"NOVEL CATIONIC AMPHIPHILES WITH QUINIC ACID HEAD-GROUPS FOR

DNA VACCINATION"

Field of the Invention The present invention relates to a novel series of glycomimicking cationic amphiphiles containing quinic acid head-groups and a process for the preparation thereof. The invention provides novel compositions containing the said amphiphiles with remarkable gene transfer properties. The area of medical science that is likely to benefit most from the present invention is DNA Vaccination.

Background of the invention

The present invention relates to the field of immunology in general and DNA vaccination in particular. DNA vaccination, the systemic administration of antigen encoded DNA, is an emerging approach for the treatment of complex disorders including cancer, infectious diseases and allergies (Gurunathan, S. et al. Annu. Rev. Immunol. 2000; 18: 1297- 1306, Liu, M. A. J. Int. Med. 2003;25i:402-410). Since the pioneering report by Wolff et al. in 1991 on the use of naked DNA in transfecting muscle cells in vivo (Wolff, J. A. et al. Science 1991 ;247: 1465-1468), several studies have shown that intramuscular injection of a specific antigen encoded naked plasmid DNA can trigger humoral and cell mediated protective immunity against the antigen (Whalen, R. G. et al. Clin. Immunol Immunopathol. 1995;75:1-12, Ulmer, J. B. et al. Vaccine 1994;12:1541-1544, Liu, M. A. et al. Vaccine 1997;15:909-912, Gurunathan, S. et al. Annu. Rev. Immunol. 2000;18:1297-1306, Liu, M. A. J. Int. Med. 2003 ;253:402-4104-6). However, recent clinical trials have revealed that the immune response induced by a topical injection of naked DNA is insufficient (Roy, M. J. et al. Vaccine, 2000; 19:764-778, Rosenberg, S. A. et al. Hum. Gene Ther. 2003; 14:709-714). Studies have shown that transfection and subsequent activation of antigen presenting cells (APC) such as dendritic cells (DC) and macrophages are key events in the development of immunity following genetic immunization (Akbari, O. et al. J. Exp. Med. 1999; 189: 169- 178, Chattergon, M. A. et al. J. Immunol. 1998; 160: 5707-5718). Mountain and co-workers have subsequently demonstrated that immunization of mice with monocyte-derived dendritic cells transfected with a complex of cationic peptide and a gene encoding tumor associated

antigens protected the mice from a lethal challenge with melanoma cells (Irvine, A. S. et al. Nat. Biotechnol. 2000; 18:1273-1278). Antigen presenting cells such as dendritic cells and macrophages process the antigenic protein through their proteasome complexes into small peptide fragments. These peptide fragments are then presented to the immune cells (CD8+ and CD4+ T cells) via MHC class I and MHC class II molecules resulting in the induction of cytotoxic T lymphocyte (CTL) and humoral responses (Steinman, R. M. Annu. Rev. Immunol. 1991; 9: 271-296, Banchereau, R. M. and Steinman, R. M. Nature 1998;392:245- 252).

Attempts have been made to increase the potency of immune response through direct transfection of APC by delivering the antigen encoding DNA via cationic liposomes (Gregoriadis, G. et al. FEBS Lett. 1997; 402:107-110, Klavinskis, L. S. et al. Vaccine 1997;7J: 818-820, Perrie, Y. et al. Vaccine 2001; 19:3301-3310, Hattori, y. et al. Biochem. Biophys. Res. Comm. 2004; 317:992-999). Cationic liposomes owing to their non-toxic and bio-compatible nature offer great advantage over other means of DNA delivery. A greater degree of control can be exercised over the lipids'structure on a molecular level and the products can be highly purified. Use of cationic liposomes does not require any special expertise in handling and preparation techniques. Cationic liposomes can be covalently grafted with receptor specific ligands for accomplishing targeted gene delivery. Such multitude of distinguished favorable clinical features are increasingly making cationic liposomes as the non-viral transfection vectors of choice for delivering genes into body cells.

The following references are examples of cationic liposomes and their formulations that are known in the art to be useful for enhancing the intracellular delivery of genetic materials.

Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 7413-7417 reported the first use of a hjghly efficient cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl ammonium chloride(DOTMA) as the non- viral DNA transfer reagent. U.S. Pat-Nos. 4,897,355 and 4,946,787 (1990) reported the synthesis and use of N- [.omega..(.omega.-l)-dialkyloxy]-and N-[..omega..(.omega.-l)-dialkenyloxy]-alk-l-yl-

N,N,N-tetrasubstituted ammonium amphiphiles and their pharmaceutical formulations as efficient transfection vectors.

Leventis, R.and Silvius, J.R Biochim. Biophys. Acta. 1990; 1023: 124-132 reported the interactions of mammalian cells with lipid dispersions containing novel metabolizable cationic amphiphiles.

U.S. Pat. No. 5,264,618 (1993) reported the synthesis and use of additional series of highly efficient cationic lipids for intracellular delivery of biologically active molecules.

Feigner et al. J. Biol. Chem. 1994; 269: 2550-2561 reported enhanced gene delivery and mechanistic studies with a novel series of cationic lipid formulations. U.S. Pat. No. 5,283,185 (1994) reported the synthesis and use of 3P[N-(N ¹ _^ N ¹ - dimethylaminoethane)carbamoyl]cholesterol, termed as "DC-Chol"for delivery of a plasmid carrying a gene for chloramphenicol acetyl transferase into cultured mammalian cells.

U.S.Pat.No.5,283,185(1994) reported the use of N-[2-[[2,5-bis[(3-aminopropyl)amino]-l-

Oxopentyl] aminoethyl] -N,N-dimethyl-2,3 -bis-(9-octadecenyloxy)- 1 -Propanaminium tetra(trifluoroacetate), one of the most widely used cationic lipids in gene delivery. The pharmaceutical formulation containing this cationic lipid is sold commercially under the trade name "Lipofectamine".

Solodin et al. Biochemistry 1995; 34: 13537-13544 reported a novel series of amphilic imidazolinium compounds for in vitro and in vivo gene delivery. Wheeler et al. Proc. Natl. Acad.Sci. U.S.A. 1996; 93: 11454-11459 reported a novel cationic lipid that greatly enhances plasmid DNA delivery and expression in mouse lung.

