Purified, Proteolytically Mature HIV Type 1 SOSIP gp140 Envelope Trimers ABSTRACT

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1 AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 23, Number 6, 27, pp Mary Ann Liebert, Inc. DOI: 1.189/aid Purified, Proteolytically Mature HIV Type 1 SOSIP gp14 Envelope Trimers SAI PRASAD N. IYER, 1 MICHAEL FRANTI, 1 AMMIE A. KRAUCHUK, 1 DANIELLE N. FISCH, 1 AMADOU A. OUATTARA, 1 KENNETH H. ROUX, 2 LAURA KRAWIEC, 1 ANTU K. DEY, 3 SIMON BEDDOWS, 3 PAUL J. MADDON, 1 JOHN P. MOORE, 3 and WILLIAM C. OLSON 1 ABSTRACT HIV type 1 (HIV-1) envelope is a noncovalent trimer of gp12 gp41 heterodimers, and its lability has hindered structural studies. SOSIP gp14 is a soluble, proteolytically mature form of the HIV-1 envelope wherein gp12 gp41 interactions are stabilized via a disulfide bond and gp41 contains an additional trimer-stabilizing point mutation. We describe the isolation of a substantially pure preparation of SOSIP gp14 trimers derived from KNH1144, a subtype A isolate. Following initial purification, the only significant contaminant was higher-order gp14 aggregates; however,.5% Tween 2 quantitatively converted these aggregates into trimers. The surfactant effect was rapid, dose dependent, and similarly effective for a subtype B SOSIP gp14. Surfactant-treated SOSIP gp14 retained favorable antigenicity and formed compact trimers nm in size as determined by electron microscopy. This report provides the first description of homogeneous, cleaved HIV-1 envelope trimers. These proteins may be useful as vaccine immunogens and for studying structure function relationships within the HIV-1 envelope glycoproteins. INTRODUCTION AN ESTIMATED 4 MILLION PEOPLE were living with HIV worldwide at the end of 25, with over 4 million newly infected cases in that year. 1 Among the hardest hit areas is sub- Saharan Africa, with over 25 million people living with HIV and about 1% dying of AIDS-related illnesses each year. Prophylactic measures, such as an HIV vaccine, have the potential to curtail the spread of HIV infection globally. Ideally, an HIV vaccine would protect against infection. One approach has been to target the HIV entry pathway, specifically focusing on the viral envelope spike. The HIV-1 envelope (Env) glycoproteins are derived from a gp16 precursor protein, which is cleaved intracellularly by members of the furin family of endoproteases to yield a gp12 surface glycoprotein and a gp41 transmembrane glycoprotein. 2,3 The fusogenic form of Env is a trimer of gp12 gp41 heterodimers that are associated via noncovalent interactions. HIV-1 gp12 binds CD4 and a coreceptor molecule on susceptible host cells and thereby triggers gp41-mediated fusion of the viral and cellular membranes. 4 A small number of monoclonal antibodies (mabs) have been identified that neutralize a broad range of HIV-1 genetic subtypes. 5 In addition, sera from some HIV-1-infected individuals display broadly neutralizing activity. Broadly neutralizing mabs all bind the trimeric viral spike, 5 and vaccines that mimic the native spike have the potential to elicit a broadly neutralizing response. HIV-1 Env trimers are technically challenging to produce in purified form. Oligomeric gp14 polyproteins have been generated both by mutating the gp12 gp41 protease cleavage site and by expressing a wild-type polyprotein under conditions that do not foster cleavage. 6 1 Trimeric forms of uncleaved gp14 have been additionally stabilized by the introduction of either heterologous trimerization domains or gp41 gp41 disulfide bonds We previously described recombinant, proteolytically processed SOSIP gp14 proteins that incorporate three modifications to the native Env sequence: (1) truncation at the 1 Progenics Pharmaceuticals, Inc., Tarrytown, New York Department of Biological Science and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida , 3 Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York

2 818 gp41 ectodomain, (2) incorporation of a gp12 gp41 intersubunit disulfide bond, and (3) a point mutation in gp41 to enhance trimer stability. 15,16 The SOSIP gp14s also may contain a hexaarginine mutation at the gp12 gp41 cleavage site to promote efficient processing in transfected cell lines. 17,18 This approach has enabled the expression of proteolytically mature SOSIP gp14 proteins in a trimeric conformation. 15,16,18,19 Initial studies examined SOSIP gp14 derived from HIV-1 JR-FL, a prototypic subtype B primary isolate. In some rabbits, HIV-1 JR-FL SOSIP gp14 elicited antibodies that neutralized HIV-1 JR-FL and certain other primary HIV-1 isolates. 18 In addition, we recently examined SOSIP gp14 proteins derived from a panel of contemporary subtype A HIV-1 isolates from sub-saharan Africa. 2 The SOSIP gp14 protein from one particular strain, KNH1144, formed unusually stable trimers. 2 In this report, we describe the isolation and characterization of substantially pure SOSIP gp14 trimers. The purification strategy exploited our prior observation that nonionic surfactants efficiently convert KNH1144 SOSIP gp14 aggregates to trimers. 