2014 DNA Vaccine Conference

Monday, July 21, 2014

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7:30am

Registration

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Sign up for an immersive DNA Vaccine Conference experience

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7:30am - 8:45am

Continental Breakfast in Exhibit/Poster Area

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Meet other conference attendees, network, and fuel up for a day filled with groundbreaking research and poignant panels.

Plenary Speaker Session

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8:45am - 9:10am

Chairman's Welcome to DNA Vaccines 2014, Dr. David Weiner

Speaker

Dr. David B. Weiner
Professor of Pathology and Laboratory Medicine, University of Pennsylvania

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9:10am - 9:50am

MERS Virus - a Novel Emerging Challenge in Infectious Disease

Speaker

Dr. Heinz Feldmann
Chief, Laboratory of Virology, National Institute of Allergy and Infectious Diseases

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9:50am - 10:10am

Next Steps for Control of TB

Speaker

Dr. David Hokey
Senior Director of Immunology & Animal Studies, Aeras

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10:10am - 10:30am

Multivalent Approach to Vaccine Design: Requirement for DNA Priming Prior to Ad Vector Boost to Induce Sterile Immunity Against Malaria with a Multi-antigen Vaccine

Speaker

Dr. Martha Sedegah
Senior Scientist and Director of the Clinical Immunology Research Lab , US Naval Medical Research Center

Abstract

We performed two malaria vaccine clinical trials; the first used a heterologous prime (DNA) and adenovirus-5 (Ad) boost (DNA/Ad trial), each containing the same malaria antigens, circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1), and achieved sterile protection in four of 15 immunized volunteers. In the second trial, the Ad vaccine alone without the DNA-prime (AdCA trial), given as a single dose was not protective. A two-dose regimen of the Ad vaccine expressing CSP was also not protective. We therefore concluded that DNA-priming was essential to induce protection. In our efforts to better understand protective immune responses in the DNA/Ad trial, and to explore the effect of DNA-priming on the Ad immune responses, we found that the DNA/Ad vaccine induced higher CD8+ T cells interferon-gamma (IFN-γ) and effector IFN-γ responses in the protected compared to the non-protected volunteers. Interestingly the magnitude of these activities was similar in non-protected volunteers in the AdCA alone trial. Upon further analysis, we found that protection was associated with predominantly monofunctional CD8+ T cell IFN-γ effector memory responses that exceeded central memory responses, and were predominantly directed to single 9mer epitopes within AMA1, later matched to HLA B*57 and A*03 restricted epitopes. In summary, our data showed that DNA-priming was essential for the induction of protective immunity against malaria.

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10:30am - 11:00am

Coffee/Networking Break in Exhibit/Poster Area

Infectious Diseases Session

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11:00am - 11:30am

Novel Recombinant Vaccines Based on Replication-defective Flavivirus Vectors

Speaker

Dr. Maryann Giel-Moloney
Deputy Director – Virology Discovery Group Cambridge, Sanofi Pasteur

Abstract

M. Giel-Moloney, A.P. Goncalvez, F. David, M. DiasFigueiredo, B. Feilmeier, T. Mebatsion, J. Catalan, M. Vaine, C. Ventura, T.U. Vogel, R. Stanislaus, R. Oomen, H. Kleanthous, K.V. Pugachev

Institution: Sanofi Pasteur, 38 Sidney Street, Cambridge, MA 02139

The RepliVax® vaccine platform (RV) is based on flavivirus RNA genomes that are rationally attenuated by deletion and do not generate a productive infection similar to replication-competent virus.  RV vectors are highly attenuated, capable of inducing robust antibody and T cell responses, and efficacious as shown for vaccine candidates against several flaviviruses, including Tick-borne encephalitis (TBE).  A single dose of RV-TBE induced a robust neutralizing antibody response in monkeys which was more durable than 3 doses of an inactivated TBE vaccine control.  RV-TBE induced similar genes and temporal expression patterns in macaques as has been observed for YF-17D vaccinated humans.  In addition to developing RV for flavivirus vaccine candidates, we have engineered West Nile (WN)-based RV vectors to express non-flavivirus immunogens.  The full length rabies virus G gene was cloned into the RV-WN genome and the chimeric virus replicated to high titers (8 logs) in helper cells and expression of RabG was stably maintained through multiple rounds of in-vitro passaging. We evaluated RV-RabG in various animal models and the vaccine provided durable protection in mice and dogs induced protective levels of neutralizing antibodies in pigs.  Conventionally, RV is administered as virus packaged in helper cells. Alternatively it can be given as nucleic acids (RNA or DNA). DNA immunization in mice with RV-WN plasmids induced neutralizing antibody titers comparable to vaccination with a single dose of RV-WN virus. Collectively, the data demonstrate RepliVax® is a vaccine platform with the potential to develop either traditional virion-based or alternatively nucleic acid based vaccines against both flavivirus or non-flavivirus pathogens.  

® RepliVax is a registered trademark of the Board of Regents of the University of Texas System

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11:30am - 11:50am

Bioterrorism

Speaker

Dr. Connie Schmaljohn
Chief Scientist, U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID)

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11:50am - 12:10pm

Pandemic Control

Speaker

Drew Hannaman
Vice President, Research and Development, Ichor Medical Systems, Inc.

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12:10pm - 12:30pm

Ebola Protection by SAM RNA

Speaker

Dr. Pamela Glass
Chief, Viral Biology Department, USAMRIID

Abstract

Protection of guinea pigs from Zaire ebolavirus (ZEBOV) challenge after vaccination with a SAM® vaccine expressing the ZEBOV envelope glycoprotein (GP)

Ebola virus infection causes an acute hemorrhagic fever following infection with a mortality rate that can approach 90% in some outbreaks.  Currently, there are no approved therapeutics or vaccines for human use against filovirus infections.  A number of vaccine candidates are in development including DNA, viral vector based, virus-like particles, and subunit vaccines.  While each platform has had successes, many face obstacles which threaten advancement through licensure.  DNA vaccines are a flexible platform and are broadly effective in small animal models, yet they have generally lacked potency in human clinical trials.  Recombinant viral vector technologies have the advantage of efficient delivery of the nucleic acid payload, but their utility is often hampered by anti-vector immunity, production limitations, and safety concerns.  While safe, VLP and subunit vaccines are often less immunogenic than live-attenuated vaccines and production limitations can exist.  Messenger RNA (mRNA) vaccines were previously considered unrealistic.  However, many of the obstacles have been surmounted, and recently there has been a revival in the use of non-amplifying mRNA vaccines for cancer, allergy, and gene therapy.  In collaboration with Novartis, a SAM® Ebola vaccine expressing ZEBOV GP was examined for efficacy against lethal guinea pig (gp)-adapted ebolavirus challenge.  The SAM vaccine platform, now in pre-clinical development, is based on a synthetic, self-amplifying mRNA, delivered by a synthetic non-viral delivery system.  For these experiments, the Ebola SAM vaccine was formulated with a cationic nanoemulsion (CNE) or lipid nanoparticle (LNP) delivery system for intramuscular delivery.  Two different doses and dosing schedules were examined.  Complete protection from lethal gp-adapted ebolavirus challenge by both the intraperitoneal and aerosol routes was achieved in animals vaccinated with the SAM Ebola vaccines formulated with both non-viral delivery systems.

