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Journal of Virology, November 2000, p. 10256-10259, Vol. 74, No. 21
0022-538X/00/$04.00+0
Incorporation of HLA-DR into the Envelope of Human
Immunodeficiency Virus Type 1 In Vivo: Correlation with Stage of
Disease and Presence of Opportunistic Infection
Stephen D.
Lawn1,2 and
Salvatore T.
Butera1,*
HIV and Retrovirology
Branch1 and
Tuberculosis/Mycobacteriology Branch,2
Division of AIDS, STD, and TB Laboratory Research, Centers for
Disease Control and Prevention, Public Health Service, U.S.
Department of Health and Human Services, Atlanta, Georgia
Received 22 May 2000/Accepted 14 August 2000
 |
ABSTRACT |
Human immunodeficiency virus type 1 (HIV-1) bearing HLA-DR in its
envelope was detected in plasma from all patients with chronic HIV-1
infection (n = 16) and was present at higher levels in
patients with active tuberculosis coinfection (n = 6).
Intriguingly, however, HLA-DR was not detectable in HIV-1 from patients
during primary viremia (n = 6), suggesting the
possibility of virus replication in less-activated cells.
| |
TEXT |
Human lymphocyte antigen class II
(HLA-DR) is incorporated into the envelope of human immunodeficiency
virus type 1 (HIV-1) as it buds from the host cell plasma membrane.
This aspect of HIV-1 maturation is seen among genotypically and
phenotypically diverse strains, including laboratory-adapted strains
and primary isolates (1, 2, 3, 6, 11, 15, 17). Compared to
other highly expressed mononuclear cell surface proteins, HLA-DR is
avidly incorporated into the viral envelope (3, 11, 20), possibly as a result of a selective process (reviewed in reference 20). Following incorporation into the HIV-1
envelope, HLA-DR retains functionality. In addition to its role in
antigen presentation, HLA-DR serves as an adhesion molecule whose
natural receptor is CD4. Thus, it is possible that the presence of
HLA-DR in the HIV-1 envelope increases virus-cell interactions and,
importantly, this may serve as the mechanism whereby the infectivity of
virions bearing this molecule is enhanced (4, 5). This
phenomenon, together with the ability of HIV-1-associated HLA-DR to
facilitate superantigen presentation (16), may influence
HIV-1 pathogenesis.
Although numerous studies have demonstrated that HLA-DR is
incorporated into the envelope of HIV-1 propagated in vitro (1, 2,
3, 6, 15, 17), studies of incorporation of host protein by HIV-1
that is present in clinical plasma samples have been hindered
technically by the interaction of virus with various serum proteins
(11). Such phenomena may explain why, in a study by Sarloos
et al. (17), virion-associated HLA-DR was detected in
only three of eight plasma samples from HIV-infected persons. More
recently, we have described an algorithm for the purification of HIV-1
from clinical specimens, thus facilitating the detection of
virion-associated host molecules (11). By using an
immunomagnetic capture technique, we found that the proportion of
HLA-DR-bearing HIV-1 that is detectable in plasma decreases during
treatment of tuberculosis (TB) and that this correlates with disease
resolution (10). Furthermore, analysis of the HIV-1 pool
replicating at an anatomic site of inflammation revealed that a greater
proportion of that virus contained HLA-DR in the envelope than did
virus present in the systemic circulation (12). Together,
these data suggest that incorporation of HLA-DR by HIV-1 in vivo
may correlate with the state of immunological activation of the cells
supporting viral replication. In support of this, upregulation of
surface expression of HLA-DR resulting from activation of U937
monocytoid cells in vitro enhances the incorporation of HLA-DR by
HIV-1 replicating within those cells (6). This phenomenon,
however, has not been studied with primary mononuclear cells.
