Study On VLP (Virus-Like Particles) Vaccine Of HIV-1 Based On The Prevalent HIV-1 Strains In China

As acquired immunodeficiency syndrome (AIDS) continues to take its toll globally, the development of a safe and effective vaccine against HIV is critical to worldwide efforts to control the epidemic. Although educational and counseling efforts have had some success, it has become evident that prevention activities are not sufficirent to contain the spread of disease Thus, nvestigators continue to design and test novel ways to present HIVproteins to the immune system and evaluate new antigerVadjvant and various formations although no study has convincingly demonstrated which immune mechanism will protect against HIV infection. VLPs(Virus-like Particles) vaccine, the second gereration genetic egineering vaccine , is the main objective of our studies to develop candidate vaccine targeted at the prevalent HIV strains in China.1. To clone the structural genes (gp120 and gag) of HIV-1, three HIV-1 positive blood samples from Guangdong and Guangxi, whose subtype had been determined as Subtype E, and one HIV-1 positive blood sample, from Henan, whose subtype had been determined as Subtype B by sequence analysis of partial env gene were chosen to be amplified by nested-PCR. We got full-length fragments of gp120 and gag and incorporated them into pFastBac1 and plin8Pr55, respectively. Sequence analysis and phylogenetic analysis showed that the gp120 genes of Guangdong and Guangxi samples fell into Subtype E while the gag gene from one Guangdong sample fell into Subtype A. The gag gene from the Henan sample fell into Subtype B. All the cloned gag and gp120 genes have the complete open-reading frames and have some special and functional domains. The genetic characteristics of the two structural genes indicated that they could be used to construct VLPs (Virus-like Particles) vector of Subtype A and expression vector of subunit vaccine of Subtype E.2. Consturction of HIV-1 Virus-like Particles candidate vaccines based on the prevalent HIV-1 strains in China. HIV-1 VLP vectors of subtype A and subtype B were constructed by inserting the polyclonal sites instead of 54 bp (Subtype A) or 45bp (Subtype B) sequence in the site between p7 and p6 of the gag genes mentioned above. DNA fragments of V3 regions of gp120 (2 Clade E, 2 Clade B and 1 Clade C) were amplified. Gag- V₃ chimeric genes were obtained by inserting V₃ into the respective gag-VLP vectors. Then, gag genes, gag-VLP vector genes, gag-V3 chimeric genes were exised from plasmids based on plin8Pr55 and inserted in the downstream of the polyhedron promoter in the donor plasid pFastBac1 The recombinant plasmid pFastBac1 was transformed into DHIOBac E coli. Cells for recombinanation between homologous sequences in the donor plasmid and the bacmid. Colonies containing recombinant bacmid were identified by disruption of the Lac Za gene. High molecular weight mini-prep DNA was prepared from selected E coli colonies containing the recombinant bacmid, and then the recombinant was used to transfect insect line Sf9. Using the Bac-to-Bac baculoviruses expression system, we obtained the recombinant baculoviruses containing gag gene, gag-VLP vector gene, and gag-V₃ chimeric gene or gp120 gene. This included gag recombinant baculoviruses (1 Clade A and 1 Clade B),gag-VLP recombinant baculoviruses ( 1 Clade A and 1 Clade B), gp120 recombinant baculoviruses (3 Clade E) and gag-V₃ recombinant baculoviruses (2 Clade A/E , 2 Clade B/E, 2 Clade A/B, 2 Clade B/B, 1 Clade A/C and 1 Clade B/C). Westem-blot analysis showed that insect cells infected with gag, gag-VLP and gag-V3 recombinant baculoviruses expressed HIV-1 gag antigen and insect cells infected with...