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Journal of Virology, November 1998, p. 9092-9100, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Studies of the Neutralizing Activity and Avidity of
Anti-Human Immunodeficiency Virus Type 1 Env Antibody Elicited by
DNA Priming and Protein Boosting
J. F. L.
Richmond,1
S.
Lu,2
J. C.
Santoro,1
J.
Weng,1
Shiu-Lok
Hu,3
D. C.
Montefiori,4 and
H. L.
Robinson1,*
Department of
Pathology1 and
Department of Infectious
Diseases,2 University of Massachusetts School of
Medicine, Worcester, Massachusetts;
Washington Regional Primate
Research Center, University of Washington, Seattle,
Washington3; and
Department of Surgery
and Center for AIDS Research, Duke University Medical Center,
Durham, North Carolina4
Received 11 May 1998/Accepted 10 August 1998
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ABSTRACT |
DNA vaccination is an effective means of eliciting strong antibody
responses to a number of viral antigens. However, DNA immunization alone has not generated persistent, high-titer antibody and
neutralizing antibody responses to human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env). We have previously reported that DNA-primed anti-Env antibody responses can be augmented by boosting with Env-expressing recombinant vaccinia viruses. We report here that
recombinant Env protein provides a more effective boost of DNA-initiated antibody responses. In rabbits primed with Env-expressing plasmids, protein boosting increased titer, persistence, neutralizing activity, and avidity of anti-Env responses. While titers increased rapidly after boosting, avidity and neutralizing activity matured more
slowly over a 6-month period following protein boosting. DNA priming
and protein immunization with HIV-1 HXB-2 Env elicited neutralizing
antibody for T cell line-adapted, but not primary isolate, viruses. The
most effective neutralizing antibody responses were observed after
priming with plasmids which expressed noninfectious virus-like
particles. In contrast to immunizations with HIV-1 Env, DNA
immunizations with the influenza virus hemagglutinin glycoprotein did
not require a protein boost to achieve high-titer antibody with good
avidity and persistence.
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INTRODUCTION |
DNA immunization effectively elicits
high-titer neutralizing antibody against influenza, measles, rabies,
and herpesviruses but has been less successful in generating
neutralizing antibody against human immunodeficiency virus type 1 (HIV-1) (reviewed by Robinson [53]). While single or
singly boosted DNA immunizations often elicit strong and long-lasting
neutralizing antibody responses comparable with those seen in virally
infected and convalescent animals (52, 40, 66), multiple DNA
immunizations are typically required to elicit even modest titers of
HIV-1-neutralizing antibody (1, 4, 15-17, 33-36, 47, 51, 58, 63,
64). Furthermore, antibody responses elicited by DNA immunization
(47, 51) or protein subunit immunization (21, 38)
with Env are transient; titers rise and fall with successive
immunizations.
Work with the simian immunodeficiency virus (SIV) system allows direct
comparison of anti-Env antibody titers elicited by DNA immunization or
viral infection. DNA immunization of macaques with SIV Env elicits
neutralizing titers which are, at best, only 5 to 10% of those in
SIV-infected macaques (53). If DNA immunization is to play a
meaningful role in the development of the antibody component of a HIV-1
vaccine, we must identify ways of improving the titer and persistence
of these neutralizing antibody responses.
Recently, attention has focused on the avidity, as well as the
neutralizing titers, of antibody responses elicited by immunodeficiency virus infections and immunizations (8, 9, 18). Antibody responses induced by the envelope glycoprotein (Env) of the
lentiviruses SIV (8, 9) and equine infectious anemia virus
(24) mature slowly. Maturation, in this sense, is defined as
development of significant avidity, high neutralizing titers, and some
degree of cross-neutralizing activity. While antibody titers rise
within several weeks of infection and neutralizing antibody specific for the autologous SIV peaks within several months, the avidity of
polyclonal antisera increases more slowly, reaching maximal levels
between 6 and 8 months after infection. Increased avidity is coincident
with a broadening of protective neutralizing antibody responses to
heterologous viruses (8, 9). Slow maturation of antibody and
development of cross-neutralizing antibody, over 8 to 12 months, is
also observed in HIV-infected patients (44). In contrast,
the avidity of antibody responses to infection with nonlentiviruses,
such as hepatitis C virus (65), varicella-zoster virus
(28), and rubella virus (31), is fairly rapid;
high-avidity responses are seen in a period of weeks to a few months
after infection.
While affinity is an absolute thermodynamic measure of the strength of
interaction determined at equilibrium, avidity can be defined as a more
relative measure of the strength of interaction which is a function of
antigenic valence and structure, antibody bivalence, the concentrations
of antibody and antigen, and affinity. Affinity of polyclonal antisera
cannot be determined. The relative avidity of polyclonal antisera can
be estimated by using so-called avidity enzyme-linked immunosorbent
assays (ELISAs) in which the ability of chaotropic agents (such as urea
or sodium thiocyanate) to disrupt antigen-antibody interactions is
determined (2, 7, 22, 37).