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12:30pm - 1:30pm

Lunch

Chronic Infections

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1:30pm - 2:10pm

NAB for HIV

Speaker

Dr. Dennis Burton
Professor, Department of Immunology and Microbial Science , The Scripps Research Institute

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2:10pm - 2:30pm

Dr. Matti Sällberg - Karolinska Institutet

Speaker

Dr. Matti Sällberg
Professor and Biomedical Scientist, Karolinska Institutet

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2:30pm - 2:45pm

Closed Linear DNA Encoding Influenza HA Produced by a Completely Synthetic Process Effectively Induces Protective Immunity Against Live Viral Challenge with PR8 Virus in Mice

Speaker

Dr. Lisa Caproni
Touchlight Genetics Ltd

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2:45pm - 3:00pm

Easy Needle-free Intradermal Delivery and Vector Optimization of a Broad Protective Polyvalent Influenza-A DNA Vaccine for Pigs and Humans

Speaker

Dr. Anders Fomsgaard
Statens Serum Institute

Abstract

Marie Borggrena, Jens Nielsena, and Anders Fomsgaard a,b, for the UNISEC Consortiumc

aVirus R&D Laboratory (ViFU), Statens Serum Institut, DK-2300 Copenhagen, Denmark
bInfectious Disease Research Unit, Clinical Institute, University of Southern Denmark
cPartly funded by EU contribution FP7-HEALTH towww.UNISECconsortium.eu (“Universal influenza vaccine secured”)

Influenza vaccines inducing a broad cross-reactive immune response would be of great advantage for improved protection against both seasonal and emerging influenza viruses in humans and pigs. We have developed an alternative influenza vaccine based on DNA expressing 6 selected influenza proteins of pandemic origin (H1,N1,H3,N2,NP,M)1. Intradermal immunisation with electroporation induced HI antibodies >40 HAI/ml between 7-10 days after second vaccination in pigs and induced protection against challenge with virus homologous and heterologous to the HA/NA DNA vaccine. Subsequently, we aimed to optimize and ease delivery suitable for pig herds using needle-free delivery to the skin. We successfully adapted our DNA formulation to the IDAL (Intra Dermal Application of Liquids) device (MSD) designed for pigs and obtained antibody responses comparable with those obtained by i.d. electroporation when tested in the rabbit model. We further aimed to enhance the DNA vaccine performance, production yield, and safety by changing our 1st generation DNA vaccine vector backbones (pSSI and wrg7079) to the antibiotic-free 3nd and 4rd generation vectors NTC8385 and NTC9385 (Nature Technologies)2, respectively. The improvements encourage for clinical trials.

1Bragstad K et al. Vaccine 2013;31:2281-228
2Williams JA. Vaccines 2013;1:225-249;doi:10.3390/vaccines1030225

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3:00pm - 3:30pm

Coffee/Networking Break in Exhibit/Poster Area

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3:30pm - 3:50pm

Therapy Approaches for HIV

Speaker

Dr. Pablo Tebas
Professor of Medicine; Director and Principal Investigator, AIDS Clinical Trial Unit Research Site , University of Pennsylvania

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3:50pm - 4:10pm

Cutaneous and Mucosal HIV Vaccination

Speaker

Dr. Robin Shattock
Professor of Mucosal Infection and Immunity, Imperial College London

Abstract

It is generally accepted an effective prophylactic HIV-1 vaccine it is likely to require both humoral and cellular responses and may be critically dependent upon the generation of neutralising antibodies. This presentation reports the use of different DNA plasmid technologies to efficiently express a model trimeric CN54-gp140 envelope glycoprotein and priming strategies using concurrent intradermal (ID) and intramuscular (IM) vaccination. To augment vaccine-elicited T and B cell responses, the impact of DNA transfection has been assessed in combination with different boosting regimes using viral vectors and/or homologous protein. Concurrent ID and IM vaccination resulted in significantly elevated IFN-* T cell and high avidity vaccine antigen-specific IgG B cell responses in mice, a hallmark of B cell maturation. Sequential protein boosting of the concurrent DNA strategy further augmented antigen-specific IgG responses but had little impact on T cell reactivity. A compressed regime where DNA and protein were co-administered had limited impact on response but provided significant regime shortening. Route optimisation has been explored to maximize the impact of protein boosting and demonstrated that a subcutaneous inoculation increased antigen-specific antibody avidity to a greater extent than either intramuscular, intranasal or transcutaneous administration. The fully optimised vaccine regime of a concurrent DNA intramuscular and intradermal prime strategy followed by a subcutaneous protein boost vaccination was also capable of eliciting humoral responses with high antibody affinty and that were capable of neutralising HIV-1 pseudoviruses from diverse clades (A, B and C) in an alternative larger animal model, the rabbit. These strategies are currently being evaluated in human phase I trials.

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4:10pm - 4:30pm

T Cell Immunology in the HIV Infected Lymph Node: Opportunities and Challenges for Therapeutic Vaccine Strategies

Speaker

Dr. Michael Betts
Associate Professor, Microbiology, University of Pennsylvania

Abstract

The lymph node (LN) is a centrally important tissue for latent HIV reservoirs in HAART treated HIV infected subjects. As such, HIV cure-based strategies designed to invoke CD8+ T cell cytotoxicity (CTL)-mediated killing of latently infected CD4+ T cells must consider not only function and magnitude, but also tissue localization. Previous data from mouse models has suggested that while CD8+ T cells can be readily found in LN, their functional properties are temporally regulated such that cytolytic effector function is only fully realized after LN egress. To address this in the context of HIV infection, we have directly examined the functional properties of LN-derived CD8+ T cells from uninfected, chronic, and HAART treated HIV infected subjects. Our data reveal distinct differences in CD8+ T cell function and localization within the LN of HIV infected and uninfected subjects, and further differences between treated and untreated HIV infected subjects. In particular, after initiation of HAART, there is a rapid decrease in the proportion of LN CD8+ T cells that express key markers associated with cytolytic function. Together our results indicate that therapeutic vaccination strategies designed to re-invigorate HIV-specific CD8+ T cell function will need to remodel the trafficking and cytolytic properties of these cells for effective HIV reservoir clearance in the LN.

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4:30pm - 4:45pm

Expanded Epitope Recognition by Vaccination with DNA Encoding Novel Immunogens Comprising HIV Conserved Elements

Speaker

Dr. Barbara Felber
Principal Investigator, NCI,

Abstract

Barbara K. Felber, Xintao Hu, Viraj Kulkarni, Antonio Valentin, Margherita Rosati, Candido Alicea, Morgane Rolland, Sylvie Le Gall, Niranjan Y. Sardesai, Beatriz Mothe, Christian Brander, James I. Mullins, George N. Pavlakis

Background: HIV sequence diversity and potential “decoy” epitopes are hurdles in the development of an effective AIDS vaccine. To target immune responses towards invariable viral regions we engineered DNA-based immunogens encoding conserved elements (CE) of HIV-1 selected on the basis of stringent conservation, functional importance, and association with immune control.

