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Naked DNA and vaccine design
NEWS
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C O M M E N ' f
Naked DNA and vaccine design Thomas J. Braciale ver the past decade, the revolution in molecular and structural biology has opened up the immune system to detailed analysis. The genes encoding critical molecules of the immune system have been identified and the structure of these genes has been determined. Immunologists now not only have detailed information on the structure of the immunoglobutin receptor on B cells, but also information on the structure and function of the T cell antigen receptor complex and the molecules encoded by genes of the major histocompatibility complex (MHC). Indeed, the three dimensional structure of both the M H C class I and, more recently, the M H C class II molecules has been solved by X-ray crystallographic analysis 1,2.These structural studies have directly established that the antigen receptor on T cells recognizes short peptide fragments of foreign antigenic proteins, such as bacterial and viral polypeptides, bound to MHC class I and class II molecules on the surface of antigenpresenting cells. This new insight into the nature of antigen recognition by T cells has led to the development of many novel strategies to manipulate the immune system and to treat autoimmune diseases. One area where the revolution in immunology has not, as yet, had its full impact is in vaccine development. At the moment, Jenner's approach of immunizing with a live, attenuated virus remains the most effective means of producing sustained immunity to most viruses. Part of the reason why live viruses are effective vaccines is that they efficiently activate the CD8 + precursor cells that give rise to the major antiviral effector cells: the MHC class I restricted cytolytic T cells. The reason for this lies in the cell biology of the synthesis, assembly and transport of the MHC class I molecule. M H C class I mol-
O
ecules capture viral peptides during their synthesis and assembly in the endoplasmic reticulum (ER). The MHC molecules transport the bound viral peptides from the ER to the cell surface, where the complex is displayed to the antigen receptor on CD8 ÷cytolytic T cell precursors and activated effector cytolytic T cells. To charge nascent MHC class I molecules, viral polypeptides must gain access to the cytoplasm of the cell. Here, the viral polypeptides are fragmented and then transported as peptide fragments into the ER. The most efficient way for viral polypeptides to gain access to the cytoplasm of an antigenpresenting cell for proteolytic fragmentation is for them to be expressed de n o v o in infected cells. Recently, a novel vaccination strategy has been developed using naked DNA encoding a viral polypeptide as an immunogen. This strategy is based on the fortuitous observation by Wolff et al. 3 that direct intramuscular inoculation of mice with plasmid DNA containing the gene for a marker protein driven by a strong eukaryotic promoter resulted in persistent expression of the gene in striated muscle at the site of injection. This finding extended earlier work on the uptake and expression of viral and eukaryotic DNA by direct DNA inoculation into experimental animals". The result of Wolff et al. has prompted researchers at Merck Research Laboratories and Vical to use the same strategy of DNA uptake and expression as a method of vaccination against influenza virus. As reported recently, Ulmer et al. v have injected mice with DNA encoding the type A influenza A/PR/8/34 nucleocapsid protein (NP) linked to the human cytoT.J. Braciale is in the Beirne Carter Center for Immunology Research, University of Virginia Health Sciences Center, MR4 Box 4012, Charlottesville, VA 22908, USA.
megalovirus promoter. Mice that had been so treated mounted a serum antibody response to the NP, and were primed for memory cytotoxic T cell (CTL) response to the NP. Most importantly, the mice injected with the DNA encoding the NP were protected against the morbidity and mortality of subsequent intranasal infection with a heterotypic strain of influenza virus. The development of antibodies to the NP by these mice after DNA injection implies that sufficient antigenic mass was produced in v i v o to stimulate antibody production. The NP is the major internal constituent of the influenza virion and is structurally conserved among type A influenza strains of different subtypes. Antibody to the NP is not, however, neutralizing, so that the protection against lethal challenge afforded by DNA vaccination is not antibody mediated but is probably caused by the in v i v o priming of NP-specific CTLs. The NP is a major target of the influenza-specific CTLs in the mouse strain used in this study (BALB/c). Although NP-specific CTLs can eliminate infectious virus from the lungs of infected mice 8 and promote recovery from lethal infections (T.J. Braciale, unpublished), earlier attempts to demonstrate resistance to infectious influenza challenge by immunizing with viral eukaryotic vectors expressing the NP gene, such as recombinant vaccinia viruses, did not succeed despite the induction of NP-specific CTLs in the immunized animals 9. The failure of these earlier attempts to demonstrate NP-specific protection probably reflects the transient nature of the CTL response to foreign antigen. Activated killer T cells arise from noncytolytic T cell precursors, which differentiate over a period of days into activated effectors after contact with viral peptide-MHC complexes. These killer T cells remain cytolytic only for a short time (5-10 d) before
© 1993 Elsevier Science Publishers Ltd (UK) 0966 842X/93/$06.00 TRENDS
, N M,CROB,OLO
.'
