Glycobiology Advance Access published November 7, 2014 1 

Crystal structure of the HIV neutralizing antibody 2G12 in complex with a bacterial oligosaccharide analog of mammalian oligomannose

Robyn L. Stanfield1, Cristina De Castro2, Alberto M. Marzaioli2, Ian A. Wilson1,# and Ralph Pantophlet3,# 1

© The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e‐ mail: [email protected] 

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Department of Integrative Structural and Computational Biology, Scripps CHAVI-ID, and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 2 Department of Chemical Sciences, University of Napoli Federico II, Complesso Universitario Monte Sant'Angelo, 80126 Napoli, Italy 3 Faculty of Health Sciences and Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada # Correspondence Address to: Ian A. Wilson ([email protected]) or Ralph Pantophlet ([email protected])



ABSTRACT Human Immunodeficiency Virus-1 (HIV-1) is a major public health threat that continues to infect millions of people worldwide each year. A prophylactic vaccine remains the most cost-effective way of globally reducing and eliminating the spread of the virus. The HIV envelope spike, which is the target of many vaccine design efforts, is densely mantled with carbohydrate and several potent broadly neutralizing antibodies to HIV-1

However, immunizing with recombinant forms of the envelope glycoprotein does not typically elicit anti-carbohydrate antibodies. Thus, studies of alternative antigens that may serve as a starting point for carbohydrate-based immunogens are of interest. Here, we present the crystal structure of one such anti-carbohydrate HIV neutralizing antibody (2G12) in complex with the carbohydrate backbone of the lipooligosaccharide from Rhizobium radiobacter strain Rv3, which exhibits a chemical structure that naturally mimics the core high-mannose carbohydrate epitope of 2G12 on HIV-1 gp120. The structure described here provides molecular evidence of the structural homology between the Rv3 oligosaccharide and highly abundant carbohydrates on the surface of HIV-1 and raises the potential for the design of novel glycoconjugates that may find utility in efforts to develop immunogens for eliciting carbohydrate-specific neutralizing antibodies to HIV.

Key Words: 2G12, HIV vaccine, molecular mimicry, oligomannose, Rhizobium radiobacter

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recognize carbohydrate on the envelope spike as a major part of their epitope.



INTRODUCTION HIV-1 continues to be a serious public health threat with 34 million people currently infected; less than a quarter of those have access to anti-retroviral therapies (UNAIDS 2012). Although these anti-retroviral drugs are highly effective at controlling the virus, they are not capable of complete eradication, leaving dormant viral reservoirs that can become active when drugs are discontinued (Donahue, D.A. and Wainberg, M.A. 2013).

viral reservoirs in infected individuals, there remains a critical need for an effective HIV1 vaccine to protect against infection. 2G12 is one of the original four broadly neutralizing antibodies (bnAbs) to HIV-1 and is still the only Ab that exclusively recognizes carbohydrate on the surface of HIV-1 (Calarese, D.A., Lee, H.K., et al. 2005, Calarese, D.A., Scanlan, C.N., et al. 2003, Kunert, R., Ruker, F., et al. 1998, Murin, C.D., Julien, J.P., et al. 2014, Sanders, R.W., Venturi, M., et al. 2002, Scanlan, C.N., Pantophlet, R., et al. 2003, Stiegler, G., Armbruster, C., et al. 2002). 2G12 has reasonable breadth due to its recognition of a conserved epitope on gp120 that consists of oligomannose-type glycans at positions 295, 332, 339, and 392. Neighboring glycans at 386 and 448 may influence the recognition process but do not appear to directly bind to 2G12 (Murin, C.D., Julien, J.P., et al. 2014). These glycans are densely clustered on the gp120 surface and are derived from the host cell during protein synthesis. For many years, 2G12 was the only antiHIV-1 antibody known to interact with carbohydrate; indeed, it was long thought that the dense patch of glycans on gp120 and the underlying protein surface were essentially inert to immune recognition (Wyatt, R., Kwong, P.D., et al. 1998). However, recent

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Thus, despite significant advances in therapeutic approaches and attempts to purge



exciting developments, spurred largely by advances in technologies that enable highthroughput screening of memory B cell cultures, have revealed that many extremely potent and broadly neutralizing anti-HIV-1 antibodies can be isolated from infected individuals that recognize glycans as a major component of their epitope along with surrounding protein segments (for review see (Kong, L., Stanfield, R.L., et al. 2014)). These antibodies include the PGT121 family that recognizes one or more

