Advanced Drug Delivery Reviews 57 (2005) 333 – 355

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Immunity in response to particulate antigen-delivery systems

Tazio Storni, Thomas M. Kqndig, Gabriela Senti, P3l Johansen*
Department of Dermatology, University Hospital of Zurich, Gloriastrasse 31, 8091 Zurich, Switzerland
Received 29 March 2004; accepted 1 September 2004
Available online 12 October 2004

Abstract

Adjuvants and antigen-delivery systems are essential in inducing and modifying immune responses, and despite the variety
of materials available for such use, mechanisms by which they support immunity appear to be little known. A common
denominator for most antigen-delivery systems is their particulate nature. Together with a certain depot effect, it is the
particulate nature that primarily decides whether the antigen-delivery system will be successful in inducing an immune
response. If this first requirement is fulfilled, the chemical composition of the vaccine decides which type of immune response
will develop, e.g. which isotype of antibodies the B cells will produce, and which cytokines the T cells will secrete, and can be
controlled by combining the antigen with immunomodulatory or co-stimulatory molecules. It is our goal to provide an overview
of the cellular and molecular factors involved in the induction of immunity and how such factors may influence the potency of
an adjuvant or a vaccine. Such factors should then be implemented in the design of new vaccines or in tuning the properties of
existing vaccines in order to reach the properties that are necessary for successful vaccination.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Innate immunity; Adaptive immunity; Viruses; Bacteria; Particles; Vaccine design

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
2. Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
2.1. Innate immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
2.2. Adaptive immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
2.2.1. Antigen presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
2.2.2. T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
2.2.3. B cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
2.3. Immunological memory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
2.4. The mucosal immune system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
3. Infectious pathogens as models for vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342

* Corresponding author. Tel.: +41 1 255 1111; fax: +41 1 255 4418.
E-mail address: pal.johansen@usz.ch (P. Johansen).

0169-409X/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2004.09.008
334 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355

3.1. Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342

3.2. Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
4. Adjuvants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
5. Designing pharmaceutical antigen-delivery systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
5.1. Targeting professional antigen-presenting cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
5.2. Concomitant delivery of antigen and co-stimulatory signals. . . . . . . . . . . . . . . . . . . . . . . . . 345
5.3. Immunomodulating activities of synthetic polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
5.4. Antigen dose and structure affect the type of immunity induced . . . . . . . . . . . . . . . . . . . . . . 347
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348

1. Introduction tion or delivery system varies considerably between

Although we encounter microorganisms on a
daily basis, this causes disease only occasionally.
Most organisms are detected and destroyed within
hours by defence mechanisms, which are not
antigen-specific and which do not require any
prolonged period of induction. These are the
mechanisms of innate immunity. Only when the
infectious agent is capable to break this early line of
defence, an adaptive immune response will develop,
including generation of antigen-specific effector cells
that specifically target the pathogen, secretion of
antibodies (B cells), through direct cytotoxic activity
(T cells), or secretion of immunological mediators
and effector molecules such as cytokines and
chemokines. Although most of these effector cells
will die within 10–14 days after infection, some cells
will survive as highly reactive plasma cells (B cells)
or memory cells (B and T cells) and prevent
subsequent infection by the same microorganism.
Along with long-lasting antibodies against a specific
pathogen, the induction of memory cells is the final
goal of preventive vaccination.
Immunity against an infectious agent by vaccina-
tion can be achieved in various ways. Firstly, the
antigen itself can be a synthetically produced peptide
representing an epitope of a pathogen protein. It can
also be the full-length protein carrying several
epitopes that are recognisable by B and T cells. Such
full-length proteins can be secreted from the pathogen
(DT, TT, PT) or produced synthetically or recombi-
nantly. During the last decade, vaccine development
has also focussed on experimental vaccines where the
gene encoding a particular protein is fused into a
DNA or RNA plasmid [1–4]. Secondly, the formula-
vaccines. DNA and some protein vaccines can be
administered in solution without adjuvant enhance-
ment of the immune response. However, most protein
and peptide vaccines show only low immunological
activity when administered in an aqueous solution and
need the help of adjuvants to induce the desired
immunological response.
In the first pioneering works of Jenner (small pox)
and Pasteur (cholera), less virulent forms of the whole
pathogen were used as a vaccine. Today, still many of
the most potent vaccines are given as a live attenuated
or killed form of the particulate microorganism. A
unique property, especially of live vaccines, is that
they often induce strong T-cell responses, a property
very much missed by protein or peptide vaccines
administered with those adjuvants that are in general
use today, e.g. aluminium salts. A drawback of live
vaccines or inactivated virus or bacteria is potential
adverse effects, as reported for whole pertussis
vaccine [5], the Sabin polio vaccine [6] and measles
vaccine [7].
During the last 25–30 years, research and develop-
ment of alternative particulate adjuvants and vaccines
has increased [8]. This has been stirred partly by the
fact that all pathogens are particulate and that particles
can target vaccine delivery to antigen-presenting cells,
and provide persistent antigen due to slow degradation
or clearance of the carrier particle. This has the
potential of obviating the requirement for multiple
vaccinations to boost the antibody response up to a
protective level. Moreover, many of the particulate
formulations tested were shown to have the potential
of inducing helper- and cytotoxic T-cell responses
[9,10], which were previously only seen induced with
attenuated or killed microorganisms and hardly seen
 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355
at all with aluminium-adjuvanted antigens. The
development of particulate vaccines has also been
motivated by safety issues, i.e., to avoid the infectious
risks with live attenuated vaccines and to avoid IgE-
mediated allergic side effects frequently caused by
aluminium salts [11]. Such a T-helper type 2 (Th2)
immunity is undesired in immunotherapy of cancer
and allergies and in vaccination against intracellular
pathogens. Other major disadvantages of aluminium
are unpredictable extent of adsorption of certain
proteins and low stability of some adsorbates [12].
Nevertheless, aluminium salts are the benchmark to
which other adjuvants must compare [13].
The success of preventive vaccination is evident
from the history of immunology, the classical example
being the eradication of small pox. The use of
therapeutic vaccines to boost immunity against
already established infections, tumours or allergies is
much less successful than preventive disease vacci-
nation [3,14,15]. Although antibodies are able to
neutralise pathogens with high efficacy, they do not
eliminate and kill cells of infected organs in a
comparably efficient way. Moreover, the immune
system faces additional hurdles during chronic dis-
eases, e.g. viruses and tumours possess various escape
mechanisms like high antigenic mutation or down-
regulation of MHC molecule expression in infected
cells, which in some instances can cause tolerance or
immune suppression [16–18]. Therefore, immuno-
therapy of chronic diseases must target the cellular
branch of the immune system, especially T-helper
cells, which are capable to modulate immune
responses including cytotoxic T cells (CTLs); CTLs
can kill altered self-cells by perforin, granzymes or
other messengers of death, and secrete cytokines such
as IFN-g and TNF-a.
Many efforts have been made to target antigens to
professional antigen-presenting cells and in particular
to dendritic cells, which are known to be the most
prominent T-cell activators [19,20]. Although peptides
and recombinant proteins are easily manufactured,
their immunogenicity is relatively low [21], mainly
because of the lack of activation of antigen-presenting
cells and the short half-life in vivo. Therefore,
proteins and peptides are generally administered in
combination with adjuvants. Classical adjuvants,
however, induce only poor maturation of antigen-
presenting cells. Furthermore, soluble proteins are
335
poorly taken up by antigen-presenting cells, whereas
peptides, which can bind directly onto MHC class-I
molecules on the cell surface, have the limitation to be
presented not only by professional antigen-presenting
cells, but also by any other types of cells, which do
not express co-stimulatory receptors or cytokines and,
thus, may induce only sub-optimal T-cell activation or
even tolerance. To avoid these drawbacks, dendritic
cells have been generated from the bone marrow or
from peripheral blood monocytes and pulsed in vitro
with peptide or protein [22] together with agents to
mature antigen-presenting cells [23]. Although the
process of dendritic-cell isolation, peptide loading and
vaccine administration is complex and requires well-
trained personnel, this technique represents a good
model for particulate antigen-delivery systems with
respect to efficient immunological recognition, anti-
gen presentation and lymphocyte activation.
In the following, we will review the immunological
events and requirements in the generation of adaptive
immunity, including how immunity is induced by the
bnature’s own particulate antigen-delivery systemsQ,
e.g. bacteria and viruses. Further, possible mecha-
nisms in which detecting and processing of particles
by the immune system can be improved are discussed
in light of developing vaccine carriers or delivery
systems.
2. Immunity
The early phase of the host response to an infection
depends on the innate part of the immune system
including a variety of resistance mechanisms that
recognise and respond to the presence of a pathogen.
Innate immunity is followed by adaptive immunity,
which includes proliferation of pathogen-specific B or
T lymphocytes and the induction of B- and T-cell
memory that provides a long-term protection from
disease. Although the innate immune system lacks
many features and the specificity of adaptive system,
it can distinguish non-self from self, and is crucial for
the stimulation of adaptive immunity.
2.1. Innate immunity
The first line of defence against intruding micro-
organisms includes mechanisms such as fever,
336 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355