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reverting to quiescent, noncytolytic memory T cells. Acute immunization with a viral expression vector such as vaccinia would therefore result in the transient generation of NP-specific CTLs, but would not stimulate resistance to subsequent intranasal challenge with influenza virus. NP-specific memory T cells would need to differentiate into activated CTLs over a period of days before virus clearance could be achieved. At that time, after virus challenge, virus infection would be well established in the respiratory tract. The naked DNA strategy employed by Ulmer et aL not only generates the NP gene product in a form capable of stimulating CTLs, but presumably also retains NPspecific CTLs or their precursors in an activated state, perhaps because of persistent NP gene expression at the site of DNA injection. The findings of Ulmer et al. raise the possibility that vaccination with DNA encoding antigenically conserved influenza A proteins could provide 'heterotypic' protection against epidemic influenza in humans. These results, along with similar observations by Robinson et al. on the induction in chickens of homotypic protective immunity to influenza using an influenza hemagglutinin-expressing plasmid 1°, suggest that naked DNA immunization may be a feasible strategy for many proteins. The advantages are clear. First, one can stimulate CTLs and antibodies to a specific viral polypeptide (or cohort of polypeptides), without introducing an infectious viral genome into the recipient. This is of considerable importance in designing vaccines for HIV, the hepatitis viruses and other viruses with a high morbidity index or propensity for long-term persistence in the body. Also, since different allelic forms of MHC molecules bind only a small subset of foreign antigenic peptides, based on the structure of their peptidebinding grooves, the T cell response of any individual to a foreign antigen will be dictated by the allelic form of the M H C molecules that the individual inherits. In outbred human populations, which display many different alleles of a particular M H C gene product, it is
important to have the largest possible array of immunogenic peptides from an antigen available for 'sampling' by the M H C molecules of an individual. In principle, DNAbased vaccines could incorporate the coding sequence for several proteins of a virus and could provide a large number of peptides for sampling by many different MHC alleles. This is an obvious advantage of DNA-based vaccines over synthetic-peptide-based vaccines, which can only allow sampling by one or a few M H C alleles. Finally, stability and ease of production make DNA-based vaccines extremely attractive for mass immunization, particularly in the developing countries. Enthusiasm for the potential of naked plasmid DNA as a vaccine must be tempered by several caveats. First, currently it is not clear which cell type must express the plasmid to stimulate an effective cellular and humoral immune response. The striated muscle cells, which are known to be capable of expressing foreign genes at the site of DNA injection, seem unlikely candidates because of their low level of expression of M H C molecules and their lack of the accessory molecules necessary for lymphocyte activation. Uptake and expression of the plasmid DNA by professional antigen-presenting cells, such as dendritic cells, would seem to be a more likely mechanism to account for the immunogenicity of these plasmids. Furthermore, it is not certain that these vaccines would lead to antigen persistence, since cells expressing the foreign gene product should ultimately be destroyed by the immune system. In contrast, if the antigen persists, chronic persistent expression of a foreign molecule and the associated
immune response could ultimately result in immune dysregulation and autoimmune injury. Finally, it is not clear that any vaccine using foreign DNA molecules at high copy number could meet current regulatory guidelines for recombinant DNA work in most developed countries. The recent success of Ulmer et al. 7 and Robinson et al. 1° in producing protective immunity against virus infection by DNA-mediated immunization opens up new avenues of research and development. Naked DNA immunization could prove to be an extremely powerful tool for basic immunological studies. Whether this strategy will yield new forms of vaccine for clinical use awaits further investigation of this novel and exciting approach.
