Tracking Global Diversity viral subtypes and their implication for HIV viral load and resistance tes

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Tracking Global Diversity - viral subtypes and
their implication for HIV viral load and
resistance testing - The Sentry Study
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• Professor Clive Loveday
• International Clinical Virology Centre
• Buckinghamshire
• UK

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Early sequencing (1985) of HIV-1
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suggested we would progress rapidly with our
understanding and clinical interventions in the
disease
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HIV-1 replication
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• However
• HIV-1 was found to be characterized by
  error-prone replication
• Host accumulates genetically diverse
  sub-populations of viruses over time
• An associated rapid replication rates meant this
  served as a mechanism for virus evolution
• In practice a broad genetic heterogeneity was
  seen within hosts, between hosts, and in
  communities
• The emergence of HIV-1 subtypes was also a
  consequence of this genetic variability

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Subtype distribution mid-1980s
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Showed genetically distinct viruses contributing
to the epidemic in Africa and the rest of the
world
B
B
A,C,D E,F,G,H
B
B
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Subtype distribution 1993
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With increase in international travel and flow of
immigrants, HIV-1 subtype infections became more
widely distributed
B,A,C,D,E,F,G,H
B,A,D,E
C.B.E
A,C,D E,F,G,H
E,B
B,E,F,C
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HIV-1 subtypes
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• HIV-1 subtypes classified by phylogenetic
  analysis of sequences revealed at least 10
  different subtypes
• They were biologically successful and contributed
  to the epidemic
• Recombination was observed where multiple
  subtypes co-existed in a community
• This event has given rise to biologically
  successful new viruses Circulating Recombinant
  Forms - CRFs in communities

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Classification of HIV
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HIV-11
HIV-21
Group M2
Group N
Group O
Clades A, B3, C, D, F, F2, G, H, J, K
Recombinants Common AE, AF, AG Uncommon AGHK,
FD, AFGHJK, AB, BC
1 HIV-1 most common, but HIV-2 now circulating
outside Africa, especially India 2 Most
infections due to group M viruses 3 Clade B
9899 USA, 90 Europe
McCutchan F. 13th IAC, Durban, 2000. 165
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Current HIV-1 subtype distribution
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• East, South Sub-Saharan Africa A, C, A/G,
  D, F, G, H, J, A/E, B, CRFs
• South SE Asia C, A/E, B, B/C
• Central and S America B, F, C
• North America B,A/E, C, A/G, D
• Europe B, A/B, A, A/E, C, D, F, G, H, J, A/G
• Australia B

McCutchan F. 13th IAC, Durban, 2000. 165
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Implications for multiple subtypes contributing
to the epidemic
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• Disease pathogenesis
• Clinical responses
• Natural resistance
• Vaccine efficacy
• Assay performance

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ICVC subtype cohort and the sentry project
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• Derived from 41 district general hospitals in
  London and around the UK
• lt500 patients with 6 years of follow-up
• On-going studies to evaluate range and
  distribution of NB subtypes, the virological
  approaches to characterization, the relative
  responses to therapy and impact of variable
  molecular epidemiology on virological assays (the
  Sentry study)

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Serological characterization of subtypes in the
ICVC cohort (n1721)
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Indeterminate 19
Subtype B 60
Subtype non-B 21
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Distribution of subtypes in the ICVC cohort in UK
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• 121/152 patients sequenced to date
• SUBTYPE A 44 (36.4)
• SUBTYPE B 22 (18.2)
• SUBTYPE C 32 (26.0)
• SUBTYPE D 15 (12.0)
• SUBTYPE F 1 (0.8)
• SUBTYPE G 3 (2.4)
• SUBTYPE H 1 (1.0)
• SUBTYPE AE 2 (1.6)
• SUBTYPE AG 1 (0.8)

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ICVC Subtype Cohort and the Sentry Project
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• NB Subtype prevalence gt20
• B most prevalent then A and C
• Recombinants found in approximately 9
• High proportion NB in females relative to B
• Association with Africa as country of origin
• Equally distributed in London and rurally
• High prevalence of NB viruses in recent infections

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Clinical responses of B vs NB to HAART (Case
controlled study)
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• Proportion undetectable (lt400c/ml) by time
• weeks
• lt24 24 48
• NB 34 (68) 39 (78) 31/44 (70.4)
• B 38 (76) 40 (80) 35/45 (77)

