TIBTECH- JANUARY 1991 [Vol. 9]
7
assess this risk injected DNA extracted from transformed Chinese hamster ovary (CHO), cell lines. The DNA was non-tumourigenic even when injected in the same species. The selective breeding of plants and animals both for the serious purpose of human food and the relatively frivolous one of winning competitions has continued since records began. Little harm - to humans - has arisen from this activity. Now genetic engineering promises more rapid and focused improvements in the age-old and economically vital processes of fermentation, cereal production, grox~h of fruit and vegetables and animal
husbandry. All responsible scientists and regulators alike have a duty to explain what they are dning, to demystify the science and provide the reassurance that will certainly be demanded. Man is not 'controlling, subjecting or perverting life'. He has the power to make genetic changes in microorganisms, plants and animals of great potential benefit. This power must be u.sed, and be seen to be used, wisely.
References
MICHAEL GE1SOW
1 Pursel, V. G., Pinkert, C. A., Miller, K. F., Bolt, D. J., Cambell, R. G., Biodigm, 115 Main Street, East Palmiter, R.D., Brinster, R.L. and Bridgeford, Nottingham NG13 8,~H, Hammer, R.D. (1989) Science 244, UK.
Progress towards rabies control Morag Ferguson Although safe and efficacious tissue-culture-derived rabies vaccines are available in developed countries, much of the w o r l d still depends on vaccines d e r i v e d f r o m n e u r a l tissue which w e r e i n t r o d u c e d h a l f a c e n t u r y ago. C o n s i d e r a b l e a d v a n c e s have been m a d e in our understanding of the molecular biology of r a b i e s virus, and genetically engineered recombinant viruses ( v a c c i n i a - r a b i e s v i r u s glycoprotein) have been developed. These may facilitate the control of rabies in some species by o r a l v a c c i n a t i o n c a m p a i g n s . world: in foxes in Europe and in skunks and raccoons in the USA. However, some countries, mainly islands and those with physical barriers at their frontiers, are free from rabies. The majority of cases of human rabies result from dog bites and the control of the dog population is therefore important. In countries where universal immunization of dogs has been introduced, the decline of rabies in man has paralleled the decline of disease in M. Ferguson is at the Division of dogs.
Rabies is a viral infection of the central nervous system and is transmitted by the bite of infected animals. Not all bites by laboratoryproven rabid animals result in the development of rabies since the virus may fail to become established. However, if clinical signs do develop, the disease is almost invariably fatal. Rabies can occur in all mammals and is enzootic in many parts of the
Virology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hefts EN6 3QG, UK.
1281-1284 2 Palmiter, R. D., Brinster, R. L., Manner, R.E., Trumb,~uer, M.E., Rosenfeld, M. G., Birnber, N. C. and Evans, R. M. (1982) Nature 300, 611-615 3 Chen, T. T. and Powers, D. A. (1990) Trends Biotechnm'. 8, 209-215 4 Palmiter, R. D. ai:d Brinster, R. L. (1986) Annu. Rev. Genet. 20, 465-499 5 Biotechnologies and Food: Assuring the Safety of Foods Produced by Genetic Modification, ,International Food Biotechnology Council, Washington DC, USA.)
The structure of rabies virus Rabies virus can be isolated from the b:'ain or saliva of an infected
~) 1991. Elsevier Science Publishers Ltd (UK) 0167 - 9430190/$2.00
animal by intracerebral inoculation of mice. By repeated passage of wild ('street') isolates, the biological properties of the virus become altered so that the incubation period becomes less variable ('fixed'). The rabies virus is a bullet-shaped particle 170 x 75 nm and has the characteristic morphology of the family Rhabdoviridae (Fig. 1). The virus contains a helical nucleocapsid core comprising singlestranded RNA, nucleoprotein, phosphoprotein and the virion transcriptase. This core is surrounded by an envelope which consists of lipids derived from the host-cell plasma membrane. 'Spikes' projecting from the surface of the virus are composed of a glycoprotein which is the major factor responsible for the induction of virus neutralizing antibodies and which confers protection against lethal infection 1. The sequence of the gene encoding the glycoprotein has been determined 2 and this has enabled an analysis of the antigenic structure of the glycoprotein to be carried out 3. Monoclonal antibodies directed against the glycoprotein of rabies virus have been used to select antigenic variants resistant to neutralization and several independent antigenic sites have been mapped on the glycoprotein. Amino acid substitutions at a single residue within one of these sites have been shown to affect pathogenicity 4. The antigenic diversity of strains isolated from different species and at different geographical locations has
8
TIBTECH - JANUARY 1991 [Vol. 9]
•- - F i g . 1
been investigated using monoclonal antibodies directed against both the glycoprotein and nucleoprotein. Such 'street' strains, laboratoryadapted strains and other members of the Lyssavirus group (which, in addition to rabies virus, includes Lagos Bat Virus, Mokola and Duvenhage, the latter two of which can cause illness indistinguishable from rabies in man), can also be characterized using monoclonal antibodies~, 6. Vaccination against rabies In 1685, Louis Fa~[e~ . . . . . . ' ' ' " rabies-infected rabbit spinal cord for the first ~mmunization of a person bitten by a rabid animal. During the following 50 years, phenol-inactivated rabies vaccines derived from nervous tissue of various species were developed and such vaccines are still in use in many developing countries even though they result in an unacceptably high number of neurological side effects. In 1950, rabies virus was adapted to grow in embryonated duck eggs and an inactivated lyophilized vaccine containing infected embryo suspension was developed. Although this vaccine contained little encephalitogenic activity, its high content of avian proteins led to some minor allergic reactions. In addition, duck embryo vaccine was of low potency and had to be administered subcutaneously, in daily doses, for 14-21 days. The most significant advance in the development of vaccines for human use was the adaptation of the Pasteur strain of virus to grow in human diploid ceils. Inactivated vaccines containing virus produced in these cells is highly effective and now in use throughout the world. However this vaccine is expensive to produce and, even if sufficient vaccine were available, people in many developing countries would be unable to afford it. Safe and efficacious vaccines containing inactivated sucrose-gradient-purified virus, grown either on primary chick embryo fibroblast cells or in Vero cells grown in large capacity bioreactors, have been introduced. Mass vaccination of dogs as a method of rabies control began in 1919 using inactivated vaccines of nervous-tissue origin. Although such vaccines are still in use in some
Electron micrograph of negatively stained preparation of rabies virus (family: Rhabdoviridae). The bar represents 100 nm.
