Production and delivery of recombinant
subunit vaccines
Christin Andersson
Royal Institute of Technology
Department of Biotechnology
Stockholm 2000
CHRISTIN ANDERSSON
 Christin Andersson
Department of Biotechnology
Royal Institute of Technology (KTH)
SE-100 44 Stockholm
Sweden
Printed at Högskoletryckeriet KTH, Stockholm 2000
ISBN 91-7170-633-X
RECOMBINANT SUBUNIT VACCINES
Christin Andersson (2000) Production and delivery of recombinant subunit vaccines
Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden
ISBN 91-7170-633-X
Abstract
Recombinant strategies are today dominating in the development of modern subunit vaccines. This
thesis describes strategies for the production and recovery of protein subunit immunogens, and how
genetic design of the expression vectors can be used to adapt the immunogens for incorporation into
adjuvant systems. In addition, different strategies for delivery of subunit vaccines by RNA or DNA
immunization have been investigated.
Attempts to create general production strategies for recombinant protein immunogens in such a way
that these are adapted for association with an adjuvant formulation were evaluated. Different
hydrophobic amino acid sequences, being either theoretically designed or representing transmembrane
regions of bacterial or viral origin, were fused on gene level either N-terminally or C-terminally to
allow association with iscoms. In addition, affinity tags derived from Staphylococcus aureus protein A
(SpA) or streptococcal protein G (SpG), were incorporated to allow efficient recovery by means of
affinity chromatography. A malaria peptide, M5, derived from the central repeat region of the
Plasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in these
studies. Furthermore, strategies for in vivo or in vitro lipidation of recombinant immunogens for iscom
incorporation were also investigated, with a model immunogen ∆SAG1 derived from Toxoplasma
gondii. Both strategies were found to be functional in that the produced and affinity purified fusion
proteins indeed associated with iscoms. The iscoms were furthermore capable of inducing antigenspecific antibody responses upon immunization of mice, and we thus believe that the presented
strategies offer convenient methods for adjuvant association.
Recombinant production of a respiratory syncytial virus (RSV) candidate vaccine, BBG2Na, in baby
hamster kidney (BHK-21) cells was investigated. Semliki Forest virus (SFV)-based expression vectors
encoding both intracellular and secreted forms of BBG2Na were constructed and found to be
functional. Efficient recovery of BBG2Na could be achieved by combining serum-free production
with a recovery strategy using a product-specific affinity-column based on a combinatorially
engineered SpA domain, with specific binding to the G protein part of the product.
Plasmid vectors encoding cytoplasmic or secreted variants of BBG2Na, and employing the SFV
replicase for self-amplification, was constructed and evaluated for DNA immunization against RSV.
Both plasmid vectors were found to be functional in terms of BBG2Na expression and localization.
Upon intramuscular immunization of mice, the plasmid vector encoding the secreted variant of the
antigen elicited significant anti-BBG2Na titers and demonstrated lung protective efficacy in mice. This
study clearly demonstrate that protective immune responses to RSV can be elicited in mice by DNA
immunization, and that differential targeting of the antigens expressed by nucleic acid vaccination
could significantly influence the immunogenicity and protective efficacy.
We further evaluated DNA and RNA constructs based on the SFV replicon in comparison with a
conventional DNA plasmid for induction of antibody responses against the P. falciparum Pf332derived antigen EB200. In general, the antibody responses induced were relatively low, the highest
responses surprisingly obtained with the conventional DNA plasmid. Also recombinant SFV suicide
particles induced EB200-reactive antibodies. Importantly, all immunogens induced an immunological
memory, which could be efficiently activated by a booster injection with EB200 protein.
Keywords: Affibody, Affinity chromatography, Affinity purification, DNA immunization, Expression plasmid,
Fusion protein, Hydrophobic tag, Iscoms, Lipid tagging, Malaria, Mammalian cell expression, Recombinant
immunogen, Respiratory Syncytial Virus, Semliki Forest virus, Serum albumin, Staphylococcus aureus protein
A, Subunit vaccine, Toxoplasma gondii
 Christin Andersson
CHRISTIN ANDERSSON
RECOMBINANT SUBUNIT VACCINES
LIST OF PUBLICATIONS
This thesis is based on the following papers, which in the text will be referred to by their
Roman numerals:
I.
Andersson, C., Sandberg, L., Murby, M., Sjölander, A., Lövgren-Bengtsson, K. and
Ståhl, S. General expression vectors for production of hydrophobically tagged
immunogens for efficient iscom incorporation. J. Immunol. Methods, 222: 171-182
(1999)
II.
Andersson, C., Sandberg, L., Wernérus, H., Johansson, M., Lövgren-Bengtsson, K.
and Ståhl, S. Improved systems for hydrophobic tagging of recombinant immunogens
for efficient iscom incorporation. J. Immunol. Methods, 238: 181-193 (2000)
III.
Andersson, C., Wikman, M., Lövgren-Bengtsson, K., Lundén, A. and Ståhl, S. In
vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation.
J. Immunol. Methods (2000) Submitted
IV.
Andersson, C., Hansson, M., Power, U., Nygren, P-Å. and Ståhl, S. Mammalian cell
production of an RSV candidate vaccine recovered using a product specific affinity
column. FEBS Letters (2000) Submitted
V.
Andersson, C., Liljeström, P., Ståhl, S. and Power, U. Protection against respiratory
syncytial virus (RSV) elicited in mice by plasmid DNA immunization encoding a
secreted RSV G protein derived antigen. FEMS Immunol. Med. Microbiol. (2000) In
press
VI.
Andersson, C., Vasconcelos, N-M., Sievertzon, M., Haddad, D., Liljeqvist, S.,
Berglund, P., Liljeström, P., Ahlborg, N., Ståhl, S. and Berzins, K. Comparative
immunization study using DNA and RNA constructs encoding a part of Plasmodium
falciparum antigen Pf332. (2000) Submitted
CHRISTIN ANDERSSON
RECOMBINANT SUBUNIT VACCINES
CHRISTIN ANDERSSON
TABLE OF CONTENTS
INTRODUCTION
1
1. Vaccines
1
1.1. Subunit vaccines
3
1.2. Recombinant subunit vaccines
4
2. Protein immunogens
7
2.1. Synthetic peptides
7
2.2. Recombinant expression of protein immunogens
8
2.2.1. Gene construct
8
2.2.2. Production hosts
9
2.2.3. Gene fusion strategies
11
2.2.4. Affinity tags
12
2.2.5. Tailor-made affinity ligands
14
2.2.6. Increasing immunogenicity
by using gene fusion strategies
15
3. Adjuvant systems for recombinant protein antigens
4. Live delivery of subunit vaccines
17
23
4.1. Bacterial delivery systems
23
4.2. Viral delivery systems
24
5. Nucleic Acid Vaccines
26
5.1. DNA vaccines
26
5.2. Improving immune responses to DNA vaccines
30
5.2.1.
Adjuvants and delivery systems for DNA vaccines
31
5.2.2. Codelivery of stimulatory substances
32
5.2.3. Immunostimulatory DNA sequences
32
5.3. Safety of DNA vaccines
33
5.4. RNA Vaccines
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RECOMBINANT SUBUNIT VACCINES
PRESENT INVESTIGATION
6. Recombinant production of protein immunogens
6.1. Recombinant strategies for
iscom incorporation (I, II, III)
37
37
6.1.1. N-terminal hydrophobic tags (I)
37
6.1.2. C-terminal hydrophobic tags (II)
43
6.1.3. Lipid tagging of protein immunogens (III)
45
6.2. Recovery of a vaccine candidate
produced in mammalian cells (IV)
7. Nucleic acid vaccines
48
53
7.1. Protection against RSV elicited in mice
by plasmid DNA immunization (V)
53
7.2. Immunization with RNA and DNA encoding a malarial antigen (VI)
56
8. Concluding remarks
9. Abbreviations
60
10. Acknowledgements
11. References
66
64
68
CHRISTIN ANDERSSON
RECOMBINANT SUBUNIT VACCINES
INTRODUCTION
The use of vaccines has had great impact on the ability to control and prevent infectious
diseases. The first vaccines consisted of whole pathogens, killed or attenuated, but today the
recombinant subunit approach, i.e. to use only a defined subunit of the pathogen, is
dominating the vaccine research in the search for new effective vaccines. The ability to use
small, defined parts of a pathogen and produce that subunit in a non-pathogenic host will
increase the safety of future vaccines. Subunit vaccine candidates typically consist of surface
proteins or polysaccharides. The use of recombinant DNA technology has simplified the
production of subunit vaccines. Protein-based subunit vaccines can consist of synthetic
peptides, recombinant proteins or gene fragments (DNA or RNA) encoding the protein
immunogens. Live delivery vehicles can furthermore be evaluated for delivery of both protein
subunits and nucleic acid vaccines. This thesis will describe some aspects on the production
of protein immunogens and how expression systems can be designed to adapt the proteins for
association to adjuvant systems. Furthermore, nucleic acid immunization approaches (DNA
and RNA) have been evaluated or subunit immunogens from respiratory syncytial virus
(RSV) and malaria.
1. Vaccines
Vaccination has been an important tool to combat infectious diseases over the past 200 years.
The first human vaccine was developed in 1798 when Edward Jenner successfully prevented
smallpox infection in milkmaids. Today, prevention of bacterial and viral infections through
vaccination is very beneficial in reducing disease mortality and healthcare costs. Successful
development of vaccines has resulted in that many major diseases, such as diphtheria,
poliomyelitis and measles nowadays are kept under control, and that smallpox is even totally
eradicated (Mäkelä, 2000).
The first vaccines were mainly based of either live but attenuated or inactivated, killed
microbes. Also today, most of the vaccines used routinely in childhood vaccination programs
are whole-organism vaccines consisting of attenuated or killed bacteria or viruses (Plotkin,
1993). Vaccine types and their characteristics are presented in Table 1.
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CHRISTIN ANDERSSON
Table 1: Different vaccine types and some of their characteristics
Vaccine type
Characteristics
Whole cell vaccines
Live, attenuated bacteria or
viruses
+ Humoral and cellular immune responses
- Risk of reversion to virulent strain
- Undefined composition
Killed, inactivated bacteria or
viruses
+ No risk of infection (replication deficient, non-infectious)
- Less powerful than live vaccines
- Undefined composition
Subunit vaccines
Purified components from
pathogen
+ Well-defined composition
- Requires large-scale cultivation of pathogen
- Poor immunogens, need of adjuvant
Synthetic peptides
+ Well-defined composition
+ No risk of pathogenicity
- Short half-life
- Need of adjuvant
Recombinant proteins
+ Well-defined composition
+ No risk for pathogenicity
+ Possibility for cost-efficient production and purification
- Primarily humoral immune response
- Need of adjuvant
Live vectors: Bacterial
+ Humoral and cellular immune responses
+ Possibility to develop oral vaccines
- Risk of reversion to virulent forms for attenuated pathogens
Live vectors: Viral
+ Humoral and cellular immune responses
- Risk of reversion to virulent forms for attenuated pathogens
Nucleic Acid vaccines: DNA
+ Cellular and humoral immune responses
+ Cost-efficient production
+ In vivo amplification systems available
- Inefficient transfection
- Risk of integration into host genome not completely excluded
Nucleic Acid vaccines: RNA
+ No risk of integration into host genome
+ Do not have to enter nucleus for translation
+ In vivo amplification systems available
- Unstable
- Quite expensive production
Live, attenuated vaccines are usually quite effective in stimulating both humoral as well as
cellular immune responses (Ellis, 1999). These vaccines might have a certain capacity to
replicate in the host, but are attenuated in their pathogenicity in order not to cause disease.
However, due to its replicating ability, live attenuated vaccines can potentially revert to a
virulent wild-type strain, e.g. by recombination, resulting in disease. Another aspect of using
2
RECOMBINANT SUBUNIT VACCINES
live attenuated vaccines is the risk of transmission between individuals. These facts are major
concerns, especially if the unwanted disease develops in immunocompromised hosts.
Killed or inactivated vaccines, however, is in general safer than live, attenuated vaccines. The
vaccine cannot replicate in the host and is non-infectious. However, a drawback with using
killed, inactivated vaccines is that they are less effective than live attenuated vaccines in
inducing protective immunity. To improve the immunogenicity of the killed or inactivated
vaccines, they often need to be coadministered with an adjuvant and administered several
times. The classical, whole cell vaccines (live, attenuated and killed, inactivated vaccines)
need to be improved, especially in terms of safety. The side effects of using such vaccines,
including risk of infection or transmission, need to be reduced. These vaccines also contain
undefined substances of bacterial or viral origin, which might make the vaccines highly
immunogenic and potent, but could also be involved in the induction of side-effects. With
increasing demands from regulatory authorities on well-defined drugs, it will be increasingly
difficult to get new vaccines of this class accepted for human use.
New technology and better understanding of cell biology and immunology has accelerated the
vaccine development during the recent years. It is now possible to use only a small, defined
part of the pathogen in a vaccine with capacity to induce an immune response to the whole
pathogen, thus without risk of infection. By using subunits of the pathogen, the safety of the
vaccine is obviously increased. The subunit part used is often a surface molecule of the
pathogen, able to induce immunity on its own.
1.1. Subunit vaccines
Vaccines based on the subunit approach may consist of natural substances, directly harvested
from cultivations of the pathogen. Such nonrecombinant subunit vaccines may consist of
bacterial polysaccharides, detoxified toxins or viral surface proteins. Several subunit vaccines
consisting of natural surface polysaccharides purified from cell cultures have been developed
(Lindberg, 1999) e.g. for Neisseria meningitidis (Gotschlich et al, 1969) and Streptococcus
pneumoniae (Smit et al., 1977; Hilleman et al., 1981). One drawback with polysaccharidebased vaccines is that they are poor immunogens in infants and children (Lindberg, 1999). To
improve the problems with poor immunogenicity of polysaccharide vaccines, the concept of
conjugate vaccines were introduced. A subunit conjugate vaccines have been licensed for
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CHRISTIN ANDERSSON
Haemophilius influenzae type b (Hib), where polysaccharides have been coupled to a tetanus
toxoid carrier (Schneerson et al., 1986). A recent strategy to improve polysaccharide-based
vaccines, through the use of peptides that mimic the capsular polysaccharide of N.
meningitidis, have been evaluated (Grothaus et al., 2000). Purified toxins, chemically
detoxified, e.g. by formalin, have been used in vaccines to diphtheria, tetanus and pertussis
(Ellis, 1999). Subunit vaccines are safer than whole-cell vaccines, but there are drawbacks,
such as the requirement of large-scale cultivation of pathogens, which is normally costly and
not without risk. The subunit vaccines also need to be administered with an adjuvant to
increase immunogenicity. Polysaccharide-based and non-recombinant subunit vaccines will
not be further discussed in this thesis.
1.2. Recombinant subunit vaccines
The first protein-based vaccines also relied on natural (non-recombinant) sources of antigens.
For example, a highly active vaccine to hepatitis B consisted of purified hepatitis B surface
antigen (HBsAg) from human plasma, see review by Hilleman, 2000. The recombinant
subunit approach is today dominating the vaccine research in the search for new vaccines.
Identification of antigens involved in inducing protective immunity and isolation of the gene
encoding these proteins makes it possible to use recombinant DNA technology or synthetic
peptides to produce sufficient quantities of the antigen for vaccine studies. The first vaccine to
be produced utilizing recombinant DNA technology was licensed in 1986 when the HBsAg
was successfully expressed in yeast (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 use of recombinant DNA technology has made the development of subunit vaccines more
efficient. The basics of this technology is to transfer a gene encoding an antigen, responsible
for inducing immune responses sufficient for protection, to a non-pathogenic host, thereby
making the production of the antigen safer and generally more efficient. Recombinant subunit
vaccines can be delivered as purified recombinant proteins, as proteins delivered using live
non-pathogenic vectors (bacterial or viral) or as nucleic acid molecules encoding the antigen
(Table 1). There are several advantages in using recombinant subunit vaccines. No pathogen
is present in the production and purification procedure, thus making the production procedure
4
RECOMBINANT SUBUNIT VACCINES
less hazardous. By using recombinant DNA technology, the production and purification
procedure can be carefully designed to obtain high yields of a well-defined product.
Recombinant strategies for production of subunit vaccines will be further discussed later in
this thesis, see section 2.2.
Recombinant strategies have also been employed for detoxification of toxins. Engineered
inactivation of toxin can be obtained by mutational replacement of specific amino acids in the
enzymatically active part of the toxin. Pertussis toxoid produced by Bordetella pertussis with
specific mutations in its toxin gene is included as a component in an acellular pertussis
vaccine (Del Giudice and Rappuoli, 1999). Chimeric composite immunogens can also be
created by fusion of different toxins, such as the cholera toxin B subunit (CTB)-E. coli heatlabile toxin B subunit (LTB) hybrid molecules, which are candidate oral vaccines against both
enterotoxic E. coli (ETEC) infections and cholera (Lebens et al., 1996).
Although the use of protein immunogens, synthetic peptides or nucleic acid vaccines offer
several advantages, e.g. reduced toxicity, they are generally poor immunogens when
administered alone. This is particularly true for vaccines based on proteins or peptides, and to
make these vaccines more efficient, the use of potent and safe immunological adjuvants might
be needed. Adjuvant systems will be described later, see section 3.
The recombinant subunit approaches are being used for the development of new vaccines and
improvement of already existing vaccines. Many recombinant vaccines are now being tested
in preclinical and clinical trials, and some recombinant vaccines are already on the market
(Walsh, 2000; Table 2).
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CHRISTIN ANDERSSON
Table 2: Selected examples of recombinant vaccines currently approved in the US or EU
(Modified from Walsh, 2000)
Vaccine
Description
Application
Recombivax
rHBsAg produced in S.
cerevisiae
Hepatitis B prevention Merck
Comvax
Combination vaccine,
Vaccination of infants
containing rHBsAg
against H. influenzae
produced in S. cerevisiae type B and hepatitis B
as one component
Merck
Tritanrix-HB
Combination vaccine,
Vaccination against
containing rHBsAg
hepatitis B, diphteria,
produced in S. cerevisiae tetanus, and pertussis
as one component
SmithKline Beecham 1996 (EU)
Twinrix, adult and
pediatric forms
Combination vaccine,
Immunization against
containing rHBsAg
hepatitis A and B
produced in S. cerevisiae
as one component
SmithKline Beecham 1996 (EU)
(adult)
Primavax
Combination vaccine,
Immunization against
containing rHBsAg
diphteria, tetanus, and
produced in S. cerevisiae hepatitis B
as one component
Pasteur Merieux
MSD
1998 (EU)
Procomvax
Combination vaccine,
Immunization against
containing rHBsAg
H. influenzae type B
produced in S. cerevisiae and hepatitis B
as one component
Pasteur Merieux
MSD
1999 (EU)
Lymerix
rOspA, a surface
Lyme disease vaccine
lipoprotein of B.
burgdorferi, produced in
E. coli
SmithKline Beecham 1998 (US)
6
Company
Approved
1986 (US)
1996 (US)
1997 (EU)
(pediatric)
RECOMBINANT SUBUNIT VACCINES
2. Protein immunogens
2.1. Synthetic peptides
Subunit vaccine candidates can be produced by chemical synthesis of short polypeptides. The
advantages of peptide vaccines are several (summarized in Table 1), such as a chemically
well-defined product and a simple preparation (Babiuk, 1999). Synthetic peptides representing
parts of higher parasites, bacteria or viruses have indeed been used for immunizations in
humans (Klipstein et al., 1985; Herrington et al., 1987; Kahn et al., 1992; Millet et al., 1993).
