The Tat Protein Enhances CTL Responses and Therapeutic Immunity of Gag-Specific Exosome-Targeted T Cell-Based Gag / Tat-Texo Vaccine in Transgenic HLA-A 2 Mice

Human immunodeficiency virus type-1 (HIV-1) chronic infection causes millions of deaths each year. We previously developed a novel HIV-1 Gag-spe cific exosome (EXO)-targeted T cell-based vaccine (Gag-Texo) using ConAstimulated polyclonal CD8 T (ConA-T) cells armed with Gag-specific dendritic cell (DC)-released EXOs, and showed that Gag-Texo stimulated more efficient cytotoxic T lymphocyte (CTL) responses than DCs. Tat HIV-1 early regulatory protein possesses immunomodulatory and adjuvant properties. To enhance Gag-Texo immunogenicity, we generated Tat-engineered OVA/Tat Texo and Gag/Tat-Texo vaccines using ConA-T cells armed with EXOs release by DCs infected with recombinant OVA/Tatand Gag/Tat-expressing adenoviruses (AdVOVA/Tat and AdVGag/Tat). We then assessed vaccination-stimulated CTL responses in naïve mice, and therapeutic immunity in transgenic HLA-A2 mice bearing Gag/HLA-A2-expressing BL6-10OVA/A2 melanoma lung metastases. We demonstrate that the OVA/Tat-Texo vaccine enhances functional OVA-specific CTL responses, compared to the OVA-Texo vaccine, and broadens CTL responses recognizing the cryptic OVA epitope in C57BL/6 mice. Furthermore, we determine that the Gag/Tat-Texo not only stimulates more efficient CTL responses than Gag-Texo, but also induces enhanced therapeutic immunity. We show that, 30% of Gag/Tat-Texo-immunized mice are free of tumor lung-metastases, compared to all Gag-Texo-immunized mice displaying lung-metastasis. In addition, the average number of tumor lung metastases colonies (32/lung) in the Gag/Tat-Texo-immunized mice was also *Xu and Wu made the same contribution to the study. How to cite this paper: Xu, A.Z., Wu, J., Xie, Y.F., Chibbar, R., Fleywald, A. and Xiang, J. (2017) The Tat Protein Enhances CTL Responses and Therapeutic Immunity of Gag-Specific Exosome-Targeted T CellBased Gag/Tat-Texo Vaccine in Transgenic HLA-A2 Mice. World Journal of Vaccines, 7, 11-25. https://doi.org/10.4236/wjv.2017.72002 Received: September 2, 2016 Accepted: May 24, 2017 Published: May 27, 2017 Copyright © 2017 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/

