Association between CCR5 Genotype and the Clinical Course of HIV1 Infection

1
Association between CCR5 Genotype and the
Clinical Course of HIV-1 Infection

• de Roda Husman, A Koot, M Cornelissen, M
  Keet, I Brouwer, M Broersen, SM Bakker, M
  Roos, M Prins, M de Wolf, F Coutinho,R
  Miedema, F Goudsmit, J Schuitemaker, H. 1997.
  Association betweenCCR5 Genotype and the Clinical
  Course of HIV-1 Infection. Annals of Internal
  Medicine 12(10)882-890.
• By Alice Cioara

2
Purpose

• Analyze the role of CCR5 alone and in relation to
  established progression markers in the clinical
  course of HIV-1 infection in participants from
  Amsterdam Cohort Studies

3
What has recently been associated with the
progression of HIV-1

• Viral, immune and host genetic factors may
  influence the clinical course of HIV-1 Infection
• High viral load
• Presence of syncytium-inducing HIV-1
• Low T-lymphocyte function
• Certain human leukocyte antigens (HLA) types

4
Co-receptors for HIV-1 that have been recently
identified

• Syncytium-inducing
• T cell line-adapted HIV-1 variants use C-X-C
  chemokine receptor 4 (CXCR4)
• Non-syncytium inducing
• Macrophagetropic variants use C-C chemokine
  receptor 5 (CCR5)
• Primary syncytium-inducing viruses use both

5
HIV Co-receptors

• Members of the 7 transmembrane family of
  chemokine receptors
• Physiological role transmit intracellular
  signal following interaction with chemoattractant
  cytokine (chemokines)

6
What are Chemokines?

• Superfamily composed of 20 different leukocyte
  chemoattractants
• Small proinflammatory proteins that recruit and
  activate leukocytes and inhibit the viral
  co-receptor function of chemokine receptors
• 2 categories
• CC (beta)
• CXC (alpha)

7
Alpha-Chemokine Receptor

• Used by T-tropic strains of HIV and by many
  clinical isolates, in later stages of the HIV
  disease.
• T-tropic viruses also called synctia-inducing
• Use CXCR4 as primary co-receptor

8
Beta Chemokine Receptor

• Identified as the major entry cofactor for most
  primary or clinical isolates of HIV.
• Also termed Macrophage-tropic (M-tropic) or
  non-syncytia-inducing
• Now designated as R5 isolated

9
Predicted Structure of CCR5

• (McNicholl et al., 1997)

10
HIV Entry

• Entry begins with binding of the viral envelope
  glycoprotein to both the CD4 receptor and one of
  several chemokine receptors and ends with fusion
  of viral and cell membranes.
• (McNicholl et al., 1997)

11
Recapitulation of HIV Entry

• Primary binding site for HIV is the CD4 molecule
  and the interaction is mediated by the viral
  surface gp120.
• HIV-1 attachment to CD4 creates a high-affinity
  binding site for CCR5 leading to membrane fusion
  and virus entry.
• CCR5 when expressed along with CD4 allows cell
  lines resistant to most primary HIV-1 isolates to
  be infected, therefore, CCR5 is the principal
  cofactor for entry

12
Hypothesis

• Persons who have been exposed to HIV-1 on
  multiple occasion, but remain uninfected seem to
  be homozygous for a 32-nucleotide deletion in the
  CCR5 gene

13
Structure of CCR5(delta32)

• (McNicholl, 1997)

14
CCR5(delta32)

• A 32-base-pair deletion within the coding region
  results in a frame shift
• Experiment by Samson et al concluded
• White blood cells from an individual homozygous
  for the null allele were found to be highly
  resistant to infection by Mtropic HIV-1 strains
• Mutant allele of CCR5 is present at a high
  frequency in caucasian populations, but absent in
  black and Japanese populations.