For example, it has been shown that a 12 amino acid long peptide from the Plasmodium
falciparum sporozoite is safe and induces humoral responses in healthy volunteers
(Herrington et al., 1987). Although some side effects encountered with other vaccine types,
e.g. whole cell vaccines, may be circumvented, one major disadvantage of peptide vaccines is
their low level of immunogenicity. One reason for this may be due to major histocompatibility
complex (MHC) restriction i.e. one particular peptide can only be recognized with a
particulate human leukocyte antigen (HLA) haplotype (Rammensee et al., 1995). Another
reason could be that the peptides are rapidly degraded or excreted in vivo (Babiuk, 1999).
However, recent advances in chemical synthesis i.e. to introduce changes in the amine bond,
have lead to the development of pseudopeptides that are more stable and as biologically active
as normal peptides (Babiuk, 1999). Pseudopeptides have for example shown to induce
neutralizing antibodies to foot and mouth disease virus (Briand et al., 1997). Another major
drawback with synthetic peptides is that only linear epitopes can be utilized in the vaccine.
Antibody responses are often directed to conformational epitopes, but those are difficult to
mimic using synthetic peptides.
A problem related to the use of synthetic peptides is that although high antibody levels are
elicited to synthetic peptide-carrier complex, a major part of the antibodies are directed
towards the carrier (Herzenberg et al., 1980; Schutze et al., 1985). A further possible
disadvantage when using single peptide vaccines is that the immune response will be raised
only to one small epitope. Mutations in that specific region may allow the pathogen to evade
the immune system. However, this may be circumvented by using multiple antigenic peptides
(MAP) (Tam, 1996). Nevertheless, certain subunit vaccine candidates have progressed into
human clinical trials, e.g. a malaria vaccine candidate, consisting of a mixture of polymerized
synthetic peptides (Patarroyo et al., 1988).
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CHRISTIN ANDERSSON
2.2. Recombinant expression of protein immunogens
The advantages of producing recombinant vaccine antigens in heterologous hosts are many.
Recombinant DNA technology has made it possible to combine the ease of growing the nonpathogenic host with all the possibilities to upregulate the protein production and to design an
effective purification scheme (Koths, 1995; Mäkelä, 2000). There are several approaches to
design and optimize the production and purification (Geisse et al., 1996; Makrides, 1996;
Ståhl et al., 1999).
2.2.1. Gene construct
The gene constructs to be used for expression can be optimized in many aspects. By selecting
the minimal region required to elicit a strong immune response the length of the DNA
fragment to be inserted into the expression vectors could be minimized. However, the product
must retain the conformation required to elicit sufficient immune responses (Dertzbaugh,
1998). The gene can also be truncated for the purpose of removing sequences encoding toxic
peptides (Dertzbaugh, 1998). Heterologous genes containing codons that are rare in E. coli
may not be efficiently expressed, and therefore the codon usage can in certain cases be
adapted to the host of choice (Hannig and Makrides, 1998). Promoter sequences may also be
altered to be more efficient in the production host (Suarez et al., 1997)
The gene construct can be designed either for secretion of the expressed protein into the
growth medium or the periplasm machinery (Abrahamsén et al., 1986; Moks et al., 1987;
Hansson et al., 1994) or for intracellulary production of the protein. There are some
advantages of using secreted production e.g. protection of the product from cytoplasmic
proteases or simplification of the purification process due to few host cell proteins present in
the periplasm or culture medium. Another advantage connected with a secretion strategy is the
stimulation of disulfide bond formation (Ståhl et al., 1999), which might be important for the
correct folding of certain proteins. Intracellular production on the other hand can be used
when expressing proteins with tendency to aggregate inside cells, or when expressing proteins
containing transmembrane regions (Sjölander et al., 1993b; Robert et al., 1996). Intracellular
expression might lead to the formation of inclusion bodies, i.e. protein aggregates, which
requires solubilization and refolding to obtain the product in a soluble and active form
(Babiuk, 1999). Some advantages of inclusion body formation are that the product normally is
8
RECOMBINANT SUBUNIT VACCINES
protected from proteolytic degradation, and that high production levels often are obtained.
Levels up to 50 % of total cell protein content have been reported (Rudolph, 1996).
2.2.2. Production hosts
The choice of expression system used will be dependent of the characteristics of the protein to
be expressed. Several issues have to be considered, e.g. (i) expression levels, (ii) the need for
post-translational modifications, (iii) scale-up consideration, and (iv) production costs
(Dertzbaugh, 1998). The heterologous host used may affect the immunogenicity and
protective efficacy of the product. For example, an antigen may be expressed at high levels in
a bacterial expression system, but may only fold partially into its native confirmation, or the
antigen needs might need to be post-translationally modified to elicit protective immunity
(Dertzbaugh, 1998).
There are today four major host cell systems commonly used to produce recombinant
proteins; (i) bacterial expression systems, (ii) yeast expression systems, (iii) insect cell
expression systems, and (iv) mammalian expression systems. Each host cell system has
individual advantages and disadvantages (Table 3).
Table 3: Examples of production hosts for recombinant protein expression
Host
Characteristics
Bacteria
+ Easy to grow and manipulate
+ High expression levels
+ Inexpensive
- Do not perform post-translational modifications
- Proteolysis
- Correct folding can be problematic to obtain
Yeast
+ Capable of certain post-translational modifications
+ High cell densities
+ Easy to grow and manipulate
- Proteolysis
Insect cells
+ Make a variety of post-translational modifications
+ High expression levels compared to mammalian cells
Mammalian cells
+ Post-translational modifications
+ Stable antigen-producing cell lines can be constructed
- Time consuming
- Expensive
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CHRISTIN ANDERSSON
Bacterial expression systems are the most convenient to use. Expression levels of recombinant
proteins in bacteria are in general high. Bacterial expression systems are suitable to use when
no post-translational modifications of the protein are required. Several bacterial systems are
available, but Escherichia coli is the dominating and also the best characterized. Many
alternatives for optimization of protein expression in bacteria have been reported e.g. choice
of strain, transcriptional and translational regulation, and also the possibility to include signal
sequences that will target the protein to periplasm or culture media (Makrides, 1996). Since
foreign proteins may be rapidly degraded in the bacterial host, the host can be engineered to
reduce the proteolysis (Murby et al., 1996; Babiuk, 1999). Other bacterial expression system
that sometimes may be of advantage to use are Salmonella typhimurum (Martin-Gallardo et
al., 1993; Liljeqvist et al., 1996), Vibrio cholerae (Viret et al., 1996) and Bacillus brevis
(Ichikawa et al., 1993; Nagahama et al., 1996). Since bacterial systems offer significantly
lower production cost, any measures are taken to solve potential production problems before a
more sophisticated expression system is evaluated.
The most studied yeast expression system is Saccharomyces cerevisiae. Yeast is well-adapted
for secreting proteins into the culture supernatant, which facilitates purification. Yeast is
further known to grow to very high cell densities, which compensates for the expression
levels that are somewhat lower than for E. coli (Dertzbaugh, 1998). The first recombinant
vaccine for human use, the surface antigen from Hepatitis B, is expressed in Saccharomyces
cerevisiae (Valenzuela et al., 1982). More recently, the yeast Pichia pastoris has become
popular to use. Protein expression levels are higher in Pichia pastoris but the growth rate is
lower, which may be a problem when expressing unstable proteins (Dertzbaugh, 1998).
Pichia pastoris has been used to produce vaccine antigens of bacterial (Clare et al., 1991) as
well as viral (Zhu et al., 1997) origin. Yeast, being a eukaryotic expression system performs
certain post-translational modifications such as glycosylations. However, glycosylations
performed by yeast differ from glycosylation patterns performed by mammalian cells (De
Wilde et al., 1990). If glycosylations are important for the desired characteristics of the
protein, a mammalian expression system may be needed.
Baculovirus-based expression systems are based on the ability of a virus from the
Baculoviridae family to infect insect cells. Heterologous proteins can be efficiently produced
in both insect cells and larvae (Geisse et al., 1996). The advantage of using baculovirus
system is the high expression levels that can be obtained, when compared to mammalian
10
RECOMBINANT SUBUNIT VACCINES
expression systems. Furthermore, insect cells have the ability to perform a variety of posttranslational modifications, however as in the case of yeast systems, the glycosylation pattern
differ from that of mammalian cells. The baculovirus system is considered to be safe to use,
since the baculovirus is unable to infect vertebrates (Carbonell et al., 1985). Furthermore, the
promoters used in the baculovirus system are inactive in most mammalian cells (Carbonell et
al., 1985).
Mammalian expression systems (Geisse et al., 1996) perform post-translational modifications,
e.g. glycosylations, phosphorylations and the addition of fatty acid chains. There are several
different cell types that are commonly used in recombinant protein production, e.g. Chinese
hamster ovary (CHO) cells, baby hamster kidney (BHK) cells and human embryonic kidney
(HEK) cells (Geisse et al., 1996). All of them can be used to establish a stable transfected cell
line and is therefore suitable for scale up and long term production. An alternative to the timeconsuming procedures of selecting a stable cell line, transient expression strategies can be
used for rapid production of proteins e.g. expression in COS cells (SV40 virus transformed
simian cells (CV-1)) using a Simian virus 40 (SV40)-based system (Gluzman, 1981). Also,
many of the currently used viral vectors can be used for transient expression in mammalian
cells e.g. vectors from adenovirus or alphavirus (Fussenegger et al., 1999).
Many viral proteins have been expressed using mammalian cell lines. However, to date, the
recombinant vaccines licensed for human use are produced in yeast or bacteria (Walsh, 2000;
Table 2). This is mostly due to the ease of growing and manipulating bacteria and yeast, and
that the expression levels are much higher than in mammalian cells. An additional cost
connected with mammalian cell production is that the product needs to be validated free of
virus or oncogenic substances originating from the mammalian cell line used to produce the
vaccine (Hesse and Wagner, 2000)
2.2.3. Gene fusion strategies
Gene fusion strategies are commonly used to design and optimize production and purification
processes (Ståhl et al., 1997 and 1999). Table 4 gives some examples of how the overall
properties of a fusion protein can be altered by introducing a carefully chosen gene fusion
partner. For example, fusing an antigen to a fusion partner with higher solubility will increase
the solubility of the overall protein, thereby minimizing the inclusion body formation when
11
CHRISTIN ANDERSSON
expressing the protein intracellulary (Samuelsson et al., 1991 and 1994). Improvement of
proteolytic stability can be obtained in a similar fashion, by fusing the target protein to
proteolytically stable fusion partners (Hammarberg et al., 1989; Murby et al., 1991 and 1996).
Production of a target protein as a multicopy protein, with a subsequent specific cleavage
reaction is a way of increasing the product yield. For example, expression of the human
proinsulin C-peptide as a heptameric fusion protein by an enzymatic cleavage procedure,
resulting in significantly improved yield of native C-peptide monomers (Jonasson et al., 1998
and 2000). Another example of using gene fusion strategies to adapt the product for a certain
purification strategy is by fusing the target protein to hydrophobic amino acid residues in
order to alter the partitioning of fusion proteins in aqueous two-phase systems (Köhler et al.,
1991; Hassinen et al., 1994) thereby increasing the recovery of fusion protein. Gene fusion
strategies have also been employed to obtain increased immune responses and to adapt the
target immunogen to certain adjuvant systems (see section 2.2.6).
The purposes of making gene fusion constructs are many (Ståhl et al., 1997 and 1999; Table
4) and one important application is to enable affinity purification of the target protein by using
affinity fusion tags.
2.2.4. Affinity tags
Using affinity fusion partners will make efficient recovery of the expressed recombinant gene
product possible. Many well-defined affinity tags have been described in the literature, e.g.
domains from staphylococcal protein A (SpA) or streptococcal protein G (SpG), GlutathioneS-tranferase (GST) and histidine tags (for reviews, see Nilsson et al., 1997; Ståhl et al., 1999;
Makrides, 1996). Different types of interactions can be used e.g. enzyme-substrates, bacterial
receptors-serum proteins, polyhistidines-metal ions and antibody-antigen interactions (Uhlén
et al., 1992). No single affinity fusion strategy is ideal for all expression and purification
situations, and different alternatives might need to be evaluated. Affinity fusion partners are
mostly used to enable simpler protein purification procedures, but can also be used for
detection and immobilization purposes (for extensive review, see Nilsson et al., 1997). In this
thesis, the affinity tags covered in detail will be the IgG-binding Z tag (Nilsson et al., 1987),
derived from SpA, and the albumin-binding ABP and BB tags, derived from SpG (Ståhl and
Nygren, 1997).
12
RECOMBINANT SUBUNIT VACCINES
Table 4: Selected examples of fusion partners and their use
Effect on target protein
Fusion partner
Reference
Secretion
Signal peptide
Abrahamsén et al., 1986
Hansson et al., 1994
Multimerization
C-peptide
Jonasson et al., 1998
Solubility
Protein A domain
Samuelsson et al., 1991 and 1994
Hydrophobicity
Isoleucin-tryptophan rich tag
Köhler et al., 1991
Affinity
Various affinity tags
For reviews, see Nilsson et al.,
1997 and Ståhl et al., 1999
Immunopotentiating
Cholera toxin B (CTB)
Sun et al., 1994
Holmgren et al., 1994
Carrier-related properties
albumin binding protein (ABP or
BB) of streptococcal protein G
(SpG)
Sjölander et al., 1997
Libon et al., 1999
Increased half-life
BB of SpG
Nygren et al., 1991
Improved association with
iscoms
Membrane-spanning regions from
viral or bacterial surface proteins
I, II
Improved association with
iscoms
In vivo lipidation signals
III
Improved production
Simplified purification
Improved immunogenic
properties
Improved adjuvant association
Protein A, a surface protein from Staphylococcus aureus (SpA), has been widely used as
affinity fusion partner due to its strong affinity to the Fc part of immunoglobulin G (Uhlén et
al., 1983). SpA consists of a signal sequence (S) followed by five homologous IgG binding
domains (E, D, A, B, C), all capable of binding IgG (Moks et al., 1986) and a cell surface
anchoring domain, XM (Schneewind et al., 1995; Navarre and Schneewind, 1999). An
engineered IgG-binding domain, Z, was derived from the B domain of SpA and was shown to
have retained IgG binding properties and to be resistant to cleavage with hydroxylamin and
CNBr (Nilsson et al., 1987). The characteristics of SpA and Z, being proteolytically stable and
highly soluble, makes them well suited as affinity fusion partners to enable purification of
recombinant proteins (Ståhl et al., 1999). The Z tag have been used in many studies as affinity
tag, mostly in its divalent form, ZZ, since it has been shown that ZZ has a ten times higher
13
CHRISTIN ANDERSSON
affinity for its ligand IgG compared to the monovalent Z domain (Nilsson et al., 1994). The
high solubility of the Z domain has shown to increase the overall solubility of fusion proteins
(Samuelsson et al., 1991 and 1994).
The streptococcal protein G (SpG) is a surface protein with affinity to both IgG and albumin
of different mammalian species, including humans (Nygren et al., 1990). The binding sites for
IgG and serum albumin are shown to be structurally separated (Nygren et al., 1988). The
complete albumin-binding region is suggested to contain three albumin-binding motifs, each
of about 5 kDa in size. Tags comprising of one, two or two-an-a-half serum albumin binding
motifs have been constructed (ABD, ABP and BB, respectively), successfully produced and
used in different applications (Nilsson et al., 1997; Ståhl and Nygren, 1997; Ståhl et al.,
1999). The albumin binding proteins derived from SpG are proteolytically stable, highly
soluble, and are possible to produce in high yields (Larsson et al., 1996; Nilsson et al., 1996;
Murby et al., 1995). Both the SpA-derived Z domain and the SpG-derived serum albumin
binding proteins have been extensively used to facilitate affinity chromatography purification
of protein immunogens (Ståhl et al., 1989; Sjölander et al., 1990, 1993c and 1997; Murby et
al., 1994; Berzins et al., 1995; Power et al., 1997; Libon et al., 1999).
2.2.5. Tailor-made affinity ligands
In order to create new binding proteins to be used as affinity ligands, the use of combinatorial
strategies to create large protein libraries is an emerging research area. Combinatorial methods
are attractive because they allow direct selection of suitable target-specific binding molecules
from large libraries of related proteins. Libraries of other molecules than proteins or peptides
have also been constructed, e.g. small organic molecules (Gordon et al., 1994) and DNA or
RNA (Gold et al., 1995; Burke and Gold, 1997), but this will not be further discussed in this
thesis.
A method of emerging interest is the display of libraries of peptides or proteins on
filamentous phage surfaces (Smith, 1985; Clackson and Wells, 1994). This concept involves
construction of a protein library containing partially randomized sequences, fusion by genetic
means of the new variants to phage protein III or phage protein VIII, and subsequently
displaying the library on phage particles. Rounds of selection of displayed peptides or proteins
with desired properties from such phage libraries can be performed, with intermediate
14
RECOMBINANT SUBUNIT VACCINES
enrichment steps of the selected phages in E. coli. A useful selection method is to use solid
phase surfaces covered with the target molecule and allowing the phages to bind, theoretically
via interactions of the displayed protein and the target molecule. The phage particle would
thus constitute the link between genotype and phenotype, which allows identification of the
protein from the DNA sequence.
As mentioned above, the IgG-binding Z-domain, derived from SpA, has several features,
which makes it suitable in affinity purification applications such as being proteolytically
stable, of small size and highly soluble. Structural analysis has shown that the Z domain is
composed of three α-helices and that the IgG-binding surface is shared between two of the
three helices. This binding surface is of approximately 600Å2, similar to the size of many
antibody-antigen interactions. These data suggest that if the amino acids involved in the IgGbinding were randomly substituted, new variants of the Z-domain with novel binding
properties could be found (Nygren and Uhlén, 1997). Theoretically, these new protein
domains would share the stability properties and the α -helical structure of the original Zdomain. Recently, such libraries of the Z-domain were created (Nord et al., 1995 and 1997).
