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 MEDICAL INTELLIGENCE UNIT 26

Ronald W. Ellis

New Vaccine
ELLIS

Technologies
MIU
26
New Vaccine Technologies
 MEDICAL
INTELLIGENCE
UNIT 26

New Vaccine
Technologies

Ronald W. Ellis, Ph.D.

BioChem Pharma
(to become Shire Biologics after soon-expected merger)
Northborough, Massachusetts, U.S.A.

LANDES BIOSCIENCE EUREKAH.COM

GEORGETOWN, TEXAS AUSTIN, TEXAS
U.S.A. U.S.A.
 NEW VACCINE TECHNOLOGIES
Medical Intelligence Unit

Eurekah.com
Landes Bioscience
designed by Lana K. Moore

Copyright ©2001 Eurekah.com

All rights reserved.
No part of this book may be reproduced or transmitted in any form or by any means, electronic or
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Printed in the U.S.A.
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ISBN 1-58706-050-7 (hard cover version)

ISBN 1-58706-080-9 (soft cover version)

While the authors, editors and publisher believe that drug selection and dosage and the specifications
and usage of equipment and devices, as set forth in this book, are in accord with current recommend-
ations and practice at the time of publication, they make no warranty, expressed or implied, with
respect to material described in this book. In view of the ongoing research, equipment development,
changes in governmental regulations and the rapid accumulation of information relating to the
biomedical sciences, the reader is urged to carefully review and evaluate the information provided
herein.

Library of Congress Cataloging-in-Publication Data

New Vaccine Technologies / [edited by] Ronald W. Ellis
p.;cm.-- (Biotechnology intelligence unit)
Includes bibliographical references and index.
ISBN 1-58706-050-7 (alk. paper)
1. Vaccines--Biotechnology. I. Ellis, Ronald W. II. Series.
[DNLM: 1. Vaccines. 2. Biotechnology. QW 805 N5324 2001]
TP248.65.V32N49 2001
615'372--dc21 00-063630
 DEDICATION

Dedicated to my wife Danielle and children Jacob and Miriam for their
love, patience and support, and to the memory of my father.
 CONTENTS
Preface ................................................................................................. XI

1. New Technologies for Making Vaccines ................................................. 1

Ronald W. Ellis
Introduction .......................................................................................... 1
Live Vaccines ......................................................................................... 4
Subunit/Inactivated Vaccines ................................................................ 8
DNA (Nucleic Acid) Vaccines ............................................................. 14
Formulation of Antigens ..................................................................... 15
Conclusion .......................................................................................... 16
2. Clinical Issues for New Technologies ................................................... 21
Luc Hessel
Introduction ........................................................................................ 21
Definition of the Medical Needs ......................................................... 22
Moving from Preclinical to Clinical Development ............................... 22
Demonstration of the Proof-of-Principle ............................................. 23
Design and Implementation of a Clinical Development Plan .............. 23
Continuous Assessment of Safety and Efficacy .................................... 27
Specific Issues ...................................................................................... 28
Conclusion .......................................................................................... 30
3. Regulatory Issues .................................................................................. 33
Marion F. Gruber, Paul G. Richman and Julianne C. M. Clifford
Introduction ........................................................................................ 33
Federal Regulations Pertaining to Vaccines .......................................... 33
Premarketing Phase ............................................................................. 35
Postmarketing Phase ............................................................................ 41
4. In-Licensing Issues and Vaccine Technologies...................................... 44
Dale R. Spriggs
Introduction ........................................................................................ 44
Why Do Companies In-License? ......................................................... 44
How Do Companies Evaluate Licensing Opportunities? ..................... 46
How Can an Inventor Make the Technology More Attractive? ........... 48
Framework of a Licensing Agreement .................................................. 49
What Does the Future Hold? .............................................................. 50
CODA ................................................................................................ 50
5. Live Vaccines ........................................................................................ 51
Alan R. Shaw
Smallpox Vaccine ................................................................................ 51
Japanese Encephalitis Vaccines ............................................................ 52
Yellow Fever Vaccines ......................................................................... 53
Poliovirus Vaccines .............................................................................. 54
Measles Vaccines ................................................................................. 57
Rubella Vaccines ................................................................................. 59
 Mumps Vaccines ................................................................................. 61
Trivalent Measles-Mumps-Rubella Vaccines ....................................... 64
Quadrivalent Measles-Mumps-Rubella-Varicella Vaccines .................. 67
Varicella Vaccines ................................................................................ 67
Rotavirus Vaccines .............................................................................. 70
Live Attenuated Influenza Vaccines ..................................................... 73

6. Recombinant Live Attenuated Viral Vaccines ....................................... 90

Richard R. Spaete
Advantages and Concerns Associated with the Use
of Live Attenuated Vaccines ............................................................ 90
Vaccine Efforts for Herpesviruses ........................................................ 91
Prospects for the Future ....................................................................... 97

7. Live Viral Vectors ............................................................................... 101

Elizabeth B. Kauffman, Michel Bublot, Russell R. Gettig,
Keith J. Limbach, Steven E Pincus and Jill Taylor
DNA Viruses ..................................................................................... 102
RNA Viruses ..................................................................................... 114
8. Inactivated Virus Vaccines .................................................................. 134
Andrew D. Murdin, Benjamin Rovinski, Suryaprakash Sambhara
Introduction ...................................................................................... 134
Current Inactivated Virus Vaccines ................................................... 134
Issues Affecting the Use of Inactivated Virus Vaccines ....................... 137
Conclusion ........................................................................................ 146
9. Live Attenuated Bacterial Vaccines ..................................................... 152
Kevin P. Killeen and Victor J DiRita
Introduction ...................................................................................... 152
Types of Vaccines .............................................................................. 152
Live Attenuated Bacterial Vaccines .................................................... 155
Live Attenuated Mycobacterium Bovis
(BCG Tuberculosis Vaccine) ......................................................... 155
Immune Correlates of Protection ...................................................... 158
Live Attenuated Salmonella typhi (Typhoid Fever Vaccines
and Vaccine Candidates) ............................................................... 158
Live Attenuated Shigella Sp (Shigellosis Vaccine Candidates) ............ 161
Live Virulence-Attenuated Vibrio cholerae (Cholera Vaccines) ........... 164
Animal Model ................................................................................... 165
Vaccine Efforts .................................................................................. 165

10. Live Attenuated Bacterial Vectors ....................................................... 172

Sims K. Kochi and Kevin P. Killeen
Introduction ...................................................................................... 171
Salmonella Vectors ............................................................................ 172
bacille Calmette-Guérin .................................................................... 175
 Vibrio Vectors ................................................................................... 176
Listerial Vectors ................................................................................. 178
Next Generation Bacterial Vectors .................................................... 179
DNA Delivery ................................................................................... 180
Summary ........................................................................................... 182
11. Protein-Based Vaccines ...................................................................... 186
Sheena M. Loosmore, Gavin R. Zealey and Raafat E.F. Fahim
Introduction ...................................................................................... 186
Pediatric Vaccines .............................................................................. 186
Adult Vaccines .................................................................................. 189
Vaccines Against Nosocomial Infections ............................................ 194
Cancer Vaccines ................................................................................ 194
Vaccines Against Autoimmune Diseases ............................................ 197
Current Technologies ........................................................................ 198
Emerging Technologies ..................................................................... 199
Summary ........................................................................................... 201

12. Peptide Vaccines ................................................................................ 214

Damu Yang, Gregory E. Holt, Michael P. Rudolf, Markwin P. Velders,
Remco M. P. Brandt, Eugene D. Kwon and W. Martin Kast
Introduction ...................................................................................... 214
Molecular Basis for the Development of Peptide Vaccines ................. 214
Advantages and Disadvantages of Peptide-Based Vaccines ................. 215
Adjuvants and Delivery Systems ........................................................ 215
Design of Peptide Vaccines: Synthetic Peptides as B-Cell Vaccines.... 216
Peptide-Based T-Cell Vaccines. Identification of Peptide Epitopes
Recognized by T Cells ................................................................... 217
Synthetic Peptides as T-Cell Vaccines ............................................... 217
Recombinant Vaccines Expressing T-Cell Epitopes ........................... 218
Adoptive Cellular Therapy ................................................................ 219
Summary and Perspectives ................................................................ 220
13. Polysaccharide Vaccines ..................................................................... 227
Stephen Freese
Polysaccharide Immunity .................................................................. 227
Issues in Designing a Conjugate ........................................................ 227
Issues in Making a Conjugate ............................................................ 229
Applications ...................................................................................... 232
14. DNA Vaccines .................................................................................... 240
Daniel E. McCallus, Catherine J. Pachuk, Shaw-guang Lee
and C. Satishchandran
Introduction ...................................................................................... 240
Gene Expression ................................................................................ 241
Mechanisms of Immunostimulation .................................................. 242
 Routes of Administration .................................................................. 245
Intracellular Delivery of DNA Vaccines ............................................ 248
Safety of Nucleic Acid Vaccines ......................................................... 251
Future Directions of DNA Vaccines .................................................. 253
Summary ........................................................................................... 256
15. Plant-Derived Vaccines ...................................................................... 263
Amanda M. Walmsely and Charles J. Arntzen
Introduction ...................................................................................... 263
Mucosal Vaccines .............................................................................. 263
Production of Plant-Derived Vaccines ............................................... 264
Plant-Derived Vaccines ..................................................................... 265
Summary ........................................................................................... 270
Future Use of Plant-Delivered Vaccines ............................................ 270

16. Biological Aspects and Prospects for Adjuvants

and Delivery Systems ......................................................................... 274
Bror Morein and Ke-Fei Hu
Introduction ...................................................................................... 274
Innate Immunity: The Gateway to an Acquired Immune Response .. 277
APCs Instruct the Acquired Immune System .................................... 280
Immune Modulation is Based on Cross-Talk Between Innate
Immunity and Helper T Cells ....................................................... 281
The Collaboration Between the Complement System and B Cells:
Roles for Adjuvants ....................................................................... 282
Immune Modulation For CTL .......................................................... 283
Delivery Systems ............................................................................... 284
Vaccines for Newborns and Elderly Require
Suitable Strong Adjuvants ............................................................. 285
The Present Situation and Future Aspects of Adjuvants
and Delivery Systems ..................................................................... 286

17. Transcutaneous Immunization ........................................................... 292

Gregory M. Glenn
Introduction ...................................................................................... 292
Barriers and Targets for TCI ............................................................. 292
Adjuvants and TCI ............................................................................ 294
Immune Responses to Transcutaneous Immunization ....................... 295
Mucosal Responses ............................................................................ 296
Diversity of Antigens ......................................................................... 298
Delivery Options Using Transcutaneous Immunization .................... 299
Optimization for Enhancement of the Immune Response ................. 299
Human Studies ................................................................................. 302
Conclusions ....................................................................................... 302

Index .................................................................................................. 305

 EDITOR
Ronald W. Ellis, Ph.D.
BioChem Pharma
(to become Shire Biologics after soon-expected merger)
Northborough, Massachussetts, U.S.A.
Chapter 1

CONTRIBUTORS
Charles J. Arntzen Russell R. Gettig
Boyce Thompson Institute for Plant Arbovirus Unit
Research Griffin Lab
Ithaca, New York, U.S.A. Slingerlands, New York, U.S.A.
Chapter 15 Chapter 7

Remco M. P. Brandt Gregory M. Glenn

Cardinal Bernardin Cancer Center IOMAI Corporation
Loyola University Chicago Washington, District of Columbia,
Maywood, Illinois, U.S.A. U.S.A.
Chapter 12 Chapter 17

Michel Bublot Marion Gruber

Arbovirus Unit Office of Vaccines Research and Review
Griffin Lab Division of Vaccines and Related
Slingerlands, New York, U.S.A. Products Applications
Chapter 7 Rockville, Maryland, U.S.A.
Chapter 3
Julianne C.M. Clifford
Office of Vaccines Research and Review Luc Hessel
Division of Vaccines and Related Medical Department
Products Applications Pasteur Merieux MSD
Rockville, Maryland, U.S.A. Lyon Cedex, France
Chapter 3 Chapter 2

Victor J. DiRita Gregory E. Holt

Department Microbiology and Cardinal Bernardin Cancer Center
Immunology Loyola University Chicago
Unit for Laboratory Animal Medicine Maywood, Illinois, U.S.A.
University of Michigan Medical School Chapter 12
Ann Arbor, Michigan, U.S.A.
Chapter 9 Ke-Fei Hu
Department of Virology
Rafaat Fahim National Veterinary Institute
Connaught Laboratories, Ltd. Uppsala, Sweden
Willowdale, Ontario, Canada Chapter 16
Chapter 11
W. Martin Kast
Stephen Freese Cardinal Bernardin Cancer Center
Wyeth-Lederle Vaccines Loyola University Chicago
West Henrietta, New York, U.S.A. Maywood, Illinois, U.S.A.
Chapter 13 Chapter 12
Elizabeth Kauffman Andrew Murdin
Arbovirus Unit Aventis Pasteur Ltd.
Griffin Lab Willowdale, Ontario, Canada
Slingerlands, New York, U.S.A. Chapter 8
Chapter 7
Catherine J. Pachuk
Kevin Killeen Wyeth-Lederle Vaccines and Pediatrics
AVANT Immunotherapeutics Inc. Malvern, Pennsylvania, U.S.A.
Needham, Massachusetts, U.S.A. Chapter 14
Chapter 9, 10
Steven E Pincus
Sims K. Kochi Arbovirus Unit
AVANT Immunotheraputics, Inc. Griffin Lab
Needham, Massachusetts, U.S.A. Slingerlands, New York, U.S.A.
Chapter 10 Chapter 7

Eugene D. Kwon Paul C. Richman

Cardinal Bernardin Cancer Center Office of Vaccines Research and Review
Loyola University Chicago Division of Vaccines and Related
Maywood, Illinois, U.S.A. Products Applications
Chapter 12 Rockville, Maryland, U.S.A.
Chapter 3
Shaw-guang Lee
Wyeth-Lederle Vaccines and Pediatrics Benjamin Rovinski
Malvern, Pennsylvania, U.S.A. Aventis Pasteur Ltd.
Chapter 14 Willowdale, Ontario, Canada
Chapter 8
Keith J. Limbach
Arbovirus Unit Michael P. Rudolf
Griffin Lab Cardinal Bernardin Cancer Center
Slingerlands, New York, U.S.A. Loyola University Chicago
Chapter 7 Maywood, Illinois, U.S.A.
Chapter 12
Sheena M. Loosmore
Connaught Laboratories, Ltd. Suryaprakash Sambhara
Willowdale, Ontario, Canada Aventis Pasteur Ltd.
Chapter 11 Willowdale, Ontario, Canada
Chapter 8
Daniel McCallus
Wyeth-Lederle Vaccines and Pediatrics C. Satishchandran
Malvern, Pennsylvania, U.S.A. Wyeth-Lederle Vaccines and Pediatrics
Chapter 14 Malvern, Pennsylvania, U.S.A.
Chapter 14
Bror Morein
Department of Virology Alan Shaw
National Veterinary Institute Virus and Cell Biology
Uppsala, Sweden Merck Research Laboratory
Chapter 16 West Point, Pennsylvania, U.S.A.
Chapter 5
Richard Spaete Amanda Walmsely
Aviron Boyce Thompson Institute for Plant
Mountain View, California, U.S.A. Research
Chapter 6 Ithaca, New York, U.S.A.
Chapter 15
Dale Spriggs
Project Planning and Management Damu Yang
BioChem Pharma, Inc. Cardinal Bernardin Cancer Center
Northborough, Massachussetts, U.S.A. Loyola University Chicago
Chapter 4 Maywood, Illinois, U.S.A.
Chapter 12
Jill Taylor
Arbovirus Unit Gavin R. Zealey
Griffin Lab Connaught Laboratories, Ltd.
Slingerlands, New York, U.S.A. Willowdale, Ontario, Canada
Chapter 7 Chapter 11