In this study, we examined the magnitude, persistence, avidity, and
neutralizing activity of antibody elicited by priming rabbits with
plasmids expressing various forms (and combination of forms) of HIV-1
Env and by boosting these responses with recombinant gp160. We compared
these anti-Env antibody responses with antibody responses produced by
DNA immunization of rabbits with a plasmid expressing influenza virus
hemagglutinin type 1 (H1). While DNA immunization with influenza virus
H1 elicited persistent high-titer antibody and neutralizing antibody,
DNA immunization with HIV-1 Env did not. Furthermore, while the avidity
of anti-H1 antibody was relatively strong, that of anti-Env antibody
was weak. Protein boosting with rgp160 increased the titers, the
persistence, and the avidity of anti-Env antibody. We conclude that DNA
priming with HIV-1 Env is an effective means of priming anti-Env
antibody responses but requires protein boosting to elicit high-titer
and high-avidity antibody.
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MATERIALS AND METHODS |
Animals.
Female New Zealand White rabbits, 8 to 10 weeks old
and 4 to 5 lb each, were purchased from Millbrook Farms (Amherst,
Mass.) and housed in accordance with U.S. Department of Agriculture
regulations. Rabbits were anesthetized with a 1:1 (vol/vol) mixture of
ketamine-anased and bled by ear vein puncture.
Plasmids and protein expression.
Vaccine plasmids which
express HXB-2 envelope glycoprotein gp120 (pJW4303/HXB-2gp120;
abbreviated pHXB2gp120) and gp140 (pJW4303/HXB-2gp140; abbreviated
pHXB2gp140) have been described by Mustafa et al. (47).
Plasmids expressing gp160 (pCMVdHXB-2env; abbreviated pHXB2env) and
defective virion constructs (pCMVHXB-2dpol; abbreviated pHXB2dpol) were
modifications of those described by Lu et al. (34); in both
cases, a downstream intron was removed from the rat preproinsulin
polyadenylation sequence of the mammalian expression plasmid
pBC/IL-2/CMV (34). A plasmid expressing A/PR/8/34 influenza virus hemagglutinin (pJW4303/H1 [pH1]) has been described by Robinson et al. (52). A plasmid which expresses human growth hormone (hGH), pWR61602, was provided by Joel Haynes, formerly of Geniva Inc.
(Middleton, Wis.). Plasmids were grown in Escherichia coli HB101 and were purified with Qiagen (Santa Clarita, Calif.)
anion-exchange resins. Lipofectamine (Life Technologies, Grand Island,
N.Y.)-mediated transient transfection of Cos cells and an HIV-1
antigen-specific ELISA were used to quantitate expression of Env from
plasmids (47). The hGH-expressing plasmid was included in
each transfection as an internal control, and hGH expression was
determined by using a commercial ELISA (Boehringer-Mannheim,
Indianapolis, Ind.).
Indirect immunofluorescence assays were used to determine the
localization of Env in transfected Cos cells. Cos monolayers were grown
on coverslips prior to transfection and fixed with 4% paraformaldehyde
or 100% methanol after transfection and prior to staining either with
polyclonal rabbit anti-HXB-2 Env (elicited by DNA vaccination with
various forms of HXB-2 Env) and goat anti-rabbit antibody conjugated
with fluorescein isothiocyanate (Sigma, St. Louis, Mo.) or with mouse
antihemagglutinin monoclonal antibodies (a kind gift of Walter Gerhard,
Wistar Institute) and goat anti-mouse antibody conjugated with
fluorescein isothiocyanate (Sigma). Fluorescence was observed with an
Axioscope microscope (Carl Zeiss, Inc., Thornwood, N.Y.).
DNA priming.
Rabbits were primed by gene gun immunization.
Gold beads, 0.95 µm in diameter (Geniva Inc.), were loaded with DNA
at either 0.25 µg of DNA/mg of gold (Env-expressing plasmids) or 0.5 µg of DNA/mg of gold (H1-expressing plasmid). Each shot delivered 0.5 mg of gold and either 0.12 µg of Env-expressing DNA or 0.25 µg of
H1-expressing DNA. Thirty-six shots, carrying a total of 4.5 µg of
HIV Env-expressing plasmid or 9 µg of H1-expressing plasmid, were
delivered to nonoverlapping areas of the shaved abdominal skin of
anesthetized rabbits by using a helium-discharge Acell II gene gun
(Geniva) at 375 to 450 lb/in2. Rabbits were primed three
times at 1-month intervals.
rgp160 boosting.
