FIELD OF INVENTION
The present invention relates to a novel recombinant plasmid comprising a novel combination of foreign genes of respiratory syncytial virus (RSV), preferably in a single cassette. The present invention further provides a recombinant Respiratory syncytial virus (RSV) vaccine prepared from the novel plasmid.
BACKGROUND OF THE INVENTION
Respiratory syncytial virus (RSV) is the single most important causative agent of severe respiratory illness in infants and young children and the major cause of infantile bronchiolitis. Bronchiolitis is the most frequent cause of hospitalization of infants and young children in industrialized countries (Reed G, et al., J Infect Dis. 1997 Apr;175(4):807-13.) Respiratory syncytial virus (RSV) is the most significant cause of viral lower respiratory tract illness (LRI) in infants and children worldwide, leading to hospitalization of 70, 000 to 1,26,000 infants for pneumonia or bronchiolitis every year in US alone. Global Respiratory syncytial virus (RSV) disease burden is estimated at 64 million cases and 1, 60,000 deaths every year.
Respiratory syncytial virus (RSV) is a member of the family Paramyxoviridae within the Pneumovirus genus. Respiratory syncytial virus (RSV) is an enveloped virus with genomic size of 15222 nucleotides (Collins, 1991, pp. 103-162, D. W. Kingsbury (ed.) Plenum Press, New York). The genome of Respiratory syncytial virus (RSV) encodes 10 mRNAs (Collins et al., 1984, J. Virol. 49: 572-578). Genome of Respiratory syncytial virus (RSV) is negative sense RNA molecule encoding 11 viral proteins, among which the nucleoprotein (N), the fusion protein (F), the surface glycoprotein (G), the matrix protein (M) and several non-structural proteins including the L protein (replicase) and virulence factors NS1 and NS2 are important (Mirza Romero Valdovinos, et al., Intervirology Vol. 46, No. 3,2003). RNA molecule tightly associates with viral N protein to form a nucleocapsid wrapped inside the viral envelope, from which protrude viral proteins F, G and SH. Fusion (F) protein of Respiratory syncytial virus (RSV) plays a vital role for fusion of virion with host cell and infection with neighboring cells through the formation of syncytia (Smith et al.,
Oxford Journals>Life Sciences & Medicine>PEDS>Volumel5, Issue5>Pp. 365-371). Respiratory syncytial virus (RSV) primarily infects cells through heparin binding domains on G protein; In case of mutant viruses lacking G protein infection can be carried out by attachment through heparin binding domains on F protein (Karron et al., PNAS December 9, 1997 vol. 94 no. 25 13961-13966). M protein of respiratory syncytial virus (RSV) plays a significant role in virus assembly through specific interactions with viral nucleocapsids and envelope glycoproteins in the cytoplasm as well as with the host cell membrane (Ghildyal et al., Biochemistry, 2005, 44 (38), pp 12887-12895). NS1 and NS2 proteins of Respiratory syncytial virus (RSV) cooperatively antagonize interferon response (Gotoh et al., Microbiol Immunol. 2001;45(12):787-800.)
Respiratory syncytial virus (RSV) has two major subgroups A and B, primarily based on differences associated with attachment glycoprotein G. G protein is around 90 kD surface protein and has extensive sequence variability and can differ up to 50% at amino acid level among different Respiratory syncytial virus (RSV) strains (Johnson et al., Proc. Natl. Acad. Sci. USA, Vol. 84, pp. 5625-5629, August 1987, Biochemistry). Interactions between fractalkine (CX3CL1) and its receptor, CX3CR1, mediate leukocyte adhesion, activation, and trafficking (Harcourt et al., The Journal of Immunology, 2006, 176: 1600-1608). G protein of Respiratory syncytial virus (RSV) also has an evolutionary conserved CX3C chemokine motif, which is capable of interacting with CX3CR1 and antagonizing activities of fractalkine (CX3CL1) (Tripp RA. Et al.,Viral Immunol. 2004; 17(2): 165-81). Fusion protein F is a 70 kD protein and is highly conserved (amino acid sequence homologue is around 90%) among different Respiratory syncytial virus (RSV) strains and depicts its high degree of antigenic relatedness (Johnson et al., J. gen. Virol. (1988), 69, 2623-2628).
In spite of continued research in this field since the past several decades, currently no commercialized vaccine is available against Respiratory syncytial virus (RSV). In case of children at high risk of disease e.g. certain premature infants with lung and heart condition, serious illness during seasons of high RSV infection can be prevented by monthly shots with a drug called Palvizumab.
Vaccine development for Respiratory syncytial virus (RSV) infection has been complicated due to the fact that host response plays a significant part in pathogenesis of disease. Formalin-inactivated Respiratory syncytial virus (RSV) vaccination attempts in children in 1960s failed as vaccinated children suffered from more severe disease on subsequent exposure to the virus as compared to unvaccinated controls. This formalin-inactivated vaccine led to exacerbated disease including pulmonary eosinophilia following a natural Respiratory syncytial virus (RSV) infection in children (Olson et al., Department of Microbiology, University of Iowa, IA 52242, USA).
Humoral immunity through antibodies plays a critical role in protection against Respiratory syncytial virus (RSV) infection. Passive prophylaxis with specific antibodies like Palvizumab have proven to be effective in prevention of severe lower respiratory tract infection caused by Respiratory syncytial virus (RSV) in infants with bronchopulmonary dysplasia, history of premature birth and children with hemodynamically significant congenital heart disease (Cardena et al., Expert Rev Anti Infect Ther. 2005 Oct;3(5):719-26).
