A review of production technologies for influenza virus vaccines

Embryonated eggs from a certified source are obtained and used 9—12 days after fertilization. The eggs are candled to locate the air sac. The egg is pierced under aseptic conditions, and the seed-virus is inoculated into the air-space with a syringe. The hole is then sealed with wax. The procedure can be carried out on a laminar flow bench. The inoculated egg is incubated for two to three days in a humidified atmosphere.

The top of the egg is cut off, the membrane pierced with a pipette and clear allantoic fluid is removed. This is then clarified by centrifugation to remove cell debris. Harvests from the eggs are pooled and sterility tested for three to four days. The degree and method of purification used depends on the type of vaccine being produced. Whole virus. The pooled harvest is concentrated and purified by ultracentrifugation on a sucrose gradient.

After harvesting from the gradient, the virus is diluted, and inactivated either by formaldehyde or betapropriolactone BPL. The concentration of the inactivating agent needs to be determined and validated for each strain.

A filtration step may be included to remove egg debris. Because relatively little purification is involved, the yield of whole-virus vaccine per egg is higher roughly 2-fold than with the other types of IIV. Split virus. The procedure above is followed to the point of harvesting from the sucrose gradient. The optimal conditions for each strain need to be determined but typically this step can take two to three days.

The preparation is then sucrose-gradient purified once more and the HA-rich fraction is harvested. Alternatively, the detergent may be removed by diafiltration. The product is inactivated and sterile filtered. Compared to the whole-virus preparation, split vaccines are better characterized, contain less ovalbumin and are claimed to be less reactogenic.

However the yield of HA can vary significantly from strain to strain and year to year, and from one manufacturer to the next between 0. Subunit vaccine. This is prepared in a similar manner to split virus; however, different, more extensive purification steps are used in place of the second sucrose-gradient purification.

This results in the isolation of relatively pure HA with minimal contaminating N, matrix protein, nucleoprotein and lipid. Whether whole, split or subunit vaccine is being produced, a final dilution is made in the formulation buffer. Typically, no adjuvant or stabilisers are used, although preservatives such as thiomersal are frequently added Nicholson, Use of adjuvants would require well-characterized material to ensure consistent formulation.

Furthermore, adsorption of alum adjuvants is influenced by the lipid to protein ratio, which varies between the different types of IIV. It should be noted that the methods described above are suitable for production of accine candidates for pre-clinical testing and phase I clinical trials. They can provide a useful guide for the development of industrial process, but the final methods used for industrial production would be dependent on, and a result of the know-how of the company developing the process.

The infrastructure requirements for the process are straightforward. Inoculation and harvesting of eggs can be performed manually. Minimum requirements would be:. Establishing such a facility, developing the process, producing and releasing a batch of vaccine suitable for use in a phase I clinical trial would take approximately months Figure 1 , depending on the specific expertise and prior experience of the manufacturer.

In order to produce sufficient inactivated vaccine for routine seasonal immunization against influenza or pandemic preparedness, the process requires automation and scale-up at all stages. A realistic goal would be a facility capable of producing 20 million doses of trivalent IIV in a four to five month production cycle.

Egg supply. In optimized conditions, each egg can produce approximately between 45 and 90 ug of a classical seasonal antigen, which corresponds to one to two doses when formulated as a trivalent vaccine. As noted above, the yield for H5N1 strains may be significantly less. Manufacture of 20 million doses of trivalent seasonal influenza vaccine requires 15 million to 20 million fertilized eggs over the production cycle. Supplying eggs in these numbers requires careful planning and a long lead-time months in order to reach the numbers required.

Furthermore, the supply has to be carefully controlled and timed because the eggs have to be inoculated at a fixed point after fertilization. The egg supply needs to be secure in terms of quantity and quality of the eggs. Automatic inoculators, incubators and harvester are required to speed up the process and increase capacity. Automatic inoculators can operate at approximately 10, eggs per hour, enabling a plant to process up to , eggs per day.

However, this also means that less care is taken with the process because there is no longer the opportunity to inspect each egg for contamination. Careful cleaning and pooling procedures are needed to prevent the contamination of one egg being spread by a harvester or inoculator to many other eggs, resulting in the loss of a whole bulk harvest.

Infrastructure and equipment. A large plant will be required to house the necessary equipment, including equipment for the initial upstream process steps:.