U.S.Pat No. 5,527,928 (1996) reported the synthesis and the use of N,N,N,N-tetramethyl-

N,N-bis (hydroxy ethyl)-2,3-di(oleolyoxy)-l,4-butanediammonim iodide i.e pharmaceutical formulation as transfection vector. U.S.Pat.No. 5.698,721 (1997) reported the synthesis and use of alkyl O-phosphate esters of diacylphosphate compounds such as phosphatidylcholine or posphatidylethanolamine for intracellular delivery of macromolecules.

U.S.Pat. Nos. 5,661,018; 5,686,620and 5,688,958 (1997) disclosed a novel class of cationic phospholipids containing phosphotriester derivatives of phosphoglycerides and sphingolipids efficient in the lipofection of nucleic acids.

U.S. Pat.No. 5,614,503 (1997) reported the synthesis and use of an amphiphatic transporter

for delivery of nucleic acid into cells, comprising an essentially nontoxic, biodegradable cationic compound having a cationic polyamine head group capable of binding a nucleic acid and a cholesterol lipid tail capable of associating with a cellular membrane. U.S.Pat.No. 5,705,693 (1998) disclosed the method of preparation and use of new cationic lipids and intermediates in their synthesis that are useful for transfecting nucleic acids or peptides into prokaryotic or eukaryotic cells. These lipids comprise one or two substituted arginine, lysine or ornithine residues, or derivatives thereof, linked to a lipophilic moiety. U.S.Pat. No.5, 719,131 (1998) has reported the synthesis of a series of novel cationic amphiphiles that facilitate transport of genes into cells. The amphiphiles contain lipophilic groups derived from steroids, from mono or dialkylamines, alkylamines or polyalkylamines. US. Patent No. 5,527,928, (1996) reported on the synthesis and transfection biology of a novel cationic lipid namely, N, N, N', N'-tetramethyl-N, N'-bis (2-hydroxyethyl)-2,3- di(oleoyloxy)- 1 ,4-butaneammonium iodide. US Patent 6,541,649 (2003) disclosed novel cationic amphiphiles containing N- hydroxyalkyl head-group and its formulation for intracellular delivery of genetic materials.

US Patent 6, 503, 945 (2003) disclosed novel cationic amphiphiles containing N- hydroxyalkyl head-group and its formulation for intracellular delivery of genetic materials.

US Patent 7,157,439 (2007) disclosed methods and compositions for improving and/or controlling wound healing by applying a wound care device comprising HoxD3 and HoxA3 and/or HoxB3 novel cationic amphiphiles containing N-hydroxyalkyl head-group and its formulation for intracellular delivery of genetic materials.

Other references may be made to:

Behr, J.P. et al. Proc. Natl. Acad. Sci. USA, 1989; 86: 124-132.

Levantis, R. et al. Biochim. Biophys. Res. Commun. 1991; 179: 280-285. Akao, T. et al. Biochem. MoI. Biol. Int. 1994; 34: 915-920.

Feigner, J. H. et al. Proc. Natl. Acad. Sci. USA. 1996; 93: 11454-11459.

Bennett, M. J. et al. J. Med. Chem. 1997; 40: 4069-4078.

Blessing, T. et al. J. Am. Chem. Soc. 1998; 120: 8519-8520.

Wang, J. et al. J. Med. Chem. 1998; 41: 2207- 2215. Lim, Y. et al. J. Am. Chem. Soc. 1999; 121: 5633-5639.

Lim, Y. et al. J. J. Am. Chem. Soc. 2000; 122: 6524-6525.

Zhu, J. et al. J. Am. Chem. Soc. 2000; 122: 3252-3253.

Lynn, D. M.; Langer, R. J. Am. Chem. Soc. 2000; 122: 10761-10768.

Ferrari, M. E.; Rusalov, D.; Enas, J.; Wheeler, C. J.; Nuc. Acid. Res. 2001, 29, 1539-1548. Banerjee, R.; Das, P. K.; Srilakshmi, G.V.; Chaudhuri, A.; Rao, N. M. J. Med.

Chem.1999, 42, 4292-4299.

Banerjee, R.; Mahidhar, Y. V.; Chaudhuri, A.; Gopal, V.; Rao, N. M.

J. Med. Chem. 2001, 44, 4176-4185.

Singh, S. R.; Mukherjee, K.; Banerjee, R.; Chaudhuri, A.; Hait, S. K.; Moulik, S. P.; Ramadas, Y.; Vijayalakshmi, A.; Rao, N. M. Chem. Eur. J. (in press).

Floch, V.; Bolc'h, G. Le.; Gable-Guillaume, C; Bris, N. Le.; Yaouanc, J-J.; Abbayes, H.

Des.; Fe'rec, C; Cle'ment, J-C. Eur. J. Med. Chem., 1998, 33, 923-934.

Solodin, L; Brown, C; Bruno, M.; Chow, C; Jang, E-H.; Debs, R.; Heath, T.

Biochemistry, 1995, 34, 13537-13544. Karmali, P. P.; Majeti, B. K.; Bojja S.; and Chaudhuri, A. Bioconjugate Chemistry 2006, 17,

159-171.

Majeti, B. K.; Karmali, P. P.; Reddy, B. S.; Chaudhuri, A. J. Med. Chem. 2005, 48, 3784- 3795.

Sen, J. and Chaudhuri, A. J. Med. Chem. 2005, 48, 812-820. Sen, J. and Chaudhuri, A. Bioconjugate Chemistry 2005, 16, 903-912.

Majeti, B. K.; Karmali, P. P.; Chaudhuri, A. Bioconjugate Chemistry 2005, 16, 676-684. Mukherjee, K. M., Sen, J. and Chaudhuri, A. FEBS Letters 2005, 579, 1291-1300.

Mahidhar, Y. V., Rajesh, M.; Madhavendra, S. S.; Chaudhuri, A. J. Med. Chem. 2004, 47, 5721-5728. Mahidhar, Y. V., Rajesh, M.; Chaudhuri, A. J. Med. Chem. 2004, 47, 3938-3948. Valluripalli, V. K. and Chaudhuri, A. FEBS Letters 2004, 571, 205-211.

Singh, R. S.; Goncalves, C; Sandrin, P.; Pichon, C; Midoux, P.; Chaudhuri, A. Chemistry and Biology 2004, 11, 713-723.

Majeti, B. K.; Singh, R. S.; Yadav, S. K.; Reddy, S. B.; Ramkrishna, S.; Diwan, P. V.; Madhavendra, S. S.; Chaudhuri, A. Chemistry and Biology 2004, 11, 427-437.

