exemplified by the Sabin polio vaccine, which is. *Author to whom all correspondence and reprint requests should be addressed. Department of Microbiology ...
REVIEW
Manipulation of the Semliki Forest Virus Genome and Its Potential for Vaccine Construction Gregory J. Atkins,* Brian J. Sheahan, and Peter Liljestr6m Abstract The Semliki Forest virus (SFV) expression vector consists of a plasmid based on the SFV infectious clone. Foreign genes may be inserted into the structural coding region, transcribed as RNA, and expressed in cell culture after transfection. RNA containing inserted sequences may be packaged into virions using a helper system. This allows efficient infection and expression without chemical transfection, but only one round of multiplication is possible. The biosafety of the system has been increased by the introduction of multiple mutations, specifying a maturation defect, into the helper. Potential vaccines can be constructed by insertion of genes coding for antigenic proteins into the vector. Following insertion of the influenza virus nucleoprotein (NP) into the SFV vector, immunity was induced following injection of packaged or naked RNA into mice. The SFV vector is a "suicide" expression vector that has great potential for the construction of vaccines for both human and veterinary use. Index Entries: Semliki Forest virus; alphavirus; togavirus; vaccine; vector; biosafety; pathogenesis. Abbreviations: SFV, Semliki Forest virus; cDNA, complementary DNA.
expression of proteins from cloned genes, have been reviewed recently (2,3). This vector system is based on an infectious clone of SFV (Fig. 1) (4). The infectious clone consists of a cDNA copy of the virus genome, constructed from the sequence of a prototype strain of SFV (5-7). A bacteriophage promoter, such as SP6, is inserted upstream of the viral sequence and RNA is transcribed in vitro using SP6 RNA polymerase. The plasmid containing these sequences is labeled pSP6-SFV4. RNA transcribed from this plasmid DNA is transfected into cultured cells. Either electroporation or chemical transfection (using lipofectin) is employed. The positive-stranded RNA corresponds to the virus genome and is able to initiate a productive viral multiplication cycle on transfection. Conventional virus vaccines are typically of the live attenuated or killed inactivated types. This is exemplified by the Sabin polio vaccine, which is
1. Introduction The use of animal virus expression vectors has three main objectives: to induce the synthesis of desired proteins from cloned sequences, to construct novel vaccines, and to attempt gene therapy. The first systems to be used were based on large DNA viruses such as baculoviruses and vaccinia virus, where foreign genes could be inserted into the viral genome and expressed along with viral genes. Later, other DNA viruses such as adenovirus and herpes virus were used. Positive-stranded RNA viruses such as poliovirus and alphaviruses have been used by transcribing RNA containing cloned sequences from a complementary (c)DNA copy, and retrovirus vectors have been used in initial attempts at gene therapy (1). Here we wish to review the potential of one RNA virus expression system, based on Semliki Forest virus (SFV), for vaccine construction. Other uses of this system, such as the high level
*Author to whom all correspondence and reprint requests should be addressed. Department of Microbiology, Moyne Institute, Trinity College, Dublin 2, Ireland. Molecular Biotechnology9
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Humana Press Inc. All rightsof any nature whatsoever reserved. 1073-6085/1996/5:1/33-38/56.50
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5'
,
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;,
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Fig. 1. Diagram of the Semliki Forest virus infectious clone. DNA is replicated from the origin (ori). To produce infectious RNA, the plasmid is linearized with the restriction enzyme SpeI, and RNA transcribed using bacteriophage SP6 polymerase and the SP6 promoter. The SFV nonstructural genes nsP1, nsP2, nsP3, and nsP4, and the structural genes El, E2, E3 (the 3 envelope genes), and C (the capsid protein gene) are shown in order of transcription. 6K is a small protein that is not incorporated into virions. The location of the SQL mutation, at the cleavage site of the E2 and E3 envelope proteins from the p62 precursor, is indicated. a live vaccine given orally and is in use in most countries, and the Salk polio vaccine (the original vaccine), which is a chemically inactivated vaccine given by injection. Live attenuated vaccines have the advantage that they stimulate good immunity, but can cause disease in immunocompromised hosts or by reversion. Inactivated vaccines generally are less immunogenic, but are safer than attenuated vaccines. One vaccine produced using an expression vector, that against hepatitis B virus, whose surface antigen is expressed in yeast, is currently in use. None of the viral expression vectors described have yet produced a licensed vaccine.