(Julien, J.P., Cupo, A., et al. 2013, Julien, J.P., Sok, D., et al. 2013, Garces, F., Sok, D., et al. 2014), the PGT128 family that recognizes oligomannose-type glycans at positions 301 and 332 on gp120 (Pejchal, R., Doores, K.J., et al. 2011), the PGT135 family that recognizes oligomannose-type glycans at positions 332, 392, and 295 and 386 depending on the isolate (Kong, L., Lee, J.H., et al. 2013), PG9, PG16, PGT145 and related antibodies that recognize glyco-epitopes around positions 160 and either 156 or 173 in the gp120 V1/V2 region (McLellan, J.S., Pancera, M., et al. 2011, Pancera, M., McLellan, J.S., et al. 2010, Pancera, M., Shahzad-Ul-Hussan, S., et al. 2013, Pejchal, R., Walker, L.M., et al. 2010), the PGT151 family and antibody 35O22 that recognize a quaternary epitope at the gp120/gp41 interface, likely involving glycans at gp41 residues 611 and 637 (PGT151) (Blattner, C., Lee, J.H., et al. 2014) and glycans at gp120 residues 88, 230, and 241 and gp41 residue 625 (35O22) (Huang, J., Kang, B.H., et al. 2014), and antibody 8ANC195 that binds at the interface between gp41 and the gp120 CD4 binding site, contacting gp120 glycans at positions 234 and 276 (Scharf, L., Scheid, J.F., et al. 2014). Thus far, crystal and electron-microscopy (EM) structures of these glycan-dependent antibodies in complex with their HIV-derived antigens reveal

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oligomannose- and/or complex-type glycans at the base of the V3 variable region



at least two main areas of glycan-dependent recognition on gp120, centered on and around dense patches of glycans located at positions 332 and 160, as well as the two distinct gp120/gp41 interface epitopes recognized by the PGT151 family of antibodies and antibodies 35O22 and 8ANC195. Indeed, the recent crystal and EM structures of an HIV-1 gp140 trimer (Julien, J.P., Cupo, A., et al. 2013, Lyumkis, D., Julien, J.P., et al. 2013, Pancera, M., Zhou, T., et al. 2014) suggest that the majority of the trimer surface

involve carbohydrate components. 2G12 is unique in that it is domain-swapped via its variable heavy (VH) chain domains, resulting in a linear IgG with the two Fab regions tethered side-by-side (Calarese, D.A., Scanlan, C.N., et al. 2003). The antigen binding sites on the two closely spaced Fab regions are about 33 Å apart, and two non-conventional sites centered at the interface of the intertwined VH domains can also potentially interact with carbohydrate, thus yielding an inherently high-avidity binding site for clustered glycans such as found on the gp120 high-mannose patch around glycan 332. While 2G12 uses this unusual dimer-of-Fabs assembly to interact with multiple glycan chains, the more recently discovered anti-glycan antibodies such as PGT128, PGT135, PG9, PG16, PGT151, 35O22 and 8ANC195 are all ‘normal’ insofar that they are non-domain swapped IgG’s; these antibodies achieve high avidity by interacting with multiple glycans within their combining site and using long complementarity determining region (CDR) loops, especially CDR H3, to penetrate the glycan shield and reach the protein surface below, thereby contacting complex epitopes made up of both glycan and protein (Kong, L., Stanfield, R.L., et al. 2014).

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is covered by glycosylation, so that many if not most neutralizing epitopes are likely to



Understanding the structure and location of a broadly neutralizing epitope and translating that knowledge into an effective immunogen against that region have proven difficult; however, recent results with respiratory syncytial virus (Correia, B.E., Bates, J.T., et al. 2014, McLellan, J.S., Chen, M., et al. 2013) provide optimism that structurebased immunogen design can be successful. Carbohydrates, in the form of glycoconjugates, have of course proven effective as vaccines for various bacterial

