Co-crystal structures of antibody N60-i3 and antibody JR4 in complex with gp120 define more ...

39 Pages·2015·3.08 MB·English

JVI Accepted Manuscript Posted Online 17 June 2015
J. Virol. doi:10.1128/JVI.01232-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1 Co-crystal structures of antibody N60-i3 and antibody JR4 in complex with gp120
2 define more Cluster A epitopes involved in effective antibody-dependent effector
3 function against HIV-1
4 Neelakshi Gohain1,2*; William D. Tolbert1,2*, Priyamvada Acharya3, Lei Yu1,4, Tongyun Liu1,4,
5 Pingsen Zhao1,4, Chiara Orlandi1,4, Maria L. Visciano1,4, Roberta Kamin-Lewis1.4; Mohammad M.
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6 Sajadi1,5,6; Loïc Martin7; James E. Robinson8, Peter D. Kwong3; Anthony L. DeVico1,5, Krishanu o
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7 Ray2, George K. Lewis1,4; Marzena Pazgier1,2. n
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8 1Institute of Human Virology and Departments of 2Biochemistry and Molecular Biology of ed

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9 University of Maryland School of Medicine, Baltimore, MD 21201, USA o
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10 3Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National h
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11 Institutes of Health, Bethesda, MD 20892, USA //
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12 4Department of Microbiology and Immunology and 5Medicine of University of Maryland School i.a
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13 of Medicine, Baltimore, MD 21201, USA .
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14 6Medical Care Clinical Center, VA Maryland Health Care Center, Baltimore, MD 21201, USA /

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15 7CEA, iBiTecS, Service d’Ingénierie Moléculaire des Protéines, F-91191 Gif-sur-Yvette, France A
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16 8Department of Pediatrics, Tulane University Medical Center, New Orleans, LA 70112, USA ril
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17 * These authors contributed equally to this work 01
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18 #To whom correspondence should be addressed: b
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19 Email: [email protected] u
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20 725 West Lombard Street, Baltimore, MD 21201, USA t
21 Tel: [phone]
22 Fax: [phone]
23 Running title: ADCC Epitope Structures in the Cluster A gp120 Region
24 Word count: Abstract = 228 words; Text (excluding references and legends) = 6,487 words
25 Abstract
26 Accumulating evidence indicates a role for Fc receptor (FcR)-mediated effector functions of
27 antibodies, including antibody-dependent cell-mediated cytotoxicity (ADCC), in prevention of
28 HIV-1 acquisition and in post-infection control of viremia. Consequently, an understanding of the
29 molecular basis for Env epitopes that constitute effective ADCC targets is of fundamental
30 interest for humoral anti-HIV-1 immunity and for HIV-1 vaccine design. A substantial portion of D
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31 FcR-effector function of potentially protective anti-HIV-1 antibodies is directed toward non-
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32 neutralizing, transitional, CD4-induceable (CD4i) epitopes associated with the gp41 reactive a
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33 region of gp120 (Cluster A epitopes). Our previous studies defined the A32-like epitope within d

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34 the Cluster A region and mapped it to the highly conserved and mobile layers 1 and 2 of the m

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35 gp120 inner domain within C1-C2 regions of gp120. Here we elucidate additional Cluster A tt
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36 epitope structures, including an A32-like epitope, recognized by human mAb N60-i3 and a /jv
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37 hybrid A32-C11-like epitope, recognized by rhesus macaque mAb JR4. These studies define for s
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38 the first time a hybrid A32-C11-like epitope and map it to elements of both the A32-like sub- o
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39 region and the 7 layered β-sheet of the gp41-interactive region of gp120. These studies provide o
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40 additional evidence that effective antibody-dependent effector function to the Cluster A region A
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41 depends on precise epitope targeting – a combination of epitope footprint and mode of antibody il
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42 attachment. All together these findings help in understanding how Cluster A epitopes are
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43 targeted by humoral responses
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44 Importance g
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45 HIV/AIDS has claimed the lives of over 30 million people. Although antiretroviral drugs can s
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46 control viral replication no vaccine has yet been developed to prevent the spread of the disease.
47 Studies of natural HIV-1 infection, SIV or SHIV infected non-human primates (NHPs) and HIV-1-
48 infected humanized mice models, passive transfer studies in infants born to HIV-infected
49 mothers and the RV144 clinical trial have linked FcR-mediated effector functions of anti-HIV-1
50 antibodies with post-infection control of viremia and/or blocking viral acquisition. With this report
51 we provide additional definition of the molecular determinants for Env antigen engagement
52 which lead to effective antibody-dependent effector function directed to the non-neutralizing
53 CD4-dependent epitopes in the gp41 reactive region of gp120. These findings have important
54 implications for the development of an effective HIV-1 vaccine.
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56 Introduction D
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57 Antibodies (Abs) must bind conserved domains on viral envelope glycoproteins (Env) during key
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58 points in retroviral replication in order to broadly protect against human immunodeficiency virus a
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59 (HIV-1) infection. Their contribution to protection may result from a variety of antiviral d

