Generation of long-lived bone marrow plasma cells secreting antibodies specific for HIV-1 gp41

63 Pages·2016·7.67 MB·English

JVI Accepted Manuscript Posted Online 27 July 2016
J. Virol. doi:10.1128/JVI.01089-16
Copyright © 2016 Donius et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Running Head: rmAbs from LLPC induced by MPER/liposome immunization
1 Title: Generation of long-lived bone marrow plasma cells secreting antibodies specific
2 for HIV-1 gp41 MPER in the absence of polyreactivity
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4 Running title: rmAbs from LLPC induced by MPER/liposome immunization
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6 Luke R. Donius1*, Yuxing Cheng1*, Jaewon Choi1, Zhen-Yu J. Sun2, Melissa Hanson3, o
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7 Michael Zhang 3, Todd M. Gierahn 6, Susanna Marquez4, Mohammed Uduman4, Steven lo
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8 H. Kleinstein4,5, Darrell Irvine3,6,7, J. Christopher Love6, Ellis L. Reinherz1 and Mikyung e
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9 Kim1# ro
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11 1Laboratory of Immunobiology and Dept. of Medical Oncology, Dana-Farber Cancer //
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12 Institute, 35 Binney St., Boston, MA 02115; 2Dept. of Biological Chemistry & Molecular as
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13 Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115; 3Dept. o
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14 of Materials Science and Engineering, and Biological Engineering, 77 Massachusetts o
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15 Ave., Massachusetts Institute of Technology, Cambridge, MA 02139; 4Dept. of p
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16 Pathology, Yale School of Medicine, New Haven, CT 06520; 5Dept. of Immunology, 5
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17 Yale School of Medicine; Interdepartmental Program in Computational Biology and 1
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18 Bioinformatics, Yale University, New Haven, CT 06520; 6Koch Institute for Integrative y

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19 Cancer Research at MIT, 500 Main St., Cambridge, MA 02139; 7Howard Hughes e
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20 Medical Institute, Chevy Chase, MD 20815
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22 *equal contribution
23 #Email address of corresponding author: [email protected];
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Running Head: rmAbs from LLPC induced by MPER/liposome immunization
24 YC present address: Pfizer, 610 Main St., Cambridge MA 02139; JC present address is
25 Bristol Myers Squibb, 38 Jackson Road, Devens MA 01434; MH present address is
26 Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris, France; MZ present address
27 is Univ. of Maryland, 1000 Hilltop Circle, Baltimore MD 21250
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29 Word count: Abstract 223; Text (excluding references, tables, footnotes, legends) 7861 o
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Running Head: rmAbs from LLPC induced by MPER/liposome immunization
30 Abstract
31 An effective preventive vaccine is highly sought after in order to stem the current HIV-1
32 pandemic. Both conservation of contiguous gp41 membrane proximal external region
33 (MPER) amino acid sequences across HIV-1 clades and the ability of anti-MPER
34 broadly neutralizing antibodies (BNAbs) to block viral hemifusion/fusion, establish the
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35 MPER as a prime vaccination target. In earlier studies, we described the development o
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36 of an MPER vaccine formulation that takes advantage of liposomes to array the MPER lo
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37 on a lipid bilayer surface, paralleling its native configuration on the virus membrane, e
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38 while also incorporating molecular adjuvant and CD4 T cell epitope cargo. Here we o
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39 demonstrate that several immunizations with these MPER/liposomes induce high levels
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40 of bone marrow long-lived plasma cell (LLPC) antibody production. Single cell //
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41 immunoglobulin gene retrieval analysis shows that these plasma cells are derived from s
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42 a germline repertoire of B cells with a diverse representation of immunoglobulin genes, o
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43 exhibiting antigen-driven positive selection. Characterization of LLPC recombinant o
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44 monoclonal antibodies (rmAbs) indicates that antigen recognition is achieved through p
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45 convergence on a common epitopic focus utilizing various complementarity determining 5
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46 region H3 (CDRH3) lengths. Importantly, the vast majority of rmAbs produced from 1
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47 these cells lack polyreactivity, yet manifest antigen specificity in the context of lipids, y