countries, cell-culture vaccines containing virus inactivated with ~-propionolactone, or modified live virus strains which are attenuated for the target species are widely used. Rabies vaccines for both human and animal use are currently manufactured using a limited number of laboratory strains of virus. Despite the antigenic variation among 'street' strains and the laboratory strain,~ which are used for the production of vaccine, the vaccines are effective in preventing disease when used prophylactically or for post-exposure immunization. The majority of vaccine failures can be attributed to events during the treatment which the patient received, such as delays in initiating vaccination or inadequate primary wound treatment 7. Immunization studies in mice indicate that rabies vaccines in current use are of reduced effectiveness against certain rabies-related viruses. However, it appears that rabies vaccines will protect against Danish Bat virus, a strain which is enzootic in bats in Northern Europe and which is similar to Duvenhage vim@. Development of new vaccines During the past decade, significant advances have been made in our understanding of the molecular biology of rabies virus 9. Considerable interest has therefore focused on the development of inexpensive subunit
vaccines containing glycoprotein produced by recombinant DNA technology 1°. However, studies of polypeptide expressed in E . c o l i proved disappointing and it appears that the secondary and tertiary structure of the glycoprotein is essential for biological activity. The expression of immunologically reactive rabies glycoprotein in baculovirus has recently been reported and this system could lead to the development of a low-cost subunit vaccine~L Other novel ways of presenting the glycoprotein have been investigated including Iscoms (Immune-stimulating complexesp 2 and immunosomes (viral antigen anchored to preformed liposomesp 3. Both of these approaches were effective in inducing protective neutralizing antibodies in mice. Incorporation of ribonucleoprotein as well as glycoprotein in liposomes resulted in levels of protection similar to that achieved by whole-virus vaccine. In addition, although liposomes containing ribonucleoprotein alone did not confer protection in mice and raccoons following challenge by the intracerebral route, these animals were protected against peripheral intramuscular challenge 14. The mechanism by which these animals are protected is unknown but may involvo T-cell immunity. The use of anti-idiotypic antibodies prepared against murine monoclonal antibodies specific for rabies virus glycoprotein has also been examined, and virus-neutralizing antibodies were obtained following immunization of mice with antiidiotypic antibodies15. Recombinant virus vaccines The use of recombinant virus vaccines in which the rabies glycoprotein is expressed in an infectious vector has also been investigated because presentation of the glycoprotein in its native form is more likely to be achieved by this method. Although neutralizing antibodies have been obtained using an adenovirus-rabies glycoprotein recombinant virus in dogs and mice inoculated by parenteral or oronasal routes 16, the use of vaccinia virus as a vector has considerable potential and has attracted most attention. The main advantages of asing vaccinia as a vector are (1) the |o~r production cost, (2) its ease of administration,
TIBTECH - JANUARY 1991 [Vol. 9]
--Table I
Species in which vaccinia-rabies glycoprotein recombinant virus has been tested a Laboratory and domestic animals
Target species
Mouse
Raccoon
Non-target species European badger
(Procyon Iotor) Syrian hamster
Red fox
Wild boar
Rabbit
Fox (adults and cubs) Striped skunk
Wood mouse
(Vulpes vulpes)
Dog
(Mephitis mephitis) Cat Sheep
(Mustela putorius furo)
(Arvicola terrestis) Common buzzard
(Sue scrofa)
(Buteo buteo)
(Apodermus sylvaticus) Yellow-necked mouse
(Apodermus flavicollis) Bank vole
(Clethrionomys glareolus) Common vole
Ferret
Water vole
(Meles meles)
(Microtus arvalis) Field vole
(Microtus agrestis)
Kestrel
(Falco tinnunculus) Carrion crow
(Corvus corone) Magpie
(Pica pica) Jay
(Garrulus glandarius) Opossum
(Didelphus virginiana)
Cattle a From Refs22 and 23.
(3) the stability of the vaccine and {4) the extensive experience of this virus as a vaccine 17. The use of vaccinia virus as an expression system has been studied extensively and has been reviewed recently TM. The glycoprotein of the EvelynRokitnicki-Abelseth {ERA) strain of rabies virus has been expressed in vaccinia virus; the antigenicity of the recombinant protein was found to be similar to that of native glycoprotein in radio-immunoassays using a large panel of monoclonal antibodies directed against rabies-virus glycoprotein TM. Protection of laboratory animals has been achieved using a va:ciniarabies glycoprotein recombinant virus both as 'live' and as 'inactivated' vaccine 19.2°. Although the use of such viruses is likely to be restricted initially to animals, the potential spread to humans and the consequences of the resultant infection must be considered; a wide range of complications was experienced when this virus was used to vaccinate humans against smallp o x 21. However, mechanisms for the attenuation of vaccinia virus have been investigated and vectors which incorporate attenuating modifications have been constructed 18.2~. Concern has also been expressed that recombination may take place between genetically engineered viruses and wild poxviruses and it has been suggested that potential interactions between engineered viruses and wild viruses present in target
and non-target species in a selection of habitats should be investigated 21.
following oral vaccination was detected in male-female pairs of raccoons, and in an uninoculated female fox which was housed with a particularly aggre'--~i,,e male fox 22.
