Production and delivery of recombinant subunit

Christin Andersson

Royal Institute of Technology Department of Biotechnology Stockholm 2000 CHRISTIN ANDERSSON

ã Christin Andersson Department of Biotechnology Royal Institute of Technology (KTH) SE-100 44 Stockholm Sweden

Printed at Högskoletryckeriet KTH, Stockholm 2000

ISBN 91-7170-633-X RECOMBINANT SUBUNIT VACCINES

Christin Andersson (2000) Production and delivery of recombinant subunit vaccines Department of Biotechnology, Royal Institute of Technology (KTH), Stockholm, Sweden ISBN 91-7170-633-X

Abstract Recombinant strategies are today dominating in the development of modern subunit vaccines. This thesis describes strategies for the production and recovery of subunit immunogens, and how genetic design of the expression vectors can be used to adapt the immunogens for incorporation into adjuvant systems. In addition, different strategies for delivery of subunit vaccines by RNA or DNA have been investigated. Attempts to create general production strategies for recombinant protein immunogens in such a way that these are adapted for association with an adjuvant formulation were evaluated. Different hydrophobic amino acid sequences, being either theoretically designed or representing transmembrane regions of bacterial or viral origin, were fused on gene level either N-terminally or C-terminally to allow association with iscoms. In addition, affinity tags derived from Staphylococcus aureus protein A (SpA) or streptococcal protein G (SpG), were incorporated to allow efficient recovery by means of affinity chromatography. A peptide, M5, derived from the central repeat region of the Plasmodium falciparum blood-stage Pf155/RESA, served as model immunogen in these studies. Furthermore, strategies for in vivo or in vitro lipidation of recombinant immunogens for iscom incorporation were also investigated, with a model immunogen DSAG1 derived from Toxoplasma gondii. Both strategies were found to be functional in that the produced and affinity purified fusion 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 presented strategies offer convenient methods for adjuvant association. Recombinant production of a respiratory syncytial virus (RSV) candidate , BBG2Na, in baby hamster kidney (BHK-21) cells was investigated. Semliki Forest virus (SFV)-based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Efficient recovery of BBG2Na could be achieved by combining serum-free production with a recovery strategy using a product-specific affinity-column based on a combinatorially engineered SpA domain, with specific binding to the G protein part of the product. Plasmid vectors encoding cytoplasmic or secreted variants of BBG2Na, and employing the SFV replicase for self-amplification, was constructed and evaluated for DNA immunization against RSV. Both plasmid vectors were found to be functional in terms of BBG2Na expression and localization. Upon intramuscular immunization of mice, the plasmid vector encoding the secreted variant of the antigen elicited significant anti-BBG2Na titers and demonstrated lung protective efficacy in mice. This study clearly demonstrate that protective immune responses to RSV can be elicited in mice by DNA immunization, and that differential targeting of the expressed by nucleic acid could significantly influence the and protective efficacy. We further evaluated DNA and RNA constructs based on the SFV replicon in comparison with a conventional DNA plasmid for induction of antibody responses against the P. falciparum Pf332- derived antigen EB200. In general, the antibody responses induced were relatively low, the highest responses surprisingly obtained with the conventional DNA plasmid. Also recombinant SFV suicide particles induced EB200-reactive antibodies. Importantly, all immunogens induced an immunological memory, which could be efficiently activated by a booster injection with EB200 protein.

Keywords: Affibody, Affinity chromatography, Affinity purification, DNA immunization, Expression plasmid, Fusion protein, Hydrophobic tag, Iscoms, Lipid tagging, Malaria, Mammalian cell expression, Recombinant immunogen, Respiratory Syncytial Virus, Semliki Forest virus, Serum albumin, Staphylococcus aureus protein A, , Toxoplasma gondii ã Christin Andersson CHRISTIN ANDERSSON RECOMBINANT SUBUNIT VACCINES

LIST OF PUBLICATIONS

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

I. Andersson, C., Sandberg, L., Murby, M., Sjölander, A., Lövgren-Bengtsson, K. and Ståhl, S. General expression vectors for production of hydrophobically tagged immunogens for efficient iscom incorporation. J. Immunol. Methods, 222: 171-182 (1999)

II. Andersson, C., Sandberg, L., Wernérus, H., Johansson, M., Lövgren-Bengtsson, K. and Ståhl, S. Improved systems for hydrophobic tagging of recombinant immunogens for efficient iscom incorporation. J. Immunol. Methods, 238: 181-193 (2000)

III. Andersson, C., Wikman, M., Lövgren-Bengtsson, K., Lundén, A. and Ståhl, S. In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation. J. Immunol. Methods (2000) Submitted

IV. Andersson, C., Hansson, M., Power, U., Nygren, P-Å. and Ståhl, S. Mammalian cell production of an RSV candidate vaccine recovered using a product specific affinity column. FEBS Letters (2000) Submitted

V. Andersson, C., Liljeström, P., Ståhl, S. and Power, U. Protection against respiratory syncytial virus (RSV) elicited in mice by plasmid DNA immunization encoding a secreted RSV G protein derived antigen. FEMS Immunol. Med. Microbiol. (2000) In press

VI. Andersson, C., Vasconcelos, N-M., Sievertzon, M., Haddad, D., Liljeqvist, S., Berglund, P., Liljeström, P., Ahlborg, N., Ståhl, S. and Berzins, K. Comparative immunization study using DNA and RNA constructs encoding a part of Plasmodium falciparum antigen Pf332. (2000) Submitted CHRISTIN ANDERSSON RECOMBINANT SUBUNIT VACCINES CHRISTIN ANDERSSON

TABLE OF CONTENTS

INTRODUCTION 1 1. Vaccines 1 1.1. Subunit vaccines 3 1.2. Recombinant subunit vaccines 4 2. Protein immunogens 7 2.1. Synthetic peptides 7 2.2. Recombinant expression of protein immunogens 8 2.2.1. Gene construct 8 2.2.2. Production hosts 9 2.2.3. Gene fusion strategies 11 2.2.4. Affinity tags 12 2.2.5. Tailor-made affinity ligands 14 2.2.6. Increasing immunogenicity 15 by 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 RECOMBINANT SUBUNIT VACCINES

PRESENT INVESTIGATION

6. Recombinant production of protein immunogens 37 6.1. Recombinant strategies for iscom 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 candidate produced in mammalian cells (IV) 48 7. Nucleic acid vaccines 53 7.1. Protection against RSV elicited in mice by plasmid DNA immunization (V) 53 7.2. Immunization with RNA and DNA encoding a malarial antigen (VI) 56 8. Concluding remarks 60 9. Abbreviations 64 10. Acknowledgements 66 11. References 68 CHRISTIN ANDERSSON RECOMBINANT SUBUNIT VACCINES

INTRODUCTION

The use of vaccines has had great impact on the ability to control and prevent infectious diseases. The first vaccines consisted of whole pathogens, killed or attenuated, but today the recombinant subunit approach, i.e. to use only a defined subunit of the pathogen, is dominating the vaccine research in the search for new effective vaccines. The ability to use small, defined parts of a pathogen and produce that subunit in a non-pathogenic host will increase the safety of future vaccines. Subunit vaccine candidates typically consist of surface proteins or . 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 successfully prevented smallpox in milkmaids. Today, prevention of bacterial and viral 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 , 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 or + Humoral and cellular immune responses viruses - Risk of reversion to virulent strain - Undefined composition Killed, inactivated bacteria or + No risk of infection (replication deficient, non-infectious) viruses - Less powerful than live vaccines - Undefined composition Subunit vaccines

Purified components from + Well-defined composition pathogen - Requires large-scale cultivation of pathogen - Poor immunogens, need of adjuvant Synthetic peptides + Well-defined composition + No risk of pathogenicity - Short half-life - Need of adjuvant Recombinant proteins + Well-defined composition + No risk for pathogenicity + Possibility for cost-efficient production and purification - Primarily humoral immune response - Need of adjuvant Live vectors: Bacterial + Humoral and cellular immune responses + Possibility to develop oral vaccines - Risk of reversion to virulent forms for attenuated pathogens Live vectors: Viral + Humoral and cellular immune responses - Risk of reversion to virulent forms for attenuated pathogens Nucleic Acid vaccines: DNA + Cellular and humoral immune responses + Cost-efficient production + In vivo amplification systems available - Inefficient transfection - Risk of integration into host genome not completely excluded Nucleic Acid vaccines: RNA + No risk of integration into host genome + Do not have to enter nucleus for translation + In vivo amplification systems available - Unstable - Quite expensive production

Live, attenuated vaccines are usually quite effective in stimulating both humoral as well as cellular immune responses (Ellis, 1999). These vaccines might have a certain capacity to replicate in the host, but are attenuated in their pathogenicity in order not to cause disease. However, due to its replicating ability, live attenuated vaccines can potentially revert to a virulent wild-type strain, e.g. by recombination, resulting in disease. Another aspect of using

2 RECOMBINANT SUBUNIT VACCINES live attenuated vaccines is the risk of transmission between individuals. These facts are major concerns, especially if the unwanted disease develops in immunocompromised hosts.

Killed or inactivated vaccines, however, is in general safer than live, attenuated vaccines. The vaccine cannot replicate in the host and is non-infectious. However, a drawback with using killed, inactivated vaccines is that they are less effective than live attenuated vaccines in inducing protective . 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 - 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 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 in human use.

The use of recombinant DNA technology has made the development of subunit vaccines more efficient. The basics of this technology is to transfer a gene encoding an antigen, responsible for inducing immune responses sufficient for protection, to a non-pathogenic host, thereby making the production of the antigen safer and generally more efficient. Recombinant subunit vaccines can be delivered as purified recombinant proteins, as proteins delivered using live non-pathogenic vectors (bacterial or viral) or as nucleic acid molecules encoding the antigen (Table 1). There are several advantages in using recombinant subunit vaccines. No pathogen is present in the production and purification procedure, thus making the production procedure

4 RECOMBINANT SUBUNIT VACCINES less hazardous. By using recombinant DNA technology, the production and purification procedure can be carefully designed to obtain high yields of a well-defined product. Recombinant strategies for production of subunit vaccines will be further discussed later in this thesis, see section 2.2.

Recombinant strategies have also been employed for detoxification of toxins. Engineered inactivation of toxin can be obtained by mutational replacement of specific amino acids in the enzymatically active part of the toxin. Pertussis toxoid produced by Bordetella pertussis with specific mutations in its toxin gene is included as a component in an acellular (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. Hepatitis B prevention Merck 1986 (US) cerevisiae Comvax Combination vaccine, Vaccination of infants Merck 1996 (US) containing rHBsAg against H. influenzae produced in S. cerevisiae type B and hepatitis B as one component Tritanrix-HB Combination vaccine, Vaccination against SmithKline Beecham 1996 (EU) containing rHBsAg hepatitis B, diphteria, produced in S. cerevisiae tetanus, and pertussis as one component Twinrix, adult and Combination vaccine, Immunization against SmithKline Beecham 1996 (EU) pediatric forms containing rHBsAg hepatitis A and B (adult) produced in S. cerevisiae 1997 (EU) as one component (pediatric) Primavax Combination vaccine, Immunization against Pasteur Merieux 1998 (EU) containing rHBsAg diphteria, tetanus, and MSD produced in S. cerevisiae hepatitis B as one component Procomvax Combination vaccine, Immunization against Pasteur Merieux 1999 (EU) containing rHBsAg H. influenzae type B MSD produced in S. cerevisiae and hepatitis B as one component Lymerix rOspA, a surface vaccine SmithKline Beecham 1998 (US) lipoprotein of B. burgdorferi, produced in E. coli

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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 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 . 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 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

8 RECOMBINANT SUBUNIT VACCINES protected from proteolytic degradation, and that high production levels often are obtained. Levels up to 50 % of total cell protein content have been reported (Rudolph, 1996).

