- Email: [email protected]
217. HIV Latency: Overcoming Transcriptional Block (Initiation and Elongation)
INFECTIOUS DISEASES AND VACCINES I (EP). In the present studies, EP-mediated delivery of DNA vaccine candidates for treatment of chronic HBV infection was evaluated in the woodchuck hepatitis virus (WHV) animal model. Initial studies in WHV-naive woodchucks confirmed enhanced reporter gene expression following EP-mediated delivery when compared to conventional intramuscular administration. Immunogenicity of DNA vaccine candidates encoding WHV antigens delivered with EP was previously demonstrated in rodents and rabbits (Luxembourg et al. 2008). EP-based delivery of a construct encoding the WHV surface antigen into WHV-naive woodchucks resulted in dose-dependent T cell and antibody responses to WHsAg that were enhanced compared to conventional intramuscular injection. In addition, EP-based vaccination induced a shift in the Th1/Th2 balance with a skew toward Th1 immune responses that is considered beneficial for therapeutic vaccination in chronic HBV infection. These encouraging results provided the impetus for evaluating EP-mediated delivery of WHV DNA vaccine candidates in woodchucks chronically infected with WHV to provide proof of concept for this approach in humans with chronic HBV infection. Given the emerging consensus supporting the combination of immunotherapy and antiviral therapy in chronic viral infections, incorporation of antiviral therapy in these studies was considered a key component of the study design. Response to therapeutic immunization following EP-mediated delivery of DNA vaccines encoding WHV antigens with or without antiviral therapy in chronically infected woodchucks is currently being evaluated.
216. Porcine Adenovirus 3 or Human Adenovirus 5 Vectors Expressing HA and NP Does Not Improve Cross-Clade Protection Against Avian Influenza H5N1 Ami Patel,1,2 Suresh K. Tikoo,3 Yan Li,1 Gary P. Kobinger.1,2 1 Public Health Agency of Canada, Winnipeg, MB, Canada; 2 Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada; 3Vaccine and Infectious Disease Organization/University of Saskatchewan, Saskatoon, SK, Canada.
Experimental vectors offer unique alternatives to traditional vaccines for improving protection against emerging infectious diseases. Adenovirus-based vectors such as porcine adenovirus 3 (PAV3) and human adenovirus 5 (AdHu5) have demonstrated protective efficacy and generation of robust immune responses against several infectious disease agents including H5N1 avian influenza virus. Combining multiple Ad-based vaccines expressing variable and conserved H5N1 antigens could potentially improve crossprotection against antigenically different H5N1 viruses. The current study evaluates protection and immune responses following coadministration of either PAV3 or AdHu5-based vaccines expressing two different influenza antigens against matched and divergent H5N1 viruses. Optimized expression cassettes containing the A/ Hanoi/30408/2005 (H05) hemagglutinin (HA) or nucleoprotein (NP) genes were inserted into replication incompetent PAV3 (PAV3-HA, PAV3-NP) or AdHu5 (AdHu5-HA, AdHu5-NP) vectors. Vaccine efficacy was first monitored in BALB/c mice following lethal homologous H05 challenge. Groups of mice were also challenged with two heterologous H5N1 viruses representing different clades: A/Hong Kong/483/1997 (HK97) and A/Indonesia/05/2005 (In05). Although both AdHu5-HA and PAV3-HA shared comparable protective efficacy against matched H05 virus, significantly better protection against divergent HK97 or In05 virus was observed in lethally challenged mice immunized with the PAV3-HA vaccine. To assess if co-administration of multiple Ad-based vaccines could improve cross-protection, (PAV3-HA+PAV3-NP) or (AdHu5-HA+AdHu5NP) were combined at a single immunization site. Interestingly, more severe signs of disease were observed against H05 following addition of the NP antigen to either vaccine. Reduced protection was also observed following vaccination with (PAV3-HA+PAV3-NP) and S84
(AdHu5-HA+AdHu5-NP) against HK97. No detectable difference in protection was observed against In05. Antibody and T-cell responses were also monitored to assess immune responses post-immunization. Higher hemagglutination inhibition antibody titres against HK97 and In05 were detected following PAV3-HA vaccination. Addition of NP did not significantly change antibody titres. Interestingly, T-cell responses against HA were equivalent following co-administration of (PAV3-HA+PAV3-NP) or (AdHu5-HA+AdHu5-NP) but reduced activation was detected against the NP antigen. Overall, the data suggests that combining adenovirus vectors expressing HA+NP does not enhance protection against lethal challenge with matched or divergent H5N1 viruses. An intriguing finding is that the addition of NP can interfere with protection provided against some H5N1 virus isolates. Further assessment of underlying immune mechanisms and the role of antigen competition will be necessary to expand cross-clade protection against antigenically diverse H5N1 viruses.
