Sex steroid hormone influences replication of major HIV subtypes

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1 Sex steroid hormone influences replication of major HIV subtypes Viswanath Ragupathy, Ph.D CBER/FDA Bethesda, MD st International Workshop on HIV & Women from Adolescence through Menopause Washington DC, Jan -,, 20

2 Background According to UNAIDS, almost half of all HIV- infected individuals worldwide are women. Studies comparing the course of HIV- infection between women and men have demonstrated considerable sex differences in the manifestations of HIV- disease (Nat.Med 2009). HIV- infected women tend to have lower viral loads early in HIV- infection but progress faster to AIDS for a given viral load than men (Gandhi et al 2002). Is HIV progression sex-linked? A genetic variant on the X chromosome may explain why some HIV-infected women are slower to develop full-blown AIDS than men. Single nucleotide polymorphism (SNP) of 6-7% located on the X chromosome significantly reduced rate of CD4 cell loss throughout the disease's progression and a lower viral load (The scientist 3 th Aug 2009) Women appear to be more susceptible to HIV- infection, but have lower plasma viral RNA levels and a more diverse population of HIV- variants.

3 Cont.. It has also been reported that there may be subtype specific differences in rates of disease progression (Laeyendecker, O. et al. CROI meeting abstracts, 2006) beta-estradiol attenuates HIV- replication in both primary human PBL and antiretroviral efficacy of D4T in PBL cultures (Zhang M et al 2008). Esterdiol and Progesterone regulate HIV - replication in peripheral blood cells at mid-proliferative phase (low hormone) increased replication, and midsecretary phase (high hormone) decreased HIV- replication (Asin et al 2008). Hormonal contraception verses HIV acquisition studies resulted in conflict results (Hormonal Contraception & HIV An Update 2004) Thus wish explore the pathogenesis of different HIV variants in the cells from male and female donors in the presence or absence of sex steroid hormones.

4 Objectives To study the replication capacity of HIV clades sex specific variations in replication of clades effect of sex hormones on replication of clades HIV replication kinetics within and between males and females in PBMC, Dc & macrophages

5 Methods Fresh/Frozen donor PBMC of 6 Female & 6 males were cultured with RPMI (5%FBS & IL2 media and seeded at a concentration of million cells/ml in a 24 well plate. For Dendritic Cells (DC) and Macrophages fresh monocytes were obtained from buffy coat of either gender and cultured, differentiated with RPMI (5%FBS) media containing IL4/MCSF. HIV subtype ASI, BNSI, CNSI, DSI, FSI, CRF0, CRF02, MN strain and HIV-2, Group O & N ( strains) were infected at a concentration of ng p24 per million cells. After 2 hours of incubation at 37c, 5%co2, cells were washed and cultured for 4 days. Dc s s co-cultured cultured with autologus PBMC. HIV replication was measured with quantization of p24/p27 (Perkin Elmer/Zeptometric kits) values in culture sups at,7 and 4.

6 Methods Cont Female hormone concentrations used: Esterdiol Progesterone Testosterone : nm/ml and.75 micro molar/ml : 2.5nM/ml and 64nm/ml : 3.5nM/ml Male hormone concentrations used: Esterdiol Progesterone Testosterone : 40pM/ml and 40pM/ml : pm/ml : 2ng/ml and ng

7 Results Invitro HIV Clade dynamics PBMC Least square Least Sq means Mean high moderate low Sub B Sub C CRF0 GR-N Sub D Sub A MN

8 Results cont. PBMC Subtype F, CRF02, Group O did not establish good infection. Among the viruses kinetics studied Sub B > Sub C > CRF0, Sub A & D > MN in both the genders. However the rates were varied between the gender.

9 Results cont.. HIV clade dynamics between gender Least Sq Mean Female Females cells ~60% more susceptible to infection Male

10 Progesterone down regulates HIV MN Strain (clade B) in cells from Women but not in Men Strain MN (W2) Day 2 0. No Hor Low.Est High Est Low Prog High Prog Test Strain MN (M) Strain MN (W) Day 2 Day 2 No Hor Low.Est High Est Low Prog High Prog Test No Hor Low.Est High Est Prog Low Test High Test

11 Progesterone/estrogen has opposite effects with HIV CRF0_AE in cells from Women and Men 0 CRF0_AE (W2) Day 2 0. No Hor Low.Est High Est Low Prog High Prog Test 00 CRF0_AE (M) Day 2 0 No Hor Low.Est High Est Prog Low Test High Test