Live attenuated RSV vaccine
Live attenuated RSV vaccine development has been hampered by inability to achieve appropriate balance between attenuation and immunogenicity. A cold passaged (cp) Respiratory syncytial virus (RSV) derivative caused mild illness in young children and even when the strain was further attenuated by chemical mutagenesis to produce cpts 248/404 strain, it was still reactogenic in 1-2 month old infants (Wright et al., J Infect Dis. 2000 Nov; 182(5): 1331-42. Epub 2000 Sep 22). A recombinant live attenuated temperature sensitive Respiratory syncytial virus (RSV) vaccine by Medlmmune is under clinical phase II for prevention of lower respiratory tract disease caused by Respiratory syncytial virus (RSV).
Subunit Respiratory syncytial virus (RSV) vaccines
Subunit vaccine strategy against Respiratory syncytial virus (RSV) has also been an area of focus and various subunit vaccines have been evaluated in clinical trials.
Purified F proteins like PFP (purified fusion protein) have been used as a candidate for subunit vaccine. These purified proteins (e.g. PFP-2) have proved to be safe and immunogenic in healthy older adults (Falsey et al., Vaccine, Volume 14, Issue 13, September 1996, Pages 1214-1218) and children with bronchopulmonary dysplasia (Groothuis et al., J Infect Dis. 1998 Feb;177(2):467-9). Co-purified F, G and M proteins from Respiratory syncytial virus (RSV) A has been tested in healthy adult volunteers in presence of adjuvant e.g. alum or polyphosphazene (PCPP). Neutralizing antibodies against subgroups A and B were detected in 76-93% of the vaccinated population, but reduced gradually after one year, indicating the need of annual immunization (ison et al., Antiviral Res. 2002 Aug;55(2):227-78.). Moderate immunogenicity and gradual reduction of neutralizing antibodies against the Respiratory syncytial virus (RSV) subgroups A and B are the major drawbacks associated with subunit Respiratory syncytial virus (RSV) vaccines.
Live chimeric and recombinant vaccines
Live chimeric and recombinant vaccines are also a potential candidate against Respiratory syncytial virus (RSV) infection. A chimeric bovine/human (B/H) strain of Parainfluenza virus (B/H PIV-3) was engineered by substituting in a BPIV-3 genome the HPIV-3 F and HN genes and the F/NH intergenic sequences to their bovine equivalent. The resulting B/H chimeric virus retained the attenuated phenotype of BPIV-3 and was highly immunogenic in rhesus monkeys. This new strain retained the attenuated phenotype of BPIV-3 and was highly immunogenic in rhesus monkeys (Schmidt et al., J Virol. 2000 Oct;74(19):8922-9). This new B/H PIV-3 strain was then used as a vector to express the F, or F and G open reading frames of RSV subgroup A and B, hence providing a vaccine candidate for both RSV and PIV-3 infections (Schmidt et al., J Virol. 2001 May;75(10):4594-603, J Virol. 2002 Feb;76(3): 1089-99). This live attenuated nasal RSV/PIV-3 candidate vaccine (MEDI 534TM) was shown to be safe and well tolerated in Phase I clinical studies conducted by MedImmune in the USA in adults and seropositive children 1 -9 years of age. Other viral vectors have also been used to deliver Respiratory syncytial virus (RSV) F or G protein gene sequences. Recombinant adenovirus and vaccinia virus expressing Respiratory syncytial virus
(RSV) F protein or G protein or both F and G protein have been constructed and tested in animal models like chimpanzees, but have shown mediocre immunogenicity (Hsu et al., J Infect Dis. 1992 Oct;166(4):769-75). Viral vectors for the delivery of Respiratory syncytial virus (RSV) associated genes is a great opportunity and looks to be very promising as it is not associated till now with prominent disadvantages as virus reverting back to its virulent form. This technology can also open new horizons in Respiratory syncytial virus (RSV) vaccination as reduced antibody titer against the Respiratory syncytial virus (RSV) virus making annual immunization a necessity can be overcome by the viral delivery.
Hence there is a need for new and effective vaccine strategies for RSV infection.
SUMMARY OF INVENTION
The present invention discloses a novel cloning system for generating a recombinant plasmid comprising specific genes from Respiratory syncytial virus (RSV).
In accordance with these and other objects, the present invention relates to a vector or plasmid comprising at least one, or at least two, or at least three or most preferably at least four genes from Respiratory syncytial virus (RSV). The genes of from Respiratory syncytial virus (RSV) may be further inserted between right and left flank regions of non-essential site preferably Del III of modified vaccinia Ankara (MVA) genome.
Specifically, the present invention relates to a vector or plasmid comprising of a cassette of one or more genes selected from Fusion (F) gene, Attachment Glycoprotein (G) gene, Nl and N2 epitopes from Nucleoprotein (N) gene from Respiratory syncytial virus (RSV), wherein the said genes are inserted between right and left flank regions of non-essential site preferably Del III of modified vaccinia Ankara (MVA) genome, wherein each of the said foreign genes are under the transcriptional control of an individual single copy or multiple copies of the same promoter or multiple promoters.
The plasmids of the invention can be used for preparing a recombinant modified vaccinia Ankara (MVA) virus comprising and capable of simultaneously expressing a cassette of at least one, or at least two, or at least three or most preferably at least four
foreign genes from Respiratory syncytial virus (RSV), wherein the said genes are inserted at a non-essential site preferably Del III, within the MVA genome, wherein each of the said foreign genes are under the transcriptional control of an individual single copy or multiple copies of the same promoter or multiple promoters.
