Determination of viral DNA in purified T cells and monocytes/macrophages in the cervix and blood of HIV+ women

Manyu Prakash1,2,3, Steve Patterson2, Frances Gotch2 and Moses S. Kapembwa3. 1Nuffield Department of Clinical Medicine, Oxford University. 2Department of Immunology, Imperial College at Chelsea & Westminster Hospital, London. 3Department of GU/HIV Medicine, Imperial College at Northwick Park & St. Marks Hospitals. Harrow, Middlesex (UK).

Background: Heterosexual transmission is the predominant route of HIV-1 infection. To further understand the dynamics of sexual transmission and levels of virus persistence following the initiation of anti-HIV therapy, CD4+ bearing cells capable of harbouring virus were isolated and the level of viral DNA quantitated. Material and Methods: Cervical brushings and peripheral blood mononuclear cells (PBMC) were investigated in parallel from HIV+ women at differing stages of disease. Using dynabeads, purification of T cells (CD3) and monocytes/macrophages (CD14) was performed. The DNA was extracted from the resulting pellet. Using nested PCR, the level of viral DNA was determined by amplification of the gag sequence, and the cell number was calculated from the b-globin copy number. Furthermore, four-colour flow cytometry was used to determine the relative proportion of leukocyte subpopulations. Results: In the cervical epithelium, macrophages constituted the predominant leukocyte subset, whereas in blood T cells were prevalent. Viral DNA in blood resided mostly in T cells, however, comparable levels of viral DNA was identified in T cells and macrophages in the cervix irrespective of patient classification. The level of viral DNA in cervical preparations exceeded the levels observed in blood per 10,000 purified cells. This increase was evident in women not on therapy (p<0.05), and those on therapy with an undetectable and particularly those with a high virus load (p<0.05). Conclusion: These data suggest differences exist in the immunophentypic profile of leukocytes that reside within the cervix and blood that is likely a consequence of the differing microenvironments. Furthermore, the higher viral DNA levels observed in the cervix suggest an important site for the transfer of virus from women to men irrespective of systemic viral load and CD4 count.