REVIEW Design and production of recombinant subunit vaccines

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98 M. Hansson, P.-A� . Nygren and S. Sta� hl lational modifications and the presentation of the antigen to the host’s immune system, they provide antigen synthesis in the host in a similar way to that which occurs during a natural infection. One drawback of DNA vaccines that has been extensively discussed is the potential risk for integration into the host’s genome. This risk could be minimized by using RNA instead of DNA for immunization. Due to the low stability of mRNA, preparation and administration of RNA vaccines are not entirely simple. These limitations could be circumvented by constructing RNA or DNA vectors based on parts of alphavirus genomes [32,33], carrying a gene encoding the foreign antigen and a gene encoding an alphaviral replicase. Upon transfection of such a construct, the replicase gene will be translated and the produced replicase will mass-replicate the antigen-encoding RNA. The transfected cell will express large amounts of the foreign protein for a short period of time, followed by cell death [32]. In a recent study, mice immunized with an alphaviral DNA vector encoding influenza antigens developed humoral and cellular responses at higher levels than mice that received a conventional DNA-vaccine vector [33]. In addition, protective immunity against influenza challenge was elicited in the immunized mice. Nucleic acid vaccines, based on either DNA or self-replicating RNA, will without doubt find a role in the future of vaccine development (for reviews see [30,34]). Recombinant production of protein immunogens Gene construction Recombinant-DNA techniques offer several ways to construct genes coding for the immunogens to be produced. The use of the PCR enables direct isolation of the gene from its natural source, but requires knowledge of the target DNA sequence. In the PCR amplification, suitable restriction sites can be introduced for direct cloning into a desired expression vector. Building up genes de novo using synthetic oligonucleotides can sometimes be preferable [35–37], e.g. if only the protein sequence is known, or if the GC content of the original gene would differ significantly from that of the host selected for expression. It may also be desirable to adapt the codons used to the chosen host [37], since heterologous genes rich in codons that are used rarely by, for example, Escherichia coli, may not be expressed efficiently in E. coli [38]. Host vector systems The choice of expression system depends upon many factors, including (i) the requirements for post-translational � 2000 Portland Press Ltd modification, (ii) the proteolytic stability of the target protein, (iii) whether the protein is secretable, (iv) the possibility of renaturation of a protein produced in a misfolded form and (v) the acceptable costs for the final product. There are four major expression hosts that are commonly used to produce vaccine antigens; bacterial, yeast, insect and mammalian expression systems. In addition, transgenic plant expression systems have started to emerge, with the aim of utilizing the plant both for production of the subunit vaccine and for vaccine delivery via the edible plant [39]. A plant-based subunit vaccine has indeed been tested in human volunteers. Transgenic potatoes expressing LTB, the binding subunit of the heat-labile toxin of enterotoxigenic E. coli, was consumed by volunteers who then developed significant LTB-specific antibody (IgG and IgA) responses [40]. Despite a costly development phase, transgenic plants might become interesting as future vaccine production� delivery vehicles, but their efficacy needs to be proven further. Bacterial systems can express antigens at very high levels and are suitable for expressing vaccine antigens that do not require significant post-translational modifications. E. coli is the most commonly used bacterium for production of heterologous proteins, being easy to manipulate, genetically and physiologically well defined, and yielding high expression levels [41]. A multitude of vectors and strains are available, making it possible to design a suitable expression system. Expression of recombinant antigens in bacterial systems other than E. coli may sometimes be advantageous [42]. Salmonella typhimurium [43,44], Vibrio cholerae [45] and Bacillus brevis [46,47] are some examples of other bacteria that have been used for expression of antigens for vaccine production purposes. One particular feature in favour of Gram-positive bacteria is that the risk of contaminating lipopolysaccharides is avoided. Among the eukaryotic expression systems, the baker’s yeast Saccharomyces cerevisiae is the most commonly used [48]. Yeast may not express proteins at the levels that can be obtained in E. coli, but Saccharomyces can easily be grown to high cell densities. The first human subunit vaccine produced by recombinant means was the hepatitis B vaccine, which consists of a major surface antigen (HBsAg) and which is produced in Sacc. cerevisiae [49]. In later years the yeast Pichia pastoris became a very promising production host due to high production levels [48]. Yeast cells possess some of the eukaryotic possibilities for post-translationally modifying proteins, e.g. phosphorylation and glycosylation, but, since the glycosylations differ from the glycosylations in mammalian cells, it can potentially also be disadvantageous [50]. Baculovirus-based expression systems take advantage of the ability of a virus to infect arthropod cells, and heterologous proteins can be efficiently produced in both insect cells and larvae [51], but to a significantly higher cost
