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AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 24, Number 2, 2008 © Mary Ann Liebert, Inc. DOI: 10.1089/aid.2007.0158

Focused Dampening of Antibody Response to the Immunodominant Variable Loops by Engineered Soluble gp140 SUGANYA SELVARAJAH,1 BRIDGET A. PUFFER,2,* FANG-HUA LEE,2 PING ZHU,3 YUXING LI,4 RICHARD WYATT,4 KENNETH H. ROUX,3 ROBERT W. DOMS,2 and DENNIS R. BURTON1

ABSTRACT Immunization studies with modified gp120 monomers using a hyperglycosylation strategy, in which undesired epitopes are masked by the selective incorporation of N-linked glycans, were described in a previous paper (Selvarajah S, et al., J Virol 2000;79:12148–12163). In this report, we applied the hyperglycosylation strategy to soluble uncleaved gp140 trimers to improve the antigenic and immunogenic profile in the context of a trimeric conformation of the immunogen. The JR-FL gp140 gene was added upstream of a soluble trimerization domain of chicken cartilage matrix (CART) protein and expressed predominantly as a trimer and called gp140-CART wild-type. In the hyperglycosylated gp140-CART mCHO(V) mutant, four extra sugar attachment motifs on the variable loops helped mask epitope recognition by monoclonal antibodies specific to the variable loops. The gp140-CART mCHO(V) mutant and gp140-CART wild-type soluble trimer protein were used to immunize rabbits. The gp140-CART mCHO(V) immune sera had reduced antibody response to the variable loops compared to gp140-CART wild-type immune sera as shown by peptide reactivity, competition assays, and the reduced ability of sera to neutralize SF162 virus (a variable loop neutralization-sensitive virus). The antibody response to the CD4 binding site was retained in the gp140-CART mCHO(V) mutant immune sera similar to gp140-CART wild-type immune sera. The results demonstrate that the strategy of hyperglycosylation is clearly useful in the context of a compact form of Env immunogen such as the soluble gp140 trimer in dampening responses to variable loops while maintaining responses to an important epitope, the CD4 binding site. However, the results also show that in order to elicit broadly neutralizing antibodies that target conserved epitopes, the soluble gp140 trimer immunogen template will require further modifications. INTRODUCTION

O

NE APPROACH TO HIV-1 IMMUNOGEN DESIGN focuses on the generation of the recombinant trimeric envelope (Env).1–13 The essential concept is that immunizing with a close mimic of the functional trimer will improve the chances of eliciting neutralizing antibodies since trimers are the target of such antibodies.14–16 Soluble gp140 trimers are typically used as the prototype immunogen.1–13 Soluble gp140 trimers containing the ectodomain of gp41 covalently linked to gp120 have been generated by fusing GCN4

trimerization domains or T4 bacteriophage fibritin trimeric (FT) motifs to the C-terminus of soluble, uncleaved gp140 glycoproteins.13,17 Immunization with YU2 gp140-GCN4/FT trimers was shown to be more efficient at eliciting neutralizing antibodies than YU2 gp120 monomers.7,18–20 In addition, the elicitation of neutralizing antibodies by YU2 gp140-GCN4 was enhanced when the immunization regime included selected adjuvants.19 In a slightly different approach, Semliki forest virus and virus-like particles have been generated containing uncleaved YU2 gp140-GCN4 glycoproteins or full-length Env, respectively.10,21,22

1Departments

of Immunology and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037. of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104. 3Department of Biological Science and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306. 4Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892. *Present address: Integral Molecular, Philadelphia, Pennsylvania 19104. 2Department

301

302 Soluble uncleaved gp140 trimers with variable loop deletions have also been used extensively in immunization studies.1,5,12,23–25 Soluble gp140 trimers with a deletion in the crown of the V2 loop have been used to immunize macaques and have been shown to elicit homologous neutralizing antibodies.5,23,25 However, the antibodies mainly target the V1 variable loop.5 Some heterologous neutralizing antibodies were also elicited in that study; however, they were weak in potency, narrow in specificity, and failed to prevent heterologous virus infection in immunized macaques.5,25 In a study aimed at circumventing the problems with uncleaved gp140, cysteine residues have been incorporated into gp120 and gp41.3 This prevents dissociation of the two subunits through the formation of an inter-subunit disulfide bridge upon expression of cleaved gp140. However, a mutation (I559P) that destabilizes the six-helix bundle formation was needed for the expression of this molecule in a stable trimeric form (SOSIP).26 A recent study showed that the SOSIP-cleaved trimers had a slightly improved ability at generating neutralizing antibodies compared to uncleaved g140 trimers (without trimerization domains for trimer stability).27 Soluble gp140 trimer antigens have been designed based on envelope sequences from different clades and strains of HIV1.8,9,28,29 Most engineered soluble trimers, probed by monoclonal antibody (mAb) reactivity, have antigenic profiles similar but not identical to the native envelope trimer expressed on the surface of infected cells and presumably also virions. It is therefore thought that further modifications are required for the soluble trimer to better resemble the native envelope spike antigenically. Such modifications are under intensive study.30–34 Recent insights into structure from cryo-electron microscope tomography studies of the native Env trimer on the viral surface may also help in future design work.35–37 However, even if recombinant trimers with conformations very close to those of functional trimers become available, they will still suffer from limitations as immunogens arising from the immunodominance of variable loops. Thus they are likely to primarily elicit strain-specific neutralizing antibodies directed to these loops whereas a vaccine should seek to elicit broadly neutralizing antibodies directed to conserved regions. We have recently published results from immunization studies with modified gp120 monomers using a hyperglycosylation strategy in which undesired epitopes are masked by the selective incorporation of N-linked glycans.34 The mCHO gp120 mutant was designed to incorporate seven extra glycans (four on the variable loops and three on the gp120 core) relative to the wild-type gp120 protein.38 Serum mapping studies on mCHO gp120 immune sera showed that the mutant gp120 failed to elicit a range of antibodies against both the gp120 core as well as the immunodominant variable loops.34 These preliminary results demonstrated that dampening of responses to multiple epitopes on gp120 can be achieved by hyperglycosylation. Because of the compact nature of the gp140 trimer (in comparison to the gp120 monomer) there is less exposed surface on each monomeric subunit. Consequently, the masking of epitopes by the strategy of hyperglycosylation might be easier in the context of soluble trimers, requiring fewer glycosylations to mask epitopes. In this report we asked the following questions: (1) can the hyperglycosylation strategy be applied to soluble uncleaved gp140 trimers to improve the antigenic profile while still maintaining the trimeric conformation of the im-