Preferably the recombinant cloning system of the invention can be used for generating recombinant vaccines against Respiratory syncytial virus (RSV) virus.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig.l: Diagrammatic representation of the construction of plasmid, pMVA2.RSV5
Fig.2: Diagrammatic representation of the construction of plasmid, pMVA2.RSVGa
DESCRIPTION OF INVENTION
The present invention is not limited to the particular process steps and materials disclosed herein, but are extended to equivalents thereof as would be recognized by those skilled in the relevant arts. It should be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.
Definitions
Modified Vaccinia Ankara (MVA) virus
Modified vaccinia Ankara (MVA) virus is a 177kb dsDNA orthopoxvirus. It is a highly attenuated strain of vaccinia virus which was developed by > 570 serial passages in chicken embryo fibroblasts ( CEFs ), during which it suffered six major deletions of DNA (namely deletion I, II, III, IV, V, and VI) totaling 31000 base pairs (Antoine et al., 1998). Deletion III (Del III) site is one of the most commonly site in the MVA genome, used for the insertion and expression of the foreign sequences The complete genomic sequence of the modified vaccinia Ankara (MVA) virus and a comparison
with other orthopoxviruses can be found in Virology 244:365-396. As a result of the serial passages, MVA virus has become severely host cell restricted to avian cells and replicates poorly in human and most other mammalian cells. Because of extreme attenuation, no adverse effects were reported even when high doses of MVA were given to immunodeficient non-human primates (Stittelaar et al., 2001). There is exhaustive clinical experience using MVA for primary vaccination of over 120,000 humans against smallpox. During extensive field studies, including high risk patients, no side effects were associated with the use of MVA vaccine (Mayr et al., 1987; Stickl et al., 1974). Although MVA is highly attenuated, but it still maintains good immunogenicity. (Meyer et al, 1991).
No MVA replication in human cells has been noticed since no assembly of mature infectious virions takes place following infection. Nevertheless, MVA is shown to express viral and recombinant genes at high levels even in non-permissive cells (mammalian cell lines) and thus has been considered as an efficient and exceptionally safe gene expression vector (Sutter and Moss, 1992). To further exploit the use of MVA, foreign genes have been introduced by DNA recombination into the MVA strain without altering the genome of the MVA virus.
Blu-MVA
Blu-MVA is a recombinant MVA virus having ß-gal gene inserted in the Del III site in its genome.
Enhanced Green Fluorescent Protein
EGFP, as used herein, refers to enhanced green fluorescent protein, a powerful reporter molecule used for monitoring gene expression, protein localization and protein-protein interaction. Several mutant variants of GFP are now available differing in absorption, emission spectra and quantum yield. Enhanced GFP (EGFP) is one such mutant of GFP containing a Phe-64-Leu and Ser-65-Thr mutation. It is being used extensively as it offers higher-intensity emission after blue light excitation with respect to wild type GFP. EGFP has emerged as an ideal molecule for fluorescence-activated cell sorting and other studies (Cinelli et al 2000).
Promoters
A promoter is a region of DNA that facilitates the transcription of a particular gene. Promoters are typically located near the genes they regulate, on the same strand and upstream (towards the 5' region of the sense strand). For expression of heterologous genes in modified vaccinia Ankara (MVA) virus, it is essential to use poxvirus promoters because cellular and other viral promoters are not recognized by the MVA transcriptional apparatus. Strong late promoters like p11 or pCAE are preferable when high levels of expression are desired in MVA.
Kozak sequence
Kozak sequence is the consensus sequence for initiation of translation in vertebrates. This sequence in an mRNA molecule is recognized by the ribosome as the translational start site, from which a protein is coded by that mRNA molecule
Non-essential regions of modifiedvacciniaAnkara (MVA) virus
Non-essential regions according to the present invention may be selected from (i) naturally occurring deletion sites in the MVA genome with respect to the genome of the vaccinia virus or (ii) intergenic regions of the MVA genome. The term intergenic region refers preferably to those parts of the viral genome located between two adjacent genes that comprise neither coding nor regulatory sequences. However, the insertion sites for the incorporation of the heterologous nucleic acid according to the invention (non-essential region) are not restricted to these preferred insertion sites since it is within the scope of the present invention that the integration may be anywhere in the viral genome as long as it is possible to obtain recombinants that can be amplified and propagated in at least one cell culture system. Thus, a non-essential region may also be a non-essential gene or genes, the functions of which may be supplemented by the cell system used for propagation of MVA.
Present Invention
Respiratory syncytial virus (RSV) is an enveloped virus, which encodes expression of around 10 viral proteins. It encodes three transmembrane glycoproteins i.e. the attachment glycoprotein (G), the fusion protein (F) and the small hydrophobic protein (SH). It also encodes two matrix proteins (Ml and M2); three proteins associated with the nucleocapsid, N, P and L; two non-structural proteins NS1 and NS2.
F and G are antigenically most important proteins of Respiratory syncytial virus (RSV) because they stimulate the production of majority of protective immune responses. The G protein with its heparin binding domains is responsible for virus binding to the cell surface receptors. G protein depicts the most extensive antigenic and genetic diversity both between and within major antigenic groups of Respiratory syncytial virus. Antibodies are produced against the G protein but it does not stimulate significant cytotoxic T-lymphocyte responses. The Fusion (F) protein, a 70 kD protein mediates the fusion of the virus and the cell membrane allowing the entrance of virus nucleoplasmid into the cell cytoplasm and the initiation of infected cell. F protein of Respiratory syncytial virus is highly conserved and demonstrates a homologue of around 90% among various strains along with high degree of antigenic relatedness. Fusion protein stimulates both humoral and cytotoxic T-lymphocyte responses.
