christin andersson
TRANSCRIPT
Production and delivery of recombinant
subunit vaccines
Christin Andersson
Royal Institute of Technology
Department of Biotechnology
Stockholm 2000
CHRISTIN ANDERSSON
Christin Andersson
Department of BiotechnologyRoyal Institute of Technology (KTH)SE-100 44 StockholmSweden
Printed at Högskoletryckeriet KTH, Stockholm 2000
ISBN 91-7170-633-X
RECOMBINANT SUBUNIT VACCINES
Christin Andersson (2000) Production and delivery of recombinant subunit vaccinesDepartment of Biotechnology, Royal Institute of Technology (KTH), Stockholm, SwedenISBN 91-7170-633-X
Abstract
Recombinant strategies are today dominating in the development of modern subunit vaccines. Thisthesis describes strategies for the production and recovery of protein subunit immunogens, and howgenetic design of the expression vectors can be used to adapt the immunogens for incorporation intoadjuvant systems. In addition, different strategies for delivery of subunit vaccines by RNA or DNAimmunization have been investigated.
Attempts to create general production strategies for recombinant protein immunogens in such a waythat these are adapted for association with an adjuvant formulation were evaluated. Differenthydrophobic amino acid sequences, being either theoretically designed or representing transmembraneregions of bacterial or viral origin, were fused on gene level either N-terminally or C-terminally toallow association with iscoms. In addition, affinity tags derived from Staphylococcus aureus protein A(SpA) or streptococcal protein G (SpG), were incorporated to allow efficient recovery by means ofaffinity chromatography. A malaria peptide, M5, derived from the central repeat region of thePlasmodium falciparum blood-stage antigen Pf155/RESA, served as model immunogen in thesestudies. Furthermore, strategies for in vivo or in vitro lipidation of recombinant immunogens for iscomincorporation were also investigated, with a model immunogen ∆SAG1 derived from Toxoplasmagondii. Both strategies were found to be functional in that the produced and affinity purified fusionproteins indeed associated with iscoms. The iscoms were furthermore capable of inducing antigen-specific antibody responses upon immunization of mice, and we thus believe that the presentedstrategies offer convenient methods for adjuvant association.
Recombinant production of a respiratory syncytial virus (RSV) candidate vaccine, BBG2Na, in babyhamster kidney (BHK-21) cells was investigated. Semliki Forest virus (SFV)-based expression vectorsencoding both intracellular and secreted forms of BBG2Na were constructed and found to befunctional. Efficient recovery of BBG2Na could be achieved by combining serum-free productionwith a recovery strategy using a product-specific affinity-column based on a combinatoriallyengineered SpA domain, with specific binding to the G protein part of the product.
Plasmid vectors encoding cytoplasmic or secreted variants of BBG2Na, and employing the SFVreplicase for self-amplification, was constructed and evaluated for DNA immunization against RSV.Both plasmid vectors were found to be functional in terms of BBG2Na expression and localization.Upon intramuscular immunization of mice, the plasmid vector encoding the secreted variant of theantigen elicited significant anti-BBG2Na titers and demonstrated lung protective efficacy in mice. Thisstudy clearly demonstrate that protective immune responses to RSV can be elicited in mice by DNAimmunization, and that differential targeting of the antigens expressed by nucleic acid vaccinationcould significantly influence the immunogenicity and protective efficacy.
We further evaluated DNA and RNA constructs based on the SFV replicon in comparison with aconventional DNA plasmid for induction of antibody responses against the P. falciparum Pf332-derived antigen EB200. In general, the antibody responses induced were relatively low, the highestresponses surprisingly obtained with the conventional DNA plasmid. Also recombinant SFV suicideparticles induced EB200-reactive antibodies. Importantly, all immunogens induced an immunologicalmemory, which could be efficiently activated by a booster injection with EB200 protein.
Keywords: Affibody, Affinity chromatography, Affinity purification, DNA immunization, Expression plasmid,Fusion protein, Hydrophobic tag, Iscoms, Lipid tagging, Malaria, Mammalian cell expression, Recombinantimmunogen, Respiratory Syncytial Virus, Semliki Forest virus, Serum albumin, Staphylococcus aureus proteinA, Subunit vaccine, Toxoplasma gondii
Christin Andersson
CHRISTIN ANDERSSON
RECOMBINANT SUBUNIT VACCINES
LIST OF PUBLICATIONS
This thesis is based on the following papers, which in the text will be referred to by theirRoman numerals:
I. Andersson, C., Sandberg, L., Murby, M., Sjölander, A., Lövgren-Bengtsson, K. andStåhl, S. General expression vectors for production of hydrophobically taggedimmunogens for efficient iscom incorporation. J. Immunol. Methods, 222: 171-182(1999)
II. Andersson, C., Sandberg, L., Wernérus, H., Johansson, M., Lövgren-Bengtsson, K.and Ståhl, S. Improved systems for hydrophobic tagging of recombinant immunogensfor efficient iscom incorporation. J. Immunol. Methods, 238: 181-193 (2000)
III. Andersson, C., Wikman, M., Lövgren-Bengtsson, K., Lundén, A. and Ståhl, S. Invivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation.J. Immunol. Methods (2000) Submitted
IV. Andersson, C., Hansson, M., Power, U., Nygren, P-Å. and Ståhl, S. Mammalian cellproduction of an RSV candidate vaccine recovered using a product specific affinitycolumn. FEBS Letters (2000) Submitted
V. Andersson, C., Liljeström, P., Ståhl, S. and Power, U. Protection against respiratorysyncytial virus (RSV) elicited in mice by plasmid DNA immunization encoding asecreted RSV G protein derived antigen. FEMS Immunol. Med. Microbiol. (2000) Inpress
VI. Andersson, C., Vasconcelos, N-M., Sievertzon, M., Haddad, D., Liljeqvist, S.,Berglund, P., Liljeström, P., Ahlborg, N., Ståhl, S. and Berzins, K. Comparativeimmunization study using DNA and RNA constructs encoding a part of Plasmodiumfalciparum antigen Pf332. (2000) Submitted
CHRISTIN ANDERSSON
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 15by using gene fusion strategies
3. Adjuvant systems for recombinant protein antigens 17
4. Live delivery of subunit vaccines 23
4.1. Bacterial delivery systems 23
4.2. Viral delivery systems 24
5. Nucleic Acid Vaccines 26
5.1. DNA vaccines 26
5.2. Improving immune responses to DNA vaccines 30
5.2.1. Adjuvants and delivery systems for DNA vaccines 31
5.2.2. Codelivery of stimulatory substances 32
5.2.3. Immunostimulatory DNA sequences 32
5.3. Safety of DNA vaccines 33
5.4. RNA Vaccines 33
RECOMBINANT SUBUNIT VACCINES
PRESENT INVESTIGATION
6. Recombinant production of protein immunogens 37
6.1. Recombinant strategies foriscom incorporation (I, II, III) 37
6.1.1. N-terminal hydrophobic tags (I) 37
6.1.2. C-terminal hydrophobic tags (II) 43
6.1.3. Lipid tagging of protein immunogens (III) 45
6.2. Recovery of a vaccine candidateproduced in mammalian cells (IV) 48
7. Nucleic acid vaccines 53
7.1. Protection against RSV elicited in miceby plasmid DNA immunization (V) 53
7.2. Immunization with RNA and DNA encoding a malarial antigen (VI) 56
8. Concluding remarks 60
9. Abbreviations 64
10. Acknowledgements 66
11. References 68
CHRISTIN ANDERSSON
RECOMBINANT SUBUNIT VACCINES
1
INTRODUCTION
The use of vaccines has had great impact on the ability to control and prevent infectious
diseases. The first vaccines consisted of whole pathogens, killed or attenuated, but today the
recombinant subunit approach, i.e. to use only a defined subunit of the pathogen, is
dominating the vaccine research in the search for new effective vaccines. The ability to use
small, defined parts of a pathogen and produce that subunit in a non-pathogenic host will
increase the safety of future vaccines. Subunit vaccine candidates typically consist of surface
proteins or polysaccharides. The use of recombinant DNA technology has simplified the
production of subunit vaccines. Protein-based subunit vaccines can consist of synthetic
peptides, recombinant proteins or gene fragments (DNA or RNA) encoding the protein
immunogens. Live delivery vehicles can furthermore be evaluated for delivery of both protein
subunits and nucleic acid vaccines. This thesis will describe some aspects on the production
of protein immunogens and how expression systems can be designed to adapt the proteins for
association to adjuvant systems. Furthermore, nucleic acid immunization approaches (DNA
and RNA) have been evaluated or subunit immunogens from respiratory syncytial virus
(RSV) and malaria.
1. Vaccines
Vaccination has been an important tool to combat infectious diseases over the past 200 years.
The first human vaccine was developed in 1798 when Edward Jenner successfully prevented
smallpox infection in milkmaids. Today, prevention of bacterial and viral infections through
vaccination is very beneficial in reducing disease mortality and healthcare costs. Successful
development of vaccines has resulted in that many major diseases, such as diphtheria,
poliomyelitis and measles nowadays are kept under control, and that smallpox is even totally
eradicated (Mäkelä, 2000).
The first vaccines were mainly based of either live but attenuated or inactivated, killed
microbes. Also today, most of the vaccines used routinely in childhood vaccination programs
are whole-organism vaccines consisting of attenuated or killed bacteria or viruses (Plotkin,
1993). Vaccine types and their characteristics are presented in Table 1.
CHRISTIN ANDERSSON
2
Table 1: Different vaccine types and some of their characteristics
Vaccine type Characteristics
Whole cell vaccines
Live, attenuated bacteria orviruses
+ Humoral and cellular immune responses- Risk of reversion to virulent strain- Undefined composition
Killed, inactivated bacteria orviruses
+ No risk of infection (replication deficient, non-infectious)- Less powerful than live vaccines- Undefined composition
Subunit vaccines
Purified components frompathogen
+ Well-defined composition- Requires large-scale cultivation of pathogen- Poor immunogens, need of adjuvant
Synthetic peptides + Well-defined composition+ No risk of pathogenicity- Short half-life- Need of adjuvant
Recombinant proteins + Well-defined composition+ No risk for pathogenicity+ Possibility for cost-efficient production and purification- Primarily humoral immune response- Need of adjuvant
Live vectors: Bacterial + Humoral and cellular immune responses+ Possibility to develop oral vaccines- Risk of reversion to virulent forms for attenuated pathogens
Live vectors: Viral + Humoral and cellular immune responses- Risk of reversion to virulent forms for attenuated pathogens
Nucleic Acid vaccines: DNA + Cellular and humoral immune responses+ Cost-efficient production+ In vivo amplification systems available- Inefficient transfection- Risk of integration into host genome not completely excluded
Nucleic Acid vaccines: RNA + No risk of integration into host genome+ Do not have to enter nucleus for translation+ In vivo amplification systems available- Unstable- Quite expensive production
Live, attenuated vaccines are usually quite effective in stimulating both humoral as well as
cellular immune responses (Ellis, 1999). These vaccines might have a certain capacity to
replicate in the host, but are attenuated in their pathogenicity in order not to cause disease.
However, due to its replicating ability, live attenuated vaccines can potentially revert to a
virulent wild-type strain, e.g. by recombination, resulting in disease. Another aspect of using
RECOMBINANT SUBUNIT VACCINES
3
live attenuated vaccines is the risk of transmission between individuals. These facts are major
concerns, especially if the unwanted disease develops in immunocompromised hosts.
Killed or inactivated vaccines, however, is in general safer than live, attenuated vaccines. The
vaccine cannot replicate in the host and is non-infectious. However, a drawback with using
killed, inactivated vaccines is that they are less effective than live attenuated vaccines in
inducing protective immunity. To improve the immunogenicity of the killed or inactivated
vaccines, they often need to be coadministered with an adjuvant and administered several
times. The classical, whole cell vaccines (live, attenuated and killed, inactivated vaccines)
need to be improved, especially in terms of safety. The side effects of using such vaccines,
including risk of infection or transmission, need to be reduced. These vaccines also contain
undefined substances of bacterial or viral origin, which might make the vaccines highly
immunogenic and potent, but could also be involved in the induction of side-effects. With
increasing demands from regulatory authorities on well-defined drugs, it will be increasingly
difficult to get new vaccines of this class accepted for human use.
New technology and better understanding of cell biology and immunology has accelerated the
vaccine development during the recent years. It is now possible to use only a small, defined
part of the pathogen in a vaccine with capacity to induce an immune response to the whole
pathogen, thus without risk of infection. By using subunits of the pathogen, the safety of the
vaccine is obviously increased. The subunit part used is often a surface molecule of the
pathogen, able to induce immunity on its own.
1.1. Subunit vaccines
Vaccines based on the subunit approach may consist of natural substances, directly harvested
from cultivations of the pathogen. Such nonrecombinant subunit vaccines may consist of
bacterial polysaccharides, detoxified toxins or viral surface proteins. Several subunit vaccines
consisting of natural surface polysaccharides purified from cell cultures have been developed
(Lindberg, 1999) e.g. for Neisseria meningitidis (Gotschlich et al, 1969) and Streptococcus
pneumoniae (Smit et al., 1977; Hilleman et al., 1981). One drawback with polysaccharide-
based vaccines is that they are poor immunogens in infants and children (Lindberg, 1999). To
improve the problems with poor immunogenicity of polysaccharide vaccines, the concept of
conjugate vaccines were introduced. A subunit conjugate vaccines have been licensed for
CHRISTIN ANDERSSON
4
Haemophilius influenzae type b (Hib), where polysaccharides have been coupled to a tetanus
toxoid carrier (Schneerson et al., 1986). A recent strategy to improve polysaccharide-based
vaccines, through the use of peptides that mimic the capsular polysaccharide of N.
meningitidis, have been evaluated (Grothaus et al., 2000). Purified toxins, chemically
detoxified, e.g. by formalin, have been used in vaccines to diphtheria, tetanus and pertussis
(Ellis, 1999). Subunit vaccines are safer than whole-cell vaccines, but there are drawbacks,
such as the requirement of large-scale cultivation of pathogens, which is normally costly and
not without risk. The subunit vaccines also need to be administered with an adjuvant to
increase immunogenicity. Polysaccharide-based and non-recombinant subunit vaccines will
not be further discussed in this thesis.
1.2. Recombinant subunit vaccines
The first protein-based vaccines also relied on natural (non-recombinant) sources of antigens.
For example, a highly active vaccine to hepatitis B consisted of purified hepatitis B surface
antigen (HBsAg) from human plasma, see review by Hilleman, 2000. The recombinant
subunit approach is today dominating the vaccine research in the search for new vaccines.
Identification of antigens involved in inducing protective immunity and isolation of the gene
encoding these proteins makes it possible to use recombinant DNA technology or synthetic
peptides to produce sufficient quantities of the antigen for vaccine studies. The first vaccine to
be produced utilizing recombinant DNA technology was licensed in 1986 when the HBsAg
was successfully expressed in yeast (Valenzuela et al., 1982). This new vaccine efficiently
elicited protective antibodies upon vaccination of chimpanzees (McAleer et al., 1984), and
soon this vaccine replaced the plasma derived hepatitis B vaccine in human use.
The use of recombinant DNA technology has made the development of subunit vaccines more
efficient. The basics of this technology is to transfer a gene encoding an antigen, responsible
for inducing immune responses sufficient for protection, to a non-pathogenic host, thereby
making the production of the antigen safer and generally more efficient. Recombinant subunit
vaccines can be delivered as purified recombinant proteins, as proteins delivered using live
non-pathogenic vectors (bacterial or viral) or as nucleic acid molecules encoding the antigen
(Table 1). There are several advantages in using recombinant subunit vaccines. No pathogen
is present in the production and purification procedure, thus making the production procedure
RECOMBINANT SUBUNIT VACCINES
5
less hazardous. By using recombinant DNA technology, the production and purification
procedure can be carefully designed to obtain high yields of a well-defined product.
Recombinant strategies for production of subunit vaccines will be further discussed later in
this thesis, see section 2.2.
Recombinant strategies have also been employed for detoxification of toxins. Engineered
inactivation of toxin can be obtained by mutational replacement of specific amino acids in the
enzymatically active part of the toxin. Pertussis toxoid produced by Bordetella pertussis with
specific mutations in its toxin gene is included as a component in an acellular pertussis
vaccine (Del Giudice and Rappuoli, 1999). Chimeric composite immunogens can also be
created by fusion of different toxins, such as the cholera toxin B subunit (CTB)-E. coli heat-
labile toxin B subunit (LTB) hybrid molecules, which are candidate oral vaccines against both
enterotoxic E. coli (ETEC) infections and cholera (Lebens et al., 1996).
Although the use of protein immunogens, synthetic peptides or nucleic acid vaccines offer
several advantages, e.g. reduced toxicity, they are generally poor immunogens when
administered alone. This is particularly true for vaccines based on proteins or peptides, and to
make these vaccines more efficient, the use of potent and safe immunological adjuvants might
be needed. Adjuvant systems will be described later, see section 3.
The recombinant subunit approaches are being used for the development of new vaccines and
improvement of already existing vaccines. Many recombinant vaccines are now being tested
in preclinical and clinical trials, and some recombinant vaccines are already on the market
(Walsh, 2000; Table 2).
CHRISTIN ANDERSSON
6
Table 2: Selected examples of recombinant vaccines currently approved in the US or EU(Modified from Walsh, 2000)
Vaccine Description Application Company Approved
Recombivax rHBsAg produced in S.cerevisiae
Hepatitis B prevention Merck 1986 (US)
Comvax Combination vaccine,containing rHBsAgproduced in S. cerevisiaeas one component
Vaccination of infantsagainst H. influenzaetype B and hepatitis B
Merck 1996 (US)
Tritanrix-HB Combination vaccine,containing rHBsAgproduced in S. cerevisiaeas one component
Vaccination againsthepatitis B, diphteria,tetanus, and pertussis
SmithKline Beecham 1996 (EU)
Twinrix, adult andpediatric forms
Combination vaccine,containing rHBsAgproduced in S. cerevisiaeas one component
Immunization againsthepatitis A and B
SmithKline Beecham 1996 (EU)(adult)
1997 (EU)(pediatric)
Primavax Combination vaccine,containing rHBsAgproduced in S. cerevisiaeas one component
Immunization againstdiphteria, tetanus, andhepatitis B
Pasteur MerieuxMSD
1998 (EU)
Procomvax Combination vaccine,containing rHBsAgproduced in S. cerevisiaeas one component
Immunization againstH. influenzae type Band hepatitis B
Pasteur MerieuxMSD
1999 (EU)
Lymerix rOspA, a surfacelipoprotein of B.burgdorferi, produced inE. coli
Lyme disease vaccine SmithKline Beecham 1998 (US)
RECOMBINANT SUBUNIT VACCINES
7
2. Protein immunogens
2.1. Synthetic peptides
Subunit vaccine candidates can be produced by chemical synthesis of short polypeptides. The
advantages of peptide vaccines are several (summarized in Table 1), such as a chemically
well-defined product and a simple preparation (Babiuk, 1999). Synthetic peptides representing
parts of higher parasites, bacteria or viruses have indeed been used for immunizations in
humans (Klipstein et al., 1985; Herrington et al., 1987; Kahn et al., 1992; Millet et al., 1993).
For example, it has been shown that a 12 amino acid long peptide from the Plasmodium
falciparum sporozoite is safe and induces humoral responses in healthy volunteers
(Herrington et al., 1987). Although some side effects encountered with other vaccine types,
e.g. whole cell vaccines, may be circumvented, one major disadvantage of peptide vaccines is
their low level of immunogenicity. One reason for this may be due to major histocompatibility
complex (MHC) restriction i.e. one particular peptide can only be recognized with a
particulate human leukocyte antigen (HLA) haplotype (Rammensee et al., 1995). Another
reason could be that the peptides are rapidly degraded or excreted in vivo (Babiuk, 1999).
However, recent advances in chemical synthesis i.e. to introduce changes in the amine bond,
have lead to the development of pseudopeptides that are more stable and as biologically active
as normal peptides (Babiuk, 1999). Pseudopeptides have for example shown to induce
neutralizing antibodies to foot and mouth disease virus (Briand et al., 1997). Another major
drawback with synthetic peptides is that only linear epitopes can be utilized in the vaccine.
Antibody responses are often directed to conformational epitopes, but those are difficult to
mimic using synthetic peptides.
A problem related to the use of synthetic peptides is that although high antibody levels are
elicited to synthetic peptide-carrier complex, a major part of the antibodies are directed
towards the carrier (Herzenberg et al., 1980; Schutze et al., 1985). A further possible
disadvantage when using single peptide vaccines is that the immune response will be raised
only to one small epitope. Mutations in that specific region may allow the pathogen to evade
the immune system. However, this may be circumvented by using multiple antigenic peptides
(MAP) (Tam, 1996). Nevertheless, certain subunit vaccine candidates have progressed into
human clinical trials, e.g. a malaria vaccine candidate, consisting of a mixture of polymerized
synthetic peptides (Patarroyo et al., 1988).
CHRISTIN ANDERSSON
8
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
RECOMBINANT SUBUNIT VACCINES
9
protected from proteolytic degradation, and that high production levels often are obtained.
Levels up to 50 % of total cell protein content have been reported (Rudolph, 1996).
2.2.2. Production hosts
The choice of expression system used will be dependent of the characteristics of the protein to
be expressed. Several issues have to be considered, e.g. (i) expression levels, (ii) the need for
post-translational modifications, (iii) scale-up consideration, and (iv) production costs
(Dertzbaugh, 1998). The heterologous host used may affect the immunogenicity and
protective efficacy of the product. For example, an antigen may be expressed at high levels in
a bacterial expression system, but may only fold partially into its native confirmation, or the
antigen needs might need to be post-translationally modified to elicit protective immunity
(Dertzbaugh, 1998).
There are today four major host cell systems commonly used to produce recombinant
proteins; (i) bacterial expression systems, (ii) yeast expression systems, (iii) insect cell
expression systems, and (iv) mammalian expression systems. Each host cell system has
individual advantages and disadvantages (Table 3).
Table 3: Examples of production hosts for recombinant protein expression
Host Characteristics
Bacteria + Easy to grow and manipulate+ High expression levels+ Inexpensive- Do not perform post-translational modifications- Proteolysis- Correct folding can be problematic to obtain
Yeast + Capable of certain post-translational modifications+ High cell densities+ Easy to grow and manipulate- Proteolysis
Insect cells + Make a variety of post-translational modifications+ High expression levels compared to mammalian cells
Mammalian cells + Post-translational modifications+ Stable antigen-producing cell lines can be constructed- Time consuming- Expensive
CHRISTIN ANDERSSON
10
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
RECOMBINANT SUBUNIT VACCINES
11
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
CHRISTIN ANDERSSON
12
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).
RECOMBINANT SUBUNIT VACCINES
13
Table 4: Selected examples of fusion partners and their use
Effect on target protein Fusion partner Reference
Improved production
Secretion Signal peptide Abrahamsén et al., 1986Hansson et al., 1994
Multimerization C-peptide Jonasson et al., 1998
Solubility Protein A domain Samuelsson et al., 1991 and 1994
Simplified purification
Hydrophobicity Isoleucin-tryptophan rich tag Köhler et al., 1991
Affinity Various affinity tags For reviews, see Nilsson et al.,1997 and Ståhl et al., 1999
Improved immunogenicproperties
Immunopotentiating Cholera toxin B (CTB) Sun et al., 1994Holmgren et al., 1994
Carrier-related properties albumin binding protein (ABP orBB) of streptococcal protein G(SpG)
Sjölander et al., 1997Libon et al., 1999
Increased half-life BB of SpG Nygren et al., 1991
Improved adjuvant association
Improved association withiscoms
Membrane-spanning regions fromviral or bacterial surface proteins
I, II
Improved association withiscoms
In vivo lipidation signals III
Protein A, a surface protein from Staphylococcus aureus (SpA), has been widely used as
affinity fusion partner due to its strong affinity to the Fc part of immunoglobulin G (Uhlén et
al., 1983). SpA consists of a signal sequence (S) followed by five homologous IgG binding
domains (E, D, A, B, C), all capable of binding IgG (Moks et al., 1986) and a cell surface
anchoring domain, XM (Schneewind et al., 1995; Navarre and Schneewind, 1999). An
engineered IgG-binding domain, Z, was derived from the B domain of SpA and was shown to
have retained IgG binding properties and to be resistant to cleavage with hydroxylamin and
CNBr (Nilsson et al., 1987). The characteristics of SpA and Z, being proteolytically stable and
highly soluble, makes them well suited as affinity fusion partners to enable purification of
recombinant proteins (Ståhl et al., 1999). The Z tag have been used in many studies as affinity
tag, mostly in its divalent form, ZZ, since it has been shown that ZZ has a ten times higher
CHRISTIN ANDERSSON
14
affinity for its ligand IgG compared to the monovalent Z domain (Nilsson et al., 1994). The
high solubility of the Z domain has shown to increase the overall solubility of fusion proteins
(Samuelsson et al., 1991 and 1994).
The streptococcal protein G (SpG) is a surface protein with affinity to both IgG and albumin
of different mammalian species, including humans (Nygren et al., 1990). The binding sites for
IgG and serum albumin are shown to be structurally separated (Nygren et al., 1988). The
complete albumin-binding region is suggested to contain three albumin-binding motifs, each
of about 5 kDa in size. Tags comprising of one, two or two-an-a-half serum albumin binding
motifs have been constructed (ABD, ABP and BB, respectively), successfully produced and
used in different applications (Nilsson et al., 1997; Ståhl and Nygren, 1997; Ståhl et al.,
1999). The albumin binding proteins derived from SpG are proteolytically stable, highly
soluble, and are possible to produce in high yields (Larsson et al., 1996; Nilsson et al., 1996;
Murby et al., 1995). Both the SpA-derived Z domain and the SpG-derived serum albumin
binding proteins have been extensively used to facilitate affinity chromatography purification
of protein immunogens (Ståhl et al., 1989; Sjölander et al., 1990, 1993c and 1997; Murby et
al., 1994; Berzins et al., 1995; Power et al., 1997; Libon et al., 1999).
2.2.5. Tailor-made affinity ligands
In order to create new binding proteins to be used as affinity ligands, the use of combinatorial
strategies to create large protein libraries is an emerging research area. Combinatorial methods
are attractive because they allow direct selection of suitable target-specific binding molecules
from large libraries of related proteins. Libraries of other molecules than proteins or peptides
have also been constructed, e.g. small organic molecules (Gordon et al., 1994) and DNA or
RNA (Gold et al., 1995; Burke and Gold, 1997), but this will not be further discussed in this
thesis.
A method of emerging interest is the display of libraries of peptides or proteins on
filamentous phage surfaces (Smith, 1985; Clackson and Wells, 1994). This concept involves
construction of a protein library containing partially randomized sequences, fusion by genetic
means of the new variants to phage protein III or phage protein VIII, and subsequently
displaying the library on phage particles. Rounds of selection of displayed peptides or proteins
with desired properties from such phage libraries can be performed, with intermediate
RECOMBINANT SUBUNIT VACCINES
15
enrichment steps of the selected phages in E. coli. A useful selection method is to use solid
phase surfaces covered with the target molecule and allowing the phages to bind, theoretically
via interactions of the displayed protein and the target molecule. The phage particle would
thus constitute the link between genotype and phenotype, which allows identification of the
protein from the DNA sequence.
As mentioned above, the IgG-binding Z-domain, derived from SpA, has several features,
which makes it suitable in affinity purification applications such as being proteolytically
stable, of small size and highly soluble. Structural analysis has shown that the Z domain is
composed of three α-helices and that the IgG-binding surface is shared between two of the
three helices. This binding surface is of approximately 600Å2, similar to the size of many
antibody-antigen interactions. These data suggest that if the amino acids involved in the IgG-
binding were randomly substituted, new variants of the Z-domain with novel binding
properties could be found (Nygren and Uhlén, 1997). Theoretically, these new protein
domains would share the stability properties and the α -helical structure of the original Z-
domain. Recently, such libraries of the Z-domain were created (Nord et al., 1995 and 1997).
Thirteen amino acids involved in the IgG binding of the Z-domain was randomly substituted
to any of the 20 amino acids. The libraries containing the Z-variants were fused to protein III
on filamentous phage M13, and subsequently displayed on phage particles (Nord et al., 1995
and 1997). Several novel binders, so called affibodies, have been selected by phage-display
technology from these libraries, including binders to Taq polymerase, human insulin, a human
apolipoprotein variant (Nord et al., 1997) and to an RSV G protein derived protein G2Na
(Hansson et al., 1999).
