Previous Article | Next Article 
J Virol, January 1998, p. 585-592, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Simian Immunodeficiency Virus (SIV)
Envelope-Specific Fabs with High-Level Homologous Neutralizing
Activity: Recovery from a Long-Term-Nonprogressor SIV-Infected
Macaque
Joakim
Glamann,1
Dennis R.
Burton,2
Paul W. H. I.
Parren,2
Henrik J.
Ditzel,2
Karen A.
Kent,3
Caroline
Arnold,3
David
Montefiori,4 and
Vanessa M.
Hirsch1,*
Immunodeficiency Viruses Section, Laboratory
of Infectious Diseases, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Rockville, Maryland
208521;
Departments of Immunology and
Molecular Biology, The Scripps Research Institute, La Jolla, California
920372;
NIBSC, Potters Bar, Herts,
United Kingdom3; and
Department of
Surgery, Duke University Medical Center, Durham, North Carolina
277104
Received 22 July 1997/Accepted 19 September 1997
 |
ABSTRACT |
An antibody phage display library was constructed from RNA
extracted from lymph node cells of a simian immunodeficiency virus (SIV)-infected long-term-nonprogressor macaque. Seven gp120-reactive Fabs were obtained by selection of the library against SIV monomeric gp120. Although each of the Fabs was unique in sequence, there were two
distinct groups based on epitope recognition, neutralizing activity in
vitro, and molecular analysis. Group 1 Fabs did not neutralize SIV and
bound to a linear epitope in the V3 loop of the SIV envelope. In
contrast, two of the group 2 Fabs neutralized homologous,
neutralization-sensitive SIVsm isolates with high efficiency but failed
to neutralize heterologous SIVmac isolates. Based on competition
enzyme-linked immunosorbent assays with mouse monoclonal antibodies of
known specificity, these Fabs reacted with a conformational epitope
that includes domains V3 and V4 of the SIV envelope. These neutralizing
and nonneutralizing Fabs provide valuable standardized and renewable
reagents for studying the role of antibody in preventing or modifying
SIV infection in vivo.
| |
INTRODUCTION |
Simian immunodeficiency virus (SIV)
infection of macaques is a relevant and widely used animal model for
human immunodeficiency virus type 1 (HIV-1) infection. SIV-infected
macaques develop an AIDS-like syndrome characterized by declining
peripheral CD4+ cell counts, opportunistic infections, and
wasting (11). As also observed in HIV-1-infected
individuals, the duration of clinical latency in SIV-infected macaques
varies widely, dependent upon the virulence of the infecting virus, as
well as undefined host factors, which include the effectiveness of the
immune response. The importance of host factors is underscored by the
observation that animals experimentally infected with molecularly
cloned SIV also exhibit considerable variation in disease outcome
(15). While the majority of animals infected with pathogenic
SIV isolates develop AIDS by 1 to 2 years postinfection, a small
fraction of infected animals remain clinically healthy and appear to be
the SIV macaque equivalent of human HIV-1 infected long-term
nonprogressors (LTNP) (6, 35, 43). Although a vigorous
humoral and cellular immune response accompanies both progressive and
nonprogressive SIV infections, the mechanisms by which host immunity
actually controls or protects against infection is not known. A role
for neutralizing antibodies is suggested by the rapid death of animals that do not mount a SIV-specific antibody response (15, 53). However, the temporal association of measurable effector cytolytic CD8
suppression with down modulation of viral replication in the postacute
phase of infection is also consistent with a role of cell-mediated
immunity in controlling infection (27, 52).
The role of neutralizing antibody can be directly assessed through
passive immunoprophylaxis experiments utilizing the SIV-infected macaque model. However, in considering SIV-infected macaques as a model
to study the role of neutralizing antibody, one must take into
consideration the fact that the immunodominant epitopes of the SIV and
HIV envelope glycoproteins may differ. For example, although the V3
loop is an immunodominant region of both the HIV-1 and SIV envelope
glycoproteins (31, 44), there are major differences in the
characters of the V3 immune responses to these two viruses. This region
of the HIV-1 envelope is antigenically variable and appears to
represent a linear, type-specific, principal neutralizing domain of
T-cell line-adapted isolates (18, 36) but not of primary
isolates (48). Like primary HIV-1 isolates, the V3 analog of
the SIV envelope is generally highly conserved and does not appear to
constitute a neutralization epitope for SIV. Conformational epitopes
may be the target of more broadly neutralizing antibody responses.
Neutralizing antibodies to conformational epitopes are prevalent in
sera from HIV-1-infected individuals (34, 45) and probably
also in sera of SIV-infected macaques (19). Thus, 90% of
the neutralizing activity in the serum of SIV-infected macaques is
absorbed by a 45-kDa fragment of gp120 that encompasses domains V3 to
V5 (20). In terms of the specific conformational epitopes of
the two viruses, the majority of neutralizing monoclonal antibodies
generated to HIV-1 appear to bind a conformational epitope involving
the CD4 binding domain, whereas, this region has not been identified as
a neutralizing domain of SIV. However, the repertoire of monoclonal
antibodies generated to the SIV envelope is considerably less extensive
than that of monoclonal antibodies generated to the HIV-1 envelope. Two
neutralizing epitopes on SIV gp120 have been defined by mouse
monoclonal antibodies, a linear V2 epitope and a conformational epitope
that includes residues in the V3 and V4 domains (3, 22, 23,
25) but appears to be distinct from the CD4 binding site (8,
19, 20). The apparent importance of conformational epitopes as
critical targets for neutralization of both HIV-1 and SIV suggests that
the SIV-infected macaque model would make a relevant system for
dissection of the role of antibody in protection.
A number of immunoprophylaxis trials have been conducted with serum or
plasma collected from SIV-infected animals but have yielded conflicting
results. In two studies, infusion of a plasma pool from SIVmac-infected
animals or a cocktail of four neutralizing mouse monoclonal antibodies
(13, 24) failed to protect any of the recipient macaques
from a homologous SIV challenge. However, two other trials reported
partial protection against a homologous intravenous virus challenge
following administration of a plasma pool from SIVsm- or
SIVmne-infected macaques (29, 38) and one reported partial
protection against a heterologous SIVsm/B670 challenge (9).
Protection of two of three neonatal macaques from oral challenge as a
result of transplacentally acquired SIV antibodies also suggests a
beneficial role for antibodies (49). Finally, in a
postexposure immunotherapy trial, administration of purified
immunoglobulin G (IgG) (SIVIG) from an SIV-infected LTNP macaque had a
beneficial effect in modifying subsequent disease progression
(14). Unfortunately, the neutralizing antibodies in these
various pools differed significantly in character and breadth and
therefore the conflicting results of many of these trials cannot be
resolved.
The SIV-infected macaque model offers a unique system for dissection of
the antiviral antibody response at the molecular level and assessment
of the role of protective epitopes by in vivo experimentation. For this
reason, we initiated a study to produce SIV envelope monoclonal
antibodies with high-level neutralizing activity that could serve as
standardized, renewable reagents. Since the humoral immune response to
SIV infection mounted by LTNP might be predictive of an effective
immune response and based upon the beneficial clinical effect of
purified SIVIG from such a macaque (14), this nonprogressor
macaque was chosen as a source of immune cells to extract RNA for the
purpose of generating a combinatorial phage display library. The
present report describes the recovery by phage display technology of
envelope-specific Fabs with high-level homologous neutralizing
activity.
| |
MATERIALS AND METHODS |
Animal.
