Adenovirus vectors for gene expression Murty V.R. Chengalvala, Michael D. Lubeck, Bernard J. Selling, Robert J. Natuk, Kuo-Hom L. Hsu, Bruce B. Mason, Pranab K. Chanda, Ramesh A. Bhat, Bheem M. Bhat, Satoshi Mizutani, Alan R. Davis and Paul P. Hung Wyeth-Ayerst
Research, Philadelphia, Pennsylvania, USA
Adenoviruses possess a combination of features that make them highly suitable as vectors for expression of heterologous genes. Non-conditional and non-defective adeno-vectors have been constructed to obtain high level expression of a number of foreign genes and some of them have been shown in animal models to exhibit excellent promise as vaccine candidates. Current Opinion in Biotechnology 1991, 2:718-722
Introduction
species with an identical 5' untranslated leader sequence, the tripartite leader (tpl).
In recent years, utilization of infectious mammalian virus vectors, especially adenovirus (Ad) and vaccinia virus has received increasing attention for the expression of foreign proteins. Adenoviruses appear to be particularly attractive for this purpose, in that their molecular biology and DNA structure have been very well characterized, so that foreign genes can be inserted with relative ease resulting in stable recombinants. Moreover, the ability of Ads to infect a wide spectrum of cells to yield high copy number of the genome as well as the strength of some adenoviral promoters to direct the synthesis of large amounts of foreign proteins make Ads highly suitable as expression vectors.
Design and construction of recombinant adenovirus vectors Adenoviruses exhibit two phases of gene expression, early and late, which are separated by the onset of DNA replication. During early gene expression, over 30 mRNAs have been identified that are transcribed from five noncontiguous regions (El, E2a, E2b, E3 and E4) but specific functions have been ascribed to only a few of their translation products [1]. During the late phase, early promoters become less active as the majority of the transcription originates at the major late promoter (MLP), generating polycistronic transcripts that are processed by alternative splicing pathways resulting in different mRNA
One important constraint associated with the utilization of Ads as an expression vector is their DNA packaging limit. A maximum of 2 kb of foreign DNA can be inserted into the wild-type vires genome; inserts larger than this are either subsequently deleted or result in incomplete encapsidation. To permit packaging of larger DNA inserts many investigators have employed Ad deletion mutants in which part of the dispensable E3 region and/or E1 region is substituted with foreign genes [2]. Recombinants with E1 region deletioi~.s have been successfully propagated in adenovirus-transformed cell lines that constitutively express E1 proteins. On the other hand, the E3 coding region has been shown to be non-essential for virus replication in cell culture; hence non-conditional helper-independent Ad vectors can be generated and are often preferred over E1 deletion vectors. Adenoviruses with large E3 deletions may accommodate insertions of up to 4 kb and potential deletions in the dispensable E4 region may even permit larger inserts [3]. It is possible to insert foreign genes without additional control elements, so that genes may be expressed by endogenous adenoviral regulatory elements. This greatly simplifies the engineering of constructs and decreases the possibility that some additional trans-acting factors may be necessary to achieve optimum transcription [4]. Insertion of foreign genes behind the E3 promoter can resuk in significant production of gene products during the early phase of infection [5]. As large amounts of Ad structural proteins are produced late in the Ad replication cycle, attempts have been made to create Ad vectors
Abbreviations Ad--adenovirus; gB--glycoprotein B; HBV--hepatitis B virus; HIV--human immunodeficiency virus; HSV --herpes simplex virus; ITR--inverted terminal repeat; MLP--major late promoter; RSV--respiratory syncytial virus; tk--thymidine kinase; tpl--tripartite leader.
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Adenovirus vectors for gene expression Chengalvala et al.
in which heterologous proteins are also expressed under control of the late acting Ad regulatory elements. The foreign gene is positioned behind a cloned copy of the Ad MLP in an expression cassette that can be inserted at any of three different locations in the Ad genome: the E1 region, the E3 region, and between the E4 region and right hand inverted terminal repeat (ITR). In general, such cassettes are engineered to contain, in sequence, the Ad MLP, Ad tpl and foreign gene, which are then followed by polyadenylation and processing signals [6]. The tpl increases the efficiency of translation late in infection; the translation efficiency of the inserted gene is dependent upon the proximity of its initiation codon to the tpl [7]. Sequences within the first intervening region between leaders 1 and 2 of the tpl may contribute significantly to expression of appended genes during late stages of infection by increasing transcription initiation from MLP [8°]. To generate infectious recombinant Ad, a linearized plasmid consisting of a portion of Ad genome and cloned foreign gene, is transfected into cultured cells along with a fragment of Ad DNA containing overlapping sequences, such that the transfected fragments together encompass the entire Ad genome. In vivo homologous recombination at the overlapping site results in the formation of viable virus [9]. Recombinant Ads have been developed as high level expression vectors for a number of important proteins, and have also been extensively used to study the functional importance of expressed proteins [10,11°]. Adenovirus vectors have also been adapted for studying tissue specific expression of genes and establishing stable cell lines expressing foreign genes [ 12].
Adenovirus vectors as vaccines Adenoviruses have been identified as promising live recombinant vaccine vectors. An impressive safety record for Ad4 and Ad7 has emerged during the past two decades as a result of their use as vaccines for prevention of acute respiratory disease in humans. Oral administration of these Ad vaccines in enteric-coated capsules resuits in an asymptomatic intestinal infection that induces protective immune responses to Ad. Oral administration of recombinant Ads to primates has recently been shown to induce immune responses to the foreign protein that is being expressed by the Ad vector, suggesting the use of orally administered recombinant Ad as a general approach to vaccination.
