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Adaptation of High-Growth Influenza H5N1 Vaccine Virus in Vero Cells: Implications for Pandemic Preparedness

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Authors:
Yufen Tseng at National Health Research Institutes
Yufen Tseng
Alan Yung-Chih Hu at Tantii Laboratory Inc.
Alan Yung-Chih Hu
  • Tantii Laboratory Inc.
Mei-Liang Huang
Mei-Liang Huang
Mei-Liang Huang
  • This person is not on ResearchGate, or hasn't claimed this research yet.
Wei-Zhou Yeh at Industrial Technology Research Institute
Wei-Zhou Yeh
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Abstract and Figures

Current egg-based influenza vaccine production technology can't promptly meet the global demand during an influenza pandemic as shown in the 2009 H1N1 pandemic. Moreover, its manufacturing capacity would be vulnerable during pandemics caused by highly pathogenic avian influenza viruses. Therefore, vaccine production using mammalian cell technology is becoming attractive. Current influenza H5N1 vaccine strain (NIBRG-14), a reassortant virus between A/Vietnam/1194/2004 (H5N1) virus and egg-adapted high-growth A/PR/8/1934 virus, could grow efficiently in eggs and MDCK cells but not Vero cells which is the most popular cell line for manufacturing human vaccines. After serial passages and plaque purifications of the NIBRG-14 vaccine virus in Vero cells, one high-growth virus strain (Vero-15) was generated and can grow over 10(8) TCID(50)/ml. In conclusion, one high-growth H5N1 vaccine virus was generated in Vero cells, which can be used to manufacture influenza H5N1 vaccines and prepare reassortant vaccine viruses for other influenza A subtypes.
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Adaptation of High-Growth Influenza H5N1 Vaccine Virus in Vero Cells: Implications for Pandemic Preparedne
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... Độ pha loãng Hiệu giá virus thu được (TCID 50 /ml) Tại các đời cấy thứ 8 và 9, hiệu giá virus thu được đạt cao nhất sau 96 giờ gây nhiễm khi các tế bào đã bị huỷ hoại gần như hoàn toàn (hình 3). Việc chọn chủng bằng phương pháp plaque assay đã được nhiều nhóm nghiên u ứng dụng trong việc chọn ra những chủng thuần có khả năng tăng sinh tốt đã thích hi với hệ thống nuôi cấy qua quá trình chọn lọc [11,12]. ...
... tương đồng 100% so với mẫu virus ban u (B5-203P-2013) (hình 5). Việc chọn chủng bằng phương pháp plaque assay đã được nhiều nhóm nghiên cứu ứng dụng trong việc chọn ra những chủng thuần có khả năng tăng sinh tốt đã thích nghi với hệ thống nuôi cấy qua quá trình chọn lọc [11,12]. ...
... tương đồng 100% so với mẫu virus ban đầu (B5-203P-2013) (hình 5). 7 Việc chọn chủng bằng phương pháp plaque assay đã được nhiều nhóm nghiên cứu ứng dụng trong việc chọn ra những chủng thuần có khả năng tăng sinh tốt đã thích nghi với hệ thống nuôi cấy qua quá trình chọn lọc [11,12]. ...
Selection of candidate strains for hand, foot and mouth disease vaccine against enterovirus 71
Article
  • Dec 2022
The study aimed to select candidate strains from Vietnam for the development of the hand, foot and mouth disease (HFMD) vaccine against enterovirus 71 (EV71). Annual EV71 virus surveillance data of Pasteur Institute in Ho Chi Minh city from 2003 to 2013 was used to predict evolutionary trends. One hundred VP1 sequences of Vietnam strains from the GenBank database were used to evaluate the identity of acid amin sequences and identify the consensus sequences at key epitopes. As a result, the subgenogroup B5 strain named B5-203P-2013 was selected. The strain was adapted on Vero cells by multiple passages and plaque isolated. The strain B5-203P-2013-P10.1 was selected as a candidate strain for the HFMD vaccine due to possessing genetic stability, hightiter of 108 TCID50/ml and cross-neutralising antibody responses against other subgenogroups B5, C4, C5.
