VERO CELLS FOR VACCINE PRODUCTION.

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COMPILED BY: PRITAM BAGWE AND NAGENDRA GOWDA. INSTITUTE OF CHEMICAL TECHNOLOGY, MATUNGA, MUMBAI. SUBJECT : PHARMACEUTICAL BIOTECHNOLOGY. DATE : 23/11/2016.

INTRODUCTION:• Derived from the epithelial kidney cells of an African

green monkey (Cercopithecus aethiops) in the 1960s by Y. Yasumura and Y. Kawakita at the Chiba University in Chiba, Japan.

• The vero cell lineage is continuous and aneuploid.

Fluorescent image of confluent Vero cells; DAPI-stained nuclei appear blue, and actin filaments stained with rhodamine conjugated phalloidin appear red.

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VERO CELLS AND ITS APPLICATIONS• Vero cells are one of the most common mammalian continuous cell lines used in research.

• This anchorage-dependent cell line has been used extensively in virology studies,

• The propagation and study of intracellular bacteria (e.g., Rickettsia spp) and parasites (e.g., Neospora),

• Assessment of the effects of chemicals, toxins and other substances on mammalian cells at the molecular level.

• In addition, Vero cells have been licensed in the United States for production of both live (rotavirus, smallpox) and inactivated (poliovirus) viral vaccines, and

• Throughout the world Vero cells have been used for the production of a number of other viruses, including Rabies virus, Reovirus and Japanese encephalitis virus.

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GROWTH AND CULTURE CHARACTERISTICS:PROPAGATION OF VERO CELL CULTURE FROM FROZEN STOCKS

• For long term storage, Vero cells are kept either in liquid nitrogen or at -80°C.

• After recovery from frozen stock, Vero cells usually take 2-3 passages to reach their regular growth rate.

• Add Dulbecco’s modified Essential Medium (DMEM), supplemented with 10% heat inactivated fetal bovine serum (FBS), filter sterilized.

• Incubate flasks at 37°C in an incubator with 5% CO2.

• Actively growing Vero cell cultures double approximately every 24 hours.4

SUBCULTURING • Remove medium, and rinse the cells with 0.25% trypsin, 0.53 mM EDTA solution by

adding 1 to 2 mL of trypsin-EDTA solution. • Incubate the flask at 37°C until the cells detach (2 mins) . • Add fresh culture medium, aspirate and dispense into new culture flasks. • Subcultivation Ratio: A subcultivation ratio of 1:8 is recommended. • Medium Renewal: 2 to 3 times per week.

T-Flask

CYROPRESERVATION

• The cells are suspended in DMEM medium supplemented with 10% FBS.

• 10% DMSO is added as cryoprotectant. • The DMSO and FBS help preserve the cells during

the freezing and thawing processes.5

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Revive the frozen cell population

Maintain them in the culture (aseptic technique)

sub-culture (passaging)

cryopreservation

VACCINES AND CELL CULTURES• Traditionally, few available viral vaccines were produced in animal systems,

such as

• calf skin for smallpox, • rabbit spinal cord for rabies and • mouse brain for Japanese encephalitis, or • in embryonated eggs in the case of influenza and yellow fever viruses.

• A major concern, is the fact that when viruses are cultivated through extended passages in hens eggs,

• there is an evolutionary process in the allantoic cavity of the egg

• resulting in the selection of a virus subpopulation, antigenically and biochemically distinct from the original inoculum.

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• Developments in the history of virus vaccine development:

• Demonstration by Enders, Weller and Robbins in 1949 that poliovirus could be grown in cell culture: development of poliovirus vaccines.

• Human viruses could be grown in vitro in a relatively safe and easy manner in monolayer cell cultures: led to significant advances in virus vaccine development.

• The first success was the growth of Lansing type II poliovirus in human cell monolayer culture.

• These discoveries permitted Salk to grow large quantities of poliovirus in primary monkey testicular and kidney cells for the development of the inactivated poliovirus vaccine (IPV).

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• This first generation of cell culture vaccines utilized • primary cell culture or cultures that had been subjected to a very limited number of

subcultures owing to the very short lifespan of such cultures.

• All of these early primary cell culture systems suffered from the disadvantage of inconsistent starting material and concerns about contamination with a number of potential adventitious agents.

• In addition, they are not easy to use in large-scale production such as bioreactor technology.

• They also need a more demanding growth medium and are difficult to propagate under serum-free conditions.

• In contrast, CCLs have the potential for an infinite lifespan.9

• Number of other CCLs, such as Madin-Darby canine kidney (MDCK) cells and PER-C6, are currently being used in the development and manufacture of human vaccines.

• Vero cells are the most widely accepted CCLs by regulatory authorities for the manufacture of viral vaccines.

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ADVANTAGES OF SERUM FREE MEDIA:

serum has some major disadvantages.

It is undefined with respect to its chemical composition.

It can be a source of adventitious agents and their by-products.

Serum also presents a variable performance of cell growth and has a substantial cost.

Serum Free Medium (SFM)

Important amino acids, some trace elements, growth factor, hormone, transport protein and adhesion factor are added.

Adhesion factor added are main components of intercellular substance and serum, such as fibronectin, collagen, and laminin.

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• CULTURE MODE: • Attachment culture: • Vero cells, are all anchorage dependent.

• Vero cells have experienced a development process from experimental spinner bottles to small industrial scale roller bottles, however, such culture system afford a limited growth area, and is not suitable for large scale culture.

• Microcarrier culture developed in 1967 solved such problem well.

• Microcarrier means beads with the diameter of 60~ 250 microns and cells could adhere to its surface, and suspended to grow together with microcarrier in bioreactor.

