Christin Andersson

Production and delivery of recombinant

subunit vaccines

Christin Andersson

Royal Institute of Technology

Department of Biotechnology

Stockholm 2000

CHRISTIN ANDERSSON

Christin Andersson

Department of BiotechnologyRoyal Institute of Technology (KTH)SE-100 44 StockholmSweden

Printed at Högskoletryckeriet KTH, Stockholm 2000

ISBN 91-7170-633-X

RECOMBINANT SUBUNIT VACCINES

Christin Andersson (2000) Production and delivery of recombinant subunit vaccinesDepartment of Biotechnology, Royal Institute of Technology (KTH), Stockholm, SwedenISBN 91-7170-633-X

Abstract

Recombinant strategies are today dominating in the development of modern subunit vaccines. Thisthesis describes strategies for the production and recovery of protein subunit immunogens, and howgenetic design of the expression vectors can be used to adapt the immunogens for incorporation intoadjuvant systems. In addition, different strategies for delivery of subunit vaccines by RNA or DNAimmunization have been investigated.

Attempts to create general production strategies for recombinant protein immunogens in such a waythat these are adapted for association with an adjuvant formulation were evaluated. Differenthydrophobic amino acid sequences, being either theoretically designed or representing transmembraneregions of bacterial or viral origin, were fused on gene level either N-terminally or C-terminally toallow 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 ofaffinity chromatography. A malaria peptide, M5, derived from the central repeat region of thePlasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in thesestudies. Furthermore, strategies for in vivo or in vitro lipidation of recombinant immunogens for iscomincorporation were also investigated, with a model immunogen ∆SAG1 derived from Toxoplasmagondii. Both strategies were found to be functional in that the produced and affinity purified fusionproteins indeed associated with iscoms. The iscoms were furthermore capable of inducing antigen-specific antibody responses upon immunization of mice, and we thus believe that the presentedstrategies offer convenient methods for adjuvant association.

Recombinant production of a respiratory syncytial virus (RSV) candidate vaccine, BBG2Na, in babyhamster kidney (BHK-21) cells was investigated. Semliki Forest virus (SFV)-based expression vectorsencoding both intracellular and secreted forms of BBG2Na were constructed and found to befunctional. Efficient recovery of BBG2Na could be achieved by combining serum-free productionwith a recovery strategy using a product-specific affinity-column based on a combinatoriallyengineered 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 SFVreplicase 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 theantigen elicited significant anti-BBG2Na titers and demonstrated lung protective efficacy in mice. Thisstudy clearly demonstrate that protective immune responses to RSV can be elicited in mice by DNAimmunization, and that differential targeting of the antigens expressed by nucleic acid vaccinationcould significantly influence the immunogenicity and protective efficacy.

We further evaluated DNA and RNA constructs based on the SFV replicon in comparison with aconventional DNA plasmid for induction of antibody responses against the P. falciparum Pf332-derived antigen EB200. In general, the antibody responses induced were relatively low, the highestresponses surprisingly obtained with the conventional DNA plasmid. Also recombinant SFV suicideparticles induced EB200-reactive antibodies. Importantly, all immunogens induced an immunologicalmemory, 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, Recombinantimmunogen, Respiratory Syncytial Virus, Semliki Forest virus, Serum albumin, Staphylococcus aureus proteinA, Subunit vaccine, Toxoplasma gondii

Christin Andersson

CHRISTIN ANDERSSON


LIST OF PUBLICATIONS

This thesis is based on the following papers, which in the text will be referred to by theirRoman numerals:

I. Andersson, C., Sandberg, L., Murby, M., Sjölander, A., Lövgren-Bengtsson, K. andStåhl, S. General expression vectors for production of hydrophobically taggedimmunogens 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 immunogensfor efficient iscom incorporation. J. Immunol. Methods, 238: 181-193 (2000)

III. Andersson, C., Wikman, M., Lövgren-Bengtsson, K., Lundén, A. and Ståhl, S. Invivo 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 cellproduction of an RSV candidate vaccine recovered using a product specific affinitycolumn. FEBS Letters (2000) Submitted