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... Most wild-type influenza viruses and egg-derived high-growth reassortants (HGRs, >10 7.5 TCID50/mL) may not grow efficiently in Vero cells and need adaptations [6], which is time-consuming and not feasible for emergency response. Since the 2017-2018 season, the World Health Organization (WHO) has provided MDCK cell-derived wild-type viruses to be used as seasonal influenza CVVs for MDCK cell-based production. ...
... To increase production efficiency, it is desirable to develop high-growth influenza viruses as master donor viruses (MDVs) for the Vero cell-based platform. Our laboratory previously adapted an egg-derived high-growth H5N1 clade 1 CVV in Vero cells (Vero-15) [6] (Figure 1, strategy one) and applied Vero-15 as an MDV to generate high-growth reassortant viruses for avian influenza viruses (AIVs). Our previous data shows that Vero-15 could generate HGRs of H5N1 clade 2 viruses but not American-lineage H5N2 viruses [7]. ...
... The six internal genes from NIBRG-14 were from the egg-adapted high growth control master donor virus, PR8. The PB2-S360Y mutation was from Vero cell-adapted NIGRG-14, Vero-15 [6]. The Vero cell-derived NS genes were from Vero cell-adapted viruses, Vero-15, Vero-16, RG6-Vero, RG30-Vero, and RG32A-Vero (Table 1). ...
Development and Evaluation of Vero Cell-Derived Master Donor Viruses for Influenza Pandemic Preparedness
Article
Full-text available
  • Oct 2020
The embryonated egg-based platform currently produces the majority of seasonal influenza vaccines by employing a well-developed master donor virus (MDV, A/PR/8/34 (PR8)) to generate high-growth reassortants (HGRs) for A/H1N1 and A/H3N2 subtypes. Although the egg-based platform can supply enough seasonal influenza vaccines, it cannot meet surging demands during influenza pandemics. Therefore, multi-purpose platforms are desirable for pandemic preparedness. The Vero cell-based production platform is widely used for human vaccines and could be a potential multi-purpose platform for pandemic influenza vaccines. However, many wild-type and egg-derived influenza viruses cannot grow efficiently in Vero cells. Therefore, it is critical to develop Vero cell-derived high-growth MDVs for pandemic preparedness. In this study, we evaluated two in-house MDVs (Vero-15 and VB5) and two external MDVs (PR8 and PR8-HY) to generate Vero cell-derived HGRs for five avian influenza viruses (AIVs) with pandemic potentials (H5N1 clade 2.3.4, H5N1 clade 2.3.2.1, American-lineage H5N2, H7N9 first wave and H7N9 fifth wave). Overall, no single MDV could generate HGRs for all five AIVs, but this goal could be achieved by employing two in-house MDVs (vB5 and Vero-15). In immunization studies, mice received two doses of Vero cell-derived inactivated H5N1 and H7N9 whole virus antigens adjuvanted with alum and developed robust antibody responses.
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... Trong các hệ thống bình khuấy spinner nuôi cấy, hạt Cytodex 1 thường được cấy với mật độ tối đa 5 g/l môi trường và đạt được năng suất tế bào xấp xỉ 2x10 6 tế bào/ml trong 4 hoặc 5 ngày nuôi cấy [7], tương đương với năng suất của chúng tôi đạt được trong khi nghiên cứu này sử dụng mật độ hạt thấp hơn là 2 g/l. ...
... Trong nghiên cứu này, với hệ thống Wave bioreactor, chúng tôi sử dụng mật độ hạt 5 g/l và đạt mật độ tế bào trung bình 3,2x10 6 tế bào/ml, tương đương với nghiên cứu sử dụng hệ thống lắc Wave bioreactor tương tự trước đây [9]. 7 mật độ tế bào trung bình 3,2x10 6 tb/ml, tương đương với nghiên cứu sử dụng hệ thống lắc Wave bioreactor tương tự trước đây [9]. ...