15
Methods

• 961 asympmtomatic men between October 1984 and
  March 1986 living in Amsterdam area and had at
  least two homosexual contacts in the preceding 6
  months were selected

16
Methods Continued
17

• Studies showed infection in seroprevalent
  homosexual men must have occurred an average of
  1.5 years before entry in this study.
• Seroconversion was set at 1.5 years before study.
• One study sample 131 persons who had
  seroconversion 233 seroprevalent persons

18
Methods Continued
19

• By January 1st 1996
• 189 developed AIDS
• (mean follow-up 5.9 years)
• 94 did not develop AIDS
• (mean follow-up 10.1)
• 81 lost to follow-up
• ( mean follow-up 2.0)

20

• A nested control study was done using the same
  group of participants to identify factors that
  may be correlated with long-term survival.
• 23 remained disease-free for at least 9 years
• Mean CD4 T-lymphocyte count of gt400 cells/mm3
  (in 8th and 9th year)
• Mean CD4 T-lymphocyte count of 534 cells/mm3
• Each long term survivor was matched up with two
  progressors

21

• Use of Polymerase Chain Reaction for CCR5
  Genotyping
• 343 of 364 - Samples of DNA for CCR5 genotyping
• DNA isolated from cryopreserved peripheral blood
  mononuclear cells and 100 mg of DNA was analyzed
• Products of PCR were analyzed by using 2 agarose
  gel electrophoresis and ethidium bromide
  staining.

22
Virologic Assays

• Co-cultivation of HIV-1 positive peripheral blood
  mononuclear cells with MT2 performed every 3
  months
• Serum viral load measured by using a quantitative
  HIV-1 RNA nuclei acid-based sequence
  amplification
• Serum levels of HIV-1 RNA analyzed after log10
  transformation
• of RNA copies below threshold were set at 10
  (3).0 copies/mL.

23
Immunology Assays

• Antibodies of HIV-1 detected using a commercial
  recombinant HIV-1/-2 enzyme immunoassay and
  reconfirmed with Western blot IgG assay
• Enumeration of CD4 and CD8 T lymphocytes done
  by using flow cytoflurometry.
• With January 1988 reactivity of T lymphocytes
  in response to stimulation with CD3 monoclonal
  antibodies in vitro was routinely determined in
  whole-blood cultures

24
Statistical Analysis

• Fisher exact test compare HIV-1 seronegative
  with HIV-1 seropositive for CCR5 genotype
  distribution
• In case study conditional logistic regression
  performed to estimate the chance that a
  CCR5(delta32) heterozygote would be a longterm
  survivor.

25
Statistical Analysis

• Mann-Whitney U test compares CCR5(delta32)
  heterozygotes and CCR5 wild-type homozygotes.
• Slope of the decrease of CD4 T lymphocytes
  determined separately by fitting a simple
  regression line to his CD4 T-lymphocyte count.

26
Statistical Analysis

• Kaplan-Meier estimate the cumulative incidence
  of conversion to syncytium-inducing HIV-1
  variants in relation to CCR5 genotype.
• Duration of AIDS-free survival in relation to
  CCR5 genotype period of only non-syncytium-inducin
  g variants were present
• Kaplan-Meier and Cox proportional hazard analysis
  study predictive value of CCR5 alone or in
  combination with serum viral RNA load, CD4 T
  lymphocyte count, T- lymphocyte function, and
  syncytium-inducing phenotype.

27
Statistical Analysis

• Evaluated predictive value of the markers by
  fitting separate Cox models at 2,4,6 and 8 years
  after seroconversion.
• Markers also analyzed as time-dependent
  covariates.
• Number Crunching Statistical Systems used for
  statistical analyses.

28
Results

• CCR5 Genotype Distribution
• 80 - of HIV-1 seropositive participants were
  homozygous for CCR5 wild type genotype
• 20 - heterozygous for CCR5(delta32)
• No participant homozygous for CCR5 (delta32)

29
CCR5 Genotype and Clinical Course of HIV-1
Infection

• (De Roda Husman et al., 1997)

30
CCR5 Genotype and Clinical Course of HIV-1
Infection

31
CCR5 Genotype and Kinetics of CD4 T-lymphocyte
Counts

32
CCR5 Genotype and Serum Viral Load

33
Genotype and HIV-1 Biological Phenotype

34
Cox Proportional Hazard Analysis

35
Cumulative Incidence of AIDS

• (De Roda Husman et al., 1997)

36
Conclusion

• Observed strong correlation between heterogynous
  for CCR5(delta32) and prolonged AIDS-free
  survival
• CCR5 genotype predicted disease progression
  independent of viral RNA load, CD4 count,
  T-lymphocyte function and biological phenotype.

