Changing clinical scenario in Chandipura virus infection.

Chandipura virus (CHPV) (Vesiculovirus: Rhabdoviridae) garnered global attention as an emerging neurotropic pathogen inflicting high mortality in children within 24 h of commencement of symptoms. The 2003-2004 outbreaks in Central India witnessed case fatality rates ranging from 56-75 per cent in Andhra Pradesh and Gujarat with typical encephalitic symptoms. Due to the acute sickness and rapid deterioration, the precise mechanism of action of the virus is still unknown. Recent studies have shown increased expression of CHPV phosphoprotein upto 6 h post infection (PI) demonstrating CHPV replication in neuronal cells and the rapid destruction of the cells by apoptosis shed light on the probable mechanism of rapid death in children. Phlebotomine sandflies are implicated as vectors due to their predominance in endemic areas, repeated virus isolations and their ability to transmit the virus by transovarial and venereal routes. Significant contributions have been made in the development of diagnostics and prophylactics, vaccines and antivirals. Two candidate vaccines, viz. a recombinant vaccine and a killed vaccine and siRNAs targeting P and M proteins have been developed and are awaiting clinical trials. Rhabdomyosarcoma and Phlebotomus papatasi cell lines as well as embryonated chicken eggs have been found useful in virus isolation and propagation. Despite these advancements, CHPV has been a major concern in Central India and warrants immediate attention from virologists, neurologists, paediatricians and the government for containing the virus.

Immune responses in mice vaccinated with virus-like particles composed of the GP5 and M proteins of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces reproductive failure in sows and respiratory problems in pigs of all ages. Live attenuated and inactivated vaccines are used on swine farms to control PRRSV. However, their protective efficacy against field strains of PRRSV remains questionable. New vaccines have been developed to improve the efficacy of these traditional vaccines. In this study, virus-like particles (VLPs) composed of the GP5 and M proteins of PRRSV were developed, and the capacity of the VLPs to elicit antigen-specific immunity was evaluated. Serum antibody titers and production of cytokines were measured in BALB/C mice immunized intramuscularly three times with different doses (0.5, 1.0, 2.0, and 4.0 µg) of the VLP vaccine. A commercial vaccine consisting of inactivated PRRSV and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. IgG titers to GP5 were significantly higher in all groups of mice vaccinated with the VLPs than in control mice. Neutralizing antibodies were only detected in mice vaccinated with 2.0 and 4.0 µg of the VLPs. Cytokine levels were determined in cell culture supernatants after in vitro stimulation of splenocytes with the VLPs for 3 days. Mice immunized with 4.0 µg of the VLPs produced a significantly higher amount of interferon-gamma (IFN-γ) than mice immunized with the commercial inactivated PRRSV vaccine and PBS. In contrast, immunization with the commercial vaccine induced higher production of IL-4 and IL-10 in mice than mice vaccinated with VLPs. These data together demonstrate the capacity of VLPs to induce both neutralizing antibodies and IFN-γ in immunized mice. The VLP vaccine developed in this study could serve as a platform for the generation of improved VLP vaccines to control PRRSV.

Universal vaccine based on ectodomain of matrix protein 2 of influenza A: Fc receptors and alveolar macrophages mediate protection.

The ectodomain of matrix protein 2 (M2e) of influenza A virus is an attractive target for a universal influenza A vaccine: the M2e sequence is highly conserved across influenza virus subtypes, and induced humoral anti-M2e immunity protects against a lethal influenza virus challenge in animal models. Clinical phase I studies with M2e vaccine candidates have been completed. However, the in vivo mechanism of immune protection induced by M2e-carrier vaccination is unclear. Using passive immunization experiments in wild-type, FcRγ(-/-), FcγRI(-/-), FcγRIII(-/-), and (FcγRI, FcγRIII)(-/-) mice, we report in this study that Fc receptors are essential for anti-M2e IgG-mediated immune protection. M2e-specific IgG1 isotype Abs are shown to require functional FcγRIII for in vivo immune protection but other anti-M2e IgG isotypes can rescue FcγRIII(-/-) mice from a lethal challenge. Using a conditional cell depletion protocol, we also demonstrate that alveolar macrophages (AM) play a crucial role in humoral M2e-specific immune protection. Additionally, we show that adoptive transfer of wild-type AM into (FcγRI, FcγRIII)(-/-) mice restores protection by passively transferred anti-M2e IgG. We conclude that AM and Fc receptor-dependent elimination of influenza A virus-infected cells are essential for protection by anti-M2e IgG.

