Biotechnological trends towards synthetic vaccines

Immunology Letters, 19 (1988) 241-244 Elsevier IML 01106

Biotechnological trends towards synthetic vaccines F. Y. Liew Department of Experimental Immunobiology, Wellcome Biotech, Beckenham, Kent, U.K. (Received 21 June 1988; accepted 3 August 1988)

1. Introduction

There can be little doubt that when successfully used, vaccination is the most effective answer to infectious diseases. Vaccination has been mainly responsible for the eradication of smallpox and for the control of yellow fever, poliomyelitis and German measles in the human population, and of Newcastle disease, foot-and-mouth disease and Marek's disease in domestic animals. These outstanding successes were often achieved without the benefit of the knowledge of the immune mechanism governing the effectiveness of the vaccines. The earlier vaccines were live wild-type organisms. Although these have been mostly replaced by attenuated or killed organisms, some, like live Leishmania major causing cutaneous leishmaniasis (Oriental sores) is still being used in highly endemic areas [1]. Attenuation is generally achieved by growing the pathogens in an 'unnatural' host (passage); less commonly, viruses have been adapted to grow at a temperature lower than normal (cold adaptation) or have been rendered temperature sensitive. For preparing killed vaccines, the pathogens are inactivated by agents such as methanol, formalin, ~propiolactone, or more recently, an imine. However, such attenuated vaccines can elicit side effects which are frequently unacceptably harmful to the host. Even with the highly successful products such as the attenuated polio virus and smallpox vaccines, there is a small but significant number of post-vaccination incidents. Killed vaccines may also present problems in that there is always the chance that some infecKey words: Vaccine; Peptides; Biotechnology Correspondence to: E Y. Liew, Dept. of Experimental lmmunobiology), Wellcome Biotech, Beckenham, Kent BR3 3BS, U.K.

tious pathogens survive the inactivation process. These risks can be somewhat reduced by the use of subunit vaccines which attempt to enrich the active components by conventional biochemical purification. Even in their purest form, these conventional vaccines would still contain contaminating materials far exceeding, in mass, the immunogenically active ingredients. Such non-essential materials may not only cause unpredictable side effects, but they may also conceivably counteract and neutralise the induction of protective immunity by the effective antigenic determinants. There is thus a real need for a new generation of molecularly defined vaccines which would induce the desirable immune responses capable of controlling particular infectious agents. In this paper, I shall review the current trends in vaccine development in the biotechnological industry using both biosynthesis and chemical synthesis. 2. The new technology

Although the desire to improve the conventional vaccines and to obtain hitherto unavailable vaccine materials were an important impetus in the new vaccine development, one of the key factors was the emergence of a whole new set of biological techniques. These include the monoclonal antibodies, recombinant DNA and microchemistry. Monoclonal antibodies provide a powerful tool for identifying and isolating specific materials of desired immunogenicity. The rDNA technique makes possible the cloning, expression and production of large quantities of materials previously undreamt of, whilst the microchemistry facilitates rapid sequencing of both the nucleic acids and the proteins in ever-decreasing quantities. The combination of these techniques which are themselves continuing to advance rapidly, creates tremendous excitement and optimism among biomedical scientists

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for designing a new generation of effective vaccines against many infectious diseases. This is further enhanced by recent advances made in the field o f immunology in which T and B cell immunity has become even better defined. We are now beginning to understand how T and B cells recognise their respective determinants and how the various cells communicate with each other. This is clearly borne out in several of the papers contained in this volume.

