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Journal of Virology, May 2004, p. 5270-5278, Vol. 78, No. 10
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.10.5270-5278.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Immunogenicity of Constrained Monoclonal Antibody A32-Human Immunodeficiency Virus (HIV) Env gp120 Complexes Compared to That of Recombinant HIV Type 1 gp120 Envelope Glycoproteins

Hua-Xin Liao,^1* S. Munir Alam,¹ John R. Mascola,² James Robinson,³ Benjiang Ma,¹ David C. Montefiori,⁴ Maria Rhein,¹ Laura L. Sutherland,¹ Richard Scearce,¹ and Barton F. Haynes¹

Duke Human Vaccine Institute and Department of Medicine,¹ Department of Surgery, Duke University School of Medicine, Durham, North Carolina 27710,⁴ Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892,² Department of Pediatrics, Tulane University School of Medicine, New Orleans, Louisiana 70112³

Received 29 September 2003/ Accepted 16 January 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
One strategy for the generation of broadly reactive neutralizing antibodies (NA) against human immunodeficiency virus type 1 (HIV-1) primary isolates is to use immunogens that have constrained HIV-1 envelope gp120 conformations reflective of triggered envelope on the surface of virions. A major change in gp120 following binding to CD4 is the enhanced exposure of the CCR5 binding site. One inducer of CCR5 binding site epitopes on gp120 is the human anti-gp120 monoclonal antibody, A32. We have made cross-linked A32-rgp120_89.6 and A32-rgp120_BaL complexes and have compared their immunogenicities to those of uncomplexed recombinant gp120_BaL (rgp120_BaL) and rgp120_89.6. A32-rgp120_89.6 and A32-rgp120_BaL complexes had stable induced CCR5 binding site expression compared to that of uncomplexed rgp120s. However, the A32-rgp120 complexes had similar capacities in guinea pigs for induction of NA against HIV-1 primary isolates versus that of rgp120 alone. A32-rgp120_89.6 induced antibodies that neutralized 6 out of 11 HIV-1 isolates, while rgp120_89.6 alone induced antibodies that neutralized 4 out of 11 HIV-1 isolates. A32-rgp120_BaL complexes induced antibodies that neutralized 4 out of 14 HIV-1 isolates while, surprisingly, non-cross-linked rgp120_BaL induced antibodies that neutralized 9 out of 14 (64%) HIV-1 isolates. Thus, stable enhanced expression of the coreceptor binding site on constrained gp120 is not sufficient for inducing broadly neutralizing anti-HIV-1 NA. Moreover, the ability of HIV-1 rgp120_BaL to induce antibodies that neutralized 60% of subtype B HIV-1 isolates warrants consideration of using HIV-1 BaL as a starting point for immunogen design for subtype B HIV-1 experimental immunogens.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The design of immunogens that will neutralize a broad spectrum of human immunodeficiency virus type 1 (HIV-1) primary isolates is a high priority for development of a practical HIV-1 vaccine. Following binding of virion gp120 to cellular CD4, the HIV-1 envelope undergoes conformational changes that result in exposure of the coreceptor binding site leading to virion-host cell fusion (1, 24).

One potential strategy for inducing broadly reactive neutralizing antibodies (NA) is to construct immunogens that are constrained and reflect wild-type fusion intermediate Env forms, with the hope of stably exposing conserved immunogenic epitopes that otherwise would not be readily available for antibody induction. An alternative strategy for selection of Env immunogens is to select the best envelopes from among many screened for their ability to induce antibodies that broadly neutralize HIV-1 primary isolates.

In this work we describe the immunogenicity of recombinant HIV-1 gp120s complexed with the CD4 mimic, monoclonal antibody (MAb) A32. Like CD4, MAb A32 induces expression of the CCR5 binding site on rgp120, but unlike CD4, MAb A32 does not bind at the CD4 binding site (26). Thus, MAb A32 has a potential advantage over CD4 in a constrained Env complex in that A32-rgp120 complexes have exposed CD4 binding sites. Here we show that both A32-rgp120_89.6 and A32-rgp120_BaL complexes are immunogenic and induce NA against HIV-1 primary isolates. However, stable expression of the CCR5 binding site on gp120 was not sufficient for induction of broad NA, as A32-rgp120 complexes did not show marked enhanced immunogenicity for NA induction over uncomplexed rgp120s. Surprisingly, we found that monomeric recombinant gp120_BaL (rgp120_BaL) was the best immunogen tested and induced NA to 64% (9 out of 14) of HIV-1 isolates tested.

