Protective CD8+ T-cell responses to cytomegalovirus driven by rAAV/GFP/IE1 loading of dendritic cells
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Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens.
We used the rAAV system to induce specific CTLs against CVM antigens for the development of cytomegalovirus HCMV) gene therapy. As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV. Our rAAV vector induced a strong stimulation of CTLs directed against the HCMV antigen IE1. We then investigated the efficiency of the CTLs in killing IE1 targeted cells.
A significant MHC Class I-restricted, anti-IE1-specific CTL killing was demonstrated against IE1 positive peripheral blood mononuclear cells (PBMC) after one, in vitro, stimulation.
In summary, single PBMC stimulation with rAAV/IE1 pulsed DCs induces strong antigen specific-CTL generation. CTLs were capable to lyse low doses of peptides pulsed into target cells. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens.
KeywordsrAAV Vector 51Cr Release Assay Adherent Monocyte HCMV Disease Peripheral Blood Mononuclear Cell Stimulation
Over the past few years, overwhelming evidence has come to light that inflammation hidden deep in the body is a common source of heart attacks, even when clogging of the arteries by plaque is minimal . A leading cause is infection by various microbes, in particular, the human cytomegalovirus (HCMV), which historically has been linked to heart/arterial disease [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15]. Existing drugs for the treatment or prevention of HCMV disease are only partially effective, have a variety of side effects, and may fail because of drug resistant mutations [12, 16, 17]. An effective HCMV gene therapy would provide a great medical benefit and would also result in annual savings in the cost of caring for persons with HCMV disease. Although immunotherapeutic interventions are promising, current treatments to induce strong immune responses against HCMV are still inadequate. In order to develop a new method to induce strong immune responses against HCMV, we used the rAAV-based antigen loading of DCs to generate significant and specific CTL responses against HCMV antigens. Several HCMV proteins have been shown to serve as target antigens for the class I-restricted CD8+ T cell responses against HCMV, including the major immediate-early protein (IE) , glycoprotein B (gB) , and non-envelope structural virion proteins, such as the matrix protein pp65 . Among the IE proteins, two nuclear regulatory phosphoproteins, IE1 and IE2, are the first and most abundantly expressed proteins and are synthesized by differential splicing from the same complex overlapping transcription unit within the major IE (MIE) locus . Early analyses of the CTL response in seropositive individuals have suggested that the 72-kDa immediate-early protein IE1 was a dominant target for CD8+ CTLs . IE1 is the major protein produced in the immediate-early phase of the human HCMV replication cycle and has been shown to be target for CD4+ and CD8+ T cells . IE1 was the first gene product identified to elicit CTL responses in mice . The role of IE1-recognizing CD8+ T cells will be an interesting subject to study. DCs are professional antigen presenting cells that are critical to prime a cellular immune response [12, 23, 24, 25]. There is evidence of several protocols for loading DCs, based on the use of tumor antigens such as peptides, lysed tumors, whole proteins, and genes expressed on plasmids or viral vectors [26, 27]. These new technologies permit in vitro manipulation of DCs for clinical studies [12, 28, 29].
Recent studies demonstrate that recombinant rAAV-based antigen loading of DCs generates significant and rapid CTL responses in vitro [12, 19, 30]. rAAV has been widely studied in applications to transduce DCs. rAAV lacks viral coding sequences, therefore the transduced DCs only express antigen proteins and not viral proteins . Further, rAAV does not elicit an immune response in its host, therefore there is no secondary inflammation in the host due to rAAV .
In the present study, IE1 genes were cloned into AAV to test the ability of r-AAV loading of DCs to generate specific CTL responses against IE1 positive cells.
Cell culture and patients material
The HEK293 cells were maintained and propagated in complete DMEM supplemented with penicillin and streptomycin (Mediatech Inc., Herndon, VA) and 10% FBS (Gemini Bio-Products, West Sacramento, CA). Autologous peripheral blood mononuclear cells (PBMCs) and were obtained from 3 female HLA-A2 restricted healthy donors. All of the clinical materials were obtained with the patient's consent and approval by the local ethics committee.