mucosal secretions, chemical mediators and phago- Table 1

cytic cells, which fight pathogens in an unspecific and Description of Toll-like receptor (TLR) ligands
non-instructive way. In particular, macrophages and TLR-L Ligand Description (Source)
number
dendritic cells are active in eliminating the intruders
by phagocytosis, a mechanism that is followed by 1 Pam3Cys Synthetic lipoprotein
2 LTA Lipoteichoic acid
antigen presentation to generate adaptive immunity.
(St. aureus)
The uptake of a pathogen or antigen normally takes PGN Peptidoglycan
either of three pathways. Firstly, antigen-presenting (St. aureus, B. subtilis,
cells are capable to engulf particles or microorganisms E. coli)
non-specifically [24]. Secondly, phagocytes are equip- HKML Heat Killed L.
monocytogenes (Gram+)
ped with several cell-surface receptors that recognise
LPS Lipopolysaccharide
pathogen surfaces for receptor-mediated endocytosis. (E. coli)
Among such receptors are the mannan-binding lectin, 3 dsRNA Oligonucleotide
which initiates complement activation, and the macro- PolyI:C Synthetic analog of
phage mannose receptor, where cell-bound lectin dsRNA
4 LPS Lipopolysaccharide
binds certain sugar molecules found on the surface (E. coli)
of many bacteria and some viruses, including the 5 Flagellin S. thyphimurium
human immunodeficiency virus [25]. Also Fcg or Fcq 6 PGN Peptidoglycan
receptors complement secondary phagocytosis by (St. aureus, B. subtilis,
opsonisation. Another set of phagocytic receptors, E. coli)
7 ssRNA Oligonuleotide with
called scavenger receptors, recognises certain anionic
repeating G and U
polymers [26]. Thirdly, phagocytic cells can take up motives
soluble substances in pinocytic vacuoles by so-called Loxoribine Guanosine analogue
macropinocytosis [25,27]. While phagocytosis is a Imiquimod and Small synthetic antiviral
common feature of macrophages and various mono- Resiquimod imidazoqionolines
8 Imiquimod and Small synthetic antiviral
cytes, macro-pinocytosis seems to be a unique
Resiquimod imidazoqionolines
property of dendritic cells. 9 dsDNA Oligodeoxynuleotide
Unlike the receptors of adaptive immunity, the with repeating C and G
receptors of the innate immune system are not motives (CpG)
clonally distributed; a given set of receptors will be 10 Unknown Unknown
11 Unknown Unknown
present on all cells of the same type. The surfaces of
microorganisms bear pathogen-associated molecular
patterns (PAMPs), and the binding of PAMPs by important mediators of innate immunity, e.g. IL-6, IL-
receptors of the innate immune system gives rise to 12, IL-18, and IFN-a and IFN-g. Concurrently, TLR
very rapid responses, which are put into effect without signalling induces co-stimulatory molecules essential
the delay that is necessary to allow for clonal for the induction of adaptive immune responses, i.e.,
expansion of cells needed in the adaptive immune B7.1 (CD80) and B7.2 (CD86), which are cell-surface
response [28]. Recognition and signalling via recep- proteins expressed by antigen-presenting cells (Fig. 1,
tors for PAMPs was originally discovered in the fruit- inset). It is the presence of these molecules on antigen-
fly Drosophila melanogaster [29], but is now known presenting cells along with presentation of microbial
to play a key role in the defence against infection in antigens that activates the CD4 T cells required to
plants, insects, and vertebrates [30–32]. The receptors initiate most adaptive immune responses. Lack of co-
mediating these functions have been named the Toll- stimulation normally leads to tolerance [34–36].
like receptors (TLRs) and their ligation activates the Substances that induce co-stimulatory activity have
cells to respond to pathogens [33]. So far, 11 been mixed with protein antigens for many years in
mammalian TLRs have been identified (Table 1), order to enhance immunogenicity and are known as
and their stimulation activates the transcription factors adjuvants, the best being those containing microbial
NFnB and MAP kinase leading to the production of components. TLR2 and TLR6 have been shown to
 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355 337

Fig. 1. Antigen uptake and presentation. Schematic overview of the major events and signalling during uptake of antigen by antigen-presenting
cells (APCs) and subsequent presentation of the digested epitopes on MHC molecules. Immunity is primarily activated by endogenous (e.g.
heat-shock proteins, DNA and RNA damage, IL-1b, CD40L, hyaluron metabolites) or exogenous (e.g. pathogen or adjuvant) danger signals
(Signal 0). These signals are typically mediated by Toll-like receptors, enable defence by the innate immunity and are succeeded by activation of
the adaptive immunity through antigen presentation (Signal 1) and concurrent co-stimulation (Signal 2) of CD4-positive T-helper (Th) cells.
Finally, the activation of Th cells result in their secretion of cytokines and chemokines, which can directly affect viability and replication of live
pathogens, or the Th cells can further activate CD8-positive cytotoxic T cells or antibody-producing B cells; the secondary activation of other T
and B cells is further supported by co-stimulatimulatory CD40 on Th cells. In the absence of concurrent co-stimulation of CD28 on T cells by B7
molecules (CD80 and CD86) on the APCs, stimulation of T cells through the T-cell receptor complex does not induce immunity but tolerance.

