text:
|Publication number
|Publication type
|Application number
|Publication date
|Mar 9, 1999
|Filing date
|Apr 11, 1994
|Priority date
|May 8, 1991
|Fee status
|Publication number
|Reinhard Gluck, Robert Mischler
|Original Assignee
|Schweiz, Serum- & Impfinstitut Bern
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|Patent Citations (11), Non-Patent Citations (43), Referenced by (59), Classifications (33), Legal Events (5)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation-in-part application of U.S. patent application Ser. No. 07/965,246, filed Mar. 3, 1993, which claims priority from PCT patent application EP 92/01014, filed May 8, 1992, which claims priority from European patent application EP 91 10 7527.3, filed May 8, 1991, and EP 91 10 7647.9, filed May 10, 1991.
The invention relates to immunostimulating and immunopotentiating reconstituted influenza virosomes (IRIVs) and to vaccines containing them.
A large range of procedures to enhance immunogenicity has been developed over several decades by measures that retain a considerable empirical element. The most potent methods (e.g. administering the immunogen together with Freund's complete adjuvant) combine a number of the separate principles explained in the following sections:
Particles are more attractive to macrophages than soluble antigens and tilt the balance in favor of immunity rather than suppression. Particle formation can vary from a simple heat-induced aggregation to sophisticated polymerization strategies, including the self aggregation characteristic of antigens, such as the soluble antigen of hepatitis B virus. In the case of liposomes or oily droplets, there is a combined effect of particulateness and slow absorption, such as with alum precipitation.
A long history of research underlies the search for a pure, safe, effective, nontoxic small organic molecule which mimics the potentiation of the whole immune response that can be achieved with killed Mycobacterium tuberculosis bacteria or toxic microbial extracts, such as E. coli LPS. No uniformly approved satisfactory agent has been found for use in humans and a disconnection of toxicity and efficacy is difficult to achieve.
There is some evidence that the co-administration of, for example, IL-2 with an antigen can result in a greater enhancement of the immune response than the separate administration of the antigen and the interleukin (see Staruch, M. J. and Wood, D. D., J. Immunol., 130:2191,1983). Before this approach becomes feasible as an immunization strategy in humans, it requires further extensive investigations. However, it may be rendered obsolete by the suggestions included in the following sections.
The sudden application of large doses of pure protein antigens includes the risk of activating the suppressor pathways in the immune responses. Particularly if the intravenous route is used (see Nossal, G. J. V., New Generation Vaccines, Marcel Dekker, Inc., New York, Basle, eds. Woodrow, Levine, 85, 1990). Slow release from a subcutaneous depot site permits extensive access of the antigen to the widely scattered dendritic cells and macrophages, and it also ensures that antigen will still be available after the initial burst of clonal proliferation, thereby permitting some facets of a secondary response. Slow release is favored by adsorbing antigens onto aluminum hydroxide ("alum precipitation"); placing antigens into water-in-oil emulsions; incorporating antigens into liposomes; and other similar manipulations. This method is conceptually close to the one described in section A.
If a particular vaccine is highly immunogenic, the adjuvant effect of this vaccine, and also the characteristics it may possess for guiding the response toward a particular immunological pathway, may "spill over" into a response to an antigen co-administered with it. For example, killed Bordetella pertussis or Corynebacterium parvum bacteria are powerful immunogens; if a pure protein is administered with the same injection, the response to it is enhanced. Certain immunogens (for reasons that are unclear) guide the response in particular directions. For example, extracts of a parasite, such as Nippostrongylus brasiliensis, elicit powerful IgE responses. Pure proteins co-administered with the parasite extracts will also evoke an IgE response (see Nossal, G. J. V., New Generation Vaccines, Marcel Dekker, Inc., New York, Basle, eds. Woodrow, Levine, 85, 1990). Presumably, this effect is somehow connected to the production of particular lymphokines which is induced by particular agents. Said lymphokines, such as IL-4, guide isotype switch patterns. The polyclonal activating characteristics of lymphokines may also form the basis for the enhancement of immune responses in general.
The notion of genetically engineered microorganisms as antigen gene carriers was pioneered by Panicali, D. and Paoletti, E. (Proc. Natl. Acad. Sci. USA, 79:4927 1982), and Smith, G. L., Macket, M. and Moss, B. (Nature, 302:490, 1983), who genetically engineered the genome of the vaccinia virus to additionally include genes coding for important host-protective antigens of various pathogens. These are synthesized by the infected cell together with vaccinia virus particles and antigens. An improvement of this concept was introduced by Langford, C. J. , et al. (Mol Cell. Biol, 6:3191, 1986). With the idea in mind that cell surface-associated antigens are more likely to evoke a strong T cell response than secreted antigens, they linked a DNA sequence encoding the transmembrane domain of an immunoglobulin heavy chain to the gene encoding the soluble S antigen of Plasmodium falciparum, and inserted the resulting hybrid gene into the genome of a vaccinia virus. The construct caused a significantly enhanced immunogenicity. Live Salmonella, BCG and measles virus have also been successfully used for the expression of foreign antigen. Thus, the advantages of a live attenuated vaccine can be combined with those of a vaccine based on viruses containing recombinant DNA.
A further development of this idea is to insert genes for various interleukins into genetically engineered vaccinia viruses already carrying genes for important antigens. For example, the immune response to vaccinia virus itself can be markedly enhanced by the insertion of the IL-2 gene into the viral genome, permitting immunodeficient mice to recover from an otherwise fatal infection (Ramshas, I. A. , Andrew, M. E. , Philips, M., et al, (Nature, 329:545, 1987).
Surface-active agents such as saponin or Quil A\ in immunostimulating complexes (iscoms) have been used in a number of experimental and veterinary vaccines. They improved the immunogenicity of several antigens, especially of viral membrane proteins.
All of the above-mentioned "adjuvanting methods" have several disadvantages. Alum precipitation is disadvantageous because of the undesirable side effects such as local reactions, and its proinflammatory and encephalopathogenic potential. Surface active agents display a number of side reactions: they are irritating, proinflammatory, they bind to cholesterol and lyse cells. Interleukins can provoke systemic reactions and, therefore, routine use in mass vaccination may be undesirable.
Safety concerns prevented the use of genetically engineered microorganisms as carriers of genes for important antigens in man. Co-exhibition of the antigen with a highly immunogenic agent is only feasible with a limited class of small peptides. Rendering the antigen particulate often goes in parallel with a significant loss of the amount of antigen. The immunostimulatory effect of liposome-associated antigen on the humoral response is a widely recognized phenomenon, but immunopotentiation is limited and the mechanism by which this potentiation occurs is not totally elucidated at present.
Thus, the technical problem underlying the present invention is to provide immunostimulating and immunopotentiating agents which do not display the above-mentioned disadvantages.
FIG. 1 is an electron micrograph of an IRIV showing reconstitution produce negatively stained with phosphotungstate before linking of the antigen to the surface of IRIVs: Mixed phospholipids are spiked with biologically fully active influenza hemagglutinin glycoprotein trimers.
FIG. 2 is a schematic diagram illustrating the principle of the procedure of preparing IRIVs (a) intact influenza virus (b) mixture of phospholipids; (c) intact, inactivated hepatitis A virions; (d) soluble influenza spike subunit antigens containing the HA and viral phospholipids; (e) phospholipid membranes without antigens; (f) covalently bound cross-linkers on the surface of HAV antigen; (g) IRIV containing the reconstituted membrane with phospholipids extracted from viruses and other phospholipids carrying the influenza spike proteins including HA and the HAV on the surface.
FIG. 3 is a bar graph comparing IRIV-HAV vaccines versus Al-HAV vaccines.
FIG. 4 is a bar graph comparing the immunogenicity of IRIV-HAV vaccines with those of Al-HAV vaccines.
FIG. 5 is a graph comparing the biological fusion activity of different reconstituted influenza vesicles.
FIG. 6 is a schematic of the preparation of an IRIV-HAV-HBx combination vaccine.
FIG. 7 is a schematic of the preparation of an IVIR traveller vaccine having HAV, HBs, diphtheria and tetanus antigens attached to the surface.
FIG. 8 is a graph showing the results of fusion tests with live influenza virus.
The solution to the above technical problem is achieved by providing the immunostimulating reconstituted influenza virosomes (IRIVs) and vaccines containing said IRIVs which are characterized in the claims. These IRIVs can be used as vehicles which actively transport desired antigens of pathogens (or entire pathogens) to antigen presenting cells, such as macrophages or B cells, which then appropriately present said antigens to the immune system so as to induce an immune response.
Accordingly, IRIVs are provided which contain the following components:
(a) a mixture of phospholipids;
(b) essentially reconstituted functional virus envelopes;
(c) an influenza hemagglutinin (HA) or a derivative thereof which is biologically active and capable of inducing the fusion of said IRIVs with cellular membranes and of inducing the lysis of said IRIVs after endocytosis by antigen presenting cells, preferably macrophages or B cells; and
(d) an antigen.