An inguinal lymph node was biopsied from a rhesus
macaque, E544, at 6 years post-SIVsmF236 challenge and was used as a
source of mRNA for construction of a combinatorial phage display Fab library. This healthy monkey was also the source of a plasma pool used
in a postexposure passive immunotherapeutic trial that demonstrated a
beneficial effect of simian antibodies (14). The lymph node biopsy was gently disrupted and stored as a viable single-cell suspension in 10% dimethyl sulfoxide and 10% fetal calf serum in
liquid nitrogen.
Construction of a lymph node 
1/
antibody phage display
library.
Total RNA isolated from 1.3 × 107 lymph
node cells (RNA Isolation Kit; Stratagene, La Jolla, Calif.) was
reverse transcribed into cDNA by using an oligo(dT) primer. Thirty
cycles of 94°C for 15 s, 52°C for 50 s, and 72°C for
90 s were performed. Macaque -chain genes were amplified with
primers specific for human light-chain genes. The Fd segment
(variable and first constant domains) of macaque heavy-chain genes was
amplified with a combination of family-specific human VH primers based
at the 5' end and a macaque isotype 1-specific primer based at the
3' end (16, 40), as listed in Table
1. A total of 27 heavy-chain and 21 -chain amplification reactions, each with a single pair of primers, were conducted.
Purification, restriction digestion, and sequential ligation of light-
and heavy-chain DNAs into the pCOMB3H phage display
vector and
transformation of electrocompetent
Escherichia coli XL-1
Blue (Stratagene) were performed essentially as previously
described
(
2,
7). Briefly, 450 ng of
-chain DNA digested
with
SacI and
XbaI was ligated into 1,500 ng of
pCOMB3H which
had been digested with
SacI and
XbaI, and the ligated product
was transformed into
E. coli XL-1 Blue. Transformants were propagated
overnight at 37°C
by solid-phase amplification (
28) in 3 liters
of 0.3%
SeaPrep agarose (FMC BioProducts, Rockland, Maine) in
TerrificBroth
(Gibco/BRL, Gaithersburg, Md.) supplemented with
1% glucose, 50 µg
of carbenicillin per ml, and 10 µg of tetracycline
per ml. Phagemid
DNA (
-chain pCOMB3H) was isolated from this
overnight culture. Nine
hundred nanograms of
1 chain digested
with
XhoI and
SpeI was ligated into 3,000 ng of similarly digested
-chain pCOMB3H, and the ligated product was transformed into
E. coli XL-1 Blue. Transformants were expanded in a volume
of
5 liters by solid-phase amplification as described above. The
final
library of 3 × 10
7 clones was stored in 12.5%
glycerol-Luria-Bertani (LB) broth
at
80°C until use.
Panning and ELISA reagents.
SIVsmH4 recombinant glycoprotein
130 (rgp130) expressed in CHO cells was a gift from Nancy Haigwood,
Bristol-Myers Squibb Pharmaceutical Research Institute (37,
42). SIVmac251 rgp120 expressed in baculovirus was purchased from
Intracel Inc. (Cambridge, Mass.), and the following reagents were
obtained through the AIDS Research and Reference Reagent Program,
National Institute of Allergy and Infectious Diseases: SIVmac239 rgp140
produced by vaccinia virus provided by the Vaccine Research and
Development Branch, Division of AIDS, National Institute of Allergy and
Infectious Diseases; HIV-2 ST rgp120 expressed in S2 drosophila cells
from Margery Chaikin, SmithKline Beecham Pharmaceuticals (4, 17, 26); and mouse monoclonal antibodies KK45 and KK46, specific for
the V3 loop of SIV gp120, from Karen Kent and Caroline Arnold (25). In all panning and enzyme-linked immunosorbent assay
(ELISA) experiments, recombinant envelope antigens were diluted to 2 to 8 µg/ml in phosphate-buffered saline (PBS) and adsorbed to EIA/RIA A/2 (ELISA) plates (Costar, Cambridge, Mass.) overnight at 4°C.
Library screening.
A total of 109 bacteria of
the library stock were thawed and grown for 90 min at 37°C in 100 ml
of 2×YT broth (Gibco/BRL) supplemented with 1% glucose, 100 µg of
carbenicillin per ml, and 10 µg of tetracycline per ml. Bacteria were
pelleted and resuspended in 100 ml of 2×YT broth with 100 µg of
carbenicillin per ml and 10 µg of tetracycline per ml, and helper
phage VCS M13 (Stratagene) was added at a multiplicity of infection of
50. The culture was expanded to 1,000 ml and shaken for 5 to 6 h
at 37°C (30). Phage particles were precipitated overnight
at 4°C from the clarified supernatant with 4% polyethylene glycol
and 0.5 M NaCl, recovered by centrifugation, and resuspended in 2%
skim milk powder in PBS with 0.05% sodium azide. ELISA wells were
coated with the selection antigen, incubated overnight, and blocked
with 2% skim milk powder in PBS and 0.05% sodium azide for 1 h
at room temperature prior to addition of phage particles. Unbound phage
particles were removed after 1 h by washing each well 10 times
with PBS containing 0.05% Tween 20. Bound phage particles were eluted
with 100 mM triethylamine (Sigma, St. Louis, Mo.) and immediately
neutralized with 2 M Tris (pH 7.2). Exponentially growing E. coli XL-1 Blue was infected with the eluted phage and expanded by
solid-phase amplification as described above. The eluted phage
particles (clones) were enumerated after each round of panning and when
a 5- to 10-fold increase was encountered (typically after the third
round), phagemid DNA was extracted. The gene III-encoding segment of
the phagemid was removed by restriction digestion with SpeI
and NheI, and the phagemid DNA was self-ligated and
transformed into E. coli XL-1 Blue. A total of 30 to 60 single colonies were picked, and each was inoculated into 200 µl of
LB broth supplemented with 1% glucose and 100 µg of carbenicillin
per ml in 96-well microtiter plates and grown overnight at 30°C. Fab
production was induced by growing the colonies in 200 µl of LB broth
with 100 µg of carbenicillin per ml and 1 mM
isopropyl-
-D-thiogalactopyranoside (IPTG; Gold
Biotechnology, St. Louis, Mo.) for 5 h. Crude bacterial
supernatants were assayed directly by ELISA for reactivity with the
same antigen used for selection.
Large-scale Fab production and purification.
To facilitate
purification of Fabs, a six-residue histidine tail (6×HIS) was added
to the C terminus of the heavy chain by overlapping PCR. Primers 5'-CAG
GTG CAG CTG CTC GAG TCG GG-3' and 5'-TCC CAG ATC TGC GGC CGC TTA AAT
TAA TTA ATG GTG ATG G-3' were used to amplify the heavy-chain gene from
Fab clone 201. The primary PCR product was reamplified with primers
5'-CAG GTG CAG CTG CTC GAG TCG GG-3' and 5'-TAA TTA ATG GTG ATG GTG ATG
GTG GCT AGC ACC ACC ACA TGT TTT G-3'. The resulting fragment was cut with restriction enzymes XhoI and NotI and
ligated back into Fab clone 201 which had been linearized with
XhoI and NotI to create Fab 201-6×HIS. Fab clone
201-6×HIS was subsequently used as a cloning vector to introduce
6×HIS tails into Fab clones 101, 102, 202, 203, and 204 by subcloning
the SacI-to-SauI fragment.
