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José M. Escribano - One of the best experts on this subject based on the ideXlab platform.
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In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors
Journal of Virological Methods, 2017Co-Authors: Eva Guijarro-pardo, Silvia Gómez-sebastián, José M. EscribanoAbstract:Trichoplusia ni Insect Larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to Insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of Larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in Insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the Insect Larvae (Trichoplusia ni) by feeding, formulated in the Insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in Insect Larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected Larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a detailed comparison of production yields reached by injection vs oral infections for different recombinant proteins. In conclusion, these results open the possibility of future industrial scaling-up production of recombinant proteins in Insect Larvae by reducing manual operations.
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Rotavirus A-specific single-domain antibodies produced in baculovirus-infected Insect Larvae are protective in vivo
BMC biotechnology, 2012Co-Authors: Silvia Gómez-sebastián, Andrés Wigdorovitz, María C. Nuñez, Lorena Garaicoechea, Carmen Alvarado, Marina Mozgovoj, Rodrigo Lasa, Alan Kahl, Viviana Parreño, José M. EscribanoAbstract:Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli, yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System (IBES® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni Larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in Insect Larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from Larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after Insect processing (1 L extract represents about 125 g of Insect biomass or about 375 Insect Larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo, neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. Our results open up the possibility of using Insects as living biofactories (IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that Insects have been used as living biofactories to produce a VHH molecule.
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Rotavirus A-specific single-domain antibodies produced in baculovirus-infected Insect Larvae are protective in vivo
BMC Biotechnology, 2012Co-Authors: Silvia Gómez-sebastián, Andrés Wigdorovitz, María C. Nuñez, Lorena Garaicoechea, Carmen Alvarado, Marina Mozgovoj, Rodrigo Lasa, Alan Kahl, Viviana Parreño, José M. EscribanoAbstract:Background Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli , yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. Results Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System ( IBES ® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni Larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in Insect Larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from Larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after Insect processing (1 L extract represents about 125 g of Insect biomass or about 375 Insect Larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo , neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. Conclusions Our results open up the possibility of using Insects as living biofactories ( IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that Insects have been used as living biofactories to produce a VHH molecule.
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Insect Larvae biofactories as a platform for influenza vaccine production.
Protein expression and purification, 2011Co-Authors: E. Gómez-casado, Silvia Gómez-sebastián, María C. Nuñez, R. Lasa-covarrubias, S. Martínez-pulgarín, José M. EscribanoAbstract:Abstract Increased production capacity is one of the most important priorities for seasonal and pandemic influenza vaccines. In the present study, we used a baculovirus-Insect Larvae system (considered small, living biofactories) to improve the production of recombinant influenza virus H1N1 hemagglutinin (HA). Insect Larvae produced four-fold more HA protein than Insect cells per biomass unit (1 g of fresh Larvae weight). A single infected Trichoplusia ni larva produced up to 113 μg of soluble and easily purified recombinant HA, an amount similar to that produced by 1.2 × 10 8 Sf21 Insect cells infected by the same baculovirus. The use of the KDEL endoplasmic reticulum retention signal fused to the HA protein further increased recombinant protein production. Larvae-derived HA was immunogenically functional in vaccinated mice, inducing the generation of hemagglutination inhibition antibodies and a protective immune response against a lethal challenge with a highly virulent virus. The productivity, scalability and cost efficiency of small, living biofactories based on Insect Larvae suggest a broad-based strategy for the production of recombinant subunit vaccines against seasonal or pandemic influenza as an alternative to fermentation technologies.
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Recombinant West Nile virus envelope protein E and domain III expressed in Insect Larvae protects mice against West Nile disease.
Vaccine, 2011Co-Authors: Julio Alonso-padilla, José M. Escribano, Nereida Jiménez De Oya, Ana-belén Blázquez, Estela Escribano-romero, Juan-carlos SaizAbstract:In this study, West Nile virus (WNV) envelope (rE) protein and its domain III (rDIII) were efficiently expressed in a cost-effective system based on Insect Larvae as non-fermentative living biofactories. Mice immunized with the partially purified rE or rDIII elicited high antibodies titers that neutralized viral infectivity in cell culture and in suckling mice. All vaccinated animals were fully protected when challenged with neurovirulent WNV NY99. Passive transfer of protective antibodies from immunized mothers to their offspring occurred both by transplacental and lactation routes. These results indicate that the Insect-derived antigens tested may constitute potential vaccine candidates to be further evaluated.
