key: cord-340240-dk48pdqa authors: Kuo, Tsun-Yung; Lin, Meei-Yun; Coffman, Robert L; Campbell, John D; Traquina, Paula; Lin, Yi-Jiun; Liu, Luke Tzu-Chi; Cheng, Jinyi; Wu, Yu-Chi; Wu, Chung-Chin; Tang, Wei-Hsuan; Huang, Chung-Guei; Tsao, Kuo-Chien; Shih, Shin-Ru; Chen, Charles title: Development of CpG-adjuvanted stable prefusion SARS-CoV-2 spike antigen as a subunit vaccine against COVID-19 date: 2020-08-11 journal: bioRxiv DOI: 10.1101/2020.08.11.245704 sha: doc_id: 340240 cord_uid: dk48pdqa The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 is a worldwide health emergency. The immense damage done to public health and economies has prompted a global race for cures and vaccines. In developing a COVID-19 vaccine, we applied technology previously used for MERS-CoV to produce a prefusion-stabilized SARS-CoV-2 spike protein by adding two proline substitutions at the top of the central helix (S-2P). To enhance immunogenicity and mitigate the potential vaccine-induced immunopathology, CpG 1018, a Th1-biasing synthetic toll-like receptor 9 (TLR9) agonist was selected as an adjuvant candidate. S-2P was combined with various adjuvants, including CpG 1018, and administered to mice to test its effectiveness in eliciting anti-SARS-CoV-2 neutralizing antibodies. S-2P in combination with CpG 1018 and aluminum hydroxide (alum) was found to be the most potent immunogen and induced high titer of spike-specific antibodies in sera of immunized mice. The neutralizing abilities in pseudotyped lentivirus reporter or live wild-type SARS-CoV-2 were measured with reciprocal inhibiting dilution (ID50) titers of 5120 and 2560, respectively. In addition, the antibodies elicited were able to cross-neutralize pseudovirus containing the spike protein of the D614G variant, indicating the potential for broad spectrum protection. A marked Th-1 dominant response was noted from cytokines secreted by splenocytes of mice immunized with CpG 1018 and alum. No vaccine-related serious adverse effects were found in the dose-ranging study in rats administered single- or two-dose regimens with up to 50 μg of S-2P combined with CpG 1018 alone or CpG 1018 with alum. These data support continued development of CHO-derived S-2P formulated with CpG 1018/alum as a candidate vaccine to prevent COVID-19 disease. Introduction 37 38 COVID-19 was first identified as a cause of severe pneumonia cases in December 2019 in association with 39 a seafood market in Wuhan, China [1] . The viral agent was identified as a novel SARS-like coronavirus (SARS-40 CoV-2) most closely related to bat coronavirus [1] . In the six months since its first appearance, SARS-CoV-2 41 has become the largest pandemic since the 1918 influenza with nearly 20 million infected and over 700,000 Coronaviruses are among the largest known enveloped RNA viruses and cause respiratory illnesses in 55 humans ranging from the common cold to SARS, MERS, as well as the current COVID-19 pandemic [7] . Similar to SARS-CoV, the spike (S) protein of SARS-CoV-2 is the receptor for attachment and cell entry via 57 the cellular receptor hACE2 [1] . Researchers are also adapting antigen design strategies used for SARS-CoV Subunit vaccines such as the spike protein are often poorly immunogenic by themselves and therefore 67 typically require adjuvants to enhance their ability to produce an immune response [ In this study, we present data from preclinical studies aimed at developing a COVID-19 candidate subunit 84 vaccine using CHO cell-expressed SARS-CoV-2 S-2P antigen combined with various adjuvants. We have 85 shown that S-2P, when mixed with CpG 1018 and aluminum hydroxide adjuvants, was most effective in 86 inducing antibodies that neutralized pseudovirus and wild-type live virus while minimizing Th2-biased 87 responses with no vaccine-related adverse effects. The plasmid expressing SARS-CoV-2 (strain Wuhan-Hu-1 GenBank: MN908947) S protein ectodomain Ltd. Animal studies