Doctoral Thesis from the Department of Immunology, The Wenner-Gren Institute,

Doctoral Thesis from the Department of Immunology, The Wenner-Gren Institute,
Stockholm University, Stockholm, Sweden
HEAT SHOCK PROTEINS AS VACCINE ADJUVANTS
Qazi Khaleda Rahman
STOCKHOLM 2005
SUMMARY
New efficient vaccines against infectious diseases are in demand. Some important factors
impeding the vaccine development are the poor immunogenicity and the MHC restriction of the
immune responses to a number of antigens. The use of novel vaccine adjuvants or carrier proteins,
which are known to enhance the immunogenicity of the subunit antigens and provide T-cell help, can
circumvent these problems. The potential of heat shock proteins (HSPs) to function as adjuvants when
fused to or co-delivered with protein antigens, make them attractive vaccine candidates. In this thesis
we have evaluated the potency of heat shock protein 70 (HSP70) as a possible vaccine adjuvant and
studied the mechanisms behind the adjuvanticity.
The first article aims to evaluate the carrier effect of glutathione-S-transferase (GST) on a
malarial antigen EB200 that induces a MHC restricted response in mice. Immunization of CBA and
C57BL/6 mice, high and low responders to EB200, respectively, with the GST-EB200 fusion protein
elicited EB200 specific antibody responses in both strains of mice, which indicated that MHC
restriction was broken in C57BL/6 mice. However, the antibody affinity and the magnitude of the
response were lower in the C57BL/6 mice compared with that in CBA. To improve the response, the
efficacy of various adjuvants like alum, HSP70 from Trypanosoma cruzi, and the adjuvant combination
(HSP70 and cholera toxin) was evaluated. The results indicated that cholera toxin and HSP70 act
synergistically and improve the immunogenicity of EB200 antigen by increasing the affinity and
magnitude of the response.
HSP belongs to a family of conserved molecules and the maximum homology lies on the Nterminal region of the protein, therefore there is a risk that use of a complete molecule would give rise
to autoimmunity. Thus, in our second study we first evaluated the adjuvant effect of the less conserved
portion of HSP70 derived from Plasmodium falciparum (Pf70C). We found that the Pf70C exhibited
similar adjuvant properties as the whole molecule. We further analyzed the adjuvant potential of Pf70C
against EB200 formulated as a chimeric DNA vaccine construct. These constructs alone failed to
generate substantial levels of EB200 specific antibodies in mice. However, the DNA immunization
efficiently primed the immune system. This was evident as the subsequent boosting with the
corresponding recombinant fusion proteins Pf70C-EB200 elicited strong EB200 specific Th-1 antibody
responses. In contrast, no such priming effect was observed for ex vivo IFN-g production, however
stimulation with the Pf70C-EB200 fusion protein induced an enhanced secretion of IFN-g in vitro.
During the infection process, the synthesis of bacterial HSP is up-regulated, which is known to
sensitize T cells in the infected host. Since a high degree of homology exists within the phylogenetic
families of HSPs, we postulated that exposure of mice to microorganisms could prime the immune
system for evolutionary diverse HSPs and for any antigen coupled to them. We tested this hypothesis
by priming mice with different microorganisms such as BCG, Mycobacterium vaccae or Chlamydia
pneumoniae and boosted with a recombinant fusion protein Pf70C-EB200 or with a panel of HSPs. We
found that BCG and M. vaccae but not C. pneumoniae could provide priming of the immune system to
induce secondary IgG responses to Pf70C as well as to other HSPs tested. The priming effect was also
observed when the EB200 antigen was coupled to Pf70C. Analysis of the IgG1 and IgG2a profiles and
IFN-g production induced against the HSPs revealed a mixture of Th1/Th2 type of responses. We also
observed that HSP70 specific sera cross-reacted some extent with certain autoreactive antigens.
However, no deposits were observed in the kidneys of HSP treated animals.
Finally, we investigated the role of TLR2 and TLR4 on HSP70-mediated adjuvanticity. We
found that HSPs displayed different degrees of adjuvanticity regarding both the strength and the profile
of the induced immune response. Also, they possessed different requirements for signaling through
TLRs. While HSP70 from T. cruzi induced antigen-specific humoral responses in wild type as well as
in both the TLR2 and TLR4 knockout mice, the response was diminished in the TLR4 knockout mice
when both the whole and C-terminal fragment of HSP70 from Mycobacterium tuberculosis was used.
However, the C-terminal fragment of P. falciparum HSP70 elicited responses only in wild type mice
but not in TLR2 or TLR4 knockout mice indicating that the adjuvant function differ for
phylogenetically related HSPs. Taken together our data suggest that HSPs can be promising candidates
in future vaccines.
ISBN 91-7155-060-7 pp 1-71
Akademitryck AB, Valdemarsvik
The thesis is published electronically at the Stockholm University website
ã Qazi Khaleda Rahman
Stockholm 2005
‘Imagination is more important than knowledge, for knowledge is limited
while imagination embraces the entire world.’
Albert Einstein
ORIGINAL PAPERS
This thesis is based on the following papers, which will be referred to in the text
by their roman numerals
Paper I
Khaleda Rahman Qazi, Klavs Berzins, Manuel Carlos López and Carmen
Fernández. Breaking the non-responsiveness of C57BL/6 mice to the malarial antigen
EB200-The role of carrier and adjuvant molecules. Scand. J. Immunol. 2003. 58: 395403.
Paper II
Khaleda Rahman Qazi*, Maria Wikman*, Nina-Maria Vasconcelos, Klavs Berzins,
Stefan Ståhl and Carmen Fernández. Enhancement of DNA vaccine potency by
linkage of Plasmodium falciparum malarial antigen gene fused with a fragment of
HSP70 gene. Vaccine. 2005. 23:1114-1125.
* Equally contributed to the work.
Paper III
Khaleda Rahman Qazi, Mousumi Rahman Qazi, Esther Julián, Mahavir Singh,
Manuchehr Abedi-Valugerdi and Carmen Fernández. Exposure to mycobacteria
primes the immune system for evolutionary diverse heat shock proteins. Submitted to
Infection and Immunity.
Paper IV
Khaleda Rahman Qazi, Wulf Oehlmann, Mahavir Singh, Manuel Carlos López and
Carmen Fernández. Mechanisms for Heat Shock Protein 70 mediated adjuvanticity.
Manuscript.
TABLE OF CONTENTS
I) GENERAL BACKGROUND .................................................. ... ...9
Introduction…………………………………………………………………9
Vaccines.................................................................................................9
Brief historical perspective .................................................................10
Characteristics of an ideal vaccine .....................................................11
Rational for development of vaccines.................................................13
Immune responses...............................................................................13
Innate immune responses ................................................................13
TLRs………………………………………………………...14
Adaptive immune responses.............................................................16
Antigen processing and presentation………………………..17
Humoral responses…………………………………………..18
Cellular responses…………………………………………...19
Immunological memory...................................................................21
Vaccine technologies ........................................................ 22
Live attenuated vaccines.....................................................................22
Killed whole organisms.......................................................................23
Subunit vaccines..................................................................................23
Polysaccharides…………………………………………………………. 24
Recombinant proteins……………………………………………………. 24
Synthetic peptides………………………………………………………… 25
New generation vaccines.................................................................... 26
DNA vaccines……………………………………………………………...26
mRNA vaccines……………………………………………………………30
Live recombinant vaccine delivery systems…………………………… 30
Improvement of the potency of subunit vaccines.................... 31
Adjuvants ............................................................................................32
Role of adjuvants in the immune responses .......................................33
Classification of adjuvants..................................................................33
Most commonly used adjuvants .........................................................34
Freund’s adjuvant……………………………………………………….. .34
ISCOMs……………………………………………………………………..34
CpG………………………………………………………………………….35
Bacterial toxins…………………………………………………………….36
Alum………………………………………………………………...37
II) RELATED BACKGROUND ........................................................37
Heat shock proteins (HSPs).............................................. 38
HSP70 as adjuvant and carrier ..........................................................38
HSP70 receptors and mechanism of adjuvanticity............................40
Role of LPS in HSPs activity ..............................................................43
HSP70 in association with autoimmunity ..........................................44
Plasmodium antigen EB200 ............................................................46
III) THE PRESENT STUDY..............................................................46
Aims .......................................................................... 46
Results and Discussion ................................................... 47
Paper I .................................................................................................48
Paper II ...............................................................................................49
Paper III ..............................................................................................50
Paper IV ..............................................................................................53
Concluding remarks ...................................................... 54
IV) ACKNOWLEDGEMENTS .........................................................57
V) REFERENCES .............................................................................59
APPENDIX: PAPERS I-IV
ABBREVIATIONS
APC
CCR
CD
CD40L
CMV
CpG
CT
CTA
CTB
CTL
DC
ER
Fas
FCA
FIA
GM-CSF
HSP
IFN
IL
IRAK
IRF
ISCOM
LOX
MAPK
MHC
MPL
MTB
MyD88
NF-kB
NK
PAMP
PKR
PRR
Th
TCM
TEM
TLR
TNF
TRAF
TREM
Antigen presenting cell
Chemokine receptor
Cluster of differentiation
CD40 ligand
Cytomegalovirus
Cytidine phosphate guanosine
Cholera toxin
Cholera toxin A subunit
Cholera toxin B subunit
Cytotoxic T lymphocyte
Dendritic cell
Endoplasmic reticulum
FS-7 associated surface antigen
Freund’s complete adjuvant
Freund’s incomplete adjuvant
Granulocyte-macrophage colony stimulating factor
Heat shock protein
Interferon
Interleukin
IL-1 receptor associated kinase
Interferon regulatory factor
Immunostimulating complex
Lectin-like oxidized low-density lipoprotein receptor
Mitogen activated protein kinase
Major histocompatibility complex
Monophosphoryl lipid
Mycobacterium tuberculosis
Myeloid differentiation factor 88
Nuclear factor-kB
Natural killer
Pathogen associated molecular pattern
RNA-dependent protein kinase
Pattern recognition receptor
T helper
Central memory T cell
Effector memory T cell
Toll-like receptor
Tumor necrosis factor
TNF-receptor associated factor
Triggering receptor expressed on myeloid cell
Heat shock proteins as vaccine adjuvants
9
I) GENERAL BACKGROUND
Introduction
Infectious diseases have always been scourge for humans. They are
responsible for approximately 25% of global mortality, especially in children younger
than five years [Kieny 2004]. Nowadays, modern technologies provide many
opportunities to prevent infectious diseases by vaccination. Vaccination mainly
capitalizes the immune system’s ability to respond rapidly to microorganisms upon a
second encounter. Large-scale and comprehensive national immunization programs,
and the considerable successes that were achieved in the eradication of smallpox and
the reduction of polio, measles, pertussis, tetanus and meningitis, were among the
most notable achievements of the 20th century. Unfortunately, vaccines are still
missing for a number of diseases like malaria, tuberculosis and AIDS, that are still
major causes of morbidity and mortality. Moreover, some of the existing vaccines do
not induce complete protection. Therefore, the development of effective vaccines
towards those diseases, as well as the improvement of efficacy and safety of existing
vaccines is needed. In this thesis, the adjuvant properties of heat shock proteins have
been studied.
Vaccines
Vaccines have been described as ‘weapons of mass protection’. They remain
the most efficacious and valuable tools in the prevention of infectious diseases,
provided that they are administered prophylactically in anticipation of pathogen
exposure [Cohen and Marshall 2001, Curtiss 2002]. The ultimate goal of a vaccine is
to develop long-lived immunological protection, whereby the first encounter with a
pathogen is ‘remembered’ by the immune system. Vaccination leads to enhanced
responses that either completely prevent infection or greatly reduce the severity of the
disease. Therefore, the important step in a rational design of a vaccine is to understand
the immune correlates of protection. From a mechanistic perspective, vaccines select,
activate and expand memory B and T cells, which are then poised to respond rapidly
and specifically to a subsequent exposure of the pathogen. Today, prevention of
bacterial and viral infections through vaccination is beneficial in reducing disease
morbidity and health care costs.
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Qazi Khaleda Rahman
Brief historical perspectives
The concept of immunity was first described by the Greek historian
Thucydides in Athens during the fifth century BC, where he first mentioned immunity
to an infection called plague. In describing a plague, he wrote that only those who had
recovered from the plague could nurse the sick, because they would not contract the
disease a second time. The first recorded attempts to induce immunity deliberately
were performed by the Chinese and Turks in the fifteen-century, making children
resistant to small pox by having them inhale powders made from the skin lesions of
patients recovering from the disease [Ki Che Leung 1996]. Variolation, i.e.
transmission of virulent matter to induce a natural disease and the immunity against it,
was brought from Constantinople to England by Lady Mary Montague, in 1718 [ Fitz
1911] who performed this method on her children. Variolation grew in popularity in
Britain after its introduction.
During the latter half of the 18th century an English surgeon called Edward Jenner
noticed that milkmaids who had recovered from cowpox never contracted the more
serious smallpox. On the basis of this observation, he injected the material from a
cowpox pustule into the arm of an 8-years old boy called James Phipps who
occasionally worked for Jenner. When this boy was later intentionally inoculated with
smallpox, the disease did not develop [Baxby 1981]. Jenner’s landmark treatise on
vaccination was published in 1798. Eventually, the English Parliament passed a law in
1840 making vaccination compulsory.
In 19th century a major step in microbiology was made exclusively by Louis Pasteur,
Robert Koch and Joseph Lister. They opened the door to the germ theory in medicine,
and to the development of vaccines for many diseases. Pasteur discovered the
possibility to artificially modify the virulence of an infectious agent and to induce
protection against it, which was a major step in preventive medicine [Geison 1995].
