Characterization and Immunogenicity of a Novel Mosaic M...
Transcript of Characterization and Immunogenicity of a Novel Mosaic M...
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Characterization and Immunogenicity of a Novel Mosaic M HIV-1 gp140 1
Trimer 2 3
Joseph P. Nkolola1*, Christine A. Bricault1*, Ann Cheung1, Jennifer Shields1, James Perry1, 4
James M. Kovacs2, Elena Giorgi3, Margot van Winsen4, Adrian Apetri4, Els C.M. Brinkman-van 5
der Linden4, Bing Chen2, Bette Korber3, Michael S. Seaman1, Dan H. Barouch1,5** 6 7
1Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, 8 Boston, MA, 02215, USA 9
2Division of Molecular Medicine, Children’s Hospital, Boston, MA 02115, USA; 10 Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA 11
3Theoretical Biology and Biophysics, Los Alamos National Laboratory, and the New 12 Mexico Consortium, Los Alamos, New Mexico, 87506, USA 13
4Crucell Vaccine Institute, 2301 CA Leiden, The Netherlands 14
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5Ragon Institute of MGH, MIT and Harvard, Boston, MA 02114, USA 16 17
Running Title: Characterization & Immunogenicity of Mosaic Env trimer 18 19
(*Authors contributed equally to this study) 20
[Abstract Word Count = 184 Text Word Count = 4,998] 21 22 **Corresponding Author: Dan H. Barouch 23 Address: Center for Virology and Vaccine Research 24
Beth Israel Deaconess Medical Center 25 E/CLS-1043, 330 Brookline Avenue 26
Boston, MA 02215, USA 27 E-mail: [email protected] 28 Tel No: (617) 735-4485 29 Fax No: (617) 735-4527 30
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JVI Accepts, published online ahead of print on 25 June 2014J. Virol. doi:10.1128/JVI.01739-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Abstract 32
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The extraordinary diversity of the human immunodeficiency virus type 1 (HIV-1) Envelope 34
(Env) glycoprotein poses a major challenge for the development of an HIV-1 vaccine. One 35
strategy to circumvent this problem utilizes bioinformatically optimized mosaic antigens. 36
However, mosaic Env proteins expressed as trimers have not been previously evaluated for their 37
stability, antigenicity, and immunogenicity. Here we report the production and characterization 38
of a stable HIV-1 mosaic M gp140 Env trimer. The mosaic M trimer bound CD4 as well as 39
multiple broadly neutralizing monoclonal antibodies, and biophysical characterization suggested 40
an intact and stable trimer. The mosaic M trimer elicited higher neutralizing antibody (nAb) 41
titers against clade B viruses than a previously described clade C (C97ZA.012) gp140 trimer in 42
guinea pigs, whereas the clade C trimer elicited higher nAb titers than the mosaic M trimer 43
against clades A and C viruses. A mixture of the clade C and mosaic M trimers elicited nAb 44
responses that were comparable to the better component of the mixture for each virus tested. 45
These data suggest that combinations of relatively small numbers of immunologically 46
complementary Env trimers may improve nAb responses. 47
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Importance 49
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The development of an HIV-1 vaccine remains a formidable challenge due to multiple 51
circulating strains of HIV-1 worldwide. This study describes a candidate HIV-1 Env protein 52
vaccine whose sequence has been designed by computational methods to address HIV-1 53
diversity. The characteristics and immunogenicity of this Env protein are described, both alone 54
and mixed together with a clade C Env protein vaccine. 55
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Introduction 57
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The generation of HIV-1 Env glycoprotein immunogens that can elicit binding and neutralizing 59
antibodies (nAbs) against diverse, circulating HIV-1 strains is a major goal of HIV-1 vaccine 60
development (2, 19, 28, 39, 41). The surface Env glycoprotein, which is the primary target of 61
neutralizing antibodies, comprises the gp120 receptor-binding subunit and the gp41 fusion 62
subunit, and it is present as a trimeric spike (gp120/gp41)3 on the virion surface. During the 63
course of natural HIV-1 infection, nearly all individuals induce anti-Env antibody responses but 64
generally with poor neutralization breadth (18, 21, 25). It has been reported that approximately 65
10–25% of HIV-1-infected individuals have the ability to produce broadly neutralizing 66
antibodies (bnAbs) (40). However, a recent evaluation of a large global panel of sera from 67
infected individuals showed that many individuals make nAb responses against a significant 68
fraction of viruses (14). 69
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One strategy to address HIV-1 sequence diversity involves the construction of bioinformatically 71
optimized ‘mosaic’ antigens (9), which are in silico recombined HIV-1 sequences designed for 72
improved coverage of global HIV-1 diversity. Several proof-of-concept immunogenicity studies 73
in nonhuman primates have demonstrated that vector-encoded mosaic antigens can augment the 74
depth and breadth of cellular immune responses and also improve antibody responses when 75
compared to consensus and/or natural sequence antigens (3, 34, 35, 42). We have also recently 76
reported the protective efficacy of vector-based HIV-1 mosaic antigens against acquisition of 77
SHIV- SF162P3 challenges in rhesus monkeys (4). However, the generation of HIV-1 mosaic 78
Env trimers as protein immunogens has not previously been described. 79
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In this study, we report the production and characterization of a mosaic M gp140 trimer. The 80
mosaic M gp140 trimer bound CD4 as well as mutiple bnAbs, including VRC01, 3BNC117, 81
PGT121, PGT126, PGT145, PG9, and PG16, and biophysical studies suggested an intact and 82
stable trimer. The mosaic M gp160 also exhibited functional capacity to infect target cells. 83
Immunogenicity studies in guinea pigs showed that the mosaic M gp140 elicited high binding 84
antibody titers, cross-clade tier 1 TZM.bl nAbs, and detectable tier 2 A3R5 nAbs that were a 85
different spectrum than those elicited by our clade C gp140 trimer. The nAb response elicited by 86
a mixture of the mosaic M gp140 and our clade C gp140 proved superior to either trimer alone, 87
and the combination induced nAb responses comparable to the better single immunogen in the 88
mixture for each virus tested. 89
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Methods 91
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Production and expression of mosaic HIV-1 Env proteins 93
The mosaic M Env gene sequences have been described previously (3) (42) (4). The mosaic 94
gp140s were engineered to contain point mutations to eliminate cleavage and fusion activity (3, 95
9). To maximize expression in human cell lines, human codon optimized mosaic M gp140s were 96
synthesized by GeneArt (Life Technologies) with a C-terminal T4 bacteriophage fibritin ‘fold-97
on’ trimerization domain. A polyhistidine motif was included to facilitate protein purification in 98
one version of the protein. Genes were cloned into the SalI-BamHI restriction sites of a pCMV 99
eukaryotic expression vector, inserts were verified by diagnostic restriction digests, DNA was 100
