1. DNA methylation as an epigenetic marker in HIV-2 disease in West Africa?
Alberta Davis
MRC Laboratories, Gambia
18th January 2013

2. HIV pathogenicity and clinical outcomes
AIDS: causal agents; retroviruses
HIV-1
HIV-2
HIV-1 “elite” controllers, long term non progressors (LTNPs), exposed uninfected. (Saksena et al 2007)
HIV-2 infection is less progressive (low VL, low transmission, slow decline of CD4 T cells, prolonged survival).
20% of HIV-2 infected individuals exhibit high VL and a clinical presentation indistinguishable from AIDS in HIV-1. (Esbjörnsson et al, 2012)

3. Determinants of progression to AIDS

4. Host determinants CCR5 receptor: Viral entry into CD4 T cells.
mutation confers resistance to infection with HIV.
Variable CCR5 expression in HIV-1 is associated with matrices of infection and pathogenesis.
Low and protective levels of CCR5 on CD4+ T cells is exhibited in HIV-2 infections (Shea et al 2004).
IL-2 –CCR5 are coregulated genes and CCR5 can influence signaling events during T cell activation (Camargo et al 2009).

5. Epigenetics and its regulation of genes
Epigenetic mechanisms:
Heritable but reversible without change in DNA sequence
DNA methylation in CpG dinucleotide = gene repression

6. hypothesis
“ The DNA methylation status of regulatory regions of CCR5 and IL-2 serve as a determinant of differential HIV-2 pathogenicity”
Objective
To determine the methylation status at CCR5 cis-regulatory CpG sites and IL-2 gene loci in progressive and non progressive infections
Evaluate the frequencies of CCR5 genetic variants

7. Methods HIV-2 rich cohorts – Guinea Bissau and Gambia.
Interrogated patient database based on previous studies of HIV-2 non progression (Berry et al 2002, Schim van der Loeff et al 2010).
Categorised samples into various groups based on CD4 and Viral load
Progressor: CD4 <200 cells/ul and VL > 10,000 copies/ml
Non progressor: CD4 >500 cells/ul and VL < 100 copies/ml

8. Overview of patient profiles
Demographic and virologic characteristics of 36 HIV-2 subjects n %
Characteristic
Categories/units
Age: Median(range)
years
45 (21-73)
CD4: Median(range)
counts
573 ( )
Viral load: Median(range)
22146 ( )
Sex
Male 15
41.7
Female 21
58.3
Ethnicity
Mandinka 13
36.1
Manjago 9 25
Other 14
38.8
CD4 counts
Low (< 200 cells/ul)
High (> 500 cell/ul)
Viral load counts
Low (< 100 copies/ml)
High (> 10,000 copies/ml)
CD4/VL
H/H
H/L 12
33.3
L/H
L/L 3
8.3

9. Specific CpG in CCR5 cis regulatory sites
Bisulphite modification and pyrosequencing (CCR5 and IL-2)

10. Association between DNA methylation and CD4
CD4 > 500 (n = 21)
CD4 > 200 (n = 15)

11. Correlation CD4 and methylation of CCR5 and IL-2

12. Progressor (L/H) and non progressor (H/L) phenotypes show different methylation patterns
L/H (n = 12)
H/L (n = 12)

13. Conclusion
The CD4 count and VL load could be influenced by methylation levels.
Viral control in non progressors is being achieved by low CCR5 expression and maintenance of CD4+ T cells which produce IL-2.
Progressors are less methylated at CCR5 regulatory regions but more methylated at the IL-2 locus than non progressors.
No significant difference was found at CCR5 promoter 2 possibly because Pr2 is active only upon T cell activation

14. Recruitment of patient and blood sampling in Guinea Bissau
Duration in the cohort (1989, 1997, 2003, 2006)
HIV-2 non progressor,HIV-2 progressor,HIV-1 asymptomatics, Healthy controls.
CCR5 and IL-2 DNA methylation
CCR5 and IL-2 mRNA expression levels
CCR5 allelic discrimination
Immunophenotyping by flow cytometry
Multiplex cytokine analysis by Bioplex-cytokine analysis

15. Acknowledgements UTHSCSA Prof Sunil K. Ahuja Dr He Weijing
Komathy Jayasekar
Una Aluyen
Shivali Chag
MRC Unit
Dr Assan Jaye
Pa Saidou Chaw, MD
Dr Alfred Ngwa
Ramou-Sarge Njie
Gilleh Thomas
James Jafali

16. THANK YOU!