Potent Neutralization of Vaccinia Virus by Divergent Murine Antibodies Targeting a Common Site ...

52 Pages·2014·0.9 MB·English

JVI Accepts, published online ahead of print on 16 July 2014
J. Virol. doi:10.1128/JVI.01491-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Potent Neutralization of Vaccinia Virus by Divergent Murine
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Antibodies Targeting a Common Site of Vulnerability in L1 Protein
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3 Running title: potent vaccinia virus neutralizing antibodies
4 Thomas Kaever1, Xiangzhi Meng6, Michael H. Matho2, Andrew Schlossman2, Sheng Li3, Inbal
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5 Sela-Culang4, Yanay Ofran4, Mark Buller5, Ryan W. Crump5, Scott Parker5, April Frazier1, o
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6 Shane Crotty1, Dirk M. Zajonc2, Bjoern Peters1, Yan Xiang6^ nlo
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7 1Division of Vaccine Discovery, 2Division of Cell Biology, La Jolla Institute for Allergy and d

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8 Immunology (LJI), La Jolla, CA 92037, USA. m

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9 3Medicine and Biomedical Sciences Graduate Program, University of California at San Diego, p:
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10 La Jolla, CA 92093, USA. i.
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11 4The Goodman Faculty of Life Sciences, Nanotechnology Building, Bar Ilan University, Ramat .o
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12 Gan 52900, Israel. o
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13 5Saint Louis University School of Medicine, 1100 S. Grand Blvd., St. Louis, MO 63104, USA. c
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14 6Department of Microbiology and Immunology, University of Texas Health Science Center at e
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15 San Antonio, San Antonio, TX 78229, USA. ,

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17 ^ Corresponding author: g
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18 Yan Xiang, [email protected]
19 Abstract: 243 words
20 Full text: 7,984 words
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21 Abstract
22 Vaccinia virus (VACV) L1 is an important target for viral neutralization and has been
23 included in multicomponent DNA or protein vaccines against orthopoxviruses. To further
24 understand the protective mechanism of the anti-L1 antibodies, we generated five murine anti-L1
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25 monoclonal antibodies (mAbs), which clustered into 3 distinct epitope groups. While two groups w
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26 of anti-L1 failed to neutralize, one group of 3 mAbs potently neutralized VACV in an isotype- lo
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27 and complement-independent manner. This is in contrast to neutralizing antibodies against major d

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28 VACV envelope proteins such as H3, D8 or A27, which failed to completely neutralize VACV m

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29 unless the antibodies are of complement-fixing isotypes and complement is present. Compared to tt
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30 non-neutralizing anti-L1 mAbs, the neutralization antibodies bound to the recombinant L1 jv
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31 protein with a significantly higher affinity and could also bind to virions. By using a variety of sm
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32 techniques including the isolation of neutralization escape mutants, hydrogen/deuterium r
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33 exchange mass spectrometry, and X-ray crystallography, the epitope of the neutralizing n

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34 antibodies was mapped to a conformational epitope with Asp35 as the key residue. This epitope ec
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35 is similar to the epitope of 7D11, a previously described potent VACV neutralizing antibody. b
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36 The epitope was recognized mainly by CDR1 and CDR2 of the heavy chain, which are highly 2
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37 conserved among antibodies recognizing the epitope. These antibodies, however, had divergent 2
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38 light chain and heavy chain CDR3 sequences. Our study demonstrates that the conformational
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39 L1 epitope with Asp35 is a common site of vulnerability for potent neutralization by a divergent g
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40 group of antibodies. t
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41 Importance
42 Vaccinia virus, the live vaccine for smallpox, is one of the most successful vaccines in
43 human history but presents a level of risk that has become unacceptable for the current
44 population. Studying the immune protection mechanism of smallpox vaccine is important for
45 understanding the basic principle of successful vaccines and the development of next generation, D
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46 safer vaccines for highly pathogenic orthopoxviruses. We studied antibody targets in smallpox n
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47 vaccine by developing potent neutralizing antibodies against vaccinia virus and comprehensively d
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48 characterizing their epitopes. We found a site in vaccinia virus L1 protein as the target of a group fr
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49 of highly potent murine neutralizing antibodies. The analysis of antibody:antigen complex h
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50 structure and the sequences of the antibody genes shed light on how these potent neutralizing :/
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51 antibodies are elicited from immunized mice. i.a
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52 Introduction
53 Variola virus and monkeypox virus are orthopoxviruses that are highly pathogenic to
54 humans, considered to be a potential bioterrorism agent (26) and an emerging pathogen (45),
55 respectively. A related orthopoxvirus, vaccinia virus (VACV), serves as the vaccine against these
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56 pathogens. Live VACV immunization is capable of eliciting neutralizing antibodies against a w
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57 variety of targets on two antigenically distinct forms of virions, the intracellular mature virions lo
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58 (MV) and the extracellular enveloped virions (EV) (15, 16). Vaccinia vaccine is arguably the d

