Hydroxitirosol, a new generation of microbicides targeting HIV through antiviral and anti-inflammatory pathways

Final Report Summary - AIM-HIV (Hydroxitirosol, a new generation of microbicides targeting HIV through antiviral and anti-inflammatory pathways)

Executive Summary:
Background and objectives
The goal of the AIM-HIV project was to provide the proof of concept that 5-hydroxytyrosol (5HT), a phenol derivative with known anti-oxidant properties, could be used as microbicide in humans. To achieve this objective, the project was organized in six work-packages (WP). WP5 and WP6 grouped work related with management and dissemination of the results respectively, whereas WP1-WP4 addressed the following scientific and technical questions:
1. Does 5HT inhibits HIV-1 replication against a broad range of HIV-1 variants and which is its mechanism of action? (WP1)
2. Does 5HT displays anti-inflammatory properties that could contribute to control of HIV-1 transmission and replication? (WP2)
3. How should we prepare 5HT as a useful drug for microbicide use? Which is its pharmacological profile and its tolerance in animal models? (WP4)
4. Is 5HT treatment applied locally capable to impede HIV-1 transmission in a relevant animal model as non-human primates? (WP3)
Results
1. Antiviral properties. 5HT displayed antiviral activity against a broad spectrum of HIV-1 variants including transmitted-founder viruses and different HIV-1 subtypes in the 5-50 micromolar range with very low toxicity in cell cultures (>1000 micromolar). 5HT was active against HIV-1 and SIV in their natural targets, CD4 lymphocytes and macrophages and in highly relevant cellular models including the immune synapse and tissue culture explants from uterine biopsies. Finally, 5HT was active against viral clones resistant to all classes of current antiretroviral drugs: NRTI, NNRTI, INTI, PrI and the inhibitor of HIV entry maraviroc.
2. Anti-inflammatory activity. Overall the anti-inflammatory activity of 5HT was less intense than expected from previous results. The most consistent data were the 5HT inhibition of TNF-α production in THP-1 cell line and the decrease of CCL2 production and CD38 activation markers. Current analysis of transcriptome changes induced by 5HT can provide more relevant data.
3. Mechanism(s) of action of 5HT. We concluded that 5HT represents a new class of drug as it targets different steps of the HIV-1 cycle through interaction with both viral and cellular factors. 5HT inhibited viral integration and decreased HIV expression through interference with transcription factors, mainly NF-kB, and NFAT1, acting at the regulatory LTR sequences of the virus,
4. Tolerance tests performed in rabbits, stability testing and pharmacokinetic studies in NHP using optimized gel formulation showed an excellent profile for 5HT. Gel formulations of 5HT at 200nM (3,8%) alone or combined with tenofovir at 1% concentration were adopted for NHP clinical trials.
5. Two clinical trials in non-human primates (NHP) were performed. A pilot study using 5HT alone did not show any protective effect as compared to placebo. At this point, based on the strong synergy observed between 5HT and tenofovir in vitro we decided to perform a new study in NHP combining both drugs. Result from the combined microbicide trial (tenofovir+5HT) was disappointing because synergism was lacking in vivo and no increased in protection from SHIV infection was observed in comparison with treatment with a tenofovir-only gel.
Final conclusion
AIM-HIV was a hypothesis-driven project with the final objective of characterizing the antiviral and anti-inflammatory properties of 5HT and providing a proof a concept about its potential use as a new class of microbicide in the clinical setting. We addressed all these objectives with rigorous scientific approaches and we challenged the activity of the molecule in vivo using probably the most demanding animal model currently available –multiple SHIV challenge protocol in NHP- to check its microbicidal activity. We consider that the negative results obtained in the NHP trials preclude the use of 5HT for further clinical development and that 5HT is not a microbicidal candidate to prevent HIV infection despite their antiviral and anti-inflammatory activities characterized in vitro.
Despite this negative result, 5HT represents a “first in class” hit displaying new antiviral and anti-inflammatory properties. Further research should address the generation of new prototypes through chemical modification of the basic polyphenol structure of 5HT in order to increase its antiviral potency in vitro and in vivo and/or to improve its PK parameters using slow-delivery devices as vaginal rings.
Project Context and Objectives:
Project context:
Worldwide the majority of HIV infections are transmitted through sexual intercourse. Several strategies have been proposed to decrease the rate of sexual transmission of HIV: sex education, increase the availability and use of male and female condoms, circumcision, early treatment of infected people (test and treat strategy) and the development of vaccines. Besides, pre-exposure prophylaxis, including microbicides, is an alternative strategy under study at present. The microbicides are compounds, usually in form of gel or creams that could be applied inside the vagina (or rectum) to protect against HIV transmission.
In many countries across the world access to prevention and treatment services is limited. Highly active antiretroviral therapy (HAART), if given to all HIV-positive individuals, could bring the pandemic under control. However, in spite of the huge increase in the access to treatment (at the end of 2014, 10.7 million people living with HIV in sub-Saharan Africa had access to antiretroviral therapy), this represents the 41% of people living with HIV in the region. Besides, women are particularly vulnerable because of socioeconomic factors and gender inequalities. Although the male condom is highly effective in preventing the sexual transmission of HIV, in many settings, women have little negotiating power to use a condom and protect themselves against HIV. Diminishing HIV transmission among women is a key step towards undermine the virus presence since it will not be transmitted to the new generation of children and to their partners, and microbicides could be a potentially important preventive tool for these women.
The Objectives and the Consortium:
AIM-HIV is the acronym of “Anti-Inflammatory Microbicides against HIV”. The project was funded by the 7th Framework Program (FP7) for Research and Technological Development of the European Community, and coordinated by José Alcamí from Instituto de Salud Carlos III, based in Madrid, Spain. The main objective of the AIM-HIV project was to provide an effective and low-cost new microbicide based on 5-Hydroxytyrosol (alone or in combination with other antiviral molecules) to be dispensed as a vaginal gel, in order to reduce HIV infection in countries with high incidence of HIV transmission, with special attention to sub-Saharan Africa. AIM-HIV project main target were young women of reproductive age who are at a particular risk of HIV acquisition, addressing by this mean one of the main gaps that exists in prevention of HIV in poverty areas. Preventing HIV transmission among potential mothers will also prevent the acquisition among the new generation of newborns, contributing to decrease the rate of HIV infected people worldwide by providing an affordable and feasible option for HIV prevention that is self-initiated and self- managed.
The specific objectives of the project were:
• To investigate the antiviral activity of 5HT by using innovative cellular and mucosal models and techniques,
• To depict the mechanism of action of 5HT based on acting on a new anti-HIV target,
• To identify the immune response of 5HT and HIV infection and the anti-inflammatory capacity of the compound,
• To assess the efficacy and pharmacokinetics of 5HT in animal models granting the success of the new microbicide in clinical trials,
• To investigate the synergism of 5HT with other microbicides and antiretroviral drugs,
• To obtain the most appropriate formulation of 5HT,
• To establish a proof of concept for the use of 5-HT as microbicide for HIV prevention,
• To design exploitation strategies that take into account the specific regulatory and social characteristics,
• To develop an optimized, low-cost and highly stable microbicide for its subsequent distribution in African countries guaranteeing the validation and exploitation of the results,
• To settle the bases for the future development of combinatorial strategies: microbicides- vaccines.
The consortium created for carrying out the project was composed by a SME company, with strong research and development capacities and broad experience in the field of hydroxytyrosol, SEPROX Biotech S.L. (Spain), and three European research groups with a profound knowledge on HIV pathogenesis: Instituto de Salud Carlos III (Spain), Commissariat à l’énergie atomique et aux energies alternatives (France) and Università Vita-Salute San Raffaele (Italy):
To achieve the objectives, the project was organized in four scientific work packages (WP). In addition, a management and a dissemination work packages were set up:
• WP1. Antiviral activity of 5HT and synergism with other microbicides (Leader: Institute of Health Carlos III).
• WP2. Immune function and inflammatory potential of 5HT and other antiretroviral drugs (Leader: Vita-Salute San Raffaele University).
• WP3. Evaluation of the efficacy and pharmacokinetics of 5HT using non-human primate (NHP) models (Leader: Atomic Energy and Alternative Energies Commission).
• WP4. Optimized 5HT formulation (Leader: Seprox Biotech).
• WP5. Project management (Leader: Institute of Health Carlos III).
• WP6. Dissemination and exploitation (Leader: Institute of Health Carlos III and Seprox Biotech).
Each WP included different tasks that were developed in a coordinate way by one or several members of the consortium and that were completed following a detailed timetable.
