Download Relative Potency of Protease Inhibitors in Monocytes/Macrophages

413
Relative Potency of Protease Inhibitors in Monocytes/Macrophages Acutely and
Chronically Infected with Human Immunodeficiency Virus
Carlo-Federico Perno, Fonda M. Newcomb,
David A. Davis, Stefano Aquaro, Rachel W. Humphrey,*
Raffaele Caliò, and Robert Yarchoan
HIV and AIDS Malignancy Branch, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland; Department of Experimental
Medicine and Biochemical Sciences, University of Rome Tor Vergata,
Rome, Italy
The activity of three human immunodeficiency virus (HIV) protease inhibitors was investigated
in human primary monocytes/macrophages (M/M) chronically infected by HIV-1. Saquinavir, KNI272, and ritonavir inhibited the replication of HIV-1 in vitro, with EC50s of Ç0.5 – 3.3 mM. However,
only partial inhibition was achievable, even at the highest concentrations tested. Also, the activity
of these drugs in chronically infected M/M was Ç7- to 26-fold lower than in acutely infected M/M
and Ç2- to 10-fold lower than in chronically infected H9 lymphocytes. When protease inhibitors
were removed from cultures of chronically infected M/M, production of virus rapidly returned to
the levels found in untreated M/M. Therefore, relatively high concentrations of protease inhibitors
are required to suppress HIV-1 production in chronically infected macrophages, and such cells may
be a vulnerable point for the escape of virus in patients taking these drugs.
The essential role of the human immunodeficiency virus
type 1 (HIV-1) protease in the viral life cycle has led to the
development of a number of inhibitors of the HIV-1 protease
[1 – 12]. Several of these drugs have been found to be quite
active in patients, especially in combination with dideoxynucleosides, and their introduction into clinical practice has
resulted in an improved outlook for HIV-1 – infected patients.
Other protease inhibitors are currently undergoing preclinical
and clinical evaluation. Also, studies are ongoing to test
whether potent combination regimens that include one or more
of these compounds can eradicate HIV from the body
[13 – 15].
HIV-1 protease inhibitors act during the late stages of HIV1 replication. These compounds inactivate the HIV-1 – encoded
aspartyl protease and prevent cleavage of Gag and Gag-Pol
polyproteins and, by this action, inhibit the production of mature infectious HIV-1 virions from cells already infected with
HIV-1 [1, 2, 16]. In this respect, they differ from reverse transcriptase inhibitors, which act before formation of the provirus
and prevent acute infection [17, 18]. The use of protease inhibitors in combination with reverse transcriptase inhibitors thus
provides the ability to block viral replication at both early and
Received 2 December 1997; revised 18 March 1998.
Presented in part: 5th Conference on Retroviruses and Opportunistic Infections, Chicago, 1 – 5 February 1998 (abstract 639).
Financial support: Collaborative Research and Development Agreement between the National Cancer Institute and Japan Energy Co.; Italian Project on
AIDS from the Italian Ministry of Health.
Reprints or correspondence: Dr. Robert Yarchoan, Bldg. 10, Rm. 12N226, 10
Center Dr., MSC 1906, Bethesda, MD 20892-1906 ([email protected]).
* Present affiliation: Pharmaceutical Division, Bayer Corp., West Haven,
Connecticut.
The Journal of Infectious Diseases 1998;178:413–22
q 1998 by the Infectious Diseases Society of America. All rights reserved.
0022–1899/98/7802–0015$02.00
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late stages of replication, and such combinations have proven
highly effective in a number of clinical trials, with plasma
HIV-1 frequently becoming undetectable by RNA polymerase
chain reaction [11, 12, 19, 20]. Such potent inhibition of HIV1 replication has also been found to be associated with a decrease in the rate of development of HIV-1 resistance [21]. At
the same time, the concern remains that if one or more drugs
in a combination regimen have relatively less anti – HIV-1 activity in a particular cell type or anatomic compartment, this
may permit continued HIV-1 replication in that cell or compartment, which may lead to the emergence of resistant virus.
Therefore, it is important to understand the activity of HIV-1
protease inhibitors in various cell types and in various potential
organs in which HIV-1 might be sequestered.