2. Semliki Forest Virus Semliki Forest virus is an enveloped, positivestranded RNA virus of the genus Alphavirus and the family Togaviridae. It is a mosquito-transmitted virus and several isolates have been made, mainly from central Africa. It is able to infect most types of animal cell, including mammalian,
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+42S RNA I1 VIRIONS Encapsidation
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Fig. 2. Schematic diagram of the RNA and protein species involved in Semliki Forest virus multiplication. avian, and insect cells. This broad host range may be an advantage when designing potential vaccines. The virion consists of a nucleocapsid made up of the capsid (core, c) protein plus the viral RNA. Surrounding this is the lipid-containing envelope derived from the host cell plasma membrane. Inserted into this envelope are spikes consisting of the three envelope glycoproteins E 1, E2, and E3. The molecular biology of alphavirus gene expression, multiplication, and evolution has been recently reviewed (8). 2.1. Virus Multiplication The virus multiplication cycle is initiated by interaction of the E2 envelope protein with the receptor. The virus enters the cell by endocytosis and is uncoated by a process that involves pHdependent fusion of the envelope with lysosomal membranes, a process that involves the E1 protein. The virion RNA is labeled 42S and can be directly translated into four nonstructural proteins that are concerned with RNA synthesis (Fig. 2). These nonstructural proteins are labeled nspl, nsp2, nsp3, and nsp4 and are formed from a polyprotein precursor by a process of post-translational cleavage. The positive-stranded 42S RNA is transcribed to a negative strand, which can in turn be transcribed to two positive-stranded RNA species. New 42S RNA can be either translated, used as a template for negative-strand RNA synthesis, or encapsidated. Encapsidation involves
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recognition by the capsid protein of an encapsidation signal located in the nonstructural region of the genome. However, a second RNA species, labeled 26S, is formed by utilization of an internal promoter on the 42S negative strand. This comprises about one third of the viral genome and codes for the viral structural proteins only (i.e., the C, El, E2, and E3 proteins). The amplification of one region of the genome through the synthesis of 26S subgenomic RNA has been exploited in the design of the vector system and is an important feature of it. The viral structural proteins are also formed by post-translational cleavage. However, the capsid protein has autoproteinase activity and is able to self-cleave from the nascent envelope proteins during translation, a process that can be exploited in the vector system. Mature virus particles are formed by budding from cell membranes, and cleavage of the precursor to the E2 and E3 proteins, labeled p62, occurs during this process.
induces central nervous system demyelination in infected mice, which is immune-mediated. It has therefore been used as a model for human demyelinating disease such as multiple sclerosis (15). All strains of SFV are lethal for neonatal mice. The SFV4 strain of SFV, produced by transcription of the infectious clone pSP6-SFV4, is lethal when given i.n. to adult mice, although only a proportion of mice die when the virus is given i.p. (16). The virus that was sequenced for the construction of the infectious clone, termed the prototype strain, is another independent isolate of SFV (17). Its pathogenicity for mice is identical to the SFV4 strain (16). One other aspect of SFV pathogenicity is the ability of the avirulent A7 strain to infect the developing fetus and induce abortion; derivatives of A7, such as ts 22, are teratogenic (13,18,19). Thus, SFV infection of pregnant mice is one of the few good models of viral teratogenesis and fetal infection (20).