Silvia Gómez-sebastián - One of the best experts on this subject based on the ideXlab platform.
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In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors
Journal of Virological Methods, 2017Co-Authors: Eva Guijarro-pardo, Silvia Gómez-sebastián, José M. EscribanoAbstract:Trichoplusia ni Insect Larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to Insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of Larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in Insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the Insect Larvae (Trichoplusia ni) by feeding, formulated in the Insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in Insect Larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected Larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a detailed comparison of production yields reached by injection vs oral infections for different recombinant proteins. In conclusion, these results open the possibility of future industrial scaling-up production of recombinant proteins in Insect Larvae by reducing manual operations.
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Rotavirus A-specific single-domain antibodies produced in baculovirus-infected Insect Larvae are protective in vivo
BMC biotechnology, 2012Co-Authors: Silvia Gómez-sebastián, Andrés Wigdorovitz, María C. Nuñez, Lorena Garaicoechea, Carmen Alvarado, Marina Mozgovoj, Rodrigo Lasa, Alan Kahl, Viviana Parreño, José M. EscribanoAbstract:Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli, yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System (IBES® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni Larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in Insect Larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from Larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after Insect processing (1 L extract represents about 125 g of Insect biomass or about 375 Insect Larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo, neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. Our results open up the possibility of using Insects as living biofactories (IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that Insects have been used as living biofactories to produce a VHH molecule.
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Rotavirus A-specific single-domain antibodies produced in baculovirus-infected Insect Larvae are protective in vivo
BMC Biotechnology, 2012Co-Authors: Silvia Gómez-sebastián, Andrés Wigdorovitz, María C. Nuñez, Lorena Garaicoechea, Carmen Alvarado, Marina Mozgovoj, Rodrigo Lasa, Alan Kahl, Viviana Parreño, José M. EscribanoAbstract:Background Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli , yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. Results Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System ( IBES ® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni Larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in Insect Larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from Larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after Insect processing (1 L extract represents about 125 g of Insect biomass or about 375 Insect Larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo , neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. Conclusions Our results open up the possibility of using Insects as living biofactories ( IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that Insects have been used as living biofactories to produce a VHH molecule.
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Antibody recognition of the glycoprotein g of viral haemorrhagic septicemia virus (VHSV) purified in large amounts from Insect Larvae
BMC research notes, 2011Co-Authors: P. Encinas, E. Gómez-casado, Silvia Gómez-sebastián, María C. Nuñez, Jose Angel Escribano, Amparo Estepa, Julio CollAbstract:There are currently no purification methods capable of producing the large amounts of fish rhabdoviral glycoprotein G (gpG) required for diagnosis and immunisation purposes or for studying structure and molecular mechanisms of action of this molecule (ie. pH-dependent membrane fusion). As a result of the unavailability of large amounts of the gpG from viral haemorrhagic septicaemia rhabdovirus (VHSV), one of the most dangerous viruses affecting cultured salmonid species, research interests in this field are severely hampered. Previous purification methods to obtain recombinant gpG from VHSV in E. coli, yeast and baculovirus grown in Insect cells have not produced soluble conformations or acceptable yields. The development of large-scale purification methods for gpGs will also further research into other fish rhabdoviruses, such as infectious haematopoietic necrosis virus (IHNV), spring carp viremia virus (SVCV), hirame rhabdovirus (HIRRV) and snakehead rhabdovirus (SHRV). Here we designed a method to produce milligram amounts of soluble VHSV gpG. Only the transmembrane and carboxy terminal-deleted (amino acid 21 to 465) gpG was efficiently expressed in Insect Larvae. Recognition of G21-465 by s-mercaptoethanol-dependent neutralizing monoclonal antibodies (N-MAbs) and pH-dependent recognition by sera from VHSV-hyperimmunized or VHSV-infected rainbow trout (Oncorhynchus mykiss) was demonstrated. Given that the purified G21-465 conserved some of its most important properties, this method might be suitable for the large-scale production of fish rhabdoviral gpGs for use in diagnosis, fusion and antigenicity studies.
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Insect Larvae biofactories as a platform for influenza vaccine production.