The "mixture of phospholipids" of feature (a) contains natural or synthetic phospholipids or a mixture thereof. At least it contains two different compounds selected from the group of glycero-phospholipids, such as phosphatidylcholine or phosphatidylethanolamine, and cholesterol. Phosphatidylcholine and phosphatidylethanolamine are preferred, in particular in a ratio of 4:1. In preferred embodiments of the present invention, the ratio of said mixture of phospholipids (a) to said essentially reconstituted functional virus envelopes (b) is about 1:1 to about 20:1. Most preferably it is about 10:1.
The term "essentially reconstituted functional virus envelopes" refers to reconstituted influenza virus envelopes which are essentially devoid of the components which naturally occur inside of (below) the influenza virus envelope's membrane part. In a preferred embodiment the essentially reconstituted functional virus envelopes exhibit the form of a unilamellar bilayer. An example of such a lacking component is the matrix protein of the natural influenza virus envelope.
The term "biologically active HA or derivative thereof" as components of the IRIVs of the present invention refers to HAs or derivatives which substantially display the full biological activity of nature HA and are thus capable of mediating the adsorption of the IRIVs of the present invention to their target cells via sialic acid-containing receptors. Furthermore, such HA components can be recognized by circulating anti-influenza antibodies. This biological activity is an essential feature of the IRIVs of the present invention.
Thus, the function of the HA component of the IRIVs of the present invention may be explained as follows:
(1) it binds to a sialic-acid (N-acetyineuramininc acid) containing receptor on a target cell to initiate the virosome-cell interaction;
(2) it mediates the entry of the IRIVs into the cytoplasm by a membrane-fusion event and thus finally leads to the release of the transported antigen; and
(3) it serves as a "recognition antigen" since most humans can be considered "primed" to HA due to prior exposure through disease or vaccination.
Thus, the essential feature of the IRIVs is that they carry on their surface beside the antigen said biologically active viral glycoprotein (HA) or derivative thereof. This component of the IRIVs of the present invention induces their immediate fusion with cellular cytoplasmic membranes and a quick release of the transported antigen, e.g., into the membranes of said cells. Thus, an undesired long stay of the transported antigen in the endocytosomes where they may be unspecifically degraded is avoided.
The fact that an antigen should be palatable for macrophages and other accessory cells is paramount. For this purpose, the particulate nature of the IRIV is advantageous since it is, like all microorganisms, a particulate entity. The presence of antibody in human bodies (in the case of influenza, all human beings have antibodies against influenza antigen. These antibodies originate either from a previous influenza infection or from a vaccination) speeds entry of antigens recognized by said antibodies not only into macrophages, but also into lymphoid follicles, in which antigens are retained long-term in an extracellular location on the surface of follicular dendritic cells. This process of entering macrophages and lymphoid follicles is called opsonization.
Binding by antibody has another consequence for the immunogenicity of antigens. Whereas a given antigen, A, in solution will only bind to B cells exhibiting antibody molecules of the specificity anti-A on their surface, immune complexes can adhere to any B cell via the Fc-receptor. Due to the capacity of B cells in afferent lymph vessels to enter B cell areas of lymph nodes, this unspecific binding via the Fc receptor is probably one route, in a natural infection, by which said antigen is transported to lymphoid follicles and elsewhere in lymphatic tissue (Nossal, G. J. V., New Generation Vaccines, ed. Woodrow, G. C. and Levine, M. M., Marcel Dekker, Inc., 85, 1990). The mechanism would be an adjunct to the transport by monocytes. Hence, the presence of influenza antigens on the surface of the IRIVs favors the immunological mechanism of opsonization.
In one embodiment, the IRIVs of the present invention contain the complete HA which is synthesized as a single polypeptide chain of 550 amino acids which is subsequently cleaved by removal of arginine 329 into two chains, HA1 (36,334 daltons) and HA2 (25,750 daltons). These chains are optionally covalently linked by a disulfide bond involving the cysteine in HA1 position 14 and the cysteine in H position 137 and the two-chain monomers are associated noncovalently to form trimers on the surface of IRIVs. These HA1 or HA2 peptides can be obtained from natural or synthetic sources or by genetic engineering.
In a preferred embodiment, the present invention relates to IRIVs wherein said antigen is derived from a pathogen including parts thereof. Preferred examples of such pathogens are a virus, a bacterium, a parasite, anti-idiotypic (Antild) antibodies mimicking said viruses, bacteria or parasites, antibodies against said viruses, bacteria or parasites, or a toxin. Examples of viruses are hepatitis A, B, C, D or E virus, Polio virus, HIV, Rabies virus, Influenza virus or Parainfluenza virus. Examples of bacteria are Pseudomonas, Kiebsiella, E. coli Salmonella typhi, Haemophilus influenzae, Bordetelia pertussis, Clostridium tetani, or Corynebacterium diphtheriae. An example of a parasite is Plasmodium falciparum.
In a particularly preferred embodiment of the present invention, said pathogen is a hepatitis A virus (HAV). A particularly preferred HAV is the HAV strain RG-SB XA112, which was deposited under the requirements of the Budapest Treaty at the Collection Nationale de Cultures de Microorganismes (CNCM) of the Pasteur Institute on Apr. 11, 1991, under the accession number 1-1080. This deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty) on Apr. 11, 1991. This assures maintenance of a viable culture for 30 years from the date of deposit. The organism will be made available at Collection Nationale de Cultures de Microorganismes (CNCM) under the terms of the Budapest Treaty which assures permanent and unrestricted availability of the progeny of the culture to the public of any U.S. or foreign parent application, whichever comes first and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. �122 and the Commissioner's rules pursuant thereto (including 37 CFR �1.14 with particular reference to 886 OG 638).
In another preferred embodiment, the IRIVs of the present invention contain the structural proteins of hepatitis A virus, VP1, VP2, VP3, VP4 or the core protein of said HAVs as the antigen.
In a further preferred embodiment of the IRIVs of the present invention, the antigen is located in the membrane. The inactivated HAV antigen or subunit thereof is optionally coupled to the surface of the IRIV. This can be achieved by covalently binding the antigen with a suitable crosslinker molecule (e.g., N-succinimidyl 3-(2-pyridyidithio) propionate, SPDP) or by spontaneous lipophilic binding to various phospholipids or glycolipids in the membrane of the IRIV .
In a further preferred embodiment the antigen, preferably the inactivated HAV antigen or subunit thereof, is adsorbed onto the surface of IRIVs in a non-covalent manner.
In another preferred embodiment of the present invention, said antigen is located inside of the IRIVs. Several HAV particles of the strain RG-SB or soluble subunits thereof are enclosed by the reconstituted membrane of the IRIV. The antigen is in a soluble state within the core fluid of the IRIV.
In a further preferred embodiment of the present invention, said HA derivative is the influenza HA fusion peptide. Since the fusion of the antigen carrier, IRIV, with the cell membrane of the immuno-competent cells is a fundamental principle of the present invention, the influenza HA fusion peptide alone is sufficient to induce the release of the antigen. This is because the release occurs at the pH value which is characteristic of the interior of endocytosomes. The pH in the endocytosomes, e.g., in macrophages, has been determined to be about 5.0 (Wiley, D. C. and Skehel, J. J. (Ann. Rev. Biochem., 56:365, 1987). At pH 5, the influenza HA fusion peptides on the surface of the IRIVs are activated in the same way as in case of the natural influenza virus.
In another embodiment, the present invention relates to a vaccines containing an IRIV of the present invention. The vaccines can contain IRIVs having a combination of different antigens on the surface of each IRIV, such as the combination IRIV, or the vaccine can contain a plurality of different types of IRIVs, each having a different antigen displayed on its surface so as to make a "cocktail" vaccine. Optionally, these vaccines additionally contain a suitable pharmaceutically acceptable carrier and/or diluent. These vaccines can be administered in conventional routes and dosages.
The following examples illustrate the manner in which the invention can be practiced. It is understood, however, that the examples are for the purpose of illustration and the invention is not to be regarded as limited to any of the specific materials or conditions therein.
(A) A dispersion of phosphatidylcholine (e.g., lecithin, SIGMA) (75%), phosphatidylethanolamine (SIGMA) (20%) and cholesterol (SIGMA) (5%) (all phospholipids 1-2% (w/v)=0.013-0.027M) in 0.1M NaCl containing 0.01M Tris/HCl, pH 7.3 was prepared by mixing these compounds with a VIRTIS homogenizer. Sodium cholate recrystallized as the acid from aceton/water 4:1 (v/v) was added to the milky dispersion in a final concentration of at least 0.03M (1.3%) which is required to disintegrate the multilamellar structures present in unsonicated phospholipid dispersions.