Karmali, P. P.; Valluripalli, V. K.; Chaudhuri, A. J. Med. Chem. 2004, 47, 2123-2132. Singh, R. S. and Chaudhuri, A. FEBS Letters 2004, 556, 86-90. Kumar, V. V.; Pichon, C; Refregiers, M.; Guerin, B.; Midoux, P.; Chaudhuri, A. Gene Therapy 2003, 10, 1206-1215.

Valluripalli, V. K.; Singh, R. S; Chaudhuri, A. Curr. Med. Chem. 2003, 10, 1297-1306.

Very recently, in US Patent 7,166,298 (2007), Jessee J. A. and Hearl W. G. disclosed a method for genetic immunization using compositions comprising cationic lipids and antigen encoded DNA. In the field of genetic immunization, liposome-entrapped DNA has been shown to be preferentially uptaken by antigen presenting cells (APC) in the draining lymph nodes (Perrie, Y. et al. Vaccine 2001 ; 19:3301-3310) in addition to protecting the DNA from nuclease degradation.

Objectives of the invention

The main object of the present invention is to provide novel glycomimicking cationic amphiphiles containing quinic acid head-groups for delivering genetic materials into our body cells.

Yet another object of the present invention is to provide a process for the preparation of novel glycomimicking cationic amphiphiles containing quinic acid head-groups.

Summary of the invention

Accordingly the present invention relates to A quinic acid head-group containing glycomimicking cationic amphiphile having the generic structure A

wherein each of R ₁ and R ₂ is independently hydrogen or a lipophilic moiety containing at least eight carbon atoms and is optionally selected from 8-22 carbon containing alkyl, mono-, di- and tri-unsaturated alkenyl (Cg-C ₂₂ ) provided both R ¹ and R ² are not hydrogen;

R ₃ is independently hydrogen or alkyl (C ₁ -C ₅ , straight or branched); n is an integer between 1 and 7;

X is optionally selected from chlorine, bromine and iodine atom.

In an embodiment of the present invention each of Rj and R ₂ is independently hydrogen or an aliphatic hydrocarbon chain.

In yet another embodiment both Ri and R ₂ are aliphatic hydrocarbon chains. In yet another embodiment both R ₁ and R ₂ are the same and saturated alkyl group

(C _12-18 ).

In yet another embodiment both Ri and R ₂ are the same and are mono-unsaturated alkenyl group (Ci _2- I ₈ ).

In yet another embodiment R ₃ is an alkyl group and both R ₁ and R ₂ are aliphatic hydrocarbon chains.

In yet another embodiment R ₃ is a hydrogen atom and both Ri and R ₂ are aliphatic hydrocarbon In yet another embodiment each Rl and R ₂ group containing about 8-

22 linked carbon atoms is independently a mono-unsaturated alkenyl (C g _-22 ) group.

The present invention further provides a process for the preparation of quinic acid head-group containing glycomimicking cationic amphiphile having the generic structure A

^IV 3 wherein each of Ri and R ₂ is independently hydrogen or a lipophilic moiety containing at least eight carbon atoms and is optionally selected from 8-22 carbon containing alkyl, mono-, di- and tri-unsaturated alkenyl (Cg-C ₂₂ ) provided both R ¹ and R ² are not hydrogen;

R ₃ is independently hydrogen or alkyl (C ₁ -Cs, straight or branched); n is an integer between 1 and 7;

X is optionally selected from chlorine, bromine and iodine atom. the said process comprising the steps of:

(a) preparing 1,3,4,5 tetraacetoxycyclohexane carboxylic acid from quinic acid by reacting it with perchloric acid in a mixture of acetic acid and acetic anhydride, at a temperature of 50-60 ⁰ C, for a period of 10-12hrs,

(b) coupling a mixed primary-tertiary lipophilic aliphatic alkyl amine containing saturated or unsaturated aliphatic hydrocarbon chains with 1,3,4,5 tetraacetoxycyclohexane carboxylic acid in polar aprotic solvent, in presence of amide bond forming reagents to obtain the corresponding aliphatic hydrophobic amide intermediate;

(c) quaternizing the hydrophobic amide obtained in step (b) with excess of appropriate alkyl iodides to obtain the corresponding quaternized amphophilic ammonium iodides intermediate;

(d) subjecting the quaternized amphiphilic intermediate obtained above in step c to ion exchange chromatography halide ion exchange resins and mixed polar organic solvent as the eluent. In yet another embodiment the saturated or unsaturated aliphatic hydrocarbon chains of the mixed primary-tertiary amine is selected from the group consisting of 8-22 carbon atoms. In yet another embodiment the polar aprotic solvent used in step (b) is selected from

the group consisting of dimethyl formamide, dimethylsulphoxide, pyridine and triethyl amine.

In yet another embodiment the reaction of step (b) is carried out at a temperature between O ⁰ C to 80 ⁰ C. In yet another embodiment the quaternization of the intermediate hydrophobic amide obtained in step (b) is carried out at a temperature between -25 ⁰ C to 30 ⁰ C.

In yet another embodiment the amide forming reagents used are ethylenedicarbodiimide (EDCI) and N-Hydroxybenzotriazole (HOBt).

In yet another embodiment the halide ion exchange resins used in step (d) is selected from the chloride and bromide ion exchange resins.

In yet another embodiment the organic solvent used as ingredients of the polar eluent in step (d) is selected from the group consisting of methanol, ethanol, chloroform, dichloro methane and ethyl acetate.

The present invention further provides a formulation comprising at least a catioinic amphiphile of formula A, a co-lipid, and a polyanionic compound along with physiologically acceptable additives.

A formulation as claimed in claim 19, wherein the catioinic amphiphile of formula A used is

wherein each of Ri and R ₂ is independently hydrogen or a lipophilic moiety containing at least eight carbon atoms and is optionally selected from 8-22 carbon containing alkyl, mono-, di- and tri-unsaturated alkenyl (Cs-C ₂₂ ) provided both R ¹ and R are not hydrogen; R ₃ is independently hydrogen or alkyl (C ₁ -C ₅ , straight or branched); n is an integer between 1 and 7;

X is optionally selected from chlorine, bromine and iodine atom.

In yet another embodiment the catioinic amphiphile used is in pure form or in combination with helper lipids.

In yet another embodiment the co lipid used is selected from the group consisting of phosphatidylethanolamine, phosphatidylglycerol and cholesterol.

