Characterization of a trimeric MPER containing HIV-1 gp41 antigen

Virology 390 (2009) 221–227

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Virology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y v i r o

Characterization of a trimeric MPER containing HIV-1 gp41 antigen Andreas Hinz a, Guy Schoehn a,b, Heribert Quendler c, David Lutje Hulsik a, Gabi Stiegler c, Hermann Katinger c, Michael S. Seaman e, David Montefiori d, Winfried Weissenhorn a,⁎ a

Unit for Virus Host Cell Interaction, UMI 3265 UJF-EMBL-CNRS, 6 rue Jules Horowitz, 38042 Grenoble cedex 9, France Institut de Biologie Structurale Jean-Pierre Ebel, UMR 5075 CEA-CNRS-UJF, 41 rue Jules Horowitz, 38027 Grenoble cedex 1, France Institute of Applied Microbiology, Department of Biotechnology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria d Laboratory for AIDS Research and Development, Department of Surgery, Duke University Medical Center, 2812 Erwin Road, Durham, NC 27705, USA e Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA b c

a r t i c l e

i n f o

Article history: Received 3 February 2009 Returned to author for revision 17 March 2009 Accepted 15 May 2009 Available online 18 June 2009 Keywords: HIV-1 gp41 mAb 2F5 mAb 4E10 Membrane fusion

a b s t r a c t The membrane-proximal external region (MPER) of gp41 is considered as a prime target for the induction of neutralizing antibodies, since it contains the epitopes for three broadly neutralizing antibodies (2F5, 4E10 and Z13). Here we present a novel gp41 construct (HA-gp41) comprising gp41 HR2 and MPER fused to two triple-stranded coiled-coil domains at both ends. HA-gp41 is trimeric, has a high helical content in solution and forms rod-like structures as revealed by negative staining electron microscopy. Immunization of rabbits with HA-gp41 induced antibodies directed against MPER, which failed to exert significant neutralization capacity against envelopes from primary isolates. Thus trimerisation of MPER regions does not suffice to induce a potent neutralizing antibody response specific for conserved regions within gp41. © 2009 Elsevier Inc. All rights reserved.

Introduction A major challenge in HIV-1 vaccine research remains to identify an immunogen that has the capacity to induce high titers of broadly neutralizing antibodies. Highlighting the problem, to date only a very limited number of envelope glycoprotein (Env) specific antibodies have been identified that are broadly cross-reactive (Burton et al., 2004). Monoclonal antibody (mAb) b12 recognizes the conserved CD4 binding site on gp120 (Burton et al., 1994), mAb 2G12 binds carbohydrates on gp120 (Trkola et al., 1996), and mAbs 2F5, 4E10 and Z13 are directed against highly conserved adjacent epitopes in the membrane-proximal external region (MPER) of the Env glycoprotein subunit gp41 (Muster et al., 1993; Nelson et al., 2007; Stiegler et al., 2001; Zwick et al., 2001). Although a variety of gp41 antigens have been studied as immunogens (Lenz et al., 2005; Mantis et al., 2001; Qiao et al., 2005) and much interest and resources have been invested to develop immunogens that are capable of inducing 2F5- and/or 4E10-like immune responses (Karlsson Hedestam et al., 2008; Zwick, 2005), no such activities have yet been reported (Qiao et al., 2005). In contrast, phage display technologies have been used successfully to

