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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3105-3114
Virological and Immunological Features of Long-Term Human
Immunodeficiency Virus-Infected Individuals Who Have Remained
Asymptomatic Compared With Those Who Have Progressed to Acquired
Immunodeficiency Syndrome
By
Edward Barker,
Carl E. Mackewicz,
Gustavo Reyes-Terán,
Akihiko Sato,
Sharon A. Stranford,
Sue H. Fujimura,
Cindy Christopherson,
Sheng-Yung Chang, and
Jay A. Levy
From the Department of Medicine, University of California, San
Francisco; Departamento de Investigacion en Microbiologia, Instituto
Nacional de Enfermeda des Respiratorias, Colonia Seccion, Mexico City,
Mexico; Shionogi Institute for Medical Science, Osaka,
Japan; and Roche Molecular Systems Inc, Alameda, CA.
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ABSTRACT |
Infection with the human immunodeficiency virus (HIV) leads to a
decrease in CD4+ T cells and disease progression within a
decade of seroconversion. However, a small group of infected people,
despite being infected by HIV for 10 or more years, remain clinically
asymptomatic and have stable CD4+ cell counts without
taking antiretroviral medication. To determine why these individuals,
known as long-term survivors (LTS), remain healthy, the hematological
profiles, viral load and properties, HIV coreceptor genotype, and
anti-HIV immune responses of these people were compared with those of
individuals who have progressed to disease (Progressors) over the same
time period. Unlike Progressors, LTS have a low circulating viral load
and a low number of HIV-infected cells. These differences in the levels
of the viral load were not associated with a dominant biologic viral
phenotype, varying growth kinetics of the virus, mutation in the
cellular CCR5 gene, or the presence of neutralizing antibodies.
Importantly, the difference in viral load could be explained by the
enhanced ability of CD8+ cells from LTS to suppress HIV
replication.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
STUDIES SUGGEST THAT 75% of human
immunodeficiency virus (HIV) infections will lead to either a
symptomatic clinical state or acquired immunodeficiency syndrome (AIDS)
within 10 years.1-3 Typically, these individuals have a
range of immune defects, high viral loads, and decreasing
CD4+ cell counts.4 However, about 20% of
infected people will remain asymptomatic for more than 10 years2 and a quarter of these will have stable
CD4+ cell counts above 500 cells/µL without taking any
antiretroviral medication.5 This latter group of
asymptomatic individuals, who have been classified as long-term
survivors (LTS)6 or long-term nonprogressors,7,8 represent an important group to study because of their potentially unique virological and immunological features. A number of parameters have been examined, but only a few
characteristics have been found associated with a long-term healthy
state after HIV infection. These include a correlation of a long-term
asymptomatic clinical state with a low viral load,7,8 a
32-bp deletion in the CCR5 coreceptors gene,9 or
differences in the biological features of the virus (eg, noncytopathic
and cytopathic).10-12 Determination of attributes unique to
LTS may provide insight into approaches to prevent the development of AIDS. The current studies were undertaken to evaluate several virological and immunological parameters in a well-defined group of LTS
compared with an age-, race-, and sex-matched group of Progressors
infected for the same time period (10 years).
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MATERIALS AND METHODS |
Study subjects.
All HIV seropositive individuals in this study were infected with HIV
for 10 years or longer. During the initial visit a medical history was
taken. Subjects were monitored every 2 to 3 months for changes in
symptoms. A physical examination and the hematological profile,
including CD4+ and CD8+ cell counts, were
assessed at the time of each visit. HIV seronegative blood donors were
provided by Irwin Memorial Blood Centers (San Francisco, CA) or
randomly selected volunteers at the University of California at San
Francisco (UCSF). All studies were approved by the Committee on Human
Research, UCSF.
Blood samples.
Blood from LTS and Progressors was collected by venipuncture into
Vacutainer tubes containing either EDTA or sodium heparin (Becton
Dickinson, Franklin Park, NJ). EDTA-treated blood was used for complete
blood and differential cell counts and flow cytometric studies. Blood
containing heparin as an anticoagulant was used for the virological and
immunological assays. All plasma samples were obtained and frozen away
( 70°C) within 2 hours of drawing the blood from the subjects
to prevent the loss of infectious virus.13
Flow cytometry.
Lymphocyte and CD8+ cell populations in the peripheral
blood of LTS and Progressors were analyzed by flow cytometry using
dual-color direct immunofluorescent staining of blood followed by red
blood cell (RBC) lysis.14 A single laser flow cytometer
(FACScan; Becton Dickinson), which discriminates forward and side light scatter, and two-color fluorescence was used with the Lysys II computer
software program (Becton Dickinson) for analysis. Lymphocyte gates were
confirmed by anti-CD45 and anti-CD14 fluorochrome antibody combination.
