Patent Grant
7901691
The present invention relates to a recombinant chimeric G-protein based rabies vaccine having SEQ ID No. 1 and its parts or variants thereof expressed in plant tissue. The invention also relates to a chimeric gene of SEQ ID No. 2 that encodes a chimeric G-protein based rabies vaccine having SEQ ID No 1. The invention also provides a method for large scale production of the chimeric G protein based rabies vaccine and a method of vaccinating a subject for rabies.
Reference may be made to WHO, 1989, WHO expert committee on Rabies: WHO Technical Report, WHO, Geneva reporting that rabies is one of the most important and widespread zoonotic diseases and is, with the exception of a few countries, a truly global problem. Rabies is a dreadful disease, one of the oldest known to mankind. It spreads due to the bite of an infected animal or by mucosal exposure. Once the symptoms appear, it proves fatal.
Reference may be made to Hohnes et al., 2002; “Genetic Constraints and the Adaptive Evolution of Rabies Virus in Nature” Virology 292, 247-257 who used a molecular evolutionary approach to investigate the adaptation of rabies virus in nature. Their analysis revealed that the DNA sequence of the nucleoprotein (N) and glycoprotein (G) genes of natural viral isolates were highly constrained, especially at nonsynonymous sites, in contrast to the higher rates of nonsynonymous evolution observed in viruses subject to laboratory passage. In evidence to this statement Charlton et al., 1997. “The long incubation period in rabies: Delayed progression of infection in muscle at the site of exposure”, Acta Neuropathologica. 94, 73-77 reported that although rabies virus has a strong neurotropism, replication in vivo does not take place only in neuronal cells. In particular, the virus replicates in muscle tissue also at the site of inoculation before entering the peripheral and central nervous system as well as the salivary gland and other non nervous tissues. Such a process has been documented by Morimoto et al., 1996. “Characterization of a unique variant of bat rabies virus responsible for newly emerging human cases in North America” Proc. Natl. Acad. Sci. USA 93, 5653-5658 who state that in vitro substitutions in the viral glycoprotein (G) sequence accumulate in cell culture of rabies virus and change the tropism for nervous tissues, thereby changing virulence. This shows an adaptive process of virus which was also highlighted in study of Kissi et al., 1999 “Dynamics of rabies virus quasispecies during serial passages in heterologous hosts”, J. Gen. Virol. 80, 2041-2050 who observed substantial genetic variation in the G protein coding gene from viruses passaged through different host species.
Reference may be made to Patrick et al., 1987. U.S. Pat. No. 4,707,356 wherein, a peptide vaccine requires identifying an antigenic determinant on the virus which has a sequence that is highly conserved among the various strains. A segment of the rabies virus coat glycoprotein was discovered which has a sequence homologous with the conserved sequence of the segment of the curaremimetic, snake—venom neurotoxins which includes the segment through which the toxins are thought to bind to the acetylcholine receptor binding—site (the AchR) at neuromuscular junctions. Lentz et al. reported in Science 226, 847-848 (1984) “Amino acid sequence similarity between rabies virus glycoprotein and snake venom curaremimetic neurotoxins” that rabies virus accumulates at the neuromuscular junction by binding to the acetylcholine receptors at such junction. Findings reported by Lenz et al. are that the binding of rabies virus at the neuromuscular junction can be blocked by pre-incubation of tissue including such junctions with the curaremimetic, snake—venom neurotoxin, alpha—bungarotoxin, which is known to bind tightly to the acetylcholine (Ach) binding site of the AchR.
Reference may be made to Dietzschold et al., 1979. “Rabies virus strain. A comparative study by polypeptide analysis of vaccine strain with different pathogenic patterns” Virology 98 63-75 wherein five constituent polypeptides of the rabies vaccine strains ERA, HEP, CVS and PM (Stereotype I) and Mokola (Serotype 3) were examined by tryptic peptide analysis and revealed general similarity between the nucleoproteins of Mokola and four serotype 1 strains while overall comparison of the tryptic vaccine strains indicates that CVS and PM are more closely related to each other than to ERA or HEP.
Reference may be made to Slater Aug. 9, 1977 U.S. Pat. No. 4,040,904 “Novel rabies virus vaccine and processes” wherein, the ERA strain of the rabies virus was derived from SAD virus strain, originally isolated from a rabid dog and propagated in mouse brain and hamster kidney cells and then adapted to primary pig kidney tissue culture. A sample of the ERA strain of rabies virus was deposited with the American Type Culture Collection, Washington, D.C. on Oct. 29, 1964 and was recorded there as number VR 332. Vaccines containing the ERA strain adapted to primary pig kidney tissue culture are widely used in immunizing various animal species including dogs, cats and cattle against rabies. According to the invention, an improved attenuated strain of rabies virus having a significantly reproducible cytopathic activity is provided. The improved rabies virus strain used in this invention is prepared from the ERA strain of rabies virus. The ERA rabies strain has been identified by Abelseth, M. K. in “Propagation of Rabies virus in Pig Kidney Cell Culture”, Can. Vet. J. 584-87 (1964) and Abelseth, M. G., “An Attenuated Rabies Vaccine for Domestic Animals Produced in Tissue Culture”, Can. Vet. J. 5279-286 (1964) was derived from the rabies virus described by Fenje, P., in “Propagation of Rabies Virus in Cultures of Hamster Kidney Cells”. Can J. Microbiol, 6 379-484.
Reference may be made to Thoulouze et al., 1997, “Rabies virus infects mouse and human lymphocytes and induces apoptosis” in Journal of Virology 71: 10: 7372-7380, who showed that the rabies virus infects both mouse spleen lymphocytes and the human T-lymphocyte cell line. Jurkat found that attenuated rabies virus strain ERA infects ConA-activated splenocytes and T-cell lines more efficiently than CVS which is a highly neurovirulent rabies virus strain and reported that in contrast to CVS, ERA rabies virus and other attenuated viruses stimulate a strong immune response and can be efficient live vaccines. Both inactivated and attenuated viruses are used for immunization but the cost of production of inactivated virus is a major problem. Secondly, such a vaccine should be completely inactivated and for such purposes it is pre-requisite that candidate vaccine should be avirulent strain of the virus but it should be immunogenic and genetically stable. (Hooper et al., 1998, Collaboration of antibody and inflammation in the clearance of rabies virus from the CNS” in Journal of Virology; 72: 3711-9).