Protein expression and purification, 2011Co-Authors: E. Gómez-casado, Silvia Gómez-sebastián, María C. Nuñez, R. Lasa-covarrubias, S. Martínez-pulgarín, José M. EscribanoAbstract:Abstract Increased production capacity is one of the most important priorities for seasonal and pandemic influenza vaccines. In the present study, we used a baculovirus-Insect Larvae system (considered small, living biofactories) to improve the production of recombinant influenza virus H1N1 hemagglutinin (HA). Insect Larvae produced four-fold more HA protein than Insect cells per biomass unit (1 g of fresh Larvae weight). A single infected Trichoplusia ni larva produced up to 113 μg of soluble and easily purified recombinant HA, an amount similar to that produced by 1.2 × 10 8 Sf21 Insect cells infected by the same baculovirus. The use of the KDEL endoplasmic reticulum retention signal fused to the HA protein further increased recombinant protein production. Larvae-derived HA was immunogenically functional in vaccinated mice, inducing the generation of hemagglutination inhibition antibodies and a protective immune response against a lethal challenge with a highly virulent virus. The productivity, scalability and cost efficiency of small, living biofactories based on Insect Larvae suggest a broad-based strategy for the production of recombinant subunit vaccines against seasonal or pandemic influenza as an alternative to fermentation technologies.
María C. Nuñez - One of the best experts on this subject based on the ideXlab platform.
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Rotavirus A-specific single-domain antibodies produced in baculovirus-infected Insect Larvae are protective in vivo
BMC biotechnology, 2012Co-Authors: Silvia Gómez-sebastián, Andrés Wigdorovitz, María C. Nuñez, Lorena Garaicoechea, Carmen Alvarado, Marina Mozgovoj, Rodrigo Lasa, Alan Kahl, Viviana Parreño, José M. EscribanoAbstract:Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli, yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System (IBES® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni Larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in Insect Larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from Larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after Insect processing (1 L extract represents about 125 g of Insect biomass or about 375 Insect Larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo, neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. Our results open up the possibility of using Insects as living biofactories (IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that Insects have been used as living biofactories to produce a VHH molecule.
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Rotavirus A-specific single-domain antibodies produced in baculovirus-infected Insect Larvae are protective in vivo
BMC Biotechnology, 2012Co-Authors: Silvia Gómez-sebastián, Andrés Wigdorovitz, María C. Nuñez, Lorena Garaicoechea, Carmen Alvarado, Marina Mozgovoj, Rodrigo Lasa, Alan Kahl, Viviana Parreño, José M. EscribanoAbstract:Background Single-domain antibodies (sdAbs), also known as nanobodies or VHHs, are characterized by high stability and solubility, thus maintaining the affinity and therapeutic value provided by conventional antibodies. Given these properties, VHHs offer a novel alternative to classical antibody approaches. To date, VHHs have been produced mainly in E. coli , yeast, plants and mammalian cells. To apply the single-domain antibodies as a preventive or therapeutic strategy to control rotavirus infections in developing countries (444,000 deaths in children under 5 years of age) has to be minimized their production costs. Results Here we describe the highly efficient expression of functional VHHs by the Improved Baculovirus Expression System ( IBES ® technology), which uses a baculovirus expression vector in combination with Trichoplusia ni Larvae as living biofactories. Two VHHs, named 3B2 and 2KD1, specific for the inner capsid protein VP6 of Group A rotavirus, were expressed in Insect Larvae. The IBES® technology achieved very high expression of 3B2 and 2KD1, reaching 2.62% and 3.63% of the total soluble protein obtained from Larvae, respectively. These expression levels represent up to 257 mg/L of protein extract after Insect processing (1 L extract represents about 125 g of Insect biomass or about 375 Insect Larvae). Larva-derived antibodies were fully functional when tested in vitro and in vivo , neutralizing Group A rotaviruses and protecting offspring mice against rotavirus-induced diarrhea. Conclusions Our results open up the possibility of using Insects as living biofactories ( IBES® technology) for the cost-efficient production of these and other fully functional VHHs to be used for diagnostic or therapeutic purposes, thereby eliminating concerns regarding the use of bacterial or mammalian cells. To the best of our knowledge, this is the first time that Insects have been used as living biofactories to produce a VHH molecule.
