Adjuvant-dependent effects on the safety and efficacy of inactivated SARS-CoV-2 vaccines during heterologous infection by a SARS-related coronavirus

Abstract

Inactivated whole virus SARS-CoV-2 vaccines adjuvanted with aluminum hydroxide (Alum) are among the most widely used COVID-19 vaccines globally and have been critical to the COVID-19 pandemic response. Although these vaccines are protective against homologous virus infection in healthy recipients, the emergence of novel SARS-CoV-2 variants and the presence of large zoonotic reservoirs provide significant opportunities for vaccine breakthrough, which raises the risk of adverse outcomes including vaccine-associated enhanced respiratory disease (VAERD). To evaluate this possibility, we tested the performance of an inactivated SARS-CoV-2 vaccine (iCoV2) in combination with Alum against either homologous or heterologous coronavirus challenge in a mouse model of coronavirus-induced pulmonary disease. Consistent with human results, iCoV2 + Alum protected against homologous challenge. However, challenge with a heterologous SARS-related coronavirus, Rs-SHC014-CoV (SHC014), up to at least 10 months post-vaccination, resulted in VAERD in iCoV2 + Alum-vaccinated animals, characterized by pulmonary eosinophilic infiltrates, enhanced pulmonary pathology, delayed viral clearance, and decreased pulmonary function. In contrast, vaccination with iCoV2 in combination with an alternative adjuvant (RIBI) did not induce VAERD and promoted enhanced SHC014 clearance. Further characterization of iCoV2 + Alum-induced immunity suggested that CD4⁺ T cells were a major driver of VAERD, and these responses were partially reversed by re-boosting with recombinant Spike protein + RIBI adjuvant. These results highlight potential risks associated with vaccine breakthrough in recipients of Alum-adjuvanted inactivated vaccines and provide important insights into factors affecting both the safety and efficacy of coronavirus vaccines in the face of heterologous virus infections.

Figure 1. Inactivated SARS-CoV-2 + aluminum hydroxide (iCoV2 + Alum) vaccination protects mice from pathogenic SARS-CoV-2 challenge but causes adjuvant-dependent type 2 inflammation.

Mice were vaccinated with inactivated SARS-CoV-2 formulated with either Alum or RIBI adjuvants and challenged 4 weeks post-boost with SARS-CoV-2 MA10. Pulmonary tissue specimens were collected at 5 DPI. (a) Post-boost serum samples were collected prior to challenge and neutralizing antibody titers against SARS-CoV-2 (D614G) were measured using a luminescence-based microneutralization assay. Log-transformed results reported as IC₈₀. Combined results from 4 independent replicates.(b) Body weights were measured daily and reported as percent relative to baseline. (c) Clinical disease was evaluated daily using the clinical scoring system below. (d) Pulmonary viral titers were quantified by plaque assay and log-transformed results reported as log₁₀ pfu. (e, f) H&E-stained pulmonary specimens were analyzed by a blinded pathologist to evaluate ALI (e) and DAD (f). (g, h) Pulmonary eosinophils (EPX⁺ cells) were measured using immunohistochemical staining (representative micrographs, 100X magnification in g) and quantified (h). (a, d-f, h) Individual data points represent biological replicates. Results analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons correction (alpha = 0.05). Solid horizontal lines overlaying data represent group means. Error bars represent group SD. Dotted lines represent assay LOD. Solid horizontal lines above data represent pairwise comparisons with P values. Results from 2 (e, f, h), 3 (d), or 4 (a) independent replicates (effects reproduced in each replicate). (b) Reported as group mean ± SD. Analyzed by two-way ANOVA with Tukey’s multiple comparisons. (b, c) Representative results from a single experiment. Number of animals per group: iFLU + Alum (12), iCoV2 + Alum (19), iCoV2 + RIBI (4). Clinical scoring system: 0 = normal (blue), 1 = piloerection (orange), 2 = piloerection + kyphosis (red), 3 = piloerection, kyphosis and reduced movement (purple), 4 = markedly reduced movement and/or labored breathing (gray) and 5 = moribund, dead or euthanized (black). iFLU = inactivated influenza virus vaccine; iCoV2 = inactivated SARS-CoV-2 vaccine; Alum = aluminum hydroxide adjuvant; RIBI = Sigma Adjuvant System adjuvant; DPI = days post-infection; IC₈₀ = 80% serum reciprocal inhibitory dilution titer; pfu = plaque-forming units; MA10 = mouse-adapted SARS-CoV-2; EPX = Eosinophil peroxidase; SD = standard deviation; LOD = limit of detection