2.2.6. Increasing immunogenicity by using gene fusion
Fusion partners can be used to increase the immunogenicity of the antigen. The SpG-derived
fusion partner BB described above has shown to have immunopotentiating properties when
used as fusion partner to the immunogen used for immunization (Sjölander et al., 1993c and
1997; Power et al., 1997; Libon et al., 1999). In immunization experiments of mice with a
malaria antigen fused to the BB tag, antibody responses were raised in mice not responding to
the malarial antigen alone (Sjölander et al., 1997). Furthermore, in a study with an RSV
antigen, the antibody response was higher to the antigen when fused to BB than when
CHRISTIN ANDERSSON
16
immunizing with the antigen alone (Libon et al., 1999). The immunopotentiating properties of
BB could be either due to T-cell epitopes present in BB (Sjölander et al., 1997) or due to the
serum albumin binding properties resulting in an increased half-life of the antigen or even a
combination of the both.
Another commonly used protein fusion partner with carrier-related properties is the surface
antigen of Hepatitis virus B (HBsAg). A fusion protein composed of a repetitive sequence
derived from the circumsporozoite (CS) protein of Plasmodium falciparum and HBsAg
strategies was produced in yeast cells and used in vaccination experiments in humans
resulting in long lasting antibody responses (Vreden et al., 1991). Fusion to the HBsAg have
also been shown to enhance the humoral as well as the cellular immune response to human
immunodeficiency virus 1 (HIV-1)-derived immunogens both when delivered as fusion
proteins (Schlienger et al., 1992; Mancini et al., 1994) and as plasmid DNA (Fomsgaard et al.,
1998). Furthermore, a strategy to fuse by genetic means the target antigen to cholera toxin
subunits in order to obtain targeting to receptors in the mucosa, has been described
(Hajishengallis et al., 1995). Antigens can also be expressed as fusion proteins to B cell or T
cell epitopes to increase humoral or cellular immune responses (Löwenadler et al., 1992). In
addition to expressing single proteins, it is possible to develop chimeric genes containing the
important epitopes from a number of pathogens (Whitton et al., 1993).
Recombinant proteins are in general relatively poor immunogens when administered alone
and the coadministration of an adjuvant is crucial to elicit a strong immune response. Fusion
strategies have been investigated to ensure proper association of fusion proteins to an
adjuvant. An example of this strategy, that will be discussed below, is to use hydrophobic
tagging of protein immunogens to improve the association of the immunogen to a
hydrophobic adjuvant system e.g. iscoms (I, II, III)
RECOMBINANT SUBUNIT VACCINES
17
3. Adjuvant systems for recombinant protein antigens
Subunit vaccines consisting of peptide or recombinant proteins are normally poor
immunogens when administered alone. Whole cell vaccines are heterogeneous and contain
many immunostimulatory substances, e.g. bacterial DNA or enterotoxins. Pure recombinant
protein antigens, lacking these natural immunostimalutory substances, therefore require co-
administration of an adjuvant to increase the immune response. Adjuvants are defined as
substances that together with the antigen stimulate the antigen-specific immune response.
Extensive research has been done in this field and it has been shown that many substances can
achieve this, but so far only aluminum salts (Gupta, 1998) and MF59 (an oil emulsion) are
adjuvants approved for human use (Singh and O'Hagan 1999). Therefore the development of
novel adjuvants (Morein et al., 1996) and strategies for efficient association of the antigen to
the adjuvant, are important topics in present vaccinology research. Table 5 presents selected
examples of adjuvants that have been evaluated in animal studies.
Table 5: Selected examples of vaccine adjuvants (modified from Singh and O'Hagan 1999)
Type of adjuvant Examples Reference
Mineral salts Aluminum hydroxideAluminium phosphateCalcium phosphate
Gupta, 1998Gupta, 1998Wang et al., 2000
Immunostimulatory adjuvants CytokinesSaponines (e.g. QS21)MDP derivatesCpG oligonucleotideslipopolysaccharides (LPS)monophosporyl lipid A (MPL)
Baca-Estrada et al., 1997Barr et al., 1998.Namba et al., 1997Klinman et al., 1999Ogawa et al., 1997Baldridge and Crane, 1999
Lipid particles and emulsions Freund incomplete adjuvantSAFMF59LiposomesImmunostimulating complexes (iscoms)
Cochleates
Chang et al., 1998Hjort et al., 1997Heineman et al., 1999Gregoriadis et al., 1999Barr et al., 1998I, II, IIIGould-Fogerite et al., 1998
Microparticulate adjuvants PLG microparticlesVirus-like particles
O'Hagan et al., 1998Coste et al., 2000
Mucosal adjuvants Heat-labile enterotoxin (LT)Cholera toxin (CT)
Park et al., 2000Matuso et al., 2000
CHRISTIN ANDERSSON
18
Adjuvants can be used in several ways; they can increase the duration of immune responses,
modulate antibody specificity, isotype and subclass distribution, stimulate cell mediated
immune responses or decrease the required dose of antigen and thus reduce the cost for the
vaccine (Singh and O'Hagan 1999). The mechanisms of adjuvant action are relatively poorly
understood. Most likely, activation of the immune response by adjuvants is a result of a
cascade of events such as induced cytokine production or activation of antigen-presenting
cells (APCs) e.g by increased expression of costimulatory molecules or MHC complexes
(Singh and O'Hagan 1999). The adjuvant to choose will totally depend on what type of
immune responses that is required for protective immunity. Different adjuvants usually induce
different types of responses, and a first choice would be to evaluate the adjuvant that is
predicted to give an immune response similar to what is postulated for protective immunity.
In general, adjuvants are immunostimulatory substances, particulate adjuvants or a
combination of the both (Table 5). Immunostimulatory substances are thought to
predominantly effect the cytokine expression by activating expression of costimulatory
molecules or major histocompatibility complex (MHC) molecules, while particulate
adjuvants, which have comparable dimensions to pathogens, probably are targets for uptake
by antigen-presenting cells. See Table 6 for a general comparison.
Table 6: A general comparison of dimensions of pathogens and particulate adjuvants (modifiedfrom Singh and O'Hagan 1999)
Natural pathogen Particulate adjuvant
Bacteria 0.5-3 µm Microparticles 100 nm-10 µm
Poxvirus 250 nm Liposomes 50 nm-1 µm
Herpesvirus 100-200 nm Virosomes 50 nm - 10 µm
HIV 100 nm MF59 200 nm
Poliovirus 20-30 nm Iscoms 40 nm
One group of immunostimulatory adjuvants is lipopolysaccharide (LPS) derived from Gram-
negative bacteria, and the most well characterized is monophosphoryl lipid A (MPL)
(Baldridge and Crane, 1999) derived from Salmonella minnesota. MPL have shown to induce
a Th1 type of response, but seems not to be a potent adjuvant for antibody production
RECOMBINANT SUBUNIT VACCINES
19
(Gustafson and Rhodes, 1992; Sing and O'Hagan, 1999). In certain studies, MLP has been
used in combination with e.g. alum or QS21 (Thoelen et al., 1998). However, when using
adjuvant combinations, the individual contribution of the included adjuvants is difficult to
establish. MPL has for example been evaluated in vaccine studies for cancer, herpes, hepatitis
B virus, malaria and human papilloma virus (Sing and O'Hagan, 1999), and has also been
investigated as adjuvant for DNA vaccines (Sasaki et al., 1997).
A second group of immunostimulatory adjuvants are the saponins, a heterogenous group of
sterol glycosides and triterpenoid glycosides, present in a wide range of plant species (Barr et
al., 1998). Saponins were included in a veterenary vaccine in the 1950s (Barr et al., 1998) but
later work showed that the saponins derived from Quillaja saponaria (Chilean soap bark tree)
were the most effective as adjuvant (Dalsgaard, 1970). Variable results have been reported
from the use of saponins, probably partly due to confusion with respect to the source of the
saponin and the variable content of saponins in extract. However, in 1978, Quillaja saponins
were purified, and a fraction with high adjuvant activity, termed Quil A, was defined
(Dalsgaard, 1978) and since then, Quil A has been used in a number of veterinary vaccines
(Dalsgaard et al., 1990). However, Quil A have been associated with some toxicity in vitro,
and several attempts have been made to purify Quil A (for review, see Barr et al., 1998). In
the 1990s, a fraction of Quil A saponins with low toxicity was isolated, denoted QS21
(Kensil, 1991). QS21 has the ability to induce antibody production and cytotoxic T cells
(CTL) (reviewed in Kensil, 1996). Studies evaluating QS21 as an adjuvant have been
performed for cancer, HIV-1, influenza, herpes, malaria and hepatitis B (Sing and O'Hagan,
1999). Human clinical trials have been performed using QS21 as adjuvant in for example
vaccines for malignant melanoma (Livingston et al., 1994) and in a DNA vaccine study for
HIV-1 (Sasaki et al., 1998)
Recent research has shown that sequences in bacterial DNA, but not vertebrate DNA has
direct immunostimulatory effect on cells in vitro (Tokunaga et al., 1984; Messina et al.,
1991). The effect is due to the presence of unmethylated CpG dinucleotides that is methylated
in vertebrate DNA (Krieg et al., 1995; Bird et al., 1987). It is thought that the CpG motifs in
bacterial DNA function as a danger signal and thus stimulating immune responses (Krieg,
1996). The exact mechanism for the immunostimulatory effect is not known, but it has been
shown that CpG motifs stimulate macrophages and dendritic cells (Jakob et al., 1998). CpG,
which can be in the form of plasmid DNA or synthetic oligonucleotides, has been evaluated as
CHRISTIN ANDERSSON
20
a mucosal adjuvant (Moldoveanu et al., 1998; McCluskie and Davis, 1999) and seems to have
a maximized effect when conjugated with protein antigens (Klinman et al., 1999).
Immunostimulatory sequences can also be used in combination with DNA vaccines, as will be
discussed later. To date, the effect of CpG immunization has only been widely used in animal
models, and its potential and safety in humans need to be evaluated.
Immunostimulatory substances are thought to induce cytokine production, and as an
alternative, cytokines can be used directly to modulate or increase the immune response. For
example, the use of cytokines has shown to be very promising for immunotherapy of cancer
(Salgaller and Lodge, 1998). Cytokines as adjuvants will not be discussed in detail here.
Particulate adjuvants, for example oil emulsions, microparticles, iscoms, liposomes and virus-
like particles, have comparable dimensions to typical pathogens the immune system normally
encounters and are therefore thought to be suitable for uptake by antigen-presenting cells. See
Table 6 for a general comparison.
MF59, an oil-in-water emulsion (Ott et al., 1995) is besides alum, the only adjuvant approved
for human use. It has been described to interact with antigen presenting cells at the site of
injection and in lymph nodes (Dupuis et al., 1998). This adjuvant has shown to enhance the
immunogenicity of an influenza vaccine (Higgins et al., 1996; Cataldo and Nest, 1997) and to
be a more potent adjuvant than alum for a hepatitis B vaccine in baboons (Traquina et al.,
1996). To date, clinical trials in humans have been performed for several vaccines using
MF59 as adjuvant, e.g. to HIV (Kahn et al., 1994; Nitayaphan et al., 2000) and herpes simplex
virus (Langenberg et al., 1995; Drulak et al., 2000).
Liposomes, another group of particulate adjuvants, have been used as delivery system for
antigens alone or in combination with another adjuvant (Gregoriadis, 1990; Alving, 1995).
Liposomes are spherical bilayers of 50 -1000 nm in diameter (Langner and Kral, 1999). Some
advantages of using liposomes are that the compounds are protected from degradation and that
the circulation time of the antigen is increased (Langner and Kral, 1999). Liposomes have
been used as delivery system for proteins and peptides (Alving, 1995) as well as for DNA
(Perrie and Gregoriadis, 2000). Cochleates, a modified liposomal structure has also been
evaluated in animal models (Gould-Fogerite et al., 1998).
Biodegradable polymeric microparticles, a group of adjuvants that have been investigated for
vaccine delivery, and among these, the poly(lactide-co-glycosides) (PLGs) are the primary
RECOMBINANT SUBUNIT VACCINES
21
candidates for adjuvant development (O'Hagan et al., 1991a and b). PLG particles have been
used in humans for many years as suture material and controlled-release delivery systems
(Sing and O'Hagan, 1999) and only recently, PLG particles have been shown to have adjuvant
properties to encapsulated antigens (O'Hagan et al., 1991a and b). Microparticles have been
demonstrated to be effective in eliciting CTLs (Maloy et al., 1994; Nixon et al., 1996) and
have been suggested as adjuvants for DNA vaccines (Hedley et al., 1998). A unique feature of
the microparticles are their ability to control the release of antigen, therefore it could be
possible to develop a vaccine that have a built-in boosting system. This is very attractive,
especially in the developing countries, since it might simplify vaccination logistics
Viral proteins expressed in heterologous hosts may associate in the culture media and form
virus like particles (VLPs). When antigens and viral proteins are coexpressed, the VLPs
entrap the antigen and can be used as an adjuvant system. For example, recombinant VLPs
from Saccharomyces cerevisiae, carrying a string of 15 epitopes from Plasmodium species
have been shown to induce CTL responses in mice (Gilbert et al., 1997). Similar VLPs have
also been shown to induce CTLs to SIV in macaques (Klavinskis et al., 1996) and to be safe
and immunogenic in humans (Martin et al., 1993).
In immunostimulating complexes, iscoms, Quil A, a saponin derived from Q. saponaria
(described above) has been incorporated into cage-like structures (Morein et al., 1984). The
iscoms are of about 40 nm in diameter and consists of cholesterol, phospholipids and the
target antigen (Morein et al., 1984; Barr et al., 1998). Iscoms have been used as adjuvant in
several animal models (Morein et al., 1995; Barr et al., 1998; Sjölander and Cox, 1998).
Protective immunity induced by iscoms has for example been demonstrated against influenza
virus in mice (Lövgren et al., 1990) and horses (Mumford et al., 1994). The mechanism of the
iscoms mode of action is not clearly understood, but some studies suggest that increased
cellular uptake may contribute to the adjuvant activity (Barr et al., 1998).
The saponin initially used in iscoms, Quil A, is quite heterogenous. In order to determine the
immunological properties and toxicity of chemically defined components of Quil A, purified
immunostimulatory fractions (QH-A, QH-B and QH-C) from Quil A was investigated, both as
free saponins and incorporated into iscoms (Rönnberg et al., 1995). Immunizations of mice
showed that all investigated combinations enhanced the antibody response equally, but the
combination of QH-A and QH-C in a ration of 7:3 (Iscoprep™703, Iscotec AB, Sweden)
where the toxic fraction QH-B was excluded, was both effective and non-toxic. The purified
CHRISTIN ANDERSSON
22
components QH-A, QH-B and QH-C have been characterized in several other studies
(Rönnberg et al., 1997; Johansson and Lövgren-Bengtson, 1999). Numerous of other purified
components from Quil A have also been investigated, both in iscoms and as free components,
reviewed in Barr et al., 1998.
One problem with iscoms is that the incorporation of the antigen need to be adapted for each
antigen, and may require modifications of the antigen. For several adjuvants, such as
liposomes, cochleates, non-ionic block polymers and iscoms, the protein antigens are
normally incorporated by means of hydrophobic interactions (Morein et al., 1996), and the
physical assocation has been demonstrated to be essential for the adjuvant to exert its
immunostimulating capacity. For iscoms, it has been shown that membrane-antigens
containing hydrophobic transmembrane sequences, are efficiently incorporated (Morein et al.,
1995). Proteins derived from membranes of a variety of viruses, bacteria and parasites have
successfully been incorporated into iscoms through hydrophobic interactions (for review, see
Barr et al., 1998). In contrast, hydrophilic antigens require modifications to be incorporated
into iscoms. Partial denaturation by using low pH (Pyle et al., 1989; Morein et al., 1990; Heeg
et al., 1991) or high temperature (Höglund et al., 1989) have been used to expose internal
hydrophobic sequences of otherwise hydrophilic proteins. Covalent addition of fatty acids is
also a useful method used to incorporate soluble proteins into iscoms, e.g. in a recombinant
HIV-1 gp120 candidate vaccine (Reid, 1992; Browning et al., 1992). Other strategies that
have been investigated include chemical conjugation of a peptide (Lövgren et al., 1987) or
proteins to pre-formed iscoms containing influenza envelope protein or matrix alone (Morein
et al., 1995; Sjölander et al., 1991; Larsson et al., 1993). Novel strategies, for inclusion of
hydrophobic protein sequences or even lipids in the protein antigen will be presented in this
thesis (I, II, III).
RECOMBINANT SUBUNIT VACCINES
23
4. Live delivery of subunit vaccines
Live vectors for delivery of heterologous subunit antigens have been suggested to be a more
cost-effective strategy than to produce and formulate protein subunit vaccines (Liljeqvist and
Ståhl, 1999). No extensive purification would be required and several live delivery systems
have been shown to elicit strong long-lasting immunity without the need for adjuvants. Both
bacteria and viruses have been investigated for delivery of foreign antigens. The knowledge of
molecular biology and genetics has resulted in the development of new attenuation strategies,
and thus genetically defined attenuated strains of bacteria or virus. By the use of recombinant
DNA technology, the genes encoding the heterologous antigen to be delivered can be inserted
into the non-pathogenic or attenuated live vector.
4.1. Bacterial delivery systems
There are two major approaches used for developing live bacterial delivery systems for
recombinant subunit vaccines, either to use genetically attenuated pathogenic bacteria or to
engineer non-pathogenic, commensal or food-grade bacteria. Both concepts require
engineering of the bacteria so that it would express the foreign target antigen, but when using
attenuated bacteria, vaccination to the original disease could simultaneously be performed.
Attenuated bacteria have been used as live recombinant vectors for heterologous antigens, e.g.
systems based on Salmonella thyphi (Levine et al., 1990), an enteric bacteria that has been
utilized to raise mucosal immunity against numerous foreign polypeptides. The oral bacteria
Streptococcus gordonii and the gut bacteria Lactococcus lactis, both commensal non-
pathogens from mucosal surfaces have been extensively studied (Dertzbaugh, 1998). S.
gordonii has been shown to elicit secretory IgA and serum IgG in mice when used to deliver
the M6 protein of S. pyogenes to the mucosal immune system (Pozzi et al., 1992; Medaglini et
al., 1995). Protection in mice after immunization with L. lactis engineered to express the C
fragment of tetanus toxin has also been reported (Wells et al., 1993).
When using naturally intracellular bacteria, the heterologous antigen can be either expressed
intracellularly or as exposed at the cell surface. However, when using commensal or food-
grade bacteria, it has been suggested that surface exposure of the heterologous antigen is
important to elicit antigen-specific immune responses (Nguyen et al., 1995; Ståhl and Uhlén,
CHRISTIN ANDERSSON
24
1997). Heterologous surface expression of antigens have been reported for several bacterial
systems evaluated as live delivery vehicles (Georgiou et al., 1993), for example gram-positive
bacteria and mycobacteria (Fischetti et al., 1996; Georgiou et al., 1997; Ståhl and Uhlén,
1997; Stover et al., 1993). Recently, protective immunity to respiratory syncytial virus (RSV)
could be evoked in mice by intranasal immunization with Staphylococcus carnosus carrying
surface-exposed RSV peptides (Cano et al., 2000). S. carnosus is a non-pathogenic bacteria
traditionally used in meat-fermentation processes.
4.2. Viral delivery systems
Recombinant viruses have been studied as candidate vaccines, both as vaccines against the
original diseases, and as viral vectors used to deliver heterologous antigens or genes. One
advantage of using viral vaccines is the ability to elicit both humoral and cellular immune
responses towards the delivered target antigen, as a result of intracellular expression of the
heterologous antigens.
Vaccinia virus was earlier used in vaccination against smallpox. The introduction of genes
encoding foreign proteins into a vaccinia vector was first demonstrated in 1982, and vaccinia
virus has since then been extensively studied as live vaccine vector (Paoletti, 1996).
Recombinant vaccinia virus was originally produced by homologous recombination, but today
alternative strategies have been developed, enabling more efficient gene construction
procedures.
Immunization experiments with recombinant vaccinia vectors expressing viral and non-viral
antigens have been reported to elicit protective immunity in animal disease models (Liljeqvist
and Ståhl, 1999). An oral rabies vaccine consisting of recombinant vaccinia expressing the
rabies virus glycoprotein has been given to wild animals, leading to drastic decrease of the
incidence of rabies in Belgium (Brochier et al. 1995).
Recombinant vaccinia virus based on wild-type vaccinia has primarily been investigated as
veterinary vaccines, mostly due to safety concerns. For human use, highly attenuated, non-
replicative vaccinia vectors have been constructed, e.g. the NYVAC strain, with deletion of 18
open-reading frames from the original genome (Tartaglia et al., 1992; Paoletti et al., 1996),
and the modified vaccinia virus Ankara strain (MVA) (Sutter and Moss, 1992). Recombinant
RECOMBINANT SUBUNIT VACCINES
25
NYVAC and MVA vectors encoding pathogen-derived antigens have been evaluated
extensively in various disease models (Perkus et al., 1995; Caroll and Moss, 1997).
Other viral vaccine vectors that have been investigated are certain poxviruses, unable to
replicate in mammalian cells, e.g. fowlpox and canarypox viruses, and ALVAC, a
recombinant canarypox virus (Perkus et al., 1995). Adenoviral vaccine vectors, not pathogenic
in humans, can be made replication-competent or deficient, and can be administered orally
(Imler, 1995). Several viral antigens have been expressed and delivered by adenoviral vectors,
resulting in humoral, cell-mediated or mucosal immune responses (Fooks et al., 1995; Mittal
et al., 1996; Xiang et al., 1996). Non-infectious self-assembled virus-like particles have also
been used as delivery systems for antigens. Fusion of a viral epitope to the N-terminus of the
VP2 capsid protein of Parvovirus led to the assembly of parvovirus-like particles, which were
able to deliver the foreign epitopes into the cytosol, resulting in stimulated cellular immunity
(Sedlik et al., 1997).
Although a number of human clinical trials with different viral recombinant vectors have been
performed, no vaccine based on live viral delivery is yet available on the market. The main
applications for virus-based vectors would initially be for certain specific diseases, such as
vaccines for HIV and cancer, as well as veterinary vaccines.
CHRISTIN ANDERSSON
26
5. Nucleic Acid Vaccines
Nucleic acid vaccines represent a rather recent approach to the control of infectious agents.
These novel vaccines consist of DNA (as plasmid) or RNA (as mRNA), although the use or
RNA has not yet been as well studied. Tables 7A and 7B show selected examples of
immunization studies using DNA or RNA, respectively.
5.1. DNA vaccines
DNA vaccines consist of designed eukaryotic expression plasmids. The gene encoding the
antigen (or antigens) of interest is placed under the control of a strong viral promoter,
recognized by the mammalian host. Inoculation of plasmid DNA vaccines into muscle or skin
results in uptake of the DNA into cells, expression of the encoded antigen and as a result, a
raised antigen-specific immune response. To enable bacterial propagation of the plasmid, it
also contains an E. coli origin of replication. The administration of plasmid DNA encoding a
specific protein antigen was first shown to induce expression of the encoded protein in mouse
muscles (Wolff et al., 1990). Soon, it became clear that immunization with plasmid DNA also
could elicit antibodies to the encoded antigen (Tang et al., 1992). Protective immunity against
e.g. influenza (Ulmer et al., 1993) and malaria (Sedegah et al., 1994) has also been shown.
Extensive work has been done on DNA vaccines the past decade, and DNA vaccines have
been shown to be well tolerated, capable of stimulation of a broad immune response,
including cytotoxic T-lymphocytes (CTLs), and generating lasting immune responses
(Yankauckas et al., 1993; Davis et al., 1993). The efficacy of DNA vaccination has been
reported in small and large animal models for infectious diseases, cancer and autoimmune
diseases (Donnelly et al., 1997b; Kowalczyk and Ertl, 1999), of which selected examples are
shown in Table 7A. Vaccines for diseases such as cancer (Restifo et al., 1999), HIV (Calarota
et al., 1998) and malaria (Hoffman et al., 1997; Wang et al., 1998a and b; Le et al., 2000) are
at present being evaluated in human clinical trials.
RECOMBINANT SUBUNIT VACCINES
27
Table 7A: Selected examples of immunization studies using DNA (modified from Kowalczykand Ertl, 1999.
Pathogen Encoded antigen Reference
Viruses
Ebola virus nucleoprotein (NP), glycoprotein (GP)NP, GP
Vanderzanden et al., 1998Xu et al., 1998
Hepatitis B virus hepatitis B surface antigen (HBsAg)HBsAg
Davis et al., 1997Prince et al., 1997
Herpes simplex virus 1 glycoprotein B, glycoprotein Dglycoprotein B
McClements et al., 1997Daheshia et al., 1998
HIV-1 nef, rev, tat, glycoprotein160, p24nef, rev, tatenvgag
Hinkula et al., 1997Calarota et al., 19981
Sasaki et al., 1998Qiu et al., 2000
Influenza hemagglutinin (HA)HA, NP, matrix protein (M1)HA
Ban et al., 1997Donnelly et al., 1997aUlmer et al., 1998
Measles HA, NP Cardoso et al., 1998
Papilloma E1, E2 Han et al., 2000
Rabies G proteinG protein
Wang et al., 1997Lodmell et al., 1998
Respiratory syncytial virus F proteinG protein
Li et al., 1998Li et al., 2000
Bovine RSV G protein Schrijver et al., 1998
Rotavirus viral protein 4 (VP4), VP6, VP7 Chen et al., 1997
Bacteria
Borrelia burgdorferi Outer surface lipoprotein A (OspA) Simon et al., 1996
Clamydia trachomatis major outer membrane protein (MOMP) Zhang et al., 1999
Clostridium tetani tetanus toxin C fragment Saikh et al., 1998
Mycoplasma turbeculosis Antigen 85 (Ag85)heat shock protein 65 (hsp65)
Ulmer et al., 1997Bonato et al., 1998
Salmonella typhii Outer membrane protein C Lopez-Macias et al., 1996
Parasites
Plasmodium falciparum circumsporozoite protein (CSP)CSP, Exp-1, surface sporozoite protein2(SSP2), LSA-1CSP
Wang et al., 1998a1
Wang et al., 1998b
Le et al., 20001
Plasmodium yoelii CSP Gramzinski et al., 1997
Trypanosoma cruzii trans-sialidase (TS) Costa et al., 1998
1 Human trials
CHRISTIN ANDERSSON
28
Table 7B: Selected examples of immunization studies using RNA or plasmid DNA encodingself-replicating RNA
Pathogen Encoded antigen System used Reference
Viruses
SIV glycoprotein 160 (gp160) rSFV viral particles Mossman et al., 1996
HIV envgp160
rSFV viral particlesrSFV viral particles
Brand et al., 1998Berglund et al., 1997
Influenza hemagglutinin (HA)nucleoprotein (NP)
NPNP
rSFV RNArSFV RNA andrSFV viral particlesrSFV viral particlesrSindbis viral particles
Dalemans et a., 1995Zhou et al., 1994
Berglund et al., 1999Tsuji et al., 1998
Japanese encephalitisvirus
E protein, nonstructuralprotein (NS) 1 and NS2a
rSindbis plasmid Pugachev et al., 1995
Looping ill virus envelope proteins (prME),NS1envelope proteins (prME),NS1
rSFV viral particles
rSFV plasmid andrSFV viral particles
Fleeton et al., 1999
Fleeton et al., 2000
Herpes virus gB rSindbis plasmid Hariharan et al., 1998
Parasites
Plasmodium yoelii epitope derived fromcircumsporoziote protein
rSindbis viral particles Tsuji et al., 1998
Plasmid DNA can be administered in several ways, but so far the most common immunization
route is intramuscular injection of plasmid in saline. Other routes that have been investigated
used include intradermal (i.d.) (Schrijver et al., 1998), intravenous (i.v) (Liu et al., 1997;
Böhm et al., 1998), nasal (Sasaki et al., 1998; Ban et al., 1997) or even ocular routes
(Daheshia et al., 1998). Comparative studies have shown that the route of administration can
have great impact on the elicited immune response (Barry and Johnston, 1997; Pertmer et al.,
1996). When using the intradermal route, plasmid can be either injected with a syringe or
administered with a gene gun (Pertmer et al., 1995; Dégano et al., 1998). Using the gene gun,
plasmid DNA is precipitated onto an inert particle (usually gold beads) and forced into the
cells by a helium blast. The gene gun strategy is more efficient in delivering the plasmid into
the cells, and the amount of DNA needed for gene-gun immunization can be as low as a few
nanograms (Pertmer et al., 1995; Dégano et al., 1998).
Delivery routes to the mucosal surface, for example direct delivery of nucleic acids to the
nasal or oral tract have been demonstrated to have the capacity to induce mucosal immune
RECOMBINANT SUBUNIT VACCINES
29
responses (Ban et al., 1997). Other delivery methods to mucosal surfaces is the use of
intracellular bacteria as carriers for plasmid DNA (Sizemore et al., 1995), the use of
incorporation of DNA into microspheres (Jones et al., 1997) and the use of lipid-based DNA
delivery systems (Felgner et al., 1995; Gregoriadis et al., 1997). More effective systems for
delivering the DNA to the nucleus also need to be further evaluated. Adjuvants and delivery
systems evaluated in DNA vaccine studies are discussed in section 5.2.1.