⁎ Corresponding author. Unit for Virus Host Cell Interaction, UMI 3265 UJF-EMBLCNRS, 6 rue Jules Horowitz, 38042 Grenoble cedex 9, France. Fax: +33 476 209400. E-mail address: [email protected] (W. Weissenhorn). 0042-6822/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2009.05.015

isolate gp41 specific antibodies with limited neutralization activity (Gustchina et al., 2007; Louis et al., 2005; Luftig et al., 2006; Nelson et al., 2007), and patient sera contain antibodies overlapping with and potentially masking gp41 MPER (Alam et al., 2008). Monoclonal antibody (mAb) 2F5 recognizes a linear sequence that adopts an extended conformation containing a short beta turn (Ofek et al., 2004) while the adjacent epitope of mAb 4E10 forms a helical structure in complex with 4E10 (Cardoso et al., 2005) and in solution (Schibli et al. 2001; Sun et al., 2008). Since linear peptides comprising both epitopes are immunogenic but do not induce neutralizing antibodies several strategies to constrain the recognized antigen conformation have been developed with little success in generating MPER-specific neutralizing antibody responses (Barbato et al., 2003; Brunel et al., 2006; Cardoso et al., 2007; Law et al., 2007; McGaughey et al., 2003). Both mAbs 2F5 and 4E10 contain long CDR3 regions, which were shown to be important for neutralization in case of 2F5 (Zwick et al., 2004). Furthermore, both mAbs have been suggested to interact with lipids and may thus recognize self-antigens (Alam et al., 2007; Brown et al., 2007; Haynes et al., 2005a; Sanchez-Martinez et al., 2006a; Sanchez-Martinez et al., 2006b); this notion was further reinforced by the potential embedding of the 4E10 epitope in a lipid bilayer (Sun et al., 2008). However, some controversy about this mode of action prevails (Scherer et al., 2007) consistent with failures to induce neutralizing antibody responses by proteoliposomes containing membrane anchored gp41 including MPER and helical region 2

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(HR2) in mice (Lenz et al., 2005), rabbits and macaques (Wagner et al., 2007; Quendler et al., unpublished results). Since gp41 can at least adopt three conformations, a native one within Env trimers, a postulated fusion-intermediate (pre-hairpin) and a post-fusion conformation (Bures et al., 2000; Harrison, 2005; Lamb and Jardetzky, 2007; Weissenhorn et al. 2007), the question remains what kind of Env antigen mAbs 2F5 and 4E10 recognize; native Env versus a fusion-intermediate conformation of gp41 (de Rosny et al., 2004; Finnegan et al., 2002; Zwick et al., 2001). Notably Frey and colleagues have reported that both mAbs do not bind native soluble Env trimers but interact with a potential fusionintermediate conformation (Frey et al., 2008) consistent with proposed epitope exposure and MPER recognition by both mAbs during the membrane fusion process (Dimitrov et al., 2007). Here we present a novel gp41 antigen, HA-gp41, that contains the MPER and HR2 regions flanked by two trimerization domains, an Nterminal fragment of influenza virus hemagglutinin subunit 2 (HA2) (Mantis et al., 2001), and a C-terminal trimeric GCN4 variant (Harbury et al., 1993; Weissenhorn et al., 1997a). We show that HA-gp41 forms elongated, rod-shaped and mostly helical trimers in solution that bind both mAbs 2F5 and 4E10 with nanomolar affinities. Immunization of rabbits with HA-gp41 induced high titers of HA-gp41 specific antibodies that recognized MPER peptides but failed to exert significant neutralization activity. Results Antigen characterization Chimeric HA-gp41, composed of an N-terminal triple-stranded coiled-coil region derived from the influenza virus hemagglutinin subunit 2 (HA2), gp41 HR2 and MPER, and a trimeric GCN4 coiled-coil region (Fig. 1A) eluted from a preparative Sephacryl 200 gel filtration column at a molecular weight of ∼ 100 kDa, between the elution positions of two marker proteins of 160 kDa and 66 kDa (Fig. 1B). Consistent with the presence of two trimerization domains, chemical cross-linking of HA-gp41 revealed the appearance of two new bands on an SDS-PAGE upon incubation with increasing amounts of EGS, migrating at ∼40 kDa and ∼60 kDa, respectively (Fig. 1C). Circular dichroism demonstrated an overall α-helical content (Fig. 2A), which was estimated to be ∼ 80% based on the analysis with K2D (www.cryst. bbk.ac.uk), and a temperature dependent unfolding at ∼55 °C (Fig. 2B). Further analysis by negative staining electron microscopy showed rodlike structures containing short flexible extensions (Fig. 2C). Twodimensional averaging revealed an approximate length of 115 Å+/ −10 Å (Fig. 2D) and a thickness of 45+/−5 Å, with one end showing the trimer symmetry of the particle in the top view (Fig. 2E). The side view of the averaged particle indicated further that one end of the particle is well defined, while the other end appeared to be smeared out (Fig. 2D). Thus a flexibly linked region of one end might have been lost due to the averaging procedure. Since the triple-stranded coiled-coil structures of HA2 and GCN4 used in the fusion protein account for ∼100 Å, gp41 contributes at least 25 Å, consistent with a larger gp41 contribution of ∼45 Å present in the gp41-inter construct (Frey et al., 2008). The binding constants of Fabs derived from mAbs 2F5 and 4E10 to HA-gp41 were determined by surface plasmon resonance (SPR) (Figs. 3A and B). The association rates (ka) were 2.96 × 105 M–1s–1 for the 2F5 Fab and 1.24 × 105 M–1s–1 for the 4E10 Fab, and the dissociation rate (kd) was 6× faster for 4E10 (1.07 × 10− 4 s–1) than for 2F5 (6.62 × 10− 4 s–1). Accordingly the affinity (KD) of 2F5 was elevated by a factor of 10 (0.36 nM) when compared to 4E10 (5.34 nM) (Table 1). Immunogenicity of HA-gp41 Five rabbits were immunized four times with HA-gp41 and Freund's complete adjuvant at days 0, 21, 42 and 63, followed by a