A. Z. Xu et al.

Introduction
HIV-1 that causes millions of deaths each year primarily acts by infecting memory CD4 + CCR5 + T cells, and becomes resistant to antiviral drug therapy (ART).This results in latent pro-viral reservoirs and a life-long infection [1].As wellestablished in natural control of HIV-1 (i.e."elite" control), potent CTLs can control it by killing virus-infected cells [2], leading to renewed interests in therapeutic vaccination for stimulating CTL responses decaying pro-viral reservoirs after pharmacologically purging the reservoirs [3].Purging the reservoir itself is not sufficient to cause death of infected cells that remain in a resting state [4], whereas boosting CTL responses efficiently eliminates latent pro-viral reservoirs [4] [5], warranting a search for potent therapeutic vaccines.
Structural proteins of HIV-1, Gp120 and Gag, have become major targets for inducing HIV-1-specific CTL responses in vaccine development [6] [7].Previous vaccines (peptides, DNA or viral vectors) often failed in stimulation of therapeutic immunity in HIV-1 patients [8] possibly due to critical defects in CD4 + T cells and DCs [1].Immune cell-based treatment approaches become a third pillar of therapeutics [8].However, efficacies of DC vaccinations, which often require CD4 + T cell help [9] remain poor [10].Functional CTL exhaustion becomes another chief issue, leading to ineffective HIV-1 control in patients [11] [12].
We previously generated an HIV-1 Gp120-specific exosome (EXO)-targeted Gp120-Texo vaccine by using ConA-T-stimulated mousepolyclonal T cells armed with Gp120-specific DC (DC Gp120 )-released EXO (EXO Gp120 ) [13] [14].We demonstrated that the Gp120-Texo vaccine was more immunogenic than the DC Gp120 vaccine and was capable of stimulating CD4 + T cell-independent Gp120specific CTL responses, leading to protective and long-term immunity against Gp120/HLA-A2-expressing B16 melanoma (BL6-10 Gp120/A2 ) in transgenic HLA-A2 mice [13] [14].Compared to the HIV-1 structural protein Gp120, HIV-1 Gag has several important advantages as a target antigen for developing HIV-1-specific vaccines.Thus, the Gag vaccine stimulates persistent and broader CTL responses against conserved Gag epitopes in animal models [15] [16] [17].HLA-B57 HIV-1-infected individuals have also been found to have autologous CTL responses against four conserved Gag epitopes, which are associated with the reduced virus replication and more efficient viral control [2].In addition, effective CTL responses against Gag have been found to correlate with the suppression of HIV-1 replication in HIV-1 patients [18] [19] [20] [21].Since Gag has become one of the most attractive target candidates for HIV-1 vaccine development, we recently generated a Gag-specific DC (DC Gag )-released EXO-targeted Gag-Texo vaccine, and demonstrated that Gag-Texo stimulated efficient Gagspecific CTL responses, leading to a complete protective immunity, but only some degree of therapeutic immunity against Gag/HLA-A2-expressing BL6-10 Gag/A2 melanoma in transgenic HLA-A2 mice [22].Like other lentiviruses, HIV-1 encodes a trans-activating regulatory protein (Tat), which is essential for efficient transcription of the viral genome [23].Since the HIV-1 early regulatory protein Tat possesses immunomodulatory and adjuvant properties that can be advantageous in vaccine development [23], we assumed that Tat may further enhance the immunogenicity of our Gag-Texo vaccine.
To assess our assumption, we produced recombinant adenoviruses AdV OVA/Tat and AdV Gag/Tat expressing OVA plus Tat and Gag plus Tat, and generated OVA/ Tat-Texo and Gag/Tat-Texo vaccines by using ConA-stimulatedmouse CD8 + T cells with the uptake of AdV OVA/Tat or AdV Gag/Tat -transfected DC (DC OVA/Tat and DC Gag/Tat )-released EXOs, respectively.We immunized wild-type C57BL/6 and transgenic HLA-A2 mice intravenously with the OVA/Tat-Texo or Gag/Tat-Texo vaccine, and assessed their ability to stimulate OVA-and Gag-specific CTL responses and immunity against OVA-and Gag/HLA-A2-expressing B16 melanoma.This demonstrated that the transgene Tat-engineered OVA/Tat-Texo vaccine not only enhanced OVA-specific CTL responses, but also broadened OVA-specific CTL responses recognizing the cryptic OVA epitope in C57BL/6 mice, compared to the OVA-Texo vaccine.In addition, the transgene Tat-engineered Gag/Tat-Texo vaccine stimulated more efficient therapeutic immunity against Gag/HLA-A2-expressing tumors in transgenic HLA-A2 mice.
Two gene fragments HIV-1 Gag and Tat were obtained from NIH AIDS Research and Reference Reagent Program, NIH, Bethesda, MD.Wild-type female C57BL/6 (B6) and transgenic (Tg) HLA-A2 mice (33,584) were obtained from the Jackson Laboratory (Bar Harbor, MA).All mice were treated according to Animal Care Committee Guidelines of the University of Saskatchewan.

Construction of Recombinant Adenoviruses Expressing OVA or Gag and Tat
Recombinant adenoviral vectors AdV OVA/Tat and AdV Gag/Tat expressing OVA and HIV-1 Gagand Tat were constructed by inserting a fused gene fragment containing a IRES fragment (including the enhancer and poly A regions) [25], to generate AdVs AdV OVA/Tat or AdV Gag/Tat expressing transgene OVA and Tat or Gag and Tat [26].AdVs were amplified in 293 cells, and purified by a series of cesium chloride ultracentrifugation gradients [26].

Dendritic Cell and Exosome Preparations
Bone marrow-derived dendritic cells (DCs) were obtained by culturing the wildtype B6 or transgenic HLA-A2 mousebone marrow cells in culture medium containing GM-CSF (20 ng/ml) and IL-4 (20 ng/ml) for six days as previously described [22].DCs derived from B6 and HLA-A2 mice were infected with AdV OVA or AdV OVA/Tat and AdV Gag or AdV Gag/Tat , respectively, and termed DC OVA or DC OVA/Tat and DC Gag or DC Gag/Tat .DC OVA , DC OVA/Tat , DC Gag and DC Gag/Tat released exosomes (EXO OVA , EXO OVA/Tat , EXO Gag and EXO Gag/Tat ) were then purified from AdV-transfected DC culture supernatants by differential ultracentrifugation [22].

