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Alphavirus expression vectors and their use as recombinant vaccines: a minireview
GENE Gene 190 (1997) 191-195
Alphavirus expression vectors and their use as recombinant vaccines: a minireviewl Ioannis Tubulekas, Peter Berglund, Marina Fleeton, Peter Liljestrdm * Department of Biosciences, Karolinska Institute, S-141 57 Huddinge, Sweden Received
8 March
1996; revised 16 May 1996; accepted
18 May 1996; Received
by S.E. Hasnain
Abstract Alphavirus vectors have become widely used in basic research to study the structure and function of proteins and for protein production purposes. Development of a variety of vectors has made it possible to deliver foreign sequences as naked RNA or DNA, or as suicide virus particles produced using helper vector strategies. Preliminary reports also suggest that these vectors may be useful for in vivo applications where transient, high-level protein expression is desired, such as recombinant vaccines. The initial studies have already shown that alphavirus vaccines can induce strong humoral and cellular immune responses with good immunological memory and protective effects. Keywords:
Semliki Forest virus; Self-replicating
RNA; Transfection;
1. Introduction Alphaviruses are arthropod-borne Togaviruses which replicate in a large number of animal hosts ranging from mosquitos to avian and mammalian species (Strauss and Strauss, 1994). The alphaviral genome consists of a single-stranded RNA, which is of positive polarity encoding its own replicase. Thus, productive replication can be initiated either by infection of the cell or by transfection of the genomic RNA into the cytoplasm of the cell. In both cases, vigorous replication combined with high-level production of viral structural proteins leads to rapid production of new virus particles, which can be as many as lo5 per cell. The structural proteins of the virus are expressed from a subgenomic RNA which is colinear with the last third of the genomic * Corresponding author. Current address: Microbiology and Tumor Biology Center (MTC), Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden. Tel. +46 8 7286306; Fax +46 8 319587; e-mail: [email protected] 1Presented at the International Conference on ‘Eukaryotic Expression Vector Systems: Biology and Applications’. National Institute of Immunology, New Delhi, India; 4-8 February 1996. Abbreviations: cDNA, DNA complementary to RNA; CMV, cytomegalovirus(es); CTL, cytotoxic T lymphocyte; Env, envelope; HIV, human immunodeficiency virus; IU, international unit(s); NP, nucleoprotein; ORF, open reading frame; re-, recombinant; SFV, Semliki Forest virus(es); SIV, simian immunodeficiency virus. 0378-l 119/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII SO378-1119(96)00679-S
Helper vector; Nucleic acid vaccine; Togaviruses
sequences. This has allowed the manipulation of the subgenomic sequences without affecting the replication capacity of the system.
2. Alphavirus vectors Full-length, infectious cDNA clones of many alphaviruses have been constructed (Rice et al., 1987; Davis et al., 1989; Kuhn et al., 1991; LiljestCm et al., 1991; Kinney et al., 1993). Of these, the clones of Semliki Forest virus (SFV) (Liljestriim and Garoff, 1991, 1994; Berglund et al., 1993, 1996; Liljestriim, 1994; Olkkonen et al., 1994) and Sindbis virus (SIN) (Xiong et al., 1989; Bredenbeek and Rice, 1992; Bredenbeek et al., 1993; Schlesinger, 1993; Piper et al., 1994) have been further developed into general expression vectors (see Fig. 1). Foreign sequences can be placed in front or after the structural genes of the virus in the context of an otherwise wild-type genome. Such constructs tend to be unstable upon passaging and have the additional drawback that large amounts of viral structural proteins are produced. Therefore, the most preferable strategy is to replace the virus structural genes with the gene of interest. Due to the efficient production of the foreign mRNA by the viral transcriptase, large amounts of foreign protein is produced. In addition, the kinetics of translation may be significantly increased if a transla-
192
I. Tubulekas et al. / Gene 190 (1997) 191-195
pew
Folalg
+RNA _
+RNA -
-
-RNA m SP6transcription +lWA
-
Fig. 1. Three strategies of expressing foreign sequences with alphaviral vectors. (i) The re-RNA can either be packaged using a helper vector into virus particles which then are used to infect cells for expression of the heterologous sequences. (ii) Alternatively, the DNA plasmid is used for in vitro transcription of re-DNA, which then is directly transfected into the cell. (iii) Finally, if the alphavirus replicon is placed under a PolII promoter, the plasmid can directly be transfected into the cell where transcription occurs in the nucleus and further replication in the cell cytoplasm. In all three cases, the viral replicase (REP) uses the subgenomic promoter (small rightward arrow) on the minus strand RNA for the transcription of the subgenomic RNA which encodes the heterologous (Foreign) protein.
tional enhancer, situated in the beginning of the capsid ORF is utilized (Frolov and Schlesinger, 1994, 1996; Sjiiberg et al., 1994).
