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Anti-idiotype vaccines
TIBTECH- MARCH 1989 [Vol. 7]
Anti-idiotype vaccines Yasmin Thanavala Gene cloning and expression can provide substantial amounts of antigenic material for vaccines. So too can systems producing antibodies that functionally mimic antigens - anti-idiotype antibodies. This article reviews the progress towards the development of anti-idiotype vaccines for the prevention of viral, bacterial and parasitic diseases. There may be particular niches for antiidiotype antibodies in vaccines that would otherwise involve nonprotein or glycoprotein antigens, or single antigenic determinants. On the other hand, recent findings on the relationships between antiidiotype antibodies and the antigens they mimic suggest alternative applications of this technology. The idea of vaccination against infectious disease was first conceived and successfully applied for smallpox (see Box). It was, however, not until 8 May 1980 that World Health Organization delegates unanimously accepted that smallpox had been erradicated globally. There was almost a century between Jenner's use of the cowpox virus to protect against smallpox and Louis Pasteur's development of microbial attenuation by passage in a new host which led to vaccines for rabies and anthrax. Vaccination has rid the world of smallpox, tamed rabies, tetanus, diptheria, whooping cough and reduced the threat of tuberculosis, pneumonia and meningitis. However, there are still a number of diseases for which no vaccines exist, as attested by over 200 million cases of schistosomiasis, at least 5 million cases of leprosy, 20 million cases of trypanosomiasis and an estimated 1800 million people who suffer from malaria. The advent of monoclonal antibodies, gene cloning techniques and synthetic peptide production has opened up new avenues in the art and science of vaccine development. This review deals with vaccine development based on the network concept of the immune system, idiotypic complementarity and idiotypic mimicry.
Background, basic concepts and theoretical considerations The area on an antibody molecule that makes contact with an antigen is called the paratope: it is synonymous with the antigen-combining site. In pioneering studies, Slater et al. 1 and Oudin and Michel 2 immunized animals w i t h an immunoglobin molecule and raised antibodies that reacted with all parts of the immunizing immunoglobin. However, after absorption of their antisera with pooled normal immunoglobulin, they were left with antibodies reacting solely with the hypervariable regions. The term idiotype was used to describe the unique set of determinants on the hypervariable region (a single determinant being called an idiotope) which were recognized by the antiBox I
On Cow Pox There is a disease to which the commonly happens that it is communicated to the cows, and
Yasmin Thanavala is at the Department of Molecular Immunology, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, N Y 14263, USA. O 1989, Elsevier Science Publishers Ltd (UK)
0167 - 9430/89/$02.00
sera raised to it. The antisera were referred to as anti-idiotypic reagents. Subsequent work has shown that antibodies of different specificities may share an idiotype. Idiotypes restricted to a single immunoglobulin are called private idiotypes, and those common to more than one immunoglobulin, public or crossreactive idiotypes (CRI). Advances in understanding immunoglobulin gene regulation have revealed that the tremendous diversity of antibodies stems in part from recombinational events between various germ-line genes and in part from subsequent somatic mutations. The germ-line gene products will appear in several different antibodies, presumably accounting for cross-reactive idiotypes; random somatic mutations presumably produce private idiotypes. An idiotope may or may not be associated with the same structural region as the paratope. Thus there are antigen binding site-related (paratopic) or non-site-related (non-paratopic) idiotopes. The binding of antiidiotypic antibodies (often abbreviated to Ab2 - second antibody) to idiotopes associated with the paratope is inhibited by antigen. In other words, anti-idiotypic antibodies and antigens can both be bound by the same antigen. To an extent the anti-idiotype mimics the antigen. The current interest in the molecular mimicry of viral, bacterial, parasite and tumor antigens using anti-idiotypic antibodies is based on the concept set forth by Niels Jerne 3 - the network theory of the immune response. Jerne envisaged the immune system as a complex network of cells held together via complementary recognition which was mediated by idiotypic antibodies. The network (Fig. 1) would, thus, consist of idiotype-anti-idiotype interactions between lymphocytes, with all lymphocytes being in a state of dynamic equilibrium as a result of these interactions. External antigen, by perturbing this equilibrium, would provoke an immune response. One of the predictions of Jerne's theory is that for every exogenous antigen there is an antibody which is its 'internal image' counterpart within the immune system.
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MARCH 1989 [Vol. 7]
~~"~
Fig. 1
Anti- id i otype (internal image) ,. ~,nugenreactive _
(
txldl
)
Anti-idiotype /"
~x
Anti-
An idiotypic network. Within an idiotypic network there will be cells which produce antibodies that bind a particular antigen. Other cells produce antibodies that bind the idiotypic determinants (often associated with binding function) of those antibodies: these are the anti-idiotypic antibodies. Here, o: is used to indicate "anti" Some, but not all anti-idiotypic antibodies may be qnternal image" antibodies that can compete with antigen for binding to the Abl antibodies. Antibodies raised against internal image antibodies may confer immunity to the microorganism that bears the original antigen. Since antibody nomenclature can become confusing in anti-idiotypic networks, immunologists have devised a simpler scheme. Antibodies raised against an antigen are known as Abl; anti-idiotypic antibodies against Abl are known as AbE; those against Ab2 are known as Ab3. There are also three subclasses of anti-idiotypic antibodies: 0:, fl and 7. Ab20: do not inhibit antigen binding to AbT; Ab2fl and Ab27 both do, but only Ab213 functionally mimic the original antigen.
Results from several laboratories (using a variety of antigens) have verified the existence of internal image antibodies. They mimic the original external antigen functionally and can, therefore, serve as surrogate antigens. Consequently, they have the potential of being used in vaccine programs and in receptor identification and purification; they may also play a role in the etiology and regulation of some autoimmune diseases. Conceptual changes in vaccine development have been occurring for several years. While the effectiveness of currently available vaccines is not the issue here, the small but existing risk factors of certain essential vaccines are becoming unacceptable. In one instance, vaccination against whooping cough, the risks have resulted in a decline in the vaccination rate. Particularly where large segments of a population require vaccination, there is a real need for yet safer vaccines. The network hypothesis suggests an elegant alternative for developing vaccines 4'5 that are not based on using nominal antigenic material. In developing a strategy for antiidiotypic vaccines, the researcher
takes advantage of the fact that the repertoire of nominal antigens is mimicked by idiotypic structures on immunoglobulins and possibly also on receptors and products of T cells.
