Repeated and spontaneous switching of the chemokine co-receptors used by HIV-1 is rare in vivo

HIV-1 uses CD4 cells as its primary receptor and a chemokine co-receptor as its secondary receptor to gain entry into cells [1]. Initial studies suggested that the dissemination of HIV-1 from lymphoid organs to non-lymphoid tissues was associated with an expansion in the number of chemokine co-receptors being used by the virus [2,3]. The rapid progression to AIDS and death of patients whose viral isolates changed from R5 (non-syncytium inducing) to R5X4 (syncytium inducing) supported this conclusion [4]. The factors that promote this switch in viral phenotype have not been established. We investigated whether repeated and spontaneous switching of the chemokine co-receptors being used by the virus is more common in vivo than has been recognized in the cross-sectional studies that have been performed [5]. Blood was collected every 3–4 months from 62 HIV-1-positive patients attending the outpatient clinics at Hammersmith Hospital during the 7 years from 1992 to 1999. Preservative-free heparin (20 IU/ml) was used up to 1994, and acid citrate dextrose (Vacutainer, Beckton Dickinson, NJ, USA) was used thereafter. Samples were processed within 4 h of collection. Peripheral blood mononuclear cells were separated and co-cultured with peripheral blood mononuclear cells from HIV-seronegative donors as previously described [6]. Cell-free culture supernatants were harvested on day 14 and stored in liquid nitrogen. Viral isolates with a p24 antigen of 2 ng/ml or greater (EIA; Beckman Coulter; High Wycombe, UK) were used to phenotype the virus using U87.CD4 transformed cells expressing each of the chemokine co-receptors CCR1, CCR2b, CCR3, CCR5 and CXCR4 (MRC AIDS Reagent Project; NIBSC, Potters Bar, UK). Cells were plated at 4 × 104/ml in 48-well trays and 1000 TCID50 units or more of a low passage (< 2) primary viral isolate was added [6]. Virus was removed by washing at 24 h and the cells stained immunohistochemically for p24 antigen at day 4. A positive result required the presence of five or more p24 antigen-positive multinucleated giant cells. The 62 patients were followed for an average of two consecutive years during the course of the study. The mean number of virus isolations per patient was 5.4 ± 2.9 (range 2–26). During this time, patients were treated with antiretroviral drugs as they became available, and in accordance with clinical guidelines at that time. Most patients were found either to retain the chemokine co-receptor phenotype identified on first isolation of the virus, or to make a single stable switch during the course of their long-term follow-up. Forty-four patients with a R5 phenotype and 11 patients with an R5X4 phenotype retained their phenotype without any changes. Five patients switched from an R5 to a stable R5X4 phenotype. In only two patients did we find unequivocal evidence of repeated and spontaneous switching of the viral chemokine co-receptor phenotype without definitive evidence of an acute opportunistic infection (Fig. 1). Both had late-stage AIDS with CD4 cell counts of less than 50 × 109/l. In the first case (HH1), the patient's disseminated cytomegalovirus infection was difficult to control despite maintenance therapy with ganciclovir and foscarnet. In the second case (HH5), the patient had widely disseminated Kaposi's sarcoma that was associated with human herpesvirus type 8 infection. It is therefore possible that chronic, recurrent viral infections were responsible for the repeated changes in chemokine co-receptor tropism identified in these two patients. Expansion of their chemokine co-receptor tropism to a stable R5X4R3 phenotype only occurred in the few months before they died, despite continuous treatment with antiretroviral and antiviral agents.Fig. 1.: Repeated and spontaneous changes in chemokine co-receptor use seen in low-passage primary viral isolates from two patients while they were clinically well. A cross denotes the death of the patient.Cilliers et al. [7] recently reported the results of a clinical study whose aim was to determine the chemokine co-receptor tropism of primary viral isolates from South African patients presenting for the first time with late-stage AIDS. Their viral isolates used CCR5 and/or CXCR4 as well as CCR3, CCR2b, CCR1, CCR8, V28 and Bob. However, most of these patients also had a major opportunistic infection that resulted in their death. These observations suggest that a stable expansion of the tropism of HIV-1 to include the use of multiple chemokine co-receptors is a pre-terminal event in the course of the disease. Therefore, in patients who are clinically well, as defined by their clinical condition and a normal complement reactive protein, the only physiologically important chemokine co-receptors being used by blood-derived isolates of HIV-1 are CCR5 and CXCR4. In such patients, repeated and spontaneous switching of chemokine co-receptor use occurs rarely, if at all. Acknowledgements The authors are grateful to all the patients who participated in this study. Claire Veryard Caroline Javan Sunil Shaunak

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