Adeno-hepatitis B vaccines Adeno-vectored hepatitis B virus (HBV) vaccines offer an attractive alternative to currently available injectable hepatitis B vaccines, which are too expensive to allow worldwide immunization. Recombinant Ads expressing the major surface a n t i g e n - - H B s A g - - o f HBV were constructed
by substituting part of the E3 region for surface antigen gene. The level of expression of HBsAg could be increased by at least 35-fold, rivaling the production of Ad capsid proteins, by placing the foreign gene under the control of MLP in a terminal cassette rather than under control of the E3 promoter [5,6,8°]. HBsAg produced by cells infected with these viruses is assembled and secreted into the medium as 22 nm particles that are very similar in size and structure to serum derived HBsAg particles. Recently, we evaluated the replication of Ad4- and Ad7vectored hepatitis B vaccines, as well as the capacity to induce humoral responses to HBsAg, in dogs [13°°]. Lung inoculations of dogs (by trans-tracheal injection) elicited large antibody responses to HBsAg and Ad. Substantial secondary responses were produced upon sequential immunization with heterotypic Ad7- and Ad4recombinant hepatitis B vaccines. Fine specificity analysis of the immune response revealed that replication of adeno-hepatitis vectors induces high avidity antiHBsAg antibodies that are directed against the primary neutralizing determinant of HBsAN [13oo]. The susceptibility of chimpanzee to enteric infection by Ad7- and Ad4-recombinant HBV vaccines has also been demonstrated by our group [14]. Significant anti-HBsAg responses were seen in chimpanzees after sequential immunization with Ad7- and Ad4-HBsAg viruses. In this study, protective efficacy was shown upon challenge of two chimpanzees that had detectable anti-HBsAg responses with live HBV. One chimpanzee was protected from acute hepatitis and the other chimpanzees experienced modified HBV-induced disease. These findings demonstrate the feasibility of using oral enteric vaccinations with recombinant adenoviruses to induce protective immune responses to foreign viral gene products. In order to enhance the response to HBsAg, the HBV core antigen that contains a dominant T-cell epitope has recently been co-expressed with HBsAg in the same recombinant. The HBsAg gene was inserted in an expression cassette between the E4 region and the right hand ITR and the core antigen was placed under the control of E3 promoter in deleted E3 region. Cells infected with this virus produced significant amounts of core antigen and HBsAg [15]. When tested in the dog model, the recombinant virus produced good antibody responses against core antigen and HBsAg. It is possible that by using this approach recombinants could be created that co-express and direct assembly of particles containing both core antigen and HBsAg, thus resembling an HBV virion without its genome. As such particles are known to be highly immunogenic, it is anticipated that such recombinants would have superior immunogenicity compared with current vaccines that consist of only HBsAg particles.
Adeno-human immunodeficiency virus vaccines The major focus of acquired immune deficiency syndrome (AIDS) subunit vaccine development, has centered on the human immunodeficiency virus (HIV)envelope glycoprotein that has been shown to induce
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Expressionsystems neutralizing immune responses. Dewar e t al. [16] have developed an Ad5-HIV recombinant that expresses the entire HIV-envelope protein from the e n v gene under the control of E3 promoter. When tested in cotton rats, an animal model for Ad5 infection, this recombinant virus produced enzyme-linked immunosorbent assay antibodies against envelope protein. A double recombinant Ad7 containing both rev, which encodes a regulatory protein of HIV, and e n v genes was constructed by cloning r e v gene in the deleted E3 region a n d t h e e n v gene cassette in the terminal region [17..]. This recombinant virus expressed high levels of envelope protein, similar to those attained upon co-infection of cells with individual viruses expressing e n v or r e v genes suggesting that r e v gene product, when supplied in t r a n s could augment the synthesis of envelope protein [17"]. The biological activity of recombinant adenovirus-coded HIV envelope protein was demonstrated by the induction of syncytia formation in HeLa cells bearing CD4 cellsurface receptors upon infection with these recombinants. Lung inoculation of dogs with the Ad7-HIV e n v - - r e v virus induced anti-gp160 antibodies by 6-43 weeks post-inoculation. Booster immunization with an Ad4-HIV e n v - r e v virus substantially enhanced the antigp160, -gpl20, and -gp41 responses.
or higher than, those produced in HSV1 infected cells. Intra-peritoneal administration of the recombinant virus into mice elicited complement-dependent neutralizing antibodies against HSV1 and HSV2. Protection experiments showed that after vaccination with adeno-HSV vaccine mice survived HSV infection. Recently, Johnson's group has demonstrated that Ad-gB infected cells may be used as target cells for testing cytotoxic T-cell activity in HSVl-infected mice. The murine cytotoxic T cells were found to recognize gB protein expressed by an Ad recombinant, suggesting that adenovirus vectors do not induce generalized defects in antigen processing and presentation. This finding indicates that the use of recombinant vectors may offer an advantage over transfected cell lines in that they can be conveniently used to study cytotoxic T-cell responses in animals of different haplotypes and in humans. Similarly, Ad5 recombinants that express human cytomegalovirus proteins [22], the glycoprotein of vesicular stomatitis virus [23], or the rabies glycoprotein [24] have also been shown to be effective as vaccines in animal models. The above studies clearly demonstrate the promise of the recombinant Ad approach in inducing significant immune responses and collectively suggest its use as a general strategy for vaccine development.
Adeno-respiratory syncytial virus vaccine The failure of conventional approaches for respiratory syncytial virus (RSV) vaccination, such as the use of formalin-inactivated or live attenuated vaccines, has prompted interest in the development of subunit or live recombinant vaccines. It is anticipated that recombinant adenoviral vaccines will be able to induce both humoral and cellular immunity, making these live viruses good candidates for RSV vaccine development. Collins e t al. [18] have developed an Ad5-RSV recombinant expressing fusion glycoprotein of RSV that can induce protective efficacy in cotton rats. This approach was further extended by developing two heterotypic Ads, Ad4F and Ad7F, in which the RSV fusion glycoprotein gene was substituted for the E3 region. As cotton rats are not susceptible to lung infection by Ad4 or Ad7, dog and ferret models were evaluated for protection studies using sequential immunization with heterotypic viruses. Intra-tracheal and intranasal immunization of dogs and ferrets, respectively, induced neutralizing antibody responses and protection of the animals from RSV infection upon challenge with live RSV [19].