Are there niches for idiotype vaccines? Sometimes, where the use of conventional vaccines presents problems, internal image anti-idiotype vaccines may prove advantageous. • Where antigen is scarce: obtaining adequate amounts of antigen has been one of the main obstacles to producing appropriate vaccines for diseases such as malaria, trypanosomiasis, leprosy and leishmaniasis. In other diseases (like hepatitis B) it is not even possible to culture the causative organism in vitro. Monoclonal anti-idiotypic antibodies serving as surrogate antigen could be easily produced on a large scale. • Where isolated products are not sufficiently antigenic: products obtained by gene cloning may be of little value as vaccines if they require glycosylation or the presence of lipid or a nucleic acid core to attain the configuration of the native antigen; synthetic peptides may not adopt the
three-dimensional structure of the original antigen. Internal image antiidiotypic molecules would have the correct conformation and may, therefore, be good antigens. • Where risks of virulence are high: there are inherent hazards associated with the use of putatively killed vaccines; there are also risks that attenuated strains may revert to a virulent form. Anti-idiotypic vaccines w o u l d bear no risk of virulence or infectiousness. Internal image anti-idiotypes may also offer tremendous promise in providing immunity to toxins. Native toxin obviously cannot be used to induce immunity; any non-native, denatured molecule could lose the antigenicity necessary to produce protective neutralizing antibodies. • Where parts of an antigen may provoke an autoimmune response: determinants of microbial antigens not needed for immune protection or complexes of the organism with body components may sometimes provoke damaging autoantibodies. Whole antigen or whole organism vaccines might also induce such a response. It would, therefore, be an advantage to immunize only against selected protective antigenic determinants. Monoclonal antiidiotype vaccines, by mimicking individual epitopes, could provide this immunization. • Where the antigen was not a peptide or protein: anti-idiotypic antibodies could mimic the structure and conformation of, say, carbo= hydrate antigen epitopes. The presentation of antigenic material in a different molecular context may also break the tolerance that seems to exist to some antigens and allow the expression of other~ wise silent antibody-producing cell clones. Anti-idiotype vaccines will not be useful against organisms like influenza virus which have epitopes that change with time.
Experimental models for anti= idiotype vaccines In this section, I will briefly review various attempts to use anti-idiotypic antibodies for vaccination in several
T I B T E C H - M A R C H 1989 [Vol. 7]
models which encompass parasitic, viral and bacterial diseases. Idiotypic and anti-idiotypic antibodies have roles in the therapy of malignancies of mature B-cell origin, and T-cell vaccination may provoke an antiidiotypic network for the treatment of several autoimmune diseases, but these aspects will not be discussed. Parasites The first successful use of antiidiotypic antibodies for vaccination against any infectious agent was achieved with Trypanosoma rhodesiense, the causative agent of sleeping sickness in Africa. This disease is characterized by cycles of parasitemia with the parasite expressing a new antigenic variant each time. Antibodies directed to surface antigens of the parasite confer protection against a particular antigenic variant but not against others. Sacks and colleagues 6 raised mouse monoclonal antibodies against three variant antigens. These antibodies (Abe) were used as antigen to produce mouse allogenic (see Glossary) anti-idiotypic antibodies. One of the three anti-idiotype antisera induced protective immunity against the original antigenic variant to which the Ab~ was directed. The other two anti-idiotypes were ineffective when used by themselves, but were effective when used as an antibody cocktail. The anti-idiotypes protected all mice to varying degrees from complete protection in some to reduced parasitemia in others. The use of the anti-idiotype was, however, restricted by the Igh a allotype 7. The same group has also reported the production of an anti-idiotype which mimics a carbohydrate determinant of the cell surface glycoprotein of Trypanosoma cruzi. Though they were able to use this Ab2 to raise antibodies in a number of species, the response was never protective. In contrast, a rat monoclonal Ab2~ mimicking Schistosoma mansoni induced 50-76% protection to a challenge infection. The protective effect of the Ab3 generated could also be passively transferred (by serum) to naive rats 8. As an aside, it is worth mentioning that both in mice immunized with S. mansoni and in former patients and
patients with active schistosomiasis, anti-idiotypic T cells that respond to antibodies against a soluble preparation of schistosomal eggs have been detected 9'1°. Similarly, antiidiotypic T cells have been demonstrated in patients with Chagas disease (Trypanosoma CrUZi)11. Anti-idiotypes have potential in vaccinating chickens against coccidiosis caused by a protozoan parasite Eimeria tenella ~2. Two rabbit anti-idiotypic antibodies were prepared against monoclonal antibodies (Abl) with specificity for the surface antigenic determinants of the sporozoites of E. tenella. Immunization of young chickens with these anti-idiotypes induced antibodies (Ab3) that bound to E. tenella sporozoites and conferred partial protective immunity against experimental infection with virulent E. tenella (manifested by reduction in severity of cecal lesions).
