Table Of ContentVaccines
New Generation Immunological Adjuvants
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Volume 282 - Vaccines: New Generation Immunological Adjuvants
edited by Gregory Gregoriadis, Brenda McCormack,
and Anthony C. Allison
~
Series A: Life Sciences
Vaccines
New Generation Immunological Adjuvants
Edited by
Gregory Gregoriadis
and
Brenda McCormack
School of Pharmacy
University of London
London, England
and
Anthony C. Allison
Dawa Corporation
Belmont, California
Plenum Press
New York and London
Published in cooperation with NATO Scientific Affairs Division
Proceedings of a NATO Advanced Study Institute on
Vaccines: New Generation Immunological Adjuvants,
held June 24 - July 5, 1994,
in Cape Sounion, Greece
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Library of Congress Cataloging-in-Publication Data
Vaccines new generation immunological adjuvants I edited by Gregory
Gregoriadis and Brenda McCormack and Anthony C. All ison.
p. em. -- (NATO ASI series. Series A. Life sciences; v.
282)
"Proceedings of a NATO Advanced Study Institute on Vaccines: New
Generation Immunological Adjuvants. held June 24-July 5. 1994. in
Cape Sounion. Greece"--T.p. verso.
"Published in cooperation with NATO Scientific Affairs Division."
Includes b1bliographical references and index.
ISBN-13: 978-1-4613-8014-6 e-ISBN-13: 978-1-4613-0357-2
001: 10.1007/978-1-4613-0357-2
1. Vacc1nes--Congresses. 2. Immunological adjuvants--Congresses.
I. Gregoriadis. Gregory. II. McCormack. Brenda. III. Allison.
Anthony C. (Anthony Clifford). 1925- IV. North Atlantic Treaty
Organization. Scientific ~ffairs Division. V. NATO Advanced Study
Institute on Vaccines: New Generation Immunological Adjuvants (1994
Akra Sounion. Greece) VI. Serles.
[ONLM: 1. Vacc; nes--; mmuno 1o gy--congresses·. 2. Adjuvants.
Im~unolog1c--therapeut1c use--congresses. 3. Immunotherapy. Active
-congresses. OW 805 V11647 1996]
OR189. V28 1996
615· .372--dc20
ONLM/OLC
for Library of Congress 96-4871
CIP
ISBN-13: 978-1-4613-8014-6
© 1995 Plenum Press, New York
Softcover reprint of the hardcover 1st edition 1995
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otherwise, without written permission from the Publisher
PREFACE
During the last decade or so vaccine development has been facilitated by rapid
advances in molecular and cell biology. These have laid the foundations of a new
generation of vaccines exemplified by subunit vaccines produced through gene cloning and
by synthetic peptides mimicking small regions of proteins on the outer coat of viruses.
Such peptide~ are capable of eliciting virus-neutralizing antibodies. Unfortunately, subunit
and peptide vaccines are only weakly or non immunogenic in the absence of immunological
adjuvants that are known to augment specific cell-mediated immune responses to the
antigens and to promote the formation of protective antibodies.
This book contains the proceedings of the 4th NATO Advanced Studies Institute
(ASI) "Vaccines: New Generation Immunological Adjuvants" held at Cape Sounion Beach,
Greece, during 24 June - 5 .July 1994 and deals in depth with both theoretical and practical
aspects of vaccinology. These include the role of antigen presenting cells in the induction
of immune responses. immunopotentiation by a variety of new generation immunological
adjuvants and vaccine carriers. and recent advances and perspectives in experimental
vaccines as well as vaccinatioll with nucleic acids. We express our appreciation to
Dr. K. Dalsgaard and Dr. J.L. Virelizier for their cooperatioll in planning the ASI and to
Mrs. Concha Pening for her excellent production of the manuscripts. The ASI was held
under the sponsorship of NATO Scientific Affairs Division and generously co-sponsored by
SmithKline Beecham Pharmaceuticals (Philadelphia). Financial assistance was also
provided by Sandoz (Hellas) (Athens), Biochime (Siena), and Sequus Pharmaceuticals
(Menlo Park, California).
Gregory Gregoriadis
Brenda McCormack
Anthony C. Allison
v
CONTENTS
Adjuvants for new and improved vaccines ..................................................................... .