V. Andersson, C., Liljeström, P., Ståhl, S. and Power, U. Protection against respiratorysyncytial virus (RSV) elicited in mice by plasmid DNA immunization encoding asecreted RSV G protein derived antigen. FEMS Immunol. Med. Microbiol. (2000) Inpress

VI. Andersson, C., Vasconcelos, N-M., Sievertzon, M., Haddad, D., Liljeqvist, S.,Berglund, P., Liljeström, P., Ahlborg, N., Ståhl, S. and Berzins, K. Comparativeimmunization study using DNA and RNA constructs encoding a part of Plasmodiumfalciparum antigen Pf332. (2000) Submitted

CHRISTIN ANDERSSON


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 15by using gene fusion strategies

3. Adjuvant systems for recombinant protein antigens 17

4. Live delivery of subunit vaccines 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 33


PRESENT INVESTIGATION

6. Recombinant production of protein immunogens 37

6.1. Recombinant strategies foriscom incorporation (I, II, III) 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 candidateproduced in mammalian cells (IV) 48

7. Nucleic acid vaccines 53

7.1. Protection against RSV elicited in miceby plasmid DNA immunization (V) 53

7.2. Immunization with RNA and DNA encoding a malarial antigen (VI) 56

8. Concluding remarks 60

9. Abbreviations 64

10. Acknowledgements 66

11. References 68

CHRISTIN ANDERSSON


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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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Table 1: Different vaccine types and some of their characteristics

Vaccine type Characteristics

Whole cell vaccines

Live, attenuated bacteria orviruses

+ Humoral and cellular immune responses- Risk of reversion to virulent strain- Undefined composition

Killed, inactivated bacteria orviruses

+ No risk of infection (replication deficient, non-infectious)- Less powerful than live vaccines- Undefined composition

Subunit vaccines

Purified components frompathogen

+ 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


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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 polysaccharide-

based 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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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


5

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 heat-

labile 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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Table 2: Selected examples of recombinant vaccines currently approved in the US or EU(Modified from Walsh, 2000)

Vaccine Description Application Company Approved

Recombivax rHBsAg produced in S.cerevisiae

Hepatitis B prevention Merck 1986 (US)

Comvax Combination vaccine,containing rHBsAgproduced in S. cerevisiaeas one component

Vaccination of infantsagainst H. influenzaetype B and hepatitis B

Merck 1996 (US)

Tritanrix-HB Combination vaccine,containing rHBsAgproduced in S. cerevisiaeas one component

Vaccination againsthepatitis B, diphteria,tetanus, and pertussis

SmithKline Beecham 1996 (EU)

Twinrix, adult andpediatric forms

Combination vaccine,containing rHBsAgproduced in S. cerevisiaeas one component

Immunization againsthepatitis A and B

SmithKline Beecham 1996 (EU)(adult)

1997 (EU)(pediatric)

Primavax Combination vaccine,containing rHBsAgproduced in S. cerevisiaeas one component

Immunization againstdiphteria, tetanus, andhepatitis B

Pasteur MerieuxMSD

1998 (EU)

Procomvax Combination vaccine,containing rHBsAgproduced in S. cerevisiaeas one component

Immunization againstH. influenzae type Band hepatitis B

Pasteur MerieuxMSD

1999 (EU)

Lymerix rOspA, a surfacelipoprotein of B.burgdorferi, produced inE. coli

Lyme disease vaccine SmithKline Beecham 1998 (US)


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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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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 non-

pathogenic 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


9

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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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


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expression systems. Furthermore, insect cells have the ability to perform a variety of post-

translational 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 time-

consuming 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

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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), Glutathione-

S-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).


13

Table 4: Selected examples of fusion partners and their use

Effect on target protein Fusion partner Reference

Improved production

Secretion Signal peptide Abrahamsén et al., 1986Hansson et al., 1994

Multimerization C-peptide Jonasson et al., 1998

Solubility Protein A domain Samuelsson et al., 1991 and 1994

Simplified purification

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

Improved immunogenicproperties

Immunopotentiating Cholera toxin B (CTB) Sun et al., 1994Holmgren et al., 1994

Carrier-related properties albumin binding protein (ABP orBB) of streptococcal protein G(SpG)

