heat shock proteins as vaccine adjuvants - DiVA portal

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 N-terminal 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-γ production, however stimulation with the Pf70C-EB200 fusion protein induced an enhanced secretion of IFN-γ 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-γ 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 Qazi Khaleda Rahman Akademitryck AB, Valdemarsvik Stockholm 2005 The thesis is published electronically at the Stockholm University website

‘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: 395-

403.

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 Antigen presenting cell CCR Chemokine receptor CD Cluster of differentiation CD40L CD40 ligand CMV Cytomegalovirus CpG Cytidine phosphate guanosine CT Cholera toxin CTA Cholera toxin A subunit CTB Cholera toxin B subunit CTL Cytotoxic T lymphocyte DC Dendritic cell ER Endoplasmic reticulum Fas FS-7 associated surface antigen FCA Freund’s complete adjuvant FIA Freund’s incomplete adjuvant GM-CSF Granulocyte-macrophage colony stimulating factor HSP Heat shock protein IFN Interferon IL Interleukin IRAK IL-1 receptor associated kinase IRF Interferon regulatory factor ISCOM Immunostimulating complex LOX Lectin-like oxidized low-density lipoprotein receptor MAPK Mitogen activated protein kinase MHC Major histocompatibility complex MPL Monophosphoryl lipid MTB Mycobacterium tuberculosis MyD88 Myeloid differentiation factor 88 NF-κB Nuclear factor-κB NK Natural killer PAMP Pathogen associated molecular pattern PKR RNA-dependent protein kinase PRR Pattern recognition receptor Th T helper TCM Central memory T cell TEM Effector memory T cell TLR Toll-like receptor TNF Tumor necrosis factor TRAF TNF-receptor associated factor TREM 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.

10 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


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.


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


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


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 co-

stimulatory 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-γ 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


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-κB (NF-κB), 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-α, 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 (TLR1-

11) [reviewed in O'Neill 2004] have been identified. They have a distinct function in


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.

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

MyD88 dependent pathway

MyD88 independent pathway

IRF-3

HHSSPP7700

MyD88

IRAK

TRAF6

NF-κB

HHSSPP7700 HHSSPP6600

Uropathogenic E.coli

TRIF

IRF-3

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.

Cytokine production Costimulatory molrcule induction

IFN-inducible gene espression

Caspase activation Costimulatory

molrcule induction


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, ubiquitin-

targeted 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].


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 co-

stimulatory 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,


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


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-γ, and TNF-β 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].


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.


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 (1908-

1921) 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


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.


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


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


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.


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-γ, 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


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).


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],

Gene of interest

Transform into bacteria

Humoral response Cellular response

vaccination


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 mass-

replicate 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


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,


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


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


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-γ 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


expression on APCs [Bergstrom-Mollaoglu et al. 1992], activating murine Th cells to

secrete the Th1 type cytokines IL-2 and IFN-γ 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-γ 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 pathogen-

derived 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-α, IL-1, and IL-

12 [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-γ

[Klinman et al. 1996], which activate NK cells for enhanced IFN-γ synthesis and

increased lytic activity [Cowdery et al. 1996]. CpG DNA alone renders protection


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 µg 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


vivo and in vitro [Ågren et al. 1997], through increased production of cytokines as IL-

1 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


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


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 peptide-

binding 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],


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 cross-

presentation of chaperoned peptides, might be mediated by different sets of receptors.

CD91 is a putative receptor for HSP70, which is specifically endocytic, whereas TLR-

2 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 HSP-

chaperoned 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-antigen-

specific 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-1β, IL-12, IL-6


and TNF-α [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.

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-κB [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 CD91-

mediated internalization, by increasing their local concentration, might became able to

Adaptive immune effect of HSP70

Innate immune effect of HSP70


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-α 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 HSP70-

peptide complexes, for the delivery to MHC II processing pathway, was not mediated


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 LPS-

mediated 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'-tetra-

acetic 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 µg/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


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.

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

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.


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 pro-

inflammatory 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 self-

HSP homologues as partial agonists or altered peptide ligands and develop a


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 [Mercereau-

Puijalon 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


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.


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 low-

responder 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


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.


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-γ

production, but stimulation with the Pf70C-EB200 fusion protein induced an

enhanced secretion of IFN-γ. 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,


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.


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.


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.


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 TLR2-

TLR4- 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


• 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 Pf70C-

EB200, 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-γ 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


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.


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,


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 sona-

your 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.


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