bind to various microbial patterns like peptidoglycans
from Gram-positive bacteria, Gram-negative lipoly-
saccharide, lipoproteins, and lipopeptides, porins and
zymosan from yeast cell walls [37,38]. TLR3 is
involved in the recognition of dsRNA, suggesting that
this TLR is involved in the initiation of immune
responses mainly against viruses, which induce
dsRNA production during replication in infected cells
[39]. Lipopolysaccharide also binds to the serum
protein MD-2, which interacts with CD14. This
enables the whole protein–lipopolysaccharide com-
plex to bind to TLR4 [40,41]. TLR5 is expressed on
the inner part of intestinal epithelia and has been
shown to induce pro-inflammatory factors. The ligand
for TLR5 is the monomeric protein flagellin, a
component in the flagella of motile bacteria [42].
The natural ligand of TLR7 and TLR8 was recently
found to be ssRNA [43,44]. In addition, TLR7 and
TLR8 can bind to synthetic low molecular weight
compounds like imiquimod and resiquimod, which
then possess the capacity to induce cytokines like IL-
12 and IFN-g and have therefore potent anti-viral and
anti-tumour properties [45,46]. TLR9 is responsible
for the innate immune responses against bacterial and
viral DNA, which are rich in non-methylated CpG-
containing consensus sequences [47]. TLR10 and
TLR11 were recently discovered, but their exact
function and ligands and still to be described,
although its function seems to be linked with other
TLRs [48].
Important differences have been observed for
TLR2/4 and TLR3/9 in terms of cellular localisation.
TLR2 and 4 require the interaction of MD-2 and
CD14 and, therefore, are expressed on the cell surface
[41]. TLR3 and 9 are principally expressed intra-
cellularly [49,50]. In addition, recent studies sug-
338 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355
gested that the various TLRs induce different immune
responses implying different signalling pathways [51–
54]. The exact profile of cytokines produced varies
according to the receptors involved and this will in
turn influence the functional character of the adaptive
immune response that develops in their presence. In
this way, the ability of the innate immune system to
discriminate between different types of pathogens is
used to ensure an appropriate type of adaptive
immune response.
2.2. Adaptive immunity
Adaptive immunity is mediated and maintained by
B and T lymphocytes. B lymphocytes develop,
independent of antigen in the bone marrow, but
emerge in the periphery when the immature B cells
express immunoglobulin (Ig)M. Here, B cells further
differentiate into naRve B cells, which also express
IgD and circulate in secondary lymphoid tissues, i.e.,
lymph nodes, spleen and mucosa-associated lymphoid
tissues (MALT), where they may encounter foreign
antigen. T lymphocytes develop from a common
lymphoid progenitor in the bone marrow that also
gives raise to B lymphocytes, but those cells destined
to give rise to T cells leave the bone marrow and
migrate to the thymus for development into CD4- or
CD8-positive T cells.
2.2.1. Antigen presentation
Immature dendritic cells take up soluble or solid
antigens at peripheral tissues and are activated to
migrate into local lymphoid tissue. Here they mature
into cells that express high levels of co-stimulatory
molecules and adhesion molecules that mediate
interactions with the naRve T cells, which are contin-
uously re-circulating through these tissues [55].
Although questioned by some recent observations
[56,57], it is generally accepted that by the time
dendritic cells arrive in the lymph nodes, they have lost
their ability to capture new antigen. They are, however,
now capable to present the antigens ingested at the site
of infection. Macrophages, which are phagocytic cells,
and B cells, which bind pathogen components, may be
similarly induced through non-specific receptors to
express co-stimulatory molecules and act as antigen-
presenting cells. Macrophages are found in many areas
of the lymph node where they can actively ingest
microbes and particulate antigens to prevent them from
reaching the blood circulation. B cells, which concen-
trate in lymphoid follicles, are particularly efficient in
taking up soluble antigens such as bacterial toxins by
specific binding of antigen to surface immunoglobu-
lins. B cells are, however, very inefficient at initiating
adaptive immune responses.
Following uptake in any antigen-presenting cell,
antigens are fragmented by proteases in endosomes,
which also contain high concentrations of MHC
molecules. Antigenic peptides are then loaded onto
MHC class-II molecules before exported to the cell
surface to bind T-cell receptors of specific CD4 T-
helper cells (Fig. 1) [58]. In contrast, activation of
cytotoxic CD8 T cells is driven by recognition of
peptide on MHC class-I molecules. Here, a consistent
part of intracellularly synthesised proteins unfolds and
is degraded by cytosolic proteasomes [59]. The protein
fragments are then transported into the endoplasmatic
reticulum by the transporter-associated protein (TAP)
complex for subsequent loading onto MHC class-I
molecules followed by cell-surface expression [60].
Alternatively, exogenous antigens from microorgan-
isms or infected apoptotic and necrotic cells can be
taken up and processed by antigen-presenting cells for
cross-presentation on MHC class-I molecules in a TAP-
dependent or non-dependent way [61–65]. In the TAP-
dependent endosome-to-cytosol pathway, the captured
antigen leaks from the endosomes into the cytosol and
is processed by the proteasomes. In the TAP-independ-
ent direct endosomal loading mechanism, the antigen is
degraded by endosomal proteases and exchanged with
peptides bound on MHC class I, a process favoured by
the low pH values in endosomal vesicles [66]. While
dendritic cells can operate both cross-presentation
mechanisms, macrophages seem to be restricted to
the direct endosomal-loading pathway [67].
2.2.2. T cells
In addition to stimulation of the T-cell receptor and
its co-receptor CD3 by the foreign peptide bound to a
self MHC molecule (Signal 1; Fig. 1), antigen-specific
clonal expansion of naRve T cells requires a so-called
second or co-stimulatory signal (Signal 2; Fig. 1),
which must be delivered by the same antigen-
presenting cell on which the T cell recognises its
antigen. The most potent activators of naRve T cells are
mature dendritic cells, with one single dendritic cell is
 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355
able to activate about 100–1000 T cells [68,69]. The
best-characterised co-stimulatory molecules belong to
the B7-familiy of glycoproteins CD80 and CD86.
Ligation of CD28 on T cells by B7 molecules
enhances production of IL-2, the major T-cell growth
hormone, thereby driving clonal expansion of naRve T
cells. In contrast, inhibited binding of B7 to CD28
inhibits T-cell responses [35]. Various other co-
stimulatory molecules such as 4-1BB, OX40 and
CD27, and secreted cytokines such as IL-12, IL-1h,
IFN-a/h/g, as well as the density of surface-expressed
MHC molecules also influence the strength of signal
transduction [70–74]. Once a naRve T cell is activated,
it expresses a number of proteins that contribute to
sustaining or modifying the co-stimulatory signal that
drives expansion and differentiation. One such protein
is CD40 ligand (CD40L or CD154), which binds to
CD40 on antigen-presenting cells, thereby transmit-
ting activation signals to T or B cells. CD40 also
activates the antigen-presenting cells to express B7
molecules, thus stimulating further T-cell activation.
NaRve CD4 T cells can differentiate upon activation
into either Th1 or Th2 cells, which differ in the type of
cytokines they produce. The role of effector Th cells is
to control whole immune responses by secreting a
multitude of cytokines and by expressing surface
molecules, such as CD40L for activation of B cells,
antigen-presenting cells, CTLs and several other cells
of the immune system. Th1 cells are associated with
IFN-g, IL-12 and TNF-a whereas Th2 cells typically
produce IL-4, IL-5, IL-10 and IL-13. CD4 T-cell
polarisation depends both on the duration of antigenic
stimulation and the cytokine environment to which the
cells are exposed [74,75]. Hence, the two CD4 T-cell
subsets can regulate each other. Once one subset
becomes dominant, it is hard to shift the response to
the other subset. IL-10 can inhibit the development of
Th1 cells by acting on the antigen-presenting cell,
whereas IFN-g can prevent the activation of Th2 cells.
NaRve CD8 T cells are predestined to become
cytotoxic. Proliferating CD8 T cells acquire effector
function and migrate to the peripheral site of inflamed
tissue to eliminate virus-infected cells and release
cytokines capable to slow down viral replication.
Observations indicated that CD8 T cells undergo a so-
called programmed differentiation upon antigenic
encounter by precursor CTLs [76]. This model
implies that only naRve T cells need to receive the
339
right activation stimulus. Daughter cells divide and
differentiate into effector CTLs without further stimuli
in a pre-programmed fashion. On the other side,
proliferation can sometimes be uncoupled from
effector function [77]. This indicates that CD8 T-cell
activation and in particular differentiation may also
depend on cell extrinsic factors. CTL responses
against certain viruses and tissue grafts seem to
require the presence of CD4 T cells during priming
of naRve CD8 T cells. In these responses, both the
naRve CD8 T cell and the CD4 T cell must recognise
related antigens on the surface of the same antigen-
presenting cells [78–80], hence, a potent CTL vaccine
should contain both CD4 and CD8 epitopes. It is
thought that the actions of the CD4 T cell are needed
to compensate for inadequate co-stimulation of naRve
CD8 T cells by the antigen-presenting cell [81,82],
and to guarantee optimal fitness of the induced CTLs
[83]. This compensatory effect is currently thought to
occur by the recruitment of effector CD4 T cell that
through CD40 up-regulate co-stimulatory activity of
the antigen-presenting cell [84].
2.2.3. B cells
