Conducting HIV Treatment Research in Resource Poor Pontiano Kaleebu MD PhDUganda Virus Research InstituteENTEBBEEric Arts PhDJoint Clinical Research Centre  CFAR Uganda Laboratory CoreKAMPALA: Conducting HIV Treatment Research in Resource Poor Pontiano Kaleebu MD PhD Uganda Virus Research Institute ENTEBBE Eric Arts PhD Joint Clinical Research Centre CFAR Uganda Laboratory Core KAMPALA


Summary of presentation: Summary of presentation History of UVRI and JCRC and what is involved in setting up basic research in a developing country Examples of successfully conducted basic research and clinical trials Some challenges/frustrations Tips for collaboration


Past research practices: Sophisticated research has been limited to developed countries Mostly observational epidemiology and clinical care in Africa Very little basic science research Laboratories were collection and shipment sites A few centres of excellence Basic science not attractive due to limited training and experience of the native population Trained African scientists and clinicians migrate to better opportunities Past research practices


Why Basic research in RPC: Why Basic research in RPC Address local problems Partnership and trust Capacity building More affordable in the long-term


Uganda Virus Research Institute (UVRI): Uganda Virus Research Institute (UVRI) Established 1936 as Yellow Fever Research Institute, funded by the Rockfeller Foundation 1950- East African Virus Research Institute 1972- Ida Amin, political turmoil, break from the East African Community- became the UVRI and the departure of most staff 1986- New Government and AIDS Epidemic


Achievements: Achievements Identification of 124 strains of new viruses mostly from mosquitoes, e.g Chikungunya, O’nyong-nyong, West Nile etc Research on Yellow fever transmission Currently, the national reference lab for HIV screening; Primary WHO/UNAIDS lab for HIV characterization; Measles, Polio and Arboviruses reference centre Collaboration with a number of programmes; Rakai project, MRC, CDC, and IAVI


The Rakai project: The Rakai project Started 1988: A multi-displinary programme Looking at HIV epidemiology and factors affecting spread including STDs Now looking at HIV-1 molecular epidemiology and preparations for vaccine trials Collaboration of Columbia University, Johns Hopkins, Makerere University, UVRI and Walter Reed Army Institute of Research


MRC programme on AIDS in Uganda: MRC programme on AIDS in Uganda Started in 1988: To investigate the epidemiology of HIV-1 infection and natural history of HIV-1 associated disease in rural Uganda. Multi-disciplinary (clinical, epidemiology, social science, basic science, community activities) Over 130 publications


CDC-Uganda: CDC-Uganda Activities started in 1994 - HIV-1 molecular epidemiology studies 2000- GAP-Research in care and HIV-1 prevention Capacity building


ICSC-World Laboratory: ICSC-World Laboratory International Center for Scientific Culture (ICSC) - World Laboratory Provided considerable equipment for containment level 3 lab and molecular lab Work on etiopathogenesis of KS


Some MRC selected publications : Some MRC selected publications S. McAdam et al. AIDS 1998; 12:571-579 A.M. Elliott et al. Int J. Tuber Lung Dis 1999; 3; 239-247 Kaleebu P et al. AIDS Res Human Retroviruses 2000;16:621-625 Jones G.J. et al. AIDS 2001;15: 1657-1663 Kaleebu P. et al. AIDS 2001, 15:293-299 Hadson W.S. et al. AIDS 2001,15:467-475 Kaleebu P. et al. JID 2002;185:1244-50 Cao H. et al. JID 2003;187:887-95 RECENT ABSRTRACTS (Barcelona conference): MoOrA 1055 Kebba A. et al. (Paper accepted JID) TuPeA4352 Nankya I.L. et al. ThPeA7097 Rutebemberwa A. et al. (Paper submitted ARHR)


Important new studies in MRC: Important new studies in MRC DART (Development of ARV therapy for Africa). Multi-centre study: Uganda (JCRC,UVRI) and Zimbabwe (Harare) 3000 patients with CD4 <200 First randomisation CMO vs LCM Second randomisation (Continuous vs STI) Drugs: First line Combivir, tenofovir, Abacavir, Niverapine,