Adeno-herpes simplex virus vaccine Glycoprotein B (gB) of herpes simplex virus (HSV) type1 is an essential component of the HSV1 virus envelope that induces neutralizing antibodies in HSV1 infected animals [20]. Johnson and his colleagues [21.] constructed an Ad5-based virus, in which the gB gene was coupled to an SV40 early promoter and inserted into a deleted E3 region. This virus vector induces synthesis of gB in murine and human cells at levels equal to,
Adeno-vectors for gene therapy Recombinant Ad can be used as an efficient delivery system in a broad range of host cells. Unlike retroviral vectors that require active proliferation of target cells for expression of newly transfected genes, adenoviral vectors can transfer recombinant genes and direct their expresion in cells with little or no proliferative activity. This property suggests that adenoviral vectors may be superior to retroviral vectors for gene therapy purposes. Two recent papers are worth mentioning in this regard. The first paper describes a potential approach for gene therapy against AIDS [25"]. An Ad recombinant encoding HSV-thymidine kinase (tk) gene under the transcriptional control of the long terminal repeat of HIV-1 was constructed to express the cytotoxic t k gene only in cells synthesizing the HIV-1 regulatory protein, Tat. The specificity of tk-mediated toxicity for HW-infected cells was further increased by including the Rev response element downstream of the t k gene, which affords Rev-dependent post-transcriptional regulation of t k RN_& T h e recombinant was replication defective in human cells because the chimeric t k gene was placed in the Ad early regions, 1A and 1B, with concomitant deletion in the E1 coding sequences. When this virus was allowed to infect HIVinfected cells in the presence of ganciclovir (an analogue of guanosine), the t k gene was expressed, and the thymidine kinase selectively converted ganciclovir to its monophosphate. This was subsequently converted by cellular phosphorylases to its triphosphate, whose incorporation into DNA led to inhibition of cellular DNA synthesis, re-
Adenovirus vectors for gene expression Chengalvalaet al. suiting in cell death [23]. Although this approach represents a potential therapeutic strategy against HW infection, its success depends upon the selective delivery of such viruses to HIV-infected cells alone, so that uninfected cells are spared. A second recent paper on the use of Ad for gene therapy [26°] reported the construction of a replication-defective adenovirus vector containing the 0 t l - a n t i t r y p s i n gene. This virus infects lung epithelial cells i n v i t r o as well as i n vivo, resulting in the transfer of the %-antitrypsin gene. D e n o v o expression of % antitrypsin protein following intra-tracheal inoculation with the virus persisted for at least one week and the secreted protein was functionally active. Although this approach to gene therapy appears promising and offers hope of treating lethal hereditary disorders, it is not clear at present whether the therapy is long lasting or whether it might require repeated administration of the recombinant virus.
with a Recombinant Adenovirus. Proc Natl Acad Sci USA 1985, 82:7560-7564. 7.
BERKNERKL, SCHAFFHAUSENBS, ROBERTSTM, SHARP PA: Abundant Expression of Polyomavirus Middle T Antigen and Dihydrofolate Reductase in an Adenovirus Recombinant. J Virol 1987, 61:1213-1220.
8. •
MASONBB, DAVISAR, BHAT BM, CHENGALVALAM, LUBECKi n , ZANDLEG, KOSTEK B, CHOLODOFSKY S, DHEER SK, MOLNARKIMBER KL, ET AL.: Adenovirus Vaccine Vectors Expressing Hepatitis B Surface Antigen: I m p o r t a n c e of Regulatory Elem e n t s in the Adenovirus Major Late Intron. Virology 1990, 177:452-461. Major late transcription regulatory sequences of Ad4 and Ad7 viruses were characterized and shown to be present within the first intron between leaders 1 and 2 of tpl. 9.
HANAHAND, GLUZMAN Y: Rescue o f Functional Replication Origins from Embedded Configurations in a Plasmid Carrying the Adenovirus Genome. Mol Cell Biol 1984, 4:302-309.
10.
LAMARCHEN, MASSIE B, RICHER M, PARADIS H, LANGELIERY: High Level Expression in 293 Cells of t h e Herpes Simplex Virus Type 2 Ribonucleotide Reductase Subunit 2 Using an Adenovirus Vector. J Gen Virol 1990, 71:1785-1792.
11. •
Conclusion Adenovirus expression vectors have been widely used as research tools to study gene expression. A rapidly growing body of evidence suggests the potential usefulness of live recombinant adenoviruses as vaccines for a number of infectious diseases. The exploration of adenovirus vectors as gene delivery systems for the treatment of hereditary disorders is in the offing.
ZHU X, CHEN J, YOUNG CSH, SILVERSTELN S: Reactivation of Latent Herpes Simplex Virus by Adenovirus Recombinants Encoding Mutant IE-O G e n e Products. J virol 1990, 64:4489-4498. Adenovector approach was used to show that a functional transactirational domain of a single HSV gene product, ICP0, is sufiqcient to reactivate latent hsv. 12.
KARLSSONS, DOREN KV, SCHWEIGERSG, NIENHUISAW, GLUZMAN Y: Stable Gene Transfer and Tissue-specific Expression of a H u m a n Globin Gene Using Adenoviral Vectors. EMBO J 1986, 5:2377-2385.
13.
CHENGALVALAM, LUBECKMD, DAVIS AR, MIZUTANIS, MOLNARKIMBERK, MORINJ, HUNG PP: Evaluation of Adenovirus Type 4 and Type 7 Recombinant Hepatitis B Vaccines in Dogs. Vaccine 1991, 9:485-490. The dog is shovm to be a suitable animal model for evaluating immune responses to vaccines based on Ad4 and Ad7 vectors. •e
References and recommended reading
14.
LUBECKMI), DAVIS AR, CHENGALVALAM, NATUK RJ, MORINJE, MOLNAR-KIMBERK, MASON BB, BHAT BM, MIZUTANI S, HUNG PP, PURCELL RH: I m m u n o g e n i c i t y and Efficacy Testing in Chimpanzees of an Oral Hepatitis B Vaccine Based o n Live Recombinant Adenovirus. Proc Natl Acad Sci USA 1989, 86:6763-6767.
15.
YE "~(F~¢',MASON BB, CHENGALVALAM, CHENG S-M, ZANDLE G, LUBECK MD, LEE S-W, MIZUTAN1 S, DAVIS AR, HUNG PP: Coexpression of Hepatitis B Virus Antigens by a Non-defective Adenovirus Vaccine Vector. Arch Virol 1991, 118:11-27.