A.C. Allison
Liposome mediated immunopotentiation and immunomodulation ................................. 15
N. van Rooijen
Quil A, anti-carbohydrate antibody specificity and biological function ........................ 25
H. Snippe, E. Alonso de Velasco, A.F.M. Verheul and J.T. Poolman
Vaccine adjuvants based on gamma inulin ...................................................................... 35
P.D. Cooper
Interleukin-2 as a co-adjuvant for liposomal tetanus toxoid ........................................... 45
M. Glirsel and G. Gregoriadis
Design of vaccines for the induction of antibody responses in Th-cell
deficient individuals ...................................................................................................... 51
B. Golding, J. Inman and H. Golding
Strategies for the stimulation of Th cell subsets ............................................................ 65
H. Golding, M.B. Zaitseva, C. Lapham and B. Golding
Feline immunodeficiency virus as a vaccine model ....................................................... 85
M.J. Hosie and o. Jarrett
Rational design of vaccine mqlecules to prevent pertussis ............................................. 97
M.T. De Magistris, A. Di Tommaso, M. Pizza and R. Rappuoli
Muiticomponem viral vaccines and their use as immunogen
delivery systenls ...... ........................... ........................ ...... ........ ........ .......... ........ ...... .... 103
P. Roy
Vaccines against HPV of the uterine cervix: Problems in clinical
applicability .................................................................................................................. 117
P. Hirnle and W. Erz
Local chemotherapy of lymph node metastases: Optimization of
targeting accuracy ........................................................................................................ 123
P. Hirnle and W. Erz
Synthetic peptide vaccines: Success at last ..................................................................... 127
R.H. Meloen, J.1. Casal, K. Dalsgaard and 1.P.M. Langeveld
Peptide vaccines: New approaches to immunopotentiatlon ............................................ 135
MJ. Francis
vii
Genetic restriction of responses to peptide antigens ....................................................... 141
MJ. Francis
DNA-based immunization: Prospects for a hepatitis B vaccine ..................................... 147
H.L. Davis and R.G. Whalen
Characterization of immune responses elicited by an experimental
facilitated-DNA vaccine for human immunodeficiency virus
type-1 (HIV-l) .............................................................................................................. 161
M.J. Newman, L. Cooney, R. Carrano, J. Boyer, W.V. Williams,
B. Wang and D.E. Weiner
Recombinant self-replicating RNA vaccines ................................................................... 173
P. Liljestr5m
Participant's Photograph .................................................................................................. 181
Contributors .................................... .................................................................................. 183
Index ................................................................................................................................. 185
viii
ADJUVANTS FOR NEW AND IMPROVED VACCINES
Anthony C. Allison
Dawa Corporation
,Belmont CA U.S.A.
INTRODUCTION
This series of NATO Advanced Studies Institutes is intended to bridge the widening
gap between basic immunobiology and its application to vaccination. There has been a
rapid expansion of knowledge about subsets of lymphocytes and accessory cells, the
chemistry of immunoglobulins, cytokines, adhesion molecules and the complex interactions
required for cellular and humoral responses to antigenic stimulation. A great deal of
information has accumulated about the structure of bacterial, viral and other antigens.
Many of these can be produced by recombinant DNA technology or peptide synthesis. Yet
the practice of vaccination has changed very little during the past decade. New live virus
vaccines have been introduced, including attenuated varicella-zoster virus (Ok a strain of
VZV, Takahashi, 1990). However, this strain, and other live viruses, can produce severe
infections in immunocompromized recipients (Gershon et aI., 1984). It is now recognized
that immunodeficiency is commoner than formerly believed, for nutritional and other
reasons (Chandra, 1991). Hence live viruses and bacteria, including vectors of antigens,
may have undesirable effects in some recipients, and emphasis is now placed on optimizing
the efficiency of subunit vaccines.
Two examples will ill4strate the discrepancy between theory and practice. On one
hand, the three-dimensional structure of the haemagglutinin (HA) of influenza virus has
been established by X-ray crystallography, and epitopes recognized by antibodies and T
lymphocytes have been characterized in exquisite detail (Wilson and Cox, 1990; Burt et aI.,
1993). On the other hand, the saline HA used in vaccination cannot be regarded as a
satisfactory vaccine. In humans over the age of 65, influenza, with secondary bacterial
infections, can be a serious disease. Vaccination with HA of the prevalent strain of
influenza virus is recommended for persons in that age group. However, only a minority of
elderly recipients of HA show fourfold increases in circulating antibody titres (Arden et aI.,
1986). The need for a better influenza vaccine is obvious.