Six months after the final DNA inoculation,
primed and naive rabbits were boosted with 100 µg of recombinant
HIV-1 IIIb gp160 (rgp160) in incomplete Freund's adjuvant (Sigma).
rgp160 was produced by using the recombinant vaccinia virus, v11Kenv5
(30). rgp160 is in a presumed oligomeric form. Rabbits were
anesthetized, and protein was injected both intradermally (six times,
50 µl each) and intramuscularly (twice, 100 µl each). A second
rgp160 boost was given 6 months after the first. Sera were collected
just prior to, and roughly 2, 4, 8, and 38 weeks after, each
immunization.
Determination of HIV-1 Env-specific IgG titers.
An ELISA was
used to determine anti-Env immunoglobulin G (IgG) titers of rabbit
sera. Sera collected following DNA immunizations were assayed by using
a mixture of the gp120 and gp140 forms of the BH8 Env produced by the
recombinant vaccinia virus vCB-14 (12) as the solid-phase
antigen. Sera collected after rgp160 boosting were assayed by using
recombinant HIV-1 IIIb gp120 produced by using baculovirus (rgp120;
Intracel, Seattle, Wash.) as the solid-phase antigen, to ensure that
anti-vaccinia virus antibodies did not interfere with determination of
anti-Env antibody titers in boosted rabbits. Details of the ELISA were
described previously by Mustafa et al. (47) and Richmond et
al. (51). Both solid-phase antigens gave identical anti-Env
concentrations for preboost sera. All samples were run in duplicate at
several serial dilutions. Titers of less than 0.1 µg of Env-specific
IgG/ml of serum were not considered significant.
Determination of H1-specific antibody titers.
ELISA plates
were coated with Triton X-100-lysed influenza virus A/PR/8/34, and
rabbit sera were assayed as described by Boyle et al. (5)
with the following modifications. Sera were assayed at threefold serial
dilutions ranging from 1:500 to 1:8,900,000 for 60 min at 23°C. Bound
antisera were detected with biotinylated goat anti-rabbit IgG and
horseradish peroxidase-linked streptavidin (Vector Labs, Burlingame,
Calif.) and 3,3',5,5'-tetramethylbenzidine (Sigma). A standard curve
was constructed by using threefold serial dilutions of blood from the
fifth blood sample of rabbit R119, which contained approximately 84.5 µg of influenza virus-specific IgG/ml. This approximate concentration
was determined as described by Mustafa et al. (47) by using
lysed A/PR/8/34 influenza virus, rather than HIV-1 Env, as the
solid-phase antigen. The optical density was measured at 450 nm, and
titers were expressed as micrograms of A/PR/8/34-specific IgG/ml of
serum.
NaSCN displacement ELISA.
Sodium thiocyanate (NaSCN)
displacement ELISAs were performed by a modification of the methods of
Charoenvit et al. (7) and Luxton and Thompson
(37). ELISA plates were coated overnight with one of the
following specific antigens: concanavalin A and vCB-14 at 0.1 µg/well, concanavalin A and rgp120 at 0.1 µg/well, rp24 at 0.1 µg/well, or Triton X-100-lysed A/PR/8/34. The plates were washed with
phosphate-buffered saline (PBS)-0.1% Triton X-100 and blocked for 60 min at 23°C with whey buffer (PBS, 4% whey powder [Davisco, Le
Sueur, Minn.], 0.05% Tween 20) containing 5% nonfat dry milk powder.
Sera, serially diluted in whey buffer to equivalent initial
concentrations of Env-specific IgG, were incubated in ELISA plates for
60 min at 23°C. The plates were washed three times with PBS-Triton
X-100, once with PBS containing 0, 1, 2, 3, 4, or 5 M NaSCN for 15 to
20 min, and then six more times with PBS-Triton X-100. Bound antibody
was detected as described for the H1-specific ELISA. A standard curve
was constructed by using sera from rabbits immunized with pHXB2gp120
that were not subjected to NaSCN washing. All samples were assayed in
duplicate over a range of dilutions, and results were expressed as the
percentage of antibody bound in the absence of NaSCN.
HIV-1-neutralizing antibody assays.
Antibody-mediated
neutralization of HIV-1 IIIb was measured by using the MT-2 cell
killing assay described by Montefiori et al. (41) and
Richmond et al. (51). Neutralization of HIV-1 primary
isolates was assayed on peripheral blood mononuclear cells as
previously described (43, 51). V3-loop specificity of
neutralizing antibody was determined by preincubating serum samples
with 20 µg of the V3-loop peptide (NNTRKSIRIQRGPGRAFVTIGKIG;
amino acids 307 to 330 of IIIb Env) prior to performance of the
MT-2 cell killing assay. Neutralizing titers are defined as that
dilution of serum which resulted in 50% protection from virally
induced cell killing in MT-2 cells or 90% reduction in p24 synthesis
in peripheral blood mononuclear cells.