Bacterial cultures of 1 to 2 liters were grown in LB broth (Gibco/BRL)
with 1% glucose and 100 µg of carbenicillin per ml
at 30°C. At an
optical density at 600 nm of approximately 0.2,
the bacteria were
pelleted and resuspended in LB broth supplemented
with 100 µg of
carbenicillin per ml and 0.1 mM IPTG. Cultures
were induced for 5 h at room temperature. For Fabs with a 6×HIS
tail, the bacterial
pellet was resuspended in 20 to 40 ml of PBS-0.1
mM
phenylmethylsulfonyl fluoride and subjected to three cycles
of freezing
(
80°C) and thawing (37°C). The extract was clarified
by
centrifugation and purified by chelate chromatography on a
nitrilotriacetic acid column (Novagen, Madison, Wis.) by following
the
supplier's instructions. For Fabs without a 6×HIS-tail, the
bacterial
pellet was resuspended in 20 to 40 ml of 20 mM Tris
(pH 8.6) and
subjected to three cycles of freezing (
80°C) and
thawing (37°C).
The clarified extract was applied to a 10-ml open
DEAE-Affi-Gel Blue
column (Bio-Rad, Hercules, Calif.) and washed
with 20 column volumes of
20 mM Tris (pH 8.6). The column was
eluted stepwise in 20-ml fractions
containing 100, 250, and 500
mM NaCl. All fractions were tested in an
ELISA for reactivity
with antigen. Most macaque Fabs eluted in the 100 mM NaCl fraction
when tested by ELISA for gp120 reactivity. Purified
Fab was concentrated
and dialyzed against PBS. Fab purity was judged by
sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, and
concentration
was determined with a BCA Protein Assay Reagent kit
(Pierce, Rockford,
Ill.) with bovine serum albumin as the standard. The
concentration
of DEAE-Affi-Gel Blue-purified Fabs was estimated with
an antigen
capture ELISA using unconjugated and alkaline
phosphatase-conjugated
goat anti-human Fab antibodies (Pierce).
Nitrilotriacetic acid
column-purified Fab served as a standard.
ELISA analysis of Fab reactivity and cross-reactivity.
The
following protein antigens were diluted and adsorbed to polystyrene
plates as described above: SIV or HIV-2 gp120 at 2 µg/ml, carbonic
anhydrase at 10 µg/ml, ovalbumin at 10 µg/ml, and thyroglobulin
(Sigma) at 100 µg/ml. Plates were blocked with 3% bovine serum
albumin-PBS for 1 h, and 50 µl of crude or purified Fab was
subsequently added to the wells. Following 1 h of incubation, Fab
binding was detected with a 1:1,000 goat anti-human
F(ab')2-alkaline phosphatase conjugate (Pierce). The assay
was developed with 1 mg of p-nitrophenyl phosphate (Sigma)
per ml.
Epitope mapping.
In initial experiments, Fabs 102 and 202 were tested in a competition ELISA as described previously
(23). A panel of mouse monoclonal antibodies that define
four epitope clusters on the SIV envelope glycoprotein were used: a
conformation-dependent neutralization epitope in the V3-V4 region (KK5,
KK9, KK17, KK44, KK56, and KK57), a linear epitope in the V3 loop (KK45
and KK46), a linear epitope in the V1-V2 region (KK13), and an epitope
within gp41 (KK41). In a second series of experiments, the epitope
specificities of Fabs 101, 102, 103, 201, 202, 203, and 204 were
determined by using KK45 and 201-IgG1. Antibody 201-IgG1 is Fab 201 converted into a whole macaque IgG1 and expressed in COS cells
(unpublished data). Subsaturating concentrations of KK45 and 201-IgG1
were mixed with serial dilutions of Fabs. The mixture was transferred to plates coated with SIVmac251 gp120 at 2 µg/ml and incubated for
2 h. Binding of mouse monoclonal antibody and 201-IgG1 was detected with a goat anti-mouse Ig-alkaline phosphatase conjugate (Jackson ImmunoResearch, West Grove, Pa.) and a goat anti-human IgG
(heavy and light chains)-alkaline phosphatase conjugate, respectively. The latter conjugate does not cross-react with macaque Fabs expressed in bacteria; thus, it only detects whole macaque IgG. Fab binding was
measured in parallel by setting up duplicate dilution series.
Peptide mapping.
Twenty-mer peptides, with an overlap of 10 amino acids, spanning the whole of SIVmac251 gp120 (EVA774) were
obtained from the Reagent Program of the European Vaccine against AIDS.
The reactivity of Fab 102 against these peptides was tested in an ELISA.
Assay of SIV-neutralizing antibodies.
SIV neutralization was
measured by using a cell-killing assay as previously described
(15, 32). All neutralization experiments were performed with
purified Fabs or heat-inactivated plasma samples. SIVsmH4, SIVsmE543-3,
and SIVmac239 are molecularly cloned viruses, whereas SIVsmE660,
SIVsmB670, and SIVmac251 are uncloned virus stocks. Virus stocks were
produced in H9 cells (SIVsmH4, SIVsmB670, and SIVmac251) or CEM×174
cells (SIVsmE543-3 and SIVmac239).
Nucleic acid sequencing and analysis.
Nucleic acid
sequencing was carried out on a 373A automated DNA sequencer (Applied
Biosystems) using a Taq fluorescent dideoxynucleotide terminator cycle sequencing kit. The following sequencing primers were
used: heavy chain, 5'-ATTGCCTACGGCAGCCGCTGG-3' (HC1) and 5'-GGAAGTAGTCCTTGACCAGGC-3' (HC4); chain,
5'-ACAGCTATCGCGATTGCAGTG-3' (LC1) and
5'-CACCTGATCCTCAGATGGCGG-3' (LC4). Resulting sequences were
analyzed by using MacVector (International Biotechnologies, New Haven,
Conn.) and GeneWorks (IntelliGenetics, Campbell, Calif.) software.
Sequence similarity searches were performed with V-base, a compilation
of all available human variable-segment Ig germ line sequences obtained
from I. Tomlinson (10).
| |
RESULTS |
Macaque SIV gp120-specific Fabs.
RNA extracted from an
inguinal lymph node of SIV-infected LTNP rhesus macaque E544 was used
as the template for first-strand cDNA synthesis. With the primers shown
in Table 1, the light-chain gene and the Fd segment of the 1
heavy-chain gene were amplified and cloned into a phagemid vector. The
resulting Fab phage library was then selected against monomeric gp120
preparations of SIVsmH4, SIVmac251, or HIV-2/ST, and seven SIV
gp120-specific Fabs were isolated. Every clone, except Fab 204, was
recovered at least twice in independent panning experiments, showing
the reproducibility of the propagation procedure employed in this
study. To increase the diversity of the Fabs recovered or enrich for
rarer Fabs, two epitope-masking experiments were conducted. Masking of
the epitope recognized by Fab 201 resulted in the reisolation of group 1 clones solely. The masking of two epitopes (Fabs 101 and 201) resulted in the isolation of a variety of clones of which only one was
new, Fab 204. In addition, two Fabs (1LNRhE544 and 2LNRhE544) were
selected from the unscreened library and used as negative controls in
subsequent experiments.