217. HIV Latency: Overcoming Transcriptional Block (Initiation and Elongation)
Suresh K. Arya.1 National Cancer Institute, National Institutes of Health, Bethesda, MD.
1
Beyond developing vaccine, curtailing HIV latency is the biggest challenge. There is no cure, and there will not be any cure, unless the latently infected cells are purged from the body. One ideal solution would be to design ‘molecular scissors’ that will scan the host genome and snip out integrated viral genome(s). However, our ability to do so is not there - yet. The major cause of latency is thought to be a transcriptional block where no viral genes are expressed, the infected cell is not marked to elicit host response or be responsive to HAART. The cell with reactivated virus expression could be subjected to immuno- or chemotherapeutic elimination. The virus uses a number of mechanisms to mute transcription to achieve and maintain latency. These include epigenetically silenced chromatin sites of integration, defect in transcriptional initiation, and block in transcriptional elongation. An additional hypothesis of transcription interference states that transcription initiated at the cellular promoter reads through the viral promoter, thus precluding the assembly of transcriptional complexes on the viral promoter itself. Much of the current effort to overcome transcriptional block has focused on activation of transcriptional initiation, such as by activating the NFKB pathway with phorbol esters, by chromatin remodeling with HDAC inhibitors, and by demethylating the viral promoter with AzaCdR. Not much effort has been expended in correcting transcriptional elongation. Elongation block could result from Tat insufficiency. Tat is the viral elongation factor, required for viral transcription. As a prelude to preclinical studies with patient CD4+ cells, we tested the idea of elongation block by using cell culture model of HIV latency (J-Lat set of cells, NIH AIDS Reagent Program. Several of these cell lines carry Env-truncated full length HIV-1 genome with the GFP gene in cis, and where virus expression is scored by GFP read-out. We demonstrate that genetically introduced extra copies of Tat indeed enhanced virus expression. Tat was provided in trans with lentiviral vectors, where Tat was directed by the hCMV or hTERT promoter. Tat by itself had only minimal effect, but in combination with NFKB activator Prostratin, it further enhanced virus expression several folds. For example, for J-Lat 6.3 cells (Env-, GFP+ provirus), preliminary results of the percentage of GFP+ cells in culture were: No treatment, 0.3 ± 0.2; Tat, 0.6 ± 0.2; Prostratin, 7.1 ± 2.1; Tat + Prostratin, 34.2 ± 7.2. Preliminary results with AzaCdR are presently unclear - with AzaCdR alone, there was little or no activation. Addition of Tat did not induce significant further activation. One interpretation would be that transcriptional initiation must be induced before Tat can promote elongation, and that in these experiments AzaCdR treatment Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
CANCER - IMMUNOTHERAPY I (in contrast to Prostratin treatment) did not induce transcriptional initiation. Experiments with the HDAC inhibitor SAHA alone and in combination with Tat are in progress.
218. Production and Characterization of Monoclonal Antibodies Against Brucella melitensis Bp26 for Mapping the Antigenic Epitope
Jinlang Qiu,1 Wenjing Wang,1 Hui Zhang,2 Yuanzhi Wang,2 Ling Zhang,1 Yixin Bian,1 Hongwei Li,1 Chuangfu Chen,2 Chengyao Li.1 1 School of Biotechnology, Southern Medical University, Guangzhou, Guangdong, China; 2Animal Science and Technology College, Shihezi University, Shihezi, Xinjiang, China.
