mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits similar B cell expansion, neutralizing responses, and protection from Omicron

Abstract

SARS-CoV-2 Omicron is highly transmissible and has substantial resistance to neutralization following immunization with ancestral spike-matched vaccines. It is unclear whether boosting with Omicron-matched vaccines would enhance protection. Here, nonhuman primates that received mRNA-1273 at weeks 0 and 4 were boosted at week 41 with mRNA-1273 or mRNA-Omicron. Neutralizing titers against D614G were 4,760 and 270 reciprocal ID₅₀ at week 6 (peak) and week 41 (preboost), respectively, and 320 and 110 for Omicron. 2 weeks after the boost, titers against D614G and Omicron increased to 5,360 and 2,980 for mRNA-1273 boost and 2,670 and 1,930 for mRNA-Omicron, respectively. Similar increases against BA.2 were observed. Following either boost, 70%-80% of spike-specific B cells were cross-reactive against WA1 and Omicron. Equivalent control of virus replication in lower airways was observed following Omicron challenge 1 month after either boost. These data show that mRNA-1273 and mRNA-Omicron elicit comparable immunity and protection shortly after the boost.

Keywords: B cells; COVID-19; Omicron; SARS-CoV-2; T cells; antibody; boost; immune memory; mRNA vaccine; original antigenic sin.

Graphical abstract

Figure S1

Experimental timeline and responses to Omicron sublineages after boost, related to Figure 1 (A) 8 NHP were vaccinated with 100 μg of mRNA-1273 at weeks 0 and 4. At week 41, NHP were split into 2 groups of 4 and boosted with 50 μg of mRNA-1273 or mRNA-Omicron. Both groups, and 8 unvaccinated NHP which were given 50 μg of mRNA control, were challenged with Omicron 1 month later. (B) Sera were collected 2 weeks postboost to measure pseudoneutralizing responses to D614G and the BA.1 and BA.2 sublineages of Omicron. Circles indicate individual NHP. Boxes represent interquartile range with the median denoted by a horizontal line. Assay LOD indicated by dotted line. 4–6 NHP per group.

Figure 1

Kinetics of serum antibody responses following mRNA-1273 immunization and boost with mRNA-1273 or mRNA-Omicron (A–F) Sera were collected at weeks 6, 40 or 41, and 43 post-immunization. (A and B) IgG-binding titers to (A) variant S and (B) variant RBD expressed in AUC. (C and D) Neutralizing titers to (C) live virus and (D) lentiviral pseudovirus expressed as the reciprocal ID₅₀. (E) p values listed for week 43 titers compared with week 6 or 41 as indicated in Figures 1A–1D. (F) Avidity index for WA1 S-2P- and Omicron S-2P-binding serum antibodies. p values for comparison of avidity index at weeks 6 versus 40 were identical for both boost cohorts. Circles represent geometric means. Error bars represent geometric standard deviations. Assay limit of detection (LOD) indicated by dotted lines. Break in x-axis indicates a change in scale without a break in the range depicted. Responses to variants are color-coded as WA1 or D614G (black), Delta (blue), Beta (red), and Omicron (green). Arrows represent timepoints of immunizations. Following the boost at week 41, mRNA-1273-boosted NHP indicated by solid lines and mRNA-Omicron-boosted NHP indicated by dashed lines. 8 vaccinated NHP, split into 2 cohorts of 4 NHP postboost. See also Figure S1 for experimental schema and neutralizing responses to BA.2, Table S1 for mRNA-Omicron sequence, and Table S2 for detailed neutralizing titers.

Figure 2

Kinetics of mucosal antibody responses following mRNA-1273 immunization and boost with mRNA-1273 or mRNA-Omicron (A–D) BAL (A and C) and NW (B and D) were collected at weeks 8, 39, and 43 post-immunization. (A and B) IgG-binding titers to WA1, Delta, Beta, and Omicron expressed in AUC. (C and D) D614G, Delta, Beta, and Omicron S-2P-ACE2 binding inhibition in the presence of mucosal fluids. All samples diluted 1:5. Circles indicate individual NHP. Boxes represent interquartile range with the median denoted by a horizontal line. Dotted lines are for visualization purposes and denote 4-log₁₀ increases in binding titers (A and B) or 0% and 100% inhibition (C and D). Statistical analysis shown for week 43 responses compared with week 39 for both boost cohorts. In total, 8 controls and 8 vaccinated NHP, split into 2 cohorts postboost. See also Table S3 for list of amino acid replacements in variant-specific S-2P-ACE2 inhibition assays.

