1. ARVS AND PROGESTINS ON IMMUNE FUNCTION IN THE FEMALE GENITAL TRACT
Goodmorning everyone, my name is Sigcinile Dlamini, and I am from the University of Cape Town at the Department of Molecular and Cell Biology. Today I will be telling you briefly about my research which I am doing under the supervision of Dr Michelle Maritz and Prof Janet Hapgood. Our research mainly focuses on ARVs and progestins and their effects on immune gene expression in the female genital tract.
Sigcinile Dlamini, Dr Michelle Maritz and Prof. Janet P. Hapgood
Department of Molecular and Cell Biology, Faculty of Science, University of Cape Town

2. Global HIV prevalence and Hormonal Contraceptive (HC) use
Millions of people around the world are still affected by HIV/AIDS. This map shows that Sub Saharan Africa has the highest HIV prevalence in the world and also relates this region to high hormonal contraceptive use. This does not directly mean that there is a causal relationship between these two but it certainly does have an effect on women in this area.
Source: Butler et al, 2013

3. HIV Impact on Women
From this graph we clearly see the disproportional effect of HIV prevalence in men and women in South Africa. As seen in red, young women have a much higher HIV prevalence than men.

4. HIV Impact on Women
56% of new HIV infections occurred in women in 2015
Poverty, lack of education and gender inequality fuel HIV transmission and unplanned pregnancy
Discreet, long acting hormonal contraceptives most widely used
Women depend largely on partner cooperation to protect themselves from HIV and other STIs
56% of new HIV infections occurred in women in In developing countries factors such as poverty, lack of education, and gender inequality fuel both unplanned pregnancies and HIV transmission. Because of these women here who opt for contraception tend to use more discreet and long acting types such as injectables. Women in developing countries find themselves at a disadvantage because most are not able to negotiate safer sex or ensure their partners faithfulness so they largely depend on partner cooperation to protect themselves from HIV and other STIs

5. Multipurpose Prevention Technologies (MPT)
Technologies providing contraception and ARVs to women
Currently only the condom protects women from unwanted pregnancies and STIs
New MPTs can empower women with wider choice of prevention strategies
Female genital tract (FGT) main target site
Intravaginal delivery methods e.g. IVRs, topical gels
Therefore, the development of multipurpose prevention technologies that provide ARVs and contraception, and require no partner cooperation is a priority. Currently the only MPT available to women is the condom and this method still has the disadvantage of requiring partner cooperation. new MPTs that are women-controlled and discreet can empower women with a wider choice in prevention. These strategies aim to target the female genital tract mucosa directly, through methods such as intravaginal rings and topical gels.

6. ARVs for Pre-exposure Prophylaxis (PrEP)
ARVs are currently being investigated as candidates for pre-exposure prophylaxis or PrEP. The first ARV that was looked at for PrEP is Tenofovir.
Tenofovir is a potent analogue of the nucleotide adenine and it inhibits the action of reverse transcriptase by incorporating into a growing viral dna chain and terminating further growth of it.
Tenofovir Disoproxil Fumarate or TDF is an orally bioavailable prodrug of tenofovir, that is more commonly used as it is more absorbed than tenofovir. Both these drugs are known as nucleotide analogue reverse transcriptase inhibitors.
Dapivirine or DPV is another promising ARV that is being investigated for PrEP and is classified as a non nucleoside reverse transcriptase inhibitor that works by blocking viral DNA elongation by binding directly to the enzyme and inducing conformational changes of it’s active site.
The two ARVs we were interested in on our study were TDF and DPV.
Tenofovir (TFV)
Tenofovir disoproxil fumarate (TDF)
Dapivirine (DPV)

