Limits...
Identification of Influenza A/PR/8/34 Donor Viruses Imparting High Hemagglutinin Yields to Candidate Vaccine Viruses in Eggs.

Johnson A, Chen LM, Winne E, Santana W, Metcalfe MG, Mateu-Petit G, Ridenour C, Hossain MJ, Villanueva J, Zaki SR, Williams TL, Cox NJ, Barr JR, Donis RO - PLoS ONE (2015)

Bottom Line: The effect of these related but distinct internal PR8 genes on the growth of vaccine viruses in eggs has not been examined previously.Although no single PR8 internal gene set outperformed the others in all candidate vaccine viruses, a combination of specific PR8 backbone with individual HA/NA pairs demonstrated improved HA yield and consequently the speed of vaccine production.These findings may be important both for production of seasonal vaccines and for a rapid global vaccine response during a pandemic.

View Article: PubMed Central - PubMed

Affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.

ABSTRACT
One of the important lessons learned from the 2009 H1N1 pandemic is that a high yield influenza vaccine virus is essential for efficient and timely production of pandemic vaccines in eggs. The current seasonal and pre-pandemic vaccine viruses are generated either by classical reassortment or reverse genetics. Both approaches utilize a high growth virus, generally A/Puerto Rico/8/1934 (PR8), as the donor of all or most of the internal genes, and the wild type virus recommended for inclusion in the vaccine to contribute the hemagglutinin (HA) and neuraminidase (NA) genes encoding the surface glycoproteins. As a result of extensive adaptation through sequential egg passaging, PR8 viruses with different gene sequences and high growth properties have been selected at different laboratories in past decades. The effect of these related but distinct internal PR8 genes on the growth of vaccine viruses in eggs has not been examined previously. Here, we use reverse genetics to analyze systematically the growth and HA antigen yield of reassortant viruses with 3 different PR8 backbones. A panel of 9 different HA/NA gene pairs in combination with each of the 3 different lineages of PR8 internal genes (27 reassortant viruses) was generated to evaluate their performance. Virus and HA yield assays showed that the PR8 internal genes influence HA yields in most subtypes. Although no single PR8 internal gene set outperformed the others in all candidate vaccine viruses, a combination of specific PR8 backbone with individual HA/NA pairs demonstrated improved HA yield and consequently the speed of vaccine production. These findings may be important both for production of seasonal vaccines and for a rapid global vaccine response during a pandemic.

No MeSH data available.


Related in: MedlinePlus

Total HA yield in virus concentrates determined by IDMS and SDS- PAGE/densitometer analysis.Reassortant viruses were propagated in embryonated eggs and purified from allantoic fluid by sucrose density gradient ultracentrifugation. Each virus was propagated and purified at least twice independently using a different batch of eggs. Total HA in concentrated virus samples was determined by IDMS (A) and SDS-PAGE/densitometry (B) as described in Materials and Methods. Statistical significance: *, P < 0.05; **, P < 0.01.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4465931&req=5

pone.0128982.g003: Total HA yield in virus concentrates determined by IDMS and SDS- PAGE/densitometer analysis.Reassortant viruses were propagated in embryonated eggs and purified from allantoic fluid by sucrose density gradient ultracentrifugation. Each virus was propagated and purified at least twice independently using a different batch of eggs. Total HA in concentrated virus samples was determined by IDMS (A) and SDS-PAGE/densitometry (B) as described in Materials and Methods. Statistical significance: *, P < 0.05; **, P < 0.01.

Mentions: To further quantify the antigen yield of the 6:2R viruses, we analyzed the purified viruses by isotope dilution mass spectrometry (IDMS) and SDS-PAGE/densitometry analysis. IDMS quantification revealed clustering of viruses into HAY (>3.4 mg/100 eggs; H3N2 Bang.) and LAY (<2.8 mg/100 eggs; H7N9) groups with almost the same distribution as determined by total virion protein assays (Fig 3A). Similarly, the total HA content of 6:2R virions concentrates determined by SDS-PAGE/densitometry (Fig 3B) ranged from <1.6 mg/100 eggs (H7N9) for LAY viruses to >3.8 mg/100 eggs (H3N2 Bang.) among HAY viruses. The HA yields determined by SDS-PAGE assays were slightly lower than those obtained by IDMS, possibly due to the lower basic amino acid content of influenza HA protein relative to BSA, resulting in decreased Coomassie blue stain uptake. However, the relative HA yields of viruses with different PR8 backbones remained consistent across assays. Comparisons within LAY 6:2R sets revealed that yields from PR8-A viruses were often higher than those of PR8-B viruses (3/6 6:2R sets with P<0.05) and in some cases yields from PR8-C viruses also were higher than those of PR8-B (P<0.05).


