- Email: [email protected]
Recombinant vaccines: experimental and applied aspects
Fish & Shellfish Immunology (1999) 9, 361–365 Article ID: fsim.1999·0195 Available online at http://www.idealibrary.com on
Recombinant vaccines: experimental and applied aspects NIELS LORENZEN Danish Veterinary Laboratory, Hangøvej 2, 8200 Århus N, Denmark (Received and accepted 28 January 1999) Development of vaccines for aquaculture fish represent an important applied functional aspect of fish immunology research. Particularly in the case of recombinant vaccines, where a single antigen is usually expected to induce immunity to a specific pathogen, knowledge of mechanisms involved in induction of a protective immune response may become vital. The few recombinant vaccines licensd so far, despite much research during the last decade, illustrate that this is not a straightforward matter. However, as vaccine technology as well as our knowledge of the fish immune system is steadily improved, these fields will open up a number of interesting research objectives of mutual benefit. Recent aspects of recombinant protein vaccines, live recombinant vaccines and DNA vaccines are discussed. 1999 Academic Press
Key words:
gene technology, recombinant protein, live vectors, DNA vaccine, protective mechanisms, safety.
Introduction About ten years ago, recombinant DNA technology was considered to be the most suitable solution for development of vaccines against diseases in fish where traditional technologies had failed. This includes vaccines against many diseases of viral or parasite origin as well as some bacterially induced disorders. However, so far only one vaccine containing recombinant products is commercially available for use in aquaculture (reviewed by Leong et al., 1997). This illustrates that development of such vaccines has generally been less straightforward than first imagined. The potential of the technology is nevertheless steadily increasing, and it is probably a question of when, rather than whether, recombinant vaccines will appear as an e$cient tool for prevention of several important diseases in aquacultured fish. Recombinant DNA technology both allows construction of multivalent vaccines inducing protection against two or more pathogens simultaneously, and also makes it possible to build in adjuvant and/or targeting components. In terms of fish immunology, recombinant vaccine development also includes some interesting possibilities for functional immune response studies. Types of vaccines Research on recombinant vaccines for fish in the sense of products fully or partly based on cloned pathogen genes has so far included: E-mail: [email protected] 1050–4648/99/040361+05 $30.00/0
361
1999 Academic Press
362
N. LORENZEN
(1) Recombinant proteins/antigens expressed in prokaryotic or eucaryotic cells by fermentation under strictly controlled laboratory conditions (Gilmore et al., 1988; Lecocq-Xhonneux et al., 1994; He et al., 1997). (2) Genetically attenuated pathogens (Vaughan et al., 1993). (3) Live but nonpathogenic recombinant microorganisms carrying foreign pathogen genes (Noonan et al., 1995; Zhang & Hanson, 1996) (4) Vaccines based on naked DNA (Anderson et al., 1996; Lorenzen et al., 1998) These vaccine development strategies have all been demonstrated to work in fish to a certain extent, under experimental conditions. Recombinant protein vaccines The immunity induced by recombinant antigens produced by fermentation has often been insu$cient or inconsistent, possibly due to poor antigenicity and/or immunogenicity of the products (Lorenzen & Olesen, 1997; Leong et al., 1997). The need for delivery by injection has furthermore put limitations on the potential applications of such vaccines. At present one licensed vaccine containing recombinant protein as immunogen is available (Christie, 1997). This vaccine is against infectious pancreatic necrosis virus in Atlantic salmon and is based on inclusion of an E. coli expressed recombinant VP2 protein into a traditional antibacterial injection vaccine. Due to the lack of a good challenge model the e#ect of the recombinant component has so far only been demonstrated indirectly through monitoring the antibody response (Frost et al., 1998). In spite of its apparent lack of success, the potential of this ‘traditional’ form of recombinant vaccine should not be overlooked. Recently, it has been demonstrated that oral vaccination with plant-derived recombinant viral protein can induce protective immunity in mice (Modelska et al., 1998); although this is projected for several years in the future, the picture of a fish farmer preparing vaccine-tomatoes in his own backyard may one day turn out to be more than a fantasy. Live recombinant vaccines Live vaccines, either in the form of attenuated pathogens or in the form of microbial vectors carrying the vaccine components as reported for Aeromonas salmonicida (Vaughan et al., 1993; Noonan et al., 1995) potentially induce a stronger immune response than non-replicating products (Marsden et al., 1998) and can eventually be delivered by immersion. Practical application of such vaccines inevitably implies release of recombinant organisms into the environment. Safety concerns both in terms of the vaccinated animals and in terms of environmental aspects is probably the main reason for such vaccines not having received more attention in relation to aquaculture. The perspective of this recombinant vaccine strategy has nevertheless recently been demonstrated in practice in the case of rabies virus. By the use of a recombinant vaccinia virus carrying the gene encoding the rabies virus surface glycoprotein, a bait-vaccination programme including millions of
RECOMBINANT VACCINES: EXPERIMENTAL AND APPLIED ASPECTS
363
vaccine doses has lead to eradication of rabies from wildlife animals in large sylvatic areas in Western Europe (Pastoret & Brochier, 1998).
