Elicit: Impact of Variants on Elasomeran Mechanism

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Impact of Variants on Elasomeran Mechanism

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May 5, 2026

How do updated formulations (variants of concern) affect the mechanism of Elasomeran?

Updated mRNA-1273 formulations shift the neutralizing antibody response toward enhanced variant-specific recognition while maintaining the core B cell memory and T cell activation mechanisms of the original vaccine platform, with bivalent versions balancing variant-targeted depth against cross-reactive breadth.

Abstract

Updated mRNA-1273 formulations targeting SARS-CoV-2 variants of concern alter the immune response mechanism through differential neutralizing antibody profiles while maintaining the platform’s core immunological pathways. Monovalent variant-specific vaccines (mRNA-1273.351 targeting B.1.351, mRNA-1273.529 targeting BA.1) demonstrated superior neutralizing antibody responses against their target variants, with geometric mean ratios of 1.67-1.77 for BA.1 formulations and twofold improvements for B.1.351 formulations, but showed reduced cross-reactivity against ancestral strains with fourfold lower neutralization against WA1/2020 D614G. Bivalent formulations (mRNA-1273.211, mRNA-1273.214) balanced this trade-off by maintaining or improving ancestral strain neutralization (GMR 1.11 against D614G for BA.1-bivalent) while achieving GMT ratios ≥1 against all variants of concern tested. All updated formulations induced robust anamnestic B cell memory responses when used as boosters, with variant-modified boosters showing 34.9-61.6 fold increases in neutralization against target variants, restoring immunity that had waned 6-7 fold against wild-type and 24-69 fold against variants over 6 months. Cellular immune mechanisms, including CD4+ and CD8+ T cell responses measured in preclinical models, showed similar activation patterns to the original vaccine, with effector T cell expansion but not effector-memory phenotypes and waning within 6 months. However, longitudinal studies revealed progressive viral escape from updated vaccine-induced immunity, with newer variants continuing to evade even XBB.1.5-updated formulations, indicating that antigenic drift outpaces the vaccine update cycle despite mechanistic improvements in neutralizing breadth and depth.

Methods

We analyzed 10 sources from an initial pool of 200, using 8 screening criteria. Each paper was reviewed for 8 key aspects that mattered most to the research question. More on methods

Records from Elicit search

n = 200

Papers screened using: Vaccine Type and Formulation, Mechanistic Outcomes, Comparative or Descriptive Data, Study Design, Updated Formulation Focus, Elasomeran Relevance, Mechanistic Data Inclusion, Study Rigor

n = 200

Papers screened out

n = 190

Papers included for extraction

n = 10

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Paper search

We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.

We ran this query: “How do updated formulations (variants of concern) affect the mechanism of Elasomeran?”

The search returned 200 total results from Elicit.

We retrieved 200 papers most relevant to the query for screening.

Screening

We screened in sources based on their abstracts that met these criteria:

We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

Data extraction

We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.

Extract details about the updated mRNA vaccine formulation studied that targets variants of concern, including:

Extract all immunological mechanisms measured to assess how the updated formulation works, including:

Extract which SARS-CoV-2 variants the immune mechanisms were tested against, including:

Extract quantitative comparisons of immune mechanisms between original mRNA-1273 and updated formulations, including:

Extract vaccination context that could affect immune mechanisms, including:

Extract data on breakthrough infections and protection specifically related to updated formulations vs original, including:

Extract population characteristics that could affect immune mechanisms with updated formulations, including:

Extract data on how long the immune mechanisms persist with updated formulations compared to original, including:

Results

Characteristics of Included Studies

The systematic review identified 10 sources examining updated mRNA-1273 (Elasomeran) formulations targeting SARS-CoV-2 variants of concern. Full texts were available for 6 studies, while 4 were available as abstracts only.

Study

Full text retrieved?

Updated formulation(s)

Study type

Population

Primary focus

Ivan T Lee et al., 2023

No

BA.1-monovalent (mRNA-1273.529), BA.1-bivalent (mRNA-1273.214)

Phase 3 RCT

Adults ≥16 years, UK

3-month safety and immunogenicity of BA.1-containing boosters

Kai Wu et al., 2021

No

mRNA-1273.351, mRNA-1273.211

Preclinical (mice)

Mice

Variant vaccine neutralization as primary or booster series

A. Choi et al., 2021

Yes

mRNA-1273.351, mRNA-1273.211

Phase 2a clinical trial

Healthy adults (mean ages 47.5-63.8 years), US

Safety and immunogenicity of variant-modified boosters

K. Wu et al., 2021

Yes

mRNA-1273.351, mRNA-1273.211

Phase 2 clinical trial

Adults ≥18 years, US

Preliminary safety and immunogenicity of variant vaccine booster

M. Koch et al., 2021

No

mRNA-1273.351, mRNA-1273.211

Preclinical (mice)

Mice

Variant vaccine neutralization and cross-protection

Baoling Ying et al., 2021

Yes

mRNA-1273.351, mRNA-1273.211

Preclinical (mice)

129S2 and K18-hACE2 mice

Protective activity against ancestral and variant strains

Baoling Ying et al., 2021a

Yes

mRNA-1273.351

Preclinical (mice)

129S2 and K18-hACE2 mice

Protection against variants with suboptimal immunity

M. Echaide et al., 2023

Yes

Bivalent vaccines (Comirnaty, Spikevax) targeting BA.4-5 Omicron

Review

Multiple populations including elderly, pediatric, immunocompromised

Advantages and limitations of mRNA vaccines against variants

A. Roederer et al., 2024

Yes

XBB.1.5 monovalent booster

Prospective cohort

Adults (median 33) and elderly (median 72), COVID-naive

Evolution of viral escape from vaccine immunity

A. Pegu et al., 2021

No

None (durability study of original mRNA-1273)

Clinical immunogenicity study

Vaccine recipients

Durability of antibodies against variants

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The studies examined three main categories of updated formulations: monovalent variant-specific vaccines (mRNA-1273.351 targeting B.1.351, mRNA-1273.529 targeting BA.1, XBB.1.5 booster), bivalent original+variant vaccines (mRNA-1273.211 combining original and B.1.351, mRNA-1273.214 combining original and BA.1), and later-generation bivalent vaccines targeting Omicron BA.4-5 subvariants. The dose for clinical formulations was consistently 50 μg, while preclinical studies used high doses (5 μg) and low doses (0.25 μg) to model varying immune responses.

Immunological Mechanisms Assessed

Studies evaluated multiple immunological mechanisms to characterize how updated formulations affect the immune response to SARS-CoV-2 variants.

Mechanism type

Measurement methods

Studies assessing

Neutralizing antibodies

Pseudovirus neutralization assays (lentivirus-based, VSV-based); live virus focus-reduction neutralization test (FRNT)

All clinical and preclinical studies

Binding antibodies

ELISA measuring IgG against recombinant spike proteins

Preclinical studies

CD8+ T cell responses

H-2b-restricted immunodominant peptides, IFN-γ production

Preclinical studies

CD4+ T cell responses

H-2b-restricted immunodominant peptides, IFN-γ production; S-specific CD4 T cells

Preclinical studies and review

B cell memory

Anamnestic response to boosting

Clinical booster studies

Durability

Persistence at Day 29 and Month 3; up to 6 months post-vaccination

Clinical studies

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Neutralizing antibody responses were the primary mechanism assessed across all studies. Clinical trials used pseudovirus neutralization assays, while preclinical studies employed live virus FRNT methods. The clinical studies by A. Choi et al. validated their neutralization assays against both lentivirus-based and VSV-based pseudovirus systems. Cellular immune responses were characterized primarily in preclinical models, with CD8+ and CD4+ T cell responses measured through peptide stimulation and interferon-γ production. Human studies noted the presence of S-specific CD4 and CD8 T cells, with expansion of effector T cells but not effector-memory phenotypes. B cell memory was inferred from anamnestic responses to booster doses in clinical trials.

Variant Testing Panels

Updated formulations were tested against comprehensive panels of SARS-CoV-2 variants to assess both homologous and cross-reactive immune responses.

Study

Ancestral strain

Variants of concern tested

Variant formulation designed for

Cross-reactivity assessed

Ivan T Lee et al., 2023

D614G

BA.1 Omicron

BA.1

Yes (D614G)

Kai Wu et al., 2021

Wild-type

B.1.351 (Beta)

B.1.351

Yes (broad cross-variant)

A. Choi et al., 2021

D614G

B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta)

B.1.351

Yes (multiple VOCs)

K. Wu et al., 2021

Wild-type

B.1.351, P.1

B.1.351

Yes (P.1)

M. Koch et al., 2021

Wild-type

B.1.351 (Beta)

B.1.351

Yes (broad)

Baoling Ying et al., 2021

WA1/2020 D614G

B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta)

B.1.351

Yes (multiple)

Baoling Ying et al., 2021a

WA1/2020 D614G, D614G/N501Y

B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta)

B.1.351

Yes (multiple)

M. Echaide et al., 2023

D614G

B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), B.1.1.529 (Omicron)

BA.4-5 Omicron

Yes (multiple)

A. Roederer et al., 2024

Wuhan-Hu-1, D614G, WA1/2020

Beta (K417N/E484K/N501Y), Gamma, Delta (L452R/P681R), Omicron (BA.5, BQ.1.1, JN.1)

XBB.1.5

Yes (comprehensive panel)

A. Pegu et al., 2021

Not mentioned

B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.429 (Epsilon), B.1.526 (Iota), B.1.617.2 (Delta)

None (original vaccine)

Yes (multiple VOCs)

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All studies tested cross-reactive immunity beyond the homologous variant targeted by each updated formulation. The most challenging variant across studies was B.1.351 (Beta), which showed the greatest resistance to neutralization by both original and updated formulations. Studies testing B.1.617.2 (Delta) consistently found lower neutralizing titers against this variant compared to ancestral strains. The comprehensive panel tested by A. Roederer et al. included 131 individual mutations across 48 named variants, revealing progressive viral escape from vaccine-induced immunity over time.

Effects of Updated Formulations on Neutralizing Antibody Responses

Superiority Against Homologous Variants

Updated formulations demonstrated superior neutralizing antibody responses against their target variants compared to the original mRNA-1273 vaccine.

