Nasal vaccine against the development of atherosclerosis disease and fatty liver

Abstract

The present invention provides a novel vaccine compound of micellar nanoparticles to be administered intranasally to treat and/or prevent the disease called atherosclerosis, which results from an abnormal metabolism of circulating lipids. The novelty of the vaccine compound of the present invention is the use of Archaebacterian lipids, lysophosphatidylcholine, and phosphatidylcholine, which give nanoparticles stability and facilitates antigen presentation in its appropriate secondary peptidic conformation. A novel process for the preparation of vaccine compounds which allows obtaining homogeneous nanoparticles with high stability is also presented in this invention.

Claims

1. A vaccine composition of micellar nanoparticles for intranasal administration comprising a carboxyl terminus peptide of a cholesteryl ester transfer protein (CETP) set forth as SEQ ID NO: 1 as an immunogen, 2,3-dibiphytanyl-o sn-glycerol (calarcheol) from archaebacteria Thermus aquaticus, lysophospholipid, and phosphatidylcholine in a pharmaceutically acceptable vehicle.

2. The vaccine composition according to claim 1, wherein the 2,3-dibiphytanyl-o-sn-glycerol (calarcheol) is obtained from cell membranes of archaebacteria Thermus aquaticus.

3. The vaccine compound according to claim 1, wherein the 2,3-dibiphytanyl-o-sn-glycerol (calarcheol) is used as scaffolding in micellar nanoparticles providing stability and improving absorption by mucosae.

4. The vaccine composition according to claim 1, wherein the lysophospholipid is lysophosphatidylcholine.

5. The vaccine composition according to claim 4, wherein the lysophosphatidylcholine induces formation and stabilization of an -helix functional secondary structure on the carboxyl-terminus peptide of CETP, which facilitates the function of lipid transfer on CETP.

6. The vaccine composition according to claim 1, wherein the lysophosphatidylcholine is 1-lauril-2-hydroxi-sn-3-phosphocholine (Lyso C.sub.12).

7. The vaccine composition according to claim 1, wherein the lipid mixture representing 50% of its components allows the CETP Y-helix antigen to preserve its -helix functional secondary structure on the surface of the nanoparticles.

8. The vaccine composition according to claim 1, to treat and/or prevent in mammals the development of the disease called atherosclerosis.

9. The vaccine composition according to claim 1, wherein the phosphatidylcholine is L--phosphatidylcholine.

10. A vaccine composition of micellar nanoparticles for intranasal administration comprising: a carboxyl terminus peptide of a cholesteryl ester transfer protein (CETP) set forth as SEQ ID NO: 1 as an immunogen, 2, 3-dibiphytanyl-o-sn-glycerol (calarcheol from archaebacteria Thermus aquaticus, 1-lauryl-2-hydroxy-sn-glycero3-phosphocholine (Lyso C.sub.12) and L phosphatidylcholine in a pharmaceutically acceptable vehicle.

11. A process for preparing a vaccine composition of micellar nanoparticles to be administered intranasally comprising: A. synthesizing and purifying an immunogen peptide set forth as SEQ ID NO: 1; b. isolating cell membranes from Thermus aquaticus; c. isolating 2,3-dibiphytanyl-o-sn glycerol (calarcheol) membrane lipids from the Thermus aquaticus cell membranes; d. incorporating L phosphatidylcholine, 1-lauryl-2-hydroxy-sn-glycero 3-phosphocholine (Lyso C.sub.12) and the lipds isolated in step c) to form micelles; e. incorporating the immunogen peptide from step a) to the micelles prepared in step d).

12. The process according to claim 11, wherein re-suspension of lipids performed at pH 9.5 is carried out before step d), which is the optimal to favor the solubility of the peptide and keep it in monomeric state.

13. The process according to claim 11, wherein the immunogenic peptide is incorporated in incubation at 25 C.

14. The process according to claim 11, wherein a lipid:peptide compound is integrated to a vehicle pharmaceutically acceptable for nasal administration.

15. The process according to claim 11, wherein the method produces a micellar-nanoparticled compound of a homogeneous size with high stability, favoring the preservation of the -helix functional secondary structure of the antigen, which triggers a specific immunologic response specifically aimed at the C-terminus end of CETP.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the histological appearance of liver from rabbits fed on normal diet during 30 days. Sinusoid permeability and inside them Kupffer cells were clearly identified. Also, the laminar o mural distribution of normal hepatocytes and their organization in classical lobule, portal lobule, and acinus is well preserved. Portal triads and the associated connective tissue show no abnormalities. Notice the macroscopic aspect and the normal color of the liver of rabbits fed on normal diet at the time of necropsy (upper left angle). Hematoxylin and eosin stain (H&E). Total magnification: A) 100 B) 1000 C) 1000 D) 1000.

(2) FIG. 2 shows histological sections of liver from rabbits fed on normal diet and administration of placebo for 30 days. In general, the microscopic structure of liver was similar to the one described for rabbits fed on normal diet. Some centrilobular hepatocytes showed minimal steatotic hepatocytes based on the presence of scarce vacuoles. The macroscopic aspect of the liver was also similar to the one of rabbits fed on normal diet. Hematoxylin and eosin stain (H&E). Total magnification: A) 100 B) 400 C) 1000 D) 1000.

