Patent Grant
9005925
Measles virus is a member of the order mononegavirales, i.e., viruses with a non-segmented negative-strand RNA genome. The non segmented genome of measles virus (MV) has an antimessage polarity which results in a genomic RNA which is neither translated in vivo or in vitro nor infectious when purified.
Transcription and replication of non-segmented (−) strand RNA viruses and their assembly as virus particles have been studied and reported especially in Fields virology (3rd edition, vol. 1, 1996, Lippincott-Raven publishers—Fields B N et al). Transcription and replication of measles virus do not involve the nucleus of the infected cells but rather take place in the cytoplasm of said infected cells. The genome of the measles virus comprises genes encoding six major structural proteins from the six genes (designated N, P, M, F, H and L) and an additional two-non structural proteins from the P. gene. The gene order is the following: 3′, N, P (including C and V), M, F, H, and L large polymerase protein at the 5′ end. The genome further comprises non coding regions in the intergenic region M/F; this non-coding region contains approximately 1000 nucleotides of untranslated RNA. The cited genes respectively encode the leader peptide (I gene), the proteins of the nucleocapsid of the virus, i.e., the nucleoprotein (N), the phosphoprotein (P), and the large protein (L) which assemble around the genome RNA to provide the nucleocapsid. The other genes encode the proteins of viral envelope including the hemagglutinin (H), the fusion (F) and the matrix (M) proteins.
The measles virus has been isolated and live attenuated vaccines have been derived from the Edmonston MV isolated in 1954 (Enders, J. F., and T. C. Peebles. 1954. Propagation in tissue cultures od cytopathogenic agents from patients with measles. Proc. Soc. Exp. Biol. Med. 86:277-286.), by serially passages performed on primary human kidney or amnion cells. The used strains were then adapted to chicken embryo fibroblasts (CEF) to produce Edmonston A and B seeds (Griffin, D., and W. Bellini. 1996. Measles virus, p. 1267-1312. In B. Fields, D. Knipe, et al. (ed.), Virology, vol. 2. Lippincott-Raven Publishers, Philadelphia). Edmonston B was licensed in 1963 as the first MV vaccine. Further passages of Edmonston A and B on CEF produced the more attenuated Schwarz and Moraten viruses (Griffin, D., and W. Bellini. 1996. Measles virus, p. 1267-1312. In B. Fields, D. Knipe, et al. (ed.), Virology, vol. 2. Lippincott-Raven Publishers, Philadelphia) whose sequences have recently been shown to be identical (Parks, C. L., R. A. Lerch, P. Walpita, H. P. Wang, M. S. Sidhu, and S. A. Udem. 2001. Analysis of the noncoding regions of measles virus strains in the Edmonston vaccine lineage. J Virol. 75:921-933; Parks, C. L., R. A. Lerch, P. Walpita, H. P. Wang, M. S. Sidhu, and S. A. Udem. 2001. Comparison of predicted amino acid sequences of measles virus strains in the Edmonston vaccine lineage. J Virol. 75:910-920). Because Edmonston B vaccine was reactogenic, it was abandoned in 1975 and replaced by the Schwarz/Moraten vaccine which is currently the most widely used measles vaccine in the world (Hilleman, M. 2002. Current overview of the pathogenesis and prophylaxis of measles with focus on practical implications. Vaccine. 20:651-665). Several other vaccineu strains are also used: AIK-C, Schwarz F88, CAM70, TD97 in Japan, Leningrad-16 in Russia, and Edmonston Zagreb. The CAM70 and TD97 Chinese strains were not derived from Edmonston. Schwarz/Moraten and AIK-C vaccines are produced on CEF. Zagreg vaccine is produced on human diploid cells (WI-38).
The live attenuated vaccine derived from the Schwarz strain is commercialized by Aventis Pasteur (Lyon France) under the trademark Rouvax®.
In a noteworthy and pioneer work, Martin Billeter and colleagues cloned an infectious cDNA corresponding to the antigenome of Edmonston MV and established an original and efficient reverse genetics procedure to rescue the corresponding virus (Radecke, F., P. Spielhofer, H. Schneider, K. Kaelin, M. Huber, K. Dötsch, G. Christiansen, and M. Billeter., 1995. Rescue of measles viruses from cloned DNA. EMBO Journal. 14:5773-5784 and WO 97/06270 incorporated herewith by reference).
However, sequence comparison (see below) revealed that the genome cloned in this vector diverged from the Edmonston B sequence. It was closer to Edmonston-wt, an early passage on Vero cells of Edmonston isolate (Parks, C. L., R. A. Lerch, P. Walpita, H. P. Wang, M. S. Sidhu, and S. A. Udem. 2001. Analysis of the noncoding regions of measles virus strains in the Edmonston vaccine lineage. J Virol. 75:921-933; Parks, C. L., R. A. Lerch, P. Walpita, H. P. Wang, M. S. Sidhu, and S. A. Udem. 2001. Comparison of predicted amino acid sequences of measles virus strains in the Edmonston vaccine lineage. J Virol. 75:910-920), and had 10 amino acid substitutions not related to any Edmonston subgroup. Moreover, despite the fact that this vector is immunogenic in mice expressing CD46 and lacking the IFN type I receptor (19), the inventors show in the following experimental work that it is not immunogenic in non-human primates when inoculated at the standard dose of 104 TCID50. Therefore, this vector developed from a vaccine strain abandoned 25 years ago, and whose sequence diverged so much, does not appear suitable as vaccination vector, especially in human, while it certainly helps to understand some aspects of MV replication.