Constructing the AAV/IE1 genome and generation of virus stocks
The AAV/IE1 genome was constructed as a plasmid as previously described [28, 30]. Briefly, the IE1 gene was amplified by PCR from plasmid pCGN-IE1, which was kindly provided by Dr. Thomas Shenk at the Department of Molecular Biology, Princeton University. PCR amplification for IE1 was carried out using the following primer pair: upstream, 5'-GGTACCATGGAGTCCTCTGCCAAGA-3'; downstream, 5'-CTCGAGGACCTTGTACTCATTACACATTG-3'. AAV/IE1 virus stocks were generated using complementary plasmids ins96-0.8 or pSH3, using HEK293 cells as described previously [28, 30, 32]. Lysates of HEK293 cells were used as virus-negative controls for mock infections.
HEK293 cells were spun in a cytospin column (5 × 104 cells/slide), fixed with SlideRite (Fisher, USA), and air dried overnight. Each sample was permeabilized (P) in PBS 1×/0.1% Triton X-100 for 15 minutes at 4°C not permeabilized (NP). Results were analyzed using an Olympus IX71 inverted microscope equipped with a Fluoview 300 confocal laser system.
Real-time PCR for virus stock titration
The titer of virus stocks was determined by real-time PCR as previously described . Briefly, we used the plasmid AAV/IE1 for the real-time PCR standards, respectively. Concentration was measured by absorbance at 260 nm.
Generation and infection of monocyte-derived DCs
Autologous DCs (2 × 105 adherent monocytes) were generated and infected (0.5 mL virus [109 eg/mL]) as previously described [28, 30]. Recombinant granulocyte macrophage-colony-stimulating factor (GM-CSF) (R&D Systems, Minneapolis, MN, USA), at a final concentration of 800 IU/mL, was included in the medium throughout the culture. To induce monocytes into DCs, human interleukin-4 (IL-4) (R&D Systems, Minneapolis, MN, USA) at 1000 IU/mL was added on day 3, after infection.
Generation of autologous 1E1-positive target cells
Non-adherent PBMCs, isolated from healthy donors, were infected with AAV/IE1 virus at a multiplicity of infection of 100, 4 days before the 51Cr release assay.
Lipofection using DOTAP
The recombinant IE1 protein was made as previous described . Lipofection was performed using the cationic liposome-mediated transfection reagent, DOTAP (Roche Diagnostics, Indianapolis, IN). IE1 protein was mixed with the DOTAP reagent and serum-free media at ratios following the manufacturer's recommendations. The cells were then incubated in serum-free media containing the lipofection mix for 4–6 hours. Final IE1 concentration was 100 nM for the DCs and PBMCs. After 4–6 hours of incubation, serum-supplemented DMEM was added to cells. After 24 hours, all of the lipofection media was replaced with fresh growth media for cells.
Generation and testing of 1E1-specific CTLs
CTL were generated from 3 normal donors (HLA matched). Experiments were performed in quadruplicate (experiments were preformed 4 times independently with different ratios of responders to DCs from 5:1; 10:1; 20:1; 40:1 data not provided) [23, 24]. For each experiment, the non-adherent PBMCs were washed and re-suspended in AIM-V at 10 to 20 × 106 cells per well in 6-well culture plates with AAV/IE1-loaded autologous DCs (optimal ratios of responders to DCs from 20:1). The cultures were supplemented with GM-CSF (800 U/mL) and recombinant human IL-2 (10 U/mL). After 7 days of co-culture, the cells were used for cytotoxicity assays in a 6-hour 51Cr assay, as previously described [16, 23, 24]. To determine the CTLs' HLA restriction, HLA-class I (W6/32) of antibodies, at a concentration of 25 μg/mL, were pre-incubated with the target cells for 30 minutes before addition of the stimulated T-cells. K562 cells were used as targets to observe natural killer (NK) cell activity. In all of these CTL killing assays, spontaneous release of chromium never exceeded 25% of the maximum release [23, 24].
Flow cytometry analysis
This protocol was adapted from that described by Pala et al. and modified [24, 28]. Cell surface marker analysis of T cells and DCs was conducted using fluorescence-activated cell scanning (FACS) (FACScan; BD Biosciences-PharMingen, Franklin Lakes, NJ), as described previously [24, 28].
All results are expressed as mean ± SD. Data were analyzed using nonparametric analysis of variance (ANOVA). Differences were considered significant if P < 0.05.
