Consensus HIV-1 subtype A integrase and its raltegravir-resistant variants: Design and characterization of the enzymatic properties.

Model studies of the subtype B and non-subtype B integrases are still required to compare their susceptibility to antiretroviral drugs, evaluate the significance of resistance mutations and identify the impact of natural polymorphisms on the level of enzymatic reactivity. We have therefore designed the consensus integrase of the HIV-1 subtype A strain circulating in the former Soviet Union territory (FSU-A) and two of its variants with mutations of resistance to the strand transfer inhibitor raltegravir. Their genes were synthesized, and expressed in E coli; corresponding His-tagged proteins were purified using the affinity chromatography. The enzymatic properties of the consensus integrases and their sensitivity to raltegravir were examined in a series of standard in vitro reactions and compared to the properties of the integrase of HIV-1 subtype B strain HXB2. The consensus enzyme demonstrated similar DNA-binding properties, but was significantly more active than HXB-2 integrase in the reactions of DNA cleavage and integration. All integrases were equally susceptible to inhibition by raltegravir and elvitegravir, indicating that the sporadic polymorphisms inherent to the HXB-2 enzyme have little effect on its susceptibility to drugs. Insensitivity of the mutated enzymes to the inhibitors of strand transfer occurred at a cost of a 30-90% loss of the efficacies of both 3'-processing and strand transfer. This is the first study to describe the enzymatic properties of the consensus integrase of HIV-1 clade A and the effects of the resistance mutations when the complex actions of sporadic sequence polymorphisms are excluded

Consensus HIV-1 FSU-A integrase gene variants electroporated into mice induce polyfunctional antigen-specific CD4+ and CD8+ T cells

Our objective is to create gene immunogens targeted against drug-resistant HIV-1, focusing on HIV-1 enzymes as critical components in viral replication and drug resistance. Consensus-based gene vaccines are specifically fit for variable pathogens such as HIV-1 and have many advantages over viral genes and their expression-optimized variants. With this in mind, we designed the consensus integrase (IN) of the HIV-1 clade A strain predominant in the territory of the former Soviet Union and its inactivated derivative with and without mutations conferring resistance to elvitegravir. Humanized IN gene was synthesized; and inactivated derivatives (with 64D in the active site mutated to V) with and without elvitegravir-resistance mutations were generated by site-mutagenesis. Activity tests of IN variants expressed in E coli showed the consensus IN to be active, while both D64V-variants were devoid of specific activities. IN genes cloned in the DNA-immunization vector pVax1 (pVaxIN plasmids) were highly expressed in human and murine cell lines (>0.7 ng/cell). Injection of BALB/c mice with pVaxIN plasmids followed by electroporation generated potent IFN-γ and IL-2 responses registered in PBMC by day 15 and in splenocytes by day 23 after immunization. Multiparametric FACS demonstrated that CD8+ and CD4+ T cells of gene-immunized mice stimulated with IN-derived peptides secreted IFN-γ, IL-2, and TNF-α. The multi-cytokine responses of CD8+ and CD4+ T-cells correlated with the loss of in vivo activity of the luciferase reporter gene co-delivered with pVaxIN plasmids. This indicated the capacity of IN-specific CD4+ and CD8+ T-cells to clear IN/reporter co-expressing cells from the injection sites. Thus, the synthetic HIV-1 clade A integrase genes acted as potent immunogens generating polyfunctional Th1-type CD4+ and CD8+ T cells. Generation of such response is highly desirable for an effective HIV-1 vaccine as it offers a possibility to attack virus-infected cells via both MHC class I and II pathway

Dynamics of bioluminescence at the sites of the IN gene and luciferase reporter gene co-administration.

<p><i>In vivo</i> monitoring of luciferase activity at days 4, 9, 15 and 21 after the administration of plasmids encoding the consensus IN (IN_a), inactivated consensus IN (IN_in), inactivated consensus IN with elvitegravir resistance mutations (IN_in_e3), or empty vector pVax1, each mixed with Luc reporter gene (1∶1). Images demonstrate two representative injection sites per group followed throughout the immunization. The scale to the right represents the strength of luminescent signal in pixels/sec/cmˆ2/sr (<b>A</b>). Kinetics of the luciferase expression over time (four mice in each group; two independent experiments) (<b>B</b>).</p

Peptides and peptide pools used in <i>in vitro</i> T-cell stimulation tests.

*<p>Choice of peptides done based on the epitopes mapped to these regions earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone.0062720-Rodriguez1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone.0062720-Casimiro1" target="_blank">[36]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone.0062720-Wilson1" target="_blank">[51]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone.0062720-Propato1" target="_blank">[52]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone.0062720-Sabbaj1" target="_blank">[53]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone.0062720-Watanabe1" target="_blank">[54]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone.0062720-LubongSabado1" target="_blank">[95]</a>. Pool_CTL contains peptides representing human CTL epitopes mapped to the given regions.</p>**<p>IN series includes peptides recognized by human T cells; and MIN, by T cells of H2-K^d- restricted BALB/c mice.</p>***<p>Peptides of pool_CTL with mutations of resistance to elvitegravir where applicable.</p

Expression of IN variants in eukaryotic cells.