A number of groups have demonstrated the ability of various
protease inhibitors to block HIV-1 production in chronically
infected T cell lines, in acute or chronically infected monocytelike cell lines, and in acutely infected monocytes/macrophages
(M/M) [2, 22 – 28]. It has been reported that protease inhibitors
have anti-HIV activity in chronically infected M/M [29], but
little information is presently available regarding the relative
activity of protease inhibitors in chronically infected M/M compared with other cell types. M/M and M/M-derived cells are
important target cells for HIV-1 infection, may produce virus
over a relatively long period of time, and appear to be the main
reservoir of HIV-1 in the central nervous system [30 – 32]. It
has been postulated that HIV-1 – infected M/M can persist for
a relatively long time, with a t1/2 of 1 – 4 weeks, after introduction of highly effective antiretroviral therapy [33]. With this
background, we undertook an investigation about the antiviral
effect of three protease inhibitors in chronically infected
M/M. The protease inhibitors used in this study were ritonavir,
saquinavir, and KNI-272, the latter being a transition-state mimetic tripeptide inhibitor of the HIV-1 protease that is currently
being investigated in clinical trials [34, 35].
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Materials and Methods
M/M and lymphocytes. Primary M/M were prepared and purified as described [36]. Briefly, peripheral blood mononuclear cells
(PBMC) were obtained from healthy HIV-1–negative donors by
the Department of Transfusion Medicine, Warren G. Magnuson
Clinical Center (Bethesda, MD). PBMC were separated over a
ficoll gradient and seeded in 48-well plates at 1.5 1 106 cells/well
in 1 mL of RPMI 1640 (Life Technologies, Gaithersburg, MD)
containing 20% heat-inactivated, low-endotoxin, Mycoplasma-free
fetal bovine serum (Hyclone Laboratories, Logan, UT), 4 mM Lglutamine (Life Technologies), 50 U/mL penicillin, and 50 mg/
mL streptomycin (Life Technologies) (hereinafter referred to as
complete medium). For some experiments, PBMC obtained by the
Blood Bank of the Frascati Hospital, Rome, were used. Complete
medium was used in all experiments; no human serum was added
in any experiment described herein. Five days after plating and
culturing the PBMC at 377C in a humidified atmosphere enriched
with 5% CO2, nonadherent cells were carefully removed with repeated washings with warmed RPMI 1640 as previously described
[36], leaving a monolayer of adherent cells, which were finally
incubated in complete medium. Cells treated under these conditions have previously been shown to be ú95% M/M, as determined
by nonspecific esterase staining and morphology [37].
H9/LAI, a CD4 T cell line chronically infected by HIV-1LAI,
was used in some experiments. To be consistent with the procedures used with M/M, the same complete medium containing 20%
fetal bovine serum was also used for H9/LAI cells, both for cell
passages and to assess the effect of antiviral drugs.
Another CD4 T cell line, MT-2, was used for experiments of
de novo (acute) infection. These cells manifest syncytium formation and cytopathic effect when infected by HIV-1, and these have
been shown to correlate with the production of HIV-1 p24 into
the supernatants [38]. MT-2 cells were also grown and passaged
in the complete medium described above. Previous studies have
shown that the activity of protease inhibitors determined in T cell
lines such as MT-2 and H9 was similar to that determined in
peripheral blood lymphocytes in cultures supplemented with fetal
calf serum [8, 39–42].
HIV-1 isolates. Three different isolates of HIV-1 were used
in this study. Unless specifically noted otherwise, a monocytotropic
isolate of HIV-1, HIV-1Ba-L, was used in all experiments involving
primary M/M. Virus was expanded and titered in primary
M/M as previously described [36]. In selected experiments, another
monocytotropic isolate of HIV-1, SRA1433, which was isolated
from the cerebrospinal fluid of an HIV-1–infected child, was used
(gift of S. Sei, NCI, NIH) [43]. After isolation, SRA1433 was
passaged and titered in M/M. Finally, a prototypic lymphocytotropic isolate, HIV-1LAI, was used to infect MT-2 cells. The same
virus is harbored in H9/LAI cells.
Anti–HIV-1 drugs. Three protease inhibitors were used in
these experiments: saquinavir (gift of I. Duncan, Roche Research
Centre, Hertfordshire, UK); KNI-272 (gift of H. Hayashi, Japan
Energy Corp., Tokyo); and ritonavir (provided by Abbott Laboratories, Abbott Park, IL). Stock solutions of each drug were made
in dimethyl sulfoxide (DMSO; Calbiochem, La Jolla, CA) and
stored at 0707C until used.
