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Biomedical subjects

O Taboga

Publications and source records attributed to O Taboga.

10 recordsLinked to original sources
PubMed2003

Bovine herpes virus gD protein produced in plants using a recombinant tobacco mosaic virus (TMV) vector possesses authentic antigenicity.

A tobacco mosaic virus (TMV)-based vector was utilized for expression of a cytosolic form of the bovine herpesvirus type 1 (BHV-1) protein glycoprotein D (gDc). Nicotiana benthamiana plants were harvested 7 days after inoculation with RNA transcripts derived from the TMV-gDc recombinant virus. Recombinant gDc protein of expected electrophoretic mobility accumulated in inoculated leaves to a concentration of about 20 micrograms/g of fresh leaf tissue. Oil-based vaccines were formulated with crude foliar extracts to immunize mice parentally. After a single injection, animals developed a sustained and specific response to both the isolated gD and native virus particles. Cattle vaccinated with the same gDc containing extracts developed specific humoral and cellular immune responses directed against both the viral gD and BHV-1 particles. Most importantly, animals vaccinated with the plant-produced gDc showed good levels of protection after challenge with the virulent BHV-1. Virus excretion was drastically reduced in these animals, reaching levels comparable to animals vaccinated with a commercial BHV-1 vaccine. The positive immunological characterization obtained for the gDc, indicated that an important part of the natural conformation was retained in the plant recombinant protein.

Animals↗
PubMed2002

BHV-1 DNA vaccination: effect of the adjuvant RN-205 on the modulation of the immune response in mice.

It is well documented that adjuvants improve the immune response generated by traditional viral vaccines, but less is known about the effects of adjuvants on the immune response elicited by DNA vaccines. In this study, we have investigated the use of RN-205 (immunomodulator containing a membrane rich in lipopolysaccharide from gram-negative bacteria) as an adjuvant and analyzed the humoral and cellular specific immune responses elicited by DNA vaccines based on the bovine herpesvirus-1 (BHV-1) glycoprotein D (gD). The comparison of the antibody response induced in mice by a mixture of the three different versions of DNA gD (membrane-anchored, secreted and cytosolic) formulated with or without RN-205 showed that the immunomodulator did not affect the total specific humoral response. The cellular immune response induced in mice immunized with vaccines plus RN-205 was higher than that obtained in mice vaccinated without RN-205, not only in the indexes of proliferation tests but in the number of IL-4 and gammaIFN secreting cells. When total spleen cells were marked with specific monoclonal antibodies against surface markers, a significant increase in the macrophage population of all the groups receiving RN-205 was observed. CD8 and CD4 positive cells were also increased but to a lesser extent. Our results indicate that the incorporation of RN-205 into DNA vaccines induces an increase of the cellular specific immune response in mice.

Adjuvants, Immunologic↗
PubMed2000

Association of bovine DRB3 alleles with immune response to FMDV peptides and protection against viral challenge.

We have analysed the influence of bovine MHC (BoLA) polymorphism on the immune response and degree of protection induced by peptide vaccines against foot-and-mouth disease (FMD) in cattle. The peptides used for animal immunisation were: A (VP1(138-156)), AT (peptide A linked to VP1(21-40)) and ACT (peptide A, linked to VP1(196-209) and VP1(21-40)). Sixteen different DRB3 types were found among the 46 cattle analysed by PCR-RFLP typing. No absolute correlation was observed, for any type, with the serum neutralising titres (SNT) values and the protection induced. However, among the most common haplotypes present, associations were observed between expression of different types with the levels of SNT and/or protection induced by peptides A and ACT. Thus, types DRB3.2*1, 3 and 7 were associated with increased levels of protection. In contrast, types DRB3.2*12 and 18 were associated non-protection, and DRB3.2*12 was also associated with low SNT titres. Overall, the results indicate that the polymorphism in BoLA class II molecules affects both the immune response and protection induced by potential FMD peptide vaccines.

Alleles↗
PubMed2000

Protective immunity against foot-and-mouth disease virus induced by a recombinant vaccinia virus.

We report the construction of a recombinant vaccinia virus expressing the precursor for the four structural proteins of FMD virus (FMDV) (P1) strain C3Arg85 using a procedure for isolation of recombinant vaccinia viruses based solely on plaque formation. Adult mice vaccinated with this recombinant vaccinia virus elicited high titers of neutralizing antibodies against both the homologous FMDV and vaccinia virus, measured by neutralization assays. Liquid phase blocking sandwich enzyme-linked immunosorbent assays (ELISAs) using whole virus as antigen showed high total antibody titers against homologous FMDV, similar to those induced by the conventional inactivated vaccine. When ELISAs were carried out with heterologous strains A79 or O1Caseros as antigens, sera from animals vaccinated with the recombinant virus cross-reacted. Mice boosted once with the recombinant vaccinia virus were protected against challenge with infectious homologous virus. These results indicate that recombinant vaccinia viruses are efficient immunogens against FMDV when used as a live vaccine in a mouse model.

Animals↗
PubMed2000

Presentation of antigenic sites from foot-and-mouth disease virus on the surface of baculovirus and in the membrane of infected cells.

