Single-Cycle Virus Variants
To reduce the risks associated with ongoing replication and evolution of live-attenuated SIV strains, single-cycle SIV variants
(scSIV) have been designed. The initial scSIV variant was designed to use an artificial tRNA primer for reverse transcription
that was exclusively present in the modified producer cell line, but not in other cells.20 In addition, attenuating deletions were introduced in the vif, vpr, vpx, and nef genes. After a single intravenous injection
in rhesus macaques, peak viral RNA levels of 103 to 104 copies/mL plasma were observed, indicating efficient expression of scSIV in the vaccinee. However, the vaccine doses used
could not protect macaques from subsequent intravenous challenges with the pathogenic wild-type virus. A second approach for
producing scSIV was based on Gag-Pol complementation of an SIV genome that is deficient for Pol expression as a result of
a combination of mutations in the frameshift site that controls Pol translation.17 Four macaques were inoculated intravenously with three concentrated doses of scSIV, and viral loads peaked between 104 and 105 RNA copies/mL.18 On challenge, all animals became infected, but two of these animals were able to contain their viral loads below 2,000 RNA
copies/mL as late as 35 weeks into the chronic phase of infection. These observations are encouraging and endorse future studies
aimed at improving the protection.
Conditional Replicating Virus Variants
To improve the safety of a live-attenuated HIV-SIV vaccine strain, one must be able to shut-off virus replication once the
vaccine has done its job. We introduced a genetic switch in the HIV-1 genome to control its replication. HIV gene expression
and replication are naturally controlled by the viral Tat protein that binds to the 5' trans-acting responsive (TAR) region
in the nascent RNA transcript to enhance transcription.26 For constructing a conditionally live HIV variant, this Tat–TAR regulatory mechanism was inactivated by mutation and functionally
replaced by components of the doxycycline(dox)-inducible gene expression system (Tet-On system).27 This E.coli-derived/ gene-expression system is controlled by the rtTA protein, and binding of dox triggers a conformational switch that allows
binding this protein to tet operator (tetO) elements and activation of transcription from the downstream positioned promoter.
Thus, we introduced tetO elements in the LTR promoter and the rtTA gene in place of the nef gene. Transcription of this HIV-rtTA
construct is activated by binding the dox-rtTA complex to the tetO-LTR promoter, and this virus replicates exclusively when
dox is administered. After vaccination with this virus, replication can be temporarily activated by transient dox administration
to the extent needed for induction of protective responses. The initial HIV-rtTA construct has been improved significantly
by spontaneous virus evolution in prolonged cell culture infections.28–34 We have shown efficient and dox-dependent virus replication not only in vitro in T cell lines, but also ex vivo in human lymphoid tissue.35 An equivalent SIVmac variant was recently constructed that yielded promising results in vaccination tests in rhesus macaques
Is Double Control Needed?
We realize that safety remains a major concern for drug-controlled virus variants. In fact, we constructed an HIV-1 variant
that depends not only on dox for gene expression, but also on the T20 peptide for cell entry.36 T20 (Fuzeon) is a 36-mer peptide that mimics part of the HR2 domain of the envelope protein that is intrinsically involved
in entry of the virus into the cell.37 We described the evolution of a T20-dependent HIV-1 variant in a patient on T20 therapy.38 This virus acquired two substitutions that created a hyperactive envelope protein. Further analysis revealed that the T20
peptide can rescue this hyperfusogenic protein by preventing the premature conformational switch, thus restoring virus infectivity
and replication.39 Introducing these two mutations in HIV-rtTA resulted in a virus that replicates exclusively in the combined presence of
dox and T20.36 Subsequent withdrawal of these inducers efficiently blocks viral replication and prevents ongoing evolution.
Dox-Controlled Virus for Virotherapy
We explored the possibility to use viruses based on HIV-1 strains that use CD4 and CXCR4 for cell entry as a therapeutic virus
against malignancies such as T-lymphoblastic leukemia/lymphoma, NK leukemia, and some myeloid leukemias.40
The dox-controllable HIV-rtTA approach was combined with the design of a minimized HIV-1 variant for developing a virotherapy
for cancer.21 This mini-HIV-rtTA variant lacks several nonessential genes and has lost the ability to replicate in normal primary cells,
but this variant is still able to replicate in leukemic T-cell lines.41 This virus can efficiently and exclusively remove leukemic cells from a mixed culture with untransformed cells. In a therapeutic
setting, the minimized virus can be used to target leukemic cells in the presence of dox. This will result in a self-limiting
viral infection because the target cells are killed by the virus. Withdrawing dox provides an additional safety feature to
block ongoing replication after the leukemic cells are removed.