Thirteen amino acids involved in the IgG binding of the Z-domain was randomly substituted
to any of the 20 amino acids. The libraries containing the Z-variants were fused to protein III
on filamentous phage M13, and subsequently displayed on phage particles (Nord et al., 1995
and 1997). Several novel binders, so called affibodies, have been selected by phage-display
technology from these libraries, including binders to Taq polymerase, human insulin, a human
apolipoprotein variant (Nord et al., 1997) and to an RSV G protein derived protein G2Na
(Hansson et al., 1999).
2.2.6. Increasing immunogenicity by using gene fusion
Fusion partners can be used to increase the immunogenicity of the antigen. The SpG-derived
fusion partner BB described above has shown to have immunopotentiating properties when
used as fusion partner to the immunogen used for immunization (Sjölander et al., 1993c and
1997; Power et al., 1997; Libon et al., 1999). In immunization experiments of mice with a
malaria antigen fused to the BB tag, antibody responses were raised in mice not responding to
the malarial antigen alone (Sjölander et al., 1997). Furthermore, in a study with an RSV
antigen, the antibody response was higher to the antigen when fused to BB than when
15
CHRISTIN ANDERSSON
immunizing with the antigen alone (Libon et al., 1999). The immunopotentiating properties of
BB could be either due to T-cell epitopes present in BB (Sjölander et al., 1997) or due to the
serum albumin binding properties resulting in an increased half-life of the antigen or even a
combination of the both.
Another commonly used protein fusion partner with carrier-related properties is the surface
antigen of Hepatitis virus B (HBsAg). A fusion protein composed of a repetitive sequence
derived from the circumsporozoite (CS) protein of Plasmodium falciparum and HBsAg
strategies was produced in yeast cells and used in vaccination experiments in humans
resulting in long lasting antibody responses (Vreden et al., 1991). Fusion to the HBsAg have
also been shown to enhance the humoral as well as the cellular immune response to human
immunodeficiency virus 1 (HIV-1)-derived immunogens both when delivered as fusion
proteins (Schlienger et al., 1992; Mancini et al., 1994) and as plasmid DNA (Fomsgaard et al.,
1998). Furthermore, a strategy to fuse by genetic means the target antigen to cholera toxin
subunits in order to obtain targeting to receptors in the mucosa, has been described
(Hajishengallis et al., 1995). Antigens can also be expressed as fusion proteins to B cell or T
cell epitopes to increase humoral or cellular immune responses (Löwenadler et al., 1992). In
addition to expressing single proteins, it is possible to develop chimeric genes containing the
important epitopes from a number of pathogens (Whitton et al., 1993).
Recombinant proteins are in general relatively poor immunogens when administered alone
and the coadministration of an adjuvant is crucial to elicit a strong immune response. Fusion
strategies have been investigated to ensure proper association of fusion proteins to an
adjuvant. An example of this strategy, that will be discussed below, is to use hydrophobic
tagging of protein immunogens to improve the association of the immunogen to a
hydrophobic adjuvant system e.g. iscoms (I, II, III)
16
RECOMBINANT SUBUNIT VACCINES
3. Adjuvant systems for recombinant protein antigens
Subunit vaccines consisting of peptide or recombinant proteins are normally poor
immunogens when administered alone. Whole cell vaccines are heterogeneous and contain
many immunostimulatory substances, e.g. bacterial DNA or enterotoxins. Pure recombinant
protein antigens, lacking these natural immunostimalutory substances, therefore require coadministration of an adjuvant to increase the immune response. Adjuvants are defined as
substances that together with the antigen stimulate the antigen-specific immune response.
Extensive research has been done in this field and it has been shown that many substances can
achieve this, but so far only aluminum salts (Gupta, 1998) and MF59 (an oil emulsion) are
adjuvants approved for human use (Singh and O'Hagan 1999). Therefore the development of
novel adjuvants (Morein et al., 1996) and strategies for efficient association of the antigen to
the adjuvant, are important topics in present vaccinology research. Table 5 presents selected
examples of adjuvants that have been evaluated in animal studies.
Table 5: Selected examples of vaccine adjuvants (modified from Singh and O'Hagan 1999)
Type of adjuvant
Examples
Reference
Mineral salts
Aluminum hydroxide
Aluminium phosphate
Calcium phosphate
Gupta, 1998
Gupta, 1998
Wang et al., 2000
Immunostimulatory adjuvants Cytokines
Saponines (e.g. QS21)
MDP derivates
CpG oligonucleotides
lipopolysaccharides (LPS)
monophosporyl lipid A (MPL)
Baca-Estrada et al., 1997
Barr et al., 1998.
Namba et al., 1997
Klinman et al., 1999
Ogawa et al., 1997
Baldridge and Crane, 1999
Lipid particles and emulsions Freund incomplete adjuvant
SAF
MF59
Liposomes
Immunostimulating complexes (iscoms)
Cochleates
Chang et al., 1998
Hjort et al., 1997
Heineman et al., 1999
Gregoriadis et al., 1999
Barr et al., 1998
I, II, III
Gould-Fogerite et al., 1998
Microparticulate adjuvants
PLG microparticles
Virus-like particles
O'Hagan et al., 1998
Coste et al., 2000
Mucosal adjuvants
Heat-labile enterotoxin (LT)
Cholera toxin (CT)
Park et al., 2000
Matuso et al., 2000
17
CHRISTIN ANDERSSON
Adjuvants can be used in several ways; they can increase the duration of immune responses,
modulate antibody specificity, isotype and subclass distribution, stimulate cell mediated
immune responses or decrease the required dose of antigen and thus reduce the cost for the
vaccine (Singh and O'Hagan 1999). The mechanisms of adjuvant action are relatively poorly
understood. Most likely, activation of the immune response by adjuvants is a result of a
cascade of events such as induced cytokine production or activation of antigen-presenting
cells (APCs) e.g by increased expression of costimulatory molecules or MHC complexes
(Singh and O'Hagan 1999). The adjuvant to choose will totally depend on what type of
immune responses that is required for protective immunity. Different adjuvants usually induce
different types of responses, and a first choice would be to evaluate the adjuvant that is
predicted to give an immune response similar to what is postulated for protective immunity.
In general, adjuvants are immunostimulatory substances, particulate adjuvants or a
combination of the both (Table 5). Immunostimulatory substances are thought to
predominantly effect the cytokine expression by activating expression of costimulatory
molecules or major histocompatibility complex (MHC) molecules, while particulate
adjuvants, which have comparable dimensions to pathogens, probably are targets for uptake
by antigen-presenting cells. See Table 6 for a general comparison.
Table 6: A general comparison of dimensions of pathogens and particulate adjuvants (modified
from Singh and O'Hagan 1999)
Natural pathogen
Particulate adjuvant
Bacteria
0.5-3 µm
Microparticles
100 nm-10 µm
Poxvirus
250 nm
Liposomes
50 nm-1 µm
Herpesvirus
100-200 nm
Virosomes
50 nm - 10 µm
HIV
100 nm
MF59
200 nm
Poliovirus
20-30 nm
Iscoms
40 nm
One group of immunostimulatory adjuvants is lipopolysaccharide (LPS) derived from Gramnegative bacteria, and the most well characterized is monophosphoryl lipid A (MPL)
(Baldridge and Crane, 1999) derived from Salmonella minnesota. MPL have shown to induce
a Th1 type of response, but seems not to be a potent adjuvant for antibody production
18
RECOMBINANT SUBUNIT VACCINES
(Gustafson and Rhodes, 1992; Sing and O'Hagan, 1999). In certain studies, MLP has been
used in combination with e.g. alum or QS21 (Thoelen et al., 1998). However, when using
adjuvant combinations, the individual contribution of the included adjuvants is difficult to
establish. MPL has for example been evaluated in vaccine studies for cancer, herpes, hepatitis
B virus, malaria and human papilloma virus (Sing and O'Hagan, 1999), and has also been
investigated as adjuvant for DNA vaccines (Sasaki et al., 1997).
A second group of immunostimulatory adjuvants are the saponins, a heterogenous group of
sterol glycosides and triterpenoid glycosides, present in a wide range of plant species (Barr et
al., 1998). Saponins were included in a veterenary vaccine in the 1950s (Barr et al., 1998) but
later work showed that the saponins derived from Quillaja saponaria (Chilean soap bark tree)
were the most effective as adjuvant (Dalsgaard, 1970). Variable results have been reported
from the use of saponins, probably partly due to confusion with respect to the source of the
saponin and the variable content of saponins in extract. However, in 1978, Quillaja saponins
were purified, and a fraction with high adjuvant activity, termed Quil A, was defined
(Dalsgaard, 1978) and since then, Quil A has been used in a number of veterinary vaccines
(Dalsgaard et al., 1990). However, Quil A have been associated with some toxicity in vitro,
and several attempts have been made to purify Quil A (for review, see Barr et al., 1998). In
the 1990s, a fraction of Quil A saponins with low toxicity was isolated, denoted QS21
(Kensil, 1991). QS21 has the ability to induce antibody production and cytotoxic T cells
(CTL) (reviewed in Kensil, 1996). Studies evaluating QS21 as an adjuvant have been
performed for cancer, HIV-1, influenza, herpes, malaria and hepatitis B (Sing and O'Hagan,
1999). Human clinical trials have been performed using QS21 as adjuvant in for example
vaccines for malignant melanoma (Livingston et al., 1994) and in a DNA vaccine study for
HIV-1 (Sasaki et al., 1998)
Recent research has shown that sequences in bacterial DNA, but not vertebrate DNA has
direct immunostimulatory effect on cells in vitro (Tokunaga et al., 1984; Messina et al.,
1991). The effect is due to the presence of unmethylated CpG dinucleotides that is methylated
in vertebrate DNA (Krieg et al., 1995; Bird et al., 1987). It is thought that the CpG motifs in
bacterial DNA function as a danger signal and thus stimulating immune responses (Krieg,
1996). The exact mechanism for the immunostimulatory effect is not known, but it has been
shown that CpG motifs stimulate macrophages and dendritic cells (Jakob et al., 1998). CpG,
which can be in the form of plasmid DNA or synthetic oligonucleotides, has been evaluated as
19
CHRISTIN ANDERSSON
a mucosal adjuvant (Moldoveanu et al., 1998; McCluskie and Davis, 1999) and seems to have
a maximized effect when conjugated with protein antigens (Klinman et al., 1999).
Immunostimulatory sequences can also be used in combination with DNA vaccines, as will be
discussed later. To date, the effect of CpG immunization has only been widely used in animal
models, and its potential and safety in humans need to be evaluated.
Immunostimulatory substances are thought to induce cytokine production, and as an
alternative, cytokines can be used directly to modulate or increase the immune response. For
example, the use of cytokines has shown to be very promising for immunotherapy of cancer
(Salgaller and Lodge, 1998). Cytokines as adjuvants will not be discussed in detail here.
Particulate adjuvants, for example oil emulsions, microparticles, iscoms, liposomes and viruslike particles, have comparable dimensions to typical pathogens the immune system normally
encounters and are therefore thought to be suitable for uptake by antigen-presenting cells. See
Table 6 for a general comparison.
MF59, an oil-in-water emulsion (Ott et al., 1995) is besides alum, the only adjuvant approved
for human use. It has been described to interact with antigen presenting cells at the site of
injection and in lymph nodes (Dupuis et al., 1998). This adjuvant has shown to enhance the
immunogenicity of an influenza vaccine (Higgins et al., 1996; Cataldo and Nest, 1997) and to
be a more potent adjuvant than alum for a hepatitis B vaccine in baboons (Traquina et al.,
1996). To date, clinical trials in humans have been performed for several vaccines using
MF59 as adjuvant, e.g. to HIV (Kahn et al., 1994; Nitayaphan et al., 2000) and herpes simplex
virus (Langenberg et al., 1995; Drulak et al., 2000).
Liposomes, another group of particulate adjuvants, have been used as delivery system for
antigens alone or in combination with another adjuvant (Gregoriadis, 1990; Alving, 1995).
Liposomes are spherical bilayers of 50 -1000 nm in diameter (Langner and Kral, 1999). Some
advantages of using liposomes are that the compounds are protected from degradation and that
the circulation time of the antigen is increased (Langner and Kral, 1999). Liposomes have
been used as delivery system for proteins and peptides (Alving, 1995) as well as for DNA
(Perrie and Gregoriadis, 2000). Cochleates, a modified liposomal structure has also been
evaluated in animal models (Gould-Fogerite et al., 1998).
Biodegradable polymeric microparticles, a group of adjuvants that have been investigated for
vaccine delivery, and among these, the poly(lactide-co-glycosides) (PLGs) are the primary
20
RECOMBINANT SUBUNIT VACCINES
candidates for adjuvant development (O'Hagan et al., 1991a and b). PLG particles have been
used in humans for many years as suture material and controlled-release delivery systems
(Sing and O'Hagan, 1999) and only recently, PLG particles have been shown to have adjuvant
properties to encapsulated antigens (O'Hagan et al., 1991a and b). Microparticles have been
demonstrated to be effective in eliciting CTLs (Maloy et al., 1994; Nixon et al., 1996) and
have been suggested as adjuvants for DNA vaccines (Hedley et al., 1998). A unique feature of
the microparticles are their ability to control the release of antigen, therefore it could be
possible to develop a vaccine that have a built-in boosting system. This is very attractive,
especially in the developing countries, since it might simplify vaccination logistics
Viral proteins expressed in heterologous hosts may associate in the culture media and form
virus like particles (VLPs). When antigens and viral proteins are coexpressed, the VLPs
entrap the antigen and can be used as an adjuvant system. For example, recombinant VLPs
from Saccharomyces cerevisiae, carrying a string of 15 epitopes from Plasmodium species
have been shown to induce CTL responses in mice (Gilbert et al., 1997). Similar VLPs have
also been shown to induce CTLs to SIV in macaques (Klavinskis et al., 1996) and to be safe
and immunogenic in humans (Martin et al., 1993).
In immunostimulating complexes, iscoms, Quil A, a saponin derived from Q. saponaria
(described above) has been incorporated into cage-like structures (Morein et al., 1984). The
iscoms are of about 40 nm in diameter and consists of cholesterol, phospholipids and the
target antigen (Morein et al., 1984; Barr et al., 1998). Iscoms have been used as adjuvant in
several animal models (Morein et al., 1995; Barr et al., 1998; Sjölander and Cox, 1998).
Protective immunity induced by iscoms has for example been demonstrated against influenza
virus in mice (Lövgren et al., 1990) and horses (Mumford et al., 1994). The mechanism of the
iscoms mode of action is not clearly understood, but some studies suggest that increased
cellular uptake may contribute to the adjuvant activity (Barr et al., 1998).
The saponin initially used in iscoms, Quil A, is quite heterogenous. In order to determine the
immunological properties and toxicity of chemically defined components of Quil A, purified
immunostimulatory fractions (QH-A, QH-B and QH-C) from Quil A was investigated, both as
free saponins and incorporated into iscoms (Rönnberg et al., 1995). Immunizations of mice
showed that all investigated combinations enhanced the antibody response equally, but the
combination of QH-A and QH-C in a ration of 7:3 (Iscoprep™703, Iscotec AB, Sweden)
where the toxic fraction QH-B was excluded, was both effective and non-toxic. The purified
21
CHRISTIN ANDERSSON
components QH-A, QH-B and QH-C have been characterized in several other studies
(Rönnberg et al., 1997; Johansson and Lövgren-Bengtson, 1999). Numerous of other purified
components from Quil A have also been investigated, both in iscoms and as free components,
reviewed in Barr et al., 1998.
One problem with iscoms is that the incorporation of the antigen need to be adapted for each
antigen, and may require modifications of the antigen. For several adjuvants, such as
liposomes, cochleates, non-ionic block polymers and iscoms, the protein antigens are
normally incorporated by means of hydrophobic interactions (Morein et al., 1996), and the
physical assocation has been demonstrated to be essential for the adjuvant to exert its
immunostimulating capacity. For iscoms, it has been shown that membrane-antigens
containing hydrophobic transmembrane sequences, are efficiently incorporated (Morein et al.,
1995). Proteins derived from membranes of a variety of viruses, bacteria and parasites have
successfully been incorporated into iscoms through hydrophobic interactions (for review, see
Barr et al., 1998). In contrast, hydrophilic antigens require modifications to be incorporated
into iscoms. Partial denaturation by using low pH (Pyle et al., 1989; Morein et al., 1990; Heeg
et al., 1991) or high temperature (Höglund et al., 1989) have been used to expose internal
hydrophobic sequences of otherwise hydrophilic proteins. Covalent addition of fatty acids is
also a useful method used to incorporate soluble proteins into iscoms, e.g. in a recombinant
HIV-1 gp120 candidate vaccine (Reid, 1992; Browning et al., 1992). Other strategies that
have been investigated include chemical conjugation of a peptide (Lövgren et al., 1987) or
proteins to pre-formed iscoms containing influenza envelope protein or matrix alone (Morein
et al., 1995; Sjölander et al., 1991; Larsson et al., 1993). Novel strategies, for inclusion of
hydrophobic protein sequences or even lipids in the protein antigen will be presented in this
thesis (I, II, III).
22
RECOMBINANT SUBUNIT VACCINES
4. Live delivery of subunit vaccines
Live vectors for delivery of heterologous subunit antigens have been suggested to be a more
cost-effective strategy than to produce and formulate protein subunit vaccines (Liljeqvist and
Ståhl, 1999). No extensive purification would be required and several live delivery systems
have been shown to elicit strong long-lasting immunity without the need for adjuvants. Both
bacteria and viruses have been investigated for delivery of foreign antigens. The knowledge of
molecular biology and genetics has resulted in the development of new attenuation strategies,
and thus genetically defined attenuated strains of bacteria or virus. By the use of recombinant
DNA technology, the genes encoding the heterologous antigen to be delivered can be inserted
into the non-pathogenic or attenuated live vector.
4.1. Bacterial delivery systems
There are two major approaches used for developing live bacterial delivery systems for
recombinant subunit vaccines, either to use genetically attenuated pathogenic bacteria or to
engineer non-pathogenic, commensal or food-grade bacteria. Both concepts require
engineering of the bacteria so that it would express the foreign target antigen, but when using
attenuated bacteria, vaccination to the original disease could simultaneously be performed.