We describe the construction of recombinant baculoviruses displaying on their surface and in the membrane of infected cells the small, immunodominant antigenic site (site A) or the large polyprotein (P1) coding for the four structural proteins of foot-and-mouth disease virus (FMDV). The coding sequences were inserted in the amino-terminus of gp64, the major glycoprotein of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). Following infection of insect cells with the recombinant baculoviruses, the cellular localization of the chimaeric proteins as well as their presence in the surface of extracellular viruses was assessed by immunofluorescence microscopy and Western blot. The antigenicity of the recombinant viruses was studied by competitive ELISAs, which showed that although both recombinant viruses were able to compete with FMDV-specific monoclonal antibodies (MAbs), their patterns of reactivity were different. The results suggest that this eukaryotic display system could be an alternative method of presentation of foreign antigens in a multimeric form as a new approach to biosynthetic vaccines.

Animals↗
PubMed1999

Expression of the Mycobacterium bovis P36 gene in Mycobacterium smegmatis and the baculovirus/insect cell system.

In the present study we evaluated different systems for the expression of mycobacterial antigen P36 secreted by Mycobacterium bovis. P36 was detected by Western blot using a specific antiserum. The P36 gene was initially expressed in E. coli, under the control of the T7 promoter, but severe proteolysis prevented its purification. We then tried to express P36 in M. smegmatis and insect cells. For M. smegmatis, we used three different plasmid vectors differing in copy number and in the presence of a promoter for expression of heterologous proteins. P36 was detected in the cell extract and culture supernatant in both expression systems and was recognized by sera from M. bovis-infected cattle. To compare the expression level and compartmentalization, the MPB70 antigen was also expressed. The highest production was reached in insect cell supernatants. In conclusion, M. smegmatis and especially the baculovirus expression system are good choices for the production of proteins from pathogenic mycobacteria for the development of mycobacterial vaccines and diagnostic reagents.

Animals↗
PubMed1997

Foot-and-mouth disease virus-infected but not vaccinated cattle develop antibodies against recombinant 3AB1 nonstructural protein.

Foot-and-mouth disease (FMD) vaccines induce antibodies against structural and some nonstructural proteins present in vaccine preparations. To differentiate between FMDV-infected and vaccinated animals, we developed immunochemical assays capable of detecting antibodies against a FMDV nonstructural protein. Recombinant nonstructural 3AB1 protein was expressed in E.coli and in insect cells and used to detect anti-3AB1 antibodies. ELISA and Western blot analysis showed that sera from cattle infected with FMDV reacted with recombinant 3AB1 protein whereas sera from cattle which had been vaccinated against FMDV, mock-infected, or infected with different bovine viruses did not recognize the 3AB1 protein. In contrast, anti-virus infection associated antigen (VIAA) antibodies were present in both FMDV-infected and vaccinated animals. Detection of anti-3AB1 antibodies in sera of experimentally infected cattle obtained between 7 and 560 days postinfection indicated that immunological tests based on the detection of recombinant 3AB1 protein could be used for the diagnosis of FMDV infection.

Animals↗
PubMed1997

A large-scale evaluation of peptide vaccines against foot-and-mouth disease: lack of solid protection in cattle and isolation of escape mutants.

A large-scale vaccination experiment involving a total of 138 cattle was carried out to evaluate the potential of synthetic peptides as vaccines against foot-and-mouth disease. Four types of peptides representing sequences of foot-and-mouth disease virus (FMDV) C3 Argentina 85 were tested: A, which includes the G-H loop of capsid protein VP1 (site A); AT, in which a T-cell epitope has been added to site A; AC, composed of site A and the carboxy-terminal region of VP1 (site C); and ACT, in which the three previous capsid motifs are colinearly represented. Induction of neutralizing antibodies, lymphoproliferation in response to viral antigens, and protection against challenge with homologous infectious virus were examined. None of the tested peptides, at several doses and vaccination schedules, afforded protection above 40%. Protection showed limited correlation with serum neutralization activity and lymphoproliferation in response to whole virus. In 12 of 29 lesions from vaccinated cattle that were challenged with homologous virus, mutant FMDVs with amino acid substitutions at antigenic site A were identified. This finding suggests the rapid generation and selection of FMDV antigenic variants in vivo. In contrast with previous studies, this large-scale vaccination experiment with an important FMDV host reveals considerable difficulties for vaccines based on synthetic peptides to achieve the required levels of efficacy. Possible modifications of the vaccine formulations to increase protective activity are discussed.

Amino Acid Sequence↗
PubMedDate not supplied

[Expression and characterization of 2 bovine viral diarrhea virus structural proteins (]BVDV)].

The BVDV glycoproteins gp48 and gp53 were expressed in the baculovirus eukaryotic system. Both recombinant proteins were recognized in western blot analysis by monoclonal antibodies and polyclonal serum. Immunofluorescent test demonstrated that gp53 was localized on the cell surface whereas gp48 was in the cytoplasm. The expressed proteins were extracted by non-denaturing detergent treatment. Rabbit antiserum raised against gp53 recombinant protein efficiently neutralized the virus. These results demonstrate that the recombinant proteins have immunological properties similar to those of the native viral protein and that they can be useful as diagnostic reagents.

Animals↗