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Monitoring of Specific Antibodies to Human Immunodeficiency Virus Structural
Proteins: Clinical Significance
By Jean-Pierre Allain, Yves Laurian, Mark H. Einstein, Brian P. Braun, Stephen R. Delaney, John E. Stephens,
Cynthia K. Daluga, Stephen J. Dahlen, and Kevin M. Knigge
Levels of antibodies to six major structural proteins of human
immunodeficiency virus type 1 (gp120, gp41, p66, p31, p24,
and p17) were assessed in serial samples from 22 persons
with severe hemophilia (16 asymptomatic and 6 who developed acquired immunodeficiency syndrome [AIDS] or AIDSrelated complex) with an automated dot blot assay using
purified recombinant antigens. High and sustained levels of
antibody to gp120, gp41, and p31 were found in all patients
irrespective of their clinical condition for 4 to 6 years after
seroconversion. In contrast, immune response to p66 and
p17 was significantly lower in symptomatic patients. Over
time, the levels of these two antibodies, as well as anti-p24,
decreased and tended to become undetectable. Abnormal
immune response and low levels of antibody to p66 and p17
are early indications of rapid clinical progression.
0 1991 by The American Society of Hematology.
H
infection, some clinical AIDS cases have been observed
where no antibody to HIV was detectable by either enzyme
immunoassay or Western blot.” In addition, the presence of
antibody to some HIV regulatory or envelope proteins was
detected before seroconversion but some of these observations have not been confirmed.”-22
The purpose of this investigation was to assess the
development of antibodies to the six main structural gene
products of HIV-1, env a 1 2 0 and gp41, pol p66 and p31,
and gag p24 and p17, with an automated dot blot assay
using purified recombinant proteins as specific capture
antigens since the time of seroconversion. The observation
of different antibody patterns in persons with hemophilia
who remained asymptomatic or developed AIDS during the
first 5 years post-seroconversion provided a new approach
to the assessment of prognosis and pathophysiology of HIV
disease.
UMAN IMMUNODEFICIENCY virus (HIV) infection is serologically characterized by the development of antibodies to structural and, in most cases, to
regulatory gene products.I4 For lack of appropriate methods of quantitation, antibodies are identified qualitatively
by Western immuno blotting or radioimmunoprecipitation,
which shows mostly antibodies to env and gag gene products.’ However, semiquantitation of antibodies to specific
structural gene products has been performed by end point
dilution with Western blot? Significant differences between
asymptomatic and acquired immunodeficiencyvirus (AIDS)
patients were found for antibodies to gpll0, p65, p55, p31,
p24, and p17 but not for anti-gp41. Antibodies to the core
protein p24 have been further studied with more quantitative methods such as competitive enzyme-linked immunosorbent assay (ELISA); p24 antigen-binding capacity,’ or
antigen sandwich assaf because their absence or their
disappearance during the natural course of HIV infection
was related to clinical progres~ion.~.~.’~
Antibodies to gp41 and gp120 were also semiquantified
with a competitive assay or Western blot and appeared
highly immunogenic and constantly detected irrespective of
clinical condition?*”,’*Antibodies to p27 nef, tat, sor, and
rev proteins were also studied but not in a quantitative
fashion and detectability ranged from 40% to 60%.4,13-’6
Only the absence of antibody to the gag gene product, p17,
or a low level of antibody to gpll0 and/or p65 were found to
be of prognostic interest, but no commercial assay was
available to further investigate these potential marker^.^^^','^
These studies were cross-sectional in subjects seropositive
for unknown periods of time, making the differentiation
between low level of antibody production and antibody
decline over time impossible. Although seropositivity for
antibody to HIV-1 is mandatory for the diagnosis of HIV
From the Abbott Laboratories, Diagnostics Division, Abbott Park,
IL; and Intemational Hemophilia Training Center, Hopital de Bicetre,
Kremlin-Bicetre, France.
Submitted July 30, 1990; accepted October 25, 1990.