Yang et al., 1992 in Journal of General Virology; 73: 895-900, “Basis of neurovirulent rabies virus variant Av01 with sterotaxic brain inoculation in mice” reported that pathogenicity is not only a function of the virus but is also largely dependent on the site of infection and the immune status of the host. Even the most attenuated rabies viruses can potentially cause a lethal encephalomyelitis, which suggests that even inactivated and attenuated viruses are not reliable source for vaccination.
Reference may be made to Coslett et al., 1980, “The structural proteins of rabies virus and evidence for their synthesis from separate monocistronic RNA species” in Journal of General Virology, 49: 161-180 who reported that the genome of rabies virus is organized similar to that of vesicular stomatitis virus encoded by five major proteins, the nucleoprotein (N), phosphoprotein (NS) and polymerase (L) protein which, together with the genomic RNA form a nucleocapsid which is enveloped by a membrane (M) containing the transmembrane glycoprotein G. Conzehnann et al., 1990 in “Molecular cloning and complete nucleotide sequence of the attenuated rabies virus SAD B 19” Virology 175: 485-499 cloned and sequenced the complete nucleotide sequence of viral genome of SAD B 19 rabies virus strain which comprises 11, 928 nucleotides and encodes the five viral proteins N, NS M, G and L. The five cistrons are separated by intergenic regions of 2, 5, 5, and 24 nucleotides respectively.
Reference may be made to Gaudin et al, 1991 “Rabies Virus Glycoprotein is a Trimer” Virology 187, 627-632. They studied the oligomeric structure of glycoprotein both on the viral surface and after solubilization with detergents. The study shows that native quaternary structure of the glycoprotein is trimeric. However most detergents used in the study solubilized G in a monomeric form and only CHAPS a zwitterionic detergent, allowed solubilization of G in its native trimeric structure, this was determined using electron microscopy and sedimentation analysis of detergent solubilized G. The CHAPS solubilized G had a sedimentation coefficient of 9 S while other detergents solubilized G in a 4 S monomeric form. This study confirmed the results obtained by Whitt et al., 1991 “Membrane fusion activity, oligomerization and assembly of the rabies virus glycoprotein” in Virology 185, 681-688 wherein, a cross—linking reagent was used to study G expressed in HeLa cells from cloned cDNA. Electron microscopy also indicated that the native molecule has a “head” and a “stalk” and provided the basis for a low—resolution model of the glycoprotein structure.
Reference may be made to Dietzschold et al., 1983 “Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus” Proc. Natl. Acad. Sci. USA; 80: 70-74 who reported that the amino acid arginine at 333 position of the glycoprotein plays a critical role in the pathogenicity of the rabies virus. Benmansour et al., 1991 “Antigenicity of rabies virus glycoprotein” Journal of Virology: 4198-4203 evaluated the relative importance of antigenic sites by describing more precisely the antigenicity of the protein by using monoclonal antibodies and neutralization—resistant (MAR) mutants. The 266 neutralizing monoclonal antibodies were identified in the study of which 97% belonged to sites II and III which were initially defined by Lafon et al., 1990 in “Human monoclonal antibodies specific for the rabies virus glycoprotein and N protein” Journal of General Virology; 71: 1689-1696.
Reference may be made from Cox et al., 1977 “Rabies virus glycoprotein 11 Biological and serological characterization” Infection Immunity 16, 743-759 wherein the glycoprotein of rabies virus was identified as a major antigen that induces protective immunity and induces the production of virus—neutralizing antibodies and confers immunity against a lethal challenge infection by the rabies virus. Although protection against rabies virus infection is probably the result of many host effector interactions, as studied by Turner 1985, “Immune response after rabies vaccination: basic aspects” Ann. Inst. Pasteur Virol. 136E: 453-460, the rabies virus G protein represents a logical choice for the development of a subunit vaccine that can be used for immunization against rabies in humans and animals. Reference in this respect can also be made from Cox et al., 1980, “Preparation and characterization of rabies hemagglutinin” Infection Immunity 30, 572-577.
Reference may be made to Swamy et al., 1984, “Neurological complication due to beta—propiolactone (BPL)—inactivated antirabies vaccination” Journal of the Neurological Sciences 63: 1: 111-128; who studied the neuroparalytic accident in patients due to antirabies vaccination with BPL vaccine which proves that the virus was not completely inactivated by beta-propiolactone and can be a high risk for the health because basically the inactivated rabies vaccines consist of suspensions of virus containing central nervous tissue of infected animals, such as rabbits or sheep or of suspensions of infected duck fetuses. Such type of vaccines also have a second drawback that, due to the high content of foreign proteins, they may cause undesired side effects at the point of injection as well as of general nature. When rabies vaccines from the central nervous tissue are used, the patient may even suffer neurocomplications with permanent damage, all the more since a series of injections is required for sufficient protection against rabies. Moreover, animal tissue based vaccines may carry other infective particles like prions and viruses like HIV, Fowl pox, Madcow etc.
