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Nov.  2020
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ZENG Yi, SI Yong Feng, LAN Gui Ping, WANG Zhan, ZHOU Ling, TANG Min Zhong, SJ O`Brien, LAN Jiao, ZHOU Xiang Yang, WANG Yong Li, TANG Juan, ZHOU Zhi Xiang, DU Hai Jun, LIN Hui. LMP2-DC Vaccine Elicits Specific EBV-LMP2 Response to Effectively Improve Immunotherapy in Patients with Nasopharyngeal Cancer[J]. Biomedical and Environmental Sciences, 2020, 33(11): 849-856. doi: 10.3967/bes2020.115
Citation: ZENG Yi, SI Yong Feng, LAN Gui Ping, WANG Zhan, ZHOU Ling, TANG Min Zhong, SJ O`Brien, LAN Jiao, ZHOU Xiang Yang, WANG Yong Li, TANG Juan, ZHOU Zhi Xiang, DU Hai Jun, LIN Hui. LMP2-DC Vaccine Elicits Specific EBV-LMP2 Response to Effectively Improve Immunotherapy in Patients with Nasopharyngeal Cancer[J]. Biomedical and Environmental Sciences, 2020, 33(11): 849-856. doi: 10.3967/bes2020.115

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

doi: 10.3967/bes2020.115
Funds:  Mega Project of Research on the Prevention and Control of HIV/AIDS, Viral Hepatitis Infectious Diseases [Grant No:2018ZX10102001]; the Key Science and Technology Program of Guangxi Zhuang Autonomous Region [Grant No. 14124003-3]; the National High Technology Research and Development Program of China [Grant No. 2007AA021107]; and the National Basic Research Program of China [973 Program, Grant No. 2011CB504800]
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  •   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 × 105 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.
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  • [1] Laboratory of tumor viruses of cancer institute, laboratory of tumor viruses of institute of epidemiology, department of radiotherapy of cancer institute, laboratory of cell biology of cancer institute. Establishment of an epitheloid cell line and a fusiform cell line from a patient with nasopharyngeal carcinoma. Sci Sin, 1978; 21, 127−34.
    [2] Chua MLK, Wee JTS, Hui EP, et al. Nasopharyngeal carcinoma. Lancet, 2016; 387, 1012−24. doi:  10.1016/S0140-6736(15)00055-0
    [3] Young LS, Dawson CW, Clark D, et al. Epstein-Barr virus gene expression in nasopharyngeal carcinoma. J Gen Virol, 1988; 69, 1051−65. doi:  10.1099/0022-1317-69-5-1051
    [4] Lee SP, Tierney RJ, Thomas WA, et al. Conserved CTL epitopes within EBV latent membrane protein 2: a potential target for CTL-based tumor therapy. J Immunol, 1997; 158, 3325−34.
    [5] Pan Y, Zhang J, Zhou L, et al. In vitro anti-tumor immune response induced by dendritic cells transfected with EBV-LMP2 recombinant adenovirus. Biochem Biophys Res Commun, 2006; 347, 551−7. doi:  10.1016/j.bbrc.2006.05.214
    [6] Wang Z, Yang S, Zhou L, et al. Specific cellular immune responses in mice immunized with DNA, adeno-associated virus and adenoviral vaccines of Epstein-Barr virus-LMP2 alone or in combination. Sci China Life Sci, 2011; 54, 263−6. doi:  10.1007/s11427-011-4147-0
    [7] Mo WN, Tang AZ, Zhou L, et al. Analysis of Epstein-Barr viral DNA load, EBV-LMP2 specific cytotoxic T-lymphocytes and levels of CD4+CD25+ T cells in patients with nasopharyngeal carcinomas positive for IgA antibody to EBV viral capsid antigen. Chin Med J (Engl), 2009; 122, 1173−8.
    [8] Hart DN. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood, 1997; 90, 3245−87. doi:  10.1182/blood.V90.9.3245
    [9] Knight SC, Stagg AJ. Antigen-presenting cell types. Curr Opin Immunol, 1993; 5, 374−82. doi:  10.1016/0952-7915(93)90056-X
    [10] Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol, 1991; 271−96.
    [11] Meixlsperger S, Leung CS, Rämer PC, et al. CD141+ dendritic cells produce prominent amounts of IFN-α after dsRNA recognition and can be targeted via DEC-205 in humanized mice. Blood, 2013; 121, 5034−44. doi:  10.1182/blood-2012-12-473413
    [12] Schlitzer A, McGovern N, Teo P, et al. IRF4 transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses. Immunity, 2013; 38, 970−83. doi:  10.1016/j.immuni.2013.04.011
    [13] Zeng Y. Serological massive surveys and vaccines for controlling nasopharygeal carcinoma. 7th Annual Global Virus Networks Meeting 2015, May, 17. www.gvn.org/events/gvn-meetings/.
    [14] Sabado RL, Balan S, Bhardwaj N. Dendritic cell-based immunotherapy. Cell Res, 2017; 27, 74−95. doi:  10.1038/cr.2016.157
    [15] Zhou ZX, Li D, Guan SS, et al. Immunotherapeutic effects of dendritic cells pulsed with a coden-optimized HPV 16 E6 and E7 fusion gene in vivo and in vitro. Asian Pac J Cancer Prev, 2015; 16, 3843−7. doi:  10.7314/APJCP.2015.16.9.3843
    [16] Palucka K, Banchereau J. Cancer immunotherapy via dendritic cells. Nat Rev Cancer, 2012; 12, 265−77. doi:  10.1038/nrc3258
    [17] Verdijk P, Aarntzen EH, Lesterhuis WJ, et al. Limited amounts of dendritic cells migrate into the T-cell area of lymph nodes but have high immune activating potential in melanoma patients. Clin Cancer Res, 2009; 15, 2531−40. doi:  10.1158/1078-0432.CCR-08-2729
    [18] Prince HM, Wall DM, Ritchie D, et al. In vivo tracking of dendritic cells in patients with multiple myeloma. J Immunother, 2008; 31, 166−79. doi:  10.1097/CJI.0b013e31815c5153
    [19] Zuo JM, Zhou L, Chen ZJ, et al. Induction of cytotoxic T lymphocyte responses in vivo after immunotherapy with dendritic cells in patients with nasopharyngeal carcinoma. J.Microbiol immunol, 2006; 4, 41−8.
    [20] Lin JC, Wang WY, Chen KY, et al. Quantification of plasma Epstein-Barr virus DNA in patients with advanced nasopharyngeal carcinoma. N Engl J Med, 2004; 350, 2461−70. doi:  10.1056/NEJMoa032260
    [21] Lo YM, Chan AT, Chan LY, et al. Molecular prognostication of nasopharyngeal carcinoma by quantitative analysis of circulating Epstein-Barr virus DNA. Cancer Res, 2000; 60, 6878−81.
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LMP2-DC Vaccine Elicits Specific EBV-LMP2 Response to Effectively Improve Immunotherapy in Patients with Nasopharyngeal Cancer

