003 1-3998/92/3 10 I-0022$03.00/0 PEDIATRIC RESEARCH Copyright O 1991 International Pediatric Research Foundation, Inc. Vol. 31, No. 1, 1992 Printed in U. S.A. A Human Milk Factor Inhibits Binding of Human Immunodeficiency Virus to the CD4 Receptor DAVID S. NEWBURG, RAPHAEL P. VISCIDI, ANDREA RUFF, AND ROBERT H. YOLKEN Department ofBiochemistry, E. K. Shriver Center, Waltham, Massachusetts 02254, and Department of Neurology, Harvard Medical School, Boston, Massachusetts 021 15 [D.S.N.]; Department of Pediatric Infectious Diseases, Johns Hopkins School of Medicine, Baltimore, MD 21205 [R.P. V., R.H. Y.]; and Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland 21205 [A.R.] ABSTRACT. Perinatal transmission of human immuno- deficiency virus (HIV) from infected mothers to their chil- dren occurs at rates reported as 20-50%. The role of breast feeding in perinatal transmission of viral infections has not been well established. We studied 34 milk and colostral samples obtained from HIV-seropositive and HIV-sero- negative women to determine if they contained anti-HIV activity. We found that all the samples contained a factor that inhibited the binding of HIV epitope-specific MAb to recombinant CD4 receptor molecules. The titers of inhibi- tory activity ranged from 1:200 to 1:10 000 and did not differ between HIV-seropositive and HIV-seronegative mothers. This milk factor also inhibited the binding of gp120 to CD4. Neither human sera nor bovine milk exhib- ited appreciable inhibitory activity. Fractionation of human milk indicated that the inhibitory activity was confined to the macromolecular fraction; little activity was found in isolated milk lipids or oligosaccharides. Chromatographic procedures indicated that the active macromolecule has an isoelectric point of 9.3-9.6. The active material did not bind to concanavalin A, however, the activity was partially destroyed by chemical and enzymatic treatments that re- moved sulfated residues. The active material may thus be a sulfated protein, glycoprotein, mucin, or glycosaminogly- can that inhibits the binding of CD4 to HIV envelope glycoproteins. The role of this factor in the natural history of HIV infection in infants and children should be the subject of additional investigations. (Pediatr Res 31: 22- 28, 1992) Abbreviations HIV, human immunodeficiency virus aq., aqueous solution DEAE, diethylaminoethyl PAS, periodic acid Schiff reagent sIgA, secretory IgA Infants born to mothers infected with HIV are at high risk for HIV infection. The reported rates of vertical transmission from HIV-infected mothers range from 20 to 50% (1-5). A better Received December 14, 1990; accepted August 19, 199 1. Reprint requests: David Newburg, E. K. Shriver Center, 200 Trapelo Road, Waltham, MA 02254. Supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (1 ROI DK40540), the National Institute of Child Health and Human Development (HD 13021), and the Massachusetts Department of Mental Retardation contract (3403-8403-306). understanding of the factors that influence disease transmission is essential to development of strategies to prevent AIDS trans- mission from HIV-infected mothers to their children. Breast feeding is known to affect the transmission of viral infections from mother to child. Human milk contains a number of Ig and non-Ig factors that can inhibit the replication of pathogenic microbial agents (6-9). Conversely, human milk can contain viruses, such as cytomegalovirus and human T-cell leu- kemia virus, that can initiate neonatal infection (1 0- 12); indeed, anecdotal reports have described the isolation of HIV from breast milk and the apparent infection of infants feeding on the milk of mothers who recently had sero-converted (13- 15). The possi- ble association between breast feeding and HIV transmission poses a medical dilemma, because breast-fed infants generally display lower rates of other infections than do infants fed bovine milk-based formula (16-20). The dilemma is particularly acute in the case of infants living in the less developed countries, because numerous studies have documented increased rates of infant mortality when bovine milk formula is substituted for human milk as the principal source of neonatal nutrition (21- 24). An improved understanding of factors in human milk that may modulate HIV infection might lead to the reassessment of the potential risks and benefits of breast feeding in populations with higher rates of maternal HIV infection. Milk may modulate infection in neonates through several mechanisms. In addition to specific Ig and cell-mediated immune responses, human milk contains macromolecules that inhibit the binding of pathogenic agents to their host receptors. Such pro- tective activity has been attributed to milk glycolipids (25) and oligosaccharides (26-28). The existence of analogous inhibitors of virus-receptor interactions could be of particular importance in the case df HIV infection, in light of the antigenic variation of neutralizing epitopes on the viral envelope and the relative conservation of the sites involved in receptor interactions (29- -,\ 3 I). The CD4 molecule is the major receptor for HIV binding to T-helper lymphocytes and, possibly, to other cells that support HIV replication (32-34). Because the viral envelope is heavily glycosylated, it is likely that glycosyl groups on the envelope are important determinants of viral-cellular interactions, and that exogenous glycoconjugates can modulate HIV interaction with its receptor (35-40). The CD4 receptor molecule has been exten- sively characterized by means of its reactions with HIV envelope glycoprotein (gp120) and with MAb that bind at specific CD4 epitopes (3 1, 4 1, 42), including the gp 120 binding site. We used a solid-phase assay system to demonstrate that whole human milk inhibits the interaction of CD4 with MAb that bind at or near the site of HIV attachment and inhibits the binding of recombinant gp120 to CD4. We then purified milk samples,