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59 most successfully vaccine in human history, having led to the eradication of smallpox (25). m

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60 However, it was also associated with a relatively high rate of adverse events (23). Consequently, tt
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61 safer multicomponent DNA or protein vaccines that include a subset of MV and EV antigens jv
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62 have been developed, and they showed protection against orthopoxvirus challenges in mice and sm
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63 nonhuman primates (8, 21, 27, 51). While many MV antigens have been shown to be r
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64 neutralization targets (42, 52), the MV antigen that is invariably included in these subunit n

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65 vaccines is L1. L1 is an immunodominant neutralizing antibody target in mice, though it is a less ec
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66 common target in humans (5). It is a 250-amino-acid myristoylated protein with a C-terminal b
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67 transmembrane domain that spans residues 186 to 204 (22, 47). L1 associates with the virus- 2
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68 encoded multiprotein entry-fusion complex (EFC) and plays an essential role in viral entry (6). 2
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69 Despite the importance of L1 as a neutralizing target and subunit vaccine component, y

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70 relatively little is known about its neutralizing epitopes and the corresponding paratopes. A e
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71 conformational epitope with Asp35 as the key residue is recognized by several murine
72 monoclonal antibodies (mAbs) (29), which potently neutralize MV. The sequence of one of the
73 mAbs, 7D11, has been reported, and a structure of the Fab domain of 7D11 bound to L1 has
74 been determined (48). A linear epitope (residues 118-128) of L1 is recognized by several
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75 antibodies, which neutralized MV with reduced potency compared to 7D11 (2). In an effort to
76 gain a more comprehensive understanding of neutralizing epitopes on L1 and the neutralizing
77 mechanism of anti-L1 antibodies, we developed additional mAbs against L1, examined their
78 neutralizing abilities in vitro and in vivo, and determined their epitopes and the corresponding
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79 paratopes.
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80 lo
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81 Materials and Methods f
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82 Viruses and antibodies. VACVWR stocks were grown on HeLa cells in T175 flasks, infecting at ht
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83 a multiplicity of infection of 0.5. Cells were harvested at 60 h, and virus was isolated by rapidly //
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84 freeze-thawing the cell pellet three times in a volume of 2.3 ml RPMI plus 1% fetal calf serum a
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85 (FCS). Cell debris was removed by centrifugation. Clarified supernatant was frozen at -80°C as .o
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86 virus stock. VACVWR stocks were titered on Vero cells (~2 x 108 PFU/ml). VACVACAM2000 was o
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87 obtained from the CDC. Strain Mos-3-P2 (Moscow strain) of ectromelia virus, as described in e
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88 Chen et. al (11), was used for ectromelia in vivo studies. Monoclonal antibodies used in the study m
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89 are: anti-B5, B126 (4); anti-A10, BG3.1 (41); anti-H3, JH4 (41), #41 (38); anti-A27, 1G6,12G2 r

