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Dual function of the hemagglutinin H5 fused to chicken CD154 in a potential strategy of DIVA against avian influenza disease: preliminary study.

Pose AG, Rodríguez ES, Méndez AC, Gómez JN, Redondo AV, Rodríguez ER, Ramos EM, Gutiérrez AÁ, Moltó MP, Roche DG, Ugalde YS, López AM - Open Vet J (2015)

Bottom Line: As the strategy of DIVA requires at least two proteins, we obtained a variant of the nucleoprotein (NP49-375) in E. coli.The results showed that the segment CD154 in the chimeric protein HACD did not interfere with the recognition of the molecule HA by its specific antibodies.No antibody detection was observed in the sera from different species of birds or the sera of chickens infected with other avian viral diseases.

View Article: PubMed Central - PubMed

Affiliation: Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), P. O. Box 6162, Havana 10600, Cuba.

ABSTRACT
In this study we demonstrated that the vaccine candidate against avian influenza virus H5N1 based on the hemagglutinin H5 (HA) fused to the chicken CD154 (HACD) can also be used for differentiating infected from vaccinated animals (DIVA). As the strategy of DIVA requires at least two proteins, we obtained a variant of the nucleoprotein (NP49-375) in E. coli. After its purification by IMAC, the competence of the proteins NP49-375 and HACD as coating antigens in indirect ELISA assays were tested by using the sera of chickens immunized with the proteins HA and HACD and the reference sera from several avian influenza subtypes. Together with these sera, the sera from different species of birds and the sera of chickens infected with other avian viral diseases were analyzed by competition ELISA assays coated with the proteins NP49-375 and HACD. The results showed that the segment CD154 in the chimeric protein HACD did not interfere with the recognition of the molecule HA by its specific antibodies. Also, we observed variable detection levels when the reference sera were analyzed in the ELISA plates coated with the protein NP49-375. Moreover, only the antibodies of the reference serum subtype H5 were detected in the ELISA plates coated with the protein HACD. The competition ELISA assays showed percentages of inhibition of 88-91% for the positives sera and less than 20% for the negative sera. We fixed the cut-off value of these assays at 25%. No antibody detection was observed in the sera from different species of birds or the sera of chickens infected with other avian viral diseases. This study supported the fact that the ELISA assays using the proteins NP49-375 and HACD could be valuable tools for avian influenza surveillance and as a strategy of DIVA for counteracting the highly pathogenic avian influenza virus H5N1 outbreaks.

No MeSH data available.


Related in: MedlinePlus

Establishment of competition ELISA assays for a potential strategy of DIVA directed to the avian influenza virus H5N1. (A) Competition ELISA assays coated with 2.5 μg/mL of the proteins NP49-375 and HACD. The reference sera from different avian influenza virus subtypes diluted 1/200 and the sera from chickens immunized with the proteins HA and HACD diluted 1/1000 were tested. Duplicated samples from the sera of ten immunized chickens per experimental group and six replicates of each reference serum were evaluated. (B) Antibody detection in duplicated samples of the sera from birds of different species. Plates coated with 2.5 μg/mL of the protein NP49-375 used the monoclonal antibody anti-NP5 diluted 1/20 000 as detection antibody. Plates coated with 2.5 μg/mL of the protein HACD used the monoclonal antibody anti-HA2 diluted 1/20 000 as detection antibody (C). Antibody detection in the sera from chickens infected with distinct avian viral diseases. Plates were coated as above and the same detection antibodies were used. Sera were diluted 1/50. Six replicates per serum from infected chickens were used. Results were expressed as the percent of inhibition. PS: Reference H5N2 serum. NS: Reference negative serum. IBD: Infectious Bursal Disease. EDS: Egg Drop Syndrome. NDV: Newcastle Disease Virus. AP: Avian Parvovirus.
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Figure 7: Establishment of competition ELISA assays for a potential strategy of DIVA directed to the avian influenza virus H5N1. (A) Competition ELISA assays coated with 2.5 μg/mL of the proteins NP49-375 and HACD. The reference sera from different avian influenza virus subtypes diluted 1/200 and the sera from chickens immunized with the proteins HA and HACD diluted 1/1000 were tested. Duplicated samples from the sera of ten immunized chickens per experimental group and six replicates of each reference serum were evaluated. (B) Antibody detection in duplicated samples of the sera from birds of different species. Plates coated with 2.5 μg/mL of the protein NP49-375 used the monoclonal antibody anti-NP5 diluted 1/20 000 as detection antibody. Plates coated with 2.5 μg/mL of the protein HACD used the monoclonal antibody anti-HA2 diluted 1/20 000 as detection antibody (C). Antibody detection in the sera from chickens infected with distinct avian viral diseases. Plates were coated as above and the same detection antibodies were used. Sera were diluted 1/50. Six replicates per serum from infected chickens were used. Results were expressed as the percent of inhibition. PS: Reference H5N2 serum. NS: Reference negative serum. IBD: Infectious Bursal Disease. EDS: Egg Drop Syndrome. NDV: Newcastle Disease Virus. AP: Avian Parvovirus.

