key: cord-0859845-6ubk3rv7 authors: McKay, Paul F.; Hu, Kai; Blakney, Anna K.; Samnuan, Karnyart; Bouton, Clément R.; Rogers, Paul; Polra, Krunal; Lin, Paulo J.C.; Barbosa, Christopher; Tam, Ying; Shattock, Robin J. title: Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine induces equivalent preclinical antibody titers and viral neutralization to recovered COVID-19 patients date: 2020-04-25 journal: bioRxiv DOI: 10.1101/2020.04.22.055608 sha: a33d15f6f4a93234644e0b5e6e72ab85cb72b0ca doc_id: 859845 cord_uid: 6ubk3rv7 The spread of the SARS-CoV-2 into a global pandemic within a few months of onset motivates the development of a rapidly scalable vaccine. Here, we present a self-amplifying RNA encoding the SARS-CoV-2 spike protein encapsulated within a lipid nanoparticle as a vaccine and demonstrate induction of robust neutralization of a pseudo-virus, proportional to quantity of specific IgG and of higher quantities than recovered COVID-19 patients. These data provide insight into the vaccine design and evaluation of immunogenicity to enable rapid translation to the clinic. The spread of the SARS-CoV-2 into a global pandemic within a few months of onset motivates the development of a rapidly scalable vaccine. Here, we present a self-amplifying RNA encoding the SARS-CoV-2 spike protein encapsulated within a lipid nanoparticle as a vaccine and demonstrate induction of robust neutralization of a pseudo-virus, proportional to quantity of specific IgG and of higher quantities than recovered COVID-19 patients. These data provide insight into the vaccine design and evaluation of immunogenicity to enable rapid translation to the clinic. The unprecedented and rapid spread of SARS-CoV-2 into a global pandemic, with the current estimated number of confirmed cases >2.2 million people, 1 has motivated the need for a rapidly producible and scalable vaccine. Coronaviruses are positive-sense, single stranded RNA viruses that cause disease pathology ranging from the common cold to pneumonia. 2, 3 Despite being listed on the WHO blueprint priority list, there are currently no licensed vaccines for SARS or MERS. 4 However, previous studies have elucidated the need to stabilize coronavirus spike proteins in their pre-fusion conformation in order to serve as a vaccine immunogen. 5 Self-amplifying RNA (saRNA) encapsulated in lipid nanoparticles (LNP) is a highly relevant platform for producing vaccines in the context of a global pandemic as it's possible to encode any antigen of interest 6, 7 and requires a minimal dose compared to messenger RNA (mRNA). 8 The first RNA therapeutic, which is formulated in LNP, was approved in 2018 and has set the precedent for clinical safety of LNP-formulated RNA. 9 Here, we compare the immunogenicity of saRNA encoding a pre-fusion stabilized SARS-CoV-2 spike protein encapsulated in LNP in a preclinical murine model to the immune response generated by a natural infection in recovered COVID-19 patients. We characterize both the humoral and cellular response as well as the neutralization capacity of a pseudotyped SARS-CoV-2 virus. After confirming expression of the pre-fusion stabilized SARS-CoV-2 spike protein in vitro (Supplementary Figure 1) , mice were immunized with saRNA encoding the SARS-CoV-2 spike protein encapsulated in LNP with doses ranging from 0.01 to 10 μ g ( Figure 1a ). Mice received two injections, one month apart, and electroporated plasmid DNA (pDNA) was used as a positive control while saRNA encoding the rabies glycoprotein (RABV) in pABOL was used as a negative control. After 6 weeks, we observed remarkably high quantities of SARS-CoV-2 specific IgG in mouse sera in a dose-responsive manner, ranging from 10 5 -10 6 ng/mL ( Figure 1b ). The groups that received doses of 10 and 1 μ g of saRNA LNP were significantly higher than the mice that received 10 μ g of electroporated pDNA, with p=0.0036 and 0.0020, respectively. All of the saRNA LNP-vaccinated mice, even the 0.01 μ g group, had higher quantities of SARS-CoV-2 specific IgG compared to patients that had recovered from COVID-19, which had a mean titer of 10 3 ng/mL and a range of 10 1 -10 5 ng/mL. Importantly both the pDNA and saRNA LNP immunizations induced a Th1-biased response in mice (Supplementary We then sought to characterize how antibodies generated by immunization compared to those generated by a natural SARS-CoV-2 infection as far as capacity to neutralize a SARS-CoV-2 pseudotyped virus ( Figure 1c ). We observed highly efficient viral neutralization that varied in a linear dose-dependent manner for the mice vaccinated with saRNA LNP, with IC 50 values ranging from 5x10 3 to 10 5 . The groups that received 10 or 1 μ g of saRNA LNP were significantly higher than the electroporated pDNA positive control group, both with p<0.0001. Here we characterized the immunogenicity of a SARS-CoV-2 saRNA LNP vaccine compared to