16.
DEWARRE, NATARAJANV, VASUDEVACHARIMB, SALZMANNP: Synthesis and Processing of H u m a n Immunodeliciency Virus Type 1 Envelope Proteins Encoded by a Recombinant Hum a n Adenovirus. J Virol 1989, 63:129-136.
Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •• of outstanding interest 1.
LEVlNEAJ: The Adenovirus Early Proteins. Curr Topics Microbiol lmmunol 1984, 110:143-167.
2.
BERKNERKL: Development of Adenovirus Vectors for t h e Expression of Heterologous Genes. Biotechniques 1988, 6:616-629.
3.
TIKCHONENKOTI: Adenoviruses as Vectors for t h e Transfer of Genetic Information and for t h e Construction of N e w Type Vaccines. Adv Exp Med Biol 1989, 257:193-204.
4.
HAJ-AHMADY, GRAHAMFL: Development of Helper-independent H u m a n Adenovirus Vector and its Use in t h e Transfer of the Herpes Simplex Virus Thymidine Kinase Gene. J Virol 1986, 57:267-274.
5.
MORINJE, LUBECKMD, MASON BB, MOLNAR-K1MBERKL, DHEER SK, BHAT BM, CHANDA PK, NATUK RJ, CHENGALVALAMVR, MIZUTANI S, DAVIS AR, HUNG PP: Recombinant Adenovirus Vaccines for Hepatitis B Virus. In New Generation Vaccines edited by Woodrow GC, Levine MM [book]. New York: Marcel Dekker inc 1990, pp 448-457.
6.
DAVIS AR, KOSTEK B, MASON BB, HSIAO CL, MORINJE, DHEER SK, HUNG PP: Expression of Hepatitis B Surface Antigen
17. ee
CHANDAPK, NATUK RJ, MASON BB, BHAT BM, GREENBERG L~ DHEERSK, MOLNAR-KIMBERKL, MLZUTANIS, LUBECKMD, DAVIS AR, HUNG PP: High Level Expression of Envelope Glycoproteins of the H u m a n Immunodeficiency Virus Type 1 in Presence of rev Gene Using Helper-independent Adenovirus Type 7 Recombinants. Virology 1990, 175:535-547. HIV rev gene product when supplied in t r a m augmented the expression of HIV-1 env gene encoded by recombinant adenovirus vector. 18.
COLLINSPL, DAVIS AR, LUBECK MD, MIZUTANI S, HUNG PP, PRINCE GA, CAMARGOE, PURCELLRH, CHANOCK RM, MURPHY BR: Evaluation of t h e Protective Efficacy of Recombinant Vaecinia Viruses and Adenoviruses that Express Respiratory Syncytial Virus Glycoproteins. In Vaccines 90: Modern Approaches to New Vaccines Including Prevention of AIDS
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Expressionsystems edited by Brown F et al. [book]. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press 1990, pp 79-83. 19.
20.
Hsu K-H, LUBECKMD, DAVIS A, BHAT R, SELLINGB, BHAT B, MIZUTANI S, HUNG P: Immunogenicity and Protective Efficacy of Adenovirus Vectored Respiratory Syncytial Virus Vaccine. In Vaccines 91: Modern Approaches to N e w Va6 cines Including Prevention of AIDS edited by Brown F et al. [book]. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 1991, pp 293-297. MCDERMOTtMR, GRAHAMFL, HANKE T, JOHNSON DC: Protection of Mice Against Lethal Challenge with Herpes Simplex Virus by Vaccination with an Adenovirus Vector Expressing HSV Glycoprotein B. Virol 1989, 169:244-247.
21.
W1TMERLA, ROSENTHALKL, GRAHAMFL, FRIEDMANHM, WEE A, JOHNSONDC: Cytotoxic T Lymphocytes Specific for Herpes Simplex Virus (HSV) Studied Using Adenovirus Vectors Expressing HSV Glycoproteins. J Gen Virol 1990, 71:387-396. Cells infected with recombinant adenovirus vectors expressing foreign genes were used as target cells in studying cytotoxic T-cell responses. •
22.
23.
MARSHALLGS, RICC1ARDIRP, RANDO RF, PUCKJ, GE R, PLOTKIN SA, GONCZOL E: An Adenovirus Recombinant that Expreses the Human Cytomegalovirus Major Envelope Glycoprotein and Induces Neutralizing Antibodies. J Infect Dis 1990, 162:11771181. PREVEC L, SCHNEIDER M, ROSENTHAL KL, BELBECK LW, DERBYSHIREJB, GRAHAMFL: Use of Human Adenovirus-based
Vectors for Antigen Expression in Animals. J Gen Viro11989, 70:429-434. 24.
PREVECL, CAMPBELLJB, CHRISTIE BS, BELBECKL, GRAHAMFL: A Recombinant Human Adenovirus Vaccine Against Rabies. J Infect Dis 1990, 161:27-30.
25. ,
VENKATESHLK, ARENS MQ, SUBRAMANIANT, CHINNADURAIG: Selective Induction of Toxicity to Human Cells Expressing Human Immunodeficiency Virus Type 1 Tat by a Conditionally Cytotoxic Adenovirus Vector. Proc Natl Acad Sci USA 1990, 87:874643750. This paper showed the possibility of engineering a cytotoxic adenovirus that could selectively kill HIV-l-infected cells and its potential use for fighting AIDS. 26. •
ROSENFELDM, SIEGFRIED W, YOSHIMURA K, YONEYAMA R, FUKAYAMAM, STIER LE, PAAKKO PK, GILARDI P, STRATFORDPERRICAUDETLD, PERRICAUDETM, ETAL.: Adenovirus-mediated Transfer of a Recombinant alpha 1-Antitrypsin Gene to the Lung Epithelium I n Vivo. Science 1991, 252:431-434. Adenovirus was used as a gene transfer vector for use in the treatment of hereditary disorders.