doi: 10.3967/bes2020.115
Funds:  Mega Project of Research on the Prevention and Control of HIV/AIDS, Viral Hepatitis Infectious Diseases [Grant No:2018ZX10102001]; the Key Science and Technology Program of Guangxi Zhuang Autonomous Region [Grant No. 14124003-3]; the National High Technology Research and Development Program of China [Grant No. 2007AA021107]; and the National Basic Research Program of China [973 Program, Grant No. 2011CB504800]

Abstract:   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 × 105 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.

ZENG Yi, SI Yong Feng, LAN Gui Ping, WANG Zhan, ZHOU Ling, TANG Min Zhong, SJ O`Brien, LAN Jiao, ZHOU Xiang Yang, WANG Yong Li, TANG Juan, ZHOU Zhi Xiang, DU Hai Jun, LIN Hui. LMP2-DC Vaccine Elicits Specific EBV-LMP2 Response to Effectively Improve Immunotherapy in Patients with Nasopharyngeal Cancer[J]. Biomedical and Environmental Sciences, 2020, 33(11): 849-856. doi: 10.3967/bes2020.115
Citation: ZENG Yi, SI Yong Feng, LAN Gui Ping, WANG Zhan, ZHOU Ling, TANG Min Zhong, SJ O`Brien, LAN Jiao, ZHOU Xiang Yang, WANG Yong Li, TANG Juan, ZHOU Zhi Xiang, DU Hai Jun, LIN Hui. LMP2-DC Vaccine Elicits Specific EBV-LMP2 Response to Effectively Improve Immunotherapy in Patients with Nasopharyngeal Cancer[J]. Biomedical and Environmental Sciences, 2020, 33(11): 849-856. doi: 10.3967/bes2020.115
    • The current standard treatment of nasopharyngeal carcinoma (NPC) is radiation therapy or a combination of radiation and chemotherapy. Although the initial rate of response and the five-year survival rate for early stage patients with NPC are promising when compared to other malignancies, recurrences and post-treatment metastases often appear in advanced-stage NPC patients. Patients with NPC recurrence and metastasis have poor prognosis, and no curative therapeutic options are available. There is substantial evidence that Epstein-Barr virus (EBV) plays an important role in NPC development[1,2]. A limited array of EBV antigens, including latent membrane proteins (LMP) 1 and 2, and EBV nuclear antigen 1 (EBNA1), are expressed in NPC cells[3]. Of these, LMP2 epitopes are the most frequently recognized by CD8+ cytotoxic T lymphocytes (CTLs)[4], and CTLs specific to LMP2 have been shown to efficiently kill NPC cells both in vivo and in vitro[57]. The results of these studies suggest that patients with NPC could benefit from immunotherapy.