Studying fowl cholera, he and his colleagues found that the virulence of the bacteria
(Pasteurella multocida) of this disease could be permanently attenuated when
cultured for long periods, and inoculation of that attenuated culture protected the
chicken from the disease. His first publication, in 1880, could be considered as a
revolution in medicine where he named the attenuated strain a vaccine (from the latin
Heat shock proteins as vaccine adjuvants
11
vacca, meaning cow), in honor of Jenner’s work with cowpox inoculation. Pasteur
extended these findings to other diseases, demonstrating that administration of heat
attenuated anthrax bacilli to sheep provides protection. Then, Pasteur managed to
develop a vaccine against the well-known disease, rabies. He treated an Alsatian boy,
badly bitten by a dog, with the attenuated form of rabies. Later on, thousands of bitten
people, inoculated according to the Pasteur’s protocol, did not die of rabies. This
success gave him enormous reputation as a benefactor of humanity. Pasteur did not
have a complete understanding of how the vaccination worked, the immunological
memory or the function of the lymphocytes, which had to wait another half century.
The experimental work of Emil von Behring, Shibashaburo Kitasato and Elie
Metchnikoff in 1890 gave the first insights into the mechanisms of immunity. von
Behring won the Nobel Prize for the discovery of serum antibodies in 1901. Finally,
with Burnet’s clonal selection theory (1957) and the discovery of T and B
lymphocytes (1965), the key mechanisms of the immunity became clear.
Characteristics of an ideal vaccine
Several factors must be kept in mind in developing a successful vaccine. Many
licensed vaccines have one or more ideal characteristics, but none manifests all of
them. A good vaccine must satisfy a number of stringent criteria:
I) A good vaccine should stimulate a strong, protective and long lasting immune
response. Key to the development of vaccines that elicit enduring protection is the
induction of strong, long-lived immunological T and B cell memory to antigens that
correlate with protection; is the ability to recall previous exposures to antigen and to
mount enhanced, accelerated effector responses [Agematsu et al. 2000, Kaech et al.
2002, Sprent 2002, Esser et al. 2003]. Some wild type infections (measles) and
vaccines (17D yellow fever) confer enduring, even lifelong, immunity after a single
immunizing event. Research in non-human primates and in humans, using new
immunological and flow cytometry techniques, is identifying the cells responsible for
maintaining T and B cell memory and long-lived protection after vaccination.
Measurement of the specificity, subsets, magnitude and longevity of T and B memory
responses elicited by immunization may guide vaccine development by providing
immunological correlates of long-lived protection.
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Qazi Khaleda Rahman
II) A good vaccine should induce the right sort of immune responses. The immune
responses correlated with protection, induced by most current vaccines seem to be
mediated by long-lived humoral immune responses through the production of
antibodies. However, in humans and in many experimental rodent models of
intracellular infection, such as malaria, leishmaniasis, tuberculosis and HIV infection,
cellular immune responses have been shown to be crucial in mediating protection.
Therefore, the development of a successful vaccine against those diseases will be
facilitated by a thorough understanding of how cellular immune responses are
generated and maintained in vivo.
III) An ideal vaccine should show an impeccable safety profile in all populations,
including young infants, elderly and immunocompromised subjects. Despite the
success of vaccination in eliminating disease and death, the public acceptance of even
minor side effects of vaccination is very low. This was illustrated by a gradual cease
of pertussis vaccination in Great Britain during 1970s where over 100,000 children
caught pertussis as a consequence, and some died or contracted chronic neurological
damages (Armstead 2003). Scientific reports on diphtheria-tetanus-pertussis (DTP)
vaccination causing asthma, and mumps measles rubella (MMR) vaccination causing
Crohn’s disease or autism, have been contradicted in several follow-up studies
[Andreae et al. 2004, Benke et al. 2004]. The challenge faced in developing new
vaccines is to achieve strong immunogenicity without increasing reactogenicity.
IV) A single dose of vaccine should confer robust, long-lived immunity. Only a few
live vaccines have achieved this goal. In contrast to the results with live vaccines, it
has been difficult to promote long-lived immunity with a single dose of non-living
antigen vaccines. One goal of vaccine development is to rectify this using new
adjuvants and antigen delivery systems.
V) An ideal vaccine should be affordable by the population at which they are aimed
and should be formulated to resist high and low temperatures to facilitate distribution.
Ideally, vaccines should have uncomplicated, economical large-scale manufacturing
processes, because simplicity of manufacture has long-term implications for vaccine
supply and cost which can be affordable by all populations. ‘Glassification’
technologies that dry vaccines in the presence of sugars such as trehalose or other
Heat shock proteins as vaccine adjuvants
13
stabilizers render vaccines resistant to high and low temperatures. This technology has
the potential to relieve pressures on the ‘cold chain’ in developing countries [Levine
and Sztein 2004].
Rationale for development of vaccines
The rationale for vaccine design initially involves identification of
immunological correlates of protection – the immune effector mechanisms
responsible for protection against diseases and the subsequent selection of an antigen
that is able 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.
According to current thinking, a productive immune response is defined by the
generation of clonally expanded antigen-specific T and/or B cells. The antigen is
initially recognized by specific T-cell receptors on naïve T cells or cell-membrane
bound immunoglobulins on B cells. This stimulus is defined as signal 1. In addition,
the delivery of costimulatory molecules or cytokines (signal 2) provided by the
antigen presenting cell (APC) contributes to the priming of T helper cells [Lafferty
1975] and their subsequent delivery of antigen-specific help for B cells and cytotoxic
T cells.
Immune responses
Long-lived immunological memory, which is the ultimate goal of vaccination,
can be achieved by activating the innate and adaptive arms of the immune responses.
Innate immune responses
Innate immune responses are defined as the non-specific host defences that
exist prior to exposure to an antigen and considered as the body’s first line of defence.
The innate response acts early and rapidly after infection (within minutes), detecting
and responding to broad cues from invading pathogens. Recognition of pathogens by
the innate immune system leads to the rapid mobilization of immune effector and
regulatory mechanisms that provide the host with three critical advantages: i)
initiating the immune response and providing the inflammatory and co-stimulatory
context for antigen recognition; ii) mounting a first line of defence, thereby holding
the pathogen in check during the maturation of the adaptive response; and iii) steering
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Qazi Khaleda Rahman
the adaptive immune system towards the cellular or humoral responses most effective
against the particular infectious agent.
The first response to microorganisms is an inflammatory reaction, characterized by
cell migration, alterations in vascular permeability and the secretion of soluble
mediators, such as cytokines, chemokines and interferons (IFNs). Pathogens are
phagocytosed or endocytosed and subsequently destroyed or degraded, then the innate
immune cells, macrophages or dendritic cells (DCs) are activated resulting in a series
of events [Pulendran et al. 2001]. This leads to the upregulation of cell surface costimulatory molecules such as CD80/86, CD40 and of major histocompatibility
complex (MHC) class I and II and production of pro-inflammatory cytokines TNF,
IL-1 and effector cytokines IL-12, IFN-g by the innate immune cells. All this has a
profound effect on the activation of the adaptive responses. Natural killer cells (NK
cells), on the other hand, can recognize certain cells that lack the ‘self’ MHC class I
molecule and kills therefore that cells [Kärre 1997, Brutkiewicz and Welsh, 1995,
Hoglund et al. 1997]. This is a useful ability, not the least in the fight against viruses
that try to escape the immune system by becoming invisible inside host cells by
down-regulating MHC class I molecules.
The receptors of innate immunity called pattern recognition receptors (PRRs) can
recognize broad structural motifs that are highly conserved and unique to microbes
[Janeway 1989]. The ability to recognize and combat invaders displaying such
molecules is a strong feature of innate immunity. Among these receptors, are the
families of Toll like receptors (TLRs) [Rock et al. 1998, reviewed in O'Neill 2004],
which is discussed below.
TLRs
The innate immune system has developed a series of diverse and evolutionary
conserved families of PRRs [Medzhitov and Janeway 1997] that recognize specific
pathogen associated molecular patterns (PAMPs), thereby allowing the innate
immune system to distinguish self-molecules from pathogen associated non-self
structures and initiate the host defense response (Medzhitov and Janeway 1998,
Janeway and Medzhitov 2002]. PAMPs represent the molecular signatures of
Heat shock proteins as vaccine adjuvants
15
potentially noxious substances and may be perceived as a ‘danger signal’ [Matzinger
and Guerder 1989] by the innate immune system [Janeway 1989 (a), Janeway 1989
(b), Janeway 1989 (c), Janeway 1992, Fearon and Locksley 1996]. Many of the
immunostimulatory adjuvants are derived from PAMPs including LPS, HSPs, CpG,
lipoprotein, flagellin etc.
Among the PRRs, the TLRs constitute a structurally conserved family of receptors,
which exhibit homology to the Drosophila Toll system [Medzhitov et al. 1997]. TLRs
are broadly expressed on macrophages, dendritic cells, epithelial cells and B- (TLR4
and 9) and T-cells (TLR2). TLRs are transmembrane proteins with an extracellular
domain containing leucine-rich repeats that recognize conserved motifs on pathogens,
and a cytoplasmic domain similar to the corresponding domain of the interleukin-1
receptor involved in signal transduction [Aderem and Ulevitch 2000, Akira et al.
2001, Hallman et al. 2001]. Binding to PAMPs by TLRs causes the adapter protein
MyD88 to be recruited to the receptor complex, which in turn promotes its association
with
the
IL-1R-associated
kinase
(IRAK).
This
is
followed
by
the
autophosphorylation of IRAK, which dissociates from the receptor complex and
interacts with tumor-necrosis-factor-receptor-associated factor-6 (TRAF-6). TRAF-6
leads to activation of the nuclear factor-kB (NF-kB), mitogen activated protein
kinases (MAPKs) and p38 kinase in APCs. This results in upregulation of cell surface
expression of co-stimulatory (CD80/86) and MHC molecules on APCs, expression of
cytokines (such as IL-6, TNF-a, IL-12), chemokines and trigger many other events
associated with DC maturation. These events lead to initiation of antigen-specific
adaptive immune responses [Medzhitov and Janeway 2000, Akira S et al. 2001].
TLR4-mediated responses may also involve a MyD88 independent pathway, where
the phosphorylation of transcription factor IRF-3 leads to the activation of type I
interferons [Kawai et al. 2001, Toshchakov et al. 2002, Hoshino et al. 2002]. The
capacity of TLRs to alter the phenotype of the cell on which they are expressed,
makes them attractive candidates for the initiators of the entire program of host
defence, be it innate or acquired.
To date, at least 11 mammalian genes encoding mammalian TLR molecules (TLR111) [reviewed in O'Neill 2004] have been identified. They have a distinct function in
16
Qazi Khaleda Rahman
pathogen recognition and constitute good targets for rational adjuvant development.
Figure 1 illustrates some ligands recognized by the TLR family. Other PRR molecules
of the innate immune system known to recognize many pathogen products include
CD14 [Takeda et al. 2003], Dectin1 [Gantner et al. 2003], Triggering receptor
expressed on myeloid cells (TREM1 and 2) [Bouchon et al. 2001], RNA-dependent
kinase (PKR) [Cella et al. 1999], and CD91 [Basu et al. 2001]. All play important
roles in activating the cells of the innate immune system.
HSP60
HSP70
Uropathogenic
E.coli
HSP70
MyD88
TRIF
IRAK
TRAF6
IRF-3
NF-kB
MyD88 dependent
pathway
IRF-3
Cytokine production
Costimulatory
molrcule induction
IFN-inducible gene
espression
Caspase activation
Costimulatory
molrcule induction
MyD88 independent
pathway
Figure 1: Summary of ligands recognized by TLR family and their signaling pathways.
This figure is adapted from the figure in Akira et al. 2003.
Adaptive immune responses
The main feature of the adaptive immune responses is their capacity to recognize and
selectively eliminate specific pathogens. This is due to the vast ability of the adaptive
Heat shock proteins as vaccine adjuvants
17
immune system to genetically create receptors with different specificities. These
receptors are expressed by specialized cells called B and T lymphocytes, which are
the key cells involved in adaptive immunity. Adaptive immunity exhibits specificity,
diversity, memory and self/nonself recognition of the antigens. The initiation of
adaptive immunity requires the cooperation between lymphocytes and APCs. APCs
are the specialized cells, including macrophages, B-cells and DCs, that first
internalize the antigen, process it and then display or present a part of that antigen to
helper T cells (Th) together with MHC molecules. The immunological importance of
MHC molecules in adaptive immunity was discovered as T cells were found to
recognize viral peptides in the context of self MHC class I molecules [Zinkernagel
and Doherty 1974]. MHC molecules have important roles as restriction elements for T
cells. The classical MHC subclasses, I and II, are highly polymorphic complexes.
Together with the highly diverse rearranged T- and B- cell receptors this constitutes a
capacity to respond to a vast variety of antigens. The conversion of antigens into
MHC-associated peptide fragments is called antigen processing and presentation. The
following section briefly describes how antigenic peptides are processed and
presented to T cells in the context of MHC.
Antigen processing and presentation
There are two ways in which antigen loading onto MHC can occur.
Endogenous antigens are produced within the host cell (such as viral or tumour
proteins), and are complexed with MHC class I through intracellular processing
pathways. This pathway involves proteasomal degradation of cytosolic, ubiquitintargeted proteins. The endogenous antigens are degraded into peptide fragments
which, are translocated to the endoplasmic reticulum (ER) by the transporters
associated with antigen processing (TAP) complex, where loading of MHC class I
molecules occur. The peptide-class I MHC complex is then transported to the cell
surface via the constitutive secretory pathway [reviewed in Gromme and Neefjes
2002, Williams et al. 2002]. MHC class I molecules may also be loaded with peptides
derived from extracellular proteins in a process called MHC class I cross presentation
[Yewdell et al. 1999].
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Qazi Khaleda Rahman
Exogenous antigens are produced outside of the host cell and enter the cell by
endocytosis or phagocytosis. Endocytosed/phagocytosed exogenous antigens and
pathogens are degraded within the acidic environment of phagolysosomes, and the
generated peptides bind to the cleft within the class II MHC molecules. The complex
then travels to the cell surface.
Two types of adaptive immune responses, humoral and cellular, mediated by B and T
lymphocytes respectively, are discussed below.
Humoral responses
Humoral responses are mediated by plasma cells secreting antibodies.
Antibodies mainly recognize extracellular pathogens as well as toxins, and function as
the effectors of the humoral response by binding to antigen and neutralizing it or
facilitating its elimination. Antibodies can exert their effect to eliminate the pathogens
in various ways, e.g. by mediating phagocytosis, by complement mediated lysis, or by
neutralizing toxins or viral particles by coating them.
