Pathogenic Mechanisms of HIV Transmission by Breastfeeding

1. Pathogenic Mechanisms of HIV Transmission by Breastfeeding Philippe Van de Perre Montpellier University Hospital, University of Montpellier 1 Research Unit 4205 IAS 2007, Sydney

2. Mechanism(s) of breastfeeding transmission of HIV: the moving target A complex and biologically active source of infection An evolving host

3. Compartmentalization of HIV-1 in breast milk

4. Blood Colostrum M2 m1 M1 Mother # 25 Viral DNA M2 m1 M2 Viral RNA m3 m4 Mother # 35 M3 Viral DNA m4 M5 Viral RNA Mother # 65 m6 m7 M8 Viral DNA M7 M9 Viral RNA P Becquart et al; Virology 2002

5. Further evidence for HIV compartmentalization in breast milk • Different distribution of HIV quasispecies in right versus left breast, and in some women in different fractions of milk: suggest local micro environmental selection pressure (P Becquart, Virology 2007) • In half of NVP-treated women: different expression of resistance profiles between plasma and breast milk (E Lee, J Infect Dis 2005)

6. Portal of entry

7. Mucosal lesion Alternative Receptor (Gal Cer) Fc receptor Transcytosis M cells Free virus Lumen Infected Cells Epithelial cells Dendritic Cells Sub mucosa T Lymphocytes Monocytes/macrophages

8. From MNeutra, 1999

9. From MNeutra, 1999

10. Apical Chamber Epithelial Cells Membrane Basal Chamber Target cells Polarised HIV-1 infected cell Gal Cer Transcytosis in an enterocyte Macrophages, lymphocytes and dendritic cells in the lamina propria M Bomsel, 1997

11. Transcytosis of HIV-1 across human enterocytes • Concept of viral synapse • HIV-1 gp41 recognises a membrane agrin (heparan sulfate proteoglycan) that favour interaction with GalCer and mediate transcytosis through an integrin associated mechanism A Alfsen, 2005

12. Oral inoculation of macaques with SIV • * Non traumatic inoculationof tonsils with cell-free and cell-associatedSIV • No infection of the epithelial cells • * Infection of CD4+ T cells close to M cells in the tonsil crypts C Stahl-Henning, 1999

13. Breastfeeding transmission of HIV-1: by free virions or by HIV-infected cells?

14. Cell-free and cell-associated HIV-1 are both responsible for breast milk transmission I Koulinska, 2006 Cell-free virus Cell-associated virus indetermined < 9 m post p 2 8 6 > 9 m post p 11 8 5 Total 13 16 11 HIV-1 Transmission p=0.03

15. 18 A Breast milk cells plus red blood cells of healthy control Plasma Spin Enriched CD4+ T cells Ficoll Ficoll - Hypaque Blood Hypaque Red blood cells and rosetted cells Unwanted cells are cross-linked - to red blood cells B Irradiated Anti - CD28 cells antibodies Day 0 Quantification of HIV-1 DNA by real-time PCR Resting CD4+ T cells Anti - CD3 antibodies Day 5 Enumeration of the HIV-1-Ag SCs by ELISPOT assay HIV-1 Activated antigens CD4+ T cells Day 10 Detection of p24 antigen in supernatants by ELISA

16. Proportion of latently infected cells able to enter viral cycle (P Becquart, 2006) Blood Breast milk HIV-1 DNA copies 6.948 4.788 per 106 T CD4+ cells (2.351-23.043) (2.590-47.294) HIV-1 Ag secreting cells 45 (9-108)* 500 (205-934)* per 106 T CD4+ cells 1 to 3 copies / infected cell % of HIV-1 infected T CD4+ cells 0,9 - 1,8% 10,4 -32,4% able to enter viral cycle * Wilcoxson rank sum test for paired samples, P < 0.01

17. Macrophages and dendritic cells in breast milk Breast Milk macrophages • Are morphologically distinct from their possible precursor PBMF • Spontaneously produce GM-CSF • Express DC-SIGN gene and protein • - Differentiate into CD1+ dendritic cells after incubation with IL4 M Ichikawa, 2003

18. Soluble factors in breast milk

19. Soluble factors and innate immune factors with potential anti-HIV activity? • Lactoferrin (suggested in vitro) (MC Hamsen, 1995) • Lewis factorX (binding to DC-SIGN) (MA Naarding, 2005) • SLPI (suggested in vitro) (SM Wahl, 1997) • a Defensins ( L Kuhn, 2005; R Bosire, 2007)) • Lysozyme ? • Complement ? • Mucines ? • Prostaglandins ? • Interleukins? • “The anti-infective activity of human milk • is potentially greater than the sum of its • microbicidal components” (CE Isaacs, 2004)

20. Anti-HIV IgG, IgA and IgM in breast milk (Kigali, Rwanda 1988-1991) % HIV+ mothers with +WB in milk sample IgG IgA IgM 100% 50% Time post partum 0% D 15 M 6 M 18 P Van de Perre, 1993

21. Perspectives

22. Free Virus - Macrophages, Infected Lymphocytes « Reservoir Cells » Soluble factors? ? Macrophages Activated Infected Lymphocytes « Reservoir Cells » - Mucosal compartment CTL? « Reservoir Cells » ? Macrophages Infected Lymphocytes Free Virus Infection of T CD4+ quiescente Cells Systemic Compartment Mammary Compartment

23. Why is the majority of infants exposed to HIV through breastfeeding escaping infection? Daily ingestion of 322,000 free viral particles and about 25,000 infected cells(RD Semba, 1999; RW Nduati, 1995) • Presence of HIV-specific MHC class I-restricted CD8+ • CTLs in breast milk(S Sabbaj, 2002; BL Lohman, 2003)? • HIV specific antibodies? • Other immune factors ?

24. Surprise to come? G Smolenski, et al. LC / MS MS • 2903 detected peptides • 143 peptides corresponding to 53 genes • Host defense/immune related, enzyme, structural, transport,DNA binding and signal transduction proteins. Anadditional 13% of the proteins are of unknown or unclassifiedfunction.

25. Use of maternal and/or infants antiretrovirals during breastfeeding ? • Maternal HAART during lactation: • But… • Breast milk CD4+ T lymphocytes expressing a high level of • CCR5 and CXCR4 are preserved despite HAART (AP Kourtis, 2007); • Cell-associated HIV persists despite maternal HAART • (R Shapiro, 2005)

26. Alternative interventions to be tested? • Periexposure prophylaxis in breastfed infants ? • Postnatal mucosal vaccine? • Pasteurisation? Heat treatment? • Microbicides? • Freezing / thawing?