of virus per liter of milk is low, so cumulative exposure of the infant to breast milk over time is more likely to lead to transmission of the virus in mothers that shed more cell-free viral particles in their breast milk.8 Both colostrum9 and milk10C13 contain HIV-specific IgG and IgA as well as innate immune constituents, each of which shows inhibition of HIV inhibition = BNC105 25) was 23 years (range 20C30). Median values for CD4+ T-cells were 410/L (range 123C934), plasma HIV RNA 85,992 copies/mL (range <400C750,000) and breast milk HIV RNA 77 copies/mL (range BNC105 4C43,363). Mothers whose newborns tested positive for HIV by PCR between birth and six months were termed transmitters and mothers of newborns screening negative were termed non-transmitters. Exclusion criteria and study approval is as previously explained.20 Breast milk samples Breast milk samples were obtained between 4 and 14 weeks postnatally. Mothers manually expressed breast milk after washing with water. Samples were centrifuged at 3000 rpm for 15 min at room temperature, visible lipid removed and lipid-poor milk (heretofore milk) aliquoted. Milk was not heat-inactivated. Cells were washed twice with phosphate buffered saline (PBS), re-suspended in 0.5 mL PBS, and frozen at ?70 C. Neutralization With modifications of reported assays,21 we incubated HIV subtype A (92UG031) or subtype D (92UG005) (AIDS Research and Reference Reagent Program, NIAID, NIH:U-NAIDS Network for HIV Isolation and Characterization) at 50 TCID50 per well with milk diluted 1:4C1:16 in RPMI 1640 (Invitrogen; Carlsbad, CA), 5% BNC105 IL-2 (Roche, Basel, Switzerland), heat-inactivated 10% FBS (Hyclone, Logan, UT) and gentamicin (Invitrogen) for 30 min at 37 C. This combination was added for 2 h to peripheral blood mononuclear cells (PBMCs) activated Rabbit Polyclonal to PKC delta (phospho-Tyr313) for 3 days with phytohemagglutinin (PHA)(Sigma, St. Louis, MO), washed and incubated at 37 C with media changed on day 4. We tested supernatants on day 6 for p24 by ELISA (Perkin Elmer, Waltham, MA). Percent neutralization was calculated as 100 ? ([p24 with computer virus and milk/p24 with computer virus alone] 100). We decided breast milk toxicity by counting viability of PHA-activated PBMCs at 2 h and 4 days by trypan blue exclusion after incubation with milk diluted 1:4 for 2 h at 37 C and washing. Samples showing toxicity were treated with Lipid Removal Agent (LRA, 2 mg in 0.5 mL milk; Suppelco, St. Louis, MO) for 4 h at 4 C, centrifuged, and reassayed. Fractionation and purification of antibodies in breast milk We prepared purified IgG milk fractions by incubating milk (500 L) and 350 L magnetic protein G beads (Miltenyi-Biotec, Auburn, CA) as altered from the manufacturers protocol. After collecting the non-IgG flow-through, the antibody-bound beads were washed with radioimmunoprecipitation (RIPA) buffer, low salt buffer and PBS, and IgG was eluted with pH shift buffer (0.1 M Triethylamine and 0.1% triton-X, pH 11.0) and adjusted to pH 7 with 1 M MES (pH 3.0). We restored the 500 L volume with PBS, buffer exchanged with PBS to avoid toxicity using Amicon ultra-0.5 centrifugal filter devices (Millipore; Billerica, MA). For IgA, we incubated milk (500 L) with 400 L of washed SSL7 agarose (Invivogen, San Diego, CA) in a spin filter (Pierce, Rockford, IL) rocked overnight at 4 C. The non-IgA flow-through was collected, the antibody-bound agarose washed with buffer (10 mM sodium phosphate, 150 mM sodium chloride, pH 7.2), and IgA eluted with 500 L of 0.1 M glycine buffer (pH 2.7), adjusted to pH 7 using 1 M tris-HCL (pH 9.0) and buffer exchanged. Purified IgG and.