Electron Microscopic Analysis
EXOs were fixed in 4% paraformaldehyde.The pellets were then loaded onto carbon-coated formvar grids.The EXO sample-loaded grids were stained with saturated aqueous uranyl, and then examined with a JEOL 1200EX electron microscope at 60 kV.

Flow Cytometric Analysis
DCs were stained with a panel of Abs and analyzed by flow cytometry [13].To

Cytotoxicity Assay
The in vivo cytotoxicity assay was performed as previously described [22].Briefly, splenocytes were harvested from naive B6 or HLA-A2mouse spleens and in-
The mice were sacrificed 3 weeks after tumor cell injection, and metastatic tumor colonies were counted in lungs in a blind fashion.Metastases on freshly isolated lungs appeared as discrete black pigmented foci that were easily distinguishable from normal lung tissues and confirmed by histological examination.
Metastatic foci too numerous to count were assigned an arbitrary value of >300.

Statistic Analyses
Statistical analyses were performed using the Student t test for comparison of variables from different groups.A value of p < 0.05 was considered to be statistically significant.

Generation of the Transgene Tat-Engineered OVA/Tat-Texo Vaccine
We used a recombinant DNA technology to construct a recombinant OVA and Tat (OVA/Tat)-expressing adenovirus (AdV OVA/Tat ) (Figure 1).We then infected We then generated the OVA-Texo and OVA/Tat-Texo vaccines using ConAstimulated B6 polyclonal CD8 + T (ConA-T) cells armed with DC OVA -or DC OVA/Tat -released EXOs, respectively, by incubating ConA-T cells with EXOs for 2 hours, as previously described [13].

Generation of the Transgene Tat-Engineered Gag/Tat-Texo Vaccine
We also constructed recombinant Gag and Tat (Gag/Tat)-expressing adenovirus (AdV Gag/Tat ) by using recombinant DNA technology (Figure 1).We infected transgenic HLA-A2 mouse bone marrow-derived DCs with AdV Gag and AdV Gag/Tat , and generated DC Gag and DC Gag/Tat .We showed that DC Gag/Tat expressed cell sur-

The Transgene Tat-Engineered Gag/Tat-Texo Vaccine Enhances CTL Responses in Transgenic HLA-A2 Mice
To assess vaccine's immunogenicity, we immunized transgenic HLA-A2 mice with the Gag/Tat-Texo vaccine or the control Gag-Texo vaccine, and measured CTL responses 6 days after immunization by flow cytometry, using FITC-anti-CD8 and PE-anti-CD44 antibodies for double staining [13] [14].This approach revealed that both Gag-Texo and Gag/Tat-Texo vaccines were capable of efficient stimulation of proliferation of CTLs expressing T cell activation marker CD44 (p < 0.05) (Figure 4(c)).Next, we assessed the ability of Gag/Tat-Texo to induce the differentiation of stimulated CD8 + T-cells into effector CTLs.We adoptively transferred Gag 76-84 peptide-pulsed splenocytes that had been strongly labeled with CSFE (CFSE high ), as well as the control HER2 peptide-pulsed splenocytes that had been weakly labeled with CFSE (CFSE low ) at 1:1 ratio, into recipient mice that had been pre-vaccinated with either Gag/Tat-Texo or Gag-Texo.
The transfer was performed at day seven following the vaccinations.Sixteen hours after the cell transfer, mouse splenocytes were analyzed by flow cytometry.
We found that there was substantial loss (53% and 68%) of the CFSE high cells in the Gag/Tat-Texo-and Gag-Texo-immunized HLA-A2 mice (Figure 4

