key: cord-0836137-18xzle7o authors: Kalnin, Kirill V.; Plitnik, Timothy; Kishko, Michael; Zhang, Jinrong; Zhang, Donghui; Beauvais, Adrien; Anosova, Natalie G.; Tibbitts, Timothy; DiNapoli, Joshua M.; Huang, Po-Wei D.; Huleatt, James; Vincent, Deanne; Fries, Katherine; Karve, Shrirang; Goldman, Rebecca; Gopani, Hardip; Dias, Anusha; Tran, Khang; Zacharia, Minnie; Gu, Xiaobo; Boeglin, Lianne; Chivukula, Sudha; Swearingen, Ron; Landolfi, Victoria; Fu, Tong-Ming; DeRosa, Frank; Casimiro, Danilo title: Immunogenicity of novel mRNA COVID-19 vaccine MRT5500 in mice and non-human primates date: 2020-10-14 journal: bioRxiv DOI: 10.1101/2020.10.14.337535 sha: 97ffc0f276411a62b00a0a664c4904bc3032cdf4 doc_id: 836137 cord_uid: 18xzle7o An effective vaccine to address the global pandemic of coronavirus disease 2019 (COVID-19) is an urgent public health priority1. Novel synthetic mRNA and vector-based vaccine technologies offer an expeditious development path alternative to traditional vaccine approaches. Here we describe the efforts to utilize an mRNA platform for rational design and evaluations of mRNA vaccine candidates based on Spike (S) glycoprotein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus causing COVID-19. Several mRNA constructs expressing various structural conformations of S-protein, including wild type (WT), a pre-fusion stabilized mutant (2P), a furin cleavage-site mutant (GSAS) and a double mutant form (2P/GSAS), were tested in a preclinical animal model for their capacity to elicit neutralizing antibodies (nAbs). The lead 2P/GSAS candidate was further assessed in dose-ranging studies in mice and Cynomolgus macaques. The selected 2P/GSAS vaccine formulation, now designated MRT5500, elicited potent nAbs as measured in two types of neutralization assays. In addition, MRT5500 elicited TH1-biased responses in both mouse and non-human primate species, a result that helps to address a hypothetical concern regarding potential vaccine-associated enhanced respiratory diseases associated with TH2-biased responses. These data position MRT5500 as a viable vaccine candidate for clinical development against COVID-19. CoV-2 S-protein contains two proline substitutions (2P), at amino acid positions 986 and 987, There is a unique feature of SARS-CoV-2 S protein which possesses a polybasic furin cleavage 56 site at the junction of S1 and S2 subunits. This feature is believed to have emerged during viral 57 transmission from zoonotic host to human [25] [26] [27] , and is key to SARS-CoV-2 high transmissibility 58 in humans 28, 29 . Although robust SARS-CoV-2 infection of human lungs requires a multibasic Approximately 45% of the total S protein monomers presented within intact SARS-CoV-2 87 virions have been reported as cleaved at the furin cleavage site 31 ; however, it is not clear which The COVID-19 vaccine hypothesis has been centered around induction of neutralizing 91 antibodies (nAbs) that either block the interaction of the RBD with ACE2, or that prevent the 92 fusion process involving S protein transition from pre-to post-fusion conformation 37,38 . Although 93 the pre-fusion conformation is known to be critical for eliciting a neutralizing response 18,19 , the 94 impact of the furin cleavage site in eliciting neutralizing antibodies requires additional studies. To test the potential contribution of this site, we mutated the furin cleavage site, composed of the 96 polybasic residues RRAR, to GSAS from amino acid position 682 to 685 30,35 . Four constructs 97 were synthesized as mRNA to represent either wild type (WT), stabilized pre-fusion mutant (2P) 98 20 , furin cleavage site mutant (GSAS) or a double mutant (2P/GSAS) of SARS-CoV-2 S gene. These constructs were transfected into a human cell line and their expression levels were verified 100 by Western Blot (Fig. 1a) . As expected, endogenous cleavage of WT and 2P constructs, but not 101 GSAS or 2P/GSAS proteins, was observed (Fig. 1a) , which yielded a band of approximately 90 102 kDa representing S2. In order to determine the potential impact of 