SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness

Abstract

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.

Extended Data Figure 1.. Transmembrane-anchored MERS-CoV S-2P (S-2P_TM) mRNA elicits more potent neutralizing antibody responses than secreted MERS-CoV S-2P and S WT mRNA.

C57BL/6J mice were immunized at weeks 0 and 4 with (a) 0.4, 2, or 10 μg of MERS-CoV S-2P_TM (red) or MERS S-2P_secreted (red hashed) or (b) 0.016 μg, 0.08 μg, or 0.4 μg of MERS-CoV S-2P or MERS-CoV S WT_TM (black) mRNA. Sera were collected 4 weeks post-boost and assessed for neutralizing antibodies against MERS-CoV m35c4 pseudovirus. Dotted line = assay limit of detection. Immunogens were compared at each dose level

Extended Data Figure 2.. Timeline for mRNA-1273’s progression to clinical trial.

The morning after novel coronavirus (nCoV) sequences were released, spike sequences were modified to include prefusion stabilizing mutations and synthesized for protein production, assay development, and vaccine development. Twenty-five days after viral sequences were released, clinically-relevant mRNA-1273 was received to initiate animal experiments. Immunogenicity in mice was confirmed 15 days later. Moderna shipped clinical drug product 41 days after GMP production began, leading to the Phase 1 clinical trial starting 66 days following the release of nCoV sequences.

Extended Data Figure 3.. In vitro expression of SARS-CoV-2 spike mRNA on cell surface.

293T cells were transfected with mRNA expressing SARS-CoV-2 wild-type spike (black) or S-2P (red), stained with ACE2 (a,c) or CR3022 (b,d), and evaluated by flow cytometry 24 post-transfection. Mock-transfected (PBS) cells served as a control.

Extended Data Figure 4.. Dose-dependent mRNA-1273-elicited antibody responses reveal strong positive correlation between binding and neutralization titers.

BALB/cJ mice were immunized at weeks 0 and 3 weeks with various doses (0.0025 – 20 μg) of mRNA-1273. (a-b) Sera were collected 2 weeks post-boost and assessed for SARS-CoV-2 S-specific IgG by ELISA (a) and neutralizing antibodies against homotypic SARS-CoV-2 pseudovirus (b). (a-b) All doses were compared to 20 μg dose.

Extended Data Figure 5.. A single dose of mRNA-1273 elicits robust antibody responses.

BALB/cJ mice were immunized with 0.1 (blue), 1 μg (red), or 10 μg (purple) of mRNA-1273. Sera were collected 2 (open circles) and 4 (closed circles) weeks post-immunization and assessed for SARS-CoV-2 S-specific total IgG by ELISA (a) and neutralizing antibodies against homotypic SARS-CoV-2 pseudovirus (b). (c) S-specific IgG2a and IgG1 were also measured by ELISA, and IgG2a to IgG1 subclass ratios were calculated. Dotted line = assay limit of detection. (a-b) Doses were compared 4 weeks post-boost, and timepoints were compared within each dose level.

Extended Data Figure 6.. mRNA-1273 and SAS-adjuvanted S-2P protein elicit both IgG2a and IgG1 subclass S-binding antibodies.

BALB/cJ (a-c) or C57BL/6J (d-f) mice were immunized at weeks 0 and 3 with 0.01 (green), 0.1 (blue), or 1 μg (red) of mRNA-1273 SARSCoV-2 S-2P protein adjuvanted with SAS. Sera were collected 2 weeks post-boost and assessed by ELISA for SARS-CoV-2 S-specific IgG1 and IgG2a or IgG2c for BALB/cJ and C57BL/6J mice, respectively. Endpoint titers (a-b, d-e) and endpoint titer ratios of IgG2a to IgG1 (c) and IgG2c to IgG1 (f) were calculated. For mice for which endpoint titers did not reach the lower limit of detection (dotted line), ratios were not calculated (N/A). IgG1 and IgG2a/c (a-b, d-e) and immunogens (c, f) were compared at each dose level.