12 HIV clades differentially regulated by hormones in cells from Women and Men 00 0 Clade A (W2) Day 2 0 CRF0_AE (W) Day 2 No Hor Low.Est High Est Low Prog High Prog Test 0. No Hor Low.Est High Est Low Prog High Prog Test 0 Clade C (M) Day Clade C (M2) Day 2 No Hor Low.Est High Est Prog Low Test High Test No Hor Low.Est High Est Prog Low Test High Test

13 Sex Hormones suppress/stimulate HIV replication in certain donors 00 Clade B (M3) Day 2 0 No Hor Low.Est High Est Prog Low Test High Test 00 0 Clade B (W) Day 2 0 Clade C (W2) Day 2 No Hor Low.Est High Est Low Prog High Prog Test No Hor Low.Est High Est Low Prog High Prog Test

14 Jurkat/H9 cells To study HIV clades infectivity with & w/o hormone MN (HIV MN strain) Strain Day Low High 0 No Hormone Low Estrogen High Estrogen Low Progesterone High Progesterone Low Testosterone High Testosterone

15 Previous studies had reported that the R5/X4 determines the infectivity, we wish to determine R5/X4 expression levels with and w/o hormone.

16 Effect of Hormones on CD4-CCR5/CxCR4 PBMC Counts CD4 CxCR4 CCR No Hormone Estrogen Progesterone Testosterone Mix CD4/X4/R5 Change No change in X4/R5 Change

17 Effect of Hormones on CD4-CCR5/CxCR4 T-cell lines

18 Effect of Hormones on CD4/CCR5/CxCR %Max CD4 CCR5 CXCR No Hor Est Prog Test Est/Prog

19 Conclusions Part PBMC cells from female donor cells showed higher susceptibility to infection than male cells. Sex hormones affect HIV replication kinetics and possibly at the receptor level (CD4, R5 or X4) Cell lines were less sensitive for infectivity of HIV clades at p24 concentrations used for primary cells.

20 Monocyte derived dendritic (MDDC) cells in transmission of HIV subtypes between the gender

21 Background Majority of HIV transmissions world wide is through sexual route(70 (70-80%). The probability of sexual HIV- transmission from female-to male (0 04%( 04% per act [95% CI ]) 4]) and male-to to-female (0 08%( 08% per act [95% CI ]) in high income countries, Low-income countries female-to to-male (0 38% per act [95% CI ]) ]) and male-to to-female (0 30%( per act [95% CI ]) (Lancet( Infect Dis 2009) DCs are thought to be involved in the sexual transmission of HIV-. In humans, two main DC subsets have been identified: CDc+ myeloid DC (MDC) and CDc- CD23+ plasmacytoid DC (PDC). Several in vivo studies indicate that intraepithelial and submucosal MDC are the first cell types that are the target of HIV/SIV after intravaginal infection. Thus, MDC play a crucial role in sexual HIV- transmission through viral capture in the mucosa and subsequent transfer to T cells. Hence we wish to study HIV clades transmission kinetics with and w/o sex hormones.

22 Adsorption and Transmission events through MDDC HIV Capture Trafficking Transmission Persistent infection

23 Results DC 2hrs post infection M W HIV-I/II, p24/p27 pg/ml HIV-2 CRF02_AG Clade C Clade B Group O Uninfected

24 HIV replication kinetics in DC, w/o PBMC co culture HIV-/2 p24/p27 pg/ml W CRF02 Sub C Sub B HIV-2 HIV-/2 p24/p27 pg/ml W2 CRF02 Sub C Sub B HIV-2 GR-O M M2 HIV-/2 p24/p27 pg/ml 0. CRF02 Sub C Sub B HIV-2 GR-O HIV-/2 p24/p27 pg/ml CRF02 Sub C Sub B HIV-2 GR-O

25 Donor to Donor variations of HIV infection in DC HIV kinetics in DC 7 days post infection Donors HIV-I/II, p24/p27 pg/ml W W2 W3 W4 W5 Dc s from Women - CRF02_AG Clade C Clade B HIV-2 Group O HIV kinetics in DC 7 days post infection Donors 5 M Dc s from Men HIV-I/II, p24/p27 pg/ml M2 M3 M4 M5 - CRF02_AG Clade C Clade B HIV-2 Group O ~ -3 log differences in infectivity

