WO2005012535A2 - Method for producing life attenuated viruses and use of viral particles thus produced. - Google Patents

Abstract

The invention is in the field of anti-viral therapy and the production of viral vaccines. More specifically, the present invention relates to life attenuated viruses, a method for the production of said viruses, to the use of such attenuated viruses as prophylactic or therapeutic vaccines and to a vaccine comprising an attenuated virus produced by a method of the invention. A method for the production of attenuated viruses comprising the steps of attenuating a virus by inactivating an early gene and by introduction into the viral genome of a nucleotide construct which is upon expression in a target cell of the virus, able to produce one transcript or multiple transcripts capable of forming a double-stranded RNA with nucleotide sequence homology to said early gene of the virus.

Description

Title: Method for producing life attenuated viruses and use of viral particles thus produced.

FIELD OF THE INVENTION The invention is in the field of prophylactic anti- viral vaccines and anti- viral therapies. More specifically, the present invention relates to a method for the generation of stable attenuated viruses, which have the capacity to inhibit the replication of cognate virulent viruses in target cells and to the use of such attenuated viruses as prophylactic or therapeutic vaccines.

BACKGROUND OF THE INVENTION Vaccines The two most important contributions to public health of the last century are sanitation and vaccination (active immunization), which together have dramatically reduced deaths from infectious disease. To date, effective vaccines comprise inactivated viruses or micro-organisms or fragments thereof, life attenuated viruses, recombinant antigens produced in cell culture, recombinant antigens produced from viral vectors and synthetic peptides. Inactivated viruses represent the classical vaccination approach, wherein the virus is propagated on a cell culture line, and killed with the use of an inactivating agent such as formalin, β-propiolactone, and ethylenimines. The goal is to destroy the infectivity of the virus, while maintaining its immunogenicity. The disadvantage is that the use of these vaccines necessitates multiple booster shots. The use of live attenuated vaccines overcomes this problem. Another major advantage of using live attenuated viruses in vaccination strategies is that in general strong and effective adaptive immune responses are mounted. Most of the live attenuated vaccines in use today are derived from serial passage in cultured cells, including human diploid cells (e.g. fetal lung tissue or other fibroblasts), monkey kidney cells, and chicken embryos, among others. Adaptation of the virus to growth in the cultured cells is accompanied by a gradual loss of virulence for the natural host. This procedure relies on the (random) accumulation of point mutations to confer avirulence. More exact and directed mutations can be attained through genetic engineering. The use of site-directed mutagenesis for the production of temperature sensitive mutants, deletion mutants and live recombinant viruses is now commonly employed to achieve stable viral attenuation. The first problem with live attenuated viruses is the genetic stability of the attenuated phenotype of a vaccine strain, since most viruses are highly variable and are capable to revert into strains with a virulence compared to that of the original virus. The second problem is that the loss of infectivity or virulence of the attenuated virus coincides with the loss of specific genetic information required for virus replication and thus with a difficulty to produce such viruses in sufficient amounts for in vivo applications. With a live attenuated HIV vaccine a major problem is that HIV infection causes destruction of the target cells, e.g. T helper lymphocytes which play a central role in adaptive immunity. The presence or emergence of wildtype HIV particles in vaccinated patients will eventually lead to a loss of immunity, despite the enhanced immune response as a consequence of the vaccination. HIV belongs to the lentivirus sub-family within the Retroviridae. The virus particles consist of two single stranded genomic RNA molecules wrapped up with protein to nucleocapsids. Lipid envelopes bearing glycoprotein projections surround the nucleocapsids. Virions enter T helper lymphocytes by binding of gpl20 to CD4 receptors. Upon entrance, nucleocapsids are released in the cytoplasm where the reverse transcriptase (included in the nucleocapsids) produces a cDNA copy of the genomic RNA. The reverse transcriptase and RNAseH, also included in the nucleocapsids produce linear double stranded DNA copies of the genomic RNA, which are targeted to the nucleus where they are integrated as proviral DNA into the nuclear DNA by a viral integrase, which is also included in the nucleocapsids. The proviral genomes are flanked by long terminal repeats (LTR's). The 5' LTR contains enhancer and promoter sequences essential for transcription of viral genes. The 3' -LTR contains the polyadenylation signals, required for polyadenylation of the viral transcripts. The provirus comprises three structural genes: gag; encoding the nucleocapsid proteins, pol; encoding the viral enzymes, and env; encoding the membrane glycoproteins. The mature proteins are formed after proteolytic cleavage of the primary translation products. h addition, the provirus contains another five genes encoding regulatory proteins involved in virion infectivity, viral assembly and release, viral mRNA translocation and genome activation. The rev, tat and nef regulatory proteins are encoded by the early, multiple-spliced mRNA molecules, whereas the structural proteins and other non-structural proteins are encoded by the late single-spliced (env, vif, vpr and vpu) and non-spliced (pol and gag) mRNA molecules. The provirus is activated by antigen binding or by infection with other viruses. Provirus activation leads to expression of genes encoding the regulatory proteins, including the tat and rev proteins. Tat will further strengthen the 5' -LTR promoter, whereas rev will promote expression of the structural proteins and the viral enzymes. Viral nucleocapsids are formed in the cytoplasm and finally, virus particles will bud from the virus-infected CD4 cells. In this process, the virus kills T helper cells, which have higher than threshold CD4 receptor levels. The other infected cell types remain intact. HrV-infected persons will gradually loose their CD4-bearing T-lymphocytes. HIV infections therefore will at a certain point in time lead to the development of the typical AIDS symptoms. Uninfected CD4 T-lymphocytes lose their capacity to respond to new antigens; general antibody levels decline; T-lymphocyte maturation in the thymus is inhibited; cytokine imbalances occur, leading to many immunological abnormalities and AIDS patients finally die from infections with progressive pathogens and from exhaustion. To date despite numerous efforts using all the vaccine types as outlined herein, a prophylactic or therapeutic vaccine for HIV has not become available. Only life attenuated HIV strains turned out to mount an adaptive immune response which is sufficient to initially reduce the viral load in patients. However, HIV is highly variable and attenuated strains have the capacity to rapidly revert to virulent strains. For these reasons, attenuated HIV strains have so far not been considered to be used as prophylactic vaccines. Other viruses for which efficient vaccines have not become available are herpesviruses and papillioma viruses (Whitley, B. and Roizman, B., J. Clin. Invest. 2002, 109, 145-151). These viruses are characterised by the fact that they are capable of replicating in cell types or tissues which are inaccessible by the adaptive immune system. Recently it has been proposed to use RNA silencing (RNAi) to combat virus infection. RNA silencing Gene expression in eukaryotic cells is controlled by several regulatory mechanisms acting at the transcriptional level in the nucleus or at the post-transcriptional level in the cytoplasm. Recently a novel regulatory mechanism has been identified referred to as RNA silencing (Ding S.W. 2000, Curr. Opin. Biotechnol. 11 : 152-156), in which RNA molecules instead of proteins serve as signalling and target molecules. RNA silencing has been proven to be induced by over-expressed and double stranded RNA (dsRNA) molecules and results in sequence-specific RNA degradation in cells from higher eukaryotes. Over recent years, silencing has been described in an increasing variety of organisms and referred to as quelling in fungi or RNAi in plants and animals. The key step in the induction of RNA silencing is the formation of dsRNA by host RNA-dependent RNA polymerases (RdRp) which is recognized and cleaved by a dsRNA-specific RNAse III- type nuclease, denoted DICER to yield small (18-26 nucleotides long) short interfering RNAs (siRNAs). The siRNAs have the capacity to specifically bind to complementary mRNA molecules and thereby enable the host-encoded RdRps to produce a second generation of dsRNA molecules, which are again cleaved by an RNAse Ill-type nuclease to yield secondary siRNAs. Genetic studies revealed additional genes and proteins, such as helicases, active in the RNA silencing pathway. All these proteins are associated with cytoplasmic nuclease complexes; denoted RNA induced silencing complexes (RISC) of approximately 500 kD in size (G. Hutvagner & P.D. Zamore, Curr. Opin. In Gen. & Dev, 12: 225-232, 2002). It is thought that next to gene regulation, RNA silencing has also been developed by higher eukaroytes to prevent replication of transposable elements and viruses. In this respect, RNA silencing can be observed as an intracellular innate antiviral defence mechanism, h the use of RNA silencing to combat viral infection it has been successfully tried to provide target cells for the virus with a genetic construct which produces a double stranded RNA that is homologous to a key sequence of the virus (e.g. Coburn, G.A. and Collen, R.R., J. Virol, 76, 9225-9231., 2002; Haasnoot, J. et al, J. Biomed. Sci, 10, 607- 616., 2003; Lee, N.S. et al, Nat. Biotechnol, 20, 500-505., 2002). However, it has been found that escape variants with a single or few point mutations in the target nucleotide sequence, rapidly emerge (Boden, D. et al, J. Virol., 2003, 77, 11531-11535; Das, A.T., et al, J. Virol., 2004, 78, 2601-2605). To combat viruses that infect cells of the immune system, or viruses that infect cells that are inaccessible for the immune system, there is a need to combine the immunogenic advantages of vaccination with an attenuated virus with the advantages of the RNA silencing approach. For this, an attenuated virus should be made by inactivating one or more genes, while said attenuated virus should be provided with a sequence which allows the formation of double stranded RNAs capable of inducing an RNA silencing response specific to the viral genome itself. Further, as part of the ongoing battle between parasite and host, many viruses, carry functions to suppress the intracellular innate defence mechanism of RNA silencing. The present inventors have earlier found that certain non- structural viral proteins, such as the non-structural protein (NSs) of the Bunyaviridae, the non-structural protein (NS3) of the genus Tenui virus the non structural protein (E3L) of the Poxviridae, the non structural protein (VP35) of the Filoviridae and the non structural protein (NS1) of the Orthomyxoviridae, are capable of acting both as RNA silencing suppressor (RSS) proteins (thus suppressing the RNA silencing mechanism) and as viral expressional enhancer proteins (thus increasing the expression of a heterologous nucleotide sequence/transgene) in vitro and in vivo (see co-pending WO 04/035796).