Address reprint requests to Jean-fiereAllain, MD, PhD, Dept. 940,
Diagnostics Division, Abbott Laboratories, North Chicago, IL 60064.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-497119117705-0004$3.0010
1118
MATERIALS AND METHODS
A patient population of 22 males with severe hemophilia A (20)
or B (2) was selected on the basis of the availability of serum
samples collected before seroconversion and during the next 3 to 6
years at 6-month intervals or less. Some of these patients have been
previously described.6 They ranged in age from 10 to 45 (median
23) and seroconverted between 1982 and 1984. Seroconversion was
characterized by a repeatably reactive commercial enzyme immunoassay (Pasteur ELAVIA-1; Diagnostic Pasteur, Paris, France)
confirmed by Western blot as the presence of antibody to HIV-1
envelope and one other structural gene product. Patients were
followed-up 36 to 70 months after seroconversion (mean 53,
median 54 months). At the end of 1988, 16 hemophiliacs were
asymptomatic and six had severe AIDS-related complex (ARC) (1)
or AIDS (5). One year later, two more patients had developed
AIDS and by January, 1990, five had died.
Automated dot blot assay apparatus. The MATRIX Analyzer
(Abbott Laboratories, North Chicago, IL) is a self-contained assay
system designed to automatically incubate, wash, and interpret
reactivity to multiple a n a l y t e ~ . ~ ~
Assay specifications. Recombinant protein sequences from
HIV-1 IIIB p17, p24, p31, p41, and p66 have been expressed in
Escherichia coli and purified from inclusion bodies in the presence
of sodium dodecyl sulfate (SDS). Recombinant HIV-1 SFl2 gp120
was purchased from Chiron Corporation (Emeryville, CA). The
recombinant antigens, greater than 90% pure by scanning gel
densitometry and amino-terminal sequencing, are quantified, then
passively absorbed onto discrete regions of bonded nitrocellulose.
As a procedural control, antihuman IgG (heavy and light chain
specific) (Kirkegaard and Perry Laboratories, Gaithersburg, MD),
is also absorbed onto the nitrocellulose array. The nitrocellulose
Blood, Vol77,No5(March 1). 1991: pp1118-1123
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1119
ANTI-HIV MONITORING
array is then mounted into a plastic reaction vessel. Under
standard assay conditions, the nitrocellulose-absorbed analytes
capture specific human Ig and react with the reagents to produce
distinct visual spots on the solid phase.
The assay is performed within the reaction vessel by diluting 10
FL of serum or plasma into 1 mL of sample diluent. The reaction
vessel is mixed and incubated at 35°C within the MATRIX
Analyzer for 1 hour, then washed with Tris-buffered saline (TBS).
At the end of the wash cycle, 1 mL of probe, consisting of
antihuman (heavy and light chain) specific antibody, coupled to
long-chain biotin (Kirkegaard and Perry Laboratories) is added to
the reaction vessel and incubated for 1 hour at 3YC, followed by
washing with TBS. At the subsequent step, 1 mL of conjugate
containing antibiotin-specific antibody, coupled to alkaline phosphatase is added to the reaction vessel and incubated for 1 hour at
35°C. After washing with TBS, 1 mL of substrate containing
5-bromo-4-chloro-3-indolyl
phosphate (BCIP) is added to the
reaction vessel and incubated for 30 minutes at 35°C.
HIV-specific reactivity is measured as reflectance at 660 nm,
then converted to density of reflectance (Dr) according to KubelkaM ~ n kNonspecific
.~~
binding is substracted, and the resulting net
Dr value for each antigen is divided by an antigen-specific cutoff
value. A sample to cutoff (S/Co) ratio of 1.0 or greater indicates
reactivity to that particular recombinant HIV protein.
Semiquantitativep24 antibudy enzyme immunoassay. The ability
of the automated dot blot assay to quantify antibody level was
compared with an enzyme immunoassay designed to measure p24
HIV-1 antibody titer. In brief, highly purified recombinant p24
antigen was expressed in E coli, purified to 95%, and coated onto
the solid phase. After 2 hours of incubation at 40°C with four serial
dilutions of each sample (15, 1:25,1:125, and 1:625), the presence
of p24 antibody was detected with the same p24 antigen labeled
with horseradish peroxidase. After 1 hour of incubation and repeat
washing, the substrate (0-phenylenediamine) was added and 30
minutes later the color development was blocked by H,SO,. Color
intensity was measured with a spectrophotometer at 492 nm. The
antibody titer was derived from the intercept of the regression line
fitting all dilutions and a calculated cutoff. It was then mathematically transformed into nanograms per milliliter of p24 antigen
binding capacity.
CD4+ cell counts were obtained by immunofluorescence using
anti-CD4 monoclonal antibody (MoAb) or by flow cytometly.