2 Here we report that Tween 2 rapidly converts KNH1144 SOSIP aggregates to trimers in a concentration-dependent, temperature-independent manner, and this surfactant effect was extended to SOSIP gp14 derived from HIV , a subtype B isolate of vaccine interest. 21 Surfactant-treated SOSIP gp14 had favorable antigenic characteristics, and electron microscopy studies revealed compact trimers that resemble virion-associated Env. Our studies provide a source of homogeneous, proteolytically mature HIV-1 envelope trimers for further structural and vaccine research. MATERIALS AND METHODS Expression and purification of SOSIP gp14 and gp12 The KNH1144 SOSIP gp14 construct with the hexaarginine (R6) mutation and the furin plasmid have been described. 2 SOSIP gp14 and monomeric gp12 proteins were expressed using PPI4, which has been described previously. 22 For a typical 8-liter preparation, human embryonic kidney (HEK) 293T cells were seeded into triple flasks (Corning Life Sciences, Acton, MA) at a density of cells/flask and cultured in Dulbecco s modified Eagle medium (DMEM)/1% fetal bovine serum (FBS)/1% pen-strep (Invitrogen, Carlsbad, CA), with 1% L-glutamine 24 h prior to transfection. On the day of transfection, 27 g KNH1144 SOSIP envelope DNA was mixed with 9 g furin DNA (per flask) in Opti-MEM. Polyethyleneimine (PEI) (Polysciences Inc., Warrington, PA) was added stepwise (2 mg PEI:1 mg total DNA) and vortexed between each addition. 23 The PEI/DNA solutions were incubated for 2 min at ambient temperature and then added to the flasks. Cells were incubated for 6 h at 32 C, 5% CO 2, washed with warmed Dulbecco s phosphate buffered saline (PBS) and then incubated in exchange media (DMEM/.5% bovine serum albumin (BSA)/1% pen-strep) for 48 h at 32 C, 5% CO 2. Supernatants were collected, and a protease inhibitor cocktail (Sigma, St. Louis, MO) was added according to the manufacturer s instructions. Harvested supernatants were clarified using a.45- m filter and concentrated 5-fold. KNH1144 SOSIP gp14 trimers were purified by ammonium sulfate precipitation followed by affinity, size-exclusion, and ion-exchange chromatography. Concentrated cell culture supernatant was precipitated with an equal volume of 3.8 M ammonium sulfate. Ammonium sulfate was added with constant stirring, and samples then were immediately centrifuged at 4 rpm, 4 C, for 45 min. The supernatant was diluted 4-fold with Dulbecco s PBS without calcium, magnesium, or phenol red (PBS ) at ph 7.25 and filtered using a.45- m vacuum filter. The sample was then loaded onto a Galanthus nivalis agglutinin (GNA) lectin (Vector Laboratories, Burlingame, CA) column equilibrated with PBS. The column was washed with PBS until OD 28 reached baseline, followed by a second wash with.5 M NaCl PBS at ph 7.25 in order to remove BSA and other contaminant proteins. The column was then eluted with 1 M methyl- -mannopyranoside (MMP, Sigma) in PBS starting with flowing one-half column volume (CV) and pausing the purification for 1 h. Flow was then restarted, and eluate fractions were pooled and concentrated using a Vivaspin 1,- Da molecular weight cutoff (MWCO) concentrator (Vivascience, Edgewood, NY) spun at 1 g. The concentrate was applied over a Superdex 2 size-exclusion chromatography (SEC) column (GE Healthcare, Piscataway, NJ) equilibrated in 2 mm Tris, ph 8, 2 mm NaCl (TN-2), and resolved at.4 ml/min. Trimer-containing fractions were pooled and diluted to 75 mm NaCl with 2 mm Tris, ph 8. The diluted SEC pool was then applied over a 1 ml HiTrap diethyl aminoethyl (DEAE) FF column (GE Healthcare), equilibrated in 2 mm Tris, ph 8, 75 mm NaCl (TN-75). The column was washed with TN-75 until the OD 28 reached baseline and then eluted with 2 mm Tris, 3 mm NaCl, ph 8. To maximize trimer yield, the DEAE flow-through was reapplied over the column (equilibrated in TN-75), and typically 2 3% more trimer was recovered in this manner. Trimer-containing fractions were pooled and the concentration was determined by densitometry on a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) gel using JR-FL gp12 as a standard. KNH1144 gp12 was expressed and purified using GNA lectin, Q Sepharose (GE Healthcare), and Superdex 2 size-exclusion chromatographies essentially as described. 19 HIV is a CCR5-tropic, subtype B strain isolated from an acutely infected patient within 1 month of seroconversion. 21 A construct expressing 5768 SOSIP gp14 containing the R6 mutation was generated as described previously. 16, SOSIP gp14 was purified as described above for KNH1144 SOSIP with the following exceptions: the 293T culture supernatants were concentrated 63-fold prior to precipitation with ammonium sulfate, and the SEC run buffer was 2 mm histidine, 15 mm, ph 6.. DEAE was not used to purify 5768 SOSIP gp14. SDS PAGE and BN PAGE IYER ET AL. SDS PAGE analyses were performed using 4 12% Bis-Tris NuPage gels (Invitrogen). Blue native PAGE (BN PAGE) was performed as described. 19 Thyroglobulin (67 kda) and ferritin (44 kda) were used as molecular weight standards. Coomassie staining was performed using either the SimplyBlue SafeStain or Easy-to-Use Coomassie G-25 Stain (Invitrogen). Silver staining was performed with the SilverQuest kit (Invitrogen).