Genetic vaccines Vaccination with DNA in the form of plasmids carrying pathogen genes under the control of eukaryotic promoters has recently been demonstrated to induce protective immunity to a number of diseases in mammals (Donelly et al., 1997). In fish, high levels of protection against infections by infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV) can be induced by intramuscular injection of viral genes encoding surface glycoproteins (Anderson et al., 1996; Lorenzen et al., 1998). But why does this kind of vaccination work so well? The in situ expression of the vaccine components ensures correct protein folding and thereby circumvents central problems encountered when expressing recombinant proteins in culture. Furthermore, expression of the antigen within the host cells resembles the immune stimulation exerted by a natural virus infection and presumably allows an optimal antigen processing and subsequent MHC- associated presentation of the resulting peptides. These appear to be reasonable answers, but as discussed by Tighe et al. (1998), the real explanation appears less straightforward and far more complicated, including, inter alia, adjuvant e#ects of non-methylated bacterial DNA sequences, alternative route MHC class I priming, etc., at least in the case of mammals. Far less is known about the mechanisms activated by DNA vaccines in fish. But the relative simplicity of the vaccine concept, combined with the very high e$cacy in terms of protection reported, makes genetic immunization an ideal tool for future functional studies of the fish immune system. This was illustrated in a study by Boudinot et al. (1998), where up-regulation of expression of MHC class II and Mx gene expression was observed following injection of a DNA construct mediating expression of the VHSV glycoprotein gene. From the perspective of field application, the potential of DNA vaccines o#ers several desirable features. They stimulate development of long-lasting immunity without apparent side e#ects are noninfectious, stable, and easy and cheap to produce under reproducible quality control measures. Provided practical delivery methods for fish can be developed, genetic immunization may in this way be a future tool not only for immune response studies but also for prevention of disease outbreaks in commercial aquaculture.
Safety Concerning recombinant vaccines in general, all kinds of safety aspects should be carefully considered before use in commercial aquaculture. As pointed out by Traavik (1997), vaccines which include recombinant DNA (i.e. live recombinant vaccines, DNA-vaccines) have a number of theoretical human and environmental safety implications related to the potential spread
364
N. LORENZEN
of recombinant nucleotide sequences both in the organism and in nature. Such aspects need to be addressed not only from a scientific but also from ethical and regulatory points of view. References Anderson, E. D., Mourich, D. V., Fahrenkrug, S., LaPatra, S., Shepard, J. & Leong, J. C. (1996). Genetic immunization of rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus. Molecular Marine Biology and Biotechnology 5, 114–122. Boudinot, P., Blanco, M., de Kinkelin, P. & Benmansour, A. (1998). Combined DNA immunization with the glycoprotein gene of viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus induces double-specific protective immunity and nonspecific response in rainbow trout. Virology 249, 297–306. Christie, K. E. (1997). Immunization with viral antigens: Infectious pancreatic necrosis. In: Fish Vaccinology. R. Gudding, A. Lillehaug, P. J. Midtlyng, F. Brown, eds.). Developments