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.
Porcine circovirus causes the post-weaning multi-systemic wasting syndrome. Despite the existence of commercial vaccines, the development of more effective and cheaper vaccines is expected. The usage of chimeric antigens allows serological differentiation between naturally infected and vaccinated animals. In this work, recombinant pentameric vaccination protein particles spontaneously assembled from identical subunits-chimeric fusion proteins derived from circovirus capsid antigen Cap and a multimerizing subunit of mouse polyomavirus capsid protein VP1 were purified and characterized using asymmetric flow field-flow fractionation (AF4) coupled with UV and MALS/DLS (multi-angle light scattering/dynamic light scattering) detectors. Various elution profiles were tested, including constant cross-flow and decreasing cross-flow (linearly and exponentially). The optimal sample retention, separation efficiency, and resolution were assessed by the comparison of the hydrodynamic radius ( R h ) measured by online DLS with the R h values calculated from the simplified retention equation according to the AF4 theory. The results show that the use of the combined elution profiles (exponential and constant cross-flow rates) reduces the time of the separation, prevents undesirable sample-membrane interaction, and yields better resolution. Besides, the results show no self-associations of the individual pentameric particles into larger clusters and no sample degradation during the AF4 separation. The R g / R h ratios for different fractions are in good correlation with morphological analyses performed by transmission electron microscopy (TEM). Additionally to the online analysis, the individual fractions were subjected to offline analysis, including batch DLS, TEM, and SDS-PAGE, followed by Western blot. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03323-6.
Porcine circovirus type 2 is the main causative agent of post-weaning multisystemic wasting syndrome, which affects the immune system of swine and causes widespread epidemics in livestock farms resulting in significant piglet mortality and economic losses every year. Although several commercial vaccines were developed, the efficiency and safety need to be improved. Therefore, we have engineered the chimeric complex containing PCV2bCap protein based on virus like particles (VLPs) and the mouse polyomavirus (MPyV) as VLPs represent modern and safe alternative of classical vaccine with high B cells stimulating activity. The ability of this complex to induce an immune response in both mouse and pig models in vivo were evaluated. Firstly, experimental mice were divided into 4 groups and immunized with sterile buffer and VP1-PCV2bCap with different adjuvants, the immune response was monitored for 10 weeks. Robust immune response was detected after the first immunization and gradually increased after the second and third dose, especially in mice immunized by recombinant protein with Emulsigen (10%) as an adjuvant. Subsequently, to confirm the vaccine efficacy in a target organism, 8-week-old piglets were immunized with VP1-PCV2bCap protein with Emulsigen (10%). The levels of anti-PCV2b specific IgG antibodies were significantly increased in piglets after the second immunization. Finally, strong neutralizing activity of these antibodies was confirmed in PK-15 cells infected with PCV2 Stoon 1010. VP1-PCV2bCap protein complex appears as a promising candidate vaccine for preventing disease associated with PCV2 infection in pigs.
Porcine circovirus type 2 (PCV2) is the main causative agents of post-weaning multi-systemic wasting syndrome leading to significant economic losses in swine population. Although vaccination is the most effective strategy to decrease morbidity and mortality rate, the virus persist in immunized populations and causes subclinical infections with symptoms similar to porcine circovirus diseases. To develop new effective vaccine against PCV2 infection, we prepared chimeric complex VP1-PCV2bCap containing PCV2bCap protein fused with mouse polyomavirus (MPyV). Complex was expressed in baculovirus expression system and purified by nickel agarose. From eluate containing large aggregates of purified proteins, fraction containing pantameres of VP1-PCV2bCap protein was separated by Asymmetrical flow field-flow fractionation and fractions were analyzed by electron microscopy with nano-gold labeling of PCV2Cap portion of the fused protein. 6 weeks old BALB/c mice were subcutaneously vaccinated by 50ug of purified chimeric protein alone or in combination with incomplete Freund adjuvants (50%) or Emulsigen (10%). Immunity response was monitored by indirect ELISA. After the first immunization strong immunity response that gradually increased after the second and third immunization was detected in all mice immunized by chimeric protein. The best immunity response was detected in mice immunized by vaccine with Emulsigen (10%) as adjuvant. Purified chimeric protein VP1-PCV2bCap has a high potential as a DIVA (differentiating infected form vaccinated animals) vaccine against PCV2b infections in pigs. The biggest benefit of this type of vaccine is possibility to distinguish between naturally infected and vaccinated animals
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