Safety and Immunogenicity of aerosolized adenovirus-vectored COVID-19 vaccine and intramuscular mRNA vaccine bivalent boosters: a randomized open-label clinical trial

Abstract

Both SARS-CoV-2 mRNA and mucosal vaccines induce protective immunity against COVID-19 but showed different immune profiles. We conducted a longitudinal head-to-head analysis of the safety and immunogenicity of the aerosolized adenovirus-vectored and mRNA COVID-19 vaccines. 450 participants were enrolled and randomly assigned into three groups to be vaccinated with an aerosolized Ad5-vectored bivalent vaccine (wild-type and BA.5, Ad5-CoV5T), an intramuscular bivalent mRNA vaccine (mbO5), and an aerosolized wild-type Ad5-vectored vaccine (Ad5-nCoV). The primary outcomes were adverse reactions within 28 days and anti-XBB.1.5-specific neutralizing antibody titers at day 28 after vaccination. The secondary outcome assessed safety within 30 min, serious adverse event within 6 months, and the persistence of anti-XBB.1.5/BA.5-specific neutralizing antibodies during the 6 months. Both the vaccines were well tolerated, but participants vaccinated with mbO5 reported more adverse reactions (73.3% mbO5 vaccinees vs. 28.7% aerosol vaccinees). No serious adverse events were recorded. The Ad5-CoV5T vaccine induced a superior anti-XBB.1.5-specific neutralizing titer than Ad5-nCoV at day 28 (geometric mean titer ratio of 1.48, 95% CI 1.12-1.97), while the mbO5 vaccine induced the highest antibody titer. The neutralizing antibodies were declined at month 6 and were similar across the three groups. In the pre-specified exploratory analysis, the mbO5 and the aerosolized vaccines induced comparable antigen-specific memory B cells but the latter stimulated higher frequency of IgA isotype and higher expression of CXCR3. This trial met the main hypothesis; the findings may provide insights for the development of the next-generation COVID-19 vaccines. Clinical Trials.gov identifier: NCT05886790.

Fig. 1. Trial profile.

Ad5-CoV5T = The participants who received one dose of bivalent Ad5-vectored COVID-19 vaccine (including 5 × 10⁹ VPs of Ad5-nCoV and 5 × 10⁹ VPs of Ad5-nCoV-BA.5) at day 0; mbO5 = The participants who received one dose of intramuscular bivalent mRNA COVID-19 vaccine (comprised of 15 μg WT SARS-CoV-2 spike mRNA and 15 μg BA.5 spike mRNA, formulated with lipid nanoparticle) at day 0; Ad5-nCoV = The participants who received one dose of aerosol Ad5-nCoV of 1 × 10¹⁰ VPs at day 0. a, 2 participants loss to the safety follow-up at 6 months; b, 1 participant loss to the sample collection follow-up at 6 months, and 1 participant loss to the safety follow-up at 6 months. c, 4 participants in Ad5-CoV5T group, 1 participant in mbO5 group, and 3 participants in Ad5-nCoV group were infected with SARS-CoV-2 during the 6-month follow-up period, and excluded from the long-term immunogenicity analysis.

Fig. 2. Neutralizing antibody and antigen-specific memory B cell responses.

Participants were vaccinated with Ad5-CoV5T, mbO5 or Ad5-nCoV on day 0, serum samples were collected at week 0, 2, 4, 12, and 24 for antibody detection, PBMCs were collected at week 0, 4, and 24 for RBD or spike-specific memory B cell detection. Serum SARS-CoV-2 XBB.1.5 (a) and BA.5.2 (b) live virus neutralizing antibody titers over time. Dotted lines indicate the limit of detection for the assay, titers below the limit were assigned a value of 2. Bars show geometric mean values with the 95% confidence interval (CI). c Frequencies of SARS-CoV-2 RBD- or spike-specific (WT⁺ or BA.5⁺ or XBB.1.5⁺) memory B cells over time. Bars show geometric mean values with the 95% CI. d Multi-reactivity profiles of RBD-specific memory B cells. Frequencies (e) and percents (f) of WT⁺ BA.5⁺ XBB.1.5⁺, WT⁺ BA.5⁺ XBB.1.5^-, WT⁺ BA.5^- XBB.1.5⁺, WT^- BA.5⁺ XBB.1.5⁺, WT⁺ BA.5^- XBB.1.5^-, WT^- BA.5⁺ XBB.1.5^- and WT^- BA.5^- XBB.1.5⁺ RBD-specific memory B cells over time. Plots show geometric mean values with the 95% CI in (e) and mean values with the 95% CI in (f). Statistics in (a, b, e, f) were calculated using unpaired nonparametric two-tailed Kruskal-Wallis test with Dunn’s correct for multiple comparisons. Statistics in (c) were calculated using paired nonparametric two-tailed Friedman test with Dunn’s correct for multiple comparisons. N = 26 for Ad5-CoV5T group, n = 29 for mbO5 group and n = 27 for Ad5-nCoV group. The neutralizing antibody detection was measured in duplicate, the antigen-specific memory B cell detection was measured in a single tube per sample due to the limited number of PBMCs. P-values marked in red in (e, f) indicate comparisons between Ad5-CoV5T group and mbO5 group, p-values marked in blue indicate comparisons between Ad5-nCoV group and mbO5 group.