3. Delivery of re-alphavirus nucleic acids A central issue for any systems is the efficiency by which re-molecules can be delivered into a cell. For the alphavirus system the most simple is of course to
transfect in vitro-transcribed RNA by means of electroporation or lipofection (Liljestrom and Garoff, 1994). However, while these methods work well under cell culture conditions (up to 100% efficiency), they have to be optimized for each cell type which can be time consuming. Therefore, helper vectors have been developed which carry the viral structural genes but which lack the replicase genes including the genomic packaging signal (Bredenbeek et al., 1993; Liljestrijm and Garoff, 1991). High titre virus stocks (up to 10” IU perml)
I. Tubulekas et al. 1 Gene 190 (1997) 191-195
are produced by co-transfection with re-RNA and helper RNA into cultured cells and incubating for 24 h. The very broad host range of these viruses makes this strategy a very useful one, since the stocks can easily be used to infect many different kinds of cells both in vitro and in vivo. The system is suicidal since helper RNAs, lacking the packaging signal, are not encapsidated and therefore new virions will not form in the new host cell. However, a problem is the fact that the two RNA species may recombine (by strand-switching of the replicase) during the packaging procedure (Weiss and Schlesinger, 1991; Berglund et al., 1993; Raju et al., 1995) which can generate wild-type genomes resulting in the spread of virus. To alleviate this problem one strategy has been to use a helper which encodes a mutated spike protein gene (Berglund et al., 1993). The mutation results in production of a spike protein which does not undergo normal maturation (protease processing) with the result that any virus produced is noninfectious (Salminen et al., 1992). The virus stock is made infectious by brief treatment of protease. While this strategy does not reduce recombination per se, it greatly reduces the spread of replication-proficient virus, which can form only if a recombination event is combined, on the same molecule, with a reversion of the conditional mutation. To date no such viruses have been found. The latest development of the alphaviral expression system is the use of a DNA-based strategy where the re-alphavirus replicon sequences have been put under a eukaryotic promoter such as the CMV immediate early promoter (pCMV) (Herweijer et al., 1995; Dubensky et al., 1996; I.T. and P.L., unpublished data). In this system, DNA is directly transfected into the cell where the recombinant construct is transcribed by the host RNA polymerase. The RNA is transported into the cytoplasm where the viral replicase is produced and then takes over the replication of the RNA into new copies and also to transcribe the foreign sequences. The benefits of this strategy are lower costs (no in vitro transcription required), a more stable molecule, and no risk of producing replication-proficient virus. Preliminary results from cell culture experiments have shown that the DNA system gives at least lo-fold higher expression levels as compared to conventional naked DNA delivery methods (using pCMV plasmids) (Herweijer et al., 1995; Dubensky et al., 1996; I.T. and P.L., unpublished data).
4. Vaccine studies One of the most obvious in vivo applications for alphavirus vectors is for developing re-vaccines. Early studies using replicative constructs, co-expressing both the antigen of interest and the viral structural genes, showed that good immune responses indeed can be obtained (Hahn et al., 1992; London et al., 1992).