Extended Data Figure 7.. mRNA-1273 elicits Th1-skewed responses compared to S protein adjuvanted with alum.

BALB/c mice were immunized at weeks 0 and 2 weeks with 1 (red) or 10 μg (purple) of mRNA-1273 or 10 μg of SARS-CoV-2 S protein adjuvanted with alum hydrogel (orange). (a-b) Sera were collected 2 weeks post-boost and assessed by ELISA for SARS-CoV-2 S-specific IgG1 and IgG2a. Endpoint titers (a) and endpoint titer ratios of IgG2a to IgG1 (b) were calculated. (c-d) Splenocytes were also collected 4 weeks post-boost to evaluate IFN-γ IL-4, IL-5, and IL-13 cytokine levels secreted by T cells re-stimulated with S1 (c) and S2 (d) peptide pools, measured by Luminex. Dotted line = assay limit of detection. IgG1 and IgG2a/c (a) were compared at each dose level. (c-d) For cytokines, all comparisons were compared to PBS-immunized mice.

Extended Data Figure 8.. mRNA-1273 protects mice from upper and lower airway SARSCoV-2 infection, 13 weeks post-boost.

BALB/cJ mice were immunized at weeks 0 and 3 with 0.01 (green), 0.1 (blue), or 1 μg (red) of mRNA-1273. Age-matched naive mice (gray) served as controls. Thirteen weeks post-boost, mice were challenged with mouse-adapted SARS-CoV-2. Two days post-challenge, at peak viral load, mouse lungs (a) and nasal turbinates (b) were harvested from 5 mice per group for analysis of viral titers. Dotted line = assay limit of detection. All dose levels were compared.

Extended Data Figure 9.. Flow cytometry panel to quantify SARS-CoV-2 S-specific T cells in mice.

(a) A hierarchical gating strategy was used to unambiguously identify single, viable CD4+ and CD8+ T cells. Gating summary of SARS-CoV-2 S-specific (b-c) CD4 (b-c) and (d-e) CD8 (d-e) T cells elicited by 1.0 and 0.01 μg mRNA-1273 immunization. Antigen-specific T cell responses following peptide pool re-stimulation were defined as CD44^hi/cytokine⁺. Concatenated files shown were generated using the same number of randomly selected events from each animal across the different stimulation conditions using FlowJo software, v1

Figure 1.. MERS-CoV S-2P mRNA protects mice from lethal challenge.

288/330^+/+ mice were immunized at weeks 0 and 3 with 0.01 (green), 0.1 (blue), or 1 μg (red) of MERS-CoV S-2P mRNA. Mock-immunized mice were immunized with PBS (gray). Two weeks post-boost, sera were collected from 3 mice per group and assessed for neutralizing antibodies against MERS m35c4 pseudovirus (a). Four weeks post-boost, 12 mice per group were challenged with a lethal dose of mouse-adapted MERS-CoV (m35c4). Following challenge, mice were monitored for weight loss (b). Two days post-challenge, at peak viral load, lung viral titers (c) and hemorrhage (0 = no hemorrhage, 4 = severe hemorrhage in all lobes) (d) were assessed from 5 animals per group. Dotted line = assay limit of detection. (a, c-d) All dose levels were compared. (b) For weight loss, all comparisons are against PBS-immunized mice.

Figure 2.. mRNA-1273 elicits robust binding and neutralizing antibody responses in multiple mouse strains.

BALB/cJ (a, d), C57BL/6J (b, e), or B6C3F1/J (c, f) mice were immunized at weeks 0 and 3 weeks with 0.01 (green), 0.1 (blue), or 1 μg (red) of mRNA-1273. Sera were collected 2 weeks post-prime (open circles) and 2 weeks post-boost (closed circles) and assessed for SARS-CoV-2 S-specific IgG by ELISA (a-c), and, for post-boost sera, neutralizing antibodies against homotypic SARS-CoV-2 pseudovirus (d-f). Dotted line = assay limit of detection. (a-c) Timepoints were compared within each dose level, and doses were compared post-boost.

