Journal of Virology, June 2001, p. 5421-5424, Vol. 75, No. 11
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.11.5421-5424.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Immunoglobulin G (IgG) and IgA, but also
Nonantibody Factors, Account for In Vitro Neutralization of Human
Immunodeficiency Virus (HIV) Type 1 Primary Isolates by Serum and
Plasma of HIV-Infected Patients
Renaud
Burrer,1,*
Dominique
Salmon-Ceron,2
Sophie
Richert,1,
Gianfranco
Pancino,3
Gabriella
Spiridon,2
Sandrine
Haessig,1
Virginie
Roques,1
Francoise
Barre-Sinoussi,3
Anne-Marie
Aubertin,1 and
Christiane
Moog1
INSERM U544, Institut de Virologie, 67000 Strasbourg,1 and Maladies
Infectieuses, Hopital Cochin,2 and
Institut Pasteur,3 Paris, France
Received 19 October 2000/Accepted 23 February 2001
 |
ABSTRACT |
The factors present in serum and plasma samples of human
immunodeficiency virus (HIV)-infected patients that are responsible for
the neutralization of four HIV type 1 (HIV-1) primary isolates in vitro
have been analyzed. Purification of immunoglobulins (Ig) by affinity
chromatography showed that the activities were mostly attributable to
IgG and less frequently to IgA. For two samples, we have shown that the
high-level and broad-spectrum inhibitory activity was essentially
caused by non-Ig factors interfering with the measurement of
antibody-specific neutralizing activity.
| |
TEXT |
Although the role of neutralizing
antibodies (nAbs) in the control and prevention of human
immunodeficiency virus type 1 (HIV-1) infection has long been the
subject of controversy, recent reports showing protection of macaques
against pathogenic simian-human immunodeficiency virus (SHIV) infection
after passive transfer of nAbs (1, 10, 15) have reinforced
the idea that humoral immunity can be beneficial and should be
stimulated by an efficient vaccine. However, insufficient knowledge of
the mechanisms of HIV neutralization and of the epitopes targeted by
Abs neutralizing a broad range of field isolates asks for further
investigations. For that purpose, sera from infected patients represent
a valuable source of naturally induced nAbs of great diversity. In this
study, we have analyzed serum and plasma samples from infected patients for neutralizing activity against different primary isolates (PI), to
select those containing nAbs with broad-spectrum activity. Purification of both immunoglobulin G (IgG) and IgA was performed to verify that neutralizing activity was antibody mediated. The inhibitory activity of the two most potent samples was shown to be
caused by non-Ig factors that may interfere with the measurement of
antibody-specific neutralizing activity.
Neutralizing activities of whole serum and plasma.
Peripheral
blood mononuclear cells (PBMC), purified by Ficoll gradient and
stimulated for 3 days with phytohemagglutinin A (PHA; Sigma), were used
as target cells for the replication and study of the neutralization of
different HIV-1 PI. Isolates Bx08 and Bx17 were kindly provided by H. Fleury (Bordeaux, France), 11105C was isolated from an infected
individual in the Central African Republic, and Kon was
provided by F. Barin (Tours, France). All PI were propagated once
or twice, exclusively on PHA-stimulated PBMC, to obtain viral stocks.
Large volumes of serum (100 ml) or plasma obtained by plasmapheresis
(500 ml) were collected from 24 infected patients (13 serum and 11 plasma samples; approval was obtained from the Comité Consultatif
de Protection des Personnes dans la Recherche Biomédicale). These
patients (19 European and 5 African) were either untreated
or treated with one or two nucleoside analogs. The mean duration of
infection was 7.3 years (range, 2 to 17 years), and the mean CD4 cell
number was 740/mm3 (range, 147 to
1,800/mm3). The serum sample designated reference
serum 2, available from the National Institutes of Health AIDS
Research and Reference Reagent Program, was provided by the Agence
Nationale de Recherche sur le SIDA. This serum sample is commonly used
as a reference because of its broad neutralizing activity
(19). A serum sample collected from an HIV-negative
patient was also included in the study. All serum and plasma samples
were heat inactivated before use (30 min at 56°C).