sequenced, and expression testing was performed using 10 µg of DNA with Lipofectamine (Life 101
Technologies) in 293T cells. Stable cell lines for NatC (22) and MosM gp140 Env trimers were 102
generated by Codex Biosolutions. 103
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For protein production, the stable cell lines were grown in Dulbecco’s Modified Eagle Medium 105
(DMEM) (supplemented with 10% FBS, penicillin/streptomycin and puromycin) to confluence 106
and then were changed to Freestyle 293 expression medium (Invitrogen) supplemented with the 107
same antibiotics. Cell supernatants were harvested at 96–108 hours after medium change and the 108
His-tagged gp140 proteins purified by Ni-NTA (Qiagen) and size-exclusion chromatography as 109
previously described (22, 31). The synthetic gene for full-length MosM gp120 was generated 110
from the MosM gp140 construct. The synthetic gene for full-length MosM gp160 used in the 111
TZM.bl assay was synthesized by GeneArt (Life Technologies) and cloned into a 112
pcDNATM3.1/V5-His-TOPO vector (Invitrogen). 113
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The MosM gp140 without a polyhistidine tag was inserted into the pcDNA2004(Neo) vector 115
(GeneArt), which is optimized for expression in PER.C6 cells. Stable MosM gp140 expressing 116
PER.C6 suspension cells were used to produce MosM gp140 trimer without a polyhistidine tag 117
either in batch or fed batch mode. The MosM gp140 was Galanthus Nivalis Lectin (Vector Labs) 118
purified from the supernatant followed by gel filtration chromatography. 119
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Biophysical characterization of MosM gp140 121
The biophysical properties of MosM gp140 without a polyhistidine tag protein produced in 122
PER.C6 cells were determined by SEC-MALS, SEC-QELS, DLS, far-UV CD, SDS-PAGE and 123
DSC as detailed below. 124
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Size Exclusion Chromatography – Multi Angle Light Scattering (SEC-MALS) / Quasi Elastic 126
Light Scattering (SEC-QELS) 127
Size exclusion chromatography was performed using an analytical column (TSKgel 128
G3000SWxl, Tosoh Bioscience) equilibrated with 150 mM sodium phosphate, 50 mM sodium 129
chloride at pH 7.0. Typically, 125 µg of protein was injected and separated at a flow rate of 1 130
mL/min. For molar mass determination, in-line UV (Agilent 1260 Infinity MWD, Agilent 131
Technologies), refractive index (Optilab T-rEX, Wyatt Technology) and 8-angle static light 132
scattering (Dawn HELEOS, Wyatt Technology) detectors were used. Replacement of one of the 133
static light scattering detectors by a dynamic light scattering detector (DynaPro NanoStar, Wyatt 134
Technology) enabled determination of hydrodynamic radii simultaneously. The stability of the 135
MosM gp140 protein was studied by SEC-MALS upon incubation for 30 minutes at 50, 60, 70, 136
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80 or 90°C. Astra Software, including the protein conjugate analysis function, was used for data 137
analysis. 138
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For determination of hydrodynamic radii in batch mode, a dynamic light scattering detector was 140
used (DynaPro Platereader, Wyatt Technology). Light scattering intensity was detected at 25°C 141
at a concentration of 1 mg/ml during 20 acquisitions of 5 seconds each. Hydrodynamic radii 142
were determined using the regularization function in the Dynamics software. 143
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Far-UV Circular Dichroism (far-UV CD) 145
Secondary structure analysis was performed using a circular dichroism spectrometer (Model 420, 146
AVIV Biomedical, Inc) equipped with a recirculating chiller (Thermocube 200/300/400, Solid 147
State Cooling Systems). Far-UV CD spectra were recorded with rectangular 1 mm path length 148
quartz cuvettes at concentrations of 3-4 μM, using a 0.5 nm (determination intrinsic properties) 149
or 1 nm (stability study) step width and an averaging time of 4 seconds. Far-UV CD was 150
recorded after incubation at 40, 50, 60, 70, 80 and 90°C for 10 minutes. After correction of the 151
signal for baseline drift and contribution of buffer components, the molar residual ellipticity 152
(MRE, in deg cm2 dmol-1) was calculated based on the following equation: (0.1 * θλ)/(d * M * # 153
of amino acids), where θλ is the observed ellipticity in milli degrees, d is the path length of the 154
cuvette in cm and M the molar concentration. 155
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Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 157
Samples were prepared under non-reducing conditions by addition of LDS sample buffer 158
(NP0007, Life Technologies) and incubation for 10 min at 70°C or 30 minutes at 98°C. For 159
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reducing conditions, LDS sample buffer and a DTT-based reducing reagent (NP0004, Life 160
Technologies) were added and samples were incubated for 10 minutes at 98°C. Samples (10 161
µg/lane) and HiMark™ Pre-stained Protein Standard (LC5699, Life Technologies) were applied 162
on the gel (EA03752BOX, Life technologies). The gel was stained with Coomassie Blue. 163
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Differential Scanning Calorimetry (DSC) 165
Measurements were performed using a differential scanning calorimeter (MicroCal VP-Capillary 166
DSC System, GE Healthcare). Samples were buffer exchanged to phosphate buffered saline and 167
diluted to 3-4 µM. Scans were recorded from 25°C to 110°C at a scan rate of 60°C/hr. Data was 168
processed using MicroCal VP-Capillary DSC Control Software 2.0. 169
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Recombinant adenovirus serotype 26 vector 171
Replication-incompetent, E1/E3-deleted recombinant adenovirus serotype 26 (rAd26) vector 172
expressing MosM gp140 was prepared as previously described (3). 173
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Western blot immunodetection 175
Supernatants (20 µl) obtained 48-hours post transient transfection of 293T cells with pCMV- 176
MosM, MosM.3.2 or MosM.3.3 gp140 expression constructs were separately mixed with 177
reducing sample buffer (Pierce), heated for 5 minutes at 100ºC and run on a precast 4-15% SDS-178
PAGE gel (Biorad). Protein was transferred to a PVDF membrane using the iBlot dry blotting 179
system (Invitrogen) and membrane blocking performed overnight at 4°C in PBS-T [Dulbeco’s 180
Phosphate Buffered Saline + 0.2% V/V Tween 20 (Sigma) + 5% W/V non-fat milk powder]. 181
Following overnight blocking, the PVDF membrane was incubated for 1 hour with PBS-T 182
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containing a 1:2000 dilution of monoclonal antibody penta His-HRP (Qiagen), washed 5 times 183
with PBS-T and developed using the Amersham ECL plus western blotting detection system (GE 184
Healthcare). For western blot immunodetection using 2F5 and 4E10 monoclonal antibodies, 185
clade A (92UG037.8) gp140 (22, 31) and MosM gp140 proteins were processed as above and 186
detected with an anti-human IgG (Jackson ImmunoResearch). 187
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Surface plasmon resonance 189
Surface plasmon resonance binding assays were conducted on a Biacore 3000 (GE Healthcare) at 190