193
However, use of such multiplying re-virus is probably not feasible for biosafety reasons and since strong immune responses against the vectors are obtained. The use of suicide vectors is clearly to be preferred. Several features of the alphavirus system may be beneficial for vaccine design: (i) intracellular antigen expression mimics that during cognate infection, suggesting that optimal priming of the immune system can be achieved; (ii) multi-subunit, multi-epitope expression alleviates the need to define epitopes and/or haplotypes; (iii) high levels of antigen expression results in strong immune responses; (iv) vector structural proteins are not expressed minimizing an immune response against the vector itself; (v) no widespread immunity against the vector; (vi) broad host range. For delivery and expression of the antigen, any of the aforementioned delivery methods could be used. The most efficient way is to use re-SFV particles, although some biosafety questions still need to be considered. A preferred system would be to inject naked RNA directly, without packaging into particles using the helper vector. The efficacy of this approach will be dependent on the stability and transfectability in vivo of such RNA. The DNA/RNA-layered system may prove very useful in this instance. First of all, its use does not employ any step using a helper vector, and thus should form a safe system since it cannot form any infectious particles. Several studies using SFV suicide vectors for vaccination have been completed. Infection of mice with re-SFV expressing the influenza A virus nucleoprotein (NP) resulted in a strong humoral and cellular immune response with prolonged memory (Zhou et al., 1994, 1995; Liljestrdm, 1995). CTLs were CD8+ positive and class-I restricted. It was recently shown that indeed naked RNA delivery can result in substantial expression of foreign sequences in vivo (Johanning et al., 1995). Experiments employing injection of naked SFV-NP RNA into the quadriceps muscle of mice also resulted in significant immune responses (Zhou et al., 1994). Recent studies have shown that the transient alphavirus vectors express foreign proteins for up to l-2 weeks in vivo (Herweijer et al., 1995; Johanning et al., 1995; M.F. and P.L., unpublished data), a time span that would appear to be sufficient to mount a strong immune response. However, more work along these lines is required to complete the picture. In primate models, the envelope proteins of HIV-l (P.B., M. Quesada-Rolander, P. Putkonen, G. Biberfeld, R. Thorstensson and P.L., submitted) and SIV PBj14 (Mossman et al., 1996) were expressed, yielding moderate to low levels of antibodies and low levels of cellular immune responses. While the Env protein is known to be an antigen of low immunogenicity, it was surprising that the cellular immune responses were so low. Nevertheless, when the monkeys were challenged with SHIV-4 (the HIV study) or PBj14 (the SIV study), a
194
I. Tubulekas et al. / Gene 190 (1997) 191-195
strong reduction in viral load was observed, suggesting that although clear immune correlates were lacking, vaccination had nevertheless been successful. The SIV PBj study was particularly illustrative; the challenge employed the highly virulent PBj14 strain of SIV which kills the animal within 2 weeks. Indeed, while 75% of controls (unimmunized) were killed upon challenge, all SFV-env immunized animals survived, although they initially had become infected by the challenge virus. It was even somewhat surprising that such a good protection had been obtained since only the highly variable Env antigen had been produced. Follow-up studies are ongoing combining the Gag, Pol, Env and Nef antigens, with the goal of mounting a broad humorai and cellular immune response which would result in both reduced initial viral loads and in strong prolonged cellular responses which eventually would clear the infection.
Acknowledgement
P.L. is supported by grants from the Swedish Medical Research Council, the Swedish Research Council for Engineering Sciences, the Swedish Cancer Research Foundation, the EU EVA Programme, and the World Health Organization (WHO).