Culture of EV71 virus in wave bioreactor system for production of hand, foot and mouth disease vaccine
Article
  • Sep 2022
The study was carried out to establish an EV71 virus culture system on Vero cells in a shake culture system (Wave bioreactor) incorporating microcarriers for the production of hand, foot and mouth (HFM) vaccine caused by the EV71 virus. The results of the investigation of 2 types of microcarriers (Cytodex 1 and Cytodex 3), showed that Cytodex 1 gave better efficiency when culturing Vero cells in suspension conditions. The combination with Cytodex 1 on the Wave bioreactor system gave Vero cell culture efficiency of over 3x106cells/ml. The optimal infectious dose of EV71 virus on Vero cells was determined to be 0.0001 TCID50/ml. The optimisation of EV71 virus culture conditions on the Wave bioreactor system has obtained suitable results for the production of vaccine with virus titres 108TCID50/ml on the fifth day after infection. The procedure of production of EV71 virus on Vero cell in Wave bioreactor system was applicable in vaccine production with virus titres 108 TCID50/ml on the fifth day after infection.
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... Therefore, a new master donor virus was required for the Vero cell-based platform. In 2010, a Vero cell-adapted high-growth H5N1 vaccine virus, Vero-15, was generated in our laboratory [15]. Vero-15 is the Vero-adapted NIBRG-14 (A/Vietnam/1194/2004 (H5N1) × PR8) virus with internal genes derived from the PR8 strain. ...
... Avian influenza viruses, A/Chicken/CY/A2628/2012 (H5N2)-(E7, LPAI) and A/Chicken/YL/0502/2012 (H5N2)-(R3, HPAI), were provided by Animal Health Research Institute (AHRI). Vero-15 virus was the Vero cell-adapted NIBRG-14 (A/Vietnam/1194/2004 (H5N1) × PR8) virus from the National Health Research Institute (NHRI) [15]. ...
Development of American-Lineage Influenza H5N2 Reassortant Vaccine Viruses for Pandemic Preparedness
Article
Full-text available
  • Jun 2019
Novel low-pathogenic avian influenza (LPAI) H5N2 viruses hit poultry farms in Taiwan in 2003, and evolved into highly pathogenic avian influenza (HPAI) viruses in 2010. These viruses are reassortant viruses containing HA and NA genes from American-lineage H5N2 and six internal genes from local H6N1 viruses. According to a serological survey, the Taiwan H5N2 viruses can cause asymptomatic infections in poultry workers. Therefore, a development of influenza H5N2 vaccines is desirable for pandemic preparation. In this study, we employed reverse genetics to generate a vaccine virus having HA and NA genes from A/Chicken/CY/A2628/2012 (E7, LPAI) and six internal genes from a Vero cell-adapted high-growth H5N1 vaccine virus (Vero-15). The reassortant H5N2 vaccine virus, E7-V15, presented high-growth efficiency in Vero cells (512 HAU, 107.6 TCID50/mL), and passed all tests for qualification of candidate vaccine viruses. In ferret immunization, two doses of inactivated whole virus antigens (3 μg of HA protein) adjuvanted with alum could induce robust antibody response (HI titre 113.14). In conclusion, we have established reverse genetics to generate a qualified reassortant H5N2 vaccine virus for further development.
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... Vero cells support the replication of various viruses as they are deficient in type I interferon production (42). This susceptibility to viruses can help to elucidate the mechanism underlying viral infection and also help develop highly productive vaccine strains through adaptation (43)(44)(45)(46)(47). For adaptation, we chose two different SARS-CoV-2 strain types, that is, the Wuhan and the Europe strain types. ...