A lipopeptide based on the M2 and HA proteins of influenza A viruses induces protective antibody.

A conserved 15 amino-acid residue sequence of the ectodomain of the M2 protein of influenza A virus (M2e) induces a strong antibody (Ab) response when incorporated into a synthetic lipopeptide vaccine candidate containing a T-helper epitope from influenza A hemagglutinin and the dendritic cell-targeting lipid moiety S-[2,3-bis(palmitoyloxy)propyl]cysteine (Pam2Cys). Abs elicited by the truncated M2e sequence were specific for the M2 protein of influenza A virus and were also capable of binding to cells that were infected with influenza A viruses of different subtypes. The Ab titres against the lipopeptide were similar in magnitude to those elicited by the full-length (23 residue) M2e peptide when administered in Freund's adjuvant. Abs to the truncated M2e sequence were also able to significantly reduce the viral load in airways of BALB/c mice after challenge with live influenza virus.

[Research progress of anti-influenza virus agents].

Influenza is a major threat to millions of people worldwide. Vaccines and antiviral agents are two main options available to reduce the impact of the influenza virus, while anti-influenza agents are the most effective means to prevent the transmission of the highly contagious virus and to treat the epidemics of disease. At present, four anti-influenza agents have been approved by the FDA for the treatment of influenza, including two M2 protein ion channel inhibitors-amantadine and rimantadine and two neuraminidase inhibitors-zanamivir and oseltamivir. Arbidol hydrochloride, launched in Russia, is a potent inhibitor of influenza virus, too. Neuraminidase inhibitors could be classified generally by structure into six different kinds: sialic acid derivatives, benzoic acid derivatives, cyclohexene derivatives, cyclopentane derivatives, pyrrolidine derivatives and natural products. In this paper, recent progress in the research of the action mechanisms and structure-activity relationships of these anti-influenza virus agents were reviewed.

LMP2-DC Vaccine Elicits Specific EBV-LMP2 Response to Effectively Improve Immunotherapy in Patients with Nasopharyngeal Cancer.

OBJECTIVE: To evaluate the safety and effectiveness of a vaccine based on latent membrane protein 2 (LMP2) modified dendritic cells (DCs) that boosts specific responses of cytotoxic T lymphocytes (CTLs) to LMP2 before and after intradermal injection in patients with nasopharyngeal carcinoma (NPC). METHODS: DCs were derived from peripheral blood monocytes of patients with NPC. We prepared LMP2-DCs infected by recombinant adenovirus vector expressing LMP2 (rAd-LMP2). NPC patients were immunized with 2 × 10 5 LMP2-DCs by intradermal injection at week 0 and after the second and fourth weeks. Specific responses to LMP2 were detected by enzyme-linked immunospot (ELISPOT) assay at week 0 and at the fifth and eighth weeks. Local clinicians performed the follow-up and tracking of patients. RESULTS: We demonstrated that DCs derived from monocytes displayed typical DC morphologies; the expression of LMP2 in the LMP2-DCs vaccine was confirmed by immunocytochemical assay. Twenty-nine patients with NPC were enrolled in this clinical trial. The LMP2-DCs vaccine was well tolerated in all of the patients. Boosted responses to LMP2 peptide sub-pools were observed in 18 of the 29 patients with NPC. The follow-up data of 29 immunized patients from April, 2010 to April 2015 indicated a five-year survival rate of 94.4% in responders and 45.5% in non-responders. CONCLUSION: In this pilot study, we demonstrated that the LMP2-DCs vaccine is safe and effective in patients with NPC. Specific CTLs responses to LMP2 play a certain role in controlling and preventing the recurrence and metastasis of NPC, which warrants further clinical testing.

A promising cancer gene therapy agent based on the matrix protein of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) plays a key role in inducing cell apoptosis during infection. To investigate whether M protein-mediated apoptosis could be used in cancer therapy, its cDNA was amplified and cloned into eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid or the control empty plasmid pcDNA3.1(+) was mixed with cationic liposome and introduced into various tumor cell lines in vitro, including lung cancer cell LLC, A549, colon cancer cell CT26 and fibrosarcoma cell MethA. Our data showed that the M protein induced remarkable apoptosis of cancer cells in vitro compared with controls. Fifty micrograms of plasmid in a complex with 250 microg cationic liposome was injected intratumorally into mice bearing LLC or MethA tumor model every 3 days for 6 times. It was found that the tumors treated with M protein plasmid grew much more slowly, and the survival of the mice was significantly prolonged compared with the mice treated with the control plasmid. In MethA fibrosarcoma, the tumors treated with M protein plasmid were even completely regressed, and the mice acquired longtime protection against the same tumor cell in rechallenge experiments. Both apoptotic cells and CD8(+) T cells were widely distributed in M protein plasmid-treated tumor tissue. Activated cytotoxic T lymphocytes (CTLs) were further detected by means of (51)Cr release assay in the spleen of the treated mice. These results showed that M protein of VSV can act as both apoptosis inducer and immune response initiator, which may account for its extraordinary antitumor effect and warrant its further development in cancer gene therapy.