25°C utilizing HBS-EP running buffer (GE Healthcare). Immobilization of soluble two-domain 191
CD4 (10) (1,500 RU) or protein A (ThermoScientific) to CM5 chips was performed following 192
the manufacturer (GE Healthcare) recommendations. Immobilized IgGs were captured at 300-193
750 RU. Binding experiments were conducted at a flow rate of 50 µl/min with a 2-minute 194
association phase and 5-minute dissociation phase. Regeneration was conducted with a single 195
injection of 35 mM NaOH and 1.3 M NaCl at 100µl/min followed by a 3-minute equilibration 196
phase in HBS-EP buffer. Injections over blank surfaces were subtracted from the binding data 197
for analyses. Binding kinetics were determined using BIAevaluation software (GE Healthcare) 198
and the Langmuir 1:1 binding model with exception of PG16 which was determined using the 199
bivalent analyte model. All samples were run in duplicate and yielded similar kinetic results. 200
Soluble two-domain CD4 and PG9 and PG16 Fabs were produced as described previously (22) 201
(10). 17b hybridoma was provided by James Robinson (Tulane University, New Orleans, LA) 202
and purified as previously described (22). VRC01 was obtained through the NIH AIDS Reagent 203
Program. VRC01 was provided by John Mascola (VRC, NIH, Bethesda, MD) (46). 3BNC117 204
was provided by Michel Nussenzweig (Rockefeller University, New York, NY). PGT121, 205
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PGT126 and PGT145 were provided by Dennis Burton (The Scripps Research Institute, La Jolla, 206
CA). 2F5, 4E10, PG9, PG16 were obtained commercially (Polymun Scientific). GCN gp41-inter 207
(11) was used as a positive control in 4E10 and 2F5 SPR analyses. 208
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Animals and immunizations 210
Outbred female Hartley guinea pigs (Elm Hill) (n=10/group) were housed at the Animal 211
Research Facility of Beth Israel Deaconess Medical Center under approved Institutional Animal 212
Care and Use Committee (IACUC) protocols. Guinea pigs were immunized intramuscularly 213
(i.m.) with either MosM or NatC gp140 Env trimers or both (100 µg/animal) at weeks 0, 4, 8 in 214
500 µl injection volumes divided between the right and left quadriceps. The adjuvant was either 215
15% (vol/vol) oil-in-water Emulsigen (MVP Laboratories)/PBS and 50 μg of immunostimulatory 216
di-nucleotide type B oCpG DNA (5’-TCGTCGTTGTCGTTTTGTCGTT-3’) (Midland Reagent 217
Company) (22) or the ISCOM-based Matrix M (Novavax). The groups of animals receiving the 218
mixture of NatC and MosM gp140s included 50 µg of each trimer for a total of 100 µg per 219
animal. In heterologous prime-boost regimens, a rAd26 vector expressing MosM gp140 (1 x 1010 220
virus particles [vp]/animal) was administered i.m. at week 0 in 500µl PBS/sucrose, and animals 221
were boosted at weeks 8, 12, and 16 with either MosM, NatC or bivalent NatC + MosM gp140 222
Env protein trimers (100 µg/animal) in Adju-Phos alum (Brenntag, Denmark) adjuvant. Serum 223
samples were obtained from the vena cava of anesthetized animals 4 weeks after each 224
immunization. 225
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ELISA binding assays 227
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Serum binding antibody titers against MosM or NatC gp140 Env trimers were determined by 228
endpoint ELISAs as previously described (22, 31). Endpoint titers were defined as the highest 229
reciprocal serum dilution that yielded an absorbance >2-fold over background values. For the 230
detection of MPER epitopes in the MosM trimer, ELISA plates were coated with either 2F5 or 231
4E10 IgG, antigen added, and detected utilizing anti-his tag HRP mAb (Abcam). 2F5 and 4E10 232
were obtained commercially (Polymun Scientific) and GCN gp41-inter (11) used as a positive 233
control in 4E10 and 2F5 ELISAs. 234
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TZM.bl neutralization assay 236
Neutralizing antibody responses against tier 1 HIV-1 Env pseudoviruses were measured using 237
luciferase-based virus neutralization assays with TZM.bl cells as previously described (22, 27, 238
31). These assays measure the reduction in luciferase reporter gene expression levels in TZM-bl 239
cells following a single round of virus infection. The 50% inhibitory concentration (IC50) was 240
calculated as the serum dilution that resulted in a 50% reduction in relative luminescence units 241
compared with the virus control wells after the subtraction of cell control relative luminescence 242
units. The panel of 11 tier 1 viruses analyzed included easy-to-neutralize tier 1A viruses 243
(SF162.LS, MW965.26) and an extended panel of tier 1B viruses (DJ263.8, Bal.26, TV1.21, 244
MS208, Q23.17, SS1196.1, 6535.3, ZM109.F, ZM197M) (27) (37). Murine leukemia virus 245
(MuLV) negative controls were included in all assays. 246
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A3R5 neutralization assay 248
Additional nAb responses were evaluated using the A3R5 assay as previously described (22). 249
Briefly, serial dilutions of serum samples were performed in 10% RPMI growth medium (100 250
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μL per well) in 96-well flat-bottomed plates. IMC HIV-1 expressing Renilla luciferase (8) was 251
added to each well in a volume of 50 μl, and plates were incubated for 1 hour at 37°C. A3R5 252
cells were then added (9×104 cells per well in a volume of 100 μl) in 10% RPMI growth medium 253
containing diethylaminoethyl-dextran (11 μg/ml). Assay controls included replicate wells of 254
A3R5 cells alone (cell control) and A3R5 cells with virus (virus control). After incubation for 4 255
days at 37°C, 90 μl of medium was removed from each assay well, and 75 μl of cell suspension 256
was transferred to a 96-well white, solid plate. Diluted ViviRen Renilla luciferase substrate 257
(Promega) was added to each well (30 μl), and after 4 minutes the plates were read on a Victor 3 258
luminometer. The A3R5 cell line was provided by R. McLinden and J. Kim (US Military HIV 259
Research Program, Rockville, MD). Tier 2 clade B IMC Renilla luciferase viruses included 260
SC22.3C2.LucR, SUMA.LucR and REJO.LucR. Tier 2 clade C IMC Renilla luciferase viruses 261
included Du422.1.LucR.T2A.ecto, Ce2010_F5.LucR.T2A.ecto, and Ce1086_B2.LucR.T2A.ecto, 262
and were provided by C. Ochsenbauer (University of Alabama at Birmingham, Birmingham, 263
AL). Viral stocks were prepared in 293T/17 cells as previously described (8). 264
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Statistical analyses 266
For both the TZM.bl and A3R5 data describe above, the neutralization scores used for plotting 267
and for statistical considerations are provided as the IC50 titer of the post-vaccination serum with 268
pre-vaccination background subtracted as previously described (24). All reactivity that were 269
below the level of assay detection (<20) were assigned the value 20 for plotting and statistical 270
purposes. Titers within a 3-fold range are considered "concordant", thus we considered titer 271
differences of the geometric mean group response that were > 0.5 log as indicative of robust 272
differences. Statistical analyses were performed using the statistical package R (http://www.r-273
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project.org/). Non-parametric Wilcoxon tests were used to compare distributions of values 274