26 HIV-I p24 pg/ml 0 Clade B W Estrogen down regulates certain clades in DC to autologus t-cell transmission 0 Clade C M2 No Hor Low Est High Est Low Prog High Prog Test Group O W No Hor Low.Est High Est Low Test High Test Prog HIV-2 W5 HIV-I p24 pg/ml No Hor Low Est High Est Low Prog High Prog Test Clade C W 0. 0 No Hor Low.Est High Est Low Prog High Prog Test Clade B W2 HIV-I p24 pg/ml 0 0. No Hor Low Est High Est Low Prog High Prog Test No Hor Low.Est High Est Low Prog High Prog Test

27 Progesterone down regulates certain clades in DC to autologus t-cell transmission HIV-2 M HIV-2 p27 pg/ml No Hor Low.Est High Est Low Test High Test Prog Clade C W4 No Hor Low.Est High Est Low Prog High Prog Test

28 Testosterone down regulates certain clades in DC to autologus t-cell transmission 0 Clade C M3 Clade B M2 No Hor Low.Est High Est Low Test High Test Prog No Hor Low.Est High Est Low Test High Test Prog HIV-2 M2 0 Clade C M HIV-2 p27 pg/ml 0. No Hor Low.Est High Est Low Test High Test Prog No Hor Low.Est High Est Low Test High Test Prog

29 In some donors two hormones down regulates certain clades in DC to autologus t-cell transmission HIV-2 W4 0. No Hor Low.Est High Est Low Prog High Prog Test Clade B W5 No Hor Low.Est High Est Low Prog High Prog Test

30 In certain donors hormones do not impact in DC to autologus t-cell transmission 0 Clade C W5 No Hor Low.Est High Est Low Prog High Prog Test

31 Hormones modulate HIV replication in Monocyte derived Macrophages (MDMs) of either gender 20 yr old male Clade B yr old female Clade B Day 3 Day Day 3 Day 6 Day Day p24 pg/ml 800 p24mpg/ml Estrogen.75 mm Estrogen nm Estrogen 40 pm Estrogen 40 pm Progesterone 64 nm Progesterone 32 nm Progesterone 2.5 nm Progesterone pm Testosterone ng/ml Testosterone 5 ng/ml Testosterone 2 ng/ml Testosterone ng/ml Control Estrogen.75 mm Estrogen nm Estrogen 40 pm Estrogen 40 pm Progesterone 64 nm Progesterone 32 nm Progesterone 2.5 nm Progesterone pm Testosterone ng/ml Testosterone 5 ng/ml Testosterone 2 ng/ml Testosterone ng/ml Control 4 yr old female clade C yr male Clade C Day 3 Day 6 Day Day 3 Day 6 Day Estrogen 75 nm Estrogen nm Estrogen 40 pm Estrogen 40 pm Progesterone 64 nm Progesterone 32 nm Progesterone 2.5 nm Progesterone pm Testosterone ng/ml Testosterone 5 ng/ml Testosterone 2 ng/ml Testosterone ng/ml Control Estrogen 75 nm Estrogen nm Estrogen 40 pm Estrogen 40 pm Progesterone 64 nm Progesterone 32 nm Progesterone 2.5 nm Progesterone pm Testosterone ng/ml Testosterone 5 ng/ml Testosterone 2 ng/ml Testosterone ng/ml 0 0 Control

32 Conclusions part 2 Significant donor to donor variation was observed. Similar to PBMC clades B and C transmit efficiently compared with other clades HIV-2 2 and Group O did not show productive infection in certain donor cells Estrogen/Progesterone modulation of HIV- replication in MDMs was more consistent in female than male donors. Hormone treatment had impact on transmission kinetics but was not consistent among various clades and donors Further studies are needed to evaluate these observations and their in vivo significance.

33 Acknowledgements Lab of Molecular Virology/CBER Indira Hewlett, Ph.D Krishna Devadas, Ph.D Owen Wood, Ph.D Sherwin Lee, BS Andy Dayton, M.D., Ph.D Office of Biostatistics and Epidemiology Funding Ghanshyam Gupta FDA s Office of Women s Health Jiangqin Zhao, M.D., Ph.D Shixing Tang, M.D., Ph.D Armeta D, BS

Modulation of HIV replication by steroid hormones in monocyte derived macrophages

Modulation of HIV replication by steroid hormones in monocyte derived macrophages Krishnakumar Devadas, Ph.D. Laboratory of Molecular Virology Division of Emerging and Transfusion Transmitted Diseases