M Chengvala, M Lubeck, B Selling, R Natuk, K-H Hsu, B Mason, P Chanda, R Bhat, B Bhat, S Mizutani, A Davis, P Hung, Wyeth-Ayerst Research Department of Microbiology and Biotechnology, PO Box 8299, Philadelphia, Pennsylvania 19101, USA.
Research, Philadelphia, Pennsylvania, USA
Adenoviruses possess a combination of features that make them highly suitable as vectors for expression of heterologous genes. Non-conditional and non-defective adeno-vectors have been constructed to obtain high level expression of a number of foreign genes and some of them have been shown in animal models to exhibit excellent promise as vaccine candidates. Current Opinion in Biotechnology 1991, 2:718-722
Introduction
species with an identical 5' untranslated leader sequence, the tripartite leader (tpl).
In recent years, utilization of infectious mammalian virus vectors, especially adenovirus (Ad) and vaccinia virus has received increasing attention for the expression of foreign proteins. Adenoviruses appear to be particularly attractive for this purpose, in that their molecular biology and DNA structure have been very well characterized, so that foreign genes can be inserted with relative ease resulting in stable recombinants. Moreover, the ability of Ads to infect a wide spectrum of cells to yield high copy number of the genome as well as the strength of some adenoviral promoters to direct the synthesis of large amounts of foreign proteins make Ads highly suitable as expression vectors.
Design and construction of recombinant adenovirus vectors Adenoviruses exhibit two phases of gene expression, early and late, which are separated by the onset of DNA replication. During early gene expression, over 30 mRNAs have been identified that are transcribed from five noncontiguous regions (El, E2a, E2b, E3 and E4) but specific functions have been ascribed to only a few of their translation products [1]. During the late phase, early promoters become less active as the majority of the transcription originates at the major late promoter (MLP), generating polycistronic transcripts that are processed by alternative splicing pathways resulting in different mRNA
One important constraint associated with the utilization of Ads as an expression vector is their DNA packaging limit. A maximum of 2 kb of foreign DNA can be inserted into the wild-type vires genome; inserts larger than this are either subsequently deleted or result in incomplete encapsidation. To permit packaging of larger DNA inserts many investigators have employed Ad deletion mutants in which part of the dispensable E3 region and/or E1 region is substituted with foreign genes [2]. Recombinants with E1 region deletioi~.s have been successfully propagated in adenovirus-transformed cell lines that constitutively express E1 proteins. On the other hand, the E3 coding region has been shown to be non-essential for virus replication in cell culture; hence non-conditional helper-independent Ad vectors can be generated and are often preferred over E1 deletion vectors. Adenoviruses with large E3 deletions may accommodate insertions of up to 4 kb and potential deletions in the dispensable E4 region may even permit larger inserts [3]. It is possible to insert foreign genes without additional control elements, so that genes may be expressed by endogenous adenoviral regulatory elements. This greatly simplifies the engineering of constructs and decreases the possibility that some additional trans-acting factors may be necessary to achieve optimum transcription [4]. Insertion of foreign genes behind the E3 promoter can resuk in significant production of gene products during the early phase of infection [5]. As large amounts of Ad structural proteins are produced late in the Ad replication cycle, attempts have been made to create Ad vectors
Abbreviations Ad--adenovirus; gB--glycoprotein B; HBV--hepatitis B virus; HIV--human immunodeficiency virus; HSV --herpes simplex virus; ITR--inverted terminal repeat; MLP--major late promoter; RSV--respiratory syncytial virus; tk--thymidine kinase; tpl--tripartite leader.
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Adenovirus vectors for gene expression Chengalvala et al.
in which heterologous proteins are also expressed under control of the late acting Ad regulatory elements. The foreign gene is positioned behind a cloned copy of the Ad MLP in an expression cassette that can be inserted at any of three different locations in the Ad genome: the E1 region, the E3 region, and between the E4 region and right hand inverted terminal repeat (ITR). In general, such cassettes are engineered to contain, in sequence, the Ad MLP, Ad tpl and foreign gene, which are then followed by polyadenylation and processing signals [6]. The tpl increases the efficiency of translation late in infection; the translation efficiency of the inserted gene is dependent upon the proximity of its initiation codon to the tpl [7]. Sequences within the first intervening region between leaders 1 and 2 of the tpl may contribute significantly to expression of appended genes during late stages of infection by increasing transcription initiation from MLP [8°]. To generate infectious recombinant Ad, a linearized plasmid consisting of a portion of Ad genome and cloned foreign gene, is transfected into cultured cells along with a fragment of Ad DNA containing overlapping sequences, such that the transfected fragments together encompass the entire Ad genome. In vivo homologous recombination at the overlapping site results in the formation of viable virus [9]. Recombinant Ads have been developed as high level expression vectors for a number of important proteins, and have also been extensively used to study the functional importance of expressed proteins [10,11°]. Adenovirus vectors have also been adapted for studying tissue specific expression of genes and establishing stable cell lines expressing foreign genes [ 12].
Adenovirus vectors as vaccines Adenoviruses have been identified as promising live recombinant vaccine vectors. An impressive safety record for Ad4 and Ad7 has emerged during the past two decades as a result of their use as vaccines for prevention of acute respiratory disease in humans. Oral administration of these Ad vaccines in enteric-coated capsules resuits in an asymptomatic intestinal infection that induces protective immune responses to Ad. Oral administration of recombinant Ads to primates has recently been shown to induce immune responses to the foreign protein that is being expressed by the Ad vector, suggesting the use of orally administered recombinant Ad as a general approach to vaccination.