References Berglund, P., Sjiiberg, M., Atkins, G.J., Sheahan, B.J., Garoff, H. and Liljestrom, P. (1993) Semliki Forest virus expression system: production of conditionally infectious recombinant particles. Bio/Technology 11, 916-920. Berglund, P., Tubulekas, I. and Liljestriim, P. (1996) Alphaviruses as vectors for gene delivery. Trends Biotechnol. 14, 130-134. Bredenbeek, P.J. and Rice, C.M. (1992) Animal RNA virus expression systems. Semin. Virol. 3, 297-310. Bredenbeek, P.J., Frolov, I., Rice, CM. and Schlesinger, S. (1993) Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs. J. Viral. 67, 6439-6446. Davis, N.L., Willis, L.V., Smith, J.F. and Johnston, R.E. (1989) In vitro synthesis of infectious Venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant. Virology 171, 189-204. Dubensky, T.W., Driver, D.A., Polo, J.M., Belli, B.A., Latham, E.M., Ibanez, C.E., Chada, S., Brumm, D., Banks, T.A., Mento, S.J., Jolly, D.J. and Chang, S.M.W. (1996) Sindbis virus DNA-based expression vectors: utility for in vitro and in vivo gene transfer. J. Virol. 70, 508-519. Frolov, I. and Schlesinger, S. (1994) Translation of Sindbis virus mRNA ~ effects of sequences downstream of the initiating codon. J. Virol. 68, 8111-8117. Frolov, Y. and Schlesinger, S. (1996) Translation of Sindbis virus mRNA: analysis of sequences downstream of the initiating AUG codon that enhance translation. J. Virol. 70, 1182-1190. Hahn, C.S., Hahn, Y.S., Braciale, T.J. and Rice, C.M. (1992) Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation. Proc. Natl. Acad. Sci. USA 89, 2679-2683. Herweijer, H., Latendresse, J.S., Williams, P., Zhang, G.F., Danko,
I., Schlesinger, S. and Wolff, J.A. (1995) A plasmid-based selfamplifying Sindbis virus vector. Hum. Gene Ther. 6, 1161-l 167. Johanning, F.W., Conry, R.M., Lobuglio, A.F., Wright, M., Sumerel, L.A., Pike, M.J. and Curie], D.T. (1995) A Sindbis virus mRNA polynucleotide vector achieves prolonged and high level heterologous gene expression in vivo. Nucleic Acids Res. 23, 1495-1501. Kinney, R.M., Chang, G.-J., Tsuchiya, K.R., Sneider, J.M., Roehrig, J.T., Woodward, T.M. and Trent, D.W. (1993) Attenuation of Venezuelan equine encephalitis virus strain TC-83 is encoded by the 5’-noncoding region and the E2 envelope glycoprotein. J. Virol. 67, 1269-1277. Kuhn, R.J., Niesters, H.G.M., Hong, Z. and Strauss, J.H. ( 1991) Infectious RNA transcripts from Ross River virus cDNA clones and the construction and characterization of defined chimeras with Sindbis virus. Virology 182, 430441. Liljestriim, P. (1994) Alphavirus expression systems. Curr. Opin. Biotechnol 5, 495-500. Liljestrom, P. (1995) Recombinant self-replicating RNA vaccines. In: Gregoriadis, G., McCormack, B. and Allison, AC. (Eds.), Vaccines: New-Generation Immunological Adjuvants, Vol. 282. Plenum Press, New York, NY, 173-180. Liljestriim, 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. Liljestrom, P. and Garoff, H. (1994) Expression of proteins using Semliki Forest virus vectors. In: Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Smith, J.A., Seidman, J.G. and Struhl, K. (Eds.), Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley Interscience, New York, NY, pp. 16.20.116.20.16. Liljestrom, P., Lusa, S., Huylebroeck, D. and Garoff, H. (1991) In vitro mutagenesis of a full-length cDNA clone of Semliki Forest virus: the 6,000-molecular-weight membrane protein modulates virus release. J. Virol. 65, 4107-4113. London, S.D., Schmaljohn, A.L., Dalrymple, J.M. and Rice, C.M. (1992) Infectious enveloped RNA virus antigenic chimeras. Proc. Natl. Acad. Sci. USA 89, 207-211. Mossman, S., Bex, F., Berglund, P., Arthos, J., O’Neil, S.P., Riley, D., Maul, D.H., Bruck, C., Momin, P., Burny, A., Fultz, P.N., Mullins, J.I., Liljestriim, P. and Hoover, E.A. (1996) Protection against lethal SIVsmmPBjl4 disease by a recombinant Semliki Forest virus gpl60 vaccine and by gpl20 subunit vaccine. J. Virol. 70, 1953-1960. Olkkonen, V.M., Dupree, P., Simons, K., Liljestrom, P. and Garoff, H. ( 1994) Expression of exogenous proteins in mammalian cells with the Semliki Forest virus vector. Methods Cell Biol. 43, 43-53. Piper, R.C., Slot, J.W., Li, G.P., Stahl, P.D. and James, D.E. (1994) Recombinant Sindbis virus as an expression system for cell biology. Methods Cell Biol. 43, 55-78. Raju, R., Subramaniam, S.V. and Hajjou, M. (1995) Genesis of Sindbis virus by in vivo recombination of nonreplicative RNA precursors. J. Virol. 69, 7391-7401. Rice, CM., Levis, R., Strauss, J.H. and Huang, H.V. (1987) Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J. Virol. 61, 3809-3819. Salminen, A., Wahlberg, J.M., Lobigs, M., Liljestrbm, P. and Garoff, H. (1992) Membrane fusion process of Semliki Forest virus II: cleavage dependent reorganization of the spike protein complex controls virus entry. J. Cell Biol. 116, 349-357. Schlesinger, S. (1993) Alphaviruses - vectors for the expression of heterologous genes. Trends Biotechnol. 11, 18-22. Sjbberg, 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, 112771131.