2.1. rDNA derived subunit vaccines These are the 'firsts' of the new generation o f vaccines. The techniques are now well known and so far, two such vaccines are commercially available. These are the Escherichia coli vaccine for pigs, with extra plasmids coding for the K88 surface antigen, and the yeast-produced vaccine against hepatitis B virus based on the common surface antigen a. T h e next series o f vaccines based on rDNA subunits are likely to be those against malaria, pertussis, influenza, CMV, RSV and even AIDS. All these vaccines still have many hurdles to overcome and it is extremely unlikely that all will prove successful. Malaria and AIDS are discussed in detail elsewhere in this volume [2]. These diseases are currently enjoying great attention from research institutes and biotechnology companies for different reasons. For malaria, it is the sheer scale o f the disease and the humanitarian reasons which make it an important target. In the case of AIDS, it is because of its potential threat and also because it is fashionable. There is tremendous pressure for improvement of the current pertussis vaccine in order to reduce the alleged serious side effects. Influenza virus, CMV and RSV vaccines all have great potential commercial return as well as intellectual challenge. The limitations of rDNA-derived subunit vaccines, even when successful, are still considerable. These are macromolecules which frequently require extensive and costly down-stream purification processes. They are proteins and hence require elaborate cold-chains for distribution and administration, which are particularly difficult to maintain in the case of tropical diseases. The subunit vaccines also contain multiple determinants, many o f which are likely to be non-active or may even be detrimental to the host's defence mechanism. In spite of these potential weaknesses, they are likely to be the new 242

vaccines for many years to come. 2.2. Heteroiogous carriers There are currently two experimentally wellestablished carriers: vaccinia and Salmonella mutants. The vaccinia system has been explored in considerable detail and several experimental vaccines have been successfully constructed [3]. However, this system is likely to encounter considerable objections on the grounds of the rare but serious complications that can be induced by the current vaccinia vaccine. Research in this area is now concentrating on the specific deletion o f genes that are not essential for virus replication in tissue culture but decrease virulence in animals [3]. In addition, insertion of human IL-2 gene into the vaccinia virus also fulfils this expectation [4]. However, until such time as a suitably attenuated vaccinia mutant, which can still serve as an effective carrier, is obtained, vaccinia virus is unlikely to secure general approval from the licensing authorities as a heterologous carrier except in the case of extremely serious diseases where other forms o f vaccination are unavailable. Salmonella typhi auxotrophic mutants which have deletion at the AroA gene, causing requirement for aromatic metabolites (including paminobenzoate) are now serious candidate vaccines for typhoid [5]. Experimentally, S. typhimurium AroA- strains have been used successfully as heterologous carriers for a range of foreign antigens, including K88 adhesion fimbriae, heat labile toxin B and a 65 kDa mycobacteria antigen. Mice immunised with these constructs expressed both humoral and cell-mediated immunity to Salmonella typhimurium as well as the foreign antigens. The major advantage of this system is the possibility of administering these antigens orally and stimulating the secretory and systemic immunity without untoward side effects. However, at present it can only be successfully constructed with respect to bacterial antigens and only in the murine experimental model. This area is receiving intensive attention in the biotech industry for further development and optimisation. 2.3. Anti-idiotypic vaccines This concept, which is based on Jerne's network

hypothesis [6] and aims at stimulating the immune responses with Ab2 or antigen-mirror-image antiidiotypic antibodies, has several outstanding potentials [7], the most important of which are the avoidance of using antigens altogether and the possibility of inducing immunity against carbohydrate materials. However, this field is perhaps the least active in the biotechnology industry, since it is generally felt that the specificity and the low immunogenicity are such that they are unlikely to fulfil the stringent requirements of the polymorphic human population. 2.4. Peptides vaccines Synthetic peptide vaccines have received much current attention because of some exciting advances in the T cell receptor recognition pattern [8, 9] and the elucidation of the three-dimensional structure of M H C class I molecules [10]. These provide the possibility of predicting various defined antigenic determinants capable of selectively activating various subsets of T cells [11]. The peptide vaccines offer several obvious advantages [12]. The product is chemically defined; it is generally stable indefinitely; there can be no infectious agent present, and there is little need for a large-scale production plant. Down-stream processing requirement is also absent. Furthermore, they provide the opportunity to use a delayed-release mechanism without damage to the peptide vaccines. In addition, it is possible, at least in theory, to stimulate appropriate immune responses by design. The major disadvantages are, however, the problems of genetic restriction and weak immunogenicity. The former has been demonstrated in several experimental models and clinical systems, whilst the latter necessitates the use of large ill-defined macromolecules as carriers, hence considerably weakening the advantages attributed to peptide vaccines per se. Two recent reports addressed these questions and in one [13] it was demonstrated that the genetic restriction could be overcome, at least in the case of foot-and-mouth disease virus (FMDV), by linking the peptide to defined helper epitopes from ovalbumin or sperm whale myoglobulin. In the other report [14] it was demonstrated that FMDV peptide (0.2/~g), when expressed with the hepatitis B core protein, elicited levels of neutralising antibody comparable to those induced by the whole inactivated virus. The antibody protected guinea-