between vaccine groups, Fisher’s exact test was used to compare the relative proportions of 275
responses that were above the level of detection in different vaccine groups, and the R package 276
lme4 (http://lme4.r-forge.r-project.org/lMMwR/lrgprt.pdf) was used to model the relative impact 277
of different variable on neutralization sensitivity levels. 278
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Results 280
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Expression and stability of the mosaic M gp140 trimer 282
We have previously described the Mosaic M Env gene sequences (3, 4). MosM.3.1 Env (whose 283
nomenclature has been simplified here to MosM Env) is more closely related to clade B natural 284
strains, whereas MosM.3.2 Env is most closely related to clade C natural strains, and MosM.3.3 285
is not particularly associated with any clade but captured complementary forms of epitopes that 286
are common throughout the M group and not already represented in MosM and MosM3.2 287
(Figure 1). The mosaic forms, in combination, optimize potential coverage of linear epitopes in 288
the population, but MosM combined with the natural isolate C97ZA.012 (22, 31, 33) (whose 289
nomenclature has been simplified here to NatC) enhanced potential linear epitope coverage to 290
levels approaching using two mosaic trimers (Figure 1). 291
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Eukaryotic pCMV DNA vectors expressing HIV-1 MosM, MosM.3.2 and MosM.3.3 gp140 were 293
transfected into 293T cells, and protein expression and stability were assessed after 48 hours. 294
Western blot analysis revealed a band of the expected size only for MosM gp140 (Figure 2A), 295
suggesting that MosM gp140 was more stable than MosM.3.2 gp140 and MosM.3.3 gp140. Size 296
exclusion chromatography (SEC) using material generated from a stable 293T cell line 297
expressing the MosM gp140 demonstrated a monodisperse peak, confirming the homogeneity of 298
the trimer (Figure 2B). To evaluate the preliminary stability of the MosM trimer, 100 µg protein 299
underwent a freeze-thaw cycle or was stored for 7 days at 4ºC and re-evaluated by SEC and 300
showed no signs of aggregation, dissociation, or degradation (Figure 2C). Given the reduced 301
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stability of MosM.3.2, and the good theoretical coverage of MosM combined with NatC, we 302
focused on the combination of MosM and NatC for the rest of this study. 303
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Biophysical characterization of the MosM gp140 trimer 305
The mass and size of MosM gp140 produced in stable PER.C6 cell clones were assessed under 306
native conditions by size exclusion chromatography (SEC) coupled with online multi-angle light 307
scattering (MALS) and dynamic light scattering (DLS) detectors. The molar mass of the 308
glycoprotein was determined by SEC-MALS to be 454 ± 36 kDa, which indicated that the MosM 309
gp140 was trimeric (Figure 3A). By applying protein conjugate analysis, the estimated 310
molecular weight of the protein component was 258 ± 20 kDa, which corresponds within 311
experimental error of the theoretical molecular weight of an unglycosylated protein trimer. The 312
amount of glycosylation was estimated to be 198 ± 18 kDa, which showed that the level of 313
glycosylation in MosM trimer is significantly higher than previously reported for the BG505 314
SOSIP.664 trimer (17). The tight packing of the MosM gp140 was further confirmed by both 315
batch and online DLS measurements, which showed that the estimated hydrodynamic radius of 316
the protein was 8.2-8.8 nm, similar to the reported 8.1 nm hydrodynamic radius of the BG505 317
SOSIP.664 trimer (17) accounting for the increased glycosylation (Figure 3B). The secondary 318
structure of the MosM trimer was assessed by far-UV circular dichroism (CD) (Figure 3C) and 319
FTIR (data not shown). As expected, both far-UV CD and FTIR confirmed the presence of 320
alpha-helical and beta-sheet secondary structure elements. The mean residue molar ellipticity 321
was assessed from the far-UV CD spectrum as ~ -11,000 deg cm2 dmol-1. 322
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The thermal stability of the MosM trimer was assessed by differential scanning calorimetry 324
(DSC), far-UV CD, and SEC-MALS. No unfolding transitions were detected by DSC upon 325
applying a temperature ramp up to 110 °C (Figure 4A). Furthermore, no significant changes in 326
secondary structure were detected upon exposure of the protein to temperatures up to 90 °C as 327
shown by the unaltered far-UV CD spectra (Figure 4B). The robustness of the protein was also 328
confirmed by FTIR (not shown). The SEC-MALS experimental data indicated that the trimer 329
molecule underwent a partial dimerization to a hexamer at temperatures above 70 °C but no 330
further aggregation or dissociation (Figure 4C). In particular, based on the quantitative 331
recoveries from the SEC column, no higher molecular weight entities were formed at elevated 332
temperatures. Furthermore, the hydrodynamic radius of the trimer did not change upon its 333
exposure to elevated temperatures, thus confirming its remarkable stability. 334
335
The high stability of the MosM trimer was also shown by the difficulty unfolding the protein 336
even in SDS-PAGE loading buffer at elevated temperatures. The MosM trimer could still be 337
observed on a non-reduced gel after the protein was incubated with SDS containing buffer at 70 338
°C (Figure 4D). The trimer band disappeared while some amounts of dimer were still detected 339
when the sample was incubated for 30 min at 98 °C in SDS loading buffer on a non-reduced gel, 340
indicating that the trimer units were not held together via disulfide bridges (Figure 4D). 341
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Antigenicity of the mosaic M gp140 trimer 343
Surface plasmon resonance (SPR) studies were next performed to determine whether the MosM 344
trimer could bind CD4 and broadly neutralizing monoclonal antibodies. The MosM trimer bound 345
CD4 at a high affinity, demonstrating that the CD4 binding site (CD4bs) is present and 346
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accessible (Figure 5A). We next evaluated the ability of the MosM trimer to bind the 347
monoclonal antibody (mAb) 17b in the presence and absence of bound CD4. 17b recognizes a 348
CD4-induced (CD4i) epitope exposed by CD4 binding and the formation of the bridging sheet 349
and co-receptor binding site in gp120 (7, 23). The MosM trimer showed modest 17b binding in 350
the absence of CD4, but there was a clear increase following CD4 binding as expected (Figure 351
5B). We next observed that the broadly neutralizing CD4bs-specific mAbs VRC01 and 352
3BNC117 (36, 46) bound the MosM gp140 with high affinity (Figure 5C, 5D). Moreover, the 353
broadly neutralizing N332 glycan-dependent mAbs PGT121 and PGT126 also bound the trimer 354
with high affinity (Figure 6A, 6B). PG9 and PG16 bind preferentially to intact, correctly folded 355
Env trimers and target V2 glycans (29, 43). PG9 bound the MosM gp140 trimer with high 356
affinity (5.8 x 10-8M), and the corresponding MosM gp120 monomer with a 22-fold lower 357