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90 (manuscript in preparation); anti-D8, JE10(41), LA5(37). ,
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91 Hybridoma generation and characterization. The generation and characterization of the b
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92 hybridomas were performed as described (41) except for some changes in the immunization u
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93 procedure described below. BALB/c mice were initially infected intranasally with 5x103 plaque- t
94 forming-units (PFU) of VACVWR. Subsequently, the mice were boosted twice with
95 intraperitoneal (i.p.) injection of 100 µg of recombinant L1 proteins together with incomplete
96 Freund's adjuvant. Recombinant L1 proteins were either Glutathione S-transferase (GST) fusion
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97 of L1(1-185) or his-tagged L1(1-185). Both forms of recombinant L1 proteins were expressed in
98 E. coli., and the his-tagged L1(1-185) protein was refolded as described below. The hybridomas
99 were screened with ELISA against recombinant L1 proteins and with immunofluorescence assay
100 of VACVWR infected cells as described (41).
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101 L1 expression and purification. VACV-L1 (residues 1-185) expression construct was kindly w
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102 provided by David Garboczi and expression and refolding was carried out as reported (47). lo
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103 Refolded protein was purified by Ni-NTA affinity chromatography (5 mL HisTrap FF Column, d

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104 GE Healthcare) by eluting with 0.2 M Imidazole, 20 mM Tris pH 8.0, 0.3 M NaCl. The eluted m

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105 protein was then further purified by gel filtration chromatography using a Superdex 200 size tt
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106 exclusion column (GE Healthcare). Proper protein folding was verified by ELISA using three jv
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107 different anti-L1 antibodies. sm
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108 Cross-Blocking ELISA. Refolded L1(1-185) was prepared at 0.5 µg/ml and used to coat Nunc /

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109 Polysorbent flat-bottom 96-well plates with 100 µl per well. Plates were incubated overnight at D
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110 4°C and washed four times with PBS plus 0.05% Tween 20. 100 µl of blocking buffer (PBS + 10% e
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111 fetal bovine serum) was added to the plate and incubated for 90 min at room temperature. e
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112 Blocking buffer was discarded, and 100 µl of antibodies of interest were added to the plate at 10 8
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113 µg/ml and incubated for 90 min. Horseradish peroxidase (HRP)-conjugated antibodies of interest 1
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114 (Innova Biosciences Lightning-Link HRP conjugation kit) were prepared at 0.5 µg/ml and added y

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115 to the plates for 20 min. The plates were developed using o-phenylenediamine (OPD), and e
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116 optical density (OD) at 490 nm was read on a SpectraMax 250 (Molecular Devices).
117 Flow Cytometry-based in vitro neutralization. Vero E6 cells (1x105 cells/well) were seeded in
118 96-well Costar plates (Corning Inc., Corning, NY) and incubated for 5 h to adhere. Subsequently,
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119 cells were infected with 12.5 µL purified VACV-GFP at 1x106 PFU/mL (final PFU of 1.25x104)
120 and 12.5 µL mAbs at 80 µg/mL (final concentration of 20 µg/mL) for 12 h at 37°C and 5% CO2
121 in a total volume of 50 µL in the presence (2% final concentration) or absence of sterile baby
122 rabbit complement (CEDARLANE). Samples were prepared in duplicates. Cells were
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123 subsequently tested using flow cytometry as described previously (4).
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124 Plaque reduction neutralization tests (PRNT). Vero E6 cells were seeded into 6-well Costar lo
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125 plates (Corning Inc., Corning, NY) and used within 2 days of reaching confluence. Purified d

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126 antibodies were prepared in duplicates at 20 µg/mL and five-fold serial dilutions were performed m

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127 in PBS. Ten µL of sonicated VACVWR at 1x105 PFU/mL was added to 970 µL for every titrated ttp
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128 antibody sample, resulting in 1x103 PFU/sample final concentration. For samples to be tested in /jv
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129 the presence of complement, 20 µL of complement was added, resulting in a final concentration sm
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130 of 2%. PBS was used instead for other samples. Samples were incubated for 1h at 4°C, shaking. r
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131 Medium was aspirated from 6-well plates and 1 mL of samples mixtures was added to each well n

