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  Pharmaceutical Patents  

 

Title:  Infectious recombinant paramyxovirus from antigenomic cDNA
United States Patent: 
7,851,214
Issued: 
December 14, 2010

Inventors:
 Billeter; Martin A. (Zurich, CH), Spielhofer; Pius (Buron, CH), Kalin; Karin (Gif-su-Yvette, FR), Radecke; Frank (Ulm, DE), Schneider; Henriette (Zurich, CH)
Assignee:
  Crucell Switzerland AG (Bern, CH)
Appl. No.:
 11/320,487
Filed:
 December 27, 2005


 

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Abstract

The present invention relates, in general, to a methodology or the generation of nonsegmented negative-strand RNA viruses (Pringle, 1991) from cloned deoxyribonucleic acid (cDNA). Such rescued viruses are suitable for use as vaccines, or alternatively, as plasmids in somatic gene therapy applications. The invention also relates to cDNA molecules suitable as tools in this methodology and to helper cell lines allowing the direct rescue of such viruses. Measles virus (MV) is used as a model for other representatives of the Mononegavirales, in particular the family Paramyxoviridae.

Description of the Invention

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates, in general, to a methodology for the generation of nonsegmented negative-strand RNA viruses (Pringle, 1991) from cloned deoxyribonucleic acid (cDNA). Such rescued viruses are suitable for use as vaccines, or alternatively, as vectors in somatic gene therapy applications. The invention also relates to cDNA molecules suitable as tools in this methodology and to helper cell lines allowing the direct rescue of such viruses. Measles virus (MV) is used as a model for other representatives of the Mononegavirales, in particular the family Paramyxoviridae.

The invention provides the technology for construction of recombinant vaccine strains, in particular MV vaccine strains containing coding regions for the expression of epitopes or entire protein from other viruses, bacteria, or parasites. It also demonstrates that chimeric MV strains containing heterologous envelope proteins can be constructed suitable for targeting cells not containing an MV receptor. Thus, in principle, plasmids based on the genome of MV, packaged in envelopes containing proteins for targeting special cell types can be constructed, encoding gene products either lacking in genetically defective individuals or toxic for targeted malignant cells.

By straightforward replacement of the MV-specific helper cell lines described in this invention by cell lines expressing the cognate proteins encoded by other representatives of the Mononegavirales to be rescued, any other member of this viral order replicating in vertebrate cells can be used for the purpose of live vaccines or of vectors for gene therapy instead of MV.

2. Background Information

Measles Virus

MV is a member of the family Paramyxoviridae. Its genetic information is encoded on a single RNA strand of negative polarity, comprising 15894 nucleotides. The genome is sequentially transcribed from the 3' terminus to yield, in addition to a leader RNA, 6 major capped and polyadenylated messenger ribonucleic acid (RNA) species, each of which encodes one major protein. The genome map is shown in FIG. 1 (see Original Patent), indicating the genes specifying as the principal products N (nucleocapsid protein), P (phosphoprotein), M (matrix protein), F (fusion protein), H (hemagglutinin) and L (large protein=polymerase). Several additional RNA and protein species, in part mentioned in the Table of FIG. 1 (see Original Patent) complicate this simple picture, but they are not relevant here.

MV is a major cause of acute febrile illness in infants and young children. According to estimates of the World Health Organisation (WHO), one million young children die every year from measles. This high toll arises primarily in developing countries, but in recent years also industrialised countries such as the USA have been affected again by measles epidemics, primarily due to incomplete adherence to immunisation programs (Clements and Cutts, 1995). At present, several live attenuated MV vaccine strains are in use (including the Schwarz, Moraten and Edmonston-Zagreb strains), almost all derived from the original Edmonston strain (Enders and Peebles, 1954) by multiple passage in non human cells (Enders, 1962). For a recent discussion of MV vaccinology including future trends see Norrby (1995). Measles vaccine is usually administered at 15 months of age or, in developing countries, already at 6 months, and has proved to be highly effective, usually providing life-long immunity against MV reinfection eliciting morbidity. To date, the genetic alterations responsible for attenuation of these vaccine strains remain unknown. The proven safety of measles vaccine, combined with its high and long-lasting efficiency, predestines it as an ideal plasmid for the expression of heterologous genes. Such a vaccine may prove as efficient in eliciting long-lasting immune protection against other pathogenic agents as against the vector virus itself. Another possible candidate as vaccination vector is Mumps virus, a distant relative of MV, which is also highly efficaceous and safe as attenuated live vaccine.