7. ARVs for Pre-exposure Prophylaxis (PrEP)
Trial
ARV
Population
Adherence
Overall Decrease in HIV incidence
TDF2 study
TDF/FTC
Oral
Heterosexual
men and women in Botswana
79%
63%
CAPRISA 004 study
TFV
1% microbicide gel
Women in South Africa
61%
39%
ASPIRE study
Dapivirine
Intravaginal Ring
Women in South Africa, Malawi, Uganda, Zimbabwe
70%
37% (excl. low adherence sites)
The efficacy and delivery of these ARVs for PrEP has been investigated in a number of clinical trials and the drug that has shown the greatest effectiveness for protection against HIV has been TDF combined with the drug emtricitabine when delivered as an oral tablet. This combination drug which is marketed as Truvada showed 63% protection for heterosexual men and women in Botswana in the TDF2 study.
Tenofovir showed 39% protection against infection in the CAPRISA study when it was delivered as a topical vaginal microbicide gel, while the recent ASPIRE study investigating the efficacy of DPV showed moderate protection of 37% against infection in women in southern Africa when it was delivered in an intravaginal ring. This was after excluding sites that had low adherence.
Adherence to PrEP is important inorder to obtain higher protection and has been a factor that has hindered the progress of other clinical trials such as the VOICE and FEM-PrEP which found no effect on protection with oral and topical TDF mainly due to low adherence.
Adherence to PrEP important to obtain higher protection
Source: Baeten et al, 2016: Hladik et al, 2015; Thigpen et al, 2012

8. Hormonal Contraceptives (HCs)
Progestin-only injectables most commonly used in Sub-Saharan Africa
Medroxyprogesterone acetate (MPA) - 3 month injection
Norethisterone enanthate (NET-EN) - 2 month injection
Studies on MPA have associated it with
Increased HIV acquisition
Disruption of epithelial barrier in FGT
MPA has been found to have immunomodulatory effects in ex-vivo studies of the FGT
The most widely used hormonal contraceptives in Sub-Saharan Africa are the progestin only injectables, medroxyprogesterone acetate known as MPA or Depo-Provera, and Norethisterone enanthate known as NET. MPA is a 3 month injection, while NET is a 2 month injection.
Over the past few years studies on MPA have associated it with increased risk of HIV acquisition as well as disruption of the natural epithelial barrier in the female genital tract that protects against infections.
In the quest to find a possible causal relationship between MPA and HIV risk, ex-vivo studies have shown that MPA has immunomodulatory effects in the female genital tract.
Medroxyprogesterone
acetate
Source: Brind et al, 2015; Quispe Calla et al, 2016; Goldfien et al, 2015

9. FGT immune expression after MPA and ARV exposure
In vitro study showed MPA unlike NET-A, decreases mRNA expression and protein levels of the pro-inflammatory genes RANTES, IL-6 and IL-8 in endocervical cells
Mechanism occurs via the glucocorticoid receptor
Recent clinical data suggest that HIV negative women using DMPA have increased levels of select pro-inflammatory cytokines including:
IL-1α, IL-1β, IL-8, RANTES, IL-2
TFV regulates cytokine expression in a cell and site specific manner
Enhanced IL-8 and TNF-α in endometrium and ectocervix epithelial cells
Decreased IL-8 in endometrium and cervix
Some examples of such studies include a study FROM OUR LAB that found that MPA, unlike NET, decreases the expression of pro-inflammatory cytokines such as RANTES, IL-8 and IL-6 in endocervical cells by acting as a partial glucocorticoid receptor agonist SIMILAR TO THE full agonist DEXAMETHASONE or DEX. Recent clinical data has shown contrasting results that HIV negative women using DMPA have increased levels of select pro-inflammatory cytokines including, IL-8, RANTES and also IL-2. This inconsistent data has left a lot of controversy on the idea that MPA increases HIV risk by being pro or anti-inflammatory.
A study looking at Tenofovir used for PrEP has also found that it regulates the cytokines IL-8 and TNF-alpha in a cell and site specific manner and this points to the idea that ARVs are also able to regulate immune function in the female genital tract, All the data available has only looked at Local immune expression in the FGT after treatment with an ARV or a progestin alone.
Source: Govender et al, 2014; Francis et al, 2016; Fichorova et al, 2014; Biswas et al, 2014

10. Research Question
What are the effects of DPV and TDF alone and in combination with MPA on select immune function genes in cells of the FGT?
With the use of MPTs delivering ARVs and hormonal contraceptives simultaneously likely to increase We decided to ask what the effects of DPV and TDF alone and in combination with MPA are on select immune function genes in cells of the FGT?