Study

Updated formulation

Target variant

Comparison to original mRNA-1273

GMR or fold-change

Statistical significance

Ivan T Lee et al., 2023

BA.1-monovalent

BA.1 Omicron

Superior at Month 3

GMR 1.77 (96% CI 1.55-2.02)

Yes

Ivan T Lee et al., 2023

BA.1-bivalent

BA.1 Omicron

Superior at Month 3

GMR 1.67 (96% CI 1.54-1.81)

Yes

A. Choi et al., 2021

mRNA-1273.351 (50 μg)

B.1.351

Numerically greater titers

34.9-fold increase

Not confirmed

A. Choi et al., 2021

mRNA-1273.211 (50 μg)

B.1.351

Numerically greater titers

61.6-fold increase

P<0.0001

K. Wu et al., 2021

mRNA-1273.351

B.1.351

Superior neutralization

1400 vs 864 ID50 GMT

35-fold increase

Baoling Ying et al., 2021

mRNA-1273.351

B.1.351

Twofold higher GMTs

Twofold vs mRNA-1273

Superiority noted

Baoling Ying et al., 2021a

mRNA-1273.351

B.1.351

Twofold higher GMTs

Twofold vs mRNA-1273

Superior against B.1.351

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The BA.1-containing boosters showed 67-77% higher neutralizing antibody titers against Omicron BA.1 compared to the original vaccine at 3 months post-booster. For B.1.351-targeted formulations, the variant-specific mRNA-1273.351 vaccine induced 35-fold increases in neutralization titers post-boost, with geometric mean titers approximately twofold higher than the original vaccine. The bivalent mRNA-1273.211 formulation showed particularly strong responses, with a 61.6-fold increase in neutralization against B.1.351.

Cross-Variant Protection

Updated formulations maintained varying levels of cross-protection against non-target variants.

Formulation

Ancestral strain response

Non-target variant responses

Direction of effect

BA.1-monovalent

GMR 0.80 (95% CI 0.71-0.90) vs D614G

Non-inferior against D614G

Maintained

BA.1-bivalent

GMR 1.11 (96% CI 1.03-1.18) vs D614G

Non-inferior against D614G

Slightly improved

mRNA-1273.351

Fourfold lower GMTs vs WA1/2020 D614G

Lower against historical strains

Trade-off

mRNA-1273

Better against WA1/2020

Less reduction vs B.1.351

Broader baseline

mRNA-1273.211

46.4-fold increase vs D614G

GMT ratio ≥1 against all VOCs/VOIs

Broadest protection

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The bivalent formulations demonstrated the most balanced cross-variant protection. The BA.1-bivalent vaccine showed slight improvement over the original vaccine against the ancestral D614G strain (GMR 1.11), while the BA.1-monovalent showed a modest 20% reduction (GMR 0.80), both meeting non-inferiority criteria. The B.1.351-targeted monovalent vaccine (mRNA-1273.351) showed a trade-off, with fourfold lower neutralization against ancestral strains but twofold higher neutralization against B.1.351. In contrast, the bivalent mRNA-1273.211 formulation achieved broad cross-variant neutralization with GMT ratios ≥1 against all variants of concern tested and showed a 46.4-fold increase against the wild-type D614G virus.

Booster Effects on Waning Immunity

Booster doses with updated formulations restored and enhanced neutralizing antibody titers that had declined over time.

Study

Pre-booster titers

Post-booster titers

Magnitude of increase

Timing

A. Choi et al., 2021

Waned significantly vs peak at Month 1

mRNA-1273: 16.7-fold vs D614G

Significant (P<0.0001)

~6 months post-primary

A. Choi et al., 2021

Low/undetectable vs B.1.351, P.1, B.1.617.2

mRNA-1273.211: 61.6-fold vs B.1.351

Significant (P<0.0001)

~6 months post-primary

K. Wu et al., 2021

Low/below quantification 6-8 months post-primary

Similar to or higher than peak post-primary at 2 weeks post-boost

Robust memory response

2 weeks post-boost

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Prior to booster administration, neutralizing antibodies against variants had waned substantially from peak levels observed one month after the primary series. Neutralization titers against B.1.351, P.1, and B.1.617.2 were either low or undetectable at approximately 6 months post-primary vaccination. Both original and variant-modified boosters induced anamnestic responses indicative of robust B cell memory, with post-booster titers reaching or exceeding peak titers measured after the primary series. Two weeks after booster vaccination, neutralization titers increased to levels similar to or higher than peak titers after primary vaccination for both wild-type and variant viruses.

Effects on Cellular Immune Responses

Preclinical studies characterized T cell responses to updated formulations, while clinical data on cellular immunity were limited.

Mechanism

Findings with updated formulations

Comparison to original

CD8+ T cells

Responses measured via H-2b peptides and IFN-γ production

Not directly compared

CD4+ T cells

Responses measured via H-2b peptides and IFN-γ production

Not directly compared

Effector T cells

Expansion observed with mRNA vaccines

Similar to original

Effector-memory T cells

Not expanded by mRNA vaccines

Limitation of platform

T cell durability

Generally lost 6 months post-vaccination

Similar to original

IL-17 production

Elevated concentrations indicating inflammatory response

Shared mechanism

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Both original and updated mRNA formulations induced CD4+ and CD8+ T cell responses that were detectable in preclinical models. However, clinical observations indicated that mRNA vaccines activate S-specific CD4 and CD8 T cells but do not expand effector-memory phenotypes, a limitation that appears consistent across formulations. T cell responses generally waned within 6 months post-vaccination, though T cells with stem cell memory phenotypes could potentially persist longer. The inflammatory signature remained similar between original and updated formulations, with elevated IL-17 concentrations observed across mRNA vaccines.

Durability of Immune Mechanisms

The persistence of immune responses varied by formulation type and dose level.

Study

Formulation

Time point assessed

Neutralizing antibody persistence

Comparison to peak

Ivan T Lee et al., 2023

BA.1-monovalent, BA.1-bivalent

Month 3 post-booster

Superior vs mRNA-1273 against BA.1

Maintained superiority

A. Choi et al., 2021

Original mRNA-1273

6 months post-primary

6-7 fold decrease vs D614G; 24-69 fold decrease vs B.1.351/P.1

Peak at Month 1

K. Wu et al., 2021

mRNA-1273.351

6-8 months post-primary

Low/undetectable before boost

Waning evident

A. Roederer et al., 2024

XBB.1.5 booster

After multiple doses

Improved persistence vs original

Better against newer variants

M. Echaide et al., 2023

Original formulations

Up to 6 months

Detectable but decreased

Peak at 4 weeks

A. Pegu et al., 2021

Original mRNA-1273

6 months

Persisted but B.1.351 dropped considerably

Variant-specific waning

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The BA.1-containing boosters maintained superior neutralizing antibody responses against Omicron BA.1 compared to the original vaccine through 3 months post-booster. With the original mRNA-1273 vaccine, neutralizing antibody titers showed a 6-7 fold decrease against wild-type D614G and a more dramatic 24-69 fold decrease against B.1.351 and P.1 variants over 6 months following the primary series. The rate of waning was particularly pronounced for the B.1.351 variant, with neutralizing antibodies dropping considerably by 6 months. Peak antibody responses occurred at 4 weeks post-vaccination for original formulations and remained detectable up to 6 months, albeit at reduced levels.

Updated formulations showed improved persistence against their target variants. The XBB.1.5 booster demonstrated better maintenance of neutralization against newer variants compared to original formulations, with the first booster administered 8-9 months after primary vaccination and the second booster 4-6 months later. However, neutralization activity declined over time even with updated boosters, particularly against emerging variants like JN.1.

Protection Against Breakthrough Infections

Data on breakthrough infection rates with updated formulations compared to original vaccines were limited, with most studies focusing on immunogenicity rather than clinical outcomes.

Aspect

Findings

Study population

B.1.617.2 breakthrough

Higher rates with low-dose formulations

K18-hACE2 mice

Disease severity

Viral pneumonia with inflammation in breakthrough cases

Preclinical models

Dose-response relationship

Low-dose (0.25 μg) showed breakthroughs vs high-dose (5 μg)

129S2 and K18-hACE2 mice

Variant-specific protection

Updated formulations offered better protection vs homologous strains

Preclinical models

Omicron protection

Updated bivalent vaccines increased protection vs monovalent

Clinical populations

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Preclinical studies revealed that breakthrough infections occurred primarily with the B.1.617.2 (Delta) variant, particularly when using low-dose vaccine formulations designed to model suboptimal immune responses. These breakthrough infections led to viral pneumonia with inflammation and airspace consolidation in K18-hACE2 mice. The effectiveness of variant-specific vaccines appeared superior against homologous strains, with updated formulations inducing greater antibody responses and conferring more protection against their target variants. Updated bivalent vaccines targeting Omicron variants offered increased protection against new Omicron subvariants compared to monovalent vaccines, though the original vaccines showed a significant decrease in protection against Omicron variants, with efficacy dropping to 30% after three doses.

Synthesis

The findings reveal systematic patterns in how updated mRNA-1273 formulations alter immunological mechanisms, with heterogeneity explained by specific variant targets, formulation strategy (monovalent vs bivalent), and the evolutionary timing of SARS-CoV-2 variants tested.

Variant-Specific vs Broad Protection Trade-offs

Monovalent variant-specific vaccines (mRNA-1273.351, mRNA-1273.529) consistently showed superior neutralization against their target variants—with geometric mean ratio increases of 1.67-1.77 for BA.1 formulations and twofold improvements for B.1.351 formulations—but demonstrated reduced neutralization against ancestral strains, with fourfold lower geometric mean titers against WA1/2020 D614G. This pattern reflects the antigenic distance between variant spike proteins and the immunodominant epitopes recognized by vaccine-induced antibodies. In contrast, bivalent formulations (mRNA-1273.211, mRNA-1273.214) maintained or improved ancestral strain neutralization while still boosting variant-specific responses. The BA.1-bivalent vaccine achieved a GMR of 1.11 against D614G, and mRNA-1273.211 showed a 46.4-fold increase against wild-type virus with GMT ratios ≥1 against all variants of concern. This breadth advantage comes at a modest cost: bivalent vaccines showed slightly lower peak titers against target variants (GMR 1.67) compared to monovalent formulations (GMR 1.77) for BA.1, suggesting antigenic competition or epitope masking when both ancestral and variant spike proteins are co-delivered.

