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Coronavirus disease 2019 (COVID-19) convalescents living in regions with low vaccination rates rely on post-infection immunity for protection against re-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluate humoral and Tcell immunity against five variants of concern (VOCs) in mild-COVID-19 convalescents at 12months after infection with ancestral virus. In this cohort, ancestral, receptor-binding domain (RBD)-specific antibody and circulating memory B cell levels are conserved in most individuals, and yet serum neutralization against live B.1.1.529 (Omicron) is completely abrogated and significantly reduced for other VOCs. Likewise, ancestral SARS-CoV-2-specific memory Tcell frequencies are maintained in >50% of convalescents, but the cytokine response in these cells to mutated spike epitopes corresponding to B.1.1.529 and B.1.351 (Beta) VOCs were impaired. These results indicate that increased antigen variability in VOCs impairs humoral and spike-specific Tcell immunity post-infection, strongly suggesting that COVID-19 convalescents are vulnerable and at risk of re-infection with VOCs, thus stressing the importance of vaccination programs.

Additional file 10. Symptom questionnaire sent to patients to assess presence of long COVID associated symptoms.

Additional file 1: Figure S1: Stability of anti-Spike and anti-RBD antibody titres over time. (A-E) Anti-Spike and (F-J) anti-RBD IgG, IgG1, IgG3, IgM and IgA titres plotted as a function of time. End point titres are reported as log10 area under the curve (AUC). The blue line represents the line of best fit from a linear regression analysis. The shaded areas represent the 95% confidence interval. The P value shown is from the linear regression. Red dashed lines represent the mean AUC + 2 SD in healthy controls for each isotype.

Additional file 3: Figure S3: Adjusting for differences in immune cell populations. We repeated the differential expression analysis multiple times, each time adjusting for differences in the frequency of major immune cell populations among individuals. Each panel shows the enrichment of selected pathways (same as those shown in Fig. 4F) among genes identified as being significantly up-regulated in each analysis (FDR 1.25-fold). None = no immune cell population adjustment.

Additional file 11. Flow cytometry gating strategy.

Additional file 2: Figure S2: Expression of genes in two pathways identified as downregulated in COVID-19 convalescents and healthy controls. (A) Reactome pathway R-HSA-76002 ���Platelet activation, signaling and aggregation��� and (B) KEGG pathway hsa00190 ���Oxidative phosphorylation���. Oxidative phosphorylation genes are sub-divided into nuclear and mitochondrially encoded, with the same x axis order of samples in each panel. Only differentially expressed genes (FDR 1.25-fold) within each pathway are shown. (C) Serum CRP levels in samples collected from healthy controls (HC) and COVID-19 convalescent individuals at 12, 16 and 24 weeks post infection.

Although endothelial cell (EC) infection is not widespread during dengue virus (DENV) infection in vivo, the endothelium is the site of the pathogenic effects seen in severe DENV disease. In this study, we investigated DENV infection of primary EC and defined factors that influence infection in this cell type. Consistent with in vivo findings where EC infection is infrequent, only 3%–15% of EC became productively DENV-2-infected in vitro. This low level infection could not be attributed to inhibition by heparin, EC donor variation, heterogeneity, or biological source. DENV-infection of EC was associated with induction of innate immune responses, including increased STAT1 protein, STAT1- phosphorylation, interferon (IFN)-β, OAS-1, IFIT-1/ISG56, and viperin mRNA. Antibody blocking of IFN-β inhibited the induction of OAS1, IFIT1/ISG56, and viperin while shRNA knockdown of viperin enhanced DENV-infection in EC. DENV-infection of EC resulted in increased activity of sphingosine kinase 1, a factor important in maintaining vascular integrity, and altered basal and stimulated changes in barrier integrity of DENV-infected EC monolayers. Thus, DENV productively infects only a small percentage of primary EC but this has a major influence on induction of IFN-β driven innate immune responses that can restrict infection while the EC themselves are functionally altered. These changes may have important consequences for the endothelium and are reflective of pathogenic changes associated with vascular leakage, as seen in DENV disease. Refereed/Peer-reviewed

Tumor necrosis factor alpha (TNF-α) has an antiviral role in some infections but in dengue virus (DENV) infection it is linked to severe pathology. We have previously shown that TNF-α stimulation cannot activate nuclear factor κB (NF-κB) to the fullest extent in DENV-2-infected cells. Here, we investigate further responses of DENV-2-infected cells to TNF-α, focussing particularly on cell death and pro-survival signals. TNF-α stimulation of productively DENV-2-infected monocyte-derived macrophages or HEK-293 cells induced caspase-3-mediated cell death. While TNF-α induced comparable degradation of the inhibitor of NF-κB alpha (IκB-α) and NF-κB activation in mock-infected and DENV-2-infected cells early in infection, later in infection and coinciding with TNF-α-induced cell death, TNF-α-stimulated IκB-α degradation and NF-κB activation was reduced. This was associated with reduced levels of sphingosine kinase-1 (SphK1) activity in DENV-2-infected cells; SphK1 being a known mediator of TNF-α-stimulated survival signals. Transfection experiments demonstrated inhibition of TNF-α-stimulated NF-κB activation by expression of DENV-2 capsid (CA) but enhancement by DENV-2 NS5 protein. DENV-2 CA alone, however, did not induce TNF-α-stimulated cell death or inhibit SphK1 activity. Thus, productively DENV-2-infected cells have compromised TNF-α-stimulated survival pathways and show enhanced susceptibility to TNF-α-stimulated cell death, suggesting a role for TNF-α in the killing of healthy productively DENV-2-infected cells. Additionally, the altered ability of TNF-α to activate NF-κB as infection progresses is reflected by the opposing actions of DENV-2 CA and NS5 proteins on TNF-α-stimulated NF-κB activation and could have important consequences for NF-κB-driven release of inflammatory cytokines.