Depending on the nature of the antigen, B-cell activation proceeds by two different
routes, one dependent upon helper T cells (Th cells) and the other independent of Th
cells. In the first case, when recognizing antigens such as proteins, B cells need costimulatory signals provided by Th cells to be able to elicit a response. This type of
antigen is known as thymus dependent (TD) antigen. Naïve B cells circulate through
blood, lymph nodes and spleen until they encounter antigens. Antigens are often
brought by macrophages and DCs from the T cell area of the spleen or lymph nodes.
After encountering the specific antigen, the initiation of B-cell activation takes place
by clustering the B-cell antigen receptors (membrane IgM on naïve B cells) by the
binding of multivalent antigen. This leads to increased expression of class II MHC
and the costimulatory B7 (CD80/86) molecules. Antigen-antibody complexes are
internalized by B cell receptor-mediated endocytosis, processed into peptides and
presented on the membrane as peptide-MHC II complexes. The immunological
synapse formed between the B- and T-cell involves interaction of the peptide-MHC
complex and CD40 on B cells with the T cell receptor and CD40L (CD154) expressed
on the T cell surface respectively, triggering a signaling cascade, leading to the
secretion of cytokines by Th cells. The cytokine signals stimulate B-cell proliferation,
Heat shock proteins as vaccine adjuvants
19
differentiation into antibody secreting plasma cells and memory B cells and induce
antibody isotype switching, IgM to IgG, IgA and IgE as well as affinity maturation.
Certain antigens can activate B-cells without the help of T-cells. These antigens are
called thymus independent (TI) antigens, and are further divided into TI-1 and TI-2
type of antigens. Most TI-1 antigens are polyclonal B-cell activators (mitogens); i.e.
they are able to activate and differentiate B cells regardless of their antigenic
specificity. Some pathogen associated molecular pattern (PAMP) found in the
bacterial cell wall such as lipopolysaccharide (LPS), peptidoglycan and lipoprotein
are TI-1 antigens. The responses against LPS have been studied extensively and it has
been shown that B-cells in mice express a specific receptor known as TLR-4, capable
of recognizing LPS [Poltorak et al. 1998, Hoshino et al. 1999]. TI-2 antigens are
characterized by their repetitive structure, e.g. bacterial cell wall polysaccharides
(dextran and levan) or polymeric proteins (bacterial flagellin) [reviewed in Coutinho
et al. 1974, reviewed in Coutinho et al. 1975, Fernández and Möller 1977, Manheimer
et al. 1984].
Cellular responses
Cellular immune responses are mediated through activation of naïve T cells by
the recognition of foreign peptide fragments bound to self-MHC molecules together
with the simultaneous delivery of a co-stimulatory signal by specialized APCs [Dustin
and Cooper 2000]. The best-defined costimulators for T cells are the B7 proteins,
expressed by the professional APCs (B-cells, macrophages, DCs), which are
recognized by CD28 on T cells. Failure to provide a CD28 based costimulatory signal
leads to T cell anergy (unresponsiveness) [Harris and Ronchese 1999]. Following
activation, T cells express a new surface antigen, CTLA-4 that binds tightly to B7
molecules, arresting T cell activation [Harris and Ronchese 1999]. APCs express
several costimulatory molecules, including B7.1 (CD80) and B7.2 (CD86), to signal T
cells and to induce clonal expansion of antigen-specific T cells. T cell responses to
antigen together with the costimulators, triggering synthesis of cytokines and other
effector molecules that lead to cellular proliferation, differentiation into effector and
memory cells. Activated T cells are subdivided into two major types of effector cells,
according to their expression of CD4 or CD8 membrane molecules. CD4+ T cells
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Qazi Khaleda Rahman
recognize antigen derived mainly from endocytosed proteins that is combined with
class II MHC molecules and function largely as Th cells, whereas CD8+ T cells
recognize cytosolic protein that is combined with class I MHC molecules and function
largely as cytotoxic T cells (CTL) [reviewed in Parkin and Cohen 2001].
Mostly, various effector T cells carry out specialized functions, such as cytokine
secretion, B cell help (CD4+ Th cells) and cytotoxic killing activity (CD8+ CTLs).
Some CD4+ cells can act as killer cells and some CD8+ CTLs have been shown to
secrete a variety of cytokines. The cytokines that are produced during the
inflammatory innate response, direct the deviation of T cells into at least two
functionally distinct subsets, Th1 and Th2, distinguished by the different panels of
cytokines they secrete [Seder and Paul 1994]. IL-4 [Le Gros et al. 1990, Swain et al.
1990] and IL-6 [Ricón et al. 1997] are instrumental in the generation of Th2
responses. IL-12, which is mainly produced by dendritic cells and macrophages,
drives Th1 differentiation. This selection appears to depend on the origin of the
activated DC that interacts with the CD4+ cells [Satthaporn and Eremin 2001]. The
Th1 subset secretes IL-2, IFN-g, and TNF-b and promote mainly cellular immunity,
whereas Th2 cells produce IL-4, IL-5, IL-6, IL-10 and IL-13, that favor antibody
production and class switching, and also inhibit Th cells from entering the Th1 path
[Murphy and Reiner 2002]. In vivo, murine Th1 type immune responses are
associated with the B cell responses characterized by IgG2a synthesis, whereas IgG1
antibodies are associated with Th2 type of responses. The Th1 to Th2 balance
determines the onset and outcome of a wide variety of immune disorders that include
autoimmune and allergic diseases.
Activation of CD8+ T cells results in the production of CTLs. Following recognition
of MHC-I antigen complexes, CTLs bind to target T cells and insert perforins into
their cell membrane, delivering granzymes into the cell cytoplasm and initiating a
process leading to target cell apoptosis. In addition, CD8+ T cells can kill infected
cells by a process of Fas-mediated lysis [Edwards et al. 1999].
Heat shock proteins as vaccine adjuvants
21
Immunological memory
The hallmark of the adaptive immune response is the capacity to remember
previous contacts with the microorganisms. Immunological memory confers the
ability to mount more rapid and more robust responses to subsequent antigenic
encounters [Gray 1993] and reflects the pre-existence of a clonally expanded
population of antigen-specific lymphocytes. Memory cells are phenotypically and
functionally distinct from naïve cells and have less stringent requirements for
activation and differentiation into CTL or plasma cells.
Memory B cells are responsible for generating the anamnestic antibody production of
higher affinity that occur after re-exposure to antigen, which is important for
eliminating the pathogen and toxic antigens not cleared by pre-existing circulating
antibodies. They have a lower threshold of activation, can be stimulated to secrete
very large amounts of class-switched Igs and are able to readily contribute to rapid
and productive B and T cell interactions, stimulating efficient antigen dependent
CD4+ T cell responses without requiring an immediate pre-activation step [Bar-Or et
al. 2001]. Stimulation through CD40L and IL-4, together with sustained expression of
Bcl-6, prevents terminal differentiation [Fearon et al. 2001, Calame 2001]. These cells
become memory B-cells, residing in secondary lymphoid organs. In contrast,
triggering of IL-2, IL-6, IL-10 and the B cell receptor, but not CD40L, induces
degradation of Bcl-6, and the expression of the B-lymphocyte-induced maturation
protein 1 (Blimp-1), leading to differentiation into plasma cells [Shapiro-Shelef and
Calame 2004]. A small fraction of the plasma cells are rescued from apoptosis, and
become long-lived plasma cells residing in the bone marrow [Manz et al. 1997].
Memory B cells play a role in replenishing the pool of long-lived plasma cells for
continuous maintenance of long-term serum antibody levels in the absence of
pathogens [Slifka and Ahmed 1998, Manz and Radbruch 2002, Bernasconi et al.
2002, Manz et al. 2002]. Two principle mechanisms have been suggested for the
maintenance, either by activation by antigen trapped by follicular DCs or by
activation by polyclonal stimuli and bystander T cell help [Gray and Skarvall 1988,
Bernasconi et al. 2002]. Long-lasting high affinity antibody responses may be the
crucial factor for designing vaccines that provide effective long-term immunity.
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Qazi Khaleda Rahman
The memory T-cell compartment consists of both CD4+ and CD8+ T cells that can
rapidly acquire effector functions to kill infected cells and/or to secrete inflammatory
cytokines inhibiting the replication of the pathogen. Two functionally distinct memory
T cell subsets are proposed on their ability to produce effector cytokines and surface
expression of chemokine receptor CCR7 [Sallusto et al. 1999, Sallusto et al. 2000]; 1)
CCR7- effector memory T cells (TEM) present in the blood, spleen and non lymphoid
tissues that will rapidly respond to antigen by producing effector molecules or 2)
CCR7+ central memory T cells (TCM) present in lymph nodes, spleen and blood that
are slower in making cytokines or becoming killer cells than the TEM cells. Both the
humoral and cellular immune responses need to be mobilized for the optimal control
of pathogens.
Vaccine technologies
Despite the fact that vaccine development presently encompasses technologies
ranging from the centuries-old approach of modifying pathogens to advanced genetic
manipulations of the immune system itself, all vaccines have in common the intention
of inducing an immune response designed to prevent infection or limit the effect of
infection. In latter sections we will discuss different approaches used today to produce
a wide variety of vaccines and also provide a glimpse into future scientific rationale
for vaccine development.
Live attenuated vaccines
The aim of attenuation is to diminish the virulence of the pathogen, while
retaining its immunogenicity. Many successful live viral and bacterial vaccines, such
as attenuated poliovirus, measles virus, rubella virus, yellow fever and Salmonella
typhi strain Ty21a, were produced by repetitive in vitro passage in cell culture or by
nonspecific mutagenesis [reviewed in Levine and Sztein 2004]. Now precise deletion
mutations in the virulence genes can be introduced into wild-type organisms, resulting
in rational attenuation. Live, attenuated bacteria were first shown by Louis Pasteur to
confer specific immunity. Attenuation was achieved successfully by Calmette and
Guérin with a bovine strain (Mycobacterium bovis) which, during 13 years (19081921) of culture in vitro, changed to an avirulent form, now known as BCG (bacillus
Calmette Guérin). BCG has been shown to perfectly protect against tuberculosis. The
Heat shock proteins as vaccine adjuvants
23
advantages of this strategy are that some important antigenic determinants can be
retained by attenuated strains that can elicit both humoral and cellular immunity.
Also, because of their capacity for transient growth, such vaccines provide prolonged
exposure to immune system, resulting in effective immune responses and production
of memory cells. Several risks, however, are associated with such vaccines.
Attenuated viruses or bacteria may through genetic mutation, either lose their potency
(so that the vaccine is ineffective), or regain their ability to cause disease. Inactivation
may be incomplete and hazardous side effects may be caused by the actual vaccine
(e.g. Bordetella pertussis) or by contaminants. Moreover, attenuated vaccines impose
a risk in immunocompromized individuals and in pregnant mothers. It is known that
standard measles vaccines cause immunosuppression, demonstrable by transient
anergy against recalled antigens [Fulginiti et al. 1968].
Killed whole organisms
To avoid the risk of live vaccines, the use of killed organisms as vaccine has
been introduced. These vaccines are made from the entire organism, killed by heating
or by adding chemicals such as formaldehyde to make them harmless. This renders
the microbes incapable of causing disease, but preserves some immunogenic
properties of the microorganisms, so that they are still able to stimulate the immune
system. It is a relatively crude approach. The limitations of these kinds of vaccines are
that they are not as potent as live vaccines. The immunogenicity usually has to be
enhanced by coadministration with adjuvants, and multiple doses are necessary for
obtaining long-term protective immunity. The production of such vaccines requires
large-scale culturing of the pathogen, which can be associated with both safety risks
and problems as cost efficient production. Typhoid, cholera, influenza and the stalk
poliomyelitis vaccine are examples of killed whole organism vaccines.
Subunit vaccines
Subunit vaccines represent technologies from the chemical purification of
components of the pathogen grown in vitro (such as surface glycoproteins
hemagglutinin and neuraminidase of influenza or the polysaccharide capsules of
Streptococcus pneumoniae or inactivated toxins) to the use of recombinant DNA
technology to produce a single viral protein (such as hepatitis B surface antigen).
Since subunit vaccines cannot replicate in the host, there is no risk of pathogenicity.
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Qazi Khaleda Rahman
Polysaccharides
Polysaccharide vaccines consist of bacterial polysaccharides or viral capsules
directly harvested from cultures of the pathogen. Polysaccharide vaccine antigens are
used against Streptococcus pneumoniae [Hilleman et al. 1981] and Neisseria
meningitidis [Gotschlich et al. 1969] infections and consist of natural surface
polysaccharide purified from cell cultures. The limitation with polysaccharide-based
vaccines is their inability to activate Th cells. Thus B cells are activated in a TI
manner, resulting in no class switch, no affinity maturation and no memory cells
development. It has been suggested that vaccination with polysaccharide antigens
early in life may not be a convenient strategy, because of the induction of negative
memory response that might impair the development of further optimal response to
the same antigen [Sánchez et al. 2001]. Polysaccharides are poor immunogens in
infants and children, whereas the immune responses to carbohydrates may mature
later in life. To improve the problems with poor immunogenicity of polysaccharide
vaccines, the concept of conjugate vaccines was introduced [Tai et al. 1987, Ellis
1999]. This strategy involves the coupling of a polysaccharide antigen to a protein
carrier that transform the antigen into a TD antigen, capable of eliciting protective
IgG and memory responses even in very young children. Subunit conjugate vaccines
have been licensed for Pneumococcus, Neisseria meningitidis and Haemophilus
influenzae type b (Hib), where polysaccharides have been covalently linked to protein
carriers, such as tetanus toxoid or diphtheria toxoid [Wuorimaa and Kayhty 2002,
Kristensen et al. 1996].