Adeno-hepatitis B vaccines Adeno-vectored hepatitis B virus (HBV) vaccines offer an attractive alternative to currently available injectable hepatitis B vaccines, which are too expensive to allow worldwide immunization. Recombinant Ads expressing the major surface a n t i g e n - - H B s A g - - o f HBV were constructed
by substituting part of the E3 region for surface antigen gene. The level of expression of HBsAg could be increased by at least 35-fold, rivaling the production of Ad capsid proteins, by placing the foreign gene under the control of MLP in a terminal cassette rather than under control of the E3 promoter [5,6,8°]. HBsAg produced by cells infected with these viruses is assembled and secreted into the medium as 22 nm particles that are very similar in size and structure to serum derived HBsAg particles. Recently, we evaluated the replication of Ad4- and Ad7vectored hepatitis B vaccines, as well as the capacity to induce humoral responses to HBsAg, in dogs [13°°]. Lung inoculations of dogs (by trans-tracheal injection) elicited large antibody responses to HBsAg and Ad. Substantial secondary responses were produced upon sequential immunization with heterotypic Ad7- and Ad4recombinant hepatitis B vaccines. Fine specificity analysis of the immune response revealed that replication of adeno-hepatitis vectors induces high avidity antiHBsAg antibodies that are directed against the primary neutralizing determinant of HBsAN [13oo]. The susceptibility of chimpanzee to enteric infection by Ad7- and Ad4-recombinant HBV vaccines has also been demonstrated by our group [14]. Significant anti-HBsAg responses were seen in chimpanzees after sequential immunization with Ad7- and Ad4-HBsAg viruses. In this study, protective efficacy was shown upon challenge of two chimpanzees that had detectable anti-HBsAg responses with live HBV. One chimpanzee was protected from acute hepatitis and the other chimpanzees experienced modified HBV-induced disease. These findings demonstrate the feasibility of using oral enteric vaccinations with recombinant adenoviruses to induce protective immune responses to foreign viral gene products. In order to enhance the response to HBsAg, the HBV core antigen that contains a dominant T-cell epitope has recently been co-expressed with HBsAg in the same recombinant. The HBsAg gene was inserted in an expression cassette between the E4 region and the right hand ITR and the core antigen was placed under the control of E3 promoter in deleted E3 region. Cells infected with this virus produced significant amounts of core antigen and HBsAg [15]. When tested in the dog model, the recombinant virus produced good antibody responses against core antigen and HBsAg. It is possible that by using this approach recombinants could be created that co-express and direct assembly of particles containing both core antigen and HBsAg, thus resembling an HBV virion without its genome. As such particles are known to be highly immunogenic, it is anticipated that such recombinants would have superior immunogenicity compared with current vaccines that consist of only HBsAg particles.
Adeno-human immunodeficiency virus vaccines The major focus of acquired immune deficiency syndrome (AIDS) subunit vaccine development, has centered on the human immunodeficiency virus (HIV)envelope glycoprotein that has been shown to induce
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Expressionsystems neutralizing immune responses. Dewar e t al. [16] have developed an Ad5-HIV recombinant that expresses the entire HIV-envelope protein from the e n v gene under the control of E3 promoter. When tested in cotton rats, an animal model for Ad5 infection, this recombinant virus produced enzyme-linked immunosorbent assay antibodies against envelope protein. A double recombinant Ad7 containing both rev, which encodes a regulatory protein of HIV, and e n v genes was constructed by cloning r e v gene in the deleted E3 region a n d t h e e n v gene cassette in the terminal region [17..]. This recombinant virus expressed high levels of envelope protein, similar to those attained upon co-infection of cells with individual viruses expressing e n v or r e v genes suggesting that r e v gene product, when supplied in t r a n s could augment the synthesis of envelope protein [17"]. The biological activity of recombinant adenovirus-coded HIV envelope protein was demonstrated by the induction of syncytia formation in HeLa cells bearing CD4 cellsurface receptors upon infection with these recombinants. Lung inoculation of dogs with the Ad7-HIV e n v - - r e v virus induced anti-gp160 antibodies by 6-43 weeks post-inoculation. Booster immunization with an Ad4-HIV e n v - r e v virus substantially enhanced the antigp160, -gpl20, and -gp41 responses.
or higher than, those produced in HSV1 infected cells. Intra-peritoneal administration of the recombinant virus into mice elicited complement-dependent neutralizing antibodies against HSV1 and HSV2. Protection experiments showed that after vaccination with adeno-HSV vaccine mice survived HSV infection. Recently, Johnson's group has demonstrated that Ad-gB infected cells may be used as target cells for testing cytotoxic T-cell activity in HSVl-infected mice. The murine cytotoxic T cells were found to recognize gB protein expressed by an Ad recombinant, suggesting that adenovirus vectors do not induce generalized defects in antigen processing and presentation. This finding indicates that the use of recombinant vectors may offer an advantage over transfected cell lines in that they can be conveniently used to study cytotoxic T-cell responses in animals of different haplotypes and in humans. Similarly, Ad5 recombinants that express human cytomegalovirus proteins [22], the glycoprotein of vesicular stomatitis virus [23], or the rabies glycoprotein [24] have also been shown to be effective as vaccines in animal models. The above studies clearly demonstrate the promise of the recombinant Ad approach in inducing significant immune responses and collectively suggest its use as a general strategy for vaccine development.
Adeno-respiratory syncytial virus vaccine The failure of conventional approaches for respiratory syncytial virus (RSV) vaccination, such as the use of formalin-inactivated or live attenuated vaccines, has prompted interest in the development of subunit or live recombinant vaccines. It is anticipated that recombinant adenoviral vaccines will be able to induce both humoral and cellular immunity, making these live viruses good candidates for RSV vaccine development. Collins e t al. [18] have developed an Ad5-RSV recombinant expressing fusion glycoprotein of RSV that can induce protective efficacy in cotton rats. This approach was further extended by developing two heterotypic Ads, Ad4F and Ad7F, in which the RSV fusion glycoprotein gene was substituted for the E3 region. As cotton rats are not susceptible to lung infection by Ad4 or Ad7, dog and ferret models were evaluated for protection studies using sequential immunization with heterotypic viruses. Intra-tracheal and intranasal immunization of dogs and ferrets, respectively, induced neutralizing antibody responses and protection of the animals from RSV infection upon challenge with live RSV [19].