Dose-Dependent Breakthrough Risk

The preclinical studies using high (5 μg) versus low (0.25 μg) doses revealed a critical threshold effect for protection. High-dose formulations of all vaccines—whether original mRNA-1273, variant-specific mRNA-1273.351, or bivalent mRNA-1273.211—conferred protection against weight loss and lung pathology across all tested variants including B.1.617.2 (Delta). However, low-dose formulations showed breakthrough lung infections and pneumonia specifically with B.1.617.2 in K18-hACE2 mice, despite producing approximately tenfold lower neutralizing activity. Notably, a minimum neutralizing titer of approximately 5000 was required to prevent lung infection, and low-dose vaccines fell below this threshold for certain variants. This dose-response relationship suggests that as immunity wanes over time in vaccinated populations—mimicking the reduced magnitude seen with low-dose vaccination—breakthrough infections become more likely, particularly with antigenically distant variants like B.1.617.2 that showed four-to-fivefold lower neutralizing responses across all vaccine types. The clinical relevance is supported by the observation that neutralizing antibodies declined 6-7 fold against D614G and 24-69 fold against B.1.351 and P.1 over 6 months following primary vaccination, potentially dropping below protective thresholds for some individuals and variants.

Temporal Evolution and Escape Dynamics

The longitudinal analysis across 2020-2024 revealed progressive viral escape from both original and updated vaccine-induced immunity. While many individual mutations emerging between 2020-2022 escaped sera from primary vaccination, few escaped boosted sera. However, newer variants demonstrated progressive loss of neutralization regardless of vaccine doses received. The bivalent booster improved neutralization against contemporaneous strains but not against JN.1, and the XBB.1.5 booster significantly increased titers against newer variants except JN.1. This pattern cannot be explained by simple waning immunity, as participants received up to 6 mRNA vaccines including updated formulations. Instead, the data suggest that SARS-CoV-2 evolution is outpacing vaccine updates. Each seasonal booster improves titers against contemporaneous strains, but novel variants continue to evade updated mRNA vaccines, demonstrating that antigenic drift occurs faster than the vaccine development-deployment cycle. The mechanistic basis likely involves progressive accumulation of mutations in immunodominant epitopes: the comprehensive testing panel of 131 mutations across 48 variants showed that newer variants like JN.1 carry combinations of mutations that collectively confer escape, even from updated boosters targeting XBB.1.5.

Booster-Mediated Memory Recall

The ability of updated formulation boosters to rescue waning immunity provides insight into B cell memory mechanisms. Six to eight months after primary vaccination, neutralizing antibodies against variants like B.1.351 and P.1 were low or undetectable. However, booster doses—whether with original mRNA-1273 or variant-modified formulations—increased neutralization titers to levels similar to or higher than peak titers after primary vaccination within two weeks, with statistically significant increases observed for mRNA-1273 and mRNA-1273.211 (P<0.0001). Importantly, the original mRNA-1273 booster induced a 16.7-fold increase against wild-type D614G, demonstrating robust homologous recall, while variant-modified boosters showed particularly strong responses: mRNA-1273.351 induced a 34.9-fold increase against B.1.351 and mRNA-1273.211 showed a 61.6-fold increase. These anamnestic responses indicative of robust B cell memory suggest that memory B cells formed during primary vaccination can be reactivated by booster antigens, even when those antigens differ antigenically from the priming strain. The magnitude of the response (up to 61.6-fold) implies affinity maturation and clonal expansion of memory cells, with cross-reactive memory B cells responding to shared epitopes between variants. This mechanism explains why both homologous and heterologous boosters effectively restore immunity, though variant-matched boosters achieve higher peak titers against their specific targets through preferential activation of variant-specific clones.

References

A. Pegu, S. O'connell, S. Schmidt, S. O'Dell, C. A. Talana, and 27 more\ (2021).Durability of mRNA-1273 vaccine–induced antibodies against SARS-CoV-2 variants. Science

Kai Wu, A. Choi, M. Koch, Sayda M. Elbashir, LingZhi Ma, and 20 more\ (2021).Variant SARS-CoV-2 mRNA vaccines confer broad neutralization as primary or booster series in mice. bioRxiv

A. Choi, M. Koch, Kai Wu, L. Chu, LingZhi Ma, and 17 more\ (2021).Safety and immunogenicity of SARS-CoV-2 variant mRNA vaccine boosters in healthy adults: an interim analysis. Nature Medicine

M. Echaide, Luisa Chocarro de Erauso, A. Bocanegra, E. Blanco, G. Kochan, and 1 more\ (2023).mRNA Vaccines against SARS-CoV-2: Advantages and Caveats. International Journal of Molecular Sciences

K. Wu, A. Choi, M. Koch, L. Ma, A. Hill, and 15 more\ (2021).Preliminary Analysis of Safety and Immunogenicity of a SARS-CoV-2 Variant Vaccine Booster. medRxiv

M. Koch, Sayda M. Elbashir, Angela Woods, C. Henry, Charis Palandjian, and 14 more\ (2021).Variant SARS-CoV-2 mRNA vaccines confer broad neutralization as primary or booster series in mice. Vaccine

Baoling Ying, Bradley M. Whitener, L. VanBlargan, Ahmed O. Hassan, S. Shrihari, and 15 more\ (2021).Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains. Science Translational Medicine

A. Roederer, Yi Cao, K. S. Denis, M. Sheehan, Chia Jung Li, and 8 more\ (2024).Ongoing evolution of SARS-CoV-2 drives escape from mRNA vaccine-induced humoral immunity. medRxiv

Baoling Ying, Bradley M. Whitener, L. VanBlargan, Ahmed O. Hassan, S. Shrihari, and 15 more\ (2021).Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains. bioRxiv

Ivan T Lee, Catherine A Cosgrove, P. Moore, M. Bula, Philip A. Kalra, and 23 more\ (2023).2363. Three-Month Safety and Immunogenicity of Bivalent SARS-CoV-2 Omicron-Containing Booster Vaccines: Interim Results From a Phase 3, Randomized, Observer-Blind, Active-Controlled Trial. Open Forum Infectious Diseases

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Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains

Baoling Ying, Bradley M. Whitener, L. VanBlargan, Ahmed O. Hassan, S. Shrihari, Chieh-Yu Liang, Courtney E. Karl, Samantha R. Mackin, Rita E. Chen, Natasha M. Kafai, S. Wilks, Derek J. Smith, J. Carreño, Gagandeep Singh, F. Krammer, A. Carfi, Sayda M. Elbashir, D. Edwards, Larissa B. Thackray, M. Diamond

Science Translational Medicine·

2021·

49 citations

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Updated Formulation

- Specific vaccine name/code: mRNA-1273.351, mRNA-1273.211 - Variant(s) targeted: B.1.351/Beta - Type of formulation: mRNA-1273.351 is monovalent variant-specific; mRNA-1273.211 is bivalent original+variant - Dose amount: Not specified for updated formulations; low-dose (0.25 μg) used in some experiments - Difference from original mRNA-1273: Targets specific variants of concern (B.1.351)

Immunological Mechanisms

- Neutralizing antibody responses: Measured using a focus-reduction neutralization test (FRNT) against live viruses. - Binding antibody responses: Evaluated using ELISA to measure IgG responses against recombinant spike proteins. - T-cell responses: CD8+ and CD4+ T cell responses were assessed using H-2b-restricted immunodominant peptides, with a focus on interferon-γ production. - Cellular immune responses: Not mentioned. - Durability measures: Not mentioned. - Mechanistic pathway analysis: Not mentioned.

Variant Testing Panel

- Ancestral/original strain: WA1/2020 (Wuhan-Hu-1 with D614G substitution) - Specific variants of concern tested: B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta) - Nomenclature used: WHO names (WA1/2020), Pango lineages (B.1.1.7, B.1.351, B.1.617.2) - Testing was done against the variant the updated formulation was designed for (B.1.351 for mRNA-1273.351) and other variants to assess cross-reactivity

Comparative Mechanism Results

- Geometric mean titers/ratios with confidence intervals: mRNA-1273.351 showed about fourfold lower GMTs against WA1/2020 D614G and twofold higher GMTs against B.1.351 compared to mRNA-1273. - Fold-change differences in antibody levels: Lower doses (0.25-g) resulted in about 10-fold lower neutralizing activity. - Statistical significance of differences: Not explicitly mentioned in terms of p-values or confidence intervals. - Superiority, non-inferiority, or inferiority results: Variant-specific vaccines (mRNA-1273.351) showed superiority in inducing greater antibody responses against homologous strains. - Cross-variant protection differences: mRNA-1273 showed less reduction in neutralization against B.1.351, indicating broader cross-variant protection. - Breadth vs depth of immune responses: Variant-specific vaccines induced greater antibody responses against homologous strains. - Direction of effect: mRNA-1273.351 showed improved protection against B.1.351, while mRNA-1273 performed slightly better against B.1.617.2.

Vaccination Regimen

- Primary vaccination series vs booster dose administration: Primary immunization followed by a booster dose. - Number of prior doses before updated formulation: Not mentioned. - Timing between doses (interval from last dose): 3 weeks. - Previous vaccine types received (homologous vs heterologous): Not mentioned. - Concurrent administration with other vaccines: Not mentioned. - Schedule used: 2-dose primary series.

Breakthrough Protection

- Breakthrough infection rates by variant: Higher with B.1.617.2, especially with low-dose formulations. - Disease severity in breakthrough cases: Development of pneumonia in K18-hACE2 mice with B.1.617.2. - Viral load differences during breakthrough: Lower neutralizing responses against B.1.617.2. - Protection against symptomatic vs asymptomatic infection: Not explicitly discussed. - Effectiveness estimates comparing updated vs original formulations: Variant-specific vaccines induce greater antibody responses and confer more protection against homologous strains. - Duration of protection differences: Not explicitly discussed, but boosting may be necessary to prevent breakthrough events.