Sjölander et al., 1997Libon et al., 1999

Increased half-life BB of SpG Nygren et al., 1991

Improved adjuvant association

Improved association withiscoms

Membrane-spanning regions fromviral or bacterial surface proteins

I, II

Improved association withiscoms

In vivo lipidation signals III

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

CHRISTIN ANDERSSON

14

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


15

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 IgG-

binding 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 Z-

domain. 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

CHRISTIN ANDERSSON

16

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)


17

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 co-

administration 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 hydroxideAluminium phosphateCalcium phosphate

Gupta, 1998Gupta, 1998Wang et al., 2000

Immunostimulatory adjuvants CytokinesSaponines (e.g. QS21)MDP derivatesCpG oligonucleotideslipopolysaccharides (LPS)monophosporyl lipid A (MPL)

Baca-Estrada et al., 1997Barr et al., 1998.Namba et al., 1997Klinman et al., 1999Ogawa et al., 1997Baldridge and Crane, 1999

Lipid particles and emulsions Freund incomplete adjuvantSAFMF59LiposomesImmunostimulating complexes (iscoms)

Cochleates

Chang et al., 1998Hjort et al., 1997Heineman et al., 1999Gregoriadis et al., 1999Barr et al., 1998I, II, IIIGould-Fogerite et al., 1998

Microparticulate adjuvants PLG microparticlesVirus-like particles

O'Hagan et al., 1998Coste et al., 2000

Mucosal adjuvants Heat-labile enterotoxin (LT)Cholera toxin (CT)

Park et al., 2000Matuso et al., 2000

CHRISTIN ANDERSSON

18

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 (modifiedfrom 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 Gram-

negative 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


19

(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

CHRISTIN ANDERSSON

20

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 virus-

like 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


21

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

CHRISTIN ANDERSSON

22

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).


23

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 non-

pathogens 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 food-

grade 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,

CHRISTIN ANDERSSON

24

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, non-

replicative 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


25

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.

CHRISTIN ANDERSSON

26

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.


27

Table 7A: Selected examples of immunization studies using DNA (modified from Kowalczykand Ertl, 1999.

Pathogen Encoded antigen Reference

Viruses

Ebola virus nucleoprotein (NP), glycoprotein (GP)NP, GP

Vanderzanden et al., 1998Xu et al., 1998

Hepatitis B virus hepatitis B surface antigen (HBsAg)HBsAg

Davis et al., 1997Prince et al., 1997

Herpes simplex virus 1 glycoprotein B, glycoprotein Dglycoprotein B

McClements et al., 1997Daheshia et al., 1998

HIV-1 nef, rev, tat, glycoprotein160, p24nef, rev, tatenvgag

Hinkula et al., 1997Calarota et al., 19981

Sasaki et al., 1998Qiu et al., 2000

Influenza hemagglutinin (HA)HA, NP, matrix protein (M1)HA

Ban et al., 1997Donnelly et al., 1997aUlmer et al., 1998

Measles HA, NP Cardoso et al., 1998

Papilloma E1, E2 Han et al., 2000

Rabies G proteinG protein

Wang et al., 1997Lodmell et al., 1998

Respiratory syncytial virus F proteinG protein

Li et al., 1998Li et al., 2000

Bovine RSV G protein Schrijver et al., 1998

Rotavirus viral protein 4 (VP4), VP6, VP7 Chen et al., 1997

Bacteria

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., 1997Bonato et al., 1998

Salmonella typhii Outer membrane protein C Lopez-Macias et al., 1996

Parasites

Plasmodium falciparum circumsporozoite protein (CSP)CSP, Exp-1, surface sporozoite protein2(SSP2), LSA-1CSP

Wang et al., 1998a1

Wang et al., 1998b

Le et al., 20001

Plasmodium yoelii CSP Gramzinski et al., 1997

Trypanosoma cruzii trans-sialidase (TS) Costa et al., 1998

1 Human trials

CHRISTIN ANDERSSON

28

Table 7B: Selected examples of immunization studies using RNA or plasmid DNA encodingself-replicating RNA

Pathogen Encoded antigen System used Reference

Viruses

SIV glycoprotein 160 (gp160) rSFV viral particles Mossman et al., 1996

HIV envgp160

rSFV viral particlesrSFV viral particles

Brand et al., 1998Berglund et al., 1997

Influenza hemagglutinin (HA)nucleoprotein (NP)