Figure S2

B cell-gating strategy, related to Figure 3 Representative flow cytometry plots showing gating strategy for B cells in Figures 3, 4, S3, and S5. Cells were gated as singlets and live cells on forward and side scatter and a live/dead aqua blue stain. CD3-, CD4-cells were then gated on absence of CD14 and CD16 expression and positive expression of CD20 and CD19. Memory B cells were selected based on lack of IgD or IgM. Finally, pairs of variant S-2P probes were used to determine binding specificity. Probe-binding cells were further characterized as having a phenotype consistent with CD27-negative resting memory (CD27-RM), tissue-like memory (TLM), activated memory (AM), or resting memory (RM) cells according to expression of CD27 and CD21.

Figure 3

Memory B cell specificities following immunization and boosting (A–D) Representative flow cytometry plots showing single variant-specific (top left and bottom right quadrant) and dual-variant-specific (top right quadrant) memory B cells at weeks 0, 6, 41, and 43 post-immunization. Event frequencies per gate are expressed as a percentage of all class-switched memory B cells. Cross-reactivity shown for (A) WA1 and Omicron S-2P, (B) Delta and Omicron S-2P, (C) WA1 and Delta S-2P, and (D) WA1 and Beta S-2P. See also Figure S2 for B cell-gating strategy.

Figure 4

Similar expansion of cross-reactive S-2P-specific memory B cells following boost with mRNA-1273 or mRNA-Omicron (A–D) Pie charts indicating the proportion of total S-binding memory B cells that are cross-reactive (dark gray) or specific for the indicated variants (black or light gray) for all NHP (geomean) at weeks 6, 41, and 43 post-immunization. Where applicable, memory B cells specific only for WA1 or Delta are represented by the light gray segment. Cross-reactivity shown for (A) WA1 and Omicron S-2P, (B) Delta and Omicron S-2P, (C) WA1 and Delta S-2P, and (D) WA1 and Beta S-2P. 4–7 NHP per group. (E) Pie charts indicating the proportion of total S-2P-binding memory B cells (geomean) that have a phenotype consistent with resting memory (pattern), activated memory (black), tissue-like memory (dark gray), or CD27-negative resting memory (light gray) B cells at weeks 6, 41, and 43 post-immunization. Analysis shown here for memory B cells that bind to WA1 and/or Omicron S-2P. 4–7 NHP per group. See also Figure S3 for frequencies of cross-reactive S-2P memory B cells, Figure S4 for serum epitope reactivity, Figures S5 and S6 for primary responses after variant-matched vaccination in naive NHP, and Figures S7, S8, and S9 for T cell responses after boosting.

Figure S3

Expansion of memory B cells that recognize unique WA1 epitopes only occurs after homologous mRNA-1273 boosting, related to Figure 4 (A–D) Frequencies of memory B cells with indicated specificities as a percentage of total class-switched memory B cells (both S-2P-binding and non-S-2P-binding) were normalized to the corresponding frequencies from each individual NHP at week 6 post-immunization. Cross-reactivity shown for (A) WA1 and Omicron S-2P, (B) Delta and Omicron S-2P, (C) WA1 and Delta S-2P, and (D) WA1 and Beta S-2P. Specificities not shown if memory B cell populations were indistinguishable from background staining. Circles indicate individual NHP. Boxes represent interquartile range with the median denoted by a horizontal line. A frequency of 1 indicates parity. 4–7 NHP per group.