As a control, the nucleoside reverse transcription inhibitor zidovudine was used in each experiment. Also, in each experiment,
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appropriate controls were run that used concentrations of DMSO
comparable to those present in the most concentrated samples.
Assessment of antiviral drug activity: chronically infected
M/M. Two days after separation (i.e., 7 days after plating),
M/M were challenged with HIV-1Ba-L or SRA1433 by adding 300
TCID50/mL. Twenty-four hours later, all wells were carefully
washed to remove excess virus; M/M were then cultured in complete medium under the same conditions and fed every 5 days
with fresh complete medium. HIV-1 p24 release in the supernatants
was assessed every 3 days starting from day 7. On the basis of
our previous experience [37, 44], chronic infection is generally
established Ç7–10 days after viral challenge (some variation being
detectable among different donors). In the experiments described
herein, drug treatment began 14 days after infection, when HIV1 p24 production reached a plateau.
Fourteen days after viral challenge (hereinafter called day 0),
M/M were carefully washed at least twice to remove any virus
present in the supernatants, replenished with fresh complete medium containing various concentrations of saquinavir, KNI-272,
ritonavir, or zidovudine, and cultured under the same conditions
as described before. Each drug concentration was run in triplicate
or quadruplicate, while positive controls were run in sextuplicate.
Starting from day 0, all M/M-containing wells were washed daily
and replenished with fresh medium containing the appropriate drug
concentration, according to the experimental protocol.
In selected experiments devoted to assessing the activity of the
protease inhibitors following their removal, the cells were extensively washed and the medium completely changed at day 5–6
after the beginning of treatment. Half of the wells were subsequently cultured without drugs while the other half were cultured
in the presence of drugs as before.
At each established time point, Ç1 mL of each supernatant was
harvested and replaced with new medium with appropriate drugs
as before. At the end of the experiment, the M/M were harvested
and lysed for Western blot analysis (see below). Virus production
was assessed at each time point by analysis of HIV-1 p24 production with a commercially available RIA (DuPont, Wilmington,
DE). Previous experiments have shown that this assay has little
cross-reactivity with uncleaved Gag and Gag-Pol (Humphrey RW,
Perno CF, Yarchoan R, unpublished data).
Assessment of drug activity: acutely infected M/M. The procedure for assessing drug activity in acutely infected M/M was similar to that described for chronically infected M/M, with the exception that antiviral drugs were added 30 min before viral challenge.
Twenty-four hours after challenge, M/M were washed to remove
the virus inoculum, complete medium containing the appropriate
drug was replaced, and the M/M were then cultured for the duration
of the experiments. Supernatants were collected at day 14 or 15 for
assessment of virus production by analysis of HIV-1 p24 antigen as
described before. M/M were harvested and lysed for Western blot
analysis (see below). To assess drug toxicity in M/M, cells were
treated for 7–14 days in the presence of different concentrations
of saquinavir, KNI-272, and ritonavir. Cells were gently detached
from the wells as described elsewhere, and cell viability was visually assessed by trypan blue exclusion [36].
Assessment of drug activity: chronically infected H9/LAI cells.
Exponentially growing H9/LAI cells were washed at least twice
with a large excess of medium and were plated in 48-well plates
(as done for M/M) at a concentration of 3 1 104 cells/well in 1
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HIV Protease Inhibitors in Macrophages
mL of complete medium. At the same time of plating, various
concentrations of each drug were added. At established time points,
100 mL of each supernatant was removed to assess HIV-1 p24
production by RIA. Additional supernatants were collected for
Western blot. At the end of the experiment, the cells were then
harvested for Western blot analysis (see below).
Assessment of drug activity: acutely infected MT-2 cells. Exponentially growing MT-2 cells were plated in wells of a 48-well
plate in the presence or absence of various concentrations of drugs
and challenged 30 min later with 300 TCID50/mL HIV-1LAI. Viral
replication was assessed by syncytium formation assay and by
HIV-1 p24 RIA. The concentration of HIV-1 p24 antigen added
at the time of infection was subtracted from the amount assayed
at later time points.