Mentions: Competition ELISA assays using the reference sera and the sera of chickens immunized with the proteins HA and HACD were employed to perform the strategy of DIVA. The competition ELISA assays also involved the sera from different species of birds including flamingo, gamecocks, parakeets, rosellas, ducks and turkeys, which were already tested as negative by the hemagglutination inhibition assay using antigens of the subtypes H5, H7 and H9. Likewise, we evaluated the sera of chickens infected with Infectious Bursal Disease, Egg Drop Syndrome, Newcastle Disease Virus and Avian Parvovirus. As expected, the pattern for the percent of inhibition detected in the reference sera when the ELISA plates were coated with the protein NP49-375 was consistent with the mODs observed in the indirect ELISA coated with the same protein (Fig. 7A). There were variable values ranged from 34.8 to 88.3. No antibodies against the protein NP49-375 were detected in the sera from chickens immunized with the proteins HA and HACD. The percent of inhibition was around 6.5. In the plates coated with the protein HACD the pattern of the percent of inhibition for the reference sera was also much related to the mODs observed in the indirect ELISA. The values ranged from 4.8 to 14.6, except for the serum H5N2 and the sera from the chickens immunized with the proteins HA and HACD. They showed percentages of inhibition of 86.2, 88.0 and 89.6 respectively. In the sera from birds of different species it seemed there were no antibodies against the proteins NP49-375 or HACD (Fig. 7B). The percentages of inhibition for the plates coated with the protein NP49-375 ranged from 7.2 to 15.5 and for the plates coated with the protein HACD the values ranged from 13.5 to 18.9. These results were similar to those observed with the sera of chickens infected with other avian viral diseases (Fig. 7C). The values ranged from 4.3 to 10.1 in the plates coated with the protein NP49-375 and from 14.0 to 17.4 in the plates coated with the protein HACD. None of the sera which were expected not to have antibodies against both proteins exceeded the 20% of inhibition, but there were values very close to this percent of inhibition in the ELISA plates coated with the two assayed proteins. Therefore, the cut-off value of each competition ELISA was determined at 25%.


Dual function of the hemagglutinin H5 fused to chicken CD154 in a potential strategy of DIVA against avian influenza disease: preliminary study.

Pose AG, Rodríguez ES, Méndez AC, Gómez JN, Redondo AV, Rodríguez ER, Ramos EM, Gutiérrez AÁ, Moltó MP, Roche DG, Ugalde YS, López AM - Open Vet J (2015)

Establishment of competition ELISA assays for a potential strategy of DIVA directed to the avian influenza virus H5N1. (A) Competition ELISA assays coated with 2.5 μg/mL of the proteins NP49-375 and HACD. The reference sera from different avian influenza virus subtypes diluted 1/200 and the sera from chickens immunized with the proteins HA and HACD diluted 1/1000 were tested. Duplicated samples from the sera of ten immunized chickens per experimental group and six replicates of each reference serum were evaluated. (B) Antibody detection in duplicated samples of the sera from birds of different species. Plates coated with 2.5 μg/mL of the protein NP49-375 used the monoclonal antibody anti-NP5 diluted 1/20 000 as detection antibody. Plates coated with 2.5 μg/mL of the protein HACD used the monoclonal antibody anti-HA2 diluted 1/20 000 as detection antibody (C). Antibody detection in the sera from chickens infected with distinct avian viral diseases. Plates were coated as above and the same detection antibodies were used. Sera were diluted 1/50. Six replicates per serum from infected chickens were used. Results were expressed as the percent of inhibition. PS: Reference H5N2 serum. NS: Reference negative serum. IBD: Infectious Bursal Disease. EDS: Egg Drop Syndrome. NDV: Newcastle Disease Virus. AP: Avian Parvovirus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4663798&req=5