Basic science studies under DART: Basic science studies under DART Base-line resistance Monitor virological response Development of resistance in CMO and LCM Development of resistance in STI Efficacy of a new combination (Tenofovir/combivir) Immune reconstitution under STI/Continuous


IAVI-UVRI: IAVI-UVRI International AIDS Vaccine Initiative Started 2001 Phase I vaccine trial Following international ethical standards Data handling and QA Laboratory-QA/QC; Core lab in London Immunogenicity- Elisopot and flowcytometer


A phase I vaccine trial: A phase I vaccine trial NIH/CWRU/JCRC/UVRI Safety and immunogenicity of Live recombinant ALVAC HIV vCP205 (Aventis Pasteur) funded by NIH First vaccine trial in Africa Trial followed GLP and GCP Accreditation-CAP, WHO etc Capacity building Immunogenicity and safety data (Cao et al. JID 2003; 187: 887-95)


Techniques at UVRI: Techniques at UVRI Viral culture and isolation (HIV, Measles, Polio) Intracellular cytokine assay by flow cytometry Elispot assays Whole blood cytokine assays Real time PCR Proliferation assays Polymerase Chain Reaction and Heteroduplex mobility assay Viral load assays (Roche and branched DNA) Standard Chemistry, heamatology assays UK certified laboratory, external QA programmes


Local training: Local training Our programme has trained PhDs in Immunology and virology- students perform their work at UVRI (Register with Imperial College, London). MSc of Makerere University


Frustrations: Frustrations Political instability (Uganda currently stable) Lack of local financial resources Lack of trained personnel and brain drain Power and Internet services (at UVRI now much better) Logistics e.g Reagent shipments Equipment mantainance/service contracts Ethics and regulatory issues Literature/information


Tips for collaboration: Tips for collaboration Openness and equal partnership Understand local research needs Balance sponsor and local interests Respect and understand local culture


Summary: Summary With good training and investment in infrastructure, good clinical and basic research can be conducted in RPC We need partners in research with plans to develop capacity


Slide21: Uganda – Case Western Reserve University Collaboration Initiated by the late Frederick Robbins (Nobel Laureate) in 1988 Over 50 research projects have been completed or are ongoing Involves direct collaboration between at least 20 US and Ugandan principle investigators, currently employees over 200 Ugandans on site, and is supported by approximately $8 million/yr Collaboration projects include: studies of HIV-1 pathogenesis vertical transmission neurodevelopment of HIV-infected children HIV and tuberculosis interaction IND phase I/II trials of promising immunoadjuvants for TB treatment HIV seroincidence preparation for AIDS vaccines the first phase I HIV preventive vaccine trial on the African continent (recombinant canarypox vector vaccine - ALVAC HIV vCP205)


Slide22: Grants and Contracts Supporting Uganda-Case Collaboration Tuberculosis Research Unit – NIAID, NIH contract Fogarty AIDS International Training and Research Program – NIAID, NIH grant Hormonal contraception and the risk of HIV acquisition – NICHD, NIH contract Center for AIDS Research – NIAID, NIH grant Oral Combination Chemotherapy in AIDS-Related Non-Hodgkin’s Lymphoma in Africa - NCI, NIH grant Six R01 grants and several other awards from various agencies


Slide23: JOINT CLINICAL RESEARCH CENTRE Kampala, Uganda Director – Dr. Peter Mugyenyi


Slide24: CWRU/UHC CFAR International Core (CCIC) Proposed structure and organization


Slide25: Joint Clinical Research Centre, Mengo Hill, Kampala, Uganda CFAR-JCRC Virology Laboratory JCRC Medical Clinic


Slide26: Joint Clinical Research Centre Main site of laboratory analyses on patient samples for CFAR and other Case-related activities


