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Protective Efficacy in Sheep of Adenovirus-Vectored Vaccines against Bluetongue Virus Is Associated with Specific T Cell Responses.

Martín V, Pascual E, Avia M, Peña L, Valcárcel F, Sevilla N - PLoS ONE (2015)

Bottom Line: Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia.This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5-BTV-VP7.These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación en Sanidad Animal (CISA-INIA), Instituto Nacional de Investigación Agraria y Alimentaria, Valdeolmos, Madrid, Spain.

ABSTRACT
Bluetongue virus (BTV) is an economically important Orbivirus of the Reoviridae family that causes a hemorrhagic disease in ruminants. Its control has been achieved by inactivated-vaccines that have proven to protect against homologous BTV challenge although unable to induce long-term immunity. Therefore, a more efficient control strategy needs to be developed. Recombinant adenovirus vectors are lead vaccine candidates for protection of several diseases, mainly because of their potency to induce potent T cell immunity. Here we report the induction of humoral and T-cell mediated responses able to protect animals against BTV challenge by recombinant replication-defective human adenovirus serotype 5 (Ad5) expressing either VP7, VP2 or NS3 BTV proteins. First we used the IFNAR(-/-) mouse model system to establish a proof of principle, and afterwards we assayed the protective efficacy in sheep, the natural host of BTV. Mice were completely protected against BTV challenge, developing humoral and BTV-specific CD8+- and CD4+-T cell responses by vaccination with the different rAd5. Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia. This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5-BTV-VP7. These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection.

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Expression of BTV-8 -NS3, -VP2 and -VP7 proteins from recombinant adenoviruses.(A) RT-PCR amplification from total RNA obtained from Ad5-BTV-NS3, Ad5-BTV-VP2, Ad5-BTV-VP7 and Ad5-DsRed infected HEK293A cells. Total RNA was assayed with a reverse transcription-PCR to detect NS3 (685 bp), VP2 (1042 pb) or VP7 (1084 bp) gene expression. Input RNA samples used are indicated above each lane. The bands corresponding to the proteins detected are indicated on the right (arrows). Primers pairs used are provided upon request. Molecular size markers (Kbp) are at right. As control, the expression of the cellular β-actin gene was determined by amplifying a 426 bp fragment of the corresponding gene by using actin-specific primers. (B) After adenovirus infection of HEK293A cells, expression of BTV-8 -NS3 (24 kDa) and -VP2 (112 kDa) proteins were analysed by Western blot using sera from BTV-8 infected mice or sheep. Input protein samples used are indicated above each lane. The bands corresponding to the proteins detected are indicated on the right (arrows). Molecular size markers (kDa) are at right.
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pone.0143273.g002: Expression of BTV-8 -NS3, -VP2 and -VP7 proteins from recombinant adenoviruses.(A) RT-PCR amplification from total RNA obtained from Ad5-BTV-NS3, Ad5-BTV-VP2, Ad5-BTV-VP7 and Ad5-DsRed infected HEK293A cells. Total RNA was assayed with a reverse transcription-PCR to detect NS3 (685 bp), VP2 (1042 pb) or VP7 (1084 bp) gene expression. Input RNA samples used are indicated above each lane. The bands corresponding to the proteins detected are indicated on the right (arrows). Primers pairs used are provided upon request. Molecular size markers (Kbp) are at right. As control, the expression of the cellular β-actin gene was determined by amplifying a 426 bp fragment of the corresponding gene by using actin-specific primers. (B) After adenovirus infection of HEK293A cells, expression of BTV-8 -NS3 (24 kDa) and -VP2 (112 kDa) proteins were analysed by Western blot using sera from BTV-8 infected mice or sheep. Input protein samples used are indicated above each lane. The bands corresponding to the proteins detected are indicated on the right (arrows). Molecular size markers (kDa) are at right.

Mentions: Recombinant adenoviruses encoding NS3 (Ad5-BTV-NS3), VP7 (Ad5-BTV-VP7) and VP2 (Ad5-BTV-VP2) from BTV-8 were generated. To determine the expression of BTV proteins by recombinant adenoviruses, Vero and ST cells were infected and immunostained using serum from BTV infected sheep. Specific labelling corresponding to NS3, VP2 and VP7 proteins was detected in Ad5-BTV-NS3, Ad5-BTV-VP2, Ad5-BTV-VP7 infected cells, respectively (Fig 1A and 1B). As expected, no signal was observed in either Ad5-DsRed (parental control recombinant adenovirus) infected or uninfected cells using either antibody (S1 Fig). To confirm that infected HEK293A cells express the BTV-8 -NS3, -VP2 and -VP7 genes, we analysed the presence of the corresponding transcripts in these cells by RT-PCR, which were readily detected (Fig 2A). To detect the expression of BTV-8 -NS3, -VP2 and -VP7 proteins, western blot analysis using polyclonal sera from BTV-8 infected mice was performed. Specific labelling corresponding to BTV-8 -NS3 (26 KDa) and -VP2 (111 KDa) proteins was detected in Ad5-NS3 and Ad5-VP2 infected cells, respectively (Fig 2B). As expected, no signal was observed in Ad5-DsRed infected cells. The VP7 protein has a 37 KDa expected size. In this case and with our antisera is not possible to detect the VP7 expressed in the HEK293A infected with Ad5-VP7.


Protective Efficacy in Sheep of Adenovirus-Vectored Vaccines against Bluetongue Virus Is Associated with Specific T Cell Responses.

