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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

Quantification of viral protein as compared to the high growth A(H1N1)pdm09 reassortant X-181A. (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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fig01: Quantification of viral protein as compared to the high growth A(H1N1)pdm09 reassortant X-181A. (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: The HA content in virus purified from eggs is a critical attribute of any candidate vaccine virus to be used in manufacturing vaccine for an urgent pandemic response. Although the infectivity and HA titers of a virus in the allantoic fluid can give an indication of the total virus concentration, these parameters are not reliable indicators of HA content in purified viruses. To better estimate the HA content of the A(H7N9) PR8 reassortants, we analyzed the total viral protein and HA antigen yield of inactivated virions purified from allantoic fluid by differential and equilibrium ultracentrifugation in sucrose gradients. In this study, both IDCDC-RG32A and IDCDC-RG32B viruses yielded approximately 8 mg virus protein/100 eggs (based on an average of 9 mL of allantoic fluid collected per egg). This yield is lower than the minimal requirement for vaccine manufacturing, representing only 45% of the total protein yield obtained under identical conditions from an A(H1N1)pdm09 reassortant virus X-181A, with an average yield for a seasonal influenza CVV (Figure1A). To quantify the HA antigen yield, we analyzed purified virus samples by isotope dilution mass spectrometry (IDMS).11 Four peptides of H7 HA (two located on HA1 and two on HA2) were used as specific target peptides that are stoichiometric representatives of the HA protein, as previously described.12 Multiple target peptides were used to ensure that enzymatic digestion of the protein was complete and reproducible as well as to verify accuracy of the measurement.11,12 The IDMS data indicated no significant HA protein yield differences between purified IDCDC-RG32A and IDCDC-RG32B viruses, with an average of 2·9 ± 0·2 mg HA/100 eggs. In contrast, the antigen yield of X-181A averaged 5·6 ± 1·2 mg HA/100 eggs, approximately a twofold greater yield than the H7N9 PR8 reassortants (Figure1B). Therefore, the antigen yields of both IDCDC-RG32A and IDCDC-RG32B viruses were considered suboptimal for vaccine manufacturing in eggs.


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)

Quantification of viral protein as compared to the high growth A(H1N1)pdm09 reassortant X-181A. (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

fig01: Quantification of viral protein as compared to the high growth A(H1N1)pdm09 reassortant X-181A. (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: The HA content in virus purified from eggs is a critical attribute of any candidate vaccine virus to be used in manufacturing vaccine for an urgent pandemic response. Although the infectivity and HA titers of a virus in the allantoic fluid can give an indication of the total virus concentration, these parameters are not reliable indicators of HA content in purified viruses. To better estimate the HA content of the A(H7N9) PR8 reassortants, we analyzed the total viral protein and HA antigen yield of inactivated virions purified from allantoic fluid by differential and equilibrium ultracentrifugation in sucrose gradients. In this study, both IDCDC-RG32A and IDCDC-RG32B viruses yielded approximately 8 mg virus protein/100 eggs (based on an average of 9 mL of allantoic fluid collected per egg). This yield is lower than the minimal requirement for vaccine manufacturing, representing only 45% of the total protein yield obtained under identical conditions from an A(H1N1)pdm09 reassortant virus X-181A, with an average yield for a seasonal influenza CVV (Figure1A). To quantify the HA antigen yield, we analyzed purified virus samples by isotope dilution mass spectrometry (IDMS).11 Four peptides of H7 HA (two located on HA1 and two on HA2) were used as specific target peptides that are stoichiometric representatives of the HA protein, as previously described.12 Multiple target peptides were used to ensure that enzymatic digestion of the protein was complete and reproducible as well as to verify accuracy of the measurement.11,12 The IDMS data indicated no significant HA protein yield differences between purified IDCDC-RG32A and IDCDC-RG32B viruses, with an average of 2·9 ± 0·2 mg HA/100 eggs. In contrast, the antigen yield of X-181A averaged 5·6 ± 1·2 mg HA/100 eggs, approximately a twofold greater yield than the H7N9 PR8 reassortants (Figure1B). Therefore, the antigen yields of both IDCDC-RG32A and IDCDC-RG32B viruses were considered suboptimal for vaccine manufacturing in eggs.

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