Pathogenic Mechanisms of HIV Transmission by Breastfeeding

Philippe Van de Perre

Montpellier University Hospital, University of Montpellier 1

Research Unit 4205

IAS 2007, Sydney

Mechanism(s) of breastfeeding transmission of HIV: the moving target

An evolving host A complex and biologically active source of infection

Cells/ml

Weeks of lactation

103

104

105

2 4 6 8 10 12AS Goldman et al. J Pediatr 1982

Macrophages

Lymphocytes

Compartmentalization of HIV-1 in breast milk

Blood Colostrum

Mother # 25

Mother # 35

Mother # 65

M2m1

M2m1

m6

m4

m3m4

M2

M1

M7

m7M8

M5

M9

Viral DNA

Viral DNA

Viral DNA

Viral RNA

Viral RNA

Viral RNA

M3

P Becquart et al; Virology 2002

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)

Portal of entry

Mucosal lesion

AlternativeReceptor(Gal Cer)

Fcreceptor

Transcytosis M cells

T LymphocytesMonocytes/macrophages

DendriticCells

Ep

ithel

ial c

ells

Free virus

InfectedCells

Lum

en

Su

b m

uco

sa

From M Neutra, 1999

From M Neutra, 1999

Polarised HIV-1 infected cell

Gal Cer

Transcytosis in an enterocyte

Macrophages, lymphocytes and dendritic cells in the lamina propria

M Bomsel, 1997

ApicalChamber

BasalChamber

Target cells

EpithelialCellsMembrane

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

Oral inoculation of macaques with SIV

* Non traumatic inoculation of tonsils with cell-free

and cell-associated SIV

• No infection of the epithelial cells

* Infection of CD4+ T cells close to M cells in the tonsil crypts

C Stahl-Henning, 1999

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

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

p=0.03

HIV-1 Transmission

SpinPlasma

FicollHypaque

Unwanted cells are cross-linked-to red blood cells

Breast milk cells plus

red blood cells of healthy control

Blood

EnrichedCD4+ TcellsFicoll -Hypaque

Redbloodcellsandrosettedcells

Irradiatedcells

Resting

CD4+ T cells

Day 5

Day 10

Day 0

Anti -CD3antibodies

Anti -CD28antibodies

Activated

CD4+ T cells

Quantification of HIV-1 DNA

by real-time PCR

Enumeration of the HIV-1-Ag SCs

by ELISPOT assay

HIV-1antigens

Detection of p24 antigen in

supernatantsby ELISA

A

B

18

Proportion of latently infected cells able to enter viral cycle

Blood Breast milk

HIV-1 DNA copies 6.948 4.788per 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

* Wilcoxson rank sum test for paired samples, P < 0.01

% of HIV-1 infected T CD4+ cells 0,9 - 1,8% 10,4 -32,4%able to enter viral cycle

1 to 3 copies / infected cell

(P Becquart, 2006)

Macrophages and dendritic cells in breast milk

-Are morphologically distinct from their possible precursor PBM

- Spontaneously produce GM-CSF

- Express DC-SIGN gene and protein

- Differentiate into CD1+ dendritic cells after incubation with IL4

M Ichikawa, 2003

Breast Milk macrophages

Soluble factors in breast milk

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)

• 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)

Anti-HIV IgG, IgA and IgM

in breast milk (Kigali, Rwanda 1988-1991)

50%

100%

0% D 15 M 6 M 18

% HIV+ mothers with +WB

in milk sample

Time post partum

IgG

IgA

IgM

P Van de Perre, 1993

Perspectives

MacrophagesMacrophagesInfected Lymphocytes Infected Lymphocytes

-

Free Virus Free Virus

Systemic CompartmentSystemic Compartment Mammary CompartmentMammary Compartment

MacrophagesMacrophagesActivated Infected Activated Infected

LymphocytesLymphocytes

« Reservoir Cells »« Reservoir Cells »

Macrophages,Macrophages,Infected LymphocytesInfected Lymphocytes

« Reservoir Cells »« Reservoir Cells »

Mucosal compartmentMucosal compartment

Free VirusFree Virus

Soluble Soluble factors?factors???

??« Reservoir Cells »« Reservoir Cells »

Infection of T CD4+ quiescente CellsInfection of T CD4+ quiescente Cells

CTL?

-

Why is the majority of infants exposed to HIV

through breastfeeding escaping infection?

• 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 ?

Daily ingestion of 322,000 free viral particles and about 25,000 infected cells (RD Semba, 1999; RW Nduati, 1995)

Surprise to come?

• 2903 detected peptides

• 143 peptides corresponding to 53 genes

Host defense/immune related, enzyme, structural, transport, DNA binding and signal transduction proteins. An additional 13% of the proteins are of unknown or unclassified function.

LC / MS MS

G Smolenski, et al.

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)

Alternative interventions to be tested?

• Periexposure prophylaxis in breastfed infants ?

• Postnatal mucosal vaccine?

• Pasteurisation? Heat treatment?

• Microbicides?

• Freezing / thawing?