Current Topics in Microbiology 226 and Immunology Editors R.W. Compans, Atlanta/Georgia M. Cooper, Birmingham/Alabama J.M. Hogle, Boston/Massachusetts· Y. Ito, Kyoto H. Koprowski, Philadelphia/Pennsylvania· F. Melchers, Basel M. Oldstone, La Jolla/California· S. Olsnes, Oslo M. Potter, Bethesda/Maryland· H. Saedler, Cologne P.K. Vogt, La Jolla/California· H. Wagner, Munich Springer Berlin Heidelberg New York Barcelona Budapest Hong Kong London Milan Paris Santa Clara Singapore Tokyo DNA Vaccination/ Genetic Vaccination Edited by H. Koprowski and D.B. Weiner With 31 Figures and 14 Tables , Springer HILARY KOPROWSKI, M.D. Professor Thomas Jefferson University Department of Microbiology and Immunology Center for Neurobiology Rm. M-85, Jefferson Alumni Hall 1020 Locust Street Philadelphia, PA 19197-6799 USA DAVID B. WEINER, Ph.D. Associate Professor University of Pennsylvania School of Medicine Hospital of the University of Pennsylvania Department of Pathology and Laboratory Medicine 500 Stellar-Chance Bldg. 422 Curie Boulevard Philadelphia, P A 19104-6100 USA Cover Illustration: Plasmid delivery to a host tissue results in tissue specific protein production and specific activation of T cells, B cells and antigen presenting cells generating specific immunity (by courtesy of Bin Wang and Michael Chattergoon). Cover Design: design & production GmbH, Heidelberg ISBN-13: 978-3-642-80477-9 e-ISBN-13: 978-3-642-80475-5 DOl: 10.1007/978-3-642-80475-5 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. 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Typesetting: Scientific Publishing Services (P) Ltd, Madras SPIN: 10503929 27j3020jSPS - 5 4 3 2 I 0 - Printed on acid-free paper Preface Genetic / DNA immunization represents a novel approach to vaccine and immune therapeutic development. The direct injec tion of nucleic acid expression cassettes into a living host results in a limited number of its cells becoming factories for production of the introduced gene products. This host-inappropriate gene expression has important immunological consequences, resulting in the specific immune activation of the host against the gene delivered antigen. The recent demonstration by a number of laboratories that the induced immune responses are functional in experimental models against both specific infectious diseases and cancers is likely to have dramatic consequences for the develop ment of a new generation of experimental vaccines and immune therapies. This technology has the potential to enable the pro duction of vaccines and immune-based therapies that are not only effective immunologically but are accessible to the entire world (rather than just to the most developed nations). Vaccine Development Vaccination against pathogenic microorganisms represents one of the most important advances in the history of medicine. Vaccines, including those against polio, measles, mumps, rubella, hepatitis A, hepatitis B, pertussis and other diseases, have dramatically improved and protected more human lives than any other avenue of modern medicine. The vaccine against smallpox, for example, has been so successful that it is now widely believed that this malicious killer, responsible for more deaths in the twentieth century than World Wars I and II combined, has been removed from the face of the earth. Traditional vaccination has relied on two specific types of microbiological preparations for producing material suitable for immunization and the generation of a pro tective immune response. These two broad categories involve either live infectious material which has been manufactured in a weakened or attenuated state, thus preventing the vaccine from VI Preface inducing disease, or alternatively, nonlive, inactivated, or subunit preparations. Live attenuated vaccines, such as the polio and smallpox vaccines, for example, stimulate protective immune re sponses as they replicate in the host. However they are limited in their ability to replicate in humans and, accordingly, do not normally induce crippling or lethal disease in inoculated indi viduals. Since these live preparations are actually produced in the host as the attenuated virus replicates within the host cells, they have several unique immunological properties associated with this in vivo replication. The viral proteins produced within the host are leaked or perhaps shed into the extracellular space sur rounding the infected cells and are then picked up, internalized, and digested by scavenger cells that patrol the body. These in clude both macro phages and the several forms of dendritic cells, as well as B cells; collectively they function to expand immune response. These antigen presenting cells (APCs) then recirculate small fragments of the antigen to their surface, attached to MHC II or class II antigens. This complex of foreign peptide antigen plus host MHC class II antigens forms part of the specific signal with which APCs themselves, along with the MHC peptide complex, trigger the action of a central population of immune cells, the T helper lymphocytes cells. The second part of the ac tivation signal comes from the APCs themselves: they display on their cell surface, in addition to MHC-antigen complexes, co stimulatory molecules. Together they can drive T cell expansion and activation through interaction with their respective ligands, the T cell receptor complex (TCR) and the costimulatory recep tors CD28/CTLA4, present on the T cell surface. Activated T helper cells secrete soluble molecules that serve as powerful ac tivators of immune cells. In addition, as viral proteins are pro duced within cells of the host, small fragments of viral proteins are drawn to the cell surface, chaperoned by the host