Plasticity of the HEK-293 cells, related to the culture media, as platform to produce a subunit vaccine against classical swine fever virus.

Classical swine fever (CSF) is a contagious disease that causes a high mortality to domestic and wild pigs. Its causative agent is an enveloped Pestivirus named Classical Swine Fever Virus (CSFV). Due to the huge economic affectations produced by this disease to porcine industry, several vaccines have been developed using principally the CSFV E2 glycoprotein. Recently, a subunit vaccine based on this structural protein of the CSFV fused to the porcine CD154 molecule as immunomodulator named E2-CD154 was assayed by us. This chimeric protein was produced in the Human Embryonic Kidney (HEK-293) cell line. In this work, the growth and the expression profiles of HEK-293 E2-CD154 cells in four commercially available culture media were studied. The oligosaccharide structures in the N-glycosylation patterns of the E2-CD154 protein produced by this cell line in 10 L fermenters with two different culture media were also analyzed. In addition, the neutralizing antibody response generated in mice vaccinated with these antigens was assayed. Our results suggest that the culture media CDM4HEK293 and SFM4HEK293 which are recommended for HEK-293 growth are the best choice to growth the cell clone expressing the E2-CD154 protein. The glycosylation pattern and the neutralizing antibody response generated by the E2-CD154 protein were independent of the culture medium used which demonstrates the high reproducibility and consistency among protein batches produced by HEK-293 cells even in different culture conditions.

Determinación in silico de las propiedades inmunogénicas de la glicoproteína E de Zika virus / In silico determination of immunogenic properties of glycoprotein E from Zika Virus

Introducción: La infección por Zika virus (ZIKV) ha sido asociada a múltiples complicaciones y nuevas formas de transmisión. La descripción del genoma y la estructura cristalizada permiten desarrollar análisis moleculares, incluyendo las propiedades inmunológicas. Objetivos: En este trabajo, se analiza a la glicoproteína E de ZIKV, con el fin de determinar su utilidad en la creación de una vacuna proteica recombinante. Métodos: Se analizó la glicoproteína E, por medio del software DNASTAR, en base a su antigenicidad de epítopos de células B y MHC-II, estructura secundaria, hidrofilizada, flexibilidad y accesibilidad a solvente en el virión maduro e hidratado. Resultados: Se identificaron 14 sitios antigénicos para células B, de los cuales, 7 comparten su antigenicidad para MHC-II. Al tomar en cuenta los demás parámetros analizados, los sitios se reducen a 3, con longitudes de 13, 9 y 5 aminoácidos. Conclusiones: La glicoproteína E de ZIKV podría desencadenar una respuesta inmune T-dependiente, por tanto, ser útil para la creación de una vacuna proteica recombinante.

Introduction: Zika virus (ZIKV) infection have been associated with multiple complications and new ways of transmission. The description of the genome and the crystalized structure allow the performance of molecular analysis, including immunological properties. Objectives: In this paper, we analyze glycoprotein E from ZIKV to determine its utility in the development of a recombinant protein vaccine. Methods: The protein was analyzed with the software DNASTAR, through the following properties: B cells and MHC-II antigenicity, secondary structure, hydrophilicity, flexibility and solvent-accessibility in the mature and hydrated virion. Results: We identified 14 antigenic sites with B-cells antigenicity, 7 of which shared the antigenicity for MHC-II. Considering other parameters analyzed, sites were reduced to 3, with length of 13, 9 and 5 amino acids. Conclusions: Glycoprotein E, from ZIKV, could trigger a T-dependent immune response, and therefore, may be useful in the creation of a recombinant protein vaccine.

A single dose of the novel chimeric subunit vaccine E2-CD154 confers early full protection against classical swine fever virus.