3 SOSIP gp14 ENVELOPE TRIMERS 819 Western blot analysis was performed with mab ARP3119 antibody (CA13, Centralised Facility for AIDS Reagents, Herts, United Kingdom). Effect of Tween 2 on Env aggregates Aliquots (1 g) of purified SOSIP gp14 were treated with Tween 2 and then analyzed by BN PAGE and Coomassie staining. In a first study, KNH1144 SOSIP gp14 was treated with Tween 2 at concentrations ranging from to.1% (v/v) for 1 h at ambient temperature ( 23 C) prior to analysis. Alternatively, samples were incubated with Tween 2 at a final concentration of.5 % (v/v) for 5 min and 1 min at ambient temperature prior to analysis. Temperature effects were studied by incubating samples in.5% (v/v) Tween 2 at C (on ice), ambient temperature, or 37 C for 1 min SOSIP gp14 was treated with.5% Tween 2 for 1 min at ambient temperature. Similarly, 1 g of purified gp12 monomer or.5 g purified 2 -macroglobulin (A2M, Sigma, St. Louis, MO) was either untreated or incubated with Tween 2 at a final concentration of.5% for 1 min at ambient temperature and then analyzed by BN PAGE. A2M is a homotetrameric glycoprotein comprised of 185 kda subunits. Analytical size-exclusion chromatography Analytical runs were performed at 4 C on an AKTA fast protein liquid chromatography (FPLC) system (GE Healthcare). Each run was performed at least twice. A Superdex 2 1/3 GL column was equilibrated in 2 mm Tris, ph 8,.5 M NaCl (TN-5) and calibrated with the following molecular weight standard proteins (GE Healthcare): thyroglobulin 669, Da, ferritin 44, Da, BSA 67, Da, and RNase A 13,7 Da. A standard curve was generated by plotting the observed retention volumes of the standard proteins against the log values of their predicted molecular weights. Purified KNH1144 SOSIP gp14 (8 1 g) was treated with Tween 2 at a final concentration of.5% for 1 3 min at ambient temperature. Samples were then applied over the Superdex 2 column equilibrated with TN-5 containing.5% Tween 2 (TNT-5) and resolved at.4 ml/min, collecting.4 ml fractions. Trimer-containing fractions were then analyzed by BN PAGE, followed by silver stain. Fractions were also separated by BN PAGE, followed by Western blot analysis with ARP 3119 antibody. In addition, 1 14 g of purified KNH1144 gp12 or JR-FL gp14 was applied over the Superdex 2 column equilibrated in TN-5 and resolved at a flow rate of.4 ml/min. Lectin ELISA and immunoprecipitation Human mabs b6, 24 b12, 25 and 2G12, 26 and HIVIg 27 were obtained from Dr. Dennis Burton (The Scripps Research Institute, La Jolla, CA) or Dr. Herman Katinger (University of Natural Resources and Applied Life Sciences, Vienna, Austria). For the lectin-capture ELISA, anti-env antibodies 2G12, b6, b12, and HIVIg were used. The CD4 IgG2 fusion protein was prepared as previously described. 28 ELISA plates were coated overnight at 4 C with Lens culinaris (lentil) lectin (Sigma) at 1 g/ml concentration, and the assay was performed at ambient temperature thereafter. Plates were washed with PBS twice and blocked with SuperBlock (Pierce). Excess blocking agent was washed off with PBS. Samples were added at.3 g/ml to the plates for four h. The plates were washed 4 times with PBS and incubated with anti-env antibodies or CD4 IgG2 starting at 1 g/ml in PBS/5% milk. Then 4 serial dilutions were performed and incubations were performed for 3 h. Plates were washed six times and goat antihuman IgG (H L) alkaline phosphatase conjugate secondary antibody (Jackson Immuno- Research) was added at 1/4 dilution in PBS/5% milk. Plates were washed four times and developed using the AMPAK detection system (Dako Cytomation, Carpinteria, CA) as per the manufacturer s instructions. Immunoprecipitation studies were performed as previously described 2 with the above mabs and with mabs to the CD4-binding site (15e 29 and D2 6 ) and to CD4-induced epitopes (17b, 3 48d, 3 and X5 31 ). DEAE anion-exchange chromatography Purified KNH1144 SOSIP gp14 trimers, treated either with or without.5% Tween 2, in TN-75 buffer were applied to a 1-ml DEAE HiTrap FF column (equilibrated in TN-75) at.25 ml/min at ambient temperature, washed with TN-75 at.5 ml/min, and eluted with TN-3. Equal amounts of flowthrough, wash, and elution fractions were analyzed via BN PAGE, followed by Coomassie G-25 stain. Electron microscopy Negative-stain electron microscopy was performed as previously described. 