Increased neutralization and IgG epitope identification after MVA-MERS-S booster vaccination against Middle East respiratory syndrome

Abstract

Vaccine development is essential for pandemic preparedness. We previously conducted a Phase 1 clinical trial of the vector vaccine candidate MVA-MERS-S against the Middle East respiratory syndrome coronavirus (MERS-CoV), expressing its full spike glycoprotein (MERS-CoV-S), as a homologous two-dose regimen (Days 0 and 28). Here, we evaluate the safety (primary objective) and immunogenicity (secondary and exploratory objectives: magnitude and characterization of vaccine-induced humoral responses) of a third vaccination with MVA-MERS-S in a subgroup of trial participants one year after primary immunization. MVA-MERS-S booster vaccination is safe and well-tolerated. Both binding and neutralizing anti-MERS-CoV antibody titers increase substantially in all participants and exceed maximum titers observed after primary immunization more than 10-fold. We identify four immunogenic IgG epitopes, located in the receptor-binding domain (RBD, n = 1) and the S2 subunit (n = 3) of MERS-CoV-S. The level of baseline anti-human coronavirus antibody titers does not impact the generation of anti-MERS-CoV antibody responses. Our data support the rationale of a booster vaccination with MVA-MERS-S and encourage further investigation in larger trials. Trial registration: Clinicaltrials.gov NCT03615911.

Fig. 1. Study design and trial profile.

Twenty-three individuals completed a homologous primary immunization with two vaccinations (Day (D) 0 (D0) and D28) of either 1 × 10⁷ plaque-forming units (PFU) (low dose (LD), blue) or 1 × 10⁸ PFU MVA-MERS-S (high dose (HD), red), and were followed-up for 180 days, which concluded this part of the study (End of Study). Participants were invited to return for an additional third vaccination as a booster 1 year ±4 months after prime. 10 participants (3 from the LD, 7 from the HD group) were re-enrolled and received a dose of 1 × 10⁸ PFU MVA-MERS-S. Safety and tolerability were assessed on Boost (B) Days (B:D) 0 (B:D0, baseline), B:D1, B:D3 (not depicted), B:D7, B:D14, and B:D28; humoral immunogenicity was assessed on B:D0, B:D7, B:D14, and B:D28 (End of Study). All 10 participants completed the extension trial and were included in the analyses. Created with BioRender.com.

Fig. 2. Biologic monitoring.

Graphs represent changes in a leukocyte, b neutrophil, c thrombocyte, and d lymphocyte counts, as well as e C-reactive protein (CRP) levels, in all booster study participants (n = 10) after booster vaccination. We observed an increase in a leukocyte (p = 0.014) and, by extension, b neutrophil counts (p = 0.002) as well as e CRP levels (p = 0.031) and a decrease in c thrombocyte (p = 0.049) and d lymphocyte (p = 0.002) counts on boost (B) Day 1 (B:D1) compared to B:D0. By B:D3, leukocyte and neutrophil counts had decreased (p = 0.004 for both neutrophil and leukocyte count on B:D1 vs B:D3 and B:D0 vs. BD3). These changes from baseline were transient and not clinically significant, but indicate biologic activity after vaccination. CRP levels of <5 mg/L (below the limit of detection) were set to 5 mg/L for visualization. Boxes indicate 25–75 percentile; whiskers are min. to max.; medians are shown as horizonal lines within the boxes. ULN = upper limit of normal. LLN = lower limit of normal. *p < 0.05, **p < 0.005, differences assessed using a two-sided Wilcoxon matched-pairs signed rank test. Source data are provided as a Source Data file.

Fig. 3. Increase in anti-MERS-CoV-S-specific binding antibodies after booster vaccination.

Anti-MERS-CoV-S-specific binding antibodies were measured as optical density values (y-axes) by two distinct ELISAs a in-house ELISA, b EUROIMMUN ELISA) at multiple timepoints after first (Day (D) 0 (D0), D35, D42, D84, D180) and booster (B) (B:D0, B:D7, B:D14, B:D28) vaccinations (x-axes). Former low dose (LD, n = 3) and high dose (HD, n = 7) vaccinees are depicted in blue and red, respectively, controls in gray (n = 2 in (a) and n = 4 in (b)). Boxes indicate 25–75 percentile; whiskers are min. to max.; medians are shown as horizonal lines within the boxes. Arrows indicate vaccinations. The horizontal dashed line in (a) indicates the cut-off level for positivity. Source data are provided as a Source Data file.