Construction of AAV/IE1 Recombinant Viruses
Titration of AAV/IE1 virus stocks using real-time PCR assays
AAV/IE1-transduced DCs express 1E1
AAV/IE1-transduced DCs stimulated AAV/IE1-specific CTLs
To achieve effective antivirus responses, recent emphasis has been placed on approaches that stimulate strong cellular immune responses, which are mediated by T-cells and particularly by CTLs. CTLs are believed to be the critical immune effector arm in mediating potential antivirus immunity. CD8+ CTLs play a major role in protection against HCMV and in maintenance of its latency [35, 36, 37, 38]. It has been hypothesized that antigen gene delivery into DCs [23, 24] may be more efficient for generating CTLs than by antigen delivery as a lipofected, exogenous protein [23, 24, 28]. Although there is some controversy as to AAV effectiveness in transducing DCs and other hematopoietic cells, donor monocytes/DCs have been shown to be successfully transduced with AAV-2 [23, 24, 28, 30]. Furthermore, in various studies, AAV has been shown to be an effective gene-delivery system for immortalized tissue-cultured cells and primary hematopoietic cells [34, 39, 40, 41]. The AAV vectors were found to transduce up to 85% of DCs [12, 19, 23, 24]. The transduced DCs displayed higher levels of CD80, CD83, CD86, and CD1a over controls. In fact, the DC-loading technique was found to be highly effective in generating significant CTLs with only one DC-T-cell co-incubation and in a time frame of only 1 week. We confirm that rAAV-infected monocytes with GM-CSF only and then adding IL-4 after 3 days induces DCs' differentiation [23, 24]. Previous studies showed that rAAV-loading DCs can rapidly generate antigen-specific CTLs against viral antigens . The IE1 protein has been proposed as a target for immunotherapy. The IE genes are the first ones to be expressed in the replicative cycle, and their expression does not depend on prior viral protein synthesis. Together with some virion proteins, the IE products activate viral genes and alter the infected cell to generate an appropriate milieu that favors viral replication . Human cytomegalovirus (HCMV) IE1, the most abundant IE product, plays an accessory role in the IE2-mediated activation of HCMV early and late genes [43, 44]. Interaction of HCMV IE1 with a number of cellular regulatory proteins has also been described previously . In addition to their regulatory activities, HCMV IE1 is involved in perturbing a variety of other cellular processes, including cell cycle regulation [46, 47], apoptosis , and cell architecture. The IE1 protein of HCMV is a major source of CD8 T-cell epitopes for HLA molecules represented in a large proportion of the human population, and plays a significant role in the control of HCMV disease . The previous study led to the identification of several new classes of I MHC-restricted CTL epitopes against IE1 antigens . This result was confirmed by another study in which several IE1 HLA class I epitopes were detected and no IE1 class II epitopes were identified .
Here we have demonstrated that rAAV-loading of DCs with IE1 can generate antigen-specific CTLs in substantial numbers, only 1 week after stimulation. Based on this and our previous studies, we hypothesize that the AAV vector causes a fundamental change in DC performance, perhaps by modifying their co-stimulatory ligand expression, resulting in more efficient generation of antigen-specific CTLs . We hypothesized that the AAV/IE1 would be superior to IE1 protein in stimulating CTL killing. Our experiments show that AAV/IE1 was much more efficient in stimulating the killing of target cells than IE1 protein (P < 0.05). Our controls (Figures 5, 6, 7) show strong antigen specificity and MHC class I restriction. For example, Figure 5 shows that autologous PBMCs were not targeted for killing unless these target were preloaded with the antigen. Without loading the antigen, there is no significant killing. Furthermore, K562 cells are shown in Figures 4, 5, 6 to be insignificant targets.
This same report  suggested that IE1 is directly related to CTL killing and the importance of MHC class I molecules as a restriction element in HCMV. Our results prove a direct link between the IE1 protein and CTL recognition. We believe it is likely that there are multiple reasons why AAV loading of DCs is effective. One reason is the high transduction frequency we have observed. A second reason could be the increased expression of CD80, CD86, and CD40 that may also contribute to generating the robust CTL response.
In summary, our results demonstrate that the delivery of IE1 antigen by an AAV vector is a good strategy for generating anti-IE1 CTLs. Our data suggest that AAV-based antigen loading of DCs is highly effective for generating a CTL response against HCMV.
This project was supported by the Institutional Research Program of the Texas Tech University Health Sciences Center, the Southwest Cancer Treatment and Research Center Program, the Laura W. Bush Institute for Women's Health and Center for Women's Health and Gender-Based Medicine, Texas Tech University Health Sciences Center. Cardiovascular TTUHSC Seed Grant.
The authors thank Teri Fields for her assistance in editing this manuscript. W. Martin Kast holds the Walter A. Richter Cancer Research Chair.
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