32,33 Because this technique is incompatible with Tween 2, 2 l of purified KNH1144 SOSIP gp14 (.5 mg/ml in TN-3 with.5% Tween 2) was dialyzed against BSB (.1 M H 3 BO 3,.25 M Na 2 B 4 O 7,.75 M NaCl, ph 8.3) and subsequently depleted of Tween 2 using the Mini Detergent-OUT kit (Calbiochem, La Jolla, CA), as described by the manufacturer. Two micrograms of Tween 2-depleted protein were diluted in 2 l BSB, and affixed to a carbon support membrane. The membrane was stained with 1% uranyl formate, and mounted on 6 mesh copper grids for analysis. Electron micrographs were recorded at 1, magnification at 1 kv on a JOEL JEM 12 electron microscope. Trimers were measured and analyzed statistically using Image-Pro Plus software ( RESULTS Expression and purification of SOSIP gp14 trimers Our purification scheme for KNH1144 SOSIP gp14 involved ammonium sulfate precipitation followed by three chromatography steps: GNA lectin affinity, Superdex 2 size exclusion, and DEAE anion exchange. While the GNA lectin column efficiently and selectively captured KNH1144 SOSIP gp14, significant dissociation of trimers into dimers and monomers occurred during elution with MMP (data not shown). Monomers and dimers were effectively removed using SEC and DEAE chromatography, respectively. Purified KNH1144 SOSIP gp14 migrated as a single 14- kda gp14 band on a nonreducing SDS PAGE gel (Fig. 1A).

4 82 IYER ET AL. FIG. 1. SDS PAGE and BN PAGE. Purified KNH1144 SOSIP gp14 trimer and gp12 monomer were analyzed by gel electrophoresis. (A) SDS PAGE was performed under reducing and nonreducing conditions. Proteins were visualized by Coomassie G-25 stain (Lanes 1 3) or via ARP3119 Western blot (Lane 4). Lane 1: gp12, reduced. Lane 2: SOSIP gp14, reduced. Lanes 3 and 4: SOSIP gp14, nonreduced. (B) BN PAGE analysis was performed in the presence and absence of Tween 2, as indicated; and proteins were visualized with Coomassie G-25 stain. Lane 1: Molecular weight markers. Lane 2: SOSIP gp14 without Tween 2. Lane 3: SOSIP gp14 with Tween 2. Lane 4: gp12 without Tween 2. Lane 5: gp12 with Tween 2. The absence of a significant gp12 band under nonreducing conditions indicates that the intersubunit disulfide bond is intact. No disulfide-linked SOSIP gp14 aggregates were detected by either Coomassie blue staining or Western blotting under nonreducing conditions. When reduced, KNH1144 SOSIP gp14 yielded a 12-kDa gp12 band in SDS PAGE (Fig. 1A). The absence of a gp14 band under reducing conditions indicates that the purified protein is fully cleaved at the gp12 gp41 proteolytic site. Purified KNH1144 gp12 migrated as a single band of approximately 12 kda in SDS PAGE (Fig. 1A). Common serum contaminants, such as A2M and BSA, collectively comprised 5% of the purified KNH1144 gp12 and SOSIP gp14 proteins. When analyzed by BN PAGE, purified KNH1144 SOSIP gp14 migrated predominantly as a trimer, consistent with our prior results for partially purified protein. 2 Dimeric and monomeric forms of gp14 were not evident. However, an additional 67-kDa band was observed in the absence of Tween 2 (Fig. 1B), and this band was determined by Western blotting to be high-molecular-weight (HMW) aggregates of SOSIP gp14 (data not shown). The relative abundance of HMW aggregates ranged from 1% to 4% among the different lots of purified product. Surfactant treatment Treatment of purified KNH1144 SOSIP gp14 with mild surfactant (.5% Tween 2) quantitatively converted the HMW aggregates into trimers (Fig. 1B). There was no appreciable dissociation of trimers into monomers or dimers, consistent with our preliminary observations. 2 Despite its nonionic nature, Tween 2 modestly but measurably increased the mobility of the trimers in BN PAGE (Fig. 1B). One possible explanation for the faster migration of Tween-treated SOSIP trimers is that the surfactant causes SOSIP to adopt a more compact conformation. As a second possible explanation, Coomassie blue may bind to Tween 2 molecules that are bound to SOSIP, giving SOSIP a greater overall net charge within the BN PAGE gel. These possibilities are not mutually exclusive. Surfactant treatment also increased the staining intensity of SOSIP gp14. Tween 2 had no obvious effect on the mobility of gp12 in BN PAGE (Fig. 1B) but modestly increased its staining intensity in some cases (data not shown). Since surfactant treatment provided a facile means to obtain homogeneous trimers, we further characterized this process. A preparation containing 3% HMW aggregates was treated with Tween 2 at concentrations ranging from.1% to.1% (v/v). Concentrations of.1% to.1% dissociated