Periplasma protein bp26 was identified as an important diagnosis antigen in Brucellosis. However, current diagnostic assays can not identify the animals wildly infected by Brucella species from the vaccinated. Molecular marker within vaccine has been considered as a way to solve the weakness of Brucella vaccines. This study was to reveal antigenic epitopes of bp26 in order to genetic modification of a current Brucella vaccine M5-90. A total of 29 monoclonal antibodies (mAb) against Brucella melitensis bp26 were produced and then tested by Peptide-ELISA, Western-Blot, and Dot-ELISA. MAbs were classified into three statuses: recognition for linear, semiconformational and conformational epitopes. To map epitopes, 28 of 16 mers overlapping peptides spanning the entire protein bp26 were synthesized. By Peptide-ELISA, 11 mAb clones reacted with three overlapping peptides, in which clone 3D7, 3H5 and 4D9 recognized peptide 11 (P11), clone 2A4, 2H9, 3H6 and 5F12 recognized P12 and 1G1, 5A5, 5B12 and 7C6 recognized both P12 and P13, respectively. For fine mapping the mAbs reactive epitopes, 12 of shorter overlapping peptides (9 mers) from P11 and P12 were synthesized. Competitive inhibition ELISA (CIEIA) was performed to determine the epitopes recognized by mAb 3H5, 2A4 and 5A5. The results showed that two linear epitopes were discovered by recognition with 3H5 or 2A4 that both reacted to the sera from vaccinated and infected sheep. These two linear epitopes could be potentially used for molecular markers of Brucella vaccines. *equal contribution.
219. GLA Synergize with Dendritic Cell-Directed LV for an Improved Immune Response Liang Xiao,1 Matt Lin,1 Jocelyn Kim,2 Lili Yang,2 Pin Wang.1 Mork Family Department of Chemical Engineering and Material Science, University of Southern California, Los Angeles, CA; 2 Division of Biology, California Institute of Technology, Los Angeles, CA.
1
Lentivectors(LVs) enveloped with Sinbis virus glycoprotein have been shown to target dendritic cells (DCs) in vitro and in vivo. These LVs can successfully generate both T cell and B cell immune responses against antigens expressed by the encoded transgene in mouse studies. DC-directed LVs have been shown to activate DCs by slightly upregulating the surface markers. In order to improve the immune response generated by this vector system, combinations of targeting LVs with TLRs agonist can be a promising strategy. GLA (glucopyranosyl lipid A), unlike LPS and MPL that are extracted from bacteria, is a novel and chemically synthesized agonist for TLR-4. Thus GLA may pose less safety concerns while maintaining its TLR-4 stimulation effect. In this study, we found that GLA-stimulated BMDC in vitro greatly enhanced the surface activation markers and the secretion of some important cytokines and chemokines; this activation is strictly TLR-4 dependent, as a TLR4 KO BMDC does not display this activation. We further assessed the effect of immunization by combining targeting LVs with GLA; this combination exhibited a significantly greater response of CD8 and CD4 T cells than that of the targeting LVs alone. Combination with GLA also enhanced the titers of different isotypes of antibodies Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
against the antigen. Taken together, an increase in T cell and B cell responses resulted in better tumor protection in both the preventive and the therapeutic EG.7 tumor challenge model. In a CD4 depletion experiment, we found that improved cytotoxic CD8 T cell response to GLA is CD4 T cell-dependent. Furthermore, the downstream signaling pathway of TLR-4 bound with GLA is both MyD88 and TRIF dependent; these conclusions were established by observing the surface activation markers and cytokine and chemokines secretions. However the MyD88 pathway is favored more as shown by the secretion of IL-6, TNF-α and IL1-β being almost MyD88-dependent in a knockout BMDC study. In a later immunization study, using MyD88 and TRIF KO mice, we observed that GLA’s adjuvant effect is MyD88-dependent. Conversely, combination with GLA in TRIF KO mice can still improve T cell response. The results of this study show that immune response generated by the DC-directed LVs can be greatly improved by the use of GLA adjuvant targeting TLR-4 and the adjuvanting effect is mediated through a MyD88 biased pathway.