B cells are responsible for the recognition of
pathogen-derived protein in its native form. They
can directly interact with viruses and bacteria through
immunoglobulin receptors with an almost infinite
number of unique specificities. Mature B cells express
surface IgM and IgD as well as various accessory
molecules, e.g. CD19, CD21, CD23 and CD37. The
surface immunoglobulins that serve as the antigen
receptor play two roles in B-cell activation. First, they
transmit signals directly to the cell’s interior upon
binding antigen (Signal 1). Second, they deliver the
antigen to intracellular sites where it is digested and
returned to the B-cell surface as peptides bound to
MHC class II molecules. The peptide-MHC class-II
complex can be recognised by antigen-specific CD4
helper T cells, stimulating them to make proteins such
as CD40 ligand (Signal 2), which in turn cause B cells
to proliferate and differentiate into antibody-secreting
cells. The more an antigen is ordered and repetitive,
the more the IgM on the B cell surface become cross-
linked, thereby increasing Signal 1 strength [85]. The
formation of clusters of cross-linked immunoglobulin
receptors generates delivery of a strong Signal 1 that
is potent enough by itself to induce the production of
340 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355
IgM antibodies of activated B cells. Less ordered and
repetitive antigens require additional stimuli like
lipopolysaccharide or other TLR ligands and the help
of CD4 T cells. T-helper cells are also crucial for
somatic hypermutation and immunoglobulin isotype
switching [86].
The linked recognition of antigen by B cells and
CD4 T-helper cells, presumably mediated by an
antigen-presenting dendritic cell, is followed by
somatic hypermutation and specific antibody produc-
tion [87]. However, B and T-helper cells do not need
to recognise the same epitopes on the antigen. For
complex antigens or pathogens, the two cells might
not even recognise the same protein. Nevertheless, it
is crucial that the peptide recognised by the T cell is a
physical part of the antigen recognised by the B cell,
which can thus produce the appropriate peptide after
internalisation of the antigen bound to its B-cell
receptors. Apart from its importance in the regulation
and manipulation of the humoral immune response,
e.g. in ensuring self-tolerance, linked recognition is
important in the design of vaccines. Haemophilus
influenzae type b is a bacterial pathogen that can
cause meningitis. Antibodies against its capsular
polysaccharide mediate protective immunity. While
adults generate thymus-independent antibody
responses to these polysaccharide antigens very
efficiently, such responses are weak in the immature
immune system of the infant. In vaccines, therefore,
the polysaccharide of Hemophilus influenzae type b is
chemically linked to tetanus or diphtheria toxoid. B
cells that bind the polysaccharide component of the
vaccine can be activated by helper T cells specific for
peptides of the linked toxoid.
Re-recognition of peptide-MHC class-II complexes
on B cells triggers Th2 cells to synthesise molecules
that can further activate B cells. One important T-cell
effector molecule is CD40L, which binds CD40 on B
cells and is involved in all phases of the B-cell
response [88,89]. CD40L and IL-4 are thought to be
synergistic in driving the clonal expansion that
precedes antibody production [90]. IL-4 is secreted
in a polar fashion by Th2 cells and acts selectively on
antigen-specific B cells. The B cells start proliferating,
and after several rounds of proliferation, aided by the
two additional T-cell-derived cytokines IL-5 and IL-6,
B cells further differentiate into antibody-secreting
plasma cells.
When activated B cells receive signals from CD4
Th cells, they form so-called germinal centers, which
purpose is to enhance the later part of the primary
immune response [86,87]. Some germinal center cells
(centroblasts) first differentiate into centrocytes and
then into plasma cells. The centrocytes are rescued by
binding to follicular dendritic cells or Th cells, which
possess antigen–antibody complexes or express
CD40L, respectively [87]. This process is thought to
be necessary for the affinity maturation of the B cells
[86]. Low antigen-affinity B cells undergo apoptosis.
The centroblasts continue to divide rapidly, but
successively begin to secrete antibodies at a high
rate. These cells are destined to become non-dividing,
terminally differentiated plasma cells and represent an
intermediate stage of differentiation. Upon migration
to the bone marrow, a subset of them survive for a
long period of time providing a source of high-affinity
antibody. Other germinal center cells differentiate into
long-lived memory B cells. It is still a matter of debate
if memory B cells require antigen to survive,
proliferate and develop into plasma cells or not
[91,92]. Early studies showed that follicular dendritic
cells are capable to display antibody-captured antigen
in its native form for years and therefore may become
an important source of antigen, indicating a role for
Ag presence in the maintenance of memory B cell
responses [93,94]. Also, mice lacking mature follic-
ular dendritic cells and B-cell follicles, adoptively
transferred with memory B cells could not maintain
elevated titers of specific antibodies, indicating a role
for follicular dendritic cells presenting antigen-anti-
body complexes in the development of plasma cells
from memory B cells and in pathogen antibody-
mediated protection [95,96]. More recently, memory
B cells were shown to survive for long periods
without the presence of antigen [92].
2.3. Immunological memory
Immunological memory, perhaps the most impor-
tant consequence of adaptive immunity, is the capacity
of the immune system to respond more rapidly and
effectively to pathogens that have been encountered
previously and reflects the pre-existence of a clonally
expanded population of antigen-specific lymphocytes.
Memory responses also differ qualitatively from
primary responses. The characteristics of antibodies
 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355
produced in secondary and subsequent responses are
distinct from those produced in the primary response
to the same antigen. Other differences are their
responsiveness to a secondary contact with pathogens,
with respect to cytokine secretion profile, expression
of other effector proteins, e.g. granzyme B, perforin,
and Fas ligand (CD95L), proliferation rate and
distribution. Specific memory is maintained by dis-
tinct populations of long-lived memory cells, and
there has been a lengthy discussion on whether the
survival and function of memory cells are dependent
on residual antigen or not [97–99].
A memory B cell is one that has undergone
immunoglobulin isotype switching and somatic
hypermutation [89]. This differentiation process starts
late in the primary immune response and takes place
in the germinal centers. CD4 T-helper cells trigger the
differentiation of naRve B lymphocytes into memory
cells through CD40 ligation. The process of differ-
entiation induced by this signal is irreversible, but
necessary and sufficient to induce B-cell memory
[100]. The generation of memory B cells is pro-
gressive, starting late in the expansion phase of the
primary immune response, well before the antigen is
eliminated; in contrast to T cells, which are considered
effectors until all antigen is gone, the moment when
antigen is eliminated is not taken into account when
defining B-cell memory. Memory B cells divide very
slowly if at all and do not secrete antibody. However,
upon re-encountering the same pathogen (or antigen
in a booster vaccine), the memory cells start dividing
at a high rate and differentiate into antibody-secreting
plasma cells.
T-cell activation and division may issue from
different types of stimulation and lead to different
functional outcomes. Memory T cells are increased in
frequency and have distinct activation requirements
and cell-surface proteins that distinguish them from
effector T cells. After immunisation, the number of T
cells reactive to a given antigen increases markedly as
effector T cells are produced, and then falls back to
persist at a level significantly (100- to 1000-fold)
above the initial frequency. These cells carry cell-
surface proteins more characteristic of effector cells
than of naRve T cells. Two subsets of memory T cells
may be distinguished, the so-called central memory T
cells and the effector memory T lymphocytes
[101,102]. Central memory T cells display a lymph
341
node-homing phenotype (CD62L and CCR7 posi-
tive). They are long-lived in the absence of antigen
and are able to face systemic pathogenic infections. In
contrast, effector memory T cells may be dependent
on the presence of antigen, home to peripheral tissues,
produce IFN-g and perforin to protect against
peripheral infection and are relatively short lived.
Effector memory T cells probably develop from
effector T cells [103]. The decreasing-potential
hypothesis states that early effector T cells have the
highest potential to become effector memory cells,
whereas T cells that have encountered antigen later
during the immune response have the tendency to
become anergic or die by apoptosis [104–106].
Hence, the definition of memory T cells is not nearly
as clear-cut as with B cells. Nonetheless, in immune
individuals, secondary and subsequent responses are
mediated solely by memory lymphocytes and not by
naRve lymphocytes [107,108]. Therefore, it would be
important to know the capacity of vaccines to induce
memory responses and to analyse the frequency and
phenotype of the generated effector and central
memory lymphocytes.
2.4. The mucosal immune system
The immune system can be divided into a series of
functional anatomical compartments, of which the two
most important ones are the peripheral lymphoid
system, e.g. spleen and lymph nodes, and the mucosal
lymphoid system. Specific homing mechanisms for
lymphocytes to each of these compartments maintain
a separate population of lymphocytes in each com-
partment. The mucosa-associated lymphoid tissues