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60 mechanisms, including direct neutralization of virus and Fc receptor-dependent effector m

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61 functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-mediated tt
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62 phagocytosis (1-4). Antibodies that directly neutralize HIV can provide protection as evinced in /jv
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63 several nonhuman primate studies with passively transferred monoclonal antibodies (mAbs) (5- s
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64 8); although their role in preventing natural HIV transmission remains equivocal (reviewed in o
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65 (9)). On the other hand, a growing body of evidence indicates that direct neutralizing activity is o
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66 not an absolute requirement for humoral protection against HIV infection. The RV144 vaccine A
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67 trial in humans (10-13), vaccine trials in non-human primates (14-17), early passive il
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68 immunization studies against Simian Immunodeficiency Virus (SIV) using polyclonal sera (18,
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69 19) and a breast milk transmission study of mother-infant pairs (20, 21) have linked Fc receptor-
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70 mediated effector functions with control or prevention of infection, often in the absence of g
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71 neutralization. Finally, the Fc effector functions’ contribution to the blocking of viral entry, s
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72 suppression of viremia and the therapeutic activity of several different anti-Env broadly
73 neutralizing Abs (bnAbs) was confirmed recently in both a mouse model of HIV-1 entry and a
74 model of mAb-mediated therapy using HIV-1-infected humanized mice (22). Overall, these
75 findings suggest that a vaccine capable of generating both neutralizing and non-neutralizing
76 humoral responses will provide the broadest measure of protection at the population level.
77 While the neutralizing epitopes have been examined in much detail (23-34), relatively little is
78 known about epitopes that are targets for antibodies acting through Fc receptor-dependent
79 effector functions, their degree of overlap with neutralizing epitopes, the immunological rules
80 underlying their selection during anti-Env antibody responses, or their precise locus of action
81 (e.g., transmission blocking or post-infection viral control). While neutralization and Fc receptor-
82 dependent processes of antibodies can be coincident for a given specificity, as has been D
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83 reported for antibodies targeting the gp120 variable loops, the co-receptor binding site, or the
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84 V2 loop region (35-38), they can be also be dissociated. Epitopes on both gp120 and gp41 are a
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85 known that are targeted by antibodies lacking of neutralizing activity but capable of potent Fc- d

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86 mediated effector function (reviewed in (38) and (37)). In this group, non-neutralizing, CD4- m

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87 inducible epitopes in the C1-C2 region of gp120 (A32-like epitopes) have recently gotten much tt
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88 attention as potent ADCC targets (39-42). RV144 analyses implicated this gp120 region as a /jv
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89 target of ADCC responses that correlated with reduced infection. In addition, a number of mAbs s
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90 specific for A32-blockable epitopes were recovered from vaccinated subjects (43) which o
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91 mediated cross-clade ADCC activity and synergized with V2-specific mAbs to mediate ADCC o
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92 against the tier 2 isolate AE.CM235 (44). Lastly, the protective vaccine efficacy due to ADCC A
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93 responses of C1-region specific mAbs was greatly attenuated by the presence of IgA mAbs il
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94 incapable of NK cell-mediated effector function but competing for the same Env binding sites
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95 (45).
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96 Previously, we designated C1-C2 epitopes mapping to the gp41 reactive face of gp120 as g
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97 “Cluster A”, the canonical examples being mAb A32 and C11 (41). These epitopes are exposed s
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98 after envelope trimers engage target cell CD4 and persist on freshly infected cell surfaces for
99 extended periods of time post-infection (46-48). They are also exposed on the surfaces of
100 persistently infected cells. In general, Cluster A epitopes are naturally immunogenic as HIV-1
101 infected individuals frequently elicit C1-C2 specific antibodies (39, 40, 42, 49). We and others
102 have shown that these epitopes become major targets for ADCC responses during HIV-1
103 infection (39, 41, 42, 50) and ADCC responses to this region are also subject to immune escape
104 early in infection (51). Recently, it was also shown that exposure of Cluster A epitopes are
105 modulated by down regulation of CD4 on the surface of the infected target cell by host factors
106 Nef and Vpu (42, 52). This points toward the possibility of Nef and Vpu evolving as viral
107 defenses against the exposure of these epitope targets during virion release and as an ADCC
108 evasion mechanism preventing antibody mediated clearance of virus-infected cells (42, 52). D
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109 We previously reported that Cluster A is comprised of at least three epitope sub-regions, as
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110 defined by ELISA competition with mAbs A32 and C11 for binding to CD4 triggered gp120 (41). a
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111 One subgroup only competes with A32 (A32-like epitopes), the second only competes with C11 d