Clinical examination was done by the attending physician every 6
months and the Centers for Disease Control (CDC) classification
of HIV disease was used. In this study, only CDC IV a (ARC) and
IV c (AIDS) are reported as end points of clinical progression.
The x2 test was used to analyze dichotomous results.
I
0
:
a
10.00
n
u
l - 4
1
0.05
O.1°
0
6
12
18
24
30
36
42
48
54
60
66
72
78
84
TIME FROM SEROCONVERSION (MONTHS)
Fig 1. Time course of antibodiesto HIV-1 gp120 (circles)and gp4l
levels (triangles) in 22 persons with hemophilia. Each data point
corresponds to the mean log ratio of reflectance (Dr) in 16 asymptomatic (solid symbol) and six symptomatic (open symbol) patients in
each time interval from seroconversion. Standard deviation (SD) of
ratios in the symptomatic group at the various time intervals ranged
from 25 to 119 and 7 to 116for antibody to gp120 and 41, respectively.
In the asymptomatic group SDs ranged between 12 and 76 (gp120)
and 8 and 175 (gp41).
interval it was averaged so that only one value per patient
per interval was used. Two clearly distinct patterns of
immune response were seen depending on the antigen
considered and the clinical group. Three antigens, gp120,
p41, and p31, corresponding to antibodies to the external
and transmembrane envelope glycoproteins and the endonuclease elicited an equally strong and sustained immune
response in asymptomatic and symptomatic clinical groups
(Figs 1 and 2).
Significant differences between the two groups were seen
with the other three antibodies to p66, p24, and p17.
Antibody response to HIV-1 reverse transcriptase (p66)
was significantly lower in the symptomatic group at all time
points (P < .Ol). Beyond 30 months postseroconversion,
the relative concentration of this antibody decreased progressively in the symptomatic group and was below a ratio
of 25 in four of the six patients followed-up for 4 years or
RESULTS
The two clinical groups studied were composed of six
patients who developed HIV-related clinical symptoms and
16 who did not during the 3 to 6 years after seroconversion.
The two groups did not differ significantly in age, date of
seroconversion, or duration of follow-up and CD4+ cell
count. All were initially HIV antigen negative, but over the
follow-up period, 3 of 6 patients who became symptomatic,
and 6 of 16 remaining asymptomatic, developed HIV
antigenemia. The natural course of immune response to
HIV-1 structural proteins was compared in the two clinical
groups as the mean log ratio of reflectance over a cutoff
established for each recombinant antigen (Figs 1 through
3). The mean ratio was calculated within intervals of 6
months. When a patient had multiple samples within each
I
0.05
O.1° 0
6
12
18
24
30
36
42
48
54
€€I
66
72
78
TIME FROM SEROCONVERSION(MONTHS)
Fig 2. Time course of antibodies to HIV-1 p66 (circle) and p31
(triangle) levels in 22 persons with hemophilia. SDs of ratios ranged
28 to 198 and 12 to 172 for anti-p66 and p31, respectively, in the
symptomatic (open symbol) group, and 88 to 160 and 39 to 175 in the
asymptomatic (solid symbol) group.
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1120
ALLAIN ET AL
0
6
12
18
24
30
36
42
54
48
60
66
72
78
was significantly higher in the symptomatic group (0.63% of
baseline value/mo v 1.67%/mo, respectively, P < .01).
The hypothesis that the disappearance of antibody to p24
and p17 might reflect the formation of an increasing level of
specific immune complexes has been p r ~ p o s e d . ~To
~~,'~
address this question, the relation between the immune
response development, the kinetics of disappearance of
each antibody, and the occurrence of HIV antigen was
examined (Table 1). In six of nine cases, the maximum ratio
of anti-p24 preceded the detection of HIV antigen. In
contrast, in five of nine patients, the peak of anti-pl7 was
observed concomitantly or later than HIV antigen first
appearance. In the 22 patients studied, the maximum ratio
of p24 and p17 antibodies was reached simultaneously in
six, anti-p24 maximum ratio preceded the peak of anti-pl7
in seven, and anti-pl7 preceded the peak of anti-p24 in nine
patients. The average interval between the two peaks when
observed at different times was 22 months. However, over
time after the maximum antibody level was reached, variations of p66, p24, and p17 antibody ratios varied similarly in
14 patients (both stable eight, both declining six) and
differed in eight patients. In these eight patients, variations
in anti-p66 and p17 levels were concordant in four, p66 and
p24 in two, and p24 and p17 in two patients.
