Recombinant antibodies to tick-borne encephalitis virus

A gene encoding the dimeric form of single-chain antibody fragments (scFv) was designed on the basis of the artificial gene coding monomeric scFv to tick-borne encephalitis glycoprotein E. The sequence encoding histidine oligomer was added to 3'-ends of the genes encoding the monomeric and dimeric forms of scFv antibodies. Escherichia coli strains were constructed for the production of monomeric and dimeric antibodies. These antibodies were purified using Ni-NTA resin. The specificity of the purified monomeric and dimeric antibodies in the binding reaction with tick-borne encephalitis virus was shown by ELISA and Western-blot analysis. The identity of glycoprotein E epitope bound by monomeric and dimeric scFv and parental monoclonal antibodies E6B was confirmed by competitive immunoassay.     

Glycoproteins E2 of the Venezuelan and Eastern equine encephalomyelitis viruses contain multiple cross-reactive epitopes

Summary Enzyme immunoassay (EIA) with sixty types of monoclonal antibodies (MAbs) was used to study cross-reactive epitopes on the attenuated and virulent strains of the Eastern equine encephalomyelitis (EEE) and Venezuelan equine encephalomyelitis (VEE) viruses. All three structural proteins of the EEE and VEE viruses were demonstrated to have both cross-reactive and specific antigenic determinants. The glycoprotein E1 of EEE and VEE viruses possesses three cross-reactive epitopes for binding to MAbs. The glycoprotein E2 has a cluster of epitopes for 20 cross-reacting MAbs produced to EEE and VEE viruses. Cross-reactive epitopes were localised within five different sites of glycoprotein E2 of VEE virus and within four sites of that of the EEE virus. There are no cross-neutralising MAbs to the VEE and EEE viruses. Only one type of the protective Mabs was able to cross-protect mice against lethal infection by the virulent strains of the VEE and EEE viruses. Eight MAbs blocked the hemagglutination activity (HA) of both viruses. Antigenic alterations of neutralising and protective sites were revealed for all attenuated strains of the VEE and EEE viruses. Comparative studies of the E2 proteins amino acid sequences show that the antigenic modifications observed with the attenuated strains of the VEE virus may be caused by multiple amino acid changes in positions 7, 62, 120, 192 and 209–213. The escape-variants of the VEE virus obtained with cross-reactive MAbs 7D1, 2D4 and 7A6 have mutations of the E2 protein at positions 59, 212–213 and 232, respectively. Amino acid sequences in these regions of the VEE and EEE viruses are not homologous. These observations indicate that cross-reactive MAbs are capable of recognising discontinuous epitopes on the E2 glycoprotein.     

Localization of tick-borne encephalitis virus protein E antigenic determinant recognized by antihemagglutinating monoclonal antibodies using a phage-display peptide library

Bacteriophages bearing peptides reacting with antihemagglutinating monoclonal antibodies (MAb) 10H10 to tick-borne encephalitis (TBE) virus protein E were selected from a phage-display peptide library by affinity selection and enzyme immunoassay. The library contained randomized peptides that are 6 amino acids long, fused with protein pIII and exposed on the surface of the bacteriophage. No significant homology between the sequences of selected peptides and TBE virus protein E was detected. Computer software was created to locate the conformation epitopes on the surface of protein E. Amino acids R73, C74, T76, M77, N103, C105, L107, and S112 were found to form a discontinuous epitope recognized by MAb 10H10. These amino acids are remote in the protein sequence but close in the tertiary structure and form a whole epitope located in the structural domain II of protein E. Presumably the localized amino acids bind to the cellular receptor for TBE virus.     