Immunization routes The use of vaccinia-rabies glycoprotein recombinant virus for the immunization of a wide range of Oral vaccination of wildlife animals by various routes has now The most effective method of been examined 22. In anticipation of reducing rabies infections in the use of such recombinant virus for humans is to reduce the number of the oral vaccination of foxes in infections in animals. In Europe, Europe, this route of administration rabies is maintained mainly within has been extensively investigated in the fox population and, as the use of laboratory studies of both target and poison and trapping failed to control non-target species which may com- the disease, vaccination of foxes was pete with target species for vaccine considered, Some laboratory strains baits {Table 1} 23 . The site of primary are avirulent when inoculated by multiplication of recombinant vi- peripheral routes and are pathogenic ruses in foxes following oral vac- only following intracerebral inocucination has been investigated -~sing lation. In addition, there is a wide the polymerase chain reaction (PCR) variation in the dose of virus retechnique and these studies have quired to induce infection in various shown that virus is present in the species. In 1971, Baer eta]. 25 demontonsils, buccal mucosa and soft strated that the ERA strain, which palate during the 48 hours im- was innocuous for foxes after intramediately following inoculation 24. muscular injection, induced protecAlthough there was initial concern tive levels of virus-neutralizing antithat previous immunization or ex- bodies when placed on their tongues. posure to vaccinia virus might inter- This observation opened up the fere with the response to a second prospect of using oral vaccination inoculation with hybrid viruses, as a means of controlling rabies in studies in rabbits demonstrated that wildlife. Although suitable for the titres of neutralizing antibodies in- oral immunization of foxes, the creased significantly after booster ERA strain is not effective in other species. inoculations 22. The spread of virus between inoculated and uninoculated animals Bait choice Considerable efforts have been has also been extensively studied in many species. In general, horizontal made to develop a bait suitable for transmission has not been observed the administration of the ERA virus although evidence of contact transfer strain as a vaccine. Such baits must
10
be attractive to the target species but sufficiently unattractive as to be rejected by other species, including man. In addition, the target species must not hoard the bait so that the virus is inactivated prior to consumption. A variety of uncontaminated baits were tested in field trials for uptake by the target species before baits containing virus were distributed. The first field trials took place in Switzerland using chicken heads as baits, and the advance of rabies into Alpine valleys was halted by achieving immunity in 60% of foxes 26. Baits consisting of fat and fishmeal (which can be produced in large quantities by machine) were developed in the Federal Republic of Germany (FRG), and trials using these baits have been carried out in Italy, Austria, Belgium, France and Luxembourg as well as the FRG. Recombinant vaccine trials
The first trials involving the use of recombinant vaccinia viruses for the oral immunization of foxes against rabies were carried out in a military zone in Belgium in 198727. After 15 days, 64% of the 250 baits distributed had disappeared or the capsule containing the vaccine had been perforated. However, of 145 small animals trapped in the baited area, only four had eaten baits as determined by uptake of tetracycline which was incorporated into the baits. No adverse effect on wildlife was observed. Because of the small area covered by this trial the efficacy of the vaccine could not be assessed and a larger trial has been carried out in Luxembourg in an area of low human-population density 28. In the seven months following the distribution of the vaccine, 16 out of 26 foxes collected in the vaccination area had had contact with bait and this was considered an encouraging outcome of a single vaccination campaign.
Regulation of oral vaccination campaigns Prior to the .implementation of the first oral vaccination campaigns with live rabies virus vaccines, extensive consultations were co-ordinated by the World Health Organisation (WHO), for scientists to consider the safety and use of live vaccines in the field. Guidelines for the administration of oral vacci-
TIBTECH - JANUARY 1991 [Vol. 9]
nation campaigns were prepared which dealt with information for the public and personnel involved in the campaign, the timetable of events, detailed plans for surveillance of target and non-target species and the collection and testing of specimens 29. The trials with the vaccinia-rabies glycoprotein recombinant virus in Belgium were carried out according to the WHO rules, though additional approval was required because the release of a genetically engineered organism was involved. Final approval was given by the National Council of Health, an advisory body to the Belgian Ministry of Health. After much delay, permission has recently been granted by US health officials in Virginia for a field trial to be carried out on an isolated island off the east coast of the USA 3°. In this trial, the immunogenicity of a live vaccinia-rabies glycoprotein vaccine in raccoons will be assessed. Although many developed countries and the European Commission now have regulations and mechanisms for the approval of the release of genetically engineered organisms, many developing countries have yet to formulate such regulations. A considerable amount of adverse publicity was generated during a trial, conducted by the Pan American Health Organization, which involved the use of vaccinia-rabies glycoprotein recombinant virus in cattle under controlled conditions on a farm in Argentina. It was halted prematurely by the Argentinian government who claimed that appropriate authorization for the trial had not been requested 31.a2. The release of genetically engineered microorganisms is of gr~at concern to scientists, ecologists and the lay public. A globally acceptable regulatory framework is required and, to facilitate this, scientific evidence must be accumulated concerning the properties of DNA and the interaction of microorganisms with their environment. Consideration of such issues has already begun al and hopefully advances in biotechnology will soon enable us to tackle problems such as rabies.
Conclusions The development of safe and effective tissue-culture-derived rabies vaccines has reduced the incidence of neurological side effects associ-
ated with rabies vaccination. It is hoped that reductions in the cost of vaccine through large-scale production or local production facilitated by technology transfer agreements will facilitate increased supplies of such vaccines. This should enable safe tissue-culture-derived vaccines to be available to the populations of developing countries who are at greatest risk of infections. Effective control of fox rabies in Europe by oral immunization using attenuated virus strains has been shown to be possible in trials. Although suitable strains for use in species in which rabies is enzootic in other countries have not been identified, the use of vaccinia-rabies glycoprotein recombinant viruses in oral vaccination campaigns may be feasible. Such recombinant vaccines may eventually be of value in the immunization of domestic animals and pets, but more data on the safety of such vaccines must be obtained before this approach can be evaluated. Vaccinia-rabies glycoprotein recombinants administered orally may also be of use for the immunization of dogs in rural and urban areas of developing countries where many dogs are considered as 'community dogs' and there is no individual who would take responsibility for the administration of conventional vaccines. This approach should reduce the incidence of rabies in the human population of these areas.