SELVARAJAH ET AL. munogen and (2) can antibody responses to specific epitopes on the gp140-trimer antigen, especially the immunodominant variable loops, be suppressed as for monomeric gp120?

MATERIALS AND METHODS Materials The mAbs used in this study were 17b, 48d, A32, C11, and 39F kindly provided by Robinson J;39–43 447-52D and 697-D kindly provided by M. Gorny and Susan Zolla-Pazner;44–47 8.22.2 kindly provided by A. Pinter;48 2G12, 2F5, and 4E10 kindly provided by H. Katinger;49–54 and F425-B4e8 kindly provided by L. Cavacini;55 b12 and b6 were produced in house;56–60 D7324 was purchased from Cliniqa (Fallbrook, CA); and sCD4 was purchased from Progenics (NY). Plasmid pSVIIIexE7pA-HXBc2 was a gift from J. Sodroski. The JRFL, JR-CSF, ADA, and 89.6 Env genes were cloned into the same plasmid pSVIIIexE7pA as described previously.54 pCAGGS-SF162 was kindly provided by J. Binley. PCAGGSMN was kindly provided by J. Moore.27 The following reagents were obtained from the NIH AIDS Research and Reference Reagent Program: F105 (contributed by M. Posner and L. Cavacini),61,62 pNL4-3.Luc.R-E- (contributed by Nathaniel Landau), U87.CD4.CCR5 and U87.CD4.CXCR4 cells (contributed by H. Deng and D. Littman), and HIV-1 consensus subtype B Env (15-mer) peptides—a complete set of 210 peptides.

Plasmids and mutagenesis The plasmid pCMV-Tag4A-tpa JR-FL gp140-CART wild type (used to produce JR-FL gp140-CART wild-type in 293T cells) is derived from plasmid pCMV-Tag4A (Stratagene, La Jolla, CA) and contains a tissue plasminogen activator leader sequence immediately upstream of the Env gene to ensure secretion of the gp140 envelope glycoprotein into the culture supernatant. The cloning of the PCMV-Tag4A-tpa plasmid encoding the tissue plasminogen activator protein (Tpa) has been described previously.38 The Env gene, JR-FL, was obtained by polymerase chain reaction (PCR) amplification by using as a template a plasmid (pSyngp140JR-FL) (obtained from the NIH AIDS Research and Reference Reagent Program and contributed by Eun-Chung Park and Brian Seed) encoding the codonoptimized gp140 gene of this HIV-1 isolate. The soluble trimerization domain of the chicken cartilage matrix protein (CMPcc) fragment was obtained by PCR amplification from a plasmid pET-CMPcc (kindly provided by Richard A. Kammerer, Ph.D., Wellcome Trust Centre for Cell-Matrix Research, Manchester, UK).63,64 The JR-FL gp140 codon-optimized gene was added upstream of the soluble trimerization domain CMPcc by overlap PCR. The PCR product was cloned into pCMV-Tag4A-tpa by using restriction enzymes BssHII and XhoI according to standard protocols. The “REKR” furin cleavage site was mutated to “SEKS” using the site-directed mutagenesis Quickchange kit (Stratagene). The DNA construct is named pCMV-Tag4A-tpa JR-FL gp140-CART wild-type and the protein expressed in 293T cells as gp140-CART wild-type in this report. Site-directed mutagenesis to substitute gp140-CART wild-type residues to incorporate N-glycosylation sequence motifs to make the gp140-CART mCHO(V) mutant construct was per-

ANTIBODY RESPONSE TO VARIABLE LOOPS BY gp140 formed by using Quikchange (Stratagene). DNA sequencing prior to use verified that the plasmids and mutations generated in the present study were correct. The YU2 gp140-FT plasmid construct was kindly provided by J. Sodroski and X. Yang.