Regarding management structure there were set up three main committees: the Steering Committee, the Scientific Advisory Board and the Social Awareness Advisory Committee (a figure related to this issue is included in this final summary report):
The Steering Committee was formed by the leaders of the consortium: José Alcamí on behalf of ISCIII, Guido Poli and Elisa Vicenzi on behalf of USR, Roger Le Grand on behalf of CEA and Eduardo Gómez Acebo on behalf of SEPROX. This Committee was devoted to the continuous monitoring of the work plan and was responsible for ensuring the good overall development of the project.
The Scientific Advisory Board (SAB) was appointed to review the scientific strategy, the overall progress and the scientific achievements of the AIM-HIV project, assuring high levels of excellence with regard to scientific aspects. The SAB was invited to the progress meeting of the project giving an essential feed-back to the Steering Committe. The SAB members who attended the meeting were: Clara Menéndez (Research Professor of Barcelona Centre for International Health Research), Felipe García (Team leader of the Infectious Diseases Service Hospital Clínic, Barcelona) on behalf of Jose María Gatell, Robin Shattock (Professor of mucosal infection and immunity and Research team leader in the Center for Infection at St George’s University of London) and Anna-Lena Spetz (Professor, Department of Molecular Biosciences, The Wenner-Gren Institute).
The Social Awareness Advisory Committee (SAAC) was set up in order to guarantee that research and development activities responded the end users’ needs and assure that their view was correctly reflected in the project. The institutions listed below were invited to be part of this committee: National Laboratory for Public Health (Gabon), Foundation for Community Development (Mozambique), Barcelona Centre for international Health Research (Spain) and Centre International de Recherches Médicales de Franceville (Gabon). The members of the SAAC were informed of the achievements obtained during the project.
Project Results:
The goal of the AIM-HIV project was to provide the proof of concept that 5-hydroxytyrosol (5HT), a phenol derivative with known anti-oxidant properties, could be used as microbicide in humans. Previous data suggested that 5HT displayed anti-HIV activity but these results were never studied in depth. Besides, it was previously described the anti-inflammatory properties of 5HT and our current knowledge on HIV-1 transmission and pathogenesis strongly suggest that decreasing the inflammatory activity in the immune system results in impaired HIV replication and transmission. All these data, together with preliminary results obtained by our groups, prompted us to propose the hypothesis that 5HT represents a new class of drug displaying both anti-HIV and anti-inflammatory properties that could be a first in class microbicide for prevention of HIV-1 transmission.
To achieve the objectives the project was organized in six work-packages (WP). The first four WPs that are considered in this section addressed the following scientific and technical questions:
1. Does 5HT inhibit HIV-1 replication against a broad range of viruses and which is its mechanism of action? (WP1)
2. Does 5HT display anti-inflammatory properties that could contribute to control of HIV-1 transmission and replication? (WP2)
3. How should we prepare 5HT as a useful drug for microbicide use? Which is its pharmacological profile and its tolerance in animal models? (WP4)
4. Is 5HT treatment applied locally capable to impede HIV-1 transmission in a relevant animal model as non-human primates? (WP3)
WP5 and WP6 grouped additional work related with management and dissemination of the results respectively. Each WP included different tasks that were developed in a coordinate way by one or several members of the Consortium and were completed following a detailed timetable. Main results obtained for each task in technical/scientific WPs are summarized in this section.

WP1: Antiviral activity of 5-Hydroxytyrosol (5HT) and synergism with other microbicides.
Task 1.1 Determination of the antiviral activity of 5HT in relevant culture models of transmission and primary infection. (ISCIII, USR, SEPROX).
All clinical samples were collected from blood healthy donors that signed an informed consent given its permission to use their peripheral blood mononuclear cells in “in vitro” experiments.
Preliminary data: 5HT activity against HIV-1 and SIV in their natural targets
In preliminary experiments we demonstrated the antiviral activity of 5HT against HIV-1 in the natural targets for infection: CD4 lymphocytes and macrophages. 5HT inhibited HIV-1 in lymphocytes from peripheral blood (PBLs) with an inhibitory concentration (IC50) in the micromolar range between 10 and 50 µM depending on the experiments. Cell viability was not affected by 5HT concentrations as high as 1000 µM in this system.
We also determined the activity of 5HT against the Simian Immunodeficiency Virus (SIV) because the project included a clinical trial in non-human primates (NHP) to demonstrate the activity of 5HT as microbicide in vivo. Both a wild type SIV strains (SIVmac251) and a recombinant virus carrying the HIV-1 envelope in the context of an SIV background SHIV (RT-SHIV162P3) were tested. 5HT inhibited replication of both strains of SIV at similar concentrations as HIV-1.
Antiviral activity of 5HT activity in different cell systems
The immune synapse. DC-SIGN dependent transmission:
Once the virus cross the epithelia, dendritic cells are involved in primary propagation to CD4 lymphocytes and the lectin DC-SIGN plays a major role in this process in which the virus is transferred from dendritic cells to lymphocytes. To analyse the activity of 5HT in this scenario named the immune synapse HIV propagation was evaluated using coculture of PBLs with DC-SIGN expressing cells (Raji-DC- SIGN). 5HT was active against DC-SIGN-mediated HIV infection which is essential for its microbicide activity.
Anti-HIV activity of 5HT in the context of transmission across the mucosa: two chamber model:
For the two-chamber model, endometrial Hec-1A and colorectal Caco-2 were seeded in the upper chamber of transwell microplates. PBMC obtained from healthy donors and previously activated with PHA+IL2 were cultured in the lower chamber. The virus (JR-Renilla, an R5 clone carrying the luciferase reporter) was added to the upper chamber and must cross through the epithelial layer to reach its target in the low chamber. 5HT was able to inhibit virus mucosal transmission across the two different types of epithelial cell lines, endometrial Hec-1A and colorectal Caco-2.
Anti-HIV activity of 5HT in culture tissue explants (CTE):
We checked 5HT activity in a model very close to the viral infection in vivo. Cervical tissue explants (CTE) were obtained from HIV seronegative women undergoing planned therapeutic hysterectomy and infected with a HIV-1 R5 laboratory strain (HIV1-BaL). Virus replication was evaluated by both p24 Gag release into the culture supernatant in the presence of different doses of 5HT. Out of 6 CTE donors tested, 3 were supportive of HIV-1 replication (“productive CTE”) whereas 3 were not (“non-productive CTE”). Incubation of productive CTE with increasing concentrations of 5HT resulted in a progressive decrease of virus replication. Of note is the fact that HIV-1 replication was not increased in non-productive CTE. In conclusion, 5HT inhibited HIV-1 infection in HIV-productive CTE and it did not promote its replication in non-productive CTE.
Task 1.2 Antiviral activity of 5HT against resistant viruses: tenofovir resistant viruses, FTC-resistant, multi-resistant NRTI, NNRTI-resistant and integrase resistant. (ISCIII, USR, SEPROX).
The capacity of a new drug to inhibit replication of HIV-1 against resistant variants represents an important added value. In particular, we have focused on virus resistant to compounds that have been used or can be potentially employed as microbicides: tenofovir, 3TC/FTC, maraviroc and raltegravir. To this aim we have generated by site-directed mutagenesis viral clones resistant to all these drugs. Besides, we have generated the pol gene of viral strains from patients carrying multiple mutations of resistance to NRTI, NNRTI and protease inhibitors. We have analyzed the activity of 5HT against this broad collection of resistant HIV-1 variants and we have shown that 5HT is active against all these resistant clones and recombinant viruses tested.
Task 1.3. Antiviral activity of 5HT against founder viruses and HIV subtypes. (ISCIII, USR, SEPROX).
One important issue is to demonstrate the activity of 5HT against different HIV-1 strains including different subtypes and transmitted-founder viruses (TFM) that carry specific properties in the viral envelope. Recombinant viruses carrying the HIV-1 envelope of TFM and different HIV-1 clades (A, B, C, D, F) were generated by cloning the full-length envelope (gp160) in the JR-Ren (R5 tropic) backbone. 5HT inhibited with very similar IC50 HIV-1 infection of TFM and different HIV-1 subtypes in PBLs.
Task 1.4- Synergism between 5HT and other compounds with potential activity as microbicides (tenofovir, FTC). (ISCIII, USR, SEPROX).
Current trials using microbicides only show partial protection. One strategy to improve these results is the combination of different microbicides. We have analysed the potential synergism of 5HT with other drugs developed as microbicides. Of note, the combination of tenofovir and 5HT shows a strong synergic effect in PBLs in vitro (combination index <0.3).
Task 1.5 Mechanism of action of 5HT (ISCIII, USR, SEPROX).
Inhibition of viral entry
Previous publications from other groups (Lee-Huang S et al. Biochem Biophys Res Commun. 2007;354:879-84) claimed that 5HT could inhibit HIV-1 replication by two different mechanisms: binding to gp41 protein thus inhibiting the fusion process. Our data show that 5HT is able to diminish viral replication in vitro in infections performed with a VSV pseudotyped HIV, lacking the fusion domain of the HIV-1 envelope.
