In vitro activation of HIV RNA expression in peripheral blood lymphocytes as a marker to predict the stability of non-progressive status in long-term survivors

Objectives: We investigated a selected group of 11 non-progressor, HIV-infected individuals 20 months prior to this study and found that they all had undetectable levels of viral RNA expression in their peripheral blood lymphocytes (PBL). Phorbol 12-myristate 13-acetate (PMA) and phytohaemagglutinin (PHA) stimulation of PBL produced easily detectable amounts of HIV RNA in only two out of five of these patients. Here we report the results of the virological and clinical follow-up of nine non-progressors from this group. We verified the stability of their non-progressive status and attempted to correlate it to specific virological markers. Methods: Proviral DNA in lymphocytes was tested by semi-quantitative polymerase chain reaction (PCR). Detection of unspliced (US) and multiple spliced (MS) HIV RNA species in unstimulated and stimulated lymphocytes was performed by reverse transcriptase-PCR (RT-PCR). The amount of p24 antigen released into the media of lymphocyte cultures was measured using a standard procedure. Lymphocyte populations were depleted of CD8 cells by immunomagnetic purging. Results: Follow-up of nine of these subjects showed that the patients who previously showed viral RNA activation following lymphocyte stimulation in vitro, developed a clinical and immunological progression characterized by CD4 count decline and lymphadenopathy. In contrast, all the other subjects maintained progression-free status throughout the follow-up period, with no detectable levels of HIV RNA in the PBL. Notably, this group of subjects showed no activation of viral RNA expression following stimulation of either undepleted or CD8-depleted lymphocytes in vitro. Conclusion: The group of non-progressors studied was found to be heterogeneous regarding the stability of the non-progressive status during the follow-up period. Our results suggest that the activation of HIV RNA expression following PMA-PHA treatment of lymphocytes in vitro is an early marker for future progression of the disease.