A recombinant adenoviral vector with a specific tropism to CD4-positive cells: a new tool for HIV-1 inhibition

Gene therapy can be an option to overcome the side effects of chemotherapy and prevent the development of drug-resistant HIV viruses in HIV-infected patients. The need to develop a safe and efficient vector for gene transfer is always necessary and an appropriate option might be adenovirus (Ad). The use of Ad vectors in gene delivery applications is limited due to the semi-specific tropism. A strategy to overcome this tropism limitation may be the modification of the fiber protein domain involved in the viral binding to cells. Therefore, we designed an Ad5 vector with a specific tropism to CD4 + cells containing an expression system limited to HIV-infected cells. We replaced the knob region of Ad5 fiber protein with the extracellular region of the HIV-1 envelope. We also used a specific Tat-inducible promoter to express two anti-HIV-1 shRNAs. Tropism of recombinant Ad5 was assayed by a comparison of the shRNA expression level in CEM and PBMC cells (as CD4 + cells) and HEK293 cells (as CD4 cells). HIV-1 inhibition was assayed by the determination of p24 antigen in the HIV-infected CEM cells transduced with the recombinant Ad5 vector. Our results showed that the shRNA expression was significantly higher in CEM and PBMC cells than HEK293 cells when transduced with recombinant Ad5 vector. This new Ad5 vector also inhibited HIV-1 proliferation in a Tat-inducible manner. Our new recombinant Ad5 vector has a specific tropism to CD4-positive cells that can effectively suppress the HIV-1 replication.


Introduction
The acquired immune deficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) is one of the acute problems of today's world that should be considered. Given the promising results of the drugs used to treat this disease, they are not without problems due to side effects such as drug-related toxicity. In addition, drug failure due to virus resistance is common in long-term antiviral chemotherapy [1][2][3]. Thus, there is a need to develop new alternative therapies to cure patients with chronic HIV infection. One of these advanced therapies is genetic-based approaches such as the use of suicide genes [4,5], dominant-negative proteins [6,7], and oligonucleotide-based materials [8,9]. Some of these therapies have shown promising results in inhibiting HIV replication, and some of those have continued to a clinical trial. Given the main problem in controlling HIV infection is the ability of the virus to resist drugs and avoid treatment, so the need to design strategies to control virus replication seems essential. These strategies can include targeting the different viral products in the early and late steps of the virus life cycle, designing RNA interference (RNAi) against the conserved regions on virus transcripts, or a combination of the two strategies. These strategies have shown that they can remarkably reduce the escape of the mutated virus [10,11].
Posttranscriptional silencing by RNAi has been used in many studies to develop new approaches in antiviral therapy [12,13]. One of the major RNAis is small interfering RNA (siRNA), which is a double-stranded RNA molecule comprising 22-23 nucleotides and two nucleotide protrusions at both ends of 3′ and is modified by the Dicer complex. Dicer is an endoribonuclease that activates the RNA-induced silencing complex [14,15]. The siRNA can be transfected as synthetic or as an expression plasmid with sense and antisense strands separately. Another strategy to construct siRNA is short-stranded RNA (shRNA), a 50-60 nucleotide single-stranded RNA that is modified to a siRNA molecule [16][17][18].
One of the best approaches in gene therapy for HIVinfected cells is to use a selective inducible promoter for the expression of inhibitory genes. In some previous studies, some of these promoters have been developed, including the native HIV long terminal repeat (LTR) [19][20][21][22][23], chimeric LTR-hsp [22,23], and chimeric CK-TAR [24,25]. All these promoters are induced by the trans-activating regulatory protein (Tat) produced by HIV [26]; thus, they express their controlled genes specifically in HIV-infected cells. The CK-TAR promoter can be safer compared to native LTR and LTR-hsp because it lacks the risk of LTR remodeling. However, in an shRNA expression cassette, the CK-TAR promoter cannot generate an shRNA site with the desired transcription start site. Also, it had a relative amount of basal expression in the absence of Tat protein where it reduces its eligibility in selective expression [25]. To overcome these risks, we recently developed a Tatinducible promoter named the CkRhsp, with neutralized LTR mobilization and a favorable transcriptional start site to express an anti-HIV shRNA [27].
In the last decade, adenoviral (Ad) vectors have been frequently used for gene delivery. They have been well studied and can be produced to high titer. The Ad vectors also efficiently express a transgene in both dividing and nondividing cells and can be conserved in cells as episomal form [28][29][30]. The results of almost all clinical trials with this vector indicated that Ad vectors are safe and well-tolerated [31]. Despite the versatility of recombinant Ad vectors in gene therapy approaches, the diversity in the use of these vectors in gene transfer applications is limited due to their semi-specific tropism and is best suited for a specific organ rather than a specific cell type [32]. One strategy to overcome this tropism restriction could be to modify the domains of the proteins involved in binding the virus to the cell through which the vector is allowed to interact with the receptors of a particular cell. Ads bind to a specific receptor in eukaryotic cells via the knob domain at the end of their fiber protein. Thus, maybe protein engineering of fiber protein will alter the natural tropism of Ads [33,34].
In this study, we decided to design an Ad5 vector with a specific tropism to CD4-positive cells. Thus, we replaced the Knob region of the Ad5 fiber protein with the extracellular domain of the HIV-1 envelope protein. To obtain a restricted expression system for HIV-infected cells, we used a Tat-inducible promoter called CkRhsp to express two anti-HIV-1 shRNAs, as we have reported previously [27].