Top Abstract Introduction Materials and Methods Results Discussion References

 
HIV-1 gp120 proteins. Recombinant vaccinia viruses (rVVs) that express HIV-1 (subtype B) 89.6 gp120 (VBD-2) and HIV-1IIIB (VPE-50) were obtained from Pat Earl and Bernard Moss (National Institutes of Health [NIH], Bethesda, Md.) (19). rVV that expresses group M consensus (CON6) rgp120 (11) was generated as described previously (19). Briefly, a DNA fragment encoding CON6 gp120 was produced by introducing stop codons after the gp120 cleavage site (REKR) by PCR and was cloned into a transfer vector, pSC65 vector (from Bernard Moss) at SalI and KpnI restriction enzyme sites (3). BSC-1 cells were seeded at 2 x 10⁵ in each well in a 6-well plate and were infected with wild-type vaccinia virus (WR) at a multiplicity of infection of 0.1 PFU/cell, and 2 h after infection pSC65-derived plasmids containing CON6 env genes were transfected into the VV-infected cells by using Lipofectamine 2000 based on the protocol recommended by the manufacturer (Invitrogen, Carlsbad, Calif.). rVV that expresses the CON6 env gene was selected and confirmed by PCR and sequencing analysis as described previously (19). HIV-1 rgp120_89.6, HIV-1 rgp120_IIIB, and CON6 rgp120 were expressed in 293T cells infected with VBD-2, VPE-50, and CON6 gp120 rVV, respectively. Serum-free tissue culture supernatants of 293T cells were harvested 3 days after infection with rVVs as a source for purification of rgp120 proteins. rgp120 proteins in the supernatants were all purified by agarose Galanthus nivalis lectin chromatography (Vector Labs, Burlingame, Calif.) and were stored at –70°C until use. Purified rgp120 proteins were quality controlled with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining and Western blot. rgp120 proteins of HIV-1 isolates of BaL (subtype B), 96ZM651 (subtype C), and JRFL (subtype B) were obtained from QBI Inc., Bethesda, Md., through the NIH AIDS Reagent Repository Program.

Cross-linking of MAb A32 and rgp120. HIV-1 rgp20_89.6 or rgp120_BaL was chemically cross-linked to MAb A32 by using the water soluble N-hydroxysuccinimide-ester dithiobis-sulfosuccinimidylporpionate (DTSSP) (Pierce, Rockford, Ill.) based on the protocol recommended by the manufacturer. Briefly, MAb A32 was mixed with a final concentration of 1 mg of rgp120/ml and was incubated at 4°C for 2 h in a 2.25-fold molar excess over MAb A32 to ensure saturation of the bivalent MAb and to have only excess rgp120 following cross-linking. DTSSP was added to the mixture at either 100-fold molar excess for rgp120_89.6 or 200-fold molar excess for rgp120_BaL at 0°C for 2 h. The reaction was stopped by adding 1 M Tris at a final concentration of 50 mM and was further incubated at 20°C for 30 min.

Cross-linked A32-rgp120 complexes were separated from uncomplexed gp120 proteins by size exclusion chromatography over a HR200 Superdex column and by using an AKTA fast protein liquid chromatography (FPLC) system (Uppsala, Sweden). Fractions were analyzed on 4 to 20% Tris-Glycine gels (Invitrogen) under reducing and nonreducing conditions by Coomassie staining, Western blot analysis, and Biacore binding assay. Western blots were developed with a saturating concentration of HIV-1 envelope protein-specific MAb T8 that reacts with most subtype B gp120s at the C1 region (gift of P. Earl) to detect rgp120, and anti-human immunoglobulin (Ig) (1:4,000; Sigma, St. Louis, Mo.) to detect MAb A32. Surface plasmon resonance binding assays were performed on a Biacore 3000 (Uppsala, Sweden). Monoclonal antibodies (17b, T8 A32) were immobilized on a CM5 sensor chip by using standard amine coupling chemistry.