Recombinant proteins
The advent of recombinant DNA technology and protein engineering allows
the design and production of recombinant subunit vaccines (Ellis 1999). The epitopes
recognized by neutralizing antibodies are usually found in just one or a few proteins
present on the surface of the pathogenic organism. Isolation of the genes encoding
such epitope-carrying protein immunogens, cloned into a suitable expression vector
and their expression in bacterial, yeast or mammalian cells, make the basis of
recombinant subunit vaccine development [Dertzbaugh 1998, Liu 1998, Babiuk 1999,
Liljeqvist and Ståhl 1999]. The first such recombinant protein vaccine approved for
human use is the hepatitis B vaccine, which was developed by cloning the gene for
Heat shock proteins as vaccine adjuvants
25
the major surface antigen of hepatitis B virus (HbsAg) and expressing in yeast cells
[Valenzuela et al. 1982]. This new vaccine efficiently elicited protective antibodies
upon vaccination of chimpanzees [McAleer et al. 1984], and soon this vaccine
replaced the plasma derived hepatitis B vaccine in human use.
The main advantage of using single proteins displaying immunodominant epitopes is
the possibility of inducing protective immunity without having side effects and
immune reactions caused by other parts of the pathogenic organism. Also, large-scale
production and purification of a well-defined product can also be achieved. However,
there are several limitations of recombinant proteins; a) they are generally poor
immunogens when administered alone and thus unable to induce effector T-cell
responses, such as the CD8+ CTLs, that are necessary for elimination of the
intracellular pathogens, b) they do not carry a sufficient capacity of turning on the
innate response, thus requiring adjuvant help, c) they often elicit only strain specific
protection, d) MHC restriction also limits the ability of the these vaccines to mount an
appropriate cell-mediated response [Good et al. 1988, Quakyi et al. 1989, Carter et al.
1989] and coupling to certain protein carriers may be needed.
Most importantly, recombinant strategies have also been employed for detoxification
of toxins. Engineered inactivation of toxins can be obtained by mutational
replacement of specific amino acids in the enzymatically active part of the toxin.
Pertussis vaccine is produced by specific mutation in the toxin gene from the
Bordetella pertussis [Del Giudice and Rappuoli 1999].
Chimeric composite immunogens can also be created by fusion of different toxins,
such as cholera toxin B subunit (CTB)-Escherichia coli heat labile toxin B subunit
(LTB) hybrid molecules, which are candidate oral vaccines against both enterotoxic
Escherichia coli infection and cholera [Lebens et al. 1996].
Synthetic peptides
Subunit vaccines can be produced by chemical synthesis of short polypeptides.
Synthetic peptides represent parts or complete antigens or selected epitopes that can
be identified from a pathogen’s proteomic sequence, which can induce protective
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Qazi Khaleda Rahman
immunity. This excludes epitopes, which might induce undesired suppression [Mutis
et al. 1994] or nonprotective antibodies [Wrightsman et al. 1994]. Synthetic peptides
offer some advantages; a) the possibility for large-scale production and purification,
b) the possibility of including the desired antigenic determinants by chemical design,
c) the combination of selected B- and T-cell epitopes in various ways to optimize the
resulting immune response in subunit synthetic vaccine. The drawbacks of the small
peptides are that they can be rapidly degraded or excreted in vivo. Also, because of the
size limits of the synthetic peptides, the immune response will be raised only to one
small epitope, that may not be cross-reactive with the native protein. Insufficient
duration of the induced immune responses to peptides also remains a difficulty. The
use of multiple antigen peptide (MAP) could circumvent the problem of the size limits
of the peptides as well as eliminates the need for a carrier. MAP consists of linear
peptide antigens conjugated to a polylysine core [Tam 1988]. It is a unique
presentation system that provides peptide epitopes in multiple copies with high
density of the desired epitopes. Moreover, the design enables circumvention of
immune responses limited by genetic restriction, since non-immunogenic B-cell
epitopes may be combined with T helper epitopes of universal character [Tam et al.
1990, Chai et al. 1992].
New generation vaccines
Modern technologies offer rational strategies for the development of the
newest generation of vaccines, including the DNA (as plasmid) or RNA (mRNA)
vaccines and the live recombinant delivery systems.
DNA vaccines
DNA vaccines are bacterial plasmids carrying genes encoding pathogen or
tumor antigens, which are engineered for optimal expression in eukaryotic cells. The
gene encoding the antigen is placed under the control of a strong mammalian viral
promoter (for this, virally derived promoters, such as from cytomegalovirus (CMV) or
simian virus 40, provide the greatest gene expression) to drive the expression of the
gene of interest directly in the injected mammalian host. To enable bacterial
propagation and to achieve large copy number and high yields, it also contains an E.
Heat shock proteins as vaccine adjuvants
27
coli origin of replication. The antigen-encoding gene will be expressed by the vaccine
upon delivery of the plasmid DNA (Figure 2).
The direct intramuscular inoculation of plasmid DNA encoding several different
reporter genes was first shown to induce protein expression within the muscle cells
[Wolff et al. 1990]. Subsequently, it was shown that DNA vaccines could protect
mice or chickens, from influenza infection [Ulmer et al. 1993, Robinson et al. 1993,
Ulmer et al. 1998]. Immunization of BALB/c mice with plasmid DNA encoding
influenza A nucleoprotein, resulted in the induction nucleoprotein-specific antibodies,
and protection from a subsequent challenge with a heterologous strain of influenza A
virus. The efficacy of DNA vaccination has been reported in small and large animal
models for infectious diseases, e.g. malaria [Hoffman et al. 1997, Le et al. 2000], HIV
infection [Calarota et al. 1998] and cancer [Boyd et al. 2003]. Irrespective of whether
the plasmid encodes a cytoplasmic, membrane bound or secreted antigen,
intramuscularly injected plasmids induce a predominantly Th1 response, with high
levels of IL-2 and IFN-g, a strong cytotoxic T cell response and antibodies
predominantly of the IgG2a subclass [Pertmer et al. 1996, Feltquate et al. 1997,
Haynes 1999]. Repeated immunization with plasmids encoding secreted antigens can,
however, generate more IgG1 than IgG2a antibodies. In contrast, intradermally (using
gene gun) introduced DNA elicits a Th2 like response in animals, with IL-4 producing
CD4+ cells and high levels of IgG1 antibodies [Torres et al. 1997, Boyle et al. 1997
(a), Boyle et al. 1997 (b)].
The processes by which plasmids are internalized and located to the cell nucleus still
remain to be elucidated. It has been suggested that plasmids could enter myocytes via
T-tubules, independently of disruption of the plasma membrane [Wolff et al. 1992].
Cellular uptake of DNA plasmids is a major limiting factor for their immunogenicity.
Intramuscular injection of plasmids immediately followed by electroporation
increases transfection both in vitro and in vivo [Neumann et al. 1982, Widera et al.
2000, Dupuis et al. 2000]. The majority of transfected cells expressing foreign protein
after in vivo plasmid injection are myocytes, although APCs participate in taking up
plasmids by phagocytosis. In the latter case, the DNA seems to be degraded within the
endosomes, and therefore does not lead to antigen expression, processing and
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Qazi Khaleda Rahman
presentation by APCs [Dupuis et al. 2000]. DNA entry into the cytoplasm is
facilitated by adsorption of DNA onto cationic microparticles to form lipoplexes,
which are thought to destabilize the endosomal membrane (Singh et al. 2000). The
delivery of the plasmid DNA with gene gun is a highly efficient way of obtaining
transfection of myocytes and APCs, but it is relatively a cost effective method [Tang
et al. 1992, Condon et al. 1996].
DNA-based vaccines are particularly interesting for several reasons:
a) DNA vaccines have the ability to elicit cellular as well as humoral immunity
[Haynes 1999];
b) they mimic the effects of live attenuated vaccines in their ability to induce
MHC class I restricted CD8+ T-cell responses, which may be advantageous
compared with conventional protein-based vaccines, while mitigating some of
the safety concerns associated with live vaccines;
c) the encoded protein is expressed in the host in its natural form, there is no
denaturation or modification, the immune response is therefore directed to the
antigen exactly as it is expressed by the pathogen, especially for viral
infections [Kowalczyk and Ertl 1999];
d) it is relatively simple to combine diverse immunogens into a single
preparation, thus decreasing the number of vaccinations required;
e) they cause prolonged expression of the antigen, which generates significant
immunological memory and protection, providing important basis for
designing vaccines against HIV, malaria or tuberculosis;
f) DNA vaccines are highly stable, can be manufactured with high purity and
large scale, in a relatively low cost-effective manner and be stored with
relative ease, eliminating the need for a ‘cold chain’;
g) specific sequence motifs called CpG, present in the prokaryotic DNA seem to
act as adjuvant, activating the innate arm of the immune system (this will be
described later in the context of adjuvants).
Heat shock proteins as vaccine adjuvants
29
Gene of interest
Transform into
bacteria
vaccination
Humoral response
Cellular response
Figure 2: Construction of a DNA based vaccine.
The main concern about subunit DNA vaccines is their limited potency, since
myocytes [Wolff et al. 1990] and keratinocytes, which appear to be the predominantly
transfected cell types after intramuscular or intradermal injection of plasmid DNA,
lack the costimulatory molecules necessary to induce a primary immune response.
Moreover, they do not have the intrinsic ability to propagate in vivo as viral vaccines
do. The cytoplasmic localization of the expressed proteins in the muscle cells also
limits the exposure of antigens to the immune cells. Furthermore, for bacterial
proteins, the mammalian post-transcriptional modifications may result in antigens that
differ from the bacterial versions, resulting in reduced immunogenicity. There are
several approaches to increase the potency of DNA vaccines, such as modification of
the mode of delivery [Charo et al. 1999], coadministration of immunostimulatory
genes or DNA [Roman et al. 1997, Krieg et al. 1998, Widera et al. 2000],
coadministration of chemokine [ Kim et al. 2003] or cytokine genes, as GM-CSF
[Haddad et al. 2000, Kumar et al. 2002], IL-12 [Katae et al. 2002] or IL-2 [Bu et al.
2003] encoding genes or costimulatory genes as B7 [Kim et al. 1997],
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coadministration of an immunostimulatory adjuvant or gene encoding cholera toxin or
heat labile enterotoxin [Arrington et al. 2002]. One of the most promising and
attractive strategies to enhance the DNA vaccine potency, is the design of chimeric
DNA constructs e.g. by linking HSP encoding genes with the gene encoding the
protein of interest [Hsu et al. 2001, Planelles et al. 2001]. This system illustrates the
versatility of the DNA vaccination and offers exciting prospects for preclinical and
clinical immunotherapy protocol.
mRNA vaccines
Nucleic acid vaccination through the delivery of RNA has been investigated to
a lesser extent than the DNA vaccination. Naked mRNA may be highly attractive,
owing to lower potential risk of integration into the host genome. The first
applications of the delivery of mRNA were shown to induce CTL to the influenza
virus nucleoprotein in mice when delivered in liposomes [Martinon et al. 1993].
Liposome mediated transfection of mouse fibroblasts with mRNA encoding human
carcinoembryonic antigen [Conry et al. 1995] resulted in a transient production of
antibodies, but the antibody levels declined rapidly, reflecting a short lived protein
expression in vivo. The inherent instability of RNA is a limitation, although the recent
demonstration that RNA can directly transfect DCs may provide a better immunologic
rationale for such an approach. This limitation could be circumvented by constructing
RNA vectors based on parts of alphavirus (Sindbis virus and Semliki Forest virus)
genomes [Tubulekas et al. 1997, Berglund et al. 1998], carrying a gene encoding a
foreign antigen and a gene encoding a alphavius replicase. Upon transfection of such
a construct, the replicase gene will be translated and the produced replicase will massreplicate the antigen-encoding RNA. The transfected cell will express large amounts
of the foreign protein for a short period of time, even when only a few cells are
transfected. Although RNA vectors have been used successfully for immunization, it
does not seem very promising as a method for large-scale vaccination because of the
difficulty and expense of large-scale production.
Live recombinant vaccine delivery systems
Attenuated viruses and bacteria can be modified for use as carriers by inserting
genes encoding a protein from a different pathogen into their genome. In this case the
Heat shock proteins as vaccine adjuvants
31
carrier virus or bacterium enables the delivery of the antigen-encoding gene to the
host, where the antigen is then expressed. By using a carrier virus or bacterium one
can deliver genes from pathogens, which themselves might be considered unsafe, as
an attenuated vaccine (e.g. HIV). Recombinant live vaccine-delivery vectors would
potentially be easier and less costly to produce, since they do not require extensive
purification processes, and since they may be able to elicit long-lasting immunity
without the need for adjuvants. The best-studied bacterial delivery systems are based
on attenuated bacteria such as Salmonella typhi [Darji et al. 1997] and Shigella
[Sizemore et al. 1995], expressing heterologous antigens [Chatfield et al. 1993,
Hackett 1993, Chatfield et al. 1995, Georgiou et al. 1997]. The attractive quality of
these bacteria includes their ability to be administered mucosally. Moreover, being
intracellular pathogen, they are capable of eliciting cellular immune responses to the
antigen delivered. BCG also represents a candidate vector for live recombinant
vaccines, inducing strong cellular and humoral responses against foreign antigens
expressed by recombinant BCG [Aldovini and Young 1991, Stover et al. 1993,
Gheorghiu et al. 1994]. Listeria monocytogenes is also being evaluated for a delivery
vector (Goossens et al. 1995, Dietrich et al. 1998). Among the live viral vectors,
modified vaccinia Ankara [Paoletti 1996] and adenoviral vaccine vectors [Imler
1995], that can carry multiple foreign genes, have been extensively studied. An
attenuated vaccinia vector expressing seven different malarial antigens has been
constructed and demonstrated to induce Plasmodium specific antibody responses in
Rhesus macaques (Tine et al. 1996). One advantage of using viral vectors is the
ability to elicit both humoral and cellular immune responses towards the delivered
target antigen, as a result of intracellular expression of the heterologous antigens, a
desired property of the immune responses protecting against viral or parasitic
diseases.
