Summary
A SARS-CoV-2 vaccine is needed to control the global COVID-19 public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a Phase 2 clinical trial with a trajectory towards Phase 3 efficacy evaluation.
Since its emergence in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has accounted for over 7 million cases of Coronavirus Disease 2019 (COVID-19) worldwide in less than 7 months1. SARS-CoV-2 is the third novel betacoronavirus in the last 20 years to cause substantial human disease; however, unlike its predecessors SARS-CoV and MERS-CoV, SARS-CoV-2 transmits efficiently from person-to-person. In absence of a vaccine, public health measures such as quarantining newly diagnosed cases, contact tracing, and mandating face masks and physical distancing have been instated to reduce transmission2. It is estimated that until 60-70% population immunity is established, it is unlikely for COVID-19 to be controlled well enough to resume normal activities. If immunity remains solely dependent on infection, even at a 1% mortality rate, >40 million people could succumb to COVID-19 globally3. Therefore, rapid development of vaccines against SARS-CoV-2 is critical for changing the global dynamic of this virus.
The spike (S) protein, a class I fusion glycoprotein analogous to influenza hemagglutinin (HA), respiratory syncytial virus (RSV) fusion glycoprotein (F), and human immunodeficiency virus (HIV) gp160 (Env), is the major surface protein on the CoV virion and the primary target for neutralizing antibodies. S proteins undergo dramatic structural rearrangement to fuse virus and host cell membranes, allowing delivery of the viral genome into target cells. We previously showed that prefusion-stabilized protein immunogens that preserve neutralization-sensitive epitopes are an effective vaccine strategy for enveloped viruses, such as RSV4–8. Subsequently, we identified 2 proline substitutions (2P) at the apex of the central helix and heptad repeat 1 that effectively stabilized MERS-CoV, SARS-CoV and HCoV-HKU1 S proteins in the prefusion conformation9–11. Similar to other prefusion-stabilized fusion proteins, MERS S-2P protein is more immunogenic at lower doses than wild-type S protein11. The 2P has been widely transferrable to other beta-CoV spike proteins, suggesting a generalizable approach for designing stabilized prefusion beta-CoV S vaccine antigens. This is fundamental to the prototype pathogen approach for pandemic preparedness12,13.
Coronaviruses have long been predicted to have a high likelihood of spill over into humans and cause future pandemics14,15. As part of our pandemic preparedness efforts, we have studied MERS-CoV as prototype pathogen for betacoronaviruses to optimize vaccine design, to dissect the humoral immune response to vaccination, and identify mechanisms and correlates of protection. Achieving an effective and rapid vaccine response to a newly emerging virus requires the precision afforded by structure-based antigen design but also a manufacturing platform to shorten time to product availability. Producing cell lines and clinical grade subunit protein typically takes more than 1 year, while manufacturing nucleic acid vaccines can be done in a matter of weeks16,17. In addition to advantages in manufacturing speed, mRNA vaccines are potently immunogenic and elicit both humoral and cellular immunity18–20. Therefore, we evaluated mRNA formulated in lipid nanoparticles (mRNA/LNP) as a delivery vehicle for the MERS S-2P and found that transmembrane-anchored MERS S-2P mRNA elicited better neutralizing antibody responses than secreted MERS S-2P (Extended Data Fig. 1a). Additionally, consistent with protein immunogens, MERS S-2P mRNA was more immunogenic than MERS wild-type S mRNA (Extended Data Fig. 1b). Immunization with MERS S-2P mRNA/LNP elicited potent neutralizing activity down to a 0.1 μg dose and protected hDPP4 transgenic (288/330+/+21) mice against lethal MERS-CoV challenge in a dose-dependent manner, establishing proof-of-concept that mRNA expressing the stabilized S-2P protein is protective. Notably, the sub-protective 0.01 μg dose of MERS S-2P mRNA did not cause exaggerated disease following MERS-CoV infection, but instead resulted in partial protection against weight loss followed by full recovery without evidence of enhanced illness (Fig. 1).
