A Recombinant Adenoviral Vector With a Specific Tropism to CD4-positive Cells: a New Tool for HIV-1 Inhibition

doi:10.21203/rs.3.rs-996637/v1

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A Recombinant Adenoviral Vector With a Specific Tropism to CD4-positive Cells: a New Tool for HIV-1 Inhibition

https://doi.org/10.21203/rs.3.rs-996637/v1

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published 30 Jan, 2022

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Gene therapy can be an option to overcoming the side effects of chemotherapy and preventing 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 the gene delivery applications is limited due to the semi-specific tropism. A strategy to overcome this tropism limitation may be the modification of 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 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 comparison of shRNA expression level in CEM and PBMC cells (as CD4+ cells) and HEK293 cells (as CD4- cells). HIV-1 inhibition was assayed by determination of p24 antigen in the HIV-infected CEM cells transduced with the recombinant Ad5 vector. Our results showed that shRNA expression was significantly higher in CEM and PBMC cells than HEK293 cells when were 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.

Translational Medicine

Medical Genetics

Gene therapy

Adenovirus

Human Immunodeficiency Virus -1

CD4 Receptor

Viral Tropism

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 [1–3]. 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 early and late steps of the virus life cycle, designing RNAs 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 actives 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 [16–18].

One of the best approaches in gene therapy for HIV-infected 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) [19–23], chimeric LTR-hsp [22, 23], and chimeric CK-TAR [24, 25]. All these promoters are induced by 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 that reduces its eligibility in selective expression [25]. To overcome these risks, we recently developed a Tat-inducible 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 non-dividing cells and can be conserved in cells as episomal form [28–30]. 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 order of CkRhsp promoter, shRNA and minimal polyadenylation signal with a length of 1.3 kilobases (kb) (GeneRay, Shanghai, China). They were then cloned into pShuttle plasmid (Agilent, Santa Clara, USA) digested by XhoI/BglII (Thermo Fisher, Massachusetts, 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, Massachusetts, USA) and named pShuttle-sh/Δknob/gp plasmid. Also, a pShuttle-sh/Δknob/gp 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 Knob domain to the CD4 receptors. The resultant plasmids were linearized by PmeI enzyme (Thermo Fisher, Massachusetts, 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, Massachusetts, USA) to confirm by size in agarose electrophoresis. The detailed structures of recombinant Ad5 vectors are shown in Figure 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 titers of 10⁷ to 10⁸ 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 1µg/ml Polyethylenimine for 20 hours at 37°C. Cell culture medium was then replaced with fresh complete Dulbecco's Modified Eagle's medium (DMEM) medium (Sigma-Aldrich, Missouri, USA). Viruses were harvested at 48 and 72-, and 96-hours post-transfection and tittered using p24 Elisa kit (Clontech, California, USA) in an Elisa plate reader (BioTek, Vermont, USA) according to the manufacturer’s instructions. HIV titers ranged from 5×10⁸ to 7×10⁸ infectious units/ml. One picogram (pg) p24 is approximately equivalent to 175000 infectious viruses, which was calculated using the following formula: 1 pg p24 / [36 (fiber numbers in one Ad virus) [35] × 24 kDa (p24 weight)] × 0.25 (Ad infectious units rate) [36] ≈ 175000 infectious units.

HIV-1 gp140 Elisa assay

To confirm the presence of HIV-1 gp140 protein on the recombinant Ad5 vector, the level of this peptide was measured in 50 µL of the recombinant Ad5-sh/Δknob/gp vector and compared with its value in the same volume of Ad5-sh/Δknob vector by HIV-1 gp120 Elisa Kit (antibodies-online GmbH, Aachen, Germany) in accordance with the manufacturer's instructions.

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 to produce TAT protein to induce shRNA expression, cells were seeded in a 6-well tissue culture plate at a density of 2 × 10⁵ cell/well with 1ml DMEM medium. After 20 hours, cells were infected with HIV-1 in a multiplicity of infection (MOI) of 1 for 20 hours at 37°C [37]. The medium was then replaced with 2 ml of fresh DMEM containing recombinant Ad5 vectors in an MOI of 10. After 6 hours, the medium was then replaced with 2 ml of fresh DMEM.