Figure 3.. Immunizations with mRNA-1273 and S-2P protein, delivered with TLR4 agonist, elicit S-specific Th1-biased T cell responses.

B6C3F1/J mice were immunized at weeks 0 and 3 with 0.01, 0.1, or 1 μg of mRNA-1273 or SAS-adjuvanted SARS-CoV-2 S-2P protein. Sera were collected 2 weeks post-boost and assessed by ELISA for SARS-CoV-2 S-specific IgG1 and IgG2a/c. Endpoint titers (a-b) and endpoint titer ratios of IgG2a/c to IgG1 (c) were calculated. For mice for which endpoint titers did not reach the lower limit of detection (dotted line), ratios were not calculated (N/A). (d-g) Seven weeks post-boost, splenocytes were isolated from 5 mice per group and re-stimulated with no peptides or pools of overlapping peptides from SARS-CoV-2 S protein in the presence of a protein transport inhibitor cocktail. After 6 hours, intracellular cytokine staining (ICS) was performed to quantify CD4+ and CD8+ T cell responses. Cytokine expression in the presence of no peptides was considered background and subtracted from the responses measured from the S1 and S2 peptide pools for each individual mouse. (d-e) CD4+ T cells expressing IFN-γ, TNFα, IL-2, IL-4 and IL-5 in response to the S1 (d) and S2 (e) peptide pools. (f-g) CD8+ T cells expressing IFN-γ, TNF-α, and IL-2 in response to the S1 (f) and S2 (g) peptide pools. IgG1 and IgG2a/c (a-b) and immunogens (c) were compared at each dose level. (d-g) For each cytokine, all comparisons were compared to naïve mice.

Figure 4.. mRNA-1273 protects mice from upper and lower airway SARS-CoV-2 infection.

(a-b) BALB/cJ mice were immunized at weeks 0 and 3 with 0.01 (green), 0.1 (blue), or 1 μg (red) of mRNA-1273. Mock-immunized mice were immunized with PBS x2. Five weeks post-boost, mice were challenged with mouse-adapted SARS-CoV-2. (c) BALB/cJ mice were also immunized with a single dose of 0.1 (blue),1 (red), or 10 (purple) μg of mRNA-1273 and challenged 7 weeks post-immunization. Two days post-challenge, at peak viral load, mouse lungs (a,c) and nasal turbinates (b) were harvested from 5 mice group for analysis of viral titers. Dotted line = assay limit of detection. (d) At day 2 and 4 post-challenge, lungs from 5 mice per group were fixed in 10% formalin, paraffin-embedded, cut in 5 μm sections, and stained with hematoxylin and eosin. Photomicrographs (4× and 10×) are representative of lung sections from groups of mice in which virus infection was detected. At day 2, lungs from mock-immunized mice demonstrated moderate to severe, predominantly neutrophilic, inflammation that was present within, and surrounding, small bronchioles (arrowheads); the surrounding alveolar capillaries were markedly expanded by infiltrating inflammatory cells. In the 0.01 μg two-dose group, inflammation was minimal to absent. In the 0.1 μg two-dose group, occasional areas of inflammation intimately associated with small airways (bronchioles) and their adjacent vasculature (arrowheads) were seen, primarily composed of neutrophils. In the single-dose 0.1 μg group, there were mild patchy expansion of the alveolar septae by mononuclear and polymorphonuclear cells. At day 4, lungs from mock-immunized mice exhibited moderate to marked expansion of the alveolar septae (interstitial pattern) with decreased prominence of the adjacent alveolar spaces. In the 0.01 μg two-dose group, inflammation was minimal to absent. Lungs in the 0.1 μg two-dose group showed mild, predominantly lymphocytic inflammation, intimately associated with bronchioles and adjacent vasculature (arrowheads). In the single-dose 0.1 μg group there was mild, predominantly lymphocytic, inflammation around bronchovascular bundles (arrowheads).