In addition to the antibody response generated by F and G glycoproteins, human cytotoxic T-cells recognize the F protein and stimulate immune response against infected cell. Certain internal proteins (M, N1 and N2) of Respiratory syncytial virus (RSV) cause cell mediated immunity by stimulation of cytotoxic T cells (CTLs) against Respiratory syncytial virus (RSV) infected cells. Following Respiratory syncytial virus (RSV) infection, matrix (M) protein, nucleoprotein (N) protein, small hydrophobic protein (SH) are produced, which can stimulate cytotoxic T-lymphocyte responses.
In a first embodiment the present invention discloses a cloning system for generating a recombinant plasmid comprising of at least one, or at least two, or at least three or most preferably at least four foreign genes from Respiratory syncytial virus (RSV). Recombinant plasmid may further have right and left flank regions of non-essential site preferably Del III of modified vaccinia Ankara (MVA) genome. The genes cloned from the Respiratory syncytial virus (RSV) may be under the control of any suitable promoter. The genes may be under the control of single or multiple copies of same or
different promoters. P11 is the most preferred promoter under the control of which the Respiratory syncytial virus (RSV) genes may be expressed. In one of the preferred embodiment, all the genes are under the control of their individual P11 promoter. In another preferred embodiment all the genes are under the control of a single P11 promoter.
According to another embodiment of the invention, the recombinant plasmid may also have a marker gene cloned along with the Respiratory syncytial virus (RSV) genes. In a preferred embodiment, the marker gene used may be green fluorescent protein gene (GFP). In a further preferred embodiment, the marker gene used may be enhanced green fluorescent protein gene (EGFP). The marker gene may be under the control of one of the P11 promoter controlling the regulation of Respiratory syncytial virus (RSV) genes. In a preferred embodiment, the GFP marker may be under the control of a separate P11 promoter.
Recombinant plasmid may further have more sequences from modified vaccinia Ankara (MVA) virus genome that would help in homologous recombination of recombinant plasmid into the genome of modified vaccinia Ankara (MVA) virus at any naturally occurring deletion.
In a preferred embodiment the present invention comprises construction of a plasmid comprising genes selected from group of Fusion (F) gene, Attachment Glycoprotein (G) gene, Nucleoprotein (N) gene, Matrix (M) gene from Respiratory syncytial virus (RSV).
Preferably in a further embodiment of the present invention the Nucleoprotein (N) gene from Respiratory syncytial virus (RSV) can be N1 epitope of Nucleoprotein (N) gene or N2 epitope of Nucleoprotein (N) gene, or both Nl epitope and N2 epitope, wherein Nl epitope corresponds to amino acid sequence from 255-263 of N gene (Accession No.: AY911262) and N2 epitope corresponds to amino acid sequence from 306-314 of N gene (Accession No.: AY911262).
Another preferred embodiment the present invention is related to construction of a plasmid comprising Fusion (F) gene, attachment glycoprotein (G) gene, Nl epitope of
Nucleoprotein (N) gene, and N2 epitope of Nucleoprotein (N) gene from Respiratory syncytial virus (RSV).
Another preferred embodiment the present invention is related to construction of a plasmid comprising Fusion (F) gene, attachment glycoprotein (G) gene, Nucleoprotein (N) gene, and Matrix (M) gene from Respiratory syncytial virus (RSV).
Another preferred embodiment the present invention is related to construction of a plasmid comprising Fusion (F) gene, attachment glycoprotein (G) gene, N1 epitope of Nucleoprotein (N) gene, N2 epitope of Nucleoprotein (N) gene, Matrix (M) from Respiratory syncytial virus (RSV).
In one of the preferred embodiments of the present invention the attachment glycoprotein (G) gene from Respiratory syncytial virus (RSV) can be from A (Ga) subgroup or B (Gb) subgroup. Major difference between the two Respiratory syncytial virus (RSV) subgroups A and B lies in the antigenicity of the attachment G glycoprotein. This specific sequence can be either in natural form or can be mutated.
In a further specific embodiment of the present invention, the attachment G protein is a modified G protein. The Respiratory syncytial virus (RSV) G protein is synthesized in two forms: as an anchored type II integral membrane protein and as N terminally deleted form which lacks essentially the entire membrane anchor and is secreted (Hendricks et al., J. Virol. 62:2228-2233 (1988)). These two different forms are produced by translation initiation at two different start sites. Membrane anchored form initiates at the first AUG (start codon) of the G protein's open reading frame (ORF) and the secreted form initiates at the second AUG (start codon) of G protein's open reading frame (ORF) at codon 48 and is further processed by proteolysis (Roberts et al., J. Virol. 68: 4538-4546 (1994)). Presence of this second AUG translation initiation site is highly conserved being present in all strains of human, bovine and ovine Respiratory syncytial virus (RSV) sequenced till date. It is highly desirable to abate or reduce the expression of secreted form of G protein because it has been suggested that soluble form of G protein might mitigate host cell immunity by acting as a decoy to tap neutralizing antibodies. Soluble form of G protein has also been implicated in playing role in imbalanced stimulation of immune response by preferential stimulation of a Th2-biased response, which in turn appears to be associated with enhanced
immunopathology upon subsequent exposure to Respiratory syncytial virus (RSV). The sequence of attachment glycoprotein (G) protein may be modified by side directed mutagenesis to express complete anchored protein and reduce the expression of N-terminally deleted secreted G protein.