SIV gp120-specific Fabs form two distinct groups.
The
specificity of the selected Fabs was assessed by ELISA using a panel of
SIV and HIV-2 recombinant envelope proteins. As shown in Fig.
1, all seven Fabs were envelope specific,
with no evidence of cross-reactivity to a panel of randomly chosen,
unrelated protein antigens. Three of the Fabs were broadly reactive,
binding to recombinant gp120 from SIVsmH4, SIVmac251, SIVmac239, and
HIV-2/ST, whereas the reactivity of the other four Fabs was restricted
to the SIV envelope preparations and did not include HIV-2 rgp120, suggesting that two distinct groups of Fabs had been recovered. Significantly, one of the Fabs with broad reactivity had been obtained
by panning with HIV-2/ST gp120. Based upon this binding specificity,
the Fab clones were numbered according to these two groups as 101, 102, and 103 for the broadly reactive Fabs and 201 to 204 for those specific
for SIV envelopes alone.
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 1.
Graphical representation of ELISA results. Reactivity of
Fabs (10 µg/ml) to monomeric rgp120 from various SIV and HIV-2
isolates and a panel of irrelevant protein antigens. Fabs 101, 102, 103, 201, 202, 203, and 204 were selected by panning of the library
against monomeric SIV rgp120, whereas 1LNRh544 is a randomly picked
clone from the unscreened library.
|
|
Sequence analysis confirmed the grouping based upon antigen binding. As
shown in Fig.
2, the deduced amino acid
sequence of
the VH-D-JH and V
-J
segments illustrated relatedness
among the
Fabs within each group. Most notably, the heavy-chain CDR3
(HCDR3)
regions within a group were conserved in terms of amino acid
sequence
and length. Within group 1, the heavy-chain sequences of Fabs
101, 102, and 103 shared 77 to 87% identity overall, with the
greatest
divergence observed in CDR2, FR3, and CDR3. The
chains
of clones
101 and 102 were identical, with the exception of two
amino acid
differences at the amino terminus due to the use of
different primers
for amplification. The
chain of clone 103
was only slightly more
divergent, differing from the latter two
clones by six amino acid
substitutions.
View larger version (27K):
[in this window]
[in a new window]
|
FIG. 2.
Alignment of the deduced amino acid sequences of the
variable domains of the heavy and chains. Substitutions relative to
either Fab 101 or 201 are shown in single amino acid code. Identical
residues are indicated by dashes. Amino acids are numbered in
accordance with Kabat et al. (21), and complementary
determining regions (CDR1, CDR2, and CDR3) and framework regions (FR1,
FR2, FR3, and FR4) are shown above the sequence alignments.
|
|
Within group 2, Fabs 201 and 202 were highly related, differing in only
nine residues in the heavy chain and one residue in
the kappa chain
(due to the use of a different PCR primer). These
clones had identical
HCDR3 sequences. Fab 204 was the most divergent
clone within this
group, differing in 36 residues from either
Fab 201 or 202. Despite the
overall divergence, each of the Fabs
within this group showed
remarkably little variation in the HCDR3;
most of the amino acid
differences were seen within HCDR1, HCDR2,
and FR3. An additional
unique feature of each of the group 2 Fabs
was the presence of two
cysteine residues in HCDR3 that probably
are bridged by a disulfide
bond, thus forming an extra loop.
The striking length and sequence uniformity of the CDR3 regions within
each group of Fabs suggested that each group might
represent somatic
variants of a common precursor. However, there
were a number of
differences at the nucleotide level at the VH-D
junctional regions of
the group 1 Fabs (Fig.
3), despite
conserved
amino acid sequences, indicating that the clones probably
arose
from independent recombinational events. In group 2, Fabs 201
and
202 had identical VH-D junctional regions and were probably
somatic
variants, but Fabs 203 and 204 appeared distinct and also
may have
arisen from independent precursors.
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 3.
Alignment of the nucleotide sequences of the VH-D
junctions from various SIV rgp120-specific Fab clones. Three codons on
either side of the VH-D junction of Fab 101 or 201 are shown.
Substitutions in clones 102, 103, 202, 203, and 204 are indicated by
the appropriate nucleotides, and identity is indicated by dashes.
|
|
The occurrence of specific Fabs with relatively conserved HCDR3 regions
but considerable variability throughout the rest of
the VH gene has
been described previously for anti HIV-1 gp41
Fabs (
5). This
could arise from multiple crossover events occurring
during PCR
amplification of antibody genes in library construction.
A more likely
explanation is that it represents convergent evolution
driven by an
HCDR3 motif giving high-affinity binding to a molecular
feature on
gp120.
To tentatively establish the germ line origin of the Fab clones, we
relied upon the extensive homology between macaque and
human Igs to
conduct similarity searches of all known human Ig
genes. As shown in
Table
2, all of the heavy-chain sequences
exhibited homology to the human VH4 family of germ line segments.
All
of the group 1 Fabs were most related to VH4.39. The heavy
chains of
Fabs 201 and 202 showed the best match to human VH segment
4.42 (
50), whereas Fabs 203 and 204 showed the best alignment
to
human VH segments DP-67 (
51) and DP-71 (
47),
respectively.
However, in all four homology searches, a high score was
obtained
for human VH segment DP-67. Significant similarity to any of
the
human D segments was not found by searching V-base or all of
GenBank.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Homology of macaque heavy and kappa light chains of
various SIV-specific Fabs with human Ig germ
line genesa
|
|
Macaque Fabs react with two distinct regions of SIV gp120.
To
identify the epitopes recognized by these macaque Fabs, we tested their
ability to compete for binding in an ELISA with mouse monoclonal
antibodies of known specificity. We utilized a series of mouse
monoclonal antibodies that were recovered by Kent et al. following
immunization with gp120 of SIVmac (23, 25), which shares
82% identity with SIVsm, the strain that induced the Fabs. As seen in
Fig. 4a, Fab clone 102 competed with the binding of MAbs KK45 and KK46, which both recognize a linear epitope in
the V3 loop. Peptide mapping indicated that Fab 102 reacted with two
overlapping peptides (EVA774.30 and EVA774.31 (1) in the V3
loop (data not shown). Thus, it appeared that the epitope recognized by
Fab 102 was contained within 10-mer peptide VTIMSGLVFH (amino acids 322 to 331), of which the last seven residues are perfectly conserved in
SIVmac, SIVsm, and HIV-2, thereby accounting for the cross-reactivity
of this Fab with these three envelope preparations. As expected based
on sequence similarity, each of the group 1 Fabs also competed with V3
loop-specific monoclonal antibody KK45 (Fig. 4c). In contrast, Fab 202 competed with two neutralizing monoclonal antibodies, KK5 and KK9 (Fig.
4b), that target another epitope cluster (KK5, KK9, KK17, KK44, KK56,
and KK57) (22, 23, 25) and recognize a
conformation-dependent epitope in the V3-to-V4 region of the SIV
envelope (8). Whole antibody 201-IgG1 inhibited the binding
of all group 2 Fabs, consistent with recognition of a common region by
these Fabs (Fig. 4d).
View larger version (27K):
[in this window]
[in a new window]
|
FIG. 4.