Markwin P. Velders
Cardinal Bernardin Cancer Center
Loyola University Chicago
Maywood, Illinois, U.S.A.
Chapter 12
 PREFACE
Vaccines are one of the most cost-effective interventions in health-care. Vaccination is
estimated to have been responsible for 10-15 years of the increase in the average human lifespan
during the 20th century, an increase probably second in impact only to that of clean water. In addition
to considerable morbidity, there are over 10 million deaths annually worldwide attributable to
infectious diseases. A large number of these deaths can be prevented by wider use of existing vaccines,
while most of these deaths would be preventable by the development of effective new vaccines.
There is an increasingly broad array of new technologies that are being employed for developing
vaccines. Such technologies are based on breakthrough discoveries in the fields of immunology,
biochemistry, molecular biology and related areas. The broad applications of such discoveries should
result in the development of many new vaccines that have not been feasible previously. Alternatively
it may be possible to improve existing vaccines in terms of their safety and efficacy. There are about
40 new vaccines (not including competing versions of the same product) that were developed and
introduced during the 20th century. It is noteworthy that almost half of these new vaccines were
introduced during the 1980s and 1990s, with many of these based on new technologies such as
recombinant proteins and conjugates. Therefore, the development of new vaccine technologies
offers yet further potential for considerably reducing worldwide mortality and morbidity from
infectious diseases.
Beyond the applications of vaccines to infectious diseases, it should be noted that there are
increasing efforts to develop vaccines for the treatment or prevention of chronic diseases such as
cancer, autoimmunity and allergy. There currently is one vaccine licensed for the treatment of a
cancer (BCG vaccine for bladder cancer), with numerous other cancer vaccines of multiple designs
in various stages of preclinical and clinical development. Such therapeutic vaccines, in conjunction
with other therapeutic modalities, offer the prospect for improving health and recovery from a range
of chronic diseases.
There are two general categories for vaccines, active and passive. Active vaccines stimulate
the production of both antibodies and/or of immune system cells with memory and effector functions
(e.g., cytotoxic T-cells). Passive vaccines are antibody preparations that are used in cases where
developing an active vaccine is not feasible or where there is a need for immediate immunity due
to acute exposure to a virus or bacteria. Passive vaccines, which do not stimulate immunological
memory, historically have been polyclonal human antibodies from individuals with the requisite
antibody specificities. However, most new passive vaccines are based on monoclonal antibodies
that are human or humanized, given recent advances in molecular biology that have enabled the
production of such antibodies.
This book focuses upon the applications of new technologies to active vaccines for the
prevention of human infectious diseases, which represent all but one of the available licensed vaccines.
There are many challenges in fully applying and developing new vaccine technologies. Most
of these technologies can be divided into five general categories: 1) discovery of new leads and candidate
antigens; 2) production; 3) design of the overall vaccine; 4) formulation of the final product; and
5) administration modality for human use.
Vaccine discovery has relied historically on a range of technologies. Live attenuated vaccines
have been based on isolating and growing the virus or bacteria in vitro. In the case of inactivated
vaccines, the in vitro-cultivated microorganism is chemically treated to destroy its infectivity. Vaccine
antigens have been identified through an approach akin to proteomics, viz., the study of the proteins
(and polysaccharides and other antigens) associated with viruses and bacteria. These antigens may
be identified by means of antibodies raised against the whole microorganism or in acute or convalescent
sera following infection. Alternatively, the microorganism is grown and biochemically fractionated
to identify antigens. Such approaches also have been taken to identifying candidate antigens for
diseases such as cancer and allergy. More recently, genomics-based approaches have been applied to
identifying vaccine antigens, whereby the complete sequence of the microorganism is derived and
annotated. Candidate vaccine antigens then are identified by homology to known vaccine antigens
or by structures (hydrophobic signal sequence) that would direct the antigen to the cell surface. This
approach has been applied to diverse bacteria such as non-typeable Haemophilus influenzae,
Helicobacter pylori, and Neisseria meningitidis, from which novel antigens have been identified and
then validated in animal studies as candidate vaccine antigens. Based on human genomics and the
study of disease-specific gene expression, novel candidate vaccine antigens are being discovered and
developed for cancer and other diseases.
There have been tremendous advances in production technologies for vaccines. Highly
productive recombinant expression systems have been developed and optimized for a broad range
of prokaryotic and eukaryotic cells. Large-scale fermentation equipment and processes as well as
growth media have been developed that enable the attaining of high cell densities for high levels of
accumulation of viruses or recombinant antigens. New large-scale filtration and chromatography
modalities have enabled the efficient processing of large biomasses in order to isolate highly purified
antigens. Advances in biochemistry and analytical chemistry/biochemistry have enabled macromol-
ecules to be very well characterized and stably formulated. Continued technical advances in all these
areas offer the prospect for even more efficient and reproducible large-scale production of vaccines.
Vaccines can be divided into three general categories: live, subunit/inactivated, and DNA (Chap-
ter 1). There are several subcategories of specific designs within each of these three general groups, as
described in Chapters 5-15. One or more of these specific designs may be applicable for developing a
vaccine for a particular virus, bacteria or disease. Each design has different potential advantages and
disadvantages in terms of production, immunobiology, potential safety and efficacy, and ability to be
analytically and biologically characterized. All of these factors need to be weighed when selecting a
design early in a development program. Given the long timeframe and large expense for development,
such decisions assume significant weight. Table 1.1 lists the status of development of vaccines made by
each specific approach, whether licensed or in clinical or preclinical evaluations.
The clinical development plan for a vaccine based on novel technologies is similar to that of
a traditional vaccine (Chapter 2). Nevertheless, there are several clinical issues that must be considered
for evaluating vaccines made by new technologies, especially approaches such as live vectors, DNA,
adjuvants and delivery systems. Certain approaches may present new safety-related issues that
require significant monitoring, especially during initial clinical studies. It is also important that a
new vaccine technology be validated for proof-of-principle early in the clinical development program
before significant resources are applied to its development. The clinical endpoint and surrogate
markers of protection should be understood in order to facilitate development, especially for a new
vaccine target.
Regulatory issues may present special challenges for technologies with which there has been
little or no experience (Chapter 3), since specific standards for criteria such as safety, purity and
potency of new vaccines may not exist. In that sense, the review by regulatory agencies of vaccines
based on new technologies often is done case-by-case in an indication-based and product-specific
fashion. Another important consideration is the risk (perceived or actual) relative to potential
benefit for the particular vaccine, one which differs for (e.g.) a prophylactic vaccine for infants vs. a
therapeutic cancer vaccine. Guidance Documents, such as Points-to-Consider monographs, prepared
by the US FDA and the International Conference on Harmonization may provide useful guidelines
regarding regulatory needs or desires to groups preparing IND and license applications.
It has been increasingly uncommon that any single organization has all the technologies at
its disposal to be able to develop a safe and effective vaccine of a particular type. Therefore,
in-licensing and business development have been areas of increasing activity for vaccines (Chap-
ter 4). The licensor is usually an academic or government laboratory or a small biotech company.
The technology available for licensing may be an antigen, vector, method for discovery or screen-
ing of antigens, production method, adjuvant or delivery system, formulation, device, or combi-
nation of such inventions for which the necessary financial, technical and physical resources are not
available in that group. In order to receive the best value in a licensing agreement, it is important that
the licensor develop its technology and associated patent portfolio to the point where it has added as
much value to it as possible within a useful timeframe.
There are three general categories of live viral vaccines (Chapters 5-7). Live attenuated vac-
cines are derived by means of the passage of a virus in cell culture until its pathogenicity has
been sufficiently attenuated for humans, but with the retention of sufficient infectivity in
vivo to stimulate protective immunity (Chapter 5). Such passaging is empirical in terms of the
number of passages and cell types used for attenuation. Furthermore, the mutations found to be
associated with such attenuated viruses are generally random. In most cases, the precise mutations
responsible for the attenuation phenotype are unknown. Since many human viruses lack useful
animal models for virus replication and virulence, it is necessary to test such vaccines extensively in
humans for safety until the vaccine virus is judged to be sufficiently attenuated. Nevertheless, these
vaccines have been very successful in terms of control of disease. Smallpox, the first human disease
ever eliminated from the earth, was eradicated through the use of a live vaccine. This also will be the
case for polio, which should be eradicated within the next few years, and possibly for measles in the
following decade.
In order to make the technique of attenuation less empirical in nature, recombinant
technology can be used to introduce mutations or deletions in key genes responsible for patho-
genicity. Such live recombinant viral vaccines have well-defined molecular changes (Chapter 6).
These mutations may exert their attenuating effects by limiting in vivo replication potential or
considerably reducing or eliminating virulence. By making multiple changes, one can assure that
there is no possibility or a very low probability for the vaccine virus to revert to virulence. These
mutations also can be exploited as immunological or molecular markers for distinguishing the
mutated virus from its wild-type counterpart. Even though attenuated human vaccines of this type
have been only in early clinical evaluations, a live attenuated recombinant animal vaccine was
licensed in the 1980s for the prevention of pseudorabies infections of pigs.
Live viral vaccines can be engineered to express that encode vaccines from other pathogens
(usually viruses), thereby functioning as live vectored vaccines. Several classes of viruses have been
developed as viral vaccine vectors (Chapter 7), including poxviruses, adenoviruses, herpesviruses,
and alphaviruses. The advantage of such live vectors is that the vaccine antigen encoded by the
transgene (inserted into the viral genome) is processed intracellularly as part of a live virus infection, by
which it may stimulate both antibody and cellular immunity. The main challenges to successful
development include achieving appropriate expression levels of the transgene, assuring adequate
attenuation of the virus vector while retaining sufficient infectivity, and obviating potential host
immunity to the vector. While it is possible that this development can yield a dual vaccine against
both the vectored virus and the virus encoding the transgene, most of the common vectors are not
vaccine targets in their own right. Virus vectors also may be used to prime the immune system, to be
followed by a booster with a recombinant protein as in the case of HIV vaccines in clinical studies.
Many viruses can be grown to high titer in cell cultures. Such viruses become the starting
material for purification and inactivation. Many such inactivated viral vaccines (Chapter 8) have
multiple repeat surface epitopes, which are composed of repeat units of viral structural proteins. As
a consequence, these vaccines are among the most potent immunogens ever characterized. For
example, immunization with a single 50-ng dose of a hepatitis A vaccine was shown to protect
against clinical disease. Many of these inactivated vaccines (e.g., influenza, polio) have been used for
decades, with an excellent track record of safety and efficacy.
There are very few examples of empirically attenuated bacterial vaccines. The only two such
licensed vaccines are the BCG vaccine for tuberculosis and bladder cancer (attenuated by >200
passages in vitro) and Salmonella typhi vaccine for typhoid fever (attenuated by random chemical
mutagenesis). The mutations associated with these attenuations remain unknown. Thus, recombinant
technology has been applied to making defined mutations in bacterial genes responsible for patho-
genesis in order to derive live recombinant vaccines (Chapter 9). Two or more mutations are made in
order to assure the lack of reversion to pathogenicity. While only one such vaccine has been licensed
to date (cholera), this approach continues to be applied, especially for enteric pathogens.
Several bacterial species have been developed into live vectored bacterial vaccines (Chapter
10) expressing proteins from other pathogens (usually bacteria) according to similar principles as for
live viral vectors. Appropriate attenuation of the live bacterial vector involves both the reduction of
pathogenicity and the maintenance of sufficient in vivo infection/replication potential to assure
effective immunization against the protein antigen encoded by the transgene. The transgene may be
integrated into the bacterial chromosome or may be encoded by a plasmid. As a result, expression of
the vaccine antigen is in the context of that for the whole bacteria, thus providing for a potentially
broader immune response to the bacteria encoding the transgene. The promoter for expression of
the transgene may be prokaryotic (in which case expression of the protein is as a typical bacterial
protein) or eukaryotic (in which case the protein may be expressed as for a DNA vaccine [Chapter
14]). Such vaccines are in early clinical studies.
Subunit vaccines consist of proteins, peptides or polysaccharides that carry protective epitopes.
While the first examples of licensed vaccines were with proteins isolated directly from bacteria (e.g.,
diphtheria, tetanus and pertussis) or viruses (e.g., hepatitis B and pertussis), most recent applications
have involved the recombinant expression of such proteins (Chapter 11). Recombinant protein
antigens that have been developed into licensed vaccines have been expressed in diverse cells such as
Saccharomyces cerevisiae, Vibrio cholerae, Bordetella pertussis, and Escherichia coli, with new candidate
vaccines also being expressed in mammalian and insect cells as well as in whole plants (Chapter 15)
or animals. Hybrid or chimeric recombinant protein antigens also have been designed and developed as
candidate vaccines. Recombinant vaccine antigens are isolated to a high level of purity and typically
are very well characterized analytically. These antigens require multiple doses for eliciting
both protective immunity and immunological memory. While some proteins are sufficiently
immunogenic to be formulated on their own, most require adjuvants (Chapter 16) for being
sufficiently immunogenic to elicit protective immunity.
There are cases where the full-length polypeptide with protective epitope(s) is not optimal as a
vaccine antigen. For instance, the polypeptide may have immunodominant epitopes that do not
elicit effective immunity, or there may be a need to focus the immune response toward a particular
protective epitope. In such instances, a peptide-derived vaccine may enable the immune response to
be focused on a single key epitope (Chapter 12). Some peptide vaccines are based on a B-cell epitope
that stimulates a protective antibody response. In this case, the B-cell epitope peptide is linked to a
T-cell epitope peptide or a carrier protein that provides for T-cell help for the immune response.
Other peptide vaccines are based on a T-cell epitope that stimulates a cell-mediated immune
response such as cytotoxic T lymphocytes (CTL), as is being applied to novel vaccines for the
prevention or therapy of cancer or chronic infections.
There are many bacteria (both Gram- and Gram+) that are encapsulated with polysaccharides.
These capsular polysaccharides carry the major seroreactivity of the bacterial species or subspecies
and as such are the object of protective antibodies, such that the polysaccharides are effective vaccine
antigens (Chapter 13). In a few cases, there is a single polysaccharide serotype for the particular
pathogen (e.g., Haemophilus influenzae type b [Hib] for invasive H. influenzae type b meningitis),
such that a single polysaccharide type can be developed into a monovalent vaccine. However, in
most cases, there are multiple capsular polysaccharide serotypes (about 90 for Streptococcus
pneumoniae), and such vaccines need to be multivalent in order to have a high enough rate of overall
efficacy. The first generation of these vaccines consisted of purified polysaccharides. These
vaccines usually are effective in eliciting protective immunity in adults and children over about 2
years of age. For preventing diseases in <2-year-old children or in adults with other underlying dis-
eases, polysaccharides are conjugated to carrier proteins for increasing their immunogenicity. Both
monovalent (Hib) and multivalent (S. pneumoniae) polysaccharide conjugate vaccines have been
developed and licensed.
The most recent of the major technologies for vaccine design is DNA (Chapter 14). This
field began with the serendipitous observation that purified plasmid DNA injected intramuscularly
could stimulate antibody- and cell-based immune responses. This field has evolved further in terms
of the development of formulations to improve DNA uptake and expression, the optimized design
of plasmid molecules, the exploration of new routes of administration, and the use of nonreplicating
viruses or bacteria to deliver DNA to cells. Clinical studies have been performed both for DNA
vaccines per se as well as for such vaccines as priming doses followed by boosting with protein
antigen-based vaccines.
A wide range of prokaryotic and eukaryotic cell types have been developed into host cells for
the expression of recombinant proteins as vaccine antigens. One of the most recent such
developments has been the engineering of whole plants as recombinant expression systems (Chap-
ter 15). In some cases, the recombinant protein may be purified from the plant and formulated as a
vaccine antigen. This approach offers the advantage of the relatively inexpensive production of a
large biomass as feedstock for the purification of the vaccine antigen. In other cases, it may be
possible to eat the recombinant plant itself, e.g., tomato or spinach, as a user-friendly route of
immunization. Initial clinical studies have been conducted with such vaccines.
Adjuvants and delivery systems are the basis of most of the new technologies in vaccine
formulation (Chapter 16). Aluminum salts have been used as vaccine adjuvants (which modulate
immune responses) throughout the 20th century. However, these salts often are not potent enough
for adjuvanting protein-based vaccines to elicit strong enough immune responses. Therefore, a range
of novel chemical and biochemical molecules as well as proteins have been evaluated as adjuvants,
many of which have advanced to clinical studies. While many of these adjuvants are more potent
than aluminum salts in animal studies and some more potent in clinical studies, their tolerability has
not always been good enough to permit full clinical development. Nevertheless, within the last year
an oil-in-water emulsion became the first new approved adjuvant (MF59 for inactivated influenza
vaccine). This development augurs well for the development and approval of other new adjuvants.
Delivery systems, which generally do not modulate immune responses, provide for the physical
targeting of the active vaccine component to particular cells of the immune system. These vehicles
may function by mechanisms such as depot effects, slow or pulsatile release, and presentation to
mucosal surfaces.
One of the key considerations in the administration of vaccines is the route of uptake, for
which there have been investigations of new routes besides injection in order to increase the rate of
compliance as well as to potentially induce mucosal immune responses more efficiently. The only
route other than injection used in any currently licensed vaccines is oral, as employed for whole virus
or bacteria vaccines (polio, cholera). There have been clinical investigations into formulations of
inactivated or subunit antigens in delivery systems for oral or nasal administration. Live attenuated
cold-adapted influenza vaccine has been developed for intranasal administration and has been shown
to be well-tolerated and efficacious in large clinical trials.
Transcutaneous immunization (Chapter 17) has been demonstrated for several different types
of vaccines, including proteins, viruses and DNA. The coadministration on skin of a vaccine
active-component with a mucosally-active toxoid (cholera toxin or E. coli heat-labile toxin) results
in transcutaneous uptake and recognition by antigen-presenting cells. This mode of administration
can result in the stimulation of both serum and mucosal immune responses, as has been observed in
initial clinical studies. If this technology proves to be successful clinically, it would provide
for relative ease of administration and consequent improved compliance with vaccination programs.
Combination vaccines, which are covered in depth in other books and reviews, are an impor-
tant technology for administration. Combination vaccines are defined as the physical mixture of one
or more vaccines during the manufacturing process or at the time of administration. In cases where
vaccines indicated for the same age-group can be combined, the use of combination vaccines would
result in fewer needlesticks. This would make multiple immunizations easier for subjects (and the
parents of immunized children!) as well as for health-care practitioners. In this way, combination
vaccines represent another technology for improving compliance with vaccination programs.
While recent technologies have expanded the horizons for new and improved vaccines, con-
siderable financial and staff resources must be available to support full vaccine development. From
the time that an initial lead has been identified, it can take 10 years and well over $100 million to
develop a new vaccine. Furthermore, the success rate from the time of entry to development to
availability on the market is only ca. 10-15%. Therefore, given this long timeframe, large cost and
high risk, it is very important to design and implement a Product Development Plan early during
this time-period in order to map out all the technologies and resources (money, people, facilities)
necessary for optimizing the likelihood of success of the program.
I hope that New Vaccine Technologies will serve as a comprehensive reference on the major
aspects of new approaches to developing vaccines. Since vaccination remains the most cost-effective
and one of the most practical ways for preventing infectious diseases (and potentially for treating
some diseases), the development and widespread applications of new technologies should spawn
new vaccines that have not been approachable technically, with consequent impact on reducing
morbidity and mortality worldwide. This book should prove useful for scientists, developers of
vaccines and biotechnology products, clinicians, regulators, and health-care practitioners.
I am very grateful for the many collaborations I have been fortunate to have had over the last
17 years with innumerable coworkers in vaccines in BioChem Pharma, Merck, and Astra as well as
with many colleagues in diverse collaborating groups. The loving support and encouragement of my
wife Danielle and children Jacob and Miriam have been very important to me throughout my career
and preparation of this book. Most importantly, I thank all the authors for their outstanding contri-
butions that should make this book a key reference in the field of vaccine technologies.