Complete blood counts and differentials were performed by the Clinical
Laboratories at UCSF using standard procedures.
Isolation of peripheral blood mononuclear cells, CD4+
cells, and CD8+ cells.
Peripheral blood mononuclear cells (PBMC) were obtained by
Ficoll-Hypaque (Sigma, St Louis, MO) gradient centrifugation of heparinized venous blood.15 The CD4+ and
CD8+ cells were isolated from PBMC by positive selection
using magnetic beads bearing anti-CD4 and anti-CD8 monoclonal
antibodies (MoAbs) (Dynal, Lake Success, NY).16 Beads were
removed from the cells by Detach-a-bead (Dynal) according to the
manufacturer's instructions. The purity of the cells obtained by the
immunomagnetic bead isolation procedure was  95% CD4+,
<1% CD8+, <1% CD19+, <1%
CD56+, and <1% CD14+ for CD4+
selected cells and 95% CD8+, <1% CD4+,
<1% CD19+, <1% CD56+, and <1%
CD14+ for CD8+ selected cells as determined by
flow cytometry.14
Culture medium and reagents.
RPMI 1640 medium (BioWhittaker; Walkersville, MD) supplemented with
10% heat-inactivated (56°C, 30 minutes) fetal bovine serum (FBS)
(Gemini Bioproducts, Calabasas, CA), 2 mmol/L glutamine (BioWhittaker),
100 U/mL penicillin, and 100 µg/mL streptomycin (BioWhittaker) was
used as culture medium. PBMC and purified CD4+ cells (3 × 106/mL) were activated with 3 µg/mL of
phytohemagglutinin (PHA; Sigma) in culture medium containing 10%
natural IL-2 (T-stim [Collaborative Biomedical Products, Bedford, MA]
with  20 U/mL of IL-2) or 10 U/mL of recombinant human IL-2
(Collaborative Biomedical Products). To help facilitate infection of
PBMC and CD4+ cells, polybrene (Sigma; 2 µg/mL) in the
culture medium was added to the cells (3 × 106/mL) 30 minutes before inoculation of virus.
Virus levels in the plasma of LTS and Progressors.
HIV RNA levels in the plasma of LTS and Progressors were determined
using quantitative-competitive polymerase chain reaction (QC-PCR),
described previously.17 The lowest level of detection of
virus load in the plasma using this assay is 50 viral RNA copies/mL. The level of infectious virus in the plasma of LTS and Progressors was
determined as reported.13 The p24 antigen levels in the plasma of the study subjects were measured by p24-specific
enzyme-linked immunosorbent assay (ELISA) according to the methods
outlined by the manufacturer (Coulter, Miami, FL).
Infectious center assay.
The frequency of infected cells in the peripheral blood of LTS and
Progressors was determined using the infectious center assay.18,19 The results are presented as the frequency of
infected cells in total PBMC which induced a positive reverse
transcriptase (RT) activity20 (104 cpm/mL of
culture fluid) in the cultured target human PBMC.
Isolation and determination of the biological phenotype of primary
HIV isolates.
Two tissue culture systems (termed A-culture and B-culture) were used
to measure the production of HIV from PBMC of LTS and Progressors.21 Briefly, in the A-culture, the PBMC of
HIV-infected individuals were activated by PHA for 3 days followed 4 days later by the addition of PHA-stimulated PBMC from HIV-seronegative
donors. In the B-culture, the PBMC from HIV-infected subjects (not
activated in vitro) were cocultured with 3 × 106
PHA-stimulated PBMC from uninfected donors. Primary virus isolates obtained from both these cultures were expanded in the PBMC from normal
donors as described elsewhere.22 The 50% tissue culture infectious dose (TCID50) of a virus isolate was determined
as described.23 The biological phenotype of the virus
isolated from LTS and Progressors was determined by inoculation onto
MT-2 cells.11,24 The virus was classified as
syncytium-inducing (SI) phenotype if the inoculated MT-2 cells had a
diameter of greater than 3 normal cells and the culture had RT
activity20 of >104 cpm/mL of culture fluid.
Detection of the CCR5  32 mutation.
Cells from each clinical specimen were resuspended in extraction buffer
(10 mmol/L KCl, 10 mmol/L Tris-HCl, pH 8.3, 0.05% Tween 20, 0.05%
NP40, 0.1 mg/mL Proteinase K) to a final cell density of 2 × 103 cells/µL and incubated at 100°C for 30 minutes.