Identification of Influenza A/PR/8/34 Donor Viruses Imparting High Hemagglutinin Yields to Candidate Vaccine Viruses in Eggs.

Johnson A, Chen LM, Winne E, Santana W, Metcalfe MG, Mateu-Petit G, Ridenour C, Hossain MJ, Villanueva J, Zaki SR, Williams TL, Cox NJ, Barr JR, Donis RO - PLoS ONE (2015)

Total HA yield in virus concentrates determined by IDMS and SDS- PAGE/densitometer analysis.Reassortant viruses were propagated in embryonated eggs and purified from allantoic fluid by sucrose density gradient ultracentrifugation. Each virus was propagated and purified at least twice independently using a different batch of eggs. Total HA in concentrated virus samples was determined by IDMS (A) and SDS-PAGE/densitometry (B) as described in Materials and Methods. Statistical significance: *, P < 0.05; **, P < 0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465931&req=5

pone.0128982.g003: Total HA yield in virus concentrates determined by IDMS and SDS- PAGE/densitometer analysis.Reassortant viruses were propagated in embryonated eggs and purified from allantoic fluid by sucrose density gradient ultracentrifugation. Each virus was propagated and purified at least twice independently using a different batch of eggs. Total HA in concentrated virus samples was determined by IDMS (A) and SDS-PAGE/densitometry (B) as described in Materials and Methods. Statistical significance: *, P < 0.05; **, P < 0.01.
Mentions: To further quantify the antigen yield of the 6:2R viruses, we analyzed the purified viruses by isotope dilution mass spectrometry (IDMS) and SDS-PAGE/densitometry analysis. IDMS quantification revealed clustering of viruses into HAY (>3.4 mg/100 eggs; H3N2 Bang.) and LAY (<2.8 mg/100 eggs; H7N9) groups with almost the same distribution as determined by total virion protein assays (Fig 3A). Similarly, the total HA content of 6:2R virions concentrates determined by SDS-PAGE/densitometry (Fig 3B) ranged from <1.6 mg/100 eggs (H7N9) for LAY viruses to >3.8 mg/100 eggs (H3N2 Bang.) among HAY viruses. The HA yields determined by SDS-PAGE assays were slightly lower than those obtained by IDMS, possibly due to the lower basic amino acid content of influenza HA protein relative to BSA, resulting in decreased Coomassie blue stain uptake. However, the relative HA yields of viruses with different PR8 backbones remained consistent across assays. Comparisons within LAY 6:2R sets revealed that yields from PR8-A viruses were often higher than those of PR8-B viruses (3/6 6:2R sets with P<0.05) and in some cases yields from PR8-C viruses also were higher than those of PR8-B (P<0.05).

Bottom Line: The effect of these related but distinct internal PR8 genes on the growth of vaccine viruses in eggs has not been examined previously.Although no single PR8 internal gene set outperformed the others in all candidate vaccine viruses, a combination of specific PR8 backbone with individual HA/NA pairs demonstrated improved HA yield and consequently the speed of vaccine production.These findings may be important both for production of seasonal vaccines and for a rapid global vaccine response during a pandemic.

View Article: PubMed Central - PubMed

Affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.

ABSTRACT
One of the important lessons learned from the 2009 H1N1 pandemic is that a high yield influenza vaccine virus is essential for efficient and timely production of pandemic vaccines in eggs. The current seasonal and pre-pandemic vaccine viruses are generated either by classical reassortment or reverse genetics. Both approaches utilize a high growth virus, generally A/Puerto Rico/8/1934 (PR8), as the donor of all or most of the internal genes, and the wild type virus recommended for inclusion in the vaccine to contribute the hemagglutinin (HA) and neuraminidase (NA) genes encoding the surface glycoproteins. As a result of extensive adaptation through sequential egg passaging, PR8 viruses with different gene sequences and high growth properties have been selected at different laboratories in past decades. The effect of these related but distinct internal PR8 genes on the growth of vaccine viruses in eggs has not been examined previously. Here, we use reverse genetics to analyze systematically the growth and HA antigen yield of reassortant viruses with 3 different PR8 backbones. A panel of 9 different HA/NA gene pairs in combination with each of the 3 different lineages of PR8 internal genes (27 reassortant viruses) was generated to evaluate their performance. Virus and HA yield assays showed that the PR8 internal genes influence HA yields in most subtypes. Although no single PR8 internal gene set outperformed the others in all candidate vaccine viruses, a combination of specific PR8 backbone with individual HA/NA pairs demonstrated improved HA yield and consequently the speed of vaccine production. These findings may be important both for production of seasonal vaccines and for a rapid global vaccine response during a pandemic.

No MeSH data available.


Related in: MedlinePlus