Este estudio esta basado en la caracterizacion laboratorial de cinco virus de la Enfermedad de Gu... more Este estudio esta basado en la caracterizacion laboratorial de cinco virus de la Enfermedad de Gumboro (IBDV) aislados en Turquia tras unos episodios de enfermedad en las zonas de Adapazari, Bandirma, Bolu e Izmir. Las sospechas de que se trataba de virus muy virulentos de Gumboro (vvIBDV) se han confirmado experimental y molecularmente. En pollos libres de germenes especificos (SPF), el 100% de los animales murieron y/o sufrieron lesiones graves en la bolsa de Fabricio (BF) al ser inoculados con los diferentes aislados. Se ha comprobado a nivel molecular que los virus contienen una diana de restriccion para la endonucleasa SspI en el fragmento de la VP2 amplificado lo que los clasifica como muy virulentos. La correcta vacunacion con cepas de IBDV de tipo intermedio ha sido eficaz para el control experimental de los virus indicados.
A porcine circovirus (PCV) was isolated from tissues of pigs with wasting syndromes from Spain, D... more A porcine circovirus (PCV) was isolated from tissues of pigs with wasting syndromes from Spain, Denmark and N. Ireland. The antigenic profiles of these viruses were determined by indirect immunofluorescence assays using polyclonal antisera and monoclonal antibodies (mAbs) prepared against previously isolated PCVs. A rapid and convenient PCR-based test was developed and used for the genotyping of these PCV isolates. These PCV isolates were found to be antigenically and genomically similar to previously reported isolates of PCV from pigs with wasting disease (PCV2), but distinct from the isolate of PCV from continuous PK/15 cell cultures (PCV1).
The immunogenicity and protective capability of several baculovirus-expressed infectious bursal d... more The immunogenicity and protective capability of several baculovirus-expressed infectious bursal disease virus (IBDV)-derived assemblies as VP2 capsids, VPX tubules and polyprotein (PP)-derived mixed structures, were tested. Four-week-old chickens were immunised subcutaneously with one dose of each particulate antigen. VP2 icosahedral capsids induced the highest neutralising response, followed by PP-derived structures and then VPX tubules. All vaccinated animals were protected when challenged with a very virulent IBDV (vvIBDV) isolate, however the degree of protection is directly correlated with the levels of neutralising antibodies. VP2 capsids elicited stronger protective immunity than tubular structures and 3 g of them were sufficient to confer a total protection comparable to that induced by an inactivated vaccine. Therefore, VP2 capsids represent a suitable candidate recombinant vaccine instead of virus-like particles (VLPs) for IBDV infections. Our results also provide clear evidence that the recombinant IBDV-derived antigens are structure-dependent in order to be efficient as vaccine components.
This paper reports on a serological and virological survey for swine influenza virus (SIV) in den... more This paper reports on a serological and virological survey for swine influenza virus (SIV) in densely populated pig areas in Spain. The survey was undertaken to examine whether the H1N2 SIV subtype circulates in pigs in these areas, as in other European regions. Six hundred sow sera from 100 unvaccinated breeding herds across Northern and Eastern Spain were examined using haemagglutination inhibition (HI) tests against H1N1, H3N2 and H1N2 SIV subtypes. Additionally, 225 lung samples from pigs with respiratory problems were examined for the presence of SIV by virus isolation in embryonated chicken eggs and by a commercial membrane immunoassay. The virus isolates were further identified by HI and RT-PCR followed by partial cDNA sequencing. The HI test on sera revealed the presence of antibodies against at least one of the SIV subtypes in 83% of the herds and in 76.3% of the animals studied. Of the 600 sow sera tested, 109 (18.2%), 60 (10%) and 41 (6.8%) had SIV antibodies to subtype H1N2 alone, H3N2 alone and H1N1 alone, respectively. Twelve H3N2 viruses, 9 H1N1 viruses and 1 H1N2 virus were isolated from the lungs of pigs with respiratory problems. The analysis of a 436 nucleotide sequence of the neuraminidase gene from the H1N2 strain isolated further confirmed its identity. Demonstrably, swine influenza is still endemic in the studied swine population and a new subtype, the H1N2, may be becoming established and involved in clinical outbreaks of the disease in Spain.