(MALT) lining the gut are known as gut-associated
lymphoid tissue (GALT) and consist of the tonsils and
the adenoids, the Peyer’s patches of the small
intestine, the appendix, and solitary lymphoid follicles
of the large intestine and rectum. The overlying layer
of the Peyer’s patches contains specialised epithelial
cells. These microfold or M cells take up molecules
and particles from the gut lumen by endocytosis or
phagocytosis. Some intestinal pathogens infect enter-
ocytes, the absorptive cells that line much of the
intestine. Enterocytes do not act as passive victims of
infection, but signal infection by releasing cytokines
and chemokines such as IL-8, MCP-1, MIP-1a and h,
and RANTES that are chemo-attractants for neutro-
342 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355
phils, monocytes, eosinophils and T lymphocytes. In
this way, the onset of infection triggers an influx of
inflammatory cells and lymphocytes, leading to the
induction of an immune response to the antigens of
the infectious agent. For a more comprehensive
review on the mucosal immunity and vaccines, refer
the two papers in this issue by Hirst et al. and by
Alpar et al.
3. Infectious pathogens as models for vaccines
The mammalian body is susceptible to infection by
many pathogens, which must first get in contact with
the host and then establish a focus of infection in
order to cause infectious disease. To establish an
infection, the pathogen must first colonise the skin or
the internal mucosal surfaces of the respiratory,
gastrointestinal, or urogenital tracts and then over-
come or bypass the innate immune defences mecha-
nisms associated with the epithelia and underlying
tissues. If it succeeds in doing this, it will provoke an
adaptive immune response that will take effect after
several days and will usually clear the infection.
Pathogens differ greatly in their means of patho-
genesis, requiring an equally diverse set of defensive
responses from the host immune system. In the
following, we will discuss how particular pathogens
induce immunity and how this knowledge can be used
in the design and development of vaccines and
vaccine delivery systems, which should adopt some
of the characteristics of pathogens in order to improve
potency.
3.1. Viruses
The immunological mechanisms in anti-viral
defence are diverse. Antibody titers, their avidity
and affinity, CTL phenotypes, their activity and
frequency, as well as anti-viral cytokines typically
secreted by T-helper cells and CTLs may all
contribute to immunity at some point. Cytopathic
viruses, e.g. polio, measles, vaccinia and smallpox,
are best controlled by B cells and neutralising
antibodies, as well as by cytokines that induce
metabolic changes in neighbouring cells, rendering
them less prone to viral replication [109]. Cytopathic
virus infections have to be rapidly controlled because
of the high damage the virus inflicts on the host. For
this reason, the infection usually has an acute phase
and resolves in either immunity or in death of the
host. In contrast, non-cytopathic viruses (e.g. LCMV
and cytomegalovirus) are best cleared by CTLs
capable to migrate to the peripheral tissues to
eliminate virus-infected cells [109]. A drawback of
this defence mechanism is that CTLs themselves can
cause severe damage to the host. This highlights
another important aspect of viral infection, which is
the kinetics of virus replication. If the virus is rapidly
controlled by CTLs, the self-inflicted damage is
marginal. In contrast, if the virus replicates fast and
can infect a large number of cells, the tissue damage
may become more severe [110]. Interestingly, during
generalised and prolonged non-cytopathic viral infec-
tions, e.g. LCMV or hepatitis C virus, a detrimental
outcome may be observed [16,17]. In fact, when the
host is confronted with systemically distributed and
high amounts of antigen, virtually all antigen-specific
T lymphocytes become simultaneously activated and
die off rapidly. If the viral infection persists, the
antigen may spread into the thymus and induce
negative selection of antigen-specific maturing T
cells, a phenomenon that leads to a hole in the T-
lymphocyte repertoire. In this case, CTLs can no
longer harm virus-infected cells and the host can
survive as chronic virus carrier.
Most anti-viral vaccines that are currently in use
consist of killed or live attenuated viruses. Inactivated
or killed viral vaccines consist of viruses typically
fixed with aldehydes or h-propiolactone so that they
are unable to replicate, e.g. rabies, polio and influenza
vaccines. The chemical or physical inactivation of
pathogens in some instances may have the drawback
that the used agents change the protein structure or
even denature the viral epitopes [111]. As a con-
sequence, the immune system may not recognise the
original virus structure. Live-attenuated viral vaccines
are generally far more potent, also because they elicit
a greater number of relevant effector mechanisms,
including T cells. Killed viruses cannot produce
proteins in the cytosol, so peptides from the viral
antigens are normally not presented by MHC class-I
molecules, and thus, CTLs are typically not generated
by these vaccines. Attenuated viral vaccines are now
in use against polio, measles, mumps, rubella, and
varicella.
 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355
Attenuation is typically achieved by growing the
virus in non-human cell cultures. In the course of such
selection, the viruses become less capable to grow in
human cells. Such attenuated strains replicate poorly
in human hosts. Ideally, replication is just enough to
induce immunity, but not disease. Although attenuated
virus strains contain multiple mutations in genes
encoding several of their proteins, it might be possible
for a pathogenic virus strain to re-emerge by a further
series of mutations, e.g. the Sabin 3 polio vaccine,
especially in immuno-deficient recipients, in whom
they often behave as virulent opportunistic infections
[112]. However, novel recombinant DNA technolo-
gies are now used to replace wild-type genes in the
virus genome. The advantage of this approach is that
mutations can be engineered so that reversion to wild
type is virtually impossible.
After the first reports on the efficacy of DNA
vaccines in animal models [2], it soon became evident
that the efficiency had to be improved if technology
should have any chance surviving the transfer into a
clinical setting, as very large amounts of DNA
plasmid would be needed for human vaccinations.
One strategy to resolve this problem was to grow the
DNA plasmid in a mutant microorganism, which
would not grow in vivo upon administration. This
could replace the labour intensive process of growing
the DNA vaccine as a plasmid in E. coli and
subsequently extracting and purifying the plasmid
for direct administration. Hence, viral vectors were
engineered to carry heterologous peptides or proteins
from other microorganisms. Adenoviruses, alfavi-
ruses, fowlpox, canarypox and vaccinia viruses have
all demonstrated their potential to serve as an non-
pathogenic carrier of heterologous antigens [113–
116]. Genes encoding protective antigens from several
different organisms could be placed in a single
vaccine strain. This approach makes it possible to
immunise individuals against several pathogens at
once, but such a vaccine could not be used twice
because the vector used for vaccination itself gen-
erates long-lasting immunity that would neutralise its
effectiveness during a second administration.
3.2. Bacteria
Similar approaches are being used for anti-bacterial
vaccine development. For example, a plasmid encod-
343
ing for Listeria monocytogenes antigens was engi-
neered into an auxotrophic mutant of Salmonella
typhimurium, i.e., the mutant was dependent on an
external supply of an essential nutrient that the wild-
type would be capable of biosynthesising [117,118].
After oral administration, the live attenuated bacteria
then carried the plasmid to the M cells that cover the
Peyer’s Patches of the gut (the M cells are the natural
target cells of S. thyphimurium). Invasion by this route
delivers salmonella bacteria straight to the lymphoid
system of the host. From the M cells, the bacteria
enter macrophages and dendritic cells, where they die
because of their mutation, thereby liberating multiple
copies of the DNA vaccine right where it is needed,
i.e., inside the antigen-presenting cell. By this means,
the antigen is more efficiently targeted to the immune
system and it curtails the need of administering large
doses of plasmid. Attenuated strains of Salmonella
have also been used as carriers of heterologous genes
encoding tetanus toxoid and antigens from other
organisms such as Bacillus anthracis, Yersinia pestis,
and Schistosoma mansoni. Each of these has been
used as an oral vaccine to protect mice against
experimental challenge with the respective pathogens
[119–121].
S. thyphimurium as well as attenuated strains of L.
monocytogenes and Bacille Calmette-Guerin (BCG)
represent effective vehicles for delivery of heterolo-
gous antigens due to their preferred intracellular
replication in professional antigen-presenting cells
[24,122–124]. The sub-cellular localisation within
antigen-presenting cells differs among these bacteria.
L. monocytogenes resides within the cytoplasmic
compartment of antigen-presenting cells, whereas S.
thyphimurium and BCG persist in the phagosomal
vacuole. The preferred intracellular localisation for
these vector strains determines the trafficking of
bacterial antigens through different MHC-processing
pathways, i.e., S. thyphimurium and BCG to the MHC
class-II pathway with the subsequent stimulation of
CD4 T cells, especially Th1 cells [125], and L.
monocytogenes to the MHC class-I pathway for
stimulation of CD8 T cells. Although this is the way
the named bacteria theoretically would induce
immunity, experimental data suggest that recombinant
variants of the bacteria that express foreign proteins
can induce both CD4- and CD8-type immune
responses [126,127].
344 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355

