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1.  HIV Env conserved element DNA vaccine alters immunodominance in macaques 
Human Vaccines & Immunotherapeutics  2017;13(12):2859-2871.
ABSTRACT
Sequence diversity and immunodominance are major obstacles in the design of an effective vaccine against HIV. HIV Env is a highly-glycosylated protein composed of ‘conserved’ and ‘variable’ regions. The latter contains immunodominant epitopes that are frequently targeted by the immune system resulting in the generation of immune escape variants. This work describes 12 regions in HIV Env that are highly conserved throughout the known HIV M Group sequences (Env CE), and are poorly immunogenic in macaques vaccinated with full-length Env expressing DNA vaccines. Two versions of plasmids encoding the 12 Env CE were generated, differing by 0–5 AA per CE to maximize the inclusion of commonly detected variants. In contrast to the full-length env DNA vaccine, vaccination of macaques with a combination of these 2 Env CE DNA induced robust, durable cellular immune responses with a significant fraction of CD8+ T cells with cytotoxic phenotype (Granzyme B+ and CD107a+). Although inefficient in generating primary responses to the CE, boosting of the Env CE DNA primed macaques with the intact env DNA vaccine potently augmented pre-existing immunity, increasing magnitude, breadth and cytotoxicity of the cellular responses. Fine mapping showed that 7 of the 12 CE elicited T cell responses. Env CE DNA also induced humoral responses able to recognize the full-length Env. Env CE plasmids are therefore capable of inducing durable responses to highly conserved regions of Env that are frequently absent after Env vaccination or immunologically subdominant. These modified antigens are candidates for use as prophylactic and therapeutic HIV vaccines.
doi:10.1080/21645515.2017.1339852
PMCID: PMC5718827  PMID: 28678607
conserved epitopes; cytotoxic T cells; DNA vaccine; Env; HIV; immunization; prime-boost; rhesus macaque; subdominant epitopes; variable epitopes
2.  31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016): part one 
Lundqvist, Andreas | van Hoef, Vincent | Zhang, Xiaonan | Wennerberg, Erik | Lorent, Julie | Witt, Kristina | Sanz, Laia Masvidal | Liang, Shuo | Murray, Shannon | Larsson, Ola | Kiessling, Rolf | Mao, Yumeng | Sidhom, John-William | Bessell, Catherine A. | Havel, Jonathan | Schneck, Jonathan | Chan, Timothy A. | Sachsenmeier, Eliot | Woods, David | Berglund, Anders | Ramakrishnan, Rupal | Sodre, Andressa | Weber, Jeffrey | Zappasodi, Roberta | Li, Yanyun | Qi, Jingjing | Wong, Philip | Sirard, Cynthia | Postow, Michael | Newman, Walter | Koon, Henry | Velcheti, Vamsidhar | Callahan, Margaret K. | Wolchok, Jedd D. | Merghoub, Taha | Lum, Lawrence G. | Choi, Minsig | Thakur, Archana | Deol, Abhinav | Dyson, Gregory | Shields, Anthony | Haymaker, Cara | Uemura, Marc | Murthy, Ravi | James, Marihella | Wang, Daqing | Brevard, Julie | Monaghan, Catherine | Swann, Suzanne | Geib, James | Cornfeld, Mark | Chunduru, Srinivas | Agrawal, Sudhir | Yee, Cassian | Wargo, Jennifer | Patel, Sapna P. | Amaria, Rodabe | Tawbi, Hussein | Glitza, Isabella | Woodman, Scott | Hwu, Wen-Jen | Davies, Michael A. | Hwu, Patrick | Overwijk, Willem W. | Bernatchez, Chantale | Diab, Adi | Massarelli, Erminia | Segal, Neil H. | Ribrag, Vincent | Melero, Ignacio | Gangadhar, Tara C. | Urba, Walter | Schadendorf, Dirk | Ferris, Robert L. | Houot, Roch | Morschhauser, Franck | Logan, Theodore | Luke, Jason J. | Sharfman, William | Barlesi, Fabrice | Ott, Patrick A. | Mansi, Laura | Kummar, Shivaani | Salles, Gilles | Carpio, Cecilia | Meier, Roland | Krishnan, Suba | McDonald, Dan | Maurer, Matthew | Gu, Xuemin | Neely, Jaclyn | Suryawanshi, Satyendra | Levy, Ronald | Khushalani, Nikhil | Wu, Jennifer | Zhang, Jinyu | Basher, Fahmin | Rubinstein, Mark | Bucsek, Mark | Qiao, Guanxi | MacDonald, Cameron | Hylander, Bonnie | Repasky, Elizabeth | Chatterjee, Shilpak | Daenthanasanmak, Anusara | Chakraborty, Paramita | Toth, Kyle | Meek, Megan | Garrett-Mayer, Elizabeth | Nishimura, Michael | Paulos, Chrystal | Beeson, Craig | Yu, Xuezhong | Mehrotra, Shikhar | Zhao, Fei | Evans, Kathy | Xiao, Christine | Holtzhausen, Alisha | Hanks, Brent A. | Scharping, Nicole | Menk, Ashley V. | Moreci, Rebecca | Whetstone, Ryan | Dadey, Rebekah | Watkins, Simon | Ferris, Robert | Delgoffe, Greg M. | Peled, Jonathan | Devlin, Sean | Staffas, Anna | Lumish, Melissa | Rodriguez, Kori Porosnicu | Ahr, Katya | Perales, Miguel | Giralt, Sergio | Taur, Ying | Pamer, Eric | van den Brink, Marcel R. M. | Jenq, Robert | Annels, Nicola | Pandha, Hardev | Simpson, Guy | Mostafid, Hugh | Harrington, Kevin | Melcher, Alan | Grose, Mark | Davies, Bronwyn | Au, Gough | Karpathy, Roberta | Shafren, Darren | Ricca, Jacob | Merghoub, Taha | Wolchok, Jedd D. | Zamarin, Dmitriy | Batista, Luciana | Marliot, Florence | Vasaturo, Angela | Carpentier, Sabrina | Poggionovo, Cécile | Frayssinet, Véronique | Fieschi, Jacques | Van den Eynde, Marc | Pagès, Franck | Galon, Jérôme | Hermitte, Fabienne | Smith, Sean G. | Nguyen, Khue | Ravindranathan, Sruthi | Koppolu, Bhanu | Zaharoff, David | Schvartsman, Gustavo | Bassett, Roland | McQuade, Jennifer L. | Haydu, Lauren E. | Davies, Michael A. | Tawbi, Hussein | Glitza, Isabella | Kline, Douglas | Chen, Xiufen | Fosco, Dominick | Kline, Justin | Overacre, Abigail | Chikina, Maria | Brunazzi, Erin | Shayan, Gulidanna | Horne, William | Kolls, Jay | Ferris, Robert L. | Delgoffe, Greg M. | Bruno, Tullia C. | Workman, Creg | Vignali, Dario | Adusumilli, Prasad S. | Ansa-Addo, Ephraim A | Li, Zihai | Gerry, Andrew | Sanderson, Joseph P. | Howe, Karen | Docta, Roslin | Gao, Qian | Bagg, Eleanor A. L. | Tribble, Nicholas | Maroto, Miguel | Betts, Gareth | Bath, Natalie | Melchiori, Luca | Lowther, Daniel E. | Ramachandran, Indu | Kari, Gabor | Basu, Samik | Binder-Scholl, Gwendolyn | Chagin, Karen | Pandite, Lini | Holdich, Tom | Amado, Rafael | Zhang, Hua | Glod, John | Bernstein, Donna | Jakobsen, Bent | Mackall, Crystal | Wong, Ryan | Silk, Jonathan D. | Adams, Katherine | Hamilton, Garth | Bennett, Alan D. | Brett, Sara | Jing, Junping | Quattrini, Adriano | Saini, Manoj | Wiedermann, Guy | Gerry, Andrew | Jakobsen, Bent | Binder-Scholl, Gwendolyn | Brewer, Joanna | Duong, MyLinh | Lu, An | Chang, Peter | Mahendravada, Aruna | Shinners, Nicholas | Slawin, Kevin | Spencer, David M. | Foster, Aaron E. | Bayle, J. Henri | Bergamaschi, Cristina | Ng, Sinnie Sin Man | Nagy, Bethany | Jensen, Shawn | Hu, Xintao | Alicea, Candido | Fox, Bernard | Felber, Barbara | Pavlakis, George | Chacon, Jessica | Yamamoto, Tori | Garrabrant, Thomas | Cortina, Luis | Powell, Daniel J. | Donia, Marco | Kjeldsen, Julie Westerlin | Andersen, Rikke | Westergaard, Marie Christine Wulff | Bianchi, Valentina | Legut, Mateusz | Attaf, Meriem | Dolton, Garry | Szomolay, Barbara | Ott, Sascha | Lyngaa, Rikke | Hadrup, Sine Reker | Sewell, Andrew Kelvin | Svane, Inge Marie | Fan, Aaron | Kumai, Takumi | Celis, Esteban | Frank, Ian | Stramer, Amanda | Blaskovich, Michelle A. | Wardell, Seth | Fardis, Maria | Bender, James | Lotze, Michael T. | Goff, Stephanie L. | Zacharakis, Nikolaos | Assadipour, Yasmine | Prickett, Todd D. | Gartner, Jared J. | Somerville, Robert | Black, Mary | Xu, Hui | Chinnasamy, Harshini | Kriley, Isaac | Lu, Lily | Wunderlich, John | Robbins, Paul F. | Rosenberg, Steven | Feldman, Steven A. | Trebska-McGowan, Kasia | Kriley, Isaac | Malekzadeh, Parisa | Payabyab, Eden | Sherry, Richard | Rosenberg, Steven | Goff, Stephanie L. | Gokuldass, Aishwarya | Blaskovich, Michelle A. | Kopits, Charlene | Rabinovich, Brian | Lotze, Michael T. | Green, Daniel S. | Kamenyeva, Olena | Zoon, Kathryn C. | Annunziata, Christina M. | Hammill, Joanne | Helsen, Christopher | Aarts, Craig | Bramson, Jonathan | Harada, Yui | Yonemitsu, Yoshikazu | Helsen, Christopher | Hammill, Joanne | Mwawasi, Kenneth | Denisova, Galina | Bramson, Jonathan | Giri, Rajanish | Jin, Benjamin | Campbell, Tracy | Draper, Lindsey M. | Stevanovic, Sanja | Yu, Zhiya | Weissbrich, Bianca | Restifo, Nicholas P. | Trimble, Cornelia L. | Rosenberg, Steven | Hinrichs, Christian S. | Tsang, Kwong | Fantini, Massimo | Hodge, James W. | Fujii, Rika | Fernando, Ingrid | Jochems, Caroline | Heery, Christopher | Gulley, James | Soon-Shiong, Patrick | Schlom, Jeffrey | Jing, Weiqing | Gershan, Jill | Blitzer, Grace | Weber, James | McOlash, Laura | Johnson, Bryon D. | Kiany, Simin | Gangxiong, Huang | Kleinerman, Eugenie S. | Klichinsky, Michael | Ruella, Marco | Shestova, Olga | Kenderian, Saad | Kim, Miriam | Scholler, John | June, Carl H. | Gill, Saar | Moogk, Duane | Zhong, Shi | Yu, Zhiya | Liadi, Ivan | Rittase, William | Fang, Victoria | Dougherty, Janna | Perez-Garcia, Arianne | Osman, Iman | Zhu, Cheng | Varadarajan, Navin | Restifo, Nicholas P. | Frey, Alan | Krogsgaard, Michelle | Landi, Daniel | Fousek, Kristen | Mukherjee, Malini | Shree, Ankita | Joseph, Sujith | Bielamowicz, Kevin | Byrd, Tiara | Ahmed, Nabil | Hegde, Meenakshi | Lee, Sylvia | Byrd, David | Thompson, John | Bhatia, Shailender | Tykodi, Scott | Delismon, Judy | Chu, Liz | Abdul-Alim, Siddiq | Ohanian, Arpy | DeVito, Anna Marie | Riddell, Stanley | Margolin, Kim | Magalhaes, Isabelle | Mattsson, Jonas | Uhlin, Michael | Nemoto, Satoshi | Villarroel, Patricio Pérez | Nakagawa, Ryosuke | Mule, James J. | Mailloux, Adam W. | Mata, Melinda | Nguyen, Phuong | Gerken, Claudia | DeRenzo, Christopher | Spencer, David M. | Gottschalk, Stephen | Mathieu, Mélissa | Pelletier, Sandy | Stagg, John | Turcotte, Simon | Minutolo, Nicholas | Sharma, Prannda | Tsourkas, Andrew | Powell, Daniel J. | Mockel-Tenbrinck, Nadine | Mauer, Daniela | Drechsel, Katharina | Barth, Carola | Freese, Katharina | Kolrep, Ulrike | Schult, Silke | Assenmacher, Mario | Kaiser, Andrew | Mullinax, John | Hall, MacLean | Le, Julie | Kodumudi, Krithika | Royster, Erica | Richards, Allison | Gonzalez, Ricardo | Sarnaik, Amod | Pilon-Thomas, Shari | Nielsen, Morten | Krarup-Hansen, Anders | Hovgaard, Dorrit | Petersen, Michael Mørk | Loya, Anand Chainsukh | Junker, Niels | Svane, Inge Marie | Rivas, Charlotte | Parihar, Robin | Gottschalk, Stephen | Rooney, Cliona M. | Qin, Haiying | Nguyen, Sang | Su, Paul | Burk, Chad | Duncan, Brynn | Kim, Bong-Hyun | Kohler, M. Eric | Fry, Terry | Rao, Arjun A. | Teyssier, Noam | Pfeil, Jacob | Sgourakis, Nikolaos | Salama, Sofie | Haussler, David | Richman, Sarah A. | Nunez-Cruz, Selene | Gershenson, Zack | Mourelatos, Zissimos | Barrett, David | Grupp, Stephan | Milone, Michael | Rodriguez-Garcia, Alba | Robinson, Matthew K. | Adams, Gregory P. | Powell, Daniel J. | Santos, João | Havunen, Riikka | Siurala, Mikko | Cervera-Carrascón, Víctor | Parviainen, Suvi | Antilla, Marjukka | Hemminki, Akseli | Sethuraman, Jyothi | Santiago, Laurelis | Chen, Jie Qing | Dai, Zhimin | Wardell, Seth | Bender, James | Lotze, Michael T. | Sha, Huizi | Su, Shu | Ding, Naiqing | Liu, Baorui | Stevanovic, Sanja | Pasetto, Anna | Helman, Sarah R. | Gartner, Jared J. | Prickett, Todd D. | Robbins, Paul F. | Rosenberg, Steven A. | Hinrichs, Christian S. | Bhatia, Shailender | Burgess, Melissa | Zhang, Hui | Lee, Tien | Klingemann, Hans | Soon-Shiong, Patrick | Nghiem, Paul | Kirkwood, John M. | Rossi, John M. | Sherman, Marika | Xue, Allen | Shen, Yueh-wei | Navale, Lynn | Rosenberg, Steven A. | Kochenderfer, James N. | Bot, Adrian | Veerapathran, Anandaraman | Gokuldass, Aishwarya | Stramer, Amanda | Sethuraman, Jyothi | Blaskovich, Michelle A. | Wiener, Doris | Frank, Ian | Santiago, Laurelis | Rabinovich, Brian | Fardis, Maria | Bender, James | Lotze, Michael T. | Waller, Edmund K. | Li, Jian-Ming | Petersen, Christopher | Blazar, Bruce R. | Li, Jingxia | Giver, Cynthia R. | Wang, Ziming | Grossenbacher, Steven K. | Sturgill, Ian | Canter, Robert J. | Murphy, William J. | Zhang, Congcong | Burger, Michael C. | Jennewein, Lukas | Waldmann, Anja | Mittelbronn, Michel | Tonn, Torsten | Steinbach, Joachim P. | Wels, Winfried S. | Williams, Jason B. | Zha, Yuanyuan | Gajewski, Thomas F. | Williams, LaTerrica C. | Krenciute, Giedre | Kalra, Mamta | Louis, Chrystal | Gottschalk, Stephen | Xin, Gang | Schauder, David | Jiang, Aimin | Joshi, Nikhil | Cui, Weiguo | Zeng, Xue | Menk, Ashley V. | Scharping, Nicole | Delgoffe, Greg M. | Zhao, Zeguo | Hamieh, Mohamad | Eyquem, Justin | Gunset, Gertrude | Bander, Neil | Sadelain, Michel | Askmyr, David | Abolhalaj, Milad | Lundberg, Kristina | Greiff, Lennart | Lindstedt, Malin | Angell, Helen K. | Kim, Kyoung-Mee | Kim, Seung-Tae | Kim, Sung | Sharpe, Alan D. | Ogden, Julia | Davenport, Anna | Hodgson, Darren R. | Barrett, Carl | Lee, Jeeyun | Kilgour, Elaine | Hanson, Jodi | Caspell, Richard | Karulin, Alexey | Lehmann, Paul | Ansari, Tameem | Schiller, Annemarie | Sundararaman, Srividya | Lehmann, Paul | Hanson, Jodi | Roen, Diana | Karulin, Alexey | Lehmann, Paul | Ayers, Mark | Levitan, Diane | Arreaza, Gladys | Liu, Fang | Mogg, Robin | Bang, Yung-Jue | O’Neil, Bert | Cristescu, Razvan | Friedlander, Philip | Wassman, Karl | Kyi, Chrisann | Oh, William | Bhardwaj, Nina | Bornschlegl, Svetlana | Gustafson, Michael P. | Gastineau, Dennis A. | Parney, Ian F. | Dietz, Allan B. | Carvajal-Hausdorf, Daniel | Mani, Nikita | Velcheti, Vamsidhar | Schalper, Kurt | Rimm, David | Chang, Serena | Levy, Ronald | Kurland, John | Krishnan, Suba | Ahlers, Christoph Matthias | Jure-Kunkel, Maria | Cohen, Lewis | Maecker, Holden | Kohrt, Holbrook | Chen, Shuming | Crabill, George | Pritchard, Theresa | McMiller, Tracee | Pardoll, Drew | Pan, Fan | Topalian, Suzanne | Danaher, Patrick | Warren, Sarah | Dennis, Lucas | White, Andrew M. | D’Amico, Leonard | Geller, Melissa | Disis, Mary L. | Beechem, Joseph | Odunsi, Kunle | Fling, Steven | Derakhshandeh, Roshanak | Webb, Tonya J. | Dubois, Sigrid | Conlon, Kevin | Bryant, Bonita | Hsu, Jennifer | Beltran, Nancy | Müller, Jürgen | Waldmann, Thomas | Duhen, Rebekka | Duhen, Thomas | Thompson, Lucas | Montler, Ryan | Weinberg, Andrew | Kates, Max | Early, Brandon | Yusko, Erik | Schreiber, Taylor H. | Bivalacqua, Trinity J. | Ayers, Mark | Lunceford, Jared | Nebozhyn, Michael | Murphy, Erin | Loboda, Andrey | Kaufman, David R. | Albright, Andrew | Cheng, Jonathan | Kang, S. Peter | Shankaran, Veena | Piha-Paul, Sarina A. | Yearley, Jennifer | Seiwert, Tanguy | Ribas, Antoni | McClanahan, Terrill K. | Cristescu, Razvan | Mogg, Robin | Ayers, Mark | Albright, Andrew | Murphy, Erin | Yearley, Jennifer | Sher, Xinwei | Liu, Xiao Qiao | Nebozhyn, Michael | Lunceford, Jared | Joe, Andrew | Cheng, Jonathan | Plimack, Elizabeth | Ott, Patrick A. | McClanahan, Terrill K. | Loboda, Andrey | Kaufman, David R. | Forrest-Hay, Alex | Guyre, Cheryl A. | Narumiya, Kohei | Delcommenne, Marc | Hirsch, Heather A. | Deshpande, Amit | Reeves, Jason | Shu, Jenny | Zi, Tong | Michaelson, Jennifer | Law, Debbie | Trehu, Elizabeth | Sathyanaryanan, Sriram | Hodkinson, Brendan P. | Hutnick, Natalie A. | Schaffer, Michael E. | Gormley, Michael | Hulett, Tyler | Jensen, Shawn | Ballesteros-Merino, Carmen | Dubay, Christopher | Afentoulis, Michael | Reddy, Ashok | David, Larry | Fox, Bernard | Jayant, Kumar | Agrawal, Swati | Agrawal, Rajendra | Jeyakumar, Ghayathri | Kim, Seongho | Kim, Heejin | Silski, Cynthia | Suisham, Stacey | Heath, Elisabeth | Vaishampayan, Ulka | Vandeven, Natalie | Viller, Natasja Nielsen | O’Connor, Alison | Chen, Hui | Bossen, Bolette | Sievers, Eric | Uger, Robert | Nghiem, Paul | Johnson, Lisa | Kao, Hsiang-Fong | Hsiao, Chin-Fu | Lai, Shu-Chuan | Wang, Chun-Wei | Ko, Jenq-Yuh | Lou, Pei-Jen | Lee, Tsai-Jan | Liu, Tsang-Wu | Hong, Ruey-Long | Kearney, Staci J. | Black, Joshua C. | Landis, Benjamin J. | Koegler, Sally | Hirsch, Brooke | Gianani, Roberto | Kim, Jeffrey | He, Ming-Xiao | Zhang, Bingqing | Su, Nan | Luo, Yuling | Ma, Xiao-Jun | Park, Emily | Kim, Dae Won | Copploa, Domenico | Kothari, Nishi | doo Chang, Young | Kim, Richard | Kim, Namyong | Lye, Melvin | Wan, Ee | Kim, Namyong | Lye, Melvin | Wan, Ee | Kim, Namyong | Lye, Melvin | Wan, Ee | Knaus, Hanna A. | Berglund, Sofia | Hackl, Hubert | Karp, Judith E. | Gojo, Ivana | Luznik, Leo | Hong, Henoch S. | Koch, Sven D. | Scheel, Birgit | Gnad-Vogt, Ulrike | Kallen, Karl-Josef | Wiegand, Volker | Backert, Linus | Kohlbacher, Oliver | Hoerr, Ingmar | Fotin-Mleczek, Mariola | Billingsley, James M. | Koguchi, Yoshinobu | Conrad, Valerie | Miller, William | Gonzalez, Iliana | Poplonski, Tomasz | Meeuwsen, Tanisha | Howells-Ferreira, Ana | Rattray, Rogan | Campbell, Mary | Bifulco, Carlo | Dubay, Christopher | Bahjat, Keith | Curti, Brendan | Urba, Walter | Vetsika, E-K | Kallergi, G. | Aggouraki, Despoina | Lyristi, Z. | Katsarlinos, P. | Koinis, Filippos | Georgoulias, V. | Kotsakis, Athanasios | Martin, Nathan T. | Aeffner, Famke | Kearney, Staci J. | Black, Joshua C. | Cerkovnik, Logan | Pratte, Luke | Kim, Rebecca | Hirsch, Brooke | Krueger, Joseph | Gianani, Roberto | Martínez-Usatorre, Amaia | Jandus, Camilla | Donda, Alena | Carretero-Iglesia, Laura | Speiser, Daniel E. | Zehn, Dietmar | Rufer, Nathalie | Romero, Pedro | Panda, Anshuman | Mehnert, Janice | Hirshfield, Kim M. | Riedlinger, Greg | Damare, Sherri | Saunders, Tracie | Sokol, Levi | Stein, Mark | Poplin, Elizabeth | Rodriguez-Rodriguez, Lorna | Silk, Ann | Chan, Nancy | Frankel, Melissa | Kane, Michael | Malhotra, Jyoti | Aisner, Joseph | Kaufman, Howard L. | Ali, Siraj | Ross, Jeffrey | White, Eileen | Bhanot, Gyan | Ganesan, Shridar | Monette, Anne | Bergeron, Derek | Amor, Amira Ben | Meunier, Liliane | Caron, Christine | Morou, Antigoni | Kaufmann, Daniel | Liberman, Moishe | Jurisica, Igor | Mes-Masson, Anne-Marie | Hamzaoui, Kamel | Lapointe, Rejean | Mongan, Ann | Ku, Yuan-Chieh | Tom, Warren | Sun, Yongming | Pankov, Alex | Looney, Tim | Au-Young, Janice | Hyland, Fiona | Conroy, Jeff | Morrison, Carl | Glenn, Sean | Burgher, Blake | Ji, He | Gardner, Mark | Mongan, Ann | Omilian, Angela R. | Conroy, Jeff | Bshara, Wiam | Angela, Omilian | Burgher, Blake | Ji, He | Glenn, Sean | Morrison, Carl | Mongan, Ann | Obeid, Joseph M. | Erdag, Gulsun | Smolkin, Mark E. | Deacon, Donna H. | Patterson, James W. | Chen, Lieping | Bullock, Timothy N. | Slingluff, Craig L. | Obeid, Joseph M. | Erdag, Gulsun | Deacon, Donna H. | Slingluff, Craig L. | Bullock, Timothy N. | Loffredo, John T. | Vuyyuru, Raja | Beyer, Sophie | Spires, Vanessa M. | Fox, Maxine | Ehrmann, Jon M. | Taylor, Katrina A. | Korman, Alan J. | Graziano, Robert F. | Page, David | Sanchez, Katherine | Ballesteros-Merino, Carmen | Martel, Maritza | Bifulco, Carlo | Urba, Walter | Fox, Bernard | Patel, Sapna P. | De Macedo, Mariana Petaccia | Qin, Yong | Reuben, Alex | Spencer, Christine | Guindani, Michele | Bassett, Roland | Wargo, Jennifer | Racolta, Adriana | Kelly, Brian | Jones, Tobin | Polaske, Nathan | Theiss, Noah | Robida, Mark | Meridew, Jeffrey | Habensus, Iva | Zhang, Liping | Pestic-Dragovich, Lidija | Tang, Lei | Sullivan, Ryan J. | Logan, Theodore | Khushalani, Nikhil | Margolin, Kim | Koon, Henry | Olencki, Thomas | Hutson, Thomas | Curti, Brendan | Roder, Joanna | Blackmon, Shauna | Roder, Heinrich | Stewart, John | Amin, Asim | Ernstoff, Marc S. | Clark, Joseph I. | Atkins, Michael B. | Kaufman, Howard L. | Sosman, Jeffrey | Weber, Jeffrey | McDermott, David F. | Weber, Jeffrey | Kluger, Harriet | Halaban, Ruth | Snzol, Mario | Roder, Heinrich | Roder, Joanna | Asmellash, Senait | Steingrimsson, Arni | Blackmon, Shauna | Sullivan, Ryan J. | Wang, Chichung | Roman, Kristin | Clement, Amanda | Downing, Sean | Hoyt, Clifford | Harder, Nathalie | Schmidt, Guenter | Schoenmeyer, Ralf | Brieu, Nicolas | Yigitsoy, Mehmet | Madonna, Gabriele | Botti, Gerardo | Grimaldi, Antonio | Ascierto, Paolo A. | Huss, Ralf | Athelogou, Maria | Hessel, Harald | Harder, Nathalie | Buchner, Alexander | Schmidt, Guenter | Stief, Christian | Huss, Ralf | Binnig, Gerd | Kirchner, Thomas | Sellappan, Shankar | Thyparambil, Sheeno | Schwartz, Sarit | Cecchi, Fabiola | Nguyen, Andrew | Vaske, Charles | Hembrough, Todd
Journal for Immunotherapy of Cancer  2016;4(Suppl 1):1-106.
doi:10.1186/s40425-016-0172-7
PMCID: PMC5123387
3.  DNA Prime-Boost Vaccine Regimen To Increase Breadth, Magnitude, and Cytotoxicity of the Cellular Immune Responses to Subdominant Gag Epitopes of Simian Immunodeficiency Virus and HIV 
The Journal of Immunology Author Choice  2016;197(10):3999-4013.