Vesicular stomatitis virus matrix protein gene enhances the antitumor effects of radiation via induction of apoptosis.

Vesicular stomatitis virus (VSV) matrix (M) protein can directly induce apoptosis by inhibiting host gene expression when it is expressed in the absence of other viral components. Previously, we found that the M protein gene complexed to DOTAP-cholesterol liposome (Lip-MP) can suppress malignant tumor growth in vitro and in vivo; however, little is known regarding the biological effect of Lip-MP combined with radiation. The present study was designed to determine whether Lip-MP could enhance the antitumor activity of radiation. LLC cells treated with a combination of Lip-MP and radiation displayed apparently increased apoptosis compared with those treated with Lip-MP or radiation alone. Mice bearing LLC or Meth A tumors were treated with intratumoral or intravenous injections of Lip-MP and radiation. The combined treatment significantly reduced mean tumor volumes compared with either treatment alone in both tumor models and prolonged the survival time in Meth A tumor models and the intravenous injection group of LLC tumor models. Moreover, the antitumor effects of Lip-MP combined with radiation were greater than their additive effects when compared with the expected effects of the combined treatment in vivo. This study suggests that Lip-MP enhanced the antitumor activity of radiation by increasing the induction of apoptosis.

Long-term Survival in Glioblastoma with Cytomegalovirus pp65-Targeted Vaccination.

Purpose: Patients with glioblastoma have less than 15-month median survival despite surgical resection, high-dose radiation, and chemotherapy with temozolomide. We previously demonstrated that targeting cytomegalovirus pp65 using dendritic cells (DC) can extend survival and, in a separate study, that dose-intensified temozolomide (DI-TMZ) and adjuvant granulocyte macrophage colony-stimulating factor (GM-CSF) potentiate tumor-specific immune responses in patients with glioblastoma. Here, we evaluated pp65-specific cellular responses following DI-TMZ with pp65-DCs and determined the effects on long-term progression-free survival (PFS) and overall survival (OS).Experimental Design: Following standard-of-care, 11 patients with newly diagnosed glioblastoma received DI-TMZ (100 mg/m2/d × 21 days per cycle) with at least three vaccines of pp65 lysosome-associated membrane glycoprotein mRNA-pulsed DCs admixed with GM-CSF on day 23 ± 1 of each cycle. Thereafter, monthly DI-TMZ cycles and pp65-DCs were continued if patients had not progressed.Results: Following DI-TMZ cycle 1 and three doses of pp65-DCs, pp65 cellular responses significantly increased. After DI-TMZ, both the proportion and proliferation of regulatory T cells (Tregs) increased and remained elevated with serial DI-TMZ cycles. Median PFS and OS were 25.3 months [95% confidence interval (CI), 11.0-∞] and 41.1 months (95% CI, 21.6-∞), exceeding survival using recursive partitioning analysis and matched historical controls. Four patients remained progression-free at 59 to 64 months from diagnosis. No known prognostic factors [age, Karnofsky performance status (KPS), IDH-1/2 mutation, and MGMT promoter methylation] predicted more favorable outcomes for the patients in this cohort.Conclusions: Despite increased Treg proportions following DI-TMZ, patients receiving pp65-DCs showed long-term PFS and OS, confirming prior studies targeting cytomegalovirus in glioblastoma. Clin Cancer Res; 23(8); 1898-909. ©2017 AACR.