Activation of the immune system plays a key role in the natural
history of HIV-1 infection, increasing during the course of disease
(18) and in the presence of opportunistic infections (13, 19, 21). The aim of this study was to determine whether the incorporation of HLA-DR by HIV-1 in plasma samples from
infected persons correlates with the clinical stage of disease and
whether this is also affected by the presence of opportunistic
bacterial infection.
HLA-DR incorporation by both macrophage- and lymphocyte-derived
HIV-1 in vitro.
We have previously shown that HLA-DR is
incorporated into the envelope of dualtropic HIV-1Ba-L
following propagation in either purified macrophages or lymphocytes
(11). In the same studies, host cell-derived CD44 was also
found to be incorporated at high levels by both macrophage- and
lymphocyte-derived viral stocks in vitro (11). Furthermore,
CD44 was detected in the envelope of HIV-1 derived from a panel of six
chronically infected, CD44-expressing, transformed cell lines
(unpublished data) as well as in the envelope of virus present in
samples of blood plasma (11), cervicovaginal fluid
(12), and pleural fluid (unpublished data) obtained from HIV-infected persons. We therefore selected anti-CD44 antibody capture
to be used as a positive control and as a comparative index of virus
capture by anti-HLA-DR antibody.
Prior to analyzing HIV-1 in clinical plasma samples in the
present study, we determined the relative efficiencies of capture of in vitro HIV-1 stocks with antibodies to CD44 and HLA-DR (Table 1) using the immunomagnetic capture
technique described previously (11). Although the extent of
anti-CD44 antibody capture of macrophage- and lymphocyte-derived virus
stocks was similar, the proportion of the macrophage-derived
virus captured using anti-HLA-DR antibody was greater than that of
lymphocyte-derived virus (Table 1), possibly reflecting a higher level
of expression of HLA-DR by macrophages.
Patient samples.
Next, we proceeded to analyze HIV-1 purified
from anonymous plasma samples from 22 patients categorized into four
groups based on clinical status (Table
2). Seroconversion panels of samples from
patients with primary HIV-1 infection were obtained commercially (Boston Biomedica, Inc., West Bridgewater, Mass.), and sera obtained at
the peak of viremia just prior to seroconversion (determined by Western
analysis) were selected. Samples from characterized patients with
chronic HIV-1 infection but no clinical or microbiological evidence of
opportunistic infections were categorized into two groups according to
the CD4+ T-lymphocyte count. Paired samples were also
obtained from patients with smear-positive pulmonary TB at the time of
diagnosis and at a later time point during treatment of TB when the
patients' sputum smear stains were negative for acid-fast bacilli
(described in reference 10). None of the 22 patients
was receiving antiretroviral treatment. HIV-1 from these 22 plasma
samples was analyzed using a purification algorithm and immunomagnetic
virus capture technique as described previously in full
(11).
Incorporation of HLA-DR by HIV-1 at different stages of
disease.
HIV-1 was captured from plasma samples of all 22 patients
using anti-CD44 antibody, and levels were broadly similar among the
four patient groups (Table 2). This suggests that CD44 is incorporated
at a high level in the HIV-1 envelope throughout the progression of
HIV-1 infection to AIDS. Since this molecule plays a physiological role
in lymphocyte homing, we have previously speculated that a high level
of incorporation of CD44 into the HIV-1 envelope may assist in
trafficking of virus to lymphoid tissue (11).
The efficiency of HIV-1 capture from plasma samples using
anti-HLA-DR antibody was lower than that seen with anti-CD44
antibody
(Table
2), as was observed on analysis of the in vitro-derived
HIV-1 stocks (Table
1). Significant capture of HIV-1 from plasma
using
anti-HLA-DR antibody (more than threefold greater than the
negative
control) was detected in all patients with chronic HIV-1
infection and
no opportunistic infection (
n = 10) and was similar
in
the two groups that were stratified by CD4 lymphocyte count
(Table
2;
Fig.