Slide27: CFAR/JCRC Virology Laboratory


Slide28: TBRU/JCRC Immunology Laboratory


Slide29: Ugandan-CWRU collaboration   CWRU CFAR-JCRC HIV/Virology Core Laboratory


Slide30: LABORATORY EQUIPMENT AND INFRASTRUCTURE Approximately 1000 sq. feet in the Joint Clinical Research Centre. The centre is located on Mengo Hill, Kampala, Uganda. Laboratory space is shared with CTL/Immunology laboratory (Director – Huyen Cao, MD) The HIV/virology laboratory contains all of the necessary equipment for virus tissue culture and molecular virology/biology analyses (e.g. PCR amplifications, DNA sequencing, radioactive reverse transcriptase assays). Equipment purchased by CFAR is highlighted. HIV/Virology Visible Genetic Automated DNA sequencer Sorval refridgerated micro-ultracentrifuge Agarose and polyacrylamide gel electrophoresis set-upS PCR thermocyclers CTL/Immunology Beta scintillation counter Flow cytometry ELISPOT reader


Slide31: CFAR Uganda laboratory core services


Slide32: CURRENT PROJECTS USING CFAR FACILITIES Antiretroviral drug resistant in Ugandans infected with non-clade B HIV-1 isolates and treated with antiretrovirals PI – Eric Arts, Cissy Kityo, Peter Mugyenyi Funded by Fogarty International AIDS Training Grant Prevelance of nevirapine and zidovudine resistance substitutions in mothers and their infants PI – Chris Whalen, Francis Bajunirwe, and Eric Arts Funded by Fogarty International AIDS Training Grant Analysis of viral fitness in Zimbabwean and Ugandan women newly infected with non-clade B HIV-1 PI – Eric Arts Contract with NICHD, NIH and R01 from NIAID, NIH HIV-1 shedding in genital secretions PI – Robert Salata Contract with NICHD, NIH Prevelance and subtyping of HPV in HIV-infected Ugandans PI – Robert Salata, Fred Kambugu Contract with NICHD, NIH


Slide33: Identification and analysis of KSHV in HIV-infected Ugandans treated for NHL PI – Rolf Renne, Scot Remick, Edward Mbidde Funded by NCI, NIH Impact of tuberculosis on HIV-1 heterogeneity and disease progression PI – Zahra Toossi, Eric Arts, Harriet Mayanja-Kizzi Funded by NHLBI, NIH and NIAID, NIH Investigating the sensitivity of HIV-1 isolates in Uganda to inhibition by RANTES derivatives and an analyses of CCR5 polymorphisms PI – Michael Lederman, Peter Zimmerman, Eric Arts Grant from NIAID, NIH Measuring the expression of level of Beta defensins in vaginal tract of HIV negative and positive women PI – Miguel Quinones-Mateu Grant from NICHD, NIH CURRENT PROJECTS USING CFAR FACILITIES


Slide34: HIV-1 DRUG RESISTANCE


Slide37: Drug resistant mutations in JCR55 L74V, K103N, M184V in RT None in PR Predicted phenotypic drug resistance High level resistance to ddI, ddC, 3TC, EFV, NVP Moderate level resistance to ABC, DLV


Slide38: Emergence of ARV resistance in ARV-treated Ugandans at the JCRC Over 50% of ARV-treated patients at the JCRC harbor drug resistant virus


Slide39: EFFECTS OF TB ON HIV-1 DIVERSITY


Slide40: Effect of TB on HIV-1 heterogeneity in co-infected Ugandans HIV-1 diversity was 2 to 3-fold greater in blood of HIV/TB as compare to that of HIV-infected Ugandans matched for CD4 cell count Collins et al., J. AIDS 2000 Increased HIV-1 diversity upon co-infection with TB is due to expansion of HIV-1 in the TB-affected organ (pleura or lung) and migration of diverse HIV-1 clones from these compartments into the blood. Collins et al., J. Virol. 2002


Slide41: Impact of TB on HIV-1 heterogeneity Active M.tb replication leads to immune activation, release of cytokines and chemokines that can upregulate HIV-1 replication Collins et al., AIDS Rev. 2002