Identification of epitope specificity of macaque Fabs by
competition ELISA with mouse monoclonal antibodies of defined
specificity. Competition for binding of mouse monoclonal antibodies
with a dilution series of Fab 102 (a) and Fab 202 (b) as the
competitor. (c) Competition of Fabs 101, 102, 103, and 104 with V3
loop-specific monoclonal antibody KK45 as measured by the binding of
the mouse monoclonal antibody. (d) Competition of Fabs 201, 202, 203, and 204 with antibody 201-IgG1 as measured by binding of the whole
macaque antibody. OD, optical density.
|
|
Neutralizing activity of macaque Fabs.
All seven SIV-specific
Fabs, including the two randomly chosen Fabs from the unscreened
library, were purified and tested for neutralizing activity in vitro.
Only Fabs 201 and 202 demonstrated neutralizing activity against the
homologous molecularly cloned virus SIVsmH4, as illustrated in Fig.
5. Although related to these clones, Fab
clones 203 and 204 did not exhibit neutralizing activity. Subsequently,
Fabs 201 and 202 were evaluated against a broader panel of viruses
belonging to the SIVsm and SIVmac lineage. As shown in Table
3, the homologous virus SIVsmB670 was
readily neutralized by Fabs 201 and 202 at a concentration of about 40 ng/ml, whereas heterologous viruses such as SIVmac251 and SIVmac239 were refractory to neutralization in vitro. A recently characterized neutralization-resistant infectious clone, SIVsmE543-3 (15), that also belongs to the SIVsm lineage, and shares 92% envelope identity with SIVsmH4, was resistant to neutralization by Fab 201 or
202 (data not shown). The type specificity of neutralization by these
macaque Fabs was similar to the neutralizing profile observed for SIVIG
purified from this donor macaque; the polyclonal SIVIG neutralized
homologous SIVsmE660 and SIVsmB670 at a concentration of approximately
1 µg/ml but required a 40-fold higher concentration to neutralize the
heterologous virus SIVmac251 (Table 3).
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 5.
Neutralization titration curve of macaque Fabs against
SIVsmH4. The Fab concentration is plotted versus the resulting
protection from virus-induced killing in CEM×174 cells. Two Fabs (201 and 202) neutralized SIV in a dose-dependent fashion, whereas all other
Fabs, including two irrelevant Fabs, 1LNRh544 and 2LNRh544 (negative
[neg] controls); from the unscreened library, failed to neutralize
SIV, even at high concentrations.
|
|
| |
DISCUSSION |
This study demonstrates the usefulness of the antibody phage
display library approach for recovering antigen-specific macaque antibodies from a SIV-infected animal and describes the first fully
homologous macaque Fabs to be generated by this technique. In the
present study, seven unique SIV gp120-specific macaque Fabs were
obtained by screening a lymph node cDNA library from a healthy
long-term survivor of SIV infection (rhesus macaque E544). Based on
nucleotide sequence similarity, pattern of gp120 reactivity, and
epitope-mapping analysis, as defined by competition with previously
characterized mouse monoclonal antibodies, the Fab clones formed two
distinct groups. One group of Fabs recognized a nonneutralization,
linear epitope in the V3 loop, whereas the other recognized a
neutralization, conformation-dependent epitope in the V3-to-V4 region
of gp120. Neutralizing Fabs 201 and 202 were highly effective in
neutralizing virus isolates very closely related to the virus that had
infected this macaque (SIVsmH4 and SIVsmB670) but failed to neutralize
the heterologous virus SIVmac251. Not unexpectedly, the Fabs also
failed to neutralize a homologous, molecularly cloned,
neutralization-resistant SIVsm (SIVsmE543-3 [15]).
These findings underscore the observation that a biologically important
neutralization epitope recognized by SIV-infected animals is within the
V3-to-V4 region (8, 20, 22, 23, 25).
The specificity of binding of the Fabs to various gp120 preparations
was not predictive of the ability to neutralize the isolates from which
these envelopes were cloned. For example, group 2 Fabs bound to SIVmac
and SIVsm gp120 preparations but neutralized only SIVsm isolates. This
disparity between the ability to bind monomeric gp120 and
neutralization might be due to differences in affinity for the
oligomeric envelope as expressed on virions. This disparity has
precedents in other viral systems, including HIV-1. For example, affinity of monoclonal antibodies for the mature oligomeric form of the
envelope glycoprotein is more predictive of neutralization of HIV-1
primary isolates than is binding to monomeric forms of this envelope
antigen (12, 33, 39, 41, 46). Interestingly, the
preferential ability of the Fabs to neutralize homologous isolates was
similar to the profile of a contemporaneous IgG preparation purified
from this macaque, suggesting that the neutralizing Fabs recovered from
this combinatorial library were representative of the antibody response
of this animal. Although this library is unlikely to represent the full
antibody repertoire, this technique shows promise as a method for the
recovery of macaque SIV-specific neutralizing Fabs. Potential factors
that could limit the repertoire are the restricted size of the library
(3 × 107 clones) and the exclusive use of kappa light
chains and 1 heavy chains. Finally, since this is a systemic
infection, the actual choice of tissue for generation of the library
could affect the overall representation of clones. However, in an
independently screened bone marrow library prepared from the same
animal, all but one of the Fab clones grouped both biologically and
genetically with those obtained from the lymph node library (data not
shown), suggesting that the tissue source was not a major determinant of the antibody repertoire representation.
In summary, the SIV envelope Fabs generated from a SIV-infected LTNP
provide further insight into the epitopes recognized by such animals.
Only two epitopes were recognized by this particular animal, one of
which constituted a neutralization epitope. A third Fab generated from
the bone marrow library recognized a third epitope in the V1 region of
gp120 which did not induce neutralizing antibodies (data not shown).
This animal exhibited tightly restricted viral replication and, perhaps
as a consequence, had a low-to-moderate serum neutralizing antibody
titer which was fairly type specific. The spectrum of epitopes
recognized by sera of SIV-infected progressor macaques may be more
extensive. We are currently generating IgG1 macaque monoclonal
antibodies which express the representative Fabs of each group. Such
macaque monoclonal antibodies should prove valuable in defining the
role of neutralizing antibody in preventing or ameliorating SIV
infection in vivo. Additionally, the availability of molecularly cloned
SIV stocks that are sensitive or resistant to neutralization by this
antibody will facilitate studies intended to define the in vivo role of
neutralizing antibodies that are identified by in vitro assays.
| |
ACKNOWLEDGMENTS |
We thank Robert Chanock for his support and for critical reading
of the manuscript, Russ Byrum and Anna Hahn for technical support, and
Mats Persson for sharing macaque Ig sequence information data prior to
publication.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: NIAID Twinbrook
II Facility, 12441 Parklawn Dr., Rockville, MD 20852. Phone: (301) 496- 2976. Fax: (301) 480-2618. E-mail:
vhirsch{at}atlas.niaid.nih.gov.
| |
REFERENCES |
| 1.
|
Almond, N.,
A. Jenkins,
A. Slade,
H. A. M. Cranage, and P. Kitchin.
1992.
Population sequence analysis of simian immunodeficiency (32H reisolate of SIVmac251): a virus stock used for international vaccine studies.
AIDS Res. Hum. Retroviruses
8:77-88[Medline].
|
| 2.
|
Barbas, C. F., III,
A. S. Kang,
R. A. Lerner, and S. J. Benkovic.
1991.