Improvement of the potency of subunit vaccines
Most traditional licensed vaccines, particularly live attenuated or killed whole
cell, contain many immunostimulatory components, e.g. bacterial DNA, enterotoxin
or HSPs (that is PAMPs), necessary for activating an integrated protective immune
responses. However, the trend in vaccine development is to move towards safer and
better-defined subunit vaccines, produced as highly purified recombinant proteins,
32
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lacking natural immunostimulatory substances and do not evoke strong immune
responses. Moreover, for the development of vaccines against pathogens, causing
chronic infections, e.g. human immunodeficiency virus (HIV), hepatitis C virus,
tuberculosis and malaria, the induction of cell-mediated immunity is likely to be
necessary besides humoral responses. Subunit vaccines have generally proven to be
ineffective at inducing cell-mediated immunity. Therefore, potent adjuvants and novel
vaccine strategies are required to make the vaccine sufficiently immunogenic to
initiate a potent immune response [Fearon 1997, Janeway 1989]. In addition, the
innate immune system directs the balance of humoral and cell mediated immunity
[Fearon and Locksley 1996], and adjuvants can control the type of acquired immune
response induced [Yip et al. 1999].
Adjuvants
Adjuvants (derived from the latin word adjuvare, meaning help or aid) are
defined as a group of structurally heterogenous compounds that enhance or modulate
the immunogenicity of the poorly immunogenic vaccine proteins or peptides [Gupta et
al. 1993, Vogel 1995]. The role of innate immunity in stimulating adaptive immune
responses is the basis of the action of adjuvants. Thus, they often form an essential
part of vaccines. In vaccine development the choice of the adjuvant is often as
important as the selection of the vaccine antigens themselves, which is sufficient to
mimic natural infection or traditional vaccine. The concept of adjuvants arouse in the
1920s from observations such as those of Ramon et al. who noted that horses that
developed an abscess at the inoculation site of diphtheria toxoid generated higher
specific antibody titers. They subsequently found that an abscess generated by the
injection of unrelated substances, along with the diphtheria toxoid, increased the
immune response against the toxoid [Ramon 1959]. The most appropriate adjuvant for
a given vaccine antigen will depend to a large extent on the type of immune response
that is required for protective immunity. Moreover, some adjuvants are strikingly
potent, but also very harmful to the host. Therefore, the potency of an adjuvant often
conflicts with host safety and tolerability.
Adjuvants can be used for various purposes; a) to enhance the immunogenicity of
recombinant antigens, b) to reduce the amount of antigens or the number of
Heat shock proteins as vaccine adjuvants
33
immunizations needed for protective immunity, c) to improve the efficacy of vaccine
in newborns, the elderly or immunocompromised persons or, d) as antigen delivery
systems for the uptake of antigens by the mucosa [Marx et al. 1993, Douce et al.
1995, McElrath 1995].
Role of adjuvants in the immune responses
Precisely, how adjuvants augment the immune response is not known, but they
appear to exert different effects to improve the immune response to vaccine antigens,
as such they:
a)
Improve antigen delivery to APCs, increase cellular infiltration,
inflammation, and trafficking to the injection site,
b)
Promote the activation state of APCs by upregulating costimulatory
signals or MHC expression, inducing cytokine release
c)
Enhance antigen processing and presentation by APCs and enhance
the speed, magnitude and duration of the immune response,
d)
Modulate antibody avidity, affinity as well as the magnitude,
isotype or subclass induction,
e)
Stimulate cell-mediated immunity and lymphocyte proliferation
nonspecifically.
Classification of adjuvants
Adjuvants can be classified according to their source, mechanism of action or
physicochemical properties [Vogel 1998]. Edelmann [reviewed in Allison and Byars
1991] classified adjuvants into three groups based on their principal mechanisms of
action; a) Immunostimulatory adjuvants, being substances that increase the immune
response to the antigen by directly activating APCs through specific receptors e.g.
TLRs, known as adjuvant receptors [Kaisho and Akira 2002], b) carriers, being
immunogenic proteins that provide T-cell help, and c) particulate or vehicle adjuvants
(vaccine delivery systems), serve as a matrix for antigens, mainly function to localize
vaccine components and to target vaccines to APCs. So, delivery systems are used to
promote the interaction of both antigens and immunostimulators with the key cells of
the innate immune system. Immunostimulatory adjuvants provide the inflammatory
34
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context necessary for optimal antigen-specific immune activation by activating APCs
and amplifying the innate immune response.
Most commonly used adjuvants
Adjuvants, currently licensed for human use include alum, squalane oil/water
emulsion (MF59), influenza virosomes, and some cytokines as IFN-g and IL-2. A
number of adjuvants are currently under
investigation as DNA motifs,
monophosphoryl lipid A, cholera toxin (CT), E. coli heat labile toxin (LT), Flt3 ligand
(a pleotropic glycoprotein), immunostimulating complexes (ISCOMs), liposomes,
saponins, non-ionic block copolymers. Some of the most common adjuvants are
described in the following section.
Freund’s adjuvants
In 1940, Jules Freund developed a powerful immunogenic adjuvant composed
of a mixture of mineral oil, a surfactant (Aracel A), and heat killed Mycobacterium
tubercuosis (MTB), which is known as Freund’s complete adjuvant (FCA). This
adjuvant functions to prolong antigen persistence. A muramyle dipeptide, a
component of the mycobacterial cell wall activates macrophages, making FCA very
potent. FCA is considered as a gold standard for immunologists as it is highly
effective at enhancing vaccine responses in animals. But, it is not used for human
vaccination because of the problem associated with its use such as ulcerating tissue
necrosis [Claassen et al. 1992]. Freund’s incomplete adjuvant (FIA) does not contain
the mycobacteria and was licensed for use in an influenza vaccine but it is no longer
used in humans because of the toxic effect of the surfactant, which causes tissue
necrosis.
ISCOMs
Immunostimulating complexes (ISCOMs) are a versatile delivery system and
the concept was first described in 1984 [Morein et al. 1984]. ISCOM is a 40 nm cage
like lipid carrier composed of a glycoside, Quillaja saponin, and cholesterol. The
assembly of the ISCOM structure and the incorporation of the antigen is facilitated by
the addition of phospholipid and is mainly mediated by hydrophobic interactions.
ISCOMs have a strong immunomodulatory capacity, increasing the MHC class II
Heat shock proteins as vaccine adjuvants
35
expression on APCs [Bergstrom-Mollaoglu et al. 1992], activating murine Th cells to
secrete the Th1 type cytokines IL-2 and IFN-g and upregulate IgG2a antibody
responses [Villacres-Eriksson et al. 1992, Villacres-Eriksson et al. 1997, Sjölander et
al. 1998]. It has the capacity to deliver antigen to the MHC class I presentation
pathway, and induces CTL responses after parenteral and mucosal administration
[Villacres et al. 1998, Morein et al. 1998, Jones et al. 1988]. Immunization with
gp120 ISCOMs has been shown to stimulate both IFN-g and IL-4 production in
primates and provide protection against HIV-1 infection [Verschoor et al. 1999].
Thus, ISCOMs also induce a concomitant Th2 response [Maloy et al. 1995], resulting
in balanced Th1/Th2 response.
CpG (cytidine-phosphate-guanosine)
Unmethylated CpG dinucleotide motifs present in bacterial DNA (uncommon
in mammalian DNA) are strong stimulators of immune responses in mammalian
hosts. CpGs in the context of selective flanking sequences are thought to be
recognized by cells of the innate immune system to allow discrimination of pathogenderived DNA from self-DNA [Bird et al. 1987]. These DNA sequences stimulate the
immune system through a specific receptor, TLR-9, which is intracellularly expressed
in human and mouse B-cells and plasmacytoid DCs [Krug et al. 2001, Kadowaki et
al. 2001, Ahmad-Nejad et al. 2002]. Within minutes of exposure of B cells or
plasmacytoid DCs to CpG motifs, they interact with TLR-9, leading to the activation
of cell signaling pathways. These culminate in the expression of MHC and
costimulatory molecules, promote the secretion of Th1 polarizing cytokines as
macrophage inflammatory protein-1, IFN-inducible protein-10, TNF-a, IL-1, and IL12 [Davis et al. 1998, Sun et al. 1998, Krieg 2002] and IgG2a and IgG2b antibody
production [Kumar et al. 2004]. The immune effects of CpG include direct triggering
of B cells, causing proliferation and polyclonal immunoglobulin synthesis, and low
CpG concentrations promote antigen specific immunoglobulin synthesis by
synergistically acting in concert with the B cell antigen receptor [Krieg et al. 1995,
Liang et al. 1996]. CpG also induces the production of type I IFNs and IFN-g
[Klinman et al. 1996], which activate NK cells for enhanced IFN-g synthesis and
increased lytic activity [Cowdery et al. 1996]. CpG DNA alone renders protection
36
Qazi Khaleda Rahman
against a variety of allergens and infectious agents by non-antigen-dependent
mechanisms [Sur et al. 1999, Klinman et al. 1999, Gramzinski et al. 2001, Bohle
2002], and enhances the protective effects of antigen-specific immunity [Near et al.
2002, Uhlmann and Vollmer 2003]. The adjuvant effect of CpG appears to be
maximized by the conjugation to plasmid protein antigens [Klinman et al. 1999], or
their formulation with delivery systems [Singh et al. 2001].
Bacterial toxins
Labile toxins from E. coli and CT from Vibrio cholerae are potent [Lycke
1997] and can induce both systemic and mucosal immune responses when
administered via the parenteral, mucosal or intraperitoneal routes. CT treatment
increases the MHC class II expression on APCs and directly affects B-cell
differentiation [Anastassiou et al. 1990]. Structurally CT is an AB5-complex, which
consists of a pentamer of B-subunit (CTB) surrounding a single A subunit that
contains a linker to the pentamer via the A2 fragment (CTA2) and enzymatically
(ADP-ribosyltransferase) active A1-fragment (CTA2) [Burnette et al. 1994, Rappuoli
et al. 1999]. Two mechanisms of adjuvanticity have been suggested for CT, one
associated with the structural binding properties of the AB5-complex, and the other
dependent on the ADP-ribosylating function of the A1-subunit [Snider 1995, Lycke
1997]. Unfortunately, CT is very toxic to humans, only 5 mg of CT orally resulted in
overt diarrhoea in human volunteers [Levine 1984]. The toxicity is associated with
both the binding of the B-subunit to the GM1-ganglioside receptor (present on all
nucleated cells) and the ADP-ribosyltransferase activity of the A1 subunit.
Recently, it has been shown that a nontoxic form of the CT could be achieved by
redirecting the full enzymatic activity of the CTA1-subunit to target B cells through
the expression of CTA1-encoding gene as a fusion protein together with a dimer (DD)
of an Ig-binding fragment of Staphylococcus aureus protein A [Ågren et al. 1997,
Ågren et al. 1998]. By doing this, the enzymatic activity of CTA1 in CTA1-DD
fusion protein is retained, while preventing the A1 subunit from binding to cells
(epithelial and nerve cells) [Ågren et al. 2000], where it could exert a more
generalized toxic effect. Both CT and CTA1-DD have been shown to bind directly to
B cells, and strongly enhance the expression of costimulatory molecules (CD80/86) in
Heat shock proteins as vaccine adjuvants
37
vivo and in vitro [Ågren et al. 1997], through increased production of cytokines as IL1 and IL-6 [McGee et al. 1993, Bromander et al. 1995, Cong et al. 1997, Eriksson and
Lycke 2003]. CTA1-DD enhances T-cell priming and germinal center reactions
following administration, resulting in augmented specific antibody responses [Ågren
et al. 1999, Lycke 2001]. Recently it has been shown that CTB subunit can act as a
carrier of antigens, and markedly increase and partially direct the DC vaccine induced
immune response with respect to Th1 and Th2 responses (Eriksson et al. 2003)
Alum
Alum are aluminum-based mineral salts (generically called alum) [Gupta
1998], which were first introduced by Glenny in 1926. They precipitated diphtheria
toxoid with potassium alum and found that the precipitate elicited the formation of
antitoxin antibodies more effectively than did the unprecipitated toxoid. Aluminium
salts are insoluble, gel like precipitates of aluminium hydroxide or aluminium
phosphate. Immunogen is bound by electrostatic interactions to pre-formed gel or
during gel formation in situ [Levine et al. 1955]. Alum has been widely used in
human and veterinary vaccines since 1930 and has a good safety record. Alum
induces strong Th2 type of responses, and recent work in vitro indicated that alum
upregulated costimulatory signals on human monocytes and promoted the release of
IL-4 [Ulanova et al. 2001]. Unfortunately, alum is a poor adjuvant for cell-mediated
immunity and can induce IgE antibody responses, which are associated with allergic
reactions in some subjects [Gupta 1998]. The administration of alum containing
vaccines might be associated with the emergence of macrophagic myofasciitis
(MMF), an inflammatory myopathy described recently [Gherardi 2003].
II) RELATED BACKGROUND
It is obvious from different studies that the currently licensed vaccine
adjuvants are not sufficiently effective for the induction of efficient and appropriate
immune responses. Several adjuvants including microbial components have been
evaluated for their ability to induce efficient immune responses in animal models as
well as in preclinical/clinical studies. HSPs are one of the widely studied vaccine
candidates. Our study mainly aims to the evaluation of the adjuvant effect of HSPs in
38
Qazi Khaleda Rahman
different immunization strategies and to explore the mechanism behind its
adjuvanticity.
Heat shock proteins (HSPs)
HSPs are highly conserved molecules, found in prokaryotes, eukaryotes and
even in plants. These proteins undertake crucial functions in maintaining cell
homeostasis and are essential for life since they behave as chaperons [Smith et al.
1998]. HSPs are expressed both constitutively (cognate proteins) and under stressful
conditions (inducible forms). Constitutively expressed HSPs appear to serve as
molecular chaperons, recognizing and binding to nascent polypeptide chains and
partially folded intermediates of proteins, preventing their aggregation and
misfolding. HSPs also participate in protein synthesis, suitable protein folding,
assembly, trafficking and degradation [Lindquist and Craig 1988, Jaattela 1999, Fink
1999, Hartl and Hayer 2002]. Under stress situations, including environmental (heat
shock, exposure to heavy metals or UV radiation), pathological (infections or fever,
malignancies, inflammation or autoimmunity) or physiological stress (growth factor
deprivation, cell differentiation, hormonal stimulation or tissue development)
[Jacquier-Sarlin et al. 1994, Moseley 1998, Feder and Hofmann 1999], HSP synthesis
is markedly increased to protect cells from damage [Gething et al. 1995, Laroia et al.