(3) FIG. 3 shows histological sections of liver from rabbits fed on high cholesterol and triglyceride diet for 30 days. The high diet consisted of cholesterol 1% and corn oil 10% added to the balanced food special for rabbits. The main histopathologic changes identified were different degrees of steatosis, both microvacuolar and macrovacuolar, from mild to severe and, in some cases, diffuse, affecting great areas of hepatic parenchyma. In mild to moderate steatosis, centrilobular hepatocytes are mainly affected, which show a microvacuolar cytoplasmic appearance, with no displacement of nuclei to the cellular periphery. In severe steatosis, the sections showed centrilobular hepatocytes and hepatocytes from the paracentral region of the lobule (about 70% of the lobule) with a great overlapping of the two main morphological patterns: microvacuolar and macrovacuolar steatosis in which lipid drops are greater and coalesce until producing a great vacuole whose nucleus and cytoplasm are moved to the peripheral area of the cell. Notice the macroscopic creamy appearance of the liver, clearly different from FIGS. 1 and 2, in which animal were fed on normal diet only and placebo, respectively. Total magnification: A) 100 B) 1000 C) 400 D) 1000 E) 400 F) 1000. H&E.

(4) FIG. 4 shows histological sections of liver from rabbits under treatment with a diet high in cholesterol and triglycerides for 15 days, followed by the administration of the intranasal vaccine developed in the present invention. The high diet consisted of cholesterol 1% and corn oil 10% added to the balance food for rabbits. The experiment lasted 30 minutes. The histopathologic changes observed in these animals were similar, although to a lesser degree and extension, to the ones described in the rabbits fed on cholesterol-rich diet without vaccination (FIG. 3). In most cases, only the cells close to the centrilobular veins were affected. Nevertheless, some groups of hepatocytes with macrovacuolar steatosis with a very specific distribution were also observed. Notice that the macroscopic appearance of the livers of rabbits fed on cholesterol-diet and which later received the intranasal vaccine have characteristics intermediate between animal controls and the most affected only fed on cholesterol-rich diet (H&E). Total magnification: A) 100 B) 1000 C) 1000 D) 1000 E) 1000.

(5) FIG. 5 shows histological sections of liver from rabbits under a diet high in cholesterol and triglycerides with simultaneous administration of the intranasal vaccine of the present invention for 30 days. The high diet consisted of cholesterol 1% and corn oil 10% added to the balanced food special for rabbits. In this group of animals, centrilobular hepatocytes showed mild microvacuolar steatosis, with a minimal increase in cell size not reaching the advanced hepatocyte vacuolization as happened in the case of rabbits only fed on cholesterol-rich diet. Periportal and paracentral hepatocytes had morphology similar to controls (FIG. 1). See macroscopically the appearance and color of liver from rabbits on high-cholesterol diet and simultaneous administration of the vaccine (H&E). Total magnification: A) 100 B) 100 C) 400 D) 1000 E) 400.

(6) FIG. 6 shows the histological appearance of the liver of rabbits fed on normal diet, without vaccination or vehicle. The histological characteristics are the ones described in FIG. 1. Additionally, collagen fibers are identified, stained blue, near the portal triad (A) and, to a lesser degree, around the central or centrilobular vein (B). Masson's trichrome stain. Total magnification 200.

(7) FIG. 7 shows histological sections of liver from rabbits fed on normal diet, without vaccination, but which received vehicle. Notice the histological similarity of the hepatic parenchyma and the distribution of collagen fibers with the group of control rabbits in FIG. 6. Masson's trichrome stain. Total magnification 200.

(8) FIG. 8 shows the histological sections of the liver of rabbits fed on high cholesterol and triglyceride diet, without vaccination or vehicle. A) Hepatocytes near the portal triad show a pattern similar to the one of controls (FIGS. 6 and 7); yet, neighboring sinusoids show perisinusoidal fibrosis (blue fibers, arrows). B) Hepatocytes around the central vein show steatosis and some of them are ballooned; additionally, these animals showed perivenular fibrosis (around central veins of hepatic lobules) and a marked perisinusoidal fibrosis. Masson's trichrome stain. Total magnification 200.

(9) FIG. 9 shows the histological sections of the liver of rabbits treated with high cholesterol and triglyceride diet, which later received the vaccine. A) portal triad, the connective tissue and the surrounding hepatocytes show structural characteristics similar to the ones of controls. B) The central vein and related sinusoids still show fibrosis, although less than that of animals which did not receive the vaccine (FIG. 8). Central hepatocytes were less damaged; some show microvacuolar simple steatosis and ballooned hepatocytes are scarce. Masson's trichrome stain. Total magnification 200.

(10) FIG. 10 shows the histological sections of the liver of rabbits fed on high cholesterol and triglyceride diet along with the intranasal vaccine. A) The histological appearance of the portal triad, connective tissue and adjacent parenchyma is practically normal. B) Both centrilobular vein fibrosis and perisinusoidal fibrosis have diminished noticeably compared to what was seen in rabbits fed on high cholesterol and triglyceride diet (FIG. 8). Many centrilobular hepatocytes are still ballooned; few of them have microvesicular steatosis and some show cytoplasm damage. Masson's trichrome stain. Total magnification 200.

(11) FIG. 11 shows the histological analysis of abdominal aortas, cross-sectioned, of rabbits treated with normal diet (A), with normal diet and vehicle (B), with high cholesterol and triglycerides diet, without intranasal vaccine or vehicle, (C) and rabbits on high cholesterol and triglycerides diet before administration of intranasal vaccine (D). Notice the development of neointima in rabbits that ingested high cholesterol and triglycerides diet with no administration of intranasal vaccine (C, blue arrows and neointima thickness with black bars). Nevertheless, the rabbits on high cholesterol and triglycerides diet that later received the intranasal vaccine developed neontimas significantly thinner (C), similar to the ones of rabbits on normal diet and vehicle (B). Rabbits on normal diet did not show changes in the tunica intima (A). Hematoxylin and eosin stain (H&E). Total magnification: 160.