References 1 Cox, J. H., Dietzschold, B. and Schneider, L. G. (1977) Infect. lmmun. 16, 754-759 2 Anilionis, A., Wunner, W. H. and Curtis, P. J. (1981) Nature 294, 275-278
3 Lafon, M., Wiktor, T. J. and Macfarlan, R. I. (1983) J. Gen. Virol. 64, 843-851 4 Seif, I., Coulon, P., Rollin, P. E. and Flamand, A. (1985) J. Virol. 53, 926-934 5 Dietzschold, B., Rupprecht, C. E., Tollis, M., Lafon, M., Mattei, J., Wiktor, T. J. and Koprowski, H. (1988) Rev. Infect. Dis. 10 (Suppl.), $785-$798 6 Wiktor, T. J., Flamand, A. and Koprowski, H. (1980) J. Virol. Methods 1, 33-46 7 Anonymous (1988) Lancet i, 917-918 8 Fekadu, M., Shaddock, J. H., Sanderlin, D. W. and Smith, J. S. (1988) Vaccine 6, 533-539
TIBTECH- JANUARY 1991 [Vol. 9]
11
9 Wunner, W. H., Larson, J. K., Dietzschold, B. and Smith, C. L. (1988) Rev. Infect. Dis. 10 (Suppl.), $771-$784 10 Wunner, W. H., Dietzschoid, B., Curtis, P. J. and Wiktor, T. J. (1983) J. Gen. Virol. 64, 1649-1656 11 Prehaud, C., Takehara, K., Flamand, A. and Bishop, D. H. L. (1989) Virology 173, 390-399 12 Morein, B., Sundquist, B., Hoglund, S., Dalsgaard, K. and Osterhaus, A. (1984) Nature 308, 457-460 13 Perrin, P., Thibodeau, L. and Sureau, P. (1985) Vaccine 3, 325-332 14 Dietzschold, B., Wang, H., Rupprecht, C. E., Celis, E., Tollis, M., Ertl, H., Heber-Katz, E. and Koprowski, H. (1987) Proc. Natl Acad. Sci. USA 84, 9165-9169 15 Reagan, K. J., Wunner, W. H., Wiktor, T. J. and Koprowski, H. (1983) J. Virol. 48, 660-666 16 Prevec, L., Campbell, J. B., Christie, B. S., Belbeck, L. and Graham, F. L. (1990) J. Infect. Dis. 161, 27-30 17 Memorandum (1985) Bull. WHO 63, 471-477 18 Miner, J. N. and Hruby, D. E. (1990)
Trends Biotechnol. 8, 20--25 Debbie, J. G. (1971) Am. ]. Epidemiol. 19 Kieny, M. P., Lathe, R., Drillien, R., 93, 487-490 Spehner, D., Skory, S., Schmitt, D., 26 Wandeler, A. I. (1988) in Rabies Wiktor, T., Koprowski, H. and (Campbell, J. B. and Charlton, K. D., Lecocq, J. P. (1984) Nature 312, eds), pp. 365-380, Kluwer Academic 163-166 Publishers 20 Wiktor, T. J., Macfarlan, R. I., Reagan, 27 Pastoret, P. P., Brochier, B., Languet. K. J., Dietzschold, B., Curtis, P. J., B., Thomas, l., Paquot, A., Bauduin, Wunner, W. H., Kieny, M. P., Lathe, B., Kieny, M. P., Lecocq, J. P., De R., Lecocq, J. P., Mackett, M., Moss, Bruyn, J., Costy, F., Antoine, H. and B. and Koprowski, H. (1984) Proc. Desmettre, P. (1988) Vet. Rec. 123, Natl Acad. Sci. USA 81, 7194--7198 481-483 21 Kaplan, C. (1989) Arch. Virol. 106, 28 Brochier, B., Thomas, I., Bauduin, B., 127-139 Leveau, T., Pastoret, P. P., Languet, 22 Rupprecht, C. E. and Kieny, M. P. B., Chappuis, G., Desmettre, P., (1988) in Rabies (Campbell, J. B. and Blancou, J. and Artois, M. (1990) Charlton, K. M., eds), pp. 335-364, Vaccine 8, 101-104 Ki~wer Academic Publishers 29 Debbie, J. G. ahd Bogel, K. (1988) 23 Brow:bier, B., Blancou, J., Thomas, I., Rev. Infect. Dis. 10 (Suppl.), Languet, B., Artois, M., Kieny, M. P., $670-$671 Lecocq, ~. P., Costy, F., Desmettre, P., 30 Anonymous (1990} Science 249, 982 Chappuis, G. and Pastoret, P. P. 31 Sussman, M., Collins, C. H., Skinner, (1989) J. W~Idl. Dis. 25, 540-547 F. A. and Stewart-Tull, D. E. (eds) 24 Thomas, I., B~ochier, B., Languet, B., (1988) Proceedings of the First InterBlancou, J., Peharpre, D., Kieny, national Conference on the Release M. P., Desmettre, P., Chappuis, G. of Genetically Engineered Microand Pastoret, P. P. (1990) J. Gen. organisms, Academic Press Virol. 71, 32-42 32 Koprowski, H. (1988) Rev. Infect. Dis. 25 Baer, G. M. Abels,eth, M. K. and 10 (Suppl.), $810-$813
Extending the chemistry of enzymes and abzymes Donald Hilvert Selective chemical modification can be used to create novel proteins, particularly enzymes and antibodies, with altered specificRies and catalytic activities in vitro. Modification strategies now being developed should soon yield a wide spectrum of novel biomolecules whose activities are optimized for specific industrial processes or therapeutic applications. Post-translational modification confers a number of advantageous properties to proteins in vivo 1, Chemical crosslinking of amino acid side chains, for example, can enhance the stability and overall structural
D. Hilvert is at the Departments of Chemistry and Molecular Biology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA.
integrity of these molecules. Selective chemical reactions can also be exploited to increase the proteolytic resistance or to alter the solubility and viscosity profiles of individual proteins. More generally, chemical modification of proteins represents a powerful tool for altering signal transduction mechanisms and controlling biological function and chemical reactivity within the cell. Chemical modification is also a valuable strategy for altering and
(~ 1991, Elsevier Science Publishers Ltd (UK) 0167 - 9430/90152.00
extending the activities of proteins in vitro 2. Although enzymes are being used increasingly in chemistry and biology, their properties are not always optimal. Covalent chemical modification of specific functional groups can increase their stability, improve their solubility, mask antigenicity, alter patterns of inhibition and activation, and change pH optima or substrate specificity. Taken a step further, these methods enable entirely ,.Jew enzymatic activities to be engir,,ee~ed into naturally occurring proteins via post-translational modification. Given our limited ability to construct large, functional proteins from their constituent amino acids, existing enzymes and antibodies are valuable starting points for the p:eparation of new active sites. Selective chemical reactions can be used, for example, to introduce nonnatural amino acids or catalytic cofactors directly into pre-existing protein binding pockets. If properly designed, the resulting hybrid molecules should combine the chemical properties of the prosthetic group with the binding specificity of the template protein. Chemical mutation of protein active sites in this way complements and extends other
7
assess this risk injected DNA extracted from transformed Chinese hamster ovary (CHO), cell lines. The DNA was non-tumourigenic even when injected in the same species. The selective breeding of plants and animals both for the serious purpose of human food and the relatively frivolous one of winning competitions has continued since records began. Little harm - to humans - has arisen from this activity. Now genetic engineering promises more rapid and focused improvements in the age-old and economically vital processes of fermentation, cereal production, grox~h of fruit and vegetables and animal
husbandry. All responsible scientists and regulators alike have a duty to explain what they are dning, to demystify the science and provide the reassurance that will certainly be demanded. Man is not 'controlling, subjecting or perverting life'. He has the power to make genetic changes in microorganisms, plants and animals of great potential benefit. This power must be u.sed, and be seen to be used, wisely.