In early January 2020, a novel CoV (nCoV) was identified as the cause of a respiratory virus outbreak occurring in Wuhan, China. Within 24 hours of the release of the SARS-CoV-2 isolate sequences (then known as “2019-nCoV”) on January 10th, the 2P mutations were substituted into S positions aa986 and 987 to produce prefusion-stabilized SARS-CoV-2 S (S-2P) protein for structural analysis22 and serological assay development23,24 in silico without additional experimental validation. Within 5 days of sequence release, current Good Manufacturing Practice (cGMP) production of mRNA/LNP expressing the SARS-CoV-2 S-2P as a transmembrane-anchored protein with the native furin cleavage site (mRNA-1273) was initiated in parallel with preclinical evaluation. Remarkably, this led to the start of a first in human Phase 1 clinical trial on March 16, 2020, 66 days after the viral sequence was released, and a Phase 2 began 74 days later on May 29, 2020 (Extended Data Fig. 2). Prior to vaccination of the first human subject, expression and antigenicity of the S-2P antigen delivered by mRNA was confirmed in vitro (Extended Data Fig. 3), and immunogenicity of mRNA-1273 was documented in several mouse strains. The results of those studies are detailed hereafter.
Immunogenicity was assessed in six-week old female BALB/cJ, C57BL/6J, and B6C3F1/J mice by immunizing intramuscularly (IM) twice with 0.01, 0.1, or 1 μg of mRNA-1273 at a 3-week interval. mRNA-1273 induced dose-dependent S-specific binding antibodies after prime and boost in all mouse strains (Fig. 2a-c). Potent neutralizing activity was elicited by 1 μg of mRNA-1273, reaching 819, 89, and 1115 reciprocal IC50 geometric mean titer (GMT) for BALB/cJ, C57BL/6J, and B6C3F1/J mice, respectively (Fig. 2d-f). These levels are similar to the neutralization activity achieved by immunizing with 1 μg of SAS-adjuvanted S-2P protein (Extended Data Table 1). To further gauge immunogenicity across a wide dose range, BALB/cJ mice were immunized with 0.0025 – 20 μg of mRNA-1273 revealing a strong positive correlation between dose-dependent mRNA-1273-elicited binding and neutralizing antibody responses (Extended Data Fig. 4). BALB/cJ mice that received a single dose of mRNA-1273 were evaluated in order to ascertain the utility for a one-dose vaccine regimen. S-binding antibodies were induced in mice immunized with one dose of 1 or 10 μg of mRNA-1273, and the 10 μg dose elicited neutralizing antibody activity that increased between week 2 and week 4, reaching 315 reciprocal IC50 GMT (Extended Data Fig. 5a-b). These data demonstrate that mRNA expressing SARS-CoV-2 S-2P is a potent immunogen and neutralizing activity can be elicited with a single dose.
Next, we evaluated the balance of Th1 and Th2, because vaccine-associated enhanced respiratory disease (VAERD) has been associated with Th2-biased immune responses in children immunized with whole-inactivated virus vaccines against RSV and measles virus25,26. A similar phenomenon has also been reported in some animal models with whole-inactivated SARS-CoV vaccines27. Thus, we first compared levels of S-specific IgG2a/c and IgG1, which are surrogates of Th1 and Th2 responses respectively, elicited by mRNA-1273 to those elicited by SARS-CoV-2 S-2P protein adjuvanted with the TLR4-agonist Sigma Adjuvant System (SAS). Both immunogens elicited IgG2a and IgG1 subclass S-binding antibodies, indicating a balanced Th1/Th2 response (Fig. 3a-c; Extended Data Fig. 6). The S-specific IgG subclass profile following a single dose of mRNA-1273 (Extended Data Fig. 5c) was similar to that observed following two doses. In contrast, Th2-biased antibodies with lower IgG2a/IgG1 subclass response ratios were observed in mice immunized with SARS-CoV-2 S protein formulated in alum (Extended Data Fig. 7a-b). Following re-stimulation with peptide pools (S1 and S2) corresponding to the S protein, splenocytes from mRNA-1273-immunized mice secreted more IFN-γ than IL-4, IL-5, or IL-13 whereas SARS-CoV-2 S protein with alum induced Th2-skewed cytokine secretion (Extended Data Fig. 7c-d). 7 weeks post-boost, we also directly measured cytokine patterns in vaccine-induced memory T cells by intracellular cytokine staining (ICS); mRNA-1273-elicited CD4+ T cells re-stimulated with S1 or S2 peptide pools exhibited a Th1-dominant response, particularly at higher immunogen doses (Fig. 3d-e). Furthermore, 1 μg of mRNA-1273 induced a robust CD8+ T cell response to the S1 peptide pool (Fig. 3f-g). The Ig subclass and T cell cytokine data together demonstrate that immunization with mRNA-1273 elicits a balanced Th1/Th2 response in contrast to the Th2-biased response seen with S protein adjuvanted with alum, suggesting that mRNA vaccination avoids Th2-biased immune responses that have been linked to VAERD.