were conducted in the Testing Facility for Biological Safety, TFBS Bioscience Inc., Taiwan. All animal work was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The Testing Facility's IACUC animal study protocol approval numbers are TFBS2020-006 and TFBS2020-010. Neutralization titers of wild-type virus and pseudovirus and total anti-S IgG titers were all found to be highly 236 correlated with Spearman's rank correlation coefficients greater than 0.8 ( Figure 5 ). We have successfully shown robust immunogenicity elicited by adjuvanted SARS-CoV-2 S-2 (Figures 1, 285 2, S1, and S2). Much stronger neutralizing antibody responses were detected in mice when 1 g or 5 g of S- 2P protein was adjuvanted with 10 g of CpG 1018 and 50 g of aluminum hydroxide than with either adjuvant 287 alone (Figure 1 ). S-2P in conjunction with CpG 1018 and aluminum hydroxide induced potent anti-S antibodies 288 that were effective against wild-type virus (Figures 2 and 3) . We have shown that high degrees of correlation Although moderate IL-4 production was detected in mice receiving 5 g of S-2P combined with CpG 1018 303 and aluminum hydroxide, the IFN-γ/IL-4 ratio was 16-fold higher than those receiving 5 g of S-2P adjuvanted 304 with aluminum hydroxide alone. These results suggested that CpG 1018, even in the presence of aluminum 305 hydroxide could steer the immune response away from Th2 to a Th1 response. Moreover, these mice produce a 306 limited amount of IL-5, which is a key mediator in eosinophil activation and major regulator of eosinophil 307 accumulation in tissues [21] . Previous studies showed that the lung-infiltrating eosinophils were a common 308 indication of Th2-biased immune responses seen in animal models testing SARS-CoV vaccine candidates [22] . The finding that IL-5 production was inhibited by the S-2P adjuvanted with CpG 1018 plus aluminum hydroxide Discovery 353 of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin ArcGIS Dashboards. Gisanddata.maps.arcgis.com. 2020 [retrieved 05 Responding to Covid-19-a once-in-a-century pandemic? Funding the development and manufacturing of COVID-19 vaccines: The 360 need for global collective action DRAFT Landscape of COVID-19 Candidate Vaccines -31 Target Product Profiles for COVID-19 Vaccines -17 Severe acute respiratory syndrome-related coronavirus-The species and its viruses, a statement 368 of the Coronavirus Study Group Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen Cryo-EM 373 structure of the 2019-nCoV spike in the prefusion conformation Recent advances of vaccine adjuvants for infectious diseases. Immune network Vaccine adjuvants: Understanding the structure and mechanism of 377 adjuvanticity. Vaccine Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the 380 SARS virus. PloS one COVID-19 vaccine design: the Janus face of immune enhancement Development of the CpG adjuvant 1018: a case study Culture-based virus isolation to evaluate potential infectivity of clinical specimens tested for COVID-19 Effect of mucosal and systemic 389 immunization with virus-like particles of severe acute respiratory syndrome coronavirus in mice Co-administration of a CpG adjuvant (VaxImmuneTM, CPG 7909) with CETP vaccines increased 393 immunogenicity in rabbits and mice. Human vaccines SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 elicits immunogenicity in baboons 396 and protection in mice. bioRxiv Mammalian cell culture 398 for production of recombinant proteins: A review of the critical steps in their biomanufacturing Tracking changes in SARS-CoV-2 Spike: evidence that D614G increases infectivity of 402 the COVID-19 virus Murine eosinophil 404 differentiation factor. An eosinophil-specific colony-stimulating factor with activity for human cells Immunization with SARS coronavirus vaccines leads to 407 pulmonary immunopathology on challenge with the SARS virus Systems and their Potential Applications in Hepatitis B Vaccines