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Current and Future Recombinant Viral Vaccines for Poultry
ADVANCESIN VETERINARYMEDICINE,VOL. 41
Current and Future Recombinant Viral Vaccines for Poultry MARK W. JACKWOOD
Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602 I. Introduction a n d B a c k g r o u n d II. Virus Vectors A. Fowlpox Virus B. H e r p e s v i r u s of Turkeys III. F u t u r e R e c o m b i n a n t Vaccines for P o u l t r y A. S u b u n i t Vaccines a n d Synthetic Peptides B. Nucleic Acid Vaccines IV. S u m m a r y References
I. Introduction and Background Biotechnology has changed the way scientists approach the development of new vaccines. The ability to manipulate genes directly (biotechnology) has allowed scientists to create nonpathogenic vaccines capable of inducing a protective immune response. The poultry industry leads the way in the development and use of these recombinant vaccines. The first commercially available recombinant viral vector vaccine was produced for poultry.
II. Virus Vectors Virus vectors are nonpathogenic viruses carrying a foreign gene inserted into a region of the viral genome that is not required for viral 517 Copyright 9 1999 by AcademicPress All rights of reproduction in any form reserved. 1093-975X/99 $25.00
518
MARK W. JACKWOOD
replication. When the virus vector infects the host, the foreign protein is expressed and the host immune system responds to the virus vector as well as the foreign protein. There are several advantages to using a virus vector. Virus vectors are live viruses that replicate in the host and induce an immune response usually with only a single vaccination. In addition, the amount and the presentation of the foreign protein is such t h a t a strong protective response is induced in the vaccinated animal. Another advantage to viral vectors as vaccines is t h a t more t h a n one foreign gene can be inserted into the vector, allowing for the construction of a multivalent vaccine. The main disadvantage of virus vector vaccines are t h a t they are expensive to develop. A. FOWLPOX VIRUS
The first commercially available virus vector vaccine was a fowlpox virus. Fowlpox Virus is a double-stranded DNA virus in the Poxviridae family, genus Avipoxvirus. Avian pox is the disease caused by fowlpox virus, and numerous vaccine strains have been shown to be safe and effective over many years of use. Scientists have found t h a t inactivation of the thymidine kinase gene in fowlpox virus does not affect the replication of that virus. Thus, foreign genes inserted into t h a t region of the genome have no affect on the ability of the vaccine viruses to infect, replicate, or induce an immune response in the host. The first commercially available fowlpox virus vaccine vector contains the hemagglutinin neuraminidase (HN) and fusion (F) genes from Newcastle disease virus (NDV). Newcastle disease virus is an orthomixovirus that has a single-stranded negative sense RNA genome. That virus causes an upper respiratory tract disease in poultry. The HA and F proteins, located on the surface of the virus, have been shown to induce neutralizing antibodies t h a t protect the host from disease. To construct the fowlpox virus vaccine vector, copy DNA was prepared to the NDV genes coding for the HA and F proteins and t h a t cDNA was inserted into a transfer plasmid. The transfer plasmid contained fowlpox virus nucleic acid sequences flanking the inserted NDV genes, and was used to insert the HA and F genes into a vaccine strain of fowlpox virus using a technique called homologous recombination. When the fowlpox virus vaccine vector containing the NDV genes coding for the HA and F proteins is used to vaccinate poultry, the birds respond immunologically to the poxvirus as well as the NDV proteins. The major advantage of the fowlpox virus vector for NDV is t h a t there is absolutely no chance of an upper respiratory reaction. That is
RECOMBINANT VIRAL VACCINES FOR POULTRY
519
because the pox virus replicates in the skin not in the upper respiratory tract where live NDV vaccines replicate. Furthermore, since the pox virus vector was originally a vaccine strain, it is a safe and effective vaccine for pox. The major disadvantage of the fowlpox virus vector for NDV is that maternal antibodies to NDV interfere with the immune response. The vaccine works best in maternal antibody negative birds. Currently there are two commercially available fowlpox virus vectors for NDV. Both contain the HN and F genes of NDV. VectorVax FPN (Hoechst-Roussel Vet, Somerville, NJ) is a lyophilized product that was licensed in July 1994. Trovac-NDV (SELECT Laboratories, Gainesville, GA) is a liquid N 2 frozen product that was licensed in October 1995. Other fowlpox virus vectored vaccines likely to be licensed in the near future will contain genes from avian influenza virus, infectious laryngotracheitis virus (ILTV), and possibly avian immune modulator genes (cytokines). B. HERPESVIRUS OF TURKEYS Another virus vector being developed for poultry is based on herpesvirus of turkeys (HVT), an alpha herpesvirus used as a vaccine against Marek's disease (Morgan et al., 1993). Several nonessential regions have been identified in the unique short and unique long region of the viral genome of HVT. Genes from NDV, avian influenza virus, Marek's disease virus, and ILTV are being inserted into HVT to develop a virus vector against those viruses. Because HVT causes a persistent systemic infection and stimulates both humoral as well as cell-mediated immunity it is hoped that a single vaccination with that vector will induce long-lasting immunity.
