The Optimization Of Attentuated VTT Vaccine Vectors By Genetic Engineering And The Immunological Study Of IMV Membrane Protein Encoded By H3L ORF

Resurgence of the study of vaccinia virus is mainly due to two reasons:first, because of the threat of terrorism, the smallpox virus may be used as a biological weapon; and second, the vaccinia virus was developed as a powerful expression vector. Concerning the study of vaccinia virus, we focus on the safety and efficincy of the Tiantan strain, which was historically used to eradicate the smallpox in China.For safety, we generated a modified VTT strain (MVTTzcI) through genetic modification and clonal selection. MVTTzcI was characterized for its host cell range in vitro. The preclinical safety and efficacy profiles of MVTTzcI were evaluated in vivo using murine models. MVTTzcI was generated from VTT by removing the hemagglutinin gene and an 11,944-bp genomic fragment from C2L to F3L. Despite replication-competency, unlike VTT, MVTTzcI did not cause death after intracranial injection or body weight loss after intranasal inoculation in mice. Moreover, MVTTzcI did not replicate in mouse brain and was safe in immunodeficient SCID mice. Importantly, MVTTzcI was able to induce potent neutralizing antibodies via the intranasal (i.n.) route of immunization. One time i.n. immunization protected animals from the challenge of the pathogenic vaccinia WR strain.For efficiency, we focused on the possible pre-existing immunity in people who has been vaccined in history. We tested a chorhort of normal people and found the VTT specific neutralization activity is common among them who were born before 1981. The VTT genome open reading frame H3L encodes an IMV (intracellular muture virion) membrane protein which is a major neutralizing antibody target in human. Therefore we constructed an H3L deleted Tiantan strain (MVTTâ–³H3L) and found the mutated strain has a reduced sensitivity to human serum neutralization.