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Antibody recognition of the glycoprotein g of viral haemorrhagic septicemia virus (VHSV) purified in large amounts from Insect Larvae
BMC research notes, 2011Co-Authors: P. Encinas, E. Gómez-casado, Silvia Gómez-sebastián, María C. Nuñez, Jose Angel Escribano, Amparo Estepa, Julio CollAbstract:There are currently no purification methods capable of producing the large amounts of fish rhabdoviral glycoprotein G (gpG) required for diagnosis and immunisation purposes or for studying structure and molecular mechanisms of action of this molecule (ie. pH-dependent membrane fusion). As a result of the unavailability of large amounts of the gpG from viral haemorrhagic septicaemia rhabdovirus (VHSV), one of the most dangerous viruses affecting cultured salmonid species, research interests in this field are severely hampered. Previous purification methods to obtain recombinant gpG from VHSV in E. coli, yeast and baculovirus grown in Insect cells have not produced soluble conformations or acceptable yields. The development of large-scale purification methods for gpGs will also further research into other fish rhabdoviruses, such as infectious haematopoietic necrosis virus (IHNV), spring carp viremia virus (SVCV), hirame rhabdovirus (HIRRV) and snakehead rhabdovirus (SHRV). Here we designed a method to produce milligram amounts of soluble VHSV gpG. Only the transmembrane and carboxy terminal-deleted (amino acid 21 to 465) gpG was efficiently expressed in Insect Larvae. Recognition of G21-465 by s-mercaptoethanol-dependent neutralizing monoclonal antibodies (N-MAbs) and pH-dependent recognition by sera from VHSV-hyperimmunized or VHSV-infected rainbow trout (Oncorhynchus mykiss) was demonstrated. Given that the purified G21-465 conserved some of its most important properties, this method might be suitable for the large-scale production of fish rhabdoviral gpGs for use in diagnosis, fusion and antigenicity studies.
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Insect Larvae biofactories as a platform for influenza vaccine production.
Protein expression and purification, 2011Co-Authors: E. Gómez-casado, Silvia Gómez-sebastián, María C. Nuñez, R. Lasa-covarrubias, S. Martínez-pulgarín, José M. EscribanoAbstract:Abstract Increased production capacity is one of the most important priorities for seasonal and pandemic influenza vaccines. In the present study, we used a baculovirus-Insect Larvae system (considered small, living biofactories) to improve the production of recombinant influenza virus H1N1 hemagglutinin (HA). Insect Larvae produced four-fold more HA protein than Insect cells per biomass unit (1 g of fresh Larvae weight). A single infected Trichoplusia ni larva produced up to 113 μg of soluble and easily purified recombinant HA, an amount similar to that produced by 1.2 × 10 8 Sf21 Insect cells infected by the same baculovirus. The use of the KDEL endoplasmic reticulum retention signal fused to the HA protein further increased recombinant protein production. Larvae-derived HA was immunogenically functional in vaccinated mice, inducing the generation of hemagglutination inhibition antibodies and a protective immune response against a lethal challenge with a highly virulent virus. The productivity, scalability and cost efficiency of small, living biofactories based on Insect Larvae suggest a broad-based strategy for the production of recombinant subunit vaccines against seasonal or pandemic influenza as an alternative to fermentation technologies.
María Victoria Miranda - One of the best experts on this subject based on the ideXlab platform.
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Expression of recombinant glutamic acid decarboxylase in Insect Larvae and its application in an immunoassay for the diagnosis of autoimmune diabetes mellitus.
Scientific reports, 2019Co-Authors: Aldana Trabucchi, Alexandra Marisa Targovnik, María Victoria Miranda, Silvina S. Bombicino, Juan Ignacio Marfía, Adriana Victoria Sabljic, Natalia Ines Faccinetti, Luciano Lucas Guerra, Rubén F. Iacono, Silvina N. ValdezAbstract:Autoimmune Diabetes Mellitus (DM) is a chronic disease caused by the selective destruction of insulin producing beta cells in human pancreas. DM is characterized by the presence of autoantibodies that bind a variety of islet-cell antigens. The 65 kDa isoform of glutamate decarboxylase (GAD65) is a major autoantigen recognized by these autoantibodies. Autoantibodies to GAD65 (GADA) are considered predictive markers of the disease when tested in combination with other specific autoantibodies. In order to produce reliable immunochemical tests for large scale screening of autoimmune DM, large amounts of properly folded GAD65 are needed. Herein, we report the production of human GAD65 using the baculovirus expression system in two species of Larvae, Rachiplusia nu and Spodoptera frugiperda. GAD65 was identified at the expected molecular weight, properly expressed with high yield and purity in both Larvae species and presenting appropriate enzymatic activity. The immunochemical ability of recombinant GAD65 obtained from both Larvae to compete with [35S]GAD65 was assessed qualitatively by incubating GADA-positive patients’ sera in the presence of 1 μM of the recombinant enzyme. All sera tested became virtually negative after incubation with antigen excess. Besides, radiometric quantitative competition assays with GADA-positive patients’ sera were performed by adding recombinant GAD65 (0.62 nM–1.4 µM). All dose response curves showed immunochemical identity between proteins. In addition, a bridge-ELISA for the detection of GADA was developed using S. frugiperda-GAD65. This assay proved to have 77.3% sensitivity and 98.2% of specificity. GAD65 could be expressed in Insect Larvae, being S. frugiperda the best choice due to its high yield and purity. The development of a cost effective immunoassay for the detection of GADA was also afforded.