      DCs are the most potent antigen-presenting cells, are widely distributed throughout the body, and have the ability to stimulate both native and secondary immune responses to antigen(Ag)-specific T cells[810]. Genetic modification of DCs with genes encoding immunoregulatory molecules provides a potential approach for Ag-specific T cell-mediated immunity by selectively targeting antigen-specific T cells[11,12]. A cellular vaccine based on DCs pulsed with LMP2-antigen has previously been reported at the Global Virus Networks meeting in 2015[13]. The results showed that the vaccine induced specific CTLs were sufficient for NPC control and treatment.

      With these recent studies in mind, we designed and conducted a pilot clinical study to test the biological effects of LMP2-DCs in 29 patients with NPC after conventional radiotherapy and chemotherapy. We report here the promising immunological and clinical results.

    • Autologous mononuclear cells were isolated by the Ficoll-Hypaque density gradient centrifugation (Pharmacia, 30 min, 4 °C, 800 ×g from peripheral blood). Peripheral blood monocytes (PBMCs) were cultured in six-well plates at 5 × 106 cells/well (37 °C, 5% CO2) for 2 h. Supernatant PBMCs were removed, and adherent monocytes were cultured for 7 d in six-well plates in 3 mL/well with 500 U/mL IL-4 (Pepretech) and 500 U/mL GM-CSF (Pepretech) in a DC medium (Cell-GRO® DC). On day 3, half of the medium was replaced with fresh DC medium. The morphology of monocytes was observed by optical microscope.

      The immature DCs were aspirated and harvested (250 ×g, 10 min) on day 7 and then transferred to new 6-well plates at 106 cells/well. DCs were then infected with rAd-LMP2 (Shen Zhen Tsinghua Yuanxing Pharmaceutical Co, Ltd, MOI100) for 2 h. DCs expressing LMP2 (LMP2-DCs) were induced to maturity by replacing cells with fresh medium containing IL-4 (500 U/mL), GM-CSF (500 U/mL), and tumor necrosis factor-alpha (TNF-α 400 U/mL Pepretech) for another two days. Mature LMP2-DCs (mLMP2-DCs) were stained with immunofluorescence antibodies CD83, CD86, and HLA-DR (BD Bioscience) and then analyzed using flow cytometry. The mLMP2-DCs were harvested by centrifugation at 250 × g for 10 min.

      The pellets of mLMP2-DCs (250 ×g, 10 min) were collected and washed with PBS three times and then resuspended in freezing medium containing 10% dimethyl sulfoxide (Sigma), 20% autologous serum, and 70% DC medium 2 × 106/mL (0.5 mL/dosage). Cells were frozen at −80 °C overnight using a Cryo 1 °C Freezing Container (Nalgene) filled with isopropanol. The frozen cells were then transferred to liquid nitrogen.

    • Cell smears were prepared from mLMP2-DCs and fixed in cold acetone at 4 °C for 15 min. The slides were allowed to dry in air and were incubated with anti-LMP2 monoclonal antibody (Santa Cruz Biotechnology, diluted 1:100 with phosphate buffer saline, PBS) at 37 °C for 40 min. After primary antibody incubation, the slides were washed three times with PBS. Biotin-conjugated anti-human IgG antibody (diluted 1:100 with PBS) was added to the slide at 37 °C for 40 min, and the slides were washed three times with PBS. Horseradish/peroxidase-conjugated anti-biotin IgG antibody (diluted 1:100 with PBS) was added to the slides at 37 °C for 40 min. The slides were incubated with diaminobenzidine (Sigma) and H2O2 for 10 min and washed in distilled water. The slides were then air dried and examined under an optical microscope.