Figure 2. Inactivated SARS-CoV-2 + aluminum hydroxide (iCoV2 + Alum) vaccination causes enhanced subclinical disease and type 2 inflammation during infection by a SARS-related coronavirus.

iFLU = inactivated influenza virus vaccine; iCoV2 = inactivated SARS-CoV-2 vaccine; Alum = aluminum hydroxide adjuvant; RIBI = Sigma Adjuvant System adjuvant; DPI = days post-infection; IC₈₀ = 80% serum reciprocal inhibitory dilution titer; pfu = plaque-forming units; EPX = Eosinophil peroxidase; WBP = whole body plethysmography; SEM = standard error of the mean; SD = standard deviation; LOD = limit of detection; PenH = Enhanced Pause; Rpef = Rate of Peak Expiratory Flow; TP = Time of Pause Vaccinated mice were challenged with Rs-SHC014-CoV (SHC014) at either 4 weeks (a-g) or 10.5 months (h) post-boost. Pulmonary function was measured 1–4 DPI using WBP and pulmonary tissue was collected from vaccinated mice at 2 and 5 DPI (results displayed for 5 DPI if not specified). (a) Post-boost serum samples were collected prior to challenge and neutralizing antibody titers against SHC014 were measured using a luminescence-based microneutralization assay. Log-transformed results reported as IC₈₀. Combined results from 4 independent replicates. (b) Pulmonary function of vaccinated mice was measured by whole-body plethysmography before (baseline) and during SHC014 infection. PenH, Rpef, and TP were measured 1–4 DPI. (c) Pulmonary viral titers at 2 and 5 DPI were quantified by plaque assay and log-transformed results reported as log₁₀ pfu. (d, e) H&E-stained pulmonary specimens were analyzed by a blinded pathologist to evaluate ALI (d) and DAD (e). (f, g) Pulmonary eosinophils (EPX⁺ cells) at 5 DPI were measured using immunohistochemical staining (representative micrographs, 100X magnification in f) and quantified (g). (h) Pulmonary viral titers, ALI and DAD were measured in samples collected at 5 DPI following challenge at 10.5 months post-boost vaccination. (a, c-e, g, h) Individual data points represent biological replicates. Results analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons correction (alpha = 0.05). Solid horizontal lines overlaying data represent group means. Error bars represent group SD. Dotted lines represent assay LOD. Solid horizontal lines above data represent pairwise comparisons with P values. Results from 1 (c, h), 3 (d, e, and g), or 4 (a) independent replicates (if multiple replicates, effects reproduced in each replicate). (b) Combined results from 2 independent replicates Reported as group mean ± SEM. Analyzed by two-way ANOVA with Tukey’s multiple comparisons (alpha = 0.05). Asterisks (*) denote P values (pairwise comparison between proximate experimental value and control iFLU + Alum value) using the following scheme: (*) = 0.01 – 0.05, (**) = 0.001 – 0.01, (***) 0.0001 – 0.001, (****) < 0.0001. Number of animals per group: iFLU + Alum (12), iCoV2 + Alum (24), iCoV2 + RIBI (12).

Figure 3. Inactivated SARS-CoV-2 vaccine (iCoV2) adjuvants promote divergent immune gene expression patterns during infection by a SARS-related coronavirus (Rs-SHC014-CoV).

iFLU = inactivated influenza virus vaccine; iCoV2 = inactivated SARS-CoV-2 vaccine; Alum = aluminum hydroxide adjuvant; RIBI = Sigma Adjuvant System adjuvant; Ccl11 = C-C motif chemokine 11; Ccl24 = C-C motif chemokine 24; Il4 = Interleukin 4; Il5 = Interleukin 5; Il13 = Interleukin 13; DPI = days post-infection; SEM = standard error of the mean; SD = standard deviation; RNA-Seq = RNA Sequencing; NS = not significant; FC = fold-change Vaccinated mice were challenged with Rs-SHC014-CoV (SHC014) 4 weeks post-boost. Pulmonary tissue was collected from vaccinated mice at 2 or 5 DPI. (a) Type 2 cytokine gene expression from whole-tissue pulmonary specimens was measured using quantitative RT-PCR. Results reported as fold change normalized to Gapdh expression using DDCt. Individual data points represent biological replicates. Results analyzed by two-way ANOVA with Tukey’s multiple comparisons test (alpha = 0.05). Solid horizontal lines overlaying data represent group means. Error bars represent group SD. Solid horizontal lines above data represent pairwise comparisons with P values. Representative results from a single experiment. (b, c) RNA-Seq was performed with whole pulmonary tissue. Volcano plots showing differential expression between iCoV + Alum relative to iCoV + RIBI at 2 DPI (b) and 5 DPI (c) following SHC014 infection. Fold-change is shown along the X-axis (with 1.5 log₂ fold-change thresholds represented by vertical dashed lines) and significance along the Y-axis (with FDR adjusted q < 0.05 thresholds represented by horizontal dashed lines). Key genes are highlighted in the upper right (iCoV + Alum expression > iCoV + RIBI) and upper left (iCoV + Alum expression < iCoV + RIBI) quadrants. Representative results from a single experiment.