III. Future Recombinant Vaccines for Poultry A. SUBUNIT VACCINES AND SYNTHETIC PEPTIDES
Several other approaches to recombinant vaccines for poultry are currently being pursued. Subunit vaccines, which are the immunogenic proteins of disease agents, are being pursued using baculovirus or the T7 transient expression systems. Synthetic peptides are short amino acid chains containing only the neutralizing epitope of an immunogenic protein from a disease agent. They can be synthesized in the laboratory and must be linked to a
520
MARK W. JACKWOOD
carrier molecule, such as bovine s e r u m a l b u m i n of keyhole limpet hemocyanin.
B. NUCLEIC
ACID VACCINES
P e r h a p s the m o s t p r o m i s i n g new r e c o m b i n a n t vaccine technology is the d e v e l o p m e n t of nucleic acid vaccines. Nucleic acid vaccines, also called gene vaccines or D N A vaccines, are u s u a l l y a bacterial p l a s m i d c o n t a i n i n g a cloned gene from a disease agent. E u k a r y o t i c p r o m o t e r s allow the gene to be expressed w h e n the DNA is injected directly into the animal. Generally nucleic acid vaccines are injected intram u s c u l a r l y , t a k e n up by the skeletal muscle fibers, a n d the i m m u -
FIG. 1. Infectious bronchitis virus nucleic acid vaccine containing the $1 glycoprotein gene (Ark S1) from the Arkansas serotype of the virus. The plasmid contains the ampicilin resistance gene (Amp), the cytomegalovirus immediate early promoter (CMV IE promoter) including the cap site at position 678bp, and the rat preproinsulin polyadenylation and processing signals (Rat preproinsulin Poly-A signals) including the poly-A site at 3318 bp. The numbers following restriction enzyme sites are in base pairs.
521
RECOMBINANT VIRAL VACCINES FOR POULTRY TABLE I DNA VACCINE TITRATIONAND PROTECTION OF CHICKENS AGAINST ARK IBV CHALLENGEa Vaccine given at Group 1 2 3 4 5 6
14 days of age
35 days of age
50 ~g pMJAS1 100 ~g pMJAS1 150 ~g pMJAS1 150 ~g pBC12 d Live Ark TE buffer
100 ~g pMJAS1 200 ~g pMJAS1 300 ~g pMJAS1 300 ~g pBC12 Live Ark TE buffer
Clinical signs at 7 days postchallenge b
Average tracheal lesion scoresc
6/6 2/5 0/5 6/6 0/6 6/6
2.71A 2.26 B 2.168 2.53 A 1.168 2.038
aSee text for details; data for nonchallenged birds are not presented. bNumber of birds with clinical signs/number of birds examined. cNumbers within the column with different superscripts are statistically different (p<0.1). dpBC12, the pBC12/CMV/IL-2 plasmid.