2.2.2. Production hosts

The choice of expression system used will be dependent of the characteristics of the protein to be expressed. Several issues have to be considered, e.g. (i) expression levels, (ii) the need for post-translational modifications, (iii) scale-up consideration, and (iv) production costs (Dertzbaugh, 1998). The heterologous host used may affect the immunogenicity and protective efficacy of the product. For example, an antigen may be expressed at high levels in a bacterial expression system, but may only fold partially into its native confirmation, or the antigen needs might need to be post-translationally modified to elicit protective immunity (Dertzbaugh, 1998).

There are today four major host cell systems commonly used to produce recombinant proteins; (i) bacterial expression systems, (ii) yeast expression systems, (iii) insect cell expression systems, and (iv) mammalian expression systems. Each host cell system has individual advantages and disadvantages (Table 3).

Table 3: Examples of production hosts for recombinant protein expression Host Characteristics

Bacteria + Easy to grow and manipulate + High expression levels + Inexpensive - Do not perform post-translational modifications - Proteolysis - Correct folding can be problematic to obtain Yeast + Capable of certain post-translational modifications + High cell densities + Easy to grow and manipulate - Proteolysis Insect cells + Make a variety of post-translational modifications + High expression levels compared to mammalian cells Mammalian cells + Post-translational modifications + Stable antigen-producing cell lines can be constructed - Time consuming - Expensive

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Bacterial expression systems are the most convenient to use. Expression levels of recombinant proteins in bacteria are in general high. Bacterial expression systems are suitable to use when no post-translational modifications of the protein are required. Several bacterial systems are available, but Escherichia coli is the dominating and also the best characterized. Many alternatives for optimization of protein expression in bacteria have been reported e.g. choice of strain, transcriptional and translational regulation, and also the possibility to include signal sequences that will target the protein to periplasm or culture media (Makrides, 1996). Since foreign proteins may be rapidly degraded in the bacterial host, the host can be engineered to reduce the proteolysis (Murby et al., 1996; Babiuk, 1999). Other bacterial expression system that sometimes may be of advantage to use are Salmonella typhimurum (Martin-Gallardo et al., 1993; Liljeqvist et al., 1996), Vibrio cholerae (Viret et al., 1996) and Bacillus brevis (Ichikawa et al., 1993; Nagahama et al., 1996). Since bacterial systems offer significantly lower production cost, any measures are taken to solve potential production problems before a more sophisticated expression system is evaluated.

The most studied yeast expression system is Saccharomyces cerevisiae. Yeast is well-adapted for secreting proteins into the culture supernatant, which facilitates purification. Yeast is further known to grow to very high cell densities, which compensates for the expression levels that are somewhat lower than for E. coli (Dertzbaugh, 1998). The first recombinant vaccine for human use, the surface antigen from Hepatitis B, is expressed in Saccharomyces cerevisiae (Valenzuela et al., 1982). More recently, the yeast Pichia pastoris has become popular to use. Protein expression levels are higher in Pichia pastoris but the growth rate is lower, which may be a problem when expressing unstable proteins (Dertzbaugh, 1998). Pichia pastoris has been used to produce vaccine antigens of bacterial (Clare et al., 1991) as well as viral (Zhu et al., 1997) origin. Yeast, being a eukaryotic expression system performs certain post-translational modifications such as glycosylations. However, glycosylations performed by yeast differ from glycosylation patterns performed by mammalian cells (De Wilde et al., 1990). If glycosylations are important for the desired characteristics of the protein, a mammalian expression system may be needed.

Baculovirus-based expression systems are based on the ability of a virus from the Baculoviridae family to infect insect cells. Heterologous proteins can be efficiently produced in both insect cells and larvae (Geisse et al., 1996). The advantage of using baculovirus system is the high expression levels that can be obtained, when compared to mammalian

10 RECOMBINANT SUBUNIT VACCINES expression systems. Furthermore, insect cells have the ability to perform a variety of 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

11 CHRISTIN ANDERSSON expressing the protein intracellulary (Samuelsson et al., 1991 and 1994). Improvement of proteolytic stability can be obtained in a similar fashion, by fusing the target protein to proteolytically stable fusion partners (Hammarberg et al., 1989; Murby et al., 1991 and 1996). Production of a target protein as a multicopy protein, with a subsequent specific cleavage reaction is a way of increasing the product yield. For example, expression of the human proinsulin C-peptide as a heptameric fusion protein by an enzymatic cleavage procedure, resulting in significantly improved yield of native C-peptide monomers (Jonasson et al., 1998 and 2000). Another example of using gene fusion strategies to adapt the product for a certain purification strategy is by fusing the target protein to hydrophobic amino acid residues in order to alter the partitioning of fusion proteins in aqueous two-phase systems (Köhler et al., 1991; Hassinen et al., 1994) thereby increasing the recovery of fusion protein. Gene fusion strategies have also been employed to obtain increased immune responses and to adapt the target immunogen to certain adjuvant systems (see section 2.2.6).

The purposes of making gene fusion constructs are many (Ståhl et al., 1997 and 1999; Table 4) and one important application is to enable affinity purification of the target protein by using affinity fusion tags.

2.2.4. Affinity tags

Using affinity fusion partners will make efficient recovery of the expressed recombinant gene product possible. Many well-defined affinity tags have been described in the literature, e.g. domains from staphylococcal protein A (SpA) or streptococcal protein G (SpG), 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).

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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., 1986 Hansson 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 immunogenic properties Immunopotentiating Cholera toxin B (CTB) Sun et al., 1994 Holmgren et al., 1994 Carrier-related properties albumin binding protein (ABP or Sjölander et al., 1997 BB) of streptococcal protein G Libon et al., 1999 (SpG) Increased half-life BB of SpG Nygren et al., 1991

Improved adjuvant association

Improved association with Membrane-spanning regions from I, II iscoms viral or bacterial surface proteins Improved association with In vivo lipidation signals III iscoms

Protein A, a surface protein from Staphylococcus aureus (SpA), has been widely used as affinity fusion partner due to its strong affinity to the Fc part of immunoglobulin G (Uhlén et al., 1983). SpA consists of a signal sequence (S) followed by five homologous IgG binding domains (E, D, A, B, C), all capable of binding IgG (Moks et al., 1986) and a cell surface anchoring domain, XM (Schneewind et al., 1995; Navarre and Schneewind, 1999). An engineered IgG-binding domain, Z, was derived from the B domain of SpA and was shown to have retained IgG binding properties and to be resistant to cleavage with hydroxylamin and CNBr (Nilsson et al., 1987). The characteristics of SpA and Z, being proteolytically stable and highly soluble, makes them well suited as affinity fusion partners to enable purification of recombinant proteins (Ståhl et al., 1999). The Z tag have been used in many studies as affinity tag, mostly in its divalent form, ZZ, since it has been shown that ZZ has a ten times higher

13 CHRISTIN ANDERSSON affinity for its ligand IgG compared to the monovalent Z domain (Nilsson et al., 1994). The high solubility of the Z domain has shown to increase the overall solubility of fusion proteins (Samuelsson et al., 1991 and 1994).

The streptococcal protein G (SpG) is a surface protein with affinity to both IgG and albumin of different mammalian species, including humans (Nygren et al., 1990). The binding sites for IgG and serum albumin are shown to be structurally separated (Nygren et al., 1988). The complete albumin-binding region is suggested to contain three albumin-binding motifs, each of about 5 kDa in size. Tags comprising of one, two or two-an-a-half serum albumin binding motifs have been constructed (ABD, ABP and BB, respectively), successfully produced and used in different applications (Nilsson et al., 1997; Ståhl and Nygren, 1997; Ståhl et al., 1999). The albumin binding proteins derived from SpG are proteolytically stable, highly soluble, and are possible to produce in high yields (Larsson et al., 1996; Nilsson et al., 1996; Murby et al., 1995). Both the SpA-derived Z domain and the SpG-derived serum albumin binding proteins have been extensively used to facilitate affinity chromatography purification of protein immunogens (Ståhl et al., 1989; Sjölander et al., 1990, 1993c and 1997; Murby et al., 1994; Berzins et al., 1995; Power et al., 1997; Libon et al., 1999).

2.2.5. Tailor-made affinity ligands

In order to create new binding proteins to be used as affinity ligands, the use of combinatorial strategies to create large protein libraries is an emerging research area. Combinatorial methods are attractive because they allow direct selection of suitable target-specific binding molecules from large libraries of related proteins. Libraries of other molecules than proteins or peptides have also been constructed, e.g. small organic molecules (Gordon et al., 1994) and DNA or RNA (Gold et al., 1995; Burke and Gold, 1997), but this will not be further discussed in this thesis.

A method of emerging interest is the display of libraries of peptides or proteins on filamentous phage surfaces (Smith, 1985; Clackson and Wells, 1994). This concept involves construction of a protein library containing partially randomized sequences, fusion by genetic means of the new variants to phage protein III or phage protein VIII, and subsequently displaying the library on phage particles. Rounds of selection of displayed peptides or proteins with desired properties from such phage libraries can be performed, with intermediate

14 RECOMBINANT SUBUNIT VACCINES enrichment steps of the selected phages in E. coli. A useful selection method is to use solid phase surfaces covered with the target molecule and allowing the phages to bind, theoretically via interactions of the displayed protein and the target molecule. The phage particle would thus constitute the link between genotype and phenotype, which allows identification of the protein from the DNA sequence.

As mentioned above, the IgG-binding Z-domain, derived from SpA, has several features, which makes it suitable in affinity purification applications such as being proteolytically stable, of small size and highly soluble. Structural analysis has shown that the Z domain is composed of three a -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 a -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

15 CHRISTIN ANDERSSON immunizing with the antigen alone (Libon et al., 1999). The immunopotentiating properties of BB could be either due to T-cell epitopes present in BB (Sjölander et al., 1997) or due to the serum albumin binding properties resulting in an increased half-life of the antigen or even a combination of the both.

Another commonly used protein fusion partner with carrier-related properties is the surface antigen of Hepatitis virus B (HBsAg). A fusion protein composed of a repetitive sequence derived from the circumsporozoite (CS) protein of Plasmodium falciparum and HBsAg strategies was produced in yeast cells and used in vaccination experiments in humans resulting in long lasting antibody responses (Vreden et al., 1991). Fusion to the HBsAg have also been shown to enhance the humoral as well as the cellular immune response to human virus 1 (HIV-1)-derived immunogens both when delivered as fusion proteins (Schlienger et al., 1992; Mancini et al., 1994) and as plasmid DNA (Fomsgaard et al., 1998). Furthermore, a strategy to fuse by genetic means the target antigen to cholera toxin subunits in order to obtain targeting to receptors in the mucosa, has been described (Hajishengallis et al., 1995). Antigens can also be expressed as fusion proteins to B cell or T cell epitopes to increase humoral or cellular immune responses (Löwenadler et al., 1992). In addition to expressing single proteins, it is possible to develop chimeric genes containing the important epitopes from a number of pathogens (Whitton et al., 1993).

Recombinant proteins are in general relatively poor immunogens when administered alone and the coadministration of an adjuvant is crucial to elicit a strong immune response. Fusion strategies have been investigated to ensure proper association of fusion proteins to an adjuvant. An example of this strategy, that will be discussed below, is to use hydrophobic tagging of protein immunogens to improve the association of the immunogen to a hydrophobic adjuvant system e.g. iscoms (I, II, III)

16 RECOMBINANT SUBUNIT VACCINES

3. Adjuvant systems for recombinant protein antigens

Subunit vaccines consisting of peptide or recombinant proteins are normally poor immunogens when administered alone. Whole cell vaccines are heterogeneous and contain many immunostimulatory substances, e.g. bacterial DNA or enterotoxins. Pure recombinant protein antigens, lacking these natural immunostimalutory substances, therefore require 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 hydroxide Gupta, 1998 Aluminium phosphate Gupta, 1998 Calcium phosphate Wang et al., 2000 Immunostimulatory adjuvants Cytokines Baca-Estrada et al., 1997 Saponines (e.g. QS21) Barr et al., 1998. MDP derivates Namba et al., 1997 CpG oligonucleotides Klinman et al., 1999 lipopolysaccharides (LPS) Ogawa et al., 1997 monophosporyl lipid A (MPL) Baldridge and Crane, 1999 Lipid particles and emulsions Freund incomplete adjuvant Chang et al., 1998 SAF Hjort et al., 1997 MF59 Heineman et al., 1999 Liposomes Gregoriadis et al., 1999 Immunostimulating complexes (iscoms) Barr et al., 1998 I, II, III Cochleates Gould-Fogerite et al., 1998 Microparticulate adjuvants PLG microparticles O'Hagan et al., 1998 Virus-like particles Coste et al., 2000 Mucosal adjuvants Heat-labile enterotoxin (LT) Park et al., 2000 Cholera toxin (CT) Matuso et al., 2000

17 CHRISTIN ANDERSSON

Adjuvants can be used in several ways; they can increase the duration of immune responses, modulate antibody specificity, isotype and subclass distribution, stimulate cell mediated immune responses or decrease the required dose of antigen and thus reduce the cost for the vaccine (Singh and O'Hagan 1999). The mechanisms of adjuvant action are relatively poorly understood. Most likely, activation of the immune response by adjuvants is a result of a cascade of events such as induced cytokine production or activation of antigen-presenting cells (APCs) e.g by increased expression of costimulatory molecules or MHC complexes (Singh and O'Hagan 1999). The adjuvant to choose will totally depend on what type of immune responses that is required for protective immunity. Different adjuvants usually induce different types of responses, and a first choice would be to evaluate the adjuvant that is predicted to give an immune response similar to what is postulated for protective immunity.