In a still further preferred embodiment of the present invention, attachment glycoprotein (G) is a modified Ga gene (Ga gene is G gene of Respiratory syncytial virus subgroup A), in which a modification is done at nucleotide position 143 from thymine (T) to cytosine (C) which leads to change in amino acid at position 48 from methionine (ATG) to threonine (ACG). This modification prevents the expression of secreted G protein.
In yet another preferred embodiment of the present invention, attachment glycoprotein (G) is a modified Gb gene (Gb gene is G gene of Respiratory syncytial virus subgroup B), in which a modification is done at four nucleotide positions namely 143, 520, 521, and 522. A modification is done at nucleotide position 143 from thymine (T) to cytosine (C) of G gene from subgroup B of Respiratory syncytial virus (RSV), which leads to change in amino acid at position 48 from methionine (ATG) to threonine (ACG). This modification prevents the expression of secreted G protein. Modified protein Gb is further prepared by modifying already modified Gb gene sequence (at nucleotide position 143) from B subgroup, at nucleotide positions 520, 521 and 522 by modifying them from adenine (A), guanine (G), and thymine (T) to thymine (T), cytosine (C) and guanine (G) respectively . These three mutations in the sequence of Gb gene are done to increase the stability of G protein as it is prone to polymerase errors (Journal of Virology, Feb 1993, pl090- 1093).
Hence in the present invention a site directed mutagenesis is carried out at nucleotide position 143 of G gene of both A and B subgroup of Respiratory syncytial virus (RSV), which leads to modification of methionine (ATG) to threonine (ACG) at amino acid position 48. This amino acid change form methionine to threonine prevents the expression of secreted form of G protein as translation initiation start codon for methionine is changed to threonine. This mutation would have qualitative and quantitative effect on host immune response, rather than to directly attenuate the virus.
In another embodiment, the present invention relates to a recombinant plasmid comprising one gene from Respiratory syncytial virus (RSV).
In a preferred embodiment, the present invention relates to a recombinant plasmid comprising one gene from Respiratory syncytial virus (RSV) that is a modified Ga gene or modified Gb gene.
In a further preferred embodiment the present invention relates to a recombinant plasmid comprising one gene from Respiratory syncytial virus (RSV) that is a modified Ga.
In another preferred embodiment the present invention relates to a novel plasmid deposited at Microbial Type Culture Collection and Gene Bank under the accession number MTCC 5677.
In another preferred embodiment the present invention relates to a novel plasmid deposited at Microbial Type Culture Collection and Gene Bank under the accession number MTCC 5678.
In another embodiment, the present invention relates to a recombinant plasmid comprising of at least one, or at least two, or at least three or most preferably at least four genes from Respiratory syncytial virus (RSV), wherein one of the genes is a modified Ga or modified Gb protein.
Another preferred embodiment the present invention is related to construction of a plasmid comprising Fusion (F) gene, modified attachment glycoprotein (Ga) gene, N1 epitope of Nucleoprotein (N) gene, and N2 epitope of Nucleoprotein (N) gene from Respiratory syncytial virus (RSV).
Another preferred embodiment the present invention is related to construction of a plasmid comprising Fusion (F) gene, modified attachment glycoprotein (Gb) gene, Nl epitope of Nucleoprotein (N) gene, and N2 epitope of Nucleoprotein (N) gene from Respiratory syncytial virus (RSV).
In a second embodiment, plasmids of the present invention can be further used for preparation of a recombinant modified vaccinia Ankara (MVA) virus comprising and capable of simultaneous expression of a cassette comprising of at least one, or at least
two, or at least three or most preferably at least four genes from Respiratory syncytial virus (RSV), wherein the said genes are inserted at a non-essential site preferably Del III, within the modified vaccinia Ankara (MVA) genome. Further recombinant modified vaccinia Ankara (MVA) virus, may have each of the said genes under the transcriptional control of an individual single copy or multiple copies of the same promoter or multiple promoters. Recombinant modified vaccinia Ankara (MVA) virus comprising plasmids of the present invention can be prepared as described in WO2010134094A1, which related to preparation of recombinant modified vaccinia Ankara (MVA) virus using plasmid comprising multiple influenza genes.
The genes cloned from the Respiratory syncytial virus (RSV) may be under the control of any suitable promoter. The genes may be under the control of single or multiple copies of same or different promoters. P11 is the most preferred promoter under the control of which the Respiratory syncytial virus (RSV) genes may be expressed. In one of the preferred embodiment, all the genes are under the control of their individual P11 promoter. In another preferred embodiment all the genes are under the control of a single P11 promoter.
According to another embodiment of the invention, the recombinant modified vaccinia Ankara (MVA) virus may also have a marker gene cloned along with the Respiratory syncytial virus (RSV) genes. In a preferred embodiment, the marker gene used may be green fluorescent protein gene (GFP). In a further preferred embodiment, the marker gene used may be enhanced green fluorescent protein gene (EGFP). The marker gene may be under the control of one of the P11 promoter controlling the regulation of Respiratory syncytial virus (RSV) genes. In a preferred embodiment, the GFP marker may be under the control of a separate Pll promoter.
In a preferred embodiment, the present invention relates to recombinant modified vaccinia Ankara (MVA) virus comprising and capable of simultaneously expressing a cassette of genes selected from group comprising Fusion (F), attachment glycoprotein (G), Nucleoprotein (N) from Respiratory syncytial virus (RSV).