Anilionis et al. 1981, “Structure of the glycoprotein gene in rabies virus” Nature: 294: 275-278 cloned the glycoprotein coding gene, using mRNA extracted from rabies virus infected BHK cells, purified by oligo (dT) cellulose chromatography and sucrose density centrifugation and determined complete nucleotide sequence of the glycoprotein cDNA. Subsequently, Yelverton et al., 1983, in “Rabies virus glycoprotein analogs: biosynthesis in Escherichia coli” Science: 219: 614-620 expressed the glycoprotein in Escherichia coli, but the protein was not immunologically active. This is because the antigenic determinants dependent on post translation modifications such as carbohydrate side—chain attachment, i.e. the introduction of the authentic carbohydrates, as it happens in eukaryotic cells is not achieved in Escherichia coli cells. Various other heterologous systems have been used to express the rabies glycoprotein. Two species of yeasts have been tried extensively for the expression of rabies glycoprotein. These are Saccharomyces cerevisiae (Kelper et al., 1993, Sakamoto et al., 1999) and Pichia pastoris (patent no. WO9000191, 1990). Polypeptides of 65-68 kDa, which migrated at the same molecular weight as authentic viral rabies G protein species, were synthesized by Saccharomyces yeast transformants as detected by immunoblotting with rabies specific antiserum. Reference may be made to Sakamoto et al., 1999, “Studies on the structures and antigenic properties of rabies virus glycoprotein analogues produced in yeast cells”, Vaccine: 17: 205-218 wherein two forms of rabies virus glycoprotein produced in the G-cDNA—transfected yeast cells were identified. These were designated as YGI (66 kDa) and YGII (56 kDa). The YG1 reacted with polyclonal anti-G antibodies but did not react with conformational epitope—specific MAb. While the protein expressed in Saccharomyces did not protect animals challenged with rabies virus, the protein expressed in Pichia were claimed to provide protection (WO 90000191 dated 1990). However, the details have not been published, nor has the Pichia system been used and may therefore not be a system of choice. The same G-cDNA was expressed in animal cells, a single form was produced. The results concluded that most G protein molecules were not processed normally in yeast cells. The crucial role of rabies glycoprotein in protection was determined by Foley et al., 2000 “A recombinant rabies virus expressing vesicular stomatitis virus glycoprotein fails to protect against rabies virus infection” in Proc. Natl. Acad. Sci.: 97; 26: 14680-14685, constructed a recombinant RV (rRV) in which rabies virus glycoprotein ectodomain and transmembrane domains were replaced with the corresponding regions of vesicular stomatitis virus (VSV) glycoprotein and immune response was studied and compared to parental rRV strain containing rabies virus glycoprotein. Similar immune responses against the internal viral proteins of both viruses indicated successful infection but all mice who received the rRV vaccine survived the challenge, whereas immunization with the domain substituted rRV-VSV-G did not induce protection. Which confirmed the critical and crucial role of glycoprotein of rabies virus and also demonstrated that immune response and immunoprotection against challenge with live rabies virus are two different phenomenon.
Expression of rabies glycoprotein gene by baculoviral vectors in insect cells gives high yields of protein to the extent of 18% of total cellular protein, 48 h post infection. In one study (Prehaud D H et al, 1989) the gene encoding the G protein of CVS strain was placed under the control of the AcNPV polyhedrin promoter and expressed at high levels by the derived recombinant virus using a Spodoptera fugiperda cell-line. The insect derived protein exhibited slightly faster electrophoretic mobility due to differences in the glycan components. Vaccination by insect derived glycoprotein, followed by challenged to mice gave delayed mortality, i.e., a low level of protection against rabies.
In another study, Rupprecht et al (1993) demonstrated that a glycoprotein (ERA strain) derived from recombinant baculovirus—infected insect cells was efficacious as an oral vaccine in raccoons. In view of relatively high costs of the insect and mammalian cell-systems these are not the systems of choice for G protein expression as a strategy to develop vaccine against rabies.
Some of the recombinant Pox virus rabies glycoprotein based vaccines licensed and marketed in US since 2000 are (i) Purevax for Feline rabies (cats)—monovalent, live canary pox vector (Merial Inc.) and (ii) Raboral V-RG for raccoons-oral live vaccinia vector. Recombinant Poxviruses (USPTO 5266313 dated 1986; USPTO 05348741 dated 1994) have many useful features as vectors for the expression of genes that carry immunizing antigens from other viruses. These are easy to produce and induce cellular and humoral immunity. However, there is concern about the safety of vaccinia virus, if used widely for men and animals.
A Canarypox virus Alvac-RG (vCP65) was used as a vector in non-avian species (Taylor et al., 1995) for expressing the rabies glycoprotein G gene (USPTO 5843456 dated 1998) to address the concern about the spread of vaccinia virus to non-target population, especially immunocompromised individuals. Avipox virus was also developed as a vector for expressing rabies glycoprotein gene. Avipoxvirus (canarypox) recombinants (USPTO 6340462 dated 2002) undergo abortive replication in nonavian cells, yet can achieve expression of extrinsic gene products and their presentation to the immune system. In vitro studies have shown that no replication of the virus can be detected on six human-derived cell lines, nor can the virus be readily adapted to replicate on non-avian cells. Expression of the rabies G was detected on all cell lines analyzed in the absence of productive viral replication. The safety and efficacy of the recombinant (Alvac-RG; vCP65) were tested in several animal species, then it was subjected to a phase 1 clinical trial. This study showed the potential of non-replicating poxviruses as vectors for vaccination in human beings (Fries et al., 1996). Recombinant were immunogenic by the intramuscular and subcutaneous routes. They were also immunogenic when given orally.
The viral vectors including posvirus vectors provide a convenient vehicle for the delivery of the vaccines. This also reduces the costs involved in purifying the proteins from cultures. However, owing to safety considerations, viral vectors meant for humans go through tougher scrutiny for approval by the regulatory bodies.
Cell-cultured based vaccines for rabies are limited to growing inactivated strains of the virus in cell cultures. These include, the relatively expensive Human Diploid Cell Vaccines (HDCV), purified Vero Cell Rabies vaccine (Verorab) and the more economical primary chick embryo cell culture vaccine (PCEV-Rabipur). These vaccines comprise the virus grown in cell cultures. Current biotechnological approaches aim at expressing the coat protein gene of the rabies virus to develop a safe RGP that could be deployed as an active vaccine.
The expression of proteins or safe ‘subunit vaccines’ in cell-cultures is a routine procedure in recent years. Examples of such useful host cell-lines are VERO and HeLa cells, Chinese Hamster Qvary (CHO) cell lines, and W 138, Baby Hamster Kidney (BHK), COS-7 and MDCK cell-lines. Stable expression of rabies virus glycoprotein was shown in Chinese Hamster Ovary cells (Burger et al., 1991). A full length, glycosylated protein of 67 K that co-migrated with the G-protein isolated from virus-infected cells, was obtained.
The rabies glycoprotein expressed in different cell-lines was similar to the native protein in terms of size and immunoreactivity. The recombinant protein thus produced provided an insight into the biology of the protein and gave a clue to the parameters to be taken into consideration while choosing an appropriate expression system. The proteins were of analytical grade and can be purified to a high degree of purity. However, the process based on animal cell lines is very expensive for industrial scale. In order to decide upon an economically viable alternative, the expression system should combine in itself the options for fermentor level scaling up (as in bacteria) and the option for producing a fully glycosylated protein that is closely similar to the native form (as in cell-lines). Expression in plants is a promising alternative in this respect.