In yet another embodiment the colipid used is selected from sterol group or a neutral phosphatidyl ethanolamine or neutral phosphatidyl choline. In yet another embodiment co-lipid used is preferentially selected from 1,2-

Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE) or cholesterol

In yet another embodiment the molar ratio of the catioinic amphiphile to colipid used is in the range of 1:1 to 3:1.

In yet another embodiment the preferred molar ratio of catioinic amphiphile to colipid is 1:1.

In yet another embodiment the polyanionic compound used is selected from the group consisting of nucleic acid that encodes for a therapeutically important immunogen, protein, nucleic acid, an oligonucleotide, a peptide or a protein and drug. In yet another embodiment the nucleic acid used is selected from the group consisting of a circular or linear plasmid, a ribonucleic acid, a ribosomal RNA, antisense polynucleotide of RNA or DNA and polynucleotide of genomic DNA, cDNA or mRNA

In yet another embodiment the polyanion used is single or in combination. In yet another embodiment the said formulation is administered intravenous, intramuscular, or intraperitonial mode.

In yet another embodiment the said formulation is administered intracellularly in the range of 25 to 100 microlitres.

In yet another embodiment the said formulation is administered to cells at a ratio 0.1 to 0.5 microgram ofDNA to 50,000 cells.

In yet another embodiment the said formulation comprises amount of amphiphile in the range of 9.0 to 0.3 microgram from a lipopeptide to DNA charge ratio ranging

The present invention further provides a method for producing immune response, comprising: (a) administering the transfection formulation as claimed in claim 19

containing at least a catioinic amphiphile of formula A and a polyanionic compound wherein the said polynucleotide encodes an immunogen to at least one mouse thereby generating at least one immunized mouse; (b) measuring the monoclonal antibodies produced in mouse body.

In still another embodiment the invention is evaluation of antigen presenting cell specific gene delivery properties of the new glycomimicking cationic lipid represented by the above- mentioned structure A.

Brief description of drawings

Figure 1 summarizes the in vitro gene delivery efficacy profiles of the glycomimicking cationic lipids 1-3 disclosed in the present invention in RAW 264.7 cells across the lipid:DNA charge ratios of 9:1-0.3:1.

Figure 2 summarizes the in vitro gene delivery efficacy profiles of the glycomimicking cationic lipids 1-3 disclosed in the present invention in NIH 3T3 cells across the lipid:DNA charge ratios of 9: 1 -0.3 : 1.

Figure 3 summarizes in vitro gene transfer efficiencies of lipid 2 in RAW 264.7 cells with cholesterol as co-lipid (at 1:1 mole ratio of lipid to co-lipid) in the presence of 50 Dg of man-BSA. Percent control values refer to the relative luciferase activities compared to values in absence of man-BSA. The relative light units obtained for cells treated with the lipoplexes of lipid 2 in the absence of man-BSA were taken to be 100.

Figure 4 summarizes in vitro gene transfer efficiencies of lipid 2 in NIH3T3 cells with cholesterol as co-lipid (at 1:1 mole ratio of lipid to co-lipid) in the presence of 50 μg of man- BSA. Percent control values refer to the relative luciferase activities compared to values in absence of man-BSA. The relative light units obtained for cells treated with the lipoplexes of lipid 2 in the absence of man-BSA were taken to be 100.

Figure 5 shows the β-gal specific antibody titer values from mice immunized with lipoplexes

of lipids 1-3 (at lipid:DNA charge ratios of 6:1) and Naked DNA. Antibody titers represented here are geometric mean titres (n =4).

Detailed description of the invention Mannose receptor is a 180 kDa transmembrane protein consisting of five domains: a cystine rich amino teαninus, a fibronectin type II repeat region, eight carbohydrate recognition domains (CRD), a transmembrane domain and a cytoplasmic domain. The mannose receptor selectively binds to molecules or micro-organisms carrying sugars such as mannose, fucose, N-acetylglucosamine and glucose on their surface through the eight CRD domains (Apostolopoulos, V. et al. Curr. MoI. Med. 2001; 1:469-474). A major contribution to the binding is provided by the extensive network of hydrogen bonds and coordination bonds between two equatorial, vicinal hydroxyl groups (at positions 3 & 4) in D-mannose, a calcium ion, two asparagines and two glutamic acid residue of the receptor protein (Weis, W. I. et al. Nature 1992;360:127-134, Drickamer, K. Nature 1992;360:183-186). Thus, the mannose receptor plays a key role in imparting protective immunity against a host of antigenic micro-organisms expressing mannose on their cell wall. Since both dendritic cells and macrophages (antigen presenting cells, APCs) predominantly express endocytic mannose receptors on their cell surfaces (Apostolopoulos, V. et al. Curr. MoI. Med. 2001;l :469-474), the selective uptake of cationic lipid:DNA complexes (lipoplexes) by the APCs could, in principle, be enhanced by the covalent modification of the liposomal surface with APC specific ligands. This would also prevent the non-specific uptake of lipid:DNA complex hy somatic cells such as myocytes or keratinocytes. In other words, if such mannose-receptor specific cationic amphiphiles complexed with genetic material encoding antigens are injected into patients, they are likely to elicit better immune response through enhanced transfection of antigen presenting cells. To this end, the present invention relates to methods for eliciting enhanced immune responses using a novel series of mannose receptor specific glycomimicking cationic amphiphiles.

The above-mentioned properties of the mannose receptor have been amply exploited in the development of gene delivery reagents capable of targeting the macrophages and dendritic cells (Ferkol, T. et al. Proc. Natl. Acad. Sci. USA 1996/93:101-105, Diebold, S. S. et al. J.

Biol. Chem. 1999; 274:19087-19094, Kawakami, S. et al. Gene Ther. 2000;7:292-299). Hashida and co-workers have reported on the potency of mannosylated cationic liposomal formulation in enhancing immune response through the targeted delivery of DNA to APC (Hattori, y. et al. Biochem. Biophys. Res. Comm. 2004;317:992-999). Grandjean et. al have shown that synthetic lysine based clusters of carbocyclic acids such as quinic and shikimic acid act as effective ligands for the mannose receptor of dendritic cells (Grandjean, C, et al. Chembiochem, 2001;2:747-757). To this end, the present invention relates to use of a novel series of mannose-mimicking cationic amphiphiles containing quinic acid head-groups (e.g. lipids 1-3) for use in transferring genes into antigen presenting cells (APCs) as well as for use in genetic immunization. These novel glycomimicking cationic amphiphiles are likely to offer greater stability than mannose in a systemic setting since the pyranose ring is replaced by a cyclohexene or cyclohexane.

