Multivalent O-antigen polysaccharide glycoconjugate vaccines are under development to prevent invasive infections caused by pathogenic Enterobacteriaceae . Sequence type 131 (ST131) Escherichia coli of serotype O25b has emerged as the predominant lineage causing invasive multidrug-resistant extraintestinal pathogenic E. coli (ExPEC) infections. We observed the prevalence of E. coli O25b ST131 among a contemporary collection of isolates from U.S. bloodstream infections from 2013 to 2016 ( n = 444) and global urinary tract infections from 2014 to 2017 ( n = 102) to be 25% and 24%, respectively.
Extraintestinal pathogenic Escherichia coli (ExPEC) is a major health concern due to emerging antibiotic resistance. Along with O1A, O2, and O6A, E. coli O25B is a major serotype within the ExPEC group, which expresses a unique O -antigen. Clinical studies with a glycoconjugate vaccine of the above-mentioned O -types revealed O25B as the least immunogenic component, inducing relatively weak IgG titers. To evaluate the immunological properties of semisynthetic glycoconjugate vaccine candidates against E. coli O25B, we here report the chemical synthesis of an initial set of five O25B glycan antigens differing in length, from one to three repeat units, and frameshifts of the repeat unit. The oligosaccharide antigens were conjugated to the carrier protein CRM 197 . The resulting semisynthetic glycoconjugates induced functional IgG antibodies in mice with opsonophagocytic activity against E. coli O25B. Three of the oligosaccharide–CRM 197 conjugates elicited functional IgGs in the same order of magnitude as a conventional CRM 197 glycoconjugate prepared with native O25B O -antigen and therefore represent promising vaccine candidates for further investigation. Binding studies with two monoclonal antibodies (mAbs) revealed nanomolar anti-O25B IgG responses with nanomolar K D values and with varying binding epitopes. The immunogenicity and mAb binding data now allow for the rational design of additional synthetic antigens for future preclinical studies, with expected further improvements in the functional antibody responses. Moreover, acetylation of a rhamnose residue was shown to be likely dispensable for immunogenicity, as a deacylated antigen was able to elicit strong functional IgG responses. Our findings strongly support the feasibility of a semisynthetic glycoconjugate vaccine against E. coli O25B.
Single-cell RNA sequencing can to resolve transcriptional features from large numbers of individual immune cells, but techniques capable of resolving the variable regions of B cell receptors (BCR) – defining features that confer antigen specificity to B cells – remain limited, especially from widely-used 3`-barcoded libraries. Here, we report a method that for recovering paired, full-length variable region sequences of the BCRs from 3`-barcoded single-cell whole transcriptome libraries. We first verified this method could produce accurate, full-length BCR sequences. We then applied this method to profile antigen-specific B cell responses elicited against the capsular polysaccharide of Streptococcus pneumoniae serotype 3 (ST3) by glycoconjugate vaccines in infant rhesus macaques. Using our method, we defined features of the BCR associated with specificity for the ST3 antigen and showed that these sequence characteristics are present in multiple vaccinated monkeys, indicating a convergent response to vaccination. These results demonstrate the utility of our method to resolve key features of the B cell repertoire and for profiling antigen-specific responses elicited by vaccination.
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