aggregates into trimers (Fig. 2A). Trace amounts of aggregates were present at.1% Tween 2, and no conversion was observed at.1% or.1% (Fig. 2A). Therefore,.1% represents a threshold concentration for conversion. The kinetics of dissociation were studied by incubating SOSIP gp14 with.5% Tween 2 for 5 and 1 min at ambient temperature, prior to analysis by BN PAGE. As shown in Fig. 2B, aggregates were completely eliminated at both 5 min and 1 min. We next examined the effect of temperature on the dissociation of aggregates. Efficient conversion of aggregates to trimers was observed within 1 min at temperatures of C, 23 C, and 37 C (Fig. 2C). Finally, similar results were obtained when Tween 8 was used in place of Tween 2 (data not shown). Surfactant treatment was also applied to purified SOSIP gp14 derived from HIV , a subtype B isolate SOSIP gp14 trimers show intermediate stability relative to the more stable KNH1144 and less stable JR-FL SOSIP gp14 proteins. Both dimeric and monomeric gp14 bands are clearly visible in a preparation of purified 5768 SOSIP gp14 (Fig. 2D).

5 SOSIP gp14 ENVELOPE TRIMERS 821 FIG. 2. Tween 2 conversion studies. Purified SOSIP gp14 was incubated with Tween 2 as indicated and analyzed by BN PAGE. (A) Concentration effects: KNH1144 SOSIP gp14 was incubated with % (Lane 2),.1% (Lane 3),.5% (Lane 4),.1% (Lane 5),.1% (Lane 6), or.1% (Lane 7) Tween 2 for 1 h at ambient temperature. (B) Time course: KNH1144 SOSIP gp14 was treated for 5 and 1 min with.5% Tween 2 at ambient temperature. Lane 2: 5 min without Tween 2. Lane 3: 5 min with Tween 2. Lane 4: 1 min without Tween 2. Lane 5: 1 min with Tween 2. (C) Temperature effects: KNH1144 SOSIP gp14 was treated for 1 min either without Tween 2 at 23 C (Lane 2) or with.5% Tween 2 at C (Lane 3), 23 C (Lane 4), or 37 C (Lane 5). (D) Subtype B Env: 5768 SOSIP gp14 was treated for 1 min at ambient temperature in the absence (Lane 2) or presence (Lane 3) of.5% Tween 2. In all gels, Lane 1 contains molecular weight markers. Nevertheless, in the absence of surfactant, the main contaminant is HMW aggregates, and these aggregates were reduced to trace levels by.5% Tween 2 (Fig. 2D). As was observed for KNH1144 SOSIP gp14, surfactant increased the mobility of 5768 SOSIP gp14 trimers gp14 monomers and dimers also showed increased mobility. Importantly, there was no apparent increase in the amounts of the monomeric and dimeric gp14 bands, suggesting that surfactant efficiently converted aggregates to trimers for this subtype B SOSIP gp14 protein. To test if surfactant dissociated an unrelated multisubunit protein,.5% Tween 2 was added to A2M, which is a noncovalent tetramer of identical 185-kDa subunits. Treatment did not affect the oligomeric state of A2M, although there was a small increase in staining intensity of the protein (Fig. 3A). We next treated a preparation of KNH1144 SOSIP gp14 containing 8% HMW aggregates. Tween 2 was effective in converting the aggregates to trimers (Fig. 3B). Finally, a preparation composed of a mixture of KNH1144 SOSIP HMW aggregates, dimers, and monomers was treated with Tween 2. In this case, Tween 2 converted HMW aggregates to trimers but had no obvious effect on the amount of monomers or dimers (Fig. 3B). The findings indicate that Tween 2 selectively dissociates HMW aggregates into trimers. SEC analysis Superdex 2 SEC was performed as a second means to characterize KNH1144 gp12 and SOSIP gp14. In addition, monomeric JR-FL gp12 was also analyzed as a control. KNH1144 gp12 and JR-FL gp12 each eluted at an apparent molecular weight of 21 kda (data not shown), consistent with our prior findings for JR-FL gp The high apparent molecular weight in SEC is thought to reflect the extended nature of the glycan chains on Env. In initial studies with surfactant-treated KNH1144 SOSIP gp14, we observed that HMW aggregates reformed when Tween 2 was not included in the SEC run buffer. Therefore, SEC was performed in the presence of.5% Tween 2, and the column fractions were analyzed by BN PAGE and silver staining. As shown in Fig. 4, trimers comprised the major SEC elution peak. The average retention volume (9.4 ml) corre-