Determination of effective drug concentrations. Within each
experiment, geometric mean p24 production was used to determine
the EC50. The EC50 was determined by linear regression of the
log of the percentage of HIV-1 p24 production (compared with
untreated controls) versus the log of the drug concentration [45].
Western blot analysis. After cells were washed with PBS
(BioWhittaker, Walkersville, MD), they were lysed with 0.75%
Triton X-100 lysis buffer containing 300 mM NaCl, 50 mM TrisHCl, pH 7.4, 2 mL/mL DMSO, and a cocktail of protease inhibitors
containing 10 mg/mL leupeptin, 20 mg/mL aprotinin, 25 mM pnitrophenyl guanidinobenzoate, and 10 mM KNI-272. After a 10min incubation in lysis buffer at 47C, the cell lysate was clarified
by centrifugation for 10 min at 10,000 rpm. Total protein concentration was determined by the BCA assay (Pierce, Rockford, IL).
Cell lysates were resuspended in SDS sample buffer containing
50 mM dithiothreitol. Cell lysate (2 mg) was electrophoresed on a
10% Bis-Tris polyacrylamide gel (Novex, San Diego), electroblotted onto nitrocellulose, and detected with a monoclonal mouse
antibody to HIV-1 p24 (Intracel, Cambridge, MA).
To assess the effects of protease inhibitors on released virus
particles, the supernatants were harvested as described previously.
Virus was pelleted by centrifugation at 22,000 rpm for 2 h at 47C.
After washing the pellet once with PBS, the virus was repelleted
under the same conditions. Virus pellets were disrupted with SDS
sample buffer containing 50 mM dithiothreitol. Supernatants from
M/M were electrophoresed on a 10% Bis-Tris polyacrylamide gel
with MES running buffer by use of the NuPage system (Novex).
H9/LAI supernatants were electrophoresed on a 12% Tris-glycine
polyacrylamide gel (Novex). Proteins were electroblotted onto nitrocellulose and were detected with a monoclonal mouse antibody
to HIV-1 p24 (Intracel).
Statistics. The differences in the EC50 in different cell populations and under different conditions of infection were assessed by
the two-tailed unpaired t test (StatView, Berkeley, CA).
Results
We first sought to determine the effect of protease inhibitors
in M/M infected de novo with HIV-1Ba-L (i.e., treated with
drugs prior to viral challenge) for comparison to M/M chronically infected with HIV-1 (figure 1). Consistent with previous
experiments, 0.1 mM zidovudine induced Ç90% inhibition of
viral replication in these acutely infected M/M. Under the same
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415
experimental conditions, all three protease inhibitors also
showed potent activity.
We then studied the activity of these protease inhibitors in
chronically infected M/M. In these experiments, treatment with
antiviral drugs was started at day 14, a time at which productive
infection had already been established [46]. Zidovudine, at a
concentration of 20 mM (Ç500-fold greater than its EC50 in
M/M infected de novo by HIV-1) [47], did not substantially
affect the production of HIV-1 p24 protein in chronically infected M/M (figure 2A). Zidovudine has little or no activity in
cells once the HIV-1 provirus is found, and this finding provided evidence that by day 14, the majority of M/M in the
cultures were infected by HIV-1. By contrast, all three protease
inhibitors showed antiviral activity in chronically infected
M/M. However, this activity was only partial; even at the highest concentrations tested, inhibition of virus in these cells never
exceeded 95%.
The effect of protease inhibitors on the production of mature
viral proteins was also assessed by Western blot analysis on
cell lysates of chronically infected M/M exposed to these drugs
(figure 2B). The band corresponding to the mature HIV-1 p24
protein was substantially reduced in intensity in M/M samples
treated with 1 mM saquinavir, 2 mM KNI-272, and 10 mM
ritonavir, in parallel with the inhibition of HIV-1 p24 antigen
production found in the supernatants of these cells by RIA
(figure 2). Similar results were obtained by Western blot analysis on the supernatants of the same chronically infected M/M
(data not shown).
We then asked whether a similar pattern of activity in
M/M could be obtained with another monocytotropic isolate
of HIV-1. To this end, we tested the antiviral activity of KNI272 in M/M chronically infected with SRA1433, a monocytotropic isolate obtained from the cerebrospinal fluid of an HIV1 – infected child and expanded by one passage in primary
M/M [43]. The EC50 determined for saquinavir using this
strain was 0.62 mM, while the EC50 for KNI-272 was 0.95
mM. These results are quite close to the EC50 determined
using HIV-1Ba-L in the same experiment: 0.55 mM for saquinavir and 0.91 mM for KNI-272.