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• This technology has dominant advantages:

• Homogenous culture together with the advantages of suspension and attachment,

• Easy to control culture conditions and the culture process can be automated.

• Earlier microcarrier adopted synthetic polymers such as PVB and some reports indicated that Vero cells under the condition of serum free were quickly adsorbed and extended.

• In recent years, many researchers attempted to use natural polymers and their derivatives such as glutin, collagen, fibrin, chitin and etc to prepare microcarrier.

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CULTURE TECHNOLOGY:

• Vero cell culture could be classified into 3 types as follows:

Batch culture

In batch mode of operation, the medium was not changed during the whole period of culture and virus was added when cell growth attained a certain stage. The entire virus production was harvested when the viral titer was highest. In order to guarantee the growth of virus, Vero cells were usually incubated in serum free medium for several generations to adapt to the medium without serum.

Fed-batch culture

Fed-batch mode of operation was mainly applied for serum free medium suspension culture of Vero cells, as well as microcarrier. 1/3 or 2/3 of the total volume of culture was replaced every day, according to the continuous expenditure and demand of nutrients in Vero cells, which facilitated a high cell density.

Perfusion culture

In perfusion mode of operation, one volume of culture per day was changed by continuous flow medium supplied by a peristaltic pump. This technology could eliminate the toxic metabolites efficiently and promptly, maintain an excellent nutritional status for cells, and thus was desirable for high density culture.

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• The use of these cells also allows large-scale production of vaccines using microcarriers and

culture in a bioreactor.

• For several new eruptible diseases, Vero cells were used as first cell line to produce vaccines.

• For SARS attracted more attention in past several years, inactivated vaccine have been

developed by using Vero cells, and after preclinical investigation, will be undertaken phase 1

clinical trial.

• As for avian influenza H5N1 focused by many researchers, some companies have claimed

that H5N1 vaccine derived from Vero cells was of good safety and immunogenicity, and

would be undertaken phase 3 clinical trial.

• Some vaccines were described in detail as follows: 17

Rotavirus• Rotavirus is the leading cause of severe diarrheal disease in

infants and young children. • The outer layer of the virus is composed of two proteins,

VP4 and VP7. • A pentavalent live human–bovine reassortant virus,

RotaTeq®(Merck). • RotaTeq was shown to be safe and immunogenic, inducing

group-specific serum IgA antibodies. • A second Vero cell-derived rotavirus vaccine, Rotarix®

(GlaxoSmithKline), was licensed in 2007. • This vaccine is a monovalent, attenuated human rotavirus

containing the most common of the human rotavirus VP4 and VP7 antigens.

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Japanese encephalitis• Japanese encephalitis (JE) is a mosquito-borne,

epidemic inflammatory disease of the CNS found across wide areas of South-East Asia.

• new inactivated Vero cell culture-derived JE vaccine, Ixiaro® (Intercell), was developed.

• The vaccine is based on an attenuated strain of JEV, SA14-14-2, which has been adapted to grow on Vero cells.

• The purified finished product is adjuvanted with 0.1% aluminium hydroxide

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Rabies• Rabies virus (RABV) is the type species of the genus Lyssavirus within the family

Rhabdoviridae. • It causes inflammation of the brain. Early symptoms can include fever and tingling,

followed by violent movements, uncontrolled excitement, fear of water, an inability to move parts of the body, confusion, and loss of consciousness, finally death.

• Using Vero cells adhered to microcarriers(Cytodex® 3-GE), and cultivated in a bioreactor with serum-free medium, an effective rabies vaccine was generated.

• With the aid of tangential filtration, Rabies virus can be purified by chromatography and inactivated it using beta-propiolactone.

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• RabAvert, produced by Novartis Vaccines and Diagnostics • Obtained by growing the virus strain in primary cultures of

chicken fibroblasts. • The virus is inactivated with β-propiolactone, and further

processed by zonal centrifugation in a sucrose density-gradient. • The vaccine is lyophilized after addition of 16 stabilizer solution

which consists of buffered polygeline and potassium glutamate.

• The Imovax® 9 Rabies Vaccine produced by Sanofi Pasteur. • Prepared from strain PM-1503-3M. • The virus is harvested from infected human diploid cells, MRC-5

strain, • concentrated by ultrafiltration and is inactivated by beta-

propiolactone.

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Influenza• The current vaccines are trivalent and contain an H1N1 and an H3N2 subtype of

the influenza A and B strain. • For vaccine production, the virus is inactivated in two separate steps: formalin and ultraviolet (UV) treatment. • The double-inactivated virus is then purified by continuous sucrose gradient

centrifugation followed by ultrafiltration prior to formulation.

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•Clinical trials I & II are carried out for • Severe acute respiratory syndrome • Ross River fever & Chikungunya fever •West Nile encephalitis

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REFERENCES:• Ammerman NC, Beier-Sexton M, Azad AF. Growth and maintenance of Vero cell lines.

Current protocols in microbiology. 2008 Nov:A-4E. • Morrow Jr KJ, Sha M. Vero Cell-based Vaccine Production: Rabies and Influenza Cell lines,

Media and Bioreactor Options. • Whitford WG, Fairbank A. Considerations in scale-up of viral vaccine production. BioProcess

Int. 2011;9(S8):S16-28. • Barrett PN, Mundt W, Kistner O, Howard MK. Vero cell platform in vaccine production:

moving towards cell culture-based viral vaccines. Expert review of vaccines. 2009 May 1;8(5):607-18.

• Tian Chen & Keping Chen. School of life science, Jiangsu University, Jiangsu.Vol.1.July,2009.

•http://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM312931.pdf

•http://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProducts/UCM133484.pdf

•https://www.atcc.org/Products/All/CRL-1587.aspx#culturemethod 24

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