Production, purification and analysis of an experimental DNA vaccine against rabies.
BACKGROUND
The basic and applied research efforts devoted to the development of DNA vaccines must be accompanied by manufacturing processes capable of being scaled up and delivering a clinical-grade product. This work describes a rapid process of this kind, based on hydrophobic interaction chromatography (HIC) for the production of milligram quantities of an experimental DNA rabies vaccine. Its properties and protective activity are tested in comparison with the same plasmid DNA purified with a commercial kit.


METHODS
The experimental DNA vaccine encoding the rabies virus glycoprotein was amplified in vivo in Escherichia coli. The plasmid was isolated by alkaline lysis, pre-purified and concentrated by isopropanol and (NH4)2SO4 precipitation, and purified by HIC and dialysis. Product quality was controlled by using high-performance liquid chromatography (HPLC), Southern slot blotting, agarose gel electrophoresis, the kinetic-QCL Limulus amoebocyte lysate assay, and protein assays. The expression of the rabies virus glycoprotein was tested in vitro in neuroblastoma cells. The production of rabies-virus-neutralising antibodies and the protection against an intracerebral virus challenge were tested in mice.


RESULTS
One hundred and forty-two milligrams of the plasmid, with an HPLC purity greater than 99% were obtained from 4.5 l medium. Control analysis showed that the vaccine conforms to specifications in terms of impurities (endotoxins, genomic DNA, RNA, proteins). Furthermore, the final experimental vaccine induces rabies-virus-neutralising antibodies and protects mice against a rabies virus challenge.


CONCLUSIONS
This study demonstrates that the method developed for the purification of milligram amounts of plasmid delivers an endotoxin-free, experimental rabies DNA vaccine, with protective activity similar to that obtained with the vaccine purified using a commercial kit.
Diogo
M M
MM
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Lisboa, Portugal.
Ribeiro
S C
SC
Queiroz
J A
JA
Monteiro
G A
GA
Tordo
N
N
Perrin
P
P
Prazeres
D M
DM
D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis; D002851:Chromatography, High Pressure Liquid; D004587:Electrophoresis, Agar Gel; D005260:Female; D005822:Genetic Vectors; D006023:Glycoproteins / Q000032:analysis; D007273:Injections, Intramuscular; D007276:Injections, Intraventricular; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D011818:Rabies / Q000276:immunology / Q000517:prevention & control*; D011819:Rabies Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011820:Rabies virus / Q000235:genetics / Q000276:immunology; D013997:Time Factors; D014170:Transformation, Genetic; D014407:Tumor Cells, Cultured; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000276:immunology / Q000302:isolation & purification; D014764:Viral Proteins / Q000032:analysis
D000914:Antibodies, Viral; D006023:Glycoproteins; D011819:Rabies Vaccines; D019444:Vaccines, DNA; D014764:Viral Proteins