PAGE and Western blotting Ready gel, 7.5% Tris-HCl gels (Bio-Rad, Hercules, CA), formulated without sodium dodecyl sulfate (SDS), were used for native polyacrylamide gel electrophoresis (PAGE). For electrophoresis, either supernatants from 293T cells transiently expressing wild-type or mutant glycoproteins or purified protein produced using the vaccinia virus system were used. The samples were diluted 1:2 with native sample buffer with no boiling (62.5 mM Tris–HCl, pH 6.8, 40% glycerol, 0.01% bromophenol blue) (Bio-Rad). Of this mixture, 30 l was loaded onto a 7.5% Tris–HCl separating gel. To allow for the proteins under native conditions to resolve better and run mainly based on molecular weight the electrophoretic running buffer (25 mM Tris, pH 8.3, 192 mM gycine) contained 0.05% SDS. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride membrane by tank blotting (Bio-Rad). Blots were incubated overnight with primary antibody diluted to 1 g/ml in phosphate-buffered saline (PBS) containing 3% nonfat milk, then detected with alkaline phosphatase-conjugated secondary antibody (Pierce, Rockford, IL) (at 1:1000) and developed by using BCIP (5-bromo-4-chloro3-indolylphosphate) and nitroblue tetrazolium as substrates (Sigma-Aldrich, St. Louis, MO).

Electron microscopy (EM) The EM analysis of gp140-CART trimers alone and in complex with b6 or b12 Fab was performed by negative staining

303

EM analysis as previously described.65 Ligand complexes were formed by 30 min RT incubation of the reactants at 1 g/ml in buffered saline borate (BSB). The molar ratios of the reactants were varied in different preparations to optimize trimer–ligand complex formation. The JR-FL gp140-CART wild-type trimers or their complexes were affixed to thin carbon membranes, stained with 1% uranyl formate, and mounted on 600 mesh copper grids for analysis. The sample grids were examined and photographed at a nominal magnification of 100,000 at 100 kV on a JEOL JEM 1200EX electron microscope.

ELISAs Enzyme-linked immunosorbent assay (ELISA) to determine antigenicity of purified protein was set up as described previously.34 ELISA plate wells were coated overnight at 4°C with 50 l of purified vaccinia-produced protein [gp140-CART wild type or gp140-CART mCHO(V)] at 1 g/ml in PBS. Wells were washed four times (4  200 l) with PBS containing 0.05% Tween 20 using an automated plate washer (Molecular Devices, Sunnyvale, CA) and blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature (RT). The 3% BSA was aspirated and primary antibodies added and incubated for 1 h at RT at a starting concentration of 2.5–10 g/ml and diluted 4-fold in dilution buffer. The wells were washed four times and secondary antibody goat anti-human IgG F(ab)2 horseradish peroxidase (HRP) (Pierce) diluted 1:1000 in dilution buffer (in the case of human Abs) was added and incubated for 1 h at RT. The plates were washed four times and developed by adding 50 l of TMB (3,3,5,5-tetramethylbenzidine) solution according to the manufacturer’s instructions (Pierce). The HRP reaction was stopped by adding 50 l of

FIG. 1. (A) Illustration of JR-FL gp140-CART wild-type construct. (B) Western blot probed with antibody b12 showing JRFL gp140-CART and YU2 gp140-FT protein (expressed transiently in 293T cells) that run predominantly as trimers (indicated by an arrow) compared to monomeric JR-FL gp140 or JR-FL gp120.

304 H2SO4 (2 M), and the plates were read at optical density (OD) 450 nm on a microplate reader (Molecular Devices). ELISA to determine serum binding titers was performed by coating microwells with wild-type gp120JR-FL [produced in Chinese hamster ovary (CHO) cells kindly provided by W. Olson and N. Schulke at Progenics], 50 l (1 g/ml), overnight at 4°C. Wells were washed four times (4  200 l) with PBS containing 0.05% Tween 20 using an automated plate washer (Molecular Devices) and blocked with 3% BSA for 1 h at RT. The 3% BSA was aspirated and rabbit serum at 1/2000 diluted down 2-fold (a total of eight dilutions) was added and incubated for 1 h 30 min at RT. The wells were washed four times and 50 l of goat anti-rabbit IgG F(ab)2 (Pierce) conjugated to HRP at 1:1000 dilution was added and the plate was incubated for 1 h at RT and developed as described above.

Protein production using the vaccinia system and purification using lectin Glycoproteins were produced in HEK 293T cells using recombinant vaccinia viruses engineered to express the designated protein as previously described.34 Cells were infected at a multiplicity of infection (MOI) of 5 for 1 h then washed twice with PBS and media replaced with Dulbecco’s modi-

SELVARAJAH ET AL. fied Eagle’s medium (DMEM), penicillin-streptomycin, without serum and incubated at 37°C for 48 h. Supernatants were clarified by low-speed centrifugation and filtration through a 0.22-m filter. Triton X-100 (0.1%) was added to inactivate residual vaccinia. Secreted gp120 was purified from the supernatant by column chromatography using Galanthus nivalis lectin cross-linked to agarose beads (Vector Laboratories, Burlingame, CA). Chromatography was performed on a Bio-Rad LP system with a 2 ml/min flow rate. The column was equilibrated with MES buffer (20 mM MES, 130 mM NaCl, 10 mM CaCl2, pH 7.0), supernatant applied, washed with MES buffer containing 500 mM NaCl, followed by MES buffer, and eluted in MES buffer containing 750 mM methyl manno-pyranoside. The eluate was concentrated and exchanged into MES buffer by centrifugal filtration and the final concentration determined using a BCA protein assay (Pierce). The proteins were assayed with silver staining and by Western blot.