pigs against a challenge infection. These observations are likely to have important implications for the future of peptide vaccines in general and FMDV peptide vaccine in particular. 3. The problems In spite of past successes and rapid advances in modern technology, vaccine manufacturers have not recently been in their most confident mood. The number of commercial vaccine producers in the world, particularly in the USA, has declined sharply in the past two decades. In the USA, for most vaccines there are now only one or two suppliers and many of the new biotechnological companies are concentrating on therapeutic products rather than vaccines. The reasons for such a state of affairs can be summarised as follows [15]: (1) Vaccines in general are not profitable, particularly when compared to therapeutic drugs. Since most vaccines are used in public programmes or intended for the Third World, there is immense pressure on prices. At least two companies, Lister Institute (ceased trading) and Wyeth, that played an important part in the W H O campaign for smallpox eradication, were not encouraged by the experience to partake in another such campaign. (2) Vaccine development is also very expensive and is an uncertain business. Contrary to popular belief, the major investment in a vaccine occurs not at the research phase but in the development stage: field trials, quality and stability control and large-scale manufacturing. Because the target populations are not always suitable or ethically available, adequate field trials to satisfy the licensing authorities are difficult to conduct. Consequently, limited use and promotion put further pressure on the return from investment. (3) The issue of liability also plays an important role, particularly in the USA. A vaccine manufacturer should rightly be charged for any negligence in production, but often he is dispiritingly held responsible for accidents which are inherent in the vaccine or incidents that are temporally but not causally related to vaccination. These are key factors which much be considered 243

TABLE l Pattern of progress in vaccine development. Phases Vaccines A B C

D

Relies on:

Conventional vaccines Empirical knowledge Living attenuated Crude killed vaccines Improved vaccines Large-scale culture including mammalian cell culture Single protein vaccines Monoclonal antibodies Pure antigens Gene cloning and sequencing Improved adjuvants Synthetic peptide Immunochemistry vaccines Computer chemistry Understanding of basis of adjuvanticity Simple synthetic Medicinal chemistry peptide vaccines Effective delivery systems active orally (Adapted from references 15 and 17).

in future vaccine d e v e l o p m e n t . F o r t u n a t e l y , there is a n increasing awareness by W H O g r o u p s a n d nat i o n a l c o n t r o l a u t h o r i t i e s a b o u t these p r o b l e m s w h i c h t h r e a t e n s u p p l y a n d the need to resolve them. There m u s t be c o m m i t m e n t to research f u n d i n g a n d r e c o g n i t i o n o f the need for a d e q u a t e p r o f i t to the m a n u f a c t u r e r s to ensure o n - g o i n g i n n o v a t i o n a n d d e v e l o p m e n t by industry. A s far as l i t i g a t i o n is concerned, g o v e r n m e n t a l c o m p e n s a t i o n systems to assess vaccine d a m a g e a n d to c o m p e n s a t e victims o f publicly supported vaccination programmes could ensure fair a n d a d e q u a t e p r o t e c t i o n for victims a n d m a n u f a c t u r e r s alike. Finally, t h e progress towards p u r e a n d m o l e c u l a r l y d e f i n e d vaccines w o u l d further m i n i m i s e the liabilities which bedeviled convent i o n a l vaccines b a s e d o n e m p i r i c i s m . T h e latest vaccine r e g u l a t i o n law to be e n a c t e d in the U S A for a g o v e r n m e n t a l c o m p e n s a t i o n scheme a n d the c o m mercial success o f the hepatitis B vaccines s h o u l d give vaccine research a n d d e v e l o p m e n t c o n s i d e r a b l e encouragement. 4. The future We are t h r o u g h the stage o f c o n v e n t i o n a l vaccines which rely o n e m p i r i c a l k n o w l e d g e - ( P h a s e A, Table 244