Response from Ulmer, Donnelly and Liu
Harriet Robinson's studies in chickens using a homologous challenge system, immune responses have been generated against HIV gp160 (Ref. 1) and HIV gp120 (Ref. 2) in mice, and protective efficacy has been recently demonstrated against bovine herpes virus in cattle 3 using
In addition to our work in mice demonstrating heterotypic and homotypic immunity against heterologous and homologous challenges with influenza virus and
Acknowledgements I thank Dr Harriet Robinson for providing unpublisheddata, Mr S. Tykodifor reviewof the manuscript and Ms V. Calhoun for excellentsecretarialsupport.
References 1 Bjorkman,P.J. et al. (1987)Nature 329, 506-512 2 Brown,J.H. et al. (1993)Nature 364, 33-39 3 Wolff,J.A. et al. (1990)Science 247, 1465-1468 4 Isreal,M.A. etal. (1979)J. Virol. 29, 990-996 5 Seeger,C. et al. (1984)Proc. Natl Acad. Sci. USA 81, 5849-5852 6 Benvenisty,N. and Reshef,L. (1986) Proc. Natl Acad. Sci. USA 83, 9551-9555 7 Ulmer,J. etal. (1993)Science259, 1745-1749 8 Lin,Y. and Askonas,B. (1981)J. Exp. Med. 154, 225-234 9 Yewdell,J. et al. (1985)Proc.Natl Acad. Sci. USA 82, 1785-1789 10 Robinson,H.L.,Hunt, L.A.and Webster, R.G. (1993) Vaccine 11, 957-960
© 1993 Elsevier Science Publishers Ltd (UK) 0966 842X/93/$06.00
TRENDS
IN MICROBIOLOGY
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VoL.
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No.
9
DECEMBER
1993
A N D
C O M M E N ' f
Naked DNA and vaccine design Thomas J. Braciale ver the past decade, the revolution in molecular and structural biology has opened up the immune system to detailed analysis. The genes encoding critical molecules of the immune system have been identified and the structure of these genes has been determined. Immunologists now not only have detailed information on the structure of the immunoglobutin receptor on B cells, but also information on the structure and function of the T cell antigen receptor complex and the molecules encoded by genes of the major histocompatibility complex (MHC). Indeed, the three dimensional structure of both the M H C class I and, more recently, the M H C class II molecules has been solved by X-ray crystallographic analysis 1,2.These structural studies have directly established that the antigen receptor on T cells recognizes short peptide fragments of foreign antigenic proteins, such as bacterial and viral polypeptides, bound to MHC class I and class II molecules on the surface of antigenpresenting cells. This new insight into the nature of antigen recognition by T cells has led to the development of many novel strategies to manipulate the immune system and to treat autoimmune diseases. One area where the revolution in immunology has not, as yet, had its full impact is in vaccine development. At the moment, Jenner's approach of immunizing with a live, attenuated virus remains the most effective means of producing sustained immunity to most viruses. Part of the reason why live viruses are effective vaccines is that they efficiently activate the CD8 + precursor cells that give rise to the major antiviral effector cells: the MHC class I restricted cytolytic T cells. The reason for this lies in the cell biology of the synthesis, assembly and transport of the MHC class I molecule. M H C class I mol-
O
ecules capture viral peptides during their synthesis and assembly in the endoplasmic reticulum (ER). The MHC molecules transport the bound viral peptides from the ER to the cell surface, where the complex is displayed to the antigen receptor on CD8 ÷cytolytic T cell precursors and activated effector cytolytic T cells. To charge nascent MHC class I molecules, viral polypeptides must gain access to the cytoplasm of the cell. Here, the viral polypeptides are fragmented and then transported as peptide fragments into the ER. The most efficient way for viral polypeptides to gain access to the cytoplasm of an antigenpresenting cell for proteolytic fragmentation is for them to be expressed de n o v o in infected cells. Recently, a novel vaccination strategy has been developed using naked DNA encoding a viral polypeptide as an immunogen. This strategy is based on the fortuitous observation by Wolff et al. 3 that direct intramuscular inoculation of mice with plasmid DNA containing the gene for a marker protein driven by a strong eukaryotic promoter resulted in persistent expression of the gene in striated muscle at the site of injection. This finding extended earlier work on the uptake and expression of viral and eukaryotic DNA by direct DNA inoculation into experimental animals". The result of Wolff et al. has prompted researchers at Merck Research Laboratories and Vical to use the same strategy of DNA uptake and expression as a method of vaccination against influenza virus. As reported recently, Ulmer et al. v have injected mice with DNA encoding the type A influenza A/PR/8/34 nucleocapsid protein (NP) linked to the human cytoT.J. Braciale is in the Beirne Carter Center for Immunology Research, University of Virginia Health Sciences Center, MR4 Box 4012, Charlottesville, VA 22908, USA.