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for producing an attenuated virus such that the attenuated virus essentially retains its tropism and is capable of mounting a strong adaptive immune response in a patient and a strong innate response in an infected cell, both responses directed against the cognate virulent virus. Said attenuated virus is highly suitable as a prophylactic and/or therapeutic vaccine. Preferably the virus is a lentivirus, a herpesvirus or a papillomavirus; more preferably the virus is HIV. A further method of the present invention comprises a second step of providing the genome of the attenuated virus with a nucleic acid construct which is able to produce one or more transcripts capable of folding into double-stranded RNA, which is substantially homologous to (part of) a nucleotide sequence essential for replication of the cognate virulent virus. Upon expression in a target cell, said recombinant attenuated virus is capable of inhibiting the replication of a cognate/ homologous virulent virus. Because virus replication is attenuated in the target cell, virus replication is not detrimental to the infected cell and the infected cells will not be destructed or eliminated by the immune system. In a further aspect, the present invention relates to a method to select for a virus both ex vivo and in vivo with a stable attenuated phenotype, which in a target cell of the virus durably inhibits the replication of a cognate/ homologous virulent virus. Preferably said sequence is located in a sequence of a virulence gene or a regulatory gene, more preferably an early viral regulatory gene. In the case of HIV said gene is the nef gene. Attenuation of the vims is preferably achieved by disturbing (part of) the sequence of a virulence gene, more specifically an early regulatory gene, which has as result that the normal gene product is no longer formed. In the case of HIV said gene is the «e sequence. A specific embodiment is formed by a virus in which the attenuation is achieved by inserting into the gene a sequence which is anti-sense with respect to said gene, thereby allowing for the production of double-stranded RNA when expressed in the host cell. Also part of the invention are viruses which are produced by such a method, especially HIV life attenuated viruses, preferably the viruses denoted in this application as AS-53 or AS-21 or AS 20 or AS 15.

LEGENDS TO THE FIGURES

Figure 1: Lai-//zNef inhibits HIV- 1 replication effectively

HEK293T cells are transfected with 0.5 micrograms of pLai (L) DNA plus 0.5 micrograms of pUC19 DNA (Column L), with 0.5 micrograms of pLai-ZANef DNA (I) plus 0.5 micrograms of pUC19 DNA (column I) or with 0.5 micrograms of L DNA plus 0.5 micrograms of I DNA (Column LIT). The amount of produced virus is measured 3 days post transfection using a CA-p24 ELISA (Beckman-Coulter). Figure 2: Lai-/ zNef inhibits HIV-1 production by RNA interference HEK293T cells are transfected with 0.1 micrograms of pLai (L) DNA plus 0.9 micrograms of pUC19 DNA (Column L), with 0.1 micrograms of L DNA plus 0.9 micrograms DNA of an Influenza virus A NS1 expression plasmid (RSS, See WO 04/035796) (Column L+RSS), 0.1 micrograms of pLai- Nef DNA (I) plus 0.9 micrograms of pUC 19 DNA (Column I), with 0.1 micrograms of I DNA plus 0.9 micrograms of RSS DNA (Column I+RSS), with 0.1 micrograms of L DNA plus 0.1 micrograms of I DNA plus 0.8 micrograms of pUC19 DNA (Column LIT) or with 0.1 micrograms of L DNA plus 0.1 micrograms of I DNA plus 0.8 micrograms of RSS DNA (Column L/I+RSS) . The amount of produced vims is measured 3 days post transfection using a CA-p24 ELISA (Beckman- Coulter).