The kinetics of virus production under treatment with protease inhibitors in chronically infected M/M is depicted in figure
3. With the active doses of each drug, a decrease in virus
production was detectable by day 1. Virus suppression was
more pronounced by day 3, and starting from this time point,
substantial inhibition of virus production was detected with 1
mM saquinavir, 2 mM KNI-272, or 10 mM ritonavir.
The antiviral effect of each protease inhibitor in chronically
infected M/M was sustained for at least up to 11 days after
treatment if the drugs were maintained in culture throughout
the whole experiment (table 1). However, when the drugs were
removed from the cultures after consistent inhibition of viral
replication had been achieved (e.g., after day 5 or day 10), virus
production rapidly returned to the levels found in untreated
M/M (results not shown).
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Figure 1. Anti – HIV-1 activity of
protease inhibitors in acutely infected macrophages. Drugs were
added to macrophages 30 min before
viral challenge and kept in culture
throughout experiments. Virus production was determined by HIV-1
p24 RIA and results represent average of 2 experiments. Error bars
show range of results. AZT, zidovudine.
We wished to compare the results obtained using chronically
infected M/M with those obtained using protease inhibitors in
lymphocytes. To this end, we first assessed the antiviral activity
of saquinavir, KNI-272, and ritonavir in acutely infected MT-
2 cells. A summary of these results, along with those in acutely
and chronically infected M/M, is shown in table 2. As can be
seen, the three protease inhibitors were quite active in MT-2
cells acutely infected with HIV-1LAI , with EC50s of 0.021,
Figure 2. Effect of protease inhibitors on virus protein production
in chronically infected macrophages.
Macrophages were infected with
HIV-1 and then treated with drugs
once chronic infection was established. A, % of virus protein production in supernatants of treated versus
untreated macrophages, assessed by
HIV-1 p24 RIA. B, Western blot
analysis of chronically infected macrophage cell lysates. Cellular extracts were prepared at same time
(day 5 after beginning of treatment)
as supernatants collected for HIV-1
p24 RIA. A, 0.25% dimethyl sulfoxide (DMSO) is final concentration
found in wells containing macrophages treated with 10 mM protease
inhibitors. AZT, zidovudine.
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Figure 3. Kinetics of HIV-1 p24 production in supernatants of chronically infected macrophages treated with no drug, protease inhibitors (saquinavir; KNI-272; ritonavir), zidovudine
(AZT), or dimethyl sulfoxide (DMSO). On day 0, drugs were added to chronically infected macrophages. Data are from single experiment (each sample run in quadruplicate) representative
of 3 different experiments.
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Table 1. Effect of protease inhibitors in chronically infected monocytes/macrophages.
HIV-1-p24 level, pg/mL (mean { SE)
Drug treatment
Control
1 mM saquinavir
10 mM KNI-272
10 mM ritonavir
Day 5
26,932
8251
4427
5215
{
{
{
{
2115
321
1114
653
Day 8
10,203
3198
2000
2397
{
{
{
{
Day 11
788
100
451
120
15,795
3797
2322
1622
{
{
{
{
1012
1405
292
560
NOTE. Data are daily virus production. Drugs were kept in culture from
day 0 for duration of experiment. Data represent 1 of 3 experiments. SE is
variation among quadruplicates (controls in sextuplicates) within same experiment.
0.059, and 0.063 mM for saquinavir, KNI-272, and ritonavir,
respectively (EC50 of zidovudine in the same cells was 0.010
mM). These results are in the range of those described by other
authors using this cellular model [27] as well as in other T
lymphocytic cell lines (such as H9 cells infected de novo by
HIV-1) [35, 40]. These EC50s were Ç14- to 52-fold lower than
the EC50 determined in chronically infected M/M (0.478, 0.844,
and 3.275 mM for saquinavir, KNI-272, and ritonavir, respectively; P õ .01 for each comparison; table 2). Also, as can be
seen in table 2, the EC50s of the drugs in acutely infected
M/M were Ç7- to 26-fold lower than the EC50 determined in
chronically infected M/M (P õ .05 for saquinavir and P õ
.01 for KNI-272 and ritonavir). Thus, protease inhibitors were
substantially less active in chronically infected M/M than in
acutely infected lymphocyte cell lines or macrophages.