The present invention also relates to process for the synthesis of the said novel series of glycomimicking cationic amphiphiles and evaluation of their mannose receptor mediated gene transfer properties in cultured mouse macrophage cells (APC) as well as in cultured mouse fibroblast cells (NIH 3T3). The novel cationic amphiphiles containing glycomimicking quinic acid head-groups are potentially useful to deliver genetic materials encoding therapeutic antigens to antigen presenting cells with over expressed mannose receptors.

The distinctive novel structural features common to the glycomimicking cationic amphiphiles disclosed in the present invention include: (1) The presence of hydrophobic groups which are directly linked to the positively charged nitrogen atom and (2) the presence of mannose receptor binding polar quinic acid head-groups. It is believed that these unique structural features contribute significantly to the mannose receptor mediated gene transfer efficiencies of the glycomimicking cationic amphiphiles disclosed herein. The area of science that is likely to be benefited most from the present invention is the field of genetic immunization or DNA vaccination. According to the practice of the present invention, "cationic" means the positive charge is either on quaternized nitrogen or on a protonated nitrogen atom. The cationic characters of the present amphiphiles may contribute to the enhanced interaction of the amphiphiles with

biologically active molecules such as nucleic acids and/or with cell constituents such as plasma membrane glycoproteins. Such enhanced interaction between the cationic amphiphiles and therapeutically active biological macromolecules and/or cell membrane constituents may play a key role in successfully transporting the therapeutic molecules into the cells.

The cationic glycomimicking lipids of the present invention have certain common structural and functional groups. As such, the said cationic amphiphiles may be represented by the following generic formula (A):

A wherein each of R ¹ and R ² is independently hydrogen or a lipophilic moiety containing at least eight carbon atoms and is optionally selected from 8-22 carbon containing alkyl, mono-, di- and tri-unsaturated alkenyl (Cs-C ₂₂ ) provided both R ¹ and R ² are not hydrogen; R ³ is independently hydrogen or alkyl (C ₁ -C ₅ , straight or branched); n is an integer between 1 and 7;

X is optionally selected from chlorine, bromine and iodine atom

The cationic amphiphiles of the present invention have a lipophilic domain that facilitates the formation of lipid complexes or aggregates in aqueous solutions. The lipophilicity of the hydrophobic domains and the hydrophilicity of the polar quinic acid head-group domains are such that when the cationic lipids are confronted with aqueous solutions, lipid aggregates are formed in the presence or absence of a second compound. Exemplary lipophilic R ₁ and R ₂ groups include (1) saturated C ₈ -C ₂₂ alkyl groups and (2) unsaturated Cg-C ₂₂ alkenyl groups containing 1, 2, or 3 double bonds. In one preferred embodiment of the presently disclosed cationic lipids Ri = R ₂ = n- hexadecyl, R ₃ = methyl and n = 2, M is a sodium atom and X ^" is a chloride ion. Accordingly, the amphiphile no. 1 is a representative example of the presently described

novel RGD-lipopeptide:

Syntheses of the glycomimicking cationic lipids 1.

Scheme 1 outlines the synthetic strategy employed for preparing the representative mannose receptor specific cationic glycomimicking amphiphiles 1 described in the present invention.

Scheme I: Synthesis of Lipid 1

Qumic aαd '

' ⁺ O (CH ₂ J ₁₃

IV

Reagents: (i) Ac ₂ O ₁ AcOH, HCIO ₄ ; (ιi) EDCI, HOBt ; (Ni) MeI (excess); (iv) NaOMe ₁ MeOH; (v) Cl ^" ion exchange (amberlyst resin)

Lipid 1 was synthesized by EDCI-coupling of the mixed tertiary-primary amine intermediate II (prepared by reacting N,N-di-n-tetradecylamine with N-tert-butyloxycarbonyl protected

2-bromoethylamine in ethyl acetate in presence of anhydrous potassium carbonate followed by deprotection and neutralization as reported earlier (Kumar, V. V. et al. Gene. Ther. 2003;10:1206-1215) with tetraacetyl quinic acid (I, prepared as described in Grandjean, C. et al. J. Chem. Soc. Perkin Trans. I, 1999;2967-2975). The coupled product obtained (intermediate III) was quaternized with huge excess of methyl iodide followed by deprotection of the acetyloxy groups with methanolic sodium methoxide. The resulting quaternized product (intermediate IV) was finally subjected to chloride ion exchange chromatography as outlined in Scheme I. Structures of all the synthetic intermediates and lipid 1 shown in Schemes I were confirmed by ¹ H NMR and FAB mass spectroscopy. Structures of all the synthetic intermediates I-IV and the lipid 1 shown in Scheme 1 were confirmed by ¹ H NMR. The structure of the lipid 1 was further characterized by the molecular ion peaks in the LSIMS. Purity of lipid 1 was confirmed by reverse phase analytical HPLC using two different mobile phases.

Formulations

The invention also provides novel formulation comprising optimal amounts of the quinic acid head-group containing glycomimicking cationic amphiphiles disclosed herein, biological macromolecules and the co-lipids. One or more additional physiologically acceptable substances may be included in the pharmaceutical formulation of the invention to stabilize the formulation for storage or to facilitate successful intracellular delivery of the biologically active molecules. Co-lipids according to the practice of the present invention are useful in mixing with one or more of the glycomimicking amphiphiles. Cholesterol is an excellent co-lipid for use in combination with the presently described amphiphiles to facilitate successful intracellular delivery of the biologically active molecules. A preferred range of molar ratio of the cationic lipid to co-lipid is 1 : 1. As such, it is within the art to vary the said range to a considerably wide extent. Typically, liposomes were prepared by dissolving the glycomimicking amphiphiles and the co-lipid (Cholesterol or DOPE) in the appropriate mole ratio in a mixture of methanol and chloroform in a glass vial. The solvent was removed with a thin flow of moisture free nitrogen gas and the dried lipid film was then kept under high vacuum for 8 h. The dried lipid film was hydrated in sterile deionized water