In general, adjuvants are immunostimulatory substances, particulate adjuvants or a combination of the both (Table 5). Immunostimulatory substances are thought to predominantly effect the cytokine expression by activating expression of costimulatory molecules or major histocompatibility complex (MHC) molecules, while particulate adjuvants, which have comparable dimensions to pathogens, probably are targets for uptake by antigen-presenting cells. See Table 6 for a general comparison.

Table 6: A general comparison of dimensions of pathogens and particulate adjuvants (modified from Singh and O'Hagan 1999) Natural pathogen Particulate adjuvant

Bacteria 0.5-3 mm Microparticles 100 nm-10 mm

Poxvirus 250 nm Liposomes 50 nm-1 mm

Herpesvirus 100-200 nm Virosomes 50 nm - 10 mm

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

18 RECOMBINANT SUBUNIT VACCINES

(Gustafson and Rhodes, 1992; Sing and O'Hagan, 1999). In certain studies, MLP has been used in combination with e.g. alum or QS21 (Thoelen et al., 1998). However, when using adjuvant combinations, the individual contribution of the included adjuvants is difficult to establish. MPL has for example been evaluated in vaccine studies for cancer, herpes, hepatitis B virus, malaria and human papilloma virus (Sing and O'Hagan, 1999), and has also been investigated as adjuvant for DNA vaccines (Sasaki et al., 1997).

A second group of immunostimulatory adjuvants are the saponins, a heterogenous group of sterol glycosides and triterpenoid glycosides, present in a wide range of plant species (Barr et al., 1998). Saponins were included in a veterenary vaccine in the 1950s (Barr et al., 1998) but later work showed that the saponins derived from Quillaja saponaria (Chilean soap bark tree) were the most effective as adjuvant (Dalsgaard, 1970). Variable results have been reported from the use of saponins, probably partly due to confusion with respect to the source of the saponin and the variable content of saponins in extract. However, in 1978, Quillaja saponins were purified, and a fraction with high adjuvant activity, termed Quil A, was defined (Dalsgaard, 1978) and since then, Quil A has been used in a number of veterinary vaccines (Dalsgaard et al., 1990). However, Quil A have been associated with some toxicity in vitro, and several attempts have been made to purify Quil A (for review, see Barr et al., 1998). In the 1990s, a fraction of Quil A saponins with low toxicity was isolated, denoted QS21 (Kensil, 1991). QS21 has the ability to induce antibody production and cytotoxic T cells (CTL) (reviewed in Kensil, 1996). Studies evaluating QS21 as an adjuvant have been performed for cancer, HIV-1, influenza, herpes, malaria and hepatitis B (Sing and O'Hagan, 1999). Human clinical trials have been performed using QS21 as adjuvant in for example vaccines for malignant melanoma (Livingston et al., 1994) and in a DNA vaccine study for HIV-1 (Sasaki et al., 1998)

Recent research has shown that sequences in bacterial DNA, but not vertebrate DNA has direct immunostimulatory effect on cells in vitro (Tokunaga et al., 1984; Messina et al., 1991). The effect is due to the presence of unmethylated CpG dinucleotides that is methylated in vertebrate DNA (Krieg et al., 1995; Bird et al., 1987). It is thought that the CpG motifs in bacterial DNA function as a danger signal and thus stimulating immune responses (Krieg, 1996). The exact mechanism for the immunostimulatory effect is not known, but it has been shown that CpG motifs stimulate macrophages and dendritic cells (Jakob et al., 1998). CpG, which can be in the form of plasmid DNA or synthetic oligonucleotides, has been evaluated as

19 CHRISTIN ANDERSSON a mucosal adjuvant (Moldoveanu et al., 1998; McCluskie and Davis, 1999) and seems to have a maximized effect when conjugated with protein antigens (Klinman et al., 1999). Immunostimulatory sequences can also be used in combination with DNA vaccines, as will be discussed later. To date, the effect of CpG immunization has only been widely used in animal models, and its potential and safety in humans need to be evaluated.

Immunostimulatory substances are thought to induce cytokine production, and as an alternative, cytokines can be used directly to modulate or increase the immune response. For example, the use of cytokines has shown to be very promising for immunotherapy of cancer (Salgaller and Lodge, 1998). Cytokines as adjuvants will not be discussed in detail here.

Particulate adjuvants, for example oil emulsions, microparticles, iscoms, liposomes and 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 (Higgins et al., 1996; Cataldo and Nest, 1997) and to be a more potent adjuvant than alum for a hepatitis B vaccine in baboons (Traquina et al., 1996). To date, clinical trials in humans have been performed for several vaccines using MF59 as adjuvant, e.g. to HIV (Kahn et al., 1994; Nitayaphan et al., 2000) and herpes simplex virus (Langenberg et al., 1995; Drulak et al., 2000).

Liposomes, another group of particulate adjuvants, have been used as delivery system for antigens alone or in combination with another adjuvant (Gregoriadis, 1990; Alving, 1995). Liposomes are spherical bilayers of 50 -1000 nm in diameter (Langner and Kral, 1999). Some advantages of using liposomes are that the compounds are protected from degradation and that the circulation time of the antigen is increased (Langner and Kral, 1999). Liposomes have been used as delivery system for proteins and peptides (Alving, 1995) as well as for DNA (Perrie and Gregoriadis, 2000). Cochleates, a modified liposomal structure has also been evaluated in animal models (Gould-Fogerite et al., 1998).

Biodegradable polymeric microparticles, a group of adjuvants that have been investigated for vaccine delivery, and among these, the poly(lactide-co-glycosides) (PLGs) are the primary

20 RECOMBINANT SUBUNIT VACCINES candidates for adjuvant development (O'Hagan et al., 1991a and b). PLG particles have been used in humans for many years as suture material and controlled-release delivery systems (Sing and O'Hagan, 1999) and only recently, PLG particles have been shown to have adjuvant properties to encapsulated antigens (O'Hagan et al., 1991a and b). Microparticles have been demonstrated to be effective in eliciting CTLs (Maloy et al., 1994; Nixon et al., 1996) and have been suggested as adjuvants for DNA vaccines (Hedley et al., 1998). A unique feature of the microparticles are their ability to control the release of antigen, therefore it could be possible to develop a vaccine that have a built-in boosting system. This is very attractive, especially in the developing countries, since it might simplify vaccination logistics

Viral proteins expressed in heterologous hosts may associate in the culture media and form virus like particles (VLPs). When antigens and viral proteins are coexpressed, the VLPs entrap the antigen and can be used as an adjuvant system. For example, recombinant VLPs from Saccharomyces cerevisiae, carrying a string of 15 epitopes from Plasmodium species have been shown to induce CTL responses in mice (Gilbert et al., 1997). Similar VLPs have also been shown to induce CTLs to SIV in macaques (Klavinskis et al., 1996) and to be safe and immunogenic in humans (Martin et al., 1993).

In immunostimulating complexes, iscoms, Quil A, a saponin derived from Q. saponaria (described above) has been incorporated into cage-like structures (Morein et al., 1984). The iscoms are of about 40 nm in diameter and consists of cholesterol, phospholipids and the target antigen (Morein et al., 1984; Barr et al., 1998). Iscoms have been used as adjuvant in several animal models (Morein et al., 1995; Barr et al., 1998; Sjölander and Cox, 1998). Protective immunity induced by iscoms has for example been demonstrated against influenza virus in mice (Lövgren et al., 1990) and horses (Mumford et al., 1994). The mechanism of the iscoms mode of action is not clearly understood, but some studies suggest that increased cellular uptake may contribute to the adjuvant activity (Barr et al., 1998).

The saponin initially used in iscoms, Quil A, is quite heterogenous. In order to determine the immunological properties and toxicity of chemically defined components of Quil A, purified immunostimulatory fractions (QH-A, QH-B and QH-C) from Quil A was investigated, both as free saponins and incorporated into iscoms (Rönnberg et al., 1995). Immunizations of mice showed that all investigated combinations enhanced the antibody response equally, but the combination of QH-A and QH-C in a ration of 7:3 (Iscoprep™703, Iscotec AB, Sweden) where the toxic fraction QH-B was excluded, was both effective and non-toxic. The purified

21 CHRISTIN ANDERSSON components QH-A, QH-B and QH-C have been characterized in several other studies (Rönnberg et al., 1997; Johansson and Lövgren-Bengtson, 1999). Numerous of other purified components from Quil A have also been investigated, both in iscoms and as free components, reviewed in Barr et al., 1998.

One problem with iscoms is that the incorporation of the antigen need to be adapted for each antigen, and may require modifications of the antigen. For several adjuvants, such as liposomes, cochleates, non-ionic block polymers and iscoms, the protein antigens are normally incorporated by means of hydrophobic interactions (Morein et al., 1996), and the physical assocation has been demonstrated to be essential for the adjuvant to exert its immunostimulating capacity. For iscoms, it has been shown that membrane-antigens containing hydrophobic transmembrane sequences, are efficiently incorporated (Morein et al., 1995). Proteins derived from membranes of a variety of viruses, bacteria and parasites have successfully been incorporated into iscoms through hydrophobic interactions (for review, see Barr et al., 1998). In contrast, hydrophilic antigens require modifications to be incorporated into iscoms. Partial denaturation by using low pH (Pyle et al., 1989; Morein et al., 1990; Heeg et al., 1991) or high temperature (Höglund et al., 1989) have been used to expose internal hydrophobic sequences of otherwise hydrophilic proteins. Covalent addition of fatty acids is also a useful method used to incorporate soluble proteins into iscoms, e.g. in a recombinant HIV-1 gp120 candidate vaccine (Reid, 1992; Browning et al., 1992). Other strategies that have been investigated include chemical conjugation of a peptide (Lövgren et al., 1987) or proteins to pre-formed iscoms containing influenza envelope protein or matrix alone (Morein et al., 1995; Sjölander et al., 1991; Larsson et al., 1993). Novel strategies, for inclusion of hydrophobic protein sequences or even lipids in the protein antigen will be presented in this thesis (I, II, III).

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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,

23 CHRISTIN ANDERSSON

1997). Heterologous surface expression of antigens have been reported for several bacterial systems evaluated as live delivery vehicles (Georgiou et al., 1993), for example gram-positive bacteria and mycobacteria (Fischetti et al., 1996; Georgiou et al., 1997; Ståhl and Uhlén, 1997; Stover et al., 1993). Recently, protective immunity to respiratory syncytial virus (RSV) could be evoked in mice by intranasal immunization with Staphylococcus carnosus carrying surface-exposed RSV peptides (Cano et al., 2000). S. carnosus is a non-pathogenic bacteria traditionally used in meat-fermentation processes.

4.2. Viral delivery systems

Recombinant viruses have been studied as candidate vaccines, both as vaccines against the original diseases, and as viral vectors used to deliver heterologous antigens or genes. One advantage of using viral vaccines is the ability to elicit both humoral and cellular immune responses towards the delivered target antigen, as a result of intracellular expression of the heterologous antigens.