NPNP

rSFV RNArSFV RNA andrSFV viral particlesrSFV viral particlesrSindbis viral particles

Dalemans et a., 1995Zhou et al., 1994

Berglund et al., 1999Tsuji et al., 1998

Japanese encephalitisvirus

E protein, nonstructuralprotein (NS) 1 and NS2a

rSindbis plasmid Pugachev et al., 1995

Looping ill virus envelope proteins (prME),NS1envelope proteins (prME),NS1

rSFV viral particles

rSFV plasmid andrSFV viral particles

Fleeton et al., 1999

Fleeton et al., 2000

Herpes virus gB rSindbis plasmid Hariharan et al., 1998

Parasites

Plasmodium yoelii epitope derived fromcircumsporoziote protein

rSindbis viral particles Tsuji et al., 1998

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


29

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 protein-

based 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).

CHRISTIN ANDERSSON

30

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 antigen-

encoding 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).


31

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., 1997Ishii 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).

CHRISTIN ANDERSSON

32

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 non-

methylated 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 CpG-

dinucleotides 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).


33

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 self-

replicating, 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

CHRISTIN ANDERSSON

34

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).

Figure 1: The life cycle of the Semliki Forest virus. Upon infection of the mammalian host cell, thefull-lenght positive stranded RNA will function as mRNA. Translation of the first two thirds results inpoduciton of the replicase. The replicase will initially be responsible for production of a negativestrand 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 subgenomicRNA, corresponding to the last third of the full-lenght RNA. Translation of the subgenomic RNAresults in production of the structural proteins, needed for production of new viral particles.

(A)ncap SFV Replicase Struct.Positive, single strandRNA

Negative strandintermediate RNA

Positive strand full-length RNAPositive strand,subgenomic RNA

Structural proteins, C,p62 and E1

Replicase

Replicase Replicase

(A)ncap SFV Replicase Struct. Struct. (A)ncap

3' 5'


35

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,

CHRISTIN ANDERSSON

36

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 β-

galactosidase 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.


37

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 iscom-

matrix 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).

CHRISTIN ANDERSSON

38

Table 9: Hydrophobic tags used in I and II

Tag Sequence Origin Position

IW IWIWSIWIWSIWIW Numerous isoleucine (I) andtryptophan residues (W)

N-terminally

MI QILAIYSTVASSLVLLVSLGAISFWMCS The membrane-integration anchorsequence of the H1 hemagglutininof influenza virus A

N and C-terminally

PD LEAALAELAELAE Predicted to form an amphiphaticα-helix at low pH

N-terminally

M ENPFIGTTVFGGLSLALGAALLAG Derived from the membrane-spanning 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 PD-

Z-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 PD-

Z-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.


39

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 1-1.5million deaths from malaria per year (WHO). The causative agents in humans are four species ofPlasmodium 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 mosquitois responsible for transmission of parasites between humans. The parasites develop in the gut of themosquito and are transferred to the blood stream of the human host when the mosquito feeds on humanblood. The life cycle is divided into several stages in the mosquito the liver and the erythrocytes and, asoutlined schematically in figure A. Symptoms of malaria include a high fever as a consequence oferuption of erythrocytes. If not treated, the disease, particularly that caused by P. falciparum, progressesto 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 ofinfection have lead to insecticide-resistant mosquitoes and drug-resistant malaria parasites. Thereforethere is need for a potent malaria vaccine. Attempts have been made to construct vaccines to all stages inthe life cycle (Cox, 1991). Pre-erythrocyte vaccines would block the infection of erythrocytes, andvaccines to the sexual stage, the gametocytes, would block the transmission of the parasite betweenindividuals. An effective vaccine to reduce the clinical effetcts of malaria would be directed to the blood-stage of the parasite. The M5 antigen (Sjölander et al., 1993a) used in paper I, II and III is derived froman 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 asexualintraerythrocytic protein, expressed on erythrocytes (Mattei et al., 1992; Mattei and Scherf, 1992;Hinterberg et al., 1994).