Protective immunity was assessed in young adult BALB/cJ mice challenged with mouse-adapted (MA) SARS-CoV-2 that exhibits viral replication localized to lungs and nasal turbinates28. BALB/cJ mice that received two 1 μg doses of mRNA-1273 were completely protected from viral replication in lungs after challenge at a 5- (Fig. 4a) or 13-week intervals following boost (Extended Data Fig. 8a). mRNA-1273-induced immunity also rendered viral replication in nasal turbinates undetectable in 6 out of 7 mice (Fig. 4b, Extended Data Fig. 8b). Efficacy of mRNA-1273 was dose-dependent, with two 0.1 μg mRNA-1273 doses reducing lung viral load by ~100-fold and two 0.01 μg mRNA-1273 doses reducing lung viral load by ~3-fold (Fig. 4a). Of note, mice challenged 7 weeks after a single dose of 1 μg or 10 μg of mRNA-1273 were also completely protected against lung viral replication (Fig. 4c). Challenging animals immunized with sub-protective doses provides an orthogonal assessment of safety signals, such as increased clinical illness or pathology. Similar to what was observed with MERS-CoV S-2P mRNA, mice immunized with sub-protective 0.1 and 0.01 μg mRNA-1273 doses showed no evidence of enhanced lung pathology or excessive mucus production (Fig. 4d). In summary, mRNA-1273 is immunogenic, efficacious, and does not show evidence of promoting VAERD when given at sub-protective doses in mice.
Here, we showed that 1 μg of mRNA-1273 was sufficient to induce robust neutralizing activity and CD8 T cell responses, balanced Th1/Th2 antibody isotype responses, and protection from viral replication for more than 3 months following a prime/boost regimen similar to that being tested in humans. Inclusion of lower sub-protective doses demonstrated the dose-dependence of antibody, Th1 CD4 T cell responses, and protection, suggesting immune correlates of protection can be further elucidated. A major goal of animal studies to support SARS-CoV-2 vaccine candidates through clinical trials is to not only prove elicitation of potent protective immune responses, but to show that sub-protective responses do not cause VAERD3. Sub-protective doses did not prime mice for enhanced immunopathology following challenge. Moreover, the induction of protective immunity following a single dose suggests that consideration could be given to administering one dose of this vaccine in the outbreak setting. These data, combined with immunogenicity data from nonhuman primates and subjects in early Phase 1 clinical trials, will be used to inform the dose and regimen of mRNA-1273 in advanced clinical efficacy trials.
The COVID-19 pandemic of 2020 is the Pathogen X event that has long been predicted12,13. Here, we provide a paradigm for rapid vaccine development. Structure-guided stabilization of the MERS-CoV S protein combined with a fast, scalable, and safe mRNA/LNP vaccine platform led to a generalizable beta-CoV vaccine solution that translated into a commercial mRNA vaccine delivery platform, paving the way for the rapid response to the COVID-19 outbreak. This is a demonstration of how the power of new technology-driven concepts like synthetic vaccinology facilitate a vaccine development program that can be initiated with pathogen sequences alone11. It is also a proof-of-concept for the prototype pathogen approach for pandemic preparedness and response that is predicated on identifying generalizable solutions for medical countermeasures within virus families or genera12. Even though the response to the COVID-19 pandemic is unprecedented in its speed and breadth, we envision a response that could be quicker. There are 24 other virus families known to infect humans, and with sustained investigation of those potential threats, we could be better prepared for future looming pandemics13.