Vero cell-adapted SARS-CoV-2 strain shows increased viral growth through furin-mediated efficient spike cleavage
Article
Full-text available
  • Feb 2024
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes several host proteases to cleave the spike (S) protein to enter host cells. SARS-CoV-2 S protein is cleaved into S1 and S2 subunits by furin, which is closely involved in the pathogenicity of SARS-CoV-2. However, the effects of the modulated protease cleavage activity due to S protein mutations on viral replication and pathogenesis remain unclear. Herein, we serially passaged two SARS-CoV-2 strains in Vero cells and characterized the cell-adapted SARS-CoV-2 strains in vitro and in vivo . The adapted strains showed high viral growth, effective S1/S2 cleavage of the S protein, and low pathogenicity compared with the wild-type strain. Furthermore, the viral growth and S1/S2 cleavage were enhanced by the combination of the Δ68–76 and H655Y mutations using recombinant SARS-CoV-2 strains generated by the circular polymerase extension reaction. The recombinant SARS-CoV-2 strain, which contained the mutation of the adapted strain, showed increased susceptibility to the furin inhibitor, suggesting that the adapted SARS-CoV-2 strain utilized furin more effectively than the wild-type strain. Pathogenicity was attenuated by infection with effectively cleaved recombinant SARS-CoV-2 strains, suggesting that the excessive cleavage of the S proteins decreases virulence. Finally, the high-growth-adapted SARS-CoV-2 strain could be used as the seed for a low-cost inactivated vaccine; immunization with this vaccine can effectively protect the host from SARS-CoV-2 variants. Our findings provide novel insights into the growth and pathogenicity of SARS-CoV-2 in the evolution of cell-cell transmission. IMPORTANCE The efficacy of the S protein cleavage generally differs among the SARS-CoV-2 variants, resulting in distinct viral characteristics. The relationship between a mutation and the entry of SARS-CoV-2 into host cells remains unclear. In this study, we analyzed the sequence of high-growth Vero cell-adapted SARS-CoV-2 and factors determining the enhancement of the growth of the adapted virus and confirmed the characteristics of the adapted strain by analyzing the recombinant SARS-CoV-2 strain. We successfully identified mutations Δ68-76 and H655Y, which enhance viral growth and the S protein cleavage by furin. Using recombinant viruses enabled us to conduct a virus challenge experiment in vivo . The pathogenicity of SARS-CoV-2 introduced with the mutations Δ68-76, H655Y, P812L, and Q853L was attenuated in hamsters, indicating the possibility of the attenuation of excessive cleaved SARS-CoV-2. These findings provide novel insights into the infectivity and pathogenesis of SARS-CoV-2 strains, thereby significantly contributing to the field of virology.
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... However, studies regarding the efficacy of Vero Cell vaccines are still ongoing. [9][10][11][12][13] Nepal started its vaccination drive with Covishield, a replication-deficient Chimpanzee Adenovirusvectored vaccine (Chad-Ox1-nCOV-19) followed by the Sinopharm -inactivated Vero cell vaccine, 14 both vaccines are almost equally covered in Nepal by till August 3, 2021. About 0.47 million people were vaccinated with Covishield and almost 0.7 million people with the Vero cell vaccine till the end of March 2022. ...
Outcome of COVID-19 Infection in vaccinated and unvaccinated adults admitted to Patan Hospital, Nepal
Article
Full-text available
  • Nov 2022
Introduction: The vaccine has a positive impact on reducing the severity of the COVID-19 disease course and studies have projected 50-90% protection against severe disease. Our study aimed to find the difference in disease severity outcomes between RT-PCR positive vaccinated and unvaccinated individuals. Method: This cross-sectional study was carried out among 417 COVID-19 cases who were admitted from June 2021 to August 2021. Collected data were fed into Microsoft-excel and analyzed using SPSS software, version- 16.0. Frequency, percentage, mean and standard deviation were calculated for descriptive analysis. To find out the association of categorical variables, the Chi-square test or Fisher’s Exact test was used where appropriate. P-value <0.05 was considered significant. Result: Out of 417 cases, 23(5.5%) were fully vaccinated i.e. 10(2.4%) with Covishield and 13(3.1%) with Vero Cell, 48(11.5%) partially vaccinated i.e. 19(4.6%) with Covishield, and 29 (6.9%) with Vero Cell and 346(83%) unvaccinated. 343(82.25%) admitted patients were discharged and 74(17.75%) died during our study period. The disposition of the patient was statistically significant (p<0.001) and revealed more mortality among unvaccinated cases. There was a statistical difference between the requirement of ICU admission (p=0.032) among vaccinated and unvaccinated cases. The Absolute Risk Reduction (ARR) of mortality and requirement of ICU after complete vaccination with respect to unvaccinated COVID-19 patients was 14.11%(5.14%-23.73%) and 20.50%(8.02%-32.97%) respectively at 95% CI. Conclusion: Vaccination reduces Mortality, the requirement of ICU, and oxygen requirement among COVID-19 cases with respect to unvaccinated COVID-19 cases.