Sporozoites

Mosquito gut

Merozoites

Mosquitoblood meal

Sporozoites

Ring

Trophozoite

Gametocytes

Gametocyte

Zygote

Ookinete

Mosquitoblood meal

Human

Schizont

Scizont

Liver

MALARIA

Anopheles

CHRISTIN ANDERSSON

40

Table 10: Fusion proteins with hydrophobic/ lipophilic tags, used in I, II and III

Study Plasmid Fusion protein Mw

(kDa)

Expressionlevel (mg/l

cell culture)

Solubility* Incorporationefficiency

I 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

II 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

III 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 iscom-

incorporation 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.


41

Figure 2A-J: Analysis of the protein content in the fractions collected from sucrose gradient ultracentrifugationof 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 tagused in each study (Z, ZZ or ABP, respectively) is shown on the right y-axis ( ). The fraction number is indicatedon the x-axis. The horizontal bar in each graph indicates the cholesterol-rich fractions. Figure 2J shows theresults from a control experiment with protein, iscom matrix but without the chelating lipid.

0.15

0.2

0.25

0.3

0.35

0.4

0.45

1 5 10 15

0.05

0.15

0.25

0.35

0.45

A: IW-Z-M5

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.35

0.15

0.2

0.25

0.3

0.4

0.45

0.5

1 5 10 15

B: MI-Z-M5

0.15

0.25

0.35

0.45

0.55

0.65

0.75

0.85

0.95

1 5 10 150

0.2

0.4

0.6

0.8

1

1.2

1.4

C: PD-Z-M5

0.2

0.4

0.6

0.8

1

1 5 10 140

0.1

0.2

0.3

0.4

0.5

D: ABP-M5-M

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

1 5 10 14

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

E: ABP-M5-MI

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1 5 10 140

0.1

0.2

0.3

0.4

0.5

0.6

F: ZZ-M5-M

0.2

0.3

0.4

0.5

0.6

0.7

1 5 10 140

0.1

0.2

0.3

0.4

0.5

0.6

G: ZZ-M5-MI

0.2

0.25

0.3

0.35

0.4

0.45

0.5

1 50

0.1

0.3

0.3

0.35

0.4

0.45

1 50

0.1

0.2

0.3

0.4

0.5

10 15

I: His6-ABP- SAG1

0.2

0.3

0.4

0.5

0.6

1 50

0.1

0.2

0.3

0.4

1510

J: His6-ABP- SAG1H: lpp-His6-ABP- SAG1

0.4

0.2

0.15

0.2

0.25

10 15

CHRISTIN ANDERSSON

42

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.


43

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, ABP-

M5-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

CHRISTIN ANDERSSON

44

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 ZZ-

M5-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, ZZ-

M5-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 well-

defined iscom structures would not be formed.


45

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.

CHRISTIN ANDERSSON

46

Ingestion

Replication

Replication

Bradyzoites

Cat Other mammalsand birds

Oocyst

Intestinalepithelium

Sporozoites

Tachysoites

Gastro-intestinaltract

Tissue cyst

Oocyst

Bradyzoites

Tissue cyst

Intestinalepithelium

Oocyst

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 widerange of animals, including humans. The parasite has a two-phase lifecycle: a sexual phase, which occursin cats and other felides, and an intermediate, non-sexual phase, which can take place in any mammal orbird. The intermediate host can be infected by ingesting oocysts. The oocysts contain sporozoites that arereleased in the gut, penetrate the intestinal epithelia and start to replicate. After about two weeks, theproliferation is slowed down and tissue cysts, containing bradyzoites, are formed. Tissue cysts are mainlylocalized in muscle tissue and the central nervous system, and can be viable for a long time. If an infectedanimal is eaten by another mammal or bird, the tissue cyst is destroyed by digestive enzymes, bradyzoitesare released, penetrate the epithelia ands start to replicate. If the new host is a cat, the parasite developsinto sexual stages, and after about 3-5 days, new oocysts are excreted in the faeces, completing thelifecycle. T. gondii parasite is widely distributed around the world. In veterinary medicine, toxoplasmainfection is well known as a cause of abortion and neonatal mortality in sheep and goat. Humans canaquire the infection from food contaminated with oocysts or meat containing tissue cysts. The infection isusually without symptoms. However, if aquired during pregnancy, the result may be miscarriage ormalformation of the child (Remington, 1990). Toxoplasma infection in immunocompromised individuals,including AIDS patients, may cause a life-threatening disease (Araujo and Remington, 1987; Israelski andRemington, 1988). In the search for a vaccine against toxoplasmosis, live attenuated, killed or lysedparasites or different antigenic fraction has been used (Hermentin and Aspöck, 1988; Johnson, 1989). Forexample, immunization of mice with the major surface antigen (SAG-1) of tachyzoites incorporated intoliposomes 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 andcellular 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 agreat need for effective vaccines, both for veterinary and human use.