Preferably in a further embodiment of the present invention the Nucleoprotein (N) gene from Respiratory syncytial virus (RSV) can be N1 epitope of Nucleoprotein (N) gene, or N2 epitope of Nucleoprotein (N) gene, or both Nl epitope and N2 epitope, wherein
Nl epitope corresponds to amino acid sequence from 255-263 of N gene (Accession No.: AY911262) and N2 epitope corresponds to amino acid sequence from 306-314 of N gene (Accession No.: AY911262).
In another preferred embodiment, the present invention relates to recombinant modified vaccinia Ankara (MVA) virus comprising and capable of simultaneously expressing a cassette of genes comprising Fusion (F), attachment glycoprotein (G), Nucleoprotein (Nl), and Nucleoprotein (N2) from Respiratory syncytial virus (RSV).
In another preferred embodiment, the present invention relates to recombinant modified vaccinia Ankara (MVA) virus comprising and capable of simultaneously expressing a cassette of genes comprising Fusion (F), attachment glycoprotein (G), Nucleoprotein (N), matrix (M) from Respiratory syncytial virus (RSV).
In another preferred embodiment, the present invention relates to recombinant modified vaccinia Ankara (MVA) virus comprising and capable of simultaneously expressing a cassette of genes comprising Fusion (F) gene, attachment glycoprotein (G) gene, Nl epitope of Nucleoprotein (N) gene, N2 epitope of Nucleoprotein (N) gene, Matrix (M) from Respiratory syncytial virus (RSV).
In a still further preferred embodiment of the present invention, attachment glycoprotein gene is a modified Ga or modified Gb gene.
In another preferred embodiment, the present invention relates to recombinant modified vaccinia Ankara (MVA) virus comprising and capable of simultaneously expressing a cassette of genes comprising Fusion (F) gene, modified attachment glycoprotein (Gb) gene, Nl epitope of Nucleoprotein (N) gene, N2 epitope of Nucleoprotein (N) gene from Respiratory syncytial virus (RSV).
In another preferred embodiment, the present invention relates to recombinant modified vaccinia Ankara (MVA) virus comprising and capable of simultaneously expressing a cassette of genes comprising Fusion (F) gene, modified attachment glycoprotein (Gb) gene, Nl epitope of Nucleoprotein (N) gene, N2 epitope of Nucleoprotein (N) gene, Matrix (M) from Respiratory syncytial virus (RSV).
In an alternate embodiment the present invention relates to a recombinant modified vaccinia Ankara (MVA) virus comprising and capable of expression of a cassette comprising of one gene from Respiratory syncytial virus (RSV), wherein the said gene is inserted at a non-essential site preferably Del III, within the modified vaccinia Ankara (MVA) genome.
In another embodiment the present invention, it relates to a recombinant modified vaccinia Ankara (MVA) virus comprising and capable of expression of a cassette comprising of one gene from Respiratory syncytial virus (RSV).
In another preferred alternate embodiment the present invention relates to a recombinant modified vaccinia Ankara (MVA) virus comprising and capable of expression of a cassette comprising of one gene from Respiratory syncytial virus (RSV), wherein the said gene is modified Ga gene (modified G protein from subgroup A of Respiratory syncytial virus) or modified Gb gene (modified G protein from subgroup B of Respiratory syncytial virus) and is inserted between right and left flank regions of non-essential site preferably Del III of modified vaccinia Ankara (MVA) genome.
A further preferred alternate embodiment the present invention relates to a recombinant modified vaccinia Ankara (MVA) virus comprising and capable of expression of a cassette comprising of one gene from Respiratory syncytial virus (RSV), wherein the said gene is modified Ga gene (modified G protein from subgroup A of Respiratory syncytial virus) and the said gene is inserted between right and left flank regions of non-essential site preferably Del III of modified vaccinia Ankara (MVA) genome.
The preparation of recombinant modified vaccinia Ankara (MVA) virus can be done using known techniques in the art. Various suitable mammalian cell lines can be used for the preparation of recombinant modified vaccinia Ankara (MVA) virus. Preparation of recombinant modified vaccinia Ankara (MVA) virus may comprise the following steps:
a) Culturing a mammalian cell line in a specific growth media,
b) Cell are grown to confluency,
c) Cells are infected with Blu-modified vaccinia Ankara (MVA) virus,
d) Transfecting the cells with recombinant plasmid comprising respiratory
syncytial virus (RSV) genes under the control of P11 promoter,
e) Increase the titre of the recombinant modified vaccinia Ankara (MVA) virus by passing the progeny, and
f) Isolating the recombinant virus.
For the preparation of recombinant modified vaccinia Ankara (MVA) virus, a mammalian cell line is grown under optimum growth conditions. After growing the cell line to confluency, it is infected with Blu-modified vaccinia Ankara (MVA) virus. These infected calls are then transfected with recombinant plasmid capable of simultaneously expressing one or more Respiratory syncytial virus (RSV) genes. Transfection is done either by liposome method, or calcium phosphate method or any other method known in the art. Appropriate controls are used during the experiment. Once the cytopathic effect (cpe) is visible, transfected cells may be scraped, pelleted and freeze thawed few times so as to release the virus which may then be used to infect a fresh batch of the mammalian cells. The recombinant modified vaccinia Ankara (MVA) virus may be passaged nine times before making the stock so as to increase the titre of recombinant modified vaccinia Ankara (MVA). This stock virus may be used to confirm the integration of foreign genes in the mixed progeny by PCR, to confirm the expression of heterologous genes by Western blot and to isolate the recombinant modified vaccinia Ankara (MVA) virus by fluorescence-activated cell sorter (FACS), based on EGFP fluorescence. The recombinant modified vaccinia Ankara (MVA)virus may then be purified.