A pellet of purified influenza virus 90 A/Taiwan (0.002M of viral membrane phospholipids) was solubilized in 700 ml of 0.1M Octaethyleneglycol mono(n-dodecyl)ether (C12E8) (Nikko Chemicals, Tokyo) in a buffer containing 7.9 mg NaCl/ml, 4.4 mg/ml trisodiumcitrate dihydrate, 2.1 mg/ml 2-morpholinoethane sulfonic acid monohydrate (MES) and 1.2 mg/ml N-hydroxy-ethyl-piperazine-N'-2-ethane sulfonic acid in H2 O (pH adjusted with 1N NaOH to 7.3). The mixture was centrifuged at 170'000 �g for 30 min. and the supernatant containing the influenza spike proteins (HA) and viral phospholipids was added to the above milky phospholipid mixture.
The whole suspension was stirred for at least one hour at low temperature (4� C.). Subsequently, the suspension was applied to a Sephadex G-50 (of medium particle size) column (80�15 cm) which was equilibrated and eluted with the same buffer as used for the preparation of the phospholipid dispersion at 4� C. (flow rate 320 ml/h). The column was embedded in a water bath connected with an ultrasonification apparatus (Bransonic, Branson Europe BV, frequency 50 kHz �10%). 10 seconds of ultrasonic shocks repeated every minute yielded small unilamellar IRIVs. The sample volumes and column dimensions were such that a complete separation of IRIVs eluted at the void volume VO and cholate micelles was achieved. The retention of cholate was tested with 3H-labelled cholate (NEN Chemicals). After the first Sephadex G-50 chromatography less than 1% of cholate was retained, yielding a phospholipid/cholate molar ratio of >50. A second chromatography dialysis for 12 hours at 4� C. reduced the cholate amount below the limit of detection, yielding phospholipid/cholate ratios >500 (i.e. less than 10 cholate molecules/IRIV). Absence of residual C12E8 was tested by a conventional hemolysis test: The amount was below the limit of detection (>100 nM). The IRIVs (FIG. 1) showed a mean diameter of about 100 nm and were conjugated with the HAV antigen in the following manner: A purified and inactivated HAV suspension, strain RG-SB XA112 (CNCM 1-1080), containing 1 mg of HAV antigen, was pelleted by ultracentrifugation (4 h, 100,000�g). The IRIVs prepared above were added to the pellet. After resuspension the suspension was stirred at 20� C. over night (16 hours). The HAV antigen spontaneously adsorbed by Vander Waals forces onto the surface of IRIVs.
(B) Purified influenza virus A/Singapore/6/86 was stabilized in a buffer containing 0.1M octaethyleneglycol mono(n-dodecyl)ether (Nikko Chemicals, Tokyo, Japan), 7.9 mg/ml NaCl, 4.4 mg/ml trisodium citrate dihydrate, 2.1 mg/ml MES and 1.2 mg/ml N-hydaxylethyl-piperazine-N'-2ethane sulfonic acid, pH 7.3. This mixture was centrifuged at 100,000�g for 30 minutes and the HA-containing supernatant was saved.
Phosphatidylcholine (PC; Sigma Chemical Co., St. Louis, Mo.) and phosphatidylethanolamine (PE; Sigma) (75%:25% wt/wt) were suspended in 0.01M Tris - 0.1M NaCl, pH 7.3, and homogenized. Recrystallized sodium cholate (Sigma) was added to a final concentration of 0.02M to disintegrate multilamellar structures. To this solution was added the HA-containing supernatant and the suspension stirred for 1 h at 4� C. The suspension was applied to a Sephadex G-50 column (Pharmacia Fine Chemicals, Uppsala, Sweden) equilibrated in 0.01M Tris - 0.1M NaCl, pH 7.3. The sealed column was placed in a water bath. During elution ultrasonic shocks (50 KHz; 10 s/min) were passed through the water bath using an ultrasonification device (Bransonic, Branson Europe BV, The Netherlands). The void volume fractions, which contained the IRIV, were pooled and re-chromatographed under identical conditions. The IRIV possessed an average diameter of approximately 150 nm.
The purified, inactivated HAV suspension with a known amount of antigen was centrifuged for 4 h at 100,000�g to pellet the virus. An appropriate quantity of the IRIV suspension was added to the pellet and gently resuspended by shaking. The suspension was gently stirred at 20� C. for 48 h to allow the HAV to adsorb onto the surface of the IRIV. This bulk suspension was diluted with sterile phosphate buffered saline, pH 7.4, to a final concentration of 2 μg HAV antigen/ml and bottled.
The preparation of IRIVs with crosslinked HAV antigens is schematically shown in FIG. 2. The IRIVs were prepared according to Example 1 with the following modifications:
The HAV antigen molecules were attached to the IRIVs with a suitable crosslinker molecule. The following procedures were employed:
(A) Phosphatidylethanolamine was coupled with N-succinimidylpyridyl dithiopropionate (SPDP, Pierce) as follows: 15 mg of PE (20 μmol) was dried down in a 5 ml glass bottle. The dried PE was redissolved in 2 ml of dry chloroform (dried over a molecular sieve). Then 30 μmol of triethylamine (TEA) (3 mg), followed by 30 μmol of SPDP (10 mg) in 1 ml of dried methanol were added. The mixture was then stirred at room temperature under nitrogen for 1-2 hours until the reaction was complete (i.e., no more free PE). The reaction product was dried down on a rotary evaporator. The dried lipids were resuspended in chloroform and were immediately applied on the top of a silicic acid chromatography column, which had been prepared as follows: 2 g of silicic acid were dissolved in 10 ml of chloroform. The solution was poured into a 10 ml plastic syringe barrel plugged with glass fibre. The surplus was allowed to drain out and the syringe barrel was fitted with a plastic disposable three-way tap. After application of the lipids, the column was washed with 4 ml of chloroform. Finally, the column was eluted with 4 ml portions of a series of chloroform-methanol mixtures, first 4:0.25 v/v! followed by 4:0.5 v/v!, 4:0.75 v/v! and finally 4:1 v/v! and 2 ml fractions were collected. The pure derivative was then located by thin-layer chromatography (TLC) using silica gel plates developed with chloroform-methanol-water (65:25:4 by vol.). The derivative runs faster than free PE and the spots are visualized by phosphomolybdate or iodine.
The fractions containing the desired product were pooled and concentrated by evaporation at reduced pressure in a rotary evaporator.
(B) The HAV antigen was thiolated by the following procedure: 5 ml of purified and inactivated HAV was dissolved in 0.1M phosphate buffer (PBS (pH 7.5)) at a concentration of 5 mg/ml-1. Then, a SPDP solution at a concentration of 20 μmol-1 (6 mg/ml-1) in ethanol was mixed and 150 μ1 thereof was under stirring slowly added to 5 ml of the HAV protein solution with a Hamilton syringe to give a molar ratio of SPDP to protein of 15:1. The ethanol concentration was kept below 5% to prevent protein denaturation. The mixture was allowed to react for 30 min. at room temperature (20� C.). After the reaction was stopped, the protein was separated from the reactants by gel chromatography on Sephadex G50, equilibrated with a solution containing 0.05M sodium citrate (Na3 C6 H5 O7 . 2H2 O, 19.7 g �I-1), 0.05M sodium phosphate (Na2 HPO4. 7H2 O , 13.4 g. I-1), 0.05M sodium chloride (2.9 g . I-1) pH 7.0.
(C) The pretreated IRIVs and HAV antigens were coupled in the following manner: The IRIVs were prepared as in Example 1. Instead of PE the PE-SPDP was used.
The HAV - SPDP was reduced as follows: The pH of the HAV - SPDP - solution in citrate-phosphate buffer was adjusted to pH 5.5 by the addition of 1M HCl. 10 μl of a DTT solution, 2.5M dithiothreitol (DTT, 380 mg/ml) in 0.2M acetate buffer, pH 5.5 (165 mg of sodium acetate in 10 ml) was added for each ml of protein solution. The solution was allowed to stand for 30 min. Subsequently, the protein was separated from the DTT by chromatography on a Sephadex G-50 column equilibrated with a PBS buffer, pH 7.0. In order to prevent oxidation of thiols all buffers were bubbled with nitrogen to remove oxygen. The protein fractions were also collected under nitrogen.
Finally, the IRIVs were mixed with the thiolated protein by stirring over night at room temperature.
The IRIVs were prepared as in Example 2 with the following modifications: HAV antigen was not coupled with SPDP, but the disulphide bridges already present at the surface of the VPI protein were used as precursors for free thiol groups. This conversion to free thiol groups was carried out as follows: 5 ml of an HAV solution were prepared in 0.1M phosphate buffer, pH 7.4 at a final antigen concentration of 5 mg/ml. For each ml of this solution, 10 μl of a DTT solution (prepared as described in Example 2) were added. The mixture was allowed to stand for 30 min. Then the protein was separated from the DTT by chromatography on a Sephadex G-50 column equilibrated with a PBS buffer at pH 7.0. Protein fractions were collected under nitrogen and were mixed with the IRIVs of Example 2.
