NATIONAL AIDS RESEARCH INSTITUTE (Indian Council of Medical Research) Annual Report. April 2014-March 2015

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3 (Indian Council of Medical Research) Annual Report April 2014-March 2015 G-73, MIDC, Post Box No. 1895, Bhosari, Pune Telephone: , Fax: Website:

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5 From Director's Desk The National AIDS Research Institute (NARI) was established in 1992 with the mandate to conduct multidisciplinary research with focus on HIV & STI prevention and care and treatment for HIV infected individuals. In the initial years of its activities, NARI generated evidences that were well utilised in development of strategies by the National AIDS Control Organization (NACO). The National AIDS Control Organization successfully contained the HIV epidemic in India through rigorous implementation of comprehensive prevention and treatment strategies. NARI continues to work in close association with the program through various program support activities such as evaluation HIV test kits, external quality assurance for HIV serology and CD4 count estimations, and was also involved in HIV sentinel surveillance and integrated biological and behavioural surveillance, early infant diagnosis along with its quality research on various facets of HIV disease. During , NARI continued to be involved in conduct of HPTN 052 trial, the findings of which led to change in the HIV management and prevention guidelines worldwide. It led to evolution of concepts known as 'test and treat' and 'treatment as prevention' (TAsP). Recognising the importance of antiretroviral therapy in prevention and control of HIV epidemic and that its success depends on rigorous implementation of free ART programming, NARI focussed its research on early warning indicators, virologic suppression at one year after initiation of ART and emergence of drug resistance. It generated evidence useful for the program to strengthen its strategies. A significant proportion of HIV infected individuals consume herbal drugs, most often without informing the health care providers. Concurrent consumption of these medicines can potentially impact bioavailability of antiretroviral drugs as well as drug-drug interaction. A study was initiated to describe the herbal medicines used by HIV infected individuals. Cure research has been accorded a high priority globally with reports of isolated successes that were based on different strategies. One of our studies that will be presented in the prestigious Keystone Symposia found that Bryostatin may be a useful anti-latency agent and HIV escapes elimination through increase in IL-10 levels. Our National antiretroviral regimen is based on 2 NRTIs & 1 NNRTI. Non-nucleoside reverse transcriptase drugs are known to cause hepatotoxicity. A case-control study done on xenobiotic enzymes revealed interesting findings. Immunologic studies on long term non-progressors, Th 17, NKT cells and SAM HD1 have found some interesting leads. Efforts to identify new antiretroviral drug/s through in vitro anti-hiv testing are ongoing. A study on Mycobacterium tuberculosis identified some important leads for newer drug targets. Another study conducted on HPV infection revealed that a significant proportion of infection is caused by HPV types that are not used in HPV vaccine. A study on psycho-social needs of HIV infected adolescents revealed findings that are useful for counsellors as well program managers. I take pride in the fact that despite decline in HIV research funding, the scientists at NARI managed to published 49 research publications and contribute in development of various program strategies and guidelines nationally. The future directions of HIV research are getting intensified towards prevention & management of chronic morbidities and identification newer strategies for cure and implementation science research. In view of this, the Institute realigning its focus of research. I take this opportunity to thank the Director General of ICMR, Members of Scientific Advisory Committee and Ethics Committee, National AIDS Control Organization and all the national and international funding agencies for their support. We look forward to continuing our meaningful contribution towards the program as well as HIV research in future. Dr. R. R. Gangakhedkar Director-in-Charge

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7 I N D E X Sr. No. Title Page 1 HIV Prevention Clinical Care and Treatment Biology of HIV Infection STIs and Opportunistic Infections Product Development and Testing Health Program Research and Contributions to the National Programme Other Prevention Appendices 119 I NARI Services NARI Clinical Research and Services 121 NARI Laboratory Services 124 The Library and Information Centre 124 II Major Events / Training / Workshops 126 III Publications 130 IV Training provided to students 136 V Committees Scientific Advisory Committee 137 Ethics Committee 138 Community Advisory Board 138 VI Staff Members and Budget List of Staff Members 139 Budget for

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9 1 HIV Prevention ANNUAL REPORT n 7

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11 A.1) A randomized trial to evaluate the effectiveness of antiretroviral therapy plus HIV primary care versus HIV primary care alone to prevent the sexual transmission of HIV-1 in serodiscordant couples [HPTN 052 study] Investigators: Dr. Sanjay Mehendale (NIE), Dr. Sheela Godbole Sponsor: HIV Prevention Trials Network (HPTN) and Division of AIDS (DAIDS), National Institutes of Health, USA Study Period: st Status: Ongoing (as of 31 March 2015) Study Population: HIV serodiscordant couples in which the HIV-infected partner is ART naïve and has a CD4+ 3 cell count of cells/mm. The study is a phase III, two-arm, randomized, controlled, multi-center trial. The purpose of this study was to determine whether antiretroviral therapy (ART) can prevent the sexual transmission of HIV-1 in HIV-1 serodiscordant couples and to assess the clinical outcomes of 'early' ART initiation in the HIV infected partners. The study was conducted at 13 sites across 9 countries. Although the interim results of the study announced in 2011 (NEJM 2011) showed that 'early ART' conferred an unprecedented 96% protection by along with significant clinical benefits to the HIV infected partner; the study follow-up was continued to assess the long term impact of 'early ART' on HIV prevention as well as treatment outcomes. Randomization, treatment and follow-up: HIV-infected index cases were assigned at random in a 1:1 ratio to one of two treatment arms: Arm 1: ART upon enrollment plus HIV primary care. Arm 2: HIV primary care without initiation of ART until the participant has two consecutive measurements of a CD4+ cell count within or below the 3 range of cells/mm, or develops an AIDS-defining illness. Participants required quarterly follow up visits and come to clinic in between for clinical care or to pick up study drugs. l l NARI enrolled 175 HIV discordant couples in the study. All couples were planned to be followed until the last couple enrolled in the study completed their 60-month follow-up visit. Hence it was planned that all rd couples would be off study by 3 May After July 2011, all HIV infected persons in the delayed ART arm who had not yet initiated ART were offered ART irrespective of CD4 cell counts. Retention rate of participants in the study has consistently been above 98% every year. During the year, 654 follow-up visits were completed for about 162 HIV discordant couples. One participant in delayed arm initiated ART during the year and with this 93% of all HIV infected participants in delayed arm had initiated ART by the end of the study. The couples were informed about upcoming closure of the trial and plans were made with the approval of Ethics Committee and NACO for transition of HIV infected study participants to government ART centres of their choice. While a majority planned to continue to access ART at the NARI ART centre, other participants needed to be transferred to centres where they could access second line ART (SACEP) or places convenient to them. As of 31 March 2015, 116 index (HIV infected) and 106 HIV negative partner participants were terminated from the study. Of these 34 were transferred to second line ART centres. ANNUAL REPORT n 9

12 A.2) Exploring attitudes and behavioral issues related to anal sex and other sexual practices among HIV infected women respondents of the anal HPV prevalence study Investigators: Dr. Sheela Godbole, Mrs. Neelam Joglekar, Dr. Arati Mane Sponsor: Intramural Study Period: April 2014-October 2015 NARI conducted an exploratory study to assess the prevalence of type-specific anal HPV infection in HIV-infected women in Pune in year The prevalence of anal HPV (any subtype) was observed to be 14.74% with a 9.47 % prevalence of high risk HPV (JAIDS 2015). Anal sexual intercourse is considered to be an important predictor of anal HPV infection. The reported prevalence of anal intercourse in our study population was just 5%. However, anal sex may not be the sole cause for anal HPV and other factors like anatomic proximity of anal and vaginal orifices, non- penetrative sexual behaviours and other practices related to ano-genital hygiene could be associated with anal HPV prevalence. We conducted a qualitative sub study to understand the practices that could possibly lead to anal HPV infection among Indian population and to assess whether anal sex may have been under-reported. It aims at understanding attitudes and behaviours surrounding the practice of anal intercourse among heterosexual couples and to identify other sexual and hygiene practices that may increase the risk of anal HPV infection. In all, In-depth Interciew (IDI) of 15 participants of the previous anal HPV prevalence study and four male partners as well as four key informant interviews (KII) (two sexologists and one psychologist and one female counselor) were conducted after obtaining informed consent. Translations of the interviews, data coding and analysis are underway. A coding scheme has been developed 'a priori' based on IDI guide and repeated reading of the data. Observations based on interviews with the first five female participants are reported here. Background of the respondents: The median age of the fifteen women participants was 35 (30, 42.5) while that of the male partners was 41 years (33, 55). Five of the female participants were anal HPV positive. Three women had previously reported a history of anal sex. Five of them reported multiple partners. Five were currently married. Attitudes regarding sexual practices Vaginal sex was perceived as a Natural and pleasurable practice. It was justified by an 'anal HPV' negative widow as, [A21] This is appropriate. Gives pleasure, satisfaction. This is the only way to get progeny. Only this fulfils need/desire. Oral sex was perceived to be dirty and nauseating. An anal HPV positive divorced participant reasoned it out as [A 28] Not right; harmful, weird people do it. The semen can spread in our body through our mouth thus it is harmful. However oral sex was reported to be practiced by many respondents. Anal sex was reported by two women who had answered 'no' in the previous survey, suggesting underreporting in the previous survey. Other women clarified that their husbands had requested anal sex but they had denied them. Most women reported anal sex, while they were unable to have vaginal intercourse due to various reasons. Almost all female participants expressed negative attitudes towards anal sex. It was perceived as dirty and painful. One anal HPV negative widow said, [A21] once my husband tried for anal sex it pained me a lot, still he was trying forcefully. I had lot of pain. I felt like shouting loudly. In case of one anal HPV negative married participant it was ANNUAL REPORT n 10

13 practiced because she has undergone a surgery. She said, [A001] I had undergone family planning operation. I was feeling weak. Husband kept anal sexual relations forcefully. Other reasons for anal sex were reported to be menstruation and practiced by sex workers. One anal HPV negative married participant said that, [A16] some girls have extra marital relations or in business of sex. They tell that some people/customer perform anal sex forcefully. A.3) A study of sexual networks of single male migrants in Pune city Guide: Dr. Seema Sahay, Ph. D. student: Neelam Joglekar Sponsor: Intramural Study Period: This study aims at generating data on sexual networks of the migrants in context of HIV to inform strategic decisions on HIV programming for migrants in Pune. In this study, psychosocial factors and contextual vulnerabilities of migration are being explored. The study comprises of three phases: I) Formative phase II) Mapping III) Quantitative data collection phase. Figure 1.1: Data collection method I: Formative Phase (Oct 2013-July 2014) Semi structured questionnaire development In-depth Interviews with Migrants n=16 During reporting period=4 Key informant interview with stakeholders n =8 During reporting period=4 II: Mapping (Initiated in Dec 2014) Primary recruit (one from each slum) Total =8 slums During reporting period, one pilot mapping exercise completed using digital map and information gathered for the slum using structured mapping tool III: Quantitative Phase (not initiated) Sampling : Respondent driven sampling (RDS) ANNUAL REPORT n 11

14 Preliminary findings from formative phase (Migrants: n=16) Median age of migrants was 28 years (21-38 Years). Mean age at migration was 22 (15-36 years). Median duration of migration was 6 years. Median duration of stay in Pune was 4 years (4 months to 8 years). Median number of destinations visited was 2 (1-4). One each was from following categories: student, marketing executive, hotel worker, barber, and a transgender, two each were skilled contract laborers, two each industrial workers, supervisors and engineers, and three were construction workers. One migrant reported staying alone in a rented flat. Of 16 respondents, 13 reported staying in a shared accommodation with other migrant colleagues. Two migrants reported staying with family members. Migration Process: Push factors facilitated the decision to migrate in case of 12/ 16 migrants. These push factor include poverty of the family, need to earn money, having economic responsibility for family members, dependency on agriculture, no opportunity for out of farm earnings in the place of origin. Family disharmony was the reason for migration in case of two migrants. No interest in education and migration of the friends boosted their decision to migrate. Pull factors were the facilitators for migration in case of three migrants. They migrated for good opportunities for higher studies or better job. One person was a transgender (TG), and migrated to western India as TGs are treated with respect in this region. The respondent said, [MM9] we know that we people (Transgender) are respected in Mumbai and Pune side. Barriers to cope up with new surroundings: Language proficiency and cultural identity with culture from destination place are considered as important factors affecting coping migrants with the environment at destination. Almost all migrants (n=16) in the study faced language barrier, problem with different taste of food, problem with communicating with people. A migrant student said, [MM1] this when we talk in regional language, we always talk very softly. Another migrant who had problems with food said, [MM2] Mix vegetable and rice. We don't put spices at all. (We put) only turmeric, red chili powder and cumin seeds. Whatever you get in 'mess' does not taste in our way hence we cook for ourselves. One more migrant faced problem because of political movement against migrants. He said, [MM6] Thus my brother said that if you find it good then only, you stay here. That time =name of local political leader= was driving Bhaiyya people away. Some facilitators for better adjustment at destination were also reported by couple of migrants. They were cleanliness, pleasant weather, and respectful behavior by local people. Sexual behavior: Sexual debut took place at destination places for 13/16 migrants. A transgender (TG) said, [MM9] Then afterwards, that boy also started loving me very much. Then I used to go with them for outing. Then afterwards in third years the feelings became stronger and when I had that (sex) with him, I did not use a condom. Three migrants did not report any sexual relationships and two of them reported that they do not follow wrong path in order to maintain family's position. Migrants reported sex with FSW often initiated their sexual activity at destination because of peer pressure. Staying in group of people from place of origin with elderly relatives and intention to return back might have a role in preventing migrants from having sexual relationships with HRG partners which needs to be explored in the quantitative study. ANNUAL REPORT n 12

15 A.4) NARI - AIDS Rural Research Initiative in Maharashtra (N-ARRIM) Investigators: Dr. Seema Sahay, Dr. Asha Jadhav, KIMSU (Karad), Mrs. Tapati Datta, IAVI (New Delhi) Collaborators: Krishna Institute of Medical Sciences University, Karad, International AIDS Vaccine Initiative Sponsor: Indian Council of Medical Research and International AIDS Vaccine Initiative Study Period: Project N-ARRIM was initiated with the objective of building partnerships with local communities/ stakeholders and assessing the research needs in the community and build HIV research capacity in consultation with local stakeholders at rural medical colleges. The study also aimed at conducting rapid assessment and map most at risk and hard to reach population groups in the area for future studies on characterization of HIV, studies on genetic diversity of Indian strains and immune response to HIV. Study process: Two sites (Udgir and Karad) were identified for establishment of rural centres in Maharashtra. The former site was dropped owing to challenges pertaining to the partner, the Life Care Foundation at Udgir who later communicated their unwillingness for establishing this centre. The latter site has been established in collaboration with the Krishna Institute of Medical Sciences University (KIMSU) at Karad. KIMSU is a NAAC accredited medical college having bed strength of 1000 beds providing health care services in the rural area since last 30 years. Multiple stakeholders' consultations were made and visits to the community and local NGOs' catchment communities were also made before delineating the vision of N-ARRIM centre at the Karad. These consultations culminated in the following vision: l l l Building a private-public partnership model between NARI (GO), KIMSU (Site Institution) and IAVI (NGO) Dedicated for HIV research and envisioning expansion of HIV related research activities in rural and semiurban pockets of Satara District Avoiding duplication of ongoing services, the project objectives and activities were to be done through regular interface and consultation with the local bodies of governance - State AIDS Control Societies (SACS), District AIDS Prevention and Control Units (DAPCU), and other relevant local stakeholders In order to build the essence of a community owned and research driven project, engagement with local stakeholders and key populations was considered pivotal N-ARRIM centre: It has two components: 1) Community and, 2) Clinic. KIMSU provided a dedicated space of approximately 1500 sq. ft. for N-ARRIM project at Krishna hospital premises. The laboratory set was established in the Microbiology department of the institute. The essential laboratory equipments and instruments were procured as per ICMR guidelines and were installed in the Microbiology department of the institute. ANNUAL REPORT n 13

16 A brief about clinic: The physical infrastructure of N-ARRIM centre has been established in the premises of Krishna Institute of Medical Sciences University (KIMSU) and inaugurated on December 1, It is envisaged that the center will have the facilities of promoting research on HIV. The centre has been established as per NACO's guidelines for voluntary counseling and testing centre and there are ongoing efforts to link it to NACO ICTC and ART centers. After initiation of the center, activities of the VCTC were initiated. A total of 362 individuals visited N- ARRIM VCT between May 2013 and March Of the 260 individuals who got tested for HIV at N-ARRIM clinic, 27 were HIV positive. A brief about community mobilisation: The linkages were established with local stakeholders including Public Health Dept., Zilla Parishad, Satara, Civil Hospital, Satara, all SDH, RH and PHCs, DAPCU, ICTC's and ART centres of Satara district. In addition, linkages were established with NGOs and CBOs. Active involvement of Secretary, DHR, Director, DHS, Govt. of Maharashtra and ADHS, DHS, Govt. of Maharashtra N-ARRIM, was sought. N-ARRIM as a program was presented to these officials and we received assurance of support from DHS, Govt. of Maharashtra. Using Programme Based Approach (PBA) N-ARRIM aimed at community ownership of the project N-ARRIM in Karad. Hence community consultation is the key strategy of N-ARRIM for outreach and community engagement. The process of CAB formation was started with visits to the direct and indirect stakeholders. The contacts included visits to NGO's such as Sangram, NSP+ (Network of positive People in Satara), community based organizations such as 'Link worker scheme' and 'Veshya Mukti Sanghatana' in Karad and other blocks in Satara district as well. Also efforts have been made to approach government officials in Satara district, which includes sub-district hospital, Karad, rural hospital, Patan block and 6 Medical Officers of Primary Health Centers (PHCs) in Karad and Patan block. Twenty one names were short listed at Karad for prospective CAB, comprising of people from diverse vocations and professional background - Positive people, organizations working with key population groups, legal experts, academicians, members of women's groups, representatives of labour unions, ART in-charge, community workers, members of Lions' and Rotary Clubs etc. The criterion for selection of the CAB members was people from all walks of the community, having infected or affected by HIV/AIDS, contributing for the upliftment of the HIV/AIDS affected, professionals and intellectuals. A total of eight members form the N-ARRIM CAB. Simultaneously along with CAB formation process, NARRIM team strategically planned for situational analysis of entire Satara district to understand community health needs. The purpose of the situational analysis was to build an ANNUAL REPORT n 14

17 in-depth understanding of HIV and broader public health scenario at the project intervention site, which, in turn will provide critical inputs to the project uptake. The focus of the situational analysis was to collect secondary information on various aspects from government departments and understand the scope for undertaking research activities in Satara district. This included administrative information, demographic and socio-economic information, mapping of the existing health infrastructure, health services and it's functioning in the district. Data collection also entailed understanding and analyzing the status of RCH indicators, HIV/AIDS, TB and Malaria visà-vis the national targets and global goals. The situational analysis of Satara district gave us a lead to pursue mapping exercise to understand the high risk and vulnerable pockets within the district. In addition, Satara is a Category 'A' designated district in Maharashtra (high HIV prevalence) as per NACO. Based on HRG mapping using key informant and HRG site verification, the HRG population have been estimated in six major high risk sites at Satara district. Table below shows the estimated high risk populations of transgenders, males having sex with male and female sex workers. Table 1.1: Identification of high risk spots in Satara district Sr. No. Major high Estimated size risk spots 1 Satara *FSW 150 to 200, # TG / MSM 50 to 60 2 Karad FSW 250 to 300, TG / MSM 100 to Khandala Folk Dancers 200 to 300, Female Sex Workers 120 to Phaltan Female Sex Workers 70 to 85, TG / MSM 20 to 25 5 Wai Female Sex Workers 65 to 80, TG / MSM 10 to 15 6 Koregaon Female sex Workers 35 to 45, TG/MSM 10 to 15 * FSW Female Sex Workers # TG /MSM Transgender / Men having Sex with Men Preliminary findings of HRG mapping 1) Sexual practices among adolescent age group reported were considerably high. The reasons speculated were common such as impact of media such as cinema, television, use of mobile phones and degradation of the value system in the society and lack of morality. However, this also indicated a need for sexual health education for the rural adolescents. Studies could be taken up to understand the vulnerabilities of the rural youth and design contextual education interventions. 2) High proportion of risky behaviors among married individuals was reported. The reasons recounted were: lifestyle changes, e.g. high amount of stress, tensions, need of change of partner, lack of sexual satisfaction from spouse, lack of fear, psychological and emotional need, lowering of social and ethical values in the society, excessive earnings etc. Socio-behavioral studies to assess the psychosocial and psychosexual needs of rural married couples could be designed to understand the vulnerabilities, risk behaviors and intervene accordingly. ANNUAL REPORT n 15

18 In line with our objective no. 3 of N-ARRIM project of strengthening capacities of the collaborative partner, two community based studies were initiated by KIMSU, Karad. The situational analysis using MDG goals helped in identifying research priorities in Satara district which helped in moving ahead. Project N-ARRIM took another step and conducted mapping of high risk groups in Satara district which led to identification of hot spots and new high risk groups. Based on these finding in the next planned phase of project N-ARRIM, a community based open cohort study is being proposed and has been submitted to the council. Acheivements: l l l l First ever Rural Research Center with a specific focus on HIV / STIs has been established at Karad, District, Satara in Maharashtra Initiated community mobilization and sensitization with the help of this rural research center First ever 'rural' Community Advisory Board (CAB) with a focus on HIV infection has been formed and CAB is actively contributed towards NARRIM aims The situational analysis and HRG mapping have given leads for further expansion of the scope of research among rural population. The scope would encompass HIV, HIV-TB co-infection, HPV and cervical cancer ANNUAL REPORT n 16

19 2 Clinical Care and Treatment ANNUAL REPORT n 17

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21 B.1) Centre for excellence in HIV-TB co-infection Investigators: Dr. Manisha Ghate and Dr. Madhuri Thakar Sponsor: Department of Biotechnology Study Period: HIV infection is considered to predispose the host to active Tuberculosis generally by impairing the hosts' immune system through killing of CD4 T lymphocytes. There is increasing evidence of significant heterogeneity within the CD4 T lymphocyte population, both in terms of function and susceptibility to Tuberculosis infection. Furthermore, it also emerges that each pathogen, can activate a very distinct set of functionally important CD4 T lymphocytes, which directly impact the ability of these cells to be infected and killed by HIV. The overall goal of this study is the identification of novel correlates of protective immunity to HIV and TB infections by comparative analysis of the immune response in subjects infected with one versus both pathogens that can be translated into novel biomarkers, which may aid patient monitoring, treatment and vaccine design. This is a core grant of the Centre of Excellence (COE) application with collaborating institutions including, Indian Institute of Science, Bengalure (responsible for co-ordination), St. Johns Research Institute, Bengalure, National AIDS Research Institute, Pune, Genomics Laboratory, Center for Cellular and Molecular Platforms, Bengalure, National Institute of Immunology, New Delhi. The titles and specific objectives of the four component award that form this consortium grant are as follows: l l l l Is an altered IF response genes profile in CD4 T cells and neutrophils a distinguishing marker of HIV/TB co infection? Is an elevated IFN response associated with impaired homeostatic signals in CD4 T cells and does this distinguish HIV/TB co-infection versus infection with each pathogen alone? What is the molecular profile and phenotype of HIV and M.Tb. specific cells compared to cells of other specificity in HIV-TB co infection? Is there evidence that HIV preferentially infects M.Tb. specific cells compared to cells of other specificity in vivo and does this correlate with expression of known markers of cellular HIV permissiveness? How does this differ in HIV-TB co infection versus infection with each pathogen? Validate bio-signatures identified in discovery phase for clinical utility in distinguishing: latent versus active M.Tb. infection and correlates of HIV disease progression versus HIV/TB co-infection. NARI is involved in two work packages: Biomarker Discovery and Validation of the Mucosal Immune Response following HIV-TB co-infection and Developing a Clinical Core for In-Depth Studies of the Correlates of Protective Immunity to HIV-TB co-infection. The patients were screened for their exposure to TB using Interferon gamma release assay (IGRA) and were enrolled on the basis of the IGRA result. Of the 12 patients screened so far, 8 were IGRA negative, 3 patients were IGRA positive and one showed borderline reactivity. Of the 12, six patients fulfilled eligibility criteria and were enrolled in the study. The screening and enrolment of the study participants in each group is underway. ANNUAL REPORT n 19

22 B.2) Pilot study of early warning indicators (EWIs) for HIV drug resistance monitoring and quality care indicators in four programme ART clinics in Pune Investigators: Dr. Manisha Ghate and Dr. Raman Gangakhedkar Sponsor: Intramural Study Period: The National free ART program has completed a decade and it is expected that the number of patients with treatment failure will be on the rise. Since the development of HIV drug resistance (HIVDR) is a paramount concern in mature ART programmes, monitoring processes to optimize prevention of HIV drug resistance and ensure the success of first line ART is important. HIVDR may necessitate switch from non-nucleoside reverse transcriptase (NNRTI) based first-line regimens to more expensive and less tolerated boosted protease inhibitor-based second-line regimens, lead to limited drug options available in the programme and increases the overall programme cost. Due to non-availability of routine viral load and HIV drug resistance (HIVDR) testing in resource limited settings, the World Health Organization (WHO), as part of its global strategy for prevention and assessment of HIVDR, developed a set of early warning indicators (EWIs) that assess ART clinic, patient and program related factors associated with HIVDR emergence. These indicators are based on the data that are routinely collected in patients' medical and pharmacy records, are comparatively inexpensive and may alert national ART program planners to processes which may be adjusted to minimize the emergence of HIVDR. In order to strengthen monitoring of the quality of ART services, the National AIDS Control Organization (NACO) has recommended regular monitoring of Quality of Care Indicators in HIV care (QCI). This includes regularity in CD4 count testing, timely initiation of ART and duration of ART initiation in HIV TB co-infected patients after initiation of anti tubercular therapy (ATT) in addition to the EWIs that are deemed most important for monitoring implementation of the ART programme. QCI monitoring provides data for the ART programme to improve patient care at ART sites and minimize HIV drug resistance. The monitoring also allows comparison of quality of ART services across time with respect to improvements and evolution. We assessed feasibility and usefulness of WHO HIVDR EWI monitoring and Quality of Care Indicators (QCI) in HIV care at four ART programme clinics in Pune city located in the high prevalence state of Maharashtra as an ART program evaluation tool. This study was a part of 'Effectiveness of antiretroviral therapy in free ART centres in Pune city'. ANNUAL REPORT n 20