Immunization regimens and adjuvants. Outbred Hartley guinea pigs were immunized with either 100 µg of rgp120 or 200 µg of rgp120-A32 complexes in either CFA/IFA (Sigma), Ribi-CWS Adjuvant (Corixa-Sigma, St. Louis, Mo.), or RC529-SE+ mutant cholera toxin (CT) subcutaneously (the gifts of John Eldridge; Wyeth, Pearl River, N.Y.) every 3 weeks for five immunizations. Serum samples were collected 10 days after each immunization and were stored at –30°C until use in enzyme-linked immunosorbent assay (ELISA) and neutralization assays.

Neutralization assays. HIV-1 isolates (ADA, JRCSF, JRFL, BaL, SF162, and 89.6) were obtained from the NIH AIDS Research and Reference Reagent Program (Division of AIDS, National Institute of Allergy and Infectious Diseases). The viruses were expanded by two or three cycles of growth on phytohemagglutinin and interleukin-2-stimulated peripheral blood mononuclear cell (PBMC). Viruses BL01 and BR07 were provided by Dana Gabuzda of the Dana-Farber Cancer Institute. Both are chimeric infectious molecular clones of NL4-3 that contain the full-length env genes from primary HIV-1 isolates (20). After initial plasmid transfection of 293 cells, these viruses were expanded in PBMC as described above.

Serum NA levels were measured either by flow cytometic enumeration of p24 antigen-positive cells following a single round of infection (16) or by using a syncytium inhibition assay with aldrithiol-2 (AT-2)-inactivated virions (21) or a luciferase-based multiple-round HIV-1 infection assay (2). The single-round flow cytometric assay for intracellular p24 antigen was performed as described previously (16). In the luciferase-based assay, NA were measured as a function of a reduction in luciferase activity in 5.25.EGFP.Luc.M7 cells kindly provided by Nathaniel R. Landau (Salk Institute, La Jolla, Calif.) (2). Five hundred 50% tissue culture infectious doses (TCID₅₀) of cell-free virus were incubated with the indicated serum dilutions in a volume 150 µl (1 h at 37°C) in triplicate in 96-well flat-bottom culture plates. The 5.25.EGFP.Luc.M7 cells were suspended at a density of 5 x 10⁵/ml in media containing diethylaminoethyl dextran (10 µg/ml). Cells (100 µl) were added until 10% of cells in control wells (no test serum sample) were positive for green fluorescent protein expression by fluorescence microscopy. At this time the cells were concentrated twofold by removing one-half of the volume of medium. A 50-µl suspension of cells was transferred to 96-well white solid plates (Costar, Cambridge, Mass.) for measurement of luciferase activity by using Bright-Glo substrate (Promega, Madison, Wis.) on a Wallac 1420 Multilabel Counter (PerkinElmer Life Sciences, Boston, Mass.). Neutralization titers in the luciferase assays were those where >50% virus infection was inhibited and were considered positive if the postimmune bleed titer minus the preimmune bleed titer was >30 and the postbleed titer was three times higher than the prebleed titer. For analysis of breadth, those isolates that were neutralized by at least 2 sera per group were considered positive.

The syncytium inhibition fusion-from-without assay utilized HIV-1 AT-2-inactivated virions from HIV-1 subtype B strains ADA and AD8 (the gift of Larry Arthur and Jeffrey Lifson, Frederick Research Cancer Facility, Md.). Twofold serially diluted immune and control sera starting at 1:10 dilution were incubated with optimal dilution (1:80, which results in approximately 80 syncytia in the assay) of AT-2-inactivated HIV-ADA or ADA (from Larry Arthur) for 1 h at 37°C. After 1 h of incubation the mixtures of serum and virus were incubated with Sup T1 cells (100,000 cells per well of 96-well half-area microtiter plates) overnight in a 37°C, 5% CO₂ incubator. The presence of characteristic syncytia was evaluated by inverted phase-contrast microscopy 16 h after virus addition. Numbers of syncytia were counted per view at 100x magnification under a phase-contrast light microscope. Serum titer was determined as a serum sample at a given dilution giving 90% inhibition of syncytium formation compared to that of prebleed sera.