For these reasons, the inventors have decided that a measles vector aimed at children needs to be developed from an approved vaccine strain and have accordingly cloned an infectious cDNA starting from viral particles of the widely used Schwarz/Moraten strain of measles virus. This cDNA may allow the production of Schwarz/Moraten vaccine stocks without having to rely on the availability of seed stocks. It may also be used as a recombinant vaccination vector based on an approved and efficient vaccine strain, grown on CEF for safety reasons, and worldwide used. Such a vector may also be of interest for adult populations in certain circumstances where a need therefore exists.
The invention relates to a cDNA molecule which encodes the nucleotide sequence of the full length antigenomic (+)RNA strand of a measles virus (MV) originating from an approved vaccine strain.
The expression <<encodes>> in the above definition encompasses the capacity of the cDNA to allow transcription of a full length antigenomic (+)RNA, said cDNA serving especially as template for transcription. Accordingly, when the cDNA is a double stranded molecule, one of the strands has the same nucleotide sequence as the antigenomic (+) strand RNA of the measles virus, except that
<<U>> nucleotides are substituted by <<T>> in the cDNA.
The cDNA molecule according to the above definition allows the production, when placed in appropriate conditions, of an infectious antigenomic (+)RNA capable of producing infectious particles of the measles virus.
The cDNA obtained has especially the original 5′- and 3′-ends of the native antigenomic (+) strand of the viral RNA. In addition, the obtained cDNA complies with the rule of 6 which is required in order to express infectious viral particles.
The <<rule of six>> which is expressed in the fact that the total number of nucleotides present in the cDNA amounts to a multiple of six, rule which allows sufficient replication of genome RNA of the measles virus. It has been described in the above cited reference Fields Virology on page 1197.
The cDNA molecule of the invention which is derived from an MV approved vaccine strain can be obtained from the Schwarz or the Moraten strain.
These strains have been disclosed in several publications and used for the preparation of the currently used vaccines. The inventors propose especially the use of the Schwartz strain which is available from Aventis Pasteur (France).
According to another particular embodiment of the invention, the cDNA molecule is placed under the control of a heterologous expression control sequence.
The insertion of such a control for the expression of the cDNA, is favorable when the expression of this cDNA is sought in cell types which do not enable full transcription of the cDNA with its native control sequences.
According to a particular embodiment of the invention, the heterologous expression control sequence comprises the T7 promoter and T7 terminator sequences. These sequences are respectively located 5′ and 3′ of the coding sequence for the full length antigenomic (+)RNA strand of MV and from the adjacent sequences around this coding sequence.
In a particular embodiment of the invention, the cDNA molecule which is defined hereabove is modified i.e., comprises additional nucleotide sequences or motifs or comprises deletions or substitutions within said cDNA.
In a preferred embodiment, the cDNA molecule of the invention further comprises, at its 5′-end, adjacent to the first nucleotide of the nucleotide sequence encoding the full length antigenomic (+)RNA strand of the MV approved vaccine strain, a GGG motif followed by a hammerhead ribozyme sequence and which comprises, at its 3′-end, adjacent to the last nucleotide of said nucleotide sequence encoding the full length anti-genomic (+)RNA strand, the sequence of a ribozyme. The Hepatitis delta virus ribozyme (δ) is appropriate to carry out the invention.
The GGG motif placed at the 5′ end, adjacent to the first nucleotide of the above coding sequence improves the efficiency of the transcription of said cDNA coding sequence. As a requirement for the proper assembly of measles virus particles is the fact that the cDNA encoding the antigenomic (+)RNA complies with the rule of six, when the GGG motif is added, a ribozyme is also added at the 5′ end of the coding sequence of the cDNA, 3′ from the GGG motif, in order to enable cleavage of the transcript at the first coding nucleotide of the full length antigenomic (+)RNA strand of MV.
Thus, in case where the GGG motif is added to improve efficiency of transcription, two ribozymes are added in order to ensure the cleavage of the coding sequence for the full length antigenomic (+)RNA strand of the MV.
According to the present invention, the expression “cDNA” encompasses a DNA molecule obtained by reverse transcription of an RNA molecule, including but not limited to an mRNA molecule.
Any other technique for the preparation of DNA, starting from the material disclosed in the present invention or using the disclosed features relating to the cDNA of the invention can be used, including techniques involving synthesis or PCR.