Viruses The principle of idiotypic vaccination has been applied quite extensively for viral infections. Reovirus anti-idiotypic antibodies have been used as vaccines. But they have also been used to study both the molecular biology of the reovirus receptor, and receptor perturbation by virus and anti-idiotypes, which alters cell growth and function. Greene and colleagues found that a monoclonal internal image antiidiotype that is directed to a syngenic monoclonal Abl (see Glossary) which reacts with the reovirus hemagglutinin 13 could induce type-specific reovirus neutralizing responses, viral-specific cytolytic T cells and delayed type hypersensitivity ~4. Neonatal mice whose mothers had received anti-idiotypes were completely resistant to disease and lethality on challenge with type 3 reovirus; litter mate controls uniformly developed hydrocephalus ~5. The internal image anti-idiotypes mimicked the binding of the reovirus hemagglutinin to a panel of target cells, and prior incubation of these cells with Ab2 inhibited viral binding and infectivity. This Ab2 has been successfully used both to isolate and to characterize the reovirus receptor which has now been identified as the
[3-adrenoceptor16. With poliovirus type II, small amounts of virus-neutralizing antibodies could be induced by a monoclonal anti-idiotype, but the antibody levels were not sufficient to protect mice against subsequent virus challenge 17. Similar results were observed using rabbit antiidiotypic antibodies against murine monoclonal antibodies to the rabies virus glycoprotein: i.e. immunization with anti-idiotype resulted in neutralizing antibodies to the virus but they were not sufficient to protect the mice against rabies infection 18. Monoclonal antibodies against a Sendai virus-specific helper T cell clone were effective in eliciting both B cell and T cell immunity. Thus, immunization of mice with the antiidiotypes afforded protection against a subsequent challenge infection with Sendai virus 19. Importantly, specific cytolytic T cells were induced which were not MHC restricted (see Glossary) in their activity, unlike the T cells activated by the virus itself2°. With tobacco mosaic virus (TMV), rabbit anti-idiotypes coupled to lipopolysaccharide induced a marked humoral response in BALB/c mice 21. The anti-TMV antibodies produced were idiotypically cross-reactive with the idiotype used to generate the anti-idiotypic antibody. With hepatitis B virus (HBV), Kennedy, Dreesman and colleagues have induced an antibody response in mice 22 and a protective response in chimpanzees 23 using polyclonal rabbit anti-idiotypic antibodies. The rabbit anti-idiotype recognized a highly conserved interspecies idiotype. Our approach 24 has been to raise mouse monoclonal antiidiotypes to a syngenic monoclonal Abe. Two internal image antiidiotypes have been fully characterized 24 and shown to mimic the group-specific a determinant of hepatitis B surface antigen (HBsAg). The anti-idiotype reacted with sera from mice, rabbits, goats, swine and humans immunized with HBsAg 25. The Ab2~ anti-idiotypes also inhibited the binding of monoclonal and polyclonal anti-HBsAg (antihepatitis B surface antigen) sera to linear and cyclic synthetic peptides
TIBTECH - MARCH 1989 [Vol. 7]
Bacteria
corresponding to the a determinant, thus confirming them as good candidate internal image anti-idiotypes 26. Using our anti-idiotypes, we have been able to generate anti-HBs responses in inbred strains of mice, in wild mice and in mice that are nonresponders to HBsAg itself. In addition, we are also able to effectively stimulate HBsAg-specific purified T cells obtained from peripheral blood lymphocytes of human donors who are immune to HBV either through natural infection or through vaccination 27. We have also studied a limited number of HBV carriers. They do not raise an anti-HBsAg response upon HBV infection and are thus serologically HBsAg positive and anti-HBsAg negative. In in vitro T cell proliferation assays, we could induce good stimulation of their T ceils using our internal image antiidiotypic antibodies but not antigen itself. Mouse monoclonal anti-idiotypes that bear the internal image of a human cytomegalovirus neutralization epitope have been used in syngenic immunization experiments to raise anti-anti-idiotypic antibodies (Ab3) which had virus-neutralizing activity 28. Not all experiments have produced protective responses. Indeed when polyclona] anti-idiotypic antibodies were used to modulate herpes simplex virus infection, they led to increased pathogenicity and shorter survival times of treated mice 29. The anti-idiotypic approach has also been tried in human immunodeficiency virus (HIV) infections. A mouse monoclonal anti-idiotype raised against anti-Leu3a mimics the CD4 receptor; it binds to recombinant gp160 and a molecule of 110-120 kDa in immunoblot analysis of HIVinfected cell lysates 3°. This monoclonal antibody also partially neutralized HIV infection of human T cells in vitro.
Rees et al. a~ raised a polyclonal rabbit anti-idiotypic antibody and used this to study proliferative responses of human T cells from tuberculosis patients and from BCGvaccinated healthy subjects. Responsiveness among patients to the antiidiotype correlated with responsiveness to a 38 kDa antigen of Mycobacterium tuberculosis 31. The induction of proliferation to both antigen and anti-idiotype was dependent on accessory cells and restricted by determinants encoded by the MHC (major histocompatibility complex). In addition, T cell reactivity was not dependent on the conformational integrity of the anti-idiotype binding site 32. Two Listeria monocflogenes specific CD4+ class II restricted clones were used to raise syngenic antisera 33. Injection of these anticlonotypic antibodies (with adjuvant) gave specific protection (in a genetically unrestricted manner) against the L. m o n o c y t o g e n e s strain recognized by the original T cell clone. Anti-idiotypes can also mimic polysaccharide antigens ~4. Monoclonal anti-idiotypes to the capsular polysaccharide of Neisseria m e n i n gitidis serogroup C could evoke antibodies specific to the meningococcal C polysaccharide in syngenic (having virtually identical genotypes) and xenogenic (different genotypes) species.
Consequences I would like to finish by briefly discussing observations from three laboratories 35-37 where mRNA sequence information of the antiidiotype clones has revealed areas of homology between the anti-idiotypes and the antigens they mimic. For instance, the amino acid sequences of reovirus hemagglutinin and the variable region of the light chain of a corresponding monoclonal internal image anti-idiotype share an area of homology (five identical amino acids and three conservative substitutions). In the antibody this area of homology lies in one of the complementarity determining regions (CDR2) 35. Similarly, on sequencing two monoclonal anti-idiotypes that mimic the rabbit immunoglobulin al
allotype (see Glossary), VanCleave et aL 37 observed that within the CDR2 of the heavy-chain V regions there was an area of homology with the rabbit al allotype, but in the opposite direction with respect to the carbon backbone. The ability of the internal image reversed sequence to produce an al-like antigenic determinant was tested by making synthetic peptides corresponding to the area of homology in both the anti-idiotype and the rabbit al allotype. Both peptides could completely inhibit the binding of rabbit immunoglobulin to anti-a1 antibody. Such results open the possibility of precisely identifying within the sequence of the anti-idiotypes those residues that mimic an original antigen. Synthetic peptide vaccines could then be constructed based on sequence data from anti-idiotypes. This could remove the need to use mouse monoclonal antibodies for vaccination.
Acknowledgements For their excellent secretarial assistance, I would like to thank both Cheryl Zuber and Donna Ovak.
