Robust Virus-Specific Adaptive Immunity in COVID-19 Patients with SARS-CoV-2 Δ382 Variant Infection

Abstract

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that have become dominant as the pandemic progresses bear the ORF8 mutation together with multiple spike mutations. A 382-nucleotide deletion (Δ382) in the ORF7b and ORF8 regions has been associated with milder disease phenotype and less systemic inflammation in COVID-19 patients. However, its impact on host immunity against SARS-CoV-2 remains undefined. Here, RNA-sequencing was performed to elucidate whole blood transcriptomic profiles and identify contrasting immune signatures between patients infected with either wildtype or Δ382 SARS-CoV-2 variant. Interestingly, the immune landscape of Δ382 SARS-CoV-2 infected patients featured an increased adaptive immune response, evidenced by enrichment of genes related to T cell functionality, a more robust SARS-CoV-2-specific T cell immunity, as well as a more rapid antibody response. At the molecular level, eukaryotic initiation factor 2 signaling was found to be upregulated in patients bearing Δ382, and its associated genes were correlated with systemic levels of T cell-associated and pro-inflammatory cytokines. This study provides more in-depth insight into the host-pathogen interactions of ORF8 with great promise as a therapeutic target to combat SARS-CoV-2 infection.

Keywords: Adaptive immune response; Antibody response; CD4+ T cell response; CD8+ T cell response; COVID-19; ORF8; SARS-CoV-2; Transcriptome.

Fig. 1

Whole blood transcriptome analysis in COVID-19 patients. RNA-seq of whole blood from COVID-19 patients (n = 25) at acute (SARS-CoV-2 PCR-positive, median 8 days PIO) and recovered (SARS-CoV-2 PCR-negative, median 21 days PIO) phases and healthy controls (n = 6) was performed. a Volcano plot indicating DEGs between blood samples collected at acute and recovered phases in patients with WT SARS-CoV-2 infection (n = 13), with thresholds of p-value < 0.01 and |FC|> 2. Numbers of over-expressed and under-expressed genes are indicated. b Top IPA canonical pathways showing differential expression of genes related to IFN and PRR signaling in WT SARS-CoV-2 infection. Pathways are ranked by − log(p-value), and the color scheme is based on predicted activation Z-scores, with activation in red and undetermined directionality in gray. DEGs related to the IFN pathway are indicated on the bar graph. c Heatmap of DEGs related to IFN and PRR signaling between COVID-19 patients infected with WT (n = 14) and Δ382 SARS-CoV-2 (n = 11) at acute and recovered phases and healthy controls. Heatmap is scaled based on log₂RPKM values, with blue and red indicating low and high expressions, respectively. WT, wildtype; DEGs, differentially expressed genes; FC, fold change; FDR, false discovery rate; PCR, polymerase chain reaction; PIO, post-illness onset; HC, healthy controls; RPKM, reads per kilobase per million reads mapped; IFN, interferon; PRR, pattern recognition receptor

Fig. 2

Effects of 382-nt deletion in SARS-CoV-2 ORF8 genome (Δ382) on whole blood transcriptome of COVID-19 patients. RNA-seq of whole blood from COVID-19 patients infected with WT (n = 14) and Δ382 SARS-CoV-2 (n = 11) at the acute phase of infection (SARS-CoV-2 PCR-positive; median 8 days PIO) was performed. Only samples with RNA integrity number > 6 were sent for sequencing and included in the analysis a PCA of COVID-19 patients and healthy controls based on DEGs, with p-value < 0.01 and |FC|> 2. b Heatmap of 358 DEGs, scaled based on log₂RPKM values, with blue and red colors indicating low and high expressions, respectively. c Top canonical pathways and upstream regulators identified by IPA based on the DEGs. Bar graphs are ranked by significance, with red indicating positive predicted activation Z-scores and gray indicating undetermined directionality. d An integrated network of HSP90B1 and TCR and their targeted genes. Stimulation of HSP90B1 and TCR leads to overexpression of the downstream genes. e GO pathway term enrichment networks of DEGs using Cytoscape add-on ClueGO. Each of the GO terms is statistically significant (Benjamini–Hochberg correction < 0.05). The filled colored circles (nodes) represent a statistically significant enriched parent GO term. The lines (edges) between nodes show overlapping genes within terms, with node size representing the term enrichment significance. The overview chart shows the distribution of the functionally grouped GO terms. The cut-off for terms in the functionally grouped networks was set at p-value < 0.05. WT, wildtype; PCA, principal component analysis; DEGs, differentially expressed genes; FC, fold change; RPKM, reads per kilobase per million reads mapped; PCR, polymerase chain reaction; IPA, Ingenuity Pathway Analysis; HSP90B1, heat shock protein 90 kDa beta member 1; TCR, T cell receptor; GO, gene ontology

