Induction of Neutralizing Antibodies and Gag-Specific Cellular Immune Responses to an R5 Primary Isolate of Human Immunodeficiency Virus Type 1 in Rhesus Macaques

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.

Clade B primary isolate Bx08 was isolated from an HIV-1-infected French individual 8 months postseroconversion (48) and was kindly provided by H. J. A. Fleury (Bordeaux, France). The other clade B primary isolates used in this study were Bal, JR-FL, P15, and P27. Bal and JR-FL were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (Bal was contributed by Suzanne Gartner, Mikulas Popovic, and Robert Gallo; JR-FL was contributed by Irvin Chen). Bal was isolated from explanted human infant lung tissue (21), JR-FL was isolated from frontal lobe brain tissue of a patient with AIDS dementia (53), and P15 and P27 were isolated during early seroconversion by PBMC coculture (58). Bal, JR-FL, P15, and P27 all use the CCR5 coreceptor (10, 18). Stocks of primary isolates were produced in PBMC as previously described (46). All virus stocks were made cell free by 0.45-μm (pore size) filtration and stored in aliquots at −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 II_a (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-1_LAI env gene was replaced with that of HIV-1_Bx08. To this end, a 2,440-bp fragment containing the entire gp160-encoding gene of HIV-1_Bx08 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-1_Bx08 under the control of the cytomegalovirus promoter. The construct, pCMV3Bx08, is derived from plasmid pCMVgDtat⁻vpr⁻Bx08, 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 gp120_Bx08 signal peptide sequence with the human herpes simplex virus glycoprotein D signal peptide sequence; (iv) replacement of the Amp^r-encoding gene with the Kan^r-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-1_LAI Gag and Pro, together with HIV-1_Bx08 gp120 fused to a 28-amino-acid fragment of the transmembrane domain of HIV-1_LAI 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 gp120_LAI was replaced with its cognate sequences from HIV-1_Bx08. 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-1_Bx08 Env and HIV-1_LAI 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 DNA_Bx08, respectively, together with 50 μg of VLP_Bx08 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 DNA_Bx08, respectively, at weeks 0 and 4 and were boosted with ALVAC_Bx08 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 VLP_Bx08 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 ALVAC_Bx08 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.

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-1_Bx08 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-γ 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-1_HXB2 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-γ 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-γ 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.

Biologic and immunologic properties of HIV-1_Bx08.

Coreceptor usage was determined by infection of cells that coexpressed CD4 and either CCR5 or CXCR4. HIV-1_Bx08 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-1_IIIB was able to infect U87-CD4-CXCR4 and MT-2 cells but not U87-CD4-CCR5 cells.

The neutralization sensitivity of HIV-1_Bx08 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-1_Bx08 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 VLP_Bx08 either alone (group 5) or in combination with the high and low doses of DNA_Bx08 (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 DNA_Bx08 (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 DNA_Bx08 (group 4). Little or no Env-specific antibody was detected at this time.

Western blot reactivities had intensified by 2 weeks following the final immunization (week 46) where, in addition to anti-Gag antibodies, all immunized animals showed evidence of Pro-specific seroconversion (Fig. 1B). We noted that the anti-Gag and anti-Pro reactivity was relatively weak in animals RAd5 (group 3) and REl5 (group 4). Env-specific antibodies (for gp160 and gp41 but not for gp120) were detected at this time in animals in groups 1, 2, and 5 only. The lack of detection of gp120-specific antibodies in these animals could be due to genetic and antigenic dissimilarities in the gp120 of Bx08 compared to the IIIB gp120 antigen used in the Western blot assay strips. All of the animals immunized with control DNA and boosted with ALVAC_Bx08 were seronegative for Env at week 46 (Fig. 1B). A boosting effect at weeks 26 and 46 (2 weeks after the third and fourth inoculations, respectively) was seen in each animal, where evidence of seroconversion remained present 12 weeks after the final inoculation (data not shown).

Neutralization of HIV-1_Bx08 with sera from immunized macaques.

Neutralization of HIV-1_Bx08 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 DNA_Bx08 plus VLP_Bx08), four of the four animals in group 2 (low-dose DNA_Bx08 plus VLP_Bx08), three of the four animals in group 3 (high-dose DNA_Bx08 plus ALVAC_Bx08 boosting), and three of the four animals in group 5 (VLP_Bx08). These neutralization titers ranged from 1:2 to 1:48. The highest titer was achieved in an animal (RYl5) immunized four times with VLP_Bx08. 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.

As shown in Fig. 2B, no HIV-1_Bx08-specific neutralization was detected with sera from animals immunized with the low dose of DNA_Bx08 plus ALVAC_Bx08 (group 4) or control DNA plus ALVAC_Bx08 (group 6). Sera from these animals reduced p24 synthesis by −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 DNA_Bx08 plus ALVAC_Bx08 (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-1_MN (titers of 1:29 and 1:50, respectively). Nine serum samples that neutralized HIV-1_Bx08 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).

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-1_Bx08. For example, at least three serum samples that neutralized HIV-1_Bx08 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-1_Bx08. 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).

Two serum samples that contained antibodies reactive with the Bx08 V3 peptide and neutralized HIV-1_Bx08 were tested for neutralizing activity in the presence and absence of Bx08 V3 peptide. Similar experiments have shown this to be a very effective means by which to determine whether a portion of the neutralizing antibodies are V3 specific (10, 16). The results of these assays showed that the Bx08 V3 peptide (50 μg/ml) was not able to outcompete the neutralizing activity of these serum samples (Fig. 4).

Analysis of IFN-γ secretion.

PBMC from the vaccinated and control animals were tested for the ability to secrete IFN-γ upon stimulation with two pools of overlapping peptides representing the Gag sequence of HIV-1_HXB2. Peptide pool 1 spanned amino acids 1 to 260, while peptide pool 2 spanned amino acids 251 to 500. The number of IFN-γ 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.

All groups of animals primed with DNA_Bx08 showed clear positive Gag-specific ELISpot responses following the third immunization, while the groups of animals receiving only VLP_Bx08 or ALVAC_Bx08 failed to score positive. Following the first two inoculations with DNA_Bx08, the groups receiving the low dose of DNA_Bx08 had more animals with >20 ELISpots (five of eight animals) than did the groups receiving the high dose of DNA_Bx08 (none of eight animals). Following the first boost with either VLP_Bx08 or ALVAC_Bx08, all DNA_Bx08-primed groups contained animals that were positive, with 12 of 16 animals scoring >20 ELISpots. Animals that scored positive typically did so with both peptide pools (28 of 42 positive samples). The second boost did not markedly increase the magnitude or frequency of the ELISpot responses. At the end of the experiment, the frequency of Gag-specific ELISpot-positive animals was only slightly lower (11 of 16 animals) than at 2 weeks after the first boost (12 of 16 animals). Boosting with ALVAC_Bx08 was no more effective than boosting with DNA_Bx08 combined with VLP_Bx08. By comparison, a typical response to corresponding SIV Gag peptide pools using cells from SIV-infected rhesus macaques is 50 to 150 ELISpots, with most animals responding to both peptide pools (data not shown).