TiPS - May 2990 [Vol. II]
194 that BARK is selectively expressed at synapses. 0
0
0
The cloning of BARK will open way to discovering how many receptor kinases exist, defining their species and tissue c&&ithe bution, and dete~ining structural ~Jationshi~ between the different classes of kinase. More information is also needed on the specificity of receptor kinases: for example, a kinase might act on all receptors coupled to a particular second messenger system (e.g. adenylyl cyclase) or on all receptors activated by a particular agonist (e.g. adrenoceptors). Knowledge of the sequences of the receptor kinases will provide information on the structural basis of their specificity. Other questions relate to whether an arrestin-like protein exists that is specific for @ARK (and if so what its mechanism of action is), and to the elucidation of mechanisms of long-term desensitization. From the answers to these questions should emerge a much fuller understanding of the molecular mechanisms involved in regulating receptor function. It should then be possible to develop strategies for modulatingdesensitization mechanisms in a way that will ultimately lead to prolonged and augmented therapeutic actions of drugs.
12 13 14
the
References 1 Lefkowitz, R. J. and Caron, M. G. (1988) 1. Viol. Ckeni. 263, 4993-4996 2 Dohlman, H. G., Caron, M. G. and Lefkowitz, R. J. (1987) Biockemistry 26, 2657-2664 3 Benovic, J. L., Bouvier, M., Caron, M. G. and Lefkowitz, R. J. (1988) Anm, Rev. CeN Eiot. 4,4m28 4 Clark, R. B. (1986) Adv. Cyclic Nucleotide Protein Pkaspko~latjon Res. 20, 155-209 5 Sibiey, D. R., Benovic, J. L., Caron, M.G.and Lefkowitz, R. J. fl987) C&48, 913-922 6 Hausdorff, W. P. et al. (1989) J_ Viol. Chem. 264.12657-12665 7 Lohse, M. J., Benovk, J. L., C~ron, M. G. and Lefkowitz, R. J. (1990) J. Biol. Chem. 265,3202-3211 8 Toews, M. L., Waldo, G. L., Harden, T. K. and Perkins, J. P. (1984) J. Biol. Chem --1 .-... . . . .._..
. a”,, Il‘Prr-.l”II” 9 Hertel,C., Coulter, S. J. and Perkins, J. P. (1985) I. Biol. Ckem. 260,
12547-12553 10 Cheung.A. H., Sigal, I. S., Dixon, R. A. F. and Strader. C. D. (1989) &&of. Pka~acol. 34.132-138 11 Binoti~, J. L., DeBlasi, A., Stone, W. C.,
15 16
17 18
Caron, M. G. and Lefkowitz, R. J. (1989) Science 246,23%240 Benovic, J. L. et al. (1985) J. Eiol. Ckem. 260,7094-7101 Benovic, J. L. et al. (1987) Proc. Nat1 Acad. Sci. USA 84.8879-8882 Dohlman, H. G., Bouvier, M., Benovic, J. L., Caron, M. G. and Lefkowitz, R. J. (1987) 1, Biol. Ckem. 262,1428Z-14288 Liggett, S. 8. et nf. (1989) Mol. Pkarmacol. 36.641646 Clark, R. 8.. Friedman, J., Dixon, R. A. F. and Strader, C. D. (1989) Mol. P~la~ffc~f. 36,343-34.8 Bouvier, M. et a!. (1989) 1. Biol. Cbem. 264,16786-16792 Blake, A. D., Mumford, R. A., Strout, H. V., Slater, E. E. and Strader, C. D. (1987) Biochenr.Biophys.Res. Commun.
147,168-I73 19 Benovic, J. L., Stone, W. C., Caron, M. G. and Lefkowitz, R. J. (1989) J. Biol. Chem. 264,6707-6710 20 Lohse, M. J., Lefkowitz, R. J., Caron, M. G. and Benovic, J. L. (1989) Proc. Nafl Acad. Sci. USA 86,3011-3015 21 Clark, R. B., Friedman, J., Johnson, J. A. and Kunkel, M. W. (1987) FASEB J. 1, 289-297 22 Gilman, A. G. (1987) Annu. Rev. Biockem. 56,615-649 23 Kwatra, M. M. and Hosey, M. M. (1986) J. Biot. Chem. 261,1242~12~2 24 Vaughan, R. A. and Devreotes, P. N. (1988) J. Biof. Chem. 263, 14538-14543 25 Reneke, J. E., Bhuner, K. J., Courchesne, W. E. and Thomer, J. (19S8) Cell 55, 221-234
Gilles J. Riveau and Franqoise M. Audibert Recent advances in immunology and biotechnology have opened the way for new approaches to vaccine design. Gilles Riveau and Francoise Audibert discuss progress in the design of synthetic peptide antigens for vaccines against pathogens, and discuss the possibility that such vaccines could also be used to control the activity of endogenous mediators. For both clinical and economic reasons, there is a strong incentive to develop new strategies for the production of vaccines. Approaches currently being investigated are the production of vaccines based on recombinant DNA techniques, anti-idiotype antibodies or synthetic peptides. The synthetic peptide approach is particularly attractive because, in addition to being safe ar.d nontoxic in use, such vaccines would be simple and inexpensive to produce. ~courag~ent for this approach was provided by the demonstration by Anderer in the 1960s that antibodies obtained by immunization with peptides corresponding to the C-terminal region of the tobacco mosaic virus coat protein precipitate and neutralize the virus’. The next step was the demonstration in 1981 that a comG. 1. Riveau is a CNRS Research Associate and F. M. Audiberf is INSERM Director of Research, Laboratoire ~~mmanopharmacologie ~~~rirne~tufe (WR 405}, 25 rue de i’Ecote de Mddecine, 75270 Paris Cedex 06, Frurree.
