Germinal center dynamics in the mediastinal lymph node distinguish time-dependent and variant-specific immunity during SARS-CoV-2 secondary exposure

Abstract SARS-CoV-2 secondary infections remain a persistent challenge even though individuals have previous immunity from natural infection or vaccination. Yet, the cellular mechanisms that determine whether a secondary exposure is rapidly controlled by pre-existing immunity and results in sterilizing protection or progresses to productive infection and disease remain poorly understood. Using homologous and heterologous secondary-challenge experimental strategies in C57BL/6J mice, we show that protection against a homologous SARS-CoV-2 Alpha secondary challenge in previously infected mice is time-limited, remaining durable through 56 days post-primary infection, with breakthrough infection on day 84 post-primary infection. This loss of protection was not associated with waning neutralizing antibody titers but instead correlated with progressive contraction of T follicular helper cells and germinal center B cells in the mediastinal lymph node. In contrast, heterologous Beta secondary exposure in Alpha-previously infected mice consistently failed to mount a robust germinal center response despite mobilizing comparable or greater numbers of immune cells than homologous re-challenge. Instead, RNA-seq and flow cytometry analyses revealed that this response was associated with an inflammatory extrafollicular B cell response characterized by reduced Bcl6 and Aicda expression and concomitant robust upregulation of Il6 . Taken together, these findings identify the germinal center recall response in the mediastinal lymph node as an important cellular correlate of SARS-CoV-2 secondary immunity and carry direct implications for the timing of booster immunization and the design of cross-variant vaccines.