Ronald W. Ellis
CHAPTER 1

New Technologies for Making Vaccines

Ronald W. Ellis

Introduction

T
he past two decades have witnessed an explosion in the number of technological and
immunological approaches for making new vaccines. These developments have flowed
from advances in a broad range of scientific fields. Some of the earliest applications of
the newer technologies were to improving previously existing vaccines. However, most recent
applications have been directed toward the development of new vaccines for diseases not
previously approachable. The protective immunity elicited by a vaccine ideally would be life-
long and robust after one or a few doses with minimal side effects (reactogenicity). Available
vaccines and those under development fall short of this ideal, thus stimulating new research
in the field.
There are two broad categories of vaccines, active and passive. An active vaccine stimulates
the host’s immune system to produce specific antibodies or cellular immune responses or both,
which would protect against or eliminate a disease. A passive vaccine is a preparation of anti-
bodies that neutralizes a pathogen and is administered before or around the time of known or
potential exposure. Most references to the term vaccine are to active vaccines, which are the
object of the vast majority of research and development activities in the field as well as the
subject of this chapter. Although it is desirable or essential to administer a passive vaccine in
specific instances (particularly if no active vaccine is available or sometimes for immuno-
compromised individuals), establishing lasting immunity through the administration of an
active vaccine is a very important means of preventive medicine.
This chapter summarizes the major technologies, key issues and immunological objectives
for making different kinds of active vaccines. The status of development of vaccines made by
each approach is identified, whether licensed or in clinical or preclinical evaluations (Table 1.1).
Only a few salient examples of each approach along with most common licensed vaccines are
given with one or two accompanying references. While most examples are prophylactic vaccines
for viruses and bacteria, there also is research into prophylactic vaccines for parasites and fungi
and therapeutic vaccines for infectious diseases, cancer and autoimmunity. The technologies
and examples presented should provide a strong framework for the reader to appreciate the
diverse approaches to the research and development of new vaccines.
There are three general categories of active vaccines. A live vaccine is a microorganism
that can replicate in the host or can infect cells, thereby functioning as an immunogen without
causing its natural disease. A subunit or inactivated vaccine is an immunogen that cannot
replicate in the host. A (DNA) nucleic acid vaccine, which cannot replicate in humans, is
taken up by cells, in which it directs the synthesis of vaccine antigen(s).

New Vaccine Technologies, edited by Ronald W. Ellis. ©2001 Eurekah.com.

2 New Vaccine Technologies

Table 1.1. Status of development of representative human vaccines made by different

technologies

STATUS OF DEVELOPMENT*
Type of Vaccine** Preclinical Clinical Licensed Example# Reference
Evaluation*** Evaluation§ Product§§

A. Live
1.Classical strategies -Viral
a. Attenuation in x Poliovirus 2
cell culture x Measles virus 3
x Mumps virus 4
x Rubella virus 5
x Varicella-zoster virus (VZV) 6
b. Variants from x Smallpox (vaccinia virus) 7
other species x Rotavirus 8
c. Reassorted genomes x Rotavirus a 9,10
d. Temperature-selected x Influenza virus 13
mutants
2. Recombinant virus x Herpes simplex virus (HSV) 16
3. Recombinant viral vector x Vaccinia virus b 18,19
x Adenovirus c 21
4. Classical strategies-Bacterial x Tuberculosis 1
(bacille Calmette-Guérin (BCG))
x Typhoid fever 14,15
(Salmonella typhi)
5. Recombinant bacteria x Cholera (Vibrio cholerae) 17
6. Recombinant bacterial vector x Salmonella typhi d 23
x V. cholerae d 24
x Shigella flexneri d 25
B. Subunit/inactivated vaccines
1. Whole pathogen
a. Inactivated bacteria x Pertussis (Bordetella pertussis) 26
x Cholera 27
x Enterotoxigenic 28
Escherichia coli
b. Inactivated virus x Poliovirus 30
x Influenza virus 31
x Rabies virus 32
x Japanese encephalitis virus 33
x Hepatitis A virus 35
2. Protein-based
a. Natural x Hepatitis B virus (HBV) 39
x Pertussis 40-42
b. Chemically inactivated x Tetanus (Clostridium tetani) 43
x Diphtheria 44
(Corynebacterium diphtheriae)
x Pertussis 45
continued on next page
New Technologies for Making Vaccines 3

Table 1.1. Status of development of representative human vaccines made by different

technologies (continued)

STATUS OF DEVELOPMENT*
Type of Vaccine** Preclinical Clinical Licensed Example# Reference
Evaluation*** Evaluation§ Product§§

c. Genetically inactivated x Pertussis 46

x Diphtheria 47
d. Recombinant x HBV 48
polypeptide x Lyme disease 50
(Borrelia burgdorferi)
x HSV 51
x Human papilloma virus 52
x Yeast Ty e 55
x Rotavirus 53

3. Peptide based
a. Fusion protein x Malaria f 58
b. Conjugate x Malaria g 60
x Pseudomonas aeruginosa g 57
c. Complex peptide x HIV 61
d. T-cell epitope x HBV 62
x cancer 63

4. Polysaccharide-based
a. Plain polysaccharide x Haemophilus influenza type b 64
(Hib)
x Meningococcal 65
(Neisseria meningitidis)
x Pneumococcal 66
(Streptococcus pneumoniae)
b. Conjugate x Hib h 67
x Pn h 68

5. Anti-idiotype (antibodies) x Cancer i 71,72

x HBV 70

C. DNA-based
1. DNA- naked x Influenza 74
2. Facilitated DNA x Hepatitis B 76
x HIV 78
3. Viral delivery x Canarypox virus j 81
4. Bacterial delivery x S. flexneri 83
x S. typhimurium 84

continued on next page

4 New Vaccine Technologies

Table 1.1. Status of development of representative human vaccines made by different

technologies (continued)

*These categories are presented in the same outline as in the text.

**This denotes the single most advanced status achieved by each example.
***Not yet evaluated in a human clinical trial.
§ In clinical trial but not yet licensed.
§§ Licensed in one or more major countries in the world.
# These are representative examples for each vaccine strategy, with one or two key illustrative
references.
a Licensed then withdrawn from distribution by the manufacturer.
b Expressing more than 50 different foreign polypeptides.
c Expressing at least six different foreign polypeptides.
d Examples of foreign polypeptides include toxoids from E. coli, V. cholerae, and C. tetani.
e Examples of foreign polypeptides include those encoded by HIV-1 gag and env genes.
f Fusion partner is HBsAg.
g Conjugate carrier is TT.
h Conjugate carriers are TT, DT, CRM197 and OMPC.
i Specificities are for human tumor carbohydrate and a human colorectal carcinoma antigen.
j Expressing rabies virus glycoproteins.

The strategic decision for developing a live, subunit/inactivated or nucleic acid-based

vaccine should be made after considering the epidemiology, pathogenesis and immunobiology
of the infection or disease in question as well as the technical feasibility of the various approaches.
Epidemiology dictates the target population for the vaccine. The age and state of health of
this population usually favors certain strategies as more appropriate for eliciting protective
immunity. For example, minimal reactogenicity is very important for a vaccine intended for
healthy infants, and certain types of vaccines are useless for infants because they do not elicit
protective immunity. However, the degree of reactogenicity is less important in cases such as a
therapeutic cancer vaccine. Knowledge of immunobiology can aid in identifying the nature of
protective immunity that should be elicited by the vaccine; certain immune responses may be
protective and others useless to the prevention or treatment of a particular infection. For example,
the clearance of the natural infection may correlate with the appearance of antibodies against a
particular microbial antigen; this would define that antigen as a candidate vaccine immuno-
gen. Alternatively, the study of immunobiology is greatly facilitated or enabled by developing
an experimental animal model, the availability of which enables candidate vaccines to be tested
and optimized for protective efficacy before bringing the best one(s) forward for clinical evalua-
tion. Historically, only a limited range of technical approaches has been feasible for a particular
vaccine. Nevertheless, considering the expanding number of technical approaches, it may be pos-
sible in the future to custom-design many vaccines for optimal efficacy and tolerability.

Live Vaccines
Some live vaccines come very close to meeting the criteria for an ideal vaccine by being
able to elicit lifelong protection with minimal reactogenicity using one or two doses. Such
vaccines may be feasible in cases where the natural infection confers lifelong protection on the
host. These vaccines consist of microorganisms (usually viruses) that replicate in the host in a
fashion like that of the natural microorganism so that the vaccine may elicit an immune
response similar to that elicited by the natural infection. The live vaccine is attenuated, meaning
that its disease-causing capacity is eliminated by biological or technical manipulations. Care
should be taken to ensure that the live vaccine is neither underattenuated (retaining
New Technologies for Making Vaccines 5

pathogenicity even to a limited extent) nor overattenuated (no longer infectious enough to
function as a vaccine). Live vaccines usually elicit both humoral immunity (antibodies) as well
as cellular immunity (e.g., cytotoxic T lymphocytes (CTL)).
Although these properties per se might make live vaccines highly desirable, this is not
technically feasible for most vaccines currently under development. A live vaccine may be
incompletely attenuated and consequently cause its natural disease at a low frequency or be
completely attenuated and incompletely immunogenic. Because a live vaccine can replicate, it
may be possible for it to revert to its more naturally pathogenic form. Live vaccine strains can
be transmitted from the vaccine to an unvaccinated individual, which can be quite serious if
the recipient is immunodeficient or is undergoing cancer chemotherapy. In some cases, the natural
viral infection per se fails to produce a protective immune response, such that an attenuated virus
(without further engineering) would not be expected to produce a protective response.

Classical Strategies
The term classical refers to technical strategies that do not utilize rDNA technology. The
production of live viral vaccines relies on propagating the virus efficiently in cell culture.

Attenuation in vitro
It has not been readily possible to develop live attenuated bacterial vaccines by classical
strategies because there has been relatively little success with in vitro culture of bacteria for
attenuation while maintaining immunogenicity. There also may be little competitive or
selective pressure for bacteria to become less virulent during in vitro passage; bacteria could
stop expressing virulence factors in vitro, then turn on their expression in vivo. The one widely
available live bacterial vaccine based on serial in vitro passage is for tuberculosis. This vaccine
consists of a live attenuated strain of Mycobacterium bovis, known as bacille Calmette-Guérin
(BCG),1 which was attenuated by 231 successive in vitro subculturings over 13 years. The
available BCG vaccines vary in tolerability, immunogenicity and rate of protective efficacy in
clinical trials. BCG vaccines have been inoculated into more than 1 billion people worldwide
and have generally acceptable tolerability profiles. One would anticipate that the techniques of
rDNA technology would be applied to attenuating a new bacterial strain. Therefore, by current
technical and regulatory standards, it seems highly unlikely that a new live bacterial vaccine
attenuated by a classical strategy alone will be developed.
The first classical strategy for viruses became possible during the 1950s with the ability to
propagate viruses in cell culture. The approach is empirical, in that the wild-type virus isolated
from a human infection is passaged in vitro through one or more cell types with the goal of
attenuating its pathogenicity. In such cases, there may be selective pressure to produce less
damage to cells. The mechanism by which mutation(s) are introduced during the course of
attenuation is not well understood. In some cases (e.g., poliovirus2), it has been possible to
demonstrate attenuation in a primate species, whereas attenuation has been proven in
most cases only through the course of extensive clinical trials. The success of this empirical
approach, which has been applied to both an oral vaccine (oral poliovirus vaccine2 (OPV))
and to injected (parenteral) vaccines (measles,3 mumps,4 rubella,5 varicella6), has been born
out by the number of available licensed vaccines. The reactogenicity of such vaccines has been
low enough that some of them (polio, measles) are widely accepted worldwide for routine
pediatric use. By means of intensive immunization programs with OPV, polio is well on its way
to worldwide eradication.

Variants from Other Species

An animal virus that causes a veterinary disease similar to a human disease can be isolated
and cultivated, as was done for smallpox vaccine vaccine (derived from cowpox virus). The
6 New Vaccine Technologies

anticipated outcome is that the animal virus will be attenuated for humans yet will be suffi-
ciently related immunologically to the natural human virus to elicit protective immunity. The
immunization program was applied worldwide using vaccinia virus7 and resulted in the com-
plete eradication of smallpox worldwide by the mid-1970s, the only infectious disease ever
eradicated. This program is a tribute to an effective control strategy and to the tireless efforts of
countless individuals. Based on this model, first-generation vaccines for rotavirus consisted of
animal-derived viruses.8 However, these rotavirus vaccines were not reproducibly efficacious as
human vaccines.

Reassorted Genomes
A reassortant virus derived following coinfection of a culture with two different viruses
with segmented genomes contains genes from both parental viruses. To improve the efficacy of
animal rotaviruses, reassortant rotaviruses were isolated containing mostly animal rotavirus
genes, which confer the attenuation phenotype for humans, as well as the gene(s) for a human
rotavirus surface protein, which elicits serotype-specific neutralizing antibodies for human
rotavirus.9,10 These reassortant rotaviruses have elicited higher efficacy rates as vaccine candidates
than their parental animal viruses. A quadrivalent reassortant rhesus rotavirus vaccine was
licensed in 1998. However, due to an increased rate of intussuseption (1:10,000) observed
immediately following immunization, the vaccine was withdrawn from use. This withdrawal
highlights safety as a key challenge for the development of new live vaccines. The same approach
has been applied to influenza vaccines, in which a newly chosen influenza virus provides the genes
that encode the immunogenic surface glycoproteins (hemagglutinin and neuraminidase), and
an attenuated virus provides all other genes and, with them, the attenuation phenotype.11 Such
reassortant influenza viruses can be adapted to grow in mammalian cell lines such as MDCK12
as a cell substrate to replace the use of chicken eggs.

Temperature-Sensitive Mutants
Viral mutants can be selected according to their growth properties at different temperatures.
These viruses have been referred to as temperature-sensitive (ts), being unable to grow at
elevated temperatures, or cold-adapted (ca), having been selected for growth in vitro at lower
than physiological (37°C) temperatures, i.e., down to 25°C. The idea behind this approach is
that ca viruses will be less vigorous in their in vivo growth than their wild-type parental virus,
hence less virulent and phenotypically attenuated. A ca influenza vaccine has been developed.13

Chemical Mutagenesis
Another technique for creating an attenuated strain has been chemical mutagenesis fol-
lowed by selection. The Ty21a strain of Salmonella typhi was derived in this fashion14 and
licensed for preventing typhoid fever based on its record of safety and efficacy over several
years.15

Recombinant Microorganisms

Viral
The increased stability of the attenuation phenotype results from making the modifications
or deletions in viral genes extensive enough that reversion through back-mutation is impossible
or highly unlikely. In contrast, attenuated viruses derived by classical strategies may have only
point mutations and therefore the capability to revert.
A deletion was made in a herpes simplex virus (HSV) gene encoding a glycoprotein required
for infectivity. This glycoprotein in supplied to the virus in trans by the cell line during in vitro
New Technologies for Making Vaccines 7

cultivation so that the resultant virus can initiate infection in vivo but not spread, which pro-
vides for its molecular attenuation.16

Recombinant Bacteria
The engineering of bacteria for attenuation is more complex than for viruses, given the
much larger size of bacterial genomes. The strategy is to identify the gene(s) responsible for the
bacterial virulence or colonization and survival and to either eliminate the gene (preferred) or
to abolish or modulate its in vivo expression. As for viruses, there can be a balance between
virulence and activity as a vaccine, which means that it is possible to overattenuate a bacterial
strain to the point that it no longer replicates sufficiently to elicit an effective immune response.
Attenuation of V. cholerae strains has been accomplished by the rDNA-directed deletion
of genes that encode virulence factors (such as cholera toxin (CT)).17 Live attenuated cholera
vaccine candidates prepared in this fashion have been evaluated clinically and one has been
licensed. In order to assure attenuation by reducing the probability of reversion, it is desirable
to delete two or more independent genes or genetic loci that contribute to virulence.

Recombinant Vectors
The second application of rDNA technology to the development of new live vaccines has
been the engineering of viruses as vectors for “foreign” polypeptides from other pathogens. The
goal of creating such vectors is to present the foreign antigen to the immune system in the
context of a live infection so that the immune system responds to the antigen as a live immunogen
and thereby develops broader immunity (humoral and cellular) to the corresponding human
pathogen. The recombinant polypeptide is expressed within the infected cell and either is
transported to the cell surface to stimulate antibody production or is broken down into
peptide fragments that are transported to the cell surface where they elicit CTL responses.
This strategy also has the potential advantage of amplification of the immunogenic signal
when the live vector replicates.

Viral
The prototype viral vector is vaccinia virus. Dozens of different recombinant polypeptides
have been expressed in vaccinia virus.18 At least 25 models for different infections have shown
that vaccination of animals can protect against the pathogen encoding the recombinant poly-
peptide. Recombinant vaccinia viruses expressing tumor antigens also have been shown to be
protective in rodent tumor model challenge studies. Given the known sequelae to immuniza-
tion for smallpox, which are more serious in immunocompromised individuals, vaccinia virus
itself has been engineered to reduce its virulence without compromising its efficacy as a live
viral vector.19 Cytokines can influence the nature of magnitude of the immune response. In
order to selectively manipulate the type of immune response to a vaccine antigen in the context
of a live vector vaccination, a recombinant vector has been constructed which expresses both a
cytokine as well as a recombinant vaccine antigen.20 Fowlpox and canarypox viruses are being
developed as live vectors that can infect human cells but not produce infectious viral progeny.
This inability to spread makes these viral vectors also classifiable as DNA-based vaccines (see
Viral Delivery in this Chapter).
Other mammalian viruses have been engineered into live vectors. Adenovirus strains, which
have been used extensively as vaccines in military recruits to prevent acute respiratory disease,
have been engineered to express foreign polypeptides and have elicited protective immunity in
several viral challenge models in animals.21 Optimizing recombinant polypeptide expression
remains an important technical objective for all these live viral vectors.
8 New Vaccine Technologies

RNA viruses can be engineered in similar fashion. Sindbis and other alphaviruses have
received extensive attention due to their broad host range, ability to infect nondividing cells,
and potential high-level expression per cell.22 On this basis, Sindbis has been developed into a
nucleic acid-based vaccine (see Viral Delivery in this Chapter).

Bacterial
Pathogenic bacteria can be engineered into live recombinant vectors for the expression of
foreign polypeptide antigens. The most common applications have been to engineer enteric
pathogens so that they can induce mucosal immunity against the foreign polypeptide upon
oral delivery. In the field of developing live bacterial vectors, S. typhi has been the focus of the
most effort in terms of strain development, immunology, molecular development; and clinical
testing.23 V. cholerae,24 and S. flexneri25 also have been engineered into oral recombinant vec-
tors for clinical evaluations. The challenges for these live attenuated vectors are both to retain
sufficient virulence for replication in the gut and expression of appropriate levels of foreign
polypeptides as well as to achieve sufficient attenuation to assure good tolerability. The ability
of some of these bacterial species to replicate intracellularly may augment the ability of expressed
foreign polypeptides to elicit cellular immune responses against their respective pathogens.