Figure 3: Generation of stable life attenuated HIV-1 in Sup-Tl cells transfected with Lai- /ANefDNA Panel A: two batches of Sup-Tl cells are electroporated with 1 microgram of pLai-lhNef DNA each. The cells are cultured for 73 days and passaged 2 times per week. At indicated time points after electrop oration total RNA is isolated from part of the cells, which is used as a template in an RT-PCR reaction using Eup and Ldo as primers. The resulting DNA fragments are resolved on 1% agarose gels and the DNA fragments corresponding with the different escape variants are indicated by the arrows. Panel 2: Schematic representation of the inserted inverted repeat in pLai-//zNef. The location of the primers Eup and Ldo used in the RT-PCR assay described under Panel A are indicated by the thin arrows. The length of the expected PCR fragment using pLai-//zNef RNA as a template is indicated.

Figure 4: The stable life attenuated HIV-1 strains derived from deletions in Lai-//zNef Schematic representation of the inserted inverted repeat in pLai-//jNef and of the same region in the different virus variants. The boxes marked with deleted indicate the length of the region in the inverted repeat, which is deleted in the respective variant. The escape variants are numbered according to the length of the inverted repeat which is maintained. ++4- indicates a high level of replication with syncitium formation. + indicates a low level of replication without syncitium formation and - indicates the absence of replication. Figure 5: The vims valiants AS 53/15 and AS 21/20 inhibit Lai replication effectively Sup-Tl cells are infected with Lai (LAI) virus, with Lai vims plus vims variant AS 53/15 (LAI/AS 53/15) and with Lai vims plus vims variant AS 21/20 (LAI/AS 21/20). The cells are cultured for 33 days and passaged 2 times per week. At indicated time points after electroporation total RNA is isolated from part of the cells, which is used as a template in an RT-PCR reaction using Eup and Ldo as primers. The resulting DNA fragments are resolved on 1% agarose gels.

DETAILED DESCRIPTION OF THE INVENTION The term "attenuated virus", as used herein, indicates a weakened vims that is no longer virulent or that is less virulent. The term "early genes" as used herein indicates those viral genes that are expressed early in the infection cycle and regulate and are essential for the expression of structural viral genes. Examples of such early genes are for instance the genes shown in Table 1. The term "target cell" as used herein indicates a eukaryotic cell, preferably an animal (including human), or plant or fungus (including both mold and yeast phenotypes) cell, including specialized tissue cells and progenitor cells thereof, which is capable of being infected or transfected by a life attenuated vims of the invention. The term "producer cell" or "producer cell-line" as used herein refers to a cell or cell-line, respectively, suitable for replication, propagation, and/or production, of attenuated vims particles. The term "host cell" as used herein generally indicates a cell used for the expression of a viral genome, or propagation of a vims and, in the context of the present invention, includes both the target and producer cell. The term an "isolated nucleic acid sequence " means a nucleic acid sequence that is free of the nucleotide sequences that flank the nucleic acid sequence in the naturally-occurring genome of the organism from which it is derived. The term therefore includes, for example, any recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or into the genomic DNA of a prokaryote or eukaryote from which it has not been derived; or which exists as a separate molecule (e.g., an RNA, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequences. A coding sequence is "under the control" of regulatory sequences in a cell when a DNA-dependent RNA polymerase transcribes the coding sequence into mRNA or micro-RNA or other RNA encoded by the coding sequence. As used herein the term "nucleic acid construct" is intended to indicate any nucleic acid molecule of cDNA, genomic DNA, synthetic DNA or RNA origin. The term "construct" is intended to indicate a nucleic acid segment which may be single- or double- stranded, and which may be based on a complete or partial naturally occurring nucleotide sequence encoding a polypeptide of interest. The construct may optionally contain other nucleic acid segments. The term "nucleic acid construct" may be synonymous with the term "expression cassette" when the nucleic acid construct contains all the regulatory sequences required for expression of a coding sequence of the present invention. The term "coding sequence" as defined herein is a sequence which is transcribed into mRNA and optionally translated into a polypeptide when placed under the control regulatory sequences. The boundaries of the coding sequence in, for instance, DNA viruses are generally determined by a translation start codon ATG at the 5 '-terminus and a translation stop codon at the 3'- terminus. A coding sequence can include, but is not limited to, RNA, DNA, cDNA, and recombinant nucleic acid sequences. The term "regulatory sequences" is defined herein to include all components which are necessary or advantageous for expression of a coding sequence. Each regulatory sequence may be native or foreign to the coding sequence. Such regulatory sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequences include a promoter, and transcriptional and translational stop signals. The regulatory sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the regulatory sequences with the coding region of the nucleic acid sequence encoding a polypeptide. The term "promoter" is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes. The terms "nucleotide sequence homology" or "sequence homology" or "homologous sequence" as used herein denote the presence of homology between two or more polynucleotides. Polynucleotides have "homologous" sequences if the sequence of nucleotides in their sequences is the same when aligned for maximum correspondence. Sequence comparison between two or more polynucleotides is generally performed by comparing portions of two sequences over a comparison window to identify and compare local regions of sequence similarity. The comparison window is generally from about 20 to 200 contiguous nucleotides. The "percentage of sequence homology" for polynucleotides, such as 50, 60, 70, 80, 90, 95, 98, 99 or 100 percent sequence homology may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by: (a) determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions; (b) dividing the number of matched positions by the total number of positions in the window of comparison; and (c) multiplying the result by 100 to yield the percentage of sequence homology. Optimal alignment of sequences for comparison may be conducted by computerized implementations of known algorithms, or by inspection. Readily available sequence comparison and multiple sequence alignment algorithms are, respectively, the Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. 1990. J. Mol. Biol. 215:403; Altschul, S.F. et al. 1997. Nucleic Acid Res. 25:3389-3402) and ClustalW programs both available on the internet. Other suitable programs include GAP, BESTFIT and FASTA in the Wisconsin Genetics Software Package (Genetics Computer Group (GCG), Madison, WI, USA). "Subject" as used herein includes, but is not limited to, an organism; a mammal, including, e.g., a human, non-human primate, mouse, pig, cow, goat, cat, rabbit, rat, guinea pig, hamster, horse, monkey, sheep, or other non-human mammal; and a non- mammal, including, e.g., a non-mammalian vertebrate, such as a bird (e.g., a chicken or duck) or a fish, and a non-mammalian invertebrate. The inventors of the present invention have surprisingly found, as shown in the example section, that pLai-//zNef is expressed in a cell, since transcripts derived from this plasmid inhibit the production of Lai particles very effectively by RNA silencing. However, genome length transcripts, which can be packaged into virus particles are not produced from this plasmid. Upon introduction of pLai-ZANef in HIV-1 target cells (Sup- Tl) vims variants emerge, which have truncated inverted repeats in the Nef region. Vims variants lacking an inverted repeat in the Nef region but which have the capacity to induce syncitium formation do not emerge. Apparently, the obtained vims variants with truncated inverted repeats prevent the formation of virus variants lacking an inverted repeat. This demonstrates that the followed procedure is well suited for selection of attenuated HIV-1 strains with highly effective inhibitory inverted repeats in the Nef region, capable of conferring durable inhibition of HIV-1 replication.