The neutralization assay combines serial dilutions of PI with serial
dilutions of serum or plasma. Briefly, 25 µl of four fourfold dilutions of virus in quadruplicate wells was
incubated for 1 h at 37°C, with 25 µl of serial serum-plasma
dilutions, in a 96-well filtration plate (Durapor-Dv, 1.25-µm pore
size; Millipore, Molsheim, France), before addition of 25 µl of PHA-stimulated PBMC (from a pool of five seronegative donors)
at a concentration of 4.106 PBMC/ml. After
24 h at 37°C, 100 µl of RPMI 1640 containing 10% fetal calf
serum and 20 IU of interleukin-2 per ml was added to each well.
Extensive washings (three washings of 200 µl of RPMI 1640 each) were performed by filtration on day 4 to remove free virus and
antibodies. Cells were then cultured in complete medium (200 µl)
until day 7 postinfection, at which time p24 was measured in the
supernatants by enzyme-linked immunosorbent assay (ELISA) (Innotest;
Innogenetics, Ghent, Belgium) to determine HIV-positive cultures. The viral titer (50% tissue culture infective dose) was
determined in the presence (Vn) and in the
absence (V0) of the serum, according
to the Reed and Muench method. The neutralization titer was
defined as the reciprocal of the serum dilution resulting in a 90%
decrease of the viral titer
(Vn/V0 = 0.1). This assay has been slightly modified from that previously
described (12, 16) to achieve a 5- to 10-fold increase in sensitivity.
Monoclonal antibodies (MAbs) 2F5 and 2G12 (gifts of H. Katinger) and
IgG1b12 (gift of D. Burton and P. Parren) were obtained through the
National Institute of Biological Standards and Control Central
Facility for AIDS Reagents. These standard PI-neutralizing antibodies
(18) were included to allow comparison of the
neutralization sensitivity of our four PI (Table
1). The three MAbs neutralize Bx08 at
high concentrations (60 to 100 µg/ml). Bx17 and 11105C are not
neutralized by 2G12 at 100 µg/ml, the highest concentration tested,
and Kon is not neutralized by the three MAbs.
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TABLE 1.
Neutralizing activities of MAbs and whole serum and
plasma samples determined for four HIV-1
primary isolatesa
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|
Serum and plasma samples were analyzed for neutralizing activity of PI
Bx08, Bx17, 11105C, and Kon. Of the 24 serum and plasma samples tested,
21 had a measurable neutralizing activity (Table 1). Sixteen serum and
plasma samples neutralized virus Bx08, and 10 of them displayed a high
rate of activity (defined as a neutralizing titer of >25). Bx17,
11105C, and Kon were, respectively, neutralized by 14, 18, and 5 of the
samples tested and by 4, 3, and 4 samples with a neutralizing titer of
>25. Ten serum and plasma samples, in addition to reference serum 2, had a high activity for at least one of the PI. It is noteworthy that
the six samples (including reference serum 2) neutralizing Kon
have an activity against the three other PI and that serum sample
19 and plasma sample 26 were able to strongly neutralize the
four viruses. Thirteen serum and plasma samples were chosen to
analyze activities after Ig purification.
Neutralizing activities of purified IgG and IgA fractions.