(12) FIG. 12 shows the serum levels of triglycerides and cholesterol in the treatment groups. A) Triglyceride concentrations after quarantine. B) Triglyceride levels after one month treatment. C) Total cholesterol concentrations after quarantine. D) Total cholesterol levels after one month treatment. Group 1 correspond to the control group on normal diet with no vehicle or vaccine; group 2 on normal diet+vehicle; group 3 on high cholesterol and triglyceride diet, group 4 on high cholesterol and triglyceride diet two weeks before starting vaccine administration; and in group 5 high cholesterol and triglyceride diet started at the same time as vaccine administration. LDL levels (mg/dl) group 1, 88.2; average for group 2, 93.7; average for group 3, 121.1; average for group 4, 73.1; average for group 5, 70.0. HDL levels (mg/dl) group 1, 34.1; average for group 2, 36.3; average for group 3, 29.2; average for group 4, 44.6; average for group 5, 49.8.

DETAILED DESCRIPTION OF THE INVENTION

(13) CETP is a hydrophobic glycoprotein joining HDL in blood plasma and promotes the transfer of cholesteryl esters and triglycerides among lipoproteins (Plump A. S., Masucci-Magoulas L., Bruce C., Bisgaier C. L., Breslow J. L., Tall A. R. Increased atherosclerosis in ApoE and LDL receptor gene knock-out mice as a result of human cholesterylester transfer protein trans gene expression. Arterioscler Thromb Vasc Biol 1999; 19: 1105-1110), process called lipid heterointerchange. Nevertheless, CETP has also been reported as participating in phospholipid heterointerchange (bidirectional transfer of the same lipid), although the net transfer is mainly carried out by Phospholipid transfer protein (PLTP) (Albers J J, Vuletic S, Cheung M C. Role of plasma phospholipid transfer protein in lipid and lipoprotein metabolism. Biochim Biophys Acta. 2012; 1821: 345-357). The movement of cholesteryl esters starts from HDL particles towards triglyceride-rich lipoproteins such as LDL and VLDL; CETP also transfers triglycerides from LDL and VLDL towards HDL, which causes a change in the composition, size, and spherical structure of HDL (Rye K. A., Hime N. J., Barter P. J. The influence of cholesteryl ester transfer protein on the composition, size, and structure of spherical, reconstituted high density lipoproteins. J Biol Chem 1995; 270:189-196).

(14) Extensive studies on CETP polymorphims and genetic deficiencies of this protein suggest a direct relation between CETP, HDL-cholesterol levels and cardiovascular disease; nevertheless, many aspects of CETP biological functions have not been discovered yet, neither the molecular bases related to the joining and transfer of lipids (Hall J., Qiu X. Structural and biophysical insight into cholesteryl ester-transfer protein. Biochem Soc Trans 2011; 39: 1000-10005).

(15) CETP is constituted of 476 residues, has a molecular weight of 66 kDa, residues Asp.sub.88, Asp.sub.240, Asp.sub.341 y Asn.sub.396 are glycosylated, it has five free cysteines and a high content of hydrophobic residues compared to other plasmatic proteins (about 44%). The tridimensional structure of CETP with a resolution of 2.2 was reported by the beginning of 2007. In general terms, the crystal reflects a long structure boomerang-like shaped, with dimensions of 1353035 and a folding similar to that of BPI (bactericidal/permeability-increasing protein). The structural description of CETP may be in four domains: a barrel on each side of the protein, called N-barrel and C-barrel, a central connecting -sheet between both barrels, and a C-terminus extension called X-helix, which is absent in BPI protein; each barrel has -sheets highly packaged along with two helices (A and B in N-barrel, and A and B in C-barrel).

(16) The crystallographic structure reveals a 60 -ling tunnel with a volume of 2560 . According to this tridimensional structure model, CETP may accommodate two molecules of cholesteryl ester inside and a phospholipid molecule associated to each gate of the tunnel, oriented in such a way that the fatty acid chains are towards the interior of the tunnel and the polar head groups located in interphase with water. The tunnel gates are big enough to let lipids in, one of them is protected by the X-helix in the region of the N-barrel and the through two structures called moving flaps (1 y 2) in the region of C-barrel. Likewise, mutagenesis and structural studies suggest that triglyceride and cholesteryl ester molecules (neutral lipids) move along the tunnel passing through the narrow central region with dimensions of 10 amplitude and 5 high (Qiu X., Mistry A., Ammirati M. J., Chrunyk B. A., Clark R. W., Gong Y., Culp J. S., et al. Crystal structure of cholesteryl ester transfer protein reveals a long tunnel and four bound lipid molecules. Nat Struct Mol Biol 2007; 14: 106-113).

(17) Currently, in our laboratory we have found that an alternative or simultaneous possibility to facilitate lipid transport among lipoproteic particles is by forming micellar structures associated to the C-terminus region of CETP (X-helix) when this region is in the structure of -helix (Garcia-Gonzlez V.; Mas-Oliva J. Structural Arrangement that Supports Lipid Transfer in the cholesteryl-ester transfer protein (CETP). USA-Mxico Workshop in Biological Chemistry: Multidisciplinary Approaches to Protein Folding, Mexico City, Mexico, 25-27 Mar. 2009).

(18) The resolution of the tridimensional structure of CETP allowed establishing that CETP joins just one lipoprotein at a time through its concave surface. This is a strong basis for the proposal that it operates by a carrying mechanism, in which it accepts neutral lipids from a donating particle, transports them through the aqueous phase and releases them in acceptor lipoproteins (Hamilton J. A., Deckelbaum R. J. Crystal structure of CETP: new hopes for raising HDL to decrease risk of cardiovascular disease? Nat Struct Mol Biol 2007; 14: 95-97).