    • We performed a pilot study in patients with NPC to determine the safety, immunogenicity, and clinical efficacy of a mLMP2-DCs vaccine. Eligible cases were histopathologically confirmed cases of NPC (stage I-IV), in which the patient’s age was ≤ 70 years and the patient was at least three months post-chemotherapy and -radiotherapy and at least three months into remission with no acute diseases and normal function of liver, kidney, and marrow. Enrollment was allowed at least three months after conventional radiotherapy and chemotherapy. The demographic information regarding all patients is summarized in Supplementary Table S1 available in www.besjournal.com. PBMCs were harvested for LMP2-DCs vaccine manufacturing at the Clinical Cell and Vaccine Production Facility at the People's Hospital of Guangxi Zhuang Autonomous Region. Vaccines were cryopreserved at −196 °C, thawed and washed before administration. Patients with NPC received three doses of the vaccine (≥ 5 × 105 DCs/dose) intradermally every two weeks. The procedure is shown in Figure 1. Patients with NPC were evaluated every two weeks. Safety was determined using the China Food and Drug Administration Grading Standard of Adverse Events in Clinical Trials (http://www.cde.org.cn/zdyz.do?method=list). Clinical indications, including breathing, heart rate, blood pressure, and body temperature were monitored. The temperature was documented when fever occurred (body’s temperature over 38 °C). Subjects underwent full examination (blood, urine, hepatorenal function, electrocardiogram) pre- and post-immunotherapy.

      Patient IDSexAgeStage at
      enrollment
      Time after radiotherapy and
      chemotherapy (months)
      1M37I9
      2F39I3
      3M37I9
      4M49II6
      5F61II3
      6F37II3
      7M34II6
      8F34II7
      9F53II3
      10F34II5
      11F61II3
      12F30II8
      13F51II3
      14M44II3
      15M66III8
      16M36III4
      17M24III9
      18F35III6
      19M39III3
      20M36III6
      21F37III5
      22M55III3
      23F60III9
      24F33III4
      25M28IV7
      26M33IV3
      27M36IV9
      28F50IV8
      29M30IV3

      Table S1.  Characteristics of NPC patients in this study

      Figure 1.  Steps of the study. Yellow triangle: border of different trial stage; Black triangle: time of immunotherapy; Orange triangle: time of LMP2 specific cellular immunity test.

      This study was approved by Institutional Review Board of the People's Hospital of Guangxi Zhuang Autonomous Region on September 24, 2008. All the procedures meet the ethics requirement. All patients gave written informed consent prior to initiation of any study procedures.

    • PBMCs were isolated from immunized patients by Ficoll-Hypaque density gradient centrifugation at week 0 and at 5 and 8 weeks 800 ×g, 15 min non-stop). Specific responses of CTLs to LMP2 were analyzed using human IFN-γ Elispot kit (BD Bioscience). The kit was used according to the manufacturer's protocol. PBMCs were plated at 2 × 105 cells/well, and six sub-peptide pools (Supplementary Table S2 available in www.besjournal.com) screened from the LMP2 pool were added to the wells at a final concentration of 5 μg/mL per peptide for overnight stimulation. Wells containing media alone were used as negative controls, and cells incubated with 1 μg/mL PhosPhomolybdic Acid (PMA) and ionomycin (Ion) (Da Kewei Co, Ltd) were used as positive controls. The chromogenic reaction was terminated by distilled water as soon as spots were ready for counting. The plate was allowed to dry in air for 10–30 min. The number of spots per well were read using Bioreader 4000 PRO, and statistic data analysis (GraphPad Prism 5) was performed. A positive response was defined as a two-fold or greater increase in the number of spot-forming cells per million PBMC in immunized wells compared to pre-immunized wells. Additionally, we tested the plasma for changes of the EBV DNA genome copies and titers of IgA antibody to VCA. If the titers of IgA were elevated more than four-fold compared to levels in pre-immunization, the difference was considered significant.

