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.
- Klíčová slova
- broadly neutralizing antibodies, hepatitis C, mimotope, myomedins, protein mimicry, protein scaffolds, vaccine,
- MeSH
- epitopy imunologie MeSH
- Hepacivirus * imunologie MeSH
- hepatitida C - protilátky * imunologie krev MeSH
- hepatitida C * imunologie MeSH
- lidé MeSH
- ligandy MeSH
- myši MeSH
- neutralizující protilátky * imunologie MeSH
- proteiny virového obalu * imunologie MeSH
- široce neutralizující protilátky * imunologie MeSH
- vakcíny proti virové hepatitidě imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- epitopy MeSH
- glycoprotein E2, Hepatitis C virus MeSH Prohlížeč
- hepatitida C - protilátky * MeSH
- ligandy MeSH
- neutralizující protilátky * MeSH
- proteiny virového obalu * MeSH
- široce neutralizující protilátky * MeSH
- vakcíny proti virové hepatitidě MeSH
Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bβ chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.
- Klíčová slova
- ABD scaffold, binding protein, combinatorial library, fibrin, fibrinogen Bβ chain, liposome, metallochelation, thrombus imaging, thrombus targeting,
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The development of an effective vaccine preventing HIV-1 infection is hindered by the enormous antigenic variability and unique biochemical and immunological properties of HIV-1 Env glycoprotein, the most promising target for HIV-1 neutralizing antibody. Functional studies of rare elite neutralizers led to the discovery of broadly neutralizing antibodies. METHODS: We employed a highly complex combinatorial protein library derived from a 5 kDa albumin-binding domain scaffold, fused with support protein of total 38 kDa, to screen for binders of broadly neutralizing antibody VRC01 paratope. The most specific binders were used for immunization of experimental mice to elicit Env-specific antibodies and to test their neutralization activity using a panel of HIV-1 clade C and B pseudoviruses. FINDINGS: Three most specific binders designated as VRA017, VRA019, and VRA177 exhibited high specificity to VRC01 antibody. Immunized mice produced Env-binding antibodies which neutralize eight of twelve HIV-1 Tier 2 pseudoviruses. Molecular modelling revealed a shape complementarity between VRA proteins and a part of VRC01 gp120 interacting surface. INTERPRETATION: This strategy based on the identification of protein replicas of broadly neutralizing antibody paratope represents a novel approach in HIV-1 vaccine development. This approach is not affected by low immunogenicity of neutralization-sensitive epitopes, variability, and unique biochemical properties of HIV-1 Env used as a crucial antigen in the majority of contemporary tested vaccines. FUND: Czech Health Research Council 15-32198A, Ministry of Health, Czech Republic.
- Klíčová slova
- Albumin-binding domain scaffold, Antibody paratope mimetics, Combinatorial protein library, HIV-1 vaccine, Neutralizing antibody, Protein docking,
- MeSH
- antigeny virové chemie imunologie MeSH
- epitopy chemie imunologie MeSH
- HIV infekce imunologie virologie MeSH
- HIV obalový protein gp120 imunologie MeSH
- HIV protilátky krev imunologie MeSH
- HIV-1 imunologie MeSH
- imunoglobulin G krev imunologie MeSH
- konformace proteinů MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- molekulární modely MeSH
- myši MeSH
- neutralizující protilátky krev imunologie MeSH
- sekvence aminokyselin MeSH
- vakcíny proti AIDS imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antigeny virové MeSH
- epitopy MeSH
- HIV obalový protein gp120 MeSH
- HIV protilátky MeSH
- imunoglobulin G MeSH
- neutralizující protilátky MeSH
- vakcíny proti AIDS MeSH