megalovirus promoter. Mice that had been so treated mounted a serum antibody response to the NP, and were primed for memory cytotoxic T cell (CTL) response to the NP. Most importantly, the mice injected with the DNA encoding the NP were protected against the morbidity and mortality of subsequent intranasal infection with a heterotypic strain of influenza virus. The development of antibodies to the NP by these mice after DNA injection implies that sufficient antigenic mass was produced in v i v o to stimulate antibody production. The NP is the major internal constituent of the influenza virion and is structurally conserved among type A influenza strains of different subtypes. Antibody to the NP is not, however, neutralizing, so that the protection against lethal challenge afforded by DNA vaccination is not antibody mediated but is probably caused by the in v i v o priming of NP-specific CTLs. The NP is a major target of the influenza-specific CTLs in the mouse strain used in this study (BALB/c). Although NP-specific CTLs can eliminate infectious virus from the lungs of infected mice 8 and promote recovery from lethal infections (T.J. Braciale, unpublished), earlier attempts to demonstrate resistance to infectious influenza challenge by immunizing with viral eukaryotic vectors expressing the NP gene, such as recombinant vaccinia viruses, did not succeed despite the induction of NP-specific CTLs in the immunized animals 9. The failure of these earlier attempts to demonstrate NP-specific protection probably reflects the transient nature of the CTL response to foreign antigen. Activated killer T cells arise from noncytolytic T cell precursors, which differentiate over a period of days into activated effectors after contact with viral peptide-MHC complexes. These killer T cells remain cytolytic only for a short time (5-10 d) before
© 1993 Elsevier Science Publishers Ltd (UK) 0966 842X/93/$06.00 TRENDS
, N M,CROB,OLO
.'
323
VOL.
1
NO.
9
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ER 1 9 9 3
NEWS
A N D
C O M M E N T
reverting to quiescent, noncytolytic memory T cells. Acute immunization with a viral expression vector such as vaccinia would therefore result in the transient generation of NP-specific CTLs, but would not stimulate resistance to subsequent intranasal challenge with influenza virus. NP-specific memory T cells would need to differentiate into activated CTLs over a period of days before virus clearance could be achieved. At that time, after virus challenge, virus infection would be well established in the respiratory tract. The naked DNA strategy employed by Ulmer et aL not only generates the NP gene product in a form capable of stimulating CTLs, but presumably also retains NPspecific CTLs or their precursors in an activated state, perhaps because of persistent NP gene expression at the site of DNA injection. The findings of Ulmer et al. raise the possibility that vaccination with DNA encoding antigenically conserved influenza A proteins could provide 'heterotypic' protection against epidemic influenza in humans. These results, along with similar observations by Robinson et al. on the induction in chickens of homotypic protective immunity to influenza using an influenza hemagglutinin-expressing plasmid 1°, suggest that naked DNA immunization may be a feasible strategy for many proteins. The advantages are clear. First, one can stimulate CTLs and antibodies to a specific viral polypeptide (or cohort of polypeptides), without introducing an infectious viral genome into the recipient. This is of considerable importance in designing vaccines for HIV, the hepatitis viruses and other viruses with a high morbidity index or propensity for long-term persistence in the body. Also, since different allelic forms of MHC molecules bind only a small subset of foreign antigenic peptides, based on the structure of their peptidebinding grooves, the T cell response of any individual to a foreign antigen will be dictated by the allelic form of the M H C molecules that the individual inherits. In outbred human populations, which display many different alleles of a particular M H C gene product, it is