To corroborate that 5HT activity was not related to viral entry, a virion-based fusion assay was performed (BlaM-Vpr system). 5HT was not able to inhibit fusion of BlaM.vpr virions to cells even at the higher concentration tested (200 uM) and its effect was specific. In conclusion we could not confirm that 5HT inhibited HIV infection at the fusion level.
Inhibition of integration
In our preliminary experiments we were unable to demonstrate any effect of 5HT on HIV-1 integration using different models. Using more specific and sensitive techniques for the detection of HIV-1 integration (quantitative Alu-LTR real time PCR) in activated PBMC infected for five days we demonstrate that 5HT was able to diminish the number of HIV-1 integrated copies at 10 and 50 µM, suggesting that integrase could be one of 5HT targets in the viral cycle.
Inhibition of viral transcription
To assess the potential impact of 5HT on HIV post-integration events, cells were transfected with luciferase expression vectors under the control of a full-length proviral HIV-DNA (NL4.3-Luc). Once demonstrated that transcription of a full infectious provirus was inhibited by 5HT, we analyzed if the compound was acting on the regulatory sequences encompassed in the HIV-LTR by transfecting a luciferase-expression vector under the control of the viral enhancer promoter (LTR). Finally, we assessed the transcription factors that drive LTR activity (NF- B, NFAT) through transfection of luciferase vectors in MT2 cells and PBLs under the control of consensus sequences for these transcription factors (Sp1, NF- B, NFAT).
In these experiments we demonstrated that 5HT displays an anti-transcriptional activity on the HIV-LTR that is mediated mainly through inhibition of NF- B and NFAT.
Role of 5HT in the modulation of HIV latency in chronically infected cell lines
In an attempt to better define the potential impact of 5HT on HIV infection and inflammation, we have investigated its potential capacity of modulating the latent state of virus infection in 3 cell lines carrying integrated HIV proviruses (U1, promonocytic, ACH-2 and J-Lat 8.4 and 10.6 of T lymphocytic origin).
No inductive effect on the undetectable levels of virus production from the 3 cell lines was observed in the presence of 5HT. Furthermore, 5HT inhibited PMA- and TNF-α induced HIV-1 expression in J-Lat 10.6 and ACH-2 cell lines, whereas no virus production in the presence or absence of 5HT was observed.
Summary. WP1: Antiviral activity of 5-Hydroxytyrosol (5HT) and synergism with other microbicides.
1. 5HT displays antiviral activity against a broad spectrum of HIV-1 variants including transmitted-founder viruses and different HIV-1 subtypes in the 5-50 micromolar range with very low toxicity in cellular cultures (>1000 micromolar).
2. 5HT is active against viral clones resistant to the NRTI tenofovir, lamivudine, emtricitabine and the viral integrase inhibitor raltegravir, Besides, 5HT is active against maraviroc-resistant virus and recombinant viruses carrying multiple mutations of resistance to NNRTI, NRTI and protease inhibitors.
3. 5HT is active against viral infection of cell lines but also against the natural targets of HIV-1 infection, CD4 lymphocytes and macrophages. Besides 5HT is active in cellular models including the immune synapse (DC-SIGN mediated transmission) and tissue culture explants from uterine biopsies.
4. 5HT displays a strong synergism with tenofovir.
5. We have shown that 5HT displays unique mechanisms of action. It targets viral proteins as the HIV-1 integrase but also cellular factors, mainly transcription factors that drives HIV-1 reactivation from the state of proviral latency.
Overall, 5HT presents an excellent antiviral profile and can be considered a “first in class” of a new category of anti-HIV drugs that interferes with both HIV-1 integration and viral transcription.

WP2: Immune function and inflammatory potential of 5HT
Task 2.1 Study of toxicity and potential release of pro- and anti-inflammatory cytokines and chemokines. (ISCIII, USR).
Anti-inflammatory activity of 5HT in THP-1 cells
Previous studies have shown that 5HT had anti-inflammatory activity in THP-1 cells by inhibiting tumor necrosis factor-α TNF-α expression (Zhang X et al. Naunyn Schmiedebergs Arch Pharmacol. 2009 Jun;379(6):581-6.). We have confirmed these results in LPS-stimulated THP-1 cells.
Anti-inflammatory activity of 5HT in whole blood assay.
The whole blood assay (WBA) was used to evaluate the potential interference of 5HT on the release of pro- or anti-inflammatory cytokines and chemokines. The panel included pro-inflammatory cytokines included IL-1α, IL-1β, IL-6, TNF-α, INF-γ, anti-inflammatory cytokines IL-1RA, IL-10 and chemokines CXCL8, CCL2, CCL3, CCL4. Basal levels of cytokines and chemokines were low and increased after LPS stimulation. Incubation with increasing concentrations of 5HT (2 to 200 µM) did not perturb either basal or LPS-induced cytokine and chemokine levels of all analytes but CCL2. Indeed, a significant reduction of CCL2 production was observed in LPS-treated WBA in the presence of 200 µM 5HT.
Anti-inflammatory activity of 5HT in monocytes
Monocytes were incubated with serial dilutions of 5HT, from 2 to 2,000 µM, for 24 h in the presence and absence of LPS. Firstly, we observed that in unstimulated conditions, the levels of cytokines expression was quite homogenous, However, LPS-induced cytokines and chemokines varied significantly among different donors. Two donors were defined as poor LPS-responders whereas other 2 responded efficiently to 100 pg/ml of LPS. Nevertheless, when we averaged the poor responders vs. the average of the good responders, TNF-α and IL-6 secretion was reduced (IL-6>TNF-α) in good LPS responders, but it was induced in poor LPS responders by 5HT. CXCL8 was induced in poor LPS responders, but not in good LPS responders. In contrast, 5HT consistently inhibited LPS-induced CCL2 expression in all 4 donors at 200 µM treatment suggesting a potential anti-inflammatory activity.
Task 2.2 Determination of the influence of 5HT in HIV spreading. (ISCIII, CEA, USR, SEPROX).
Preliminary studies assessing the role of sexual hormones on HIV infection.
The project was approved by the ethical committee of USR and all patients gave their written informed consent to use samples from hysterectomy in the research studies here described.
Some studies have indicated that hormonal contraceptives increase the risk of transmission and acquisition of HIV infection in women. For this reason, before the study of the influence of 5HT in HIV spreading, it is important to study if the modulation of sexual hormones during the menstrual cycle may alter the sexual HIV-1 transmission and replication in cervical tissue explants (CTE). For this purpose, CTE from HIV seronegative women undergoing planned therapeutic hysterectomy were obtained from 23 different donors in either fertile or menopause phase and infected with a HIV-1 R5 laboratory strain (HIV-Bal). A post-hoc histological analysis on fresh tissue blocks revealed that all productive CTE were established from women who were in their secretory phase of the menstrual cycle at the time of surgery. Therefore, these preliminary results support the hypothesis that the menstrual cycle phases strongly interfere with female genital tract susceptibility to productive HIV-1 infection. Indeed, only tissues obtained from women in their secretory phase of the menstrual cycle were susceptible to R5 HIV-1 infection.
We then treated CTE with increasing concentrations of 5HT from 2 to 2,000 µM. Three days after treatment the percentage of CD4+ and CCR5+ T cells, cell death and markers of T cell activation including CD38, CD69 and CD25 were evaluated on the surface of CD4+ T cells. 5HT did not perturb either the proportion of CD4+ T cells or the proportion of CCR5+ T cells. The expression of both early and late markers of T cell activation, i.e. CD69 and CD25, respectively, were not altered by 5HT. As 5HT does not induce the markers of T cell activation, we do not expect an increase of HIV-1 infection in CTE cultures.
Cytokine and chemokine production in human cervical tissue explants (CTE) in the presence of 5HT
Prior to testing cytokine and chemokine release, an evaluation of cytotoxicity was performed in 5HT treated CTE. No cell death was observed at concentrations <400 µM. 5HT did not either increase or decrease the basal levels of IL-6 and chemokines tested by ELISA in the supernatants of treated-CTE. These results suggest that 5HT is devoid of pro-inflammatory activities up to 400 µM. However, no clear anti-inflammatory activity was detected in the CTE. An important caveat concerns this peculiar model in that cytokine and chemokine release is strongly influenced by the tissue damage consequent to tissue processing.
Effect of 5HT on markers of activation and proliferation of T lymphocytes isolated from CTE
In addition, we tested the potential impact of 5HT on markers of activation and proliferation of T lymphocytes isolated from human cervical tissue of HIV-seronegative women. 5HT (200 µM) did not modify the cell surface markers of T cell activation CD25, CD69 or HLADR after 3 days of incubation. Interestingly enough, 5HT decreased CD38 expression in both CD4 and CD8 T cells, although this difference was only statistically significant for CD8 cells.