affinity (1.3 x 10-6M) and substantially lower magnitude (Figure 6C). The PG9 Fab bound to the 358
MosM gp140 trimer with a similar affinity (5.5 x 10-8M) as the complete PG9 IgG (data not 359
shown), confirming the high affinity PG9 binding. Similarly, PG16 bound the MosM gp140 360
trimer with a higher affinity than the corresponding gp120 monomer (Figure 6D), although the 361
off-rate was faster for PG16 compared to PG9. The trimer-specific mAb PGT145 also bound the 362
MosM gp140 trimer (data not shown). 363
364
We also assessed 2F5 and 4E10 binding to membrane proximal external region (MPER) 365
epitopes. 2F5 and 4E10 bind to linear epitopes (5, 32). The 2F5 epitope was present in the linear 366
MosM gp140 sequence as confirmed by sequence alignment (Figure 7A) and Western blot 367
analyses (Figure 7B). However, by SPR and ELISA, the intact MosM gp140 trimer was unable 368
to bind 2F5, suggesting that the MPER epitope is not accessible in the trimer structure, 369
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presumably as a result of being buried (32) (Figure 7C, 7D), similar to our previous findings 370
with the NatC trimer (22). Alternatively, it is possible that the absence of a lipid membrane (1, 371
13) in the MosM gp140 trimer may have reduced 2F5 binding. 372
373
Taken together, the biochemical, biophysical, and antigenicity data suggest that the MosM gp140 374
is an intact and likely well-folded trimer. This is based on the expected molecular mass and 375
hydrodynamic radius of an intact trimer, its remarkable stability, and its capacity to bind all CD4 376
binding site-, V3 glycan-, and V2 glycan-specific broadly neutralizing mAbs that we have tested, 377
except for 2F5 and 4E10 as expected. These findings are surprising, since it has been reported 378
that most uncleaved gp140 trimers are in an open or partially dissociated conformation (16, 26). 379
380
Functionality of mosaic M gp140 trimer 381
We next evaluated whether the MosM Env protein was functional. Full-length MosM gp160 was 382
used to generate pseudovirions to assess infectivity in TZM.bl cells (22, 27, 30, 31). We 383
observed that, over a broad titration range, MosM gp160 Env readily infected TZM.bl target 384
cells over-expressing CD4 and co-receptors CCR5/CXCR4 (Figure 8). These data show that the 385
synthetic MosM Env has the functional ability to infect TZM.bl target cells. 386
387
Immunogenicity of the mosaic M gp140 trimer 388
Guinea pigs (n=10/group) were immunized three times at monthly intervals with 100 µg MosM 389
gp140 trimer, 100 µg NatC gp140 trimer (22, 31), or a mixture of 50 µg of both trimers. Half the 390
animals were immunized with ISCOM-based Matrix M adjuvant, and half were immunized with 391
CpG/Emulsigen adjuvants (15, 22). High titer, binding antibodies by ELISA were elicited by all 392
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the vaccination regimens with comparable kinetics (Figure 9). These responses were detectable 393
after a single immunization and increased after the second and third immunization. Peak binding 394
antibody titers ranged from 5-7 logs. Binding antibody titers elicited by the NatC and MosM 395
trimers were higher against their respective homologous antigens (P<0.05), whereas the bivalent 396
MosM and NatC mixture induced comparable responses to both antigens (Figure 9). ELISA 397
titers were similar using trimers with or without the His tag as coating antigens (data not shown). 398
399
We next assessed serum nAb responses elicited in these animals after the 3rd vaccination using 400
both TZM.bl and A3R5 nAb assays (22, 27, 30, 31). For the TZM.bl assays, we included a multi-401
clade panel of tier 1 pseudoviruses comprising easy-to-neutralize tier 1A viruses (SF162.LS, 402
MW965.26) and an extended panel of intermediate tier 1B viruses (DJ263.8, Bal.26, TV1.21, 403
MS208, Q23.17, SS1196.1, 6535.3, ZM109.F, ZM197M) (27, 37). Background pre-vaccination 404
titers were negative or low in all animals (<20; data not shown). We observed a trend for slightly 405
increased responses with the Matrix M adjuvant as compared with CpG/Emulsigen adjuvant (p = 406
0.05 and 0.06 for the TZM.bl and A3R5 assay, respectively) (Figure 10). 407
408
The nAb responses elicited by the bivalent MosM + NatC trimer mixture were comparable to the 409
better of the two immunogens in the cocktail for each virus tested (Figure 11A). Thus, there 410
was no apparent loss of potency due to dilution or competition in the MosM + NatC mixture, and 411
the overall response to the MosM + NatC cocktail was thus superior to either immunogen alone. 412
A statistical breakdown of the data, comparing response level distributions by a non-parametric 413
Wilcoxon rank sum test is provided in Table 1 and summarized in Figure 11B. Specifically, for 414
2 of the 11 viruses using the TZM.bl assay, the responses were indistinguishable between the 415
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three vaccine groups, although responses to both of these viruses were borderline (ZM197M, 416
6535). For 6 out of the 11 viruses, the responses to the individual MosM and NatC monovalent 417
immunogens were significantly different, and the MosM + NatC combined vaccine was 418
comparable to the higher of the two monovalent vaccines in each case. A similar trend was 419
observed for SF162.LS. The MosM + NatC combination yielded a more potent response than 420
both monovalent vaccines for one virus (MS208), whereas the NatC gp140 slightly out-421
performed the bivalent MosM + NatC vaccine for one virus (ZM109F). Overall, these data 422
suggest that the MosM and NatC gp140 Env trimers were immunologically complementary and 423
could be effectively combined as a mixture that was superior to either trimer alone in terms of 424
nAb coverage. Less clear differences were observed for tier 2 viruses in A3R5 neutralization 425
assays (Figure 12). 426
427
To evaluate the complexity of the interactions between variables that might impact neutralization 428
sensitivity, each assay was analyzed separately using an inverse Gaussian generalized model. 429
Animals and viruses were included in the model as random effects. For fixed effects, we started 430
testing the larger possible model, which included vaccine, clade, adjuvant, and all their possible 431
interactions, and then scaled down the model in a step-wise manner, eliminating one variable at 432
the time until we minimized the Akaike Information Coefficient (AIC). We initially fit the whole 433
dataset with 11 viruses and both adjuvants (Table 2). The best model had a significant 434
interaction between vaccine and clade (P<2.2e-16). This statistical analysis confirms that the 435
MosM trimer, which is more clade B-like (Figure 1A), elicits stronger responses to the clade B 436
viruses, whereas the NatC trimer elicits stronger responses to clade C as well as clade A viruses. 437
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A significant effect from the adjuvant (P=0.0074) was also observed, indicating the higher 438
potency of the Matrix M adjuvant. 439
440
Finally, we assessed nAb responses following priming with a replication-incompetent rAd26 441
vector expressing the MosM gp140 protein, followed by three protein boosts with the NatC, 442
MosM, or bivalent MosM + NatC gp140 trimers (Figure 13; Table 3). This experiment suggests 443
