Why STI-associated genital tract inflammation still matters in HIV transmission

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2/1/21 Why STI-associated genital tract inflammation still matters in HIV transmission

Efficiency of transgressing mucosal barrier low with only few foci of virus detected

Lab, IDM, University of Cape Town, Cape Town, South Africa Head of the CAPRISA Mucosal

does genital tract inflammation place women at higher risk for HIV infection?

infections to draining lymph nodes and then, systemically Li et al. 29 Haase et al.

212 The Battlefield Window of opportunity: Manage inflammation (STIs) to block HIV

SIV-specific effector CD8+ T cell SIV-infected target cell Effector target conjugate

increased risk for acquiring HIV Women who later became HIV-infected had pre-infection

1 2/1/21 Why STI-associated genital tract inflammation still matters in HIV transmission A healthy female genital mucosa presents a relatively effective barrier to HIV entry during sex Miller et al. 2 Protection by mucous barrier Window of opportunity: 6 days to intervene! Efficiency of transgressing mucosal barrier low with only few foci of virus detected Jo-Ann Passmore PhD Medical Scientist NHLS Head of the Genital Mucosal STI/HIV (GEMS) Lab, IDM, University of Cape Town, Cape Town, South Africa Head of the CAPRISA Mucosal Lab, CAPRISA, NRF-DST Centre of Excellence in HIV Prevention, Durban, South Africa How does genital tract inflammation place women at higher risk for HIV infection? The battlefield is fuelled by inflammation allowing spread of local genital tract infections to draining lymph nodes and then, systemically Li et al. 29 Haase et al. 21 Haase et al. 212 Li et al. 29 Haase et al. 21 Haase et al. 212 The Battlefield Window of opportunity: Manage inflammation (STIs) to block HIV infection! SIV-specific effector CD8+ T cell SIV-infected target cell Effector target conjugate SIV-specific effector CD8+ T cell SIV-infected target cell Effector target conjugate Part 1 Women with genital tract inflammation (cytokines) prior to HIV infection were at increased risk for acquiring HIV Women who later became HIV-infected had pre-infection genital inflammation IL-1α TNF-α IL-1β IL-1 MIP-1β MIP-1α IP-1 Max Later became HIV-infected (n=8) Remained HIV-uninfected (n=8) Min CID 21 epub 1.193/cid/civ298 Dr Lindi Masson (UCT), Dr Lenine Liebenberg (CAPRISA) 1

2/1/21 Genital tract chemokines (MIP-1α, MIP-1β, IP-1, ) were associated with HIV acquisition Cytokine HIV Negative Women (n=8) HIV Seroconvertors (N=8) Pair-Matched Odds Ratio (9% CI) % detectable

( 2-2 1) 874 Per 1 log1 pg/ml increase log1 pg/ml log1 pg/ml in individual women was IP-1 1 88 ( 94 2 47) 2 26 (1 4-2 86) 1 94 (1 19-3 17) 8* 2 81 relatively (2 41-3 28) constant 3 18 (2 82-3 92)

