key: cord-0907110-0441ezu8 authors: Vogel, Annette B.; Kanevsky, Isis; Che, Ye; Swanson, Kena A.; Muik, Alexander; Vormehr, Mathias; Kranz, Lena M.; Walzer, Kerstin C.; Hein, Stephanie; Güler, Alptekin; Loschko, Jakob; Maddur, Mohan S.; Tompkins, Kristin; Cole, Journey; Lui, Bonny G.; Ziegenhals, Thomas; Plaschke, Arianne; Eisel, David; Dany, Sarah C.; Fesser, Stephanie; Erbar, Stephanie; Bates, Ferdia; Schneider, Diana; Jesionek, Bernadette; Sänger, Bianca; Wallisch, Ann-Kathrin; Feuchter, Yvonne; Junginger, Hanna; Krumm, Stefanie A.; Heinen, André P.; Adams-Quack, Petra; Schlereth, Julia; Kröner, Christoph; Hall-Ursone, Shannan; Brasky, Kathleen; Griffor, Matthew C.; Han, Seungil; Lees, Joshua A.; Mashalidis, Ellene H.; Sahasrabudhe, Parag V.; Tan, Charles Y.; Pavliakova, Danka; Singh, Guy; Fontes-Garfias, Camila; Pride, Michael; Scully, Ingrid L.; Ciolino, Tara; Obregon, Jennifer; Gazi, Michal; Carrion, Ricardo; Alfson, Kendra J.; Kalina, Warren V.; Kaushal, Deepak; Shi, Pei-Yong; Klamp, Thorsten; Rosenbaum, Corinna; Kuhn, Andreas N.; Türeci, Özlem; Dormitzer, Philip R.; Jansen, Kathrin U.; Sahin, Ugur title: A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates date: 2020-09-08 journal: bioRxiv DOI: 10.1101/2020.09.08.280818 sha: 98b2386315c67ac4cde876446ad146451f704be9 doc_id: 907110 cord_uid: 0441ezu8 To contain the coronavirus disease 2019 (COVID-19) pandemic, a safe and effective vaccine against the new severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is urgently needed in quantities sufficient to immunise large populations. In this study, we report the design, preclinical development, immunogenicity and anti-viral protective effect in rhesus macaques of the BNT162b2 vaccine candidate. BNT162b2 contains an LNP-formulated nucleoside-modified mRNA that encodes the spike glycoprotein captured in its prefusion conformation. After expression of the BNT162b2 coding sequence in cells, approximately 20% of the spike molecules are in the one-RBD ‘up’, two-RBD ‘down’ state. Immunisation of mice with a single dose of BNT162b2 induced dose level-dependent increases in pseudovirus neutralisation titers. Prime-boost vaccination of rhesus macaques elicited authentic SARS-CoV-2 neutralising geometric mean titers 10.2 to 18.0 times that of a SARS-CoV-2 convalescent human serum panel. BNT162b2 generated strong TH1 type CD4+ and IFNγ+ CD8+ T-cell responses in mice and rhesus macaques. The BNT162b2 vaccine candidate fully protected the lungs of immunised rhesus macaques from infectious SARS-CoV-2 challenge. BNT162b2 is currently being evaluated in a global, pivotal Phase 2/3 trial (NCT04368728). Page 6 (100 µg dose level). For comparison, the S1-binding IgG GMC of a panel of 38 SARS-CoV-2 145 convalescent human sera was 631 U/mL, substantially lower than the GMCs of the immunised 146 rhesus macaques after one or two doses. 147 Fifty percent virus neutralisation GMTs, measured by an authentic SARS-CoV-2 neutralisation 148 assay 25 that produced IFNγ, IL-2, and TNF but a low frequency of CD4 + T cells that produced IL-4, 160 indicating a TH1-biased response (Fig. 3e,f) . BNT162b2 also elicited S-specific 161 IFNγ + producing CD8 + T cells (Fig. 3g) . 162 Six rhesus macaques that had received two immunisations with 100 µg BNT162b2 and three 163 age-matched macaques that had received saline were challenged 55 days after Dose 2 with 164 1.05 × 10 6 plaque forming units of SARS-CoV-2 (strain USA-WA1/2020), split equally 165 between intranasal and intratracheal routes, as previously described 26 . Three additional non-166 immunised, age-matched rhesus macaques (sentinels) were mock-challenged with cell culture 167 medium. Nasal and oropharyngeal (OP) swabs were collected and bronchoalveolar lavage 168 (BAL) was performed at the times indicated, and samples were tested for SARS-CoV-2 RNA 169 (genomic RNA or subgenomic transcripts) by reverse-transcription quantitative polymerase 170 chain reaction (RT-qPCR; Fig. 4 ). All personnel performing clinical, radiological, 171 histopathological, or RT-qPCR evaluations were blinded to the group assignments of the 172 macaques. 173 Viral RNA was detected in BAL fluid from 2 of the 3 control-immunised macaques on Day 3 174 after challenge and from 1 of 3 on Day 6 ( Fig. 4a ). At no time point sampled was viral RNA Page 7 detected in BAL fluid from the BNT162b2-immunised and SARS-CoV-2 challenged macaques and control-immunised rhesus macaques after challenge is highly statistically significant (by a 178 nonparametric test, p=0.0014). 