Vaccinia virus was earlier used in vaccination against smallpox. The introduction of genes encoding foreign proteins into a vaccinia vector was first demonstrated in 1982, and vaccinia virus has since then been extensively studied as live vaccine vector (Paoletti, 1996). Recombinant vaccinia virus was originally produced by homologous recombination, but today alternative strategies have been developed, enabling more efficient gene construction procedures.

Immunization experiments with recombinant vaccinia vectors expressing viral and non-viral antigens have been reported to elicit protective immunity in animal disease models (Liljeqvist and Ståhl, 1999). An oral consisting of recombinant vaccinia expressing the rabies virus glycoprotein has been given to wild animals, leading to 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

24 RECOMBINANT SUBUNIT VACCINES

NYVAC and MVA vectors encoding pathogen-derived antigens have been evaluated extensively in various disease models (Perkus et al., 1995; Caroll and Moss, 1997).

Other viral vaccine vectors that have been investigated are certain poxviruses, unable to replicate in mammalian cells, e.g. fowlpox and canarypox viruses, and ALVAC, a recombinant canarypox virus (Perkus et al., 1995). Adenoviral vaccine vectors, not pathogenic in humans, can be made replication-competent or deficient, and can be administered orally (Imler, 1995). Several viral antigens have been expressed and delivered by adenoviral vectors, resulting in humoral, cell-mediated or mucosal immune responses (Fooks et al., 1995; Mittal et al., 1996; Xiang et al., 1996). Non-infectious self-assembled virus-like particles have also been used as delivery systems for antigens. Fusion of a viral epitope to the N-terminus of the VP2 capsid protein of Parvovirus led to the assembly of parvovirus-like particles, which were able to deliver the foreign epitopes into the cytosol, resulting in stimulated cellular immunity (Sedlik et al., 1997).

Although a number of human clinical trials with different viral recombinant vectors have been performed, no vaccine based on live viral delivery is yet available on the market. The main applications for virus-based vectors would initially be for certain specific diseases, such as vaccines for HIV and cancer, as well as veterinary vaccines.

25 CHRISTIN ANDERSSON

5. Nucleic Acid Vaccines

Nucleic acid vaccines represent a rather recent approach to the control of infectious agents. These novel vaccines consist of DNA (as plasmid) or RNA (as mRNA), although the use or RNA has not yet been as well studied. Tables 7A and 7B show selected examples of immunization studies using DNA or RNA, respectively.

5.1. DNA vaccines

DNA vaccines consist of designed eukaryotic expression plasmids. The gene encoding the antigen (or antigens) of interest is placed under the control of a strong viral promoter, recognized by the mammalian host. 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.

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Table 7A: Selected examples of immunization studies using DNA (modified from Kowalczyk and Ertl, 1999. Pathogen Encoded antigen Reference

Viruses

Ebola virus nucleoprotein (NP), glycoprotein (GP) Vanderzanden et al., 1998 NP, GP Xu et al., 1998 Hepatitis B virus hepatitis B surface antigen (HBsAg) Davis et al., 1997 HBsAg Prince et al., 1997 Herpes simplex virus 1 glycoprotein B, glycoprotein D McClements et al., 1997 glycoprotein B Daheshia et al., 1998 HIV-1 nef, rev, tat, glycoprotein160, p24 Hinkula et al., 1997 nef, rev, tat Calarota et al., 19981 env Sasaki et al., 1998 gag Qiu et al., 2000 Influenza hemagglutinin (HA) Ban et al., 1997 HA, NP, matrix protein (M1) Donnelly et al., 1997a HA Ulmer et al., 1998 Measles HA, NP Cardoso et al., 1998

Papilloma E1, E2 Han et al., 2000

Rabies G protein Wang et al., 1997 G protein Lodmell et al., 1998 Respiratory syncytial virus F protein Li et al., 1998 G protein Li 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) Ulmer et al., 1997 heat shock protein 65 (hsp65) Bonato et al., 1998 Salmonella typhii Outer membrane protein C Lopez-Macias et al., 1996

Parasites

Plasmodium falciparum circumsporozoite protein (CSP) Wang et al., 1998a1 CSP, Exp-1, surface sporozoite protein2 Wang et al., 1998b (SSP2), LSA-1 CSP Le et al., 20001 Plasmodium yoelii CSP Gramzinski et al., 1997

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

1 Human trials

27 CHRISTIN ANDERSSON

Table 7B: Selected examples of immunization studies using RNA or plasmid DNA encoding self-replicating RNA Pathogen Encoded antigen System used Reference

Viruses

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

HIV env rSFV viral particles Brand et al., 1998 gp160 rSFV viral particles Berglund et al., 1997 Influenza hemagglutinin (HA) rSFV RNA Dalemans et a., 1995 nucleoprotein (NP) rSFV RNA and Zhou et al., 1994 rSFV viral particles NP rSFV viral particles Berglund et al., 1999 NP rSindbis viral particles Tsuji et al., 1998 Japanese encephalitis E protein, nonstructural rSindbis plasmid Pugachev et al., 1995 virus protein (NS) 1 and NS2a Looping ill virus envelope proteins (prME), rSFV viral particles Fleeton et al., 1999 NS1 envelope proteins (prME), rSFV plasmid and Fleeton et al., 2000 NS1 rSFV viral particles Herpes virus gB rSindbis plasmid Hariharan et al., 1998

Parasites

Plasmodium yoelii epitope derived from rSindbis viral particles Tsuji et al., 1998 circumsporoziote protein

Plasmid DNA can be administered in several ways, but so far the most common immunization route is intramuscular injection of plasmid in saline. Other routes that have been investigated used include intradermal (i.d.) (Schrijver et al., 1998), intravenous (i.v) (Liu et al., 1997; Böhm et al., 1998), nasal (Sasaki et al., 1998; Ban et al., 1997) or even ocular routes (Daheshia et al., 1998). Comparative studies have shown that the route of administration can have great impact on the elicited immune response (Barry and Johnston, 1997; Pertmer et al., 1996). When using the intradermal route, plasmid can be either injected with a syringe or administered with a gene gun (Pertmer et al., 1995; Dégano et al., 1998). Using the gene gun, plasmid DNA is precipitated onto an inert particle (usually gold beads) and forced into the cells by a helium blast. The gene gun strategy is more efficient in delivering the plasmid into the cells, and the amount of DNA needed for gene-gun immunization can be as low as a few nanograms (Pertmer et al., 1995; Dégano et al., 1998).

Delivery routes to the mucosal surface, for example direct delivery of nucleic acids to the nasal or oral tract have been demonstrated to have the capacity to induce mucosal immune

28 RECOMBINANT SUBUNIT VACCINES responses (Ban et al., 1997). Other delivery methods to mucosal surfaces is the use of intracellular bacteria as carriers for plasmid DNA (Sizemore et al., 1995), the use of incorporation of DNA into microspheres (Jones et al., 1997) and the use of lipid-based DNA delivery systems (Felgner et al., 1995; Gregoriadis et al., 1997). More effective systems for delivering the DNA to the nucleus also need to be further evaluated. Adjuvants and delivery systems evaluated in DNA vaccine studies are discussed in section 5.2.1.

DNA vaccines have some advantages over traditional vaccines. Immunologically, due to endogenously production of the antigen, DNA vaccines induce the same immune response as a natural infection (Kowalczyk and Ertl, 1999), resulting in the induction of a cellular response, generally not elicited with protein-based vaccines. In a comparative study using the SV40 large tumor antigen, plasmid DNA showed to be superior to immunization with proteins or virus in inducing antigen-specific CTLs (Schirmbeck et al., 1996). Moreover, DNA immunizations circumvents problems like incorrect folding and lack of post-translational modifications that has been associated with protein subunit vaccines. Compared to 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 . Vaccines based on DNA is also easy to distribute since the DNA is extremely stable and does not require cold chains, which is difficult to maintain in developing countries (Kowalczyk and Ertl, 1999).

Some comparative studies involving DNA vaccines have been performed e.g. DNA encoding human wild type p53 induced protective immune responses in mice, but the same gene delivered with a viral vector did not (Hurpin et al., 1998). However, others have experienced that the efficacy of nucleic acid vaccines is very low and highly variable (reviewed in Manickan et al., 1997). Thus, one emerging opinion is, that since the antibody response raised from DNA vaccines is generally slow and not very potent, and unless they are improved, DNA vaccines might serve best as priming agents in combination vaccines. (Kowalczyk and Ertl, 1999).

29 CHRISTIN ANDERSSON

5.2. Improving immune responses to DNA vaccines

A large number of approaches have been used in attempts to improve the efficiency of DNA vaccines (Rodriguez and Whitton, 2000). One strategy is to optimize of the many sequence elements included in the plasmid used, e.g. promoter, introns, enhancers and poly-adenylation signals (Böhm et al. 1996; Danko et al. 1997; Montgomery et al., 1993). The 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).

30 RECOMBINANT SUBUNIT VACCINES

5.2.1. Adjuvants and delivery systems forDNA vaccines

To enhance or modulate immune responses elicited by DNA vaccines, different adjuvants or delivery systems can be used. Table 8 presents selected examples used in immunization studies.

Table 8: Selected examples of adjuvants and delivery systems used for plasmid DNA

Adjuvant /delivery system Reference

Aluminum adjuvant Ulmer et al., 2000

Cationic liposomes Gregoriadis et al., 1997 Ishii et al., 1997 Cationic lipid mixtures Norman et al., 1997

Cationic microparticles Singh et al., 2000

Monophosphoryl lipid A (MPL) Sasaki et al. 1997

Poly(DL-lactide-co-glycolide) microparticle Jones et al. 1997

QS 21 Sasaki et al. 1998

Bacterial delivery systems

Listeria monocytogenes Dietrich et al., 1998

Salmonella typhimurium Darji et al., 1997

Shigella flexneri Sizemore et al., 1997

Live, attenuated bacteria have been used as carriers for plasmid DNA, for example attenuated Shigella flexneri that has the capacity to invade mammalian cells upon infection. Delivery of plasmid to the cytoplasm of infected cells using S. flexneri as delivery system (Sizemore et al., 1995) has been demonstrated. Furthermore, high serum antibody titers in mice immunized intranasally with such S. flexneri delivery system (Sizemore et al., 1997) has been shown. Delivery of plasmid DNA encoding model antigens to macrophages was studied in vitro using Listeria monocytogenes as suicide vector (Dietrich et al., 1998) and successful expression and antigen presentation could be shown. The intracellular pathogen Salmonella typhimurium has also been used for DNA delivery (Darji et al., 1997).

31 CHRISTIN ANDERSSON

5.2.2. Codelivery of stimulatory substances

In order to increase or modulate the vaccine-induced immune response, natural signal substances from the immune system can be used, codelivered as either active substances or as plasmid DNA. Codelivery of cytokines has show to modulate the immune response in a number of ways (Kowalczyk and Ertl, 1999), e.g. codelivery of GM-CSF was shown to increase the immune response to a rabies antigen (Ertl and Xiang, 1996), but in the same system, codelivery of IFN-g 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).

32 RECOMBINANT SUBUNIT VACCINES

5.3. Safety of DNA vaccines

DNA vaccines have thus far not shown to induce any severe side-effects. There is little evidence for integration of DNA into the host genome (Nichols et al., 1995) and no autoimmune reactions or persistent anti-DNA antibodies have been observed. Some studies have reported production of anti-DNA antibodies, however with very low and transient titers (Mor et al., 1997).

5.4. RNA Vaccines

Nucleic acid vaccination through the delivery of RNA has been investigated to a lesser extent than the DNA vaccination. The first applications of delivery of mRNA showed to induce CTL to the influenza virus nucleoprotein in mice when mRNA was delivered in liposomes (Martinon et al. 1993). Liposome-mediated transfection of mouse fibroblasts with mRNA encoding human carcioembryonic antigen (Conry et al., 1995) resulted in a transient production of antibodies. The rapidly declining antibody levels could reflect a short-lived protein expression in vivo. One major advantage of RNA vaccines over DNA vaccines would be that no risk of host-genome integration of the delivered gene exists. However, a disadvantage of using RNA-based vaccines is that the preparation and administration of RNA is troublesome because of the low stability of the RNA. This might lead to rapid in vivo degradation, and thus a short-lived expression of the encoded gene. Furthermore, certain reagents needed for RNA preparation, e.g. RNA polymerases and capping agents, are rather costly.