Subunit/Inactivated Vaccines
Such vaccines have advantages that relate to their inability to multiply within the host.
Generally they are well tolerated, especially for the majority of such vaccines that undergo
purification to remove other macromolecules. Given the broad range of available approaches,
it also is generally more feasible technically to produce a subunit or inactivated vaccine.
Immuno-genicity may be enhanced by its administration with an adjuvant or delivery system
(see Formulation of Antigens in this Chapter). Nevertheless, a development program should be
undertaken with the realization that multiple doses, often followed by booster doses, most
often are necessary for attaining long-term protective immunity. These vaccines usually function
by stimulating humoral immune responses as well as by priming for immunological memory.
In certain cases, especially when administered with certain adjuvants and delivery systems,
nonlive vaccines may stimulate CTL immunity.

Whole Pathogen
The earliest approach to making inactivated vaccines relied on the use of whole bacteria or
viruses with the objective of eliciting the formation of antibodies to many antigens, some of
which would neutralize the pathogen.

Bacteria
These vaccines are prepared by cultivating the bacteria, collecting the cells, and inactivating
them with heat or with chemical agents such as thimerosal or phenol.26 The final vaccine does not
undergo further purification. Owing to their biochemically highly crude nature, which includes
virtually all bacterial cellular components, the reactogenicity of such vaccines when given
parenterally (e.g., Bordetella pertussis) is usually greater than that of other types of vaccines. On
the other hand, inactivated whole-cell V. cholerae27 and enterotoxigenic Escherichia coli (ETEC)28
vaccines have been well-tolerated by the oral route. Oral inactivated whole-cell cholera (WCC)
vaccine, which lacks CT (and its toxic effects), has been shown to be very well tolerated and to
have a rate of efficacy of ca. 60% for three years in a high-risk population.27 In order to elicit
antibodies that would neutralize CT and increase efficacy, the recombinant B subunit of CT
(CTB) which lacks toxin activity is independently expressed, purified, and added back to the
WCC vaccine. This combined WCC + rCTB vaccine was shown to have a somewhat higher
rate of efficacy than WCC vaccine alone.29
New Technologies for Making Vaccines 9

Virus
Some inactivated viral vaccines have been available for decades and are generally very
well tolerated. Because viruses generally are shed into the cell culture media when grown in
vitro, cell-free media from infected cultures are collected. The large size of the virus particles
relative to other macromolecules in the media enables the particles to be enriched readily
by simple purification techniques that exploit the size of the particles. Examples include
poliovirus,30 influenza virus,31 rabies virus32 and Japanese encephalitis virus.33 Alternatively in
the case of killed hepatitis A virus (HAV) vaccine, infected cells are lysed and virus particles are
purified.34 The virus particles are inactivated chemically, typically by treatment with forma-
lin, and then may be adjuvanted by an aluminum salt. The key epitope(s) on the surface of
many nonenveloped small viruses that elicits a protective immune response (protective epitope)
is often conformational, being formed by the highly ordered assembly of structural proteins
into precise structures. For most of the listed viruses for which inactivated vaccines have been
developed and licensed, it has not been possible to readily mimic the conformation of such
epitopes by other technologies, e.g., recombinant polypeptides. Inactivated viral vaccines tend
to be highly potent immunologically, e.g., one dose of hepatitis A vaccine is protective at a
dosage of 50 ng.35 Thus, this classical strategy, which has had an excellent track record of
producing well-tolerated and efficacious vaccines, remains the technology of choice for many
viral vaccines.

Protein-Based
Developing a protein-based vaccine is a preferred strategy for many pathogens in which a
polypeptide contains protective epitopes, given the abovementioned issues regarding inactivated
vaccines. Protein-based approaches have relied on genetic, biochemical, and immunological
techniques to identify protective epitopes and their corresponding polypeptides as candidate
vaccine antigens.
More recently, genomics technology has enabled the identification of new vaccine antigens
in lieu of prior available biochemical or antigen data. Once the complete sequence (or portions
thereof ) of the genomic DNA or RNA are available, open reading frames (ORFs) are identified.
The derived amino acid sequence can be inspected for structural features, such as homologies
with proteins from other related pathogens that are vaccine candidates or a hydrophobic
N-terminal sequence that suggests surface localization. The genes are expressed in a recombinant
host cell (typically E. coli) and the recombinant polypeptide is purified and used to immunize
animals to derive polyclonal antibodies for identifying whether the hypothetical protein is
produced by the pathogen. Antisera also can be used in biological assays (neutralization of
viruses, opsonization of bacteria) to see whether the protein may be an attractive vaccine
candidate. The new protein also can be used for immunization and challenge in an animal
model. Some of the earliest applications of genomics technology to viruses were for the
discovery of hepatitis C virus (HCV)36 and hepatitis E virus (HEV),37 which resulted in the
direct definition of candidate vaccine antigens. The genomic approach was applied to Neisseria
meningitidis in which a number of candidate vaccine antigens were defined.38

Natural
The first protein-based vaccines relied on natural sources of antigens. In this regard, the
first-generation hepatitis B vaccine was unique among active vaccines in that it utilized a human
tissue source (plasma) for the vaccine antigen. Liver cells of individuals chronically infected with
hepatitis B virus (HBV) shed excess viral surface protein, i.e., hepatitis B surface antigen (HBsAg),
into blood as 22 nm virus-like particles (VLP) with protective epitopes. To develop a vaccine,
plasma was harvested from long-term chronic carriers of hepatitis B, HBsAg purified and the
10 New Vaccine Technologies

final preparation subjected to 1-3 inactivation techniques (depending on the manufacturer) to

kill HBV and any other potential human pathogens.39
Proteins purified from cultures of B. pertussis are combined to formulate acellular pertussis
(aP) vaccines, which eventually should replace whole-cell pertussis vaccine for routine pediatric
vaccinations in many developed countries. Depending on the number of different protein
antigens, these aP vaccines are referred to as one-, two-, three-, four-, or five-component vaccines
and have been licensed based on recent efficacy studies.40-42 These vaccines all contain pertussis
toxoid (PT) as a component, whose preparation is described below.

Chemical Inactivation
Many bacteria produce protein toxins that are responsible for the pathogenesis of infection.
It had been recognized for many decades that, when a toxin was pathogenic after infection,
antitoxins (antisera enriched in toxin-specific antibodies) that were effective in neutralizing
toxin activity in vivo could prevent or ameliorate symptoms of certain bacterial infections. This
precedent established the basis for bacterial toxins to be formulated as active vaccines. The
toxin molecules are purified from bacterial cultures (e.g., Corynebacterium diphtheriae (D),
Clostridium tetani (T), B. pertussis (P)) and then detoxified by incubation with a chemical such
as formalin or glutaraldehyde. Detoxified toxins, referred to as toxoids, thus represent two of
the vaccines (D,T) in the diphtheria, tetanus and pertussis (DTP) combination vaccine.43,44
PT45 combined with other pertussis antigens comprise the aP vaccines.

Genetic Inactivation
The chemical toxoiding procedure has possible disadvantages, including the alteration of
protective epitopes with ensuing reduced immunogenicity and potential reversion to a biologically
active toxin. To produce a stable PT, codons for amino acids required for toxin bioactivity
(adenosine diphosphate (ADP) ribosyl transferase) were mutated. The altered gene was substituted
for the native gene in the parental organism, which then produces immunogenic but stably
inactivated PT. As a refinement of this strategy, two mutations were introduced into PT to
assure the lack to reversion;46 this double mutant PT (which also is treated with formalin under
milder conditions to improve its immunogenicity or stability) is a component of a aP vaccine.40
In a related application, mutated cultures of C. diphtheriae were screened for the secretion of
enzymatically inactive yet antigenic toxin molecules. Subsequent cloning and sequencing of
one such mutated toxin gene identified a single amino acid mutation at the enzymatic active
site (also an ADP-ribosyl transferase). This genetic toxoid (CRM197)47 is the protein carrier for
a licensed H. influenzae type b (Hib) conjugate vaccine (Section II.D). This technology also has
been applied to V. cholerae toxin (CT) and ETEC heat-labile toxin (LT) to produce candidate
mucosal adjuvants (see Formulation of Antigens-Adjuvants p. 15).

Recombinant Polypeptides
The first application of rDNA technology to the production of a vaccine was for hepatitis
B. Given the precedent of plasma-derived HBsAg as a well-tolerated and efficacious vaccine,
the S gene encoding HBsAg was expressed in bakers’ yeast S. cerevisiae,48 which express 22-nm
HBsAg particles within cells. HBsAg is a VLP in that its surface is similar to that of HBV
virions. The yeast-derived vaccine, which is available worldwide in large supply, has largely
supplanted the equally efficacious and well-tolerated plasma-derived vaccine. HBsAg also has
been expressed in transgenic tobacco leaves and potato tubers; the purified HBsAg was
immunogenic.49
There are innumerable ongoing research and development applications of rDNA technology
to produce proteins as vaccine candidates. The major Borrelia burgdorferi surface protein (OspA),
expressed in E. coli as a recombinant lipoprotein,50 has been licensed as a vaccine for Lyme
New Technologies for Making Vaccines 11

disease. Recombinant-derived HSV glycoproteins expressed in Chinese hamster ovary (CHO)

cells and formulated as vaccines were tested in clinical trials.51
Large particles most often are more immunogenic than individual polypeptides. Further-
more, as in the case of HBsAg VLPs, particles usually elicit antibodies to conformational
epitopes on the particle, while isolated surface polypeptides of the particle might not elicit the
production of such antibodies. The human papilloma virus (HPV) virion is a highly ordered
structure whose major protein is L1. Expression of L1 in eukaryotic cells (e.g., S. cerevisiae)
results in the formation of L1 VLPs, which after immunization elicit antibodies that bind to
virions.52 Recombinant rotavirus53 and parvovirus54 VLPs also have been expressed as potential
parenteral vaccines.
Many host cells have been used for the expression of heterologous recombinant genes. In
addition to the previously mentioned (E. coli, S. cerevisiae and CHO), expression systems have
been developed for cells from other bacterial and yeast species and other mammalian continu-
ous cell lines (CCLs), e.g., African green monkey kidney (Vero). Whole animals and plants also
can be employed as hosts for recombinant expression. In general, smaller proteins that do not
require posttranslational modifications can be expressed efficiently in authentic form in microbial
expression systems. In contrast, polypeptides that require posttranslational modifications for
immunogenicity such as glycosylation for proper immunogenicity are expressed in mammalian
CCLs capable of correctly performing such modifications.

Carrier
A novel approach to recombinant vaccines is the use of yeast Ty particles as killed carriers
for foreign proteins. Yeast Ty is a particle assembled in S. cerevisiae that cannot replicate in
mammals. It is possible to express a gene encoding a foreign protein in conjunction with Ty
genes such that the foreign proteins assemble with Ty proteins into mixed particles.55 Because
the foreign proteins are expressed on the surface of these large particles, their immunogenicity
as vaccine antigens might be enhanced.

Peptide-Based
In many cases, it has been possible to identify B-cell epitopes within a polypeptide against
which neutralizing antibodies are directed. Many B-cell epitopes are conformational, being
formed by the juxtaposition in three-dimensional space of amino acid residues from different
portions of the polypeptide, which means that such epitopes require the full polypeptide for
their proper immunogenic presentation. In contrast, other peptide epitopes are linear in na-
ture, being fully antigenic as short linear sequences in the range of 6-20 consecutive amino acid
residues in the polypeptide. Some linear epitopes are only weakly immunogenic when pre-
sented in the context of the full polypeptide. In other cases, natural peptides would be effective
vaccine antigens if they were rendered sufficiently immunogenic. Linear B-cell epitopes of this
type have been defined for the malarial circumsporzoite (CS) protein (repetitive 4-amino acid
sequence)56 and for the Pseudomonas aeruginosa pilus protein.57 Both of these polypeptides
contain linear epitopes that are recognized by antibodies that neutralize the respective patho-
gens, yet the whole polypeptides elicit such antibodies only weakly. It is interesting to speculate
that this may represent a mechanism by which these and other pathogens have evolved to escape
immunological surveillance by rendering their neutralization epitopes less immunogenic.
The application of the following strategies (fusion protein, conjugate, complex peptide)
to weakly immunogenic linear epitopes has resulted in immunogenic presentations that elicit
substantially increased titers of neutralizing antibody compared with those elicited by the epitope
presented in the context of its natural full-length polypeptide. Nevertheless, the most effective
strategy in terms of ultimate clinical utility remains to be established on a case-by-case basis.
12 New Vaccine Technologies

Fusion Protein
The immunogenicity of linear epitopes can be increased by making a genetic fusion of
defined epitopes to a carrier protein that forms a large particle to improve the immune presen-
tation of the peptide. Two commonly used protein fusion partners of this type are HBsAg58
and hepatitis B core antigen,59 a 28-nm particle encoded by hepatitis B virus. Fusions have
been made at the N-terminus, the C-terminus, or the internal portion of the polypeptide
sequence of the protein partner, depending on which location affords the best immunogenic
presentation while maintaining efficient particle formation.

Conjugate
The peptide can be chemically conjugated to a carrier protein. The peptide sequence is
synthesized chemically with a reactive amino acid residue through which conjugation occurs to
the carrier protein. The most commonly used carrier proteins in conjugates are bacterial pro-
teins that humans commonly encounter such as tetanus toxoid (TT), for which a conjugate
with the malarial CS epitope has been tested clinically.60

Complex Peptide
Multimers of the peptide sequence can be synthesized for linkage together in repeated
arrays, as applied to the malarial CS and HIV-1 gp120 peptide epitopes.61

T-Cell Epitopes
Peptide epitopes recognized by CTL may be useful immunogens for the prophylaxis of
infections by agents such as HIV or immunotherapy for chronic diseases such as hepatitis B.
CTL peptide epitopes generally are poor immunogens. Thus, for an immunotherapeutic hepatitis B
vaccine, a CTL epitope from the HBV core protein was modified by covalent linkage to a
T-helper epitope (from tetanus toxoid) as well as two palmitic acid molecules.62 This vaccine
was shown in clinical studies to be immunogenic for eliciting HBV-specific CTL and memory
CTL. Melanoma-specific T-cell epitopes as peptides have been used to pulse dendritic cells in
vitro for delivery to the patient, with some observations of tumor regression.63

Polysaccharide-Based
There are many bacteria with an outer polysaccharide (Ps) capsule. In many if not most of
the encapsulated bacteria studied, antibodies directed against capsular Ps are protective against
infection. These observations have established capsular Ps as vaccine antigens.

Plain Ps
Native capsular Ps contain up to hundreds of repeat units distinctive for each bacterial
species and antigenic subtype in which each monomer consists of a combination of mono-
saccharides, phosphate groups and small organic moieties. The Ps is shed by the organism
during its growth and is harvested from the culture medium. These Ps preparations are usually
immunogenic in adults and children over 2 years of age and elicit antibodies that may mediate
the opsonization of the organism thereby protecting against infection. Ps vaccines have been
licensed for Hib64 (monovalent for serotype b), Neisseria meningitidis65 (quadrivalent) and
Streptococcus pneumoniae66 (23-valent). The shortcoming of these vaccines is that Ps, being T-cell–
independent (TI) immunogens, are poorly immunogenic or nonimmunogenic in children younger
than 2 years, and they do not elicit immunological memory in older children and adults.

Conjugate
Although infants and children younger than two years old do not recognize TI immunogens
efficiently, they can respond immunologically to T-cell-dependent (TD) immunogens such as
New Technologies for Making Vaccines 13

proteins. The chemical conjugation of Ps to a carrier protein converts the Ps from a TI to a TD

immunogen. As a consequence, Ps-protein conjugate vaccines can elicit protective IgG and
immunological memory in infants and young children. This strategy is particularly important
for encapsulated bacteria such as Hib and S. pneumoniae (pneumococcal; Pn) owing to the
preponderance of invasive diseases caused by these bacteria in children younger than two years
old, in whom a Ps vaccine is ineffective. There are four different licensed Hib conjugate vaccines,67
all with different carrier proteins (TT, DT, CRM197 and an outer membrane protein complex
from N. meningitidis Group B) of different sizes and immunological character, distinct Ps chain
lengths and distinct conjugation chemistries. Given these differences, the four vaccines display
one or more differences in the following immunological properties: response of 2-month-old
infants to the first dose of vaccine, responses of four- and six-month-old infants to the second
and third doses, response of children older than one year to a booster dose, kinetics of decay of
antibody levels, peak of antibody titer and age at which protection from clinical disease first
can be shown.
Pn bacteria consist of ca. 90 serotypes, as reflected in distinct capsular Ps structures. For
designing a pediatric Pn conjugate vaccine, seven serotypes have been recognized as responsible
for 60-75% of the major pediatric Pn diseases (acute otitis media, pneumonia, meningitis). A
heptavalent vaccine was recently licensed.68 Other vaccines being tested in advanced clinical
trials consist of a mixture of up to 11 individual Pn Ps conjugates.69

Anti-Idiotypic Antibodies
The idiotype (Id), that is, idiotypic determinant, represents unique antigenic determinants
associated with the hypervariable region of the antibody molecule. An antibody-1 (Ab-1) can
be defined as an antibody recognizing a particular antigen, e.g., vaccine candidate. The Id on
Ab-1 itself can act as an immunogen; the antibodies that bind to the Id on Ab-1 are referred to
as anti-idiotypic antibodies (anti-Id) or Ab-2. The paratope on Ab-1 is the binding site for
the particular antigen; thus, the binding site of an anti-paratope antibody is a molecular “mimic”
of the original antigen. If the paratope and the Id on Ab-1 represent the same or overlapping sites,
then the Ab-2 and particular antigen both bind at that site and thus have similar conformations
(Ab-1 is the image of both the antigen and Ab-2). By virtue of the antibody-binding site of
Ab-2 mimicking the conformation of the particular antigen, Ab-2 molecules themselves can be
used as vaccine candidates in which an epitope (the Id) is presented on a carrier molecule
(whole Ab-2). It was shown that vaccination of chimpanzees with anti-Id that mimicked HBsAg
protected the animals from infection with HBV.70
Numerous technologies exist for using an antigen as a vaccine candidate, either directly or
by augmenting its immunogenicity as described earlier. Furthermore, an antibody molecule
(Ab-2) is not necessarily a desirable immunological carrier for an antigen (anti-Id). Hence, the
situations in which the use of anti-Id would be the preferred vaccine strategy are quite limited
in number. Certain tumor antigens cannot be recognized immunologically by the host, because
these antigens are self-antigens, often being expressed in low levels in the host. Nevertheless,
the Ab-2 that is the mimic of the tumor antigen, yet not necessarily identical in structure to the
antigen (hence not a self-antigen), can elicit an immune response against the tumor antigen.71
When the tumor antigen is a defined Ps that cannot be isolated or synthesized in quantities
sufficient for vaccine studies, an anti-Id of the mimic of the Ps can be a useful cancer vaccine
candidate.72 The ultimate utility of anti-Id as a vaccine strategy remains to be established.
Furthermore, to obtain the highest degree of specificity as a vaccine candidate, one would
derive a monoclonal antibody (MAb) as an anti-Id and make it into a recombinant human or
humanized MAb.
14 New Vaccine Technologies

DNA (Nucleic Acid) Vaccines

It was shown that after cells in vivo take up DNA encoding vaccine antigen(s), the antigens
can be secreted or can be associated with the cell surface in a way that would trigger a humoral
or cellular immune response. Furthermore, the uptake of DNA can be facilitated by chemical
formulation or delivery by a virus or bacteria. The latter approaches fit the definition of a
DNA-based vaccine as one that cannot replicate in humans. “Naked”, facilitated and
virally-delivered DNA vaccines recently have entered clinical studies.