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48 shaping MPER-specific paratopes through selective pressure. Together these findings e
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49 demonstrate that the MPER is a vaccine target with minimal risk of generating off-target
50 autoimmunity.
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51 Importance
52 A useful vaccine must generate desired long-term, antigen-specific antibody responses
53 devoid of polyreactivity or autoreactivity. The common polyreactive features of some
54 HIV-1 BNAbs have raised concern about elicitation of anti-MPER antibodies. Utilizing
55 single LLPC repertoire analysis and biophysical characterization of anti-MPER rmAbs,
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56 we show that their fine specificities require a structural fitness of the antibody combining o
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57 site involving heavy and light chain variable domains shaped by somatic hypermutation lo
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58 and affinity maturation of B cells in the germinal center. Perhaps more importantly, our e
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59 results demonstrate that the majority of MPER-specific antibodies are not inherently o
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60 polyspecific and/or autoreactive, suggesting that polyreactivity of the MPER-specific
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61 antibodies are separable from their antigen specificity. //
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62 s
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63 Introduction o
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64 To date no widely applicable cure for HIV-1 is known and current preventive efforts o
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65 have not proven completely effective. Successful vaccination would be a powerful p
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66 means to fight the global HIV-1 pandemic. Unlike infectious diseases against which 5
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67 vaccines induce highly protective immunity (1), broad and potent neutralization of HIV-1 1
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68 strains have not been elicited through vaccination with HIV-1 protein envelope (Env) y

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69 subunits or inactivated virus. However, discovery of numerous broadly neutralizing e
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70 antibodies (BNAbs) capable of blocking viral binding or entry to host cells has
71 suggested that vaccination is a promising strategy (2-4).
72
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73 The HIV-1 envelope spike protein, comprised of trimeric gp41 and gp120 subunits, is
74 the only viral target exposed on the virion membrane surface and therefore is the
75 singular focus for an antibody-based vaccine. The first HIV-1 BNAb discovered, 2F5,
76 was specific for the membrane proximal external region (MPER) and more recently, the
77 MPER-specific neutralizing antibody list has grown to include 4E10, Z13e1, m66, m66.6,
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78 10E8, and CAP206-CH12 (5-12). The BNAb list has also widened over time with o
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79 identification of a variety of other targets including the CD4-binding site, V1/V2-glycan- lo
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80 containing epitope, V3-glycan-containing epitope, and gp120/gp41-bridging epitopes. e
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81 These BNAbs have been discovered through the recovery of single memory B cells o
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82 from infected individuals and recombinant monoclonal antibody (rmAb) production
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83 [reviewed in (2, 13, 14)]. Nevertheless, as one of the most highly-conserved regions on //
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84 the envelope spike the MPER remains an exemplary vaccine target (9, 15, 16). s
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85 o
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86 The MPER is a hydrophobic and tryptophan-rich segment of 22 amino acids in length o
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87 located immediately external to the transmembrane (TM) domain of gp41 (15, 17). p
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88 Structurally, the MPER consists of two alpha-helices connected by a linker in a helix- 5
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89 hinge-helix motif in a lipid environment (16, 18). We have previously shown that the 1
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90 BNAbs 2F5 and 4E10 mediate extraction of their epitopic residues on the MPER helices y

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91 from the lipid membrane (18-20). Very recently, the first micelle-embedded trimer spike e
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92 structure that included the MPER and TM regions was elegantly solved using cryo-EM,
93 and this structure suggests that in a 10E8-bound conformation the MPER is lifted up off
94 the membrane (21). A recent crystallographic analysis has identified lipid as an integral
95 component of the 4E10 BNAb and implied a similar MPER segment extraction geometry
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96 out of the membrane (22). Functionally, the MPER has been shown to be required for
97 both hemifusion and fusion processes preceding viral entry (15-17, 23-25) presumably
98 through its strong interaction with membrane. Therefore, antibodies elicited by
99 vaccination that bind with high affinity to the MPER on the trimer would impede or block
100 MPER function and manifest neutralizing activity.
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101 o
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102 Extensive biochemical and structural analysis of MPER-specific BNAbs have suggested lo
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103 the obligate role of membrane environment in MPER immunogen design, both to e
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104 configure native MPER structure as well as to induce potent BNAbs (18, 19, 22, 26-35). o
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105 Such requirements are likely explanations for the lack of anti-MPER neutralizing
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106 antibodies elicited through vaccination with free MPER peptides, MPER epitope //
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107 mimetics or when grafted onto protein scaffolds [(28, 36-38) and reviewed in (39)]. s
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108 Nonetheless, while liposome-based MPER vaccines induce strong MPER antibody o
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109 responses (40-45), the chemical modifications used for membrane anchoring and o
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110 prevention of proteolysis, such as palmitic acid adducts and amide capping, respectively, p
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111 impact antigenic determinants even without inducing alterations in the MPER segment 5
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112 structure (42). These challenges for membrane-arrayed MPER segment immunogen 1
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113 design still await innovative solutions. Moreover, it has been proposed that sequence y