HIV sequence diversity and the propensity of eliciting immunodominant responses targeting variable regions of the HIV proteome are hurdles in the development of an effective AIDS vaccine. An HIV-derived conserved element (CE) p24gag plasmid DNA (pDNA) vaccine is able to redirect immunodominant responses to otherwise subdominant and often more vulnerable viral targets. By homology to the HIV immunogen, seven CE were identified in SIV p27Gag. Analysis of 31 rhesus macaques vaccinated with full-length SIV gag pDNA showed inefficient induction (58% response rate) of cellular responses targeting these CE. In contrast, all 14 macaques immunized with SIV p27CE pDNA developed robust T cell responses recognizing CE. Vaccination with p27CE pDNA was also critical for the efficient induction and increased the frequency of Ag-specific T cells with cytotoxic potential (granzyme B+ CD107a+) targeting subdominant CE epitopes, compared with the responses elicited by the p57gag pDNA vaccine. Following p27CE pDNA priming, two booster regimens, gag pDNA or codelivery of p27CE+gag pDNA, significantly increased the levels of CE-specific T cells. However, the CE+gag pDNA booster vaccination elicited significantly broader CE epitope recognition, and thus, a more profound alteration of the immunodominance hierarchy. Vaccination with HIV molecules showed that CE+gag pDNA booster regimen further expanded the breadth of HIV CE responses. Hence, SIV/HIV vaccine regimens comprising CE pDNA prime and CE+gag pDNA booster vaccination significantly increased cytotoxic T cell responses to subdominant highly conserved Gag epitopes and maximized response breadth.
doi:10.4049/jimmunol.1600697
PMCID: PMC5096510  PMID: 27733554
4.  Comparative Analysis of SIV-specific Cellular Immune Responses Induced by Different Vaccine Platforms in Rhesus Macaques 
To identify the most promising vaccine candidates for combinatorial strategies, we compared five SIV vaccine platforms including recombinant canary pox virus ALVAC, replication-competent adenovirus type 5 host range mutant RepAd, DNA, modified vaccinia Ankara (MVA), peptides and protein in distinct combinations. Three regimens used viral vectors (prime or boost) and two regimens used plasmid DNA. Analysis at necropsy showed that the DNA-based vaccine regimens elicited significantly higher cellular responses against Gag and Env than any of the other vaccine platforms. The T cell responses induced by most vaccine regimens disseminated systemically into secondary lymphoid tissues (lymph nodes, spleen) and effector anatomical sites (including liver, vaginal tissue), indicative of their role in viral containment at the portal of entry. The cellular and reported humoral immune response data suggest that combination of DNA and viral vectors elicits a balanced immunity with strong and durable responses able to disseminate into relevant mucosal sites.
doi:10.1016/j.clim.2014.09.005
PMCID: PMC4252823  PMID: 25229164
cellular immune response; prime-boost vaccination; DNA; protein; pox virus ALVAC MVA
7.  DNA Vaccination by Intradermal Electroporation Induces Long-lasting Immune Responses in Rhesus Macaques 
Journal of medical primatology  2014;43(5):329-340.
Background
A desirable HIV vaccine should induce protective long-lasting humoral and cellular immune responses.
Methods
Macaques were immunized by env DNA, selected from a panel of recently transmitted SIVmac251 Env using intradermal electroporation as vaccine delivery method and magnitude, breadth and longevity of humoral and cellular immune responses.
Results
The macaques developed high, long-lasting humoral immune responses with neutralizing capacity against homologous and heterologous Env. The avidity of the antibody responses was also preserved over 1 year of follow-up. Analysis of cellular immune responses demonstrated induction of Env-specific memory T cells harboring granzyme B, albeit their overall levels were low. Similar to the humoral responses, the cellular immunity was persistent over the ~1 year of follow-up.
Conclusion
These data show that vaccination by this intradermal DNA delivery regimen is able to induce potent and durable immune responses in macaques.
doi:10.1111/jmp.12123
PMCID: PMC4176517  PMID: 24810337
DNA vaccine; HIV; SIV; binding antibody; neutralizing antibody; avidity; cellular responses; durability of responses
8.  Humoral immunity induced by mucosal and/or systemic SIV-specific vaccine platforms suggest novel combinatorial approaches for enhancing responses 
Clinical immunology (Orlando, Fla.)  2014;153(2):308-322.
Combinatorial HIV/SIV vaccine approaches targeting multiple arms of the immune system might improve protective efficacy. We compared SIV-specific humoral immunity induced in rhesus macaques by five vaccine regimens. Systemic regimens included ALVAC-SIVenv priming and Env boosting (ALVAC/Env); DNA immunization; and DNA plus Env co-immunization (DNA&Env). RepAd/Env combined mucosal replication-competent Ad-env priming with systemic Env boosting. A Peptide/Env regimen, given solely intrarectally, included HIV/SIV peptides followed by MVA-env and Env boosts. Serum antibodies mediating neutralizing, phagocytic and ADCC activities were induced by ALVAC/Env, RepAd/Env and DNA&Env vaccines. Memory B cells and plasma cells were maintained in bone marrow. RepAd/Env vaccination induced early SIV-specific IgA in rectal secretions before Env boosting, although mucosal IgA and IgG responses were readily detected at necropsy in ALVAC/Env, RepAd/Env, DNA&Env and DNA vaccinated animals. Our results suggest combined RepAd priming with ALVAC/Env or DNA&Env regimen boosting might induce potent, functional, long-lasting systemic and mucosal SIV-specific antibodies.
doi:10.1016/j.clim.2014.05.008
PMCID: PMC4102324  PMID: 24907411
Simian Immunodeficiency Virus; poxvirus-, adenovirus-, and DNA-based vaccines; mucosal and systemic humoral immunity; memory B cells; functional antibody activities
9.  Intramuscular delivery of heterodimeric IL-15 DNA in macaques produces systemic levels of bioactive cytokine inducing proliferation of NK and T cells 
Gene therapy  2014;22(1):76-86.
Interleukin-15 (IL-15) is a common γ-chain cytokine that plays a significant role in the activation and proliferation of T and NK cells and holds great potential in fighting infection and cancer. We have previously shown that bioactive IL-15 in vivo comprises a complex of the IL-15 chain with the soluble or cell-associated IL-15 receptor alpha (IL-15Rα) chain, which together form the IL-15 heterodimer. We have generated DNA vectors expressing the heterodimeric IL-15 by optimizing mRNA expression and protein trafficking. Repeated administration of these DNA plasmids by intramuscular injection followed by in vivo electroporation in rhesus macaques resulted in sustained high levels of IL-15 in plasma, with no significant toxicity. Administration of DNAs expressing heterodimeric IL-15 also resulted in an increased frequency of NK and T cells undergoing proliferation in peripheral blood. Heterodimeric IL-15 led to preferential expansion of CD8+NK cells, all memory CD8+ T cell subsets and effector memory CD4+ T cells. Expression of heterodimeric IL-15 by DNA delivery to the muscle is an efficient procedure to obtain high systemic levels of bioactive cytokine, without the toxicity linked to the high transient cytokine peak associated with protein injection.
doi:10.1038/gt.2014.84
PMCID: PMC4289118  PMID: 25273353
10.  Dose-dependent inhibition of Gag cellular immunity by Env in SIV/HIV DNA vaccinated macaques 
Human Vaccines & Immunotherapeutics  2015;11(8):2005-2011.