The IRIVs were prepared according to Example 1 with the following modification. 1 mg of purified and inactivated HAV in suspension was added to a pellet of purified influenza virus 90 A/Taiwan (0.002M of viral membrane phospholipids) and incorporated into the IRIVs by the method described in Example 1.
HAV-IRIVs were prepared according to Examples 1, 2, 3 or 4 and diluted in PBS, pH 7.4 to a final concentration of 500 ng HAV protein ml-1. This bulk solution was sterile filtrated through a membrane filter of pore size 0.2 μm (Millipore). A preservative (thiomersal) was added to a final dilution of 10 -4. Aliquots of 0.6 ml of the final bulk vaccine were filled into vaccine vials under sterile conditions. Safety and potency tests were performed according to international regulations.
The antigen-binding sites of antibody molecules (Ab1), also known as idiotypes, have been shown to induce the production of antibodies (anti-idiotypes, or Ab2). Because inoculation of animals with some Ab2 results in the production of antibodies (Ab3) that resemble Ab1 in their ability to bind antigen, it has been assumed that the binding sites of Ab2 act as a "mirror image" of the antigenic determinants originally recognized by Ab1 (and subsequently by Ab3 Jerne, N. K. , Ann. Immunol, Paris, 125 C:373, 1974!). The major advantage of using anti-id antibodies (Ab2) for eliciting antigen-specific antibodies (Ab3) is that the vaccine recipient is never in contact with infectious agents or materials containing foreign genes.
The anti-idiotype IRIV vaccine against hepatitis C was prepared as follows: Sheep were immunized with an Ab1 (dissolved of a concentration of 1 mg/ml in PBS) according to the following schedule: On day 0 the animals received 4 doses of 2 ml i.m. at different sites (thighs). On days 7, 14 and 28 they received 2 doses of 2 ml into both hind legs. On day 42 350 ml of blood was collected from each sheep. The serum fraction was separated and further purified by conventional techniques (for a review, see Gluck, R. and Labert, D., Laboratory techniques in rabies, WHO, 4th edition, 1991).
The purified anti-Id hepatitis C antibody was cleaved by digestion with pepsin and the resulting F(ab') 2 fragment was reduced with DTT (see Example 2) to yield 2 Fab' fragments. These Fab' fragments contained free sulfhydryl groups which reacted directly with the IRIVs of Example 2. This preparation was diluted to a protein concentration of 50 μg/ml with PBS, pH 7.4, and portioned in 0.6 ml aliquots in vaccine vials.
(A) Hepatitis A virus (HAV) was purified after growth on MRC-5 human diploid cells (available from the American Type Culture Collection under accession number ATCC CCL 171). The virus was inactivated by treatment with formaldehyde (0.05%) at 37� C. for 10 days. Two vaccine series were tested. Vaccine series 1 consisted of inactivated virus linked to IRIVs according to Example 1 (A) (IRIV-HAV). Vaccine of series 2 was an alum-absorbed preparation containing 0.4% Al(OH)3 (Al-HAV).
Both vaccines contained 150 ng of HAV antigen per 0.5 ml dose. Seronegative adult volunteers (two groups of 15 persons each) received two intramuscular injections on day 0 and a booster injection on day 7 into the deltoid region. No systemic reaction or alterations in the blood chemistry were detected. With respect to local reactions, IRIV preparations provoked a significantly lower percentage of reactions than the alum-absorbed vaccine. The results of these experiments are summarized in FIG. 3.
It was also found that the IRIV preparations were more immunogenic than the alum preparation. To test the anti-HAV immune response, blood samples were taken from the volunteers on days 21 and 28 after the last injection. Sera were tested for HAV specific antibodies using a commercially available RIA (Abbott). The results are summarized in FIG. 4. The numbers on the columns represent the range of the anti-HAV antibody titer. Thus, the range of the anti-HAV antibody titers for the IRIV and alum-adsorbed vaccine formulations on day 21 was 82-988 and 69-844, respectively. The geometric mean titer (GMT) (range) for the IRIV and alum-adsorbed vaccine formulations on day 28 was 453 mlU/ml (92-1210) and 361 mlU/ml (60-929), respectively. Thus, the IRIV preparations of the present invention are superior to alum-adsorbed vaccines.
(B) In a phase I clinical study with 120 human volunteers it could be demonstrated that one single IRIV adjuvanted hepatitis A vaccine dose induced protective antibody titers against hepatitis A which were 7 times higher than the antibody titer after the alum formulation. Up to now such a high immunopotentiation in man has never been achieved with any other liposomal, virosomal or immunosomal formulation due to the obvious lack of fully biologically active fusion peptides.
A total of 120 HAV seronegative (<10 mlU/ml) healthy adults were randomized to receive either fluid, alum-adsorbed, or IRIV vaccine according to Example 1 (B). The vaccine (0.5 ml) was administered intramuscularly into the deltoid region. Volunteers were observed for approximately 30 minutes after vaccination for immediate-type reactions. Each volunteer was asked to record all adverse reactions on a report sheet for the 4 days following immunization. Serum samples for antiHAV antibody determinations were taken at the time of immunization and 14 days later.
Each vaccine formulation contained 1 μg of HAV antigen per 0.5 ml dose. One dose of the IRIV-HAV formulation also contained 10 μg of influenza HA and 125 μg total phospholipids. All three vaccines were found to be sterile and nontoxic for animals by standard test methods. In addition, all 3 formulations elicited a good anti-HAV antibody response in laboratory animals.
Each formulation was administered intramuscularly to 40 healthy adult volunteers seronegative for HAV antibody. The groups were well matched in regard to age and sex. Adverse reactions associated with immunization are shown in Table I. Pain at the injection site was the most frequently reported complaint with all the vaccines. Such discomfort was classified as moderate by one vaccinee (2.5%) who received the fluid formulation, 9 (23%) who were immunized with the alum-adsorbed vaccine, and one (2.5%) who received the IRIV preparation. Severe pain was reported by one subject who received the alum-adsorbed vaccine. All other subjects who reported a "painful" reaction graded it as mild. Immunization with the alum-adsorbed vaccine was associated with a significantly (P<0.01) higher incidence of both pain and swelling/induration as compared to either the fluid or IRIV formulations. No systemic reactions attributable to vaccination were noted.
The anti-HAV antibody response engendered 14 days after vaccination is shown in Table II. Immunization with the fluid vaccine yielded a geometric mean titer (GMT) of 15.7 mlU/ml with 30% of subjects seroconverting (>20 mlU/ml). While the alum-adsorbed vaccine induced both a modestly higher GMT (21.3 mlU/ml) and seroconversion rate (44%), neither was significantly greater than that obtained with the fluid vaccine. In contrast, the IRIV vaccine formulation elicited a far more vigorous antibody response. The GMT of 139.8 was significantly (P<0.0001) higher compared to either of the other two vaccines. All but one vaccinee possessed >100 mlU/ml. Of greater importance was the fact that all vaccinees seroconverted by day 14 compared to less than 50% for the other vaccine formulations (P<0.005).
TABLE I______________________________________Adverse Reactions Associated with ImmunizationLocal reactions (%) Swelling/ Systemic reactions (%)Vaccine Pain Induration Redness Fever Headache Malaise______________________________________Fluid 42* 0∥ 0 0 0 0Al(OH)3 - 88+ 23� 0 0 0 0adsorbedIRIV 25� 5** 0 0 0 0______________________________________ + vs * or �: P < 0.01 � vs ∥ or **: P < 0.01
TABLE II______________________________________Immunogenicity of Fluid, Al(OH)3 -Adsorbed, and IRIV-AdjuvantedHepatitis A Vaccines Geometric mean titer Seroconversion rateVaccine (range) in mIU/ml No. ≧20Formulation Day 0 Day 14 mIU/total (%)______________________________________Fluid <10 15.7 (<10-100)* 12/40 (30%)∥Al(OH)3 -adsorbed <10 21.3 (<10-100)+ 18/40 (44%)�IRIV-adjuvanted <10 139.8 (25-300)� 40/40 (100%)**______________________________________ Subjects received a single dose of vaccine on day 0. � vs * or +: P < 0.0001 ** vs ∥ or �: P < 0.005
To study the role of the influenza viral membrane components in the fusion reaction in detail, it is necessary to be able to manipulate these components. For this purpose a method is required for the isolation and reconstitution of the viral spike proteins, producing reconstituted virosomes with full biological fusion activity. Most of the methods that have been used to reconstitute viral envelopes are based on solubilization of the viral membrane with a detergent.
Several reconstituted influenza virus envelopes have been prepared using different methods which are described in the literature:
A! A virosome according to Huang, R. T. C., et al, (Virology, 97:212-217, 1979), which was prepared using detergents with a high critical micelle concentration (c.m.c.) e.g., octylglucosidel!.
