Article pubs.acs.org/jpr
Elevated Concentrations of Milk β2-Microglobulin Are Associated with Increased Risk of Breastfeeding Transmission of HIV‑1 (Vertical Transmission Study) Alain Mangé,†,‡ Edouard Tuaillon,§,∥,⊥ Johannes Viljoen,⊥,# Nicolas Nagot,§,⊥ Sophie Bendriss,§,⊥ Ruth M. Bland, # Marie-Louise Newell,# Philippe Van de Perre,§,∥,⊥,○ and Jérôme Solassol†,‡,▽,○ †
University of Montpellier 1, EA 2415, 641 av. du Doyen Gaston Giraud, 34093 Montpellier, France Department of Clinical Oncoproteomic, ICM Val d’Aurelle, 208 Avenue des Apothicaires, 34298 Montpellier, France § University of Montpellier 1, 5 Bd Henri IV, 34295 Montpellier, France ∥ Department of Bacteriology and Virology, CHU Arnaud de Villeneuve, 371, av. du Doyen Gaston Giraud, 34295 Montpellier, France ⊥ INSERM, U1058, 60 Rue de Navacelles, 34394 Montpellier, France # Africa Centre for Health and Population Studies, University of KwaZulu-Natal, R618 en rouite to Hlabisa, Somkhele A2074 Rd, Mtubatuba 3935, South Africa ▽ Department of Biopathology, CHU Arnaud de Villeneuve, 371 av. du Doyen Gaston Giraud, 34295 Montpellier, France ‡
S Supporting Information *
ABSTRACT: There is increasing evidence to support a relationship between human immunodeficiency virus (HIV-1) transmission through breastfeeding and milk host factors. We analyzed skim milk proteome to further determine the contribution of host factors to the risk of mother-to-child transmission of HIV-1. Quantitative mass spectrometry analysis was performed on nine case−control pairs of HIV+ transmitter/ nontransmitter mothers, and specific biochemical assays on two selected proteins were assessed in an independent validation set of 127 samples. 33 identified proteins were differentially expressed between HIV+ transmitter and nontransmitter mothers. Among them, β2-microglobulin was significantly higher in the maternal transmitter than in the nontransmitter groups (p value = 0.0007), and S100A9 was significantly higher in the early maternal transmitter cases (before 4 months of age) compared with the nontransmitters (p value = 0.004). β2-Microglobulin correlated with milk and plasma HIV viral load and CD4+ cell count, whereas S100A9 correlated with the estimated timing of infection of the infant through breastfeeding. Finally, β2-microglobulin concentration in milk could accurately predict the risk of HIV-1 postnatal transmission by breastfeeding (p value < 0.0001, log-rank test). In conclusion, milk β2-microglobulin and S100A9 are host factors that are found to be associated with mother-to-child transmission of HIV-1. KEYWORDS: HIV transmission, milk, proteomics
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from mother to child.10 However, despite advances in HIV-1 transmission prevention, mechanisms of HIV-1 transmission through breastfeeding remains poorly understood. We, and others, have shown that high levels of cell-free HIV-1 RNA as well as cell-associated HIV-1 proviral DNA in breast milk increase the risk of breastfeeding transmission, suggesting a role of both cell-free and cell-associated HIV-1 in breastfeeding transmission.6,11−13 The length and feeding mode of breastfeeding have also been shown to determine the infant’s risk of postnatal transmission of HIV-1.9 Finally, host factors present in breast milk may also play important roles in an infant’s susceptibility to infection with HIV-1. For example, erythropoietin in human milk
INTRODUCTION
Human milk is key for infant nutrition. It also protects the integrity of the infant’s gut by delivering immunological and nonimmunological innate factors with antimicrobial or antiinflammatory properties. However, despite its beneficial effects, breast milk can also be a vehicle for the transmission of viruses to the infant, including cytomegalovirus, human T-lymphotropic virus type I, and human immunodeficiency virus type 1 (HIV-1).1−7 Mother-to-child transmission of HIV has become a rare event in well-resourced settings, but around 330000 children were newly infected with HIV in 2010, with 90% of these in subSaharan Africa.8 The risk of postnatal transmission of HIV-1 through breastfeeding, in the absence of targeted interventions, has been estimated to be 3.2 per 100 child-years of breastfeeding.9 Recent advances in prophylactic HIV-1 treatment have led to significant decreases in perinatal transmission of HIV-1 © 2013 American Chemical Society
Received: June 18, 2013 Published: October 21, 2013 5616
dx.doi.org/10.1021/pr400578h | J. Proteome Res. 2013, 12, 5616−5625
Journal of Proteome Research
Article
Figure 1. Flow chart illustrating the main steps of the study.