Study Population

- Age groups studied: Young adult mice (7-to 9-week-old) - Prior SARS-CoV-2 infection status: Naive - Immunocompromised status: Immunocompetent - Time since last vaccination or infection: Not applicable (first-time vaccination) - Geographic location: Not applicable (laboratory setting) - Sample size for mechanism analyses: Not explicitly stated

Mechanism Durability

- Time points measured: 3 weeks after boosting - Rate of antibody waning over time: Lower serum IgG responses at lower doses - Persistence differences between updated vs original formulations: Variant-specific vaccines induced greater antibody responses against homologous strains - Memory response durability: Implied by protection against weight loss and lung pathology - When peak responses occurred: At higher doses - Whether mechanism differences were maintained over time: Continued protection against certain strains correlated with neutralizing titers

Although mRNA vaccines encoding the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevent COVID-19, the emergence of new viral variants jeopardizes their efficacy. Here, we assessed the immunogenicity and protective activity of historical (mRNA-1273, designed for Wuhan-1 spike protein) or modified (mRNA-1273.351, designed for B.1.351 spike protein) Moderna mRNA vaccines in 129S2 and K18-hACE2 mice. Mice were immunized with either high-dose or low-dose formulations of the mRNA vaccines, where low-dose vaccination modeled suboptimal immune responses. Immunization with formulations at either dose induced neutralizing antibodies in serum against ancestral SARS-CoV-2 WA1/2020 and several virus variants, although serum titers were lower against the B.1.617.2 (Delta) virus. Protection against weight loss and lung pathology was observed with all high-dose vaccines against all viruses. However, low-dose formulations of the vaccines, which produced lower magnitude antibody and T cell responses, showed breakthrough lung infections with B.1.617.2 and development of pneumonia in K18-hACE2 mice. Thus, in individuals with reduced immunity after mRNA vaccination, breakthrough infection and disease may occur with some SARS-CoV-2 variants. Description mRNA-1273–based vaccines protect against historical and variant SARS-CoV-2 strains in 129S2 and K18-hACE2 mice. A variant vaccine The success of mRNA vaccines against SARS-CoV-2 is being challenged by the emergence of variants of concern (VOC). To address this, it may be necessary to develop vaccines that encode spike proteins from these VOC. Here, Ying et al. compared two SARS-CoV-2 mRNA vaccines, the mRNA-1273 vaccine, which is now in use globally, and a variant version, mRNA-1273.351. The authors showed that mRNA-1273, mRNA-1273.351, and a mix of the two, mRNA-1273.211, conferred protection against SARS-CoV-2 in two different mouse models when administered at a higher dose. Both vaccines also conferred some degree of protection at a lower dose of vaccine, which the authors use to recapitulate suboptimal vaccine responses. Together, these results support the continued development of mRNA vaccines for SARS-CoV-2.

INTRODUCTION

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the coronavirus disease 2019 . More than 341 million infections and 5.5 million deaths have been recorded worldwide (https://covid19.who.int) since the start of the pandemic. The extensive morbidity and mortality associated with the COVID-19 pandemic made the development of SARS-CoV-2 vaccines a global health priority. In a period of less than 1 year, several highly effective vaccines targeting the SARS-CoV-2 spike protein encompassing multiple platforms [lipid nanoparticle-encapsulated mRNA, inactivated virion, or viral-vectored vaccine platforms (1)] gained Emergency Use Authorization or Food and Drug Administration approval and were deployed with billions of doses given worldwide (https://covid19. who.int). The currently used vaccines were all designed against the spike protein of strains that were circulating early in the pandemic. In localities with high rates of vaccination, markedly reduced numbers of infections, hospitalizations, and deaths were initially observed.

Despite the success of COVID-19 vaccines and their potential for curtailing the pandemic, the continued evolution of more transmissible SARS-CoV-2 variants of concern (VOC) jeopardizes the efficacy of vaccination campaigns. These VOC include B.1.1.7 (Alpha), B.1.351 (Beta), B.1.1.28 (Gamma), and B.1.617.2 (Delta), all of which have substitutions in the spike protein (2). Experiments in cell culture suggest that neutralization by vaccine-induced serum is diminished against variants expressing mutations in the spike gene at positions L452, E484, and elsewhere (3)(4)(5)(6)(7)(8). Moreover, viral-vectored (ChAdOx1 nCoV-19 and Ad26.CoV2) and protein nanoparticle (NVX-CoV2373)based vaccines showed reduced activity (10 to 60%) against symptomatic infection caused by the B.1.351 variant in clinical trials in humans (9)(10)(11), whereas mRNA-based vaccines (such as BNT162b2) retained substantial (about 75%) efficacy against the B.1.351 variant in humans with almost complete protection against severe disease (12).

Immunization of humans with two 100-g doses of the lipid nanoparticle-encapsulated mRNA-1273 vaccine encoding a prolinestabilized full-length SARS-CoV-2 spike glycoprotein corresponding to the historical Wuhan-Hu-1 virus conferred 94% efficacy against symptomatic COVID-19 in clinical trials performed in the United States (13). More recent data in nonhuman primates show that vaccination with two doses of mRNA-1273 results in an effective immune response that controls upper and lower respiratory tract infection after challenge with the SARS-CoV-2 B.1.351 variant (14). As an alternative approach, several manufacturers have designed modified vaccines that target specific VOC, including B.1.351, for possible immunization or boosting. A mRNA-1273.351 vaccine recently was generated, which encodes a proline stabilized full-length SARS-CoV-2 spike glycoprotein from the B.1.351 virus. Here, we evaluated the immunogenicity and protective activity of lipidencapsulated mRNA-1273 and mRNA-1273.351 Moderna vaccines in the context of challenge of wild-type 129S2 and human ACE2 (hACE2) transgenic (K18-hACE2) mice with historical and emerging SARS-CoV-2 strains including several key VOC.

mRNA vaccines are immunogenic in 129S2 mice

We first tested preclinical versions of the Moderna mRNA-1273 and mRNA-1273.351 vaccines encoding sequenced-optimized prefusionstabilized spike proteins of Wuhan-1 and B.1.351, respectively, in immunocompetent 129S2 mice. These animals are permissive to infection by some SARS-CoV-2 variants (including B. 1.1.7, B.1.1.28, and B.1.351) or mouse-adapted strains (15)(16)(17) that encode an N501Y mutation, which enables engagement of endogenous murine angiotensin converting enzyme 2 (ACE2) (18). Infection of 129S2 mice with SARS-CoV-2 results in mild to moderate lung infection and clinical disease with subsequent recovery (15,17). To assess the immunogenicity of the vaccines, groups of 7-to 9-week-old female 129S2 mice were immunized and boosted 3 weeks later by an intramuscular route with 5-g (high) or 0.25-g (low) doses of mRNA-1273, mRNA-1273.351, mRNA-1273.211 [1:1 mixture (total of 5 or 0.25 g) of mRNA-1273 and mRNA-1273.351], or a control untranslated mRNA (Fig. 1A ); we included the mRNA-1273.211 mixture because it is being tested in humans [NCT04927065 (19)]. A lower vaccine dose (0.25 g) arm was included as a model for suboptimal responders and for evaluating correlates of protection, as we expected a greater frequency of breakthrough infections after SARS-CoV-2 challenge of this group. Serum samples were collected 3 weeks after boosting, and immunoglobulin G (IgG) responses against recombinant spike proteins of ancestral (Wuhan-1) or variant (B.1.1.7, B.1.351, or B.1.617.2) viruses (20) were evaluated by enzyme-linked immunosorbent assay (ELISA) (Fig. 1B ). As expected, the control mRNA did not generate spike-specific IgG (values below the limit of detection), whereas antibody responses against the spike proteins from all other mRNA vaccines were robust. For the 5-g dose, mean end-point titers of serum ranged from 619,650 to 1,503,560 against the different spike proteins with little variation between the mRNA vaccines. For the 0.25-g dose, about fivefold lower serum IgG responses were observed with mean end-point titers ranging from 126,900 to 382,725, again with little difference between the mRNA vaccines. Responses to different spike proteins for each vaccine generally were similar. Overall, both doses and all spike protein-based mRNA vaccines generated antispike protein IgG responses in 129S2 mice.