Fig. 1. HA-gp41 forms trimers. (A) Schematic representation of gp41 and HA-gp41. (B) Size exclusion chromatography of HA-gp41 shows that HA-gp41 elutes as a larger complex ∼100 kDa. (C) Chemical cross-linking reveals trimer formation; two new bands migrating at ∼ 40 and 60 kDa appear upon incubation with increasing amounts of EGS (0, 1, 2, 5 and 10 mM). (D) Native gel electrophoresis of HA-gp41. Lane 1 HA-gp41; HA-gp41 incubated with 10% (lane 2), 25% (lane 3) and 50% (lane 4) Freund's adjuvant.

final bleed at day 77. Native gel electrophoresis of HA-gp41 alone revealed a single band (Fig. 1D); this pattern did not change upon incubation with increasing concentrations of Freund's adjuvant suggesting that the trimeric structure of HA-gp41 was still intact in the presence of Freund's adjuvant. Reciprocal titers of the sera against the antigen HA-gp41 were determined by ELISA and varied between 1:830,677 and 1:147,662 (Table 2). HA-gp41 specific IgG antibodies were purified from sera by affinity chromatography and both serum and IgGs were employed to detect neutralization activity. All sera and purified IgGs were found to be negative in a TZM-b1 assay, using Tier 1 and Tier 2 viruses. In contrast, when sera and affinity purified IgGs were tested in a PBMC assay, significant neutralization of a few PBMCgrown B-clade primary isolates such as Tier 2 viruses PVO.4, 6535.3 and QH0692.42 and Tier 1 SF162.LS was observed. However, since the samples turned out to be contaminated with endotoxin the significance of neutralization is highly questionable (D. Montefiori, unpublished results) and most likely due to the presence of endotoxins in the samples tested. In order to better evaluate potential neutralization, a third assay format was used that employs the low CCR5-expressing cell line 5.25.EGFP.Luc.M7 that was suggested to be more sensitive for certain antibodies including MPER-specific neutralizing antibodies (Choudhry et al., 2006). However, no significant neutralization of the sera against three clade B Tier 1 viruses could be observed. Thus the conclusion from the current study is that HA-gp41 did not induce neutralizing activity. The purified IgGs were further tested for their western blot reactivity against the T-20 peptide containing a part of HR2 and the 2F5 epitope as well as an MPER peptide containing only the 2F5 and the 4E10 epitopes (Fig. 4A). The western blot revealed that both peptides are recognized