Methods: Macaques were immunized by IM injection followed by electroporation with DNA plasmids expressing CE or complete immunogens (gag, env), alone or in heterologous prime-boost regimens. Immune responses elicited by CE from gag (HIV, SIV) and env (HIV) were compared to those obtained upon vaccination with DNA expressing the complete antigens.

Results: Macaques vaccinated with CE gag or CE env DNA developed robust CE-specific cytotoxic T cells (Granzyme B+, CD107+). CE-specific T cells were found only in ~50% of animals vaccinated with DNA encoding the complete antigen and these responses targeted fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length antigen increased both magnitude and breadth of the CE responses. 

Although designed as CTL vaccine, p24CE DNA vaccine induced Ab responses able to recognize p55gag and targeting a broad range of linear epitopes. In contrast, antibodies induced by p55gag DNA vaccination failed to recognize p24CE or linear CE epitopes. Interestingly, boosting of p24CE DNA primed macaques with p55gag DNA increased Abs recognizing HIV p24gag as well as p24CE proteins, thereby inducing broadest immunity.

Conclusions: Combination of conserved elements and full-length immunogen provides a novel strategy to increase the magnitude and breadth of cellular and humoral immunity while targeting efficiently the conserved regions of the virus. This allows for the development and expansion of subdominant responses and greater breadth of immune response.

Panel Session I: DNA Vaccines - Scientific and Technological Drivers

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Moderator

Dr. Niranjan Y. Sardesai - Inovio

Panelists

Dr. Darrel Irvine - Massachusetts Institute of Technology
Dr. Holbrook Kohrt - Stanford
Dr. George Pavlakis - National Cancer Institute
Dr. Connie Schmaljohn - USAMRIID
Dr. Jeffrey Ulmer - Novartis

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5:30pm - 7:00pm

Reception and Poster Session

Tuesday, July 22, 2014

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8:00am - 9:00am

Continental Breakfast in Exhibit/Poster Area

Plenary Lecture

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9:00am - 9:45am

Chondroitin Sulphate Proteoglycan 4 (CSPG4), an Attractive Target of Antibody-based Immunotherapy in Various Types of Solid Tumors

Speaker

Dr. Soldano Ferrone
Professor, Massachusetts General Hospital, Harvard Medical School

Abstract

Like CD44 (CSPG8), the tumor cell membrane-bound CSPG4, also known as high molecular weight-melanoma associated antigen (HMW-MAA), or neuron-glial antigen 2 (NG2), is a member of the CSPG family. CSPGs are key bioactive molecules that play a major role in tumor growth, migration and neo angiogenesis. CSPG4 is a unique glycoprotein-proteoglycan complex consisting of a 250 kDa N-linked glycoprotein and a 450 kDa proteoglycan component. It is composed of three major structural components: the extracellular domain (consisting of 3 subdomains), the transmembrane region, and the cytoplasmic C-terminal domain (CTD).

Flow cytometry analysis of established cancer cell lines stained with mAbs and immunohistochemical staining of surgically excised tumors from patients have shown that CSPG4 is expressed on glioma, squamous cell carcinoma of the head and neck (SCCHN), esophageal squamous cell carcinoma,  triple negative breast cancer (TNBC), melanoma, mesothelioma, renal cell carcinoma, chordoma, chondrosarcoma, osteosarcoma, and soft tissue sarcomas. On most malignant cells CSPG4 has a high expression with limited intra- and inter-lesional heterogeneity. It is noteworthy that CSPG4 is expressed not only on differentiated malignant cells, but also on cancer initiating cells (CICs). Therefore targeting CSPG4 may eliminate not only differentiated cancer cells, but also CICs. In contrast CSPG4 has a restricted distribution in normal tissues. It is expressed on activated pericytes in the tumor microenvironment. As a result neo angiogenesis and growth of tumor cells, even those which do not express CSPG4 are inhibited. 

The structural and functional properties of CSPG4 are highly conserved through phylogenetic evolution. The high degree of homology, but not complete identity among CSPG4s in various animal species accounts for their ability to overcome unresponsiveness to self-CSPG4 in xenogeneic hosts. This mechanism accounts for the ability of DNA encoding human CSPG4 in combination with electroporation to elicit humoral immunity to self-CSPG4 in dogs with melanoma. In agreement with results obtained in patients with melanoma immunized with CSPG4 mimics, this immunity appears to have clinical significance, since it is associated with a favorable clinical course of the disease.

Most, if not all the available CSPG4-specific mAb are not effective in mediating complement- and cell-dependent lysis of tumor cells. On the other hand, the few CSPG4-specific mAb used have been very useful to generate chimeric antigen receptors to redirect T cells to tumors expressing this tumor antigen. Furthermore CSPG4-specific mAbs are very effective in inhibiting both in vitro and in vivo signal transduction pathways associated with tumor cell proliferation, survival and migration. These results provide a mechanism(s) for the ability of CSPG4-specific mAbs to inhibit tumor growth, and more importantly disease recurrence and metastatic spread in immunodeficient mice grafted with human melanoma, TNBC or mesothelioma cells. 

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9:45am - 10:05am

Prime Boost Strategies for Immune Protection

Speaker

Dr. Shan Lu, MHA
Professor of Medicine, University of Massachusetts Medical School

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10:05am - 10:25am

DNA and Protein Co-immunization Improves the Magnitude, Longevity, and Mucosal Dissemination of Immune Responses

Speaker

Dr. George Pavlakis
Principal Investigator, Human Retrovirus Section, Vaccine Branch , National Cancer Institute

Abstract

Optimal DNA vaccination in primates including humans has been shown to induce strong, broad and durable cellular immune response and also good antibody titers, especially in macaques. To increase effectiveness and versatility of DNA vaccines we combined them with other vaccine modalities. One method with emerging advantages is the simultaneous vaccination with DNA and protein. Co-immunization strategy of DNA & protein induced rapid and high humoral responses while maintaining robust cellular responses typically obtained with DNA vaccines. The vaccine induced Ab against both homologous and heterologous Env; high binding titers against scaffolded V1/V2 env region; efficient dissemination to mucosal sites; high Env-specific IgG in saliva and Env-specific IgG and IgA in rectal mucosa. Analysis of cellular responses revealed the presence of cytotoxic memory T cells against several viral proteins. These cellular responses disseminated systemically as demonstrated by their presence not only in blood and lymphoid tissues, but also in bone marrow, liver, lung (effector site) and, importantly, rectal and vaginal mucosa. The longevity of the cellular responses induced by this co-immunization regimen was significantly improved, with SIV-specific T cells detected >5 yrs after the vaccination.

Thus, intramuscular DNA & protein co-delivery increases the magnitude and longevity of systemic and mucosal humoral immune responses in immunized macaques and is proposed as a practical and efficient method for human vaccination.

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10:25am - 11:00am

Coffee/Networking Break in Exhibit/Poster Area

Special EU Session

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11:00am - 11:20am

Introduction of Collaborative Projects and UE Research Programme Horizon 2020

Speaker

Dr. David Gancberg
EU Project and Scientific Officer, Novel Medical Developments, European Commission

Abstract

Through its Framework Programmes for Research and Technological Development, the European Union has played an important role in supporting collaborative efforts in nucleic-acid-based research, development of vaccines and immunotherapy, with the ultimate goal of bringing these technologies to the clinic and possibly to the market. Lately, the new research and innovation programme, Horizon 2020, has put a special emphasis on clinical trials and involvement of small and medium-sized enterprises (SME), with nearly €80 billion of funding available over 7 years (2014 to 2020) – in addition to the private investment that this money will attract. By coupling research and innovation, Horizon 2020 is helping to achieve this with its emphasis on excellent science, industrial leadership and tackling societal challenges. The goal is to ensure Europe produces world-class science, removes barriers to innovation and makes it easier for the public and private sectors to work together in delivering innovation.