A, C, Y and W-135, and Streptococcus pneumoniae (Astronomo, R.D. and Burton, D.R. 2010, Taylor, C.E., Cobb, B.A., et al. 2012), so that investigating carbohydrate-based conjugates for prevention of HIV-1 infection is a highly attractive area for investigation. It was recently discovered that Rhizobium radiobacter strain Rv3, a gram-negative bacterium previously classified as Agrobacterium tumefaciens (Farrand, S.K., Van Berkum, P.B., et al. 2003, Young, J.M., Kuykendall, L.D., et al. 2001), expresses a lipopolysaccharide on its surface with a distal segment that is chemically analogous to the D1 arm of oligomannose, which constitutes the core epitope of 2G12 (Clark, B.E., Auyeung, K., et al. 2012) (Fig. 1). Rhizobia are commonly regarded as associated with plants; some species, such as R. radiobacter, are phytopathogens that can cause crown gall disease in certain plants (reviewed in (Escobar, M.A. and Dandekar, A.M. 2003)). R. radiobacter is increasingly recognized also as an opportunistic pathogen in human nosocomial infections associated with the use of intravenous catheters (Amaya, R.A. and Edwards, M.S. 2003, Chen, C.Y., Hansen, K.S., et al. 2008, Edmond, M.B., Riddler, S.A., et al. 1993, Paphitou, N.I. and Rolston, K.V. 2003). The lipopolysaccharide of R. radiobacter Rv3 is naturally devoid of an O-antigenic portion

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pathogens, including Haemophilus influenza type b, Neisseria meningitidis serogroups



commonly found in the lipopolysaccharides of many other gram-negative bacteria, and is thus termed a lipooligosaccharide (LOS); thus, the Rv3 LOS consists solely of a lipid A moiety and a core oligosaccharide (OS) region (Fig. 1), which in turn contains inner and outer core segments (De Castro, C., Holst, O., et al. 2012). The D1-arm like portion of the Rv3 LOS differs from the D1 arm of oligomannose where the anomeric configuration of the first branching mannose (Man2 in Fig. 1) is α in Rv3 instead of β as

lipopolysaccharide-specific Kdo (3-deoxy-octulosonic acid) residue in Rv3 instead of to the chitobiose core in oligomannose (De Castro, C., Molinaro, A., et al. 2008). Immunization of mice with heat-killed Rv3 bacteria has been shown previously to result in serum antibodies with capacity to bind monomeric HIV-1 gp120 with modest affinity (Clark, B.E., Auyeung, K., et al. 2012); however, those sera failed to neutralize HIV-1 strains. In order to better understand the antigenic similarity between the OS backbone of the Rv3 LOS and mammalian oligomannoses and the role, if any, played by chemical differences between the Rv3 OS and oligomannose, we determined the crystal structure of Fab 2G12 in complex with the Rv3 OS to an effective resolution of 2.0 (Urzhumtseva, L., Klaholz, B.P., et al. 2013; Weiss, M. 2001). To the best of our knowledge, these data provide the most conclusive evidence so far for the unique resemblance between a bacterial OS and mammalian oligomannose.

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found in oligomannose. The connection of this Man2 unit also differs and is to the



RESULTS Oligosaccharide NMR structure. After chromatographic purification, the Rv3 core OS was isolated from the LOS in 16% yield. Its proton NMR spectra (Supplementary Fig. 1) displayed 5 main anomeric signals in the low field region (δ 5.4-4.9), a crowded carbinolic region ( 4.3-3.5), and two couples of methylene protons (δ 2.4-1.7) related to

protons and carbon chemical shifts (Supplementary Table 1) was afforded by integrating information from the homo- and heteronuclear 2D NMR spectra; their values did not diverge from those of the mannose residues reported for the full-length OS isolated after alkaline deacetylation (Clark, B.E., Auyeung, K., et al. 2012). The only main difference was attributed to Man2, as it was affected by the anomeric status of the nearby Kdo1, which could freely interconvert among its α or β anomers. Indeed H-1 of Man2 was found at δ 5.02 when linked at O-4 of the α isomer of Kdo1 or at δ 4.96 for the β anomer (labeled as Man2* in Supplementary Fig. 1). Analysis of the HSQC-TOCSY spectrum (Supplementary Fig. 1B) immediately allowed identification of the different mannose units in the OS. The two terminal mannose, Man5 and Man6, could be assigned as their carbon chemical shifts were below 75 ppm and are not shifted by linkage to any further sugar units. On the contrary, C-6 of Man2 was shifted at low field (δ 66.6) because of its linkage to Man6, while, Man3 and Man4 each displayed one low field carbon signal (δ 80.0 and δ 79.8, respectively) that were identified as C-2 in both cases with the aid of the other NMR spectra. Analysis of HMBC spectrum (Supplementary Fig. 1C) confirmed the sequence of the hexasaccharide that is expected after mild acid hydrolysis of the LPS (Fig. 1, highlighted structure).