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... However, the yield of egg-derived CVVs directly cultured in mammalian cells is usually low [9], which increases the cost of production and delays the supply of influenza vaccine during pandemics. To increase the yield of egg-derived CVVs in cell-based production platforms, further adaptation is usually required [10]. We produced inactivated whole-virion H5N1 and H7N9 vaccines in characterized adherent MDCK (aMDCK) cells in a PIC/ S GMP bioproduction plant at the National Health Research Institutes (NHRI), Taiwan [11,12]. ...
Development of high-growth influenza H7N9 prepandemic candidate vaccine viruses in suspension MDCK cells
Article
Full-text available
  • Dec 2020
  • J BIOMED SCI
Background: Influenza vaccine manufacturers traditionally use egg-derived candidate vaccine viruses (CVVs) to produce high-yield influenza viruses for seasonal or pandemic vaccines; however, these egg-derived CVVs need an adaptation process for the virus to grow in mammalian cells. The low yields of cell-based manufacturing systems using egg-derived CVVs remain an unsolved issue. This study aimed to develop high-growth cell-derived CVVs for MDCK cell-based vaccine manufacturing platforms. Methods: Four H7N9 CVVs were generated in characterized Vero and adherent MDCK (aMDCK) cells. Furthermore, reassortant viruses were amplified in adherent MDCK (aMDCK) cells with certification, and their growth characteristics were detected in aMDCK cells and new suspension MDCK (sMDCK) cells. Finally, the plaque-forming ability, biosafety, and immunogenicity of H7N9 reassortant viruses were evaluated. Results: The HA titers of these CVVs produced in proprietary suspension MDCK (sMDCK) cells and chicken embryos were 2- to 8-fold higher than those in aMDCK cells. All H7N9 CVVs showed attenuated characteristics by trypsin-dependent plaque assay and chicken embryo lethality test. The alum-adjuvanted NHRI-RG5 (derived from the fifth wave H7N9 virus A/Guangdong/SP440/2017) vaccine had the highest immunogenicity and cross-reactivity among the four H7N9 CVVs. Finally, we found that AddaVax adjuvant improved the cross-reactivity of low pathogenic H7N9 virus against highly pathogenic H7N9 viruses. Conclusions: Our study indicates that cell-derived H7N9 CVVs possessed high growth rate in new sMDCK cells and low pathogenicity in chicken embryo, and that CVVs generated by this platform are also suitable for both cell- and egg-based prepandemic vaccine production.
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... In previous reports [10,28], MDCK and Vero cells could produce influenza viruses using SFM in spinner flasks. In this study, the same cells were used for the production of H5N1 influenza clade II and H7N9 vaccine strains using SFM in the BelloCell systems, as well as in spinner flasks. ...
Evaluation of novel disposable bioreactors on pandemic influenza virus production
Article
Full-text available
Since 1997, the highly pathogenic influenza H5N1 virus has spread from Hong Kong. According to the WHO bulletin report, the H5N1 virus is a zoonotic disease threat that has infected more than 850 humans, causing over 450 deaths. In addition, an outbreak of another new and highly pathogenic influenza virus (H7N9) occurred in 2013 in China. These highly pathogenic influenza viruses could potentially cause a worldwide pandemic. it is crucial to develop a rapid production platform to meet this surge demand against any possible influenza pandemic. A potential solution for this problem is the use of cell-based bioreactors for rapid vaccine production. These novel bioreactors, used for cell-based vaccine production, possess various advantages. For example, they enable a short production time, allow for the handling highly pathogenic influenza in closed environments, and can be easily scaled up. In this study, two novel disposable cell-based bioreactors, BelloCell and TideCell, were used to produce H5N1 clade II and H7N9 candidate vaccine viruses (CVVs). Madin-Darby canine kidney (MDCK) cells were used for the production of these influenza CVVs. A novel bench-scale bioreactor named BelloCell bioreactor was used in the study. All culturing conditions were tested and scaled to 10 L industrial-scale bioreactor known as TideCell002. The performances of between BelloCell and TideCell were similar in cell growth, the average MDCK cell doubling time was slightly decreased to 25 hours. The systems yielded approximately 39.2 and 18.0 μg/ml of HA protein with the 10-liter TideCell002 from the H5N1 clade II and H7N9 CVVs, respectively. The results of this study not only highlight the overall effectiveness of these bioreactors but also illustrate the potential of maintaining the same outcome when scaled up to industrial production, which has many implications for faster vaccine production. Although additional studies are required for process optimization, the results of this study are promising and show that oscillating bioreactors may be a suitable platform for pandemic influenza virus production.