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Strauss, J.H. and Strauss, E.G. (1994) The alphaviruses: gene expression, replication, and evolution. Microbial. Rev. 58, 491-562. Weiss, B.G. and Schlesinger, S. (1991) Recombination between Sindbis virus RNAs. J. Virol. 65, 4017-4025. Xiong, C., Levis, R., Shen, P., Schlesinger, S., Rice, C.M. and Huang, H.V. (1989) Sindbis virus: an efficient, broad host range vector for gene expression in animal cells. Science 243, 118881191.
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Zhou, X., Berglund, P., Rhodes, G., Parker, S.E., Jondal, M. and Liljestrom, P. (1994) Self-replicating Semliki Forest virus RNA as recombinant vaccine. Vaccine 12, 1510-1514. Zhou, X., Berglund, P., Zhao, H., Liljestriim, P. and Jondal, M. (1995) Generation of cytotoxic and humoral immune responses by nonreplicative recombinant Semliki Forest virus. Proc. Nat]. Acad. Sci. USA 92, 3009-3013.
Alphavirus expression vectors and their use as recombinant vaccines: a minireviewl Ioannis Tubulekas, Peter Berglund, Marina Fleeton, Peter Liljestrdm * Department of Biosciences, Karolinska Institute, S-141 57 Huddinge, Sweden Received
8 March
1996; revised 16 May 1996; accepted
18 May 1996; Received
by S.E. Hasnain
Abstract Alphavirus vectors have become widely used in basic research to study the structure and function of proteins and for protein production purposes. Development of a variety of vectors has made it possible to deliver foreign sequences as naked RNA or DNA, or as suicide virus particles produced using helper vector strategies. Preliminary reports also suggest that these vectors may be useful for in vivo applications where transient, high-level protein expression is desired, such as recombinant vaccines. The initial studies have already shown that alphavirus vaccines can induce strong humoral and cellular immune responses with good immunological memory and protective effects. Keywords:
Semliki Forest virus; Self-replicating
RNA; Transfection;
1. Introduction Alphaviruses are arthropod-borne Togaviruses which replicate in a large number of animal hosts ranging from mosquitos to avian and mammalian species (Strauss and Strauss, 1994). The alphaviral genome consists of a single-stranded RNA, which is of positive polarity encoding its own replicase. Thus, productive replication can be initiated either by infection of the cell or by transfection of the genomic RNA into the cytoplasm of the cell. In both cases, vigorous replication combined with high-level production of viral structural proteins leads to rapid production of new virus particles, which can be as many as lo5 per cell. The structural proteins of the virus are expressed from a subgenomic RNA which is colinear with the last third of the genomic * Corresponding author. Current address: Microbiology and Tumor Biology Center (MTC), Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden. Tel. +46 8 7286306; Fax +46 8 319587; e-mail: [email protected] 1Presented at the International Conference on ‘Eukaryotic Expression Vector Systems: Biology and Applications’. National Institute of Immunology, New Delhi, India; 4-8 February 1996. Abbreviations: cDNA, DNA complementary to RNA; CMV, cytomegalovirus(es); CTL, cytotoxic T lymphocyte; Env, envelope; HIV, human immunodeficiency virus; IU, international unit(s); NP, nucleoprotein; ORF, open reading frame; re-, recombinant; SFV, Semliki Forest virus(es); SIV, simian immunodeficiency virus. 0378-l 119/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII SO378-1119(96)00679-S
Helper vector; Nucleic acid vaccine; Togaviruses
sequences. This has allowed the manipulation of the subgenomic sequences without affecting the replication capacity of the system.
