RESUMO
We have recently communicated the oral and parental immunogenicity of the structural protein VP1 of foot and mouth disease virus (FMDV) expressed in different transgenic plants. Those results clearly indicated the necessity of increasing the expression of the foreign genes in the transgenic plant to avoid additional steps toward the purification and/or concentration of the antigen of interest. Here, we report the production of transgenic potatoes plants containing the VP1 gene cloned under the regulatory activity of either a single (pRok2) or a double (pRok3) copy of the S35 cauliflower mosaic virus (CaMV 35S) promoter, as a strategy for increasing the level of VP1 gene expression. The presence of the VP1 gene in the plants was confirmed by polymerase chain reaction (PCR) and its specific transcription activity was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that, although the immunized animals presented a FMDV VP1 specific antibody response and protection against the experimental challenge, no significant differences were demonstrated in the immunizing activity of plant extracts obtained from the pRok2 or pRok3 transformed plants. These results confirm those previously obtained using other plant species allowing the possibility of using plants as antigen expression vectors, and demonstrated that at least in the potato system, the use of double CaMV 35S promoter does not cause a significant increase in the level of the VP1 expressed.
Assuntos
Anticorpos Antivirais/sangue , Aphthovirus/imunologia , Capsídeo/imunologia , Febre Aftosa/imunologia , Plantas Geneticamente Modificadas/imunologia , Solanum tuberosum/imunologia , Animais , Capsídeo/genética , Capsídeo/metabolismo , Proteínas do Capsídeo , Febre Aftosa/prevenção & controle , Genes Virais , Imunização , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/imunologia , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Transformação Genética , Vacinas ViraisRESUMO
The utilization of transgenic plants expressing recombinant antigens to be used in the formulation of experimental immunogens has been recently communicated. We report here the development of transgenic plants of alfalfa expressing the structural protein VP1 of foot and mouth disease virus (FMDV). The presence of the transgenes in the plants was confirmed by PCR and their specific transcription was demonstrated by RT-PCR. Mice parenterally immunized using leaf extracts or receiving in their diet freshly harvested leaves from the transgenic plants developed a virus-specific immune response. Animals immunized by either method elicited a specific antibody response to a synthetic peptide representing amino acid residues 135-160 of VP1, to the structural protein VP1, and to intact FMDV particles. Additionally, the immunized mice were protected against experimental challenge with the virus. We believe this is the first report demonstrating the induction of a protective systemic antibody response in animals fed transgenic plants expressing a viral antigen. These results support the feasibility of producing edible vaccines in transgenic forage plants, such as alfalfa, commonly used in the diet of domestic animals even for those antigens for which a systemic immune response is required.
Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Capsídeo/imunologia , Febre Aftosa/prevenção & controle , Plantas Geneticamente Modificadas , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Administração Oral , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/genética , Capsídeo/genética , Proteínas do Capsídeo , Injeções Intraperitoneais , Masculino , Medicago sativa , Camundongos , Camundongos Endogâmicos BALB C , Transcrição Gênica , Transformação Genética , VacinaçãoRESUMO
We previously demonstrated that the immunization of cattle with a synthetic peptide representing the amino acid sequence of foot and mouth disease virus (FMDV) type O1 Campos VP1 residues 135-160 (p135-160), containing immunodominant T and B epitopes, was able to induce a strong neutralizing antibody (NA) response. The epitope mapping of p135-160 identified T and B epitopes in the area restricted to amino acid residues 135-144 (Zamorano et al. 1994, Virology 201; 1995, Virology 212). We are now reporting that, although immunization with a synthetic peptide covering amino acids 135-144 (p135-144) failed to elicit an anti-FMDV response, a synthetic peptide representing a tandem duplication of the VP1 epitope 135-144 (p135-144 x 2) was very efficient in inducing a strong NA response in cattle. Both the antibody and T cell responses elicited by p135-144 x 2 were highly specific for the VP1 135-144 sequence since no reactivity was detected against synthetic peptides representing the 140-160 sequence of VP1. Additionally, both responses to B and T epitopes were long lasting in the immunized cattle. These results constitute a good example of the improvement of the immune response by rational handling of precisely identified B and T epitopes. To our knowledge, this is the shortest native amino acid sequence to induce a significant NA response to FMDV in cattle.
Assuntos
Aphthovirus/imunologia , Linfócitos B/imunologia , Capsídeo/imunologia , Capsídeo/farmacologia , Ativação Linfocitária/imunologia , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/farmacologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Proteínas do Capsídeo , Bovinos , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Epitopos Imunodominantes/imunologia , Dados de Sequência Molecular , Testes de Neutralização , Sequências Repetitivas de Ácido NucleicoRESUMO
It has been reported recently that genes encoding antigens of bacterial and viral pathogens can be expressed in plants in a form in which they retain native immunogenic properties. The structural protein VP1 of foot-and-mouth disease virus (FMDV), which has frequently been shown to contain critical epitopes, has been expressed in different vectors and shown to induce virus-neutralizing antibodies and protection in experimental and natural hosts. Here we report the production of transformed plants (Arabidopsis thaliana) expressing VP1. Mice immunized with leaf plant extracts elicited specific antibody responses to synthetic peptides representing amino acid residues 135 to 160 of VP1, to VP1 itself, and to intact FMDV particles. Additionally, all of the immunized mice were protected against challenge with virulent FMDV. To our knowledge, this is the first study showing protection against a viral disease by immunization with an antigen expressed in a transgenic plant.