Vaccine adjuvants

Viral vaccines in the medical practice

8 June 2010, Cluj-Napoca
Klmn Bartha PhD bartha.kalman@oek.antsz.hu
Zsuzsanna Pauliny MD zsuzsanna.pauliny@oek.antsz.hu

National Centre for Epidemiology Budapest,

Hungary

Why we need adjuvants?

Traditional vaccines based on attenuated live organisms already

have them their invasiveness provides efficient delivery to
antigen-presenting cells and
Various naturally occuring components of the pathogens
stimulate the innate immune system
The majority of recent vaccines represent highly purified
subunit components of pathogens, they lack most of the
features of the original pathogens, such as immunostimulatory
components, and the ability to replicate and produce high level
of antigens,
Therefore, they are usually poorly immunogenic and need
adjuvants to improve immunogenicity.
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Key elements (components) of

effective vaccines

(there is a nice confusion in the literature some authors use simply adjuvants some distinct clearly delivery
Antigen(s) against which adaptive
immune
responses are elicited
systems and
potentiators)

Delivery systems to ensure that the vaccine antigen is delivered to the right
place at the right time. It means the role of a delivery system is to enhance
the amount of antigen reaching the cells responsible for immune response
induction.

Adjuvants / (Immune potentiators) to stimulate the innate immune system

What is the difference between adjuvants and immune potentiators?

Pathogen-associated molecular patterns (PAMP-s) and related compounds are
called immune potentiators, allowing a clear distinction between them and
particulate adjuvants such as microparticles, emulsions, liposomes and virus-like
particles.
MPL is the only immune potentiator has been approved for human use in
prophylactic vaccines yet
(MPL) - monophosphoryl lipid A
(LPS) - lipopolysaccharide
Bacterial DNA CpG containing optimised oligo sequences

The lipopolisaccharide (LPS) component of Gramnegative bacteria has been shown to act as a potent
immune potentiator however, the profound toxicity
and pyrogenicity of LPS prevents its use in humans.

Alternatively, a chemically modified LPS derived from

Salmonella minnesota R595, called monophosphoryl
lipid A (MPL), exhibits potent adjuvant activity with
essentially no toxicity.
MPL has been shown to be an effective immune
potentiator for the induction of both humoral and
cell-mediated immunity in which MPL can induce both
Th1- and Th2-type immune responses in the systemic
and mucosal compartments of the immune system.

 Currently

licensed adjuvants were developed

using empirical methods. They are not optimal
for many of the challenges in vaccination
today.

In

particular, the historical emphasis on

humoral immune responses has led to the
development of adjuvants with the ability to
enhance antibody response.

As

a consequence, most commonly used

adjuvants are effective at elevating serum
antibody titers, but do not elicit significant
Th1 responses or CTLs.
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Innate/adaptive immune responses

The immune system has evolved two main functions:

to react quickly (within minutes) to molecular patterns

found in microbes, and
to develop slowly (over days to weeks), precisely
targeted specific adaptive immune responses.

The faster acting innate immune responses provide a

necessary first line of defense because of the
relatively slow nature of adaptive immunity.
In contrast, adaptive immunity uses selection and clonal
expansion of immune cells harboring made-to-order
somatically rearranged receptor genes (T- and B-cell
receptors) recognising antigens from the pathogen,
thereby providing specificity and long-lasting
immunological memory.
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Innate immune response

Innate immune responses, among their many effects,

lead to a rapid burst of inflammatory cytokines and
activation of antigen-presenting cells (APCs) such as
macrophages and dendritic cells. These nonclonal
responses also lead to a conditioning of the immune
system for subsequent development of specific adaptive
immune responses.

To distinguish pathogens from self-components, the

innate immune system uses a wide variety of relatively
invariable receptors that detect evolutionary conserved
signatures from pathogens (pathogen-associated
molecular patterns, PAMPs).
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Immunological background I.

The addition of such microbial components to experimental vaccines

leads to the development of robust and durable adaptive immune
responses.

The mechanism behind this potentiation of immune responses was

not well understood as long as some of the pattern-recognition
receptors (PRRs) involved in the innate immune responses to PAMPs
were not identified.
PRRs are differentially expressed on a wide variety of immune cells,
including neutrophils, macrophages, dendritic cells, natural killer
cells, B cells and in some nonimmune cells too, such as epithelial and
endothelial cells.
Engagement of PRRs leads to the activation of some of these cells
and secretion of cytokines and chemokines, as well as maturation and
migration of other cells.
In tandem, this creates an inflammatory environment that leads to
the establishment of the adaptive immune response.

Immunological background II.

PRRs consist of nonphagocytic receptors, such as

Toll-like receptors (TLRs) and nucleotid-binding
oligomerization domain (NOD) proteins, and receptors
that induce phagocytosis, such as scavenger
receptors, mannose receptors and -glucan receptors.