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Fig. 3. Both mAbs 2F5 and 4E10 bind HA-gp41 with high affinity. HA-gp41 was immobilized on a Ni-NTA chip and sensorgrams are shown for (A) the 2F5 Fab (the concentrations of the analyte were 100, 50, and 10 nM), and (B) the 4E10 Fab (concentrations of the analyte were 100, 50, and 10 nM). Binding kinetics were evaluated with a 1:1 Langmuir model and the fits are shown. The rate and binding constants are summarized in Table 1.

by mAb 2F5 as well as serial dilutions of the IgGs obtained from the rabbit sera; only rabbit serum #2 showed rather weak reactivity with the peptides (Fig. 4B). This indicated that MPER was immunogenic in the context of the trimer construct. To further evaluate the presence of antibodies that cross-react with the 2F5 and 4E10 epitopes we performed a competition ELISA. HA-gp41 was directly coated on the plates and detection of biotinylated 2F5 or 4E10 was competed with increasing amounts of serum IgG from rabbit # 4 and # 5. This revealed that both sera contained antibodies which interfered with 2F5 and 4E10 binding (Fig. 4C) thus indicating the presence of antibodies that at least overlap with either the 2F5 or the 4E10 epitopes. Discussion Broadly neutralizing antibodies directed against the conserved gp41 MPER are thought to be a key element of a potential HIV-1 Fig. 2. Circular dichroism and negative staining electron microscopy reveal rod-shaped helical structures. (A) Circular dichroism analysis of HA-gp41 and temperature dependent unfolding (B) of HA-gp41 recorded at 220 nm. (C) Electron micrograph of HA-gp41 negatively stained by using sodium silicotungstate at pH 9.0. The arrows indicate the rod-like structure of the protein and the asterisks an extra part that seems to be flexibly linked to the rod. (D) Selected side views of the rod-like structure. The average structure resulting from 180 selected side views is shown on the right. (E) Selected top views of the rod-like structure. The average structure resulting from 40 top views is shown on the right showing a triangular shape.

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Table 1 Fabs of the neutralizing antibodies 2F5 and 4E10 interact with HA-gp41 with high affinity as determined by SPR.

2F5 Fab 4E10 Fab

ka (M− 1s− 1)

kd (s− 1)

KD (nM)