pletely synthetic peptide derived from diphthe~a toxin could induce protective immunity2. The production of synthetic peptides has been greatly simplified in recent years by the development of solid-phase synthesis technology3. The design of synthetic immunogenic peptides for use in vaccines requires the epitopes on the virus or bacterium that elicit neutralizing antibodies and/or cell-mediated immunity to be identified. Where the threedimensional structure of a protein is known, computer analysis can be helpful in predicting regions that are exposed on the surface of the protein and therefore likely to be immunogenic. Alternatively, regions that show sequence variability between isolates have been chosen as synthetic peptides on the grounds that the variability is due to high selective pressure imposed by their availability for interaction with antibodies. However, such peptides would only be expected to confer immunity against the stram of mi~org~ism from which their sequence
@ 1990, Elsevim Science Publishers Ltd. (UK} 0165- 6147/9U/$O2.00
TiPS -May
2990 [Vol. 111
was derived. In most cases suitable peptides have been identified experimentally by synthesizing and testing several peptides corresponding to different regions of the protein. Many epitopes on a protein will be made up of amino acids that are brought together in the threedimensional conformation and not arranged in a linear sequence. B cells tend to recognize this type of conformational epitope. However, in general, T cells recognize linear peptide sequences on proteins presented in association with major histocompatibility complex molecules. Synthetic peptides are therefore more likely to evoke responses involving T cells than those involving B cells. Howevpr, an ideal vaccine should prime both B cells and T cells. It may be possible to use information on spatial arrangements of amino acids to design peptides that mimic conformational epitopes mad2 up of discontinuous amino acid sequences4. Better understanding of the interactions between the MHC complex molecules and antigens will be required, however, before it will be possible to identify suitable epitopes on anything other than an empirical basis. Another issue that must be addressed in designing vaccines is the use of adjuvants. In addition to enhancing the level of the response, these can also be used to modify the type of response evoked: for some pathogens a cellmediated response is required; for others, mucosal immunity or antibody production are most appropriate.
Viral diseases Viruses cause many major diseases in mammals. Those causing influenza, hepatitis, poliomyelitis and now AIDS are particularly important targets for vaccine manufacturers. Outbreaks of animal diseases such as foot-andmouth disease in cattle represent an economic problem that justifies the search for an effective vaccine. The first completely synthetic immunogen to be used successfully was a peptide (designated P2) corresponding to amino acids 88-108 of the MS-2 coliphage coat protein. When covalently linked to the synthetic carrier A-L [poly(Ala)-poly(Lys)] and administered to guinea-pigs in
B cells. Lymphocytes that expand clonally after antigenic stimulation, to become antibody-secretory cells and/or B memory cells.
Helper T cells. Lymphocytes that interact with B cells and provide helper
signals for their proliferation and differentiation into antibody-secretory cells.
Epitope (antigenic determinant). Structures on the antigen responsible for the activation of either B cells or T cells. Both types of epitope should be present in an immunogenic structure. A B-cell epitope binds to the antibody produced by a specific B cell: a T-cell epitope is recognized by receptors on the surface of activated T cells. The epitope is thus the region that comes into contact with the antibodies raised, and with receptor on immune T cells. Epitopic suppression. Epitope-specific regulatory system that selectively suppresses the antibody production to an individual epitope linked to a carrier when the host has been preimmunized against this carrier. Genetic restriction. The ability of an animal to develop an immune response to an antigen is under control of genes located in the major histocompatibility complex (h4HC) (H-2 in mouse, HLA in humans).
Freund’s complete adjuvant, this peptide elicited phage-neutralizing antibodies. Conjugation in this structure of a low molecular weight peptidoglycan from Bacillus megaterium allowed the same response to be obtained in the absence of Freund’s adjuvant5. The peptidoglycan was replaced with a synthetic analogue, muramyl dipeptide (MDP) without loss of function: neutralizing antibodies were obtained when the completely synthetic MDP-PZ-A-L was administered in water to rabbit&. Foot-and-mouth disease virus has provided an excellent model system for the development of synthetic vaccines because its structure is simple, and the antigenicity of its componer?ts well understood. It contains four structural proteins, and it is the 213 amino acid VP1 polypeptide that bears the determinants for cellular and induction of attachment neutralizing antibodies. Of seven synthetic peptides corresponding to different regions of VP1 that were injected into guinea-pigs, only those derived from positions 130-160 or 190-213 elicited neutralizing antibodies7; these are the regions of VP1 that vary most between different -solates of the virus. A single inmculation with a peptide correspc nding to amino acid positions 141--160 provides better protection against subsequent challenge with foot-
and-mouth disease virus than pdy7 the complete VP1 polypepThis synthetic vaccine has been improved so that it should be able to compete commercially with the inactivated virus vaccine in the not too distant future. The most promising advances have been the construction of dimers and tetramers of the synthetic pepide, and incorporation of the peptide into the core of hepatitis B virus2. Furthermore, the genetically determined non-responsiveness of some individuals to the VP1 peptide has been overcome by incorporating epitopes from proteins (not from foot-and-mouth disease virus) that are known to stimulate helper T cells (see Ref. 8 for review). To be effective against all strains of the virus, a vaccine would need to contain peptides corresponding to this immunogenic, variable region of the VI’1 polypeptide from each serotype. The peptide vaccine has the additional advantages that it can be prepared safely without special facilities, has a long shelf-life and is easy to store, and that there is no risk of incomplete inactivation. The most important of the viruses that carry out reverse transcription are the hepadnaviruses causing hepatitis B, and the retrovirus human immunodeficiency virus (HIV) which causes AIDS. Developing viruses against HIV presents far greater technical and
TiPS - May 1990 [Vol. 111
1%
Th-2R
CS-T3
FJ. 1. Structureof the circum.spomzoitepmteinof P. falciparum. Th-2R and CS-T3 are apttopesthat stimulate/?elp?rTcells. Repeats. the B-cell-stimulatingimmunodominant region contafningthe repeated fU4NP sequence. Charged regions are indicated by a grey tfnt ([7). (After Ref. 15.)
theoretical pitfalls than developing vaccines against hepatitis 8. Firstly, HIV can integrate its genetic material into the genome of the host cell, and remain latent. particularly for postThus, exposure vaccination, a very high level of immunity must be achieved from the primary immunization and a cell-mediated immune remust be induced9Js. sponse Secondly, there is a lack of experimental models, and it is difficult to produce the virus on a large scale. Promising results are being obtained, however. For example, the regions of the HIV structure able to raise B-cell and T-cell responses are being defined”. Useful lessons have been learnt from developments in hepatitis B vaccination. Indeed, a recombinant vaccine (the first to be licensed) has been developed that offers improvements over the classical vaccine based on the hepatitis B surface antigen12. These artificial vaccines avoid the risks and costs inherent in the use of human blood products. Epitopes that induce protective immunity to murine and feline retroviruses have been found on virus-encoded envelope glycoproteins, and recent evidence suggests that the neutralizing epitope of HIV resides on the 160 kDa envelope glycoprotein (gp160). Thus, the development of a vaccine for AIDS has centred on gp160 and its components gp120 (which is external) and gp41 (Ref. 13). There is high antigenic variation in gp160 (up to 25% of the amino acids may vary between different strains). However, certain of its conserved amino acid sequences can also be recognized neutralizing antibodiesia. by Indeed, monoclonal antibodies have been raised against synthetic peptides mimicking a conserved hydrophilic region of the gp41 of
HIV-l (amino acids 735-752). These antibodies neutralized different HIV-l isolates by affecting early steps in adsorption and penetration of HIV-l. The gp41 envelope antigen is probably involved in membrane fusion following the binding of virions to the CD4 receptor molecule of T lymphocytes, which is mediated by gp120. It is likely that successful HIV vaccines will incorporate several viral antigens because the variable neutralization epitopes on gp120 may not allow cross-protection against most strains. However, these results suggest that vaccines might usefully incorporate the conserved gp41 epitope that elicits neutralizing antibodies. Parasitic diseases There is still no vaccine against any human parasite. Many of the problems in developing parasite vaccines are due to antigen diversity and variability, and the fact that the life cycle of most parasites consists of several stages, requiring multiple immune effector mechanisms to be involved in evoking host resistance. One hundred million malarial infections, and one million deaths amongst children occur annually in Africa (see Ref. 15 for review). A vaccine against Plnsmodium fulciparum is therefore a crucial goal, and the pioneering work of Nussenzweig16 suggested that the sporozoite stage should be the target. The circumsporozoite (CS) proteins, which cover the surface of the parasite, contain speciesspecific immunodominant epitopes formed by tandem repeat sequences of amino acids (Fig. 1). The dominant CS epitope of P. fdciparum consists of repeated units of Asn-Ala-Asn-Pro (NANP) and has been identified as the major sporozoite surface antigen.