2. Alphavirus vectors Full-length, infectious cDNA clones of many alphaviruses have been constructed (Rice et al., 1987; Davis et al., 1989; Kuhn et al., 1991; LiljestCm et al., 1991; Kinney et al., 1993). Of these, the clones of Semliki Forest virus (SFV) (Liljestriim and Garoff, 1991, 1994; Berglund et al., 1993, 1996; Liljestriim, 1994; Olkkonen et al., 1994) and Sindbis virus (SIN) (Xiong et al., 1989; Bredenbeek and Rice, 1992; Bredenbeek et al., 1993; Schlesinger, 1993; Piper et al., 1994) have been further developed into general expression vectors (see Fig. 1). Foreign sequences can be placed in front or after the structural genes of the virus in the context of an otherwise wild-type genome. Such constructs tend to be unstable upon passaging and have the additional drawback that large amounts of viral structural proteins are produced. Therefore, the most preferable strategy is to replace the virus structural genes with the gene of interest. Due to the efficient production of the foreign mRNA by the viral transcriptase, large amounts of foreign protein is produced. In addition, the kinetics of translation may be significantly increased if a transla-
192
I. Tubulekas et al. / Gene 190 (1997) 191-195
pew
Folalg
+RNA _
+RNA -
-
-RNA m SP6transcription +lWA
-
Fig. 1. Three strategies of expressing foreign sequences with alphaviral vectors. (i) The re-RNA can either be packaged using a helper vector into virus particles which then are used to infect cells for expression of the heterologous sequences. (ii) Alternatively, the DNA plasmid is used for in vitro transcription of re-DNA, which then is directly transfected into the cell. (iii) Finally, if the alphavirus replicon is placed under a PolII promoter, the plasmid can directly be transfected into the cell where transcription occurs in the nucleus and further replication in the cell cytoplasm. In all three cases, the viral replicase (REP) uses the subgenomic promoter (small rightward arrow) on the minus strand RNA for the transcription of the subgenomic RNA which encodes the heterologous (Foreign) protein.
tional enhancer, situated in the beginning of the capsid ORF is utilized (Frolov and Schlesinger, 1994, 1996; Sjiiberg et al., 1994).
3. Delivery of re-alphavirus nucleic acids A central issue for any systems is the efficiency by which re-molecules can be delivered into a cell. For the alphavirus system the most simple is of course to
transfect in vitro-transcribed RNA by means of electroporation or lipofection (Liljestrom and Garoff, 1994). However, while these methods work well under cell culture conditions (up to 100% efficiency), they have to be optimized for each cell type which can be time consuming. Therefore, helper vectors have been developed which carry the viral structural genes but which lack the replicase genes including the genomic packaging signal (Bredenbeek et al., 1993; Liljestrijm and Garoff, 1991). High titre virus stocks (up to 10” IU perml)
I. Tubulekas et al. 1 Gene 190 (1997) 191-195
are produced by co-transfection with re-RNA and helper RNA into cultured cells and incubating for 24 h. The very broad host range of these viruses makes this strategy a very useful one, since the stocks can easily be used to infect many different kinds of cells both in vitro and in vivo. The system is suicidal since helper RNAs, lacking the packaging signal, are not encapsidated and therefore new virions will not form in the new host cell. However, a problem is the fact that the two RNA species may recombine (by strand-switching of the replicase) during the packaging procedure (Weiss and Schlesinger, 1991; Berglund et al., 1993; Raju et al., 1995) which can generate wild-type genomes resulting in the spread of virus. To alleviate this problem one strategy has been to use a helper which encodes a mutated spike protein gene (Berglund et al., 1993). The mutation results in production of a spike protein which does not undergo normal maturation (protease processing) with the result that any virus produced is noninfectious (Salminen et al., 1992). The virus stock is made infectious by brief treatment of protease. While this strategy does not reduce recombination per se, it greatly reduces the spread of replication-proficient virus, which can form only if a recombination event is combined, on the same molecule, with a reversion of the conditional mutation. To date no such viruses have been found. The latest development of the alphaviral expression system is the use of a DNA-based strategy where the re-alphavirus replicon sequences have been put under a eukaryotic promoter such as the CMV immediate early promoter (pCMV) (Herweijer et al., 1995; Dubensky et al., 1996; I.T. and P.L., unpublished data). In this system, DNA is directly transfected into the cell where the recombinant construct is transcribed by the host RNA polymerase. The RNA is transported into the cytoplasm where the viral replicase is produced and then takes over the replication of the RNA into new copies and also to transcribe the foreign sequences. The benefits of this strategy are lower costs (no in vitro transcription required), a more stable molecule, and no risk of producing replication-proficient virus. Preliminary results from cell culture experiments have shown that the DNA system gives at least lo-fold higher expression levels as compared to conventional naked DNA delivery methods (using pCMV plasmids) (Herweijer et al., 1995; Dubensky et al., 1996; I.T. and P.L., unpublished data).
