MZB1 expression levels are associated with long-term maintenance of high anti-HBs titers: single-cell RNA-seq with validation by nested case-control and intervention studies.

Background: Approximately 5-10% of healthy, immunocompetent individuals are low- or non-responders to the HepB vaccine. The mechanism remains obscured. Among responders, immunity may wane over time, becoming undetectable in some. However, in some vaccinees, high anti-HBs titers persist for decades. Investigating the underlying causes may in turn provide clues to solving low- or non-response. Methods: Single-cell RNA sequencing of blood cells was performed on ten subjects maintaining high anti-HBs titers (>1000 mIU/ml) for over 30 years post-vaccination and ten subjects who had seroconverted. The validation included a nested case-control study (n=102) and an interventional study (n=79). Subjects in the latter study received the HepB vaccine (20 μg) according to a 0-1-6 schedule. Blood samples were collected pre-vaccination, first-and -third dose. Results: MZB1 expression was significantly upregulated in the B cells of subjects with high anti-HBs titers (P.adj=2.1e-12). Subjects with long-term maintenance of high anti-HBs titers had significantly higher serum MZB1 concentrations (7.1 ± 2.2 ng/mL) than anti-HBs-negative subjects (0.96 ± 0.1 ng/mL) (p < 0.0001). High concentration of MZB1 correlated strongly with a high titer of anti-HBs (r=0.892). The HepB vaccine induces MZB1 prior to anti-HBs. Among individuals who only achieved anti-HBs positivity after the third dose, MZB1 concentrations were already significantly elevated following the first dose (p=0.01) despite still being negative for anti-HBs at that point. The higher the concentration of serum MZB1 protein rises, the better the response to HepB vaccine. The pre-vaccination MZB1 concentrations of non-responders did not increase significantly post-third dose (p=0.548). Conclusions: Upregulated expression of MZB1 is associated with long-term maintenance of high anti-HBs titers. Serum MZB1 levels are associated with the magnitude of responses to the HepB vaccine, suggesting that serum MZB1 is a valuable predictive biomarker for anti-HBs responses. Further investigation into MZB1 and its upstream regulatory pathways may inform the development of novel adjuvants aimed at achieving sustained antibody titers, which could potentially offer insights into overcoming low or non-responses.