6 822 A IYER ET AL. A2M B Aggregates Trimers Dimers Monomers FIG. 3. Effects of Tween 2 on A2M and additional forms of KNH1144 SOSIP gp14. (A) Purified 2 -macroglobulin (A2M) was either left untreated (Lane 2) or incubated with.5% Tween 2 (Lane 3). Reactions were analyzed by BN PAGE and Coomassie stain. Lane 1 contains molecular weight markers. (B) Tween 2 effect on HMW aggregate and dimer fractions. A sample containing 8% HMW aggregates or a sample of mixed oligomers (HMW aggregates, dimers, and monomers) was untreated or was treated with Tween 2 prior to analysis by BN PAGE and Coomassie G-25 stain. Lane 1: Molecular weight markers. Lane 2: Untreated HMW aggregates. Lane 3: HMW aggregates treated with Tween 2. Lane 4: Untreated mixed oligomers. Lane 5: Mixed oligomers treated with Tween 2. sponds to an apparent molecular weight of 52 kda, similar to that reported previously for JR-FL SOSIP gp14 trimers. 16 The SEC and BN PAGE data concur that surfactant-treated KNH1144 SOSIP gp14 is trimeric. Effect of surfactant on antigenicity Previously, we reported that unpurified KNH1144 SOSIP gp14 was immunoprecipitated by the neutralizing agents 2G12, b12, and CD4 IgG2, and by the nonneutralizing mab b6. 2 In preliminary experiments, we immunoprecipitated purified trimers with 2G12, IgG1b12, CD4 IgG2, b6, D2, 15e, 17b, X5, and 48d. Consistent with our observations on unpurified material, purified KNH1144 SOSIP trimers were efficiently immunoprecipitated with 2G12, IgG1b12, CD4 IgG2, and b6. In addition, both trimers and gp12 monomers were immunoprecipitated with mabs 17b and X5 in the presence but not the absence of soluble CD4, but neither trimers nor monomers were immunoprecipitated with mab 48d under any circumstance. The CD4 binding site mabs 15e and D2 did not immunoprecipitate either KNH1144 SOSIP gp14 trimers or gp12 monomers (data not shown).

7 SOSIP gp14 ENVELOPE TRIMERS 823 TG Ferr BSA A optical density, mau B Trimers We next extended these studies using a more quantitative ELISA method, and we also assessed the effect of surfactant on antigenicity. In an initial experiment, a sample containing 8% HMW aggregates was analyzed by lectin-capture ELISA in the presence or absence of.5% Tween 2, which quantitatively converted the HMW aggregates to trimers (see Fig. 3B). Following treatment, we observed increased binding of b6, b12, and CD4 IgG2, all of which bind the CD4-binding site on Env (Fig. 5A). In contrast, surfactant had a minimal effect on the binding of 2G12 and a modest effect on the binding of HIVIg. These results indicate that the CD4 binding site region of gp12 may be partially occluded within the HMW aggregates, but the 2G12 epitope is not. We performed similar lectin ELISAs on SOSIP gp14 trimers that contained modest amounts of HMW aggregates ( 15%). As shown in Fig. 5B, Tween 2 did not significantly affect the binding of 2G12, b6, b12, or CD4 IgG2 to trimeric KNH1144 SOSIP. These studies indicate that surfactant does not appreciably alter the antigenicity of KNH1144 SOSIP trimers. The binding of 2G12, b12, and CD4 IgG2 indicates that these broadly conserved neutralization epitopes are present in appropriate conformations in KNH1144 SOSIP trimers in the presence or absence of Tween 2. However, b6 binding indicates that the trimers do not perfectly mimic the conformation of the native envelope spike, and studies have been initiated to address this issue ml (retention volume) FIG. 4. SEC analysis. KNH1144 SOSIP gp14 trimer was resolved on a Superdex 2 1/3 GL column in TN-5 buffer containing.5% Tween 2 (TNT-5), and column fractions were analyzed by BN PAGE and silver staining. (A) A 28 trace of the run. Arrows represent the retention volumes of the protein standards [thyroglobulin (TG), ferritin (Ferr), and BSA] used to calibrate the column. (B) Silver-stained BN PAGE gel of the column fractions. Lane 1: molecular weight markers. Lanes 2 8 sequential.4 ml column fractions from 8.2 ml retention volume. 8 Effects of Tween 2 on the ionic properties of KNH1144 SOSIP gp14 and A2M We used DEAE anion-exchange chromatography to examine the effect of Tween 2 on the ionic properties of SOSIP gp14 and A2M. For this, KNH1144 SOSIP gp14 was spiked with A2M, and the mixture either was treated with Tween 2 or was left untreated prior to DEAE chromatography. Column fractions were analyzed via BN PAGE. As expected, untreated SOSIP gp14 trimers bound to the DEAE column and were recovered in the eluate (Fig. 6A). In the presence of surfactant, however, KNH1144 SOSIP gp14 flowed through the column (Fig. 6B). In contrast, A2M bound DEAE in the presence or absence of Tween 2 (Fig. 6). In similar experiments, Tween 2 did not affect binding of BSA to DEAE (data not shown). Therefore, Tween 2 selectively affects both the oligomeric state and the charge properties