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.
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.
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).
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
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
Table 1. Cost categories for influenza vaccines manufactured with different production technologies.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.