Analysis of the Kinetics of Latent HIV-1 Present in the
Resting CD4+ T-Lymphocytes of HIV-1-Infected Children Treated with Highly Active
Antiretroviral Therapy
D Persaud, T Pierson, C Ruff, K Chadwick, J Margolick, A
Ruff, N Hutton, RF Silicano
Human immunodeficiency virus type 1 (HIV-1) persists in a latent
state in the resting CD4+ T lymphocytes of infected adults even after prolonged
treatment with highly active antiretroviral therapy (HAART). Available
antiretroviral therapies are ineffective in eradicating virus from these
cells. The existence of a latent reservoir for HIV-1 in infected children
has not been established. In this study, a latent reservoir for HIV-1 is
demonstrated in infected children, and the size and decay rate of this reservoir
described.
Methods: A quantitative viral culture assay was
carried out on purified populations of restring CD4+ T lymphocytes isolated from
the blood of twenty-one HIV-1-infected children with various states of HIV-1
infection and various degrees of suppression of viral replication. The
assay was used to determine the frequency of cells carrying latent,
replication-competent HIV-1 among resting CD4+ T lymphocytes of infected
children. Correlations between the frequencies of latently-infected,
resting CD4+ T lymphocytes and viral load, CD4 percentage, and antiretroviral
treatment regimen were analyzed. Longitudinal analysis of the effects of
HAART on the kinetics of latent HIV-1 in resting CD4+ T lymphocytes in children
was performed in 7 of the 20 children who had suppression of viral replication
to undetectable levels (<400 copies/mL) for one year or more. In
addition, sequence analysis of the HIV-1 pol gene from the viral isolates
cultured from the resting CD4+ T lymphocytes was performed to determine the
evolution or persistence of mutations coding for drug resistance in the reverse
transcriptase and protease regions of the HIV-1 pol gene.
Results: Replication-competent HIV-1 was recovered
from resting CD4+ T lymphocytes of 17/21 children from whom sufficient cells
were obtained for culture. The mean frequency of latently-infected,
resting CD4+ T lymphocytes was 10.0 per 1×106 lymphocytes (range
<1 to 2,503 per1x106 cells). The frequency of
latently-infected resting CD4+ T lymphocytes correlated directly with plasma
HIV-1 RNA levels (r=.86, p<.0001), and inversely with the CD4 percentage
(r=.60, p<.005). Replication-competent HIV-1 persisted in the resting
CD4+ T lymphocytes of the 7 children who had suppression of viral replication to
undetectable levels of plasma HIV-1 RNA for one year or more of HAART. The
mean frequency of latently-infected resting C4+ T lymphocytes in this cohort of
7 children was 04 per 1 x 106 resting CD4+ T lymphocytes. In 3
children, replication-competent HIV-1 was recovered from resting CD4+ T
lymphocytes 21 to 37 months after a fully suppressive regiment of HAART.
Two of these children had substantial increases in their CD4+ T lymphocytes with
HAART. Further evidence for the persistence of latent HIV-1 was provided
by the detection of pol mutations selected for by prior non-suppressive
regiments of zidovudine and didanosine in viral isolates from latently infected
cells in 5 of the 6 children who were subsequently treated with other fully
suppressive regiments of HAART for one year or more. However, except for a
single V32L mutation in one child on ritonavir, no new primary mutations coding
for phenotypic drug resistance to the protease or reverse transcriptase
inhibitor in the suppressive regimen were detected.
Conclusions: This study demonstrated that HIV-1 persists
latently in replication-competent forms in the resting CD4+ T lymphocytes of
HIV-1 infected children despite prolonged treatment with HAART, and after immune
reconstitution. Replication-competent virus with mutations coding for drug
resistance generated by previous therapy with non-suppressive regiments persists
in this compartment despite more than one year of a fully suppressive regiment
of HAART. This reservoir may prove to be a major obstacle to HIV-1 eradication
in children and may serve to store drug resistant mutants selected by previous
non-suppressive therapy, thereby rendering untenable the notion of reusing drugs
that were part of failed regiments.