References
MICHAEL GE1SOW
1 Pursel, V. G., Pinkert, C. A., Miller, K. F., Bolt, D. J., Cambell, R. G., Biodigm, 115 Main Street, East Palmiter, R.D., Brinster, R.L. and Bridgeford, Nottingham NG13 8,~H, Hammer, R.D. (1989) Science 244, UK.
Progress towards rabies control Morag Ferguson Although safe and efficacious tissue-culture-derived rabies vaccines are available in developed countries, much of the w o r l d still depends on vaccines d e r i v e d f r o m n e u r a l tissue which w e r e i n t r o d u c e d h a l f a c e n t u r y ago. C o n s i d e r a b l e a d v a n c e s have been m a d e in our understanding of the molecular biology of r a b i e s virus, and genetically engineered recombinant viruses ( v a c c i n i a - r a b i e s v i r u s glycoprotein) have been developed. These may facilitate the control of rabies in some species by o r a l v a c c i n a t i o n c a m p a i g n s . world: in foxes in Europe and in skunks and raccoons in the USA. However, some countries, mainly islands and those with physical barriers at their frontiers, are free from rabies. The majority of cases of human rabies result from dog bites and the control of the dog population is therefore important. In countries where universal immunization of dogs has been introduced, the decline of rabies in man has paralleled the decline of disease in M. Ferguson is at the Division of dogs.
Rabies is a viral infection of the central nervous system and is transmitted by the bite of infected animals. Not all bites by laboratoryproven rabid animals result in the development of rabies since the virus may fail to become established. However, if clinical signs do develop, the disease is almost invariably fatal. Rabies can occur in all mammals and is enzootic in many parts of the
Virology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hefts EN6 3QG, UK.
1281-1284 2 Palmiter, R. D., Brinster, R. L., Manner, R.E., Trumb,~uer, M.E., Rosenfeld, M. G., Birnber, N. C. and Evans, R. M. (1982) Nature 300, 611-615 3 Chen, T. T. and Powers, D. A. (1990) Trends Biotechnm'. 8, 209-215 4 Palmiter, R. D. ai:d Brinster, R. L. (1986) Annu. Rev. Genet. 20, 465-499 5 Biotechnologies and Food: Assuring the Safety of Foods Produced by Genetic Modification, ,International Food Biotechnology Council, Washington DC, USA.)
The structure of rabies virus Rabies virus can be isolated from the b:'ain or saliva of an infected
~) 1991. Elsevier Science Publishers Ltd (UK) 0167 - 9430190/$2.00
animal by intracerebral inoculation of mice. By repeated passage of wild ('street') isolates, the biological properties of the virus become altered so that the incubation period becomes less variable ('fixed'). The rabies virus is a bullet-shaped particle 170 x 75 nm and has the characteristic morphology of the family Rhabdoviridae (Fig. 1). The virus contains a helical nucleocapsid core comprising singlestranded RNA, nucleoprotein, phosphoprotein and the virion transcriptase. This core is surrounded by an envelope which consists of lipids derived from the host-cell plasma membrane. 'Spikes' projecting from the surface of the virus are composed of a glycoprotein which is the major factor responsible for the induction of virus neutralizing antibodies and which confers protection against lethal infection 1. The sequence of the gene encoding the glycoprotein has been determined 2 and this has enabled an analysis of the antigenic structure of the glycoprotein to be carried out 3. Monoclonal antibodies directed against the glycoprotein of rabies virus have been used to select antigenic variants resistant to neutralization and several independent antigenic sites have been mapped on the glycoprotein. Amino acid substitutions at a single residue within one of these sites have been shown to affect pathogenicity 4. The antigenic diversity of strains isolated from different species and at different geographical locations has
8
TIBTECH - JANUARY 1991 [Vol. 9]
•- - F i g . 1
been investigated using monoclonal antibodies directed against both the glycoprotein and nucleoprotein. Such 'street' strains, laboratoryadapted strains and other members of the Lyssavirus group (which, in addition to rabies virus, includes Lagos Bat Virus, Mokola and Duvenhage, the latter two of which can cause illness indistinguishable from rabies in man), can also be characterized using monoclonal antibodies~, 6. Vaccination against rabies In 1685, Louis Fa~[e~ . . . . . . ' ' ' " rabies-infected rabbit spinal cord for the first ~mmunization of a person bitten by a rabid animal. During the following 50 years, phenol-inactivated rabies vaccines derived from nervous tissue of various species were developed and such vaccines are still in use in many developing countries even though they result in an unacceptably high number of neurological side effects. In 1950, rabies virus was adapted to grow in embryonated duck eggs and an inactivated lyophilized vaccine containing infected embryo suspension was developed. Although this vaccine contained little encephalitogenic activity, its high content of avian proteins led to some minor allergic reactions. In addition, duck embryo vaccine was of low potency and had to be administered subcutaneously, in daily doses, for 14-21 days. The most significant advance in the development of vaccines for human use was the adaptation of the Pasteur strain of virus to grow in human diploid ceils. Inactivated vaccines containing virus produced in these cells is highly effective and now in use throughout the world. However this vaccine is expensive to produce and, even if sufficient vaccine were available, people in many developing countries would be unable to afford it. Safe and efficacious vaccines containing inactivated sucrose-gradient-purified virus, grown either on primary chick embryo fibroblast cells or in Vero cells grown in large capacity bioreactors, have been introduced. Mass vaccination of dogs as a method of rabies control began in 1919 using inactivated vaccines of nervous-tissue origin. Although such vaccines are still in use in some
Electron micrograph of negatively stained preparation of rabies virus (family: Rhabdoviridae). The bar represents 100 nm.