DNA vaccines have some advantages over traditional vaccines. Immunologically, due to
endogenously production of the antigen, DNA vaccines induce the same immune response as
a natural infection (Kowalczyk and Ertl, 1999), resulting in the induction of a cellular
response, generally not elicited with protein-based vaccines. In a comparative study using the
SV40 large tumor antigen, plasmid DNA showed to be superior to immunization with proteins
or virus in inducing antigen-specific CTLs (Schirmbeck et al., 1996). Moreover, DNA
immunizations circumvents problems like incorrect folding and lack of post-translational
modifications that has been associated with protein subunit vaccines. Compared to protein-
based vaccines, the cost of production is also drastically reduced due to simplified production
and purification (Ferriera et al., 2000). Another advantage of DNA vaccines compared to live
attenuated vaccines is that there is no associated risk of reversion to virulence. Vaccines based
on DNA is also easy to distribute since the DNA is extremely stable and does not require cold
chains, which is difficult to maintain in developing countries (Kowalczyk and Ertl, 1999).
Some comparative studies involving DNA vaccines have been performed e.g. DNA encoding
human wild type p53 induced protective immune responses in mice, but the same gene
delivered with a viral vector did not (Hurpin et al., 1998). However, others have experienced
that the efficacy of nucleic acid vaccines is very low and highly variable (reviewed in
Manickan et al., 1997). Thus, one emerging opinion is, that since the antibody response raised
from DNA vaccines is generally slow and not very potent, and unless they are improved,
DNA vaccines might serve best as priming agents in combination vaccines. (Kowalczyk and
Ertl, 1999).
CHRISTIN ANDERSSON
30
5.2. Improving immune responses to DNA vaccines
A large number of approaches have been used in attempts to improve the efficiency of DNA
vaccines (Rodriguez and Whitton, 2000). One strategy is to optimize of the many sequence
elements included in the plasmid used, e.g. promoter, introns, enhancers and poly-adenylation
signals (Böhm et al. 1996; Danko et al. 1997; Montgomery et al., 1993). The antigen-
encoding gene can be optimized in several ways, like eliminating sequences encoding
intracellular domains or transmembrane sequences (Chen et al., 1998) or reduction of the size
of the antigen-encoding sequence by identifying the immunodominant epitopes, leaving only
defined epitopes for B or T cells. The latter approach has been used with T cell epitopes
(McCabe et al., 1995; Casares et al., 1997). In some cases, the full length antigen is too toxic
or immunosuppressive for the host, and insertion of only the immunodominant epitopes into a
highly immunogenic core sequence will activate the immune system in a proper way (Barry
and Johnston, 1997; Levy et al., 1993). Strategies to design effective DNA vaccines could
also be to create multi-epitope DNA vaccines, in which antigen-encoding genes or genes
encoding specific epitopes could be fused together, to simultaneously immunize for several
diseases (Suhriber, 1997).
Immune responses after DNA immunization might be improved by altering the immunization
schedule, for example fewer immunizations and longer resting periods between
immunizations have shown to develop three to tenfold higher antibody levels (Fuller et al.,
1997). As mentioned above choice of route can have impact on the elicited immune response,
e.g. immunization at mucosal surfaces induce mucosal immunity (Ban et al., 1997). Another
strategy would be to target DNA vaccines to cell-specific ligands. Attempts have been made
to specifically target a DNA vaccine to the antigen-presenting cells e.g. by using DNA
encapsulated in mannose-coated liposomes (Toda et al., 1997) for the purpose of targeting
receptors on dendritic cells. Even targeting to the nucleus to increase the transport of DNA to
the nucleus has been investigated, for example inclusion of nuclear localization sequences in
DNA complexes has been shown to be increase transfection efficiency in vitro (Kaneda et al.,
1989).
RECOMBINANT SUBUNIT VACCINES
31
5.2.1. Adjuvants and delivery systems forDNA vaccines
To enhance or modulate immune responses elicited by DNA vaccines, different adjuvants or
delivery systems can be used. Table 8 presents selected examples used in immunization
studies.
Table 8: Selected examples of adjuvants and delivery systems used for plasmid DNA
Adjuvant /delivery system Reference
Aluminum adjuvant Ulmer et al., 2000
Cationic liposomes Gregoriadis et al., 1997Ishii et al., 1997
Cationic lipid mixtures Norman et al., 1997
Cationic microparticles Singh et al., 2000
Monophosphoryl lipid A (MPL) Sasaki et al. 1997
Poly(DL-lactide-co-glycolide) microparticle Jones et al. 1997
QS 21 Sasaki et al. 1998
Bacterial delivery systems
Listeria monocytogenes Dietrich et al., 1998
Salmonella typhimurium Darji et al., 1997
Shigella flexneri Sizemore et al., 1997
Live, attenuated bacteria have been used as carriers for plasmid DNA, for example attenuated
Shigella flexneri that has the capacity to invade mammalian cells upon infection. Delivery of
plasmid to the cytoplasm of infected cells using S. flexneri as delivery system (Sizemore et al.,
1995) has been demonstrated. Furthermore, high serum antibody titers in mice immunized
intranasally with such S. flexneri delivery system (Sizemore et al., 1997) has been shown.
Delivery of plasmid DNA encoding model antigens to macrophages was studied in vitro using
Listeria monocytogenes as suicide vector (Dietrich et al., 1998) and successful expression and
antigen presentation could be shown. The intracellular pathogen Salmonella typhimurium has
also been used for DNA delivery (Darji et al., 1997).
CHRISTIN ANDERSSON
32
5.2.2. Codelivery of stimulatory substances
In order to increase or modulate the vaccine-induced immune response, natural signal
substances from the immune system can be used, codelivered as either active substances or as
plasmid DNA. Codelivery of cytokines has show to modulate the immune response in a
number of ways (Kowalczyk and Ertl, 1999), e.g. codelivery of GM-CSF was shown to
increase the immune response to a rabies antigen (Ertl and Xiang, 1996), but in the same
system, codelivery of IFN-γ was shown to reduce the immune response (Xiang et el., 1997).
An important costimulatory molecule in the immune system is B7, a receptor expressed on
activated APCs that binds T-cell ligands. The gene encoding B7 was shown to enhance the
immune response only when it was inserted in the same plasmid as the gene encoding the
antigen, suggesting that the antigen-specific and costimulatory signals must originate from the
same cell (Conry et al., 1996). Codelivery of B7 with an HIV-1 DNA vaccine dramatically
increased the CTL response in mice (Kim et al., 1997) as well as in chimpanzees (Kim et al.,
1998).
5.2.3. Immunostimulatory DNA sequences
Plasmid DNA without adjuvant has in certain studies been shown capable of inducing strong
immune responses to the encoded antigen (Kowalczyk and Ertl, 1999). As been discussed
before in section 3, this may partly be due to the presence of immunostimulatory DNA
sequences (ISS) in the plasmid DNA (Krieg et al. 1995; Weiner et al. 1997). ISS are non-
methylated DNA sequences containing CpG-dinucleotides that can activate the non-specific,
innate immune response, mainly by activating monocytes, NK cells, dendritic cells (DC) and
B-cells in an antigen-independent manner (Krieg and Kline, 2000). Methylation of the CpG-
dinucleotides has been shown to abolish the immunogenicity of the DNA vaccine (Lipford et
al., 1997). CpG motifs can either be co-administered with the plasmid DNA in the form of
oligonucleotides or the number of ISS in the plasmid can be increased. Ongoing research in
this area will reveal the importance of species-specificity, restrictions of the DNA sequences
outside the CpG-sequences and the potential for CpG as adjuvant for use in humans (Lipford
et al., 1997; Krieg and Kline, 2000).
RECOMBINANT SUBUNIT VACCINES
33
5.3. Safety of DNA vaccines
DNA vaccines have thus far not shown to induce any severe side-effects. There is little
evidence for integration of DNA into the host genome (Nichols et al., 1995) and no
autoimmune reactions or persistent anti-DNA antibodies have been observed. Some studies
have reported production of anti-DNA antibodies, however with very low and transient titers
(Mor et al., 1997).
5.4. RNA Vaccines
Nucleic acid vaccination through the delivery of RNA has been investigated to a lesser extent
than the DNA vaccination. The first applications of delivery of mRNA showed to induce CTL
to the influenza virus nucleoprotein in mice when mRNA was delivered in liposomes
(Martinon et al. 1993). Liposome-mediated transfection of mouse fibroblasts with mRNA
encoding human carcioembryonic antigen (Conry et al., 1995) resulted in a transient
production of antibodies. The rapidly declining antibody levels could reflect a short-lived
protein expression in vivo. One major advantage of RNA vaccines over DNA vaccines would
be that no risk of host-genome integration of the delivered gene exists. However, a
disadvantage of using RNA-based vaccines is that the preparation and administration of RNA
is troublesome because of the low stability of the RNA. This might lead to rapid in vivo
degradation, and thus a short-lived expression of the encoded gene. Furthermore, certain
reagents needed for RNA preparation, e.g. RNA polymerases and capping agents, are rather
costly.
Development of a new generation of RNA vaccines, derived from members of the Alphavirus
family e.g. Sindbis virus and Semliki Forest virus (SFV), have some interesting fetures that
makes them suitable for development of RNA vaccines (Tubulekas et al., 1997). The
alphaviruses have a very broad host range since they infect various mammalian, avian and
insect cells, (Zhou et al., 1994). Also, the RNA genomes of the alphaviruses are self-
replicating, which will make up for e.g. low transfection efficiencies of DNA vaccines. The
mechanism of SFV-based vaccines is shown in Figure 1.
In the alphaviruses, the genes encoding the structural proteins, needed for virus particle
formation, are located in the last third of the genome. When constructing RNA-based vaccine
vectors, those genes are replaced for a gene encoding an antigen. The RNA vector now carry
CHRISTIN ANDERSSON
34
the gene encoding an alphavirus replicase (e.g. SFV replicase) together with the gene
encoding the foreign antigen. Transfection of a mammalian cell with such an RNA construct
will first lead to translation of the replicase. The replicase will be responsible for production
of new full-length RNA genomes and shorter subgenomic RNA molecules, via a negative
strand intermediate. The subgenomic RNA molecule corresponds to the last third of the full
length RNA. Translation of the subgenomic RNA will lead to production of the antigen. Mass
replication of the RNA will theoretically produce up to 200 000 copies of RNA in a single cell
within 4 hours, and the gene product can be as much as 25% of total cell protein (Rolls et al.,
1994).
Figure 1: The life cycle of the Semliki Forest virus. Upon infection of the mammalian host cell, thefull-lenght positive stranded RNA will function as mRNA. Translation of the first two thirds results inpoduciton of the replicase. The replicase will initially be responsible for production of a negativestrand RNA. Using the negative RNA as template, the replicase will mass-produce full-length RNA.The replicase will also recognize an internal promoteer sequence, and start mass-produce subgenomicRNA, corresponding to the last third of the full-lenght RNA. Translation of the subgenomic RNAresults in production of the structural proteins, needed for production of new viral particles.
(A)ncap SFV Replicase Struct.Positive, single strandRNA
Negative strandintermediate RNA
Positive strand full-length RNAPositive strand,subgenomic RNA
Structural proteins, C,p62 and E1
Replicase
Replicase Replicase
(A)ncap SFV Replicase Struct. Struct. (A)ncap
3' 5'
RECOMBINANT SUBUNIT VACCINES
35
The Semliki Forest virus vector systems were first used as a tool for mammalian production
of recombinant proteins (Liljeström and Garoff, 1991, Liljeström, 1994; IV) but has lately
been evaluated in nucleic acid vaccine approaches (Table 7B).
An alphavirus based RNA vaccine can be delivered alone or as packed in a virus particle
(Tubulekas et al., 1997). To achieve packaging in a viral particle, cotransfection of a helper
RNA molecule encoding the genes for the structural proteins is required for production of
viral particles. Only the full-length RNA molecule, lacking the genes encoding the structural
proteins, contains a packaging signal, and will be packed in the produced recombinant virus
particles. The sub-genomic RNA lacks the packaging signal and will thus not be packed in
viral particles. Transfection of cells with these recombinant virus particles will initially lead to
delivery of the full-length RNA to the host cell cytoplasm. However, since the gene encoding
the structural proteins, needed for production of new viral particles, is removed, transfection
of the recombinant virus will not result in production of new viral particles.
RNA alone, encoding the alphavirus replicase and the target antigen, has proven to give in
vivo expression of the antigen (Johanning et al., 1995) resulting in strong immune responses
to the encoded antigen, e.g. influenza virus nucleoprotein in mice (Zhou et al., 1994).
Recombinant SFV particles with packed RNA encoding influenza virus nucleoprotein have
been shown to induce strong immune responses in mice (Zhou et al., 1995), and were recently
demonstrated capable of inducing protective immunity in mice (Berglund et al., 1998). The
immunizations of primates with RNA packed in SFV particles have been demonstrated to
induce strong immune responses to HIV-1 (Berglund et al., 1997) and SIV (Mossman et al.,
1996).
As mentioned above, the low stability of naked RNA makes the preparation and
administration of RNA somewhat troublesome. Those problems have been overcome by the
development of a plasmid DNA vaccine, based on the Sindbis genome (Herweijer et al.,
1995) or the SFV genome (Berglund et al., 1998). When using conventional DNA vectors,
containing a eukaryotic promoter, e.g. the cytomegalovirus promoter (CMV), the amount of
RNA transcribed is totally dependent on the promoter. However, when using SFV-based
plasmids, transcription of RNA and subsequent translation will lead to the production of the
SFV replicase. From this point on, the replicase will be responsible for copying the RNA, and
the alphaviral replication cycle will continue as for the RNA-based system (Figure 1). Thus,
CHRISTIN ANDERSSON
36
plasmids employing the alphaviral self-replication properties on RNA level could be seen as
RNA-vaccines delivered as a DNA precursor, and was presented in Table 7B.
Plasmids derived from alphaviral genomes have been used in expression studies, e.g. high
expression levels of a luciferase reporter gene in cell lines after transfection of a Sindbis
replicon-based DNA plasmid have been reported (Herweijer et al., 1995; Dubensky et al.,
1996). Similar studies using an SFV-based plasmid have shown high level expression of β-
galactosidase in BHK-21 cells upon transfection (Berglund et al., 1998). Plasmids with
alphaviral self-replication properties have also been used in immunization studies in animals
(Table 7B). Immunization of mice with the SFV-based plasmid containing the gene encoding
influenza nucleoprotein induced humoral and cellular immune responses as well as protection
in mice (Berglund et al., 1998). A similar plasmid derived from the genome of the Sindbis
virus have been shown to effectively induce humoral and cellular immune responses as well
as protection to herpes simplex virus in mice (Hariharan et al., 1998). Some attempts to
campare the RNA and DNA based alphaviral systems have been performed. For exemple, in
immunization of mice with plasmid and recombinant viral particles louping ill antigens, the
viral particles were more effective in inducing protective immune responses (Fleeton et al.,
2000). However, more extensive studies have to be performed in order to evaluate the
differences between the systems.
RECOMBINANT SUBUNIT VACCINES
37
PRESENT INVESTIGATION
6. Recombinant production of protein immunogens
6.1. Recombinant strategies for iscom incorporation (I, II, III)
Subunit vaccine research is dependent on efficient production of protein antigens, and
recombinant strategies are today dominating (Liljeqvist and Ståhl, 1999; Ståhl et al., 1999).
As discussed earlier gene fusions are frequently used to improve or alter the characteristics of
the proteins to make the production more efficient (Ståhl et al., 1997; Ståhl et al., 1999). In
this thesis, recombinant strategies for production of protein immunogens furnished with a
hydrophobic tag to improve incorporation into iscoms have been evaluated (I, II, III). For
several adjuvants, such as liposomes, cochleates, non-ionic block polymers and
immunostimulating complexes (iscoms), the protein antigens are normally incorporated by
hydrophobic interactions (Morein et al., 1996). For iscoms, it has been shown that antigens
containing hydrophobic amino acid sequences are efficiently incorporated (Morein et al.,
1995) while hydrophilic antigens instead could be incorporated by partial denaturation to
expose hydrophobic sequences, or by chemical conjugation to preformed iscoms or iscom-
matrix alone (Morein et al., 1995).
Here, two protein sequences with different properties were combined; a hydrophobic tag, to
make direct incorporation into iscoms possible and an affinity tag, to enable efficient affinity
purification of the fusion proteins. As hydrophobic tags, either hydrophobic amino acid
residues (I, II), added N-terminally or C-terminally, or lipids, added by in vivo or in vitro
strategies (III) were investigated.
6.1.1. N-terminal hydrophobic tags (I)
Initially, three different hydrophobic tags, IW, MI and PD (Table 9), were evaluated for their
efficiency to direct iscom incorporation. The first tag IW, composed of numerous isoleucine
(I) and tryptophan residues (W), was selected because similar tags have been shown to
improve the partitioning of recombinant proteins in aqueous two-phase systems (Köhler et al.,
1991; Hassinen et al., 1994).
CHRISTIN ANDERSSON
38
Table 9: Hydrophobic tags used in I and II
Tag Sequence Origin Position
IW IWIWSIWIWSIWIW Numerous isoleucine (I) andtryptophan residues (W)
N-terminally
MI QILAIYSTVASSLVLLVSLGAISFWMCS The membrane-integration anchorsequence of the H1 hemagglutininof influenza virus A
N and C-terminally
PD LEAALAELAELAE Predicted to form an amphiphaticα-helix at low pH
N-terminally
M ENPFIGTTVFGGLSLALGAALLAG Derived from the membrane-spanning region of SpA
C-terminally
The second tag MI represents the membrane-integration anchor sequence of the H1
hemagglutinin of influenza virus A (Feldmann et al., 1988). Influenza hemagglutinins have
previously been efficiently incorporated into iscoms (Sjölander et al., 1991). The third tag PD,
was designed to form an amphiphatic α-helix at low pH (Parente et al., 1990a, b). As affinity
tag in the three expression systems, the Z domain from Sthaphylococcus aerus protein A
(SpA) was used (Nilsson et al., 1987). In this study, a 53 amino acid malaria immunogen, M5
(Sjölander et al., 1993a) derived from the central repeat region of the Plasmodium falciparum
blood-stage antigen Pf155/RESA (Coppel et al., 1984; Perlmann et al., 1984) (Figure A)
served as model antigen.
General expression vectors were constructed, pT7IWZmp8, pT7MIZmp8 and pT7PDZmp8
designed for intracellular production in E. coli under control of the tightly regulated T7
promoter system (Studier et al., 1990). The target immunogen M5 was introduced into the
multiple cloning site and the three fusion proteins IW-Z-M5, MI-Z-M5 and PD-Z-M5 (Table
10) respectively, were expressed. All three fusion proteins could be recovered as full-length
fusion proteins after affinity purification on IgG-Sepharose. Probably due to the
hydrophobicity of the proteins, insoluble aggregates of IW-Z-M5 and MI-Z-M5 (but not PD-
Z-M5) were formed during the protein production (Table 10). For all three fusion proteins,
rather low expression levels were obtained, in the range of 1 mg per liter cell culture.
Iscom preparations were performed of all three fusion proteins (IW-Z-M5, MI-Z-M5 and PD-
Z-M5, respectively) as described before (Morein et al., 1984; Lövgren et al., 1987). Briefly,
proteins were mixed with cholesterol (including a small amount of 3H-cholesterol for
subsequent analysis of cholesterol content), phosphatidyl choline and Quil A.
RECOMBINANT SUBUNIT VACCINES
39
Figure A: The life cycle of the Plasmodium falciparum parasite in humans and in the mosquito.
At present, at least 300 million people are infected by malaria globally and there are between 1-1.5million deaths from malaria per year (WHO). The causative agents in humans are four species ofPlasmodium protozoan parasites; P. falciparum, P. vivax, P. ovale and P. malariae. Of these, P.falciparum accounts for the majority of infections and is the most lethal. The female Anopheles mosquitois responsible for transmission of parasites between humans. The parasites develop in the gut of themosquito and are transferred to the blood stream of the human host when the mosquito feeds on humanblood. The life cycle is divided into several stages in the mosquito the liver and the erythrocytes and, asoutlined schematically in figure A. Symptoms of malaria include a high fever as a consequence oferuption of erythrocytes. If not treated, the disease, particularly that caused by P. falciparum, progressesto severe malaria. Severe malaria is associated with death. Traditional methods for controlling malaria,like the use of insecticides against the mosquito and also drug treatment for prevention or cure ofinfection have lead to insecticide-resistant mosquitoes and drug-resistant malaria parasites. Thereforethere is need for a potent malaria vaccine. Attempts have been made to construct vaccines to all stages inthe life cycle (Cox, 1991). Pre-erythrocyte vaccines would block the infection of erythrocytes, andvaccines to the sexual stage, the gametocytes, would block the transmission of the parasite betweenindividuals. An effective vaccine to reduce the clinical effetcts of malaria would be directed to the blood-stage of the parasite. The M5 antigen (Sjölander et al., 1993a) used in paper I, II and III is derived froman immunodominant part of the P. falciparum ring-infected erythrocyte surface antigen Pf155/RESA(Perlman et al., 1984). EB200, used in paper IV, is derived from Pf332, a highly repetitive, late asexualintraerythrocytic protein, expressed on erythrocytes (Mattei et al., 1992; Mattei and Scherf, 1992;Hinterberg et al., 1994).
Sporozoites
Mosquito gut
Merozoites
Mosquitoblood meal
Sporozoites
Ring
Trophozoite
Gametocytes
Gametocyte
Zygote
Ookinete
Mosquitoblood meal
Human
Schizont
Scizont
Liver
MALARIA
Anopheles
CHRISTIN ANDERSSON
40
Table 10: Fusion proteins with hydrophobic/ lipophilic tags, used in I, II and III
Study Plasmid Fusion protein Mw
(kDa)
Expressionlevel (mg/l
cell culture)
Solubility* Incorporationefficiency
I pT7IWZM5mp8 IW-Z-M5 17.7 > 1 insoluble high
pT7MIZM5mp8 MI-Z-M5 18.7 > 1 insoluble high
pT7PDZM5mp8 PD-Z-M5 17.1 > 1 soluble no
II pT7ABPM5M ABP-M5-M 25.5 40 n.d.1 low
pT7ABPM5MI ABP-M5-MI 24.9 6 n.d. high
pT7ZZM5M ZZ-M5-M 28.0 40 n.d. high
pT7ZZM5MI ZZ-M5-MI 27.3 66 n.d. high
III pMLHABP∆SAG1 lpp-His6-ABP-∆SAG1 42.4 7 insoluble high
PAff8∆SAG1 His6-ABP-∆SAG1 41.6 102 soluble high
* Dominating fraction indicated, 1 not determined
The mixture was dialysed and subjected to ultracentrifugation in sucrose gradient. Analysis of
individual fractions of the gradient was performed to evaluate the iscom association
efficiency. The fractions were analyzed for; (i) cholesterol content, the result is indicated with
a horizontal bar in Figure 2, (ii) protein content by Bradford analysis, and (iii) protein content
by a Z-specific ELISA and an M5-specific ELISA. The results are summarized in Figure 2.
The comigration of protein and cholesterol in the sucrose gradient has been considered
indicative of successful iscom association.
Two of the hydrophobic tags evaluated in the first study (I) were found to mediate efficient
iscom association; IW and MI, (Figure 2A and B, Table 10) as the protein analyses showed
the highest protein content in the cholesterol-rich fractions. However, analysis of iscom-
incorporation experiment of the fusion protein PD-Z-M5, indicated that there was no or very
little incorporation of the protein immunogen into iscoms, since almost all protein was found
in the upper fractions of the sucrose gradient. These results are normally seen with hydrophilic
antigens. Since the PD tag was predicted to be pH-dependent, the iscom incorporation
experiments was performed at various pHs (pH 3-8) but with the same negative results. These
results were further supported by the fact that the fusion protein PD-Z-M5 showed
hydrophilic properties during the protein expression and purification procedure.
RECOMBINANT SUBUNIT VACCINES
41
Figure 2A-J: Analysis of the protein content in the fractions collected from sucrose gradient ultracentrifugationof iscom preparations performed in paper I, II and III. The respective fusion protein is shown above each graph.Total protein content is shown on the left y-axis ( ) and the results from an ELISA, specific for the affinity tagused in each study (Z, ZZ or ABP, respectively) is shown on the right y-axis ( ). The fraction number is indicatedon the x-axis. The horizontal bar in each graph indicates the cholesterol-rich fractions. Figure 2J shows theresults from a control experiment with protein, iscom matrix but without the chelating lipid.
0.15
0.2
0.25
0.3
0.35
0.4
0.45
1 5 10 15
0.05
0.15
0.25
0.35
0.45
A: IW-Z-M5
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.35
0.15
0.2
0.25
0.3
0.4
0.45
0.5
1 5 10 15
B: MI-Z-M5
0.15
0.25
0.35
0.45
0.55
0.65
0.75
0.85
0.95
1 5 10 150
0.2
0.4
0.6
0.8
1
1.2
1.4
C: PD-Z-M5
0.2
0.4
0.6
0.8
1
1 5 10 140
0.1
0.2
0.3
0.4
0.5
D: ABP-M5-M
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0.75
0.8
0.85
1 5 10 14
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0.55
E: ABP-M5-MI
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 5 10 140
0.1
0.2
0.3
0.4
0.5
0.6
F: ZZ-M5-M
0.2
0.3
0.4
0.5
0.6
0.7
1 5 10 140
0.1
0.2
0.3
0.4
0.5
0.6
G: ZZ-M5-MI
0.2
0.25
0.3
0.35
0.4
0.45
0.5
1 50
0.1
0.3
0.3
0.35
0.4
0.45
1 50
0.1
0.2
0.3
0.4
0.5
10 15
I: His6-ABP- SAG1
0.2
0.3
0.4
0.5
0.6
1 50
0.1
0.2
0.3
0.4
1510
J: His6-ABP- SAG1H: lpp-His6-ABP- SAG1
0.4
0.2
0.15
0.2
0.25
10 15
CHRISTIN ANDERSSON
42
While the fusion proteins IW-Z-M5 and MI-Z-M5 formed aggregates during expression, the
protein PD-Z-M5 was found to be soluble upon expression in E. coli. The results are
summarized in Table 10. Furthermore, the iscom formation was supported by electron
microsopy inspection of the cholesterol-rich fractions from iscom preparation. The iscom
preparations of the proteins IW-Z-M5 and MI-Z-M5 contained typical iscom structures
(Figure 3), while iscom preparations of the protein PD-Z-M5, in contrast, contained no iscom
structures.
To evaluate the immunogenic properties of the iscom preparations, immunization studies were
performed in mice. Sera from mice immunized with the IW-Z-M5 and MI-Z-M5 iscom
preparations, respectively, contained M5-specific IgG-titers of approximately 1:1000 (mean)
after the first immunization. After the booster immunization, the M5-specific antibody levels
increased 10-fold.
Figure 3: A typical iscom structure, as evaluated by electron microscopy analysis.
RECOMBINANT SUBUNIT VACCINES
43
6.1.2. C-terminal hydrophobic tags (II)
In an effort to improve the system for hydrophobic tagging of immunogens for efficient iscom
incorporation, new, improved general expression vectors were designed. The major drawback
with the previously presented system (I) was the overall low production levels of the proteins.
It has been suggested that translation initiation sequences containing hydrophobic amino acids
will decrease production levels (Makrides, 1996) and in order to increase the production
levels of the fusion proteins, the hydrophobic tag was instead placed in the C-terminus of the
fusion proteins (II). As hydrophobic tags, the most successful hydrophobic tag from the
previous study, the membrane-integration MI tag from influenza hemagglutinin (Feldmann et
al., 1988) was included (Table 9). The second tag used in this study, denoted M, is derived
from the membrane-spanning region of Staphylococcus aureus protein A (SpA) (Navarre and
Schneewind, 1994; Schneewind et al., 1995) (Table 9). The rational for selecting the latter
was that a hydrophobic tag of bacterial origin might be more efficiently produced in E. coli.
To make the affinity purification process more efficient, the divalent ZZ tag was utilized as
affinity tag, since it has been shown (Nilsson et al., 1994), that ZZ has a ten times higher
affinity for its ligand IgG, than the monovalent Z domain. The ZZ domains are also highly
soluble, and it has been shown in other studies that ZZ as fusion partner can increase the
overall solubility of fusion proteins (Samuelsson et al., 1991 and 1994). This might be of
importance to minimize the formation of unwanted hydrophobic aggregates during the
purification and iscom incorporation processes. As an alternative affinity tag, the serum
albumin-binding protein ABP (Larsson et al., 1996), derived from streptococcal protein G
(Nygren et al., 1988), was included. The ABP tag is also known to be highly soluble, and
binds human serum albumin (HSA), which makes it suitable as tag for affinity purification
procedures (Ståhl et al., 1999).