Adeno-herpes simplex virus vaccine Glycoprotein B (gB) of herpes simplex virus (HSV) type1 is an essential component of the HSV1 virus envelope that induces neutralizing antibodies in HSV1 infected animals [20]. Johnson and his colleagues [21.] constructed an Ad5-based virus, in which the gB gene was coupled to an SV40 early promoter and inserted into a deleted E3 region. This virus vector induces synthesis of gB in murine and human cells at levels equal to,
Adeno-vectors for gene therapy Recombinant Ad can be used as an efficient delivery system in a broad range of host cells. Unlike retroviral vectors that require active proliferation of target cells for expression of newly transfected genes, adenoviral vectors can transfer recombinant genes and direct their expresion in cells with little or no proliferative activity. This property suggests that adenoviral vectors may be superior to retroviral vectors for gene therapy purposes. Two recent papers are worth mentioning in this regard. The first paper describes a potential approach for gene therapy against AIDS [25"]. An Ad recombinant encoding HSV-thymidine kinase (tk) gene under the transcriptional control of the long terminal repeat of HIV-1 was constructed to express the cytotoxic t k gene only in cells synthesizing the HIV-1 regulatory protein, Tat. The specificity of tk-mediated toxicity for HW-infected cells was further increased by including the Rev response element downstream of the t k gene, which affords Rev-dependent post-transcriptional regulation of t k RN_& T h e recombinant was replication defective in human cells because the chimeric t k gene was placed in the Ad early regions, 1A and 1B, with concomitant deletion in the E1 coding sequences. When this virus was allowed to infect HIVinfected cells in the presence of ganciclovir (an analogue of guanosine), the t k gene was expressed, and the thymidine kinase selectively converted ganciclovir to its monophosphate. This was subsequently converted by cellular phosphorylases to its triphosphate, whose incorporation into DNA led to inhibition of cellular DNA synthesis, re-
Adenovirus vectors for gene expression Chengalvalaet al. suiting in cell death [23]. Although this approach represents a potential therapeutic strategy against HW infection, its success depends upon the selective delivery of such viruses to HIV-infected cells alone, so that uninfected cells are spared. A second recent paper on the use of Ad for gene therapy [26°] reported the construction of a replication-defective adenovirus vector containing the 0 t l - a n t i t r y p s i n gene. This virus infects lung epithelial cells i n v i t r o as well as i n vivo, resulting in the transfer of the %-antitrypsin gene. D e n o v o expression of % antitrypsin protein following intra-tracheal inoculation with the virus persisted for at least one week and the secreted protein was functionally active. Although this approach to gene therapy appears promising and offers hope of treating lethal hereditary disorders, it is not clear at present whether the therapy is long lasting or whether it might require repeated administration of the recombinant virus.
with a Recombinant Adenovirus. Proc Natl Acad Sci USA 1985, 82:7560-7564. 7.
BERKNERKL, SCHAFFHAUSENBS, ROBERTSTM, SHARP PA: Abundant Expression of Polyomavirus Middle T Antigen and Dihydrofolate Reductase in an Adenovirus Recombinant. J Virol 1987, 61:1213-1220.
8. •
MASONBB, DAVISAR, BHAT BM, CHENGALVALAM, LUBECKi n , ZANDLEG, KOSTEK B, CHOLODOFSKY S, DHEER SK, MOLNARKIMBER KL, ET AL.: Adenovirus Vaccine Vectors Expressing Hepatitis B Surface Antigen: I m p o r t a n c e of Regulatory Elem e n t s in the Adenovirus Major Late Intron. Virology 1990, 177:452-461. Major late transcription regulatory sequences of Ad4 and Ad7 viruses were characterized and shown to be present within the first intron between leaders 1 and 2 of tpl. 9.
HANAHAND, GLUZMAN Y: Rescue o f Functional Replication Origins from Embedded Configurations in a Plasmid Carrying the Adenovirus Genome. Mol Cell Biol 1984, 4:302-309.
10.
LAMARCHEN, MASSIE B, RICHER M, PARADIS H, LANGELIERY: High Level Expression in 293 Cells of t h e Herpes Simplex Virus Type 2 Ribonucleotide Reductase Subunit 2 Using an Adenovirus Vector. J Gen Virol 1990, 71:1785-1792.
11. •
Conclusion Adenovirus expression vectors have been widely used as research tools to study gene expression. A rapidly growing body of evidence suggests the potential usefulness of live recombinant adenoviruses as vaccines for a number of infectious diseases. The exploration of adenovirus vectors as gene delivery systems for the treatment of hereditary disorders is in the offing.
ZHU X, CHEN J, YOUNG CSH, SILVERSTELN S: Reactivation of Latent Herpes Simplex Virus by Adenovirus Recombinants Encoding Mutant IE-O G e n e Products. J virol 1990, 64:4489-4498. Adenovector approach was used to show that a functional transactirational domain of a single HSV gene product, ICP0, is sufiqcient to reactivate latent hsv. 12.
KARLSSONS, DOREN KV, SCHWEIGERSG, NIENHUISAW, GLUZMAN Y: Stable Gene Transfer and Tissue-specific Expression of a H u m a n Globin Gene Using Adenoviral Vectors. EMBO J 1986, 5:2377-2385.
13.
CHENGALVALAM, LUBECKMD, DAVIS AR, MIZUTANIS, MOLNARKIMBERK, MORINJ, HUNG PP: Evaluation of Adenovirus Type 4 and Type 7 Recombinant Hepatitis B Vaccines in Dogs. Vaccine 1991, 9:485-490. The dog is shovm to be a suitable animal model for evaluating immune responses to vaccines based on Ad4 and Ad7 vectors. •e
References and recommended reading
14.
LUBECKMI), DAVIS AR, CHENGALVALAM, NATUK RJ, MORINJE, MOLNAR-KIMBERK, MASON BB, BHAT BM, MIZUTANI S, HUNG PP, PURCELL RH: I m m u n o g e n i c i t y and Efficacy Testing in Chimpanzees of an Oral Hepatitis B Vaccine Based o n Live Recombinant Adenovirus. Proc Natl Acad Sci USA 1989, 86:6763-6767.
15.
YE "~(F~¢',MASON BB, CHENGALVALAM, CHENG S-M, ZANDLE G, LUBECK MD, LEE S-W, MIZUTAN1 S, DAVIS AR, HUNG PP: Coexpression of Hepatitis B Virus Antigens by a Non-defective Adenovirus Vaccine Vector. Arch Virol 1991, 118:11-27.
16.
