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. 2024 Jan 26;9(1):20.
doi: 10.1038/s41541-023-00801-z.

MVA-based vaccine candidates encoding the native or prefusion-stabilized SARS-CoV-2 spike reveal differential immunogenicity in humans

Leonie Mayer #  1   2   3 , Leonie M Weskamm #  4   5   6 , Anahita Fathi  4   5   6   7 , Maya Kono  4   5   6 , Jasmin Heidepriem  8 , Verena Krähling  9   10 , Sibylle C Mellinghoff  11   12 , My Linh Ly  4   5   6 , Monika Friedrich  4   5   6 , Svenja Hardtke  4   5   6 , Saskia Borregaard  13 , Thomas Hesterkamp  14 , Felix F Loeffler  8 , Asisa Volz  15   16 , Gerd Sutter  17   18 , Stephan Becker  9   10 , Christine Dahlke #  4   5   6 , Marylyn M Addo #  19   20   21
Affiliations

MVA-based vaccine candidates encoding the native or prefusion-stabilized SARS-CoV-2 spike reveal differential immunogenicity in humans

Leonie Mayer et al. NPJ Vaccines. .

Abstract

In response to the COVID-19 pandemic, multiple vaccines were developed using platforms such as viral vectors and mRNA technology. Here, we report humoral and cellular immunogenicity data from human phase 1 clinical trials investigating two recombinant Modified Vaccinia virus Ankara vaccine candidates, MVA-SARS-2-S and MVA-SARS-2-ST, encoding the native and the prefusion-stabilized SARS-CoV-2 spike protein, respectively. MVA-SARS-2-ST was more immunogenic than MVA-SARS-2-S, but both were less immunogenic compared to licensed mRNA- and ChAd-based vaccines in SARS-CoV-2 naïve individuals. In heterologous vaccination, previous MVA-SARS-2-S vaccination enhanced T cell functionality and MVA-SARS-2-ST boosted the frequency of T cells and S1-specific IgG levels when used as a third vaccination. While the vaccine candidate containing the prefusion-stabilized spike elicited predominantly S1-specific responses, immunity to the candidate with the native spike was skewed towards S2-specific responses. These data demonstrate how the spike antigen conformation, using the same viral vector, directly affects vaccine immunogenicity in humans.

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Conflict of interest statement

All authors declare no financial or non-financial competing interests.