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the α and β anomeric forms of the Kdo1 residue at the reducing end. Attribution of all



Overall crystal structure. The 2G12 Fab is dimeric, as seen previously (Calarese, D.A., Scanlan, C.N., et al. 2003), with the expected swap of its VH domains. Two Rv3 glycans bind to 2G12, one in each classic antibody combining site, contacting residues from CDRs H3, H1, and L3 (Fig. 2). All six sugars of the OS have strong electron density (Fig. 3A), with the B-values increasing as Rv3 exits the binding site. The overall carbohydrate geometry is good, with linkage torsion angles falling within normal

Lutteke, T. and von der Lieth, C.W. 2004) (Supplementary Fig. 2). The mannose that is bound most deeply in the binding site is Man5, which makes 10 hydrogen bonds to the Fab (Table I). Other sugars making contact to the Fab are Man4 (6 hydrogen bonds), Man3 (2 hydrogen bonds), and Man2 (2 hydrogen bonds). The Kdo1 residue does not contact the Fab, while Man6 makes just one hydrogen bond. There are also 67 van der Waals interactions between the glycan and Fab. The two combining sites in the dimer of Fabs are very similar. When the variable domains of each Fab monomer are superimposed (Cα atoms of K1-107 and M1-113 superimposed onto L1-107 and H1113) the root mean squared deviation (RMSD) for the superimposed Cα atoms is 0.30 Å, and the RMSD for all carbohydrate atoms is 0.81 Å. Hydrogen bonds in the two combining sites are also very well conserved (Table 1). Comparison to 2G12 in complex with GlcNAc2Man9. The crystal structure of 2G12 in complex with the Rv3 sugar is very similar to that of 2G12 in complex with GlcNAc2Man9 (PDB code 1OP5 (Calarese, D.A., Scanlan, C.N., et al. 2003), resolution 3.0 Å). When the variable regions are superimposed by Cα atoms (using residues L2-L107, H1-H113, K2-K107, and M1-M113 for the superposition), the overall RMSD is 0.73 Å, with

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distributions as evaluated with pdb-care (Lutteke, T., Bohne-Lang, A., et al. 2006,

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RMSD’s for CDRs L1, L2, L3, H1, H2, H3 of 0.63 Å, 1.03 Å, 0.33 Å, 0.57 Å, 0.63 Å, and 0.90 Å, respectively. The Rv3 residues Man2-Man5 superimpose well with the equivalent Bma3 (β-D-mannose 3) and Man4-Man6 of GlcNAc2Man9 in 1OP5 (sugar in chain A), with RMSD for all atoms of 1.02 Å (Fig. 3B). In the original crystal structure for 2G12 in complex with GlcNAc2Man9 (PDB code 1OP5), crystal packing contacts allow the D2/D3 arms of the sugar to occupy two symmetry-related clefts located between the

interaction. The 2G12-Rv3 structure does not have electron density in these extra pockets at the VH-VH’ interface, which is not surprising as the Rv3 OS has no D2/D3 arm-like branches. When comparing 2G12-Rv3 with previously determined structures for 2G12 in complex with carbohydrate ranging in complexity from Man2 to GlcNAc2Man9, both Fab and sugar structures are highly similar, with the only differences occurring in the bacterial sugars that differ from those in GlcNAc2Man9. However, the different anomeric configuration at Man2 and the presence of the Kdo residue are not important for the binding interaction of Rv3 OS in the 2G12 binding site, making the Rv3 OS an excellent mimic of the naturally occurring GlcNAc2Man9 sugar.