      Sub-peptide poolSequences of amino
      acids in LMP2
      Purity (%)
      5MGSLEMVPM95
      5LPVIVAPYL95
      5PYLFWLAAI95
      5FTASVSTVV95
      5IEDPPFNSL95
      5RRRWRRLTV95
      5RRWRRLTVC95
      5RRLTVCGGIMF95
      5TVCGGIMFL95
      5SSCSSCPLSKI95
      5ILLARLFLY95
      5TYGPVFMCL95
      5LTAGFLIFL95
      5LIVDAVLQL95
      6LFWLAAIAASCF75
      6AAIAASCFTASV75
      6STVVTATGLALS75
      6LALSLLLLAAVA75
      7LTAVVTFFAICL75
      7FNSLLFALLAAA75
      7LFALLAAAGGLQ75
      7CGGIMFLACVLV75
      7MFLACVLVLIVD75
      8CVLVLIVDAVLQ75
      8TVVSMTLLLLAF75
      8MTLLLLAFVLWL75
      8LLTLAAALALLA75
      8LTTMFLLMLLWT75
      10AGILFILAILTE75
      10GLLTMVAGAVWL75
      10MVAGAVWLTVMS75
      10SAWILTAGFLIF75
      10FLIFLIGFALFG75
      11IYVLVMLVL95
      11LLWTLVVLL95
      11WTLVVLLI95
      11FLYALALLL95
      11CLGGLLTMV95
      11VMSNTLLSAW95
      11LLSAWILTA95

      Table S2.  Peptide mixes from LMP2

    • The benefit of LMP2-DCs to patients with NPC was investigated through follow-up and tracking of the study subjects. Admission data was reviewed every three months and close follow-up of individual subjects was performed every half year after immunotherapy was completed. Follow-up included phone inquiry to determine physical condition and clinical monitoring of NPC progression.

    • Patients with NPC began to enroll in this study in April 2009. We completed the study in October 2010. Twenty-nine patients with NPC (age 24–66) were enrolled at the hospital for treatment with intradermal injections and agreed to participate in follow up for five years.

    • Monocytes isolated from PBMCs were cultured and induced to DC differentiation in vitro. During differentiation, cells progressively acquired DC morphological features. Cells were loosely adherent and irregularly netted. We observed the formation of cell mass, increased cell size, folding on the cellular surface, and the formation of cytoplasmic projections (Figure 2). Immunocytochemical assay, using a LMP2 monoclonal antibody, was performed to confirm the expression of LMP2 in 100 MOI rAd-LMP2 infected DCs. An optical microscope was used to illuminate DCs expressing LMP2 (Figure 3).

      Figure 2.  Morphology of mature DCs expressing LMP2. Magnification, 400×.

      Figure 3.  LMP2-expression in monocyte-derived DC infected by rAd-LMP2 with 100 MOI. (A) Negative control; (B) LMP2-DCs. Magnification, 200×.

    • We tested LMP2-pulsed matured DCs for vaccine production in a pilot study of patients with NPC after conventional radiotherapy and chemotherapy. Elutriated monocytes were obtained from the PBMCs of the study subjects and cultured in six-well plates with IL-4 and GM-CSF for 7 d. On day 8 of culturing, DCs were pulsed with rAd-LMP2 for 2 h, followed by treatment with TNF-α, IL-4, and GM-CSF for two days. mLMP2-DCs were harvested and cryopreserved before administration. At the same time, the expression of CD83, CD86, and DC-DR on DC surface was confirmed by flow cytometry. Expression of CD molecules on the surface of DCs showed considerable variation (from 10.57% to 99.87%) in 22 patients with NPC, which may be due to the different responses to LMP2 (Supplementary Table S3 available in www.besjournal.com); i.e., NPC (as indicated by CD83, CD86, and DR molecules) was not detected in seven of the patients (Patients No 4, 15, 16, 17, 18, 19, 25). mLMP2-DCs vaccines were administered through intradermal injection. Each of the study subjects received three vaccinations. In total, 87 intradermal vaccinations were performed. All vaccines were well tolerated, with only local rigor and swelling at the injection site, which subsided the following day without treatment. Weight, blood pressure, heart rate, respiratory signs, and other clinical monitoring data were within the normal range before and after injection, and no serious side effects were observed.

      Patients IDDC-CD83 (%)DC-CD86 (%)DC-DR (%)
      199.3299.7699.61
      267.2447.5466.86
      364.0744.2457.27
      4MissedMissedMissed
      584.4594.9293.74
      699.3984.8888.09
      760.6552.1361.61
      817.0252.6737.26
      955.1742.3453.40
      1076.7777.4892.56
      1149.5351.0881.82
      1274.7665.7578.67
      1369.80 71.8667.83
      1499.8699.8799.52
      15MissedMissedMissed
      16MissedMissedMissed
      17MissedMissedMissed
      18MissedMissedMissed
      19MissedMissedMissed
      2091.5297.5397.45
      2153.10 59.5187.67
      2268.5157.6372.53
      2354.9938.2552.29
      2498.9186.7296.93
      25MissedMissedMissed
      2683.2894.2292.25
      2710.5760.1247.38
      2829.1336.80 20.92
      2980.3172.40 80.28
        Note. The positive rates of CD83, CD86, and DC-DR on DC surface were also detected by flow cytometry. Expression of CD molecules on the surfaces of DCs varied widely in patients with NPC.

