Flow cytometric (A) and whole-cell ELISA (B) analyses of binding of recombinant Stx1B by <i>L</i>. <i>lactis</i> cells displaying S1B variants or ABDwt on their surface.

<p>(A) Alexa488-conjugated Stx1B was used for detection. MFI: Mean fluorescence intensity. (B) Mouse antiStx1B antibody and HRP-conjugated anti mouse antibody were used for detection of Stx1B. A₄₅₀: Absorbance at 450 nm. Vertical bars denote standard deviation. MFI or A₄₅₀ values of S1B binders were compared to those of the ABDwt control using Student’s t test. *: p<0.05, ** p<0.01, *** p<0.001.</p

Development of Recombinant <i>Lactococcus lactis</i> Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit - Fig 7

<p>(A) SDS PAGE analysis of lysates of <i>L</i>. <i>lacti</i>s cells expressing S1B22, S1B26, ABDwt and H6-ABDwt (ABDwtH), all in fusion with Usp45 secretion signal and the LysM-containing cA domain, and stained with Coomassie brilliant blue. ABD fusion proteins are high-lighted with arrows. (B) Flow cytometric analysis of ABD surface display, detection with FITC-conjugated human serum albumin. The MFI value of ABDwt was compared with that of the control using Student’s t test. *** p<0.001. Cont.: control containing empty plasmid pNZ8148.</p

Influence of S1B binders on Stx1B transport into HeLa cells.

<p>A, B: Flow cytometric analysis of HeLa cells demonstrating shift in fluorescence intensity (A) and mean fluorescence intensity (MFI; B) upon 1h incubation of HeLa cells with mixtures of S1B22, S1B26 or ABDwt (all in fusion with TolA-Avi and H6) and Alexa Fluor 488-labeled Stx1B. Cont: unstained HeLa cells. Stx1B: HeLa cells incubated with Stx1B-Alexa Fluor 488 alone. C, D: Fluorescence microscopy images of HeLa cells incubated with Alexa Fluor 488 labelled Stx1B (green) with or without pre-incubation with S1B22 and S1B26. DAPI staining (blue) was used to label nuclei. Cells were stained either with PKH26 membrane labeling dye (red; C) and Golgi apparatus was detected with mouse monoclonal Golgin-97 antibody and secondary polyclonal goat anti-mouse antibody conjugated with Alexa Fluor 633 (purple; D). Bars = 10 μm.</p

Sequence similarity analysis (A) and binding affinity (B) of 17 S1B binders selected after five rounds of ribosome display.

<p>A: The sequence of the parental ABD wild-type domain was used as a root of the tree and is highlighted as a square, while S1B variants selected for more detailed analysis are highlighted as circles. B: S1B binders-containing cell lysates were incubated with immobilized Stx1B (grey bars) or BSA (white bars) and detected with HRP-conjugated streptavidin. Error bars denote standard deviations.</p

Strains, plasmids, gene and primers used in this study.

<p>Strains, plasmids, gene and primers used in this study.</p

SPR analysis of binding of S1B22, S1B26 and ABDwt in fusion with TolA-Avi and H6 at 1 μM concentration to immobilized recombinant Stx1B.

<p>SPR analysis of binding of S1B22, S1B26 and ABDwt in fusion with TolA-Avi and H6 at 1 μM concentration to immobilized recombinant Stx1B.</p

DataSheet_1_Myomedin replicas of gp120 V3 loop glycan epitopes recognized by PGT121 and PGT126 antibodies as non-cognate antigens for stimulation of HIV-1 broadly neutralizing antibodies.docx

IntroductionImprinting broadly neutralizing antibody (bNAb) paratopes by shape complementary protein mimotopes represents a potential alternative for developing vaccine immunogens. This approach, designated as a Non-Cognate Ligand Strategy (NCLS), has recently been used for the identification of protein variants mimicking CD4 binding region epitope or membrane proximal external region (MPER) epitope of HIV-1 envelope (Env) glycoprotein. However, the potential of small binding proteins to mimic viral glycan-containing epitopes has not yet been verified.MethodsIn this work, we employed a highly complex combinatorial Myomedin scaffold library to identify variants recognizing paratopes of super candidate bNAbs, PGT121 and PGT126, specific for HIV-1 V3 loop epitopes.ResultsIn the collection of Myomedins called MLD variants targeted to PGT121, three candidates competed with gp120 for binding to this bNAb in ELISA, thus suggesting an overlapping binding site and epitope-mimicking potential. Myomedins targeted to PGT126 designated MLB also provided variants that competed with gp120. Immunization of mice with MLB or MLD binders resulted in the production of anti-gp120 and -Env serum antibodies. Mouse hyper-immune sera elicited with MLB036, MLB041, MLB049, and MLD108 moderately neutralized 8-to-10 of 22 tested HIV-1-pseudotyped viruses of A, B, and C clades in vitro.DiscussionOur data demonstrate that Myomedin-derived variants can mimic particular V3 glycan epitopes of prominent anti-HIV-1 bNAbs, ascertain the potential of particular glycans controlling neutralizing sensitivity of individual HIV-1 pseudoviruses, and represent promising prophylactic candidates for HIV-1 vaccine development.</p