“Naked” DNA
One strategy has been to inject intramuscularly a solution of DNA encoding a vaccine
antigen.73 Cells take up the plasmid DNA, transcribe it and synthesize the antigen, which may
be processed in a similar way as in a live viral infection. The advantages of using DNA are the
relative technical ease of preparation and the ability to direct the synthesis of multiple copies of
mRNA, hence an expected amplification of both antigen synthesis and immune response.
Such vaccines have been shown to be effective in many animal models of infection, especially
virus models.74 As a novel route of delivery, naked DNA has been applied to mouse skin from
which it is taken up by hair follicles to stimulate an immune response.75 While naked DNA
does elicit the production of specific antibodies, it is particularly proficient at eliciting cellular
immune responses.

Formulated DNA
Facilitation can be at the level of cellular uptake, expression or immunological activation.
One strategy has been the incorporation of DNA into microprojectiles that then are “fired”
into cells, which produce the encoded antigen. This “gene gun” technique has been reported to
be potent at eliciting immune responses and has undergone initial clinical use.76 For improving
the efficiency of uptake, DNA has been coated with cationic lipids, lipospermines or other
molecules which neutralize their charge and have lipid groups for facilitating membrane transfer.77
Such formulations also are being researched for alternate routes of administration (e.g., oral,
nasal) which may elicit mucosal immunity. The anesthetic bupivacaine given in conjunction
with DNA has been shown to enhance DNA uptake and expression.78 ADP-ribosylating
exotoxins given together with DNA and applied to the skin can stimulate transcutaneous
immunization.79 The base composition of the DNA may affect its potency in that unmethylated
CpG dinucleotides have been shown to induce B-cell proliferation and immunoglobulin secretion
and to adjuvant responses to DNA vaccines.80

Viral Delivery
The above nucleic acid-based vaccines all result in the deposition into a cell of a plasmid.
For delivery of DNA by fowlpox or canarypox virus, the expression cassette for the recombinant
protein is integrated into the viral genome. These avian poxviruses can infect mammalian
(human) cells but not produce infectious virus;81 hence this can be considered a nucleic
acid-based approach. This single round of self-limiting infection may be sufficient to elicit
broad immunity to a pathogen whose recombinant polypeptide is expressed by these avian
poxviruses in infected cells, while reactogenicity should be minimal, given the inability of the
virus to spread within the host. A variation on the design of the expression plasmid is to use a
virus-based DNA expression system that can amplify the level of RNA and protein expression
as occurs in a live virus infection, as developed for Sindbis virus vectors.82

Bacterial Delivery
Bacteria that replicate intracellularly can be engineered to deliver plasmid DNA into cells
for the expression of recombinant proteins. S. flexneri has been attenuated by making a deletion
New Technologies for Making Vaccines 15

mutant in an essential gene (asd). While such a strain can be propagated in vitro in the presence
of diaminopimelic acid (DAP) and can invade cells, it cannot replicate in vivo, where DAP is
not available. A plasmid harboring a eukaryotic promoter and recombinant gene was transformed
into this strain. The resultant recombinant S. flexneri strain was shown to be able to invade
mammalian cells in vitro and to express the plasmid-encoded protein as a potential vaccine
antigen.83 Since S. flexneri replicates in the intestine and stimulates mucosal immunity, this
vector may be delivered orally for delivering DNA to cells where mucosal immunity is stimulated.
Other attenuated strains of bacterial species, e.g., Salmonella,84 that can invade mammalian
cells but not divide also can deliver recombinant plasmids orally for expressing recombinant
proteins as vaccine antigens.

Formulation of Antigens
The immunological effectiveness of vaccines (other than live) may be enhanced by their
formulation, which refers to the final form of the vaccine to be administered in vivo. In addi-
tion to the “active substance” (antigen or DNA), the formulation may contain an adjuvant
and/or delivery system in addition to excipients. The adjuvant is a substance that stimulates an
increased humoral and/or cellular immune response to a coadministered antigen. The delivery
system is a vehicle for assuring the presentation of the vaccine to cells of the immune system or
for stabilizing and releasing the antigen over an extended period of time. There may be overlap
in structure and function between adjuvants and delivery systems. Many future vaccines are
expected to contain new adjuvants and delivery systems. This topic has been addressed exten-
sively in reviews by others.85,86

Adjuvants
Aluminum salts, such as hydroxide or phosphate, are currently the only adjuvants widely
licensed for human use. This adjuvant has been used for decades in vaccines injected into more
than 1 billion people worldwide. The vaccine antigen binds stably to the aluminum salt by
ionic interactions and forms a macroscopic suspension in solution.87 This adjuvant preferen-
tially promotes a Th2-type immune response, i.e., antibody-based, and thus is not useful in
applications where inducing a cell-mediated immune response is needed for protection. While
aluminum salts have been useful for certain vaccines (e.g., hepatitis B, pertussis), for other
vaccine antigens they are not potent enough for inducing antibody responses which are high
enough to be optimally effective. Aluminum salts have not been shown to be useful for presen-
tation of vaccines by the oral or intranasal routes. Therefore, many chemicals, biochemicals
from natural sources, and proteins with immune-system activity (cytokines88) have been
researched as potential adjuvants. The adjuvanticity of virtually all known formulations is asso-
ciated with local or systemic side-effects which may be mechanism-based or nonspecific. The
ideal adjuvant needs to achieve a balance between degree of side-effects and immune-enhancement.
Certain bacterial toxins with ADP-ribosylating activity have received considerable atten-
tion as mucosal adjuvants in terms of molecular engineering. In particular, CT was shown to
be active as a mucosal adjuvant for a coadministered antigen89 when presented by the oral,
nasal, vaginal or rectal routes, as was shown subsequently for the heat-labile toxin (LT) of
ETEC. These toxins are composed of a catalytic A subunit and a pentameric B subunit that
binds to GM1 ganglioside on many cell types. However, both CT and LT are toxic in humans,
especially by the oral route through which they induce diarrhea. To dissociate the toxicity and
adjuvanticity of CT and LT, point mutations have been made which result in reduced or elimi-
nated ADP-ribosylating activity, reduced toxicity, and the apparent retention of adjuvanticity
in mice.90 An alternative approach has been to eliminate the B subunit and substitute a syn-
thetic dimeric peptide derived from Staphylococcus aureus Protein A (DD) which binds to
16 New Vaccine Technologies

immunoglobulin (Ig). The fusion protein of the CTA subunit with the DD domain binds to
Ig+ cells, appears devoid of toxicity, retains ADP-ribosylating activity, and is active as an adju-
vant in mice.91 The tolerability and effectiveness of these engineered adjuvants needs to be
validated in humans.

Delivery Systems
Besides presenting an antigen or DNA to cells of the immune system, a delivery system
may perform other key functions. There may be a depot effect whereby the antigen is main-
tained in an appropriate in vivo site for continual immune stimulation. There may be an
enhancement of vaccine stability in vivo. For mucosally-delivered vaccines, the delivery system
may enable efficient presentation and uptake by M cells, followed by transcytosis into Peyer’s
patches and presentation to lymphocytes for the induction of mucosal immunity.92 For certain
formulations, the vaccine may be maintained in vivo inside a physical structure for a signifi-
cant period of time, during which it is released slowly or in pulsatile fashion such that it may
function as a one dose vaccine. No delivery systems have been widely licensed. Gaining clinical
and pharmaceutical experience with new delivery systems and adjuvants remains a key goal in
the field.

Conclusion
Technological developments in the past decade have rapidly expanded the number of
general strategies for making new vaccines. In the next decade the number of approaches will
continue to expand and technical aspects further refined, such that most antigens could be
presented in a highly immunogenic form in the context of a live or subunit vaccine. Protein
antigens alternatively can be expressed through a nucleic acid-based vaccine. Further under-
standing of gene function in viral and bacterial pathogens should enable live vaccines to be
more stably and predictably attenuated as vaccines and as live vectors for immunizing against
other pathogens. Adjuvant technologies should advance to the point where formulations which
are more potent than aluminum salts, yet as well tolerated, gain widespread use for subunit/
inactivated vaccines and where oral delivery of purified proteins for immunization becomes
feasible. Similarly, formulations of DNA may improve the potency of DNA vaccines and its
ability to be delivered by routes that elicit mucosal immunity.
As all these technological advances proceed, it is likely that the limiting factor in developing
new vaccines for human use will continue to be a more comprehensive understanding of
immunology. Some areas in which increased knowledge would have a practical payoff for
vaccine development are the immunobiology of pathogens, the type and specificity of immune
response required for persistent protection against disease, the attainment of mucosal immunity
and the optimal vaccination strategy to achieve this protection. There also should be significant
developments in applications to noninfectious diseases, such as cancer and autoimmune diseases.

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CHAPTER 2

Clinical Issues for New Vaccine Technologies

Luc Hessel

Introduction

V
accination as a means of preventing infectious diseases arguably has had the greatest
impact on human health of any medical intervention.1 Since the pioneer work of
Jenner and Pasteur, the development of vaccines has been the consequence of the
uninterrupted introduction of a series of new technologies. The discovery of toxoids, purification
of polysaccharides, cell culture enabling virus culture, controlled methods for attenuation of
viruses, conjugation of polysaccharides to proteins, production of protein vaccines in
genetically-engineered cells and reassortment of viruses have been among the basic technologies
used so far in the development of vaccines.1,2 Application of the tools of modern biotechnology
has resulted in an array of vaccine candidates coming from many sources and created the
promise of prevention or treatment for many more infectious and chronic diseases.3,4 It has
also revolutionized the capability to engineer and produce vaccines that are potentially safer,
more effective, easier to produce and less costly.5 The forthcoming new technologies form a
continuum in the innovative process that has always been characteristic of vaccine develop-
ment. Different new technologies are currently considered with more attention, such as
1. genetically-engineered vaccines,
2. live vectors,
3. nucleic acid vaccines,
4. new delivery systems or
5. new adjuvants.1,2
This biotechnology revolution poses a tremendous challenge for traditional vaccine develop-
ment to provide adequate and timely assessments so that maximal benefits might be reaped
from these advances.6 Indeed, successful development of vaccines is a time-intensive process
requiring years of commitment from a network of scientists and a continuum of regulatory and
manufacturing entities.7
The vaccine development process leading to licensure is pyramidal and selective.4 It is
step-wise and bridges basic research, development, large-scale production in an approved facility,
and clinical evaluation to establish safety and protective efficacy.7 Although not basically
different than for conventional vaccines, the clinical contribution to the development of new
vaccines is of special importance at many stages of the discovery process and can be schematically
defined as follows:
1. definition of the medical needs,
2. choice of the rationale and elaboration of the strategy based on preclinical development,
3. demonstration of proof-of-principle in early clinical research,
4. design and implementation of an integrated clinical development plan and
5. continuous assessment of safety and efficacy.

New Vaccine Technologies, edited by Ronald W. Ellis. ©2001 Eurekah.com.

22 New Vaccine Technologies

After a review of the core essence of the work, some specific clinical issues raised by each of
the new technologies will be addressed.

Definition of the Medical Needs

The ultimate goal of vaccine research is to develop vaccines that are useful and effective.
Vaccines must address public health needs and be a logical means of controlling the disease of
interest. In theory, the field of application of vaccines is extremely broad, including at least all
diseases caused by microorganisms. Recently the field of vaccines has been expanded to other
fields such as cancer, allergy, autoimmune diseases, contraception and drug addictions.4,8-11
Choices have to be made, guided by epidemiological studies, experiments and observations
evaluating the burden of the disease as well as the existence of alternative prophylactic
measures and the availability of curative treatments.12 The exercise is not easy. In addition to
ubiquitous diseases of major importance such as pneumococcal infections or HIV, special
consideration is given to tropical diseases which have a tremendous impact on health, with
hundreds of thousand of deaths annually in children.4 Thus, a strong scientific base and
rationale, a firm quantification of disease burden to be prevented (including mortality, acute
morbidity, long-term sequelae and the associated direct and indirect economic costs), and
clear identification of target populations are central to the successful development of new
vaccines. The review of the development of selected vaccines recently performed by the
National Vaccine Advisory Committee in the USA clearly showed that vaccine development
moved forward expeditiously when the scientific base was well established, whereas develop-
ment efforts often stalled when the science was less mature due to a lack of clear direction and
endpoints.7

Moving from Preclinical to Clinical Development

Choice of the Rationale
Once the new target has been defined, the difficult step is to understand the basis for
natural protection against the pathogen, which will enable the identification of the relevant
immunological approach for developing a candidate vaccine. In some cases the nature of the
immune response needed for protection is well known and the antigen is clearly identified
(e.g., polysaccharide vaccines, neutralizing antibodies against viral diseases). In many cases,
however, there is no clear evidence of surrogate markers of protection. The identification of
candidate immunogens relies on several approaches, including a thorough analysis of the human
immune response to disease and a clear understanding of the biology of the causative organism.
The techniques used for these approaches are more and more complex, and interpretation of
results is not easy. Thus, laboratory animal assays are essential tools at several stages of the
research, development and production of improved and novel vaccines, delivery systems or
adjuvants.13 Demonstration of immunogenicity in animals is an absolute prerequisite to clinical
studies of a product. Preclinical studies in animals also include extensive toxicology studies in
order to demonstrate the absence of major safety issues. In most areas of vaccine research,
animal models are developed that contribute to the characterization of protective immunity,
the study of the safety and immunogenicity of various formulations and the preclinical evaluation of
the protective efficacy.13 In spite of their limitations, animal studies are still irreplaceable and taken
with caution they are of great help.

Choice of a Strategy
Once the antigen has been identified, there often are several ways to produce or express
it; these include attenuation of the live microorganism to lose virulence while maintaining
Clinical Issues for New Vaccine Technologies 23

immunogenicity, a protein purified from the microorganism itself, a recombinant protein

made in bacteria, yeast or mammalian cells, the antigen expressed by a recombinant live
attenuated bacterium or virus or a plasmid DNA construct. Every approach has its own
merits and limits in terms of the type of elicited immune response, ease of production, ease of
controls and risks for the environment. All these aspects will have an impact on the objectives
and methodology of the clinical trials to be performed as well as on the overall acceptability of
the vaccine by regulatory agencies. Sometimes, as illustrated by the first generation of respiratory
syncytial virus (RSV) formalin-inactivated parenteral vaccines, the legacy of safety concerns
has a marked impact on subsequent vaccine development.14
Thus, before moving to clinical trials in man, the vaccine candidate must have been
designed with a sound scientific rationale. Based on either known or likely protective antigens
or on a live strain with genetic deletion of known virulence factors, there must be an expectation
of efficacy and safety formally demonstrated in an appropriate model using a dose and a route
of administration that will be proposed in clinical trials. Animal studies demonstrating the
immunogenicity and, if an appropriate model exists, the efficacy of the vaccine candidate against
laboratory- or wild-type pathogens represent the ideal approach. It is also highly desirable that
the vaccine be prepared in a formulation as close as possible to the final manufacturing process,
including antigen preparation, adjuvants, volume, etc. The critical scale-up from bench-scale
to pilot lots and then to large-scale production is often a particularly vulnerable point in the
development process of new vaccines.7 Thus, early establishment of the product profile and
characteristics will considerably help the regulatory process.