Assembly of combinatorial antibody libraries on phage surfaces: the gene III site.
Proc. Natl. Acad. Sci. USA
88:7978-7982[Abstract/Free Full Text].
|
| 3.
|
Benichou, S.,
R. Legrand,
N. Nakagawa,
T. Taure,
F. Traincard,
G. Vogt,
D. Dormont,
P. Tiollais,
M.-P. Kieny, and P. Madaule.
1992.
Identification of a neutralizing domain in the external envelope glycoprotein of simian immunodeficiency virus.
AIDS Res. Hum. Retroviruses
8:1165-1170[Medline].
|
| 4.
|
Benos, D. J.,
B. H. Hahn,
J. K. Bubien,
S. K. Ghosh,
N. A. Mashburn,
M. A. Chaikin,
G. M. Shaw, and E. N. Benveniste.
1994.
Envelope glycoprotein gp120 of human immunodeficiency virus type 1 alters ion transport in astrocytes: implications for AIDS dementia complex.
Proc. Natl. Acad. Sci. USA
91:494-498[Abstract/Free Full Text].
|
| 5.
|
Binley, J. M.,
H. J. Ditzel,
C. F. Barbas III,
N. Sullivan,
J. Sodroski,
P. W. Parren, and D. R. Burton.
1996.
Human antibody responses to HIV type 1 glycoprotein 41 cloned in phage display libraries suggest three major epitopes are recognized and give evidence for conserved antibody motifs in antigen binding.
AIDS Res. Hum. Retroviruses
12:911-924[Medline].
|
| 6.
|
Buchbinder, S. P.,
M. H. Katz,
N. A. Hessol,
P. M. O'Malley, and S. D. Holmberg.
1994.
Long-term HIV-1 infection without immunological progression.
AIDS
8:1123-1128[Medline].
|
| 7.
|
Burton, D. R.,
C. F. Barbas III,
M. A. A. Persson,
S. Koening,
R. M. Chanock, and R. A. Lerner.
1991.
A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals.
Proc. Natl. Acad. Sci. USA
88:10134-10137[Abstract/Free Full Text].
|
| 8.
|
Choi, W. S.,
C. Collignon,
C. Thiriart,
D. P. W. Burns,
E. J. Stott,
K. A. Kent, and R. C. Desrosiers.
1994.
Effects of natural sequence variation on recognition by monoclonal antibodies that neutralize simian immunodeficiency virus infectivity.
J. Virol.
68:5395-5402[Abstract/Free Full Text].
|
| 9.
|
Clement, J. E.,
R. C. Montelaro,
M. C. Zink,
A. M. Amedee,
S. Miller,
A. M. Trichel,
B. Jagerski,
D. Hauer,
L. N. Martin,
R. P. Bohm, and M. Murphey-Corb.
1995.
Cross-protective immune responses induced in rhesus macaques by immunization with attenuated macrophage-tropic simian immunodeficiency virus.
J. Virol.
69:2737-2744[Abstract].
|
| 10.
|
Cook, G. P., and I. M. Tomlinson.
1995.
The human immunoglobulin VH repertoire.
Immunol. Today
16:237-242[Medline].
|
| 11.
|
Desrosiers, R. C.
1990.
The simian immunodeficiency viruses.
Annu. Rev. Immunol.
8:557-578[Medline].
|
| 12.
|
Fouts, T. R.,
J. M. Binley,
A. Trkola,
J. E. Robinson, and J. P. Moore.
1997.
Neutralization of the human immunodeficiency virus type 1 primary isolate JR-FL by human monoclonal antibodies correlates with antibody binding to the oligomeric form of the envelope glycoprotein complex.
J. Virol.
71:2779-2785[Abstract].
|
| 13.
|
Gardner, M.,
A. Rosenthal,
M. Jennings,
J. Yee,
L. Antipa, and E. Robinson.
1995.
Passive immunization of rhesus macaques against SIV infection and disease.
AIDS Res. Hum. Retroviruses
11:843-854[Medline].
|
| 14.
|
Haigwood, N. L.,
A. Watson,
W. F. Sutton,
J. McClure,
A. Lewis,
J. Ranchis,
B. Travis,
G. Voss,
N. Letvin,
S.-L. Hu,
V. M. Hirsch, and P. R. Johnson.
1996.
Passive immune globulin therapy in the SIV/macaque model: early intervention can alter disease profile.
Immunol. Lett.
51:107-114[Medline].
|
| 15.
|
Hirsch, V.,
D. Adger-Johnson,
B. Campbell,
S. Goldstein,
C. Brown,
W. R. Elkins, and D. C. Montefiori.
1997.
A molecularly cloned, pathogenic, neutralization-resistant simian immunodeficiency virus, SIVsmE543-3.
J. Virol.
71:1608-1620[Abstract].
|
| 16.
|
Huse, W. D.,
L. Sastry,
S. A. Iverson,
A. S. Kang,
M. Alting-Mees,
D. R. Burton,
S. J. Benkovic, and R. A. Lerner.
1989.
Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda.
Science
246:1275-1281[Abstract/Free Full Text].
|
| 17.
|
Ivey-Hoyle, M.,
J. S. Culp,
M. A. Chaikin,
B. D. Hellmig,
T. J. Matthews,
R. W. Sweet, and M. Rosenberg.
1991.
Envelope glycoproteins from biologically diverse isolates of human immunodeficiency viruses have widely different affinities for CD4.
Proc. Natl. Acad. Sci. USA
88:512-516[Abstract/Free Full Text].
|
| 18.
|
Javaherian, K.,
A. J. Langlois,
C. McDanal,
K. L. Ross,
L. I. Eckler,
C. L. Hellis,
A. T. Profy,
J. R. Rusche,
D. P. Bolognesi,
S. D. Putney, and T. J. Matthews.
1989.
Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein.
Proc. Natl. Acad. Sci. USA
86:6768-6772[Abstract/Free Full Text].
|
| 19.
|
Javaherian, K.,
A. J. Langlois,
D. C. Montefiori,
K. A. Kent,
K. A. Ryan,
P. D. Wyman,
J. Stott,
D. P. Bolognesi,
M. Murphey-Corb, and G. J. Larosa.
1994.
Studies of the conformation-dependent neutralizing epitopes of simian immunodeficiency virus envelope protein.
J. Virol.
68:2624-2631[Abstract/Free Full Text].
|
| 20.
|
Javaherian, K.,
A. J. Langlois,
S. Schmidt,
M. Kaufmann,
N. Cates,
J. P. M. Langedijk,
R. H. Meloen,
R. C. Desrosiers,
D. P. W. Burns,
D. P. Bolognesi,
G. J. LaRosa, and S. D. Putney.
1992.
The principal neutralization determinant of simian immunodeficiency virus differs from that of human immunodeficiency virus type 1.
Proc. Natl. Acad. Sci. USA
89:1418-1422[Abstract/Free Full Text].
|
| 21.
|
Kabat, E. A.,
T. T. Wu,
H. M. Perry,
K. S. Gottesman, and C. Foeller.
1991.
.
Sequences of proteins of immunological interest, 5th ed.
U.S. Department of Health and Human Services, Bethesda, Md.
|
| 22.
|
Kent, K.,
C. Powell,
T. Corcoran,
A. Jenkins,
N. Almond,
I. Jones,
J. Robinson, and E. J. Stott.
1993.