Four new expression vectors were thus constructed pT7ABPM, pT7ABPMI, pT7ZZM and
pT7ZZMI, and a gene fragment encoding the same model immunogen M5 as in the previous
study was introduced into the vectors. The four encoded fusion proteins ABP-M5-M, ABP-
M5-MI, ZZ-M5-M and ZZ-M5-MI (Table 10), were produced intracellularly in E. coli under
control of the phage T7 promoter (Studier et al., 1990). The fusion proteins could be affinity
purified on either HSA-Sepharose (ABP-fusions) or IgG-Sepharose (ZZ-fusions) and all four
proteins were recovered as full-length fusion proteins. Compared to the initial study, the
expression levels were now significantly improved, being in the range of 6-66 mg per liter of
CHRISTIN ANDERSSON
44
cell culture for the four fusion proteins, ABP-M5-M, ABP-M5-MI, ZZ-M5-M and ZZ-M5-MI
(Table 10).
Iscom incorporation experiments of the fusion proteins ABP-M5-M, ABP-M5-MI, ZZ-M5-M
and ZZ-M5-MI, respectively, were performed as described above. Again, the iscom
preparations were subjected to a sucrose gradient centrifugation. Collected fractions were
analyzed for cholesterol content as described above, and for (i) total protein content according
to Bradford (Bradford, 1976) and, (ii) the presence of the affinity tags in ABP-or ZZ-specific
ELISA experiments. The results are summarized in Figure 2D-G.
As discussed earlier, comigration of protein and cholesterol indicates successful iscom
association of the protein. All four proteins ABP-M5-M, ABP-M5-MI, ZZ-M5-M and ZZ-
M5-MI could be found in the cholesterol-rich fractions of the sucrose gradient as shown by
both protein analyses with high protein content in the cholesterol-rich fractions. When
analyzing the protein content in fraction of iscom preparation of ABP-M5-M, the total protein
analysis (Bradford) indicated that only a fraction of the fusion protein was found in the
cholesterol-rich fractions while the ABP-specific ELISA showed a more equal distribution
between the cholesterol-rich fractions and the upper fractions. The reason for this discrepancy
is not known. Nevertheless, electron microscopy inspection of all four iscom preparations
verified the presence of the typical iscom structures.
Immunization of mice with iscom preparations containing ABP-M5-M, ABP-M5-MI, ZZ-
M5-M and ZZ-M5-MI, respectively resulted in M5-specific serum IgG titers of about 1:100
(mean) after the first immunization and a 10 to 100-fold increase after a booster
immunization. Interestingly, despite the apparent lower incorporation of the fusion protein
ABP-M5-M into iscoms, mice immunized with ABP-M5-M-iscoms also demonstrated high
levels of M5-specific antibodies. To further evaluate this, a control immunization, using the
hydrophobically tagged antigens (ABP-M5-M) simply admixed with iscom matrix was
performed. This immunization resulted in significant induction of M5-specific antibodies
(mean titers 1:1000), indicating that the introduction of a hydrophobic tag into a normally
hydrophilic immunogen, could be sufficient to allow the formation of hydrophobic
aggregates, most probably also including iscom-matrix components, capable of inducing
significant antibody responses. A drawback with this strategy could however be that the well-
defined iscom structures would not be formed.
RECOMBINANT SUBUNIT VACCINES
45
6.1.3. Lipid tagging of protein immunogens (III)
As an alternative to using hydrophobic amino acid residues as hydrophobic tags, a strategy
based on lipid tags was investigated. In order to develop a general system for production of
lipid-tagged immunogens, a novel expression vector, pMLHABP, was constructed. The
expression is under the control of the tac promoter (Amann et al., 1988). The vector encodes
the 20 amino acid signal peptide and the nine N-terminal amino acid residues (lpp) of the
major lipoprotein of E. coli and this sequence is found to be sufficient for lipidation (Ghrayeb
and Inouye, 1984; Laukkanen et al., 1993). A dual affinity tag, consisting of a hexahistidyl
(His6) peptide and the serum albumin binding protein ABP, was used. Introduction of the
His6-ABP dual affinity tag would offer alternative strategies for affinity purification of
expressed fusion proteins, by either immobilized metal ion affinity chromatography (IMAC)
(Hochuli et al., 1988) or HSA-Sepharose columns (Ståhl et al., 1999).
To make comparison possible, an in vitro lipidation strategy was also investigated. This
strategy utilizes a metal-chelating lipid, which could be linked to hexahistidyl peptides and
would thus not depend on the E. coli-lipidation machinery. To produce recombinant
immunogens for in vitro lipidation via a chelating peptide, the expression vector pAff8c
(Larsson et al., 2000) was used. This vector encodes the same dual affinity tag, His6-ABP, as
pMLHABP. The expression is under control of the T7 promoter (Studier et al., 1990). As
model immunogen in both systems, a 238 amino acid immunogen ∆SAG1, derived from the
central region of the major surface antigen SAG1 (amino acid residues 61-298 of the native
SAG1) (Burg et al., 1988; Harning et al., 1996) of Toxoplasma gondii (Figure B) (Grimwood
and Smith, 1992), was used.
The constructed plasmids, pMLHABP∆SAG1 and pAff8∆SAG1, thus encode the fusion
proteins, lpp-His6-ABP-∆SAG1 and His6-ABP-∆SAG1, respectively (Table 10). The fusion
proteins were evaluated for in vivo and in vitro lipidation experiments and subsequently also
in iscom incorporation experiments.
CHRISTIN ANDERSSON
46
Ingestion
Replication
Replication
Bradyzoites
Cat Other mammalsand birds
Oocyst
Intestinalepithelium
Sporozoites
Tachysoites
Gastro-intestinaltract
Tissue cyst
Oocyst
Bradyzoites
Tissue cyst
Intestinalepithelium
Oocyst
Figure B: The life cycle of the Toxoplasma gondii parasite in cats and other mammals, including humans.
Toxoplasmosis is caused by Toxoplasma gondii , an obligate intracellular parasite that can infect a widerange of animals, including humans. The parasite has a two-phase lifecycle: a sexual phase, which occursin cats and other felides, and an intermediate, non-sexual phase, which can take place in any mammal orbird. The intermediate host can be infected by ingesting oocysts. The oocysts contain sporozoites that arereleased in the gut, penetrate the intestinal epithelia and start to replicate. After about two weeks, theproliferation is slowed down and tissue cysts, containing bradyzoites, are formed. Tissue cysts are mainlylocalized in muscle tissue and the central nervous system, and can be viable for a long time. If an infectedanimal is eaten by another mammal or bird, the tissue cyst is destroyed by digestive enzymes, bradyzoitesare released, penetrate the epithelia ands start to replicate. If the new host is a cat, the parasite developsinto sexual stages, and after about 3-5 days, new oocysts are excreted in the faeces, completing thelifecycle. T. gondii parasite is widely distributed around the world. In veterinary medicine, toxoplasmainfection is well known as a cause of abortion and neonatal mortality in sheep and goat. Humans canaquire the infection from food contaminated with oocysts or meat containing tissue cysts. The infection isusually without symptoms. However, if aquired during pregnancy, the result may be miscarriage ormalformation of the child (Remington, 1990). Toxoplasma infection in immunocompromised individuals,including AIDS patients, may cause a life-threatening disease (Araujo and Remington, 1987; Israelski andRemington, 1988). In the search for a vaccine against toxoplasmosis, live attenuated, killed or lysedparasites or different antigenic fraction has been used (Hermentin and Aspöck, 1988; Johnson, 1989). Forexample, immunization of mice with the major surface antigen (SAG-1) of tachyzoites incorporated intoliposomes or mixed with Quil A resulted in protection against lethal infection (Bülow and Boothroyd,1991; Khan et al., 1991). It has also been shown that surface antigens (SAG-1, SAG-2 and others)incorporated into iscoms elicited protective immunity in mice (Lundén et al., 1993) and both humoral andcellular immune response in sheep (Lundén et al., 1995).The severe effects of toxoplasma infection in animals and in humans are well known. Therefore there is agreat need for effective vaccines, both for veterinary and human use.
TOXOPLASMA
Ingestion
RECOMBINANT SUBUNIT VACCINES
47
Both fusion proteins were expressed in E. coli, and could be recovered by affinity purification
using ABP-mediated affinity chromatography on HSA columns (Ståhl et al., 1999; Ståhl et
al., 1989). The fusion protein lpp-His6-ABP-∆SAG1, designed to be lipidated in vivo was,
after disruption of cells, predominantly found in the fraction containing insoluble proteins and
cell debris, indicating membrane association. The intracellularly produced fusion protein His6-
ABP-∆SAG1, intended for in vitro lipidation, was mainly soluble upon expression in E. coli.
The results from the expression in E. coli are summarized in Table 10.
For the in vivo lipidated fusion protein lpp-His6-ABP-∆SAG1, iscoms were prepared as
described above simply by mixing the fusion protein with cholesterol, phosphatidyl cholin
and Quil A, followed by dialysis. The fusion protein His6-ABP-∆SAG1, intended to be
incorporated via interaction of the His6 tag with a Cu2+-chelating lipid (IDA-TRIG-DSGE),
was directly added to pre-formed iscom matrix containing Cu2+ and the chelating lipid. A
control preparation, by simple mixing the fusion protein His6-ABP-∆SAG1 with pre-formed
iscom matrix without addition of the chelating lipid, was prepared (Figure 2J). After a sucrose
gradient ultracentrifugation of the three preparations, the fractions of the gradient were
analyzed for presence of cholesterol, as described above, and for protein content. The protein
analyses performed were; (i) total protein content, according to Bradford (Bradford, 1976)
and, (ii) an ELISA analysis, specific for the affinity tag ABP. The results are summarized in
Figure 2H-J.
A significant portion of the lipidated immunogens, both the in vivo lipidated lpp-His6-ABP-
∆SAG, as wells as the in vitro lipidated His6-ABP-∆SAG1 was found in the cholesterol-rich
fractions of the sucrose gradient, indicating association with iscoms. For the non-lipidated
His6-ABP-∆SAG1 control, only a small fraction was detected the cholesterol fractions,
indicating that lipidation was required for iscom association. Electron microscopy inspection
of the iscom preparations verified the presence of the typical iscom structures.
Mice immunized twice with the three different preparations, respectively, responded after the
first immunization with serum IgG antibodies reactive to His6-ABP-∆SAG1 with titers of
approximately 1:1000. The booster immunization resulted in a 100-fold increase. However,
we could also detect antibody responses in sera from the mice immunized with His6-ABP-
∆SAG1, simply admixed with iscom matrix lacking the chelating lipid. This was surprising
since the fusion protein was not found to a significant extent in the cholesterol-rich iscom
CHRISTIN ANDERSSON
48
fractions after sucrose gradient ultracentrifugation (Figure 2). The results from another control
immunization, consisting of mice immunized with the His6-ABP-∆SAG1 fusion protein in
PBS, showed also significant titers of His6-ABP-∆SAG1 reactive antibodies after a booster
immunization. The results from the control groups indicate that the ∆SAG1 immunogen is
very immunogenic in itself. An earlier study showed that poorly immunogenic hydrophilic
antigens normally fail to elicit antibody responses when administered mixed with iscom
matrix or with PBS alone (II).
Taken together, both the concepts of including hydrophobic amino acid sequences into
recombinant immunogens (I, II), and the presented strategies to obtain in vivo and in vitro
lipidation of recombinant immunogens result in association with the iscoms. Comparing the
two lipidation strategies, one advantage of using the in vivo lipidation strategy is that only the
typical iscom forming material is needed, while the in vitro strategy requires the addition of a
chelating lipid. On the other hand, the expression levels obtained with the in vitro strategy are
much higher and in addition, this strategy offers a much simpler method for iscom formation
since iscom matrix with pre-associated chelating lipid can be prepared in bulk. The general
applicability of the presented strategies needs to be further evaluated for various immunogens.
6.2. Recovery of a vaccine candidate produced in mammalian cells (IV)
A promising vaccine candidate to human respiratory syncytial virus (RSV) (Figure C)
produced in E. coli has been demonstrated to induce protection to virus challenge in rodent
models (Power et al., 1997) and has now progressed into phase III human clinical trials
(http://www.cipf.com). The candidate vaccine, BBG2Na consists of a serum albumin binding
region BB derived from streptococcal protein G (SpG) (Nygren et al., 1988) and amino acids
130-230 of the G glycoprotein of RSV subgroup A (Power et al., 1997). The immunogenic
properties of the E. coli-produced and non-glycosylated BBG2Na is interesting considering
that the native RSV G protein is heavily glycosylated (Collins et al., 1996). Therefore, a
mammalian expression system for production of glycosylated BBG2Na was created in order
to enable comparative immunological studies of non-glycosylated and glycosylated BBG2Na.
Expression vectors based on the positive-strand RNA alphavirus Semliki Forest virus (SFV),
was used to achieve transient expression of BBG2Na in baby hamster kidney cells.
RECOMBINANT SUBUNIT VACCINES
49
Figure C: An overview of the respiratory syncytial virus. The surface proteins are outlined in the picture.
Respiratory syncytial virus (RSV) is the most important cause of viral lower respiratory tract infectionduring infancy and early childhood worldwide. RSV was first isolated in 1956 and has since then beenassociated with lower tract illness during infancy and childhood in many parts of the world (Collins et al,1996). It has also been found that RSV infection is an important agent of disease in immuno-suppressedadults and in elderly peoples (Collins et al., 1996). Outbreaks of RSV disease are abrupt and can last up to 5months, and usually peaks in winter. There are two major strains, A and B, both of them circulatesimultaneously during outbreak, and it has been suggested that group A strains are associated with moresevere disease (Domachowske and Rosenberg, 1999).RSV is an enveloped, negative-sense RNA virus with a genome of about 15 kb encoding 11 viral proteins(Collins et al., 1996). The virus particle is about 60 to 100 nm in diameter and consists of a nucleocapsidsurrounded by a lipid envelope with two major surface proteins, F and G. The F protein is thought tomediate virus penetration while the RSV G protein is thought to mediate virus attachment to cells (Peroulis,1999; Collins et al., 1996). Both F and G protein induce neutralizing antibodies (Dudas et al., 1998). It hasbeen demonstrated that monoclonal antibodies to G protein inhibit virus entry into cell. (Karron et al., 1997).Studies have shown that the G glycoprotein is a major viral antigen and the glycosylated G protein isprotective upon immunization in several animal models (Power et al, 1997). It has been suggested that theglycosylation protect the virus from the host immune system (Collins et al., 1996). A vaccine candidate BBG2Na is a fusion protein consisting of an albumin-binding region fromstreptococcal protein G (BB) and RSV G protein fragment. (G2N) has been extensively studied. Studies withBBG2Na in animal models, mice and cotton rats, has shown that high immune response and also protectionto viral challenge can be obtained when immunizing with a nonglycosylated fragment of the G protein.Administering this E. coli produced fusion protein, BBG2Na, to mice resulted in a strong immune responseof these mice and subsequent protection from RSV infections. Lungs and nasal tracts of immunized micewere efficiently protected to subsequent RSV challenges (Power et al., 1997). Maternal immunizationinduces protection of offspring of test animals, and passive transfer of the maternal antibodies was sufficientto induce a high protective immune response. (Brandt et al., 1997; Siegrist et al, 1999).
Still there is no vaccine for RSV although the virus and the severe effects of infection are well known.
Surface proteinsFGSH
Nucleocapsid-associated proteinsNPL
Matrix proteinMM2
Nonstructural proteinsNS1NS2
RESPIRATORY SYNCYTIAL VIRUS
CHRISTIN ANDERSSON
50
Such vectors have been used to express heterologous proteins in a wide range of host cells,
i.e. insect, avian and mammalian cells (Liljeström, 1994; Liljeström and Garoff, 1991). For
example, biologically active full-length RSV G protein was successfully expressed using an
SFV-based vector (Peroulis et al., 1999).
These SFV vectors were furnished with SP6 phage-derived RNA promoter to enable in vitro
transcription of RNA, and the gene encoding BBG2Na is introduced in the position where the
genes encoding the SFV structural proteins normally reside (Liljeström, 1994; Liljeström and
Garoff, 1991). The positive-strand RNA from such vectors encodes its own RNA-RNA
replicase, responsible for amplificaiton of RNA in vivo. Two different vectors encoding
BBG2Na were created, both utilizing the SFV self-amplification system. The first vector,
pSFV3-l-BBG2Na encodes a cytoplasmic version of BBG2Na, denoted BBG2Nacyt, and the
second vector, pSFV3-spl-BBG2Na, were a signal peptide (sp) (Berglund et al., 1997) was
included upstream of the BBG2Na gene, encodes a secreted version of BBG2Na, denoted
BBG2Nasec. RNA was transcribed in vitro from both vectors and transfection of BHK-21 cells
was performed by electroporation with the RNA constructs encoding BBG2Nacyt and
BBG2Nasec, respectively. Western blot analysis of culture supernatants and cell lysates from
cells expressing the two variants of BBG2Na, was performed. BB-specific antibodies could
detect BBG2Nacyt in the cell lysate and not in the culture supernatant and BBG2Nasec was, as
expected, predominantly found in the culture supernatant. Furthermore, immunofluorescence
microscopy analysis showed a difference in the localization patterns for BBG2Nacyt and
BBG2Nasec. The cells expressing BBG2Nacyt showed an even distribution in the cytoplasm,
while in cells expressing BBG2Nasec, the product was detected in a network-like pattern in the
cytoplasm adjacent to the nucleus. The latter staining pattern is typical of endoplasmic
reticulum and golgi complex localization (Kamrud et al., 1998), and is consistent with
BBG2Nacyt being directed to the secretion machinery. The differential localization of the two
BBG2Na variants indicated that BBG2Nacyt was found predominantly in the cytoplasm while
BBG2Nasec was targeted to the secretion machinery.
Since our aim was to produce the secreted form of BBG2Na, we wanted to develop a recovery
strategy for BBG2Nasec. The first evaluated purification method to recover BBG2Nasec from
culture media, was to use human serum albumin (HSA) columns, thus enabling BB-mediated
recovery (Murby et al., 1995; Ståhl et al., 1999). We initially evaluated HSA affinity
purification using HSA-Sepharose columns. However, analysis of the purification procedure
RECOMBINANT SUBUNIT VACCINES
51
showed that only very low amounts of BBG2Nasec could be recovered. The reason for this is
most probably due to the presence of BSA in the culture medium (originating from the fetal
calf serum (FCS)). The BSA will then compete with HSA for the binding to the BB-tag since
the BB-tag has a lower but still significant affinity for BSA, as compared to HSA (Nygren et
al., 1990). The purification strategy is schematically shown in Figure 4.
In order to improve the recovery, a batch-wise HSA affinity recovery procedure was
investigated, and by this strategy, a certain amount of BBG2Nasec could indeed be recovered
(approximately 4 µg per 10 ml culture supernatant). BBG2Nasec could, however, be detected
in the unbound material indicating inefficient recovery, again probably due to the BSA
competition. Furthermore, the affinity-purified material contained significant levels of BSA
contamination. The results are summarized in table 11.
To decrease the amount of copurified BSA in the eluted material, BBG2Nasec was produced
by an alternative cultivation protocol. Cultivation of the BHK-21 cells was performed using
culture media containing FCS. However, after transfection of the cells, culture media without
FCS was used. By excluding FCS during the protein production, the amount of BSA in the
start material for the purification should be significantly decreased. BBG2Nasec was purified
from the culture supernatant from the alternative production scheme using HSA-Sepharose
columns. Detectable but still only small amounts of BBG2Nasec could be obtained (0.5 µg per
10 ml culture supernatant).
Figure 4: Overview of the two affinity purification strategies used in paper IV. The potentialinterference of BSA is illustrated.
HSA-purification Affibody mediatedpurification
BSA
ZRSV1
ZRSV1
G2NBBHSA
BSA
G2NBB
BSA
G2NBB
CHRISTIN ANDERSSON
52
Table 11: Protein production characteristics of BBG2Nasec
Production/affinity purification mode Recovered BBG2Naseca
(µg/10 ml)Puritya (%)
With FCS/HSA-Sepharoseb 4 ≈ 50
Without FCS/HSA-Sepharose 0.5 ≈ 20
With FCS/ZRSV1 affibody-Sepharose 6 ≈ 30
Without FCS/ ZRSV1 affibody-Sepharose 2 > 90
aEstimated from Commassie-stained SDS-PAGE gels after affinity recovery. bPerformed in batch mode insteadof column.
The purified material also contained copurified BSA, probably remaining from the initial cell
cultivation. These results indicate that the yields and purity of BBG2Nasec purified using the
HSA affinity procedures were insufficient (Table 11).
To circumvent the problems of BSA competition, a column with a product-specific affinity
ligand, which is specific for the RSV G protein part of BBG2Nasec, was created. The ligand, a
recently described RSV G protein-specific binding molecule, ZRSV1 affibody (Hansson et al.,
1999) was created through phage display-mediated selection from a combinatorial library of
the Z protein domain derived from SpA as described above (see section 2.2.5). ZRSV1 binds
amino acids 164-186 of the RSV G protein of both E. coli-produced BBG2Na and
glycosylated RSV G protein from whole virus (Hansson et al., 1999). Dimeric versions of
affibodies have in certain cases been shown to have an apparent higher affinity (Gunneriusson
et al., 1999) and therefore a dimeric construct of the ZRSV1 was created resulting in the fusion
protein ZZ-diZRSV1.
The affibody fusion protein ZZ-diZRSV1 was coupled as ligand to an activated Sepharose
column to create a product-specific affinity column. Purification of culture supernatant from
BBG2Nasec-expressing BHK-21 cells showed that full-length BBG2Nasec could be recovered
(6 µg per 10 ml cell culture supernatant) (Table 11). Still, significant amounts of co-purified
BSA were present, probably as a result of interaction of the BB tag of BBG2Nasec with BSA
(Figure 4). Again, the production of BBG2Nasec using serum-free media was evaluated.
Production of BBG2Nasec in culture medium with BSA omitted during protein production and
recovery using the G2Na-specific affibody column, resulted in a product yield of
RECOMBINANT SUBUNIT VACCINES
53
approximately 2 µg BBG2Nasec per 10 ml culture supernatant with only very low amounts of
BSA contamination (Table 11).
We thus succeeded in creating a system for production and recovery of BBG2Nasec. The
possible differences in antigenicity and immunogenicity between BBG2Na produced in
mammalian cells and the vaccine candidate, produced in E. coli can thus be initiated.
7. Nucleic acid vaccines
7.1. Protection to RSV elicited in mice by plasmid DNA immunization (V)
As described above, respiratory syncytial virus (RSV) is a major cause of lower-respiratory
tract infections in young infants and elderly (Collins et al., 1996). The bacterially produced
protein subunit vaccine BBG2Na (Murby et al., 1995) has been able to induce protection in
mice and cotton rats against RSV challenge (Power et al., 1997; Corvaia et al., 1997; Brandt
et al., 1997; Plotnicky-Gilquin et al., 1999 and 2000; Siegrist et al., 1999), and this RSV
vaccine candidate has progressed into phase III human clinical trials (http://www.cipf.com).
Therefore, it would be of interest to evaluate a nucleic acid vaccine strategy using BBG2Na as
antigen (V). The purpose would be to investigate if the same immunogen could elicit
protection when delivered as a nucleic acid vaccine. Nucleic acid based vaccination studies
using genes encoding RSV proteins have been performed e.g. using RSV F protein (Li et al.,
1998) or RSV G glycoprotein (Li et al., 2000), and protective effects have been shown in
rodents.
The SFV system, used as expression system in a previous study (IV) have been evaluated in
nucleic acid vaccination strategies using naked RNA, virus coated-RNA or plasmid DNA
(Tubulekas et al., 1997; Berglund et al., 1998). Therefore, we constructed SFV-based
plasmids designed for DNA immunization encoding BBG2Na in a cytoplasmic (BBG2Nacyt)
or a secreted (BBG2Nasec) form. The plasmids, denoted pBK-SFV3l2-BBG2Na and pBK-
SFV3l2-spBBG2Na, respectively are schematically shown in Figure 5. Both vectors encode
the SFV replicase, responsible for amplification, and the transcription is under control of the
eukaryotic cytomegalovirus (CMV) immediate early promoter (Berglund et al., 1998).
CHRISTIN ANDERSSON
54
Figure 5: Plasmid vectors, transcription products and encoded fusion proteins. (A) The two expression vecors,pBBG2Nacyt and pBBG2Nasec, encoding the SFV replicase (enabling in vivo amplification) and the cytoplasmicand secreted variants of BBG2Na, respectively. The transcription is under control of the CMV immediate earlypromoter (pCMV). (B) The positive strand RNAs, transcribed by the transfected cells, will be self-amplified in thecytoplasm of transfected cells, since they encode the SFV replicase responsible for the RNA-dependent RNA-replication. (C) The encoded gene products from the plasmid vectors, BBG2Nacyt and pBBG2Nasec, the latterproduct furnished with a signal peptide (sp) for direction to the secretion machinery. The indicated theoreticalmolecular masses are after processing of the signal peptide are indicated.
To investigate the functionality of the plasmids, BHK-21 cells were transfected with the two
plasmids, respectively. Analysis of culture supernatants and cell lysates by Western blot
demonstrated that BBG2Nacyt was detected only in the cell lysate, while BBG2Nasec was
found both in the culture supernatant and the cell lysate. Further expression studies were
performed using immunofluorescence microscopy analysis. The same difference in
localization patterns for BBG2Nacyt and BBG2Nasec as described in earlier studies (IV) was
shown. The BBG2Nacyt was detected in the cytoplasm while BBG2Nasec was detected in a
network-like pattern in the cytoplasm adjacent to the nucleus, a staining pattern typical of
endoplasmic reticulum and golgi complex localization (Kamrud et al., 1998). The results from
A
B
Neo
pCMV
Ori
SFV-Repl. BBG2Na
BBG2Nacyt BBG2Nasec
Neo
pCMV
C
(A)ncap(A)ncap
pBBG2Nacyt pBBG2Nasec
38.2 kDa 38.3 kDa
Ori
SFV-Repl. sp BBG2Na
SFV-Repl. BBG2Na SFV-Repl. sp BBG2Na
BBG2Na sp BBG2Na
RECOMBINANT SUBUNIT VACCINES
55
the expression studies suggest that the plasmids are functional in terms of BBG2Na
expression and localization of the encoded proteins.
In order to investigate the potential of the plasmids as vaccines, mice were immunized
intramuscularly with the plasmids pBBG2Nacyt, pBBG2Nasec or a control plasmid, pControl
(encoding only the SFV replicase and E. coli β-galactosidase). None of the mice demonstrated
detectable RSV-specific antibodies. However, while sera from mice immunized with
pBBG2Nacyt did not contain detectable BBG2Na-specific antibodies, all five mice immunized
with pBBG2Nasec developed BBG2Na-reactive antibody responses. Evaluation of the lung
protective efficacy against RSV challenge induced by these plasmids, showed clearly better
results in the group immunized with pBBG2Nasec, where sterilizing lung protection was
obtained in three out of five mice. None of the mice immunized with pBBG2Nacyt or pControl
showed significant virus reduction. The results are summarized in Table 12.
Table 12: Lung protective efficacya to virus challenge expressed as virus titersb(log10) afterintramuscular immunization of mice with plasmids, pBBG2Nacyt , pBBG2Nasec or pControl
and challenge with 105 TCID50 RSV-A.
Immunization group Mouse number Mouse protected/total
1 2 3 4 5 Mean
pBBG2Nacyt 4.95 4.45 4.45 4.45 4.57±0.25 0/4
pBBG2Nasec ≤1.45 ≤1.45 ≤1.45 3.95 3.95 2.45±1.37 3/5
pControl 4.45 4.2 4.95 4.45 4.45 4.5±0.27 0/5
aThe lungs were, in accordance with earlier studies (Power et al., 1997) considered protected (underlinedfigures) when virus titers were reduced by 2 log10 relative to the mice immunized with the control strain.bViraltiters for the challenged mice are expressed as log10 pfu/g of lung tissue, 5 days post challenge. The detectionlimit for the assay is 101.45.
Interestingly, there was an indication of correlation between the virus reduction and the anti-
BBG2Na IgG titer for individual mice in the group immunized with pBBG2Nasec. The
BBG2Na-specific antibody titers in sera from non-protected mice in the BBG2Nasec group
were just above the limit of detection, (102.43) while the protected mice had higher antibody
titers (102.9 to 103.9). Possible explanations for the differences in immunogenic properties
could be different expression levels from the plasmids or differential antigen presentation. The
CHRISTIN ANDERSSON
56
Western blot analysis indicates that BBG2Nasec was expressed at somewhat higher levels, but
more extensive studies would be required to fully assess the differences in expression levels.