Cancer - Immunotherapy I 220. Chemotherapy in Combination with T-Cell Therapy Results in Significant Antitumor Activity and Improved Clinical Outcomes for EBVAssociated Nasopharyngeal Carcinoma
Marissa Teo,1 John Chia,1 Peter Wang,1 David Tai,1 Joanna Ng,1 ChitLai Chee,1 Fei Gao,2 SzeHuey Tan,2 SooFan Ang,1 Yatanar Soe,1 Stephen Gottschalk,3 HanChong Toh.1 1 Medical Oncology, National Cancer Centre, Singapore, Singapore; 2Clinical Trials and Epidemiological Sciences, National Cancer Centre, Singapore, Singapore; 3Pediatrics and Immunology, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX.
Advanced nasopharyngeal carcinoma (NPC) is overall incurable. Presence of EBV makes adoptive immunotherapy with EBV-specific cytotoxic T cells (EBV-CTL) an attractive therapeutic option. This phase II clinical study aims to evaluate safety and efficacy of standard cytoreductive combination chemotherapy followed by serial infusions of autologous EBV-CTL. Patients with metastatic or locally recurrent NPC received, as first line therapy, 4 cycles of gemcitabine and carboplatin on days 1,8,15 every 4 weeks, followed by 6 cycles of EBV-CTL prepared using autologous lymphoblastoid cell lines as antigen presenting cells(induction dose of 1x108/m2 weekly x2 followed by maintenance of 1x108/m2 every 8 weeks x4). 38 de novo metastatic(n=19), locoregionally recurrent(n=9) or both(n=10) NPC patients were enrolled, making it the largest phase II clinical study of chemo/immunotherapy for NPC. 35 patients completed 4 cycles of gemcitabine and carboplatin. 33 patients completed at least 3 cycles of CTL and 5 patients came off study due to rapid progressive disease. CTL lines were predominantly CD8+ (mean87.62%), and contained a mixture of effector memory (mean43.85%), late effector memory (mean14.63%), and central memory T cells (mean1.66%). All 33 CTL lines contained T cells specific for immunodominant EBV antigens EBNA3s and/or BZLF1. 29 CTL lines contained T cells specific for EBV antigens predominantly expressed in NPC: LMP2(n=24), LMP1(n=7), and EBNA1(n=4). Infusions were well tolerated with no dose limiting toxicity. Objective response rate (ORR includes CR and PR) after chemotherapy was 62.2% and clinical benefit rate (CBR includes CR, PR and SD) was 94.6%. ORR post CTL infusion was 24.2% and CBR was 66.7%. Overall response rate was 64.9% with CBR of 97.3%. Intent to treat analysis of all 38 patients revealed a median progression-free survival (PFS) of 7.73 months. The 2-year OS for this trial to date, was higher (62.6%;95% CI: 41.6-77.8 vs 46.4%; 95%CI:27.6-63.3) when compared to historic controls (n=28) treated at our center with optimal 4-drug chemother apy(paclitaxel,gemcitabine,carboplatin and 5-Fluorouracil). Plasma S85
216. Porcine Adenovirus 3 or Human Adenovirus 5 Vectors Expressing HA and NP Does Not Improve Cross-Clade Protection Against Avian Influenza H5N1 Ami Patel,1,2 Suresh K. Tikoo,3 Yan Li,1 Gary P. Kobinger.1,2 1 Public Health Agency of Canada, Winnipeg, MB, Canada; 2 Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada; 3Vaccine and Infectious Disease Organization/University of Saskatchewan, Saskatoon, SK, Canada.