cell MHC I antigens. These complexes are recognized by a second class of T cells, the killer or cytotoxic T cells. This recognition, coupled with secondary stimulation provided by professional APCs and helped by cytokine production from the stimulated helper T cells, either individually or collectively, is responsible for the development of the mature effector T cytotoxic cells capable of destroying viral factories, i.e., infected cells. In addition, in most instances live infection induces an additional benefit, that of lifelong immunity. In contrast, killed or nonlive vaccines composed of whole or even fragments of viruses, when inoculated as vaccines, are not produced within host cells and upon administration mostly wind up in the extracellular space and not within cells. Accordingly they provide protection by directly generating T helper and hu- Preface VII moral immune responses against the pathogenic immunogen and are therefore particularly useful in protecting against infection, especially when antibodies are responsible for microbe inactiva tion. In the absence of the cellular production of the foreign antigen, these vaccines are usually devoid of the ability to induce significant T cytotoxic responses. The lymphokine profiles of these antigens are also often different from those induced by live attenuated vaccines. In addition these vaccines are not actually produced in the host and therefore are not customized by the host. The immunity induced by these vaccines frequently wanes during the lifespan of the inoculated hosts and may often require repeated immunizations or boosting to achieve lifelong immuni ty. However, nonlive vaccines offer some important advantages over live vaccines. They are generally easier to manipulate and usually easier to produce than live vaccines. Furthermore they enjoy an inherent advantage: we can design them to contain only the specific antigenic target of the pathogen that is involved in the development of protective immunity and exclude all other viral components. The latter, namely, can deflect the focus of the im mune response and actually have unwanted immunological cross reactivities that can cause problems for the vaccinated host. Accordingly, live attenuated preparations are clearly the vaccines of choice in terms of the diverse immune responses they produce. However they pose risks of reversion to a more pathogenic form: during their replication process they can sometimes convert back into an actual disease-causing virus. Furthermore they may retain the unwanted characteristic, even in the attenuated state, of inducing disease in persons with weak or compromised immune systems. This includes persons suffering from genetic or drug induced immune deficiency, receiving cancer chemotherapy, or suffering from a separate infection that weak ens their immunity (AIDS patients, the elderly). An individual vaccinated with a live attenuated virus can pass that attenuated virus on to others, thus presenting risks of inadvertent spread to an unknowing host (something health care workers need to keep in mind). Mothers have become vaccinated with the live attenu ated polio vaccine simply by changing the diapers of their re cently vaccinated infants. In contrast, safely manufactured nonlive vaccines that have specific immunity-inducing abilities have no risk of inducing infection or disease due to a need to maintain a replicating phenotype. They can be designed to ex clude those portions of a pathogen that may induce unwanted cross-reactive immune responses. Accordingly they have been the vaccines of choice, based on both their safety and specificity and certain manufacturing considerations. VIII Preface Recent work from a number of laboratories (including that of the editors) has demonstrated that inoculation of a DNA plasmid directly into a host with subsequent expression of the encoded peptide sequences in vivo results in presentation of the specific encoded protein(s) to the immune system. Since DNA vaccines are nonreplicating and are produced within the host cells they can be constructed to function with the safety advantages of a subunit nonlive vaccine and yet to mimic immune potentiating aspects of a live attenuated vaccine. This represents a distinct advantage for initial vaccine and analysis as DNA vaccination represents a rapid system for directly testing subunit vaccination strategies without viral production or protein purification pro cedures. The latter can significantly slow down the investigation process. Furthermore, DNA vaccination facilitates a diverse ar ray of immune analysis from a single immunization platform. It may be particularly advantageous that such a system can deliver an antigen which could be presented for development of both the helper T cell and the cytotoxic T cell arms of the immune system in the absence of live vector construction. Using direct DNA immunization the genes cloned into the expression vectors can be manipulated to present single proteins or an extended genome with only the genes that might lead to pathogenesis removed. Accordingly, this approach, if translatable into success in the clinic, may present the best of both prior vaccine modalitieswith the added benefit of decreased turnaround times for vaccine de velopment. History of DNA Inoculation in Vivo The ability of genetic material to deliver genes for therapeutic purposes has been appreciated for some time. Some of the earliest experiments describing the transfer of DNA into the cells of a living animal were reported by Stasmey (1950), Parchkis (1955), and Ito (1958). These reports described the ability of chromatin preparations, crude preparations of DNA isolated from tumors, to induce formation