Preliminary binary ethylenimine (BEI) inactivated bovine papillomavirus (BPV) vaccine trial against cutaneous warts in bull calves: a pathological assessment

A preliminary prophylactic and therapeutic vaccine trial was successfully conducted in bull calves with binary ethylenimine (BEI) inactivated saponized crude cutaneous warts bovine papillomavirus 1 and 2 (CW BPV-1 & -2) vaccine. In Prophylactic Group of vaccinated animals no temperature, untoward reaction or nodule formation was seen at vaccine inoculation site. After challenge with BPV inoculum, cutaneous warts (CWs) failed to develop in this group indicating that vaccine was effective. In Therapeutic Group, before vaccination in two animals CWs were experimentally induced. In one animal, luxuriant cauliflower-like and in other linear slow growth was observed. After vaccination, CWs in both animals became dried and growth was arrested. It appeared that tumours were regressing which was confirmed by further clinical observations as very little tumour-scar was left at termination of experiment.Histopathologically, both cases were diagnosed as regressing occult/ early fibroblastic type papilloma. Mononuclear cellular infiltration/ aggregates around hair follicles and in tumour stroma were noted. As compared to first case (cauliflower- like/ fast growing) in second case (linear growth/ fast regressing), in addition to other findings mononuclear cellular inflammatory reaction were more pronounced. The clinical regression results are in accordance with histopathological findings of degree of infiltration of lymphocytes and this trial showed that both type use of BEI inactivated saponized crude CW BPV-1&-2 vaccine gave encouraging results with fast regression/ prevention of CWs.

Preliminary binary ethylenimine (BEI) inactivated bovine papillomavirus (BPV) vaccine trial against cutaneous warts in bull calves: a pathological assessment

A preliminary prophylactic and therapeutic vaccine trial was successfully conducted in bull calves with binary ethylenimine (BEI) inactivated saponized crude cutaneous warts bovine papillomavirus 1 and 2 (CW BPV-1 & -2) vaccine. In Prophylactic Group of vaccinated animals no temperature, untoward reaction or nodule formation was seen at vaccine inoculation site. After challenge with BPV inoculum, cutaneous warts (CWs) failed to develop in this group indicating that vaccine was effective. In Therapeutic Group, before vaccination in two animals CWs were experimentally induced. In one animal, luxuriant cauliflower-like and in other linear slow growth was observed. After vaccination, CWs in both animals became dried and growth was arrested. It appeared that tumours were regressing which was confirmed by further clinical observations as very little tumour-scar was left at termination of experiment.Histopathologically, both cases were diagnosed as regressing occult/ early fibroblastic type papilloma. Mononuclear cellular infiltration/ aggregates around hair follicles and in tumour stroma were noted. As compared to first case (cauliflower- like/ fast growing) in second case (linear growth/ fast regressing), in addition to other findings mononuclear cellular inflammatory reaction were more pronounced. The clinical regression results are in accordance with histopathological findings of degree of infiltration of lymphocytes and this trial showed that both type use of BEI inactivated saponized crude CW BPV-1&-2 vaccine gave encouraging results with fast regression/ prevention of CWs.(AU)

CONSTRUÇÃO DE VLPs (VIRUS-LIKE PARTICLES) A PARTIR DA PROTEÍNA RECOMBINANTE L1 DE PAPILOMAVÍRUS BOVINO TIPO 2 EXPRESSA EM CÉLULAS BSC-40 POR MEIO DE VETOR VIRAL CONSTITUIDO PELO VÍRUS VACCÍNIA

