Conducting HIV Treatment Research in Resource Poor Pontiano Kaleebu MD PhD Uganda Virus Research Ins

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Conducting HIV Treatment Research in Resource
Poor Pontiano Kaleebu MD PhDUganda Virus
Research InstituteENTEBBEEric Arts PhDJoint
Clinical Research Centre CFAR Uganda Laboratory
CoreKAMPALA
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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

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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

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Why Basic research in RPC

• Address local problems
• Partnership and trust
• Capacity building
• More affordable in the long-term

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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

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Achievements

• Identification of 124 strains of new viruses
  mostly from mosquitoes, e.g Chikungunya,
  Onyong-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

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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

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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

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CDC-Uganda

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

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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

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Some MRC selected publications

• S. McAdam et al. AIDS 1998 12571-579
• A.M. Elliott et al. Int J. Tuber Lung Dis 1999
  3 239-247
• Kaleebu P et al. AIDS Res Human Retroviruses
  200016621-625
• Jones G.J. et al. AIDS 200115 1657-1663
• Kaleebu P. et al. AIDS 2001, 15293-299
• Hadson W.S. et al. AIDS 2001,15467-475
• Kaleebu P. et al. JID 20021851244-50
• Cao H. et al. JID 2003187887-95
• 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)

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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 lt200
• First randomisation CMO vs LCM
• Second randomisation (Continuous vs STI)
• Drugs First line Combivir, tenofovir, Abacavir,
  Niverapine,

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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

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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

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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)

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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

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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

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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

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Tips for collaboration

• Openness and equal partnership
• Understand local research needs
• Balance sponsor and local interests
• Respect and understand local culture

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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

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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)

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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-Hodgkins Lymphoma in Africa - NCI, NIH
  grant
• Six R01 grants and several other awards from
  various agencies

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JOINT CLINICAL RESEARCH CENTRE Kampala,
Uganda Director Dr. Peter Mugyenyi
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CWRU/UHC CFAR International Core (CCIC) Proposed
structure and organization
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Joint Clinical Research Centre, Mengo Hill,
Kampala, Uganda
CFAR-JCRC Virology Laboratory
JCRC Medical Clinic
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Joint Clinical Research Centre Main site of
laboratory analyses on patient samples for CFAR
and other Case-related activities
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CFAR/JCRC Virology Laboratory
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TBRU/JCRC Immunology Laboratory
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Ugandan-CWRU collaboration   CWRU
CFAR-JCRC HIV/Virology Core Laboratory
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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

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CFAR Uganda laboratory core services
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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
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CURRENT PROJECTS USING CFAR FACILITIES
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
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HIV-1 DRUG RESISTANCE
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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
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Emergence of ARV resistance in ARV-treated
Ugandans at the JCRC
Over 50 of ARV-treated patients at the JCRC
harbor drug resistant virus
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EFFECTS OF TB ON HIV-1 DIVERSITY
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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
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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
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HIV-1 FITNESS AND DISEASE PROGRESSION
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Worldwide Distribution of HIV-1 subtypes
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Relationship between HIV-1 subtype, fitness and
disease progression
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?
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Intrasubtype competitions
Y axis wins
X axis wins
Paired T test, p gt 0.5
Paired T test, p gt 0.5
B vs. B
C vs. C
There is no significant difference in
intrasubtype competition involving B or C isolates
Ball et al., J. Virol. 2003
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Intersubtype competitions
B wins
C wins
C vs. B
C vs. B
Paired T test, p lt 0.0001
Subtype C isolates are significantly less fit
than subtype B isolates
Ball et al., J. Virol. 2003
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• 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

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• 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

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Processing of GS/VF Samples
Blood (Red Top)
Endocervical/Vaginal Swabs (RNA Later)
Aliquot Serum Freeze 70oC
Syphilis Testing (JCRC)
Aliquot and Freeze 70oC
Sample processing (pelleting and RNA extraction)
Repository
HSV Testing (JCRC)
Viral loads
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Processing of GS/VF Samples
Blood (EDTA)
Measurement CD4/CD8 lymph. (JCRC)
Isolate PBMCs (Buffy coat)
DBS
Repository
Repository
Viral Propagations Co-culture
Extract DNA
PCR and sequencing
Harvest virus for viral fitness
Clone for fitness studies
Subtyping
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HIV-1 subtyping
EDTA blood
External PCR with EnvB-ED14
PBMC isolation
DNA extraction
Nested PCR with E80-E125
DNA sequencing with fluorescent tagged primers
and using the Visible Genetic Sequencer
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Phylogenetic analyses
Likely lab contamination complex of A,E,G, and
J. Subtype A in env and gag
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GS Sample Analysis of fitness
Blood (EDTA)
Viral Propagations Co-culture
Isolate PBMCs (Buffy coat)
Harvest virus for viral fitness
Repository
Extract DNA
Determination of virus titer
Clone for fitness studies
Competitions with reference isolates
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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
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There are no Amersham, Invitrogen, BioRad or any
other biotech even remotely interesting in
installing a freezer
Establish good shipping routes and dont 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