LMP2-DC Vaccine Elicits Specific EBV-LMP2 Response to Effectively Improve Immunotherapy in Patients with Nasopharyngeal Cancer / 生物医学与环境科学（英文）

Objective@#To evaluate the safety and effectiveness of a vaccine based on latent membrane protein 2 (LMP2) modified dendritic cells (DCs) that boosts specific responses of cytotoxic T lymphocytes (CTLs) to LMP2 before and after intradermal injection in patients with nasopharyngeal carcinoma (NPC).@*Methods@#DCs were derived from peripheral blood monocytes of patients with NPC. We prepared LMP2-DCs infected by recombinant adenovirus vector expressing LMP2 (rAd-LMP2). NPC patients were immunized with 2 × 10 @*Results@#We demonstrated that DCs derived from monocytes displayed typical DC morphologies; the expression of LMP2 in the LMP2-DCs vaccine was confirmed by immunocytochemical assay. Twenty-nine patients with NPC were enrolled in this clinical trial. The LMP2-DCs vaccine was well tolerated in all of the patients. Boosted responses to LMP2 peptide sub-pools were observed in 18 of the 29 patients with NPC. The follow-up data of 29 immunized patients from April, 2010 to April 2015 indicated a five-year survival rate of 94.4% in responders and 45.5% in non-responders.@*Conclusion@#In this pilot study, we demonstrated that the LMP2-DCs vaccine is safe and effective in patients with NPC. Specific CTLs responses to LMP2 play a certain role in controlling and preventing the recurrence and metastasis of NPC, which warrants further clinical testing.

Disease-driven T cell activation predicts immune responses to vaccination against melanoma.

Tumor vaccines may induce activation and expansion of specific CD8 T cells which can subsequently destroy tumor cells in cancer patients. This phenomenon can be observed in approximately 5-20% of vaccinated melanoma patients. We searched for factors associated with T cell responsiveness to peptide vaccines. Peptide antigen-specific T cells were quantified and characterized ex vivo before and after vaccination. T cell responses occurred primarily in patients with T cells that were already pre-activated before vaccination. Thus, peptide vaccines can efficiently boost CD8 T cells that are pre-activated by endogenous tumor antigen. Our results identify a new state of T cell responsiveness and help to explain and predict tumor vaccine efficacy.

Two phase I studies of low dose recombinant human IL-12 with Melan-A and influenza peptides in subjects with advanced malignant melanoma.

Preclinical studies have shown that low dose IL-12 can potentiate cytotoxic lymphocyte responses. Since previous trials have demonstrated significant toxicity from high dose recombinant human IL-12 (rhIL-12), we sought to determine an optimal biological dose for rhIL-12 at lower doses when combined with peptide antigens. Two studies were undertaken. The rhIL-12 was administered at doses of 0 (placebo), 10, 30 and 100 ng/kg, subcutaneously in one study and intravenously in the other. Apart from IL-12 dosing, the studies were identical. Subjects had evaluable stage III or IV melanoma which expressed Melan-A by RT-PCR or immunohistochemistry. Melan-A (26-35) (EAAGIGILTV) and influenza matrix (58-66) (GILGFVFTL) peptides were administered intradermally on weeks 1, 2, 3, 4 and 9. Twenty-eight subjects were enrolled, of whom 24 were evaluable for clinical and immunological responses. Therapy was well tolerated, the main adverse event being influenza-like symptoms. Immunological monitoring included the evaluation of cutaneous reactions and assays for antigen-specific T-cells. Clinical responses included a complete response in a subject with small volume subcutaneous disease, a partial response in a subject with hepatic metastases, and mixed responses in pulmonary, pleural and nodal disease. Biopsies of accessible tumors showed infiltration with CD4+ and CD8+ lymphocytes capable of lysing Melan-A peptide-pulsed targets in vitro. No clear dose-dependent effect of rhIL-12 could be determined. The rhIL-12 given either s.c. or i.v. was well tolerated at doses of 10-100 ng/kg. Clinical and immunological activity has been observed in this study where peptides were administered either with or without low dose rhIL-12.

Cytomegalovirus pp65 antigen-guided preemptive therapy with ganciclovir in solid organ transplant recipients: a prospective, double-blind, placebo-controlled study.

BACKGROUND: The aim of this study was to evaluate pp65 antigen-guided antiviral therapy in preventing human cytomegalovirus (HCMV) infection in solid organ transplant recipients. METHODS: Ten kidney and two liver transplant recipients with asymptomatic HCMV infection were randomized either for i.v. ganciclovir or placebo treatment in a prospective, double-blind study. All patients were positive by HCMV pp65 antigen test at levels >5 positive cells/2 x 10(5) investigated cells. RESULTS: No cases of HCMV end-organ disease occurred. In contrast to patients on placebo (5/7), none of the patients on ganciclovir (0/5) developed HCMV-associated symptoms (P=0.01). However, because of the small number of patients, all three high-risk patients (donor seropositive, recipient seronegative) were randomized to placebo and all three developed symptoms. CONCLUSIONS: Preemptive antiviral therapy guided by the pp65 antigen test seems to have a beneficial effect on preventing HCMV-associated symptoms in kidney and liver transplant recipients.