Attenuated bacteria have been used as live recombinant vectors for heterologous antigens, e.g.
systems based on Salmonella thyphi (Levine et al., 1990), an enteric bacteria that has been
utilized to raise mucosal immunity against numerous foreign polypeptides. The oral bacteria
Streptococcus gordonii and the gut bacteria Lactococcus lactis, both commensal nonpathogens from mucosal surfaces have been extensively studied (Dertzbaugh, 1998). S.
gordonii has been shown to elicit secretory IgA and serum IgG in mice when used to deliver
the M6 protein of S. pyogenes to the mucosal immune system (Pozzi et al., 1992; Medaglini et
al., 1995). Protection in mice after immunization with L. lactis engineered to express the C
fragment of tetanus toxin has also been reported (Wells et al., 1993).
When using naturally intracellular bacteria, the heterologous antigen can be either expressed
intracellularly or as exposed at the cell surface. However, when using commensal or foodgrade bacteria, it has been suggested that surface exposure of the heterologous antigen is
important to elicit antigen-specific immune responses (Nguyen et al., 1995; Ståhl and Uhlén,
23
CHRISTIN ANDERSSON
1997). Heterologous surface expression of antigens have been reported for several bacterial
systems evaluated as live delivery vehicles (Georgiou et al., 1993), for example gram-positive
bacteria and mycobacteria (Fischetti et al., 1996; Georgiou et al., 1997; Ståhl and Uhlén,
1997; Stover et al., 1993). Recently, protective immunity to respiratory syncytial virus (RSV)
could be evoked in mice by intranasal immunization with Staphylococcus carnosus carrying
surface-exposed RSV peptides (Cano et al., 2000). S. carnosus is a non-pathogenic bacteria
traditionally used in meat-fermentation processes.
4.2. Viral delivery systems
Recombinant viruses have been studied as candidate vaccines, both as vaccines against the
original diseases, and as viral vectors used to deliver heterologous antigens or genes. One
advantage of using viral vaccines 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.
Vaccinia virus was earlier used in vaccination against smallpox. The introduction of genes
encoding foreign proteins into a vaccinia vector was first demonstrated in 1982, and vaccinia
virus has since then been extensively studied as live vaccine vector (Paoletti, 1996).
Recombinant vaccinia virus was originally produced by homologous recombination, but today
alternative strategies have been developed, enabling more efficient gene construction
procedures.
Immunization experiments with recombinant vaccinia vectors expressing viral and non-viral
antigens have been reported to elicit protective immunity in animal disease models (Liljeqvist
and Ståhl, 1999). An oral rabies vaccine consisting of recombinant vaccinia expressing the
rabies virus glycoprotein has been given to wild animals, leading to drastic decrease of the
incidence of rabies in Belgium (Brochier et al. 1995).
Recombinant vaccinia virus based on wild-type vaccinia has primarily been investigated as
veterinary vaccines, mostly due to safety concerns. For human use, highly attenuated, nonreplicative vaccinia vectors have been constructed, e.g. the NYVAC strain, with deletion of 18
open-reading frames from the original genome (Tartaglia et al., 1992; Paoletti et al., 1996),
and the modified vaccinia virus Ankara strain (MVA) (Sutter and Moss, 1992). Recombinant
24
RECOMBINANT SUBUNIT VACCINES
NYVAC and MVA vectors encoding pathogen-derived antigens have been evaluated
extensively in various disease models (Perkus et al., 1995; Caroll and Moss, 1997).
Other viral vaccine vectors that have been investigated are certain poxviruses, unable to
replicate in mammalian cells, e.g. fowlpox and canarypox viruses, and ALVAC, a
recombinant canarypox virus (Perkus et al., 1995). Adenoviral vaccine vectors, not pathogenic
in humans, can be made replication-competent or deficient, and can be administered orally
(Imler, 1995). Several viral antigens have been expressed and delivered by adenoviral vectors,
resulting in humoral, cell-mediated or mucosal immune responses (Fooks et al., 1995; Mittal
et al., 1996; Xiang et al., 1996). Non-infectious self-assembled virus-like particles have also
been used as delivery systems for antigens. Fusion of a viral epitope to the N-terminus of the
VP2 capsid protein of Parvovirus led to the assembly of parvovirus-like particles, which were
able to deliver the foreign epitopes into the cytosol, resulting in stimulated cellular immunity
(Sedlik et al., 1997).
Although a number of human clinical trials with different viral recombinant vectors have been
performed, no vaccine based on live viral delivery is yet available on the market. The main
applications for virus-based vectors would initially be for certain specific diseases, such as
vaccines for HIV and cancer, as well as veterinary vaccines.
25
CHRISTIN ANDERSSON
5. Nucleic Acid Vaccines
Nucleic acid vaccines represent a rather recent approach to the control of infectious agents.
These novel vaccines consist of DNA (as plasmid) or RNA (as mRNA), although the use or
RNA has not yet been as well studied. Tables 7A and 7B show selected examples of
immunization studies using DNA or RNA, respectively.
5.1. DNA vaccines
DNA vaccines consist of designed eukaryotic expression plasmids. The gene encoding the
antigen (or antigens) of interest is placed under the control of a strong viral promoter,
recognized by the mammalian host. Inoculation of plasmid DNA vaccines into muscle or skin
results in uptake of the DNA into cells, expression of the encoded antigen and as a result, a
raised antigen-specific immune response. To enable bacterial propagation of the plasmid, it
also contains an E. coli origin of replication. The administration of plasmid DNA encoding a
specific protein antigen was first shown to induce expression of the encoded protein in mouse
muscles (Wolff et al., 1990). Soon, it became clear that immunization with plasmid DNA also
could elicit antibodies to the encoded antigen (Tang et al., 1992). Protective immunity against
e.g. influenza (Ulmer et al., 1993) and malaria (Sedegah et al., 1994) has also been shown.
Extensive work has been done on DNA vaccines the past decade, and DNA vaccines have
been shown to be well tolerated, capable of stimulation of a broad immune response,
including cytotoxic T-lymphocytes (CTLs), and generating lasting immune responses
(Yankauckas et al., 1993; Davis et al., 1993). The efficacy of DNA vaccination has been
reported in small and large animal models for infectious diseases, cancer and autoimmune
diseases (Donnelly et al., 1997b; Kowalczyk and Ertl, 1999), of which selected examples are
shown in Table 7A. Vaccines for diseases such as cancer (Restifo et al., 1999), HIV (Calarota
et al., 1998) and malaria (Hoffman et al., 1997; Wang et al., 1998a and b; Le et al., 2000) are
at present being evaluated in human clinical trials.
26
RECOMBINANT SUBUNIT VACCINES
Table 7A: Selected examples of immunization studies using DNA (modified from Kowalczyk
and Ertl, 1999.
Pathogen
Encoded antigen
Reference
Ebola virus
nucleoprotein (NP), glycoprotein (GP)
NP, GP
Vanderzanden et al., 1998
Xu et al., 1998
Hepatitis B virus
hepatitis B surface antigen (HBsAg)
HBsAg
Davis et al., 1997
Prince et al., 1997
Herpes simplex virus 1
glycoprotein B, glycoprotein D
glycoprotein B
McClements et al., 1997
Daheshia et al., 1998
HIV-1
nef, rev, tat, glycoprotein160, p24
nef, rev, tat
env
gag
Hinkula et al., 1997
Calarota et al., 19981
Sasaki et al., 1998
Qiu et al., 2000
Influenza
hemagglutinin (HA)
HA, NP, matrix protein (M1)
HA
Ban et al., 1997
Donnelly et al., 1997a
Ulmer et al., 1998
Measles
HA, NP
Cardoso et al., 1998
Papilloma
E1, E2
Han et al., 2000
Rabies
G protein
G protein
Wang et al., 1997
Lodmell et al., 1998
Viruses
Respiratory syncytial virus F protein
G protein
Li et al., 1998
Li et al., 2000
Bovine RSV
G protein
Schrijver et al., 1998
Rotavirus
viral protein 4 (VP4), VP6, VP7
Chen et al., 1997
Borrelia burgdorferi
Outer surface lipoprotein A (OspA)
Simon et al., 1996
Clamydia trachomatis
major outer membrane protein (MOMP)
Zhang et al., 1999
Clostridium tetani
tetanus toxin C fragment
Saikh et al., 1998
Mycoplasma turbeculosis
Antigen 85 (Ag85)
heat shock protein 65 (hsp65)
Ulmer et al., 1997
Bonato et al., 1998
Salmonella typhii
Outer membrane protein C
Lopez-Macias et al., 1996
circumsporozoite protein (CSP)
CSP, Exp-1, surface sporozoite protein2
(SSP2), LSA-1
CSP
Wang et al., 1998a1
Wang et al., 1998b
Plasmodium yoelii
CSP
Gramzinski et al., 1997
Trypanosoma cruzii
trans-sialidase (TS)
Costa et al., 1998
Bacteria
Parasites
Plasmodium falciparum
1
Le et al., 20001
Human trials
27
CHRISTIN ANDERSSON
Table 7B: Selected examples of immunization studies using RNA or plasmid DNA encoding
self-replicating RNA
Pathogen
Encoded antigen
System used
Reference
SIV
glycoprotein 160 (gp160)
rSFV viral particles
Mossman et al., 1996
HIV
env
gp160
rSFV viral particles
rSFV viral particles
Brand et al., 1998
Berglund et al., 1997
Influenza
hemagglutinin (HA)
nucleoprotein (NP)
rSFV RNA
rSFV RNA and
rSFV viral particles
rSFV viral particles
rSindbis viral particles
Dalemans et a., 1995
Zhou et al., 1994
Japanese encephalitis E protein, nonstructural
virus
protein (NS) 1 and NS2a
rSindbis plasmid
Pugachev et al., 1995
Looping ill virus
envelope proteins (prME),
NS1
envelope proteins (prME),
NS1
rSFV viral particles
Fleeton et al., 1999
rSFV plasmid and
rSFV viral particles
Fleeton et al., 2000
gB
rSindbis plasmid
Hariharan et al., 1998
epitope derived from
circumsporoziote protein
rSindbis viral particles
Tsuji et al., 1998
Viruses
NP
NP
Herpes virus
Berglund et al., 1999
Tsuji et al., 1998
Parasites
Plasmodium yoelii
Plasmid DNA can be administered in several ways, but so far the most common immunization
route is intramuscular injection of plasmid in saline. Other routes that have been investigated
used include intradermal (i.d.) (Schrijver et al., 1998), intravenous (i.v) (Liu et al., 1997;
Böhm et al., 1998), nasal (Sasaki et al., 1998; Ban et al., 1997) or even ocular routes
(Daheshia et al., 1998). Comparative studies have shown that the route of administration can
have great impact on the elicited immune response (Barry and Johnston, 1997; Pertmer et al.,
1996). When using the intradermal route, plasmid can be either injected with a syringe or
administered with a gene gun (Pertmer et al., 1995; Dégano et al., 1998). Using the gene gun,
plasmid DNA is precipitated onto an inert particle (usually gold beads) and forced into the
cells by a helium blast. The gene gun strategy is more efficient in delivering the plasmid into
the cells, and the amount of DNA needed for gene-gun immunization can be as low as a few
nanograms (Pertmer et al., 1995; Dégano et al., 1998).
Delivery routes to the mucosal surface, for example direct delivery of nucleic acids to the
nasal or oral tract have been demonstrated to have the capacity to induce mucosal immune
28
RECOMBINANT SUBUNIT VACCINES
responses (Ban et al., 1997). Other delivery methods to mucosal surfaces is the use of
intracellular bacteria as carriers for plasmid DNA (Sizemore et al., 1995), the use of
incorporation of DNA into microspheres (Jones et al., 1997) and the use of lipid-based DNA
delivery systems (Felgner et al., 1995; Gregoriadis et al., 1997). More effective systems for
delivering the DNA to the nucleus also need to be further evaluated. Adjuvants and delivery
systems evaluated in DNA vaccine studies are discussed in section 5.2.1.
DNA vaccines have some advantages over traditional vaccines. Immunologically, due to
endogenously production of the antigen, DNA vaccines induce the same immune response as
a natural infection (Kowalczyk and Ertl, 1999), resulting in the induction of a cellular
response, generally not elicited with protein-based vaccines. In a comparative study using the
SV40 large tumor antigen, plasmid DNA showed to be superior to immunization with proteins
or virus in inducing antigen-specific CTLs (Schirmbeck et al., 1996). Moreover, DNA
immunizations circumvents problems like incorrect folding and lack of post-translational
modifications that has been associated with protein subunit vaccines. Compared to proteinbased vaccines, the cost of production is also drastically reduced due to simplified production
and purification (Ferriera et al., 2000). Another advantage of DNA vaccines compared to live
attenuated vaccines is that there is no associated risk of reversion to virulence. Vaccines based
on DNA is also easy to distribute since the DNA is extremely stable and does not require cold
chains, which is difficult to maintain in developing countries (Kowalczyk and Ertl, 1999).
Some comparative studies involving DNA vaccines have been performed e.g. DNA encoding
human wild type p53 induced protective immune responses in mice, but the same gene
delivered with a viral vector did not (Hurpin et al., 1998). However, others have experienced
that the efficacy of nucleic acid vaccines is very low and highly variable (reviewed in
Manickan et al., 1997). Thus, one emerging opinion is, that since the antibody response raised
from DNA vaccines is generally slow and not very potent, and unless they are improved,
DNA vaccines might serve best as priming agents in combination vaccines. (Kowalczyk and
Ertl, 1999).
29
CHRISTIN ANDERSSON
5.2. Improving immune responses to DNA vaccines
A large number of approaches have been used in attempts to improve the efficiency of DNA
vaccines (Rodriguez and Whitton, 2000). One strategy is to optimize of the many sequence
elements included in the plasmid used, e.g. promoter, introns, enhancers and poly-adenylation
signals (Böhm et al. 1996; Danko et al. 1997; Montgomery et al., 1993). The antigenencoding gene can be optimized in several ways, like eliminating sequences encoding
intracellular domains or transmembrane sequences (Chen et al., 1998) or reduction of the size
of the antigen-encoding sequence by identifying the immunodominant epitopes, leaving only
defined epitopes for B or T cells. The latter approach has been used with T cell epitopes
(McCabe et al., 1995; Casares et al., 1997). In some cases, the full length antigen is too toxic
or immunosuppressive for the host, and insertion of only the immunodominant epitopes into a
highly immunogenic core sequence will activate the immune system in a proper way (Barry
and Johnston, 1997; Levy et al., 1993). Strategies to design effective DNA vaccines could
also be to create multi-epitope DNA vaccines, in which antigen-encoding genes or genes
encoding specific epitopes could be fused together, to simultaneously immunize for several
diseases (Suhriber, 1997).
Immune responses after DNA immunization might be improved by altering the immunization
schedule, for example fewer immunizations and longer resting periods between
immunizations have shown to develop three to tenfold higher antibody levels (Fuller et al.,
1997). As mentioned above choice of route can have impact on the elicited immune response,
e.g. immunization at mucosal surfaces induce mucosal immunity (Ban et al., 1997). Another
strategy would be to target DNA vaccines to cell-specific ligands. Attempts have been made
to specifically target a DNA vaccine to the antigen-presenting cells e.g. by using DNA
encapsulated in mannose-coated liposomes (Toda et al., 1997) for the purpose of targeting
receptors on dendritic cells. Even targeting to the nucleus to increase the transport of DNA to
the nucleus has been investigated, for example inclusion of nuclear localization sequences in
DNA complexes has been shown to be increase transfection efficiency in vitro (Kaneda et al.,
1989).
30
RECOMBINANT SUBUNIT VACCINES
5.2.1. Adjuvants and delivery systems forDNA vaccines
To enhance or modulate immune responses elicited by DNA vaccines, different adjuvants or
delivery systems can be used. Table 8 presents selected examples used in immunization
studies.
Table 8: Selected examples of adjuvants and delivery systems used for plasmid DNA
Adjuvant /delivery system
Reference
Aluminum adjuvant
Ulmer et al., 2000
Cationic liposomes
Gregoriadis et al., 1997
Ishii et al., 1997
Cationic lipid mixtures
Norman et al., 1997
Cationic microparticles
Singh et al., 2000
Monophosphoryl lipid A (MPL)
Sasaki et al. 1997
Poly(DL-lactide-co-glycolide) microparticle
Jones et al. 1997
QS 21
Sasaki et al. 1998
Bacterial delivery systems
Listeria monocytogenes
Dietrich et al., 1998
Salmonella typhimurium
Darji et al., 1997
Shigella flexneri
Sizemore et al., 1997
Live, attenuated bacteria have been used as carriers for plasmid DNA, for example attenuated
Shigella flexneri that has the capacity to invade mammalian cells upon infection. Delivery of
plasmid to the cytoplasm of infected cells using S. flexneri as delivery system (Sizemore et al.,
1995) has been demonstrated. Furthermore, high serum antibody titers in mice immunized
intranasally with such S. flexneri delivery system (Sizemore et al., 1997) has been shown.
Delivery of plasmid DNA encoding model antigens to macrophages was studied in vitro using
Listeria monocytogenes as suicide vector (Dietrich et al., 1998) and successful expression and
antigen presentation could be shown. The intracellular pathogen Salmonella typhimurium has
also been used for DNA delivery (Darji et al., 1997).
31
CHRISTIN ANDERSSON
5.2.2. Codelivery of stimulatory substances
In order to increase or modulate the vaccine-induced immune response, natural signal
substances from the immune system can be used, codelivered as either active substances or as
plasmid DNA. Codelivery of cytokines has show to modulate the immune response in a
number of ways (Kowalczyk and Ertl, 1999), e.g. codelivery of GM-CSF was shown to
increase the immune response to a rabies antigen (Ertl and Xiang, 1996), but in the same
system, codelivery of IFN-γ was shown to reduce the immune response (Xiang et el., 1997).
An important costimulatory molecule in the immune system is B7, a receptor expressed on
activated APCs that binds T-cell ligands. The gene encoding B7 was shown to enhance the
immune response only when it was inserted in the same plasmid as the gene encoding the
antigen, suggesting that the antigen-specific and costimulatory signals must originate from the
same cell (Conry et al., 1996). Codelivery of B7 with an HIV-1 DNA vaccine dramatically
increased the CTL response in mice (Kim et al., 1997) as well as in chimpanzees (Kim et al.,
1998).