An additional possible explanation of the differences in immunogenicity could be that the
presentation of BBG2Na to the immune system could be inefficient. The SFV replicase has
been shown to induces apoptosis in cells (Glasgow et al., 1998) and others have suggested
that apoptotic cells are poor antigen presenters (Barker et al., 1999). Therefore, BBG2Nacyt,
expressed inside such cells could be poorly immunogenic, while the secreted BBG2Na
encounter direct contact with antigen presenting cells and might therefore be efficiently
presented to the immune system. However, the reasons for the different immunogenicity of
the two BBG2Na variants have to be further investigated.
In this study, we demonstrate that protection to RSV challenge can be elicited in mice by
immunizing with DNA encoding a subunit RSV vaccine candidate, BBG2Na, at present
evaluated in human clinical trials as a prokaryotically produced protein immunogen. The
results also indicate that targeting of the antigens expressed by nucleic acid vaccination could
have important effects.
7.2. Immunization with DNA or RNA encoding a malarial antigen (VI)
In order to evaluate the differences in immune responses elicited by the different SFV-
systems, we constructed plasmid vectors to be used either for in vitro transcription of RNA or
direct for immunization of mice. The constructed plasmid designed for transcription of SFV-
based RNA encodes the SFV replicase. The RNA was delivered either as naked RNA or
packaged in SFV suicide particles. The plasmids designed for direct immunization of mice
were of two different types, both containing a cytomegalovirus promoter, but one containing
and one lacking the SFV replicase. The antigen used in this study was a malarial antigen,
EB200 derived from the erythrocyte membrane associated P. falciparum antigen Pf332
(Mattei et al., 1992), and the gene encoding the antigen was introduced into all plasmid
constructs (Figure 6).
The constructed plasmid vector, denoted pSFV3-spl-EB200, designed for in vitro
transcription of RNA in the same fashion as described above (IV) contains the gene encoding
the SFV replicase and the gene encoding EB200 preceded by a sequence encoding a signal
peptide (Berglund et al., 1997). The signal peptide has been used in previous studies (IV and
RECOMBINANT SUBUNIT VACCINES
57
Figure 6: Schematic representation of the plasmids used for in vitro transcription or immunization. (A) Thevector pSFV3-spl-EB200 encoding the SFV replicase (enabling in vivo amplification) and the secreted variant ofEB200. The transcription is under control of the phage SP6 RNA polymerase promoter (pSP6). The positivestrand RNA, suitable for transfection of mammalian cells, are transcribed and capped in vitro, and carry 3' poly-A-tails, which are transcribed from poly-A sequences in the plasmid vector. (B) The vectors pBK-SFV3L2-EB200 and pBK-SFV3L2-spEB200 encode the cytoplasmic or the secreted variant of EB200. The transcriptionis under control of the CMV major immediate-early (IE1) enhancer. (C) The expression vector pCMVintBL-EB200. The CMV major immediate-early (IE1) enhancer, promoter and intron A and the tPA signal sequenceprecede the gene fragment to be expressed (EB200). The vector contains the simian virus 40 (SV40) origin ofreplication, intron and polyadenylation signals from the SV40 T-antigen gene.
V), and has been shown functional in terms of directing the product to the secretion
machinery. The encoded product was denoted EB200sec. Transcription was under control of
the SP6 RNA polymerase promoter, thus enabling in vitro transcription of RNA (Liljeström
and Garoff 1991) as described before (IV).
A
Neo
pCMV
SFV Replicase EB200sp
pBK-SFV3L2-spEB200
Ori E
B
bla
pSP6
SFV Replicase EB200sp
pSFV3-spl-EB200
C
Neo
pCMV
SFV Replicase EB200
pBK-SFV3L2-EB200
Ori E
Ori E
Bla
SV40Ori
EB200
pCMVintBL-EB200HCMVIE1
enh/pr
Ori E
HCMV int SV40 int p(A)
SFV Replicase EB200spcap A(n)
RNA
StPA
CHRISTIN ANDERSSON
58
Plasmid vectors for DNA immunization, pBK-SFV3L2-EB200 and pBK-SFV3L2-spEB200,
encoding the SFV replicase and two variants of the antigen EB200; one cytoplasmic variant
denoted EB200cyt, and one secreted variant denoted EB200sec, respectively, were also
constructed. Again, the signal peptide would enable secretion of the encoded product,
EB200sec. Transcription in both plasmids is under control of the CMV immediate early
promoter, commonly used in plasmid DNA vaccine vectors due to efficient transcription.
Another plasmid, pCMVintBL-EB200, lacking the SFV replicase was also constructed, in
which the gene encoding EB200 is preceded by the tissue plasminogen activator tPA signal
sequence for production of a secreted variant of EB200. In this plasmid, the number of RNA
copies transcribed would only be the result of transcription controlled by the CMV promoter.
To perform the immunizations using RNA in viral particles, in vitro transcription of RNA
encoding the P. falciparum-derived antigen EB200 or a control antigen BBG2Na (IV, V) was
performed. These RNA-constructs were also prepared in a virus-coated fashion by packaging
into into recombinant SFV (rSFV) particles using a two-helper RNA system (Smerdou et al.,
1999).
Mice were immunized intramuscularly with the different RNA and DNA constructs, see Table
13, and antibody responses were monitored.
Table 13: Immunization groups used in study VI
Group Molecule used inimmunizations
Plasmid Encoded protein
A RNA pSFV3-spl-EB200a EB200sec
B RNA pSFV3-spl-BBG2Naa BBG2Nasec
C Plasmid DNA pBK-SFV3L2-EB200 EB200cyt
D Plasmid DNA pBK-SFV3L2-spEB200 EB200sec
E Plasmid DNA pCMVintBL-EB200 EB200sec
F Plasmid DNA pCMVintBL none
G Protein in FIA c - E. coli produced EB200
H rSFV particles pSFV3-spl-EB200b EB200sec
I rSFV particles pSFV3-spl-BBG2Nab BBG2Nasec
J rSFV particles pSFV3-lacZb β-galactosidase
aRNA transcribed from the plasmid was used for immunizations, bRNA transcribed from the plasmid waspacked in SFV viral particles used for immunizations, c Freunds incomplete adjuvant
RECOMBINANT SUBUNIT VACCINES
59
All three groups of mice immunized with plasmids encoding EB200 (group C, D and E)
developed GST-EB200 reactive antibodies. Only background levels of antibody reactivity to
GST were found, suggesting that the major part of the reactivity of these sera was directed
against EB200. Unexpectedly, both groups and of mice immunized with SFV-RNA (group A
and B), encoding EB200sec or control antigen BBG2Nasec, developed high and similar levels
of antibody reactivity to GST-EB200. However, most of this reactivity appeared not to be
directed against EB200, as both these groups showed an even higher reactivity with GST. The
reason for the induction of these apparently non-specific antibodies is not known. Importantly,
the RNA packaged in SFV suicide particles (group H and I) did not elicit this kind of antibody
reactivity.
The antibody responses obtained in this study were relatively low, surprisingly with the
highest responses obtained with the conventional DNA plasmid (group E). However,
immunizations with plasmids (group C, D and E) as well as with rSFV particles (group H)
induced an efficient immunological memory response, as demonstrated by the efficient
boosting of the anti-EB200 antibody response with recombinant EB200 protein. The efficient
priming of immune responses by naked DNA immunizations has been observed previously
when boosting with the corresponding protein (Haddad et al., 1999) or with recombinant
vaccinia virus expressing the protein (Schneider et al., 1998; Sedegah et al., 1998). Our low
responses to EB200 with the SFV based vectors appear to be due to the inherent properties of
the antigen, as our contol vectors coding for the protein BBG2Na (group I) or LacZ gave high
levels of antibodies to the respective protein.
In conclusion, we have in this study shown that DNA plasmids expressing the gene for the
EB200 fragment of the P. falciparum vaccine candidate antigen Pf332 induce an antibody
response to the antigen in mice. A conventional plasmid was more efficient than SFV-based
plasmids in inducing antibodies to EB200. Also recombinant SFV suicide particles expressing
EB200 induced such antibodies, although at lower levels. Importantly, all these immunogens
induced an immunological memory, which could be efficiently activated by a booster
injection with EB200 protein.
CHRISTIN ANDERSSON
60
8. Concluding remarks
Recombinant strategies are today common for the development of novel subunit vaccines. In
this thesis, recombinant methods have been employed to enable studies regarding; (i) the
generation of general expression systems for production of recombinant subunit immunogens
furnished with a hydrophobic tag, either hydrophobic amino acids or a lipid, allowing
association to iscoms, (ii) the development of a mammalian cell production and recovery
system for an RSV vaccine candidate, and (iii) construction and evaluation of nucleic acid
immunization strategies for RSV and malaria.
Robust systems for E. coli production of protein immunogens were created, in which
hydrophobic tags were fused to the target immunogen. This would enable direct association of
the produced immunogen to an adjuvant system, e.g. iscoms. Initially, three different
hydrophobic tags were evaluated, as fused N-terminally. A malaria peptide, M5, was used as
model antigen. The produced fusion proteins could be efficiently recovered by the
incorporation of also an affinity tag, the SpA-derived Z-domain, in the expression vectors, and
for two hydrophobic tags out of three, efficient association to iscoms was demonstrated.
Since the expression levels were rather low from the first generation expression vectors, a
second set of expression vectors were designed and constructed. In these vectors, one of the
hydrophobic tags was included that were found to be functional in the previous study, i.e. MI,
representing the membrane-integration region of influenza virus hemagglutinin. An additional
tag, M, representing the membrane-spanning region of staphylococcal protein A, was also
included in the study. In these vectors the hydrophobic tags were placed C-terminally, in order
not to disturb the transcription and translation initiation, to thus potentially improve
expression levels. The SpA-derived ZZ tag and the SpG-derived ABP tags were included in
the vectors to allow affinity purification, and the M5 malaria peptide served as model
immunogen. The second generation expression vectors were indeed found to increase
expression levels ten to fifty-fold, and the affinity-purified hydrophobically tagged
immunogens could efficiently be incorporated into iscoms.
A third study was performed in which lipid tagging of the recombinant immunogens was
evaluated. First, a system which utilized the E. coli lipidation system was designed. The
second system exploited a chelating lipid that was known to interact with hexahistidyl tags,
RECOMBINANT SUBUNIT VACCINES
61
and the recombinant immunogen was thus produced as fused to a His6 peptide. In both
systems the SpG-derived ABP tag was included for affinity purification purposes. A 238
amino acid segment ∆SAG1 from Toxoplasma gondii, served as model immunogen in this
study. The two types of lipid-tagged immunogens were both found to associate with iscoms.
The in vivo lipidation strategy has the advantage that only the typical iscom constituents are
needed, while the in vitro strategy requires a chelating lipid. In contrast, the expression vector
connected with the in vitro strategy gave much higher expression levels and in addition, a
straightforward method for iscom association, since iscoms-matrix with pre-incorporated
chelating lipid can be prepared in bulk. In all three studies, we confirmed the immunological
properties of the iscoms containing the recombinant immunogen, and we could in all cased
observe antigen-specific antibodies.
BBG2Na, a promising E. coli-produced vaccine candidate to human respiratory syncytial
virus (RSV) has been demonstrated to induce protection to virus challenge in rodent and
monkey models and has now progressed into phase III human clinical trials. In the presented
study, we wanted to create a mammalian expression system to produce BBG2Na, in order to
make comparative studies of E. coli produced and mammalian cell produced BBG2Na
possible. Expression systems for mammalian cell production of cytoplasmic and secreted
variants of BBG2Na, was thus created. The expression vectors were based on the SFV
operon, and RNA transcribed in vitro, encoding the SFV-replicase for self-amplification, was
used for transfection of the BHK-21 mammalian cells. The two different products BBG2Nacyt
and BBG2Nasec were both found to be expressed, and properly targeted to their intended
subcellular localization. Since the secreted variant, BBG2Nasec, was found to be expressed as
a full-length product, a suitable recovery scheme was evaluated. Two different modes of
affinity chromatography were evaluated, the first taking advantage of the serum albumin-
binding properties of the product, and the second, utilizing an SpA-based affibody created by
combinatorial engineering and selected for its specific binding to the G2Na part of the
product. For both strategies, significant co-purification of BSA, present in the culture medium
during the production, was demonstrated. A strategy was then evaluated in which BSA was
excluded in the culture medium during protein production, thus having it present only during
cultivation of the untransfected cells. The significantly reduced levels of BSA in the culture
medium made it possible to recover BBG2Nasec with a much higher degree of purity, and
when using the G2Na-specific affibody column for the affinity chromatography, the purity of
CHRISTIN ANDERSSON
62
BBG2Nasec, exceeded 90%, and the product could not be detected in the flow-through
fractions, indicating quantitative recovery. Comparative studies of the E. coli produced
BBG2Na and mammalian cell produced BBG2Na, are presently being conducted.
In an additional investigation, plasmid vectors for DNA immunization against RSV was
constructed and evaluated for their efficacy. Two different vectors were constructed encoding
variants of the RSV candidate vaccine BBG2Na; one designed for cytoplasmic localization
(BBG2Nacyt) and one for targeting to the secretion machinery (BBG2Nasec) of the transfected
cells. The two plasmid vectors, pBBG2Nacyt and pBBG2Nasec, were found to be functional in
terms of BBG2Na expression and localization of the encoded proteins. Upon intramuscular
immunization of mice, the plasmid vector pBBG2Nasec, encoding the secreted variant of the
antigen, elicited significant anti-BBG2Na titers and demonstrated lung protective efficacy (in
three out of five mice). Irrespective of the reasons for the differential immunogenicity
between the two variants, we clearly demonstrated by this study that protective immune
responses to RSV can be elicited in mice by DNA immunization, and the obtained results
further indicate that differential targeting of the antigens expressed by nucleic acid
vaccination could significantly influence the immunogenicity and protective efficacy of the
encoded heterologous antigens.
In a second nucleic acid vaccination study, we evaluated DNA- and RNA constructs based on
the SFV replicon in comparison with a conventional DNA plasmid for induction of antibody
responses against the P. falciparum malaria vaccine candidate antigen Pf332. The included
RNA constructs encoded the SFV replicase and the Pf332-derived antigen EB200, and were
delivered as either naked RNA or packaged in non-replicative SFV particles. Plasmid DNA
vectors were constructed encoding the same antigen, and vectors employing or lacking the
SFV-replicase were included in the immunization study. In general, the antibody responses
induced were relatively low, the highest responses surprisingly obtained with the conventional
DNA plasmid. Our low responses to EB200 with the SFV based vectors appear to be related
with inherent properties of the antigen, as our control vectors coding for the protein BBG2Na
or LacZ gave high levels of antibodies to the respective protein. Our immunizations with
naked RNA gave confusing results, with the induction of antibodies showing high background
reactivity with the fusion-tag GST of the recombinant EB200 protein used for antibody
analysis, as well as with the control antigen BBG2N. The reason for the appearance of these
RECOMBINANT SUBUNIT VACCINES
63
apparently non-specific sticky antibodies is not known. Importantly, the same RNA when
packaged in SFV suicide particles did not induce this kind of antibody reactivity.
In conclusion, we have in this study shown that DNA plasmids expressing the gene for the
EB200 fragment of the P. falciparum vaccine candidate antigen Pf332 induce an antibody
response to the antigen in mice. A conventional plasmid was in our hands more efficient than
SFV-based plasmids in inducing antibodies to EB200. Also recombinant SFV suicide
particles expressing EB200 induced such antibodies, although at lower levels. Importantly, all
these immunogens induced an immunological memory, which could be efficiently activated
by a booster injection with EB200 protein.
Taken together, it is evident that recombinant DNA technology will have a major impact on
future strategies for the prevention of infectious diseases. The design, production and delivery
of recombinant subunit vaccines will be the basis of modern vaccinology. The studies
summarized in this thesis demonstrate that new methods will be available for the production
and recovery of protein subunit immunogens, and that these can be further adapted by genetic
design of the expression vectors for incorporation into adjuvant systems. In addition, various
strategies for delivery of subunit vaccines by RNA or DNA immunization will be available
for the development of future vaccines.
CHRISTIN ANDERSSON
64
Abbreviations
ABD albumin binding domain derived from streptococcal protein GABP albumin binding protein derived from streptococcal protein GALVAC a recombinant canarypox virusAPC antigen presenting cellB7 a B-cell ligandBB albumin binding region derived from streptococcal protein GBHK baby hamster kidney cellsBSA bovine serum albuminCHO chinese hamster ovary cellsCMV cytomegalovirusCpG a cytosine-guanine dinucleotide, p indicates the phosphate bondCS circumsporozoiteCTB cholera toxin B subunitCTL cytotoxic T lymphocyteEB200 a malaria antigen fragment, derived from Pf332ELISA enzyme-linked immunosorbent assayETEC enterotoxic E. coliFCS foetal calf serumFIA Freunds incomplete adjuvantG2Na a part of the respiratory syncytial virus G proteinGM-CSF granulocyte-macrophage colony stimulating factorGST glutathione-S-transferaseHA hemagglutininHEK human embryonic kidney cellsHBsAg Hepatitis B surface antigenHib Haemophilus influenzae type BHis6 hexahistidyl tagHIV human immunodeficiency virusHLA human leukocyte antigenHSA human serum albuminIg immunoglobulinIFN interferonIL interleukinIscoms immunostimulating complexesISS immunostimulatory sequencesIW hydrophobic amino acid sequence (isoleucines and trypophanes)kDa kiloDaltonLPS lipopolysaccharidesLTB E. coli heat-labile toxin B subunitM5 a malaria peptide derived from the central region of Pf155/RESAM membrane-spanning region of SpAMAP multiple antigenic peptidesMHC major histocompatibility complexMI membrane-spanning region of hemagglutinin A from influenza virus
RECOMBINANT SUBUNIT VACCINES
65
MPL monophosporyl lipid AMVA modified vaccinia virus Ankara strainNP nucleoproteinNYVAC attenuated vaccinia virus strainPAGE polyacrylamide gel electrophoresisPD hydrophobic tag, designed to be pH-dependentPCR polymerase chain reactionPLG poly (lactide-co-glycosides)QS-21 a fraction of Quil AQuil A a saponin derived from Quillaja saponariaRSV respiratory syncytial virusSAG1 surface antigen 1 from Toxoplasma gondiiSFV Semliki Forest virusSDS sodium dodecyl sulphateSpA staphylococcal protein ASpG streptococcal protein GSV40 simian virus 40tPA tissue plasminogen activatorVLP virus-like particlesVP viral proteinXM cell-surface-anchoring region of staphylococcal protein AZZ IgG-binding domains derived from staphylococcal protein A
CHRISTIN ANDERSSON
66
Acknowledgements
First of all I would like to thank my supervisor, Prof. Stefan Ståhl. You have, in good and bad times,always been supportive and encouraging and somehow always incredibly optimistic. Thank you forhelping me finish this thieis.
I would also like to thank Prof. Mathias Uhlén for leading the DNA corner group in an inspiring wayand for being such a visionary person.
My collaboration partners have contributed a great deal to the work presented in this thesis. I wouldlike to send my gratitude to: Karin Lövgren-Bengtsson at SLU in Uppsala for introducing me to theworld of iscoms. Anna Lundén at SVA in Uppsala for collaboration in the SAG-1 iscom project andall help with the thesis. Klavs Berzins, Diana Haddad, Niklas Ahlborg and Nina-Maria Vasconcelos atStockholms University for collaboration in the EB200 project. Ultan Power, CIPF, for all collaborationin the RSV project. Peter Liljeström and Peter Berglund, MTC, Karolinska Institute, for all help in theSFV projects. Sissela Liljeqvist, for completing the EB200-story.
I want to thank all colleagues at the department of Biotechnology at KTH, over the years, for creatinga friendly and stimulating working atmosphere. During my years at the department, I have reallyenjoyed all theatre evenings, dinners, travels (Paris, Sandhamn, Gotland, Berlin, Väddö), Laser Domebattles, Christmas parties, fredagsöl and fun company during lunches and coffee breaks. I am going tomiss you all.There are some persons I would like to give special thanks to:
Maria Murby, my exjobbs-supervisor for introducing me to the lab (together with Sissela).
Monica and Pia for taking care of all administrative matters, and Monica also for simultaneouslykeeping track of Mathias.
Sophia, P-Å , Jocke and Stefan for dealing with every-day problems, such as lack of space, and carbonboxes in "korridoren" with a never-ending patience.
My DNA-heroes, Bertil and Jocke W, for teaching me all I know about DNA sequencing, hairpin-loops, GC content and Tm.
Marianne, Pelle and Torbjörn, my "bollplank" when it comes to protein-related problems, for alwayshaving time to answer my questions.
The brave K90-troup, Torbjörn, Afshin, Nina, Jenny O, Bahram and Eric B. For the first time in tenyears, I have to manage without you.
My three exjobbare or projectarbetare, Maria, Maria and Henrik, for giving me a hard time awnseringall your questions.
All persons in Stora labbet for all fun things during the years, especially for winning the Laser Domebattle (thank you Malin). I would like to thank my closest neighbors Jenny, Kristofer, Johanna, Amelieand Henrik, for being such nice persons to work around, Anna B and Deirdre for bringing DNA intoStora labbet, Susanne, my singing-colleague, Torbjörn and My for giving me something else to thinkabout when I was writing (the fermentor) and of course, Olof and Erik in "mellanstadiet", for justbeing two the crazy persons you are.Also, I would like to thank the rest of the KTH Vaccine Research Center, Maria Wikman, for being agood friend and an excellent shopping partner.
RECOMBINANT SUBUNIT VACCINES
67
Thank you all my friends for being there for me. Tove, Jenny, Malin, Nina, Anna W, Malin C andSonja, for dinners, midsummer at Muskö, Advents-dinners with singing, and much more.I would especially like to thank, Henrik, my END-Note hero, for everything, Jenny O, for dinners andmysterious parties, Janne, for being the happiest person I know, and for showing me Åbo, Brita, forsailing trips, hours on the phone or at some café, and Ulla, my best friend for 28 years, for being theperson you are and for supporting me (See you in Rome!).
So at last, my beloved family.Moster Annica, for letting me live with you for my first month in Stockholm, and for showing me how"tunnelbanan" works.Fredrik Skeppstedt for computer assistance and for taking care of my sister.Pernilla, for all romantic "kits", and for friendship, and Victor for being a darling.
My brothers Anders, Lars and Fredrik and my sister Sara for all fun we have shared during all theseyears, for being loving, and always supporting me and believing in me.
Tack Mor och Far för att ni lärt mig att tro på mig själv och att våga gå min egen väg.
This work was supported by grants from by Pierre Fabre Médicaments, France.
CHRISTIN ANDERSSON
68
References
Abrahamsén, L., Moks, T., Nilsson, B. and Uhlén, M. Secretion of heterologous gene products to theculture medium of Escherichia coli. Nucleic Acids Res. 14 (1986) 7487-7500.
Alving, C.R. Liposomal vaccines: clinical status and immunological presentation for humoral andcellular immunity. Ann. N Y Acad. Sci. 754 (1995) 143-152.
Amann, E., Ochs, B. and Abel, K.J. Tightly regulated tac promoter vectors useful for the expression ofunfused and fused proteins in Escherichia coli. Gene 69 (1988) 301-315.
Araujo, F.G. and Remington, J.S. Toxoplasmosis in immunocompromised patients [editorial]. Eur. J.Clin. Microbiol. 6 (1987) 1-2.
Babiuk, L.A. Broadening the approaches to developing more effective vaccines. Vaccine 17 (1999)1587-1595.
Baca-Estrada, M.E., Foldvari, M., Snider, M., van Drunen Littel-van den Hurk, S. and Babiuk, L.A.Effect of IL-4 and IL-12 liposomal formulations on the induction of immune response to bovineherpesvirus type-1 glycoprotein D. Vaccine 15 (1997) 1753-1760.
Baldridge, J.R. and Crane, R.T. Monophosphoryl lipid A (MPL) formulations for the next generationof vaccines. Methods 19 (1999) 103-107.
Ban, E.M., van Ginkel, F.W., Simecka, J.W., Kiyono, H., Robinson, H.L. and McGhee, J.R. Mucosalimmunization with DNA encoding influenza hemagglutinin. Vaccine 15 (1997) 811-813.
Barker, R.N., Erwig, L., Pearce, W.P., Devine, A. and Rees, A.J. Differential effects of necrotic orapoptotic cell uptake on antigen presentation by macrophages. Pathobiology 67 (1999) 302-305.
Barr, I.G., Sjölander, A. and Cox, J.C. ISCOMs and other saponin based adjuvants. Adv. Drug. Deliv.Rev. 32 (1998) 247-271.
Barry, M.A. and Johnston, S.A. Biological features of genetic immunization. Vaccine 15 (1997) 788-791.
Berglund, P., Fleeton, M.N., Smerdou, C. and Liljeström, P. Immunization with recombinant SemlikiForest virus induces protection against influenza challenge in mice. Vaccine 17 (1999) 497-507.
Berglund, P., Quesada-Rolander, M., Putkonen, P., Biberfeld, G., Thorstensson, R. and Liljeström, P.Outcome of immunization of cynomolgus monkeys with recombinant Semliki Forest virus encodinghuman immunodeficiency virus type 1 envelope protein and challenge with a high dose of SHIV-4virus. AIDS Res. Hum. Retroviruses 13 (1997) 1487-1495.
Berglund, P., Smerdou, C., Fleeton, M.N., Tubulekas, I. and Liljeström, P. Enhancing immuneresponses using suicidal DNA vaccines. Nat. Biotechnol. 16 (1998) 562-565.
Berzins, K., Adams, S., Broderson, R., Chizzolini, C., Hansson, M., Lövgren, K., Millet, P., Morris,C.L., Perlmann, H., Perlmann, P., Sjölander, A., Ståhl, S., Sullivan, J.S., Troye-Blomberg, M.,Wåhlin-Flyg, B. and Collins, W. Immunogenicity in Aotus monkeys of ISCOM formulated repeat
RECOMBINANT SUBUNIT VACCINES
69
sequences from the Plamodium falciparum asexual blood stage antigen Pf155/RESA. Vaccineresearch 4 (1995) 121-133.
Bird, A.P., Taggart, M.H., Nicholls, R.D. and Higgs, D.R. Non-methylated CpG-rich islands at thehuman alpha-globin locus: implications for evolution of the alpha-globin pseudogene. Embo J. 6(1987) 999-1004.
Bonato, V.L., Lima, V.M., Tascon, R.E., Lowrie, D.B. and Silva, C.L. Identification andcharacterization of protective T cells in hsp65 DNA-vaccinated and Mycobacterium tuberculosis-infected mice. Infect. Immun. 66 (1998) 169-175.
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of proteinutilizing the principle of protein-dye binding. Anal. Biochem. 72 (1976) 248-254.
Brand, D., Lemiale, F., Turbica, I., Buzelay, L., Brunet, S. and Barin, F. Comparative analysis ofhumoral immune responses to HIV type 1 envelope glycoproteins in mice immunized with a DNAvaccine, recombinant Semliki Forest virus RNA, or recombinant Semliki Forest virus particles. AIDSRes. Hum. Retroviruses 14 (1998) 1369-1377.
Brandt, C., Power, U.F., Plotnicky-Gilquin, H., Huss, T., Nguyen, T., Lambert, P.H., Binz, H. andSiegrist, C.A. Protective immunity against respiratory syncytial virus in early life after murinematernal or neonatal vaccination with the recombinant G fusion protein BBG2Na. J. Infect. Dis. 176(1997) 884-891.
Briand, J.P., Benkirane, N., Guichard, G., Newman, J.F., Van Regenmortel, M.H., Brown, F. andMuller, S. A retro-inverso peptide corresponding to the GH loop of foot-and-mouth disease viruselicits high levels of long-lasting protective neutralizing antibodies. Proc. Natl. Acad. Sci. U S A 94(1997) 12545-12550.
Brochier, B., Costy, F. and Pastoret, P.P. Elimination of fox rabies from Belgium using a recombinantvaccinia-rabies vaccine: an update. Vet. Microbiol. 46 (1995) 269-279.
Browning, M., Reid, G., Osborne, R. and Jarrett, O. Incorporation of soluble antigens into ISCOMs:HIV gp120 ISCOMs induce virus neutralizing antibodies. Vaccine 10 (1992) 585-590.
Bulow, R. and Boothroyd, J.C. Protection of mice from fatal Toxoplasma gondii infection byimmunization with p30 antigen in liposomes. J. Immunol. 147 (1991) 3496-3500.
