Tenascin-C is an innate broad-spectrum, HIV-1-neutralizing protein in breast milk

Abstract

Achieving an AIDS-free generation will require elimination of postnatal transmission of HIV-1 while maintaining the nutritional and immunologic benefits of breastfeeding for infants in developing regions. Maternal/infant antiretroviral prophylaxis can reduce postnatal HIV-1 transmission, yet toxicities and the development of drug-resistant viral strains may limit the effectiveness of this strategy. Interestingly, in the absence of antiretroviral prophylaxis, greater than 90% of infants exposed to HIV-1 via breastfeeding remain uninfected, despite daily mucosal exposure to the virus for up to 2 y. Moreover, milk of uninfected women inherently neutralizes HIV-1 and prevents virus transmission in animal models, yet the factor(s) responsible for this anti-HIV activity is not well-defined. In this report, we identify a primary HIV-1-neutralizing protein in breast milk, Tenascin-C (TNC). TNC is an extracellular matrix protein important in fetal development and wound healing, yet its antimicrobial properties have not previously been established. Purified TNC captured and neutralized multiclade chronic and transmitted/founder HIV-1 variants, and depletion of TNC abolished the HIV-1-neutralizing activity of milk. TNC bound the HIV-1 Envelope protein at a site that is induced upon engagement of its primary receptor, CD4, and is blocked by V3 loop- (19B and F39F) and chemokine coreceptor binding site-directed (17B) monoclonal antibodies. Our results demonstrate the ability of an innate mucosal host protein found in milk to neutralize HIV-1 via binding to the chemokine coreceptor site, potentially explaining why the majority of HIV-1-exposed breastfed infants are protected against mucosal HIV-1 transmission.

Fig. 1.

TNC is a primary HIV-1–neutralizing protein in breast milk. (A) Anion-exchange chromatography with a linear gradient of 1 M NaCl (green line) of the high molecular mass fraction of milk revealed three distinct protein peaks (blue line). The IC₅₀ of protein peaks against HIV-1 env variant C.Du156 is reported. The unique 250-kDa band on reducing SDS/PAGE in the neutralizing protein fraction (peak 3, black arrow) was identified as TNC by high-resolution LC/MS/MS. Analysis of the smaller protein bands in peak 3 (blue arrows) confirmed matching identity with those contained in inactive fractions (lactoferrin and heavy chain of Ig). (B) Milk of two uninfected women was depleted of TNC by mAb 81C6 and control polyclonal human IgG immunoprecipitation and confirmed by Western blot and SDS/PAGE. Depletion of TNC reduced neutralization activity of the milk samples against HIV-1 C.Du156 Env pseudovirus compared with control IgG-treated milk samples. Line graphs indicate mean percent virus neutralization and SD of two assays performed in duplicate.

Fig. 2.

TNC neutralizes HIV-1 in a dose-dependent manner in both TZM-bl reporter cells and primary PBMCs. (A) Percent neutralization of HIV-1 pseudovirus Du156 in TZM-bl reporter cells with increasing concentrations of TNC. (B and C) Percent neutralization of clade B T/F HIV-1 variant CH40 (B) and CH77 (C) Renilla luciferase (LucR) reporter HIV-1 infectious molecular clones in TZM-bl cells (squares) and primary PBMCs (circles) with increasing concentration of TNC. Assays were performed in duplicate.

Fig. 3.

TNC captures HIV-1 virions and blocks HIV-1 interaction with colonic epithelial cells. (A) Purified TNC captures HIV-1 virions expressing Env from chronic clade B (B.BAL), chronic clade C (C.Du156), clade B T/F (B.CH40), and clade C infant T/F (C.BF1677) strains with similar potency to anti–HIV-1 Env mAb 2G12 (positive control). Bars indicate the mean percent virus capture, with lines indicating the SD. Asterisks indicate significantly lower (P < 0.05) virion capture compared with TNC, using a two-tailed Mann–Whitney U test (Prism 5; GraphPad Software) to compare the results of two assays performed in triplicate. (B) Purified TNC inhibits infectious T/F C.1086 HIV-1 virus binding to a monolayer of HT-29 colonic epithelial cells in a dose-dependent manner. Results are displayed as mean percent virus inhibition of virion epithelial cell binding compared with virus-only wells of two experiments performed in quadruplicate, with lines indicating the SD.

Fig. 4.

TNC binds to HIV-1 gp120 at a CD4-inducible epitope that overlaps the chemokine coreceptor binding site via a charge–charge interaction. (A) Interaction of TNC and B.MN gp120 is potently blocked by preincubation of the Env protein with anti-V3 loop mAbs 19B and F393F. Percent blocking is in parentheses. (B) Binding to both the C clade T/F HIV-1 C.1086 Env gp120 and gp140 proteins is enhanced by preincubation of Env proteins with soluble CD4. (C) TNC binding to B.MN gp120 captured by soluble CD4 is abolished by prebinding of mAb 17B, an mAb directed against the chemokine coreceptor binding site. Approximately 1,200 response units (RUs) of 17B mAb bound to B.MN gp120 are captured on the immobilized CD4 surface. (D) Binding of CD4-captured TNC to MN gp120 (100 μg/mL) is abrogated in 250 mM NaCl buffer. All data are representative of at least two experiments.