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Journal of Virology, July 2001, p. 5879-5890, Vol. 75, No. 13
Department of Surgery, Duke University
Medical Center, Durham, North Carolina 277101;
Yerkes Regional Primate Research Center, Atlanta, Georgia
303292; Aventis Pasteur, Marcy
l'Etoile, France3; and Aventis Pasteur,
Willowdale, Ontario, Canada4
Received 6 December 2000/Accepted 23 March 2001
The ability to generate antibodies that cross-neutralize diverse
primary isolates is an important goal for human immunodeficiency virus
type 1 (HIV-1) vaccine development. Most of the candidate HIV-1
vaccines tested in humans and nonhuman primates have failed in this
regard. Past efforts have focused almost entirely on the envelope
glycoproteins of a small number of T-cell line-adapted strains of the
virus as immunogens. Here we assessed the immunogenicity of
noninfectious virus-like particles (VLP) consisting of Gag, Pro
(protease), and Env from R5 primary isolate HIV-1Bx08.
Immunogens were delivered to rhesus macaques in the form of either
purified VLP, recombinant DNA and canarypox (ALVAC) vectors engineered to express VLP, or a combination of these products. Seroconversion to
Gag and Pro was detected in all of the immunized animals. Antibodies that could neutralize HIV-1Bx08 were detected in animals
that received (i) coinoculations with DNABx08 and
VLPBx08, (ii) DNABx08 followed by
ALVACBx08 boosting, and (iii) VLPBx08 alone.
The neutralizing antibodies were highly strain specific despite the
fact that they did not appear to be directed to linear epitopes in the
V3 loop. Virus-specific cellular immune responses also were generated, as judged by the presence of Gag-specific gamma interferon
(IFN- Accumulating evidence supports a
positive role for neutralizing antibodies and
CD8+ cytotoxic T lymphocytes (CTL) in the
antiviral immune response to human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) (33, 44).
The ability to generate antibodies that neutralize a broad spectrum of
primary HIV-1 isolates has proven to be one of the more difficult
challenges for vaccine development (44). Major targets for
neutralizing antibodies are the surface gp120 and transmembrane gp41
envelope glycoproteins of the virus (12). Antibodies that
bind these viral glycoproteins with adequate affinity and appropriate
specificity can prevent HIV-1 from entering host cells (32, 41,
45, 71, 78, 83, 84) and have provided potent protection against
infection in passive-antibody experiments in animal models (2,
20, 22, 35, 38, 76). Attempts at inducing cross-reactive
neutralizing antibodies through vaccination have, however, had limited
success. Most attempts have focused on the envelope glycoproteins of a
small number of T-cell line-adapted (TCLA) strains of virus. Although
the envelope glycoproteins of those strains generate high titers of
neutralizing antibodies, those antibodies mostly target strain-specific
epitopes in the third variable cysteine-cysteine loop (V3 loop)
of gp120 (10, 45) and have been highly specific for TCLA
strains of virus (23, 50). Importantly, the antibodies
have failed to neutralize primary isolates (4, 5, 10, 37)
and often possess little or no detectable neutralizing activity against heterologous TCLA strains (5, 10).
The B-cell response leading to HIV-1-specific neutralizing antibody
production could be fundamentally different for primary isolates
compared to TCLA strains. For example, whereas TCLA strains are highly
sensitive to neutralization by V3 loop-specific antibodies (26,
68), the V3 loop on primary isolates is occluded by N-linked glycans and tertiary folds on the native gp120 molecule (30, 62,
73-75, 90, 91), making it a poor target for neutralizing antibodies (9, 77, 86). The envelope glycoproteins of TCLA strains and many, but not all, primary isolates also exhibit different coreceptor preferences for virus entry. Generally speaking, primary isolates utilize either CCR5 (R5), CXCR4 (X4), or both coreceptors (R5/X4) whereas all TCLA strains have the X4 phenotype (6, 7,
67). For reasons that are poorly understood, most transmitted strains of HIV-1 have an R5 phenotype (63, 87) and,
although no association has been found between coreceptor preference
and neutralization sensitivity (31, 43, 81), it seems
prudent to target R5 strains when designing candidate HIV-1 vaccines.
