The Scientific Future of DNA for Immunization

1 Copyright © 1997 American Society for Microbiology American Academy of Microbiology 1325 Massachusetts Avenue, NW Washington, DC 20005-4171 E-mail: [email protected] Fax: (202) 942-9380

2 The Scientific Future of DNA for Immunization

Prepared by:

Harriet L. Robinson, Ph.D. Harold S. Ginsberg, M.D. Heather L. Davis, Ph.D. Stephen A. Johnston, Ph.D. Margaret A. Liu, M.D.

A report from The American Academy of Microbiology Available on-line at http://www.asmusa.org/acasrc/aca1.htm

3 C O L L O Q U I U M S T E E R I N G C O M M I T T E E

Harold S. Ginsberg, M.D. (Co-Chair) National Institutes of Health

Harriet L. Robinson, Ph.D. (Co-Chair) University of Massachusetts Medical School

Heather L. Davis, Ph.D. Loeb Medical Research Institute

Stephen A. Johnston, Ph.D. University of Texas, Southwestern Medical Center

Margaret A. Liu, M.D. Chiron Corporation

B O A R D O F G O V E R N O R S American Academy of Microbiology

Rita R. Colwell, Ph.D., Sc.D. (Chair) University of Maryland Biotechnology Institute

Joseph M. Campos, Ph.D. Children’s National Medical Center

R. John Collier, Ph.D. Harvard Medical School

Julian E. Davies, Ph.D. University of British Columbia

Harold S. Ginsberg, M.D. National Institutes of Health

Martha M. Howe, Ph.D. University of Tennessee, Memphis

Mary E. Lidstrom, Ph.D. University of Washington

Eugene W. Nester, Ph.D. University of Washington

Mary Jane Osborn, Ph.D. University of Connecticut Health Center

Moselio Schaechter, Ph.D. San Diego State University

Melvin I. Simon, Ph.D. California Institute of Technology

4 C O L L O Q U I U M P A R T I C I P A N T S

Abul K. Abbas, MBBS Margaret A. Liu, M.D. Harvard Medical School Chiron Corporation Boston, Massachusetts Emeryville, California

M. Teresa Aguado, Ph.D. Pierre Meulien, Ph.D. World Health Organization Pasteur Mérieux Serums and Vaccines Geneva, Switzerland March L’Etoile, France

Heather L. Davis, Ph.D. Bernard Moss, M.D., Ph.D. Loeb Medical Research Institute National Institutes of Health Ottawa, Canada Bethesda, Maryland

Hildegund C.J. Ertl, M.D. Carol A. Nacy, Ph.D. Wistar Institute, University of Pennsylvania Rockville, Maryland Philadelphia, Pennsylvania Terence A. Partridge, Ph.D. Harold S. Ginsberg, M.D. Royal Postgraduate Medical School National Institutes of Health London, England Rockville, Maryland David S. Pisetsky, M.D., Ph.D. Joel R. Haynes, Ph.D. Duke University Medical Center Agracetus Inc. Durham, North Carolina Middleton, Wisconsin Ian Ramshaw, Ph.D. Maurice R. Hilleman, Ph.D. Australian National University Merck Institute for Therapeutic Research Canberra, Australia West Point, Pennsylvania Harriet L. Robinson, Ph.D. Vanessa Hirsch, D.Sc. University of Massachusetts Medical School National Institutes of Health Worcester, Massachusetts Rockville, Maryland Michael Sheppard, Ph.D. Stephen L. Hoffman, M.D., D.T.M.H. Pfizer Central Research Naval Medical Research Institute Lincoln, Nebraska Rockville, Maryland Frederick R. Vogel, Ph.D. Stephen A. Johnston, Ph.D. National Institutes of Health University of Texas, Southwestern Medical Center Bethesda, Maryland Dallas, Texas Britta Wahren, Ph.D. Dennis Klinman, M.D., Ph.D. National Bacteriology Laboratory Food and Drug Administration Stockholm, Sweden Rockville, Maryland Robert G. Webster, Ph.D. Myron M. Levine, M.D., D.T.M.H. St. Jude’s Children’s Hospital University of Maryland School of Medicine Memphis, Tennessee Baltimore, Maryland David B. Weiner, Ph.D. University of Pennsylvania Philadelphia, Pennsylvania

5 6 EXECUTIVE SUMMARY

novel approach to the development of needed vaccines A uses DNA for immunization. DNA represents the genetic blueprint for life. When DNA is used for immunization, the DNA in plasmid form provides the code for the vaccinating protein. The actual production of the immunizing protein takes place in the DNA-inoculated host, initiating both humoral and cellular immunity. DNA vaccines are administered in saline using hypodermic needles or by propelling DNA-coated gold beads into skin using gene guns. Recent results obtained in animal models indicate that this new technology may revolutionize the vaccination of humans. Protective immunity has been achieved for such major killers as diarrhea-causing viruses, tuberculosis-inducing bacteria, and malaria-inducing parasites.

These new DNA vaccines also hold promise for being safer, less expensive, and easier to produce and administer than conventional vaccines. This report is based on a colloquium of experts convened to consider this new and extremely promising technology.

7 INTRODUCTION

Historical Importance of Vaccination infectious agents remain major killers and and the Need for New, Improved debilitators (see Figure 1), despite worldwide Vaccines improvements in sanitation and vaccination. Emerging infections, the reemergence of historical scourges, and microorganisms as yet he widespread use of vaccines has uncontrolled continue to pose major risks to resulted in the global eradication of world health. smallpox,1 the near elimination of Tpoliomyelitis and measles from the United Emerging diseases. Increased population States,2 and dramatic reductions in cases of density, the soaring frequency of travel, and the diphtheria, tetanus, whooping cough, mumps, juxtaposition of species in changing natural and rubella (German measles) (see Table 1).2,3 environments promote the emergence and No other medical procedure has had such dissemination of infectious agents (see Figure profound and long-lasting effects on world 2).4,5,6 HIV-1, the virus that induces AIDS, health. No other medical procedure has unrecognized in human populations before resulted in the actual eradication of disease or 1984, has become the eighth leading cause of rivaled the cost effectiveness of vaccines. Each death in the United States and now infects an development of a vaccine has proved a triumph estimated 24,000,000 adults worldwide. of humankind against disease. Nonetheless, Horrifying viruses that cause hemorrhagic fevers, such as the Ebola virus of central Africa and the Hanta viruses of mice, have scrambled Table 1. Comparison of Maximum and Current for brief, but unsuccessful, toeholds in human 1 Morbidity for Vaccine-Preventable Diseases populations. Although multi-drug therapies are Disease Maximum Year 1992 Percentage providing new hope for stemming the inexo- Cases Change rable progression of HIV-1 infected humans to Diptheria 206,939 1921 4 -99.99 AIDS, relatively few nations can provide these Measles 894,134 1941 2,237 -99.75 expensive and regimented treatments. True Mumps2 152,209 1968 2,572 -98.31 control of AIDS awaits an effective vaccine. Pertussis 265,269 1934 4,083 -98.46 The ultimate promise for containment of such Polio (paralytic) 21,269 1952 43 -99.98 emergent agents as Ebola and Hanta also lies in Rubella4 57,686 1969 160 -99.72 vaccine development. CRS5 20,000 1964-65 11 -99.95 Reemerging diseases. Microorganisms not Tetanus6 1,560 1923 45 -97.12 eliminated by drugs or vaccines pose continuing Haemophilus threats of reemergent pandemics.4 Dreaded, influenzae type b 20,0007 1984 1,412 -92.94 drug resistant forms of tuberculosis have Hepatitis B 26,611 1985 16,126 -39.40 emerged not only in humans but in cattle and 1Adapted from (11). deer.7 Tuberculosis is responsible for more 2First reportable in 1968. deaths in adults than any other single infectious 3Subject to change due to retrospective case evaluation of late reporting. agent. The combination of increasing numbers 4First reportable in 1966. of immunocompromised individuals with HIV 5Congenital rubella syndrome estimated for the 1964–65 pandemic. infections and the emergence and spread of 6Cases first reportable in 1947. Maximum based on number of deaths. multi-drug resistant tubercule bacilli has led to 7First reportable in 1991. Estimate based on five U.S. population-based studies, 1976–84. a dramatic worsening of the impact of this disease. Malaria is a major killer of both