Synthetic peptides representing this epitope, when coupled to a foreign carrier protein, raised antibodies able to block sporozoite invasion of human hepatocytes in vitro 17. However, in clinical trials, this construct evoked only a weak antibody response in humans; this was possibly due to epitopic suppression with regard to the carrierls. Another clinical trial using a repetitive structure obtained by recombinant DNA techniques produced similar results. In an attempt to raise antibodies against the NANP synthetic epitope in the absence of carrier, NANP was polymerized by treatment with carbodiimideig. Although this produced a good response in mice, the approach is not robust because of the existence of strong genetic restriction2s-zz. An experimental anti-malarial vaccine has been produced that incorporates an epitope that binds to helper T cells*sJ4. This epitope (CS-T3) corresponds (with the exception of two Cys+Ala substitutions) to a site located on the Cterminal part of the non-repetitive region of the circumsporozoite protein, at residues 378-398. This suggests that this peptide could associate with many different MHC class II molecules=. When coupled to the repetitive epitope (NAN& (which stimulates B cells), this epitope was very effective in raising anti-NANP and anti-sporozoite antibodies. This construct shows less genetic restriction, and is capable of inducing a memory response during an infection with the pathogen. Vaccination of humans against asexual blood stages of P. falciparum has also been achieved using synthetic peptides without carrier. In particular, polymeric synthetic hybrid proteins, based partly on fragments related to p. falciparum merozoite-specific proteins, delayed or suppressed the development of parasitaemia in immunized human volunteer+. If antigens from both sporozoite and merozoite stages and perhaps even from the sexual stages were incorporated into a vaccine, it might afford better protection. Bacterial diseases Two examples illustrate the approaches in development of synthetic vaccines for bacterial
TiPS - May 1990 [Vol. 2 II diseases. One of the first synthetic vaccines capable of inducing protection against a bacterial infection was obtained in 1981 using a copy of sequence found in the Strepfococcus pyogenes M protein27. Of all the surface components of Streptococcus pyogenes, only the M protein evokes protective immunity. However, attempts to vaccinate humans against streptococcal infections have been hampered by several problems: the production of antibodies crossreacting with heart tissue after vaccination; and the high number of serotypes due to antigenic varia:ioi,s of the M protein. The synthetic approach has allowed epitopes inducing protective immunity to be distinguished from those that determine heart crossreactivity, and has led to the development of a multivalent vaccine containing protective synthetic peptides derived from various M serotypes. The peptides in this vaccine formed a hybrid complex that raised protective immune rerelated sponses against the streptococci without evoking tissue-crossreactive immunity2*. Other polyvalent vaccines in which a streptococcal peptide is associated with synthetic peptides derived from different pathogens have also been described29. The major antigenic determinant of the M24 protein contains five Lys residues, which allow up to five proteins to be covalently linked onto the backbone. In one successful construct, three synthetic peptides of different specificities (diphtheria toxin, circumsporozoite protein and hepatitis B surface antigen) were conjugated to the streptococcal peptide. Immunization of guinea-pigs with this construct induced biologically active and specific sensitized T cells against each of the four peptides; thus antigenic competition does not impair the antigenic properties of totally synthetic peptide vaccines30. Enterotoxigenic Escherichia coli is a major health problem, particularly in developing countries where it is the commonest cause of acute diarrhoea. Enterotoxigenic strains produce two antigenically distinct toxins - heat-labile and heat-stable - either singly or together; thus vaccines need to contain both antigens to provide complete protection. A synthetic
197 44 amino acid peptide was produced that contained the 26 amino acids of the nontoxic B subunit of the heat-labile toxin which binds the intestinal receptors: and the 18 amino acids forming the immunogenic part of the heat-stable toxin31. This completely synthetic peptide vaccine is nontoxic and immunogenic for B subunits of both toxins. In a clinical trial, this vaccine was given orally to thirteen volunteers who developed antibodies against both toxin components. Antibodies were detected both in the sera and, at a higher concentration, in jejunal aspirates3*. The antibodies neutralized the secretory activity of both toxins.
Control of endogenous mediators A new field for synthetic vaccines is the regulation of the anabolism and activity of endogenous mediators by antibody production. Renin, a very specific aspartate proteinase, cleaves angiotensinogen to release the decapeptide angiotensin I. Renin plays a key role in the angiotensin-aldosterone cascade, which regulates blood pressure homeostasis and
85
06
6.7
88
89 Fig. 2. Conformation of an antigenic peptide(Cy&O-Cys89) similar to part of the human renin flap. Bold lines are covalent links; fine lines, hydrogen bonds; broken lines, observed interstrand nuclear Overhauser effects.