Rescue of RNA Virus from Cloned DNA

The study of the replication cycle of a number of RNA viruses has been greatly facilitated by the availability of DNA clones from which infectious virus can be rescued, thus allowing the application of reverse genetics. Initially, the bacteriophage Q.beta. (Taniguchi et al., 1978) and polio virus (Racaniello and Baltimore, 1981), and subsequently Sindbis virus (Rice et al., 1987) were expressed from cloned cDNA. To date, a large variety of positive-strand RNA viruses, primarily infecting vertebrates and plants, can be rescued from cloned DNA (for a recent review see Boyer and Haenni, 1994). In addition, proviral DNA of retroviruses is infectious. However, attempts to obtain infectious virus from cDNA clones of negative-strand RNA viruses have met with great difficulties. This is due to two properties of these viruses: (i) neither genomic nor antigenomic RNAs are infectious, because they do not serve as mRNAs; and (ii) both transcription and replication require ribonucleocapsids, i.e., rod-like nucleoprotein complexes (RNPs), containing the genomic RNA and several proteins with structural and/or enzymatic function.

Rescue from cloned DNA has been achieved several years ago in the case of influenza virus, a negative-strand RNA virus containing eight genome segments. Their RNPs which are small in size and loosely structured as revealed by the susceptibility of their RNA component to RNase, can be assembled in vitro from RNA and the required viral proteins, N and the polymerase components. Initially, an artificial RNA has been used carrying as a reporter the chloramphenicol acetyltransferase (CAT) coding sequence embedded in the noncoding terminal segments of an influenza virus genome subunit (Luytjes et al., 1989). Later, single authentic or altered genome subunit RNAs transcribed in vitro from cloned DNA were used (Enami and Palese, 1991). The assembled RNPs replicated and transcribed upon transfection into influenza-infected cells, as monitored by CAT production and by rescue of a reasserted influenza virus, respectively. Purification of virus containing the introduced subunit from the vast excess of non-reassorted virus in some cases can be accomplished by selection, for example, using a specific neutralising antibody directed against the protein encoded by the cognate subunit of the helper virus.

In contrast, for the viruses with a nonsegmented negative-strand RNA genome, grouped together in the order Mononegavirales (Pringle, 1991) the much more tightly structured and longer RNPs, containing in addition to the N protein the assembly and polymerase cofactor phosphoprotein (P) and the viral RNA polymerase (large protein, L) have been refractory to functional reassociation in vitro. Therefore, many laboratories approached the rescue of representatives of the Mononegavirales starting out with subgenomic RNAs containing only essential sections of the viral genomes, using viruses to provide the helper proteins required to intracellularly encapsidate and replicate these mini-replicons. First, naturally arising subgenomic RNAs, competing with the viral replication and thus known as defective interfering particle (DI) RNAs (Re, 1991) were used, being substituted later by artificial DI RNAs containing reporter genes, transcribed from appropriately constructed plasmids. These mini-replicons, first devised by the group of M. Krystal (Park et al., 1991) according to the replicon used for the initial influenza rescue model (Luytjes et al., 1989), carry a CAT coding sequence inserted into viral noncoding terminal regions of Sendai virus (SeV) and have been used successfully also for respiratory syncytial virus (Collins et al., 1993; Collins et al., 1991), human parainfluenza virus 3 (Dimock and Collins, 1993), rabies virus (RV) (Conzelmann and Schnell, 1994) and MV (Sidhu et al., 1995).