Fig. 4. Increase in MERS-CoV neutralizing antibodies after booster vaccination.

MERS-CoV neutralization was measured by a ≥80% plaque reduction neutralization test (PRNT₈₀) and b, c virus neutralization test (VNT). Neutralizing antibody levels were measured in reciprocal titers (y-axes) at multiple timepoints after first (Day (D) 0 (D0), D35, D42, D84, and D180) and booster (B) (B:D0, B:D7, B:D14, and B:D28) vaccinations (x-axes). Arrows indicate vaccinations. Horizontal dashed lines indicate the cut-off levels for positivity. a+b Boxes indicate 25–75 percentile; whiskers are min. to max.; medians are shown as horizonal lines within the boxes. Former low dose (LD, n = 3) and high dose (HD, n = 7) vaccinees are depicted in blue and red, respectively, controls in gray (n = 4). c VNT titers after prime vaccination measured longitudinally in the five individuals who did not develop nAb after prime vaccinations. Source data are provided as a Source Data file.

Fig. 5. Identification of four immunogenic MERS-CoV-S epitopes.

a Schematic representation of the MERS-CoV-S protein. The N-terminal domain (NTD), receptor-binding domain (RBD), S1/S2 cleavage site (S1/S2), fusion peptide (FP), S2‘ cleavage site (S2‘), heptad repeat 1 and 2 (HR1, HR2), transmembrane domain (TM) and cytoplasmic domain (CD) are illustrated. b Microarray of 15-mer peptides spanning the complete MERS-CoV-S protein with a 13 amino acid (AA) overlap. Immunogenic B-cell peptides are marked with red lines. c–f IgG binding to the respective peptides on MERS-CoV-S was measured in fluorescence intensity (as arbitrary fluorescence units, AFU), depicted as transformed values (areas sinus hyperbolicus (asinh), y axis), in all booster study participants (n = 10). Mean levels of peptide-binding IgG at baseline (Day (D) 0 (D0), white boxes) and 28 days after booster vaccination (Boost Day (B:D) 28 (B:D28), gray boxes) were compared using a two-sided Wilcoxon matched-pairs signed rank test and are depicted for each peptide (x-axis) within the immunogenic epitopes c AA 535-553 (AA 535–549, p = 0.014; AA 537–551, p = 0.002; AA 539–553, p = 0.002), d AA 887–913 (AA 887–901, p = 0.027; AA 889–903, p = 0.006; AA 891–905, p = 0.002; AA 897–911, p = 0.002; AA 899–913, p = 0.002), e AA 1225–1247 (AA 1225–1239, p = 0.002; AA 1227–1241, p = 0.002; AA 1229–1243, p = 0.002; AA 1231–1245, p = 0.002; AA 1233–1247, p = 0.004) and f AA 1333–1353 (AA 1333–1347, p = 0.002; AA 1335–1349, p = 0.002; AA 1337–1351, p = 0.002; AA 1339–1353, p = 0.002). Boxes indicate 25–75 percentile; whiskers are min. to max.; medians are shown as horizonal lines within the boxes. *p < 0.05, **p < 0.005. Source data are provided as a Source Data file.

Fig. 6. Immunity to endemic HCoV compared to anti-MERS-CoV immunity.

Reciprocal anti-CoV spike protein antibody titers of a human (H) Coronavirus (CoV) (HCoV)-OC43, b HCoV-229E, c HCoV-HKU1, d HCoV-NL63, e MERS-CoV, and f SARS-CoV (used as control antigen) were measured on Day (D) 0 (D0), pre-vaccination) and D42 via immunofluorescence assay in all participants who completed the primary immunization schedule with MVA-MERS-S on D0 and D28 (n = 23) and unvaccinated controls (n = 6). Low dose (n = 12) and high dose (n = 11) vaccinees are depicted in blue and red, respectively, unvaccinated controls in gray. A titer of <40 (dotted line) was considered negative. While we observed increased anti-MERS-CoV titers on Day 42 compared to baseline, there was no significant increase of anti-HCoV titers. Boxes indicate 25–75 percentile; whiskers are min. to max.; medians are shown as horizonal lines within the boxes. e ***p = 0.001 (LD) and p = 0.001 (HD), assessed via two-sided Wilcoxon matched-pairs signed rank test. Source data are provided as a Source Data file.