Building and validating the large-scale facility would require at least two years Figure 1. Laboratory-scale production of IIV in eggs should be readily achievable. Cox, R. A phase I clinical trial of a PER. Vaccine 27 , — Couch, R.

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The elements of this include the Vaccine Safety Datalink VSD , started in to collect information from electronic medical records; the FDA Sentinel initiative launched in and its Post-licensure Rapid Immunization Safety Monitoring PRISM programme activated in , which integrates administrative and claims data from hospitals and insurance companies, and Medicaid and Medicare databases.

Although observational data are inherently biased, they can support data obtained from randomized controlled trials, especially in cases of accelerated approval as may be expected during a public health crisis like an influenza pandemic.

For example, during the pandemic, accelerated licensure was granted to a high-dose trivalent influenza vaccine independent of the H1N1 outbreak for use in the elderly based on superior induction of HAI as a surrogate of efficacy Part of the licensure agreement was for the manufacturer to carry out post-marketing efficacy studies.

A 31,person randomized controlled trial subsequently showed that the high-dose influenza vaccine demonstrated clear superior efficacy relative to the standard-dose vaccine Using the Medicare database, an observational study design to control for bias in health-seeking behaviour and other factors provided supportive data that efficacy was achieved Subjects with laboratory-proven influenza are assigned as cases and those who test negative are designated controls.

The frequency of vaccination in each group can be used to accurately estimate vaccine efficacy , especially if factors like the method of diagnosis, vaccine type and influenza strain are specified. Despite moderate-to-low efficacy, cumbersome manufacturing processes and long lead times for annual strain reformulation, the current production system of seasonal influenza vaccines has been relatively unchanged over the past 40 years.

Advances across the fields of structural biology, influenza virology and immunity have set the stage for major advances towards improved seasonal and universal influenza vaccines. Meaningful and lasting advances in the influenza vaccine field are now achievable, but they depend upon leveraging expertise, communication and cooperation from stakeholders across many disciplines, from funding agencies to basic scientists, epidemiologists, regulators, manufacturers and the public.

Seasonal influenza vaccine production remains an enormous challenge for manufacturers as the vaccines must be produced and released 6 months after the WHO announces the vaccine strains for the following season in a given hemisphere.

Currently, there are three different production technologies approved for influenza vaccines: egg-based, cell-based and recombinant proteins.

The majority of the licensed vaccines are made using embryonic chicken eggs, and even though this production system has remained unchanged for decades, it is still the only method that can meet the current annual need of seasonal influenza vaccine for the global population.

Five hundred million doses are generated annually but could potentially produce 1. Vaccines produced from cell culture were first approved by the FDA in ref. This recombinant protein-based approach allows a process that does not require virus propagation and can be run on a large scale once the appropriate infrastructure is in place.

Together with the recent advances in high-cell density, perfusion continuous flow processing , this approach opens the door to producing next-generation subunit protein vaccines and meeting the increasing demand for safe, affordable and effective influenza vaccines.

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This study identifies human monoclonal antibodies that protect against lethal virus challenge from both influenza B lineages and shows that one antibody, CR, recognizes a conserved HA stem epitope and protects against both influenza A and influenza B viruses. Antibody recognition of a highly conserved influenza virus epitope.

This study delineates the crystal structures of HA complexed with a broadly neutralizing antibody, CR, and identifies the highly conserved neutralizing epitope in the HA stem.

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Sui, J. Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. This study isolates a family of broadly neutralizing antibodies, including F10, that recognize a highly conserved epitope within the HA stem and shows that these antibodies are protective against both highly pathogenic H1N1 and H5N1 viruses in animal models. Throsby, M. Wu, Y. A potent broad-spectrum protective human monoclonal antibody crosslinking two haemagglutinin monomers of influenza A virus.

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Chen, M. Broadly neutralizing DNA vaccine with specific mutation alters the antigenicity and sugar-binding activities of influenza hemagglutinin. Florek, N. A modified vaccinia Ankara vaccine vector expressing a mosaic H5 hemagglutinin reduces viral shedding in rhesus macaques. Kamlangdee, A. Broad protection against avian influenza virus by using a modified vaccinia Ankara virus expressing a mosaic hemagglutinin gene.

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This study shows in two phase I studies that DNA priming followed by a monovalent inactivated vaccine boost improved the neutralizing antibody response, and demonstrates that HA stem-directed antibodies can be induced by vaccination in humans.

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