8 Figure 1. Major Causes of Death in the World

A. All ages, 1995 estimates B. Children under 5 years of age, 1992 estimates

hook worm dengue

Ieishmaniasis meningitis

pertussis (whooping cough) tuberculosis (children)

tetanus (neonatal) acute respiratory infections (viral)

AIDS pertussis

measles neonatal tetanus

hepatitis B virus viral diarrhea

malaria malaria

tuberculosis measles

diarrhea bacterial diarrhea Diseases in order of increasing importance of increasing Diseases in order acute importance of increasing Diseases in order respiratory acute respiratory infections infections (bacteria)

012 345 012 345 Millions of deaths per year Millions of deaths per year

Figure 2. New Infectious Diseases in Humans and Animals Since 1976. Countries where cases first appeared or were identified

1988 Salmonella enteritidis PT4 United Kingdom

1986 Bovine spongiform 1982 encephalopathy* E.coli United Kingdom 0157:H7 1989 1980 United States 1980 Hepatitus C Hepatitus D Human T-cell 1981 United States (Delta) 1976 lymphotropic virus 1 AIDS Italy Cryptosporidiosis Japan United States United States 1977 1976 1991 Hantaan virus Legionnaires‘ Disease Venezuelan Republic of Korea haemorrhagic fever United States 1992 Venezuela Vibrio cholerae 0139 India

1994 1976 Brazilian Ebola haemorrhagic fever haemorrhagic fever 1994 Brazil Zaire Human and equine morbilivirus Australia

*Animal cases only.

World Health Organization. 1993. Ten Years of Progress 1984–1993. The Programme for Vaccine Development. 9 children and adults. Emergence of resistance of drug resistant tuberculosis threatens to become the parasites to antimalarial drugs and of the the norm, and enterobacteria that cause critical mosquito vectors to insecticides, the deteriora- diarrheal diseases often prove resistant to tion of the health care systems of countries in antibiotic treatment.10 These increases in turmoil, and the refugee crisis resulting in the resistance stem from excessive use of antibiotics movement of non-immune individuals to malari- in patient populations, as well as the use of ous areas have resulted in malaria reemerging agents with clinical applications in agriculture where it was once under control and worsening and aquaculture. In Denmark in 1993, human in areas where it has always been a serious use of a new glycopeptide antibiotic (vancomy- problem. Viruses, such as the influenza viruses, cin) totaled 22kg, while animals consumed can emerge from animal reservoirs.8,9 Pan- 19,000kg of a closely related compound demic influenza viruses emerge after (avoparcin).10 reassortment between human and avian influ- enza viruses in pigs and are transmitted via the Endemic infections. Most microbial agents pig to humans. represent pathogens for which no effective The spread of antibiotic resistance in bacteria vaccines exist (see Table 3). The annual financial represents an ecological disaster (see Table 2). cost of common infectious diseases in the United Penicillin resistant pneumococci jeopardize the States, according to the National Science and treatment of serious childhood diseases. Multi- Technology Council, totals $120 billion per year.

Table 2. The Most Serious Antibiotic-Resistant Bacteria*

Organism Diseases Resistance To

Enterobacteriaceae Bacteremia, pneumonia, urinary tract, Aminoglycosides, beta-lactams, trimethoprim, surgical wound infections vancomycin, chloramphenicol

Haemophilis influenzae Pneumonia, sinusitis, epiglottis, Beta-lactame, tetracycline, chloramphenicol, meningitis, ear infections ampicillin, trimethoprim + sulphonamide

Mycobacterium spp. Tuberculosis Aminoglycosides, isoniazid, ethambutol, pyrazinamide, rifampin

Neisseria gonorrhoeae Gonorrhoea Beta-lactams, penicillins, spectinomycin, tetracycline

Ampicillin, chloramphenicol, trimethoprim + Shigella dysenteriae Severe diarrhoea sulphonamide, tetracycline

Aminoglycosides, beta-lactams, tetracycline, Pseudomonas aeruginosa Bacteremia, pneumonia, urinary tract chloramphenicol, ciprofloxacin, culphonamides infections Chloramphenicol, rifampin, cipfloxacin, clindamycin, Staphyloccus pneumoniae Bacteremia, pneumonia, surgical erythromycin, beta-lactams, tetracycline, wound infections trimethoprim

Chloramphenicol, penicillins, erythromycin Streptococcus pneumoniae Meningitis, pneumonia Penicillins, clindamycin Bacteroides spp. Anaerobic infections, septicaemia Penicillins, aminoglycosides, vancomycin, Enterococcus spp. Catheter infections, blood poisoning erythromycin, tetracycline

* Reprinted with permission from Nature. Davies, J. 1996. Bacteria on the rampage. Nature 383:219-220. Macmillan Magazines Limited.

10 Over $23 billion of this is in direct medical costs and lost productivity due to intestinal infections; influenza infections cost over $17 billion.4 Table 3. Within the United States, the average sick leave Pathogens For Which Vaccines is eight of the 261 working days per year; Are Needed employees suffering “colds” likely spend twice that many days ill on the job. Borrelia burgdoferi Endemic infections can also cause long-term Chlamydia sp. chronic infections. Chronic infections can Coccidioides immitis predispose the host to death from other dis- Cryptococcus neoformans eases—such as the opportunistic infections seen Cytomegalovirus in AIDS—or certain cancers. About 1.2 million Dengue (20%) cancer cases in developing countries and entamoeba histolytica 363,000 (9%) in developed countries are Enterotoxigenic Deficiency virus HIV-1 associated with chronic infectious agents (see Figure 3).4 Of the 527,000 new cases per year of Epstein-Barr virus (EBV) liver cancer, 60% are caused by hepatitis B virus, Group A streptococcus and 22% by hepatitis C virus; 550,000 cases a Haemophilis influenzae nontypable year of stomach cancer are attributed to the Hepatitis C virus (HCV) bacterium Helicobacter pylori, a causative agent Hepatitis D of duodenal ulcers and gastritis. Human papil- Hepatitis E loma viruses types 16 and 18 are associated with Herpes simples virus types 1 and 2 83% of the 529,000 cases per year of cervical Histoplasma capsulatum cancer. Human Immune Deficiency virus HIV-1 Human Immune Deficiency virus HIV-2 Vaccines and Vaccination Human papillomavirus Nature of a vaccine. Vaccines mobilize the Legionella pneumophila immune system (white blood cells and lymphoid Leishmania sp. tissues) to combat and control infectious dis- Moraxella catarrhalis eases.11 Put simply, a vaccine is a nondisease Mycoplasma pneumoniae causing mimic of an infectious agent. Vaccina- Neisseria gonorrheae tion is the administration of a vaccine and the Parainfluenza virus elicitation by the vaccine of an immune response Plasmodium spp. capable of controlling a specific infectious agent. Pseudomonas aeruginosa Successful vaccination protects both individuals Pseudomona cepacia and populations. Individuals are protected Respiratory syncytial virus against the development of disease; populations Rickettsia rickettsii are protected against the spread of the disease- Rotavirus causing agent. Schistomosoma mansoni Antigens and the expansion of the immune Shigella (all species) response. Immunization with a vaccine primes Toxoplasma gondii the immune system to recognize and contain an Treponema pallidum invading infection. White blood cells (lympho- cytes) that reside in tissues, or patrol the body in the blood and lymph, recognize and respond to infectious agents as foreign. These blood cells recognize specific structural shapes (epitopes)