Figure 4. Heterologous boost vaccination partially reduces enhanced disease from vaccination with inactivated SARS-CoV-2 + aluminum hydroxide (iCoV2 + Alum).

iFLU = inactivated influenza virus vaccine; iCoV2 = inactivated SARS-CoV-2 vaccine; Alum = aluminum hydroxide adjuvant; RIBI = Sigma Adjuvant System adjuvant; DPI = days post-infection; Rpef = Rate of Peak Expiratory Flow; ALI = Acute lung injury; DAD = Diffuse alveolar damage; S2P = full-length pre-fusion stabilized SARS-CoV-2 Spike protein; WBP = Whole body plethysmography Mice were initially vaccinated with inactivated SARS-CoV-2 and aluminum hydroxide adjuvant (iCoV2 + Alum). Approximately 9.5 months post-boost vaccination, mice were administered either a third dose of iCoV2 + Alum (homologous second boost) or were boosted with a heterologous vaccine formulation (recombinant full-length pre-fusion stabilized Spike protein [S2P] + RIBI) 4 weeks prior to challenge with Rs-SHC014-CoV (SHC014). Pulmonary function was measured 1–4 DPI using WBP and pulmonary tissue was collected 5 DPI. (a) Pulmonary function of vaccinated mice was measured by whole-body plethysmography before (baseline) and during SHC014 infection. Rpef was measured 1–4 DPI. (b) Pulmonary viral titers were quantified by plaque assay and log-transformed results reported as log₁₀ pfu. (c, d) H&E-stained pulmonary specimens were analyzed by a blinded pathologist to evaluate ALI (c) and DAD (d). (e) Eosinophils (EPX⁺ cells) were detected using immunohistochemistry and measured using a blinded semi-quantitative eosinophil scoring system described below. (a) Results from a single experiment. Reported as group mean ± SEM. Analyzed by two-way ANOVA with Tukey’s multiple comparisons (alpha = 0.05). Asterisks (*) denote P values (pairwise comparison between proximate experimental value and control iFLU + Alum value) using the following scheme: (*) = 0.01 – 0.05, (**) = 0.001 – 0.01, (***) 0.0001 – 0.001, (****) < 0.0001. Number of animals per group: iFLU + Alum, iFLU + Alum (5); iCoV2 + Alum, iFLU + Alum (14), iCoV2 + Alum, iCoV2 + Alum (10); iCoV2 + Alum, S2P + RIBI (9). (b-d) Individual data points represent biological replicates. Results analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons correction (alpha = 0.05). Solid horizontal lines overlaying data represent group means. Error bars represent group SD. Dotted lines represent assay LOD. Solid horizontal lines above data represent pairwise comparisons with P values. A sagittal section through the left lobe was scanned by a blinded analyst for eosinophils using the following 5-point scoring system: 0 = no eosinophils; 1 = rare, scattered eosinophils; 2 = small clusters of eosinophils surrounding 1 or 2 airways; 3= small to moderately sized clusters surrounding multiple airways; 4= large clusters of eosinophils surrounding multiple airways/within the parenchyma; 5= large clusters of eosinophils involving most of the lung iCoV2 + Alum – iFlu + Alum control group data are repeated from Figure 2. This group served as the iCoV2 + Alum group for the final 10.5-month post-boost vaccination (Figure 2) and the control group for comparison to later secondary boost vaccination (Figure 4).

Figure 5. Inactivated SARS-CoV-2 (iCoV2) vaccine immune serum promotes cross-protection with modest pathology during heterologous infection.