We then assessed the antiviral activity of these drugs in
H9/LAI, a CD4 T cell line chronically infected with HIV-1
(figure 4). As previously reported, 20 mM zidovudine gave no
substantial inhibition of virus production in this cellular model
[46]. By contrast, inhibition of virus production was detectable
by day 3 after treatment with all of the protease inhibitors
tested and was sustained at least for 5 days (figure 4). The
EC50s of the protease inhibitors in H9/LAI cells were 0.048,
0.442, and 0.945 mM for saquinavir, KNI-272, and ritonavir,
respectively (table 2). The EC50s were 9.9-, 1.9-, and 3.4-fold
lower than those found in chronically infected M/M with saquinavir, KNI-272, and ritonavir, respectively (P õ .01 for KNI272 and P õ .05 for saquinavir and ritonavir). Moreover,
ú99% inhibition of HIV-1 p24 production was reached in
H9/LAI cells at concentrations of 0.2, 2, and 10 mM saquinavir,
KNI-272, and ritonavir, respectively (figure 4); by contrast, this
degree of inhibition of viral replication could not be achieved in
chronically infected M/M, even at 5-fold-greater concentrations
of saquinavir and KNI-272 (figure 2).
It should be noted that these results were obtained with the
same RIA used for chronically infected M/M, thus ruling out
the possibility that the p24 detected in supernatants of M/M
treated with the highest concentrations of protease inhibitors
(up to 10 days after treatment; figures 2, 3) was simply a result
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of a cross-reaction of p55 with the anti-p24 antibody used in
p24 RIA. Thus, these results indicated that the protease inhibitors were Ç2- to 10-fold less active in chronically infected
M/M than in chronically infected lymphocytes (table 2). Also,
while the results were not statistically significant for each drug,
the drugs tended to be slightly (Ç1.9- to 2.4-fold) less active
in acutely infected M/M than in acutely infected lymphocytes
(table 2).
The Western blots of lysates of H9/LAI cells treated with
protease inhibitors are also shown in figure 4. When the cell
lysates were examined, the inhibition of HIV-1 p24 antigen
release into the supernatants correlated with the disappearance
of the p24 band in the immunoblots. Similar results were obtained in Western blots of the supernatants of these H9/LAI
cells treated with protease inhibitors (data not shown). Again,
the concentrations of protease inhibitors found active in this
assay with H9/LAI cells are lower than those effective in chronically infected M/M under the same experimental conditions
(figure 2).
Toxicity of these drugs in both M/M and lymphocytes was
assessed in parallel with the above experiments. No evidence
of cell killing was found in concentrations of protease inhibitors
up to 10 mM. Neither toxicity nor substantial alteration of p24
production could be detected with 0.25% DMSO (figures 3,
4), which is the final concentration present in cell samples
treated with 10 mM KNI-272 and ritonavir. Thus, the antiviral
activity observed in these experiments can be attributed to the
effect of these protease inhibitors.
Discussion
We report herein that HIV-1 production is inhibited in vitro
by each of three different protease inhibitors in chronically inTable 2. Activity of protease inhibitors in monocytes/macrophages
and lymphocyte lines.
EC50, mM (mean { SE)
Cells
Monocytes/Macrophages
Chronically infected
Acutely infected
Lymphocytes
Chronically infected
H9/LAI cells
Acutely infected
MT-2 cells
Saquinavir
KNI-272
Ritonavir
0.478 { 0.106 0.844 { 0.051 3.275 { 0.442
0.052 { 0.004* 0.113 { 0.005† 0.125 { 0.009†
0.048 { 0.001* 0.442 { 0.008† 0.945 { 0.435*
0.021 { 0.001† 0.059 { 0.003† 0.063 { 0.002†
NOTE. For culture of chronically infected monocytes/macrophages, chronically infected lymphocytes, and acutely infected lymphocytes, EC50s were
calculated using supernatants collected 5 days after introduction of drugs; for
cultures of acutely infected monocytes/macrophages, EC50s were calculated
using supernatants collected after 14 days (to allow for infection to become
established). EC50s are based on 3 – 5 experiments each with 2 – 4 replicates.