l) a n d o f i m p r o v e d vaccines b a s e d o n p u r i f i e d antigens ( P h a s e B). We are now into the stage o f s u b u n i t vaccines derived f r o m m o n o c l o n a l a n t i b o d i e s a n d gene c l o n i n g (Phase C). T h e t r a n s i t i o n f r o m s u b u n i t vaccines to synthetic p e p t i d e vaccines ( P h a s e D) has a l r e a d y b e g u n the e x p e r i m e n t a l stage, a n d s o m e vaccines have reached P h a s e 1 clinical trials. T h e ultim a t e vaccine, however, will take the f o r m o f simple synthetic p e p t i d e s which are active o r a l l y (Phase E). A realistic example is p r o v i d e d by C a p t o p r i l , an antihypertensive agent b a s e d o n i n h i b i t i o n o f a n g i o t e n s i n - c o n v e r t i n g enzyme. T h e first such inhibitors were p e p t i d e s extracted f r o m snake venom. K n o w l e d g e o f their structures led to the synthesis o f a h y b r i d m o l e c u l e which r e t a i n e d the required activity, b u t was o r a l l y active. Hence, s u b s t i t u t i o n at selected residues with u n n a t u r a l D - i s o m e r s [16] which c o n f e r a high degree o f stability a g a i n s t enz y m e d e g r a d a t i o n m a y be c o n s t r u c t e d to achieve effective a b s o r p t i o n a n d hence m i m i c p a r e n t a l vaccin a t i o n . This u l t i m a t e vaccine is at least t h e o r e t i c a l l y possible. References [ 1] Greenblatt, C. L. (1980) in: New Developments with Human and Veterinary Vaccines, pp. 259-285, Alan. R. Liss, Inc., New York. [2] Essex, M. E. (1988) Immunology Letters 19, 000-000. [3] Moss, B., Fuerst, T. R., Flexner, C. and Hugin, A. (1988) Vaccine 6, 161. [4] Ramshaw, I. A., Andrew, M. E., Phillips, S. M., Boyle, D. B. and Coupar, B. E. H. (1987) Nature 329, 545. [5l Hoiseth, S. K. and Stocker, B. A. D. (1981) Nature 291,238. [6] Jerne, N. K. (1974) Ann. Immunol. 125C, 373. [7] Moller, G. (Ed.) (1986) Immunol. Rev. 90, 1. [8] Berzofsky, J. A. (1988) Vaccine 6, 89. [9] Rothbard, J. B. and Taylor, W. R. (1988) EMBO J. 7, 93. ll0] Bjorkman, P.J., Super, M.A., Samraoui, B., Bennett, W. S., Strominger, J. L. and Wiley, D. C. (1987) Nature 329, 506. ll 1] Zanetti, M., Sercarz, E. and Sulk, J. (1987) Immunol. Today 8, 18. [12] Brown, E (1988) Vaccine 6, 180. [13] Francis, M. J., Hastings, G. Z., Syred, A. D., McGinn, B., Brown, E and Rowlands, D. J. (1987) Nature 300, 168. [14] Clarke, B. E., Newton, S. E., Carroll, A. R., Francis, M. J., Appleyard, G., Syred, A. D., Highfield, P. E., Rowlands, D. J. and Brown, E (1987) Nature 330, 381. [15] Liew, E Y. (1985) Clin. Exp. Immunol 63, 225. [16] Beddell, C. R., Clark, R. B., Hardy, G. W., Lowe, L. A., Ubatuba, E B., Vane, J. R., Wilkinson, S., Chang, K-J., Cuatrecasas, P. and Miller, R. J. (1977) Proc. R. Soc. London 198, 249. [17] Vane, J. R. and Cuatrecasas, E (1984) Nature 312, 303.