The induction of a balanced immune response targeting the major structural proteins, Gag and Env of HIV, is important for the development of an efficacious vaccine. The use of DNA plasmids expressing different antigens offers the opportunity to test in a controlled manner the influence of different vaccine components on the magnitude and distribution of the vaccine-induced cellular and humoral immune responses. Here, we show that increasing amounts of env DNA results in greatly enhanced Env antibody titers without significantly affecting the levels of anti-Env cellular immune responses. Co-immunization with Env protein further increased antibody levels, indicating that vaccination with DNA only is not sufficient for eliciting maximal humoral responses against Env. In contrast, under high env:gag DNA plasmid ratio, the development of Gag cellular responses was significantly reduced by either SIV or HIV Env, whereas Gag humoral responses were not affected. Our data indicate that a balanced ratio of the 2 key HIV/SIV vaccine components, Gag and Env, is important to avoid immunological interference and to achieve both maximal humoral responses against Env to prevent virus acquisition and maximal cytotoxic T cell responses against Gag to prevent virus spread.
doi:10.1080/21645515.2015.1016671
PMCID: PMC4635869  PMID: 26125521
cellular immunity; DNA electroporation; HIV vaccines; humoral immunity; vaccine optimization
11.  DNA is an efficient booster of dendritic cell-based vaccine 
Human Vaccines & Immunotherapeutics  2015;11(8):1927-1935.
DC-based therapeutic vaccines as a promising strategy against chronic infections and cancer have been validated in several clinical trials. However, DC-based vaccines are complex and require many in vitro manipulations, which makes this a personalized and expensive therapeutic approach. In contrast, DNA-based vaccines have many practical advantages including simplicity, low cost of manufacturing and potent immunogenicity already proven in non-human primates and humans. In this study, we explored whether DC-based vaccines can be simplified by the addition of plasmid DNA as prime or boost to achieve robust CD8-mediated immune responses. We compared the cellular immunity induced in BALB/c and C57BL/6 mice by DC vaccines, loaded either with peptides or optimized SIV Env DNA, and plasmid DNA-based vaccines delivered by electroporation (EP). We found that mature DC loaded with peptides (P-mDC) induced the highest CD8+ T cell responses in both strains of mice, but those responses were significantly higher in the C57BL/6 model. A heterologous prime-boost strategy (P-DC prime-DNA boost) induced CD8+ T cell responses similar to those obtained by the P-DC vaccine. Importantly, this strategy elicited robust polyfunctional T cells as well as highest antigen-specific central memory CD8+ T cells in C57BL/6 mice, suggesting long-term memory responses. These results indicate that a DC-based vaccine in combination with DNA in a heterologous DC prime-DNA boost strategy has potential as a repeatedly administered vaccine.
doi:10.1080/21645515.2015.1020265
PMCID: PMC4635890  PMID: 26125100
DNA vaccine; mice; dendritic cells; peptide
12.  A human immune data-informed vaccine concept elicits strong and broad T-cell specificities associated with HIV-1 control in mice and macaques 
Background
None of the HIV T-cell vaccine candidates that have reached advanced clinical testing have been able to induce protective T cell immunity. A major reason for these failures may have been suboptimal T cell immunogen designs.
Methods
To overcome this problem, we used a novel immunogen design approach that is based on functional T cell response data from more than 1,000 HIV-1 clade B and C infected individuals and which aims to direct the T cell response to the most vulnerable sites of HIV-1.
Results
Our approach identified 16 regions in Gag, Pol, Vif and Nef that were relatively conserved and predominantly targeted by individuals with reduced viral loads. These regions formed the basis of the HIVACAT T-cell Immunogen (HTI) sequence which is 529 amino acids in length, includes more than 50 optimally defined CD4+ and CD8+ T-cell epitopes restricted by a wide range of HLA class I and II molecules and covers viral sites where mutations led to a dramatic reduction in viral replicative fitness. In both, C57BL/6 mice and Indian rhesus macaques immunized with an HTI-expressing DNA plasmid (DNA.HTI) induced broad and balanced T-cell responses to several segments within Gag, Pol, and Vif. DNA.HTI induced robust CD4+ and CD8+ T cell responses that were increased by a booster vaccination using modified virus Ankara (MVA.HTI), expanding the DNA.HTI induced response to up to 3.2% IFN-γ T-cells in macaques. HTI-specific T cells showed a central and effector memory phenotype with a significant fraction of the IFN-γ+ CD8+ T cells being Granzyme B+ and able to degranulate (CD107a+).
Conclusions
These data demonstrate the immunogenicity of a novel HIV-1 T cell vaccine concept that induced broadly balanced responses to vulnerable sites of HIV-1 while avoiding the induction of responses to potential decoy targets that may divert effective T-cell responses towards variable and less protective viral determinants.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-015-0392-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12967-015-0392-5
PMCID: PMC4336696  PMID: 25879820
HIV-1 T-cell immunogen; HIV-1 specific CTL; HLA; Immunogenicity; Subdominant; Viral fitness; CTL escape; T-helper epitope; Population coverage
13.  DNA and Protein Co-Immunization Improves the Magnitude and Longevity of Humoral Immune Responses in Macaques 
PLoS ONE  2014;9(3):e91550.
We tested the concept of combining DNA with protein to improve anti-HIV Env systemic and mucosal humoral immune responses. Rhesus macaques were vaccinated with DNA, DNA&protein co-immunization or DNA prime followed by protein boost, and the magnitude and mucosal dissemination of the antibody responses were monitored in both plasma and mucosal secretions. We achieved induction of robust humoral responses by optimized DNA vaccination delivered by in vivo electroporation. These responses were greatly increased upon administration of a protein boost. Importantly, a co-immunization regimen of DNA&protein injected in the same muscle at the same time induced the highest systemic binding and neutralizing antibodies to homologous or heterologous Env as well as the highest Env-specific IgG in saliva. Inclusion of protein in the vaccine resulted in more immunized animals with Env-specific IgG in rectal fluids. Inclusion of DNA in the vaccine significantly increased the longevity of systemic humoral immune responses, whereas protein immunization, either as the only vaccine component or as boost after DNA prime, was followed by a great decline of humoral immune responses overtime. We conclude that DNA&protein co-delivery in a simple vaccine regimen combines the strength of each vaccine component, resulting in improved magnitude, extended longevity and increased mucosal dissemination of the induced antibodies in immunized rhesus macaques.
doi:10.1371/journal.pone.0091550
PMCID: PMC3953433  PMID: 24626482
14.  Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques 
PLoS ONE  2014;9(1):e86254.
HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24gag elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55gag increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist.
doi:10.1371/journal.pone.0086254
PMCID: PMC3900501  PMID: 24465991
16.  IL-12 DNA as molecular vaccine adjuvant increases the cytotoxic T cell responses and breadth of humoral immune responses in SIV DNA vaccinated macaques 
Human Vaccines & Immunotherapeutics  2012;8(11):1620-1629.
Intramuscular injection of macaques with an IL-12 expression plasmid (0.1 or 0.4 mg DNA/animal) optimized for high level of expression and delivered using in vivo electroporation, resulted in the detection of systemic IL-12 cytokine in the plasma. Peak levels obtained by day 4–5 post injection were paralleled by a rapid increase of IFN-γ, indicating bioactivity of the IL-12 cytokine. Both plasma IL-12 and IFN-γ levels were reduced to basal levels by day 14, indicating a short presence of elevated levels of the bioactive IL-12. The effect of IL-12 as adjuvant together with an SIVmac239 DNA vaccine was further examined comparing two groups of rhesus macaques vaccinated in the presence or absence of IL-12 DNA. The IL-12 DNA-adjuvanted group developed significantly higher SIV-specific cellular immune responses, including IFN-γ+ Granzyme B+ T cells, demonstrating increased levels of vaccine-induced T cells with cytotoxic potential, and this difference persisted for 6 mo after the last vaccination. Coinjection of IL-12 DNA led to increases in Gag-specific CD4+ and CD4+CD8+ double-positive memory T cell subsets, whereas the Env-specific increases were mainly mediated by the CD8+ and CD4+CD8+ double-positive memory T cell subsets. The IL-12 DNA-adjuvanted vaccine group developed higher binding antibody titers to Gag and mac251 Env, and showed higher and more durable neutralizing antibodies to heterologous SIVsmE660. Therefore, co-delivery of IL-12 DNA with the SIV DNA vaccine enhanced the magnitude and breadth of immune responses in immunized rhesus macaques, and supports the inclusion of IL-12 DNA as vaccine adjuvant.
doi:10.4161/hv.21407
PMCID: PMC3601136  PMID: 22894956
in vivo electroporation; HIV; SIVsmE660; SIVmac239; antibody; neutralizing antibody; avidity; central memory; transitional memory; effector memory
17.  Vaccination with Vaxfectin® adjuvanted SIV DNA induces long-lasting humoral immune responses able to reduce SIVmac251 Viremia 
Human Vaccines & Immunotherapeutics  2013;9(10):2069-2080.
We evaluated the immunogenicity and efficacy of Vaxfectin® adjuvanted SIV DNA vaccines in mice and macaques. Vaccination of mice with Vaxfectin® adjuvanted SIV gag DNA induced higher humoral immune responses than administration of unadjuvanted DNA, whereas similar levels of cellular immunity were elicited. Vaxfectin® adjuvanted SIVmac251 gag and env DNA immunization of rhesus macaques was used to examine magnitude, durability, and efficacy of humoral immunity. Vaccinated macaques elicited potent neutralizing antibodies able to cross-neutralize the heterologous SIVsmE660 Env. We found remarkable durability of Gag and Env humoral responses, sustained during ~2 y of follow-up. The Env-specific antibody responses induced by Vaxfectin® adjuvanted env DNA vaccination disseminated into mucosal tissues, as demonstrated by their presence in saliva, including responses to the V1-V2 region, and rectal fluids. The efficacy of the immune responses was evaluated upon intrarectal challenge with low repeated dose SIVmac251. Although 2 of the 3 vaccinees became infected, these animals showed significantly lower peak virus loads and lower chronic viremia than non-immunized infected controls. Thus, Vaxfectin® adjuvanted DNA is a promising vaccine approach for inducing potent immune responses able to control the highly pathogenic SIVmac251.
doi:10.4161/hv.25442
PMCID: PMC3906391  PMID: 23820294
HIV; Rhesus macaques; SIVmac239; SIVsmE660; V1 and V2 antibodies; adjuvant; antibody; avidity; mucosal immunity; neutralizing antibody; rectal fluid; saliva; systemic immunity
18.  Comparison of intradermal and intramuscular delivery followed by in vivo electroporation of SIV Env DNA in macaques 
Human Vaccines & Immunotherapeutics  2013;9(10):2081-2094.
A panel of SIVmac251 transmitted Env sequences were tested for expression, function and immunogenicity in mice and macaques. The immunogenicity of a DNA vaccine cocktail expressing SIVmac239 and three transmitted SIVmac251 Env sequences was evaluated upon intradermal or intramuscular injection followed by in vivo electroporation in macaques using sequential vaccination of gp160, gp120 and gp140 expressing DNAs. Both intradermal and intramuscular vaccination regimens using the gp160 expression plasmids induced robust humoral immune responses, which further improved using the gp120 expressing DNAs. The responses showed durability of binding and neutralizing antibody titers and high avidity for > 1 y. The intradermal DNA delivery regimen induced higher cross-reactive responses able to neutralize the heterologous tier 1B-like SIVsmE660_CG7V. Analysis of cellular immune responses showed induction of Env-specific memory responses and cytotoxic granzyme B+ T cells in both vaccine groups, although the magnitude of the responses were ~10x higher in the intramuscular/electroporation group. The cellular responses induced by both regimens were long lasting and could be detected ~1 y after the last vaccination. These data show that both DNA delivery methods are able to induce robust and durable immune responses in macaques.
doi:10.4161/hv.25473
PMCID: PMC3906392  PMID: 23811579
DNA vaccine; HIV; SIVmac239; SIVmac251; SIVsmE660; avidity; binding antibody; effector memory; in vivo electroporation; intradermal; intramuscular; neutralizing antibody; transitional memory; transmitted Env
19.  HIV-1 p24gag Derived Conserved Element DNA Vaccine Increases the Breadth of Immune Response in Mice 
PLoS ONE  2013;8(3):e60245.
Viral diversity is considered a major impediment to the development of an effective HIV-1 vaccine. Despite this diversity, certain protein segments are nearly invariant across the known HIV-1 Group M sequences. We developed immunogens based on the highly conserved elements from the p24gag region according to two principles: the immunogen must (i) include strictly conserved elements of the virus that cannot mutate readily, and (ii) exclude both HIV regions capable of mutating without limiting virus viability, and also immunodominant epitopes located in variable regions. We engineered two HIV-1 p24gag DNA immunogens that express 7 highly Conserved Elements (CE) of 12–24 amino acids in length and differ by only 1 amino acid in each CE (‘toggle site’), together covering >99% of the HIV-1 Group M sequences. Altering intracellular trafficking of the immunogens changed protein localization, stability, and also the nature of elicited immune responses. Immunization of C57BL/6 mice with p55gag DNA induced poor, CD4+ mediated cellular responses, to only 2 of the 7 CE; in contrast, vaccination with p24CE DNA induced cross-clade reactive, robust T cell responses to 4 of the 7 CE. The responses were multifunctional and composed of both CD4+ and CD8+ T cells with mature cytotoxic phenotype. These findings provide a method to increase immune response to universally conserved Gag epitopes, using the p24CE immunogen. p24CE DNA vaccination induced humoral immune responses similar in magnitude to those induced by p55gag, which recognize the virus encoded p24gag protein. The inclusion of DNA immunogens composed of conserved elements is a promising vaccine strategy to induce broader immunity by CD4+ and CD8+ T cells to additional regions of Gag compared to vaccination with p55gag DNA, achieving maximal cross-clade reactive cellular and humoral responses.
doi:10.1371/journal.pone.0060245
PMCID: PMC3610668  PMID: 23555935
20.  The p40 Subunit of Interleukin (IL)-12 Promotes Stabilization and Export of the p35 Subunit 
The Journal of Biological Chemistry  2013;288(9):6763-6776.