(2.96 ± 0.42) E5 (1.24 ± 0.74) E5

(1.07 ± 0.02) E− 4 (6.62 ± 3.66) E− 4

0.36 ± 0.05 5.34 ± 3.19

vaccine (Burton et al., 2004; Karlsson Hedestam et al., 2008; Walker and Burton, 2008) since three broadly neutralizing antibodies 2F5, 4E10 and Z13 recognize linear epitopes within MPER (Muster et al., 1993; Nelson et al., 2007; Parker et al., 2001; Stiegler et al., 2001; Zwick et al., 2001). Although the main structural principles for epitope recognition have been elucidated (Cardoso et al., 2007; Cardoso et al., 2005; Ofek et al., 2004), the nature of the antigen, which can elicit such antibody specificities, is still elusive. Multiple approaches to generate antigens that present both epitopes as peptides, constrained peptides or within protein scaffolds have failed to produce neutralizing antibodies in animal models (reviewed in Montero et al. (2008)). A number of studies suggested that 2F5 and 4E10 react poorly if at all with native envelope trimers (Binley et al., 2003; Cavacini et al., 2002; Zwick et al., 2001) and might only interact with an intermediate conformation of gp41 that is induced by receptor binding (de Rosny et al., 2004; Frey et al., 2008). We thus set out to define a trimeric fusion-intermediate conformation of gp41 that could represent the native target for mAbs 2F5 and 4E10. This intermediate conformation, HA-gp41, contains MPER and HR2 fused to two triple-stranded coiledcoil regions at both ends and follows a similar principle as applied in case of gp41-inter, published by Frey et al., which contains MPER, HR2 and the cystein-loop region constrained between two triple-stranded coiled-coil regions (Frey et al., 2008). HA-gp41 interacts with Fabs derived from mAbs 2F5 and 4E10 with high nanomolar affinities similar to those reported for gp41-inter (Frey et al., 2008). Both HA-gp41 and gp41-inter have similar helical contents indicating that at least parts of HR2 and/or MPER must adopt α-helical secondary structures. HA-gp41 and gp41-inter fold into elongated rod-like structures with a similar length contribution of the gp41 part; ∼25 Å in case of HA-gp41 versus 45 Å in case of gp41-inter. The difference in length is probably due to the fact that gp41-inter contains the Cys-loop region and thus ∼100 gp41specific amino acids (Frey et al., 2008) while HA-gp41 contains only 60 gp41-specifc amino acids (Fig. 1A). However albeit their slight differences in design, both structures, gp41-inter (Frey et al., 2008) and HA-gp41 might resemble the proposed fusion-intermediate conformation of gp41 (Weissenhorn et al., 1997b). It is also notable that HA-gp41 does not contain the immunodominant region (cluster I) preceding the Cys-loop region, which induces most of the gp41-specific antibodies in patient sera (Gnann et al. 1987; Xu et al., 1991). HA-gp41 was shown to be immunogenic and produced gp41specific antibodies, which recognized epitopes overlapping with 2F5 and 4E10 epitopes as judged by western blot analysis and competition ELISA. However, the sera and purified HA-gp41-specific antibodies showed no significant neutralization activity against Tier 1 and Tier 2 clade B isolates in the TZM-b1 reporter cell line assay (Montefiori, 2005). We next determined significant neutralization activity of the sera and purified HA-gp41-specific antibodies using a PBMC based assay format. We reasoned that detection of neutralization might be due to the differences between both assays (Brown et al., 2008; Polonis et al., 2008); the PBMC assay probably reflects a slower kinetic of infection based on lower co-receptor surface concentrations Table 2 Titers of HA-gp41 IgG were determined by ELISA.

HA-gp41

#1

#2

#3

#4

#5

nt

209,036

147,662

404,868

830,677

The titer of rabbit serum #1 could not be determined.

(Choudhry et al., 2006). This might be more favourable to detect neutralizing MPER-specific antibodies which act during the conformational changes leading to membrane fusion. However, later on during the course of the study all samples (sera and purified HA-gp41specific antibodies) were tested positive for the presence of endotoxins. Thus, because of the proven effect of endotoxins on the outcome of PBMC based neutralization (Verani et al., 1997), we consider the current neutralization results as negative unless proven differently with endotoxin-free sera. The employment of the low CCR5-expressing cell line 5.25.EGFP.Luc.M7 in a luciferase assay (Montefiori, 2004) confirmed further that no significant neutralization activity could be detected in the sera induced by HA-gp41 immunization. The failure to generate neutralizing antibodies targeting the MPER employing HA-gp41 might be due to the lack of lipid determinants that have been suggested to be important for 2F5 and 4E10 neutralization via their CDR3 regions, which are only partially employed for epitope binding (Cardoso et al., 2005; Ofek et al., 2004). Due to their close binding mode to the membrane, the CDRs have been implicated in membrane interaction (Grundner et al., 2002; Haynes et al., 2005a; Ofek et al., 2004; Sanchez-Martinez et al., 2006a). Based on this hypothesis the problem to induce 2F5 and 4E10like antibody responses was attributed to the difficulty of inducing autoreactive antibodies (Haynes et al., 2005b). Although the NMR structure of the isolated peptide epitope indicated the potential of