Figure S4

Serum epitope reactivity following boost, related to Figure 4 (A and B) Relative serum reactivity was measured as fractional competition of total measured serum antibody S-2P binding competed by single monoclonal antibody (mAb) targeting epitopes on WA1 (A) or Omicron (B) S-2P at week 43 post-immunization. Antigenic sites are defined by mAbs B1-182 (RBD-A), A19-46.1 (RBD-D), A19-61.1 (RBD-F), S309 (RBD-G), A23-97.1 (RBD-H), and A23-80.1 (RBD-J). Circles indicate individual NHP. Boxes represent interquartile range with the median denoted by a horizontal line. Dotted lines are for visualization purposes and denote 0% competition. 4 NHP per boost group.

Figure S5

Memory B cell specificities following mRNA-Omicron immunization in naive primates, related to Figure 4 (A) Naive NHP were immunized with two doses of mRNA-Omicron (100 μg) at weeks 0 and 4. Representative flow cytometry plots for two different NHP showing single variant-specific (top left and bottom right quadrant) and dual-variant-specific (top right quadrant) memory B cells at weeks 0 (pre-vaccination) and 2 and 6 post-immunization. Event frequencies per gate are expressed as a percentage of all class-switched memory B cells. Cross-reactivity shown for WA1 and Omicron S-2P, Delta and Omicron S-2P, WA1 and Delta S-2P, and WA1 and Beta S-2P. (B) Frequencies of memory B cells that bound Omicron S-2P but not WA1 S-2P as a percentage of total CD19⁺, CD20⁺ B cell population. Symbol colors are as follows: red (2 doses of 100 μg mRNA-1273 at weeks 0 and 4 and a 50 μg mRNA-1273 boost at week 41), blue (2 doses of 100 μg mRNA-1273 at weeks 0 and 4 and a 50 μg mRNA-Omicron boost at week 41), orange (1 dose of 100 μg mRNA-Omicron at week 0), and cyan (2 doses of 100 μg mRNA-Omicron at weeks 0 and 4). All analysis conducted 2 weeks after the final immunization for each cohort specified above. Circles indicate individual NHP. Boxes represent interquartile range with the median denoted by a horizontal line. 4–6 NHP per group.

Figure S6

Primary responses to variant-matched vaccines, related to Figure 4 Naive NHP were immunized with two doses of mRNA-1273 (100 μg), mRNA-Beta (50 μg), or mRNA-Omicron (100 μg) at weeks 0 and 4. Sera were collected at week 6 to measure pseudoneutralizing responses to D614G, Delta, Beta, and Omicron. Circles indicate individual NHP. Boxes represent interquartile range with the median denoted by a horizontal line. Assay LOD (not shown) is 20. 8 NHP per cohort.

Figure S7

T cell-gating strategy, related to Figure 4 Representative flow cytometry plots showing gating strategy for T cells in Figures S8 and S9. Cells were gated as singlets and live cells on forward and side scatter and a live/dead aqua blue stain. CD3+ events were gated as CD4+ or CD8+ T cells. Total memory CD8+ T cells were selected based on expression of CCR7 and CD45RA. Finally, SARS-CoV-2 S-specific memory CD8+ T cells were gated according to co-expression of CD69 and IL-2, TNF or IFNγ. The CD4+ events were defined as naive, total memory, or central memory according to expression of CCR7 and CD45RA. CD4+ cells with a T_H1 phenotype were defined as memory cells that co-expressed CD69 and IL-2, TNF or IFNγ. CD4+ cells with a T_H2 phenotype were defined as memory cells that co-expressed CD69 and IL-4 or IL-13. T_FH cells were defined as central memory CD4+ T cells that expressed CXCR5, ICOS, and PD-1. T_FH cells were further characterized as IL-21+, CD69+ or CD40L+, CD69+.