that the mixture of the MosM and NatC trimers was superior to either trimer alone also in the 444
context of a prime-boost vaccine regimen. 445
446
447
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Discussion 448
449
In this study, we assessed the biophysical properties, antigenicity, and immunogenicity of a 450
novel, bioinformatically optimized mosaic M gp140 Env trimer. Although this Env sequence was 451
developed by a sliding linear optimization algorithm (3), the MosM gp140 trimer protein proved 452
remarkably stable and intact. It exhibited the hydrodynamic radius and antigenicity of a correctly 453
folded Env trimer, and it demonstrated marked thermostability and binding to multiple broadly 454
neutralizing mAbs. These results are surprising, since it has been recently reported that most Env 455
gp140 sequences do not form stable trimers and often result in open or partially dissociated 456
conformations (16, 26). Moreover, our uncleaved mosaic M trimer appears to share several key 457
biophysical and antigenic properties with the cleaved BG505 SOSIP.664 trimer, which is 458
believed to be a well-formed trimer (16, 26). 459
460
The NatC trimer was predicted to be a good immunologic complement to the MosM trimer in 461
terms of theoretical global coverage (Figure 1). In guinea pigs, the MosM trimer elicited nAbs 462
primarily against clade B viruses, whereas the NatC trimer (22, 31) induced nAbs primarily 463
against clades A and C viruses (Figure 11; Tables 1-2). Mixing the MosM trimer with the NatC 464
trimer resulted in a cocktail that induced nAb responses that were superior to those obtained 465
using either trimer alone. These findings suggest that it may be possible to improve nAb 466
responses with a relatively small number of Env immunogens. However, the development of 467
Env immunogens that can generate cross-clade, tier 2 nAb responses remains a major unsolved 468
challenge for the HIV-1 vaccine field. 469
470
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Our studies extend previous efforts in the field to develop HIV-1 Env immunogens aimed at 471
increasing nAb breadth. Prior studies have reported the generation of chronic, 472
transmitter/founder, and consensus Envs (24), DNA/Ad prime-boost regimens with multiple 473
diverse Env immunogens (38), polyvalent HIV-1 gp120 Env cocktails delivered by DNA/prime 474
protein-boost regimens (45), and polyvalent DNA/Ad Env immunizations (6). These preclinical 475
studies have highlighted potential benefits of Env cocktails. The feasibility and utility of 476
multivalent Env vaccination has also been explored in clinical trials (20, 44). The current study 477
extends these prior studies and shows that a bivalent combination of the MosM and NatC trimers 478
resulted in improved nAb responses compared with either trimer alone in guinea pigs. 479
480
In conclusion, our studies show the production and immunogenicity of a stable, intact mosaic M 481
gp140 Env trimer. Our data suggest that the mosaic M trimer can immunologically complement 482
a natural clade C Env trimer, and that the combination of the two trimers result in improved nAb 483
responses compared with either trimer alone. These data suggest that multivalent mixtures of 484
carefully selected trimers may represent a promising strategy to improve nAb breadth. 485
486
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Acknowledgements 487
488
The authors thank H. Peng, J. Chen, H. Inganäs, D. Tomkiewicz, H. Verveen, H. van Nes, G. 489
Perdok, K. Hegmans, J. Meijer, R. van Schie, N. Kroos, R. Janson, M. Pau, M. Weijtens, H. 490
Schuitemaker, A. Eerenberg, J. Goudsmit, J. Robinson, J. Mascola, M. Nussenzweig and D. 491
Burton for generous advice, assistance, and reagents. VRC01 was obtained through the NIH 492
AIDS Research and Reference Reagent Program. We acknowledge support from NIAID grants 493
AI078526, AI084794, AI096040; Bill and Melinda Gates Foundation grants OPP1033091, 494
OPP1040741; and the Ragon Institute of MGH, MIT and Harvard. The authors declare no 495
financial conflicts of interest. 496
497 498
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46. Wu, X., Z. Y. Yang, Y. Li, C. M. Hogerkorp, W. R. Schief, M. S. Seaman, T. Zhou, 690 S. D. Schmidt, L. Wu, L. Xu, N. S. Longo, K. McKee, S. O'Dell, M. K. Louder, D. L. 691 Wycuff, Y. Feng, M. Nason, N. Doria-Rose, M. Connors, P. D. Kwong, M. Roederer, 692 R. T. Wyatt, G. J. Nabel, and J. R. Mascola. 2010. Rational design of envelope 693 identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 329:856-694 861. 695
696
697
698
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Figure Legends 699
700
Figure 1. Phylogeny and theoretical coverage of mosaic Env immunogens. (A) 701
Phylogenetic tree illustrating sequence associations of gp140 vaccine candidate sequences and 702
representative sequences from different clades. The subtype reference alignment 703
(http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html) from the Los Alamos HIV 704
database is used here to provide context to illustrate the subtype associations of the different 705
mosaic sequences. Representative sequences of major clades are shown, with the clade indicated 706
by the letter, and vaccine candidates studied here are indicated in bold italics. The mosaics were 707
intended to be used in combination, and when combined they maximize M group epitope 708
coverage, but slightly favor B and C subtypes as these clades are most heavily sampled. This tree 709
was generated using PhyML [(12); 710
http://www.hiv.lanl.gov/content/sequence/PHYML/interface.html], using a HIVb model and 711
parameters estimated from the data. (B) Potential epitope coverage of different potential 712
vaccines and vaccine combinations. We tested the potential epitope coverage (linear 9-mers), 713
restricted to only one Env per person, or a total of 4,186 sequences: 1,501 clade B, 1,031 clade 714
C, 226 clade A, and 1,428 other subtypes and recombinants, grouped together in the category 715
labeled “O” for “other”. Because HIV database sampling is biased towards B and C clades, the 716
coverage is naturally slightly better for these subtypes than for other subtypes; still, using 717
combinations of mosaics give relatively good potential epitope coverage of all subtypes [(9); 718
http://www.hiv.lanl.gov/content/sequence/MOSAIC/epicover.html]. Vaccine combinations are 719
labeled across the bottom. The average fraction of 9-mers per natural strain perfectly matched 720
by the vaccines are indicated by the red bars; the fraction with an 8/9 match or better, by orange; 721
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and a 7/9 match or better by yellow. The only mosaic protein that was stable and well expressed 722
as a trimer, MosM, fortuitously turns out to be an excellent complement to the natural C clade 723
protein (NatC), C97ZA.012, that we had previously targeted for vaccine design because of it 724
expression, stability, antigenic and immunogenic attributes when expressed as a trimer (compare 725
the MosM + MosM.3.2 to MosM + NatC). The coverage of 9-mers for MosM and NatC, the 726
series of coverage graphs on the far right, approaches the coverage of 9-mers for MosM and 727
MosM.3.2, which were optimized for 9-mer coverage. 728
729