Fig. 4. Epitope recognition by the rabbit sera obtained upon HA-gp41 immunization. (A) Sequences of peptides T-20 and MPER used in the western blot analysis. Hydrophobic (asterisk) and polar (circle) residues contacting either mAb 2F5 (black) or 4E10 (blue) are indicated. (B) Western blot analysis of affinity purified IgG. Staining with mAb 2F5 reveals two bands corresponding to the T-20 and MPER peptides. Purified IgGs were employed in 1:4 dilutions per lane. (C) Rabbit sera compete with 2F5 and 4E10 binding to HA-gp41 as detected by ELISA. The relative absorbance of labeled 2F5 and 4E10 are plotted against the serum dilution, indicating recovery of the signal at high serum dilutions (squares 4E10 and circles 2F5 reactivities for two rabbit sera).

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membrane interaction (Sun et al., 2008), presentation of MPER in a membrane environment previously failed to produce neutralizing antibodies in mice (Lenz et al., 2005), guinea pigs (Kim et al., 2007), rabbits and macaques (Quendler, unpublished results). These results thus argue against the potential positive effect of a lipidic environment for the elucidation of 2F5 and 4E10-like antibodies. Thus both mAbs might not recognize self-antigens (Scherer et al., 2007). HA-gp41 immunization produced antibodies specific for MPER, which is consistent with the presence of such antibodies in patient sera (Alam et al., 2008; Penn-Nicholson et al., 2008). These antibodies either contribute to neutralization (Binley et al., 2008; Shen et al., 2009; Sather et al., 2009) or are considered to be mostly nonneutralizing and might mask MPER (Alam et al., 2008). However, since we do not observe significant neutralization, the epitopes recognized by the sera might only overlap with that of 2F5 and 4E10 or even m44 that was suggested to target HR2 with no requirements for lipids (Zhang et al., 2008) without displaying the same fine specificity. Furthermore HA-gp41 might induce preferentially antibodies masking MPER, which could partially explain the failure to obtain significant neutralizing activity. In summary, our results suggest that the trimeric conformation represented by HA-gp41 was not beneficial to obtain neutralization activity indicating that either MPER was not presented in the correct trimer context or lacked important additional factors such as a membrane environment. Finally, it is possible that Freund's adjuvant might impede efficient epitope exposure since it might interfere with the hydrophobic nature of the epitopes targeted although the trimer structure was most likely not affected by incubation with Freund's adjuvant. It is thus necessary to determine whether more efficient immunization strategies can be employed using HA-gp41 alone or in combination with a specific adjuvant or prime-boost regimen to broaden the generation of neutralizing antibodies by directing the immune response specifically towards the MPER.

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150 mM NaCl, 50 μM EDTA, 0.05% Surfactant P20) at a flow rate of 30 μl/min. HA-gp41 was immobilized on channel 2 of a Ni-NTA chip at a concentration of 10 μg/ml for 2 min, channel 1 was used as control. Fabs of 2F5 and 4E10 were passed over the chip surface at concentrations of 100, 50, and 10 nM for 3 min, dissociation was recorded for 15 min. Afterwards, the chip was regenerated with 0.35 M EDTA in buffer C for 1 min at a flow rate of 50 μl/min and 100 mM NaOH each, respectively, followed by recharging with 500 μM NiCl2 in buffer C for 2 min at 30 μl/min. Binding kinetics were evaluated using the BiaEvaluation software package (Biacore). Circular dichroism Circular dichroism (CD) measurements were carried out on a Jasco J-810 spectro-polarimeter (JascoLtd, Tokyo, Japan) equipped with a temperature control. Spectra were recorded in buffer D (10 mM sodium phosphate pH 7.4, 100 mM NaF) at a protein concentration of 8 μM. Thermal unfolding of HA-gp41 was monitored by recording the mean residue ellipticity at 220 nm as a function of the temperature. The resulting spectra were analyzed with the DiChroWeb software package available at www.cryst.bbk.ac.uk. Electron microscopy Samples were applied to the clean side of carbon on mica (carbon/ mica interface) and negatively stained with 1% sodium silicotungstate pH 9.0. A grid was placed on top of the carbon film, and subsequently air-dried. Micrographs were taken under low-dose conditions with a Jeol 1200-EX II microscope at 100 kV and a calibrated magnification of 39,750 times (based on the helical pitch of Tobacco Mosaic Virus). Selected negatives were digitized on a Zeiss scanner (Photoscan TD) with a pixel size of 7 μm (1.75 Å at the sample scale). Image processing and model representation

