characterization and immunogenicity of a novel mosaic m...

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1 Characterization and Immunogenicity of a Novel Mosaic M HIV-1 gp140 1 Trimer 2 3 Joseph P. Nkolola 1* , Christine A. Bricault 1* , Ann Cheung 1 , Jennifer Shields 1 , James Perry 1 , 4 James M. Kovacs 2 , Elena Giorgi 3 , Margot van Winsen 4 , Adrian Apetri 4 , Els C.M. Brinkman-van 5 der Linden 4 , Bing Chen 2 , Bette Korber 3 , Michael S. Seaman 1 , Dan H. Barouch 1,5** 6 7 1 Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, 8 Boston, MA, 02215, USA 9 2 Division of Molecular Medicine, Children’s Hospital, Boston, MA 02115, USA; 10 Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA 11 3 Theoretical Biology and Biophysics, Los Alamos National Laboratory, and the New 12 Mexico Consortium, Los Alamos, New Mexico, 87506, USA 13 4 Crucell Vaccine Institute, 2301 CA Leiden, The Netherlands 14 15 5 Ragon 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 31 JVI Accepts, published online ahead of print on 25 June 2014 J. Virol. doi:10.1128/JVI.01739-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved. on July 15, 2018 by guest http://jvi.asm.org/ Downloaded from

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1

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

15

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

31

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

33

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

48

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Importance 49

50

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

56

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Introduction 57

58

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

70

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

90

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Methods 91

92

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

104

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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114

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

120

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

125

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

139

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

144

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

156

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

164

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

170

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

174

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

188

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

209

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

226

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

235

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

247

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

265

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

281

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

292

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

304

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

323

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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

342

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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43. Walker, L. M., S. K. Phogat, P. Y. Chan-Hui, D. Wagner, P. Phung, J. L. Goss, T. 673 Wrin, M. D. Simek, S. Fling, J. L. Mitcham, J. K. Lehrman, F. H. Priddy, O. A. 674 Olsen, S. M. Frey, P. W. Hammond, G. P. I. Protocol, S. Kaminsky, T. Zamb, M. 675 Moyle, W. C. Koff, P. Poignard, and D. R. Burton. 2009. Broad and potent 676 neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. 677 Science 326:285-289. 678

44. Wang, S., J. S. Kennedy, K. West, D. C. Montefiori, S. Coley, J. Lawrence, S. Shen, 679 S. Green, A. L. Rothman, F. A. Ennis, J. Arthos, R. Pal, P. Markham, and S. Lu. 680 2008. Cross-subtype antibody and cellular immune responses induced by a polyvalent 681 DNA prime-protein boost HIV-1 vaccine in healthy human volunteers. Vaccine 26:3947-682 3957. 683

45. Wang, S., R. Pal, J. R. Mascola, T. H. Chou, I. Mboudjeka, S. Shen, Q. Liu, S. 684 Whitney, T. Keen, B. C. Nair, V. S. Kalyanaraman, P. Markham, and S. Lu. 2006. 685 Polyvalent HIV-1 Env vaccine formulations delivered by the DNA priming plus protein 686 boosting approach are effective in generating neutralizing antibodies against primary 687 human immunodeficiency virus type 1 isolates from subtypes A, B, C, D and E. Virology 688 350:34-47. 689

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 1

A. B.

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Figure 2

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Figure 3

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Figure 4

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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

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PG9 IgG / MosM gp140

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PG9 IgG / MosM gp120

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PG16 IgG / MosM gp140

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PG16 IgG / MosM gp120

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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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Figure 10 

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Figure 11A 

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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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Figure 12 

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Figure 13

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