DEWARRE, NATARAJANV, VASUDEVACHARIMB, SALZMANNP: Synthesis and Processing of H u m a n Immunodeliciency Virus Type 1 Envelope Proteins Encoded by a Recombinant Hum a n Adenovirus. J Virol 1989, 63:129-136.
Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •• of outstanding interest 1.
LEVlNEAJ: The Adenovirus Early Proteins. Curr Topics Microbiol lmmunol 1984, 110:143-167.
2.
BERKNERKL: Development of Adenovirus Vectors for t h e Expression of Heterologous Genes. Biotechniques 1988, 6:616-629.
3.
TIKCHONENKOTI: Adenoviruses as Vectors for t h e Transfer of Genetic Information and for t h e Construction of N e w Type Vaccines. Adv Exp Med Biol 1989, 257:193-204.
4.
HAJ-AHMADY, GRAHAMFL: Development of Helper-independent H u m a n Adenovirus Vector and its Use in t h e Transfer of the Herpes Simplex Virus Thymidine Kinase Gene. J Virol 1986, 57:267-274.
5.
MORINJE, LUBECKMD, MASON BB, MOLNAR-K1MBERKL, DHEER SK, BHAT BM, CHANDA PK, NATUK RJ, CHENGALVALAMVR, MIZUTANI S, DAVIS AR, HUNG PP: Recombinant Adenovirus Vaccines for Hepatitis B Virus. In New Generation Vaccines edited by Woodrow GC, Levine MM [book]. New York: Marcel Dekker inc 1990, pp 448-457.
6.
DAVIS AR, KOSTEK B, MASON BB, HSIAO CL, MORINJE, DHEER SK, HUNG PP: Expression of Hepatitis B Surface Antigen
17. ee
CHANDAPK, NATUK RJ, MASON BB, BHAT BM, GREENBERG L~ DHEERSK, MOLNAR-KIMBERKL, MLZUTANIS, LUBECKMD, DAVIS AR, HUNG PP: High Level Expression of Envelope Glycoproteins of the H u m a n Immunodeficiency Virus Type 1 in Presence of rev Gene Using Helper-independent Adenovirus Type 7 Recombinants. Virology 1990, 175:535-547. HIV rev gene product when supplied in t r a m augmented the expression of HIV-1 env gene encoded by recombinant adenovirus vector. 18.
COLLINSPL, DAVIS AR, LUBECK MD, MIZUTANI S, HUNG PP, PRINCE GA, CAMARGOE, PURCELLRH, CHANOCK RM, MURPHY BR: Evaluation of t h e Protective Efficacy of Recombinant Vaecinia Viruses and Adenoviruses that Express Respiratory Syncytial Virus Glycoproteins. In Vaccines 90: Modern Approaches to New Vaccines Including Prevention of AIDS
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Expressionsystems edited by Brown F et al. [book]. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press 1990, pp 79-83. 19.
20.
Hsu K-H, LUBECKMD, DAVIS A, BHAT R, SELLINGB, BHAT B, MIZUTANI S, HUNG P: Immunogenicity and Protective Efficacy of Adenovirus Vectored Respiratory Syncytial Virus Vaccine. In Vaccines 91: Modern Approaches to N e w Va6 cines Including Prevention of AIDS edited by Brown F et al. [book]. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 1991, pp 293-297. MCDERMOTtMR, GRAHAMFL, HANKE T, JOHNSON DC: Protection of Mice Against Lethal Challenge with Herpes Simplex Virus by Vaccination with an Adenovirus Vector Expressing HSV Glycoprotein B. Virol 1989, 169:244-247.
21.
W1TMERLA, ROSENTHALKL, GRAHAMFL, FRIEDMANHM, WEE A, JOHNSONDC: Cytotoxic T Lymphocytes Specific for Herpes Simplex Virus (HSV) Studied Using Adenovirus Vectors Expressing HSV Glycoproteins. J Gen Virol 1990, 71:387-396. Cells infected with recombinant adenovirus vectors expressing foreign genes were used as target cells in studying cytotoxic T-cell responses. •
22.
23.
MARSHALLGS, RICC1ARDIRP, RANDO RF, PUCKJ, GE R, PLOTKIN SA, GONCZOL E: An Adenovirus Recombinant that Expreses the Human Cytomegalovirus Major Envelope Glycoprotein and Induces Neutralizing Antibodies. J Infect Dis 1990, 162:11771181. PREVEC L, SCHNEIDER M, ROSENTHAL KL, BELBECK LW, DERBYSHIREJB, GRAHAMFL: Use of Human Adenovirus-based
Vectors for Antigen Expression in Animals. J Gen Viro11989, 70:429-434. 24.
PREVECL, CAMPBELLJB, CHRISTIE BS, BELBECKL, GRAHAMFL: A Recombinant Human Adenovirus Vaccine Against Rabies. J Infect Dis 1990, 161:27-30.
25. ,
VENKATESHLK, ARENS MQ, SUBRAMANIANT, CHINNADURAIG: Selective Induction of Toxicity to Human Cells Expressing Human Immunodeficiency Virus Type 1 Tat by a Conditionally Cytotoxic Adenovirus Vector. Proc Natl Acad Sci USA 1990, 87:874643750. This paper showed the possibility of engineering a cytotoxic adenovirus that could selectively kill HIV-l-infected cells and its potential use for fighting AIDS. 26. •
ROSENFELDM, SIEGFRIED W, YOSHIMURA K, YONEYAMA R, FUKAYAMAM, STIER LE, PAAKKO PK, GILARDI P, STRATFORDPERRICAUDETLD, PERRICAUDETM, ETAL.: Adenovirus-mediated Transfer of a Recombinant alpha 1-Antitrypsin Gene to the Lung Epithelium I n Vivo. Science 1991, 252:431-434. Adenovirus was used as a gene transfer vector for use in the treatment of hereditary disorders.
M Chengvala, M Lubeck, B Selling, R Natuk, K-H Hsu, B Mason, P Chanda, R Bhat, B Bhat, S Mizutani, A Davis, P Hung, Wyeth-Ayerst Research Department of Microbiology and Biotechnology, PO Box 8299, Philadelphia, Pennsylvania 19101, USA.