In all these systems, the essential helper proteins were provided either by the homologous viruses or by the vaccinia vector vTF7-3 encoding phage T7 RNA polymerase (Fuerst et al., 1986) to drive T7-specific transcription of transfected plasmids encoding the required proteins N, P and L as pioneered by Pattnaik et al., (1990). These investigations using mini-replicons have allowed important insights into the noncoding regulatory regions of the corresponding viral genomes and antigenomes (for a recent discussion see Wertz et al., 1994). Adopting the same experimental set up, the rescue of VSV, as RV a member of the Rhabdoviridae, has now also been reported (Lawson et al., 1995).

An important drawback of that method (as well as the method reported for the rescue of negative-strand RNA viruses with a segmented genome) is the involvement of a helper virus which has to be separated from the rescued virus and which can interfere with the replication of the virus to be rescued. For RV and VSV, both belonging to the rigidly structured Rhabdoviridae and replicating to high titers, this is not an important problem. However, in case of loosely structured, polymorphic virions typical for the members of the family Paramyxoviridae and in case of viruses yielding only relatively low titers, the presence of a helper virus would render the recovery of rescued viruses difficult and may well preclude their rescue altogether.

Accordingly, the technical problem underlying the present invention was to provide genetic material useful for the generation of non-segmented negative-strand RNA viruses, preferably of the family Paramyxoviridae and most preferably of measles virus and a system for the recovery of such viruses with reasonable efficiency. The solution to said technical problem is provided by the embodiments characterised in the claims.

Thus the present invention relates to a cDNA molecule for the production of negative-strand RNA virus comprising (a) the entire (+)-strand sequence of a non-segmented negative-strand RNA virus of the family Paramyxoviridae from which anti-genomic RNA transcripts bearing the authentic 3'-termini can be transcribed; operatively linked to (b) an expression control sequence.

Accordingly, the present invention relates to a cDNA molecule for the production of any negative-strand RNA virus of the family Paramyxoviridae. Preferably said anti-genomic RNA transcripts also bear the authentic 5'-termini.

As has been further found in accordance with the present invention, effective production of measles virus which is a negative-strand RNA virus of the family Paramyxoviridae, is only obtained if the replicons specified by said cDNA molecule consist of an integral multiple of six nucleotides. This phenomenon will also be referred to as the "rule of six" throughout this application. The cDNA molecules of the present invention can conveniently be used for the rescue of negative strand RNA viruses of the family Paramyxoviridae.

In a preferred embodiment of the present invention, in said cDNA molecule, the expression control sequence (b) is an RNA polymerase promoter.

The present invention further relates to a plasmid containing the cDNA molecule of the invention. The plasmid of the present invention is capable of propagation and preferably also expressing the cDNA molecule of the invention as an antigenomic RNA.

In a preferred embodiment, said plasmid contains an expressible DNA fragment which replaces a preferably homologous DNA region of said cDNA molecule, or provides additional genetic information.

As was also found in accordance with the present invention, in the case of MV-based replicons the rule of six must be obeyed, if a foreign--homologous or heterologous--expressible DNA fragment is inserted into the plasmid containing the cDNA of the invention. In other words, any newly created replicon specified by appropriately constructed cDNA molecules will only be capable of yielding reasonable amounts of the desired product, if it obeys the rule of six.

In a most preferred embodiment, said plasmid is characterised in that the expressible DNA fragment is inserted into or adjacent to a region of said cDNA encoding a viral protein, said insertion being effected in a manner maintaining the reading frame to create a fusion protein and permitting the expression of said DNA fragment under the control of the signal sequences of said viral protein. In accordance with the present invention it is anticipated that in various cases appropriate C-terminal extensions of viral proteins will not interfere with their functionality.

In variation to the above described preferred embodiment and also comprised by the present invention, the expressible DNA fragment is expressed in such a manner downstream of a viral protein coding region to avoid formation of a fusion protein, but nevertheless allowing expression of the downstream coding sequence either by a stop/restart mechanism where the last A residue of the upstream termination triplett coincides with that of the start codon of the downstream coding region, or by placing an internal ribosome entry site (IRES) between the two coding regions; see example 12, second paragraph.

In a further most preferred embodiment, said plasmid is characterised in that the expressible DNA fragment is inserted into a non-coding region of said cDNA and flanked by viral signal sequences or heterologous signal sequences controlling the expression of the RNA fragment specified by said DNA fragment; see example 12, first paragraph.