Much of what is known about the neutralization epitopes on primary
isolates comes from studies of three human monoclonal antibodies (MAbs), immunoglobulin G1b12 (IgG1b12), 2G12, and 2F5. Each of these
MAbs can neutralize diverse primary isolates (12) and, when combined, exhibit synergistic neutralizing activity
(36). A vaccine that generates antibodies equal to the
combination of these specificities is highly desirable and deserving of
intense investigation. Unfortunately, the corresponding epitopes have proven to be poorly immunogenic in natural infections and in
experimentally immunized animals. Other neutralization epitopes are
present on primary isolates that are less conserved. The latter
epitopes account for the sporadic, low-level neutralization of
heterologous isolates by sera from infected individuals (46, 58,
89). They might also account for the potent neutralization of
primary isolates by autologous serum samples that are obtained months or years after the time of virus isolation (29, 48, 56, 58,
89), which would require the cognate epitopes to be immunogenic and well exposed on the virus surface. The presence of neutralization epitopes that are both immunogenic and antigenic on primary isolates affords opportunities for vaccine development that have received little attention.
Part of the reason why the latter epitopes have received little
attention for vaccine development relates to their high degree of
variability, which may not be suitable for cross-reactive neutralizing antibody induction. Given the paucity of information on the
immunogenicity of primary isolate Env, however, it would be premature
to conclude that no monovalent Env will generate cross-reactive
neutralizing antibodies. Also, should monovalent primary isolate Env be
capable of generating antibodies that neutralize the autologous primary isolate, it might ultimately make it feasible to formulate a composite (polyvalent) Env vaccine that will target a broad spectrum of primary isolates.
Few studies have examined the immunogenicity of primary-isolate
envelope glycoproteins. VanCott et al. (85) showed that immunization with the gp120 protein from the R5 primary isolate HIV-1CM235 generated antibodies in baboons that neutralized TCLA strains, including the TCLA version of the vaccine strain, but not the
autologous primary isolate. Similar results were obtained by Beddows et
al. (4), who immunized healthy, HIV-1-seronegative volunteers with the gp120 protein from R5/X4 strain HIV-1W61D. Only
Berman et al. (8) have succeeded at generating antibodies that neutralized an R5 primary isolate; this was done by immunizing rabbits with the gp120 protein from HIV-1CM244.
In addition to neutralizing antibodies, increasing evidence supports a
critical role for virus-specific CD8+ T cells in
protective immunity against HIV-1 (11, 15, 55, 61, 64, 69, 70,
92). CD8+ T-cell responses to HIV-1 can be
both cytotoxic (11, 15, 55, 61, 69, 92) and virus
suppressive (80, 88). Recent reports of the effect of
CD8+ cell depletion on viremia and
CD4+ cell counts in SIV- or SIV-HIV-infected
macaques (27, 40, 72) has given additional in vivo
relevance to the importance of the CD8+ antiviral
response in the control of this viral infection. Even more recently,
vaccine-induced, virus-specific CTL strongly correlated with viremia
control and the preservation of CD4+ lymphocytes
in the highly pathogenic SIV-HIV 89.6P-macaque model (3).
The immunogenicity of Gag, Pro, and Env from R5 primary isolate
HIV-1Bx08 was investigated here in rhesus
monkeys. Immunogens were delivered in the form of either purified
noninfectious virus-like particles (VLP), recombinant DNA and canarypox
vectors engineered to express VLP, or a combination of delivery modes.
Our results reveal the priming of Gag-specific cellular responses by
DNA-based immunization. We also demonstrate that envelope glycoprotein
from an R5 primary isolate can be used to generate antibodies in
nonhuman primates that are capable of neutralizing the autologous
primary isolate.
Cells and viruses.
MT-2 is a CD4+
human lymphoblastoid cell line that is highly permissive to cytopathic
infection with TCLA strains of HIV-1 (24). Stocks of HIV-1
strains IIIB, MN, and SF2 were produced in H9 cells as previously
described (47). MT-2 and H9 cells were maintained at
37°C in 5% CO2 in growth medium consisting of
RPMI 1640 medium supplemented with 12% heat-inactivated fetal bovine
serum (FBS) and 50 µg of gentamicin/ml. Peripheral blood mononuclear
cells (PBMC) were purified from buffy coats of healthy HIV-1-seronegative individuals and stimulated for 1 day with
phytohemagglutinin P as described previously (46). Growth
medium for PBMC consisted of RPMI 1640 medium supplemented with 20%
heat-inactivated FBS, 4% interleukin-2, and 50 µg of gentamicin/ml.