B! A virosome according to Kawasaki, K., et al. (Biochem. Biophys. Acta, 733:268-290, 1983), which was prepared using detergents with a low c.m.c. (e.g., Triton X-100).
C! A virosome according to Hosoka, Y., et al. (Virol, 46:1014-1017, 1983), which was prepared using Nonidet P-40 as detergent.
D! An IRIV as it has been described in Example 1.
In addition, the following controls have been prepared:
E! A purified influenza virus suspension as positive control.
F! PBS-NaCl, pH 7.4, as negative control.
From each reconstituted influenza virus envelope solution and the influenza virus control a concentration of 10 μg/ml hemagglutinin in bicarbonate-free RPMI 1640 medium, supplemented with 10 mM NaCl (pH 7.4) was prepared. The negative control (PBS-NaCl) was diluted in the same medium 1:10. For the vesicle binding and fusion experiment MRC-5 human diploid fibroblasts were grown in 12-well cluster dishes (NUNC). The cells were seeded at 34,000 cells/ml per well and were used 3 days later. At this time, they were approximately 70-80% confluent.
0.5 ml of the reconstituted envelope solutions were added per well, for each preparation 20 wells. The vesicles were allowed to bind to the cells for 30 minutes at room temperature. During this incubation period the dishes were spun twice for 3 minutes at 500 g with a 180� rotation between spins. The centrifugation step enhances the binding of vesicles about 3-fold. After this 30-minute period the cells were washed four times with PBS-NaCl, pH 7.4, to remove unbound vesicles. For the fusion experiment, the fusion activity of the five preparations was induced by adding a fusion medium (RPMI 1640, supplemented with 10 mM succinate, 0.2% bovine serum albumin and 35 mM NaCl, pH 5.0) to each well (0.5 ml/well). After one minute, in two wells per preparation the fusion reaction was stopped by replacing the fusion medium with ethanol absolute. This was done every minute until 10 minutes had passed. The cells were then stained according to the method of May Grunwald-Giemsa:
The cells were then covered with an alcoholic MayGrunwald solution (Fluka No. 63590). After 5 minutes the cells were quickly washed with a phosphate buffer, pH 6.5, and stained with a Giemsa solution (Fluka No. 48900), diluted 1:10 with the same phosphate buffer. After another 10 minutes the cells were washed with running water. Under the microscope the cytoplasm of the cells appeared in a light blue color, the membranes in a dark blue color and the nuclei in a dark red color.
Under the microscope 10 sight fields were evaluated for counting the fused cells (containing at least two nuclei) and were calculated for each preparation and time interval. The FIG. 5 shows the mean value of two wells: It was obvious that only the IRIV preparation shared a fusion activity which was comparable to the influenza virus control. Preparation B! yielded a fusion activity which was only around 30% compared to the positive control. The other preparations did not show any fusion activity (as the negative control did). From these results it can be concluded that only the described IRIVs show fully biological fusion activity, whereas the other methods for influenza envelope reconstitution do not yield vesicles with fusion activity and lead from a considerable to a complete loss of fusion activity.
Two groups of 6 volunteers each, positive for antibodies to human Hepatitis B antigen produced recombinantly in yeast cells (yHBs) were immunized. One group of volunteers was immunized with IRIVs having adsorbed both HAV and yHBs. A dose of the IRIVs contained 500 RIA Units of HAV and 10 μg of yHBs. The other group was immunized with two separate vaccines, one the hepatitis.
A vaccine of Example 3 above containing 500 RIA units of HAV per dose, and the other a conventional alum adsorbed yHBs vaccine containing 0.25 mg per dose of yHBs.
The IRIV were prepared as described in Examples 1, 2, and 3 with the exception that two antigens were attached to the surface of the IRIVs as shown in FIG. 6. The coupling procedure for each was as described in Example 3.
The human hepatitis B surface antigen produced recombinantly in yeast cells (Saccharomyces cerevisiae) (yHBs) were grown in stirred tank fermentors. Because the recombinant antigen accumulates intracellularly, the yeast cells were harvested, washed, and disrupted by high pressure. The antigen then was purified by conventional biochemical techniques to greater than 99% purity for protein. The yHBs was reduced and coupled to IRIVs in the same manner as described for HAV in Example 3.
The results in Table III show that the IRIV-yHBs vaccine induced a higher GMT than the conventional Alum-yHBs vaccine. The antibody titers against HAV were comparable in both groups. Furthermore, the clinical acceptability of the IRIV vaccine was significantly better than that of the conventional vaccine: 67% of the volunteers vaccinated with the alum-based vaccine reported adverse reactions at the site of injection as compared to only 9% of the volunteers immunized with the IRIV vaccine.
TABLE III______________________________________COMPARISON OF THE IMMUNOGENICITY OF IRIVADJUVANTED yHBs ANTIGEN WITH A COMMERCIAL ALUMADJUVANTED VACCINEEffect of Booster A B A B Efficiency G.M.T. G.M.T. Factor IU/ml IU/ml Mean of eachVaccine Day 0 Day 28 single case______________________________________IRIV-HBs 343 13204 107Alum-HBs 292 6373 30______________________________________ Each group consisted of 6 volunteers. One vaccine dose contained 10 μg of yHBs antigen.
To produce a IRIV supercombination, HAV (500 RIA units), yHBs (10 μg), diphtheria antigen (2 International Units) and tetanus antigen (20 International Units) were simultaneously coupled to the surface of IRIVs as described in Example 3 (see FIG. 7). These IRIVs were used to vaccinate 15 volunteers. For comparison, a second group of 23 volunteers were vaccinated first with IRIV-HAV (500 RIA Units), and then separately with standard diphtheria/tetanus vaccine (4 and 40 IU, respectively) adsorbed onto alum-HGsAG (20 μg) on alum basis (Alumdite).
The results are shown in Tables IVa, IVb, and IVc and may be summarized as follows: The immune response to diphtheria and tetanus antigens was significantly higher when they were coupled to the IRIVs, whereas the data for IRIV-adsorbed HAV were comparable in both groups. Antibodies against yHBs were not tested in this study. Again, the tolerance to the IRIV-based vaccines was significantly better than that to the alum-based vaccines as shown in Table V. The IRIV supercombination, therefore, can be used to immunize with administration of a single dose to simultaneously raise protective neutralizing antibodies against Hepatitis A, Hepatitis B, tetanus and diphtheria.
TABLE IVa______________________________________ Day 0 Day 0 Day 25! 8 Day 25! 8______________________________________HAV antibodies IRIV IRIV IRIV IRIV (ILVI)! (IU/ml) Super Single Super Singlen 15 23 15 23Min 1 2 10 20Max 10 16 3559 963Range 9 14 3549 943Median 4 7 247 161Arithmetic Mean 4.3 6.9 531.9 262.7Standard Deviation 2.1 3.6 309.5 248.4GMT 3.8 6.0 198.15 166.5Seroconversion 0 0 14 23% seroconversion 0 0 93.3 100Mann Whitney U test p = 0.01 p = 0.71______________________________________
TABLE IVb______________________________________ Day 0 Day 0 Day 25! 8 Day 25! 8______________________________________ HAV antibodies! (ILVI)!αDT antibodies IRIV Alum IRIV alpha DT!(IU/ml) Super D I!iTe Super antibodies (ILVT)! Alum DiTen 15 23 15 23Min 0.03 0.1 0.2 0.2Max 2.7 6.4 10.1 4.7 Ra!Range 2.6 6.3 9.9 4.5Median 0.6 0.4 4.6 0.5Arithmetic Mean 0.7 0.8 4.6 1.0Standard Deviation 0.6 1.3 -- 0.9GMT 0.51 0.43 3.22 0.75Seroconversion 14 23 15 23 23!% seroconversion 93.3 100 100 100Mann Whitney U test p = 0.43 p = 0.000007______________________________________
TABLE IVc______________________________________ Day 0 Day 0 Day 25! 8 Day 25! 8______________________________________α-TTantibodies IRIV Alum IRIV Alum (ILVI)! (IU/ml) Super yHBs! Super yHBs! DiTe DiTen 15 23 15 23Min 0.2 0.5 10.4 4.6Max 39.3 23 148.0 25.6Range 39.2 22.5 137. 5! 8 21Median 8.1 4 52.3 16Arithmetic Mean 11.3 6.3 54.3 14.9Standard Deviation 11.6 6.2 32.8 6.9GMT 6.59 433 45.16 13.1Seroconversion 15 23 15 23% seroconversion 100 100 100 100Mann Whitney U test p = 0.07 p = 0.00002______________________________________
TABLE V______________________________________COMARISON OF SIDE REACTIONSCOMBINED COMMERCIAL DI, TE, HEPATITIS AAND HEPATITIS B VACCINES VERSUS AN IIVSUPERCOMBI WITH THE SAME ANTIGENSide Reactions Commercial Vaccines IRIV Supercombi______________________________________Pain grade 2 or 3 82% 24%Induration 41% 23%Redness 37% 9%Redness averagearea left: 3800 mm2 2 mm2 right: 1034 mm2Swelling 48% 27%Genoral symptoms(Headache, nausea) 78% 28%______________________________________
Utilizing a fusion test described by Luscher and Gluck (Antiviral Research, 14:39-50, 1990), IRIVs prepared according to the method of Example 1 were compared with live influenza virus in fusion activity with model membranes. FIG. 8 shows the kinetics of fluorescence de-quenching with DOPC-cholesterol liposomes. The increase in fluorescence is expressed in % florescence de-quenching (FDQ), calculated according to Luscher and Gluck.