4. Vaccine studies One of the most obvious in vivo applications for alphavirus vectors is for developing re-vaccines. Early studies using replicative constructs, co-expressing both the antigen of interest and the viral structural genes, showed that good immune responses indeed can be obtained (Hahn et al., 1992; London et al., 1992).
193
However, use of such multiplying re-virus is probably not feasible for biosafety reasons and since strong immune responses against the vectors are obtained. The use of suicide vectors is clearly to be preferred. Several features of the alphavirus system may be beneficial for vaccine design: (i) intracellular antigen expression mimics that during cognate infection, suggesting that optimal priming of the immune system can be achieved; (ii) multi-subunit, multi-epitope expression alleviates the need to define epitopes and/or haplotypes; (iii) high levels of antigen expression results in strong immune responses; (iv) vector structural proteins are not expressed minimizing an immune response against the vector itself; (v) no widespread immunity against the vector; (vi) broad host range. For delivery and expression of the antigen, any of the aforementioned delivery methods could be used. The most efficient way is to use re-SFV particles, although some biosafety questions still need to be considered. A preferred system would be to inject naked RNA directly, without packaging into particles using the helper vector. The efficacy of this approach will be dependent on the stability and transfectability in vivo of such RNA. The DNA/RNA-layered system may prove very useful in this instance. First of all, its use does not employ any step using a helper vector, and thus should form a safe system since it cannot form any infectious particles. Several studies using SFV suicide vectors for vaccination have been completed. Infection of mice with re-SFV expressing the influenza A virus nucleoprotein (NP) resulted in a strong humoral and cellular immune response with prolonged memory (Zhou et al., 1994, 1995; Liljestrdm, 1995). CTLs were CD8+ positive and class-I restricted. It was recently shown that indeed naked RNA delivery can result in substantial expression of foreign sequences in vivo (Johanning et al., 1995). Experiments employing injection of naked SFV-NP RNA into the quadriceps muscle of mice also resulted in significant immune responses (Zhou et al., 1994). Recent studies have shown that the transient alphavirus vectors express foreign proteins for up to l-2 weeks in vivo (Herweijer et al., 1995; Johanning et al., 1995; M.F. and P.L., unpublished data), a time span that would appear to be sufficient to mount a strong immune response. However, more work along these lines is required to complete the picture. In primate models, the envelope proteins of HIV-l (P.B., M. Quesada-Rolander, P. Putkonen, G. Biberfeld, R. Thorstensson and P.L., submitted) and SIV PBj14 (Mossman et al., 1996) were expressed, yielding moderate to low levels of antibodies and low levels of cellular immune responses. While the Env protein is known to be an antigen of low immunogenicity, it was surprising that the cellular immune responses were so low. Nevertheless, when the monkeys were challenged with SHIV-4 (the HIV study) or PBj14 (the SIV study), a
194
I. Tubulekas et al. / Gene 190 (1997) 191-195
strong reduction in viral load was observed, suggesting that although clear immune correlates were lacking, vaccination had nevertheless been successful. The SIV PBj study was particularly illustrative; the challenge employed the highly virulent PBj14 strain of SIV which kills the animal within 2 weeks. Indeed, while 75% of controls (unimmunized) were killed upon challenge, all SFV-env immunized animals survived, although they initially had become infected by the challenge virus. It was even somewhat surprising that such a good protection had been obtained since only the highly variable Env antigen had been produced. Follow-up studies are ongoing combining the Gag, Pol, Env and Nef antigens, with the goal of mounting a broad humorai and cellular immune response which would result in both reduced initial viral loads and in strong prolonged cellular responses which eventually would clear the infection.
Acknowledgement
P.L. is supported by grants from the Swedish Medical Research Council, the Swedish Research Council for Engineering Sciences, the Swedish Cancer Research Foundation, the EU EVA Programme, and the World Health Organization (WHO).
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