Anti-gp120 serum antibody assay. Binding antibody titers against a panel of HIV-1 rgp120 proteins were measured in a standard ELISA. HIV-1 rgp120s from various HIV-1 isolates, including subtype B isolates of 89.6, JRFL, IIIB, BaL, subtype C isolates of 96ZM651, and CON6. HIV-1 rgp120 proteins were all used at a concentration of 2 µg/ml (200 ng/well) to coat 96-well ELISA microtiter plates. Antibody endpoint binding titers were determined as the reciprocal of the highest dilution of the serum assayed against corresponding recombinant HIV-1 envelope proteins, giving optical density readings of experiment versus control of 3.0.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Enhanced binding of MAb 17b to A32-rgp120 complexes. MAb A32 broadly reacts with a wide spectrum of HIV-1 primary isolates from different subtypes (26). By using surface plasmon resonance assays we found MAb A32 reacted with both rgp20_89.6 and rgp120_BaL (Fig. 1A and B). HIV-1 MAb 17b constitutively bound to rgp20_89.6 but was nonreactive with rgp120_BaL (Fig. 1C and D). Incubation of MAb A32 whole IgG as well as Fab fragments with HIV-1 rgp120 proteins induced markedly enhanced MAb17b binding to rgp20_89.6 (Fig. 1C) and induced de novo MAb 17b binding to rgp120_BaL (Fig. 1D).

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FIG. 1. Reactivity of HIV-1 MAb A32 with HIV-1 rgp12089.6 and rgp120BaL proteins in Biacore assays. MAb A32 was covalently immobilized to a CM5 sensor chip (Biacore), and rgp12089.6 (A) and rgp120BaL (B) proteins were injected over each surface. Overlay of curves of Biacore analysis shows that anti-HIV MAb A32 (solid line) reacted with both rgp12089.6 (A) and rgp120BaL (B) proteins. Curves in dotted lines represent background binding to an irrelevant IgG. To determine induction of 17b MAb binding to rgp12089.6 (C) and rgp120BaL (D), HIV-1 rgp12089.6 (C) and HIV-1 rgp120BaL (D) proteins were flowed over 17b MAb, which was immobilized on a Biacore chip and was tested for the ability to bind HIV-1 MAb 17b in the presence or absence of MAb A32 whole IgG or Fab fragment. The x axis shows the duration of time (in seconds), and the y axis shows the response unit of MAb 17b binding in the Biacore assay. HIV-1 rgp12089.6 constitutively bound MAb 17b (C), indicating the availability of the CCR5 binding site, while rgp120BaL did not bind to MAb 17b (D). Both whole IgG and Fab fragment of MAb A32 markedly upregulated (for rgp12089.6) (C) or induced (for rgp120BaL) (D) MAb 17b binding.

Production of A32-rgp120 complexes with the CCR5 binding site stably exposed. The presence of stably exposed or induced CCR5 binding sites on constrained HIV-1 envelope proteins indicates induction of conformational changes in gp120 following CD4 or MAb A32 binding (1, 13, 23, 26). While the CCR5 binding site itself might not be a target for broadly reactive NA (8), in this study the conformational exposure of the 17b binding site was monitored as an indicator of A32-induced conformational changes in rgp120. Our postulate was that A32-triggered rgp120 might possess conserved conformational epitopes against which broadly reactive NA could be made. Thus, we produced A32-HIV-1 rgp120 complexes by cross-linking MAb A32 and either gp120_89.6 or rgp120_BaL with DTSSP. To purify A32-rgp120 complexes and to remove uncross-linked rgp120, A32-gp120_89.6 and A32-rgp120_BaL stable complexes were purified on Superdex HR200 gel filtration columns. Figure 2 shows that the higher molecular sizes of A32-rgp20_89.6 (Fig. 2A) and A32-rgp120_BaL (Fig. 2B) complexes were eluted first and could be separated from uncross-linked rgp120. We next examined each fraction off the Superdex HR200 FPLC column for constitutive binding activity to MAb 17b immobilized on a Biacore chip. We found that all upregulated binding to 17b was indeed in the A32-rgp120 complex in peak 1 (Fig. 2A) fractions containing A32-gp120_89.6 complexes. Similarly, analysis of the A32-rgp120_BaL complexes showed stable induced MAb 17b binding (Fig. 2B).

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FIG. 2. FPLC elution pattern of the cross-linked A32-gp120 complexes on a Superdex H200 column. MAb A32-rgp12089.6 (A) and A32-rgp120BaL (B) complexes were made as described in Materials and Methods and fractionated on a Superdex HR200 column. Fractions were collected and assayed for the ability to bind MAb 17b, immobilized on a Biacore CM5 sensor chip. Circles represent binding levels of each fraction to immobilized 17b MAb, while the UV trace from the FPLC run is shown with a solid line. Also shown is that the enhanced level of MAb 17b binding was obtained in fractions under peak 1 (molecular size of about 440 kDa) that contained the cross-linked rgp12089.6 (A) and rgp120BaL (B). In contrast, peak 2 containing excess gp120 did not show upregulated MAb 17b binding.

