The frequency of side reactions observed with the first-generation vaccine was the main driving force behind further development. In this context, Immuno started a collaborative project with Christian Kunz and me to develop an improved product that would meet higher standards and comply with modern requirements.
Our scientific competence and potential for improving the vaccine was founded by the early initiative of Christian Kunz to invest in basic research on TBE virus. He personally sponsored my PhD thesis on the growth, purification and molecular characterization of the virus. Together we published our new data on the properties of TBE virus, and especially its envelope glycoprotein as the main protective antigen, in a series of publications [
11‐
19]. Most importantly, in 1976 we were able to demonstrate that highly purified inactivated virus preparations did not exhibit any pyrogenicity in the established rabbit animal model, even at several-fold higher concentration of the viral antigen than present in the current vaccine. This important finding suggested that the fever reactions observed with the first-generation vaccine were not due to the immunogen itself (i.e., the inactivated virus) but likely caused by contaminants from the cell substrate and insufficient purity achieved during the production process. We therefore concluded that it should be possible to reduce reactogenicity substantially by developing a vaccine that contained the inactivated virus in a highly purified form.
Inspiration from outside TBE virus research
In the mid-1970s, new influenza vaccines were considered a gold standard and represented the state of the art for guidance. We became familiar with the development of these vaccines and obtained deep insights into their design through our connections to researchers at the Sandoz Research Institute in Vienna. They had developed a next-generation influenza vaccine, based on the viral envelope proteins only [
20], and Christian Kunz had conducted clinical trials on its immunogenicity and reactogenicity [
21]. Throughout my PhD thesis, the personal interaction with the influenza scientists at the Sandoz Research Institute was inspiring and very helpful, fostering my understanding of modern virus research and making me familiar with a multitude of techniques that I could apply to my work on TBE virus.
One of these techniques was continuous-flow zonal ultracentrifugation, which was used for influenza virus purification in the course of vaccine development and production. This technology allows the simultaneous concentration and purification of viruses in a continuous process that is scalable for industrial production. In January 1977, I got the opportunity to carry out two experimental runs with the ultracentrifuge at the Sandoz Research Institute using inactivated TBE virus I had produced in our laboratories after growing the virus in primary chick embryo cells. Analyses of these experiments at the institute were encouraging, although many questions remained unresolved and needed detailed investigation in a specifically dedicated project.
The second-generation TBE vaccine project
Several aspects of our work on TBE virus were crucial for exploring alternative procedures towards an improved TBE vaccine. These included the demonstration that the inactivated virus (constituting the immunogen of the vaccine) was not pyrogenic at concentrations needed (see above), as well as methods for detecting and quantifying the inactivated viral antigen. The latter aspect was especially important for designing new processes of vaccine production. In the first-generation vaccine produced at the MRE in Porton, partial purification was carried out with infectious virus suspensions, and standardization of the antigen concentration relied on infectivity measurements during the procedure; however, purification of a highly pathogenic infectious virus in a large-scale industrial process would require an enormous operating expense for biosafety containment and therefore should best be avoided.
Based on these considerations, Immuno ventured into a project for vaccine improvement, which included the evaluation of continuous-flow zonal ultracentrifugation for large-scale industrial purification of inactivated TBE virus. For that purpose, in 1977, they organized a mission for me and Johann Fauma (scientist at Immuno and in charge of TBE vaccine production) to the MRE in Porton, their contract partner for producing the bulk material of the vaccine then available. We obtained access to a continuous-flow zonal ultracentrifuge at the MRE and technical support, including the provision of the same starting material for our experimental ultracentrifugation runs that was also used for the conventional production, i.e., supernatants from infected chick embryo cells before purification. I was primarily in charge of defining the scientific questions to be asked and establishing the details of the work plan, and, in close coordination, Johann Fauma primarily took care of aspects relevant to industrial production and the Immuno agenda at Porton.
For the project, we conducted two series of experiments, the first in July and the second in November 1977. All samples collected from these experiments were sent to Vienna through Immuno for detailed analyses, using previously established methods for quantifying both the viral antigen and impurities throughout the purification process, as well as for measuring protective potency in a mouse animal model. The results (including recovery rates of viral antigen and immunogenicity) were highly convincing and Immuno swiftly invested in the new technology and implemented continuous-flow zonal ultracentrifugation for production at its industrial plant in Vienna. Already in 1979, the zonally purified vaccine replaced the old one on the Austrian market [
22].
The newly developed method combined several innovative steps so that Immuno could file a patent application in 1978. The patent was granted in Austria in 1980, followed by several other countries, including Germany, Czechoslovakia, Great Britain, Switzerland, France, Italy, Slovenia, Sweden, and the Soviet Union. Details of the process were published by Heinz et al. in 1980 [
23], demonstrating that the purity of the second-generation vaccine was more than 95-fold higher than that of the first-generation vaccine. Clinical trials yielded excellent data on immunogenicity combined with a substantial reduction of reactogenicity, as documented by the corresponding publication of Kunz et al. in 1980 [
8].
For my published contributions in the development of a highly purified TBE vaccine as academic partner of Christian Kunz, I was awarded two national prizes in Austria: 1. The Merck Sharp&Dohme (MSD) Vaccine Prize 1983 for the design of immunoassays to quantify inactivated TBE virus [
19]. 2. The HERBA Prize 1983 for the publication on the use of continuous-flow zonal ultracentrifugation for the preparation of a highly purified TBE vaccine [
23].
More than 15 years after development of the new vaccine, in 1996, the international company Baxter took over Immuno, and in 2014, Baxter’s vaccine portfolio was acquired by Pfizer, which remained the producer of this TBE vaccine until today. During these years, changes were made to the industrial manufacturing process, including a switch from mouse brain-derived virus to chick embryo cell-derived virus for inoculating production cultures of primary chick embryo cells, and the removal of merthiolate as a preservative [
24]. Continuous-flow zonal ultracentrifugation, however, represents the heart of the production and purification process until today and is integrated in a high-tech setting of modern state-of-the-art vaccine manufacturing at the Pfizer production plant in Orth/Donau near Vienna. A similar vaccine but using different stabilizers and based on a German TBE virus isolate, grown in primary chick embryo cells and also purified by continuous-flow zonal ultracentrifugation, was introduced in Germany by the Behringwerke AG in 1991 [
24].
Success of the highly purified vaccine on large-scale application in Austria
Evaluation of the effectiveness of TBE vaccination under real world conditions was enabled by the meticulous analysis of every single case of TBE in Austria and the documentation of its vaccination history [
7]. This work was started by Christian Kunz and, after his retirement in 1995, continued by the institute he had founded until today. Patient data were combined with the figures on vaccination coverage in Austria, revealing a field effectiveness well above 90% [
7]. The high rate of protection resulted in a continuous decline of TBE cases parallel to the increase of vaccination coverage. In contrast, the incidence of TBE in the unvaccinated population [
25] as well as in neighboring countries with a comparably low vaccination rate remained unchanged [
4,
25]. Referring to an Austrian success story [
3] thus appears to be justified in this context.