Rabbit immunization New Zealand White female rabbits, weighing 3–5 kg, 4 per immunogen, were immunized with purified protein. Pre-bleeds were taken prior to immunization to be used as controls. Each

FIG. 2. Negative stain electron micrographs of JR-FL gp140-CART trimers (expressed using recombinant vaccine virus in 293T cells) showing a field of molecules alone (left panel) as well as examples of trimers bound to antibodies b12 (right upper panel showing two b12 mAbs each binding with a single Fab arm to a different trimer) and b6 (right lower panel showing two Fab arms of a single b6 mAb cross-linking two trimers). Bar  50 nm.

ANTIBODY RESPONSE TO VARIABLE LOOPS BY gp140 rabbit was injected with 200 g of protein in 1 ml 1  Ribi adjuvant (MPLTDMCWS; Corixa, Seattle, WA) subcutaneously (0.5 ml), intramuscularly (0.1 ml  2) and intradermally (0.05 ml  6 sites) in the case of the gp140-CART wild-type and gp140-CART mCHO(V) rabbits. The boosts were carried out at 4 weeks intervals and bleeds taken 10 days post-injection. A total of five boosts were carried out. Animal housing and immunization procedures adhered to the IACUC (Institutional Animal Care and Use Committee).

Neutralization assay In this assay, virus competent for a single round replication was produced by cotransfection of 293T cells with pSVIIIexE7JR-FL, JR-CSF, ADA, 89.6, or HXBc2 or pCAGGS-SF162, MN or VSV-G, and pNL4-3.luc.R-E-. The degree of virus neutralization by antibody was determined by measuring luciferase activity. Briefly, 3  104 U87.CD4.CCR5 or CXCR4 (for HXBc2) cells in 200 l of medium [DMEM containing 10% fetal bovine serum (FBS), 1 g of puromycin/ml, 300 l of G418/ml, glutamine, and penicillin–streptomycin] were added to microplate wells (96-well flat bottom; Corning Inc., Corning, NY) and incubated for 24 h at 37°C in 5% CO2. Sixty microliters of medium containing an amount of virus previously determined to yield 100,000 relative light units (RLUs) was mixed with 60 l of sera diluted 1:2 2-fold, incubated for 2 h at 37°C in 5% CO2. After 2 h, the medium was aspirated from the plates with U87 cells and 100 l serum and virus were added and the culture was incubated 2 h at 37°C in 5% CO2. After the incubation, serum and virus were aspirated and the wells were washed with 100 l media twice, 200 l of fresh media was added, and the plates were incubated for a further 3 days. On the third day, medium was aspirated from wells and 60 l of luciferase cell culture lysis reagent (Promega, Madison, WI) was added. The wells were scraped and the lysate was mixed by pipetting. The plate was centrifuged at 1800  g for 10 min at 4°C. Twenty microliters was transferred to an opaque assay plate (Corning), luciferase reagent (Promega) was added, and the luciferase activity was measured on a luminometer (Orion, Berthold Detection Systems, Oak Ridge, TN). Serum and virus were incubated for only 2 h with cells since this was found to give the least nonspecific activity from serum (especially effects of enhancement). In addition, each immune serum was always compared to the activity measured with the given prebleed serum diluted in the same fashion to control for nonspecific neutralization activity.

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Serum binding to linear peptides A linear peptide set for V1/V2 and V3 (32, 1-mers) based on the consensus clade B sequence was obtained from the NIH AIDS Research and Reference Reagent Program. The soluble peptides were dissolved in PBS and insoluble peptides in 10% dimethyl sulfoxide (DMSO). Microwell plates were coated at a concentration of 4 nmol per well in 50 l coating buffer23: 15 mM Na2CO3, 35 mM NaHCO3, 3 mM NaN3, pH 9.6, overnight at 4°C. The following day the plates were washed (PBS containing 0.05% Tween 20) and blocked with 3% BSA for 1 h at RT. BSA was aspirated and 50 l of serum diluted 1/100 in dilution buffer (PBS containing 1% BSA and 0.02% Tween 20) was added and incubated for 1 h at RT. The wells were washed and a goat anti-rabbit IgG Fc fragment (Jackson ImmunoResearch, West Grove, PA) conjugated to AP diluted

Serum competition ELISAs The ELISA format for competition with biotinylated antibodies was set up as described previously.34 Briefly, 1 g/ml of g120JR-FL was captured with D7324 sheep anti-gp120 antibody for 2 h at RT. It was then washed and rabbit polyclonal immune sera added at a single concentration of 1/100 dilution for 2 h at RT. Excess solution was aspirated and biotinylated mAbs 10 g/ml diluted 4-fold was added for 1 h and 30 min at RT. Plates were washed and streptavidin conjugated to alkaline phosphatase (Vector Laboratory, Burlingame, CA) diluted 1:300 was added and the plates were incubated for 1 h at RT. The plates were washed and developed with AP substrate and the OD was measured at 405 nm.