<p>Western blotting of lysates of HeLa cells transfected with the pVaxIN_a (lane 1), pVaxIN_a_e3 (lane 2), pVaxIN_in (lane 3), pVaxIN_in_e3 (lane 4), or empty vector pVax1 (lane 5); recombinant IN of HXB2 carrying 6His-tag (34 kDa) loaded in the amounts of 0.5, 2.5, 5 and 10 ng/well (lanes 7 to 10, respectively). Blot was stained with the rabbit polyclonal anti-IN antibodies, stripped, and re-stained with the monoclonal anti-actin antibodies. Molecular mass markers as defined by the protein ladder (Page Ruler Prestained Protein Ladder, Fermentas; lane 6) are given to the left (<b>A</b>). Average amount of the IN variants expressed per transfected human (HeLa, HEK293) or mouse (NIH3T3) cell (results of two independent runs, each done in duplicate) (<b>B</b>).</p

Average radiance at the sites of the IN/Luc-reporter genes co-injection correlates to IN-specific cytokine response.

<p>Inverse correlation of the bioluminescence represented by the average radiance (BLI) at the injection site on day 21 after the immunization to: % CD4+T cells secreting IFN-γ, IL-2, IFN-γ/IL-2, and IFN-γ/IL-2/TNF-α (<b>A</b>); % CD8+T cells secreting IFN-γ, TNF-α, IFN-γ/TNF-α and IFN-γ/IL-2/TNF-α (<b>B</b>). Correlations of BLI on days 4, 9, 15 and 21 to the triple IFN-γ/IL-2/TNF-α secretion by CD4+ T cells by day 23 (<b>C</b>). Results of the BLI and FACS analysis of the data collected in two independent experiments (2 times × 4 mice in each group) were analyzed by the Spearman rank-order test (Statistica AXA 10).</p

Integrase activities: 3′-processing and DNA strand transfer by IN variants.

<p>Products of 3′-processing and strand transfer of the synthetic DNA duplexes with ³²P-labeled B-strands (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone-0062720-t001" target="_blank">Table 1</a>) by the consensus HIV-1 clade A integrase (IN_a), its inactivated variant (IN_in), and the inactivated variant with elvitegravir resistance mutations (IN_in_e3) were separated by gel electrophoresis and quantified using Image-QuantTM 4.1 software. The 3′-processing assay: U5 substrate in the absence of integrases (lane 1) and in the presence of IN_a, IN_in, and IN_in_e3 (lanes 2, 3, and 4, respectively) (<b>A</b>). The strand transfer reaction: U5-2 substrate in the absence of integrases (lane 1) and in the presence of IN_a, IN_in, and IN_in_e3 (2, 3, and 4, respectively); T – the strand transfer products (<b>B</b>). Incubation of the non-specific DNA Ran in the absence of integrases (lane 1), and in the presence of IN_a, IN_in, and IN_in_e3 (2, 3, and 4, respectively) (<b>C</b>). Activities of HXB2 integrase, 3′-processing (1), strand transfer (2) (<b>D</b>). Tests were performed with 100 nM integrases and 10 nM DNA. Products were separated in denaturing 20% PAAG with 7M urea (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#s4" target="_blank">Methods</a> for details). Data are representative of two independent experiments.</p

Catalytic activities of the recombinant integrases.

<p>Catalytic activities of the recombinant integrases.</p

Expression of integrase variants in <i>E.coli</i> BL21(DE3).

<p>SDS-PAGE analysis of the purified consensus HIV-1 clade A integrase (IN_a, lane 1), its inactivated variant (IN_in, lane 2) and the inactivated IN variant with elvitegravir resistance mutations (IN_in_e3, lane 3) eluted from the Ni-NTA-agarose column with 500 mM imidazole, followed by staining with Coomassie Blue (<b>A</b>). Western blotting of integrase preparations (diluted 1∶50) after SDS-PAGE and transfer, using polyclonal rabbit anti-IN antibodies (<b>B</b>). Data are representative of three independent experiments.</p

IFN-γ/IL-2 Fluorospot assay of the splenocytes of mice immunized with IN gene variants.

<p>The results of IFN-γ/IL-2 Fluorospot performed on splenocytes of mice immunized with plasmids encoding consensus IN (IN_a), inactivated consensus IN (IN_in), inactivated consensus IN with mutations conferring resistance to elvitegravir (IN_in_e3), or empty vector. Splenocytes were stimulated <i>in vitro</i> with a Luc-derived peptide (LUC), and individual or pooled IN-derived peptides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone-0062720-t003" target="_blank">Table 3</a>) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#s4" target="_blank">Methods</a>. IN-specific <i>in vitro</i> secretion of IFN-γ (<b>A</b>), IL-2 (<b>B</b>), and dual secretion of IFN-γ/IL-2 (<b>C</b>). Responses represent the average number of signal-forming units (sfu) per mln cells in two independent experiment runs, each done in duplicate,+SD.</p