Most preferably, the expressible DNA fragment is placed upstream of the N gene. As has been found in accordance with the present invention, the positioning of said expressible DNA fragment at the 5' end of the MV sequence results in a particularly strong expression thereof; see also Example 14.

Examples of this embodiment, creating additional transcription units, are provided by the plasmids specifying MVs expressing the heterologous CAT reading frame shown in FIG. 10 (see Original Patent).

A further preferred embodiment of the invention relates to a plasmid comprising a genomic ribozyme sequence immediately adjacent to the 3' terminal nucleotide of said cDNA molecule and optionally downstream of said genomic ribozyme sequence at least one terminator, preferably the T7 terminator.

The inclusion of a ribozyme sequence into the plasmid of the invention leads to the faithful cleavage of the RNA transcript, thus greatly enhancing the yield of transcripts bearing the correct 3' termini which, in the case of MV, must obey the rule of six.

The person skilled in the art is, naturally, capable of devising other means that result in the generation of the authentic 3' termini. Such means include the use or incorporation of restriction sites at the DNA level, or of tripplehelical DNAs.

In a most preferred embodiment of the plasmid of the invention said genomic ribozyme sequence is the hepatitis delta virus genomic ribozyme sequence.

The invention relates in a further preferred embodiment to a plasmid bearing said cDNA which is capable of replicating in a prokaryotic host. A preferred example of such a prokaryotic host is E. coli. Illustrations of this preferred example are all cDNA constructs giving rise to modified MVs as shown in FIGS. 2 and 10 (see Original Patent) demonstrating plasmids replicating to high copy number in E. coli.

Additionally, the present invention relates in a preferred embodiment to a plasmid bearing said cDNA(s) which is capable of replicating in a eukaryotic host.

The invention envisages the replication and expression (i.e. transcription, followed by translation of the transcripts formed) of the rescued vector, i.e. the packaged RNA particles (RNPs), in any suitable eukaryotic, preferably vertebrate, host cell. Preferred host cells are those with a high replication and expression capacity. Most preferred are those host cells that allow an easy recovery of rescued viruses for further replication and subsequent formulation in vaccines.

The invention relates in another preferred embodiment to a plasmid wherein said expressible DNA fragment is a DNA fragment being homologous or heterologous with respect to the negative-strand RNA virus and encoding at least one immunogenic epitope.

In a further preferred embodiment of the present invention in said plasmid said expressible DNA fragment encodes at least one immunogenic epitope of at least one pathogen, preferably an envelope protein, at least one gene product lacking in genetically defective individuals or toxic for targeted malignant cells.

This most preferred embodiment of the invention allows for the construction of plasmids as a basis for vaccines that effectively induce an immune response against one or preferably various different pathogens. In the case that the expressible DNA fragment encodes an envelope protein of a different virus than measles virus or of another pathogen, a measles virus based plasmid can be used to target specific cell types usually not recognised by measles virus. Said cell types can then selectively be targeted by rescued viruses specified by the plasmid of the invention and confer to said cell type, for example, a molecule that said cell type is in need of or a toxin, if said cell type is to be eliminated. Naturally, said molecule or toxin is also to be encoded by said plasmid. The person skilled in the art is capable of devising further applications of this basic principle for which the plasmid of the invention can be used.

Also, said plasmid can encode a product lacking in genetically defective individuals. The rescued virus can then be used for gene therapy of said genetically defective individuals.

Further, malignant cells can be targeted by the rescued virus which is based on the plasmid of the invention and molecules toxic for said malignant cells may be delivered.

In a further most preferred embodiment of the present invention, in said plasmid said expressible DNA fragment is derived from a virus, a bacterium, or a parasite.

A further preferred embodiment of the invention relates to a plasmid wherein said expressible DNA fragment encodes an immunogenic epitope being capable of eliciting a protective immune response.

In a further preferred embodiment, the cDNA molecule or the plasmids according to the invention are based on an RNA virus which is measles virus or mumps virus.

The invention relates further to a prokaryotic or eukaryotic host cell transformed with a plasmid according to the invention. Preferred host cells have been discussed above.