11 helper cells secrete growth factors for lymphoid Figure 3. Cancers Associated with Infectious cells (called lymphokines) that stimulate the Diseases, 1995 Estimates Stomach activation and function of B- and T-cells. The Heliobacter cytolytic T-cells recognize and either directly, or pylori 1.0 indirectly, kill cells infected by a microorganism. Like B-cells, each T-cell has receptors specific for Infection associated one and only one epitope. However, unlike B- 0.8 cells, whose immunoglobulin receptors recognize Liver Cervical Hepatitis B & C Papilloma determinants in native forms of protein, T-cell 0.6 virus virus receptors recognize fragments of proteins that Bladder Lymphoma are displayed on the cell surface by major 0.4 Shistosomiasis Epstein-Barr virus histocompatibility complexes (MHC). Millions of cases 0.2 Major histocompatibility (MHC) antigens. A central problem for the immune system is its 0.0 deployment against invading organisms and not Cancers in order of increasing importance against one’s self. It accomplishes this by “re- stricting” its destructive activities to foreign World Health Organization. 1993. antigens that the host cell MHC proteins present to T-cells. The co-discoverers of this phenom- within molecules (antigens) of the infectious enon, Peter Doherty and Rolf Zinkernagel, agent. Each microbe has unique structures, and received the 1996 Nobel Prize in Physiology and thus unique epitopes. The priming of an immune Medicine. response expands and activates “naive” white cells There are two different types of MHC (those that have not previously seen an immuno- proteins, Class I and Class II. Both of these gen) to become “effector” cells that actively present proteolytically degraded fragments of combat the infection. Each naive cell has the proteins to T-cells (see Figure 4). Class I mol- potential for seeing one—and only one—epitope, ecules present fragments of proteins to cytolytic a recognition system often likened to a key T-cells. Class II molecules present protein fitting into a lock. Only those cells that recog- fragments to T-helper cells. In most instances, nize their cognate epitope become effector cells. Class I presents foreign proteins synthesized in a cell. For presentation by Class II, the foreign Antibody-producing B cells. Recognition of protein either can be synthesized in the cell or specific immunological epitopes can expand taken up by the cell from the outside. If an antibody producing white blood cells, called antigen is synthesized in a cell and presented by B-cells. Antibodies are proteins that bind to both Class I and Class II molecules, both anti- foreign antigens. Each B-cell makes one and only body-producing B-cells and cytolytic T-cells are one antibody. This antibody has the potential to raised. However, if an antigen originated outside bind to one conformational or linear epitope of a cell and is expressed only by Class II, the within an antigen. Each antibody binds to its specific immune response is largely limited to specific epitope. Lymphocytes that produce T-helper cells and antibody production. antibody are called B-cells because scientists first recognized them in the bursa of Fabricius, a Memory responses. As an infection is contained lymphoid organ of chickens. and the inducing antigen eliminated, the immune system creates “memory” B- and T-cells. T-helper cells and cytolytic T-cells. Recognition Memory cells represent inactive yet rapid- of specific antigens also expands lymphocytes response patrols on guard for the reappearance called T-cells (or thymus dependent cells). These of their specific antigen. The power of vaccina- T-cells can be “helper” or “cytolytic” cells. The tion lies in the memory response. Under the best

12 Figure 4

MHC I, Synthesis of immunogen in cell

Nucleus

vaccine plasmid

Synthesis of vaccinating immunogen in cell

Proteasome digestion of the immunogen and loading of peptide fragments onto MHC I molecules in the endoplasmic reticulum

Transport of peptide-MHC I complexes to cell surface

MHC II, Synthesis of immunogen in cells or Presentation of peptide MHC I internalization of immunogen from outside of cell complexes to CTL

Internalization of vaccinating immunogen into an endosome Synthesis of vaccinating immunogen in cell Nucleus vaccine plasmid

MHC II blocked by invariant chain Digestion of the immunogen and the invariant chain by lysosomal proteases

Loading of peptide fragments of the immunogen onto MHC II and transport to cell surface

Presentation of peptide-MHC II complexes to T-helper cells

13 case scenario, vaccines require only one adminis- Live attenuated vaccines. Jenner’s cowpox tration to provide life-long protection. And, if vaccine represented the first use of a live, given worldwide, vaccinations can achieve attenuated organism for vaccination. In Jenner’s eradication, eliminating both the disease and the case, attenuation resulted from using a non- need for further vaccination. human virus in a human.13 The cowpox virus (which may have come from a horse) grows The Development of Human Vaccines sufficiently in the human to induce immune responses. However, it does not grow sufficiently The roots of vaccination trace back to early to cause disfigurement beyond the site of inocu- Western and Eastern civilizations. Roman lation; only in rare instances did it cause death.12 scholars suggested that livers of mad dogs could The use of live attenuated organisms has since protect against rabies, and 16th century Taoist proved effective in preventing diseases such as healers used crusts from smallpox to protect rabies, yellow fever, poliomyelitis, measles, against smallpox disease.12 An 18th century mumps, rubella, and most recently chicken pox British physician, Edward Jenner, made the first (see Table 4, column 1). Since World War II, scientific demonstration of vaccination.13 In viruses for vaccines have been attenuated by 1796, he used cowpox (taken from the lesion on serial passage of the human virus in cell culture a milk maid’s hand) to inoculate an 8-year-old of nonhuman origin—for example, chicken boy. The boy developed a localized infection that embryo or monkey kidney cells. During a rapidly healed. Approximately two months later, successful attenuation (and not all efforts at Jenner inoculated (challenged) the boy with attenuations are successful), the virus loses smallpox virus. The boy was completely resis- virulence while retaining the antigens that tant to the disease. The cowpox virus had stimulate protective immunity. The current sufficient antigens in common with the deadly “Jeryl Lynn” mumps vaccine represents one of smallpox to provide protective memory. After several attempts at attenuation, with success the British Royal Society rejected Jenner’s report occurring only after a 16-passage attenuation of “lest he damage his reputation,” Jenner privately a fresh isolate cultured from a case of clinical published a paper. The news that one could mumps in the 6-year-old daughter of the devel- prevent the then leading cause of adult death by oper.14 inoculation with the cowpox led to the rapid acceptance of this revolutionary medical tech- Killed whole vaccines. A second type of vaccine, nique.12 killed whole organisms, emerged in the late 19th Despite this initial success, the development of century. At first, developers used heat to inacti- further vaccines awaited another century, vate (kill) the Salmonella typhi and Vibrio gaining momentum only as fundamental work cholera bacteria that cause typhoid fever and by Louis Pasteur and Robert Koch demonstrated cholera respectively15 (see Table 4). Initially that microorganisms (not poisons) cause infec- tested in British troops in India and the Boer tious diseases and that specific microorganisms War, these heat-inactivated vaccines became cause specific diseases. The realization that mandatory for virtually all soldiers by the first specific microorganisms caused specific diseases World War. Since World War II, chemicals have provided the rationale for using a non-patho- replaced heat to inactivate viruses used in killed genic form of a microbe for vaccination. Cur- whole vaccines. The poliovirus vaccine that rently human vaccines utilize four different Jonas Salk developed offers one example of a forms of non-pathogenic agents: live attenuated chemically (formaldehyde) inactivated vaccine. microorganisms, killed whole microorganisms, purified components from microorganisms Purified component vaccines. A third type of (subunits), and genetically engineered compo- vaccine comprising purified components from nents of microorganisms (see Table 4). pathogenic organisms was developed in the early 20th century to prevent tetanus and diphtheria