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Production of biologically active feline interferon beta in Insect Larvae using a recombinant baculovirus.
3 Biotech, 2018Co-Authors: Mariana Bernadett Arregui, Alexandra Marisa Targovnik, Federico Javier Wolman, Gregorio Juan Mc Callum, Ignacio Smith, Marcela Solange Villaverde, María Victoria MirandaAbstract:Feline interferon beta is a cytokine that belongs to the type I IFN family, with antitumor, antiviral and immunomodulatory functions. In this work, recombinant feline interferon beta (rFeIFNβ) was expressed in Insect Larvae that constitute important agronomic plagues. rFeIFNβ accumulated in the hemolymph of Spodoptera frugiperda Larvae infected with recombinant baculovirus and was purified by Blue-Sepharose chromatography directly from larval homogenates on day 4 post-infection. rFeIFNβ was recovered after purification with a specific activity of 1 × 106 IU mg-1. By this method, we obtained 8.9 × 104 IU of purified rFeIFNβ per larva. The product was biologically active in vitro, with an antiviral activity of 9.5 × 104 IU mL-1, as well as a potent antitumor activity comparable to that of the commercial FeIFNω. The glycosylation of rFeIFNβ was confirmed by peptide-N-glycosidase F digestion. Our findings provide a cost-effective platform for large-scale rFeIFNβ production in laboratory research or veterinary medicine applications.
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Expression and purification of recombinant feline interferon in the baculovirus-Insect Larvae system
Process Biochemistry, 2014Co-Authors: Alexandra Marisa Targovnik, Mariana Bernadett Arregui, Osvaldo Cascone, Oscar Taboga, Marcela Solange Villaverde, Mariela Noemi Fogar, Gerardo C. Glikin, Liliana María Elena Finocchiaro, María Victoria MirandaAbstract:Abstract Feline interferons (FeIFNs) are cytokines with antiviral, antitumor and immunomodulatory functions used as therapeutic agents in a variety of veterinary diseases. In this work, FeIFN-α7 and FeIFN-α7xArg containing eight residues of arginine were expressed in Sf9 cells and Insect Larvae. At 4 days post-infection (dpi), the concentrations of FeIFN-α7 and FeIFN-α7xArg in suspension culture were (1.28 ± 0.15) × 106 U ml−1 and (1.3 ± 0.2) × 106 U ml−1 respectively. The maximum expression levels of FeIFN-α7 and FeIFN-α7xArg were (3.7 ± 0.2) × 106 U ml−1 and (3.5 ± 0.4) × 106 U ml−1 at 2 dpi in Rachiplusia nu Larvae and (1.1 ± 0.2) × 106 U ml−1 and (1.0 ± 0.15) × 106 U ml−1 at 5 dpi in Spodoptera frugiperda Larvae respectively. R. nu was a better host for FeIFN-α7 and FeIFN-α7xArg expression. The 8xArg tag did not affect the biological activity of FeIFN-α7 and was useful to promote the FeIFN-α7xArg adsorption on ion exchange chromatography (IEC), allowing its purification in a single step from supernatant culture and R. nu Larvae. FeIFN-α7xArg was purified from the larval extract with a yield of 70% and a purification factor of 25 free of viruses. We conclude that R. nu Larvae are new low-cost hosts for the expression of recombinant FeIFN-α7.