Additionally, the invention relates to a helper cell capable of expressing an RNA replicon from a cDNA molecule of the invention, said cDNA molecule being comprised in the plasmid of the invention or a plasmid comprising a cDNA molecule for the production of negative-strand RNA virus of a family of the order Mononegavirales which is not a member of the family of the Paramyxoviridae, said cDNA molecule comprising the entire (+)-strand sequence, operatively linked to an expression control sequence, and optionally an expressible DNA fragment which replaces a preferably homologous DNA region of said cDNA molecule or provides additional genetic information, said expressible DNA fragment encoding preferably at least one immunogenic epitope of at least one pathogen, which most preferably is capable of eliciting a protective immune response, said cell further being capable of expressing proteins necessary for transcription, encapsidation and replication of said RNA.

Apart from the features described above, the cDNA molecule for the production of negative-strand RNA virus of a family of the order Mononegavirales which is not a member of the family of the Paramyxoviridae may also have in certain embodiments the characteristics of the cDNA molecules of the invention that were discussed herein above, optionally in conjunction with the plasmids of the invention.

In view of the problems the prior art was confronted with for rescuing non-segmented negative-strand RNA viruses, in accordance with the present invention paradigmatic cell lines providing as helper functions T7 RNA polymerase and MV N and P protein were developed. Rescue of MVs can be directly monitored after transfection with plasmids specifying antigenomic RNAs and MV L mRNA. In principle, analogous helper cell lines can be generated for any of these viruses; thus this rescue approach is applicable for all Mononegavirales replicating in vertebrate cells.

Thus, in a preferred embodiment of the helper cell according to the invention said proteins necessary for encapsidation, transcription and replication of said RNA are an RNA polymerase, preferably T7 RNA polymerase and optionally T3 RNA polymerase, and N and P protein, preferably of the virus to be rescued. In accordance with the present invention, said proteins are expressed from stably transfected expression plasmids, henceforth defined as genomic expression.

Since the rescue system now developed, in contrast to the one used for rescue of RV (Schnell et al., 1994), VSV (Lawson et al., 1995) and very recently also for SeV (D. Kolakofsky, personal communication), does not rely on any helper virus, there is no need to separate the rescued virus from the vast excess of any helper virus. Elimination of vaccinia virus from rescued virus is accomplished by a simple filtration step in the case of the rigidly structured virions of Rhabdoviridae but would involve more complex purification schemes in case of pleomorphic Paramyxoviridae, particularly those not replicating to high titers such as MV. Furthermore, for viruses impaired in replication and/or budding by the vaccinia virus, rescue using the prior art systems might fail altogether. Another possible drawback of the prior art systems based on the vaccinia helper virus is the high frequency of DNA recombinations occurring in the cytoplasm of vaccinia virus infected cells which might cause recombination of the plasmid bearing the anti-genomic sequence with the plasmids encoding N, P and L protein required for the helper function; this may lead to rescue of viruses containing N, P and L sequences derived in part from the helper plasmids rather than from the plasmid bearing the antigenomic sequence. The helper cell system circumvents all of these problems and should in principle be applicable for the rescue of any of the Mononegavirales replicating in vertebrate cells.

It may not be necessary for the rescue of any single representative of Mononegavirales, to establish a helper cell line expressing the cognate N and P protein (in addition to T7 polymerase). Mini-replicon constructs containing the noncoding terminal regions (NCTs) of canine distemper virus (CDV) which is like MV a morbillivirus, differing from MV in 35% of the nucleotides in the NCTs, replicate in the MV-specific helper cells at an efficiency approaching that of the homologous MV mini-replicon. Thus, possibly CDV could be rescued with the 293-3-46 cells, which were developed in accordance with the present invention and more generally, any helper cell line might be able to rescue a number of not too distantly related Mononegavirales. This will probably depend on the compatibility of the proteins elicited by the related viruses, which was shown not to be the case for SeV-specific N and P and PIV3-specific L (Curran and Kolakofsky, 1991).