Monkey kidney Vero cells used for Bx08 VLP production were grown and
passaged biweekly in Dulbecco's modified Eagle's medium (Flow
Laboratories, McLean, Va.) supplemented with 10% heat-inactivated FBS,
glutamine (2 mM), penicillin (50 IU/ml), and streptomycin (50 µg/ml).
Stably transfected cells secreting VLP were maintained in the presence of 0.5 mg of Geneticin (Gibco BRL, Grand Island, N.Y.) per ml.
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.13.5879-5890.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Induction of Neutralizing Antibodies and
Gag-Specific Cellular Immune Responses to an R5 Primary Isolate of
Human Immunodeficiency Virus Type 1 in Rhesus Macaques
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
)-producing cells. These cellular immune responses required the
inclusion of DNABx08 in the immunization modality, since
few or no IFN--producing cells were detected in animals that
received either VLPBx08 or ALVACBx08 alone. The
results demonstrate the feasibility of generating neutralizing
antibodies and cellular immune responses that target an R5 primary
HIV-1 isolate by vaccination in primates.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
80°C until use. Coreceptor usage of Bx08 was
assessed in MT-2, U87-CD4-CCR5, and U87-CD4-CXCR4 cells
(25) by using p24 production as a measure of infection.
sCD4 and MAbs. Recombinant soluble human CD4 (sCD4) containing the full-length extracellular domain of human CD4 was obtained from Progenics Pharmaceuticals (Tarrytown, N.Y.). Human MAbs IgG1b12, 2G12, and 2F5 have been shown to neutralize diverse primary isolates (14, 19, 28, 59, 82). IgG1b12 recognizes an epitope in the CD4-binding domain of gp120 that is sensitive to mutations in V2 and C3 (42). 2G12 recognizes an epitope comprising residues within C2-V4 of gp120 that involved sites of N glycosylation (82). 2F5 recognizes a linear epitope in the ectodomain of gp41 having the amino acid sequence ELDKWA (51).
DNA vectors. The nucleotide and amino acid numbering used throughout for HIV was that of Myers et al. (52). All pseudovirion-expressing vectors were constructed from p83-19 (57, 65), which was, in turn, derived from plasmid pMTHIVd25 (66). Plasmid p83-19 was engineered to incorporate several mutations to enhance safety, such as deletion of a 25-bp DNA fragment (nucleotides 753 to 777; LAI sequence) containing viral RNA packaging sequences (1, 34) and deletion of a 1.9-kbp DNA fragment containing most of the pol gene, effectively eliminating the coding sequences for reverse transcriptase and integrase. However, the deletion left intact the gene encoding the viral protease (pro), resulting in the expression of particles with processed Gag antigens. In p83-19, transcription of the HIV-1 coding sequences is regulated by the inducible human metallothionein IIa (MT) promoter and a simian virus 40 polyadenylation sequence. In order to produce pseudovirions with the Env glycoprotein from the Bx08 primary isolate, the HIV-1LAI env gene was replaced with that of HIV-1Bx08. To this end, a 2,440-bp fragment containing the entire gp160-encoding gene of HIV-1Bx08 was amplified by PCR from cells infected with this isolate. The PCR product was then used to replace the corresponding region in p83-19, giving rise to plasmid p133B1.
In order to produce a plasmid DNA vector for immunization studies, a construct was prepared incorporating the env gene from HIV-1Bx08 under the control of the cytomegalovirus promoter. The construct, pCMV3Bx08, is derived from plasmid pCMVgDtat
vprBx08, which
was, in turn, engineered from p83-19 after several modifications. These
included (i) replacement of the MT promoter with a 1.6-kbp DNA fragment
containing the human cytomegalovirus immediate-early gene promoter,
enhancer, and intron A sequences; (ii) deletion of the coding sequences
for HIV-1 Tat and Vpr; (iii) replacement of the HIV-1
gp120Bx08 signal peptide sequence with the human
herpes simplex virus glycoprotein D signal peptide sequence; (iv)
replacement of the Ampr-encoding gene with the
Kanr-encoding gene; and (v) replacement of the
simian virus 40 polyadenylation sequence with that of the bovine growth
hormone-encoding gene. The nucleotide sequences of all constructs were
confirmed by DNA sequencing.