Martín V, Pascual E, Avia M, Peña L, Valcárcel F, Sevilla N - PLoS ONE (2015)

Expression of BTV-8 -NS3, -VP2 and -VP7 proteins from recombinant adenoviruses.(A) RT-PCR amplification from total RNA obtained from Ad5-BTV-NS3, Ad5-BTV-VP2, Ad5-BTV-VP7 and Ad5-DsRed infected HEK293A cells. Total RNA was assayed with a reverse transcription-PCR to detect NS3 (685 bp), VP2 (1042 pb) or VP7 (1084 bp) gene expression. Input RNA samples used are indicated above each lane. The bands corresponding to the proteins detected are indicated on the right (arrows). Primers pairs used are provided upon request. Molecular size markers (Kbp) are at right. As control, the expression of the cellular β-actin gene was determined by amplifying a 426 bp fragment of the corresponding gene by using actin-specific primers. (B) After adenovirus infection of HEK293A cells, expression of BTV-8 -NS3 (24 kDa) and -VP2 (112 kDa) proteins were analysed by Western blot using sera from BTV-8 infected mice or sheep. Input protein samples used are indicated above each lane. The bands corresponding to the proteins detected are indicated on the right (arrows). Molecular size markers (kDa) are at right.
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pone.0143273.g002: Expression of BTV-8 -NS3, -VP2 and -VP7 proteins from recombinant adenoviruses.(A) RT-PCR amplification from total RNA obtained from Ad5-BTV-NS3, Ad5-BTV-VP2, Ad5-BTV-VP7 and Ad5-DsRed infected HEK293A cells. Total RNA was assayed with a reverse transcription-PCR to detect NS3 (685 bp), VP2 (1042 pb) or VP7 (1084 bp) gene expression. Input RNA samples used are indicated above each lane. The bands corresponding to the proteins detected are indicated on the right (arrows). Primers pairs used are provided upon request. Molecular size markers (Kbp) are at right. As control, the expression of the cellular β-actin gene was determined by amplifying a 426 bp fragment of the corresponding gene by using actin-specific primers. (B) After adenovirus infection of HEK293A cells, expression of BTV-8 -NS3 (24 kDa) and -VP2 (112 kDa) proteins were analysed by Western blot using sera from BTV-8 infected mice or sheep. Input protein samples used are indicated above each lane. The bands corresponding to the proteins detected are indicated on the right (arrows). Molecular size markers (kDa) are at right.
Mentions: Recombinant adenoviruses encoding NS3 (Ad5-BTV-NS3), VP7 (Ad5-BTV-VP7) and VP2 (Ad5-BTV-VP2) from BTV-8 were generated. To determine the expression of BTV proteins by recombinant adenoviruses, Vero and ST cells were infected and immunostained using serum from BTV infected sheep. Specific labelling corresponding to NS3, VP2 and VP7 proteins was detected in Ad5-BTV-NS3, Ad5-BTV-VP2, Ad5-BTV-VP7 infected cells, respectively (Fig 1A and 1B). As expected, no signal was observed in either Ad5-DsRed (parental control recombinant adenovirus) infected or uninfected cells using either antibody (S1 Fig). To confirm that infected HEK293A cells express the BTV-8 -NS3, -VP2 and -VP7 genes, we analysed the presence of the corresponding transcripts in these cells by RT-PCR, which were readily detected (Fig 2A). To detect the expression of BTV-8 -NS3, -VP2 and -VP7 proteins, western blot analysis using polyclonal sera from BTV-8 infected mice was performed. Specific labelling corresponding to BTV-8 -NS3 (26 KDa) and -VP2 (111 KDa) proteins was detected in Ad5-NS3 and Ad5-VP2 infected cells, respectively (Fig 2B). As expected, no signal was observed in Ad5-DsRed infected cells. The VP7 protein has a 37 KDa expected size. In this case and with our antisera is not possible to detect the VP7 expressed in the HEK293A infected with Ad5-VP7.

Bottom Line: Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia.This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5-BTV-VP7.These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigación en Sanidad Animal (CISA-INIA), Instituto Nacional de Investigación Agraria y Alimentaria, Valdeolmos, Madrid, Spain.

ABSTRACT
Bluetongue virus (BTV) is an economically important Orbivirus of the Reoviridae family that causes a hemorrhagic disease in ruminants. Its control has been achieved by inactivated-vaccines that have proven to protect against homologous BTV challenge although unable to induce long-term immunity. Therefore, a more efficient control strategy needs to be developed. Recombinant adenovirus vectors are lead vaccine candidates for protection of several diseases, mainly because of their potency to induce potent T cell immunity. Here we report the induction of humoral and T-cell mediated responses able to protect animals against BTV challenge by recombinant replication-defective human adenovirus serotype 5 (Ad5) expressing either VP7, VP2 or NS3 BTV proteins. First we used the IFNAR(-/-) mouse model system to establish a proof of principle, and afterwards we assayed the protective efficacy in sheep, the natural host of BTV. Mice were completely protected against BTV challenge, developing humoral and BTV-specific CD8+- and CD4+-T cell responses by vaccination with the different rAd5. Sheep vaccinated with Ad5-BTV-VP2 and Ad5-BTV-VP7 or only with Ad5-BTV-VP7 and challenged with BTV showed mild disease symptoms and reduced viremia. This partial protection was achieved in the absence of neutralizing antibodies but strong BTV-specific CD8+ T cell responses in those sheep vaccinated with Ad5-BTV-VP7. These data indicate that rAd5 is a suitable vaccine vector to induce T cell immunity during BTV vaccination and provide new data regarding the relevance of T cell responses in protection during BTV infection.

Show MeSH
Related in: MedlinePlus