A constante preocupação com a saúde humana e animal tem resultado na evolução de metodologias científicas que utilizam ferramentas biotecnológicas que objetivam o desenvolvimento de novas técnicas, produtos, processos ou adaptações, além de possibilitar a compreensão dos mecanismos biológicos dos organismos. A papilomatose bovina é uma infecção viral que pode ser considerada negligenciada por parte das empresas do setor privado que não buscam soluções para o controle e profilaxia, principalmente porque ocorre com maior frequência em animais de pequenos rebanhos, principalmente dedicados à exploração leiteira. O papilomavírus bovino (BPV), agente etiológico dessa doença, é um vírus não cultivável in vitro. Esse é o principal aspecto biológico que dificulta o desenvolvimento de trabalhos de pesquisa com esse vírus e que impõe a obrigatoriedade da utilização de recursos biotecnológicos para o melhor conhecimento dessa infecção e para o desenvolvimento de métodos de diagnóstico, controle e profilaxia. O objetivo desse estudo foi o desenvolvimento de partículas semelhantes a vírus (VLPs virus-like particles) a partir da proteína L1 recombinante de BPV2 expressa em células de mamíferos (BSC-40) infectadas com vírus vaccínia como vetor. Para facilitar as etapas de recuperação e purificação das VLPs por meio de cromatografia de afinidade foi avaliada a inserção de uma cadeia peptídica constituída por seis moléculas de histidina. A proteína L1 recombinante de BPV2 contendo uma cauda de histidina possibilitou a formação de VLPs que foram visualizadas por microscopia eletrônica de transmissão. A possibilidade do uso das VLPs em sistemas de diagnóstico e, principalmente, para a elaboração de vacinas para a profilaxia da infecção pelo BPV2 em bovinos motivou a solicitação junto ao Instituto Nacional de Propriedade Intelectual (INPI) de um pedido de patente de processo que foi registrado sob número BR 1020140329919.

The constant concern for human and animal health has resulted in the evolution of scientific methodologies using biotechnological tools that aim to develop new techniques, products, processes or adaptations, and allows wider understanding of the biological mechanisms of organisms. Bovine papillomatosis is a viral infection that can be considered neglected by the private companies which do not develop alternatives for the control and prevention, mainly because of occurrence of the disease more frequently in small dairy cattle herds. The bovine papillomavirus (BPV), the etiologic agent of this disease, does not grow in conventional cell culture. This is the main biological aspect which hampers the development of research with this virus and requires the compulsory use of biotechnological resources for better understanding of this infection and the development of methods of diagnosis, control, and prevention. The aim of this study was to develop virus-like particles (VLPs) from BPV2 recombinant L1 protein expressed in mammalian cells (BSC-40) infected with vaccinia virus as vector. In order to facilitate the recovery and purification steps of VLPs by affinity chromatography the inclusion of a peptide chain consisting of six histidine molecules in the recombinant protein was evaluated. The BPV2 recombinant L1 protein, containing a histidine tag, allowed the formation of VLPs that were visualized by transmission electron microscopy. The possibility of the use of VLPs in diagnostic systems and especially for the development of vaccines for prophylaxis of the BPV2 infection in cattle stimulate the request from the National Institute of Industrial Property (INPI) of a process patent application that was registered under number 1020140329919 BR.

Bovine papillomavirus type 4 L1 gene transfection in a Drosophila S2 cell expression system: absence of L1 protein expression

The development of a bovine papillomavirus (BPV) vaccine is an outstanding challenge. BPV protein L1 gene transfection in the Drosophila melanogaster S2 cell expression system failed to produce L1 protein notwithstanding correct L1 gene insertion. Severe genetic inbalance in the host cell line, including cytogenetic alterations, may account for the lack of protein expression.

O desenvolvimento de uma vacina para papilomavirus bovino (BPV) consiste em grande desafio. A transfecção do gene codificante da proteína L1 de BPV em sistema de células S2 de Drosophila melanogaster não logrou sucesso, apesar da correta inserção da seqüência gênica em vetor apropriado.Graves alterações genéticas na linhagem celular S2, que incluem aberrações cromossômicas, provavelmente estão relacionadas à ausência da expressão da proteína desejada.

Expressão e purificação da proteína recombinante L2 do Papilomavírus bovino tipo-2 em sistema bacteriano / Expression and purification of recombinant protein L2 of Bovine papillomavirus type-2 in bacterial system