(AlterRef. 35.)
fluid and electrolyte balance. Immunologists aimed to produce antibodies capable of recognizing the renin molecule and thus of inhibiting the renin-angiotensin system. Prediction of the different renin epitopes by computer modeling studies allowed the selection of peptides inducing antibodies that inhibit enzymatic activity33.34. The aspartate proteinases are characterized by the existence of an extended hairpin - the flap’- which protrudes from the surface of the protein (Fig. 2). The synthetic cyclic peptides (Cys8tKys89 and Cys78-Cys91) adopted a conformation similar to that of the human renin flap35 and were recognized by anti-renin antibodies. These peptides are therefore good candidates for the elaboration of the first synthetic anti-enzyme vaccine. Experiments in animals established the principle that immunity to the pregnancy hormone human chorionic gonadotropin (hCG) could block fertility at an early stage of pregnancy with no discernible alterations in the menstrual cycle36. Different kinds of vaccination to disrupt hCG function have been evaluated. These strategies involved the use of three different structures, i.e. the B subunit of ovine LH, B subunit of hCG and a synthetic peptide representing a determinant specific to the B subunit of hCG, corresponding to the carboxyl terminal amino acid region of the B subunit (positions 109-145) (Ref. 37). Phase I clinical trials have been performed with this peptide coupled to diphtheria toxin and peptide as using a muramyl adjuvanP. These trials demonstrate that vaccines containing synthetic hCG B subunit epitopes are promising antifertility agents because: (1) the target antigen is present transiently in the reproductive process; (2) because the immune response appears to be specific to the target hormone; and (3) its effect is potentially reversible. These studies indicate the feasibility of further approaches based on other endogenous target antigens. 0
0
0
Recent clinical trials have demonstrated that biologically
TiPS - May 1990 [Vol. 111
19s active antibodies can be raised in humans following administration of synthetic antigens’**3**%.HOWeve;, the level of immunity obtained was not sufficient to allow these vaccines to be used in the clinic. Better response would be &$zI~ if several synthetic pep- to several different antigenie determinants of the natural immunogen - were included. Moreover, synthetic vaccines usuaily contain epitopes that invoke either a T-cell or a B-cell response; ideally they should contain both. They are therefore poor antigens and need to be administered in appropriate vehicles and in conjunction with strong adjuvants. Optimizing these preparations is a key issue for research. References 1 Anderer, F. (1963) Bjoc~~jrn. Eiophys. 2 3
4
5
Actn 71,246-248 Audibert, F. et nl. (1981) Nattire 289, S9%594 Gehoff, E. D., Meloen. R. H. and Barteiing, S. J. (1984) Proc. Nat2 Acod. Sci. USA 81.399-2 Geysen, H. M., Rhodda, S. J. and Mason, T. J. (1985) in Synthetic Peptide> as Antigens (Cibn FourzdationSymposia, vat. 190). pp. 130-149 Langbeheim, H., Amon, R. and Sela, M. (1976) Pmt. Natl Acnd. SC;. USA 73,
6 Amon, R.. Seia. M.. Parant. M. and Chedid, L: (1986) P&c. Nat1 &ad. Sri. USA 77.6769-6772 7 BittIe, J. L. et al. (1982) Nature 298.30-34 8 Brown, F. fl988) Veccine 6.180-182 9 Salk, J. (1987) Nature 327,473-476 10 Fauci. A. (1988) Proc. Nafl Acad. Sci. USA 83.9278-9283 11 Benofsky, J. A. er al. (1988) Natwe 334, 7ofi-708 12 Zajac, B. A., West, D. J., McAleer, W. J. and Scolnick, E. M. (1986) J. Infect. 13 (Suppl. A), 39-45 13 Ada, G. (1389) Natwe 339, 331-332 14 Dalgleish. A. G. et al. (1988) Virology 165,209-21s 15 Mitchell, G. H. (1989) Parasitology 98, S29-S47 16 Nussenzweig, R, Vanderburg, J., Most, X. and Orton, C. (1967) Nature 216. 160-163 17 ZavaIa, F. ef at. (1985) Science 228, 1436-1440 18 E&get, H. M. et ~1. (1988) J. fmmunof. 140,626-633 19 Herrington, 0. A. et ~1. (1987) Nafure 328,257-2.59 20 Good, M. F. et al. (1986) 1. Exp. Med. 164, 655-660 21 Togna, A. R. et al. (1986) J. Immunol. 137, 295~2960 22 Lise, L. D. et al. (1988) Biochem. Biophys. Res. Commun. 153.31-38 23 Sin&a&a, F. et a{: (1988) Eur. 1. Zmmunol. l&633-636 24 Sinigaglia, F. et al. (1988) Nature 336,
.._ ._-
77iL7Ml
2s Margalit, H. et al. (1987) J. ~mmanoZ.138, 2213-2229
26 Patarroyo, M. E. ef al. (1988) Nature 332, 158-161 27 Beachey, E. H., Seyer, J. M., Dale, J. B., Simpson, W. A. and Kang, A. H. (1981) Natwe 292, 457-459 28 Beachey, E. H.. Seyer. J. M. and Dale, 1. B. (1987) 1. Exv. Med. 166.647-656 29 joI&, M:et Ql. 11987) Infect. Immun. 55, 149%1502 __ _ _ 30 Jolivet, M. et al. (1990) Vaccine 8,35-40 31 Hsughter., R. A., En@, R. F., Ostresh, J. M., Hoffman, S. R. and Klipstein, F. A. (198.5) Infect. fmmun. 48,735-740 32 Klipstein, F. A., Engert, R. F. and
Houghten, R. A. (1986) Law&i. 471-473 33 Galen, F-X. ef al. (1987) J. Hypertens. 5 (Suppl. 5). 511-514 34 Bouhnik, J. ef al. (1987) /. Biol. Chem. 262,2913-2918 35 hhrentz, J-A. c’ ai. (1988) Biochemistry 27,4071-4078 36 Stevens, V. C. (1976) in Development of Vuccincs for Ferfirify Regulation IWHD Symposiumf, pp. 93-110, Scriptor 37 Steven +, V. C. (1986) Immutml. Today 7, 369-374 38 Jones, W. R. et al. (1988) Lancef i, 1295-1298
for the of AIDS Erik De Clercq Eradjcafion of human immunodeficiency virus (WV1 fiiam in~ecied cells or organisms has remained an elusive goal. In p~nciple, any of the steps in fhe replicafive cycle could form the basis for ratiana~ design of chemofherapeutic agents, although in practice not all have so far proved amenable to intervention. Here, Erik De Clercq summarizes the strategies that are being employed, focusing primarily on recent promising developments in inhibition of adsorption of I-ilVonto CD4+ T cells, and inhibition of the viral reverse transcriptuse. Although any of the steps in the replicative cycle of HIV (Fig. 1) could, in principie, serve as a site of attack for chemotherapeutic agents’, only the following five have so far been identified and investigated as potential targets: 6 adsorption of the virus to the cell membrane, which depends on the interaction between the viral envelope glycoprotein gp120 and the cellular CD4 receptor (which is present on a subset of T lymphocytes and certain other immune cells); 0 transcription of the viral RNA to proviral DNA by the reverse transcriptase and degradation of the residual RNA by ribonuclease H (RNase H); 6 transactivation of the expression of the viral genes by the regulatory proteins (such as the truns-acting transcriptional activator tat); @ proteolytic cleavage of the viral precursor proteins by the viral E. De CJercq is Professor of Microbiology and Biochemistryat the Faculty of Medicine of the Kntholieke Universifeit Leuven, and I
194 that BARK is selectively expressed at synapses. 0
0
0
The cloning of BARK will open way to discovering how many receptor kinases exist, defining their species and tissue c&&ithe bution, and dete~ining structural ~Jationshi~ between the different classes of kinase. More information is also needed on the specificity of receptor kinases: for example, a kinase might act on all receptors coupled to a particular second messenger system (e.g. adenylyl cyclase) or on all receptors activated by a particular agonist (e.g. adrenoceptors). Knowledge of the sequences of the receptor kinases will provide information on the structural basis of their specificity. Other questions relate to whether an arrestin-like protein exists that is specific for @ARK (and if so what its mechanism of action is), and to the elucidation of mechanisms of long-term desensitization. From the answers to these questions should emerge a much fuller understanding of the molecular mechanisms involved in regulating receptor function. It should then be possible to develop strategies for modulatingdesensitization mechanisms in a way that will ultimately lead to prolonged and augmented therapeutic actions of drugs.