in a total volume of 1 mL at cationic lipid concentration of 1 mM for a minimum of 12 h. Liposomes were vortexed for 1-2 minutes to remove any adhering lipid film and sonicated in a bath sonicator (ULTRAsonik 28X) for 2-3 minutes at room temperature to produce multilamellar vesicles (MLV). MLVs were then sonicated with a Ti-probe (using a Branson 450 sonifier at 100% duty cycle and 25 W output power) for 1-2 minutes to produce small unilamellar vesicles (SUVs) as indicated by the formation of a clear translucent solution. Biologically active molecules that can be administered intracellularly in therapeutic amounts using the cationic amphiphiles of the present invention include ribosomal RNA, antisense polynucleotide of RNA or DNA, polynucleotide of genomic DNA, cDNA or mRNA that encodes for a therapeutically important antigen or protein. The quinic acid head-group containing cationic amphiphiles of the present invention may be blended such that one or more of the representatives thereof may be used in a combination to facilitate entry of the said biologically active molecules into cells/tissues. In a further embodiment, the cationic amphiphiles disclosed in the present invention may be used either in pure form or in combination with other lipids or helper lipids such as cholesterol, phosphatidylethanolamine, phosphatidylglycerol, etc. The said therapeutic formulation may be stored at 0-4 ⁰ C until complexed with the biologically active therapeutic molecules. Agents that prevent bacterial growth and increase the shelf life may be included along with reagents that stabilize the preparation, e.g., low concentrations of glycerol. It is specifically warned that freezing and thawing cycles could cause loss in efficiency of the formulation.

In yet another embodiment, the formulation of the quinic acid head-group containing cationic amphiphiles disclosed herein, co-lipids (cholesterol or DOPE) and the biologically active therapeutic molecules may be administered intravenously besides other routes such as intramuscular and intraperitonial. Further, the said formulations may be administered to cells at a ratio of 0.1-0.5 microgram of DNA to 50,000 cells in an in vitro system. The amount of amphiphile could be varied from a cationic lipid to DNA charge ratio of 0.3:1 to 9:1 considering one positive charges for one cationic glycomimicking amphiphile and one negative charge of a single nucleotide base.

The invention further provides a process for the preparation of the said formulation

comprising the steps of preparing a dispersion of the cationic amphiphiles disclosed in the present invention; contacting said dispersion with a biologically active molecule to form a complex between said glycomimicking cationic amphiphile and the said biologically active molecules and contacting the cells with the said complex thereby facilitating transfer of said biologically active molecules into the cells. The present invention also provides with various formulations that facilitate intracellular delivery of biologically active molecules.

Maηpose Receptor Specific Gene Delivery Properties of the presently described Cationic Amphiphiles:

The relative in vitro gene delivery efficacies of the presently described glycomimicking cationic amphiphiles were evaluated in RAW 264.7 cells (a murine macrophage cell line, antigen presenting cells) as well as in a non-antigen presenting cell such as NIH 3T3 cells (mouse fibroblast cells) across the lipid:DNA charge ratios 9:1 to 0.3:1 using cholesterol as a co-lipid. The results for the representative cationic lipids 1-3 are summarized in Figures 1 & 2. Several interesting transfection profiles were observed. In general, lipid 1-3 exhibited higher transfection efficacies than a commonly used commercially available transfection reagent DOTMA:DOPE (at 1:1 mole ratio) in both RAW 264.7 and NIH 3T3 cells (Figures 1 & 2). DOTMA was used for comparison as a non-targeting cationic lipid in these studies. Further, the gene transfer efficacies of quinic acid based amphiphiles (1-3) were found to be remarkably higher (by more than 100 fold) than that of DOTMA in both the cells (Figures 1 & 2). Interestingly, in RAW 264.7 cells, lipids 2 & 3 exhibited superior gene transfer efficacies at higher lipid:DNA charge ratios of 9: 1 & 3:1 while lipid 1 was more efficient at 3:1 & 1:1 than at 9:1 lipid:DNA charge ratio (Figure 1). However, all the lipids showed highest gene transfer efficiencies at lipid:DNA charge ratios of 3:1 or 1 :1 in NIH 3T3 cells.

Next, the transfection efficiencies of lipid 2 (as a representative example) were evaluated in both the cells in the presence of mannosylated bovine serum albumin (man-BSA) to assess whether the cellular uptake of lipoplexes (lipid 2: DNA complexes) is mediated through the mannose receptor. In RAW 264.7 cells (known to over express the mannose receptor, Apostolopoulos, V. et al. Curr. MoI. Med. 2001; 1 :469-474), the gene transfer efficacy of lipid 2 was significantly inhibited by nearly 60-65% across the lipid:DNA charge ratio of

9:1-1:1 in the presence of 50 μg of man-BSA (Figure 3). Contrastingly, the inhibition of transfection efficiency of lipid 2 in NIH 3T3 cells (control cell with no over expressed mannose receptors present in the cell surface) in presence of 50 μg of man-BSA was only 20-25% (Figure 4). Thus, the findings summarized in Figures 3 & 4 support the notion that cellular uptake of the lipid:DNA complexes for the presently described glycomimicking cationic amphiphiles are likely to be mediated via mannose receptor in antigen presenting cells.

Generation of humoral immune upon intramuscular genetic immunization in mice using antigen encoding PNA in complexation with the presently described glycomimicking cationic amphiphiles :

The potential of the presently described glycomimicking cationic amphiphiles as DNA vaccine delivery agents were evaluated by intramuscularly immunizing Balb/c mice with lipoplexes of lipids 1-3 containing 25 μg of pCMV-SPORT-β-gal (as a model antigen encoded DNA) at lipid:DNA charge ratio of 6:1. After three weeks post primary immunization, a booster dose of lipoplexes having the same amount of DNA as in the first dose was administered and the mice were bled two week after the administration of this dose. Antibodies generated against the plasmid encoded β -galactosidase protein (the model antigen) was measured by the ELISA assay. As depicted in Figure 5, mice immunized with lipoplexes of lipids 1-3 exhibited two fold higher antibody titers than the mice injected with same amount of naked DNA.