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114 similarity of endogeneous mammalian proteins with the MPER limits expansion of B e
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115 cells expressing germline-encoded MPER reactive B cell receptors (BCR) because of
116 negative selection during development (46, 47). It has further been suggested that HIV-
117 1 Env gp41 antibodies might arise from polyreactive B cells, possibly from the pool of
118 gut microbe regulating B cells (48, 49). This notion might explain why acutely HIV-1-
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119 infected subjects have antibodies whose unmutated ancestors react with bacterial or
120 host antigens but not HIV-1 envelope. The highly polyreactive nature of 2F5 and 4E10
121 MPER BNAbs has been construed as evidence for the existence of such pathway (47,
122 50, 51). These are sound proposals; however, given the ability of certain BNAbs,
123 notably 10E8, to arise independent of polyreactivity (6), it is not a sine qua non that a
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124 successful MPER immunogen must elicit antibodies with this characteristic in order to o
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125 be broadly neutralizing. lo
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126 e
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127 In this study, we have experimentally assessed the extent to which durable, affinity- o
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128 matured anti-MPER antibody responses induced by MPER/liposome vaccination exhibit
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129 polyreactivity. We demonstrate that MPER/liposome immunizations generate MPER- //
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130 specific bone marrow (BM) long-lived plasma cells (LLPC) of class-switched Abs. Single s
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131 plasma cell analysis followed by immunoglobulin gene rescue, sequencing, and o
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132 analysis of expressed rmAb reveal that BM LLPC use a diverse collection of o
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133 immunoglobulin genes to target the same epitope specificity but with functionally distinct p
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134 characteristics. Affinity maturation of multiple distinct B cells post-immunization and 5
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135 boosting yields MPER-epitope specificity through structural fitness and convergence on 1
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136 a common antigenic determinant, accompanied by little, if any, polyreactivity in 39 of 44 y

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137 rmAbs analyzed. These characteristics demonstrate that MPER-specificity is not e
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138 defined by polyreactivity for the vast majority of rmAbs that recognize membrane
139 embedded MPER by vaccination.
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141 Materials and Methods
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142 Mice and MPER/liposome Immunizations
143 BALB/c mice were obtained from Taconic Biosciences (Hudson, NY). All mice used
144 were 8-10 weeks of age at time of initial immunization. Mice were housed in a specific
145 pathogen-free facility and maintained in accordance with procedures and protocols
146 approved by the Dana-Farber Cancer Institute and Harvard Medical School Animal
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147 Care and Use Committee Institutional Review Board. Immunization liposomes were o
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148 made as before (42) by drying the following components under a nitrogen stream and lo
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149 placing them under vacuum overnight: N- or C-terminally palmitoylated MPER peptides e
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150 (Npalm-MPER or Cpalm-MPER), monophosphoryl lipid A (MPLA) from Salmonella o
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151 enterica serotype minnesota (Sigma-Aldrich, St.Louis, MO), and the lipids 1,2-dioleoyl-
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152 sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine //
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153 (DMPC), and 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) (Avanti Polar s
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154 Lipids Inc, Alabaster, AL) at a 2:2:1 ratio. Liposomes were formed with encapsulated o
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155 LACK1 peptide by rehydrating with phosphate-buffered saline (PBS) containing 1000 o
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156 mg/ml LACK1 to a final 25.2 mg/ml liposome concentration. Final liposomes p
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157 incorporated MPER at 1:200 (MPER:lipid molar ratio) and 175 μg/ml MPLA with a total 5
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158 lipid concentration of 25mg/ml. MPER/liposomes were sized by 6X vortexing for 30 1
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159 seconds at 5 minute intervals, 6X flash freezing in liquid nitrogen and thawing at 37oC, y

