In order to determine the frequency of CD4+
T cells carrying HIV DNA from the study participants, genomic DNA was prepared from highly purified CD4+
T cells and subjected to real-time PCR specific for HIV proviral DNA (limit of detection 2.6 copies of HIV DNA per µg of genomic DNA or 150 000 cell equivalent) [10
]. The median copy number of HIV proviral DNA for all study participants examined was 417.1 (range <2.6–8804.4) per 106
T cells (). The median copy number of HIV proviral DNA in the study participants who had initiated ART within 6 months of infection was significantly lower (4.6 copies per 106
T cells) compared to those who had initiated ART during the chronic phase of infection (949.4 copies per 106
T cells) (P
= 0.003). Of note, no measurable HIV proviral DNA was detected in four of nine early treated (44.4%) and four of 35 chronic treated (11.4%) individuals.
Frequencies of HIV proviral DNA (a) and infectious virus (b) in CD4+ T cells of HIV-infected individuals receiving effective ART for prolonged periods of time
In order to examine the frequency of CD4+
T cells carrying infectious virus, a HIC assay [11
], which allows examination of large numbers of cells, was conducted using highly enriched CD4+
T cells from the eight infected individuals in whose cells no measurable HIV proviral DNA had been detected. As shown in , infectious virus was recovered in all infected individuals using the HIC assay on CD4+
T cells. In one particular individual, the first HIC assay involving 3 × 108
T cells failed to produce replication-competent virus (7.6 years after initiation of ART). The infectious viral burden was estimated to be below 0.0012 per 106
T cells, using the assumption that one additional well would have resulted in HIV p24-positive outcome. However, a subsequent HIC assay conducted 10.5 years after initiation of ART using 1.56 × 109
T cells resulted in one out of 156 wells being positive for infectious virus. This translated into an infectious viral burden of 0.00064 per 106
T cells or one infected cell per 1.7 × 109
T cells. This is the lowest infectious HIV burden recorded to date in our laboratory and is considerably lower than the previously reported average frequency of 0.059 infectious units per 106
T cells in HIV-infected individuals having received ART for more than 7.6 years [10
]. When the coculture data were stratified by time of initiation of ART, the frequency of cells carrying infectious virus in infected individuals who initiated therapy within 6 months of infection was significantly lower (median: 0.0074 infectious units per 106
T cells, range 0.00064–0.0173) than that of infected individuals who initiated therapy during the chronic phase of infection (median 0.0666 infectious units per 106
T cells, range 0.0345–0.0847) (P
Colonoscopy was performed on two infected individuals in order to measure levels of HIV in GALT. As shown in , real-time PCR conducted on CD8-depleted cells from sigmoid colon biopsies from one infected individual (who initiated ART during the chronic phase of infection) in whom HIV proviral DNA was undetectable in peripheral blood CD4+ T cells, but in whom infectious virus was recovered, revealed readily detectable HIV DNA (89 copies of DNA per million cells). However, HIV DNA was undetectable in CD8-depleted cells isolated from the sigmoid colon biopsies of the infected individual (who initiated ART during the early phase of infection) in whom HIV proviral DNA was undetectable and the level of infectious virus was extraordinarily low (one infected cell per 1.7 × 109 CD4+ T cells) in peripheral blood CD4+ T cells (), suggesting that a profound reduction in the size of viral reservoir was achieved in this study participant.
Immunologic and virologic profiles of selected HIV-infected individuals in whom sigmoid colon biopsies were preformed.
In order to investigate whether discontinuation of ART in the individual who was started on ART early in the course of infection and who had an extraordinarily low HIV reservoir would result in rebound of plasma viremia, and what the kinetics of such a rebound would be if it occurred, we discontinued ART in the patient upon his consent and monitored plasma viremia. As shown in , plasma viremia was not detected for the first 50 days following discontinuation of therapy. Subsequently, plasma viremia rebounded to 1593 copies of HIV RNA per ml followed by spontaneous suppression to an undetectable level. However, plasma viremia then rebounded again to 8684 copies of HIV RNA per ml on day 143 at which point ART was re-initiated.
Levels of plasma viremia following discontinuation and re-initiation of ART