12 13 14
the
References 1 Lefkowitz, R. J. and Caron, M. G. (1988) 1. Viol. Ckeni. 263, 4993-4996 2 Dohlman, H. G., Caron, M. G. and Lefkowitz, R. J. (1987) Biockemistry 26, 2657-2664 3 Benovic, J. L., Bouvier, M., Caron, M. G. and Lefkowitz, R. J. (1988) Anm, Rev. CeN Eiot. 4,4m28 4 Clark, R. B. (1986) Adv. Cyclic Nucleotide Protein Pkaspko~latjon Res. 20, 155-209 5 Sibiey, D. R., Benovic, J. L., Caron, M.G.and Lefkowitz, R. J. fl987) C&48, 913-922 6 Hausdorff, W. P. et al. (1989) J_ Viol. Chem. 264.12657-12665 7 Lohse, M. J., Benovk, J. L., C~ron, M. G. and Lefkowitz, R. J. (1990) J. Biol. Chem. 265,3202-3211 8 Toews, M. L., Waldo, G. L., Harden, T. K. and Perkins, J. P. (1984) J. Biol. Chem --1 .-... . . . .._..
. a”,, Il‘Prr-.l”II” 9 Hertel,C., Coulter, S. J. and Perkins, J. P. (1985) I. Biol. Ckem. 260,
12547-12553 10 Cheung.A. H., Sigal, I. S., Dixon, R. A. F. and Strader. C. D. (1989) &&of. Pka~acol. 34.132-138 11 Binoti~, J. L., DeBlasi, A., Stone, W. C.,
15 16
17 18
Caron, M. G. and Lefkowitz, R. J. (1989) Science 246,23%240 Benovic, J. L. et al. (1985) J. Eiol. Ckem. 260,7094-7101 Benovic, J. L. et al. (1987) Proc. Nat1 Acad. Sci. USA 84.8879-8882 Dohlman, H. G., Bouvier, M., Benovic, J. L., Caron, M. G. and Lefkowitz, R. J. (1987) 1, Biol. Ckem. 262,1428Z-14288 Liggett, S. 8. et nf. (1989) Mol. Pkarmacol. 36.641646 Clark, R. 8.. Friedman, J., Dixon, R. A. F. and Strader, C. D. (1989) Mol. P~la~ffc~f. 36,343-34.8 Bouvier, M. et a!. (1989) 1. Biol. Cbem. 264,16786-16792 Blake, A. D., Mumford, R. A., Strout, H. V., Slater, E. E. and Strader, C. D. (1987) Biochenr.Biophys.Res. Commun.
147,168-I73 19 Benovic, J. L., Stone, W. C., Caron, M. G. and Lefkowitz, R. J. (1989) J. Biol. Chem. 264,6707-6710 20 Lohse, M. J., Lefkowitz, R. J., Caron, M. G. and Benovic, J. L. (1989) Proc. Nafl Acad. Sci. USA 86,3011-3015 21 Clark, R. B., Friedman, J., Johnson, J. A. and Kunkel, M. W. (1987) FASEB J. 1, 289-297 22 Gilman, A. G. (1987) Annu. Rev. Biockem. 56,615-649 23 Kwatra, M. M. and Hosey, M. M. (1986) J. Biot. Chem. 261,1242~12~2 24 Vaughan, R. A. and Devreotes, P. N. (1988) J. Biof. Chem. 263, 14538-14543 25 Reneke, J. E., Bhuner, K. J., Courchesne, W. E. and Thomer, J. (19S8) Cell 55, 221-234
Gilles J. Riveau and Franqoise M. Audibert Recent advances in immunology and biotechnology have opened the way for new approaches to vaccine design. Gilles Riveau and Francoise Audibert discuss progress in the design of synthetic peptide antigens for vaccines against pathogens, and discuss the possibility that such vaccines could also be used to control the activity of endogenous mediators. For both clinical and economic reasons, there is a strong incentive to develop new strategies for the production of vaccines. Approaches currently being investigated are the production of vaccines based on recombinant DNA techniques, anti-idiotype antibodies or synthetic peptides. The synthetic peptide approach is particularly attractive because, in addition to being safe ar.d nontoxic in use, such vaccines would be simple and inexpensive to produce. ~courag~ent for this approach was provided by the demonstration by Anderer in the 1960s that antibodies obtained by immunization with peptides corresponding to the C-terminal region of the tobacco mosaic virus coat protein precipitate and neutralize the virus’. The next step was the demonstration in 1981 that a comG. 1. Riveau is a CNRS Research Associate and F. M. Audiberf is INSERM Director of Research, Laboratoire ~~mmanopharmacologie ~~~rirne~tufe (WR 405}, 25 rue de i’Ecote de Mddecine, 75270 Paris Cedex 06, Frurree.
pletely synthetic peptide derived from diphthe~a toxin could induce protective immunity2. The production of synthetic peptides has been greatly simplified in recent years by the development of solid-phase synthesis technology3. The design of synthetic immunogenic peptides for use in vaccines requires the epitopes on the virus or bacterium that elicit neutralizing antibodies and/or cell-mediated immunity to be identified. Where the threedimensional structure of a protein is known, computer analysis can be helpful in predicting regions that are exposed on the surface of the protein and therefore likely to be immunogenic. Alternatively, regions that show sequence variability between isolates have been chosen as synthetic peptides on the grounds that the variability is due to high selective pressure imposed by their availability for interaction with antibodies. However, such peptides would only be expected to confer immunity against the stram of mi~org~ism from which their sequence
@ 1990, Elsevim Science Publishers Ltd. (UK} 0165- 6147/9U/$O2.00
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was derived. In most cases suitable peptides have been identified experimentally by synthesizing and testing several peptides corresponding to different regions of the protein. Many epitopes on a protein will be made up of amino acids that are brought together in the threedimensional conformation and not arranged in a linear sequence. B cells tend to recognize this type of conformational epitope. However, in general, T cells recognize linear peptide sequences on proteins presented in association with major histocompatibility complex molecules. Synthetic peptides are therefore more likely to evoke responses involving T cells than those involving B cells. Howevpr, an ideal vaccine should prime both B cells and T cells. It may be possible to use information on spatial arrangements of amino acids to design peptides that mimic conformational epitopes mad2 up of discontinuous amino acid sequences4. Better understanding of the interactions between the MHC complex molecules and antigens will be required, however, before it will be possible to identify suitable epitopes on anything other than an empirical basis. Another issue that must be addressed in designing vaccines is the use of adjuvants. In addition to enhancing the level of the response, these can also be used to modify the type of response evoked: for some pathogens a cellmediated response is required; for others, mucosal immunity or antibody production are most appropriate.