The cell lysate was then diluted 10-fold with specimen extraction
buffer without Proteinase K and stored at 70°C. Each
100-µL polymerase chain reaction (PCR) consisted of a cell lysate
from 104 cells or 50 ng genomic DNA in 50 mmol/L KCl, 10 mmol/L Tris-HCl, pH 8.3, 2 mmol/L MgCl2, 0.1 mmol/L dATP,
dGTP, dCTP, 0.2 mmol/L dUTP, 0.4 µmol/L each primer, SYC658 and
SYC659, 5 U AmpliTaq (Perkin-Elmer Corp, Norwalk, CT), and 2 U Amperase
UNG (Perkin-Elmer). PCR conditions were 50°C for 2 minutes, 95°C for 1 minute, followed by 30 cycles of 95°C for
20 seconds and finally holding at 72°C, using the Perkin-Elmer
Thermocycler 9600. A 177-bp and a 145-bp PCR product were
amplified from wild-type (+) allele and 32 allele, respectively. The
CCR5 32 mutation was determined by analyzing 5 µL of each PCR
reaction on a 3% NuSieve (FMC Bioproducts, Rockland, ME)
and 1% agarose gel. The frequency of homozygous mutations in the CCR5
gene in the uninfected population has been reported to be
1%.25
Antibody neutralization assay.
The ability of plasma to neutralize primary HIV isolates was determined
as described.26 The neutralization of HIV was assessed in
duplicate wells using primary virus isolates (100 TCID50)
mixed with serially diluted autologous or heterologous plasma. Plasma from some HIV-infected subjects was tested against more than one heterologous virus isolate. Plasma from HIV-seronegative donors was
used as negative controls. Neutralization of virus by plasma was
considered positive if a 66% reduction was observed in the RT
activity, compared with the RT activity of control cultures containing
plasma from HIV uninfected individuals.
CD8+ cell noncytotoxic anti-HIV response.
The extent to which the CD8+ cells from an infected subject
can suppress HIV replication was determined by an acute infection assay27 using the cytopathic, syncytium-inducing, and
 -chemokine-insensitive HIV-1SF33 strain.28
This assay was chosen to measure CD8+ cell antiviral
activity instead of an endogenous assay (ie, using naturally infected
cells)16 because of the difficulties of obtaining CD4+ cells from Progressors. Briefly, PHA (3 µg/mL)
stimulated CD4+ cells from HIV-seronegative donors were
infected with 10,000 × TCID50 of
HIV-1SF33. After 1 hour of incubation, the
CD4+ cells were washed three times and mixed with
CD8+ cells that were isolated from PBMC stimulated with PHA
for 3 days before the acute assay was conducted. The acute assay was performed in the presence of 100 U/mL of recombinant human
interleukin-2 (IL-2; Collaborative Biomedical Products). The ability of
CD8+ cells to suppress HIV replication in 5 × 105 infected CD4+ cells was
evaluated over a range of CD8+:CD4+ cell ratios
from 0.25:1 to 4:1 (twofold dilutions) in 24-well tissue culture plates
(Falcon, Lincoln Park, NJ). Culture fluid samples taken every 3 days
were monitored for RT activity. The percent suppression was determined
by comparing the RT activity in the culture fluids of the
CD8+ and CD4+ cell cocultures with the RT
activity of fluids from the infected CD4+ cells cultured
alone at the time of peak virus production (6 days following initiation
of the assay).16,27 The RT activity from the
control-infected cultures was always 105 cpm/mL of
culture fluid.
Statistical analyses.
The Mann-Whitney U-test was used to determine statistical significance
of all studies except the evaluation of neutralizing antibodies and
32 mutations in CCR5 which used the Fisher Exact Test. Spearman's
coefficient of rank correlation was used to compare the
CD8+ cell antiviral response with the viral load of LTS and
Progressors. P values of  .05 were considered statistically
significant.
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RESULTS |
Characteristics of the study subjects.
The subjects involved in this study were between 30 and 65 years old
with a median age of 44. Most were white and nearly all were male. All
of the subjects were infected for at least 10 years spanning the same
period in time (±2 years) (Table 1).
During their infection period, LTS remained clinically healthy with a stable CD4+ cell count above 500 cells/µL (Table 1 and
Fig 1A). All Progressors at the time of the study had an
AIDS diagnosis based on the 1993 Centers for Disease Control (USA) case
criteria29 with <200 CD4+
cells/µL (Fig 1). Progressors either had a slow decrease
in CD4+ cell counts since infection (Fig 1B) or a high
level of CD4+ cells for about 8 years followed by a
decrease (Fig 1C). None of the LTS had received antiretroviral drugs
(Table 1). Seventeen of the 21 Progressors were taking antiretroviral
medication (nucleoside reverse transcriptase inhibitors), which were
taken alone or in combination (Table 1). None of the subjects was
taking HIV protease inhibitors or nonnucleoside reverse transcriptase
inhibitors. Subjects from these groups of LTS and Progressors were
chosen at random for all the studies described.
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| Fig 1.
CD4+ cell counts of LTS and
individuals who progressed to AIDS (Progressors). Examples of
CD4+ cell counts over a 10-year period in the peripheral
blood of LTS (A) and Progressors (B and C) are provided. The hatched
region represents the range of CD4+ cell counts of
uninfected individuals.