23 ART clinic NATIONAL AIDS RESEARCH INSTITUTE Table 2.1: Early Warning Indicators (EWI) for four ART clinic in Pune city EWI1: On time pill pick up % (number EWI 2: Retention in ART care at 12 EWI 3: Pharmacy stock out % of EWI 4: Pharmacy dispensing practices % of patients) months % (number months with no of patients with mono or of patients) pharmacy stock outs, dual therapy (number of (12 months) patients) Target: Red: < 80% Target: Red: < 75% Target: Red: < 100% Target: Red: > 0% Amber: 80-90% Amber: 75-85% Green: 100% Green: 0% Green: >85% Green:>85% Clinic A 84 (201) 82 (187) 83 (12) 0 (203) Clinic B 77 (216) 67 (232) 42 (12) 0 (224) Clinic C 94 (200) 83 (160) 100 (12) 0 (201) Clinic D 81 (216) 74 (190) 92 (12) 0 (217) All four ART clinics studied met the WHO EWI target (100%) for ART prescribing practices. The target for EWIs 'on time pill pick-up' and 'pharmacy stock outs' could be achieved in one clinic and none of the clinics could meet the EWI target for retention in care at 12 months. The overall performance on quality care indicators fell below target in some clinics. Efforts are required for improving retention in care, timely pill pick up and ensuring clinic-level drug supply continuity. B.3) Effectiveness of Antiretroviral Therapy in four free ART Centres in Pune Investigators: Dr.R.R.Gangakhedkar; Dr. MV Ghate; Dr. Smita Kulkarni; Dr. Swarali Kurle; Dr. Nitin Gaekwad; Dr. Dileep Kadam; Dr. Shankar; Dr. RS Paranjape, Dr. B.B. Rewari Study Period: May June 2015 A cross-sectional study to assess effectiveness of ART was conducted among 846 eligible consecutive patients who were initiated on ART between months in four free ART centres in Pune. The overall frequency of virological failure at one year was 12.3% (95% CI %). NARI ART centre had the lowest treatment failure of 4.4% among four ART centre (N.S.). The risk of virological failure was found to be 2.19 (95% CI ) and 4.39 (95% CI: ) folds among those who do not live with their partner and those who were not adherent to ART respectively. Only 7/40 patients who failed did not reveal any drug resistant mutation against NRTI & NNRTI indicating late detection of virologic failure even at one year. Of the 80 tested with genotypic drug resistance assays, 18 patients revealed K65R mutation. These findings also indicate that virologic monitoring should be considered in monitoring patients on first line for early detection of treatment failure without impacting second-line antiretroviral therapeutic options. Intensified efforts for counselling among those who do not live with their partners need to be undertaken in the program. ANNUAL REPORT n 21

24 B.4) Comparison of Montreal Cognitive Assessment (MOCA) battery with two test short screening battery for screening of neurocognitive impairment Investigators: Dr. Manisha Ghate and Dr. Dileep Kadam Sponsor: Intramural Study Period: HIV Associated Neurocognitive Disorder (HAND) is one of the important conditions that may affect the quality of life of PLHIVs. The severity of dementia has reduced with the introduction of HAART but studies on HAND in treated patients have documented high persisting rates of mild-to-moderate neurocognitive impairment (NCI). The main problems with recognition of HAND are: many patients with HAND have mild symptoms, patients may not report symptoms from lack of awareness and clinicians may not have relevant training for diagnosis and management of HAND, practical difficulties with routine screening for HAND in busy clinic settings and limited access to formal neuropsychological testing. Therefore, there is a need for brief screening tools that are sensitive to HIV-related cognitive dysfunction and easily administered in OPD set ups. Therefore this study aims to compare the Montreal Cognitive Assessment with a short two test battery derived from a comprehensive, validated battery developed at UCSD and tested at NARI in a previous study. The study was initiated in February 2015 at NARI and BJMC. A total of 37 patients were screened and 15 were enrolled till March B.5) Psychosocial needs among HIV infected adolescents in Pune, India Guide: Dr. Seema Sahay, Ph.D. student: Archana Verma Sponsor: Senior Research Fellowship (ICMR) Study period: Longevity of children born with HIV is being witnessed as a positive outcome of free ART roll out program in India. A large number of infected youth with perinatal HIV and ARV exposure provides a strong public health mandate to monitor their development and routinely assess their mental health needs. In this study, a mixed method approach is being used to understand the mental health needs of perinatally HIV infected adolescents in the context of Indian culture. The study has 2 phases: 1) Qualitative research phase, and 2) Quantitative research phase. The first phase of the study i.e. the qualitative research phase is ongoing. Using purposive sampling technique a total ANNUAL REPORT n 22

25 of 35 respondents were recruited from different geographic regions of India. The categories of respondents who participated in this phase were: Primary caregivers of HIV infected and uninfected adolescents, health care providers who deal with adolescents, HIV infected adolescents and HIV uninfected adolescents. The data collection has been completed and data processing is ongoing. The transcribed, translated and typed interviews are being entered in QSR NUD*IST software version 6.0 and primary code list is being prepared following the grounded theory principles. Preliminary findings (qualitative phase): The emerging themes are described as follows: Critical 'age group' requiring focused support: All the respondents acknowledged the burden of psychological distress among HIV infected adolescents. Health care providers emphasized the need for psychological support for this population. This population was aptly described by an HCP: these are the adolescents; they need lot of other support as compared to the adults as you know some of them are in schools, some of them are in college, some of them are in their age where they go to get married or couple of them who are married and they are in the reproductive stage. So they need a variety of supportive counseling [HCP-DO-15]. Parental communication about RSH issues: There are cultural and religious barriers for parent-adolescent communication on RSH issues. In our religion these things [/sexual issues/] are being curtailed, do not do. Stated by a father of uninfected adolescents [CG-HN-02]. But HIV is seen as the trigger for communication on RSH issues with adolescents. so if a girl is normal and you will make relations with her then it will be wrong, means her loss will also happen. That's why I told to son [about sexual relations] personally, did not tell anything to daughter. responded a mother of HIV infected son & uninfected daughter [CG-HP-07]. Mental Health needs: Adolescence itself is a phase of emotional and physical turmoil and HIV infection adds additional burden. The mental health needs of adolescents get linked to HIV, its transmission and how to live with HIV. Various issues have been identified by the respondents that result in emotional distress among infected adolescents as follows: i. Suspicion: Adolescents might have suspicion about parents after knowing the modes of transmission of HIV. A son asked his mother: Mother, this HIV has happened to me because of you or because of father? You have this [/HIV/], means you got from father or from outside? Figure 2.1: Conceptual framework for issues surrounding the mental health needs of adolescents Body Image (stunted physical growth and delayed puberty) Social Networking (peers) Social Stigme & discrimination Secrecy about illness Death of dear ones & life long illness Affected Mental health and social development of adolescents Parent-Adolescent Relationships Exploration of Sexual Relations Hopelessness from life Daily medication Future aspirations (career, intimate relations, marriage etc.) ANNUAL REPORT n 23

26 Similarly, caregivers, being unaware of parents' status voice suspicion on infected adolescents. A grandmother [CG-HP-04] of infected girl asked: So we asked to =XXX= [/granddaughter's name/] that how has it [HIV] happened? With whom did you make sexual contact? We told her directly. Is someone is there or someone forced you then she said no. Anger : Adolescents found that their parents are responsible for their ill health and because of them they cannot fulfill their dreams. ii. She becomes angry that because of him [/father/] this [/HIV/] happened to me. She doesn't feel good and never takes his name [/never talked about father/], never ask anything Isolation: Both captive and self isolation has been reported by the parents and adolescents. Parents prevent socialization of their infected children because of fear of social stigma. A mother of HIV infected son shared:...do not send because of the disease... daughter goes with him [/husband/], [I] do not send son because this tablet [/ART/] is going on [/participant send daughter to relatives with father as she is uninfected but does not send son as he has HIV infection/]. [CG-HP-01] Adolescents reported that knowing their HIV status leads to isolation: I used to feel lonely that why only I have this disease, why not others have earlier how much I was close with my friends which I am not now because after knowing about this disease [HIV] I have kept distance from my friends [AD-HP-01] Impact on Future aspirations i. Studies and Career: Frequent sickness and hospitalisation affect academics as a student reported: ii. iii. iv. th nd rd I had entered in 10 std and that time I was very much sincere means got 2, 3 rank in class. Then was felt that I have to remain absent from school because of this hospital something different had started happening in my life... I was getting tensed that something is affecting my studies, means... how long it [/HIV/] will go on this for lifelong means I was thinking in a different way. Marriage: This is an important social institution and both parents and adolescents, all think about its possibilities and perhaps would need guidance. A girl lamented: I am crazy about my marriage from my childhood, means marriage was my dream... means all girls have this dream, like that I have same dream about my marriage. I felt that now my marriage will not happen, means I was not aware that as I am [HIV infected] Another important social responsibility and need of procreation emerged and it seemed to matter to many adolescents and parents likewise: Because of father you got this [/HIV/] and I also got this. Now will my children also get this? [asked an infected son to mother] Gender relationship: A period of new relationship especially with opposite sex gets marred and affects mental health: I was not having boyfriend but many boys used to propose me. It is not allowed in the house so I never paid attention on that... and I also have this illness so why to spoil the other boy's life. Health care provision /health system infrastructure issues that may have impact on mental health program related needs ANNUAL REPORT n 24

27 Adolescence is a transition phase between childhood and adulthood and hence they neither fit in paediatric settings nor in adult settings. But in our health care settings there is no provision for dealing with this adolescence phase. This is a lacuna in our health system as emphasized by health care providers, Once they are 18 they go to the adult [OPD] then it become very difficult suddenly they are in an adult OPD on their own. In paediatric OPD they are all cocooned, nurtured and suddenly in the adult OPD on their own they are waiting in the queue, people don't have time for them so that transition becomes little difficult for them [HCP-PD-20] Adolescence is the transition phase of life which becomes complex in the presence of lifelong illness resulting in the psychological distress. There is a need of adolescent friendly health care settings where they can get psychological support for coping up the distress. The concerns related to marriage and having offspring need to be discussed during counseling of parents as well as adolescents. Strategies should be designed to encourage the adolescents for fulfilling their life's goals and to improve the quality of life. A survey, which is the second phase of the study is being planned among HIV infected and uninfected adolescents to assess their mental health and related needs. B.6) Use of herbal drugs and dietary supplements by HIV infected people and its effect on antiretroviral therapy. Investigator: Dr. Abhijit Kadam, Dr. Manisha Ghate, Dr. Vishnu Joglekar (Ayurvedic consultant) and Dr. R.R. Gangakhedkar Sponsor: Intramural Study Period: Use of herbal drugs is increasing in chronic disorders such as diabetes, hypertension and HIV is not an exception. There is limited data regarding the use of herbal drugs and dietary supplements by People Living with HIV/AIDS (PLHA) in India. Herbal drugs and dietary supplements are easily available, less expensive and perceived as safe by the people. Primary objectives: 1. To assess the frequency of use of herbal drugs and dietary supplements by HIV infected individuals who are not on ART 2. To understand the reasons behind use of herbal drugs and dietary supplements who are not on ART This is a cross-sectional study carried out at NARI clinic. A semi-structured questionnaire was used for evaluating pattern of use of herbal drugs and dietary supplements. A total of 389 HIV infected individuals who were not on ART were screened. Thirty one (8%) patients have reported to be using herbal drugs and/or dietary supplements in the last six months. ANNUAL REPORT n 25

28 Figure 2.2: Frequency of use of one or more herbal drugs &/or dietary supplements (Nutraceuticals) No. of patients one herbal drug more than one Herbal drug 2 2 Herbal drug and Dietary supplement Dietary supplement Table 2.2: Most commonly used herbal drugs and reasons given by the patient Name of herbal drugs Reason given by patient for use Number of patients Noni juice (Morinda citrofilia) To increase immunity 4 Panch Tulsi Ras/tulsi ark To increase immunity 2 Aloe vera juice To increase immunity, appetite 3 Chyawan prash To increase appetite, for good health 5 Greenish Brown powder with For HIV and related symptoms, to increase unknown ingredients immunity 10 Liquid with unknown ingredients To increase immunity 5 Shilajeet powder To increase CD4 count 3 ANNUAL REPORT n 26

29 Ten patients have reported consuming a greenish brown colored powder packaged in a small transparent polythene bag. These powders were compounded and dispensed unlabelled by ayurvedic practitioners for HIV and HIV related symptoms. Patients were unaware of the contents of the powder and were satisfied with the use of these powders. Similarly, five patients reported consumption of herbal liquid with unknown ingredients. Only 7 out of 31 patients had informed to HIV care provider about the use of herbal drugs. Further, HIV infected patients who are on ART will be screened and enrolled for use of herbal drugs and dietary supplements. B. 7) Improving Cervical Cancer Prevention among HIV-Infected Women Using Novel HPV Based Biomarker Assays: An 'Intramural-to-India' study Investigators: Dr. Ramesh Paranjape, Dr. Arati Mane, Dr. Arun Risbud, Dr. Seema Sahay, Dr. Dhanwanti Inamdar, Dr. Shubhangi Kalgutkar, Dr. Ramesh Bhosale Collaborators: National Institute of Epidemiology (NIE), Chennai and the Jawaharlal Nehru Medical College (JNMC), Belgaum, TATA Memorial Hospital Mumbai, BJ Medical College, Pune Sponsor: National Cancer Institute (NCI), USA and Indian Council of Medical Research (ICMR) Study Period: While Human Papillomavirus (HPV) DNA testing is a highly sensitive screening method for cervical cancer, it cannot differentiate between the majority of benign infections and few persistent infections linked to cervical precancer. Given the high prevalence of carcinogenic HPV DNA and higher risk for cervical precancer and cancer among HIV-infected women, there is a substantial need for screening tests that are both adequately sensitive as well as specific, so as to maximize detection while reducing false-positive referrals. Objectives: The primary objective is to evaluate the clinical performance of two novel biomarker assays (i) INK4a immunocytostaining by p16 /Ki-67 and (ii) testing for HPV E6/E7 mrna for detection of histologicallyconfirmed cervical intraepithelial neoplasia grade 2/3 or more severe (>CIN2 and >CIN3) among HIV-infected women. Secondary objectives include studying the association of risk factors and biomarkers with specific HPV genotypes and the interaction of HIV and HPV in cervical disease categories and in the context of immunosuppression. A total of 1025 HIV-infected women attending HIV care and treatment clinics at these three sites; NARI, Pune (n=408), NIE, Chennai (n=403), and Belgaum (n=200) were enrolled in the study. Participants were administered informed consent and a short risk-factor questionnaire. Participants underwent routine HIV care exam, including blood collection for serologic verification of HIV status and CD4 measurements. This was followed by per- speculum examination and cervical specimen collection for biomarker evaluation (p16 INK4a /Ki-67 immunocytostaining and E6/E7 mrna testing), HPV DNA genotyping and liquid cytology. All women then underwent digital colposcopy assessment along with cervical biopsies of all visible lesions, thus maximizing ANNUAL REPORT n 27

30 opportunities for detection of CIN2/CIN3 lesions. In the reporting year, the enrollment of participants at all sites and testing for HPV was completed. The data for NARI site is presented here. Population characteristics: A total of 408 participants were enrolled at the NARI, Pune site. The median age of the participants was 36 years (IQR: 33-40). A total of 161 (39.5%) were married and co-habiting with their husband, 41 (10.1%) reported to have more than 2 lifetime sexual partners, 99 (24.2%) were illiterate, while 49 (12%) reported 3 family income of less than Rs.2500/- per month. The median CD4+ count was 468 cells/mm (IQR: ). Majority of the women were receiving antiretroviral therapy (96.9%). Clinical findings: The analysis of colposcopic results was possible in 397 cases, while conventional cytology analysis was possible 320 cases. Cervical histopathologic analysis after cervical biopsy in women presenting with visible cervical lesions was done for 152 women consenting for the procedure. Figure 2.3 depicts the cervical disease status on colposcopy, cytology and histopathology. HPV DNA detection and genotyping: HPV DNA detection and typing was done for 402 samples by the Linear Array (LA-HPV) genotyping assay (Roche Molecular Systems). 'Any HPV' genotype positivity was detected in 162/402 (40.3%) cases. The overall distribution of the HPV types is depicted in the figure 2.4. Carcinogenic 'highrisk' (HR) HPV were present in 117/402 (29.1%) cases, of which a single HR-HPV genotype were present in 93/117 (79.5%) and multiple (i.e., two or more) HR-HPV genotypes were seen in 24/117 (20.5%) cases. Non-carcinogenic 'low-risk' or 'unknown-risk' HPV were present 128/402 (31.8%) cases. Figure 2.3: Cervical disease status on colposcopy, cytology and histopathology *[Abbreviations used in the report: CIN=cervical intraepithelial neoplasia; LSIL= low-grade squamous intraepithelial lesion; HSIL=high-grade squamous intraepithelial lesion; ASC-US=atypical squamous cells of undetermined significance; ASC-H=atypical squamous cells- cannot rule out high-grade] ANNUAL REPORT n 28

31 Figure 2.4: Overall distribution of the HPV types HPV16 was the commonest genotype present in 30.9% cases, followed by HPV62 (9.8%) and HPV35 & HPV 56 (8.6% each).the HR-HPV types detected in high-grade cervical disease (i.e., CIN 2+ lesions) in decreasing order of frequency were HPV16, HPV52, HPV33, HPV39 and HPV56. Figure 2.5 shows the HR-HPV distribution in histopathologically-confirmed high grade cervical (CIN2+) lesions. The HPV positivity percentage by histopathologically-confirmed CIN disease grades and invasive cervical cancer (ICC) is presented in Table 2.3 Table 2.3 : Correlation of HPV positivity with cervical disease status Cervical disease status Any HPV HR-HPV HPV16 I. COLPOSCOPY No CIN (n=132) 28% 17% 4% CIN1(n=154) 41% 27% 14% CIN2(n=66) 44% 39% 12% CIN3(n=44) 70% 61% 32% ICC(n=1) 100% 100% 100% II. CYTOLOGY Normal (n=77) 34% 21% 9% ASCUS (n=45) 33% 29% 13% LSIL (n=176) 41% 30% 11% HSIL/ASC-H (n=22) 73% 64% 41% III. HISTOPATHOLOGY No CIN (n=1) CIN1 (n=75) 40% 35% 15% CIN2 (n=51) 49% 37% 18% CIN3 (n=11) 82% 82% 45% ICC (n=4) 100% 100% 100% ANNUAL REPORT n 29

32 The current findings indicate the presence of HR-HPV types that are not covered in the currently available HPV vaccines in severe cervical disease among HIV infected women. A trend of increasing HPV positivity (any as well as HR-HPV and HPV16) with increasing cervical disease severity on colposcopy, cytology and histopathology was observed. The current findings indicate the presence of HR-HPV types that are not covered in the currently available HPV vaccines in severe cervical disease among HIV infected women. ANNUAL REPORT n 30

33 3 Biology of HIV Infection ANNUAL REPORT n 31

34

35 C.1) Identification of HIV modulated cell signaling pathways in context with persistence of HIV after activation Investigators: Dr. Ashwini Shete, Dr. Vijay Nema, Dr. Madhuri Thakar, Dr. Sampada Dhayarkar Sponsor: DBT (RGYI) Study Period: Immune responses are thought to play an important role in elimination of latently infected CD4 cells after HIV reactivation. The reactivated cells have been shown to secrete IL-10, an immunosuppressive cytokine, which might interfere with the elimination of infected cells by inhibiting CTLs. Understanding mechanisms responsible for IL- 10 secretion by these cells would be important for devising strategies for effective blockade of IL-10. Also other cytokines secreted by the cells after treatment with latency reversing agents (LRAs) would be crucial in deciding the fate of the infected cells as well as would influence the safety profile of the agents. Hence it is also important to study pattern of cytokine response after treatment with different LRAs. The phosphorylated signals belonging to different pathways in HIV infected CD4 T cells at baseline and after activation were reported in the previous year. During the reporting year, inhibitors of the pathways were used to determine their downstream effect on IL-10 secretion after overnight stimulation of these cells with HIV-1 Env. Different inhibitors, PD98059 (50µM), Tipifarnib (10µM) and cyclosporin A (5µM), were used to inhibit ERK, T- bet and FoxP3, respectively. PBMCs were treated with the inhibitors for 2 hours and then stimulated with HIV-1 Env for overnight) and expression of IL-10 was determined by flow cytometry (Figure 3.1). Although ERK phosphorylation has been reported to be involved in IL-10 secretion by monocytic cells, ERK inhibition caused only 1.4 fold reduction in IL-10 secretion suggesting role of other pathways in IL-10 secretion by infected CD4 T cells (Figure 3.1 a). As against this, T-bet inhibition using Tipifarnib resulted in 6 fold reduction in IL-10 secretion (Figure 3.1 b) indicating involvement of T-bet dependent pathway in IL-10 secretion by infected CD4 T cells stimulated with Env. Inhibition of FoxP3 resulted in higher IL-10 secretion by infected CD4 T cells (Figure 3.1 c) ruling out its involvement in IL-10 secretion by these cells. ANNUAL REPORT n 33

36 Figure 3.1: Effect of PD98059, Tipifarnib and Cyclosporin A treatment on IL-10 expression by CD4 T cell % of CD4 cells Expressing IL-10 a % of CD4 cells Expressing IL-10 b Treated PBMCs % of CD4 cells Expressing IL-10 Treated PBMCs c Treated PBMCs The dot plot shows percentages of CD4+T cells expressing intracellular IL-10 (plotted on Y axis) after overnight stimulation of PBMCs with HIV-1 Env in presence or absence of PD98059 (a), Tipifarnib (b), Cyclosporin A (c) as shown on X axis. Cytokine pattern after treatment of PBMCs for 24 hours with different anti-latency agents: anti CD3/CD28, Bryostatin-1, HIV-1 Env and Valproic acid was determined by Bioplex assay using Th1/Th2 cytokine kit (Figure 3.2). Anti-CD3 stimulation caused secretion of all estimated cytokines, whereas Bryostatin-1 treatment caused increased TNF-α secretion & Env stimulation caused predominant secretion of IL-10 and GM-CSF. Valproic acid did not lead to secretion of any of the estimated cytokines by PBMCs. ANNUAL REPORT n 34

37 Figure 3.2 Th1/Th2 Cytokine pattern after stimulation with different anti latency agents Fold increase in cytokines calculated by dividing cytokine levels in stimulated cells by those in unstimulated controls is plotted on Y axis and cytokines assessed in the assay are plotted on X axis. Different anti-latency agents used, anti CD3 antibody (CD3), Bryostatin-1, Valproic acid (VPA) and HIV-1 Env (Env) are as shown in the legend. Effect of PD98059 (50µM), Tipifarnib (10µM) and cyclosporin A (5µM) was also determined on IL-10 secretion by whole PBMCs after HIV-1 Env stimulation (figure 3.3). In case of whole PBMCs reduction in IL-10 secretion was maximum after ERK inhibition suggesting possible predominant role of monocytes in IL-10 secretion as reported by other investigators. Figure 3.3: Effect of PD98059, Tipifarnib and Cyclosporin A treatment on IL-10 secretion by PBMCs Inhibition of IL-10 secretion by PBMCs was determined in presence of inhibitors of different pathways. Fold inhibition of IL-10 secretion (Y axis) was calculated by dividing IL-10 levels in Env stimulated PBMCs without inhibitor by those stimulated in presence of the inhibitors as mentioned on X axis. ANNUAL REPORT n 35

38 The results indicate involvement of T- bet dependent pathway in IL-10 secretion by CD4+ T cells suggesting that T- bet blocking strategy might have therapeutic implications in the treatment of HIV infection. Also, cytokine response elicited after HIV reactivation using different anti-latency agents would have implications in understanding type of immune response elicited by them helping in deciphering their role in immune mediated elimination of the cells as well as their toxicity profile. C.2) Phenotypic and functional characterization of Th17 cells during long term non-progression of HIV Investigators: Dr. Vandana Saxena, Dr. Ashwini Shete, Dr. Manisha Ghate, Dr. Sheela Godbole, Dr. Madhuri Thakar Sponsor: Intramural Study period: Recent studies have shown that two subsets of CD4+ cells i.e. Th17 and Treg cells are also involved in HIV disease progression. Th17 cells are associated with slow disease progression while Treg cells with disease progression, indicative of a probable role of Th17 cells in the control of HIV disease which is not yet precisely defined. Hence this study is initiated to study the functional role of Th17 cells and their gene expression profile in patients with slow disease progression or LTNPs. So far we have enrolled 19 LTNPs (HIV infected, asymptomatic, ART naïve and who have maintained the CD4 count of 500 or more for over of a period of 7 years), 12 HIV progressors (HIV infected, ART naive individuals with CD4 count between and viral load of > 10,000 RNA copies/ml) and 12 HIV negative healthy donors as control groups. The gene expression profile of Th17 and Treg cells was studied by real- 6 time Q-PCR. 1-2x10 peripheral blood mononuclear cells (PBMCs) from LTNPs, progressors and healthy controls (12 from each group) were used for RNA extraction and cdna synthesis, followed by real time Q-PCR assay for various genes related to Th17 and Treg cells i.e. transcription factor and specific cytokine genes namely IL-10, IL- 17, IL-22, FoxP3. We found that the expression of Treg specific cytokine i.e. IL-10 was lower in PBMCs of LTNPs as compared with HIV progressors (Figure 3.4a). Also the expression of FoxP3 which is transcription factor for Treg cells was lower in LTNPs than progressors while its expression remained unchanged in both LTNPs and progressors in comparison with healthy donors (Figure 3.4b). The expression of IL-17 and IL-22 genes which are associated with Th17 cells was similar between LTNPs and progressors (Figure 3.4c, d). ANNUAL REPORT n 36

39 Figure 3.4: Relative gene expression profile of IL-10, FoxP3, IL-17 and IL-22 6 Relative gene expression profile of IL-10 (a), FoxP3 (b), IL-17 (b) and IL-22 (d). RNA was extracted from 1-2X10 PBMCs of study participants followed by cdna synthesis and real time Q-PCR using SYBR Green. β-actin gene was used as housekeeping gene for relative gene expression analysis. Statistical analysis was done by Mann-Whitney test using Graphpad Prism5 software. 'ns' denotes non-significant, * denotes p value of <0.01and ** denotes p value of < This study is ongoing and the analysis of expression of other cytokine genes i.e. IL-21, IL-23 and transcription factor ROR-γT etc is in progress. C.3) Characterization of tetherin/bst-2 gene alterations in HIV infected long term non-progressors (LTNPs) Investigators: Dr. Dhanashree Jagtap, Dr. Madhuri Thakar Sponsors: Intramural Study Period: Tetherin/BST-2 is a recently identified host restriction factor which tethers newly formed HIV-1 virions to the surface of HIV infected cells and prevents HIV egress and further infection. Long Term Non-Progressors (LTNPs) are rare unique individuals who are infected with HIV but control the infection without antiretroviral therapy (ART). It is not known whether polymorphisms of tetherin/bst-2 gene are associated with natural control of HIV-1 infection. Till date, no variations in tetherin/bst-2 gene have been reported in Indian HIV positive individuals. Characterization of tetherin/bst-2 gene promoter region in three groups of individuals among Indian population was undertaken to understand whether there are any differences among these groups. For this, DNA was extracted 3 from blood/pbmcs of 34 HIV infected LTNPs (CD4 counts >500 cells/mm for more than 7 years and not on ART) and 77 healthy controls. Also, DNA was extracted from stored PBMCs of 17 clinical progressors of HIV infection ANNUAL REPORT n 37

40 3 (CPI; CD4 counts <200 cells/mm for 2 years and subsequently on ART). This DNA was then used for BST-2 promoter amplification and sequencing. Analysis of the data revealed that BST-2 promoter is highly polymorphic (Table 3.1). Table 3.1: Frequency distribution of BST-2 promoter variants in controls, LTNPs and CPI individuals Three novel insertions namely -17 Ins CTTTCAGC, -203 Ins A and -443 Ins CGCCCCCAGACCCAGGCCC were found in Indian population. Four novel substitutions at positions -58 G>A, -236 G>A, -260 G/A, -268 C>T and three reported substitutions at positions -245C>T, -305A>G and -380 G>A were also found. The -443 Ins was present in 12% HIV clinical progressors and 9% controls while this insertion was absent in LTNPs. The BST-2 gene promoter sequence data obtained from the above groups was submitted to NCBI (Accession numbers KP to ). Sequencing and analysis of the intron-exon regions and 3' UTR region of the BST-2 gene is in progress. C.4) Prospective Long Term Non-Progressors' Cohort Investigators: Dr. R.R. Gangakhedkar, Dr. Madhuri Thakar Sponsor: Intramural NARI has developed a cohort of long term non-progressors (LTNPs): a population of HIV-1 infected persons who have controlled their HIV infection successfully for at least 7 years, without antiretroviral treatment. This cohort is very important to understand the correlates of slow disease progression of HIV disease. For efficient identification ANNUAL REPORT n 38

