Short Papers. Potential use of non-replicating vectors as recombinant vaccines Derrick Baxby* and Enzo Paoletti *'~; Avipoxviruses, members of the Poxvirus family, are naturally restricted in that productive replication takes place only in avian species. Recent work has described the construction of Avipox recombinants usingfowlpox and canarypox viruses. Preparation of recombinant fowlpox viruses which express immunogens from avian pathogens and successful vaccination of poultry have been reported. Recombinant fowlpox and canarypox viruses which express immunogens from mammalian pathogens have also been described and have been demonstrated to provide protective immunity on inoculation in non-avian species. This is a surprisin9 result. Such non-replicatin9 expression vectors provide the possibility of developin9 safe, effective vaccines which combine the advantages of killed and live vaccines. Keywords: Avipoxvirus ; recombinant ; mammals ; immunity
INTRODUCTION The introduction of recombinant vaccinia virus in 19821 provided a new strategy for vaccine development. In addition to providing a magnificent research tool, particularly to address questions in immunology 2, a large number of recombinant vaccinia viruses which express immunogens of both veterinary and human pathogens have been described 3. Indeed, measurable progress in the application of recombinant vaccinia virus vaccines has been demonstrated. Extensive field testing of a recombinant vaccinia virus expressing the rabies glycoprotein has been carried out in Europe and to a lesser extent in North America 4"5. These studies have demonstrated both safety and efficacy in targeted intervention against wildlife rabies. Studies in humans utilizing a vaccinia recombinant expressing immunogens from human immunodeficiency virus have also been initiated in the United States 6. The basic technologies used to construct vaccinia virus recombinants have been modified and extended to other members of the Poxvirus family, particularly to members of the Avipox genus. In contrast to vaccinia virus which has a broad host range in terms of replication in susceptible species, members of the Avipox genus are natu'r~lly restricted in that productive replication takes place only in susceptible avian species. No productive replication of avipox virus in non-avian species has been reported and despite extensive field use of attenuated avipox vaccines in poultry and other avians, no adverse effects in humans, other species or to the environment have been documented. Recent reports v's have described the construction of *Department of Medical Microbiology, Duncan Building, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK. tVirogenetics Corporation, 465 Jordan Road, Rensselaer Technology Park, Troy, New York 12180, USA. ++To whom correspondence should be addressed. (Received 13 May 1991 ; revised 12 August 1991 ; accepted 26 August 1991) 0264410x/92/01000802 © 1992 Butterworth-Heinemann Ltd 8
Vaccine, Vol. 10, Issue 1, 1992
recombinant fowlpox viruses which express specific immunogens from pathogens of poultry. These recombinant fowlpox viruses demonstrated clear utility as potential vaccines for avian influenza 7 and Newcastle disease virus infection 9'1°. In attempts to determine the molecular basis of the natural restriction of Avipox viruses in terms of productive replication to avian species, fowlpox virus recombinants were constructed with a reporter gene derived from a mammalian pathogen. Hence, recombinant fowlpox viruses were constructed to express the rabies glycoprotein. These fowlpox virus recombinants were shown to replicate productively in cell cultures derived from susceptible avian species and to express faithfully the rabies glycoprotein in both susceptible avian cell cultures as well as in cell cultures derived from non-avian species. Significantly, the fowlpox virus recombinants did not either replicate productively or become adapted for replication in mammalian cell culture 11-15. These latter results support the known natural history of the host restriction of avipoxviruses. Surprisingly, when the fowlpox virus recombinant expressiiag the rabies glycoprotein was inoculated into non-avian species the animals seroconverted to the extrinsic rabies immunogen, produced rabies virus neutralizing antibodies and, significantly, induced an immune response which was shown to protect against a lethal rabies virus challenge in mice, cats and dogs 11-~ 5 Induction of an immunological response was demonstrated in a number of non-avian species and was not dependent on the route of inoculation 1~. No evidence for productive replication of the fowlpox-rabies recombinant virus in vivo was demonstrated. Although rabies virus neutralizing antibodies were detected and protection against a lethal rabies virus challenge was demonstrated, the vaccine dose of fowlpox recombinant virus was higher than the dose required when using a replicationcompetent vaccinia rabies recombinant virus.