Viral diseases Viruses cause many major diseases in mammals. Those causing influenza, hepatitis, poliomyelitis and now AIDS are particularly important targets for vaccine manufacturers. Outbreaks of animal diseases such as foot-andmouth disease in cattle represent an economic problem that justifies the search for an effective vaccine. The first completely synthetic immunogen to be used successfully was a peptide (designated P2) corresponding to amino acids 88-108 of the MS-2 coliphage coat protein. When covalently linked to the synthetic carrier A-L [poly(Ala)-poly(Lys)] and administered to guinea-pigs in
B cells. Lymphocytes that expand clonally after antigenic stimulation, to become antibody-secretory cells and/or B memory cells.
Helper T cells. Lymphocytes that interact with B cells and provide helper
signals for their proliferation and differentiation into antibody-secretory cells.
Epitope (antigenic determinant). Structures on the antigen responsible for the activation of either B cells or T cells. Both types of epitope should be present in an immunogenic structure. A B-cell epitope binds to the antibody produced by a specific B cell: a T-cell epitope is recognized by receptors on the surface of activated T cells. The epitope is thus the region that comes into contact with the antibodies raised, and with receptor on immune T cells. Epitopic suppression. Epitope-specific regulatory system that selectively suppresses the antibody production to an individual epitope linked to a carrier when the host has been preimmunized against this carrier. Genetic restriction. The ability of an animal to develop an immune response to an antigen is under control of genes located in the major histocompatibility complex (h4HC) (H-2 in mouse, HLA in humans).
Freund’s complete adjuvant, this peptide elicited phage-neutralizing antibodies. Conjugation in this structure of a low molecular weight peptidoglycan from Bacillus megaterium allowed the same response to be obtained in the absence of Freund’s adjuvant5. The peptidoglycan was replaced with a synthetic analogue, muramyl dipeptide (MDP) without loss of function: neutralizing antibodies were obtained when the completely synthetic MDP-PZ-A-L was administered in water to rabbit&. Foot-and-mouth disease virus has provided an excellent model system for the development of synthetic vaccines because its structure is simple, and the antigenicity of its componer?ts well understood. It contains four structural proteins, and it is the 213 amino acid VP1 polypeptide that bears the determinants for cellular and induction of attachment neutralizing antibodies. Of seven synthetic peptides corresponding to different regions of VP1 that were injected into guinea-pigs, only those derived from positions 130-160 or 190-213 elicited neutralizing antibodies7; these are the regions of VP1 that vary most between different -solates of the virus. A single inmculation with a peptide correspc nding to amino acid positions 141--160 provides better protection against subsequent challenge with foot-
and-mouth disease virus than pdy7 the complete VP1 polypepThis synthetic vaccine has been improved so that it should be able to compete commercially with the inactivated virus vaccine in the not too distant future. The most promising advances have been the construction of dimers and tetramers of the synthetic pepide, and incorporation of the peptide into the core of hepatitis B virus2. Furthermore, the genetically determined non-responsiveness of some individuals to the VP1 peptide has been overcome by incorporating epitopes from proteins (not from foot-and-mouth disease virus) that are known to stimulate helper T cells (see Ref. 8 for review). To be effective against all strains of the virus, a vaccine would need to contain peptides corresponding to this immunogenic, variable region of the VI’1 polypeptide from each serotype. The peptide vaccine has the additional advantages that it can be prepared safely without special facilities, has a long shelf-life and is easy to store, and that there is no risk of incomplete inactivation. The most important of the viruses that carry out reverse transcription are the hepadnaviruses causing hepatitis B, and the retrovirus human immunodeficiency virus (HIV) which causes AIDS. Developing viruses against HIV presents far greater technical and
TiPS - May 1990 [Vol. 111
1%
Th-2R
CS-T3
FJ. 1. Structureof the circum.spomzoitepmteinof P. falciparum. Th-2R and CS-T3 are apttopesthat stimulate/?elp?rTcells. Repeats. the B-cell-stimulatingimmunodominant region contafningthe repeated fU4NP sequence. Charged regions are indicated by a grey tfnt ([7). (After Ref. 15.)
theoretical pitfalls than developing vaccines against hepatitis 8. Firstly, HIV can integrate its genetic material into the genome of the host cell, and remain latent. particularly for postThus, exposure vaccination, a very high level of immunity must be achieved from the primary immunization and a cell-mediated immune remust be induced9Js. sponse Secondly, there is a lack of experimental models, and it is difficult to produce the virus on a large scale. Promising results are being obtained, however. For example, the regions of the HIV structure able to raise B-cell and T-cell responses are being defined”. Useful lessons have been learnt from developments in hepatitis B vaccination. Indeed, a recombinant vaccine (the first to be licensed) has been developed that offers improvements over the classical vaccine based on the hepatitis B surface antigen12. These artificial vaccines avoid the risks and costs inherent in the use of human blood products. Epitopes that induce protective immunity to murine and feline retroviruses have been found on virus-encoded envelope glycoproteins, and recent evidence suggests that the neutralizing epitope of HIV resides on the 160 kDa envelope glycoprotein (gp160). Thus, the development of a vaccine for AIDS has centred on gp160 and its components gp120 (which is external) and gp41 (Ref. 13). There is high antigenic variation in gp160 (up to 25% of the amino acids may vary between different strains). However, certain of its conserved amino acid sequences can also be recognized neutralizing antibodiesia. by Indeed, monoclonal antibodies have been raised against synthetic peptides mimicking a conserved hydrophilic region of the gp41 of
HIV-l (amino acids 735-752). These antibodies neutralized different HIV-l isolates by affecting early steps in adsorption and penetration of HIV-l. The gp41 envelope antigen is probably involved in membrane fusion following the binding of virions to the CD4 receptor molecule of T lymphocytes, which is mediated by gp120. It is likely that successful HIV vaccines will incorporate several viral antigens because the variable neutralization epitopes on gp120 may not allow cross-protection against most strains. However, these results suggest that vaccines might usefully incorporate the conserved gp41 epitope that elicits neutralizing antibodies. Parasitic diseases There is still no vaccine against any human parasite. Many of the problems in developing parasite vaccines are due to antigen diversity and variability, and the fact that the life cycle of most parasites consists of several stages, requiring multiple immune effector mechanisms to be involved in evoking host resistance. One hundred million malarial infections, and one million deaths amongst children occur annually in Africa (see Ref. 15 for review). A vaccine against Plnsmodium fulciparum is therefore a crucial goal, and the pioneering work of Nussenzweig16 suggested that the sporozoite stage should be the target. The circumsporozoite (CS) proteins, which cover the surface of the parasite, contain speciesspecific immunodominant epitopes formed by tandem repeat sequences of amino acids (Fig. 1). The dominant CS epitope of P. fdciparum consists of repeated units of Asn-Ala-Asn-Pro (NANP) and has been identified as the major sporozoite surface antigen.