Vaccine efficacy of such antigen-delivery systems
is corroborated by their ability to induce strong Th1-
like cytokine responses. By using bacterial and viral
vectors, the antigens are not only targeted to the
appropriate pathways of MHC, but also stimulate the
innate immune system to provide the adequate
cytokine milieu and appropriate expression of co-
stimulatory molecules [128]. Non-proteinaceous
immunogenic components of these antigen carrier
systems, e.g. phospholigands for recognition by gy T
cells or glycolipids for stimulating T cells via CD1
binding, not only provide potential target antigens, but
also represent moieties with additional immuno-
stimulating properties. Such components could theo-
retically be used as adjuvants in other pharmaceutical
antigen-delivery systems.
4. Adjuvants
The design of vaccines primarily requires the
identification of immunological correlates of protec-
tion, i.e., the immune effector mechanism(s) respon-
sible for protection against disease, and the
subsequent selection of an antigen that is capable to
elicit the desired adaptive response. Once this
appropriate antigen has been identified, it is essential
to deliver it effectively to the host’s immune system
[129,130].
Traditionally, vaccines come in several forms: live-
attenuated, replicating pathogens and non-replicating,
inactivated pathogens or their subunits. The latter,
non-viable category is the safest one, but because of
poor or no immunogenicity, it often requires adjuvants
to elicit an adequate immune response. In the absence
of adjuvant, a lack of responsiveness may occur, and
naRve antigen-specific T cells may recognise the
antigen but are not sufficiently activated or they even
become tolerised. Adjuvants are defined as a group of
structurally heterogeneous compounds, used to evoke
or increase an immune response to an antigen [131].
Classically recognised examples include oil emul-
sions, saponins, aluminium or calcium salts, non-ionic
block polymer surfactants, derivatives of lipopolysac-
charide (LPS), mycobacteria and many others
[8,132,133]. Theoretically, each molecule or sub-
stance that is capable to favour or amplify a particular
step in the cascade of immunological events, ulti-
mately leading to a better immunological response,
can be defined as an adjuvant. Obviously, the first step
is very important. However, the in vivo molecular and
cellular mechanisms required for the generation of an
effective immune response, which depends critically
on co-injection of adjuvant, are poorly understood.
The structural requirements of adjuvants are similarly
poorly understood. Adjuvants have therefore been
surrounded by obscurity and called bthe immunolo-
gist’s dirty little secretQ [28].
Schijns classified adjuvants functionally according
to five recently proposed concepts of immunogenicity
[134] (Table 2): firstly, the geographical concept of
immune reactivity and, secondly, the theory of depot
effect, both emphasizing the importance of antigen
localisation for a period of time after immunisation

