INTRODUCTION: The persistent rise in new Hepatitis C virus (HCV) infections threatens WHO efforts to eliminate HCV infection by 2030. Although direct-acting antiviral (DAA) drugs are efficacious, access remains limited, reinfections occur, and perinatal infections continue to pose long-term complications. Therefore, an effective anti-HCV vaccine is urgently needed. METHODS: We employed a highly complex combinatorial Myomedin-loop scaffold library to identify variants binding to paratopes of HCV E2-specific broadly neutralizing antibodies (bNAbs) HC-1AM and HC84.26.WH.5DL. The selected binders, named SHB and WIN, respectively, represent non-cognate mimotopes of the aforementioned bNAbs. These binders were subsequently used as immunogens in experimental mice to elicit serum antibodies capable of binding to HCV E2 and neutralize HCV pseudotyped viruses. RESULTS AND DISCUSSION: The non-cognate mimotopes SHB and WIN competed with the E2 glycoprotein for bNAbs binding and, after immunizing experimental mice, elicited E2- and HCV-pseudovirus-specific antibodies. WIN- and SHB-immunized mice exhibited neutralization against 15 HCV pseudoviruses with varying neutralization sensitivities. The most potent binders WIN028 and WIN047, were modified with a C-terminal His-tag, allowing the generation of WIN proteoliposome and subsequent use in experimental mice immunizations. Hyperimmune sera exhibited improved binding to HCV E2 and neutralized 60% of the tested HCV pseudoviruses. The broad neutralization of HCV pseudoviruses achieved by hypperimmune sera from SHB- and WIN-immunized mice highlights the potential of this approach in the HCV vaccine design.

• Keywords
• broadly neutralizing antibodies, hepatitis C, mimotope, myomedins, protein mimicry, protein scaffolds, vaccine,
• MeSH
• Epitopes immunology   MeSH
• Hepacivirus * immunology   MeSH
• Hepatitis C Antibodies * immunology   blood   MeSH
• Hepatitis C * immunology   MeSH
• Humans MeSH
• Ligands MeSH
• Mice MeSH
• Antibodies, Neutralizing * immunology   MeSH
• Viral Envelope Proteins * immunology   MeSH
• Broadly Neutralizing Antibodies * immunology   MeSH
• Viral Hepatitis Vaccines immunology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Epitopes MeSH
• glycoprotein E2, Hepatitis C virus MeSH Browser
• Hepatitis C Antibodies * MeSH
• Ligands MeSH
• Antibodies, Neutralizing * MeSH
• Viral Envelope Proteins * MeSH
• Broadly Neutralizing Antibodies * MeSH
• Viral Hepatitis Vaccines MeSH

BACKGROUND: The treatment of non-small cell lung cancer (NSCLC) patients is correlated with the efficacy of immune checkpoint blockade therapy (ICB) targeting programmed cell death ligand 1 (PD-L1) or its cognate receptor (PD-1) on cancer cells or infiltrating immune cells. Analysis of PD-L1/PD-1 expression in tumor tissue represents a crucial step before PD-L1/PD-1 blocker usage. METHODS: We used directed evolution of protein variants derived from a 13 kDa Myomedin loop-type combinatorial library with 12 randomized amino acid residues to select high-affinity binders of human PD-L1 (hPD-L1). After the ribosome display, individual clones were screened by ELISA. Detailed analysis of binding affinity and kinetics was performed using LigandTracer. The specificity of Myomedins was assessed using fluorescent microscopy on HEK293T-transfected cells and cultured cancer cells in vitro, formalin-fixed paraffin-embedded (FFPE) sections of human tonsils, and FFPE tumor samples of NSCLC patients. RESULTS: Seven identified PD-L1 binders, called MLE, showed positive staining for hPD-L1 on transfected HEK293T cells and cultured MCF-7 cells. MLE031, MLE105, MLE249, and MLE309 exhibited high affinity to both human and mouse PD-L1-transfected HEK293T cells measured with LigandTracer. The diagnostic potential of MLE variants was tested on human tonsillitis tissue and compared with diagnostic anti-PD-L1 antibody DAKO 28-8 and PD-L1 IHC 22C3 pharmDx antibody. MLE249 and MLE309 exhibited an excellent overlap with diagnostic DAKO 28-8 (Pearson´s coefficient (r) = 0.836 and 0.731, respectively) on human tonsils on which MLE309 exhibited also excellent overlap with diagnostic 22C3 antibody (r = 0.876). Using three NSCLC tissues, MLE249 staining overlaps with 28-8 antibody (r = 0.455-0.883), and MLE309 exhibited overlap with 22C3 antibody (r = 0.534-0.619). Three MLE proteins fused with Fc fragments of rabbit IgG, MLE249-rFc, MLE309-rFc and MLE031-rFc, exhibited very good overlap with anti-PD-L1 antibody 28-8 on tonsil tissue (r = 0.691, 0.610, and 0.667, respectively). Finally, MLE249-rFc, MLE309-rFc and MLE031-rFc exhibited higher sensitivity in comparison to IHC 22C3 antibody using routine immunohistochemistry staining system Ventana, which is one of gold standards for PD-L1 diagnosis. CONCLUSIONS: We demonstrated the development of MLE Myomedins specifically recognizing hPD-L1 that may serve as a refinement tool for clinical PD-L1 detection.

