Background: Future AIDS vaccine candidates should generate strong, broad and long-lasting CTL and neutralizing antibody response. To ensure the safety and efficacy for AIDS vaccine, VLPs with native conformation could be the another candidate. We produced the replicative and non-replicative Human Immunodeficiency Virus-like Particles (HIVLPs) containing high quantities of native envelope (ENV) proteins from a single Semliki Forest Virus (SFV) replicon originally developed as a viral expression system.

Methods: The gag and env gene were cloned in a SFV replicon on the same plasmid with separate subgenomic promoters. The RNA transcripts containing two genes were transfected into mammalian cells and the immature VLPs were identified in the supernatant with Western blot analysis and electron microscopy (EM). To produce mature VLPs, we cloned gagpol and env genes into a single SFV replicon with separate subgenomic promoters. After sucrose density gradient ultracentrifugation of the supernatant, equal fractions of the gradient were collected and assayed for p24 content by ELISA and Western blot analysis. To produce the replicative VLPs, we inserted the packaging signal sequence of HIV-1 into the upstream of SP6 or 26S subgenomic promoter of SFV replicon. We analyzed the HIVLP-associated nucleic acids by RT-PCR and real time PCR. The immunity of these HIVLPs was tested in mouse model.

Results: Immature and mature HIVLPs composed with Gag with adequate incorporation of Env proteins were produced from a single SFV replicon. EM analysis showed that these HIVLPs were 100 to 120 nm in diameter, and the mature gp120-gp41 complexes were displayed on their surfaces. The proteinase activity was necessary to produce the processed Env protein efficiently as well as to produce the processed Gag protein. We observed that the 2 SFV-derived RNA species (full-length and subgenomic) were detected in these VLPs. As the real time PCR result, the smaller RNAs were packaged preferentially in HIVLPs. In Balb/c mice study, these HIVLPs also elicit HIV-specific CTL response and humoral immunity.

Conclusions: Our studies represent a useful system for the production of the native and mature HIVLPs for the purpose of the prophylactic vaccine in future and gene delivery.