Table 2
Classification of adjuvants based on their functional properties
Mode of action Examples Key event(s)
Facilitation of antigen uptake, transport Liposome, VLPs, aluminium salts, Antigen localisation in the lymph node
and presentation by APCs polymeric nano- and microparticles,
ISCOMs, QuilA
Depot effect Oil emulsions, gels, polymeric nano- and Prolonged antigen presentation
microparticles,
Signal 0 Cytokines, TLR ligands (e.g. LPS, CpG) Signalling through PAMP receptors on
innate immune cells
Signal 2 Cytokines, co-stimulatory molecules APC polarisation, T- and B-cell help
(e.g. B7 analogues, anti-CD28
antibodies)
Danger signal (the Matzinger theory) Oil-emulsions, surfactants, aluminium Tissue destruction/stress, DNA and RNA
salts, cytokines, interferons, CD40L damage, hyaluron metabolites, IL-1b,
analogues, heat shock proteins heat shock proteins, CD40L
 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355
[135]; thirdly, the paradigm that adjuvants act as
Signal 0, which precedes the induction of the epitope
Signal 1 and co-stimulatory Signal 2 and, which is
characterised by pathogen recognition through
PAMPs [28]; fourthly, the role of Signal 2 molecules
as natural adjuvants in the activation of naive T-helper
cells, the master coordinators of subsequent T-cell-
dependent immune responses; finally, the hypothesis
that immunity is activated by exogenous and even-
tually endogenous danger signals [136,137]. Accor-
ding to the danger model, immune activation occurs
upon recognition of host-derived danger signals from
stressed cells in damaged tissues—not from an
invading pathogen. These signals have yet to be
defined molecularly, they may include cell debris
from necrotic cells, heat shock proteins, nucleotides,
reactive oxygen intermediates, cytokines, etc.
5. Designing pharmaceutical antigen-delivery
systems
Pharmaceutical vaccine formulations presently
being tested in various experimental and clinical
models have one basic common denominator: they
are of particulate nature. In contrast to most soluble
antigens, which are ignored by the immune system,
particles are recognised as foreign and as danger.
Hence, the starting-point when designing a new
vaccine delivery system is often that a particulate
structure is required. This is easily obtained by
aggregation or cross-linking of antigen [1,138,139]
or, conventionally, by adsorption or precipitation of
and antigen on aluminium salts. Alternatively, the
antigen can be chemically attached to a pre-formed
particular carrier [140], or it is chemically or physi-
cally distributed in or on particles in a more (in
liposomes and virus-like particles) or less (in
polymeric microspheres) organised way [140–144].
Any type of particle typically facilitates the recog-
nition of the vaccine by professional antigen-present-
ing cells as well as the total or partial uptake of the
vaccine into these cells. As a result of activation, the
antigen-presenting cells normally start their migration
from their peripheral location towards the nearest
draining lymphatic organ. However, prior to the
uptake into antigen-presenting cells, the formulation
itself influences the property of the recruited pha-
345
gocytic cells. The chemical and physical composition
of the vaccine will partly define the quality and type
of cells attracted to the site of the vaccine entry.
5.1. Targeting professional antigen-presenting cells
Among the cells picking up and transporting
antigens to the lymph nodes, the dendritic cells that
are resident in all tissues are by far the most potent
activators of T cells. They migrate to secondary
lymphoid tissues and present the antigens they
originally ingested at the peripheral site of infec-
tion/injection. Hence, in ideal priming, vaccines
should preferentially be directed to the dendritic
cells. This can either be achieved by administration
of the vaccine to a tissue which contains a relative
high frequency of such cells, e.g. Langerhans cells
in the epidermis of the skin, or by guiding the
vaccine to dendritic cells using targeting molecules
such as the myeloid marker CD11c, the adhesion
molecule DC-SIGN [145], the putative antigen-
uptake receptor DEC-205 [146,147], or the T-cell
analogues of ICAM-1, ICAM-2 and ICAM-3. By
such means, it was also shown that CTLs can bee
primed after a single injection, bypassing require-
ment for adjuvant, CD4 T cell help and CD40
signalling [148].
5.2. Concomitant delivery of antigen and co-stimula-
tory signals
The binding of PAMPs by TLRs on antigen-
presenting cells initiates the adaptive immune response
with maturation of antigen-presenting cells, up-regu-
lation of co-stimulatory molecules (e.g. of the B7
family) and migration to the draining lymph nodes.
The ligation of TLRs through PAMPs or their
immunostimulatory analogues in vaccines (Table 1)
further induces the production of several important
mediators of innate immunity, such as cytokines and
chemokines, including co-stimulatory molecules
essential for the induction of adaptive immune
responses. For instance, by combining antigen-loaded
virus-like particles with CpG, a vaccine possessing the
most important immunological features of viruses was
engineered, with the immune system perceiving the
virus-like particles as dangerous pathogens against
which a strong immune response should be mounted
346 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355
[77]. By this way, professional antigen-presenting cells
were shown to take up particles for antigen processing
and presentation to specific T cells for several days.
Efficient maturation of the antigen-presenting cells
was guaranteed by the packaged CpGs, which trigger
endosomal TLR-9. This vaccine formulation induced
high frequencies of specific CD8 T cells protective
against cytopathic and non-cytopathic viruses as well
as an established fibrosarcoma tumour [149]. The data
imply that effective stimulation of the innate immune
system is a key factor for the induction of strong and
protective T-cell immune responses. Having received
both the antigen and the co-stimulatory signal, T cells
can initiate adaptive immune responses. Whereas most
PAMPs bind to receptors on the surface of antigen-
presenting cells, some viruses are thought to be
recognised trough the TLR3 and TLR9 inside the
cells [49,50,150] as a consequence of the production of
double-stranded RNA or DNA, respectively, in the
course of their replication. Hence, when using TLR3
and TLR9 ligands in particulate vaccines, one must
assure that the same antigen-presenting cell takes up
both antigen and TLR ligand. Viral infections also
induce the infected cells to produce IFN-a, which
subsequently can activate dendritic cells to express co-
stimulatory molecules. Antigen-delivery systems that
are capable of doing the same will also have the
general potential to stimulate adaptive immunity,
especially CTLs.
Because activation of naRve T cells requires the
simultaneous delivery of the antigen-specific and the
co-stimulatory signals, only activated professional
antigen-presenting cells can usually initiate T-cell
responses. If the vaccine by itself is not capable to
stimulate expression of molecules necessary for T-cell
activation, a combined delivery of antigen and co-
stimulation within the same pharmaceutical formula-
tion could improve the priming of lymphocytes. Here,
DNA vaccines offer an opportunity as they permit the
concomitant delivery of pathogen-derived genes
together with genes encoding for stimulatory cyto-
kines, e.g. GM-CSF, IL-2, IFN-g, and IL-12 [3,151–
154]. The feasibility of this concept has been demon-
strated with recombinant viral vectors and liposomes,
which provided co-stimulatory molecules such as
those of the B7 family [155–158], 4-1BB ligand
[159], and NF-nB (RANK/RANK) ligand [116,160].
However, a particulate vaccine combining an antigen
and a co-stimulatory molecule is not effective if the
molecule dissociates prior to encountering antigen-
presenting cells. Furthermore, to encounter a CD4 T
cell, the antigen-presenting cell must migrate to a
nearby lymph node, and this migration is stimulated by
cytokines such as TNF-a [161,162].
Once a naRve T cell is activated, it expresses
proteins that contribute to clonal expansion and
differentiation. One such protein is CD40 ligand,
which binds to CD40 on antigen-presenting cells. This
binding transmits activating signals to the T cell and
also activates the antigen-presenting cells to express
B7 molecules, thus stimulating further T-cell prolifer-
ation, and CD40 ligation is a prerequisite for the
generation of B cell memory [100]. There are several
reports on experimental and clinical vaccines where
CD40 antibodies or CD40L are used to promote the
clonal expansion and differentiation of lymphocytes
[116,163,164]. When a T cell has differentiated into
an effector cell, encounter with its specific antigen
results in immune attack without the need for co-
stimulation. This applies to all classes of effector T
cells, and its importance is easy to understand in the
case of cytotoxic CD8 T cells, which must be able to
act on any cell infected with a virus, whether or not
the infected cell can express co-stimulatory mole-
cules. Hence, for a boosting vaccine, it is not
necessarily required to provide co-stimulation. This
is also illustrated by the ambiguity in adjuvant
requirements for the boosting of immune responses
against tetanus and diphtheria [11]; in contrast to the
primary administration of soluble antigen, which can
cause tolerance, once an immune response is primed,