Burg, J.L., Perelman, D., Kasper, L.H., Ware, P.L. and Boothroyd, J.C. Molecular analysis of the geneencoding the major surface antigen of Toxoplasma gondii. J. Immunol. 141 (1988) 3584-3591.
Burke, D.H. and Gold, L. RNA aptamers to the adenosine moiety of S-adenosyl methionine: structuralinferences from variations on a theme and the reproducibility of SELEX. Nucleic. Acids Res. 25(1997) 2020-2924.
Böhm, W., Kuhröber, A., Paier, T., Mertens, T., Reimann, J. and Schirmbeck, R. DNA vectorconstructs that prime hepatitis B surface antigen-specific cytotoxic T lymphocyte and antibodyresponses in mice after intramuscular injection. J. Immunol. Methods 193 (1996) 29-40.
Böhm, W., Mertens, T., Schirmbeck, R. and Reimann, J. Routes of plasmid DNA vaccination thatprime murine humoral and cellular immune responses. Vaccine 16 (1998) 949-954.
CHRISTIN ANDERSSON
70
Calarota, S., Bratt, G., Nordlund, S., Hinkula, J., Leandersson, A.C., Sandström, E. and Wahren, B.Cellular cytotoxic response induced by DNA vaccination in HIV-1-infected patients. Lancet 351(1998) 1320-1325.
Cano, F., Plotnicky-Gilquin, H., Nguyen, T.N., Liljeqvist, S., Samuelson, P., Bonnefoy, J., Ståhl, S.and Robert, A. Partial protection to respiratory syncytial virus (RSV) elicited in mice by intranasalimmunization using live staphylococci with surface-displayed RSV-peptides. Vaccine 18 (2000) 2743-2752.
Carbonell, L.F., Klowden, M.J. and Miller, L.K. Baculovirus-mediated expression of bacterial genes indipteran and mammalian cells. J. Virol. 56 (1985) 153-160.
Cardoso, A.I., Sixt, N., Vallier, A., Fayolle, J., Buckland, R. and Wild, T.F. Measles virus DNAvaccination: antibody isotype is determined by the method of immunization and by the nature of boththe antigen and the coimmunized antigen. J. Virol. 72 (1998) 2516-2518.
Carroll, M.W. and Moss, B. Poxviruses as expression vectors. Curr. Opin. Biotechnol. 8 (1997) 573-577.
Casares, S., Brumeanu, T.D., Bot, A. and Bona, C.A. Protective immunity elicited by vaccination withDNA encoding for a B cell and a T cell epitope of the A/PR/8/34 influenza virus. Viral Immunol. 10(1997) 129-136.
Cataldo, D.M. and Van Nest, G. The adjuvant MF59 increases the immunogenicity and protectiveefficacy of subunit influenza vaccine in mice. Vaccine 15 (1997) 1710-1715.
Chang, J.C., Diveley, J.P., Savary, J.R. and Jensen, F.C. Adjuvant activity of incomplete Freund'sadjuvant. Adv. Drug Deliv. Rev. 32 (1998) 173-186.
Chen, S.C., Fynan, E.F., Robinson, H.L., Lu, S., Greenberg, H.B., Santoro, J.C. and Herrmann, J.E.Protective immunity induced by rotavirus DNA vaccines. Vaccine 15 (1997) 899-902.
Chen, Y., Hu, D., Eling, D.J., Robbins, J. and Kipps, T.J. DNA vaccines encoding full-length ortruncated Neu induce protective immunity against Neu-expressing mammary tumors. Cancer Res. 58(1998) 1965-1971.
Clackson, T. and Wells, J.A. In vitro selection from protein and peptide libraries. Trends. Biotechnol.12 (1994) 173-184.
Clare, J.J., Rayment, F.B., Ballantine, S.P., Sreekrishna, K. and Romanos, M.A. High-level expressionof tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of thegene. Biotechnology (N Y) 9 (1991) 455-460.
Collins, P.L., McIntosh, K. and Chanock, R.M. Respiratory Syncytial Virus. In: Fields, B.N., Knipe,D.M. and Howley, P.M. (Eds.), Fields Virology. Lippincott-Raven, Philadelphia, 1996, pp. 1313-1351.
Conry, R.M., LoBuglio, A.F., Wright, M., Sumerel, L., Pike, M.J., Johanning, F., Benjamin, R., Lu, D.and Curiel, D.T. Characterization of a messenger RNA polynucleotide vaccine vector. Cancer Res. 55(1995) 1397-1400.
Conry, R.M., Widera, G., LoBuglio, A.F., Fuller, J.T., Moore, S.E., Barlow, D.L., Turner, J., Yang,N.S. and Curiel, D.T. Selected strategies to augment polynucleotide immunization. Gene Ther. 3(1996) 67-74.
RECOMBINANT SUBUNIT VACCINES
71
Coppel, R.L., Cowman, A.F., Anders, R.F., Bianco, A.E., Saint, R.B., Lingelbach, K.R., Kemp, D.J.and Brown, G.V. Immune sera recognize on erythrocytes Plasmodium falciparum antigen composedof repeated amino acid sequences. Nature 310 (1984) 789-792.
Corvaïa, N., Tournier, P., Nguyen, T.N., Haeuw, J.F., Power, U.F., Binz, H. and Andreoni, C.Challenge of BALB/c mice with respiratory syncytial virus does not enhance the Th2 pathway inducedafter immunization with a recombinant G fusion protein, BBG2NA, in aluminum hydroxide. J. Infect.Dis. 176 (1997) 560-569.
Costa, F., Franchin, G., Pereira-Chioccola, V.L., Ribeirao, M., Schenkman, S. and Rodrigues, M.M.Immunization with a plasmid DNA containing the gene of trans-sialidase reduces Trypanosoma cruziinfection in mice. Vaccine 16 (1998) 768-774.
Coste, A., Sirard, J.C., Johansen, K., Cohen, J. and Kraehenbuhl, J.P. Nasal immunization of micewith virus-like particles protects offspring against rotavirus diarrhea. J. Virol. 74 (2000) 8966-8971.
Cox, F.E. Malaria vaccines--progress and problems. Trends Biotechnol 9 (1991) 389-94.
Daheshia, M., Kuklin, N., Manickan, E., Chun, S. and Rouse, B.T. Immune induction and modulationby topical ocular administration of plasmid DNA encoding antigens and cytokines. Vaccine 16 (1998)1103-1110.
Dalemans, W., Delers, A., Delmelle, C., Denamur, F., Meykens, R., Thiriart, C., Veenstra, S.,Francotte, M., Bruck, C. and Cohen, J. Protection against homologous influenza challenge by geneticimmunization with SFV-RNA encoding Flu-HA. Ann N Y Acad. Sci. 772 (1995) 255-256.
Dalsgaard, K. A study of the isolation and characterization of the saponin Quil A. Evaluation of itsadjuvant activity, with a special reference to the application in the vaccination of cattle against foot-and-mouth disease. Acta Vet. Scand. Suppl. (1978) 7-40.
Dalsgaard, K. Thinlayer chromatographic fingerprinting of commercially available saponins. Dan.Tidsskr. Farm. 44 (1970) 327-331.
Dalsgaard, K., Hilgers, L. and Trouve, G. Classical and new approaches to adjuvant use in domesticfood animals. Adv. Vet. Sci. Comp. Med. 35 (1990) 121-160.
Danko, I., Williams, P., Herweijer, H., Zhang, G., Latendresse, J.S., Bock, I. and Wolff, J.A. Highexpression of naked plasmid DNA in muscles of young rodents. Hum. Mol. Genet. 6 (1997) 1435-1443.
Darji, A., Guzman, C.A., Gerstel, B., Wachholz, P., Timmis, K.N., Wehland, J., Chakraborty, T. andWeiss, S. Oral somatic transgene vaccination using attenuated S. typhimurium. Cell 91 (1997) 765-775.
Davis, H.L., Brazolot Millan, C.L., Mancini, M., McCluskie, M.J., Hadchouel, M., Comanita, L.,Tiollais, P., Whalen, R.G. and Michel, M.L. DNA-based immunization against hepatitis B surfaceantigen (HBsAg) in normal and HBsAg-transgenic mice. Vaccine 15 (1997) 849-852.
Davis, H.L., Michel, M.L. and Whalen, R.G. DNA-based immunization induces continuous secretionof hepatitis B surface antigen and high levels of circulating antibody. Hum. Mol. Genet. 2 (1993)1847-1851.
CHRISTIN ANDERSSON
72
De Wilde, M.J. Yeast as a host for the production of macromolecules of prophylactic or therapeuticinterest in human health care. Bioprocess Technol. 5 (1990) 479-504.
Dégano, P., Sarphie, D.F. and Bangham, C.R. Intradermal DNA immunization of mice againstinfluenza A virus using the novel PowderJect system. Vaccine 16 (1998) 394-398.
Del Giudice, G. and Rappuoli, R. Genetically derived toxoids for use as vaccines and adjuvants.Vaccine 17 Suppl. 2 (1999) S44-52.
Dertzbaugh, M.T. Genetically engineered vaccines: an overview. Plasmid 39 (1998) 100-113.Dietrich, G., Bubert, A., Gentschev, I., Sokolovic, Z., Simm, A., Catic, A., Kaufmann, S.H., Hess, J.,Szalay, A.A. and Goebel, W. Delivery of antigen-encoding plasmid DNA into the cytosol ofmacrophages by attenuated suicide Listeria monocytogenes. Nat. Biotechnol. 16 (1998) 181-185.
Domachowske, J.B. and Rosenberg, H.F. Respiratory syncytial virus infection: immune response,immunopathogenesis, and treatment. Clin. Microbiol. Rev. 12 (1999) 298-309.
Donnelly, J.J., Friedman, A., Ulmer, J.B. and Liu, M.A. Further protection against antigenic drift ofinfluenza virus in a ferret model by DNA vaccination. Vaccine 15 (1997a) 865-868.
Donnelly, J.J., Ulmer, J.B., Shiver, J.W. and Liu, M.A. DNA vaccines. Annu. Rev. Immunol. 15(1997b) 617-648.
Drulak, M.W., Malinoski, F.J., Fuller, S.A., Stewart, S.S., Hoskin, S., Duliege, A.M., Sekulovich, R.,Burke, R. and Winston, S. Vaccination of seropositive subjects with CHIRON CMV gB subunitvaccine combined with MF59 adjuvant for production of CMV immune globulin. Viral Immunol.13(2000) 49-56.
Dubensky, T.W., Jr., Driver, D.A., Polo, J.M., Belli, B.A., Latham, E.M., Ibanez, C.E., Chada, S.,Brumm, D., Banks, T.A., Mento, S.J., Jolly, D.J. and Chang, S.M. Sindbis virus DNA-basedexpression vectors: utility for in vitro and in vivo gene transfer. J. Virol. 70 (1996) 508-519.
Dudas, R.A. and Karron, R.A. Respiratory syncytial virus vaccines. Clin. Microbiol. Rev. 11 (1998)430-439.
Dupuis, M., Murphy, T.J., Higgins, D., Ugozzoli, M., van Nest, G., Ott, G. and McDonald, D.M.Dendritic cells internalize vaccine adjuvant after intramuscular injection. Cell. Immunol. 186 (1998)18-27.
Ellis, R.W. New technologies for making vaccines. Vaccine 17 (1999) 1596-1604.
Ertl, H.C.J. and Xiang, Z.Q. DNA vaccines to rabies virus: effects of cytokines on the immuneresponse. Vaccines 96 (1996) 83-86.
Feldmann, H., Kretzschmar, E., Klingeborn, B., Rott, R., Klenk, H.D. and Garten, W. The structure ofserotype H10 hemagglutinin of influenza A virus: comparison of an apathogenic avian and amammalian strain pathogenic for mink. Virology 165 (1988) 428-437.
Felgner, P.L., Tsai, Y.J., Sukhu, L., Wheeler, C.J., Manthorpe, M., Marshall, J. and Cheng, S.H.Improved cationic lipid formulations for in vivo gene therapy. Ann. N Y Acad. Sci. 772 (1995) 126-139.
RECOMBINANT SUBUNIT VACCINES
73
Ferreira, G.N., Monteiro, G.A., Prazeres, D.M. and Cabral, J.M. Downstream processing of plasmidDNA for gene therapy and DNA vaccine applications. Trends Biotechnol. 18 (2000) 380-388.
Fischetti, V.A., Medaglini, D. and Pozzi, G. Gram-positive commensal bacteria for mucosal vaccinedelivery. Curr. Opin. Biotechnol. 7 (1996) 659-666.
Fleeton, M.N., Liljeström, P., Sheahan, B.J. and Atkins, G.J. Recombinant Semliki Forest virusparticles expressing louping ill virus antigens induce a better protective response than plasmid-basedDNA vaccines or an inactivated whole particle vaccine. J. Gen. Virol. 81 Pt 3 (2000) 749-758.
Fleeton, M.N., Sheahan, B.J., Gould, E.A., Atkins, G.J. and Liljeström, P. Recombinant Semliki Forestvirus particles encoding the prME or NS1 proteins of louping ill virus protect mice from lethalchallenge. J. Gen. Virol. 80 (1999) 1189-1198.
Fomsgaard, A., Nielsen, H.V., Bryder, K., Nielsen, C., Machuca, R., Bruun, L., Hansen, J. and Buus,S. Improved humoral and cellular immune responses against the gp120 V3 loop of HIV-1 followinggenetic immunization with a chimeric DNA vaccine encoding the V3 inserted into the hepatitis Bsurface antigen. Scand. J. Immunol. 47 (1998) 289-295.
Fooks, A.R., Schadeck, E., Liebert, U.G., Dowsett, A.B., Rima, B.K., Steward, M., Stephenson, J.R.and Wilkinson, G.W. High-level expression of the measles virus nucleocapsid protein by using areplication-deficient adenovirus vector: induction of an MHC-1-restricted CTL response andprotection in a murine model. Virology 210 (1995) 456-465.
Fuller, D.H., Corb, M.M., Barnett, S., Steimer, K. and Haynes, J.R. Enhancement ofimmunodeficiency virus-specific immune responses in DNA-immunized rhesus macaques. Vaccine 15(1997) 924-926.
Fussenegger, M., Bailey, J.E., Hauser, H. and Mueller, P.P. Genetic optimization of recombinantglycoprotein production by mammalian cells. Trends Biotechnol. 17 (1999) 35-42.
Geisse, S., Gram, H., Kleuser, B. and Kocher, H.P. Eukaryotic expression systems: a comparison.Protein. Expr. Purif. 8 (1996) 271-282.
Georgiou, G., Poetschke, H.L., Stathopoulos, C. and Francisco, J.A. Practical applications ofengineering gram-negative bacterial cell surfaces. Trends Biotechnol. 11 (1993) 6-10.
Georgiou, G., Stathopoulos, C., Daugherty, P.S., Nayak, A.R., Iverson, B.L. and Curtiss, R., 3rdDisplay of heterologous proteins on the surface of microorganisms: from the screening ofcombinatorial libraries to live recombinant vaccines. Nat. Biotechnol. 15 (1997) 29-34.
Ghrayeb, J. and Inouye, M. Nine amino acid residues at the NH2-terminal of lipoprotein are sufficientfor its modification, processing, and localization in the outer membrane of Escherichia coli. J. Biol.Chem. 259 (1984) 463-467.
Gilbert, S.C., Plebanski, M., Harris, S.J., Allsopp, C.E., Thomas, R., Layton, G.T. and Hill, A.V. Aprotein particle vaccine containing multiple malaria epitopes. Nat. Biotechnol. 15 (1997) 1280-1284.
Glasgow, G.M., McGee, M.M., Tarbatt, C.J., Mooney, D.A., Sheahan, B.J. and Atkins, G.J. TheSemliki Forest virus vector induces p53-independent apoptosis. J. Gen. Virol. 79 (1998) 2405-2410.
Gluzman, Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell 23(1981) 175-182.
CHRISTIN ANDERSSON
74
Gold, L., Polisky, B., Uhlenbeck, O. and Yarus, M. Diversity of oligonucleotide functions. Annu. Rev.Biochem 64 (1995) 763-797.
Gordon, E.M., Barrett, R.W., Dower, W.J., Fodor, S.P. and Gallop, M.A. Applications ofcombinatorial technologies to drug discovery. 2. Combinatorial organic synthesis, library screeningstrategies, and future directions. J. Med. Chem. 37 (1994) 1385-1401.
Gotschlich, E.C., Liu, T.Y. and Artenstein, M.S. Human immunity to the meningococcus. 3.Preparation and immunochemical properties of the group A, group B, and group C meningococcalpolysaccharides. J. Exp. Med. 129 (1969) 1349-1365.
Gould-Fogerite, S., Kheiri, M.T., Zhang, F., Wang, Z., Scolpino, A.J., Feketeova, E., Canki, M. andMannino, R.J. Targeting immune response induction with cochleate and liposome-based vaccines.Adv. Drug. Deliv. Rev. 32 (1998) 273-287.
Gramzinski, R.A., Maris, D.C., Doolan, D., Charoenvit, Y., Obaldia, N., Rossan, R., Sedegah, M.,Wang, R., Hobart, P., Margalith, M. and Hoffman, S. Malaria DNA vaccines in Aotus monkeys.Vaccine 15 (1997) 913-915.
Gregoriadis, G. Immunological adjuvants: a role for liposomes. Immunol. Today 11 (1990) 89-97.
Gregoriadis, G., McCormack, B., Obrenovic, M., Saffie, R., Zadi, B. and Perrie, Y. Vaccineentrapment in liposomes. Methods 19 (1999) 156-162.
Gregoriadis, G., Saffie, R. and de Souza, J.B. Liposome-mediated DNA vaccination. FEBS Lett. 402(1997) 107-110.
Grimwood, J. and Smith, J.E. Toxoplasma gondii: the role of a 30-kDa surface protein in host cellinvasion. Exp. Parasitol. 74 (1992) 106-111.
Grothaus, M.C., Srivastava, N., Smithson, S.L., Kieber-Emmons, T., Williams, D.B., Carlone, G.M.and Westerink, M.A. Selection of an immunogenic peptide mimic of the capsular polysaccharide ofNeisseria meningitidis serogroup A using a peptide display library. Vaccine 18 (2000) 1253-1263.
Gunneriusson, E., Nord, K., Uhlén, M. and Nygren, P.-Å. Affinity maturation of a Taq DNApolymerase specific affibody by helix shuffling. Protein Eng. 12 (1999) 873-878.
Gupta, R.K. Aluminum compounds as vaccine adjuvants. Adv. Drug Deliv. Rev. 32 (1998) 155-172.
Gustafson, G.L. and Rhodes, M.J. Bacterial cell wall products as adjuvants: early interferon gamma asa marker for adjuvants that enhance protective immunity. Res. Immunol. 143 (1992) 483-488;discussion 573-574.
Haddad, D., Liljeqvist, S., Ståhl, S., Hansson, M., Perlmann, P., Ahlborg, N. and Berzins, K.Characterization of antibody responses to a Plasmodium falciparum blood- stage antigen induced by aDNA prime/protein boost immunization protocol. Scand. J. Immunol. 49 (1999) 506-514.
Hajishengallis, G., Hollingshead, S.K., Koga, T. and Russell, M.W. Mucosal immunization with abacterial protein antigen genetically coupled to cholera toxin A2/B subunits. J. Immunol. 154 (1995)4322-4332.
RECOMBINANT SUBUNIT VACCINES
75
Hammarberg, B., Nygren, P.-Å., Holmgren, E., Elmblad, A., Tally, M., Hellman, U., Moks, T. andUhlén, M. Dual affinity fusion approach and its use to express recombinant human insulin-like growthfactor II. Proc. Natl. Acad. Sci. U S A 86 (1989) 4367-4371.
Han, R., Reed, C.A., Cladel, N.M. and Christensen, N.D. Immunization of rabbits with cottontailrabbit papillomavirus E1 and E2 genes: protective immunity induced by gene gun-mediatedintracutaneous delivery but not by intramuscular injection. Vaccine 18 (2000) 2937-2944.
Hannig, G. and Makrides, S.C. Strategies for optimizing heterologous protein expression inEscherichia coli. Trends Biotechnol. 16 (1998) 54-60.
Hansson, M., Ringdahl, J., Robert, A., Power, U., Goetsch, L., Nguyen, T.N., Uhlén, M., Ståhl, S. andNygren, P.-Å. An in vitro selected binding protein (affibody) shows conformation-dependentrecognition of the respiratory syncytial virus (RSV) G protein. Immunotechnology 4 (1999) 237-252.
Hansson, M., Ståhl, S., Hjorth, R., Uhlén, M. and Moks, T. Single-step recovery of a secretedrecombinant protein by expanded bed adsorption. Biotechnology (N Y) 12 (1994) 285-288.
Hariharan, M.J., Driver, D.A., Townsend, K., Brumm, D., Polo, J.M., Belli, B.A., Catton, D.J., Hsu,D., Mittelstaedt, D., McCormack, J.E., Karavodin, L., Dubensky, T.W., Jr., Chang, S.M. and Banks,T.A. DNA immunization against herpes simplex virus: enhanced efficacy using a Sindbis virus-basedvector. J. Virol. 72 (1998) 950-958.
Harning, D., Spenter, J., Metsis, A., Vuust, J. and Petersen, E. Recombinant Toxoplasma gondiisurface antigen 1 (P30) expressed in Escherichia coli is recognized by human Toxoplasma-specificimmunoglobulin M (IgM) and IgG antibodies. Clin. Diagn. Lab. Immunol. 3 (1996) 355-357.
Hassinen, C., Kohler, K. and Veide, A. Polyethylene glycol-potassium phosphate aqueous two-phasesystems. Insertion of short peptide units into a protein and its effects on partitioning. J. Chromatogr. A668 (1994) 121-128.
Hedley, M.L., Curley, J. and Urban, R. Microspheres containing plasmid-encoded antigens elicitcytotoxic T-cell responses. Nat. Med. 4 (1998) 365-368.
Heeg, K., Kuon, W. and Wagner, H. Vaccination of class I major histocompatibility complex (MHC)-restricted murine CD8+ cytotoxic T lymphocytes towards soluble antigens: immunostimulating-ovalbumin complexes enter the class I MHC-restricted antigen pathway and allow sensitization againstthe immunodominant peptide. Eur. J. Immunol. 21 (1991) 1523-1527.
Heineman, T.C., Clements-Mann, M.L., Poland, G.A., Jacobson, R.M., Izu, A.E., Sakamoto, D.,Eiden, J., Van Nest, G.A. and Hsu, H.H. A randomized, controlled study in adults of theimmunogenicity of a novel hepatitis B vaccine containing MF59 adjuvant. Vaccine 17 (1999) 2769-2778.
Hermentin, K. and Aspock, H. Efforts towards a vaccine against Toxoplasma gondii: a review.Zentralbl. Bakteriol. Mikrobiol. Hyg. [A] 269 (1988) 423-436.
Herrington, D.A., Clyde, D.F., Losonsky, G., Cortesia, M., Murphy, J.R., Davis, J., Baqar, S., Felix,A.M., Heimer, E.P., Gillessen, D., Nardin, E., Nussenzweig, R.S., Nussenzweig, V., Hollingdale, M.R.and Levine, M.M. Safety and immunogenicity in man of a synthetic peptide malaria vaccine againstPlasmodium falciparum sporozoites. Nature 328 (1987) 257-259.
CHRISTIN ANDERSSON
76
Herweijer, H., Latendresse, J.S., Williams, P., Zhang, G., Danko, I., Schlesinger, S. and Wolff, J.A. Aplasmid-based self-amplifying Sindbis virus vector. Hum. Gene. Ther. 6 (1995) 1161-1167.
Herzenberg, L.A. and Tokuhisa, T. Carrier-priming leads to hapten-specific suppression. Nature 285(1980) 664-667.
Hesse, F. and Wagner, R. Developments and improvements in the manufacturing of humantherapeutics with mammalian cell cultures. Trends Biotechnol. 18 (2000) 173-180.
Higgins, D.A., Carlson, J.R. and Van Nest, G. MF59 adjuvant enhances the immunogenicity ofinfluenza vaccine in both young and old mice. Vaccine 14 (1996) 478-484.
Hilleman, M.R. Vaccines in historic evolution and perspective: a narrative of vaccine discoveries.Vaccine 18 (2000) 1436-1447.
Hilleman, M.R., Carlson, A.J., Jr., McLean, A.A., Vella, P.P., Weibel, R.E. and Woodhour, A.F.Streptococcus pneumoniae polysaccharide vaccine: age and dose responses, safety, persistence ofantibody, revaccination, and simultaneous administration of pneumococcal and influenza vaccines.Rev. Infect. Dis. 3 Suppl. (1981) S31-42.
Hinkula, J., Lundholm, P. and Wahren, B. Nucleic acid vaccination with HIV regulatory genes: acombination of HIV-1 genes in separate plasmids induces strong immune responses. Vaccine 15(1997) 874-878.
Hinterberg, K., Scherf, A., Gysin, J., Toyoshima, T., Aikawa, M., Mazie, J.C., da Silva, L.P. andMattei, D. Plasmodium falciparum: the Pf332 antigen is secreted from the parasite by a brefeldin A-dependent pathway and is translocated to the erythrocyte membrane via the Maurer's clefts. Exp.Parasitol. 79 (1994) 279-291.
Hjorth, R.N., Bonde, G.M., Piner, E.D., Goldberg, K.M. and Levner, M.H. The effect of Syntexadjuvant formulation (SAF-m) on humoral immunity to the influenza virus in the mouse. Vaccine 15(1997) 541-546.
Hochuli, E., Bannwarth, W., Döbeli, H., Gentz, R. and Stüber, D. Genetic approach to facilitatepurification of recombinant proteins with a novel metal chelate adsorbent. Bio/Technology 11 (1988)887-893.
Hoffman, S.L., Doolan, D.L., Sedegah, M., Aguiar, J.C., Wang, R., Malik, A., Gramzinski, R.A.,Weiss, W.R., Hobart, P., Norman, J.A., Margalith, M. and Hedström, R.C. Strategy for developmentof a pre-erythrocytic Plasmodium falciparum DNA vaccine for human use. Vaccine 15 (1997) 842-845.
Holmgren, J., Czerkinsky, C., Lycke, N., and Svennerholm, A.-M. Strategies for the induction ofimmune responses at mucosal surfaces making use of cholera toxin B subunit as immunogen, carrier,and adjuvant. Am. J. Trop. Med. Hyg. 50 (1994) 42-54.
http://www.cipf.com
Hurpin, C., Rotarioa, C., Bisceglia, H., Chevalier, M., Tartaglia, J. and Erdile, L. The mode ofpresentation and route of administration are critical for the induction of immune responses to p53 andantitumor immunity. Vaccine 16 (1998) 208-215.
RECOMBINANT SUBUNIT VACCINES
77
Höglund, S., Dalsgaard, K., Lövgren, K., sundqvist, B., Osterhaus, A. and Morein, B. ISCOMs andimmunostimulation with viral antigens. Subcellar biochemistry 15 (1989) 39-68.
Ichikawa, Y., Yamagata, H., Tochikubo, K. and Udaka, S. Very efficient extracellular production ofcholera toxin B subunit using Bacillus bRev.is. FEMS Microbiol. Lett. 111 (1993) 219-224.
Imler, J.L. Adenovirus vectors as recombinant viral vaccines. Vaccine 13 (1995) 1143-1151.
Ishii, N., Fukushima, J., Kaneko, T., Okada, E., Tani, K., Tanaka, S.I., Hamajima, K., Xin, K.Q.,Kawamoto, S., Koff, W., Nishioka, K., Yasuda, T. and Okuda, K. Cationic liposomes are a strongadjuvant for a DNA vaccine of human immunodeficiency virus type 1. AIDS Res. Hum. Retroviruses13 (1997) 1421-1428.
Israelski, D.M. and Remington, J.S. Toxoplasmic encephalitis in patients with AIDS. Infect. Dis. Clin.North Am. 2 (1988) 429-445.
Jakob, T., Walker, P.S., Krieg, A.M., Udey, M.C. and Vogel, J.C. Activation of cutaneous dendriticcells by CpG-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1responses by immunostimulatory DNA. J. Immunol. 161 (1998) 3042-3049.
Johanning, F.W., Conry, R.M., Lo Buglio, A.F., Wright, M., Sumerel, L.A., Pike, M.J. and Curiel,D.T. A Sindbis virus mRNA polynucleotide vector achieves prolonged and high level heterologousgene expression in vivo. Nucleic. Acids Res. 23 (1995) 1495-1501.
Johansson, M. and Lövgren-Bengtsson, K. Iscoms with different quillaja saponin components differ intheir immunomodulating activities. Vaccine 17 (1999) 2894-900
Johnson, A.M. Toxoplasma vaccines. In: Wright, I.G. (Ed.), Veterinary protozoan and hemoparasitevaccines. CRC press, Boca Raton, Florida, 1989, pp. 177-202.
