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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.

A vaccine that induces potent, broad and sustained cell-mediated immunity, resulting in effective memory has the potential to restrict hepatitis C (HCV) virus infection. Early, multi-functional CD4+ and CD8+ T cell responses against non-structural protein 3 (NS3) have been associated with HCV clearance. Necrotic cells generate strong immune responses and represent a major antigenic source used by dendritic cells (DC) for processing and presentation, but there is conflicting evidence as to their immunogenicity in vaccination. Immunization with DC loaded with viral antigens has been done in the past, but to date the immunogenicity of live vs. necrotic DC vaccines has not been investigated. We developed a DC2.4 cell line stably expressing HCV NS3, and compared the NS3-specific responses of live vs. necrotic NS3 DC. Vaccination of mice with necrotic NS3 DC increased the breadth of T-cell responses and enhanced the production of IL-2, TNF-α, and IFN-γ by effector memory CD4+ and CD8+T cells, compared to mice vaccinated with live NS3 DC. A single dose of necrotic NS3 DC vaccine induced a greater influx and activation of cross-presenting CD11c+ CD8α+ DC and necrosis-sensing Clec9A+ DC in the draining lymph nodes. Furthermore, using a hydrodynamic challenge model necrotic NS3 DC vaccination resulted in enhanced clearance of NS3-positive hepatocytes from the livers of vaccinated mice. Taken together, the data demonstrate that necrotic DC represent a novel and exciting vaccination strategy capable of inducing broad and multifunctional T cell memory.