Material and methods Protein expression and purification HA-gp41 is composed of the influenza virus hemagglutinin subunit 2 region (amino acids 372–417) followed by a linker composed of amino acids Gly-Ser-Thr, the clade B HR2 and MPER regions (residues 629–683, HXB2c numbering) and a trimeric GCN4 isoleucine zipper motif. The cDNA was cloned into pETM-11 (EMBL, Heidelberg) using standard PCR techniques. HA-gp41 was expressed in Escherichia coli Rosetta 2 (DE3). Cells were grown to an OD600 of 0.8 and induced with 0.5 mM IPTG at 37 °C. After 4 h cells were harvested by centrifugation and lysed in buffer A (20 mM Tris pH 8, 100 mM NaCl). HA-gp41 from the soluble fraction was purified on a Ni2+ affinity column in buffer A and eluted with 250 mM imidazole. A final purification step included gel filtration chromatography on a Sephacryl 200 HiPrep column (GE Healthcare) in PBS. Inclusion bodies of HA-gp41 were purified using standard protocols. Briefly, inclusion bodies were washed three times with buffer A containing 0.5% Triton X-100, followed by a detergent-free wash in buffer A, and solubilized in buffer B (50 mM acetate pH 4.5, 8 M guanidine). Inclusion bodies were stored at − 80 °C at concentrations between 20 and 30 mg/ml. HA-gp41 was refolded at concentrations of 2 to 3 μM at 4 °C by rapid dilution into PBS buffer, followed by overnight incubation. The refolded protein was further purified as described above for the soluble fraction. The total yield of soluble and refolded HA-gp41 is about 2 mg per liter E. coli culture. Surface plasmon resonance All experiments were performed in duplicate with a Biacore X instrument (Biacore. Inc.) at 25 °C in buffer C (10 mM HEPES pH 7.4,

2D averaging of particles was performed as follows: A total of 180 side views and 40 top views were selected using the X3d program (Conway and Steven, 1999) and averaged together after crosscorrelation using SPIDER (Frank et al., 1996). The average size of the rods was determined to be 115 Å +/− 10 Å × 45 +/− 5 Å. The significant variation of the rod length is most likely due to the averaging procedure and the overall shape of the rods. The images show some ends of the rod out of the plane. The averaging procedure thus produced an image where one end of the rod is well defined while the other one appears smeared out indicating that parts of the end were averaged out. Chemical cross-linking and native gel electrophoresis Samples from gel filtration were treated with 1, 2, 5 and 10 mM EGS (ethylen-glycol bis(succinimidyl-succinate)) for 15 min at room temperature. The reaction was quenched with 50 mM Tris pH 8.0 and samples were analyzed by SDS-PAGE. 20 μg of HA-gp41 was incubated with Freund's complete adjuvants at 10% (v/v), 25% (v/v) and 50% (v/v) for 30 min and separated on a 12% native polyacrylamide gel. Bands were visualized by Coomassie brilliant blue staining. Immunization Five New Zealand rabbits of mixed sexes (3 females and 2 males) were immunized 4 times with 20−30 μg antigen at days 0, 21, 42 and 63. Briefly, equal volumes of HA-gp41 and Freund's complete adjuvants (280 μL each) were mixed and the resulting suspension was injected subcutaneously and intramuscularly (2 times each per rabbit). Pre-immunization samples of the blood serum (1 ml) were

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collected one day before the first immunization from the ear vein 12 h after the removal of food. The terminal bleeding was done on day 77. Sera were stored at −20 °C until further use.