in Biological Standardization 90, 191–200. Donelly, J. J., Ulmer, J. B. & Liu, M. A. (1997). DNA vaccines. Life Sciences 60, 163–172. Frost, P., Börsheim, K. & Endresen, C. (1998). Analysis of the antibody response in Atlantic salmon against VP2 of infectious pancreatic necrosis virus (IPNV). Fish & Shellfish Immunology 8, 447–456. Gilmore, R. D. Jr., Engelking, H. M., Manning, D. S. & Leong, J. C. (1988). Expression in Escherichia coli of an epitope of the glycoprotein of infectious haematopoietic necrosis virus protects against challenge. Bio/Technology 6, 295–300. He, J., Yin, Z., Xu, G., Gong, Z., Lam, T. J. & Sin, Y. M. (1997). Protection of goldfish against Ichthyophthirius multifiliis by immunization with a recombinant vaccine. Aquaculture 158, 1–10. Lecocq-Xhonneux, F., Thiry, M., Dheur, I., Rossius, M., Vanderheijden, N., Martial, J. & de Kinkelin, P. (1994). A recombinant viral haemorrhagic septicaemia virus glycoprotein expressed in insect cells induces protective immunity in rainbow trout. Journal of General Virology 75, 1579–1587. Leong, J. C., Anderson, E., Bootland, L. M., Chiou, P.-W., Johnson, M., Kim, C., Mourich, D. & Trobridge, G. (1997). Fish vaccine antigens produced or delivered by recombinant DNA technologies. In Fish vaccinology (R. Gudding, A. Lillehaug, P. J. Midtlyng & F. Brown, eds). Developments in Biological Standardization 90, 267–277. Lorenzen, N. & Olesen, N. J. (1997). Immunization with viral haemorrhagic septicaemia antigens. In Fish Vaccinology (R. Gudding, A. Lillehaug, P. J. Midtlyng, F. Brown, eds.) Developments in Biological Standardization 90, 201–209. Lorenzen, N., Lorenzen, E., Einer-Jensen, K., Heppell, J., Wu, T. & Davis, H. (1998). Protective immunity to VHS in rainbow trout (Oncorhynchus mykiss, Walbaum) following DNA vaccination. Fish & Shelfish Immunology 8, 261–270. Marsden, M. J., Vaughan, L. M., Fitzpatrick, R. M., Foster, T. J. & Secombes, C. J. (1998). Potency testing of a live, genetically attenuated vaccine for salmonids. Vaccine 16, 1087–1094. Modelska, A., Dietzschold, B., Sleysh, N. Fu, F. Z., Steplewski, K., Hooper, D. C., Koprowski, H. & Yusibov, V. (1998). Immunization against rabies with plantderived antigen. Proceedings of The National Academy of Sciences USA 95, 2481–2485. Noonan, B., Enzmann, P. J. & Trust, T. J. (1995). Recombinant infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus glycoprotein epitopes expressed in Aeromonas salmonicida induce protective immunity in rainbow trout (Oncorhynchus mykiss). Applied and Environmental Microbiology 61, 3586–3591. Pastoret, P. P. & Brochier, B. (1998). Epidemiology and elimination of rabies in western Europe. Veterinary Journal 156, 83–90.
RECOMBINANT VACCINES: EXPERIMENTAL AND APPLIED ASPECTS
365
Tighe, H., Corr, M., Roman, M. & Raz, E. (1998). Gene vaccination: plasmid DNA is more than just a blueprint. Immunology Today 19, 89–97. Traavik, T. (1997). Environmental issues of recombinant vaccines. In Fish Vaccinology (R. Gudding, A. Lillehaug, P. J. Midtlyng, & F. Brown, eds.) Developments in Biological Standardization 90, 381. Vaughan, L. M. Smith, P. R. & Foster, T. J. (1993). An aromatic-dependent mutant of the fish pathogen Aeromonas salmonicida is attenuated in fish and is e#ective as a live vaccine against the salmonid disease furunculosis. Infection and Immunity 61, 2172–2181. Zhang, H. G. & Hanson, L. A. (1996). Recombinant channel catfish virus (Ichtalurid herpesvirus 1) can express foreign genes and induce antibody production against the gene product. Journal of Fish Diseases 19, 121–128.