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has been suggested to be a protective agent against mother-to-child transmission of HIV-1 through breastfeeding.14 Not all infants who are breastfed by HIV-1-infected mothers become infected, even in the absence of maternal or infant prophylactic treatment.15 Thus, identifying potential factors in breast milk that are protective or that facilitate transmission of HIV-1 is an important area of research to prevent mother-tochild in resource-poor settings. Proteins are a major contributor of immunologically active components in human milk. Human breast milk has been analyzed over the past decade using comprehensive proteomic approaches, increasing the number of identified proteins.16−19 Among them, proteins involved in immunity and inflammation have been estimated to be 24% of the proteins identified in human milk, signifying the critical role that milk plays in protecting infants from infection.18,19 However, despite recent progress in the inventory of proteins in human milk, there is still limited knowledge on the defense proteome. In addition, a global protein profiling assay has, to our knowledge, not yet been investigated to specifically identify host factors associated with HIV-1 transmission via breast milk. Here we conducted a case−control study to identify factors associated with increased or decreased risk of HIV-1 transmission by breastfeeding in a cohort of women from South Africa using a quantitative proteomic approach.
METHODS
Patient Selection
For this case−control study, breast milk samples were collected from a nonrandomized intervention cohort (the Vertical Transmission Study - VTS) of women attending nine antenatal clinics in KwaZulu-Natal, South Africa.5,20,21 This study aimed to examine breastfeeding and HIV-1 transmission (in the absence of prophylactic HIV-1 treatment) in a community with a high prevalence of HIV-1 infection. The VTS and breast milk analyses were approved by the Biomedical Research Ethics Committee of the University of KwaZulu-Natal. Breast milk samples were collected by gentle bimanual expression at 6 weeks postpartum and monthly thereafter and stored as whole breast milk (−80 °C). Postnatal transmission in infants was defined as HIV-1 acquisition (positive PCR) between 6 and 28 weeks of age in a previously uninfected child. Venous samples were taken from women at enrollment for plasma RNA load assessment and CD4 cell count. For the discovery set analysis, nine case−control pairs, with information on breast milk viral load and postnatal transmission status, were selected from the VTS cohort. For cases, the last breast milk sample available before the estimated timing of infant transmission was considered for analysis. Controls were 5617
dx.doi.org/10.1021/pr400578h | J. Proteome Res. 2013, 12, 5616−5625
Journal of Proteome Research
Article
Figure 2. Gene Ontology (GO) classification of the 212 identified proteins. Cellular compartments (A) and molecular functions (B) according to GO classification of the identified proteins from nine skim milk samples of HIV-1 transmitter and nontransmitter mothers.
for further analysis: two or more unique peptides with a high confidence score (>99%), a p value for protein quantification assigned by the ProteinPilot software 99% confidence score and 5% local FDR. This represents 27% of the total proteins identified in all experiments. Interestingly, among them, 58 proteins had not previously been identified in human milk. MS and MS/MS analysis results are summarized in the Supporting Information. (See Supplemental Tables S2, S3, and S4 in the Supporting Information.) The biological processes and the molecular functions of all of the 212 proteins identified in the breast skimmed milk were classified using a GO classification system. The subcellular distribution of identified proteins was 39% cytoplasmic proteins, 30% extracellular space proteins, 21% membrane proteins, and 10% nucleus proteins (Figure 2A). Skim proteins were associated with a broad range of pathways, molecular functions, and biological processes (Figure 2B and Supplemental Table S5 in the Supporting Information). Functionally, 46% of proteins were involved in immune response, defense, and inflammation
(Figure 2B). The other proteins were associated with various cellular functions including cellular metabolism, signaling, lipid transport and metabolism, complement and coagulation cascade, response to nutrient level, or regulation of cell growth and cell adhesion (Figure 2B). Identification and Characterization of the Differentially Expressed Proteins
To identify the differentially expressed proteins related to postnatal HIV-1 transmission through breastfeeding, we compared protein profiles of each case−control pair. First, we selected proteins that correspond to our biological selection criteria: (1) proteins identified and quantified in all three iTRAQ labelings; (2) proteins identified with at least two peptides with a high confidence; and (3) proteins with a differential expression of at least 1.3-fold relative to the control sample and a p value
