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

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

p19-targeted ABD-derived protein variants inhibit IL-23 binding and exert suppressive control over IL-23-stimulated expansion of primary human IL-17+ T-cells

Interleukin-23 (IL-23), a heterodimeric cytokine of covalently bound p19 and p40 proteins, has recently been closely associated with development of several chronic autoimmune diseases such as psoriasis, psoriatic arthritis or inflammatory bowel disease. Released by activated dendritic cells, IL-23 interacts with IL-23 receptor (IL-23R) on Th17 cells, thus promoting intracellular signaling, a pivotal step in Th17-driven pro-inflammatory axis. Here, we aimed to block the binding of IL-23 cytokine to its cell-surface receptor by novel inhibitory protein binders targeted to the p19 subunit of human IL-23. To this goal, we used a combinatorial library derived from a scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived p19-targeted variants, called ILP binders. From 214 clones analyzed by ELISA, Western blot and DNA sequencing, 53 provided 35 different sequence variants that were further characterized. Using in silico docking in combination with cell-surface competition binding assay, we identified a group of inhibitory candidates that substantially diminished binding of recombinant p19 to the IL-23R on human monocytic THP-1 cells. Of these best p19-blockers, ILP030, ILP317 and ILP323 inhibited IL-23-driven expansion of IL-17-producing primary human CD4+ T-cells. Thus, these novel binders represent unique IL-23-targeted probes useful for IL-23/IL-23R epitope mapping studies and could be used for designing novel p19/IL-23-targeted anti-inflammatory biologics

ABD-Derived Protein Blockers of Human IL-17 Receptor A as Non-IgG Alternatives for Modulation of IL-17-Dependent Pro-Inflammatory Axis

Interleukin 17 (IL-17) and its cognate receptor A (IL-17RA) play a crucial role in Th17 cells-mediated pro-inflammatory pathway and pathogenesis of several autoimmune disorders including psoriasis. IL-17 is mainly produced by activated Th-17 helper cells upon stimulation by IL-23 and, via binding to its receptors, mediates IL-17-driven cell signaling in keratinocytes. Hyper-proliferation of keratinocytes belongs to major clinical manifestations in psoriasis. To modulate IL-17-mediated inflammatory cascade, we generated a unique collection of IL-17RA-targeting protein binders that prevent from binding of human IL-17A cytokine to its cell-surface receptor. To this goal, we used a highly complex combinatorial library derived from scaffold of albumin-binding domain (ABD) of streptococcal protein G, and ribosome display selection, to yield a collection of ABD-derived high-affinity ligands of human IL-17RA, called ARS binders. From 67 analyzed ABD variants, 7 different sequence families were identified. Representatives of these groups competed with human IL-17A for binding to recombinant IL-17RA receptor as well as to IL-17RA-Immunoglobulin G chimera, as tested in enzyme-linked immunosorbent assay (ELISA). Five ARS variants bound to IL-17RA-expressing THP-1 cells and blocked binding of human IL-17 cytokine to the cell surface, as tested by flow cytometry. Three variants exhibited high-affinity binding with a nanomolar Kd value to human keratinocyte HaCaT cells, as measured using Ligand Tracer Green Line. Upon IL-17-stimulated activation, ARS variants inhibited secretion of Gro-α (CXCL1) by normal human skin fibroblasts in vitro. Thus, we identified a novel class of inhibitory ligands that might serve as immunosuppressive IL-17RA-targeted non-IgG protein antagonists

Increasing Affinity of Interferon-γ Receptor 1 to Interferon-γ by Computer-Aided Design

We describe a computer-based protocol to design protein mutations increasing binding affinity between ligand and its receptor. The method was applied to mutate interferon-γ receptor 1 (IFN-γ-Rx) to increase its affinity to natural ligand IFN-γ, protein important for innate immunity. We analyzed all four available crystal structures of the IFN-γ-Rx/IFN-γ complex to identify 40 receptor residues forming the interface with IFN-γ. For these 40 residues, we performed computational mutation analysis by substituting each of the interface receptor residues by the remaining standard amino acids. The corresponding changes of the free energy were calculated by a protocol consisting of FoldX and molecular dynamics calculations. Based on the computed changes of the free energy and on sequence conservation criteria obtained by the analysis of 32 receptor sequences from 19 different species, we selected 14 receptor variants predicted to increase the receptor affinity to IFN-γ. These variants were expressed as recombinant proteins in Escherichia coli, and their affinities to IFN-γ were determined experimentally by surface plasmon resonance (SPR). The SPR measurements showed that the simple computational protocol succeeded in finding two receptor variants with affinity to IFN-γ increased about fivefold compared to the wild-type receptor

p19-Targeting ILP Protein Blockers of IL-23/Th-17 Pro-Inflammatory Axis Displayed on Engineered Bacteria of Food Origin

IL-23-mediated Th-17 cell activation and stimulation of IL-17-driven pro-inflammatory axis has been associated with autoimmunity disorders such as Inflammatory Bowel Disease (IBD) or Crohn’s Disease (CD). Recently we developed a unique class of IL-23-specific protein blockers, called ILP binding proteins that inhibit binding of IL-23 to its cognate cell-surface receptor (IL-23R) and exhibit immunosuppressive effect on human primary blood leukocytes ex vivo. In this study, we aimed to generate a recombinant Lactococcus lactis strain which could serve as in vivo producer/secretor of IL-23 protein blockers into the gut. To achieve this goal, we introduced ILP030, ILP317 and ILP323 cDNA sequences into expression plasmid vector containing USP45 secretion signal, FLAG sequence consensus and LysM-containing cA surface anchor (AcmA) ensuring cell-surface peptidoglycan anchoring. We demonstrate that all ILP variants are expressed in L. lactis cells, efficiently transported and secreted from the cell and displayed on the bacterial surface. The binding function of AcmA-immobilized ILP proteins is documented by interaction with a recombinant p19 protein, alpha subunit of human IL-23, which was assembled in the form of a fusion with Thioredoxin A. ILP317 variant exhibits the best binding to the human IL-23 cytokine, as demonstrated for particular L.lactis-ILP recombinant variants by Enzyme-Linked ImmunoSorbent Assay (ELISA). We conclude that novel recombinant ILP-secreting L. lactis strains were developed that might be useful for further in vivo studies of IL-23-mediated inflammation on animal model of experimentally-induced colitis

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

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

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

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

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