84
TIME FROM SEROCONVERSION(MONTHS)
Fig 3. Time course of antibodies to HIV-1 p24 (circle) and p17
(triangle) levels in 22 persons with hemophilia. SDs of ratios ranged
2.5 to 184 and 1.5 to 88 for anti-p24 and p17, respectively, in the
symptomatic (open symbol) group, and 48 to 187 and 11 to 92 in the
asymptomatic (solid symbol) group.
more (Fig 2). Nine of 16 asymptomatic hemophiliacs
maintained a stable ratio of anti-p66 during the 4 years of
follow-up. As shown in Fig 3, the initial immune response to
p24 was of lower magnitude in the symptomatic group; the
antibody titer was maintained for 2 years and subsequently
declined to undetectability. Beyond 3years after seroconversion, the difference in ratio was significant at all time points
(P < .01). The most striking difference between the two
groups was seen with anti-pl7 (Fig 3). While asymptomatic
patients progressively developed a strong antibody response, patients who became symptomatic had a very low
antibody response to p17 (2) or essentially no detectable
antibody production (4). The difference between the two
groups of patients was significant at all time points beyond
12 months postseroconversion (P < .01). Only one patient
who developed symptoms had detectable p17 antibody
within the first 2 years of follow-up.
The ability of the dot blot assay to semiquantitate levels
of antibody was assessed by comparing p24 antibody ratios
and results obtained with a more quantitative assay using a
different format. Within a range of ratio 2 to 500 the
correlation coefficient between the two assays was 0.783
(data not shown). A comparison of variations of CD4+ cells
DISCUSSION
In the abundant literature regarding antibody response
to HIV-1 proteins and the variations of antibody levels, two
main hypotheses have been proposed. One is the immune
complex hypothesis according to which some antibodies
(anti-p24 and p17) have a low level of detectability and tend
to decrease in concentration over time in relation to the
formation of complexes with free viral antigensJ6.I6The
second hypothesis stems from the fact that HIV causes
immunodeficiency and could therefore impair the mechanisms of antibody production." The first hypothesis has
been supported by several lines of evidence. (1) Specific
p24-anti-p24 immune complexes have been found in p24
antibody negative
(2) Anti-p24 disappearance
often precedes the detection of its complementary p24
antigen.63"(3) Both absence of p24 antibody and presence
of HIV antigen are correlated with plasma viremia and
Table 1. Temporal Occurrence of Maximum Ratio of Antibody to p24 and p17 HIV Antigen and Symptoms in Nine HemophiliacsWho Became
HIV Antigen Positive
Time Interval From Seroconversion(mol
Maximum
Reflectance Ratio
Patient
13
14
18
11
15
17
19
20
23
~ 2 4
14.4
0.8
4.3
154.8
19.8
97.0
63.8
2.0
69.0
Peak of Antibody to
pi7
P24
P17
HIVAntigen
Positivity
0.4
0.3
0.6
32.5
4.6
255.0
24.3
41.0
109.1
11
26
34
11
11
17
4
60
14
11
54
34
11
11
40
4
51
39
26
27
36
42
11
14
29
30
30
'Patient 23 developed AIDS in 1989,6 months after the end of the study.
Symptoms
26
49
48
61,
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1121
ANTI-HIV MONITORING
clinical p r o g r e ~ s i o n .In~ ~contrast,
~~
besides the reported
decline of anti-gp41 in AIDS associated with absence of
anti-p24, there is little evidence to be credited to the second
hypothesis?’ In addition, the fact that antibody disappearance is restricted to antibodies to the two core proteins
rather than to all antibody species would be difficult to
explain in the context of a global dysfunction of immune
mechanisms.
The data presented provide a global view of the natural
history of antibodies to most HIV-1 structural proteins.
Two groups of antibody seem to emerge. One group
includes antibodies to external and transmembrane glycoproteins and endonuclease p31. As suggested previously by
end-point dilutions on Western blot, the immune response
to these antigens is strong and sustained in all patients,
irrespective of the clinical outcome.’* However, in a previous report from the same group, a lower titer of antibody to
gp120 was observed in subjects who progressed clinically
from asymptomatic to ARC or AIDS? In our study, no
evidence of a decline was seen in five patients with AIDS
and one with ARC at the time or after clinical diagnosis
(Figs 1 and 2). The discrepancy might be explained by the
selection of a population of progressors who had seroconverted considerably earlier than the nonprogressor group,
although both groups were asymptomatic at baseline, while
our group was unselected and studied since seroconversion.