nogenic protein encoded by the cloned gene is expressed. When the bird mounts an immune response to the expressed protein, that immature response has been shown to be protective and persistent. One of the first nucleic acid vaccines was described and developed by Fynan et al. (1993) to avian influenza virus. Those scientists cloned the H7 (hemagglutinin) gene into an expression plasmid containing the cytomegalovirus immediate early promoter and showed that birds were protected from a lethal challenge with H7 influenza virus. Nucleic acid vaccines have also been developed to a number of other avian diseases including one developed in our laboratory (Jackwood et al., 1995) to infectious bronchitis virus (IBV). That vaccine contained the base plasmid described by Cullen (1986) and the IBV S1 gene subunit of the immunogenic spike glycoprotein (see Fig. 1). In vitro transfection studies showed that the S1 glycoprotein could be expressed in COS cells. Following vaccination of specific pathogen-free chickens twice at 14 and 35 days of age with 150 and 300 ~g of DNA, respectively, the birds were shown to be protected from the disease following challenge with the homologous serotype of the virus (Table I).
IV. Summary The use of biotechnology to create recombinant viral vaccines holds many promises for the future. But, to be practical, new vaccines must
522
MARK W. JACKWOOD
have a selective advantage over traditional vaccines. A vaccine that is novel because it is a recombinant vaccine is not enough. Recombinant vaccines m u s t be safer, or more efficacious, or less expensive to produce in order for t h e m to gain a niche in the marketplace. REFERENCES Cullen, B. R. (1986). Trans-activation of human immunodeficiency virus occurs via a bimodal mechanism. Cell 46, 973-982. Fynan, E. F., Robinson, H. L., and Webster, R. G. (1993). Use of DNA encoding influenza hemagglutinin as an avian influenza vaccine. D N A Cell Biol. 12, 785-789. Jackwood, M. W., Hilt, D. A., and Moore, K. M. (1995). Vaccination of SPF leghorn chickens with a plasmid vector containing the S1 glycoprotein gene of infectious bronchitis virus. Proc. 132nd Annu. Meet. Am. Vet. Med. Associ., Pittsburg, PA, p. 143. Morgan, R. W., Gelb, J., Jr., Pope, C. R., and Sondermeijer, P. J. A. (1993). Efficacy in chickens of a herpesvirus of turkeys recombinant vaccine containing the fusion gene of Newcastle disease virus: Onset of protection and effect of maternal antibodies. Avian Dis. 37, 1032-1040.
Current and Future Recombinant Viral Vaccines for Poultry MARK W. JACKWOOD
Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602 I. Introduction a n d B a c k g r o u n d II. Virus Vectors A. Fowlpox Virus B. H e r p e s v i r u s of Turkeys III. F u t u r e R e c o m b i n a n t Vaccines for P o u l t r y A. S u b u n i t Vaccines a n d Synthetic Peptides B. Nucleic Acid Vaccines IV. S u m m a r y References
I. Introduction and Background Biotechnology has changed the way scientists approach the development of new vaccines. The ability to manipulate genes directly (biotechnology) has allowed scientists to create nonpathogenic vaccines capable of inducing a protective immune response. The poultry industry leads the way in the development and use of these recombinant vaccines. The first commercially available recombinant viral vector vaccine was produced for poultry.