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112 (C11-like epitopes), and the third competes with both A32 and C11 (hybrid A32-C11-like m

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113 epitopes). Recently, we defined the A32-like epitope sub-region at atomic level by describing tt
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114 structures of Fab fragments of two A32-like antibodies in complexes with the CD4-triggered /jv
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115 gp120 cores (53). These studies mapped the A32-like epitope to the mobile layers 1 and 2 of s
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116 the gp120 inner domain within the C1-C2 regions. They also pointed toward a role of precise o
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117 epitope targeting and mode of antibody binding in the of Fc-mediated effector functions of o
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118 antibodies against HIV-1. Here we elucidate two more epitope structures within Cluster A region A
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119 and provide a more comprehensive understanding of how these epitopes are recognized by a il
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120 human mAb and a Rhesus macaque mAb, both capable of potent ADCC function.
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121 Materials and Methods:
122 Protein purification JR4 and N60-i3 monoclonal antibodies (mAbs) were purified by HiTrap
123 protein A column (GE Healthcare) chromatography from 293T supernatants prepared by
124 transfecting plasmids encoding the heavy- and light-chain genes of the respective Abs. The
125 Fabs of both the mAbs were prepared from the purified IgGs (10 mg/ml) by proteolytic digestion
126 with immobilized papain (Pierce, Rockford, IL) and purified using a protein A column to remove D
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127 Fc (GE Healthcare, Piscataway,NJ) followed by gel filtration chromatography on a Superdex
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128 200 16/60 column (GE Healthcare,Piscataway, NJ). The elution peak of each of the Fabs a
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129 corresponded to a molecular weight of approximately 50 kDa and was collected and d

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130 concentrated for use in the crystallization trials. m

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131 For crystallographic studies, gp120 coree, of clade A/E strain 93TH057 (lacking N-, C- termini, ttp
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132 variable loops 1, 2 & 3) and the CD4-mimetic miniprotein M48 [F23M47] or M48U1 (54, 55) was /jv
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133 used to prepare the ternary complexes of JR4 and N60-i3, respectively. gp12093TH057 coree was s
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134 prepared and purified as previously described (53). Deglycosylated gp120 core was first o
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135 mixed with CD4 mimetic peptide M48 or M48U1 at a molar ratio of 1:1.5 and purified through gel o
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136 filtration chromatography using a Superdex 200 16/60 column (GE Healthcare, Piscataway, NJ). A
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137 After concentration, the gp12093TH057 coree -M48 or gp12093TH057 coree -M48U1 complex was il 7
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138 mixed with a 20% molar excess of JR4 Fab or N60-i3 Fab respectively and passed again
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139 through the gel filtration column equilibrated with 25 mM Tris-HCl buffer pH 7.2 with 0.35 M
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140 NaCl for the JR4 Fab-gp120 core -M48 complex and with 0.15 M NaCl for the N60-i3 g
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141 Fab-gp120 core -M48-U1 complex. The purified complexes were concentrated to ~10 s
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142 mg/ml for crystallization experiments.
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144 Crystallization Initial crystal screens were done in robotic vapor-diffusion sitting drop trials
145 using commercially available sparse matrix crystallization screens then reproduced and
146 optimized using the hanging-drop, vapor diffusion method (drops of 0.5 μl protein and 0.5 μl
147 precipitant solution equilibrated against 700 μl of reservoir solution). JR4 Fab crystals were
148 obtained from a condition containing 0.2 M ammonium sulfate, 1.0 M sodium cacodylate
149 trihydrate pH 6.5, and 30% w/v PEG 5,000. Prior to freezing, the crystals were transferred into
150 the crystallization condition containing 15% v/v of glycerol. Crystals of JR4 Fab-gp120
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151 core -M48 were grown from 16.6% PEG400, 13.3% PEG3350, 0.1 M MgCl and 0.1 M Tris pH
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152 8.5 and soaked in mother liquor supplemented with 20% MPD prior to being frozen for data D
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153 collection. Crystals of N60-i3 Fab-gp120 -M48-U1 were grown from 10-16% PEG 8000 or
93TH057 n
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154 PEG 10,000, 0.065 M NaCl and 0.1 M Tris-HCl pH 8.5 at 22°C and cryoprotected in 18% MPD, a
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155 16% PEG 8000 or 10,000, 0.1 M Tris-HCl pH 8.5, and 0.065 M sodium chloride. d

