The presence of broadly neutralizing anti-SARS-CoV-2 RBD antibodies elicited by primary series and booster dose of COVID-19 vaccine.

Antibody-mediated immunity plays a key role in protection against SARS-CoV-2. We characterized B-cell-derived anti-SARS-CoV-2 RBD antibody repertoires from vaccinated and infected individuals and elucidate the mechanism of action of broadly neutralizing antibodies and dissect antibodies at the epitope level. The breadth and clonality of anti-RBD B cell response varies among individuals. The majority of neutralizing antibody clones lose or exhibit reduced activities against Beta, Delta, and Omicron variants. Nevertheless, a portion of anti-RBD antibody clones that develops after a primary series or booster dose of COVID-19 vaccination exhibit broad neutralization against emerging Omicron BA.2, BA.4, BA.5, BQ.1.1, XBB.1.5 and XBB.1.16 variants. These broadly neutralizing antibodies share genetic features including a conserved usage of the IGHV3-53 and 3-9 genes and recognize three clustered epitopes of the RBD, including epitopes that partially overlap the classically defined set identified early in the pandemic. The Fab-RBD crystal and Fab-Spike complex structures corroborate the epitope grouping of antibodies and reveal the detailed binding mode of broadly neutralizing antibodies. Structure-guided mutagenesis improves binding and neutralization potency of antibody with Omicron variants via a single amino-substitution. Together, these results provide an immunological basis for partial protection against severe COVID-19 by the ancestral strain-based vaccine and indicate guidance for next generation monoclonal antibody development and vaccine design.

High Prevalence of Carbapenem-Resistant Enterobacterales Producing OXA-48 among Carbapenem-Resistant Isolates in a Regional Hospital in Central Taiwan.

In response to the increasing number of carbapenem-resistant Enterobacterales (CRE), we investigated carbapenemase-producing Klebsiella pneumoniae and non-K. pneumoniae epidemiology and genetics. We collected 76 clinical Enterobacterales and 4 stool surveillance Escherichia coli isolates resistant to ertapenem or imipenem. Using polymerase chain reaction (PCR) and DNA sequencing, we assessed carbapenemases, extended-spectrum ß-lactamases, and AmpC ß-lactamases. Molecular typing via pulsed-field gel electrophoresis (PFGE) and conjugation experiments were conducted to examine resistance gene transfer. Among the 80 isolates, 96.2% harbored at least one carbapenemase gene, with blaOXA-48 in 87.5%. KPC-2 and IMP-8 carbapenemases were found in 15.0 and 22.5% of the isolates, respectively, with 27.5% having 2 or more carbapenemase genes. The PFGE analysis revealed the presence of diverse genotypes. PCR-based plasmid replicon typing identified IncA/C as the most prevalent type among K. pneumoniae isolates (26/29), and IncF and IncFIB among E. coli isolates (22/28). Conjugal transfer was successful for plasmids encoding OXA-48, CTX-M-3, CTX-M-14, CMY-2, and other ß-lactamases, except the KPC-2 gene. In conclusion, our study highlights high carbapenemase prevalence in CRE, primarily OXA-48. Multiple carbapenemases within strains were common, and PFGE showed diverse patterns in these carbapenem-resistant isolates.

Structure of the heterotrimeric membrane protein complex FtsB-FtsL-FtsQ of the bacterial divisome.

The synthesis of the cell-wall peptidoglycan during bacterial cell division is mediated by a multiprotein machine, called the divisome. The essential membrane protein complex of FtsB, FtsL and FtsQ (FtsBLQ) is at the heart of the divisome assembly cascade in Escherichia coli. This complex regulates the transglycosylation and transpeptidation activities of the FtsW-FtsI complex and PBP1b via coordination with FtsN, the trigger for the onset of constriction. Yet the underlying mechanism of FtsBLQ-mediated regulation is largely unknown. Here, we report the full-length structure of the heterotrimeric FtsBLQ complex, which reveals a V-shaped architecture in a tilted orientation. Such a conformation could be strengthened by the transmembrane and the coiled-coil domains of the FtsBL heterodimer, as well as an extended ß-sheet of the C-terminal interaction site involving all three proteins. This trimeric structure may also facilitate interactions with other divisome proteins in an allosteric manner. These results lead us to propose a structure-based model that delineates the mechanism of the regulation of peptidoglycan synthases by the FtsBLQ complex.

Structural basis for a conserved neutralization epitope on the receptor-binding domain of SARS-CoV-2.

Antibody-mediated immunity plays a crucial role in protection against SARS-CoV-2 infection. We isolated a panel of neutralizing anti-receptor-binding domain (RBD) antibodies elicited upon natural infection and vaccination and showed that they recognize an immunogenic patch on the internal surface of the core RBD, which faces inwards and is hidden in the "down" state. These antibodies broadly neutralize wild type (Wuhan-Hu-1) SARS-CoV-2, Beta and Delta variants and some are effective against other sarbecoviruses. We observed a continuum of partially overlapping antibody epitopes from lower to upper part of the inner face of the RBD and some antibodies extend towards the receptor-binding motif. The majority of antibodies are substantially compromised by three mutational hotspots (S371L/F, S373P and S375F) in the lower part of the Omicron BA.1, BA.2 and BA.4/5 RBD. By contrast, antibody IY-2A induces a partial unfolding of this variable region and interacts with a conserved conformational epitope to tolerate all antigenic variations and neutralize diverse sarbecoviruses as well. This finding establishes that antibody recognition is not limited to the normal surface structures on the RBD. In conclusion, the delineation of functionally and structurally conserved RBD epitopes highlights potential vaccine and therapeutic candidates for COVID-19.

Characterization of the Genetic Background of KPC-2-Producing Klebsiella pneumoniae with Insertion Elements Disrupting the ompK36 Porin Gene.

Carbapenemase-producing combined porin loss is one of the primary mechanisms for carbapenem resistance. Although mutations in ompK35 and ompK36 genes have often been identified in carbapenem-resistant Klebsiella pneumoniae, reports on the porin protein gene disruption by insertion sequence (IS) elements are varied. The ompK36 porin protein gene disruption by IS elements and OmpK36 production loss in six blaKPC-2-carrying K. pneumoniae isolates were detected in this study. IS903, ISEc68, and IS1 insertions were noted in the 3, 2, and 1 isolates, respectively. The six K. pneumoniae isolates showed five different pulsed-field gel electrophoresis patterns and belonged to four multilocus sequence typing types, ST4, ST11, ST15, and ST39. This study increases our understanding of the genetic background of KPC-2 carbapenemases in porin-defective clinical isolates and the contribution of OmpK36 production loss to carbapenem resistance.