The detection and quantitative determination of antigens to the human immunodeficiency virus types 1 and 2]

Three modifications of ELISA test system for HIV antigen detection are described. They are based on IgG from HIV-1 and HIV-2-infected human sera and monoclonal antibodies against HIV-1 p24 used as immunosorbents. The peroxidase/anti-HIV-IgG conjugate was used in all the test systems. A possibility of quantitative detection of viral antigen in native culture fluids, lysates, and purified virus preparations was demonstrated. The test system for HIV-1 antigen detection cannot be used for HIV-2 antigen detection and vice versa. The diagnostic value of HIV-1 p24 antigen detection consists in the possibility of earlier AIDS identification and monitoring of the disease at various stages. The sensitivity of "p24" assay is 0.5 ng/ml.     

Genetically delivered antibody protects against West Nile virus

Gene-based delivery of recombinant antibody genes is a promising therapeutic strategy offering numerous advantages including sustained antibody levels, better safety profile and lower production cost. Here we describe generation of a recombinant antibody Fc-9E2 comprising a fusion protein between human Fc of IgG1 and a single-chain Fv derived from a hybridoma 9E2 secreting a mAb neutralizing West Nile virus (WNV). Fc-9E2 was shown to retain parental mAb's specificity and WNV-neutralizing capacity. Adenovirus-mediated in vivo delivery of the antibody gene resulted in sustained Fc-9E2 serum levels leading to abrogation of lethal WNV infection in an animal model.     

Cystatin C and Cystatin SN As Possible Soluble Tumor Markers in Malignant Uveal Melanoma

BackgroundThe aim of the study was to determine the concentration of endogenous cystatin C and cystatin SN, as potential tumor biomarkers, in the serum and biological fluids of the eye in both healthy controls and patients with uveal melanoma.Patients and methodsThe concentration of both cystatins was determined in the intraocular fluid (IOF), tear fluid, and serum of patients with uveal melanoma and compared to baseline measurements in IOF, tears, serum, cerebral spinal fluid, saliva and urine of healthy controls.ResultsThe concentration of cystatin C in all the biological matrices obtained from healthy controls significantly exceeded the concentration of cystatin SN and was independent of gender. Cystatin C concentrations in the tear fluid of patients with uveal melanoma (both the eye with the malignancy, as well as the contralateral, non-affected eye), were significantly greater than cystatin C concentrations in the tear fluid of healthy controls and was independent of tumor size. The concentration of cystatin SN in IOF of patients with uveal melanoma was significantly less than the corresponding concentration of cystatin SN in healthy controls.ConclusionsThe ratio of cystatins (CysC:CysSN) in both the serum and tear fluid, as well as the concentration of cystatin SN in IOF, would appear to strongly suggest the presence of uveal melanoma. It is further suggested that multiple diagnostic criteria be utilized if a patient is suspected of having uveal melanoma, such as determination of the cystatin C and cystatin SN concentrations in serum, tears, and IOF, ocular fundus and ultrasound imaging, and biopsy with histopathological evaluation.Key words: uveal melanoma, cystatins, biomarkers, diagnosis     

PET/CT Imaging of NSCLC with a αvβ6 Integrin-Targeting Peptide

Molecular Imaging and Biology - Targeted therapies are regarded as promising approaches to increase 5-year survival rate of non-small cell lung cancer (NSCLC) patients. Here, we investigated the...     

Tick-borne virus diseases of human interest in Europe

Several human diseases in Europe are caused by viruses transmitted by tick bite. These viruses belong to the genus Flavivirus, and include tick-borne encephalitis virus, Omsk haemorrhagic fever virus, louping ill virus, Powassan virus, Nairovirus (Crimean-Congo haemorrhagic fever virus) and Coltivirus (Eyach virus). All of these viruses cause more or less severe neurological diseases, and some are also responsible for haemorrhagic fever. The epidemiology, clinical picture and methods for diagnosis are detailed in this review. Most of these viral pathogens are classified as Biosafety Level 3 or 4 agents, and therefore some of them have been classified in Categories A-C of potential bioterrorism agents by the Centers for Disease Control and Prevention. Their ability to cause severe disease in man means that these viruses, as well as any clinical samples suspected of containing them, must be handled with specific and stringent precautions.