In vitro evolution of a highly replicating, doxycycline-dependent HIV for applications in vaccine studies

Abstract

A major concern associated with the use of vaccines based on live-attenuated viruses is the possible and well documented reversion to pathogenic phenotypes. In the case of HIV, genomic deletions or mutations introduced to attenuate viral pathogenicity can be repaired by selection of compensating mutations. These events lead to increased virus replication rates and, eventually, disease progression. Because replication competence and degree of protection appear to be directly correlated, further attenuation of a vaccine virus may compromise the ability to elicit a protective immune response. Here, we describe an approach toward a safe attenuated HIV vaccine. The system is not based on permanent reduction of infectivity by alteration of important viral genomic sequences, but on strict control of replication through the insertion of the tetracycline (Tet) system in the HIV genome. Furthermore, extensive in vitro evolution was applied to the prototype Tetcontrolled HIV to select for variants with optimized rather than diminished replication capacity. The final product of evolution has properties uniquely suited for use as a vaccine strain. The evolved virus is highly infectious, as opposed to a canonically attenuated virus. It replicates efficiently in T cell lines and in activated and unstimulated peripheral blood mononuclear cells. Most importantly, replication is strictly dependent on the nontoxic Tetanalogue doxycycline and can be turned on and off. These results suggest that this in vitro evolved, doxycycline-dependent HIV might represent a useful tool toward the development of a safer, live-attenuated HIV vaccine.

Autoregulatory lentiviral vectors allow multiple cycles of doxycycline-inducible gene expression in human hematopoietic cells in vivo

“…Recently, we have generated a conditional-live HIV-1 variant in which life cycle is dependent on the Tet-On gene regulation system (HIVrtTA). 44,45 The modifications introduced in the HIV-rtTA genome changed the viral replication control from a constitutive autoregulatory loop driven by the Tat-TAR axis into an inducible autoregulatory loop regulated by dox. In the presence of dox, rtTA can bind to tetO and activate transcription from the downstream transcription initiation site.…”

Viral Evolution as a Tool to Improve the Tetracycline-regulated Gene Expression System

“…Proviral DNA sequences were PCR amplified from total cellular DNA, using the 5Ј primer nef-seq1 (positions 56 to 37 upstream of the rtTA translation start site) and the 3Ј U3 primer anti-U3-att (positions 63 to 44 downstream of the rtTA translation stop codon). PCR fragments were digested with XcmI and SmaI and cloned into the corresponding sites in the shuttle vector pBlue3ЈLTRext-⌬U3-rtTA-2⌬tetO, which is identical to the vector pBlue3ЈLTRext-⌬U3-rtTA-K8-TAR* (18) but with the optimized 2⌬tetO promoter configuration (21,22). Sequence analysis was performed by big dye terminator cycle sequencing (PE Biosystems).…”

HIV-1 evolution: frustrating therapies, but disclosing molecular mechanisms

“…However, an intriguing sequence duplication was selected in the LTR promoter, which doubled the number of Sp1 binding sites from three to six and greatly improved virus replication. Multiplication or deletion of repeat sequence motifs is more frequently observed and seems to be a popular virus evolution strategy Marzio et al 2001;Leonard et al 2008;Berkhout 2009). Evolution experiments may also help in the development of more safe HIV-1 vaccine candidates.…”