11. Aims Aim1: Investigate the effects of DPV and TDF alone on:
gene expression of the pro-inflammatory IL-8 and anti- inflammatory GILZ
cell viability
Aim 2: Investigate the effects of DPV and TDF in combination with MPA or the glucocorticoid DEX on:
Model: TZM-bl cell line
Cervical epithelial cell line
To investigate this we firstly aimed to look at the effects of DPV and TDF alone on gene expression of the pro-inflammatory cytokine IL-8 and the anti-inflammatory cytokine GILZ and on cell viability.
The second aim was to look at the effects of DPV and TDF in combination with MPA or the glucocorticoid DEX on gene expression of IL-8 and GILZ and on cell viability.
The model we used for our study were TZMbl cells which are cervical epithelial cells.

12. Experimental Approach
MPA
Incubate cells with TDF or DPV alone and in combination with MPA or DEX
Determine in vitro effects in cervical TZMBL cells
Cell viability
MTT assay
Gene expression
RT qPCR analysis
TDF
DPV
DEX
Our Experimental approach was to treat the cells with the ARVs DPV or TDF alone or treat them in combination with DEX and MPA. Then we would harvest the cells and isolate RNA and perform qPCR after certain timepoints. To assess cell viability we did an MTT assay which allows us to measure the metabolic state of the cells or reduction in cell number due to the ARVs.

13. Aim 1: What are the effects of DPV on IL-8 mRNA expression?
24 hr
48 hr
To investigate the effects of DPV on IL-8 expression, TZM-bl cells were treated with concentrations ranging from 0.1 nM to 1 µM for 24 hrs on the left and 48 hours on the right. After 24 hrs we saw a massive increase of IL-8 mRNA at 1 µM of DPV. After 48 hrs we saw the same effect and the response was much greater than at 24hrs. This effect at high concentrations of DPV was interesting because this was within the range of concentrations that have been used in studies evaluating DPV for delivery in the FGT.
High concentrations of DPV (1 μM) enhance expression of IL-8 after 24 and 48 hrs of treatment
Concentrations of ARVs in the micromolar range have been used for PrEP

14. Aim 1: What are the effects of TDF on IL-8 mRNA expression?
When we looked at IL-8 expression after treating the cells with TDF at concentrations between 100 nM and 1 µM TZM-bl cell we saw that after 24 hrs there was no change in expression of IL-8 but only after 48 hrs we started seeing an increase in expression of IL-8 at 1 and 10 µM TDF. From these results we found that both TDF and DPV appear to induce a pro-inflammatory response
High concentrations of TDF (1 and 10 μM) appear to enhance expression of IL-8 after 48 hrs of treatment

15. Aim 1: What are the effects of DPV and TDF on GILZ mRNA expression?
This result was further strengthened by our observation of GILZ which is an anti-inflammatory cytokine. Treatment of cells with varying concentrations of both DPV and TDF showed that the ARVs had no effect on GILZ.
Concentrations of up to 1 μM DPV and 10 μM TDF have no apparent effect on the expression of the anti-inflammatory gene, GILZ

16. Aim 1: What are the effects of DPV and TDF on cell viability?
When we investigated the effect of DPV and TDF on cell viability we found that only high concentrations of DPV, but not TDF seemed to reduce the viability of the cells. From this we concluded that DPV may be cytotoxic to the cells at such high concentrations.
Only high concentrations of DPV, but not TDF seem to reduce the viability of the cells