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160 and extruding by passing through 100nm pore-size polycarbonate membrane 21 times. e
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161 Mice were immunized intradermally with 50 μl per hind flank. Unless noted,
162 immunizations were administered on three occasions at 21 day intervals.
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164 Microengraving and Single B cell Isolation
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165 Npalm-MPER/liposomes consisting of MPER at a 1:50 molar ratio with DOPC and
166 DOPG lipids (4:1) at a concentration of 2mg/ml were rehydrated with vortexing,
167 freeze/thawing, and extrusion as above. Poly-lysine-coated glass slides (25x75x1mm)
168 were incubated, rocking at room temperature, submerged in 100 μg/ml liposomes in
169 PBS overnight. The liposome solution was poured off and slides were blocked for 1
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170 hour in a 3% milk 0.05% tween-20 PBS solution with rocking at room temperature. o
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171 Polymethylsiloxane microwell molds were prepared as described in detail by Ogunniyi lo
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172 et al. (52). Molds were subjected to plasma cleaning under vacuum to charge and e
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173 sterilize the surface, and then were blocked with 1% bovine serum albumin (BSA) in o
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174 PBS for 20 minutes. The blocking solution was rinsed from slides with purified water.
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175 CD138+ plasma cells were sorted from the BM of femurs and tibias from one mouse //
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176 using a Miltenyi Biotec (Bergisch Gladbach, Germany) magnetic bead labeling and s
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177 enrichment kit. The mold was removed from blocking buffer and aspirated followed by o
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178 three washes with 10% fetal bovine serum containing 1% penicillin/streptomycin in o
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179 RPMI1640 (growth media). The growth media was aspirated and plasma cells p
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180 (~100,000 in 300 μl growth media) were dropwise distributed evenly across the mold. 5
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181 The mold was aspirated and growth media was distributed onto the mold surface. The 1
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182 mold was then aspirated and growth media plus 100ng/ml IL-6 was distributed on the y

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183 mold before the MPER/liposome-labeled slide was placed on the mold and clamped in e
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184 place in a hybridization chamber for 1 hour at 37oC. Following incubation the slide was
185 washed, blocked, and secondary antibody (goat anti-mouse IgG-AF647/anti-IL6-AF488)
186 hybridization was carried out in a Tecan HS 400 Pro slide washer (Mannedorf,
187 Switzerland). Microarray analysis of slides was performed using a GenePix 4400
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Running Head: rmAbs from LLPC induced by MPER/liposome immunization
188 (Molecular Devices LLC, Sunnyvale, CA) at 488nm and 635nm. The mold with plasma
189 cells was stored submerged in growth media at 4oC overnight. Microarray analysis and
190 identification of wells containing MPER-specific cells was performed using GenePix Pro
191 6 analysis software. The mold with plasma cells was stored submerged in growth media
192 at 4oC overnight, and single plasma cells were isolated by micromanipulator into 96-well
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193 plates containing a solution of 1 μl RNasin Plus RNase Inhibitor (Promega, Madison, o
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194 WI) and 9 μl UltraPure DNase-/RNase-free H2O (Life Technologies, Grand Island, NY). lo
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195 Plates were sealed and stored at -80oC for later RNA isolation/cDNA synthesis. e
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196 o
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197 MPER-specific Single B cell cDNA Generation, PCR, and Cloning
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198 Our method for cloning and expression of mouse immunoglobulin genes from single B //
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199 cells was adopted from the method developed by Tiller et al. (53) and utilized the s
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200 primers and vectors they described. In brief, 96-well plates with single B cells were o
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201 thawed on ice and 5 μl of reverse transcription primer mix was added to each well (1 μl o
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202 random primers, 1 μl 10mM dNTP, 1pmol 1st polymerase chain reaction (PCR) reverse p
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203 heavy- and kappa light-chain (IgH and IgLk) primers, DNase/RNase-free water). Plate 5
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204 was heated to 65oC for 5 minutes in a thermocycler and then cooled on ice for 1 minute. 1
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205 SuperScript3 reverse transcription mix containing 5X reverse transcription buffer, DTT, y

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206 RNase Out, SuperScript3, and DNase/RNase-free water was added to final 30 μl e
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207 volume and cDNA synthesis was performed by running a ramped thermocycler
208 program: 25oC, 5 minutes; 50oC, 60 minutes; 55oC, 60 minutes; and 70oC, 15 minutes.
209 A nested PCR strategy was used to amplify IgH or IgLk chain immunoglobulin genes
210 from cDNA. First and second round nested PCR were performed using high-fidelity Q5
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