REVIEW Design and production of recombinant subunit vaccines

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96 M. Hansson, P.-A� . Nygren and S. Sta� hl Table 1 A comparison of some properties of different vaccine types Vaccine type Advantages Drawbacks Live vaccines (attenuated) One or few doses normally required Controlled attenuation normally required Long-lasting protection Risk of reversion to pathogenicity Both humoral and cellular responses Certain risk of transmission Poorly defined composition Killed vaccines No risk of reversion to pathogenicity Multiple doses typically required No risk of transmission Poorly defined composition Antigen must be produced by cultivation of a pathogen Mainly humoral responses Adjuvants normally needed Subunit vaccines (non-recombinant) Defined composition Various delivery systems available Subunit vaccines (recombinant) No risk of pathogenicity since the pathogenic organism is not present Defined composition Various delivery systems available Simplified large-scale production Further engineering possible Administration (FDA) and World Health Organization (WHO), for exact definitions of the vaccine preparations, it will probably become increasingly difficult to get new vaccines of this class accepted for human use. Subunit vaccines take advantage of the possibility of using only part of the infectious micro-organism to raise a protective immune response, and since subunit vaccines cannot replicate in the host, there is no risk of pathogenicity. The composition of a subunit vaccine can normally be clearly defined, which is a significant advantage in terms of safety considerations and minimization of side-effects. In order to elicit a vigorous immune response, subunit vaccines often require multiple doses, as well as the use of adjuvants. Subunit vaccines can be based on peptides, proteins or polysaccharides that have been shown to contain protective epitopes. Many of the cell-surface carbohydrates of pathogenic bacteria, e.g. capsular polysaccharides, are important antigenic determinants for vaccine development. Subunit conjugate vaccines [2,3] based on such polysaccharides, e.g. Haemophilus influenzae type b (Hib) vaccines, which are commonly delivered coupled to protein carriers such as the tetanus toxoid [4], will not be described further in this review. Immunogenic components can be isolated directly from pathogenic organisms, e.g. bacterial polysaccharides are often shed during growth and can be enriched from the culture medium. However, the production of subunit vaccines often requires purification of the immunogens from large quantities of the pathogenic organism, which is not without risk and significant cost. Recombinant-DNA technology allows controlled production of protein-subunit vaccines in heterologous hosts. Such strategies have several advantages, e.g. safe and cost- � 2000 Portland Press Ltd Antigen must be produced and purified by cultivation of a pathogen Multiple doses typically required Adjuvants needed Multiple doses typically required Adjuvants needed Table 2 Recombinant subunit vaccines and examples of their advantages (+) and drawbacks (�) Recombinant vaccine Advantages/drawbacks Protein immunogens + No risk of pathogenicity since the pathogenic organism is not present + Efficient production systems available � Multiple doses required � Adjuvants needed Live delivery systems + May induce both humoral and cellular responses + Adjuvants normally not needed � Risk of reversion when using attenuated pathogens as carriers Bacterial + Surface display of antigens possible + Mucosal administration possible Viral + Efficient induction of cellular responses Nucleic acid vaccines + No risk of pathogenicity + Simple production + May induce both humoral and cellular responses � Variable immune responses � Inefficient transfection DNA + Dedicated delivery systems exist � Inefficient delivery � Risk of integration into host chromosomes must be considered � Moderate and variable immune responses RNA + No risk of integration into host chromosomes + No need to enter the nucleus for translation + In vivo amplification systems available � Cumbersome large-scale production � Unstable efficient production systems can be used. Recombinant strategies further offer the possibility of delivering protein subunits with the help of live delivery systems, bacterial or viral, or even as antigen-encoding genes, so-called nucleic acid vaccines (Table 2).