Development of a new generation of RNA vaccines, derived from members of the Alphavirus family e.g. Sindbis virus and Semliki Forest virus (SFV), have some interesting fetures that makes them suitable for development of RNA vaccines (Tubulekas et al., 1997). The alphaviruses have a very broad host range since they infect various mammalian, avian and insect cells, (Zhou et al., 1994). Also, the RNA genomes of the alphaviruses are 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

33 CHRISTIN ANDERSSON the gene encoding an alphavirus replicase (e.g. SFV replicase) together with the gene encoding the foreign antigen. Transfection of a mammalian cell with such an RNA construct will first lead to translation of the replicase. The replicase will be responsible for production of new full-length RNA genomes and shorter subgenomic RNA molecules, via a negative strand intermediate. The subgenomic RNA molecule corresponds to the last third of the full length RNA. Translation of the subgenomic RNA will lead to production of the antigen. Mass replication of the RNA will theoretically produce up to 200 000 copies of RNA in a single cell within 4 hours, and the gene product can be as much as 25% of total cell protein (Rolls et al., 1994).

Positive, single strand cap SFV Replicase Struct. (A)n RNA

Replicase Negative strand 3' 5' intermediate RNA

Replicase Replicase

Positive strand full- cap SFV Replicase Struct. (A)n cap Struct. (A)n length RNA Positive strand, subgenomic RNA

Structural proteins, C, p62 and E1

Figure 1: The life cycle of the Semliki Forest virus. Upon infection of the mammalian host cell, the full-lenght positive stranded RNA will function as mRNA. Translation of the first two thirds results in poduciton of the replicase. The replicase will initially be responsible for production of a negative strand RNA. Using the negative RNA as template, the replicase will mass-produce full-length RNA. The replicase will also recognize an internal promoteer sequence, and start mass-produce subgenomic RNA, corresponding to the last third of the full-lenght RNA. Translation of the subgenomic RNA results in production of the structural proteins, needed for production of new viral particles.

34 RECOMBINANT SUBUNIT VACCINES

The Semliki Forest virus vector systems were first used as a tool for mammalian production of recombinant proteins (Liljeström and Garoff, 1991, Liljeström, 1994; IV) but has lately been evaluated in nucleic acid vaccine approaches (Table 7B).

An alphavirus based RNA vaccine can be delivered alone or as packed in a virus particle (Tubulekas et al., 1997). To achieve packaging in a viral particle, cotransfection of a helper RNA molecule encoding the genes for the structural proteins is required for production of viral particles. Only the full-length RNA molecule, lacking the genes encoding the structural proteins, contains a packaging signal, and will be packed in the produced recombinant virus particles. The sub-genomic RNA lacks the packaging signal and will thus not be packed in viral particles. Transfection of cells with these recombinant virus particles will initially lead to delivery of the full-length RNA to the host cell cytoplasm. However, since the gene encoding the structural proteins, needed for production of new viral particles, is removed, transfection of the recombinant virus will not result in production of new viral particles.

RNA alone, encoding the alphavirus replicase and the target antigen, has proven to give in vivo expression of the antigen (Johanning et al., 1995) resulting in strong immune responses to the encoded antigen, e.g. influenza virus nucleoprotein in mice (Zhou et al., 1994). Recombinant SFV particles with packed RNA encoding influenza virus nucleoprotein have been shown to induce strong immune responses in mice (Zhou et al., 1995), and were recently demonstrated capable of inducing protective immunity in mice (Berglund et al., 1998). The immunizations of primates with RNA packed in SFV particles have been demonstrated to induce strong immune responses to HIV-1 (Berglund et al., 1997) and SIV (Mossman et al., 1996).

As mentioned above, the low stability of naked RNA makes the preparation and administration of RNA somewhat troublesome. Those problems have been overcome by the development of a plasmid DNA vaccine, based on the Sindbis genome (Herweijer et al., 1995) or the SFV genome (Berglund et al., 1998). When using conventional DNA vectors, containing a eukaryotic promoter, e.g. the cytomegalovirus promoter (CMV), the amount of RNA transcribed is totally dependent on the promoter. However, when using SFV-based plasmids, transcription of RNA and subsequent translation will lead to the production of the SFV replicase. From this point on, the replicase will be responsible for copying the RNA, and the alphaviral replication cycle will continue as for the RNA-based system (Figure 1). Thus,

35 CHRISTIN ANDERSSON plasmids employing the alphaviral self-replication properties on RNA level could be seen as RNA-vaccines delivered as a DNA precursor, and was presented in Table 7B.

Plasmids derived from alphaviral genomes have been used in expression studies, e.g. high expression levels of a luciferase reporter gene in cell lines after transfection of a Sindbis replicon-based DNA plasmid have been reported (Herweijer et al., 1995; Dubensky et al., 1996). Similar studies using an SFV-based plasmid have shown high level expression of b- 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.

36 RECOMBINANT SUBUNIT VACCINES

PRESENT INVESTIGATION

6. Recombinant production of protein immunogens

6.1. Recombinant strategies for iscom incorporation (I, II, III)

Subunit vaccine research is dependent on efficient production of protein antigens, and recombinant strategies are today dominating (Liljeqvist and Ståhl, 1999; Ståhl et al., 1999). As discussed earlier gene fusions are frequently used to improve or alter the characteristics of the proteins to make the production more efficient (Ståhl et al., 1997; Ståhl et al., 1999). In this thesis, recombinant strategies for production of protein immunogens furnished with a hydrophobic tag to improve incorporation into iscoms have been evaluated (I, II, III). For several adjuvants, such as liposomes, cochleates, non-ionic block polymers and immunostimulating complexes (iscoms), the protein antigens are normally incorporated by hydrophobic interactions (Morein et al., 1996). For iscoms, it has been shown that antigens containing hydrophobic amino acid sequences are efficiently incorporated (Morein et al., 1995) while hydrophilic antigens instead could be incorporated by partial denaturation to expose hydrophobic sequences, or by chemical conjugation to preformed iscoms or 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).

37 CHRISTIN ANDERSSON

Table 9: Hydrophobic tags used in I and II

Tag Sequence Origin Position

IW IWIWSIWIWSIWIW Numerous isoleucine (I) and N-terminally tryptophan residues (W) MI QILAIYSTVASSLVLLVSLGAISFWMCS The membrane-integration anchor N and C- sequence of the H1 hemagglutinin terminally of influenza virus A PD LEAALAELAELAE Predicted to form an amphiphatic N-terminally a -helix at low pH M ENPFIGTTVFGGLSLALGAALLAG Derived from the membrane- C-terminally spanning region of SpA

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 a -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.

38 RECOMBINANT SUBUNIT VACCINES

MALARIA

Anopheles Human Mosquito blood meal Liver Sporozoites

Sporozoites Schizont

Ookinete

Merozoites Ring Zygote Mosquito gut

Scizont Gametocyte Trophozoite Mosquito blood meal Gametocytes

Figure A: The life cycle of the Plasmodium falciparum parasite in humans and in the mosquito.

At present, at least 300 million people are infected by malaria globally and there are between 1-1.5 million deaths from malaria per year (WHO). The causative agents in humans are four species of Plasmodium protozoan parasites; P. falciparum, P. vivax, P. ovale and P. malariae. Of these, P. falciparum accounts for the majority of infections and is the most lethal. The female Anopheles mosquito is responsible for transmission of parasites between humans. The parasites develop in the gut of the mosquito and are transferred to the blood stream of the human host when the mosquito feeds on human blood. The life cycle is divided into several stages in the mosquito the liver and the erythrocytes and, as outlined schematically in figure A. Symptoms of malaria include a high fever as a consequence of eruption of erythrocytes. If not treated, the disease, particularly that caused by P. falciparum, progresses to severe malaria. Severe malaria is associated with death. Traditional methods for controlling malaria, like the use of insecticides against the mosquito and also drug treatment for prevention or cure of infection have lead to insecticide-resistant mosquitoes and drug-resistant malaria parasites. Therefore there is need for a potent malaria vaccine. Attempts have been made to construct vaccines to all stages in the life cycle (Cox, 1991). Pre-erythrocyte vaccines would block the infection of erythrocytes, and vaccines to the sexual stage, the gametocytes, would block the transmission of the parasite between individuals. An effective vaccine to reduce the clinical effetcts of malaria would be directed to the blood- stage of the parasite. The M5 antigen (Sjölander et al., 1993a) used in paper I, II and III is derived from an immunodominant part of the P. falciparum ring-infected erythrocyte surface antigen Pf155/RESA (Perlman et al., 1984). EB200, used in paper IV, is derived from Pf332, a highly repetitive, late asexual intraerythrocytic protein, expressed on erythrocytes (Mattei et al., 1992; Mattei and Scherf, 1992; Hinterberg et al., 1994).

39 CHRISTIN ANDERSSON

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

Study Plasmid Fusion protein Mw Expression Solubility* Incorporation (kDa) level (mg/l efficiency cell culture) 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 pMLHABPDSAG1 lpp-His6-ABP-DSAG1 42.4 7 insoluble high

PAff8DSAG1 His6-ABP-DSAG1 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.

40 RECOMBINANT SUBUNIT VACCINES

A: IW-Z-M5 B: MI-Z-M5 C: PD-Z-M5

0.45 0.45 0.5 0.45 0.95 1.4 0.4 0.45 0.85 1.2 0.4 0.35 0.35 0.75 0.4 1 0.35 0.3 0.65 0.25 0.35 0.25 0.8 0.3 0.55 0.3 0.2 0.6 0.45 0.25 0.15 0.15 0.25 0.4 0.1 0.35 0.2 0.05 0.2 0.05 0.25 0.2

0.15 0.15 0 0.15 0 1 5 10 15 1 5 10 15 1 5 10 15 D: ABP-M5-M E: ABP-M5-MI

1 0.5 0.85 0.55

0.8 0.5 0.4 0.8 0.75 0.45 0.7 0.3 0.4 0.65 0.6 0.35 0.6 0.2 0.3 0.55 0.4 0.25 0.1 0.5 0.45 0.2 0.2 0 0.4 0.15 1 5 10 14 1 5 10 14

F: ZZ-M5-M G: ZZ-M5-MI

0.9 0.6 0.7 0.6

0.8 0.5 0.6 0.5 0.7 0.4 0.4 0.6 0.5 0.3 0.3 0.5 0.4 0.2 0.2 0.4 0.3 0.3 0.1 0.1

0.2 0 0.2 0 1 5 10 14 1 5 10 14

H: lpp-His6-ABP- SAG1 I: His6-ABP- SAG1 J: His6-ABP- SAG1

0.5 0.4 0.45 0.5 0.6 0.4

0.45 0.4 0.4 0.5 0.3 0.3 0.4 0.35 0.3 0.4 0.35 0.2 0.3 0.2 0.2 0.3 0.25 0.3 0.1 0.1 0.1 0.25 0.2 0.2 0.2 0 0.15 0 0 1 5 10 15 1 5 10 15 1 5 10 15 Figure 2A-J: Analysis of the protein content in the fractions collected from sucrose gradient ultracentrifugation of iscom preparations performed in paper I, II and III. The respective fusion protein is shown above each graph. Total protein content is shown on the left y-axis ( ) and the results from an ELISA, specific for the affinity tag used in each study (Z, ZZ or ABP, respectively) is shown on the right y-axis ( ). The fraction number is indicated on the x-axis. The horizontal bar in each graph indicates the cholesterol-rich fractions. Figure 2J shows the results from a control experiment with protein, iscom matrix but without the chelating lipid.