A papilomatose bovina é uma doença infectocontagiosa de ocorrência mundial, que assola o rebanho brasileiro, sem qualquer atitude efetiva de controle, e que tem como enfermidades associadas a tumores de bexiga hematúria enzoótica e tumores de trato digestório superior caraguatá, responsáveis por sensíveis perdas para a pecuária. Várias tentativas vacinais têm sido empreendidas com finalidades profiláticas ou terapêuticas, porém sem resultados eficazes. Esta situação se deve a aspectos relacionados à estrutura viral que dificultam uma manipulação eficiente para produção de produtos vacinais. Para que tais dados possam ser obtidos, faz-se necessário uma melhor compreensão da ação das proteínas recombinantes. A clonagem em vetores bacterianos para a expressão e purificação dessas proteínas serve a diferentes propósitos. Entre eles, a produção de insumos imunológicos, como testes diagnósticos ou mesmo vacinas. O presente projeto visou à expressão e purificação da proteína de capsídeo recombinante L2 de BPV-2. A proteína foi expressa em bactéria e tentou-se a purificação por coluna de afinidade; entretanto, problemas na purificação não possibilitaram a conclusão deste objetivo. Todas as abordagens e protocolos utilizados nesse trabalho são discutidos para contribuição ao conhecimento do processo

The bovine papillomatosis is an infectious disease of worldwide occurrence, plaguing the Brazilian herd, without any effective attitude control, and whose illnesses associated with bladder tumors /"enzootic hematuria/" and upper digestive tract tumors /"caraguatá/" sensitive responsible for losses to livestock. Several attempts have been undertaken vaccine with prophylactic or therapeutic purposes, but without effective results. This is due to issues related to viral structure that hinder efficient manipulation for production of vaccine products. In order to obtain such information, it is necessary better understanding of the action of recombinant proteins. The bacterial cloning vectors for the expression and purification of such proteins serve different purposes. Among them, the production of immune inputs, such as diagnostic tests or vaccines. This project aimed the expression and purification of recombinant L2 capsid protein of BPV-2. The protein was expressed in bacteria and purification was carried out by affinity column. However, difficulties in the purification process, impaired the full completion of this objective. All the attempted approaches and protocols were discussed and potential solutions proposed

Expressão e purificação da proteína recombinante L2 do Papilomavírus bovino tipo-2 em sistema bacteriano / Expression and purification of recombinant protein L2 of Bovine papillomavirus type-2 in bacterial system

A papilomatose bovina é uma doença infectocontagiosa de ocorrência mundial, que assola o rebanho brasileiro, sem qualquer atitude efetiva de controle, e que tem como enfermidades associadas a tumores de bexiga hematúria enzoótica e tumores de trato digestório superior caraguatá, responsáveis por sensíveis perdas para a pecuária. Várias tentativas vacinais têm sido empreendidas com finalidades profiláticas ou terapêuticas, porém sem resultados eficazes. Esta situação se deve a aspectos relacionados à estrutura viral que dificultam uma manipulação eficiente para produção de produtos vacinais. Para que tais dados possam ser obtidos, faz-se necessário uma melhor compreensão da ação das proteínas recombinantes. A clonagem em vetores bacterianos para a expressão e purificação dessas proteínas serve a diferentes propósitos. Entre eles, a produção de insumos imunológicos, como testes diagnósticos ou mesmo vacinas. O presente projeto visou à expressão e purificação da proteína de capsídeo recombinante L2 de BPV-2. A proteína foi expressa em bactéria e tentou-se a purificação por coluna de afinidade; entretanto, problemas na purificação não possibilitaram a conclusão deste objetivo. Todas as abordagens e protocolos utilizados nesse trabalho são discutidos para contribuição ao conhecimento do processo (AU)

The bovine papillomatosis is an infectious disease of worldwide occurrence, plaguing the Brazilian herd, without any effective attitude control, and whose illnesses associated with bladder tumors \"enzootic hematuria\" and upper digestive tract tumors \"caraguatá\" sensitive responsible for losses to livestock. Several attempts have been undertaken vaccine with prophylactic or therapeutic purposes, but without effective results. This is due to issues related to viral structure that hinder efficient manipulation for production of vaccine products. In order to obtain such information, it is necessary better understanding of the action of recombinant proteins. The bacterial cloning vectors for the expression and purification of such proteins serve different purposes. Among them, the production of immune inputs, such as diagnostic tests or vaccines. This project aimed the expression and purification of recombinant L2 capsid protein of BPV-2. The protein was expressed in bacteria and purification was carried out by affinity column. However, difficulties in the purification process, impaired the full completion of this objective. All the attempted approaches and protocols were discussed and potential solutions proposed (AU)