Characterisation of neutralising epitopes on simian immunodeficiency virus envelope using monoclonal antibodies, p. 167-172.
Huitieme colloque des cent gardes.
Fondation Marcel Merieux, Lyon, France.
|
| 23.
|
Kent, K. A.,
L. Gritz,
G. Stallard,
M. P. Crangage,
C. Collignon,
C. Thiriart,
T. Corcoran,
P. Silvera, and E. J. Stott.
1991.
Production and characterisation of monoclonal antibodies to simian immunodeficiency virus envelope glycoproteins.
AIDS
5:829-836[Medline].
|
| 24.
|
Kent, K. A.,
P. Kitchen,
K. H. G. Mills,
M. Page,
F. Taffs,
T. Corcoran,
P. Silvera,
B. Flanagan,
C. Powell,
J. Rose,
C. Ling,
A. M. Aubertin, and E. J. Stott.
1994.
Passive immunization of cynomolgus macaques with immune sera or a pool of neutralizing monoclonal antibodies failed to protect against challenge with SIVmac251.
AIDS Res. Hum. Retroviruses
10:189-194[Medline].
|
| 25.
|
Kent, K. A.,
E. Rud,
T. Corcoran,
C. Powell,
C. Thiriart,
C. Collignon, and E. J. Stott.
1992.
Identification of two neutralizing and 8 non-neutralizing epitopes on simian immunodeficiency virus envelope using monoclonal antibodies.
AIDS Res. Hum. Retroviruses
8:1147-1151[Medline].
|
| 26.
|
Kong, L. I.,
S. Lee,
J. C. Kappes,
J. S. Parkin,
D. Decker,
J. A. Hoxie,
B. H. Hahn, and G. M. Shaw.
1988.
West African HIV-2-related human retrovirus with attenuated cytopathicity.
Science
240:1525-1529[Abstract/Free Full Text].
|
| 27.
|
Koup, R. A.,
J. T. Safrit,
Y. Cao,
C. A. Andrews,
G. McLeod,
W. Borkowsky,
C. Farthing, and D. D. Ho.
1994.
Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome.
J. Virol.
68:4650-4655[Abstract/Free Full Text].
|
| 28.
|
Kriegler, M.
1990.
.
Gene transfer and expression: a laboratory manual. W. H.
Freeman & Company, New York, N.Y.
|
| 29.
|
Lewis, M. G.,
W. R. Elkins,
F. McCutchan,
R. E. Benveniste,
C. Y. Lai,
D. C. Montefiori,
D. S. Burke,
G. A. Eddy, and A. Shafferman.
1993.
Passively transferred antibodies directed against conserved regions of SIV envelope protect macaques from SIV infection.
Vaccine
11:1347-1355[Medline].
|
| 30.
|
Marks, J. D.,
H. R. Hoogenboom,
T. P. Bonnert,
J. McCafferty,
A. D. Griffiths, and G. Winter.
1991.
Human antibodies from V-gene libraries displayed on phage.
J. Mol. Biol.
222:581-597[Medline].
|
| 31.
|
Miller, M. A.,
M. Murphey-Corb, and R. C. Montelaro.
1992.
Identification of broadly reactive continuous antigenic determinants of simian immunodeficiency virus glycoproteins.
AIDS Res. Hum. Retroviruses
8:1153-1164[Medline].
|
| 32.
|
Montefiori, D. C.,
W. E. Robinson, Jr.,
S. S. Schuffman, and W. M. Mitchell.
1988.
Evaluation of antiviral drugs and neutralizing antibodies to human immunodeficiency virus by rapid and sensitive microtiter infection assay.
J. Clin. Microbiol.
26:231-235[Abstract/Free Full Text].
|
| 33.
|
Moore, J. P.,
Y. Cao,
L. Qing,
Q. J. Sattentau,
J. Pyati,
R. Koduri,
J. Robinson,
C. F. Barbas III,
D. R. Burton, and D. D. Ho.
1995.
Primary isolates of human immunodeficiency virus type 1 are relatively resistant to neutralization by monoclonal antibodies to gp120, and their neutralization is not predicted by studies with monomeric gp120.
J. Virol.
69:101-109[Abstract].
|
| 34.
|
Moore, J. P., and D. D. Ho.
1993.
Antibodies to discontinuous or conformationally sensitive epitopes on the gp120 glycoprotein of human immunodeficiency virus type 1 are highly prevalent in sera of infected humans.
J. Virol.
67:863-875[Abstract/Free Full Text].
|
| 35.
|
Munoz, A.,
A. J. Kirkby,
Y. D. He,
J. B. Margolick,
B. R. Visscher,
C. R. Rinaldo,
R. A. Kaslow, and J. P. Phair.
1995.
Long-term survivors with HIV-1 infection: incubation period and longitudinal patterns of CD4+ lymphocytes.
J. Acquired Immune Defic. Syndr.
8:496-505.
|
| 36.
|
Palker, T. J.,
M. E. Clark,
A. J. Langlois,
T. J. Matthews,
K. J. Winhold,
R. R. Randall,
D. P. Bolognesi, and B. F. Haynes.
1988.
Type-specific neutralization of the human immunodeficiency virus with antibodies to env-encoded synthetic peptides.
Proc. Natl. Acad. Sci. USA
85:1932-1936[Abstract/Free Full Text].
|
| 37.
|
Planelles, V.,
N. L. Haigwood,
M. L. Marthas,
K. A. Mann,
C. Scandella,
W. D. Lidster,
J. R. Schuster,
R. van Kuyk,
P. A. Marx,
M. B. Gardner, and P. A. Luciw.
1991.
Functional and immunological characterization of SIV envelope glycoprotein produced in genetically engineered mammalian cells.
AIDS Res. Hum. Retroviruses
7:889-896[Medline].
|
| 38.
|
Putkonen, P.,
R. Thorstensson,
L. Ghavamzadeh,
J. Albert,
K. Hild,
G. Biberfeld, and E. Norrby.
1991.
Prevention of HIV-2 and SIVsm infection by passive immunization in cynomolgus monkeys.
Nature
352:436-438[Medline].
|
| 39.
|
Roben, P.,
J. P. Moore,
M. Thali,
J. Sodroski,
C. F. Barbas III, and D. R. Burton.
1994.
Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding sites of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1.
J. Virol.
68:4821-4828[Abstract/Free Full Text].
|
| 40.
|
Samuelsson, A.,
F. Chiodi,
P. Ohman,
P. Putkonen,
E. Norrby, and M. A. Persson.
1995.
Chimeric macaque/human Fab molecules neutralize simian immunodeficiency virus.
Virology
207:495-502[Medline].
|
| 41.
|
Sattentau, Q. J., and J. P. Moore.
1995.
Human immunodeficiency virus type 1 neutralization is determined by epitope exposure on the gp120 oligomer.
J. Exp. Med.
182:185-196[Abstract/Free Full Text].
|
| 42.
|
Scandella, V.,
J. Kilpatrick,
W. Lidster,
C. Parker,
G. K. Moore,
K. A. Mann,
P. Brown,
S. Coates,
B. A. Chapman,
F. Maziarz,
K. S. Steimer, and N. L. Haigwood.
1993.
Non-affinity purification of recombinant gp120 for use in AIDS vaccine development.