Recombinant vaccines: experimental and applied aspects NIELS LORENZEN Danish Veterinary Laboratory, Hangøvej 2, 8200 Århus N, Denmark (Received and accepted 28 January 1999) Development of vaccines for aquaculture fish represent an important applied functional aspect of fish immunology research. Particularly in the case of recombinant vaccines, where a single antigen is usually expected to induce immunity to a specific pathogen, knowledge of mechanisms involved in induction of a protective immune response may become vital. The few recombinant vaccines licensd so far, despite much research during the last decade, illustrate that this is not a straightforward matter. However, as vaccine technology as well as our knowledge of the fish immune system is steadily improved, these fields will open up a number of interesting research objectives of mutual benefit. Recent aspects of recombinant protein vaccines, live recombinant vaccines and DNA vaccines are discussed. 1999 Academic Press
Key words:
gene technology, recombinant protein, live vectors, DNA vaccine, protective mechanisms, safety.
Introduction About ten years ago, recombinant DNA technology was considered to be the most suitable solution for development of vaccines against diseases in fish where traditional technologies had failed. This includes vaccines against many diseases of viral or parasite origin as well as some bacterially induced disorders. However, so far only one vaccine containing recombinant products is commercially available for use in aquaculture (reviewed by Leong et al., 1997). This illustrates that development of such vaccines has generally been less straightforward than first imagined. The potential of the technology is nevertheless steadily increasing, and it is probably a question of when, rather than whether, recombinant vaccines will appear as an e$cient tool for prevention of several important diseases in aquacultured fish. Recombinant DNA technology both allows construction of multivalent vaccines inducing protection against two or more pathogens simultaneously, and also makes it possible to build in adjuvant and/or targeting components. In terms of fish immunology, recombinant vaccine development also includes some interesting possibilities for functional immune response studies. Types of vaccines Research on recombinant vaccines for fish in the sense of products fully or partly based on cloned pathogen genes has so far included: E-mail: [email protected] 1050–4648/99/040361+05 $30.00/0
361
1999 Academic Press
362
N. LORENZEN
(1) Recombinant proteins/antigens expressed in prokaryotic or eucaryotic cells by fermentation under strictly controlled laboratory conditions (Gilmore et al., 1988; Lecocq-Xhonneux et al., 1994; He et al., 1997). (2) Genetically attenuated pathogens (Vaughan et al., 1993). (3) Live but nonpathogenic recombinant microorganisms carrying foreign pathogen genes (Noonan et al., 1995; Zhang & Hanson, 1996) (4) Vaccines based on naked DNA (Anderson et al., 1996; Lorenzen et al., 1998) These vaccine development strategies have all been demonstrated to work in fish to a certain extent, under experimental conditions. Recombinant protein vaccines The immunity induced by recombinant antigens produced by fermentation has often been insu$cient or inconsistent, possibly due to poor antigenicity and/or immunogenicity of the products (Lorenzen & Olesen, 1997; Leong et al., 1997). The need for delivery by injection has furthermore put limitations on the potential applications of such vaccines. At present one licensed vaccine containing recombinant protein as immunogen is available (Christie, 1997). This vaccine is against infectious pancreatic necrosis virus in Atlantic salmon and is based on inclusion of an E. coli expressed recombinant VP2 protein into a traditional antibacterial injection vaccine. Due to the lack of a good challenge model the e#ect of the recombinant component has so far only been demonstrated indirectly through monitoring the antibody response (Frost et al., 1998). In spite of its apparent lack of success, the potential of this ‘traditional’ form of recombinant vaccine should not be overlooked. Recently, it has been demonstrated that oral vaccination with plant-derived recombinant viral protein can induce protective immunity in mice (Modelska et al., 1998); although this is projected for several years in the future, the picture of a fish farmer preparing vaccine-tomatoes in his own backyard may one day turn out to be more than a fantasy. Live recombinant vaccines Live vaccines, either in the form of attenuated pathogens or in the form of microbial vectors carrying the vaccine components as reported for Aeromonas salmonicida (Vaughan et al., 1993; Noonan et al., 1995) potentially induce a stronger immune response than non-replicating products (Marsden et al., 1998) and can eventually be delivered by immersion. Practical application of such vaccines inevitably implies release of recombinant organisms into the environment. Safety concerns both in terms of the vaccinated animals and in terms of environmental aspects is probably the main reason for such vaccines not having received more attention in relation to aquaculture. The perspective of this recombinant vaccine strategy has nevertheless recently been demonstrated in practice in the case of rabies virus. By the use of a recombinant vaccinia virus carrying the gene encoding the rabies virus surface glycoprotein, a bait-vaccination programme including millions of
RECOMBINANT VACCINES: EXPERIMENTAL AND APPLIED ASPECTS
363
vaccine doses has lead to eradication of rabies from wildlife animals in large sylvatic areas in Western Europe (Pastoret & Brochier, 1998).