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MDCK-Adaptive Mutation of A169S Changes Glycosylation Pattern of Hemagglutinin and Enhances MDCK-Based H7N9 Vaccine Virus Production without Loss of Antigenicity and Immunogenicity
Article
Full-text available
  • Mar 2024
The adaptation of egg-derived H7N9 candidate vaccine virus (CVV) in the mammalian cell line is an approach to developing a high-growth virus strain for the mass production of vaccine manufacturing. The adaptive mutations that occur in hemagglutinin (HA) are critical to the activity and potency of the vaccine virus. Previously, we identified a new mutation of A169S in the HA protein of an MDCK-adapted H7N9 vaccine virus (A/Anhui/2013, RG268); however, whether and how this mutation affects vaccine potency remain to be investigated. In this study, we serially passaged RG268 in MDCK cells and found that the HA titer and the TCID50 of the passaged virus RG268-M5 were 4-fold (HA units/50 μL) and 3.5-fold (log10 TCID50/mL) higher than those of the original CVV. By inspecting tandem MS spectra, we identified a new glycosylation site at N167 near the receptor binding site of the HA protein of RG268-M5. Flow cytometry results revealed that RG268-M5 could efficiently infect MDCK cells and initiate viral protein replication as well as that of RG268. Though the new glycosylation site is in the antigenic epitope of viral HA protein, the HI assay result indicated that the antigenicity of RG268-M5 was similar to RG268. Additionally, immunizing mice with RG268-M5 mixed aluminum hydroxide could induce potent antibody responses against the homologous and heterologous H7N9 viruses in vitro whereas the titers were comparable with those from the RG268 group. These results provide in-depth structural information regarding the effects of site-specific glycosylation on virus properties, which have implications for novel avian influenza vaccine development.
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Comparative assessment of the replication efficiency of dengue, yellow fever, and chikungunya arboviruses in some insect and mammalian cell lines
Article
Full-text available
  • Apr 2019
  • REV SOC BRAS MED TRO
INTRODUCTION: Insect cell cultures play an essential role in understanding arboviral replication. However, the replicative efficiency of some of these viruses such as dengue (DENV), yellow fever (YFV), and chikungunya (CHIKV) in a new cellular substrate (Lulo) and in the other two recognized cell lines has not been comparatively assessed. METHODS: Vero, C6/36, and Lulo cell lines were infected with DENV, YFV, and CHIKV. The viral progeny was quantified through plaque assays and quantitative reverse transcription-polymerase chain reaction, while for DENV2, the findings were confirmed by immunofluorescence antibody assay. RESULTS: The higher DENV2 titer (from multiplicity of infection 0.001) was obtained on day four post-infection in C6/36 and on day six in Vero cells, while the Lulo cell line was almost impossible to infect under the same conditions. However, C6/36 showed the highest values of viral RNA production compared to Vero cells, while the quantification of the viral RNA in Lulo cells showed high levels of viral genomes, which had no correlation to the infectious viral particles. CONCLUSIONS: C6/36 was the most efficient cell line in the alpha and flavivirus production, followed by Vero cells. Thus, Lulo cells may be a useful substrate to study the mechanisms by which cells evade viral replication.