Immunomonitoring measures in relapsing-remitting multiple sclerosis.

Forty-five patients with relapsing-remitting multiple sclerosis (MS) were examined to determine intracellular cytokine profiles and the surface phenotype of circulating lymphocytes during active, recovery, and stable stages. Active stage patients were characterized by decreases in CD4(+)IL-4(+) Th2 as well as CD4(+)IFN-gamma(+) Th1 cells, when compared with stable stage patients and 16 healthy controls. CCR4(+) Th2 cells were persistently decreased at every MS stage as compared to the controls. CD4(+)CD29(+) and CD4(+)CXCR3(+) cells were closely correlated with IFN-gamma-producing cells. These findings suggest that simultaneous flow cytometry for these two types of measurements can provide information concerning current immune status in MS.

EBV LMP1, a viral mimic of CD40, activates dendritic cells and functions as a molecular adjuvant when incorporated into an HIV vaccine.

HIV-1 does not significantly activate cellular immunity, which has made it difficult to use attenuated forms of HIV-1 as a vaccine. In contrast, EBV induces robust T cell responses in most infected individuals, perhaps as this virus contains LMP1, a viral mimic of CD40, which is a key activating molecule for DCs and macrophages. Consequently, studies were conducted using LMP1 and LMP1-CD40, a related construct formed by replacing the intracellular signaling domain of LMP1 with that of CD40. Upon electroporation into DCs, LMP1 and LMP1-CD40 mRNAs were sufficient to up-regulate costimulatory molecules and proinflammatory cytokines, indicating that these molecules can function in isolation as adjuvant-like molecules. As a first step toward an improved HIV vaccine, LMP1 and LMP1-CD40 were introduced into a HIV-1 construct to produce virions encoding these proteins. Transduction of DCs and macrophages with these viruses induced morphological changes and up-regulated costimulatory molecules and cytokine production by these cells. HIV-LMP1 enhanced the antigen-presenting function of DCs, as measured in an in vitro immunization assay. Taken together, these data show that LMP1 and LMP1-CD40 are portable gene cassettes with strong adjuvant properties that can be introduced into viruses such as HIV, which by themselves, are insufficient to induce protective cellular immunity.

Ex vivo expansion and prophylactic infusion of CMV-pp65 peptide-specific cytotoxic T-lymphocytes following allogeneic hematopoietic stem cell transplantation.

Cytomegalovirus reactivation and infection post-allogeneic hematopoietic stem cell transplant continue to cause morbidity and mortality. Current pharmacologic therapies are limited by side effects. Adoptive transfer of ex vivo generated cytomegalovirus-specific T cells has the potential to restore immunity, prevent cytomegalovirus, and circumvent the need for pharmacologic therapies. We have generated donor-derived cytomegalovirus-specific cytotoxic T cells using dendritic cells pulsed with the HLA-A2 restricted nonapeptide NLVPMVATV (NLV) derived from the cytomegalovirus-pp65 protein. These cytotoxic T cells have been given prophylactically to 9 recipients aged 4 to 65 years on or after day 28 post-allogeneic hematopoietic stem cell transplant. Only 2 of 9 recipients received T cell depletion in vivo or in vitro. There were no immediate adverse reactions to the infusions. During 97-798 days of follow-up, 2 recipients developed cytomegalovirus reactivation; neither developed cytomegalovirus disease or required pharmacotherapy. Three recipients developed acute graft versus host disease after infusion. Two recipients died, 1 from thrombotic thrombocytopenia purpura secondary to cyclosporine, 1 from complications of graft versus host disease. A transient increase in numbers of cytomegalovirus-specific T cells demonstrated by NLV-tetramer binding was seen in 6 recipients. Prophylactic adoptive transfer of NLV-specific T cells is safe and may be effective in preventing cytomegalovirus reactivation.

Transcriptional activation of NF-kappa B activity by Epstein-Barr virus (EBV) LMP1 as a selective therapeutic strategy for EBV-associated diseases.