A method of the present invention for the production of attenuated vims particles of the invention, comprises a producer cell-line. Producer cells used in a method according to the invention for production of the attenuated vims particles can be of any suitable cell type. Most commonly, mammalian producer cells are used but other cells, such as insect cells are not excluded. Many suitable producer cell lines are known in the ait. The skilled person is also capable of making suitable producer cell-lines by, for example stably introducing a nucleic acid construct encoding packaging components into a cell line. Examples of suitable producer cell lines are the Sup-Tl (human, lymphoma, lymphoblastic, T cell) cell line, the JURKAT (human, peripheral blood, leukemia, T cell) cell line, the HEK293 (human embryonal kidney) cell line or HEK293T cell line or PER C6 cell line. Also suitable are the VERO, the MDCK (NBL-2) (dog, cocker spaniel, kidney) and other cell lines. Initially a HIV mutant DNA construct was made with a deletion of 150 basepairs in the 5 'region of the Nef coding domain and which harboured a 300 nucleotides anti-sense sequence in its nef gene, located next to the sense sequence thereof, thereby disrupting the He/ gene, so that no functional gene product could be established in a host cell. The experiments show that this DNA constmct could indeed inhibit the wild- type HIV-1 vims production when co-infected (fig. 1). However, it appeared that this DNA construct was unable to produce a replicating recombinant vims in tissue culture. The postulated reason for this is that the possession of a 300 nucleotides anti-sense and sense sequences which are able to form a long hairpin double stranded RNA structure in the host cell, would prevent the production of genome length RNA molecules, thereby preventing the production of mutant vims particles. When, however, the DNA construct was introduced into host cells which were subsequently cultured for a longer time, it was found that the supernatants contained vims variants, which apparently were able to replicate. Upon sequence verification of these vims variants it appeared that these variants surprisingly still contained a sequence which could form a double-stranded RNA, but that the anti-sense sequence thereof (and thus the length of the hairpin structure) was shorter than the originally introduced 300 nucleotides. Four distinct variants were found: one variant with a 53 nucleotides anti-sense sequence left, one with a 21 nucleotides anti-sense sequence left, one with a 20 nucleotides anti-sense sequence left and one with a 15 nucleotides anti-sense sequence left, denoted as AS 53, AS 21, AS 20 and AS 15, respectively. It was further observed that apparently AS 53 is out competed eventually by AS 15 after still prolonging the tissue culture (fig. 3). Further, it can be observed from fig. 3 that apparently the HIV-1 variants have a preference for certain spots where the anti-sense nucleotides are deleted, since the gels that are depicted in fig. 3 show several distinct bands, whereas if the deletion of anti-sense nucleotides would be a random process, a smear resulting from different lengths of the variants should have been visible on the gel of Fig. 3. Further, it seems that both AS 15 and AS 21/20 are stable variants, since they are present for at least 33 days in the tissue culture cells. Testing of AS 53/15 and AS 21/20 in co-infection experiments wherein the host cells were also challenged with a virulent wild-type HIV-1 strain, showed that the variants still were able to inhibit replication of the wild type vims. Moreover, in contrast to recent publications describing RNA interference-inducing constructs (Boden et al, and Das et al, supra), it appeared that even after prolonged tissue culture no escape from this RNA interference- mediated inhibition was observed (fig. 5). It can thus be learnt that it is surprisingly possible to obtain a vims variant, which is able to inhibit replication of wild type virulent viruses through RNA silencing, and which, very surprisingly, at the same time is capable of self-replication. This thus probably means that there is an optimal length of the sequence which is able to form a double stranded RNA, whereby shortening that sequence would increase the capability of self-replication and whereby deletion of that sequence would render the RNA of the variant target for RNA silencing induced by the provirus. Thus, the attenuated vims variants do not revert to wild type, but maintain a hairpin structure in their genomes to protect themselves from proviras-induced RNA silencing. The life attenuated vimses contain hairpin structures and are thereby protected from RNA silencing induced by a nucleotide sequence encoding one or multiple transcripts capable of folding into double stranded RNA, which is homologous the nucleotide sequence of said hairpin structure. It is believed that this phenomenon will be encountered in all vimses, whereby a distinction should be made between DNA vimses and RNA vimses. RNA vimses, such as HIV, replicate their genome with the help of, amongst others, reverse transcriptase, which is prone to introduce mutations in the reverse transcribed DNA. On the other hand, DNA viruses, especially double stranded DNA viruses replicate like pro- and eukaryotic organisms, i.e. they have DNA polymerases which are able to correct errors during the replication process, the so-called proof-reading activity. Hence, RNA vimses will display a larger mutation frequency, which in this particular case means that the variants in which the originally inserted anti-sense sequence will be shortened will be more abundant than with DNA viruses. Nevertheless, life attenuated viruses according to the invention are characterised by the fact that one of the genes has been made inoperable, e.g. by deleting or inserting a sequence of nucleotides and by the fact that they contain a nucleotide sequence which is identical and or highly homologous with the sequence of the gene which is disrupted, and which is able to form a double-stranded RNA upon expression in a host cell. Nucleic acid sequences comprised in a nucleic acid constmct producing the