Subunit vaccines are important improvements over conventional attenuated or killed vaccines in many aspects including safety and production systems. The expression of foreign proteins in plants has become an attractive alternative in recent years as it has the potential of producing recombinant proteins in large quantities and at low cost. Recent advances in genetic engineering have provided the requisite tools to transform plants to express foreign gene. Agrobacterium tumefaciens have proven to be efficient and highly versatile vehicles for the expression of industrially valuable foreign genes into the plant tissue, as described in Hood et al. (1999) “Plant—based production of xenogenic proteins” Current Opinion in Biotechnology 10: 382-386. Vaccine for human and animal disease prevention comprise the most competitive area for plant based production of xenogenic proteins. Utilization of plants as expression vectors for the production of foreign proteins has captured attention in recent years. Viral proteins (HbsAg, Norwalk virus capsid protein, rabies virus glycoprotein, FMDV structural protein VP1), bacterial toxins (LTB), antibody molecules and several other industrially and therapeutically important proteins have been expressed successfully in plants (Tacket et al., 2000; Kong et al., 2001). In most cases the expressed proteins are fully functional as antigens or in ligand recognition. Importantly, they are effective in eliciting in eliciting specific immune responses. The production of immunogens in plants might be an economic alternative to animal cell based production systems for the development of vaccine. The greatest advantage of using plant systems for the expression of therapeutically important proteins is the absence of human or animal pathogens like HIV, Fowl Fox, Mad Calf, prions etc. in the protein preparations made from plants. Yet another possibility is of utilizing the plant material directly as a feed, thus generating an edible vaccine. Although there is a lot of scope for studies on high—level protein accumulation, post—translational protein modifications and downstream processing, enough progress has been made to arouse interest in plants as robust and commercially viable systems.
The simplistic requirements of plants for sunlight, water and minerals makes them an inexpensive means of correctly processing and expressing proteins that can be quite complex. The traditional subunit vaccines are expensive to produce and not heat stable necessitating a ‘cold-chain’ en rote from manufacturer to vaccination. This limits their availability and use in low-funded health care systems of developing countries. However, proteins expressed in plant parts are often stable for years, as for example, the seed proteins.
A bacterial antigen (E. coli enterotoxin) produced in transgenic plants was shown to effectively immunize mice when the crude protein extracts from the transgenic plant tissue were administered orally, as shown by Curtiss and Cardineau, 1997 in “Oral immunization by transgenic plants” U.S. Pat. No. 5,686,079 and Haq et al., 1995 in “Oral immunization with a recombinant bacterial antigen produced in plants” Science 268: 714-716. The work of Haq and co-workers was followed by human clinical trials to show that humans do develop an immune response to antigen delivered in uncooked food as referred by Tackett et al., 1998 “Immunogenicity in humans of a recombinant bacterial antigen delivered in transgenic potato”. Nature Medicine 4: 607-609.
Reference may be made to McGarvey et al., 1995 “Expression of the rabies virus glycoprotein in transgenic tomatoes” Bio/technology 13: 1484-1487 who engineered tomato plants (Lycopersicon esculentum) to express a gene for the rabies glycoprotein (G-protein) under the control of the 35S′ promoter of cauliflower mosaic virus. The protein was expressed in tomato and showed molecular weight of 62 and 60 kDa in western blot after immunoprecipitation, as compared to 66 kDa observed for G protein from virus grown in BHK cells. The amount of G protein immunoprecipitated was found to be approximately 1-10 ng/mg of soluble protein i.e. at 0.0001% to 0.001% of soluble protein. The low expression level may have been due to using a poorly designed gene. For example, a native G protein coding gene was used along with its native signal peptide The inventors did not examine antigenicity of the G protein and therefore it is not possible to comment on biological activity and utility of the G protein expressed in tomato plant or the gene designed in that study, specially for therapeutic purpose.
Plant derived immune response against diseases such as mink enteritis and rabies were reported by expressing viral epitopes on the surface of plant viruses, followed by infection of susceptible host with the recombinant modified virus, reference can be made from Modelska et al., 1998 “Immunization against rabies with plant—derived antigen” Proc. Natl. Acad. Sci. USA 95: 2481-2485 and Yusibov et al., 2002, “Expression in plants and immunogenicity of plant virus—based experimental rabies vaccine”. The plant virus was purified from the tissue and administered to the test animals. Although this system is very effective, the size of the antigen polypeptide that can be expressed on surface of a vector virus is limited to 37 amino acids. Hence epitope mapping of the antigen is needed for this approach. Such thorough knowledge of the antigen is not generally available, especially with newly discovered diseases where the expression of full-length proteins may be the only option. Also several epitopes need to be identified and joined together since a single epitope may not give acceptable protection against challenge by the pathogenic virus. Furthermore, containment could be considered as a significant problem at the agricultural level, especially when environmentally stable viruses like TMV are used.
One object of the present invention is to provide a recombinant chimeric G-protein based rabies vaccine having SEQ ID No 1 and its parts or variants thereof.
Another object of the present invention is to provide a chimeric gene of SEQ ID No. 2, encoding chimeric G-protein based rabies vaccine having SEQ ID No 1.
Still another object of the present invention is to provide a method for generating large scale chimeric G-protein based rabies vaccine.
Yet another object of the present invention is to provide a method of vaccinating a subject for rabies.
In some embodiments of the present invention, the G-protein based rabies vaccine comprises 1-26 amino acid residues of PR-S signal peptide at N-terminal, followed by 27-32 six hexahistidine tag residues (SEQ ID NO: 55), 33-36 amino acid residues of tetrapeptide of factor Xa proteolytic cleavage site, 37-541 amino acid residues of mature glycoprotein G of ERA strain of rabies virus, 542-547 amino acid residues of six amino acid long for retention of the chimeric G protein in endoplasmic reticulum at C-terminus extreme of the chimeric protein.
Accordingly the present invention provides a recombinant chimeric G-protein having SEQ ID No. 1 and its parts or variants thereof.
In an embodiment of the present invention, the length of the chimeric G-protein comprises 547 amino acids or its parts and variants thereof.
In a further embodiment of the present invention, the chimeric G-protein comprises amino acid residues of PR-S signal peptide at N-terminal at position 1 to 26, followed by six hexahistidine tag (SEQ ID NO: 55) residues at 27-32 position, amino acid residues of tetrapeptide of factor Xa proteolytic cleavage site at 33-36 position, amino acid residues of mature glycoprotein G or ERA strain of rabies virus at 37 to 541 position and, six amino acid long residue at C-terminus extreme of the chimeric protein at position 542 to 547 for retention of the chimeric G protein in endoplasmic reticulum.
