Absence of HIV associated with erythrocytes in vivo.

Daniel S. Fierer, M.D., Jesus Vargas, Ph.D., Niraj Patel, M.D., Geoffrey Clover, M.D., and Ayele Bandele, B.S. Division of Infectious Diseases, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10025 (USA).

Background: Despite administration of potent combination antiretroviral medication, evidence of persistent HIV replication can be found in essentially all treated patients.  In a recent report¹, HIV was found to be tightly associated with erythrocytes at levels significantly higher than those found in the plasma of the same patient; the differences were especially marked in patients who had plasma viral loads (VL) < 50 copies/ml.  As these findings, if confirmed, would have a significant impact on current models of HIV pathogenesis, we attempted to replicate these findings. Material and Methods: Patients were recruited from the outpatient HIV clinic at Mt. Sinai.  The erythrocyte fraction from freshly drawn blood was washed and depleted of CD3+ cells with Dynabeads CD3 (Dynal).  FACS analysis was performed on the fresh pre- and post-depletion erythrocyte fractions using TriTEST CD3/CD4/CD45 reagent with TruCOUNT tubes and MultiSET software on a FACSCalibur flow cytometer (all Becton Dickinson) to enable quantification of residual CD3+/CD4+ cells.  Erythrocyte VL was measured from lysed erythrocyte aliquots representing 1 ml of whole blood (VL analysis performed by the Mt. Sinai clinical diagnostic laboratory using Roche Amplicor HIV-1 Monitor Test v.1.5; sensitivity 50 copies/ml). Results: Twelve patients with long-term (median 48 mo.) suppression of plasma VL to < 50 copies/ml and a wide range of CD4+ counts (21 to 1,653 cells/ml, median 515  cells/ml) were enrolled:  11 were receiving anti-retroviral therapy (ART) and 1 “long-term non-progressor” (LTNP) was not receiving ART.  Dynabeads depletion of CD3+/CD4+ cells was extremely efficient (median 99.4% depletion).  VL assay of the lysed CD3-depleted erythrocytes showed only 2 of 12 patients’ specimens with OD₄₅₀ readings significantly over background, for calculated VL of 42 and 52 copies/ml blood for those samples; all other patient’s specimens had OD₄₅₀ levels indistinguishable from background. Conclusion: We have found no evidence that a significant quantity of HIV binds tightly to erythrocytes in patients with long-term suppression of plasma viremia.  The previously reported finding suggesting this association may have represented contamination of erythrocytes by CD4+ cells containing integrated HIV DNA.

¹Hess, C et al., Lancet 2002;359:2230.