Synthetic peptides representing this epitope, when coupled to a foreign carrier protein, raised antibodies able to block sporozoite invasion of human hepatocytes in vitro 17. However, in clinical trials, this construct evoked only a weak antibody response in humans; this was possibly due to epitopic suppression with regard to the carrierls. Another clinical trial using a repetitive structure obtained by recombinant DNA techniques produced similar results. In an attempt to raise antibodies against the NANP synthetic epitope in the absence of carrier, NANP was polymerized by treatment with carbodiimideig. Although this produced a good response in mice, the approach is not robust because of the existence of strong genetic restriction2s-zz. An experimental anti-malarial vaccine has been produced that incorporates an epitope that binds to helper T cells*sJ4. This epitope (CS-T3) corresponds (with the exception of two Cys+Ala substitutions) to a site located on the Cterminal part of the non-repetitive region of the circumsporozoite protein, at residues 378-398. This suggests that this peptide could associate with many different MHC class II molecules=. When coupled to the repetitive epitope (NAN& (which stimulates B cells), this epitope was very effective in raising anti-NANP and anti-sporozoite antibodies. This construct shows less genetic restriction, and is capable of inducing a memory response during an infection with the pathogen. Vaccination of humans against asexual blood stages of P. falciparum has also been achieved using synthetic peptides without carrier. In particular, polymeric synthetic hybrid proteins, based partly on fragments related to p. falciparum merozoite-specific proteins, delayed or suppressed the development of parasitaemia in immunized human volunteer+. If antigens from both sporozoite and merozoite stages and perhaps even from the sexual stages were incorporated into a vaccine, it might afford better protection. Bacterial diseases Two examples illustrate the approaches in development of synthetic vaccines for bacterial
TiPS - May 1990 [Vol. 2 II diseases. One of the first synthetic vaccines capable of inducing protection against a bacterial infection was obtained in 1981 using a copy of sequence found in the Strepfococcus pyogenes M protein27. Of all the surface components of Streptococcus pyogenes, only the M protein evokes protective immunity. However, attempts to vaccinate humans against streptococcal infections have been hampered by several problems: the production of antibodies crossreacting with heart tissue after vaccination; and the high number of serotypes due to antigenic varia:ioi,s of the M protein. The synthetic approach has allowed epitopes inducing protective immunity to be distinguished from those that determine heart crossreactivity, and has led to the development of a multivalent vaccine containing protective synthetic peptides derived from various M serotypes. The peptides in this vaccine formed a hybrid complex that raised protective immune rerelated sponses against the streptococci without evoking tissue-crossreactive immunity2*. Other polyvalent vaccines in which a streptococcal peptide is associated with synthetic peptides derived from different pathogens have also been described29. The major antigenic determinant of the M24 protein contains five Lys residues, which allow up to five proteins to be covalently linked onto the backbone. In one successful construct, three synthetic peptides of different specificities (diphtheria toxin, circumsporozoite protein and hepatitis B surface antigen) were conjugated to the streptococcal peptide. Immunization of guinea-pigs with this construct induced biologically active and specific sensitized T cells against each of the four peptides; thus antigenic competition does not impair the antigenic properties of totally synthetic peptide vaccines30. Enterotoxigenic Escherichia coli is a major health problem, particularly in developing countries where it is the commonest cause of acute diarrhoea. Enterotoxigenic strains produce two antigenically distinct toxins - heat-labile and heat-stable - either singly or together; thus vaccines need to contain both antigens to provide complete protection. A synthetic
197 44 amino acid peptide was produced that contained the 26 amino acids of the nontoxic B subunit of the heat-labile toxin which binds the intestinal receptors: and the 18 amino acids forming the immunogenic part of the heat-stable toxin31. This completely synthetic peptide vaccine is nontoxic and immunogenic for B subunits of both toxins. In a clinical trial, this vaccine was given orally to thirteen volunteers who developed antibodies against both toxin components. Antibodies were detected both in the sera and, at a higher concentration, in jejunal aspirates3*. The antibodies neutralized the secretory activity of both toxins.
Control of endogenous mediators A new field for synthetic vaccines is the regulation of the anabolism and activity of endogenous mediators by antibody production. Renin, a very specific aspartate proteinase, cleaves angiotensinogen to release the decapeptide angiotensin I. Renin plays a key role in the angiotensin-aldosterone cascade, which regulates blood pressure homeostasis and
85
06
6.7
88
89 Fig. 2. Conformation of an antigenic peptide(Cy&O-Cys89) similar to part of the human renin flap. Bold lines are covalent links; fine lines, hydrogen bonds; broken lines, observed interstrand nuclear Overhauser effects.
