West Nile virus (WNV) is a member of the Flavivirus genus, which includes several human pathogens transmitted by arthropod vectors. The maintenance in nature of WNV is possible through a bird-mosquito life cycle, in which mosquitoes act as transmission vectors and birds as virus amplification hosts. Humans are not able to contribute to WNV transmission cycle, therefore are defined accidental or dead end hosts. The 20% of WNV infected patients develop a mild disease with influenza like symptoms known as West Nile fever, while in the 1% of cases the viral infection results in a severe neuroinvasive disease, which can lead to death. The most widely used approaches for WNV diagnosis relay on the detection of specific antibodies against viral antigens and are generally based on ELISA format assays. The weak points of the immunodiagnostic tests are the false positive results deriving by cross-reactive antibodies. The rise of such antibodies is caused by the structural similarity of flaviviruses. The diagnosis becomes particularly complicate in geographical areas interested by the co-circulation of more than one flavivirus or vaccination programs leading to the immune system pre-exposure to flaviviral antigens, as occurs in some European areas interested by the co-circulation of WNV and Tick-Borne Encephalitis virus (TBEV). In addition, the vaccination against TBEV is extensively applied in north Eastern Europe countries. In order to improve the detection of WNV infection, the needed reagents for immunodiagnostic applications have been produced. Such reagents include viral antigens and highly specific antibodies against the antigens. As most of commercial kit for WNV diagnosis provide the use of the inactivated virus, requiring specialized equipment and extensive production procedures, a fast and easy protocol has been developed for viral antigens expression and purification. The quality of the produced antigens, in terms of purity, integrity and antigenicity, has been confirmed by SDS PAGE, Western Blot and ELISA assays with murine immunized sera. The isolation of antibodies specifically targeting the WNV antigens has been based on two strategies: the selection of a naïve phage library and the construction and screening of immunized phage libraries from murine samples. In addition to libraries construction, the murine samples have been used for immune repertoires analysis by Next Generation Sequencing. Both the approaches have led to the identification of highly sensitive and specific antibodies. Two of them have been characterized, defining their binding and affinity properties for the target through epitope mapping, ELISA and Western Blot assays. All the collected data suggest the high potentiality of the produced reagents for WNV immunodiagnostic applications.

West Nile virus (WNV) is a member of the Flavivirus genus, which includes several human pathogens transmitted by arthropod vectors. The maintenance in nature of WNV is possible through a bird-mosquito life cycle, in which mosquitoes act as transmission vectors and birds as virus amplification hosts. Humans are not able to contribute to WNV transmission cycle, therefore are defined accidental or dead end hosts. The 20% of WNV infected patients develop a mild disease with influenza like symptoms known as West Nile fever, while in the 1% of cases the viral infection results in a severe neuroinvasive disease, which can lead to death. The most widely used approaches for WNV diagnosis relay on the detection of specific antibodies against viral antigens and are generally based on ELISA format assays. The weak points of the immunodiagnostic tests are the false positive results deriving by cross-reactive antibodies. The rise of such antibodies is caused by the structural similarity of flaviviruses. The diagnosis becomes particularly complicate in geographical areas interested by the co-circulation of more than one flavivirus or vaccination programs leading to the immune system pre-exposure to flaviviral antigens, as occurs in some European areas interested by the co-circulation of WNV and Tick-Borne Encephalitis virus (TBEV). In addition, the vaccination against TBEV is extensively applied in north Eastern Europe countries. In order to improve the detection of WNV infection, the needed reagents for immunodiagnostic applications have been produced. Such reagents include viral antigens and highly specific antibodies against the antigens. As most of commercial kit for WNV diagnosis provide the use of the inactivated virus, requiring specialized equipment and extensive production procedures, a fast and easy protocol has been developed for viral antigens expression and purification. The quality of the produced antigens, in terms of purity, integrity and antigenicity, has been confirmed by SDS PAGE, Western Blot and ELISA assays with murine immunized sera. The isolation of antibodies specifically targeting the WNV antigens has been based on two strategies: the selection of a naïve phage library and the construction and screening of immunized phage libraries from murine samples. In addition to libraries construction, the murine samples have been used for immune repertoires analysis by Next Generation Sequencing. Both the approaches have led to the identification of highly sensitive and specific antibodies. Two of them have been characterized, defining their binding and affinity properties for the target through epitope mapping, ELISA and Western Blot assays. All the collected data suggest the high potentiality of the produced reagents for WNV immunodiagnostic applications.

IDENTIFICATION AND PRODUCTION OF RECOMBINANT ANTIBODIES TARGETING WEST NILE VIRUS THROUGH NAÏVE AND IMMUNIZED PHAGE-DISPLAY LIBRARIES

RIZZO, SERENA
2019-03-29

Abstract

West Nile virus (WNV) is a member of the Flavivirus genus, which includes several human pathogens transmitted by arthropod vectors. The maintenance in nature of WNV is possible through a bird-mosquito life cycle, in which mosquitoes act as transmission vectors and birds as virus amplification hosts. Humans are not able to contribute to WNV transmission cycle, therefore are defined accidental or dead end hosts. The 20% of WNV infected patients develop a mild disease with influenza like symptoms known as West Nile fever, while in the 1% of cases the viral infection results in a severe neuroinvasive disease, which can lead to death. The most widely used approaches for WNV diagnosis relay on the detection of specific antibodies against viral antigens and are generally based on ELISA format assays. The weak points of the immunodiagnostic tests are the false positive results deriving by cross-reactive antibodies. The rise of such antibodies is caused by the structural similarity of flaviviruses. The diagnosis becomes particularly complicate in geographical areas interested by the co-circulation of more than one flavivirus or vaccination programs leading to the immune system pre-exposure to flaviviral antigens, as occurs in some European areas interested by the co-circulation of WNV and Tick-Borne Encephalitis virus (TBEV). In addition, the vaccination against TBEV is extensively applied in north Eastern Europe countries. In order to improve the detection of WNV infection, the needed reagents for immunodiagnostic applications have been produced. Such reagents include viral antigens and highly specific antibodies against the antigens. As most of commercial kit for WNV diagnosis provide the use of the inactivated virus, requiring specialized equipment and extensive production procedures, a fast and easy protocol has been developed for viral antigens expression and purification. The quality of the produced antigens, in terms of purity, integrity and antigenicity, has been confirmed by SDS PAGE, Western Blot and ELISA assays with murine immunized sera. The isolation of antibodies specifically targeting the WNV antigens has been based on two strategies: the selection of a naïve phage library and the construction and screening of immunized phage libraries from murine samples. In addition to libraries construction, the murine samples have been used for immune repertoires analysis by Next Generation Sequencing. Both the approaches have led to the identification of highly sensitive and specific antibodies. Two of them have been characterized, defining their binding and affinity properties for the target through epitope mapping, ELISA and Western Blot assays. All the collected data suggest the high potentiality of the produced reagents for WNV immunodiagnostic applications.
29-mar-2019
SBLATTERO, DANIELE
31
2017/2018
Settore BIO/13 - Biologia Applicata
Università degli Studi di Trieste
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/2991028
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