RNA silencing-inducing transcripts are preferably fully identical to a (portion of) the viral gene that is to be inhibited. However, RNA sequences with insertions, deletions and single point mutations relative to the targeted viral sequence (i.e. not identical but homologous to the viral sequence) may also be effective for inhibition. This is especially valuable in circumstances where the vims is prone to genomic variation. Even if the vims' genomic sequence shows variation, the inhibiting effect of the nucleic acid construct pertains. Sequence identity may be determined by sequence comparison and alignment algorithms known in the art (see Gribskov, M. and J. Devereux (Eds.), Sequence Analysis Primer, Stockton Press,New York, 1991, and references cited therein), and calculating the percent difference between the nucleotide sequences, for instance by using the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g. University of Wisconsin Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory nucleotide sequence and the portion of the targeted viral gene is preferred. Alternatively, the duplex or double stranded region of the RNA transcript or transcripts encoded by the nucleic acid construct may be defined functionally as a (double stranded) nucleotide sequence that upon denaturing is capable of hybridising with a portion of the transcript of the targeted gene (e.g., under conditions of 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at a temperature of 50°C to 65°C and hybridisation for 12-16 hours, followed by washings). As disclosed herein, 100% sequence identity between the RNA silencing nucleic acid construct and the targeted gene is not required to practice the present invention, and is difficult to obtain in the event that the targeted viral sequence changes or has changed due to genomic variation of the vims. Thus the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence. As used in the present invention, sequence homology between the dsRNA transcripts and one or more viral genes, which are essential for replication of the virus and which are to be inhibited, is essentially substantial, meaning that said homology is suitably greater than 80%, preferably greater than 90%, more preferably greater than 95%, even more preferably greater than 98%, and most preferably greater than 99%. To produce one or more transcripts capable of forming a dsRNA, a nucleic acid constmct is prepared that comprises both a sense and an anti-sense nucleotide sequence in opposite polarities (i.e. so that the anti-sense sequence is the reverse complement of the sense sequence) wherein both sense and anti-sense sequences having a length of at least 15 nucleotides. The length of the predicted dsRNA transcripts should be at least 15 base pairs, but preferably larger, such as 20, 30, 40, 50, 100, 200, 300 or 400 base pairs. It should, however, be noted that the length of the nucleotide sequence should still allow the attenuated virus to replicate. It is believed that for DNA vimses a greater length is tolerated than for RNA vimses. For RNA vimses, in this particular case HIV, it has been shown in the experiments that a length of 53 nucleotides for the anti-sense nucleotides, which thus means a length of about 106 nucleotides for the sequence encoding the dsRNA transcript, is tolerated, because the AS-53 variant is still able to replicate. Further, in order to provide for a transcript that effectively inhibits gene expression of a viral gene, either the sense or antisense strand of the nucleic acid constmct is complementary or exhibits substantial nucleotide sequence homology to a said viral gene. Although a length of 18 base-pairs has been described to be minimally required for a dsRNA to effectively block the expression of a target gene, the experiments show that a length of 15 basepairs is also sufficient to completely block replication of the wild-type vims. The nucleic acid constmct from which the RNA silencing-inducing dsRNA is transcribed should therefore comprise at least 30 nucleotides . Because it is generally recognised that to increase the chances that RNA silencing is effective, and to allow for variation in the wild-type sequences, preferably larger stretches of DNA, with a length of 40, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 nucleotides should be used, it would be advisable to conduct experiments to investigate which length would still give rise to stable attenuated vimses which are able to self-replicate. A person skilled in the art could easily perform such experiments and find the optimal sequence length using routine experimentation. The RNA transcribed from the RNA silencing nucleic acid constmct should form into a dsRNA or RNA duplex. Suitably therefore, the RNA comprises inverted repeats, or is of a palindromic structure but preferably comprises anti-sense and sense sequences. The dsRNA may be in the form of a hairpin structure or panhandle folding, whereby the sense and anti-sense regions are separated by introns or lariat structures (Moore et al. 1993 The RNA World, eds. Gesteland, R. F. & Atkins, J. F. Cold Spring Harbor Lab. Press, Plainview, NY, pp. 303-357). Preferably, the two RNA strands that fomi the RNA duplex are held together by a spacer region that forms a loop. More preferably, the two RNA strands that form the RNA duplex are held together by a spacer region that is left when the intron is spliced out. The sense and anti-sense nucleotide sequences can be connected tlirough a spacer nucleotide sequence of any length (such as an intron sequence) or can be joined directly, in which case the bases ate the connection will not pair because of conformational restraints and thus will form a spacer by nature. If an intact intron sequence is used as spacer this intron may be removed during mRNA foiixiation by endogenous processes in the nucleus of the cell. The order of the sense and anti-sense sequence is not essential, although an anti-sense-sense orientation is preferred for reasons that such a transcript is unlikely to code for a functional gene transcript, even when the sequences are changed or shortened by mutational activity during replication of the vims. It is also possible to combine more than one sense-anti-sense combination in one and the same constmct. The simple form can be depicted as: prom — AS - spac - S — term, wherein prom represents the viral promoter, S the targeted viral DNA sequence, AS the targeted viral DNA sequence in opposite polarity compared to S, "spac" a spacer sequence and "term" the transcriptional temiinator DNA sequence. Also the following constmcts can be applied: prom - AS 1 - spac - SI - spac - AS2 - spac - S2 - term, or prom - AS2 - spac - AS1 - spac - SI - spac - S2 - term. Variations in the composition of the constmct are possible, as long as the transcripts of said constructs may fold partially or fully into dsRNA. For transcription from a nucleic acid constmct, a regulatory sequence, such as a promoter, enhancer, temiinator, splice donor and acceptor, or polyadenylation site may be used to transcribe the DNA. The RNA strand(s) may or may not be polyadenylated and the RNA strand(s) may or may not be capable of being translated into a polypeptide by the cell's translational apparatus. Preferably, the constructs are not translated into a polypeptide.

Attenuated vimses according to the invention may be selected from the group of lentiviruses, herpesviruses and papillomaviruses. Suitable lentiviruses for use as attenuated vims in a method of the invention include bovine lentivirases, such as bovine immunodeficiency vims and Jembrana disease virus, equine lentiviruses such as equine infectious anemia vims, feline lentivirases such as feline immunodeficiency virus (FIV), panther lentivirus and puma lentiviras, ovine/caprine lentivirases such as Brazilian caprine lentivirus, caprine arthritis-encephalitis virus, caprine lentiviras, Maedi-Visna virus, ovine lentivirus and Visna lentivirus and the primate lentiviras group including such viruses as human immunodeficiency virus (HIV) and simian iirmiunodeficiency virus (SIV). Preferably the virus is human immunodeficiency virus (HIV). The genes to be disrupted and targeted for several of the viruses in the above mentioned groups are depicted in Table 1.