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Hematological and lymphocyte profiles of LTS and Progressors.
LTS had complete blood counts (CBC) within the range found in healthy
HIV seronegative individuals (Table 2). In
contrast, except for platelets (187 × 109/L), the CBC
of Progressors were below normal and differed significantly from that
of LTS (P < .02). Although the differential cell counts of
these two clinical groups were still within the normal range, LTS had
higher neutrophil, lymphocyte, monocyte, and basophil counts than the
Progressors (Table 2). The eosinophil count was lower in LTS than
Progressors but not significantly (P = .63).
Within the lymphocyte population, the percent of CD19+
cells (B lymphocytes) was similar among LTS, Progressors, and
HIV-seronegative donors (Table 3). A lower
percentage of CD56+/CD16+ lymphocytes (natural
killer cells) was noted in the LTS compared with the Progressors, but
this difference was not statistically different (P = .11). A
higher percentage of T cells was found in LTS compared to Progressors
and seronegative donors (mean percent CD3+ cells = 81%
v 71% [P = .03]). The higher percentage of
CD3+ cells in LTS compared with the HIV seronegative donors
reflected the increased percentage of CD8+ cells (mean
CD8+/CD3+ cells of 46% v 28%,
respectively). The percent of CD4+ T cells in the
lymphocyte population was significantly higher (P < .01) in
LTS (mean percent CD4+/CD3+ cells = 35%)
compared with Progressors (mean percent
CD4+/CD3+ cells = 4%). In addition, the mean
absolute CD4+ cell count of LTS (834 cells/µL) was
significantly higher (P < .01) than that of Progressors (51 cells/µL) (Table 3). In contrast to the CD4+ cells, the
percentage of CD8+ T cells in LTS (mean percent
CD8+/CD3+ cells = 46%) was significantly lower
(P = .03) than Progressors (mean percent
CD8+/CD3+ cells = 63%), although the mean
absolute CD8+ cell counts for both groups (1,122 v
812 cells/µL) did not differ significantly (P = .15).
Of the various CD8+ cell subpopulations analyzed
(Table 4), only the percent of
CD38+ cells differed significantly between LTS and
Progressors. In LTS, the mean percent of CD8+ which were
CD38+ was 62%, similar to the mean percent of
CD8+ cells which were CD38+ in HIV uninfected
donors (65%) (Table 4). Of the CD8+ cells from
Progressors, 96% expressed the CD38 molecule which was statistically
different from LTS (P < .01). Moreover, a lower percentage of
CD8+ cells expressed HLA-DR in LTS compared with
Progressors but this difference was not statistically significant
(P = .06). In summary, aside from differences in the percentage
of CD8+ cells, the LTS in contrast to the Progressors had a
significantly lower number of CD8+ cells expressing the
CD38 molecule.
Quantification of virus in the plasma of LTS and Progressors.
Virus levels in plasma were measured by two procedures:
QC-PCR,17 which measures virus particles by the level of
HIV RNA, and an infection assay,13 which measures the level
of infectious virus particles in the plasma. Using the QC-PCR
procedure, the LTS were shown to have significantly lower levels
(P < .01) of HIV RNA in the plasma (mean of 9.5 × 103 copies/mL) compared with Progressors (mean
of 3.6 × 105 RNA copies/mL)
(Fig 2). Two of the 12 LTS tested had <5 × 102 RNA copies/mL. All Progressors had levels 9.8 × 104 RNA copies/mL.
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| Fig 2.
Level of viral RNA detected in plasma of LTS and
individuals who progressed to AIDS (Progressors). Plasmas from the
peripheral blood of 10 LTS and 13 Progressors were recovered and stored
at 70°C within 2 hours of venipuncture. Each plasma was then
evaluated for the level of virus using quantitative-competitive
PCR.17 Each point represents the level of HIV in the plasma
from a different individual. Bars represent the mean value for the
plasma of each group of individuals tested. The level of HIV in the
plasma of LTS and Progressors was statistically different (P < .01) using the Mann-Whitney U test.
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As measured by the infection center assay, the LTS did not have
detectable infectious virus in their plasma whereas HIV was detectable
in the plasma of all Progressors (Table 5).
The levels of infectious virus in the plasma of the Progressors varied
(from titers of 2 to 32) (Table 5).
Another means of determining the level of HIV in the plasma is by
measuring the level of HIV p24 protein. In the plasma of 11 LTS tested
the viral p24 antigen was below the limit of the assay used in our
study (<5 pg of p24 HIV antigen/mL) and was found to be present in 4 of 7 Progressors (Table 5). The amount of p24 antigen found in the
plasma from the 4 Progressors ranged from 74.4 to 448.9 pg/mL. Overall,
our studies of virus particles in the plasma showed that in comparison
to Progressors, LTS had a lower level of cell-free virus in their
plasma and none was infectious in cell culture.