of KNH1144 SOSIP gp14. Electron microscopy of KNH1144 SOSIP gp14 We next studied the appearance of purified SOSIP gp14 using negative-stain electron microscopy. As shown in Fig. 7, KNH1144 SOSIP gp14 displayed a regular compact morphology with approximate 3-fold symmetry. As is typical, different areas on the grid display different staining characteristics including areas of shallow and deep stain. To estimate the size of the trimers, 78 trimers from the deep-stain regions (Fig. 7) were scored, yielding a diameter estimate of 1 nm 1.8 nm. In addition, 7 spikes in the shallow-stain regions (data not shown) were also scored; the resulting diameter estimate was nm. The shallow stain slightly overestimates protein size, whereas the deep stain slightly underestimates it. Therefore, the true diameter is likely to be nm, in line with that observed for virion-associated simian immunodeficiency virus (SIV) Env spikes as measured using both similar negative-staining electron microscopy procedure and cryoelectron microscopy methods. 34,35 The electron microscopy, SEC, and BN PAGE data are consistent in demonstrating that purified KNH1144 SOSIP gp14 is a trimer. DISCUSSION We describe the isolation and initial characterization of proteolytically mature HIV-1 envelope trimers. To our knowledge, KNH1144 SOSIP gp14 represents the first HIV-1 envelope to be produced and isolated as essentially homogeneous cleaved trimers. Electron microscopy and antigenicity studies demonstrate that the purified protein forms compact trimers that reproduce a number of features of the native envelope spike. Isolation of homogeneous trimer was enabled by the finding that mild treatment with nonionic surfactant quantitatively converted HMW aggregates to trimers, and the surfactant effect was generalizable to another subtype B HIV-1 Env. The purified, cleaved Env trimers are being utilized for further structural analyses and for vaccine research. Compared to other subtype A and subtype B SOSIP gp14 proteins, 16,18 2 KNH1144 SOSIP trimerized more efficiently, and the trimers proved stable to purification. The stability of the KNH1144 Env was identified empirically; however, its sequences may prove useful in understanding the determinants of

8 824 IYER ET AL. A G12 b6 HIVig CD4-igG b12 With Tween 2 Without Tween B 2.4 2G b CD4-igG2 1.2 b12 With Tween 2 Without Tween FIG. 5. Effect of Tween 2 on antigenicity. Lectin ELISA of untreated and Tween 2-treated KNH1144 SOSIP gp14. Untreated or Tween 2-treated proteins were bound to lectin-coated ELISA plates and probed with 2G12, b6, b12, CD4 IgG2, and HIVIg. The y-axis represents the colorimetric signal at OD492 and the x-axis represents antibody concentration in g/ml. (A) Fraction containing 8% HMW aggregates pretreatment. (B) Fraction containing 1 15% HMW aggregates pretreatment. HIVIg was not tested with this fraction due to a limited availability of material. Env trimerization, at least in the context of soluble gp14 proteins. Initial studies have demonstrated that trimer stability is influenced by residues in the N-terminal heptad-repeat region of gp41, and the stability of JR-FL gp14 trimers was improved by substitution with five residues from this region of KNH In the absence of surfactant, purified KNH1144 SOSIP gp14 contained 1 4% HMW aggregates in addition to trimers. Fortuitously, the HMW SOSIP aggregates were noncovalently associated and were rapidly and quantitatively converted to trimers in the presence of.1% Tween 2. In contrast, JR-FL SOSIP gp14 16 and other recombinant HIV-1 gp14 proteins 1,13,14,37 form HMW aggregates that include disulfide-linked species, which reflect aberrant linkages between the 2 cysteines within gp12 and gp41. Such aggregates are dissociated only under reducing and denaturing conditions, and can be challenging to separate from the trimers. In addition, Tween 2 dissociated the HMW aggregates present in purified SOSIP gp14 derived from the subtype B isolate HIV Therefore, surfactant treatment may have utility in iso-