2 2/1/21 Genital tract chemokines (MIP-1α, MIP-1β, IP-1, ) were associated with HIV acquisition Cytokine HIV Negative Women (n=8) HIV Seroconvertors (N=8) Pair-Matched Odds Ratio (9% CI) % detectable (n) % detectable (n) Per detection of cytokine MIP-1β 43 1% (2) 7 9% (44) 3 17 ( ) 3* MIP-1α 1 % (9) 44 8% (26) 3 9 ( ) 6* 7 9% (44) 89 7% (2) 2 64 ( 87 8 ) % (34) 6 3% (3) 1 6 ( 2-2 1) 874 Per 1 log1 pg/ml increase log1 pg/ml log1 pg/ml in individual women was IP ( ) 2 26 ( ) 1 94 ( ) 8* 2 81 relatively ( ) constant 3 18 ( ) over 1 8 (1 9 time ) 22 IL-1α 1 97 ( ) 2 22 ( ) 1 6 ( ) ( ) 1 43 ( ) 1 47 ( 8-2 4) 164 IL-1-37 ( ) - 14 ( ) 1 21 ( ) ( ) 87 ( ) 1 23 ( 7 2 1) 419 IL-1β 27 ( ) 2 ( ) 1 22 ( ) 26 TNF-α - 68 (-1 1-1) - 62 (-1-31) 1 16 ( ) 498 predicted HIV acquisition with an Odds Ratio of 3.2 HIV+ HIV- Total present* absent Total OR (9% CI) 3.2 ( ) p-value 14 *Women with or more pro-inflammatory cytokines or chemokines (MIP-1a, MIP-1b,, IP-1, TNF-a, MCP- 1,, IL-1a, IL-1b) above the 7 th percentile Significant after adjusting for age, urban/rural, condom use, hormonal contraceptives, number of sex acts, number of returned used applicators, HSV-2 status Proteomics data from CAPRISA4 CVLs is providing further confirmation that genital inflammation and barrier repair predict HIV infection risk in the trial Lyle McKinnon (and Adam Burgener) STI 214, 9 (8), 8-87 In the CAPRISA2 cohort, women infected with chlamydia, gonorrhoea or trichomonas had elevated genital tract proinflammatory cytokines.. Dr Lyle McKinnon, CAPRISA S :3-11:4am Rm M3 Mezzanine Dr Lindi Masson (UCT) - Poster Potential causes of genital inflammation in CAPRISA4 Demographic and behavioral characteristics present (n=2) absent (n=91) p-value % (n) % (n) Age in years [median(iqr)] 22 (2-24) 22 (2-2).4 Assigned to use tenofovir gel 36. (9) 4.7 (37).82 Women who reside in a rural setting 64. (16) 69.2 (63).63 Completed high school 44. (11) 4.7 (37).82 Given birth previously 72. (18) 8.2 (73).41 Married 8. (2) 4 4 (4).61 Stable partner 92. (23) 92 3 (84) 1. Reported sexual intercourse per month [median(iqr)] (4-6) 4 (3-7).4 Number of sexual partners in lifetime [median(iqr)] 2 (2-3) 2 (1-3).2 Reported always using a condom during sex 48 (12) 26.4 (24). HSV-2 status during study Baseline positive 6. (14) 8.9 (3). Acquired new infection 2. () 11 1 (1) Remained negative 24. (6) 3. (27) Clinical signs of an STI 24 (6) 13 3 (12).23 Genital discharge at sampled visit Genital ulcer at sampled visit () 2 2 (2) 1. Contraceptive choice at sampled visit Injectable (Depo-Provera or Nur-Isterate) 8. (2) 82.4 (7).73 Oral contraceptive pill 2. () 1 4 (14) Tubal ligation/hysterectomy () 2 2 (2) Intravaginal insertions within 3 days of sampled visit 4. (1) 4.4 (4) 1. Intravaginal insertions at any point during trial 2. () 17.6 (16).77 Potential causes of genital inflammation in CAPRISA4 STI (by PCR) present (n=2) Only 2% of HIV infections could be attributed to (or were a result of) an STI T. vaginalis was the most strongly predictive of genital inflammation Microbiome analysis ongoing absent (n=68) % (n) % (n) Trichomonas vaginalis 4. (8) 2.6 (14).9 Chlamydia trachomatis 2. (4) 16.2 (11).74 Neisseria gonorrhoeae. (1) 4.4 (3) 1. HSV-2. (1) 2.9 (2).4 Any one of the above STIs. (1) 33.8 (23).2 Brent Williams Ian Lipkin 2

2/1/21 Overlap between genital versus plasma cytokine signatures to predict HIV infection in CAPRISA4 Women with genital inflammation did not have similarly elevated plasma cytokine concentrations

76) 1.87 (.1-3.).9 IL-1.24 (.24-8.12).24 (.24-.88).78.89 (.2-1.93).86 (.41-1.67).1 TNF-α 3.72 (2.4-.92) 3.62 (2.6-.47).73 IP-1 147.4 (99.8-246.13) 168.9 (114.26-23.6).44 11.49 (122.68-218.63) 13.