Infection with hepatitis B virus (HBV) is still a major global health problem. In
North America and Europe the infection is especially important in susceptible groups of
persons, including intravenous drug abusers. In many Asian and African countries the
frequency of HBV infection and of carriers remains high. Persistent HBV infection is
associated with chronic hepatitis, cirrhosis and hepatocellular carcinoma (Beasley and
Hwang, 1984). In Asian countries HBV is transmitted by mothers to their infants during
the neonatal period, and vaccination of infants can prevent such early infections, which
often persist (Beasley et aI., 1983). The HBV vaccine'as currently used consists of three
doses in HBsAg in alum adjuvant. The antigen can be serum-derived or produced by
Vaccines: New-Generation Immunological Adjuvants
Edited by G. Gregoriadis et aI., Plenum Press, New York, 1995
recombinant technology. Recombinant HBsAg is expensive to produce, and reducing the
dose required to elicit protection by a factor of ten would increase the availability of the
vaccine. Administering two doses instead of three would make vaccination more
convenient. Augmenting responses is also needed: currently used vaccines produce
seroconversion in more than 90% of healthy North American and European adults, but
responses are lower in newborn children, the elderly, and intravenous drug users. Some
persons, especially of certain HLA haplotypes, have low inherited responsiveness to HBsAg
(Walker et a!., 1981; Alper et a!., 1989), as do mice of some haplotypes (Milich et a!.,
1983).
Improvement of currently used subunit vaccines and the development of new
vaccines requires three components: methods for the production of subunit antigens,
optimization of carriers and the introduction into human vaccines of adjuvants that elicit
better and more consistent immune responses, even when these are suboptimal because of
the age or genetic constitution of the recipient. Other possibilities have also attracted
attention, including delayed release of antigens to overcome the need for more than one
injection, and DNA vaccines. Such approaches are still in the experimental stage, but will
be discussed at this conference.
DEFINITIONS
An adjuvant is a substance or procedure which augments specific immune responses
to antigens. A carrier is an immunogenic molecule which, when bound to a second
molecule, augments immune responses to the latter. Examples of carriers are the protein
components of glycoconjugates that increase antibody responses to bacterial capsular
polysaccharides and proteins bound to peptides which increase anti-peptide responses. A
carrier is thus distinguished from a vehicle, which is a two-phase system that transports
antigens from injection sites to lymphoid tissues. Examples of vehicles are liposomes and
microfluidized squalene emulsions. Adjuvants frequently contain immunomodulators, which
induce the production of cytokines and augment immune responses. Examples are muramyl
peptides, lipopolysaccharides and derivatives, and certain cationic detergents. The
combination of an immunomodulator and a vehicle, to optimize activity, is an adjuvant
formulation.
ANTIGENS AND CARRIERS
The prototype recombinant antigen is the surface antigen of hepatitis B virus
(HBsAg) cloned and expressed in yeast in a form physically and antigenically resembling
the 22 nm particles in serum (Murray et aI., 1984; Valenzuela et a!., 1982). Recombinant
HBsAg has been shown to have immunogenicity in humans comparable to that of HSsAg
derived from serum (Scolnick et a!., 1984), and it is now approved for human use by
regulatory authorities in several countries. In principle it is possible to produce a wide
range of antigens by recombinant DNA technology, although optimal expression systems
vary with different antigens. For example, HBsAg in not readily produced in Escherichia
coli, which is, however, a good expression system for the nucleocapsid antigen (HBcAg) of
the same virus (McKay et a!., 1981) in the form of particles physically and antigenically
resembling those produced during natural infection. Using suitable expression systems, e.g.,
-E. coli, yeast, baculovirus, or mammalian cells, it is possible to produce protein antigens
with conformations similar to those naturally occurring in many infectious agents. For
example, herpes simplex virus (HSV-2) glycoproteins Band D expressed in mammalian
cells are typically glycosylated and, inoculated with suitable adjuvants, efficiently protect
guinea pigs from genital HSV -2 infections (Byars et aI., 1994).