Short p a p e r : D. B a x b y a n d E. Paoletti
More recently, other members of the Avipox genus have been evaluated for their utility as vaccines for non-avian species. A recombinant canarypox virus which expressed the rabies glycoprotein has recently been described x6. In a number of experiments the properties of the canarypox recombinant were similar to the fowlpox virus recombinant noted above. Hence, the rabies glycoprotein G was authentically expressed in cell culture derived from non-avian species and no evidence of productive viral replication was demonstrated either in vitro in non-avian cell culture or in vivo in non-avian animal hosts. Significantly, however, the protective vaccinating dose of the recombinant canarypox rabies virus required to protect vaccinated mice or the natural target species (cats and dogs) from lethal rabies virus challenge was much lower than the dose required when a fowlpox vector was used and was not statistically different from the immunizing protective dose required when a replicating vaccinia virus recombinant which expressed the same rabies glycoprotein was tested in the same species x6. This relative potency of the canarypox recombinant is not peculiar to the rabies glycoprotein antigen. Similar observations have been made using a number of other extrinsic immunogens from a number of different classes of mammalian pathogens (data in preparation for publication ). In theory, and now with good experimentally based support, these non-replicating avipox virus vectors provide significant advantages as recombinant vectorbased vaccines. These vectors are capable of expressing an extrinsic antigen in an authentic fashion, of presenting the antigen in a native configuration on the inoculated cell membrane, thus providing for the induction of both humoral and cellular immune responses, and of inducing protective immunity in a wide range of species without apparent productive replication of the vector itself. This last property should result in minimal or non-existent side effects due to vaccination. The vector, in the absence of replication, should not disseminate within the vaccinee, disseminate from a vaccinated individual to a nonvaccinated contact or contaminate the general environment, thus providing a significant safety margin. Additional beneficial properties allowing for the construction of polyvalent recombinants and thermostability should be maintained in these vectors. It is clear that this novel approach may be extended to other viral vectors including vaccinia, and further, that the breadth of applicability can only be addressed by future work. Nevertheless, an interesting alternative
approach is provided which can be compared with more conventional replication-competent vectors as well as with more classical vaccine approaches.
REFERENCES 1
2
3 4 5 6 7 8 9
10
11 12 13
14
15
16
Panicali, D. and Paoletti, E. Construction of poxviruses as cloning vectors: Insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus. Proc. Nat/Acad. Sci. USA 1982, 79, 4927 Bennink, J.R. and Yewdell, J.W. Recombinant vaccinia viruses as vectors for studying T lymphocyte specificity and function. In: Current Topics in Microbiology and Immunology (Eds Moyer, R.W. and Turner, P.C.) Springer-Verlag, 1990, p. 153 Tartaglia, J., Pincus, S. and Paoletti, E. Poxvirus-based vectors as vaccine candidates. Crit. Rev. Immunol. 1990, 10, 13 Brochier, B., Thomas, I., Bauduin, B., Leveau, T., Pastoret, P.-P., Lanquet, B. et al. Use of vaccinia-rabies recombinant virus for the oral vaccination of foxes against rabies. Vaccine 1990, 8, 101-104 Desmettre, P., Languet, B., Chappius, G., Brochier, B., Thomas, I., Lecocq, J.-P. at al. Use of vaccinia rabies recombinant for oral vaccination of wildlife. Vet. Microbiol. 1990, 23, 227-236 Koff, W.C. and Fauci, A.S. Human trials of AIDS vaccines: current status and future directions. AIDS 1989, 1, 5125 Taylor, J., Weinberg, R., Kawaoka, Y., Webster, R. and Paoletti, E. Protective immunity against avian influenza induced by a fowlpox virus recombinant. Vaccine 1988, 6, 504 Boyle, D.B. and Coupar, B.E.H. Construction of recombinant fowlpox viruses as vectors for poultry vaccines. Virus Res. 1988, 10, 343 Taylor, J., Edbauer, C., Rey-Senelonge, A., Bouquet, J-F., Norton, E., Goebel, S. e t a / . Newcastle disease virus fusion protein expressed in a fowlpox virus recombinant confers protection in chickens. J. Virol. 