Influenza vaccination reveals and partly reverses sex dimorphic immune imprints associated with prior mild COVID-19

Viral infections can have profound and durable functional impacts on the immune system. There is an urgent need to characterize the long-term immune effects of SARS-CoV-2 infection given the persistence of symptoms in some individuals and the continued threat of novel variants including the recent rapid acceleration in infections. As the majority of COVID-19 patients experienced mild disease, here we use systems immunology approaches to comparatively assess the post-infection immune statuses (mean: 151 [5th – 95th percentile: 58 – 235] days after diagnosis) and subsequent innate and adaptive responses to seasonal influenza vaccination (as an “immune challenge”) in 33 previously healthy individuals after recovery from mild, non-hospitalized COVID-19, against 40 age- and sex-matched healthy controls with no history of COVID-19. Sex-specific, temporally stable shifts in signatures of metabolism, T-cell activation, and innate immune/inflammatory processes suggest that mild COVID-19 can establish new, stable post-infection immunological set-points. Strikingly, COVID-19-recovered males had more robust innate, influenza-specific plasmablast, and antibody responses after vaccination, as well as an increase in CD71hi B-cells (including influenza-specific subsets) before vaccination compared to healthy males. Intriguingly, by day 1 post-vaccination in COVID-19-recovered subjects, the expression of numerous innate defense/immune receptor genes (e.g., Toll-like receptors) in monocytes increased and “shifted” away from their post-COVID-19 repressed state toward that of healthy controls at baseline (before vaccination), and these changes tended to persist to day 28 especially in females, hinting that the acute inflammatory responses induced by vaccination could partly reset the dysregulated immune states established by prior mild COVID-19. Our study reveals sex-dimorphic immune imprints and in vivo functional impacts of mild COVID-19, suggesting that prior COVID-19 could shift future responsiveness to vaccination and in turn, vaccines could help retrain the immune system back towards normality after COVID-19, both in antigen-agnostic manners. Overall design: 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). Samples from days -7, 0, 1, 7, and 28 were selected and drawn directly into the Tempus™ Blood RNA Tube (Thermo Fisher Scientific, Waltham, MA) according to manufacturer's protocol. The RNA samples were isolated in groups of 12-22 samples per batch based on age, gender, and patient group. Each RNA isolation batch had a technical control, which was tempus blood collected from one healthy donor, who was also a part of this study. RNA from this sample was isolated with almost every batch except for a few due to technical issues. For each sample, 500ng of total RNA was used to isolate mRNA via poly(A) selection. All libraries were sequenced on the NovaSeq 6000 instrument across five plates. A total 11 samples were re-sequenced on a NextSeq 500 instrument to replace some of the initial samples with low output. Technical replicates were placed on each plate to control for plate variability.