41 3 of LTNPs, the patients with 3 year follow up with NARI and having stable CD4 count above 500 cells/mm are being enrolled in the cohort of 'potential LTNPs'. The enrolled participants are followed up at 6 monthly. They receive clinical care as required and samples are collected. They receive CD4 cell counts and viral load tests every 6 month. Peripheral blood mononuclear cells (PBMCs) and plasma samples are stored for the characterization of host (genetic and immune responses) and viral characterization of these individuals. Till date, 132 patients have been enrolled as potential LTNPs in this cohort, out of which 17 patients were enrolled in the reporting year. Of these 132 participants, 37 participants have been identified as True LTNPs (participants with documented HIV infection but 3 remained asymptomatic, ART naïve and have stable CD4 count >500 cells/mm for more than 7 years). These true LTNPs are being studied for various immune responses against HIV such as presence of ADCC antibodies, Th17 CD4+ T cell characterization, and characterization of NK and NKT cells. The interim findings of these studies have been reported individually in this report. C.5) Role of SAMHD1 in HIV-1 infection of Human Dendtritic Cells Investigators: Dr. Swarali Kurle Sponsor: Intramural Study Period: Although CD4+T cells are the major target cells for HIV, other immune cells such as monocytes, Dendritic cells (DCs), Macrophages also contribute in the viral transmission and establishment of initial infection. Some host proteins known as restriction factors also influence the HIV infection. Already identified host proteins like APOBEC3G, TRIM5α and tetherin are such restriction factors. However, a new restriction factor known as SAMHD1 ('Sterile Alfa Motif' domain and Histidine-Aspartate domain containing protein 1) has been recently identified. SAMHD1 has been shown to block the HIV infection of cells of myeloid origin (monocytes, macrophages as well as DCs) and quiescent CD4+ T cells. HIV-2 accessory protein Vpx has been known to degrade the SAMHD1. DCs are known professional antigen presenting cells which initiate primary immune response by activating CD4+ and CD8+ T cells. SAMHD1-mediated restriction of HIV-1 infection of DCs may hamper this intrinsic immunity in vivo. Hence, we hypothesize that silencing SAMHD1 may lead to HIV-1 infection of DCs which in turn could induce HIV-specific T cell immunity. Hence the present study was proposed with the following objectives: 1. Down regulating SAMHD1 may allow HIV-1 infection of human DCs. 2. Characterizing the HIV-specific immune response induced by HIV-1 infected Monocyte derived dendtritic cells (MDDCs) in vitro. The down regulation of SAMHD1 with the help of HIV-2 accessory protein Vpx was standardized during the first year of the study using whole blood from healthy HIV uninfected individuals. For this, monocytes were isolated (RosetteSep Human monocyte enrichment cocktail) and cultured in presence of IL-4 and GM-CSF to get immature DCs (idcs). The idcs were then nucleofected with a plasmid prod9+vpx (10µg). The idcs were then (within 24 ANNUAL REPORT n 39

42 hours) stained for intracellular SAMHD1 using the FITC labeled polyclonal antibody (Cat# bs-8060r-fitc). The figure 3.5 below depict down regulation of SAMHD1 in idc from three different donors. Figure 3.5: Down regulation of SAMHD1 by Vpx The expression of SAMHD1 in idcs. The idcs were permeabilized and stained with FITC labeled Anti-SAMHD1antibody. The expression of SAMHD1 in idcs ( blue histograms) was then compared with idcs transfected with prod9+vpx (idc+vpx) ( Red histograms). The percent reduction in thesamhd1 expression is shown below each histogram. The down regulation of SAMHD1 with pro9+vpx ranged between 41.2% and 52.5%. The CD8+T cell response after stimulation with these DCs will be characterized further. C.6) Role of Natural Killer T lymphocyte (NKT) cells in HIV-1 infection Guide: Dr. Madhuri Thakar, Ph.D. Student: Dharmendra Singh The innate immune system provides the first line of defense against invading pathogens. CD1d restricted Natural Killer T (NKT) cells are unconventional T cells, operating on the border between innate and adaptive immune system and comprise about 0.2% of the cells in peripheral blood T cells in humans. NKT cells are known to work efficiently via NKG2D mediated activitation of NK cells and the action of NKT is modulated by myeloid dendritic cells (mdc). Based on the available information we hypothesize that NKT cells are functionally competent in HIV infected individuals with slow/no disease progression. Hence this study is proposed to characterize NKT cells in HIV infected individuals with slow/ no disease progression (LTNPs) and the HIV infected inviduals before and after ART initiation. Activated NKT cells can also interact directly with other cells of the immune system and can induce the maturation of DC into antigen presenting cells (APC) and B cells into antibody secreting plasma cells. These mechanisms require coordination between NKT cells and mdcs. ANNUAL REPORT n 40

43 The objectives of the study are: 1. Identification and phenotypic characterization of NKT cells in HIV infected individuals with slow or no disease progression (LTNPs), patients on ART (before and after ART initiation), as well as HIV uninfected controls. 2. To assess the functionality of NKT cells in terms of cytokine production, proliferation and production of cytolytic granules. 3. To assess NKT-mDC interaction during HIV-1 infection. 4. To determine the functional significance of expression of NKG2D and other NKRs ( NK cell receptors) on NKT cells in HIV infection Characterization of NKT cells: This study was initiated in the reporting year. Samples were collected from participant in following groups: Group 1: Natural Progressors (N=50). Asymptomatic HIV infected patients having CD4 count between 350 and cells/mm and ART naïve. Group 2: Long Term Non Progressors (LTNPs) (N=35): The HIV infected individuals with follow up for at least 3 seven years, should have CD4 count above 500 cells/mm, and should be asymptomatic and ART naïve for the follow up period Group 3: HIV negative healthy individuals (N=40) 3 Group 4: (For longitudinal study) Patients on ART: (N=50): Patients with CD4 count<350 cells/mm, eligible to initiate on ART. The patients were enrolled before ART initiation and followed up for one year at every six months The enrollment for cross sectional study has been completed and the follow up of the participants enrolled in Group 4 is ongoing. Table 3.2 provides demographic details of the enrolled patients. Table 3.2: Demographic details of the study participants Population N Median Gender and Median Median Log viral load: age in years No of CD4 (Range) CD8 (Range) Median (Range) participants (Range) Progressors Male: ( (30-48) Female:28 ( ) ( ) LTNPs Male: ( ( ) (29-74) Female: 23 ( ) 1450) HCs Male: NA (18-53) Female: 15 ( ) ( ) Patients: just Male: ( ) prior to ART (22-49) Female: 25 (48-348) ( ) initiation ANNUAL REPORT n 41

44 The protocol for quantification and phenotypic characterization of NKT cells was standardized using specific markers of NKT cells such as CD3, CD4, V alpha 24, V beta 11, IFN γ, and CD107a expression. The LIVE/DEAD marker was also included in the analysis. Since we planned to use frozen PBMCs for NKT analysis, we first assessed whether there was any difference in results between experiments on fresh and frozen cells. Thus, samples from 10 volunteers were collected, processed for paired fresh and frozen samples. The results are given in figure 3.6. The experiments showed that the percentages obtained from fresh and frozen PBMCs were similar and protocol can be used successfully on revived frozen cells. Figure 3.6: Quantification of NKT cells in Fresh and Frozen PBMCs in the same sample S. N o Fresh PBMC Frozen PBMC % 0.24% 0.41% 0.22% Flowcytometry analysis of lymphocyte derived from PBMCs, in the gating strategy for identification and quantification of NKT cells. (A).identification of NKT cells frequencies on Fresh PBMCs. (B) identification of total NKT cells frequencies on frozen PBMCs. After the standardization of the protocols, the frozen PBMCs from 10 LTNPs, 10 progressors, and 10 healthy controls were processed for NKT cell characterization using NKT specific markers. Activation and exhaustion status of the NKT cells was assessed on the basis of CD38 and CD69 and PD1 expression respectively. Preliminary data is shown in figure 3.7. The data showed that the NKT cells are depleted in HIV infection and also higher percentage of NKT cells expressed CD38 and CD69 in progressors as compared to LTNP and healthy controls. ANNUAL REPORT n 42

45 Figure 3.7: Expression of CD38, CD69 and PD1 in NKT cells (a) Representative flow cytometric analysis showing the total percent of NKT cells in different groups, quantification of CD4+NKT and CD4- NKT cells (b). (C) percent of exhaustion markers (PD1) on NKT cells and (d) percent of activation markers CD38 (left) panel and CD69 (right panel) on NKT cells in different groups. (Group1: Progressor. Group2: LTNPs and Group 3: Healthy controls) C.7) Antibody-Dependent Cellular Cytotoxicity (ADCC): A New Frontier in Vaccine Research Investigator: Dr. Madhuri Thakar, Dr. Ashwini Shete, Dr. Swarali Kurle, Dr. Manisha Ghate and Dr. Ramesh Paranjape Sponsor: DBT (Indo-Australian Biotechnology Fund) Collaborator: Prof. Stephen Kent, Department of Microbiology, University of Melbourne, Australia. Study Period: Antibody-Dependent Cellular Cytotoxicity (ADCC) mediating antibodies are known to kill the infected cells by utilizing innate immune cells such as Natural Killer (NK) cells. The ADCC antibodies are found to be associated with low viral replication in people with HIV-infection. The immune correlate analyses of RV 144 HIV vaccine trial demonstrated an important role for non-neutralizing ADCC antibodies contributing to the vaccine efficacy. However the information on the epitopes recognized by anti-hiv ADCC antibodies is not available. Also the role of ANNUAL REPORT n 43

46 ADCC antibodies in patients on long term ART and in a potential cure of HIV is not known. It is now known that the HIV remains latent in the CD4+ memory cells in spite of successful ART. It is likely that immune responses, possibly ADCC antibodies, will be needed to mop up the HIV that is reactivated and thus ADCC antibodies might contribute to the functional cure in HIV. We further hypothesize that isolation of potent HIV-specific ADCC antibodies directed against multiple epitopes will have a potential of use as immune therapeutics in HIV infection. Techniques to obtain human HIV-specific monoclonal antibodies are available, but have not been explored for ADCC antibodies. The potential use of these monoclonal antibodies at mucosal level will be an important step towards the development of mucosal immunotherapeutic. To study and validate the hypotheses, samples from large cohorts of HIV positive individuals who control HIV infection without ART (LTNPs) and with ART in diverse geographical settings with different genetic constitution and also infected by different HIV-1 clades are required. The HIV-1 subtype C is the most prevalent subtype found in India and Australia. Hence, this collaborative study was proposed with the following objectives l l l l l To evaluate and map ADCC responses in HIV positive individuals with controlled virus multiplication known as Long term non-progressors (LTNPs) from Australia and India To assess and map ADCC antibody responses in Australian and Indian cohorts of HIV-positive individuals receiving Antiretroviral Treatment (ART). To assess the functional ability of ADCC antibodies in serum from HIV positive individuals to clear latent HIV in vitro To generate monoclonal HIV-specific ADCC antibodies directed against multiple epitopes from both Indian and Australian HIV positive individuals and test these antibodies in cervical explants cultures with a view to future use as immunotherapeutic at systemic and mucosal level. To assess HIV positive individuals in Australia and India for ADCC antibodies against Influenza virus During the first year of the study, the protocol for assessing the ADCC antibody response was standardized. The flow cytometry based assay was used to assess the HIV antibody mediated activation of NK cells in presence of HIV antigens which lead to secretion of Interferon (IFN)-γ and CD107a by the NK cells. The ADCC response was measured as percent NK cells secreting both IFN-γ and CD107a in response to HIV antigens. Thirty five LTNPs and 8 healthy controls were enrolled in the study. The ADCC response was assessed in 27 LTNPs and 8 HIV uninfected healthy individuals against various HIV-1 antigens such as Proteins (Env B and Env C), and pools of overlapping peptides of Env B, Env C, HIV-1 C Gag, HIV-1 B Pol and peptide pool of Tat, Nef, Rev and Vif antigens. Peptides from SIV Env were included as non-specific control. The negative and positive controls included were only DMSO (used for reconstitution of the peptides) and Anti-CD16 antibody respectively. The baseline ADCC response was determined using samples from healthy, HIV uninfected donors. The ADCC response in LTNPs was considered positive when the % of NK cells secreting IFN-γ and expressing CD107 was more than the mean of the response from healthy donors +2SD. The ADCC responses assessed in 27/35 LTNPs so far are presented in figure 3.8. ANNUAL REPORT n 44

47 Figure 3.8: ADCC response against HIV antigens in LTNPs and health controls Vertical scatter plots showing ADCC response against HIV peptide pools /proteins in healthy controls measured in terms of % NK cells which are IFN-γ+CD107a+. To determine the baseline of ADCC responses, ADCC response against HIV peptide pools /proteins in healthy HIV- controls (n=10) was determined. The orange colored data points from LTNP group in all graphs represent the value of ADCC response in terms of NK cell percentages equal to or more than mean +2SD of baseline ADCC responses. The results of this preliminary analysis showed presence of ADCC antibodies in plasma samples from LTNPs and the response was predominant against Env peptides and protein (Both HIV-1 C and B Env). The response against the Env C peptides was seen in 6/27, Env C protein in 8/27, Env B peptides 12/27 and Env B protein 11/27 LTNPs. The response against Gag C, Pol B and Ta-Rev-Nef-Vif peptides were seen in 5, 3 and 3 of the 27 LTNPs respectively. It was observed that the magnitude of the response was also higher in case of Env as compared to other antigens. The laboratory is currently engaged in standardizing the in vitro experiments to assess the functional ability of ADCC antibodies in serum from HIV positive individuals to clear latent HIV. The mapping epitopes recognized by ADCC antibodies is underway. C.8) Studies on the role of Tripartite Motif (TRIM) Proteins in HIV infection Investigators: Dr. R. S. Paranjape; PhD student: Ipsita Choudhary Sponsor: Intramural Study period: Tripartite motif (TRIM) proteins constitute a host protein family characterized by a RING finger domain, one or two B-box domains, a coiled-coil domain and a variable C-terminus. Recent studies have shown that TRIM family proteins have anti-retroviral properties upon interferon stimulation. However, the contribution of this family of ANNUAL REPORT n 45

48 proteins to protection against HIV infection or to the control of disease progression in HIV infected patients is largely unknown. Hence, this study has been undertaken to investigate the role of TRIM proteins in HIV-1 infected individuals. The objectives of the study are: l To identify Type 1 (IFN α and ß) and Type 2 (IFN-?) Interferon induced members of TRIM family with potential antiviral activity against HIV-1 in human cell lines. l l To study if type I IFN (α and ß) serves as a synergist with type II IFN (IFN γ) in the induction of TRIM genes for its activity against HIV virus. To study if the expression of identified TRIM proteins up-regulated on IFN induction has any role in HIV-1 infected individuals with controlled disease progression and understand if polymorphism in the upregulated TRIM genes has any effect on HIV-1 susceptibility. In the reporting year, standardization time dose experiments for Interferon stimulation of cell lines were carried out. A pilot study was also done to understand which TRIM genes are up regulated in HIV infected progressors (n=14) and long term non progressors (LTNPs) who have broken down after many years of successful virus control (n=06). A total of 14 TRIM genes which have been reported to have anti-hiv activity in human cell lines were screened. It was observed that out of 14 genes, there was significant up regulation of TRIM5 and TRIM22 genes in both the groups. Further, the association of the variants of TRIM5 α genes with HIV disease progression in Indian patients was studied. DNA extracted from stored PBMCs of LTNPs, (n=32), healthy controls (n=50) and from progressors (n=16) and the exons of the TRIM5a gene were amplified under the same PCR condition. Figure 3.9: Standardization of PCR for Exons of TRIM5 α gene 1% agarose gel showing different amplicons of Exon 2, 3, 4, 5, 6, 7 & 8 for TRIM5α gene ANNUAL REPORT n 46

49 The amplified products of the exons for all the three study groups were purified by ExoSAP-IT for PCR product clean up kit followed by DNA sequencing in ABI PRISM 3100 Genetic Analyzer. Exon2 and 3 spanning the RING, B-Box and Coiled coil domains and Exon 5 spanning the linker region were sequenced and the data has been presented in Table 3.3 below. Table 3.3: Frequency distribution of TRIM5α variants in the study groups Genotypes LTNP Progressors (n=16) Healthy controls (n=32) (% Variants) (n=50) 127C>T (rs , H43Y) Allele CC 26 (72.2%) 16 (100%) 38 (76%) CT 04 (12.5%) 0 12 (24%) TT 02 (6.2%) G>A (rs , R136Q) Allele GG 28 (87.5%) 13 (81.2%) 39 (78%) GA 03 (9.3%) 03 (18.7%) 11 (22%) AA 01 (3.1%) G>A (rs , G249D) Allele GG 20 (62.5%) 14 (87.5%) 40 (80%) GA 11 (34.3%) 02 (12.5%) 10 (20%) AA 01 (3.1%) 0 0 (rs , V112F) Allele GG 21 (65.6%) 08 (50%) 36 (72%) GT 09 (28.1%) 07 (43.7%) 14 (28%) TT 02(6.2%) 01 (6.2%) 0 Based on the available data, there was no association of any variant with the disease progression. The study is ongoing to understand the effect of IFN- α secretion on the expression of TRIM genes. C.9) Plasmacytoid Dendritic cells in HIV-1 Infection Guide: Dr. R. S. Paranjape Ph.D. Student: Ashwini Dhamanage Plasmacytoid dendritic cells (pdcs) play a crucial role in innate immune response against viruses. They exert their action mainly through secretion of type I Interferons in large quantity and through various cytokines and chemokines which attract and activate other immune cells. But it has been observed that in early stages of HIV-1 infection, pdcs are impaired in number and functionality from peripheral blood with functional impairment in IFNα secretion. Since the immune control of HIV multiplication in early HIV infection may influence the disease progression, understanding the mechanism of decline in numbers and functions of pdcs is necessary. The reason for impaired IFN-α production in pdcs could be related to its signaling pathway after recognition of HIV ssrna ANNUAL REPORT n 47

50 through TLR-7, which is followed by translocation of IRF-7 protein into the nucleus to initiate IFN- α production. We are exploring whether HIV-1 infection blocks the step of IRF-7 translocation into the nucleus, affecting functionality of pdcs. Decline in numbers of pdcs from peripheral blood in recent HIV-1 infection has been linked to apoptosis of pdcs in many studies. Hence we hypothesize that impairment of PI3K/AKT pathway; the survival factor for pdcs, causes death of pdcs from peripheral blood in presence of HIV-1. Standardizations: 1. To determine minimum time required for detection of intracellular IFN- α after stimulation of pdcs for determination of time of intranuclear localization of IRF-7- IRF-7 translocation from cytoplasm to nucleus initializes IFN-α production. Hence knowing minimum time required for intracellular IFN-α production can help in deciding intranuclear detection time of IRF-7 after stimulation. This time of IRF-7 detection can then be used to find out whether HIV blocks translocation of IRF-7 into nucleus during HIV-1 stimulation. To standardize this, PBMCs were stimulated with Imiquimod (TLR-7 agonist-positive control for IFN-α synthesis) for 2, 4 and 6 hr and intracellular IFN- α was detected by flow cytometry. It has been observed that pdcs produced detectable IFN- α as early as in 2 hrs. Hence translocation of IRF-7 in to the nucleus is expected to be seen at 2 hrs after stimulation. 2. Standardization of Multiplicity of Infection (M.O.I.) of HIV-1 to be used in all objectives: a. To see the intracellular IFN-α in plasmacytoid dendritic cells after infection with different M.O.I. of HIV-1 Purified pdcs from normal healthy individuals were stimulated with 0.1, 0.5 and 1 MOI of HIV-1 and intracellular IFN-α was detected by flow cytometry at 8 hours after infection. While Imiquimod stimulated pdcs produced IFN-α, the HIV stimulated pdcs (all three M.O.I.) inhibited IFN-α production. Since MOI contained viral load (2.8*10 viral copies) similar to in vivo conditions, it was decided to use 0.5 MOI as infection inoculums for all experiments. b. To see the effect of 0.5 M.O.I. HIV-1 on IFN-α secretion beyond eight hours: Purified pdcs from normal healthy individuals were stimulated with Imiquimod for 8 hrs and with 0.5 M.O.I. of HIV-1 for 24 and 48 hours. By 48 hours, pdcs resumed IFN-α secretion, however, not to baseline levels. Thus indicating that HIV-1 causes delayed as well as impaired IFN- α production as compared to Imiquimod. c. To see the effect on IFN-α production by Imiquimod in presence of 0.5 M.O.I of HIV. PBMCs from normal healthy individuals were stimulated with either 1) Imiquimod (20ug/ml) or 2) 8 8 simultaneously with Imiquimod (20ug/ml) and 2.8*10 HIV-1 viral copies or 3) 2 hrs with 2.8*10 HIV-1 viral copies and then 8 hrs with Imiquimod (20ug/ml). The supernatants were collected after specified time and analyzed for amount of IFN-α by ELISA. ANNUAL REPORT n 48

51 Unstimulated Stimulation Stimulation Stimulation with Stimulation with 8 8hrs* with with Imiquimod+ 2.8*10 HIV-1 viral Imiquimod 8 2.8* *10 HIV-1 copies for 2 hours -8hrs* HIV-1 viral viral copies - and then with copies-8hrs* 8hrs* incubation with Imiquimod * IFN-α Nil 1019 Nil (pg/ml) * Average of four independent experiments with four different donors These results show that when cells were incubated with HIV-1 along with Imiquimod, there was no inhibition in IFN-α production, but when cells were incubated for two hours with HIV-1 and then stimulated with Imiquimod, 8 IFN-α production is inhibited (191 compared to 1019 pg/ml). This confirms that 0.5 M.O.I. (2.8*10 viral copies) inhibit IFN-α production even stimulated by Imiquimod. 1. Staining procedures for pakt identification in pdcs after stimulation with positive control The functionality of PI3K/AKT pathway depends on AKT phosphorylation, hence detection of AKT phosphorylation by flowcytometry is a prerequisite to assess this pathway in HIV-1 treated pdcs. Detection of phospho-proteins by flow cytometry is affected by methods of permeabilizaion used during staining by antiphospho-protein antibodies. Hence to find out correct permeabilizaion procedure for detection of phospho-akt is very important. To standardize this, PBMCs from normal healthy individuals were stimulated with Imiquimod (positive control) for 90 min and then permeabilized with Fix/Perm buffer or with 50% Perm buffer III or with 100% Perm buffer III. Then cells were stained with surface staining antibodies and intracellular pakt antibody. The cells were acquired on FACS Aria. It was seen that Perm III was harsh, while 50% Perm III and cytofix/cytoperm buffers were gentle on surface markers of cells. But 50% Perm III treatment was not able to detect pakt from stimulated samples as compared to cytofix/cytoperm buffer. Hence it was decided to use cytofix/cytoperm buffer as permeabilizing agent during pakt staining. The study objectives are 1. To study whether the HIV-1 inhibits the translocation of IRF-7 from cytoplasm to nucleus causing suppression of interferon α synthesis by pdcs. 2. To study whether HIV-1 inhibits the PI3K/akt pathway in pdcs causing suppression of interferon α synthesis. 3. To find out whether inactive PI3K/akt pathway is the cause of pdcs apoptosis after HIV-1 infection. 4. To find out whether an availability of IFN α is the cause of inactive PI3K pathway and ultimately the reason for pdc apoptosis. Table 3.4: Results of the standardization experiments ANNUAL REPORT n 49

52 C.10) Characteristics of cellular effector mechanisms against HIV at cervicovaginal level in Indian women. Investigator: Dr. Madhuri Thakar, Dr. Sheela Godbole, Dr. Seema Sahay, Dr. Manisha Ghate, Dr. R.R. Gangakhedkar, Dr. Ashwini Shete, Dr. R.S. Paranjape. Sponsor: DBT-ICMR Study Period: The major mode of HIV transmission is through heterosexual contact. However the genital mucosal immune response has not been very well characterized. We hypothesize that the innate immune factors such as cytokines and antimicrobial peptides secreted by a variety of the mucosal cells in the female genital tract (FGT) influence the activity of innate NK cells, pdcs and HIV specific CD4 and CD8+ T cells; resulting in modulation of HIV-1 shedding. The increased viral shedding could increase the chances of secondary transmission. Hence, this study is planned to assess the impact of the innate immune secretory factors on the effector immune response (NK cells, pdcs, CD4 and CD8+ T cells) in the genital mucosa and its influence on the viral shedding in Indian women infected with HIV. In this study we planned to enroll women participants in the same phase of menstrual cycle (proliferative phase of menstrual cycle at the time of sample collection) and who do not have symptomatic sexually transmitted infection (STI). Fifty participants will be enrolled in each of the following groups: a) Group I (N=50): HIV positive women, ART naïve b) Group II (N=50): HIV negative women with high risk behavior for HIV acquisition c) Group III (N=50): HIV negative women with low risk behavior This study is ongoing. Thus far, we have enrolled and analyzed the samples from a total of 37 women participants including Group I: HIV positive (n=29), Group II: HIV negative with high risk behavior (n=1) and Group III: HIV negative with low risk behavior (n=7). The demographic details of all the study participants are mentioned in Table 3.5. Group I: HIV +ve women Table 3.5: Demographic details of the enrolled women participants in the study Age in years 3 Mean (range) (cells/mm ) Median (range) Median (range) Median (range) (N=29) (24-48) 450 ( ) 4.42 ( ) 2.94 ( ) II: HIV -ve women with high risk behavior (N=1) 40 NA NA NA III: HIV -ve women with low risk CD4 cell count Plasma Viral Load CVL Viral Load (Log RNA copies) behavior (N=7) (35-44) NA NA NA (Log RNA copies) ANNUAL REPORT n 50

53 From all the enrolled women, cytobrush, cervicovaginal lavage (CVL) and blood samples were collected. From the 0 blood, plasma and PBMCs were separated on Ficoll hypaque density gradient and stored at -80 C and liquid nitrogen respectively for further investigations. CVL samples were spun down and supernatants were stored at C and further used to assess the levels of secretory innate immune factors such as cytokines (IL-2, IFN-, TNF-, IL-6, IL-10, IL-8 & TGF- β1) and chemokines (GM-CSF, MCP-1, MCP-1, MIP-1β, RANTES and MIP-). From the cytobrush samples, mucosal cells were separated and stained for various immune cell markers and analyzed on FACSAria (Figure 3.10). Based on Forward (FSC) and side scattered graphs, mucosal mononuclear cells (MMC) +ve +ve +ve were identified. These cells were further identified CD16/56 NK cells and CD3 T lymphocytes. CD3 T cells were further identified as CD8+ T cells and CD4+ T cells using specific antibodies. +ve +ve Figure 3.10: Identification of mucosal mononuclear cells (MMC), NK cells, CD3 T cells, CD4 T cells +ve and CD8 T cells from the cytobrush samples -ve +ve To identify dendritic cells (DCs), MMCs were stained with Lin-1 and HLA-DR antibodies and Lin-1 HLA-DR -ve +ve cells were identified as DCs. DCs were further identified as CD11c CD123 cells plasmacytoid DCs (pdcs) and +ve -ve CD11 CD123 cells myloid DCs (mdcs) as shown in figure ve +ve Figure 3.11: Identification of mucosal mononuclear cells (MMC), NK cells, CD3 T cells, CD4 T cells +ve and CD8 T cells from the cytobrush samples ANNUAL REPORT n 51