1999, Jaattela 1999]. HSPs are classified based on their homology, related function
and molecular mass. The most studied HSP families are HSP60, HSP70 and HSP90
[Fink 1999].
HSP70 as adjuvant and carrier
The immunological functions of HSPs began to emerge in the 1980s, when it
was observed that homogenous preparations of certain HSPs that were isolated from
cancer cells elicited immunity to cancers [Srivastava 1998]. That study was first
carried out with the HSP gp96 [Blachere et al. 1993], but similar results were later
obtained with HSP70 [Udono and Srivastava 1993, Ciupitu et al. 2002], HSP90
[Udono and Srivastava 1994], calreticulin [Basu and Srivastava 1999], HSP170 and
HSP110 [Wang et al. 2001]. Among those HSPs, the HSP70 family is well
characterized and attracts much attention because of its versatile functions in the
Heat shock proteins as vaccine adjuvants
39
immune system. It is considered as the ‘workhouse’ of the chaperons, because of its
promiscuity to assist in folding new polypeptide chains [Beckmann et al. 1990,
Liberek et al. 1991, Hartl 1996]. Besides the chaperone activity, HSP70 molecules
can function as endogenous as well as exogenous adjuvants [Vabulas et al. 2002,
Asea et al. 2002]. HSP70s prepared from tumor cells or virus-infected cells are
capable of eliciting CD8+ CTL responses in vivo and in vitro against a variety of
antigens expressed in the cells from which these immunogenic proteins have been
purified [reviewed in Srivastava 2002]. Extremely small quantities of HSP70 bound
peptide, around 120 pM, can generate a CTL response in vivo, whereas 2000-fold
higher concentrations of free peptide was unable to do so [reviewed in Minton 2004,
Javid et al. 2004]. However, an HSP70 mutant with markedly decreased peptidebinding affinity due to a point mutation in the peptide binding domain, could still
induce the production of pro-inflammatory cytokines by DCs, but did not lead to CTL
generation. Thus, the delivery of antigen can be separated from DC stimulation
[MacAry et al. 2004]. In vivo immunogenicity of tumor-derived HSP70-peptide
complexes have been extensively demonstrated in murine, rat and human tumors, and
HSP-based vaccination has proven efficacious in both prophylactic and therapeutic
settings [Srivastava and Maki 1991, Udono and Srivastava 1993, Blachere et al. 1997,
Melcher et al. 1998, Noessner et al. 2002]. Extracellular HSP70 can complex with
antigenic peptides and simultaneously activate professional APCs. This interaction
triggers a cascade of events, including re-presentation of chaperoned peptides to MHC
I restricted CD8+ and MHC II restricted CD4+ T cells, secretion of proinflammatory
cytokines and phenotypic and functional maturation of DCs [Asea et al. 2000,
Castellino et al. 2000, Basu et al. 2001, Harmala et al. 2002, Tobian et al. 2004 (a,b)].
These properties combine to make HSP70 a potent adjuvant that integrates innate and
adaptive immune responses.
HSP70 contains strong T-cell epitopes and serves as a carrier of antigens, effectively
inducing antigen specific B cells as well as CD4+ and CD8+ T-cell responses without
requiring an adjuvant [Barrios et al. 1992, 1994, Del Giudice 1994, Suzue and Young
1996, Roman and Moreno 1996, Rico et al. 1998 and 1999, Udono et al. 2001].
Fusing mycobacterial HSP70 to HIV-1 gag p24 [Suzue and Young 1996 (a), Suzue
and Young 1996 (b)], or synthetic malarial antigen (NANP)40 [Barrios et al. 1992],
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Qazi Khaleda Rahman
enhanced the immunogenicity of the antigens and obviated the need for adjuvant.
Mice immunized with a membrane protein (KMP11) covalently fused to HSP70 from
Trypanosoma cruzi elicited a CTL response against the Jurkat-A2/Kb cells expressing
the KMP11 protein [Marañón et al. 2001]. Moreover, HSP70 has been used as a
carrier for group C meningococcal oligosaccharide, inducing antibodies against
oligosaccharide in mice [Perraut et al. 1993]. Furthermore, chimeric proteins formed
by antigens coupled to the C-terminal fragment of HSP70 from MTB [Wang et al.
2002, Lehner et al. 2004], and N-terminal fragment from Leishmania infantum [Rico
et al. 1999] induced humoral and cell mediated immune responses to the coupled
antigens.
HSP70 receptors and mechanism of adjuvanticity
The existence of receptors on APCs, specifically mediating the cellular
internalization of HSPs was postulated in 1994 by Srivastava [Srivastava et al. 1994].
The first HSP70 receptor was identified in 2000, eliciting considerable interest in this
area [Binder et al. 2000]. It has been suggested that the signaling and crosspresentation of chaperoned peptides, might be mediated by different sets of receptors.
CD91 is a putative receptor for HSP70, which is specifically endocytic, whereas TLR2 and TLR-4 are implied as the signaling receptors [reviewed in Binder et al. 2004].
The adjuvanticity of HSP70 is based on the specific interaction of HSPs with the
receptors present on professional APCs (DCs and macrophages) having two distinct
consequences: 1) stimulation of an innate response (regardless of chaperoned
peptides) and 2) activation of adaptive immune events through representation of HSPchaperoned peptides to MHC molecules, therefore integrating innate and adaptive
immune events. HSP70 activates DCs through binding to its cognate receptor
CD14/TLR-4 or TLR-2 complexes, expressed on those cells. This is a non-antigenspecific event and important for efficient priming of T cells. TLR4/2 receptor
mediated binding initiates signaling cascades in immature DCs [Suzue et al. 1997,
Castellino et al. 2000] causing them to differentiate and migrate from the periphery to
the draining lymph nodes. This leads to several activities, including up-regulation of
MHC and costimulatory (CD86/83) molecules, induction of chemokine secretion,
production of NO and secretion of inflammatory cytokines such as IL-1b, IL-12, IL-6
Heat shock proteins as vaccine adjuvants
41
and TNF-a [Asea et al. 2000, Moroi et al. 2000, Kuppner et al. 2001]. HSP70 can
also interact specifically with the CD91 receptor [Basu et al. 2001], that mediates
endocytosis and results in cross-presentation of HSP70-associated peptides to both
CD8+ and CD4+ T lymphocytes [Udono et al. 2001]. This alternate MHC I antigen
processing and cross-presentation is mediated via cytosolic mechanisms in dendritic
cells and vacuolar mechanisms in macrophages [Tobian et al. 2004 (b)]. Therefore,
the remarkable immunogenicity and adjuvanticity of HSP70 may be ascribed to two
crucial features: HSP70 as cross-priming adjuvant and as direct activators of
professional APCs. Figure 3 illustrates the HSP70-APC interaction that integrates
innate and adaptive immune events.
Adaptive immune
effect of HSP70
Innate immune
effect of HSP70
Figure 3: Role of HSP70 in innate and adaptive immunity (This figure is adapted
from Srivastava 2002).
TLR2/4 are the major receptors involved in transducing HSP70-mediated signaling
through activation of the MyD88/NF-kB [Asea et al. 2002, Vabulas et al. 2002].
However, it is not yet clear how activation by HSP70 internalized via CD91 occurs. It
has been postulated that HSPs, transported in the endocytic vesicles by CD91mediated internalization, by increasing their local concentration, might became able to
42
Qazi Khaleda Rahman
trigger signaling through the TLR2 and TLR4 present in these vesicles [Vabulas et al.
2002].
In addition to TLR2 and TLR4, other cell surface receptors, such as CD40 have been
found to be potentially involved in transducing activation signals of HSP70 to APCs,
[Wang et al. 2001, Becker et al. 2002]. The idea of the involvement of CD40 in the
interaction of HSP70 with APCs draws indirect support from different studies. Millar
et al. (2003) reported that immunization with lymphocytic choriomeningitis virus
derived antigenic peptide together with recombinant HSP70 can break tolerance to the
peptide expressed as a self-antigen in transgenic mice. This tolerance breaking
activity is not seen in CD40-/- mice. Lazarevic et al [Lazarevic et al. 2003] observed
that CD40-/- mice succumb to MTB infection, whereas CD40L-/- mice are MTB
resistant. Nolan et al. (2004) demonstrated that CD40 can be activated independent of
CD154 in poly microbial sepsis and this activation in sepsis may be in part mediated
via HSP70. Both human [Becker et al. 2002] and MTB HSP70 [Wang et al. 2001]
have been shown to bind the CD40 receptor, and function as Th1 type adjuvant.
Interaction of MTB HSP70 with CD40 causes human DCs to release IL-12 and CC
chemokines such as RANTES, MIP1-a and function as Th1 type adjuvant [Wang et
al. 2001]. Interestingly, the binding site within the HSP was found to be different for
different HSP70 families. It has been demonstrated that the human NH2-terminal
ATPase domain of HSP70 binds one site (exoplasmic domain of CD40), whereas the
microbial C terminal peptide binding domain binds another site of the CD40 molecule
[Wang et al. 2001, Becker et al. 2002, Wang et al. 2002]. Moreover, binding of
human HSP70 to CD40 has a dual role in addition to stimulating activation of p38.
Human HSP70 mediates the uptake of peptides bound to its substrate binding domain
and delivers it into the MHC class I pathway. This process cannot be served by
microbial HSP70, considering that its substrate-binding site is occupied by CD40. A
scavenger receptor LOX-1, expressed by macrophages and immature DCs has also
been identified as a receptor for HSP70. This receptor is involved in HSP-mediated
cross-presentation of antigen but not in APC activation [Delneste et al. 2002,
Theriault et al. 2005]. Recently, Tobian et al. have shown that the uptake of HSP70peptide complexes, for the delivery to MHC II processing pathway, was not mediated
Heat shock proteins as vaccine adjuvants
43
by CD91 receptor and was independent of MyD88 and CD40 signaling [Tobian et al.
2004 (a)].
Role of LPS in HSPs activity
Whether the stimulatory effects mediated by the HSPs is due to the presence
of LPS or not [Wallin et al. 2002, Bausinger et al. 2002] have been under debate for
last few years, since some of the functional activities of the two molecules are similar
and imposes a burden of proof on molecules suggested to be HSPs receptors. Several
experimental findings suggested that HSP70-mediated effects are independent of LPS
action. It has been shown that treatment of HSP preparations with polymyxin B that is
a potent inhibitor of LPS does not reduce their activity [Asea et al. 2000, Dybdahl et
al. 2002, Wang et al. 2002]. On the other hand, treatment with heat or proteinase K
abrogates the ability of HSP to stimulate cells in vitro, but does not inhibit LPSmediated stimulation [Rico et al. 1999, MacAry et al. 2004]. Since the stimulating
activity of HSP70 is dependent on calcium flux, the intracellular calcium chelator
BAPTA-AM (BAPTA stands for bis-o- aminophenoxy ethane- N,N,N',N'-tetraacetic acid) has been used to differentiate between LPS and HSP70 functions (Asea et
al. 2000, Wang et al. 2001, MacAry et al. 2004). Furthermore, CD40 and CD91 are
ascribed as the putative receptors for HSP70 [Basu et al. 2001, Wang et al. 2001] and
antibodies to CD14 but not those to CD40, suppress the effect of LPS stimulation
[Wang et al. 2001]. However, there is growing evidence suggesting the involvement
of LPS in the immunomodulatory effect of HSPs. Wallin et al. (2002) have found that
highly purified HSP70 at a concentration of 200-300 mg/ml failed to stimulate murine
DCs, whereas HSP70 preparations containing tiny amounts of LPS induced DC
stimulation and such preparations were heat sensitive and were not inhibitable by
polymyxin B. According to the observations of Gao and Tsan (2003), the LPS
contamination of recombinant HSP70 is responsible for its CD14/TLR-4 mediated
effects on monocytes and DCs and the highly purified, LPS free recombinant HSP70
has lost the capacity to induce the expression of any of the 96 common cytokine genes
in murine macrophages [Gao and Tsan 2004]. The binding of HSP70 to ANA-1
macrophages has also been shown to markedly increase after stimulation with LPS
[Becker et al. 2002]. A recent finding sheds new light into the role of LPS in HSPs
activity. It has been reported that the capacity of HSPs to activate innate immune cells
44
Qazi Khaleda Rahman
depends on LPS, and that macrophage stimulation by HSP60 and HSP70 is not due to
free LPS, but to LPS tightly bound to intact HSP molecules [Triantafilou et al. 2001,
Habich et al. 2005]. Following LPS stimulation, HSP70 and HSP90 form a cluster
with TLR4 within lipid microdomain, transferring the TLR4-MD2 complex onto the
cell surface, and assist further in the trafficking and targeting of LPS to the Golgi
apparatus [Triantafilou and Triantafilou 2002 and 2004]. Therefore, the binding of
HSP70 within the lipid raft might be the mechanism of HSP70 delivery and release to
the plasma membrane. Figure 4 depicts the hypothetical model of the signal
transduction complex formation of LPS with HSP70 and HSP90 proposed by
Triantafilou and Triantafilou 2002.
LPS binding protein (LBP)
binds and catalyzes the
transfer of LPS to
membrane bound CD14
LPS is released from CD14
in the lipid bilayer, and the
intercalated LPS forms
complex with chemokine
receptor 4, HSP70 & 90.
TLR4 complexed with
MD-2, other TLRs are
further recruited into the
activation cluster
triggering multiple
signalling cascades.
Figure 4: Schematic representation of signal transduction complex formation of LPS
that contains HSP70 and HSP90 (Adapted from Triantafilou and Triantafilou 2002).
HSP70 in association with autoimmunity
One peculiar aspect of HSPs is their sequence conservation, leading to
homologies between bacterial and mammalian members of the same HSP family.
Therefore, immunization with bacterial HSP might lead to the induction of immune
responses against self-HSP, which may end up in autoimmune reactions. Although
very little is known about the involvement of HSP70 in autoimmunity, some studies
Heat shock proteins as vaccine adjuvants
45
have shown that recognition of HSP70 by antibodies and T cells induce autoimmune
conditions [Abulafia-Lapid et al. 2003]. HSP70 from the malaria parasite P.
falciparum has been shown to react with the homologous human HSP [Mattei et al.