2.2. SFV Pathogenicity
3. Molecular Analysis of SFV Pathogenicity
SFV is a minor human pathogen in Africa, where it causes a flu-like illness. The original SFV isolation was made in Uganda (9), but several more independent isolates have been made, and a human outbreak of this disease in Central African Republic has been described (10). A laboratory infection, in which a laboratory worker died, has also been described. This accident was reported in 1979 (11), and despite extensive use of SFV in many laboratories since then, no further fatal infections have been reported although a small number of documented laboratory infections have occurred (12). It is probable that human infection by SFV is incidental to the normal life cycle of the virus, although the natural host is unknown. SFV has been exploited for many years as a model of viral neuropathogenesis (13,14). Virulent strains of SFV such as L10 (derived from the original isolate) kill adult mice through a lethal encephalitis when given intraperitoneally (i.p.), subcutaneously, or intranasally (i.n.). Avirulent strains such as A7 (an independent isolate) do not kill adult mice when given by these routes, but induce immunity to virulent SFV challenge. A7-SFV
Mutants of the virulent L10 strain of SFV have been isolated that are less virulent. Two of these, M9 and M136, induce immune-mediated demyelination (13). Mutants of A7, such as ts 22, are teratogenic. Although these mutations have not yet been mapped, the pathogenicity of mutants of SFV4 of known sequence location has been studied. The mutant mL has uncleaved p62; this mutant multiplies slowly but nevertheless induces demyelination following i.n. infection. Another mutant in the E2 protein, mut 64, kills adult mice when given i.n. but i.p. infected mice survive, although the fetuses are lethally infected (16). Sequencing of the avirulent A7 strain of SFV recently has been completed (Tarbatt, Sheahan, and Atkins, unpublished). The structural protein region of A7 contains several amino acid substitutions compared to SFV4. Also, the 3' noncoding region of A7 is longer than that of SFV4 and contains divergent as well as repeated sequences (21,22). The significance of these differences for the control of pathogenesis is not yet clear. However, one amino acid substitution in the E2 protein of SFV4, present in A7, does affect pathogenesis
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SFV RNA
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Fig. 3. Schematic diagram of the Semliki Forest virus vector system. RNA is transcribed from the appropriate cDNA clones, then electroporated into cultured cells. The foreign gene that has been cloned in this example is Lac Z.
but does not generate the full A7 phenotype (21). The significance of other sequence differences between SFV4 and A7 for pathogenesis remains to be investigated through the construction of chimeric virus and the use of site-directed mutagenesis.
4. The SFV Vector System In its original form, this consisted of two separate derivatives of the SFV infectious clone (Fig. 3) (23). In one, most of the structural protein region of the genome is deleted, and replaced with a short BamHI-SmaI-XmaI polylinker sequence to facilitate the insertion of foreign sequences. Following this sequence, a stop cassette consisting of three stop codons in all three reading frames is inserted. Thus, foreign sequences may be cloned between the polylinker and the stop cassette. Three forms of this vector plasmid have been constructed; in one (pSFV 1) the polylinker is situated just downstream of the 26S promoter and transcription initiation site. In another (pSFV2), the cloning site is positioned immediately after the ribosomal binding site for the SFV capsid gene. In the third (pSFV3), the initial ATG codon of the capsid gene is included. Thus, the gene amplification mechanism involved in the synthesis of 26S RNA is utilized. If necessary, a ribosomal binding site and/or initial met codon for a cloned protein sequence can also be included. Thus, cloned sequences are amplified in two ways. To maintain self-replication, the complete nonMOLr
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structural region (encoding the replicase complex) and 3' noncoding region are retained in the vector. Also, the 26S RNA amplification system is retained. Thus, very high levels of expression can be obtained. Expression may be increased even further by incorporation of the first 102 bases of the capsid protein gene resulting in a fusion protein with the heterologous insert. Since the capsid protein has autoproteinase activity, the heterologous protein will be released by cleavage when translated (24). The second component consists of a helper plasmid that provides a packaging system. This has a deletion of much of the nonstructural coding region of the virus, including the packaging signal that is recognized by the capsid protein. The helper RNA cannot therefore itself be packaged although it can provide structural proteins for the vector RNA. Thus if RNA transcribed from the vector and helper plasmids is dually transfected into susceptible host cells, particles containing recombinant RNA and foreign sequences can be produced. These particles can infect fresh cells, which will lead to expression of the cloned foreign sequences, without the need for transfection. However, fully infectious virus particles should not be produced, since the helper virus lacks a packaging signal and the vector lacks the structural protein genes. Hence the recombinant particles will undergo one round of multiplication only, and this property could be used to advantage in the construction of prototype vaccines.
4,1. Biosafety Considerations As discussed earlier, SFV is a human pathogen and therefore a safety consideration in the use of SFV as a vector in vaccine construction is whether fully infectious SFV particles could be produced. In the original vector system, described earlier, the two component plasmids have deletions and so cannot form infectious virus as they lack some genetic information. However, the packaging system involves dual electroporation of cells with the two RNA species, and low level recombination between alphavirus RNA species has been described (25). Therefore, it is at least theoretically possible for a recombination event to occur Volume 5, 1996
SFV and V a c c i n e C o n s t r u c t i o n
between the vector and helper RNA species, to produce fully infectious RNA. In an attempt to prevent the formation of infectious particles by recombination, a cleavage deficiency was introduced into the envelope region of the helper virus used for packaging. This cleavage defect is in the E2/E3 cleavage from the p62 precursor, and results in the production of virus particles with uncleaved p62. However, the cleavage may be carried out in vitro by treatment with protease, producing fully infectious particles. The cleavage defect is produced by a triple mutation, termed SQL, and therefore is unlikely to revert. This was incorporated into the helper plasmid. Particles containing packaged RNA produced with this helper can be rendered infectious by treatment with chymotrypsin, which cleaves at a leucine residue introduced at the cleavage site (26).