For the establishment of new helper cell lines for other viruses which are also envisaged by the present invention, the following considerations might be helpful. The constitutive expression of the T7 RNA polymerase and the MV proteins N and P did not impair the long term stability of the 293-3-46 cell line, as mentioned in the examples attached hereto. Thus, inducible expression of these proteins, for example, by the approaches described by the group of Bujard (for a review see Gossen et al., 1993) will probably not be necessary, although it cannot be excluded that the N and P proteins of other viruses are more deleterious for cell growth than those of MV. Titration of the plasmids used for transfection proved essential, showing that a ratio of about 1:1000 of L-encoding and antigenome-producing plasmid, respectively, was optimal, in agreement with the deleterious effect of high VSV L expression for VSV replication noted by Schubert et al. (1985). An alternative mode of transiently supplying L, using a plasmid containing a CMV promoter/enhancer and an intron upstream rather than downstream of the L coding region to permit some export of the long L mRNA from the nucleus, was also successful in rescue, but the efficiency was not better than with the standard method of cytoplasmic T7-dependent L expression and more than a hundred times more L encoding plasmid was optimal for rescue. In view of these experiences, the decision not to include an L encoding plasmid for the generation of helper cells, thus allowing expression of L at adjustable ratios, was probably advantageous. Nevertheless, it should be mentioned that a cell line stably expressing SeV-derived N, P and L which mediates long term replication of natural SeV DIs has been described (Willenbrink and Neubert, 1994). It is important to note that this cell line differs fundamentally from the helper cells defined in the present invention by its lack of T7 polymerase. As a consequence, no rescue of a virus and not even of a minireplicon from cloned DNA is feasible with this cell line.

In a further preferred embodiment of said helper cell said cell is transfected with at least one of said above described plasmids, said plasmids containing variant antigenomic cDNA of a representative of the Mononegavirales, and is additionally stably transfected with a plasmid comprising DNA encoding the cognate viral L protein.

Thus, instead of selecting for a helper cell that also encodes per se the viral polymerase (L protein), said L protein is transfected into said helper cell on a different plasmid, preferably by cotransfection. Further, a skilled person using the teachings of the present invention is able to create a suitable helper cell line expression also L protein, in which case cotransfection is not necessary.

In a most preferred embodiment of said helper cell, the genes encoding said N, P and L proteins are derived from measles or mumps virus.

In a further most preferred embodiment said helper cell is derived from the human embryonic kidney cell line 293 (ATCC CRL 1573). A preferred example of such a cell is clone 293-3-46 described in the examples.

The invention further relates to an infectious negative-strand RNA virus strain belonging to the order Mononegavirales isolated from the helper cell of the invention.

It must be recalled that five years ago, in an erroneous account, MV rescue was reported by our laboratory (Ballart et al., 1990 and EP-A 0 440 219), using the same basic principle. At that time, the experiments were based on microinjection of initiation complexes, consisting of T7 RNA polymerase and plasmids specifying MV genomes or antigenomes, into a particular cell line containing defective but replicating MV genomes. However, the rescue by microinjection experiments, unfortunately carried out by only one collaborator, could not be repeated, and all purportedly rescued viruses did not contain the genetic tag, as summarised in a commentary to these extremely sad and devastating events (Aldhous, 1992). It is now clear that rescue of MV could not be expected with that experimental setup for several reasons, in particular due to additional nucleotides at both ends of the generated RNAs and due to a cloning mistake rendering the RNA incompatible with the rule of six (Calain and Roux, 1993; the present invention).