54 Here, we present our early interim finding. However due to small sample size available so far, the results are not yet conclusive. So far only one woman has been enrolled in Group II i.e. HIV non-infected with high risk behavior), we have excluded this group in our further analysis and present the interim data on analysis of Group I and III only. We found in HIV +ve women i.e. Group I, the mean frequency of plasmacytoid DC (major producer of type-i IFNs) was lower in comparison with HIV-ve women with low risk behavior (Figure 3.12a). In CD3+T cell analysis, we found that the mean frequency of CD8+ T cells was higher in the HIV +ve women than HIV-ve women (Figure 3.12b). But the mean level of degranulation marker expressed on CD8 T cells remained unchanged between the two groups (Figure 3.12c). Figure 3.12: Frequency of pdc, CD8 T and CD107a+ve CD8 T cells in the cytobrush samples between Group I and Group III Characterization of DCs (a) and CD8+ T (b) cells in the mucosal mononuclear cells of HIV infected and HIV non-infected women. CD107a was used as a marker to assess the cytotoxic potential or degranulation of CD8+ve T cells (c) TGF-β levels were determined by ELISA in the CVL supernatants and it was found that the level of TGF- β was higher in the CVLs of HIV +ve women (Group I) than the HIV ve women with low risk behavior (Group III) as shown in figure 3.13a) confirming our previous finding. Levels of secreted cytokines and chemokines were also determined by bioplex assay but we did not find any change for other cytokines (data not shown) except the mean levels of IL-17 which was higher in HIV +ve women (Group I) in comparison to the HIV-ve women with low risk behavior (Figure 3.13b). ANNUAL REPORT n 52

55 Figure 3.13: Estimation of TGF-β and IL-17 levels in the CVL samples of Group I & Group III women Levels of TGF-β (a) and IL-17 (b) in the cervicovaginal lavages samples of HIV infected and HIV non-infected women as determined by ELISA and Bioplex respectively. The data presented here are based on the interim findings of the study and on a smaller sample. This study is ongoing and we are enrolling more study participants in all the three study groups, performing the assays and analyzing the data. Since Th17 cells and IL-17 have been associated with the mucosal immunity, we are further planning to characterize the Th17 cells in the mucosal samples. C.11) Molecular probing of the Mycobacterium tuberculosis isolates with respect to their resistance pattern and conservation pattern of new drug targets Investigator: Dr. Vijay Nema, Dr. Arun R. Risbud, Dr. Srikanth Tripathy, Mr. Mycal Pereira Sponsor: Extramural funded by ICMR Study Period: Drug resistance remain a challenge in tuberculosis treatment and hence exploring the new targets and ascertaining that they remain conserved in all types of clinical isolates (susceptible as well as resistant to the existing drug regimen) is imperative. This study was done in order to assess mutations in the genes which are the targets of currently used antitubercular drugs and the genes which are proposed to be the targets of future drugs. Genes like icl, pknb, pcaa and devs were probed being the targets of newly proposed drugs and genes like rpob,katg,inha,embb,pnca,rpsl,rrs,gyra were studied as the targets of currently used drugs. Isolates were obtained from three geographical areas in the country viz. Pune (two sites), Chennai and Varanasi. ANNUAL REPORT n 53

56 Table 3.6: Isolates obtained under the study for analysis Sr. No. Source No. of isolates MDR/ Mono-resistant/ poly-resistant but not MDR 1. NARI, Pune 29 (N1-N29) 3/6/5 2. STDC, Pune 20 (S1-S20) 4/4/2 3. NIRT, Chennai 41 (C1-C41) 17/1/8 4. BHU, Baranasi 26 (B1-B26) 14/1/3 These were spoligotyped to link them with their origin and clade type. For Pune we could get isolates from (29) and from (20), however these numbers are not enough for comparison. In brief, CAS1-Delhi spoligotype dominated in both sample types that corroborates with the data available in SITVIT WEB about the distribution of spoligotypes in India. Of 49 isolates obtained from Pune, 11 isolates were orphans (22%). This percentage is high and reflects both the current absence of knowledge on the genetic diversity of Indian M. tuberculosis strains and the microevolutionary genetic driving forces active in TB-epidemic dynamics. Two isolates with beijing-1 spoligotypes were obtained from recent isolates but none from the older ones. The isolates obtained from NIRT Chennai were collected from the nearby areas during A total of 41 isolates were studied. Spoligotype profile of these isolates was very different from what was obtained in isolates collected from Pune region. Orphan spoligotype was observed to be the common most spoligotype (14/41) with EAI3_IND as the second in prevalance among susceptible isolates. However 4 isolates with Beijing-1 spoligotype were obtained and 75% of them (3/4) were MDRs. CAS1-Delhi spoligotype was least dominant as opposed by the isolates from Pune region. The isolates obtained recently from BHU were all collected during An indicator of increasing virulence in these isolates is the prevalence of high number of isolates with Beijing-1 spoligotype (8/26) wherein all except one isolate are drug resistant. Surprisingly an isolate showed H1/SIT47 spoligotype which has not been included in the list of spoligotypes frequently found in India. Rather it has been reported to be one of the prevalent spoligotype patterns in Turkey. The results of sequencing were compared and linked with drug resistance using TBDReam database for the drugs being used in current treatment. For new targets a bioinformatics approach was used to find those areas which would be responsible for ligand/drug binding. These hypothetical hot spots were mutated and a docking study with known inhibitors was done. Sequencing of existing drug targets and their analysis provided following inferences: rpob: Apart from the reported high confidence mutations directly co-related with resistance, a new mutation was detected at 440(GTG-GCG) in one isolate. However, isolate remains phenotypically susceptible. Mechanistic studies may be done in future. rpsl and rrs Mutations associated with streptomycin resistance in M. tuberculosis have been identified in the rpsl and rrs ANNUAL REPORT n 54

57 genes, which encode the ribosomal protein S12 and 16S rrna, respectively. In this study, we could see a few mutations indicating streptomycin resistance which was confirmed by phenotypic resistance data. Apart from the reported high confidence mutations directly co-related with resistance, a new deletion was also detected. Isolate remains phenotypically susceptible. Interestingly, two isolates on BLAST search they turned out to be non-tubercular mycobacterium (NTM) species. As compared to rpsl, rrs remain conserved in almost all isolates, except in three isolates. katg and inha: A mutation in katg at position 944 is reported to be a high confidence mutation directly co-related with resistance. However, in one isolate reported to be susceptible by the provider we found the same mutation. This can be confirmed in future studies and if found to be true case, mechanism of susceptibility despite this mutation may be explored. A noteworthy finding here is that a mutation at 280 nucleotide position (TCG/GCG) in inha was found to induce resistance to isoniazid in absence of any mutation in katg gene. This has been reported earlier and informs that study of mutations in katg only may not be a useful tool for molecular detection of resistance against isoniazid. embb: embb remain the major contributor in the function of arabinosyl transferases. Mutations found in this gene are 916(ATG-GTG), 918(ATG-ATA) (Reported); in addition to a new mutation. pnca: We consider that the analysis of the pnca gene provides rapid and useful information regarding PZA susceptibility in M. tuberculosis and may therefore contribute to early optimization of treatment. Furthermore, pnca sequencing may be a reliable alternative to phenotypic PZA DST techniques, which sometimes give irreproducible results and discordance among findings from different laboratories. We found many reported and unreported mutations in this gene. However lack of phenotypic data did not allow us to correlate their phenotypic significance. gyra: Resistance to fluoroquinolones is estimated phenotypically with the use of Ofloxacin as a representative drug. Apart from various reported mutations in ofloxacin resistant isolates, polymorphism at nucleotide positions 61 and 284 as compared to Rv0006 was found in all isolates except a very few (7/106). There are reports available about this polymorphism and their co-relation with no phenotypic resistance is well established. In our isolates some of them have showed resistance in phenotypic assays and have mutation at 61 and 284 positions. This indicates towards an external cause of resistance that may be efflux pump or anything else related and needs to be explored further. However this polymorphism has not been reported frequently in earlier studies and this could indicate a silent micro-evolution in these isolates with no predictive direction at this time. We tried to explore further why mutations at position 61(Glu/Gln) and 284(ser/thr) translating into different amino acids does not have change resulting in drug resistance. The bioinformatics analysis as below has revealed that change in amino acid does not impact the overall structure (Figure 3.14 a and b) as well as the drug binding (Figure 3.15 and Figure 3.16). ANNUAL REPORT n 55

58 Figure 3.14 (a): Wild Ligand binding domain Figure 3.14 (b): Mutated binding domain (Ser 95Th) Hydrogen bonding pattern in Ser95toThr95 mutated (3IFZ) grya protein where Thr95 binds to three amino acids Ser91, Ile92 and Met99. After mutation from Ser95toThr95, the hydrogen bonding changes. Thr95 binds with residues namely Ile92 with two H-bonds having distance 2.33 and 2.96, Ser91 with one H-bond having distance 3.16 and Met99 with two H-bonds having distance 3.06 and However this is not enough to drop the bound ligand i.e. Ofloxacin in our case as shown in docking studies below: Figure 3.15: Elaborated structure of Wild gyra docked with ofloxacin ANNUAL REPORT n 56

59 Showing different interacting molecules. Below is the energy calculations of the binding with wild type protein. Estimated free energy of binding is -5.58Kcal/mol. 3.16: Docking study of mutated Ser95toThr95 (3IFZ) grya) protein to the ligand ofloxacin (Nuc. Positon: 284). The change in energy is shown in the table below. Estimated free energy of binding is -5.34Kcal/mol and is not significantly different from wild type. Sequencing of putative drug targets and looking for their conservation provided following inferences: pknb: As no drug is currently being used to target this gene, no database has any information about the mutations in this gene target. Hence a bioinformatics analysis was done to see the amino acid changes in substrate binding site and to assess if it can bring the changes in future drug binding. Results of bioinformatics analysis: Asp-198 an important residue: Asp198 forms total of 5 H-bonds with residues namely His130, His 136, Arg137, Ala195, and Leu202. These 5 interactions provide stability and shape to the pknb protein, especially the active substrate binding site. We mutated this residue with the incorporation of alanine in place of aspartic acid as is customary for mutation analysis. Mutation of only this particular residue (Asp-198) can cause deleterious effect on the shape and stability of the protein. When Asp-198 is mutated with the Alanine amino-acid, there is direct loss of ANNUAL REPORT n 57

60 three H-bonds. We explored if any of the clinical isolate harbored this mutation and found that it was absent from all. Further the wild type and mutated structures were docked with the known inhibitor 'Mitoxantrone' and AMP-PCP (Adenylylmethylenediphosphonate disodium salt) to see if this structural change is translated into the change in binding of inhibitor also and to what extent (Figure 3.17). Figure 3.17: Comparison between wild type and mutated structure of pknb for AMP-PCP binding Binding of AMP-PCP with the wild type structure (D198A) Binding of AMP-PCP with the mutated structure As the structure is destablized, there is no interactin of AMP-PCP with mutated structure. A similar exercise was done for other amino acids which are found to be important for substrate binding i.e., G21, M22, S23, V25, K40, V95, N143, D156 and a dual mutation having V95 and N143 with AMP-PCP as substrate and was repeated with Mitoxantrone as substrate with the same amino acids. The exercise done with hypothetical mutations in pknb assured us that the prediction about the amino acids participating in ligand binding was accurate and mutation in these amino acids would have significant change in binding efficiency of the protein. Hence a model is ready for the analysis of mutations and their effect if they are found in clinical isolates in due course of time or under new drug pressure. Surprisingly no mutation could be detected after sequencing around 150 isolates containing isolates from all three geographical locations, susceptible and resistant to various first and second line drugs and with different spoligotypes. Hence the target seems to be highly conserved in clinical isolates of M.tuberculsis prevalent in all geographical locations as considered in this study. icl and other targets: As like any other new target, icl also is not known to harbor mutations which may impact drug binding. In our study we observed that no isolate had any mutation in the sequences probed. This highlighted the conserved profile of these genes even in the isolates which are otherwise resistant to many of the currently used drugs along with those which remain susceptible. ANNUAL REPORT n 58

61 Salient findings of the study: l l l l Results of this study have provided some interesting leads about the resistance pattern in the targets of currently used drugs. For instance, it can be inferred that there has been an exposure of floroquinolones to most of the isolates. Despite being on the first line and irrespective of their origin, many isolates have shown some or the other mutation in gyra gene that can be assigned to empirical treatments using floroquinolones much before the diagnosis of tuberculosis. Some of these mutations were not translated into resistance profile, but have indicated the drug pressure and microevolution taking place in response to it. Identification of two NTM (Mycobacterium abscessus and Mycobacterium sp. JDM601) during rrs gene sequence analysis informed that our current diagnostic pipeline does not differentiate between such species and further work is required. The findings of this study have generated useful information about the hot spots of the mutations in the genes which are the targets of the current drug regimen. New drug discovery pipeline targeting the genes which were thoroughly probed in this study is moving in the right direction as the genes remain conserved in all isolates irrespective of their resistance to first line drugs, their spoligotype and their place of origin as far as the regions covered in this study. C.12) The role of mirnas in the pathogenesis of HIV/AIDS and their Utility as biomarkers of disease progression and therapy failure Investigators: Dr.R.R.Gangakhedkar (NARI) and Dr. Shahid Jameel, (ICGEB), New Delhi Sponsors: Department of Biotechnology (DBT) Study Period: The role of mirnas in the pathogenesis of HIV/AIDS is poorly understood. The published literature suggests that mirna signatures can be used as biomarkers of HIV disease progression. An exploratory study at International Center of Genetic Engineering and Biotechnology (ICGEB), New Delhi has showed that mir-150 and mir-146b levels can differentiate the HIV-infected asymptomatic and ART treated persons from ART-naïve symptomatic patients and those failing therapy. The present study has been planned to validate these finding in larger sample size. The cross sectional study included samples from different groups such as healthy controls; HIV-positive asymptomatic (CD4 Count Range , Median-568), HIV-positive symptomatic, therapy naïve (CD4 Count Range , Median-238), HIV-positive on ART for at least 12 months (CD4 Count Range , Median- 385) (n=50 per group )and Patients failing first line ART (n=25) (CD4 Count Range , Median-110). In a longitudinal study, the ART treated patients will be followed up at ART initiation visit and after ART (N=10) (CD4 Count Range , Median-486). Similarly, 10 patients who have been on therapy for at least 7 years (CD4 Count Range , Median-653) will also be followed up to assess the mirnas as biomarkers of response to therapy and to assess the mirnas as biomarkers of therapy failure. The study was initiated in 2013, the enrollment has been completed and in cross sectional study In longitudinal study, 10 HIV infected therapy naïve and 10 HIV infected individuals who are on ART at least 7 years have been ANNUAL REPORT n 59

62 enrolled. Every three month follow up for two years for all patients in longitudinal study have been completed. The samples were processed for absolute quantification of mir150 and mir146b by Real Time PCR Figure 3.18: mirna 150 quantification in HIV infected patients mirna 150 Copies / ng RNA Healthy controls ART >1year HIV symptomatic HIV asymptomatic ART 1st line failure Dot plot showing levels of mir-150 in HIV infected patients. Different groups of patients are plotted on X-axis and levels of mir- 150 are plotted on Y axis. The error bars indicate median and interquartile range for each group. The levels of mirna -150 in the samples from asymptomatic HIV infected patients were significantly higher than those in symptomatic HIV infected patients (P<0.0001) (Figure 3.18). The levels were higher in patients who were on ART for more than one year as compared to those who were ART naïve and symptomatic indicating response to treatment, although not significanlty. However, the levels were also higher in patients showing first line failure. Figure 3.19: mirna 146b quantification in HIV infected patients mirna 146b Copies / ng RNA Healthy controls ART >1 year HIV symptomatic HIV asymptomatic ART 1st line failure Dot plot showing levels of mir-146b in HIV infected patients. Different groups of patients are plotted on X-axis and levels of mir-146b are plotted on Y axis. The error bars indicate median and interquartile range for each group. ANNUAL REPORT n 60

63 The levels of mirna -146b in the samples from asymptomatic HIV infected patients were higher than those in symptomatic HIV infected patients although not significantly. However the levels in patients on ART for more than 1 year were not higher than the symptomatic HIV infected patients (Figure 3.19). C.13) Genetic Susceptibility of Xenobiotic Drug Metabolizing Enzyme Gene and Drug transport Gene in ARV associated Hepatotoxicity Investigators: Dr. Hari Om Singh and Dr. R.R. Gangakhedkar Sponsors: Intramural Study Period: Xenobiotic Drug metabolizing enzyme helps defend our body from environmental toxin and drug toxicity. It acts in two phases i.e. Phase I and Phase II. Phase I (CYP) is multigene family of microsomal isozyme, it plays an important role in metabolism of various drugs and phase II enzyme (GST& NAT) is multifactorial enzyme and it plays an important role in cellular detoxification. We enrolled a total of 34 HIV infected patients with hepatotoxicity with NNRTI, 132 HIV infected individuals without hepatotoxicity under NNRTI regimens and 153 unrelated healthy individuals in this study. With a case control design, polymorphisms of phase II and Phase I drug metabolizing enzyme gene were genotyped by polymerase chain reaction and restriction enzyme length polymorphism. The conclusions from the study are summarized below 1) Impact of GSTM1, GSTT1 and GSTP1 gene polymorphism and risk of ARV associated hepatotoxicity in HIV infected individuals and its modulation GSTT1 null and GSTM1 null genotypes independent and in combination may predict development of hepatotoxicity. HIV infected individuals using alcohol and nevirapine both by independent GSTT1 null and in combination, GSTT1null, GSTM1 null genotypes had enhanced risk of development of hepatotoxcity. GSTM1, GSTT1 null and GSTP1 genotypes may not have impact on modulation of toxicity in hepatotoxicity among patients in the present study. 2) CYP1AI and CYP2C9 gene polymorphisms and risk for hepatotoxicity among HIV infected individuals and its modulation in Hepatotoxicity patients Individuals with CYP2C9*2 430CT and CYP1A1m1 3801TT genotype among advanced stage and initial stage are likely to develop hepatotoxicity. Individuals with CYP2C9*2 3801TC and 3801TC+CC genotype and using Nevirapine may develop hepatotoxicity. HIV infected Individuals with CYP2C9*2 3801TT genotypes and usage of Efavirenz may have higher chance to develop the hepatotoxicity. Neither independent genotypes of CYP2C9*2 430C/T, CYP2C9*3 1075A/C and CYP1A1m1 3801T/C nor haplotypes of (CYP2C9*2/ CYP2C9*3) gene were associated with the risk development of hepatotoxicity in HIV infected individuals and its modulation in hepatotoxicity patients. 3) CYP2B6 gene polymorphisms and risk for development of hepatotoxicity in HIV infected individuals and its modulation Individual with intermediate metaboliser genotype 516GT and 516GT+TT of CYP2B6 among independent ANNUAL REPORT n 61

64 alcohol user and combined alcohol and nevirapine user are associated with higher rise of development of hepatotoxicity. Interaction of CYP2B6 516TT genotype with nevirapine regimen showed higher risk of hepatotoxicity in HIV infected individuals though not significant. CYP2B6 516G/T polymorphism is neither associated with risk of hepatotoxicity nor its modulation. The level of efavirenz and nevirapine exposure in plasma was not estimated in the present study. C.14) Genetic susceptibility of APOBEC3G Gene in HIV infected patients Investigators: Dr. HariOm Singh, Dr. R.R. Gangakhedkar Sponsors : Extramural-ICMR Study Period : APOBEC3G is a host cellular protein. It plays an important role in the resistance of replication of HIV by creating G/A hypermutaion and rupturing the reading frame of viral gene. Thus this protein plays an important role in innate antiviral immunity. In this report a case-control study undertaken, a total of 156 HIV infected patients and 156 unrelated healthy individuals were enrolled in this study. Polymorphism for APOBEC3G and APOBEC3B gene were genotyped by polymerase chain reaction and restriction enzyme length polymorphism. The conclusions from the study are summarized below Genetic variants of APOBEC3G-90C/G and -571G/C polymorphism and risk of acquiring HIV infection Individuals with APOBEC3G-571GC genotype and haplotype CC of APOBEC3G-90C/G and APOBEC3G- 571G/C may have higher susceptibility for acquiring HIV infection. Limitation of the study is that the healthy controls may not have been exposed to similar risky behavior and hence this association should be studied in a larger sample size. 1) Genetic variants of APOBEC3BI/D polymorphism and risk of acquiring HIV infection APOBEC3B deletion is associated with the risk of acquiring HIV infection suggests a possible role for APOBEC3B gene in innate immunity against HIV. These results might help to understand of genetic factors influencing HIV infection and outcome. Interaction of APOBEC3B genotype with advanced stage of HIV infection may also influence the risk of progression. It might also have potential implication in prevention and clinical care of HIV infection and progression of HIV. Study of AIDS restriction genes polymorphism may help to screen the susceptible population for acquiring HIV infection and its progression. The role of editing of the HIV genome in such defense systems should be further investigated to understand the natural antiviral mechanisms and to develop an antiviral strategy against HIV. Hence, future studies should critically evaluate the role of gene-environment interactions related to AIDS restriction genes. ANNUAL REPORT n 62

65 C.15) Genetic variants of matrix metalloproteinase enzymes gene in HIV associated neurological disease Investigators: Dr. Hari Om Singh, Dr. R.R. Gangakhedkar Sponsors: Extramural-ICMR Study Period: Matrix metalloproteinase (MMP) enzymes are a family of structurally similar enzyme zinc containing endopetidases which degrade the collagen, proteoglycon of extracellular matrix and rupture the blood brain barrier and synoptic complex. Thus play an important role in neuro-inflamatory diseases. In is cross-sectional study, we enrolled a total of 50 patients of HIV associated neurological disease (HAND), 130 HIV infected individuals without HAND and 150 unrelated healthy individuals. Polymorphisms for MMPs gene were genotyped by PCR-RFLP. The conclusions from the study are summarized below 1) Impact of promoter polymorphism MMP-3( A>6A) on HAND MMP and MMP gene polymorphism may not have impact on modulation of pathogenesis of HAND patients. 2) MMP-7(-181A>G) promoter polymorphism and modulation of pathogenesis of HAND Our data suggests that MMP GG genotype and G allele was neither associated with modulation of pathogenesis of HAND. 3) Role of genetic polymorphism of matrix metalloproteinase enzyme in HIV related disease In the present review, a brief description on classification, regulation of MMP and TIMP, effect of different MMPs and TIMPs gene polymorphism on different HIV related disease and expression of MMPs and TIMPs gene in HIV related disease has been provided. Studies till date have suggested that MMPs polymorphism (MMP-1, MMP-2 MMP3, and MMP9) playing role in modulation of HIV related disease. C.16) Development of a National HIV Drug Resistance Database by Generating and Utilizing Clinical, Genotypic and Phenotypic Characterization Data of the Prevalent Drug Resistant Strains of Indian HIV-1 Clade C Investigators: Dr. Swarali Kurle, Dr. R.R. Gangakhedkar Investigators at large : Dr. Srikanth Tripathy & Dr. R.S. Paranjape Sponsor: ICMR Although the rapid scaling up of ART program has shown benefits of reducing AIDS related deaths, the biggest threat for the success of an ART program, is the development of drug resistance which is a major challenge for successful management of HIV/AIDS. Hence, monitoring drug resistance is crucial for clinical management of HIV infected patients receiving the ART. Drug resistance data on Indian HIV variants is very limited. Developing a ANNUAL REPORT n 63

66 drug resistance database for Indian HIV is important to inform the management of ART regimen algorithm. This study is primarily aimed at developing HIVDR database on pol gene sequences and mutations through the network of ART centers across India. The study also aims to develop technological tools for phenotypic drug resistance and replication fitness studies of a subset of Indian HIV-1 Clade C resistant viruses and will also develop a preliminary database for phenotypic drug resistance in India. A National HIV drug resistance database in the form of web resource will be developed for submission, analysis and retrieval of genotypic and phenotypic data related to HIV drug resistance in the country. To achieve the aims of the project; a network of 12 institutes involved in HIV-1 drug resistance testing in India was formed in the first year of the project. The standardized and validated in-house assays for drug resistance genotyping were disseminated to these institutes and staff was trained to perform the assays. The samples collected at these collaborating centres were sent to NARI Pune, in the form of either plasma (AFMC, Pune and Prayas, Pune) or Dried Blood Spots (DBS). During year a total of 231 samples were collected across all centres and the HIVDR genotyping for the pol gene was completed for 210 samples (Table 3.7). Table 3.7: Status of Sample collection and genotyping across different centers in the network Name of the Site Samples Samples collected Genotyped NARI, Pune AFMC, Pune * YRGCARE, Chennai * NIRT, Chennai * JALMA, Agra 6 5 AIIMS, New Delhi 0 0 PGIMER, Chandigarh 22 9 STM, Kolkata 5 4 # BHU, Banaras 0 0 * SMS, Jaipur Prayas, Pune Total Indian HIV-1 predominantly belongs to subtype C. The samples genotyped under this study were found to be mainly of the subtype C except five strains of which three belonged to subtype A1, one was A1C recombinant and one strain was C/CRF01_AE (Figure 3.20). ANNUAL REPORT n 64

67 Figure 3.20: Phylogenetic analysis of the HV-1 strains genotyped for HIV drug resistance The genotyping data of 169 samples from patients with first line regimen failure showed presence of mutations leading to high level resistance to NRTI and NNRTI (Figure 3.21). The frequencies of mutations (presented as percentage of samples showing mutation) leading to resistance to nucleoside reverse transcriptase inhibitors (NRTIs) is shown as non-thymidine Analogue Mutations (Non TAMs), TAMs and mutations with major drug resistance (MDR). Overall 68.6% of the samples showed development of M184VI which reduces the susceptibility to Lamivudine by >100 fold. The frequency of TAM ranged between 2.37% to 28.99%. Among the mutations associated with NNRTI resistance, K103N/S was the most common mutation found in 60% of the samples followed by Y181C (58% samples) and G190ASEQ (48% samples). ANNUAL REPORT n 65

68 Figure 3.21: Frequency of mutations associated with high level resistance to NRTI and NNRTI in patients failing first line ART regimen Frequency of mutations associated with high level resistance to NRTI and NNRTI in patients (n=169) failing first line ART regimens. The mutations associated with NRTI resistance have been separately indicated as Non TAMs, TAMs and MDR (Major Drug Resistance). The HIV drug resistance genotyping of 37 samples collected from the patients who failed second line ART regimens showed (Figure 3.22) presence of major mutations associated with resistance to Protease Inhibitors (PI). M46L mutation was seen in 37.84% of samples which is selected by most of protease inhibitors such as Indinavir, Nelfinavir, Fosamprenavir, Atazanavir, Lopinavir etc. and is known to reduce the susceptibility to Atazanavir, Fosamprenavir, Indinavir, Nelfinavir and Lopinavir. ANNUAL REPORT n 66

69 Figure 3.22: Frequency of mutations associated with high level resistance to NRTI, NNRTI and PI in patients failing second line ART regimen Frequency of mutations associated with high level resistance to NRTI, NNRTI and PI in patients (n=37) failing second line ART regimens. To represent the resistance patterns from the North-East Indian HIV, samples from Assam will also be included. The HIV drug resistance database will be made accessible to all researchers as an interactive web resource for submission, analysis and interpretation of drug resistance. C.17) Studies on in-vitro replication fitness of antiretroviral drug resistant strains of the Indian HIV-1 clade C Guide: Dr. Srikanth P. Tripathy, Ph.D. student: Nitin Hingankar, SRF, ICMR This study aims to develop HIV-1 subtype C specific replication fitness assay to study replication fitness properties of drug resistant strains of the Indian HIV-1 Clade C. It includes generation of a panel of reverse transcriptase drug resistant clones of the Indian HIV-1 Clade C and assessment of their replication fitness. Further, this study aims to compare the impact of select drug resistance mutations on replication fitness of the Indian HIV-1 Clade C and HIV- 1 subtype B. Differences in the pol gene and overall genetic background between HIV-1 subtype B and Indian Clade C can lead to distinct fitness properties on acquiring resistance mutations. Therefore, assessment of the replication fitness with subtype specific assays is necessary which would help generate more accurate information on fitness. Knowledge ANNUAL REPORT n 67