countries, cell-culture vaccines containing virus inactivated with ~-propionolactone, or modified live virus strains which are attenuated for the target species are widely used. Rabies vaccines for both human and animal use are currently manufactured using a limited number of laboratory strains of virus. Despite the antigenic variation among 'street' strains and the laboratory strain,~ which are used for the production of vaccine, the vaccines are effective in preventing disease when used prophylactically or for post-exposure immunization. The majority of vaccine failures can be attributed to events during the treatment which the patient received, such as delays in initiating vaccination or inadequate primary wound treatment 7. Immunization studies in mice indicate that rabies vaccines in current use are of reduced effectiveness against certain rabies-related viruses. However, it appears that rabies vaccines will protect against Danish Bat virus, a strain which is enzootic in bats in Northern Europe and which is similar to Duvenhage vim@. Development of new vaccines During the past decade, significant advances have been made in our understanding of the molecular biology of rabies virus 9. Considerable interest has therefore focused on the development of inexpensive subunit
vaccines containing glycoprotein produced by recombinant DNA technology 1°. However, studies of polypeptide expressed in E . c o l i proved disappointing and it appears that the secondary and tertiary structure of the glycoprotein is essential for biological activity. The expression of immunologically reactive rabies glycoprotein in baculovirus has recently been reported and this system could lead to the development of a low-cost subunit vaccine~L Other novel ways of presenting the glycoprotein have been investigated including Iscoms (Immune-stimulating complexesp 2 and immunosomes (viral antigen anchored to preformed liposomesp 3. Both of these approaches were effective in inducing protective neutralizing antibodies in mice. Incorporation of ribonucleoprotein as well as glycoprotein in liposomes resulted in levels of protection similar to that achieved by whole-virus vaccine. In addition, although liposomes containing ribonucleoprotein alone did not confer protection in mice and raccoons following challenge by the intracerebral route, these animals were protected against peripheral intramuscular challenge 14. The mechanism by which these animals are protected is unknown but may involvo T-cell immunity. The use of anti-idiotypic antibodies prepared against murine monoclonal antibodies specific for rabies virus glycoprotein has also been examined, and virus-neutralizing antibodies were obtained following immunization of mice with antiidiotypic antibodies15. Recombinant virus vaccines The use of recombinant virus vaccines in which the rabies glycoprotein is expressed in an infectious vector has also been investigated because presentation of the glycoprotein in its native form is more likely to be achieved by this method. Although neutralizing antibodies have been obtained using an adenovirus-rabies glycoprotein recombinant virus in dogs and mice inoculated by parenteral or oronasal routes 16, the use of vaccinia virus as a vector has considerable potential and has attracted most attention. The main advantages of asing vaccinia as a vector are (1) the |o~r production cost, (2) its ease of administration,
TIBTECH - JANUARY 1991 [Vol. 9]
--Table I
Species in which vaccinia-rabies glycoprotein recombinant virus has been tested a Laboratory and domestic animals
Target species
Mouse
Raccoon
Non-target species European badger
(Procyon Iotor) Syrian hamster
Red fox
Wild boar
Rabbit
Fox (adults and cubs) Striped skunk
Wood mouse
(Vulpes vulpes)
Dog
(Mephitis mephitis) Cat Sheep
(Mustela putorius furo)
(Arvicola terrestis) Common buzzard
(Sue scrofa)
(Buteo buteo)
(Apodermus sylvaticus) Yellow-necked mouse
(Apodermus flavicollis) Bank vole
(Clethrionomys glareolus) Common vole
Ferret
Water vole
(Meles meles)
(Microtus arvalis) Field vole
(Microtus agrestis)
Kestrel
(Falco tinnunculus) Carrion crow
(Corvus corone) Magpie
(Pica pica) Jay
(Garrulus glandarius) Opossum
(Didelphus virginiana)
Cattle a From Refs22 and 23.
(3) the stability of the vaccine and {4) the extensive experience of this virus as a vaccine 17. The use of vaccinia virus as an expression system has been studied extensively and has been reviewed recently TM. The glycoprotein of the EvelynRokitnicki-Abelseth {ERA) strain of rabies virus has been expressed in vaccinia virus; the antigenicity of the recombinant protein was found to be similar to that of native glycoprotein in radio-immunoassays using a large panel of monoclonal antibodies directed against rabies-virus glycoprotein TM. Protection of laboratory animals has been achieved using a va:ciniarabies glycoprotein recombinant virus both as 'live' and as 'inactivated' vaccine 19.2°. Although the use of such viruses is likely to be restricted initially to animals, the potential spread to humans and the consequences of the resultant infection must be considered; a wide range of complications was experienced when this virus was used to vaccinate humans against smallp o x 21. However, mechanisms for the attenuation of vaccinia virus have been investigated and vectors which incorporate attenuating modifications have been constructed 18.2~. Concern has also been expressed that recombination may take place between genetically engineered viruses and wild poxviruses and it has been suggested that potential interactions between engineered viruses and wild viruses present in target
and non-target species in a selection of habitats should be investigated 21.
following oral vaccination was detected in male-female pairs of raccoons, and in an uninoculated female fox which was housed with a particularly aggre'--~i,,e male fox 22.