Impact of 5HT on gene expression
In order to carry out a global approach to study the impact of 5HT on cellular factors, we decided to perform transcriptome analyses after treatment with 5HT in CD4, monocyte-derived macrophages and dendritic cells which are the main target of HIV-1 infection, To date, we have sequenced and analyzed the mRNA libraries using the BaseSpace platform from Illumina. Specifically, we have used the application TopHat to align sequences and CuffLinks to compare gene expression in the different samples.
In a preliminary analysis of results in mature dendritic cells, we have observed changes in the pathways related with cellular adhesion molecules (CAMs) and cytokine-receptor interactions. Regarding immature dendritic cells, the most altered pathways under 5HT treatment are the glutathione metabolism and several interleukins.
Task 2.3. Determination of inflammatory potential of tested compounds. (ISCIII, CEA, USR, SEPROX).
Studies regarding inflammatory potential of 5HT in the non-human primate (NHP) CTE were planned. These experiments expected to be performed at CEA in collaboration with USR. The histocultures methodology was transferred from CEA to USR but it was verified that this was both impractical and costly. Therefore, it was decided to abandon such an effort in order to concentrate on more achievable targets.
Summary WP2: Immune function and inflammatory potential of 5HT
1. 5HT decreases TNF-α production in the THP-1 monocytic cell line.
2. In the whole blood assay 5HT was devoid of pro-inflammatory activity. 5HT did not modify the basal or LPS-induced production of cytokines IL-1α, IL-1β, IL-6, TNF-α, INF-γ, IL-1RA, IL-10 and chemokines CXCL8, CCL3, CCL4.
3. A significant reduction of the LPS-induced CCL2 levels in WBA and LPS-stimulated monocytes was obtained with the 200 µM treatment suggesting a potential anti-inflammatory activity.
4. In human cervical explants, 5HT did not modify either the proportion of CD4 or CCR5+ lymphocytes or the activation markers CD25, CD69 or DR. Levels of IL6, CCL2, CCL20 or CXCL12 were not altered by 5HT treatment. An important caveat concerns this peculiar model in that cytokine and chemokine release is strongly influenced by the tissue damage consequent to tissue processing.
5. A decrease in the activation marker CD38 was induced by 5HT in CD4 lymphocytes isolated from CTE.
Overall the anti-inflammatory of 5HT was less intense than expected from previous data. A decrease in CCL2 production and CD38 activation markers were the most consistent data. Current analysis of transcriptome changes induced by 5HT can provide more relevant data on this objective of the project.

WP4: Optimized 5HT formulation
Task 4.1 Initial HTs formulation and stability studies. (ISCIII, CEA, USR, SEPROX).
Hydroxytyrosol is a phenylethanoid, a simple polyphenolic phytochemical with antioxidant properties. Like other polyphenols can be unstable and may suffer many reactions during processing and storage of products containing them. This fact is well known in the industry and has consequences affecting the quality of final products.
Its orto-diphenolic structure confers hydroxytyrosol its chemical behaviour which can be summarized as unstable towards alkaline media and molecules with strong basic properties, towards oxidizing elements, including oxygen or light as possible initiators of radical reactions, enzymes and microorganisms, and towards heavy metals which can be quelated by phenolic oxygens. Increases in concentration of hydroxytyrosol might have its own effect into formulations.
All these aspects could also be increased by temperature and thus be affected by manufacture processes in which relatively high temperatures could be used. To ensure the safety and applicability of formulations experimental conditions were chosen to cover the development of the final product from room temperature to 90º C and changes in pH up to physiological 7.4.
Manufacture conditions (results of heating) with different preservatives
The inclusion of lactic or sorbic acid has neutral or weakly beneficial effects with or without sodium metabisulfite but in all cases formulations without sodium metabisulfite develop colour.
Handling and storage conditions (results at room temperature)
Best protected formulations show less than five percent of degradation. The presence of lactic or sorbic acid additionally to the lowest concentration of metabisulfite generally increases stability at all concentrations with respect to the control. These formulations need 0.10% of sodium metabisulfite.
Application and use conditions (stability at pH 7.0):
The vaginal pH of animals for pharmacokinetic and infection assays is 7.0 higher than 4-5 for humans. For application purposes in these assays representative formulations of the three selected concentrations of hydroxytyrosol (30, 100 and 200 mM) including appropriate quantities of preservatives were tested changing the pH to 7.0 by dilution in saline phosphate buffer (100 mM, NaCl 37 mM) and heating at 37 degrees.
Less than five percent of decomposition is registered in best protected formulations after the period of heating. Colour development was observed since the second hour in the highest concentrated gels and control. Low and medium concentrated gels show moderated development of colour until four to five hours.
Task 4.2. Final product formulation. (ISCIII, CEA, USR, SEPROX).
Final preferred formulation
In view of the synergic effects of combined mixtures of tenofovir and hydroxytyrosol in vitro, the final formulation chosen among those developed was the one including 1% tenofovir and 3.08% hydroxytyrosol with the corresponding amount of preservatives.
Preparation of sample gels
Each ingredient was carefully weighted and added to the vessel with the aid of small amounts of buffer. Before the addition of tenofovir at least 80% of the volume must be added to increase tenofovir solubility to the maximum possible. After all the components were homogenized into the 95% of the volume the mixture was automatically adjusted to pH 4.68 with 0.5 M sodium bicarbonate solution.
Combined stability tests at different temperatures and mix ratios
Although final formulation includes hydroxytyrosol at 200 mM, two different concentrations of hydroxytyrosol were considered (30 and 200 mM) for stability tests before efficacy assays in animals. Five reference gels were prepared containing tenofovir alone (TF 1%), hydroxytyrosol alone (5HT 30 and 5HT 200 mM) and mixtures of both (TF 1%, 5HT 30 mM and TF 1%, 5HT 200 mM). All of them were tested in order to check the effects of temperature on each ingredient separately and combined.
Experiments were extended over 5 to 6 months for temperatures between 4º C and 40º C and 1 to 2 months for higher temperatures. Due to the prolonged heating period some weight loss of samples was expected, especially for temperatures over 60º C. However, the experimental model used showed good correlation between concentration decrease of both tenofovir and 5HT, and temperature for weight losses lower than 5%. Losses of weight higher than 5% reflected abnormal results in almost 96% of cases.
The presence of 1% tenofovir in the formulation slightly increases the decomposition rate of hydroxytyrosol in the interval of temperatures between 4º C and 40º C. However, less than 5% of decomposition is registered after 6 months, predicting a t90 after linear regression of the data of 17.6 months at room temperature for the highest concentration of 5HT. In contrast to what is happening in the interval of temperatures between 4ºC and 40ºC, at higher temperatures (60ºC and 75ºC) combination of 5HT with tenofovir increased the stability of 5HT. The combination does not affect tenofovir concentrations at different temperatures. The highest degradation for 5HT when combined with tenofovir was obtained at lower concentration of 5HT. At 90º C the weight loss by evaporation in samples (only 30% of samples below the 5%) was too high and fast enough to reliably quantify the effects of temperature over the decomposition of tenofovir or 5HT.
Osmolarity of formulations
Osmolarity of formulations were also measured on formulation containing hydroxytyrosol alone or in combination with tenofovir. Values were lower than that of the original TF1% gel used in CAPRISA004 (3111 mOsmol/Kg) and the reformulated one for better tolerance with only 5% glycerine content (836 mOsmol/Kg) [Charlene S. Dezzutti et al, Reformulated tenofovir gel for use as a dual compartment microbicide. Journal of Antimicrobial Chemotherapy, 2012, 67: 2139-2142].
Task 4.3. Tolerance studies. (ISCIII, SEPROX).
The tolerability of hydroxytyrosol formulations was evaluated at Harlan Laboratories S.A. (Barcelona, Spain). The study was the title: HYDROXYTYROSOL GEL: Vaginal Tolerance after Repeated Administration for 14 days in Rabbits (Harlan Study Number: S47252).
Test methods were designed to be compatible with the procedures indicated by the accepted guidelines and recommendations:
• Note for guidance on non-clinical local tolerance testing of medicinal products (European Agency for the Evaluation of Medicinal Products, 2001)
• Tests for irritation and delayed-type hypersensitivity (ISO 10993-10: 2010)
• The conduct of the study was reviewed, as part of the Harlan Laboratories S.A. ethical review process.
Materials and methods
The tolerability of hydroxytyrosol GEL after administration at the concentrations of 30, 100 and 200 mM for 14 consecutive days was evaluated. The degree of irritation in the vaginal mucosa was also determined.
