Current inactivated influenza vaccines are made by growing the virus in embryonated eggs. The allantoic fluid is harvested,
and the virus in it is killed with formalin. The killed virus is enriched by centrifugation and then disrupted with detergent,
and is generally followed with a second formalin kill step. The result is the bulk vaccine, of which HA comprises up to 20%
of the protein component.12
We have discovered a means of producing just the "globular head" of HA, the segment of the protein that contains all of the
structures recognized by neutralizing antibodies. This globular head can be produced as a fusion protein at the C-terminus
of flagellin in E. coli, and can be purified to endotoxin-free homogeneity. Although the globular head moiety is a complex structure with disulfide
bonds that must be configured and folded correctly for optimum immunogenicity, we have found that the purified protein reacts
strongly with serum from convalescent animals as well as with monoclonal antibodies directed at the neutralizing epitopes,
suggesting that it is appropriately structured.
In addition, vaccinating mice with two doses of the HA globular head fusion can raise antibodies that recognize native HA
on infected cells and also have the ability to block the virus's ability to agglutinate red blood cells, a classic assay for
quality of HA antigens. Mice vaccinated with 0.3 or 3.0 μg of globular head fusion protein are protected 100% from death following
a challenge with virus that kills 90% of naïve mice (Figure 3).7 This vaccine is also highly immunogenic in rabbits and ferrets (unpublished data). As seen with other proteins, the globular
head alone, or mixed with flagellin, is not immunogenic. In the last 12 months, we have made a variety of HA vaccines with
different serotypes, including H1 PR8, H1 New Caledonia, H1 Solomon Islands, H3 Wisconsin, H5 Vietnam, H5 Indonesia, H5 Anhui,
B Malaysia, B Lee, and B Shanghai. All are well-expressed in E. coli (20–25% of total cell protein on induction) and the first clinical trial is underway. A new vaccine seed strain may be made
and initial protein produced in a month, providing a rapid response to an emerging virus.
The speed, simplicity, and efficiency of production of these vaccines in E. coli provides an attractive alternative to the present egg-derived influenza vaccine. A typical protein yield is >2 g/L of fermentation
broth, and this may be optimized further. Compared to the average of about 6–8 mg of HA recovered per liter of allantoic fluid
of infected eggs, and 0.6 to 6 mg of HA per liter from mammalian cell culture, an E. coli-based vaccine approach is highly attractive (unpublished data).
The US Department of Health and Human Services has stated that its goal is to have within the US the capacity to make enough
monovalent pandemic influenza vaccine to give two doses to the entire US population, or 600 million doses. Current egg-based
systems are unable to meet this demand, and even with recent enormous investments in expanded egg facilities and new cell-culture
efforts, there will be a shortfall. As described here, an E. coli-based system could produce the required vaccine in one 5,000-L fermenter cycle. Moreover, because the methodologies are standard,
the production process could easily be transferred to other countries or regions. Given the projected worldwide impact of
a pandemic, local or regional manufacture of this vaccine may be able to provide countries or regions without a current flu
vaccine, the means to be self-sufficient. With this kind of production capability, one can contemplate vaccinating the global
population effectively and rapidly, if the necessary public health infrastructure could be developed.
Alan Shaw, PhD, is the president and CEO of VaxInnate, Cranbury, NJ, 609.860.2260, email@example.com