Activated CD4 T Cells Spontaneously Producing HIV1 in Breast Milk from Women Treated with Antiretrov

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Activated CD4 T Cells Spontaneously Producing
HIV-1 in Breast Milk from Women Treated with
Antiretroviral Drugs

• Diane Valea, Edouard Tuaillon, Yassine Al Tabaa,
  François Rouet , Pierre-Alain Rubbo, Nicolas
  Meda, Vincent Foulongne, Karine Bollore,
  Jean-Pierre Vendrell and Philippe Van de Perre

-Centre Muraz, Bobo-Dioulasso, Burkina
Faso -University Montpellier 1, EA 4205, France
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Background (1)

• Mechanisms of breast milk transmission of HIV-1
  remain poorly understood
• In breast milk, HIV-1 may be present as three
  different forms
• Cell-free virus (CFV) measured as HIV-1 RNA in
  lactoserum,
• Cell-associated virus (CAV) measured as HIV-1 RNA
  released in supernatants by activated cells,
• Integrated provirus measured as HIV-1 DNA.

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Background (2)

• Some babies breastfed by HIV-1 infected mothers
  taking HAART get infected despite HIV RNA being
  undetectable in their mothers plasma and breast
  milk (Manigart O et al, J Infect Dis 2004 Thomas
  T et al, CROI 2008, abstr. 45 aLB)
• In vitro, HIV-1 infectivity is known to be 100 to
  1000 times higher in CAV than in CFV stocks
  (Dimotrov DS et al, J Virol 1993)

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Objective

• To study activated CD4T cells producing
  spontaneously HIV-1 possibly responsible for
  HIV-1 postnatal transmission through
  breastfeeding
• Sample collection from HIV-1 infected lactating
  women receiving short regimen PMTCT prophylaxis
  or HAART,
• Characterization/enumeration of freshly purified
  CD4 T-cells using an ELISPOT assay,
• HIV-1 RNA quantification in supernatants after
  overnight cell-culture.

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Methods (1) Study population and sample
collection

• 15 HIV-1 infected lactating women (9 on short
  perinatal regimen, 6 on HAART),
• 70 ml of milk and 20 ml of blood for each woman,
• Mean duration of lactation, 42.2 days (range 9-91
  days).

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Methods (2) Characterization of CD4T cells in
breast milk and blood
A
Breast milk cells plus red blood cells from
healthy control
Spin
Enriched CD4 T cells
Ficoll-
-
hypaque
Red blood cells and
Rosetted (unwanted cells)
Characterization of CD4T cells by flow cytometry
B
Day 0
HIV-1 RNA quantification in supernatants by real
time RT-PCR
Quiescent CD4 T cells
Day 1
Enumeration of the HIV-1-Ag -SCs by ELISPOT
assay
Day 1
Quiescent spontaneous activated CD4 T cells
HIV-1
antigens HIV-1 RNA
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Results (1) cells surface markers expression on
breast milk and blood T lymphocytes
amean with range in parenthesis bNS,not
significantly
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Results (2) Detection of ex-vivo CD4T
lymphocytes secreting spontaneously HIV-1 Ag in
milk and blood
N7
N8
N7
N8
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Results (3) Quantification of Cell-associated-HIV
-1 RNA in culture supernatants
N7
N8
N7
N8
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Sammury (1)

• Most of breast milk CD4 T cells expressed low
  level of surface-CD45RA, high levels of HLA-DR
  and CD38 markers, characterizing spontaneously
  activated memory cells.
• Our data
• are consistent with the physiological role of
  breast milk as a source of immune active cells,
• suggest a minimal blood contamination since
  peripheral blood contain a large proportion of
  naive cells.

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Sammury (2)

• Presence of HIV-1-AgSCs in breast milk from all
  women, regardless plasma HIV-1 RNA levels.
• Similar number of immunospots in aviremic vs.
  viremic women.
• Presence of cell-associated HIV-1 RNA in breast
  milk supernatants. But, higher HIV-1 RNA levels
  in supernatant of blood versus breast milk cell
  culture.

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Conclusions

• HIV-1 RNA detection in breast milk from aviremic
  individuals suggests a residual virus
  replication. This reservoir may represent a
  potential source of HIV-1 transmission via
  breastfeeding from women successfully treated
  with ARVs.
• Alternative prevention strategies against
  breastfeeding transmission of HIV-1 should be
  urgently evaluated.

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Centre Muraz, Bobo-Dioulasso, Burkina
Faso Diane Valea François Rouet Nicolas
Meda
University Montpellier 1, EA 4205,Montpellier,
France Edouard Tuaillon Yassine Al
Tabaa Pierre-Alain Rubbo Vincent
Foulongne Karine Bollore Jean-Pierre Vendrell
Philippe Van de Perre
This study was supported by the Agence Nationale
de Recherches sur le Sida et les hépatites ANRS