Horizon 2020 is open to everyone, with a simple structure (3 pillars: Excellence in Science, Industrial Leadership and Societal challenges) that reduces red tape and time so participants can focus on what is really important. This approach makes sure new projects get off the ground quickly – and achieve results faster. In particular, Horizon 2020 is open to the USA-based participants.

The Horizon 2020 societal challenge of ‘health, demographic change and wellbeing’ for the years 2014 and 2015 includes 34 topics in the ‘personalising health and care’ focus area call (15 in 2014 only, 17 in 2015 only and 2 which are open in both years) and 16 topics in the ‘co-ordination activities’ call (11 in 2014 and 5 in 2015). The total budget available is approximately EUR 1.21bn. Topics (for 2015) of interest for the community present at the meeting (DNA/RNA vaccines, immunotherapy) will be presented. Several ongoing projects funded by the European Union in the field will be presented in dedicated sessions (cancer immunotherapy, vaccines for infectious diseases, and this technology session

REFERENCES

ec.europa.eu/programmes/horizon2020/en

ec.europa.eu/programmes/horizon2020/en/h2020-section/health-demographic-change-and-wellbeing

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11:20am - 11:40am

Dr. Karl-Josef Kallen - CureVac

Speaker

Dr. Karl-Josef Kallen
Principal Scientific Fellow, CureVac, GmbH

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11:40am - 12:00pm

Mutanome Engineered RNA Immune-therapy

Speaker

Dr. Sandra Heesch
Senior Project Manager, BioNTech AG

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12:00pm - 12:20pm

Therapeutic TriMix/mRNA Based Vaccine in Chronic HIV-1 Infected Patients Receiving Antiretroviral Therapy

Speaker

Felipe Garcia Alcaide
Team Leader, Infectious Diseases Service & Professor of Medicine, University and Hospital Clinic Barcelona

Abstract

Currently, over 30 million people worldwide are infected with HIV, most of them living in developing countries. Although combined antiretroviral therapy (cART) has proven to be highly effective to prevent clinical progression and death, by itself it is unable to eradicate the infection, thus necessitating therapy throughout life. Therefore, for an effective control of the HIV epidemic new cost-effective and viable therapeutic strategies need to be evaluated.

Therapeutic vaccinations have emerged as one of the most promising strategies that could restore HIV-specific T-cell responses in HIV infected patients and help them control viral replication without cART. A dendritic cell-based vaccine was able to significantly control viral load in vaccinated subjects with mean peak reduction of viral load of 94%. However functional cure was not observed in any of the patients, possibly due to the induction of insufficiently strong anti-viral immune responses. Additional data in mouse tumour models suggested that intranodal immunization with a messenger RNA (mRNA) based therapeutic vaccine (tumour associated antigen-TriMix-mRNA) has the potential to efficiently augment the induction of tumour-specific immune responses compared to a mRNA electroporated DC-based vaccine, suggesting that this strategy could provide the necessary tool to be tested in humans.

The iHIVARNA strategy proposes that direct vaccination with mRNA encoding HIV antigens could be a novel and promising approach in HIV immunotherapy and that to be effective, mRNA vaccines would need to fulfil two characteristics: 1) HIV antigens encoded by mRNA should induce responses to the most vulnerable viral targets and be selected using a rational design; and 2) mRNA should deliver activation stimuli in addition to HIV antigens to DCs.

The principal aim of iHIVARNA is to successfully immunize antiretroviral-treated HIV-infected patients with 3 injections of the candidate universal HIV-TriMix-mRNA as an mRNA-based therapeutic vaccine with the final objective of achieving a functional cure of HIV infection.

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12:20pm - 1:30pm

Lunch

Technology Session I

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1:30pm - 2:00pm

Novel System for mRNA Vaccine Delivery

Speaker

Dr. Darrell Irvine
Professor of Materials Science & Engineering and Biological Engineering, Massachusetts Institute of Technology

Abstract

Toward DNA- and RNA-Based Vaccines Enabled by Synthetic Materials and Synthetic Biology

Nucleic acid vaccines based on DNA or RNA offer many potential advantages for vaccination, but strategies to enhance the delivery and function of such nucleic acid vaccines are still a topic of intense study. Here we will describe two approaches for enhancing nucleic acid vaccines using tools from synthetic materials and synthetic biology.  In the first segment, a new strategy for transcutaneous vaccination we have termed “microneedle tattooing” will be discussed.  In this approach, we construct multi-layered polymer thin film coatings on skin patch microneedle arrays.  The polymer film heterostructure is comprised of a biodegradable polyelectrolyte multilayer carrying proteins, DNA, or RNA, assembled via a layer-by-layer self-assembly process onto an underlying pH-responsive release layer. Application of microneedles to skin leads to rapid dissolution of the release layer and implantation of intact multilayer films into the epidermis, where the film structure allows control over subsequent release of therapeutic cargos over days to weeks.  When applied to the delivery of DNA vaccines, we show that this approach achieves efficient transfection in both mouse and non-human primate skin, leading to immune responses comparable to the current gold standard for DNA vaccines, in vivo electroporation.  Thus, this technology may provide a route to dry skin patch vaccines that do not require refrigeration, can be self-applied, and achieve efficient transfection in humans. 

In the second part of this talk, we will describe our efforts to apply methods from synthetic biology and self-replicating alphavirus-derived replicons to create engineered RNA circuits.  To enable exogenous control over the timing and magnitude of vaccine antigen/adjuvant expression, we first generated replicons where a vaccine or reporter payload is expressed under subgenomic promoters in response to the presence of the small molecule antibiotic trimethoprim (TMP).  When packaged in lipid nanoparticles, these replicons achieved reporter expression 10-fold higher than naked RNA lasting more than 1 month intramuscularly in mice; expression of model antigens led to robust and boostable CD8+ T-cell responses sustained for more than 100 days.  TMP-triggered gene expression allowed gene expression to be toggled by 50-fold, suggesting the ability of engineered replicons to allow “on/off” expression behavior regulated by this small-molecule drug.  These constructs will enable the design of novel vaccine regimens where antigen/adjuvant genes can be expressed in stages, as well as the generation of single-shot vaccines where boosting is achieved by swallowing a pill containing the small molecule regulator.

