Background:  We have developed a single-cycle lentviral vector simian immunodeficiency virus (SIV)_smH4i-SC27.1 that is capable of expressing all of the viral gene products as a potential SIV/HIV-1 vaccine candidate. The strategy we have used is based on the idea that with a sufficient number of mutations to functionally important residues it is possible to preclude reversion to replication competency while still allowing expression of all viral protein targets.

Methods:  This vector was created by incorporating 54 mutations into 27 codons dispersed among the viral env, vif and nef genes to block protein function, attenuate viral replication, and reduce the ability to manipulate the host immune system. To complement the Env and Nef defects, plasmid DNA encoding the SC27.1 genome was co-transfected with a plasmid expressing the VSV G glycoprotein to produce infectious pseudotyped particles that would allow entry and avoid the effect of the nef mutations on viral infectivity. Pseudotyped SC27.1 viral particles have similar or lower titers than wild type parental virus derived from the SIV_smmh4i infectious clone.

Results:  The effect of the env mutations were seen as a distinct phenotype of cells infected with the pseudotyped virus compared to the parental virus, with the latter but not the former inducing syncytia. In addition, the Env protein from SC27.1 was truncated and unprocessed, giving a gp145 product that was expressed on the cell surface but not packaged into virions. The Vif defect was complemented by producing particles from APOBEC3G-negative 293T cells. Pseudotyped SC27.1 lost most of its infectivity if a plasmid expressing APOBEC3G was included during virus production in the 293T cells, validating the Vif protein defect. Gag expression was demonstrated in both producer cell lysates and in supernatant particles. To address the problem of an antibody response to the VSV G protein in a vaccination strategy, the genes for 2 additional serotypes of the VSV G protein were used to create expression plasmids that were then used to produce pseudotyped virus particles. No cross-neutralization activity was observed among the 3 pseudotyped particles in an in vitro neutralization assay. Repeated passage of the VSV G pseudotyped SC27.1 particles demonstrated that infectivity was limited to a single cycle with no evidence of reversion.

Conclusions:  These studies validate the design of this vaccine vector, which will now move to a prime-boost and challenge experiment to test its efficacy as a vaccine.