30 young adult New Zealand White rabbits of 11-13 weeks of age were distributed into five groups of six females. A group of animals was administered Placebo HEC GEL and another group, water for injection (Blank control group). The different treatments were administered topically (1 mL/animal/day) by means of a catheter, to the vaginal mucosa of the rabbits.
Clinical signs were observed daily and mortality/viability was recorded twice daily. Body weight was recorded immediately before the first, fourth, seventh and tenth administration, and prior to sacrifice.
The day before treatment start, a vaginal smear sample was taken from the vaginal mucosa using a sterile swab. The swab was kept in Stuart Agar Gel Medium at 20 ± 5 ºC until shipment. At the end of the study and after the sacrifice of the animals, a sample of the vaginal mucosa was collected in order to carry out a microbiological count and vaginal pH measurement. The samples were kept at room temperature (20 ± 5 ºC) until shipment. Samples were evaluated for microbiological composition.
After sacrifice, necropsy of the vagina was carried out. A macroscopic examination of the genital apparatus of each animal was performed. Sections of the distal, medial and proximal zones of the vagina were collected, fixed in 10% formalin and then stained with hematoxylin and eosin for microscopic examination.
Results
Mortality/Viability:
All animals survived the schedule treatment period. No mortality was recorded.
Clinical signs:
No treatment-related clinical signs were observed. Throughout the experimental phase of the study, blood was observed in the urine of some animals from all groups and was therefore not related to treatment with 5HT GEL. Animals presenting this finding were observed not to have blood in urine during the 14 days of treatment but on specific days throughout the experiment.
Vaginal pH:
Group mean vaginal pH ranged from 7.46 ± 0.30 (Group 1, Blank Control) to 7.80 ± 0.48 (Group 2, Placebo HEC GEL). The pH observed was 7.73 ± 0.27 in Group 3 (5HTl GEL 30 mM), 7.66 ± 0.39 in Group 4 (5HTl GEL 100 mM), and 7.55 ± 0.35 in Group 5 (5HT GEL 200 mM). No treatment-related alterations in this parameter were observed. No statistically significant differences were observed (Dunnett’s test, p<0.05%).
Body weight:
No effects on body-weight evolution were observed during the 14 days of administration. No statistically significant differences were observed (Dunnett’s test, p<0.05%).
Macroscopic findings:
Slightly to moderately reddish vaginal mucosa was noted in medial, proximal and/or distal areas (mainly in distal area) of three animals from Group 1, three animals from Group 2, four animals from Group 3, all the animals from Group 4, and five animals from Group 5. No statistically significant differences were observed between groups (Fisher Exact Test, p>0.05%). No test-item related macroscopic findings were observed.
Microscopic findings:
Microscopic observation revealed minimal vascular congestion on the submucosa of the proximal, medial and/or distal parts of the vagina in four animals from Group 1, in three Group-2 animals, in four Group-3 animals, in all Group-4 animals and in five Group-5 animals.
The microscopic findings recorded in the five experimental groups were considered within the range of normal background lesions that may be seen in rabbits of this strain and age in this type of study. They were therefore considered incidental, reflecting the usual individual variability.
Microbiological assessment:
A change in the microflora of the vagina was observed, probably due to the administration procedure as no treatment-related findings were detected. In vaginal exudate samples (taken before treatment start), the predominant bacteria in almost 100% of the animals was Streptococcus sp. whereas in the mucosa samples (collected after 14 days of treatment) only two animals were observed to have this bacteria. Moreover, Klebsiella pneumoniae was present in 1/3 of the animals after treatment whereas this bacterium was only detected in one animal prior to treatment start.
Discussion
Mean irritation values of 1.3 for Group 2, 2.0 for Group 3 and 2.3 for Groups 4 and 5 were obtained, which would indicate that the Placebo and the test item induce minimal irritation after their repeated application to the vaginal mucosa of rabbits. However, Group 1 (Blank Control) had a mean irritation value of 1.7 thus making the mean irritation value (MIV) -0.6 for Group 2, 0.3 for Group 3 and 0.6 for Groups 4 and 5 (after subtracting the control group average value). This result would indicate that the test item induces no effects. The alterations observed, such as vascular congestion, edema and leukocyte infiltration, may be considered normal in the vaginal submucosa and the degree of the alterations recorded among all groups is minimal or mild. Likewise, same alterations and similar degree were observed in Blank Control.
Task 4.4. Pilot batch. (ISCIII, SEPROX).
In view of the lack of efficacy of 5HT alone in the prevention of the infection in vivo, and the observed synergic effect of tenofovir and 5HT in vitro, the final formulation chosen among those developed was the one including 1% tenofovir and 3.08% hydroxytyrosol with the corresponding amount of preservatives.
Citric acid buffer solution 5 mM, pH 5.5
Lactic acid: 0.1 %,
Sodium metabisulfite: 0.1%,
Glycerol: 2.5 %
tenofovir: 1%,
Hydroytyrosol: 3.08% (200 mM)
Final pH 4.68
Osmolarity 558.33+/-8.96
The final product formulation was established after performing the stability tests and the tolerance studies in rabbits, in which no morphological alterations or irritative response in the vagina were observed. The pilot batch was manufactured on three lots of 2 Kg of gel.
Task 4.5 Regulatory aspects. (SEPROX).
At this time all the basic work for the preparation of the Investigational Medicinal Product Dossier (IMPD) document for the European Medicines Agency (EMA) is already performed. A pre-IMPD meeting was envisaged to analyze the existing work and eventually submit the preclinical dossier. However taking into account the results obtained in the NHP trial, we have to evaluate if it is worth to continue with the development of 5HT as a candidate microbicide.
Summary WP4: Optimized 5HT formulation
1. In view of the synergic effects of combined mixtures of tenofovir and 5HT in vitro, the final formulation chosen was the one including 1% tenofovir and 3.08% 5HT with the corresponding amount of preservatives. Values measured for osmolarity are lower than that of the original TF1% gel used in CAPRISA004 and the reformulated one for better tolerance with only 5% glycerine content.
2. The stability test were performed with five reference gels containing tenofovir alone (TF 1%), 5HT alone (5HT 30 and 5HT 200 mM) and mixtures of both (TF 1%, 5HT 30 mM and TF 1%, 5HT 200 mM) at temperatures of 4º C, room temperature, 40º C, 60º C, 75º C and 90º C. Experiments were extended over 5 to 6 months for temperatures between 4º C and 40º C and 1 to 2 months for higher temperatures. Due to the prolonged heating period some weight loss of samples was expected, especially for temperatures over 60º C. However, in combined gels, the experimental model used showed good correlation between concentration decrease of both tenofovir and 5HT and temperature, with weight losses lower than 5% for a 5HT concentration of 200mM.
3. The presence of 1% tenofovir in the formulation slightly increases the decomposition rate of hydroxytyrosol in the interval of temperatures between 4º C and 40º C but always below 5% of total weight. In general, the highest degradation for 5HT was obtained with the lowest 5HT concentration.
4. Under the conditions of this experiment, the topical administration of the test item hydroxytyrosol GEL at three different concentrations (30, 100 and 200 mM) caused no morphological alterations in the vagina. The mean irritation value (MIV) obtained was zero as compared with the control group indicating no irritative response in the vaginal mucosa of the rabbit
5. All the tasks of WP4 have been performed including new aspects as the preparation of a pilot batch including 5HT+tenofovir and stability studies of both compounds in the combination. These data were important to perform the second trial in NHP and close collaboration between CEA and SEPROX was required to address these issues in a very short time.
6. Different steps regarding the last phase of the project (pilot batch generation, preparation of the dossier for regulatory authorities) were performed or initiated. However taking into account the results obtained in the NHP trial, we have to evaluate if it is worth to continue with the development of 5HT as a candidate microbicide.
WP3: Evaluation of the efficacy and pharmacokinetics of 5HT using non-human primate (NHP) models
All the experiments performed in animal models were approved by the CEA Ethical Committee and also obtained the authorization of the Ministry of Research as only the Ministry can authorize the projects conducted in animals besides the approval of the local Ethical Committee.
Task 3.1. Providing AIM-HIV partners with biological material for ex vivo and in vitro studies. (ISCIII, CEA, USR).
CEA aims to provide partners of the consortium with mucosal secretions and tissue samples for testing 5HT stability, for ex vivo PK and PD studies including ex vivo challenges against a variety of viral strains and for assessing the anti-inflammatory potential of 5HT. These samples include 1) cervico-vaginal fluids; 2) tissue sections of female genital tract; 3) cervico-vaginal tissue explants.
Taks 3.2. Study of pharmacokinetics and pharmacodynamics of 5HT microbicide formulations in NHP. (ISCIII, CEA, USR, SEPROX).