(19) Biochemical studies have proved that CETP shows a high affinity for 10 nm-diameter nascent discoid HDL particles (Kd=20-120 nM), this size coincides with the one observed on the concave curvature of CETP, suggesting that it may join an individual particle of HDL through its concave surface with a modest movement of the X-helix and the moving flap 1. In order to adapt to lipoproteins of greater sizes, such as LDL, VLDL, a conformational change in the helices of the N- and C-barrels must take place. Likewise, the protein surface has several polar and hydrophobic residues evenly distributed, which suggest that the interactions with lipoprotein surfaces are equally distributed (Jiang X. C., Bruce C., Cocke T., Wang S., Boguski M., Tall A. R. Point mutagenesis of positively charged amino acids of cholesteryl ester transfer protein: conserved residues within the lipid transfer/lipopolysaccharide binding protein gene family essential for function. Biochemistry 1995; 34: 7258-7263) (Desrumaux C., Athias A., Masson D., Gambert P., Lallemant C., Lagrost L. Influence of the electrostatic charge of lipoprotein particles on the activity of the human plasma phospholipid transfer protein. J Lipid Res 1998; 39: 131-142).

(20) Therefore, one of the aims of this invention is to create a vaccine that inhibits CETP activity and increases HDL levels using the amino acid sequence H486 to S496 of CETP; that is, the last eleven residues of the protein. This sequence includes three of the four key residues to maintain the lipid joining and transfer capacity; these are L.sub.488, F.sub.491 y L.sub.495. The sequence of the synthetic peptide has homology with no other CETP epitopes or other mammal proteins, it shows a high homology, though, with CETP C-terminus of many species: 100% rabbit, human and monkey; and 90% hamster. Since the peptide is formed by just eleven residues, it presents just one window for recognition by the immunologic system.

(21) The efficiency of the immune response is determined by the administration route. In general, the advantages of a vaccine lie in that they are affordable, highly specific, and, in general, have few adverse effects. Nevertheless, an intramuscularly injected vaccine has the risks of contamination and lesions due to the use of needles; in addition, it requires trained personnel for its administration, thus increasing its cost. That is why in this invention nasal application is proposed, so that not requiring trained personnel for its administration, its cost does not increase. On the other hand, since it is a noninvasive route, the lesions generated by the use of needle, as well as the risk of contamination are avoided. It also more practical and painless, providing a greater acceptance by users, because it may be administered to people of any age in a faster way and without the fear provoked by injections.

(22) Currently some intranasal vaccines approved by FDA (Food and Drug Administration) exist, among them the trivalent vaccine against influenza caused by influenza virus subtypes A and B, commercially known as FluMist. This vaccine may be administered to people between 2 and 49 years of age and does not require trained personnel (McDonald J., Moore D. FluMistvaccine: Questions and answerssummary. Paediatr Child Health 2011; 16: 31). The side effects that normally appear are fever, nasal congestion, and nasal flow.

(23) When manufacturing a nasal vaccine, it must be considered that this route, as a rule, induces poor immunologic responses in the absence of stimulants or delivery vehicles (Hobson P., Barnfield C., Barnes A., Klavinskis L. S. Mucosal immunization with DNA vaccines. Methods 2003; 31:217-224). Hence, an appropriate administration system must be developed. Previous studies have described a quitosan-based system (a chitin-derived polysaccharide) widely studied due to its compatibility, biodegradability, and low toxicity. It also has the property to condense DNA, which allows DNA protection from degradation and the improvement of mucosal administration. In this study, plasmid pCR-X8-HBc-CETP (pCETP), encoding for CETP B cell epitope, exhibiting the central particle of hepatitis B virus condensed with quitosan was used to form quitosan/pCETP aggregates. Intranasal immunization with this preparation showed a long-term immune response in vivo, stimulating the production of anti-CETP antibodies, modulates lipoproteic profile in plasma and delays the deformation process of atherosclerotic plaques in rabbits. These results prove that intranasal vaccination is equivalent to intramuscular vaccination as for immunogenicity and suggest that intranasal vaccination may be a noninvasive convenient route for the administration of DNA vaccines.

(24) On the other hand, some CETP inhibitors have been developed and are in clinical trial phase, while others are currently in the preclinical phase (Zhao L., Jin W., Rader D., Packard C., Feuerstein G. A translational medicine perspective of the development of torcetrapib: Does the failure of torcetrapib development cast a shadow on future development of lipid modifying agents, HDL elevation strategies or CETP as a viable molecular target for atherosclerosis? A case study of the use of biomarkers and Translational Medicine in atherosclerosis drug discovery and development. Biochem Pharmacol 2009; 78:315-325), although it is likely that such drugs may continue presenting tolerance and adverse reactions. Hence, the use of a vaccine in which booster doses were administered temporarily, might lead to a better tolerance by the patient and, thus, to a reduction in atherosclerosis risks.