FIG. 3. Western blot (PAGE gel) showing gp140-CART wild-type protein (lane 1) and mutants gp140-CART mCHO(V) (on V1-N141, E150, V2-K171, and V3-P313) (lane 2) and gp140-CART GDMR mCHO(V) (V1-N141, V2-K171, and V3P313) (lane 3) protein (produced using the vaccinia virus protein expression system). The blot was probed with human mAb b12. Arrows (→) indicate trimer protein and asterisks (*) indicate monomer protein bands.

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1:1000 in dilution buffer was added for 1 h at RT. Plates were washed again and developed with AP substrate and the OD was read at 405 nm.

RESULTS Construction and characterization of soluble gp140-CART wild-type trimer The JR-FL gp140 codon-optimized gene was added upstream of a soluble trimerization domain of chicken cartilage matrix protein (CMPcc, denoted as CART in this report) and named

gp140-CART wild-type, described in detail in Materials and Methods (shown in Fig. 1A). The novel CART trimerization domain is composed of homotrimeric coiled-coil monomers that are closely held together by two cysteines contributed from each monomer to form a ring of three interchain disulfide bonds. The “REKR” furin cleavage site was mutated to “SEKS.” The gp140-CART wild-type protein was transiently expressed in 293T cells as a high-molecular-weight trimer/oligomer similar to a previously characterized YU2 gp140-FT trimer protein (Fig. 1B).13 Interestingly, even the unpurified gp140-CART wild-type protein shown in Fig. 1B is seen to migrate in a Tris–HCl gel (with 0.05% SDS containing buffer) mainly as a

FIG. 4. Antigenicity of purified vaccinia produced gp140-CART wild-type (left panel) and gp140-CART mCHO(V) (right panel) protein. Top panel shows ELISA binding graph to the gp140-CART wild-type and gp140-CART mCHO(V) by CD4bs mAbs b12, b6, and F105, and gp120 glycan-specific 2G12. The middle panel represents the binding of V3-specific mAbs 44752D and F425-b4e8 and V2-specific mAbs 697-D and 8.22.2. The bottom panel shows mAb binding by gp41 membrane proximal external region (MPER)-specific antibodies 2F5 and 4E10. The x-axis represents OD at 450 nm and the y-axis the antibody concentration in g/ml.

ANTIBODY RESPONSE TO VARIABLE LOOPS BY gp140 trimer/oligomer band with trace amounts of monomers, demonstrating the stability of the gp140 protein linked to the novel CART trimerization domain. However, for further characterization and immunization studies, purified protein was needed in amounts larger than readily available from transient transfections and therefore the protein was produced using the vaccinia virus system in 293T cells and purified using the Galanthus nivalis lectin (described in Materials and Methods). The purified protein was used in all experiments described below. Figure 2 shows electron micrographs (EMs) of gp140-CART wild-type trimers alone and small complexes of gp140-CART wild-type trimers bound to antibodies b12 and b6. In some preparations larger complexes composed of multiple trimers and Abs were observed for both mAbs (not shown). The neutralizing antibody b12 but not the nonneutralizing antibody b6 is expected to bind to functional trimers. However, as shown by Pancera et al., b6 can bind to uncleaved soluble trimers.66

Design and characterization of gp140-CART mCHO(V) mutant; masking epitopes on the variable loops To investigate whether it is possible to use the hyperglycosylation strategy on soluble uncleaved gp140 trimers to improve the antigenic profile while still maintaining the trimeric conformation of the immunogen, we designed a mutant named gp140-CART mCHO(V). The gp140-CART mCHO(V) mutant has four extra sugar attachment motifs (NXT) on the variable loops (on V1-N141, E150, V2-K171, and V3-P313). By incorporating sugar attachment motifs on the V1/V2 and V3 variable loops, our hope was to mask the respective epitopes in the gp140-CART antigen. The gp140-CART mCHO(V) protein, similar to the gp140-CART wild-type protein, is also seen to TABLE 1.

RELATIVE ANTIGENICITY

OF

307

run as a trimer and the extra sugars do not destabilize the trimer protein (Fig. 3). The resolution of the high-molecular-weight trimer protein band is too low on a 7.5% gel to be able to clearly detect the molecular size differences between the gp140-CART mCHO(V) mutant protein and gp140-CART wild-type protein (Fig. 3). However, the molecular size differences are better discernible in the small amounts of gp140-CART mCHO(V) monomer protein band (Fig. 3) that undoubtedly runs at a higher molecular weight compared to the gp140-CART wild-type monomer protein. In addition, the monomer protein band of a different mutant gp140-CART GDMR mCHO(V) protein with only three extra sugar attachment motifs (V1-N141, V2K171, and V3-P313), compared to four on the gp140-CARTmCHO(V) protein, runs at a slightly lower molecular weight, but higher than the gp140-CART wild-type monomer protein band. These results suggest that the engineered extra sugar attachment motifs in the gp140-CART mCHO(V) mutant protein are indeed glycosylated. The apparent binding affinities for a number of mAbs for the gp140-CART wild-type and gp140-CART mCHO(V) mutant protein were determined as described previously.34 The CD4 binding site (CD4bs) antibodies b12, b6, and F105 as well as CD4-IgG2 bound equivalently to both gp140-CART wild-type and gp140-CART mCHO(V) (Fig. 4 and Table 1). The binding of CD4-induced antibodies, 17b and 48d, in the absence and presence of sCD4 was also analyzed and the gp140-CART mCHO(V) mutant was recognized marginally better than the gp140-CART wild-type in the absence of sCD4. However, the binding of 17b and 48d in the presence of sCD4 remained the same for the gp140-CART mCHO(V) mutant and gp140-CART wild-type (Fig. 4 and Table 1). Antibody 2G12, specific to sugars on gp120, and 2F5 and 4E10 specific to the membrane proximal external region (MPER) of gp41 also bound equally well