Demonstration of the Proof-of-Principle

Once preclinical development has been completed, it is time to turn to clinical testing.
The scope of the challenge may be limited. If the protective antigen or the type of immune
response responsible for protection is well known, it will be necessary only to demonstrate that
the product developed at the laboratory level achieves expectations. This is the objective of
classical phase I studies. If the product is safe and raises the expected immune response, the
decision may be taken quickly to bring the product to full development.
In some other cases, the validity of the approach is not ensured even when safety and
immunogenicity have been established, and the proof-of-principle will be qualified only after
protective efficacy results are known. This especially applies when
1. animal models do not exist or are not relevant,
2. clinical or immunological markers are not available and
3. safety issues are central to the acceptability of the vaccine. In this case human challenge
studies may be recommended as long as they are ethical and do not endanger volun-
teers’ health.
Human challenge tests can represent a good marker of the actual efficacy of the vaccine candi-
date against laboratory or wild pathogens and across serotypes. They will also contribute to
identifying immunological correlates of protection, to comparing protection conferred by the
vaccine candidate to that of a clinical infection, and to giving some information of the duration
of immunity. Human challenge tests represent very useful tools for the early screening of vac-
cine candidates, but their use remain limited by several issues relating to their reproducibility,
to the possible lack of correlation between experimental disease and natural infection and to
general and specific ethical concerns.15
However, in a few cases when the disease is very common, preliminary efficacy data can be
obtained at a very early stage. This is the case for some respiratory and diarrheal diseases such as
rotavirus diarrhea or RSV infection where the incidence is so high that studies in small
numbers of children may allow to estimate the value of the approach.
24 New Vaccine Technologies

Design and Implementation of a Clinical Development Plan

General Design
The goal of prelicensure studies is to show that the vaccine is safely tolerated in terms of
local and systemic reactions and to demonstrate that it confers protection against the target
disease.12 The Clinical Development Plan (CDP) describes and justifies in a logical and phased
way the clinical studies to be done in order to answer well-defined questions for documenting
the safety, immunogenicity and efficacy of the vaccine and delineating its conditions of use in
order to obtain the marketing approval in the proper indications. The CDP must take into
account numerous aspects of the future vaccine, especially its regulatory and marketing strategy
as well as industrialization issues and include all questions to be clinically addressed in the
Product License Application (PLA). It also needs to anticipate short- and long-term evaluation
to be considered after the vaccine has been licensed, including concomitant administration
with other vaccines, antibody persistence and need for booster injections, administration in
special populations and development of surveillance systems to address long-term safety
issues.7,12 Phase I trials usually enroll 10-100 adult volunteers to assess initial safety tolerability
and acceptable vaccine dosage in humans. Phase II trials (100-1,000 persons) seek to expand
knowledge about the safety and immunogenicity of the vaccine, to select the optimal formu-
lation of a candidate product, and to identify the most suitable schedule for vaccine adminis-
tration (including dosage, route of administration, optimal interval between primary series and
boosters, if needed) for subsequent evaluation in phase III efficacy studies.16 Phase III trials
aim to show that the candidate vaccine is efficacious in conferring protection on a targeted,
at-risk population under controlled conditions. Safety issues are also examined to the extent
that the sample size and study duration permit. As with any clinical trial, issues such as case
definition, case finding, trial design and sample size (which can range from 1,000 to 100,000
subjects according to the incidence of the disease) must be considered carefully.17 Because
sample sizes for these pivotal studies are large, they often require several years to complete enroll-
ment and follow-up.18 Although not basically different from those relating to conventional vaccines,
special attention must be paid to some methodological aspects when designing a CDP for vaccines
using novel technologies (see below).

Objectives
Many aspects must be considered when preparing a CDP. They include clear definitions
of objectives and how they will be addressed. Precise objectives are central to the development
of a proper methodology, the establishment of relevant clinical and statistical hypotheses and
the determination of the number of subjects to be included. According to the type of technology
or delivery system used, considerations include specific regulatory requirements as well as manu-
facturing issues in terms of stability and consistency of the vaccine lots and expected safety
issues, e.g., person-to-person transmission or environmental risk for genetically-engineered
live vector vaccines. Similarly, objectives will be different when the vaccine is intended for pre-
or postexposure prophylaxis as opposed to a therapeutic vaccine. What is expected from the
vaccine candidate should also be defined, e.g., an improved immunogenicity or safety profile
compared to an existing vaccine or an extension of the target population such as non- or
poor-responders, immunocompromised, neonates, etc. Whereas most vaccines are aimed at
controlling the spread of a disease or even eradicating it, future vaccines may control the
progression of an existing disease, e.g., cancer.
Clinical Issues for New Vaccine Technologies 25

Regulatory Issues
The development of novel vaccine technologies led to considerable changes in the international
regulations in order to address safety issues linked to the use of genetically-modified organisms (GMOs),
live vectors and any new means of influencing immunological mechanisms through adjuvants.
In addition to the relevant guidance established by the International Conference on Harmoni-
zation (ICH), precise guidelines have been defined in the USA and Europe which have to be
taken into consideration in the design of the CDP for a new vaccine.19 In Europe, the recently
issued CPMP Note for Guidance on Clinical Evaluation of New Vaccines gives very precise
methodological requirements on the assessment of efficacy, immunogenicity and safety during
both the pre- and postlicensure periods.20 The guideline insists also on the fact that
nonconventional vaccines require special attention with regard to vectors, immune responses,
immunological mechanisms and safety considerations. Indeed, special requirements and laws
have been defined regarding certain technologies like GMOs. Directive 90/220/EEC addresses
the potential risk to human health and to the environment, including plants and animals,
which may be associated with investigational products containing GMOs. An environmental
risk assessment (ERA) (which does not include naked DNA) is requested for any biological
entity capable of replicating or of transferring genetic material. An updated Note for Guidance
has been issued in December 1999 on the quality, preclinical and clinical aspects of gene transfer
medicinal products. In addition to international guidelines many national regulations and ad hoc
committees have been established which may influence the content of a CDP. For instance, in the
USA, the FDA also has issued guidance for industry for the evaluation of combined vaccines,21
plasmid DNA vaccines,22 and human somatic cell therapy and gene therapy.23

Clinical Endpoints and Surrogate Markers

The ultimate clinical endpoint may be to prevent infection (e.g., HIV), the final disease
(e.g., AIDS), severe disease (e.g., pertussis), disease progression (therapeutic vaccines) or survival
(rabies). It is not always possible to directly address such primary endpoints for some vaccines
due to the epidemiology of the disease, long-term clinical endpoints or multiple endpoints.
Should prevention of CMV infection be focused on prevention of congenital infections or of
secondary infection among women of childbearing age?24
When it is not feasible to obtain clinical endpoints, appropriate immunological data,
determined by an adequately validated method, may be used to support evidence of efficacy.20
Indeed the clinical efficacy of vaccines depends on the elicited immune response, whether
humoral, mucosal or cellular. Surrogate markers are parameters for predicting efficacy without
knowing the exact level of protection. They are mainly biological markers of humoral and/or
cellular immunity. Serological surrogates are mostly used and defined as a predefined antibody
concentration correlating with clinical protection.20 This especially applies when a reference
vaccine exists and is routinely used. This may also apply to safety issues that could be related to
potential autoimmune mechanisms. While sometimes scientifically well-grounded (e.g., anti-
body protective levels against Haemophilus influenzae type b or hepatitis B surface antigen), it
may be very difficult in many instances to establish a precise correlation between an antibody
level and clinical protection.25 This especially applies to pertussis or rotavirus infections.
Attempts to establish such correlations might be difficult when the measured antibodies are not
specific enough (ELISA), although they could remain relevant for comparison with a reference
vaccine. Antibody assays are especially acceptable when the function of the antibody is known.
Neutralizing or bactericidal activities or measurement of avidity may represent valid surrogate
markers of efficacy as long as it contributes directly to protection. Assessment of cellular
immunity is also subject to many limitations, since the assays require very demanding biological
monitoring, are not always well standardized, often rely on cytokine induction, are variable
26 New Vaccine Technologies

and irreproducible and very expensive. Mucosal immunity might be an important parameter
to be measured when the vaccine-preventable disease is a local infection, or starts in the
mucosa and invades (e.g., upper respiratory infections), or sexually transmitted diseases.26
Unfortunately, the methods for measuring local immune responses are not well standardized
and the collection and storage of such samples is problematic.25 Other surrogate markers such
as the evaluation of bacterial or viral load may also be extremely useful in the development of
therapeutic vaccines.
Overall, any information on the characteristics of the immune response according to the
known or presumed activities of the vaccine represents a very valuable tool for establishing vaccine
efficacy;20 these include the level, class, subclass and function of specific antibody produced,
the induction of mucosal or cell-mediated immunity, formation of neutralizing antibodies,
cross-reactive antibodies, etc.

Study Design
As described above, the traditional design for developing a new vaccine consists in
establishing the safety and immunogenicity of the product through phase I, II and III
studies. This sequential approach may not always be applicable for novel technology vaccines.
Phase I studies may not be possible when the candidate vaccine has no relevance for healthy
adult volunteer, e.g., cancer vaccines. In other instances (e.g., live vectors), it may need a
confined environment and long surveillance. Phase II studies may not be conducted as
expected in the target population. This applies to cancer vaccines that are basically indicated
at early stages of cancer when the immune system is still able to develop adequate protective
responses. However, due to potential safety issues and ethical concerns, inclusion criteria may
be restricted to advanced disease where an actual therapeutic effect might not be apparent.
Selection of volunteers or patients can be an issue when sero-eligibility is required or when
the pharmacological properties of the vaccine relies on HLA specificity or other immune
characteristics of the host. Defining the relevant control group is critical when a placebo is
not acceptable or when no control vaccine is available, especially for therapeutic or postexposure
vaccines. Concomitant administration of other vaccines according to routine recommendations
can constitute a confounding factor for safety and immunogenicity evaluation. Overall, the
methodological approach for a relevant clinical study design mainly relies on the quality and
accuracy of the objectives, the existence of validated clinical or biological endpoints and on a
clear statistical hypothesis.

Statistical Issues
The design of clinical trials obviously depends on its objectives but will be influenced by
the statistical hypothesis, the tests to be used and the power requested to meet them. This will
affect the number of subjects to be included, the choice of the control vaccine and of the
evaluation criteria to be chosen as primary or secondary endpoints. The criteria for determining
sample size in each trial must be based on methodological and statistical considerations, clinical,
immunological and epidemiological issues and tailored to the clinical hypothesis.20 When
immunogenicity is the sole primary endpoint to evaluate efficacy, appropriate randomization
of the target population should be ensured. Controlled trials, ideally placebo-controlled
double-blind, have been the gold standard in vaccine development for many decades. It
provides the ideal means to demonstrate the efficacy of a new vaccine by showing its superiority
to a placebo or a passive control vaccine. As more and more effective vaccines become available,
the objective of clinical investigation changes. Comparative trials can seek equivalence or
noninferiority of the candidate vaccine rather than its superiority to an effective standard
reference in active controlled trials.27 The fundamental assumption of such trials is that showing
Clinical Issues for New Vaccine Technologies 27

noninferiority is evidence of efficacy. This especially applies to vaccines combining multiple

antigens already available as separate vaccines and to the use of new technologies to improve
the safety of the manufacturing process of already existing vaccines without increasing their
efficacy. In this design, the choice of the control vaccine is critical. It has to be a well-accepted
standard vaccine across different geographical regions and one for which efficacy has been
satisfactorily and consistently proven. This methodological approach may have an important
impact of the size of the study population as it requires a much larger sample size than a
placebo-controlled superiority trial.27 In noninferiority trials, the purpose it to show that the
efficacy of the test treatment is not inferior to that of the reference treatment by more than a
minimum clinically relevance difference (δ), which has to be predefined at the trial design
stage. Typically, the sample size should be such that, if the treatments are equivalent or the test
vaccine is superior, there is a high probability (>80%) that the lower limit of a 95% confidence
interval for the difference in protection rate will not fall below the predefined δ.20,27 One key
issue is to define the clinical relevance of differences in serological responses in order to avoid
disqualifying a candidate vaccine because it does not meet narrow statistical differences. In-
deed, the choice of the δ value depends on the expected seroprotection/conversion rates and
will strongly influence the size of the study population. In individual trials, δ can often be set
to about 10 percentage points but will need to be smaller for very high protection rates and
larger with lower protection rates.20

Ethical Issues
Ethical issues represent a major aspect in the design of a clinical development plan for
novel technology vaccines. They are obviously critical for testing candidate vaccines in spe-
cial groups, e.g., for cancer vaccines where survival may be the primary endpoint, as well as for
HIV vaccines, and when placebo controls are used or challenge tests requested. 15,20
Genetically-engineered live vectors may evoke long-term safety concerns, generating demand-
ing clinical and biological long-term monitoring. Developing vaccines for infants or neonates
also represents an ethical challenge, as recently illustrated with a reassortant rotavirus vaccine.28
The content of informed consent forms therefore become an important aspect of the proto-
cols. Setting up data safety monitoring committees in charge of reviewing and validating ad-
verse events, independently from the investigators and the sponsor, represent the best way to
address such issues.

Continuous Assessment of Safety and Efficacy

In many respects, safety is a critical issue for novel technology vaccines. This applies for
both prophylactic and therapeutic vaccines, especially if there is an existing alternative for
prevention or cure of the given disease. As the result of safety considerations, certain vaccines
(e.g., live virus or bacterial vaccines) may require considerable additional information relating
to the vaccine strain, including comparison of the properties of the shed vaccine isolates versus
the parent vaccine strain, the pattern of shedding, transmissibility to contacts,29 and its potential
to survive in the environment or to enter the food chain.30 Similarly special attention on safety
has been developed for genetically-engineered live vectors using GMOs. New adjuvants
targeted at stimulating a specific immune response will justify paying attention to specific
safety issues such as autoimmune diseases or potentially rare and distant adverse events (AEs).31,32
In the latter case, it may be difficult to link AEs to the vaccine when such diseases are of
unknown origin or incidence. Development of new delivery systems, such as jet injectors and
mucosal administration, also will lead to defining specific approaches for new potential safety
issues. Safety will be addressed both during the pre- and postlicensure phases.
28 New Vaccine Technologies

In the prelicensure evaluation phase, potential safety hazards ideally will have been identi-
fied in preclinical laboratory tests, e.g., genetic stability, as well as animal testing where applicable.
Clinical assessment will remain the key factor, in particular for defining proper control groups,
identifying relevant markers for autoimmunity and addressing the risk of person-to-person
transmission. When GMOs are used, ERA also will be required. Prelicensure evaluation never-
theless will be limited to a relatively small number of subjects, usually not exceeding 10,000
followed for short periods of time. Ensuring the safety of new vaccines as they become licensed
and are given to larger numbers of persons is critical to ensuring the trust of the public and
stability of the immunization program.7
Therefore, post marketing surveillance (PMS) is essential to assess rare, unexpected, distant
AEs.33 PMS is traditionally based on spontaneous reporting of AEs following immunization by
health-care workers as a tool to generate signals to be further investigated in epidemiological
studies. In an attempt to overcome the deficiencies of passive reporting systems, “active” sur-
veillance through prospective or retrospective observational studies is becoming more frequently
used in the immediate postlicensure period. They allow the assessment of rare AEs that cannot
be described in the selected and relatively small population involved in the clinical develop-
ment of the vaccine. This requires a procedure for identifying all clinically significant events
that occur within defined postvaccination periods.12,33 Most recent regulations even consider
these PMS studies as conditional for granting a license.20 New tools must be developed to
allow a more systematic and cheaper way of addressing long-term safety issues linked to the use
of novel technology vaccines. This especially includes establishing national or international
disease registries that could be linked to vaccination registries for allowing the determination of
possible changes in the epidemiology of a disease or a group of disorders and the implementation
of the use of a new vaccine.12,33

Specific Issues
According to the novel technology used, some specific issues will apply to clinical
development of new vaccines, as described in more detail in the relevant chapters of this book.

Live Vectors
By using live vectors multiplying in the organism and expressing foreign genes, one can
deliver protective antigens from pathogens that themselves might be considered as unsafe as an
attenuated vaccine.1 Nevertheless, this technology raises different clinical issues that need to be
addressed at different times of the development process. The rational choice of the vector is
closely dependent on the pathophysiology of the wild organism.34 Live Salmonella recombinants
are widely explored because of their potential ability to reach the lymphoid tissue of the gut
and thus deliver the antigens to immunocompetent cells. Similarly Shigella are considered
potential vectors because of the possibility of passing the gut mucosa. BCG or BCG-like
organisms are selected as potential vectors because of their capacity to trigger T-cell responses
and also because of the adjuvant effect of the bacterial membrane. Many viruses are also
explored as live vectors expressing foreign antigens; among the most studied are poxviruses,
adenoviruses and more recently flaviviruses. The very first point to address clinically is the
attenuation of the live vector that does not allow for any residual effect. Even if the balance
between attenuation and antigen expression has been found, the immunogenicity of the vector
itself is another frequent issue. It may be previous immunity (this is definitely the case for
vaccinia immunity which interferes with pox-recombinants) or it may be that the first contact
with the vectored vaccine will elicit a stronger response to the vector than to the foreign gene,
thus jeopardizing the response to a further injection of the same live vector. These points
represent key evaluation criteria to be addressed very early in phase I and II trials.
Clinical Issues for New Vaccine Technologies 29

Recombinant DNA technology using attenuated viral or bacterial strains falls under
regulatory requirements regarding GMOs. They will justify specific extensive safety evaluation
linked to the residual virulence of the vector strain, as well as an environmental analysis of virus
replication and survivability of the vaccine strain in various environments.35 Person-to-person
transmission will request special attention as well as documentation of the stability and
potential reversion of the strain to a virulent one through shedding and immunological markers.
Therefore all possible tests on suitable animal models (including SCID/transgenic animals)
as well as in vitro tests will be necessary before moving to clinical trials. Phase I/II studies could
need to be conducted in confined environments and have an ERA performed. The clinical
evaluation of the consistency of manufacturing also represents an important part of the clinical
development.

Cancer Vaccines
Understanding the interactions between the immune system and cancer cells, especially
the molecular mechanisms of immune recognition and immune regulations, so that antitumor
immunity could be amplified forms the basis for the development of effective means to treat
cancer patients. In contrast to prophylactic vaccination against infectious agents, in which the
generation of humoral immunity is the most important feature, cancer vaccine development
focuses mainly on the generation of antigen-specific T-cell responses.9 Whatever the fundamental
approach is, tumor antigens or cells, cytokines or dendritic cells, the development of cancer
vaccines represents a real challenge, as clinical studies in man are often the only way of
supporting proof-of-concept for many vaccine candidates. Animal models are not always
available or relevant for selecting antigens. Correlation between immunological markers and
clinical efficacy is difficult to establish. In addition, phase I study in healthy volunteers are
generally not applicable, such that phase II studies often will be the first approach for supporting
the concept. But they cannot be easily conducted in the final target population (minimal vs.
advanced disease). Clinical endpoints are not easy to define or acceptable (e.g., survival). Con-
trol groups are difficult to define due to concomitant therapies or surgery and lack of standard-
ized therapeutic protocols (impact on stratification). Finally, AE reporting could be very de-
manding in a population at high risk of experiencing many AEs unrelated to vaccination that
will have to be followed up for a long period of time.