The power of recombinant technology is illustrated by the identification of
hepatitis C virus (Choo et aI., 1989), the major blood-transmitted variety of non-A, non-B
hepatitis. A recombinant nonstructUl:al protein of HeV is becoming routinely used for
blood tests, and a recombinant surfade antigen is a vaccine candidate.
An example of the improvement of a vaccine by the use of a protein carrier is the
2
glycoconjugate of Haemophilus influenzae type B. This vaccine has shown efficacy in
children (Kayhty et aI., 1991), and is now widely used. Several other bacterial capsular
polysaccharide-protein conjugates are being developed (Lifely, 1993).
Small peptides are not immunogenic, even when administered with adjuvants.
Combining a T- and B-cell epitope in a linear peptide is insufficient for good
immunogenicity. This has been improved by having several repeats of peptides in synthetic
constructs (Francis et aI., 1991; Tam, 1988). However, the most immunogenic peptides
have been those included in genetic constructs of a self-assembling protein, such as the core
antigen of hepatitis B virus (Clarke et aI., 1990) or the Ty virus-like particles of yeast
(Gilmour et aI., 1989). The repeating units of the assembled carrier protein stimulate
T-Iymphocytes efficiently, and the use of an efficacious adjuvant can further increase both
cell-mediated and humoral responses to the co-expressed peptides. The possibilities are
discussed further by Francis at this meeting.
It remains to be determined whether this carrier strategy can overcome an inherent
disadvantage of peptide vaccines: the genetic restriction of responses to peptides. In an
outbred popuJation it is likely that some individuals will be low responders to any peptide.
Since self-assembling protein carriers have multiple epitopes, genetic restriction may be
overcome to some extent. An adjuvant fomlulation might also help.
AFFINITIES AND ISOTYPES OF ANTIBODIES
Traditionally the efficacy of adjuvants has been judged by the levels of antibodies
elicited (using a convenient test, such as ELISA or hemagglutination). While these assays
have provided useful information, they should be supplemented by other measures of the
quantity and quality of antibodies elicited. Preferably, antibody levels should be quantified
by tests relevant to function, such as neutralization of bacterial toxins or viruses, killing of
tumor cells or induced cytostasis. Because of potential problems with solid-phase assays, at
least some measurements of antibody levels using fluid-phase assays should be made. In
addition to the quantities of antibodies elicited by a vaccine, two properties of the
antibodies are likely to be important for protection: their affinity for antigen and their
isotype. To neutralize a virus or bacterial toxin, antibodies should bind them with
sufficiently high affinity. If the complexes are not removed by phagocytic cells, antibodies
must bind to a virus or toxin with an affinity of at least the same order as the natural
receptor. Measurements o(affinities by dissociation from antigen bound to a surface, using
low pH or chaotropic agents, have limitations. In the author's laboratory methods have
been developed for measurement of the quantities and affinities of antibodies in the fluid
phase (Kenney et aI., 1990).
Another important property of antibodies is their isotype. Antibodies of the
immunoglobulin G (IgG) class pass from the vascular to the extravascular compartment
more easily than those of the IgM class; only the former are transferred across the placenta
or by milk to fetuses and newborn animals. Antibodies of some isotypes efficiently activate
complement, bind to high-affinity receptors on monocytes and act synergistically with
antibody-dependent effector cells (ADCC), to produce cytotoxicity. Examples of IgG2a
antibodies in mice and IgG 1 antibodies in humans, both of which bind to high-affinity FcyI
receptors (Unkeless et aI., 1988). Studies with isotype-switch variants of murine
monoclonal antibodies (which have the same Fab regions, so binding to antigen is
comparable) show that IgG2a antibodies confer better protection against tumors than those
of other isotypes (Kaminsky et aI., 1986). Studies with 'reshaped' human antibodies,
genetically constructed to have antigen-binding hypervariable regions like those of rodent
monoclonals, confirm the superiority of the human IgG I isotype in ADCC-mediated lysis
(Reichmann et aI., 1988). The desirability of developing an adjuvant formulation that
preferentially elicits high-affinity antibodies of the IgG2a isotype in mice and IgG I in
humans is apparent.
3
