key: cord-0968507-gsl8yj3t authors: Veenstra, T.; Injeti, E.; Pauley, B. title: In vitro Characterization of SARS-CoV-2 Protein Translated from the Moderna mRNA-1273 Vaccine date: 2022-03-02 journal: nan DOI: 10.1101/2022.03.01.22271618 sha: 801484855ccfb69fc38d32f18c7402c6da3dbefb doc_id: 968507 cord_uid: gsl8yj3t Extensive research around mRNA vaccines and their proposed utility during the current COVID-19 pandemic resulted in many publications concerning the SARS-Cov-2 spike protein and angiotensin converting enzyme-2 receptor-binding domain of the virus, but none describe the characteristics of the full-length protein obtained from the modified/synthetic mRNA that is part of the Moderna and Pfizer-BioNTech vaccines. In this paper, we provide the first data characterizing the actual proteins produced by mouse and human cells in culture that had been incubated up to 30 minutes with the commercial vaccine produced by Moderna (i.e., Spikevax). The mRNA vaccine continues to produce proteins up to 12-14 days after introduction to the cells. The molecular weight of the SARS-CoV-2 encoded protein ranges from 135-200 kilodaltons depending on the extent of glycosylation. The current coronavirus (COVID-19) pandemic has resulted in several FDA approved emergency use authorizations (EUA) to slow the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1 -3] . Messenger ribonucleic acid (mRNA) vaccines were at the forefront of these EUAs based on their efficacy and how they could be formulated to enter cells after intramuscular injection [4] [5] [6] . RNA delivery to cells had been a challenge for decades, however, the development of lipid nanoparticles and the modification with N1-methylpseudouridine greatly enhanced the efficacy of both Moderna's (Spikevax) and Pfizer-BioNTech's (Comirnaty) mRNA vaccines [7] . In addition, modification of the mRNA with multiple N1methyl-pseudouridine bases enhanced the stability of the mRNA enabling it to survive the intra and extracellular environments long enough to be translated into a functional, intact protein [8] . One knowledge gap concerning these vaccines was the length of time the mRNA survives in vivo and is translated into protein. Another unknown is the size of the actual protein(s) that is translated and what does the actual protein look like after it is made within mammalian cells in culture. In this study, we evaluated the length of time and the size of the protein that the Spikevax vaccine produces in vitro. The results show that the vaccine readily enters the cells and produces a variety of protein isoforms within 24 hours. These results suggest the variability in immune response may be due to the translation of different protein isoforms that cause differences in the antibodies produced within the vaccinated or COVID-19 infected subject. hundred microliters of room temperature Spikevax vaccine (Moderna, Cambridge, MA) was added to the cells, which were then incubated for 30 minutes with slow rocking at room temperature. After 30 minutes, 10% (v/v) fetal bovine serum was added, and the cells were placed in an incubator maintained at 37 °C and 4.5% CO2. Due to the nature of the lipid, cholesterol, and polyethylene glycol lipid shell that surrounds the mRNA, no other agent was required to assist the entry of the vaccine into cells. This incubation time did not affect cell viability, which remained above 90% based on trypan blue exclusion and cell counting using a CytoSMART cell counter (CytoSMART Technologies LLC, Skillman, NJ). At selected time points, cells were collected and lysed in Triton lysis buffer containing a protease inhibitor cocktail. The cell supernatants were also collected and stored at -20 °C for further analysis. Protein concentrations were measured using a Bradford microplate assay (Biorad, Hercules, CA). Enzyme-linked immunoassay (ELISA) reactivity was evaluated against the commercially available SARS-CoV-2 protein (R&D Systems, Minneapolis, MN). Briefly, Immulon TM 2 high protein binding ELISA plates (ICT; Davis, CA) were coated with SARS-CoV-2 proteins at 100 ng/well in phosphate buffered saline (PBS) at 4 °C overnight. Wells were rinsed twice with PBS and blocked using 3% blotting-grade protein blocker (Biorad). Antibodies were incubated in concentration ranges from 1-10 μg/ml and detected using donkey anti-human secondary antibodies, (Jackson ImmunoResearch Laboratories; West Grove, PA) labeled with horse radish peroxidase (HRP) at a 1:5,000 dilution. These secondary antibodies are supplied pre-adsorbed to numerous species' IgGs to reduce non-specific binding. After incubation with 3,3',5,5'tetramethylbenzidine (TMB) substrate for 10 minutes, 50 μl of 2 N sulfuric acid was added and the absorbance of the TMB substrate at 450 nm was measured using a Promega plate reader. The binding of the collected human saliva and REGEN-COV2 antibodies to cell lysates and supernatants was evaluated using an in-house developed ELISA. To test for the presence of autoantibodies, the ELISA was performed by coating the wells of the plate with the mRNA vaccine All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 2, 2022. ; https://doi.org/10.1101/2022.03.01.22271618 doi: medRxiv preprint and probing them with the patient samples (Groups A-D) along with the commercial antibodies. No positive signals were observed in the ELISA showing that vaccinated patients do not develop autoantibodies against the vaccine. Cell lysates were quantitated and combined with 4x Laemmli buffer and heated at 100 °C Enhanced chemiluminescence (ECL) was employed to detect proteins, using an Azure 600 imager (Azure Biosystems, Dublin, CA). (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 2, 2022. ; https://doi.org/10.1101/2022.03.01.22271618 doi: medRxiv preprint Emergency use authorization of Covid vaccines -safety and efficacy follow-up considerations Communicating effectively about emergency use authorization and vaccines in the COVID-19 pandemic Effectiveness of COVID-19 booster vaccines against covid-19 related symptoms, hospitalisation and death in England Early clinical trial data and real-world assessment of COVID-19 vaccines: insights from the Society of Infectious Disease Pharmacists Prevention and attenuation of Covid-19 with the BNT162b2 and mRNA-1273 vaccines mRNA vaccine: a potential therapeutic strategy The critical contribution of pseudouridine to mRNA COVID-19 vaccines Expression of the vav oncogene in somatic cell hybrids