We characterized serum antibody responses functionally by assaying inhibition of SARS-CoV-2 infectivity using a focus-reduction neutralization test (FRNT) (21). We tested a panel of serum samples from each group of vaccinated mice against several fully infectious SARS-CoV-2 strains, including an ancestral Washington strain with a single D614G substitution (WA1/2020 D614G) or one with both D614G and N501Y substitutions (WA1/2020 D614G/N501Y), a B.1.1.7 isolate encoding an E484K mutation (B.1.1.7/E484K), a B.1.351 isolate, and a B.1.617.2 isolate (Fig. 1 , C to L). Because of the limited amount of serum recovered from live animals, we started dilutions at 1:180. As expected, serum from all control mRNA-immunized mice did not inhibit infection of the SARS-CoV-2 strains (Fig. 1 , C to L). For the 5-g dose, all three spike protein-based mRNA vaccines (mRNA-1273, mRNA-1273.351, and mRNA-1273.211) induced robust serum neutralizing antibody responses (Fig. 1 , C to G). In general, these titers were similar with the exception of about fourfold lower geometric mean titers (GMTs) against WA1/2020 D614G and about twofold higher GMTs against B.1.351 induced by mRNA-1273.351 compared to the mRNA-1273 and mRNA-1273.211 vaccines. Lower neutralizing responses (about four-to fivefold) were seen against the B.1.617.2 strain by all three mRNA vaccines (Fig. 1G ). For the 0.25-g vaccine dose, we observed about 10-fold lower titers of serum neutralizing activity against each of the viruses (Fig. 1 , H to L). We also noted the following: (i) the mRNA-1273.351 vaccine induced lower titers of neutralizing antibody against WA1/ 2020 D614G and WA1/2020 D614G/N501Y than the mRNA-1273 vaccine (Fig. 1 , H and I); (ii) the mRNA-1273.211 mixture induced neutralizing antibodies that were equivalent to one of the two vaccine components; (iii) serum from mRNA-1273-vaccinated mice showed less reduction in neutralization against B.1.351 than anticipated based on prior studies in humans and C57BL6 mice (Fig. 1K ) (6,7); and (iv) serum neutralizing antibody titers from all vaccinated mice were lower against B.1.617.2 than other strains, although responses from animals administered mRNA-1273 were slightly higher (Fig. 1L ). Overall, these differences were visualized best in a comparative analysis of the inhibitory activity of each serum sample for the 5-g (fig. S1, A Using the neutralization data from mRNA vaccinated 129S2 mice, we created antigenic maps to visualize the relationships between the WA1/2020 D614G, WA1/2020 D614G/N501Y, B.1.1.7/E484K, B.1.351, and B.1.617.2 SARS-CoV-2 strains (Fig. 1 , M and N). Neutralization titers obtained after 5-or 0.25-g dosing with mRNA-1273 and mRNA-1273.351 vaccines were used to position the serum relative to each virus using antigenic cartography (a modification of multidimensional scaling for binding assay data), such that higher neutralization titers are represented by shorter distances between serum and the virus. Each gridline, or antigenic unit, of the map corresponds to a twofold difference in neutralization titer of a given virus. Three antigen clusters were observed: (i) WA1/2020 D614G and WA1/2020 D614G/N501Y grouped together; (ii) viruses containing E484K mutations (B.1.1.7/E484K and B.1.351) had a similar antigenic position; and (iii) B.1.617.2 was the most distant antigenically, which is consistent with the lower serum neutralization titers induced by all of the mRNA vaccines against this VOC. In addition to providing a visual representation of the antigenic relationships observed in Fig. 1 (C to L), the antigenic maps also show some differences between the 5-and 0.25-g groups, particularly the movement of the B.1.617.2 virus leftward. In the 5-g dosing, the B.1.617.2 strain escaped serum antibodies from both mRNA-1273 and mRNA-1273.351 vaccines similarly (roughly equidistant position), whereas in the lower 0.25-g dosing, the leftward position of B.1.617.2 indicates a greater distance and antigenic escape from serum generated by the mRNA-1273.351 than the mRNA-1273 vaccine.

mRNA vaccines confer protection against SARS-CoV-2 in 129S2 mice

We tested the protective activity of the different mRNA vaccines in 129S2 mice. Three weeks after boosting, mice were challenged by an intranasal route with WA1/2020 D614G/N501Y, B.1.1.7/E484K, or B.1.351. The WA1/2020 D614G and B.1.617.2 viruses were not used for challenge in this model because they lack the mouse-adapting N501Y substitution and cannot infect conventional laboratory mice (16). Compared to the control mRNA vaccine, the 5-g or 0.25-g doses of mRNA-1273, mRNA-1273.351, or mRNA-1273.211 vaccines all prevented weight loss between 2 and 4 days post-infection (dpi), although protection for some mice was not observed after immunization with the mRNA-1273 vaccine and challenge with B.1.351 or B.1.1.7/E484K viruses (Fig. 2, A and B ).

At 4 dpi, mice were euthanized, and nasal washes, lungs, and spleens were collected for viral burden analysis. In the nasal washes or lungs from control mRNA-vaccinated 129S2 mice, high amounts (about 10 RNA (about 10 2 to 10 4 copies of N per mg) were measured in the spleen (fig. S2A ). In general, the mRNA-1273, mRNA-1273.351, and the mRNA-1273.211 vaccines conferred robust protection against infection in nasal washes, lungs, and spleens by the challenge SARS-CoV-2 strains, although some breakthrough was noted. After the 5-g dose immunization with mRNA-1273, moderate B.1.1.7/E484K infection was detected in nasal washes in five of eight mice, although viral RNA was absent from the lungs. Three of eight mice immunized with the mRNA-1273.211 mixture also showed breakthrough in the lungs, albeit at greater than 100-fold lower quantities than the control vaccine. In comparison, the 5-g dose of mRNA-1273.351 was protective in the nasal wash and lungs against all viruses, with little, if any, viral RNA measured.

As expected, the 0.25-g dose of the mRNA vaccines showed less protective efficacy against SARS-CoV-2 challenge. Protection was conferred by the 0.25-g dose against WA1/2020 D614G/N501Y and B.1.1.7/E484K challenge in the nasal washes at 4 dpi, except for the mRNA-1273.351 vaccine against B.1.1.7/E484K challenge (Fig. 2C ). In comparison, after B.1.351 challenge, eight of eight mice immunized with mRNA-1273 showed viral RNA in nasal washes, with three of eight showing amounts that approached those seen in control-vaccinated mice. Protection was generated against B.1.351 by mRNA-1273.351 or the mRNA-1273.211 mixture vaccines, although breakthrough infections were detected. In the lungs, protection against infection with WA1/2020 D614G/N501Y was generated by all three mRNA vaccines (Fig. 2D ). However, some infection was seen after B.1.1.7/E484K or B.1.351 challenge especially with the 0.25-g dose vaccine formulations. For example, six of eight mice immunized with 0.25 g of mRNA-1273 had moderate to high amounts of B.1.351 viral RNA in their lungs at 4 dpi.

We assessed for correlations between vaccine-induced neutralizing antibody titers and protection against SARS-CoV-2 infection in the lung after virus challenge. Serum titers of neutralizing antibodies exhibited an inverse association with quantities of SARS-CoV-2 RNA in the lung (Fig. 2E ) with a minimum neutralizing titer of about 5000 required to prevent infection in the lung at 4 dpi. Most of the breakthrough infections occurred with the B.1.351 challenge at the 0.25-g dose of vaccines. For reasons that remains unclear, the threshold for complete protection in the lung after challenge with WA1/ 2020 D614G/N501Y was lower (two-to sevenfold) than against the other viruses. Moreover, when we compared body weight change at 4 dpi with neutralizing titers, only animals challenged with B.1.351 showed a linear correlation (fig. S2B ), possibly because of the greater number of breakthrough infections in this group.

We also assessed the effect of the mRNA vaccines on lung disease at 4 dpi in 129S2 mice. For these studies, we analyzed lung sections from the group of mice that received the lower 0.25-g vaccine dose and the B.1.351 challenge virus, as this combination resulted in the greatest number of breakthrough infections. As expected, mice immunized with the control mRNA vaccine and challenged with B.1.351 developed mild pneumonia characterized by immune cell accumulation in perivascular and alveolar locations, vascular congestion, and interstitial edema. In contrast, animals immunized with mRNA-1273, mRNA-1273.351, or mRNA-1273.211 vaccines did not show these pathological changes (Fig. 3 ). Thus, immunization with even the low dose of the mRNA vaccines was sufficient to mitigate SARS-CoV-2-induced lung injury in immunocompetent 129S2 mice challenged with some VOC.

mRNA vaccines are immunogenic in K18-hACE2 transgenic mice

We next evaluated the mRNA-1273 and mRNA-1273.351 vaccines in K18-hACE2 transgenic mice, which are highly susceptible to severe infection and disease after intranasal inoculation by many SARS-CoV-2 strains ( 22) including isolates containing or lacking mouse-adapting mutations, such as N501Y (17). Because of a limited availability of K18-hACE2 mice and the need to test two control viruses (WA1/ 2021 D614G and WA1/2021 D614G/N501Y), we tested mRNA-1273 and mRNA-1273.351 but not the mRNA-1273.211 mixture vaccine. Groups of 7-week-old female K18-hACE2 mice were immunized and boosted 3 weeks later by intramuscular route with 5-or 0.25-g doses of mRNA-1273, mRNA-1273.351, or control mRNA vaccine (Fig. 4A ). Serum samples were collected 3 weeks after boosting, and IgG responses against recombinant spike proteins (Wuhan-1, B.1.1.7, B.1.351, or B.1.617.2) were evaluated by ELISA (Fig. 4B ). Antibody responses against the different spike proteins were robust although slightly lower (about two to threefold) than that seen in 129S2 mice Downloaded from https://www.science.org at Cambridge University on June 22, 2023 from all control mRNA-immunized mice did not inhibit infection of the SARS-CoV-2 strains (Fig. 4 , C to L). In general, neutralizing antibody titers induced by 5-or 0.25-g mRNA vaccine dosing were lower (about three-to sixfold) in immunized K18-hACE2 than from 129S2 mice. For the 5-g dose, although both mRNA-1273 and mRNA-1273.351 vaccines induced robust serum neutralizing antibody responses, we observed the following (Fig. 4 , C to G, and fig. S3 ): (i) the mRNA-1273.351 vaccine induced lower titers of neutralizing antibody against WA1/2020 D614G and WA1/2020 D614G/ N501Y than the mRNA-1273 vaccine (Fig. 4 , C and D); (ii) a reciprocal pattern was observed against viruses containing E484K mutations. The mRNA-1273.351 vaccine induced higher titers of neutralizing antibody against B.1.1.7/E484K and B.1.351 than the mRNA-1273 vaccine (Fig. 4, E and F ); and (iii) no differences in neutralizing activity were observed with the mRNA-1273 and mRNA-1273.351 vaccines against the B.1.617.2 strain. Although responses were elevated, they were lower than against other strains (Fig. 4G ). Similar patterns were observed for the 0.25-g dose (Fig. 4 , H to L), although about 10-fold lower titers of neutralizing activity were induced by each vaccine against each of the viruses. Because of this, we started our dilution series at 1:60 for serum derived from animals immunized with the 0.25-g dose of mRNA vaccines. In general, the pattern of neutralization paralleled results with the higher dose, with the mRNA-1273 vaccine performing better against historical WA1/2020 and WA1/2020 D614G/N501Y viruses (Fig. 4 , H to I). However, serum from mice vaccinated with mRNA-1273 or mRNA-1273.351 vaccines neutralized B.1.617.2 less efficiently (Fig. 4L ), with several data points at the limit of detection 4E ). In the 0.25-g dose group, however, the B.1.1.7/E848K strain moves to a location more equidistant from both serum groups, suggesting more escape from neutralization by mRNA-1273.351induced serum (Fig. 4J ).