Production of noninfectious VLP for immunogenicity studies. Vero cells were transfected at passage 141 with plasmid p133B1 by the calcium phosphate method as specified previously (57), and stable transfectants expressing noninfectious HIV-1-like particles were established. VLP were isolated from the supernatants of a stable clone (DD19) after induction of the Vero cells with heavy metals (57). Expression of Gag and Env was verified by Western blot assay using, respectively, an anti-HIV-1 p24 mouse MAb (NEA-9306; Dupont Canada, Inc., Markham, Ontario, Canada) and MAb K3A, which recognizes gp120 envelope proteins from different clades (kindly supplied by Ronald Kennedy). Fully assembled, Env-containing particles were isolated from the supernatants of the stably engineered Vero DD19 cell clone by ultracentrifugation through a glycerol cushion and purified by sucrose gradient fractionation (66). The p24 contents of the various particle species were determined by a p24-specific enzyme immunoassay (Coulter Immunology, Hialeah, Fla.). The cell line secreted approximately 800 to 950 µg of p24 per liter.
Construction of recombinant canary poxvirus vCP1579. Recombinant vCP1579 was engineered to express HIV-1LAI Gag and Pro, together with HIV-1Bx08 gp120 fused to a 28-amino-acid fragment of the transmembrane domain of HIV-1LAI gp41, and a synthetic polypeptide containing Pol and Nef CTL epitopes. This recombinant is similar to vCP1452 (10, 79), except that the gene fragment encoding gp120LAI was replaced with its cognate sequences from HIV-1Bx08. Due to the absence of the gp41 ectodomain, the expressed envelope glycoprotein would be expected to consist of membrane-anchored gp120. The construct was generated in several steps. Briefly, the vector-modifying sequences from the pMPC6H6K3E3 insertion vector, encoding E3L and K3L, were inserted into the C6 locus of recombinant vCP1566. Recombinant vCP1566 was, in turn, generated by insertion of an expression cassette encoding a synthetic polypeptide containing Pol and Nef CTL epitopes into the C5 locus of vCP1453. Recombinant vCP1453 was obtained after coinsertion of the genes encoding the HIV-1Bx08 Env and HIV-1LAI Gag-Pro products into the ALVAC genome at the C3 locus.
Macaque immunizations.
Rhesus macaques were divided into six
groups with four animals in each group, as summarized in Table
1. Briefly, animals in groups 1 and 2 were inoculated with either a high dose (3 mg) or a low dose (600 µg)
of DNABx08, respectively, together with 50 µg
of VLPBx08 in QS21 (100 µg) at weeks 0, 4, 24, and 44. Animals in groups 3 and 4 were inoculated with either the high
dose or the low dose of DNABx08, respectively, at
weeks 0 and 4 and were boosted with ALVACBx08 at
weeks 24 and 44 (4 × 108 PFU at week 24 and
4 × 108 PFU at week 44). Animals in group 5 were inoculated with 50 µg of VLPBx08 in QS21
adjuvant at weeks 0, 4, 24, and 44. Animals in group 6 received 3 mg of
control DNA at weeks 0 and 4 and were boosted with
ALVACBx08 at weeks 24 and 44 (same doses as
above). All immunogens were administered in a volume of 2 ml
intramuscularly. The QS21 adjuvant was prepared and filter sterilized
before filling in of single-dose vials and stored at 20°C.
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Western blot. Antibodies specific for HIV-1 antigens were assessed with a commercial HIV-1 Western blot kit (Genetic Systems, Sanofi Diagnostics Pasteur, Inc.) that is based on the LAI strain of HIV-1.