Discussion
CD8 + CTLs capable of recognizing some conserved and cross-strain epitopes [17] [28] play a critical role in controlling HIV-1 proliferation [29], acute phase of infection and disease progression even in the absence of neutralizing Abs [7] [30].Dendritic cells (DCs), the most potent antigen presenting cells (APCs) capable of stimulating efficient CTL responses have been used for vaccine development [31].DCs expressing HIV-1 Gp120 and Gag have been used as vaccines to stimulate HIV-1-specific CTL responses in animal models [32] [33].HIV-1specific DC vaccines have been also assessed in clinical trials [33] [34].However, these vaccines stimulated only very low levels of CTL responses and immunity in patients [35], warranting the search for other more efficient vaccination strategies.
Among HIV early regulatory proteins (Tat, Rev and Nef), Tat protein possesses immunomodulatory and adjuvant properties that can be advantageous in vaccine development [23].For example, the Tat protein can efficiently enter DCs via binding to RGD-binding integrin receptor and activate the proteasomes, leading to increased antigen presentation and DC maturation [36] [37], direct DCs to induce predominantly Th1 responses, favoring stimulation of CTL immunity [38] and enhance, as well as broaden CTL responses to Gag and Gp120 in DNA vector-or protein-based vaccinations [39] [40] [41] [42].
We recently demonstrated that the OVA-Texo vaccine directly converted CTL exhaustion independent of the host CD4 + T cell help via its CD40L signaling activation of the mTORC1 pathway, and the Gag-Texo vaccine stimulated Gagspecific therapeutic immunity against Gag-expressing BL6-10 Gag melanoma lung metastases in AdV OVA -induced chronic infection [43].In this study, we provide the first evidence that Tat enhances CTL responses and therapeutic immunity in T cell-based vaccines.We demonstrate that the transgene Tat-engineered OVA/ Tat-Texo vaccine not only enhances OVA-specific CTL responses, but also broadens their recognition to the cryptic OVA epitope.This is further supported by the evidence that Tat also enhances Gag-specific CTL responses derived from the Gag/Tat-Texo vaccination, suggesting that Gag/Tat-Texo-stimulate Gagspecific CTL responses may also eradicate HIV-1-infected cells if the vaccine is applied to HIV-1 patients.Importantly, the Gag/Tat-Texo vaccine further stimulates more efficient therapeutic immunity than Gag-Texo in transgenic HLA-A2 mice bearing 6 day-established HLA-A2/Gag-expressing BL6-10 Gag/A2 melanoma lung metastases.We thus, assume that our transgene Tat-engineered EXO-targeted T cell-based vaccine might more efficiently convert CTL res-ponses and stimulate Gag-specific therapeutic immunity in chronic infection.To assess our assumption, similar vaccination experiments using the OVA/Tat-Texo and Gag/Tat-Texo vaccines, are underway in our laboratory in our recently described chronic infection model [43].

Conclusion
Our novel transgene Tat-engineered Gag/Tat-Texo vaccine capable of directly stimulating efficient Gag-specific CTL responses and potent therapeutic immunity in transgenic HLA-A2 mice may be useful as a new immunotherapeutic vaccine for controlling virus in HIV-1 patients with CD4 + T cell deficiency, who are being subjected to the antiviral drug therapy.
was located between the two gene fragments OVA and Tat or Gag and Tat, into the pShuttle vector (Stratagene, La Jolla, CA) by recombinant technology.The recombinant AdV vectors were linealized by PacI digestion, and then transfected into 293 cells using Lipofectamine 2000 (Invitrogene, Carlsbad, CA) ConA-activated CD8 + T (ConA-T) cells were enriched by passage through nylon wool columns (C & A Scientific, Manassas, VA), and then purified by negative selection using anti-mouse CD8 paramagnetic beads (DYNAL Inc., Lake Success, NY).OVA-Texo or OVA/Tat-Texo and Gag-Texo or Gag/Tat-Texo vaccines were generated by incubation of ConA (1 µg/ml)-stimulated polyclonal A. Z. Xu et al.
cubated with either high (3.0 µM, CFSE high ) or low (0.6 µM, CFSE low ) concentrations of carboxy-fluorescein succinimidyl ester (CFSE), to generate differentially labeled target cells.The CFSE high cells were pulsed with OVA 257-264 or Gag 76-84 peptide, whereas the CFSE low cells were pulsed with irrelevant Mut1 or HER2 peptide, and served as internal controls.These peptide-pulsed target cells (10 × 10 6 cells/mouse) were i.v.co-injected at 1:1 ratio into OVA/Tat-Texo-immunized B6 or Gag/Tat-Texo-immunized HLA-A2 mice six days after the immunization.Sixteen hours later, the residual OVA-specific or Gag-specific CFSE high and irrelevant control CFSE low target cells remaining in the recipients' spleens were analyzed by flow cytometry.