Author Contributions
K.S.C., D.K.E., S.R.L., O.M.A, S.B.B., R.A.G., S.H., A.S., C.Z., A.T.D., K.H.D., S.E., C.A.S., A.W., E.J.F., D.R.M, K.W.B., M.M., B.M.N., G.B.H., K.B., D.G.D., L.M., I.R., W.P.K, S.S., L.W., Y.Z., J.C., L.S., L.A.C., E.P., R.J.L., N.E.A., E.N., M.M., V.P., C.L., M.K.L., W.S., K.G., K.L., E.S.Y., A.W., G.A., N.A.D.R., G.S.J., H.B., M.N., T.J.R., M.R.D., I.N.M., K.M.M., J.R.M., R.S.B., A.C., and B.S.G. designed, completed, and/or analyzed experiments. N.W., D.W., and J.S.M. contributed new reagents/analytic tools. K.S.C., K.M.M, and B.S.G. wrote the manuscript. All authors contributed to discussions in regard to and editing of the manuscript.
Competing Interest Declaration
K.S.C., N.W., J.S.M., and B.S.G. are inventors on International Patent Application No. WO/2018/081318 entitled “Prefusion Coronavirus Spike Proteins and Their Use.” K.S.C., O.M.A., G.B.H., N.W., D.W., J.S.M, and B.S.G. are inventors on US Patent Application No. 62/972,886 entitled “2019-nCoV Vaccine”. R.S.B. filed an invention report for the SARS-CoV-2 MA virus (UNC ref. #18752).
Additional Information
Correspondence and requests for materials should be addressed to Barney S. Graham, bgraham{at}nih.gov and Andrea Carfi, andrea.carfi{at}modernatx.com.
Methods
MERS-CoV S-2P and SARS-CoV-2 S-2P mRNA synthesis and lipid nanoparticle formulation
For each vaccine, T7 RNA polymerase-mediated transcription was used in vitro to synthesize the mRNA from a linearized DNA template, which flanked the immunogen open-reading frames with the 5′ and 3′ untranslated regions and a poly-A tail as described previously 29. mRNA was then purified, diluted in citrate buffer to the desired concentration and encapsulated into lipid nanoparticles (LNP) by ethanol drop nanoprecipitation. At molar ratio of 50:10:38.5:1.5 (ionizable lipid:DSPC:cholesterol:PEG-lipid), lipids were dissolved in ethanol and combined with a 6.25-mM sodium acetate buffer (pH 5) containing mRNA at a ratio of 3:1 (aqueous:ethanol). Formulations were dialyzed against phosphate-buffered saline (pH 7.4) for at least 18 hr, concentrated using Amicon ultracentrifugal filters (EMD Millipore), passed through a 0.22-μm filter and stored at −20°C until use. All formulations underwent quality control for particle size, RNA encapsulation, and endotoxin. LNP were between 80 – 100 nm in size, with > 90% encapsulation on mRNA and < 10 EU/mL endotoxin.
MERS-CoV and SARS-CoV Protein Expression and Purification
Vectors encoding MERS-CoV S-2P11 and SARS-CoV S-2P22 were generated as previously described with the following small amendments. Proteins were expressed by transfection of plasmids into Expi293 cells using Expifectamine transfection reagent (ThermoFisher) in suspension at 37°C for 4-5 days. Transfected cell culture supernatants were collected, buffer exchanged into 1X PBS, and protein was purified using Strep-Tactin resin (IBA). For proteins used for mouse inoculations, tags were cleaved with addition of HRV3C protease (ThermoFisher) (1% wt/wt) overnight at 4 °C. Size exclusion chromatography using Superose 6 Increase column (GE Healthcare) yielded final purified protein.
Design and Production of Recombinant Minifibritin Foldon Protein
A mammalian codon-optimized plasmid encoding foldon inserted minifibritin (ADIVLNDLPFVDGPPAEGQSRISWIKNGEEILGADTQYGSEGSMNRPTVSVLRNVEVLDKNIGI LKTSLETANSDIKTIQEAGYIPEAPRDGQAYVRKDGEWVLLSTFLSPALVPRGSHHHHHHSAWS HPQFEK) with a C-terminal thrombin cleavage site, 6x 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 (NTA) 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
HEK293T/17 (ATCC #CRL-11268), Vero E6 (ATCC), Huh7.5 cells (provided by Deborah R. Taylor, US Food and Drug Administration), and ACE-2-expressing 293T cells (provided by Michael 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 titers were cultured in DMEM supplemented with 10% Fetal Clone II and 1% anti/anti at 37C and 5% CO2. Vero E6 cells used in PRNT assays were cultured in DMEM supplemented with 10% Fetal Clone II and amphotericin B [0.25 μg/ml] at 37C and 5% CO2. Expi293 cells were maintained in manufacturer’s suggested media.