3 2/1/21 Overlap between genital versus plasma cytokine signatures to predict HIV infection in CAPRISA4 Women with genital inflammation did not have similarly elevated plasma cytokine concentrations Plasma cytokine concentrations (pg/ml) present (n=23) absent (n=83) IL-1β.7 (.1-.23).3 (.1-.14).27 IL-1α.68 ( ).68 ( ).3.86 ( ).73 (.19-9).42.1 (.1-.8).1 (.1-.31) ( ) 1.87 (.1-3.).9 IL-1.24 ( ).24 ( ) ( ).86 ( ).1 TNF-α 3.72 ( ) 3.62 ( ).73 IP ( ) ( ) ( ) ( ).41 MIP-1β 1.63 ( ) ( ).2 MIP-1α 4.22 ( ) 1.96 ( ).13 Dr Lenine Liebenberg, CAPRISA; S pm M1-2, Mezzanine No correlation between genital tract and plasma cytokines Plasma cytokine concentrations (pg/ml) Spearman Rho p-value South African Press coverage about genital inflammation and HIV risk in women. IL-1β IL-1α IL TNF-α IP MIP-1β.61.3 MIP-1α Lindi Masson handling some very awkward questions about genital inflammation from the South African public in interviews on several radio news shows in June. Discussion (Part 1) Women who had genital inflammation were at increased risk of HIV infection Elevated concentrations of 4 chemokines (MIP-1α, MIP-1β, IP-1 and ) were associated with increased risk of HIV infection These chemokines are likely to recruit potential HIV target cells MIP-1β, in particular, bind to the HIV co-receptor CCR and specifically recruit CCR+ HIV target cells that potentially enhance HIV infection. In macaques, production of these and other inflammatory cytokines has been shown to be essential for recruitment of CD4+ T cell targets needed for SIV replication Part 2 Women who acquired HIV during CAPRISA4 had high frequencies of CD68+ and CD4+ target cells within the stratified squamous epithelium (from vaginal biopsies) in collaboration with Prof Thomas Hope and Dr Ann Carias Northwestern University, Chicago Sinaye Ngcapu, CAPRISA Lenine Liebenberg, CAPRISA 3

CD68 m/phages CD4 T cells Beta estimate (SE) Beta estimate (SE) IL-12p7 IL-1b TNF-a IL-12p4 IL-18 IL-1a MIF TNF-b TRAIL IL-12p7 IL-1b TNF-a IL-12p4 IL-18 IL-1a MIF TNF-b TRAIL Cytokine concentration

LIF M-CSF SCF SCGF-b SDF-1a b-ngf FGF-Basic G-CSF IL-9 PDGF-BB VEGF HGF IL-3 LIF M-CSF SCF SCGF-b SDF-1a b-ngf IFN-g IL-13 IL-1 IL-17A IL-2 IL-4 IL- IL-2Ra IFN-g IL-13 IL-1 IL-17A IL-2 IL-4 IL-

(2-64) cells/mm 2 Median depth 113 (82-189) um Median density 22.

(Northwestern) Sinaye Ngcapu (CAPRISA) Relationship between target cell density in biopsies and genital tract (CVL) cytokines 3 2 1-1 -2-3 4 2-2 -4 Pro-inflammatory Chemokines Growth Factors Adaptive