Immunization routes The use of vaccinia-rabies glycoprotein recombinant virus for the immunization of a wide range of Oral vaccination of wildlife animals by various routes has now The most effective method of been examined 22. In anticipation of reducing rabies infections in the use of such recombinant virus for humans is to reduce the number of the oral vaccination of foxes in infections in animals. In Europe, Europe, this route of administration rabies is maintained mainly within has been extensively investigated in the fox population and, as the use of laboratory studies of both target and poison and trapping failed to control non-target species which may com- the disease, vaccination of foxes was pete with target species for vaccine considered, Some laboratory strains baits {Table 1} 23 . The site of primary are avirulent when inoculated by multiplication of recombinant vi- peripheral routes and are pathogenic ruses in foxes following oral vac- only following intracerebral inocucination has been investigated -~sing lation. In addition, there is a wide the polymerase chain reaction (PCR) variation in the dose of virus retechnique and these studies have quired to induce infection in various shown that virus is present in the species. In 1971, Baer eta]. 25 demontonsils, buccal mucosa and soft strated that the ERA strain, which palate during the 48 hours im- was innocuous for foxes after intramediately following inoculation 24. muscular injection, induced protecAlthough there was initial concern tive levels of virus-neutralizing antithat previous immunization or ex- bodies when placed on their tongues. posure to vaccinia virus might inter- This observation opened up the fere with the response to a second prospect of using oral vaccination inoculation with hybrid viruses, as a means of controlling rabies in studies in rabbits demonstrated that wildlife. Although suitable for the titres of neutralizing antibodies in- oral immunization of foxes, the creased significantly after booster ERA strain is not effective in other species. inoculations 22. The spread of virus between inoculated and uninoculated animals Bait choice Considerable efforts have been has also been extensively studied in many species. In general, horizontal made to develop a bait suitable for transmission has not been observed the administration of the ERA virus although evidence of contact transfer strain as a vaccine. Such baits must
10
be attractive to the target species but sufficiently unattractive as to be rejected by other species, including man. In addition, the target species must not hoard the bait so that the virus is inactivated prior to consumption. A variety of uncontaminated baits were tested in field trials for uptake by the target species before baits containing virus were distributed. The first field trials took place in Switzerland using chicken heads as baits, and the advance of rabies into Alpine valleys was halted by achieving immunity in 60% of foxes 26. Baits consisting of fat and fishmeal (which can be produced in large quantities by machine) were developed in the Federal Republic of Germany (FRG), and trials using these baits have been carried out in Italy, Austria, Belgium, France and Luxembourg as well as the FRG. Recombinant vaccine trials
The first trials involving the use of recombinant vaccinia viruses for the oral immunization of foxes against rabies were carried out in a military zone in Belgium in 198727. After 15 days, 64% of the 250 baits distributed had disappeared or the capsule containing the vaccine had been perforated. However, of 145 small animals trapped in the baited area, only four had eaten baits as determined by uptake of tetracycline which was incorporated into the baits. No adverse effect on wildlife was observed. Because of the small area covered by this trial the efficacy of the vaccine could not be assessed and a larger trial has been carried out in Luxembourg in an area of low human-population density 28. In the seven months following the distribution of the vaccine, 16 out of 26 foxes collected in the vaccination area had had contact with bait and this was considered an encouraging outcome of a single vaccination campaign.
Regulation of oral vaccination campaigns Prior to the .implementation of the first oral vaccination campaigns with live rabies virus vaccines, extensive consultations were co-ordinated by the World Health Organisation (WHO), for scientists to consider the safety and use of live vaccines in the field. Guidelines for the administration of oral vacci-
TIBTECH - JANUARY 1991 [Vol. 9]
nation campaigns were prepared which dealt with information for the public and personnel involved in the campaign, the timetable of events, detailed plans for surveillance of target and non-target species and the collection and testing of specimens 29. The trials with the vaccinia-rabies glycoprotein recombinant virus in Belgium were carried out according to the WHO rules, though additional approval was required because the release of a genetically engineered organism was involved. Final approval was given by the National Council of Health, an advisory body to the Belgian Ministry of Health. After much delay, permission has recently been granted by US health officials in Virginia for a field trial to be carried out on an isolated island off the east coast of the USA 3°. In this trial, the immunogenicity of a live vaccinia-rabies glycoprotein vaccine in raccoons will be assessed. Although many developed countries and the European Commission now have regulations and mechanisms for the approval of the release of genetically engineered organisms, many developing countries have yet to formulate such regulations. A considerable amount of adverse publicity was generated during a trial, conducted by the Pan American Health Organization, which involved the use of vaccinia-rabies glycoprotein recombinant virus in cattle under controlled conditions on a farm in Argentina. It was halted prematurely by the Argentinian government who claimed that appropriate authorization for the trial had not been requested 31.a2. The release of genetically engineered microorganisms is of gr~at concern to scientists, ecologists and the lay public. A globally acceptable regulatory framework is required and, to facilitate this, scientific evidence must be accumulated concerning the properties of DNA and the interaction of microorganisms with their environment. Consideration of such issues has already begun al and hopefully advances in biotechnology will soon enable us to tackle problems such as rabies.
Conclusions The development of safe and effective tissue-culture-derived rabies vaccines has reduced the incidence of neurological side effects associ-
ated with rabies vaccination. It is hoped that reductions in the cost of vaccine through large-scale production or local production facilitated by technology transfer agreements will facilitate increased supplies of such vaccines. This should enable safe tissue-culture-derived vaccines to be available to the populations of developing countries who are at greatest risk of infections. Effective control of fox rabies in Europe by oral immunization using attenuated virus strains has been shown to be possible in trials. Although suitable strains for use in species in which rabies is enzootic in other countries have not been identified, the use of vaccinia-rabies glycoprotein recombinant viruses in oral vaccination campaigns may be feasible. Such recombinant vaccines may eventually be of value in the immunization of domestic animals and pets, but more data on the safety of such vaccines must be obtained before this approach can be evaluated. Vaccinia-rabies glycoprotein recombinants administered orally may also be of use for the immunization of dogs in rural and urban areas of developing countries where many dogs are considered as 'community dogs' and there is no individual who would take responsibility for the administration of conventional vaccines. This approach should reduce the incidence of rabies in the human population of these areas.