References 1 Slater, R. J., Ward, S. M. and Kunkel, H. G. (1955) J. Exp. Med. 101, 85-108 2 0 u d i n , J. and Michel, M. (1963) C. R. Acad. Sci. 257, 805-808 3 Jerne, N. K. (1974) Ann. Immunol. (Inst. Pasteur) 125C, 373-389 4 Nisonoff, A. and Lamoyi, E. (1981) Clin. Immunol. lmmunopatho]. 21, 397-406 5 Roitt, I. M., Cooke, A., Male, D. K. et a]. (1981) Lancet i, 1041-1045 6 Sacks, D. L., Esser, K. M. and Sher, A. (1982) J. Exp. Med. 155, 1108-1119 7 Sacks, D. L. and Sher, A. (1983) J. Immunol. 131, 1511-1515 8 Grzych, J. M., Capron, M., Lambert, P. H. et al. (1985) Nature 316, 74--76 9 Powell, M. R. and Colley, D. G. (1987) Cell. Immunol. 104,377-385 10 Parra, J. C., Lima, M. S., Gazzinelli, G. and Colley, D. G. (1988) J. Immuno]. 140, 2401-2405 11 Gazzinelli, R. T., Morato, M. F., Nunes, R. B. eta]. (1988) J. Immunol. 140, 3167-3172 12 Bhogal, B. S., Nollstadt, K. H., Karkhanis, Y. D., Schmatz, D. M. and Jacobson, E. B. (1988) Infect. Immun. 56, 1113-1119
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13 Noseworthy, J. H., Fields, B. N., Dichter, M.A. et al. (1983) J. Immunol. 131, 2533-2538 14 Sharpe, A. H., Gaulton, G. N., McDade, K.K., Fields, B.N. and Greene, M. I. (1984) J. Exp. Med. 160, 1195-1205 15 Gaulton, G. N. and Greene, M. I. (1987) in Anti-idiotypes, Receptors and Molecular Mimicry (Linthicum, D, S. and Farid, N.R., eds), pp. 301-309, Springer Verlag 16 Co, M. S., Gaulton, G. N., Tominga, A. et al. (1985)Proc. Natl Acad. Sci. USA 82, 5315-5318 17 Uytdehaag, G. C. M. and Osterhaus, A. D. M. E. (1985) J. Immunol. 134, 1225-1229 18 Reagan, K. J., Wunner, W. H., Wiktor, T. J. and Koprowski, H. (1983)J. Viro]. 48,660-666 19 Ertl, H. C. J., McCarthy, E., Homans, E. and Finberg, R.W. (1985) in ItighTechnology Routes to Virus Vaccines (Dreesman, G. R., Bronson, J. G. and Kennedy, R.C., eds), pp. 125-141, American Society for Microbiology []
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20 Ertl, H. C. J. and Finberg, R. W. (1984) Proc. Natl Acad. Sci. USA 81, 2850-2854 21 Francotte, M. and Urbain, J. (1984) J. Exp. Med. 160, 1485-1494 22 Kennedy, R. C. and Dreesman, G. R. (1984) J. Exp. Med. 159, 655-665 23 Kennedy, R. C., Eichberg, J. W., Lanford, R. E. and Dreesman, G. R. (1986) Science 232,220-223 24 Thanavala, Y. M., Bond, A., Tedder, R., Hay, F. C. and Roitt, I. M. (1985) Immunology 55, 197-204 25 Thanavala, Y. M., Bond, A., Hay, F. C. and Roitt, I.M. (1985) J. Immunol. Methods 83, 227-232 26 Thanavala, Y. M., Brown, S. E., Howard, C. R., Roitt, I. M. and Steward, M. W. A. (1986) J. Exp. Med. 164, 227-236 27 Pride, M., Thakur, A. N., Ogra, P. A., Evans, R.L. and Thanavala, Y.M. (1988) FASEB Abstr. No. 1067 28 Keay, S., Rasmussen, L. and Merigan, T. C. (1988)J. Immunol. 140,944-948 29 Kennedy, R. C., Adler-Storthz, K., Burns, J. W., Henkel, R. D. and []
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Insect cell culture technology in baculovirus expression systems lain R. Cameron, Robert D. Possee and David H. L. Bishop The ability to produce large quantities of proteins relatively cheaply and easily, by expressing genes in heterologous systems, is a useful tool in modern biology and medicine. High-level protein expression systems have been developed from a number of prokaryotic and eukaryotic organisms. Insect baculovirus-based systems have shown great promise recently for the production of biologically active proteins. In this review we discuss the application of insect cell culture to baculovirus expression systems. Protein expression systems based on the A u t o g r a p h a californica nuclear polyhedrosis virus (AcNPV, an insect baculovirus) have wide applicability as an alternative to prokaryotic or other eukaryotic expression systems. lain Cameron, Robert Possee and David Bishop are at the NERC Institute of Virology, Mansfield Road, Oxford OX1 3SR, UK.
The vectors are not d e p e n d e n t on helper virus, standard virological methods are e m p l o y e d and the viruses are readily grown, maintained and m a n i p u l a t e d in vitro using cell lines derived frolil Spodoptera frugiperda (the fall armyworm). Baculoviruses are not pathogenic for vertebrates or plants. Baculoviruses contain a very late hyper-expressed gene w h i c h is not
© 1989, Elsevier Science Publishers Ltd (UK) 0167- 9430/89/$02.00
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Dreesman, G.R. (1984) J. Virol. 50, 951-953 Chanh, T. C., Dreesman, G. R. and Kennedy, R.C. (1987) Proe. Nat] Acad. Sci. USA 84, 3891-3895 Rees, A. D. M., Praputpittaya, K., Scoging, A. et al. (1987) Immunology 60, 389-393 Rees, A. D. S., Scoging, A., Dobson, N. et al. (1987) Eur. J. Immunol. 17, 197-201 Westerink, J., Campagnari, A. A., Wirth, M.A. and Apicella, M.A. (1988) Infect. Immun. 56, 1120-1127 Kaufmann, S. H. E., Eichmann, K., Muller, I. and Wrazel, L.J. (1985) J. Immuno]. 134, 4123-4127 Bruck, C., Co, M. S., Slaoui, M. et al. (1986) Proc. Natl Acad. Sci. USA 83, 6578-6582 Roth, C., Rocca-Serra, J., Somme, G., Fougereau, M. and Theze, J. (1985) Proc. Natl Acad. Sci. USA 82, 4788-4792 VanCleave, V. H., Naeve, C. W. and Metzger, D.W. (1988) J. Exp. Med. 167, 1841-1848 []
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essential for viral replication. The normal product of this gene is the p o l y h e d r i n protein, the subunit from w h i c h the crystalline occlusion body, or polyhedron, is formed. The p o l y h e d r o n contains infectious virus particles and is responsible for horizontal transmission of virus in the wild. Genes incorporated into the DNA genome of baculoviruses and expressed u n d e r the control of the strong viral p o l y h e d r i n promoter have enabled a b u n d a n t quantities (up to 50% of total cellular protein) of m a n y foreign proteins to be p r o d u c e d w h i c h are biologically active 1-3. The recent d e v e l o p m e n t of virus vectors expressing more t h a n one foreign product (multiple expression vectors; Fig. 1) indicates that prot e i n - p r o t e i n interactions such as the assembly processes of viruses 4 or m u l t i - c o m p o n e n t enzymes 5 could also be studied. Indeed it is possible to insert a DNA copy of the entire RNA genome (6.6 kb) of polio virus into the baculovirus: non-infectious viral particles are p r o d u c e d w h i c h m a y be useful for vaccination (T. Urakawa, pers. commun.). Baculovirus expression systems are relatively easy to use and can (in
Anti-idiotype vaccines Yasmin Thanavala Gene cloning and expression can provide substantial amounts of antigenic material for vaccines. So too can systems producing antibodies that functionally mimic antigens - anti-idiotype antibodies. This article reviews the progress towards the development of anti-idiotype vaccines for the prevention of viral, bacterial and parasitic diseases. There may be particular niches for antiidiotype antibodies in vaccines that would otherwise involve nonprotein or glycoprotein antigens, or single antigenic determinants. On the other hand, recent findings on the relationships between antiidiotype antibodies and the antigens they mimic suggest alternative applications of this technology. The idea of vaccination against infectious disease was first conceived and successfully applied for smallpox (see Box). It was, however, not until 8 May 1980 that World Health Organization delegates unanimously accepted that smallpox had been erradicated globally. There was almost a century between Jenner's use of the cowpox virus to protect against smallpox and Louis Pasteur's development of microbial attenuation by passage in a new host which led to vaccines for rabies and anthrax. Vaccination has rid the world of smallpox, tamed rabies, tetanus, diptheria, whooping cough and reduced the threat of tuberculosis, pneumonia and meningitis. However, there are still a number of diseases for which no vaccines exist, as attested by over 200 million cases of schistosomiasis, at least 5 million cases of leprosy, 20 million cases of trypanosomiasis and an estimated 1800 million people who suffer from malaria. The advent of monoclonal antibodies, gene cloning techniques and synthetic peptide production has opened up new avenues in the art and science of vaccine development. This review deals with vaccine development based on the network concept of the immune system, idiotypic complementarity and idiotypic mimicry.