41 CHRISTIN ANDERSSON

While the fusion proteins IW-Z-M5 and MI-Z-M5 formed aggregates during expression, the protein PD-Z-M5 was found to be soluble upon expression in E. coli. The results are summarized in Table 10. Furthermore, the iscom formation was supported by electron microsopy inspection of the cholesterol-rich fractions from iscom preparation. The iscom preparations of the proteins IW-Z-M5 and MI-Z-M5 contained typical iscom structures (Figure 3), while iscom preparations of the protein PD-Z-M5, in contrast, contained no iscom structures.

To evaluate the immunogenic properties of the iscom preparations, immunization studies were performed in mice. Sera from mice immunized with the IW-Z-M5 and MI-Z-M5 iscom preparations, respectively, contained M5-specific IgG-titers of approximately 1:1000 (mean) after the first immunization. After the booster immunization, the M5-specific antibody levels increased 10-fold.

Figure 3: A typical iscom structure, as evaluated by electron microscopy analysis.

42 RECOMBINANT SUBUNIT VACCINES

6.1.2. C-terminal hydrophobic tags (II) In an effort to improve the system for hydrophobic tagging of immunogens for efficient iscom incorporation, new, improved general expression vectors were designed. The major drawback with the previously presented system (I) was the overall low production levels of the proteins. It has been suggested that translation initiation sequences containing hydrophobic amino acids will decrease production levels (Makrides, 1996) and in order to increase the production levels of the fusion proteins, the hydrophobic tag was instead placed in the C-terminus of the fusion proteins (II). As hydrophobic tags, the most successful hydrophobic tag from the previous study, the membrane-integration MI tag from influenza hemagglutinin (Feldmann et al., 1988) was included (Table 9). The second tag used in this study, denoted M, is derived from the membrane-spanning region of Staphylococcus aureus protein A (SpA) (Navarre and Schneewind, 1994; Schneewind et al., 1995) (Table 9). The rational for selecting the latter was that a hydrophobic tag of bacterial origin might be more efficiently produced in E. coli. To make the affinity purification process more efficient, the divalent ZZ tag was utilized as affinity tag, since it has been shown (Nilsson et al., 1994), that ZZ has a ten times higher affinity for its ligand IgG, than the monovalent Z domain. The ZZ domains are also highly soluble, and it has been shown in other studies that ZZ as fusion partner can increase the overall solubility of fusion proteins (Samuelsson et al., 1991 and 1994). This might be of importance to minimize the formation of unwanted hydrophobic aggregates during the purification and iscom incorporation processes. As an alternative affinity tag, the serum albumin-binding protein ABP (Larsson et al., 1996), derived from streptococcal protein G (Nygren et al., 1988), was included. The ABP tag is also known to be highly soluble, and binds human serum albumin (HSA), which makes it suitable as tag for affinity purification procedures (Ståhl et al., 1999). Four new expression vectors were thus constructed pT7ABPM, pT7ABPMI, pT7ZZM and pT7ZZMI, and a gene fragment encoding the same model immunogen M5 as in the previous study was introduced into the vectors. The four encoded fusion proteins ABP-M5-M, 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

43 CHRISTIN ANDERSSON cell culture for the four fusion proteins, ABP-M5-M, ABP-M5-MI, ZZ-M5-M and ZZ-M5-MI (Table 10). Iscom incorporation experiments of the fusion proteins ABP-M5-M, ABP-M5-MI, ZZ-M5-M and ZZ-M5-MI, respectively, were performed as described above. Again, the iscom preparations were subjected to a sucrose gradient centrifugation. Collected fractions were analyzed for cholesterol content as described above, and for (i) total protein content according to Bradford (Bradford, 1976) and, (ii) the presence of the affinity tags in ABP-or ZZ-specific ELISA experiments. The results are summarized in Figure 2D-G. As discussed earlier, comigration of protein and cholesterol indicates successful iscom association of the protein. All four proteins ABP-M5-M, ABP-M5-MI, ZZ-M5-M and 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.

44 RECOMBINANT SUBUNIT VACCINES

6.1.3. Lipid tagging of protein immunogens (III) As an alternative to using hydrophobic amino acid residues as hydrophobic tags, a strategy based on lipid tags was investigated. In order to develop a general system for production of lipid-tagged immunogens, a novel expression vector, pMLHABP, was constructed. The expression is under the control of the tac promoter (Amann et al., 1988). The vector encodes the 20 amino acid signal peptide and the nine N-terminal amino acid residues (lpp) of the major lipoprotein of E. coli and this sequence is found to be sufficient for lipidation (Ghrayeb and Inouye, 1984; Laukkanen et al., 1993). A dual affinity tag, consisting of a hexahistidyl

(His6) peptide and the serum albumin binding protein ABP, was used. Introduction of the

His6-ABP dual affinity tag would offer alternative strategies for affinity purification of expressed fusion proteins, by either immobilized metal ion affinity chromatography (IMAC) (Hochuli et al., 1988) or HSA-Sepharose columns (Ståhl et al., 1999). To make comparison possible, an in vitro lipidation strategy was also investigated. This strategy utilizes a metal-chelating lipid, which could be linked to hexahistidyl peptides and would thus not depend on the E. coli-lipidation machinery. To produce recombinant immunogens for in vitro lipidation via a chelating peptide, the expression vector pAff8c

(Larsson et al., 2000) was used. This vector encodes the same dual affinity tag, His6-ABP, as pMLHABP. The expression is under control of the T7 promoter (Studier et al., 1990). As model immunogen in both systems, a 238 amino acid immunogen DSAG1, 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, pMLHABPDSAG1 and pAff8DSAG1, thus encode the fusion proteins, lpp-His6-ABP-DSAG1 and His6-ABP-DSAG1, respectively (Table 10). The fusion proteins were evaluated for in vivo and in vitro lipidation experiments and subsequently also in iscom incorporation experiments.

45 CHRISTIN ANDERSSON

TOXOPLASMA Cat Other mammals and birds

Intestinal Gastro-intestinal Intestinal epithelium tract epithelium Ingestion

Oocyst Oocyst Oocyst

Replication Sporozoites

Tachysoites

Tissue cyst Tissue cyst Replication

Bradyzoites Bradyzoites

Ingestion

Figure B: The life cycle of the Toxoplasma gondii parasite in cats and other mammals, including humans.

Toxoplasmosis is caused by Toxoplasma gondii, an obligate intracellular parasite that can infect a wide range of animals, including humans. The parasite has a two-phase lifecycle: a sexual phase, which occurs in cats and other felides, and an intermediate, non-sexual phase, which can take place in any mammal or bird. The intermediate host can be infected by ingesting oocysts. The oocysts contain sporozoites that are released in the gut, penetrate the intestinal epithelia and start to replicate. After about two weeks, the proliferation is slowed down and tissue cysts, containing bradyzoites, are formed. Tissue cysts are mainly localized in muscle tissue and the central nervous system, and can be viable for a long time. If an infected animal is eaten by another mammal or bird, the tissue cyst is destroyed by digestive enzymes, bradyzoites are released, penetrate the epithelia ands start to replicate. If the new host is a cat, the parasite develops into sexual stages, and after about 3-5 days, new oocysts are excreted in the faeces, completing the lifecycle. T. gondii parasite is widely distributed around the world. In veterinary medicine, toxoplasma infection is well known as a cause of abortion and neonatal mortality in sheep and goat. Humans can aquire the infection from food contaminated with oocysts or meat containing tissue cysts. The infection is usually without symptoms. However, if aquired during pregnancy, the result may be miscarriage or malformation of the child (Remington, 1990). Toxoplasma infection in immunocompromised individuals, including AIDS patients, may cause a life-threatening disease (Araujo and Remington, 1987; Israelski and Remington, 1988). In the search for a vaccine against toxoplasmosis, live attenuated, killed or lysed parasites or different antigenic fraction has been used (Hermentin and Aspöck, 1988; Johnson, 1989). For example, immunization of mice with the major surface antigen (SAG-1) of tachyzoites incorporated into liposomes or mixed with Quil A resulted in protection against lethal infection (Bülow and Boothroyd, 1991; Khan et al., 1991). It has also been shown that surface antigens (SAG-1, SAG-2 and others) incorporated into iscoms elicited protective immunity in mice (Lundén et al., 1993) and both humoral and cellular immune response in sheep (Lundén et al., 1995). The severe effects of toxoplasma infection in animals and in humans are well known. Therefore there is a great need for effective vaccines, both for veterinary and human use.

46 RECOMBINANT SUBUNIT VACCINES

Both fusion proteins were expressed in E. coli, and could be recovered by affinity purification using ABP-mediated affinity chromatography on HSA columns (Ståhl et al., 1999; Ståhl et al., 1989). The fusion protein lpp-His6-ABP-DSAG1, 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-DSAG1, 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-DSAG1, 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-DSAG1, intended to be 2+ incorporated via interaction of the His6 tag with a Cu -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-DSAG1 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-

DSAG, as wells as the in vitro lipidated His6-ABP-DSAG1 was found in the cholesterol-rich fractions of the sucrose gradient, indicating association with iscoms. For the non-lipidated

His6-ABP-DSAG1 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-DSAG1 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- DSAG1, simply admixed with iscom matrix lacking the chelating lipid. This was surprising since the fusion protein was not found to a significant extent in the cholesterol-rich iscom

47 CHRISTIN ANDERSSON fractions after sucrose gradient ultracentrifugation (Figure 2). The results from another control immunization, consisting of mice immunized with the His6-ABP-DSAG1 fusion protein in

PBS, showed also significant titers of His6-ABP-DSAG1 reactive antibodies after a booster immunization. The results from the control groups indicate that the DSAG1 immunogen is very immunogenic in itself. An earlier study showed that poorly immunogenic hydrophilic antigens normally fail to elicit antibody responses when administered mixed with iscom matrix or with PBS alone (II).

Taken together, both the concepts of including hydrophobic amino acid sequences into recombinant immunogens (I, II), and the presented strategies to obtain in vivo and in vitro lipidation of recombinant immunogens result in association with the iscoms. Comparing the two lipidation strategies, one advantage of using the in vivo lipidation strategy is that only the typical iscom forming material is needed, while the in vitro strategy requires the addition of a chelating lipid. On the other hand, the expression levels obtained with the in vitro strategy are much higher and in addition, this strategy offers a much simpler method for iscom formation since iscom matrix with pre-associated chelating lipid can be prepared in bulk. The general applicability of the presented strategies needs to be further evaluated for various immunogens.

6.2. Recovery of a vaccine candidate produced in mammalian cells (IV)

A promising vaccine candidate to human respiratory syncytial virus (RSV) (Figure C) produced in E. coli has been demonstrated to induce protection to virus challenge in rodent models (Power et al., 1997) and has now progressed into phase III human clinical trials (http://www.cipf.com). The candidate vaccine, BBG2Na consists of a serum albumin binding region BB derived from streptococcal protein G (SpG) (Nygren et al., 1988) and amino acids 130-230 of the G glycoprotein of RSV subgroup A (Power et al., 1997). The immunogenic properties of the E. coli-produced and non-glycosylated BBG2Na is interesting considering that the native RSV G protein is heavily glycosylated (Collins et al., 1996). Therefore, a mammalian expression system for production of glycosylated BBG2Na was created in order to enable comparative immunological studies of non-glycosylated and glycosylated BBG2Na.

Expression vectors based on the positive-strand RNA alphavirus Semliki Forest virus (SFV), was used to achieve transient expression of BBG2Na in baby hamster kidney cells.

48 RECOMBINANT SUBUNIT VACCINES

RESPIRATORY SYNCYTIAL VIRUS

Surface proteins F G SH

Nucleocapsid-associated proteins N P L

Matrix protein M M2

Nonstructural proteins NS1 NS2

Figure C: An overview of the respiratory syncytial virus. The surface proteins are outlined in the picture.

Respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract infection during infancy and early childhood worldwide. RSV was first isolated in 1956 and has since then been associated with lower tract illness during infancy and childhood in many parts of the world (Collins et al, 1996). It has also been found that RSV infection is an important agent of disease in immuno-suppressed adults and in elderly peoples (Collins et al., 1996). Outbreaks of RSV disease are abrupt and can last up to 5 months, and usually peaks in winter. There are two major strains, A and B, both of them circulate simultaneously during outbreak, and it has been suggested that group A strains are associated with more severe disease (Domachowske and Rosenberg, 1999). RSV is an enveloped, negative-sense RNA virus with a genome of about 15 kb encoding 11 viral proteins (Collins et al., 1996). The virus particle is about 60 to 100 nm in diameter and consists of a nucleocapsid surrounded by a lipid envelope with two major surface proteins, F and G. The F protein is thought to mediate virus penetration while the RSV G protein is thought to mediate virus attachment to cells (Peroulis, 1999; Collins et al., 1996). Both F and G protein induce neutralizing antibodies (Dudas et al., 1998). It has been demonstrated that monoclonal antibodies to G protein inhibit virus entry into cell. (Karron et al., 1997). Studies have shown that the G glycoprotein is a major viral antigen and the glycosylated G protein is protective upon immunization in several animal models (Power et al, 1997). It has been suggested that the glycosylation protect the virus from the host immune system (Collins et al., 1996). A vaccine candidate BBG2Na is a fusion protein consisting of an albumin-binding region from streptococcal protein G (BB) and RSV G protein fragment. (G2N) has been extensively studied. Studies with BBG2Na in animal models, mice and cotton rats, has shown that high immune response and also protection to viral challenge can be obtained when immunizing with a nonglycosylated fragment of the G protein. Administering this E. coli produced fusion protein, BBG2Na, to mice resulted in a strong immune response of these mice and subsequent protection from RSV infections. Lungs and nasal tracts of immunized mice were efficiently protected to subsequent RSV challenges (Power et al., 1997). Maternal immunization induces protection of offspring of test animals, and passive transfer of the maternal antibodies was sufficient to induce a high protective immune response. (Brandt et al., 1997; Siegrist et al, 1999).

Still there is no vaccine for RSV although the virus and the severe effects of infection are well known.

49 CHRISTIN ANDERSSON

Such vectors have been used to express heterologous proteins in a wide range of host cells, i.e. insect, avian and mammalian cells (Liljeström, 1994; Liljeström and Garoff, 1991). For example, biologically active full-length RSV G protein was successfully expressed using an SFV-based vector (Peroulis et al., 1999).

These SFV vectors were furnished with SP6 phage-derived RNA promoter to enable in vitro transcription of RNA, and the gene encoding BBG2Na is introduced in the position where the genes encoding the SFV structural proteins normally reside (Liljeström, 1994; Liljeström and Garoff, 1991). The positive-strand RNA from such vectors encodes its own RNA-RNA replicase, responsible for amplificaiton of RNA in vivo. Two different vectors encoding BBG2Na were created, both utilizing the SFV self-amplification system. The first vector, pSFV3-l-BBG2Na encodes a cytoplasmic version of BBG2Na, denoted BBG2Nacyt, and the second vector, pSFV3-spl-BBG2Na, were a signal peptide (sp) (Berglund et al., 1997) was included upstream of the BBG2Na gene, encodes a secreted version of BBG2Na, denoted

BBG2Nasec. RNA was transcribed in vitro from both vectors and transfection of BHK-21 cells was performed by electroporation with the RNA constructs encoding BBG2Nacyt and

BBG2Nasec, respectively. Western blot analysis of culture supernatants and cell lysates from cells expressing the two variants of BBG2Na, was performed. BB-specific antibodies could detect 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

50 RECOMBINANT SUBUNIT VACCINES

showed that only very low amounts of BBG2Nasec could be recovered. The reason for this is most probably due to the presence of BSA in the culture medium (originating from the fetal calf serum (FCS)). The BSA will then compete with HSA for the binding to the BB-tag since the BB-tag has a lower but still significant affinity for BSA, as compared to HSA (Nygren et al., 1990). The purification strategy is schematically shown in Figure 4.

In order to improve the recovery, a batch-wise HSA affinity recovery procedure was investigated, and by this strategy, a certain amount of BBG2Nasec could indeed be recovered

(approximately 4 mg 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 mg per 10 ml culture supernatant).

HSA-purification Affibody mediated purification

BSA BSA ZRSV1 HSA BB G2N BB G2N ZRSV1

BSA

BB G2N

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

51 CHRISTIN ANDERSSON

Table 11: Protein production characteristics of BBG2Nasec

Production/affinity purification mode Recovered BBG2Naseca Puritya (%) (µg/10 ml)

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 instead of column.

The purified material also contained copurified BSA, probably remaining from the initial cell cultivation. These results indicate that the yields and purity of BBG2Nasec purified using the HSA affinity procedures were insufficient (Table 11).

To circumvent the problems of BSA competition, a column with a product-specific affinity ligand, which is specific for the RSV G protein part of BBG2Nasec, was created. The ligand, a recently described RSV G protein-specific binding molecule, ZRSV1 affibody (Hansson et al., 1999) was created through phage display-mediated selection from a combinatorial library of the Z protein domain derived from SpA as described above (see section 2.2.5). ZRSV1 binds amino acids 164-186 of the RSV G protein of both E. coli-produced BBG2Na and glycosylated RSV G protein from whole virus (Hansson et al., 1999). Dimeric versions of affibodies have in certain cases been shown to have an apparent higher affinity (Gunneriusson et al., 1999) and therefore a dimeric construct of the ZRSV1 was created resulting in the fusion protein ZZ-diZRSV1.

The affibody fusion protein ZZ-diZRSV1 was coupled as ligand to an activated Sepharose column to create a product-specific affinity column. Purification of culture supernatant from

BBG2Nasec-expressing BHK-21 cells showed that full-length BBG2Nasec could be recovered (6 mg 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

52 RECOMBINANT SUBUNIT VACCINES

approximately 2 mg 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).

53 CHRISTIN ANDERSSON

A

SFV-Repl. BBG2Na SFV-Repl. sp BBG2Na

pCMV pCMV pBBG2Nacyt pBBG2Nasec

Neo Neo Ori Ori B cap (A) cap SFV-Repl. BBG2Na n SFV-Repl. sp BBG2Na (A)n

C BBG2Nacyt BBG2Na BBG2Nasec sp BBG2Na

38.2 kDa 38.3 kDa

Figure 5: Plasmid vectors, transcription products and encoded fusion proteins. (A) The two expression vecors, pBBG2Nacyt and pBBG2Nasec, encoding the SFV replicase (enabling in vivo amplification) and the cytoplasmic and secreted variants of BBG2Na, respectively. The transcription is under control of the CMV immediate early promoter (pCMV). (B) The positive strand RNAs, transcribed by the transfected cells, will be self-amplified in the cytoplasm of transfected cells, since they encode the SFV replicase responsible for the RNA-dependent RNA- replication. (C) The encoded gene products from the plasmid vectors, BBG2Nacyt and pBBG2Nasec, the latter product furnished with a signal peptide (sp) for direction to the secretion machinery. The indicated theoretical molecular masses are after processing of the signal peptide are indicated.

To investigate the functionality of the plasmids, BHK-21 cells were transfected with the two plasmids, respectively. Analysis of culture supernatants and cell lysates by Western blot demonstrated that BBG2Nacyt was detected only in the cell lysate, while BBG2Nasec was found both in the culture supernatant and the cell lysate. Further expression studies were performed using immunofluorescence microscopy analysis. The same difference in localization patterns for BBG2Nacyt and BBG2Nasec as described in earlier studies (IV) was shown. The BBG2Nacyt was detected in the cytoplasm while BBG2Nasec was detected in a network-like pattern in the cytoplasm adjacent to the nucleus, a staining pattern typical of endoplasmic reticulum and golgi complex localization (Kamrud et al., 1998). The results from

54 RECOMBINANT SUBUNIT VACCINES the expression studies suggest that the plasmids are functional in terms of BBG2Na expression and localization of the encoded proteins.

In order to investigate the potential of the plasmids as vaccines, mice were immunized intramuscularly with the plasmids pBBG2Nacyt, pBBG2Nasec or a control plasmid, pControl (encoding only the SFV replicase and E. coli b-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.

a b Table 12: Lung protective efficacy to virus challenge expressed as virus titers (log10) after

intramuscular immunization of mice with plasmids, pBBG2Nacyt , pBBG2Nasec or pControl and challenge with 105 TCID50 RSV-A. Immunization group Mouse number Mouse protected /total 1 2 3 4 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 (underlined b figures) when virus titers were reduced by 2 log10 relative to the mice immunized with the control strain. Viral titers for the challenged mice are expressed as log10 pfu/g of lung tissue, 5 days post challenge. The detection limit for the assay is 101.45.

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

55 CHRISTIN ANDERSSON

Western blot analysis indicates that BBG2Nasec was expressed at somewhat higher levels, but more extensive studies would be required to fully assess the differences in expression levels. An additional possible explanation of the differences in immunogenicity could be that the presentation of BBG2Na to the immune system could be inefficient. The SFV replicase has been shown to induces apoptosis in cells (Glasgow et al., 1998) and others have suggested that apoptotic cells are poor antigen presenters (Barker et al., 1999). Therefore, BBG2Nacyt, expressed inside such cells could be poorly immunogenic, while the secreted BBG2Na encounter direct contact with antigen presenting cells and might therefore be efficiently presented to the immune system. However, the reasons for the different immunogenicity of the two BBG2Na variants have to be further investigated.

In this study, we demonstrate that protection to RSV challenge can be elicited in mice by immunizing with DNA encoding a subunit RSV vaccine candidate, BBG2Na, at present evaluated in human clinical trials as a prokaryotically produced protein immunogen. The results also indicate that targeting of the antigens expressed by nucleic acid vaccination could have important effects.

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

In order to evaluate the differences in immune responses elicited by the different 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

56 RECOMBINANT SUBUNIT VACCINES

A SFV Replicase sp EB200

pSP6 RNA Ori E pSFV3-spl-EB200 cap SFV Replicase sp EB200 A(n) bla B SFV Replicase EB200 SFV Replicase sp EB200

pCMV pCMV pBK-SFV3L2-EB200 pBK-SFV3L2-spEB200

Neo Neo Ori E Ori E

C HCMV int SV40 int p(A)

StPA EB200

HCMV pCMVintBL-EB200 IE1 enh/pr Bla Ori E SV40 Ori

Figure 6: Schematic representation of the plasmids used for in vitro transcription or immunization. (A) The vector pSFV3-spl-EB200 encoding the SFV replicase (enabling in vivo amplification) and the secreted variant of EB200. The transcription is under control of the phage SP6 RNA polymerase promoter (pSP6). The positive strand RNA, suitable for transfection of mammalian cells, are transcribed and capped in vitro, and carry 3' 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 transcription is 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 sequence precede the gene fragment to be expressed (EB200). The vector contains the simian virus 40 (SV40) origin of replication, intron and polyadenylation signals from the SV40 T-antigen gene.

V), and has been shown functional in terms of directing the product to the secretion

machinery. The encoded product was denoted EB200sec. Transcription was under control of the SP6 RNA polymerase promoter, thus enabling in vitro transcription of RNA (Liljeström and Garoff 1991) as described before (IV).

57 CHRISTIN ANDERSSON

Plasmid vectors for DNA immunization, pBK-SFV3L2-EB200 and pBK-SFV3L2-spEB200, encoding the SFV replicase and two variants of the antigen EB200; one cytoplasmic variant denoted EB200cyt, and one secreted variant denoted EB200sec, respectively, were also constructed. Again, the signal peptide would enable secretion of the encoded product,

EB200sec. Transcription in both plasmids is under control of the CMV immediate early promoter, commonly used in plasmid DNA vaccine vectors due to efficient transcription. Another plasmid, pCMVintBL-EB200, lacking the SFV replicase was also constructed, in which the gene encoding EB200 is preceded by the tissue plasminogen activator tPA signal sequence for production of a secreted variant of EB200. In this plasmid, the number of RNA copies transcribed would only be the result of transcription controlled by the CMV promoter.

To perform the immunizations using RNA in viral particles, in vitro transcription of RNA encoding the P. falciparum-derived antigen EB200 or a control antigen BBG2Na (IV, V) was performed. These RNA-constructs were also prepared in a virus-coated fashion by packaging into into recombinant SFV (rSFV) particles using a two-helper RNA system (Smerdou et al., 1999).

