Body fluids are constantly replenished with a population of genetically diverse cell-free DNA (cfDNA) fragments, representing a vast reservoir of information reflecting real-time changes in the host and metagenome. As many body fluids can be collected non-invasively in a one-off and serial fashion, this reservoir can be tapped to develop assays for the diagnosis, prognosis, and monitoring of wide-ranging pathologies, such as solid tumors, fetal genetic abnormalities, rejected organ transplants, infections, and potentially many others. The translation of cfDNA research into useful clinical tests is gaining momentum, with recent progress being driven by rapidly evolving preanalytical and analytical procedures, integrated bioinformatics, and machine learning algorithms. Yet, despite these spectacular advances, cfDNA remains a very challenging analyte due to its immense heterogeneity and fluctuation in vivo. It is increasingly recognized that high-fidelity reconstruction of the information stored in cfDNA, and in turn the development of tests that are fit for clinical roll-out, requires a much deeper understanding of both the physico-chemical features of cfDNA and the biological, physiological, lifestyle, and environmental factors that modulate it. This is a daunting task, but with significant upsides. In this review we showed how expanded knowledge on cfDNA biology and faithful reverse-engineering of cfDNA samples promises to (i) augment the sensitivity and specificity of existing cfDNA assays; (ii) expand the repertoire of disease-specific cfDNA markers, thereby leading to the development of increasingly powerful assays; (iii) reshape personal molecular medicine; and (iv) have an unprecedented impact on genetics research.

Neutrophil granulocytes are a central component of the innate immune system. In recent years, they have gained considerable attention due to newly discovered biological effector functions and their involvement in various pathological conditions. They have been shown to trigger mechanisms that can either promote or inhibit the development of autoimmunity, thrombosis, and cancer. One mechanism for their modulatory effect is the release of extracellular vesicles (EVs), that trigger appropriate signaling pathways in immune cells and other target cells. In addition, activated neutrophils can release bactericidal DNA fibers decorated with proteins from neutrophil granules (neutrophil extracellular traps, NETs). While NETs are very effective in limiting pathogens, they can also cause severe damage if released in excess or cleared inefficiently. Since NETs and EVs share a variety of neutrophil molecules and initially act in the same microenvironment, differential biochemical and functional analysis is particularly challenging. This review focuses on the biochemical and functional parallels and the extent to which the overlapping spectrum of effector molecules has an impact on biological and pathological effects.

Background: Systematic studies providing differentiated insight into the contribution of immunity directed against conserved and non-conserved epitopes of SARS-CoV-2 Spike on long-term protection are rare and insufficiently guide future pan-variant vaccine research. The present observational cohort study aimed to evaluate the correlation of neutralizing antibody levels and cellular immunity against the Spike protein with symptomatic Omicron breakthrough infection. Methods: Neutralizing antibody levels against multiple (sub)variants were analyzed 6 months following the second wild-type mRNA vaccination and 6 months after booster in 107 subjects using a multiplex surrogate virus neutralization assay. To assess cellular immunity, cytokine mRNA expression levels were determined after peptide pool stimulation in whole blood samples of a study subgroup. Results: Neutralizing antibody titers were found to serve as a reasonably reliable correlate of protection prior to booster immunization. However, the predictive power of neutralizing antibody titers was diminished after boosting. This loss appears to be due to a critical remodeling of the antibody repertoire—a process that was dose-dependent on pre-boost humoral immunity. Vaccination against Omicron infection was most effective when a balanced immune response to both conserved and non-conserved epitopes of the viral Spike protein was induced. While neutralizing antibodies against receptor-binding domain epitopes affected by mutations were specifically associated with protection from symptomatic variant infection, cellular immunity was most effective when targeting conserved Spike epitopes. Conclusions: Optimal long-term protection against Omicron infection requires balanced immunity to both conserved and non-conserved epitopes of the viral Spike protein. The limited availability of cross-neutralizing antibodies targeting non-conserved epitopes and their inherently lower efficacy renders them a limiting factor as humoral immunity wanes over time. Future pan-SARS-CoV-2 variant vaccines that primarily target conserved epitopes may therefore provide less effective long-term protection against symptomatic variant infection than vaccines targeting a broader epitope spectrum including both conserved and non-conserved epitopes.

The objective of this study was to investigate the features of immune protection against SARS-CoV-2 infection in a single cohort during the 6–17 months following booster immunization with an mRNA-based vaccine. The results illustrate the influence of humoral and cellular immunity on the efficacy of the vaccine. Notably, neutralizing antibody titers were found to serve as a reasonably reliable correlate of protection prior to booster immunization. However, this predictive power was largely lost after boosting. The loss appears to be due to the critical remodeling of the humoral immune response following booster immunization. Our findings support the hypothesis that immunity to both conserved and non-conserved epitopes of the viral Spike protein's receptor-binding domain (RBD) is crucial for optimal long-term protection against Omicron infection. While immunity to conserved epitopes may provide cross-variant protection, antibodies targeting non-conserved RBD epitopes play a pivotal role in achieving maximum protection. These observations highlight the critical role of repeated immunization in shaping the immune response landscape and reinforce the necessity of considering both humoral and cellular immune components, alongside intended use considerations, when assessing vaccine efficacy and developing future immunization strategies.