Background, basic concepts and theoretical considerations The area on an antibody molecule that makes contact with an antigen is called the paratope: it is synonymous with the antigen-combining site. In pioneering studies, Slater et al. 1 and Oudin and Michel 2 immunized animals w i t h an immunoglobin molecule and raised antibodies that reacted with all parts of the immunizing immunoglobin. However, after absorption of their antisera with pooled normal immunoglobulin, they were left with antibodies reacting solely with the hypervariable regions. The term idiotype was used to describe the unique set of determinants on the hypervariable region (a single determinant being called an idiotope) which were recognized by the antiBox I
On Cow Pox There is a disease to which the commonly happens that it is communicated to the cows, and
Yasmin Thanavala is at the Department of Molecular Immunology, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, N Y 14263, USA. O 1989, Elsevier Science Publishers Ltd (UK)
0167 - 9430/89/$02.00
sera raised to it. The antisera were referred to as anti-idiotypic reagents. Subsequent work has shown that antibodies of different specificities may share an idiotype. Idiotypes restricted to a single immunoglobulin are called private idiotypes, and those common to more than one immunoglobulin, public or crossreactive idiotypes (CRI). Advances in understanding immunoglobulin gene regulation have revealed that the tremendous diversity of antibodies stems in part from recombinational events between various germ-line genes and in part from subsequent somatic mutations. The germ-line gene products will appear in several different antibodies, presumably accounting for cross-reactive idiotypes; random somatic mutations presumably produce private idiotypes. An idiotope may or may not be associated with the same structural region as the paratope. Thus there are antigen binding site-related (paratopic) or non-site-related (non-paratopic) idiotopes. The binding of antiidiotypic antibodies (often abbreviated to Ab2 - second antibody) to idiotopes associated with the paratope is inhibited by antigen. In other words, anti-idiotypic antibodies and antigens can both be bound by the same antigen. To an extent the anti-idiotype mimics the antigen. The current interest in the molecular mimicry of viral, bacterial, parasite and tumor antigens using anti-idiotypic antibodies is based on the concept set forth by Niels Jerne 3 - the network theory of the immune response. Jerne envisaged the immune system as a complex network of cells held together via complementary recognition which was mediated by idiotypic antibodies. The network (Fig. 1) would, thus, consist of idiotype-anti-idiotype interactions between lymphocytes, with all lymphocytes being in a state of dynamic equilibrium as a result of these interactions. External antigen, by perturbing this equilibrium, would provoke an immune response. One of the predictions of Jerne's theory is that for every exogenous antigen there is an antibody which is its 'internal image' counterpart within the immune system.
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~~"~
Fig. 1
Anti- id i otype (internal image) ,. ~,nugenreactive _
(
txldl
)
Anti-idiotype /"
~x
Anti-
An idiotypic network. Within an idiotypic network there will be cells which produce antibodies that bind a particular antigen. Other cells produce antibodies that bind the idiotypic determinants (often associated with binding function) of those antibodies: these are the anti-idiotypic antibodies. Here, o: is used to indicate "anti" Some, but not all anti-idiotypic antibodies may be qnternal image" antibodies that can compete with antigen for binding to the Abl antibodies. Antibodies raised against internal image antibodies may confer immunity to the microorganism that bears the original antigen. Since antibody nomenclature can become confusing in anti-idiotypic networks, immunologists have devised a simpler scheme. Antibodies raised against an antigen are known as Abl; anti-idiotypic antibodies against Abl are known as AbE; those against Ab2 are known as Ab3. There are also three subclasses of anti-idiotypic antibodies: 0:, fl and 7. Ab20: do not inhibit antigen binding to AbT; Ab2fl and Ab27 both do, but only Ab213 functionally mimic the original antigen.
Results from several laboratories (using a variety of antigens) have verified the existence of internal image antibodies. They mimic the original external antigen functionally and can, therefore, serve as surrogate antigens. Consequently, they have the potential of being used in vaccine programs and in receptor identification and purification; they may also play a role in the etiology and regulation of some autoimmune diseases. Conceptual changes in vaccine development have been occurring for several years. While the effectiveness of currently available vaccines is not the issue here, the small but existing risk factors of certain essential vaccines are becoming unacceptable. In one instance, vaccination against whooping cough, the risks have resulted in a decline in the vaccination rate. Particularly where large segments of a population require vaccination, there is a real need for yet safer vaccines. The network hypothesis suggests an elegant alternative for developing vaccines 4'5 that are not based on using nominal antigenic material. In developing a strategy for antiidiotypic vaccines, the researcher
takes advantage of the fact that the repertoire of nominal antigens is mimicked by idiotypic structures on immunoglobulins and possibly also on receptors and products of T cells.