Before performing the PK studies of 5HT in macaques, stability tests of 5HT in gel formulations were done by SEPROX. Based on the concentrations of hydroxytyrosol chosen for the PK studies (concentrations of 30mM, 100mM and/or 200mM), the final formulation of the compound was defined
Pharmacokinetic studies were conducted in cynomolgus macaques following vaginal application of 5HT gel formulations. We have tested two HEC-based gel formulations containing 30 mM and 200 mM of 5HT.
The objectives of these PK studies were to quantify and to compare 5HT concentrations in vaginal fluid and serum samples collected 1, 2, 4, 8 and 24 hours after a single-dose vaginal administration of 5HT-loaded HEC gel containing 30 mM or 200 mM of 5HT. 5HT was kindly provided by partner 4 (SEPROX) and gel formulations were prepared at Faculty of Pharmacy, Chatenay-Malabry, France. The composition gels was similar: HEC 1.5%, glycerol 2.5%, lactic acid 0.1% and sodium metabisulfite 0.1% excepting for 5HT concentration that was 0.465 (w/w) and 3.08% (w/w) for 30mM and 200mM respectively.
Four female cynomolgus macaques were used in this study. Animals were not treated with depo-medroxyprogesterone acetate. The study was realized in two steps, as follows: after the first treatment with the 5HT gel formulation at 30 mM, the four female cynomolgus macaques were re-included, after extensive washing of the vaginal vault with PBS and a wash-out of 6 days, for testing the pharmacokinetic of the 5HT gel formulation at 200 mM. After the intravaginal (IVAG) administration of each HEC gel formulation on day 0, vaginal fluids were collected using Weck-Cel® spears at baseline, 1, 2, 4, 8 and 24 hours after dosing for quantifying 5HT. Blood was sampled at baseline, 1, 2, 4, 8 and 24 hours after vaginal application of the gel for quantifying 5HT in serum.
Sample analysis was performed using high performance liquid chromatography (HPLC) coupled to tandem mass spectrometer (LC-MS/MS). Mean 5HT concentrations in vaginal fluids and sera over a period of 24 hours after each gel dosing were determined. 5HT concentrations in vaginal fluid and serum were generally highest at 1 hour after gel application and then declined until the end of the monitoring period, dropping by 1-2 orders of magnitude over 24 hours. As expected, vaginal fluid and serum levels of 5HT were greater with the 200 mM gel compared to the 30 mM gel. Mean maximum vaginal fluid and serum concentrations were 1 log higher with the 200 mM gel compared to the 30 mM gel. We determined that 5HT levels measured at 1 hour after 30 mM and 200 mM gel dosing were 300 and 3000-fold above the in vitro IC50 value against the viral strain SIVmac251 (IC50 SIVmac251 9.247µM), respectively. Overall, the data support the use of 5HT gel formulation at 200 mM in efficacy studies.
Task 3.3. Testing microbicide efficacy in NHP against vaginal challenge with SIV. (CEA, SEPROX).
It was initially decided to use the viral strain SIVmac251 to assess the efficacy of 5HT gel formulation in NHP model of HIV sexual transmission. 5HT is active against SIVmac251 with IC50 value in the micromolar range (IC50SIVmac251 9.247µM). However, SHIV162P3 and RT-SHIV162P3 macaque models for evaluation of candidate microbicides were developed. The RT-SHIV macaque model was used in the CHAARM project (EU FP7 project) to assess the efficacy of microbicides formulations after vaginal challenges with repeated low doses of virus. In order to conduct a combination microbicide trial in NHP with 5HT and tenofovir and to compare results to those obtained with other candidate microbicides and because similar IC50 values were found for both viruses (IC50RT-SHIV162P3 27.97 µM), we decided to use the vaginal RT-SHIV162P3 macaque model for challenge studies.
According to the decision tree generated for testing the 5HT and tenofovir combination in NHP, a pilot study was conducted to first assess the efficacy of the 5HT gel formulation at 200 mM against repeated low-dose intravaginal challenges with the RT-SHIV162P3 virus.
The results of this pilot study showed that 5HT did not provide any protection against vaginal RT-SHIV162P3 challenge. Using the same vaginal challenge protocol, a combination trial with 5HT and tenofovir was conducted with six female cynomolgus macaques to determine if 5HT can improve the antiviral activity of tenofovir in vivo. 50% of protection was observed in animals treated with the gel formulation containing 200 mM of 5HT and 1% tenofovir (5HT/TFV gel-treated animals. We previously observed similar level of protection with 1% tenofovir gel alone in a vaginal repeated low-dose challenge model with the SHIV162P3 virus. Overall, these data indicated that 5HT has no protective effect to prevent vaginal transmission of RT-SHIV162P3 virus.
Summary WP3: Evaluation of the efficacy and pharmacokinetics of 5HT using non-human primate (NHP) models
1. PK data were promising as at the highest dose tested (200 mM) levels in vaginal fluids 1, 10 and 24 hours after gel application were largely above the IC50. Besides, 5HT was detected in blood which demonstrate the permeability of the drug in the conditions tested.
2. Taking into account in vitro data showing a strong synergism with tenofovir we modified the initial plan and a decisional tree in which a first trial in NHP with 5HT alone would be performed.
3. The first pilot trial failed to demonstrate any effect of 5HT alone in NHP challenged with RT-SHIV162P3
4. According to the results in this trial we will move towards a 5HT-only protocol to a 5HT-tenofovir combination protocol. For ethical and statistical considerations we decided to perform a pilot study with 6 macaques and move for a larger trial if results were successful. Unfortunately the level of protection of the combination 5HT+tenofovir was the same as tenofovir-only animals.
This was a disappointing result but gives an answer to one the major objectives of the project, to provide a proof of concept about the potential use of 5HT as microbicide. With these data we consider that no additional assays in NHP are recommended.
Coordinator´s final comments
To study the mechanism(s) of action of 5HT, both its antiviral and anti-inflammatory activities, was a major objective of the whole consortium. This issue has been studied in depth and final results represent one major achievement of the project. We can conclude that 5HT represents a “first in class” drug as it targets different steps of the HIV-1 cycle through its impact on both viral and cellular factors. As stated in the project, previous work claimed for a role of 5HT in the fusion and integration processes of HIV infection. Our data do not confirm any effect of 5HT on the process of viral entry/fusion. On the contrary we have shown the interference of 5HT in viral integration. Decreased integration could be due to inhibition of earlier steps in the viral cycle as reverse transcription or nuclear transport. To rule out this possibility specific experiments are planned but the main result is that 5HT is acting at a pre-integration step in the HIV cycle.
Regarding antiviral and anti-inflammatory properties we have shown that 5HT decreases HIV expression through interference with transcription factors acting at the regulatory Long Terminal Repeat sequences of the virus, mainly NF-kB, Sp1 and NFAT1. A large study using Next Generation Sequencing techniques has been performed to assess the role of 5HT on gene expression in HIV target cells. Preliminary data point to changes in the pathways related with cellular adhesion molecules, cytokine, cytokine receptors and glutathione metabolism. All these pathways have been previously described to modulate HIV replication. Detailed analysis will provide new data on the metabolic pathways modified by 5HT and their impact on the HIV cycle and inflammatory mechanisms.
Tolerance tests performed in rabbits and pharmacokinetic studies in NHP showed an excellent profile for 5HT and the 200nM dose was adopted for NHP clinical trials. A pilot study using 5HT alone did not show any effect as compared to placebo. At this point, based on the strong synergy observed between 5HT and tenofovir “in vitro” we decided to perform a new study in NHP combining both drugs. This decision requested a big effort from SEPROX and CEA to test the stability of the gel combination and to prepare the new gel, a goal that was achieved thanks to the excellent cooperation between both partners.
Result from the combined microbicide trial (tenofovir+5HT) have been disappointing because no protection with the 5HT-only gel was observed and synergism was lacking when both drugs were combined in vivo in comparison with treatment with a tenofovir-only gel.
Despite the fact that regulatory agencies do not contemplate results in animal models (non-human primates and BLT-reconstituted SCID mice) for approval of microbicide entry in clinical phases we consider that the negative results obtained in the NHP trials preclude the use of 5HT for further clinical development.
AIM-HIV has been a hypothesis-driven project with the final objective of providing a proof a concept about the microbicidal activity of 5HT, the characterization of the antiviral and anti-inflammatory properties of this compound and its potential use as a new class of microbicide in the clinical setting. We have addressed all these objectives with rigorous scientific approaches and we challenged the activity of the molecule “in vivo” using probably the most demanding animal model currently available –multiple SHIV challenge protocol in NHP- to check its microbicidal activity. We can conclude that 5HT is not a microbicidal candidate to prevent HIV infection despite their antiviral and anti-inflammatory activities that have been characterized “in vitro”.