than in microbial expression systems. Advantages of baculovirus over other expression systems are that proteins are expressed at very high levels, and since these cells are eukaryotic, they are able to make many of the posttranslational modifications of mammalian cells, even though the glycosylations are not the same as in mammalian systems. Several mammalian expression systems for production of recombinant proteins have been developed [51], but most vaccines requiring eukaryotic post-translational modifications are still being expressed in yeast. The main reasons are the higher product yield in yeast and the ease with which yeast can be manipulated and grown. The major advantage of a mammalian expression system is that it will post-translationally modify the expressed product with the highest degree of fidelity compared with the other expression systems, and expressed viral proteins will thus be glycosylated in a human-like fashion. Drawbacks of mammalian expression systems are the laborious and time-consuming generation of stable cell lines, the high cultivation costs, and the problems associated with viral and bacterial (e.g. Mycoplasma) infections. The choice of a suitable production system thus depends on several factors, e.g. the biochemical and biological properties of the protein of interest, the amount of recombinant protein needed and the requirements for posttranslational modification. Concerning the latter point, bacterial antigens do not normally need such modifications and would thus be likely to be expressed successfully in bacteria. Parasitic antigens originating from eukaryotic organisms may have such requirements, and a eukaryotic expression system might be needed in cases where the posttranslational modifications are necessary for eliciting protective immune responses. Nevertheless, a normally highly glycosylated antigen can be found to be perfectly immunogenic when produced in bacteria [50]. Normally when setting up a production scheme for a target subunit immunogen, the least-complicated bacterial expression systems are carefully investigated before more complicated and more costly host�vector systems are evaluated. Strategies for improved bioprocesses Recombinant-DNA techniques and the expression of heterologous proteins in E. coli and other hosts have provided a source of proteins of a quantity and quality that, previously, was difficult or impossible to achieve through their isolation from natural sources. In addition, gene technology can be used to engineer proteins to facilitate bioprocessing. Geneproduct design can influence the yield and the localization of the product, as well as adapting the gene product by fusion technology to specific unit operations suitable for large-scale downstream processing. Since cost-efficient production of subunit vaccines is of utmost importance, it is advisable to Recombinant subunit vaccines 99 use careful genetic design, when possible, to simplify and improve the overall production process. Secretion By taking advantage of a signal peptide (secretory leader), normally fused to the N-terminus of the target protein, efficient secretion of the protein can be achieved. For example, export of a protein from the cytoplasm into the periplasm of E. coli offers several advantages [52,53]. Secretion of the target protein to the periplasm functions as an initial purification step and simplifies the downstream purification considerably, since the periplasm contains approx. 100 host proteins [54] compared with more than 4000 proteins in the cytoplasm [55]. The number of proteases in the periplasm is also drastically fewer [56], which reduces the risk of degradation, and the oxidizing environment in the periplasm favours disulphide-bond formation, which thus stimulates a potentially correct folding of cysteine-containing target proteins. In addition to secretion into the periplasmic space of the E. coli cell, the protein can in some cases be recovered from the culture medium [57–59]. The mechanism for the release of proteins into culture medium by E. coli cells is not known in detail, but is probably due to non-specific leakage from the cells [60,61]. Recovery of proteins from the culture medium has the advantages that cell disruption is not needed and that there is a low number of host proteins present [59]. A potential drawback may be the dilution of the protein, which could potentially complicate the purification in large-scale bioprocesses. Improved recovery by engineering of protein properties The addition of charged amino acid residues or mutation of amino acids of a protein to alter the isolectric point (pI), can improve the purification by allowing ion-exchange chromatography [62]. Fusion of hydrophobic tails to the target protein has been shown to alter the partitioning of the fusion protein in aqueous two-phase systems, enabling the use of such a system as a primary purification step for efficient recovery of the target protein [63,64]. Hydrophobic tagging of protein immunogens has the additional advantage, as will be discussed further below, that it could allow direct adjuvant incorporation of purified subunit vaccines through hydrophobic interaction [65,66]. An illustrative example of how genetic design can be used to achieve integration of unit operations was presented in the process design for production of a malaria subunitvaccine candidate [59,67]. The fusion protein ZZ-M5, constituting the malaria subunit-vaccine candidate, was produced in E. coli using a secretion strategy. The M5 polypeptide is derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155�RESA, and the ZZ region represents an IgG-binding affinity tag derived from Staphylococcus aureus Protein A (SpA). More than 65% � 2000 Portland Press Ltd
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REVIEW Design and production of recombinant subunit vaccines

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