DISCUSSION The recent crystal and cryo-electron microscopy structures of a cleaved, stabilized gp140 trimer in complex with Fabs (Julien, J.P., Cupo, A., et al. 2013, Lyumkis, D., Julien, J.P., et al. 2013, Pancera, M., Zhou, T., et al. 2014) have clearly shown that the

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two paired heavy chains, suggesting that 2G12 may have up to 4 sites for sugar

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HIV-1 gp140 trimer is almost completely coated with carbohydrate. This dense arrangement of high-mannose as well as some complex sugars presents an attractive target for vaccine design because it is not typical of mammalian glycosylation (Scanlan, C.N., Offer, J., et al. 2007). Thus, studies of both natural and synthetic carbohydrates are of interest to vaccine design studies. In a previous study (Clark B.E., Auyeung K., et al. 2012), it was shown that the Rv3 LOS backbone is antigenically analogous to the D1

gp120 better than soluble synthetic oligomannosides or Man9GlcNAc2 and the level of inhibition observed with the bacteria matched that of 2G12 inhibition by soluble monomer gp120 based on the yield of LOS extracted from bacteria. The Rv3 sugar not only has high structural homology to the D1 arm of Man9GlcNAc2, as shown in this study, but may be attractive for vaccine design because it also contains bacterialderived sugars such as Kdo that may trigger qualitatively different antibody responses. Whether incorporation of non-mammalian carbohydrate moieties can help to trigger or boost antibody responses to oligomannose sugars on HIV needs to be explored further. A previous attempt to use synthetic non-self glycans comprised of the D1 oligomannosides with fructose termini as immunogens failed to evoke antibodies capable of recognizing gp120, even though these antibodies showed excellent binding to the D1 arm when presented in a range of other glycoconjugate formats (Doores, K.J., Fulton, Z., et al. 2010). Molecular mimicry of mammalian host structures by foreign polysaccharides is of course not new (Caplan, S.N., Berkman, E.M., et al. 1977); for example, antibodies to the LOS of certain Campylobacter jejuni strains can cross-react with gangliosides expressed on

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arm in mammalian oligomannose; soluble Rv3 LOS inhibited the binding of 2G12 to

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mammalian cells due to chemico-structural homology (Ang, C.W., Noordzij, P.G., et al. 2002) and the potential for autoimmunity due to mimicry between Helicobacter pylori lipopolysaccharides and host Lewis blood group antigens has been noted (Appelmelk, B.J., Simoons-Smit, I., et al. 1996). While unnaturally glycosylated yeast have been investigated for their ability to elicit anti-oligomannose neutralizing antibodies (AgrawalGamse, C., Luallen, R.J., et al. 2011, Luallen, R.J., Lin, J., et al. 2008), we are not

eliciting neutralizing antibodies to HIV (or any other virus). Therefore, the Rv3 structure presented here provides impetus for further exploration. Attempts to elicit oligomannose-specific broadly neutralizing antibodies to HIV using synthetic oligomannosides have so far not been successful (Astronomo, R.D. and Burton, D.R. 2010, Astronomo, R.D., Lee, H.K., et al. 2008, Dudkin, V.Y., Orlova, M., et al. 2004, Geng, X., Dudkin, V.Y., et al. 2004, Joyce, J.G., Krauss, I.J., et al. 2008, Kabanova, A., Adamo, R., et al. 2010, Li, H. and Wang, L.X. 2004, Mandal, M., Dudkin, V.Y., et al. 2004, Ni, J., Song, H., et al. 2006, Wang, L.X., Ni, J., et al. 2004) indicating that simply presenting dense arrays of oligomannosides that in essence are identical to natural oligomannoses is insufficient to yield the desired responses. The posit here is that the bacterial origin of the Rv3 glycan will serve as a better stimulant for the elicitation of antibodies capable of cross-recognizing oligomannose clusters on the surface of HIV. While the domain-swapped dimer structure of 2G12 is highly effective at contacting closely spaced glycans such as those on the gp140 trimer, 2G12 is so far the only antibody known with such a structural configuration. However, the recent discovery of ‘normal’, non-domain swapped neutralizing antibodies to HIV that recognize mixed

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aware of any report in which bacterial-based antigenic mimicry has been applied to

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glycan/protein epitopes, means that an effective immunogen to produce a high affinity response towards glycans does not necessarily need to elicit domain swapping such as in 2G12. Thus, the structures of these antibodies have revealed other ways of achieving high-affinity carbohydrate recognition, but with the caveat that, in addition to carbohydrate, they also bind peptide segments of the HIV glycoprotein surface. In this regard it is worth noting that the Rv3 structure also allows identification of locations at

