Download Use of Tissue Culture—Amplified Human Immunodeficiency Virus

173
CORRESPONDENCE
Use of Tissue Culture–Amplified Human
Immunodeficiency Virus Type 1 to Study
Evolutionary Changes In Vivo
To the Editor—We would like to comment on the recent report
by Dykes et al. [1] regarding the evolution of the human immunodeficiency virus (HIV) type 1 env gene after treatment
with nonnucleoside reverse-transcriptase inhibitors (NNRTIs)
and to provide a note of caution regarding the use of tissue
culture–amplified HIV-1 to study short-term viral evolution in
vivo. Determining the effect of drug-resistance selection on the
evolution of a linked gene, such as env, can provide information
regarding the effective population size of HIV-1 in vivo and its
potential compartmentalization into anatomical sites where antiviral drug activity may vary [2, 3]. Dykes et al. [1] found no
significant correlation between NNRTI therapy and changes in
env sequence variants using DNA heteroduplex tracking assays
and therefore concluded that these treatments had little impact
on HIV-1 env evolution.
We offer 5 possible explanations for why their conclusion differs from those of previous studies showing significant changes
in envelope gene quasi species during the early phase of virus
load rebound after failed protease inhibitor monotherapies [3–5].
First, as noted by Dykes et al. [1], because the level of drug
activity in various tissues may differ between classes of drugs, it
is possible that the effect of NNRTI selection on the evolution
of the env loci differed from that of protease inhibitor resistance
selection. Second, Dykes et al. [1] analyzed the proviral quasi
species in peripheral blood mononuclear cells (PBMC), whereas
the other studies analyzed the plasma viral RNA quasi species
[3, 4]. Proviral DNA from PBMC represent HIV-1 variants integrated at unknown times in the past; for some proviruses, this
integration might have occurred as early as the acute infection
stage [6–8]. Plasma viral RNA load decreases within hours of
initiating antiviral therapies and is the first viral compartment to
show the appearance of drug resistance mutations [6]. Therefore,
viral RNA variants in plasma reflect the actively replicating virus
population, whereas PBMC proviral DNA represent largely archival HIV-1 variants [6–8]. Third, the proviral quasi species
analyzed by Dykes et al. were generated after in vitro activation
of PBMC and tissue culture amplification of HIV-1. Although
tissue culture amplification selects for the outgrowth of replication-competent variants, tissue culture is also known to rapidly
perturb and often reduce the diversity of the original PBMC
proviral quasi species [9, 10]. Fourth, only data collected at 2
time points within a 4-month period were analyzed. The rapid,
but mostly transient, changes previously detected would not have
been apparent with such sparse sample collection [3]. Last, sufficient (i.e., reproducible) sampling by polymerase chain reaction
(PCR) of the quasi species was not verified, nor was the PCR
input copy number quantified; thus, the extent to which the het-
eroduplex tracking assay patterns reflected the composition of
the quasi species under study is questionable [3, 11].
Therefore, because the variants analyzed were proviruses derived in vitro by tissue culture amplification, they may not
reflect the virus population evolving in vivo under NNRTI selection. The analysis of plasma viral RNA would have been
more relevant for this particular study. The origin of the drugresistant genotypes listed by Dykes et al. (table 1 [1]) was not
described in the text, but if these drug-resistant genotypes were
derived from plasma viral RNA, they may differ from the genotypes of the PBMC-derived samples used for the envelope analysis. Thus, because the original time of formation of the PBMC
proviruses reactivated by Dykes et al. cannot be readily determined, the extent to which their sequences reflect the evolutionary changes taking place after the addition of NNRTI to
failing regimen is uncertain.
Laurence Doukhan and Eric L. Delwart
Blood Centers of the Pacific and Department of Medicine,
University of California, San Francisco
References
1. Dykes C, Mootsikapun P, Dexter A, et al. Analysis of env sequence evolution
in human immunodeficiency virus–infected patients receiving therapy with
nonnucleoside reverse-transcriptase inhibitors. J Infect Dis 2000; 182:316–20.
2. Leigh-Brown A, Richman DD. HIV-1: gambling on the evolution of drug
resistance. Nat Med 1997; 3:268–71.
3. Delwart EL, Heng P, Neumann A, Markowitz M. Rapid, transient changes
at the env locus of plasma HIV-1 populations during the emergence of
protease inhibitor resistance. J Virol 1998; 72:2416–21.
4. Ibanez A, Clotet B, Martinez MA. Human immunodeficiency virus type 1
population bottleneck during indinavir therapy causes a genetic drift in
the env quasispecies. J Gen Virol 2000; 81:85–95.
5. Nijhuis M, Boucher CA, Schipper P, Leitner T, Schuurman R, Albert J.
Stochastic processes strongly influence HIV-1 evolution during suboptimal
protease-inhibitor therapy. Proc Natl Acad Sci USA 1998; 95:14441–6.
6. Wei X, Ghosh SK, Taylor ME, et al. Viral dynamics in human immunodeficiency virus type 1 infection. Nature 1995; 373:117–26.
7. Finzi D, Hermankova M, Pierson T, et al. Identification of a reservoir for HIV1 in patients on highly active antiretroviral therapy. Science 1997; 278:
1295–330.
8. Chun TW, Carruth L, Finzi D, et al. Quantification of latent tissue reservoirs
and total body viral load in HIV-1 infection. Nature 1997; 387:183–8.
9. Delwart EL, Pan H, Sheppard HW, et al. Slower evolution of HIV-1 quasispecies during progression to AIDS. J Virol 1997; 71:7498–508.
10. Meyerhans A, Cheynier R, Albert J, et al. Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations. Cell 1989; 58:
901–10.
11. Delwart EL, Gordon CJ. Tracking changes in HIV-1 envelope quasispecies
using DNA heteroduplex analysis. Methods 1997; 12:348–54.
Reprints or correspondence: Dr. Eric L. Delwart, Blood Centers of the Pacific,
Dept. of Medicine, University of California, San Francisco, 270 Masonic Ave.,
San Francisco, CA 94118 ([email protected]).
The Journal of Infectious Diseases 2001; 183:173
q 2001 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2001/18301-0026$02.00