Bivalent SARS-CoV-2 mRNA vaccines increase breadth of neutralization and protect against the BA.5 Omicron variant in mice

Abstract

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in the Omicron lineage has resulted in diminished Coronavirus Disease 2019 (COVID-19) vaccine efficacy and persistent transmission. In this study, we evaluated the immunogenicity and protective efficacy of two, recently authorized, bivalent COVID-19 vaccines that contain two mRNAs encoding Wuhan-1 and either BA.1 (mRNA-1273.214) or BA.4/5 (mRNA-1273.222) spike proteins. As a primary two-dose immunization series in mice, both bivalent vaccines induced greater neutralizing antibody responses against Omicron variants than the parental, monovalent mRNA-1273 vaccine. When administered to mice as a booster at 7 months after the primary vaccination series with mRNA-1273, the bivalent vaccines induced broadly neutralizing antibody responses. Whereas most anti-Omicron receptor binding domain antibodies in serum induced by mRNA-1273, mRNA-1273.214 and mRNA-1273.222 boosters cross-reacted with the antecedent Wuhan-1 spike antigen, the mRNA-1273.214 and mRNA-1273.222 bivalent vaccine boosters also induced unique BA.1-specific and BA.4/5-specific responses, respectively. Although boosting with parental or bivalent mRNA vaccines substantially improved protection against BA.5 compared to mice receiving two vaccine doses, the levels of infection, inflammation and pathology in the lung were lowest in animals administered the bivalent mRNA vaccines. Thus, boosting with bivalent Omicron-based mRNA-1273.214 or mRNA-1273.222 vaccines enhances immunogenicity and confers protection in mice against a currently circulating SARS-CoV-2 strain.

Extended Data Figure 1.

VSV pseudovirus neutralization

Extended Data Figure 2.

Lentivirus pseudotype neutralization

Extended Data Figure 3.

VSV pseudotype neutralization

Extended Data Figure 4.

Lentivirus pseudotype neutralization

Extended Data Figure 5.

Pre and post-boost comparisons

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Pre-boost

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Post-boost

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RBD

Figure 1.. Robust antibody responses in BALB/c mice after a primary immunization series with preclinical versions of monovalent and bivalent mRNA vaccines.

Six-to-eight-week-old female BALB/c mice were immunized twice over a three-week interval with PBS or 1 μg total dose of preclinical versions of mRNA-1273 [Wuhan-1 spike], mRNA-1273.529 [BA.1 spike], mRNA-1273.045 [BA.4/5 spike], mRNA-1273.214 [benchside 1:1 mixture of mRNA-1273 + mRNA-1273.529], or mRNA-1273.222 [benchside 1:1 mixture of mRNA-1273 + mRNA-1273.045]. Immediately before (day 21) or two weeks after (day 35) the second vaccine dose, serum was collected. a. Scheme of immunization and blood draws. b. Serum antibody binding to Wuhan-1, BA.1, or BA.4/5 spike proteins by ELISA at Day 21 and Day 35 (n = 8 mice per group, one experiment, tops of boxes show geometric mean titers (GMT), which are indicated above each column; dotted lines show the limit of detection [LOD]). c. Neutralizing activity of serum at day 35 against VSV pseudoviruses displaying the spike proteins of Wuhan-1 D614G, BA.1, BA.2.75, or BA.4/5 (n = 8 mice per group, one experiment, tops of boxes show GMT, which are indicated above each column; dotted lines show LOD). d. Neutralizing activity of serum at day 35 against pseudotyped lentiviruses displaying the spike proteins of Wuhan-1, BA.1, or BA.4/5 (n = 8, boxes illustrate geometric mean values, dotted lines show the LOD). GMT values are indicated above the columns. Statistical analysis. b. One-way ANOVA with Tukey’s multiple comparison post-test; comparisons are between all groups except for the PBS control, which is shown for reference. c-d. One-way Kruskal-Wallis ANOVA with Dunn’s multiple comparison post-test; comparisons are between all groups. Exact P values are indicated, and only significant comparisons are shown. Primary data are provided as a Source Data file.

Figure 2.. Robust neutralizing antibody responses in BALB/c mice after primary series immunization with clinically representative versions of mRNA-1273, mRNA-1273.214, and mRNA-1273.222.

Six-to-eight-week-old female BALB/c mice were immunized twice over a three-week interval with PBS or 1 μg total dose of clinically representative versions of mRNA-1273, mRNA-1273.214 [1:1 mixture in the vial of separately formulated mRNA-1273 and mRNA-1273.529], or mRNA-1273.222 [1:1 mixture in the vial of separately formulated mRNA-1273 and mRNA-1273.045]. Two weeks after (day 35) the second vaccine dose, serum was collected. a. Scheme of immunization and blood draws. b. Neutralizing activity of serum at day 35 against VSV pseudoviruses displaying the spike proteins of Wuhan-1 D614G, BA.1, BA.2.75, or BA.4/5 (n = 16 mice per group, one experiment, tops of boxes show GMT, which are indicated above each column; dotted lines show LOD). c. Neutralizing activity of serum at day 35 against pseudotyped lentiviruses displaying the spike proteins of Wuhan-1 DG14G, BA.1, or BA.4/5 (n = 15 for mRNA-1273 and mRNA-1273.214, n = 16 for mRNA-1273.222, one experiment, tops of boxes shows GMT, which are indicated above each column; dotted lines show LOD). Statistical analysis. b-c. One-way Kruskal-Wallis ANOVA with Dunn’s multiple comparison post-test; comparisons are between all groups. Exact P values are indicated, and only significant comparisons are shown. Primary data are provided as a Source Data file.

