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

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)

Determinants of progression to AIDS

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).

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

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

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

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 ( 10 - 2720) Viral load: Median(range) 22146 ( 100 - 607000) 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

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

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

Correlation CD4 and methylation of CCR5 and IL-2

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

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

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

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

THANK YOU!