1). Although the efficiency of
anti-HLA-DR antibody
capture was low, our results are an important
advance from those
of a previous study, in which HIV-1 from plasma
samples of five
out of eight HIV-infected persons could not be captured
using
this antibody (
17). It is likely that the virus
purification
algorithm that we employed substantially overcame the
inhibition
of HIV-1 capture that is potentially attributable to the
presence
of acute-phase proteins, anti-HIV-1 antibodies
(
11), soluble
HLA-DR (
22), and
anti-HLA-DR autoantibodies (
7).
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FIG. 1.
HIV-1 was analyzed in plasma samples obtained from 22 patients who were categorized into four groups as shown in Table 2.
Virions (6 × 104) from each patient were distributed
equally among anti-CD44, anti-HLA-DR, and negative control antibody
captures. For each patient sample, specific HIV-1 capture by anti-CD44
and anti-HLA-DR antibodies was calculated by subtracting the amount
of HIV-1 bound by the negative control antibody. Data for each patient
are presented as a percentage of the amount of HIV-1 capture using
anti-CD44 antibody. Significant amounts of HIV-1 were captured from all
patients with chronic HIV-1 infection, and amounts were similar among
those with CD4 counts in the ranges of 200 × 106/liter to 500 × 106/liter and
<200 × 106/liter. The greatest anti-HLA-DR HIV-1
capture was from samples of patients with active TB coinfection.
However, there was no significant anti-HLA-DR antibody capture of
HIV-1 over background levels from plasma samples of patients with
primary HIV-1 infection (Primary HIV+). Data indicating percent HIV-1
capture by each antibody are displayed in Table 2.
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|
In marked contrast to capture of HIV-1 by anti-CD44 antibody, capture
using anti-HLA-DR antibody was not uniform among samples
obtained
at different stages of HIV-1 infection. Most striking
was the complete
absence of a significant level of capture of
HIV-1 from plasma samples
of any of the patients with primary
HIV-1 infection (
n = 6), despite substantial virus capture using
anti-CD44 antibody
(Table
2; Fig.
1). Although it has been proposed
that HIV-1 replicates
predominantly in immunologically activated
cells in vivo, there is now
evidence that both simian immunodeficiency
virus and HIV-1 also
replicate substantially in inactive, HLA-DR-negative
cells in vivo,
most notably during acute retroviral infection
(
23). Our
findings are consistent with and support this observation.
Immediately following seroconversion, the establishment of an
immune
response might lead to HIV-1 replication in mononuclear
cells
that have a higher degree of activation, possibly leading
to the
ability to detect HLA-DR in the HIV-1 envelope during the
transition from the acute phase to chronic HIV-1 infection. However,
we
were unable to analyze HIV-1 plasma samples at time points
immediately
following seroconversion because the amount of virus
present in the
plasma was insufficient for
analysis.
Although we were unable to detect HLA-DR in the envelope of HIV-1
present in plasma samples that were obtained during primary
HIV-1
infection (
n = 6), this molecule may have been
incorporated
at levels below the limit of detection of the assay.
Indeed, it
is likely that a critical threshold density of the host
molecule
in the HIV-1 envelope is required to enable the virus to be
captured
by our technique. Increasing activation of the cells
supporting
HIV-1 replication may well result in increases in both the
frequency
of virions bearing HLA-DR and the density of the molecule
in the
envelope, thereby increasing the proportion of virus that can
be
captured using antibodies to this
molecule.
Effects of active TB coinfection on incorporation of HLA-DR by
HIV-1 in vivo.
The percentage of specific anti-HLA-DR antibody
capture of HIV-1 present in plasma of patients with untreated
smear-positive pulmonary TB (n = 6) was 3.1-fold
greater than the capture of virus from patients with chronic HIV-1
infection but no opportunistic infection (n = 10) (P < 0.001) (Table 2; Fig. 1). Since active TB infection in
HIV-infected persons is associated with a marked systemic immune
activation (13, 21), the presence of this opportunistic
infection is likely to increase mononuclear cell surface expression of
HLA-DR, which is a highly inducible molecule. Our results may
reflect this, with upregulation of HLA-DR on cells supporting viral
replication resulting in increased incorporation of HLA-DR into
progeny HIV-1 particles. In addition to this, we have previously shown
that the presence of active TB coinfection leads to a substantial
production of cell-free HIV-1 derived from macrophages (10).