14 Table 4. The Development of Human Vaccines

Live Killed Whole Purified Proteins or Genetically Attenuated Organisms Polysaccharides Engineered

18th Century Smallpox 1798 19th Century Rabies 1885 Typhoid 1896 Cholera 1896 Plague 1897 Early 20th Century Bacille Calmette-Guerin Pertussis 1926 Diptheria 1923 1927 (tuberculosis) (whole cell) Yellow Fever 1935 Influenza 1936 Tetanus 1927 Rickettsia 1938 Post-World War II (cell culture) Polio (oral) Polio (injected) Pneumococcus Hepatitis B Measles Rabies (new) Meningococcus recombinant Mumps Japanese B Haemophilus Rubella encephalitis influenzae (PRP)* Adenovirus (Type 4) Hepatitis A Hepatitis B (plasma- Typhoid Tickborne derived) (salmonella Ty21a) encephalitis H. influenzae PRP*- Varicella protein (conjugate) Typhoid (Vi) Acellular pertussis

*PRP: capsular polysaccharide (polyribosylribitol phosphate)

(Table 4, column 3). The bacteria that cause saccharides from 23 strains of pneumonia- tetanus and diphtheria secrete disease causing inducing pneumococcal bacteria vaccinates toxins. Inactivated forms of these toxins, called against invasive pneumococcal infections.2 toxoids, can raise “neutralizing” (inactivating) A vaccine composed of capsular polysaccharides antibodies against the toxin. With improvements from six strains of the bacterium Hemophilus in fermentation (growth of bacteria) and in the influenzae b coupled to carrier proteins (to purification of macromolecules, inactivated increase immunogenicity) prevents hemophilus toxins could be produced in bacterial cultures b-induced pneumonia and meningitis.2,16 The and used as vaccines. Following the introduction carrier-coupled vaccine is effective in infants as of the diphtheria toxoid vaccine, cases of well as adults. Prior to the introduction of the diphtheria declined from more than 200,000 per Hemophilus influenzae b vaccine, meningitis year in the United States in 1921 to 2 per year in caused by this bacterium ranked as the leading 1984. The recent reappearance of diphtheria in cause of acquired learning disabilities in the the former Soviet Union, where social and United States. economic upheavals led to a lapse in adequate immunization,2 emphasizes the continuing Genetically engineered vaccines. Genetically importance of this vaccination. engineered vaccines, which use proteins pro- Since World War II, purified polysaccharides duced by recombinant DNA technology, repre- from bacterial pathogens have served as highly sent a late 20th century approach to vaccine effective vaccines. A combination of poly- development. Recombinant DNA technology

15 allowed the production of proteins for agents that would not grow in culture. The recombi- Figure 5. DNA Vaccine nant hepatitis B virus vaccine, whose immuniz- ing protein is produced by DNA introduced into yeast or mammalian cell cultures,2,17 represents Gene for an an example of this type of vaccine. Prior to the gene immunogen genetically engineered production of the hepati- tis B virus surface antigen, this protein had been purified from the blood of infected carriers. g ene r The advent of recombinant DNA technology e t o Insert gene into an

m

also enabled the development of live recombi- p

o

o r

l

p

y expression plasmid nant and live attenuated vaccines. In live recom- A

binant vaccines, one or more genes encoding plasmid critical determinants for immunity are intro- duced into a benign, yet live vector. Live recom- binant vaccines now use a number of different viruses or bacteria as vectors.18 These recombi- Transform bacterial cells, nant virus vectors possess many advantages of grow bacteria, live attenuated vaccines, while expressing only purify plasmid DNA selected genes of a pathogen. Recombinant DNA technology also allows construction of live- attenuated vaccines by directly mutating viru- Immunize with lence genes within the genome of a microorgan- immunogen-expressing ism. No live genetically engineered vaccine has plasmid yet won approval for human use. Some, how- ever, are in human testing and several are licensed for veterinary uses.

A New Technology, DNA Vaccines protein in eukaryotic cells. The construction of Recently, a new approach to vaccination opened bacterial plasmids with vaccine inserts is accom- up with the demonstration that direct inocula- plished using recombinant DNA technology. tion with DNA that encodes a foreign antigen Once constructed, the vaccine plasmid is intro- can initiate protective immune responses. DNA duced (transformed) into bacteria. There the represents the genetic information of all cellular growth of the bacteria produces many plasmid life. It provides the code (blueprint) for the copies. The plasmid DNA is purified from the synthesis of the macromolecules that make up bacteria, using relatively simple techniques for living organisms. DNA vaccines containing the separating small circular plasmid DNAs from genes of foreign proteins provide cells in the the much larger bacterial DNA and other vaccinated host with the code for the synthesis bacterial impurities. The purified DNA (a stable of the immunizing antigens (see Figure 5). molecule) is the vaccine.

Construction of a DNA vaccine. Vaccine DNAs Administration of DNA vaccines. DNA vaccines consist of bacterial plasmids (small circular can be injected in saline solutions into muscle or DNAs that can replicate in bacteria). Each skin using a syringe and needle.19,20 DNA vaccine consists of a plasmid bearing an insert vaccines can also be given by coating the DNA (see Figure 5). The insert comprises sequences onto microscopic gold beads and then using a for the vaccinating protein as well as control “gene gun” to fire the beads into cells.21 The elements (termed promoter and poly A in Figure saline injections deliver DNA into extracellular 5) that allow expression of the vaccinating spaces, whereas gene guns bombard DNA-

16 Table 5. Immunogenicity and Efficacy of DNA Vaccines Successfully Used in Animal Models Pathogen Protein(s) Antibodies CTL Protection Reference(s)

VIRUSES ␣ ␣ cytomegalovirus pp89 + + + 22 ␣ ␣ encephalitis virus prM/E + ND + 23 ␣ ␣ hepatitis B virus HBsAg, HBcAG + + + 24–27 ␣ ␣ herpes virus, bovine gD + ND + 28 ␣ ␣ herpes simplex virus 1,2 gB, gD + equivocal + 24–33 ␣ ␣ simian immunodeficiency Gag, Pol, + + +/- 34–37 (for ␣ ␣ viruses and simian/ Env, Vif, Vpr, review see 38) ␣ ␣ human immunodeficiency Vpu, Rev, ␣ ␣ virus chimeras Tat, Nef ␣ ␣ influenza HA, NP + + + 39–43 ␣ ␣ lymphocytic choriomenin- NP + + + 44–46 gitis virus ␣ ␣ measles HA, F, NP + + ND 47,48 ␣ ␣ papilloma L1 + ND + 49 ␣ ␣ rabies G + + + 50 ␣ ␣ rotavirus VP6 + ND + 51,52 BACTERIA ␣ ␣ mycoplasma pulmonis library + ND + 53 ␣ ␣ mycobacterium tuber- culosis Ag85, hsp65 + + + 54,55 PARASITES ␣ ␣ malaria CSP, HEP17 + + + 56,57

ND: not done

coated gold beads directly into cells. The im- Choosing the name “DNA vaccines.” The mune responses raised by injection and bom- World Health Organization, among the first to bardment require different amounts of DNA and realize that something new was happening in can raise different types of T-cell help (see vaccinology, convened a meeting in May of 1994 below). to hear its pioneers present their results. On the second day of the meeting, a vote was taken on a Animal trials with DNA vaccines. Animal trials name for the new technique from a list of of DNA vaccines have revealed the ability of this candidates: genetic immunization, polynucle- new technique to raise protective immunity otide vaccines, gene vaccines, and nucleic acid against a number of agents for which we need vaccines. Voters split; however, the majority new or improved vaccines (see Table 5). Preclini- chose nucleic acid vaccines, with subterms DNA cal studies also have revealed that fairly similar vaccines or RNA vaccines. The rationale for doses of DNA are effective in essentially all choosing the name nucleic acid vaccines focused animal species (mice, chickens, cows, and on public perception. To gain wide acceptance, monkeys). Thus, this novel method of immuni- the new technology’s name needed to convey its zation offers high promise for the realization of purpose as a protective vaccine without suggest- much needed vaccines. ing that it modified the genetic information of the recipient.