In vitro mRNA Expression
HEK293T cells were transiently transfected with mRNA encoding SARS-CoV-2 WT S or S-2P protein using a TranIT mRNA transfection kit (Mirus). After 24 hr, the cells were harvested and resuspended in FACS buffer (1X PBS, 3% FBS, 0.05% sodium azide). To detect surface protein expression, the cells were stained with 10 μg/mL ACE2-FLAG (Sigma) or CR302230 in FACS buffer for 30 min on ice. Thereafter, cells were washed twice in FACS buffer and incubated with FITC anti-FLAG (Sigma) or Alexafluor 647 goat anti-human IgG (Southern Biotech) in FACS buffer for 30 min on ice. Live/Dead aqua fixable stain (Invitrogen) were utilized to assess viability. Data acquisition was performed on a BD LSRII Fortessa instrument (BD Biosciences) and analyzed by FlowJo software v10 (Tree Star, Inc.)
Mouse Models
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 of the Vaccine Research Center, Moderna Inc., or University of North Carolina at Chapel Hill. For immunogenicity studies, 6-8-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 of 1X PBS, and mice were inoculated IM into the same hind leg for both prime and boost. For all SARS-CoV-2 S-P protein vaccinations, mice were inoculated IM, with SAS, as previously detailed11. For S + alum immunizations, SARS-CoV-2 S protein (Sino Biological) + 250 μg alum hydrogel was delivered IM. For challenge studies to evaluate MERS-CoV-2 vaccines, 16-20-week-old 288/330+/+mice21 were immunized. Four weeks post-boost, pre-challenge sera were collected from a subset of mice, and remaining mice were challenged with 5×105 PFU of a mouse-adapted MERS-CoV EMC derivative, m35c431. On day 3 post-challenge, lungs were harvested, and hemorrhage and viral titer were assessed, per previously published methods32. For challenge studies to evaluate SARS-CoV-2 vaccines, BALB/cJ mice were challenged with 105 PFU of mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA). On day 2 post-challenge, lungs and nasal turbinates were harvested for viral titer assessment, per previously published methods28.
Histology
Lungs from mice were collected at the indicated study endpoints and placed in 10% neutral buffered formalin (NBF) 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 hematoxylin and eosin (H&E). 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.
Enzyme-linked Immunosorbent Assay (ELISA)
Nunc Maxisorp ELISA plates (ThermoFisher) were coated with 100 ng/well of protein in 1X PBS at 4°C for 16 hr. Where applicable, to eliminate fold-on-specific binding from MERS S-2P- or SARS-CoV-2 S-2P protein-immune mouse serum, 50 μg/mL of fold-on protein was added for 1 hr at room temperature (RT). After standard washes and blocks, plates were incubated with serial dilutions of heat-inactivated (HI) sera for 1 hr at RT. Following washes, anti-mouse IgG, IgG1, or IgG2a or IgG2c–horseradish peroxidase conjugates (ThermoFisher) were used as secondary Abs, and 3,5,3′5′-tetramethylbenzidine (TMB) (KPL) was used as the substrate to detect Ab responses. Endpoint titers were calculated as the dilution that emitted an optical density exceeding 4X background (secondary Ab alone).
Pseudovirus Neutralization Assay
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)
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.