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Vero cell platform in vaccine production: Moving towards cell culture-based viral vaccines
Article
Full-text available
The development of cell culture systems for virus propagation has led to major advances in virus vaccine development. Primary and diploid cell culture systems are now being replaced by the use of continuous cell lines (CCLs). These substrates are gaining increasing acceptance from regulatory authorities as improved screening technologies remove fears regarding their potential oncogenic properties. The Vero cell line is the most widely accepted CCL by regulatory authorities and has been used for over 30 years for the production of polio and rabies virus vaccines. The recent licensure of a Vero cell-derived live virus vaccine (ACAM2000, smallpox vaccine) has coincided with an explosion in the development of a range of new viral vaccines, ranging from live-attenuated pediatric vaccines against rotavirus infections to inactivated whole-virus vaccines against H5N1 pandemic influenza. These developments have illustrated the value of this cell culture platform in the rapid development of vaccines against a range of virus diseases.
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Growth Determinants for H5N1 Influenza Vaccine Seed Viruses in MDCK Cells
Article
Full-text available
H5N1 influenza A viruses are exacting a growing human toll, with more than 240 fatal cases to date. In the event of an influenza pandemic caused by these viruses, embryonated chicken eggs, which are the approved substrate for human inactivated-vaccine production, will likely be in short supply because chickens will be killed by these viruses or culled to limit the worldwide spread of the infection. The Madin-Darby canine kidney (MDCK) cell line is a promising alternative candidate substrate because it supports efficient growth of influenza viruses compared to other cell lines. Here, we addressed the molecular determinants for growth of an H5N1 vaccine seed virus in MDCK cells, revealing the critical responsibility of the Tyr residue at position 360 of PB2, the considerable requirement for functional balance between hemagglutinin (HA) and neuraminidase (NA), and the partial responsibility of the Glu residue at position 55 of NS1. Based on these findings, we produced a PR8/H5N1 reassortant, optimized for this cell line, that derives all of its genes for its internal proteins from the PR8(UW) strain except for the NS gene, which derives from the PR8(Cambridge) strain; its N1 NA gene, which has a long stalk and derives from an early H5N1 strain; and its HA gene, which has an avirulent-type cleavage site sequence and is derived from a circulating H5N1 virus. Our findings demonstrate the importance and feasibility of a cell culture-based approach to producing seed viruses for inactivated H5N1 vaccines that grow robustly and in a timely, cost-efficient manner as an alternative to egg-based vaccine production.
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Global production of seasonal and pandemic (H1N1) influenza vaccines in 2009-2010 and comparison with previous estimates and global action plan targets
Article
  • Jul 2010
Immunization against influenza is considered among the most important interventions in reducing the public health impact of seasonal epidemic and pandemic influenza infections. However, there are marked differences across countries with regards to production, supply and access to influenza vaccines. A global action plan (GAP) to increase supply of pandemic influenza vaccine was developed by the World Health Organization in May 2006 to reduce the anticipated gap between potential vaccine demand and supply during an influenza pandemic. To quantify the increase in global influenza vaccine production capacity and actual production in response to the influenza A(H1N1) 2009 pandemic, 3 years after the development of the GAP, the WHO conducted a survey of vaccine producers from December 2009 through February 2010, and compared the results of this survey with results from surveys conducted in 2006-2007 and May 2009.
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Vaccine production capacity for seasonal and pandemic (H1N1) 2009 influenza
Article
  • Jul 2009
The first influenza pandemic of the 21st century, due to a new strain of A(H1N1) virus, was declared on 11 June 2009 by the Director-General of the World Health Organization. Fortunately, the international community, including influenza vaccine manufacturers, has been increasing its preparedness for such an event, triggered by the need to stem the spread of the highly pathogenic avian influenza A(H5N1) virus over recent years. Today, the development of a pandemic influenza vaccine in the fastest possible time is a global priority. However, two major issues need to be taken into consideration: how long will it take to produce sufficient pandemic vaccine doses to immunize the global population at risk, including poor populations that have no resources to purchase the vaccine; and how will pandemic vaccine production affect availability of trivalent vaccine for the forthcoming 2009-2010 influenza season. To address these questions, WHO carried out a survey in May 2009 among influenza vaccine manufacturers on their planned seasonal and pandemic production with a view to developing recommendations on the distribution and use of pandemic influenza vaccine.