In another embodiment, the present invention provides a recombinant chimeric G-protein based rabies vaccine having SEQ ID No. 1 and its parts or variants thereof expressed in plant tissue. In a further embodiment, the chimeric G-protein is of length 547 amino acids in the vaccine. In yet a further embodiment, the chimeric G-protein comprises 1-26 amino acid residues of PR-S signal peptide at N-terminal, followed by 27-32 six hexahistidine tag (SEQ ID NO: 55) residues, 33-36 amino acid residues of tetrapeptide of factor Xa proteolytic cleavage site, 37-541 amino acid residues of mature glycoprotein G of ERA strain of rabies virus, 542-547 amino acid residues of six amino acid long for retention of the chimeric G protein in endoplasmic reticulum at C-terminus extreme of the chimeric protein.
In yet another embodiment of the present invention, the recombinant chimeric G-protein gene encodes a protein having immunoprotective activity against live rabies virus.
In still another embodiment of the present invention, the vaccine provides 100% immunoprotection against rabies virus.
In still another embodiment of the present invention, the vaccine controls rabies in humans, pets and wild life.
Further, the present invention also provides a chimeric gene of SEQ ID No. 2, encoding chimeric G-protein based rabies vaccine.
In an embodiment of the present invention, the chimeric gene of SEQ ID No. 2 comprises 1.67 kb.
Present invention also provides a set of 52 primers of SEQ ID No. 3 to 54 is useful for the synthesis of chimeric gene of SEQ ID 2.
In an embodiment of the present invention, all oligonucleotides are synthesized chemically and fused enzymatically to obtain desired double stranded DNA.
Further, the present invention also provides an economical method for generating large scale chimeric G-protein based rabies vaccine by expressing novel chimeric gene SEQ ID No. 2, for control rabies, wherein the said process comprising the steps of:
In an embodiment of the present invention, the strain of rabies virus is selected from the group consisting of ERA strain, CVS strain etc.
In another embodiment of the present invention, the PR-S signal peptide is replaced with another comparable signal peptide, e.g., PR-1 or any other signal peptide, depending on the plant expression system for transport of the protein to endoplasmic reticulum.
In yet another embodiment of the present invention, the protein is of length 547 amino acids.
In still another embodiment of the present invention, the hexa histidine residue can be replaced with any other peptide selected from the group consisting of cellulose binding domain, streptavidin agarose binding domain, glutathione-S-transferase fusion etc. having affinity to a matrix for purification.
In still another embodiment of the present invention, four amino acid residues for cleavage can be replaced with any endo-protease selected from the group consisting of bovine enterokinase, thrombin, factor Xa etc.
In still another embodiment of the present invention, a polypeptide of four amino acid residues is introduced at the downstream of histidine residue to allow enzymatic cleavage of the histidine residues after purification of chimeric G-protein on metal chelate affinity column.
In still another embodiment of the present invention, other amino acid residues of similar applications, such as Asp Asp Asp Asp Lys ↓ (SEQ ID NO: 56), Leu Val Pro Arg ↓ Gly Ser (SEQ ID NO: 57), Ile Glu Glu Arg ↓ (SEQ ID NO: 58), may be used to replace cleavage site amino acid residues of protein vaccine of SEQ ID No.1.
In still another embodiment of the present invention, a hexapeptide may be introduced at the C-terminus extreme of the chimeric protein for its retention in lumen of endoplasmic reticulum. In further embodiments, other retention signal may be equally good for targeting and accumulation of the chimeric G-protein in vacuole, golgibody, microtubules and microsomes or any other organelle.
In still another embodiment of the present invention, recombinant chimeric G-protein gene encodes a protein having immunoprotective activity against live rabies virus.
In still another embodiment of the invention, the vaccine controls rabies in humans, pets and wild life.
In still another embodiment of the present invention, plants for expressing recombinant G-protein gene is selected from group consisting of tobacco, corn, legumes like chickpea, pigeonpea, groundnut, soybean, vegetables like tomato, potato, musk melon, water melon, spinach, cauliflower, cabbage, chili, capsicum, carrot, and/or other plants and lower plant selected from any algae.
In still another embodiment of the present invention, the novel chimeric G protein may be partially purified from total soluble protein from the leaf of the transgenic tobacco plants.
In still another embodiment of the present invention, the protein is 2-4 fold more immunogenic as compared to inactivated virus. For example, as shown in example 2 in table 3 and
In still another embodiment of the present invention, the vaccine expression is about 0.2% of total soluble protein in plant.
In yet another embodiment, the present invention also provides a method of vaccinating a subject for rabies, wherein the said method comprising administering of pharmaceutical effective amount of recombinant chimeric G-protein gene vaccine. In a further embodiment the vaccine is produced and expressed in transgenic plants, optionally along with pharmaceutical acceptable additive(s) to the subject.
In an embodiment of the present invention, the subject is human, pets and wild life. In yet another embodiment of the present invention, the administration of the plant expressed vaccine is through routes commonly known in the art, including, but not limited to, oral, intrapertional etc.
In still another embodiment of the present invention, the recombinant chimeric G-protein gene vaccine is administered at concentration ranging from ng to μg quantities, depending upon purity of the protein. In a preferred embodiment μg amounts may be administered. See examples 2 and 3.
In still another embodiment of the present invention, the vaccine is safe for the administration.
In still another embodiment of the present invention, the vaccine is expected to be stable for a long time, when expressed in different plant parts and tissues, specially seeds, tubers, roots, leaves etc. For example, proteins stored in seeds are known to be stable for several years.
In still another embodiment of the present invention, the antibody titre of the protein is in the range of 0.365±0.20 after IInd booster dose and 0.833±0.20 after IIrd booster dose. See example 2.
In still another embodiment of the present invention, the said vaccine provides 100% immunoprotection.
Present invention relates to a chimeric G-protein of ERA strain of rabies virus and a synthetic gene to encode a strategically designed recombinant protein. Chimeric G-protein, 547 amino acid residues long. The chimeric G-protein may be strategically designed by replacing the native signal polypeptide (from position 1 to 19) of ERA strains of rabies (EMBL: RHRBGP) by that of PR-S of tobacco (from position 1 to 26). ERA (Evelyn Rokitniki Abelseth) is a well known rabies vaccine strain which is a derivative of wild type rabies virus strain SAD (Street Albama Duffering). SAD strain belongs to serotype I under which most of the classical rabies virus strains are grouped. It has been determined that ERA strain of rabies functions as the most efficient vaccine at mouse intracerebral lethal dose 50% (MCLD50). It produces the titer to the magnitude of 105-107 MICLD50/0.03 ml in various tissue culture cell line. Hence the G protein gene of ERA strain was selected for the study. Hence, to develop a plant based vaccine. ERA is one of the well studies strains of rabies virus. Genes corresponding to other strains like CVS etc. will also be appropriate for developing plant based vaccine technology.
