Context
On May, 8th 1980 smallpox was declared to be eradicated. Small stocks of the virus remained in secure laboratories and some other laboratories were allowed to maintain stocks of up to 20% of the genome sequence for the purpose of international research. No vaccination of populations has been carried out since eradication was declared. The live viral vaccine indeed caused rare but serious adverse reactions, especially in persons with deficiencies of the immune system, and common local reactions. Epidemiological studies have demonstrated that vaccination protects against smallpox for less than five years after primary vaccination and that substantial but waning immunity can persist for ten years or longer. Protection may last longer when revaccination is performed.
Because of the smallpox eradication, the vaccination secondary effects, the orthopox viruses stability in aerosol, low infecting dose, high contagion (1 person may contaminate 20 others), it is to be feared that the smallpox virus is used as a biological weapon. To complete the only antiviral therapeutic possibility that Cidofovir represents, the search for new active inhibitors on the Orthopoxvirus is necessary.
The Vaccine virus is the Poxvirus prototype; it reproduces exclusively in the cytoplasm of the host cells and code for the majority of proteins necessary to its replication and transcription. Numbers of these proteins were identified even A20R, the central protein of the replication complex, more particularly targeted during our study.
Replication complex
Replication complex of the vaccine virus. E9L: DNA Polymerase, D4R: Uracil DNA Glycosylase, D5R: Nucleoside Triphosphatase, H5R: Triphosphatase.
Production strategy
The first part of the programme consisted in identifying the production conditions of the replication complex proteins. Our goal was to obtain these proteins in sufficient quantity to carry out various applications and particularly to solve their structure.
The selected strategy was to clone and express in Escherichia coli system A20R, D5R and E9L proteins from the poxvirus as well as protein homologues in variola, monkeypox, cowpox, camelpox for which cDNA were available.
The second strategy selected was to clone A20R, E9L et D5R proteins in bacculovirus and use insect cell expression system.
Aptamer approach
As the project was evolving, the objective was also to select peptides (aptamers) able to inhibit the protein interactions of the Poxvirus replication complex. This strategy still aimed at obtaining an antiviral activity.
Aptamers are molecules that can interact with a specific target protein and inhibit this protein functionally at an intracellular level. A20R protein has been chosen as an aptamer target because of its interactions with 4 others proteins involved in the Vaccine replication complex.
11 aptamers have been selected because of their interaction properties with A20R protein and because of their capacity to inhibit the Vaccine replication complex.