Mice were immunized intramuscularly with the different RNA and DNA constructs, see Table 13, and antibody responses were monitored.

Table 13: Immunization groups used in study VI Group Molecule used in Plasmid Encoded protein immunizations a A RNA pSFV3-spl-EB200 EB200sec

a B RNA pSFV3-spl-BBG2Na 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

b H rSFV particles pSFV3-spl-EB200 EB200sec

b I rSFV particles pSFV3-spl-BBG2Na BBG2Nasec

J rSFV particles pSFV3-lacZb b-galactosidase

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

58 RECOMBINANT SUBUNIT VACCINES

All three groups of mice immunized with plasmids encoding EB200 (group C, D and E) developed GST-EB200 reactive antibodies. Only background levels of antibody reactivity to GST were found, suggesting that the major part of the reactivity of these sera was directed against EB200. Unexpectedly, both groups and of mice immunized with SFV-RNA (group A and B), encoding EB200sec or control antigen BBG2Nasec, developed high and similar levels of antibody reactivity to GST-EB200. However, most of this reactivity appeared not to be directed against EB200, as both these groups showed an even higher reactivity with GST. The reason for the induction of these apparently non-specific antibodies is not known. Importantly, the RNA packaged in SFV suicide particles (group H and I) did not elicit this kind of antibody reactivity.

The antibody responses obtained in this study were relatively low, surprisingly with the highest responses obtained with the conventional DNA plasmid (group E). However, immunizations with plasmids (group C, D and E) as well as with rSFV particles (group H) induced an efficient immunological memory response, as demonstrated by the efficient boosting of the anti-EB200 antibody response with recombinant EB200 protein. The efficient priming of immune responses by naked DNA immunizations has been observed previously when boosting with the corresponding protein (Haddad et al., 1999) or with recombinant vaccinia virus expressing the protein (Schneider et al., 1998; Sedegah et al., 1998). Our low responses to EB200 with the SFV based vectors appear to be due to the inherent properties of the antigen, as our contol vectors coding for the protein BBG2Na (group I) or LacZ gave high levels of antibodies to the respective protein.

In conclusion, we have in this study shown that DNA plasmids expressing the gene for the EB200 fragment of the P. falciparum vaccine candidate antigen Pf332 induce an antibody response to the antigen in mice. A conventional plasmid was more efficient than SFV-based plasmids in inducing antibodies to EB200. Also recombinant SFV suicide particles expressing EB200 induced such antibodies, although at lower levels. Importantly, all these immunogens induced an immunological memory, which could be efficiently activated by a booster injection with EB200 protein.

59 CHRISTIN ANDERSSON

8. Concluding remarks

Recombinant strategies are today common for the development of novel subunit vaccines. In this thesis, recombinant methods have been employed to enable studies regarding; (i) the generation of general expression systems for production of recombinant subunit immunogens furnished with a hydrophobic tag, either hydrophobic amino acids or a lipid, allowing association to iscoms, (ii) the development of a mammalian cell production and recovery system for an RSV vaccine candidate, and (iii) construction and evaluation of nucleic acid immunization strategies for RSV and malaria.

Robust systems for E. coli production of protein immunogens were created, in which hydrophobic tags were fused to the target immunogen. This would enable direct association of the produced immunogen to an adjuvant system, e.g. iscoms. Initially, three different hydrophobic tags were evaluated, as fused N-terminally. A malaria peptide, M5, was used as model antigen. The produced fusion proteins could be efficiently recovered by the incorporation of also an affinity tag, the SpA-derived Z-domain, in the expression vectors, and for two hydrophobic tags out of three, efficient association to iscoms was demonstrated.

Since the expression levels were rather low from the first generation expression vectors, a second set of expression vectors were designed and constructed. In these vectors, one of the hydrophobic tags was included that were found to be functional in the previous study, i.e. MI, representing the membrane-integration region of influenza virus hemagglutinin. An additional tag, M, representing the membrane-spanning region of staphylococcal protein A, was also included in the study. In these vectors the hydrophobic tags were placed C-terminally, in order not to disturb the transcription and translation initiation, to thus potentially improve expression levels. The SpA-derived ZZ tag and the SpG-derived ABP tags were included in the vectors to allow affinity purification, and the M5 malaria peptide served as model immunogen. The second generation expression vectors were indeed found to increase expression levels ten to fifty-fold, and the affinity-purified hydrophobically tagged immunogens could efficiently be incorporated into iscoms.

A third study was performed in which lipid tagging of the recombinant immunogens was evaluated. First, a system which utilized the E. coli lipidation system was designed. The second system exploited a chelating lipid that was known to interact with hexahistidyl tags,

60 RECOMBINANT SUBUNIT VACCINES

and the recombinant immunogen was thus produced as fused to a His6 peptide. In both systems the SpG-derived ABP tag was included for affinity purification purposes. A 238 amino acid segment DSAG1 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

61 CHRISTIN ANDERSSON

BBG2Nasec, exceeded 90%, and the product could not be detected in the flow-through fractions, indicating quantitative recovery. Comparative studies of the E. coli produced BBG2Na and mammalian cell produced BBG2Na, are presently being conducted.

In an additional investigation, plasmid vectors for DNA immunization against RSV was constructed and evaluated for their efficacy. Two different vectors were constructed encoding variants of the RSV candidate vaccine BBG2Na; one designed for cytoplasmic localization

(BBG2Nacyt) and one for targeting to the secretion machinery (BBG2Nasec) of the transfected cells. The two plasmid vectors, pBBG2Nacyt and 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

62 RECOMBINANT SUBUNIT VACCINES apparently non-specific sticky antibodies is not known. Importantly, the same RNA when packaged in SFV suicide particles did not induce this kind of antibody reactivity. In conclusion, we have in this study shown that DNA plasmids expressing the gene for the EB200 fragment of the P. falciparum vaccine candidate antigen Pf332 induce an antibody response to the antigen in mice. A conventional plasmid was in our hands more efficient than SFV-based plasmids in inducing antibodies to EB200. Also recombinant SFV suicide particles expressing EB200 induced such antibodies, although at lower levels. Importantly, all these immunogens induced an immunological memory, which could be efficiently activated by a booster injection with EB200 protein.

Taken together, it is evident that recombinant DNA technology will have a major impact on future strategies for the prevention of infectious diseases. The design, production and delivery of recombinant subunit vaccines will be the basis of modern vaccinology. The studies summarized in this thesis demonstrate that new methods will be available for the production and recovery of protein subunit immunogens, and that these can be further adapted by genetic design of the expression vectors for incorporation into adjuvant systems. In addition, various strategies for delivery of subunit vaccines by RNA or DNA immunization will be available for the development of future vaccines.

63 CHRISTIN ANDERSSON

Abbreviations

ABD albumin binding domain derived from streptococcal protein G ABP albumin binding protein derived from streptococcal protein G ALVAC a recombinant canarypox virus APC antigen presenting cell B7 a B-cell ligand BB albumin binding region derived from streptococcal protein G BHK baby hamster kidney cells BSA bovine serum albumin CHO chinese hamster ovary cells CMV cytomegalovirus CpG a cytosine-guanine dinucleotide, p indicates the phosphate bond CS circumsporozoite CTB cholera toxin B subunit CTL cytotoxic T lymphocyte EB200 a malaria antigen fragment, derived from Pf332 ELISA enzyme-linked immunosorbent assay ETEC enterotoxic E. coli FCS foetal calf serum FIA Freunds incomplete adjuvant G2Na a part of the respiratory syncytial virus G protein GM-CSF granulocyte-macrophage colony stimulating factor GST glutathione-S-transferase HA hemagglutinin HEK human embryonic kidney cells HBsAg Hepatitis B surface antigen Hib Haemophilus influenzae type B His6 hexahistidyl tag HIV human immunodeficiency virus HLA human leukocyte antigen HSA human serum albumin Ig immunoglobulin IFN interferon IL interleukin Iscoms immunostimulating complexes ISS immunostimulatory sequences IW hydrophobic amino acid sequence (isoleucines and trypophanes) kDa kiloDalton LPS lipopolysaccharides LTB E. coli heat-labile toxin B subunit M5 a malaria peptide derived from the central region of Pf155/RESA M membrane-spanning region of SpA MAP multiple antigenic peptides MHC major histocompatibility complex MI membrane-spanning region of hemagglutinin A from influenza virus

64 RECOMBINANT SUBUNIT VACCINES

MPL monophosporyl lipid A MVA modified vaccinia virus Ankara strain NP nucleoprotein NYVAC attenuated vaccinia virus strain PAGE polyacrylamide gel electrophoresis PD hydrophobic tag, designed to be pH-dependent PCR polymerase chain reaction PLG poly (lactide-co-glycosides) QS-21 a fraction of Quil A Quil A a saponin derived from Quillaja saponaria RSV respiratory syncytial virus SAG1 surface antigen 1 from Toxoplasma gondii SFV Semliki Forest virus SDS sodium dodecyl sulphate SpA staphylococcal protein A SpG streptococcal protein G SV40 simian virus 40 tPA tissue plasminogen activator VLP virus-like particles VP viral protein XM cell-surface-anchoring region of staphylococcal protein A ZZ IgG-binding domains derived from staphylococcal protein A

65 CHRISTIN ANDERSSON

Acknowledgements

First of all I would like to thank my supervisor, Prof. Stefan Ståhl. You have, in good and bad times, always been supportive and encouraging and somehow always incredibly optimistic. Thank you for helping me finish this thieis. I would also like to thank Prof. Mathias Uhlén for leading the DNA corner group in an inspiring way and for being such a visionary person.

My collaboration partners have contributed a great deal to the work presented in this thesis. I would like to send my gratitude to: Karin Lövgren-Bengtsson at SLU in Uppsala for introducing me to the world of iscoms. Anna Lundén at SVA in Uppsala for collaboration in the SAG-1 iscom project and all help with the thesis. Klavs Berzins, Diana Haddad, Niklas Ahlborg and Nina-Maria Vasconcelos at Stockholms University for collaboration in the EB200 project. Ultan Power, CIPF, for all collaboration in the RSV project. Peter Liljeström and Peter Berglund, MTC, Karolinska Institute, for all help in the SFV projects. Sissela Liljeqvist, for completing the EB200-story.

I want to thank all colleagues at the department of Biotechnology at KTH, over the years, for creating a friendly and stimulating working atmosphere. During my years at the department, I have really enjoyed all theatre evenings, dinners, travels (Paris, Sandhamn, Gotland, Berlin, Väddö), Laser Dome battles, Christmas parties, fredagsöl and fun company during lunches and coffee breaks. I am going to miss you all. There are some persons I would like to give special thanks to:

Maria Murby, my exjobbs-supervisor for introducing me to the lab (together with Sissela).

Monica and Pia for taking care of all administrative matters, and Monica also for simultaneously keeping track of Mathias.

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

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

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

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

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

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

66 RECOMBINANT SUBUNIT VACCINES

Thank you all my friends for being there for me. Tove, Jenny, Malin, Nina, Anna W, Malin C and Sonja, for dinners, midsummer at Muskö, Advents-dinners with singing, and much more. I would especially like to thank, Henrik, my END-Note hero, for everything, Jenny O, for dinners and mysterious parties, Janne, for being the happiest person I know, and for showing me Åbo, Brita, for sailing trips, hours on the phone or at some café, and Ulla, my best friend for 28 years, for being the person you are and for supporting me (See you in Rome!).

So at last, my beloved family. Moster Annica, for letting me live with you for my first month in Stockholm, and for showing me how "tunnelbanan" works. Fredrik Skeppstedt for computer assistance and for taking care of my sister. Pernilla, for all romantic "kits", and for friendship, and Victor for being a darling.

My brothers Anders, Lars and Fredrik and my sister Sara for all fun we have shared during all these years, for being loving, and always supporting me and believing in me.

Tack Mor och Far för att ni lärt mig att tro på mig själv och att våga gå min egen väg.

This work was supported by grants from by Pierre Fabre Médicaments, France.

67 CHRISTIN ANDERSSON

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