Experimental vectors offer unique alternatives to traditional vaccines for improving protection against emerging infectious diseases. Adenovirus-based vectors such as porcine adenovirus 3 (PAV3) and human adenovirus 5 (AdHu5) have demonstrated protective efficacy and generation of robust immune responses against several infectious disease agents including H5N1 avian influenza virus. Combining multiple Ad-based vaccines expressing variable and conserved H5N1 antigens could potentially improve crossprotection against antigenically different H5N1 viruses. The current study evaluates protection and immune responses following coadministration of either PAV3 or AdHu5-based vaccines expressing two different influenza antigens against matched and divergent H5N1 viruses. Optimized expression cassettes containing the A/ Hanoi/30408/2005 (H05) hemagglutinin (HA) or nucleoprotein (NP) genes were inserted into replication incompetent PAV3 (PAV3-HA, PAV3-NP) or AdHu5 (AdHu5-HA, AdHu5-NP) vectors. Vaccine efficacy was first monitored in BALB/c mice following lethal homologous H05 challenge. Groups of mice were also challenged with two heterologous H5N1 viruses representing different clades: A/Hong Kong/483/1997 (HK97) and A/Indonesia/05/2005 (In05). Although both AdHu5-HA and PAV3-HA shared comparable protective efficacy against matched H05 virus, significantly better protection against divergent HK97 or In05 virus was observed in lethally challenged mice immunized with the PAV3-HA vaccine. To assess if co-administration of multiple Ad-based vaccines could improve cross-protection, (PAV3-HA+PAV3-NP) or (AdHu5-HA+AdHu5NP) were combined at a single immunization site. Interestingly, more severe signs of disease were observed against H05 following addition of the NP antigen to either vaccine. Reduced protection was also observed following vaccination with (PAV3-HA+PAV3-NP) and S84
(AdHu5-HA+AdHu5-NP) against HK97. No detectable difference in protection was observed against In05. Antibody and T-cell responses were also monitored to assess immune responses post-immunization. Higher hemagglutination inhibition antibody titres against HK97 and In05 were detected following PAV3-HA vaccination. Addition of NP did not significantly change antibody titres. Interestingly, T-cell responses against HA were equivalent following co-administration of (PAV3-HA+PAV3-NP) or (AdHu5-HA+AdHu5-NP) but reduced activation was detected against the NP antigen. Overall, the data suggests that combining adenovirus vectors expressing HA+NP does not enhance protection against lethal challenge with matched or divergent H5N1 viruses. An intriguing finding is that the addition of NP can interfere with protection provided against some H5N1 virus isolates. Further assessment of underlying immune mechanisms and the role of antigen competition will be necessary to expand cross-clade protection against antigenically diverse H5N1 viruses.
217. HIV Latency: Overcoming Transcriptional Block (Initiation and Elongation)
Suresh K. Arya.1 National Cancer Institute, National Institutes of Health, Bethesda, MD.
1
Beyond developing vaccine, curtailing HIV latency is the biggest challenge. There is no cure, and there will not be any cure, unless the latently infected cells are purged from the body. One ideal solution would be to design ‘molecular scissors’ that will scan the host genome and snip out integrated viral genome(s). However, our ability to do so is not there - yet. The major cause of latency is thought to be a transcriptional block where no viral genes are expressed, the infected cell is not marked to elicit host response or be responsive to HAART. The cell with reactivated virus expression could be subjected to immuno- or chemotherapeutic elimination. The virus uses a number of mechanisms to mute transcription to achieve and maintain latency. These include epigenetically silenced chromatin sites of integration, defect in transcriptional initiation, and block in transcriptional elongation. An additional hypothesis of transcription interference states that transcription initiated at the cellular promoter reads through the viral promoter, thus precluding the assembly of transcriptional complexes on the viral promoter itself. Much of the current effort to overcome transcriptional block has focused on activation of transcriptional initiation, such as by activating the NFKB pathway with phorbol esters, by chromatin remodeling with HDAC inhibitors, and by demethylating the viral promoter with AzaCdR. Not much effort has been expended in correcting transcriptional elongation. Elongation block could result from Tat insufficiency. Tat is the viral elongation factor, required for viral transcription. As a prelude to preclinical studies with patient CD4+ cells, we tested the idea of elongation block by using cell culture model of HIV latency (J-Lat set of cells, NIH AIDS Reagent Program. Several of these cell lines carry Env-truncated full length HIV-1 genome with the GFP gene in cis, and where virus expression is scored by GFP read-out. We demonstrate that genetically introduced extra copies of Tat indeed enhanced virus expression. Tat was provided in trans with lentiviral vectors, where Tat was directed by the hCMV or hTERT promoter. Tat by itself had only minimal effect, but in combination with NFKB activator Prostratin, it further enhanced virus expression several folds. For example, for J-Lat 6.3 cells (Env-, GFP+ provirus), preliminary results of the percentage of GFP+ cells in culture were: No treatment, 0.3 ± 0.2; Tat, 0.6 ± 0.2; Prostratin, 7.1 ± 2.1; Tat + Prostratin, 34.2 ± 7.2. Preliminary results with AzaCdR are presently unclear - with AzaCdR alone, there was little or no activation. Addition of Tat did not induce significant further activation. One interpretation would be that transcriptional initiation must be induced before Tat can promote elongation, and that in these experiments AzaCdR treatment Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
CANCER - IMMUNOTHERAPY I (in contrast to Prostratin treatment) did not induce transcriptional initiation. Experiments with the HDAC inhibitor SAHA alone and in combination with Tat are in progress.
