Crystal structure of the HIV neutralizing antibody

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Nov 7, 2014 - the crystal structure of one such anti-carbohydrate HIV neutralizing antibody (2G12) in .... Amaya and Edwards 2003; Paphitou and Rolston 2003; Chen et al. ... the mannose residues reported for the full-length OS isolated after ..... Ang CW, Noordzij PG, de Klerk MA, Endtz HP, van Doorn PA, Laman JD.
Glycobiology, 2015, vol. 25, no. 4, 412–419 doi: 10.1093/glycob/cwu123 Advance Access Publication Date: 7 November 2014 Original Article

Original Article

Crystal structure of the HIV neutralizing antibody 2G12 in complex with a bacterial oligosaccharide analog of mammalian oligomannose Robyn L Stanfield2, Cristina De Castro3, Alberto M Marzaioli3, Ian A Wilson2,1, and Ralph Pantophlet4,1 2

Department of Integrative Structural and Computational Biology, Scripps CHAVI-ID, and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA, USA, 3Department of Chemical Sciences, University of Napoli Federico II, Complesso Universitario Monte Sant’Angelo, 80126 Napoli, Italy, and 4Faculty of Health Sciences and Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada 1

To whom correspondence should be addressed: e-mail: [email protected] (I.A.W.); [email protected] (R.P.)

Received 10 October 2014; Revised 2 November 2014; Accepted 3 November 2014

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

Introduction Human immunodeficiency virus-1 (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 and Wainberg 2013). Thus, despite significant advances in therapeutic approaches and attempts to purge

viral reservoirs in infected individuals, there remains a critical need for an effective HIV-1 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 (Kunert et al. 1998; Sanders et al. 2002; Stiegler et al. 2002; Calarese et al. 2003; Scanlan et al. 2003; Calarese et al. 2005; Murin et al. 2014). 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

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Structure of HIV Fab 2G12 with a bacterial oligosaccharide and 392. Neighboring glycans at 386 and 448 may influence the recognition process but do not appear to directly bind to 2G12 (Murin 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 anti-HIV-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 et al. 1998). However, recent exciting developments, spurred largely by advances in technologies that enable high-throughput 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 et al. 2014). These antibodies include the PGT121 family that recognizes one or more oligomannose- and/or complex-type glycans at the base of the V3 variable region (Julien, Cupo, et al. 2013; Julien, Sok, et al. 2013; Garces et al. 2014), the PGT128 family that recognizes oligomannose-type glycans at positions 301 and 332 on gp120 (Pejchal et al. 2011), the PGT135 family that recognizes oligomannose-type glycans at positions 332, 392 and 295 and 386 depending on the isolate (Kong 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 (Pancera et al. 2010; McLellan et al. 2011; Pancera et al. 2013; Pejchal 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 et al. 2014) and glycans at gp120 residues 88, 230 and 241 and gp41 residue 625 (35O22) (Huang 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 et al. 2014). Thus far, crystal and electron microscopy (EM) structures of these glycandependent antibodies in complex with their HIV-derived antigens reveal 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, Cupo, et al. 2013; Lyumkis et al. 2013; Pancera et al. 2014) suggest that the majority of the trimer surface is covered by glycosylation, so that many if not most neutralizing epitopes are likely to 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 et al. 2003). The antigen-binding sites on the two closely spaced Fab regions are ∼33 Å apart, and two nonconventional 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 et al. 2014).

413 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 (McLellan et al. 2013; Correia et al. 2014) provide optimism that structure-based immunogen design can be successful. Carbohydrates, in the form of glycoconjugates, have of course proven effective as vaccines for various bacterial pathogens, including Haemophilus influenza type b, Neisseria meningitidis serogroups A, C, Y and W-135 and Streptococcus pneumoniae (Astronomo and Burton 2010; Taylor 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 (Young et al. 2001; Farrand et al. 2003), 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 et al. 2012) (Figure 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 and Dandekar 2003). R. radiobacter is increasingly recognized also as an opportunistic pathogen in human nosocomial infections associated with the use of intravenous catheters (Edmond et al. 1993; Amaya and Edwards 2003; Paphitou and Rolston 2003; Chen et al. 2008). The lipopolysaccharide of R. radiobacter Rv3 is naturally devoid of an O-antigenic portion 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 (Figure 1), which in turn contains inner and outer core segments (De Castro 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 Figure 1) is α in Rv3 instead of β as found in oligomannose. The connection of this Man2 unit also differs and is to the lipopolysaccharide-specific Kdo (3-deoxy-octulosonic acid) residue in Rv3 instead of the chitobiose core in oligomannose (De Castro 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 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 (Weiss 2001; Urzhumtseva et al. 2013). 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.

Fig. 1. Sequence comparison of Man9GlcNAc2 and the Rv3 coreOS. The yellow highlighted fragment corresponds to the hexasaccharide used for crystallization.

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Results Oligosaccharide NMR structure After chromatographic purification, the Rv3 core OS was isolated from the LOS in 16% yield. Its proton NMR spectra (Supplementary data, Figure S1) displayed five main anomeric signals in the low field region (δ 5.4–4.9), a crowded carbinolic region (δ 4.3–3.5) and two couples of methylene protons (δ 2.4–1.7) related to the α and β anomeric forms of the Kdo1 residue at the reducing end. Attribution of all protons and carbon chemical shifts (Supplementary data, Table SI) 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 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 data, Figure S1). Analysis of the HSQC-TOCSY spectrum (Supplementary data, Figure S1B) 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 0.0σF) 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 and Cowtan 2004), carbohydrate geometry was evaluated with pdb-care (Lutteke and von der Lieth 2004; Lutteke et al. 2006) and superpositions carried out using the McLachlan algorithm (McLachlan 1982) as implemented in the program ProFit (Martin, ACR, http://www.bioinf.org.uk/software/profit/). Waters were added using the ‘Find Water’ function in Coot and monitored visually using the ‘Check/Delete waters’ function, also in Coot. Figures were generated with Molscript and Bobscript (Kraulis 1991; Esnouf 1999). The Fab is numbered with Kabat nomenclature (Kabat et al. 1991) and the Rv3 sugar units are numbered as in Figure 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.

Supplementary Data Supplementary data for this article are available online at http:// glycob.oxfordjournals.org/.

Funding This work was supported by the National Institutes of Health (AI084817 to 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 Operativo Regionale Campania FSE 2007-2013 (Project crème to C.D.C. and A.M.M.); 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).

Conflict of interest statement None declared.

Abbreviations bnAbs, broadly neutralizing antibodies; CDR, complementarity determining region; EM, electron microscopy; HIV-1, human immunodeficiency virus-1; LOS, lipooligosaccharide; OS, oligosaccharide; PBS, phosphate-buffered saline; RMSD, root mean squared deviation.

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