4 CD68 m/phages CD4 T cells Beta estimate (SE) Beta estimate (SE) IL-12p7 IL-1b TNF-a IL-12p4 IL-18 IL-1a MIF TNF-b TRAIL IL-12p7 IL-1b TNF-a IL-12p4 IL-18 IL-1a MIF TNF-b TRAIL Cytokine concentration [(log 1 (pg/ml)] IP-1 MIP-1a MIP-1b RANTES CTACK GRO-a IFN-a2 IL-16 MCP-3 MIG Eotaxin IP-1 MIP-1a MIP-1b RANTES CTACK GRO-a IFN-a2 IL-16 MCP-3 MIG Eotaxin FGF-Basic G-CSF IL-9 PDGF-BB VEGF HGF IL-3 LIF M-CSF SCF SCGF-b SDF-1a b-ngf FGF-Basic G-CSF IL-9 PDGF-BB VEGF HGF IL-3 LIF M-CSF SCF SCGF-b SDF-1a b-ngf IFN-g IL-13 IL-1 IL-17A IL-2 IL-4 IL- IL-2Ra IFN-g IL-13 IL-1 IL-17A IL-2 IL-4 IL- IL-2Ra IL-1 IL-1ra IL-1 IL-1ra 2/1/21 CD4 target cell density in CAPRISA4 vaginal biopsies CD68 cell density in CAPRISA4 vaginal biopsies Median density 3. (2-64) cells/mm 2 Median depth 113 (82-189) um Median density 22.6 (16-3) cells/mm 2 Median depth 18 ( ) um E-cadherin Nuclei CD4 + T cells Background Nuclei CD68 + cells Tom Hope, Ann Carias (Northwestern) Sinaye Ngcapu (CAPRISA) Tom Hope, Ann Carias (Northwestern) Sinaye Ngcapu (CAPRISA) Relationship between target cell density in biopsies and genital tract (CVL) cytokines Pro-inflammatory Chemokines Growth Factors Adaptive CD68+ macrophage - density - in vaginal biopsies -2 were -2 positively associated with chemokines and growth factor concentrations in matching CVLs Not so for CD4+ cells Anti- Inflam. Relationship between target cell depth in biopsies and genital tract (CVL) cytokines The depth of CD4+ target cells in the vaginal epithelium was positively associated with chemokines and inflammatory cytokines in CVL BUT depth of CD68+ target cells were associated negatively with inflammatory cytokines, chemokines and growth factors Part 3 Exploring younger age and adolescence as important risk factor for HIV infection in women 1 - =-.3 p=.6 adj. p= =-.3 p=.7 adj. p= Age [log 1 (years)] IP-1 =-.2 p=.2 adj. p= EDCTP Mucosal Primer The WISH study (Women s Initiative in Sexual Health) Adolescent genital immune activation and inflammation Study in 3 adolescent females (16-22 year olds) from Masipumelele, Cape Town (DTHF Youth Centre) and Soweto, Johannesburg (PHRU) Median age 23 (range 18-39) Heather Jaspan (UCT, UW) Linda-Gail Bekker (DTHF) 4

2/1/21 EDCTP Mucosal Primer The WISH study (Women s Initiative in Sexual Health) Cytokines (luminex, 48, Biorad) T cell activation (cytobrush CD38, HLA-DR, CCR, Ki67 on CD4+ and CD8+ by FACS, ex