218. Production and Characterization of Monoclonal Antibodies Against Brucella melitensis Bp26 for Mapping the Antigenic Epitope
Jinlang Qiu,1 Wenjing Wang,1 Hui Zhang,2 Yuanzhi Wang,2 Ling Zhang,1 Yixin Bian,1 Hongwei Li,1 Chuangfu Chen,2 Chengyao Li.1 1 School of Biotechnology, Southern Medical University, Guangzhou, Guangdong, China; 2Animal Science and Technology College, Shihezi University, Shihezi, Xinjiang, China.
Periplasma protein bp26 was identified as an important diagnosis antigen in Brucellosis. However, current diagnostic assays can not identify the animals wildly infected by Brucella species from the vaccinated. Molecular marker within vaccine has been considered as a way to solve the weakness of Brucella vaccines. This study was to reveal antigenic epitopes of bp26 in order to genetic modification of a current Brucella vaccine M5-90. A total of 29 monoclonal antibodies (mAb) against Brucella melitensis bp26 were produced and then tested by Peptide-ELISA, Western-Blot, and Dot-ELISA. MAbs were classified into three statuses: recognition for linear, semiconformational and conformational epitopes. To map epitopes, 28 of 16 mers overlapping peptides spanning the entire protein bp26 were synthesized. By Peptide-ELISA, 11 mAb clones reacted with three overlapping peptides, in which clone 3D7, 3H5 and 4D9 recognized peptide 11 (P11), clone 2A4, 2H9, 3H6 and 5F12 recognized P12 and 1G1, 5A5, 5B12 and 7C6 recognized both P12 and P13, respectively. For fine mapping the mAbs reactive epitopes, 12 of shorter overlapping peptides (9 mers) from P11 and P12 were synthesized. Competitive inhibition ELISA (CIEIA) was performed to determine the epitopes recognized by mAb 3H5, 2A4 and 5A5. The results showed that two linear epitopes were discovered by recognition with 3H5 or 2A4 that both reacted to the sera from vaccinated and infected sheep. These two linear epitopes could be potentially used for molecular markers of Brucella vaccines. *equal contribution.
219. GLA Synergize with Dendritic Cell-Directed LV for an Improved Immune Response Liang Xiao,1 Matt Lin,1 Jocelyn Kim,2 Lili Yang,2 Pin Wang.1 Mork Family Department of Chemical Engineering and Material Science, University of Southern California, Los Angeles, CA; 2 Division of Biology, California Institute of Technology, Los Angeles, CA.