In a third embodiment of the present invention can further be used to develop a method for immunization of humans/animals/birds in need of a prophylactically effective amount of the recombinant modified vaccinia Ankara (MVA) virus. Said method according to the invention may comprise a composition which may also optionally contain carriers, additives, antibiotics, preservatives, diluents, salts, buffers, stabilizers, solubilizers and other materials well known in the art. It is necessary that these are "pharmaceutically acceptable" which means that these are non-toxic material that does
not interfere with the effectiveness of the biological activity of the modified vaccinia Ankara (MVA) virus according to the invention. The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition may further contain other agents which either enhance the activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to be applied for the method for immunization according to the invention to produce a synergistic effect or to minimize side effects. Techniques for formulation and administration of modified vaccinia Ankara (MVA) virus according to the invention may be found in "Remington's Pharmaceutical Sciences" (Muck Publishing Company, Easton, PA, latest edition).
The present invention can be further used to develop a method of treatment of an infection caused by an Respiratory syncytial virus (RSV), by administration to a subject, the immunogenic composition or vaccine comprising a recombinant modified vaccinia Ankara (MVA) virus comprising genes from Respiratory syncytial virus (RSV).
The method for immunization according to the present invention will make use of a prophylactically effective amount of the recombinant modified vaccinia Ankara (MVA) virus. Vaccines of the invention may be used to treat both children and adults. A prophylactically effective dose further refers to that amount of the compound/ingredient sufficient to result in inhibition of establishment of infection or amelioration of symptoms.
The detailed example which follows is intended to contribute to a better understanding of the present invention. However, it is not intended to give the impression that the invention is confined to the subject matter of the example.
EXAMPLE
1. Preparation of plasmids having commercially synthesized respiratory syncytial virus (RSV) genes F, Gb, N1, N2, marker Enhanced Green Fluorescent protein (GFP), Left and right flank gene of MVA
The Fusion (F, Accession No.: AY911262), Gb (modification of G gene at nucleotide positions 143, 520, 521, and 522) from Respiratory syncytial virus (RSV) subgroup B (G (Gb)), Accession No.: AFO13254), Nl epitope of Nucleoprotein (N) gene (N1, amino acids 255-263 of Accession No.: AY911262), and N2 epitope of Nucleoprotein (N) gene (N2, amino acids 306-314 of Accession No.: AY911262) genes of Respiratory syncytial virus (RSV), enhanced green fluorescent protein gene (EGFP, Accession No.: U57609), Flanking genes of Del III region of MVA (Accession No.: U94848) were procured after synthesis from a commercial source. Each of the genes had promoter p11 and ribosomal binding site kozak sequence in front of the gene. The artificially synthesized genes were cloned in separate plasmids. The artificially synthesized genes were cloned to generate plasmids plasmid 1, plasmid 2, plasmid 3, plasmid 4, plasmid 5, plasmid 6, and plasmid 7 to clone EGFP, N2 gene, Nl gene, Right flank gene of MVA, Left flank gene of MVA, F gene, and Gb (modification of G gene at nucleotide positions 143, 520, 521, and 522) respectively.
2. Preparation of plasmids having commercially Respiratory syncytial virus (RSV)
gene (Ga) and marker Enhanced Green Fluorescent protein (EGFP)
The Ga (modification of G gene at nucleotide positions 143) gene from Respiratory syncytial virus (RSV) subgroup A (G (Ga)), Accession No.: AY911262) gene of Respiratory syncytial virus (RSV) and enhanced green fluorescent protein gene (EGFP, Accession No.: U57609) were procured after synthesis from a commercial source. Each of the genes had promoter p11 and ribosomal binding site kozak sequence in front of the gene. The artificially synthesized gene for Ga (modification of G gene at nucleotide positions 143) gene was cloned in plasmid 8 and cassette with EGFP, Right flank gene of MVA, Left flank gene of MVA were together cloned in plasmid 9.
3. Construction of recombinant plasmid pMVA2.RSV5
A novel recombinant plasmid was constructed through a number of cloning steps (Figure 1). The final recombinant plasmid has the F, modified Gb, Nl, N2 genes from Respiratory syncytial virus (RSV) and the EGFP marker gene, each under the control of separate P11 promoter. The stretch of the genes in the recombinant plasmid are flanked from both the sides by "left flank" and "right flank" sequences of the Del III site of modified vaccinia Ankara (MVA) virus that will aid in the homologous recombination
of the recombinant plasmid in the Del III site of the modified vaccinia Ankara (MVA) viral genome.
4. Construction of recombinant plasmid pMVA2.RS VGa
A novel recombinant plasmid was constructed through a number of cloning steps (Figure 2). The final recombinant plasmid has the modified Ga gene from Respiratory syncytial virus (RSV) and the EGFP marker gene, each under the control of separate P11 promoter. The stretch of the genes in the recombinant plasmid are flanked from both the sides by "left flank" and "right flank" sequences of the Del III site of modified vaccinia Ankara (MVA) that will aid in the homologous recombination of the recombinant plasmid in the Del III site of the modified vaccinia Ankara (MVA) viral genome.

WE CLAIM:
1. A novel plasmid comprising at least one, or at least two, or at least three or at least four genes from the Respiratory syncytial virus (RSV), wherein genes are selected from the group comprising of Fusion (F), attachment glycoprotein (G), Nucleoprotein (N), Matrix (M).
