Live vaccines have long been known to trigger far more vigorous immune responses than their killed counterparts1–6. This has been attributed to the ability of live microorganisms to replicate and express specialized virulence factors that facilitate invasion and infection of their hosts7. However, protective immunization can often be achieved with a single injection of live, but not dead, attenuated microorganisms stripped of their virulence factors. Pathogen associated molecular patterns (PAMPs), which serve to alert the immune system8,9, are present in both live and killed vaccines, suggesting that certain poorly characterized aspects of live microorganisms, not incorporated in dead vaccines, are particularly effective at inducing protective immunity. Here we show that the innate immune system can directly sense microbial viability through detection of a special class of viability-associated PAMPs (vita-PAMPs). We identify prokaryotic messenger RNA (mRNA) as a vita-PAMP present only in viable bacteria, recognition of which elicits a unique innate response and a robust adaptive antibody response. Notably, the innate response evoked by viability and prokaryotic mRNA was thus far considered to be reserved for pathogenic bacteria, but we show that even nonpathogenic bacteria in sterile tissues can trigger similar responses, provided they are alive. Thus, the immune system actively gauges the infectious risk by scouring PAMPs for signatures of microbial life and thus infectivity. Detection of vita-PAMPs triggers an alert mode not warranted for dead bacteria. Vaccine formulations that incorporate vita-PAMPs could thus combine the superior protection of live vaccines with the safety of dead vaccines.

Two elementary parameters for quantifying viral infection and shedding are viral load and whether samples yield a replicating virus isolate in cell culture. We examined 25,381 German SARS-CoV-2 cases, including 6110 from test centres attended by pre-symptomatic, asymptomatic, and mildly-symptomatic (PAMS) subjects, 9519 who were hospitalised, and 1533 B.1.1.7 lineage infections. The youngest had mean log10 viral load 0.5 (or less) lower than older subjects and an estimated ~78% of the peak cell culture replication probability, due in part to smaller swab sizes and unlikely to be clinically relevant. Viral loads above 109 copies per swab were found in 8% of subjects, one-third of whom were PAMS, with mean age 37.6. We estimate 4.3 days from onset of shedding to peak viral load (8.1) and cell culture isolation probability (0.75). B.1.1.7 subjects had mean log10 viral load 1.05 higher than non-B.1.1.7, with estimated cell culture replication probability 2.6 times higher.

SummaryLymphocytes circulate through lymph nodes (LN) in search for antigen in what is believed to be a continuous process. Here, we show that lymphocyte migration through lymph nodes and lymph occurred in a non-continuous, circadian manner. Lymphocyte homing to lymph nodes peaked at night onset, with cells leaving the tissue during the day. This resulted in strong oscillations in lymphocyte cellularity in lymph nodes and efferent lymphatic fluid. Using lineage-specific genetic ablation of circadian clock function, we demonstrated this to be dependent on rhythmic expression of promigratory factors on lymphocytes. Dendritic cell numbers peaked in phase with lymphocytes, with diurnal oscillations being present in disease severity after immunization to induce experimental autoimmune encephalomyelitis (EAE). These rhythms were abolished by genetic disruption of T cell clocks, demonstrating a circadian regulation of lymphocyte migration through lymph nodes with time-of-day of immunization being critical for adaptive immune responses weeks later.