Figure 3 shows SDS-PAGE gels of unpurified A32, rgp20_89.6, and A32-rgp120_89.6 complexes both before and after separation on a Superdex HR200 FPLC column. MAb A32 sized at 210,000 Da while rgp120_89.6 was 120,000 Da. Unpurified A32-rgp120_89.6 complexes contained higher-molecular-size complexes as well as excess uncomplexed rgp120. Peak 2 in the chromatogram below contained free rgp120 (fraction lane 23, 24, and 25), while A32-rgp120_89.6 complexes eluted in peak 1 (fractions 18 to 21). The weak lower-molecular-size band in fraction 21 reflected complexes comprised of one rgp120 and one A32 molecule, while the higher-molecular-size band in fractions 18 to 20 reflected saturated A32 MAb with two bound rgp120s. Under reducing conditions (the middle panel) the complexes in fractions 18 to 21 all contained rgp120 as well as 55,000 Da of Ig heavy chains and 29,000 Da of Ig light chains. In Fig. 3B, Western blot with an anti-human Ig (middle panel) and anti-gp120 mouse MAb T8 (lower panel) confirmed that the complexes in peak 1 contained both rgp120 and MAb A32. Thus, both A32-rgp120_89.6 and rgp120_BaL complexes had stable expression of the CCR5 binding site, and these complexes (lanes 18 to 21 in Fig. 3) were used in a series of immunizations in guinea pigs.

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FIG. 3. SDS-PAGE and Western blot analysis of unfractionated and FPLC-fractionated A32-rgp120 complexes. MAb A32-rgp12089.6 complexes were made as described in Materials and Methods, fractionated on a Superdex HR200 FPLC column (bottom panel), analyzed, and compared to unfractionated rgp120, A32, and A32-rgp120 by Coomassie staining (A) and Western blot (B) of the reducing (middle frame of panel A) and nonreducing (top frame of panel A) SDS-PAGE. In Western blotting run under nonreducing conditions, HIV-1 envelope-specific MAb T8 was used to detect rgp12089.6 in the A32-rgp12089.6 complexes (top frame of panel B), and anti-human Ig was used to detect human MAb A32 (middle frame of panel B). Samples in the individual lanes are corresponding the fractions off the Superdex H200 FPLC column (bottom frames of panels A and B). The analysis demonstrates that complexes made with one gp120 or two gp120s bound to MAb A32 indeed contained both MAb A32 and rgp12089.6.

Ability of antisera against A32-rgp120 complexes to react with a panel of HIV-1 rgp120s. To determine if cross-linking 89.6 or BaL rgp120s prevented induction of anti-gp120 antibodies, we screened anti-A32-rgp120 complex antisera for reactivity with a panel of four subtype B rgp120s (89.6, JRFL, IIIB, and BaL), a group M consensus Env rgp120 (CON6), and a subtype C rgp120 (96ZM651). We found that both 89.6 and BaL rgp120 complexed with A32-induced anti-gp120 antibodies equally well, with both types of antisera reacting with each rgp120 (Fig. 4).

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FIG. 4. Reactivity of guinea pig anti-HIV-1 sera to a panel of HIV-1 rgp120. To determine reactivity of guinea pig postimmune sera to HIV-1 gp120 envelope proteins in ELISA, a panels of HIV-1 rgp120 proteins (200 ng/well) as indicated in the X axis were coated in 96-well microtiter plates and tested for the reactivity of sera from guinea pigs immunized with either A32-rgp12089.6 or rgp120BaL. The endpoint ELISA titers of guinea pig sera are determined as described in Material and Methods and are shown in the Y axis.