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Emergence and pandemic potential of Swine-Origin H1N1 Influenza viruses
Article
  • Jul 2009
  • NATURE
Influenza viruses cause annual epidemics and occasional pandemics that have claimed the lives of millions. The emergence of new strains will continue to pose challenges to public health and the scientific communities. A prime example is the recent emergence of swine-origin H1N1 viruses that have transmitted to and spread among humans, resulting in outbreaks internationally. Efforts to control these outbreaks and real-time monitoring of the evolution of this virus should provide us with invaluable information to direct infectious disease control programmes and to improve understanding of the factors that determine viral pathogenicity and/or transmissibility.
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Trivalent MDCK cell culture-derived influenza vaccine Optaflu(R) (Novartis Vaccines)
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Annual influenza epidemics continue to have a considerable impact in both developed and developing countries. Vaccination remains the principal measure to prevent seasonal influenza and reduce associated morbidity and mortality. The WHO recommends using established mammalian cell culture lines as an alternative to egg-based substrates in the manufacture of influenza vaccine. In June 2007, the EMEA approved Optaflu, a Madin Darby canine kidney cell culture-derived influenza vaccine manufactured by Novartis Vaccines. This review examines the advantages and disadvantages of cell culture-based technology for influenza vaccine production, compares immunogenicity and safety data for Optaflu with that of currently marketed conventional egg-based influenza vaccines, and considers the prospects for wider use of cell culture-based influenza vaccines.
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Current challenges in implementing cell-derived influenza vaccines: Implications for production and regulation, July 2007, NIBSC, Potters Bar, UK
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  • Mar 2009
  • VACCINE
A meeting was held at NIBSC, UK in July 2007 to discuss the implications of progress in the use of cell culture systems for the manufacture of vaccines against influenza. Issues discussed included the effect of using eggs and different cell types in strain selection, development of seed viruses to be used in production and the nature of the reagents to be used in determining vaccine potency. Future studies to progress the field were reviewed.
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Progress with human H5N1 vaccines: A perspective from industry
Article
With the ongoing widespread circulation of avian H5N1 influenza viruses and the threat of pandemic influenza an ever-present reality, it is essential that those charged with protecting public health continue to focus intently on this urgent issue. As part of the response to this pandemic threat, research-based influenza vaccine manufacturers have made rapid progress with the research, development and testing of new vaccines and have greatly expanded their capacity to manufacture these products in the last few years. With the research and development phase now reaching completion, regulators have approved several prototype 'mock-up' pandemic vaccines and a number of 'prepandemic' H5N1 vaccines. As a consequence, the world is better prepared than it has ever been to counter an influenza pandemic. However, despite these advances, many policy, logistical and practical issues must be resolved for the population to benefit from these breakthroughs. In particular, the global community must establish the infrastructure required for population-wide immunization, secure appropriate vaccine supplies and undertake sufficient vaccine stockpiling to protect against the first wave of a pandemic.
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Pre-clinical development of cell culture (Vero)-derived H5N1 pandemic vaccines
Article
  • Jun 2008
The rapid spread of avian influenza (H5N1) and its transmission to humans has raised the possibility of an imminent pandemic and concerns over the ability of standard influenza vaccine production methods to supply sufficient amounts of an effective vaccine. We report here on a robust and flexible strategy which uses wild-type virus grown in a continuous cell culture (Vero) system to produce an inactivated whole virus vaccine. Candidate vaccines based on clade 1 and clade 2 influenza H5N1 strains, produced at a variety of manufacturing scales, were demonstrated to be highly immunogenic in animal models without the need for adjuvant. The vaccines induce cross-neutralising antibodies and are protective in a mouse challenge model not only against the homologous virus but against other H5N1 strains, including those from other clades. These data indicate that cell culture-grown, whole virus vaccines, based on the wild-type virus, allow the rapid high-yield production of a candidate pandemic vaccine.
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