17 A GENERAL CONSIDERATION OF DNA VACCINES

Advantages of DNA Vaccines Over Antibody against the natural forms of viral More Classical Vaccines proteins. The ability of DNA to produce the immunizing protein(s) in host cells yields a Introduction. DNA vaccines have a number of vaccinating protein in its “native” form. Many advantages over more classical vaccines (see viral proteins have folded structures that the Figure 6). In regard to viruses, DNA vaccines purification process can easily disrupt. Most mimic live attenuated vaccines and live recombi- antibodies recognize folded structures. If the nant vectors by producing the immunizing preparation of a subunit vaccine has resulted in material in the host. However, DNA vaccines are a misshapened protein, the antibody response unlike live viral vaccines in that they do not will recognize the disrupted structure, rather cause infections. DNA vaccines are subunit than the normal protein present on the micro- vaccines in that they express only one, or a organism. Antibodies that fail to recognize the subset of proteins, from a pathogen. Classical native form of a protein frequently prove subunit vaccines are produced in fermenters or ineffective at containing an invading micro- cell cultures and the desired antigen(s) purified. organism. Then the purified components are inoculated into the vaccinee. Recombinant virus vectors Induction of cytolytic T-cells. A distinguishing revolutionized this process by producing the feature of DNA vaccines, as opposed to more subunit vaccine in the host. DNA vaccines take classical subunit vaccines, is the production of this one step further by eliminating the need for the immunogen in host cells. This supports a virus vector. The production of subunit processing and presentation by both Class I and vaccines in hosts by DNA has a number of Class II MHC molecules (see Figure 4). By important consequences for the success of contrast, killed-whole or protein subunit vac- vaccination. It also greatly simplifies vaccine cines undergo presentation by Class II MHC development and production. molecules. These differences in presentation result in DNA vaccines raising both cytolytic T-cells and antibody, whereas more classical subunit vaccines raise antibody mostly. The Figure 6. Unique Features of DNA Vaccines importance of raising cytolytic T-cells lies in No risk for infection their ability to directly kill pathogen-containing Raise antibody against the native forms of proteins cells. The use of DNA, a nonliving agent, to raise cytolytic T-cells represents a milestone in Raise cytolytic T-cell responses vaccinology. Raise long-lived responses Facilitate the use of combination vaccines Long-lived responses. As early vaccine develop- Good stability at low and high temperatures ment progressed from live-attenuated to killed- Potential for generic production and manufacture whole organism vaccines, workers rapidly came Support the recovery of candidate vaccines from infected tissue to appreciate that raising and maintaining Allow rapid screening of multiple sequences for protective responses immunity requires multiple inoculations of Ability to prime for T-helper 1 or T-helper 2 biased responses inactivated vaccines.14 This stands in contrast to live-attenuated vaccines, where a single inocula- tion can result in long-term protection. In the

18 United States, the DPT vaccine (inactivated materials. The ability to recover microbial DNA diphtheria, pertussis, and tetanus) is adminis- sequences using PCR revolutionized vaccine tered five times between birth and 7 years of development. For known organisms for which age.11 In preclinical studies, DNA vaccines we can design PCR reagents, candidate vaccines appear similar to live vaccines, in that they have can be directly recovered from infected tissue, the potential to raise long-lived immune re- eliminating the weeks to months required to sponses. For researchers who pioneered DNA culture a microbe from a diseased tissue. The vaccines, the two most exciting results are the ability to make a vaccine directly from the tissue achievement of protection and the realization of an infected animal also allows construction of that the protection could last a long time. vaccines for microorganisms that fail to grow in cultures. Examples of such pathogens include Facilitation of combination vaccines. In the such major killers as the hepatitis B and C United States, the full course of childhood viruses, diarrhea-causing viruses, and papilloma immunizations currently requires 18 visits to a viruses. physician or clinic.11 Giving several vaccines at once could reduce this number of visits, but Library screening. The ability to use DNA as a differences in the formulations of the different vaccine allows researchers to screen genes from vaccines limits their use in combination. DNA an organism for their ability to raise protective vaccines, by having the same formulation, might responses. Even academic laboratories can con- eliminate this problem. struct and test multiple candidate vaccines, a feat once undreamt of by even the largest industrial Generic production and validation. All DNA giants using conventional vaccine development vaccines can be produced using similar fermenta- approaches. In cases where the immunologically tion, purification, and validation techniques. relevant genes remain unknown, DNA “librar- This ability to use generic production and ies,” or collections of DNA sequences, can be verification techniques vastly simplifies vaccine used to screen the entire genome of a micro- development and production. organism for sequences that raise protective immune responses. This technique is called Stability. In contrast to many conventional “expression library immunization.”23 As pro- vaccines, such as live viruses and protein sub- tective libraries are identified, these are subdi- units, DNA vaccines remain stable at both high vided and further tested to identify which gene (below boiling) and low temperatures. DNA or genes within the library provide protection. vaccines can be stored either dry or in an aque- ous solution. The good stability of DNA vaccines Manipulation of the vaccine response. DNA should facilitate distribution and administration vaccines can be used to bias an immune response and eliminate the need for “the cold chain”—the towards one of two different types of T-cell help series of refrigerators required to maintain the (see Figure 7).58,59 These two types of T-cells, viability of a vaccine during its distribution. called T-helper 1 and T-helper 2, affect the types Currently, maintaining the cold chain represents of antibody raised and the nature of the inflam- 80% of the cost of vaccinating individuals in matory cells mobilized to fight an infection. The developing nations.59 type of T-help and its associated inflammatory responses are important in determining how Recovery of candidate vaccines from diseased effectively the body clears an infection. The tissue. Of the complex molecules that make up ability of DNA immunizations to bias the type living organisms, DNA is the easiest with which of T-cell help may also support the use of this to work. A technique called polymerase chain new technology for the control of autoimmune reaction (PCR) supports the recovery and disease (an inappropriate T-helper 1 response) amplification of specific DNA sequences—even and allergy (an inappropriate T-helper 2 those present at very low levels—from biological response).