(25) One of the novel aspects of the present invention is the use of a vaccine compound constituted by lipids from Archaebacteria cell membranes (54% of total lipids). The use of these preparations has shown that they not only function as humoral adjuvants, but also promote a strong cytotoxic T-cell immune response characterized by long-term memory (Krishnan L., Sad S., Patel G. B., Sprott G. D. Archaeosomes induce long-term CD8+ cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway, in the absence of CD4+ T cell help. J Immunol 2000; 165:5177-5185). In some cases, the immune response is similar to that obtained with the potent, yet toxic, Freund's adjuvant. However, it has been proved that preparations with lipids derived from archaebacteria are not toxic (Patel G. B., Omri A., Deschatelets L., Sprott G. D. Safety of archaeosome adjuvants evaluated in a mouse model. J Liposome Res 2002; 12:353-372) (Patel G. B., Ponce A., Zhou H., Chen W. Safety of intranasally administered archaeal lipid mucosal vaccine adjuvant and delivery (AMVAD) vaccine in mice. Int J Toxicol 2008; 27:329-339). Actually, a number of successful trials using vaccines based on these lipids have already been performed (Conlan J. W., Krishnan L., Willick G. E., Patel G. B., Sprott G. D. Immunization of mice with lipopeptide antigens encapsulated in novel liposomes prepared from the polar lipids of various Archaeobacteria elicits rapid and prolonged specific protective immunity against infection with the facultative intracellular pathogen, Listeria monocytogenes. Vaccine 2001; 19:3509-3517) (Krishnan L., Dennis Sprott G.; Institute for Biological Sciences, National Research Council of Canada. Archaeosomes as self-adjuvanting delivery systems for cancer vaccines. J Drug Target 2003; 11:515-522); some even administered through intranasal route using ovalbumin as model antigen in a mice model trial which was successful (Patel G. B., Zhou H., Ponce A., Chen W. Mucosal and systemic immune responses by intranasal immunization using archaeal lipid-adjuvanted vaccines. Vaccine 2007; 25:8622-8636).

(26) Archaebacteria represent one of the three primary kingdoms or domains of living organisms. They are unicellular organisms without nuclear envelope and with a low content of deoxyribonucleic acid. They include thermophile, halophile, and acidophile organisms, collectively known as extremophiles. Some authors have proposed that they are similar to organism living in the primitive biosphere. Many of these species are methanogenic, even those found in freezing environment. Four archaea phylums are known, Euryarchaeota, Crenarchaeota, Korarchaeota y Nanoarchaea. These organisms live in extreme habitats, like hot springs, and highly-saline or highly-alkaline water or in acid conditions. It has made evident that a great number of these organisms may constitute up to 20% of the ocean biomass in soft environmental conditions (Peret J., Lpez-Garcia P., Moreira D. Ancestral lipid biosynthesis and early membrane evolution. Trends Biochem Sci 2004; 29: 469-477).

(27) The lipids from the cell membrane of these animals have an important amount of polar lipids which are unique and characteristic, based on the 2, 3-dialkylglycerol skeleton. These alkyl groups are isoprenoid and the simplest molecules derived from this type are derivatives from 2,3-dibiphytanyl-O-sn-glycerol (archeol); for instance, two isoprenoid units of 20 carbons joined at positions sn-2 and sn-3 of glycerol. These alkyl chains are generally saturated; nevertheless, some forms have double bonds in different positions. These molecules have one or two groups of polar head, which may be different with units 2, 3-sn-glycerol joined by C40 alkyl components which are also isoprenoid molecules. For instance, calarcheol (called like this because it is the predominant form in some thermophile archaebacteria), it has two C40 isoprenoid units bonded from positions 2 to 3 and from position 3 to 2 (Chong P. L. G., Archaebacterial bipolar tetraetherlipids: Physico-chemical and membrane properties. Chem Phys Lipids 2010; 163:253-265).

(28) Some lipids of this kind have two methyl groups and from one to four cyclopentane rings, whereas Crenarchaeota may have one cyclohexane ring additional to the alkyl chains. Other related molecules with up to eight cyclopentyl rings have been observed in naphthenate deposits during the processing of crude oil. These exist as both phosphor- and glycolipids (or in combination), and as the sulfated form of them. Most groups of the phospholipid polar heads are similar to the ones of organisms of primary kingdoms and include ethanolamine, L-serine, glycerol, myo-inositol, and choline in phosphodiester bond. Nevertheless, it is important to observe that some unique polar groups such as di- and trimethylaminopentanotetrol and eigiycosaminyl-myo-inositol may be found in some Archea species (Koga Y., Morii H. Recent advances in structural research on ether lipids from Archaea Including comparative and physiological aspects. Biosci Biotechn Biochem 2005; 69: 2019-2034).

(29) The present invention consists of a vaccine compound for intranasal administration, which uses a preparation of micellar nanoparticles including CETP C-terminus as immunogen, phosphatidylcholine, lysophospholipid and lipids from the cell membrane of Archaebacteria Thermus aquaticus as a successful mixture to promote an adaptive immune response in the mammal.

(30) During the development of our vaccine compound a series of conditions were assessed such as lipid concentration and composition. Thus one of the novel aspects of this invention is the use of lysophosphatidylcholine, a molecule that we identified as inducing the formation and stabilization of the -helix secondary functional structure in the Y-helix peptide (antigen of the vaccine compound). Likewise, a series of phospholipids were assessed in the compound, with different length in the fatty acid chains, modifying the electrostatic properties of the polar head, varying the concentration, even some preparations with cholesterol were assessed. Nevertheless, in all the referred conditions no change in the secondary structure of the antigen were observed, which is the C-terminus peptide of CETP, only lysophosphatidylcholine (C.sub.12) favored the presentation of the -helix structure, which is the functional conformation that facilitates the lipid transfer function in CETP. This characterization was accomplished using techniques as spectroscopy with ocular dichroism, fluorescence, and electronic microscopy.

(31) The lipid structure of Archaebacterian lipids composed by long-chain alkyl groups bound by ether bonds with glycerol is a characteristic that allows this molecules to have scaffolding functions in micellar nanoparticles, which, at the same time determines the size and increases in an important manner the stability of such micelles.

(32) After finishing a huge number of trials, the optimal concentration of vaccine components was defined, obtaining the following result, which does not mean being limitative of the present invention, but it means that this is the concentration at which the best results were obtained during the preparation of the unilamelar particles

(33) Phosphatidylcholine 3 mM.

(34) Archaebacterian lipids 6 mM.

(35) Lysophosphatidylcholine 2 mM.