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132 to adsorb for 60 min at 37°C with periodic swirling. Mix was aspirated and 2 mL of DMEM + 1% ec
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133 FBS + 1% PenStrep was then added, and the plates were incubated for 2.5 days for the plaques to b
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134 develop. The medium was aspirated, the cells were fixed and stained in one step with 0.1% 2
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135 crystal violet in 20% ethanol, and the plaques were quantified over white-light transillumination. 2
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136 The data were plotted, and a curve of best fit was applied by eye. The neutralization ability was
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137 investigated as the comparison of the samples including antibody to the mean number of plaques g
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138 of controls with no antibody. t
139 Vaccinia intranasal infections and protection studies. Female BALB/c mice were used at an
140 age of 7-8 weeks. Animal husbandry and experimental procedures were approved by the
141 Department of Laboratory Animal Care and the Animal Care Committee of the La Jolla Institute.
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142 To infect the mice, a Pipetman was used to place 10 µl of VACVWR on each nare of an
143 isofluorane-anesthetized mouse (total volume, 20 µl), and the liquid was rapidly inhaled by the
144 mouse. Mice were weighed daily to assess disease progression. After intranasal infection with
145 VACVWR, mice develop a systemic infection and exhibit severe weight loss. Mice were
146 euthanized if and when 25% weight loss occurred. A dose of 1x105 PFU of VACVWR was the Do
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147 standard lethal dose given to 7-week-old BALB/c females. For L1 mAb protection studies, mice lo
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148 were treated by i.p. injection with 100 µg of antibodies 1 day before infection. Control mice e
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149 received anti-A10 BG3.1 antibody, which is known to provide no protection. An additional r
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150 group received anti-B5 B126 as positive control. h
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151 Vaccinia intravenous infection protection studies. To infect mice, 1x105 PFU of /jv
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152 VACVACAM2000 was injected retro-orbitally. Mice were weighed daily to assess disease sm
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153 progression. Clinical score, a composite score of the pox lesion abundance on the four paws plus r
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154 the tail, was evaluated as described (38). For L1 mAb protection studies, mice were inoculated n

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155 i.p. with 100 µg of antibodies 1 day before infection. Control mice received anti-A10 BG3.1 ec
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156 antibody. An additional group received anti-H3 #41 as positive control. b
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157 Ectromelia infection and protection studies. Four to six week old female C57BL/6 mice were 8
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158 obtained from Harlan Laboratories (Indianapolis, IN) and were housed in filter-top microisolator 1
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159 cages and fed commercial mouse chow and water, ad libitum. The mice were housed in an y

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160 animal biosafety level 3 containment area. Animal husbandry and experimental procedures were e
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161 approved by the Institutional Animal Care and Use Committee of Saint Louis University. The
162 day before challenge, mice were treated i.p. with 100 µg of the specified antibody. Immediately
163 before challenge, mice were anesthetized with 0.1 ml/10 g body weight of ketamine HCl (6
164 mg/ml) and xylazine (0.5 mg/ml) by i.p. injections. 1000 PFU of ECTV (Moscow strain) in PBS
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165 without Ca2+ and Mg2+ was slowly loaded into nares (5 µl/nare). Mice were subsequently left in
166 situ for 2-3 minutes before being returned to their cages (19, 44). Mice were monitored daily for
167 mortality and morbidity – as measured by weight-change.
168 Epitope Mapping by ELISA. Overlapping 20-mer peptides for the L1 antigen were synthesized
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169 (AnaSpec) and tested for mAb binding using an ELISA. Flat-bottom 96-well microtiter plates w
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170 were coated with 100 µl of NeutrAvidin biotin-binding protein (1 mg/ml) diluted in PBS lo
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171 overnight at 4°C (ThermoScientific Pierce). Then, coated plates were washed with washing d

