Characterization Of Modified VTT Vectors And Construction Of Recombinant Vaccine Based On VTT For HIV-1

The vaccinia Tiantan strain (VTT) was widely used in China as the vaccine against smallpox and had played a critical role in variola eradication in China before 1980. We have previously characterized the phenotypic properties of VTT both in vitro and in vivo and have identified its virulence property in causing the body weight loss in mice once inoculated directly through the intracranial route. To explore the potential use of VTT as a live vaccine vector, we have generated three modified VTT strains (MVTTs) by homologous recombination with the green fluorescent protein (GFP) gene at three distinct genomic locations. These MVTTs were selected based on GFP expression and produced in Vero cells. Purified MVTTs were characterized genetically and phenotypically both in vitro and in vivo. The exact sites of recombination have been confirmed by sequencing which match perfectly with experimental design. We found that GFP gene was stable in MVTT genome after multiple rounds of replication and passages in Vero cells, suggesting these three sites are tolerable for the introduction of foreign genes and could therefore potentially serve as insertion sites for the construction of recombinant vaccines. We also tested the growth properties of these three MVTTs on 14 cells in vitro and found that they displayed significantly reduce cytopathic effect and host cell range compared to the parental VTT, but with comparable levels of GFP expression. Moreover, we have further found that these three MVTTs are about 250 to 1000 less virulent in causing the body weight loss compared to the parental VTT and no longer able to replicate in the central nerve system of infected mice. Yet, all three MVTT can elicit strong humoral and cytotoxic responses to GFP in infected mice. Taken together, we conclude that replication capacity of MVTT has higher capability in inducing immune responses in vivo. By minimizing the virulence associated with the parental VTT strain while maintaining its replication capability, we hope to create an ideal live vaccinia vector for the design and development of vaccine against wide range of infectious pathogens including HIV-1.Among the first attempts, here we generated MVTT expressing the fusion Nef-tat-rev of HIV-1 in center China. Using PCR method, we deleted the stop codon of nef, the start and stop codon of tat and the start codon of rev for the fusion expression under the control of one promoter. We found the MVTT-Nef-tat-rev can express the fusion protein. We have immunized mice using the MVTT-Nef-tat-rev and samples of mice serum of week 0, 3 and 5 as well as the spleens of the mice at the end of week5. The humoral response against the foreign gene has been detected using Elisa and ELISpot test is ongoing for the cytotoxic responses detection.