2P and GSAS mutations on immunogenicity, we To understand the potential impact of these mutations on nAbs titers, we tested the ability of 116 immune sera to neutralize the infectivity of GFP reporter pseudoviral particles (RVP) in HEK- 293T cells stably over-expressing human ACE2 40 . RVPs expressed antigenically correct SARS CoV-2 S protein and GFP reporter genes on lentiviral (HIV) core and were capable of a single 119 round of infection. Pseudoviral neutralization assay (PsVNa) allowed the determination of serum 120 dilution which can achieve 50% inhibition of RVP entry (ID50; see Materials and Methods). Contrary to binding antibodies which could be detected at D14 after the first immunization, the 122 neutralizing antibody response could only be detected after the second immunization. Also 123 noted, the spread of the nAb titers within each group were more pronounced when compared to 124 binding antibody titers, with 95% confidence intervals overlapping each other. On D35, the The selected MRT5500 formulation was evaluated in both mouse and NHP studies with a range 140 of doses covering more than 10-fold titration. The hypothesis for this study is that S-specific 141 antibodies blocking viral infection are key for protection, and our evaluation therefore focused 142 on serological responses against SARS-CoV-2 S, with a particular emphasis on neutralizing 143 titers post vaccination 8, 9, 41 . Four dose levels in mice were assessed, ranging from 0.2 to 10 µg 144 per dose. As expected, MRT5500 induced dose-dependent S-specific binding antibodies and 145 neutralizing antibodies in mice (Fig. 2) . PsVNa titers were detected in the higher dose groups (5 146 µg, 10 µg) after one vaccination, within the titers being more pronounced after the second 147 vaccination at D21 (Fig. 2b) . The PsVNa GMTs were 534, 5232, 9370 and 7472 at D35 for the Although we have used two assays to measure the neutralizing potency, the results from both 166 assays were highly correlated (Suppl. Fig. 3 and Suppl. Table 1) Vaccine associated enhanced respiratory disease (VAERD) has been a safety concern for 173 COVID-19 vaccines in development, although the concern at this stage is only a theoretical one 1 . This phenomenon has been reported for whole-inactivated virus vaccines against measles and The two GSAS containing mutants (GSAS and double mutant 2P/GSAS) resulted in nAb titers 226 that trended higher than the WT and 2P analogues (Fig. 1c) . While the nAb levels from these 227 two GSAS-containing antigens were not significantly different from one another, we believe likely, is that we tested the mRNA vaccines at a poorly differentiating dose level (0.4 µg/dose) in 238 mice (Fig. 1c) . Our results in a subsequent experiment (Fig. 2) confirmed that the saturation patients, especially those of mild and asymptomatic cases, induce antibodies that decay rapidly 255 in convalescent phase, with some drifting down to baseline within three months after diagnosis 51 . MRT5500 and its immunological effects for applications towards COVID-19. In summary, we have utilized mRNA technology for the rapid evaluation of vaccine candidates 260 for COVID-19, and our results led to the selection of a double mutant candidate which has a 261 better potential to preserve a pre-fusion conformation. The candidate MRT5500 has been shown The endpoint antibody titer for each sample was determined as the highest dilution which gave 329 OD value 3x higher than the background. Rapid COVID-19 vaccine development Outbreak of pneumonia of unknown etiology in Wuhan, 468 China: The mystery and the miracle The COVID-19 Pandemic: A Comprehensive Review of Taxonomy Diagnosis, Treatment, and Control An overview of coronaviruses including the SARS-2 coronavirus -Molecular biology, 472 epidemiology and clinical implications Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Structural basis of receptor recognition by SARS-CoV-2 Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques Evaluation of 481 the mRNA-1273 vaccine against SARS-Cov-2 in nonhuman primates 