* P õ .05 by Student’s t test compared with chronically infected monocytes/
macrophages.
†
P õ .01 by Student’s t test compared with chronically infected monocytes/
macrophages.
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419
Figure 4. Effect of protease inhibitors on viral protein production
in chronically infected H9/LAI
lymphocytic cells. Chronically infected H9/LAI cells were carefully
washed at day 0 and treated with
drugs until end of experiment. A, %
of viral protein production in supernatants of treated versus untreated
lymphocytes, assessed by HIV-1 p24
RIA. B, Western blot analysis of
chronically infected H9/LAI cell lysates. Cellular extracts were prepared at same time (day 5 after beginning of treatment) as supernatants
collected for HIV-1 p24 RIA A,
0.25% dimethyl sulfoxide (DMSO)
is final concentration found in wells
containing H9/LAI cells treated with
10 mM protease inhibitors. AZT, zidovudine.
fected M/M at clinically relevant concentrations and that this effect
correlates with the disruption of Gag and Gag-Pol polyprotein
processing. This activity was observed with two separate monocytotropic isolates of HIV-1. However, the activity of protease inhibitors in chronically infected M/M is overall several-fold lower
than that in chronically infected lymphocytes and substantially
lower than that determined in acutely infected lymphocytes [2,
22–25, 27, 28]. Moreover, we could not achieve complete inhibition of HIV-1 p24 production in chronically infected M/M, even
at the highest concentrations of drugs tested. This latter finding
agrees with recently published results by Pretzer et al. [48] determined using the protease inhibitor L-589,502 in M/M exposed to
HIV 4 days earlier. Thus, for several protease inhibitors, relatively
high concentrations are required to suppress HIV-1 production
by chronically infected M/M, and such cells may thus be relatively
more likely to escape complete HIV-1 suppression in patients
receiving these drugs.
It should be noted that antiviral assays involving acutely
infected cells reflect a drug effect amplified over several cycles
of viral replication, while the results of those involving chronically infected cells do not reflect such an amplification (because
all cells are already infected). This may account for much
of the difference between the assays involving acutely and
chronically infected cells in the present work. At the same time,
the finding of the relatively low activity of protease inhibitors in
chronically infected M/M has potential clinical applicability,
as productively infected M/M are found in HIV-infected pa-
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tients and as these cells are efficient at infecting other susceptible cells [31, 49 – 51].
It is noteworthy that the protease inhibitors were found to
be less active in chronically infected M/M than in chronically
infected lymphocytes. In both cases, the assays involve the
effects of drugs on virus production from cells already infected
with HIV-1, suggesting that this may reflect a true difference
between the cell types. It is possible that the monocytotropic
isolates used in our experiments are intrinsically less sensitive
than lymphocytotropic isolates to protease inhibitors. Previous
experiments, however, have shown that reverse transcriptase
inhibitors are equally active in macrophages infected with either lymphocytotropic or monocytotropic isolates of HIV-1
[44, 52]. In addition, the amino acid sequences of both the
reverse transcriptase and the protease of HIV-1Ba-L are nearly
identical to that of the corresponding proteins of all wild type
lymphocytotropic isolates studied, including HIV-1LAI [53, 54].
Finally, in a preliminary experiment, we have found that the
EC50 of saquinavir against HIV-1Ba-L in acutely infected primary lymphocytes is Ç0.01 mM (unpublished data), which is
quite similar to its activity in MT-2 cells acutely infected with
HIV-1LAI (table 2). Additional work needs to be done to fully
clarify this point, yet the current data suggest that variation in
the sensitivity of the HIV-1 isolates is not a major factor affecting their sensitivity to protease inhibitors.
Another possible contributing factor to these results is that
M/M produce substantially more virus progeny in culture than
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Perno et al.
do H9/LAI cells, on the basis of the amount of HIV-1 p24
released by untreated cells. Indeed, as shown in table 1 and
figure 2, between 10,000 and 35,000 pg/mL HIV-1 p24 protein
is produced daily at steady state by each well containing Ç5
1 104 M/M (the M/M do not proliferate under these culture
conditions). Similar virus production by H9/LAI cells (an actively replicating cell line) is achieved only in wells containing
Ç3 1 105 cells (data not shown). Also, HIV virions can accumulate in chronically infected M/M [55, 56]. Thus, there is
evidence that the concentrations of Gag and Gag-Pol polyproteins in chronically infected M/M are higher than in chronically
infected lymphocytes. This may reduce the relative effectiveness of protease inhibitors, similar to the relatively reduced
effectiveness of reverse transcriptase inhibitors found in experiments involving higher virus inocula [57].