Figure 3.. Neutralizing antibody responses in K18-hACE2 mice after boosting with clinically representative versions of mRNA-1273, mRNA-1273.214, and mRNA-1273.222.

Seven-week-old female K18-hACE2 mice were immunized with 0.25 μg of control mRNA or mRNA-1273 vaccine and then boosted 31 weeks later with PBS, 0.25 μg of control mRNA, or 0.25 μg of clinically representative versions of mRNA-1273, mRNA-1273.214, or mRNA-1273.222 vaccines. a. Scheme of immunizations, blood draws and virus challenge. b-c. Serum neutralizing antibody responses immediately before (b, pre-boost) and four weeks after (c, post-boost) receiving the indicated mRNA boosters or PBS as judged by focus reduction neutralization test (FRNT) with authentic WA1/2020 D614G, B.1.617.2, BA.1, and BA.5 viruses (n = 9 for mRNA-1273, n = 10 for other groups, two experiments; tops of boxes show GMT values, which are indicated above each column; dotted lines show LOD based on a 1/60 (WA1/2020 D614G, B.1.617.2) or 1/30 serum dilution (BA.1, BA.5)). d. Scheme of serum depletion of anti-receptor binding domain (RBD) antibodies. e. Serum (1/150 dilution) from mice boosted with mRNA-1273, mRNA-1273.214, or mRNA-1273.222 was incubated with empty, Wuhan-1 RBD-loaded, or BA.4/5 RBD-loaded magnetic beads. After separation of pre-clearing beads, supernatants were diluted serially and added to ELISA plates coated with Wuhan-1 or BA.4/5 RBD. Representative depletion curves performed in technical duplicate corresponding to individual mice are shown for the indicated vaccines. f. Bar graphs derived from area under the curve (AUC) analysis of the data in panel e showing the relative proportions of Wuhan-1-specific, Wuhan-1/BA.4/5 cross-reactive, and BA.4/5-specific RBD responses (n = 9 for mRNA-1273, n = 10 for mRNA-1273.214 and mRNA1273.222, two experiments, right edge of boxes illustrate mean values, and error bars indicate standard deviations). Statistical analysis. c: One-way Kruskal-Wallis ANOVA with Dunn’s multiple comparisons post-test; comparisons are between all groups except for the PBS control, which is shown for reference. Exact P values are indicated, and only significant comparisons are shown. Primary data are provided as a Source Data file.

Figure 4.. Protection of K18-hACE2 mice from BA.5 challenge after boosting with clinically representative versions of mRNA-1273, mRNA-1273.214, and mRNA-1273.222.

Seven-week-old female K18-hACE2 mice were immunized with 0.25 μg of control mRNA or mRNA-1273, boosted 31 weeks later with PBS, 0.25 μg of control mRNA, or 0.25 μg of clinically representative versions of mRNA-1273, mRNA-1273.214, or mRNA-1273.222 vaccines, and then one month later challenged via intranasal route with 10⁴ focus-forming units (FFU) of BA.5. a. Viral RNA levels at 4 dpi in the nasal washes, nasal turbinates, and lungs. b. Infectious viral load at 4 dpi in the lungs after BA.5 challenge of vaccinated and boosted mice as determined by plaque assay (a-b: n = 8 for mRNA-1273.214, n = 9 for control mRNA and mRNA-1273, n = 10 for PBS and mRNA-1273.222, two experiments, tops of boxes illustrate mean values, dotted lines show LOD based on tissue weight or volume). c. Cytokine and chemokine levels in lung homogenates at 4 dpi. Data are expressed as fold-change relative to naive mice, and log₂ values are plotted (n = 8 for mRNA-1273.214, n = 9 for control mRNA and mRNA-1273, n = 10 for PBS and mRNA-1273.222, n = 4 for naïve, two experiments, lines illustrate median values, dotted lines indicate LOD for each respective analyte based on a standard curve. Measurements are also shown in Supplementary Table S1. d. Hematoxylin and eosin staining of lung sections harvested at 4 dpi from mice immunized with control mRNA (CTRL-mRNA) or mRNA-1273 (primary two dose series) and then boosted with PBS, mRNA-1273, mRNA-1273.214, or mRNA-1273.222 vaccines. An uninfected (Naïve) animal is shown for comparison. Low (top; scale bars, 1 mm), moderate (middle, scale bars, 200 µm), and high (bottom; scale bars, 50 µm) power images are shown. Representative images of multiple lungs sections from n = 2 for each group, two experiments. Statistical analysis. a-c: one-way Kruskal-Wallis ANOVA with Dunn’s multiple comparisons post-test; comparisons are between all groups. Exact P values are indicated, and only significant comparisons are shown. Primary data are provided as a Source Data file.