This virus may contain a higher level of HLA-DR in the envelope
than lymphocyte-derived virus (Table 1), and therefore, differential
cellular expression may have contributed to the findings among TB
patients in this study.
Since the presence of HLA-DR within the viral envelope enhances
HIV-1 infectivity (
4,
5,
6), the increased incorporation
of
HLA-DR in the HIV-1 envelope that is associated with the presence
of opportunistic infections might promote the propagation of HIV-1
in
mononuclear cell pools. Such a mechanism may be important in
the
copathogenesis of HIV-1 and opportunistic infections, facilitating
HIV-1 replication and increasing the HIV-1 load in plasma (
8,
19). There is also evidence that mycobacterial superantigens
are
important in TB pathogenesis (
14), and it is possible that
HLA-DR in the HIV-1 envelope of virus from coinfected persons
facilitates presentation of these superantigens (
16),
further
contributing to mononuclear cell activation and
pathogenesis.
Analysis of HIV-1 in plasma samples prospectively obtained from
patients with TB receiving antituberculosis treatment confirms
and
expands our previous findings (
10). Treatment of TB
resulting
in sputum smear conversion was associated with both relative
and
absolute reductions in anti-HLA-DR antibody capture of virus,
with a decline to levels similar to those observed in patients
with
HIV-1 infection but no opportunistic infection (Table
2;
Fig.
2). This indicates that the increased
incorporation of HLA-DR
in the HIV-1 pool that is associated with
active TB infection
is reversible, and in view of the potential for
enhanced viral
propagation, this further highlights the importance of
early diagnosis
and prompt treatment of copathogens in HIV-infected
persons (
9).
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FIG. 2.
Paired plasma samples were obtained from patients
(n = 6) with HIV-1 infection and smear-positive
pulmonary TB at the time of diagnosis and at a later time point
following a successful response to treatment. Virus was captured as
described in the legend for Fig. 1. The mean (± the standard error)
percentage of input HIV-1 captured by each antibody is presented
normalized for the mean anti-CD44 antibody capture at each time point.
The percentage of HIV-1 captured by each antibody is presented in Table
2.
|
|
In summary, incorporation of HLA-DR into the HIV-1 envelope was
detectable in the plasma of all patients with chronic HIV-1
infection
but was strikingly undetectable in samples from those
with primary
HIV-1 infection, suggesting that viral replication
prior to
seroconversion occurs in less-activated cell pools. Active
TB infection
leads to a reversible increase in incorporation of
HLA-DR by HIV-1
in vivo and may represent an important mechanism
whereby TB and
potentially other opportunistic infections enhance
HIV-1
pathogenesis.
| |
ACKNOWLEDGMENTS |
Stephen D. Lawn was funded by the Wellcome Trust of London, United
Kingdom, and subsequently by a Research Participation Program administered by the Oak Ridge Institute for Science and Education, Oak
Ridge, Tenn.
We thank R. B. Lal and C. E. Hart for supplying clinical
samples. The Committee on Human Research, Publications and Ethics of
the School of Medical Sciences, Kumasi, Ghana, approved collection of
field samples by S. D. Lawn.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Mail-Stop G19,
HIV and Retrovirology Branch, Centers for Disease Control and
Prevention, 1600 Clifton Rd., NE, Atlanta, GA 30333. Phone: (404)
639-1033. Fax: (404) 639-1174. E-mail: stb3{at}cdc.gov.
| |
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Journal of Virology, November 2000, p. 10256-10259, Vol. 74, No. 21
0022-538X/00/$04.00+0
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