Virus production
The two shRNA cassettes used in this study, target two highly conserved sequences in the HIV-1 RNA [27]. Two shRNA expressional cassettes were synthesized in the order of CkRhsp promoter, shRNA, and minimal polyadenylation signal with a length of 1.3 kb (Generay, Shanghai, China). They were then cloned into pShuttle plasmid (Agilent, Santa Clara, USA) digested by XhoI/BglII (Thermo Fisher, MA, USA), and the pShuttle-sh plasmid was generated. The chimeric knobless Ad5 fiber gene was designed, respectively, by the N-terminal tail and the first two repeats of the shaft domain of the Ad5 fiber protein, HIV-1 gp140, and Ad5 poly-A. This gene along with the right homology arm was synthesized and cloned into pShuttle-sh plasmid digested by BbsI/BamHI (Thermo Fisher, MA, USA) and named pShuttle-sh/Δknob/gp140 plasmid. Also, a pShuttle-sh/Δknob/gp140 plasmid was prepared with two irrelevant shRNAs as a mock control. In addition, a recombinant Ad5 fiber gene without knob region along with the right homology arm was synthesized as mock control and cloned into BbsI/BamHI-digested pShuttle-sh plasmid and named pShuttle-sh/Δknob (Fig. 1). This vector is constructed to assay the binding of adenovirus coating proteins other than the knob domain to the CD4 receptors. The resultant plasmids were linearized by the PmeI enzyme (Thermo Fisher, MA, USA) and were cotransformed with a pAdEasy-1 plasmid (replication-defective Ad5 genome) into E. coli BJ5183 strain for homologous recombination according to the AdEasy Adenoviral Vector System instruction (Agilent, Santa Clara, USA). The smallest well-isolated colonies were then removed and cultured in LB-kanamycin broth. The recombinant Ad5 plasmids were extracted and digested by PacI enzyme (Thermo Fisher, MA, USA) to confirm by size in agarose electrophoresis. The detailed structures of recombinant Ad5 vectors are shown in Fig. 1. The resultant Ad plasmid was transfected into the human embryonic kidney (HEK) 293 cells (ATCC, CRL1573) to produce recombinant Ad5 virus. The virus titer was determined by plaque assay using agarose overlay according to the AdEasy Adenoviral Vector System instruction. Recombinant Ad5 viruses were produced in titer of 10 7 to 10 8 plaque-forming units (PFU)/ml. The HIV-1 virus was produced by transfection of HEK293 cells with a pNL4-3 plasmid (HIV-1 producer) (NIH AIDS Research and Reference Reagent Program, Germantown, USA) using 1 µg/ml polyethylenimine for 20 h at 37 °C. Cell culture medium was then replaced with fresh complete Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, MO, USA). Viruses were harvested at 48 and 72 and 96 h post-transfection and tittered using the p24 ELISA kit (Clontech, CA, USA). According to the manufacturer's instructions, 20 µl of lysis buffer was added to each well. A total of 200 µl of each standard curve dilution and supernatant were added to each well as triplicate, incubated at 37 °C for 60 min, and washed. One hundred microliters of biotin conjugate anti-p24 antibody was added to each well, incubated at 37 °C for 60 min, and washed. One hundred microliters of Streptavidin-HRP conjugate was added to each well, incubated at room temperature for 30 min, and washed. One hundred microliters of Substrate Solution was immediately added to each well, incubated at room temperature for 30 min. One hundred microliters of stop solution was added to each well, and the absorbance values were immediately read using an ELISA plate reader (BioTek, VT, USA) at 450 nm blanked by the negative control well. The p24 level of the samples was then quantified against a standard curve. HIV titers ranged from 5 × 10 8 to 7 × 10 8 ifu/ml according to the manufacturer's instructions.