(PSB) for access to the common platforms and Dr. B. Ferko for performing the rabbit immunization.

Purification of gp41-specific antibodies

References

5 mg HA-gp41 was coupled to 1 g CNBr-activated Sepharose (GE Healthcare) following manufacturer's instructions. Rabbit sera were diluted one time with PBS and passed over the HA-gp41 affinity column. HA-gp41 specific antibodies were eluted with 50 mM glycine pH 2.5 into a solution containing 1 M Tris HCl pH 9. Antibodies were dialyzed against PBS, sterile filtered and stored at 4 °C until further use. Neutralization assay Serum and purified IgG samples were tested against five Tier 1 viruses (clade B: SF162.LS, BAL.26, SS1196.1, DJ263; clade C MW965.26) and eight Tier 2 viruses (clade B: QHO692.42, SC422661.8, PVO.4, 6535.3, AC10.029, RHPA4259.7, TRO.11 and TRO4156.18) using the TZM-b1-assay (Montefiori, 2009). A second assay included the determination of virus neutralization as a function of reductions in p24 Gag antigen synthesis by using peripheral blood mononuclear cell (PBMC)-grown viruses (SF162.LS, 6535.3, QH0692.42 and PVO.4) and phytohemagglutinin (PHA)-stimulated PBMC as targets for infection as described (Bures et al., 2000). Serum endotoxin levels were determined using an ELISA assay and ranged from 500 to N50,000 EU/ml. A third neutralization assay was performed to establish potential neutralization by using 3 clade B viruses (SF162, Bal, WEAU3-3) in the low CCR5-expressing cell line 5.25.EGFP.Luc.M7 (Montefiori, 2004). Western blot analysis 5 μg of T-20 and MPER peptides, respectively, were separated on a 16% Tricine SDS-PAGE and transferred onto a nitrocellulose membrane for western blot analysis. 10 μg of affinity purified rabbit IgG and 10 μg of 2F5 were used for incubation and detection of peptides as shown in the first two lanes. The affinity purified rabbit IgGs were then stepwise diluted 1:4 per lane, thus using ∼0.1 μg IgGs in lane 6. The peptides were visualized with AP-conjugated anti-human (mAb 2F5) and antirabbit (serum, gp41-specific antibodies) antibodies. Competition ELISA A 96-well plate (immunosorb, Nunc) was coated with 50 μl of HAgp41 at a concentration of 2 μg/ml in PBS overnight at 4 °C. Excess HAgp41 was washed three times with PBS-Tween (0.02%) and the wells were blocked with 100 μl of 4% BSA in PBS-Tween for 1 h at room temperature. After a pre-incubation of 1:1 (v/v) mixtures of 50 μl rabbit serum (at serial dilutions of 1, 2, 4, 8, 16 and 32) and 50 μl biotinylated antibodies 2F5 or 4E10 at concentrations of 5 ng/ml for 30 min, the wells were washed three times with PBS-Tween and then incubated with the serum/antibody mixtures for 30 min at room temperature. The wells were washed three times and incubated with 50 μl of streptavidin–HRP (1:1000) for 1 h at room temperature. Excess streptavidin was washed five times and the wells were incubated with 50 μl POD solution for 10 min. The reaction was stopped by adding 50 μl 0.2 M H2SO4 and the absorption was recorded at 490 nm. Acknowledgments This work was conducted as part of the Collaboration for AIDS Vaccine Discovery with support from the Bill and Melinda Gates Foundation (W.W.). We thank the Partnership for Structural Biology

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