5 2/1/21 EDCTP Mucosal Primer The WISH study (Women s Initiative in Sexual Health) Cytokines (luminex, 48, Biorad) T cell activation (cytobrush CD38, HLA-DR, CCR, Ki67 on CD4+ and CD8+ by FACS, ex vivo) Microbiome (16S) Culture International Partnerships Robin Shattock (Imperial UK) Francesca Chiodi (Karolinska, Sweden) Thomas Hope (Northwestern USA) NHLS LAB STI LAB Lynn Morris David Lewis (NICD) Venessa Maseko Nono Mkhize Etienne Muller (NICD) CLINICAL Glenda Gray (PHRU, MRC) Janan Dietrich (PHRU) ** Dr Shaun Barnabas (DTHF, UCT) Oral presentation O :4-12:, Door 8 Mezzanine Ms Smritee Dabee (UCT) Poster P1.4; Katie Viljoen (UCT); Heather Jaspan (UCT, SHC) CLINICAL Linda-Gail Bekker (DTHF, UCT) * LAB Heather Jaspan (UCT, UW) Shaun Barnabas (UCT, DTHF) Lindi Masson (UCT) LAB Lyle McKinnon Lenine Liebenberg Sinaye Ngcapu * Making our circle bigger The EDCTP Mucosal Strategic Primer Web 3 Collaborating SA Centres 11 Collaborating SA investigators 3 international partners * ** Prevalence of STIs (particularly CT) and BV higher in adolescent women in Cape Town compared to Johannesburg, South Africa Masi Cape Town Soweto Johannesburg Chlamydia trachomatis 41.6% (62/149) 1% (1/1) <.1 Trichomonas vaginalis 7.4% (11/149) 4% (4/1).27 Neisseria gonorrhoea 11.4% (17/149) 4% (4/1).14 HSV-2 4.7% (7/149) % (/1).289 Mycoplasma genitalium 4.% (6/149) 2% (2/1) 1. Bacterial Vaginosis (Nugent >7) 48.% (71/148) 31% (31/1).793 Total 1/1 1/1 Dr Shaun Barnabas (DTHF, UCT) Oral presentation O :4-12:, Door 8 Mezzanine Concentrations of cytokines in mucosal softcup secretions Comparing the genital cytokines between Cape Town and Johannesburg Cape Town Johannesburg Unsupervised hierarchical clustering was used to visualize the variation in genital tract cytokine concentrations in individual women and to cluster women across 2 sites in South Africa according to the similarities of their cytokine expression profiles (using Qlucore Omics Explorer). Ms Smritee Dabee (UCT) Poster P1.4 b-ngf, CTACK, IFN-a2, IL-12p4, IL-16, IL-18, IL-2Ra, IL-3, IL-, LIF, MCP-3, MIF, SCF, SDF-1a, TNF-b

Inflammation (/9 inflammatory cytokines in 7 th percentile) Cumulative Spearman correlation coefficient % of CD4 + T-cells % of CD4 + T-cells % of CD4 + T-cells 2/1/21 Comparing the vaginal

Any STI BV (Nugent >7) Any STI and BV 1 p =.12 p =.147 1 1 p =.1 p =.33 p <.1 7 7 p =.46 7 p =.172 Cape Town Johannesburg 2 p =.

6 Inflammation (/9 inflammatory cytokines in 7 th percentile) Cumulative Spearman correlation coefficient % of CD4 + T-cells % of CD4 + T-cells % of CD4 + T-cells 2/1/21 Comparing the vaginal microbiome between Cape Town and Johannesburg in STI/BV negative adolescents Having an STI or BV (Nugent >7) increases the frequency of activated CD4 T cells in the female genital tract (cytobrush) Any STI BV (Nugent >7) Any STI and BV 1 p =.12 p = p =.1 p =.33 p < p =.46 7 p =.172 Cape Town Johannesburg 2 p =.1 CCR + CD38 + HLADR + CD38 + HLADR + Ki CCR + CD38 + HLADR + CD38 + HLADR + Ki67 + CCR + CD38 + HLADR + CD38 + HLADR + Ki STI-/BV- STI+ BV+ STI+BV+ Dr Katie Viljoen (UCT) Ms Smritee Dabee (UCT) Poster P1.4 Longitudinal cytobrush CD4 T cell activation T cell activation did not differ significantly between time points (spanning 4 months) T cell activation did not correlate significantly between visits Relationship between genital tract cytokines and cervical T cell activation IL-1α IL-1β IL-12p4 IL-12p7 IL-18 MIF TNF-α TNF-β TRAIL CTACK Eotaxin GRO-α IL-16 IP-1 MCP-3 MIG MIP-1α MIP-1β RANTES IFN-α2 β-ngf FGF-basic G-CSF HGF IL-3 IL-9 LIF M-CSF PDGF-BB SCF SCGF-β SDF-1α VEGF IFN-γ IL-2 IL-4 IL- IL-13 IL-1 IL-17 IL-2Rα IL-1 IL-1RA Inflammatory Chemokines Growth Factors/ Haematopoetic Adaptive Regulatory Smritee Dabee (UCT) CD4+ Ms Smritee Dabee (UCT) Poster P1.4 CD8+ Relationship between genital tract cytokines and cervical T cell activation Vaginal microbiome according to genital inflammatory cytokine score Pro-inflammatory Chemokines Growth Factors Adaptive Anti-inflammatory CCR+ CD38+ CD4+ CD38+HLADR+ HLADR+ Ki67+ CCR+ CD38+ CD8+ CD38+HLADR+ HLADR+ Ki67+ IL-1a CTACK B-NGF IFN-g IL-1 IL-1b Eotaxin FGF-basic IL-2 IL-1RA GRO-a G-CSF IL-4 IL-12p4 IL- IL-12p7 IL-16 HGF IL-13 IL-18 IP-1 IL-3 IL-1 MIF IL-17 TNF-a MCP-3 IL-9 IL-2Ra TNF-b MIG LIF TRAIL MIP-1a M-CSF MIP-1b PDGF-BB RANTES SCF IFN-a2 SCGF-b SDF-1a VEGF Dr Katie Viljoen (UCT) 6