Influenza virus-neutralizing antibody assay.
Sterile 96-well
tissue culture plates were seeded with 105 MDCK cells in
modified Eagle's medium (MEM) containing penicillin, streptomycin,
L-glutamine, and 5% fetal calf serum and grown to confluence overnight at 37°C in 5% CO2. Serial threefold
dilutions of rabbit serum, beginning at 1:50, were made in 100 µl of
MEM. One hundred 50% tissue culture infective doses
(TCID50), in 100 µl of MEM-4% bovine serum albumin-1
µg of TPCK (N-tosyl-L-phenylalanine chloromethyl ketone [Sigma]) per ml, were added to each serum sample.
Virus-antiserum mixtures were incubated at 37°C, in 5% CO2, for 60 min. Confluent monolayers were washed twice
with sterile PBS, overlaid with virus-antiserum mixtures (all
conditions were assayed in duplicate wells, including control wells
containing no antisera or neither antisera nor virus), and incubated
for 72 h. Monolayers were visually scored for cytopathic effects, and neutralizing titer was defined as the highest antiserum dilution which prevented cytopathic effects.
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RESULTS |
Vaccine plasmids and expression in transiently transfected Cos
cells.
Vaccine plasmids expressed four forms of HIV-1 HXB-2 Env
(Fig. 1a). The gp120 form represents a
monomeric CD4-binding subunit of Env and terminates at amino acid 506 (numbered to include the signal sequence). In contrast, the gp140 form
terminates at amino acid 675, includes the entire extracellular domain
of Env, and forms an oligomer. Full-length Env (gp160) includes the
transmembrane and cytoplasmic domains (Fig. 1a). The dpol plasmid
encodes full-length Env and all HIV-1 HXB-2 proteins except Pol (HXB-2
is defective for vpr, vpu, and nef)
and produces defective virus-like particles which bud from transfected
cells (34). A fifth plasmid expressed the complete
membrane-bound form of influenza virus H1 (52).
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FIG. 1.
Study design. (a) Vaccine plasmids were constructed to
express four forms of HIV-1 Env: gp120, gp140, gp160, and
noninfectious, virus-like particles. gp120 is the nonreceptor-binding
domain of Env. gp140 is the entire extracellular domain of Env and
contains oligomerization sequences. (b) The immunization schedule used
is shown.
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Both the release of Env from cells and the localization of Env within
cells were dependent on the form of Env. Nearly all
of the HXB-2 gp120
was in culture medium, while only half of the
gp140 and only 10% of
the full-length Env were present in the
medium (Table
1). About one-third of the HIV-1 antigens
expressed
from pHXB2dpol were secreted or shed into the medium (Table
1).
In agreement with prior studies (
11,
23), indirect
immunofluorescence
analyses revealed different localizations of the
different forms
of Env (data not shown). The cell-associated gp120 was
located
both in the cytosol and at the plasma membrane. In contrast,
most
gp140 was found in the perinuclear regions of the cell, while
smaller amounts were seen in the cytosol and at the cell surface.
Full-length Env and Env expressed by pHXB2dpol were observed only
in
the perinuclear regions of the cell. Like gp120, influenza
virus H1
localized both in the cytosol and at the plasma membrane
(data not
shown).
Temporal antibody responses following DNA priming with Env
antigens.
To investigate the immunogenicity of Env-expressing
plasmids, four groups of three rabbits were immunized with
Env-expressing plasmids. The first group was primed with pHXB2env (the
Env group), and the second was primed with pHXB2dpol (the dpol group).
The third group was primed with a combination of pHXB2env, pHXB2gp120, and pHXB2gp140 (the Env++ group), and the fourth group was primed with
pHXB2dpol, pHXB2gp120, and pHXB2gp140 (the dpol++ group). A fifth
group of two rabbits was immunized with the plasmid expressing influenza virus H1 (the H1 group). All rabbits were primed three times
at 1-month intervals (Fig. 1b).
Consistent with previous studies (
1,
4,
15-17,
33-36,
47,
51,
58,
63,
64), the induction of anti-Env antibody
required multiple
DNA immunizations (Fig.
2). One of three
rabbits
from the dpol group and two of three rabbits from the Env++ and
dpol++ groups seroconverted after the third DNA immunization.
None of
the rabbits in the Env group seroconverted (Fig.
2). As
noted in
previous studies (
47,
51), anti-Env titers were low
(1 to 4 µg of Env-specific IgG/ml) (Table
2)
and were not persistent
(Fig.
2).
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FIG. 2.
Temporal ELISA and IIIb-neutralizing antibody responses.