1989]. Various studies have provided evidence, suggesting involvement of HSP70 in
atherosclerosis [Kanwar et al. 2001]. Moreover, Millar et al. (2003) observed that
HSP70 could induce autoimmunity in a mouse model. They found that HSP70
induces maturation of DCs in vivo, which then stimulated T cells to division and
differentiation into immune effector cells. If the effector T cells recognize a particular
self-antigen, then organs bearing that self-antigen can be targeted for tissue
destruction.
However, several studies carried out in a variety of distinct autoimmune and
autoimmune inflammation related diseases have shown that the occurrence of disease
coincided with the generation of immunity to HSPs, and it has appeared that such
immunity can represent the response of regulatory T cell during disease [van Eden
and Young 1996]. Despite the paradigm of self-tolerance, HSP-epitopes homologous
to endogenous host HSP sequences have been implicated as T cell epitopes to endow
cross-reactive, HSP specific T cells with the capacity to regulate inflammation, such
as in experimentally induced autoimmune diseases. As a possible reflection of such
mechanisms, in a number of studies, self HSP cross-reactive T cells have been
observed to be skewed toward the production of IL-10, which can be a mediator of the
regulatory effects of such T cells. The selective up-regulation of HSP at sites of
inflammation, due to cellular stress caused by the locally produced toxic proinflammatory mediators, is possibly essential for the function of host HSP to attract
regulatory T cells and to let them exert their regulation (van Eden et al. 2003). It has
been shown that T cell responses to HSP70 modulate the arthritogenic response in
adjuvant-induced
arthritis.
Moreover,
immunization
with
HSP70
peptides
encompassing conserved epitopes led to induction of protection [Wendling et al.
2000]. It is suggested that the regulatory mechanisms induced by HSP70 are
reinforced by an immune network that connects their reactivities [Quintana et al.
2004]. Three mechanisms have been proposed for anti-inflammatory T cell induction
by HSPs; 1) Altered peptide regulation: microbial HSP reactive T cells perceive selfHSP homologues as partial agonists or altered peptide ligands and develop a
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Qazi Khaleda Rahman
regulatory phenotype; 2) Mucosal tolerance: HSP reactive T cells recognize microbial
HSP in the tolerizing gut associated lymphoid tissue (GALT) and display a tolerizing
activity when confronted with self-HSP expressed elsewhere in the body; 3) Anergy:
non-professional or non-activated APCs present constitutively self-HSP in the
absence of costimulation. The resulting self-HSP specific ‘anergic’ T cell can exert
regulatory activity following the encounter with professional or activated APC
presenting up-regulated self-HSP [van Eden et al. 2003].
Plasmodium antigen EB200
Of the more than 5300 genes identified for the P. falciparum malaria parasite
[Gardner et al. 2002], about 20 antigens are currently being investigated for the
development of vaccines. EB200 [Mattei et al. 1992] is one of the vaccine candidate
antigens, derived from a giant protein Pf332 of 750 kDa from P. falciparum. EB200 is
a 140 amino acid sequence and is expressed during the development of the
trophozoite and schizont stages [Mattei et al. 1992]. Most protein antigens from
malaria parasites identified as vaccine candidates are polymorphic in natural parasite
populations. There exists a certain degree of diversity in the Pf332 gene [MercereauPuijalon et al. 1991], but in general the EB200 fragment is conserved and stably
expressed in parasite isolates [Fandeur et al. 1996]. However, immunization of mice
with recombinant EB200 evokes a genetically restricted response. H-2d and H-2k mice
are high responders, whereas H-2b, H-2q and H-2s are low responder strains [Ahlborg
et al. 1997]. This drawback makes EB200 less potent for creating a universal vaccine,
providing that the same limitation could occur in the genetically heterogenous human
population. An important aspect of vaccine development against infectious diseases,
including malaria, is the identification of an appropriate carrier and adjuvant, which
are capable of both stimulating a protective immune response and being safe for use
by humans.
III) THE PRESENT STUDY
Aims
One major challenge in developing effective vaccines, is to design a vaccine
that can induce effective immune responses to the desired antigen with no or very
Heat shock proteins as vaccine adjuvants
47
limited side effects. Poor immunogenicity and MHC restriction hamper the potential
of many candidate antigens. The immunogenicity can be improved by using
appropriate carriers and adjuvant molecules. HSPs are highly immunogenic and
function as adjuvants that may play a crucial role in integrating innate and adaptive
immunity. Our main strategy was to evaluate the adjuvant effect of HSP70 and to
explore the possible mechanisms and effectiveness of selected members of the HSP70
family in exerting adjuvanticity in a mouse model.
The specific aims for each paper are listed below:
Paper I: In this study we aimed to understand the cause of low responsiveness of the
EB200 antigen in C57BL/6 mice, and to explore possible ways to overcome low
responsiveness by using a carrier and various adjuvant molecules, including HSP70.
Paper II: In this paper we investigated whether the less conserved C-terminal
fragment of HSP70 (Pf70C) could exert the adjuvant effect. Later on we evaluated the
immunostimulatory activity of this Pf70C delivered as a chimeric DNA construct
fused with the EB200 gene.
Paper III: During the infection process the expression of HSPs is upregulated and can
mediate T cell and B cell sensitization. Since HSPs are one of the most conserved
proteins through evolution, we wanted to see if exposure to a number of
microorganisms could prime the immune system to evolutionary diverse HSPs and to
any antigen coupled to HSP.
Paper IV: To assess the role of TLR2 and TLR4 in HSP70 mediated adjuvanticity, we
aimed to evaluate the immune response of a thymus dependent antigen, OVA,
administered together with HSP70 in TLR2 and TLR4 knockout mice.
Results and discussion
In the following section I intend to recapitulate the results presented in papers
I-IV and discuss our findings in relation to the current knowledge and previous
findings in the relevant field.
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Qazi Khaleda Rahman
Paper I: Effect of carrier and adjuvants in improving immune responses to EB200.
Although EB200 is considered to be a potential vaccine candidate antigen for
malaria in humans, it induces poor immune responses in mice of certain MHC
haplotypes [Ahlborg et al. 1997]. Such poor responsiveness of mice has been shown
to be circumvented by coupling vaccine antigens to protein carriers. In this light, we
evaluated the carrier effect of GST, an immunostimulatory protein [Ouaissi et al.
2002], in EB200 low responder C57BL/6 (H-2b) mice and compared the response
induced in high responder CBA (H-2d) strain. Our results indicate that GST, as carrier
of EB200 helps in overcoming the MHC restriction in the antibody responses to
EB200.
It was still possible that other aspects related to the B cell responses were different in
low and high responders. Therefore, we studied the B cell repertoire by generating
hybridoma cell line collections from both CBA (high responders) and C57BL/6 (low
responders) mice after immunization with GST-EB200. Analysis of the antibody
reactivity pattern in supernatants from both hybridoma collections and in the serum of
immunized mice, indicated that the antibody reactivity pattern was comparable in
both strains of mice, suggesting that the B cell repertoire in EB200 high- and lowresponder strains is similar. However, we observed differences in the individual
specificity of the antibodies in the hybridomas, when tested against a panel of 17
synthetic peptides spanning the EB200 sequence. Some hybridoma lines displayed
reactivity only with the intact EB200 molecule and the others only with the peptides.
The reactivity with the complete EB200 protein may be explained by the recognition
of conformational epitopes, while the peptide specific antibodies may have been
generated against fragments of partially degraded EB200.
Comparative analysis of the magnitude and antibody affinity pattern elicited in the
serum of C57BL/6 and CBA mice indicated that the T cell help was still not sufficient
enough to induce optimal humoral responses. A number of adjuvants with different
modes of action were chosen to improve the immune response to EB200 in C57BL/6
mice. We have shown that the combination of adjuvant as CT and HSP70 promoted
efficient immune responses in the low responder C57BL/6 mice, generating
Heat shock proteins as vaccine adjuvants
49
antibodies of similar or higher affinity than those induced in the high responder CBA
strain.
HSPs are versatile molecules, and several studies indicate that HSP chaperoned
peptides intersect with the peptide traffic that leads to antigen presentation by MHC
molecules. Thus, HSP70 may chaperon EB200-derived peptides, leading to a better
antigen presentation to MHC molecules. Another explanation to the adjuvanticity of
HSP70, might be a more efficient targeting of the innate immune system by triggering
a signal cascade through TLRs [Asea et al. 2002, Vabulas et al. 2002]. CT, the other
adjuvant used, has been found to strongly enhance antigen presentation, by induction
of IL-1 by macrophages and upregulation of costimulation [Cong et al. 1997,
Bromander et al. 1995, McGee et al. 1993]. Thus, the favorable adjuvant effect
obtained by the combination of CT and HSP70 may be explained by the
complementary properties of both adjuvants.
Paper II: DNA based priming with a P. falciparum antigen fused to HSP70.
The use of subunit recombinant proteins and synthetic peptide vaccines is very
promising and offers several advantages in comparison to other vaccines, e.g.
generation of good humoral responses and reduced toxicity. They are, however,
generally poor immunogens when administered alone, and require strong adjuvants
for eliciting appropriate immune responses. We have previously demonstrated that
HSP70 greatly enhances the immune responses to the malarial antigen EB200. Due to
the high degree of sequence homology existing within the HSPs family, there is a
potential risk that immunization with bacterial HSPs might lead to autoimmunity,
which will impose a risk for use in human vaccines. Therefore, in the present study
we evaluated the adjuvant potential of the less homologous C-terminal fragment of P.
falciparum HSP70 (Pf70C), in comparison with that of the whole HSP70 molecule of
T. cruzi (TcHSP70). Even though it is implied that the C-terminal part is less
homologous, we found that antibodies generated against both HSPs cross-reacted well
with each other and induced memory responses. Also, in this work we showed that
both TcHSP70 and Pf70C exhibited adjuvant effect when coadministered with the
antigen OVA. This indicates that the C-terminal fragment could replace the complete
protein as adjuvant.
50
Qazi Khaleda Rahman
DNA based vaccines emerged as a promising approach for vaccine development, but
one of the concerns with DNA vaccines is their limited potency in the context of
inducing humoral responses. Previous studies with DNA or RNA based immunization
of EB200 and other malarial antigens have shown that the antibody responses induced
were generally low [Andersson et al. 2001, Haddad et al. 1999]. The linkage of
antigen encoding gene to the HSP70 gene has been shown to enhance the DNA
vaccine potency [Chen et al. 2000]. On the basis of the above-mentioned results, we
aimed to assess the ability of Pf70C to modulate the immune response to EB200
delivered as a chimeric DNA vaccine. No major increase of EB200 specific antibodies
was detectable by immunizing mice with different DNA constructs containing EB200
encoding gene, not even by including Pf70C in the construct. This could be due to
inefficient priming, with the antigen expressed at too low concentrations at the site of
administration in the muscles. However, the DNA immunization efficiently primed
the immune system, generating a memory response, as indicated by the increased
production of EB200 specific IgG2a elicited by a subsequent boosting with the
recombinant fusion protein Pf70C-EB200. No priming effect was observed for IFN-g
production, but stimulation with the Pf70C-EB200 fusion protein induced an
enhanced secretion of IFN-g. Thus, our results corroborate the previous observations
that DNA-based immunizations are efficient in generating B-cell memory, even in the
absence of any substantial induction of antibody production [Laylor et al. 1999].
Furthermore, the presence of Pf70C in the chimeric construct contributed to the
generation of not only a Th2, but also a Th1 type of responses.
Paper III: Exposure to mycobacteria primes for phylogenetically diverse HSPs.
HSPs are a large family of proteins with different molecular weights and
different intracellular and cell surface localizations [Hantschel et al. 2000, Welch and
Suhan 1985, Kurucz et al. 1999]. These proteins undertake crucial functions in
maintaining cell homeostasis, which may be the reason for being conserved during
evolution. In spite of their high degree of conservation, HSPs have been shown to
behave as immunodominant antigens in many bacterial and parasitic infections
[Young 1990], and act as adjuvants and carriers of antigens. In our previous study, we
have shown that a fragment of HSP70 (Pf70C) exerts a potent carrier effect in mice,
Heat shock proteins as vaccine adjuvants
51
when conjugated to the malarial antigen EB200 (Pf70C-EB200) delivered as fusion
protein or chimeric DNA [Qazi et al. 2005].
During the infection, the synthesis of HSPs is upregulated, and is known to sensitize T
cells in the infected host [Kaufmann et al. 1990]. Since, HSP molecules are highly
conserved throughout evolution, we postulated that priming of mice with
microorganisms, would facilitate the induction of memory T- and B-cell through
HSPs and these cells can cross-react with HSPs of different origins. Moreover, T-cells
induced after priming would be recalled to help the antigen specific B cells.
We first tested our hypothesis by exposing mice to BCG followed by boosting with
the recombinant fusion protein Pf70C-EB200, to see if BGC prime for Pf70C as well
as for EB200 antigen. Later on we assessed the priming efficacy of BCG on various
evolutionary diverse HSPs of different families. We showed that both live and heat
killed BCG could prime the immune system to induce a secondary IgG response to
Pf70C. Moreover, Pf70C served as a carrier for the induction of EB200 specific IgG
antibodies. We also observed that BCG primed the immune system to induce memory
responses to phylogenetically diverse HSPs with high molecular weight (MW). No
priming was observerved against the low MW HSPs. HSP70 is one of the
immunodominant antigens in BCG and contains strong T-cell epitopes [Lehner et al.
2000], providing a helper effect in vivo when conjugated to synthetic peptides,
bacterial oligosaccharides or any subunit antigens [Barrios et al. 1992, Lussow et al.
1991, Perraut et al. 1993]. In our system, priming of mice with BCG might have led
to the induction of a pool of memory T cells, that underwent clonal expansion upon
boosting with evolutionary diverse HSPs, by recognizing conserved (cross-recognize)
epitopes on HSPs molecules. A priming effect was also exerted by heat-killed BCG,
to induce anti-Pf70C and anti-EB200 antibodies. One explanation for the
effectiveness of heat killed BCG may be that the HSPs are more protected to heat
denaturation inside the cells, so that the immunodominant epitopes remain intact after
heating.