Ability to recovered virus from the PBMC and the number of infected
cells in the peripheral blood of LTS and Progressors.
The A-culture and B-culture, two standard tissue culture techniques
used to isolate HIV from cells in the peripheral blood,21 were used to isolate virus, in vitro, from LTS and Progressors (see
Materials and Methods). None of the PBMC from 10 LTS tested produced
detectable virus in the A-culture (mean RT activity of 2.1 ± 1.3 × 103 cpm/mL) (Fig 3A).
With the B-cultures, the PBMC from 7 of 10 LTS released HIV (mean RT
activity of 110.3 ± 137.3 × 103 cpm/mL) (Fig 3B).
All Progressors produced HIV in both the A- and B-culture (mean RT
activity = 58.3 ± 35.4 and 285 ± 215.6 × 103
cpm/mL, respectively) (Fig 3A and B). In comparison with the PBMC from
Progressors, the PBMC of LTS produced significantly lower levels of
virus in B-cultures (P = .02).
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| Fig 3.
Production of HIV in culture fluids of PBMC from LTS and
individuals who progressed to AIDS (Progressors). Two isolation methods
[A-culture (A) and B-culture (B)] were used to detect HIV production
from PBMC of LTS and Progressors. In the A-culture, the PBMC from each
individual were stimulated with PHA for 3 days. Seven days later,
PHA-activated PBMC from HIV-seronegative donors were added to these
cultures. In the B-culture, PHA-activated PBMC from HIV-seronegative
donors were added to unstimulated PBMC from LTS and Progressors.
Culture fluids were monitored every 3 or 4 days for RT
activity.20 Culture fluids were considered to contain HIV
if the level of RT activity was 104 cpm/mL. The amount
of HIV in the culture fluids of A- and B-cultures of LTS and
Progressors was found to be statistically different (P < .01 and P = .02, respectively) using the Mann-Whitney U test.
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To determine the levels of infected PBMC in LTS and Progressors,
infectious center assays were used.18,19 The LTS had almost a 2 log10 lower mean frequency of HIV-infected cells (1 in
9.5 × 105) in the PBMC than did Progressors (1 in 2 × 104 [P = .01])
(Fig 4). In 7 of 17 LTS examined, the
frequency of infected cells was less than 1 in 106 PBMC. In
contrast, the lowest frequency of infected cells found in Progressors
was 1 in 105 cells (Fig 4). Thus, the number of
infected cells in the peripheral blood was at least 50 times lower in
LTS than Progressors. Overall, our studies indicate that like the
levels in the plasma (Fig 2 and Table 5), a lower frequency of the
infected peripheral blood cells are present in LTS relative to cells in
the Progressors.
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| Fig 4.
Frequency of infected cells in PBMC of LTS and
individuals who progressed to AIDS (Progressors). PBMC from LTS and
Progressors were serially diluted 10-fold and cultured with
PHA-stimulated PBMC from HIV seronegative donors. The cocultures were
evaluated for the lowest dilution of cells which produced positive
particle-associated reverse transcriptase activity20
(104 cpm/mL) in the culture fluids over a 30-day culture
period. Each point represents the result from a different individual.
Bars represent the mean value for the data obtained from the two groups
of individuals tested. The number of infected cells in the peripheral
blood of LTS and Progressors was statistically different (P = .01) using the Mann-Whitney U test.
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Biological properties of virus isolated from the peripheral blood of
LTS and Progressors.
The majority of primary virus isolates from PBMC of the LTS (9 of 10)
did not induce syncytium formation in MT-2 cells
(Table 6), indicating an non-SI (NSI)
phenotype. Infectious virus from the PBMC of 44% of the Progressors
was of the NSI phenotype whereas 56% was of the SI phenotype.
From the plasma of all eight Progressors evaluated, the infectious
virus recovered was of the SI phenotype (Table 6). Notably, the
biologic phenotype of HIV present in the plasma did not always correlate with the virus from the PBMC; HIV isolated from the plasma of
two Progressors tested had virus from their PBMC which was of the NSI
phenotype.
Despite the differences in the frequency of NSI and SI viruses obtained
from PBMC of LTS and Progressors, the replication kinetics in normal
PBMC of the primary isolates from LTS (Fig 5A) and Progressors (Fig 5B) did not differ substantially. Thus, the
biological properties of the virus isolated from LTS and Progressors may not explain the differences in the levels of virus observed between
the two groups.
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| Fig 5.