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2:00pm - 2:30pm

Protective Immunity Against Blood-stage Malaria Induced by Vaccination with a Self-amplifying mRNA Vaccine Encoding Plasmodium MIF

Speaker

Dr. Richard Bucala
Professor of Medicine, Pathology, and Epidemiology & Public Health , Yale University School of Medicine

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2:30pm - 3:00pm

Self-amplifying mRNA Vaccine and Proof of Concept Experiments in Preclinical Animal Models

Speaker

Dr. Andrew Geall
RNA Vaccine Platform Leader, Novartis Vaccines

Abstract

RNA: The new revolution in nucleic acid vaccines

Although the RNA vaccine field is in its infancy, the prospects are promising and recent advancements have demonstrated that vaccines based on mRNA have the potential to combine the positive attributes of other types of vaccines.  Naturally transient and cytosolically-restricted mRNA can now be produced at sufficient quantity and quality from a cell-free enzymatic transcription reaction for human clinical trials.  In addition, product stability, large-scale production, and purification are no longer perceived as barriers to the wide spread implementation of the technology.  We have developed a self-amplifying mRNA vaccine platform and have utilized lipid nanoparticle (LNP) and cationic nanoemuslion (CNE) non-viral delivery systems, which substantially increases vaccine potency.  The broad utility of this novel vaccine technology has been demonstrated with genes encoding vaccine candidate antigens from several pathogens and was found to elicit broad and potent protective immune responses in multiple animal models, including non-human primates.  To demonstrate the speed at which synthetic self-amplifying mRNA vaccine can be produced, we responded to the recent H7N9 influenza outbreak in China as soon as the viral sequence was posted on a web-based data sharing system.  Using rapid and accurate cell-free gene synthesis, the viral antigen was produced and incorporated into the self-amplifying mRNA vaccine, allowing the generation of a vaccine candidate within 8 days.  If self-amplifying mRNA vaccines prove safe, potent, well-tolerated, and effective in humans, this novel nucleic acid vaccine technology will enable a new generation of vaccines able to address the health challenges of the 21st century.

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3:00pm - 3:30pm

Coffee/Networking Break in Exhibit/Poster Area

Technology Session II: Oral Abstract Presentations

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3:30pm - 3:45pm

MicroArray Ballistic Drug Delivery

Speaker

Dr. Nazly Pirmoradi
Palo Alto Research Center, Inc.

Abstract

Pirmoradi, F. N.; Recht, M.; Linn, F.; Pattekar, A. V.; Volkel, A. R.; Veiseh, M.; Anderson, G. B.; Peeters, E.; Uhland, S.A.; Chow, E. M.

Palo Alto Research Center Inc. (PARC)

Particle based drug delivery uses mechanical force to penetrate into cells. Biochemical techniques for cell penetration require complex formulations, and electroporation can cause irreversible tissue damage and potential destabilization of therapeutic material. In contrast, ballistic delivery relies on physical properties of the particles, potentially enabling simpler formulations and delivery strategies. However, particle based ballistic delivery is not currently available for mainstream human use due to problems with control (e.g. sporadic bleeding, repeatability, etc.), loud noise (explosive), and low dosing capability.

We describe a disposable microarray ballistic aerosol drug delivery device enabling on-demand transdermal delivery of drug particles through needle-free injections. Our delivery platform is explosion free and has the potential for higher delivery dosages and significantly improved tissue depth and spatial control compared to previous particle delivery systems (shock-tube based ballistic liquid jet/solid particle).

The device creates continuous and finely tunable arrays of laser-like beams of drug particles that have been entrained into high speed collimated air jets. Similar to printing, the architecture allows for scanning target skin for large area/dose delivery and is scalable to smaller form factors. Drug particles can be directly targeted to physical locations, such as epidermal Langerhans cells for vaccines, and to depths restricted to nerve-less regions (painless). Our platform allows for delivery of a broad range of drugs, but it is particularly advantageous for transdermal delivery of DNA/RNA based therapies for intracellular delivery (e.g. vaccines).

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3:45pm - 4:00pm

T-Rp3, a Promising Non-viral Gene Delivery Vector

Speaker

Marianna Favaro
UNICAMP

Abstract

Favaro, MTP; de Toledo, MAS, Vinolo, MAR; Janisen, R; Souza, AP, Azzoni, AR

Although several gene delivery strategies focus on an enhanced internalization, intracellular trafficking is often neglected despite limiting the efficiency. The natural movement of dyneins along microtubules from cell’s periphery to the centrosome can be exploited to accomplish gene delivery to the nucleus, a mechanism widely used by viruses. In this work, we present a modified dynein light chain (Rp3) fusioned to a DNA binding domain, a TAT peptide and a histidine tag. This modular recombinant protein, named T-Rp3, promoted efficient gene delivery in cultured HeLa cells with Luciferase activity slightly lower than LipofectamineTM, but with low cytotoxicity. Confocal microscopy images shown pDNA delivered with T-Rp3 in the nucleus as soon as 6 hours post-transfection. Additionally, T-Rp3-pDNA complexes were able to transfect bone marrow-derived macrophages (BMM) more efficiently than LipofectamineTM. Quantification of TNF-α produced by BMM cells exposed to T- Rp3 for a period of 24 hours indicated a low inflammatory potential. Overall T- Rp3 is a promising platform to be used to enhance the efficiency of DNA vaccines. 

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4:00pm - 4:15pm

Differential Roles of Antigen Presentation and DNA Adjuvanticity in Immunogenicity of DNA Vaccine

Speaker

Dr. Kouji Kobiyama
National Institute of Biomedical Innovation

Abstract

Kouji Kobiyama1,2, Miyuki Tozuka1, Nao Jounai1, Shohei Koyama3, Cevayir Coban4, and Ken J Ishii1,2.

1 Laboratory of adjuvant innovation, National Institute of Biomedical Innovation
2 Laboratory of Vaccine Science, WPI Immunology Frontier Research Center, Osaka University
3 Department of Medical Oncology and Cancer Vaccine Center, Dana-Farber Cancer Institute; Department of Medicine, Brigham and Women's Hospital; and Harvard Medical School
4 Laboratory of Malaria Immunology, WPI IFReC, Osaka University

The DNA vaccine induces both humoral and cellular immune responses against plasmid-encoded antigen, and has already been approved as vaccines for infectious diseases of particular animals. In human, many of DNA vaccines are currently under clinical trials. We previously clarified that intracellular DNA sensing machinery through TBK1 is essential for induction of adaptive immune responses after DNA vaccination. However, a precise mode of antigen presentation and immunogenicity of DNA vaccine, especially signal transduction, has not been fully elucidated. In this study, we demonstrated that the immunogenicity of DNA vaccine expressing non-secretory antigen, such as LacZ-encoded DNA vaccine, was dependent on IRF7, but not IRF3. Interestingly, the immunogenicity of DNA vaccines expressing secretory antigen, such as OVA-encoded DNA vaccine, was independent on both IRF3 and IRF7, while both OVA- and LacZ-encoded DNA vaccine immunogenicity were totally dependent on TBK1. We further demonstrate that the differential requirements of IRF7 and type I IFN were dependent on mode of antigen presentation. Collectively, our results suggested that immunogenicity of DNA vaccine is influenced by its mode of antigen presentation as well as immunogenicity of DNA vaccine relying on intracellular DNA sensing signaling pathways.