Applications: The process of the present invention can be exploited for preparing glycomimicking cationic amphiphiles based gene transfer reagents efficient delivery of genetic materials into the antigen presenting cells in DNA vaccination. The present inventions are useful for mannose receptor specific delivery of polyanions, polypeptides or nucleopolymers into the antigen presenting cells. The present invention is directed to methods of eliciting immune responses in animals through administering complexes of the presently described cationic amphiphiles and a polynucleotide coding for an antigentic determinant. Furthermore, the present

invention is also directed to methods of eliciting active immunity against an infectious disease in animals through administering complexes of the presently described cationic amphiphiles and a polynucleotide coding for the infectious disease causing protein. The present invention is also related to the genetic immunization methods wherein the polynucleotide is an expression vector comprising a DNA sequence encoding the antigenic determinant of the infectious disease causing immunogen and wherein the transcription of the DNA is under the control of a promoter. The present invention is further directed to a genetic immunization wherein the polynucleotide is an RNA molecule encoding for an infectious immunogen. In particular, the presently disclosed glycomimicking cationic amphiphiles hold potential for future exploitation in genetic immunization in delivering DNA or RNA encoding infectious immunogen.

The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

EXAMPLE 1:

Synthesis of the cationic glycomimicking amphiphile 1 (Scheme I).

Step i (Scheme I). D (-) Quinic acid (Ig, 5.2 mmol) was suspended in a 2:1 (v/v) mixture of acetic acid-acetic anhydride (10 mL) and a drop of perchloric acid was added to it at room temperature. The reaction mixture became clear as the temperature increased to 50-60 ⁰ C after the addition of perchloric acid. The resulting solution was left stirred at room temperature for a further 12 h, diluted with ice-cold water and extracted with chloroform (3 x 100 mL). The organic layer was again washed with water (3 x 100 mL), dried over sodium sulphate, filtered and the solvent evaporated on a rotavapor. Crystallization of the residue from CHC1 ₃ /Hexane (2:8) afforded 1.7 g (91% yield) of the pure title compound 1, 3, 4, 5- tetracetoxycyclohexane carboxylic acid (Intermediate I, Scheme I) as a white solid. 1H NMR (300 MHz, CDCl ₃ ): δ/ppm = 1.9-2.0 [dd, IH, 6-H]; 2.0-2.3 [4s, 12H, 4 x - COCH ₃ ]; 2.4-2.5 [m, 2H, 6-H\ 2-H]; 2.5-2.6 [dd, IH, 2-H']; 4.95-5.05 [dd, IH, 4-H]; 5.3- 5.45 [ddd, IH, 5-H]; 5.5-5.6 [m, IH, 3-H].

Step ii (Scheme I). Solid HOBt (0.36 g, 2.65 mmol) and EDCI (0.51 g, 2.65 mmol) were added sequentially to an ice cold and stirred solution of 1, 3, 4, 5-tetracetoxycyclohexane carboxylic acid (0.96 g, 2.65 mmol, prepared above in step a) in 5 mL dry DCM/dry DMF (9:1, v/v). After half an hour, N-aminoethyl-N,N-di-n-tetradecylamine (II, Scheme I, 1.0 g, 2.21 mmol prepared as described in Kumar, V. V. et al. Gene. Ther. 2003;10:1206-1215) dissolved in dry DCM was added to the reaction mixture. The resulting solution was left stirred at room temperature overnight, diluted with excess DCM and washed sequentially with saturated sodium bicarbonate (3 x 100 mL) and water (3 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent from the filtrate removed by rotary evaporation. The residue upon column chromatographic purification with 60-120 mesh silica gel using 8-10% acetone-petroleum ether (v/v) as eluent afforded 1.43 g (80% yield) of the pure intermediate N-2-[N'-(l, 3, 4, 5- tetraacetoxycyclohexanecarbonyl)]aminoethyl-N,N-di-n-tetrade cylamine (Intermediate III, Scheme I, R _f = 0.5, 35% acetone-petroleum ether, v/v). 1H NMR (200 MHz, CDCl ₃ ):δ/ppm = 0.9 [t, 6H, CH ₃ -(CH ₂ )O-]; 1.2-1.6 [m, 44H, - CH ₂ (CH ₂ ) _U -; 4H, -N(-CH ₂ -CH ₂ -) ₂ ]; 1.8-2.0 [dd, IH, quin-6-H]; 2.0-2.2 [4s, 12H, 4 x - COCH ₃ ]; 2.3-2.6 [m, 4H, -Nt-CH ₂ -CH ₂ ")^ 2H, -N-CH ₂ -CH ₂ -NH-CO-; IH, quin-6-H; IH, quin-2-H]; 2.8-2.9 [dd, IH, quin-2-H']; 3.15-3.3 [m, 2H, -N-CH ₂ -CH ₂ -NH-CO]; 4.9-5.0 [dd, IH, quin-4-H]; 5.25-5.4 [ddd, IH. quin-5-H]; 5.6 [m, IH, quin-3-H]; 6.5 [m, IH, CO-NH]. FABMS: m/z: 796 [M+ 1 ] ⁺ (calculated for C ₄₅ H ₈₂ O ₉ N ₂ ).

Step iii (Scheme I). The intermediate III prepared above in step ii (1.3 g, 1.64 mmol) was dissolved in 8 mL chloroform/methanol (1 :1, v/v) and 5 mL methyl iodide was added to the solution. The reaction mixture was stirred at room temperature for 6 h and the solvent was removed on a rotary evaporator. The residue upon column chromatographic purification with silica gel (60-120 mesh size) and 20-25% acetone in petroleum ether (v/v) as eluent afforded 0.90 g (59% yield) of N-2-[N'-(l, 3, 4, 5- tetraacetoxycyclohexanecarbonyl)]aminoethyl-N,N-di-n-tetrade cyl,N-methyl ammonium iodide as a colourless gummy solid (Intermediate IV, Scheme I, R _f = 0.5, 50% acetone in petroleum ether, v/v). 1H NMR (200 MHz, CDCl ₃ ): δ /ppm = 0.9 [t, 6H, CH ₃ -(CH ₂ ), ₃ -]; 1.2-1.6 [m, 44H ₅ -(CH ₂ ), ,- ]; 1.7-1.9 [m, 4H, -N ⁺ C-CH ₂ -CH ₂ -);)]; 1.9-2.0 [dd, IH, quin-6-H]; 2.0-2.2 [3s, 9H, 3 x -

COCH ₃ ]; 2.4 [s, 3H, -COCH ₃ ]; 2.45-2.7 [m, 2H, quin-6-H', quin-2-H]; 2.75-2.85 [dd, IH, quin-2-H']; 3.35 [s, 3H, -N ⁺ CH ₃ ]; 3.4-3.6 [m, 4H, -N ⁺ ^CH ₂ -CH ₂ -);),]; 3.65-3.85 [m, 4H, - N ⁺ -CH ₂ -CH ₂ -NH-CO]; 4.95-5.1 (dd, IH, quin-4-H]; 5.35-5.5 [ddd, IH, quin-5-H]; 5.6 [m, IH, quin-3-H]; 8.2 [m, IH, CO-NH]. FABMS: m/z: 810 [M] ⁺ (calculated for C ₄₆ H ₈₅ O ₉ N ₂ ).