Jonasson, P., Nygren, P.-Å., Johansson, B.L., Wahren, J., Uhlén, M. and Ståhl, S. Gene fragmentpolymerization gives increased yields of recombinant human proinsulin C-peptide. Gene 210 (1998)203-210.
Jonasson, P., Nygren, P.-Å., Jörnvall, H., Johansson, B.L., Wahren, J., Uhlén, M. and Ståhl, S.Integrated bioprocess for production of human proinsulin C-peptide via heat release of an intracellularheptameric fusion protein. J. Biotechnol. 76 (2000) 215-226.
Jones, D.H., Corris, S., McDonald, S., Clegg, J.C. and Farrar, G.H. Poly(DL-lactide-co-glycolide)-encapsulated plasmid DNA elicits systemic and mucosal antibody responses to encoded protein afteroral administration. Vaccine 15 (1997) 814-817.
Kahn, J.O., Sinangil, F., Baenziger, J., Murcar, N., Wynne, D., Coleman, R.L., Steimer, K.S., Dekker,C.L. and Chernoff, D. Clinical and immunologic responses to human immunodeficiency virus (HIV)type 1SF2 gp120 subunit vaccine combined with MF59 adjuvant with or without muramyl tripeptidedipalmitoyl phosphatidylethanolamine in non-HIV-infected human volunteers. J. Infect. Dis. 170(1994) 1288-1291.
Kahn, J.O., Stites, D.P., Scillian, J., Murcar, N., Stryker, R., Volberding, P.A., Naylor, P.H., Goldstein,A.L., Sarin, P.S., Simmon, V.F. and et al. A phase I study of HGP-30, a 30 amino acid subunit of thehuman immunodeficiency virus (HIV) p17 synthetic peptide analogue sub-unit vaccine in seronegativesubjects. AIDS Res. Hum. Retroviruses 8 (1992) 1321-1325.
CHRISTIN ANDERSSON
78
Kamrud, K.I., Olson, K.E., Higgs, S., Carlson, J.O. and Beaty, B.J. Use of the Sindbis replicon systemfor expression of LaCrosse virus envelope proteins in mosquito cells. Arch. Virol. 143 (1998) 1365-1377.
Kaneda, Y., Iwai, K. and Uchida, T. Increased expression of DNA cointroduced with nuclear proteinin adult rat liver. Science 243 (1989) 375-378.
Karron, R.A., Buonagurio, D.A., Georgiu, A.F., Whitehead, S.S., Adamus, J.E., Clements-Mann,M.L., Harris, D.O., Randolph, V.B., Udem, S.A., Murphy, B.R. and Sidhu, M.S. Respiratory syncytialvirus (RSV) SH and G proteins are not essential for viral replication in vitro: clinical evaluation andmolecular characterization of a cold-passaged, attenuated RSV subgroup B mutant. Proc. Natl. Acad.Sci. U S A 94 (1997) 13961-13966.
Kensil, C.R. Saponins as vaccine adjuvants. Crit. Rev. Ther. Drug Carrier Syst. 13 (1996) 1-55.
Kensil, C.R., Patel, U., Lennick, M. and Marciani, D. Separation and characterization of saponins withadjuvant activity from Quillaja saponaria Molina cortex. J. Immunol. 146 (1991) 431-437.
Khan, I.A., Ely, K.H. and Kasper, L.H. A purified parasite antigen (p30) mediates CD8+ T cellimmunity against fatal Toxoplasma gondii infection in mice. J. Immunol. 147 (1991) 3501-3506.
Kim, J.J., Ayyavoo, V., Bagarazzi, M.L., Chattergoon, M., Boyer, J.D., Wang, B. and Weiner, D.B.Development of a multicomponent candidate vaccine for HIV-1. Vaccine 15 (1997) 879-883.
Kim, J.J., Nottingham, L.K., Sin, J.I., Tsai, A., Morrison, L., Oh, J., Dang, K., Hu, Y., Kazahaya, K.,Bennett, M., Dentchev, T., Wilson, D.M., Chalian, A.A., Boyer, J.D., Agadjanyan, M.G. and Weiner,D.B. CD8 positive T cells influence antigen-specific immune responses through the expression ofchemokines. J. Clin. Invest. 102 (1998) 1112-1124.
Klavinskis, L.S., Bergmeier, L.A., Gao, L., Mitchell, E., Ward, R.G., Layton, G., Brookes, R., Meyers,N.J. and Lehner, T. Mucosal or targeted lymph node immunization of macaques with a particulateSIVp27 protein elicits virus-specific CTL in the genito-rectal mucosa and draining lymph nodes. J.Immunol. 157 (1996) 2521-2527.
Klinman, D.M., Barnhart, K.M. and Conover, J. CpG motifs as immune adjuvants. Vaccine 17 (1999)19-25.
Klipstein, F.A., Engert, R.F. and Houghten, R.A. Mucosal antitoxin response in volunteers toimmunization with a synthetic peptide of Escherichia coli heat-stable enterotoxin. Infect. Immun. 50(1985) 328-332.
Koths, K. Recombinant proteins for medical use: the attractions and challenges. Curr. Opin.Biotechnol. 6 (1995) 681-687.
Kowalczyk, D.W. and Ertl, H.C. Immune responses to DNA vaccines. Cell. Mol. Life. Sci. 55 (1999)751-770.
Krieg, A.M. An innate immune defense mechanism based on the recognition of CpG motifs inmicrobial DNA. J Lab Clin Med 128 (1996) 128-33.Levy, J.A. Pathogenesis of human immunodeficiency virus infection. Microbiol Rev 57 (1993) 183-289.
RECOMBINANT SUBUNIT VACCINES
79
Krieg, A.M. and Kline, J.N. Immune effects and therapeutic applications of CpG motifs in bacterialDNA. Immunopharmacology 48 (2000) 303-305.
Krieg, A.M., Yi, A.K., Matson, S., Waldschmidt, T.J., Bishop, G.A., Teasdale, R., Koretzky, G.A. andKlinman, D.M. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374 (1995) 546-549.
Köhler, K., Ljungquist, C., Kondo, A., Veide, A. and Nilsson, B. Engineering proteins to enhance theirpartition coefficients in aqueous two-phase systems. Bio/Technology 9 (1991) 642-646.
Langenberg, A.G., Burke, R.L., Adair, S.F., Sekulovich, R., Tigges, M., Dekker, C.L. and Corey, L. Arecombinant glycoprotein vaccine for herpes simplex virus type 2: safety and immunogenicity[corrected] [published erratum appears in Ann Intern Med 1995 Sep 1;123(5):395]. Ann. Intern. Med.122 (1995) 889-898.
Langner, M. and Kral, T.E. Liposome-based drug delivery systems. Pol. J. Pharmacol. 51 (1999) 211-222.
Larsson, M., Brundell, E., Nordfors, L., Höög, C., Uhlén, M. and Ståhl, S. A general bacterialexpression system for functional analysis of cDNA- encoded proteins. Protein Expr. Purif. 7 (1996)447-457.
Larsson, M., Gräslund, S., Yuan, L., Brundell, E., Uhlén, M., Höög, C. and Ståhl, S. High-throughputprotein expression of cDNA products as a tool in functional genomics. J. Biotechnol. 80 (2000) 143-157.
Larsson, M., Lövgren, K. and Morein, B. Immunopotentiation of synthetic oligopeptides by chemicalconjugation to iscoms. J. Immunol. Methods 162 (1993) 257-260.
Laukkanen, M.-L., Teeri, T.T. and Keinänen, K. Lipid-tagged antibodies: bacterial expression andcharacterization of lipoprotein-single-chain antibody fusion protein. Protein engineering 6 (1993) 449-454.
Le, T.P., Coonan, K.M., Hedström, R.C., Charoenvit, Y., Sedegah, M., Epstein, J.E., Kumar, S.,Wang, R., Doolan, D.L., Maguire, J.D., Parker, S.E., Hobart, P., Norman, J. and Hoffman, S.L. Safety,tolerability and humoral immune responses after intramuscular administration of a malaria DNAvaccine to healthy adult volunteers. Vaccine 18 (2000) 1893-1901.
Lebens, M., Shahabi, V., Backström, M., Houze, T., Lindblad, N. and Holmgren, J. Synthesis ofhybrid molecules between heat-labile enterotoxin and cholera toxin B subunits: potential for use in abroad-spectrum vaccine. Infect. Immun. 64 (1996) 2144-2150.
Levine, M.M., Hone, D., Tacket, C., Ferreccio, C. and Cryz, S. Clinical and field trials with attenuatedSalmonella typhi as live oral vaccines and as "carrier" vaccines. Res. Microbiol. 141 (1990) 807-816.
Li, X., Sambhara, S., Li, C.X., Ettorre, L., Switzer, I., Cates, G., James, O., Parrington, M., Oomen,R., Du, R.P. and Klein, M. Plasmid DNA encoding the respiratory syncytial virus G protein is apromising vaccine candidate. Virology 269 (2000) 54-65.
Li, X., Sambhara, S., Li, C.X., Ewasyshyn, M., Parrington, M., Caterini, J., James, O., Cates, G., Du,R.P. and Klein, M. Protection against respiratory syncytial virus infection by DNA immunization. J.Exp. Med. 188 (1998) 681-688.
CHRISTIN ANDERSSON
80
Libon, C., Corvaia, N., Haeuw, J.F., Nguyen, T.N., Ståhl, S., Bonnefoy, J.Y. and Andreoni, C. Theserum albumin-binding region of streptococcal protein G (BB) potentiates the immunogenicity of theG130-230 RSV-A protein. Vaccine 17 (1999) 406-414.
Liljeqvist, S., Haddad, D., Berzins, K., Uhlén, M. and Ståhl, S. A novel expression system forSalmonella typhimurium allowing high production levels, product secretion and efficient recovery.Biochem. Biophys. Res. Commun. 218 (1996) 356-359.
Liljeqvist, S. and Ståhl, S. Production of recombinant subunit vaccines: protein immunogens, livedelivery systems and nucleic acid vaccines. J. Biotechnol. 73 (1999) 1-33.
Liljeström, P. Alphavirus expression systems. Curr. Opin. Biotechnol. 5 (1994) 495-500.
Liljeström, P. and Garoff, H. A new generation of animal cell expression vectors based on the semlikiforest virus replicon. Bio/Technology 9 (1991) 1356-1361.Lindberg, A.A. Glycoprotein conjugate vaccines. Vaccine 17 Suppl 2 (1999) S28-36.
Lipford, G.B., Sparwasser, T., Bauer, M., Zimmermann, S., Koch, E.S., Heeg, K. and Wagner, H.Immunostimulatory DNA: sequence-dependent production of potentially harmful or useful cytokines.Eur. J. Immunol. 27 (1997) 3420-3426.
Liu, Y., Mounkes, L.C., Liggitt, H.D., Brown, C.S., Solodin, I., Heath, T.D. and Debs, R.J. Factorsinfluencing the efficiency of cationic liposome-mediated intravenous gene delivery. Nat. Biotechnol.15 (1997) 167-173.
Livingston, P.O., Adluri, S., Helling, F., Yao, T.J., Kensil, C.R., Newman, M.J. and Marciani, D.Phase 1 trial of immunological adjuvant QS-21 with a GM2 ganglioside-keyhole limpet haemocyaninconjugate vaccine in patients with malignant melanoma. Vaccine 12 (1994) 1275-1280.
Lodmell, D.L., Ray, N.B. and Ewalt, L.C. Gene gun particle-mediated vaccination with plasmid DNAconfers protective immunity against rabies virus infection. Vaccine 16 (1998) 115-118.
Lopez-Macias, C., Lopez-Hernandez, M.A., Gonzalez, C.R., Isibasi, A. and Ortiz-Navarrete, V.Induction of antibodies against Salmonella typhi OmpC porin by naked DNA immunization. Ann. N YAcad. Sci. 772 (1995) 285-288.
Lundén, A. Immune responses in sheep after immunization with Toxoplasma gondii antigensincorporated into iscoms. Vet. Parasitol. 56 (1995) 23-35.
Lundén, A., Lövgren, K., Uggla, A. and Araujo, F.G. Immune responses and resistance to Toxoplasmagondii in mice immunized with antigens of the parasite incorporated into immunostimulatingcomplexes. Infect. Immun. 61 (1993) 2639-2643.
Lövgren, K., Kåberg, H. and Morein, B. An experimental influensza subunit vaccine (iscom):induction of protective immunity to challange infection in mice after intranasal or subcutaneosadministration. Clin. Exp. Immunol. 82 (1990) 435-439.
Lövgren, K., Lindmark, J., Pipkorn, R. and Morein, B. Antigenic presentation of small molecules andpeptides conjugated to a preformed iscom as carrier. J. Immunol. Methods 98 (1987) 137-143.
Löwenadler, B., Lycke, N., Svanholm, C., Svennerholm, A.M., Krook, K. and Gidlund, M. T and Bcell responses to chimeric proteins containing heterologous T helper epitopes inserted at differentpositions. Mol. Immunol. 29 (1992) 1185-1190.
RECOMBINANT SUBUNIT VACCINES
81
Makrides, S.C. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiol.Rev. 60 (1996) 512-38.
Maloy, K.J., Donachie, A.M., O'Hagan, D.T. and Mowat, A.M. Induction of mucosal and systemicimmune responses by immunization with ovalbumin entrapped in poly(lactide-co-glycolide)microparticles. Immunology 81 (1994) 661-667.
Mancini, M., Schlienger, K., Tiollais, P. and Michel, M.L. Immunogenicity of hybrid hepatitis Bsurface antigen particles. Int. Rev. Immunol. 11 (1994) 143-151.
Manickan, E., Karem, K.L. and Rouse, B.T. DNA vaccines -- a modern gimmick or a boon tovaccinology? Crit. Rev. Immunol. 17 (1997) 139-154.
Martin, S.J., Vyakarnam, A., Cheingsong-Popov, R., Callow, D., Jones, K.L., Senior, J.M., Adams,S.E., Kingsman, A.J., Matear, P., Gotch, F.M. and et al. Immunization of human HIV-seronegativevolunteers with recombinant p17/p24:Ty virus-like particles elicits HIV-1 p24-specific cellular andhumoral immune responses. Aids 7 (1993) 1315-1323.
Martin-Gallardo, A., Fleischer, E., Doyle, S.A., Arumugham, R., Collins, P.L., Hildreth, S.W. andParadiso, P.R. Expression of the G glycoprotein gene of human respiratory syncytial virus inSalmonella typhimurium. J. Gen. Virol. 74 (1993) 453-458.
Martinon, F., Krishnan, S., Lenzen, G., Magne, R., Gomard, E., Guillet, J.G., Levy, J.P. and Meulien,P. Induction of virus-specific cytotoxic T lymphocytes in vivo by liposome- entrapped mRNA. Eur. J.Immunol. 23 (1993) 1719-1722.
Matsuo, K., Yoshikawa, T., Asanuma, H., Iwasaki, T., Hagiwara, Y., Chen, Z., Kadowaki, S.E.,Tsujimoto, H., Kurata, T. and Tamura, S.I. Induction of innate immunity by nasal influenza vaccineadministered in combination with an adjuvant (cholera toxin). Vaccine 18 (2000) 2713-2722.
Mattei, D., Hinterberg, K. and Scherf, A. Pf11-1 and Pf332: Two giant proteins synthesized inerythrocytes infected with Plasmodium falciparum. Parasitol. Today 8 (1992) 426-428.
Mattei, D. and Scherf, A. The Pf332 gene of Plasmodium falciparum codes for a giant protein that istranslocated from the parasite to the membrane of infected erythrocytes. Gene 110 (1992) 71-79.
McAleer, W.J., Buynak, E.B., Maigetter, R.Z., Wampler, D.E., Miller, W.J. and Hilleman, M.R.Human hepatitis B vaccine from recombinant yeast. Nature 307 (1984) 178-180.
McCabe, B.J., Irvine, K.R., Nishimura, M.I., Yang, J.C., Spiess, P.J., Shulman, E.P., Rosenberg, S.A.and Restifo, N.P. Minimal determinant expressed by a recombinant vaccinia virus elicits therapeuticantitumor cytolytic T lymphocyte responses. Cancer Res. 55 (1995) 1741-1747.
McClements, W.L., Armstrong, M.E., Keys, R.D. and Liu, M.A. The prophylactic effect ofimmunization with DNA encoding herpes simplex virus glycoproteins on HSV-induced disease inguinea pigs. Vaccine 15 (1997) 857-860.
McCluskie, M.J. and Davis, H.L. CpG DNA as mucosal adjuvant. Vaccine 18 (1999) 231-237.
Medaglini, D., Pozzi, G., King, T.P. and Fischetti, V.A. Mucosal and systemic immune responses to arecombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordoniiafter oral colonization. Proc. Natl. Acad. Sci. U S A 92 (1995) 6868-6872.
CHRISTIN ANDERSSON
82
Messina, J.P., Gilkeson, G.S. and Pisetsky, D.S. Stimulation of in vitro murine lymphocyteproliferation by bacterial DNA. J. Immunol. 147 (1991) 1759-1764.
Millet, P., Campbell, G.H., Sulzer, A.J., Grady, K.K., Pohl, J., Aikawa, M. and Collins, W.E.Immunogenicity of the Plasmodium falciparum asexual blood-stage synthetic peptide vaccine SPf66.Am. J. Trop. Med. Hyg. 48 (1993) 424-431.
Mittal, S.K., Papp, Z., Tikoo, S.K., Baca-Estrada, M.E., Yoo, D., Benko, M. and Babiuk, L.A.Induction of systemic and mucosal immune responses in cotton rats immunized with humanadenovirus type 5 recombinants expressing the full and truncated forms of bovine herpesvirus type 1glycoprotein gD. Virology 222 (1996) 299-309.
Moks, T., Abrahmsén, L., Nilsson, B., Hellman, U., Sjöquist, J. and Uhlén, M. Staphylococcal proteinA consists of five IgG-binding domains. Eur. J. Biochem. 156 (1986) 637-643.
Moks, T., Abrahmsén, L., Österlöf, B., Josephson, S., Östling, M., Enfors, S.-O., Persson, I., Nilsson,B. and Uhlén, M. Large-scale affinity purification of human insulin-like growth factor I from culturemedium of Escherichia coli. Bio/Technology 5 (1987) 379-382.
Moldoveanu, Z., Love-Homan, L., Huang, W.Q. and Krieg, A.M. CpG DNA, a novel immuneenhancer for systemic and mucosal immunization with influenza virus. Vaccine 16 (1998) 1216-1224.
Montgomery, D.L., Shiver, J.W., Leander, K.R., Perry, H.C., Friedman, A., Martinez, D., Ulmer, J.B.,Donnelly, J.J. and Liu, M.A. Heterologous and homologous protection against influenza A by DNAvaccination: optimization of DNA vectors. DNA Cell. Biol. 12 (1993) 777-783.
Mor, G., Singla, M., Steinberg, A.D., Hoffman, S.L., Okuda, K. and Klinman, D.M. Do DNA vaccinesinduce autoimmune disease? Hum. Gene. Ther. 8 (1997) 293-300.
Morein, B., Ekström, J. and Lövgren, K. Increased immunogenicity of a non-amphipathic protein(BSA) after inclusion into iscoms. J. Immunol. Methods 128 (1990) 177-181.
Morein, B., Lövgren, K., Rönnberg, B., Sjölander, A. and Villacrés-Eriksson, M. Immunostimulatingcomplexes. Clinical potential in vaccine development. Clin. Immunother. 3 (1995) 461-475.
Morein, B., Lövgren-Bengtsson, K. and Cox, J. General principles of vaccinology. In: Kaufmann,S.H.E. (Ed.), Concepts in Vaccine Development. Walter de Gruyter, New York, 1996, pp. 243-263.
Morein, B., Sundquist, B., Höglund, S., Dalsgaard, K. and Osterhaus, A. Iscom, a novel structure forantigenic presentation of membrane proteins from enveloped viruses. Nature 308 (1984) 457-460.
Mossman, S.P., Bex, F., Berglund, P., Arthos, J., O'Neil, S.P., Riley, D., Maul, D.H., Bruck, C.,Momin, P., Burny, A., Fultz, P.N., Mullins, J.I., Liljeström, P. and Hoover, E.A. Protection againstlethal simian immunodeficiency virus SIVsmmPBj14 disease by a recombinant Semliki Forest virusgp160 vaccine and by a gp120 subunit vaccine. J. Virol. 70 (1996) 1953-1960.
Mumford, J.A., Jessett, D., Dunleavy, U., Wood, J., Hannant, D., Sundquist, B. and Cook, R.F.Antigenicity and immunogenicity of experimental equine influenza ISCOM vaccines. Vaccine 12(1994) 857-863.
Murby, M., Cedergren, L., Nilsson, J., Nygren, P.-Å., Hammarberg, B., Nilsson, B., Enfors, S.O. andUhlén, M. Stabilization of recombinant proteins from proteolytic degradation in Escherichia coli usinga dual affinity fusion strategy. Biotechnol. Appl. Biochem. 14 (1991) 336-346.
RECOMBINANT SUBUNIT VACCINES
83
Murby, M., Nguyen, T.N., Binz, H., Uhlén, M. and Ståhl, S. Production and recovery of recombinantproteins of low solubility. In: Pyle, L. (Ed.), Separation for Biotechnology 3. Bookcraft Ldt., Bath,UK, 1994, pp. 336-344.
Murby, M., Samuelsson, E., Nguyen, T.N., Mignard, L., Power, U., Binz, H., Uhlén, M. and Ståhl, S.Hydrophobicity engineering to increase solubility and stability of a recombinant protein fromrespiratory syncytial virus. Eur. J. Biochem. 230 (1995) 38-44.
Murby, M., Uhlén, M. and Ståhl, S. Upstream strategies to minimize proteolytic degradation uponrecombinant production in Escherichia coli. Protein. Expr. Purif. 7 (1996) 129-136.
Mäkelä, P.H. Vaccines, coming of age after 200 years. FEMS Microbiol. Rev. 24 (2000) 9-20.
Nagahama, M., Michiue, K. and Sakurai, J. Production and purification of Clostridium perfringensalpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47. FEMS Microbiol. Lett. 145(1996) 239-243.
Namba, K., Yamamura, E., Nitanai, H., Otani, T. and Azuma, I. Romurtide, a synthetic muramyldipeptide derivative, promotes megakaryocytopoiesis through stimulation of cytokine production innonhuman primates with myelosuppression. Vaccine 15 (1997) 405-413.
Navarre, W.W. and Schneewind, O. Proteolytic cleavage and cell wall anchoring at the LPXTG motifof surface proteins in gram-positive bacteria. Mol. Microbiol. 14 (1994) 115-21.
Navarre, W.W. and Schneewind, O. Surface proteins of gram-positive bacteria and mechanisms oftheir targeting to the cell wall envelope. Microbiol. Mol. Biol. Rev. 63 (1999) 174-229.
Nguyen, T.N., Gourdon, M.H., Hansson, M., Robert, A., Samuelson, P., Libon, C., Andreoni, C.,Nygren, P.-Å., Binz, H., Uhlén, M. and et al. Hydrophobicity engineering to facilitate surface displayof heterologous gene products on Staphylococcus xylosus. J. Biotechnol. 42 (1995) 207-219.
Nichols, W.W., Ledwith, B.J., Manam, S.V. and Troilo, P.J. Potential DNA vaccine integration intohost cell genome. Ann. N Y Acad. Sci. 772 (1995) 30-9.
Nilsson, J., Larsson, M., Ståhl, S., Nygren, P.-Å. and Uhlén, M. Multiple affinity domains for thedetection, purification and immobilization of recombinant proteins. J Mol Recognit 9 (1996) 585-594.
Nilsson, B., Moks, T., Jansson, B., Abrahamsén, L., Elmblad, A., Holmgren, E., Henrichson, C.,Jones, T.A. and Uhlén, M. A synthetic IgG-binding domain based on staphylococcal protein A.Protein Eng. 1 (1987) 107-113.
Nilsson, J., Nilsson, P., Williams, Y., Pettersson, L., Uhlén, M. and Nygren, P.-Å. Competitive elutionof protein A fusion proteins allows specific recovery under mild conditions. Eur. J. Biochem. 224(1994) 103-108.
Nilsson, J., Ståhl, S., Lundeberg, J., Uhlén, M. and Nygren, P.-Å. Affinity fusion strategies fordetection, purification, and immobilization of recombinant proteins. Protein. Expr. Purif. 11 (1997) 1-16.
Nitayaphan, S., Khamboonruang, C., Sirisophana, N., Morgan, P., Chiu, J., Duliege, A.M.,Chuenchitra, C., Supapongse, T., Rungruengthanakit, K., deSouza, M., Mascola, J.R., Boggio, K.,Ratto-Kim, S., Markowitz, L.E., Birx, D., Suriyanon, V., McNeil, J.G., Brown, A.E. and Michael,R.A. A phase I/II trial of HIV SF2 gp120/MF59 vaccine in seronegative thais. AFRIMS-RIHES
CHRISTIN ANDERSSON
84
Vaccine Evaluation Group. Armed Forces Research Institute of Medical Sciences and the ResearchInstitute for Health Sciences. Vaccine 18 (2000) 1448-1455.
Nixon, D.F., Hioe, C., Chen, P.D., Bian, Z., Kuebler, P., Li, M.L., Qiu, H., Li, X.M., Singh, M.,Richardson, J., McGee, P., Zamb, T., Koff, W., Wang, C.Y. and O'Hagan, D. Synthetic peptidesentrapped in microparticles can elicit cytotoxic T cell activity. Vaccine 14 (1996) 1523-1530.
Nord, K., Gunneriusson, E., Ringdahl, J., Ståhl, S., Uhlén, M. and Nygren, P.-Å. Binding proteinsselected from combinatorial libraries of an alpha- helical bacterial receptor domain. Nat. Biotechnol.15 (1997) 772-777.
Nord, K., Nilsson, J., Nilsson, B., Uhlén, M. and Nygren, P.-Å. A combinatorial library of an alpha-helical bacterial receptor domain. Protein Eng. 8 (1995) 601-608.
Norman, J.A., Hobart, P., Manthorpe, M., Felgner, P. and Wheeler, C. Development of improvedvectors for DNA-based immunization and other gene therapy applications. Vaccine 15 (1997) 801-803.
Nygren, P.-Å., Eliasson, M., Abrahmsen, L., Uhlén, M. and Palmcrantz, E. Analysis and use of theserum albumin binding domains of streptococcal protein G. J. Mol. Recognit 1 (1988) 69-74.
Nygren, P.-Å., Flodberg, P., Andersson, R., Wigzell, H. and Uhlén, M. In vivo stabilization of a humanrecombinant CD4 derivate by fusion to a serum-binding receptor. In: Chanock, M., Ginsberg, H.S.,Brown, F. and Lerner, R.A. (Eds.), Vaccines 91. Cold Spring Harbour Laboratory Press, New York,NY, 1991, pp. 363-368.
Nygren, P.-Å., Ljungquist, C., Tromborg, H., Nustad, K. and Uhlén, M. Species-dependent binding ofserum albumins to the streptococcal receptor protein G. Eur. J. Biochem. 193 (1990) 143-148.
Nygren, P.-Å. and Uhlén, M. Scaffolds for engineering novel binding sites in proteins. Curr. Opin.Struct. Biol. 7 (1997) 463-469.
O'Hagan, D.T., Jeffery, H., Roberts, M.J., McGee, J.P. and Davis, S.S. Controlled releasemicroparticles for vaccine development. Vaccine 9 (1991a) 768-771.
O'Hagan, D.T., Rahman, D., McGee, J.P., Jeffery, H., Davies, M.C., Williams, P., Davis, S.S. andChallacombe, S.J. Biodegradable microparticles as controlled release antigen delivery systems.Immunology 73 (1991b) 239-242.
O'Hagan, D.T., Singh, M. and Gupta, R.K. Poly(lactide-co-glycolide) microparticles for thedevelopment of single-dose controlled-release vaccines. Adv Drug Deliv Rev 32 (1998) 225-246.
Ogawa, T., Suda, Y., Kashihara, W., Hayashi, T., Shimoyama, T., Kusumoto, S. and Tamura, T.Immunobiological activities of chemically defined lipid A from Helicobacter pylori LPS incomparison with Porphyromonas gingivalis lipid A and Escherichia coli-type synthetic lipid A(compound 506). Vaccine 15 (1997) 1598-1605.
Ott, G., Barchfeld, G.L. and Van Nest, G. Enhancement of humoral response against human influenzavaccine with the simple submicron oil/water emulsion adjuvant MF59. Vaccine 13 (1995) 1557-1562.Paoletti, E. Applications of pox virus vectors to vaccination: an update. Proc. Natl. Acad. Sci. U S A93 (1996) 11349-11353.