The rescue efficiency, in comparison to rescue of positive-strand RNA viruses (Perrotta and Been, 1990), is low, since only 1 to 6 out of 10.sup.6 transfected cells, each exposed on average to about 2.5.times.10.sup.5 molecules of antigenomic and 80 to 800 molecules of L-encoding plasmid, trigger the formation of syncytia. Nevertheless, in comparison with the rescue method described for RV and VSV, where about 2.times.10.sup.7 cells are transfected to obtain one rescue event (Lawson et al., 1995; Schnell et al., 1994), the MV rescue compares well, particularly in view of the fact that the MV genome size is roughly 4.5 kb larger and thus in principle more difficult to rescue. Importantly, the low efficiency should not constitute a difficulty for the rescue of MV variants replicating only to titer levels even orders of magnitude lower than the Edmonston B strains, since the bottle-neck of rescue is constituted most likely by an early event. It is important to note that on cells fixed at various times after transfection, immunofluorescence indicating H or M gene expression was monitored exclusively in syncytia and there was no indication that rescue was confined to single cells. When rescue is visible directly by syncytia formation, already thousand of progeny MV genomes have arisen; impaired and thus slowly replicating virus variants might not form visible syncytia initially, but should be revealed after splitting of the transfected cell culture or upon seeding onto fresh Vero cells.

The invention further relates to a method for the production of an infectious negative-strand RNA virus belonging to the order Mononegavirales, comprising the steps of (a) transfecting the helper cell of the invention with any one of the plasmids described above and comprising antigenomic DNA from a virus belonging to the order Mononegavirales (first vector) and optionally a plasmid comprising DNA encoding the viral L protein (second vector); and (b) recovering the assembled infectious negative-strand RNA viruses.

Transfection with the second vector is not necessary, if the helper cell genomically expresses the viral L protein.

In a preferred embodiment of the method of the invention, the ratio of the first vector and the second vector is about 1000:1.

In accordance with the present invention it has been shown that the above ratio is optimal for transfection efficiency.

In further preferred embodiments of the method of the invention, said recovery is either directly effected from the transfected helper cell culture after syncytia formation or, after mixing of detached helper cells with any other cells competent of being infected and replicating the assembled RNA viruses.

The invention relates further to a vaccine comprising the RNA virus according to the invention which optionally is obtainable by the method of the invention described above, optionally in combination with a pharmaceutically acceptable carrier.

The advantages of the vaccine of the present invention will be briefly discussed below.

In the past, a variety of DNA viruses and positive-strand RNA viruses have been used as carriers to direct the expression of heterologous genes or gene segments in host cells, mainly with the aim to elicit immune protection against the pathogen from which the heterologous genetic material was derived. The main advantage of using such live vaccines is their ability to multiply and typically infect a variety of different cell types, generating the antigens of interest intracellularly which can therefore be presented efficiently to the immune system, thus facilitating the induction of both T cell help and cytotoxicity. In contrast, killed vaccines or proteins manufactured by recombinant DNA technology are much less efficient, even by administration in various particulate forms developed recently, which are more efficient than traditionally used adjuvants. In addition, such vaccines typically induce no mucosal immunity, which is very important for protection against pathogens entering by the respiratory or intestinal route. Failure to induce mucosal immunity is also typical for the immunisation approach using injection of naked DNA encoding antigens.

On the other hand, most replicating vaccines constitute a possible threat, even if they are not proliferating, such as avipox vectors in humans (Baxby and Paoletti, 1992). Complex viral vectors (e.g. based on vaccinia virus and related pox viruses, adenoviruses or herpesviruses) and bacterial vectors (e.g. based on derivatives of the agents causing tuberculosis or cholera) inherently elicit many lateral, unnecessary and/or undesired immune responses. In addition, DNA integration in the genome of infected or transfected cells bears at least the potential for malignant transformation. Multiauthored assessments of various types of vaccines have been published recently (vaccines and public health; Internat. J. of techn. Ass. in Health care 10, 1-196 1994; Science 265, 1371-1451, 1994), from which the particular benefits of small RNA-based live vaccines are evident.

Several engineered positive-strand RNA viruses have been described for potential use as vectors for immunisation purposes; early examples include poliovirus (Burke et al., 1988) and Sindbis virus (Xiong et al., 1989) and among several more recent accounts, involving larger polypeptide fragments expressed from various representatives of the Picornaviridae, just one should be mentioned here (Andino et al., 1994).