Tropism assay and HIV-1 challenge

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 hours post-transduction by the mirVana miRNA Isolation Kit (Thermo Fisher, Massachusetts, USA). The first-strand the complementary DNA (cDNA) was synthesized by stem-loop primers and using reverse transcription enzyme (Thermo Fisher, Massachusetts, USA) according to the kit instructions. 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, California, USA). The cDNA expression was normalized with U6-snRNA endogenous control as described previously [38]. The shRNA relative expression was calculated by the 2^−∆Ct [39]. HIV-1 proliferation was investigated in culture supernatants harvested 24 hours post-transduction and assayed by p24 antigen ELISA kit.

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 as 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 Elisa assay. As results of HIV-1 gp140 Elisa show, gp140 concentration in Ad5-sh/Δknob/gp vector (713.3 ± 72.19 picomol/ml) is significantly higher than 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/gp vector to CD4-positive cells, 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 (1.17 ± 0.24) (p = 0.0127) compared with HEK293 cells (0.47 ± 0.15) when were transduced with Ad5-sh/Δknob/gp vector. No significant difference was observed in shRNA expression level between CEM cells (0.31 ± 0.10) (p = 0.3476) and PBMCs (0.42 ± 0.12) (p = 0.9573) with HEK293 cells (0.42 ± 0.13) when were transduced with Ad5-sh/Δknob vector.

Recombinant Ad5 vector inhibits HIV-1 proliferation

To investigate the efficiency of HIV-1 inhibition by the new recombinant Ad5 vector, the HIV-infected cells were transiently transduced by the recombinant Ad5 vector, and p24 protein level was determined as an HIV-1 proliferation index. As results of Fig. 4 show, the p24 level is significantly lower in CD4-positive cells (CEM and PBMC) transduced with Ad5-sh/Δknob/gp vector (2341 ± 413 and 1332 ± 312 respectively) compared with untransduced cells (10324 ± 1122 and 5768 ± 654 respectively) (p < 0.001). There was no significant difference in p24 level between cells transduced with Ad5-sh/Δknob and Ad5-sh.irrel/Δknob/gp with untransduced cells.

Gene therapy can be an option for overcoming the side effects of chemotherapy and preventing the development of drug-resistant HIV [10]. The exclusive properties of HIV-1 and the existence of multiple mechanisms for disease development, make it difficult to design an effective gene therapy strategy. However, we previously reported a lentiviral vector containing a TAT-inducible expression system with specific tropism to CD4-positive cells to inhibit HIV-1 replication [27]. But the need to develop a safer and more efficient vector for gene transfer seems to be necessary, and the appropriate option may be to use an adenoviral vector. Although, in the past, efforts have been made to engineer fiber protein to modify Ad tropism [28, 32–35]. But so far, an Ad vector with a specific tropism to CD4 positive cells has not been reported. Thus, in this study, we decided to create a recombinant Ad5 vector with a modified fiber to gene transfer into CD4-positive 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 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 CD4-positive cells compared with CD4-negative cells. Indirectly analysis of the binding of the Ad5-sh/Δknob/gp and Ad5-sh/Δknob vectors showed that the binding of the Ad5-sh/Δknob/gp vector to the CD4 receptor was probably dependent on gp140, and that other vector envelope proteins other than the Knob domain play little role in binding to this receptor.

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 [40]. 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, 41]. 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.

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.

Ethical statement

Ethics approval and consent to participate

Not Applicable

Consent for publication

All authors give consent for publication.

Availability of data and materials

All data and materials support the claims in this manuscript and comply with field standards.

Competing interests

The authors declare no competing interests.

Funding

The study was supported by a grant from the research department in Golestan University of Medical Sciences, Gorgan, Iran (Grant number: G/P/35/306082).

Authors' contributions

T.F. conceived and designed the experiments. A.B. conducted the laboratory experiments. A.B. and T.F. analyzed the data. A.B. and T.F. prepared and reviewed the manuscript.

Acknowledgements

The study was supported by a grant from the research department in the Golestan University of Medical Sciences, Gorgan, Iran (Grant number: G/P/35/306082).

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A Recombinant Adenoviral Vector With a Specific Tropism to CD4-positive Cells: a New Tool for HIV-1 Inhibition

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