19 Figure 7. T-helper 1 and T-helper 2 Responses

Type of T-help Associated Antibody Associated Innate Immunity Special consideration of DNA vaccines ○○○○○○ Th1○○○○○ C1 dependent Phagocyte-mediated for the control of viruses, parasites, and IFN-γ FcγR-binding bacteria IL-2 IgG2a, IgG2b activated opsonization macrophages Unique patterns of protein modifications for

bacteria and parasites. Whereas viruses use the

○○○○○ ○○○○○○ machinery of animal cells to synthesize proteins, Th2 C1 independent Phagocyte-independent parasites and bacteria provide their own machin- IL-4 FcεR-binding ery for protein synthesis. As proteins are synthe- IL-5 IgG1, IgE activated anti- mucosal eosinophils, phagocytic responses sized they undergo modifications, the most fre- IL-6 mast cells controls quent of which is the addition of sugar groups IL-10 (glycosylation). Animal cells, bacteria, and para- sites have distinctive patterns of protein modifi- Figure 7. Two different Types of T-cell help. Th1, T-helper 1; Th2, T-helper 2. C', cation. A bacterial protein produced by an animal complement dependent; IFNγ and IL-2, distinguishing lymphokines produced by T-helper 1 cells; IL-4, IL-5, IL-6, IL-10, distinguishing lymphokines produced cell will not have the same modifications and by T-helper 2 cells. structure as the protein produced by bacterial cells. Thus, a unique advantage of DNA vaccines for immunization against viruses—production of Limitations of DNA vaccines over more the native form of the viral protein—does not classical methods of vaccination necessarily hold for vaccinations against bacte- rial or parasitic proteins that carry modifications Limited to protein antigens. DNA can be used distinct from those conferred by animal cells. to raise immune responses against the protein Despite this limitation, early work with DNA components of pathogens. Proteins are the vaccines has provided impressive results against major building blocks of all life. However, bacterial and parasitic agents.55,56,57 Develop- certain microbes have outer capsular structures ment of vaccines for these more complex organ- that are made of polymerized sugars (polysac- isms has relied on both DNA-expressed proteins charides). This limits the extent of DNA vac- that represent known targets of immune re- cines’ usage because they cannot substitute for sponses and expression library immunizations to polysaccharide-based subunit vaccines (e.g., identify as yet unidentified protective genes.53 pneumococcus). Importance of multi-valent DNA vaccines for Mucosal delivery. DNA vaccines have yet to be parasites and bacteria. For complex microor- developed for intranasal and oral delivery. Most ganisms, such as bacteria and parasites, the infectious agents enter humans through the ability to use multiple DNAs for immunization respiratory, intestinal, or genital tract. Vaccina- represents a particular strength of the DNA tion for microorganisms that cross mucosal approach.57 For example, parasites go through surfaces is most effective when induced at the different stages of infection in humans, such as entry surface because memory cells patrol the the blood, liver, and reproductive stages of a surfaces where they first encountered an antigen. malarial infection. Each of these stages involves For example, the Sabin oral poliovirus vaccine different antigens. The ability to use multiple initiates vaccination in the gut, the site of DNAs for immunization allows the use of poliovirus infection. DNA vaccines administered immunogens against each stage. This ensures to skin or muscle against influenza virus (a that those parasites that make it past stage one respiratory tract infection) and to skin against (a transit time accomplished in minutes) again rotavirus (an intestinal infection) successfully will be embattled at stages 2 and 3. The use of prime for protective mucosal responses. Whether multiple DNAs will also be important for delivery of DNA vaccines to mucosal surfaces controlling Mycobacterium tuberculosis, the will improve this protection remains to be cause of tuberculosis. In this case, multiple determined. vaccine targets should act much as multidrug therapy, limiting low-level survival and the outgrowth of vaccine-resistant variants. 20 IMPLEMENTATION OF A NEW TECHNOLOGY FOR VACCINATION

mplementing a new technology requires Inoculation with more classical forms of basic research, applied research, and prod- subunit vaccines requires microgram amounts of uct development (see Figure 8). Competitive protein to raise immune responses. By contrast, Igovernment basic research grants were seminal DNA immunizations appear to need much less to the realization and demonstration that DNA protein (on the order of 1,000 times less).21,60 could be used to raise immune responses. How such low levels of DNA-expressed proteins Applied research is now translating this basic raise such effective immune responses remains a finding into vaccines. With the identification of profound and unsolved puzzle. candidate vaccines, product development— including scale-up for manufacture, safety Target site and lymphoid tissue. Determining the testing, and marketing—ensues. Government relative roles of the target site (muscle or skin supports most basic research through grants to delivery site) and lymphoid tissues in DNA- academic institutions or its support of govern- raised responses represents a high priority for ment laboratories, such as those at the National basic research.61 Is this novel immunization Institutes of Health. Applied research receives method obeying classical rules for provoking both government and industrial support. The immune responses or are DNA-transfected final step, product development, falls largely to muscle cells or skin cells, and not lymphoid cells, private industry, with government resources presenting antigen? If lymphoid cells present provided only up to that point where a fairly antigen, how do they obtain antigen? Does DNA certain (and cost justifiable) path to an actual directly transfect these cells or do they acquire vaccine emerges. protein from transfected skin or muscle cells?

Need for basic research on DNA vaccines T-helper biases. A second priority seeks a better understanding of the T-helper preference raised The use of DNA for immunizations opens a very by DNA immunizations and what determines new area of research. We currently understand these different biases. In human warfare, appro- very little about how immune responses are raised and what factors modulate the T-helper types of these immune responses. Fortu- Figure 8. Steps in Vaccine Development nately, developing an effective Product vaccine does not require under- Basic Research Targeted Research Development standing how the vaccine works. However, gaining knowledge of Virology Preclinical Clinical Manufacture Reagents Phase I how DNA-based immunizations Immunology Safety tests Pathology Comparative tests Phase II Quality control function should provide spring- in-vivo & in-vitro Epidemiology Phase III Licensing boards for more effective applica- Solicited problem Human trials Vaccine Design solving Distribution tion of this exciting technology and also support insights into the cellular and molecular biology of immune responses. Therefore, there Government Support is a cogent practical, as well as Commercial Support intellectual, promise for basic research on DNA vaccines.

21 priate circumstances exist for deployment of the Immunization of neonates. From the point of army or the navy; for the immune system there view of logistics and disease prevention, the day are appropriate circumstances for the deploy- of birth offers the most favorable time for ment of T-helper 1, as opposed to T-helper-2 immunization. However, the presence of mater- responses. The realization that the immune nal antibody and the immaturity of the infant system has different branches to its defense immune system pose major problems to vacci- mechanisms came relatively recently62,63 (see nating neonates. At present, relatively little is Figure 7). Each branch has T-helper cells with understood about either of these phenomena and distinctive patterns of lymphokine production. In how they might interact with DNA-based murine models, where most of the work has been immunizations. Maternal antibodies neutralize done, these distinctive patterns of lymphokines the infecting virus in live attenuated vaccines 11 determine the type of antibody produced and the and, therefore, block immunization. DNA types of inflammatory cells mobilized and would not be subject to such neutralization. The activated during the memory response. immune system undergoes rapid development The method of DNA delivery,58,59 the form of immediately after birth. Will the temporal DNA-expressed antigen,47,49,64 and in some in- expression of DNA (generally initiated within stances, co-transfected lymphokines,65,66 can 12 to 24 hours of inoculation) or the longevity affect the type of T-help that DNA-based immu- of DNA expression (presumed to last for at least nizations raise. Quite impressively (but in several days) support successful immunization? agreement with how memory responses work), If responses occurred, how would these differ the T-helper bias established by a DNA immuni- from those in the adult? Could infant vaccines zation is maintained during the amplification of also deliver lymphokines or co-stimulatory its memory response.58 Thus, one can use DNA molecules, known to participate in the elicitation inoculation, or a DNA-expressed antigen, to bias of immune responses, to provide the necessary the inflammatory response that a challenge machinery for the neonate to establish protective infection will mobilize. For example, vaccines vaccine responses? for those bacterial infections best controlled by phagocytosis can be specifically designed to Vaccine vectors and delivery methods. At establish T-helper 1 memory that mobilizes present, vaccine vectors are modeled on the phagocytic defenses preferentially (see Figure 7). vectors used to achieve high-level production of And, vaccines for infections that mast cells therapeutic proteins, such as tissue plasminogen control best, such as worms, could preferentially activator. Researchers need to evaluate a host of be designed to elicit T-helper 2 responses. approaches to improve vaccine vectors. These include the improvement of antigen expression Identification of antigens that raise protective and the evaluation of the effect of immuno- responses. A third important area for research on stimulatory “CpG” DNA motifs on immuniza- 67,68,69 DNA-based immunizations involves identifying tions. Immunostimulatory CpG motifs can γ the antigens, combinations of antigens, and stimulate the production of lymphokines—IFN , α forms of antigens most effective at inducing IFN , IL-12—which in turn can affect the protective immunity against microbial infections. efficiency and, potentially, the T-helper type of 67 We have already discussed the ability to screen an immune response. different genes from an organism rapidly and the Many different delivery systems are being ability to use multiple genes for immunization. In evaluated for the administration of DNA addition to these two advantages, the use of vaccines, especially to mucosal surfaces. These recombinant DNA for immunization allows one include the use of intracellular bacteria as 70 to test different forms of antigens or antigens carriers for vaccine DNAs, the use of DNA 71 constructed to favor presentation by MHCI or containing microspheres, and the use of lipid- 72 MHC II complexes.53 based DNA delivery. More effective systems for delivering DNA to the nucleus also need evaluation.