5. Prototype Vaccine Production Using the SFV Vector Prototype vaccines may be produced by incorporating a cloned gene for a protective antigen into the SFV vector. Such a vector would be a "suicide vector," that is, it would go through one round of multiplication in the host and no further. It would thus be intermediate in properties between a conventional inactivated vaccine and a live attenuated vaccine. The SFV vector has been used to express the HIV envelope glycoproteins (27), but much of the preliminary work has been done using a suicide vector that expresses the influenza virus nucleoprotein (NP) (28). Such suicide particles induced a long-lasting CD8 § cytotoxic T-cell immune response with as little as 100 active particles. A high titre antibody response was also produced. One potential disadvantage of the suicide vector system is that it incorporates SFV proteins with the packaged RNA. To obviate this, it is possible to use naked RNA incorporating a cloned gene for an antigenic protein, i.e., unpackaged RNA. Preliminary experiments have shown that such RNA incorporating influenza virus NP gene does induce an immune response in mice, although less efficiently than packaged RNA (29).
MOtKULRRBIOTKHNOtOGV
37 6. Conclusions The SFV vector system is a safe and highly efficient system for expression of cloned genes in mammalian cells. It has great potential for the construction of vaccines for both human and veterinary use (28,29). Preliminary experiments indicate that vaccines produced using this system may have the advantage of efficient stimulation of the immune system (generally a property of conventional live attenuated vaccines) combined with inability to replicate (a property of inactivated vaccines).
Acknowledgments Studies of SFV in our laboratories are supported by the Wellcome Trust, the Health Research Board, the Multiple Sclerosis Society of Ireland (G. A. and B. S.), the Swedish Medical Research Council, Swedish Research Council for Engineering Sciences, EVA program of the EU and WHO (P. L.).
References 1. Rice,C. M. (ed.) (1992) Animal virus expressionvectors, in Seminars in Virology, vol. 3. 2. Liljestr6m, P. and Garoff, H. (1994) Expression of proteins using Semliki Forest virus vectors, in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A., Seidman, J. G., and Struhl, K., eds.), Saunders, London, section 16.20, pp. 1-16. 3. Liljestr6m,P. (1994) Alphavirus expression systems. Current Opin. Biotech. 5, 495-500. 4. Liljestr6m,P., Lusa, S., Huylebroeck,D., and Garoff, H. (1991) In vitro mutagenesisof a full-length cDNA clone of Semliki Forest virus--the small 6,000molecular-weightmembraneprotein modulates virus release. J. Virol. 65, 4107-4113. 5. Garoff, H., Frischauf, A.-M., Simons, K., Lehrach, H., and Delius, H. (1980) The capsid protein of SemlikiForest virus has clusters of basic amino acids and prolines in its amino-terminalregion. Proc. Natl. Acad. Sci. USA 77, 6376-6380. 6. Garoff, H., Frischauf, A.-M., Simons, K., Lehrach, H., and Delius, H. (1980) Nucleotide sequence of cDNA codingfor SemlikiForest virus membraneglycoproteins. Nature 288, 236-241. 7. Takkinen, K. (1986) Complete nucleotide sequence of the nonstructural protein genes of Semliki Forest virus. Nucleic Acids Res. 14, 5667-5682. 8. Strauss, J. H. and Strauss, E. G. (1994) The alphaviruses: gene expression, replication and evolution. Microbiol. Revs. 58, 491-562.