Genetic vaccines Vaccination with DNA in the form of plasmids carrying pathogen genes under the control of eukaryotic promoters has recently been demonstrated to induce protective immunity to a number of diseases in mammals (Donelly et al., 1997). In fish, high levels of protection against infections by infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV) can be induced by intramuscular injection of viral genes encoding surface glycoproteins (Anderson et al., 1996; Lorenzen et al., 1998). But why does this kind of vaccination work so well? The in situ expression of the vaccine components ensures correct protein folding and thereby circumvents central problems encountered when expressing recombinant proteins in culture. Furthermore, expression of the antigen within the host cells resembles the immune stimulation exerted by a natural virus infection and presumably allows an optimal antigen processing and subsequent MHC- associated presentation of the resulting peptides. These appear to be reasonable answers, but as discussed by Tighe et al. (1998), the real explanation appears less straightforward and far more complicated, including, inter alia, adjuvant e#ects of non-methylated bacterial DNA sequences, alternative route MHC class I priming, etc., at least in the case of mammals. Far less is known about the mechanisms activated by DNA vaccines in fish. But the relative simplicity of the vaccine concept, combined with the very high e$cacy in terms of protection reported, makes genetic immunization an ideal tool for future functional studies of the fish immune system. This was illustrated in a study by Boudinot et al. (1998), where up-regulation of expression of MHC class II and Mx gene expression was observed following injection of a DNA construct mediating expression of the VHSV glycoprotein gene. From the perspective of field application, the potential of DNA vaccines o#ers several desirable features. They stimulate development of long-lasting immunity without apparent side e#ects are noninfectious, stable, and easy and cheap to produce under reproducible quality control measures. Provided practical delivery methods for fish can be developed, genetic immunization may in this way be a future tool not only for immune response studies but also for prevention of disease outbreaks in commercial aquaculture.
Safety Concerning recombinant vaccines in general, all kinds of safety aspects should be carefully considered before use in commercial aquaculture. As pointed out by Traavik (1997), vaccines which include recombinant DNA (i.e. live recombinant vaccines, DNA-vaccines) have a number of theoretical human and environmental safety implications related to the potential spread
364
N. LORENZEN
of recombinant nucleotide sequences both in the organism and in nature. Such aspects need to be addressed not only from a scientific but also from ethical and regulatory points of view. References Anderson, E. D., Mourich, D. V., Fahrenkrug, S., LaPatra, S., Shepard, J. & Leong, J. C. (1996). Genetic immunization of rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus. Molecular Marine Biology and Biotechnology 5, 114–122. Boudinot, P., Blanco, M., de Kinkelin, P. & Benmansour, A. (1998). Combined DNA immunization with the glycoprotein gene of viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus induces double-specific protective immunity and nonspecific response in rainbow trout. Virology 249, 297–306. Christie, K. E. (1997). Immunization with viral antigens: Infectious pancreatic necrosis. In: Fish Vaccinology. R. Gudding, A. Lillehaug, P. J. Midtlyng, F. Brown, eds.). Developments