oligomannose-specific antibodies to HIV, such as those from the PGT128 family of antibodies which do not bind Rv3 heat-killed bacteria (unpublished data). Most broadly neutralizing anti-HIV-1 antibodies are highly somatically mutated; 2G12 has an estimated 38/16 amino acid mutations in the heavy/light chains (Huber, M., Le, K.M., et al. 2010). Synthetically constructed, V-gene reverted germline-like precursors of these antibodies do not bind their HIV-1 antigens (Hoot, S., McGuire, A.T., et al. 2013, Xiao, X., Chen, W., et al. 2009), begging the question of which B cells were initially activated for evolution of antibodies such as 2G12. Indeed, a previous study showed that, while able to activate engineered mouse B cell lines displaying domainexchanged or non-domain-exchanged 2G12 on their surface, neither heat-killed Rv3 bacteria or other current immunogens (i.e., heat-killed engineered yeast, recombinant monomeric and trimeric forms of the HIV-1 envelope glycoprotein, or oligodendrons) were able to activate B cell lines displaying V-gene reverted germline 2G12 (Doores K.J., Huber M., et al 2013). However, one study has posited that domain-exchange may be achievable by as few as 5-7 mutations from the germline (Huber, M., Le, K.M., et al. 2010), suggesting that it may be possible to induce such an exchange using carefully

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which to incorporate modifications to allow for recognition by the more recently identified

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designed immunogens. Furthermore, bacterial carbohydrate epitopes encompassing Kdo sugars have been shown to bind to select sets of germline gene segments in mice (Brooks C.L., Müller-Loennies S., et al. 2010, Brooks, C.L., Wimmer, K., et al. 2013, Nguyen, H.P., Seto, N.O., et al. 2003); naïve B cell receptors with affinity for a carbohydrate immunogen based on the Rv3 glycan may similarly exist in the human antibody repertoire. Alternatively, glycoconjugates incorporating tailored oligovalent

strategy, perhaps in concert with other HIV-1 glycoprotein antigens such as the gp140 SOSIP trimer (Binley, J.M., Sanders, R.W., et al. 2000, Julien, J.P., Cupo, A., et al. 2013, Lyumkis, D., Julien, J.P., et al. 2013, Sanders, R.W., Dankers, M.M., et al. 2004), to drive the elicitation of broadly neutralizing carbohydrate-specific antibodies to HIV.

MATERIALS AND METHODS Rv3 oligosaccharide isolation. Rhizobium radiobacter Rv3 LOS was isolated from cells as described previously (Clark B.E., Auyeung K., et al. 2012) and the OS moiety cleaved from the lipid A region by mild acid hydrolysis. Briefly, LOS (50 mg) was treated with 1% acetic acid (5 ml, 100°C, 2h) and the cleaved lipid A removed by centrifugation (5000 rpm, 15 min). The supernatant was chromatographed on a Bio-Gel P2 (Bio-Rad, 1.5 x 100 cm, eluent water, flow = 12 ml/h) and the eluate monitored with a refractive index detector (Knauer K-2301). Fractions giving a positive read-out at the detector were individually freeze-dried and inspected by proton NMR. Pure hexasaccharide (8

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clusters of the Rv3 OS (or derivatives thereof) may find use as part of an immunization

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mg), containing the D1-like arm, attached to one Kdo residue was isolated (Fig 1, highlighted structure) and its structure confirmed by 2D NMR.

NMR spectroscopy. NMR experiments were carried out on a Bruker DRX-600 spectrometer equipped with a cryo-probe operating at 298 K. Chemical shift of spectra

ppm). Two-dimensional spectra (DQ-COSY, TOCSY, TROESY, gHSQC, gHMBC and gHSQCTOCSY) were measured using standard Bruker software, as reported previously (Clark B.E., Auyeung K., et al. 2012). Data processing and analysis were performed with the Bruker Topspin 3 program.

Expression and purification of 2G12. 2G12 IgG was produced as previously described (Doores, K.J., Fulton, Z., et al. 2010) by transient co-transfection of 293F cells with two separate plasmids containing the respective light and heavy chain (for sequences see (Kunert, R., Ruker, F., et al. 1998)), each preceded by an Ig kappa leader sequence. One week after transfection, the media containing 2G12 IgG was collected and passed over a protein A column (5 ml HiTrap rProtein A FF; GE Healthcare; flow rate 1 ml/min) equilibrated in 1X phosphate buffered saline (PBS), and the bound IgG was eluted with 0.1 M glycine, pH 2.7, collecting 1.3 ml fractions into tubes containing 0.2 ml 1 M Tris, pH 9. The IgG was further purified by size exclusion chromatography on a Superdex 200 16/60 column with running buffer 20 mM Tris, 150 mM NaCl, pH 8.0. To digest the purified IgG, activated papain was prepared by