6 MiceFigure 2 .
Figure 2. Phenotypic analysis of DCs and EXOs.(a) DC OVA/Tat cells were stained with a panel of antibodies (Abs) (solid lines) or isotype-matched irrelevant Abs (dotted lines), and analyzed by flow cytometry.(b) Western blot analysis using lysates of DC OVA/Tat and DCs and the anti-Tat Ab.(c) Electron micrograph of EXO OVA/Tat .Scale bar: 100 nm.(d) Western blot analysis of EXO OVA/Tat using anti-CD9 Ab.One representative experiment of two is shown.

Figure 3 .
Figure 3.The OVA/Tat-Texo vaccine broadens and enhances OVAspecific CTL responses.(a) B6 mice were i.v.immunized with OVA-Texo, OVA/Tat-Texo or PBS.Six days after the immunization, tail blood samples of the immunized mice were stained with PE-tetramer I, PE-tetramer II or PE-tetramer III, and FITC-anti-CD8 Ab, and analyzed by flow cytometry.The value in each panel represents the percentage of CD8 + Tetramer + T cells in the total CD8 + T cell population analyzed.(b)In vivo cytotoxicity assay.Six days after immunization, the immunized mice were i.v.injected with a mixture of CFSE high and CFSE low -labeled splenocytes (at 1:1 ratio) that had been pulsed with OVAI and irrelevant Mut1 peptide, respectively.After 16 hrs, the spleens of immunized micewere removed, and the percentages of the residual CFSE high (H) and CFSE low (L) target cells remaining in the recipients' spleens were analyzed by flow cytometry.The value in each panel represents the percentage of CFSE high vs.CFSE low target cells remaining in the spleen.The value in parenthesis represents the standard deviation (SD).*P < 0.05 versus cohorts of OVA-Texo.One representative experiment of two is shown.
face DC marker CD11c, HLA-A2 and DC maturation markers Ia b and CD80 by flow cytometry (Figure 4(a)), indicating that DC Gag/Tat are matured immunogenic DCs.DC Gag expressed a similar pattern of these molecules (data not shown).In addition, according to Western blotting analysis, DC Gag/Tat also expressed cytoplasmic Gag and Tat (Figure 4(b)).EXOs from culture supernatants of DC Gag/Tat were purified by differential ultracentrifugation, and used to generate Gag-Texo and Gag/Tat-Texo vaccines based on ConA-stimulated HLA-A2 polyclonal CD8 + T (ConA-T) cells armed with DC Gag -or DC Gag/Tat -released EXOs, respectively, by incubating T cells with EXOs for 2 hours.

Figure 4 .
Figure 4.The Gag/Tat-Texo vaccine enhances Gag-specific CTL responses and therapeutic immunity in transgenic HLA-A2 mice.(a) Phenotypic analyses of DC Gag/Tat .DC Gag/Tat were stained with a panel of Abs (solid lines) or isotype-matched irrelevant Abs (dotted lines), and analyzed by flow cytometry.(b) Western blot analysis using lysates of DCGag/Tat and DCs and the anti-Gag and anti-Tat Abs.(c) HLA-A2 mice were i.v.immunized with Gag-Texo, Gag/Tat-Texo or PBS.Six days after the immunization, tail blood samples of the immunized mice were stained with PE-anti-CD44 and FITC-anti-CD8 Ab, and analyzed by flow cytometry.The value in each panel represents the percentage of CD8 + CD44 + T cells in the total CD8 + T cells analyzed.(d) In vivo cytotoxicity assay.Six days after immunization, the immunized mice were i.v.injected with a mixture of CFSE high and CFSE low -labeled splenocytes (at 1:1 ratio) that had been pulsed with Gag 76-84 or irrelevant Mut1 peptide, respectively.After 16 hrs, the spleens of immunized mice were removed, and the percentages of the residual CFSE high (H) and CFSE low (L) target cells remaining in the recipients' spleens were analyzed by flow cytometry.The value in each panel represents the percentage of CFSE high vs.CFSE low target cells remaining in the spleen.(e) To assess therapeutic immunity, HLA-A2 mice were i.v.injected with BL6-10Gag/A2 melanoma cells.Six days after tumor challenge, mice were vaccinated with Gag-Texo, Gag/Tat-Texo or PBS.The mice were sacrificed 3 weeks after tumor cell challenge.The average number metastatic tumor colonies were counted in lungs.The value in parenthesis represents the standard deviation (SD).**P < 0.01 versus cohorts of Gag-Texo.One representative experiment of two is shown.