We also examined T cell responses in mRNA-vaccinated K18-hACE2 mice 2 weeks after boosting (Fig. 4 , O to R) using H-2 brestricted immunodominant peptides in the spike protein for CD8 + and CD4 + T cells. After peptide stimulation ex vivo and staining for intracellular interferon- (IFN-) production, we detected a robust CD8 + T cell (2 to 4% positive) response in the spleens of animals immunized with 5 g of the mRNA-1273 or mRNA-1273.351 vaccines (Fig. 4, O and P ). The response was about 10-fold lower in animals immunized with the 0.25-g dose. Although we also detected a spike protein-specific CD4 + T cell response after immunization (0.5 to 1.5% positive) with the 5-g dose of mRNA-1273 or mRNA-1273.351 vaccines, it was lower in magnitude (Fig. 4, Q and R ). Moreover, the low 0.25-g dose mRNA-1273 or mRNA-1273.351 vaccines induced CD4 + T cell responses that were barely greater than the control mRNA vaccine.

High-dose mRNA vaccines confer protection in K18-hACE2 transgenic mice

We next evaluated the protective activity of the mRNA vaccines in K18-hACE2 mice. Three to 4 weeks after boosting, mice were challenged by intranasal route with WA1/2020 D614G, WA1/2020 D614G/N501Y, B.1.1.7/E484K, B.1.351, or B.1.617.2 strains. Compared to the control mRNA vaccine, the 5-and 0.25-g doses of mRNA-1273 and mRNA-1273.351 vaccines all prevented the weight loss occurring between 3 and 6 dpi (Fig. 5, A and B ).

At 6 dpi, mice were euthanized, and nasal washes, lungs, and brains were collected for viral burden analysis (Fig. 5 , C and D, and fig. S4A ). In the nasal washes of control mRNA-vaccinated K18-hACE2 mice, moderate amounts (about 10 5 copies of N per milliliter) of viral RNA were measured after challenge with WA1/2020 D614G, WA1/2020 D614G/N501Y, B.1.1.7/E484K, B.1.351, or B.1.617.2 strains, although some variability was observed (Fig. 5C ). In comparison, in the lungs of control mRNA-vaccinated K18-hACE2 mice, higher and more uniform amounts (about 10 7 copies of N per milligram) of viral RNA were detected after challenge with all SARS-CoV-2 strains (Fig. 5D ). The viral burden in the brains of control RNAvaccinated K18-hACE2 mice showed some variability, as seen previously (22), with many but not all animals showing substantial infection (10 8 copies of N per milliliter) (fig. S4A ). The high 5-g dose of mRNA-1273 or mRNA-1273.351 vaccines protected against infection in nasal washes, lung, and brain, with no viral breakthrough regardless of the challenge strain. After the 0.25-g dose immunization with mRNA-1273, a loss of protection against infection in the nasal washes and lungs (six of eight mice) was observed after challenge with B. We explored whether vaccine-induced neutralizing antibody titers correlated with protection after challenge with WA1/2020 D614G, WA1/2020 D614G/N501Y, B.1.1.7/E484K, B.1.351, or B.1.617.2 viruses. In general, serum neutralizing antibody titers exhibited an inverse correlation with amounts of viral RNA in the lung (Fig. 5E ) for all viruses, with more infection occurring in animals with lower neutralization titers. However, for WA1/2020 D614G, WA1/2020 D614G/N501Y, B.1.1.7/E484K, and B.1.351, some of the animals with low neutralization titers were still protected against infection in the lung. The correlation was most linear for B.1.617.2-challenged animals, with a minimum neutralizing titer of about 2000 required to completely prevent infection at 6 dpi. Most of the breakthrough B.1.617.2 infections occurred with the 0.25-g dose of mRNA vaccines. The threshold for complete protection in the lung after virus challenge varied somewhat with lower amounts required for WA1/ 2020 D614G and WA1/2020 D614G/N501Y. When we compared body weight change in K18-hACE2 mice at 6 dpi with neutralizing titers, a linear relationship was observed with all challenge viruses except B.1.351 (fig. S4B ). The best correlation was seen after B.1.617.2 challenge, with greater weight loss in mice immunized with the 0.25-g vaccine dose and having lower serum neutralizing antibody titers.

Because a proinflammatory host response to SARS-CoV-2 infection can contribute to pulmonary pathology and severe COVID-19, we assessed the ability of the mRNA vaccines to suppress cytokine and chemokine responses in the lung after virus challenge (fig. S5 ). For these studies, K18-hACE2 mice were immunized and boosted with 5 or 0.25 g of control, mRNA-1273, or mRNA-1273.351 vaccines and then challenged with WA1/2020 N501Y/D614G, B.1.351, or B.1.617.2. SARS-CoV-2 infection of control mRNA-vaccinated K18-hACE2 mice resulted in high expression of several pro-inflammatory cytokines and chemokines in lung homogenates, including granulocyte colony-stimulating factor, IFN-, interleukin-1 (IL-1), IL-6, CXCL1, CXCL5, CXCL9, CXCL10, CCL2, and CCL4. Pro-inflammatory cytokine and chemokines in the lung at 6 dpi generally were decreased in all mice vaccinated with 5-g doses of mRNA-1273 or mRNA-1273.351, regardless of the challenge virus (fig. S5, A and B ). Although this pattern was also observed for the 0.25-g dose of both mRNA vaccines, some cytokines and chemokines (such as IL-1 , IL-6, CXCL9, and CXCL10) remained elevated, especially after challenge with B.1.617.2 (fig. S5, C and D ).

We evaluated the ability of the mRNA-1273 and mRNA-1273.351 vaccines to prevent disease in K18-hACE2 mice by performing histological analysis of lung tissues from immunized animals that were challenged with WA1/2020 D614G, WA1/2020 N501Y/D614G, B.1.1.7/E484K, B.1.351, or B.1.617.2. As expected, lung sections obtained at 6 dpi from mice immunized with the control mRNA vaccine and challenged with any of the SARS-CoV-2 strains showed severe pneumonia characterized by immune cell infiltration, alveolar space consolidation, vascular congestion, and interstitial edema (Figs. 6 and 7 ). In comparison, mice immunized with the 5-g dose of mRNA-1273 or mRNA-1273.351 did not develop lung pathology, with histological findings similar to uninfected K18-hACE2 mice (Fig. 6 ). Mice immunized with the 0.25-g dose of the mRNA vaccines however, showed different results (Fig. 7 ). Mice vaccinated with mRNA-1273 showed few, if any, pathological changes after WA1/2020 D614G, WA1/2020 D614G/N501Y, or B.1.1.7/E484K challenge. Nonetheless, some mRNA-1273-vaccinated mice challenged with B.1.351 showed pulmonary vascular congestion and mild lung inflammation. Mice vaccinated with mRNA-1273.351 showed almost complete protection after WA1/2020 D614G, B.1.1.7/E484K, or B.1.351 challenge, whereas scattered inflammation and alveolar septal thickening was apparent in sections from some WA1/2020 D614G/ N501Y challenged mice. Lung sections from mice vaccinated with the lower 0.25-g dose mRNA-1273 or mRNA-1273.351 and challenged with B.1.617.2 showed evidence of viral pneumonia with prominent foci of immune cells inflammation and airspace consolidation. Thus, low doses of the original mRNA-1273 or the variant mRNA vaccines do not fully protect K18-hACE2 mice from challenge with B.1.617.2 and result in mild to moderate infection and lung pathology.

DISCUSSION

Robust vaccine-induced immune responses and sustained protective activity against emerging SARS-CoV-2 variants are needed to limit human disease and curtail the COVID-19 pandemic. A concern in the field is whether immunity generated by vaccines will lose activity against VOC with mutations or deletions in regions of the spike protein recognized by neutralizing antibodies. In the current study, we evaluated the immunogenicity and protective activity of high-and low-dose formulations of Moderna mRNA vaccines targeting historical (mRNA-1273) or variant (mRNA-1273.351) strains. The low-dose vaccine study arm was designed to model individuals with suboptimal immune responses and assess for possible strainspecific breakthrough infections.

Immunization of 129S2 or K18-hACE2 transgenic mice with mRNA-1273, mRNA-1273.351, or the mRNA-1273.211 mixture induced neutralizing antibodies against spike protein in serum against historical WA1/2020 and several key VOC. Challenge studies performed about 1 month after the second vaccine dose showed robust protection against weight loss and lung pathology with all highdose vaccines and infecting SARS-CoV-2 strains. Nonetheless, the low-dose vaccine formulations showed evidence of viral infection breakthrough and lung pathological changes consistent with pneumonia, especially with the B.1.617.2 strain, which correlated with lower strain-specific neutralizing antibody titers in mice. In general, variant-specific vaccine designs appeared to induce greater antibody responses and confer more protection against homologous virus strains.

Our experiments expand upon a preliminary immunogenicity study, which showed that vaccination of H-2 d BALB/c mice with mRNA-1273.351 resulted in high serum neutralizing antibody titers against the B.1.351 lineage, whereas the mRNA-1273.211 vaccine induced broad cross-variant neutralization (19). We performed experiments with two H-2 b -expressing strains, 129S2 and K18-hACE2 C57BL/6, and observed some similarities and differences. In K18-hACE2 mice, the mRNA-1273 vaccine, which encodes for the Wuhan-1 prefusion-stabilized spike, induced higher neutralizing titers against WA1/2020 strains but lower responses against viruses containing E484K mutations in spike (B.1.1.7/E484K and B.1.351), which agrees with recent immunization studies in nonhuman primates (14). Reciprocally, the mRNA-1273.351 vaccine, which encodes for the B. vaccines will be needed to corroborate our results obtained in small animal models. The differences in neutralizing antibody titers induced by mRNA-1273 against WA1/2020 D614G and B.1.351 in mice were smaller in magnitude than that seen in humans 1 month after boosting but were more similar to that observed 6 months after boosting (23).