subsequent administration of soluble antigen does not
cause tolerisation, but boosts the already established
immune response. The boosting doses of antigen are
rarely presented to new naRve T cells. In the
competition for antigen, memory cells and specific
antibodies are superior to naRve cells and will capture
and neutralise boosting antigens before they get to
prime naRve lymphocytes [107,108]. Such a principle
could be applied in a prime-boost vaccine were the
priming vaccine contains the antigen plus all neces-
sary co-stimulatory additives, whereas the boost
vaccine could consist of a pharmaceutical formula-
tion, which provided long-term delivery of the antigen
only. Similarly, it is conceivable that a vaccine-
delivery system may be combined with factors that
 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355
drive the differentiation of CD4 T cells into Th1 (e.g.
in HIV, malaria or tuberculosis vaccines) or Th2 (e.g.
in polio and tetanus vaccines). Th1-polarising cyto-
kines have been used in particulate antigen-delivery
systems to stimulate cell-mediated immunity
[165,166]. Especially, IL-12 may find application in
therapeutic vaccination [3,167].
5.3. Immunomodulating activities of synthetic
polymers
The introduction of a synthetic material into the
body affects different body systems, including the
defence system. Synthetic polymers have only a
limited capacity to elicit antibody formation or to
induce a cellular immune response against them.
However, synthetic polymers sometimes exhibit sig-
nificant immuno-modulating activity [168–170],
mainly concerning the activation/suppression of nat-
ural killer cells (NK cells), so called lymphokine-
activated killer cells (LAK cells) and macrophages
[171]. Some polymers can be used as effective protein
carriers, and the development of biodegradable and
biocompatible vaccine delivery systems based on
liposomes, microspheres, nanoparticles or water-solu-
ble synthetic polymers has received considerable
attention, as they can be tailored to meet the specific
physical, chemical, and immunogenic requirements of
a particular antigen [171]. As mentioned above, the
loading of peptides on MHC class-II molecules occurs
subsequent to the acidification of the antigen-contain-
ing endosomes upon fusion with lysosomes. By
preventing acidification of these vacuoles, antigen
presentation can be blocked. Drugs, such as chlor-
oquine, raise the pH of vacuoles and inhibit the
presentation of antigens that enter the cells by
endocytosis. This is also why chloroquine is a
therapeutic principle in malaria. However, antigen-
delivery systems, which assist the acidification of
endosomes, also promote the MHC class-II presenta-
tion of antigen they are carrying. This might be one
mechanism by which nano- and microparticles of
poly(lactide-co-glycolide) work. The acidic moieties
of lactic or glycolic acid monomers or soluble
oligomers, as well as the proton liberated into the
endosomes during erosion of the polymers will trigger
MHC class-II presentation [9]. On the other hand,
pharmaceutical formulations, which contain basic
347
polymers or excipients (e.g. chitosan, ethylene imine,
collagen, poly-phosphates, some substituted lignins
and dextrans, as well as anionic lipids and surfactants)
with proton scavenger properties may prevent antigen
presentation by the MHC class-II pathway. Indeed,
chitosan has been shown to promote Th1 cytokine
reponses and MHC class-I antigen presentation [172].
5.4. Antigen dose and structure affect the type of
immunity induced
The amount and sequence of the antigen that
initiates the response also influence the differentiation
of CD4 T cells into distinct effector subsets, with high
and low density of peptide on the surface of antigen-
presenting cells stimulating Th1 or Th2 cell responses,
respectively [173,174]. Hence, when stability of the
antigen is compromised, as may occur in poly(lactide-
co-glycolide) microspheres [175,176], this might also
have consequences for the Th1/Th2 skewing of the
immune response, since the relative accessibility of
different epitopes might change. Moreover, peptides
that interact strongly with the T-cell receptor tend to
stimulate Th1-like responses, whereas peptides that
bind weakly tend to stimulate Th2-like responses
[174,177]. This difference could be very important in
several circumstances. Allergy is caused by the
production of IgE antibody, which requires high
levels of IL-4, but does not occur in the presence of
IFN-g [178]. Antigens that elicit IgE-mediated allergy
are generally delivered in minute doses, and they elicit
Th2 cells that make IL-4 and no IFN-g. It is also
relevant that allergens do not elicit any of the known
innate immune responses, which produce cytokines
that tend to bias CD4 T-cell differentiation toward Th1
cells, probably because allergen should not represent a
bdangerQ. Allergens are also delivered to humans in
minute doses across a thin mucosa, such as that of the
lung. Something about this route of sensitisation
allows even potent generators of Th1 responses like
Leishmania major to induce Th2 responses.
Ordered and repetitive structures permit to elicit
significantly more potent B-cell responses [85], and
this principle has been exploited in virus-like par-
ticles, which are composed by spontaneous self-
assembling viral proteins in a quasi-crystalline struc-
ture [140,179]. B-cell receptor cross-linking by the
repetitively displayed epitopes on virus-like particles
348 T. Storni et al. / Advanced Drug Delivery Reviews 57 (2005) 333–355
activates B cells, which in turn will be able to induce
activation of Th cells. Secondary, this permits the
development of IgG secreting plasma cells and B-
memory cells. Hepatitis B s antigen (HBsAg) virus-
like particles are a successful example of an effective
vaccination approach against hepatitis B [180,181].
Moreover, responses against chosen antigenic targets
can be obtained by genetically fusing the B-cell
epitopes to exposed regions of various types of virus-
like particles, or alternatively, by chemically cross-
linking antigenic peptides or even whole proteins to
the virus-like particles [140,182]. This latter method is
currently preferred because it allows the display of
proteins in their native form, which is important for
the generation of antibodies able to bind to pathogenic
antigens in a functional conformation. Similarly,
influenza hemaglutinin and neuraminidase proteins
put into a highly repetitive order on the surface of
phospholipid liposomes (so-called virosomes) have
proven powerful in inducing immunity [143,183].
Hemaglutinin, which possess a sialic acid binding
region, binds to surface receptors of host antigen-
presenting cells and promotes up-take into the cell. By
cross-linking or integrating proteins or B-cell epitopes
to the virosomal structures, one can induce B-cell
responses against selected antigens [184]. This pro-
cedure proved particularly effective for the develop-
ment of vaccines against influenza [185] and hepatitis
A [186]. For an extended review on the properties of
liposomes and virosomes, we refer to the paper of
Wilschut and co-workers in this issue.
6. Conclusion
Vaccination techniques do not always stimulate
immunity because of the inappropriate elicitation of
immune responses. Such limitations have spurred the
development of new adjuvants and antigen-delivery
systems. Adjuvants play an important role in enhanc-
ing the efficacy of vaccines that consist of antigens
becoming more and more purified. Indeed, recombi-
nant proteins or synthetic peptides are safer than crude
inactivated microorganism, but less immunogenic.
This can be balanced by specific adjuvants. But there
are no universally active adjuvants and, apart from
often being particulate and thereby attract the attention
of professional antigen-presenting cells and other
participants of the innate immune system, their action
is not yet clear and probably rely on different
mechanisms. The adjuvants must therefore be chosen
according to several criteria, like the target species,
the antigens, the type of desired immune response, the
route of administration, or the duration of immunity.
Many papers reports on the mechanism by which a
pharmaceutical antigen-delivery system actually
works. The usual take-home message is that it is a
particle and that this is the target function for antigen
presentation. bNanoparticles because size mattersQ,
bThe smaller the betterQ or bAPC targetingQ are
frequently used as catchphrases, but do not really
represent thorough studies of the uptake and antigen-
presentation mechanism for the actual pharmaceutical
formulation. Although the most important feature of
adjuvants or antigen-delivery systems is likely to be
the particulate nature, there is a lot more to be done in
basic research on how such systems can be engineered
to trigger a certain strength and type of immune
response. With today’s knowledge in basic immunol-
ogy, the opportunities for a rational design and testing
of antigen-delivery systems should be available. The
success is perhaps only limited by the will and
opportunity to break with conventional topical board-
ers in natural and medical science. Furthermore,
although historically the best medical developments
were achieved by empirical approaches, the highly
specific medical and regulatory requirements of
modern vaccines are probably better met by rational
design than by trial and error.
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