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were recorded in D2O and expressed in δ relative to internal acetone (2.225 and 31.45

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combining 20.8 µl papain solution (Sigma P3125, 15-30 mg/ml, 16-40 U/mg), 100 µl 100 mM cysteine, 100 µl 10x papain digest buffer (10X = 1 M NaAc pH 5.5, 3 mM EDTA), and 778 µl MilliQ water) and incubating at 37°C for 15 minutes. Test digests with 0.5%, 1%, 2%, and 4% activated papain were carried out for 1, 2, 3, and 4 hours at 37 °C, and analyzed by non-reducing SDS polyacrylamide gel electrophoresis. Based on these results, 2% papain for between 3 and 4 hours was chosen for the large-scale cleavage.

2G12 (2.5 mg/ml). The mixture was incubated at 37°C for 3.5 hours. The papain was inactivated by the addition of iodoacetamide (37 mg/ml) to a final concentration of 200 mM with incubation at 37 °C for 1 hour. Fab was then separated from Fc and uncleaved IgG by protein A chromatography, as described above for the IgG purification. The flow through was collected as Fab and further purified by size exclusion chromatography (Superdex 200 16/60 in 20mM Tris, 150 mM NaCl, pH 8.0) to ensure that dimeric Fab was obtained (Calarese, D.A., Scanlan, C.N., et al. 2003). Bound Fc and uncleaved IgG fragments were eluted from the protein A column with 0.1 M glycine, pH 2.7 and discarded. Approximately 5 mg of Fab were obtained from 13.75 mg of IgG. The final product includes the complete 2G12 light chain, and heavy chain residues 1-224 (Kabat numbering), organized as a domain-swapped dimer of Fabs (two light and two heavy chains in one biological functional unit).

Crystallization. 2G12 Fab (17 mg/ml, in 20 mM Tris-HCl, 150 mM NaCl, pH 8.0) was mixed with Rv3 OS (100 mg/ml in H20) at a 1:3 molar ratio and screened for crystallization at 22ºC using the Stura Footprint Screen #1 (Stura, E.A., Nemerow, G.R.,

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Fresh activated papain was prepared as above, and 600 µl were added to 5.5 ml of

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et al. 1992), using sitting drop Cryschem plates (Hampton Research) with 0.4 μl protein mixed with 0.4 μl well solution and a 1 ml well volume. Crystals were obtained in several similar conditions with the highest resolution data collected from a crystal grown in 33% PEG 600, 0.2 M imidazole malate pH 5.5 (condition 1C). The crystal was briefly immersed in a mixture of 30% glycerol and well solution before vitrification by plunging into liquid nitrogen.

23-ID-D with a MarMosaic300 CCD detector to a high resolution limit of 1.85 Å resolution. Data collection statistics are compiled in Supplementary Table II. The diffraction data were indexed in space group P212121, with unit cell dimensions a=45.30, b=130.88, c=170.10 Å. With a calculated mass of 94,223 Da for the 2G12 dimeric Fab and 1049 Da for each of the two bound Rv3 OS moieties, the Matthews’ coefficient was calculated to be 2.6 Å3/Da for one dimeric Fab in complex with two Rv3 carbohydrates. Data were processed with HKL-2000 (Otwinowski, Z. and Minor, W. 1997) and the scaled reflections were then merged with Scala (Evans, P. 2006). Some data were lost due to overlapping reflections, so that the final data are 88% complete overall, with a completeness of 77.8% in the highest resolution shell (1.90-1.85 Å). The effective resolution of the dataset was estimated to be 2.0 Å resolution (Urzhumtseva, L., Klaholz, B.P., et al. 2013; Weiss, M. 2001). Structures were determined by the molecular replacement method using Phaser (McCoy, A.J., Grosse-Kunstleve, R.W., et al. 2007) with 2G12 coordinates from PDB file 1OP5 as a starting model. Refinement was carried out with Phenix (Afonine, P.V., Grosse-Kunstleve, R.W., et al. 2012) against

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Data collection and structure determination. Data were collected at APS beamline