HIV-1 gp140 ELISA assay
The presence of HIV-1 gp140 protein on the recombinant Ad5 vector was confirmed by the HIV-1 gp120 ELISA Kit (Antibodies-online GmbH, Aachen, Germany). In accordance with the manufacturer's instructions, 50 μl of the recombinant Ad5-sh/Δknob/gp140 vector, Ad5-sh/Δknob vectors, and standards were added to each well on a 96-well plate coated with HIV gp120, incubated for 2 h at room temperature, and washed. One hundred microliters of the detection antibody was added to each well, incubated for 2 h at room temperature, and washed. Two hundred microliters of Substrate Solution was added to each well, incubated for 20 min at room temperature. Fifty microliters of stop solution was added to each well, and the optical density of each domain. The pShuttle-sh/Δknob plasmid is similar to the pShuttlesh/Δknob/gp140 plasmid except that its fiber gene does not have the HIV-1 gp140 sequence. The recombinant Ad5-sh/Δknob/gp140 vector is the product of recombination between shuttle pShuttle-sh/Δknob/ gp140 plasmid and the Ad5 vector plasmid, pAdEasy-1. Recombinant Ad5-sh/Δknob vector is the product of recombination between shuttle pShuttle-sh/Δknob plasmid and pAdEasy-1 plasmid well was immediately determined using a microplate reader at 450 nm. The gp140 level of the samples was then quantified against a standard curve.

Cell culture
The cells used in this study included CEM cells, a human T-cell line (ATCC, CCL-119) and the peripheral blood mononuclear cells (PBMC) (as CD4 positive), and HEK293 cell line (as CD4 negative). To generate HIV-infected cells containing TAT protein, cells were seeded in a 6-well tissue culture plate at a density of 2 × 10 5 cell/well with 1 ml DMEM medium. After 20 h, cells were infected with HIV-1 in a multiplicity of infection (MOI) of 1 for 20 h at 37 °C [35]. To investigate the effect of recombinant Ad5 vector on HIV proliferation, the medium was then replaced with 2 ml of fresh DMEM containing recombinant Ad5 vectors in an MOI of 10. After 6 h, the medium was then replaced with 2 ml of fresh DMEM.

Tropism assay
To investigate the efficient tropism of the recombinant Ad5 vector to CD4-positive cells, shRNA expression was assayed as an indirect index of successful transduction. For this purpose, total RNA was isolated from cells at 24 h posttransduction by the mirVana miRNA Isolation Kit (Thermo Fisher, MA, USA). The first-strand the complementary DNA (cDNA) was synthesized by stem-loop primers and using reverse transcription enzyme (Thermo Fisher, MA, USA) according to the kit instruction. The cDNA level was then determined by forward and reverse primers and the SYBR Green Master Mix (YektaTajhiz, Tehran, Iran) as triplicate in the ABI Real-Time PCR system (Applied Biosystems, CA, USA). The cDNA expression was normalized with U6-snRNA endogenous control as described previously [36]. The shRNA relative expression was calculated by the 2 −∆Ct [37].

HIV-1 challenge
To evaluate the effective inhibition of HIV by the recombinant Ad5 vector, HIV-1 proliferation was investigated in culture supernatants harvested 24 h post-transduction by the p24 antigen ELISA kit and AccuPower HIV-1 quantitative real-time PCR (qPCR) kit (Bioneer, Daejeon, South Korea) in three replications. qPCR was performed on the RNA extracted by the viral nucleic acid extraction kit (YektaTajhiz, Tehran, Iran) against a standard curve and expressed as IU/ml.

Statistical analysis
The variables were expressed as the mean ± standard deviation. Data were analyzed by GraphPad software (GraphPad, La Jolla, USA). Mann-Whitney U test was used to compare the means of two groups. Differences of p-value less than 0.05 were considered statistically significant.

