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

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

INTRODUCTION: Imprinting 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. METHODS: In 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. RESULTS: In 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. DISCUSSION: Our 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.

• Keywords
• HIV-1, broadly neutralizing antibody, glycoprotein 120, protein engineering, protein scaffold, vaccine,
• MeSH
• Epitopes MeSH
• HIV Envelope Protein gp120 MeSH
• HIV Antibodies * MeSH
• HIV-1 * MeSH
• Mice MeSH
• Antibodies, Neutralizing MeSH
• Polysaccharides MeSH
• Broadly Neutralizing Antibodies MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Epitopes MeSH
• HIV Envelope Protein gp120 MeSH
• HIV Antibodies * MeSH
• Antibodies, Neutralizing MeSH
• Polysaccharides MeSH
• Broadly Neutralizing Antibodies MeSH

One of the proposed strategies for the development of a more efficient HIV-1 vaccine is based on the identification of proteins binding to a paratope of chosen broadly neutralizing antibody (bNAb) that will mimic cognate HIV-1 Env (glyco)protein epitope and could be used as potent immunogens for induction of protective virus-neutralizing antibodies in the immunized individuals. To verify this "non-cognate ligand" concept, we developed a highly complex combinatorial library designed on a scaffold of human myomesin-1 protein domain and selected proteins called Myomedins specifically binding to variable regions of HIV-1 broadly neutralizing antibody 10E8. Immunization of mice with these Myomedin variants elicited the production of HIV-1 Env-specific antibodies. Hyperimmune sera bound to Env pseudotyped viruses and weakly/moderately neutralized 54% of tested clade A, B, C, and AE pseudotyped viruses variants in vitro. These results demonstrate that Myomedin variants have the potential to mimic Env epitopes and could be used as potential HIV-1 vaccine components.

• Keywords
• Env glycoprotein, HIV vaccine, broadly neutralizing antibody, combinatorial library, protein mimetics, protein scaffold,
• MeSH
• Epitopes MeSH
• env Gene Products, Human Immunodeficiency Virus genetics   MeSH
• HIV Infections * prevention & control   MeSH
• HIV Antibodies MeSH
• HIV-1 * genetics   MeSH
• Mice MeSH
• Antibodies, Neutralizing MeSH
• Viral Pseudotyping MeSH
• Broadly Neutralizing Antibodies MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Epitopes MeSH
• env Gene Products, Human Immunodeficiency Virus MeSH
• HIV Antibodies MeSH
• Antibodies, Neutralizing MeSH
• Broadly Neutralizing Antibodies MeSH

Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. coli, solubility, and thermostability. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The procedure yielded scaffold-related variants with nanomolar affinity.

• Keywords
• computational saturation, directed evolution, interleukin-10, protein engineering, protein scaffold, ribosome display,
• MeSH
• Databases, Protein MeSH
• Interleukin-10 metabolism   MeSH
• Protein Conformation MeSH
• Computer Simulation MeSH
• Protein Engineering MeSH
• Proteins chemistry   genetics   metabolism   MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Ribosomes metabolism   MeSH
• Directed Molecular Evolution methods   MeSH
• Amino Acid Sequence MeSH
• Protein Stability MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Interleukin-10 MeSH
• Proteins MeSH
• Recombinant Proteins MeSH