8.6 billion USD annual global burden.

1 death every 10 minute in the world.

59,000 annual deaths.

40% of the victims are children under 15.

This is just a few figures about rabies virus, an entity 100% fatal.

Rabies is probably one of the oldest known virus, already depicted before 2300 BC in many ancient texts from Mesopotamia, Egypt, Persia and Palestine. Greek litterature fervently describes virus transmittion through the saliva of infected animals, like dogs, bats, cats, rabbits and horses.

Rabies is a fatal form of encephalomyelitis caused by viruses of the Lyssavirus genus of the Rhabdoviridae family. The rabies virus is in the form of a bullet which measures on average 180 nm long and 75 nm in diameter. The genetic information is stored in a single strand of linear RNA. This RNA of about 12,000 nucleotides encodes five proteins: the nucleoprotein (N); the matrix protein (M); the glycoprotein (G), which represents the major antigen target of the protective immune response, phosphoprotein (P) and virion transcriptase (L), mainly devoted to the transcription and replication.

In 1885, two French doctors found a solution. Pasteur and Roux produced the first vaccine for rabies by growing the virus in rabbits, and then weakening it by drying the affected nerve tissue.

Typically, vaccines are administered before exposure to an infectious agent and are otherwise not very useful after exposure has occurred. Historically, rabies is an exception, immunization being used for both prophylatic and therapeutics purposes.

Since Pasteur’s original work with vaccines containing nerve tissue, many adverse reactions and neurologic complications have been recognized. The solution to the problem of safety of rabies vaccines lay in the development of vaccines prepared from rabies virus grown in tissue culture free of neuronal tissue.

Today, 42 worlwide manufacturers are producing 90 million vials for human vaccines (more than 15 million people are vaccinated each year), and 181 million vials for dog vaccines, using different successful cell culture approaches:

• Vero-cell rabies vaccine in India (e.g., Bharat Biotech, Wockhardt, the Human Biologicals Institute) and China (e.g., Chengdu Biotechnology, Wuhan Institute, Liaoning Yisheng, Changsheng Life Sciences) on microcarrier beads, originally developed by Sanofi Pasteur,

• BHK-21 cell culture vaccine was produced by the Institute of Poliomyelitis and Virus Encephalitides (Moscow, Russia), and by several institutes in China, including the Wuhan Institute of Biological Products,

• Duck embryo vaccine by The Swiss Serum and Vaccine Institute (Bern, Switzerland) then transferred to Zydus Cadila Pharmaceuticals in India,

• MRC-5 human fibroblasts at Merck & Co., Novartis AG, Serum Institute of India, Novartis (Chiron Behring) and many others.

The technical advances leading to the development of the vaccine included the adaptation of the strain of virus to WI-38 cells, the inactivation of cell-free virus by β-propiolactone, and the concentration of virus by ultrafiltration.

Two potentially inexpensive technologies for rabies vaccine production are expression of the G protein in genetically modified plants and the generation of recombinant plant viruses, both of which might be administered orally to humans in the future.

At the end of the manufacturing process, all modern vaccines must have a potency of at least 2.5 IU/dose, as measured by the National Institutes of Health test.

NIH vaccine release test is based on mice immunization followed by intracerebral viral challenge : because rabies is present in nervous tissue (and not blood like many other viruses), the ideal tissue to test for rabies antigen remains brain.

But the NIH test has a number of huge limitations :

• the use of mice, despite the efforts to reduce animal experimentation,
• the handling of a virulent virus,
• its long duration (28 days),
• the route of challenge differs from the natural infection route,
• its high inherent variability due to in-vivo testing, poorly fitting a batch-to-batch release testing method,
• not user-friendly, requiring many manipulations.

Rabies subunit vaccines have been developed based upon the use of the mentioned glycoprotein (G). This molecule is composed of a cytoplasmic domain, a transmembrane domain, and an ectodomain, exposed as trimers at the virus surface. The ectodomain is involved in the induction of both virus neutralizing antibody (VNAb) production and protection after pre- and postexposure vaccination.

It is commonly recognized that the G molecule consists of two immunologically active parts, each potentially ables to induce both VNAb and T-helpers cells :

• the NH2-terminal half containing one important conformational and discontinuous antigenic site (aa 34 to 42 and aa 198 to 200 associated by disulfide bridges, so called « site II »)
• and the COOH-terminal half containing the other major conformational and continuous immune-dominant epitopes (aa 330 to 338, so called « site III »).

The idea was to develop a specific mouse monoclonal antibody (D1-25) that specifically recognizes the native G-protein, thanks to the binding of site III.

Subsequent ELISA test is able to discriminate native from altered G protein, providing a strong alternative to NIH test :

• No use of animals,
• Avoid any biosafety concern,
• Fast method with outcomes in a few hours,
• Robust and validated in-vitro potency test
• User friendly with few handling and minimum clear steps.

Additional special feature !

As site III is a highly preserved sequence between the different rabies virus strains, the D1-25 monoclonal antibody detects a broad spectrum of rabies strains.