22 Evaluation of therapeutic immunizations for to express lymphokines or co-stimulatory chronic infections, autoimmunity, allergy, and molecules in cancer cells to attract and activate cancer. Chronic infections occur when a host immune system cells to kill the cancer.77 cannot clear an infection. DNA immunizations may play a therapeutic role in such situations by Special considerations for applied augmenting an existing immune response or research on DNA vaccines broadening a response to include additional antigens. In instances where an infection fails to Safety issues. Regulatory agencies considering elicit cytotoxic T-cells, DNA vaccinations may the application of DNA vaccines for human use generate such cells, which in turn may destroy have identified several key areas of concern infected cells. (Figure 9). Each of these is briefly discussed Researchers also need to evaluate DNA below. immunizations for their ability to provide therapy for autoimmune and allergic diseases. Integration. Integration is the insertion of a Autoimmune disease occurs when the host DNA sequence into the chromosomal DNA of a mounts an immune response against one of its host organism. Each insertion represents a own components. For example, in multiple mutagenic event. A fraction of these mutations sclerosis, immune cells target myelin basic have the potential to cause cancer by perturbing protein in the brain. Inflammatory responses the structure or expression of genes that control supported by T-helper 1 cells mediate these cell growth and differentiation. Integrations can debilitating attacks on one’s own body. DNA- take place between identical DNA sequences in a based immunizations may selectively destroy the plasmid and a host cell or between differing immune cells supporting the autoimmune sequences, in which case the integration event is attack.73 In contrast to autoimmunity, which is referred to as an illegitimate recombination.78 associated with complement-binding IgG and Efforts to find integrations of vaccine plasmids T-helper 1-biased responses against one’s own into mouse DNA have failed to detect insertions proteins, allergy stems from IgE and T-helper of the injected plasmid.79 These studies could 2-based responses against foreign proteins. have detected one integration event for each Most allergy-causing proteins are ones to which 150,000 nuclei, a mutation rate estimated at humans undergo regular exposure, such as 1,000 times less than the spontaneous mutation house mites, cat fur, and pollen. If the antibod- rate of DNA. ies against these allergens can be changed from IgE to IgG, and the response changed from T- Tolerance. In some experimental systems, helper 2 to T-helper 1, then allergic symptoms repeated injection of small quantities of antigen will be relieved. Indeed in mice, IgE-initiated leads to the development of immunologic allergic responses have been modulated towards unresponsiveness or tolerance. Since DNA IgG and T-helper 1 using intramuscular saline immunization produces a small amount of injections of allergen encoding DNA.74,75 antigen, and the expression of antigen after Finally, DNA vaccines to control cancer merit DNA immunization appears to persist, the further development. We could prevent a remarkably large number of cancers by vacci- nating for the infections that cause the cancer— Figure 9. Safety Issues for DNA Vaccines a process already started for hepatitis B virus (see Figure 3). For those cancers unrelated to The potential integration of the plasmid DNA into the genome of microbial infection, DNA vaccines may raise transfected cells. immune responses for proteins specific to the The potential induction of immune tolerance to the vaccine antigen. cancer and thus kill cancer cells. Indeed, some The potential induction of autoimmunity. success has been obtained using DNAs to raise The potential induction of antibodies to the injected plasmid DNA. antibodies specific to certain B-cell lympho- mas.76 Success may also come from using DNAs

23 possibility exists that unresponsiveness, rather Pre-clinical and Clinical Trials than protective immunity, might result. This needs further investigation in both infants and Pre-clinical evaluation. The issues for pre- adults. clinical efficacy of DNA vaccines are the same as those for traditional vaccines; they must demon- Autoimmunity. Autoimmunity is the attack of strate immunogenicity and protective or thera- the immune system on one’s own cells. Autoim- peutic efficacy in animal models. Concerns about mune responses might occur as a result of the the relevance of results in animal models to immune-mediated destruction of cells expressing human applications exist for all vaccines. These a DNA vaccine, or by virtue of the DNA con- concerns arise from potential differences in verting a normally nonantigen presenting cell immune responses, as well as possible differences into an antigen presenting cell. However, both in disease development. Nevertheless, the basic the destruction of one’s own cells and the issue of the relevance of pre-clinical immunoge- expression of foreign antigens occur in the nicity and efficacy of DNA vaccines and tradi- course of viral and bacterial infections. Thus, tional vaccines remains the same. And this DNA vaccines may pose no greater risk of necessitates the usual consideration of the type inducing autoimmunity than natural infections. of immune response, the relevance of a disease model to clinical disease, and the significance of Anti-DNA antibodies. The induction of anti- observations in inbred laboratory animals for the DNA antibodies by plasmid DNA—for example, human population. the immune system would see the vaccine DNA as foreign—poses another safety consideration. Clinical trials. As for any biomedical agent, the Antibodies to DNA can cause disease and are critical issue is the risk-benefit ratio for a disease. associated with systemic lupus erythematosus. For prophylactic vaccination, this means that the To date, studies have not associated DNA safety and protective efficacy of the vaccine are vaccines with the induction of anti-DNA anti- very important. While safety ranks as the most bodies. This appears to reflect several factors. important component for prophylactic DNA First, purified double stranded DNA does not vaccines, the particular disease and population readily induce anti-DNA antibodies. Second, under consideration would affect the risk- nonpathogenic anti-DNA antibodies are found in benefit. To this end, it is important to evaluate most humans. These antibodies are specific for carefully the safety of DNA vaccines in pre- the DNA of particular bacterial species and do clinical and then clinical studies, while at the not cross-react with mammalian DNA, which same time considering the benefit of a protective suggests that these antibodies were generated vaccination. New technologies to evaluate safety during bacterial infections. Third, vaccination of have greatly facilitated the ability to address the lupus-prone mice with purified plasmid DNA has safety concerns described above. little effect on the levels of anti-DNA antibodies and no detrimental autoimmune effect in this Unique Aspects for animal model. Finally, vaccination of normal Product Development animals with DNA vaccines has induced few or no anti-DNA antibodies, as measured by ELISA, Generic production. One advantage of DNA immunoblot, or radioimmunoassay. Therefore, vaccines lies in the likelihood that their manufac- while it remains unknown whether DNA vac- ture will require only generic technology, since cines will induce anti-DNA antibodies in hu- vaccines for different diseases will utilize similar mans, animal studies suggest this appears plasmid backbones, differing mainly by the unlikely.80 vaccine gene inserted (see Figure 5). Thus, unlike other vaccine technologies that necessitate