Fig. 3. Circulation and mucosal IgA and IgG responses.

Participants were vaccinated with Ad5-CoV5T, mbO5 or Ad5-nCoV on day 0, nasal swabs were collected at week 0, 4, 24 and serum samples were collected at week 0, 2, 4, 12 and 24 for IgA and IgG concentrations detection, PBMCs were collected at week 0, 4 and 24 for antigen-specific memory B cell detection. Geometric mean fold rise of spike-specific IgA (a) and IgG (b) concentrations in nasal swab at 4- and 24-weeks post-vaccination. Geometric mean fold rise of spike-specific IgA (c) and IgG (d) concentrations in serum at 2-, 4-, 12- and 24-weeks post-vaccination. Plots show geometric mean values with the 95% CI; dotted lines indicate the fold rise equal to 1. e Correlation of nasal swab IgG concentrations with serum IgG concentrations at week 4. f Correlation of nasal swab IgA concentrations with serum IgA concentrations at week 4. g Frequencies of RBD⁺ IgG⁺, RBD⁺ IgA⁺ and RBD⁺ IgM⁺ memory B cells over time. h Frequencies of spike⁺ IgG⁺, spike⁺ IgA⁺ and spike⁺ IgM⁺ memory B cells over time. Plots show geometric mean values with the 95% CI. For (e, f), correlations were calculated using nonparametric two-tailed Spearman’s rank correlation. N = 26 for Ad5-CoV5T group, n = 29 for mbO5 group and n = 27 for Ad5-nCoV group. The IgA and IgG detection was measured in duplicate, the antigen-specific memory B cell detection was measured in a single tube per sample due to the limited number of PBMCs.

Fig. 4. Antigen-specific memory B cell phenotypes and subsets.

Participants were vaccinated with Ad5-CoV5T, mbO5 or Ad5-nCoV on day 0, PBMCs were collected at week 0, 4 and 24 for antigen-specific memory B cell detection. Mean fluorescence intensity (MFI) of CXCR3 (a), CD71 (b) and CXCR5 (c) on RBD-specific memory B cells or total memory B cells at week 0, 4, and 24. Bars show median values with the 95% CI. d MFI of CXCR3, CD71 and CXCR5 on spike-specific memory B cells or total memory B cells at week 4. Bars show median values with the 95% CI. Percents of activated (e), tissue-like (f), resting (g) and intermediate (h) RBD-specific memory B cell subsets at week 0, 4, and 24. Bars show median values with the 95% CI. Frequencies of activated (i), tissue-like (j), resting (k) and intermediate (l) RBD-specific memory B cell subsets at week 0, 4, and 24. Values less than 0.001 were assigned to 0.001. Lines connect the median values at different time points. Bars show median values with the 95% CI. For (a–h), statistics were calculated using unpaired nonparametric two-tailed Kruskal-Wallis test with Dunn’s correct for multiple comparisons. For (i–l), statistics between different time points within one group were calculated using paired nonparametric two-tailed Friedman test with Dunn’s correct for multiple comparisons, statistics between the different groups at week 4 were calculated using unpaired nonparametric two-tailed Kruskal-Wallis test with Dunn’s correct for multiple comparisons. N = 26 for Ad5-CoV5T group, n = 29 for mbO5 group and n = 27 for Ad5-nCoV group. The detection was measured in a single tube per sample due to the limited number of PBMCs. P values marked in red in (i, j) indicate comparisons between Ad5-CoV5T group and mbO5 group, p values marked in blue indicate comparisons between Ad5-nCoV group and mbO5 group. MBCs, total memory B cells.

Fig. 5. Spike-specific CD4⁺ and CD8⁺ T cell responses.

Participants were vaccinated with Ad5-CoV5T, mbO5 or Ad5-nCoV on day 0, PBMCs were collected at week 0, 2, 4 and 24 for spike-specific T cell detection. Frequencies of IFNγ⁺ CD4⁺ T cells (a), AIM⁺ CD4⁺ T cells (b), IFNγ⁺ CD8⁺ T cells (c) and AIM⁺ CD8⁺ T cells (d) over times in PBMCs. Data were background subtracted with the unstimulated control for each sample at each time point and values less than 0.001 were assigned to 0.001. Summary plots show median values with the 95% CI. Multifunctional profiles of spike-specific CD4⁺ T cells (e) and spike-specific CD8⁺ T cells (f). g Gating strategy of IFNγ⁺ CD4⁺ T helper subsets. The subsets were defined on the expression of chemokine receptor. h Frequencies of IFNγ⁺ CD4⁺ T helper subsets over time. Values less than 0.001 were assigned to 0.001. Lines connect the median values at different time points. Bars show median values with the 95% CI. Statistics were calculated using paired nonparametric two-tailed Friedman test with Dunn’s correct for multiple comparisons. N = 26 for Ad5-CoV5T group, n = 29 for mbO5 group and n = 27 for Ad5-nCoV group. The detection was measured in a peptide pool stimulated tube and an unstimulated control tube per sample.