Table 1 Target genes for the production of attenuated viruses

Retrovirases

HIV nef protein Herpes simplex virus ICP34.5 protein ICP27 protein ICP47 protein US 11 protein Epstein-Barr virus EBNA-2 protein EBER RNAs Herpesvirus 8 vIRF proteins Papilliomavirases Human papillioma virus E6 protein E7 protein

Attenuated viruses according to the invention can be produced by genetically engineering a wild type virus by dismpting a gene, such as indicated in Table 1, and introducing a sequence which can produce a dsRNA transcript. Preferably, the insertion of the sequence capable of producing a dsRNA transcript causes the dismption of the early gene. If the insert produces dsRNA which is of a length which does not totally abolish the replication of the attenuated virus, such a virus would be ready for use in a vaccine. However, when, such as in the case of the 300 nucleotide insert in the HIV virus as described in the Examples, such an insert would cause inhibition of replication, it would still be possible to obtain attenuated virus according to the invention by continuing the tissue culture which has been transfected with such a virus, and obtaining variant viruses from this virus from the supernatant of the culture after culturing for several days/weeks. In this way, as described in the Examples, the attenuated HIV variants AS-53, AS-21, AS- 20 and AS-15 have been obtained, which form a specific embodiment of the current invention. It is also possible to obtain the mutants AS 53, AS 21, AS 20 and AS 15 by direct genetic engineering. Therefore the sequences from Table 2, which include the mutations that have been found in the mutants AS 53, AS 21, AS 20 and AS 15 , should be engineered in the nef gene of a wild type HIV virus, such as pLai (Peden, K. et al, 1991, Virology 185:661-672). Although one of the characteristics of these attenuated vimses is that they are still capable of self-replication, this self-replication is impaired in comparison to the wild type viras. In the Examples it is shown that addition of a silencing suppressor boosts the production of viras particles in producer cells and is even able to overcome the inliibition of replication of the wild type virus when co-transfected with an attenuated virus (fig. 2). Life attenuated viruses according to the invention can be used as a vaccine for a subject who is infected with a virus or who is at risk of becoming infected with a viras. A live attenuated viral vaccine can be formulated according to techniques known in the art. Production of such live attenuated virus vaccine formulations may be accomplished using conventional methods involving propagation of the virus in cell culture as described above followed by purification. In the formulation a range of about 10⁴ pfu to about 5*10⁶ pfu per dose should be used. Many methods may be used to introduce the vaccine formulations described above, these include but are not limited to intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. It may be preferable to introduce the virus vaccine formulation via the natural route of infection of the parental attenuated virus. A vaccine of the present invention, comprising 10⁴-5*10⁶pfu of attenuated viruses could be administered once. Alternatively, a vaccine of the present invention, comprising 10⁴-5*10⁶pfu of attenuated viruses could be administered twice or three times with an interval of 2 to 6 months between doses. Alternatively, a vaccine of the present invention, comprising 10⁴- 5*10⁶pfu of attenuated viruses could be administered as often as needed to an animal, preferably a mammal, and more preferably a human being. The present invention encompasses pharmaceutical compositions comprising attenuated viruses to be used as anti-viral agents. The pharmaceutical compositions have utility as an anti-viral prophylactic and may be administered to an individual at risk of getting infected or is expected to be exposed to a virus. For example, when people run at risk of getting in contact with a source of infection, it would be indicated to administer the anti-viral pharmaceutical composition of the invention to that person to prevent viral infection and subsequent illness. In the case of HIV prophylactic infection would be indicated for prostitutes, adult movie actors and actrices, and so on. People traveling to parts of the world where a certain infectious disease is prevalent can also be treated.

Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The pharmaceutical compositions of the present invention may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, in a preferred embodiment it may be desirable to introduce the pharmaceutical compositions of the invention into the blood by any suitable route. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. In a specific embodiment, especially related to the herpesviruses and/or the papillomaviruses, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue. In yet another embodiment, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger & Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351 (1989); Howard et al., 1989, J. Neurosurg. 71:105). In yet another embodiment, a controlled release system can be placed in proximity of the composition's target organ, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533).

The vaccines and pharmaceutical compositions of the present invention comprise a therapeutically effective amount of the attenuated virus, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. These compositions can be formulated as a suppository. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. The amount of the pharmaceutical composition of the invention which will be effective in the treatment of a particular infection or condition will depend on the nature of the infection or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or infection, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for administration are generally about 10⁴-5*10⁶ pfu and can be administered once, or multiple times with intervals as often as needed. Pharmaceutical compositions of the present invention comprising 10⁴- 5*10^G pfu of attenuated viruses can be administered intranasally, intravenously, intramuscularly or subcutaneously Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. The present invention will now be illustrated by way of the following non limiting examples.

EXAMPLES

Example 1. Construction of the recombinant lentiviral vector vaccine.

A recombinant lentiviral vector vaccine to be used against HIV-1 infections can be made using the infectious cDNA clone of HIV-1, denoted pLai (Peden K., et al, 1991. Virology 185:661-672). The unique Xhol restriction site located within the 5' coding domain of the Nef cistron and the BamHl site located in the Env coding domain are used for deletion of the 5' 150 basepairs of the Nef coding region and for insertion of a 300 basepairs long DNA fragment con-esponding with the 3 'part of the Nef cistron in antisense orientation. DNA of pLAi was subjected to PCR using synthetic oligonucleotides

JvdV174: 5'-CTAGTGGATCCTTAGCACTTATCTGGGACG-3' and JvdV175: 5'- CCAGATAACTTATAGCAAAATCCTTTCCAAGCCC-3' yielding a 330 basepairs long DNA fragment and synthetic oligonucleotides JvdV176: 5'- CCCGCTCGAGTGCTGCTTGTGCCTGGCTAGAAGCACAAGAGG-3 ' and JvdV177: 5 -GCTATAAGTTATCTGGCTCAACTGGTACTAGCTTGTAGC-3, yielding a 300 basepairs long DNA fragment. Both DNA fragments are gel purified, mixed and again subjected to PCR using synthetic oligonucleotides JvdV175 and JvdV176, yielding a 630 basepairs long DNA fragment. The DNA fragment is digested with Xhol and BamHl, gel purified and cloned into the Xhol and BamHl site of pLai, yielding pLai- //zNef. pLai-/ANef has a 150 basepairs long deletion in 5 'region of the Nef coding domain and a 300 basepairs long inverted repeat within the viral vector genome, without having direct repeats in the viral vector genome other than the LTR sequences (See figures 3B and 4).

Example 2. Transfection experiments usins the recombinant lentiviral vector vaccine.