Intracellular Cytokine Staining
Mononuclear single cell suspensions from whole mouse spleens were generated using a gentleMACS tissue dissociator (Miltenyi Biotec) followed by 70 μm filtration and density gradient centrifugation using Fico/Lite-LM medium (Atlanta Biologicals). Cells from each mouse were resuspended in R10 media (RPMI 1640 supplemented with Pen-Strep antibiotic, 10% HI-FBS, Glutamax, and HEPES) and incubated for 6 hr at 37°C with protein transport inhibitor cocktail (eBioscience) under three conditions: no peptide stimulation, and stimulation with two spike peptide pools (JPT product PM-WCPV-S-1). Peptide pools were used at a final concentration of 2 μg/mL each peptide. Cells from each group were pooled for stimulation with cell stimulation cocktail (eBioscience) as a positive control. Following stimulation, cells were washed with PBS prior to staining with LIVE/DEAD Fixable Blue Dead Cell Stain (Invitrogen) for 20 min at RT. Cells were then washed in FC buffer (PBS supplemented with 2% HI-FBS and 0.05% NaN3) and resuspended in BD Fc Block (clone 2.4G2) for 5 min at RT prior to staining with a surface stain cocktail containing the following antibodies purchased from BD and Biolegend: I-A/I-E (M5/114.15.2) PE, CD8a (53-6.7) BUV805, CD44 (IM7) BUV395, CD62L (MEL-14) BV605, and CD4 (RM4-5) BV480 in brilliant stain buffer (BD). After 15 min, cells were washed with FC buffer then fixed and permeabilized using the BD Cytofix/Cytoperm fixation/permeabilization solution kit according to manufacturer instructions. Cells were washed in perm/wash solution and stained with Fc Block (5 min at RT), followed by intracellular staining (30 min at 4°C) using a cocktail of the following antibodies purchased from BD, Biolegend, or eBioscience: CD3e (17A2) BUV737, IFN-γ (XMG1.2) BV650, TNF-α (MP6-XT22) BV711, IL-2 (JES6-5H4) BV421, IL-4 (11B11) Alexa Fluor 488, and IL-5 (TRFK5) APC in 1x perm/wash diluted with brilliant stain buffer. Finally, cells were washed in perm/wash solution and resuspended in 0.5% PFA-FC stain buffer prior to running on a Symphony A5 flow cytometer (BD). Analysis was performed using FlowJo software, version 10.6.2 according to the gating strategy outlined in Extended Data Figure 9. Background cytokine expression in the no peptide condition was subtracted from that measured in the S1 and S2 peptide pools for each individual mouse.
T Cell Stimulation and Cytokine Analysis
Spleens from immunized mice were collected 2 weeks post-boost. 2 × 106 splenocytes/well (96-well plate) were stimulated in vitro with two peptide libraries, JPT1 and JPT2, (15mers with 11 aa overlap) covering the entire SARS-CoV-2 spike protein (JPT product PM-WCPV-S-1). Both peptide libraries were used at a final concentration of 1 μg/mL. After 24 hr of culture at 37°C, the plates were centrifuged and supernatant was collected and frozen at −80°C for cytokine detection. Measurements and analyses of secreted cytokines from a murine 35-plex kit were performed using a multiplex bead-based technology (Luminex) assay with a Bio-Plex 200 instrument (Bio-Rad) after 2-fold dilution of supernatants.
Statistical Analysis
Geometric means or means are represented by the heights of bars, or symbols, and error bars represent the corresponding SD. Dotted lines indicate assay limits of detection. Mann-Whitney tests were used to compare 2 experimental groups and Wilcoxon signed rank tests to compare the same animals at different time points. To compare >2 experimental groups, Kruskal-Wallis ANOVA with Dunn’s multiple comparisons tests were applied. For antibody responses in Extended Data Fig. 4c, a Spearman correlation test was used to correlate binding antibody titers to neutralizing antibody titers. * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001, **** = p-value < 0.0001.
Acknowledgements
We thank Gabriela Alvarado, Karin Bok, Kevin Carlton, Masaru Kanekiyo, Robert Seder, and additional members of all included laboratories for critical discussions, advice, and review of the manuscript. We thank Judy Stein and Monique Young for technology transfer and administrative support, respectively. We thank members of the NIH NIAID VRC Translational Research Program for technical assistance with mouse experiments. This work was supported by the Intramural Research Program of the VRC and the Division of Intramural Research, NIAID, NIH (B.S.G) and NIH NIAID grant R01-AI127521 (J.S.M.). mRNA-1273 has been funded in part with Federal funds from the Department of Health and Human Services, Office of the Assistant Secretary for Preparedness and Response, Biomedical Advanced Research and Development Authority, under Contract 75A50120C00034. PRNT assays were funded under NIH Contract HHSN261200800001E Agreement 17×198 (to J.D.C.), furnished through Leidos Biomedical Research, Inc. MERS mRNA mouse challenge studies were funded under NIH Contract HHSN272201700036I Task Rrder No. 75N93019F00132 Requisition No. 5494549 (to R.B.). K.S.C.’s research fellowship was partially funded by the Undergraduate Scholarship Program, Office of Intramural Training and Education, Office of the Director, NIH. D.R.M. was funded by NIH NIAID grant T32-AI007151 and a Burroughs Wellcome Fund Postdoctoral Enrichment Program Award.