179 From control-immunised macaques, viral RNA was detected in nasal swabs obtained on Days 180 1, 3, and 6 after SARS-CoV-2 challenge; from BNT162b2-immunised macaques, viral RNA 181 was detected only in nasal swabs obtained on Day 1 after challenge and not in swabs obtained 182 on Day 3 or subsequently (Fig. 4b) . The pattern of viral RNA detection from OP swabs was 183 similar to that for nasal swabs (Fig. 4c) . In mice, a single injection of BNT162b2 elicited high neutralizing titers and strong TH1 and TFH 198 type CD4 + and IFNγ + IL-2 + CD8 + T-cell responses. Both BNT162b2 induced CD4 + T-cell types 199 may support antigen-specific antibody generation and maturation, and potentially protection 200 from infectious challenge. Limitation and clearance of virus infection is promoted by the 201 interplay of neutralising antibodies with CD8 + T cells that eliminate intracellular virus 202 reservoirs. CD8 + T cells may also reduce the influx of monocytes into infected lung tissue, 203 which can be associated with undesirable IL-6 and TNF production and impaired antigen 204 presentation 28, 29 . The contributions of the immune effector systems to human protection from Page 8 SARS-CoV-2 is not yet understood. Therefore, it appears prudent to develop COVID-19 206 vaccines that enlist concomitant cognate B cell, CD4 + T cell, and CD8 + T-cell responses. 207 The immunogenicity of BNT162b2 in rhesus macaques paralleled its immunogenicity in mice. 208 Seven days after Dose 2 of 100 µg, the neutralising GMT reached 18-times that of a human 209 SARS-CoV-2 convalescent serum panel and remained 3.3-times higher than this benchmark 210 five weeks after the last immunisation. The strongly TH1-biased CD4 + T-cell response and 211 Purified recombinant SARS-CoV-2 S1 subunit including a histidine tag and the RBD tagged 254 with the Fc region of human IgG1 (both Sino Biological) were used in ELISA to detect SARS-255 CoV-2 S-specific IgG in mice. Purified recombinant SARS-CoV-2 S1 and RBD with a histidine 256 tag (both Sino Biological) were used for surface plasmon resonance (SPR) spectroscopy. An 257 overlapping 15-mer peptide pool of the S protein was used for ELISpot, cytokine profiling and 258 intracellular cytokine staining. An irrelevant peptide control (SPSYVYHQF, derived from gp70 259 AH-1 33 ) or a CMV peptide pool was used as control for ELISpot assays. All peptides were 260 obtained from JPT Peptide Technologies. To express P2 S for structural characterisation, a gene encoding the full length of SARS-CoV-308 2 (GenBank: MN908947) with two prolines substituted at residues 986 and 987 followed with 309 a C-terminal HRV3C protease site and a TwinStrep tag was cloned into a modified 310 pcDNA3.1(+) vector with the CAG promoter. The TwinStrep-tagged P2 S was expressed in 311 Expi293 cells. Purification of the recombinant protein was based on a procedure described 312 previously, with minor modifications 5 . Upon cell lysis, P2 S was solubilized in 1% NP-40 313 detergent. The TwinStrep-tagged protein was then captured with StrepTactin Sepharose HP 314 resin in 0.5% NP-40. P2 S was further purified by size-exclusion chromatography and eluted 315 as three distinct peaks in 0.02 % NP-40 as previously reported 5 . Peak 1, which consists of intact 316 P2 S migrating at around 150 kDa, as well as dissociated S1 and S2 subunits, which co-migrate 317 at just above 75 kDa, was used in the structural characterisation. Spontaneous dissociation of 318 the S1 and S2 subunits mostly occurs throughout the course of the protein purification, starting 319 at the point of detergent-mediated protein extraction. 320 Biolayer interferometry. The binding of detergent NP-40 solubilized, purified P2 S to human ACE2 peptidase domain 322 (ACE2 PD) and human neutralising monoclonal antibody B38 22 was performed on Octet 323 RED384 (FortéBio) at 25 ⁰C in a running buffer (RB) consisting of 25 mM Tris pH7.5, 150 mM 324 NaCl, 1 mM EDTA and 0.02% NP-40. Avi-tagged ACE2-PD was captured on streptavidin 325 coated sensors and B38 antibody was captured on sensors coated with protein G. After initial 326 baseline equilibration of 120 s, the sensors were dipped in 10 µg/mL solution of Avi-tagged 327 ACE2-PD or B38 mAb for 300 s to achieve capture levels of 1 nM using the threshold function. 