Classical swine fever is an economically important, highly contagious disease of swine worldwide. Subunit vaccines are a suitable alternative for the control of classical swine fever. However, such vaccines have as the main drawback the relatively long period of time required to induce a protective response, which hampers their use under outbreak conditions. In this work, a lentivirus-based gene delivery system is used to obtain a stable recombinant HEK 293 cell line for the expression of E2-CSFV antigen fused to porcine CD154 as immunostimulant molecule. The E2-CD154 chimeric protein was secreted into the medium by HEK293 cells in a concentration around 50mg/L in suspension culture conditions using spinner bottles. The E2-CD154 immunized animals were able to overcome the challenge with a high virulent CSF virus strain performed 7days after a unique dose of the vaccine without clinical manifestations of the disease. Specific anti-CSFV neutralizing antibodies and IFN-γ were induced 8days after challenge equivalent to 14days post-vaccination. The present work constitutes the first report of a subunit vaccine able to confer complete protection by the end of the first week after a single vaccination. These results suggest that the E2-CD154 antigen could be potentially used under outbreak conditions to stop CSFV spread and for eradication programs in CSF enzootic areas.

Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE∆) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric â-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK∆) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE∆) and TK + gE (BoHV-5 gE/TK∆) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

Immunogenicity of an inactivated bovine herpesvirus type 5 strain defective in thymidine kinase and glycoprotein E / Imunogenicidade de uma cepa inativada do herpesvírus bovino tipo 5 defectiva na timidina quinase e glicoproteína E

The immunogenicity of an inactivated, experimental vaccine based on a bovine herpesvirus type 5 strain defective in thymidine kinase and glycoprotein E (BoHV-5 gE/TKΔ) was evaluated in cattle and the results were compared with a vaccine containing the parental BoHV-5 strain (SV507/99). To formulate the vaccines, each virus (wildtype SV507/99 and BoHV-5 gE/TK∆) was multiplied in cell culture and inactivated with binary ethyleneimine (BEI). Each vaccine dose contained approximately of 10(7.5) TCID50 of inactivated virus mixed with an oil-based adjuvant (46:54). Forty calves, 6 to 9-months-old, were allocated into two groups of 20 animals each and vaccinated twice (days 0 and 22pv) by the subcutaneous route with either vaccine. Serum samples collected at day 0 and at different intervals after vaccination were tested for virus neutralizing (VN) antibodies against the parental virus and against heterologous BoHV-5 and BoHV-1 isolates. The VN assays demonstrated seroconversion to the respective homologous viruses in all vaccinated animals after the second vaccine dose (mean titers of 17.5 for the wildtype vaccine; 24.1 for the recombinant virus). All animals remained reagents up to day 116 pv, yet showing a gradual reduction in VN titers. Animals from both vaccine groups reacted in similar VN titers to different BoHV-1 and BoHV-5 isolates, yet the magnitude of serological response of both groups was higher against BoHV-5 field isolates. Calves vaccinated with the recombinant virus did not develop antibodies to gE as verified by negative results in a gE-specific ELISA, what would allow serological differentiation from naturally infected animals. Taken together, these results indicate that inactivated antigens of BoHV-5 gE/TK recombinant virus induced an adequate serological response against BoHV-5 and BoHV-1 and thus can be used as an alternative, differential vaccine candidate.