TOXOPLASMA

Ingestion


47

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 His6-

ABP-∆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

CHRISTIN ANDERSSON

48

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.


49

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 infectionduring infancy and early childhood worldwide. RSV was first isolated in 1956 and has since then beenassociated 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-suppressedadults and in elderly peoples (Collins et al., 1996). Outbreaks of RSV disease are abrupt and can last up to 5months, and usually peaks in winter. There are two major strains, A and B, both of them circulatesimultaneously during outbreak, and it has been suggested that group A strains are associated with moresevere 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 nucleocapsidsurrounded by a lipid envelope with two major surface proteins, F and G. The F protein is thought tomediate 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 hasbeen 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 isprotective upon immunization in several animal models (Power et al, 1997). It has been suggested that theglycosylation 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 fromstreptococcal protein G (BB) and RSV G protein fragment. (G2N) has been extensively studied. Studies withBBG2Na in animal models, mice and cotton rats, has shown that high immune response and also protectionto 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 responseof these mice and subsequent protection from RSV infections. Lungs and nasal tracts of immunized micewere efficiently protected to subsequent RSV challenges (Power et al., 1997). Maternal immunizationinduces protection of offspring of test animals, and passive transfer of the maternal antibodies was sufficientto 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.

Surface proteinsFGSH

Nucleocapsid-associated proteinsNPL

Matrix proteinMM2

Nonstructural proteinsNS1NS2

RESPIRATORY SYNCYTIAL VIRUS

CHRISTIN ANDERSSON

50

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 BBG2Nacyt 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 BBG2Nasec, 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


51

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).

Figure 4: Overview of the two affinity purification strategies used in paper IV. The potentialinterference of BSA is illustrated.

HSA-purification Affibody mediatedpurification

BSA

ZRSV1

ZRSV1

G2NBBHSA

BSA

G2NBB

BSA

G2NBB

CHRISTIN ANDERSSON

52

Table 11: Protein production characteristics of BBG2Nasec

Production/affinity purification mode Recovered BBG2Naseca

(µg/10 ml)Puritya (%)

With FCS/HSA-Sepharoseb 4 ≈ 50

Without FCS/HSA-Sepharose 0.5 ≈ 20

With FCS/ZRSV1 affibody-Sepharose 6 ≈ 30

Without FCS/ ZRSV1 affibody-Sepharose 2 > 90

aEstimated from Commassie-stained SDS-PAGE gels after affinity recovery. bPerformed in batch mode insteadof 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 BBG2Nasec in culture medium with BSA omitted during protein production and

recovery using the G2Na-specific affibody column, resulted in a product yield of


53

approximately 2 µg BBG2Nasec 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 pBK-

SFV3l2-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).

CHRISTIN ANDERSSON

54

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 cytoplasmicand secreted variants of BBG2Na, respectively. The transcription is under control of the CMV immediate earlypromoter (pCMV). (B) The positive strand RNAs, transcribed by the transfected cells, will be self-amplified in thecytoplasm of transfected cells, since they encode the SFV replicase responsible for the RNA-dependent RNA-replication. (C) The encoded gene products from the plasmid vectors, BBG2Nacyt and pBBG2Nasec, the latterproduct furnished with a signal peptide (sp) for direction to the secretion machinery. The indicated theoreticalmolecular 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