17. Aim 2: What are the effects of DPV and TDF in combination with MPA or DEX on IL-8 mRNA expression?
The next part of our study was to look at the effect of DPV and TDF in combination with MPA or DEX on IL-8 expression. Just to walk you through this figure, the white bars on the left show IL-8 expression after treating cells with DEX and MPA alone, the next bars in the middle show the response after treating cells with TDF in pink and DPV in green. The bars on the right show the IL-8 response after treating the cells with a combination of TDF with DEX or MPA shown in pink, or a combination of DPV with DEX or MPA.
The first interesting thing we saw from this experiment was that MPA and DEX were pro-inflammatory on their own, whereas we had expected these to be anti-inflammatory as was seen by a previous study from our lab in endocervical cells. DPV alone at 1 µM gave a proinflammatory response as we had seen earlier.
When we co-stimulated the cells with DPV and DEX or MPA this response was massively potentiated, the MPA/DPV combo shows the greatest effect, if you look at this bar compared to this last bar (point). TDF alone gave no change in the IL-8 response and after co-stimulation with DEX or MPA the response induced by DEX and MPA was also potentiated but to a much lesser extent, if you look at the white bars compared with the pink bars on the far right. From this result we found that the addition of MPA to DPV potentiates the IL-8 pro-inflammatory response.
DEX and MPA treatment on their own are pro-inflammatory
Co-treatment of cells with MPA and DPV greatly potentiates the pro- inflammatory response while MPA and TDF treatment does so to a lesser extent

18. Aim 2: What are the effects of DPV and TDF in combination with MPA or DEX on GILZ mRNA expression?
When we looked at GILZ we saw that DEX and MPA on their own induced the gene, as we had expected them to because they have both been shown to transactivate this gene, The ARVs on their own had no effect on GILZ and when we costimulated the cells with the ARVs and DEX or MPA we saw that the presence of the ARVs appeared to have no effect on the GILZ response induced by DEX and MPA.
DEX and MPA alone transactivate GILZ, unlike DPV and TDF
Addition of ARVs appears to have no effect on the GILZ response induced by DEX and MPA

19. Aim 2: What are the effects of DPV and TDF in combination with MPA or DEX on cell viability?
TZM-bl, 24hrs
When we investigated the effect of DPV and TDF in combination with DEX or MPA on cell viability we found that these combinations appeared to have no apparent effect on cell viability.
Co-treatment of cells with DEX or MPA and the ARVs TDF or DPV has no apparent effect on cell viability

20. The ARVs DPV and TDF appear to induce a pro-inflammatory response in cervical epithelial cells in a time and concentration dependent manner
High concentrations of DPV may exert cytotoxic effects directly on cervical cells
The progestin MPA and the glucocorticoid DEX appear to induce a pro- inflammatory response on their own in cervical epithelial cells
Our data suggests that the addition of the progestin MPA appears to potentiate the pro-inflammatory response induced by the ARVs
From these results the ARV TDF seems to be the better choice of microbicide to be used in combination with a progestin
Conclusion
So in Summary we found in our study that the ARVs DPV and TDF appear to induce a pro-inflammatory response in cervical epithelial cells in a time and concentration dependent manner.
We also found that high concentrations of DPV may exert cytotoxic effects directly on cervical epithelial cells.
Another interesting finding was that MPA and DEX appear to induce a pro-inflammatory response on their own in cervical epithelial cells which further adds to the knowledge that these two are able to induce anti-inflammatory and pro-inflammatory effects on select genes.
When we did our ARV progestin combination study we found that when DEX or MPA are combined with DPV the pro-inflammatory response induced by DPV appears to be massivley potentiated.
From this study we get an indication that TDF may be the better choice of ARV to be used in combination with a progestin in the development of new MPTs targeting the FGT.

21. Physiological relevance
Monitoring the effects on pro-inflammatory cytokines is a useful strategy to assess the potential safety of candidate microbicides for use in the FGT
An increased inflammatory state in the FGT may lead to a higher risk of HIV acquisition as well as other sexually transmitted infections
These results may aid in the choice of ARV for combination with progestins, to protect against HIV infection and unwanted pregnancies with minimal side effects.
The findings of this study are relevant because monitoring the effects of ARVs on pro-inflammatory cytokines is a useful strategy to assess their potential safety for use in the FGT. Having an increased inflammatory state in the FGT may lead to a higher risk of HIV acquisition as well as other infections and our study may aid in the choice of ARV for combination with progestins that will protect young women against HIV infection and unwanted pregnancies in high risk areas.

22. Acknowledgements Prof Janet P. Hapgood Dr Michelle Maritz Hapgood Lab
I would like to acknowledge and thank my Supervisors Janet Hapgood and Michelle Maritz for all the support on this project, as well as everyone in my lab.
Thank you very much for your time, I now welcome any questions.