II. Virus Vectors Virus vectors are nonpathogenic viruses carrying a foreign gene inserted into a region of the viral genome that is not required for viral 517 Copyright 9 1999 by AcademicPress All rights of reproduction in any form reserved. 1093-975X/99 $25.00
518
MARK W. JACKWOOD
replication. When the virus vector infects the host, the foreign protein is expressed and the host immune system responds to the virus vector as well as the foreign protein. There are several advantages to using a virus vector. Virus vectors are live viruses that replicate in the host and induce an immune response usually with only a single vaccination. In addition, the amount and the presentation of the foreign protein is such t h a t a strong protective response is induced in the vaccinated animal. Another advantage to viral vectors as vaccines is t h a t more t h a n one foreign gene can be inserted into the vector, allowing for the construction of a multivalent vaccine. The main disadvantage of virus vector vaccines are t h a t they are expensive to develop. A. FOWLPOX VIRUS
The first commercially available virus vector vaccine was a fowlpox virus. Fowlpox Virus is a double-stranded DNA virus in the Poxviridae family, genus Avipoxvirus. Avian pox is the disease caused by fowlpox virus, and numerous vaccine strains have been shown to be safe and effective over many years of use. Scientists have found t h a t inactivation of the thymidine kinase gene in fowlpox virus does not affect the replication of that virus. Thus, foreign genes inserted into t h a t region of the genome have no affect on the ability of the vaccine viruses to infect, replicate, or induce an immune response in the host. The first commercially available fowlpox virus vaccine vector contains the hemagglutinin neuraminidase (HN) and fusion (F) genes from Newcastle disease virus (NDV). Newcastle disease virus is an orthomixovirus that has a single-stranded negative sense RNA genome. That virus causes an upper respiratory tract disease in poultry. The HA and F proteins, located on the surface of the virus, have been shown to induce neutralizing antibodies t h a t protect the host from disease. To construct the fowlpox virus vaccine vector, copy DNA was prepared to the NDV genes coding for the HA and F proteins and t h a t cDNA was inserted into a transfer plasmid. The transfer plasmid contained fowlpox virus nucleic acid sequences flanking the inserted NDV genes, and was used to insert the HA and F genes into a vaccine strain of fowlpox virus using a technique called homologous recombination. When the fowlpox virus vaccine vector containing the NDV genes coding for the HA and F proteins is used to vaccinate poultry, the birds respond immunologically to the poxvirus as well as the NDV proteins. The major advantage of the fowlpox virus vector for NDV is t h a t there is absolutely no chance of an upper respiratory reaction. That is
RECOMBINANT VIRAL VACCINES FOR POULTRY
519
because the pox virus replicates in the skin not in the upper respiratory tract where live NDV vaccines replicate. Furthermore, since the pox virus vector was originally a vaccine strain, it is a safe and effective vaccine for pox. The major disadvantage of the fowlpox virus vector for NDV is that maternal antibodies to NDV interfere with the immune response. The vaccine works best in maternal antibody negative birds. Currently there are two commercially available fowlpox virus vectors for NDV. Both contain the HN and F genes of NDV. VectorVax FPN (Hoechst-Roussel Vet, Somerville, NJ) is a lyophilized product that was licensed in July 1994. Trovac-NDV (SELECT Laboratories, Gainesville, GA) is a liquid N 2 frozen product that was licensed in October 1995. Other fowlpox virus vectored vaccines likely to be licensed in the near future will contain genes from avian influenza virus, infectious laryngotracheitis virus (ILTV), and possibly avian immune modulator genes (cytokines). B. HERPESVIRUS OF TURKEYS Another virus vector being developed for poultry is based on herpesvirus of turkeys (HVT), an alpha herpesvirus used as a vaccine against Marek's disease (Morgan et al., 1993). Several nonessential regions have been identified in the unique short and unique long region of the viral genome of HVT. Genes from NDV, avian influenza virus, Marek's disease virus, and ILTV are being inserted into HVT to develop a virus vector against those viruses. Because HVT causes a persistent systemic infection and stimulates both humoral as well as cell-mediated immunity it is hoped that a single vaccination with that vector will induce long-lasting immunity.
III. Future Recombinant Vaccines for Poultry A. SUBUNIT VACCINES AND SYNTHETIC PEPTIDES
Several other approaches to recombinant vaccines for poultry are currently being pursued. Subunit vaccines, which are the immunogenic proteins of disease agents, are being pursued using baculovirus or the T7 transient expression systems. Synthetic peptides are short amino acid chains containing only the neutralizing epitope of an immunogenic protein from a disease agent. They can be synthesized in the laboratory and must be linked to a
520
MARK W. JACKWOOD
carrier molecule, such as bovine s e r u m a l b u m i n of keyhole limpet hemocyanin.