References 1 Cox, J. H., Dietzschold, B. and Schneider, L. G. (1977) Infect. lmmun. 16, 754-759 2 Anilionis, A., Wunner, W. H. and Curtis, P. J. (1981) Nature 294, 275-278
3 Lafon, M., Wiktor, T. J. and Macfarlan, R. I. (1983) J. Gen. Virol. 64, 843-851 4 Seif, I., Coulon, P., Rollin, P. E. and Flamand, A. (1985) J. Virol. 53, 926-934 5 Dietzschold, B., Rupprecht, C. E., Tollis, M., Lafon, M., Mattei, J., Wiktor, T. J. and Koprowski, H. (1988) Rev. Infect. Dis. 10 (Suppl.), $785-$798 6 Wiktor, T. J., Flamand, A. and Koprowski, H. (1980) J. Virol. Methods 1, 33-46 7 Anonymous (1988) Lancet i, 917-918 8 Fekadu, M., Shaddock, J. H., Sanderlin, D. W. and Smith, J. S. (1988) Vaccine 6, 533-539
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9 Wunner, W. H., Larson, J. K., Dietzschold, B. and Smith, C. L. (1988) Rev. Infect. Dis. 10 (Suppl.), $771-$784 10 Wunner, W. H., Dietzschoid, B., Curtis, P. J. and Wiktor, T. J. (1983) J. Gen. Virol. 64, 1649-1656 11 Prehaud, C., Takehara, K., Flamand, A. and Bishop, D. H. L. (1989) Virology 173, 390-399 12 Morein, B., Sundquist, B., Hoglund, S., Dalsgaard, K. and Osterhaus, A. (1984) Nature 308, 457-460 13 Perrin, P., Thibodeau, L. and Sureau, P. (1985) Vaccine 3, 325-332 14 Dietzschold, B., Wang, H., Rupprecht, C. E., Celis, E., Tollis, M., Ertl, H., Heber-Katz, E. and Koprowski, H. (1987) Proc. Natl Acad. Sci. USA 84, 9165-9169 15 Reagan, K. J., Wunner, W. H., Wiktor, T. J. and Koprowski, H. (1983) J. Virol. 48, 660-666 16 Prevec, L., Campbell, J. B., Christie, B. S., Belbeck, L. and Graham, F. L. (1990) J. Infect. Dis. 161, 27-30 17 Memorandum (1985) Bull. WHO 63, 471-477 18 Miner, J. N. and Hruby, D. E. (1990)
Trends Biotechnol. 8, 20--25 Debbie, J. G. (1971) Am. ]. Epidemiol. 19 Kieny, M. P., Lathe, R., Drillien, R., 93, 487-490 Spehner, D., Skory, S., Schmitt, D., 26 Wandeler, A. I. (1988) in Rabies Wiktor, T., Koprowski, H. and (Campbell, J. B. and Charlton, K. D., Lecocq, J. P. (1984) Nature 312, eds), pp. 365-380, Kluwer Academic 163-166 Publishers 20 Wiktor, T. J., Macfarlan, R. I., Reagan, 27 Pastoret, P. P., Brochier, B., Languet. K. J., Dietzschold, B., Curtis, P. J., B., Thomas, l., Paquot, A., Bauduin, Wunner, W. H., Kieny, M. P., Lathe, B., Kieny, M. P., Lecocq, J. P., De R., Lecocq, J. P., Mackett, M., Moss, Bruyn, J., Costy, F., Antoine, H. and B. and Koprowski, H. (1984) Proc. Desmettre, P. (1988) Vet. Rec. 123, Natl Acad. Sci. USA 81, 7194--7198 481-483 21 Kaplan, C. (1989) Arch. Virol. 106, 28 Brochier, B., Thomas, I., Bauduin, B., 127-139 Leveau, T., Pastoret, P. P., Languet, 22 Rupprecht, C. E. and Kieny, M. P. B., Chappuis, G., Desmettre, P., (1988) in Rabies (Campbell, J. B. and Blancou, J. and Artois, M. (1990) Charlton, K. M., eds), pp. 335-364, Vaccine 8, 101-104 Ki~wer Academic Publishers 29 Debbie, J. G. ahd Bogel, K. (1988) 23 Brow:bier, B., Blancou, J., Thomas, I., Rev. Infect. Dis. 10 (Suppl.), Languet, B., Artois, M., Kieny, M. P., $670-$671 Lecocq, ~. P., Costy, F., Desmettre, P., 30 Anonymous (1990} Science 249, 982 Chappuis, G. and Pastoret, P. P. 31 Sussman, M., Collins, C. H., Skinner, (1989) J. W~Idl. Dis. 25, 540-547 F. A. and Stewart-Tull, D. E. (eds) 24 Thomas, I., B~ochier, B., Languet, B., (1988) Proceedings of the First InterBlancou, J., Peharpre, D., Kieny, national Conference on the Release M. P., Desmettre, P., Chappuis, G. of Genetically Engineered Microand Pastoret, P. P. (1990) J. Gen. organisms, Academic Press Virol. 71, 32-42 32 Koprowski, H. (1988) Rev. Infect. Dis. 25 Baer, G. M. Abels,eth, M. K. and 10 (Suppl.), $810-$813
Extending the chemistry of enzymes and abzymes Donald Hilvert Selective chemical modification can be used to create novel proteins, particularly enzymes and antibodies, with altered specificRies and catalytic activities in vitro. Modification strategies now being developed should soon yield a wide spectrum of novel biomolecules whose activities are optimized for specific industrial processes or therapeutic applications. Post-translational modification confers a number of advantageous properties to proteins in vivo 1, Chemical crosslinking of amino acid side chains, for example, can enhance the stability and overall structural
D. Hilvert is at the Departments of Chemistry and Molecular Biology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037, USA.
integrity of these molecules. Selective chemical reactions can also be exploited to increase the proteolytic resistance or to alter the solubility and viscosity profiles of individual proteins. More generally, chemical modification of proteins represents a powerful tool for altering signal transduction mechanisms and controlling biological function and chemical reactivity within the cell. Chemical modification is also a valuable strategy for altering and
(~ 1991, Elsevier Science Publishers Ltd (UK) 0167 - 9430/90152.00
extending the activities of proteins in vitro 2. Although enzymes are being used increasingly in chemistry and biology, their properties are not always optimal. Covalent chemical modification of specific functional groups can increase their stability, improve their solubility, mask antigenicity, alter patterns of inhibition and activation, and change pH optima or substrate specificity. Taken a step further, these methods enable entirely ,.Jew enzymatic activities to be engir,,ee~ed into naturally occurring proteins via post-translational modification. Given our limited ability to construct large, functional proteins from their constituent amino acids, existing enzymes and antibodies are valuable starting points for the p:eparation of new active sites. Selective chemical reactions can be used, for example, to introduce nonnatural amino acids or catalytic cofactors directly into pre-existing protein binding pockets. If properly designed, the resulting hybrid molecules should combine the chemical properties of the prosthetic group with the binding specificity of the template protein. Chemical mutation of protein active sites in this way complements and extends other
