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Exploitation of the immune system of livestock to develop novel diagnostic and therapeutic tools

Boek - Dissertatie

The livestock sector is a very important pillar of the global food system. It plays a significant role in maintaining and uplifting the economic and socio-cultural outlook of developing countries. In recent times, rapidly growing human population has also led to the accelerated growth in livestock sector to ensure food security. This increase in livestock farming has posed some serious challenges in maintaining the well-being of humans and animals. In addition, globalization and climate change has resulted in emergence and re-emergence of animal and human diseases and zoonoses. These infectious diseases present a major threat to the human and animal health and welfare worldwide. Keeping this in mind, effective control and eradication of these livestock infections is not only crucial for animal health, but also in safeguarding and smooth lining the national and international food supplies and economic development. In this regard, positive sense single stranded RNA viruses belonging to the family Flaviviridae cause serious infections in animals and humans. These viruses are grouped into four main genera i.e., Flavivirus, Pestivirus, Pegivirus and Hepacivirus. Amongst these genera, pestiviruses including bovine viral diarrhea virus (BVDV), classical swine fever (CSFV) and border disease fever mainly infect livestock species. Similarly, West Nile virus (WNV), a Flavivirus, is a zoonotic virus of public health significance. It is a vector borne virus and infects mainly humans and horses, causing serious neurological disease. The control of these emerging and re-emerging infections requires the development of easy, rapid and cost effective field and laboratory diagnostic tools alongside innovative therapeutics. By looking at the threat of these viruses, it is the need of the hour to investigate the immunological organization of the host, viruses and their pathogenic profile. From the view point of developing these tools, this thesis focuses on two viruses i.e., BVDV and WNV. The immune system is a dynamic and complex biological organization. It is composed of hierarchically organized set of biomolecules, cells and biological networks that act in harmony to promote effective host defense. Ecoimmunology deals with biological organization of the individual's immune system and its evolution in a given ecological and life history contexts. In this regards, skin swelling test serves as a useful ecoimmunological tool to decipher individual's immune responsiveness following a pro-inflammatory mitogen injection. In this context, phytohemagglutinin (PHA) is considered as a universal mitogen of choice. The mechanism behind local immune reaction stimulated by these mitogens particularly PHA is poorly understood. Here, we implemented an image-based platform for the quantification of inflammatory response of the skin welling test. The experiment was conducted twice five months apart. In this study, all the intradermal injections PBS (negative control), histamine (positive control) and PHA were administered in neck of the cow after proper shaving. Thermal dynamics of the inflammatory reaction were observe using FLIR One™, a commercial infrared thermal camera especially designed for smart phone application. Additionally, skin measurements such as skin fold thickness and swelling diameter were measured using vernier caliper. In the first swelling test, It was observed that histamine caused the maximum skin swelling and thickness as compared to that of PHA. While in second skin swelling test, skin welling and thickness caused by PHA was in comparison with histamine. Thermal imaging of the inflamed skin area revealed interesting findings with maximum surface area with increased temperature was achieved by histamine with first 1.5 hours of injection as compared to delayed reaction of PHA and PBS (approximately 6 hours post injection). Another interesting finding was the pattern of heat signals with histamine showing spreading outward trend as compared to intense localized heat signals in case of PHA and PBS. These finding elucidate the quantification of immune reaction with infrared thermal imaging as a practical tool in understanding skin swelling test. BVDV causes a significant infection in cattle with a wide spectrum of clinical signs, ranging from asymptomatic to acute and severe fatal disease. In general, BVDV is endemic worldwide in bovine populations. The 12.3 kb long BVDV genome codes for a long polypeptide which is co- and post-translationally is processed into four structural (C, Erns, E1, E2,) and eight non-structural proteins (Npro, p7, NS2/3, NS4A, NS4B, NS5A and NS5B) by host and viral proteases. The immunogenic profile of few of these viral proteins including Erns, E2 and NS3 is well established and characterized. In addition, roles of Npro and NS2 in viral replication and pathogenesis have also been thoroughly studied in literature. Apart from this, there is not much information available about other BVDV's non-structural proteins. In recent times, in the family of Flaviviridae, NS4B protein has emerged as a promising target of diagnostic, prophylactic and therapeutic importance. BVDV-NS4B is also a hydrophobic protein having membrane topology. Furthermore, single point mutation (Y2441C) in BVDV-NS4B switches the BVDV from cytopathic to a non-cytopathic biotype, indicating its role in viral replication. Apart from these studies there exists a clear gap of knowledge regarding the immunogenicity of BVDV-NS4B protein during disease pathogenesis and vaccination. To characterize the immunogenic profile of BVDV-NS4B, five cows were vaccinated with live attenuated BVDV vaccine called "Bovela®". To assess the humoral response, blood sampling was performed from all the cows pre- and post-immunization for serum isolation. Virus neutralization assay (VNA) validated the presence of anti-BVDV antibodies in the post-vaccinated sera of all the five cows, where highest post-immunization titer dilutions of the serum capable of protecting the cells from virus induced cytopathic effect were found to be 1:10,240, 1: 10,240, 1: 10,240, 1:2560 and 1:5120, respectively. VNA also disclosed the pre-existing humoral immunity against BVDV in the pre-immunization sera of cow 3 and 4 with highest serum dilutions of 1: 10,240 and 1:5120, respectively, capable of protecting the cells from virus induced cytopathic effect. Using the pCI-neo vector system, the BVDV-NS4B protein was expressed in mammalian cells. The presence of BVDV-NS4B specific antibodies in the sera of vaccinated cows was evaluated through western blot and indirect ELISA. Interestingly, both western blot and ELISA elucidated the presence of anti-BVDV-NS4B antibodies in the sera of cows which already showed the presence of pre-existing humoral immunity in VNA. This is the first proof of study indicating the immunogenic nature of BVDV-NS4B protein in sero-converted animals. These findings are in agreement with the observation made for NS4B protein in other Flaviviridae members and highlight its significance as a valuable target of diagnostic, vaccination and therapeutic importance. The WNV causes a complicated disease being characterized by clinical signs ranges from asymptomatic to a severe neurological syndrome. This severe infection leads to paralysis and ultimately death. Currently, there is a need for developing better diagnostic tools for genetic surveillance, characterization of the emergent viral strains alongside early and differential diagnosis of this dangerous disease. Keeping this in mind, we exploited the antibody phage display platform to explore immune repertoire of horses for the isolation of scFv binders against WNV envelop protein. In this thesis, we described the construction of immunized horse scFv phage libraries. The sizes of newly generated Vκ-VH and Vλ-VH antibody libraries were 1.2×107 and 1.5×106, respectively. Using whole cell-based biopanning, these libraries were screened by four rounds of biopanning in order to select high affinity binders against cell surface expressed WNV envelop protein. Clear enrichment of high affinity clones was validated by increasing output titer after each round of panning, polyclonal phage ELISA, monoclonal ELISA, sequencing and expression of selected clones. According to the best of our knowledge, this is first study where design and validation of primers for the construction of horse immune libraries has been reported. This thesis presents a promising approach of generating diverse equine scFv libraries in vitro and their screening against a variety of target of equine health significance. The findings reported here will surely contribute to the further development of veterinary diagnostics and therapeutics. The findings reported here will certainly contribute in further developing and implementation of the additional control strategies for Flaviviridae related infections in livestock.
Jaar van publicatie:2021
Toegankelijkheid:Closed