Are there niches for idiotype vaccines? Sometimes, where the use of conventional vaccines presents problems, internal image anti-idiotype vaccines may prove advantageous. • Where antigen is scarce: obtaining adequate amounts of antigen has been one of the main obstacles to producing appropriate vaccines for diseases such as malaria, trypanosomiasis, leprosy and leishmaniasis. In other diseases (like hepatitis B) it is not even possible to culture the causative organism in vitro. Monoclonal anti-idiotypic antibodies serving as surrogate antigen could be easily produced on a large scale. • Where isolated products are not sufficiently antigenic: products obtained by gene cloning may be of little value as vaccines if they require glycosylation or the presence of lipid or a nucleic acid core to attain the configuration of the native antigen; synthetic peptides may not adopt the
three-dimensional structure of the original antigen. Internal image antiidiotypic molecules would have the correct conformation and may, therefore, be good antigens. • Where risks of virulence are high: there are inherent hazards associated with the use of putatively killed vaccines; there are also risks that attenuated strains may revert to a virulent form. Anti-idiotypic vaccines w o u l d bear no risk of virulence or infectiousness. Internal image anti-idiotypes may also offer tremendous promise in providing immunity to toxins. Native toxin obviously cannot be used to induce immunity; any non-native, denatured molecule could lose the antigenicity necessary to produce protective neutralizing antibodies. • Where parts of an antigen may provoke an autoimmune response: determinants of microbial antigens not needed for immune protection or complexes of the organism with body components may sometimes provoke damaging autoantibodies. Whole antigen or whole organism vaccines might also induce such a response. It would, therefore, be an advantage to immunize only against selected protective antigenic determinants. Monoclonal antiidiotype vaccines, by mimicking individual epitopes, could provide this immunization. • Where the antigen was not a peptide or protein: anti-idiotypic antibodies could mimic the structure and conformation of, say, carbo= hydrate antigen epitopes. The presentation of antigenic material in a different molecular context may also break the tolerance that seems to exist to some antigens and allow the expression of other~ wise silent antibody-producing cell clones. Anti-idiotype vaccines will not be useful against organisms like influenza virus which have epitopes that change with time.
Experimental models for anti= idiotype vaccines In this section, I will briefly review various attempts to use anti-idiotypic antibodies for vaccination in several
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models which encompass parasitic, viral and bacterial diseases. Idiotypic and anti-idiotypic antibodies have roles in the therapy of malignancies of mature B-cell origin, and T-cell vaccination may provoke an antiidiotypic network for the treatment of several autoimmune diseases, but these aspects will not be discussed. Parasites The first successful use of antiidiotypic antibodies for vaccination against any infectious agent was achieved with Trypanosoma rhodesiense, the causative agent of sleeping sickness in Africa. This disease is characterized by cycles of parasitemia with the parasite expressing a new antigenic variant each time. Antibodies directed to surface antigens of the parasite confer protection against a particular antigenic variant but not against others. Sacks and colleagues 6 raised mouse monoclonal antibodies against three variant antigens. These antibodies (Abe) were used as antigen to produce mouse allogenic (see Glossary) anti-idiotypic antibodies. One of the three anti-idiotype antisera induced protective immunity against the original antigenic variant to which the Ab~ was directed. The other two anti-idiotypes were ineffective when used by themselves, but were effective when used as an antibody cocktail. The anti-idiotypes protected all mice to varying degrees from complete protection in some to reduced parasitemia in others. The use of the anti-idiotype was, however, restricted by the Igh a allotype 7. The same group has also reported the production of an anti-idiotype which mimics a carbohydrate determinant of the cell surface glycoprotein of Trypanosoma cruzi. Though they were able to use this Ab2 to raise antibodies in a number of species, the response was never protective. In contrast, a rat monoclonal Ab2~ mimicking Schistosoma mansoni induced 50-76% protection to a challenge infection. The protective effect of the Ab3 generated could also be passively transferred (by serum) to naive rats 8. As an aside, it is worth mentioning that both in mice immunized with S. mansoni and in former patients and
patients with active schistosomiasis, anti-idiotypic T cells that respond to antibodies against a soluble preparation of schistosomal eggs have been detected 9'1°. Similarly, antiidiotypic T cells have been demonstrated in patients with Chagas disease (Trypanosoma CrUZi)11. Anti-idiotypes have potential in vaccinating chickens against coccidiosis caused by a protozoan parasite Eimeria tenella ~2. Two rabbit anti-idiotypic antibodies were prepared against monoclonal antibodies (Abl) with specificity for the surface antigenic determinants of the sporozoites of E. tenella. Immunization of young chickens with these anti-idiotypes induced antibodies (Ab3) that bound to E. tenella sporozoites and conferred partial protective immunity against experimental infection with virulent E. tenella (manifested by reduction in severity of cecal lesions).
