Editing MRNA Vaccine Protocol

ModeRNA&nbsp;SARS-CoV-2 mRNA vaccine design

&nbsp;mammalian codon-optimized plasmid encoding foldon inserted minifibritin (ADIVLNDLPFVDGPPAEGQSRISWIKNGEEILGADTQYGSEGSMNRPTVSVLRNVEVLDKNIGILKTSLETANSDIKTIQEAGYIPEAPRDGQAYVRKDGEWVLLSTFLSPALVPRGSHHHHHHSAWSHPQFEK) with a C-terminal thrombin cleavage site, 6×His tag, and Strep-TagII was synthesized and subcloned into a mammalian expression vector derived from pLEXm. The construct was expressed by transient transfection of Expi293 (ThermoFisher) cells in suspension at 37 °C for 5 days. The protein was first purified with a Ni2+-nitrilotriacetic acid resin (GE Healthcare) using an elution buffer consisting of 50 mM Tris-HCl, pH 7.5, 400 mM NaCl and 300 mM imidazole pH 8.0, followed by purification with StrepTactin resin (IBA) according to the manufacturer’s instructions.

Patents:

US 10,703,789, US 10,702,600, US 10,577,403, US 10,442,756, US 10,266,485, US 10,064,959, US 9,868,692

Patent/Published<br/>Application<br/>Applicant/Assignee Filing Date Status Invention Type<br/>US 10,703,789 Moderna June 12, 2019 Active PC: Lipids/NP +<br/>mRNA<br/>US 10,702,600 Moderna February 28, 2020 Active Betacoronavirus<br/>mRNA Vaccine<br/>US 10,577,403 Moderna June 12, 2019 Active PC: Lipids/NP +<br/>mRNA<br/>US 10,442,756 Moderna December 18, 2017 Active Lipids/NP + mRNA<br/>US 10,266,485 Moderna June 11, 2018 Active Lipids/NP + mRNA<br/>US 10,064,959 Moderna April 21, 2017 Active mRNA synthesis<br/>US 9,868,692 Moderna March 31, 2017 Active Lipids/NP + mRNA<br/>US2020/0206362 Moderna October 11, 2019 Pending PC: Lipids/NP +<br/>mRNA<br/>US2020/0164038 Moderna July 29, 2019 Pending PC: Lipids/NP +<br/>mRNA<br/>US2019/0015501 Moderna September 27, 2018 Pending Nucleic acid vaccine<br/>WO2016/118724 Moderna January 21, 2016 Published Lipids/NP + mRNA<br/>WO2016/118725 Moderna January 21, 2016 Published Lipids/NP + mRNA

VACCINE<br/>The mRNA-1273 vaccine candidate, manufactured by Moderna, encodes the S-2P antigen, consisting of the SARS-CoV-2 glycoprotein with a transmembrane anchor and an intact S1–S2 cleavage site. S-2P is stabilized in its prefusion conformation by two consecutive proline substitutions at amino acid positions 986 and 987, at the top of the central helix in the S2 subunit.8 The lipid nanoparticle capsule composed of four lipids was formulated in a fixed ratio of mRNA and lipid. The mRNA-1273 vaccine was provided as a sterile liquid for injection at a concentration of 0.5 mg per milliliter. Normal saline was used as a diluent to prepare the doses administered.

&nbsp;mammalian codon-optimized plasmid encoding foldon inserted minifibritin (ADIVLNDLPFVDGPPAEGQSRISWIKNGEEILGADTQYGSEGSMNRPTVSVLRNVEVLDKNIGILKTSLETANSDIKTIQEAGYIPEAPRDGQAYVRKDGEWVLLSTFLSPALVPRGSHHHHHHSAWSHPQFEK) with a C-terminal thrombin cleavage site, 6×His tag, and Strep-TagII was synthesized and subcloned into a mammalian expression vector derived from pLEXm. The construct was expressed by transient transfection of Expi293 (ThermoFisher) cells in suspension at 37 °C for 5 days. The protein was first purified with a Ni2+-nitrilotriacetic acid resin (GE Healthcare) using an elution buffer consisting of 50 mM Tris-HCl, pH 7.5, 400 mM NaCl and 300 mM imidazole pH 8.0, followed by purification with StrepTactin resin (IBA) according to the manufacturer’s instructions.