B. NUCLEIC
ACID VACCINES
P e r h a p s the m o s t p r o m i s i n g new r e c o m b i n a n t vaccine technology is the d e v e l o p m e n t of nucleic acid vaccines. Nucleic acid vaccines, also called gene vaccines or D N A vaccines, are u s u a l l y a bacterial p l a s m i d c o n t a i n i n g a cloned gene from a disease agent. E u k a r y o t i c p r o m o t e r s allow the gene to be expressed w h e n the DNA is injected directly into the animal. Generally nucleic acid vaccines are injected intram u s c u l a r l y , t a k e n up by the skeletal muscle fibers, a n d the i m m u -
FIG. 1. Infectious bronchitis virus nucleic acid vaccine containing the $1 glycoprotein gene (Ark S1) from the Arkansas serotype of the virus. The plasmid contains the ampicilin resistance gene (Amp), the cytomegalovirus immediate early promoter (CMV IE promoter) including the cap site at position 678bp, and the rat preproinsulin polyadenylation and processing signals (Rat preproinsulin Poly-A signals) including the poly-A site at 3318 bp. The numbers following restriction enzyme sites are in base pairs.
521
RECOMBINANT VIRAL VACCINES FOR POULTRY TABLE I DNA VACCINE TITRATIONAND PROTECTION OF CHICKENS AGAINST ARK IBV CHALLENGEa Vaccine given at Group 1 2 3 4 5 6
14 days of age
35 days of age
50 ~g pMJAS1 100 ~g pMJAS1 150 ~g pMJAS1 150 ~g pBC12 d Live Ark TE buffer
100 ~g pMJAS1 200 ~g pMJAS1 300 ~g pMJAS1 300 ~g pBC12 Live Ark TE buffer
Clinical signs at 7 days postchallenge b
Average tracheal lesion scoresc
6/6 2/5 0/5 6/6 0/6 6/6
2.71A 2.26 B 2.168 2.53 A 1.168 2.038
aSee text for details; data for nonchallenged birds are not presented. bNumber of birds with clinical signs/number of birds examined. cNumbers within the column with different superscripts are statistically different (p<0.1). dpBC12, the pBC12/CMV/IL-2 plasmid.
nogenic protein encoded by the cloned gene is expressed. When the bird mounts an immune response to the expressed protein, that immature response has been shown to be protective and persistent. One of the first nucleic acid vaccines was described and developed by Fynan et al. (1993) to avian influenza virus. Those scientists cloned the H7 (hemagglutinin) gene into an expression plasmid containing the cytomegalovirus immediate early promoter and showed that birds were protected from a lethal challenge with H7 influenza virus. Nucleic acid vaccines have also been developed to a number of other avian diseases including one developed in our laboratory (Jackwood et al., 1995) to infectious bronchitis virus (IBV). That vaccine contained the base plasmid described by Cullen (1986) and the IBV S1 gene subunit of the immunogenic spike glycoprotein (see Fig. 1). In vitro transfection studies showed that the S1 glycoprotein could be expressed in COS cells. Following vaccination of specific pathogen-free chickens twice at 14 and 35 days of age with 150 and 300 ~g of DNA, respectively, the birds were shown to be protected from the disease following challenge with the homologous serotype of the virus (Table I).
IV. Summary The use of biotechnology to create recombinant viral vaccines holds many promises for the future. But, to be practical, new vaccines must
522
MARK W. JACKWOOD
have a selective advantage over traditional vaccines. A vaccine that is novel because it is a recombinant vaccine is not enough. Recombinant vaccines m u s t be safer, or more efficacious, or less expensive to produce in order for t h e m to gain a niche in the marketplace. REFERENCES Cullen, B. R. (1986). Trans-activation of human immunodeficiency virus occurs via a bimodal mechanism. Cell 46, 973-982. Fynan, E. F., Robinson, H. L., and Webster, R. G. (1993). Use of DNA encoding influenza hemagglutinin as an avian influenza vaccine. D N A Cell Biol. 12, 785-789. Jackwood, M. W., Hilt, D. A., and Moore, K. M. (1995). Vaccination of SPF leghorn chickens with a plasmid vector containing the S1 glycoprotein gene of infectious bronchitis virus. Proc. 132nd Annu. Meet. Am. Vet. Med. Associ., Pittsburg, PA, p. 143. Morgan, R. W., Gelb, J., Jr., Pope, C. R., and Sondermeijer, P. J. A. (1993). Efficacy in chickens of a herpesvirus of turkeys recombinant vaccine containing the fusion gene of Newcastle disease virus: Onset of protection and effect of maternal antibodies. Avian Dis. 37, 1032-1040.