NA induced by A32-rgp120 complexes compared to that of rgp120 alone. Next we compared the ability of A32-rgp120_89.6 (Table 1) and A32-rgp120_BaL (Table 2) complexes to rgp120_89.6 (Table 1) and rgp120_BaL (Table 2) alone for their ability to induce anti-HIV-1 neutralizing antibodies. While A32-rgp120 complexes induced antibodies that neutralized multiple HIV primary isolates (A32-rgp120_89.6, 6 out of 11 isolates neutralized; A32-rgp120_BaL, 4 out of 11 isolates neutralized), there were no profound differences between the number of HIV isolates neutralized by antisera induced by rgp120 alone and that induced by A32-rgp120 complexes. While A32-rgp120_89.6 complexes induced antibodies that neutralized two additional HIV-1 isolates more than A32-rgp120_89.6 alone (IIIB, JRCSF), the A32-rgp120 complexes did not induce antibodies that broadly neutralized five of the more difficult-to-neutralize HIV-strains (US-1, US717, BL01, 6101, and BG1168).

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TABLE 1. Ability of MAb A32 complexed with HIV 89.6 gp120 versus 89.6 gp120 alone to induce antibodies that neutralize HIV primary isolates

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TABLE 2. Ability of MAb A32 complexed with HIV BaL gp120 versus BaL gp120 alone to induce antibodies that neutralize HIV primary isolates

To determine if A32-rgp120 complexes induced antibodies that inhibited HIV-1-induced fusion, we tested the sera listed in Tables 1 and 2 for the ability to inhibit inactivated HIV-1 induced syncytium from without by using AT-2-inactivated virions ADA and AD8 (Table 3). We found most postimmune sera had syncytium inhibiting activity, with the anti-rgp120 sera titers as high or higher than those for the anti-A32-rgp120 complex antisera.

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TABLE 3. Comparison of A32-gp120 complexes and rgp120 to induce antibodies that inhibit syncytia formation by AT-2-inactivated virions

Because the rgp120s performed as well as the A32-rgp120 complexes, we next determined the ability of antisera against 89.6 and BaL rgp120s to neutralize seven subtype B HIV-1 isolates in a luciferase-based multiple round infection NA assay (Table 4). Four isolates were the same as those tested in the single-round p24 assay (BaL, BG1168, 6101, SF162), and three were different (BX08, SS1196 and QH0692). While rgp120_89.6 antisera only strongly neutralized BX08 (2 out of 3 were serum positive), rgp120_BaL antisera strongly neutralized SF162, BX08, SS1196, and QH0692 in addition to BaL and SF162.

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TABLE 5. Ability of MAb A32 complexed with rgp12089.6 versus rgp12089.6 alone formulated in aqueous adjuvants to induce antibodies that neutralize HIV-1 primary isolates

Taken together with the data in Tables 1 and 2, rgp120_89.6 neutralized 4 out of 14 HIV-1 isolates (29%) while rgp120_BaL antisera neutralized 9 out of 14 HIV-1 isolates (64%).

Thus, for both the single-round neutralization assays and the inhibition of virion-induced fusion assays, rgp120 was equal (89.6) or superior (BaL) to A32-rgp120 complex induction of anti-HIV-1 NA.

Evaluation of the effect of adjuvants and DTSSP cross-linking on A32-rgp120 immunogenicity. We next performed two additional sets of immunizations to evaluate conditions that might affect the ability of A32-rgp120 complexes to induce broadly reactive NA. First, because of a concern that the oil adjuvants described in Tables 1 and 2 might interfere with the ability of constrained rgp120 to express important conformational neutralizing epitopes, we formulated A32-rgp120_89.6 complexes in 10 µg of CT as an adjuvant in saline and immunized SQ X3, followed by 50 µg of lipopolysaccharide plus 10 µg of CT in saline for an additional two boosts. This regimen achieved an endpoint titer of 1:204,800 against rgp120_89.6 in both animals tested, an ELISA titer that is sufficient for demonstrating the presence of NA. Table 5 shows that 1 of 2 animals immunized with A32-rgp20_89.6 complexes in saline plus CT plus LPS (no. 572) neutralized 5 of 11 HIV-1 isolates while the other serum (no. 570) neutralized none. The sera from animals immunized with rgp120_89.6 alone neutralized from 2 to 4 HIV-1 isolates (Table 4). As for Table 1, there was a suggestion of more breadth with A32-rgp120_89.6 complexes with more neutralization in serum 572 of HIV-1 IIIB and BaL than with rgp120_89.6 immune sera. However, the A32-rgp120 complex sera with saline adjuvants again did not neutralize HIV-1 isolates B61168, 6101, BL01, US17, or US717.