5.2.3. Immunostimulatory DNA sequences
Plasmid DNA without adjuvant has in certain studies been shown capable of inducing strong
immune responses to the encoded antigen (Kowalczyk and Ertl, 1999). As been discussed
before in section 3, this may partly be due to the presence of immunostimulatory DNA
sequences (ISS) in the plasmid DNA (Krieg et al. 1995; Weiner et al. 1997). ISS are nonmethylated DNA sequences containing CpG-dinucleotides that can activate the non-specific,
innate immune response, mainly by activating monocytes, NK cells, dendritic cells (DC) and
B-cells in an antigen-independent manner (Krieg and Kline, 2000). Methylation of the CpGdinucleotides has been shown to abolish the immunogenicity of the DNA vaccine (Lipford et
al., 1997). CpG motifs can either be co-administered with the plasmid DNA in the form of
oligonucleotides or the number of ISS in the plasmid can be increased. Ongoing research in
this area will reveal the importance of species-specificity, restrictions of the DNA sequences
outside the CpG-sequences and the potential for CpG as adjuvant for use in humans (Lipford
et al., 1997; Krieg and Kline, 2000).
32
RECOMBINANT SUBUNIT VACCINES
5.3. Safety of DNA vaccines
DNA vaccines have thus far not shown to induce any severe side-effects. There is little
evidence for integration of DNA into the host genome (Nichols et al., 1995) and no
autoimmune reactions or persistent anti-DNA antibodies have been observed. Some studies
have reported production of anti-DNA antibodies, however with very low and transient titers
(Mor et al., 1997).
5.4. RNA Vaccines
Nucleic acid vaccination through the delivery of RNA has been investigated to a lesser extent
than the DNA vaccination. The first applications of delivery of mRNA showed to induce CTL
to the influenza virus nucleoprotein in mice when mRNA was delivered in liposomes
(Martinon et al. 1993). Liposome-mediated transfection of mouse fibroblasts with mRNA
encoding human carcioembryonic antigen (Conry et al., 1995) resulted in a transient
production of antibodies. The rapidly declining antibody levels could reflect a short-lived
protein expression in vivo. One major advantage of RNA vaccines over DNA vaccines would
be that no risk of host-genome integration of the delivered gene exists. However, a
disadvantage of using RNA-based vaccines is that the preparation and administration of RNA
is troublesome because of the low stability of the RNA. This might lead to rapid in vivo
degradation, and thus a short-lived expression of the encoded gene. Furthermore, certain
reagents needed for RNA preparation, e.g. RNA polymerases and capping agents, are rather
costly.
Development of a new generation of RNA vaccines, derived from members of the Alphavirus
family e.g. Sindbis virus and Semliki Forest virus (SFV), have some interesting fetures that
makes them suitable for development of RNA vaccines (Tubulekas et al., 1997). The
alphaviruses have a very broad host range since they infect various mammalian, avian and
insect cells, (Zhou et al., 1994). Also, the RNA genomes of the alphaviruses are selfreplicating, which will make up for e.g. low transfection efficiencies of DNA vaccines. The
mechanism of SFV-based vaccines is shown in Figure 1.
In the alphaviruses, the genes encoding the structural proteins, needed for virus particle
formation, are located in the last third of the genome. When constructing RNA-based vaccine
vectors, those genes are replaced for a gene encoding an antigen. The RNA vector now carry
33
CHRISTIN ANDERSSON
the gene encoding an alphavirus replicase (e.g. SFV replicase) together with the gene
encoding the foreign antigen. Transfection of a mammalian cell with such an RNA construct
will first lead to translation of the replicase. The replicase will be responsible for production
of new full-length RNA genomes and shorter subgenomic RNA molecules, via a negative
strand intermediate. The subgenomic RNA molecule corresponds to the last third of the full
length RNA. Translation of the subgenomic RNA will lead to production of the antigen. Mass
replication of the RNA will theoretically produce up to 200 000 copies of RNA in a single cell
within 4 hours, and the gene product can be as much as 25% of total cell protein (Rolls et al.,
1994).
cap
SFV Replicase
Struct.
Positive, single strand
RNA
(A)n
Replicase
3'
5'
Replicase
cap
SFV Replicase
Negative strand
intermediate RNA
Struct.
Replicase
(A)n cap
Struct.
(A)n
Positive strand fulllength RNA
Positive strand,
subgenomic RNA
Structural proteins, C,
p62 and E1
Figure 1: The life cycle of the Semliki Forest virus. Upon infection of the mammalian host cell, the
full-lenght positive stranded RNA will function as mRNA. Translation of the first two thirds results in
poduciton of the replicase. The replicase will initially be responsible for production of a negative
strand RNA. Using the negative RNA as template, the replicase will mass-produce full-length RNA.
The replicase will also recognize an internal promoteer sequence, and start mass-produce subgenomic
RNA, corresponding to the last third of the full-lenght RNA. Translation of the subgenomic RNA
results in production of the structural proteins, needed for production of new viral particles.
34
RECOMBINANT SUBUNIT VACCINES
The Semliki Forest virus vector systems were first used as a tool for mammalian production
of recombinant proteins (Liljeström and Garoff, 1991, Liljeström, 1994; IV) but has lately
been evaluated in nucleic acid vaccine approaches (Table 7B).
An alphavirus based RNA vaccine can be delivered alone or as packed in a virus particle
(Tubulekas et al., 1997). To achieve packaging in a viral particle, cotransfection of a helper
RNA molecule encoding the genes for the structural proteins is required for production of
viral particles. Only the full-length RNA molecule, lacking the genes encoding the structural
proteins, contains a packaging signal, and will be packed in the produced recombinant virus
particles. The sub-genomic RNA lacks the packaging signal and will thus not be packed in
viral particles. Transfection of cells with these recombinant virus particles will initially lead to
delivery of the full-length RNA to the host cell cytoplasm. However, since the gene encoding
the structural proteins, needed for production of new viral particles, is removed, transfection
of the recombinant virus will not result in production of new viral particles.
RNA alone, encoding the alphavirus replicase and the target antigen, has proven to give in
vivo expression of the antigen (Johanning et al., 1995) resulting in strong immune responses
to the encoded antigen, e.g. influenza virus nucleoprotein in mice (Zhou et al., 1994).
Recombinant SFV particles with packed RNA encoding influenza virus nucleoprotein have
been shown to induce strong immune responses in mice (Zhou et al., 1995), and were recently
demonstrated capable of inducing protective immunity in mice (Berglund et al., 1998). The
immunizations of primates with RNA packed in SFV particles have been demonstrated to
induce strong immune responses to HIV-1 (Berglund et al., 1997) and SIV (Mossman et al.,
1996).
As mentioned above, the low stability of naked RNA makes the preparation and
administration of RNA somewhat troublesome. Those problems have been overcome by the
development of a plasmid DNA vaccine, based on the Sindbis genome (Herweijer et al.,
1995) or the SFV genome (Berglund et al., 1998). When using conventional DNA vectors,
containing a eukaryotic promoter, e.g. the cytomegalovirus promoter (CMV), the amount of
RNA transcribed is totally dependent on the promoter. However, when using SFV-based
plasmids, transcription of RNA and subsequent translation will lead to the production of the
SFV replicase. From this point on, the replicase will be responsible for copying the RNA, and
the alphaviral replication cycle will continue as for the RNA-based system (Figure 1). Thus,
35
CHRISTIN ANDERSSON
plasmids employing the alphaviral self-replication properties on RNA level could be seen as
RNA-vaccines delivered as a DNA precursor, and was presented in Table 7B.
Plasmids derived from alphaviral genomes have been used in expression studies, e.g. high
expression levels of a luciferase reporter gene in cell lines after transfection of a Sindbis
replicon-based DNA plasmid have been reported (Herweijer et al., 1995; Dubensky et al.,
1996). Similar studies using an SFV-based plasmid have shown high level expression of βgalactosidase in BHK-21 cells upon transfection (Berglund et al., 1998). Plasmids with
alphaviral self-replication properties have also been used in immunization studies in animals
(Table 7B). Immunization of mice with the SFV-based plasmid containing the gene encoding
influenza nucleoprotein induced humoral and cellular immune responses as well as protection
in mice (Berglund et al., 1998). A similar plasmid derived from the genome of the Sindbis
virus have been shown to effectively induce humoral and cellular immune responses as well
as protection to herpes simplex virus in mice (Hariharan et al., 1998). Some attempts to
campare the RNA and DNA based alphaviral systems have been performed. For exemple, in
immunization of mice with plasmid and recombinant viral particles louping ill antigens, the
viral particles were more effective in inducing protective immune responses (Fleeton et al.,
2000). However, more extensive studies have to be performed in order to evaluate the
differences between the systems.
36
RECOMBINANT SUBUNIT VACCINES
PRESENT INVESTIGATION
6. Recombinant production of protein immunogens
6.1. Recombinant strategies for iscom incorporation (I, II, III)
Subunit vaccine research is dependent on efficient production of protein antigens, and
recombinant strategies are today dominating (Liljeqvist and Ståhl, 1999; Ståhl et al., 1999).
As discussed earlier gene fusions are frequently used to improve or alter the characteristics of
the proteins to make the production more efficient (Ståhl et al., 1997; Ståhl et al., 1999). In
this thesis, recombinant strategies for production of protein immunogens furnished with a
hydrophobic tag to improve incorporation into iscoms have been evaluated (I, II, III). For
several adjuvants, such as liposomes, cochleates, non-ionic block polymers and
immunostimulating complexes (iscoms), the protein antigens are normally incorporated by
hydrophobic interactions (Morein et al., 1996). For iscoms, it has been shown that antigens
containing hydrophobic amino acid sequences are efficiently incorporated (Morein et al.,
1995) while hydrophilic antigens instead could be incorporated by partial denaturation to
expose hydrophobic sequences, or by chemical conjugation to preformed iscoms or iscommatrix alone (Morein et al., 1995).
Here, two protein sequences with different properties were combined; a hydrophobic tag, to
make direct incorporation into iscoms possible and an affinity tag, to enable efficient affinity
purification of the fusion proteins. As hydrophobic tags, either hydrophobic amino acid
residues (I, II), added N-terminally or C-terminally, or lipids, added by in vivo or in vitro
strategies (III) were investigated.
6.1.1. N-terminal hydrophobic tags (I)
Initially, three different hydrophobic tags, IW, MI and PD (Table 9), were evaluated for their
efficiency to direct iscom incorporation. The first tag IW, composed of numerous isoleucine
(I) and tryptophan residues (W), was selected because similar tags have been shown to
improve the partitioning of recombinant proteins in aqueous two-phase systems (Köhler et al.,
1991; Hassinen et al., 1994).
37
CHRISTIN ANDERSSON
Table 9: Hydrophobic tags used in I and II
Tag
Sequence
Origin
Position
IW
IWIWSIWIWSIWIW
Numerous isoleucine (I) and
tryptophan residues (W)
N-terminally
MI
QILAIYSTVASSLVLLVSLGAISFWMCS
The membrane-integration anchor
sequence of the H1 hemagglutinin
of influenza virus A
N and Cterminally
PD
LEAALAELAELAE
Predicted to form an amphiphatic
α-helix at low pH
N-terminally
M
ENPFIGTTVFGGLSLALGAALLAG
Derived from the membranespanning region of SpA
C-terminally
The second tag MI represents the membrane-integration anchor sequence of the H1
hemagglutinin of influenza virus A (Feldmann et al., 1988). Influenza hemagglutinins have
previously been efficiently incorporated into iscoms (Sjölander et al., 1991). The third tag PD,
was designed to form an amphiphatic α-helix at low pH (Parente et al., 1990a, b). As affinity
tag in the three expression systems, the Z domain from Sthaphylococcus aerus protein A
(SpA) was used (Nilsson et al., 1987). In this study, a 53 amino acid malaria immunogen, M5
(Sjölander et al., 1993a) derived from the central repeat region of the Plasmodium falciparum
blood-stage antigen Pf155/RESA (Coppel et al., 1984; Perlmann et al., 1984) (Figure A)
served as model antigen.
General expression vectors were constructed, pT7IWZmp8, pT7MIZmp8 and pT7PDZmp8
designed for intracellular production in E. coli under control of the tightly regulated T7
promoter system (Studier et al., 1990). The target immunogen M5 was introduced into the
multiple cloning site and the three fusion proteins IW-Z-M5, MI-Z-M5 and PD-Z-M5 (Table
10) respectively, were expressed. All three fusion proteins could be recovered as full-length
fusion proteins after affinity purification on IgG-Sepharose. Probably due to the
hydrophobicity of the proteins, insoluble aggregates of IW-Z-M5 and MI-Z-M5 (but not PDZ-M5) were formed during the protein production (Table 10). For all three fusion proteins,
rather low expression levels were obtained, in the range of 1 mg per liter cell culture.
Iscom preparations were performed of all three fusion proteins (IW-Z-M5, MI-Z-M5 and PDZ-M5, respectively) as described before (Morein et al., 1984; Lövgren et al., 1987). Briefly,
proteins were mixed with cholesterol (including a small amount of 3H-cholesterol for
subsequent analysis of cholesterol content), phosphatidyl choline and Quil A.
38
RECOMBINANT SUBUNIT VACCINES
MALARIA
Anopheles
Human
Mosquito
blood meal
Liver
Sporozoites
Sporozoites
Schizont
Ookinete
Merozoites
Ring
Zygote
Mosquito gut
Scizont
Gametocyte
Trophozoite
Mosquito
blood meal
Gametocytes
Figure A: The life cycle of the Plasmodium falciparum parasite in humans and in the mosquito.
At present, at least 300 million people are infected by malaria globally and there are between 1-1.5
million deaths from malaria per year (WHO). The causative agents in humans are four species of
Plasmodium protozoan parasites; P. falciparum, P. vivax, P. ovale and P. malariae. Of these, P.
falciparum accounts for the majority of infections and is the most lethal. The female Anopheles mosquito
is responsible for transmission of parasites between humans. The parasites develop in the gut of the
mosquito and are transferred to the blood stream of the human host when the mosquito feeds on human
blood. The life cycle is divided into several stages in the mosquito the liver and the erythrocytes and, as
outlined schematically in figure A. Symptoms of malaria include a high fever as a consequence of
eruption of erythrocytes. If not treated, the disease, particularly that caused by P. falciparum, progresses
to severe malaria. Severe malaria is associated with death. Traditional methods for controlling malaria,
like the use of insecticides against the mosquito and also drug treatment for prevention or cure of
infection have lead to insecticide-resistant mosquitoes and drug-resistant malaria parasites. Therefore
there is need for a potent malaria vaccine. Attempts have been made to construct vaccines to all stages in
the life cycle (Cox, 1991). Pre-erythrocyte vaccines would block the infection of erythrocytes, and
vaccines to the sexual stage, the gametocytes, would block the transmission of the parasite between
individuals. An effective vaccine to reduce the clinical effetcts of malaria would be directed to the bloodstage of the parasite. The M5 antigen (Sjölander et al., 1993a) used in paper I, II and III is derived from
an immunodominant part of the P. falciparum ring-infected erythrocyte surface antigen Pf155/RESA
(Perlman et al., 1984). EB200, used in paper IV, is derived from Pf332, a highly repetitive, late asexual
intraerythrocytic protein, expressed on erythrocytes (Mattei et al., 1992; Mattei and Scherf, 1992;
Hinterberg et al., 1994).
39
CHRISTIN ANDERSSON
Table 10: Fusion proteins with hydrophobic/ lipophilic tags, used in I, II and III
Study
I
II
III
Plasmid
Fusion protein
Mw
Solubility*
(kDa)
Expression
level (mg/l
cell culture)
Incorporation
efficiency
pT7IWZM5mp8
IW-Z-M5
17.7
>1
insoluble
high
pT7MIZM5mp8
MI-Z-M5
18.7
>1
insoluble
high
pT7PDZM5mp8
PD-Z-M5
17.1
>1
soluble
no
pT7ABPM5M
ABP-M5-M
25.5
40
n.d.1
low
pT7ABPM5MI
ABP-M5-MI
24.9
6
n.d.
high
pT7ZZM5M
ZZ-M5-M
28.0
40
n.d.
high
pT7ZZM5MI
ZZ-M5-MI
27.3
66
n.d.
high
pMLHABP∆SAG1
lpp-His6-ABP-∆SAG1
42.4
7
insoluble
high
PAff8∆SAG1
His6-ABP-∆SAG1
41.6
102
soluble
high
* Dominating fraction indicated, 1 not determined
The mixture was dialysed and subjected to ultracentrifugation in sucrose gradient. Analysis of
individual fractions of the gradient was performed to evaluate the iscom association
efficiency. The fractions were analyzed for; (i) cholesterol content, the result is indicated with
a horizontal bar in Figure 2, (ii) protein content by Bradford analysis, and (iii) protein content
by a Z-specific ELISA and an M5-specific ELISA. The results are summarized in Figure 2.
The comigration of protein and cholesterol in the sucrose gradient has been considered
indicative of successful iscom association.
Two of the hydrophobic tags evaluated in the first study (I) were found to mediate efficient
iscom association; IW and MI, (Figure 2A and B, Table 10) as the protein analyses showed
the highest protein content in the cholesterol-rich fractions. However, analysis of iscomincorporation experiment of the fusion protein PD-Z-M5, indicated that there was no or very
little incorporation of the protein immunogen into iscoms, since almost all protein was found
in the upper fractions of the sucrose gradient. These results are normally seen with hydrophilic
antigens. Since the PD tag was predicted to be pH-dependent, the iscom incorporation
experiments was performed at various pHs (pH 3-8) but with the same negative results. These
results were further supported by the fact that the fusion protein PD-Z-M5 showed
hydrophilic properties during the protein expression and purification procedure.
40
RECOMBINANT SUBUNIT VACCINES
A: IW-Z-M5
B: MI-Z-M5
0.45
C: PD-Z-M5
0.45
0.95
1.4
0.4
0.85
1.2
0.5
0.45
0.45
0.4
0.35
0.35
0.4
0.35
0.75
1
0.3
0.65
0.25
0.35
0.25
0.3
0.2
0.3
0.45
0.1
0.35
0.4
0.05
0.25
0.2
0
0.15
0.25
0.2
0.2
0.05
0.15
0.15
1
5
10
15
1
5
10
1
15
D: ABP-M5-M
0.6
0.15
0.15
0.25
0.8
0.55
5
E: ABP-M5-MI
1
0.5
0.4
0.8
0.85
0.55
0.8
0.5
0.75
0.45
0.7
0.3
0.4
0.65
0.6
0.35
0.6
0.2
0.3
0.55
0.4
0.1
0.2
5
10
0.5
0.25
0.45
0.2
0.4
0
1
0
15
10
0.15
1
14
5
10
14
G: ZZ-M5-MI
F: ZZ-M5-M
0.9
0.6
0.8
0.5
0.7
0.7
0.6
0.6
0.5
0.4
0.4
0.5
0.6
0.3
0.3
0.5
0.4
0.2
0.4
0.2
0.3
0.1
0.3
0.2
0
0.2
1
5
10
14
H: lpp-His6-ABP- SAG1
0.4
0.5
0.1
0
1
5
0.4
0.45
14
J: His6-ABP- SAG1
I: His6-ABP- SAG1
0.45
10
0.5
0.6
0.4
0.5
0.3
0.4
0.3
0.35
0.4
0.3
0.2
0.35
0.3
0.4
0.2
0.2
0.25
0.3
0.3
0.1
0.1
0.1
0.2
0.25
0.2
0.2
0
1
5
10
15
0
0.15
1
5
10
15
0
1
5
10
15
Figure 2A-J: Analysis of the protein content in the fractions collected from sucrose gradient ultracentrifugation
of iscom preparations performed in paper I, II and III. The respective fusion protein is shown above each graph.