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4:15pm - 4:30pm

DNA Vaccination Strategy Targets Epidermal Dendritic Cells, Initiating their Migration and Induction of an Accelerated Immune Response

Speaker

Dr. Kate Broderick
Director, Research and Development, Inovio Pharmaceuticals, Inc

Plotkin Award Lecture

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4:30pm - 5:30pm

2014 Award Winner: Dr. Nathalie Garçon - GlaxoSmithKline Vaccines

Speaker

Dr. Nathalie Garçon
Vice President and Head of Global Adjuvant and Technologies , GlaxoSmithKline Vaccines

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5:30pm - 7:00pm

Reception in Poster Area

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7:00pm

Dinner and Awards Presentation

Panel Session II: DNA Vaccines - Financial and Strategic Drivers

Details

Moderator

Dr. David B. Weiner - University of Pennsylvania

Panelists

Dr. David Gancberg - European Commission
Dr. Nathalie Garcon - GlaxoSmithKline
Dr. J. Joseph Kim - Inovio
Dr. Steven J. Projan - MedImmune
Dr. Daniel J. Wattendorf - DARPA

Wednesday, July 23, 2014

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8:00am - 9:00am

Continental Breakfast in Exhibit/Poster Area

Cancer Sessions

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9:00am - 9:40am

New Approaches to Cancer Immune Therapy

Speaker

Dr. Phil Greenberg
Professor, Medicine, Oncology and Immunology, University of Washington

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9:40am - 10:05am

Immune Responses in Preinvasive HPV Disease: Going to the Target Site to Get Answers

Speaker

Dr. Cornelia Trimble
Associate Professor of Gynecology and Obstetrics, Johns Hopkins School of Medicine

Abstract

Vaccines designed to prevent development of HPV-induced cancers are focused on induction of an antibody response that prevents infection with the virus. However, once exposure has occurred, which is essentially concurrent with the onset of sexual activity, these antibody responses become irrelevant, as pre-malignant and malignant cells that have integrated the viral genome into the host genome no longer express the targets of these B cell responses. To eliminate disease, T cell responses capable of recognizing E6 and/or E7, the transforming proteins of oncogenic HPV genotypes, become essential. Unfortunately, it has proven much more difficult to generate therapeutically effective T cell responses than to induce preventive antibody responses. Addressing this problem has been made more difficult due to the discrepancy between preclinical models and human clinical trials, which in part reflects the poor immunogenicity of the E6 and E7 proteins in humans. More recently, with the development of more potent vectors and better immunization strategies, detectable T cell responses are being identified in immunized humans. However, even in the presence of systemically detectable T cell responses, lesions in many/most patients have failed to regress, suggesting the presence of additional obstacles. Therefore, we have directly examined HPV-induced cervical lesions in vaccinated as well as unvaccinated individuals to better understand the reasons for success or failure of T cell responses in eradicating HPV- induced lesions.

The techniques we are using to analyze tissue include quantitative digital image analysis of immune cell infiltrates in lesions, before and after vaccination; qRT-PCR on immuno-laser capture microdissected tissue; TCR deep sequencing of tissue T cells; and flow cytometry phenotyping of tissue T cells. Additionally, we are assessing the relationship between the responses detected in the cervical tissue and the peripheral systemic responses, by measuring responses in peripheral blood lymphocytes using conventional methods including ELISPOT, intracellular cytokine staining, and flow cytometry phenotyping.

The issues to be addressed include: a) can T cells be identified in the lesional tissue that have been clonally expanded in the blood; b) are T cells that are enriched in the lesional tissue distinct from responses that can be measured in the peripheral blood; c) to what extent does vaccination increase the frequency and of expanded T cells measured pre-vaccination; d) are obstacles operative in the lesion that interfere with the efficacy of reactive T cells that do reach the tissue; and e) how can our tissue-directed analyses inform strategies to improve the therapeutic activity of T cells that localize to the tumor site. 

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10:05am - 10:30am

Induction of T cells for Cancer Immune Therapy

Speaker

Dr. Mark Bagarazzi
Chief Medical Officer, Inovio Pharmaceuticals

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10:30am - 11:00am

Coffee/Networking Break in Exhibit/Poster Area

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11:00am - 11:25am

Checkpoint Inhibitors Game Changers for Immune Therapy

Speaker

Dr. Michael McCarthy
Head of the Vaccine Platform Group in ID/Vaccines , Medimmune LLC

Abstract

Our understanding of the components which regulate immune cell activity is advancing rapidly, and provides an opportunity to intervene therapeutically across a broad range of disease indications.  Monoclonal antibodies which can either stimulate or decrease T-cell responsiveness, in particular, have generated exciting results in the clinic and are poised to dramatically alter treatment paradigms in cancer.  These checkpoint modifiers also have the potential to partner with vaccine approaches, enhancing intra-nodal priming events and combatting the immune-suppressed tumor environment.  Challenges which remain to be addressed are the specific immune-modulators to be employed, and the timing of their combined use with active therapeutic vaccination

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11:25am - 11:50am

Combination Immunotherapy - Augmenting Anti-tumor Immunity Elicited by DNA Vaccines

Speaker

Dr. Wei Xu
Senior Scientist, Roche Innovation Center

Abstract

The field of Onco-Immunology includes the development of therapies that harness the immune system to provide durable and adaptable cancer control. DNA vaccine platforms for the treatment of cancers are highly appealing owing to their favorable safety profiles, and inexpensive, simple and stable nature. However, immunization with a DNA vaccine is often accompanied by a lack of strong and durable immunogenicity, potentially resulting from the inefficient antigen processing and presentation, limited T cell priming, and the negative regulation of effector T cells in the tumor microenvironment. Thus there is ample room to improve the efficacy of DNA vaccination by combing different immunotherapy modalities, including checkpoint inhibitors, adjuvants for antigen presenting cell activation, and/or cytokine modulators. I will discuss the rationale and data supporting the selected use of immunotherapeutic combinations in preclinical models of cancer. 

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11:50am - 12:05pm

Phase 1/2 Clinical Trial of SCIB1 ImmunoBody in Stage III/IV Melanoma

Speaker

Lindy Durrant
Scancell Holdings plc

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12:05pm - 12:20pm

DNA vaccination with synthetic WT1 constructs result in the induction of WT1-specific T-cell responses that can inhibit tumor growth

Speaker

Dr. Jewell Walters
University of Pennsylvania

Abstract

Jewell N. Walters1, Jian Yan2, Jaemi S. Chu1, Amir S. Khan2 and David B. Weiner1.

1 Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104
2 Inovio Pharmaceuticals Inc, 1787 Sentry Parkway West, Building 18, Suite 400, Blue Bell, PA 19422.

Cancer antigens have emerged as possible important immunotherapeutic vaccine targets in the fight against cancer.  Wilms’ tumor gene 1 (WT1) is known to be mutated or over expressed in pediatric Wilms’ tumors, leukemia and various solid tumors including lung, pancreatic, thyroid, breast, testicular and ovarian carcinomas and in melanoma.  In recent years a number of epitope based WT1 vaccines have been developed and studied including peptide derived and dendritic cell approaches.  A major issue with such approaches has been poor in vivo immune potency likely due to the highly conserved self-antigen nature of WT1.  Here we developed two unique consensus WT1 DNA immunogens, SynConWT1-S and SynConWT1-L, and studied their immune profiles in mice.  When delivered by electroporation, we observed that SynConWT1-S induced robust IFN-ƴ responses (~1200 SFU/106 PBMCs) and SynConWT1-L induced ~800 SFU/106 PBMCs, in the absence of an immunological adjuvant.  We also show that the consensus WT1 vaccines were much more effective at driving immunity than optimized native construct or un-optimized native construct, indicating that the consensus WT1 vaccines were capable of breaking tolerance.  The functionality of antibodies produced by vaccinated mice assessed by immunofluorescence and immunoblot, illustrated that sera from vaccinated mice recognized native WT1 in vitro. We have also observed impact of this immunogen on tumor growth in vivo where vaccinated mice showed decreased tumor growth compared to the naïve group. Together these data support further study of SynConWT1 as a potential therapeutic approach to blood tumors.