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156 m

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157 Data collection and structure solution Diffraction data were collected at the Stanford tt
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158 Synchrotron Radiation Light Source (SSRL) at the beam lines BL9-2 (JR4 Fab), BL12-2 (JR4 /jv
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159 Fab-gp12093TH057 coree -M48) and BL7-1 (N60-i3 Fab-gp12093TH057 coree -M48-U1), equipped s
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160 with MAR325, PILATUS 6M PAD and ADSC Quantum 315 area detectors, respectively. All o
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161 data were processed and reduced with HKL2000 (56). Structures were solved by molecular o
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162 replacement with Phaser (57) from the CCP4 suite (58) based on the coordinates of gp120 A
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163 (PDB: 3TGT) and the JR4 Fab (PDB: 4RFE) for JR4 and N5-i5 Fab (PDB: 4H8W) for N60-i3 il
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164 and the CD4 mimetic peptide M48 (PDB: 2I6O) for JR4 and M48U1 (PDB: 4JZW) for N60-i3.
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165 Refinement was carried out with Refmac (59) and/or Phenix (60). Refinement was coupled with
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166 manual refitting and rebuilding with COOT (61). Data collection and refinement statistics are g
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167 shown in Table 1. s
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169 Structure validation and analysis The quality of the final refined models was monitored using
170 the program MolProbity (62). Structural alignments were performed using the Dali server and
171 the program lsqkab from CCP4 suite (58). PISA (63) and PIC (64) webservers were used to
172 determine contact surfaces and residues. All illustrations were prepared with the PyMol
173 Molecular Graphic suite (http://pymol.org) (DeLano Scientific, San Carlos, CA, USA).
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175 FRET-FCS competition assay Alexa488-Alexa568 donor-acceptor pairs were used for
176 competition assay using fluorescence resonance energy transfer (FRET) - fluorescence
177 correlation spectroscopy (FCS). For FRET measurements, the Fabs (C11, A32, N60i3, JR4) D
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178 were labeled with either donor (Alexa 488) or acceptor (Alexa 568) probes (Invitrogen mAb
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179 labeling kit). Briefly, the Alexa Fluor 488 or 568 reactive dye has a succinimidyl ester moiety that a
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180 reacts efficiently with primary amines of Fab to form stable dye-protein conjugates. The dye d

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181 labeled Fabs were purified using 10 KDa cutoff spin columns. Purified Alexa-488 or 568 labeled m

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182 Fabs were quantified by a UV-visible (UV-vis) spectrometer (Nanodrop 2000). Dye-to-protein tt
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183 ratios were determined by measuring absorbance at 280 nm (protein) versus 488 or 577 nm /jv
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184 (dye). The dye-to-protein ratios were between 1 and 2. We specifically aimed to keep this low s
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185 level of dye labeling as we are using a single molecule fluorescence method to minimally o
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186 perturb the functionality of the protein. FRET measurements were performed in a confocal o
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187 microscope (MicroTime 200; PicoQuant). PicoQuant Symphotime software was used to A
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188 generate the FRET histograms and for further analyses. FRET measurements were performed il
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189 after forming immune complex with full-length single chain gp120 -sCD4 (FLSC) with donor-
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190 labeled Fab and acceptor labeled Fab. In all of our measurements each Fab concentration was
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191 1 µg/ml and FLSC concentration was 1.5 µg/ml. The immune complexes were made by g
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192 incubating Fabs to FLSC at 20oC for 1 hour. Fluorescence responses from the donor and the s
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193 acceptor molecules were separated by a 50/50 beam splitter and detected by two avalanche
194 photodiode detectors (APD) using the method of time-correlated single photon counting and the
195 Time-Tagged Time-Resolved (TTTR) mode of the PicoHarp 300 board. High quality bandpass
196 (Chroma) filters were used for recording donor and acceptor fluorescence in two separate
197 detection channels. The collected single photon data was binned by a 1 msec bin in each
198 channel (donor or acceptor), which resulted in intensity-time traces and count-rate histograms.
199 Threshold values in each channel were used to identify the single molecule bursts from the
200 corresponding background signal level. Fluorescence bursts were recorded simultaneously in
201 donor and acceptor channels and FRET efficiencies were calculated using E = I /(I + γI )
A A D
202 where I and I are the sums of donor counts and acceptor counts for each burst, taking into
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203 account the possible difference in the detection efficiency (γ) in two separate channels (65). The D
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204 donor-to-acceptor distance (r) in terms of efficiency of energy transfer (E) and Förster Distance n
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205 (R0) is given by r = R0 [1/E – 1]1/6. We have used R0 value of 62 Å for the Alexa 488 (donor) and ad
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206 Alexa 568 (acceptor) pair for estimating the donor-to-acceptor distances. In addition to FRET
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207 measurements, we have also performed FCS measurements to assess in vitro binding of single m