Steps iv & v (Scheme I). The intermediate iii prepared above in step iii (0.70 g, 0.75 mmol) was dissolved in 3 mL methanol and 1 M sodium methoxide in methanol was added drop wise to raise the apparent pH ~ 9. The reaction mixture was allowed to stir at room temperature for 3 h, neutralized with Amberlite IRl 20 (H ⁺ ), filtered and the filtrate was dried over anhydrous Na ₂ SO ₄ and concentrated on a rotary evaporator. The residue upon column chromatographic purification with silica gel (60-120 mesh size) and 6-8% methanol in chloroform (v/v) as eluent followed by chloride ion exchange chromatography (using amberlyst A-26 chloride ion exchange resin) afforded 0.22 g (38% yield) of the pure N-2- [N'-(l, 3, 4, 5-tetrahydroxycyclohexanecarbonyl)]aminoethyl-N,N-di-n-tetra decyl,N-methyl ammonium chloride, (lipid 1, Scheme I, R _f = 0.5, 20% methanol-chloroform, v/v). 1H NMR (200 MHz, CDCI ₃ +CD ₃ OD): δ /ppm = 0.9 [t, 6H, CH ₃ -(CH ₂ )B-]; 1.2-1.5 [m, 44H ₉ -(CH ₂ ) _H -]; 1.8-2.05 [m, 4H, -N^-CH ₂ -CH ₂ -);,; 2H, quin-6-H, quin-6-H'; 2H, quin-2-H, quin-2-H']; 3.1 [s, 3H, -N ⁺ CH ₃ ]; 3.25-3.45 [m, 4H, -N ⁺ (-CH ₂ -CH ₂ -) ₂ ; 2H, -N ⁺ -CH ₂ -CH ₂ - NH-CO-; IH, quin-4-H]; 3.55-3.7 [m, 2H, -N ⁺ -CH ₂ -CH ₂ -NH-CO]; 3.95-4.1 [ddd, IH, quin- 5-H]; 4.2 [m, IH, quin-3-H]. FABMS: m/z: 644 [M+2] ⁺ (calculated for C ₃₈ H ₇₇ O ₅ N ₂ ).

EXAMPLE 2: Evaluation of the in vitro gene transfer efficacies in RAW 264.7 and NIH 3T3 cells.

Cells were seeded at a density of 30000 (for RAW 264.7) and 20000 cells (for NIH 3T3) per well in a 96- well plate for 18-24 h before the transfection. 0.3 (g (0.9 nmol) of plasmid DNA was complexed with varying amounts of lipids (0.3 nmol-9.0 nmol) in plain DMEM medium (total volume made up to 100 (L) for 30 minutes. The charge ratios were varied from 0.3:1 to 9:1 over these ranges of the lipids. Immediately prior to transfection, cells plated in the 96-well plate were washed with PBS (2 x 100 (L) followed by the addition of

lipoplexes. After 4 h of incubation, the medium was replaced with fresh complete medium containing 10% FBS. The luciferase reporter gene activity was estimated after 24 h. The cells were washed twice with PBS (100 (L each) and lysed in 50. (1 reporter lysis buffer. Care was taken to ensure complete lysis. 50 (L promega luciferase assay buffer was added to the lysate in a polystyrene plate and the luciferase activity per well was estimated using a Microplate Luminometer (Berthold, Germany). Protein concentration in each well was determined by the modified Lowry method (Markwell, M.A.K. et al. Anal. Bicochem. 1978;87:206-210) and luciferase activity was expressed as the relative light unit (RLU) per mg of the protein. Each transfection experiment was repeated two times on two different days. The transfection values reported were average values from two replicate transfection plates assayed on the same day. When mannosylated-bovine serum albumin (man-BSA) was used, the cells were pretreated for 30 mins at 37°C with man-BSA (50 (g), and the transfection was carried out in its presence. After 4 h at 37 ⁰ C, the medium was replaced with fresh complete medium without man-BSA and the transfection activity was evaluated after 24 h as described above.

EXAMPLE 3 Genetic Immunization of Mice and Evaluation of Humoral Response

Mice Immunization. Cationic liposomes of lipids 1-3 and pCMV-SPORT-β-gal (25 μg) were mixed at a lipid:DNA charge ratio of 6: 1 in 5% glucose solution and the resulting lipoplexes were incubated at room temperature for 15-20 minutes. Groups of 4-6 week old male Balb/c mice (n = 6) were intramuscularly injected with 300 μL of the above formed lipoplexes (150 μL into quadriceps of each hind leg). Naked DNA (25 μg) was also injected in one group. Three weeks after the primary immunization, a booster dose containing the same amount of DNA as in the primary dose was given. Two weeks later, mice were bled from the retro-orbital sinus and the sera were collected for the antibody assay.

Antibody Assay. Titres of anti-β -gal antibodies were assayed using an enzyme linked immunosorbent (ELISA) assay as described earlier (McKeever, U. et al. Vaccine

2002;20:1524-1531). A 96-well ELISA plate was coated with -β -gal protein (0.2 μg per well) overnight at 4°C using a 2 μg per mL stock solution prepared in phosphate buffered saline (PBS). Plates were washed with PBS thrice and blocked with 1% BSA in PBS at

room temperature for 2h. The plate was washed thrice with PBS containing 0.05% Tween- 20 and incubated with serially diluted mouse sera at room temperature for 2h. The plate was again washed thrice with PBS containing 0.05% Tween-20 and 100 μL of anti-mouse antibody conjugated to horse radish peroxidase (diluted 1:1000) was added per well, followed by incubation at room temperature for 2h. The antibody conjugate was removed by washing the plate thrice with PBS containing 0.05% Tween-20. The plate was then incubated in dark with 100 μL of ABTS at room temperature for 10 min and the absorbance was measured at 405 run. Antibody titres were calculated as the highest dilution to reach an OD that was at least two fold above the negative serum control at the same dilution.