Figures

Fig. 1
Fig. 1. Study design.
a Participants of five study cohorts received up to four vaccinations (V1 to V4) with different COVID-19 vaccines. The vaccines administered in this study include the two experimental rMVA-based vaccine candidates MVA-SARS-2-S (MVA-S) and MVA-SARS-2-ST (MVA-ST), as well as the licensed vaccines BNT162b2 and mRNA-1273 (together referred to as mRNA) and ChAdOx1 nCov-19 (ChAd). The different vaccines encode either the native spike protein (black) or the prefusion-stabilized spike (yellow). Blood samples were collected at different time points after vaccination, labeled as T0 (baseline), T1 (1–2 weeks), T2 (3–5 weeks), T3 (12 weeks), and T4 (17–29 weeks), referring to the time since last vaccination (V1–V4). b The humoral and cellular immune response was analyzed using different assays. See Supplementary Tables 4–8 for detailed number of samples and analyzed time points by assay and study cohort.
Fig. 2
Fig. 2. S1/S2-specific IgG response induced by MVA-S and MVA-ST immunization.
a S1 (continuous line)- and S2 (dashed line)- specific IgG responses measured at baseline and longitudinally after each vaccination. Colored lines depict median MFI (measured by bead-based multiplex immunoassay, mean of technical duplicates). Gray lines show dynamics of individual study participants. Vaccinations V1 to V4 are indicated by arrows. b S1- (left) and S2- (right) specific IgG levels induced by two doses of MVA-S (blue) or MVA-ST (red) at V2T1 in comparison to the control cohorts (green and brown). c S1- (left) and S2- (right) specific IgG levels after first mRNA vaccination in the MVA-S/mRNA cohort (blue; V3T2) in comparison to the mRNA control cohort (green; V1T2). d, e S1-specific IgG levels induced by third vaccination with MVA-ST divided into (d) dose groups (purple; LD = low dose, MD = middle dose, HD = high dose) compared to mRNA (green) or by (e) low (≤median V3T0; left) and high baseline (>median V3T0; right). Data are represented as median ± IQR (b, c) or individual data points and median (d, e). f Spearman correlation of serum neutralizing capacity (measured by SARS-CoV-2 virus neutralization test, VNT100) with S1- (left) and S2- (right) specific IgG, n = 228. Significant p-values are indicated as calculated by two-tailed Mann–Whitney U test (b, c) or Wilcoxon matched-pairs signed rank test (d, e) and adjusted for multiple comparisons using a Benjamini & Hochberg correction: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Time points after vaccination are indicated as T0 (baseline), T1 (1-2 weeks), T2 (3–5 weeks), T3 (12 weeks), and T4 (17–29 weeks) (a, b, c, d, e). P values and sample sizes are indicated in Supplementary Tables 9 and 4 to 8.
Fig. 3
Fig. 3. Dynamics of IgG subclasses and identification of immunogenic S1/S2-specific B cell epitopes.
a S1- (top) and S2- (bottom) specific IgG subclasses of the different study cohorts measured at baseline and longitudinally after each vaccination. Median MFIs (measured by bead-based multiplex immunoassay, mean of technical duplicates) of IgG1-4 are shown as differently dotted lines. Vaccinations V1 to V4 are indicated by arrows and time points after vaccination are indicated as T0 (baseline), T1 (1–2 weeks), T2 (3-5 weeks), T3 (12 weeks), and T4 (17–29 weeks). b Schematic representation of immunogenic B cell epitopes measured on peptide microarrays and identified by increased fluorescent intensity (as arbitrary fluorescence units, AFU) in the five study cohorts in one (gray) or multiple (black) individuals, aligned to a schematic depiction of the S-protein. Positive epitope binding was defined as >400 mean AFU of three successive peptides and 2.5-fold above baseline before first vaccination (if available). Time points analyzed after vaccination: mRNA (V2:T1), ChAd/mRNA (V2:T1; V3:T1), MVA-S/mRNA (V2:T1; V4:T1), MVA-ST (V2:T1), mRNA/MVA-ST (V3:T0; V3:T1). (NTD: N-terminal domain, RBD receptor-binding domain, SD1, SD2 subdomain 1 and 2, S1/S2 S1/S2 cleavage site, S2‘ S2’cleavage site, FP fusion peptide, HR1 heptad repeat 1, CH central helix, CD connector domain, HR2 heptad repeat 2, TM transmembrane domain, CT cytoplasmic tail). Sample sizes are indicated in Supplementary Tables 4–8.
Fig. 4
Fig. 4. Longitudinal analysis of S1/S2-specific B cell responses induced by MVA-S and MVA-ST immunization.