A

B

Neo

pCMV

Ori

SFV-Repl. BBG2Na

BBG2Nacyt BBG2Nasec

Neo

pCMV

C

(A)ncap(A)ncap

pBBG2Nacyt pBBG2Nasec

38.2 kDa 38.3 kDa

Ori

SFV-Repl. sp BBG2Na

SFV-Repl. BBG2Na SFV-Repl. sp BBG2Na

BBG2Na sp BBG2Na


55

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) afterintramuscular 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 5 Mean

pBBG2Nacyt 4.95 4.45 4.45 4.45 4.57±0.25 0/4

pBBG2Nasec ≤1.45 ≤1.45 ≤1.45 3.95 3.95 2.45±1.37 3/5

pControl 4.45 4.2 4.95 4.45 4.45 4.5±0.27 0/5

aThe lungs were, in accordance with earlier studies (Power et al., 1997) considered protected (underlinedfigures) when virus titers were reduced by 2 log10 relative to the mice immunized with the control strain.bViraltiters for the challenged mice are expressed as log10 pfu/g of lung tissue, 5 days post challenge. The detectionlimit for the assay is 101.45.

Interestingly, there was an indication of correlation between the virus reduction and the anti-

BBG2Na 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

CHRISTIN ANDERSSON

56

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 SFV-

systems, 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 SFV-

based 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


57

Figure 6: Schematic representation of the plasmids used for in vitro transcription or immunization. (A) Thevector pSFV3-spl-EB200 encoding the SFV replicase (enabling in vivo amplification) and the secreted variant ofEB200. The transcription is under control of the phage SP6 RNA polymerase promoter (pSP6). The positivestrand RNA, suitable for transfection of mammalian cells, are transcribed and capped in vitro, and carry 3' poly-A-tails, which are transcribed from poly-A sequences in the plasmid vector. (B) The vectors pBK-SFV3L2-EB200 and pBK-SFV3L2-spEB200 encode the cytoplasmic or the secreted variant of EB200. The transcriptionis under control of the CMV major immediate-early (IE1) enhancer. (C) The expression vector pCMVintBL-EB200. The CMV major immediate-early (IE1) enhancer, promoter and intron A and the tPA signal sequenceprecede the gene fragment to be expressed (EB200). The vector contains the simian virus 40 (SV40) origin ofreplication, 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).

A

Neo

pCMV

SFV Replicase EB200sp

pBK-SFV3L2-spEB200

Ori E

B

bla

pSP6

SFV Replicase EB200sp

pSFV3-spl-EB200

C

Neo

pCMV

SFV Replicase EB200

pBK-SFV3L2-EB200

Ori E

Ori E

Bla

SV40Ori

EB200

pCMVintBL-EB200HCMVIE1

enh/pr

Ori E

HCMV int SV40 int p(A)

SFV Replicase EB200spcap A(n)

RNA

StPA

CHRISTIN ANDERSSON

58

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 inimmunizations

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

aRNA transcribed from the plasmid was used for immunizations, bRNA transcribed from the plasmid waspacked in SFV viral particles used for immunizations, c Freunds incomplete adjuvant


59

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.

CHRISTIN ANDERSSON

60

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,


61

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 albumin-

binding 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

CHRISTIN ANDERSSON

62

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 pBBG2Nasec, 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


63

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.

CHRISTIN ANDERSSON

64

Abbreviations

ABD albumin binding domain derived from streptococcal protein GABP albumin binding protein derived from streptococcal protein GALVAC a recombinant canarypox virusAPC antigen presenting cellB7 a B-cell ligandBB albumin binding region derived from streptococcal protein GBHK baby hamster kidney cellsBSA bovine serum albuminCHO chinese hamster ovary cellsCMV cytomegalovirusCpG a cytosine-guanine dinucleotide, p indicates the phosphate bondCS circumsporozoiteCTB cholera toxin B subunitCTL cytotoxic T lymphocyteEB200 a malaria antigen fragment, derived from Pf332ELISA enzyme-linked immunosorbent assayETEC enterotoxic E. coliFCS foetal calf serumFIA Freunds incomplete adjuvantG2Na a part of the respiratory syncytial virus G proteinGM-CSF granulocyte-macrophage colony stimulating factorGST glutathione-S-transferaseHA hemagglutininHEK human embryonic kidney cellsHBsAg Hepatitis B surface antigenHib Haemophilus influenzae type BHis6 hexahistidyl tagHIV human immunodeficiency virusHLA human leukocyte antigenHSA human serum albuminIg immunoglobulinIFN interferonIL interleukinIscoms immunostimulating complexesISS immunostimulatory sequencesIW hydrophobic amino acid sequence (isoleucines and trypophanes)kDa kiloDaltonLPS lipopolysaccharidesLTB E. coli heat-labile toxin B subunitM5 a malaria peptide derived from the central region of Pf155/RESAM membrane-spanning region of SpAMAP multiple antigenic peptidesMHC major histocompatibility complexMI membrane-spanning region of hemagglutinin A from influenza virus