the immune response of a natural infection in COVID-19 recovered patients. We observed that two saRNA LNP immunizations induced remarkably high SARS-CoV-2 specific IgG antibodies in mice, with quantities that were superior to both EP pDNA and natural infection in humans, that were able to efficiently neutralize a pseudotyped virus. We also observed that the saRNA LNP vaccine induces a robust cellular response, which is partially enabled by the potent LNP formulation. We observed that the saRNA-encoded pre-fusion stabilized spike protein of SARS-CoV-2 used in these studies is highly immunogenic, yielding antibody titers >10 6 ng/mL (Figure 1 ), which is superior to what others have reported for subunit vaccines for the SARS, MERS and SARS-2 coronaviruses. 10 Furthermore, we observed higher antibody titers, viral neutralization (IC 50 ) and cellular response for LNP-formulated saRNA than electroporated pDNA, which we postulate is due to the potent LNP used in these studies, as previous comparisons between polyplex-formulated saRNA and EP pDNA have yielded similar immunogenicity. 8 This is highly useful for translation as it means a potent LNP-formulated saRNA vaccine can be injected with a widely accepted syringe and needle, and does not require electroporation instrumentation, which we envision will enable more widespread vaccination to curb the spread of SARS-CoV-2. The saRNA LNP vaccine presented in these studies elicited robust antibody and cellular responses, with a Th1 bias that we hypothesize will enable immunogenicity in humans. Ongoing studies are being carried out to characterize the potential for antibody dependent enhancement (ADE) of SARS-CoV-2 as has been observed for SARS and MERS, 11, 12 but the role of this phenomena in vaccine-induced immunity is not yet fully understood. Overall, we believe that these data inform the antigen design, formulation and preclinical evaluation of immunogenicity that will enable rapid translation of a SARS-CoV-2 vaccine to the clinic trials. We used a plasmid vector to synthesize a self-amplifying RNA (saRNA) replicon, based on a The plasmid expressing the soluble pre-fusion version of SARS-CoV-2 S was used to produce the recombinant protein using the FreeStyle™ 293 Expression System (Thermo Fisher Scientific), according to the manufacturer's instructions. Conditioned medium was clarified by centrifugation and protein was sequentially purified by a HisTrap HP column and a HiPrep 16/60 Sephacryl S-300 HR size exclusion chromatography (SEC) column (both from GE Healthcare). Purified protein was first analyzed by Native-PAGE and Western blot, and then filtered through Samples were analyzed on a LSRForterssa (BD Biosciences) with FACSDiva software (BD Biosciences). Data were analyzed using FlowJo Version 10 (FlowJo LLC). saRNA was encapsulated in LNP using a self-assembly process in which an aqueous solution of saRNA at pH=4.0 is rapidly mixed with an ethanolic lipid mixture. 14 LNP used in this study were similar in composition to those described previously 15, 16 , which contain an ionizable The antigen-specific IgG, IgG1 and IgG2a titres in mouse sera were assessed by a semiquantative ELISA as previously described. 17 In brief, MaxiSorp high binding ELISA plates A HIV-pseudotyped luciferase-reporter based system was used to assess the neutralization ability of sera from vaccinated animals and recovered patients against SARS-CoV, SARS-CoV-2, MERS-CoV and 229E-CoV, as previously described with modifications. 18, 19 In brief, CoV S- Global COVID-19 Statistics Angewandte Chemie was measured using Bright-Glo Luciferase Assay System (Promega). The IC 50 neutralization x 10 6 per well in a 200 uL total volume) and cultured for 7 days with media alone, 5 ug / mL SARS-CoV-2 recombinant protein or 5 ug / mL ConA as a positive control. For the sera samples, mice were bled 4h after injection with SARS-CoV-2 LNP vaccine or control RABV vaccine and sera were collected. The cytokine response in each well was quantified with a custom 25-plex ProcartaPlex Immunoassay Bio-Rad), according to the manufacturer's instructions Statistical differences were analyzed using either a two-way ANOVA adjusted for multiple comparisons or a Kruskal-Wallis test adjusted for multiple comparisons, with p<0.05 used to indicate significance. AUTHOR CONTRIBUTIONS PFM and RJS conceptualized the antigen design and designed the studies. PFM, KS and KH designed and performed in vitro experiments. PFM, AKB, KH and KS performed in vivo studies, aided by CRB, KP, and PR. PL, CB and YT designed and prepared the saRNA LNP. AKB analysed the data and wrote the manuscript with help from PFM We gratefully acknowledge Graham Cooke, Rachael Quinlan, Charlotte Short, and Carolina Ltd for providing funds to purchase equipment used in these studies. Raw data is available upon reasonable request from Imperial College London.