Human gastric alcohol dehydrogenase (uo-ADH) was submitted to peptide analysis at picomole scale.... more Human gastric alcohol dehydrogenase (uo-ADH) was submitted to peptide analysis at picomole scale. A total of 72 positions were determined in the protein chain, providing information on three aspects of alcohol dehydrogenase structures in general, First, the data establish !he presence of a unique clnss of the enzyme, now confirmed as class IV, expressed in gastric tissue and separate from another novel class, now termed class V. Second, the class IV gastric enzyme has active site relationships compatible with an ethanol-active, zinc-containing alcohol dehydrogenase. Third, this enzyme class is of the variable type, like that for the 'variable', classical liver alcohol dehydrogenase of class I, and in contrast to that for the 'constant' class I11 enzyme. Known human alcohol dchydrogcnasc structures now prove the presence of at least seven human genes for the enzyme and nine for the whole protein ramily. Gastric alcohol dehydrogenase; Alcohol dehydrogenase classes; Enzyme evolution; Gene duplication; Conserved and variable enzymes
An assessment of the performance of an inactivated chicken anaemia virus (CAV) vaccine following ... more An assessment of the performance of an inactivated chicken anaemia virus (CAV) vaccine following administration to specific-pathogen-free (SPF) breeder hens before the egg production period is reported. Antibodies to CAV in adult hens and maternally-derived antibodies to CAV in the progeny chicks were assayed by ELISA. Progeny of vaccinated chickens showed sufficient levels of maternally-derived antibody to CAV for up to 3 to 4 weeks of age. Following challenge at 1, 10, 20 and 30 days of age of chicks from CAV-vaccinated and non-vaccinated hens with virulent CAV, haematocrit levels and weights of thymus in vaccinates remained within normal ranges. Chicks derived from non-vaccinated hens developed anaemia and thymus atrophy following challenge. These results support the view that vaccination of breeder flocks with an inactivated vaccine to CAV could be an effective means of control of CAV-induced clinical disease.
Molecular Therapy - Methods & Clinical Development, 2019
The tuberculosis (TB) vaccine MTBVAC is the only live-attenuated Mycobacterium tuberculosis (Mtb)... more The tuberculosis (TB) vaccine MTBVAC is the only live-attenuated Mycobacterium tuberculosis (Mtb)-based vaccine in clinical development, and it confers superior protection in different animal models compared to the current vaccine, BCG (Mycobacterium bovis bacillus Calmette-Guérin). With the aim of using MTBVAC as a vector for a dual TB-HIV vaccine, we constructed the recombinant MTBVAC.HIVA 2auxo strain. First, we generated a lysine auxotroph of MTBVAC (MTBVACDlys) by deleting the lysA gene. Then the auxotrophic MTBVACDlys was transformed with the E. coli-mycobacterial vector p2auxo.HIVA, harboring the lysA-complementing gene and the HIV-1 clade A immunogen HIVA. This TB-HIV vaccine conferred similar efficacy to the parental strain MTBVAC against Mtb challenge in mice. MTBVAC.HIVA 2auxo was safer than BCG and MTBVAC in severe combined immunodeficiency (SCID) mice, and it was shown to be maintained up to 42 bacterial generations in vitro and up to 100 days after inoculation in vivo. The MTBVAC.HIVA 2auxo vaccine, boosted with modified vaccinia virus Ankara (MVA).HIVA, induced HIV-1 and Mtb-specific interferon-g-producing T cell responses and polyfunctional HIV-1-specific CD8+ T cells producing interferon-g (IFN-g), tumor necrosis factor alpha (TNF-a), and CD107a in BALB/c mice. Here we describe new tools to develop combined vaccines against TB and HIV with the potential of expansion for other infectious diseases.