      Table S3.  Detection of CD83, CD86, and DR molecules on the cell surface in LMP2-DCs

    • To analyze the effects of vaccination, specific CTL responses to LMP2 were examined pre- and post-injection for each of the three injections. CTL responses to LMP2 increased in 18 of 29 immunized NPC patients (62.1%), compared with pre-immunized subjects (Figure 4A, B). The size of responses varied from 2.0 to 8.4-fold (Figure 5A). Among these 18 patients, two (2/3, 66.7%) were in stage I of the disease, nine (9/11, 81.8%) in stage II, five (5/10, 50.0%) in stage III, and two (2/5, 40.0%) in stage IV (Figure 5B). There was a higher incidence of increased specific responses median 4.3 and 3.2-fold) in subjects at an early stage of NPC (than in advanced stage subjects (median 3.1 and 2.9-fold) (Supplementary Table S4 available in www.besjournal.com). However, 11 of the 29 patients did not show any difference in CTL responses to LMP2 (Figure 6). CTL responses to LMP2 increased (1.8-fold) in Patient No. 24 but were still below two-fold and the case was therefore defined as a non-responder. Of the 11 non-responder patients, one was in stage I, two in stage II, five in stage III, and three in stage IV. The expression of CD83, CD86, and DC-DR on the surface of DCs was lower in non-responders than in responders, which caused no CTL responses to LMP2. The titer of IgA antibody to VCA did not show obvious variation (≥ 4-fold titer changes defined as positive responses) (Supplementary Table S5 available in www.besjournal.com). A test for EBV DNA proved to be negative for both pre- and post-immunization of DC-LMP2 in all 29 patients. All the patients were clinically disease-free after treatment with chemotherapy and radiotherapy (Supplementary Table S6 available in www.besjournal.com). However, EBV DNA was detected in the plasma of one patient (No. 22) and increased along with the development of disease (578 copies/mL in 2012 and 4.6 × 104 copies/mL in 2013) (Supplementary Table S6). The size of LMP2-specific T cell responses gradually decreased with development of cancer in patients with NPC (Figure 6).

      Figure 4.  Boosted CTLs responses to LMP2 peptide sub-pools before and after immunization. (A) CTLs responses specific to LMP2 peptide sub-pools before and after immunization in early-stage NPC patients. Boosted T-cell responses to LMP2 in 11 of 14 NPC stage I and II patients. 18 of 29 NPC patients (62.1%) had boosted CTL responses to LMP2, as shown in Figures 45. However, 11 of 19 patients did not show positive CTL responses to LMP2. Of 18 patients, 2 (2/3, 66.7%) were in stage I, 9 (9/11, 81.8%) were in stage II, 5 (5/10, 50.0%) were in stage III, and 2 (2/5, 40.0%) were in stage IV. Specific CTL responses were augmented 2.0 to 8.4-fold compared with pre-immunization. Increased size of specific responses is higher in early stage (median 4.3- and 3.2-fold) than at advanced stage (median 3.1- and 2.9-fold). (B) specific responses of CTLs to LMP2 peptide sub-pool before and after immunization in advanced patients with NPC. T-cell responses to LMP2 were boosted in 7 of 15 stage III and IV patients. CTL, cytotoxic T lymphocyte; SFC, spot-forming cell.

      Figure 5.  The size of responses to DC vaccine in patients with NPC. (A) size (percent increase) of LMP2-specific T cell responses stimulated by LMP2-DCs vaccine. (B) size (percent increase) of the responses in patients at different stages of NPC. CTL, cytotoxic T lymphocyte

      Figure 6.  CTL responses to LMP2 peptide sub-pool before and after immunization. In 11 of 29 patients, there were no positive CTL responses to LMP2; rather, CTL levels were slightly elevated, unchanged, or decreased. 72.7% of the patients were in later stages of NPC (stage III and stage IV). Patient characteristics are presented in Supplementary Table S1. CTL, cytotoxic T lymphocyte; SFC, spot-forming cell.