Therefore, the expression “cDNA” used for the description of the nucleotide sequence of the molecule of the invention merely relates to the fact that originally said molecule is obtained by reverse transcription of the full length genomic (−)RNA strand of the genome of viral particles of the measles virus. This should not be viewed as a limitation for the methods used for its preparation. Purified nucleic acids, including DNA are thus encompassed within the meaning cDNA according to the invention, provided said nucleic acid, especially DNA fulfils the above-given definitions.
The invention also concerns a cDNA molecule according to one of the above definitions which is comprised in a plasmid capable of replication.
Many plasmids can be prepared in order to fulfil the requirement of the invention and the invention especially relates to plasmid pTM-MVSchw which is represented on
The invention especially also relates to a cDNA molecule which is capable of producing infectious viral particles of the MV approved vaccine strain, preferably using the previously reported rescue system involving 293-3-46 helper cells (Radecke et al. and WO 97/06270), 293-3-46 helper cells expressing proteins necessary for transcription, replication of the RNA genome-sequence of MV from said cDNA and under conditions enabling viral particles assembly.
293-3-46 cells are cited as example for the preparation of the viral particles. They can however be replaced by any other appropriate cell line suitable for constituting helper cells.
Methods for the production of such infectious particles are given in the examples of the present application.
Particular preferred cDNA molecules according to the invention are the molecules having a nucleotide sequence selected among the following sequences:
The invention of course relates to each of the particular sequences described hereabove.
A particular cDNA molecule which is preferred to carry out the invention is the molecule which comprises the insert contained in plasmid pTMMVschw deposited at the CNCM under number i-2889, wherein said insert encodes a nucleotide sequence of the full length antigenomic (+)RNA strand of the measles virus. One particular insert is the one which is comprised within the sequence defined by the following restriction sites: NotI (located at position 1 on
In a particular embodiment of the invention, the cDNA molecule is the product of the reverse transcription of the viral RNA purified from viral particles of the measles virus.
The preparation of the cDNA from viral purified RNA advantageously limits the presence of cellular components and especially cellular DNA or RNA which could be present in cells used for the cultivation of the virus. It limits especially the presence of viral genomic RNA which would be incomplete or mutated and which are present in cells, and limits the presence of viral mRNA present in large quantities in the cells.
The invention further relates to a cDNA molecule having the above defined features, which is capable of inducing an immune response against at least one antigen of a measles virus, when administered in vivo.
The invention also relates to a recombinant mononegavirales virus comprising the cDNA molecule of a measles virus according to anyone of the above definitions and a DNA sequence of a RNA virus, which recombinant virus is capable of eliciting in vivo a humoral and/or a cellular response against measles virus or against said RNA virus, or against both measles virus and RNA virus.
The invention also concerns a recombinant cDNA molecule as defined above, which further comprises a heterologous DNA sequence cloned therein in conditions enabling its expression as a heterologous amino acid sequence, said cloning being performed in such a way that the obtained recombinant cDNA complies with the rule of six.
Heterologous coding sequences especially DNA sequences are advantageously selected among sequences capable of expressing antigens or epitopes of antigens, having immunogenic properties and especially having the capacity of eliciting or favoring an immunogenic response in a host to which they are administered. Such heterologous DNA sequences can be derived for instance from antigens of pathogens.
The invention advantageously enables the insertion of such heterologous DNA sequences in a sequence which is designated an Additional Transcription Unit (ATU) especially an ATU as disclosed by Billeter et al. in WO 97/06270.
This ATU is especially represented on
When used for the performance of the invention, the ATU is advantageously located in the N-terminal sequence of the cDNA molecule encoding the full-length (+)RNA strand of the antigenome of the MV and is especially located between the P and M genes of this virus or between the H and L genes. It has been observed that the transcription of the viral RNA of MV follows a gradient from the 5′ to the 3′ end. This explains that when inserted in the 5′ end of the coding sequence of the cDNA, the ATU will enable a more efficient expression of the heterologous DNA sequence that it contains.
The invention also relates to a vector comprising a cDNA molecule as defined above including a recombinant cDNA. A particular vector is vector for cloning and/or expressing of this cDNA.
According to a preferred embodiment of the invention, the vector is a plasmid and is especially pTM-MVSchw deposited at the CNCM on Jun. 12, 2002 under No. I-2889.
Other vectors, designated pTM-MVSchw2-gfp deposited at the CNCM under no I-2890 on Jun. 12, 2002 or designated pTM-MVSchw2-GFPbis deposited at the CNCM under n° I-3034 on May 26, 2003 are encompassed within the invention.
These vectors are derived from pTM-MVSchw, and are accordingly plasmid vectors derived from Bluescript, comprising the full length sequence coding for the measles virus, strain Schwarz, placed under the control of the promoter of the T7 RNA polymerase, and further containing the gfp gene coding for the GFP protein, said gene being inserted in an ATU at position 2 (i.e., between the N and P genes of MV).
The size of pTM-MvSchw is 18967 nucleotides. The size of pTM-MVSchw2-gfp is 19800 nucleotides.