A total of six histidine residues (SEQ ID NO: 55) and a tetrapeptide representing the cleavage site of factor Xa were designed between signal peptide and mature G-protein. A hexapeptide for anchoring the chimeric protein in endoplasmic reticulum was included at the C-terminus of the chimeric protein. In this way, chimeric G-protein comprises amino acid residues 1-26 of PR-S signal peptide of tobacco, 27-32 of six histidine residues (SEQ ID NO: 55), 33-36 of cleavage site for factor Xa, 37-541 of mature G-protein of ERA strain of rabies virus and 542-547, a hexapeptide for localization in endoplasmic reticulum.
A 1.67 kb nucleotide sequence was theoretically designed to code for the above-mentioned chimeric G-protein. The gene encoding such a chimeric protein, was designed for high-level expression in plants. The plant preferred codons for each amino acid were distributed evenly to facilitate efficient translation. A translation initiation context appropriate to achieve high level of gene expression in plants (TAAACAATGAAC (SEQ ID NO: 60)) was included at 5′ extreme and two translation stop codons were introduced at the end of the reading frame of the chimeric protein. A total of 34 unique restriction sites were introduced uniformly throughout the length of the gene at intervals of 40-80 bp. The HindIII and XbaI restriction sites were created at the upstream and BamHI and SacI at the downstream of the gene to facilitate its cloning. The gene was divided into 50 overlapping oligonucleotides (48 to 50 nucleotides long) with 10 to 16 base long gaps in between. Each oligonucleotide had 13-18 nucleotide long overlap with the immediately adjacent oligonucleotides on the complementary strand. The complementary overlaps were designed to keep Tm value between 48-50° C. The oligonucleotides were synthesized on a DNA synthesizer (Gene Assembler Special, Pharmacia, Sweden) at 200 nmole scale and purified on denaturing urea-PAGE. All 50 oligonucleotides were assembled into 1.67 kb double-stranded DNA, herein said chimeric G-protein gene following the ligation-free gene synthesis method of Singh et al. (1996) and as shown in
A cassette was constructed also for the expression of the modified gene in E. coli under the control of T7lac promoter. A suitable set of primers was separately designed to amplify the DNA coding for the mature G-protein (1545 bp) and create NdeI and BamHI restriction sites. The plasmid pSA17 was amplified; DNA was digested with the restriction enzymes NdeI and BamHI and cloned in expression vector pET-19b (Novagen, Madison Wis.). The plasmid was named as pSA33 (
As used here, reference to the “chimeric G-protein” means, the chimeric fusion of PR-S signal peptide, histidine tag, cleavage site for factor Xa, mature G-protein tagged with endoplasmic reticulum retention signal, which shows a high immunoprotection activity in mice against live virus challenge.
A person skilled in this art can use any other suitable plant, animal, virus, insect, microbial signal peptide to express the chimeric G-protein. Such protein can be expressed in respective system. The histidine tag and the cleavage site for factor Xa are essentially included to facilitate purification of the chimeric G protein from plant leaf extract. These are not essential to immunoprotection and can be substituted by other alternative strategies to facilitate protein purification. After purification, such molecule can be used as subunit vaccine for the management of rabies in pets, wild life and humans. Such subunit vaccine can be given as such or in combination DNA vaccine and/or inactivated virus. Such chimeric protein can also be used as oral vaccine.
The gene and chimeric G-protein are useful according to the subject invention and include not only 547 amino acid long protein expressed in plants but also parts of the sequence, variants and mutants, which retain the high immunoprotective activity of the mature G-protein specifically exemplified herein. As used here, the terms “variants” or “variations” of genes refer to nucleotide sequences, which encode the same chimeric protein or which encode chimeric protein having lower or equivalent biological activity in terms of immune response and immunoprotection in mice and other animals. In minor variant, addition, deletion or acceptable substitution of a few amino acids may give an equally effective rabies vaccine. As used here, the term “equivalent biological activity” refers to chimeric protein having similar or essentially the same immunological and immunoprotective activity with applications in diagnostics, therapeutics and prophylaxis.
With reference to SEQ ID No 1, several of its variants are expected to be functionally active because they may lead to change from one amino acid to another closely related amino acid. Such a substitution often does not lead to any effect on activity of the protein. Similarly, deletion or addition of a few amino acids may not lead to a significant effect on function of the protein. Examples of amino acid substitutions that may not often effect function of a protein are given in
Table No. 2. Matrix presenting the acceptability of amino acid substitution in proteins. Single letter codes are given for amino acids along rows and columns.
A positive numeral in the matrix indicates that a given substitution may be acceptable. A negative numeral indicates non acceptability. For example, Cysteine (C) can be substituted by self (score=12) only but not by any other amino acid (all scores being negative, e.g. the score for cysteine to glycine G is −3 and therefore, not acceptable. On the other hand, glycine (G) can be substituted by self (score 5) but also by serine—S (score 1), alanine—A (score 1) and aspartic acid—D (score 1). the extent of positivity suggests the confidence level in the substitution being successful (Dayhoff, M. O., Schwartz, R. M., Orcutt, B. C. 1978. “A model of evolutionary change in proteins.” In “Atlas of Protein Sequence and Structure” 5(3) M. O. Dayhoff (ed.), 345-352. National Biomedical Research Foundation, Washington.
With reference to SEQ ID No2 and the corresponding primers in SEQ ID No 3 to 54, several variants of the DNA sequence will not cause a change in function of the nucleic acid. For example, Table 3 gives a list of codons that code for the same amino acid. Such codons can be substituted for one another in a gene, without any effect on the protein sequence coded by the gene.
It is well within the skill of a person trained in the art to create alternative DNA sequences encoding the same or essentially the same, protein. These variant DNA sequences are within the scope of the subject invention. As used herein, reference to “essentially the same” sequence refers to sequences, which have amino acid substitutions, deletions, additions or insertions, which do not materially affect immunoprotective activity. Fragments retaining immunoprotective activity are also included in this definition.