Enzyme-linked immunosorbent assay. Bx08-V3 peptide-specific binding antibodies were assessed in Nunc (Roskilde, Denmark) Immunoplates (MaxiSorb F96) using alkaline phosphatase-conjugated goat anti-monkey IgG as described previously (16). Plasma samples were assayed in duplicate at a 1:50 dilution, and values are given as the average A405. The Bx08-V3 peptide had the amino acid sequence TRPNNNTRKSIHIGPGRAFYTTGDIIGDIR and was made by SynPep Corporation (Dublin, Calif.). The peptide was judged to be 91.5% pure by high-pressure liquid chromatography analysis. This same peptide was used in neutralization competition assays as described below.
Neutralizing antibody assays. Neutralization of HIV-1Bx08 and other primary isolates was assessed in human PBMC by using a reduction in p24 Gag antigen synthesis as described previously (46, 58). Briefly, 500 50% tissue culture-infective dose of virus were incubated with various dilutions of serum samples for 1 h at 37°C before the addition of PBMC. Cells were washed three times with 200 µl of growth medium and resuspended in 200 µl of fresh interleukin-2 growth medium 1 day later. Culture supernatants were assessed for p24 content at a time when p24 synthesis in virus control wells (no test sample) was in a linear phase of increase, which is when optimum sensitivity is achieved in this assay (93). Neutralization titers were defined as either the dilution of serum or concentration of sCD4 and MAbs at which p24 synthesis was reduced by 80% relative to that of a negative control. These titers refer to conditions under which serum samples were incubated with virus before the addition of cells. All serum samples were heat inactivated for 1 h at 56°C prior to assay. V3 peptide neutralization competition assays were performed by preincubating serum samples with either phosphate-buffered saline (PBS) or Bx08-V3 peptide (50 µg/ml [final concentration]) for 1 h at 37°C. The serum samples were then assayed at a 1:3 dilution on the virus as described above.
Neutralization of HIV-1 strains IIIB, MN, and SF2 was measured in an MT-2 cell-killing assay by using neutral red to quantify the fraction of cells that survived virus-induced cytopathic effects (47). Briefly, 500 50% tissue culture-infective doses of virus were incubated with multiple dilutions of serum samples for 1 h at 37°C before the addition of cells. The incubation was continued until extensive syncytium formation had just occurred in wells that contained no serum sample (usually 4 to 6 days). Neutralizing antibody titers are defined as the serum dilution (before the addition of cells) at which 50% of the cells were protected from virus-induced killing. A 50% reduction in cell-killing corresponds to an approximately 90% reduction in viral Gag antigen synthesis in this assay (10, 54). Each set of assays included a positive control serum that had been assayed multiple times and had a known average titer.ELISpot assay for IFN- release from antigen-specific
PBMC.
PBMC were separated from whole blood by Lymphocyte
Separation Medium (Cellgro), washed twice, counted, and viably frozen
in freezing medium for time course studies. Cells were thawed, washed twice, resuspended at 4 × 106/ml, and
stimulated for 48 h with 2 µg of HIV-1HXB2
Gag peptide pools (National Institutes of Health AIDS Research and
Reference Reagent Program) per ml. The peptides were 20-mers
overlapping by 10 amino acids and were divided into two pools of
peptides with 25 peptides per pool. Pool 1 represented amino acids 1 to 260, while pool 2 represented amino acids 251 to 500. Ovalbumin peptide
was used as a negative control, and 50 ng of phorbol myristate acetate
(Sigma) per ml plus 1 µg of ionomycin (Sigma) per ml served as
a positive control. Ninety-six-well mixed cellulose ester filtration plates (Millipore) were coated with 50 µl of a 5-µg/ml
concentration of mouse anti-human IFN- MAb 1-D1K (Biosource
International) overnight at 4°C. Plates were then washed four times
with PBS and blocked with RPMI (Cellgro) medium supplemented with
L-glutamine, penicillin, and 10% heat-inactivated FBS
(HyClone) for 1 h at 37°C. Prestimulated cells were then added
to the coated plate in 100 µl of R10. Duplicate wells containing
either 5 × 105 or 2.5 × 105 stimulated PBMC were plated for each sample
and incubated for 16 to 20 h at 37°C. Plates were washed four
