Development of high-growth influenza H7N9 prepandemic candidate vaccine viruses in suspension MDCK cells.

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.

MDCK-Adaptive Mutation of A169S Changes Glycosylation Pattern of Hemagglutinin and Enhances MDCK-Based H7N9 Vaccine Virus Production without Loss of Antigenicity and Immunogenicity.

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.

Characterization and Immunogenicity of Influenza H7N9 Vaccine Antigens Produced Using a Serum-Free Suspension MDCK Cell-Based Platform.

Human infections with avian-origin H7N9 influenza A viruses were first reported in China, and an approximately 38% human mortality rate was described across six waves from February 2013 to September 2018. Vaccination is one of the most cost-effective ways to reduce morbidity and mortality during influenza epidemics and pandemics. Egg-based platforms for the production of influenza vaccines are labor-intensive and unable to meet the surging demand during pandemics. Therefore, cell culture-based technology is becoming the alternative strategy for producing influenza vaccines. The current influenza H7N9 vaccine virus (NIBRG-268), a reassortant virus from A/Anhui/1/2013 (H7N9) and egg-adapted A/PR/8/34 (H1N1) viruses, could grow efficiently in embryonated eggs but not mammalian cells. Moreover, a freezing-dry formulation of influenza H7N9 vaccines with long-term stability will be desirable for pandemic preparedness, as the occurrence of influenza H7N9 pandemics is not predictable. In this study, we adapted a serum-free anchorage-independent suspension Madin-Darby Canine Kidney (MDCK) cell line for producing influenza H7N9 vaccines and compared the biochemical characteristics and immunogenicity of three influenza H7N9 vaccine antigens produced using the suspension MDCK cell-based platform without freeze-drying (S-WO-H7N9), the suspension MDCK cell-based platform with freeze-drying (S-W-H7N9) or the egg-based platform with freeze-drying (E-W-H7N9). We demonstrated these three vaccine antigens have comparable biochemical characteristics. In addition, these three vaccine antigens induced robust and comparable neutralizing antibody (NT; geometric mean between 1016 and 4064) and hemagglutinin-inhibition antibody (HI; geometric mean between 640 and 1613) titers in mice. In conclusion, the serum-free suspension MDCK cell-derived freeze-dried influenza H7N9 vaccine is highly immunogenic in mice, and clinical development is warranted.

Development and characterization of standard reagents for cell-based prepandemic influenza vaccine products.

Outbreaks of infection by novel avian influenza virus strains in humans cause public health issues worldwide, and the development of vaccines against such novel strains is the most effective method for the prevention of these virus outbreaks. All types of vaccines must be tested for potency before use; thus, quantitative potency assays are needed for influenza vaccines. The single radial immunodiffusion (SRID) assay is considered the gold standard for quantification of influenza virus antigens, and the SRID reference reagents are essential for the determination of vaccine potency. However, it remains debatable whether reference reagents derived from egg-based vaccine platforms can be used to precisely quantify non-egg-derived vaccines; thus, influenza vaccine production using cell-based platforms has attracted increasing attention. To evaluate the utility of reference reagents derived from a cell-based influenza vaccine platform, we prepared cell-based reference reagents from MDCK cell-grown viruses and compared them with egg-derived reference reagents. A primary liquid standard (PLS) was purified from cell-derived candidate influenza vaccine viruses, and hemagglutinin (HA) antigen content was determined by a densitometric method. The produced PLS could be stored at 4°C for more than 10 months. We also established a simple HA protein purification method for goat antiserum preparation, and the performance of the resulting antiserum was compared to that of standard reagents obtained using different production platforms. The results of this study indicate that these reference reagents can be used for both cell-based and egg-based production platforms and that the differences between these two types of platforms are negligible.

Evaluation of novel disposable bioreactors on pandemic influenza virus production.

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.

The stability and immunogenicity of inactivated MDCK cell-derived influenza H7N9 viruses.

In recent years, cell-based influenza vaccines have gained a great interest over the egg-based vaccines. Several inactivated H7N9 vaccines have been evaluated in clinical trials, including whole-virion vaccines, split vaccines and subunit vaccines. Recently, we developed a new suspension MDCK (sMDCK) cell line for influenza viruses production. However, the properties of purified antigen from sMDCK cells remain unclear. In this study, the stability of influenza H7N9 vaccine bulk derived from sMDCK cells was investigated, and the data were compared with the vaccine antigen derived from our characterized adhesion MDCK (aMDCK) cells in serum-free medium. The influenza H7N9 bulks derived from sMDCK and aMDCK cells were stored at 2-8 °C for different periods of time, and a number of parameters selected to monitor the H7N9 vaccine antigen stability were evaluated at each interval (1, 3 and 12 months). The monitored parameters included virus morphology, hemagglutinin (HA) activity, HA concentration, antigenicity, and immunogenicity. The sMDCK-derived H7N9 bulk showed similar morphology to that of the aMDCK-derived H7N9 bulk, and there were no obvious changes after the extended storage periods. Furthermore, the HA titer, HA concentration, and antigenicity of sMDCK-derived H7N9 bulk were stable after 28 months of storage. Finally, the results of hemagglutination inhibition and neutralization tests showed that sMDCK- and aMDCK-derived H7N9 vaccines had comparable immunogenicity. These results indicated that sMDCK-derived H7N9 bulk has good stability compared to that of aMDCK-derived H7N9 bulk. Thus, the newly developed suspension MDCK cell line shows a great alternative for manufacturing cell-based influenza vaccines.