1990, 64, 1441 Edbauer, C., Weinberg, R., Taylor, J., Rey-Senelonge, A., Bouquet, J-F., Desmettre, P. and Paoletti, E. Protection of chickens with a recombinant fowlpox virus expressing the Newcastle disease virus hemagglutinin-neuraminidase gene. Virology 1990, 179, 901 Taylor, J., Weinberg, R., Languet, B., Desmettre, P. and Paoletti, E. A recombinant fowlpox virus induces protective immunity in non-avian species. Vaccine 1988, 6, 497 Taylor, J. and Paoletti, E. Fowlpox virus as a vector in non-avian species. Vaccine 1988, 6, 466 Taylor, J., Weinberg, R., Languet, B., Desmettre, P. and Paoletti, E. Fowlpox virus based recombinant vaccines. In: Technological Advances in Virus Development: UCLA Symposia on Molecular and Cellular Biology, New Series (Ed. Lasky, L. ) Alan R. Liss, New York, 1988, Vol. 84, p. 321 Taylor, J., Weinberg, R., Languet, B., Desmettre, P. and Paoletti, E. The use of fowlpox virus vectors to vaccinate non-avian species. In: The Re/ease of Genetically-engineered Micro-organisms (Eds Sussman, M., Collins, C.H., Skinner, F.A. and Stewart-Tull, D.E.) Academic Press, New York, 1988, p. 89 Taylor, J. and Paoletti, E. Pox viruses as eukaryotic cloning and expression vectors: Future medical and veterinary vaccines. In: Progress in Veterinary Microbiology and Immunology (Ed. Pandey, R.) Karger Press, 1988, p. 197 Taylor, J., Trimarchi, C., Weinberg, R., Languet, B., Guillemin, F., Desmettre, P. and Paoletti, E. Efficacy studies on a canarypexrabies recombinant virus. Vaccine 1991, 9, 190
Vaccine, Vol. 10, Issue 1, 1992
9
INTRODUCTION The introduction of recombinant vaccinia virus in 19821 provided a new strategy for vaccine development. In addition to providing a magnificent research tool, particularly to address questions in immunology 2, a large number of recombinant vaccinia viruses which express immunogens of both veterinary and human pathogens have been described 3. Indeed, measurable progress in the application of recombinant vaccinia virus vaccines has been demonstrated. Extensive field testing of a recombinant vaccinia virus expressing the rabies glycoprotein has been carried out in Europe and to a lesser extent in North America 4"5. These studies have demonstrated both safety and efficacy in targeted intervention against wildlife rabies. Studies in humans utilizing a vaccinia recombinant expressing immunogens from human immunodeficiency virus have also been initiated in the United States 6. The basic technologies used to construct vaccinia virus recombinants have been modified and extended to other members of the Poxvirus family, particularly to members of the Avipox genus. In contrast to vaccinia virus which has a broad host range in terms of replication in susceptible species, members of the Avipox genus are natu'r~lly restricted in that productive replication takes place only in susceptible avian species. No productive replication of avipox virus in non-avian species has been reported and despite extensive field use of attenuated avipox vaccines in poultry and other avians, no adverse effects in humans, other species or to the environment have been documented. Recent reports v's have described the construction of *Department of Medical Microbiology, Duncan Building, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK. tVirogenetics Corporation, 465 Jordan Road, Rensselaer Technology Park, Troy, New York 12180, USA. ++To whom correspondence should be addressed. (Received 13 May 1991 ; revised 12 August 1991 ; accepted 26 August 1991) 0264410x/92/01000802 © 1992 Butterworth-Heinemann Ltd 8
Vaccine, Vol. 10, Issue 1, 1992