70 of the relative fitness costs of various resistance mutations and mutations that confer distinct impact on fitness of resistant strains is crucial for understanding the pathogenesis of HIV-1 Indian Clade C which can be utilized in tailoring effective treatment strategies for Indian patients. Previously in the last year, a panel of 12 Non Nucleoside Reverse Transcriptase Inhibitor (NNRTI) drug resistant viruses harboring single mutations was generated and assessed for single cycle replication fitness. Multiple cycle replication fitness was determined for four viruses harboring mutations: K103N, V106A, V106M and Y181C. During :Fresh virus stocks were generated for the 8 subtype B clones and 11 Indian subtype C clones, additionally p24 antigen quantification as well as TCID50 titers of these viruses were determined as indicated in figure 3.23 to 3.26 below Figure 3.23: p24 antigen quantification of HIV-1 subtype B virus stocks Figure 3.24: TCID50 Titers of HIV-1 subtype B virus stocks. Figure 3.25: p24 antigen quantification of HIV-1 Indian Subtype C virus stocks Figure 3.26: TCID50 Titers of HIV-1 Indian Subtype C virus stocks p24 antigen concentration was determined for a panel of 6 NNRTI drug resistant viruses of the HIV-1 subtype B and Indian HIV-1 Clade C as indicated in Table 3.8. This p24 antigen concentration was used to normalize the inoculums of each virus in single cycle replication fitness assays. ANNUAL REPORT n 68

71 Table 3.8: p24 antigen quantification of virus stocks of HIV-1 subtype B and Indian HIV-1 Clade C viruses used for comparison of single cycle replication fitness. HIV-1 subtype B viruses WILD ( pnl-4-3-b-rt-in-pindiec ) Indian HIV-1 Clade C viruses WILD (pindiec-cons-in-c-rt- ) Virus harboring P24 Virus harboring P24 Mutation concentration concentration. (ng/ml) Mutation (ng/ml) V106A V106A Y181C Y181C Y188C Y188C Y188L Y188L G190A G190A M184V M184V pnl-4-3-b-rt-in pindiec-cons WILD WILD pindiec IN-C-RT- Single cycle replication fitness of these 6 freshly prepared mutant viruses of each of the Indian Clade C and HIV-1 subtype B was assessed and compared using TZM-bl cells. Figure 3.27: Comparison of single cycle replication fitness of NNRTI mutants of HIV-1 subtype B and Indian Clade C in TZM-bl cells Comparison of single cycle replication fitness of 6 NNRTI mutants of HIV-1 subtype B and Indian Clade C as indicated by % RLUs (Relative Luminescence Units) of each mutant compared to the respective WILD type virus. (Values shown are the mean of at least two independent experiments performed in at least three replicates for each virus). ANNUAL REPORT n 69

72 Each virus was infected with TZM-bl cells in a total volume of 200 µl in 96 well tissue culture plates. Culture was incubated for the 48 hours and then Relative Luminescence Units (RLUs) were measured by using 50 µl of the Brightlight Plus reagent in the Luminometer. The mean RLU reading of all the cell control wells was deducted from each dilution of sample replicate to determine the actual total RLUs for each replicate. For comparison % RLUs of each mutant was calculated by taking the RLU value of WILD type virus as 100 %. Graph was plotted to indicate the % Relative Luminescence Units of each NNRTI mutant compared to the respective WILD type which was considered 100% as shown in figure As indicated in the Figure 3.22, the results showed varied replication fitness of the NNRTI mutants indicating variable impact of resistance mutations on fitness of virus. Replication fitness of most mutants was decreased compared to wild type virus. Importantly, V106A mutant showed significant decrease in fitness in both, HIV- 1subtype B and Indian Clade C RT gene background, indicating high fitness cost of this mutation in both the subtypes. When the replication fitness of mutants was compared between HIV-1 subtype B and Indian Clade C, significant difference was observed in the fitness costs of the Y181C mutation between two subtypes. In subtype B background Y181C did not show significant difference in fitness compared to wild type, however in Indian Clade C background the fitness of Y181C was reduced considerably compared to wild type virus. Mutations Y188L and G190A also showed different fitness costs when assessed in RT background of HIV-1subtype B and Indian Clade C, although the difference was moderate. However, fitness cost of mutations V106A, Y188C and M184V was not significantly different between HIV-1subtype B and Indian Clade C. C.18) Antiretroviral Drug Resistance in HIV-1 Infected Adults Detected to Have Treatment Failure After Virological Monitoring Versus Those Detected by Immunological Monitoring Guide: Dr. R.R. Gangakhedkar and Ph. D. student: Dr. Santosh Karade Sponsors : WHO and Intramural Study Period : The emergence and spread of drug resistant mutations (DRM) in HIV is a major threat to the goal of National AIDS control programme of halting and reversing the epidemic. The evolution of drug resistance in HIV is inevitable due to, error prone nature of the reverse transcriptase. To preserve the benefits of currently available ART for longer duration, it is necessary to employ appropriate laboratory tests to monitor the progress of disease. As routine virological monitoring is limited by high cost, treatment failure is diagnosed by clinical or Immunological means in resource limited setting and targeted viral load is performed only for confirmation of failure. Since virological failure precedes immunologic and clinical failure, delays in detection of failure may lead to accumulation of thymidine analogue mutations (TAMs). TAMs are non-polymorphic mutations selected by the thymidine analogs Zidovudine (AZT) and Stavudine (d4t).the primary objective of this study is to compare HIV drug resistance mutation pattern, with special attention to TAMs, observed after detection of treatment failure by early viral load screening at 12 months of ART versus routine immunological monitoring up to 36 months of ART initiation. ANNUAL REPORT n 70

73 The study is approved by Ethics Committee of NARI. In this cross sectional study, HIV-1 infected adult individuals with virological failure at 12 months of ART initiation were recruited from four ART centres of Pune city after obtaining informed consent. As on 01 April 2015, 85 plasma samples with viral load > 1000 copies/ml were collected for drug resistance genotyping. Pol gene sequences involving partial reverse transcriptase and complete protease region from 80 individuals (42 males and 38 females) were successfully amplified and sequenced (GenBank accession numbers KR KR816097). Subtype C was most prevalent, seen in 97.5% sequences. Remaining two sequences were of subtype A1 and a novel recombinant form of CRF01_AE/C. Following first line failure, Zidovudine (AZT) was most susceptible NRTI (Figure 3.28). The prevalence of drug resistance mutations (DRMs) against NRTIs, NNRTIs and protease inhibitors (PIs) were 58.75%, 78.75% and 1.25% respectively. The commonest NRTI and NNRTI mutation observed were M184V/I (51.25%) and 101E/N/R(51.25%) respectively. Out of 80, only 14 (17.5%) sequences showed at least one thymidine analogue mutations (TAMs). Though overall prevalence of DRM was high (78.75%), restriction of TAMs (17.5%) due to early detection is an important finding. Recruitment of participants in immunological failure group for comparison of DRM pattern is ongoing. Figure 3.28: Overall resistance pattern to various NRTI and NNRTI at first line ART failure Degree of resistance is graded into five categories from susceptible(green) to high level resistance(red) based on Stanford database mutation scoring. ANNUAL REPORT n 71

74 C.19) Crosstalk between Notch-1 and EGFR during HIV-HPV Co-infections Investigators: Dr Serena D'Souza; Dr Arati Mane, Dr Vandana Saxena, Dr Sheela Godbole Sponsor: ICMR Extramural Study Period: Notch and Epidermal Growth Factor Receptor (EGFR) play an important role in cell signalling. Aberrant Notch signalling has been reported to trigger oncogenesis. The study aims to understand the implications of how the virus hijacks hosts' proteins for its own propagation in cervical cancers arising during HIV- HPV co-infections. The objectives of the project are as follows: 1. To identify the role of Notch signaling pathway(s) during HIV-HPV co-infection. 2. To determine the association between Notch 1 and EGFR expression during HPV-induced tumorigenesis in the presence of HIV infection. The study was approved by the NARI Scientific Advisory and Ethics Committee/s. In-vitro molecular studies requiring the use of molecular clones for infectivity assays were procured. Molecular clones are large plasmids containing the proviral genomes.the puc18 based pnl plasmid encoding HIV-1, 4.3 HPV-16 cloned from an invasive cervical carcinoma into an expression vector pbr322, retroviruses expressing Tat86 (RV- Tat) tagged to Green Fluorescent Protein (GFP) and MigR1 expressing the Intra Cellular Domain (ICD) of Notch 1 were transformed into Escherichia coli DH5a competent cells. The plasmid's DNA were re-extracted from the transformed cells and quantitated by nanodrop spectrophotometer. Purified plasmids DNA were obtained. TaqMan real-time PCR using type specific primers for E6 /E7 detection was performed. The PCR products were analysed on 0.8% agarose gel run under standard low-voltage electrophoresis conditions (Figure 3.29). HPV-16 E6 detection and typing were confirmed by the general primer PCR and sequence-based typing (PCR-SBT) method. Transfection studies of the amplified plasmid DNA into Foetal Buccal Mucosal Cells (FBM) procured from ACTREC, Navi Mumbai and CaSki Cells procured from NCCS, Pune will commence soon. These cell lines were also propagated and cryo preserved for further use. In vitro infectivity assays of HIV-1 Ada5 (R5 subtype B)viral supernatant having p24=1ng/ml, with MOI= 0 0.5/well and pre-adsorbed onto FBM cells (p#35) for 24 hours at 37 C, were optimized. Figure 3.29: Electrophoretic Separation of Re-extracted Amplified Plasmid DNA Electrophoretic separation of 3 different amplified plasmid DNA (MigR1, HA-Tat and pnl4-3) fragments of MigR1 ~ 1.2Kb, HA-Tat~ 1Kb and pnl 4.3~1.5Kb along with a 10Kb DNA ladder as seen on gel documentation system after UV exposure. ANNUAL REPORT n 72

75 Figure 3.30: MTT Assay for FBM Cell Viability using 25µg/ml Polybrene CC Absorbance of enzyme-linked immunosorbent assay at 550/630 nm as a measure of percent viability of FBM (5x 10 ) cells treated with differing concentrations of polybrene for enhancing in-vitro infectivity The efficiency of retrovirus infection was tested using polybrene (25µg/ml), a polycationic agent. Culture supernatants were harvested post infection in separate experiments with polybrene (days 5, 12, 14) and without polybrene (days 4, 8 and 13). Cell cytotoxicity (CC ), assayed by the MTT method was calculated from concentration effect curves after linear 50 regression analysis (Figure 3.30). p24 concentration was measured using the Mini Vidas p24 kit (biomérieux, France). It was observed that polybrene enhanced and could sustain HIV infectivity for two weeks (Figure 3.31 A & B). Figure 3.31: Polybrene enhances HIV Infectivity in FBM Cells Influence of polybrene as an enhancer of in vitro HIV-1 infectivity for FBM cells as demonstrated by p24-elisa. ANNUAL REPORT n 73

76 C.20) Genetic and neutralization properties of the envelope gene in HIV-1 and HIV-2 monotypic and dual infections Guide: Dr. Smita Kulkarni, Ph.D. student: Ms. Priyanka Khopkar Sponsors: Intramural Study Period: HIV-2 reports a lower pathogenicity than HIV-1. HIV-2 sera depict a significant capacity to neutralize autologous and heterologous viruses, indicating rare viral escape to neutralization responses (nab) in HIV-2 infection. It is known that the entry mediated via HIV envelope glycoproteins is a key factor that impacts neutralization. Therefore, delineating the characteristics of HIV-1/HIV-2 envelope gene in modulating the neutralization responses in HIV-1 and HIV-2 monotypic and dual infections may provide insights for designing anti-hiv therapeutic and vaccine modalities. The objectives of the project are: 1. To characterize env gene of viruses isolated from HIV-1 and HIV-2 monotypic and dually infected patients in order to study the genetic diversity. 2. To study autologous, heterologous and cross neutralization patterns in HIV-1 and HIV-2 monotypic and dually infected serum samples. The patient enrolments in three study groups (HIV-1 infected, HIV-2 infected; HIV-1 and 2 dually infected, 10 patients/group) have been completed recently. Subsequently, attempts have been made to isolate virus using the cocultivation method. Nine reference strains, each for HIV-1 and HIV-2 have been procured from NIH under the AIDS Research Reference Reagent program (ARRRP), stocks of these viruses have been expanded (Figure 3.32) and titrated for further use. ANNUAL REPORT n 74

77 Figure 3.32: Microscopic images of the Infectivity assay in TZM-bl cells during expansion of NIH HIV-2 strains As in the case of HIV-1, HIV-2 nabs are not available through NIH ARRRP. Hence, respective plasmids have been obtained (courtesy: Dr George Shaw, USA and Dr James Robinson, USA) for generating four HIV-2 monoclonal antibodies and HIV-2 backbone plasmid for pseudoviruses. The bacterial stocks harboring respective plasmids have been prepared using transformation experiments. Large scale plasmid expansion required for pseudovirus generation has been completed. Transfection experiments with plasmids to generate HIV-1 and HIV-2 pseudoviruses and HIV-2 monoclonal antibodies are in progress. ANNUAL REPORT n 75

78 C.21) Impact of Human T cell Leukemia Virus II on HIV-I replication Investigators: Dr Smita Kulkarni, Dr. Vidya Arankalle, PhD Student Mr. Rajkumar B. Londhe Sponsors: NARI - Intramural project. Study Period: 2013 to 2016 Epidemiological data suggests a slower HIV disease progression in individuals co infected with HIV-1 and HTLV- II. The antisense protein of HTLV-II (APH-2) negatively regulates Tax mediated transcription of HTLV-II by interacting with transcriptional and co-transcriptional factors like Cyclic Response Element Binding protein (CREB) which are also important for HIV-1 transcription. Hence this study has been designed to understand the role of HTLV-II negative regulatory protein APH-2 on HIV-1 replication. The objectives of the study are: i. To study the effect of APH-2 on HIV-1 replication by using HIV-1 LTR based luciferase assay. ii. To study the role of the host protein CREB in APH-2:HIV-1 interaction In the reporting year preliminary experimentation was completed. As infectious clones of HTLV-II are not available with NIH ARRRP, they were obtained from Dr P G Green, Ohio State University, USA. The expression vectors (pcmvha) with cloned insert for APH-2, MutAPH-2 (LXXLL domain is muted to AXXAA) and CREB were generated and expressed in 293T cell line (Figure 3.33 and Figure 3.34). Figure 3.33: Generation and Confirmation of HA-APH-2 clone Generation of HA - APH -2 Clone 293T + ph6neo Total RNA extraction cdna synthesis and Amplification. (Gene specific primers) + Top 10 E.coli Colony PCR HA -Tag APH - 2 ORF MYPYDVPDYALMAMEARIRSTEISRGTMDPQDYFRRLPLGDQGEASLTSGVPPPGEEPRRTAKGRPLGSG SVSEQVRDSRRRLLAEEQREILKLLQEREERERRRKERLREKDQKRREREERHQQLECIGLLGFDGFCDL LQGYVDFLEGEKKVLWEECERGLEELFEAIIQFESGLDIGSLIWYRGL ANNUAL REPORT n 76

79 Figure 3.34: Generation and Confirmation of Cmyc-CREB expression clone Generation of Myc -CREB clone Total RNA extraction cdna synthesis and Amplification. (Gene specific primers) 293T + c r e b 1 C M y c _ C R E B _ I n s e r t c r e b 1 C M y c _ C R E B _ I n s e r t c r e b 1 C M y c _ C R E B _ I n s e r t Top 10 E.coli Colony PCR A T G A C C A T G G A A T C T G G A G C C G A G A A C C A G C A G A G T G G A G A T G C A G C T G T A A C A G A A G C T G A T G C A G C T G T A A C A G A A G C T * * * * * * * * * * * * * * * * * * * * * G A A A A C C A A C A A A T G A C A G T T C A A G C C C A G C C A C A G A T T G C C A C A T T A G C C C A G G T A T C T G A A A A C C A A C A A A T G A C A G T T C A A G C C C A G C C A C A G A T T G C C A C A T T A G C C C A G G T A T C T * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * A T G C C A G C A G C T C A T G C A A C A T C A T C T G C T C C C A C C G T A A C T C T A G T A C A G C T G C C C A A T A T G C C A G C A G C T C A T G C A A C A T C A T C T G C T C C C A C C G T A A C T C T A G T A C A G C T G C C C A A T * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * The expression of HA-CREB clone (Figure 3.35) has been confirmed by western blot, expression and confirmation of APH-2 and MutAPH-2 protein is in progress. Figure 3.35: Confirmation of Cmyc-CREB expression Expression of Cmyc-CREB, lane 1; Negative control - 293T transfected with Moc and lane 2; 293T transfected with Cmyc- CREB expression clone ANNUAL REPORT n 77

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81 4 STIs and Opportunistic Infections ANNUAL REPORT n 79

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83 D.1) Genetic basis of azole resistance in clinical candida albicans isolates Investigator: Dr. Arati Mane and Dr. Arun Risbud Sponsor: Intramural Study Period: Candidiasis is one of the commonest opportunistic fungal infections, with Candida albicans as the commonest infecting species. The azoles, particularly Fluconazole, remain the most commonly used antifungal drugs. Increasing resistance to Fluconazole over the past decade from 0 % to upto 20% has been reported by various researchers from India. An understanding of resistance at the molecular level is essential for the development of strategies to tackle antifungal resistance and for rationale design of newer antifungals using target based molecular approaches. The objectives are as follows 1. To study the target site mutations in ERG11 and ERG 3 genes in azole susceptible (S), susceptible-dose dependent (S-DD) and resistant (R) clinical Candida albicans isolates. 2. To study the expression patterns of ERG11 gene & genes encoding efflux pumps (CDR1, CDR2 and MDR). A collection of 87 clinical C. albicans isolates [Fluconazole susceptible (n=30), susceptible-dose dependent (S- DD) (n=30) and resistant (n=27)] obtained from separate patients and from different body sites (oral, vaginal and pulmonary) and maintained in our laboratory were included in this study. Last year, we reported the drug target site mutations in ERG 11 and ERG 3 genes and the results indicated that alterations in drug target sites do not play a major role in flucoazole resistance. During the reporting year , the expression patterns of ERG11 gene and genes encoding efflux pumps (CDR1, CDR2, MDR) were studied and the results are presented here. Total RNA was extracted and used to synthesize complementary DNA using superscript VILO cdna kit. The quantitative PCRs for CDR1, CDR2, MDR1, ERG11, and ACT1 genes were performed in duplicate using customized Taqman based assays on the 7900HT Real Time PCR system. Gene over-expression was defined as more than 3 times the average fluconazole susceptible strain values. Over-expression of CDR1, CDR2, MDR and ERG11 genes in S-DD and resistant isolates as compared to susceptible isolates is depicted in Figure1. CDR1 gene: The mean relative CDR1 gene expression levels in susceptible, S-DD and resistant isolates were 0.252±0.046 (mean±standard error of mean), 1.541±0.453 and 3.158±0.758 respectively. The CDR1 expression levels were significantly greater in S-DD (p<0.0001) and resistant isolates (p<0.0001) as compared to the susceptible isolates. CDR2 gene: The mean relative CDR2 gene expression levels in susceptible, S-DD and resistant isolates were ±0.038, 0.908±0.365 and 1.560±0.525 respectively. The CDR2 expression levels were significantly greater in resistant isolates compared to susceptible ones (p=0.002). A total of 33.3% resistant isolates over expressed both CDR1 and CDR2 genes simultaneously. CDR2 was exclusively expressed with CDR1. Eight of the 9(89%) resistant isolates with CDR1 and CDR2 co-expression exhibited cross-resistance to ketoconazole and itraconazole. MDR gene: The mean relative MDR1 gene expression levels in susceptible, intermediate-susceptible and resistant ANNUAL REPORT n 81

84 isolates were ± 0.011, ± and ± respectively. There was no statistically significant difference in the MDR1 expression levels between susceptible, intermediate-susceptible and resistant isolates. ERG11 gene: The mean relative ERG11 gene expression levels in susceptible, intermediate-susceptible and resistant isolates were ± 0.077, ± and ± respectively. There was no statistically significant difference in the ERG11 expression levels between susceptible, intermediate-susceptible and resistant isolates. Figure 4.1: The expression patterns of CDR1, CDR2, MDR1 and ERG11 genes in S-DD and resistant Candida albicans isolates as compared to susceptible isolates Our findings show that drug efflux, mediated by Adenosine-5'-triphosphate (ATP)-binding cassette transporters, especially CDR1, is associated with fluconazole resistance and azole cross-resistance in C. albicans. Thus a prudent approach would be a combination therapy of azole and an efflux pump inhibitor for management of severe C. albicans infections. There is a need to prioritize research for identifying new efflux inhibitors. D.2) Role of HSV-2 in HIV-1/ HSV-2 co-infection Guide: Dr. Smita Kulkarni, Ph.D. Student: Mr. Dipen Desai Sponsor: Intramural Study Period: Epidemiological studies show that HSV-2 co-infection is associated with higher HIV-1 viral load in HIV-coinfected individuals. Therefore, in our endeavor to identify a suitable T cell line for studying the interactions between HIV-1 and HSV-2, we studied HIV-1 and HSV-2 co-infections in three cell lines (CEMccr5, PM-1 and ANNUAL REPORT n 82

85 MOLT4/R5). Of the three cell lines, a significant increase in the HIV-1 viral load (4.5 folds, p<0.01) was observed when CEMccr5 cells were co-infected with HSV-2 and HIV-1 as compared to viral load in the CEMccr5 cells infected with HIV-1 alone. These data suggested that CEMccr5 cells are best suited to study HSV-2 and HIV-1 coinfection. Subsequently, similar experiments were carried out using primary CD4 T-cells to compare the results observed in the cell lines with that in primary cells isolated from peripheral blood. Furthermore, to investigate whether productive HSV-2 infection is necessary for an increase in HIV-1 replication, co-infection experiments were performed using a replication incompetent HSV-2 ATCC VR734 virus (UV inactivated). In the co-infection experiments performed using CD4 T-cells, though statistically non-significant, an increase (2.47 fold / 0.4 log 10 RNA copies /ml) (p > 0.05) in the HIV-1 viral load was observed when the cells were infected with HSV-2 and HIV-1, as compared to CD4 T cells infected with HIV-1 alone (Figure 4.2 A). This could be attributed to the slower cell division in primary CD4 T-cells isolated from peripheral blood, than the established cell lines, indicating a rare possibility of multiple infections of CD4 T-cells with HSV-2. Nonetheless, the increase in HIV-1 viral load was noteworthy; highlighting that the influence of HSV-2 co-infection on HIV-1 replication is a natural phenomenon and not just an in-vitro effect. In co-infection experiments carried out in CEMccr5 cell line using a UV inactivated HSV-2 virus, there was significant reduction in HIV-1 viral load (0.5 log RNA copies /ml, (p < 0.05) (Figure 4.2 B). This indicates that 10 productive infection of HSV-2 is essential to influence HIV-1 viral load. Further studies to investigate intricacies in the reduction in HIV-1 viral load due to the UV-inactivated HSV-2 virus are in progress. Published literature suggests that the tegument proteins present in HSV-2, particularly the virion host shut-off RNAse (VHSRNase/UL- 41) may be responsible for inhibiting HIV-1 replication by degrading the host cellular and HIV-1 RNAs. The data obtained so far suggests that the viral infection is a fragile process which is carefully controlled by the viral proteins to ensure efficient infection by evading host cellular and immune responses. ANNUAL REPORT n 83

86 Figure 4.2: HIV-1 viral load in culture supernatants of T cells infected with HIV-1 and HSV-2 HIV-1 viral load in HIV-1 and HSV-2 co-infected CD4+ T cells (A) The experiments were performed using CD4 T-cells isolated from two separate donors. (B) CEMccr5 cells were infected with either the wild type HSV-2 ATCC VR734 strain or UVinactivated HSV-2 strain. The HIV-1 viral load estimation was carried out using Abbott m2000rt assay. The data represents Mean + SD of three independent experiments. Significance of the results was calculated using two way ANOVA. * indicates p < ns: not significant, PID: Post infection day D.3) Relation between Genetic Markers of Drug Resistance and Susceptibility Profile of Neisseria gonorrhoeae strains Investigators- Dr.Arun Risbud, Dr.Sheela Godbole, Dr.Sangeeta Kulkarni, Dr.Manju Bala, Dr.Syeda Amtul Moqueeth Collaborators: Regional STD Teaching, Training & Research Centre, V.M. Medical College & Safdarjang Hospital, New Delhi, Gandhi Medical College, Secunderabad Sponsor- Indian Council of Medical Research-Extramural Study period The gram negative bacterium, Neisseria gonorrhoeae is the etiologic agent of the sexually transmitted infection gonorrhea which classically presents as genital discharge syndrome in men and causes cervical discharge in women. Untreated gonorrhea can cause significant reproductive tract morbidity especially among women. Over the last two decades, N. gonorrhoeae strains have developed high levels of resistance against several antimicrobial ANNUAL REPORT n 84

87 agents like penicillin, tetracycline and quinolones in different countries including India. The use of syndromic management of genital discharge, adapted as a standard nationwide, adds to the challenges of obtaining genital samples for cultures. In this scenario, it is necessary to identify emerging patterns of bacterial resistance in time to prevent an explosive spread of resistant infections. One alternative approach towards monitoring resistance could be the molecular (genetic) basis for resistance, which needs to be explored. The genetic mechanism of N. gonorrhoeae resistance to antibacterials is complicated and not definitively elucidated. However acquisition of some genes and a number of mutations in structural genes and regulatory regions are considered to be unambiguously involved in development of resistance. Resistance to fluoroquinolones is conferred by mutations in the topoisomerase-encoding genes gyra, parc and mtrr genes. Mutations in pona and pena, which encode PBP1 and PBP2, respectively, confer resistance to penicillin and structural alterations in porb, which is a major outer membrane porin. Resistance to tetracycline is due to the acquisition of the tet(m) gene and mutation in rpsj gene. Knowledge of mechanism of resistance would help us to devise methods to prevent the occurrence of drug resistance against existing and new drugs. Such studies could also help in finding out new drug targets in N. gonorrhoeae and also a possibility of identification of new drugs for treating gonorrhoea. This study was undertaken to identify and analyze mutations in the tet (M), pena, gyra, mtrr, porb1, parc and por gene of N. gonorrhoeae strains and to compare these with their antibiotic resistance profile. Study Objectives: To identify and analyze mutations in the tet(m), pena, pona, gyra, mtrr porb1 parc and por gene in drug resistant and drug sensitive N. gonorrhoeae strains To study the association of specific gene mutations in N. gonorrhoeae strains with resistance to different antibiotics i.e. tetracycline, penicillin and fluoroquinolones. Salient findings: Thirty three Neisseria gonorrhoeae isolates from samples of patients attending sexually transmitted disease clinics with symptoms of urethral / cervical discharge from different parts of India [25-Delhi, 3-Pune, 5-Hyderabad] were collected in the reporting year. All the strains were confirmed by using standard biochemical techniques and by using Phadebact Monoclonal GC test. The antibiotic susceptibility pattern to Penicillin, Tetracycline, Ciprofloxain, Ceftriaxone, Cefixime, Spectomycin and Azithromycin was determined by disc diffusion methods and minimum inhibitory concentration [MIC] using E-test method. PCR and direct DNA sequencing were performed to identify mutations in the pena and pona (n=21), gyra, parc and mtrr (n=24) genes of antibiotic resistant strains. Penicillin and Tetracycline resistance phenotypes were categorized based on the plasmid or chromosomally mediated resistance to either Penicillin or Tetracycline were exhibited by 45.5% were PPNG, 11% were TRNG and 5% were CMRNG. Preliminary data about antibiotic susceptibility and mutations are presented below. All the thirty three strains showed 100% resistance to Ciprofloxacin and Nalidixic acid and 100% sensitive to Spectinomycin and Azithromycin. Four (12.1%) of the strains showed intermediate susceptibility to Ceftriaxone and Cefixime (Figure 4.3). ANNUAL REPORT n 85