A imunogenicidade de vacina experimental inativada, produzida com uma cepa do herpesvírus bovino tipo 5 defectiva nos genes da timidina quinase e glicoproteína E (BoHV-5 gE/TKΔ) foi avaliada em bovinos e o resultado foi comparado com a resposta induzida pela cepa parental do BoHV-5 (SV507/99). Para a formulação da vacina, cultivos de células infectados com cada um dos vírus (SV507/99 ou BoHV-5 gE/TKΔ) foram inativados com etilenamina binária. Cada dose de vacina continha aproximadamente 107,5 TCID50 de um dos vírus inativados emulsionado em adjuvante oleoso (46:54). Quarenta bezerros de raças cruzadas, com idade entre seis a nove meses, foram alocados em dois grupos de 20 animais cada e vacinados duas vezes (dia 0 e 22 pv) pela via subcutânea com uma das vacinas. Amostras de soro foram coletadas no dia 0 e a vários intervalos após vacinação para a pesquisa de anticorpos neutralizantes frente ao vírus homólogo ou frente a isolados de BoHV-5 e BoHV-1. Os testes de soroneutralização (SN) demonstraram que todos os animais soroconverteram após a segunda dose da vacina (títulos médios de 17,5 para o grupo SV507/99; 24,1 para o grupo BoHV-5 gE/TKΔ). Todos os animais mantiveram níveis de anticorpos neutralizantes até o dia 116 pv, no entanto foi observada uma redução gradual no títulos. A sorologia cruzada com amostras heterólogas do BoHV-5 e BoHV-1 indicou que ambos os grupos vacinais reagiram em níveis similares frente ao mesmo vírus, no entanto a magnitude da resposta sorológica foi maior frente a amostras de BoHV-5. Os animais vacinados com a cepa recombinante não desenvolveram anticorpos contra a gE detectáveis por um ELISA específico, o que permitiria a sua diferenciação sorológica de animais infectados naturalmente. Esses resultados demonstram que a vacina contendo antígenos inativados do vírus recombinante BoHV-5 gE/TKΔ induziu resposta sorológica em níveis satisfatórios, constituindo-se, assim, em alternativa a cepa ...

Immunogenicity of an inactivated bovine herpesvirus type 5 strain defective in thymidine kinase and glycoprotein E / Imunogenicidade de uma cepa inativada do herpesvírus bovino tipo 5 defectiva na timidina quinase e glicoproteína E

The immunogenicity of an inactivated, experimental vaccine based on a bovine herpesvirus type 5 strain defective in thymidine kinase and glycoprotein E (BoHV-5 gE/TKΔ) was evaluated in cattle and the results were compared with a vaccine containing the parental BoHV-5 strain (SV507/99). To formulate the vaccines, each virus (wildtype SV507/99 and BoHV-5 gE/TK∆) was multiplied in cell culture and inactivated with binary ethyleneimine (BEI). Each vaccine dose contained approximately of 107.5 TCID50 of inactivated virus mixed with an oil-based adjuvant (46:54). Forty calves, 6 to 9-months-old, were allocated into two groups of 20 animals each and vaccinated twice (days 0 and 22pv) by the subcutaneous route with either vaccine. Serum samples collected at day 0 and at different intervals after vaccination were tested for virus neutralizing (VN) antibodies against the parental virus and against heterologous BoHV-5 and BoHV-1 isolates. The VN assays demonstrated seroconversion to the respective homologous viruses in all vaccinated animals after the second vaccine dose (mean titers of 17.5 for the wildtype vaccine; 24.1 for the recombinant virus). All animals remained reagents up to day 116 pv, yet showing a gradual reduction in VN titers. Animals from both vaccine groups reacted in similar VN titers to different BoHV-1 and BoHV-5 isolates, yet the magnitude of serological response of both groups was higher against BoHV-5 field isolates. Calves vaccinated with the recombinant virus did not develop antibodies to gE as verified by negative results in a gE-specific ELISA, what would allow serological differentiation from naturally infected animals. Taken together, these results indicate that inactivated antigens of BoHV-5 gE/TK recombinant virus induced an adequate serological response against BoHV-5 and BoHV-1 and thus can be used as an alternative, differential vaccine candidate.(AU)