A porcine circovirus (PCV) was isolated from tissues of pigs with wasting syndromes from Spain, D... more A porcine circovirus (PCV) was isolated from tissues of pigs with wasting syndromes from Spain, Denmark and N. Ireland. The antigenic profiles of these viruses were determined by indirect immunofluorescence assays using polyclonal antisera and monoclonal antibodies (mAbs) prepared against previously isolated PCVs. A rapid and convenient PCR-based test was developed and used for the genotyping of these PCV isolates. These PCV isolates were found to be antigenically and genomically similar to previously reported isolates of PCV from pigs with wasting disease (PCV2), but distinct from the isolate of PCV from continuous PK/15 cell cultures (PCV1). # 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 -1 1 3 5 ( 9 9 ) 0 0 0 0 4 -8
Este estudio esta basado en la caracterizacion laboratorial de cinco virus de la Enfermedad de Gu... more Este estudio esta basado en la caracterizacion laboratorial de cinco virus de la Enfermedad de Gumboro (IBDV) aislados en Turquia tras unos episodios de enfermedad en las zonas de Adapazari, Bandirma, Bolu e Izmir. Las sospechas de que se trataba de virus muy virulentos de Gumboro (vvIBDV) se han confirmado experimental y molecularmente. En pollos libres de germenes especificos (SPF), el 100% de los animales murieron y/o sufrieron lesiones graves en la bolsa de Fabricio (BF) al ser inoculados con los diferentes aislados. Se ha comprobado a nivel molecular que los virus contienen una diana de restriccion para la endonucleasa SspI en el fragmento de la VP2 amplificado lo que los clasifica como muy virulentos. La correcta vacunacion con cepas de IBDV de tipo intermedio ha sido eficaz para el control experimental de los virus indicados.
A porcine circovirus (PCV) was isolated from tissues of pigs with wasting syndromes from Spain, D... more A porcine circovirus (PCV) was isolated from tissues of pigs with wasting syndromes from Spain, Denmark and N. Ireland. The antigenic profiles of these viruses were determined by indirect immunofluorescence assays using polyclonal antisera and monoclonal antibodies (mAbs) prepared against previously isolated PCVs. A rapid and convenient PCR-based test was developed and used for the genotyping of these PCV isolates. These PCV isolates were found to be antigenically and genomically similar to previously reported isolates of PCV from pigs with wasting disease (PCV2), but distinct from the isolate of PCV from continuous PK/15 cell cultures (PCV1).
The immunogenicity and protective capability of several baculovirus-expressed infectious bursal d... more The immunogenicity and protective capability of several baculovirus-expressed infectious bursal disease virus (IBDV)-derived assemblies as VP2 capsids, VPX tubules and polyprotein (PP)-derived mixed structures, were tested. Four-week-old chickens were immunised subcutaneously with one dose of each particulate antigen. VP2 icosahedral capsids induced the highest neutralising response, followed by PP-derived structures and then VPX tubules. All vaccinated animals were protected when challenged with a very virulent IBDV (vvIBDV) isolate, however the degree of protection is directly correlated with the levels of neutralising antibodies. VP2 capsids elicited stronger protective immunity than tubular structures and 3 g of them were sufficient to confer a total protection comparable to that induced by an inactivated vaccine. Therefore, VP2 capsids represent a suitable candidate recombinant vaccine instead of virus-like particles (VLPs) for IBDV infections. Our results also provide clear evidence that the recombinant IBDV-derived antigens are structure-dependent in order to be efficient as vaccine components.
This paper reports on a serological and virological survey for swine influenza virus (SIV) in den... more This paper reports on a serological and virological survey for swine influenza virus (SIV) in densely populated pig areas in Spain. The survey was undertaken to examine whether the H1N2 SIV subtype circulates in pigs in these areas, as in other European regions. Six hundred sow sera from 100 unvaccinated breeding herds across Northern and Eastern Spain were examined using haemagglutination inhibition (HI) tests against H1N1, H3N2 and H1N2 SIV subtypes. Additionally, 225 lung samples from pigs with respiratory problems were examined for the presence of SIV by virus isolation in embryonated chicken eggs and by a commercial membrane immunoassay. The virus isolates were further identified by HI and RT-PCR followed by partial cDNA sequencing. The HI test on sera revealed the presence of antibodies against at least one of the SIV subtypes in 83% of the herds and in 76.3% of the animals studied. Of the 600 sow sera tested, 109 (18.2%), 60 (10%) and 41 (6.8%) had SIV antibodies to subtype H1N2 alone, H3N2 alone and H1N1 alone, respectively. Twelve H3N2 viruses, 9 H1N1 viruses and 1 H1N2 virus were isolated from the lungs of pigs with respiratory problems. The analysis of a 436 nucleotide sequence of the neuraminidase gene from the H1N2 strain isolated further confirmed its identity. Demonstrably, swine influenza is still endemic in the studied swine population and a new subtype, the H1N2, may be becoming established and involved in clinical outbreaks of the disease in Spain.