The greatest loss in antibody neutralization (both 129S2 and K18-hACE2 mice) and protection (K18-hACE2 mice) consistently not apparent why neutralizing activity and protection in mice were lower against B.1.617.2 than B.1.351, although there was an inverse correlation with titers of neutralizing antibody and B.1.617.2 burden in the lung. Nonetheless, mutations in the B.1.617.2 alter key antigenic sites and can abrogate recognition by neutralizing antibodies (26). Other possible explanations for the loss of potency of antibodies against B.1.617.2 include differential display of B.1.617.2 spike proteins on the surface of infected cells and engagement of Fc effector functions (27,28) or differential ability of antibodies to block cell-to-cell spread in a strain-dependent manner (29). Our observation of B.1.617.2 infection and lung disease in low-dose mRNA-vaccinated K18-hACE2 mice corresponds to descriptions of B.1.617.2 breakthrough infections in vaccinated humans, some of which have required hospitalization (30,31).

We note several limitations in our study. First, the studies in 129S2 mice precluded challenge with B.1.617.2, as it does not infect mice because it lacks an N501Y mutation. The generation of recombinant SARS-CoV-2 strains with spike genes encoding B.1.617.2 and an N501Y mutation could overcome this limitation. Second, female 129S2 and K18-hACE2 mice were used to allow for group caging of the large cohorts required for these multiarm vaccination studies. Follow-up experiments in male mice are needed to confirm these results are not sex-biased. Third, differences in the repertoire of antibodies in mice and humans could contribute to the relative differences in neutralization potency of serum against B.1.617.2 and B.1.351 viruses. Fourth, we used historical, variant, or mixed mRNA vaccine formulations with homologous boosting schemes. Animal studies that test heterologous boosting (mRNA-1273 prime followed by mRNA-1273.351 boost) (19) also are needed to support clinical trials. Fifth, our studies focused on immunogenicity and protection in two strains of mice because of the ability to set up large animal cohorts and the tools available for analysis. These results require confirmation in other animal models of SARS-CoV-2 infection including hamsters and nonhuman primates (32). Last, we did not establish immunological correlates of vaccine protection or failure for all vaccine and challenge strain pairs. Although some relationships were more predictive (low B.1.617.2 neutralizing titers and viral burden in the lung), others were not.

Our studies in 129S2 and K18-hACE2 mice with parental and modified mRNA vaccines show robust immunogenicity and protection against multiple SARS-CoV-2 strains when high-dose immunization schemes are used, although some differences in immunity are seen, particularly with vaccines against selected variants. Although the lower dose of mRNA vaccines generally protected against matched virus challenge infection (mRNA-1273 vaccination and WA1/2020 challenge or mRNA-1273.351 vaccination and B.1.351 challenge), breakthrough events were seen with some nonmatched challenges (mRNA-1273 vaccination and B.1.351 challenge or mRNA-1273.351 vaccination and WA1/2020 challenge). As the low dose of mRNA-1273 and -1273.351 vaccines induced lower neutralizing titers and protected less against challenge with the B.1.617.2 variant, higher titers will be needed to minimize B.1.617.2 infection, transmission, and disease. Although studies in humans are required, boosting with historical or variant vaccines might be required to prevent breakthrough events for individuals with suboptimal responses. Booster doses for vaccines recently were approved for some populations in the United States and other parts of the world based on recent breakthrough infection data (33).

Study design

The goal of this study was to evaluate the immunogenicity and efficacy of a high or low dose of mRNA vaccines (mRNA-1273, mRNA-1273.351, or mRNA-1273.211) against emerging SARS-CoV-2 strains using two different mouse models (129S2 and K18-hACE2). Mice were immunized and boosted 3 weeks later, and immune responses were analyzed. About 3 weeks later, animals were challenged with different SARS-CoV-2 strains, and clinical, virological, immunological, and pathological outcomes were measured. All data collected were included without exclusion of outliers. Mice were randomly assigned to cages, and investigators performing the immunological analyses were blinded. Sample sizes were chosen on the basis of power analysis estimates and prior experience in evaluating differences in serological responses and viral infection in mice. All experiments in mice were repeated on two separate occasions.

Cells

African green monkey Vero-TMPRSS2 (34) and Vero-hACE2-TMPRRS2 (7) cells were cultured at 37°C in Dulbecco's modified Eagle medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), 10 mM Hepes (pH 7.3), 1 mM sodium pyruvate, 1× nonessential amino acids, and penicillin-streptomycin (100 U/ml) (Thermo Fisher Scientific). Vero-TMPRSS2 cells were supplemented with blasticidin (5 g/ml). Vero-hACE2-TMPRSS2 cells were supplemented with puromycin (10 g/ml). All cells routinely tested negative for mycoplasma using a polymerase chain reaction-based assay.

Viruses

The WA1/2020 recombinant strain with substitutions (D614G or N501Y/D614G) was obtained from an infectious cDNA clone as described previously (35). The B.1.351, B.1.1.7/E484K, and B.1.617.2 strains were obtained from nasopharyngeal isolates. All viruses were passaged once in Vero-TMPRSS2 cells and subjected to next-generation sequencing (7) to confirm the introduction and stability of substitutions. All virus experiments were performed in an approved biosafety level 3 facility.

Mice

Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (assurance number A3381-01). Virus inoculations were performed under anesthesia that was induced and maintained with ketamine hydrochloride and xylazine, and all efforts were made to minimize animal suffering. Heterozygous K18-hACE2 C57BL/6J mice [strain: 2B6.Cg-Tg(K18-ACE2)2Prlmn/J, catalog no. 34860) and 129S2 mice (strain: 129S2/SvPasCrl, catalog no. 287) were obtained from the Jackson Laboratory and Charles River Laboratories, respectively. Animals were housed in groups and fed standard chow diets.

Preclinical vaccine mRNA and lipid nanoparticle production process

A sequence-optimized mRNA encoding prefusion-stabilized Wuhan-Hu-1 (mRNA-1273) or B.1.351-variant (mRNA-1273.351) SARS-CoV-2 S-2P protein was synthesized in vitro using an optimized T7 Downloaded from https://www.science.org at Cambridge University on June 22, 2023 RNA polymerase-mediated transcription reaction with complete replacement of uridine by N1m-pseudouridine (36). The reaction included a DNA template containing the immunogen open-reading frame flanked by 5′ untranslated region (UTR) and 3′UTR sequences and was terminated by an encoded polyA tail. After transcription, the cap-1 structure was added using the vaccinia virus capping enzyme (New England Biolabs). The mRNA was purified by oligo-dT affinity purification, buffer exchanged by tangential flow filtration into sodium acetate (pH 5.0), sterile filtered, and kept frozen at -20°C until further use.

The mRNA was encapsulated in a lipid nanoparticle through a modified ethanol-drop nanoprecipitation process described previously (37). Ionizable, structural, helper, and polyethylene glycol lipids were briefly mixed with mRNA in an acetate buffer (pH 5.0) at a ratio of 2.5:1 (lipid:mRNA). The mixture was neutralized with tris-HCl (pH 7.5), sucrose was added as a cryoprotectant, and the final solution was sterile-filtered. Vials were filled with formulated lipid nanoparticle and stored frozen at -20°C until further use. The vaccine product underwent analytical characterization, which included the determination of particle size and polydispersity, encapsulation, mRNA purity, doublestranded RNA content, osmolality, pH, endotoxin, and bioburden, and the material was deemed acceptable for in vivo study.

Antigens

Recombinant soluble spike proteins from different SARS-CoV-2 strains were expressed as described (20,38). Briefly, mammalian cell codon-optimized nucleotide sequences coding for the soluble ectodomain of the spike protein of SARS-CoV-2 including a C-terminal thrombin cleavage site, T4 foldon trimerization domain, and hexahistidine tag were cloned into mammalian expression vector pCAGGS. The spike protein sequence was modified to remove the polybasic cleavage site (RRAR to A), and two prefusion-stabilizing proline mutations were introduced (K986P and V987P, wild-type Wuhan-Hu-1 numbering). Recombinant proteins were produced in Expi293F cells (Thermo Fisher Scientific) by transfection of DNA using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Supernatants were harvested 3 days after transfection, and recombinant proteins were purified using nickel-nitrilotriacetic acid agarose (Thermo Fisher Scientific), then buffer exchanged into phosphate-buffered saline (PBS), and concentrated using Amicon Ultracel centrifugal filters (EMD Millipore). Plates were incubated at 4°C overnight and then blocked with 200 l of 3% nonfat dry milk (AmericanBio) in PBS containing 0.1% Tween 20 (PBST) for 1 hour at room temperature. Serum samples were serially diluted in 1% nonfat dry milk in PBST and added to the plates. Plates were incubated for 2 hours at room temperature and then washed three times with PBST. Goat anti-mouse IgGhorseradish peroxidase (HRP; 1:9000; Sigma-Aldrich) was diluted in 1% nonfat dry milk in PBST before adding to the wells and incubating for 1 hour at room temperature. Plates were washed three times with PBST before the addition of peroxidase substrate (SigmaFAST o-phenylenediamine dihydrochloride, Sigma-Aldrich). Reactions were stopped by the addition of 3 M hydrochloric acid. Optical density (OD) measurements were taken at 490 nm, and end-point titers were calculated in excel using a 0.15 OD at 490-nm cutoff. Graphs were generated using GraphPad Prism v9.

Focus reduction neutralization test

Serial dilutions of serum samples were incubated with 10 2 focusforming units (FFU) of different strains of SARS-CoV-2 for 1 hour at 37°C. Antibody-virus complexes were added to Vero-TMPRSS2 cell monolayers in 96-well plates and incubated at 37°C for 1 hour. Subsequently, cells were overlaid with 1% (w/v) methylcellulose in Eagle's minimal essential medium (Thermo Fisher Scientific). Plates were harvested 30 hours later by removing overlays and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Plates were washed and sequentially incubated with an oligoclonal pool of SARS2-2, SARS2-11, SARS2-16, SARS2-31, SARS2-38, SARS2-57, and SARS2-71 (39) antispike protein antibodies and HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich, catalog no. A8924, RRID: AB_ 258426) in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin. SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot microanalyzer (Cellular Technologies).