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TABLE 4. Comparison of HIV-1 gp120 BaL and gp120 89.6 to induce antibodies that neutralize HIV-1 primary isolates

The second set of control immunizations performed was with rgp120_BaL treated with DTSSP in the absence of A32 MAb. In contrast to when A32 was used in the complex, we found that postimmune sera from three animals (H12, H13, H14) immunized four times with DTSSP-treated rgp120_BaL neutralized 0 of 11 of the HIV-1 isolates listed in Table 4 (data not shown). Taken together, these controls demonstrated that (i) the oil adjuvants described in Tables 1 and 2 did not prevent expression or recognition of conserved epitopes exposed on the surface of A32-rgp120 and epitopes, and (ii) DTSSP treatment of rgp120_BaL in the absence of MAb A32 eliminated the immunogenicity of rgp120 neutralizing determinants. Thus, the immunogenicity of rgp120 in A32 complexes was not due to DTSSP treatment per se.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study we have produced A32-rgp120 complexes with stably expressed coreceptor binding sites and have compared these complexes as immunogens with uncomplexed rgp120. Our study is important in that we determined (i) that stable enhanced expression of the coreceptor binding site is not sufficient for induction of broadly reactive NA in the absence of CD4, (ii) oil adjuvant formulation did not prevent A32-rgp120 broadly neutralizing antigen expression, and (iii) rgp120_BaL alone was the best Env immunogen tested.

Fouts et al. have reported that cross-linked CD4-rgp120_IIIB and CD4-rgp140_IIIB complexes, unlike uncross-linked rgp120 or rgp140, induced antibodies that neutralized a spectrum of HIV-1 primary isolates from multiple HIV-1 subtypes (9). Soluble CD4 in cross-linked CD4-rgp120 complexes could be important as a component for the ability to induce cross-reactive NA in multiple ways. Human CD4 induces anti-CD4 antibodies that themselves neutralize HIV-1. Indeed, absorption of anti-CD4 antibodies removed some but not all of the cross-subtype neutralizing activity induced by CD4-rgp120 complexes (5, 9). Second, CD4, when complexed to rgp120, can be part of a new neutralizing determinant in the complex and/or induce new neutralizing determinants on rgp120 following induction of conformational changes in rgp120 by CD4 (5, 6, 9).

We chose to study MAb A32-rgp120 complexes for several reasons: (i) MAb A32 reacts with many HIV-1 isolates of multiple subtypes, (ii) MAb A32 is as potent an inducer of the CD4i coreceptor binding site as is sCD4, and (iii) MAb A32 does not bind to the CD4BS, thus leaving it open for induction of anti-CD4BS antibodies (26).

While A32-rgp120 complexes with rgp120_89.6 induced slightly more neutralizing breadth than rgp120_89.6 alone, comparative studies with A32-rgp120_BaL showed that A32-rgp120_BaL complexes were less immunogenic for broadly reactive NA than uncomplexed rgp120_BaL (Tables 2 and 5). Thus, our carefully characterized A32-rgp120 complexes provide the first data demonstrating that exposure of the coreceptor binding site alone is not sufficient for induction of broadly reactive NA. Immunofluorescence studies on live HIV-infected cells have shown limited accessibility of the coreceptor binding site during fusion (8). In contrast, the fusion protein CD4-17b Fab is a potent bivalent inhibitor of most HIV-1 primary isolates (7). Clearly envelope constructs more native than A32-rgp120 monomers, such as A32-bound gp140 or gp160 trimers, need to be tested as immunogens.

DeVico et al. have shown that expression of CD4-induced neutralizing determinants defined by MAbs raised against CD4-rgp120 complexes vary in their expression on native gp120s, being constitutively expressed on some gp120s and less so on others (5). In this regard, rgp120_89.6 had constitutive low-level expression of MAb 17b (7) binding that increased after MAb A32 binding while rgp120_BaL had no constitutive MAb 17b binding. However, rgp120_BaL induced antibodies that neutralized 64% (9 of 14) of subtype B isolates tested while rgp120_89.6 was less effective, which could be due to constitutive expression of as-yet undefined conserved neutralizing determinants on rgp120_BaL. These latter data give credence to the strategy of testing multiple HIV-1 primary isolate Envs to select a best Env or best polyvalent Envs for induction of broadly reactive NA.

