Breakthrough infections by SARS-CoV-2 variants boost cross-reactive hybrid immune responses in mRNA-vaccinated Golden Syrian hamsters

Abstract

Hybrid immunity (vaccination + natural infection) to SARS-CoV-2 provides superior protection to re-infection. We performed immune profiling studies during breakthrough infections in mRNA-vaccinated hamsters to evaluate hybrid immunity induction. The mRNA vaccine, BNT162b2, was dosed to induce binding antibody titers against ancestral spike, but inefficient serum virus neutralization of ancestral SARS-CoV-2 or variants of concern (VoCs). Vaccination reduced morbidity and controlled lung virus titers for ancestral virus and Alpha but allowed breakthrough infections in Beta, Delta and Mu-challenged hamsters. Vaccination primed for T cell responses that were boosted by infection. Infection back-boosted neutralizing antibody responses against ancestral virus and VoCs. Hybrid immunity resulted in more cross-reactive sera, reflected by smaller antigenic cartography distances. Transcriptomics post-infection reflects both vaccination status and disease course and suggests a role for interstitial macrophages in vaccine-mediated protection. Therefore, protection by vaccination, even in the absence of high titers of neutralizing antibodies in the serum, correlates with recall of broadly reactive B- and T-cell responses.

Fig 1. Study design and total IgG ELISA titers post vaccination.

(A) Vaccination and SARS-CoV-2 challenge study design. Syrian golden hamsters were given 5 μg of Pfizer BNT162b2 mRNA vaccine or PBS via the intramuscular route once (n = 30 hamsters/group). Blood was collected for serology 3-weeks post-vaccination. Animals were either mock-challenged or challenged with the indicated SARS-CoV-2 variants. Morbidity was monitored daily as body weight changes and lungs, spleen and blood were collected at 5 days post infection (DPI). Figure created with Biorender.com (B) USA-WA1/2020 SARS-CoV-2 S-specific IgG ELISA titers in hamster sera 3-weeks post-vaccination. Pfizer-BNT162b2 vaccinated hamsters (n = 30) are presented in grey and unvaccinated controls (n = 30) are presented in white. Values under the Limit of Detection (10²) are set to 10 for representation purposes. (C) Microneutralization assays in hamster sera 3-weeks post- vaccination against USA-WA1/2020, Alpha, Beta, Delta and Mu variants. The assay was performed with 350 TCID₅₀ per well on VeroE6/TMPRSS2 in all cases.

Fig 2. Body weight loss in hamsters with and without suboptimal BNT162b2 vaccination after challenge with USA-WA1/2020, Alpha, Beta, Delta or Mu variants.

Hamster body weight measured after a challenge from 0 to 5 DPI. (A) Body weight loss in vaccinated (n = 5) and unvaccinated (n = 5) hamsters upon mock-challenge, 10⁴ PFU/animal of (B) ancestral WA1/2020 virus, (C) Alpha variant, (D) Beta variant, (E) Delta variant, or (F) Mu variant. Statistical analysis: n = 5/group; by Mann-Whitney U test.

Fig 3. Viral titers in the lung measured via plaque assays in VeroE6/TMPRSS2 cells and variant-specific RNA reads.

(A) Viral titers. Limit of detection (LOD) = 50 PFU/mL. (B) Viral RNA reads represented as fraction (%) of total RNA reads. Statistical analysis: n = 5/group; Mann-Whitney U test.

Fig 4. T cell activation measured by IFN-γ⁺ ELISpots from splenocytes.

N-peptide: 15-mer overlapping peptides based on the Nucleoprotein (N) sequence of SARS-CoV-2. S-peptide: 15-mer overlapping peptides based on the Spike (S) sequence of SARS-CoV-2. (A) IFN-γ-releasing splenocytes per million after stimulation with S or irrelevant Hemaglutinin (HA). Statistical analysis: Mann-Whitney U test. (B) Log₁₀ of the fold induction calculated based on number of IFN-γ-releasing splenocytes when stimulated with S or N taking HA as reference.

Fig 5. Antibody neutralization activity after challenge.

(A) Micro-neutralization assays performed in the presence of 350 TCID₅₀ per well of virus and sera collected 5 DPI. In all cases, the left panel represents neutralization activity against USA-WA1/2020 (clear: unvaccinated; solid: vaccinated) and the right panel neutralization activity against the variant of infection (clear: unvaccinated; solid: vaccinated). Statistical analysis: n = 5/group; Mann-Whitney U test. (B) Antigenic map constructed with ID50 values of unvaccinated animals. Each square in the grid represents one antigenic distance unit (C) Antigenic map constructed with ID50 values of BNT162b2 primed animals. Each square in the grid represents one antigenic distance unit.

Fig 6. Lung pathology.

(A) Radar charts representing mean pathology scores, scaled 1 to 5. In all cases, scores for uninfected unvaccinated (black) and vaccinated (red) individuals are included for comparison. Mean pathology scores of challenged unvaccinated individuals are shown in blue and challenged BNT162b2 primed individuals in orange. From left to right and top to bottom: Mock vs WA1/2020. Mock vs Alpha. Mock vs Beta. Mock vs Delta. Mock vs Mu. Histological parameters: 0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe. Overall lesion scores are scaled to 0–5 for representation. (B) Section slides with Hematoxylin and Eosin staining of lungs from one animal in each experimental group.

Fig 7. Lung RNA-seq.

(A) PCA plot showing the relationship between vaccinated and unvaccinated groups challenged with different variants. For each group, centroids are calculated. (B) Pairwise comparisons of the number of up and down-regulated genes between unvaccinated and vaccinated animals challenged with each variant (adj. p<0.05). (C) Heatmap showing gene expression of the top 20 genes involved in the defense response to virus (GO:0051607). (D) Heatmap of the 1,000 most significant genes. A common legend for A, C and D panels is included. Hierarchical clustering was performed through Ward’s clustering criterion. Relevant genes from cluster 1 and 2 are highlighted on the right.

Fig 8. Abundance of different cell subsets in the lungs of infected hamsters extrapolated from the host transcriptome.

RNA was extracted from lungs of infected hamsters at 5 DPI and subjected to bulk RNA-seq analysis. Host transcriptomes were used to impute gene expression profiles using the Cibersortx algorithm. Statistical analysis: n = 5/group; Mann-Whitney U test.