(36) Antigen (Y-helix) 2.9 mM.

(37) Therefore, the molar ratio lipid/peptide is 3.8/1, obtaining micellar nanoparticles. Likewise, trials were performed to define and standardize the methodology for manufacturing the vaccine.

(38) Considering the physicochemical properties of the antigen, like hydrophobicity, the isoelectric point and the hydrophobic moment, and with the objective of preventing the appearance of aggregation in the Y-helix peptide, after a series of trials it was determined that pH 9.5 (buffered with a solution of NaHCO.sub.3/Na.sub.2CO.sub.3 mM) is an optimal condition that allows solubility and keeps the peptide in monomeric state.

(39) On the other hand, considering the high concentration of the lipid mixture (11 mM), several conditions for the preparation of the micellar nanoparticles were assessed; nevertheless, the method was by using a 10 h drying under continuous N.sub.2 flow and an extensive sonication process, which is detailed in the methodology section. So that the molar ratio, 3.8:1 (lipid:peptide), was the best to maintain a high stability of the formula, and preserve the secondary structure in -helix of Y-helix.

(40) In conclusion, during the development of this invention, a formulation with a great number of advantages was used; while nanoparticles with a homogeneous size and good stability, in which the -helicoidal functional secondary structure of the antigen is maintained, were produced. This guarantees a directed specific immunological response against the C-terminus of CETP. Particularly, the characteristics of the Archaebacterian lipids permit a system which, unlike other adjuvants, provokes minor side toxic effects, whereas maintains the stability of the formulation and improves the absorption in the mucosa. This technological development may also be the basis for other therapeutic applications.

(41) Preparation of the Vaccine Compound

(42) Materials

(43) L--phosphatidylcholine (PC) and 1-Lauril-2-Hydroxi-snglycero 3 phosphocholine (Lyso C.sub.12) were obtained from Avanti Polar Lipids. The reagents sodium bicarbonate, sodium carbonate, sodium azide, monobasic and dibasic sodium phosphates used in the preparing of the buffer solutions and cholesterol were from Sigma-Aldrich. The preparation of Archaebacterian membrane lipids was made from the organism Thermus aquaticus (American Type Culture Collection, ATCC).

(44) Peptide Synthesis

(45) A peptide corresponding to the last 11 residues of the C-terminus end of CETP (CHLLVDFLQSLS, SEQ ID NO: 1) U.S. Pat. No. 7,749,721 (Alonso et al) was synthesized. This peptide has a cysteine residue in its amino end. The so called Y-helix was obtained with purity over 98%. The identity and the degree of purity were compared through HPLC and mass spectrometry.

(46) Dissolution and Quantification of Y-Helix

(47) For the preparation of the vaccine, a Y-helix solution was made at a concentration of 4 mg/ml in NaHCO.sub.3/Na.sub.2CO.sub.3 buffer, 50 mM (pH 9.5). The concentration was determined through the absorbance of the peptidic bond at 205 nm, using a UV-Visible HP8452A Spectrophotometer with diodes arranged.

(48) Vaccine Preparation

(49) The lipid mixture designed in this protocol permits keeping the Y-helix antigen in its -helix functional secondary structure on the surface of the nanoparticles, condition that must promote a greater immunological response once the vaccine is administered. On the other hand, the addition of phosphatidylcholine to the mixture of Archaebacterian lipids and Lysophosphatidylcholine facilitates the manufacturing of the micellar nanoparticles, since it gives them structure and stability. The following procedure has been defined according to several trials performed with the methodologies described in our laboratory.

(50) a) All the material is washed with chloroform and with H.sub.2O/Ethanol 1:1 solution.

(51) b) Specific amounts of phosphatidylcholine, Archaebacterian lipids, and C.sub.12 phospholipid (LisoC.sub.12) are homogenized in chloroform/methanol 9:1. This mixture is placed under a continuous flow of N.sub.2 in the darkness until solvents have evaporated completely (about 10 h).
c) Then, lipids are re-suspended in the carbonate buffer 50 mM (pH 9.5). Immediately after this, it is shaken vigorously for 10 minutes to incorporate the total amount of lipids into the solution.
d) The mixture is then practiced an extensive sonication process to obtain the micellar particles. This step is carried out with Branson equipment (SONIFIER 250 with a 10 mm diameter titanium probe) with pulses of 15 s for 30 s of rest, during 5 intervals of 10 minutes each under N.sub.2 flow at 4 C.
e) Once sonication is over, the micellar nanoparticles must settle for 150 minutes at 25 C. in darkness. Then they are centrifuged at 13000 rpm for 15 minutes and are filtrated through membranes with pores of 0.45 m. Samples of the particles were processed through transmission electron microscopy with the negative staining technique to confirm their correct manufacturing.
f) The peptide is incorporated to the micellar nanoparticles by soft mixing (80 rpm) and incubation at 25 C. for 20 minutes.
g) The preparation is aliquoted and kept refrigerated at 4 C., showing a high stability under this storage conditions.

(52) The final lipid concentrations were as follows: phosphatidylcholine 3 mM, Archaebacterian lipids 6 mM, and Lyso C122 mM. The final concentration of the peptide was 4 mg/ml (2.91 mM); the molar ratio of total lipids in relation to the peptide was 3.8/1. Lyso C12 is able to modulate the formulation of the -helix structure in apolipoprotein segments, as well as in the C-terminus domain of CETP; therefore, it maintains the functional structure of nanoparticles in -helix of the incorporated peptide.