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9. Smithburn, K. C. and Haddow, A. J. (1944) Semliki Forest virus. I. Isolation and pathogenic properties. J. Immunol. 49, 141-157. 10. Mathiot, C. C., Grimaud, G., Garry, P., Bouquety, J. C., Mada, A., Daguisy, A. M., and Georges, A. J. (1990) An outbreak of human Semliki Forest virus infection in Central African Republic. Am. J. Trop. Med. Hyg. 42, 386-393. 11. Williams, W. R., Kaluza, G., and Boschek, C. B. (1979) Semliki Forest virus: cause of a fatal case of human encephalitis. Science 203, 1127-1129. 12. Collins, C. H. (1988) Laboratory-Acquired Infections. Butterworths, London. 13. Atkins, G. J., Sheahan, B. J., and Dimmock, N. J. (1985) Semliki Forest virus infection of mice: a model for the genetic and molecular analysis of viral pathogenicity. J. Gen.Virol. 66, 395-408. 14. Atkins, G. J., Balluz, I. M., Glasgow, G. M., Mabruk, M. J. E. M. F., Natale, V. A. I., Smyth, J. M. B., and Sheahan, B. J. (1994) Analysis of the molecular basis of neuropathogenesis of RNA viruses in experimental animals: relevance for human disease? Neuropathol. Appl. Neurobiol. 20, 91-102. 15. Atkins, G. J., Sheahan, B. J., and Mooney, D. A. (1990) Pathogenicity of Semliki Forest virus for the rat central nervous system and primary rat neural cell cultures--possible implications for the pathogenesis of multiple sclerosis. Neuropathol. Appl. Neurobiol. 16, 57-68. 16. Glasgow, G. M., Sheahan, B. J., Atkins, G. J., Wahlberg, J. M., Salminen, A., and Liljestrtim, P. (1991) Two mutations in the envelope glycoprotein E2 of Semliki Forest virus affecting the maturation and entry patterns of the virus alter pathogenicity for mice. Virology 185, 741-748. 17. Henderson, B. E., Metselaar, D., Kirya, G. B., and Timms, G. L. (1970) Investigations into yellow fever virus and other arboviruses in the northern regions of Kenya. Bull WHO 42, 787-795. 18. Hearne, A. M., O'Sullivan, M. A., and Atkins, G. J. (1986) Infection of cultured early mouse embryos with Semliki Forest and rubella viruses. J. Gen. Virol. 67, 1091-1098. 19. Hearne, A. M., O'Sullivan, M. A., and Atkins, G. J. (1987) Isolation and preliminary characterization of
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Semliki Forest virus mutants with altered pathogenicity for mouse embryos. J. Gen. Virol. 68, 107-113. Atkins, G. J., Mabruk, M. J. E. M. F., Glasgow, G. M., Griffin, A. M., and Sheahan, B. J. (1995) Mechanisms of viral teratogenesis. Rev. Med. Virol. 5, 75-86. Glasgow, G. M., Killen, H. M., LiljestrSm, P., Sheahan, B. J., and Atkins, G. J. (1994) A single amino acid change in the E2 spike protein of a virulent strain of Semliki Forest virus attenuates pathogenicity. J. Gen. Virol. 75, 663-668. Santagati, M. G., It~ranta, P. V., Koskimies, P. R., M~itt~i, J. A., Salmi, A. A., and Hinkkanen, A. E. (1994) Multiple repeating motifs are found in the 3'terminal nontranslated region of Semliki Forest virus A7 genome. J. Gen. Virol. 75, 1499-1504. Liljestr6m, P. and Garoff, H. (1991) A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Bio/Technology 9, 1356-1361. Sjt~berg, E. M., Suomalainen, M., and Garoff, H. (1994) A significantly improved Semliki Forest virus expression system based on translation enhancer segments from the viral capsid gene. Bio/Technology 12, 1127-1131.
25. Weiss, B. G. and Schlesinger, S. (1991) Recombination between Sindbis virus RNAs. J. Virol. 65, 40174025. 26. Berglund, P., Sjt~berg, M., Sheahan, B. J., Atkins, G. J., Garoff, H., and Liljestrtim, P. (1993) Semliki Forest virus expression system: production of conditionally infectious recombinant particles. Bio/Technology 11, 916-920. 27. Paul, N. L., Marsh, M., McKeating, J. A., Schulz, T. F., LiljestrOm, P., Garoff, H., and Weiss, R. A. (1993) Expression of HIV-1 envelope glycoproteins by Semliki Forest virus vectors. AIDS Res. Hum. Retroviruses 9, 963-970. 28. Zhou, X., Berglund, P., Zhao, H., Liljestrt~m, P., and Jondal, M. (1995) Generation of cytotoxic and humoral immune responses using non-replicative recombinant Semliki Forest virus, in press. 29. Zhou, X., Berglund, P., Rhodes, G., Parker, S. E., Jondal, M., and Liljestr6m, P. (1995) Self-replicating Semliki Forest virus RNA as recombinant vaccine. Vaccine 12, 510-514.
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