times with PBS (Cellgro) and then four times with PBS containing 0.05%
Tween 20 (Sigma) to remove all cells and debris from the plates. Next,
100 µl of 1-µg/ml biotinylated anti-IFN- MAb 1-D1K (Mabtech)
diluted in PBS was added. After 2 h, the plates were washed four
times with PBS containing 0.1% Tween 20 and incubated with 100 µl of
Vectastain ABC (Vector Laboratories) per well for 1 h. Following
four washes with PBS plus 0.1% Tween 20, Stable DAB (Research
Genetics) was added to the wells and monitored for color development
(approximately 5 min). The Stable DAB reaction was stopped by
washing the wells three times with H2O. Wells
were counted visually under a dissecting microscope (Olympus) after the
plates had dried.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Biologic and immunologic properties of HIV-1Bx08. Coreceptor usage was determined by infection of cells that coexpressed CD4 and either CCR5 or CXCR4. HIV-1Bx08 was able to infect U87-CD4-CCR5 cells but not U87-CD4-CXCR4 cells or MT-2 cells (data not shown). This outcome is consistent with an R5 phenotype for this virus. As a control for the integrity of the cells used for X4 phenotype classification, HIV-1IIIB was able to infect U87-CD4-CXCR4 and MT-2 cells but not U87-CD4-CCR5 cells.
The neutralization sensitivity of HIV-1Bx08 was assessed with sCD4 and human MAbs in the PBMC assay. The virus was sensitive to all four reagents. The minimum concentration of sCD4 required to inhibit HIV-1Bx08 was 9.9 µg/ml. This concentration of sCD4 is approximately 500 to 5,000 times higher than that required to inhibit TCLA strains (10, 17, 49) but was moderately low compared to other primary isolates, most of which resist inhibition by this same preparation of sCD4 at concentrations of up to 50 µg/ml (10). The virus was also neutralized by the MAbs, where the 80% inhibition doses were 11.5 (IgG1b12), 1.2 (2G12), and 27.9 (2F5) µg/ml.HIV-1 seroconversion.
HIV-1-specific IgG was assessed by
Western blot assay at multiple time points throughout the immunization
schedule. Figure 1A shows that all of the
immunized animals possessed Gag-specific antibodies by week 6 (2 weeks
after the second inoculation). Gag-specific seroconversion was
strongest in animals that received VLPBx08 either
alone (group 5) or in combination with the high and low doses of
DNABx08 (groups 1 and 2, respectively).
Gag-specific seroconversion was variable and less potent at this time
in animals that received the high and low doses of
DNABx08 (groups 3 and 4, respectively). A similar
pattern was noted for Pro-specific antibodies (p31), with the exception
of very low levels of anti-Pro antibodies in animals inoculated with
the low dose of DNABx08 (group 4). Little or no
Env-specific antibody was detected at this time.
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Neutralization of HIV-1Bx08 with sera from immunized
macaques.
Neutralization of HIV-1Bx08 was
assessed in human PBMC with serum collected preimmunization and after
the second, third, and fourth (final) immunizations. These assays were
performed without knowledge of the animal group assignments. Figure
2A shows that neutralizing antibodies
were detected in three of the four animals in group 1 (high-dose
DNABx08 plus VLPBx08), four
of the four animals in group 2 (low-dose DNABx08
plus VLPBx08), three of the four animals in group
3 (high-dose DNABx08 plus
ALVACBx08 boosting), and three of the four
animals in group 5 (VLPBx08). These
neutralization titers ranged from 1:2 to 1:48. The highest titer was
achieved in an animal (RYl5) immunized four times with
VLPBx08. Notably, a 1:2 dilution of sera from
several animals in groups 1, 2, and 5 achieved >90% neutralization
and serum from at least one animal in each of the latter groups
achieved 99 to 100% neutralization. The potency of neutralization at a
1:2 serum dilution did not correlate with the 80% neutralization
titer.
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113% to +76% at the lowest dilution tested
(1:2), which is within the normal variation range of this assay.
Although an 80% reduction in p24 Gag antigen synthesis (fivefold
decrease in infectivity) has proven to be the most reliable
minimum cutoff for a true positive (10), two animals in
the group that received the low dose of DNABx08 plus ALVACBx08
(REl5 and RHg5) had neutralization values of 76 and 75%, respectively,
after final boosting.