Development of American-Lineage Influenza H5N2 Reassortant Vaccine Viruses for Pandemic Preparedness.

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.

A fast and efficient purification platform for cell-based influenza viruses by flow-through chromatography.

Since newly emerging influenza viruses with pandemic potentials occurred in recent years, the demand for producing pandemic influenza vaccines for human use is high. For the development of a quick and efficient vaccine production, we proposed an efficient purification platform from the harvest to the purified bulk for the cell-based influenza vaccine production. This platform based on flow-through chromatography and filtration steps and the process only involves a few purification steps, including depth filtration, inactivation by formaldehyde, microfiltration, ultrafiltration, anion-exchange and ligand-core chromatography and sterile filtration. In addition, in the proposed chromatography steps, no virus capture steps were employed, and the purification results were not affected by the virus strain variation, host cells and culturing systems. The results from different virus strains which produced by Vero or MDCK cells in different culturing systems also obtained 33-46% HA recovery yields by this platform. The overall removal rates of the protein and DNA concentration in the purified bulk were over 96.1% and 99.7%, respectively. The low residual cellular DNA concentrations were obtained ranged from 30 to 130pg per human dose (15µg/dose). All influenza H5N1 purified bulks met the regulatory requirements for human vaccine use.

Evaluation of MDCK cell-derived influenza H7N9 vaccine candidates in ferrets.

Avian-origin influenza A (H7N9) viruses emerged as human pathogens in China in early 2013 and have killed >100 persons. Influenza vaccines are mainly manufactured using egg-based technology which could not meet the surging demand during influenza pandemics. In this study, we evaluated cell-based influenza H7N9 vaccines in ferrets. An egg-derived influenza H7N9 reassortant vaccine virus was adapted in MDCK cells. Influenza H7N9 whole virus vaccine antigen was manufactured using a microcarrier-based culture system. Immunogenicity and protection of the vaccine candidates with three different formulations (300 µg aluminum hydroxide, 1.5 µg HA, and 1.5 µg HA plus 300 µg aluminum hydroxide) were evaluated in ferrets. In ferrets receiving two doses of vaccination, geometric mean titers of hemagglutination (HA) inhibition and neutralizing antibodies were <10 and <40 for the control group (adjuvant only), 17 and 80 for the unadjuvanted (HA only) group, and 190 and 640 for the adjuvanted group (HA plus adjuvant), respectively. After challenge with wild-type influenza H7N9 viruses, virus titers in respiratory tracts of the adjuvanted group were significantly lower than that in the control, and unadjuvanted groups. MDCK cell-derived influenza H7N9 whole virus vaccine candidate is immunogenic and protective in ferrets and clinical development is highly warranted.

Adaptation of high-growth influenza H5N1 vaccine virus in Vero cells: implications for pandemic preparedness.

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.

Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium.

BACKGROUND: Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system. PRINCIPAL FINDING: The current study has evaluated the performance of cell adaptation switched from serum-containing (SC) medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3 × 10(6) cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA) units/50 µL and 7.1 ± 0.3 × 10(8) pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera. CONCLUSIONS: The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.

Microcarrier-based MDCK cell culture system for the production of influenza H5N1 vaccines.

Current egg-based influenza vaccine production technology, which is labor intensive and slow, would not be able to meet demand during an influenza pandemic. Thus, interest in the emerging technology of using mammalian cells for vaccine production has been great. In this study, Madin-Darby canine kidney (MDCK) cells using microcarrier culture systems were established to produce inactivated whole-virus H5N1 vaccine. The current clade-1 influenza H5N1 vaccine virus (NIBRG-14) was provided by the UK National Institute for Biological Standards and Control. Various process parameters were first optimized in 100-mL scale spinner flasks then scaled up to a 1-L scale bioreactor system. In the 1-L scale bioreactor system, peak virus titer could reach 10(8-9)TCID50/mL using serum-containing medium. After purification and inactivation, hemagglutinin (HA) protein content reached 31.56-43.96 microg/mL in two different runs. In mice immunogenicity studies, two doses of the purified vaccine antigen adjuvanted with aluminum phosphate induced good immune responses in 0.2 and 1.0 microg HA dosages (geometric mean titers of hemagglutination-inhibition antibody: 113 and 242, respectively). This study demonstrates the feasibility of the development of MDCK cell-based inactivated influenza H5 vaccines in microcarrier culture systems and could be valuable to many countries that are planning to establish manufacturing capacity for influenza vaccines.