The difference between pTM-MVSchw2-gfp and pTM-MVSchw2-GFPbis corresponds to a mutation in the ATU sequence where a C nucleotide is substituted as illustrated on
The invention also relates to a process for the preparation of infectious measles virus particles comprising:
1) expressing the cDNA of the invention according to one of the above definitions or the vector containing such cDNA in a helper cell line which also expresses proteins necessary for transcription, replication and encapsidation of the antigenomic (+)RNA sequence of MV from said cDNA and under conditions enabling viral particles assembly and
2) recovering the expressed viral particles.
According to a particular embodiment of this process, it comprises
According to a preferred embodiment, helper cells are derived from human embryonic kidney cell line 293, which cell line 293 is deposited with the ATCC under No. CRL-1573.
According to another aspect of this process, the cells suitable for passage are CEF cells.
CEF cells can be prepared from fertilized chicken eggs as obtained from EARL Morizeau, 8 rue Moulin, 28190 Dangers, France, or from any other producer of fertilized chicken eggs.
The process which is disclosed according to the present invention is used advantageously for the production of infectious measles virus appropriate for use as vaccine compositions.
The invention thus relates to an immunogenic composition whose active principle comprises infection measles viral particles obtained by the process disclosed above.
The invention also concerns a vaccine composition. Such a vaccine composition has advantageously an active principle which comprises measles virus particles rescued from the cDNA of the vector which has been defined hereabove, which is expressed in a helper cell based rescue system.
Advantageously, such a vaccine composition is suitable for protection against measles virus. According to the embodiment where the cDNA is recombined with a heterologous DNA sequence encoding an immunogenic amino acid sequence, the vaccine composition can further be suitable for protection against the pathogen from which the immunogenic DNA sequence derives.
The invention also concerns a cell which is recombined with a cDNA molecule according to the invention or with a vector as defined above. A preferred cell is a prokaryotic cell such as E. coli or Salmonella.
Another preferred cell is a eukaryotic cell, especially a cell selected among yeasts, such as Saccharomyces Cerevisiae.
A cell within the definition of the invention, can be characterized according to a particular embodiment by the fact that this comprises nucleotide sequences expressing helper functions necessary to express an RNA polymerase and to express the N, P and L proteins of the MV virus. Such a cell can thus be used for the rescue of the viral particles.
The examples and figures which follow provide additional features for the characterization of the invention.
B: ATU sequence (SEQ ID NO: 83): small letters represent additional sequences (copy of the N-P intergenic region of measles virus) plus cloning sites. Capital letters correspond to the inserted enhanced GFP sequence. This sequence is inserted at the SpeI site (position 3373) of the cDNA sequence of the Schwarz strain of the measles virus for ATU2 and at the SpeI site (position 9174) for the ATU3. The mutation which distinguishes normal ATU from bis(in pTM-MVSchw2-gfp and pTM-MVSchw2-GFPbis) is a substituted C (Capital letter) at the end of ATU.
Sequence Comparison Between EdB-Tag and Measles Vaccine Strains
In a nice analysis previously reported (Parks, C. L., R. A. Lerch, P. Walpita, H. P. Wang, M. S. Sidhu, and S. A. Udem. 2001. Analysis of the noncoding regions of measles virus strains in the Edmonston vaccine lineage. J Virol. 75:921-933; Parks, C. L., R. A. Lerch, P. Walpita, H. P. Wang, M. S. Sidhu, and S. A. Udem. 2001. Comparison of predicted amino acid sequences of measles virus strains in the Edmonston vaccine lineage. J Virol. 75:910-920), the coding and non-coding sequences of Edmonston-derived vaccine virus strains were compared to that of a low-passage isolate of the Edmonston wild-type measles virus. The authors identified 10 amino acids substitutions shared by almost all the vaccine strains. We compared the genomic sequences of these Edmonston-derived vaccine strains and two primary isolates (Takeda, M., A. Kato, F. Kobune, H. Sakata, Y. Li, T. Shioda, Y. Sakai, M. Asakawa, and Y. Nagai. 1998. Measles virus attenuation associated with transcriptional impediment and a few amino acid changes in the polymerase and accessory proteins. J Virol. 72:8690-8696; Takeuchi, K., N. Miyajima, F. Kobune, and M. Tashiro. 2000. Comparative nucleotide sequence analyses of the entire genomes of B95a cell-isolated and vero cell-isolated measles viruses from the same patient. Virus Genes. 20:253-257) to that of the previously reported Edmonston B infectious cDNA (EdB-tag) (Radecke, F., P. Spielhofer, H. Schneider, K. Kaelin, M. Huber, K. Dötsch, G. Christiansen, and M. Billeter. 1995. Rescue of measles viruses from cloned DNA. EMBO Journal. 14:5773-5784).
Construction of a cDNA Corresponding to the Antigenome of the Schwarz Vaccine Strain of Measles Virus.
