**2. Construction of the Vaccine**

But we set to. The Scientific Director of Transgène, Jean-Pierre Lecocq, established a collaboration with researchers at the Wistar Institute (**Figure 1**) under Hilary Koprowski (Philadelphia) who had obtained, for the first time, a cDNA copy of rabies virus glycoprotein at the end of 1981 [2, 3] – the key antigenic determinant of this virus. Beginning in early 1982, we expressed the cDNA in *E. coli*, *B. subtilis*, yeast, mammalian cells (reviewed in [4]). The pressure was on, we worked 7 days a week, so did our key technical staff; we were fuelled by Martha Argerich interpreting J.S. Bach on the tape player until long after the sun set over the Vosges hills (**Box 1**). The extracts were sent to the rabies expert in Philadelphia, Tadeusz Wiktor. He systematically vaccinated lab

It is singularly appropriate that the recombinant rabies vaccine should have been developed in Strasbourg, the central town of the province of Alsace, France. Louis Pasteur, famed for the first rabies vaccine, was born in the Jura hills (the southern extension of the Vosges – the hills of Alsace) and was Professor of Chemistry at Strasbourg University (formerly known as the Université Louis Pasteur) from 1849 to 1854, where he married Marie (also known as Louise) Laurent (the daughter of the Rector of the University), who for many years acted as his scientific assistant. The first patient to be treated against rabies, Joseph Meister (9 years old), traveled from Alsace to Pasteur's laboratory (then in Paris) for treatment [1]. The first fox to be inoculated with the new recombinant vaccine was in Malzéville, over the Vosges hills just 100 km from Strasbourg.

**Box 1.** *Rabies, Pasteur, and Alsace***.** *The Early Development of the Vaccinia–Rabies Recombinant Vaccine Raboral® DOI: http://dx.doi.org/10.5772/intechopen.97344*


#### **Figure 2.**

*Construction and Activity of the Vaccinia-Based Recombinant Rabies Virus Raboral® VR-G. The recombinant was variously known as VR-G, VVTGgRAB-26D3, and VR-Gpro8. (A) Correction of a mutation in the N-terminus of the rabies glycoprotein coding sequence. Panel adapted, with permission, from [6]. (B) Protection from rabies using the live recombinant vaccine [6]. Mice were inoculated (intradermal) with 5 × 105 PFU of VR-G or wild-type vaccinia, and challenged on day 15 by intracerebral inoculation of CDC culture-adapted street rabies virus (2400 LD50 units). All vaccinated animals survived. Panel adapted, with permission, from [6]. (C). Protection of rabbits and mice: inoculation was with 2 × 108 PFU (intradermal) or 5 × 107 (footpad) of VR-G; intracerebral challenge at day 14 was with 2400 (mice) or 24 000 (rabbits) mouse LD50 units of MD5951 street rabies virus. Panel adapted, with permission, from [7]. (D) Protection using inactivated VR-G. Vaccines were inactivated with* β*-propionolactone before intraperitoneal administration into mice. Challenge at day 14 was with 240 LD50 units of MD5951. Panel adapted, with permission, from [7].*

animals (mostly mice and hamsters) with the extracts, and then challenged them with street rabies virus. None survived.

We were dismayed, and ready to give up. Two developments changed everything. First, at a chance meeting with Peter Curtis (Wistar) he advised that there was a possible problem with his cDNA sequence – there appeared to be a mutation. Second, we were impressed by the growing achievements of recombinant vaccinia virus (the basis of the smallpox vaccine), inspired by Enzo Paoletti in 1983 [5], in eliciting immunity beyond what could be obtained with bacteria, yeasts, or even mammalian cells.

The mutation in the rabies glycoprotein cDNA was indeed suspect – a Pro to Leu mutation near the beginning of the mature protein sequence at a position that (to our minds) seemed to resemble known mutations at the beginning of the oncoprotein RAS (Gly to Asp, or Gly to Val) that entirely transform the structure and activity of the protein. The first thing we did was to correct the mutation. Something we had never done before, and this took months. In the key *Nature* paper the reader will see that, because our techniques were challenged, we took advantage of an artificial site for Dam methylase (that governs mismatch repair in *E. coli*) site to tip the balance in our favor (unheard of today) (**Figure 2A**). But it worked! We wonder how many rabies glycoprotein sequences circulating in the GE world today bear that same signature alteration.

The second thing we did was to get vaccinia up and running as a vehicle. We got into collaboration with Robert Drillien and Danièle Spehner at the Institut de Virologie on the same campus, and they supplied us with a microgram of vaccinia virus DNA. The key thymidine kinase gene – required for recombinational exchange with the vaccinia genome – had already been cloned by Robert, but we lacked a promoter to drive expression. We wanted the 7.5 K gene promoter, that had been worked up by researchers in the USA, but starting with only 1 microgram of DNA (the vaccinia genome is ~190 kb in length), given the state of the technology, cloning was near to impossible. In the event we turned to calculations of insert size, vector size, and absolute concentrations based on the physicochemistry of ligation [8, 9], and brewed up an optimum ligase mix. Only four clones were obtained. Two were 7.5 K in one orientation, and two were the same promoter in the other orientation! Were we lucky? No, in retrospect we would have got nothing had we not used the mathematics of the ligation reaction to get what we needed.

From there it moved quite rapidly. Ligate the promoter to the modified rabies G coding sequence, insert the construct into the vaccinia TK gene on a plasmid – then transfect the recombinant (TK-negative) plasmid into vaccinia virus-infected cells, and select chemically using bromodeoxyuridine (that kills TK-positive viruses). And we had our first vaccinia–rabies recombinant ('26D3').

This sped off to Tad Wiktor in Philadelphia. We waited what seems like an eternity, but he reported quickly back with his preliminary results: 'this is the best rabies vaccine I have ever seen' – it protected mice against severe rabies challenge (**Figure 2B**–**D**) better than any other rabies vaccine. And so it turned out – first published in September 1984 ([6, 7]; reviewed in [10–12]).

What lessons can be drawn, if any? Our first thought is that we worked as a team, there was no academic infighting, no bickering about authorships that is too common today, and we all worked day and night to see the project through. Even technical staff were there late in the evening and at weekends. Second, collaboration is essential: we could not have done this project without the participation of scientists both near (Robert Drillien and Danièle Spehner) and far {Tadeusz Wiktor (**Figure 3A**), Peter Curtis, and Hilary Koprowski}. Third, we wonder whether Martha Argerich and J.S.

*The Early Development of the Vaccinia–Rabies Recombinant Vaccine Raboral® DOI: http://dx.doi.org/10.5772/intechopen.97344*

#### **Figure 3.**

*Pioneering Rabies Vaccination. (A) Tadeusz J. Wiktor (1920–1985), rabies expert, Wistar Institute, who first tested the recombinant for efficacy. Photo courtesy of the Wistar Institute. (B) Loading vaccine baits into a helicopter during the first wildlife vaccination campaigns (Belgium, ca 1988; image collection M.P.K.).*

Bach played a role. In this day of *P* values, we would need to run the entire project again with a different musical background.