(AlterRef. 35.)
fluid and electrolyte balance. Immunologists aimed to produce antibodies capable of recognizing the renin molecule and thus of inhibiting the renin-angiotensin system. Prediction of the different renin epitopes by computer modeling studies allowed the selection of peptides inducing antibodies that inhibit enzymatic activity33.34. The aspartate proteinases are characterized by the existence of an extended hairpin - the flap’- which protrudes from the surface of the protein (Fig. 2). The synthetic cyclic peptides (Cys8tKys89 and Cys78-Cys91) adopted a conformation similar to that of the human renin flap35 and were recognized by anti-renin antibodies. These peptides are therefore good candidates for the elaboration of the first synthetic anti-enzyme vaccine. Experiments in animals established the principle that immunity to the pregnancy hormone human chorionic gonadotropin (hCG) could block fertility at an early stage of pregnancy with no discernible alterations in the menstrual cycle36. Different kinds of vaccination to disrupt hCG function have been evaluated. These strategies involved the use of three different structures, i.e. the B subunit of ovine LH, B subunit of hCG and a synthetic peptide representing a determinant specific to the B subunit of hCG, corresponding to the carboxyl terminal amino acid region of the B subunit (positions 109-145) (Ref. 37). Phase I clinical trials have been performed with this peptide coupled to diphtheria toxin and peptide as using a muramyl adjuvanP. These trials demonstrate that vaccines containing synthetic hCG B subunit epitopes are promising antifertility agents because: (1) the target antigen is present transiently in the reproductive process; (2) because the immune response appears to be specific to the target hormone; and (3) its effect is potentially reversible. These studies indicate the feasibility of further approaches based on other endogenous target antigens. 0
0
0
Recent clinical trials have demonstrated that biologically
TiPS - May 1990 [Vol. 111
19s active antibodies can be raised in humans following administration of synthetic antigens’**3**%.HOWeve;, the level of immunity obtained was not sufficient to allow these vaccines to be used in the clinic. Better response would be &$zI~ if several synthetic pep- to several different antigenie determinants of the natural immunogen - were included. Moreover, synthetic vaccines usuaily contain epitopes that invoke either a T-cell or a B-cell response; ideally they should contain both. They are therefore poor antigens and need to be administered in appropriate vehicles and in conjunction with strong adjuvants. Optimizing these preparations is a key issue for research. References 1 Anderer, F. (1963) Bjoc~~jrn. Eiophys. 2 3
4
5
Actn 71,246-248 Audibert, F. et nl. (1981) Nattire 289, S9%594 Gehoff, E. D., Meloen. R. H. and Barteiing, S. J. (1984) Proc. Nat2 Acod. Sci. USA 81.399-2 Geysen, H. M., Rhodda, S. J. and Mason, T. J. (1985) in Synthetic Peptide> as Antigens (Cibn FourzdationSymposia, vat. 190). pp. 130-149 Langbeheim, H., Amon, R. and Sela, M. (1976) Pmt. Natl Acnd. SC;. USA 73,
6 Amon, R.. Seia. M.. Parant. M. and Chedid, L: (1986) P&c. Nat1 &ad. Sri. USA 77.6769-6772 7 BittIe, J. L. et al. (1982) Nature 298.30-34 8 Brown, F. fl988) Veccine 6.180-182 9 Salk, J. (1987) Nature 327,473-476 10 Fauci. A. (1988) Proc. Nafl Acad. Sci. USA 83.9278-9283 11 Benofsky, J. A. er al. (1988) Natwe 334, 7ofi-708 12 Zajac, B. A., West, D. J., McAleer, W. J. and Scolnick, E. M. (1986) J. Infect. 13 (Suppl. A), 39-45 13 Ada, G. (1389) Natwe 339, 331-332 14 Dalgleish. A. G. et al. (1988) Virology 165,209-21s 15 Mitchell, G. H. (1989) Parasitology 98, S29-S47 16 Nussenzweig, R, Vanderburg, J., Most, X. and Orton, C. (1967) Nature 216. 160-163 17 ZavaIa, F. ef at. (1985) Science 228, 1436-1440 18 E&get, H. M. et ~1. (1988) J. fmmunof. 140,626-633 19 Herrington, 0. A. et ~1. (1987) Nafure 328,257-2.59 20 Good, M. F. et al. (1986) 1. Exp. Med. 164, 655-660 21 Togna, A. R. et al. (1986) J. Immunol. 137, 295~2960 22 Lise, L. D. et al. (1988) Biochem. Biophys. Res. Commun. 153.31-38 23 Sin&a&a, F. et a{: (1988) Eur. 1. Zmmunol. l&633-636 24 Sinigaglia, F. et al. (1988) Nature 336,
.._ ._-
77iL7Ml
2s Margalit, H. et al. (1987) J. ~mmanoZ.138, 2213-2229
26 Patarroyo, M. E. ef al. (1988) Nature 332, 158-161 27 Beachey, E. H., Seyer, J. M., Dale, J. B., Simpson, W. A. and Kang, A. H. (1981) Natwe 292, 457-459 28 Beachey, E. H.. Seyer. J. M. and Dale, 1. B. (1987) 1. Exv. Med. 166.647-656 29 joI&, M:et Ql. 11987) Infect. Immun. 55, 149%1502 __ _ _ 30 Jolivet, M. et al. (1990) Vaccine 8,35-40 31 Hsughter., R. A., En@, R. F., Ostresh, J. M., Hoffman, S. R. and Klipstein, F. A. (198.5) Infect. fmmun. 48,735-740 32 Klipstein, F. A., Engert, R. F. and
Houghten, R. A. (1986) Law&i. 471-473 33 Galen, F-X. ef al. (1987) J. Hypertens. 5 (Suppl. 5). 511-514 34 Bouhnik, J. ef al. (1987) /. Biol. Chem. 262,2913-2918 35 hhrentz, J-A. c’ ai. (1988) Biochemistry 27,4071-4078 36 Stevens, V. C. (1976) in Development of Vuccincs for Ferfirify Regulation IWHD Symposiumf, pp. 93-110, Scriptor 37 Steven +, V. C. (1986) Immutml. Today 7, 369-374 38 Jones, W. R. et al. (1988) Lancef i, 1295-1298
for the of AIDS Erik De Clercq Eradjcafion of human immunodeficiency virus (WV1 fiiam in~ecied cells or organisms has remained an elusive goal. In p~nciple, any of the steps in fhe replicafive cycle could form the basis for ratiana~ design of chemofherapeutic agents, although in practice not all have so far proved amenable to intervention. Here, Erik De Clercq summarizes the strategies that are being employed, focusing primarily on recent promising developments in inhibition of adsorption of I-ilVonto CD4+ T cells, and inhibition of the viral reverse transcriptuse. Although any of the steps in the replicative cycle of HIV (Fig. 1) could, in principie, serve as a site of attack for chemotherapeutic agents’, only the following five have so far been identified and investigated as potential targets: 6 adsorption of the virus to the cell membrane, which depends on the interaction between the viral envelope glycoprotein gp120 and the cellular CD4 receptor (which is present on a subset of T lymphocytes and certain other immune cells); 0 transcription of the viral RNA to proviral DNA by the reverse transcriptase and degradation of the residual RNA by ribonuclease H (RNase H); 6 transactivation of the expression of the viral genes by the regulatory proteins (such as the truns-acting transcriptional activator tat); @ proteolytic cleavage of the viral precursor proteins by the viral E. De CJercq is Professor of Microbiology and Biochemistryat the Faculty of Medicine of the Kntholieke Universifeit Leuven, and I