88 Figure 4.3: Antimicrobial susceptibility of Neisseria gonorrhoeae strains from Delhi (n=25), Pune (n=3) and Hyderabad (n=5) l All the strains were resistant to Ciprofloxacin and Nalidixic acid. No resistance was observed for Spectinomycin, and Azithromycin. Penicillin susceptibility and mutations in pena and pona genes: Sequence analysis was performed for 21 of 33 N. gonorrhoeae strains. There were 3 different amino acid alterations in pena gene and four alterations in pona gene. Double mutations was also observed in N.gonorrhoeae strains. Ciprofloxacin susceptibility and mutations in gyra, parc and mtrr genes: Out of 33 N. gonorrhoeae strains, sequence analysis was performed for 24 strains. All the strains showed resistance to ciprofloxacin. Six different mutations were observed in gyra, while 5 and 4 mutations were seen in parc and mtrr genes respectively. ANNUAL REPORT n 86

89 Figure 4.4: Agarose gel electrophoresis of PCR products for gyra, parc, pena and pona genes PCR products for gyra,parc, pena and pona gene at 278bp,255bp,501bp,236bp ANNUAL REPORT n 87

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91 5 Product Development and Testing ANNUAL REPORT n 89

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93 E.1) Identification of anti-hiv leads from plant sources and determination of mechanism of action Investigators: Dr. Smita Kulkarni, NARI, Pune; Dr. Thulsasiram, NCL, Pune. Sponsors: DBT-ICMR Study Period: Earlier studies identified Terminalia paniculata and Polygonum glabrum plant extracts and their fractions as potential leads. In the previous year, the acetone and methanol extracts of T. paniculata fruits (NCL 51 and NCL 52) and P. glabrum aerial parts (NCL 53 and NCL 54) were tested for anti-hiv1 activity by TZM-bl and PBMC assay. Of these, NCL 52 showed promising activity and hence it was fractionated into 13 fractions (A to M). These fractions were tested for anti-hiv1 activity of which fractions K and L showed promising anti-hiv1 activity and were taken as leads. The other fraction, NCL 54 was directly fractionated into 11 pure compounds (A to K) which did not show anti-hiv1 activity. In the reporting year, NCL 52 fractions K and L were further fractionated into 13 and 6 sub fractions, respectively. The cytotoxicity and anti-hiv1 profiles of these sub fractions were determined and sub fractions K-2, K-3, K-13 and L-7 were obtained as leads. Extract NCL 51 was also fractionated into 12 fractions (A to L), of which fractions J and K showed prominent activity (Figure 5.1 and Figure 5.2). Fractionation of NCL 53 and anti-hiv1 testing of the fractions is in progress. For determining the mechanism of action using cell based assays; entry inhibition assay using TZM-bl cell line and fusion inhibition assay using TZM-bl and HL2/3 cell lines have been standardized, and the determination of the mechanism of action of lead extracts/fractions is in progress. Figure 5.1: Anti-HIV1 activity of NCL 51 and its fractions in TZM-bl assay IC values of NCL 51 extract in cell associated and cell free anti-hiv1 assays 50 ANNUAL REPORT n 91

94 Figure 5.2: Anti-HIV1 activity of fractions of NCL 51 IC values of fractions of NCL 51 (A-L) in cell associated and cell free anti-hiv1 assays. 50 However, the pure compounds of NCL 54 which included a new natural product were also tested for antimycobacterial, anti-malarial and anti-proliferative activities did not show anti-hiv1 activity. E.2) Identification of potential anti-hiv natural product analogs using molecular docking and medicinal chemistry approaches Investigators: Dr. Inder Pal Singh, NIPER, Mohali; Dr.Smita Kulkarni, NARI, Pune; Dr K K Bhutani, NIPER, Mohali. Sponsors: DBT-ICMR Study Period: During the previous year, analogues of γ-fagarine, IPSM-179 and IPSM 224 were identified as lead molecules. In the reporting year, molecular docking studies of these two leads were carried out which indicated that styrylquinoline nucleus may act through inhibition of integrase activity. In continuation with this, a total of 21analogues (17 analogues of IPSM-179 and IPSM224, 4 analogues of Tembamide) were synthesized and tested for inhibition of HIV-1 replication using TZM-bl assay. The cytotoxicity was assessed by the MTT assay and CC 50 was calculated. The antiviral activity was assessed against cell associated CXCR4 (HIV-1 UG070 ) and CCR5 (HIV- 1 ) tropic primary isolates and IC and TI (CC / IC ) were estimated. Of the 21 analogues tested, NP3551 was VB identified as a lead with highest TI (HIV-1 UG070: 21, HIV-1 VB59: 16) (Figure 5.3A and 5.3B). ANNUAL REPORT n 92

95 Figure 5.3A: Cytotoxicity and anti-hiv1 activity of NP-3551 in TZM-bl assay The compound NP-3551 was tested against cell associated HIV-1 primary isolates (HIV-1 and HIV-1 ). The IC values UG070 VB ranged between µg/ml. Figure 5.3B: Therapeutic index of NP-3551 (TZM-bl assay) NP-3551 was tested against cell associated HIV-1 primary isolates. The TI ranged between The activity of this lead compound was confirmed in primary cells (stimulated PBMC). Further, the mechanism of action of the three identified leads (IPSM179, IPSM224 and NP3551) was determined using cell based assays: entry inhibition using TZM-bl cell line and fusion inhibition using TZM-bl and HL2/3 cell lines. Confirmation of preliminary results is in progress. E.3) Exploring anti-hiv activity of lead HIV-1 RT inhibitors-4-thiazolidinone compounds against drug resistant strains of HIV Investigators: Dr. S.S. Kulkarni, Dr. R.S. Paranjape, NARI, Pune Sponsors: NARI-Intramural project Study Period: Rapid emergence of drug resistance is a major problem in the management of HIV infection which limits the applicability of effective anti-retroviral drugs in the ART regimen. Therefore, it is essential to design new drugs that ANNUAL REPORT n 93

96 are capable of inhibiting drug resistant strains. Our previous efforts identified four 4-Thiazolidinone derivatives, synthesized by CDRI, Lucknow as potent leads. During reporting year, in silico molecular docking studies were carried out in collaboration with NIRRH, Mumbai for comparing the binding affinity of these four lead molecules to the reverse transcriptase (RT) gene of 10 HIV-1 Nevirapine drug resistant isolates. The 2D structures of the lead compounds (1908, 1909, 1911 and 1912) drawn using ChemSketch are shown in Figure 5.4. Figure 5.4: 2D structures of the lead compounds drawn using ChemSketch The 3D structures of 10 HIV-1 Nevirapine drug resistant isolates with mutations in the non-nucleoside inhibitor binding pocket of RT gene were modeled using Prime module ver. 3.0 of Schrodinger ver The crystal structure of HIV-1 RT bound to Nevirapine (PDB id: 2HNY) was used as a template for modeling. These structures were prepared using protein preparation wizard of Schrodinger and energy was minimized using OPLS-2005 force field software (Figure 5.5). ANNUAL REPORT n 94

97 Figure 5.5: Molecular interactions of the docked complexes of compound S with RT gene of 10 HIV-1 Nevirapine drug resistant isolates The compound S is shown as ball and stick and the binding site residues are shown in thin lines using CPK colour scheme. The hydrogen bonds are shown in pink dotted lines; electrostatic and cation-π/π-π interactions are shown in green and orange lines respectively. The ChemScore obtained for the docked complexes revealed that among the four molecules; S possesses higher binding affinity for all the drug resistant isolates except DR6 (Data not shown). Further, the cytotoxicity and anti-hiv activity against 10 Nevirapine resistant isolates was assessed using TZM-bl assay and the data were expressed as CC 50 and IC 50 (Table 5.1). ANNUAL REPORT n 95

98 Table 5.1: Anti-HIV activity of lead compounds against Nevirapine resistant strains using TZM-bl assay a Isolate No IC 50 (µg/ml) NVP a CC 50 (µg/ml) DR DR DR DR DR DR DR DR DR DR a) Data represents mean of two independent assays for IC and CC, respectively The molecular interactions of compound S , when docked with the RT gene of 10 drug resistant isolates indicated that it stably interacted by forming hydrogen bonds, electrostatic and cation-π/π-π interactions with the binding site residues and identified S as a lead molecule to inhibit drug resistant strains, which was also confirmed by in-vitro analysis. Hence, based on the docking scores, S was identified as the most promising compound followed by S , S and S For other lead molecules, differential response against drug resistant isolates indicated that mutations in the drug resistant isolates might be accountable for variable degree of inhibition executed by the derivatives. ANNUAL REPORT n 96

99 6 Health Program Research and Contribution to the National Programme ANNUAL REPORT n 97

100

101 F.1) HIV Sentinel Surveillance Sponsor: National AIDS Control Organization Nodal Officers: Dr. Arun Risbud, Dr. Sheela Godbole NARI project team and SST Members: Dr.Amit Lokhande (Project Coordinator), Ms. Chitra Kadu, Mr.Umesh Saundankar, Dr.Megha Mamulwar, Mr. Rajesh Yadav, Mr Rajendra Yelgate, Mr Michael Pereira, Mrs Varsha Kale, Mrs Sangeeta Kulkarni, Dr Suchit Kamble,. Period: A. Surveillance among Antenatal Clinic Attendees [ANC] The National AIDS Research Institute is the Regional Institute (RI) for the 5 states of Maharashtra, Gujarat, Madhya Pradesh, Rajasthan and Goa and the 2 Union territories of Dadra Nagar Haveli and Daman Diu for ANC surveillance. th st The 14 round of ANC HIV sentinel surveillance (HSS) was completed at 194 sentinel sites between 1 January st 2015 and 31 March Three new ANC sentinel sites: Palghar, Maharashtra; Singrauli and Jhabua, Madhya Pradesh and one sub-site Sagwara, Dungarpur district, Rajasthan, were added this year. NARI conducted the regional training of trainers for State Surveillance Team (SST) members, key personnel from the respective State AIDS Control Societies (SACS) and other trainers. The state level site trainings were conducted by SACS through these trainers and trainings were monitored by NARI. Supervisory field visits were conducted by NARI through SST members (which included NARI team) to ensure survey quality. Double data entry was done at NARI for data forms. The samples collected were tested at pre-identified HIV State Reference Laboratories (SRL) for which the staff was trained at NARI. Quality control of HIV testing was done at the National Reference Laboratories (NRL) to which the SRLs are linked. The two-test method is followed nationally for HSS. SMS Based Reporting: The SMS based reporting system was used as a useful aid in monitoring the progress of HSS activities at individual sites. Sites were expected to text and report the number of samples collected daily to a pre-specified phone number and the data could be viewed on a daily basis by the Regional Institute (NARI) and at NACO. The challenge was to ensure that maximum number of sites complied with these reporting systems. Since there was a continued inadequacy in reporting, NARI initiated an SMS based intervention to remind sites to report sample collection on a daily basis. This was very useful and due to continuous vigilance and timely feedback from NARI, the percentage of sites reporting by SMS increased steadily and the percentage of SMS rejected by the server declined. Based on a review of the data reported via SMS, NARI team could review the progress of data collection and provide a real time feedback to site personnel by telephone and SMS. ANNUAL REPORT n 99

102 Figure 6.1: Number of sites reporting in MIS Data quality assurance processes are in progress at NARI. Therefore, only preliminary unpublished data of HSS 2015 are presented below. The overall prevalence for the region was 0.28%. Figure 6.2: HSS : HIV Prevalence (%) among Antenatal Clinic attendees in Western Indian States/UT We reviewed data for the states of Rajasthan, Gujarat, Maharashtra (except Mumbai), Mumbai, Goa, Dadra Nagar Haveli (DNH), Daman Diu (DD) and Madhya Pradesh. HIV prevalence was calculated as a ratio of infected population to population tested, for each site that was consistently sampled for three years and an average prevalence ratio was calculated for each state. To observe sampling variability and standard error of point estimate, 2 95% confidence intervals for mean were calculated. For measuring trends and their significance X test for trend was used. ANNUAL REPORT n 100

103 Figure 6.3: * Trends in ANC HIV Prevalence (%) based on consistent sites *Provisional findings a) Three year moving averages based on consistent sites are presented b) Data are presented separately for Mumbai district and Rest of Maharashtra Overall there did not seem to be a significant difference in HIV prevalence in these states and UT over the years 2010, 2013 and 2015 although there is some decline in prevalence. However when we tested for linearity in trends using chi-square test, Mumbai district (p=0.026) and Goa (p=0.0003) showed a statistically significant trend for the consecutive years; 2010, 2013 and 2015 F.2) Integrated Biological and Behavioural Surveillance (IBBS): Western India Region Study Period: March 2013 to Dec 2015 Funding: National AIDS Control Organization Nodal Officers: Dr. A.R. Risbud, Dr. Sheela Godbole NARI Study team: Ms. Sucheta Deshpande, Mrs. Neelam Joglekar, Dr. Pranil Kamble, Dr. Amit Lokhande Ms. Chitra Kadu, Mr.Umesh Saundankar, Mrs. Varsha Kale and NARI SST members- Dr. S Kamble, Mr. Rajesh Yadav, Mrs. Radhika Brahme A strategic focus of the National AIDS Control Program is to strengthen surveillance among High Risk Groups (HRG) and bridge population, given the geographically diverse low level and concentrated nature of the HIV epidemic in our country. The National Integrated Biological and Behavioral Surveillance (IBBS) is being implemented by National AIDS Control Organization (NACO), under the Ministry of Health and Family Welfare (MoHFW) to generate evidence on prevalence and risk behaviors among HRG (FSW, IDU, MSM and TG) and migrants (MIG) to support planning and prioritization of programme efforts at district, state and national levels. ANNUAL REPORT n 101

104 Specific objectives To measure and estimate the change in HIV-related risk behaviours and HIV prevalence at district and state level among key risk groups, between baseline and end line for NACP-IV To analyze and understand HIV related vulnerabilities and risk profiles among key risk groups in different regions, by linking behaviours with biological findings NARI has been playing an important role in N-IBBS, serving as a Regional Institute (RI) with oversight over N- IBBS in four states of India: Goa, Gujarat, Karnataka and Maharashtra. Various members from NARI teams have contributed to the core planning and supervisory activity of the National Working Group. The NARI region includes 54 survey domains - 16 Female sex workers (FSW), 16 Men having Sex with Men (MSM), four each from Transgender (TG) and Intravenous Drug Users (IDU) and 14 for migrants. IBBS is being implemented in 4 steps. Viz: 1. Pre-surveillance Assessment (PSA) [completed in 2014], 2 Sampling Frame Development (SFD) [Ongoing in ], 3. Main Survey, 4. Data Analysis The following activities were conducted during the reporting period: A. Trainings for IBBS Implementation 1. National and regional trainings: Figure 6.4: National and Regional Training of Trainers for N-IBBS NARI conducted three regional trainings for field research agency and State Surveillance Team (SST) members on study guidelines and monitoring techniques. In all, 32 SSTs were trained for N-IBBS and were involved in field training and monitoring of survey implementation. Table 6.1: Regional Trainings conducted by NARI Activity Organizer Period of training nd th Regional Training of Trainers (Pune) NARI 2 to 8 April 2014 th th Regional Training of Trainers (Pune) [SFD] NARI 25 to 29 Sept 2014 th th Regional Training of Trainers (Pune) [Main Survey] NARI 16 to 20 Feb 2015 ANNUAL REPORT n 102

105 After the National and regional trainings, the field research agency conducted field trainings where field teams implementing the survey were trained in study procedures and guidelines. Investigators from NARI attended these trainings in the capacity of expert trainers and observers. The trainings are being conducted in two phases. Viz: SFD component and main-survey component. First phase of training for SFD was conducted in four states between th October January 2015 and the second phase started on 20 March A. IBBS Implementation Sample Frame Development (SFD) was done during reporting year. SFD field work for HRG domains began in November 2014 and was completed in February The SFD for Migrant domains was still ongoing as of March The primary objective of SFD was to list all HRG/ MIG hotspots/ cruising sites and estimate the size of HRGs/ MIGs at each of the sites. SST members played a vital role in ensuring adherence to survey procedures and all domains were visited for supervision during the sample frame development (Table 6.2). NARI Investigators reviewed the SFD data for correctness and gave onsite orientation to field teams during visits. Table 6.2: Supervisory visits by SST members and NARI investigators Field Supervision Maharashtra Karnataka Gujarat Goa Total Total number of domains Number of supervisory visits The list of known (existing) hotspots obtained from secondary sources like NGO's and Targeted Intervention sites was made available to the field teams through a specially designed web system for IBBS; i.e. Integrated Information Management System (IIMS). During SFD, field teams updated existing hotspots as well as identified hotspots that were not listed in IIMS. HRG Size estimation was done using data collected during Sample Frame Development (SFD). Additionally secondary data on HRG sizes in different domains was also collected and reviewed. Figure 6.5 shows the distribution of hotspots identified during SFD by state and typology and previously unlisted hotspots were identified for all typologies. ANNUAL REPORT n 103

106 Figure 6.5: State wise distribution of HRG hotspots identified during SFD Across all typologies and domains estimated size from other sources was found to be higher than SFD estimates, except for few a domains in Karnataka. The data from SFD activity will be the basis for sampling for the main survey in F.3) National AIDS Control Organization supported PMC-NARI ART centre Nodal officer: Dr. Manisha Ghate A total of 3692 patients were registered for clinical care irrespective of their CD4 counts and 3242 have been initiated on ART till March 2015 at the NACO PMC NARI ART center. Of these, 702 patients were registered and 622 were enrolled between April 2014 and March The male: female ratio was 1.02: 1. Overall antiretroviral drug adherence of patients attending the centre was above 90%. The regimens administered to the patients are shown in figure 6.6. Of the enrolled patients in the reporting year, 4 (0.6%) patients were lost to follow-up, 125 (20.1%) were transferred out to other ART centres and 14 (2.3%) patients died. ANNUAL REPORT n 104

107 Figure 6.6: ARV drug regimens provided to patients at NARI ARTC Tuberculosis was the commonest opportunistic infection among patients attending the centre during April March A total 430 X-rays and 394 abdominal sonographies were done for ARTC patients during this year. F.4) Early infant diagnosis (EID) programme Nodal Officer: Dr. Dhanashree Jagtap Early Infant Diagnosis (EID) programme has been implemented across India from April 2010 and is continued till date by NACO with the aim of screening HIV exposed infants/children aged between 6 weeks to 18 months for HIV- 1 positivity. This is done by HIV-1 DNA PCR test on their Dried Blood Spot (DBS) samples collected at various ICTCs and the HIV status is confirmed by Whole Blood (WB) samples collected at various ART centres. These samples are received for HIV testing by the seven Regional Research Laboratories (RRLs) located in various states across India. The HIV DNA PCR Laboratory at NARI is one of the seven RRLs identified by NACO for HIV-1 DNA PCR testing of HIV exposed babies from Maharashtra excluding Mumbai and has completed five years of HIV-1 testing activity. Analysis of the samples tested during the five year period between April 2010 and March 2015 was undertaken. A total of 8963 babies were tested for HIV-1 DNA PCR, out of which 14.1% (1304) babies was found to be HIV-1 positive by DBS testing (Figure 6.7). ANNUAL REPORT n 105

108 Figure 6.7: Distribution of HIV positive babies tested for five years from April 2010 to March 2015 from Maharashtra under NACO EID programme at NARI Figure 6.8: Trends of HIV positivity among babies of HIV exposed mothers from Maharashtra tested under NACO EID programme at NARI from Trends of HIV-1 positive babies tested from 2010 to 2015 revealed that there was a decreasing trend of HIV positivity from 16.3% to 13.7%. F. 5) Laboratory support for the National AIDS Control Programme Investigator: Dr. Madhuri Thakar NARI as an Apex laboratory for HIV serology, that carries out a number of activities approved in NACP IV, which include providing External Quality Assessment (EQA) for HIV serology, CD4 count estimation, monitoring quality control for HIV diagnosis and CD4 count estimation, analysis of data, preparation of reports, development and distribution of SOPs and conducting training workshops and mentoring of SRLs. ANNUAL REPORT n 106

109 HIV serology: The network of laboratories consists of Apex lab, 12 NRLs, 118 SRLs and around 5000 ICTCs. EQAS is conducted twice a year wherein proficiency panels are distributed from Apex lab to NRLs, NRLs to SRLs and SRLs to ICTCs. Data collected in NACP III (2007 to 2012) from all these laboratories was compiled in the reporting year to assess the effects of EQA in HIV serology. It was observed that participation of SRLs in EQAS has improved from 98.3 to 99 % and discordance decreased from 3.4 to 1.6 %. The participation of ICTCs in EQAS has improved from 83 to 89 % and discordance decreased from 1.1% to 0.35 % (Figure 6.9) Figure 6.9: Participation and level of discordance in EQA for HIV serology The reasons for non participation of ICTCs were non-availability of technician (post vacant or the technician is on leave) (73 %), not communicating testing laboratories (20 %) and kits not available (4 %) Whereas the reasons for discordance were technical error (60 %), transcriptional error (10%), kit quality (10%), leakage of the sample during transport (2 %) and unknown reasons (18 %) (Figure 6.10) ANNUAL REPORT n 107

110 Figure 6.10: Reasons for discordance in results Confirmation of the HIV-2 diagnosis: Identification of types of HIV (HIV-1or HIV-2 or both) is important because it has implications on the treatment regimen to be provided at ART centers. Confirmation of HIV-2 diagnosis at 13 referral laboratories was continued in the reporting year. Under this activity, individuals found to be HIV-2 or HIV 1+2 reactive at ICTC are referred to HIV-2 one of the 13 referral laboratories for confirmation. Data on 710 samples (HIV-2 or HIV 1+2 reactive) collected from February 2013 to June 2014 was analyzed. Out of 710 samples, 78 were found to be HIV-1, 534 HIV-2 and 97 were HIV1+2. Ninety five percent concordance was observed between the results of ICTCs and HIV-2 referral lab in case of only HIV-2 reactive samples. This was observed especially when the ICTCs used two HIV-1 and HIV-2 differentiating kits. Hence it was recommended that the HIV-2 results confirmed by ICTCs using two differentiating kits can be given to the patients and so that the time for reporting could be reduced. However, in case of HIV1 and 2 reactive samples, the patients need to be referred to the referral lab through the designated ART centers to rule out the cross reactivity that might exist. CD4 count estimation: CD4 count is being used as a marker of disease progression in HIV infection. It is also used to decide when to initiate antiretroviral therapy and to monitor the effect of ART. Hence, it is of utmost importance that the testing laboratory should provide accurate and reproducible CD4 cell count estimation for making correct decisions in clinical settings. External Quality Assessment (EQA) scheme for CD4+ T cell count provides the means to attain the goals and objectives of the laboratory, determines means of preventing non-conformities. As an apex laboratory, NARI provides EQA to all CD4 estimating laboratories linked to NACO ART centers. In the reporting year, 241 centers participated in the CD4 EQA programme thrice a year. These centers used a variety of the equipments such as, FACSCount (N=154), FACSCalibur (N=24), Partec (Cyflow) (N=63), Beckman Coulter (N=1). Stabilized whole blood is used as a proficiency sample for CD4 count. Since the commercially available stabilized blood is very expensive, NARI developed in-house stabilized sample by stabilizing the whole blood using available ANNUAL REPORT n 108

111 stabilizing reagent. The stabilized samples were tested, validated and are now being used in the EQA for the last 5 years. This is brought down the cost of the EQA by 50%. The equipment wise performance of the centers was analyzed for the most commonly used equipments from EQA round 1 (2011) to EQA round 9(2014). (Figure 6.11 a,b,c) Figure 6.11: Equipment wise performance in the CD4 EQA CD4 EQA performance for FACSCount users(figure 8.8a), FACSCalibur users (figure 8.8b and Cyflow users(figure 8.8c). The X axis gives no of rounds and y axis shows no of with satisfactory and unsatisfactory performance ANNUAL REPORT n 109

112 The frequency of satisfactory performances for FACSCount and FACSCalibur users has not changed drastically over four years period of CD4 EQA. This is because the increase in no of equipments and turnover of the trained staff. Maximum satisfactory performances were observed in case of Cyflow users. The outlier performances overall indicated errors in pipetting, mixing of blood samples before use and improper gating in case of FACSCalibur. Accreditation as the proficiency testing (PT) providers for HIV serology and CD4 count estimation The institute has applied to NABL for accreditation as a PT provider unit under ISO for both HIV serology and CD4 count estimation. The pre assessment was completed in Sept 2014 and final assessment was completed in December The report is awaited. Consortium of National Reference Laboratories (NRL's) for kit quality testing: Consortium of four NRLs namely National AIDS Research Institute (NARI), Pune; National Center for Disease Control (NCDC), New Delhi; National Institute of Cholera and Enteric diseases (NICED), Kolkata and National Institute of Mental Health and Neurosciences (NIMHANS), Bengaluru, was established in 2009 as an initiative of NACO with NARI as the Secretariat. The consortium has been providing quality evaluated kits to be used in the National Program. Since its commencement in 2009, Consortium has evaluated approximately 600 ELISA and RAPID kits for HIV, HBV and HCV till date. A total of 174 kits were received for evaluation in the year 2014, of which 100 were for HIV, 15 for HBV and 59 for HCV diagnosis. ANNUAL REPORT n 110

113 7 Other Prevention ANNUAL REPORT n 111

114

115 G.1) Community engagement for Japanese Encephalitis (JE) and Acute Encephalitis Syndrome (AES) prevention and control: a component of ICMR's research cum intervention (RCI) project Investigator: Dr. Seema Sahay, Dr. M Gore, NIV, Pune; Mrs. Suvarna Sane; Mr. Girish Rahane; Mr. Narayan Panchal; Mrs. Neelam Joglekar Sponsors: ICMR-DHR Study Period: The aim of the project is to test an innovative, participatory model for better access to JE/ AES information, screening, and preventive health care for people residing in Gorakhpur district in Uttar Pradesh, India and at the capacity building of the community employing participatory research approaches. This is a case control intervention study. Qualitative and quantitative methods are being used to document the barriers experienced by various stakeholders in the process of community engagement, process of involvement of peer for health promotion and community capacity building for JE / AES control; changes in health knowledge and practices of the beneficiary community participating in this study and JE related health services offered to the community. In the qualitative component, 36 in-depth interviews and two focus group discussions to explore issues around community engagement for JE / AES prevention control were conducted in Gorakhpur. Community opinion leaders, members of village health and sanitation committees, health care providers, parents of vaccinated and non vaccinated children, parents of affected children, media representatives, and school teachers were included. The qualitative data collection was conducted from January 2014 to May Findings from the qualitative data are as follows: 1. Belief about causes: Respondents from the general community made a direct causal link between mosquito bites, 'dirty surroundings', 'not eating proper food', 'not maintaining cleanliness', contaminated water, foul food and Navki Beemari (colloquial term). The germ of this, the virus of this, is born from the dirt itself and that mosquitoes are born from the dirt. So that wherever the dirt, the formation of it [/disease/] is there only. [##VA/034## parent of vaccinated child] Navki Beemari was associated with local theories of causation, since few caregivers also stated that Navki Beemari in the villages is seen as an effect of bad karma, bad fate, bewitching, evil eye, or somebody 'scarring the child while he/she has gone to play'. We came with bad KARMA so we will suffer it, who else will suffer therefore people follow religion,... family goddess is upset, There was lack of God's worship by those people she [/the Goddess/] has come [/belief that evil soul enters the body/] on him/her. [##OS/035##, Traditional medical healer] a. Issues related to sanitation, hand washing and drinking water: Open defecation is a common practice; reasons being lack of toilets in the villages and other central locations such as market place, having no money to build toilets, no felt need to use toilets. b. Hand washing: A variety of materials are used for multiple personal and household purposes. Ash and detergent together serve the household need of dish washing. The ash obtained, as a by product from ANNUAL REPORT n 113