Figure 7: Establishment of competition ELISA assays for a potential strategy of DIVA directed to the avian influenza virus H5N1. (A) Competition ELISA assays coated with 2.5 μg/mL of the proteins NP49-375 and HACD. The reference sera from different avian influenza virus subtypes diluted 1/200 and the sera from chickens immunized with the proteins HA and HACD diluted 1/1000 were tested. Duplicated samples from the sera of ten immunized chickens per experimental group and six replicates of each reference serum were evaluated. (B) Antibody detection in duplicated samples of the sera from birds of different species. Plates coated with 2.5 μg/mL of the protein NP49-375 used the monoclonal antibody anti-NP5 diluted 1/20 000 as detection antibody. Plates coated with 2.5 μg/mL of the protein HACD used the monoclonal antibody anti-HA2 diluted 1/20 000 as detection antibody (C). Antibody detection in the sera from chickens infected with distinct avian viral diseases. Plates were coated as above and the same detection antibodies were used. Sera were diluted 1/50. Six replicates per serum from infected chickens were used. Results were expressed as the percent of inhibition. PS: Reference H5N2 serum. NS: Reference negative serum. IBD: Infectious Bursal Disease. EDS: Egg Drop Syndrome. NDV: Newcastle Disease Virus. AP: Avian Parvovirus.
Mentions: Competition ELISA assays using the reference sera and the sera of chickens immunized with the proteins HA and HACD were employed to perform the strategy of DIVA. The competition ELISA assays also involved the sera from different species of birds including flamingo, gamecocks, parakeets, rosellas, ducks and turkeys, which were already tested as negative by the hemagglutination inhibition assay using antigens of the subtypes H5, H7 and H9. Likewise, we evaluated the sera of chickens infected with Infectious Bursal Disease, Egg Drop Syndrome, Newcastle Disease Virus and Avian Parvovirus. As expected, the pattern for the percent of inhibition detected in the reference sera when the ELISA plates were coated with the protein NP49-375 was consistent with the mODs observed in the indirect ELISA coated with the same protein (Fig. 7A). There were variable values ranged from 34.8 to 88.3. No antibodies against the protein NP49-375 were detected in the sera from chickens immunized with the proteins HA and HACD. The percent of inhibition was around 6.5. In the plates coated with the protein HACD the pattern of the percent of inhibition for the reference sera was also much related to the mODs observed in the indirect ELISA. The values ranged from 4.8 to 14.6, except for the serum H5N2 and the sera from the chickens immunized with the proteins HA and HACD. They showed percentages of inhibition of 86.2, 88.0 and 89.6 respectively. In the sera from birds of different species it seemed there were no antibodies against the proteins NP49-375 or HACD (Fig. 7B). The percentages of inhibition for the plates coated with the protein NP49-375 ranged from 7.2 to 15.5 and for the plates coated with the protein HACD the values ranged from 13.5 to 18.9. These results were similar to those observed with the sera of chickens infected with other avian viral diseases (Fig. 7C). The values ranged from 4.3 to 10.1 in the plates coated with the protein NP49-375 and from 14.0 to 17.4 in the plates coated with the protein HACD. None of the sera which were expected not to have antibodies against both proteins exceeded the 20% of inhibition, but there were values very close to this percent of inhibition in the ELISA plates coated with the two assayed proteins. Therefore, the cut-off value of each competition ELISA was determined at 25%.

Bottom Line: As the strategy of DIVA requires at least two proteins, we obtained a variant of the nucleoprotein (NP49-375) in E. coli.The results showed that the segment CD154 in the chimeric protein HACD did not interfere with the recognition of the molecule HA by its specific antibodies.No antibody detection was observed in the sera from different species of birds or the sera of chickens infected with other avian viral diseases.

View Article: PubMed Central - PubMed

Affiliation: Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), P. O. Box 6162, Havana 10600, Cuba.

ABSTRACT
In this study we demonstrated that the vaccine candidate against avian influenza virus H5N1 based on the hemagglutinin H5 (HA) fused to the chicken CD154 (HACD) can also be used for differentiating infected from vaccinated animals (DIVA). As the strategy of DIVA requires at least two proteins, we obtained a variant of the nucleoprotein (NP49-375) in E. coli. After its purification by IMAC, the competence of the proteins NP49-375 and HACD as coating antigens in indirect ELISA assays were tested by using the sera of chickens immunized with the proteins HA and HACD and the reference sera from several avian influenza subtypes. Together with these sera, the sera from different species of birds and the sera of chickens infected with other avian viral diseases were analyzed by competition ELISA assays coated with the proteins NP49-375 and HACD. The results showed that the segment CD154 in the chimeric protein HACD did not interfere with the recognition of the molecule HA by its specific antibodies. Also, we observed variable detection levels when the reference sera were analyzed in the ELISA plates coated with the protein NP49-375. Moreover, only the antibodies of the reference serum subtype H5 were detected in the ELISA plates coated with the protein HACD. The competition ELISA assays showed percentages of inhibition of 88-91% for the positives sera and less than 20% for the negative sera. We fixed the cut-off value of these assays at 25%. No antibody detection was observed in the sera from different species of birds or the sera of chickens infected with other avian viral diseases. This study supported the fact that the ELISA assays using the proteins NP49-375 and HACD could be valuable tools for avian influenza surveillance and as a strategy of DIVA for counteracting the highly pathogenic avian influenza virus H5N1 outbreaks.

No MeSH data available.


Related in: MedlinePlus