Serum was collected from mice 4-weeks post-boost vaccination and passively transferred to naïve recipient mice 1 day prior to challenge with Rs-SHC014-CoV (SHC014). Pulmonary function was measured 1–4 DPI using WBP and pulmonary tissue was collected at 5 DPI. (a) Pulmonary function of mice was measured by whole-body plethysmography before (baseline) and during SHC014 infection. PenH, Rpef, and TP were measured 1–4 DPI. (b, c) H&E-stained pulmonary specimens were analyzed by a blinded pathologist to evaluate ALI and DAD. (d) Pulmonary viral titers were quantified by plaque assay and log-transformed results reported as log₁₀ pfu. (a) Results from a single experiment. Analyzed by two-way ANOVA with Tukey’s multiple comparisons test (alpha = 0.05). Data points represent group mean values. Error bars represent group Standard Error of the Mean (SEM). Asterisks (*) denote P values (pairwise comparison between proximate experimental value and control iFLU + Alum value) using the following scheme: (*) = 0.01 – 0.05, (**) = 0.001 – 0.01, (***) 0.0001 – 0.001, (****) < 0.0001. Number of animals per group: iFLU + Alum (6), iCoV2 + Alum (6), iCoV2 + RIBI (6). (b-d) Combined results from 2 independent replicates. Analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons correction (alpha = 0.05). Solid lines represent group mean values. Error bars represent group standard deviation (SD). Individual data points represent independent biological replicates. Solid horizontal lines above data represent pairwise comparisons with P values. DPI = days post-infection; ALI = Acute lung injury; DAD = Diffuse alveolar damage; iFLU = inactivated influenza virus vaccine; iCoV2 = inactivated SARS-CoV-2 vaccine; Alum = aluminum hydroxide adjuvant; RIBI = Sigma Adjuvant System adjuvant; PenH = Enhanced Pause; Rpef = Rate of Peak Expiratory Flow; TP = Time of Pause; CD4+ dep = CD4⁺ T cell depletion; WBP = Whole body plethysmography

Figure 6. CD4⁺ T helper cells promote inactivated SARS-CoV-2 + aluminum hydroxide (iCoV2 + Alum) vaccine associated enhanced respiratory disease (VAERD) during heterologous infection.

Anti-CD4 monoclonal antibody (GK1.5) was administered to mice 4-weeks post-double-boost vaccination before and during SHC014 challenge (days −5, −3 and 2 relative to challenge at day 0) to deplete CD4⁺ T helper cells (see Methods for explanation of double-boost method). Pulmonary function was measured 1–4 DPI using WBP and pulmonary tissue was collected at 5 DPI. (a) Pulmonary function of mice was measured by whole-body plethysmography before (baseline) and during SHC014 infection. PenH, Rpef, and TP were measured 1–4 DPI. (b, c) H&E-stained pulmonary specimens were analyzed by a blinded pathologist to evaluate ALI and DAD. (d) Pulmonary viral titers were quantified by plaque assay and log-transformed results reported as log₁₀ pfu. (e) Type 2 cytokine gene expression from whole-tissue pulmonary specimens was measured using quantitative RT-PCR. Results reported as fold change normalized to Gapdh expression using DDCt. (f) Eosinophils (EPX⁺ cells) were measured using immunohistochemical staining. Representative micrographs (40X magnification) from a single experiment. (a) Combined results from 2 independent replicates. Analyzed by two-way ANOVA with Tukey’s multiple comparisons test (alpha = 0.05). Data points represent group mean values. Error bars represent group Standard Error of the Mean (SEM). Asterisks (*) denote P values (pairwise comparison between proximate experimental value and control iFLU + Alum value) using the following scheme: (*) = 0.01 – 0.05, (**) = 0.001 – 0.01, (***) 0.0001 – 0.001, (****) < 0.0001. Number of animals per group: iFLU + Alum control depletion (12); iFLU + Alum CD4⁺ cell depletion (12); iCoV2 + Alum control depletion (13); iCoV2 + Alum CD4⁺ cell depletion (14). (b-f) Results from a single experiment. Analyzed by Kruskal-Wallis test with Dunn’s multiple comparisons correction (alpha = 0.05). Solid lines represent group mean values. Error bars represent group standard deviation (SD). Individual data points represent independent biological replicates. Solid horizontal lines above data represent pairwise comparisons with P values. DPI = days post-infection; ALI = Acute lung injury; DAD = Diffuse alveolar damage; iFLU = inactivated influenza virus vaccine; iCoV2 = inactivated SARS-CoV-2 vaccine; Alum = aluminum hydroxide adjuvant; RIBI = Sigma Adjuvant System adjuvant; PenH = Enhanced Pause; Rpef = Rate of Peak Expiratory Flow; TP = Time of Pause; CD4+ dep = CD4⁺ T cell depletion; WBP = Whole body plethysmography; EPX = Eosinophil peroxidase; AB/PAS = Alcian Blue Periodic Acid-Schiff