      Patient IDStage at enrollmentPre-vaccination
      (Spot-forming cells/million PBMC)
      Post-vaccination
      (Spot-forming cells/million PBMC)
      Percent increase (%)
      1I 270 1,576 484
      3I8402,395185
      4II5001,478196
      5II212670216
      6II3101,530394
      9II450977117
      10II9751,980103
      11II9805,760488
      12II6802,995340
      13II7701,850140
      14II5343,023466
      17III80673741
      18III35068596
      19III6751,775163
      20III160580263
      23III1,4202,80598
      26IV2931,055260
      29IV2,9106,590126

      Table S4.  Specific-CTLs responses induced by LMP2-DCs in NPC patients

      Patient IDStage at enrollmentWeek 0Week 5Week 8
      1I 320 160 160
      2I808080
      3I404040
      4II16080160
      5II204020
      6II160160160
      7II404040
      8II808080
      9II404040
      10II804080
      11II160160160
      12II202020
      13II808080
      14II808080
      15III202020
      16III404040
      17III408040
      18III404040
      19III 40 40 40
      20III320320160
      21III404040
      22III8016080
      23III404040
      24III202020
      25IV404040
      26IV804080
      27IV808080
      28IV320320320
      29IV808080

      Table S5.  IgA /VCA antibody titer in plasma

      Patient IDStage at enrollmentYear (2011)Year (2012)Year (2013)Year (2014)Year (2015)
      1INeg
      2INegNegNeg
      3INegNegNegNeg
      4IINegNegNegNegNeg
      5IINegNegNegNeg
      6II
      7IINegNegNegNegNeg
      8IINeg
      9IINegNegNeg
      10IINeg
      11II
      12IINeg
      13IINeg1.6 × 103
      14IINegNegNegNeg
      15IIINegNegNeg
      16IIINegNeg
      17IIINegNegNegNeg
      18III
      19IIINegNegNeg
      20IIINegNegNeg
      21IIINeg
      22IIINeg5784.6 × 104
      23IIINegNegNegNeg
      24III
      25IVNeg
      26IVNegNegNegNegNeg
      27IV
      28IV
      29IVNegNegNegNeg3 × 103

      Table S6.  EBV-DNA concentration in plasma

    • To evaluate the long-term therapeutic efficacy after vaccination, we followed up with the study subjects for five years (from October 2009 to December 2015). Seven patients died, of whom one (1/11) had stage II NPC, four (4/10) had stage III NPC, and two (2/5) had stage IV NPC. Only one of the deceased subjects was a responder, the other six of the seven deceased subjects were non-responders (Supplementary Table S7 available in www.besjournal.com). The five-year survival rate, from immunotherapy completion to the end of 2015, was 94.4% in responders and 45.5% in non-responders (Table 1). The survival durations were 1–4 years in the seven patients who died within five years, with durations in most patients being 2–3 years (Supplementary Table S8 available in www.besjournal.com). This suggests that CTLs responses that are specific to LMP2 play a role in controlling and preventing NPC recurrence and metastasis.

      Patient IDStage at enrollmentPre-vaccination
      (Spot-forming cells/million PBMC)
      Post-vaccination
      (Spot-forming cells/million PBMC)
      Percent increase (%)
      2I 630 285 –54.8
      7II855320–62.6
      8II575245–57.4
      15III1,2781,3001.7
      16III928495–46.7
      21III1,095570–47.9
      22III690685–0.7
      24III30054080.0
      25IV995700–29.6
      27IV525500–4.8
      28IV609300–50.7

      Table S7.  Non-specific CTLs responses induced by LMP2-DCs vaccine in NPC patients

      StageEarly stageAdvanced stage5-year survival rate (%) (n = 29)
      I (n = 3)II (n = 11)III (n = 10)IV (n = 5)
      Positive reaction295218/29 (62.1)
      Death00101/18 (5.6)
      Survival294217/18 (94.4)
      5-years survival rate (%)100.085.7
      Negative reaction125311/29 (37.9)
      Death01326/11 (54.5)
      Survival11215/11 (45.5)
      5-years survival rate (%)66.737.5
      Total death rate (%)0.09.140.040.07/29 (24.1)
      Total survival rate (%)100.090.960.060.022/29 (75.9)

      Table 1.  Specific CTLs responses in patients with NPC after immunization with LMP2-DC

      Patient IDStage at enrollmentFoldPercent increase (%)Survival period (year)Year of deathCause of death
      7IIN –54.822012metastatic carcinoma of bone
      16IIIN–62.642013, 7pulmonary metastasis
      22IIIN–57.432013, 4multiple metastasis
      23IIIN1.732013, 12pulmonary metastasis
      24IIIN–46.712011, 4multiple metastasis
      25IVN–47.932013, 8multiple metastasis
      28IVN–0.732013, 7pulmonary metastasis