Figure 2. Expression and stability of the MosM trimer. (A) Western blot showing expression 730
of HIV-1 MosM, MosM.3.2, and MosM.3.3 gp140 48 hours after transient transfection of 293T 731
cells. NatC gp140 and Clade A (92UG037.8) gp140 were used as positive controls. (B) Size-732
exclusion chromatography (SEC) profile of the purified mosaic MosM trimer with SDS-PAGE 733
of peak fractions. (C) SEC profile of the MosM trimer after freeze-thaw and incubation at 4ºC 734
for 7 days. 735
736
Figure 3. Biophysical characterization of MosM trimer. (A) SEC-MALS profile of the MosM 737
trimer. The total mass of the molecule and the mass contributions of the protein and glycan 738
components are shown for the 9.0-9.7 elution interval as determined from protein conjugate 739
analysis (B) SEC-QELS analysis. The SEC profile of the MosM trimer is shown together with 740
the hydrodynamic radii derived from the autocorrelation functions. (C) Far-UV CD analysis. The 741
mean residue molar ellipticity is shown as a function of wavelength between 200-260 nm. 742
743
744
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Figure 4. Thermal stability of the MosM trimer. (A) DSC thermal scanning of the MosM 745
trimer. Heat capacity was followed as a function of temperature up to 110°C showing no 746
dissociation (black line). For comparison, the DSC profile of a typical monoclonal antibody is 747
shown (blue line). (B) Far-UV CD spectra of the MosM trimer recorded at 25°C and after 10 748
minutes subsequent heating at 40, 50, 60, 70, 80 and 90°C, respectively. (C) SEC-MALS 749
profiles of the MosM trimer in the absence of heat stress and upon incubation for 30 minutes at 750
50, 60, 70, 80 or 90°C, respectively. Quantitative recoveries from the SEC column demonstrate 751
that no high molecular weight species were formed during incubation. (D) SDS-PAGE of the 752
MosM trimer under non-reducing conditions after heating for 10 minutes at 70°C (lane 1) or 30 753
minutes at 98°C (lane 2) and under reducing conditions after heating for 10 minutes at 98 °C 754
(lane 3). The molecular weight ladder used as standard for molecular weight determination is 755
shown on lane 4. 756
757
Figure 5. SPR binding profiles of the MosM trimer to CD4, 17b, VRC01 and 3BNC117. For 758
all bnAb binding experiments, protein A was irreversibly coupled to a CM5 chip and IgGs were 759
captured. (A) Soluble, two-domain CD4 was irreversibly coupled to a CM5 chip and MosM 760
gp140 flowed over the chip at concentrations of 62.5-1000 nM. (B) 17b IgG was captured and 761
MosM gp140 flowed over bound IgG at a concentration of 1000 nM in the presence (red trace) 762
or absence (blue trace) of CD4 bound to the immunogen. (C) VRC01 IgG or (D) 3BNC117 IgG 763
were captured and MosM gp140 flowed over the bound IgGs at concentrations of 62.5-1000 nM. 764
All sensorgrams are presented in black, kinetic fits in green. RU, response units. 765
766
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Figure 6. SPR binding profiles of the MosM trimer to glycan-dependent bnAbs PGT121, 767
PGT126 and trimer-dependent bnAbs PG9 and PG16. For all experiments, protein A was 768
irreversibly coupled to a CM5 chip and IgGs were captured. MosM gp140 was flowed over 769
bound (A) PGT121 IgG and (B) PGT126 IgG at concentrations of 62.5-1000 nM. MosM gp140 770
or gp120 was flowed over bound (C) PG9 IgG and (D) PG16 IgG. Sensorgrams are presented in 771
black, kinetic fits in green. RU, response units. 772
773
Figure 7. Lack of presentation of membrane-proximal external region epitopes by the 774
MosM trimer. (A) Sequence alignment of the 2F5 and 4E10 epitope sequences to MosM gp140 775
trimer sequence. (B) Western blot of 2F5 and 4E10 bound to (1) MosM gp140 (2) 92UG037.8 776
gp140 (positive control) and (3) MosM gp120 (negative control). (C) 2F5 and 4E10 binding 777
ELISA of MosM gp140. Clade A (92UG037.8) gp41-inter (positive control) presented as 778
squares, MosM gp140 presented as triangles. Dotted line indicates assay background threshold. 779
(D) SPR binding of protein A captured 2F5 and 4E10 to MosM gp140. 780
781
Figure 8. MosM gp160 Env pseudovirion infection of TZM.bl cells. Pseudovirions generated 782
with full-length MosM gp160 Env were used to infect target TZM.bl cells expressing CD4 and 783
co-receptors CCR5/CXCR4 in the TZM.bl assay. Broken horizontal line indicates background of 784
TZM.bl cells alone without virus (negative control). RLU, relative luminescence units. 785
786
Figure 9. ELISA antibody endpoint binding titers in guinea pigs. Sera obtained 4 weeks after 787
each immunization with NatC, MosM, or bivalent MosM + NatC gp140s in (A) Matrix M or (B) 788
CpG/Emulsigen adjuvants were assessed by ELISA against MosM and NatC gp140 trimer 789
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antigens. Data are presented as geometric mean titers at each time point +/- standard deviations. 790
The horizontal broken line indicates assay background threshold. *P < 0.05; unpaired t-test. 791
792
Figure 10. Adjuvant comparison. (A) Adjuvant comparison in the TZM.bl assay (B) Adjuvant 793
comparison in the A3R5 assay. In a two-sided test, there was a marginal significance level, 794
suggesting that Matrix M was slightly more potent than CpG/Emulsigen (P = 0.05 for the 795
TZM.bl assay, P = 0.06 for the A3R5 assays). 796
797
Figure 11. TZM.bl nAb responses in guinea pigs. Animals immunized with either NatC, 798
MosM or bivalent MosM + NatC trimers were assayed against tier 1A viruses (SF162.LS, 799
MW965.26) and an extended panel of tier 1B viruses (DJ263.8, Bal.26, TV1.21, MS208, 800
Q23.17, SS1196.1, 6535.3, ZM109.F, ZM197M). (A) Darker colors indicate the Matrix M 801
adjuvant, whereas the equivalent lighter colors indicate the CpG/Emulsigen adjuvants. Individual 802
animals are labeled with the letters of the alphabet, uppercase for animals vaccinated with the 803
Matrix M adjuvant, lowercase for CpG/Emulsigen and are ordered the same way for each virus 804
tested. The viruses are ordered such that the top row and the first 2 panels in the second two had 805
the most distinctive behavior in terms of virus sensitivity to neutralizing activity raised by each 806
vaccine (over a half log difference between the geometric means of the groups). The bottom 807
right shows a representative MuLV negative control. There were only 4 animals in the 808
CpG/Emulsigen, bivalent MosM + NatC vaccine group as one was lost during bleeding 809
procedures. (B) Summary figure comparing the nAb responses to the MosM, NatC and bivalent 810
MosM + NatC trimers to 9 clade A, B, C viruses. *P < 0.05. 811
812
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Figure 12. A3R5 nAb responses in guinea pigs. Animals immunized with either NatC, MosM 813
or bivalent MosM + NatC trimers were assayed against three tier 2 clade B viruses (SC22.3C2, 814
SUMA and REJO) and three tier 2 clade C viruses (Du422.1, Ce2010 and Ce1086_B2). 815
Individual animals are labeled with the letters of the alphabet, uppercase for animals vaccinated 816
with the Matrix M adjuvant, lowercase for CpG/Emulsigen adjuvant and are ordered the same 817
way for each virus tested. 818
819
Figure 13. ELISA and TZM.bl nAb responses with heterologous rAd26-prime, protein-820
boost regimens. (A) Sera obtained 4 weeks after a single priming immunization with rAd26-821