7 2/1/21 Discussion (Part 2) A collaborating network of South African clinical and laboratory investigators has been established, with specific expertise in mucosal assessment for conducting HIV prevention research Discussion (Part 2) A collaborating network of South African clinical and laboratory investigators has been established, with specific expertise in mucosal assessment for conducting HIV prevention research Young women, particularly in Cape Town, had unacceptably high rates of asymptomatic STIs and BV, calling for an urgent re-evaluation of how appropriate our current STI surveillance and testing guidelines are for sub-saharan Africa Both having an STI and/or BV was associated with significant increases in both inflammatory cytokines and chemokines in genital secretions and frequencies of activated CD4+ HIV target cells at the cervix This study provides an important link between genital cytokine markers of inflammation and cellular activation Acknowledgements Acknowledgements Dr Lindi Masson Lecturer, UCT Ms Smritee Dabee PhD Candidate, UCT Dr Katie Viljoen CBIO UCT Dr Lenine Liebenberg Dr Shaun Barnabas CAPRISA DTHF, Lecturer, UCT UKZN Dr Lyle McKinnon CAPRISA Associate Prof, UKZN U. Manitoba Mr Sinaye Ngcapu CAPRISA Dr Heather Jaspan Research Senior Lecturer, Associate UCT; Seattle Children s, USA UCT: Shaun Barnabas Smritee Dabee Heather Jaspan Lindi Masson Katie Viljoen Shameem Jaumdally Hoyam Gamieldien Cobus Olivier Iyaloo Mbado Jean-Mari Kriek Francesca Little PHRU: Glenda Gray Janan Dietrich NICD: David Lewis Venessa Maseko Etienne Muller Nonhlanhla Mkhize Lynn Morris MIT: Kelly Arnold DTHF Linda-Gail Bekker Imperial College London: The EDCTP Mucosal Robin Shattock (WISH and MMC) study participants CAPRISA: Lenine Liebenberg Lyle McKinnon Sinaye Ngcapu Salim Abdool Karim Quarraisha Abdool Karim Desh Archary The CAPRISA 4 and 2 study participants Northwestern: Tom Hope Ann Carias Karolinska Institute: Francesca Chiodi Columbia: Brent Williams W. Ian Lipkin Mara Couto-Rodriguez Funding: EDCTP Strategic Primer CAPRISA (CIPRA, NIAID & OAR, NIH, South African NRF, USAID, FHI, LIFElab, CONRAD) South African National Research Foundation and Dept. Science and Technology Centre of Excellence 7

STI and BV prevalence in young women in Cape Town and Soweto

STI and BV prevalence in young women in Cape Town and Soweto Shaun Barnabas Genital Mucosal and STI lab Desmond Tutu HIV Foundation University of Cape Town FAMCRU University of Stellenbosch WISH: Women