116 cooking fires, and mud, are considered more consistently and readily available and a low-cost alternative for soap for hand washing. No, ash is not costly, you only tell. This wood burns ash also like soap. [##V/018## parent of vaccinated child] c. Small/ shallow hand pumps' are the most common source of drinking water, their depth being feet. This poses a threat to the water getting contaminated by the nearby drains, contaminated ponds and toilets. The community prefers to use these hand pipes instead of government provided India mark II hand pumps having a deeper boring of feet. The reasons of preference are ownership issues (The household that has hand pump adjacent to it takes the ownership of public hand pump), taste and colour and due to its better accessibility. 2. Beliefs about vaccination: Certain misconceptions such as 'not suiting to family' or 'getting sick because of vaccine' emerge as a crucial impeding factor in achieving long term improvements in the vaccination status of the children. Distance and health seeking behaviour of the community, as well, emerged as barriers. 3. Reported personal protection modalities: Among the personal protection methods, besides commonly using mosquito nets, fumigating the house and surroundings in the evenings with dried neem leaves or mango leaves, husk, straw, or garbage was reported to be the most common way of controlling mosquitoes. 4. Beliefs that may limit access to proper treatment: Treatment seeking in the village is usually a community decision. Participants prioritized seeking treatment from 'private doctors' often the 'bangali doctors' (unqualified medical healers) or the faith based healers (quacks). Care seeking from these healers is more of a reflection of faith and some even rely on them while simultaneously seeking care from other providers. These practitioners also referred children to government health facility in case of severity or persistence of symptoms. Lower cost, traditional beliefs of disease causation, perception of disease severity are the factors affecting this behavior. Taking a child to a qualified medical practitioner seemed to incur more cost on the treatment. A child in case of minor fever, refusal to eat or drink is often looked at as affected by 'evil eye' and the child is taken to traditional healer to ward off evil. ANNUAL REPORT n 114

117 Figure 7.1: Pathways followed by the community for seeking health care 5. Need for community engagement and role of village heads Community engagement for JE/AES control through engaging school going children, government employees, general community, school teachers, and most importantly, village heads. Also, village leaders, by the general community were identified as major actors to promote healthy sanitation behaviours: This is a huge work, rather, getting this all done in the field requires huge effort and health system alone cannot take care of this so as we talked about schooling and use of schools as a setting to vaccinate them or even education that even school children... the school teachers or any other community leaders they can be of very good help in taking care of these activities by education [##OS/037##, Researcher] When the message comes in our Health department; then we can run the programme nicely in that village only through the head [/of the village/] because the head [/of the village /] knows all people, maximum people listen to him. [##HC/025##, doctor] Several village heads publicly announced that they had no knowledge about the JE vaccination schedule and that they were unaware about the vaccination program (ethnographic observation during a large community sensitization meeting). The community leaders, although identified as major actors in health promotion activities, had little knowledge that vaccine for preventing Navki Beemari was available. No. For Brain fever if any vaccine would have been there, then why would this disease happen? For this there is no medicine made. In medical doctor great are not able to catch, so what are we people. [##CL/004## community leader] Quantitative Component Between May 2014 to December 2014, a baseline survey was conducted in Gorakhpur (intervention arm) and Deoria (control arm) to assess the, knowledge, awareness, attitude, practices, coverage of immunization related to ANNUAL REPORT n 115

118 JE/ AES, understand drinking water sources, hand washing practices, use of soap, waste disposal, beliefs, gender specific practices regarding use of toilets, obstacles to use toilets, soap, health services availability, healthcare seeking behavior, perceptions, beliefs about causes of JE/AES, personal protection measures, knowledge and practices regarding JE/AES prevention. Preliminary findings show that the households have water facility and they are mostly shallow hand pumps in both control (Deoria) and intervention (Gorakhpur) arms (Table 7.1). Acceptance for Mark II hand pumps is very low. Open defecation is high. Table 7.1: Preliminary findings of sources of water at Deoria and Gorakhpur Source of water Deoria Gorakhpur Drinking water facility in house 1028 premises (96.8%) 995 (94.2%) Main source of drinking water for members of family (for most of months in the year) Shallow Hand pump/ tube well 877 (88%) 891 (85%) Mark II hand pump 120 (12%) 149 (42%) If the drinking water source is 101/120 (84%) 138/149 (93%) mark II hand pump, is it surrounded by a cement platform from all sides? How do you dispose off solid waste? Throw it in the nearby drains 96 (9.5%) 66 (6.9%) Throw it out of the window 143 (14%) 71 (7.4%) Safely dispose off in public dustbins 14 (1.4%) 29 (3%) In farm / field 738 (73%) 776 (81%) Bury in field 24 (2.4%) 22 (2.3%) The last time your child passed stools, what was done to dispose off the stools? Children always use toilet/latrine 48 (5%) 42 (4.5%) Put/rinsed into toilet/latrine 23 (2.3%) 22 (2.4%) Put/rinsed into drain or ditch 297 (29%) 268 (29%) Thrown into garbage 64 (6%) 64 (7%) Left in the open 542 (53%) 452 (49%) Washed away 10 (1%) 36 (3.9%) Buried 37 (4%) 42 (4.5%) Based on the qualitative study and the quantitative survey, a community engagement intervention module has been developed. ANNUAL REPORT n 116

119 Intervention module update: This module is translated into Hindi, to help local volunteers to bring in community mobilization to prevent and control JE/AES. Group intervention, is facilitated by the local peer volunteers; focuses on increasing community understanding of JE/AES, immunization, hygiene, sanitation and on catalyzing individual and community level action to address it. The main purpose of the community engagement module is on changing behaviors, inculcating hygienic practices and capacity building of local communities to help them identify their problems, develop strategies to tackle them and build unity to work together as a group and community. This interactive module helps local communities to participate in task-oriented activities in health and hygiene promotion context. It uses drawings, games and stories to stimulate discussions and facilitate problemsolving and decision-making. Intervention has been initiated in January 2015 and is planned to be delivered in 3 blocks of Gorakhpur district. A total of 187 of 600 peers have been selected from across one block (Bhathath) and 89 community engagement meetings have been conducted. Various stakeholder meetings are being routinely conducted with village leaders, DMO, CMO to gain support in the intervention activities. There was no intervention activity for control group. Local peer volunteer facilitating an intervention meeting Mobilizing the village community and bringing community consultative approaches for JE/AES prevention and treatment is a prerequisite for the success of the program. Education and community involvement through village leaders/ opinion leaders, school teachers for immunization might increase acceptance and uptake of vaccine and early case reporting. Hygienic behavior practices need to be encouraged among community and issues towards easy access of safe drinking water needs to be addressed. An end line survey is being planned to study the impact of this intervention. ANNUAL REPORT n 117

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121 8 Appendices ANNUAL REPORT n 119

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123 NARI services NARI Clinical Research and Services NARI has six clinics located in different parts of Pune and Pimpri Chinchwad area. These NARI clinics are located within National Institute of Virology and Kotnis Gadikana hospital and in Chest Hospital, Aundh in the PMC area. In the PCMC area, clinics are located within NARI main campus at Bhosari and Talera municipal hospital. These clinics are situated at convenient locations facilitating accessibility easy for patients availing clinical care. These clinics work in collaboration with local health authorities like Pune Municipal Corporation and Pimpri Chinchwad Municipal Corporation. NARI is operating an ART center through the clinic at Model Colony, which has been approved by the Division of AIDS (DAC) for registration and provision of free ART to the patients. A total of 3692 patients were registered for clinical care irrespective of their CD4 counts and 3242 have been initiated on ART till March Figure 8.1: Clinical care services PRE TEST Counseling HIV Diagnosis Medical Services POST TEST Counseling STI diagnosis and Care Couples Counseling ALL NARI CLINICS Chemoprophylaxis and PEP ART Adherence Counseling Referrals to DOTs centres, Specialty Clinics and Inpatient Care Nutrition and Growth Assessment Psychosocial support including counselling Positive Prevention Figure 8.2: Total number of patient visits at NARI clinics Figure 8.3: Total number of new patients at NARI clinics ANNUAL REPORT n 121

124 Investigations done at NARI clinics: 3411 patients who were diagnosed HIV positive at our clinics were given post test counseling in the year April14- March 15. Routine investigations like Complete Blood Count, Blood Sugar level, Liver Function tests, Renal Function tests and special investigations like CD4 count, Plasma viral load, HBsAg are done for proper management of patients. The figure shows the number of other non -hematological investigations done at NARI clinics (Figure 8.5). Figure 8.4: Non-hematological investigations done at NARI clinics All NARI clinic attendees receive primary care and treatment on a cost-free basis and are offered opportunities to participate voluntarily in various research studies ongoing at NARI. If they are willing and eligible for NARI research studies, they are enrolled and followed up at the respective clinics following all good clinical practice (GCP) and ethical guidelines. The number of patients screened, enrolled and followed up for various studies, projects and cohorts at the NARI clinics during are shown in below table Table 8.1: Cohort of patients at NARI clinics Name of research study /project Screening Enrollment Follow-up Visits NARI Care Protocol Long Term Non Progressors Cohort NACO Supported PMC-NARI ART Center ANNUAL REPORT n 122

125 Table 8.2: Study participants in research studies at all clinics Name of research study /project Screening Enrollment Followup ACTG 5273: Options For Second line Effective Combination Therapy ACTG 5282 Trial: Test and treat for prevention of cervical pre-cancer HPTN-052 Trial: Early ART prevents transmission of HIV-1 among discordant couples study Characteristics of cellular effectors mechanism against HIV at cervico - vaginal level in Indian women ( CVL Study ) Nevirapine versus Efavirenz based highly active antiretroviral therapy regimens in antiretroviral naïve patients with HIV and Tuberculosis infection in India NA NA 53 NA NA 11 NA NA NA NA 724 Dual infection Viral isolation study DUIN Study Anal sex behavioral sub-study 0 7 NA Genetic Variants of MMP gene study Genetic susceptibility APOBAC3G gene study Centre for excellence in HIV-TB co-infection mirna Study : Utility as biomarkers of disease progression and therapy failure Longitudinal Cross- sectional NA Comparison of phenotypic and functional characteristics of M. tuberculosis specific T cells in active and latent tuberculosis Detection of antibodies to HIV 1/2 in oral fluid (Orachek) NA Pediatric Disclosure Study NA Pediatric Neuro-Cognitive Study Comparative evaluation of ViveSt and DBS specimens for HIV-1 drug resistance genotyping (ViveST DBS) The role of natural killer T lymphocyte (NKT) and regulatory T (Regs) cells in HIV infection (NKTreg ) Use of herbal drugs and dietary supplement by PLHA 9 18 NA NA Study of Th 17 cells during long term non-progression of HIV (Th-17) 0 44 NA Comparative performance evaluation of the Cavidi ExaVir Load version 3 with the Abbott HIV-1 Real Time PCR assay using plasma specimens NA Antibody-dependent cellular cytotoxicity (ADCC) a new frontier in vaccine research (ADCC) Genetic susceptibility of xenobiotic drug metabolizing enzyme gene in ARV associated hepatotoxicity ANNUAL REPORT n 123

126 NARI laboratories services NARI laboratories provide services to the patients and study participants attending NARI clinics and ART centre. Additionally various quality assurance activities in support of the National program are also conducted by NARI laboratories as a service to the program. Table 8.3: Summary of testing services provided Name of the laboratory Name of the assay No of samples processed Serology ELISA, Rapid and Western blot for anti-hiv antibodies 4524 Virology HIV-1 viral load / HIV-1 RNA estimation 2615 Microscopy/ Culture and antibiotic sensitivity 97 HBsAg 583 Microbiology HCV 89 Syphilis serology 1230 Molecular assays (NG/CT, HPV) 1254 Mycobacterium ZN staining 16 Clinical Pathology Hematology 9714 (including NARI and Biochemistry 6984 Model Colony lab) Urine analysis 558 Urine pregnancy tests 391 Cervical cytology 1289 Immunology CD HIV-PCR laboratory (EID program) HIV-1 DNA PCR 2009 The Library and Information centre Library and Information Centre of NARI continues to support the research activities of staff and students working in the institute. Keeping in mind the research priorities of researchers at NARI, the library has built up a decent collection of about 2400 books on Virology, Immunology, Cell Biology, Microbiology, Biochemistry, Sexually Transmitted Diseases, Infectious Diseases, Social and Behavioral sciences, Epidemiology, Biostatistics and Clinical Medicine. Additionally, it has 14 thesis and 38 MSc dissertations of students, who had completed research in NARI. The library has access to the NML-ERMED Consortium, which provides access to about 370 biomedical journals online. Being an ICMR institute, it has access to J-Gate Plus database, which provides indexing and abstracting services to about 2629 journals subscribed by all the ICMR institutes clubbed together. The online platform J-Gate Plus also allows scientists to place request within ICMR institutes for journal articles required by them. Besides, the library has access to the Cochrane Database provided by ICMR. The library receives about 40 print journals on gratis. Access to the online journals both subscribed and free journals, database, and consortium ANNUAL REPORT n 124

127 has been provided through the website of the library (to be accessed through Even the Online Public Access Catalogue (OPAC) has also been made available over LAN through a link on the website. The library has eight computers dedicated for users to search resources over internet and access online resources provided by NARI and ICMR individually or through collaborations. To promote awareness against HIV/AIDS among general population, it has posters, CDs and DVDs. The library provides reference and information services on demand and anticipation as well. It provides reprography services and citation analyses on demand. It also provides audio-conferencing facilities. ANNUAL REPORT n 125

128 Major events/training/workshops NATIONAL AIDS RESEARCH INSTITUTE v Training workshop for Gorakhpur team on Community engagement in JE and AES prevention and control. NARI, Pune. [4-8th August 2014] th th A 5 day's training workshop between 4 and 8 August 2014 was conducted at NARI, Pune for implementation of community engagement intervention under RCI project, at three blocks of Gorakhpur district in Uttar Pradesh. Four participants from Gorakhpur and two from NARI participated in this workshop. Dr. Tandale from NIV, Pune, gave a talk on Vector borne Disease with special focus on Japanese encephalitis (JE). Dr. Suchit Kamble talked on the 'Importance of prevention and early treatment: Symptoms, prevention and treatment, vaccination'. Dr.R.R. Gangakhedkar explained the clinical symptoms and ways to identify JES/ AES patients. Study protocol training was given by Dr. Seema Sahay. Participants received training on community development and communications skill through interactive activities. Dr. Nita Mawar trained the participants in bioethics, specifically bringing out issues and concerns pertaining to field work in rural settings in India. Mrs. Suvarna Sane discussed at length about the survey which was to be taken up in Gorakhpur and Deoria district in the state of UP. She brought the focus of the team on clusters, households, skips and the sample size of the survey. Mrs. Joglekar took an interactive session on informed consent process especially at field level. Mrs. Radhika Brahme provided hands on training on data entry, checks and reports. The community team of NARI along with the partner NGOs gave the participants a real demonstration of peer program in the community in Pune city. Mr. Mahesh Kharat and team shared NARI's experience with community engagement program. They also provided description of documenting process indicators and managing the peer volunteers. Extensive training on each chapter of community engagement module for JE/ AES prevention and control was done through lectures, activities and question answer sessions. nd v Swachh Bharat Abhiyaan' [2 December 2014] Mahatma Gandhiji communicated a quintessential message to the nation through his efforts to educate people around him about cleanliness. He wished to see a "Clean India" where people work hand in hand to th make the country clean. In order to realize Mahatma Gandhijis dream of clean India by his 150 birth anniversary in 2019, our honorable Prime Minister Shri Narendra Modi launched the 'Swachh Bharat Abhiyaan' as a mass movement on 2nd October 2, 2014 and asked people from all walks of life to help in successful implementation of this mission. Swaachh Bharat Abhiyaan exhorts people to devote 100 hours every year towards the cause of cleanliness. In this connection Swatch Bharat Abhiyaan campaign was inaugurated at National AIDS Research Institute nd (NARI), Pune, on 2 October Dr. R.S.Paranjape, Director, National AIDS Research Institute (NARI) presented pledge to all NARI employees at 9.45am. Dr. Suchit Kamble, Scientist D & Working Chairperson of Hygiene Committee announced the action plan for Swachh Bharat Campaign at NARI for next six months. ANNUAL REPORT n 126

129 There was an interactive session to identify and prioritize the unclean and improper sites in the NARI campus for immediate and long term actions. This was followed by Kar Sewa by all employees. A Public Lecture was organized for awareness of personal hygiene, environmental cleanliness and impact on health. Dr. Suchit Kamble, gave a talk on this topic in Hindi. A quiz programme also was arranged for the audience and they were asked questions based on cleanliness & Gandhiji's thought. st Following activities were conducted under Swachh Bharat abhiyaan at NARI till 31 March 15 E-Reporting system for each sections of the Institute has been developed and implemented the for monthly reporting and action taken report of cleanliness activities. Developed model for improving the cleanliness & hygiene at schools. Prepared the Information and Education material on cleanliness and hygiene for dissemination. A poster competition on Hygiene & cleanliness related disease was organised between to for NARI employees & their children. 12 employees and one sibling of an employee participated. three best posters were awarded along with consolation prize to the sibling of the employee during NARI annual foundation day celebration on The best poster was published in NARI Bulletin. These posters are displayed in NARI Campus on the occasion of Open House on World AIDS Day and Science day. Technique of proper hand washing and its usefulness were discussed during these open house days with general public. Patients were given instructions on proper disposal of face masks, precautions to be taken while coughing, sneezing to avoid transmission of swine flu to others. th On the occasion of Gandhijis death anniversary 30 January 2015 a speech competition on Thoughts of Gandhiji was organised. This was preceded by a short talk on Microorganisms isolated from Mobile /Smart phones of Health care providers Following activities are ongoing under Swachh Bharat Abhiyaan at NARI: Organization of monthly Kar seva event at NARI campus from Oct Regular meeting of committee members every fortnight to review the activities of Swachh Bharat. Maintain log chart for cleaning of spots vulnerable to unhygienic conditions. Lectures and Seminars on Hygiene and Swachh Bharat Abhiyaan ANNUAL REPORT n 127

130 Digitization and Shredding activity to make work spaces spruced up. Innovatiove and implementable Model proposals for Swacch bharat abhiyan are developed by employees of NARI and one of that is nominated and send further to ICMR once in every two months. Each department sends e-report of cleanliness th activities at 28 of every month based on which Final report is prepared by committee. ATR on Hon'ble PM Instructions and ATR on th Swachh Bharat Campaign is prepared and uploaded on ICMR intranet portal on 28 of every month. v Training of staff members from National Public Health Laboratory, Nepal under WHO SEARO Training Support Coordinator: Dr. Smita Kulkarni, Scientist F Training conducted by: Dr. Vijay Nema, Scientist C ; Dr. Dhanashree Jagtap, cientist-b ; Mrs. Sushama Jadhav, Technical Officer; Ms. Sharda Gadhe, Technical Officer Dates: December 2014; Venue: Department of Molecular Biology, NARI Nepal has succeeded in keeping the prevalence of HIV to % in the age group of years. However, a steady introduction of new strains from various countries, as well as repeated cross-border transmission with neighboring states and expanded interventions for earlier detection and treatment of HIV among returning migrants and their families could be analyzed to observe the transmission dynamics in Nepal. This required a molecular approach for characterization of the isolates from Nepal. NARI was contacted for training National Public Health Laboratory, Nepal staff in HIV-1 sub-typing and on HIV-1 DNA PCR testing by Roche Amplicor v1.5 method. The training included the following: HIV-1 sub-typing Amplification of gag/env gene from HIV using nested PCR Confirmation of amplification using gel electrophoresis Purification of amplified PCR products using PCR cleanup kit Setting up of sequencing reaction Purification of sequencing reaction product Performing sequencing using genetic analyzer Analysis of sequences using online software to determine subtype of the HIV strain HIV-1 DNA PCR testing by Roche Amplicor v1.5 method ANNUAL REPORT n 128

131 DNA extraction from dried blood spots (DBS) & whole blood samples PCR amplification of gag gene Hybridization and detection by ELISA Analysis of results and data interpretation v Trainings Conducted for HIV Sentinel Surveillance (HSS) and Integrated Biological and Behavioral Surveillance (IBBS) As a designated Regional institute for the Western Indian states, NARI conducted several trainings for State AIDS Control Society personnel as well as Microbiologists and Community Medicine Faculty from the medical colleges who were identified as State Surveillance Team members in these states. The states /UT included: Maharashtra, Gujarat, Goa, Madhya Pradesh, Rajasthan, Diu & Daman, Dadra Nagar Haveli and Mumbai. rd th 1) Regional Training of Trainers for ANC HSS. : 3-4 Dec 2014, Chief Trainer: Dr Sheela Godbole. It was attended by 22 SACS personnel and 36 SST members. Training included - a) The Broad strategy, action plan & key changes in methodology of HSS b) Discussion and finalization of training plans of zonal trainings for all states/uts and c) Monitoring visit schedules. 2) HSS SRL Training workshop on HIV testing and reporting and quality management for HSS th conducted by NARI at Pune on 10 Feb 2015 and it was attended by 37 participants. (4 TO, 21 LTs and 12 NARI members) nd th th th 3) Regional Training of Trainers (IBBS-SFD): 2-8 April 2014 and Sept 2014 Chief Trainers: Dr Sheela Godbole (NARI), Dr Yujwal Raj (NACO) No. of Participants: 57 (14 SACS, 22 SSTs and 4 National Working Group (NWG) members and NARI members) and 73 (9 SACS, 18 SSTs, 12 NWG, 19 FRA IMRB and NARI members) 4) Regional Training of Trainers for Main IBBS Survey: 16th to 20th Feb 2015 No. of Participants: 76 (13 SACS, 27 SSTs, 2 NWG, 19 Field Research Agency (FRA) members and NARI members) attended the training. ANNUAL REPORT n 129

132 List of Publications 1. Alary M, Banandur P, Rajaram SP, Thamattoor UK, Mainkar MK, Paranjape R, Adhikary R, Duchesne T, Isac S, Moses S. Increased HIV prevention program coverage and decline in HIV prevalence among female sex workers in south India. Sex Transm Dis. June 2014;41 (6): Alexander M, Chidrawar S, Deshpande S, Paranjape R. Significant Prevalence of Heterosexual Anal Sex among FSWs in India Highlights the Need for Specifically Tailored Interventions for HIV Prevention. AIDS Res Hum Retroviruses. Oct 2014; (Suppl1): A273-A Alexander M. CD4:CD8 Ratio and Non AIDS Defining Events in Virally Suppressed HIV Infected Patients: Need to Look Beyond CD4+ T cell Counts. HIV/AIDS Res Treatment Open J. 2015;2 (1): e12- e Antwal M, Gurjar R, Chidrawar S, Pawar J, Gaikwad S, Panchal N, Kale V, Thakar M, Risbud A, Tripathy S. Clinical profile of HIV infected patients attending a HIV referral clinic in Pune, India. Indian J Med Res. Aug 2014;140 (2): Chandhiok N, Joshi SN, Gangakhedkar R. Acceptability of oral and topical HIV chemoprophylaxis in India: implications for at-risk women and men who have sex with men. Sex Health. July 2014;11 (2): Closson EF, Sivasubramanian M, Mayer KH, Srivastava A, Safren SA, Anand VR, Gangakhedkar R, Mimiaga MJ. The other side of the bridge: exploring the sexual relationships of men who have sex with men and their female partners in Mumbai, India. Cult Health Sex. Aug 2014;16 (7): Danga SK, Sahay S, Jadhav AJ, Patange RP, Patel RB. A Study Of Contraceptive Utilization Practices Among Married Couple From Rural Area Of Satara District: A Community Based Observational Study. Int J Inf Res Rev. Feb 2015;2 (2): Dhayarkar S, Chadha M, Tripathy A, Jadhav S, Deshmukh N, Mehendale S. Outbreak of waterborne hepatitis E, Pune, Maharashtra, India, Indian J Comm Family Med. Jan-Jun 2015;1 (1): Ghate M, Zirpe S, Gurav N, Paranjape R, Rewari B, and Gangakhedkar R. Transfer out Patients Receiving Antiretroviral Therapy from Programme Clinic: A Potential Leak in the HIV Treatment Cascade. World J AIDS. Dec 2014;4 (4): Ghate MV, Marcotte TD, Rangnekar H, Mayer R, Sakamoto M, Mehendale SM. Depressive Symptoms in Spouses of HIV Infected Individuals: A Study of HIV Uninfected Caregivers in Pune, India. Open J Pyschiatry. Jan 2015;5: Ghate MV, Zirpe SS, Gurav NP, Rewari BB, Gangakhedkar RR, Paranjape RS. Retention of antiretroviral naïve patients registered in HIV care in a program clinic in Pune, India. Indian J Sex Transm Dis. July 2014;35 (2): Godbole S, Sane S, Kamble P, Raj Y, Dulhani N, Venkatesh S, Reddy DC, Chavan L, Bhattacharya M, Bindoria S, Kadam D, Thakur S, Narwani P, Pereira E, Paranjape R, Risbud A. Predictors of Bisexual ANNUAL REPORT n 130

133 Behaviour among MSM Attending Intervention Sites May Help in Prevention Interventions for This Bridge to the Heterosexual Epidemic in India: Data from HIV Sentinel Surveillance. PLoS One. Sep 2014;9 (9): e Godbole SV, Mane AK, Chidrawar SR, Katti UR, Kalgutkar S, Athavale PV, Pawar JS, Ratnaparkhi MM, Alexander M, Risbud AR, Paranjape RS. Prevalence of anal human papillomavirus infection among HIVinfected women from India. J Acquir Immune Defic Syndr. Nov 2014;67 (3): e111-e Goswami P, Medhi GK, Armstrong G, Setia MS, Mathew S, Thongamba G, Ramakrishnan L, George B, Singh RK, Paranjape RS, Mahanta J. An assessment of an HIV prevention intervention among People Who Inject Drugs in the states of Manipur and Nagaland, India. Int J Drug Policy. May 2014; (pii): S0955- S Grinsztejn B, Hosseinipour MC, Ribaudo HJ, Swindells S, Eron J, Chen YQ, Wang L, Ou SS, Anderson M, McCauley M, Gamble T, Kumarasamy N, Hakim JG, Kumwenda J, Pilotto JH, Godbole SV, Chariyalertsak S, de Melo MG, Mayer KH, Eshleman SH, Piwowar-Manning E, Makhema J, Mills LA, Panchia R, Sanne I, Gallant J, Hoffman I, Taha TE, Nielsen-Saines K, Celentano D, Essex M, Havlir D, Cohen MS; HPTN 052-ACTG Study Team. Effects of early versus delayed initiation of antiretroviral treatment on clinical outcomes of HIV-1 infection: results from the phase 3 HPTN 052 randomised controlled trial. Lancet Infect Dis. April 2014;14 (4): Hu P, Herningtyas EH, Kale V, Crimmins EM, Risbud AR, McCreath H, Lee J, Strauss J, O'Brien JC, Bloom DE, Seeman TE. External quality control for dried blood spot-based C-reactive protein assay: experience from the indonesia family life survey and the longitudinal aging study in India. Biodemography Soc Biol. Apr 2015;61 (1): Jha UM, Raj Y, Venkatesh S, Dhingra N, Paranjape RS, Saggurti N. HIV epidemic among men who have sex with men in India: national scenario of an unfinished agenda. HIV/AIDS Research and Palliative Care. 2014;6 (): Kalokhe AS, Potdar RR, Stephenson R, Dunkle KL, Paranjape A, Del Rio C, Sahay S. How well does the world health organization definition of domestic violence work for India? PLoS One. Mar 2015;10 (3): e Kohli R, Mohanty S, Jadhav P, Paranjape R. Need to Raise the Level of Knowledge of PrEP and Rectal Microbicides (RM) among Men Who Have Sex with Men (MSM). AIDS Res Hum Retroviruses. Oct 2014;10 (Suppl1): A113-A Kulkarni AG, Paranjape RS, Thakar MR. Higher Expression of Activating Receptors on Cytotoxic NK Cells is Associated with Early Control on HIV-1C Multiplication. Front Immunol. May 2014;5: Kulkarni SV, Bala M, Bhattacharya J, Risbud A. Detection of mutations in mtrr gene in quinolone resistant strains of N.gonorrhoeae isolated from India. Indian J Med Microbiol. Apr-Jun 2015;33 (2): Mahajan B, Reddy GSM, Bagul NM, Mane A, Mahajan A. Identification of candida species using hichrome agar in hiv-seropositive patients with oral candidiasis. J Dent Res Sci Develop. 2014;1: ANNUAL REPORT n 131