AIDS Res. Hum. Retroviruses
9:1227-1238.
|
| 43.
|
Sheppard, H. W.,
W. Lang,
M. S. Ascher,
E. Vittinghoff, and W. Winkelstein.
1993.
The characterization of non-progressors: long-term HIV-1 infection with stable CD4 22 T-cell levels.
AIDS
7:1159-1166[Medline].
|
| 44.
|
Silvera, P.,
B. Flanagan,
K. Kent,
E. Rud,
C. Powell,
T. Corcoran,
C. Bruck,
C. Thiriart,
N. L. Haigwood, and E. J. Stott.
1994.
Fine analysis of humoral antibody response to envelope glycoprotein of SIV in infected and vaccinated macaques.
AIDS Res. Hum. Retroviruses
10:1295-1304[Medline].
|
| 45.
|
Steimer, K. S.,
C. J. Scandella,
P. V. Skiles, and N. L. Haigwood.
1991.
Neutralization of divergent HIV-1 isolates by conformation-dependent human antibodies to gp120.
Science
254:105-108[Abstract/Free Full Text].
|
| 46.
|
Sullivan, N.,
Y. Sun,
J. Li,
W. Hofman, and J. Sodroski.
1995.
Replicative function and neutralization sensitivity of envelope glycoproteins from primary and T-cell line-passaged human immunodeficiency virus type 1 isolates.
J. Virol.
69:4413-4422[Abstract].
|
| 47.
|
Tomlinson, I. M.,
G. Walter,
J. D. Marks,
M. B. Llewelyn, and G. Winter.
1992.
The repertoire of human germline VH segment reveals about fifty groups of VH segments with different hypervariable loops.
J. Mol. Biol.
227:776-798[Medline].
|
| 48.
|
Vancott, T. C.,
V. R. Polonis,
L. D. Loomis,
N. L. Michael,
P. L. Nara, and D. L. Birx.
1995.
Differential role of V3-specific antibodies in neutralization assays involving primary and laboratory-adapted isolates of HIV type 1.
AIDS Res. Hum. Retroviruses
11:1379-1390[Medline].
|
| 49.
|
Van Rompay, K. K. A.,
M. G. Otsyula,
R. P. Tarara,
D. R. Canfield,
C. Berardi,
M. B. McChesney, and M. L. Marthas.
1996.
Vaccination of pregnant macaques protects newborns against mucosal simian immunodeficiency virus infection.
J. Infect. Dis.
173:1327-1335[Medline].
|
| 50.
|
Weng, N.,
J. G. Snyder,
L. Yu-Lee, and D. M. Marcus.
1992.
Polymorphism of human immunoglobulin VH4 germ-line genes.
Eur. J. Immunol.
22:1075-1082[Medline].
|
| 51.
|
Winkler, T. H.,
H. Fehr, and J. R. Kalden.
1992.
Analysis of immunoglobulin variable region genes from human IgG anti-DNA hybridomas.
Eur. J. Immunol.
22:1719-1728[Medline].
|
| 52.
|
Yasutomi, Y.,
K. Reiman,
C. Lord,
M. Miller, and N. Letvin.
1993.
Simian immunodeficiency virus-specific CD8+ lymphocyte response in acutely infected rhesus monkeys.
J. Virol.
67:1707-1711[Abstract/Free Full Text].
|
| 53.
|
Zhang, J.,
L. N. Martin,
E. A. Watson,
R. C. Montelaro,
M. West,
L. Epstein, and M. Murphey-Corb.
1988.
Simian immunodeficiency virus/Delta-induced immunodeficiency disease in rhesus monkeys: relation of antibody response and antigenemia.
J. Infect. Dis.
158:1277-1286[Medline].
|
J Virol, January 1998, p. 585-592, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Goncalvez, A. P., Men, R., Wernly, C., Purcell, R. H., Lai, C.-J.
(2004). Chimpanzee Fab Fragments and a Derived Humanized Immunoglobulin G1 Antibody That Efficiently Cross-Neutralize Dengue Type 1 and Type 2 Viruses. J. Virol.
78: 12910-12918
[Abstract]
[Full Text]
-
Men, R., Yamashiro, T., Goncalvez, A. P., Wernly, C., Schofield, D. J., Emerson, S. U., Purcell, R. H., Lai, C.-J.
(2004). Identification of Chimpanzee Fab Fragments by Repertoire Cloning and Production of a Full-Length Humanized Immunoglobulin G1 Antibody That Is Highly Efficient for Neutralization of Dengue Type 4 Virus. J. Virol.
78: 4665-4674
[Abstract]
[Full Text]
-
Johnson, W. E., Sanford, H., Schwall, L., Burton, D. R., Parren, P. W. H. I., Robinson, J. E., Desrosiers, R. C.
(2003). Assorted Mutations in the Envelope Gene of Simian Immunodeficiency Virus Lead to Loss of Neutralization Resistance against Antibodies Representing a Broad Spectrum of Specificities. J. Virol.
77: 9993-10003
[Abstract]
[Full Text]
-
Hansen, M. H., Nielsen, H. V., Ditzel, H. J.
(2002). Translocation of an Intracellular Antigen to the Surface of Medullary Breast Cancer Cells Early in Apoptosis Allows for an Antigen-Driven Antibody Response Elicited by Tumor-Infiltrating B Cells. J. Immunol.
169: 2701-2711
[Abstract]
[Full Text]
-
Haddrick, M., Brown, C. R., Plishka, R., Buckler-White, A., Hirsch, V. M., Ginsberg, H.
(2001). Biologic Studies of Chimeras of Highly and Moderately Virulent Molecular Clones of Simian Immunodeficiency Virus SIVsmPBj Suggest a Critical Role for Envelope in Acute AIDS Virus Pathogenesis. J. Virol.
75: 6645-6659
[Abstract]
[Full Text]
-
Edinger, A. L., Ahuja, M., Sung, T., Baxter, K. C., Haggarty, B., Doms, R. W., Hoxie, J. A.
(2000). Characterization and Epitope Mapping of Neutralizing Monoclonal Antibodies Produced by Immunization with Oligomeric Simian Immunodeficiency Virus Envelope Protein. J. Virol.
74: 7922-7935
[Abstract]
[Full Text]
-
Glamann, J., Hirsch, V. M.
(2000). Characterization of a Macaque Recombinant Monoclonal Antibody That Binds to a CD4-Induced Epitope and Neutralizes Simian Immunodeficiency Virus. J. Virol.
74: 7158-7163
[Abstract]
[Full Text]
-
Schofield, D. J., Glamann, J., Emerson, S. U., Purcell, R. H.
(2000). Identification by Phage Display and Characterization of Two Neutralizing Chimpanzee Monoclonal Antibodies to the Hepatitis E Virus Capsid Protein. J. Virol.
74: 5548-5555
[Abstract]
[Full Text]
-
Ourmanov, I., Brown, C. R., Moss, B., Carroll, M., Wyatt, L., Pletneva, L., Goldstein, S., Venzon, D., Hirsch, V. M.
(2000). Comparative Efficacy of Recombinant Modified Vaccinia Virus Ankara Expressing Simian Immunodeficiency Virus (SIV) Gag-Pol and/or Env in Macaques Challenged with Pathogenic SIV. J. Virol.
74: 2740-2751
[Abstract]
[Full Text]
Top
Abstract
Introduction