1
Lentivectors(LVs) enveloped with Sinbis virus glycoprotein have been shown to target dendritic cells (DCs) in vitro and in vivo. These LVs can successfully generate both T cell and B cell immune responses against antigens expressed by the encoded transgene in mouse studies. DC-directed LVs have been shown to activate DCs by slightly upregulating the surface markers. In order to improve the immune response generated by this vector system, combinations of targeting LVs with TLRs agonist can be a promising strategy. GLA (glucopyranosyl lipid A), unlike LPS and MPL that are extracted from bacteria, is a novel and chemically synthesized agonist for TLR-4. Thus GLA may pose less safety concerns while maintaining its TLR-4 stimulation effect. In this study, we found that GLA-stimulated BMDC in vitro greatly enhanced the surface activation markers and the secretion of some important cytokines and chemokines; this activation is strictly TLR-4 dependent, as a TLR4 KO BMDC does not display this activation. We further assessed the effect of immunization by combining targeting LVs with GLA; this combination exhibited a significantly greater response of CD8 and CD4 T cells than that of the targeting LVs alone. Combination with GLA also enhanced the titers of different isotypes of antibodies Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
against the antigen. Taken together, an increase in T cell and B cell responses resulted in better tumor protection in both the preventive and the therapeutic EG.7 tumor challenge model. In a CD4 depletion experiment, we found that improved cytotoxic CD8 T cell response to GLA is CD4 T cell-dependent. Furthermore, the downstream signaling pathway of TLR-4 bound with GLA is both MyD88 and TRIF dependent; these conclusions were established by observing the surface activation markers and cytokine and chemokines secretions. However the MyD88 pathway is favored more as shown by the secretion of IL-6, TNF-α and IL1-β being almost MyD88-dependent in a knockout BMDC study. In a later immunization study, using MyD88 and TRIF KO mice, we observed that GLA’s adjuvant effect is MyD88-dependent. Conversely, combination with GLA in TRIF KO mice can still improve T cell response. The results of this study show that immune response generated by the DC-directed LVs can be greatly improved by the use of GLA adjuvant targeting TLR-4 and the adjuvanting effect is mediated through a MyD88 biased pathway.
Cancer - Immunotherapy I 220. Chemotherapy in Combination with T-Cell Therapy Results in Significant Antitumor Activity and Improved Clinical Outcomes for EBVAssociated Nasopharyngeal Carcinoma
Marissa Teo,1 John Chia,1 Peter Wang,1 David Tai,1 Joanna Ng,1 ChitLai Chee,1 Fei Gao,2 SzeHuey Tan,2 SooFan Ang,1 Yatanar Soe,1 Stephen Gottschalk,3 HanChong Toh.1 1 Medical Oncology, National Cancer Centre, Singapore, Singapore; 2Clinical Trials and Epidemiological Sciences, National Cancer Centre, Singapore, Singapore; 3Pediatrics and Immunology, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX.
Advanced nasopharyngeal carcinoma (NPC) is overall incurable. Presence of EBV makes adoptive immunotherapy with EBV-specific cytotoxic T cells (EBV-CTL) an attractive therapeutic option. This phase II clinical study aims to evaluate safety and efficacy of standard cytoreductive combination chemotherapy followed by serial infusions of autologous EBV-CTL. Patients with metastatic or locally recurrent NPC received, as first line therapy, 4 cycles of gemcitabine and carboplatin on days 1,8,15 every 4 weeks, followed by 6 cycles of EBV-CTL prepared using autologous lymphoblastoid cell lines as antigen presenting cells(induction dose of 1x108/m2 weekly x2 followed by maintenance of 1x108/m2 every 8 weeks x4). 38 de novo metastatic(n=19), locoregionally recurrent(n=9) or both(n=10) NPC patients were enrolled, making it the largest phase II clinical study of chemo/immunotherapy for NPC. 35 patients completed 4 cycles of gemcitabine and carboplatin. 33 patients completed at least 3 cycles of CTL and 5 patients came off study due to rapid progressive disease. CTL lines were predominantly CD8+ (mean87.62%), and contained a mixture of effector memory (mean43.85%), late effector memory (mean14.63%), and central memory T cells (mean1.66%). All 33 CTL lines contained T cells specific for immunodominant EBV antigens EBNA3s and/or BZLF1. 29 CTL lines contained T cells specific for EBV antigens predominantly expressed in NPC: LMP2(n=24), LMP1(n=7), and EBNA1(n=4). Infusions were well tolerated with no dose limiting toxicity. Objective response rate (ORR includes CR and PR) after chemotherapy was 62.2% and clinical benefit rate (CBR includes CR, PR and SD) was 94.6%. ORR post CTL infusion was 24.2% and CBR was 66.7%. Overall response rate was 64.9% with CBR of 97.3%. Intent to treat analysis of all 38 patients revealed a median progression-free survival (PFS) of 7.73 months. The 2-year OS for this trial to date, was higher (62.6%;95% CI: 41.6-77.8 vs 46.4%; 95%CI:27.6-63.3) when compared to historic controls (n=28) treated at our center with optimal 4-drug chemother apy(paclitaxel,gemcitabine,carboplatin and 5-Fluorouracil). Plasma S85