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Development of influenza A(H7N9) candidate vaccine viruses with improved hemagglutinin antigen yield in eggs.

Ridenour C, Johnson A, Winne E, Hossain J, Mateu-Petit G, Balish A, Santana W, Kim T, Davis C, Cox NJ, Barr JR, Donis RO, Villanueva J, Williams TL, Chen LM - Influenza Other Respir Viruses (2015)

Bottom Line: The resulting A(H7N9) CVVs needed improvement because they had titers and antigen yields that were suboptimal for vaccine manufacturing in eggs, especially in a pandemic situation.CVVs were antigenically characterized by hemagglutination inhibition (HI) assays with ferret antisera.However, HI tests indicated that the antigenic properties of two CVVs remained unchanged.

View Article: PubMed Central - PubMed

Affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.

No MeSH data available.


Related in: MedlinePlus

Yield analysis of egg-passaged reassortant viruses as compared to parental virus yield (A) Quantification of total viral protein, shown as mg total viral protein/100 eggs. (B) Quantification of HA antigen, shown as mg HA/100 eggs. Values shown are the average of at least two independent experiments with errors bars denoting standard deviation.
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fig02: Yield analysis of egg-passaged reassortant viruses as compared to parental virus yield (A) Quantification of total viral protein, shown as mg total viral protein/100 eggs. (B) Quantification of HA antigen, shown as mg HA/100 eggs. Values shown are the average of at least two independent experiments with errors bars denoting standard deviation.

Mentions: Candidate vaccine viruses used to manufacture seasonal influenza vaccines undergo approximately four to five egg passages in the course of classical reassortment with PR8, with several additional passages in eggs to achieve higher titers and establish the master and working seed stocks for manufacturing.14 Dozens of CVVs prepared since the late 1970′s validate this approach for development of high yield viruses.15 In contrast to seasonal CVVs prepared by classical reassortment in eggs, pandemic CVVs derived by reverse genetics are generally passaged only twice in eggs prior to establishment of master and working seed stocks.16 In this study, we used serial passage in eggs as an approach to improve the antigen yields of the reverse genetics derived A(H7N9) PR8 reassortant viruses. Genetic analysis of the HA and NA genes from IDCDC-RG32B V1E13 (initial growth in Vero cells followed by 13 passages in eggs) stocks revealed a mixture of Gly/Glu at codon 196 of the mature HA protein (Gly as the wild-type codon, Glu as the mutant). Although no mutations in HA or NA genes were detected in the IDCDC-RG32A V1E13 virus stock, a mixture of Asn/Asp was detected at HA codon 149 in the V1E16 passage stock. Residue 149 (158 in H3 numbering) is located at the top of the globular head and has previously been shown to undergo positive selection in subtype H3 viruses when adapted to eggs.17 Residue 196 is at the monomer interface and has been shown to modulate receptor binding in the adjacent monomer.18,19 In order to evaluate the impact of these HA mutations on antigen yield, virus stocks with homogeneous sequences were prepared by limiting dilution and designated IDCDC-RG32A.1 (with 149Asp in HA) and IDCDC-RG32B.1 (with 196Glu in HA). The total viral protein yield of IDCDC-RG32A.1 and IDCDC-RG32B.1 after sucrose density gradient separation was 14·5 ± 0·2 mg virus protein/100 eggs and 10·7 ± 1·8 mg virus protein/100 eggs, respectively. The HA antigen yield of IDCDC-RG32A.1 and IDCDC-RG32B.1 was 5·5 ± 0·3 mg HA/100 eggs and 4·7 ± 0·4 mg HA/100 eggs, respectively, approximately a 78% and 81% increase in HA yield compared to their respective parental virus (Figure2).


Development of influenza A(H7N9) candidate vaccine viruses with improved hemagglutinin antigen yield in eggs.

Ridenour C, Johnson A, Winne E, Hossain J, Mateu-Petit G, Balish A, Santana W, Kim T, Davis C, Cox NJ, Barr JR, Donis RO, Villanueva J, Williams TL, Chen LM - Influenza Other Respir Viruses (2015)

Yield analysis of egg-passaged reassortant viruses as compared to parental virus yield (A) Quantification of total viral protein, shown as mg total viral protein/100 eggs. (B) Quantification of HA antigen, shown as mg HA/100 eggs. Values shown are the average of at least two independent experiments with errors bars denoting standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4548996&req=5

fig02: Yield analysis of egg-passaged reassortant viruses as compared to parental virus yield (A) Quantification of total viral protein, shown as mg total viral protein/100 eggs. (B) Quantification of HA antigen, shown as mg HA/100 eggs. Values shown are the average of at least two independent experiments with errors bars denoting standard deviation.
Mentions: Candidate vaccine viruses used to manufacture seasonal influenza vaccines undergo approximately four to five egg passages in the course of classical reassortment with PR8, with several additional passages in eggs to achieve higher titers and establish the master and working seed stocks for manufacturing.14 Dozens of CVVs prepared since the late 1970′s validate this approach for development of high yield viruses.15 In contrast to seasonal CVVs prepared by classical reassortment in eggs, pandemic CVVs derived by reverse genetics are generally passaged only twice in eggs prior to establishment of master and working seed stocks.16 In this study, we used serial passage in eggs as an approach to improve the antigen yields of the reverse genetics derived A(H7N9) PR8 reassortant viruses. Genetic analysis of the HA and NA genes from IDCDC-RG32B V1E13 (initial growth in Vero cells followed by 13 passages in eggs) stocks revealed a mixture of Gly/Glu at codon 196 of the mature HA protein (Gly as the wild-type codon, Glu as the mutant). Although no mutations in HA or NA genes were detected in the IDCDC-RG32A V1E13 virus stock, a mixture of Asn/Asp was detected at HA codon 149 in the V1E16 passage stock. Residue 149 (158 in H3 numbering) is located at the top of the globular head and has previously been shown to undergo positive selection in subtype H3 viruses when adapted to eggs.17 Residue 196 is at the monomer interface and has been shown to modulate receptor binding in the adjacent monomer.18,19 In order to evaluate the impact of these HA mutations on antigen yield, virus stocks with homogeneous sequences were prepared by limiting dilution and designated IDCDC-RG32A.1 (with 149Asp in HA) and IDCDC-RG32B.1 (with 196Glu in HA). The total viral protein yield of IDCDC-RG32A.1 and IDCDC-RG32B.1 after sucrose density gradient separation was 14·5 ± 0·2 mg virus protein/100 eggs and 10·7 ± 1·8 mg virus protein/100 eggs, respectively. The HA antigen yield of IDCDC-RG32A.1 and IDCDC-RG32B.1 was 5·5 ± 0·3 mg HA/100 eggs and 4·7 ± 0·4 mg HA/100 eggs, respectively, approximately a 78% and 81% increase in HA yield compared to their respective parental virus (Figure2).

Bottom Line: The resulting A(H7N9) CVVs needed improvement because they had titers and antigen yields that were suboptimal for vaccine manufacturing in eggs, especially in a pandemic situation.CVVs were antigenically characterized by hemagglutination inhibition (HI) assays with ferret antisera.However, HI tests indicated that the antigenic properties of two CVVs remained unchanged.

View Article: PubMed Central - PubMed

Affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.

No MeSH data available.


Related in: MedlinePlus