328 The sensors were dipped in RB for 120 s for collecting baseline before they were dipped in a 329 concentration series of purified P2 S samples for 300 s (association phase). The sensors were 330 immersed in RB for measuring 600 s (dissociation phase). Data were reference subtracted and 331 fit to a 1:1 binding model with R 2 value greater than 0.95, to determine kinetics and affinity of 332 binding, using Octet Data Analysis Software v10.0 (FortéBio). 333 Cryo-electron microscopy sample preparation, data collection and data processing. 334 For TwinStrep-tagged P2 S, 4 μL purified protein at 0.5 mg/mL were applied to gold Quantifoil 335 R1.2/1.3 300 mesh grids freshly overlaid with graphene oxide. Sample was blotted using a 336 Vitrobot Mark IV for 4 s with a force of -2 before being plunged into liquid ethane cooled by 337 liquid nitrogen. 27,701 micrographs were collected from a two identically prepared grids on a 338 Titan Krios operating at 300 keV equipped with a Gatan K2 Summit direct electron detector in 339 super-resolution mode at a magnification of 165,000x, for a magnified pixel size of 0.435 Å at 340 the specimen level. Data were collected from each grid over a defocus range of -1.2 to -3.4 μm 341 with a total electron dose of 50.32 and 50.12 e -/Å 2 , respectively, fractionated into 40 frames 342 over a 6-second exposure for 1.26 and 1.25 e -/Å 2 /frame. On-the-fly motion correction, CTF 343 estimation, and particle picking and extraction with a box size of 450 pixels were performed in 344 Warp 38 , during which super-resolution data were binned to give a pixel size of 0.87 Å. A total 345 of 1,119,906 particles were extracted. All subsequent processing was performed in RELION 346 3.1-beta 39 . Particle heterogeneity was filtered out with 2D and 3D classification to filter out bad 347 particles, yielding a set of 73,393 particles, which refined to 3.6 Å with C3 symmetry. 3D 348 classification of this dataset without particle alignment separated out one class with a single 349 anesthetised with ketamine HCl (10 mg/kg; IM) during blood collection and immunisation, and 376 monitored for adequate sedation. 377 For mouse sera, MaxiSorp plates (Thermo Fisher Scientific) were coated with recombinant S1 379 or RBD (100 ng/100 µL) in sodium carbonate buffer, and bound IgG was detected using an 380 HRP-conjugated secondary antibody and TMB substrate (Biotrend). Data collection was 381 performed using a BioTek Epoch reader and Gen5 software version 3.0.9. For concentration 382 analysis, the signal of the specific samples was correlated to a standard curve of an isotype 383 Bound rhesus macaque or human anti-S1 antibodies present in the serum were detected with a 386 fluorescently labelled goat anti-human polyclonal secondary antibody (Jackson 387 ImmunoResearch). Data were captured as median fluorescent intensities (MFIs) using a 388 Bioplex200 system (Bio-Rad) and converted to U/mL antibody concentrations using a reference 389 standard consisting of 5 pooled human COVID-19 convalescent serum samples (obtained >14 390 days PCR diagnosis), diluted in antibody depleted human serum with arbitrary assigned 391 concentrations of 100 U/mL and accounting for the serum dilution factor. 392 Binding kinetics of antigen-specific IgGs using surface plasmon resonance spectroscopy 393 Binding kinetics of murine S1-and RBD-specific serum IgGs was determined using a Biacore 394 The SARS-CoV-2 inoculum was obtained from a stock of 2.1 × 10 6 PFU/mL previously 519 and analysed and interpreted data. A.P., S.E., D.P. and G.S. performed and analysed the S1-593 binding IgG assays. R.C., Jr. and K.J.A. performed and analysed viral RT-qPCR data. A.M., BioNTech is the Sponsor of the study, and Pfizer it its agent. BioNTech and Pfizer are 623 responsible for the design, data collection, data analysis, data interpretation, and writing of the 624 report. The corresponding authors had full access to all the data in the study and had final 625 responsibility for the decision to submit the data for publication. This study was not supported 626 by any external funding at the time of submission. 627 Supplementary Information is available for this study. 629 Correspondence and requests for materials should be addressed to Ugur Sahin. 630 Page 23 TwinStrep tag, expressed in Expi293F cells, was detergent solubilized and purified by affinity and size exclusion chromatography. Protein from the first peak of a size exclusion column, containing intact P2 S and dissociated S1 and S2 fragments, was assayed by biolayer interferometry. Sensorgram of the binding kinetics of TwinStrep-tagged P2 S to immobilised b, human ACE2-PD and c, B38 monoclonal antibody. 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