High-affinity memory B cells induced by SARS-CoV-2 infection produce more plasmablasts and atypical memory B cells than those primed by mRNA vaccines.

Although both infections and vaccines induce memory B cell (MBC) populations that participate in secondary immune responses, the MBCs generated in each case can differ. Here, we compare SARS-CoV-2 spike receptor binding domain (S1-RBD)-specific primary MBCs that form in response to infection or a single mRNA vaccination. Both primary MBC populations have similar frequencies in the blood and respond to a second S1-RBD exposure by rapidly producing plasmablasts with an abundant immunoglobulin (Ig)A+ subset and secondary MBCs that are mostly IgG+ and cross-react with the B.1.351 variant. However, infection-induced primary MBCs have better antigen-binding capacity and generate more plasmablasts and secondary MBCs of the classical and atypical subsets than do vaccine-induced primary MBCs. Our results suggest that infection-induced primary MBCs have undergone more affinity maturation than vaccine-induced primary MBCs and produce more robust secondary responses.

A Replicating Single-Cycle Adenovirus Vaccine Effective against Clostridium difficile.

Clostridium difficile causes nearly 500,000 infections and nearly 30,000 deaths each year in the U.S., which is estimated to cost $4.8 billion. C. difficile infection (CDI) arises from bacteria colonizing the large intestine and releasing two toxins, toxin A (TcdA) and toxin B (TcdB). Generating humoral immunity against C. difficile's toxins provides protection against primary infection and recurrence. Thus, a vaccine may offer the best opportunity for sustained, long-term protection. We developed a novel single-cycle adenovirus (SC-Ad) vaccine against C. difficile expressing the receptor-binding domains from TcdA and TcdB. The single immunization of mice generated sustained toxin-binding antibody responses and protected them from lethal toxin challenge for up to 38 weeks. Immunized Syrian hamsters produced significant toxin-neutralizing antibodies that increased over 36 weeks. Single intramuscular immunization provided complete protection against lethal BI/NAP1/027 spore challenge 45 weeks later. These data suggest that this replicating vaccine may prove useful against CDI in humans.

Agmatine accumulation by Pseudomonas aeruginosa clinical isolates confers antibiotic tolerance and dampens host inflammation.

PURPOSE: In the cystic fibrosis (CF) airways, Pseudomonas aeruginosa undergoes diverse physiological changes in response to inflammation, antibiotic pressure, oxidative stress and a dynamic bioavailable nutrient pool. These include loss-of-function mutations that result in reduced virulence, altered metabolism and other phenotypes that are thought to confer a selective advantage for long-term persistence. Recently, clinical isolates of P. aeruginosa that hyperproduce agmatine (decarboxylated arginine) were cultured from individuals with CF. Sputum concentrations of this metabolite were also shown to correlate with disease severity. This raised the question of whether agmatine accumulation might also confer a selective advantage for P. aeruginosa during chronic colonization of the lung. METHODOLOGY AND RESULTS: We screened a library of P. aeruginosa CF clinical isolates and found that ~5 % of subjects harboured isolates with an agmatine hyperproducing phenotype. Agmatine accumulation was a direct result of mutations in aguA, encoding the arginine deiminase that catalyses the conversion of agmatine into various polyamines. We also found that agmatine hyperproducing isolates (aguA-) had increased tolerance to the cationic antibiotics gentamicin, tobramycin and colistin relative to their chromosomally complemented strains (aguA+). Finally, we revealed that agmatine diminishes IL-8 production by airway epithelial cells in response to bacterial infection, with a consequent decrease in neutrophil recruitment to the murine airways in an acute pneumonia model. CONCLUSION: These data highlight a potential new role for bacterial-derived agmatine that may have important consequences for the long-term persistence of P. aeruginosa in the CF airways.

Generation of 13C-Labeled MUC5AC Mucin Oligosaccharides for Stable Isotope Probing of Host-Associated Microbial Communities.

Stable isotope probing (SIP) has emerged as a powerful tool to address key questions about microbiota structure and function. To date, diverse isotopically labeled substrates have been used to characterize in situ growth activity of specific bacterial taxa and have revealed the flux of bioavailable substrates through microbial communities associated with health and disease. A major limitation to the growth of the field is the dearth of biologically relevant "heavy" labeled substrates. Mucin glycoproteins, for example, comprise an abundant source of carbon in the gut, oral cavity, respiratory tract, and other mucosal surfaces but are not commercially available. Here, we describe a method to incorporate a 13C-labeled monosaccharide into MUC5AC, a predominant mucin in both gastrointestinal and airway environments. Using the lung adenocarcinoma cell line, Calu-3, polarized cell cultures grown in 13C-labeled d-glucose resulted in liberal mucin production on the apical surface. Mucins were isolated by size-exclusion chromatography, and O-linked glycans were released by ß-elimination, permethylated, and analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) techniques. We demonstrate a 98.7% incorporation of 13C in the heterogeneous O-linked oligosaccharides that make up >80% of mucin dry weight. These "heavy" labeled glycoproteins represent a valuable tool for probing in vivo activity of host-associated bacterial communities and their interactions with the mucosal barrier. The continued expansion of labeled substrates for use in SIP will eventually allow bacterial taxa that degrade host compounds to be identified, with long-term potential for improved health and disease management.