important to have the largest possible array of immunogenic peptides from an antigen available for 'sampling' by the M H C molecules of an individual. In principle, DNAbased vaccines could incorporate the coding sequence for several proteins of a virus and could provide a large number of peptides for sampling by many different MHC alleles. This is an obvious advantage of DNA-based vaccines over synthetic-peptide-based vaccines, which can only allow sampling by one or a few M H C alleles. Finally, stability and ease of production make DNA-based vaccines extremely attractive for mass immunization, particularly in the developing countries. Enthusiasm for the potential of naked plasmid DNA as a vaccine must be tempered by several caveats. First, currently it is not clear which cell type must express the plasmid to stimulate an effective cellular and humoral immune response. The striated muscle cells, which are known to be capable of expressing foreign genes at the site of DNA injection, seem unlikely candidates because of their low level of expression of M H C molecules and their lack of the accessory molecules necessary for lymphocyte activation. Uptake and expression of the plasmid DNA by professional antigen-presenting cells, such as dendritic cells, would seem to be a more likely mechanism to account for the immunogenicity of these plasmids. Furthermore, it is not certain that these vaccines would lead to antigen persistence, since cells expressing the foreign gene product should ultimately be destroyed by the immune system. In contrast, if the antigen persists, chronic persistent expression of a foreign molecule and the associated
immune response could ultimately result in immune dysregulation and autoimmune injury. Finally, it is not clear that any vaccine using foreign DNA molecules at high copy number could meet current regulatory guidelines for recombinant DNA work in most developed countries. The recent success of Ulmer et al. 7 and Robinson et al. 1° in producing protective immunity against virus infection by DNA-mediated immunization opens up new avenues of research and development. Naked DNA immunization could prove to be an extremely powerful tool for basic immunological studies. Whether this strategy will yield new forms of vaccine for clinical use awaits further investigation of this novel and exciting approach.
Response from Ulmer, Donnelly and Liu
Harriet Robinson's studies in chickens using a homologous challenge system, immune responses have been generated against HIV gp160 (Ref. 1) and HIV gp120 (Ref. 2) in mice, and protective efficacy has been recently demonstrated against bovine herpes virus in cattle 3 using
In addition to our work in mice demonstrating heterotypic and homotypic immunity against heterologous and homologous challenges with influenza virus and
Acknowledgements I thank Dr Harriet Robinson for providing unpublisheddata, Mr S. Tykodifor reviewof the manuscript and Ms V. Calhoun for excellentsecretarialsupport.
References 1 Bjorkman,P.J. et al. (1987)Nature 329, 506-512 2 Brown,J.H. et al. (1993)Nature 364, 33-39 3 Wolff,J.A. et al. (1990)Science 247, 1465-1468 4 Isreal,M.A. etal. (1979)J. Virol. 29, 990-996 5 Seeger,C. et al. (1984)Proc. Natl Acad. Sci. USA 81, 5849-5852 6 Benvenisty,N. and Reshef,L. (1986) Proc. Natl Acad. Sci. USA 83, 9551-9555 7 Ulmer,J. etal. (1993)Science259, 1745-1749 8 Lin,Y. and Askonas,B. (1981)J. Exp. Med. 154, 225-234 9 Yewdell,J. et al. (1985)Proc.Natl Acad. Sci. USA 82, 1785-1789 10 Robinson,H.L.,Hunt, L.A.and Webster, R.G. (1993) Vaccine 11, 957-960
© 1993 Elsevier Science Publishers Ltd (UK) 0966 842X/93/$06.00
TRENDS
IN MICROBIOLOGY
324
VoL.
1
No.
9
DECEMBER
1993