Figure S8

Both mRNA-1273 and mRNA-Omicron boost T cell responses to S peptides, related to Figure 4 (A–E) PBMC collected at weeks 0, 6, 41, and 44 post-immunization. Cells were stimulated with SARS-CoV-2 S1 and S2 peptide pools (WA1) and then measured by intracellular cytokine staining. (A and B) Percentage of memory CD4+ T cells with (A) T_H1 markers (IL-2, TNF, or IFNγ) or (B) T_H2 markers (IL-4 or IL-13) following stimulation. (C) Percentage of CD8+ T cells expressing IL-2, TNF, or IFNγ. (D and E) Percentage of T_FH cells that express (D) CD40L or (E) IL-21. (F–H) BAL fluid was collected at weeks 8, 39, and 43 post-immunization. Lymphocytes in the BAL were stimulated with S1 and S2 peptide pools (WA1) and responses measured by intracellular cytokine staining using T_H1 (F), T_H2 (G), and CD8 markers (H). Break in y axis indicates a change in scale without a break in the range depicted. Circles in (A–H) indicate individual NHP. Boxes represent interquartile range with the median denoted by a horizontal line. Dotted lines set at 0%. Reported percentages may be negative due to background subtraction and may extend below the range of the y axis. 8 vaccinated NHP, split into 2 cohorts postboost. 4 control NHP shown for comparison.

Figure S9

T cell responses to Omicron S peptides are conserved, related to Figure 4 (A–E) PBMC collected at week 44 post-immunization. Cells were stimulated with SARS-CoV-2 OS1 and OS2 peptide pools and then measured by intracellular cytokine staining. (A and B) Percentage of memory CD4+ T cells with (A) T_H1 markers (IL-2, TNF, or IFNγ) or (B) T_H2 markers (IL-4 or IL-13) following stimulation. (C) Percentage of CD8+ T cells expressing IL-2, TNF, or IFNγ. (D and E) Percentage of T_FH cells that express (D) CD40L or (E) IL-21. Circles in (A–E) indicate individual NHP. Boxes represent interquartile range with the median denoted by a horizontal line. Dotted lines set at 0%. Reported percentages may be negative due to background subtraction and may extend below the range of the y axis. 4 NHP per group.

Figure S10

Omicron challenge stock sequence, related to Figure 5 (A and B) Omicron stock was sequenced and aligned with Wuhan-Hu-1. (A) S gene only. Amino acid replacements listed above graphic. NTD, N-terminal domain; RBD, receptor binding domain; RBM, receptor binding motif; FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; FCS, furin cleavage site; S2′, S2′ site. (B) Whole genome.

Figure 5

Boosting with ancestral or Omicron-matched vaccine provides equivalent protection in the lungs against Omicron challenge (A–E) BAL (A and D), NS (B and E), and OS (C) were collected at the indicated times following challenge with 1 × 10⁶ PFU Omicron. (A–C) Omicron sgRNA_N copy numbers per mL of BAL (A) or per swab (B and C). (D and E) Viral titers per mL of BAL (D) or per swab (E). Circles indicate individual NHP. Boxes represent interquartile range with the median denoted by a horizontal line. Assay LOD indicated by dotted lines. Statistical analysis shown for cohorts boosted with mRNA-1273 (red text) or mRNA-Omicron (blue text) in comparison with controls at each timepoint. 8 controls and 4 vaccinated NHP per boost cohort. See also Figure S10 for Omicron challenge stock sequence.

Figure 6

Virus antigen and pathology in the lungs after challenge (A and B) 2 NHP per group were euthanized on day 8 postchallenge and tissue sections taken from lungs. (A) Left: representative images indicating detection of SARS-CoV-2 N antigen by immunohistochemistry with a polyclonal anti-N antibody. Antigen-positive foci are marked by a red arrow. Right: hematoxylin and eosin stain (H&E) illustrating the extent of inflammation and cellular infiltrates. Images at 10× magnification with black bars for scale (100 μm). (B) SARS-CoV-2 antigen and inflammation scores in the left cranial lobe (Lc), right middle lobe (Rmid), and right caudal lobe (Rc) of the lungs. Antigen scoring legend: − no antigen detected; +/− rare to occasional foci; + occasional to multiple foci; ++ multiple to numerous foci; +++ numerous foci. Inflammation scoring legend: − absent to minimal inflammation; +/− minimal to mild inflammation; + mild to moderate inflammation; ++ moderate-to-severe inflammation; +++ severe inflammation. Horizontal rows correspond to individual NHP depicted above (A).