A imunogenicidade de vacina experimental inativada, produzida com uma cepa do herpesvírus bovino tipo 5 defectiva nos genes da timidina quinase e glicoproteína E (BoHV-5 gE/TKΔ) foi avaliada em bovinos e o resultado foi comparado com a resposta induzida pela cepa parental do BoHV-5 (SV507/99). Para a formulação da vacina, cultivos de células infectados com cada um dos vírus (SV507/99 ou BoHV-5 gE/TKΔ) foram inativados com etilenamina binária. Cada dose de vacina continha aproximadamente 107,5 TCID50 de um dos vírus inativados emulsionado em adjuvante oleoso (46:54). Quarenta bezerros de raças cruzadas, com idade entre seis a nove meses, foram alocados em dois grupos de 20 animais cada e vacinados duas vezes (dia 0 e 22 pv) pela via subcutânea com uma das vacinas. Amostras de soro foram coletadas no dia 0 e a vários intervalos após vacinação para a pesquisa de anticorpos neutralizantes frente ao vírus homólogo ou frente a isolados de BoHV-5 e BoHV-1. Os testes de soroneutralização (SN) demonstraram que todos os animais soroconverteram após a segunda dose da vacina (títulos médios de 17,5 para o grupo SV507/99; 24,1 para o grupo BoHV-5 gE/TKΔ). Todos os animais mantiveram níveis de anticorpos neutralizantes até o dia 116 pv, no entanto foi observada uma redução gradual no títulos. A sorologia cruzada com amostras heterólogas do BoHV-5 e BoHV-1 indicou que ambos os grupos vacinais reagiram em níveis similares frente ao mesmo vírus, no entanto a magnitude da resposta sorológica foi maior frente a amostras de BoHV-5. Os animais vacinados com a cepa recombinante não desenvolveram anticorpos contra a gE detectáveis por um ELISA específico, o que permitiria a sua diferenciação sorológica de animais infectados naturalmente. Esses resultados demonstram que a vacina contendo antígenos inativados do vírus recombinante BoHV-5 gE/TKΔ induziu resposta sorológica em níveis satisfatórios, constituindo-se, assim, em alternativa a cepa vacinal diferencial.(AU)

Cloning and expression of Aujeszky's disease virus glycoprotein E (gE) in a baculovirus system

Aujeszky' s disease (AD) is an infectious disease causing important economic losses to the swine industry worldwide. The disease is caused by an alpha-herpesvirus, Aujeszky' s disease virus (ADV), an enveloped virus with a double stranded linear DNA genome. The ADV genome encodes 11 glycoproteins, which are major targets for the immune system of the host in response to the infection. The glycoprotein E (gE) is a non-essential protein and deletion of the gE gene has been used for the production of marker vaccines. Aiming to develop molecular reagents for the production of a gE specific ELISA test, the gE gene was amplified by PCR, cloned and expressed into a baculovirus expression system. The recombinant DNA vector pFastBac-gE-ADV was used for site-specific transposition into the recombinant baculovirus (bacmid). Colonies with recombinant bacmid-pFastBac-gE-ADV were selected by antibiotic and color selection and the presence of the gE gene was confirmed by PCR. The recombinant bacmid-pFastBac-gE-ADV was cotransfected in insect Trichoplusia ni and the presence of the recombinant DNA and gE protein were detected by PCR, SDS-PAGE and Western blotting, respectively.

A doença de Aujeszky (DA) é uma enfermidade infecto-contagiosa que causa grandes perdas econômicas ao produtor e à agroindústria suinícola em todo o mundo. É causada pelo vírus da doença de Aujeszky (VDA), um alfaherpesvírus envelopado com genoma DNA de fita dupla e linear. O genoma do VDA codifica 11 glicoproteínas, as quais são os maiores alvos do sistema imune do hospedeiro em resposta a infecção. A glicoproteína E (gE) é uma proteína não essencial e a deleção do gene da gE é muito utilizada para a produção de vacinas com marcadores. Com o objetivo de desenvolver insumos moleculares para a produção de um teste de ELISA específico para gE do VDA, a seqüência do gene da gE foi amplificada, clonada e expressa no sistema de expressão em baculovírus. O produto da amplificação foi clonado no vetor pGem®-T Easy e subclonado no plasmídeo de expressão pFastBac1. O DNA recombinante pFastBac-gE-VDA foi usado para a transposição sítio-específica no baculovírus recombinante (bacmid). Após seleção por antibióticos e cor, as colônias com o recombinante bacmid-pFastBac-gE-VDA foram selecionadas e a presença do gene da gE foi confirmada por PCR. O DNA recombinante viral, bacmid-pFastBac-gE-VDA, foi usado para cotransfecção de células de inseto Trichoplusia ni e a presença do recombinante e a proteína gE foi determinada por PCR, por SDS-PAGE e Western blotting, respectivamente.