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208 or multiple Fab fragments to FLSC. We determined translational diffusion coefficients of Alexa tt
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209 488 or 568 labeled Fabs and the corresponding immune complexes from FCS measurements. jv
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210 The FCS measurements and analyses were performed as previously reported (47). s
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211 SPR competition analysis The binding footprint of mAb N60-i3 and JR4 in relation to mAb C11 g
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212 and A32 was assessed by Surface Plasmon Resonance (SPR) competition on a Biacore T-100 n

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213 (GE Healthcare) at 25°C with buffer HBS-EP (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM r
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214 EDTA, and 0.05% surfactant P-20). Protein A was first immobilized onto the second of two flow ,

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215 cells on a CM5 chip to ~3000 response units (RU) and the first flow cell blocked with a standard 1
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216 amine coupling protocol (GE Healthcare). IgGs to be evaluated were then captured onto the y

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217 second flow cell by flowing a 5-10 nM solution of mAb at 10 µl/min flow rate for 30 seconds. e
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218 The antibody concentration was varied to give a RU in the range of 150 to 400. Single-chain
219 gp120 -sCD4 (FLSC) (66) was then passed over the same flow cell at a flow rate of 10 µl/min
BaL
220 for 30 seconds. A FLSC concentration was chosen to give a RU in the range of 150 to 400,
221 comparable to the RU for the antibody. Varying concentrations of mAb Fab were then passed
222 over both flow cells at a flow rate of 30 µl/min for 200 seconds and allowed to dissociate by
223 passing buffer over both cells at the same flow rate for 800 seconds. The cells were
224 regenerated between concentrations with a 30 second injection of 0.1 M glycine pH 3.0 at a flow
225 rate of 100 μl/min. The antibody and FLSC were then reloaded onto the second flow cell for the
226 next concentration. Blank sensorgrams were obtained by injection of HBS-EP buffer in place of
227 Fab. Sensorgrams of the concentration series (flow cell two minus one) were corrected with
228 corresponding blank. D
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229
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230 ADCC assays ADCC assays were carried out using the rapid fluorescence ADCC method (67) a
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231 modified to reduce prozone effects. All ADCC studies used CEM-NKr-CCR5 target cells d

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232 sensitized with recombinant gp120 from the HIV-1 BaL isolate or spinoculated with AT-2 m

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233 inactivated BaL HIV-1 virus (kindly supplied by Dr. Jeffrey Lifson, NCI) at 3000 RPM for 2 hrs at tt
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234 12° C. gp120-sensitized or virus-spinoculated cells were then washed twice and added to a 96- /jv
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235 well V-bottom plate (5,000 cells/well). The gp120 sensitized or virion bound target cells were s
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236 incubated with mAb dilutions for 15 mins and were washed with culture medium before the o
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237 addition of peripheral blood mononuclear effector cells from healthy donors at a final ratio of o
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238 50:1. The effector and target cells were pelleted by centrifugation and incubated for 2-3 hrs at A
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239 37°C followed by fixation and cytolysis determined by flow cytometry as described in (67). The il
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240 absolute cytotoxicity values were normalized using the mAbs C11 as described previously (41).
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242 Results g
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243 mAb Origin and Epitope Cluster A Assignment s
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244 mAb N60-i3 was isolated from B-cells of an HIV-1–infected individual and characterized for
245 initial reactivity using recombinant proteins based on the HIV-1 isolate as described
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246 previously for other Cluster A mAbs (41). mAb JR4 was derived from the peripheral blood B
247 cells of a Rhesus macaque infected with a SHIV KB9 mutant with deletions of glycosylation
248 sites in gp41. The detailed description of mAb N60-i3 and JR4 and JR4 isolation, germline gene