of tumors after injection in laboratory ro dents. Later experiments further purified the genetic material and confirmed that direct DNA gene injection in the absence of viral vectors can result in the expression of the inoculated genes in the host. If genes encoding cancer antigens were injected, cellular transformation and cancer, with a reproducible frequency, would result in the inoculated animals. Studies using purified DNA and RNA derived from viruses were also reported. Most importantly, Atanasia and colleagues (1962) demonstrated that the subcuta- Preface IX neous administration of purified polyoma virus DNA trans forming sequences as part of the viral genome to newborn hamsters resulted in the generation of anti-polyoma antibodies to the virus and the induction of tumors from the transforming genes. In a similar study, Orth and coworkers (1964) injected either newborn or 40-day-old hamsters with DNA isolated from polyoma virus grown in tissue culture. Thirty days following subcutaneous injection of purified DNA, 34 of the 35 newborn hamsters had developed antibodies and 26 developed tumors. The results described by these studies are explained by the uptake of the injected DNA by cells of the inoculated host followed by translation of the injected DNA sequences into messenger RNA. The host translation system then drives expression of viral en coded genes and their functions, which in the case of the ad ministration of transforming gene sequences, is cellular transformation. Crucial for this discussion however is the out come of this delivery of viral genes: it resulted in the presentation of these genes to the immune system, leading to the production of an immune response against the virus, as determined by the in vivo antibody production observed in these studies. Additional experiments extend these findings to recombinant DNA molecules. Israel and colleagues (1979) reported that re combinant clones of purified DNA containing head to tail dimers of the polyoma virus resulted in seroconversion of inoculated animals after injection of such DNA vectors into weanling mice. Similar experiments using plasmids containing hepatitis B genetic material were reported by Will (1984). The in vivo gene expres sion of plasmid material was further studied in regard to suc cessful insulin delivery as a gene therapy approach in living animals (Dobensky 1985). Elegant studies from the laboratory of Jon Wolff (University of Wisconsin) detailed the long-term ex pression of injected plasmids in vivo (1990). Together, these studies and others served to support the idea that purified nucleic acids could be directly delivered into a host and proteins would be produced. Such proteins could be immunogenic in some set tings. However, challenge studies in these systems have not yet been widely reported. In 1992 Tang and Johnston reported that the delivery of human growth hormone gene in an expression cassette in vivo resulted in protein expression in the inoculated animals. These authors utilized a genetic gun to shoot gold particles coated with DNA through the skin layers of mice. The inoculated animals produced detectable levels of human growth hormone. Interest ingly they reported that antibodies developed against the human growth hormone produced in the mice. These authors termed this X Preface immunization procedure genetic immunization, describing the ability of inoculated genes to be individual immunogens. These studies elegantly brought together and focused the earlier work in this area. Anti-Pathogen Immune Responses Induced By DNA Immunization In Vivo Almost simultaneously with the publication by Tang and John ston the annual vaccines meeting at Cold Spring Harbor in the fall of 1992 brought together several independent groups that collectively reported on the use of DNA immunization technol ogy for the generation of protective immune responses in vivo. These presentations, which used unique approaches and were directed at different viral targets, were not the first public pre sentations of any of the laboratories involved. However, the collective success of these independent investigators and the large amount of data they presented from diverse systems could not be ignored by the vaccine community. Specifically, Margaret Liu of Merck and her collaborators at Vical reported on the induction of immune responses to influenza A virus following intramus cular inoculation of mice with highly purified plasmid DNA en coding influenza A genes. Following three inoculations over 6 weeks using lOOg doses of plasmid DNA, the resulting immune responses to the influenza proteins included antibodies, cellular immune responses, and evidence of protection from viral chal lenge. Harriet Robinson and colleagues (University of Mass achussetts) utilized the gene gun to deliver influenza genes in plasmids in vivo; they observed that DNA doses on the nano gram level were able to induce both antibodies and cellular im mune responses. These doses were also able to protect mice from challenge in a similar influenza challenge model. One of us (Weiner) reported that direct injection of plasmids encoding core HIV genes, rev and the envelope genes induced humoral and cellular immunity against HIV-l, the virus which causes AIDS. The immune responses induced were able to prevent HIV infec tion in in vitro assays. There have been many important studies, which, together, form the basis of this emerging field. Some of the seminal pio neers in this arena have contributed chapters to this volume. Specifically, using a variety of delivery methods and DNA con structs, DNA immunization in animals has been reported to generate host immunity against herpes simplex virus (B. Rouse