Adjuvants
It is recognized that impurities had a nonnegligible effect and thus a real impact on the
efficacy of old vaccines. A weaker immunogenicity profile generally appears as the price to pay
for safer and purer vaccine so that there is now a need for strong adjuvants of immunity. The
development of new adjuvants represents a major goal for new vaccine technology. Such new
vaccine formulations should allow improved immunogenicity of antigens in the general
population or in persons whose immune system is immature (neonates) or suffers from
genetically or acquired deficiencies. The clinical development of new adjuvants illustrates a
number of difficulties mentioned above. The target population may be poor or nonresponders,
justifying prescreening of a large population. Such populations include immunodeficient patients
or neonates, thus raising special immunological evaluation and safety issues. Immunological
endpoints for adjuvants may be very specific when they are targeted at stimulating cellular or
mucosal immunity. Defining the type of immune responses (Thl vs. Th2) will also require
specific tests with demanding monitoring processes. Depending on the type of adjuvant,
specific short- and long-term, local and systemic safety issues will have to be addressed, includ-
ing potential mutagenic effects and induction of autoimmunity justifying extensive safety
surveillance.35
30 New Vaccine Technologies

DNA Vaccines
A new approach to vaccination opened up recently with the demonstration that direct
inoculation of DNA encoding a foreign antigen can initiate protective immune responses.36
DNA vaccines containing the genes of foreign proteins provide cells in the vaccinated host
with the code for the synthesis of the immunizing antigens. Such vaccines have a number of
advantages over more classical vaccines, although their use is limited to protein antigens and so
far to skin or muscle delivery. They raise antibody against the natural form of proteins, can
elicit both cytolytic T-cell and antibody responses and long-lived immunity.35 In addition they
hold promise for safer, less expensive, easier-to-produce and easier-to-administer than conventional
vaccines. Finally DNA vaccines offer a broad range of applications including new antigens
(viral, bacterial and parasitic), combination of multiple antigens and biased Th1 or Th2
responses.36
Although DNA technology will apply to vaccines for which some clinical and immunological
endpoints are well established, the applied preclinical and clinical research on DNA vaccines
will have to address very specific additional issues, including a better characterization of the
induced immune responses and special consideration for safety. Determining the relative roles
of the target site (muscle or skin), better understanding of the T-helper preference and evaluation
of vaccine vectors and delivery systems will represent specific clinical and immunological
endpoints for phase I and II studies. Regarding safety issues, regulatory agencies considering
the application of DNA vaccines for human use have already identified key areas of concern,
including mutagenic events linked to the integration of the plasmid DNA into the genome of
transfected cells, potential induction of immune tolerance to the vaccine antigen or induction
of autoimmunity and/or of antibodies to the plasmid DNA. Based on animal experiments
and current knowledge of the role of natural infections in the induction of autoimmunity,
these potential safety issues appear very unlikely to be manifest.36-38 These complicated issues
will nevertheless represent a key aspect of the pre- and postlicensure clinical development of
DNA vaccines. It will be based not just on the identification of relevant biological markers
but also mostly on the recently developed epidemiological surveillance systems mentioned
above that provide background data on rare diseases and prospective registries.12,33

Conclusion
Designing and performing clinical trials with a novel vaccine technology is a complex
process which needs to be carefully analyzed before entering into long and costly clinical trials
in order to address all specific issues that may impact on the licensibility of the vaccine.
Numerous guidelines and regulations have been issued recently which help designing relevant
clinical plans but which must be interpreted with some flexibility. Thus, early discussions with
regulatory authorities of the content of the CDP of a novel technology vaccine are key for
further acceptability of the registration file.7 Finally, one must accept that, even at the dawn of
the new millennium, the process of vaccine research and development continues to be, at least in
part, an empirical and methodic science. In the era of molecular vaccinology and immunology,
developing an effective vaccine does not necessarily mean understanding the precise mechanisms
by which most vaccines work.

Acknowledgment
The author is grateful to Dr Michel Cadoz for his helpful comments and suggestions
during the preparation of the manuscript.
Clinical Issues for New Vaccine Technologies 31

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2. Ellis RW. New technologies for making vaccines. Vaccine 1999; 17:1596-1604.
3. Levine MM. The legacy of Edward Jenner. Br Med J 1996; 312:1177-1183.
4. Division of the Microbiology and Infectious Diseases, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD. Accelerated development of vaccines, The
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5. Woodrow GC. An overview of biotechnology as applied to vaccine development. In Woodrow
GC, Levine MM eds. New generation vaccines. New York, NY: Marcel Dekker, 1990:31-41.
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7. National Vaccine Advisory Committee. Lessons learned from a review of the development of selected
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8. Linehan DC, Goedegebuure PS, Eberlain TJ. Vaccine therapy for cancer. Ann Surg Oncol
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9. Pardoll DM. Cancer vaccines. Nature Medicine 1998; 4:525-531.
10. Cookson WOCM, Moffatt MF. Alchemy for asthma. Nature Medicine 1998; 4:500-501.
11. Rose NR, Wick G, Berger P et al. Immunological hazards associated with human immunization
with self or self-like antigens. In: GGL Ada, PD Griffin, eds. Vaccines for Fertility Regulation.
The assessment of their safety and efficacy. Cambridge: Cambridge University Press 1991:1121-146.
12. Chen RT, Orenstein WA. Epidemiologic methods in immunization programs. Epidemiol Rev 1996;
18:99-117.
13. Makela H et al. Animal models for vaccines to prevent infectious diseases. The European Commission
COST/STD-3 initiative on European vaccine research. Vaccine 1996; 14:717-731.
14. Kapikian AZ, Mitchell RH, Chanock RM et al. An epidemiological study of altered clinical reac-
tivity to respiratory syncytial virus (RS) infection in children previously vaccinated with an inacti-
vated RS vaccine. Am J Epidemiol 1969; 89:405-421.
15. Levine MM. Experimental challenge studies in the development of vaccines for infectious diseases.
In: Plotkin S, Brown F, Horaud F, eds. Preclinical and Clinical Development of New Vaccines.
Dev Biol Stand. Basel: Karger 1998; 95:169-174.
16. Tacket CO, Mattheis M, Rennels MB. Initial clinical evaluation of new candidate vaccines: Phase
1 and 2 clinical trials for safety, immunogenicity and preliminary efficacy. In: Levine MM, Woodrow
GC, Kaper JB, Cobon GS, eds. New generation vaccines. New York, NY: Marcel Dekker
1997:35-46.
17. Herrington DA. Initial clinical evaluation of new vaccine candidates. In: Woodrow GC, Levine
MM eds. New generation vaccines. New York, NY: Marcel Dekker 1990:43-49.
18. Clemens JD, Naficy A, Rao MR. Long term evaluation of vaccine protection methodological issues
for phase 3 trials and phase 4 studies. In: Levine MM, Woodrow GC, Kaper JB et al, eds. New
generation vaccines. New York, NY: Marcel Dekker 1997:47-67.
19. Georges AM. Regulatory aspects of clinical trials with vaccines. In: Plotkin S, Brown F, Horaud F,
eds. Preclinical and Clinical Development of New Vaccines. Dev Biol Stand Basel: Karger
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20. Committee for Proprietary Medicinal Products. Note for Guidance on Clinical Evaluation of New
Vaccines. CPMP/EWP/463/97.
21. Guidance for industry for evaluation of combination vaccines for preventable diseases: Production,
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22. Points to consider on plasmid DNA vaccines for preventive infectious disease indications. FDA,
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23. Guidance for human somatic cell therapy and gene therapy. FDA, CBER March 1998.
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25. Kayhty H. Immunogenicity Assays and surrogate markers to predict vaccine efficacy. In: Plotkin S,
Brown F, Horaud F, eds. Preclinical and Clinical Development of New Vaccines. Dev Biol Stand.
Basel, Karger, 1998; 95:175-180.
26. Kaul D, Ogra PL. Mucosal responses to parenteral and mucosal vaccines. In: Plotkin S, Brown F,
Horaud F eds. Preclinical and Clinical Development of New Vaccines. Dev Biol Stand. Basel,
Karger, 1998; 95:141-146.
27. Hwang IK, Morikawa T. Design issues in noninferiority/equivalence trials. Drug Information Journal
1999; 33:12205-1218.
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vaccination. Pediatrics 1999; 104:575.
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36. Robinson HL, Ginsberg HS, Davis HL et al. The scientific future of DNA for immunization.
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37. Nichols WW, Ledwith BJ, Manam SV et al. Potential DNA vaccine integration into host cell
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CHAPTER 3

Vaccine Regulatory Issues

Marion F. Gruber, Paul G. Richman and Julianne C. M. Clifford

Introduction

T
he fundamental goals in developing new vaccine technologies are to improve current
vaccines for existing clinical indications and to develop new immunogens for both
pediatric and adult use. Recent progress in the field of recombinant DNA technology
and advances in basic immunology have accelerated the development of novel vaccine approaches
to modulate the immune response. Examples include non-replicating antigen delivery systems,
genetically modified vectors expressing foreign antigens, DNA vaccines, as well as the use of
novel adjuvants and different modes of vaccine administration.
The diversity and complexity of these products present new regulatory challenges, because
specific standards for the criteria of safety, purity and potency for these products may not exist
and current experience largely relies on animal models. The problem is further complicated by
the variety of regulatory submissions for prevention of viral, bacterial and parasitic diseases.
Therefore, the safety, purity and potency of these products are evaluated using a case-by-case
approach that is product-specific and indication-based. Consideration is also given to potential
risks versus the benefits of using the product in the target population. It is important to realize
that regulatory policy evolves in response to advances in technologies. Thus, the purpose of this
chapter is to provide a framework for product characterization from which preclinical and
clinical testing strategies will need to be developed according to current knowledge and
state-of-the-art technology.

Federal Regulations Pertaining To Vaccines

In the United States, vaccines are regulated as biological products by the Center for Biologics
Evaluation and Research (CBER) of the U.S. Food and Drug Administration (FDA). The CBER is
currently organized into offices with specific areas of expertise and responsibilities for regulating
biologic products made by manufacturers which are or desire to be licensed for marketing in the
United States. Preventive vaccines and therapeutic vaccines for infectious diseases are reviewed and
regulated by the Office of Vaccines Research and Review (OVRR), whereas most vaccines intended
for therapeutic indications other than infectious diseases are reviewed and regulated by the Office of
Therapeutic Research and Review (OTRR).
Current authority for the regulation of vaccines along with other biologics resides in Section
351 of the Public Health Service Act as well as specific Sections of the Food, Drug and Cosmetic
Act.1 A single set of basic regulatory approval criteria apply to vaccines, regardless of the technology
used in their manufacture. These regulations are contained in Title 21 of the Code of Federal Regu-
lations (21 CFR).2 They include a description of the production, testing and establishment stan-
dards pertaining to the manufacture of a biological product and are intended to assure that the
product is safe and meets the quality and purity characteristics that it claims to possess (Table 3.1).

New Vaccine Technologies, edited by Ronald W. Ellis. ©2001 Eurekah.com.