(53) Examples of Usage

(54) The vaccine compound was tried in White rabbits of the New Zealand species with initial weights of 2.0-2.5 kg, which were kept 12 days on normal diet in the vivarium of the Cellular Physiology Institute, UNAM, as a quarantine period. This facility meets the requirements of the Norma Oficial Mexicana (Official Mexican Regulation) NOM-062-ZOO-1999, entitled Technical Specifications for the Production, Care and Use of Laboratory Animals. In addition to observing this regulation, for animal care and management, also the Guide for the Care and Use of Laboratory Animals backed up by the National Institutes of Health (NIH) of the United States and the Declaration of Helsinki were also observed. 16 rabbits were used.

(55) The normal diet consists of rabbit-specific food 5321 from LabDiet, with the following composition: crude protein not less than 16%; crude fat not less than: 2.5%; crude fiber not more than: 18%; ash not more than: 8%; additional minerals not more than: 2.1%. In the high-cholesterol diet a mixture of cholesterol 1% and corn oil 10% were added to the normal food. All rabbits were fed ad libitum.

(56) After the quarantine period, the administration of the high-cholesterol diet to groups 3, 4 and 5 was started. After 15 days, vehicle and vaccine administration to groups 2, 4 and 5 was started. 50 l of vehicle and vaccine were nasally administered twice a week. This treatment lasted three months.

(57) The following table summarizes the animal groups used.

(58) TABLE-US-00001 TABLE 1 Groups of experimental animals used in the different trials Group Treatment characteristics Num. of animals 1 Normal diet (control) 1 2 Normal diet + vehicle 3 3 High-cholesterol diet (1%) 5 4 High-cholesterol diet + vaccine 4 5 High-cholesterol diet + vaccine 3

(59) In group 4, high-cholesterol diet started two weeks before vaccine administration.

(60) In group 5, high-cholesterol diet started at the same time of vaccine administration.

(61) After the quarantine period and after 12 hr fast, blood samples were taken from the marginal vein of the ear from the different groups of experimental rabbits, and then every 15 days until the end of treatment. The serum fraction was sent to the Biochemistry Laboratory of the Pathology/Clinical Pathology Department of Facultad de Medicina Veterinaria y Zootecnia, UNAM, for triglyceride and total cholesterol analysis.

(62) After experiments concluded, the animals were sacrificed with a lethal dose of pentobarbital sodium, then cardiac perfusion was performed with Krebs-Ringer solution (Glucose 5 mM, NaCl 1.2 mM, KCl 1.75 mM, NaHCO.sub.3 24 mM, KH.sub.2PO.sub.4 1.2 mM, MgSO.sub.4 1.2 mM, EDTA15 mM). Then, representative samples of the liver, thoracic and abdominal aorta, heart, and small intestine were collected.

(63) Histopathologic Analysis of the Liver

(64) Fragments no greater than 1 cm.sup.3 were used, which were treated with formaldehyde buffered to neutrality to 10% at 25 C. for 24 hours to continue with the normal histological technique of embedding in paraffin wax and cutting into slices. The samples were oriented to obtain transverse and longitudinal cuts from 4 to 6 m thick and stained with hematoxylin and eosin (H&E) and with Masson's trichrome stains. The observation of the sections was performed in single blind using an optical microscopy equipped with a digital camera.

(65) The livers of rabbits on normal diet, Group 1, clearly showed the laminar or normal mural organization of hepatocytes, preserving the lobular and acinar organization, as can be seen in FIG. 1A. Adjacent sinusoids were visible all the time, containing numerous circulating erythrocytes as it may be seen in FIGS. 1B, 1C, and 1D. As a rule, no pathologic change was observed in the cytology of any processed sample.

(66) The livers of the animals that received the vehicle and had the normal, Group 2, showed few noticeable changes, as can be seen in FIG. 2. The most common characteristic observed was some steatotic centrilobular hepatocytes with scarce vacuoles. Nevertheless, the sinusoids were visible and preserved the laminar and lobular structure of hepatocytes (FIGS. 2C and 2D).

(67) In contrast, the livers of rabbits fed on high cholesterol diet which received no vaccine, Group 3, showed variable degrees of steatosis, both microvacuolar and macrovacuolar, from mild to severe and, in some cases diffuse, affecting great portions of the hepatic parenchyma as can be seen in FIG. 3. In animals with mild to moderate steatosis, the component mainly affected was the centrilobular hepatocytes, which had a microvacuolar cytoplasmic appearance, although their nuclei were not displaced to the peripheral region of the cell. Some of these hepatocytes showed cytoplasm distention due to the presence of lipid drops (FIGS. 3B, 3C and 3D), which looked like vacuoles because the cell contents had been eliminated by the solvents used during the process of paraffin wax embedding and cutting (FIGS. 3E and 3F). Most hepatocytes from the lobular periphery, close to the portal triads, had normal appearance although some of them showed microvacuolar cytoplasm. The liver that macroscopically always presented a creamy color, showed centrilobular hepatocytes as well as hepatocytes of the paracentral region of the lobule, as a whole almost 70% of the lobule, with a considerable degree of overlapping between the two main morphologic patterns: microvesicular and macrovesicular, as can be seen in FIGS. 3B and 3C, where lipid drops are greater and coalesce until becoming a great drop or vacuole of fat which moves the nucleus and cytoplasm to the periphery of the cell (FIG. 3D). These hepatocytes are hardly recognizable as such, because they have a morphology similar to small adipocytes. Another characteristic found in these animals was that the bile canaliculi are distended and some bile ducts apparently show cholestasis.

(68) The combination of steatosis, the presence of polymorphonuclear leukocytes, monocytes or both, ballooned hepatocytes and areas of necrosis. A datum important to mention is that necrotized hepatocytes were extraordinarily rare, even in the rabbits with the worst hepatic damage; therefore, steatohepatitis was ruled out in the samples studied.