484 SARS-Cov-2 mRNA Vaccine Development Enabled by Prpototype Pathogen Preparedness Safety and 487 immunogenicity of ChAd0zx1 nCov1-19 vaccine against SARS-Cov-2: a preliminary report of 488 phase 1/2, single-blind Rapid development of an inactivated vaccine candidate for SARS-CoV-2 A Novel Vaccine Employing Non-Replicating Rabies 492 Virus Expressing Chimeric SARS-CoV-2 Spike Protein Domains: Functional Inhibition of 493 Viral/Nicotinic Acetylcholine Receptor Complexes DNA vaccine protection against SARS-CoV-2 in rhesus macaques Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces 498 high neutralizing antibody titers in mice Structure-based design of prefusion-stabilized SARS-CoV-2 spikes A Cryptic Site of Vulnerability on the Receptor Binding Domain of the SARS-503 Neutralization of SARS-CoV-2 by Destruction of the Prefusion Spike Distinct conformational states of SARS-CoV-2 spike protein Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer. 509 Pre-fusion structure of a human coronavirus spike protein Immunogenicity and structures of a rationally designed prefusion MERS-CoV 513 spike antigen RNA-Based COVID-19 Vaccine BNT162b2 Selected for a Pivotal Efficacy Study An mRNA Vaccine against SARS-CoV-2 -Preliminary Report Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus 521 evolution and furin-cleavage effects Author Correction: SARS-CoV-2 and bat RaTG13 spike glycoprotein structures 524 inform on virus evolution and furin-cleavage effects The SARS-CoV-2 Coronavirus and the 527 COVID-19 Outbreak COVID-19: Time to exonerate the pangolin 529 from the transmission of SARS-CoV-2 to humans Cleavage Site in the Spike Protein 532 of SARS-CoV-2 Is Essential for Infection of Human Lung Cells Structures and distributions of SARS-CoV-2 spike proteins on intact virions The furin cleavage site of SARS-COV-2 539 spike protein is a key determinant for transmission due to enhanced replication in airway cells A model of the ACE2 structure and function as a SARS-542 CoV receptor Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein Natural Polymorphisms Are Present in the Furin Cleavage Site 546 of the SARS-CoV-2 Spike Glycoprotein The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like 549 cleavage site absent in CoV of the same clade Endocytosis of the receptor-binding domain of SARS-CoV spike protein together 552 with virus receptor ACE2 SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a 554 mRNA vaccine 557 delivery using lipid nanoparticles Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-559 CoV-2 Spike Protein for Neutralization Assays DNA 561 Vaccine Protection Against SARS-CoV-2 in Rhesus Macaques A double-inactivated severe acute respiratory syndrome coronavirus vaccine 563 provides incomplete protection in mice and induces increased eosinophilic proinflammatory 564 pulmonary response upon challenge T cell mediate protective immunity against 566 emerging resperotory coronaviruses Immunization with SARS coronavirus vaccines leads to pulmonary 568 immunopathology on challenge with the SARS virus COVID-19 patients display distinct SARS-CoV-2 specific T-cell responses 571 according to disease severity Recent Trends in RSV 573 Immunoprophylaxis: Clinical Implications for the Infant An Adjuvanted, Postfusion F Protein-Based Vaccine Did Not Prevent Respiratory 576 Syncytial Virus Illness in Older Adults Phase 1/2 study of COVID-19 RNA vaccine BNT162b1 in adults Furin-mediated protein processing in infectious diseases and cancer Human Cytomegalovirus Glycoprotein B Nucleoside-Modified mRNA Vaccine 583 Elicits Antibody Responses with Greater Durability and Breadth than MF59-Adjuvanted gB 584 Protein Immunization Longitudinal 590 evaluation and decline of Antibody responses in SARS-CoV-2 infection Structural Basis for Potent Neutralization of Betacoronaviruses by Single-593 SARS-CoV-2, SARS-CoV, and MERS-COV: A comparative overview Improved Efficacy in a Fabry Disease Model Using a Systemic mRNA Liver Depot 597 System as Compared to Enzyme Replacement Therapy Molecular cloning. Laboratory manual