Absence of cross-reactive neutralizing antibodies.
Cross-neutralizing activity was assessed with three TCLA strains (IIIB,
MN, and SF2) and four heterologous R5, clade B primary isolates. In
only two cases were neutralizing antibodies detected with a TCLA strain
as measured in the MT-2 cell assay (data not shown). These cases
occurred 2 weeks after final boosting and included sera from animal
RLj5 in group 3 and animal REl5 in group 4, both having weak
neutralizing activity against HIV-1MN (titers of
1:29 and 1:50, respectively). Nine serum samples that neutralized HIV-1Bx08 were further tested for the ability to
neutralize heterologous R5 primary isolates in the PBMC assay. The
results of the latter assays were all negative (Table
2).
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Antibody specificity for the V3 loop.
Serum samples obtained 2 weeks after final boosting (week 46) were tested for the presence
antibodies that could bind a Bx08-V3 peptide. Sera from animals in
groups 1, 2, and 5 and, to a lesser extent, groups 3 and 4 tested
positive by enzyme-linked immunosorbent assay (Fig.
3). The detection of V3-specific
antibodies did not always predict the ability to neutralize
HIV-1Bx08. For example, at least three serum
samples that neutralized HIV-1Bx08 had no detectable V3-specific antibodies (RZh5, RMg5, and RWh5). In addition, two samples that were positive for V3-specific antibodies (RSg5 and
RNl5) failed to neutralize HIV-1Bx08. The
frequent detection of antibodies to the V3 loop of Bx08 is strong
evidence of the presence of gp120-specific antibodies that were not
detected by the Western blot assay (Fig. 1).
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Analysis of IFN- secretion.
PBMC from the vaccinated and
control animals were tested for the ability to secrete IFN- upon
stimulation with two pools of overlapping peptides representing the Gag
sequence of HIV-1HXB2. Peptide pool 1 spanned
amino acids 1 to 260, while peptide pool 2 spanned amino acids 251 to
500. The number of IFN- spots per 106 PBMC for
each animal at each time point is shown in Fig.
5, and the average value for each group
is shown in Fig. 6. We measured Gag-specific responses 2 weeks after the second immunization, a time
when near-peak cellular responses would be expected, and then 3 months
later (16 weeks), when memory responses from the first two
immunizations would be in effect. The PBMC were then screened 2 weeks
after each of the next two immunizations (weeks 26 and 46) to determine
boosting effects and finally at week 56 (3 months following the final
immunization) for immunologic memory for the HIV-1 antigen. The
presence of 
20 ELISpots in an assay was considered a positive test.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Despite a growing desire to generate antibodies that are capable
of neutralizing primary isolates, very few primary-isolate Envs have
been investigated as potential immunogens for inclusion in an HIV-1
vaccine. Here we show that noninfectious VLP containing the surface
envelope glycoprotein of an R5 primary isolate were capable of
generating antibodies that neutralized the vaccine strain of virus. The
antibodies could be generated by (i) coinoculation with
DNABx08 and VLPBx08, (ii)
priming with DNABx08 and boosting with
ALVACBx08, and (iii) inoculation with
VLPBx08 alone. Most notable was the fact that, in
certain cases, neutralizing antibodies were induced by recombinant
vectors without the need for protein boosting
a milestone that has not
been reported previously with respect to primary-isolate neutralization
by macaque serum.
Unlike the neutralizing antibodies generated by most of the candidate HIV-1 Env vaccines tested to date, the neutralizing antibodies generated here did not appear to be directed to linear epitopes in the V3 loop of gp120. Two lines of evidence support this conclusion. First, some serum samples with neutralizing activity had little or no antibody reactive with a Bx08 V3 loop peptide. Second, the neutralizing activity of two serum samples with strong reactivity to the Bx08 V3 loop peptide was not reduced in the presence of a high concentration (50 µg/ml) of the V3 loop peptide. These results suggest that the neutralizing antibodies targeted one or more epitopes outside the V3 loop. We note, however, that our experiments with linear peptides do not eliminate the possibility that the epitope(s) consisted of a conformational structure within V3 that was not represented by the linear peptide.