Kinetics of replication of primary HIV isolates from LTS
and individuals who progressed to AIDS (Progressors). The ability of
primary HIV isolates from 6 LTS (A) and 6 Progressors (B) to replicate
in PHA-stimulated PBMC of HIV seronegative donors was evaluated over a
2-week period. The PBMC were infected with 100 TCID50 of
virus, and replication of virus was indicated by the level of reverse
transcriptase activity in the culture fluids.20
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Prevalence of the CCR5 32 mutation in LTS and Progressors.
Differences in the level of HIV found in LTS and Progressors (Figs 2
and 3, Table 5) could be explained by the variability in the expression
of HIV coreceptors. The presence of the 32 mutation in the CCR5 gene
has been shown to be associated with a favorable clinical state in some
infected individuals.30 In this study we evaluated whether
the presence of the 32 mutation in the CCR5 gene of PBMC could
explain the different levels of HIV in LTS and Progressors (Table 7).
In the LTS group, 8 of 21 individuals were heterozygous for the 32
deletion in the CCR5 gene. In contrast, only 3 of 20 Progressors tested
had this mutation. This difference was not significantly different
(P = .12). Moreover, no statistical difference was observed between the LTS and a group of HIV seronegative donors in which 5 of 24 subjects had a 32 deletion in one copy of the CCR5 gene (P = .12). None of the infected subjects tested had mutations in both copies
of the gene encoding CCR5.
Neutralizing antibody in LTS and Progressors.
The level of neutralizing antibodies in the plasma could also explain
the different levels of virus present in the plasma and peripheral
blood cells of LTS and Progressors. The number of LTS (5 of 7) and
Progressors (5 of 6) whose plasma was capable of preventing the
replication of autologous virus was similar (Table 8). However, plasmas from 3 of the 7 LTS and none of the 6 Progressors tested was able to neutralize
autologous virus at dilutions of 1:50 or more, although the difference
observed was not significant (P = .1). The number of LTS whose
plasma was capable of neutralizing heterologous virus at dilutions of
1:10 or less was slightly greater against virus from LTS compared with
virus from Progressors (82% v 72%). Conversely, the number of
Progressors whose plasma samples were able to neutralize heterologous
virus was slightly greater if the virus was isolated from a Progressor compared with virus from an LTS (70% v 56%, respectively)
(Table 8).
CD8+ cell noncytotoxic anti-HIV activity in LTS and
Progressors.
CD8+ cells from HIV-infected individuals have the ability
to suppress HIV replication without killing the infected
cell.31 This CD8+ cell noncytotoxic antiviral
response has been shown to correlate with the clinical state of the
infected individuals.16,32 Progression to disease
correlates with a decrease in this CD8+ cell
activity.33 Here we evaluated whether the variability in
the level of HIV in LTS and Progressors can be associated with differences in the antiviral activity of their CD8+ cells.
Using the acute assay system we evaluated the extent to which
CD8+ cells can suppress HIV replication in CD4+
cells infected with the -chemokine-insensitive cytopathic
HIV-1SF33 strain. An average
CD8+:CD4+ cell ratio of 1:1 was needed for 90%
suppression of HIV replication by CD8+ cells from LTS
(Fig 6) whereas a mean ratio of 3:1 was
needed for the CD8+ cells from Progressors to suppress HIV
replication to a similar level (P < .01). The
CD8+:CD4+ cell ratio needed to suppress HIV
replication correlated directly with the viral load of the infected
individual. (Spearman coefficient of rank correlation; P < .01).
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| Fig 6.
Suppression of HIV replication by CD8+
cells from LTS and individuals who progressed to AIDS (Progressors).
The ability of CD8+ cells from 14 LTS and 14 Progressors
to suppress HIV-1SF33 replication in CD4+
cells was determined in an acute infection assay over a range of
CD8+:CD4+ cell ratios of twofold dilutions
ranging from 0.25:1 to 4:1. The level of antiviral activity of
CD8+ cells shown represents the lowest
CD8+:CD4+ cell ratio that achieved 90%
reduction of RT activity relative to that observed in the fluid of
CD4+ cells cultured alone. Each point represents the
result from a different individual. A
CD8+:CD4+ cell ratio greater than 2:1 was
used in cases where the number of CD8+ cells from
Progressors was limited such that the
CD8+:CD4+ cell ratio of 4:1 could not be
tested. For statistical analysis, the anti-HIV level was considered to
be 3:1 (see Results). Bars represent the mean values of the data
obtained for each group of individuals tested. The ratio of
CD8+:CD4+ cells needed from LTS and
Progressors to suppress HIV replication by 90% was statistically
different (P < .01) using the Mann-Whitney U test.
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Because of the limitation in the number of CD8+ cells
recovered from the stimulated PBMC of 8 of the 14 Progressors,
CD8:CD4+ cell ratios of 2:1 were used. Thus, the extent
of CD8+ cell antiviral response could not be quantified
further. For example, suppression of virus replication by 90% was
not achieved in 6 of the 8 Progressors tested at a ratio of 2:1.