Viral particles were purified from a measles Schwarz vaccine batch kindly provided by Aventis Pasteur (Lyon, France). This bulk vaccine preparation (50 ml, 3 104 TCID50/ml) was obtained by scraping of infected CEF cells, freeze-thawing cells and medium, and filtration of cellular debris. Particles were concentrated by centrifugation through a 30% sucrose cushion. Viral RNA was purified from lysed particles using a silica-gel-based membrane (QIAmp, Qiagen). Viral RNA was reverse-transcribed into cDNA using a mixture of random hexameres (pdN6, 1 μM) and a specific oligonucleotide representing the 32 first nucleotides of MV genome (MVSchwRT1 5′-ACCAAACAAAGTTGGGTAAGGATAGTTCAATC-3′ (SEQ ID NO: 65), 10 μM), as primers. In order to ensure the fidelity of reverse transcription and the yield of full-length products, the SuperScript II DNA polymerase was used (GibcoBRL). A set of six overlapping fragments covering the full viral cDNA (numbered 1 to 6 in
Recovery of Infectious Schwarz Virus from pTM-MVSchw Plasmid.
To recover the Schwarz virus from the pTM-MVSchw cDNA, we used the helper-cell-based rescue system described by Radecke et al. (Radecke, F., P. Spielhofer, H. Schneider, K. Kaelin, M. Huber, K. Dötsch, G. Christiansen, and M. Billeter. 1995. Rescue of measles viruses from cloned DNA. EMBO Journal. 14:5773-5784) and modified by Parks et al. (Parks, C. L., R. A. Lerch, P. Walpita, M. S. Sidhu, and S. A. Udem. 1999. Enhanced measles virus cDNA rescue and gene expression after heat shock. J Virol. 73:3560-3566). Human helper cells stably expressing T7 RNA polymerase and measles N and P proteins (293-346 cells, disclosed by Radecke et al (17) were transfected using the calcium phosphate procedure with pTM-MVSchw plasmid (5 μg) and a plasmid expressing the MV polymerase L gene (pEMC-La, 20 ng, disclosed by Radecke et al (17). After overnight incubation at 37° C., the transfection medium was replaced by fresh medium and a heat shock was applied (43° C. for two hours) (12). After two days of incubation at 37° C., transfected cells were transferred on a CEF cells layer and incubated at 32° C. in order to avoid any adaptation of the Schwarz vaccine that was originally selected on CEF cells and is currently grown on these cells for safety considerations. The above chicken embryo fibroblastic cells (CEF) were prepared as follows. Fertilized chicken eggs (EARL Morizeau, 8 rue Moulin, 28190 Dangers, France) was incubated at 38° C. for 9 days. Embryos were collected sterilely. Head, limbs and viscera were removed and embryos were sliced up and trypsinized for 5-10 minutes at 37° C. (Trypsine/EDTA 2.5 g/L). After filtration (70 μm) and several washes in DMEM high glucose/10% FCS, cells were seeded (5-7 106 cells/pertri dish) and incubated overnight at 37° C. Infectious virus was easily recovered between 3 and 7 days following cocultivation. Syncytia appeared occasionally in CEF, but not systematically. The Schwarz virus was also rescued by the same technique after cocultivation of transfected 293-3-46 helper cells at 37° C. with primate Vero cells (african green monkey kidney). In this case, syncytia appeared systematically in all transfections after 2 days of coculture. In order to test for viral adaptation to Vero cells, a preparation of cloned Schwarz virus rescued on Vero cells was passaged two times on Vero cells. Viral particles were purified and viral RNA was reverse-transcribed as described above with the primers used for the cloning (see above). The viral genome was fully sequenced. Two nucleotide changes out of 15894 were found between the rescued/passaged virus and the cDNA used for transfection. These mutations were found in 7 and 8 respectively out of 10 different clones of the same region, indicating a high percentage of mutation among the viral population. Moreover, both mutations resulted in amino acid changes in the fusion protein (F): G→R in position 266 and Y→S in position 365.
In contrast, the genomic sequence of the virus recovered and passaged on CEF cells at 32° C. was identical to that of the original Schwarz virus. This observation indicates that changing the host cell of Schwarz virus leads to a rapid adaptation that may affect the properties of the vaccine.
Growth Capacity of the Rescued Virus.
The capacity of the Schwarz virus rescued from cDNA to grow on CEF and Vero cells was analyzed and compared to the industrial bulk Schwarz vaccine from which it was derived (obtained from Aventis Pasteur) and to the EdB-tag virus rescued from its cDNA. Monolayers of Verocells in 6-well plates were infected with viruses at different multiplicity of infection. A various time post infection (pi), the cells were seraped into culture medium. After freezing and thawing, infectivity titers were determined by measuring the TCID50 in Vero cells. Growth curves: Monolayers of Vero cells in 6-well plates were infected with viruses at different multiplicities of infection (MOI). At various times postinfection (pi), the cells were scraped into culture medium. After freezing and thawing, infectivity titers were determined by measuring the TCID50 in Vero cells.