The second group of antibodies to p66, p24, and p17 is
more heterogenous but is characterized by a significantly
lower immune response in patients who developed HIV
disease and a subsequent decline of detectable antibodies
(Figs 2 and 3). This decline seems to occur soon after
seroconversion for anti-p24 and later, 2 to 3 years after
seroconversion, for anti-reverse transcriptase p66. In patients who did not develop HIV disease within 60 months
after seroconversion, levels of antibodies to p66 and p24
tended to remain stable and antibody to p17 seemed to
increase. Antibody pattern differences between patients
who developed AIDS and those remaining asymptomatic
can be explained by two factors: (1) the innate inability of
certain patients to mount strong immune response to
apparently critical antigens; and (2) the formation of
immune complexes removing some antibodies. The importance of the humoral immune response to p24 antibody in
the host defense against HIV has already been ~uggested.~’
Our data tend to extend this hypothesis to both anti-reverse
transcriptase and anti-pl7. A neutralizing activity of antibody to p17 has been suggested” and its absence in
hemophiliacs who progressed to AIDS would be compatible with such function. However, the major body of data
indicates that antibody to gp120 is the primary, if not
unique, agent of neutralizing activity. The functional role of
anti-pl7 remains to be confirmed. The correlation between
antibody variation and antigen release in circulation appears closer for anti-p24 than the other two antigens (Table
1). However, concordant variations of antibody to p66, p24
and p17 were observed in 14 hemophiliacs and of anti-p66
and p17 in 18 patients. The apparent discrepancies observed between these antibody variations, in particular to
p24 and p17, can be explained by different kinetics of
0
6
12
18
24
30
36
42
48
54
60
66
MONTHS POST SEROCONVERSION
Fig 4. Variations of mean CD4+ cell counts from 22 patients with
hemophilia stratified in 16 asymptomatic (0-0) and six with HIV
disease (0-0).
immune response. P24 appears to be a stronger immunogen
inducing early maximum levels of antibody while p17 tends
to elicit steadily increasing antibody levels over several
years in asymptomatic hemophiliacs. In addition, as previously ~uggested,’~
episodes of intermittent release of virus
and/or viral proteins in circulation might take place. In this
study, two patients who became HIV antigen positive had
simultaneous fluctuations of antibody ratios to p24 and p17,
suggesting underlying bursts of immune complex formation.
Contrary to the accepted concept that antibody to gp120
is the main key to containing HIV infection, our data
suggest that other antibodies are critical, either directly or
through mechanisms reflected in the levels of antibodies to
p66, p24, and ~ 1 7 .The
’ ~ role of antibodies to HIV envelope
proteins appears more qualitative than quantitative, as
suggested by studies of neutralization titers or peptide
Several other markers of clinical progression have been
described. Among them, CD4+ cell count, levels of p-2
microglobulin and serum neopterin were shown to be
strongly correlated and significantly predictive of AIDS?’
In a limited number of patients, the CD4 cell count of the
two clinical groups were similar during the first 30 months
after seroconversion (Fig 4). Later, the difference became
significant and the rate of cell decline was accelerated in
patients developing HIV disease.
The results of this study suggest that monitoring the
levels of individual antibodies to HIV-1 structural proteins
can provide early prognosis of HIV disease. HIV antigen
and CD4+ cell count become predictive later in the course
of the infection. The observations made in this study are
derived from a relatively small group of patients with
hemophilia. Similar studies should be undertaken in other
risk groups where HIV is sexually transmitted or in intravenous drug abusers to confirm the data obtained in hemophilia.
ACKNOWLEDGMENT
We are indebted to J. Pennet, MD, A. Rafowicz, MD, and A.
Blanc, MD, who participated in the clinical evaluation of some
patients, and to Ann Greenlee,who prepared the manuscript.
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1122
ALLAIN ET AL
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Monitoring of specific antibodies to human immunodeficiency virus
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JP Allain, Y Laurian, MH Einstein, BP Braun, SR Delaney, JE Stephens, CK Daluga, SJ Dahlen
and KM Knigge
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