Cell lines<br/>HEK293T/17 (ATCC CRL-11268), Vero E6 (ATCC), Huh7.5 cells (provided by D. R. Taylor, US Food and Drug Administration) and ACE2-expressing 293T cells (provided by M. Farzan, Scripps Research Institute) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 2 mM glutamine and 1% penicillin–streptomycin at 37 °C and 5% CO2. Vero E6 cells used in plaque assays to determine lung and nasal turbinate viral titres were cultured in DMEM supplemented with 10% Fetal Clone II and 1% antibiotic–antimycotic at 37 °C and 5% CO2. Vero E6 cells used in plaque-reduction neutralization test (PRNT) assays were cultured in DMEM supplemented with 10% Fetal Clone II and amphotericin B (0.25 μg ml−1) at 37 °C and 5% CO2. Lentivirus encoding hACE2-P2A-TMPRSS2 was made to generate A549-hACE2-TMPRSS2 cells, which were maintained in DMEM supplemented with 10% FBS and 1 μg ml−1 puromycin. Expi293 cells were maintained in the manufacturer’s suggested medium. BHK-21/WI-2 cells were obtained from Kerafast and cultured in DMEM with 5% FBS at 37 °C and 6–8% CO2. Cell lines were not authenticated. All cells lines were tested for mycoplasma and remained negative.

In vitro mRNA expression<br/>HEK293T cells were transiently transfected with mRNA encoding SARS-CoV-2 wild-type S or S(2P) protein using a TranIT mRNA transfection kit (Mirus). After 24 h, the cells were collected and resuspended in fluorescence-activated cell sorting (FACS) buffer (1× PBS, 3% FBS, 0.05% sodium azide). To detect surface-protein expression, the cells were stained with 10 μg ml−1 ACE2–Flag (Sigma) or 10 μg ml−1 CR302235 in FACS buffer for 30 min on ice. Thereafter, cells were washed twice in FACS buffer and incubated with FITC–anti-Flag (Sigma) or Alexa Fluor 647–goat anti-human IgG (Southern Biotech) in FACS buffer for 30 min on ice. Live/Dead aqua fixable stain (Invitrogen) were used to assess viability. Data acquisition was performed on a BD LSRII Fortessa instrument (BD Biosciences) and analysed by FlowJo software v.10 (Tree Star).

Mouse models<br/>Animal experiments were carried out in compliance with all pertinent US National Institutes of Health regulations and approval from the Animal Care and Use Committee (ACUC) of the Vaccine Research Center, Moderna Inc., or University of North Carolina at Chapel Hill. For immunogenicity studies, 6- to 8-week-old female BALB/c (Charles River), BALB/cJ, C57BL/6J or B6C3F1/J mice (Jackson Laboratory) were used. mRNA formulations were diluted in 50 μl 1× PBS, and mice were inoculated intramuscularly in the same hind leg for both prime and boost. Control mice received PBS because previous studies have demonstrated the mRNA formulations being tested do not create substantial levels of nonspecific immunity beyond a few days36,37,38. For all SARS-CoV-2 S(2P) protein vaccinations, mice were inoculated intramuscularly with SAS as previously described1. For S(2P) + alum immunizations, SARS-CoV-2 S(2P) protein + 250 μg alum hydrogel was delivered intramuscularly. For challenge studies to evaluate MERS-CoV vaccines, 16- to 20-week-old male and female 288/330+/+mice22 were immunized. Four weeks post-boost, pre-challenge sera were collected from a subset of mice, and the remaining mice were challenged with 5 × 105 PFU of a mouse-adapted MERS-CoV EMC derivative, m35c439. On day 3 post-challenge, lungs were collected and haemorrhage and viral titre were assessed according to previously published methods40. For challenge studies to evaluate SARS-CoV-2 vaccines, BALB/cJ mice were challenged with 105 PFU SARS-CoV-2 MA. This virus contains two mutations (Q498T/P499Y) in the receptor binding domain that enable binding of SARS-CoV-2 S protein to the mouse ACE2 receptor and infection and replication in the upper and lower respiratory tract32. On day 2 post-challenge, lungs and nasal turbinates were collected for viral titre assessment according to previously published methods32. Sample size for animal experiments was determined on the basis of criteria set by institutional ACUC. Experiments were not randomized or blinded.