Despite this negative result, I would like to emphasize that 5HT represents a “first in class” hit displaying new antiviral and anti-inflammatory properties. Further research should address the generation of new prototypes through chemical modification of the polyphenol structure of 5HT in order to increase its antiviral potency.
Importantly, all the procedures and experiments were performed following rigorous ethical requirements and these procedures were also reviewed by an external advisor (Dr JM Gatell) to confirm the compliance with ethical criteria.
It is important to note the help and critical assessment provided by the Scientific Advisory Board of the consortium. Advices from Professors Robin Shattock, Anne-Lenna Spetz, Felipe García and Clara Menéndez have contributed to the strength of the project. All their comments and suggestions were incorporated into the scientific program.
Finally, I want to acknowledge the efforts and collaboration of the members of the consortium to achieve in a very short time such a big amount of knowledge and sound scientific conclusions, and to acknowledge the Commission for their continuous support to the AIM-HIV project.
Potential Impact:
Potential impact
Scientific
• The discovery of the mechanism of action of 5HT: Our results suggest that 5HT activity is directed against bot viral and cellular targets. These properties made of 5HT a unique drug that interferes with both HIV-1 integration and viral transcription and probably restricts to some extent reverse transcription due to the interference with cellular factors. Therefore, 5HT has new mechanisms of action providing important insights on potential new targets and may open up the possibility to develop new molecules based on the 5HT structure with enhanced potency against the same targets.
• The study of the synergy of 5HT with other antiretroviral drugs: Our results show synergy of 5HT + tenofovir and an additive effect with lamivudine and emtricitabine in vitro. These studies are very important because the combination of antiretroviral drugs not only improves the antiviral efficacy of the compounds alone but also prevents the development of resistant viruses. If new molecules based on the 5HT structure are developed, it is very probable they keep the synergy with tenofovir.
• The clarification of the anti-inflammatory activity of 5-HT: A drug with anti-inflammatory properties add value to any anti-HIV drug.
• The study of the pharmacokinetic of 5HT in non-human primates (NHP) models gives basic information that can be useful in the future.
• The dissemination of the scientific results to ensure that the work funded by the EU citizen is available to the community. These results were disseminated mainly through contributions to international conferences, educational activities, contacts with research organizations, scientific societies, pharmaceutical companies, special interest groups and through the public website of the project. Scientific articles will be published shortly.
Translational
• Formulate 5HT in Good Manufacturing Practice conditions.
• Develop of in vitro models for the assessment of microbicide activity and toxicity (tissue explant cell model).
• Develop of in vivo models for the assessment of microbicide activity and safety.
Educational
• Public website: The goal of the public website is to inform the public, especially in the sub-Saharan area, but also in our social context, about the existence of projects like the AIM-HIV project aiming at developing new HIV preventive strategies.
• Lectures for students, academic conferences, seminars, etc.
• Training of master and PhD students that collaborate in the different laboratories involved in the project.
Social and public health impact
The epidemic has a deep socio-economic impact around the world, especially in sub-Saharan Africa, therefore is essential to strengthen prevention measures and to implement new strategies to halt HIV transmission. Microbicides are one of these new strategies.
The expected final result of the project was to provide an effective and low-cost new microbicide based on 5HT in order to reduce HIV sexual transmission in countries with high incidence of HIV transmission, with special attention to sub-Saharan Africa. The UNAIDS estimates that the number of new infections in sub-Saharan Africa in 2014 was 1.4 million. In this region, adolescent girls and young women continue to experience elevated HIV risk and vulnerability. Of the 2.8 million young people aged 15-24 years living with HIV in sub-Saharan Africa in 2014, 63% were female. Therefore, diminishing HIV transmission among women will help to avoid the transmission to the new generation of children and to their partners.
The data indicated that 5HT has no protective effect to prevent vaginal transmission of RT-SHIV162P3 virus in female cynomologous macaques. However, due to its characteristics 5HT represents a “first in class” molecule that could be chemically modified to increase its intrinsic potency. Besides, new strategies must be tested with the aim of developing a microbicide that, in combination with other strategies (condoms use, test and treat, extended treatment access), may contribute to the control of HIV epidemics and improved life expectancy and quality of life, particularly in women living in low- and middle- income countries.

Main dissemination activities
The objective of dissemination activities of the AIM-HIV project was to improve the generation of new scientific knowledge, public interest on AIM-HIV project and cross-fertilization between researchers, industry and patients.
The AIM-HIV project generated an important amount of knowledge whose dissemination and use was of great importance to the members of the consortium
Dissemination to scientists
• Scientific articles will be written and submitted to peer-reviewed scientific journals, to ensure an increased scientific and technological capability of Europe in the field of health, HIV/AIDS research, drug discovery and other related areas.
Under preparation: ISCIII is writing 2 papers at present, one about the antiviral activity and toxicity of 5HT and the other one about the mechanism of action of 5HT, and USR is preparing another paper about the inflammatory potential of 5HT.
• Contributions to international symposia: The following communications were accepted in national and international conferences:
o ISCIII and SEPROX presented a poster at the 7th IAS Conference on HIV Pathogenesis, Treatment and Prevention (IAS 2013), in June 2013. See the webpage of the conference at www.ias2013.org and the poster at aim-hiv.isciii.es:
Bedoya LM, Beltrán M, Auñón D, Gómez-Acebo E, Alcamí J. Antiviral activity of 5-Hydroxytyrosol, a microbicidal candidate. 7th IAS Conference on HIV Pathogenesis, Treatment and Prevention (IAS 2013); 2013 Jun 30- Jul 3; Kuala Lumpur, Malaysia.
o ISCIII and SEPROX presented an oral communication at the AIDS Vaccine Conference 2013 (www.vaccineenterprise.org/conference/2013/) held in Barcelona in October 2013. The title of the oral communication was “Antiviral activity of 5-Hydroxytyrosol, a microbicidal candidate” and it was presented by Dr Alcamí.
In addition, ISCIII and SEPROX presented a poster at the AIDS Vaccine Conference 2013. See http://epostersonline.com/aidsvax2013/?q=node/3504 for the poster:
Bedoya LM, Beltrán M, Auñón D Gómez-Acebo E, Alcamí J. Antiviral activity of 5-Hydroxytyrosol, a microbicidal candidate. AIDS Vaccine 2013; 2013 Oct 7-10; Barcelona, Spain.
o Two posters were accepted at the HIV Research for Prevention (HIVR4P) Conference (hivr4p.org) in October 2014. One of them was presented by ISCIII and SEPROX and the other one by USR, SEPROX and ISCIII:
Alcamí J, Bedoya LM, Obregón P, Beltrán M, Gómez-Acebo E, Auñón D, Capa L. Antiviral activity of 5-Hydroxytyrosol, a microbicidal candidate against HIV-1 transmission. HIV Research for Prevention (HIVR4P) Conference; 2014 Oct 28-31; Cape Town, South Africa.
Saba E, Origoni M, Taccagni G, Doglioni C, Auñón D, Gómez-Acebo E, Alcamí J, Poli G, Vicenzi E. Candidate Microbicide 5-hydroxytyrosol (5- HT) Inhibits Productive R5 HIV-1 Infection of Human Cervical Tissue Explants (CTE). HIV Research for Prevention (HIVR4P) Conference; 2014 Oct 28-31; Cape Town, South Africa.
o ISCIII and SEPROX presented a poster at the XVII Congreso Nacional sobre el Sida e ITS (www.seisida.es) in May 2015. See the poster at aim-hiv.isciii.es:
Alcamí J, Bedoya LM, Obregón P, Beltrán M, Gómez-Acebo E, Auñón D, Capa L. Propiedades del 5-hidroxitirosol, una nueva clase de microbicidas frente al VIH-1. XVII Congreso Nacional sobre el Sida; 2015 May 6-8; San Sebastián, Spain.
o ISCIII and SEPROX presented a poster at the 8th IAS Conference on HIV Pathogenesis, Treatment and Prevention (IAS 2015) in July 2015. See the webpage of the conference at: www.ias2015.org and aim-hiv.isciii.es for the poster.
Bedoya LM, Beltrán M, Obregón P, Gómez-Acebo E, Auñón D, Capa L, Alcami J. Antiviral activity of 5-Hydroxytyrosol, a microbicidal candidate against HIV-1 transmission. 8th IAS Conference on HIV Pathogenesis, Treatment and Prevention (IAS 2015); 2015 Jul 19-22; Vancouver, Canada.
• Seminars, lectures for students, etc.