A disadvantage of the neutralizing antibodies seen here was their minimal cross-reactivity. Specifically, the antibodies had little or no neutralizing activity against TCLA strains and heterologous R5 primary isolates. The neutralizing antibody response generated by these candidate vaccines needs to be improved to target a broad spectrum of HIV-1 antigenic variants. We also noted that the Bx08-specific neutralizing antibodies were low in titer (1:2 to 1:48); however, the small magnitude of this response may be less of a concern than the limited strain specificity. For example, results of recent passive-immunization experiments with nonhuman primates indicate that preexisting neutralization titers of 1:10 or less may provide complete or partial protection against intravenous and mucosal challenge with simian-human immunodeficiency virus (35, 38, 76). Given that natural HIV-1 transmission appears to be virus dose dependent (60), where not all exposures result in infection (39), it is conceivable that a 5- or 10-fold reduction in virus infectivity by minimally diluted serum in vitro will correspond to an undiluted potency in vivo that is effective prophylactically. A number of recombinant vaccine vectors are also known to prime B cells for a rapid secondary neutralizing antibody response generated by HIV-1 Env protein boosting (23, 79), and at least one example exists of a dramatic secondary neutralizing antibody response after a virus challenge of macaques that had been primed with recombinant modified vaccinia virus strain Ankara-SIV vaccines (54). It may be expected that DNA vectors expressing VLP will prime for similar secondary neutralizing antibody production in response to HIV-1 infection.
We also detected HIV-1 Gag-specific cellular immune responses in the
groups of animals that were inoculated with
DNABx08. All groups of animals receiving this
immunogen scored positive for IFN--producing cells, and with the
exception of a weak positive response to Gag pool 1 in one animal
(RWh5, week 56), no IFN--producing cells were detected in animals
inoculated with either VLPBx08 or
ALVACBx08 alone. Thus, as judged by the best
overall immune responses (i.e., IFN--producing cells and
HIV-1Bx08 neutralizing antibody), coinoculation
with a low dose of DNABx08 plus
VLPBx08 was the superior immunization modality
investigated (Fig. 6). The Gag peptides used for PBMC stimulation were
not completely matched to the vaccine strain, making it possible that
some strain-specific reactivities went undetected in our assay. Despite
this, it is noteworthy that IFN- production could be stimulated in
many cases with two overlapping peptide pools covering the
amino-terminal and carboxyl-terminal halves of Gag as evidence that at
least two specificities of the cellular response were induced by vaccination.
While we consider the combined humoral and cellular immune responses elicited by these vaccine candidates to be encouraging, we also readily acknowledge the limited benefit that can be expected from neutralizing antibodies that are so narrow in specificity. Nonetheless, our results point to several vaccine strategies that may be useful for delivering a more appropriate immunogen for cross-reactive neutralizing antibody induction once that immunogen has been identified. We also did not determine whether the structure of Bx08 Env incorporated into the VLP is optimal for neutralizing antibody induction. For example, it has been argued that the native oligomeric (trimeric) structure of gp120-gp41 heterodimers must be preserved for optimal immunogenicity (13). Although it is possible that the Bx08 VLP expressed by our DNA vector contained native oligomeric envelope glycoproteins, we have not confirmed this. Furthermore, the envelope glycoproteins expressed by ALVACBx08 consisted of membrane-anchored gp120 lacking the gp41 ectodomain, which may not represent the most suitable structure for optimal immunogenicity. Additional studies are required to determine the full potential of VLP as an immunogen for neutralizing antibody induction.
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ACKNOWLEDGMENTS |
|---|
We thank H. J. A. Fleury for HIV-1Bx08 and Dennis Burton, Herman Katinger, and John Mascola for MAbs. We also thank Ginger James, Lauren Rodrigues, Heike Marshall, and Lisa Murdin for excellent technical assistance.
This work was supported by funding from Aventis Pasteur and the National Institutes of Health (AI-85343).
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Surgery, Box 2926, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-5278. Fax: (919) 684-4288. E-mail: monte{at}acpub.duke.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, July 2001, p. 5879-5890, Vol. 75, No. 13
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.13.5879-5890.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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