The foregoing description of the invention is exemplary for purposes of illustration and explanation. It should be understood that various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the following claims are intended to be interpreted to embrace all such modifications.
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|Jun 6, 1994
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- Published in issue 12/2006 on page 0
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The situation is a familiar one: in a quiz, whoever presses (or, in practice, thumps) their button first lights their lamp and is given the opportunity to answer the question. This circuit demonstrates that we can do the job very simply, using just a couple of discrete (not to say antique!) components.
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Is that an 8.16 ERA???
Holy crap.Zimmerman is freaking INSANEThat play is a "web gem" by most standards - just look at the crazy amount of distance he covered. But it looks like a fairly normal "scoop-up and throw" so it won't get any recognition for being a really tremendous play.This happens with Z a lot. He covers so much ground that extraordinary plays don't look like such.
he's walked 14 batters in 14.1 innings.
This guy is a huge dude with a BB/inning. Our patience can pay off.
Kearns just saw the ball real well right there.
Daniel Cabrera is like Inspector Clouseau. Obviously an idiot, but never screws up so badly that you can finally fire him.
cabrera pitches well enough to justify keeping him around (ERA-wise), but he's so terrible that every start i expect to be a disaster.
Actually Broxton has had a bad couple of outings recently. I know because he's on my fantasy and he blew a save last week.
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Thank you, thank you, and thank you.
This has to be one of the best commentaries I've ever read. Bravo!
Girl has had enough!!!!!
So any comments to the editor regarding this article will be published after the election.
@ John 1: Yes, "silly" you; the opposing view is another article--see the link in the Editor's note at the beginning?!
The main problem with all those other initiatives you mentioned is that the folks pushing them haven't even so much as gathered half the signatures needed to get them on the ballot in over twenty years of alleged trying, which is kind of suspect since Richard Lee got his on the ballot all by himself, none of those nasty old "corporate billionaires" needed ... !
Sorry, we're done waiting around while the medical industry enjoys the high prices and no competiton of a prohibition market, and demonstrates no real inclination to move forward, other than a lot of noise *now* that their de facto monopoly is going away--and that's from the MCRSA, whether Prop 64 wins or fails ...
"The policy discourse around this issue has too often been poisoned with the same kind of misinformation and fear tactics that have been perpetuated by institutional anti-marijuana forces since the days of Reefer Madness." Absolutely goddamn right.
Cops wants Prohibition. It is a jobs program for them, and an avenue for corruption.
The argument about the children is so tired. Kids always have access to dangerous conditions and substances, as we live on planet Earth
A No Vote is about keeping the status quo of law enforcement abuse of our citizens and empowers the worst of cartels and gangsters nationwide.
Yes on 64. It's not perfect but we need to head out of the shadow of this racist and ill conceived experiment of outlawing a plant.
To quote Bill Hicks, it's like saying God made a mistake to outlaw an herb. All plants are an offering from God.
thank you 'adaminoregon' , this is common sense that the uninformed need to read, (so much backward thinking, people still believe old myths from 'drug propaganda film, The devils weed ' ! )
John 1, those polls specifically asked about prop 64 and not the general question of "do you support marijuana legalization, yes or no?" You can try to deny that all you want as prohibitionists often do end up resorting to denial. This law is just as good and in some ways better than the 4 legalization laws already approved and will also greatly weaken the federal prohibition as well. I'm not listening to the "wait for a better law" line that is almost a decade old now and will also be around again the next time a chance to legalize would come along. The only regrettable thing here is maintaining prohibition and allowing the failed policy of prohibition to continue on instead of weakening it.
Polls that specifically ask about prop 64:
Worst "legalization" scheme ever pushed at California! And pushed this time by a team of cannabis hating Big Wigs...Dr Donald The Lying Man Lyman and Newsom both detest cannabis and consider daily smokers as the addicts this type of legalization will cure. Newsom's fantasy is that his young daughter can grow up in California and never have to smell Marijuana...not once...as he promotes this Legal-Lies 64 bs. Is anyone really paying attention? Guess we'll know in 3 weeks.
Wow this letter is filled with nonsense. These arguments have been debunked for years, and even if they were true, it still doesn't mean cannabis should remain illegal for adults. Its as if these guys don't realize cannabis has been used for decades in Humboldt. It isn't being introduced now.
The reason there is crime around cannabis is because its illegal. Many of those problems go away in a regulated market. Of course it would also mean Humboldt will need a whole lot less cops. That is why these police are afraid of the change.
There is no place that has re-legalized cannabis that is ever going to go back. States are making millions of dollars. Humboldt has the potential to be the Napa Valley of cannabis. Even if this law isn't perfect, it will keep thousands of people from being hassled for a victimless crime. It will free up police to worry about real crime. It will make your county millions of dollars. It will keep more cannabis away from children then what we have now.It will create a new industry that will employ thousands of people. Vote YES.
Silly me, I thought you were going to represent 2 Different points of view in this article.
Other than one sentence in the 4th paragraph, where you said you respect those concerns.
There seems to be only one point of view in this story and it is that of someone who is in favor of Prop 64 !
You may see these so called "Polls" showing 60% support, let me tell you what that is all about, there is 60% or more support for Legalization, not for Prop 64.
WAIT CALIFORNIA WE CAN DO BETTER, THIS INITIATIVE IS GARBAGE, There are already several more initiatives written by people who are actualy part of the Cannabis Community, not a bunch of corporate lawyers, which is Obviously who wrote this one !
The main point is that cannabis has been illegal about 40 years too long. Cannabis should be grown on farmland, by businesspersons, freely traded, widely available, cheap, and taxed. Anyone wishing to grow their own, should be able to do so.
Prohibition has fostered so many local problems, and crimes against persons and the environment, which escalate every year in severity, in a county ill equipped and underfunded to deal with ANY social issue, crime, drugs or mental health.
Legal weed will cause some economic shock, but the quality of Humboldt weed and the cache of our boutique pot will not be reduced. The products of our county, with local control and compliance, could enjoy worldwide status and distribution. The Humboldt brand will continue to set the standard, our growers will be respected, and our identity will not be challenged by anyone, anywhere. Legalization will put pride in your product, while filtering out the pirates. The end result will be to legitimize your services and products, and put them permanently into the actual economy. The value is there, develop the market! Go forward without fear!
I'm not a grower, but I think these taxes are way out of hand. And $7 million annually when the deficit is in the hundreds of millions?? How would these state officials like it if their income was suddenly lowered by $15k/year for an empty promise of smoother roads?
I think this is one side of the argument. But I see a different side. The Bulgarians or Eastern Europeans are heavily funded from big money organizations. They have offered me money to run my property, all upfront(in spring) and they have made the same offer to other neighbors. They are also buying large tracts of land at above market prices. As someone who cares about the livability and character of my neighborhood, this is a problem. I don't want my hood to turn into a third world feeling, drug producing, gun toting(yes they shoot all the time) place. I will admit, I prefer loggers, hippys, and the other unique characters that have lived in these hills and I really don't like the big money people AT ALL. All races included, but the organized crime people in particular. Which happen to be Eastern European at this time.
Of course there are friendly bulgys, that's not the point! It's the bigger picture. I'm sure I could write up a great story about a wonderful plantation owner back in the good old days, but that doesn't mean it's an acceptable way to do things in the bigger picture. We need to police our rural neighborhoods ourselves or the police and f&g are going to be all over us, as they are beginning to do. Time to wake up and stop the p.correct handwringing people. That's my opinion!
Talk about too little, too late. By this time next year pot will be legalized in California and anyone will be able to buy weed at the corner grocery store. The entire "medical marijuana" industry will cease to exist once anyone can buy it over the counter without a prescription.
Slim down with pot? News to me and the rest of my fat, smokin' buddies.
Maybe I'm not smokin' enough.
If I smoke more will I finally get slim again?
Good for the Eureka City Council.
It's about time they realized it's almost 2017 with legalization, hopefully, just around the corner.
Gooberments better pull their collective heads out because the bad old days of trying to use scare tactics to put down pot are long over.
Smoke 'em if ya got 'em.