Background: The biosynthesis of IL-12p70 depends on the intracellular interaction of its p35 and p40 subunits.
Results: The p40 subunit stabilizes p35 and promotes its secretion.
Conclusion: Understanding the regulatory steps of IL-12 biosynthesis led to the generation of optimized IL-12 plasmids.
Significance: Availability of expression-optimized IL-12 DNA plasmids is important for practical applications as DNA vaccine adjuvants and in cancer immunotherapy.
IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ∼1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.
doi:10.1074/jbc.M112.436675
PMCID: PMC3585113  PMID: 23297419
Cancer Therapy; Cytokine; DNA; Gene Expression; Immunology; Interleukin; Intracellular Trafficking; Protein-Protein Interactions; Export; Posttranslational Regulation
21.  Comparison of immune responses generated by optimized DNA vaccination against SIV antigens in mice and macaques 
Vaccine  2010;29(39):6742-6754.
Optimized DNA vectors were constructed comprising the proteome of SIV including the structural, enzymatic, regulatory, and accessory proteins. In addition to native antigens as produced by the virus, fusion proteins and modified antigens with altered secretion, cellular localization and stability characteristics were generated. The DNA vectors were tested for expression upon transfection in human cells. In addition, the vectors were tested either alone or in combinations in mice and macaques, which provided an opportunity to compare immune responses in two animal models. DNA only immunization using intramuscular injection in the absence or presence of in vivo electroporation did not alter the phenotype of the induced T cell responses in mice. Although several fusion proteins induced immune responses to all the components of a polyprotein, we noted fusion proteins that abrogated immune response to some of the components. Since the expression levels of such fusion proteins were not affected, these data suggest that the immune recognition of certain components was altered by the fusion. Testing different DNA vectors in mice and macaques revealed that a combination of DNAs producing different forms of the same antigen generated more balanced immune responses, a desirable feature for an optimal AIDS vaccine.
doi:10.1016/j.vaccine.2010.12.056
PMCID: PMC3115438  PMID: 21195080
DNA vaccine; electroporation; immune response; mice; macaque; SIV; HIV; antibody; cellular immune responses
22.  Long-lasting humoral and cellular immune responses and mucosal dissemination after intramuscular DNA immunization 
Vaccine  2010;28(30):4827-4836.
Naïve Indian rhesus macaques were immunized with a mixture of optimized plasmid DNAs expressing several SIV antigens using in vivo electroporation via the intramuscular route. The animals were monitored for the development of SIV-specific systemic (blood) and mucosal (bronchoalveolar lavage) cellular and humoral immune responses. The immune responses were of great magnitude, broad (Gag, Pol, Nef, Tat, Vif), long-lasting (up to 90 weeks post 3rd vaccination) and were boosted with each subsequent immunization, even after an extended 90 week rest period. The SIV-specific cellular immune responses were consistently more abundant in BAL than in blood, and were characterized as predominantly effector memory CD4+ and CD8+ T cells in BAL and as both central and effector memory T cells in blood. SIV-specific T cells containing Granzyme B were readily detected in both blood and BAL, suggesting the presence of effector cells with cytolytic potential. DNA vaccination also elicited long-lasting systemic and mucosal humoral immune responses, including the induction of Gag-specific IgA. The combination of optimized DNA vectors and improved intramuscular delivery by in vivo electroporation has the potential to elicit both cellular and humoral responses and dissemination to the periphery, and thus to improve DNA immunization efficacy.
doi:10.1016/j.vaccine.2010.04.064
PMCID: PMC2932451  PMID: 20451642
DNA vaccine; AIDS vaccine; rhesus macaque; BAL; PBMC; rectal mucosa; humoral immune response; cellular immune response; IFN-γ; TNFα; IL-12; adjuvant; immunology
23.  Repeated DNA Therapeutic Vaccination of Chronically SIV-Infected Macaques Provides Additional Virological Benefit 
Vaccine  2010;28(8):1962-1974.
We have previously reported that therapeutic immunization by intramuscular injection of optimized plasmid DNAs encoding SIV antigens effectively induces immune responses able to reduce viremia in antiretroviral therapy (ART)-treated SIVmac251 infected Indian rhesus macaques. We subjected such therapeutically immunized macaques to a second round of therapeutic vaccination using a combination of plasmids expressing SIV genes and the IL-15/IL-15 receptor alpha as molecular adjuvant, which were delivered by the more efficacious in vivo constant-current electroporation. A very strong induction of antigen-specific responses to Gag, Env, Nef, and Pol, during ART (1.2-1.6% of SIV-specific T cells in the circulating T lymphocytes) was obtained with the improved vaccination method. Immunological responses were characterized by the production of IFN-γ, IL–2, and TNFα either alone, or in combination as double or triple cytokine positive multifunctional T cells. A significant induction of CD4+ T cell responses, mainly targeting Gag, Nef, and Pol, as well as of CD8+ T cells, mainly targeting Env, was found in both T cells with central memory and effector memory markers. After release from ART, the animals showed a virological benefit with a further ∼1 log reduction in viremia. Vaccination with plasmid DNAs has several advantages over other vaccine modalities, including the possibility for repeated administration, and was shown to induce potent, efficacious, and long-lasting recall immune responses. Therefore, these data support the concept of adding DNA vaccination to the HAART regimen to boost the HIV-specific immune responses.
doi:10.1016/j.vaccine.2009.10.099
PMCID: PMC2830913  PMID: 20188252
24.  Long-Lasting Decrease in Viremia in Macaques Chronically Infected with Simian Immunodeficiency Virus SIVmac251 after Therapeutic DNA Immunization▿  
Journal of Virology  2006;81(4):1972-1979.
Rhesus macaques chronically infected with highly pathogenic simian immunodeficiency virus (SIV) SIVmac251 were treated with antiretroviral drugs and vaccinated with combinations of DNA vectors expressing SIV antigens. Vaccination during therapy increased cellular immune responses. After the animals were released from therapy, the virus levels of 12 immunized animals were significantly lower (P = 0.001) compared to those of 11 animals treated with only antiretroviral drugs. Vaccinated animals showed a persistent increase in immune responses, thus indicating both a virological and an immunological benefit following DNA therapeutic vaccination. Several animals show a long-lasting decrease in viremia, suggesting that therapeutic vaccination may provide an additional benefit to antiretroviral therapy.
doi:10.1128/JVI.01990-06
PMCID: PMC1797580  PMID: 17135321

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