recombinant fowlpox viruses which express specific immunogens from pathogens of poultry. These recombinant fowlpox viruses demonstrated clear utility as potential vaccines for avian influenza 7 and Newcastle disease virus infection 9'1°. In attempts to determine the molecular basis of the natural restriction of Avipox viruses in terms of productive replication to avian species, fowlpox virus recombinants were constructed with a reporter gene derived from a mammalian pathogen. Hence, recombinant fowlpox viruses were constructed to express the rabies glycoprotein. These fowlpox virus recombinants were shown to replicate productively in cell cultures derived from susceptible avian species and to express faithfully the rabies glycoprotein in both susceptible avian cell cultures as well as in cell cultures derived from non-avian species. Significantly, the fowlpox virus recombinants did not either replicate productively or become adapted for replication in mammalian cell culture 11-15. These latter results support the known natural history of the host restriction of avipoxviruses. Surprisingly, when the fowlpox virus recombinant expressiiag the rabies glycoprotein was inoculated into non-avian species the animals seroconverted to the extrinsic rabies immunogen, produced rabies virus neutralizing antibodies and, significantly, induced an immune response which was shown to protect against a lethal rabies virus challenge in mice, cats and dogs 11-~ 5 Induction of an immunological response was demonstrated in a number of non-avian species and was not dependent on the route of inoculation 1~. No evidence for productive replication of the fowlpox-rabies recombinant virus in vivo was demonstrated. Although rabies virus neutralizing antibodies were detected and protection against a lethal rabies virus challenge was demonstrated, the vaccine dose of fowlpox recombinant virus was higher than the dose required when using a replicationcompetent vaccinia rabies recombinant virus.
Short p a p e r : D. B a x b y a n d E. Paoletti
More recently, other members of the Avipox genus have been evaluated for their utility as vaccines for non-avian species. A recombinant canarypox virus which expressed the rabies glycoprotein has recently been described x6. In a number of experiments the properties of the canarypox recombinant were similar to the fowlpox virus recombinant noted above. Hence, the rabies glycoprotein G was authentically expressed in cell culture derived from non-avian species and no evidence of productive viral replication was demonstrated either in vitro in non-avian cell culture or in vivo in non-avian animal hosts. Significantly, however, the protective vaccinating dose of the recombinant canarypox rabies virus required to protect vaccinated mice or the natural target species (cats and dogs) from lethal rabies virus challenge was much lower than the dose required when a fowlpox vector was used and was not statistically different from the immunizing protective dose required when a replicating vaccinia virus recombinant which expressed the same rabies glycoprotein was tested in the same species x6. This relative potency of the canarypox recombinant is not peculiar to the rabies glycoprotein antigen. Similar observations have been made using a number of other extrinsic immunogens from a number of different classes of mammalian pathogens (data in preparation for publication ). In theory, and now with good experimentally based support, these non-replicating avipox virus vectors provide significant advantages as recombinant vectorbased vaccines. These vectors are capable of expressing an extrinsic antigen in an authentic fashion, of presenting the antigen in a native configuration on the inoculated cell membrane, thus providing for the induction of both humoral and cellular immune responses, and of inducing protective immunity in a wide range of species without apparent productive replication of the vector itself. This last property should result in minimal or non-existent side effects due to vaccination. The vector, in the absence of replication, should not disseminate within the vaccinee, disseminate from a vaccinated individual to a nonvaccinated contact or contaminate the general environment, thus providing a significant safety margin. Additional beneficial properties allowing for the construction of polyvalent recombinants and thermostability should be maintained in these vectors. It is clear that this novel approach may be extended to other viral vectors including vaccinia, and further, that the breadth of applicability can only be addressed by future work. Nevertheless, an interesting alternative
approach is provided which can be compared with more conventional replication-competent vectors as well as with more classical vaccine approaches.