2. A novel plasmid as claimed in the claim 1, wherein a marker gene is cloned along with the Respiratory syncytial virus (RSV) genes.
3. A novel plasmid as claimed in the claim 2, wherein the marker gene is Enhanced Green Fluorescent Protein (EGFP) gene.
4. A novel plasmid as claimed in the claim 1, wherein the genes from the Respiratory syncytial virus (RSV) are under the control of single or multiple copies of same or different promoters.
5. A novel plasmid as claimed in the claim 4, wherein the genes from the Respiratory syncytial virus (RSV) are under the control of one promoter.
6. A novel plasmid as claimed in the claim 4, wherein the genes from the Respiratory syncytial virus (RSV) are under the control of a separate promoter.
7. A novel plasmid as claimed in the claim 5 and 6, wherein the promoter used isP11.
8. A novel plasmid as claimed in the claim 1, wherein gene for attachment glycoprotein (G) can be selected from subgroup A or subgroup B of respiratory syncytial virus (RSV).
9. A novel plasmid as claimed in the claim 1 and 8, wherein gene for attachment glycoprotein (G) is full length integrated glycoprotein (G).
10. A novel plasmid as claimed in the claim 1, 8, and 9, wherein gene for
attachment glycoprotein (G) is a modified sequence.
11. A novel plasmid as claimed in the claim 10, wherein the modified sequence is obtained by modifying nucleotide sequence at position 143 from thymine (T) to cytosine (C) which changes amino acid sequence of the said modified sequence at position 48 from methionine (M) to threonine (T).
12. A novel plasmid as claimed in the claim 11, wherein modified sequence is further modified by modifying nucleotide sequence at positions 520, 521, and 522.
13. A novel plasmid as claimed in the claim 12, wherein modified sequence is obtained by modifying nucleotide sequence at positions 520, 521 and 522 by modifying them from adenine (A), guanine (G), and thymine (T) to thymine (T), cytosine (C) and guanine (G) respectively.
14. A novel plasmid as claimed in the claim 1, wherein gene for Matrix (M) protein is either M1 or M2 or both Ml and M2.
15. A novel plasmid as claimed in the claim 1, wherein gene for Nucleoprotein (N) is either N1 epitope (amino acid no 255-263 of N protein) or N2 epitope (amino acid no 306-314 of N protein) of Respiratory syncytial virus (RSV) or both N1 epitope and N2 epitope.
16. A novel plasmid comprising Fusion (F) gene, attachment glycoprotein (G) gene, Nl epitope of Nucleoprotein gene, and N2 epitope of Nucleoprotein gene from Respiratory syncytial virus (RSV).
17. A novel plasmid as claimed in the claim 16, wherein attachment
glycoprotein (G) gene is a modified attachment glycoprotein (G) gene.
18. A novel plasmid deposited at Microbial Type Culture Collection and Gene Bank under the accession number MTCC 5677.
19. A novel plasmid deposited at Microbial Type Culture Collection and Gene Bank under the accession number MTCC 5678.
20. A host cell transformed or transfected with the novel plasmid as claimed in
the claim 1-19.
21. A recombinant modified vaccinia Ankara (MVA) virus comprising and
capable of simultaneously expressing a cassette of at least one, or at least two,
or at least three or at least four genes from Respiratory Syncytial Virus (RSV),
wherein genes are selected from the group comprising of Fusion (F), attachment
glycoprotein (G), Nucleoprotein (N), Matrix (M) and the said genes are inserted
between right and left flank regions of non-essential site preferably Del III of
modified vaccinia Ankara (MVA) genome.
22. A recombinant modified vaccinia Ankara (MVA) virus as claimed in the claim 21, which can be prepared from novel plasmid of claim 1.
23. A recombinant modified vaccinia Ankara (MVA) virus as claimed in the claim 21, wherein attachment glycoprotein (G) gene is a modified attachment glycoprotein (G) gene.
24. A recombinant modified vaccinia Ankara (MVA) virus as claimed in the claim 21, wherein the gene(s) are under the transcriptional control of separate P 11 promoters.
25. A method of preparing a recombinant modified vaccinia Ankara (MVA) virus as claimed in claims 21, comprising the steps of

a) culturing a mammalian cell line,
b) growing the cell line to confluency,
c) infecting the cells with BIu-MVA virus,
d) transfecting the cells with nucleic acid comprising the Respiratory Syncytial Virus (RSV) genes under the control of the P 11 promoter,
e) passaging the progeny virus to increase the titre of the recombinant MVA and
f) isolation of the recombinant virus.
26. The method as claimed in claim 25, wherein the mammalian cell line used is
BHK21.
27. A vaccine comprising the virus as claimed in the claims 21, further
optionally comprising other excipients, diluents and stabilizers.
28. A vaccine comprising the virus as claimed in claims 21, which is suitable
for parenteral and non-parenteral administration.
29. The vaccine as claimed in claim 28, which is suitable for administration via
intranasal, intramuscular and mucosal routes.
30. The vaccine as claimed in claim 29, wherein the said composition is
suitable for delivery via nasal route, in liquid form as nose drops or sprays, or
via inhalation, as powder or as cream or emulsion.
31. A kit comprising the vaccine as claimed in claims 27- 30, comprising a
leaflet giving details of the vaccine e.g. instructions for administration, details
of the antigens within the vaccine, etc.
32. A method of treatment of an infection caused by Respiratory Syncytial Virus (RSV), by administration of the vaccine as claimed in claims 27-30 to a subject.