HEK293 human embryonic kidney cells (Graham et al., 1977. J. Gen Virol. 36:59-74; purchased from hivitrogen) are grown at 37 degrees Celsius in DME medium. Twice a week the confluent cell cultures are diluted 10 times in DME medium and sub cultured at 37 degrees Celsius. Co-transfection of HEK293T cells with pLai and pLai- /TzNef DNA using lipofectamin ( nvitrogen) and a protocol supplied by the manufacturer, leads to production of very low amounts of viras. The amount of vims present in the supernatant is quantified using a p24 (capsid) enzyme-linked immunosorbent assay

(ELISA) according to the manufacturer's protocol (Beckman-Coulter). This demonstrates that the /ΛNef gene cassette in pLai-//?Nef effectively inhibits the expression of the early regulatory viral proteins Rev, Tat and Nef, encoded by the early multi-spliced HTV-1 mRNAs as well as all other HTV-1 mRNAs (See figure 1). This recombinant anti-HIV-1 viral vector vaccine strain: pLai-/ΛNef is attenuated in vivo through the loss of the Nef gene and both in vitro and in vivo through the presence of the anti-HIV-1 gene cassette. Using the same protocol it was shown that viral RNA silencing suppressors are able to enhance the production of HIV-1 (See figure 2) and to suppress the RNA silencing-based inliibition of Lai production by pLai-//zNef. SupTl human non-Hodgkin's T-lymphoma cells (Smith S.D., et al., 1984.

Cancer Research 44:5657) are grown at 37 degrees Celsius in RPMI 1640 medium with 2 mmol/L L-Glutamine (Gibco) supplemented with 10% Fetal bovine serum (Biochrom KG) and 100 μg/ml Zeocine (Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin (RPMI medium). Twice a week the cell cultures containing 2 times 10⁶ cells/ml are diluted 10 times in RPMI medium and sub cultured at 37 degrees Celsius. SupTl cell cultures containing 10⁶ cells/ml are electroporated with pLai-/ΛNef DNA and incubated at 37 degrees Celsius according to Beerens, N. & Berkhout, B. 2002, Journal of Virology 76: 2329-2339. After electroporation, the cells are cultured for prolonged periods of time. Total RNA is isolated from the electroporated cells using the RNeasy tissue kit (Qiagen) and subjected to PCR using synthetic oligonucleotides Eup: 5'-GGAGCCTGTGCCTCTTCAGC-3' and Ldo: 5'-GGAAAGTCCCTTGTAGCAAGC-3', yielding a DNA fragment of 1200 basepairs long for Lai-/ΛNef and of 900 basepairs long for Lai. Figure 3 demonstrates that in time Lai-/ΛNef is replaced with different virus escape variants carrying shorter antisense-sense combinations in the Nef coding domain. Different supematants of Sup-Tl cultures each containing a single species of a stable escape variant are stored at -80 degrees Celsius. A number of these escape variants have been sequence verified. The most interesting variants are AS 45, which has an inverted Nef repeat of 45 basepairs long; AS 19, which has an inverted Nef repeat of 19 basepairs long and AS 15, which has an inverted Nef repeat of 15 basepairs long (See figure 4). All these variants are able to replicate in Sup-Tl cells, however at a reduced level. Moreover, they do not cause the formation of syncitia, characteristic of infection of Sup-Tl with Lai viras.

Example 3. Co-infection experiments usins Lai and the escape variants.

Sup-Tl cells are co-infected with Lai and different escape variants. After infection, the cells are cultured for prolonged periods of time. Ten days post infection syncitium formation is observed in the cultures infected with Lai only, hi the cultures co-infected with Lai and escape variants AS 45 or AS 19 no syncitium fonnation is observed even after 33 days post infection. PCR analysis revealed that AS 45 but also AS 19 effectively inhibit Lai replication (See figure 5). This demonstrates that the inverted repeats present in the AS45 and AS 19 variants are much more active than inverted repeats expressed from a retroviral vector (Das A.T., et al, 2004. J. of Virol. 78: 2601-2605) or from an AAV vector (Boden D., et al., 2003. J. of Virol. 77:11531-11535).

TABLE 2 15 25 35 45 55 65 75 85 95

LAI-lhNEF TTCGCCACAT ACCTAGAAGA ATAAGCACAG GGCTTGGAAA GGATTTTGCT ATAAGATTAT CTGGCTCAAC TGGTACTAGC TTGTAGCACC ATCCAAAG

AS 53 Rl TAGAAGA ATAAGCACAG GGCTTGGAAA GGATTTTGCT ATAAGATTAT CTGGCTCAAC TGGTACTAGC TTGTAGCACC ATCCAAAG

AS 53 R2.1 TTCGCCACAT ACCTAGAAGA ATAAGCACAG GGCTTGGAAA GGATTTTGCT ATAAGATTAT CTGGCTCAAC TGGTACTAGC TTGTAGCACC ATCCAAAG

AS 53 R3.1 ~ —CTAGAAGA ATAAGCACAG GGCTTGGAAA GGATTTTGCT ATAAGATTAT CTGGCTCAAC TGGTACTAGC TTGTAGCACC ATCCAAAG

AS 15 ~~ —AAGCACAG GGCTTGGAAA GGATTTTGCT ATAAGATTAT CTGGCTCAAC T~

AS 21 —CGCCACAT ACCTAGAAGA ATAAGCACAG GGCTTGGAAA GGATTTTGCT ATAAGATTAT CTGGCTCAAC TGGTACT- AS 20 -CCTAGAAGA ATAACCACAG GGCTTGGAAA GGATTTTGCT ATAAGATTAT CTGGCTCAAC TGGTAC-- I I I I ^■ I [ I I I I [ I I 1 I I [ I .... I 105 115 125 135 145 155 165 175 185 195

LAI-lhNEF CAGTGGATAT CTGACCCCTG GCCCTGGTGT GTAGTTCTGC CAATCAGGGA AGTAGCCTTG TGTGTGGTAG ATCCACAGAT CAAGGATATC TTGTCTTC

AS 53 Rl

AS 53 R2. 1

AS 53 R3. 1

AS 15

AS 21

AS 20 I [ I I I I I I I [ I I I 205 215 225 235 245 255 265 275 285 295 LAI-lhNEF TGGGAGTGAA TTAGCCCTTC CAGTCCCCCC TTTTCTTTTA AAAAGTGGCT AAGATCTACA GCTGCCTTGT. AAGTCATTGG TCTTAAAGGT ACCTGAGG AS 53 Rl