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Extractive purification of recombinant peroxidase isozyme c from Insect Larvae in aqueous two-phase systems
Separation and Purification Technology, 2012Co-Authors: A. M. Targovnik, Osvaldo Cascone, María Victoria MirandaAbstract:Abstract Aqueous two-phase systems (ATPSs) have not yet been applied to purify proteins expressed in Insect Larvae infected by recombinant baculovirus. This work describes the behavior of typical contaminants in the baculovirus-Insect Larvae expression system such as larval proteins and baculovirus particles in PEG/phosphate ATPSs, in addition to the extraction and purification of the target protein (horseradish peroxidase isozyme C, HRPC). After assessing the influence of PEG molecular weight, system pH and added salt on the partition constants of HRPC and total protein of a clarified Larvae extract, two ATPSs were selected for the first extraction step: PEG 1500/phosphate, pH 7.0 with 4.0% NaCl (System 1) and PEG/phosphate, pH 5.0 in the absence of NaCl (System 2). Both systems were found to be appropriate since a clarified enzyme-enriched top phase was obtained with a yield of 99% and 90% respectively. The direct partition of Larvae homogenized with the components of Systems 1 and 2, yielded a HRPC recovery in top phase of 71.4% and 81.1% respectively, whereas total protein recovery was 5.2% and 3.3% respectively. In both systems, the top phase was clear and particulate material remained in the interphase and the bottom phase. The bulk of immunogenic proteins of the Larvae concentrated in the bottom phase of both systems. The PCR assay revealed the presence of viral DNA in both phases. It was possible to extract the HRPC back from the PEG-rich phase by adding a fresh magnesium sulfate solution to form a new ATPS, achieving a recovery in the bottom phase of 50% and 98% in Systems 1 and 2 respectively, whereas the recovery of total protein was 69% and 24% respectively. The HRPC global recovery of the two-step processes was 35.4% and 79.6% for Systems 1 and 2, with purification factors of 14.5 and 114.2 respectively. The final product was free of viral particles.
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Expression and purification of horseradish peroxidase in Insect Larvae
Process Biochemistry, 2008Co-Authors: María De Las Nieves Loustau, Osvaldo Cascone, Lucía Romero, Gustavo Levin, María L. Magri, María Gabriela López, Oscar Taboga, María Victoria MirandaAbstract:Abstract A strategy for obtaining a high-level expression of horseradish peroxidase isozyme C (HRPC) in Insect Larvae is herein described. The HRPC-6xHis coding sequence was inserted into the AcNPV genome via the pAcGP67HRPC-6xHis transfer vector to originate an AcHRPC-6xHis recombinant baculovirus of phenotype occ − . Rachiplusia nu Larvae were injected with a viral stock derived from culture supernatants of Sf9 cells infected with AcHRPC-6xHis. Enzyme concentration at day 3 post-infection was 230 ± 10 mg/kg haemolymph and 100 ± 14 mg/kg Larvae, an expression level never attained by other expression systems. HRPC-6xHis was purified from the crude larval extract or haemolymph by immobilised metal ion affinity chromatography with yields of 88.8% and 89.0% and purification factors of 18.9 and 14.0, respectively, in a single step. The purity of the final product was 90%.
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Using SDS-PAGE gel fingerprinting to identify soft-bodied wood-boring Insect Larvae to species.
Pest Management Science, 2017Co-Authors: Mark A. O’neill, Mia Denos, Daniel ReedAbstract:BACKGROUND: This paper describes the progress that we have made in assessing the feasibility of 'fingerprinting' using imaged SDS-PAGE gels of haemolymph proteins, to identify soft-bodied wood-boring Insect Larvae such as the Asian longhorn beetle, Anoplophora glabripennis (Motscholsky, 1853) (Coleoptera: Cerambycidae). Because of stringent import restrictions and difficulty in obtaining licences to work with these organisms, we opted to work with four species of scarab beetle, Mecynorhina polyphemus (Fabricius, 1781), Pachnoda sinuata (Fabricius, 1775), Eucidella shiratica (Csiki, 1909) and Eucidella shultzeorum (Kolbe, 1906) which have near identical larval morphologies. RESULTS: We show that this technology when combined with an advanced pattern matching system (Digital Automated Identification SYstem - DAISY) can classify soft-bodied Insect Larvae that are almost identical morphologically to species at a level of accuracy is in excess of 98%. The study also indicates that the technology copes well with noisy data and small training sets. CONCLUSION: The experience gained in undertaking this study gives us confidence that we will be able to develop a field deployable system in the medium term. We believe that as a high-throughput identification tool, this technology is superior to competitor technologies (e.g. fingerprinting of imaged DNA gels) in terms of speed, cost and ease of use; and therefore, is suitable for low-cost deployment in the field. © 2017 Society of Chemical Industry.