Receptors that induce phagocytosis are directly

recognise ligands on the surface of pathogenic
microbes and lead to their engulfment into phagocytic
cells such as macrophages.

Nonphagocytic receptors that recognise PAMPs

extracellularly (certain TLRs) or intracellularly (NOD
family of proteins) lead to an elaborate signal
transduction cascade.

Adjuvants for TLR-independent immune

activation

It has been shown that Toll-like receptors (TLRs), one

of the innate immune sensors, plays important roles not
only in the initial proinflammatory responses, but also
in the consequent adaptive, antigen-specific immune
responses

Conventional adjuvants such as Alum, incomplete and

complete Freunds adjuvant elicit efficient adaptive
immune responses to vaccine antigen in the absence of
TLRs
Intracellular innate receptors, such as NOD-like
receptors, retinoic-acid-inducible gene (RIG)-like
receptors and intracellular DNA receptors have been
demonstrated to activate the innate immune responses,
and possibly the adaptive-immune responses, in a TLR10
independent manner.

Adjuvants for TLR-dependent immune

activation

TLR ligands are promising candidates as vaccine adjuvants. In

experimental vaccines TLR agonists are very potent adjuvants in
capacity of activating cells expressing the TLR, in particular, dendritic
cells (DCs), which are the key antigen presenting cells.

The TLR4 ligand LPS has been experimentally shown to be a potent

adjuvant, although its extreme toxicity prevents its use in humans. The
adjuvant effect of LPS is solely dependent on TLR4-mediated, MyD88dependent signaling. Efforts to eliminate the toxicity of lipid A led to
the development of monophosphoryl lipid A (MPL) which is the only
licensed new-generation TLR ligand vaccine adjuvant.

MPL contains lipid A as a TLR4 ligand. The dependency of TLR4 on

adjuvant effect of MPL was surprisingly minor, at least for antigenspecific antibody responses, suggesting that there are yet unknown
TLR-independent adjuvant factors within the MPL compound.
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AF03

The slow process of adjuvant discovery. Alum was the first adjuvant to
be licensed in the 1920s and is still the only adjuvant approved for
human use in the USA. The squalene-based oil-in-water emulsion MF59
was first licensed in Europe for a flu vaccine (FLUAD) in 1997. The
LPS analog monophosphoryl lipid A (MPL) formulated with alum (AS04)
was first approved for an HBV vaccine (Fendrix) in Europe in 2005.
The oil-in-water emulsion AS03 was approved for a pandemic flu
vaccine (Prepandrix) in 2008. AF03 Humenza in 2009.
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Alum I.
Aluminium

based mineral salts (generally called

Alum) have been successfully used as adjuvants
in licensed vaccines for many years. Alum
typically induces Th2 immune response.

Although

it has been shown to be safe and

effective in traditional vaccines where eliciting
antibody response is necessary, it is a weak
adjuvant for protein subunits.
it fails to induce the Th1 responses
associated with the induction of gamma
interferon and cytotoxic T lymphocytes (CTL)
which are required to clear the body of
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intracellular viral infection.

Moreover,

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Proposed mechanisms of action of alum in vitro and in vivo and their possible contributions to
adjuvanticity.
In vitro, alum complexed with antigen increases antigen uptake by APC. In addition, alum
induces direct activation of Nlrp3 (Nalp3) inflammasome complex and synergizes with
LPS stimulation of TLR4 for the secretion pro-inflammatory cytokines such as IL-1b, IL18 and IL-33.
In vivo, alum induces necrosis in unidentified target cells resulting in production of uric acid,
which has the potential to stimulate Nlrp3. Alum also stimulates local recruitment of APC
and migration of APC to the draining lymph nodes. It has been proposed that alum may
also enhance local antigen persistency ("depot" effect). The contribution of all these
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activities to alum adjuvanticity and the requirement of Nlrp3 are not yet fully understood.

In summary just alum

It is very difficult to compare data obtained on the same adjuvant in
different laboratories. In the case of alum, discrepancies may also arise
from the multiple mechanisms of action, whether it is antigen delivery
to APC or immunostimulation through Nlrp3 activation.
Some antigens may be contaminated by immunostimulatory molecules,
therefore requiring only alum's antigen delivery function for an
efficient adaptive response.
On the other hand, other antigens may be easily internalized by APC
despite the absence of alum but are poorly immunogenic and therefore
may require Nlrp3-dependent alum-immunostimulatory activity. More
work needs to be performed on inflammasome-deficient mice, using
different immunization protocols and different formulations in parallel,
in order to fully understand the contribution of the inflammasome to
alum adjuvanticity.
In summary, we are just beginning to understand the molecular mechanisms
of alum, an adjuvant that has been used in humans for almost a century
without knowing how it worked; however, standardized vaccination
models are required to accurately address the differential contribution
of each of the multiple mechanisms to adjuvanticity.
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