Total protein content is shown on the left y-axis ( ) and the results from an ELISA, specific for the affinity tag
used in each study (Z, ZZ or ABP, respectively) is shown on the right y-axis ( ). The fraction number is indicated
on the x-axis. The horizontal bar in each graph indicates the cholesterol-rich fractions. Figure 2J shows the
results from a control experiment with protein, iscom matrix but without the chelating lipid.
41
CHRISTIN ANDERSSON
While the fusion proteins IW-Z-M5 and MI-Z-M5 formed aggregates during expression, the
protein PD-Z-M5 was found to be soluble upon expression in E. coli. The results are
summarized in Table 10. Furthermore, the iscom formation was supported by electron
microsopy inspection of the cholesterol-rich fractions from iscom preparation. The iscom
preparations of the proteins IW-Z-M5 and MI-Z-M5 contained typical iscom structures
(Figure 3), while iscom preparations of the protein PD-Z-M5, in contrast, contained no iscom
structures.
To evaluate the immunogenic properties of the iscom preparations, immunization studies were
performed in mice. Sera from mice immunized with the IW-Z-M5 and MI-Z-M5 iscom
preparations, respectively, contained M5-specific IgG-titers of approximately 1:1000 (mean)
after the first immunization. After the booster immunization, the M5-specific antibody levels
increased 10-fold.
Figure 3: A typical iscom structure, as evaluated by electron microscopy analysis.
42
RECOMBINANT SUBUNIT VACCINES
6.1.2. C-terminal hydrophobic tags (II)
In an effort to improve the system for hydrophobic tagging of immunogens for efficient iscom
incorporation, new, improved general expression vectors were designed. The major drawback
with the previously presented system (I) was the overall low production levels of the proteins.
It has been suggested that translation initiation sequences containing hydrophobic amino acids
will decrease production levels (Makrides, 1996) and in order to increase the production
levels of the fusion proteins, the hydrophobic tag was instead placed in the C-terminus of the
fusion proteins (II). As hydrophobic tags, the most successful hydrophobic tag from the
previous study, the membrane-integration MI tag from influenza hemagglutinin (Feldmann et
al., 1988) was included (Table 9). The second tag used in this study, denoted M, is derived
from the membrane-spanning region of Staphylococcus aureus protein A (SpA) (Navarre and
Schneewind, 1994; Schneewind et al., 1995) (Table 9). The rational for selecting the latter
was that a hydrophobic tag of bacterial origin might be more efficiently produced in E. coli.
To make the affinity purification process more efficient, the divalent ZZ tag was utilized as
affinity tag, since it has been shown (Nilsson et al., 1994), that ZZ has a ten times higher
affinity for its ligand IgG, than the monovalent Z domain. The ZZ domains are also highly
soluble, and it has been shown in other studies that ZZ as fusion partner can increase the
overall solubility of fusion proteins (Samuelsson et al., 1991 and 1994). This might be of
importance to minimize the formation of unwanted hydrophobic aggregates during the
purification and iscom incorporation processes. As an alternative affinity tag, the serum
albumin-binding protein ABP (Larsson et al., 1996), derived from streptococcal protein G
(Nygren et al., 1988), was included. The ABP tag is also known to be highly soluble, and
binds human serum albumin (HSA), which makes it suitable as tag for affinity purification
procedures (Ståhl et al., 1999).
Four new expression vectors were thus constructed pT7ABPM, pT7ABPMI, pT7ZZM and
pT7ZZMI, and a gene fragment encoding the same model immunogen M5 as in the previous
study was introduced into the vectors. The four encoded fusion proteins ABP-M5-M, ABPM5-MI, ZZ-M5-M and ZZ-M5-MI (Table 10), were produced intracellularly in E. coli under
control of the phage T7 promoter (Studier et al., 1990). The fusion proteins could be affinity
purified on either HSA-Sepharose (ABP-fusions) or IgG-Sepharose (ZZ-fusions) and all four
proteins were recovered as full-length fusion proteins. Compared to the initial study, the
expression levels were now significantly improved, being in the range of 6-66 mg per liter of
43
CHRISTIN ANDERSSON
cell culture for the four fusion proteins, ABP-M5-M, ABP-M5-MI, ZZ-M5-M and ZZ-M5-MI
(Table 10).
Iscom incorporation experiments of the fusion proteins ABP-M5-M, ABP-M5-MI, ZZ-M5-M
and ZZ-M5-MI, respectively, were performed as described above. Again, the iscom
preparations were subjected to a sucrose gradient centrifugation. Collected fractions were
analyzed for cholesterol content as described above, and for (i) total protein content according
to Bradford (Bradford, 1976) and, (ii) the presence of the affinity tags in ABP-or ZZ-specific
ELISA experiments. The results are summarized in Figure 2D-G.
As discussed earlier, comigration of protein and cholesterol indicates successful iscom
association of the protein. All four proteins ABP-M5-M, ABP-M5-MI, ZZ-M5-M and ZZM5-MI could be found in the cholesterol-rich fractions of the sucrose gradient as shown by
both protein analyses with high protein content in the cholesterol-rich fractions. When
analyzing the protein content in fraction of iscom preparation of ABP-M5-M, the total protein
analysis (Bradford) indicated that only a fraction of the fusion protein was found in the
cholesterol-rich fractions while the ABP-specific ELISA showed a more equal distribution
between the cholesterol-rich fractions and the upper fractions. The reason for this discrepancy
is not known. Nevertheless, electron microscopy inspection of all four iscom preparations
verified the presence of the typical iscom structures.
Immunization of mice with iscom preparations containing ABP-M5-M, ABP-M5-MI, ZZM5-M and ZZ-M5-MI, respectively resulted in M5-specific serum IgG titers of about 1:100
(mean) after the first immunization and a 10 to 100-fold increase after a booster
immunization. Interestingly, despite the apparent lower incorporation of the fusion protein
ABP-M5-M into iscoms, mice immunized with ABP-M5-M-iscoms also demonstrated high
levels of M5-specific antibodies. To further evaluate this, a control immunization, using the
hydrophobically tagged antigens (ABP-M5-M) simply admixed with iscom matrix was
performed. This immunization resulted in significant induction of M5-specific antibodies
(mean titers 1:1000), indicating that the introduction of a hydrophobic tag into a normally
hydrophilic immunogen, could be sufficient to allow the formation of hydrophobic
aggregates, most probably also including iscom-matrix components, capable of inducing
significant antibody responses. A drawback with this strategy could however be that the welldefined iscom structures would not be formed.
44
RECOMBINANT SUBUNIT VACCINES
6.1.3. Lipid tagging of protein immunogens (III)
As an alternative to using hydrophobic amino acid residues as hydrophobic tags, a strategy
based on lipid tags was investigated. In order to develop a general system for production of
lipid-tagged immunogens, a novel expression vector, pMLHABP, was constructed. The
expression is under the control of the tac promoter (Amann et al., 1988). The vector encodes
the 20 amino acid signal peptide and the nine N-terminal amino acid residues (lpp) of the
major lipoprotein of E. coli and this sequence is found to be sufficient for lipidation (Ghrayeb
and Inouye, 1984; Laukkanen et al., 1993). A dual affinity tag, consisting of a hexahistidyl
(His6) peptide and the serum albumin binding protein ABP, was used. Introduction of the
His6-ABP dual affinity tag would offer alternative strategies for affinity purification of
expressed fusion proteins, by either immobilized metal ion affinity chromatography (IMAC)
(Hochuli et al., 1988) or HSA-Sepharose columns (Ståhl et al., 1999).
To make comparison possible, an in vitro lipidation strategy was also investigated. This
strategy utilizes a metal-chelating lipid, which could be linked to hexahistidyl peptides and
would thus not depend on the E. coli-lipidation machinery. To produce recombinant
immunogens for in vitro lipidation via a chelating peptide, the expression vector pAff8c
(Larsson et al., 2000) was used. This vector encodes the same dual affinity tag, His6-ABP, as
pMLHABP. The expression is under control of the T7 promoter (Studier et al., 1990). As
model immunogen in both systems, a 238 amino acid immunogen ∆SAG1, derived from the
central region of the major surface antigen SAG1 (amino acid residues 61-298 of the native
SAG1) (Burg et al., 1988; Harning et al., 1996) of Toxoplasma gondii (Figure B) (Grimwood
and Smith, 1992), was used.
The constructed plasmids, pMLHABP∆SAG1 and pAff8∆SAG1, thus encode the fusion
proteins, lpp-His6-ABP-∆SAG1 and His6-ABP-∆SAG1, respectively (Table 10). The fusion
proteins were evaluated for in vivo and in vitro lipidation experiments and subsequently also
in iscom incorporation experiments.
45
CHRISTIN ANDERSSON
TOXOPLASMA
Cat
Other mammals
and birds
Intestinal
epithelium
Gastro-intestinal
tract
Intestinal
epithelium
Ingestion
Oocyst
Oocyst
Oocyst
Replication
Sporozoites
Tachysoites
Tissue cyst
Tissue cyst
Replication
Bradyzoites
Bradyzoites
Ingestion
Figure B: The life cycle of the Toxoplasma gondii parasite in cats and other mammals, including humans.
Toxoplasmosis is caused by Toxoplasma gondii, an obligate intracellular parasite that can infect a wide
range of animals, including humans. The parasite has a two-phase lifecycle: a sexual phase, which occurs
in cats and other felides, and an intermediate, non-sexual phase, which can take place in any mammal or
bird. The intermediate host can be infected by ingesting oocysts. The oocysts contain sporozoites that are
released in the gut, penetrate the intestinal epithelia and start to replicate. After about two weeks, the
proliferation is slowed down and tissue cysts, containing bradyzoites, are formed. Tissue cysts are mainly
localized in muscle tissue and the central nervous system, and can be viable for a long time. If an infected
animal is eaten by another mammal or bird, the tissue cyst is destroyed by digestive enzymes, bradyzoites
are released, penetrate the epithelia ands start to replicate. If the new host is a cat, the parasite develops
into sexual stages, and after about 3-5 days, new oocysts are excreted in the faeces, completing the
lifecycle. T. gondii parasite is widely distributed around the world. In veterinary medicine, toxoplasma
infection is well known as a cause of abortion and neonatal mortality in sheep and goat. Humans can
aquire the infection from food contaminated with oocysts or meat containing tissue cysts. The infection is
usually without symptoms. However, if aquired during pregnancy, the result may be miscarriage or
malformation of the child (Remington, 1990). Toxoplasma infection in immunocompromised individuals,
including AIDS patients, may cause a life-threatening disease (Araujo and Remington, 1987; Israelski and
Remington, 1988). In the search for a vaccine against toxoplasmosis, live attenuated, killed or lysed
parasites or different antigenic fraction has been used (Hermentin and Aspöck, 1988; Johnson, 1989). For
example, immunization of mice with the major surface antigen (SAG-1) of tachyzoites incorporated into
liposomes or mixed with Quil A resulted in protection against lethal infection (Bülow and Boothroyd,
1991; Khan et al., 1991). It has also been shown that surface antigens (SAG-1, SAG-2 and others)
incorporated into iscoms elicited protective immunity in mice (Lundén et al., 1993) and both humoral and
cellular immune response in sheep (Lundén et al., 1995).
The severe effects of toxoplasma infection in animals and in humans are well known. Therefore there is a
great need for effective vaccines, both for veterinary and human use.
46
RECOMBINANT SUBUNIT VACCINES
Both fusion proteins were expressed in E. coli, and could be recovered by affinity purification
using ABP-mediated affinity chromatography on HSA columns (Ståhl et al., 1999; Ståhl et
al., 1989). The fusion protein lpp-His6-ABP-∆SAG1, designed to be lipidated in vivo was,
after disruption of cells, predominantly found in the fraction containing insoluble proteins and
cell debris, indicating membrane association. The intracellularly produced fusion protein His6ABP-∆SAG1, intended for in vitro lipidation, was mainly soluble upon expression in E. coli.
The results from the expression in E. coli are summarized in Table 10.
For the in vivo lipidated fusion protein lpp-His6-ABP-∆SAG1, iscoms were prepared as
described above simply by mixing the fusion protein with cholesterol, phosphatidyl cholin
and Quil A, followed by dialysis. The fusion protein His6-ABP-∆SAG1, intended to be
incorporated via interaction of the His6 tag with a Cu2+-chelating lipid (IDA-TRIG-DSGE),
was directly added to pre-formed iscom matrix containing Cu2+ and the chelating lipid. A
control preparation, by simple mixing the fusion protein His6-ABP-∆SAG1 with pre-formed
iscom matrix without addition of the chelating lipid, was prepared (Figure 2J). After a sucrose
gradient ultracentrifugation of the three preparations, the fractions of the gradient were
analyzed for presence of cholesterol, as described above, and for protein content. The protein
analyses performed were; (i) total protein content, according to Bradford (Bradford, 1976)
and, (ii) an ELISA analysis, specific for the affinity tag ABP. The results are summarized in
Figure 2H-J.
A significant portion of the lipidated immunogens, both the in vivo lipidated lpp-His6-ABP∆SAG, as wells as the in vitro lipidated His6-ABP-∆SAG1 was found in the cholesterol-rich
fractions of the sucrose gradient, indicating association with iscoms. For the non-lipidated
His6-ABP-∆SAG1 control, only a small fraction was detected the cholesterol fractions,
indicating that lipidation was required for iscom association. Electron microscopy inspection
of the iscom preparations verified the presence of the typical iscom structures.
Mice immunized twice with the three different preparations, respectively, responded after the
first immunization with serum IgG antibodies reactive to His6-ABP-∆SAG1 with titers of
approximately 1:1000. The booster immunization resulted in a 100-fold increase. However,
we could also detect antibody responses in sera from the mice immunized with His6-ABP∆SAG1, simply admixed with iscom matrix lacking the chelating lipid. This was surprising
since the fusion protein was not found to a significant extent in the cholesterol-rich iscom
47
CHRISTIN ANDERSSON
fractions after sucrose gradient ultracentrifugation (Figure 2). The results from another control
immunization, consisting of mice immunized with the His6-ABP-∆SAG1 fusion protein in
PBS, showed also significant titers of His6-ABP-∆SAG1 reactive antibodies after a booster
immunization. The results from the control groups indicate that the ∆SAG1 immunogen is
very immunogenic in itself. An earlier study showed that poorly immunogenic hydrophilic
antigens normally fail to elicit antibody responses when administered mixed with iscom
matrix or with PBS alone (II).
Taken together, both the concepts of including hydrophobic amino acid sequences into
recombinant immunogens (I, II), and the presented strategies to obtain in vivo and in vitro
lipidation of recombinant immunogens result in association with the iscoms. Comparing the
two lipidation strategies, one advantage of using the in vivo lipidation strategy is that only the
typical iscom forming material is needed, while the in vitro strategy requires the addition of a
chelating lipid. On the other hand, the expression levels obtained with the in vitro strategy are
much higher and in addition, this strategy offers a much simpler method for iscom formation
since iscom matrix with pre-associated chelating lipid can be prepared in bulk. The general
applicability of the presented strategies needs to be further evaluated for various immunogens.
6.2. Recovery of a vaccine candidate produced in mammalian cells (IV)
A promising vaccine candidate to human respiratory syncytial virus (RSV) (Figure C)
produced in E. coli has been demonstrated to induce protection to virus challenge in rodent
models (Power et al., 1997) and has now progressed into phase III human clinical trials
(http://www.cipf.com). The candidate vaccine, BBG2Na consists of a serum albumin binding
region BB derived from streptococcal protein G (SpG) (Nygren et al., 1988) and amino acids
130-230 of the G glycoprotein of RSV subgroup A (Power et al., 1997). The immunogenic
properties of the E. coli-produced and non-glycosylated BBG2Na is interesting considering
that the native RSV G protein is heavily glycosylated (Collins et al., 1996). Therefore, a
mammalian expression system for production of glycosylated BBG2Na was created in order
to enable comparative immunological studies of non-glycosylated and glycosylated BBG2Na.
Expression vectors based on the positive-strand RNA alphavirus Semliki Forest virus (SFV),
was used to achieve transient expression of BBG2Na in baby hamster kidney cells.
48
RECOMBINANT SUBUNIT VACCINES
RESPIRATORY SYNCYTIAL VIRUS
Surface proteins
F
G
SH
Nucleocapsid-associated proteins
N
P
L
Matrix protein
M
M2
Nonstructural proteins
NS1
NS2
Figure C: An overview of the respiratory syncytial virus. The surface proteins are outlined in the picture.
Respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract infection
during infancy and early childhood worldwide. RSV was first isolated in 1956 and has since then been
associated with lower tract illness during infancy and childhood in many parts of the world (Collins et al,
1996). It has also been found that RSV infection is an important agent of disease in immuno-suppressed
adults and in elderly peoples (Collins et al., 1996). Outbreaks of RSV disease are abrupt and can last up to 5
months, and usually peaks in winter. There are two major strains, A and B, both of them circulate
simultaneously during outbreak, and it has been suggested that group A strains are associated with more
severe disease (Domachowske and Rosenberg, 1999).
RSV is an enveloped, negative-sense RNA virus with a genome of about 15 kb encoding 11 viral proteins
(Collins et al., 1996). The virus particle is about 60 to 100 nm in diameter and consists of a nucleocapsid
surrounded by a lipid envelope with two major surface proteins, F and G. The F protein is thought to
mediate virus penetration while the RSV G protein is thought to mediate virus attachment to cells (Peroulis,
1999; Collins et al., 1996). Both F and G protein induce neutralizing antibodies (Dudas et al., 1998). It has
been demonstrated that monoclonal antibodies to G protein inhibit virus entry into cell. (Karron et al., 1997).
Studies have shown that the G glycoprotein is a major viral antigen and the glycosylated G protein is
protective upon immunization in several animal models (Power et al, 1997). It has been suggested that the
glycosylation protect the virus from the host immune system (Collins et al., 1996).