TCID50 titration: Vero cells were seeded into 96-well plate (7500 cells/well) and infected by serial 1:10 dilutions of virus sample in DMEM/5% FCS. After incubation at 37° C. for 4-5 days for Ed-B virus and 7 days for Schwarz virus, cells were stained with crystal violet and the virus dilution that resulted in infection in 50% of test unit was determined. The 50% end point described as tissue culture infectious dose (TCID50) was calculated by the Kärber method (Karber, G. 1931. Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Arch Exp Path Pharmak. 162:480-483). Tested on Vero cells, the growth kinetics of Schwarz and EdB-tag viruses rescued from their respective cDNA were similar (
Introduction of an Additional Transcription Unit in the Schwarz cDNA.
In the previous work reporting the cloning of EdB-tag virus (17), the authors developed an original method to adapt the viral cDNA as a vector suitable for the expression of foreign transgenes. They inserted an additional transcription unit (ATU) in different positions of the viral genome. This ATU is a copy of the MV N-P intergenic region containing the cis-acting sequences necessary for MV-dependant expression of a transgene inserted into a multiple cloning sites cassette. Largely tested by the authors and ourselves, the expression of foreign transgenes inserted in this ATU was very efficient, depending on the position of insertion in the genome. The different MV genes are expressed according to a transcriptional gradient down the genome, leading to a high expression of the N gene to a low expression of the L gene (Lamb, R., and D. Kolakofsky. 1996. Paramyxoviridae: the viruses and their replication, p. 1177-1199. In B. Fileds, D. Knipe, et al. (ed.), Fields Virology. Lippincott-Raven Publishers, Philadelphia).
The insertion of the ATU takes advantage of this gradient, allowing high or low expression of the transgene, depending on the position of insertion. Moreover, in this context the foreign transgenes are expressed using the same controls and pathways as authentic MV genes.
In order to transform the Schwarz cDNA as a vector, we constructed a similar ATU that was inserted in two different positions of the cDNA (
Immunogenicity of Schwarz MV Recovered from cDNA in Macaques.
First Experiment: Comparison with Schwarz Vaccine.
The immunogenicity of the virus rescued from pTM-MVSchw plasmid and passaged two times on CEF cells was compared to the immunogenicity of Schwarz vaccine in cynomolgus macaques. The conditions for passage were the following:
Different formulations of the vaccine were tested using both the unpassaged bulk preparation from Aventis Pasteur, and the same preparation passaged two times on CEF cells. Viruses were prepared as follows: CEF cells (obtained from chick embryos incubated during 9 days) were infected at a MOI of 0.05 and incubated at 32° C. during 7 days. Viruses were purified by scraping infected cells, freeze/thawing and low speed clarification of cells debris. Stabilizing agents used in the preparation of MV vaccine were obtained from Aventis Pasteur. Different bulk vaccine preparations with and without stabilizing agents were compared at the same dose to the lyophilized final product (Rouvax, Aventis Pasteur). All vaccine preparations were titrated using the TCID50 method on Vero cells. Monkeys were injected sub-cutaneaously and blood samples were taken at different time points. In order to compare both humoral and cellular responses, the presence of anti-MV antibodies was looked for in serums by ELISA (Trinity Biotech, USA) and the presence of anti-MV T-cells was looked for by ELISPOT in PBMCs.
Second Experiment: Comparison with EdB-Tag Strain
Colony-bred rhesus (Macaca mulatta) or cynomolgus (Macaca fascicularis) macaques that were seronegative for simian type D retrovirus, simian T-cell lymphotropic virus, simian immunodeficiency virus, and measles virus were housed in accordance with the American Association for Accreditation of Laboratory Animal Care. Monkeys were inoculated subcutaneously with different doses (103-105 TCID50) of EdB-tag or Schwarz MV diluted in OptiMEM (GibcoBRL) or with 104 TCID50 of the lyophilized Rouvax MV vaccine (Aventis Pasteur, Marcy I'Etoile, France) diluted in the solution provided by the supplier. Blood samples were collected at different time after inoculation.
The presence of anti-MV antibodies in serum was looked for by ELISA (Trinity Biotech, USA) one month after vaccination. Each determination was done in triplicate on 1/20 dilution of serum samples. A mixture of 5 samples from negative monkeys was used as the negative control. To determine the immune status ratio (ISR) of each sample, the absorbance of the negative control was subtracted from the absorbance of the positive sample and the result was divided by the absorbance of a calibrator supplied in the ELISA kit, as recommended by the supplier. Only ISR values higher than 0.9 were considered as positive in this test.