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12:20pm - 1:30pm

Lunch

Tumor Targeting

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1:30pm - 1:50pm

Advanced T-cell Engineered for Cancer Therapy

Speaker

Dr. Martin Pule
Clinical Senior Lecturer Honorary Consultant in Haematology & Coordinator of ATECT – "Advanced T-cell Engineered for Cancer Therapy" , UCL Cancer Institute, UK

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1:50pm - 2:10pm

Intralesional DNA Immunotherapy: Converting Anti-PD-1 Non-Responders to Responders

Speaker

Dr. Robert Pierce
Chief Medical Officer, OncoSec

Abstract

Monoclonal antibodies (mAbs) inhibiting the T cell immune-inhibitory PD-1 pathway are demonstrating unprecedented durable responses in a variety of solid tumor types, including melanoma, NSCLC, squamous H&N and bladder (ASCO, 2014). In melanoma, the most extensively studied indication for anti-PD-1 mAbs, PD-1 responders appear to be those patients, whose tumors harbor significant numbers of pre-treatment CD8+PD1+ TILs. Conversely, patients who fail to respond to PD-1 mAbs are characterized by low numbers of pre-treatment TILs. These patients constitute the majority (approximately 60-70%) of patients with Stage IV melanoma and represent a tremendous unmet medical need. Data will be presented supporting the TIL-dependent responder hypothesis and the potential of intratumoral therapies to enhance immunogenicity. The future role of intratumoral therapies in driving a de novo TIL response and potentially converting PD-1 mAb non-responders into responders will be discussed.

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2:10pm - 2:30pm

Anti-CTLA-4 and CPG: Experience with Direct Lesion Therapy

Speaker

Dr. Holbrook Kohrt
Assistant Professor of Medicine (oncology), Stanford University Medical Center

Abstract

Stimulation of Natural Killer Cells with an Anti-CD137 Antibody Enhances the Efficacy of Trastuzumab, Cetuximab, and Rituximab in HER2-expressing Breast Cancer, EGFR+ Head and Neck Cancer, and CD20+ Lymphoma

My focus is on NK cells and translational research work that led from an initial in vitro observation to Phase 1 clinical studies. The starting observation was that primary human NK cells in the presence of a CD20-positive cell line and rituximab for 24 h upregulated CD137 (4-1BB) at their cell surface, and this upregulation was dependent on effective ADCC. The same observation was made with trastuzumab and cetuximab in the presence of HER-positive and EGFR-positive tumors, respectively. Using an agonistic anti-CD137 antibody, we demonstrate that triggering CD137 on NK cells enhanced the anti-tumor activity of rituximab, trastuzumab and cetuximab in vitro. Moving to various in vivo efficacy models in mice, my group showed that the combination of an anti-CD137 antibody with either rituximab, cetuximab or trastuzumab greatly enhanced anti-cancer activity of these thera- peutic antibodies. The next step was to study CD137 expres- sion on NK cells in patients with head and neck cancer, NHL, or breast cancer treated with cetuximab, rituximab or trastuzumab. Overall, CD137 is heterogeneously upregulated on circulating NK cells in these patients. This heterogeneity is accounted for by tumor histology, tumor burden, extent of prior antibody treatment and CD16 polymorphism. In addition, only circulating NK cells were studied and the extent of CD137 upregulation on tumor infiltrating NK cells was not investigated in these studies. Time post-treatment was also very important, as observed in pre-clinical experiments, as CD137 is transiently expressed on circulating NK cells. I will present various additional means to enhance ADCC by blocking inhibitory signals, such as CD47 on macrophages, IL-15 on monocytes or DCs, KIR or PD-1 on NK cells.  The future is bright for combination therapies that enhance immune responses.

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2:30pm - 2:50pm

Dr. Robert H. I. Andtbacka - Huntsman Cancer Institute

Speaker

Dr. Robert H. I. Andtbacka
Associate Professor, Surgical Oncology Department of Surgery , University of Utah, Huntsman Cancer Institute

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2:50pm - 3:30pm

Coffee/Networking Break in Exhibit/Poster Area

Oral Abstract Presentations

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3:30pm - 3:45pm

Preclinical Evaluation of the Immunogenicity and Safety of VGX-6150, a Therapeutic DNA Vaccine for Chronic Hepatitis C

Speaker

Dr. Moonsup Jeong
GeneOne Life Science

Abstract

1Moonsup JEONG, 1Hyojin LEE, 2Xuefei SHEN, 2John STONE, 2Jian YAN, 2Amir S. KHAN, 1Byong Moon CHO, 1Young K. PARK, 3David B. WEINER

1 GeneOne Life Science, Seoul Korea
2 Inovio Pharmaceuticals, PA USA
3 Department of Pathology and Laboratory Medicine, University of Pennsylvania, PA USA

Hepatitis C virus is a major cause of severe liver diseases and efficiently establishes persistent infection. Many studies suggest that specific T cell response is essential for clearance and control of HCV and DNA vaccine is a good candidate for inducing strong T cell response. VGX-6150 is a mixture of plasmids expressing HCV non-structural proteins and IL-28B as immune adjuvant and delivered by electroporation device, CELLECTRA®. In previous studies, although both IL-12 and IL-28B are able to bolster Th1-based immune response, we revealed the activity of IL-12 was short lived and IL-28B had long-term influence in monkey study. And, each plasmid expressing HCV non-structural proteins, NS3/4A, NS4B, and NS5A, has demonstrated to elicit strong antigen specific T cell immune response in animal. To develop DNA vaccine for chronic hepatitis C in Korea, we should mix all plasmids as one drug and conduct preclinical evaluation of it. In this study, we evaluated the interference of immune response between plasmids in VGX-6150 compared to each plasmid alone, and the adjuvant function of IL-28B compared to VGX-6150 without IL-28B plasmid. This study also evaluated the plasmids’ bio-distribution and toxicity after single or repeated administration of VGX-6150 in mice. This GLP-compliant toxicology study showed a favorable safety profile and was judged as well-tolerated. The results supported clinical study of VGX-6150 and we are currently conducting phase I study against chronic hepatitis C patients who failed SOC and DAA agents in Korea.