Env indicates DNA priming with pHXB2env, while Env++ indicates priming
with a combination of pHXB2env, pHXB2gp120, and pHXB2gp140. Similarly,
dpol indicates DNA priming with pHXB2dpol and dpol++ indicates priming
with a combination of pHXB2dpol, pHXB2gp120, and pHXB2gp140. Times of
DNA immunizations are represented by thin vertical dotted lines, while
times of protein immunizations are represented by heavier vertical
dashed lines. Open symbols represent sera collected after DNA priming,
while filled symbols represent sera collected after protein boosting.
Individual rabbits are represented by different symbols (circles,
squares, or triangles). ELISA values are arithmetic means ± standard deviations. Values on graphs are GMTs.
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In contrast to immunization with HIV-1 Env, DNA immunization with
influenza virus H1 elicited high titers of specific antibody
that
ranged from 100 to 300 µg of specific IgG per ml of serum
(Fig.
3). Furthermore, these high titers were
apparent after the
second DNA immunization, and unlike anti-Env
responses, anti-H1
antibody titers were persistent. Anti-H1 titers fell
less than
threefold over a 60-week period following the final DNA
immunization
(Fig.
3).
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FIG. 3.
Temporal anti-H1 antibody and A/PR/8/34-neutralizing
antibody responses. Antibody titers are expressed as micrograms of
A/PR/8/34-specific IgG per milliliter of serum. A neutralizing antibody
titer was defined as that dilution of serum which protected MDCK
monolayers from infection with 100 TCID50 of influenza
virus A/PR/8/34. Immunizations were at 0, 4, and 8 weeks.
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Recombinant protein boosting.
In an effort to increase
DNA-primed anti-Env antibody titers, all four Env-primed groups, as
well as a group of naive rabbits, were boosted with rgp160 protein.
rgp160 was chosen because it is likely to retain some native,
oligomeric structure. Intradermal and intramuscular boosts of 100 µg
of rgp160 in incomplete Freund's adjuvant were given twice, at 6-month
intervals (Fig. 1b).
The initial protein boost increased geometric mean titers (GMTs) of
Env-specific antibody in the DNA-primed rabbits 100- to
200-fold to a
GMT of ~100 µg/ml (Fig.
2 and Table
2). Antibody
titers increased
rapidly in the Env++, dpol, and dpol++ groups,
where titers were
similar at 2 and 4 weeks postboost in most rabbits.
In contrast,
maximal antibody responses in the Env group did not
occur until 4 weeks
after the rgp160 boost. rgp160 immunization
of naive rabbits elicited
an anti-Env GMT of 16.0 µg/ml (Table
2). Maximal titers were not
observed in naive rabbits until 4
weeks after immunization, consistent
with induction of a primary
B-cell response by rgp160 boosting. In both
DNA-primed and naive
rabbits, protein immunization produced a
persistent antibody response
that decreased by a factor of two to four
over the next 6 months
(Fig.
2).
A second protein boost was given 6 months after the first. This second
boost increased antibody titers 5- to 15-fold. Antibody
titers of
DNA-primed and protein-boosted rabbits remained higher
(GMT, 792 µg/ml) than those of animals which had not been primed
with DNA (GMT,
207 µg/ml) (Table
2). The kinetics of the antibody
response to the
second boost were similar in all groups, and titers
peaked within 2 weeks. Again, antibody responses were persistent,
with titers
decreasing only three- to fivefold over the next 6
months (Fig.
2).
Studies of antibody avidity using NaSCN displacement ELISAs.
NaSCN displacement ELISAs demonstrated that the nature of the
Env-specific antibody changed with both protein boosting and the
passage of time (Fig. 4). Antibody
elicited by pHXB2dpol priming of rabbit R110 was released at low
concentrations of NaSCN. The effective concentration of NaSCN required
to release 50% of antiserum from the ELISA plate (referred to as the
ED50) was 0.8 M. Four weeks after the first rgp160 boost,
the ED50 had increased to 1.0 M. However, 6 months later
(just prior to the second protein boost), the ED50 had
risen to 2.3 M. Four weeks after the second rgp160 boost, this value
remained largely unchanged (2.0 M). In contrast, the ED50
of serum collected from a rabbit immunized with protein only (R126) was
fairly high (2.2 M) by 4 weeks after the first immunization and was
basically unchanged 4 weeks after the second protein boost (Fig. 4).
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FIG. 4.
NaSCN displacement ELISAs of anti-Env antisera. Selected
serum samples from a rabbit primed with pHXB2dpol and boosted with
rgp160 (R110) or from a naive rabbit immunized with rgp160 (R126) were
assayed by using an Env-specific NaSCN displacement ELISA. Sera were
diluted to similar concentrations of Env-specific antibody prior to
assay, and all sera were assayed at several dilutions. R110 serum
samples were taken 2 weeks after DNA priming (open circles), 4 weeks
after the first rgp160 boost (filled squares), 6 months after the first
rgp160 boost (filled triangles), and 4 weeks after the second rgp160
boost (filled inverted triangles). R126 serum samples were taken 4 weeks (filled circles) and 6 months (filled squares) after the first
immunization and 4 weeks after the second (filled triangles) rgp160
immunization.