Recombinant subunit vaccines Recent advances in immunology and protein engineering have allowed the design and production of recombinant subunit vaccines [3,5–8]. The epitopes recognized by neutralizing antibodies are usually found in just one or a few proteins present on the surface of the pathogenic organism. Isolation of the genes encoding such epitope-carrying protein immunogens and their expression in heterologous hosts form the basis of recombinant-subunit-vaccine development. The main advantage of using single proteins displaying immunodominant epitopes as vaccines is the possibility of inducing protective immunity without having side effects and immune reactions caused by other parts of the pathogenic organism. Potential challenges in the development of subunit vaccines are that they often are poorly immunogenic and have short in vivo half-lifes. Another difficulty with subunit vaccines is that they often elicit only strain-specific protection, so, to evoke full protection to a disease caused by several related strains, combinations of immunogens from the different strains might be needed. Protein immunogens Protein production in heterologous hosts is a wellestablished technique today, and recombinant strategies can thus combine the benefits of subunit vaccines with efficient production systems using non-pathogenic expression vehicles [8,9]. As will be discussed further, the use of fusion proteins can facilitate the downstream purification of immunogens [8,10] and, in addition, fusion of the target immunogens to carrier proteins can increase both the halflife [11,12] and immunogenicity [13–15] of the immunogen. Live delivery systems Besides the possibility of producing recombinant protein immunogens in heterologous hosts, technologies to construct recombinant live viral and bacterial vaccine-delivery vectors carrying foreign immunogens have been developed. Several viruses have been investigated for their possible use as recombinant vaccines (for extensive reviews see [3,8, 16,17]). The vaccinia vector is perhaps the most wellcharacterized viral vector, and a vaccinia vector encoding a rabies glycoprotein has been used for oral immunization of wild animals [18,19]. Due to safety concerns, non-attenuated recombinant vaccinia virus is not likely to be used in humans. As alternatives, a highly attenuated recombinant vaccinia virus vector (NYVAC), which has been constructed through deletion of 18 open reading frames [20], as well as an avianpoxvirus vector (ALVAC) [21], have been developed. A NYVAC vector expressing seven different malarial antigens has been constructed and demonstrated to induce a Recombinant subunit vaccines 97 Plasmodium-specific antibody response in rhesus macaques [22]. Adenoviruses have also been investigated as live vectors [17], and are especially interesting, since they are not particularly pathogenic in humans. The major disadvantage with adenoviruses is the limited cloning capacity compared with, for example, the vaccinia virus. Several other viruses have also been studied for the possible use as live vectors [17], but no virus-based vaccine, employed for live heterologous vaccine delivery, is yet accepted for human use. The use of live bacteria as mucosal vaccines has been studied extensively, both against the corresponding disease and also as delivery systems for heterologous diseases. Mucosal vaccines are easy to administer, e.g. by the oral or nasal route, and using bacteria as a delivery vehicle makes the vaccine comparatively inexpensive to produce [23]. The best-studied bacterial delivery systems are based on attenuated Salmonella spp. as live vectors expressing heterologous antigens and numerous studies have been performed for development of oral vaccines against bacterial, viral or parasitic diseases [24–26]. Live Salmonella, being an intracellular pathogen, is generally capable of eliciting cellular immune responses to the antigen delivered, a desired property of the immune responses protecting against viral or parasitic diseases. The possibility of expressing foreign proteins on the surface of bacteria makes live bacterial vectors highly interesting. Surface display of heterologous proteins for the construction of live vectors was first described for Gramnegative bacteria (for reviews see [26,27]) and more recently systems for surface display on Gram-positive bacteria have also been described [28,29]. Surface display for the development of live bacterial vectors is described in more detail below. Nucleic acid vaccines Nucleic acid vaccines constitute a new class of recombinant subunit vaccine, consisting of, for example, plasmid DNA containing the gene encoding the antigen of interest under the control of a strong mammalian viral promoter. The antigen-encoding gene will be expressed by the vaccinee upon delivery of the plasmid DNA. DNA vaccines have been shown to generate both humoral and cellular immune responses [30] and the first report of protective efficacy of DNA immunization was against influenza [31]. Immunization of BALB�c mice with plasmid DNA encoding influenza A nucleoprotein resulted in the induction of nucleoproteinspecific antibodies, and protection from a subsequent challenge with a heterologous strain of influenza A virus [31]. In addition to the advantage of a subunit vaccine including only the antigen (or antigens) required for protective immunization, nucleic acid vaccines present several other advantages. Nucleic acid vaccines are relatively easy to construct and produce, and considering the post-trans- � 2000 Portland Press Ltd
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REVIEW Design and production of recombinant subunit vaccines

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