New Approaches to Improved Vaccine Manufacturing in Embryonated Eggs - Recombinant vector technologies can improve the yield and lower the cost of egg-based influenza vaccine production. - BioPharm


New Approaches to Improved Vaccine Manufacturing in Embryonated Eggs
Recombinant vector technologies can improve the yield and lower the cost of egg-based influenza vaccine production.

BioPharm International Supplements

Improving the Molecular Process

Several human, mammalian, and avian viruses can infect cells of the chorionallantoic membrane of chicken eggs (a monolayer of cells surrounding the fluid-filled allantoic cavity in the egg). Infection results in the accumulation of live virus particles in the allantoic fluid that can be collected after a suitable incubation period. The standard method of egg-based vaccine production consists of pre-incubation of the eggs, inoculation with a live virus (e.g., influenza, yellow fever), incubation, harvesting of allantoic fluids, downstream processing, and filling and finishing. For the classic inactived influenza vaccine, purification, inactivation, and stabilization of this harvested material yields vaccine product.

Figure 1. AdCEV vector driven protein expression in eggs. 1) Coumassie-stained SDS-PAGE MW standards (Bio-Rad). 2) Coumassie-stained SDS-PAGE 10x concentrated allantoic fluid from SPF eggs infected with AdCEV-rabies G vector; 3) Western blot of Material in column 2, reacted with anti-rabies(Vnukovo-32) mouse monoclonal antibody (Capricorn Products, ME, USA).
Fowl Adenovirus Type 1 (FAV1) is an adenovirus that can infect embryonated eggs. Viral vectors constructed by manipulating the FAV1 genome yield a novel class of vectors (AdCEV vectors, AfriVax, Inc., Seattle, WA) that can be used to produce recombinant proteins in eggs.4 FAV1 vector-driven expression in eggs has been demonstrated for recombinant human C-reactive protein, rabies glycoprotein (Figure 1), HEV glycoprotein, and a handful of other viral and human proteins.5 A similar approach using a Sendai virus-derived mini-genome system also has been used to produce recombinant viral glycoproteins in chicken eggs at yields 3–5 times higher than a vaccinia cell culture system.6 These and other results show that biologically functional human and avian recombinant proteins can be made in eggs.

Avian Adenovirus-Based Vectors Can Improve Yields From Eggs

For vaccines, the main impacts of using AdCEV vectors derive from the potential for higher yields of antigen per egg. In wild-type FAV1 infections of eggs, up to milligram amounts of viral proteins can accumulate in the allantoic fluid. This is much higher than the quantities of immunogen produced in influenza virus infected eggs (about 50 μg/egg). Recombinant flu antigens made with FAVI-based vectors should have native immunogenic characteristics, because eggs are a natural host for influenza.

Influenza immunogen production was the world's first scaffold approach to industrial protein production.1 Annually, the epitopes on the scaffold are modified by viral evolution, and vaccine selections for manufacture are recommended by regulatory agencies to match viral strains in circulation. The ability to express immunogens from vectors that can be manipulated in E. coli and only introduced into eggs at the production batch stage offers great flexibility for molecular engineering scaffolds for flu and other diseases. For example, innovative molecular chimera constructs that include TLR-stimulating components3 or that produce self-assembling virus-like particles (VLPs) have been shown to result in efficacious flu vaccines containing 10-fold less immunogen.3

The expression of such advanced immunogen constructs in eggs, with the yield improvements provided by AdCEV vectors, could result in a rapid and readily implementable solution to the global shortage of manufacturing capacity. Using expression vectors also decouples product yield from strain-to-strain variations that affect traditional flu vaccine manufacturing in eggs.

Reducing Production Costs

Table 1. Cost categories for influenza vaccines manufactured with different production technologies.7
Egg-based recombinant platforms create a new category of production system that combines the advantages of recombinant cell culture with inactivated vaccine manufacturing approaches while eliminating many of their drawbacks. A recent comparison of bulk costs per liter for different flu vaccine production systems shows that they can be divided into high, medium, and low-cost categories (Table 1).7

The cost of flu vaccines per course derives from a complex equation of molecular, manufacturing, and operational factors that affect the number of courses per liter. These include: antigen yield per liter, required antigen dosage per strain, number of strains per dose, and number of doses required per course.7 Egg-derived recombinant flu antigens could shift the cost of inactived influenza vaccine manufacturing into a lower cost category similar to, or better than, that of live attenuated flu vaccines. Efforts are underway to (i) evaluate production yields achievable for pandemic and epidemic strain vaccines made with AdCEV; (ii) reduce the quantity of antigen per dose and number of doses required; and (iii) evaluate the potential of recombinant chimeric molecular adjuvant strategies.

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