The
serum and plasma samples were first fractionated by protein A-Sepharose
affinity chromatography (Pharmacia) to separate unbound factors from
IgG1, -2, and -4 eluted with a low-pH buffer (0.1 M glycine [pH
2.7]). Efficiency of purification was evaluated by detection of Ig
isotypes in the collected fractions by specific ELISA. Briefly, 96-well
plates (Nunc Maxisorp) were coated (100 µl) with anti-human IgA, IgG,
and IgM (The Binding Site, Birmingham, United Kingdom) at a
1/1,000 dilution in 50 mM bicarbonate buffer (pH 9.6). Serial
dilutions of the affinity column fractions were incubated for 2 h
at 37°C. Bound Ig's were revealed by anti-human Ig conjugate coupled
to horseradish peroxidase (Southern Biotechnology Associates) at a
1/10,000 dilution (17). Repeated experiments indicated
that the elution was highly reproducible, allowing purification of 80 to 90% of total IgG (data not shown). Fractions were filtered through
a 0.45-µm-pore-size filter (µSTAR; Costar), and neutralization assays were performed as for whole serum and plasma, taking into account the fivefold dilution of the fractions with respect to the
original sample. The neutralizing activities of serum and plasma
samples 4, 6, 8, 14, 18, 20, 23, and 33 and reference serum 2 were
predominantly recovered in the IgG1-, -2-, and -4-containing fraction
(Table 2), with neutralizing titers
ranging from 26 to 100% of the initial value. Flowthrough
fractions displayed 12.5 to 25% of the unfractionated sample
neutralizing activity for serum and plasma samples 6, 8, and 33 and
reference serum 2. Remarkably, for the broadly neutralizing serum
sample 19 and plasma sample 26, the neutralizing activity was almost
exclusively recovered in the flowthrough fraction.
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TABLE 2.
Neutralizing titers of whole serum of plasma samples and
corresponding protein A affinity column fractions for isolates Bx08
and Bx17a
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|
The residual neutralizing activity present in the flowthrough
fractions can be attributed either to nonretained IgG1, -2, or -4, IgG3, IgA, or IgM or to nonantibody serum factors. To assess the
involvement of IgA in this activity, further separation was achieved by
Jacalin affinity chromatography (Sigma). Despite the fact that IgA2 is
not retained by this lectin, this purification was performed, as the
anti-HIV serum IgA response is almost completely restricted to the IgA1
subclass (7). The protein A flowthrough fractions
bearing inhibitory activity (samples 6, 8, 18, 19, and 26 and reference
serum 2) were loaded on Jacalin-Sepharose 4B columns, and purified IgA1
was eluted by 100 mM methyl 
-D-galactopyranoside (Sigma). Purification efficiency and an absence of contaminating IgG
were verified by ELISA as reported for protein A affinity chromatography, with an additional recording of IgA1 (anti-human IgA1
MAb, I-7262; Sigma; coated at a 1/500 dilution). Jacalin flowthrough and purified IgA1 fractions were filtered and evaluated for neutralizing activity (Table 3). In
samples 6 and 8 and reference serum 2, neutralizing activities were
associated only with IgA1. For serum sample 18, the activity detected
in the protein A flowthrough fraction was not attributable to IgA1.
Again, the activities of broadly neutralizing serum sample 19 and
plasma sample 26 were almost entirely recovered in the flowthrough
fraction.
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TABLE 3.
Neutralizing titers (PI Bx08) of protein A
flowthrough fraction (non-IgG1, -2, and -4) further separated
by Jacalin-Sepharose chromatographya
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|
Neutralizing activities of the dialyzed flowthrough
fractions.
To evaluate the contribution of antibodies remaining in
the Jacalin-Sepharose flowthroughs to the neutralizing activity
detected in these fractions, they were separated from smaller molecules by dialysis with 10-kDa (Slide-A-Lyzer; Pierce) and 50-kDa (Spectrapor; Spectrum Medical Industries) cutoff membranes. Two milliliters of
flowthrough fraction was dialyzed against 1 liter of
phosphate-buffered saline for 24 h at 4°C, with one replacement
of the phosphate-buffered saline after 12 h. Results of
neutralization assays performed with the dialyzed flowthrough
fraction of plasma sample 26 are shown in Fig.
1. Dialysis with the 50-kDa membrane led
to a drop of activity to under detectable levels. Neutralizing activity was, however, conserved after dialysis with the 10-kDa membrane, which
allowed elimination of any drug that may have remained in the serum or
plasma of treated patients. Comparable results were obtained with serum
sample 19 (data not shown). Dialysis caused no loss of Ig of any class,
as verified by specific ELISA, and did not affect the neutralizing
activity of the protein A-Sepharose-purified IgG fraction of serum
sample 8 (data not shown). The activity detected in the two strongly
and broadly neutralizing serum sample 19 and plasma sample 26 flowthrough fractions can thus unequivocally be attributed to
a nonantibody soluble factor(s), of a molecular mass(es)
between 10 and 50 kDa.