Cellular immune responses were determined by γ-IFN ELISpot assays. Frozen PBMC were thawed and incubated overnight in RPMI, 10% FCS and 4 U/ml rh-IL2 (Boehringer Mannheim). Multiscreen-HA 96-wells plates were coated overnight at 4° C. with 4 μg/ml of capture anti-γ-IFN (GZ-4, MAbTech) in PBS, washed, then incubated with 100 μl RPMI, 10% FCS for 1 h at 37° C. The medium was replaced by 5.105 PBMC in suspension in 100 μl of RPMI-10% FCS and 100 μl of stimulating agent. The stimulating agent consisted of 107 pfu of recombinant Modified Vaccine Ankara (32) MVA-HMV or MVA-wt as a control Cells were stimulated for 24 h at 37° C. Phytohemaglutinin A (2.5 μg/ml, Sigma) was used as positive control and RPMI as a negative control. The plates were washed twice with PBS, 4 times with PBS, 0.05% Tween 20 (Sigma), and twice again with PBS. A biotinylated anti-γ-IFN antibody (7-B6-1, MabTech, 100 μl, 1 μg/ml in PBS) was added and the plates were incubated for 2-4 h at 37° C. Streptravidin-Alkaline Phosphatase (AP) conjugate (Roche, 100 μl, 1/2000 dilution in PBS) was added And spots were developed with BCIP/NBT (Promega) in 1 M Tris pH 9.5, 1.5 M NaCl, 0.05 M MgCl2. After drying overnight at room temperature, spots were counted using an automated image analysis system (ELISpot Reader, Bio-Sys). The low background obtained after MVA-wt stimulation was subtracted and the results were expressed as MVA-HMV specific γ-IFN producing cells per million PBMC.
Mice immunization and characterization of humoral immune responses. FVB mice heterozygous for the CD46 transgene (33), were crossed with 129sv IFN-α/βR−/− mice which lack the type I interferon receptor (30). The F1 progeny was screened by PCR and the CD46+/− animals were crossed again with 129sv IFN-α/βR−/− mice. IFN-α/βR−/− CD46+/− animals were selected and used for immunization experiments. These mice are susceptible to MV infection (27, 29). Six-week-old female CD46+/− or CD46+/− IFN-α/βR−/− (IFNAR) mice were inoculated intraperitoneally with 104 TCID50 of the different vaccine preparations (4 mice per group). The presence of anti-MV antibodies was looked for by ELISA (Trinity Biotech, USA) in sera collected one month after vaccination. In this case, an anti mouse IgG Mab (Amersham) was used as secondary antibody. Each determination was done in triplicate. The absorbence determined with a mixture of negative mice sera was subtracted from the absorbence measured in positive mice. Because it was not possible in this case to use the ISR to compare samples, serial dilutions of mice sera were tested to determine the endpoint limit positive dilution.
Results
Comparison of Humoral Immune Responses after Vaccination of Macaques and Mice with EdB-Tag and Schwarz MV Vaccines.
EdB-tag MV is a molecularly cloned MV derived from the Edmonston B strain (16). We compared its immunogenicity in macaques with that of the Schwarz commercial MV vaccine. The EdB-tag virus was prepared in Vero cells infected at a multiplicity of infection (MOI) of 0.05. When syncytia occupied 80-90% of the culture, the cells were scraped, cells and medium were freeze/thawed and cell debris were eliminated by low speed centrifugation. The Schwarz MV, obtained from Aventis Pasteur (Marcy I'Etoile, France), was prepared in the same way from infected chick embryo fibroblasts (CEF) grown at 32° C., the temperature at which this strain has been adapted to CEF. The titers of both vaccine preparations were determined by endpoint dilution assays in Vero cells and expressed as TCID50. Different doses (103 to 105 TCID50) of EdB-tag and Schwarz MV were injected subcutaneously to macaques (2 monkeys per dose). As a control, animals were also injected with 104 TCID50 of the lyophilized commercial Schwarz vaccine (Rouvax, Aventis Pasteur). Anti-MV antibodies levels were determined by ELISA in macaques' sera collected one month after vaccination. Macaques inoculated with 103 and 104 TCID50 of the Schwarz MV had antibody levels similar to those induced by a standard dose of Rouvax vaccine (
Immunogenicity of Schwarz MV Recovered from cDNA.