in Biological Standardization 90, 191–200. Donelly, J. J., Ulmer, J. B. & Liu, M. A. (1997). DNA vaccines. Life Sciences 60, 163–172. Frost, P., Börsheim, K. & Endresen, C. (1998). Analysis of the antibody response in Atlantic salmon against VP2 of infectious pancreatic necrosis virus (IPNV). Fish & Shellfish Immunology 8, 447–456. Gilmore, R. D. Jr., Engelking, H. M., Manning, D. S. & Leong, J. C. (1988). Expression in Escherichia coli of an epitope of the glycoprotein of infectious haematopoietic necrosis virus protects against challenge. Bio/Technology 6, 295–300. He, J., Yin, Z., Xu, G., Gong, Z., Lam, T. J. & Sin, Y. M. (1997). Protection of goldfish against Ichthyophthirius multifiliis by immunization with a recombinant vaccine. Aquaculture 158, 1–10. Lecocq-Xhonneux, F., Thiry, M., Dheur, I., Rossius, M., Vanderheijden, N., Martial, J. & de Kinkelin, P. (1994). A recombinant viral haemorrhagic septicaemia virus glycoprotein expressed in insect cells induces protective immunity in rainbow trout. Journal of General Virology 75, 1579–1587. Leong, J. C., Anderson, E., Bootland, L. M., Chiou, P.-W., Johnson, M., Kim, C., Mourich, D. & Trobridge, G. (1997). Fish vaccine antigens produced or delivered by recombinant DNA technologies. In Fish vaccinology (R. Gudding, A. Lillehaug, P. J. Midtlyng & F. Brown, eds). Developments in Biological Standardization 90, 267–277. Lorenzen, N. & Olesen, N. J. (1997). Immunization with viral haemorrhagic septicaemia antigens. In Fish Vaccinology (R. Gudding, A. Lillehaug, P. J. Midtlyng, F. Brown, eds.) Developments in Biological Standardization 90, 201–209. Lorenzen, N., Lorenzen, E., Einer-Jensen, K., Heppell, J., Wu, T. & Davis, H. (1998). Protective immunity to VHS in rainbow trout (Oncorhynchus mykiss, Walbaum) following DNA vaccination. Fish & Shelfish Immunology 8, 261–270. Marsden, M. J., Vaughan, L. M., Fitzpatrick, R. M., Foster, T. J. & Secombes, C. J. (1998). Potency testing of a live, genetically attenuated vaccine for salmonids. Vaccine 16, 1087–1094. Modelska, A., Dietzschold, B., Sleysh, N. Fu, F. Z., Steplewski, K., Hooper, D. C., Koprowski, H. & Yusibov, V. (1998). Immunization against rabies with plantderived antigen. Proceedings of The National Academy of Sciences USA 95, 2481–2485. Noonan, B., Enzmann, P. J. & Trust, T. J. (1995). Recombinant infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus glycoprotein epitopes expressed in Aeromonas salmonicida induce protective immunity in rainbow trout (Oncorhynchus mykiss). Applied and Environmental Microbiology 61, 3586–3591. Pastoret, P. P. & Brochier, B. (1998). Epidemiology and elimination of rabies in western Europe. Veterinary Journal 156, 83–90.
RECOMBINANT VACCINES: EXPERIMENTAL AND APPLIED ASPECTS
365
Tighe, H., Corr, M., Roman, M. & Raz, E. (1998). Gene vaccination: plasmid DNA is more than just a blueprint. Immunology Today 19, 89–97. Traavik, T. (1997). Environmental issues of recombinant vaccines. In Fish Vaccinology (R. Gudding, A. Lillehaug, P. J. Midtlyng, & F. Brown, eds.) Developments in Biological Standardization 90, 381. Vaughan, L. M. Smith, P. R. & Foster, T. J. (1993). An aromatic-dependent mutant of the fish pathogen Aeromonas salmonicida is attenuated in fish and is e#ective as a live vaccine against the salmonid disease furunculosis. Infection and Immunity 61, 2172–2181. Zhang, H. G. & Hanson, L. A. (1996). Recombinant channel catfish virus (Ichtalurid herpesvirus 1) can express foreign genes and induce antibody production against the gene product. Journal of Fish Diseases 19, 121–128.