The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

The expression of the HER2 (human epidermal growth factor receptor 2) protein in cancer cells is a well-established cancer marker used for diagnostic and therapeutic purposes in modern treatment protocols, especially in breast cancer. The gold-standard immunohistochemical diagnostic methods with the specific anti-HER2 antibodies are utilized in the clinic to measure expression level of the membrane-bound receptor. However, a soluble extracellular domain (ECD) of HER2 is released to the extracellular matrix, thus the blood assays for HER2 measurements present an attractive way for HER2 level determination. There is a need for accurate and validated assays that can be used to correlate the concentration of the circulating HER2 protein with disease clinical manifestations. Here we describe two monoclonal antibodies binding HER2 with a unique sequence of the complementarity-determining regions that recognize HER2 ECD. Development and validation of the sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the soluble HER2 in a variety of biological samples is also presented. The assay provides HER2 quantitation within a concentrations range from 1.56 to 100 ng/ml with sensitivity at the level of 0.5 ng/ml that meets the expectations for measurements of HER2 in the blood and tumor tissue samples. The method presents satisfactory intra- and inter-assay precision and accuracy for immunochemical quantification of biomarkers in biological samples. The utility of the generated monoclonal anti-HER2 antibodies has been confirmed for use in the precise measurement of HER2 (both cell-bound and soluble) in several types of biological material, including serum, solid tumor tissue, and cell culture medium. Additionally, the developed immunochemical tools have a potential for HER2 detection, not only in a wide range of sample types but also independently of the sample storage/pre-processing, allowing for comprehensive HER2 analysis in tissue (IHC), cultured cells (immunofluorescence) and blood (ELISA).

Vaccination Failure in Eradication and Control Programs for Bovine Viral Diarrhea Infection.

Vaccination against bovine viral diarrhea (BVD) is one of the key elements to protect cattle herds from this economically important disorder. Bovine viral diarrhea virus (BVDV) is a pestivirus infecting animals at all ages with significant impact on reproductive, digestive, and respiratory systems. Financial burden caused by this pathogen prompts many farmers to introduce vaccination as the control and prophylactic measure especially when persistently infected (PI) individuals, being the main source of the virus in the herd, are removed after test-and-cull approach. The aim of the study was to compare the serological response in cattle herds where new PI calves were identified without prior removal of PI animals or despite their removal and after the introduction of whole herd vaccination against BVDV infection. Overall seroprevalence in 5 vaccinated herds was 91.7 and 83.3% using ELISA and virus neutralization test, respectively. Despite high titers for both vaccine and field strains of BVDV in analyzed herds the analysis of comparative strength of neutralization indicated that 41.4% of positive samples did not have a predominant titer against one specific subtype of BVDV. In 3 herds BVDV-1b subtype was identified while in 2 others it was BVDV-1d, while the vaccine used was based on BVDV-1a which was never identified in Poland so far. To increase the success of the BVDV eradication program, a careful approach is suggested when planning herd vaccination. Comparison of existing field strains and their similarity with vaccine strains at antigenic and genetic levels can be a useful approach to increase the effectiveness of vaccination and efficient protection of fetuses from persistent infection.

Porcine Complement Regulatory Protein CD46 Is a Major Receptor for Atypical Porcine Pestivirus but Not for Classical Swine Fever Virus.

Pestiviruses such as bovine viral diarrhea virus (BVDV) and classical swine fever virus (CSFV) belong to the family Flaviviridae and represent pathogens of outstanding veterinary relevance. Pestiviruses enter cells via receptor-mediated endocytosis. For entry in bovine cells, complement regulatory protein CD46bov serves as a cellular receptor for BVDV. In this study, the role of porcine CD46pig in cellular entry was investigated for the recently discovered atypical porcine pestivirus (APPV), CSFV, and Bungowannah virus (BuPV) in order to elucidate the observed differences in host cell tropism. A cell culture-adapted APPV variant, which shows enhanced viral replication in vitro, was generated and demonstrated a strict tropism of APPV for porcine cells. One of the porcine cell lines displayed areas of CD46pig-expressing cells and areas of nonexpressing cells, and one single cell line revealed not to express any CD46pig The CD46pig-deficient porcine lymphoma cell line, known to facilitate CSFV replication, was the only porcine cell line nonpermissive to APPV, indicating a significant difference in the entry mechanism of APPV and CSFV. Infection experiments with a set of genetically engineered CD46pig knockout cells confirmed that CD46pig is a major receptor of APPV as CD46bov is for BVDV. In contrast, it is apparently not an essential determinant in host cell entry of other porcine pestiviruses such as CSFV and BuPV. Existence of a CD46pig-independent entry mechanism illustrates that the pestiviral entry process is more diverse than previously recognized.IMPORTANCE Pestiviruses comprise animal pathogens such as classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) that cause notifiable diseases with great economic impact. Several additional pestivirus species affecting animal health were recently identified, including atypical porcine pestivirus (APPV). APPV is associated with health problems in piglets and is highly abundant in pig populations worldwide. Complement control protein CD46 serves as a receptor for diverse bacterial and viral pathogens, including particular adenoviruses, herpesviruses, measles virus (MeV), and BVDV. Porcine CD46 (CD46pig) was suggested to be a major receptor for CSFV. Here, we identified remarkable differences in relevance of CD46pig during entry of porcine pestiviruses. Resembling BVDV, efficient APPV infection in cell culture depends on CD46pig, while other porcine pestiviruses can efficiently enter and infect cells in the absence of CD46pig Thus, the study provides insights into the entry process of these pathogens and may help to understand differences in their biology.

Comparison of Schmallenberg virus sequences isolated from mammal host and arthropod vector.

Schmallenberg virus (SBV) is the member of Peribunyaviridae family, which comprises pathogens of importance for human and veterinary medicine. The virus is transmitted only between animals and mainly by biting midges of the genus Culicoides. This study was performed in order to determine SBV genetic diversity and elucidate the host-vector adaptation. All three viral segments were analysed for sequence variability and phylogenetic relations. The Polish SBV strains obtained from acute infections of cattle, congenital cases in sheep, and from Culicoides midges were sequenced using Sanger and next-generation sequencing (NGS) methods. The obtained sequences were genetically similar (99.2-100% identity) to the first-detected strain BH80/11-4 from German cattle. The sampling year and origin of Polish sequences had no effect on molecular diversity of SBV. Considering all analysed Polish as well as European sequences, ovine-derived sequences were the most variable, while the midge ones were more conserved and encompassed unique substitutions located mainly in nonstructural protein S. SBV sequences isolated from Culicoides are the first submitted to GenBank and reported.