A vaccine candidate BBG2Na is a fusion protein consisting of an albumin-binding region from
streptococcal protein G (BB) and RSV G protein fragment. (G2N) has been extensively studied. Studies with
BBG2Na in animal models, mice and cotton rats, has shown that high immune response and also protection
to viral challenge can be obtained when immunizing with a nonglycosylated fragment of the G protein.
Administering this E. coli produced fusion protein, BBG2Na, to mice resulted in a strong immune response
of these mice and subsequent protection from RSV infections. Lungs and nasal tracts of immunized mice
were efficiently protected to subsequent RSV challenges (Power et al., 1997). Maternal immunization
induces protection of offspring of test animals, and passive transfer of the maternal antibodies was sufficient
to induce a high protective immune response. (Brandt et al., 1997; Siegrist et al, 1999).
Still there is no vaccine for RSV although the virus and the severe effects of infection are well known.
49
CHRISTIN ANDERSSON
Such vectors have been used to express heterologous proteins in a wide range of host cells,
i.e. insect, avian and mammalian cells (Liljeström, 1994; Liljeström and Garoff, 1991). For
example, biologically active full-length RSV G protein was successfully expressed using an
SFV-based vector (Peroulis et al., 1999).
These SFV vectors were furnished with SP6 phage-derived RNA promoter to enable in vitro
transcription of RNA, and the gene encoding BBG2Na is introduced in the position where the
genes encoding the SFV structural proteins normally reside (Liljeström, 1994; Liljeström and
Garoff, 1991). The positive-strand RNA from such vectors encodes its own RNA-RNA
replicase, responsible for amplificaiton of RNA in vivo. Two different vectors encoding
BBG2Na were created, both utilizing the SFV self-amplification system. The first vector,
pSFV3-l-BBG2Na encodes a cytoplasmic version of BBG2Na, denoted BBG2Nacyt, and the
second vector, pSFV3-spl-BBG2Na, were a signal peptide (sp) (Berglund et al., 1997) was
included upstream of the BBG2Na gene, encodes a secreted version of BBG2Na, denoted
BBG2Nasec. RNA was transcribed in vitro from both vectors and transfection of BHK-21 cells
was performed by electroporation with the RNA constructs encoding BBG2Nacyt and
BBG2Nasec, respectively. Western blot analysis of culture supernatants and cell lysates from
cells expressing the two variants of BBG2Na, was performed. BB-specific antibodies could
detect BBG2Na cyt in the cell lysate and not in the culture supernatant and BBG2Nasec was, as
expected, predominantly found in the culture supernatant. Furthermore, immunofluorescence
microscopy analysis showed a difference in the localization patterns for BBG2Nacyt and
BBG2Nasec. The cells expressing BBG2Nacyt showed an even distribution in the cytoplasm,
while in cells expressing BBG2Na sec, the product was detected in a network-like pattern in the
cytoplasm adjacent to the nucleus. The latter staining pattern is typical of endoplasmic
reticulum and golgi complex localization (Kamrud et al., 1998), and is consistent with
BBG2Nacyt being directed to the secretion machinery. The differential localization of the two
BBG2Na variants indicated that BBG2Nacyt was found predominantly in the cytoplasm while
BBG2Nasec was targeted to the secretion machinery.
Since our aim was to produce the secreted form of BBG2Na, we wanted to develop a recovery
strategy for BBG2Nasec. The first evaluated purification method to recover BBG2Nasec from
culture media, was to use human serum albumin (HSA) columns, thus enabling BB-mediated
recovery (Murby et al., 1995; Ståhl et al., 1999). We initially evaluated HSA affinity
purification using HSA-Sepharose columns. However, analysis of the purification procedure
50
RECOMBINANT SUBUNIT VACCINES
showed that only very low amounts of BBG2Nasec could be recovered. The reason for this is
most probably due to the presence of BSA in the culture medium (originating from the fetal
calf serum (FCS)). The BSA will then compete with HSA for the binding to the BB-tag since
the BB-tag has a lower but still significant affinity for BSA, as compared to HSA (Nygren et
al., 1990). The purification strategy is schematically shown in Figure 4.
In order to improve the recovery, a batch-wise HSA affinity recovery procedure was
investigated, and by this strategy, a certain amount of BBG2Nasec could indeed be recovered
(approximately 4 µg per 10 ml culture supernatant). BBG2Nasec could, however, be detected
in the unbound material indicating inefficient recovery, again probably due to the BSA
competition. Furthermore, the affinity-purified material contained significant levels of BSA
contamination. The results are summarized in table 11.
To decrease the amount of copurified BSA in the eluted material, BBG2Nasec was produced
by an alternative cultivation protocol. Cultivation of the BHK-21 cells was performed using
culture media containing FCS. However, after transfection of the cells, culture media without
FCS was used. By excluding FCS during the protein production, the amount of BSA in the
start material for the purification should be significantly decreased. BBG2Nasec was purified
from the culture supernatant from the alternative production scheme using HSA-Sepharose
columns. Detectable but still only small amounts of BBG2Nasec could be obtained (0.5 µg per
10 ml culture supernatant).
HSA-purification
Affibody mediated
purification
BSA
BSA
ZRSV1
BB
G2N
BB
G2N
HSA
BB
G2N
ZRSV1
BSA
Figure 4: Overview of the two affinity purification strategies used in paper IV. The potential
interference of BSA is illustrated.
51
CHRISTIN ANDERSSON
Table 11: Protein production characteristics of BBG2Nasec
Production/affinity purification mode
Recovered BBG2Naseca
(µg/10 ml)
Puritya (%)
4
≈ 50
0.5
≈ 20
With FCS/ZRSV1 affibody-Sepharose
6
≈ 30
Without FCS/ ZRSV1 affibody-Sepharose
2
> 90
With FCS/HSA-Sepharoseb
Without FCS/HSA-Sepharose
aEstimated from Commassie-stained SDS-PAGE gels after affinity recovery. bPerformed in batch mode instead
of column.
The purified material also contained copurified BSA, probably remaining from the initial cell
cultivation. These results indicate that the yields and purity of BBG2Nasec purified using the
HSA affinity procedures were insufficient (Table 11).
To circumvent the problems of BSA competition, a column with a product-specific affinity
ligand, which is specific for the RSV G protein part of BBG2Nasec, was created. The ligand, a
recently described RSV G protein-specific binding molecule, ZRSV1 affibody (Hansson et al.,
1999) was created through phage display-mediated selection from a combinatorial library of
the Z protein domain derived from SpA as described above (see section 2.2.5). ZRSV1 binds
amino acids 164-186 of the RSV G protein of both E. coli-produced BBG2Na and
glycosylated RSV G protein from whole virus (Hansson et al., 1999). Dimeric versions of
affibodies have in certain cases been shown to have an apparent higher affinity (Gunneriusson
et al., 1999) and therefore a dimeric construct of the ZRSV1 was created resulting in the fusion
protein ZZ-diZRSV1.
The affibody fusion protein ZZ-diZRSV1 was coupled as ligand to an activated Sepharose
column to create a product-specific affinity column. Purification of culture supernatant from
BBG2Nasec-expressing BHK-21 cells showed that full-length BBG2Nasec could be recovered
(6 µg per 10 ml cell culture supernatant) (Table 11). Still, significant amounts of co-purified
BSA were present, probably as a result of interaction of the BB tag of BBG2Nasec with BSA
(Figure 4). Again, the production of BBG2Nasec using serum-free media was evaluated.
Production of BBG2Na sec in culture medium with BSA omitted during protein production and
recovery using the G2Na-specific affibody column, resulted in a product yield of
52
RECOMBINANT SUBUNIT VACCINES
approximately 2 µg BBG2Na sec per 10 ml culture supernatant with only very low amounts of
BSA contamination (Table 11).
We thus succeeded in creating a system for production and recovery of BBG2Nasec. The
possible differences in antigenicity and immunogenicity between BBG2Na produced in
mammalian cells and the vaccine candidate, produced in E. coli can thus be initiated.
7. Nucleic acid vaccines
7.1. Protection to RSV elicited in mice by plasmid DNA immunization (V)
As described above, respiratory syncytial virus (RSV) is a major cause of lower-respiratory
tract infections in young infants and elderly (Collins et al., 1996). The bacterially produced
protein subunit vaccine BBG2Na (Murby et al., 1995) has been able to induce protection in
mice and cotton rats against RSV challenge (Power et al., 1997; Corvaia et al., 1997; Brandt
et al., 1997; Plotnicky-Gilquin et al., 1999 and 2000; Siegrist et al., 1999), and this RSV
vaccine candidate has progressed into phase III human clinical trials (http://www.cipf.com).
Therefore, it would be of interest to evaluate a nucleic acid vaccine strategy using BBG2Na as
antigen (V). The purpose would be to investigate if the same immunogen could elicit
protection when delivered as a nucleic acid vaccine. Nucleic acid based vaccination studies
using genes encoding RSV proteins have been performed e.g. using RSV F protein (Li et al.,
1998) or RSV G glycoprotein (Li et al., 2000), and protective effects have been shown in
rodents.
The SFV system, used as expression system in a previous study (IV) have been evaluated in
nucleic acid vaccination strategies using naked RNA, virus coated-RNA or plasmid DNA
(Tubulekas et al., 1997; Berglund et al., 1998). Therefore, we constructed SFV-based
plasmids designed for DNA immunization encoding BBG2Na in a cytoplasmic (BBG2Nacyt)
or a secreted (BBG2Nasec) form. The plasmids, denoted pBK-SFV3l2-BBG2Na and pBKSFV3l2-spBBG2Na, respectively are schematically shown in Figure 5. Both vectors encode
the SFV replicase, responsible for amplification, and the transcription is under control of the
eukaryotic cytomegalovirus (CMV) immediate early promoter (Berglund et al., 1998).
53
CHRISTIN ANDERSSON
A
SFV-Repl.
SFV-Repl.
BBG2Na
pCMV
sp BBG2Na
pCMV
pBBG2Nacyt
pBBG2Nasec
Neo
Neo
Ori
Ori
B
cap
C
SFV-Repl.
BBG2Nacyt
38.2 kDa
BBG2Na
BBG2Na
(A)n
cap
SFV-Repl.
BBG2Nasec
sp BBG2Na
(A)n
sp BBG2Na
38.3 kDa
Figure 5: Plasmid vectors, transcription products and encoded fusion proteins. (A) The two expression vecors,
pBBG2Nacyt and pBBG2Nasec, encoding the SFV replicase (enabling in vivo amplification) and the cytoplasmic
and secreted variants of BBG2Na, respectively. The transcription is under control of the CMV immediate early
promoter (pCMV). (B) The positive strand RNAs, transcribed by the transfected cells, will be self-amplified in the
cytoplasm of transfected cells, since they encode the SFV replicase responsible for the RNA-dependent RNAreplication. (C) The encoded gene products from the plasmid vectors, BBG2Nacyt and pBBG2Nasec, the latter
product furnished with a signal peptide (sp) for direction to the secretion machinery. The indicated theoretical
molecular masses are after processing of the signal peptide are indicated.
To investigate the functionality of the plasmids, BHK-21 cells were transfected with the two
plasmids, respectively. Analysis of culture supernatants and cell lysates by Western blot
demonstrated that BBG2Nacyt was detected only in the cell lysate, while BBG2Nasec was
found both in the culture supernatant and the cell lysate. Further expression studies were
performed using immunofluorescence microscopy analysis. The same difference in
localization patterns for BBG2Nacyt and BBG2Nasec as described in earlier studies (IV) was
shown. The BBG2Nacyt was detected in the cytoplasm while BBG2Nasec was detected in a
network-like pattern in the cytoplasm adjacent to the nucleus, a staining pattern typical of
endoplasmic reticulum and golgi complex localization (Kamrud et al., 1998). The results from
54
RECOMBINANT SUBUNIT VACCINES
the expression studies suggest that the plasmids are functional in terms of BBG2Na
expression and localization of the encoded proteins.
In order to investigate the potential of the plasmids as vaccines, mice were immunized
intramuscularly with the plasmids pBBG2Nacyt, pBBG2Nasec or a control plasmid, pControl
(encoding only the SFV replicase and E. coli β-galactosidase). None of the mice demonstrated
detectable RSV-specific antibodies. However, while sera from mice immunized with
pBBG2Nacyt did not contain detectable BBG2Na-specific antibodies, all five mice immunized
with pBBG2Nasec developed BBG2Na-reactive antibody responses. Evaluation of the lung
protective efficacy against RSV challenge induced by these plasmids, showed clearly better
results in the group immunized with pBBG2Nasec, where sterilizing lung protection was
obtained in three out of five mice. None of the mice immunized with pBBG2Nacyt or pControl
showed significant virus reduction. The results are summarized in Table 12.
Table 12: Lung protective efficacya to virus challenge expressed as virus titersb(log10) after
intramuscular immunization of mice with plasmids, pBBG2Nacyt , pBBG2Nasec or pControl
and challenge with 105 TCID50 RSV-A.
Immunization group
Mouse number
Mouse protected
/total
1
2
3
4
pBBG2Nacyt
4.95
4.45
4.45
4.45
pBBG2Nasec
≤1.45
≤1.45
≤1.45
3.95
pControl
4.45
4.2
4.95
4.45
5
Mean
4.57±0.25
0/4
3.95
2.45±1.37
3/5
4.45
4.5±0.27
0/5
a
The lungs were, in accordance with earlier studies (Power et al., 1997) considered protected (underlined
figures) when virus titers were reduced by 2 log10 relative to the mice immunized with the control strain.bViral
titers for the challenged mice are expressed as log10 pfu/g of lung tissue, 5 days post challenge. The detection
limit for the assay is 101.45.
Interestingly, there was an indication of correlation between the virus reduction and the antiBBG2Na IgG titer for individual mice in the group immunized with pBBG2Nasec. The
BBG2Na-specific antibody titers in sera from non-protected mice in the BBG2Nasec group
were just above the limit of detection, (102.43) while the protected mice had higher antibody
titers (102.9 to 103.9). Possible explanations for the differences in immunogenic properties
could be different expression levels from the plasmids or differential antigen presentation. The
55
CHRISTIN ANDERSSON
Western blot analysis indicates that BBG2Nasec was expressed at somewhat higher levels, but
more extensive studies would be required to fully assess the differences in expression levels.
An additional possible explanation of the differences in immunogenicity could be that the
presentation of BBG2Na to the immune system could be inefficient. The SFV replicase has
been shown to induces apoptosis in cells (Glasgow et al., 1998) and others have suggested
that apoptotic cells are poor antigen presenters (Barker et al., 1999). Therefore, BBG2Nacyt,
expressed inside such cells could be poorly immunogenic, while the secreted BBG2Na
encounter direct contact with antigen presenting cells and might therefore be efficiently
presented to the immune system. However, the reasons for the different immunogenicity of
the two BBG2Na variants have to be further investigated.
In this study, we demonstrate that protection to RSV challenge can be elicited in mice by
immunizing with DNA encoding a subunit RSV vaccine candidate, BBG2Na, at present
evaluated in human clinical trials as a prokaryotically produced protein immunogen. The
results also indicate that targeting of the antigens expressed by nucleic acid vaccination could
have important effects.
7.2. Immunization with DNA or RNA encoding a malarial antigen (VI)
In order to evaluate the differences in immune responses elicited by the different SFVsystems, we constructed plasmid vectors to be used either for in vitro transcription of RNA or
direct for immunization of mice. The constructed plasmid designed for transcription of SFVbased RNA encodes the SFV replicase. The RNA was delivered either as naked RNA or
packaged in SFV suicide particles. The plasmids designed for direct immunization of mice
were of two different types, both containing a cytomegalovirus promoter, but one containing
and one lacking the SFV replicase. The antigen used in this study was a malarial antigen,
EB200 derived from the erythrocyte membrane associated P. falciparum antigen Pf332
(Mattei et al., 1992), and the gene encoding the antigen was introduced into all plasmid
constructs (Figure 6).
The constructed plasmid vector, denoted pSFV3-spl-EB200, designed for in vitro
transcription of RNA in the same fashion as described above (IV) contains the gene encoding
the SFV replicase and the gene encoding EB200 preceded by a sequence encoding a signal
peptide (Berglund et al., 1997). The signal peptide has been used in previous studies (IV and
56
RECOMBINANT SUBUNIT VACCINES
A
SFV Replicase sp EB200
RNA
pSP6
Ori E
pSFV3-spl-EB200
cap
SFV Replicase sp EB200
A(n)
bla
B
SFV Replicase
SFV Replicase sp EB200
EB200
pCMV
pCMV
pBK-SFV3L2-EB200
pBK-SFV3L2-spEB200
Neo
Neo
Ori E
C
Ori E
HCMV int
SV40 int p(A)
StPA EB200
HCMV
IE1
enh/pr
pCMVintBL-EB200
Bla
SV40
Ori
Ori E
Figure 6: Schematic representation of the plasmids used for in vitro transcription or immunization. (A) The
vector pSFV3-spl-EB200 encoding the SFV replicase (enabling in vivo amplification) and the secreted variant of
EB200. The transcription is under control of the phage SP6 RNA polymerase promoter (pSP6). The positive
strand RNA, suitable for transfection of mammalian cells, are transcribed and capped in vitro, and carry 3' polyA-tails, which are transcribed from poly-A sequences in the plasmid vector. (B) The vectors pBK-SFV3L2EB200 and pBK-SFV3L2-spEB200 encode the cytoplasmic or the secreted variant of EB200. The transcription
is under control of the CMV major immediate-early (IE1) enhancer. (C) The expression vector pCMVintBLEB200. The CMV major immediate-early (IE1) enhancer, promoter and intron A and the tPA signal sequence
precede the gene fragment to be expressed (EB200). The vector contains the simian virus 40 (SV40) origin of
replication, intron and polyadenylation signals from the SV40 T-antigen gene.
V), and has been shown functional in terms of directing the product to the secretion
machinery. The encoded product was denoted EB200sec. Transcription was under control of
the SP6 RNA polymerase promoter, thus enabling in vitro transcription of RNA (Liljeström
and Garoff 1991) as described before (IV).
57
CHRISTIN ANDERSSON
Plasmid vectors for DNA immunization, pBK-SFV3L2-EB200 and pBK-SFV3L2-spEB200,
encoding the SFV replicase and two variants of the antigen EB200; one cytoplasmic variant
denoted EB200cyt, and one secreted variant denoted EB200sec, respectively, were also
constructed. Again, the signal peptide would enable secretion of the encoded product,
EB200sec. Transcription in both plasmids is under control of the CMV immediate early
promoter, commonly used in plasmid DNA vaccine vectors due to efficient transcription.