Generally, most analyses of immune sera from animals and humans have shown little neutralizing activity for HIV-1 primary isolates (13, 14, 17, 18, 22, 24, 25). However, recent analysis of immune sera raised against either polyvalent gp120 (4), gp120 subunit peptides (15), or oligomeric gp160 (25) have demonstrated the ability to induce antibodies that neutralized select HIV-1 primary isolates. Clearly, the majority of HIV-1 primary isolates neutralized by anti-gp120 immune sera in our study are more easily neutralizable than others, and the breadth of neutralization elicited by the best immunogen, rgp120_BaL, is not sufficient to anticipate widespread utility as a vaccine. It is important to note that AT-2-inactivated HIV-1 virions (21) have been grown in vitro in T-cell lines and are readily neutralized in syncytium from without inhibition assays. Thus, the syncytium inhibition assay used in our study is useful to demonstrate the presence of inhibiting activities, but the assay appears to be more sensitive than the luciferase-base multiple-round infection assay, the single-round infection cytoplasmic p24 staining assay, and the tissue culture supernatant p24 production in PBMC assay.

Fouts et al. also compared CD4-rgp120_IIIB and CD4-rgp140_IIIB complexes and found no difference in immunogenicity (9). However, we have recently shown in a direct comparison of rgp120_ADA and purified trimeric rgp120_ADA as immunogens, that trimeric rgp140_ADA was superior to monomeric rgp120 for NA induction (M. Kim, H.-X. Liao, D. Montefiori, E. Reinherz, and B. Haynes, unpublished observations). We have begun to express and purify rgp140_BaL trimers for comparison in immunogenicity studies with rgp120_BaL as well to determine if A32-rgp140 trimer complexes have an enhanced capacity for inducing NA over uncomplexed rgp140 trimers.

A critical issue for us in this study was to determine the role that adjuvants might play in the ability of conformational epitopes on constrained envelope complexes to be immunogenic. While not an exhaustive study, we found no significant difference in the breadth of NA induced with oil (mineral oil, squalene) versus aqueous (CT and LPS) adjuvants. It was important to rule out that the adjuvant formulation was not preventing immunogenicity. Our data suggest that induction of broadly reactive NA was not prevented because of adjuvant interference with immunogenicity. However, in this regard, Van Cott et al. have shown that mineral oil adjuvants such as CFA and IFA can induce decreased recognition of conformational epitopes to some degree on rgp140 oligomers (25). This issue will remain a concern in future studies of other immunogens, such as Env trimers.

Finally, it is critical to note that each incremental improvement in induction of breadth of NA by experimental immunogens is important. The rgp120 human Phase III trial was not successful, suggesting that rgp120s may not be viable vaccine candidates in and of themselves (12). However, that rgp120_BaL alone can induce antibodies that neutralize 60% of subtype B HIV-1 isolates provides a beach head from which to work to improve the immunogenicity. Clearly, most HIV-1 isolates neutralized by anti-rgp120_BaL sera were the isolates known to be easily neutralized (e.g., IIIB, BaL, SF162, 89.6), while the more difficult to neutralize HIV isolates (e.g., BG11681, 6101, US1) were not neutralized. Thus, rgp120_BaL in and of itself is not likely to solve the HIV-1 vaccine NA immunogen problem, but as a present best Env it can serve as a starting point for design of more native Env immunogens. These strategies include formulation of trimeric rgp140_BaL immunogens and production of constrained gp140 trimer complexes. In this regard, Fouts et al. have described the production of a single-chain polypeptide analogue of the CD4-rgp120 complex utilizing the gene for rgp120_BaL (10).

Taken together, our data suggest that HIV-1 BaL rgp120 is a promising R5 envelope that can serve as a starting point for development of an HIV-1 immunogen for induction of broadly reactive NA against subtype B HIV-1 primary isolates.

 
We acknowledge Kim McClammy for expert secretarial assistance.

We acknowledge the Proteomics Core of Human Immunology Center grant AI-51445, PO-1 AI-52816. This work supported by AI-15351, HIV Team Contract grant no. N01-AI05397, AI-24030, and the NIH, NIAID, AIDS Research and Reference Reagent Program.

 
* Corresponding author. Mailing address: Box 3258 Human Vaccine Institute, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-5858. Fax: (919) 681-8992. E-mail: liao0001{at}mc.duke.edu.

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Journal of Virology, May 2004, p. 5270-5278, Vol. 78, No. 10
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.10.5270-5278.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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