I just drove the Mattole road from Ferndale into AW Way Campground for a few days, a quick stop into Honeydew for gas and EVERY person who passed us on the road waved. The waves were enthusiastic, consistent and each came with a smile. Was it my classic Ford 4x4 that provoked the locals to be friendly? Did they know of my Portuguese decent from my grandfather and great aunt who immigrated to Ferndale in the 60's? I highly doubt it. Just the most friendly warm folks I've come across in Humboldt. Pordagees, Bulgarians, eastern European who knows. What I can confirm is I had a great experience and felt welcome! Leaving Ferndale when I was a child to return to Humboldt as an adult, all i can say is Thank you!
Great article, LOVE the bridge!
I find the Bulgarians of Humboldt County to be extremely polite and amagable. I think the reason they get a bad rap is because they are good at what they do. Their professionalism in their work is extremely apparent. They take greater risks then their neighbors. Therefore greater reward. All interactions I have had have been pleasant. They might even be willing to help you out as a neighbor if your not a bigot. America is a country of immigrants, it's a melting pot of cultures. If you don't like culture start packing.
this is a great article! thank you for sharing without prejudice!
No one is "from around here" except the Natives who were wiped out in the 1800's.


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Contact | Careers | Events | News | Articles | About
Clearwater Seafoods was recognized as the province's top exporter at the 28th Nova Scotia Export Achievement Awards at the World Trade and Convention Centre today, May 15.
"Exporting plays an important role in growing our economy," said Percy Paris, Minister of Economic and Rural Development and Tourism. "On behalf of the province of Nova Scotia, I'd like to congratulate Clearwater Seafoods on this important achievement along with all the other companies for their innovation and success." Ten other Nova Scotia businesses were also recognized for excellence in exporting. Companies recognized were:
Darren Zwicker, executive vice-president LED Roadway Lighting Ltd., was the keynote speaker at today's event, which was attended by more than 250 people. "Clearwater Seafoods and the other export achievement award winners are the kinds of companies NSBI and our partners at ACOA and Economic Rural Development and Tourism work with in markets around the world," said Stephen Lund president and CEO of Nova Scotia Business Inc. "We help these companies expand and diversify their markets and identify new customers, distributors and suppliers. Last year, over 200 unique companies participated in our trade missions and programs." The Nova Scotia Export Achievement Awards are presented by Nova Scotia Business Inc., the Atlantic Canada Opportunities Agency and Trade Team Nova Scotia. For more information on the awards, visit www.exportachievementawards.com
Mel Rusinak 902.424.6390 Send a Message
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Pinterest でおしゃれアイデアをまとめましょう!
Gyro Zeppeli (ジャイロ・ツェペリ Jairo Tseperi), born Julius Caesar Zeppeli, is, alongside Johnny Joestar, the main protagonist of Part VII: Steel Ball Run. Gyro is a master of the Spin, applied with his Steel Balls, developed in the service of the King of Naples. He joins the Steel Ball Run race to win amnesty for a child he is assigned to execute.
ジョジョの冒険,ジョジョの奇妙な冒険,ジョジョSbizarre,Jojo Araki,アニメ,Pic Jojo,Jojo Stand,Jojo Exhibition,Jojo Aesthetic


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Picante Tomato Dressing
Total Time
Prep 5 mins
Cook 2 mins
Posting this to save and try later. From our local paper.
Ingredients Nutrition
- 1 medium tomatoes, cored and chopped
- 1 garlic clove, chopped
- 1 teaspoon dijon-style mustard
- 2 tablespoons fresh lime juice
- 1⁄2 teaspoon ground cumin
- 1⁄2 teaspoon chili powder
- 1 pinch cayenne pepper
- 1⁄8 teaspoon salt
- 3⁄4 cup regular tomato juice (or Picante tomato juice)
- Put tomato and garlic into food procesor and puree.
- Add remaining ingredients and blend until smooth.
- Transfer to a jar and refrigerate. Whisk before using. Makes 2/3 cup.
Most Helpful
This was nice and light. I did make one mistake: I somehow thought it called for tomato sauce, not tomato juice. So I just went ahead and made it with the sauce anyhow, as I like a thicker dressing. Didn't seem to have a real "kick" to me. Very mild spicyness, but that was fine. I do like the low calorie count and will probably make again and play with it a bit. Thanks.
We did love it!! love the combination of ingredients and spices. It had a very nice kick. Served over codfish cakes, perfect match. I will be making this again, thanks for posting!


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It's the Year of the Dragon in college hoops
Forty names, games, teams and minutiae making news in college basketball (tickets to the Matt Painter-Branden Dawson MMA cage match sold separately):
Happy Year of the Dragon, hoops fans. Perhaps this means a reversal of fortune for UAB (1) (6-12, losers of five of its past six) and Illinois-Chicago (2) (6-13, on a two-game losing streak). Both have dragon mascots – "Blaze" and "Spark," respectively – if not dragon nicknames. The only D-I dragon, Drexel (3), already has a head start on the Chinese New Year, owning a seven-game winning streak and standing 15-5 overall, tied for second in the Colonial Athletic Association.
Where have the great teams gone?
When the season began, The Minutes was convinced it would be a great one. Why? Because after a couple of seasons of a rather mediocre upper crust in college basketball, we had the potential for at least three great teams. Enough first-round NBA draft picks stuck around college to give their programs a shot at something special.
Roughly midway through the season, those teams have not lived up to their potential.
The Minutes is looking at you, North Carolina (4). You go and lose to Florida State by 33, which simply should not happen no matter how well the Seminoles are playing. And you suffer another season-ending knee injury in the backcourt (Dexter Strickland joining Leslie McDonald, who went out before the season began). Now what do we make of you, Tar Heels? Still capable of winning the ACC, but are you capable of winning it all? Strong doubts.
And The Minutes is looking at you, Ohio State (5). The loss at Kansas was understandable – the Jayhawks are better than expected, especially in the Phog, and Jared Sullinger didn't play. But the 5-2 Big Ten record is not what we envisioned. The Buckeyes haven't proven much on the road yet – beating up on Iowa and Nebraska does not offset losing to Indiana and Illinois, both of which currently are leaking oil. Ohio State has five road games remaining, four against teams ranked in Jeff Sagarin's top 45. The Bucks are slightly deeper than last season, but concerns remain about whether they might wear down in the bruising Big Ten over the next six weeks.
Maybe Syracuse is ready for a good-to-great leap into the vacuum left by Carolina and Ohio State. Even after being thumped at Notre Dame on Saturday, the Orange got a strong bounce-back victory at Cincinnati on Monday night and theoretically will get center Fab Melo back if/when he resolves his "academic issue" that kept him out of that two-game road trip. (Say this for the 'Cuse: This is a team that can make 3-point shots from a few feet behind the arc. That's a small but important detail because a lot of college defenders are accustomed to only closing out to 21 feet. When your guys can shoot from 23, that's 2 feet of extra space to make shots.)
The third team with preseason greatness potential, Kentucky (6), may fill the bill. If The Minutes had to name a national title favorite today, the Wildcats would be the choice. But they've played sporadically in recent weeks, rarely putting together the kind of crushing runs we saw from this collection of athletes in November and December. And the SEC schedule is so user-friendly right now that we won't see the 'Cats play a premier league opponent outside of Rupp Arena until Feb. 11.
Speaking of league schedules …
How league offices are shaping league races
At conference headquarters across the land, two things are givens: complaints from schools about the officiating and complaints from schools about the schedule.
In the case of the former, leagues must try to hire well – both refs and supervisors of officials – and simply hope for the best. In the case of the latter, the job is nearly impossible and always thankless.
There is annual crabbing about who has to play where on how much rest, and who has the most difficult stretches of games. In the days of full round-robin scheduling, it all used to even out in the end. Then conferences became the size of China, and it became even more arduous to schedule fairness into the question.
So The Minutes sympathizes with the suits in charge of putting together a balanced slate. But it's interesting to see how much strength-of-schedule appears to be impacting conference races at this point. Check Ken Pomeroy's SOS numbers in the top 10 leagues and you'll see the perks of an easy entry into conference play.
Big Ten (7): Co-leaders are Ohio State, Michigan State and Michigan at 5-2. The Buckeyes have played the 11th-hardest league schedule out of 12.The Wolverines have had five home games and only two on the road, and have had the ninth-hardest schedule to date. Only the Spartans have kept it real, with the No. 5 schedule and one more road game than at home.
Toughest Big Ten schedule to date: Nebraska, which is 2-6 and tied for last. So much for a honeymoon period for the Huskers, who already have played Ohio State and Wisconsin twice, plus Indiana and Michigan State.
Big 12 (8): As if perennial kingpin Kansas needed the assist, it has played the 10th-toughest schedule out of 10. The Jayhawks (7-0) have played four at home and three on the road, visiting relative lightweights Oklahoma and Texas Tech. They received another assist Monday night, when Texas A&M's Khris Middleton was injured before the game and guard Dash Harris was injured during it.