Therefore, when scoring for statistical analysis we chose to designate
the above as suppressing at a 3:1 CD8+:CD4+
cell ratio. The CD8+ cells from 2 of the 14 Progressors did
not suppress HIV replication by 90% at the highest
CD8+:CD4+ cell ratio tested, 4:1.
| |
DISCUSSION |
LTS represent a small subset of infected individuals (5%) who,
beyond the usual time to develop AIDS (10 years),1-3 remain healthy without receiving any antiviral medication.5
Socioeconomic or behavioral characteristics apparently do not account
for this long-term healthy state.2,34 Moreover, results
suggesting that long-term survival is associated with certain major
histocompatibility complex (MHC) alleles35,36 have not been
consistently found.37
In an attempt to define unique features of LTS which enable them to
remain clinically healthy, virological and immunological properties of
these individuals were evaluated and compared with those of
individuals, infected for the same length of time, but who have
progressed to disease. In the present studies, two well-characterized cohorts were used to assess a large number of factors involved in HIV
infection that may be uniquely associated with a long-term asymptomatic
clinical condition. Attempts were made to rule out any possible
influence of age, gender, ethnicity, or length of HIV infection on the
analyses by closely matching the two clinical groups (Table 1).
Emphasis was placed on hematological profiles and several virological
and immunological parameters. Other reports7,8,38 have
attempted to define some of the viral and immune components associated
with long-term survival, but either a limited number of subjects was
used, the subjects were infected for a shorter period of time (<10
years), or appropriate controls were not always included.
Aside from the amount of CD8+ cells and subset composition,
we found a hematological profile in LTS (Tables 2-4) resembling that of uninfected individuals. The increase in CD8+ cells
observed in infected individuals compared with HIV uninfected controls
appears to reflect the expansion of memory (eg, increased CD45R0+ and CD62L ) and activated (eg,
HLA-DR+) CD8+ cells (Table 4) associated with
people infected with HIV.39
The only notable difference in the subsets of CD8+ cells
between LTS and Progressors was a lower percentage of CD38+
cells. A significantly (P < .01) lower percentage of
CD38+CD8+ cells was found in LTS compared with
the Progressors. The percentage of CD8+ cells expressing
the CD38 molecule was similar between LTS and seronegative individuals.
These findings are in agreement with those of other investigators who
showed an increased percentage of CD38+CD8+
cells associated with disease progression in HIV-infected
individuals.40 The significance of a higher level of
CD8+ cells expressing CD38 in Progressors relative to LTS
is unclear at this time, but may reflect an increase in the number of
cytotoxic T lymphocytes41 that could lead to the lysis of
uninfected CD4+ cells.42
Examination of virological properties of LTS and Progressors indicated
that the former group of infected individuals has low plasma viral
loads (Fig 2, Table 5), low numbers of infected cells (Figs 3 and 4),
and is infected with a less cytopathic virus strain (Table 6). Some of
these observations have been reported by others in a limited number of
subjects.7,8,43 In fact, we showed in this study that even
when using optimal procedures to recover virus from the plasma,
infectious virus could not be isolated from the plasma of all the 12 LTS evaluated (Table 5). This finding was in contrast to the ability to
recover infectious virus from the plasma of all 12 Progressors that
were tested. The inability to recover infectious virus (Table 5) in LTS
despite the ability to detect HIV RNA (Fig 2) in the plasma most likely reflects a large number of defective particles found in
plasma.44 The differences in the levels of virus in the
infected individuals could not be explained by different growth
kinetics of the virus (Fig 5), differential amounts of neutralizing
antibodies (see below), or mutations in the CCR-5 receptor gene.
Although the frequency of LTS heterozygous for the 32 deletion in
the CCR5 gene was higher then those observed in the Progressors (Table 7), it could not account for the healthy clinical state of the LTS. The
majority of infected individuals were without this mutation (14 of 21 for LTS and 17 of 20 for Progressors) and the difference in the
frequency of the heterozygous mutation in CCR5 of LTS and Progressors
was not statistically significant (P = .12).
Although the results of our studies show a trend toward Progressors
having more SI virus isolates and LTS having more NSI viruses (Table
6), the characteristics of the dominant virus strain isolated from LTS
and Progressors did not completely account for the difference in the
levels of free virus and infected cells observed in our study (Figs 2
and 4). Although some results have suggested that disease progression
is associated with a change in virus phenotype from NSI type to an SI
type,10,11 almost half the primary viruses isolated from
PBMC of Progressors in our study were NSI (Table 6). Moreover, an SI
virus was recovered from an LTS subject. These results are in agreement
with the recent findings of other investigators who recovered NSI virus
in 50% of Progressors and an SI virus from an LTS.43 Thus,
the biologic phenotype of the virus does not always predict the
clinical outcome of HIV infection. It is noteworthy that in two
Progressors, the phenotype of the plasma and PBMC-derived viruses were
different (Table 6). These findings suggest that different compartments may harbor different virus phenotypes.