9 SOSIP gp14 ENVELOPE TRIMERS 825 A A2M gp14 trimers B A2M gp14 trimers FIG. 6. Effect of Tween 2 on KNH1144 SOSIP gp14 binding to DEAE. Purified KNH1144 SOSIP gp14 trimer was spiked with 2 -macroglobulin (A2M) and then either left untreated or treated with Tween 2. Samples were then applied over a DEAE HiTrap FF 1 ml column. Load (Lane 2), flow-through (Lanes 3 and 4), wash (Lane 5), and eluate (Lane 6) fractions were collected and analyzed by BN PAGE, followed by Coomassie G-25 stain. Lane 1 contains molecular weight markers. (A) Untreated sample. (B) Sample treated with.5% Tween 2. lating highly purified forms of SOSIP gp14 trimers derived from different strains and genetic subtypes of HIV-1. A concentration of.1% Tween 2 represented a threshold for efficient conversion of SOSIP HMW aggregates to trimers. This and higher amounts of Tween 2 had no effect on oligomerization of 2 -macroglobulin, as expected given the mild nature of this surfactant. Indeed, Tween 2 and Tween 8 commonly are used to stabilize protein formulations in order to reduce the formation of aggregates However, these surfactants are much less effective in dissociating preexisting protein aggregates. This observation highlights both the novelty of our finding and the unusually labile nature of the SOSIP HMW aggregates. The purification scheme for KNH1144 SOSIP gp14 incorporates a number of improvements over the process previously described for JR-FL SOSIP gp14. First, the lectin column was optimized by adding a static incubation in elution buffer. This incubation reduced trimer dissociation and improved trimer yield. Galanthus nivalis lectin binds 1 3 and 1 6 mannose linkages, which are internal and not terminal linkages. 43 We reasoned that SOSIP trimers are likely to bind multivalently to the lectin column, and the effective off-rate would be slower

10 826 IYER ET AL. FIG. 7. Negative stain electron micrograph gallery of KNH1144 SOSIP gp14. Bar 5 nm. than those for smaller or monovalent ligands. The extended incubation may permit a greater number of trimers to fully dissociate from the column in the absence of shear forces and concentration gradients that would be generated by buffer flow. Second, an anion-exchange column was introduced. DEAE chromatography effectively resolved SOSIP trimers from dimers that were not fully removed by SEC. Surprisingly, retention of SOSIP trimers on DEAE was profoundly altered by Tween 2. Trimers bound DEAE in the absence but not the presence of.5% Tween 2, whereas retention of contaminants was unaffected by Tween 2. The unique behavior of SOSIP gp14 over DEAE can be exploited for purification purposes. As a nonionic surfactant, Tween 2 is likely to exert an indirect effect on the charge properties of KNH1144 SOSIP gp14, and this issue is being addressed in ongoing studies. Purified KNH1144 SOSIP gp14 demonstrated favorable antigenic properties, consistent with our previous findings for unpurified material. 2 Tween 2 had no obvious effect on the

11 SOSIP gp14 ENVELOPE TRIMERS 827 antigenicity of a preparation containing predominantly pure trimers ( 15% HMW aggregates pretreatment). However, antigenicity is an imperfect predictor of immunogenicity, and a study in rabbits has been initiated to compare the immunogenicity of KNH1144 SOSIP gp14 in the presence and absence of Tween 2. Treatment of a preparation containing 8% HMW aggregates increased exposure of the CD4-binding site, suggesting that aggregates form in a manner that occludes this region of Env. Other epitopes were unaffected, indicating that dissociation of aggregates does not entail a global change in Env conformation. Collectively, the BN PAGE, SDS PAGE, and antigenicity data are consistent with a view that the HMW aggregates represent two or more native trimers that are associated via weak hydrophobic and/or other noncovalent interactions. A distinguishing feature of KNH1144 SOSIP gp14 is its compact nature. Other recombinant HIV-1 gp14 proteins appear in electron micrographs as either elongated, loosely associated subunits or a mix of loosely and tightly associated subunits. 14,44 46 In comparison, KNH1144 SOSIP gp14 more closely resembles native Env in its dimensions and configuration. We are performing immunoelectron microscopy analyses of this protein in complex with a panel of mabs to delineate the spatial relationships between known neutralization epitopes on the trimers. We will also investigate, in comparative studies, whether cleaved and uncleaved forms of KNH1144 gp14 differ in their appearance in electron microscopy. KNH1144 SOSIP gp14 and the methods described herein provide the first reliable source of homogeneous, proteolytically mature HIV-1 envelope trimers. The trimers possessed favorable antigenic and biophysical properties, and this protein may have value in further elucidating structure function relationships within the HIV-1 envelope. 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