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.

• Keywords
• ABD scaffold, binding protein, combinatorial library, fibrin, fibrinogen Bβ chain, liposome, metallochelation, thrombus imaging, thrombus targeting,
• Publication type
• Journal Article MeSH

Lyme disease, Borrelia burgdorferi-caused infection, if not recognized and appropriately treated by antibiotics, may lead to chronic complications, thus stressing the need for protective vaccine development. The immune protection is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies, associated with the Th1 immune response. Surface antigen OspC is involved in Borrelia spreading through the host body. Previously we reported that recombinant histidine tagged (His-tag) OspC (rOspC) could be attached onto liposome surfaces by metallochelation. Here we report that levels of OspC-specific antibodies vary substantially depending upon whether rOspC possesses an N' or C' terminal His-tag. This is the case in mice immunized: (a) with rOspC proteoliposomes containing adjuvants MPLA or non-pyrogenic MDP analogue MT06; (b) with free rOspC and Montanide PET GEL A; (c) with free rOspC and alum; or (d) with adjuvant-free rOspC. Stronger responses are noted with all N'-terminal His-tag rOspC formulations. OspC-specific Th1-type antibodies predominate post-immunization with rOspC proteoliposomes formulated with MPLA or MT06 adjuvants. Further analyses confirmed that the structural features of soluble N' and C' terminal His-tag rOspC and respective rOspC proteoliposomes are similar including their thermal stabilities at physiological temperatures. On the other hand, a change in the position of the rOspC His-tag from N' to C' terminal appears to affect substantially the immunogenicity of rOspC arguably due to steric hindrance of OspC epitopes by the C' terminal His-tag itself and not due to differences in overall conformations induced by changes in the His-tag position in rOspC variants.

• MeSH
• Adjuvants, Immunologic * MeSH
• Antigens, Bacterial administration & dosage   chemistry   immunology   MeSH
• Borrelia burgdorferi immunology   MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Immunization MeSH
• Lyme Disease immunology   MeSH
• Models, Animal MeSH
• Mice MeSH
• Bacterial Outer Membrane Proteins administration & dosage   chemistry   immunology   MeSH
• Proteolipids MeSH
• Antibodies, Bacterial immunology   MeSH
• Recombinant Fusion Proteins administration & dosage   chemistry   immunology   isolation & purification   MeSH
• Protein Structure, Secondary MeSH
• Antibody Specificity immunology   MeSH
• Protein Stability MeSH
• Antibody Formation immunology   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
• Adjuvants, Immunologic * MeSH
• Antigens, Bacterial MeSH
• OspC protein MeSH Browser
• Bacterial Outer Membrane Proteins MeSH
• Proteolipids MeSH
• proteoliposomes MeSH Browser
• Antibodies, Bacterial MeSH
• Recombinant Fusion Proteins MeSH

PURPOSE: The aim of this work was to demonstrate an immunostimulatory and adjuvant effect of new apyrogenic lipophilic derivatives of norAbuMDP and norAbuGMDP formulated in nanoliposomes. METHODS: Nanoliposomes and metallochelating nanoliposomes were prepared by lipid film hydration and extrusion methods. The structure of the liposomal formulation was studied by electron microscopy, AF microscopy, and dynamic light scattering. Sublethal and lethal γ-irradiation mice models were used to demonstrate stimulation of innate immune system. Recombinant Hsp90 antigen (Candida albicans) bound onto metallochelating nanoliposomes was used for immunisation of mice to demonstrate adjuvant activities of tested compounds. RESULTS: Safety and stimulation of innate and adaptive immunity were demonstrated on rabbits and mice. The liposomal formulation of norAbuMDP/GMDP was apyrogenic in rabbit test and lacking any side effect in vivo. Recovery of bone marrow after sublethal γ-irradiation as well as increased survival of mice after lethal irradiation was demonstrated. Enhancement of specific immune response was demonstrated for some derivatives incorporated in metallochelating nanoliposomes with recombinant Hsp90 protein antigen. CONCLUSIONS: Liposomal formulations of new lipophilic derivatives of norAbuMDP/GMDP proved themselves as promising adjuvants for recombinant vaccines as well as immunomodulators for stimulation of innate immunity and bone-marrow recovery after chemo/radio therapy of cancer.

• MeSH
• Acetylmuramyl-Alanyl-Isoglutamine administration & dosage   analogs & derivatives   chemistry   pharmacology   therapeutic use   MeSH
• Adaptive Immunity drug effects   MeSH
• Adjuvants, Immunologic administration & dosage   chemistry   pharmacology   therapeutic use   MeSH
• Survival Analysis MeSH
• Antigens, Fungal immunology   MeSH
• Radiation Injuries, Experimental immunology   prevention & control   MeSH
• Rabbits MeSH
• Liposomes MeSH
• Microscopy, Atomic Force MeSH
• Microscopy, Electron, Scanning MeSH
• Molecular Structure MeSH
• Mice, Inbred ICR MeSH
• Mice MeSH
• Nanoparticles MeSH
• Drug Carriers chemistry   MeSH
• Immunity, Innate drug effects   MeSH
• HSP90 Heat-Shock Proteins immunology   MeSH
• Antibodies, Fungal blood   MeSH
• Recombinant Proteins immunology   MeSH
• Microscopy, Electron, Transmission MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Acetylmuramyl-Alanyl-Isoglutamine MeSH
• Adjuvants, Immunologic MeSH
• Antigens, Fungal MeSH
• glucosaminylmuramyl-2-alanine-D-isoglutamine MeSH Browser
• Liposomes MeSH
• N-acetylmuramyl-aminobutyryl-isoglutamine MeSH Browser
• Drug Carriers MeSH
• HSP90 Heat-Shock Proteins MeSH
• Antibodies, Fungal MeSH
• Recombinant Proteins MeSH