gp140-CART mCHO(V) PROTEIN COMPARED

TO

gp140-CART WILD-TYPE PROTEINa

CD4-induced antibodies JRFL gp140 antigen gp140 CART gp140 mCHO(V)

gp140 CART gp140 mCHO(V)

17b CD4

CD4

b12

b6

F105

CD4-IgG2

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

0.57

0.8

1.00

0.9

2.00

0.72

2.8

0.64

V3 loop antibodies JRFL gp120 antigen

48d

CD4 binding site antibodies

V2 loop

C1–C4

C1–C5

Sugar

gp41 antibodies

44752D

F425B4e8

39F

697-D

8.22.2

A32

C11

2G12

2F5

4E10

1.00

1.00

1.00

1.00

1.00

1.00

No binding

1.00

1.00

1.00

0.03

0.06

0.67

0.1

0.00

2.00

No binding

1.4

1.25

0.9

aRatio of antibody binding affinities to the gp140-CART mCHO(V) mutant is shown in comparison to gp140-CART wild type. The values in italics indicate the binding affinity of the antibody for the mutant is less than 0.1  gp140-CART wild type.

308 to the gp140-CART wild-type and gp140-CART mCHO(V) mutant (Fig. 4 and Table 1). As per design, the gp140-CART mCHO(V) mutant protein was not recognized by the V3 crownspecific antibodies 447-52D and F425-b4e8 (Fig. 4 and Table 1). However, the antibody to the V3 stem 39F bound fairly well to both the gp140-CART wild-type and gp140-CART mCHO(V) mutant (Table 1). Antibodies to the V2 loop, 697D and 8.22.2 (Fig. 4 and Table 1), failed to bind the gp140CART mCHO(V) mutant, and this indicates masking of the corresponding epitopes by the three extra sugars on the V1/V2 loops of the mutant. Overall, the data indicate that the four extra sugars on the variable loops selectively mask epitopes on the V1/V2 and V3 variable loops.

Serum titers and neutralization potential of gp140-CART wild-type and gp140-CART mCHO(V) immunized rabbit sera Four rabbits per group were immunized with the gp140CART wild-type or gp140-CART mCHO(V) protein. Each rabbit was immunized with 200 g of protein in Ribi adjuvant by intradermal, intramuscular, and subcutaneous injections. Booster injections were given at an interval of 4 weeks and bleeds taken 10 days postinjection. Serum binding titers were determined against wild-type gp120JR-FL coated directly on plates. Half-maximal serum binding titers were determined from anti-gp120 binding curves of fourth bleed sera. Titers ranged from 7  104 to 23  104 (Fig. 5 and Table 2). Both groups had similar serum binding titers; the extra

SELVARAJAH ET AL. glycosylation in the gp140-CART mCHO(V) mutant group did not alter the overall serum binding titers against wild-type gp120. Sera from the fourth bleed for both immunogen groups were subjected to further analysis. Neutralization assays were performed on immune sera (Table 2). The neutralization titers of the gp140CART mCHO(V) sera were significantly reduced compared to gp140-CART wild-type sera for neutralization of SF162 virus. Since SF162 is very sensitive to neutralization by variable loop antibodies,5,7,34 this suggests the success of the hyperglycosylation strategy in dampening the antibody response to the variable loops. There was no significant improvement to the very weak neutralization of the homologous JR-FL virus by the gp140-CART mCHO(V) sera compared to gp140-CART wild-type sera (Table 2). Both sets of immune sera failed to show over 50% neutralization of viruses JR-CSF, ADA, or 89.6 at the lowest dilution (1:4) tested (Table 2). There was no significant change in the ability of the gp140-CART mCHO(V) sera compared to gp140-CART wildtype sera to neutralize HxBc2 and MN (Table 2), two TCLA (Tcell line adapted) viruses, sensitive to neutralization by CD4bs and/or CD4i antibodies.67 This result suggests that the extra sugar on the variable loops of the gp140-CART mCHO(V) mutant protein did not affect the elicitation of CD4bs and CD4i antibodies directed toward the gp120 core.