However, it must be stressed that the use of RNA viruses as vectors for vaccination purposes crucially depends on the stability of the foreign genetic material during the replication of the virus. This is not a trivial problem, because these viruses rely on a polymerase devoid of proofreading activity. Said problem has advantageously been solved by the present invention: in comparison to vaccine vectors based on positive-strand RNA viruses as mentioned above, the vaccine of the invention as exemplified by MV-based di- or multivalent vaccines show several important advantages which are valid in principle for any other member of the Paramyxoviridae such as mumps virus. First, the size of inserts is not a priori limited by a requirement to fit into an icosahedral protein shell. Second, the tight encapsidation of the genomes of Mononegavirales obviates RNA secondary structure which is very important in case of the positive-strand RNA viruses over the whole genome length to allow proper replication without annealing of the product to the template RNA strand; RNA segments encoding foreign antigens are not evolved to meet such requirements. Third, due to the modular set up of the genome, different insertion sites and expression modes, either as additional transcription units or as elongation of existing transcription units, expressing the inserted downstream reading frames by stop/restart or by an internal ribosome entry site can be envisaged, thus allowing a large range of different expression levels according to the position within the MV transcription gradient. Fourth, due to extremely low recombination frequencies, Mononegavirales can be expected to retain nonessential genetic material much more stably than positive-strand RNA-viruses. Finally, the rule of six, valid for MV as was found in accordance with the present invention and for other Paramyxovirinae (Calain and Roux, 1993), but as judged from cognate mini- and midi-replicons, not for Rhabdoviridae (Conzelmann and Schnell, 1994) or for Pneumovirinae (Collins et al., 1993), should even increase the faithful retention of foreign coding regions inserted in Paramyxovirinae in comparison to other Mononegavirales. Such an additional genetic stability can be anticipated because only one in six adventitiously arising large deletions and no small insertion or deletion of 1 to 5 nucleotides in a region nonessential for viral replication are expected to lead to viable progeny.

Further, knowledge of the nucleotide sequence variants conferring attenuation will allow one to change the coding sequences not implicated in attenuating properties according to the evolution of the viruses over the years thus permitting one to "update" the vaccines without incurring the danger of losing the quality of attenuation.

The invention additionally relates to the use of the plasmid of the invention in somatic gene therapy.

Since viral envelope proteins can be exchanged among different representatives of Mononegavirales, as shown here by the replacement of the MV envelope proteins with the VSV glycoprotein, it seems feasible to target the replion based on the replication machinery of Mononegavirales to particular cell types; thus, certain applications in somatic gene therapy can be envisaged. Advantages in comparison to existing vectors for gene therapy include their small size, thus limiting antigen reactions to a few proteins, and their complete inability to integrate into DNA and thus to transform cells.

Additionally, the invention relates to the use of the plasmid of the invention for targeting special cell types. An outline of such targeting schemes and applications has been provided above.

The invention relates further to the use of the plasmid of the invention for the functional appraisal of mutations found typically in MV variants responsible for fatal subacute sclerosing panencephalits or for the identification of mutations responsible for attenuation of Paramyxoviridae strains, preferably measles virus strains.

Finally, the invention relates to a diagnostic composition comprising at least one cDNA molecule of the invention and/or at least one plasmid of the invention.
 

Claim 1 of 14 Claims

1. An isolated batch of infectious RNA viruses for use as a vaccine, said infectious RNA viruses each comprising: (a) the entire (-)-strand sequence of a non-segmented negative-strand RNA virus of the family Paramyxoviridae which obeys the rule of six; operatively linked to (b) a foreign expressible RNA fragment, wherein the length of the genome of the infectious RNA virus is an integral multiple of six, and further wherein the infectious RNA virus is obtainable by the steps of: (1) introducing into a host cell a plasmid comprising a cDNA molecule, said cDNA molecule comprising the entire (+)-strand sequence of said negative-strand RNA virus operatively linked to an expression control sequence, which allows the synthesis of antigenomic RNA transcripts bearing the authentic 3'-termini without help of a helper virus, said host cell comprising (i) transfected nucleic acid molecules containing nucleic acid sequences that allow expression of an exogenous RNA polymerase, a viral N protein and a viral P protein, and (ii) a nucleic acid molecule containing nucleic acid sequence encoding a viral L protein; and (2) recovering and isolating the infectious RNA virus, wherein the isolated batch of infectious RNA viruses is free of helper virus.

 

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