AS 53 R2.1

AS 53 R3.1

AS 15

AS 21 S 20 ....I....I ....I....I ....|.... I ....|....| ....|....| ....|....| ....!.... I ....|.... I ....|....| ....|.... 305 315 325 335 345 355 365 375 385 395 LAI-lhNEF TGACTGGAAA ACCCACCTCC TCCTCCTCTT GTGCTTCTAG CCAGGCACAA GCAGCACTCG AGACCTGGAA AAACATGGAG CAATCACAAG TAGCAATA AS 53 Rl ——~ ~~- ~ CAA GCAGCACTCG AGACCTGGAA AAACATGGAG CAATCACAAG TAGCAATA

AS 53 R2.1 ~ --CAA GCAGCACTCG AGACCTGGAA AAACATGGAG CAATCACAAG TAGCAAA

AS 53 R3.1 —— ~ —~~~~ CAA GCAGCACTCG AGACCTGGAA AAACATGGAG CAATCACAAG TAGCAATA

I ! I I I I I I [ I I I I I [ I I I I 405 415 425 435 445 455 465 475 485 495

LAI-lhNEF GCAGCTACCA ATGCTGCTTG TGCCTGGCTA GAAGCACAAG AGGAGGAGGA GGTGGGTTTT CCAGTCACAC CTCAGGTACC TTTAAGACCA ATGACTTA

Rl GCAGCTACCA ATGCTGCTTG TGCCTGGCTA GAAGCACAAG AGGAGGAGGA GGCGGGTT

R2.1 GCAGCTACCA ATGCTGCTTG TGCCTGGCTA GAAGCACAAG AGGAGGAGGA GGTGGGTTTT CCAGTCACAC CTTAGGTACC TTTAAGACCA ATGACTTA

R3.1 GCAGCTACCA ATGCTGCTTG TGCCTGGCTA GAAGCACAAG AGGAGGAGGA GGTGGGTTTT CCAGTCACA- TTTAGGTACC TTTAAGACCA ATGACTTA

R3.2 ~ — „_ _ACAC CTCAGGTACC TTTGGGACCA _ATG_ACTT_A

R5

R6 I I I I I I I I |.... I I I [ [ I I |.... I ....I 505 515 525 535 545 555 565 575 585 595

LAI :-lh EF AGGCAGCTGT AGATCTTAGC CACTTTTTAA AAGAAAAGGG GGGACTGGAA GGGCTAATTC ACTCCCAACG AAGACAAGAT ATCCTTGATC TGTGGATC

Rl

R2 . 1 AGGCAGCTGT ATATCTTAGC CACTTTTTAA AAGAAA

R3 . 1 AGGCAGCTGG AGATCTTAGC CACTCT

R3 . 2 AGGCAGCTGC AGATCTTAGC CACTTTTTAA AAGAAAGGGG GGGACTGGAA GGGCT

R5 AGCTGT GGATCTTAGC CACTTTTTGA AAGAAAAGGG GGGACTGGAA GGGCTGGTTC ACTCCCAACG AGGACAAGAT ATCCTTGATC TGTGGATC

R6 AGCTGT GGATCTTAGC CACTTTTTGA AAGAAAAGGG GGGACTGGAA GGGCTGGTTC ACTCCCAACG AGGACAAGAT ATCCTTGATC TGTGGATC i I I I I I [....I I I I I I I [ I I I ....| 605 615 625 635 645 655 665 675 685 695

LAI :-lhNEF CCACACACAA GGCTACTTCC CTGATTGGCA GAACTACACA CCAGGGCCAG GGGTCAGATA TCCACTGACC TTTGGATGGT GCTACAAGCT AGTACCAG

Rl

R2 . 1

R3 . 1

R3 . 2

R5 CCACACGCAG GGCTGCTTCC CTGATTGGCA GAGCTGCGCA CCGGGGCCAG GGGTCGGATA TCCACTGACC TTTGGATGGT GCTACAAGCT AGTACCAG

R6 CCACACGCAG GGCTGCTTCC CTGATTGGCA GAGCTGCGCA CCGGGGCCAG GGGTCGGATA TCCACTGACC TTTGGATGGT GCTACAAGCT AGTACCAG ....|....| ....|....| ....|.... I ....|.. 705 715 725 735

LAI-lhNEF GAGCCAGATA AGGTAGAAGA GGCCAATAAA GGAGAGA Bold - asNEF

Rl

R2 . 1 Bold italics

R5 GAGCCAGATA AGGTAGAAGA GGCCAATAAA GGAGAGA

Claims

Claims

1. Life attenuated retro vims, characterized in that it is still capable of self-replication and that it comprises a sequence which is able to be expressed as a double-stranded RNA which is identical or highly homologous with a sequence comprised in the wild type vims.

2. Life attenuated viras according to claim 1, characterized in that the viras is a retrovirus, more preferably a lentivirus, more preferably the human immunodeficiency virus, most preferably HIV-1..

3. Life attenuated virus according to claim 2, characterized in that attenuation is achieved by at least disrupting the nef gene.

4. Life attenuated virus according to claim 2 or 3, characterised in that the double stranded RNA is identical to or highly homologous with a fragment of the nef gene of a wild type vims.

5. Life attenuated viras according to any of claims 1 to 4, characterised in that the sequence which is able to be expressed as a double-stranded RNA comprises more than 20 nucleotides and less than 150 nucleotides

6. Life attenuated viras according to any of claims 2 to 5, characterized in that it comprises the sequence as depicted in Table 2.

7. Life attenuated viras according to any of claims 2 to 5, characterized in that the virus is variant AS-53, AS-21, AS-20 or AS-15.

8. Method to produce a virus according to any of claims 1 to 7, characterized in that it comprises the steps of: a. constructing a life attenuated viras in which a virulence gene has been disrupted; b. inserting in said viras, at a spot that does not interfere with viras replication, a nucleotide sequence of more than 100 nucleotides which is capable of forming a double stranded RNA, when expressed in a host cell; c. transfecting a producer cell with said virus; d. culturing said transfected producer cell for at least two weeks; and e. harvesting variant viruses from the supernatant of said cell culture.

9. Method to produce a life attenuated viras according to any of claims 1 to 7, characterized in that it comprises the steps of: a. constructing a life attenuated viras in which a viralence gene has been dismipted; b. inserting in said viras, at a spot that does not interfere with viras replication, a nucleotide sequence according to Table 2; c. transfecting a producer cell with said viras; and d. harvesting viruses from the supernatant of said cell culture.

10. Use of a life attenuated viras according to any of claims lto 7 or a viras produced by a method according to claim 8 or claim 9 for therapeutic use or use as a vaccine.