Another plasmid, pCMVintBL-EB200, lacking the SFV replicase was also constructed, in
which the gene encoding EB200 is preceded by the tissue plasminogen activator tPA signal
sequence for production of a secreted variant of EB200. In this plasmid, the number of RNA
copies transcribed would only be the result of transcription controlled by the CMV promoter.
To perform the immunizations using RNA in viral particles, in vitro transcription of RNA
encoding the P. falciparum-derived antigen EB200 or a control antigen BBG2Na (IV, V) was
performed. These RNA-constructs were also prepared in a virus-coated fashion by packaging
into into recombinant SFV (rSFV) particles using a two-helper RNA system (Smerdou et al.,
1999).
Mice were immunized intramuscularly with the different RNA and DNA constructs, see Table
13, and antibody responses were monitored.
Table 13: Immunization groups used in study VI
Group
Molecule used in
immunizations
Plasmid
Encoded protein
A
RNA
pSFV3-spl-EB200a
EB200sec
B
RNA
pSFV3-spl-BBG2Naa
BBG2Nasec
C
Plasmid DNA
pBK-SFV3L2-EB200
EB200cyt
D
Plasmid DNA
pBK-SFV3L2-spEB200
EB200sec
E
Plasmid DNA
pCMVintBL-EB200
EB200sec
F
Plasmid DNA
pCMVintBL
none
G
Protein in FIA c
-
E. coli produced EB200
H
rSFV particles
pSFV3-spl-EB200b
EB200sec
I
rSFV particles
pSFV3-spl-BBG2Nab
BBG2Nasec
J
rSFV particles
pSFV3-lacZb
β-galactosidase
a
RNA transcribed from the plasmid was used for immunizations, bRNA transcribed from the plasmid was
packed in SFV viral particles used for immunizations, c Freunds incomplete adjuvant
58
RECOMBINANT SUBUNIT VACCINES
All three groups of mice immunized with plasmids encoding EB200 (group C, D and E)
developed GST-EB200 reactive antibodies. Only background levels of antibody reactivity to
GST were found, suggesting that the major part of the reactivity of these sera was directed
against EB200. Unexpectedly, both groups and of mice immunized with SFV-RNA (group A
and B), encoding EB200sec or control antigen BBG2Nasec, developed high and similar levels
of antibody reactivity to GST-EB200. However, most of this reactivity appeared not to be
directed against EB200, as both these groups showed an even higher reactivity with GST. The
reason for the induction of these apparently non-specific antibodies is not known. Importantly,
the RNA packaged in SFV suicide particles (group H and I) did not elicit this kind of antibody
reactivity.
The antibody responses obtained in this study were relatively low, surprisingly with the
highest responses obtained with the conventional DNA plasmid (group E). However,
immunizations with plasmids (group C, D and E) as well as with rSFV particles (group H)
induced an efficient immunological memory response, as demonstrated by the efficient
boosting of the anti-EB200 antibody response with recombinant EB200 protein. The efficient
priming of immune responses by naked DNA immunizations has been observed previously
when boosting with the corresponding protein (Haddad et al., 1999) or with recombinant
vaccinia virus expressing the protein (Schneider et al., 1998; Sedegah et al., 1998). Our low
responses to EB200 with the SFV based vectors appear to be due to the inherent properties of
the antigen, as our contol vectors coding for the protein BBG2Na (group I) or LacZ gave high
levels of antibodies to the respective protein.
In conclusion, we have in this study shown that DNA plasmids expressing the gene for the
EB200 fragment of the P. falciparum vaccine candidate antigen Pf332 induce an antibody
response to the antigen in mice. A conventional plasmid was more efficient than SFV-based
plasmids in inducing antibodies to EB200. Also recombinant SFV suicide particles expressing
EB200 induced such antibodies, although at lower levels. Importantly, all these immunogens
induced an immunological memory, which could be efficiently activated by a booster
injection with EB200 protein.
59
CHRISTIN ANDERSSON
8. Concluding remarks
Recombinant strategies are today common for the development of novel subunit vaccines. In
this thesis, recombinant methods have been employed to enable studies regarding; (i) the
generation of general expression systems for production of recombinant subunit immunogens
furnished with a hydrophobic tag, either hydrophobic amino acids or a lipid, allowing
association to iscoms, (ii) the development of a mammalian cell production and recovery
system for an RSV vaccine candidate, and (iii) construction and evaluation of nucleic acid
immunization strategies for RSV and malaria.
Robust systems for E. coli production of protein immunogens were created, in which
hydrophobic tags were fused to the target immunogen. This would enable direct association of
the produced immunogen to an adjuvant system, e.g. iscoms. Initially, three different
hydrophobic tags were evaluated, as fused N-terminally. A malaria peptide, M5, was used as
model antigen. The produced fusion proteins could be efficiently recovered by the
incorporation of also an affinity tag, the SpA-derived Z-domain, in the expression vectors, and
for two hydrophobic tags out of three, efficient association to iscoms was demonstrated.
Since the expression levels were rather low from the first generation expression vectors, a
second set of expression vectors were designed and constructed. In these vectors, one of the
hydrophobic tags was included that were found to be functional in the previous study, i.e. MI,
representing the membrane-integration region of influenza virus hemagglutinin. An additional
tag, M, representing the membrane-spanning region of staphylococcal protein A, was also
included in the study. In these vectors the hydrophobic tags were placed C-terminally, in order
not to disturb the transcription and translation initiation, to thus potentially improve
expression levels. The SpA-derived ZZ tag and the SpG-derived ABP tags were included in
the vectors to allow affinity purification, and the M5 malaria peptide served as model
immunogen. The second generation expression vectors were indeed found to increase
expression levels ten to fifty-fold, and the affinity-purified hydrophobically tagged
immunogens could efficiently be incorporated into iscoms.
A third study was performed in which lipid tagging of the recombinant immunogens was
evaluated. First, a system which utilized the E. coli lipidation system was designed. The
second system exploited a chelating lipid that was known to interact with hexahistidyl tags,
60
RECOMBINANT SUBUNIT VACCINES
and the recombinant immunogen was thus produced as fused to a His6 peptide. In both
systems the SpG-derived ABP tag was included for affinity purification purposes. A 238
amino acid segment ∆SAG1 from Toxoplasma gondii, served as model immunogen in this
study. The two types of lipid-tagged immunogens were both found to associate with iscoms.
The in vivo lipidation strategy has the advantage that only the typical iscom constituents are
needed, while the in vitro strategy requires a chelating lipid. In contrast, the expression vector
connected with the in vitro strategy gave much higher expression levels and in addition, a
straightforward method for iscom association, since iscoms-matrix with pre-incorporated
chelating lipid can be prepared in bulk. In all three studies, we confirmed the immunological
properties of the iscoms containing the recombinant immunogen, and we could in all cased
observe antigen-specific antibodies.
BBG2Na, a promising E. coli-produced vaccine candidate to human respiratory syncytial
virus (RSV) has been demonstrated to induce protection to virus challenge in rodent and
monkey models and has now progressed into phase III human clinical trials. In the presented
study, we wanted to create a mammalian expression system to produce BBG2Na, in order to
make comparative studies of E. coli produced and mammalian cell produced BBG2Na
possible. Expression systems for mammalian cell production of cytoplasmic and secreted
variants of BBG2Na, was thus created. The expression vectors were based on the SFV
operon, and RNA transcribed in vitro, encoding the SFV-replicase for self-amplification, was
used for transfection of the BHK-21 mammalian cells. The two different products BBG2Nacyt
and BBG2Nasec were both found to be expressed, and properly targeted to their intended
subcellular localization. Since the secreted variant, BBG2Nasec, was found to be expressed as
a full-length product, a suitable recovery scheme was evaluated. Two different modes of
affinity chromatography were evaluated, the first taking advantage of the serum albuminbinding properties of the product, and the second, utilizing an SpA-based affibody created by
combinatorial engineering and selected for its specific binding to the G2Na part of the
product. For both strategies, significant co-purification of BSA, present in the culture medium
during the production, was demonstrated. A strategy was then evaluated in which BSA was
excluded in the culture medium during protein production, thus having it present only during
cultivation of the untransfected cells. The significantly reduced levels of BSA in the culture
medium made it possible to recover BBG2Nasec with a much higher degree of purity, and
when using the G2Na-specific affibody column for the affinity chromatography, the purity of
61
CHRISTIN ANDERSSON
BBG2Nasec, exceeded 90%, and the product could not be detected in the flow-through
fractions, indicating quantitative recovery. Comparative studies of the E. coli produced
BBG2Na and mammalian cell produced BBG2Na, are presently being conducted.
In an additional investigation, plasmid vectors for DNA immunization against RSV was
constructed and evaluated for their efficacy. Two different vectors were constructed encoding
variants of the RSV candidate vaccine BBG2Na; one designed for cytoplasmic localization
(BBG2Nacyt) and one for targeting to the secretion machinery (BBG2Nasec) of the transfected
cells. The two plasmid vectors, pBBG2Nacyt and pBBG2Na sec, were found to be functional in
terms of BBG2Na expression and localization of the encoded proteins. Upon intramuscular
immunization of mice, the plasmid vector pBBG2Nasec, encoding the secreted variant of the
antigen, elicited significant anti-BBG2Na titers and demonstrated lung protective efficacy (in
three out of five mice). Irrespective of the reasons for the differential immunogenicity
between the two variants, we clearly demonstrated by this study that protective immune
responses to RSV can be elicited in mice by DNA immunization, and the obtained results
further indicate that differential targeting of the antigens expressed by nucleic acid
vaccination could significantly influence the immunogenicity and protective efficacy of the
encoded heterologous antigens.
In a second nucleic acid vaccination study, we evaluated DNA- and RNA constructs based on
the SFV replicon in comparison with a conventional DNA plasmid for induction of antibody
responses against the P. falciparum malaria vaccine candidate antigen Pf332. The included
RNA constructs encoded the SFV replicase and the Pf332-derived antigen EB200, and were
delivered as either naked RNA or packaged in non-replicative SFV particles. Plasmid DNA
vectors were constructed encoding the same antigen, and vectors employing or lacking the
SFV-replicase were included in the immunization study. In general, the antibody responses
induced were relatively low, the highest responses surprisingly obtained with the conventional
DNA plasmid. Our low responses to EB200 with the SFV based vectors appear to be related
with inherent properties of the antigen, as our control vectors coding for the protein BBG2Na
or LacZ gave high levels of antibodies to the respective protein. Our immunizations with
naked RNA gave confusing results, with the induction of antibodies showing high background
reactivity with the fusion-tag GST of the recombinant EB200 protein used for antibody
analysis, as well as with the control antigen BBG2N. The reason for the appearance of these
62
RECOMBINANT SUBUNIT VACCINES
apparently non-specific sticky antibodies is not known. Importantly, the same RNA when
packaged in SFV suicide particles did not induce this kind of antibody reactivity.
In conclusion, we have in this study shown that DNA plasmids expressing the gene for the
EB200 fragment of the P. falciparum vaccine candidate antigen Pf332 induce an antibody
response to the antigen in mice. A conventional plasmid was in our hands more efficient than
SFV-based plasmids in inducing antibodies to EB200. Also recombinant SFV suicide
particles expressing EB200 induced such antibodies, although at lower levels. Importantly, all
these immunogens induced an immunological memory, which could be efficiently activated
by a booster injection with EB200 protein.
Taken together, it is evident that recombinant DNA technology will have a major impact on
future strategies for the prevention of infectious diseases. The design, production and delivery
of recombinant subunit vaccines will be the basis of modern vaccinology. The studies
summarized in this thesis demonstrate that new methods will be available for the production
and recovery of protein subunit immunogens, and that these can be further adapted by genetic
design of the expression vectors for incorporation into adjuvant systems. In addition, various
strategies for delivery of subunit vaccines by RNA or DNA immunization will be available
for the development of future vaccines.
63
CHRISTIN ANDERSSON
Abbreviations
ABD
ABP
ALVAC
APC
B7
BB
BHK
BSA
CHO
CMV
CpG
CS
CTB
CTL
EB200
ELISA
ETEC
FCS
FIA
G2Na
GM-CSF
GST
HA
HEK
HBsAg
Hib
His6
HIV
HLA
HSA
Ig
IFN
IL
Iscoms
ISS
IW
kDa
LPS
LTB
M5
M
MAP
MHC
MI
64
albumin binding domain derived from streptococcal protein G
albumin binding protein derived from streptococcal protein G
a recombinant canarypox virus
antigen presenting cell
a B-cell ligand
albumin binding region derived from streptococcal protein G
baby hamster kidney cells
bovine serum albumin
chinese hamster ovary cells
cytomegalovirus
a cytosine-guanine dinucleotide, p indicates the phosphate bond
circumsporozoite
cholera toxin B subunit
cytotoxic T lymphocyte
a malaria antigen fragment, derived from Pf332
enzyme-linked immunosorbent assay
enterotoxic E. coli
foetal calf serum
Freunds incomplete adjuvant
a part of the respiratory syncytial virus G protein
granulocyte-macrophage colony stimulating factor
glutathione-S-transferase
hemagglutinin
human embryonic kidney cells
Hepatitis B surface antigen
Haemophilus influenzae type B
hexahistidyl tag
human immunodeficiency virus
human leukocyte antigen
human serum albumin
immunoglobulin
interferon
interleukin
immunostimulating complexes
immunostimulatory sequences
hydrophobic amino acid sequence (isoleucines and trypophanes)
kiloDalton
lipopolysaccharides
E. coli heat-labile toxin B subunit
a malaria peptide derived from the central region of Pf155/RESA
membrane-spanning region of SpA
multiple antigenic peptides
major histocompatibility complex
membrane-spanning region of hemagglutinin A from influenza virus
RECOMBINANT SUBUNIT VACCINES
MPL
MVA
NP
NYVAC
PAGE
PD
PCR
PLG
QS-21
Quil A
RSV
SAG1
SFV
SDS
SpA
SpG
SV40
tPA
VLP
VP
XM
ZZ
monophosporyl lipid A
modified vaccinia virus Ankara strain
nucleoprotein
attenuated vaccinia virus strain
polyacrylamide gel electrophoresis
hydrophobic tag, designed to be pH-dependent
polymerase chain reaction
poly (lactide-co-glycosides)
a fraction of Quil A
a saponin derived from Quillaja saponaria
respiratory syncytial virus
surface antigen 1 from Toxoplasma gondii
Semliki Forest virus
sodium dodecyl sulphate
staphylococcal protein A
streptococcal protein G
simian virus 40
tissue plasminogen activator
virus-like particles
viral protein
cell-surface-anchoring region of staphylococcal protein A
IgG-binding domains derived from staphylococcal protein A
65
CHRISTIN ANDERSSON
Acknowledgements
First of all I would like to thank my supervisor, Prof. Stefan Ståhl. You have, in good and bad times,
always been supportive and encouraging and somehow always incredibly optimistic. Thank you for
helping me finish this thieis.
I would also like to thank Prof. Mathias Uhlén for leading the DNA corner group in an inspiring way
and for being such a visionary person.
My collaboration partners have contributed a great deal to the work presented in this thesis. I would
like to send my gratitude to: Karin Lövgren-Bengtsson at SLU in Uppsala for introducing me to the
world of iscoms. Anna Lundén at SVA in Uppsala for collaboration in the SAG-1 iscom project and
all help with the thesis. Klavs Berzins, Diana Haddad, Niklas Ahlborg and Nina-Maria Vasconcelos at
Stockholms University for collaboration in the EB200 project. Ultan Power, CIPF, for all collaboration
in the RSV project. Peter Liljeström and Peter Berglund, MTC, Karolinska Institute, for all help in the
SFV projects. Sissela Liljeqvist, for completing the EB200-story.
I want to thank all colleagues at the department of Biotechnology at KTH, over the years, for creating
a friendly and stimulating working atmosphere. During my years at the department, I have really
enjoyed all theatre evenings, dinners, travels (Paris, Sandhamn, Gotland, Berlin, Väddö), Laser Dome
battles, Christmas parties, fredagsöl and fun company during lunches and coffee breaks. I am going to
miss you all.
There are some persons I would like to give special thanks to:
Maria Murby, my exjobbs-supervisor for introducing me to the lab (together with Sissela).
Monica and Pia for taking care of all administrative matters, and Monica also for simultaneously
keeping track of Mathias.
Sophia, P-Å , Jocke and Stefan for dealing with every-day problems, such as lack of space, and carbon
boxes in "korridoren" with a never-ending patience.
My DNA-heroes, Bertil and Jocke W, for teaching me all I know about DNA sequencing, hairpinloops, GC content and Tm.
Marianne, Pelle and Torbjörn, my "bollplank" when it comes to protein-related problems, for always
having time to answer my questions.
The brave K90-troup, Torbjörn, Afshin, Nina, Jenny O, Bahram and Eric B. For the first time in ten
years, I have to manage without you.
My three exjobbare or projectarbetare, Maria, Maria and Henrik, for giving me a hard time awnsering
all your questions.
All persons in Stora labbet for all fun things during the years, especially for winning the Laser Dome
battle (thank you Malin). I would like to thank my closest neighbors Jenny, Kristofer, Johanna, Amelie
and Henrik, for being such nice persons to work around, Anna B and Deirdre for bringing DNA into
Stora labbet, Susanne, my singing-colleague, Torbjörn and My for giving me something else to think
about when I was writing (the fermentor) and of course, Olof and Erik in "mellanstadiet", for just
being two the crazy persons you are.
Also, I would like to thank the rest of the KTH Vaccine Research Center, Maria Wikman, for being a
good friend and an excellent shopping partner.
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Thank you all my friends for being there for me. Tove, Jenny, Malin, Nina, Anna W, Malin C and
Sonja, for dinners, midsummer at Muskö, Advents-dinners with singing, and much more.
I would especially like to thank, Henrik, my END-Note hero, for everything, Jenny O, for dinners and
mysterious parties, Janne, for being the happiest person I know, and for showing me Åbo, Brita, for
sailing trips, hours on the phone or at some café, and Ulla, my best friend for 28 years, for being the
person you are and for supporting me (See you in Rome!).
So at last, my beloved family.
Moster Annica, for letting me live with you for my first month in Stockholm, and for showing me how
"tunnelbanan" works.
Fredrik Skeppstedt for computer assistance and for taking care of my sister.
Pernilla, for all romantic "kits", and for friendship, and Victor for being a darling.
My brothers Anders, Lars and Fredrik and my sister Sara for all fun we have shared during all these
years, for being loving, and always supporting me and believing in me.
Tack Mor och Far för att ni lärt mig att tro på mig själv och att våga gå min egen väg.
This work was supported by grants from by Pierre Fabre Médicaments, France.
67
CHRISTIN ANDERSSON
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