Histology<br/>Lungs were collected from mice at the indicated study end points and placed in 10% neutral-buffered formalin until adequately fixed. Thereafter, tissues were trimmed to a thickness of 3–5 mm, processed and paraffin embedded. The respective paraffin tissue blocks were sectioned at 5 μm and stained with haematoxylin and eosin. All sections were examined by a board-certified veterinary pathologist using an Olympus BX51 light microscope, and photomicrographs were taken using an Olympus DP73 camera.

ELISA<br/>Nunc Maxisorp ELISA plates (ThermoFisher) were coated with 100 ng per well of protein in 1× PBS at 4 °C for 16 h. Where applicable, to eliminate fold-on-specific binding from MERS-CoV S(2P) or SARS-CoV-2 S(2P) protein-immune mouse serum, 50 μg ml−1 of fold-on protein was added for 1 h at room temperature. After standard washes and blocks, plates were incubated with serial dilutions of heat-inactivated sera for 1 h at room temperature. Following washes, anti-mouse IgG, IgG1 or IgG2a and/or IgG2c–horseradish peroxidase conjugates (ThermoFisher) were used as secondary antibodies, and 3,5,3′5′-tetramethylbenzidine (TMB) (KPL) was used as the substrate to detect antibody responses. End-point titres were calculated as the dilution that emitted an optical density exceeding 4× background (secondary antibody alone).

Lentivirus-based pseudovirus-neutralization assay<br/>The pseudovirus-neutralization assay measures the inhibition of pseudovirus attachment and entry including fusion-inhibiting activity. It is a single-round virus, does not replicate, and does not express the S protein in transduced cells. Therefore, pseudovirus infection will not cause cell-to-cell fusion or plaque formation that can be measured in a classical neutralization assay using live virus. This pseudovirus neutralization assay has been shown to correlate with live virus plaque-reduction neutralization33, and because it does not require BL3 containment, was chosen as the preferred assay for measuring neutralizing activity in these studies. We introduced divergent amino acids, as predicted from translated sequences, into the CMV/R-MERS-CoV EMC S (GenBank: AFS88936) gene41 to generate a MERS-CoV m35c4 S gene39. To produce SARS-CoV-2 pseudoviruses, a codon-optimized CMV/R-SARS-CoV-2 S (Wuhan-1, GenBank: MN908947.3) plasmid was constructed. Pseudoviruses were produced by co-transfection of plasmids encoding a luciferase reporter, lentivirus backbone, and S genes into HEK293T/17 cells (ATCC CRL-11268), as previously described41. For SARS-CoV-2 pseudovirus, human transmembrane protease serine 2 (TMPRSS2) plasmid was also co-transfected42. Pseudoneutralization assay methods have been previously described1,33. In brief, heat-inactivated serum was mixed with pseudoviruses, incubated, and then added to Huh7.5 cells or ACE-2-expressing 293T cells for MERS-CoV and SARS-CoV-2 respectively. Seventy-two hours later, cells were lysed and luciferase activity (in relative light units (RLU)) was measured. Per cent neutralization was normalized considering uninfected cells as 100% neutralization and cells infected with only pseudovirus as 0% neutralization. IC50 titres were determined using a log (agonist) vs normalized-response (variable slope) nonlinear function in Prism v8 (GraphPad).