The coordinator of the AIM-HIV project, Dr. Alcamí, carried out an important dissemination work aimed at publicizing the project among scientists or future scientists, clinicians and students. The performed dissemination activities are listed below:
o Seminar titled "Changes in gene expression induced by the HIV-1 Tat protein. Role of the second exon" at CEA and USR facilities in March 2013.
o Lecture “HIV pathogenesis” at the Universidad Europea, Madrid, May 26, 2015.
o Lectures in the “Infectious Diseases Master”, Universidad de Alcalá de Henares, Madrid, October 16, 2014, and February 7, 2015.
o Lecture addressed to Infectious Diseases Residents, El Escorial, Madrid, May 27, 2015.
o Lecture in the “AIDS Master”, Barcelona, April 15, 2015.
• Establishment of contacts with international research organizations in the field of microbicides with the objective of capture learning and sharing knowledge in these fields.
o Partners of the consortium (CEA, ISCIII and USR) participated in the European network for microbicide development, CHAARM, which represented the main European forum to share and discuss progress in this field. The partners of the AIM-HIV project participated in the CHAARM meetings:
19-21 March 2013. Camogli, Italy
18-19 March 2014. Camogli, Italy
o The members of the Scientific Advisory Board (SAB) are recognized scientists belonging to important research organizations:
Robin Shattock, professor of mucosal infection and immunity, leads the Group of Mucosal Infection & Immunity of the Department of Medicine, at Imperial College in London.
Clara Menéndez, senior investigator and research professor of the Barcelona Centre for International Health Research (CRESIB). This research institute is particularly important because of their work in sub-Saharan African countries, representing a potential collaborator in future clinical trials.
José María Gatell, head of the Infectious Diseases & AIDS Unit at Hospital Clínic in Barcelona, professor of medicine at the University of Barcelona, co-director of HIVACAT program for the development of an effective vaccine against HIV, co-director of the Spanish AIDS Research Network and European leader in HIV/AIDS Clinic Research.
Anna-Lena Spetz, associate professor at the Center for Infectious Medicine, Karolinska Institute, Karolinska University Hospital Huddinge.
The SAB members and Felipe García, replacing José María Gatell, participated in the AIM-HIV Progress Meeting (January 10, 2014, Madrid), giving an important feed back assessing the scientific objectives, evaluating the different work packages and the progress towards the objectives, appraising the management, coordination and dissemination plan, and finally, giving recommendations for the final period of the project. The SAB members had access to the AIM-HIV intranet.
o A workshop was organised (June 18, 2015, Brussels) where partners of projects funded by the EC and focused on microbicides (AIM-HIV, CHAARM and MOTIF) summarized their achievements (see agenda in Annex II). Members of the European Commission and other stakeholders, like the International Partnership for Microbicides (IPM) and the European & Developing Countries Clinical Trials Partnership (EDCTP), participated in this event aim to review how to go ahead with the results generated by these projects.
In addition, a short report was written for the Aidsmap and EATG sites.
• Contacts with scientific societies
ISCIII contacted with SEISIDA, a scientific society made up of health care professionals and professionals in other related disciplines (psychologists, social workers, sociologists, etc.) and members of NGOs working in the HIV/AIDS field. The coordinator of the project provided SEISIDA information about the project and the link of the AIM-HIV public web.
Dissemination to industry
Contacts with pharmaceutical companies working in the development of microbicides such as Gilead and Conrad were established. Data generated during the project were shared, especially those regarding the synergism between 5HT and tenofovir.
Dissemination to other stakeholders
• The Social Awareness Advisory Committee (SAAC) was set up in order to guarantee that research and development activities responded the end users’ needs and assured that their view was correctly reflected in the project. The institutions listed below were invited finally to be part of this committee:
o Foundation for Community Development (FCD), Mozambique, a civil organization, without party affiliation, which aims to join forces of all sectors of society in achieving an ideal of development, democracy and social justice.
o Centre International de Recherches Médicales de Franceville (CIRMF), Gabon, an institute for research, training and support to healthcare systems. The Centre's activities are diversified and cover research in basic virology and epidemiology, parasitology, primatology, genetics, haematology and health ecology.
o Barcelona Centre for international Health Research (CRESIB), Spain, a global health research institute developed from some of the leading academic and biomedical research institutions together with the Catalan Government, as a response to the new international health challenges of the 21st century.
o National Laboratory for Public Health, Virology Unit, Libreville, Gabon. The Emerging Viral Diseases Unit conducts public health initiatives and regular diagnostic activities for emerging viral diseases in all countries in the Central African sub-region.
The link of the public website of the project, a Report and an Executive Summary were sent to the members of the SAAC.
• Given the fact that AIM-HIV deals with diseases with great socio-economic impact, information about the project was given to Spanish NGOs working in the HIV-1 field. This was achieved through an online course of CESIDA (a Spanish platform grouping different NGOs working in the HIV-1 field) in which the microbicide topic was tackled.
Dissemination to society
The goal of dissemination to society was to inform the public, especially in the sub-Saharan area, but also in our social context, about the benefits of the results of AIM-HIV project, to guarantee the adequate product acceptance in the future. To this aim, the AIM-HIV consortium developed the following dissemination activities:
• Public website: Available at the link: http://aim-hiv.isciii.es. This website, as well as the private website, was launched in July 2013. It was an open access web containing a description of the project and the consortium, general information about HIV epidemic, prevention strategies and microbicides, and specific sections about publications and news. It was structured in a way to be clearly accessible to general visitors, trying to maintain a single and direct language in order to make the project comprehensible for general population and non-specialized audience. The public website included the AIM-HIV project specific email account to help contact of anyone interested.
ISCIII was responsible for designing and updating the public website within the project course. All the consortium partners were responsible for publicizing the website to other stakeholders and providing the data requested by the administrator of the website.
• The partner´s institutional websites publicized the project:
o SEPROX (http://seprox.es/wordpress/) published an article titled “Hydroxytyrosol, SEPROX and VIH” explaining the project and containing the link to the public website.
o The ISCIII institutional webpage published the news about the launch of the project:
http://www.isciii.es/ISCIII/es/contenidos/fd-el-instituto/fd-comunicacion/fd-noticias/10-jul-2012-Presentacion-Alcami-Microbicina.shtml
Including a link to the press release performed in July 2012:
http://www.isciii.es/ISCIII/es/contenidos/fd-el-instituto/fd-comunicacion/fd-noticias/10-jul-2012-LNP-Alcami.pdf
And the presentation of the project:
http://www.isciii.es/ISCIII/es/contenidos/fd-el-instituto/fd-comunicacion/fd-noticias/10-jul-2012-Presentacion-Alcami-Microbicina.pdf
o The laboratory of Dr. Alcamí (Aids Immunopathology Lab, ISCIII) published in its webpage (http://hiv-lab.com/) a section about the AIM-HIV project (http://hiv-lab.com/index.php/participation/aim-hiv).
• Press:
o Information about the launch of the project was widely disseminated through different mass media in Spain: television, radio, national, local and specialized newspapers, and specialized websites.
o On the occasion of the World Aids Day on December 1, 2014, Dr. Alcamí granted several interviews about the AIM-HIV project:
Spanish public TV news:
http://www.rtve.es/alacarta/videos/noticias-24-horas/continua-investigacion-prevencion-del-sida-instituto-salud-carlos-iii/2883957/#aHR0cDovL3d3dy5ydHZlLmVzL2FsYWNhcnRhL2ludGVybm8vY29udGVudHRhYmxlLnNodG1sP2N0eD00NjA1MiZwYWdlU2l6ZT0xNSZvcmRlcj0mb3JkZXJDcml0ZXJpYT1ERVNDJmxvY2FsZT1lcyZtb2RlPSZtb2R1bGU9JmFkdlNlYXJjaE9wZW49dHJ1ZSZ0aXRsZUZpbHRlcj1TSURBJm1vbnRoRmlsdGVyPTEyJnllYXJGaWx0ZXI9MjAxNCZ0eXBlRmlsdGVyPSY9dW5kZWZpbmVkJg
http://www.rtve.es/alacarta/videos/telediario/2013-se-diagnosticaron-3200-nuevos-casos-sida-espana/2884112/
Internet press:
http://noticias.lainformacion.com/salud/enfermedades-viricas/el-gel-vaginal-contra-el-vih-basado-en-el-olivo-entra-en-su-fase-definitiva_cidafxuk0ZO355TkI4sap4/
http://www.republica.com/2014/11/30/el-gel-vaginal-contra-el-vih-basado-en-el-olivo-entra-en-su-fase-definitiva_854317/
Radio Uruguay:
Programme “Efecto Mariposa”, Uruguayan public radio (www.radiouruguay.com.uy; http://efectoblog.blogspot.com/):
http://www.radiouruguay.com.uy/innovaportal/v/62851/22/mecweb/angeles_en_america?parentid=44871
o Dr. Alcamí also granted the COPE radio an interview about HIV, introducing the AIM-HIV project, on May 18, 2015.
List of Websites:
http://aim-hiv.isciii.es/

aim-hiv@isciii.es

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