Fig. 3

Effects of 382-nt deletion in SARS-CoV-2 ORF8 genome on immune responses in COVID-19 patients. Transcriptomic and cytokine profiles of COVID-19 patients infected with WT (n = 14) or Δ382 SARS-CoV-2 (n = 11) at the acute phase of infection (SARS-CoV-2 PCR-positive; median 8 days PIO) were analyzed. a Expressions of genes associated with granulocytes, monocytes, lymphocytes, cytokines and T/NK cell functionality were compared between COVID-19 patients infected with WT or Δ382 SARS-CoV-2. Heatmaps of the DEGs, scaled based on log₂FC values, with blue and red colors indicating low and high expressions, respectively. b Plasma immune mediator levels of COVID-19 patients infected with WT (n = 14) or Δ382 SARS-CoV-2 (n = 11) at the acute phase of infection (SARS-CoV-2 PCR-positive; median 8 days PIO) and profiles of significant immune mediators are illustrated as scatter plots and shown as mean. Mann–Whitney U tests were conducted on the logarithmically transformed concentration values (*p < 0.05; **p < 0.01; ***p < 0.001). Respective mean concentrations of immune mediators from healthy controls (HC; n = 23) are indicated as black dotted lines. Patient samples with concentrations out of measurement range are presented as the logarithmically transformed value of LOQ and indicated as blue dotted lines. c Association between elF2 signaling and immune signatures in Δ382 SARS-CoV-2 infection. Spearman’s correlation matrix for the genes associated with eIF2 signaling, T cell functionality, neutrophil activation and plasma cytokines. Colors represent the Spearman correlation coefficients (rho) between the expression of genes related to eIF2 signaling and genes or immune mediators associated with different immune phenotypes. FC, fold change, WT, wildtype; PCR, polymerase chain reaction; PIO, post-illness onset; DEGs, differentially expressed genes; LOQ, limit of quantification

Fig. 4

Effects of 382-nt deletion in SARS-CoV-2 ORF8 genome on adaptive immune responses to COVID-19. a SARS-CoV-2 specific CD4⁺ and CD8⁺ non-T follicular helper (TFH) cells were characterized with flow cytometry-based on the expression of IFN-γ, IL-2, and TNF-⍺ upon SARS-CoV-2 peptide stimulation in WT (n = 14) or Δ382 SARS-CoV-2 (n = 14) infected patients. Statistical analyses were performed with the Mann–Whitney U test (**p < 0.01). b Antibody responses in Δ382 SARS-CoV-2 infected patients. Spike protein-specific antibody response was characterized using an S-flow assay. Plasma samples of COVID-19 patients infected with either WT (n = 20) or Δ382 SARS-CoV-2 (n = 30) were screened at 1:100 dilution against cells expressing the full-length SARS-CoV-2 spike protein, with healthy donors (n = 22) screened in parallel. IgM and IgG profiles of COVID-19 patients at timepoints ≤ 7, 8 to 14, and 15 to 30 days PIO are illustrated as violin plots. The dotted line indicates the mean + 3SD of healthy donors. Data are shown as mean ± SD of two independent experiments. For determination of anti-peptide IgG responses, plasma samples o COVID-19 patients infected with either WT (n = 20) or Δ382 SARS-CoV-2 (n = 30) were tested at 1:1000 dilution on an IgG ELISA against SARS-CoV-2 spike epitopes S21P2 and S14P5. Healthy controls (n = 22) were screened in parallel. Antibody profiles of COVID-19 patients at timepoints ≤ 7, 8 to 14, 15 to 30 days PIO are illustrated as violin plots. The dotted line indicates the mean + 3SD of healthy controls. Data are shown as mean ± SD of two independent experiments. Statistical analysis was carried out using Mann–Whitney U tests (*p < 0.05). WT, wildtype

Fig. 5

Molecular mechanisms underlying the milder disease phenotype in Δ382 SARS-CoV-2 infections. A SARS-CoV-2 variant with a 382-nucleotide deletion (Δ382) truncates ORF7b and removes the ORF8 transcription-regulatory sequence, eliminating ORF8 transcription. The ORF8 382-nt deletion has recently been associated with a milder disease phenotype. The attenuation of SARS-CoV-2 ORF8 upregulates eIF2 signaling and cellular stress responses at the acute phase of infection, potentially interrupting the downregulation of MHC-I molecules by ORF8 and also enhances the activation of both CD4⁺ and CD8⁺ T cells, evidenced by enrichment of effector cytotoxic genes and upregulation of SARS-CoV-2 specific T cell responses in Δ382 SARS-CoV-2 infected patients. Enhanced T cell responses may in turn mediate rapid and effective antibody responses in Δ382 SARS-CoV-2 infection. More pronounced cellular stress responses may further reduce systemic inflammation and dysfunctional neutrophils in Δ382 SARS-CoV-2 infected patients. Overall, the attenuation of SARS-CoV-2 ORF8 produced a molecular phenotype characterized by more pronounced cellular stress responses and a less dysregulated immune phenotype with more robust T and B cell responses. ORF, open reading frame; eIF2, eukaryotic initiation factor 2; MHC-I, major histocompatibility complex 1; CD, cluster of differentiation