• Keywords
• Cancer diagnostic, Combinatorial library, Immune checkpoint, Non-small cell lung cancer, Programmed cell death ligand 1, Protein engineering,
• MeSH
• B7-H1 Antigen * metabolism   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Lung Neoplasms * diagnosis   metabolism   pathology   MeSH
• Carcinoma, Non-Small-Cell Lung * diagnosis   metabolism   pathology   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• B7-H1 Antigen * MeSH
• CD274 protein, human MeSH Browser

Human immunodeficiency virus type 1 (HIV-1) vaccine immunogens capable of inducing broadly neutralizing antibodies (bNAbs) remain obscure. HIV-1 evades immune responses through enormous diversity and hides its conserved vulnerable epitopes on the envelope glycoprotein (Env) by displaying an extensive immunodominant glycan shield. In elite HIV-1 viremic controllers, glycan-dependent bNAbs targeting conserved Env epitopes have been isolated and are utilized as vaccine design templates. However, immunological tolerance mechanisms limit the development of these antibodies in the general population. The well characterized bNAbs monoclonal variants frequently exhibit extensive levels of somatic hypermutation, a long third heavy chain complementary determining region, or a short third light chain complementarity determining region, and some exhibit poly-reactivity to autoantigens. This review elaborates on the obstacles to engaging and manipulating the Env glycoprotein as an effective immunogen and describes an alternative reverse vaccinology approach to develop a novel category of bNAb-epitope-derived non-cognate immunogens for HIV-1 vaccine design.

• Keywords
• Broadly neutralizing antibodies, Combinatorial protein library, Glycans, HIV-1 vaccine, Non-cognate ligands, Protein mimicry,
• MeSH
• Epitopes immunology   MeSH
• env Gene Products, Human Immunodeficiency Virus immunology   MeSH
• HIV Infections immunology   MeSH
• HIV Antibodies * immunology   MeSH
• HIV-1 * immunology   MeSH
• Humans MeSH
• Ligands MeSH
• Molecular Mimicry immunology   MeSH
• Antibodies, Neutralizing * immunology   MeSH
• Polysaccharides immunology   MeSH
• AIDS Vaccines * immunology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Epitopes MeSH
• env Gene Products, Human Immunodeficiency Virus MeSH
• HIV Antibodies * MeSH
• Ligands MeSH
• Antibodies, Neutralizing * MeSH
• Polysaccharides MeSH
• AIDS Vaccines * MeSH

BACKGROUND: Programmed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging. METHODS: We designed a 13 kDa β-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1. RESULTS: Binding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7-99.3% and in vitro stability in human serum after 120 min was in the range 94.6-98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors. CONCLUSIONS: Using directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.

• Keywords
• Cancer diagnostic, Combinatorial library, Immune checkpoint, Non-small cell lung cancer, Programmed cell death 1, Protein engineering,
• MeSH
• Programmed Cell Death 1 Receptor * metabolism   MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Lung Neoplasms diagnostic imaging   pathology   metabolism   genetics   MeSH
• Carcinoma, Non-Small-Cell Lung diagnostic imaging   pathology   metabolism   MeSH
• Positron-Emission Tomography * methods   MeSH
• Protein Engineering * MeSH
• Amino Acid Sequence MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Programmed Cell Death 1 Receptor * MeSH
• PDCD1 protein, human MeSH Browser