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all Fobs (F> 0.0 σF) excluding a randomly chosen Rfree test set of 1999 reflections (2.6% and refining isotropic B values. Model building was carried out with Coot (Emsley, P. and Cowtan, K. 2004), carbohydrate geometry was evaluated with pdb-care (Lutteke, T., Bohne-Lang, A., et al. 2006, Lutteke, T. and von der Lieth, C.W. 2004), and superpositions carried out using the McLachlan algorithm (McLachlan, A.D. 1982) as implemented in the program ProFit (Martin, A.C.R.,

function in Coot and monitored visually using the “Check/Delete waters’ function, also in Coot. Figures were generated with Molscript and Bobscript (Esnouf, R.M. 1999, Kraulis, P.J. 1991). The Fab is numbered with Kabat nomenclature (Kabat, E.A., Wu, T.T., et al. 1991) and the Rv3 sugar units are numbered as in Fig 1.

Protein structure accession number. The coordinates and data for the 2G12-Rv3 structure have been deposited with the Protein Data Bank as entry 4RBP.

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http://www.bioinf.org.uk/software/profit/). Waters were added using the ‘Find Water’

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ACKNOWLEDGEMENTS This work was supported by the National Institutes of Health (AI084817to I.A.W. and R.L.S.); the International AIDS Vaccine Initiative (IAVI) Neutralizing Antibody Consortium (to I.A.W.); Scripps CHAVI- ID (UM1 AI100663 to I.A.W.); the Programma

the Canadian Institutes of Health Research (New Investigator Award 212115 to R.P.); and the Michael Smith Foundation for Health Research (Career Scholar Award 5268 to R.P.). This is manuscript number 28022 from The Scripps Research Institute. Portions of this research were carried out at Beamline 23-ID, a part of GM/CA at the Advanced Photon Source (APS). GM/CA @ APS has been funded in whole or in part with Federal funds from the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Sciences (Y1-GM-1104). Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science (contract no. DE-AC02-06CH11357).

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Operativo Regionale Campania FSE 2007-2013 (Project crème to C.D.C. and A.M.M.);

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FIGURE LEGENDS Figure 1. Sequence comparison of Man9GlcNAc2 and the Rv3 core oligosaccharide. The yellow highlighted fragment corresponds to the hexasaccharide used for crystallization.

hexasaccharide. The 2G12 light chains are shown in cyan, and the domain-swapped heavy chains are shown in red and magenta. The Rv3 hexasaccharide is shown in a ball-and-stick representation.

Figure 3. Rv3 OS electron density and comparison to oligomannose. (A) Wall-eyed stereo view of electron density for the Rv3 sugar. The simulated annealing omit Fo-Fc density for the sugar is contoured at 3.0σ. (B) Superposition of Rv3 carbohydrate (yellow) and Man9GlcNAc2 (cyan) from 1OP5.

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Figure 2. Overall structure of dimeric Fab 2G12 in complex with the Rv3

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Table I. Hydrogen bonds between Fab 2G12 and Rv3a Fab atom

Rv3 atom

Distance (Å) Fab1

Fab2

Man2 O4

2.79

2.64

Asp H100B OD2

Man2 O4

2.98

3.29

Tyr L94 OH

Man3 O3

2.85

2.63

Tyr L94 OH

Man3 O4

2.77

3.47

Ala H31 O

Man4 O2

3.27

3.21

Ala H31 O

Man4 O3

2.66

2.75

Asp H100B OD1

Man4 O5

3.38

3.68

Ser H100A O

Man4 O5

3.57

3.39

Asp H100B N

Man4 O6

3.39

3.64

Asp H100B OD1

Man4 O6

2.29

2.85

Thr H33 N

Man5 O2

2.73

2.71

Lys H95 O

Man5 O2

2.84

2.76

Lys H95 O

Man5 O3

3.03

3.04

Asp H100D OD2

Man5 O3

2.73

2.60

Ser H100A O

Man5 O4

2.70

2.84

Asn H100C N

Man5 O4

3.17

3.23

Asp H100D N

Man5 O4

2.81

3.02

Thr H33 OG1

Man5 O5

2.92

3.08

Thr H33 OG1

Man5 O6

2.65

2.66

Gly L93 O

Man5 O6

2.83

2.82

Asp H100B OD2

Man6 O4

2.27

2.68

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Asp H100B OD1

a

Hydrogen bond distances were evaluated with Contacsym (Sheriff, S., Hendrickson,

W.A., et al. 1987).

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Crystal structure of the HIV neutralizing antibody 2G12 in complex with a bacterial oligosaccharide analog of mammalian oligomannose.

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