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3:45pm - 4:00pm

An Optimized, Synthetic DNA Vaccine Encoding the Toxin A and Toxin B Receptor Binding Domains of Clostridium Difficile Induces Protective Antibody Responses in Vivo

Speaker

Dr. Michele Kutzler
Assistant Professor, Drexel University College of Medicine

Abstract

Scott M. Baliban1, Amanda Michael2, Berje Shammassian2, Shikata Mudakha2, Amir S. Khan3, Simon Cocklin4, Isaac Zetner4, Brain P. Latimer2, Laurent Bouillaut5, Meredith Hunter6, Preston Marx6, Niranjan Y. Sardesai3, Seth L. Welles7, Jeffrey M. Jacobson2, David B. Weiner8 and Michele A. Kutzler1,2

1 Department of Microbiology and Immunology, Drexel University College of Medicine (DUCOM), Philadelphia, PA
2 Department of Medicine, DUCOM, Philadelphia, PA
3 Inovio Pharmaceuticals, Blue Bell, PA
4 Department of Biochemistry, DUCOM, Philadelphia, PA
5 Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA
6 Tulane National Primate Research Center, Tulane University, Covington, LA
7 School of Public Health, Drexel University, Philadelphia, PA
8 Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA. 

Clostridium difficile–associated disease (CDAD) constitutes a large majority of nosocomial diarrhea cases in industrialized nations and is mediated by the effects of two secreted toxins, toxin A (TcdA) and toxin B (TcdB). Key toxin-neutralizing epitopes have been discovered within the carboxy-terminal receptor-binding domain (RBD) of the toxins, which has generated interest in developing the RBD as a vaccine target. While numerous platforms have been studied, very few data describe the potential of DNA vaccination against CDAD. Therefore, we created highly optimized plasmids encoding the RBD from TcdA and TcdB and immunized C57BL/6 mice and rhesus macaques intramuscularly followed by in vivo electroporation. In these animal models, vaccination induced significant levels of anti-RBD antibodies within the serum and feces that could neutralize toxins in an in vitro cytotoxicity assay. Moreover, mice that were actively immunized with the DNA vaccine or given passive transfer of immunized non-human primate sera were protected from a lethal intraperitoneal challenge of purified C. difficile toxins.  Finally, immunized mice were significantly protected following orogastric challenge with strains of C. difficile spores that were homologous (VPI 10463; n=10/10) and heterologous (hypervirulent UK1; n=4/8) strains to our vaccine antigens. These data demonstrate the robust immunogenicity and efficacy of a TcdA/B RBD-based DNA vaccine in preclinical models of acute toxin-associated disease. This work is funded by a Department of Defense Congressionally Directed Medical Research Grant W81XWH0910382 (http://cdmrp.army.mil) to MAK.

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4:00pm - 4:15pm

B16 Melanomas and their Resistance to Trp2-specific CTLs

Speaker

Dr. Jeong-Im Sin
Professor of Microbiology, Kangwon National University School of Medicine

Abstract

Department of Microbiology, School of Medicine, Kangwon National University, Chuncheon, Gangwon-do 200-701, Korea

Previously, we reported that intratumoral electroporation of IL-12 cDNA eradicated large established B16 melanomas through induction of Trp2180-188-specific CD8+ CTL responses. Subsequently, co-delivery of Trp2180-188 peptides, CpG-oligodeoxynucleotide (Toll like receptor-9 ligand) and agonistic anti-4-1BB Abs in combination dramatically increased tumor cure rates against established melanoma. However, when the tumor-cured mice were re-challenged with a parental B16 tumor cell, some mice still formed tumors while most others formed no tumor. The two tumor cells (B16-S0-1 and B16-S1-1), which were obtained from the tumor-formed mice, displayed the expression of both Trp2 and MHC class I antigens in vitro. However, they were still able to form tumors in animals previously immunized with Trp2 vaccines. In particular, UV-irradiated wild type B16 and B16-S0-1 cells but not B16-S1-1 cells were able to induce IFN-ƴ production from Trp2-specific immune cells in vitro. Furthermore, B16-S1-1 cells exhibited no increase in the expression of the ligands (Fas-ligand, CD73 and PD-L1) on the cell surface for inhibitory immune receptors, suggesting that B16-S1-1 cells may be unable to present an antigen to CTLs in conjunction with MHC class I. However, B16-S0-1 cells exhibited both an active state of Stat3 and an increased secretion of IL-6. Treatment of B16-S0-1 cells with anti-IL-6 resulted in inactivating the Stat3 pathway. However, in vivo blocking of the Stat3 pathway by treatment with plasmid DNAs encoding si-Stat3 and with anti-IL-6 failed to recover sensitivity to Trp2 vaccine-mediated tumor control, indicating that the Stat3 pathway may not be associated with tumor resistance in B16-S0-1 cells. Moreover, they expressed a lower amount of Bcl-2 proteins but showed a more increased sensitivity to chemotherapeutic drugs including cisplatin, as compared to wild type B16 cells. Based upon these in vitro and animal studies, B16 cells may obtain Trp2-specific immune escape by not only impaired antigen presentation to CTLs but also yet-unknown novel mechanism(s).

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4:15pm - 4:30pm

Genomes-to-Vaccines: Harnessing Genomics and Immunoinformatics to Produce Immunogenic and Protective Epitope-driven DNA Vaccines

Speaker

Leonard Moise

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4:30pm - 4:45pm

Gene Electrotransfer of IL-15/IL-15Ra as an Effective Immunotherapy in a Mouse Melanoma Model

Speaker

Dr. Richard Heller
Director and Professor, Frank Reidy Research Center for Bioelectrics

Abstract

Richard Heller, Cathryn M. Lundberg, Niculina Burcus, and Shawna A. Shirley
1
Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA 23508 2Medical Diagnostics and Translational Sciences, Old Dominion University, Norfolk, VA 23508

Gene electrotransfer (GET) is a reliable and effective physical method for in vivo delivery of plasmid DNA (pDNA). Several pre-clinical and clinical studies have utilized GET to deliver genes for cancer therapy. It is particularly attractive for use in anti-cancer cytokine therapy as it does not incur the side effects associated with conventional protein cytokine therapy. Delivery of plasmids encoding cytokines directly to tumors has been shown by our lab and others to induce not only local immune response, but a systemic one as well. Previously we have shown that using GET to deliver interleukin-15 (IL-15) to mouse melanoma resulted in long term-tumor regression and survival of a percentage of treated animals after challenge. In the current study we have enhanced this anti-tumor activity by delivering both IL-15 and its soluble receptor IL-15Ra.

GET was used to deliver a plasmid encoding both IL-15 and its soluble receptor IL-15Ra to established B16.F10 tumors. Multiple delivery protocols were utilized to establish varying levels of gene expression. Plasmids expressing either IL-15 alone or IL-15Ra only were used as controls. Tumor volume was monitored for changes over a period of 9 weeks. Mice that were observed to be tumor free were then challenged with B16.F10 cells on the opposite flank and monitored for an additional 50 days. In separate experiments, protection was examined using a metastatic model. Over the course of the experiment, samples were collected for histology, IL-15 expression and cytokine analysis. Tumor regression and long- term survival in a larger percentage of animals was observed when GET was used to deliver the plasmid expressing both IL-15 and IL-15Ra compared to delivery of plasmid containing IL-15 only or simultaneous delivery of separate plasmids expressing IL-15 and IL-15Ra. After challenge, mice that were treated with the IL-15/IL-15Ra plasmid showed protection in 70% of the original treated group compared to only 30% of the group that received IL-15 only and 20% in the group that received the genes on separate plasmids. These results demonstrate that using GET to deliver IL-15/IL-15Rα a local and systemic anti- tumor immune response can be generated in a melanoma model. 

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4:45pm - 5:00pm

Conference Wrap Up