Influenza vaccination reveals sex dimorphic imprints of prior mild COVID-19 [single-cell CITEseq]

Acute viral infections can have durable functional impacts on the immune system long after recovery, but how they affect homeostatic immune states and responses to future perturbations remain poorly understood. Here we use systems immunology approaches, including longitudinal multimodal single cell analysis (surface proteins, transcriptome, and V(D)J sequences), to comparatively assess baseline immune statuses and responses to influenza vaccination in 33 healthy individuals after recovery from mild, non-hospitalized COVID-19 (mean: 151 days after diagnosis) and 40 age- and sex-matched controls who never had COVID-19. At baseline and independent of time since COVID-19, recoverees had elevated T-cell activation signatures and lower expression of innate immune genes in monocytes. COVID-19-recovered males had coordinately higher innate, influenza-specific plasmablast, and antibody responses after vaccination compared to healthy male and COVID-19-recovered females, partly because male recoverees had monocytes with higher IL-15 responses early after vaccination coupled with elevated pre-vaccination frequencies of "virtual memory" like CD8+ T-cells poised to produce more IFNg upon IL-15 stimulation. In addition, the expression of the repressed innate immune genes in monocytes increased by day 1 through day 28 post-vaccination in recoverees, thus moving towards the pre-vaccination baseline of healthy controls. In contrast, these genes decreased on day 1 and returned to the baseline by day 28 in controls. Our study reveals sex-dimorphic impacts of prior mild COVID-19 and suggests that viral infections in humans can establish new immunological set-points impacting future immune responses in an antigen-agnostic manner. Blood samples were collected on 33 subjects with prior symptomatic SARS-CoV-2 infection (diagnosed by nasal PCR test) or asymptomatic infection (by antibody test), and 40 age- and sex-matched healthy controls with no history of COVID-19, at each of the following timepoints relative to seasonal influenza vaccination (day 0): days -7, 0, 1, 7, 14, 28, 70, 100. Subjects between ages 18 – 64 years were administered the Flucelvax Quadrivalent seasonal influenza vaccine (2020-2021; Seqirus Inc, Summit, NJ), whereas those aged 65 years old and above were administered the high-dose Fluzone Quadrivalent seasonal influenza vaccine (2020-2021; Sanofi Pasteur Inc, Swiftwater, PA). Peripheral blood mononuclear cells from days 0, 1, and 28 among a selected group of 24 COVID19 patients and 16 healthy controls were profiled using multi-modal single cell CITE-seq, including surface protein staining (Biolegend TotalSeq-C lyophilized panel) and mRNA, on the 10x Genomics Chromium system. Samples from different donors were pooled and different timepoints from the same donor were pooled separately so that each pool contains only one timepoint from one donor. Assignment of the individual cells to a donor and timepoint was done using a combination of SNP and antibody hashtags. SNP calls per donor were determined from bulk level RNAseq data of the individual samples. A single sample, Control, was included across the three batches as a technical control.