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FIG. 1.
Neutralization of Bx08, Bx17, and Kon by whole and
dialyzed Jacalin flowthrough fraction of plasma sample 26. ND, not done.
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Our results confirm that the majority of nAbs in the sera and plasmas
of infected patients are of IgG isotype, although neutralizing IgA1 was
detected in 4 of 21 neutralizing samples. A limited number of studies
have reported the neutralization of primary strains of HIV-1 by serum
IgA from infected patients (11) or exposed seronegative
individuals (6, 14). These results show that PI-neutralizing IgA is induced during infection. However, circulating and secreted IgA originates from different compartments, and the presence of serum neutralizing IgA does not reflect mucosal IgA status.
We detected neutralization of the four PI by purified Ig in 3 of 24 samples (samples 18, 23, and 33), which is close to the ±10%
prevalence of broadly cross-neutralizing sera observed in other studies
(3, 13). The purified IgG of these three patients
neutralized PI Kon which was not neutralized by any of the three MAbs
tested, suggesting the potential presence of as-yet-unidentified broadly neutralizing or synergistically acting antibodies in these samples. Interestingly, of these three samples, two were obtained from
black African female patients (samples 23 and 33) (Table 1), supporting
the observation made by Beirnaert et al. (3) that there
may be a correlation between broadly cross-neutralizing activity,
African origin, and gender. We have also shown that nonantibody
inhibitory activities that may significantly affect the results of in
vitro antibody-mediated neutralization studies are detected in 2 of 24 samples tested. Heat inactivation of the serum eliminated any
complement-associated antiviral effect, and alpha interferon was below
detectable levels in both serum sample 19 and plasma sample 26 (data
not shown). Nonantibody-neutralizing activities could be due to
cytokines, chemokines (e.g., RANTES, MIP-1, MIP-1
, or SDF-1), or
CD8+ T-cell antiviral factor, or more likely to a
particularly powerful combination of some of those factors. In fact,
none of these molecules can be definitively excluded after dialysis
experiments, as the molecular masses of some are too close to the
theoretical cutoff of the membranes, and some may be present as dimers.
However, the neutralizing activity detected in our experiments is
conserved at high dilutions compared to that observed either with
chemokines (4, 5) or with CD8+
T-cell antiviral factor released from CD8+ T
cells (2, 8). Moreover, additional experiments with three other PI as well as with the T-cell-line-adapted strain MN (data not
shown) have confirmed that the non-Ig factors of samples 19 and 26 are
active against both R5 and X4 strains, excluding the action of either
- or -chemokines alone. It is no longer possible to determine
whether the inhibitory factors were present in serum or plasma at the
time of collection or were subsequently produced by donor cells ex vivo
(9), but further experiments will be carried out to
precisely define the nature of the unknown inhibitory factor(s) and to
investigate the persistence of such a factor(s) in additional samples
collected from the same patients. Because of the high and potent in
vitro-inhibitory activity of those serum factors, we propose that, if
Igs are not purified, serum and plasma samples should at least be
dialyzed for performance of antibody-mediated neutralization studies.
| |
ACKNOWLEDGMENTS |
This work has been supported by grants from the Agence Nationale de
Recherche sur le SIDA and Synthelabo.
We thank S. Risch for the measurement of alpha interferon and B. Lafont
and M. C. Navas for critical reading of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut de
Virologie, INSERM U544, 3 rue K
berlé, 67000 Strasbourg, France.
Phone: (33) 3 90 24 37 38. Fax: (33) 3 90 24 37 23. E-mail:
renaud.burrer{at}ulp.u-strasbg.fr.
Present address: Laboratoire de Spectrométrie, Ecole de
Chimie, Strasbourg, France.
| |
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Journal of Virology, June 2001, p. 5421-5424, Vol. 75, No. 11
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.11.5421-5424.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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