Another random document with
no related content on Scribd:
Tendovaginitis, gonococcus of, 331
Tenorrhaphy, 324
Tenotomy, 327
Teratomas, 268
embryonal adenosarcoma, 268
of thyroid, 712
Tertiary syphilis, 132
Testicle, absence of, 1015
atrophy of, 1015
cancer of, 1017
chondroma of, 1017
congenital abnormalities of, 1014
contusions of, 1015
cystic degeneration of, 260
cysts of, 1016
epididymitis, 1016
treatment of, 1017
fibroma of, 1017
gonorrhea of, 151
hematoma of, 1015
hydrocele of, encysted, 260
injuries to, 1015
lipoma of, 1017
orchitis, 1017
treatment of, 1017
retained, 1014
treatment of, 1014
syphilis of, 138, 1016
tuberculosis of, 118, 1015
treatment of, 1016
tumors of, 1017
Tetanin, 98
Tetanotoxin, 98
Tetanus, 97
cephalicus, 99
chronic, 100
 death in, 100
diagnosis of, 101
from hysteria, 101
etiology of, 97
hydrophobicus, 99
of newborn, 97, 99
parasitic nature of, 98
postmortem appearances in, 100
prognosis of, 100
toy-pistol, 97
treatment of, 101
Tetany, bacillus of, 54
gastric, 798
Thecitis, 328
Thiersch method of skin grafting, 188
Thigh, amputation of, 1043
above knee, 1044
fracture of, 509
diagnosis of, 511
prognosis of, 511
treatment of, 512
Thoracentesis, 736, 746
Thoracic duct, injuries to, 725
treatment of, 726
viscera, injuries to, 724
walls, diseases of, 726
Thoracoplastic operations, 748
Thoracotomy, 747
drainage in, 747
irrigation in, 747
Thorax, actinomycosis of, 729
carcinoma of, 730
chondroma of, 730
fibroma of, 729
granuloma of, 729
injuries to, 721
 lipoma of, 729
malformations of, 718, 719
operations on, 746
osteoma of, 730
sarcoma of, 730
tumors of, 729
treatment of, 730
wounds of, gunshot, 230
Thrombo-arteritis, 91
Thrombophlebitis, 37, 90
Thrombosis, 34
annular, 35
causes of, 35
following abdominal operations, 784
gangrene from, 73
infective, 36, 570
marasmic, 36, 570
mechanical, 36
of mesenteric vessels, 938
obstructive, 36
parietal, 35
primary, 35
propagated, 36
sinus, 570
diagnosis of, 571
prognosis of, 571
symptoms of, 570
treatment of, 573
traumatic, 36
valvular, 35
Thrombus, calcification of, 36
decolorization of, 36
organization of, 36
softening of, 37
Thrush, oïdium albicans of, 657
Thumb, amputation of, 1029
Thymic asthma, 163
Thymus, hypertrophy of, 717, 751
inflammation of, 717
Thyroglossal duct, 710
Thyrohyoid cysts of neck, 707
Thyroid arteries, inferior, ligation of, 353
body, adenoma of, 712
bronchocele, 712
congenital affections of, 710
endothelioma of, 712
goitre of, 712
hypertrophy of, acute idiopathic, 711
intra-uterine, 711
sarcoma of, 712
struma of, 712
teratomas of, 712
tumors of, 711
dermoids, 267
Thyroidectomy, 715
Thyroidism, 82
Thyroiditis, 711
Thyroids, accessory, 710
Thyrotomy, 674, 688
Tibia, dislocations of, 543
fractures of, 518
treatment of, 521
Tibial arteries, ligation of, 360
nerve, operations on, 623
Tibiotarsal amputations, 1037
Tic douloureux, 640
Toe-nail, ingrowing, 318
Toes, amputation of, 1034
hammer, 321
treatment of, 321
Tongue, absence of, 652
actinomycosis of, 659
 bifid, 652
cysts of, retention, 659
epithelioma of, 660
treatment of, 660
gangrene of, 659
inflammation of, 658
leukoplakia of, 659
treatment of, 659
macroglossia of, 660
malformations of, 652
nevi of, 659
operations on, 661
Kocher’s, 661
Langenbeck’s, 662
Regnoli-Billroth’s, 661
Sédillot’s, 662
Whitehead’s, 661
papilloma of, 659
ranula of, 660
syphilis of, 659
-tie, 652
tuberculosis of, 659
tumors of, 659
Tonometer, use of, 177
Tonsillotomy, 663
Tonsils, absence of, 662
calculi of, 663
enlarged, 662
foreign bodies in, 663
hypertrophy, 662
infection through, 49
syphilis of, 662
tuberculosis of, 662
tumors of, 664
Torsion, control of hemorrhage by, 236
of omentum, 935
Torticollis, 457
diagnosis of, 458
pathology of, 457
treatment of, 458
Tourniquet for control of hemorrhage, 234
Toxic antiseptics, 175
Toy-pistol tetanus, 97
Trachea, operations on, 691
rupture of, 699
scabbard, 713
tumors of, 687
wounds of, 699
Tracheal tugging, 345
Tracheocele, 707
Tracheotomy, 691
Trachoma, 599
Transfixion suture, 241
Transfusion of blood, 185
Transhyoid pharyngotomy, 664
Transplantation of bone, 431
of tendons, 324
Transudates, 23
Trauma as cause of tumor, 255
Traumapnea, 724
Traumatic abscess of brain, 567
erysipelas, 93, 94
fever, 85. See Surgical fever.
hematoma of scalp, 218
hernia, 890
insanity, surgical treatment of, 582
intraventricular hemorrhage, 564
mania, 175
neuroma, 280
othematoma, 605
peritonitis, 786
spondylitis, 462
 thrombosis, 36
Treatment of abscess, 60
of bone, 426
of brain, 573
of liver, 912
of rectum, 879
of actinomycosis, 110
of acute catarrh of biliary passages, 918
cholecystitis, 921
pancreatitis, 948
after abdominal operations, 777
of adenoids of pharynx, 680
of aneurysm of abdominal aorta, 346
of angioma of veins, 367
of ankylosis, 405
of anthrax, 107
of arthritis, chronic, 386
deformans, 389
tuberculous, 398
of atrophy of muscles, 332
of biliary calculi, 926
of boils, 304
of bow-leg, 465
of bunions, 311
of burns, 301
x-ray, 304
of carbuncle, 305
of carcinoma, 295
of breast, 763
of intestines, 828
of rectum, 887
of stomach, 803
of cardiospasm, 798
of caries of hip, 454
of cerebral palsies, 478
of cervical lymph-node affections, 706
of chancre, 128
of chancroid, 145
of cholelithiasis, 926
of chondroma, 272
of chronic affections of pancreas, 951
pancreatitis, 950
prostatitis, 995
sapremia, 87
tendosynovitis, 322
of cold abscess, 114
peri-articular, 399
of compression of brain, 562
of concussion of brain, 559
of chest, 722
of spine, 629
of congenital anomalies of neck, 698
club-foot, 466
dislocation of hip, 474
of congestion, 23
of contraction of fasciæ, 320
of muscles, 332
of contusions, 212
of brain, 560
of chest, 722
of cryptorchidism, 1014
of curvature of spine, 460
of cutaneous horns, 311
of cystitis, 985
of cysts of pancreas, 952
of skin, 310
of dacryocystitis, 600
of delirium tremens, 174
of dental caries, 665
of dermatitis calorica, 299
of desmoids, 271
of dilatation of stomach, 796
of dislocations, 527
of clavicle, 529
of elbow, 536
of foot, 544
of hip, 539
of jaw, 528
of knee, 544
metacarpophalangeal, 537
of patella, 543
of shoulder, 532
of spine, 632
of duodenal ulcers, 826
of Dupuytren’s contraction, 320
of ectopia of bladder, 978
of epididymitis, 1017
of epistaxis, 681
of epithelioma of skin, 315
of tongue, 660
of erysipelas, 95
of exophthalmic goitre, 714
of exophthalmos, 594
of exstrophy of bladder, 978
of fat embolism, 40
of fibroma molluscum, 313
of fistula, 63
of rectum, 880
of floating liver, 911
of foreign bodies in esophagus, 740
in pharynx, 673
in stomach, 794
of fractures, 486
of clavicle, 493
Colles’, 504
of femur, 513
of fibula, 521
of forearm, 501
 of humerus, 495
of inferior maxilla, 490
of leg, 521
of patella, 517
of pelvis, 508
of radius, 504
of ribs, 492
of skull, base, 558
vertex, 555
of sternum, 492
of thigh, 512
of tibia, 521
of ulna, 501
of frostbite, 302
of furuncle, 304
of gallstones, 926
of gangrene, 77
of gastric ulcer, 800
of gastroptosis, 797
of glanders, 106
of glaucoma, 597
of gonorrhea, 152
complications, 152
of testicles, 151
in women, 159
of hammer-toe, 321
of hematomyelia, 634
of hematorrhachis, 634
of hematuria, 959
of hemorrhage, 234
secondary, 237
of hemorrhoids, 883
of hernia, 898
of brain, 566
femoral, 908
inguinal, 908
 postoperative, 909
umbilical, 909
ventral, 909
of Hodgkin’s disease, 378
of hydatid cysts of liver, 913
disease, 432
of hydrocele, 1018
of hydrocephalus, 579
of hydronephrosis, 973
of hydrophobia, 104
of hypertrophy of prostate, 998
of hysterical joints, 392
of injuries of eyeball, 604
of orbit, 593
to thoracic duct, 726
to upper nerve trunks, 726
of intestinal obstruction, acute, 836
chronic, 839
of intraspinal hemorrhage, 634
of intussusception, 832
of invagination of rectum, 882
of keloid, 271, 313
of keratosis of skin, 314
of knock-knee, 464
of laceration of lung, 722
of leptomeningitis, 573
of leukoplakia, 659
of lock finger, 320
of lymphangioma, 279
circumscriptum, 374
of skin, 315
of lymphangitis, 375
of Madura foot, 110
of malformation of esophagus, 738
of respiratory passages, 671
of malignant edema, 108
of mastitis, 758
of mastodynia, 758
of mediastinitis, 728
of melanoma of skin, 317
of meningitis, 572
of metatarsalgia, 470
of Morton’s disease, 471
of movable and floating kidney, 967
bodies in joints, 402
of myalgia, 331
of necrosis of bone, 428
of neoplasm of nasal cavities, 678
of neuralgia of breast, 758
of neuropathic disease of joints, 392
of orchitis, 1017
of osteo-arthritis, 389
of osteomalacia, 435
of osteomyelitis, 419
of panophthalmitis, 595
of papilloma, 283
of paralytic affections of muscles, 332
of perforations of biliary passages, 922
of perineal abscess, 1013
fistulas, 1013
of perinephritis, 961
of periostitis, 421
of perirectal abscess, 879
of peritonitis, 787
of phlegmonous affections of rectum, 879
of phlegmons of neck, 703
of piles, 883
of pneumatocele of scalp, 546
of Pott’s disease, 448
of procidentia, 882
of proctitis, 875
of prolapse of brain, 566
 of rectum, 882
of pruritus ani, 879
of pyelitis, 957
of pyelonephritis, 957
of pyemia, 92
of pyonephrosis, 961
of renal calculus, 966
colic, 964
of retained testicle, 1014
of rhinophyma, 314
of rickets, 162
of rupture of diaphragm, 722
of muscles, 330
of sacro-iliac disease, 452
of sapremia, 87
of sarcoma of bone, 441
of scalds, 301
of scleroderma, 313
of scoliosis, 460
of scurvy, 160
of septic nephritis, 957
of septicemia, 89
of sequestrum formation, 429
of shock, 179
of sinus, 63
thrombosis, 573
of snake-bites, 171
of spina bifida, 626
of spondylolisthesis, 463
of sprains, 380
of spinal column, 629
of status lymphaticus, 165
of stenosis of pylorus, 799
of strictures of intestines, 828
of larynx, 684
of rectum, 877
of subphrenic abscess, 754
of suppurative tendosynovitis, 321
of surgical kidney, 957
of sympathetic ophthalmitis, 596
of synovitis, 385
acute, 383
chronic, 386
purulent, 385
of syphilis, 140
of talipes equinovarus, 466
equinus, 471
valgus, 469
of tetanus, 101
of torticollis, 458
of traumatic insanity, 582
of trigger finger, 320
of tuberculosis, 120
of bone, 425
of breast, 759
of joints, 398
of knee, 456
of kidneys, 963
of larynx, 685
of mesentery, 939
of peritoneum, 791
of skin, 307
of testicle, 1016
of tuberculous hydrops, 399
ulcers of intestines, 827
of tumors, 258
of bladder, 992
of brain, 585
of breast, 760
of larynx, 687
of spinal cord, 622
of thorax, 730
 of typhoid ulcers of intestines, 826
of ulcerations of biliary passages, 922
of varices, 365
of varicocele, 1021
of varicose veins, 365
of vascular growths of skin, 314
of vesical calculus, 987
of warts, 312
venereal, 312
of wounds, 238
of chest, 723
contused, 212
of diaphragm, 726
gunshot, 225
of head, 565
of heart, 335
of intestines, 824
of larynx, 676
of lung, 726
open, 239
of pleura, 726
punctured, 214
of wryneck, 458
of xanthoma of skin, 314
Trephining of cranium, 587
Trichiasis, 601
Trigger finger, 320
treatment of, 320
Trismus, 97. See Tetanus.
Tropacocaine, 207
Trophoneuroses, 310
Trophoneurotic atrophy, 27
diseases of bone, 432
Tuberculides, 307
Tuberculin in tuberculosis, 121
dose of, 121
 manner of preparation, 121
Tuberculosis, 111
of abdominal wall, 783
of axilla, 751
bacillus of, 54
of bladder, 118
of bone, 116, 422
acute miliary, 423
chronic tuberculous osteomyelitis, 423
pathology of, 423
symptoms of, 423
treatment of, 425
of breast, 759
treatment of, 759
of bursæ, 118
of choroid, 595
cold abscess in, 112
diagnosis of, 113
lumbar, 114
psoas, 114
retropharyngeal, 114
treatment of, 114
colon, 869
degenerative changes in, 111
diagnosis of, 120
from epithelioma, 293
of face, 640
fungous granulation in, 115
giant cells in, 111
gummas in, 114
of joints, 393
diagnosis of, 398
pathology of, 394
symptoms of, 397
treatment of, 398
of kidney, 118, 961
 diagnosis of, 962
symptoms of, 961
treatment of, 963
of knee-joint, 456
treatment of, 456
of larynx, 684
treatment of, 685
lupus, 115
of lymph nodes, 376
cervical, 705
of lymphatic structures, 116
of mesentery, 939
treatment of, 939
method of healing, 112
absorption, 112
calcification, 112
caseation, 112
encapsulation, 112
suppuration, 112
miliary tubercles in, 111
of mouth, 657
of mucous membrane, 115
of muscles, 331
of ovary, 118
paths of infection, 119
of peritoneum, 116, 118
of rectum, 876
of seminal vesicles, 1021
of skin, 115, 306
folliculitis, 307
lupus vulgaris, 306
painful subcutaneous, 313
scrofuloderm, 306
treatment of, 307
verruca necrogenica, 306
verrucose, 306
 of spermatic cord, 1019
of stomach, 795
of tendon sheaths, 118
of testicle, 118, 1015
treatment of, 1016
of tongue, 659
of tonsils, 662
treatment of, 120
constitutional, 120
tuberculin in, 121
local, 120
amputation in, 121
Bier’s permanent hyperemia, 120
excision in, 121
ignipuncture in, 121
iodoform injections in, 120
Tuberculous cystitis, 985
hydrops, treatment of, 399
panarthritis, 395
peritonitis, 786, 790
ascitic, 791
fibrinoplastic, 791
treatment of, 791
ulcerative, 791
ulcers of intestines, 827
symptoms of, 827
treatment of, 827
Tubulo cysts, 260
allantoic, 260
of vitello-intestinal duct, 260
of Wolffian body, 260
dermoids, 266
Tumid ulcer, 67
Tumors, 255
of abdominal wall, 783
vascular, 784
albus, 393, 456
of bladder, 992
symptoms of, 992
treatment of, 992
of bone, 438
cartilaginous, 440
of brain, 582
symptoms of, 583
treatment of, 585
of breast, 760
treatment of, 760
of cheek, 641
classification of, 259
connective tissue, 269
cysts, 259
dermoids, 264
epithelium, 281
of nerve elements, 279
teratomas, 268
clinical observations of, 257
coccygeal, 627
congenital, 635
comparative pathology of, 256
connective tissue, 269
angioma, 277
chondroma, 271
exostoses, 272
fibroma, 269
lipoma, 269
lymphangioma, 278
myoma, 277
myxoma, 276
osteoma, 272
sarcoma, 272
cysts, 259
glandular, 261
 hydrocele, 261
pseudo-, 262
retention, 259
tubulo, 260
derived from epithelium, 281
adenoma, 284
carcinoma, 289
epithelioma, 286
malignant chorion, 292
suprarenal, 292
fibro-adenoma, 284
fibro-epithelioma, 282
goitre, 283
hydronephroma, 292
mucous polyp, 283
odontoma, 281
ovarian cystoma, 284
papilloma, 282
struma, 283
dermoids, 264
ovarian, 267
sequestration, 265
tubulo, 266
of diaphragm, 753
of ear, 605
embryonal hypothesis of Cohnheim, 256
of face, 641
of gall-bladder, 927
gangrene from, 74
of heart, 336
heredity of, 256
inflammation as cause of, 256
inoculation experiments on, 257
of intestines, 828
intralaryngeal, 686
intra-ocular, 594
intratracheal, 686
irritation as cause of, 255
of jaw, 668
of kidney, 969
of larynx, 686
of liver, 914
local infectivity of, 257
of lung, 732
of lymphatics, 378
metastasis of, 257
microscopic appearances of, 257
of neck, 706
of nerve elements, 279
glioma, 279
neuroma, 280
of nerves, 622
nomenclature of, 258
of omentum, 935
of optic nerve, 593
of orbit, 593
cystic, 593
vascular, 593
of pancreas, 953
parasitic theory of, 256
of rectum, 885
of salivary glands, 650
of scalp, 546
acquired, 546
congenital, 546
gaseous, 545
of skin, benign, 311
malignant, 315
of spinal cord, 621
diagnosis of, 622
symptoms of, 621
treatment of, 622
 of stomach, 801
of teeth, 665
teratomas, 268
of testicle, 1017
of thorax, 729
treatment of, 730
of thyroid, 711
of tongue, 659
of tonsils, 664
of trachea, 687
trauma as cause of, 255
treatment of, 258
of operable, 259
Typhoid fever, secondary infection in, 167
spine, 462
ulcer of intestines, 826
symptoms of, 826
treatment of, 826
of rectum, 876

U
Ulcer, 65
atheromatous, 339
of auricle, rodent, 606
of biliary passages, 921
symptoms of, 922
treatment of, 922
callous, 67
causes of, 65
constitutional, 66
local, 65
traumatic, 65
duodenal, 825
symptoms of, 825
 treatment of, 826
edematous, 67
erethistic, 66
of face, 640
fissured, 66
fistulous, 66
of foot, perforating, 310
fungous, 66
gastric, 799
diagnosis of, from appendicitis, 857
operations for, 811
symptoms of, 800
treatment of, 800
healthy, 66, 67
hemorrhagic, 66
indolent, 66
indurated, 67
inflamed, 67
of intestines, 825
cancerous, 827
diagnosis of, from appendicitis, 857
duodenal, 825
treatment of, 826
dysenteric, 827
tuberculous, 827
treatment of, 827
typhoidal, 826
symptoms of, 826
treatment of, 826
irregular, 66
irritable, 66
livid, 67
peptic, of jejunum, 816
phagedenic, 66
process of repair in, 67
of rectum, 876
 catarrhal, 876
chancroidal, 876
symptoms of, 877
syphilitic, 876
treatment of, 877
tuberculous, 876
typhoid, 876
regular, 66
rodent, 66, 67, 288
sloughing, 66
treatment of, 70
tumid, 67
undermined, 67
of urethra, 1011
venereal, 144. See Chancroid.
Ulceration, 65. See Ulcers.
Ulcerative endocarditis in septicemia, 88
gingivitis, 664
stomatitis, 657
tuberculous peritonitis, 791
Ulna, dislocations of, 536
fractures of, 501
treatment of, 501
Ulnar artery, ligation of, 356
nerve, operations on, 623
Umbilical hernia, 896
treatment of, 909
vein, phlebitis of, 362
Upper extremity, amputations of, 1029
entire, 1033
Uranoplasty, 654
Urea in auto-intoxication, 80
Ureteral catheterization, 958
Ureters, anomalies of, 955
calculus of, 973
operations on, 976
 stricture of, 973
Urethra, congenital defects of, 1004
epispadias, 1005
foreign bodies in, 1009
hypospadias, 1005
injuries of, 1008
lacerations of, 1008
strictures of, 1011
ulcers of, 1011
Urethritis, 147
gonorrheal, in women, 159
Urethrometer, 155
Otis’, 1012
Urethrotome, Otis’ dilating, 157, 1012
Urine, electroconductivity of, 959
incontinence of, 982
retention of, 982
suppression of, 982
Uveitis, 596, 598
Uvula, elongation of, 682
syphilis of, 683

V
Valvular thrombosis, 35
Vanghetti’s cinematic method of amputation, 1048
Varices, 364
congenital, 364
symptoms of, 365
treatment of, 365
Varicocele, 364, 1019
treatment of, 1021
Varicose aneurysm, 339, 342
veins, 363
extirpation of, 366
 symptoms of, 365
treatment of, 365
Variola, secondary infection in, 169
Varix, anastomotic, 364
aneurysmal, 363
compound, 367
Vascular growth of skin, 314
treatment of, 314
system, surgical diseases of, 334
syphilis of, 135
Veins, air embolism of, 363
angioma of, 366
treatment of, 367
atrophy of, 361
calcification of, 361
fatty degenerations of, 361
hemorrhoidal, phlebitis of, 362
hypertrophy of, 361
inflammation of, 361
injuries of, 217, 363
treatment of, 364
mesenteric, phlebitis of, 362
rupture of, 363
umbilical, phlebitis of, 362
varicose, 363
Velpeau’s bandage, 189
Venereal ulcer, 144. See Chancroid.
warts, treatment of, 312
Venesection, 182
Venous nevus, 367
congenital, 367
Ventral hernia, 896
treatment of, 909
Vermiform appendix, actinomycosis of, 852
anatomy of, 851
cysts of, 852
 diseases of, 851
empyema of, 860
foreign bodies in, 852
inflammation of, 851
tuberculosis of, 852
tumors of, 852
Verruca necrogenica, 306
Verrucæ, 311
Vertebral artery, ligation of, 353
Vesical calculus, 986
symptoms of, 986
treatment of, 987
diverticula, 262
Vesicants, counterirritation by, 184
Vesicovaginal fistula, 839
Villous papillomas, 282
intracystic, 282
Vincent’s angina, 703
Viscera, wounds of, 219
Vitello-intestinal duct, cysts of, 260
Volvulus, 832, 870
von Langenbeck’s incision in excision of elbow, 410
of wrist, 411
von Mosetig-Moorhof’s incision in excision of elbow, 410
Vulva, elephantiasis of, 371
gonorrhea of, 158

W
Wagner’s osteoplastic resection of cranium, 590
Wandering erysipelas, 94
liver, 910
symptoms of, 910
treatment of, 911
Wardrop’s method of treating aneurysms, 347
Warts, 282, 311
filiform, 311
seborrheic, 312
treatment of, 312
venereal, treatment of, 312
Warty horns, 283
Wasps, poisoning by, 172
Weir’s operation for saddle-nose, 644
Wens, 285
White swelling, 393
Whitehead’s operation for hemorrhoids, 884
on tongue, 661
Whitlow, 328
Witzel’s method of gastrostomy, 807
Wolffian body, cysts of, 260
Woodbury’s method of amputation of hip-joint, 1045
Woolsorters’ disease, 106. See Anthrax.
Wounds of abdominal wall, gunshot, 783
penetrating, 781
of bladder, 981
of chest, 722
treatment of, 723
contused, 211
of bloodvessels, 216
of tendons, 218
treatment of, 212
of viscera, 219
of diaphragm, 725, 753
treatment of, 726
of esophagus, 741
of face, 639
fever, aseptic, 85. See Surgical fever.
gunshot, 220
of abdomen, 232
of bladder, 233
diagnosis of, 225
 of face, 229, 639
foreign material in, 224
of head, 228, 565
of heart, 231
hemorrhage from, 223
of intestines, 823
of joints, 228, 381
key-hole, 225
of kidney, 233
localizing symptoms of, 233
multiple, 224
of neck, 229
pain from, 223
prognosis of, 225
of respiratory passages, 675
shock from, 223
of small intestines, 823
of spinal cord, 624
of spine, 230, 624
of spleen, 233
of thorax, 230
treatment of, 225
of heart, 334, 733
suture of, 335 (note)
treatment of, 335
incised, 214
of bloodvessels, 216
of skull, 552
of intestines, 823
lacerated, 212
of bloodvessels, 216
of muscles, 218
of larynx, 675
treatment of, 675
of liver, 911
of lung, 724
 treatment of, 726
of mouth, 658
of muscles, 330
of neck, 698
of nerves, 612
open, treatment of, 239
of pancreas, 945
penetrating, of joints, 381
of chest, 722
of orbit, 592
of skull, 552
of spinal cord, 624
of spine, 624
of stomach, 805
of pleura, 724
treatment of, 726
punctured, 213
Wounds, punctured, of abdomen, 214
treatment of, 213
repair of, 215
of respiratory passages, 675
of stomach, 794
of trachea, 699
treatment of, 238
drainage in, 239
Wrist, amputation of, 1031
dislocations of, 536
excision of, 411
fractures of, 507
Wryneck, 457
diagnosis of, 458
pathology of, 457
treatment of, 458
Wyeth’s exsector in excision of joints, 409
method of amputation of hip-joint, 1045