a Frequencies of IgG-secreting B cells shown as SFC/106 PBMCs (mean of technical duplicates) measured by IgG ELISpot. Colored lines depict median S1- (continuous line) and S2- (dashed line) specific responses for each cohort. Gray lines show the dynamics of individual participants. b S1- (left) and S2- (right) specific IgG-secreting B cells induced by two doses MVA-S (blue) or MVA-ST (red) at V2T1 in comparison to the control cohorts (green and brown). c S1- (left) and S2- (right) specific IgG-secreting B cells after first mRNA vaccination in the MVA-S/mRNA cohort (blue; V3T2) in comparison to first vaccination in the mRNA control cohort (green; V1T2). S1-specific IgG-secreting B cells induced by third vaccination with MVA-ST divided into (d) dose groups (purple; LD = low dose, MD = middle dose, HD = high dose) compared to mRNA (green) or by (e) low (<median V3T0; left) and high baseline (>median V3T0; right). Data are represented as median ± IQR (b, c) or individual data points and median (d, e). Significant p values are indicated as calculated by two-tailed Mann–Whitney-U test (b, c) or Wilcoxon matched-pairs signed rank test (d, e) and adjusted for multiple comparisons using a Benjamini & Hochberg correction: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (b, c). Time points after vaccination are indicated as T0 (baseline), T1 (1–2 weeks), T2 (3–5 weeks), T3 (12 weeks), and T4 (17–29 weeks) (a, b, c). P values and sample sizes are indicated in Supplementary Tables 10 and 4–8.
Fig. 5
Fig. 5. Longitudinal analysis of S1/S2-specific T cell responses induced by MVA-S and MVA-ST immunization.
a Frequencies of IFN-γ-producing T cells (SFC/106 PBMCs; mean of technical triplicates) measured by ELISpot. Colored lines depict median responses, gray lines show the dynamics of individual participants. b Schematic of the peptide pools M1-4 resembling the spike S1 and S2. Representative ELISpot wells pre (left) and post vaccination (right). c Spearman correlation of ELISpot and IFN-γ release assay. T cell response induced by two doses MVA-S (blue) or MVA-ST (red) at (d) V2T1 or (e) against individual peptide pools M1-M4 compared to the control cohorts (green and brown). f T cell responses after first mRNA vaccination in the MVA-S/mRNA cohort (blue; V3T1) compared to the mRNA control cohort (green; V1T1). T cell responses induced by third vaccination with MVA-ST divided into (g) dose groups (purple; LD = low dose, MD = middle dose, HD = high dose) compared to mRNA (green) or by (h) low ( ≤ median V3T0; left) and high baseline (>median V3T0; right). Data are represented as median ± IQR (d, f), sum of medians (e), or individual data points and median (g, h). Significant p values are indicated as calculated by two-tailed Mann–Whitney U test (d, f) or Wilcoxon matched-pairs signed rank test (g, h) and adjusted for multiple comparisons using a Benjamini & Hochberg correction: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Time points after vaccination are indicated as T0 (baseline), T1 (1–2 weeks), T2 (3–5 weeks), T3 (12 weeks), and T4 (17–29 weeks) (a, d, e, f, g, h). P values and sample sizes are indicated in Supplementary Tables 11 and 4–8.
Fig. 6
Fig. 6. Enhanced T cell polyfunctionality after previous MVA-S immunization.
a Analyzed study cohorts and time points: MVA-S/mRNA (blue) and mRNA (green) were analyzed at baseline (T0) and time points after first and second vaccination with licensed mRNA vaccines, referred to as VI and VII (T2). b Representative gating strategy of cytokine-secreting CD4+ (top) and CD8+ (bottom) memory T cells after stimulation with overlapping peptide pools covering the S-protein. c Median frequencies of single positive (IFN-γ+ or IL-2+ or TNF-α+), double positive (IFN-γ+ IL-2+ TNF-α- or IFN-γ+ IL-2- TNF-α+ or IFN-γ- IL-2+ TNF-α+), and triple positive (IFN-γ+ IL-2+ TNF-α+) T cells out of total CD4+ (top) and CD8+ (bottom) memory T cells. Results were obtained by Boolean gating of the cytokine gates shown in (b). d Frequencies of IFN-γ, IL-2, and TNF-α-positive T cells out of total CD4+ (top) and CD8+ (bottom) memory T cells at baseline and time point T2 post VI and VII. Data are represented as individual data points and median ± IQR. Significant p values are indicated as calculated by two-tailed Wilcoxon matched-pairs signed rank test and adjusted for multiple comparisons using a Benjamini & Hochberg correction: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. P values and sample sizes are indicated in Supplementary Tables 12 and 4–8.
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