Journal: ACS Central Science

Article Title: Profiling Germinal Center-like B Cell Responses to Conjugate Vaccines Using Synthetic Immune Organoids

doi: 10.1021/acscentsci.2c01473

Figure Lengend Snippet: Experimental framework for profiling GC-like B cell responses to glycoconjugate vaccine candidates. (a) Schematic of strategies for preparing designer glycoconjugate vaccine candidates (left) and evaluating their immunogenicity in mice and in synthetic immune organoids (right). Glycoengineered bacteria enable the assembly of a single O-PS repeat unit (a tetrasaccharide structure in the case of F. tularensis Schu S4) on a undecaprenol lipid carrier in the cytoplasmic membrane, which is subsequently flipped into the periplasm and polymerized to form variable-length O-PS antigens by the endogenous Wzx flippase and Wzy O-antigen polymerase Wzy, respectively. In the presence of C. jejuni PglB ( Cj PglB), the lipid-linked O-PS is site-specifically transferred to asparagine residues in recombinant carrier proteins bearing a DQNAT motif. Schematic created with BioRender.com . (b) Immunoblot analysis of purified carrier proteins derived from E. coli CLM24 cells carrying a plasmid encoding either MBP 4xDQNAT or CRM 197 4xDQNAT along with plasmid pGAB2 encoding the Ft O-PS biosynthetic pathway and with (+) or without (−) plasmid pMAF10 encoding Cj PglB as indicated. Blots were probed with anti-FLAG antibody to detect acceptor proteins (green signal) and FB11 antibody to detect Ft O-PS antigens (red signal). Images depict an overlay of anti-FLAG and FB11 blots. Arrows denote aglycosylated (agly) and multiply glycosylated (gly) forms of MBP 4xDQNAT and CRM 197 4xDQNAT . Molecular weight ( M W ) markers are indicated on the left. Results are representative of three biological replicates.

Article Snippet: Antimouse antiboides used for confocal imaging included the following: anti-CD19 polyclonal (ThermoFisher Cat # PA5-27442); anti-IgM II/41 clone (ThermoFisher Cat # 14-5790-82); antidiphtheria toxin polyclonal (Abcam Cat # ab151222); and anti- Francisella tularensis LPS FB11 clone (ThermoFisher Cat # MA1-21690).

Techniques: Immunopeptidomics, Bacteria, Membrane, Recombinant, Western Blot, Purification, Derivative Assay, Plasmid Preparation, Molecular Weight

Journal: Microorganisms

Article Title: Virulence of Francisella tularensis Subspecies holarctica Biovar japonica and Phenotypic Change during Serial Passages on Artificial Media

doi: 10.3390/microorganisms8121881

Figure Lengend Snippet: Antigenic change in lipopolysaccharide (LPS) of KU-1 variants passaged on Eugon chocolate agar in Western blots. Purified LPSs of KU-1 original and the variants passaged on Eugon chocolate agar 10, 20, and 80 times (EP10, EP20, and EP80, respectively) and LVS (lanes 1, 2, 3, 4, and 5, respectively) were detected using MAbs M15C6, M13A13, and FB11 [sheets ( A ), ( B ), and ( C ), respectively]. M: molecular weight markers (Bio-Rad) indicate 75, 50, 37, 25, 20, 15, and 10 (arrow) kDa from the top.

Article Snippet: FB11 was purchased from Biodesign International (Saco, ME, USA).

Techniques: Chocolate, Western Blot, Purification, Molecular Weight