Viruses The principle of idiotypic vaccination has been applied quite extensively for viral infections. Reovirus anti-idiotypic antibodies have been used as vaccines. But they have also been used to study both the molecular biology of the reovirus receptor, and receptor perturbation by virus and anti-idiotypes, which alters cell growth and function. Greene and colleagues found that a monoclonal internal image antiidiotype that is directed to a syngenic monoclonal Abl (see Glossary) which reacts with the reovirus hemagglutinin 13 could induce type-specific reovirus neutralizing responses, viral-specific cytolytic T cells and delayed type hypersensitivity ~4. Neonatal mice whose mothers had received anti-idiotypes were completely resistant to disease and lethality on challenge with type 3 reovirus; litter mate controls uniformly developed hydrocephalus ~5. The internal image anti-idiotypes mimicked the binding of the reovirus hemagglutinin to a panel of target cells, and prior incubation of these cells with Ab2 inhibited viral binding and infectivity. This Ab2 has been successfully used both to isolate and to characterize the reovirus receptor which has now been identified as the
[3-adrenoceptor16. With poliovirus type II, small amounts of virus-neutralizing antibodies could be induced by a monoclonal anti-idiotype, but the antibody levels were not sufficient to protect mice against subsequent virus challenge 17. Similar results were observed using rabbit antiidiotypic antibodies against murine monoclonal antibodies to the rabies virus glycoprotein: i.e. immunization with anti-idiotype resulted in neutralizing antibodies to the virus but they were not sufficient to protect the mice against rabies infection 18. Monoclonal antibodies against a Sendai virus-specific helper T cell clone were effective in eliciting both B cell and T cell immunity. Thus, immunization of mice with the antiidiotypes afforded protection against a subsequent challenge infection with Sendai virus 19. Importantly, specific cytolytic T cells were induced which were not MHC restricted (see Glossary) in their activity, unlike the T cells activated by the virus itself2°. With tobacco mosaic virus (TMV), rabbit anti-idiotypes coupled to lipopolysaccharide induced a marked humoral response in BALB/c mice 21. The anti-TMV antibodies produced were idiotypically cross-reactive with the idiotype used to generate the anti-idiotypic antibody. With hepatitis B virus (HBV), Kennedy, Dreesman and colleagues have induced an antibody response in mice 22 and a protective response in chimpanzees 23 using polyclonal rabbit anti-idiotypic antibodies. The rabbit anti-idiotype recognized a highly conserved interspecies idiotype. Our approach 24 has been to raise mouse monoclonal antiidiotypes to a syngenic monoclonal Abe. Two internal image antiidiotypes have been fully characterized 24 and shown to mimic the group-specific a determinant of hepatitis B surface antigen (HBsAg). The anti-idiotype reacted with sera from mice, rabbits, goats, swine and humans immunized with HBsAg 25. The Ab2~ anti-idiotypes also inhibited the binding of monoclonal and polyclonal anti-HBsAg (antihepatitis B surface antigen) sera to linear and cyclic synthetic peptides
TIBTECH - MARCH 1989 [Vol. 7]
Bacteria
corresponding to the a determinant, thus confirming them as good candidate internal image anti-idiotypes 26. Using our anti-idiotypes, we have been able to generate anti-HBs responses in inbred strains of mice, in wild mice and in mice that are nonresponders to HBsAg itself. In addition, we are also able to effectively stimulate HBsAg-specific purified T cells obtained from peripheral blood lymphocytes of human donors who are immune to HBV either through natural infection or through vaccination 27. We have also studied a limited number of HBV carriers. They do not raise an anti-HBsAg response upon HBV infection and are thus serologically HBsAg positive and anti-HBsAg negative. In in vitro T cell proliferation assays, we could induce good stimulation of their T ceils using our internal image antiidiotypic antibodies but not antigen itself. Mouse monoclonal anti-idiotypes that bear the internal image of a human cytomegalovirus neutralization epitope have been used in syngenic immunization experiments to raise anti-anti-idiotypic antibodies (Ab3) which had virus-neutralizing activity 28. Not all experiments have produced protective responses. Indeed when polyclona] anti-idiotypic antibodies were used to modulate herpes simplex virus infection, they led to increased pathogenicity and shorter survival times of treated mice 29. The anti-idiotypic approach has also been tried in human immunodeficiency virus (HIV) infections. A mouse monoclonal anti-idiotype raised against anti-Leu3a mimics the CD4 receptor; it binds to recombinant gp160 and a molecule of 110-120 kDa in immunoblot analysis of HIVinfected cell lysates 3°. This monoclonal antibody also partially neutralized HIV infection of human T cells in vitro.
Rees et al. a~ raised a polyclonal rabbit anti-idiotypic antibody and used this to study proliferative responses of human T cells from tuberculosis patients and from BCGvaccinated healthy subjects. Responsiveness among patients to the antiidiotype correlated with responsiveness to a 38 kDa antigen of Mycobacterium tuberculosis 31. The induction of proliferation to both antigen and anti-idiotype was dependent on accessory cells and restricted by determinants encoded by the MHC (major histocompatibility complex). In addition, T cell reactivity was not dependent on the conformational integrity of the anti-idiotype binding site 32. Two Listeria monocflogenes specific CD4+ class II restricted clones were used to raise syngenic antisera 33. Injection of these anticlonotypic antibodies (with adjuvant) gave specific protection (in a genetically unrestricted manner) against the L. m o n o c y t o g e n e s strain recognized by the original T cell clone. Anti-idiotypes can also mimic polysaccharide antigens ~4. Monoclonal anti-idiotypes to the capsular polysaccharide of Neisseria m e n i n gitidis serogroup C could evoke antibodies specific to the meningococcal C polysaccharide in syngenic (having virtually identical genotypes) and xenogenic (different genotypes) species.
Consequences I would like to finish by briefly discussing observations from three laboratories 35-37 where mRNA sequence information of the antiidiotype clones has revealed areas of homology between the anti-idiotypes and the antigens they mimic. For instance, the amino acid sequences of reovirus hemagglutinin and the variable region of the light chain of a corresponding monoclonal internal image anti-idiotype share an area of homology (five identical amino acids and three conservative substitutions). In the antibody this area of homology lies in one of the complementarity determining regions (CDR2) 35. Similarly, on sequencing two monoclonal anti-idiotypes that mimic the rabbit immunoglobulin al
allotype (see Glossary), VanCleave et aL 37 observed that within the CDR2 of the heavy-chain V regions there was an area of homology with the rabbit al allotype, but in the opposite direction with respect to the carbon backbone. The ability of the internal image reversed sequence to produce an al-like antigenic determinant was tested by making synthetic peptides corresponding to the area of homology in both the anti-idiotype and the rabbit al allotype. Both peptides could completely inhibit the binding of rabbit immunoglobulin to anti-a1 antibody. Such results open the possibility of precisely identifying within the sequence of the anti-idiotypes those residues that mimic an original antigen. Synthetic peptide vaccines could then be constructed based on sequence data from anti-idiotypes. This could remove the need to use mouse monoclonal antibodies for vaccination.
Acknowledgements For their excellent secretarial assistance, I would like to thank both Cheryl Zuber and Donna Ovak.