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In contrast to the low-avidity antibody elicited by DNA immunization
with Env, a higher-avidity antibody was induced by DNA
immunization
with influenza virus H1 (Fig.
5).
H1-specific antibody
was released by higher levels of NaSCN
(ED
50, ranging from 2.4
to 3.0 M) than antibody elicited by
Env-expressing plasmids (ED
50,
0.8 M). Anti-H1 antibody
avidity was high at the earliest time
point tested and did not change
with either time or number of
DNA inoculations (Fig.
5).
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FIG. 5.
NaSCN displacement ELISAs of anti-H1 antisera. Rabbit
119 was immunized three times with pH1. Selected serum samples were
assayed by using an A/PR/8/34-specific NaSCN-displacement ELISA. Serum
samples were collected 2 weeks after the second DNA immunization
(filled circles), at the time of the third DNA immunization (filled
squares), and 4 months (filled triangles) and 10 months (filled
inverted triangles) after the third immunization. A serum sample
collected after three DNA immunizations with pHXB2dpol (rabbit R110)
(open circles) is included for comparison.
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Temporal IIIb-neutralizing antibody responses.
The first
rgp160 boost increased both the frequency and titer of
IIIb-neutralizing antibodies. The kinetics of neutralizing antibody
responses were similar to those of ELISA antibody responses (Fig. 2).
Four weeks after the final DNA priming, serum from one of three rabbits
in each of the Env++, dpol, and dpol++ groups exhibited low-titer
IIIb-neutralizing antibody (1:48 to 1:108) (Table 2). Detectable
neutralizing antibody (titer, 1:506 to 1:1,692) was present within 2 weeks after the first protein boost in those three rabbits. Within 4 weeks after the first protein boost, all rabbits, including the naive
group, exhibited neutralizing antibody (Fig. 2). The geometric mean
neutralizing titer of DNA-primed rabbits (titer, 333.5) was
significantly greater than that of naive rabbits (titer, 93) (Table 2).
The second rgp160 boost further increased neutralizing titers in all
rabbits within 2 weeks of this boost (Fig.
2). Again,
the rabbit with
the highest neutralizing titer (R110) had displayed
DNA-primed
neutralizing activity. By 4 weeks after the second
protein boost, the
IIIb-neutralizing titers of rabbits which had
not been primed with DNA
(naive) were approaching those of the
DNA-primed rabbits (Table
2).
Protein boosts also increased the persistence of IIIb-neutralizing
antibody (Fig.
2). Over the 6-month period between the
first and second
rgp160 boosts, geometric mean neutralizing titers
decreased less than
fourfold. Six months after the second rgp160
boost, geometric mean
IIIb-neutralizing titers of the Env and
Env++ groups had decreased
twofold, while that of the naive animals
had not changed. The mean
neutralizing titers of animals in the
dpol and dpol++ groups may have
increased slightly (Fig.
2).
Quality of IIIb-neutralizing antibody.
Over the 6-month period
following each protein boost, ELISA titers decreased more quickly than
IIIb-neutralizing titers and neutralizing antibody/ELISA antibody
ratios therefore rose (Table 3). For
example, mean neutralizing antibody/ELISA antibody ratios of rabbits in
the dpol group rose from 2.5 at four weeks after the first rgp160 boost
to 5.8 at 6 months later. Similarly, neutralizing antibody/ELISA
antibody ratios in this dpol group were higher 6 months after the
second boost (ratio of 20) than immediately after the boost (ratio of
11). Throughout the experiment, the dpol group had the overall most
favorable neutralizing antibody/ELISA antibody ratio, except for the
period of time immediately following the first protein boost (Table 3).
Interestingly, rabbits immunized with protein only also had quite
favorable neutralizing antibody/ELISA antibody ratios. While
determination of neutralizing antibody titers is neither simple nor
absolutely quantitative, these data suggest that protein boosting
increased the quality, as well as the titer, of neutralizing antibody.
Breadth of neutralization.
To assess the breadth of
neutralization elicited by the DNA prime-protein boost protocol,
neutralizing assays were performed with two unrelated, T cell
line-adapted (TCLA) viruses, HIV-1 MN and SF2 (Table
4). Sera collected four weeks after the
second rgp160 boost exhibited moderate to strong neutralization of both MN and SF2. MN-neutralizing titers ranged from 1:149 to 1:3,598, while
SF-neutralizing titers ranged from 1:37 to 1:269. Sera from all
rabbits, including the rabbits immunized with protein only, exhibited
cross-neutralizing activity (Table 4).
V3 loop dependence of IIIb-neutralizing antibody.