Epstein-Barr virus (EBV) has been known to be associated with many malignant tumors, including nasopharyngeal carcinoma (NPC). Previous studies have indicated that an EBV-encoded oncoprotein, latent membrane protein 1 (LMP1), is expressed in many NPC tissues. LMP1 has been shown to stimulate HIV LTR through the two NF-kappa B binding sites within this promoter. In this study, we examined the feasibility of using this property of LMP1 as a therapeutic strategy for the treatment of NPC. This therapy consists of the preferential killing of the LMP1-expressing cells by gene transfer using the NF-kappa B-mediated herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system. The 800-bp HIV-LTR, which contains two NF-kappa B binding sites, was used to drive the HSVtk gene. Stable C33A cell clones expressing the LMP1 and the HSVtk genes were subjected to the GCV sensitivity test. Results showed that cells expressing both the LMP1 and the HSVtk genes were highly sensitive to GCV treatment. These cells were introduced into nude mice subcutaneously and tumors became palpable within 2 weeks. GCV was then introduced intraperitoneally to these mice and the sizes of the tumors were measured daily. Results showed that the tumors regressed in the group of mice carrying cells that stably expressed both the LMP1 and the HSVtk genes, but not in mice carrying cells containing LMP1 or HSVtk alone. Our data indicate that the HSVtk gene expressed from a NF-kappa B-binding motif-containing promoter that is regulated by LMP1 may be used as an in vivo gene therapy strategy of EBV LMP1-expressing cancers such as NPC.

Treatment of influenza virus-infected SCID mice with nonneutralizing antibodies specific for the transmembrane proteins matrix 2 and neuraminidase reduces the pulmonary virus titer but fails to clear the infection.

Antibodies (Abs) can contribute to the cure of a viral infection, in principle, in two ways by: (1) binding to infected cells and thereby reducing the production of progeny virus [here termed cell-targeting (CT) activity] and (2) reacting with released progeny virus and thereby inhibiting the spread of the infection [termed virus neutralizing (VN) activity]. We have previously shown that a pulmonary influenza virus infection in severe combined immunodeficient mice could be cured by treatment of these mice with hemagglutinin (HA)-specific monoclonal Abs (mAbs) that mediated both of the above activities. Although the therapeutic activity of these mAbs correlated with their VN activity, it remained unclear how much their CT activity contributed to the Ab-mediated recovery process. To clarify this point, we tested the therapeutic efficacy of two mAbs of IgG2a isotype that mediated CT but no VN activity: one specific for the viral neuraminidase and the other for matrix protein 2. Both mAbs reduced pulmonary virus titers by 100- to 1000-fold but they failed to clear the infection, even when administered in combination and at therapeutically saturating concentrations. The results suggest that CT activity contributes significantly also to the therapeutic activity of HA-specific mAbs and further support the notion that VN-activity is required for Ab-mediated virus clearance.

Adenoviral gene transfer into dendritic cells efficiently amplifies the immune response to LMP2A antigen: a potential treatment strategy for Epstein-Barr virus--positive Hodgkin's lymphoma.

The EBV-encoded LMP2A protein is consistently expressed in EBV(+) Hodgkin's lymphoma and can be targeted by CTLs. CTLs stimulated conventionally by LCLs have little activity against LMP2A(+) target cells. Here, we describe an alternative approach, based on the in vitro stimulation of CTLs with DCs genetically modified with 2 E1/E3-deleted recombinant adenoviruses, AdGFPLMP2A, encoding a fusion gene of GFP and LMP2A, and AdLMP2A, encoding LMP2A only. Transduction of DCs with AdGFPLMP2A at MOI 1,000 resulted in LMP2A expression in up to 88% of DCs. LMP2A protein was expressed in 40% of DCs transduced with AdLMP2A at an MOI of 100. Higher MOI resulted in DC death. CTL lines activated by transduced DCs had a higher frequency of LMP2A tetramer-specific CTLs than CTL lines activated by LCLs. CTLs stimulated with transduced DCs lysed both autologous fibroblasts infected with vaccinia virus LMP2A (FBvaccLMP2A) and autologous LCLs, which express LMP2A at lower levels. In contrast, CTLs generated from the same donors by stimulation with autologous LCLs showed minimal lysis of FBvaccLMP2A. Moreover, 1 donor who did not respond to LMP2A when CTLs were stimulated with LCLs became a responder when LMP2A was expressed by transduced DCs. Hence, recombinant adenoviruses encoding LMP2A effectively transduce DCs and direct the generation of LMP2A-specific CTLs. This approach will be a potent strategy in Hodgkin's lymphoma immunotherapy.