Another possible explanation for the lower activity of protease inhibitors in chronically infected M/M may be related to
differences in the redox states of these cells. Macrophages are
often under oxidative stress after viral infection [58 – 60], and
this may, in turn, affect the activity of the HIV-1 protease.
Under such conditions, the HIV-1 protease may become glutathionylated in such a way as to increase protease activity [61,
62]. Such active forms of protease may be less sensitive to
protease inhibitors, and this may conceivably account for the
relatively lower activity of protease inhibitors in these cells.
These forms of the protease would more likely be present
in chronically infected macrophages than lymphocytes, and
exposure of infected cells to reactive oxygen species may lead
to activation of HIV-1 replication [63]. Future studies will be
required to address this point. Yet another possibility is that
cellular enzymes within macrophages can degrade protease inhibitors, lowering their relative concentration within those
cells.
The EC50 of the protease inhibitors for chronically infected
M/M are at concentrations that are attainable in patients receiving these drugs. In the case of ritonavir, which has good oral
bioavailability, peak plasma levels of Ç15.5 mM and trough
levels of Ç3.6 mM are attained under treatment with the currently approved regimen of 600 mg twice daily [64]. Saquinavir
is not absorbed as well orally, and at the approved dose of 600
mg three times daily, peak plasma levels are generally below
1 mM [65]. However, peak plasma saquinavir levels of 1.8 mM
can be attained if patients are given 4-fold-higher doses, and
higher saquinavir plasma levels can also be attained if the drug
is coadministered with ritonavir [10, 66]. Finally, peak plasma
KNI-272 levels of Ç3.5 mM are obtained after oral doses of
4 mg/kg [34]. Thus, the peak plasma levels of these drugs are
only about two to three times higher than the EC50 in chronically infected M/M, and trough levels fall below the EC50.
Also, as noted above, these drugs are highly bound to plasma
proteins (especially a1-acid glycoprotein), and the drugs are
thus somewhat less active in patients than predicted by the
assays here, which use 20% fetal calf serum [67, 68]. Finally,
the development of even minimal resistance to these drugs may
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JID 1998;178 (August)
result in the peak levels being lower then the EC50. Thus,
M/M may be a vulnerable point for virus escape in patients
taking these drugs, particularly in cases of poor drug compliance or administration of low drug doses.
Another issue that should be considered is that M/M are the
main target for HIV-1 in the central nervous system [30, 31].
Although protease inhibitors are lipophilic drugs, they may not
effectively penetrate the blood-brain barrier, and their levels
in the cerebrospinal fluid may be relatively low [69]. The juxtaposition of these relatively low drug levels with the relatively
high drug levels required to completely suppress HIV-1 replication in chronically infected M/M suggests that control of HIV1 in the central nervous system by protease inhibitors may not
be as complete as in other parts of the body. Protease inhibitors
are now recommended for use in combination with reverse
transcriptase inhibitors [11], which inhibit acute infection of
M/M and penetrate the central nervous system to various degrees [17, 44]. However, some nucleoside reverse transcriptase
inhibitors have relatively poor penetration through the bloodbrain barrier [70], and more important, they have little or no
activity in M/M already infected with HIV-1. Therefore, it is
conceivable that the central nervous system may serve as a
sequestered site in which continued viral replication may occur,
thus thwarting the goal of complete eradication of HIV-1 and
potentially permitting the development of resistant virus [71].
In summary, while the HIV-1 protease inhibitors tested are
active in chronically infected M/M, the EC50s are somewhat
higher than in chronically infected lymphocytes. Anti – HIV-1
therapy has evolved to the point at which a principal goal is
to suppress HIV-1 replication as completely as possible, ideally
to below the levels of detection [11]. Therefore, it is of interest
to identify cells or anatomic sites in which HIV-1 infection
may be incompletely suppressed, possibly permitting the emergence of resistant virus. The results of this study suggest that
M/M bear further study in this regard.
Acknowledgment
We thank Hiroaki Mitsuya for helpful discussions.
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