24 unique growth and attenuation processes for Recently, the U.S. Food and Drug Administra- different vaccines, or specific purification or tion’s Center for Biologics Evaluation and chemical processes for inactivated or subunit Research released a “Points to Consider” vaccines, all DNA vaccines will rely largely on document. The process of licensing DNA the same growth and purification process. vaccines would closely follow that for traditional Large-scale fermentation conditions and vaccines in requiring a demonstration of the processes already exist for various recombinant safety and efficacy of the vaccine commensurate DNA technologies. Fermentation for manufac- with the risk-benefit ratios of the targeted ture of DNA vaccines would differ from these disease. previous efforts. In DNA vaccines, the plasmid itself—DNA—rather than a produced protein is Potential for Utilization in the desired product. Both the state of the art for Developing Countries large-scale bacterial fermentation and the generic nature of DNA vaccines offer significant advan- The crucial criteria for utilizing DNA vaccines in tages for manufacturing DNA vaccines cost developing countries are the same as those for effectively. developed countries: safety and efficacy for relevant diseases. The feasibility of DNA vac- Purification. Purifying DNA plasmid from the cines for developing countries will depend upon bacterial cellular components and any contami- the success of large-scale manufacturing pro- nants introduced during manufacture (fermenta- cesses to produce vaccines that remain stable tion) and purification should be relatively simple. under local conditions, presumably without a Acceptable limits on the presence of other cold chain. Currently, it appears that DNA components in the final product and their effect vaccines will prove both practical and affordable upon the potency or tolerability of DNA vac- for developing nations. cines are only being determined now. For example, bacterial endotoxin, present in host bacteria and known to affect both immune responses and reactogenicity, requires purifica- tion to an acceptable extent. Preliminary work indicates that the technology to purify DNA vaccines could be scaled up, but the ultimate cost per dose will depend upon current unknowns. Those include human potency and the number of constructs needed for a given vaccine.

Characterization. One can characterize a vaccine plasmid at various levels, including the actual DNA sequence of the vaccine, the form of the DNA (linear, open-circular, or super-coiled), and the presence of other materials such as inert substances of the manufacturing process. Al- though various plasmid forms may be biologi- cally active, a licensed DNA vaccine must meet specific criteria to ensure the stability and potency of its different manufactured lots.

25 SOCIETAL ISSUES

Responsibilities of Organizations for sequencing the genomes of microbial patho- and Societies gens should be encouraged, as the knowledge of DNA sequences is basic for the development of Scientific community. The primary responsibility DNA vaccines. Governmental agencies might of the scientific community in the development also establish targeted research programs on of DNA vaccines is to conduct the necessary DNA vaccines. Currently, the NIH has initia- research as quickly and efficiently as possible. tives, such as funded production facilities for This will require that individual scientists gene therapy and national centers for antigen- disseminate their findings through scientific based vaccine trials, that could provide needed presentations and publications to minimize the reagents and infrastructure for the clinical duplication of efforts and provide cross-fertiliza- testing of DNA vaccines. tion of ideas. This is particularly important for Another important role for governmental DNA-based immunization. This novel agencies involves the regulatory issues associated technology’s simplicity makes it accessible to with the approval of clinical trials and the many levels and types of scientists, even those in licensing of new products for use in humans and countries with less developed research programs animals. DNA vaccines should be treated as any and less sophisticated facilities. other new product. For some applications the potential benefits clearly outweigh the risks, and Industry. The new technology of DNA vaccines expeditious approval of clinical trials will has piqued the interest of established pharmaceu- provide important safety information for other tical giants as well as small biotechnology applications where the potential risks initially companies. Major pharmaceutical companies appear of greater importance. As trials are have established in-house research and develop- undertaken, careful surveillance should be ment programs, provided external funding to undertaken. academic scientists, and supported scientific meetings on DNA vaccines. Ideally, such support International organizations. The World Health will continue and industry will openly share Organization (WHO) has shown the greatest information and reagents to encourage continued interest in DNA vaccines of any international rapid development of DNA vaccines. Many body. At a very early stage, WHO saw the biotechnology companies were established for potential benefits for application of this technol- research and development on gene therapy, an ogy to the poorer and less developed areas of the expertise easily transferred to DNA vaccines. world. WHO has played a significant role in facilitating the development of DNA vaccines Government. Governments have and will through direct support of academic research as continue to play an important role in the devel- well as the organization and support of interna- opment of DNA vaccines. Governments can tional symposia. expedite the development of DNA vaccine WHO also has established a system to ensure technology by providing funding for basic and that vaccines, including DNA vaccines, reach applied research on DNA vaccines. The National citizens of poor as well as wealthy countries. Institutes of Health (NIH) provided the competi- The world’s nations have been divided into tiers tive research grants that fostered the inception based on population and gross national produc- and initial discovery of DNA vaccines. Funding tion (see Figure 10). Actual vaccine purchases

26 Figure 10. Sustainable Vaccine Supply—Global Targeting Strategy

Switzerland Japan

Germany USA

Canada

Greece $6000 Portugal Libya Argentina

Gabon Oman Mexico Russia Brazil Malaysia Iran Botswana Turkey D Namibia Syria Thailand Congo Jordon PNG Camaroon Senegal Cote d‘Ivoire Morocco Philippines C Angola Indonesia China $500 Mauritania Yemen Egypt Lesotho Guinea Ghana Togo Burkina Faso Sudan Pakistan Haiti B Nigeria India Myanmar Viet Nam Mali Zaire Bangladesh

Ethiopia Bhutan Laos Somalia A A. Financial support Mozambique B. Finance/Service Log GNP/Capita Gradual Hand-off Log Population 10 million 50 million C. Self Sufficient D. Rapid Independence

Source: ref. (81). for the poorest countries would be made by the Communication and the role of the media. The United Nations International Children’s media will play a large and important role in the Education Fund (UNICEF). Such a system implementation of DNA vaccines. While the would make vaccines available to those nations potential benefits of DNA vaccines are enor- that could not afford to purchase them under mous, until the true benefits are known, the other circumstances. media need to inform the public with cautious optimism. Unrealistic optimism could generate a Other Societal Issues backlash if hopes fail to become reality in a timely fashion. Thus, it is important that the Intellectual property and patents. The award- media play an active, yet cautious role during ing of patents for DNA vaccines is just begin- the development of this novel technology that ning. It is essential that those who hold key holds such high promise for world health. patents foster the cross-licensing agreements that will allow the rapid development and potentially broad use of DNA vaccines.

Environmental issues. DNA vaccines appear to pose no environmental risk. Furthermore, DNA vaccines should reduce existing environ- mental risks, by reducing endemic infections.

27 References and Suggested Reading

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31 The Scientific Future of DNA for Immunization

American Academy of Microbiology This report is based on an American Academy of Microbiology colloquium held May 31–June 2, 1996.

The colloquium was supported by the following sponsors:

Agracetus, Inc., Middleton, Wisconsin Amgen, Inc., Thousand Oaks, California Astra Arcus, Sodertalje, Sweden Bayer AG GB-Pharma, Wupertal, Germany Bayer AG, Animal Health Group, Leverkusen, Germany Bio-Rad Laboratories, Hercules, California Chiron Corporation, Emeryville, California Connaught Laboratories, North York, Ontario, Canada IRIS, Research Institute of Biocine Sclavo, Siena, Italy Lederle-Praxis Biologicals, Wayne, New Jersey Merck Research Laboratories, West Point, Pennsylvania Pasteur Mérieux Connaught, La Coquette, France Pfizer Central Research, Lincoln, Nebraska QIAGEN GMBH, Hilden, Germany Univax Biologics Inc., Rockville, Maryland Vida Labs, Dallas, Texas