Nearly all
IIIb-neutralizing antibody in sera of naive rabbits immunized with
rgp160 only was V3-loop dependent (Table 4). However, the V3-loop
specificity of neutralizing activity in DNA-primed and protein-boosted
groups was much more variable. For example, within the Env++ group, the
neutralizing activity of rabbit R107 was independent of V3-loop
specificity, while that of rabbits R108 and R109 was nearly completely
dependent on antibodies specific for the V3-loop. The V3-loop
specificity in DNA-primed rabbits did not correlate with the ability to
cross-neutralize other TCLA strains. Both sera with and without
V3-loop-dependent IIIb-neutralizing antibody were able to
cross-neutralize MN and SF-2 (Table 4).
Failure to neutralize primary isolates.
Primary isolate
neutralization studies performed on PBMC did not detect primary
isolate-neutralizing antibody in any rabbit sera. Sera were tested
against two non-syncytium-inducing (P46471 and W97464) and two
syncytium-inducing (V67970 and W179273) viruses (43). Some
rabbit sera modestly inhibited production of p24, but none demonstrated
the 90% inhibition viewed as a benchmark of neutralization (data not
shown). However, p24 production by all four primary isolates was
inhibited by greater than 90% by serum from an HIV-1-infected patient
with an atypically high neutralizing titer, demonstrating that these
primary isolates could be neutralized (data not shown).
| |
DISCUSSION |
DNA elicits low-titer, low-avidity, and transient IgG responses to
Env.
This study demonstrates that immunization of rabbits with
plasmids expressing HIV-1 Env or influenza virus H1 elicits very different humoral responses. Multiple DNA immunizations with Env were
required for seroconversion, and antibody responses to Env were
transient, low titer, and low avidity. In contrast, DNA immunization with H1 elicited a persistent, high-titer antibody response with relatively high avidity. Since both glycoproteins are expressed in the
context of the same plasmid (pJW4303) and at generally similar levels
(several nanograms) following gene gun delivery to skin
(34; also unpublished observations), we do not
believe that discordant titers can be attributed to differences in
levels of expression.
We believe that the markedly different antibody responses elicited by
DNA immunization with HIV-1 Env and influenza virus
H1 reflect
fundamental differences in these antigens and in their
interaction with
the immune system. While Env is not a classical
T cell-independent (TI)
antigen, it may exhibit some TI characteristics
noted by Binley et al.
(
3). Env is a heavily glycosylated protein
(
32,
48) and may somewhat resemble TI bacterial polysaccharide
antigens. This may affect the ability of Env to elicit T-cell
help and
may preclude development of germinal center reactions
which are
critical for antibody maturation and persistence (
38,
59).
The persistence of Env but not Gag antibody responses in
HIV-1-positive
patients with low CD4
+ counts (i.e., low T-cell help)
(
3) supports the notion that
antibody responses to Env, but
not Gag, may be TI.
The antibody responses observed in this study, to what are essentially
subunit immunizations, mirror antibody responses observed
following
infection with influenza and immunodeficiency viruses.
Infection with
influenza virus quickly elicits an effective, high-titer
neutralizing
antibody response (
5). In contrast, antibody responses
to
infection with immunodeficiency virus (
8,
9,
24,
44)
mature
more slowly. Development of significant avidity, neutralizing
titer,
and breadth of neutralizing activity lag behind the appearance
of
Env-binding antibodies. Thus, intrinsic antigenic differences
between
immunodeficiency virus Env and influenza virus H1 are
apparent both in
natural infection (
8,
9,
49,
50) and
in DNA immunization.
Protein boosting of DNA-primed responses.
Protein boosting
effectively increased the titer, avidity, and persistence of anti-Env
antibody responses (Fig. 2 and 4). In DNA-primed animals, the avidity
of the anti-Env antibody increased fairly slowly after protein boosting
and more slowly than that in rabbits immunized with protein alone (Fig.
4). This suggests that DNA priming may bias the antibody response
towards recognition of complex, discontinuous epitopes that undergo
slow affinity maturation (2). This would be consistent with
epitope specificities detected in V3-loop inhibition of neutralization
(Table 4). While neutralizing antibody elicited by protein alone was
always specific for the linear V3-loop, the specificity of neutralizing
antibody elicited by DNA priming and protein boosting appeared to be
more complex. A recent study of chimpanzees by Girard et al.
(18) has also found that the avidity of anti-Env antibodies,
primed with recombinant canarypox vaccines, was increased by boosting with recombinant protein. Protein immunizations may effectively boost
antibody responses by providing higher doses of antigen than either DNA
immunization or inoculation with recombinant vaccinia viruses.
Forms of Env.