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172 buffer (PBS, pH 7.2, plus 0.05% Tween 20) and blocked with blocking buffer (PBS, pH 7.2, plus m

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173 1% BSA plus 0.1% Tween 20) for 2 h at room temperature (RT). Plates were incubated with 100 tt
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174 µl of overlapping linear biotinylated peptides (200 ng/ml) in blocking buffer for 90 min at RT. jv
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175 Plates were washed and incubated with purified mAb at 10 µg/ml for 90 min at RT. Plates were sm
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176 washed, and the bound mAb was detected by adding a streptavidin-HRP-conjugated secondary r
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177 antibody to mouse immunoglobulin G (Invitrogen) and incubated for 60 min at RT, followed by n

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178 OPD substrate (Sigma-Aldrich). ec
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179 Peptide Truncation and Alanine Scan. Variant peptides with N- or C-terminal truncations e
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180 and/or alanine substitutions were tested for their ability to block binding to the parent 20-mer 8
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181 peptides in ELISA. 96-well plates were coated with 100 µL NeutrAvidin per well at a 1
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182 concentration of 0.5 µg/mL. Plates were incubated overnight at 4°C and washed 4 times with y

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183 PBS plus 0.05% Tween-20. 100 µL of blocking buffer (PBS + 10% FBS) were added to the e
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184 plates and incubated for 90 min at 4°C. Blocking buffer was discarded, 100 µL of biotinylated
185 20-mer peptides were added to the plate at 200 ng/mL and incubated for 90 min at 4°C.
186 Simultaneously, selected antibodies were incubated with variant peptides. We used 30 µL/well
187 of mAb at 600 ng/mL and incubated it with 30 µL/well of alanine-modified peptides at 100
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188 µg/mL for 90 min at 4°C. After washing the plates, 50 µL of the antibody/alanine peptide mix
189 was added to plate bound peptides and incubated for 20 min at 4°C. Plates were washed and 100
190 µL/well of secondary goat anti-mouse anti IgGγ HRP (1:1000 diluted in blocking buffer) were
191 added and incubated for 90 min at 4°C. A final wash step was performed and plates were
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192 developed using o-phenylenediamine and OD at 490nm was read on a SpectraMax 250
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193 (Molecular Device). lo
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194 Epitope Mapping by Deuterium Exchange Mass Spectrometry. To maximize peptide d

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195 sequence coverage, the optimized quench and proteolysis condition was determined prior to m

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196 DXMS experiments (36). For each sample, 0.4 µl of stock solution of L1 at 6.2 mg/ml was tt
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197 diluted with 7.6 µl of H2O buffer (8.3 mM Tris-HCl, 150 mM NaCl, in H2O, pH 7.2) at 0°C and jv
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198 then quenched with 12 µl of a quench solution containing 0.8% formic acid, 16% glycerol, 1.4 M sm
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199 GdnHCl and 100mM TCEP. The quenched samples were incubated on ice for 5 min and then r
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200 frozen on dry ice and stored at -80°C. Procedures for pepsin digestion from DXMS have been n

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201 described elsewhere (9). In addition, nondeuterated samples (incubated in H2O buffer mentioned ec
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202 earlier) and equilibrium-deuterated back-exchange control samples (incubated in D2O buffer b
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203 containing 0.5% formic acid overnight at 25°C) were prepared as described elsewhere (24). The 2
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204 centroids of the isotopic envelopes of nondeuterated, functionally deuterated, and fully 2
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205 deuterated peptides were measured using HDExaminer and then converted to corresponding
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206 deuteration levels with corrections for backexchange (53). g
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207 Isolation of neutralization escape mutants. A crude stock of mutant VACV was prepared from
208 cells that were infected with VACVWR in the presence of ethyl methanesulfonate (Sigma-
209 Aldrich). Ethyl methanesulfonate was present in the culture medium at 500 µg/ml from 2 h to 6 h
210 post infection. The mutant viral stock was mixed with M12B9 antibody at a final concentration
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