Recombinant Ad5 vector has a specific tropism to CD4-positive cells
The presence of HIV-1 gp140 protein on the recombinant Ad5 was confirmed by the ELISA assay. As results of HIV-1 gp140 ELISA show, gp140 concentration in Ad5-sh/Δknob/ gp140 vector (713.3 ± 72.19 picomol/ml) is significantly higher than the Ad5-sh/Δknob vector (as control without gp140) (not detectable) (p = 0.0006) (Fig. 2). These results show that the Ad5 envelope protein is efficiently engineered to pseudotype to CD4-positive cells. To investigate specific tropism of the recombinant Ad5-sh/Δknob/gp140 vector to CD4-positive cells, the shRNA expression level was determined as an indirect index of successful transduction. The results of Fig. 3 show that shRNA expression is significantly higher in CEM cells (2.35 ± 0.33) (p = 0.0009) and PBMCs

Discussion
The standard therapy option for HIV-infected patients is highly active antiretroviral therapy (HAART), which includes a combination of several drugs that target different stages of the HIV-1 life cycle. Although HAART improves the quality of life and longevity of infected people, it is a lifelong treatment that only slows the progression of the disease without treatment. Furthermore, HAART is often associated with severe side effects and poor diets that make life difficult for the patients. In addition, patients treated with HAART often experience an increasing prevalence of infections caused by weakened immune systems. These shortcomings lead to the idea of creating a more effective and less expensive alternative treatment [38].
Gene therapy can be an option for overcoming the side effects of chemotherapy and preventing the development of drug-resistant HIV strains [10]. We previously reported a lentiviral vector containing a Tat-inducible expression system with specific tropism to CD4-positive cells to inhibit HIV-1 replication. Although the vector appears to be nonproliferative and safe, there is still a risk of integration within transcriptionally active regions and tumorigenesis [27]. Therefore, the need to create a safer vector for gene transfer in vivo seemed essential, and it was thought that using an adenovirus vector could be a good idea. Although attempts have been made in the past to engineer fiber protein to modify Ad tropism [28,[32][33][34]39], no vector with a specific affinity for CD4-positive cells has been reported. Therefore, in this study, we decided to create a recombinant Ad5 vector with modified fiber to gene transfer into CD4positive cells. Given the similarity of the trimeric structure of the Ad5 fiber protein and the HIV-1 envelop protein, it was hypothesized that the replacement of the knob region of Ad5 fiber with the extracellular region of HIV-1 envelop could lead to the creation of a recombinant Ad5 vector with a specific tropism to CD4-positive cells. In designing this new vector, our theory was that the identification of the receptors of CD4 and CCR5 alone could lead to viral endocytosis in CD4-positive cells. As results in Fig. 3 show, our recombinant Ad5 vector has a significant tropism to CD4positive cells compared with CD4-negative cells. Indirect analysis of the binding of the Ad5-sh/Δknob/gp140 and Ad5-sh/Δknob vectors showed that the binding of the Ad5sh/Δknob/gp140 vector to the CD4 receptor was probably dependent on gp140, and other vector coat proteins play a small role in binding to this receptor. Remarkably, there are different results in shRNA expression and p24 level between PBMC and CEM cells (Figs. 3 and 4), which may be due to the variable percentage of cd4 cells (14-65%) in PBMC cells [40][41][42].
RNAi gene silencing is an efficient mechanism for downregulating specific genes. Thus, it can be used as a useful antiviral agent in strategies of gene therapy. One of the advantages of the RNAi-based approaches in comparison with protein-based is the low immunogenicity of the RNAi molecule [43]. As reported in our study and other studies previously, the use of multiple shRNAs to target highly conserved HIV sequences can be an effective method to inhibit virus replication in RNAi-based approaches [27,44]. Thus, in this study, we decided to use the same dual-shRNA expression system in the recombinant Ad5 vector to inhibit HIV-1 replication. This expression system is under the control of the CkRhsp promoter, which is active only in cells infected with HIV. As the results in Fig. 4 show, this expressional system can efficiently and transiently inhibit HIV replication. Although the in vitro effect of this recombinant Ad vector in preventing virus replication in this study was promising, there is a long way to go to use this vector in humans, which requires purification with a standard drug and testing in model animals.

Conclusions
In this study, we developed a novel recombinant Ad5 vector with a specific tropism to CD4-positive cells. This vector has an expression system through which can effectively and transiently suppress the HIV-1 replication. This vector can be used as a complementary HIV therapy or an anti-HIV vaccine in the future.