REFERENCES 1
2
3 4 5 6 7 8 9
10
11 12 13
14
15
16
Panicali, D. and Paoletti, E. Construction of poxviruses as cloning vectors: Insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccinia virus. Proc. Nat/Acad. Sci. USA 1982, 79, 4927 Bennink, J.R. and Yewdell, J.W. Recombinant vaccinia viruses as vectors for studying T lymphocyte specificity and function. In: Current Topics in Microbiology and Immunology (Eds Moyer, R.W. and Turner, P.C.) Springer-Verlag, 1990, p. 153 Tartaglia, J., Pincus, S. and Paoletti, E. Poxvirus-based vectors as vaccine candidates. Crit. Rev. Immunol. 1990, 10, 13 Brochier, B., Thomas, I., Bauduin, B., Leveau, T., Pastoret, P.-P., Lanquet, B. et al. Use of vaccinia-rabies recombinant virus for the oral vaccination of foxes against rabies. Vaccine 1990, 8, 101-104 Desmettre, P., Languet, B., Chappius, G., Brochier, B., Thomas, I., Lecocq, J.-P. at al. Use of vaccinia rabies recombinant for oral vaccination of wildlife. Vet. Microbiol. 1990, 23, 227-236 Koff, W.C. and Fauci, A.S. Human trials of AIDS vaccines: current status and future directions. AIDS 1989, 1, 5125 Taylor, J., Weinberg, R., Kawaoka, Y., Webster, R. and Paoletti, E. Protective immunity against avian influenza induced by a fowlpox virus recombinant. Vaccine 1988, 6, 504 Boyle, D.B. and Coupar, B.E.H. Construction of recombinant fowlpox viruses as vectors for poultry vaccines. Virus Res. 1988, 10, 343 Taylor, J., Edbauer, C., Rey-Senelonge, A., Bouquet, J-F., Norton, E., Goebel, S. e t a / . Newcastle disease virus fusion protein expressed in a fowlpox virus recombinant confers protection in chickens. J. Virol. 1990, 64, 1441 Edbauer, C., Weinberg, R., Taylor, J., Rey-Senelonge, A., Bouquet, J-F., Desmettre, P. and Paoletti, E. Protection of chickens with a recombinant fowlpox virus expressing the Newcastle disease virus hemagglutinin-neuraminidase gene. Virology 1990, 179, 901 Taylor, J., Weinberg, R., Languet, B., Desmettre, P. and Paoletti, E. A recombinant fowlpox virus induces protective immunity in non-avian species. Vaccine 1988, 6, 497 Taylor, J. and Paoletti, E. Fowlpox virus as a vector in non-avian species. Vaccine 1988, 6, 466 Taylor, J., Weinberg, R., Languet, B., Desmettre, P. and Paoletti, E. Fowlpox virus based recombinant vaccines. In: Technological Advances in Virus Development: UCLA Symposia on Molecular and Cellular Biology, New Series (Ed. Lasky, L. ) Alan R. Liss, New York, 1988, Vol. 84, p. 321 Taylor, J., Weinberg, R., Languet, B., Desmettre, P. and Paoletti, E. The use of fowlpox virus vectors to vaccinate non-avian species. In: The Re/ease of Genetically-engineered Micro-organisms (Eds Sussman, M., Collins, C.H., Skinner, F.A. and Stewart-Tull, D.E.) Academic Press, New York, 1988, p. 89 Taylor, J. and Paoletti, E. Pox viruses as eukaryotic cloning and expression vectors: Future medical and veterinary vaccines. In: Progress in Veterinary Microbiology and Immunology (Ed. Pandey, R.) Karger Press, 1988, p. 197 Taylor, J., Trimarchi, C., Weinberg, R., Languet, B., Guillemin, F., Desmettre, P. and Paoletti, E. Efficacy studies on a canarypexrabies recombinant virus. Vaccine 1991, 9, 190
Vaccine, Vol. 10, Issue 1, 1992
9