134 23. Mahajan SD, Gaekwad A, Pawar J, Tripathy S, Ghate M, Bhattacharya J, Singh HO, Schwartz SA, Paranjape R, Gangakhedkar R. Cardiac Morbidity in an HIV-1 Lipodystrophy Patient Cohort Expressing the TNF-α-238 G/A Single Nucleotide Gene Polymorphism. Curr HIV Res. 2015;13 (2): Mahajan SD, Tripathy S, Bhattacharya J, Gaekwad A, Pawar J, Ghate M, Paranjape R, Gangakhedkar R. Antiretroviral Therapy and Genetic Predisposition: Co-factors Contributing to The Lipodystrophy Syndrome. J AIDS HIV Res. Aug 2014;6 (7): Mane A, Gujar P, Chandra J, Lokhande R, Dhamgaye T, Ghorpade S, Risbud A. Pneumocystis jirovecii Infection and the Associated Dihydropteroate Synthase (DHPS) and Dihydrofolate Reductase (DHFR) Mutations in HIV-Positive Individuals from Pune, India. Mycopathologia. Feb 2015;179 (1-2): Maselko M., Joshi R., Prescotta M., Talwar G., Kulkarni S. and Pastey M. Basant, a polyherbal topical microbicide candidate inhibits different clades of both CCR5 and CXCR4 tropic, lab-adapted and primary isolates of HIV Virus-1 infection in vitro. J Virol Antivir Res. 2014;3: Mawar N, Katendra T, Bagul R, Bembalkar S, Vedamurthachar A, Tripathy S, Srinivas K, Mandar K, Kumar N, Gupte N, Paranjape RS. Sudarshan Kriya yoga improves quality of life in healthy people living with HIV (PLHIV): results from an open label randomized clinical trial. Indian J Med Res. Jan 2015;141 (1): Mayston R, Patel V, Abas M, Korgaonkar P, Paranjape R, Rodrigues S, Prince M. Determinants of common mental disorder, alcohol use disorder and cognitive morbidity among people coming for HIV testing in Goa, India. Trop Med Int Health. Mar 2015;20 (3): *Mhatre DR, Mahale SD, Khatkhatay MI, Desai SS, Jagtap DD, Dhabalia JV, Tongaonkar HB, Desai MP, Dandekar SP, Varadkar AM. Development of an ELISA for spsp94 and utility of the spsp94/spsa ratio as a diagnostic indicator to differentiate between benign prostatic hyperplasia and prostate cancer. Clin Chim Acta. June 2014;436C: Muralidharan S, Kumar A, Gangakhedkar R. Oral Health Status and Treatment Needs of Institutionalized HIV Positive and Non- HIV Positive Children and Adolescents Between 3 To 17 Years of Age in Pune Maharashtra, India: A Comparative Study. Int J Oral Care Res. Jan-Mar 2015; 2(7): Murugesan V, Makwana N, Suryawanshi R, Saxena R, Tripathi R, Paranjape R, Kulkarni S, Katti SB. Rational design and synthesis of novel thiazolidin-4-ones as non-nucleoside HIV-1 reverse transcriptase inhibitors. Bioorg Med Chem. June 2014;22 (12): Nagarajan K, Godbole S, Deshpande S, Paranjape R. Correlates of Voluntary HIV Testing and Collection of Test Results among Male Clients of FSWs in Three States of India. AIDS Res Hum Retroviruses. Oct 2014; (Suppl1): A112-A Nagarajan K, Sahay S, Mainkar MK, Deshpande S, Ramesh S, Paranjape RS. Female sex worker's participation in the community mobilization process: two distinct forms of participations and associated contextual factors. BMC Public Health. Dec 2014;14 (1): Nain S, Sharma A, Singh H, Paliwal S. Recent Advances in use of Semicarbazones as Anticonvulsant ANNUAL REPORT n 132

135 Agents: A Review. J Biomed Ther Sci. 2015;2 (1): Nema V, Nair R. Metagenomic analysis of diarrheal stool samples of HIV infected individual and HIVuninfected individual using 16SrDNA sequencing. Indian J Med Microbiol. July 2014;32 (3): Patel P, Ansari M, Bapat S, Thakar M, Gangakhedkar R, Jameel S. The microrna mir-29a is associated with human immunodeficiency virus latency. Retrovirology. Dec 2014;11: Patil RT, Gupta RM, Sen S, Tripathy SP, Chaturbhuj DN, Hingankar NK, Paranjape RS. Emergence of Drug Resistance in Human Immunodeficiency Virus Type 1 Infected Patients from Pune, India, at the End of 12 Months of First Line Antiretroviral Therapy Initiation. ISRN AIDS. April 2014: Patil S, Choudhary I, Chaudhary NK, Ringe R, Bansal M, Shukla BN, Boliar S, Chakrabarti BK, Bhattacharya J. Determinants in V2C2 region of HIV-1 clade C primary envelopes conferred altered neutralization susceptibilities to IgG1b12 and PG9 monoclonal antibodies in a context-dependent manner. Virology. July 2014; C: Praveena P., Godse A., Pawar M., Kulkarni S. and Sudarsanam D. In vitro anti-hiv activity studies on Enicostema littorale (Lam) Raynal whole plants. World J Pharm Res. Sep 2014;3 (6): Rajaram SP, Banandur P, Thammattoor UK, Thomas T, Mainkar MK, Paranjape R, Adhikary R, Duchesne T, Ramesh BM, Isac S, Moses S, Alary M. Two cross-sectional studies in south India assessing the effect of an HIV prevention programme for female sex workers on reducing syphilis among their clients. Sex Transm Infect. Nov 2014;90 (7): Ramanathan S, Nagarajan K, Ramakrishnan L, Mainkar MK, Goswami P, Yadav D, Sen S, George B, Rachakulla H, Subramanian T, Paranjape RS. Inconsistent condom use by male clients during anal intercourse with occasional and regular female sex workers (FSWs): survey findings from southern states of India. BMJ Open. Nov 2014;4: e Ramesh S, Mehrotra P, Mahapatra B, Ganju D, Nagarajan K, Saggurti N. The effect of mobility on sexual risk behaviour and HIV infection: a cross-sectional study of men who have sex with men in southern India. Sex Transm Infect. Sep 2014; 90 (6): Safren SA, Biello KB, Smeaton L, Mimiaga MJ, Walawander A, Lama JR, Rana A, Nyirenda M, Kayoyo VM, Samaneka W, Joglekar A, Celentano D, Martinez A, Remmert JE, Nair A, Lalloo UG, Kumarasamy N, Hakim J, Campbell TB; PEARLS (ACTG A5175) Study Team. Psychosocial predictors of non-adherence and treatment failure in a large scale multi-national trial of antiretroviral therapy for HIV: data from the ACTG A5175/PEARLS trial. PLoS One. Aug 2014;9 (8): e Safren SA, Thomas BE, Mayer KH, Biello KB, Mani J, Rajagandhi V, Periyasamy M, Swaminathan S, Mimiaga MJ. A pilot RCT of an intervention to reduce HIV sexual risk and increase self-acceptance among MSM in Chennai, India. AIDS Behav. Oct 2014;18 (10): Shete A, Thakar M, Mehendale SM, Paranjape RS. Is Prime Boost Strategy a Promising Approach in HIV Vaccine Development? J AIDS Clin Res. April 2014;5: Shiboski CH, Chen H, Ghannoum MA, Komarow L, Evans S, Mukherjee PK, Isham N, Katzenstein D, ANNUAL REPORT n 133

136 Asmelash A, Omozoarhe AE, Gengiah S, Allen R, Tripathy S, Swindells S; AIDS Clinical Trials Group Network and Oral HIV/AIDS Research Alliance. Role of oral candidiasis in TB and HIV co-infection: AIDS Clinical Trial Group Protocol A5253. Int J Tuberc Lung Dis. June 2014;18 (6): Shivakoti R, Christian P, Yang WT, Gupte N, Mwelase N, Kanyama C, Pillay S, Samaneka W, Santos B, Poongulali S, Tripathy S, Riviere C, Berendes S, Lama JR, Cardoso SW, Sugandhavesa P, Tang AM, Semba RD, Campbell TB, Gupta A; for the NWCS 319 and PEARLS Study Team. Prevalence and risk factors of micronutrient deficiencies pre- and post-antiretroviral therapy (ART) among a diverse multicountry cohort of HIV-infected adults. Clin Nutr. Feb 2015; (pii): S Thakar M, Patil R, Shukre S, Bichare S, Kadam P, Khopkar P, Ghate M, Paranjape R. Genital Tumor Growth Factor-β1 Levels in HIV-Infected Indian Women Are Associated with Reduced Levels of Innate Antimicrobial Products and Increased HIV Shedding. AIDS Res Hum Retroviruses. July 2014;30 (7): Yadav D, Chakrapani V, Goswami P, Ramanathan S, Ramakrishnan L, George B, Sen S, Subramanian T, Rachakulla H, Paranjape RS. Association Between Alcohol Use and HIV-Related Sexual Risk Behaviors Among Men Who Have Sex with Men (MSM): Findings from a Multi-Site Bio-Behavioral Survey in India. AIDS Behav. July 2014;18 (7): Ahead of Publication 1. Ghate M, Mehendale S, Meyer R, Umlauf A, Deutsch R, Kumar R, Thakar M, Risbud A, Kulkarni S, Sakamoto M, Alexander T, Franklin D, Letendre S, Heaton RK, Grant I, Marcotte TD. The effects of antiretroviral treatment initiation on cognition in HIV-infected individuals with advanced disease in Pune, India. Journal of Neurovirology. Published online 7 March [Ahead of Print] 2. Kantor R, Smeaton L, Vardhanabhuti S, Hudelson SE, Wallis CL, Tripathy S, Morgado MG, Saravanan S, Balakrishnan P, Reitsma M, Hart S, Mellors JW, Halvas E, Grinsztejn B, Hosseinipour MC, Kumwenda J, La Rosa A, Lalloo UG, Lama JR, Rassool M, Santos BR, Supparatpinyo K, Hakim J, Flanigan T, Kumarasamy N, Campbell TB, Eshleman SH; AIDS Clinical Trials Group (ACTG) A5175 Study Team. Pretreatment HIV Drug Resistance and HIV-1 Subtype C Are Independently Associated With Virologic Failure: Results From the Multinational PEARLS (ACTG A5175) Clinical Trial. Clin Infect Dis May 15;60(10): doi: /cid/civ102. Epub 2015 Feb Shivakoti R, Yang WT, Gupte N, Berendes S, Rosa A, Cardoso SW, Mwelase N, Kanyama C, Pillay S, Samaneka W, Riviere C, Sugandhavesa P, Santos B, Poongulali S, Tripathy S, Bollinger RC, Currier JS, Tang AM, Semba RD, Christian P, Campbell TB, Gupta A; New Work Concept Sheet 319 and The Prospective Evaluation of Antiretrovirals in Resource-Limited Settings Study Team. Concurrent Anemia and Elevated C-Reactive Protein Predicts HIV Clinical Treatment Failure, Including Tuberculosis, After Antiretroviral Therapy Initiation. Clin Infect Dis Mar 31. pii: civ265. [Epub ahead of print] ANNUAL REPORT n 134

137 4. Shivakoti R, Christian P, Yang WT, Gupte N, Mwelase N, Kanyama C, Pillay S, Samaneka W, Santos B, Poongulali S, Tripathy S, Riviere C, Berendes S, Lama JR, Cardoso SW, Sugandhavesa P, Tang AM, Semba RD, Campbell TB, Gupta A; for the NWCS 319 and PEARLS Study Team. Prevalence and risk factors of micronutrient deficiencies pre- and post-antiretroviral therapy (ART) among a diverse multicountry cohort of HIV-infected adults. Clin Nutr Feb 10. pii: S (15)00045-X. doi: /j.clnu [Epub ahead of print] 5. Sinha A, Chandhiok N, Sahay S, Deb S, Bharat S, Gupta A, Bhatt S, Kanthe V, Kumar B, Joglekar N, Paranjape R, Mehendale S. Male circumcision for HIV prevention in India: emerging viewpoints and practices of health care providers. AIDS Care May 22:1-3. [epub Ahead of Print] *Work carried out at NIRRH, Mumbai Review articles/perspectives/book chapters 1. R.R. Gangakhedkar. Prevention of Parent-to-Child Transmission of HIV: The Indian Roadmap in The Positive Child Has a Right to a Positive Life. Action Report on Pediatric HIV in India.Ed. Dr. Mamatha Lala, Mumbai, Sahay S, Kumar AB. Tribal India: A Review of Health Care Delivery in India Health and Tribes. In: TribalIndia: Challenges and Opportunities. Chapter 2, Sarup Publishers Pvt Ltd, New Delhi. 2015, PP Sahay S. Mitigating Stressors of HIV by Involving the significant others. In: HIV/AIDS in India: A Public Health Approach on Contemporary Trends. Edited by Sibnath Deb and Archana Shukla. Pp Sahay S, Verma A, Mehendale S. Male Circumcision for HIV Prevention: Care Providers' Attitude. In: HIV/AIDS in India: A Public Health Approach on Contemporary Trends. Edited by Sibnath Deb and Archana Shukla. Pp ANNUAL REPORT n 135

138 Training provided to students NATIONAL AIDS RESEARCH INSTITUTE NARI invites applications from the eligible candidates pursuing M.Sc. in any branch of Life Sciences/MA Social sciences/m.pharma/msw/m.e./m.tech. /M.B.B.S and in penultimate year of their degree, to undertake short term project as a part of their course curriculum at this institute. Additioanlly some students are trained as part of other curricula like MPH. Sr. No Name of Student Title of the Project D.N.Sri.Anannya Amity Institute Of Virology And Immunology, Amity University Uttar Pradesh, Noida Jampala Harshitha Vignan s Foundation for Science, Technology and Research, Deemed University Bhavana Rahangdale Rajiv Gandhi Institute of IT And Biotechnology, Bharati Vidyapeeth, Pune Glory Francis VIT University, Vellore Mandar Bhutkar NIV, Pune Manju Nair II year M.Sc. (Biomedical Genetics), VIT University, Vellore (Jan - Jun 2015). Comparison of Evolution of vpu and env genes of HIV-1: A Longitudinal Study Probing mutations and phylogenetic linkages in rrs, rpob, inha & katg genes of TB clinical isolates In-silico analysis of probable mutations affecting structure of isocitrate lyase in Mycobacterium tuberculosis and their impact on drug binding Screening the types and number of nucleotide changes in gyra gene and its correlation with phenotypic resistance in clinical isolates of M.tuberculosis Evaluation of HSV-2 Replication kinetics in T-cell lines Characterization of genetic signatures of BST -2 gene of progressors and slow progressors of HIV infection Department / Supervisor Dr. Vijay Nema Dr. Vijay Ne ma Dr. Vijay Nema Dr. Vijay Nema Dr. Smita Kulkarni Dr. Dhanashree Jagtap 7 Dr. Prasad Bogam IIHMR and Johns Hopkins University India Master in Public Health ( MPH ) Practicum Training Dr. Sheela Godbole ANNUAL REPORT n 136

139 Institutional Committees SCIENTIFIC ADVISORY COMMITTEE Dr. Bhan M. K. (Chairman) Secretary, Department of Biotechnology, New Delhi Dr. Bharat Shalini Professor, School of Health Systems Studies, Tata Institute of Social Sciences, Mumbai Dr. Chauhan P. S. Formerly Head, Cell Biology Division, Bhabha Atomic Research Centre, Mumbai Dr. Deshpande Alka Retired Professor and Head, Department of Medicine, J.J. Hospital, Grant Medical College, Mumbai Dr. Khan M. E. Social and Behavioral Sciences, Population Council, New Delhi Dr. Mehra N. K. Professor and Head, Department of Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, New Delhi Dr. Pandav C. S. Professor & Head, Centre for Community Medicine, All India Institute of Medical Science, New Delhi Dr. Pandey Arvind Director, National Institute of Medical Statistics, New Delhi Dr. Phadke M. A. Ex. Vice Chancellor, Maharashtra Health Sciences University, Nasik Dr. S. Rajsekaran NACO Consultant (ART Quality Management), Chennai Prof. V. Ravi Head, Department of Neurovirology, National Institute of Mental Health and Neuro Sciences, Bangalore Dr. V. K. Subburaj Department of AIDS Control and Director General, National AIDS Control Organization, New Delhi Dr. Shriniwas Retired Professor, All India Institute of Medical Sciences, New Delhi Dr. Tripathy S. P. Former Director-General, Indian Council of Medical Research Dr. Arora Rashmi, for DG, ICMR Scientist 'G' and Chief, ECD Division, Indian Council of Medical Research, New Delhi Dr. Paranjape R. S. (Member Secretary) (Upto 31st Oct. 2014) Director and Scientist 'G', National AIDS Research Institute, Pune Dr. Gangakhedkar R. R. (Member Secretary) (From 1st Dec. 2014) Director In-Charge and Scientist 'F', National AIDS Research Institute, Pune ANNUAL REPORT n 137

140 ETHICS COMMITTEE MEMBERS Dr. Padbidri V. S. (Chairperson) Director Research, KEM Hospital Research Center, Sardar Moodliar Road, Rasta Peth, Pune Mrs. Abhyankar Madhuri Director, SOFOSH, Pune Mrs. Chinchkar Varsha NGO link worker Dr. Gupte M. D. ICMR Chair Epidemiology, Ex Director, National Institute of Epidemiology, Chennai Mrs. Hiremath Nirmala TARA Mobile Creches, Pune Dr. Kakrani A. L. Prof. and Head, Department of Medicine, Dr. DY Patil, Medical College, Pimpri, Pune Dr. Pandit Vijaya Department of Pharmacology, Bharati Vidyapeeth Medical college, Pune Ms. Pawar Manisha Community Representative, Pune Prof. Pal J. K. Dept. of Biotechnology, University of Pune Dr. Sagade Jaya ILS Law College, Law College Road, Pune Justice Sathe S. R. (Retired) Dr. Shrotri Aparna Dr. Worlikar Pratibha st Dr. Paranjape R. S. Member Secretary (up to 31 October 2014) Director and Scientist 'G', National AIDS Research Institute, Pune st Dr. Gangakhedkar R. R. Member Secretary (From 1 December 2014) Director in-charge and Scientist 'F', National AIDS Research Institute, Pune COMMUNITY ADVISORY BOARD MEMBERS Dr. V. N. Karandikar (Chairperson); Director, Health Sciences, Bharati Vidyapeeth Deemed University, Bharati Vidyapeeth Bhavan, Pune Mr. George Swami, Ms. Ratna Acharya, Ms. Lata Mane, Dr. Kalpana Mutatkar, Ms. Prema Kamble-Khandagale, Adv. Surekha Potphode, Ms. Madhuri Abhyankar, Ms. Nirmala Hiremath, Dr. Madhu Bamboli, Dr. P. V. Mahajan, Dr. Sanjeevani Kulkarni, Mr. Abhay Pawar, Ms. Anuradha Karkare, Dr. Prakash Onawale, Dr. Mallika Mistri, Mr. Shridhar Loni, Ms. Vinaybala Mehta, Ms. Mangala Patil, Ms. Meena Kurlekar, Mr. Vilas Chaphekar, Ms. Tejaswi Sevekari, Mr. Irfan Karnalkar, Ms. Swati Chondhe, Adv. Asim Sarode, Ms. Mrunal Pendse, Dr. (Mrs.) Amrita Bagga, Dr. Sunita Chaudhari, Mr. Manoj Pardeshi, Ms. Sushila Joseph, Ms. Leena Rajan, Dr. Sagar Pathak, Mr. Mahendra Gaikwad, Ms. Medha Ranade, Adv. (Mrs.) Kailash Nevagi, Ms. Arati Pendse, Ms.Ashwini Tambe, Dr. Shakera Inamdar ANNUAL REPORT n 138

141 LIST OF STAFF MEMBERS Director-in-Charge & Scientist 'F' Dr. Raman R. Gangakhedkar M.B.B.S., D.C.H., M.P.H. From 01/12/2014 onwards Director-in-Charge & Scientist 'F' Scientist 'G' Scientist 'F' Scientist 'E' Scientist 'D' Scientist 'C' Director and Scientist 'G' Dr. Nita Mawar Dr. Ramesh S. Paranjape M.Sc., Ph.D. M.Sc., Ph. D. From 01/11/2014 to 30/11/2014 Up to 31/10/2014 Retired Retired Dr. Arun R. Risbud M.B.B.S., M.D., M.P.H. (Up to 14/10/2014) Dr. Seema Sahay M.Sc., Ph. D. Dr. Smita S. Kulkarni M.Sc., DMLT, Ph. D Dr. Manisha V. Ghate Dr. Madhuri R. Thakar Dr. Sheela V.Godbole M.B.B.S., D.C.H M.Sc., Ph. D. M.B.B.S., M.D., PGDEPI Dr. Suchit Kamble M.B.B.S., M.D. Dr. Vijay Nema Dr. Ashwini V. Shete Dr. Arati K. Mane M.Sc., Ph. D. M.B.B.S., M. D. M.B.B.S., M. D. Dr. Vandana Saxena Dr. Sampada D. Dhayarkar M.Sc., Ph.D. M.B.B.S. Scientist 'B' Dr. Hari Om Singh Mr. P. Murugesan Dr. Serena D' Souza M.Sc., Ph. D. M. S. W., P.G.D.C M.Sc., Ph. D. Dr. Dhanashree Jagtap Dr.Swarali Kurle Mrs. Radhika Brahme M.Sc., Ph. D. M.Sc. Ph.D. M.C.M. M.Phil, Dr. Shakuntala T.S. Dr.Abhijit V. Kadam Dr. Megha Mamulwar M.B.B.S., M.D. M.B.B.S., M.D. M.B.B.S., M.D. Technical Officer-B Mr. Rajesh Yadav M.Sc., M.Phil. (from 03/11/2014) ANNUAL REPORT n 139

142 Technical Officer-A Dr. Sangeeta V. Kulkarni Mrs. Sushma D. Jadhav Mrs. Medha M. Deshpande M.Sc., Ph. D. M. Sc. B. Com., M.A. Mrs. Rajani D. Bagul Mrs. Bharati A. Mahajan M. S. W. B. Sc., D. M. L.T. Technical Assistant Mrs. Suvarna S. Sane Mrs. Varsha A. Kale Mr. Mycal Pereira M. Sc. M. Sc. M. Sc. Ms. Shilpa C. Bembalkar Mr. Tumanlal Katendra Mrs. Shubhangi A. Bichare M. Sc. M. Sc. B. Sc., D. M. L.T. Mr. Rajendra P. Yelgate Mr. Amit P. Nirmalkar Ms. Ipsita A. Choudhary M. Sc. M. Sc. M. Sc. Mr. Ajit A. Patil Mr. Vilas P. Pawar Mrs. Shradha Y. Gaikwad B. Sc., D. M. L.T. B. Sc., Dip. X-ray & B.Sc., D.M.L.T. ECG Tech. Ms. Nilam P. Gurav Mrs. Madhuri D. Chandane Mr. Narayan U. Panchal M. Sc. M. Sc., D. M. L. T. B. Com., M. S. W. Mrs. Sunita Kumbhar Mr. Girish Rahane Ms. Suvarna Sonavale Dip. in GNM M. A., B.Ed. M.Sc. Mr. Kumar Vaidya M. Pharm Technician-C Mr. Karna S. Sahis Mrs. Ujjwala M. Ghule Mr. Vijay N. Chauware ( Up to 30/11/2014) Dip. in GNM B. Sc., D. M. L. T. Mrs. Dipali K. Kale Mrs. Vaishali Chimanpure Mrs. Varda S. Ganu B. Sc., D. M. L. T. B. Sc., D. M. L. T. B. Sc., D. M. L. T. Technician-C Mrs. Pallavi Birdawade M.Sc. D.M.L.T. Technician-B Mr. Vinayak R. Chavan Technician-A Mr. Mahibub B. Attar Assistant Library & Information Officer Dr. S. M. Shahabuddin M.L.I.Sc. Ph.D Mr. Vilas Bharde Mr. Jeevan K. Waghela ANNUAL REPORT n 140

143 Administrative Officer Mrs. A. Susila M.Com. Private Secretary Mrs. Swati M. Salunke B. A. Technical Officer A (Engg. Support) Mr. Sattar D. Gadwale D.C.E. Technical Assistant / Junior Engineer (Engineering Support) Mr. Vishal Bhosale Dip. in EE (DEE) Administrative Staff Mrs. Savita B. Sonawane Mr. Biji George Mrs. Varsha V. Malwadkar B.Com Matriculation B.Com Mrs. Asmita A. Kadhe Mr. Sunil R. Awchare Mr. Pawan V. Shivankar M.Com B.Com, M.B.A. M. Com. Multi-tasking Staff Mrs. Satyabhama M. Kamble Mrs. Bharati S. Nindankar Mr. Sachin V. Dhondge (Up to 30/06/2014) Mr. Amol Raut Mr. Chandrashekhar Mane SENIOR PROJECT STAFF Dr. Mallika Alexandar Dr. Anjali Joglekar Dr.Usha Katti Dr. Jyoti Pawar Dr. Dhanwanti Inamdar Dr. Prachi Athavale Dr. Amit Lokhande Dr. Shraddha Bapat Mrs. Sujata Zankar Medical Manager SAE coordinator & Clinician (RS-II) CRS coordinator & Clinician (RS-II) CRS coordinator & Clinician (RS-II) CRS Pathologist (RS-II) CTU Pathologist (RS-II) Project Coordinator (RS-II) CRS coordinator & Clinician (RS-I) Administrative Officer ANNUAL REPORT n 141

144 BUDGET : Intra-mural Funds Head Plan (Rs. in Lakhs) Non Plan (Rs. in Lakhs) Total (Rs. in Lakhs) Grants -in-aid-salaries Grants -in-aid- General Acquisition of Capital Assets and Other Capital Expenditure (Equipment) Acquisition of Capital Assets and Other Capital Expenditure (Capital) Total Extra-mural Funds Sponsor Amount (Rs. in Lakhs) National International Total ANNUAL REPORT n 142

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