      Table S8.  Characteristics of NPC patients who were immunized by LMP2-DCs and died within five years of cancer treatment

    • DC-based immunotherapy shows great potential for the treatment of cancer[14,15]. DC immunotherapy is conducted mostly through tumor antigen peptides or tumor gene-loaded DCs. However, few tumor-specific or associated antigens have been identified. EBV infection is closely associated with NPC and is recognized as an etiological agent. LMP2 encoded by EBV is detected in most NPC biopsies. This protein contains CD8 T cell epitopes and is considered one of the ideal target antigens for immunotherapy[5]. Therefore, LMP2 was selected as the target antigen for NPC immunotherapy in our study. Although DC vaccine could be administered by venous transfusion, lymph node, and intra-tumoral injection; fewer DCs were required for intra-tumoral injection to stimulate an efficient Th1/Th2 response[1618]. Thus, it is more practical to administer DC vaccination to tumor patients via intradermal injection. Our group has previously reported nine NPC patients who received 60Co irradiated LMP2-DCs through intradermal injection, and the CTL responses specific to LMP2 were boosted in five of these nine NPC patients[19]. rAd-LMP2 is a replication-defective adenovirus which has a good safety and may affect the function of LMP2-DCs that were irradiated by 60Co. In the present study, LMP2-DCs were directly immunized through intradermal injection instead of 60Co irradiation. Our results suggest that CTL responses specific to LMP2 in the early stage of NPC are much better than that in the advanced stage of the disease (Table 2). Therefore, we consider intradermal injection more suitable for patients with early-stage NPC than those in the advanced stage. Among the 29 patients, 11 had been for 3 months after radiotherapy or/and chemotherapy, and specific CTL responses improved in nine of these eleven patients after injection of LMP2-DCs, which indicates that immunity levels were back to normal for most of the patients three months after radiation and chemotherapy. The LMP2-specific responses of CTL in some responsive NPC patients are reduced very quickly. One of our main concerns is how to maintain LMP2-specific CTL responses at a certain level. We are currently testing if we can maintain LMP2-specific CTL responses by increasing injections and using multi-vector forms of immunity. We did not detect changes in IgA antibody titers to EBV-VCA, since our experimental design was to activate LMP2 specific cellular immune responses, not humoral immune responses. In addition, two months was not sufficient to detect the changes in IgA antibody levels.

      StageCasesLMP2 CTLs POS (%)LMP2 CTLs NEG (%)Total (%)
      I 32 (66.7)1 (33.3)100.0
      II 87 (87.5)1 (12.5)100.0
      III 53 (60.0)2 (40.0)100.0
      IV 31 (33.3)2 (66.7)100.0
      Total1913 (68.4)6 (31.6)100.0

      Table 2.  Relation between the stage of NPC and LMP2 specific-CTLs

      In previous studies it has been reported that EBV genome copies could serve as an index of treatment efficacy for early diagnosis and prognosis of NPC[20,21]. We therefore examined plasma EBV genome copies of NPC patients before and after DC immunotherapy, but the plasma data results were negative in all NPC patients, which suggests that clinical post-radiotherapy and post-chemotherapy may inhibit the replication of the EB virus. However, EBV DNA was detected in the plasma of one patient (No. 22) and increased along with the development of the disease (578 copies/mL in 2012 and 4.6 ×104 copies/mL in 2013).

      Each subject received three consecutive injections of LMP2-DCs: at week 0 and after at thesecond and fourth weeks. LMP2-specific CTL responses were examined pre-injection, at week 0, and at the fifth and eighth weeks. To further confirm the therapeutic efficacy of LMP2-DCs vaccines in treating NPC, future studies should include follow-up for long-term therapeutic effects, the launch of large multicenter clinical trials, and so on. How to best combine laboratory data with efficacious clinical treatment is a topic for future research.

    • Zeng Y designed the experiments and supervised the project. Si YF, Du HJ, and Lin H supervised the project. SJ O'Brien, Lan GP, Wang Z, Zhou L, Tang MZ, Lan J, Zhou XY, Wang YL, and Tang J designed and conducted the experiments and analyzed results. Du HJ and Zhou ZX wrote the manuscript. Zeng Y gratefully acknowledges the founding support. The authors declare no competing financial interests

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