Focused masking of responses to the variable loops in the gp140-CART mCHO(V) mutant protein We performed assays to map serum responses in order to explore the suppression of variable loop responses in the context

FIG. 5. ELISA binding to wild-type gp120 of sera from rabbits immunized with gp140-CART wild-type (black dashed line) (rabbit # 7376–7379) and gp140-CART mCHO(V) (gray lines) (rabbit # 7526–7529). Prebleed sera from one rabbit (7376 and 7526) (black lines) in each group were used as negative controls. Half-maximal serum binding titers were calculated based on the graph and are presented in Table 2.

ANTIBODY RESPONSE TO VARIABLE LOOPS BY gp140 TABLE 2.

309

NEUTRALIZATION TITERS OF RABBIT SERA FOLLOWING IMMUNIZATION gp140-CART WILD-TYPE AND gp140-CART mCHO(V)

WITH

Rabbit number and antigen gp140-CART 7376 gp140 7377 gp140 7378 gp140 7379 gp140 gp140 mCHO(V) 7526 gp140 mCHO(V) 7527 gp140 mCHO(V) 7528 gp140 mCHO(V) 7529 gp140 mCHO(V)

Half-maximum serum binding titers

Serum dilution at 50% neutralization SF162

JR-FL

JR-CSF

ADA

89.6

HXB2

MN

VSV-G

1:128 1:64 1:128 1:8

1:8 — — —

— — — —

— — — —

— — — —

1:32 1:32 1:4 1:16

1:256 1:256 1:1024 1:32

— — — —

23  10(4)











1:64

1:128



18  10(4)

1:8

1:4







1:32

1:16



17  10(4)

1:8

1:4







1:64





11  10(4)











1:32

1:256



12 20 12 7

   

10(4) 10(4) 10(4) 10(4)

of hyperglycosylated gp140 trimers. We set up an antibody competition ELISA as described previously with biotinylated human mAbs in the presence of rabbit sera and detected directly for inhibition of mAb binding.34 In this assay, biotinylated human mAb was titrated in the presence of a fixed concentration of polyclonal rabbit sera (1/100 dilution) and the human mAb binding determined with streptavidin–alkaline phosphatase. Sera from gp140-CART wild-type and mCHO(V)immunized rabbits competed equivalently for the CD4bs mAbs b12 and b6 (Fig. 6). This was also true for the CD4i mAbs 48d and 17b, which were competed equivalently by both the gp140CART wild-type and gp140-CART mCHO(V) immune sera (data not shown). In contrast, the gp140-CART wild-type immune sera competed considerably more effectively than gp140CART mCHO(V) sera with V3-specific mAbs 447-52D and F425-b4e8 (Fig. 6). However, the V2 specific antibody 697-D binding to gp120 was not inhibited by either the gp140-CART wild-type or gp140-CART mCHO(V) immune sera (Fig. 6). 2G12 competition was equivalent for the two groups of rabbit sera. This is a good control for the assay, since such antibodies (to glycan clusters recognized by 2G12) are not normally elicited upon gp140 immunization. Serum binding to a set of overlapping linear V1/V2 and V3 peptides (total number of 32 peptides) (Fig. 7) based on the consensus clade B sequence was also examined. Sera from gp140-CART wild-type immunized rabbits mainly recognized peptides from the N-terminal sequences of the V1 loop and this was also the case for the gp140-CART mCHO(V) immune sera. Both wild-type and mutant immune sera showed no detectable response to the peptides in the V1 loop where the extra sugars were incorporated at positions 141 and 150, except one peptide C-terminus of position 141, for which three of the four gp140CART mCHO(V) immune sera bound better (Fig. 7). There was no significant response to the V2 loop by the gp140-CART wild-type or mCHO(V) immune sera. This result is similar to that observed with the biotinylated monoclonal antibody com-

petition assay with the V2-specific antibody 697-D. Sera from gp140-CART wild-type immunized rabbits reacted principally with two peptides in the V3 loop, TRPNNNTRKSIHIGP (peptide #8837) and NNTRKSIHIGPGRAF (peptide #8838) (Fig. 7). However, three of the four immune sera raised against the gp140-CART mCHO(V) protein failed to recognize the second peptide (peptide #8838) and showed marginally reduced binding to the first peptide. This is in accordance with results from the biocompetition assay described above that showed that antibodies to the V3 loop were in lower abundance in the gp140CART mCHO(V) immune sera compared to gp140-CART wild-type immune sera. We also tested the gp140-CART wildtype immune sera binding to 10 15-mer consensus peptides in the membrane-proximal external domain (MPER) of the gp41 region (data not shown). The gp140-CART wild-type immune sera recognized only the first two peptides in the N-terminus of MPER and failed to recognize any of the others. In particular, there was no binding to peptides encompassing the epitopes of the broadly neutralizing 2F5 and 4E10 antibodies.