As HSPs are widely distributed in microorganisms, we reasoned that other
mycobacteria or intracellular bacteria could also provide priming of T cells.
52
Qazi Khaleda Rahman
Therefore, we tested the same protocol of priming using M. vaccae and C.
pneumoniae. We found that only M. vaccae but not C. pneumoniae primed for Pf70C
and for other diverse HSPs used for boosting. Moreover, Pf70C served as a carrier,
inducing enhanced EB200 specific response. It is not clear to us why priming with C.
pneumoniae did not work in this context. One explanation could be that the mode of
infection with Chlamydia is different from mycobacteria, or it is also possible that
during the infection process, the expression of HSPs was not upregulated sufficiently
to be recalled by the HSPs used for boosting.
The involvement of bacterial HSPs in autoimmune phenomena may be considered as
a potential caveat for including HSPs in human vaccines due to the homologies
between bacterial and human HSPs [Jindal et al. 1989]. We investigated whether
antibodies induced in mice immunized with MTB70 would cross-react with a panel of
autoreactive antigens. We found that the M. vaccae primed mice, followed by
immunization with MTB70 induced cross-reactive antibodies but the reactivity was
low. Cross-reactive antibodies are frequently detected in sera from healthy individuals
and commonly induced in primary immune responses shortly after challenge with the
antigens. Consequently, the presence of cross-reactive antibodies does not necessarily
have to be correlated with autoimmunity. It has been shown before that treatment with
HgCl2 induces in SJL mice kidney damage, promoted by the accumulation of
antibody deposits in the kidneys [al-Balaghi et al. 1996]. No immune complexes were
detected in the kidneys of the HSPs treated mice in this study.
A new concept is emerging, suggesting a new role for HSPs as sensors for internal
and external danger, which may explain the presence of HSPs in the site of injure
more as a consequence than as the cause of the reaction. Different experimental data
strongly support the view that conserved HSPs (self or foreign) are indeed negotiators
between danger and control mechanisms of autoimmunity [van Eden et al. 2003].
Thus, the priming to microbial HSP could be regarded more as a regulatory effect
than an enhancing event for autoimmunity. More studies have to be performed to
clarify this issue, but our findings that the HSP70 induced cross-reactive antibodies at
least do not accumulate in the kidney and, thus, are not apparently pathogenic, support
this idea.
Heat shock proteins as vaccine adjuvants
53
There is widespread recognition of the need for improved vaccines for control of
infectious diseases, and scientists are searching for appropriate combinations of
antigens and adjuvants or suitable carrier molecules for inclusion in subunit vaccines.
Our approach of immunization is particularly interesting for the development of
vaccine strategies, since BCG is widely used very early in life as a vaccine against
tuberculosis, and a large number of people are sensitized to mycobacteria or other
parasites through natural contact. Collectively, our results provide support and offer
rationale for the utility of HSPs in vaccine design.
Paper IV: Mechanisms of HSP70 adjuvanticity
Our previous studies have shown that recombinant HSP70 from T. cruzi
(Tc70) and from P. falciparum (Pf70C) function as adjuvants and greatly enhance the
antibody response to OVA and other thymus dependent (TD) antigens when
coadministered with them [Qazi Rahman et al. 2003, Qazi Rahman et al. 2005]. Since
HSP70 has been shown to activate professional APCs by binding to TLR2 and TLR4
expressed on APCs [Vabulas et al. 2002, Asea et al. 2002], in the present study we
extended our previous observations using other HSPs. We investigated the role of
TLR2 and TLR4 in HSP70 mediated adjuvanticity regarding the induction of antigen
specific humoral responses. We evaluated the adjuvant effect of various HSP70
molecules in TLR2 and TLR4 knockout mice. Our results revealed that within the
same family, HSP70 displayed different degrees of adjuvanticity, regarding both the
strength and the profile of the induced immune response. Furthermore, the HSPs
tested, possessed different requirements for signaling through TLR receptors. We
found that HSP70 from T. cruzi induced OVA specific humoral responses in both
TLR2 and TLR4 knockout mice, meaning that the adjuvant effect is independent of
TLR2 and TLR4 signaling. In contrast, both MTB70 and its C-terminal fragment
elicited a response in TLR2-/- but not in TLR4-/- mice, which means that TLR4 but not
TLR2 is required to stimulate the OVA specific responses. For the C-terminal
fragment of P. falciparum, the adjuvant effect was abolished in both TLR2-/- and
TLR4-/- mice, indicating that in this case, adjuvanticity is dependent on both TLR2
and TLR4 signaling. We also observed that only Tc70 potentiated the induction of a
mixture of Th1 and Th2 type of antibodies in wild type, TLR2-/- and TLR4-/- mice.
54
Qazi Khaleda Rahman
As Tc70 is an efficient adjuvant in both TLR2-/- or TLR4-/- mice, it is possible that
both TLR2 and TLR4 are redundant and function independently for Tc70 signaling.
This has been shown for Chlamydia derived HSP60 [Da Costa et al. 2004]. TLR2/4
double knockout mice were completely unable to respond in terms of CC chemokine
production, while the single knockout strain responded normally [Da Costa et al.
2004]. It may be also possible that other TLRs, different from TLR2 and TLR4, are
involved in this process. This will confer a broader and therefore, more interesting
role for HSPs as sensors of danger. In this scenario, HSPs could be able to recognize
not only LPS, but also other PAMP (pathogen associated molecular patterns) on
microorganisms. Finally, other receptors, different from TLRs, might be involved in
HSP promoted adjuvancy. Since CD40 was reported to be the signaling receptor for
HSP70 expressed on macrophages and DCs [Wang et al. 2001, Becker et al. 2002,
Lazarevic et al. 2003, Nolan et al. 2004], it is also possible that the adjuvant effect of
Tc70 may be mediated through direct binding of HSP70 to CD40. Ligation of CD40
may then activate APCs by increasing expression of costimulatory molecules [Caux et
al. 1994, Sallusto and Lanzavecchia 1994] and the production of inflammatory
cytokines [Kiener et al. 1995], which ultimately instruct the adaptive immune
response to generate antigen specific T and B cells.
Regarding the adjuvant effect of Pf70C, it is not clear to us why the effect is totally
diminished in both TLR2 and 4 knockout mice, since the wild type mice responded as
efficiently as the groups received other HSP70. Perhaps, TLR4 needs to form
functional heterodimers with TLR2 for this particular HSP70 signaling.
It remains to be elucidated the exact mechanism of Tc70 adjuvancy in vivo. Therefore,
future work will be directed toward focusing the adjuvant effect of Tc70 in TLR2TLR4- double deficient mice, CD40-/- mice, or mice knocked out in the signaling
molecules downstream to the TLRs, i.e. MyD88 or IRAK.
Concluding remarks
The studies presented in this thesis have shown the following:
·
GST, as carrier of EB200 helps in overcoming the MHC restriction in
C57BL/6 mice
Heat shock proteins as vaccine adjuvants
·
55
Combination of adjuvants as CT and HSP70 promotes efficient immune
responses in C57BL/6 mice, generating antibodies of similar or higher affinity
and magnitude than those induced in CBA mice
·
The less homologous C-terminal fragment of HSP70 (Pf70C) can also exert
potent adjuvant effect compared to the whole HSP70 molecule
·
Mice immunized with DNA vectors containing the Pf70C gene fused to the
sequence coding for the a subunit antigen EB200, can induce EB200 specific
antibodies associated with Th1 type of responses
·
Mice primed with live or heat-killed BCG or M. vaccae but not with C.
pneumoniae, followed by boosting with recombinant fusion protein Pf70CEB200, can generate secondary anti-Pf70C IgG antibodies, and Pf70C can act
as a carrier to induce EB200 specific secondary responses
·
The fusion protein Pf70C-EB200 can effectively stimulate spleen cells from
BCG and M. vaccae primed mice to produce IFN-g in vitro
·
Exposure of mice to live BCG and heat-killed M. vaccae, but not to C.
pneumoniae, can prime the immune system for HSPs of different families,
inducing mixed of Th1 and Th2 type of responses
·
HSP70 specific sera cross-react to a certain extent with some autoreactive
antigens, but no immune complex deposits are observed in the kidneys of HSP
treated animals
·
HSP70 from various origins display different degrees of adjuvanticity,
regarding both the strength and the profile of the induced immune response
·
Coadministration of Tc70 with OVA, can elicit OVA specific Th1 and Th2
type of antibodies in WT, TLR2-/- and TLR4-/- mice while MTB70 and Pf70C
can induce only Th2 type antibodies in WT, TLR2-/- and WT mice,
respectively
·
LPS cannot stimulate OVA specific Th1 type antibodies
Collectively, we observed from our studies that for the induction of Th1 type of
responses, it is not always essential for the antigen to be physically linked to HSP
molecules. Our findings are the base for a model, trying to emphasize
immunomodulatory properties of HSP70, as well to explain the mechanisms by which
the HSP70 molecule elicits its adjuvant effect. In search for new vaccine adjuvants to
56
Qazi Khaleda Rahman
modulate the potency of recombinant proteins, HSP70 arises a good candidate to be
used as an adjuvant and carrier for the application of a wide variety of infectious
diseases. Moreover, the incorporation of the HSP70 encoding gene in a DNA vaccine
vector as a chimeric construct, is an attractive strategy to augment the immune
response to fused antigens, and makes it a promising candidate for new generation
vaccines. The inclusion of HSPs in DNA vaccine constructs can also be particularly
interesting, since contamination with LPS and other products from bacteria is a major
problem with recombinant vaccines and with DNA vaccines this problem can be
avoided. In addition, the widespread exposure to microbial HSPs through natural
infection or vaccination may trigger the priming of specific T cells, and the high
frequency of HSP reactive T cells even in apparently healthy individuals, may speak
in favour of the feasibility of including HSPs in universal vaccines. Finally, the
finding that HSP70 from various sources possessed different requirements for
signaling through TLRs, sheds new light towards its adjuvanticity, and hopefully
paves the way for the development of effective vaccines against infections. Taken
together, this thesis may provide information on the importance of the improvement
of prophylactic and therapeutic approaches for infectious diseases in general, aiming
at mitigating the threat by the killer pathogens.
Heat shock proteins as vaccine adjuvants
57
IV) ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to all my colleagues, my friends and my
family who have helped and supported me during these years. I would especially like
to thank
- Professor Carmen Fernández, my supervisor, for accepting me as a PhD student, for
your infinite enthusiasm, for helping me and especially for inspiring me to become an
independent thinker. Thank you for the invaluable guidance.
- Professor Klavs Berzins, my co-supervisor, for always being there with your minute
observations and helpful advice.
- Professor Stefan Ståhl, Maria Wikman and all the other co-authors for your brilliant
collaboration, contribution and advice during the project.
- Professor Peter Perlmann and Hedvig Perlmann, for always being there to remind
me that science is a passion; I admire your perseverance.
- Seniors at the department, Professor Marita Troye-Blomberg, Eva Sverremark for
sharing your vast knowledge and for your cooperation during the study.
- Manuchehr Abedi-Valugerdi, for the great technical support, for many interesting
scientific discussions.
- Maggan Hagstedt, Ann Sjölund, Gelana Yadeta and Gunilla Tillinger for your kind
and unconditional help whenever I needed it.
- Nina-Maria Vasconcelos, for teaching me so many things within and outside the
laboratory, for being such a wonderful and considerate person.
- Jacob Minang, for your sharp intellect, your critical analysis concerning anything
happening in the universe, for never saying ‘no, I don’t know the answer’.
- Esther Julián, for always being there to help in any situation, for your determination
and sincere devotion to work - which has always inspired me.
- Caroline Ekberg, for your kindness and your precious time spent in listening to me
- All students that have come and gone during my time at the department, Cecilia
Rietz, Eva Nordström, Karin Lindroth, Izaura Ross, Ben Adu Gyan, Ahmed Bolad,
Salah Eldin Farouk, Ariane Rodríguez Muñoz, Anna Tjärnlund, Alice Nyakeriga,
Manijeh Vafa, Shiva S. Esfahani, John Arko Mensah, Shanie Saghafian, Anna-Karin
Larsson, Petra Amoudruz, Halima Balogun, Norra Bachmayer, Yvonne Sundström,
58
Qazi Khaleda Rahman
Piyatida Tangteerawatana, Camilla Rydström, Elisabeth Israelsson, Sara Gilljam,
Magdi Ali, for being good friends.
- Staffs at the animal house Eva Nygren, Solveig Sundberg and Diana for your
excellent assistance and taking good care of my mice.
- My cousin Zarina Nahar Kabir, for inspiring me with your strength of mind, for
being my social guide in Sweden, for the immense support you provided and the
sincere concern you have shown for me during my stay here.
- Dr. Atiqul Islam, my brother-in-law, for your wonderful sense of humor, and my
two sweet little nieces Shanta and Tonima, you two make my living here enriched
with love and fun.
- Reshma, Shahanaz, Babu, Sharif, Rafid, Shabab, for providing so many memorable
moments.
- Dilnewaz Ruby, Shahidul Sohel, Tamanna, for many pleasant & enriching
conversations.
- My little sister Mousumi, for being the best sister in the world, for sharing so many
happy moments, for being patient with me when I was low, for providing support and
encouragement when I needed it most.
- Subrata, bondhuboreshu, for being a precious and adorable friend.
- My aunts (especially Siddiqua Kabir) uncles, cousins and friends in Bangladesh, for
their thorough encouragement.
- My brother Sajib, sister-in-law Sabrina, my wonderful little niece Neelima sonayour giggle and laughter always make me feels happy and rejuvenated. You are truly
an angel.
- And lastly my dear Ma and Baba, for your love and support, for your pride and faith
in me - virtues which formed the basis of my inspiration and determination. Without
you I could never be what I am today.
This work was financially supported by the European Commission (QLK2-CT-2002-00846), Magnus
Bergvalls Stiftelse and Swedish Institute.
Heat shock proteins as vaccine adjuvants
59
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