MosM gp140 and 4 weeks after each NatC, MosM or MosM + NatC trimer boost were tested in 822
endpoint ELISAs against MosM and NatC trimer antigens. Data are presented as geometric mean 823
titers at each time point +/- standard deviations. The horizontal broken line indicates assay 824
background threshold. (B) Week 20 sera from animals primed with rAd26-MosM gp140 and 825
boosted 3 times with either the NatC (green bars), MosM (blue bars) or bivalent NatC + MosM 826
gp140 (pink bars) were tested in the TZM.bl assay against MW965.26, SF162.LS, Bal.26 and 827
DJ263.8. Individual animals are labeled with capital letters. The top left panel shows a 828
representative MuLV negative control. 829
830
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Table 1. A summary of the p-values from Wilcoxon rank sum tests comparing group responses to different vaccines. In this 831 analysis, we considered a two sided p-value of < 0.05 using a Wilcoxon rank test to compare the Matrix M vaccine groups shown in 832 Figures 9-11 to be indicative of a difference between groups. If the p-value was < 0.05 for a given virus when comparing the MosM 833 and NatC gp140 vaccine groups, we considered the monovalent groups to be distinctive and compared each of them to the bivalent 834 MosM + NatC vaccine group. If the p-value was > 0.05, we considered the response between the groups to be equivalent, and 835 compared the combined monovalent groups to the bivalent group (single x bivalent). The outcome is summarized in the “Pattern” 836 column on the right; ~ = response levels were approximately the same between two groups, > = one or two of the groups had higher 837 responses. If the Wilcoxon test indicated there was a difference in the distributions, and the geometric means were greater than half a 838 log different, the higher response is indicated by >>. The difference between average values of the log10 neutralization titers two 839 monovalent vaccine groups is given in the Diff column. The six groups that differ by > 0.5 log in their mean responses are indicated in 840 bold. 841 842
843
Clade Assay Virus MosM vs NatC MosM vs
[MosM + NatC] NatC vs
[MosM + NatC] Single x Bivalent Diff Pattern
B TZMbl SF162 0.095 NA NA 0.075 -0.32 MosM+NatC ~ MosM ~ NatC C TZMbl ZM109F 0.008 0.095 0.032 NA 0.49 NatC > MosM+NatC ~ MosM C TZMbl MW965 0.008 0.008 0.69 NA 1.3 MosM+NatC ~ NatC >> MosM C TZMbl TV1 0.010 0.010 0.69 NA 0.73 MosM+NatC ~ NatC >> MosM B TZMbl BaL 0.008 0.15 0.008 NA -1.4 MosM+NatC ~ MosM >> NatC A TZMbl DJ263 0.008 0.008 0.095 NA 0.94 MosM+NatC ~ NatC >> MosM B TZMbl SS1196 0.008 0.075 0.008 NA 0.81 MosM+NatC ~ MosM >> NatC A TZMbl MS208 0.075 NA NA 0.007 0.18 MosM+NatC > MosM ~ NatC A TZMbl Q23 0.044 0.010 0.14 NA 0.14 MosM+NatC ~ NatC > MosM C TZMbl ZM197M 0.40 NA NA 0.38 0.15 MosM+NatC ~ NatC ~ MosM B TZMbl 6535 0.55 NA NA 0.58 -0.11 MosM+NatC ~ NatC ~ MosM C A3R5 Du422 0.22 NA NA 0.013 0.2 MosM+NatC >> NatC ~ MosM B A3R5 SC22 0.032 0.55 0.095 NA -0.58 MosM+NatC ~ MosM >> NatC C A3R5 Ce1086 0.22 NA NA 0.013 0.27 MosM+NatC > NatC ~ MosM C A3R5 Ce2010 0.60 NA NA 0.098 0.10 MosM+NatC ~ NatC ~ MosM B A3R5 SUMA 0.75 NA NA 0.075 0.20 MosM+NatC ~ NatC ~ MosM
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Table 2. Summary of the results of an inverse-Gaussian generalized model with virus and animal as random effects. We 844 initially fit the whole dataset with 11 viruses and both adjuvants. The best model had a significant interaction between vaccine and 845 clade (P<2.2e-16) and a significant effect from the adjuvant (P=0.0074). Matrix M responses were on average 1.6 times higher than 846 CpG/Emulsigen responses (see model summary below). The summary shows the estimated effects (in logarithmic scale). Effects of 847 categorical variables are with respect to the reference category: the row labeled “AdjuvantMatrixM”, for example, shows the effect of 848 Matrix M compared to the Emulsigen adjuvant. “VaccineMosM+NatC” and “VaccineMosaicM” show the estimated effects with 849 respect to the reference category, which in this case is the NatC vaccine. The estimate indicates the relative impact, on a log scale, of 850 a given factor relative to the adjuvant CpG/Emulsigen (Matrix M is log 0.2 higher), the NatC vaccine, the virus clade A. The colon (:) 851 indicates an interaction between two variables. 852
(Intercept) Estimate Std Error t-value
Pr(>|z|)
Adjuvant Matrix M 2.2 0.18 11.97 <2e-16 *** Vaccine MosM+NatC 0.21 0.050 4.17 3.1e-05 ***
Vaccine MosM 0.089 0.084 1.06 0.29 Clade B -0.10 0.24 -0.42 0.68 Clade C 0.98 0.24 4.07 4.80e-05 ***
Vaccine MosM+NatC:CladeB 0.31 0.10 3.06 0.0022 ** Vaccine MosM:CladeB 0.71 0.10 7.36 1.82e-13 ***
Vaccine MosM+NatC:CladeC -0.18 0.10 -1.78 0.075 Vaccine MosM:CladeC -0.21 0.09 -2.42 0.015 *
853
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Table 3. Heterologous prime-boost vaccination TZM.bl assay comparison. A table summarizing comparisons the Env nAb 854 responses in guinea pigs primed with rAd26-MosM gp140 and boosted with either NatC, MosM or bivalent MosM + NatC trimers. 855 The mean response of the log titers for vaccines MosM and NatC single antigen boost vaccines for each Env differed by more than 856 half a log in each case. 857 858
Clade Virus MosM vs NatC MosM vs [MosM + NatC] NatC vs [MosM + NatC] Boost Pattern
C MW965.26 0.0079 0.014 0.19 MosM+NatC ~ NatC >> MosM
B SF162.LS 0.056 0.17 0.28 MosM+NatC ~ NatC ~ MosM
B BaL.26 0.018 0.88 0.018 MosM+NatC ~ MosM >> NatC
A DJ263.8 0.009 0.056 0.063 MosM+NatC ~ NatC > MosM
859
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Figure 5
RU RU
A. B.
RU RU
C. D.time (sec)
time (sec) time (sec)
time (sec)
MosM gp140 MosM gp140
MosM gp140 MosM gp140
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Figure 6
PGT121 IgG / MosM gp140
100 200 300 400 5000
20
40
60
80
time (sec)
RU
PGT126 IgG / MosM gp140
0 100 200 300 400 500
0
20
40
60
80
100
120
time (sec)
PG9 IgG / MosM gp140
0 100 200 300 400 5000
50
100
150
200
250
RU
PG9 IgG / MosM gp120
0 100 200 300 400 5000
50
100
150
200
250
time (sec)
PG16 IgG / MosM gp140
0 100 200 300 400 5000
50
100
150
200
PG16 IgG / MosM gp120
0 100 200 300 400 5000
50
100
150
200
time (sec)
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Figure 7
RU RU
4E10 NWFXITXXLW
Mosaic 668SLWNWFDISNWLW 680
LS I
2F5 ELDKWA
Mosaic 660LLELDKWASL669
181
115
82644837
25
2F5 4E10
181
115
82644837
25
Mos
M g
p140
Cla
de A
gp1
40M
osM
gp1
20
Mos
M g
p140
Cla
de A
gp1
40M
osM
gp1
20
4E10 NWFXITXXLW
Mosaic 668SLWNWFDISNWLW 680
LS I
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Figure 8
100 101 102 103 104 105 106 107 108 109
103
104
105
106
Virus Stock Dilution
RLU
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Figure 9
A. NatC gp140 MosM gp140 NatC + MosM gp140s
B. NatC gp140 MosM gp140 NatC + MosM gp140s
NatC gp140 bindingMosM gp140 binding
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101
102
103
104
105
106
DJ263.8
* *
101
102
103
104
105
106
SF162.LS
**
101
102
103
104
105
106
MW965.26* *
101
102
103
104
105
106
MS208
* *
101
102
103
104
105
106
Bal26
**
101
102
103
104
105
106
TV1.21
* *
NatC MosM NatC + MosM101
102
103
104
105
106
Q23.17
* *
NatC MosM NatC + MosM101
102
103
104
105
106
SS1196.1
**
NatC MosM NatC + MosM101
102
103
104
105
106
ZM109.F
* *
Clade A Clade B Clade C
ID50
Tite
r 1/X
Figure 11B
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