DISCUSSION In this report we asked two questions: (1) Can a hyperglycosylation strategy be used on soluble uncleaved gp140 trimers to improve the antigenic profile while still maintaining the trimeric conformation of the immunogen? The answer is yes, the four extra sugars on the variable loops specifically masked epitope recognition by mAbs specific to the variable loops. In addition, even with the four extra sugars the gp140-CART mCHO(V) soluble protein was still expressed as a trimer. (2) Is it possible to suppress the antibody response to specific epitopes on the gp140-trimer antigen, especially the immunodominant variable loops? The reduction in antibody response of the gp140-CART mCHO(V) immune sera compared to gp140-CART wild-type immune sera against the variable

310

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FIG. 6. Competition ELISAs between biotinylated mAbs and immune rabbit sera. The x-axis represents the concentration of biotinylated human mAb in g/ml. The y-axis represents the binding of biotinylated mAb detected with streptavidin-alkaline phosphatase at OD 405 nm. Biotinylated mAb binding was inhibited in the presence of a single dilution (1/100) of gp140-CART wild-type or gp140-CART mCHO(V) immune sera. The gp140-CART wild-type immunized rabbit sera (7376–7379) inhibiting the binding of the respective bio-mAbs are shown as black dotted lines and the gp140-CART mCHO(V) immunized rabbit sera (7526–7529) inhibiting the binding of the respective bio-mAbs are shown as gray lines. Prebleed (negative control), biotinylated antibody only (negative control) and unbiotinylated antibody (positive control) inhibiting the binding of the respective bio-mAbs are shown as thick black lines. The upper panel represents inhibition of bio-b12 (CD4bs), bio-b6 (CD4bs), or bio-2G12 (glycans) and lower panel with bio-447-52D (V3 mAb), bio-F425-B4e8 (V3 mAb), or bio-697-D (V2 mAb).

loops is clearly shown by assays based on linear peptides, competition assays, and by the reduced ability of the sera to neutralize SF162 virus. In particular, the antibody response to the V3 loop, which seems to be a major target epitope for antibody elicitation following immunization with gp140-CART wildtype, is dampened in gp140-CART MCHO(V) immune sera. The reason is likely the extra sugar on the crown of the V3 loop of gp140-CART mCHO(V) that specifically reduced antibodies to the dominant epitope on the crown of the V3 loop. In addition, based on the modeled structure for trimer by Kwong et al.,68 it is also possible that the extra sugars on the V1 and V2 loops from one gp140 protomer also help mask the V3 loop of the adjacent protomer. Competition assay with 697-D, a V2 loop antibody, and rabbit polyclonal immune sera did not show any inhibition by

either gp140-CART wild-type or gp140-CART mCHO(V) immune sera. Similar results were obtained with a linear peptide-binding assay with sera that failed to recognize peptides in the V2 loops. A recent paper by Beddows et al.2 showed that immunizing with JR-FL gp140-SOSIP antigen did not elicit V1/V2 loop-specific neutralizing antibodies. From our own previous work immunizing with JR-FL gp120 and hyperglycosylated gp120-mCHO we did not detect a large proportion of antibodies raised to the V1 or V2 loops.34 In the immunogen design reported here for soluble gp140 we masked all three variable loops with extra sugars, and the rationale is based on previous reports that have shown that when the antibody response is dampened to only one immunodominant loop (V1/V2 or V3 loop deleted gp120) the antibody response now shifts and targets epitopes on the other variable loops.5,43 In immunization

FIG. 7. ELISA of serum binding to linear overlapping V1/V2 and V3 peptides (32 15-mers). The sequences of these peptides are shown as peptide numbers as given by the NIH AIDS Research and Reference Reagent Program and the sequences corresponding to them are listed below the graphs. The upper panel represents the four (7276–7279) gp140-CART wild-type immune sera and pooled prebleed and the bottom panel shows the binding of sera from the four (7526–7529) gp140-CART mCHO(V) immune sera and pooled prebleed. The y-axis represents binding measured at OD 405 nm. The peptides that include the amino acid residues (N141, E150, K171, and P313) that were mutated in order to incorporate the sugar attachment motif are also indicated.

312

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studies with other clade B Env trimers YU2 gp140-GCN and SF162 gp140 (V2 crown deleted), a proportion of antibodies targeting epitopes in the V1 loop is elicited.5,19 Therefore, for future soluble gp140 trimer immunogen designs, with the aim of using the hyperglycosylation strategy, it will be important to mask and dampen antibody responses to all immunodominant variable loops. The antibody response to the CD4bs and the CD4i epitopes was similar in gp140-CART wild-type and gp140-CART mCHO(V) sera as shown by a competition assay with the CD4bs antibodies as b12, b6, and the CD4i antibodies 48d and 17b. In addition, the ability of the gp140-CART mCHO(V) sera to neutralize HxBc2 and MN viruses was similar to that in gp140-CART wild-type sera. Therefore, the results shown here with a gp140 trimer mutant containing the extra sugars only on the variable loops are a clear demonstration that sugars on the variable loops specifically reduced antibody response only to the immmundominant loops and not to the two major epitopes on the gp120 core. Overall, the results demonstrate that the strategy of hyperglycosylation and masking of epitopes works in the context of soluble trimers. However, these strategies will come to full fruition when better recombinant mimics of the functional trimer found on the surface of HIV become available.

6.

7.

8.

9.

10.

11.

ACKNOWLEDGMENTS We thank Philip Rudge Arca from R. Dom’s laboratory for technical support. We acknowledge support from grants from the National Institutes of Health, AI33292, AI060425 (D.R.B.) and AI055461 (K.H.R.); the International AIDS Vaccine Initiative (IAVI) Neutralizing Antibody Consortium; and the Pendleton Trust Foundation.

12.

13.

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