Slide42: HIV-1 FITNESS AND DISEASE PROGRESSION


Slide43: Worldwide Distribution of HIV-1 subtypes


Slide44: HIV-1 fitness increases during disease progression Patients that are characterized as slow progressor tend to have less fit virus than patients than patients that progress rapidly to disease Quinones-Mateu et al., J. Virol. 2000 Subtype C is now dominating the epidemic Ugandans infected with subtype D appear to progress more rapidly than those infected with subtype A Kaleebu et al., JID 2002 Can different subtypes alter the course of disease progression? Transmission? The epidemic? Relationship between HIV-1 subtype, fitness and disease progression


Slide45: Intrasubtype competitions B vs. B C vs. C Paired T test, p > 0.5 Paired T test, p > 0.5 There is no significant difference in intrasubtype competition involving B or C isolates Y axis wins X axis wins Ball et al., J. Virol. 2003


Slide46: Intersubtype competitions C vs. B C vs. B Paired T test, p < 0.0001 B wins C wins Subtype C isolates are significantly less fit than subtype B isolates Ball et al., J. Virol. 2003


Slide47: HIV-1 shedding from the genital tract To compare shedding of HIV-1 from the genital tracts to hormonal contraceptive use To determine if HIV-1 shedding/viral loads is related to HIV-1 subtype


Slide48: HIV-1 fitness study: comparing HIV-1 fitness to disease progression in patients infected with subtypes A, C, and D HIV-1 isolates To compare HIV-1 fitness upon primary infection Analyze the changes in fitness during disease progression Relate fitness and disease progression to the infecting HIV-1 subtype Determine if hormonal contraceptive use affects HIV-1 fitness in the genital tract


Processing of GS/VF Samples: Processing of GS/VF Samples Endocervical/Vaginal Swabs (RNA Later) Aliquot and Freeze –70oC Viral loads Blood (Red Top) Aliquot Serum Freeze –70oC HSV Testing (JCRC) Syphilis Testing (JCRC) Repository Sample processing (pelleting and RNA extraction)


Processing of GS/VF Samples: Processing of GS/VF Samples Blood (EDTA) Isolate PBMCs (Buffy coat) Viral Propagations Co-culture DBS Repository Measurement CD4+/CD8+ lymph. (JCRC) Harvest virus for viral fitness Repository Extract DNA PCR and sequencing Subtyping Clone for fitness studies


Slide51: EDTA blood PBMC isolation DNA extraction External PCR with EnvB-ED14 DNA sequencing with fluorescent tagged primers and using the Visible Genetic Sequencer Nested PCR with E80-E125 HIV-1 subtyping


Slide52: *Likely lab contamination **complex of A,E,G, and J. Subtype A in env and gag Phylogenetic analyses


GS Sample Analysis of fitness: GS Sample Analysis of fitness Blood (EDTA) Isolate PBMCs (Buffy coat) Viral Propagations Co-culture Harvest virus for viral fitness Repository Extract DNA Clone for fitness studies Determination of virus titer Competitions with reference isolates


Slide57: Personal observations after 6 years of research in Uganda Setting up a laboratory is extremely time consuming, difficult, but incredibly rewarding However, establishing strong collaborations with pre-existing facilities (if present) and strengthening their capabilities avoids unnecessary duplication Communication is the key to success. Obvious comment but difficult to accomplish Do not take anything for granted! Know your wattage, voltage, current, stablizers, power backups, frequency, step-down converters, and a very good electrician


Slide58: There are no Amersham, Invitrogen, BioRad or any other biotech even remotely interesting in installing a freezer Establish good shipping routes and don’t expect anything to arrive on time Where can I develop my radiographic film? How do get a radiation license? And who the hell will ship it to me? What do I do when my thermocycler dies or a bulb burns out on my plate reader? Always send a Ugandan to buy from local merchants Another yeast contamination, time to formaldehyde bomb this place When all else fails there is always Nile Special beer and Waragi