Mouse experiments

Seven-to 9-week-old female 129S2 and K18-hACE2 C57BL/6 mice were immunized and boosted 3 weeks apart with 5 or 0.25 g (total dose) of mRNA vaccines (control, mRNA-1273, mRNA-1273.351, or mRNA-1273.211) in 50 l of PBS by intramuscular injection in the hind leg. The mRNA 1273.211 combination vaccine was mixed from the mRNA-1273 and mRNA-1273.351 vaccines at a 1:1 ratio immediately before usage. Animals were bled at specified time points to obtain serum for immunogenicity analysis. Three to 4 weeks after boosting, mice were challenged with 10 5 FFU (129S2) or 10 3 to 3 × 10 4 FFU (K18-hACE2) of WA1/2020 D614G (10 4 ), WA1/2020 N501Y/ D614G (10 3 ), B.1.1.7/E484K (10 3 ), B.1.351 (10 3 ), or B.1.617.2 (3 x 10 4 ) of SARS-CoV-2 strains by the intranasal route. Different doses of viruses were used in K18-hACE2 mice to match weight loss and infection data. This approach was necessary as some viruses (WA1/ 2020 N501Y/D614G, B.1.1.7/E484K, and B.1.351) encode N501Y mutations that enhance pathogenicity in mice (15,16,40). Animals were euthanized at 4 or 6 dpi, and tissues were harvested for virological, immunological, and pathological analyses.

Measurement of viral burden

Tissues were weighed and then homogenized with zirconia beads in a MagNA Lyser instrument (Roche Life Science) in 1 ml of DMEM medium supplemented with 2% heat-inactivated FBS. Tissue homogenates were clarified by centrifugation at 10,000 rpm for 5 min and stored at -80°C. RNA was extracted using the MagMax mirVana Total RNA Isolation Kit (Thermo Fisher Scientific) on the Kingfisher Flex extraction robot (Thermo Fisher Scientific). RNA was reverse-transcribed and amplified using the TaqMan RNA-to-CT 1-Step Kit (Thermo Fisher Scientific). Reverse transcription was carried out at 48°C for 15 min followed by 2 min at 95°C. Amplification was accomplished over 50 cycles as follows: 95°C for 15 s and 60°C for 1 min. Copies of SARS-CoV-2 N gene RNA in samples were determined using a published assay (41).

Cytokine and chemokine protein measurements

Lung homogenates were incubated with Triton X-100 (1% final concentration) for 1 hour at room temperature to inactivate SARS-CoV-2.

Downloaded from https://www.science.org at Cambridge University on June 22, 2023 Homogenates were analyzed for cytokines and chemokines by Eve Technologies Corporation using their Mouse Cytokine Array/ Chemokine Array 31-Plex (MD31) platform.

Lung histology

Lungs of euthanized mice were inflated with 1 to 2 ml of 10% neutral buffered formalin using a 3-ml syringe after a catheter was inserted into the trachea. Lungs were then kept in fixative for 7 days. Tissues were embedded in paraffin, and sections were stained with hematoxylin and eosin. Images were captured using the Nanozoomer (Hamamatsu) at the Alafi Neuroimaging Core at Washington University.

Peptide restimulation and intracellular cytokine staining

Two weeks after boosting, splenocytes from vaccinated K18-hACE2 mice were stimulated ex vivo with an H-2D b -restricted CD8 + or CD4 + T cell immunodominant peptide (amino acids 262 to 270 and 62 to 76 of the spike protein, respectively; gift of K. Valentine and S. Shresta) for 16 hours at 37°C with brefeldin A (BioLegend, 420601) added for the last 4 hours of incubation. After blocking with FcR antibody (clone 93; Thermo Fisher Scientific, catalog no. 14-0161-85) cells were stained on ice in PBS with CD45 (1 g/ml) Brilliant Ultraviolet 395 (clone 30-F11; BD Biosciences, catalog no. 564279, RRID: AB_2651134), CD4 phycoerythrin (PE; clone GK1.5; BD Biosciences, catalog no. 553730, RRID: AB_395014), CD8 fluorescein isothiocyanate (FITC; clone 53-6.7; BioLegend, catalog no. 100706, RRID: AB_312745), and Fixable Aqua Dead Cell Stain (Invitrogen, L34966). Stained cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBiosciences, 00-5523). Intracellular staining was performed with anti-IFN- Alexa Fluor 647 (clone XMG1.2; BioLegend, catalog no. 505814, RRID: AB_493314) and anti-tumor necrosis factor- Brilliant Violet 605 (clone MP6-XT22; BioLegend, catalog no. 506329, RRID: AB_ 11123912). Analysis was performed on a BD LSRFortessa X-20 cytometer using FlowJo X 10.0 software.

Antigenic cartography

A target distance from an individual serum to each virus was derived by calculating the difference between the logarithm (log 2) reciprocal neutralization titer for that particular virus and the log 2 reciprocal maximum titer achieved by that serum (against any virus). Thus, the higher the reciprocal titer, the shorter the target distance. As the log 2 of the reciprocal titer was used, a twofold change in titer equates to a fixed change in target distance whatever the magnitude of the actual titers. Antigenic cartography (42) then was used to optimize the positions of the viruses and serum relative to each other on a map, minimizing the sum-squared error between map distance and target distance. Each virus is therefore positioned by multiple serum samples, and the serum samples themselves also are positioned only by their distances to the viruses. Hence, serum samples with different neutralization profiles to the virus panel are in separate locations on the map but contribute equally to positioning of the viruses.

Statistical analysis

All raw, individual level data are shown in data file S1. Statistical significance was assigned when P < 0.05 using Prism version 10 (GraphPad). Tests [one-way Kruskal-Wallis analysis of variance (ANOVA] with Dunn's post test; one or two-way ANOVA with Tukey's post test; one-way ANOVA of area under the curve with Dunnett's post test), number of animals (n), median values, and statistical comparison groups are indicated in the figure legends.

Protective activity of mRNA vaccines against ancestral and variant SARS-CoV-2 strains

Baoling Ying, Bradley Whitener, Laura A. VanBlargan, Ahmed O. Hassan, Swathi Shrihari, Chieh-Yu Liang, Courtney E. Karl, Samantha Mackin, Rita E. Chen, Natasha M. Kafai, Samuel H. Wilks, Derek J. Smith, Juan Manuel Carreo, Gagandeep Singh, Florian Krammer, Andrea Carfi, Sayda M. Elbashir, Darin K. Edwards, Larissa B. Thackray, and Michael S. Diamond Sci. Transl. Med., 14 (630), eabm3302. DOI: 10.1126/scitranslmed.abm3302

A variant vaccine

The success of mRNA vaccines against SARS-CoV-2 is being challenged by the emergence of variants of concern (VOC). To address this, it may be necessary to develop vaccines that encode spike proteins from these VOC. Here, Ying et al. compared two SARS-CoV-2 mRNA vaccines, the mRNA-1273 vaccine, which is now in use globally, and a variant version, mRNA-1273.351. The authors showed that mRNA-1273, mRNA-1273.351, and a mix of the two, mRNA-1273.211, conferred protection against SARS-CoV-2 in two different mouse models when administered at a higher dose. Both vaccines also conferred some degree of protection at a lower dose of vaccine, which the authors use to recapitulate suboptimal vaccine responses. Together, these results support the continued development of mRNA vaccines for SARS-CoV-2.

Acknowledgements

Acknowledgments: We thank M. DeGrace for help in study design and funding support; R. Webby, M. Suthar, and P.-Y.Shi for viruses used in this study; K. Valentine and S. Shresta for providing immunodominant T cell peptides; B. Mühlemann for help with antigenic cartography; and E. Winkler and O. Dmytrenko for lung sections from naïve mice.We acknowledge the Pulmonary Morphology Core at Washington University School of Medicine for tissue sectioning and slide imaging.Funding: This study was supported by the NIH (R01 AI157155, HHSN75N93019C00074, and T32 AI007172), NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract HHSN272201400008C and NIAID Centers of Excellence for Influenza Research and Response (CEIRR) contract 75N93021C00014, and the Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051.This work also was supported by the Alafi Neuroimaging Laboratory, the Hope Center for Neurological Disorders, and NIH Shared Instrumentation Grant (S10 RR0227552).Author contributions: L.A.V. and B.Y. performed and analyzed neutralization assays.B.Y., B.W., L.A.V., A.O.H., C-Y.L., C.E.K., S.M., N.M.K., S.S., R.E.C., and L.B.T. performed mouse experiments.B.W., B.Y., S.S., and C-Y.L. performed viral burden analyses.A.O.H. and B.Y. performed T cell analyses.J.M.C., G.S., and F.K. performed the ELISA experiments.S.H.W. and D.J.S. performed the antigenic cartography analysis.A.C., S.M.E., and D.K.E.provided mRNA vaccines and helped design experiments.M.S.D. and L.B.T. wrote the initial draft, with the other authors providing editorial comments.Competing interests: M.S.D. is a consultant for Inbios, Vir Biotechnology, Senda Biosciences, and Carnival Corporation, and on the Scientific Advisory Boards of Moderna and Immunome.The Diamond laboratory has received unrelated funding support in sponsored research agreements from Vir Biotechnology, Kaleido, and Emergent BioSolutions and past support from Moderna not related to these studies.A.C., S.M.E., and D.K.E. are employees of and shareholders in Moderna Inc. F.K. has consulted for Merck and Pfizer (before 2020) and is currently consulting for Pfizer, Seqirus, and Avimex.The Krammer laboratory is also collaborating with Pfizer on animal models of SARS-CoV-2.F.K. is a coinventor on a patent application for serological assays and SARS-CoV-2 vaccines (international application numbers PCT/US2021/31110 and 62/994,252).The other authors declare that they have no competing interests.Data and materials availability: All data associated with this study are present in the paper or the Supplementary Materials.All requests for resources and reagents (proteins, antibodies, cell lines, and plasmids) should be directed to and will be fulfilled directly by the corresponding authors or upon completion of a material transfer agreement (MTA).The mRNA vaccines can be obtained under an MTA with Moderna [contact: D.K.E.(Darin.Edwards@modernatx.com)].This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using this material.Submitted 9 September 2021 Accepted 22 November 2021 Published First Release 30 November 2021 Published 2 February 2022 10.1126/scitranslmed.abm3302Downloaded from https://www.science.orgat Cambridge University on June 22, 2023

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