(69) In the rabbits that received a high-cholesterol diet before administering the vaccine, Group 4, showed less changes than the ones described for the rabbits that just received a high-cholesterol diet with no vaccine administration (FIG. 4). In this case, the lesions are more localized and less extended in the lobule (FIGS. 4B, 4C, 4D). In most cases the affectation is restricted to the cells closed to the centrilobular veins (FIG. 4E). However, some groups of hepatocytes with macrovacuolar steatosis of focalized distribution were also observed, as can be seen in the different images of FIG. 4.

(70) In the samples of livers from rabbits that received the vaccine and a change in the high-cholesterol diet, simultaneously, Group 5, centrilobular hepatocytes with some microvacuole were found (FIGS. 5C, 5D and 5E), with an apparent size increase, although without hepatocyte ballooning, as in the case of rabbit that only received high-cholesterol diet, as can be seen in FIG. 3. Just like in the former groups, Periportal and paracentral hepatocytes of the lobule show a practically normal appearance, while sinusoids adjacent to the centrilobular vein seem to be somewhat occluded (FIGS. 5C, 5E). The administration of the vaccine simultaneously with the start of the high-cholesterol diet has a protective effect on the hepatic parenchyma, as can be concluded from the minimal cytoplasmic alteration of hepatocytes observed.

(71) FIG. 6 shows the histological sections of the livers of control rabbit fed on normal diet without the administration of vehicle or vaccine, Group 1, stained with Masson's trichrome technique. FIG. 6A shows a normal portal triad in the periphery of the hepatic lobule. FIG. 6B shows the normal structure of a central vein, surrounded by hepatocyte layers, into which sinusoids drain. The histological sections of livers from control animals which received the vehicle intranasally, Group 2, show normal structures as can be seen in FIGS. 7A and 7B, similar to the ones of FIG. 6.

(72) In contrast, the histological sections of livers from animals fed on a high-cholesterol and triglyceride diet, without administration of vaccine or vehicle, Group 3, show the development of an important perisinusoidal fibrosis close to the portal triad, as can be seen in FIG. 8A, and of the centrilobular vein (FIG. 8B). These changes are more frequently observed in the zone III of the hepatic acinus, corresponding to the center of the classical lobule. An association between the presence of ballooned hepatocytes and perisinusoidal fibrosis, near the central vein was frequently observed (FIG. 8B).

(73) The administration of the vaccine after the starting of high-cholesterol and triglyceride diet, Group 4, significantly improved the histological appearance of the liver as can be seen in FIGS. 9.sup.a and 9B; since they showed features similar to the ones of control animals of FIG. 6. The same histological improvement was seen in the liver samples from animals treated with the vaccine at the beginning of the high-lipid diet, Group 5 (FIGS. 10A and 10B).

(74) Histopathologic Analysis of the Abdominal Aorta

(75) The histopathologic analysis showed an evident increase in the thickness of the intima (neointima) of the animals treated just with a high-cholesterol and triglyceride diet, Group 3, as can be seen in FIG. 11C. In animals treated with either placebo (Group 2, FIG. 11B) or high-cholesterol diet before vaccine administration (Group 4, FIG. 11D), no significant thickening of the intima was observed, presenting an appearance closely similar to the observed in the aortas of animals fed on normal diet (FIG. 11A). Based on these observations, we can conclude that treatment with the vaccine formed by micellar nanoparticles is able to delay the process of atherosclerotic plaque formation.

(76) Total Cholesterol and Triglycerides

(77) After one month's treatment (the full treatment lasted three months) a decrease in the serum levels of total cholesterol and triglycerides was observed in the groups that received the vaccine (Groups 4 and 5), being more evident in the Group 5, which started the high-cholesterol diet along with the vaccine administration. The results can be seen in the graphs of FIG. 12.

(78) The results obtained using the formulation described in the present invention prove that intranasal immunization with micellar-nanoparticled vaccine preparation consisting of Archaebacterian lipids, phosphatidylcholine, lysophosphatidylcholine, and carboxyl-terminus of CETP protein, modifies the profile of plasmatic lipoproteins. The level of total cholesterol significantly decreased in the group treated with the vaccine, compared to the group just treated with a high-cholesterol diet. Also HDL-C levels increased in animals fed on high-cholesterol diet and treated with the vaccine compound of the present invention, in relation to the animals with similar diets but not treated with the vaccine compound.

(79) The most important finding using the present invention is that when plasma lipids are higher than normal, and treatment with the vaccine compound of the present invention is administered, a significant decrease in the thickness of the tunica intima of the abdominal aorta is clearly seen, which is directly related to the decrease in the formation of neointima and atherosclerotic plaques. The thickening of the intima and the area of lesions in the aorta were noticeably reduced in the groups treated with the vaccine compound, when compared to the abdominal aortas of animals that did not the vaccine. Another important aspect of the vaccine compound of the present invention is the protection against the development of non-alcoholic fatty liver. This protection is related to a decrease in both microvesicular and macrovesicular steatosis associated with a decrease in perisinusoidal and perivenular fibrosis. All our data suggest that intranasal immunization with lipid/CETP micellar nanoparticles inhibits the progression of the disease known as atherosclerosis.

(80) In conclusion, it is demonstrated that a lipid/CETP micellar-nanoparticled vaccine compound for intranasal administration was developed, which induces anti-CETP antibodies, modulates the profile of plasmatic lipoproteins, delays the process of atheroma-plaque formation in arteries, and protects against the development of non-alcoholic fatty liver. The experimental results presented in this document suggest that nasal vaccination is an appropriate route for the administration of vaccines based on the use of peptides obtained from proteins involved in the development of atherosclerosis such as the Cholesterylester Transfer Protein.