Recombinant VSV∆G-based pseudovirus neutralization assay<br/>Codon-optimized wild-type (D614) or D614G spike gene (Wuhan-Hu-1 strain, NCBI reference sequence: NC_045512.2) was cloned into pCAGGS vector. To generate VSV∆G-based SARS-CoV-2 pseudovirus, BHK-21/WI-2 cells were transfected with the spike expression plasmid and infected VSV∆G-firefly-luciferase as previously described43. A549-hACE2-TMPRSS2 cells were infected by pseudovirus for 1 h at 37 °C. The inoculum virus or virus-antibody mix was removed after infection. Eighteen hours later, an equal volume of One-Glo reagent (Promega) was added to culture medium for readout using a BMG PHERastar-FS plate reader. The neutralization procedure and data analysis are the same as mentioned above for the lentivirus-based pseudovirus neutralization assay.



Pseudovirus Neutralization Assay<br/>We introduced divergent amino acids, as predicted from translated sequences, into the CMV/R-MERS-CoV EMC S (GenBank#: AFS88936) gene33 to generate a MERS-CoV m35c4 S gene31. To produce SARS-CoV-2 pseudoviruses, a codon-optimized CMV/R-SARS-CoV-2 S (Wuhan-1, Genbank #: MN908947.3) plasmid was constructed. Pseudoviruses were produced by co-transfection of plasmids encoding a luciferase reporter, lentivirus backbone, and S genes into HEK293T/17 cells (ATCC #CRL-11268), as previously described33. For SARS-CoV-2 pseudovirus, human transmembrane protease serine 2 (TMPRSS2) plasmid was also co-transfected34. Pseudoneutralization assay methods have been previously described11. Briefly, HI serum was mixed with pseudoviruses, incubated, and then added to Huh7.5 cells or ACE-2-expressing 293T cells, for MERS-CoV and SARS-CoV-2 respectively. Seventy-two hr later, cells were lysed, and luciferase activity (relative light units, RLU) was measured. Percent neutralization was normalized considering uninfected cells as 100% neutralization and cells infected with only pseudovirus as 0% neutralization. IC50 titers were determined using a log (agonist) vs. normalized response (variable slope) nonlinear function in Prism v8 (GraphPad).

Plaque Reduction Neutralization Test (PRNT)<br/>HI sera were diluted in gelatin saline (0.3% [wt/vol] gelatin in phosphate-buffered saline supplemented with CaCl2 and MgCl2) to generate a 1:5 dilution of the original specimen, which served as a starting concentration for further serial log4 dilutions terminating in 1:81,920. Sera were combined with an equal volume of SARS-CoV-2 clinical isolate 2019-nCoV/USA-WA1-F6/2020 in gelatin saline, resulting in an average concentration of 730 plaque-forming units per mL (determined from plaque counts of 24 individual wells of untreated virus) in each serum dilution. Thus, final serum concentrations ranged from 1:10 to 1:163,840 of the original. Virus/serum mixtures were incubated for 20 min at 37 °C, followed by adsorption of 0.1 mL to each of two confluent Vero E6 cell monolayers (in 10-cm2 wells) for 30 min at 37°C. Cell monolayers were overlaid with Dulbecco’s modified Eagle’s medium (DMEM) containing 1% agar and incubated for 3 d at 37°C in humidified 5% CO2. Plaques were enumerated by direct visualization. The average number of plaques in virus/serum (duplicate) and virus-only (24) wells was used to generate percent neutralization curves according the following formula: 1 – (ratio of mean number of plaques in the presence and absence of serum). The PRNT IC50 titer was defined as the reciprocal serum dilution at which the neutralization curve crossed the 50% threshold.