Molecular Epidemiology of HIV

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Molecular Epidemiology of HIV
Francine E. McCutchan, PhD Global Molecular
Epidemiology Program
Supported by the US Military HIV Research
Program, the Division of AIDS, NIAID, NIH, and
the European Commission
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Status of the Global Epidemic

• 40 million infections worldwide
• More that 150 countries on 6 continents
• Most infections are acquired heterosexually and
  affect men and women in equal numbers
• A major cause of mortality in young adults
• Substantial rates of perinatal transmission in
  much of the developing world
• The epidemic continues essentially unabated in
  the absence of an effective vaccine

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Worldwide Distribution of HIV Infections
1.4 million
610,000
1.0 million
1.1 million
540,000
440,000
7.1 million
25.4million
1.7 million
35,000
Source UNAIDS Report on the Global Epidemic,
2004
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Origins and Epidemic Spread

• HIV-1 and HIV-2
• HIV-1 group M, N,O
• HIV-1 Group M subtypes and recombinants

• Different primates
• Different introductions
• Spread in human populations

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The origins of HIV in Africa are reflected in its
current distribution
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Sources of Variation

• HIV-1 is the most genetically variable of human
  pathogens

• Rapid replication rate
• High mutation rate
• Recombination

• HIV in the infected individual exists as a swarm
  of highly related but non-identical viral genomes
  termed a quasispecies

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Rapid replication
1010 virions per day
High mutation rate
1 substitution per genome per round
Recombination
7-30 crossovers per genome per round
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HIV-1 Group M Subtypes and Recombinants

• Nine Subtypes
• Phylogenetically distinct across the genome
• Inter-subtype Recombinants
• At least 21 Circulating Recombinant Forms (CRF)
• Numerous Unique Recombinant Forms (URF)

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Phylogenetic Criteria for Classification Subtype
vs. Recombinant
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Recombinant HIV-1

• Recombinant HIV are an important and particularly
  dynamic component of the global epidemic, and
  insight into their genesis and spread is of
  crucial importance for treatment and prevention
• Genotyping of HIV strains is complicated by
  recombination, and requires suitable approaches
• Complete sequencing of the genome of each strain
  provides an unequivocal classification
• The database of complete genome sequences has
  grown exponentially in recent years

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The HIV Sequence Database
A B C D F, G, H, J, K
Global prevalence High High High High Low
Complete Genome Sequences 62 129 209 47 25
Subtype
CRF01_AE CRF02_AE CRF03 through CRF16
High High Low
52 38 60
CRF
AC, AD, CD, ACD Complex BF CRF01_AE/B
Low Low Low Low
44 33 31 13
URF
743
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Global Prevalence and Distribution

• Subtype C accounts for almost 50 of HIV-1
  infections worldwide
• Globally prevalent strains include subtypes A, B,
  C, D, and CRF01_AE, CRF02_AG
• Many regional epidemics contain a mixture of
  subtypes, while others are dominated by a single
  subtype or CRF
• The regional epidemic patterns of HIV are varied,
  complex, and dynamic

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Regional Epidemic Patterns of Subtypes and
Recombinants
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The distribution of HIV-1s subtypes and
recombinants is as dynamic as its human host
Be a virus, see the world
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Examples of a changing global epidemic

• CRF01_AE a minor strain in Central Africa, but
  gained global importance as it entered Southeast
  Asia
• Two new BC recombinant CRF circulate in China
  along different drug trafficking routes
• A subtype A strain of low diversity, and a new
  CRF, CRF03_AB, emerged in former Soviet Republics
  after the dissolution of the Soviet Union
  destabilized the social milieu
• CRF14_BG arose among IDU in Spain and Portugal in
  recent years
• Subtype F, a rare strain in Central Africa,
  emerged in the form of BF recombinant strains,
  including a new CRF, in the Southern cone of
  South America

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The Genesis of Recombinant Strains
URF
A
URF
D
A
URF
C
A
30-40 of strains in mixed-subtype epidemics can
be unique recombinants
120 complete genomes
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Hypothesis

• Some of the individuals who harbor URF are
  themselves co-infected with two or more HIV-1
  strains
• High risk, multiply exposed cohorts may exhibit a
  higher proportion of URF and dual infection
  compared to their lower-risk counterparts
• The proportion of URF may increase over time in
  highly exposed cohorts due to ongoing re-infection

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Approach

• Develop tools for HIV-1 genotyping that are high
  throughput and capable of detection of both
  unique recombinant forms and dual infections
• Establish cohorts exposed to a similar mixture of
  subtypes and recombinants in a given geographic
  region that represent various levels of HIV-1
  incidence, prevalence, and risk
• Perform genotyping cross sectionally at baseline
  and during longitudinal follow-up
• Explore the viral dynamics and nature of the
  quasispecies in dual infections

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Multi-Region Hybridization Assays (MHAs)

• Fluorescent, subtype-specific probes spaced along
  the genome
• Real-time PCR to provide automated, high
  throughput data collection and analysis
• Validated against complete genome sequences for
  their ability to distinguish subtypes and
  recombinant forms
• Capability to detect dual infections