RECOMBINANT SUBUNIT VACCINES
85
Parente, R.A., Nadasdi, L., Subbarao, N.K. and Szoka, F.C., Jr. Association of a pH-sensitive peptidewith membrane vesicles: role of amino acid sequence. Biochemistry 29 (1990a) 8713-8719.
Parente, R.A., Nir, S. and Szoka, F.C., Jr. Mechanism of leakage of phospholipid vesicle contentsinduced by the peptide GALA. Biochemistry 29 (1990b) 8720-8728.
Park, E.J., Chang, J.H., Kim, J.S., Yum, J.S. and Chung, S.I. The mucosal adjuvanticity of twonontoxic mutants of Escherichia coli heat-labile enterotoxin varies with immunization routes. Exp.Mol. Med. 32 (2000) 72-78.
Patarroyo, M.E., Amador, R., Clavijo, P., Moreno, A., Guzman, F., Romero, P., Tascon, R., Franco,A., Murillo, L.A., Ponton, G. and et al. A synthetic vaccine protects humans against challenge withasexual blood stages of Plasmodium falciparum malaria. Nature 332 (1988) 158-161.
Perkus, M.E., Tartaglia, J. and Paoletti, E. Poxvirus-based vaccine candidates for cancer, AIDS, andother infectious diseases. J. Leukoc. Biol. 58 (1995) 1-13.
Perlmann, H., Berzins, K., Wahlgren, M., Carlsson, J., Björkman, A., Patarroyo, M.E. and Perlmann,P. Antibodies in malarial sera to parasite antigens in the membrane of erythrocytes infected with earlyasexual stages of Plasmodium falciparum. J. Exp. Med. 159 (1984) 1686-1704.
Peroulis, I., Mills, J. and Meanger, J. Respiratory syncytial virus G glycoprotein expressed using theSemliki Forest virus replicon is biologically active. Arch. Virol. 144 (1999) 107-116.
Perrie, Y. and Gregoriadis, G. Liposome-entrapped plasmid DNA: characterisation studies. Biochim.Biophys. Acta 1475 (2000) 125-132.
Pertmer, T.M., Eisenbraun, M.D., McCabe, D., Prayaga, S.K., Fuller, D.H. and Haynes, J.R. Genegun-based nucleic acid immunization: elicitation of humoral and cytotoxic T lymphocyte responsesfollowing epidermal delivery of nanogram quantities of DNA. Vaccine 13 (1995) 1427-1430.
Pertmer, T.M., Roberts, T.R. and Haynes, J.R. Influenza virus nucleoprotein-specific immunoglobulinG subclass and cytokine responses elicited by DNA vaccination are dependent on the route of vectorDNA delivery. J. Virol. 70 (1996) 6119-6125.
Plotkin, S.A. Vaccination in the 21st century. J. Infect. Dis. 168 (1993) 29-37.
Plotnicky-Gilquin, H., Goetsch, L., Huss, T., Champion, T., Beck, A., Haeuw, J.F., Nguyen, T.N.,Bonnefoy, J.Y., Corvaia, N. and Power, U.F. Identification of multiple protective epitopes(protectopes) in the central conserved domain of a prototype human respiratory syncytial virus Gprotein. J. Virol. 73 (1999) 5637-5645.
Plotnicky-Gilquin, H., Robert, A., Chevalet, L., Haeuw, J.F., Beck, A., Bonnefoy, J.Y., Brandt, C.,Siegrist, C.A., Nguyen, T.N. and Power, U.F. CD4(+) T-cell-mediated antiviral protection of the upperrespiratory tract in BALB/c mice following parenteral immunization with a recombinant respiratorysyncytial virus G protein fragment. J. Virol. 74 (2000) 3455-3463.
Power, U.F., Plotnicky-Gilquin, H., Huss, T., Robert, A., Trudel, M., Ståhl, S., Uhlén, M., Nguyen,T.N. and Binz, H. Induction of protective immunity in rodents by vaccination with a prokaryoticallyexpressed recombinant fusion protein containing a respiratory syncytial virus G protein fragment.Virology 230 (1997) 155-166.
CHRISTIN ANDERSSON
86
Pozzi, G., Oggioni, M.R., Manganelli, R. and Fischetti, V.A. Expression of M6 protein gene ofStreptococcus pyogenes in Streptococcus gordonii after chromosomal integration and transcriptionalfusion. Res. Microbiol. 143 (1992) 449-457.
Prince, A.M., Whalen, R. and Brotman, B. Successful nucleic acid based immunization of newbornchimpanzees against hepatitis B virus. Vaccine 15 (1997) 916-919.
Pugachev, K.V., Mason, P.W., Shope, R.E. and Frey, T.K. Double-subgenomic Sindbis virusrecombinants expressing immunogenic proteins of Japanese encephalitis virus induce significantprotection in mice against lethal JEV infection. Virology 212 (1995) 587-594.
Pyle, S.W., Morein, B., Bess, J.W., Jr., Akerblom, L., Nara, P.L., Nigida, S.M., Jr., Lerche, N.W.,Robey, W.G., Fischinger, P.J. and Arthur, L.O. Immune response to immunostimulatory complexes(ISCOMs) prepared from human immunodeficiency virus type 1 (HIV-1) or the HIV-1 externalenvelope glycoprotein (gp120). Vaccine 7 (1989) 465-473.
Qiu, J.T., Liu, B., Tian, C., Pavlakis, G.N. and Yu, X.F. Enhancement of primary and secondarycellular immune responses against human immunodeficiency virus type 1 gag by using DNAexpression vectors that target Gag antigen to the secretory pathway. J. Virol. 74 (2000) 5997-6005.
Rammensee, H.G., Friede, T. and Stevanoviic, S. MHC ligands and peptide motifs: first listing.Immunogenetics 41 (1995) 178-228.
Reid, G. Soluble proteins incorporate into ISCOMs after covalent attachment of fatty acid. Vaccine 10(1992) 597-602.
Remington, J.S. The tragedy of toxoplasmosis. Pediatr. Infect. Dis. J. 9 (1990) 762-763.Restifo, N.P. and Rosenberg, S.A. Developing recombinant and synthetic vaccines for the treatment ofmelanoma. Curr. Opin. Oncol. 11 (1999) 50-57.
Robert, A., Samuelson, P., Andreoni, C., Bachi, T., Uhlén, M., Binz, H., Nguyen, T.N. and Ståhl, S.Surface display on staphylococci: a comparative study. FEBS Lett. 390 (1996) 327-333.
Rodriguez, F. and Whitton, J.L. Enhancing DNA immunization. Virology 268 (2000) 233-238.
Rolls, M.M., Webster, P., Balba, N.H. and Rose, J.K. Novel infectious particles generated byexpression of the vesicular stomatitis virus glycoprotein from a self-replicating RNA. Cell 79 (1994)497-506.
Rudolph, R. Successful protein folding on an industrial scale. In: Cleland, J.L. and Craik, C.S. (Eds.),Protein Engineering: Principles and Practice. Wiley-Liss, Inc., New York, 1996, pp. 283-298.
Rönnberg, B., Fekadu, M., Behboudi, S., Kenne, L. and Morein, B. Effects of carbohydratemodification of Quillaja saponaria Molina QH-B fraction on adjuvant activity, cholesterol-bindingcapacity and toxicity. Vaccine 15 (1997) 1820-1826.
Rönnberg, B., Fekadu, M. and Morein, B. Adjuvant activity of non-toxic Quillaja saponaria Molinacomponents for use in ISCOM matrix. Vaccine 13 (1995) 1375-1382.
Saikh, K.U., Sesno, J., Brandler, P. and Ulrich, R.G. Are DNA-based vaccines useful for protectionagainst secreted bacterial toxins? Tetanus toxin test case. Vaccine 16 (1998) 1029-1038.
RECOMBINANT SUBUNIT VACCINES
87
Salgaller, M.L. and Lodge, P.A. Use of cellular and cytokine adjuvants in the immunotherapy ofcancer. J. Surg. Oncol. 68 (1998) 122-138.
Samuelsson, E., Moks, T., Nilsson, B. and Uhlén, M. Enhanced in vitro refolding of insulin-likegrowth factor I using a solubilizing fusion partner. Biochemistry 33 (1994) 4207-4211.
Samuelsson, E., Wadensten, H., Hartmanis, M., Moks, T. and Uhlén, M. Facilitated in vitro refoldingof human recombinant insulin-like growth factor I using a solubilizing fusion partner. Biotechnology(N Y) 9 (1991) 363-366.
Sasaki, S., Sumino, K., Hamajima, K., Fukushima, J., Ishii, N., Kawamoto, S., Mohri, H., Kensil, C.R.and Okuda, K. Induction of systemic and mucosal immune responses to human immunodeficiencyvirus type 1 by a DNA vaccine formulated with QS-21 saponin adjuvant via intramuscular andintranasal routes. J. Virol. 72 (1998) 4931-4939.
Sasaki, S., Tsuji, T., Hamajima, K., Fukushima, J., Ishii, N., Kaneko, T., Xin, K.Q., Mohri, H., Aoki,I., Okubo, T., Nishioka, K. and Okuda, K. Monophosphoryl lipid A enhances both humoral and cell-mediated immune responses to DNA vaccination against human immunodeficiency virus type 1.Infect. Immun. 65 (1997) 3520-3528.
Schirmbeck, R., Bohm, W. and Reimann, J. DNA vaccination primes MHC class I-restricted, simianvirus 40 large tumor antigen-specific CTL in H-2d mice that reject syngeneic tumors. J. Immunol. 157(1996) 3550-3558.
Schlienger, K., Mancini, M., Riviere, Y., Dormont, D., Tiollais, P. and Michel, M.L. Humanimmunodeficiency virus type 1 major neutralizing determinant exposed on hepatitis B surface antigenparticles is highly immunogenic in primates. J. Virol. 66 (1992) 2570-2576.
Schneerson, R., Robbins, J.B., Parke, J.C., Jr., Bell, C., Schlesselman, J.J., Sutton, A., Wang, Z.,Schiffman, G., Karpas, A. and Shiloach, J. Quantitative and qualitative analyses of serum antibodieselicited in adults by Haemophilus influenzae type b and pneumococcus type 6A capsularpolysaccharide-tetanus toxoid conjugates. Infect. Immun. 52 (1986) 519-528.
Schneewind, O., Fowler, A. and Faull, K.F. Structure of the cell wall anchor of surface proteins inStaphylococcus aureus. Science 268 (1995) 103-106.
Schneider, J., Gilbert, S.C., Blanchard, T.J., Hanke, T., Robson, K.J., Hannan, C.M., Becker, M.,Sinden, R., Smith, G.L. and Hill, A.V.S. Enhanced immunogenicity for CD8+ T cell induction andcomplete protective efficacy of malaria DNA vaccination by boosting with modified vaccinia virusAnkara. Nature Med. 4 (1998) 397-402.
Schrijver, R.S., Langedijk, J.P., Keil, G.M., Middel, W.G., Maris-Veldhuis, M., Van Oirschot, J.T. andRijsewijk, F.A. Comparison of DNA application methods to reduce BRSV shedding in cattle. Vaccine16 (1998) 130-134.
Schutze, M.P., Leclerc, C., Jolivet, M., Audibert, F. and Chedid, L. Carrier-induced epitopicsuppression, a major issue for future synthetic vaccines. J. Immunol. 135 (1985) 2319-2322.
Sedegah, M., Hedström, R., Hobart, P. and Hoffman, S.L. Protection against malaria by immunizationwith plasmid DNA encoding circumsporozoite protein. Proc. Natl. Acad. Sci. U S A 91 (1994) 9866-9870.
CHRISTIN ANDERSSON
88
Sedegah, M., Jones, T.R., Kaur, M., Hedström, R., Hobart, P., Tine, J.A. and Hoffman, S.L. Boostingwith recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine.Proc. Natl. Acad. Sci. USA 95 (1998) 7648-7653.
Sedlik, C., Saron, M., Sarraseca, J., Casal, I. and Leclerc, C. Recombinant parvovirus-like particles asan antigen carrier: a novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic Tcells. Proc. Natl. Acad. Sci. U S A 94 (1997) 7503-7508.
Siegrist, C.A., Plotnicky-Gilquin, H., Cordova, M., Berney, M., Bonnefoy, J.Y., Nguyen, T.N.,Lambert, P.H. and Power, U.F. Protective efficacy against respiratory syncytial virus following murineneonatal immunization with BBG2Na vaccine: influence of adjuvants and maternal antibodies. J.Infect. Dis. 179 (1999) 1326-1333.
Simon, M.M., Gern, L., Hauser, P., Zhong, W., Nielsen, P.J., Kramer, M.D., Brenner, C. and Wallich,R. Protective immunization with plasmid DNA containing the outer surface lipoprotein A gene ofBorrelia burgdorferi is independent of an eukaryotic promoter. Eur. J. Immunol. 26 (1996) 2831-2840.
Singh, M., Briones, M., Ott, G. and O'Hagan, D. Cationic microparticles: A potent delivery system forDNA vaccines. Proc. Natl. Acad. Sci. U S A 97 (2000) 811-816.
Singh, M. and O'Hagan, D. Advances in vaccine adjuvants. Nat. Biotechnol. 17 (1999) 1075-1081.
Sizemore, D.R., Branström, A.A. and Sadoff, J.C. Attenuated Shigella as a DNA delivery vehicle forDNA-mediated immunization. Science 270 (1995) 299-302.
Sizemore, D.R., Branström, A.A. and Sadoff, J.C. Attenuated bacteria as a DNA delivery vehicle forDNA-mediated immunization. Vaccine 15 (1997) 804-807.
Sjölander, A. and Cox, J.C. Uptake and adjuvant activity of orally delivered saponin and ISCOMvaccines. Adv Drug Deliv Rev 34 (1998) 321-338.
Sjölander, A., Hansson, M., Lövgren, K., Wahlin, B., Berzins, K. and Perlmann, P. Immunogenicity inrabbits and monkeys of influenza ISCOMs conjugated with repeated sequences of the Plasmodiumfalciparum antigen Pf155/RESA. Parasite Immunol. 15 (1993a) 355-359.
Sjölander, A., Lövgren, K., Ståhl, S., Aslund, L., Hansson, M., Nygren, P.-Å., Larsson, M., Hagstedt,M., Wåhlin, B., Berzins, K., Uhlén, M., Morein, B. and Perlmann, P. High antibody responses inrabbits immunized with influenza virus ISCOMs containing a repeated sequence of the Plasmodiumfalciparum antigen Pf155/RESA. Vaccine 9 (1991) 443-450.
Sjölander, A., Nygren, P.-Å., Ståhl, S., Berzins, K., Uhlén, M., Perlmann, P. and Andersson, R. Theserum albumin-binding region of streptococcal protein G: a bacterial fusion partner with carrier-relatedproperties. J. Immunol. Methods 201 (1997) 115-123.
Sjölander, A., Ståhl, S., Lövgren, K., Hansson, M., Cavelier, L., Walles, A., Helmby, H., Wåhlin, B.,Morein, B., Uhlén, M., Berzins, K., Perlmann, P. and Wahlgren, M. Plasmodium falciparum: Theimmune response in rabbits to the clustered asparagine-rich protein (CARP) after immunization inFreund's adjuvant or immunostimulating complexes (ISCOMs). Exp. Parasitol. 76 (1993b) 134-145.
Sjölander, A., Ståhl, S., Nygren, P.-Å., Aslund, L., Ahlborg, N., Wahlin, B., Scherf, A., Berzins, K.,Uhlén, M. and Perlmann, P. Immunogenicity and antigenicity in rabbits of a repeated sequence ofPlasmodium falciparum antigen Pf155/RESA fused to two immunoglobulin G- binding domains ofstaphylococcal protein A. Infect. Immun. 58 (1990) 854-859.
RECOMBINANT SUBUNIT VACCINES
89
Sjölander, A., Ståhl, S. and Perlmann, P. Bacterial expression systems based on protein A and proteinG designed for the production of immunogens: applications to Plasmodium falciparum malariaantigens. Immunomethods 2 (1993c) 79-92.
Smerdou, C. and Liljeström, P. Two-helper RNA system for production of recombinant Semliki forestvirus particles. J Virol 73 (1999) 1092-1098.
Smit, P., Oberholzer, D., Hayden-Smith, S., Koornhof, H.J. and Hilleman, M.R. Protective efficacy ofpneumococcal polysaccharide vaccines. Jama 238 (1977) 2613-2616.
Smith, G.P. Filamentous fusion phage: novel expression vectors that display cloned antigens on thevirion surface. Science 228 (1985) 1315-1317.
Stover, C.K., Bansal, G.P., Hanson, M.S., Burlein, J.E., Palaszynski, S.R., Young, J.F., Koenig, S.,Young, D.B., Sadziene, A. and Barbour, A.G. Protective immunity elicited by recombinant bacilleCalmette-Guerin (BCG) expressing outer surface protein A (OspA) lipoprotein: a candidate Lymedisease vaccine. J. Exp. Med. 178 (1993) 197-209.
Studier, F.W., Rosenberg, A.H., Dunn, J.J. and Dubendorff, J.W. Use of T7 RNA polymerase to directexpression of cloned genes. Methods Enzymol. 185 (1990) 60-89.
Ståhl, S., Nilsson, J., Hober, S., Uhlén, M. and Nygren, P.-Å. Affinity fusions, gene expression. In:Flickinger, M.C. and Drew, S.W. (Eds.), The encyclopedia of Bioprocess technology: Fermentation,Biocatalysis and Bioseparation. John Wiley and Sons Inc., New York, 1999, pp. 49-63.
Ståhl, S. and Nygren, P.-Å. The use of gene fusions to protein A and protein G in immunology andbiotechnology. Pathol. Biol. (Paris) 45 (1997) 66-76.
Ståhl, S., Nygren, P.-Å. and Uhlén, M. Strategies for gene fusions. Methods Mol. Biol. 62 (1997) 37-54.
Ståhl, S., Sjölander, A., Nygren, P.-Å., Berzins, K., Perlmann, P. and Uhlén, M. A dual expressionsystem for the generation, analysis and purification of antibodies to a repeated sequence of thePlasmodium falciparum antigen Pf155/RESA. J. Immunol. Methods 124 (1989) 43-52.
Ståhl, S. and Uhlén, M. Bacterial surface display: trends and progress. Trends Biotechnol. 15 (1997)185-192.
Suarez, A., Staendner, L.H., Rohde, M., Piatti, G., Timmis, K.N. and Guzman, C.A. Stable expressionof pertussis toxin in Bordetella bronchiseptica under the control of a tightly regulated promoter. Appl.Environ. Microbiol. 63 (1997) 122-127.
Suhrbier, A. Multi-epitope DNA vaccines. Immunol Cell Biol 75 (1997) 402-8.
Sun, J.-B., Holmgren, J. and Czerkinsky, C. Cholera toxin B subunit: an efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance. Proc. Natl. Acad. Sci. USA 91(1994) 10795-10799.
Sutter, G. and Moss, B. Nonreplicating vaccinia vector efficiently expresses recombinant genes. Proc.Natl. Acad. Sci. U S A 89 (1992) 10847-10851.
Tam, J.P. Recent advances in multiple antigen peptides. J. Immunol. Methods 196 (1996) 17-32.
CHRISTIN ANDERSSON
90
Tang, D.C., DeVit, M. and Johnston, S.A. Genetic immunization is a simple method for eliciting animmune response. Nature 356 (1992) 152-154.
Tartaglia, J., Perkus, M.E., Taylor, J., Norton, E.K., Audonnet, J.C., Cox, W.I., Davis, S.W., van derHoeven, J., Meignier, B., Riviere, M. and et al. NYVAC: a highly attenuated strain of vaccinia virus.Virology 188 (1992) 217-232.
Thoelen, S., Van Damme, P., Mathei, C., Leroux-Roels, G., Desombere, I., Safary, A.,Vandepapeliere, P., Slaoui, M. and Meheus, A. Safety and immunogenicity of a hepatitis B vaccineformulated with a novel adjuvant system. Vaccine 16 (1998) 708-714.
Toda, S., Ishii, N., Okada, E., Kusakabe, K.I., Arai, H., Hamajima, K., Gorai, I., Nishioka, K. andOkuda, K. HIV-1-specific cell-mediated immune responses induced by DNA vaccination wereenhanced by mannan-coated liposomes and inhibited by anti-interferon-gamma antibody. Immunology92 (1997) 111-117.
Tokunaga, T., Yamamoto, H., Shimada, S., Abe, H., Fukuda, T., Fujisawa, Y., Furutani, Y., Yano, O.,Kataoka, T., Sudo, T. and et al. Antitumor activity of deoxyribonucleic acid fraction fromMycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity. J.Natl. Cancer Inst. 72 (1984) 955-962.
Traquina, P., Morandi, M., Contorni, M. and Van Nest, G. MF59 adjuvant enhances the antibodyresponse to recombinant hepatitis B surface antigen vaccine in primates. J. Infect. Dis. 174 (1996)1168-1175.
Tsuji, M., Bergmann, C.C., Takita-Sonoda, Y., Murata, K., Rodrigues, E.G., Nussenzweig, R.S. andZavala, F. Recombinant Sindbis viruses expressing a cytotoxic T-lymphocyte epitope of a malariaparasite or of influenza virus elicit protection against the corresponding pathogen in mice. J. Virol 72(1998) 6907-6910.
Tubulekas, I., Berglund, P., Fleeton, M. and Liljeström, P. Alphavirus expression vectors and their useas recombinant vaccines: a minireview. Gene 190 (1997) 191-195.
Uhlén, M., Forsberg, G., Moks, T., Hartmanis, M. and Nilsson, B. Fusion proteins in biotechnology.Curr. Opin. Biotechnol. 3 (1992) 363-369.
Uhlén, M., Nilsson, B., Guss, B., Lindberg, M., Gatenbeck, S. and Philipson, L. Gene fusion vectorsbased on the gene for staphylococcal protein A. Gene 23 (1983) 369-738.
Ulmer, J.B., DeWitt, C.M., Chastain, M., Friedman, A., Donnelly, J.J., McClements, W.L., Caulfield,M.J., Bohannon, K.E., Volkin, D.B. and Evans, R.K. Enhancement of DNA vaccine potency usingconventional aluminum adjuvants. Vaccine 18 (2000) 18-28.
Ulmer, J.B., Donnelly, J.J., Parker, S.E., Rhodes, G.H., Felgner, P.L., Dwarki, V.J., Gromkowski,S.H., Deck, R.R., De Witt, C.M., Friedman, A., Hawe, l.A., Leander, K.A., Martinez, D., Perry, H.C.,Shiver, J.W., Montgomery, D.L. and Liu, M.A. Heterologous protection against influenza by injectionof DNA encoding a viral protein. Science 259 (1993) 1745-1749.
Ulmer, J.B., Fu, T.M., Deck, R.R., Friedman, A., Guan, L., DeWitt, C., Liu, X., Wang, S., Liu, M.A.,Donnelly, J.J. and Caulfield, M.J. Protective CD4+ and CD8+ T cells against influenza virus inducedby vaccination with nucleoprotein DNA. J. Virol. 72 (1998) 5648-5653.
RECOMBINANT SUBUNIT VACCINES
91
Ulmer, J.B., Liu, M.A., Montgomery, D.L., Yawman, A.M., Deck, R.R., DeWitt, C.M., Content, J.and Huygen, K. Expression and immunogenicity of Mycobacterium tuberculosis antigen 85 by DNAvaccination. Vaccine 15 (1997) 792-794.
Valenzuela, P., Medina, A., Rutter, W.J., Ammerer, G. and Hall, B.D. Synthesis and assembly ofhepatitis B virus surface antigen particles in yeast. Nature 298 (1982) 347-350.
Vanderzanden, L., Bray, M., Fuller, D., Roberts, T., Custer, D., Spik, K., Jahrling, P., Huggins, J.,Schmaljohn, A. and Schmaljohn, C. DNA vaccines expressing either the GP or NP genes of Ebolavirus protect mice from lethal challenge. Virology 246 (1998) 134-144.
Viret, J.F., Cryz, S.J., Jr. and Favre, D. Expression of Shigella sonnei lipopolysaccharide in Vibriocholerae. Mol. Microbiol. 19 (1996) 949-963.
Vreden, S.G., Verhave, J.P., Oettinger, T., Sauerwein, R.W. and Meuwissen, J.H. Phase I clinical trialof a recombinant malaria vaccine consisting of the circumsporozoite repeat region of Plasmodiumfalciparum coupled to hepatitis B surface antigen. Am J. Trop. Med. Hyg. 45 (1991) 533-538.
Walsh, G. Biopharmaceutical benchmarks. Nat. Biotechnol. 18 (2000) 831-833.
Wang, R., Doolan, D.L., Charoenvit, Y., Hedström, R.C., Gardner, M.J., Hobart, P., Tine, J., Sedegah,M., Fallarme, V., Sacci, J.B., Jr., Kaur, M., Klinman, D.M., Hoffman, S.L. and Weiss, W.R.Simultaneous induction of multiple antigen-specific cytotoxic T lymphocytes in nonhuman primatesby immunization with a mixture of four Plasmodium falciparum DNA plasmids. Infect. Immun. 66(1998a) 4193-4202.
Wang, R., Doolan, D.L., Le, T.P., Hedström, R.C., Coonan, K.M., Charoenvit, Y., Jones, T.R., Hobart,P., Margalith, M., Ng, J., Weiss, W.R., Sedegah, M., de Taisne, C., Norman, J.A. and Hoffman, S.L.Induction of antigen-specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine. Science282 (1998b) 476-480.
Wang, S., Liu, X., Fisher, K., Smith, J.G., Chen, F., Tobery, T.W., Ulmer, J.B., Evans, R.K. andCaulfield, M.J. Enhanced type I immune response to a hepatitis B DNA vaccine by formulation withcalcium- or aluminum phosphate. Vaccine 18 (2000) 1227-1235.
Wang, Y., Xiang, Z., Pasquini, S. and Ertl, H.C. Immune response to neonatal genetic immunization.Virology 228 (1997) 278-284.
Weiner, G.J., Liu, H.M., Wooldridge, J.E., Dahle, C.E. and Krieg, A.M. Immunostimulatoryoligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigenimmunization. Proc. Natl. Acad. Sci. U S A 94 (1997) 10833-10837.
Wells, J.M., Wilson, P.W., Norton, P.M., Gasson, M.J. and Le Page, R.W. Lactococcus lactis: high-level expression of tetanus toxin fragment C and protection against lethal challenge. Mol. Microbiol. 8(1993) 1155-1162.
Whitton, J.L., Sheng, N., Oldstone, M.B. and McKee, T.A. A "string-of-beads" vaccine, comprisinglinked minigenes, confers protection from lethal-dose virus challenge. J. Virol. 67 (1993) 348-352.
Wolff, J.A., Malone, R.W., Williams, P., Chong, W., Acsadi, G., Jani, A. and Felgner, P.L. Directgene transfer into mouse muscle in vivo. Science 247 (1990) 1465-1458.
CHRISTIN ANDERSSON
92
Xiang, Z.Q., He, Z., Wang, Y. and Ertl, H.C. The effect of interferon-gamma on geneticimmunization. Vaccine 15 (1997) 896-898.
Xiang, Z.Q., Yang, Y., Wilson, J.M. and Ertl, H.C. A replication-defective human adenovirusrecombinant serves as a highly efficacious vaccine carrier. Virology 219 (1996) 220-227.Xu, L., Sanchez, A., Yang, Z., Zaki, S.R., Nabel, E.G., Nichol, S.T. and Nabel, G.J. Immunization forEbola virus infection. Nat. Med. 4 (1998) 37-42.
Yankauckas, M.A., Morrow, J.E., Parker, S.E., Abai, A., Rhodes, G.H., Dwarki, V.J. andGromkowski, S.H. Long-term anti-nucleoprotein cellular and humoral immunity is induced byintramuscular injection of plasmid DNA containing NP gene. DNA Cell. Biol. 12 (1993) 771-776.
Zhang, D.J., Yang, X., Shen, C. and Brunham, R.C. Characterization of immune responses followingintramuscular DNA immunization with the MOMP gene of Chlamydia trachomatis mousepneumonitis strain. Immunology 96 (1999) 314-321.
Zhou, X., Berglund, P., Rhodes, G., Parker, S.E., Jondal, M. and Liljeström, P. Self-replicatingSemliki Forest virus RNA as recombinant vaccine. Vaccine 12 (1994) 1510-1514.
Zhou, X., Berglund, P., Zhao, H., Liljeström, P. and Jondal, M. Generation of cytotoxic and humoralimmune responses by nonreplicative recombinant Semliki forest virus. Proc. Natl. Acad. Sci. 92(1995) 3009-3013.
Zhu, X., Wu, S. and Letchworth, G.J.,3rd, Yeast-secreted bovine herpesvirus type 1 glycoprotein D
has authentic conformational structure and immunogenicity. Vaccine 15 (1997) 679-688.