A novel chimeric G-protein of the subject invention has been specifically exemplified herein. It should be readily apparent that the subject invention comprises variants or equivalent protein (and nucleotide sequences encoding equivalent protein) having the same or similar immunoprotective activity of the exemplified chimeric protein expressed in plant or plant cells. Equivalent protein will have amino acid homology with the exemplified protein. This amino acid homology will typically be greater than 75%, preferably be greater than 90% and most preferably be greater than 95%. The amino acid homology will be highest in critical regions (antigenic epitopes) of the chimeric protein, which account for the immunoprotective activity or are involved in the determination of three-dimensional configuration, which ultimately is responsible for the biological activity. In this regard, certain amino acid substitutions are acceptable and can be expected if these substitutions are in regions, which are not critical in biological activity or are conservative amino acid substitutions, which do not affect the three-dimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same class fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the rabies G protein. Table 4 provides a listing of examples of amino acids belonging to such classes.
In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the biological activity of the chimeric protein. It is well within the skill of a person trained in the art of protein engineering to substitute at many places, the amino acids of the chimeric G-protein with alanine since that does not often change conformation of proteins. Such substitutions are also well within the scope of the invention.
A gene encoding the chimeric G-protein of the subject invention can be introduced into a wide variety of plant virus vectors. Expression of the chimeric gene results in intracellular production and maintenance of the immunoprotective protein. Such virus can be used as a system for production and purification of chimeric G-protein. Suitable viral hosts are Badnavirus, Caulimovirus, SbCMV-like virus, CsVMV-like viruses, RTBV-like viruses Petunia vein clearing-like viruses, Mastrevirus, Curtovirus, Begomovirus, Alfamovirus or Ilarivirus (Koprowski et al., 2000:U.S. Pat. No. 6,042,832 and 2002: U.S. Pat. No. 6,448,070) Cumovirus Closterovirus, Cmivirus, Comovirus, Fabavirus, Nepovirus, Potyvirus, Rymovirus, Bymovirus, Sequivirus, Waikavirus, Carmovirus, Dianthovirus, Machlomovirus, Necrovirus, Tombusvirus, Capillovirus, Carlavirus, Enamovirus, Furovirus, Hordeivirus, Idaeovirus, Luteovirus, Marafivirus, Potexvirus, Sobemovirus, Tenuivirus, Tobamovirus, Tobravirus, Trichovirus, Tymovirus, Umbravirus, Cytorhabdovirus, Nucleorhabdovirus, Tospovirus, Alphacryptovirus, Betacryptovirus, Fijivirus, Phytoreovirus, Oryzavirs to developed production system for chimeric G protein.
A plant transformation vector was constructed for the development of transgenic plants. A plasmid pPK201 (having CaMV35S promoter with duplicated enhancer) was digested with HindIII to excise out CaMV35S promoter with the duplicated enhancer. The plasmid pSA17 (
The following examples are given by way of illustrations and therefore should not be construed to limit the scope of the present invention.
Expression of the chimeric gene in Escherichia coli
A gene coding for the chimeric G protein of 547 amino acid residues length, herein said chimeric G protein was strategically designed (SEQ ID NO. 1). The native polypeptide domain (from position 1 to 19) of G protein was replaced with PR-S signal peptide of tobacco (from position 1 to 26). Following that, a hexapeptide of histidine amino acid (SEQ ID NO: 55) a residues and tetrapeptide comprising the amino acid residues IEGR (SEQ ID NO: 61) were included at the N-terminus of the gene in that order. Following the G protein coding sequence, a hexapeptide coding for SEKDEL (SEQ ID NO: 59) for retention of the chimeric G protein in endoplasmic reticulum was included at the C-terminus extreme of the chimeric gene (SEQ ID NO. 1). A 1.67 kb nucleotide sequence was theoretically designed (SEQ ID NO. 2) to code for the above-mentioned chimeric G protein. Several 6-base cutter restriction enzyme sites were created in the designed gene. HindIII and SalI restriction sites were created at 5′-end and BamHI ,SacI at the 3′-end of the designed gene. The gene was divided into 50 overlapping oligonucleotides (40 to 58 nucleotides long). Each oligonucleotide had 13-18 nucleotide long overlap with the immediately adjacent oligonucleotides on the complementary strand (Tm between 48-50° C.). Oligonucleotides were synthesized on a DNA synthesizer (Gene Assembler Special, Pharmacia, Sweden) at 200 nmole scale and purified on denaturing urea-PAGE. All 50 oligonucleotides were assembled into the double-stranded DNA, herein said chimeric G gene following the ligation-free gene synthesis method of Singh et al. (1996). The DNA was digested with HindIII and SacI restriction enzyme and cloned in pBluescriptII SK(+) (Stratagene). The plasmid was named as pSA17 (shown in
A cassette was constructed for the expression of the chimeric toxin in E.coli under control of T7 lac promoter. For this purpose, the plasmid pSA17 was amplified by two primers to create NdeI and BamHI sites at the upstream and downstream of the amplicon and cloned in expression vector pET-19b (Novagen)at the same sites. The plasmid was named as PSA33 (shown in
The expression was carried out at 37° C. for different periods. E.coli cells were lysed in SDS PAGE loading buffer and resolved on 10% denaturing gel. The result of SDS PAGE and western show that E.coli expressed the recombinant protein of expected size 60.65 kDa which is comparable to that of molecular weight determined by a protein molecular weight analysis program of non glycosylated modified protein which hereby confirmed that the synthetic gene, herein said the gene for chimeric G-protein was developed and expressed correctly. The amino acid sequence of the chimeric G-protein designed for expression in E. coli and plants is novel and differs substantially (
Immunization of BalbC mice with plant derived vaccine.
In order to establish efficacy of the novel chimeric G-protein in plants, a plant transformation vector was constructed for the development of transgenic plants.
