The lymphocyte HIV reservoir in patients on long-term HAART is a memory of virus evolution.

OBJECTIVE: To investigate the dynamics of the lymphocyte HIV reservoir in patients on prolonged and effective highly active antiretroviral therapy (HAART). DESIGN: Nine HAART-treated patients were selected on the basis of long-term infection and long-term undetectable plasma viral RNA. Five patients had received antiretroviral therapy before HAART. We compared a polymorphic region of the env gene (C2V4), and the part of the pol gene encoding the reverse transcriptase in pre-HAART plasma and in the reservoir lymphocytes during HAART; the first plasma sample taken after structured treatment interruption was also studied in three patients. METHODS: Both regions of interest were amplified from plasma HIV RNA and cellular proviral DNA, then cloned, sequenced and subjected to phylogenetic analysis. RESULTS: Diversity of the lymphocyte reservoir was found in six of nine patients. Archiving of pre-HAART plasma clones was observed in six of nine patients. 'Wild-type' and zidovudine-resistant strains co-existed in reservoir T cells of two pre-HAART treated patients. In three patients, no resistant virus was found in the T-cell reservoir despite the detection of resistant virus in pre-HAART plasmas. However, virus archiving was documented in two of these three patients on the basis of C2V4 analysis. Latently infected T cells only partly accounted for the plasma viral load rebound after structured treatment interruption. CONCLUSIONS: The HIV lymphocyte reservoir is dynamic. Its diversity results mainly from successive archiving of circulating plasma viruses during the course of HIV infection. Archiving of resistant virus must be taken into account in therapeutic decisions.

Persistence of replication-competent HIV in both memory and naive CD4 T cell subsets in patients on prolonged and effective HAART.

OBJECTIVE: To investigate the phenotypic features of infected lymphocytes in patients on prolonged and effective highly active antiretroviral therapy (HAART). DESIGN: We examined highly purified subsets of memory and naive CD4 T lymphocytes for the presence of replication-competent virus. METHODS: In 11 highly selected HAART-treated patients, we isolated highly purified CD45RO CD45RA CD4 T cells using a magnetic bead-based procedure. In some patients, a subsequent cell separation according to CD62L expression was performed. We quantified total viral DNA in freshly isolated T-cell subsets. To verify whether the virus was replication-competent, HIV RNA was measured in supernatants following cell activation. RESULTS: HIV DNA was detectable in the CD45RO and CD45RA CD4 T-cell subsets in 100% and 90% of the patients tested, respectively. In central memory CD45ROCD62L, effector memory CD45RO+CD62L-, truly naive CD45RACD62L, and CD45RA+CD62L- CD4 T cells, HIV DNA was found in 100%, 55%, 88%, and 50% of the patients tested respectively. HIV DNA was significantly higher in the CD45RO fraction than in the CD45RA subset and in the CD45ROCD62L fraction than in the three other CD45RA/ROCD62L+/- subsets. Detectable HIV RNA was found in the culture supernatants of CD45RO and CD45RA CD4 T-cell subsets in 80% and 66% of the patients tested respectively, and in CD45ROCD62L, CD45RO+CD62L-, CD45RACD62L, and CD45RA+CD62L- CD4 T cells in 100%, 100%, 100% and 50% of the patients tested respectively. CONCLUSIONS: In patients on prolonged and effective HAART, the pool of infected CD4 T lymphocytes consists predominantly of memory cells but also contains naive cells.

In patients on prolonged HAART, a significant pool of HIV infected CD4 T cells are HIV-specific.

OBJECTIVES: To examine the antigen specificities of HIV reservoir CD4 T cells in patients on prolonged and effective highly active antiretroviral therapy (HAART). DESIGN: Five HIV-infected patients, who were highly adherent to antiretroviral treatment, were selected on the basis of long-term undetectable plasma viral RNA on unmodified HAART. To investigate the antigen specificities of infected memory CD4 T cells, we examined the capacity of recall antigens, including HIV antigens, to induce virus production by peripheral blood mononuclear cells (PBMC). METHODS: To quantify CD4 T cells infected by replication-competent virus, and to determine their antigen specificities, we used a limiting dilution-based culture assay. CD8 T cell-depleted PBMC at several cell densities were activated by using Tuberculin purified protein derivative, cytomegalovirus, or HIV-1 p24 with and without HIV-1 Nef. RESULTS: We found that the pool of infected CD4 T cells includes HIV-specific cells with apparent frequencies between 5- and 100-fold higher than those of the common specificities for cytomegalovirus or Tuberculin. CONCLUSION: Our findings suggest that a significant proportion of replication-competent HIV-infected CD4 T cells in these patients are memory cells directed against HIV determinants. This may provide a rationale for the therapeutic use of recombinant HIV antigens to reduce the pool of HIV-reservoir cells.