Previous studies have suggested that oligomeric
forms of Env are superior antigens for raising neutralizing antibody
(14, 39, 51, 57, 62) and that distinct sets of epitopes are exposed on oligomeric and monomeric forms of Env (6, 12, 57). All immunizations given in our study included one or more plasmids expressing an oligomeric form of Env (see Fig. 1). The most
effective neutralizing antibody elicited by DNA priming and protein
boosting was induced by priming with a plasmid that expressed noninfectious particles (dpol) (Table 3 and Fig. 2). It may be worthy
of note that dpol presents Env as spikes exposed to the immune system
on the surface of virus-like particles; the multivalent nature of these
particles may enhance presentation to, and stimulation of, the humoral
immune system. The addition of plasmids expressing secreted monomeric
(gp120) and oligomeric (gp140) forms of Env increased antibody, but not
neutralizing antibody, titers. These data support the finding of Moore
and Sodroski (46) that many antibodies elicited by monomeric
gp120 are specific for nonneutralizing epitopes of Env which are masked
or sequestered in native, oligomeric Env complexes.
DNA priming with full-length Env alone elicited no antibody response
but did provide some priming which may have been T-cell
help. The
majority of this membrane-bound form of Env was found
in the
endoplasmic reticulum and Golgi bodies of transfected Cos
cells and may
not have been available for stimulation of significant
antibody titers.
Intracellular localization may have resulted
from endocytosis directed
by a tyrosine-containing internalization
motif in the cytoplasmic tail
of Env; rapid internalization of
Env occurs in the absence of Gag
(
11,
13,
55). In contrast,
the more potent H1 and gp120
immunogens are found at the surface
of Cos cells as well as in the
cytoplasm. Recent manipulation
of HIV-1 Env expressed by recombinant
vaccinia virus demonstrates
that alterations of transmembrane and
cytoplasmic domains of Env
increase its expression on the surface of
vaccinia virions and
dramatically increase humoral immunogenicity
(
29). Enhanced
surface expression (or increased residence
time at the plasma
membrane) may make Env more accessible to
antigen-presenting cells
and antibody.
Neutralizing antibody.
Sera from all rabbits, either DNA
primed and protein boosted or immunized with protein alone, exhibited
high-titer neutralizing antibody with significant breadth for TCLA
strains of HIV-1 (Table 4). Cross-neutralizing titers were somewhat
higher for HIV-1 MN than for HIV-1 SF2 (data not shown). The good
cross-neutralizing activity for TCLA strains raised by protein-only
immunization may in part reflect the presumed oligomeric structure of
the rgp160 used for boosts. Unfortunately, cross-neutralizing activity
did not extend to the four primary isolates tested in this study. Previous studies have shown that priming with recombinant vaccinia virus followed by protein boosting elicited significant titers of TCLA
neutralizing antibody (19, 20, 26, 27). A recent study by
Letvin et al. (33) demonstrated that DNA immunization, followed by rgp160 boosting, elicited high titers of simian-human immunodeficiency virus HXB-2-neutralizing titers and protected macaques
from challenge with this simian-human immunodeficiency virus) with a
TCLA Env. None of these prime-boost schemes have elicited neutralizing
antibody for primary isolates of HIV-1.
These results are consistent with other studies in which antibody
elicited by TCLA Envs is able to neutralize other TCLA strains
of HIV-1
(
10,
19,
39,
42,
45,
56,
62) but not primary
isolates
(
25,
39,
51), with the notable exception of an
oligomeric
gp160 study with rabbits (
62). We have previously
observed
distinct patterns of neutralization in rabbit sera after
DNA priming
rabbits with primary isolate Env constructs and boosting
them with
recombinant vaccinia viruses which express a variety
of primary isolate
Envs (
51).
Development of a DNA prime-protein boost protocol with primary isolate
Envs which consistently elicits higher-titer, cross-reactive
neutralizing antibody for primary isolates is our next goal. The
observation that primary isolate-neutralizing antibody present
in the
HIV-1-positive patients is specific for complex, conformation-dependent
epitopes (
61) suggests that protein boosting reagents which
maintain the neutralizing epitopes of primary isolate Envs will
be
critical.
| |
ACKNOWLEDGMENTS |
We are indebted to F. Vogel, N. Miller, and A. Schultz for
discussion. We thank Helen Drake-Perrow for administrative assistance.
This research was supported in part by U.S. Public Health Service
grants R01-AI-34241 (H. Robinson) and 5-T32 AI-07272 (J. Richmond), by
a Howard Hughes Postdoctoral Research Fellowship for Physicians (S. Lu), and by contract NCI-6S-1649 (D. Montefiori).
| |
FOOTNOTES |
*
Corresponding author. Present address: Division of
Microbiology and Immunology, Yerkes Primate Research Center, 954 Gatewood NE, Atlanta, GA 30329. Phone: (404) 727-7217. Fax: (404)
727-7768. E-mail: hrobins{at}rmy.emory.edu.
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Journal of Virology, November 1998, p. 9092-9100, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