A plasmid pPK201 (having CaMV35S promoter with duplicated enhancer) was digested with HindIII to excise out CaMV35S promoter with the duplicated enhancer. The plasmid pSA17, containing the chimeric G-protein gene cloned at HindIII-SacI in Bluescript II SK+(Stratagene, La Jolla, Calif.) was prepared as described in Example 1. The chimeric gene consists of 26 amino acid residues of PRS signal peptide of tobacco followed by six histidine residues (SEQ ID NO: 55), four amino acid residues for factor Xa cleavage site, 505 amino acid residues of matured ERA rabies Glycoprotein and six amino acid residues for endoplasmic reticulum retention signal at C′ terminus of the chimeric G-protein gene in that order (SEQ ID NO. 1). A 1.67 Kb, sequence was theoretically designed using plant preferred codons to achieve high level expression of the above mentioned chimeric G-protein. A novel translational initiation context (TAAACAATGAAC (SEQ ID NO: 60)) was included at 5′ extreme and two stop codons TGA,TGA were introduced at the end of the reading frame of the chimeric G protein. The gene was divided into 50 overlapping oligonucleotides and assembled into 1.67 kb double stranded DNA, following ligation free gene synthesis method of Singh et al. (1996). HindIII and XbaI restriction sites were created at the upstream and BamHI and SacI at the downstream of the gene to facilitate cloning. The chimeric G protein was cloned at HindIII-SacI restriction sites of Bluescript II SK+, sequenced and named as pSA17. The clone pSA17 was digested by HindIII and SacI to excise the 1.67 kb fragment having a complete chimeric G-protein gene (SEQ ID NO. 2). A triple ligation was carried out for the cloning of the two fragments, that is, the HindIII fragment of clone pPK201 (having CaMV35S promoter with duplicate enhancer) and HindIII-SacI fragment having a chimeric gene. The resultant clone was name as pSA17. The above two fragments were triple ligated to pBI101 at HindIII-SacI, replacing the (-glucuronidase cassette. The plant expression cassette had a CaMV35S promoter with the duplicated enhancer at the upstream of the chimeric gene and 260 by fragment containing the polyadenylation signal from the nopaline synthase gene (Nos-ter) transcription terminator at the downstream of the chimeric gene. The expression cassette had the NPTII gene for kanamycin resistance under the control of Nos promoter. The correct orientation of CaMV35S with duplicate enhancer was confirmed by restriction analysis. The expression cassette (the synthetic chimeric-G protein gene with E-35S promoter) cloned in Ti binary vector HindIII-SacI of pBI101 with correct orientation of E-35S to chimeric-G protein was named as pSA5 (shown in
Immunoprotection Analysis of Immunized Mice
The efficacy of plant derived G protein was studied in immunoprotection by injecting the G enriched plant membrane fraction in mice and challenging it with live rabies virus. Mice were intraperitoneally injected by the G protein enriched membrane fraction of transgenic tobacco leaves and a commercially available killed rabies virus vaccine (as positive control Rabipur by Aventis Pharma Ltd.) and phosphate buffer saline (as negative control). As described in Example 2, boosters were given on 7, 14 and 28th day. At each stage a total of 25 μg of protein was injected. After the third booster, mice were intracerebrally challenged with 10×LD50 of live rabies virus and observed for the appearance of symptoms. After thirteen days of the challenge the result was noted down and are shown in the table 6 and
The results obtained after the challenge experiment clearly show that the G protein located in plant cell membrane elicits a high level of immune response and is highly immunoprotective.
Thus, the present invention provides, inter alia, a very safe vaccine as compared to inactivated virus for rabies management in human, pets and wild life. It does not require handling of a live pathogenic virus and has no risk of vaccine reverting to infective virus particles.
G-protein of rabies virus was designed theoretically to express at high level in novel hosts, including transgenic plants and plant cell lines, microbes like E. coli and yeast and animals and animal cell lines recombinant animal and plant viruses. Thus, without handling a pathogenic virus, as this an immunogenic antigen can be produced in new host cells. Chimeric G-protein was designed specifically to express at high level in plants cells. With a suitable signal peptide the G protein was made to accumulate in membranes of plant cells. Comparable signals can be used to accumulate in specific compartments. Enrichment of target protein in specific organelle like membrane allows the development of less expensive and convenient approaches for large scale preparation of the target protein in animal cell lines and yeast.
Hexa-histidine tag (SEQ ID NO: 55) was designed at N-terminus extreme to further facilitate purification of chimeric protein on immobilized metal affinity column. Since monospecific antibody against histidine tag is commercially available, the histidine tag can also be used for the identification the tagged chimeric protein during various stages of purification. Factor Xa cleavage site was included at the downstream of histidine tag to chop it off after purification, in case this is not desired in therapeutic applications. SEKDEL (SEQ ID NO: 59) retention signal was designed at the C-terminus extreme which enhanced the accumulation of chimeric G-protein in endoplasmic reticulum of cell. Compartmentalization of heterologous proteins in endoplasmic reticulum can enhance their stability and overall accumulation. This makes the purification procedure simpler and efficient. Further, membrane localization prevents the target protein from proteolytic enzymes present in cytosol. Also membrane localization can lead to functionally important modifications in the target protein.
The immune response elicited with the chimeric G-protein was several folds higher as compared to the commercially available inactivated rabies virus vaccine. The immunised animals exhibited immunoprotection against live virus challenge. High immune response of G protein localized in tobacco leaf membranes reflects novel designing of the protein since it allows the G protein to take an immunologically active conformation.
Employment of chimeric G-protein purified from plants for rabies management will be very safe as the vaccine preparations will be free of animal pathogens, like HIV, Fowlpox, Madcow, prions etc.
Chimeric G-protein can be used as an oral vaccine, if it is expressed in edible parts of the plants like leaves, grains etc. Rabies virus has earlier been shown to be immuno stimulatory/immunoprotective when given orally to experimental animals. Hence expressing functionally active rabies capsid G-protein in plant leaves opens new opportunities of developing an inexpensive vaccine that is expected to be safe and does not require cold chain, if transported in form of seeds, grain, plant storage tissue etc.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1502/DEL/2004 | Aug 2004 | IN | national |
The present application claims benefit under 35 U.S.C. §119 to U.S. provisional application No. 60/683,054 filed on May 20, 2005 and also claims benefit under 35 U.S.C. §119 to Indian Patent Application No. 1502/DEL/2004 filed on Aug. 13, 2004. The disclosures of which are hereby incorporated by reference in their entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 6673601 | Jacob et al. | Jan 2004 | B1 |
| 7235245 | Jacob et al. | Jun 2007 | B2 |
| Number | Date | Country |
|---|---|---|
| WO 9509249 | Apr 1995 | WO |
| WO 9640229 | Dec 1996 | WO |
| WO 0063242 | Oct 2000 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20090004215 A1 | Jan 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60683054 | May 2005 | US |