Because anti-HIV antibody response has been implicated as being
important in controlling the HIV infection,4 we examined the potential role of neutralizing antibodies in the plasma from LTS
and Progressors to prevent HIV replication. In contrast to other
studies,45 we used autologous virus strains to assess the
neutralizing capacity of plasma. Neutralization measured by this method
may have greater clinical relevance. An equal number of individuals
from both groups had neutralizing antibodies directed against
homologous and heterologous viral strains (Table 8). Thus, although
loss of neutralizing antibodies and appearance of neutralization escape
viruses have been associated with disease progression,46
the presence or absence of neutralizing antibodies in the present
studies did not account for the difference in the clinical outcome of
long-term HIV infection.
The extent of the CD8+ cell noncytotoxic suppression of HIV
replication (Fig 6) can provide an explanation for the low viral load
(Fig 2), stable CD4+ cell counts (Fig 1 and Table 3), and
favorable clinical outcome (Table 1) observed in LTS compared with
Progressors. This conclusion was supported by two studies described
here. In the first, A- and B-cultures of PBMC from LTS and Progressors
(Fig 3A) give an indication of the relative ability of the
CD8+ cells within the PBMC to suppress HIV
replication.31 In the A-culture, PBMC from the infected
individual are treated with the mitogen, PHA, which can activate the
CD8+ cells and increase their ability to control virus
replication.33 The absence of HIV recovery in the
A-cultures from LTS, but the presence of virus in the A-cultures of all
Progressors (Fig 3), reflects a diminished CD8+ cell
response in Progressors.21 In the B-culture, because of the
lack of PHA stimulation and the early addition of fresh permissive cells, the CD8+ cells are not usually as effective at
suppressing virus replication. In these cultures, virus was recovered
from LTS, as well as Progressors, but the level of virus produced by
the PBMC of LTS was significantly lower than that of Progressors
(P = .02). This finding could reflect the CD8+ cell
antiviral activity as well as the low number of infected cells (Fig 4).
In the second study, the acute infection assay, the purified
CD8+ cells from LTS suppressed HIV replication more
efficiently than CD8+ cells from the Progressors (Fig 6).
Moreover, the low ratio of CD8+ to CD4+ cells
needed to suppress HIV replication by 90% correlated directly with
the low viral load in the plasma of LTS (P < .01). On
average, at least three times more CD8+ cells from
Progressors compared to LTS was needed to suppress 90% of HIV
replication in CD4+ cells. In some cases, the
CD8+ cells from Progressors were unable to control HIV
replication (Fig 6).
These findings strongly support the conclusion that the ability of
CD8+ cells to suppress HIV replication is important in
controlling HIV infection, reducing the loss of CD4+ cells
and preventing progression to disease in LTS. Progressors infected for
the same length of time have a reduced capacity to control HIV
replication in culture as shown by the need for many more
CD8+ cells to suppress HIV replication (Fig 6). These
results are in agreement with previous findings demonstrating a
correlation between the ability of CD8+ cells to suppress
HIV replication and a healthy clinical state.16,33
In summary, the LTS, despite their length of infection which generally
leads to AIDS in a majority of infected individuals (>10 years), are
clinically healthy without the need for antiretroviral medication. They
usually have low levels of noncytopathic virus typically found in
asymptomatic individuals who were recently infected. But, unlike most
asymptomatic infected individuals who eventually progress to AIDS
(Progressors), LTS appear to have the ability to maintain low viral
levels, a high CD4+ cell count, and not develop disease.
The CD8+ cell noncytotoxic antiviral response seems to play
a major part in ensuring long-term survival. The results of this study
suggest that sustaining this CD8+ cell antiviral function
is important in preventing disease progression and has relevance in
studies directed at developing anti-HIV immune-based therapies.
| |
FOOTNOTES |
Submitted April 9, 1998;
accepted June 16, 1998.
Supported by a grant from the National Institutes of Health
(RO1-AI30350). G.R.-T. was the recipient of a Fogarty International Fellowship.
Address reprint requests to Jay A. Levy, MD, Department of Medicine,
Third Ave at Parnassus, University of California, San Francisco, CA
94143-1270.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Alan Landay and Janis Giorgi for their critical
review of the manuscript, John Sninsky and Shirley Kwok for their
advice and assistance on certain aspects of this study, Katharine
Bossart and Roland Orque for technical assistance, and Chris Beglinger
and Ann Murai for their assistance in the preparation of this
manuscript.
| |
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Abstract
Introduction
Abstract
32 Mutation in CCR5 From
LTS and Progressors