37
Conclusion

• RNA viral load after 2.5 years 2.6 fold
  decrease may be relevant to delayed disease
  progression
• At 2 years after seroconversion CCR5 wild-type
  homozygous have a higher rate for more rapid
  progression to AIDS even if CD4 and viral load
  are the same for both groups at that time point

38
Conclusion

• Observed slower rate of decrease in CD4 count in
  CCR5 (delta32)
• At the end of study CCR5 wild type and
  CCR5(delta32) had the same frequency of
  syncytium-inducing HIV-1 variants although the
  conversion to these variants tended to be delayed
  in CCR5(delta32)

39
References

• Samson, M Libert, F Doranz, BJ Rucker, J
  Forceille, CMuyldermans, G Verhofstede, C
  Burtonboy, G Georges, M Imai, T Rana, S Yi,
  Y Smyth, RJ Collman, RG Doms, RW Vassart, G
  Parmentier, M. 1996. Resistance to HIV-1
  infection in caucasian individuals bearing mutant
  alleles of the CCR5 chemokine receptor gene.
  Nature (6593)722-5.
• Trkola, A Dragic, T Arthos, J Binley, JM
  Olson, WC Allaway, GP Cheng-Mayer, C Robinson,
  J Maddon, PJ Moore, JP. 1996. CD4-dependent,
  antibody-sensitive interactions between HIV-1 and
  its co-receptor CCR5. Nature 384(6605)184-7.
• DSouza, MP Cairns, JS Plaeger, S. 2000.
  Current Evidence and Future Directions for
  Targeting HIV Entry Therapeutic and
  Prophylactic strategies. JAMA 284(2)215-222.
• McNicholl, JM Smith, DK Qari, Shoukat Hodge,
  T. 1997. Host Genes and HIV The Role of the
  Chemokine Receptor Gene CCR5 and its Allele
  (delta32 CCR5). Emerging Infectious Diseases
  3(3)
• Murphy, PM. 1996. Chemokine receptors
  structure, function, and role in microbial
  pathogenesis. Cytokine Growth Factor Rev 7(1)
  47-64.

40
References

• Deng, H Liu, R Ellmeier, W Choe, S Unutmaz,
  D Burkhart, M Di, Marzio, P Marmon, S Sutton,
  RE Hill, CM Davis, CB Peiper SC Schall, TJ
  Littman, DR Landau, NR. 1996. Identification
  of a major co-receptor for primary isolates of
  HIV-1. Nature (6584) 661-6.
• Berger, EA Murphy, PM Farber, JM. 1999.
  Chemokine receptors as HIV-1 coreceptors roles
  in viral entry, tropism, and disease. Annual
  Review of Immunology 17657-700.
• Choe, H Farzan M Sun, Y Sullivan, N Rollins,
  B Ponath, PD Wu, L Mackay, CR LaRosa, G
  Newman, W Gerard, N Gerard, C Sodroski, J.
  1996. The beta-chemokine receptor CCR3 and CCR5
  facilitate infection by primary HIV-1 isolates.
  Cell 85(7) 1135-48.
• Wu, L Gerard, NP Wyatt, R Choe, H Parolin, C
  Ruffing, N Borsetti, A Cardoso, AA Desjardin,
  E Newman, W Gerard, C Sodroski, J.
  CD4-induced interaction of primary HIV-1 gp120
  glycoprteins with the chemoking receptor CCR5.
  Nature 384(6605) 179-83.
• Michael, NL Chang, G Louie, LG Mascola, JR
  Dondero, D Birx, DL Sheppard, HW. 1997. The
  role of viral phenotype and CCR5 gene defects in
  HIV-1 transmission and disease progression.
  Natural Medicine 3(3)338-40.
• Rucker, J Samson, M Doranz, BJ Libert, F
  Berson, JF Yi, Y Smyth, RJ Collman, RG
  Broder, CC Vassart, G Doms, RW Parmentier, M.
  1996. Regions in beta-chemokine receptors CCR5
  and CCR2b that determine HIV-1 cofactor
  specificity. Cell 87(3)43-46.