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A Family of MHAs for Regional Application
Region East Africa West/Central Africa Southeast
Asia South America
Subtypes A, C, D, recombinants CRF02_AG,
recombinants CRF01_AE, B, recombinants B, C, BF
recombinants
Assay MHAacd MHAcrf02 MHAbce MHAbcf
Oral presentation (Kijak et.al.) in Molecular
Epidemiology and Transmission session later this
morning
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Assay Principle of the MHAacd for East Africa
Visit 1 2 3 4
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Comparative Epidemiology in East Africa Cohorts
MHA
Population High risk females Urban and rural
communities Rural communities Agricultural
Plantation
Country Tanzania Tanzania Uganda Kenya
Cohort HISIS CODE MER Kericho
Genotypes (N) 238 487 329 366 1420

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Proportions of Recombinant HIV
Using a cross sectional sample frame.
Urban High Risk
Agricultural/Rural
Rural/Urban
URF
26.4
29.5
35.9
50.8
MER
HISIS
CODE
KERICHO
A
A
URF
C
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These data are consistent with the hypothesis
that some individuals become dual infected with
more than one HIV-1 subtype, and that URF are the
result They also suggest that both high and low
risk cohorts are susceptible to dual infection,
albeit to different degrees
Increased dual infection
Expansion of URF component
Elevated risk
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Changing Distribution of Subtypes and
Recombinants during follow-up of the HISIS cohort
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Summary of Comparative Molecular Epidemiology in
East African Cohorts

• Identification of a significant fraction of both
  URF and dual infections in all cohorts studied
• Association between higher multiple exposure risk
  and higher rates of dual infection
• Association between dual infection and an
  increasing fraction of URF

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Viral Dynamics in the Dual Infected Individual
Possible outcomes of dual infection

• Persistence of two HIV-1 strains without
  detectable recombination
• Virtual elimination of one of the original
  strains by selection
• Generation of a new recombinant form that
  dominates the quasispecies, eclipsing the
  original strains
• Continuous succession of new recombinant forms
• Stable proportions of original strains and their
  recombinants over time
• Changing proportions of original strains and
  their recombinants over time

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Molecular forms of gag in a dual infection
One year follow-up, 66 clones
Form
Structure
Subtype
I II III IV V VI VII
A AC AC AC AC AC AC
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Relationships of recombinant forms in a dual
infection
Probable original strains
Derived recombinants
VI
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Temporal Fluctuation of the Viral Quasispecies
123 gag gp41
442 gag gp41
507 gag gp120 gp41
529 gag gp41
551 gag p120
0 3 6 9 12 15-21
Sample Interval (months)
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Viral Dynamics in Dual Infected Individuals

• Persistence of two HIV-1 strains without
  detectable recombination
• Virtual elimination of one of the original
  strains by selection
• Generation of a new recombinant form that
  dominates the quasispecies, eclipsing the
  original strains
• Continuous succession of new recombinant forms
• Stable proportions of original strains and their
  recombinants over time
• Changing proportions of original strains and
  their recombinants over time

X X X X X X v
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Implications of Dual Infection - Epidemiology

• Detection of dual infections is an essential
  element of HIV-1 genotyping
• Each dual infected individual could be the source
  of a series of different recombinants during the
  course of infection, amplifying the fraction of
  URF in the population
• A dual infected individual could transmit more
  than one strain simultaneously, thereby
  increasing the dual infected population directly
• Effective interventions in populations
  susceptible to dual infection may limit the
  genetic complexity of HIV strains, both URF and
  the eventual emergence of CRF

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Implications of Dual Infection - Vaccines
Why does HIV infection sometimes fail to protect
against re-infection?

• Damage to the human immune system at the time of
  initial HIV-1 infection
• Failure of cross-protective immunity resulting
  from the high genetic diversity of HIV-1
• Some combination of the above

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Implications of Dual Infection -Pathogenesis

• By 2004, studies in the US and in Africa had
  associated dual infection with increased viral
  load and/or rapid disease progression

The Lancet, 2004 Gottlieb et.al. JID, 2004
Grobler et.al. AIDS 2004 Manigart et.al.

• Yet, it is unclear which is cause and effect
• More severe initial damage to the immune system
  may predispose to rapid progression and increase
  susceptibility to re-infection
• Dual infection could generate a more diverse and
  difficult to control viral quasispecies, which
  could accelerate disease progression

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Implications of Dual Infection - Transmission

• Higher plasma viral loads associated with
  increased transmission
• Dual infection associated with higher plasma
  viral loads

It is possible that the very individuals who
become dually infected also develop a higher
plasma viral load that renders them particularly
efficient at transmission of the recombinant
forms that arise within them
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Conclusion
By specifically addressing the factors that
foster dual infection and the generation and
transmission of recombinant HIV-1, benefit may
accrue to the individual, in terms of preventing
a more rapid disease progression, and to society,
by limiting the complexity of strains that must
eventually be controlled by treatments and
vaccines. In the future, prevention of
re-infection of those already infected with HIV-1
may become an important element in the overall
strategy to gain control of the global epidemic
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Thanks to the many collaborators and to the
participants in cohort studies in Africa
Makerere University and Uganda Virus Research
Institute Nelson Sewankambo David Serwadda
Mbeya Medical Research Programme
US Military HIV Research Program
Michael Hoelscher Martina Gerhardt Leonard
Maboko Donan Mmbando Eluter Samky Oliver
Hoffmann Steffan Geis
Miguel Arroyo Sucheep Piyasirisilp Sodsai
Tovanabutra Gustavo Kijak Eric Sanders-Buell Merli
n Robb Deborah Birx
Colombia University and Johns Hopkins
University Maria Wawer Ron Gray
US Army Medical Command Kenya and Kenya Medical
Research Institute Ginamarie Foglia Monique
Wasunna Sam Martin Wilfred Langat