The immunogenicity for cynomolgus macaques of the virus rescued from pTM-MVSchw plasmid and passaged two times on CEF or Vero cells was compared to that of the industrial Schwarz vaccine. Cynomolgus macaques were used in this experiment because of the difficulty of obtaining rhesus macaques from China that were MV negative. These macaques are as sensitive to MV as rhesus macaques, as shown by several studies (28, 26). Monkeys (2 animals per preparation) were injected sub-cutaneaously with 104 TCID50 of Schwarz MV vaccine from Aventis or Schwarz MV rescued from pTM-MVSchw plasmid and grown either on CEF or Vero cells. The presence of anti-MV antibodies was determined in sera collected at different time points (
Changes in the number of total white blood cells (WBC), lymphocytes and monocytes were observed during the first month following inoculation (
Discussion
In the present work we describe cloning and rescuing the Schwarz/Moraten attenuated strain of measles virus, the constituent of two widely used measles vaccines, Aftenuavax (Merck and Co. Inc., West Point, USA) and Rouvax (Aventis Pasteur, Marcy I'Etoile, France), and of the combined measles, mumps, and rubella vaccine (MMR) (35). To be used in a pediatric clinical trial, a live attenuated MV produced from a cDNA must be as safe and efficient as the parental vaccine. Assuming that safety and efficiency depend ultimately on the genomic sequence of the attenuated strain, we cloned the MV Schwarz cDNA from viral particles prepared from an industrial batch of vaccine using procedures optimized for fidelity of cloning. As a result, the sequence of the clone that we obtained was identical to that of the parental Schwarz MV genome. To maximize yield during rescue, the viral antigenomic cDNA was placed under the control of a T7 RNA polymerase promoter with the GGG motif necessary for full efficiency. A hammerhead ribozyme was inserted between this GGG motif and the first viral nucleotide to allow the exact cleavage of the viral RNA. In order to avoid adapting the Schwarz vaccine to non-certified cells during rescue, helper cells transfected with the engineered cDNA were cocultivated with CEF, the cells on which this vaccine was selected originally and is currently prepared. The rescued virus was passaged two times on CEF and its genome was entirely sequenced. No mutation was found when the sequence was compared to that of the original virus. Moreover, the growth kinetics and the yield of the rescued virus and the original Schwarz virus on CEF were identical.
The Schwarz virus was also rescued after co-cultivation of transfected helper cells with Vero cells, which are very permissive to MV. In this case, however, two mutations appeared in the viral fusion protein (F) after two passages on Vero cells. This rapid adaptation correlated with a much more fusogenic phenotype on Vero cells. In contrast, the rescued Schwarz MV was not fusogenic on CEF (only rare syncytia could be observed in infected CEF). The two mutations occurred in the F protein (G→R in position 266 and Y→S in position 365). These mutations are present in the EdB-tag virus (see
The virus rescued from the pTM-Schw plasmid had the same immunogenicity in macaques as the parental Schwarz vaccine. It is important to emphasize that in these experiments macaques were inoculated with the low dose of virus used for human immunization. Therefore, it will be possible to conduct human clinical trials with this virus using standard vaccine doses (104 TCID50). In contrast, the previously cloned EdB-tag MV was not immunogenic in macaques and poorly immunogenic in mice transgenic for CD46, when used at the same dose as the cloned Schwarz MV.
What could be the reason for the higher immunogenicity of the Schwarz MV strain? Inducing good immunogenicity with a live attenuated viral vaccine requires replication in tissues at a level high enough to prime the immune system adequately. Several of the mutations between the Schwarz and the EdB-tag MV genomes are located in the P/V/C and L genes, suggesting difference in replication efficiency. It is possible that the Schwarz MV replicates in lymphoid cells in vivo more efficiently than the EdB-tag MV even though they replicated at the same rate in Vero cells. Efficient replication in vivo requires some evasion mechanism from the IFN-α/β response. Vero cells, on which the EdB-tag virus was adapted, do not respond to IFN-α/β stimulation. Therefore the EdB-tag MV was selected in the absence of an IFN-α/β response and might be particularly sensitive to this host defense mechanism. Indeed, it has been shown that passaging wild type MV on Vero cells changes the phenotype of the virus from non-IFN-inducer to IFN-inducer (36). Also, the fact that the Ed-tag MV was immunogenic in mice transgenic for the CD46 receptor providing they were also knock-out for the IFN-α/β, receptor suggest that this virus is particularly IFN-sensitive. Interestingly, the IFN-α/β response helps priming the specific immune response against the vaccine. Therefore a good live vaccine must at the same time induce an IFN-α/β response and evade it to some extent. For this reason selecting attenuated viral vaccines on primary cells with a strong IFN-α/β response, such as CEF, might be a good strategy.
The MV products which contribute to IFN resistance have not been identified. However, the nonstructural C protein of the closely related Sendai virus has been shown to counteract the IFN-induced antiviral state (37). The 5 mutations not related to any Edmonston subgroup that we found in the EdB-tag P/V/C gene might be responsible for its low immunogenicity in macaques. On the other hand, the two mutations generated in the F protein by passaging the Schwarz virus on Vero cells did not affect its immune potential, indicating that the fusogenic property of the viral envelope proteins may not play a significant role in immunogenicity.
The pTM-MVSchw plasmid was engineered for the expression of foreign genes by the introduction of two ATU at different positions of the genome. Rescued Schwarz recombinant MV expressed the green fluorescent protein, thus showing that this new measles vaccine functions as a vector. In conclusion, this molecular clone will allow producing MV vaccine without having to rely on seed stocks. With its ATUs, it will be possible to use it as a vector to produce recombinant vaccines based on an approved, efficient and worldwide used vaccine strain.
| Number | Date | Country | Kind |
|---|---|---|---|
| 02291551 | Jun 2002 | EP | regional |
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| 20130296541 A1 | Nov 2013 | US |
| Number | Date | Country | |
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| Parent | 11013786 | Dec 2004 | US |
| Child | 12219570 | US |
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| Parent | PCT/EP03/07145 | Jun 2003 | US |
| Child | 11013786 | US |