Human gastric alcohol dehydrogenase (uo-ADH) was submitted to peptide analysis at picomole scale.... more Human gastric alcohol dehydrogenase (uo-ADH) was submitted to peptide analysis at picomole scale. A total of 72 positions were determined in the protein chain, providing information on three aspects of alcohol dehydrogenase structures in general, First, the data establish !he presence of a unique clnss of the enzyme, now confirmed as class IV, expressed in gastric tissue and separate from another novel class, now termed class V. Second, the class IV gastric enzyme has active site relationships compatible with an ethanol-active, zinc-containing alcohol dehydrogenase. Third, this enzyme class is of the variable type, like that for the 'variable', classical liver alcohol dehydrogenase of class I, and in contrast to that for the 'constant' class I11 enzyme. Known human alcohol dchydrogcnasc structures now prove the presence of at least seven human genes for the enzyme and nine for the whole protein ramily. Gastric alcohol dehydrogenase; Alcohol dehydrogenase classes; Enzyme evolution; Gene duplication; Conserved and variable enzymes
An assessment of the performance of an inactivated chicken anaemia virus (CAV) vaccine following ... more An assessment of the performance of an inactivated chicken anaemia virus (CAV) vaccine following administration to specific-pathogen-free (SPF) breeder hens before the egg production period is reported. Antibodies to CAV in adult hens and maternally-derived antibodies to CAV in the progeny chicks were assayed by ELISA. Progeny of vaccinated chickens showed sufficient levels of maternally-derived antibody to CAV for up to 3 to 4 weeks of age. Following challenge at 1, 10, 20 and 30 days of age of chicks from CAV-vaccinated and non-vaccinated hens with virulent CAV, haematocrit levels and weights of thymus in vaccinates remained within normal ranges. Chicks derived from non-vaccinated hens developed anaemia and thymus atrophy following challenge. These results support the view that vaccination of breeder flocks with an inactivated vaccine to CAV could be an effective means of control of CAV-induced clinical disease.
Molecular Therapy - Methods & Clinical Development, 2019
The tuberculosis (TB) vaccine MTBVAC is the only live-attenuated Mycobacterium tuberculosis (Mtb)... more The tuberculosis (TB) vaccine MTBVAC is the only live-attenuated Mycobacterium tuberculosis (Mtb)-based vaccine in clinical development, and it confers superior protection in different animal models compared to the current vaccine, BCG (Mycobacterium bovis bacillus Calmette-Guérin). With the aim of using MTBVAC as a vector for a dual TB-HIV vaccine, we constructed the recombinant MTBVAC.HIVA 2auxo strain. First, we generated a lysine auxotroph of MTBVAC (MTBVACDlys) by deleting the lysA gene. Then the auxotrophic MTBVACDlys was transformed with the E. coli-mycobacterial vector p2auxo.HIVA, harboring the lysA-complementing gene and the HIV-1 clade A immunogen HIVA. This TB-HIV vaccine conferred similar efficacy to the parental strain MTBVAC against Mtb challenge in mice. MTBVAC.HIVA 2auxo was safer than BCG and MTBVAC in severe combined immunodeficiency (SCID) mice, and it was shown to be maintained up to 42 bacterial generations in vitro and up to 100 days after inoculation in vivo. The MTBVAC.HIVA 2auxo vaccine, boosted with modified vaccinia virus Ankara (MVA).HIVA, induced HIV-1 and Mtb-specific interferon-g-producing T cell responses and polyfunctional HIV-1-specific CD8+ T cells producing interferon-g (IFN-g), tumor necrosis factor alpha (TNF-a), and CD107a in BALB/c mice. Here we describe new tools to develop combined vaccines against TB and HIV with the potential of expansion for other infectious diseases.
A porcine circovirus (PCV) was isolated from tissues of pigs with wasting syndromes from Spain, D... more A porcine circovirus (PCV) was isolated from tissues of pigs with wasting syndromes from Spain, Denmark and N. Ireland. The antigenic profiles of these viruses were determined by indirect immunofluorescence assays using polyclonal antisera and monoclonal antibodies (mAbs) prepared against previously isolated PCVs. A rapid and convenient PCR-based test was developed and used for the genotyping of these PCV isolates. These PCV isolates were found to be antigenically and genomically similar to previously reported isolates of PCV from pigs with wasting disease (PCV2), but distinct from the isolate of PCV from continuous PK/15 cell cultures (PCV1). # 0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 -1 1 3 5 ( 9 9 ) 0 0 0 0 4 -8
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