References 1 Slater, R. J., Ward, S. M. and Kunkel, H. G. (1955) J. Exp. Med. 101, 85-108 2 0 u d i n , J. and Michel, M. (1963) C. R. Acad. Sci. 257, 805-808 3 Jerne, N. K. (1974) Ann. Immunol. (Inst. Pasteur) 125C, 373-389 4 Nisonoff, A. and Lamoyi, E. (1981) Clin. Immunol. lmmunopatho]. 21, 397-406 5 Roitt, I. M., Cooke, A., Male, D. K. et a]. (1981) Lancet i, 1041-1045 6 Sacks, D. L., Esser, K. M. and Sher, A. (1982) J. Exp. Med. 155, 1108-1119 7 Sacks, D. L. and Sher, A. (1983) J. Immunol. 131, 1511-1515 8 Grzych, J. M., Capron, M., Lambert, P. H. et al. (1985) Nature 316, 74--76 9 Powell, M. R. and Colley, D. G. (1987) Cell. Immunol. 104,377-385 10 Parra, J. C., Lima, M. S., Gazzinelli, G. and Colley, D. G. (1988) J. Immuno]. 140, 2401-2405 11 Gazzinelli, R. T., Morato, M. F., Nunes, R. B. eta]. (1988) J. Immunol. 140, 3167-3172 12 Bhogal, B. S., Nollstadt, K. H., Karkhanis, Y. D., Schmatz, D. M. and Jacobson, E. B. (1988) Infect. Immun. 56, 1113-1119
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13 Noseworthy, J. H., Fields, B. N., Dichter, M.A. et al. (1983) J. Immunol. 131, 2533-2538 14 Sharpe, A. H., Gaulton, G. N., McDade, K.K., Fields, B.N. and Greene, M. I. (1984) J. Exp. Med. 160, 1195-1205 15 Gaulton, G. N. and Greene, M. I. (1987) in Anti-idiotypes, Receptors and Molecular Mimicry (Linthicum, D, S. and Farid, N.R., eds), pp. 301-309, Springer Verlag 16 Co, M. S., Gaulton, G. N., Tominga, A. et al. (1985)Proc. Natl Acad. Sci. USA 82, 5315-5318 17 Uytdehaag, G. C. M. and Osterhaus, A. D. M. E. (1985) J. Immunol. 134, 1225-1229 18 Reagan, K. J., Wunner, W. H., Wiktor, T. J. and Koprowski, H. (1983)J. Viro]. 48,660-666 19 Ertl, H. C. J., McCarthy, E., Homans, E. and Finberg, R.W. (1985) in ItighTechnology Routes to Virus Vaccines (Dreesman, G. R., Bronson, J. G. and Kennedy, R.C., eds), pp. 125-141, American Society for Microbiology []
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20 Ertl, H. C. J. and Finberg, R. W. (1984) Proc. Natl Acad. Sci. USA 81, 2850-2854 21 Francotte, M. and Urbain, J. (1984) J. Exp. Med. 160, 1485-1494 22 Kennedy, R. C. and Dreesman, G. R. (1984) J. Exp. Med. 159, 655-665 23 Kennedy, R. C., Eichberg, J. W., Lanford, R. E. and Dreesman, G. R. (1986) Science 232,220-223 24 Thanavala, Y. M., Bond, A., Tedder, R., Hay, F. C. and Roitt, I. M. (1985) Immunology 55, 197-204 25 Thanavala, Y. M., Bond, A., Hay, F. C. and Roitt, I.M. (1985) J. Immunol. Methods 83, 227-232 26 Thanavala, Y. M., Brown, S. E., Howard, C. R., Roitt, I. M. and Steward, M. W. A. (1986) J. Exp. Med. 164, 227-236 27 Pride, M., Thakur, A. N., Ogra, P. A., Evans, R.L. and Thanavala, Y.M. (1988) FASEB Abstr. No. 1067 28 Keay, S., Rasmussen, L. and Merigan, T. C. (1988)J. Immunol. 140,944-948 29 Kennedy, R. C., Adler-Storthz, K., Burns, J. W., Henkel, R. D. and []
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Insect cell culture technology in baculovirus expression systems lain R. Cameron, Robert D. Possee and David H. L. Bishop The ability to produce large quantities of proteins relatively cheaply and easily, by expressing genes in heterologous systems, is a useful tool in modern biology and medicine. High-level protein expression systems have been developed from a number of prokaryotic and eukaryotic organisms. Insect baculovirus-based systems have shown great promise recently for the production of biologically active proteins. In this review we discuss the application of insect cell culture to baculovirus expression systems. Protein expression systems based on the A u t o g r a p h a californica nuclear polyhedrosis virus (AcNPV, an insect baculovirus) have wide applicability as an alternative to prokaryotic or other eukaryotic expression systems. lain Cameron, Robert Possee and David Bishop are at the NERC Institute of Virology, Mansfield Road, Oxford OX1 3SR, UK.
The vectors are not d e p e n d e n t on helper virus, standard virological methods are e m p l o y e d and the viruses are readily grown, maintained and m a n i p u l a t e d in vitro using cell lines derived frolil Spodoptera frugiperda (the fall armyworm). Baculoviruses are not pathogenic for vertebrates or plants. Baculoviruses contain a very late hyper-expressed gene w h i c h is not
© 1989, Elsevier Science Publishers Ltd (UK) 0167- 9430/89/$02.00
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Dreesman, G.R. (1984) J. Virol. 50, 951-953 Chanh, T. C., Dreesman, G. R. and Kennedy, R.C. (1987) Proe. Nat] Acad. Sci. USA 84, 3891-3895 Rees, A. D. M., Praputpittaya, K., Scoging, A. et al. (1987) Immunology 60, 389-393 Rees, A. D. S., Scoging, A., Dobson, N. et al. (1987) Eur. J. Immunol. 17, 197-201 Westerink, J., Campagnari, A. A., Wirth, M.A. and Apicella, M.A. (1988) Infect. Immun. 56, 1120-1127 Kaufmann, S. H. E., Eichmann, K., Muller, I. and Wrazel, L.J. (1985) J. Immuno]. 134, 4123-4127 Bruck, C., Co, M. S., Slaoui, M. et al. (1986) Proc. Natl Acad. Sci. USA 83, 6578-6582 Roth, C., Rocca-Serra, J., Somme, G., Fougereau, M. and Theze, J. (1985) Proc. Natl Acad. Sci. USA 82, 4788-4792 VanCleave, V. H., Naeve, C. W. and Metzger, D.W. (1988) J. Exp. Med. 167, 1841-1848 []
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essential for viral replication. The normal product of this gene is the p o l y h e d r i n protein, the subunit from w h i c h the crystalline occlusion body, or polyhedron, is formed. The p o l y h e d r o n contains infectious virus particles and is responsible for horizontal transmission of virus in the wild. Genes incorporated into the DNA genome of baculoviruses and expressed u n d e r the control of the strong viral p o l y h e d r i n promoter have enabled a b u n d a n t quantities (up to 50% of total cellular protein) of m a n y foreign proteins to be p r o d u c e d w h i c h are biologically active 1-3. The recent d e v e l o p m e n t of virus vectors expressing more t h a n one foreign product (multiple expression vectors; Fig. 1) indicates that prot e i n - p r o t e i n interactions such as the assembly processes of viruses 4 or m u l t i - c o m p o n e n t enzymes 5 could also be studied. Indeed it is possible to insert a DNA copy of the entire RNA genome (6.6 kb) of polio virus into the baculovirus: non-infectious viral particles are p r o d u c e d w h i c h m a y be useful for vaccination (T. Urakawa, pers. commun.). Baculovirus expression systems are relatively easy to use and can (in