65

MPL monophosporyl lipid AMVA modified vaccinia virus Ankara strainNP nucleoproteinNYVAC attenuated vaccinia virus strainPAGE polyacrylamide gel electrophoresisPD hydrophobic tag, designed to be pH-dependentPCR polymerase chain reactionPLG poly (lactide-co-glycosides)QS-21 a fraction of Quil AQuil A a saponin derived from Quillaja saponariaRSV respiratory syncytial virusSAG1 surface antigen 1 from Toxoplasma gondiiSFV Semliki Forest virusSDS sodium dodecyl sulphateSpA staphylococcal protein ASpG streptococcal protein GSV40 simian virus 40tPA tissue plasminogen activatorVLP virus-like particlesVP viral proteinXM cell-surface-anchoring region of staphylococcal protein AZZ IgG-binding domains derived from staphylococcal protein A

CHRISTIN ANDERSSON

66

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 forhelping me finish this thieis.

I would also like to thank Prof. Mathias Uhlén for leading the DNA corner group in an inspiring wayand for being such a visionary person.

My collaboration partners have contributed a great deal to the work presented in this thesis. I wouldlike to send my gratitude to: Karin Lövgren-Bengtsson at SLU in Uppsala for introducing me to theworld of iscoms. Anna Lundén at SVA in Uppsala for collaboration in the SAG-1 iscom project andall help with the thesis. Klavs Berzins, Diana Haddad, Niklas Ahlborg and Nina-Maria Vasconcelos atStockholms University for collaboration in the EB200 project. Ultan Power, CIPF, for all collaborationin the RSV project. Peter Liljeström and Peter Berglund, MTC, Karolinska Institute, for all help in theSFV 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 creatinga friendly and stimulating working atmosphere. During my years at the department, I have reallyenjoyed all theatre evenings, dinners, travels (Paris, Sandhamn, Gotland, Berlin, Väddö), Laser Domebattles, Christmas parties, fredagsöl and fun company during lunches and coffee breaks. I am going tomiss 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 simultaneouslykeeping track of Mathias.

Sophia, P-Å , Jocke and Stefan for dealing with every-day problems, such as lack of space, and carbonboxes in "korridoren" with a never-ending patience.

My DNA-heroes, Bertil and Jocke W, for teaching me all I know about DNA sequencing, hairpin-loops, GC content and Tm.

Marianne, Pelle and Torbjörn, my "bollplank" when it comes to protein-related problems, for alwayshaving time to answer my questions.

The brave K90-troup, Torbjörn, Afshin, Nina, Jenny O, Bahram and Eric B. For the first time in tenyears, I have to manage without you.

My three exjobbare or projectarbetare, Maria, Maria and Henrik, for giving me a hard time awnseringall your questions.

All persons in Stora labbet for all fun things during the years, especially for winning the Laser Domebattle (thank you Malin). I would like to thank my closest neighbors Jenny, Kristofer, Johanna, Amelieand Henrik, for being such nice persons to work around, Anna B and Deirdre for bringing DNA intoStora labbet, Susanne, my singing-colleague, Torbjörn and My for giving me something else to thinkabout when I was writing (the fermentor) and of course, Olof and Erik in "mellanstadiet", for justbeing two the crazy persons you are.Also, I would like to thank the rest of the KTH Vaccine Research Center, Maria Wikman, for being agood friend and an excellent shopping partner.


67

Thank you all my friends for being there for me. Tove, Jenny, Malin, Nina, Anna W, Malin C andSonja, 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 andmysterious parties, Janne, for being the happiest person I know, and for showing me Åbo, Brita, forsailing trips, hours on the phone or at some café, and Ulla, my best friend for 28 years, for being theperson 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 theseyears, 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.

CHRISTIN ANDERSSON

68

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