Mimi Ghosh, PhD

1
Intrinsic Anti-HIV Activity in Cervical-vaginal
Secretions from HIV Positive and HIV Negative
Women
Mimi Ghosh, PhD Department of Physiology Dartmouth
Medical School USA
2
The New Face of HIV/AIDS Epidemic Increasingly
Female

• Heterosexual Transmission 80 Women more likely
  (2X) to contract HIV infection during vaginal
  intercourse than men.
• Proportion of women currently living with HIV
• 61 in Sub-Saharan Africa
• 43 in the Caribbean
• 29 in Asia
• 26 in Eastern Europe
• Currently 15.4 million women living with HIV/AIDS
  worldwide

World Health Organization, CDC, UNAIDS, NIAID
3
Paradoxically, the estimated rate of HIV-1
transmission per coital act is 1122 to 11000.
Previous studies from our lab and others indicate
that secretions from human female reproductive
tract (FRT) contain a spectrum of antimicrobials,
some of which are anti-HIV. HBD2, SLPI, Mip3a
(Ghosh et al, AJRI, 2009), Elafin (Ghosh et al,
in press, Immunology)
Hypothesis Cervical-vaginal lavages (CVL)
contain endogenous factors that are protective
against pathogens such as HIV. The levels of
these factors decrease with disease progression.
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Methods

• Patient demographics
• CD4 cut-off values for HIV() women gt350 (n32),
  gt200 (n19), lt100 (n6). None of the women were
  on antiretroviral drugs.
• HIV(-) women Healthy, age-matched, n15
• HIV Viral Stocks
• IIIB (X4) and BaL (R5) (obtained from Dr. P.
  Gupta, Univ. of Pittsburgh, PA).
• NL4.3 (X4) and YU-2.c (R5) (obtained from Drs.
  Kappes and Ochsenbauer-Jambor, UAB, Alabama).
• CH077.c A Clade B infectious molecular clone
  matching the nucleotide sequence determined to be
  the transmitted/founder virus sequence of CHAVI
  subject 700010077 (Keele 2008) (Obtained from
  Drs. Kappes and Ochsenbauer-Jambor, UAB,
  Birmingham, AL).
• Measurement of HIV-1 anti-gp160 IgG antibody
  levels in CVL Specimens were tested by kinetic
  ELISA (kELISA). Duplicate wells were coated with
  MN strain gp160.
• TZM-bl assay for viral infection

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TZM-bl Cell Assay for HIV-1 Infection
Binding
Fusion

• TZM-bl cells
• Express CD4, CCR5, CXCR4
• HIV-1 LTR promoter linked to reporter cassette
  B-Gal/Luciferase driven by TAT
• Luminescence is proportional to HIV infection

Reverse Transcription
Integration
Transcription
Splicing
Translation
TAT
TAT
U3
U5
R
b-gal/luciferase
Reporter Genes
HIV-1 LTR Promoter
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CVL from healthy HIV() and HIV(-) women have
intrinsic anti-HIV-1 activity against X4/IIIB and
R5/BaL viruses
CVL collected from HIV() and HIV(-) women were
diluted 14 and incubated with X4/IIIB and R5/BaL
for 1 hr at 370C prior to adding to TZM-bl cells.
Viral infection was quantified by addition of
substrate 48 hrs later. (Similar results for
NL4.3 and YU2-c).
7
CVL from healthy HIV() and HIV(-) women have
intrinsic anti-HIV activity against transmitted
HIV-1 CH077.C
CVL collected from HIV() and HIV(-) women were
diluted 14 and incubated with CH077 for 1 hr at
370C prior to adding to TZM-bl cells. Viral
infection was quantified by addition of substrate
48 hrs later .
8
Lack of correlation between anti-HIV activity and
levels of antimicrobials in HIV(-) women
MEDIUM
HIGH
LOW
9
HIV specific IgG levels in CVL correlate
significantly with anti-HIV activity
No IgA was detected
10
Window of Vulnerability in the Female
Reproductive Tract

• Sex hormones are key regulators of all aspects of
  the immune system in the reproductive tract!
• As a part of a normal menstrual cycle, there is a
  window of vulnerability (7-10 days) during which
  the potential for viral infectivity is optimized,
  owing to hormonal suppression of parts of the
  innate, humoral and cell-mediated immune systems
  in the FRT.
• Immune suppression occurs in the upper (Fallopian
  tubes, uterus, endocervix) and lower (ectocervix
  and vagina) FRT as an integral part of the
  physiological processes that underlie successful
  reproduction.
• Moreover, immune suppression coincides with the
  recruitment of potentially infectable cells and
  the upregulation of co-receptors on target cells
  that are essential for viral uptake.
• Wira and Fahey, AIDS, 2008

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Levels of Antimicrobial factors in healthy HIV()
CVL vary with the menstrual cycle
N 23
Plt0.05
12
Amounts of antimicrobials change with disease
progression
N15
N32
N19
N6
Not on ARVs
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Conclusions

• CVL from healthy HIV() and HIV(-) women have
  intrinsic anti-HIV-1 activity against both
  laboratory and transmitted strains. A given CVL
  can be differentially inhibitory toward different
  strains.
• This activity could not be correlated with a
  single antimicrobial factor, presumably because
  multiple anti-HIV factors are secreted by cells
  of the female reproductive tract.
• In CVL from HIV() women, anti-HIV activity
  correlated with the levels of anti-HIV gp160 IgG
  antibodies.
• Levels of anti-HIV endogenous microbicides were
  higher in the CVL during the secretory phase
  compared to the proliferative phase of the
  menstrual cycle, suggesting that women are
  vulnerable to infection depending on the stage of
  their cycle.
• Reduced anti-HIV activity was observed in women
  with lower CD4 counts. Levels of antimicrobials
  in CVL decreased with HIV disease progression.
  Possible correlations with MIP3a and HBD2.

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Implications Vaccines and Microbicides

• Identification of endogenous microbicides in the
  FRT and their regulation by sex hormones may lead
  to new avenues to prevent HIV infection.
• Our findings suggest that the FRT utilizes a
  combination of endogenous antimicrobials to
  control the transmission of HIV. Our findings
  suggest that the answer to HIV transmission in
  women exists in the FRT, but that there is no
  single magic bullet!

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Acknowledgments
Dartmouth Medical School Dept. Of Physiology Wira
Lab Charles Wira, PhD John Fahey, PhD Zheng
Shen, MD
Dartmouth Medical School Dept. of Medicine Peter
Wright, MD
Brown University Susan Cu-Uvin, MD Kenneth Mayer,
MD Zhijin Wu, PhD
University of Alabama Christina
Ochsenbauer-Jambor,PhD John Kappes, PhD
This work was supported by a National Institutes
of Health Grants AI-51877 and AI-071761 (CRW).
AI40350 and AI066884, (SUC), U01-AI067854 (JCK
and COJ) Lifespan/Tufts/Brown CFAR P30AI42853,
CDC106795 (SUC and KM) CHAVI, A1067854 (PW) and
the Virology and Nucleotide Sequencing Cores of
the UAB CFAR (P30-AI27767).