Loveday C et al 8th CROI Chicago 2001
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Virological responses of B vs C to HAART (Case
controlled study)
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Proportion gt50 6/34 10/33 C 20/37 25/35 B
5
4
Mean plasma VL (log copies ml)
Subtype C n 37
3
Subtype B n 37
2
Time (weeks)
1
0
24
48
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Change in CD4 counts
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5
0
0
Subtype B
4
0
0
3
0
0
MEAN CD4 (cells/mm3)
Subtype C
2
0
0
1
0
0
Time (Weeks)
0
2
4
4
8
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Data concerning natural drug resistance in NB
viruses
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• ICVC cohort no major mutations but there were
  minor mutations in NB and patterns of discordant
  polymorphisms between B and C in RT and protease
• Israel B v C (n28) -striking differences in
  protease M36I (2593), K20K (432), A71V (3911),
  G73V (160), further key mutations K20R, M46I,
  L90M in 5 naïve subtype C
• French/African study A-K (n142) - no major
  mutations in drug naïve patients, exempt O and J
  having NNRTI mutations. However, similar did have
  distribution of minor mutations
• PHLS UK (n149) - 5 possessed low level
  resistance to PI
• South Africa C (37) - naïve, 3 harbored A98S and
  V179I in RT, an additional 3 had V118I

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Molecular tests to support patient care
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• The majority of virological assays to monitor and
  support patient clinical care are now based on
  molecular technologies which are potentially
  vulnerable to the ongoing viral Evolution (?
  TIME), and the worldwide genetic diversity of
  HIV-1 (? SPACE)
• Assays we use were originally designed in the
  late 80s using laboratory strains of HIV-1
  subtype B
• CURRENTLY WE APPLY ASSAYS FIXED IN TIME AND
  SPACE TO MEASURE VIRUSES THAT ARE GENETICALLY
  MOBILE IN TIME AND SPACE

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Molecular tests to support patient care
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• Need to understand that with the dynamics of
  HIV-1 replication there is the potential for
  viruses to evolve away from assays in the same
  way as they currently evolve away from drugs.
• A shift in primer fidelity may produce no
  detection, but equally a gradual drift may reduce
  assay performance over time
• Need for worldwide ongoing surveillance
• Need for flexibility in assay design

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Subtype diversity and divergent diagnostic PCR
assays (1994)
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• Amplicor (gag) In House (pol/gag)
• MUM 1 Positive Positive
• BABY 1 (3 days) Negative Positive (pol)
  (p24, Culture)
• MUM 2 (1993) Positive Positive
• MUM 2 (1995) Negative Positive (pol)
• BABY 3 (1/12) Positive Positive
• BABY 3 (5/12) Negative Positive (pol)

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Sequences associated with primer binding sites in
gag for mother and baby (example 1)
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• A GTT GGA GGA CAT CAA GCA GCC ATG CAA AT
• MUM - --G --G --G --- --- --- --T --- --- --
• BABY - --G --G --G --- --- --- --T --- --- --
• GAG ACC AAT GAG GAA GCT GCA GAA TGG GAT
• MUM --T --A --- --- --- --- --- --- --- --C
• BABY --T --A --- --- --- --- --- --G --- --C
• AGA GAA CCA AGG GGA ACT GAC ATA GCA
• MUM --- --- --- --A --- -G- --T --- ---
• BABY --- A-C --- -CT --- -G- --T --- ---

SK462 (5 primer)
SK102 (PROBE)
SK431 (3 primer)
Arnold C, Barlow KL, Loveday C et al ARHR (1995)
11, 999
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Quantification of NB viruses using Roche Amplicor
Monitor 1.0 vs 1.5
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Subtype B
Subtype NB
7
7
6
6
5
5
Q RT-PCR (Roche Version 1.5)
4
4
3
3
2
2
2
3
4
5
6
7
2
3
4
5
6
7
Q RT-PCR (Roche Version 1.0) log c/ml
Dann L PhD Thesis, University London, 2002
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Viral load (log c/ml) difference between Roche
1.0 and Roche 1.5 for populations of subtype B
and non-B viruses
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2
.
5
0
Subtype non-B
2
.
0
0
Subtype B
1
.
5
0
(plt0.01)
1
.
0
0
Viral load difference (log)
Median 0.03
Median 0.23
0
.
5
0
0
.
0
0
-
0
.
5
0
Devereux H, Loveday C, Burke A et al AIDS (1999)
13. 142
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Summary statement
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• Surveillance
• Performance QA/QC
• Assay development from evaluated sequences
• Vigilance

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The Problem Today
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• The full genetic complexity of HIV-1 is greater
  than (originally) anticipated F McCutchan
  (2000)

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The Sentry Study
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• Ask questions of direct clinical significance to
  patient care in a clinical cohort infected with
  field strains of HIV-1 NB viruses
• Monitor molecular epidemiology of HIV-1 NB
  subtypes in UK (Surveillance)
• Evaluate the performance of molecular assays to
  support assay development
• Surveillance of primer drift with dynamic NB
  panels over time
• Collaboration data sharing internationally