Toughest Big 12 schedule to date: Kansas State, which is 3-3 and in fifth place. The Wildcats still have six games left against the four schools considered the weakest in the league (Texas Tech, Texas A&M, Oklahoma State and Oklahoma).
Big East (9): Before Monday night's game at Cincinnati, league leader Syracuse had played the No. 15 schedule out of 16. Pomeroy rates six of the Orange's first nine opponents 75th or worse – though Syracuse has played five on the road and only four at home.
Toughest Big East schedule to date: Providence, which is 1-6 and tied for 14th in the league. St. John's is the only team the Friars have faced that doesn't look like an eventual NCAA tournament team.
[Related: Boeheim passes Rupp for fourth on wins list]
SEC (10): The top three teams in the league standings – Kentucky, Vanderbilt and Florida – have played schedules ranked 10th, 11th and 12th, respectively, out of 12. It will be Feb. 11 before the Wildcats play a league road game against a team currently ranked in Sagarin's top 75. And because they are the TV darlings of the league, gobbling up Tuesday night ESPN spots, Kentucky has zero Thursday-Saturday turnaround games. Vanderbilt's Kevin Stallings, whose team just ran that 48-hour gantlet and split against Alabama and Mississippi State, pointed that out last week. SEC officials were too busy counting bowl revenues to hear him.
Toughest SEC schedule to date: South Carolina, which is 0-4 and last in the league. As if Darrin Horn's job already wasn't hard enough, he had to open with Kentucky, Vanderbilt and Florida in succession.
ACC (11): Duke, Florida State and North Carolina State are tied for the top at 4-1. The Blue Devils have played the No. 9 ACC schedule out of 12 and the Wolfpack have played the easiest of the 12. Props to the Seminoles, rolling hard despite playing the No. 2 schedule. NC State was gifted with an opening stroll against Maryland, Georgia Tech, Wake Forest, Boston College and Miami, but still managed to lose at home to the putrid Yellow Jackets.
Toughest ACC schedule to date: Miami, which is 1-3 and tied for 10th place. Opening at Virginia and North Carolina was cruel.
Missouri Valley (12): Co-leader Wichita State has played the No. 10 schedule out of 10, and fellow co-leader Creighton has played the No. 7 schedule. The Shockers already have played softies Bradley and Southern Illinois twice each.
Toughest Valley schedule to date: Southern Illinois, which is 3-6 in the league and in eighth place. And that's after a non-conference schedule that was too difficult for the Salukis (Saint Louis, Kansas State, Xavier, Clemson).
Pac-12 (13): Leader California has played the No. 8 schedule out of 12 in America's most underachieving league. (How underachieving? The Pac-12 is 3-36 against Sagarin top 50 competition, and the three wins belong to Washington State and Oregon State – neither of which is a serious title contender.) The Golden Bears have had the luxury of playing Utah and USC.
Toughest Pac-12 schedule to date: UCLA, which is 3-4 and tied for seventh place. The Bruins have played five on the road and two at home to date.
Conference USA (14): Leader UCF has played the seventh-toughest schedule out of 12. Opening against Tulane, Houston and East Carolina has its rewards.
Toughest C-USA schedule to date: UAB, which is 1-4 and tied for 11th. The depth-shy Blazers have played each of the league's top four teams – UCF, Memphis, Marshall and Southern Miss – and have the losses to show for it.
Exceptions to the soft-schedule influence on the standings:
Atlantic 10 (15): Leader Dayton has played the second-hardest league schedule. Give credit to the Flyers under first-year coach Archie Miller for already having knocked off Xavier, Temple, Saint Louis and La Salle.
Mountain West (16): Leader San Diego State has played the second-hardest league schedule, with victories over UNLV and New Mexico. Then again, MWC teams have played only three league games, which means a limited sample size.
In praise of the passers
Scorers are the glamour boys in basketball; it always has been that way. But The Minutes has to give it up to those who give it up: the unselfish passers who make the shooters look good. In particular, six players who dished the ball with aplomb this past weekend and will be key to their teams' success the rest of the way.
Luke Loucks (17), Florida State. Loucks became an instant favorite in the final seconds Saturday at Cameron Indoor Stadium by refusing to commit a Minutes pet peeve: He passed the ball in the final seconds instead of playing the hero and jacking up a bad shot just because he had the ball in his hands on the final play.
It's become a basketball epidemic: one guy pounding the ball to the exclusion of his four teammates as the offense dies, leading to a bad shot that doesn't challenge the defense at all. And unless the shooter is Kobe Bryant, it usually misses.
But not Loucks. He went rushing up the court in a tie game with less than five seconds to play against the Blue Devils and … ah, heck, let him tell you about it:
" 'B.J.' [Bernard James] was setting a drag screen near midcourt. It ended up a little early, but it worked great. When I got free, there were a couple Duke defenders ahead of me. I was facing the shot clock and saw 2.1, so I knew I had time. I had Ryan Murphy in front of me and a few others on the wings.
"I was about to pull up and shoot a long, contested '3,' then I looked up and saw Mike [Snaer] wide open. Andre Dawkins was kind of watching the ball and lost Mike, so I passed it to him."
Loucks laughed.
"I had a lot of thoughts going on in 4.5 seconds," he said.
A lot of thoughts beats no thought. And it led to Snaer hitting the most dramatic shot of the season this side of Christian Watford's Wildcat killer in Bloomington, Ind., on Dec. 10.
Loucks admitted he did have a hankering to take the hero shot.
"I did have a little selfish thought: 'This play's designed for me to take the last shot,' " he said. "But Mike had a better shot. That's something that's helped our whole team the last couple weeks: getting open shots for others and not necessarily for ourselves."
The Minutes thanks you, Luke Loucks. So does Michael Snaer and every Florida State fan.
The Flip and Pierre Show (18). The point-guard play Saturday in Missouri's pulsating 89-88 victory over Baylor was sublime. The Tigers' Phil "Flip" Pressey had 18 points, seven assists, six steals and five rebounds. The Bears' Pierre Jackson finished with 20 points and 15 assists. Both are listed at 5 feet 10, but both probably are shorter. Neither is deterred.
Scott Machado (19), Iona. Machado, a senior, leads the nation in assists (10.2 per game) and has five games of at least 15 dimes already. He missed a triple-double by a single rebound Friday night against Rider. Not coincidentally, the Gaels are tied for the lead in the Metro Atlantic Athletic Conference.
Kendall Marshall (20), North Carolina. His role for the Tar Heels only grows with Dexter Strickland's season-ending knee injury. Marshall is second nationally in assists (9.5 per game), enjoying the luxury of finding Tyler Zeller and John Henson inside and Harrison Barnes on the wing. But Marshall helps make them look good, too. He handles with his head up and is the best at the long hit-ahead pass for hard-running big men in transition. Marshall's perimeter shooting will be critical later in the season, and he's made only one of his past 10 3-point attempts.
Draymond Green (21), Michigan State. The most clever frontcourt passer in the game had a season-high seven assists in the Spartans' rout of Purdue on Saturday. He routinely outsmarts double-teams with passes out of the post, many of which set up the next pass for an open shot. (If this were hockey and you could get multiple assists on a single basket, Green would have more than his 71 assists on the season.) When the guy they call "Day-Day" has at least five assists in a game this season, the Spartans are undefeated and win by an average of 28.6 points.
[The Dagger: Purdue coach has heated exchange with rival fans after loss]
The final frontier
Missouri's rise to No. 2 in the rankings has inflamed Tigers fans with early-onset Final Four fever. Why? Because Mizzou never has advanced that far in the NCAA tournament.
That got The Minutes wondering which teams have been the biggest heartbreakers at Madness time. Here's a look at the schools with the most NCAA tournament wins without a Final Four berth.
Missouri (22). The Tigers have 22 NCAA tourney victories over the years, but never have won a regional final. Closest call: 2009 regional final loss to Connecticut.
Boston College (23). The Eagles also have 22 NCAA victories without ever playing on the season's final weekend. Closest call: John Bagley & Co.'s 1982 regional final loss to Phi Slama Jama Houston.
Alabama (24). The Crimson Tide have 20 NCAA wins but has reached only one regional final; the Tide was crushed upon arrival by eventual national champion UConn in 2004.
Xavier (25). The Musketeers have 19 NCAA victories and two regional-final losses. Closest call: A red-hot Xavier team – coached by Thad Matta – suffered a three-point loss to Duke in 2004.
Tennessee (26). The Volunteers have 16 tournament wins but only one Elite Eight appearance. That came two years ago, and it nearly resulted in a Final Four berth until Michigan State pulled out a 70-69 victory. That would prove to be the high-water mark of the Bruce Pearl Era in Knoxville.
Gonzaga (27). The Bulldogs also have 16 tourney wins. Their closest run at a Final Four came in the tourney that put the program on the map, in 1999. The Zags lost by five points to eventual national champ UConn in a regional final.
These schools have made the Final Four, but they haven't figured out how to win it all yet. They have the most NCAA tournament wins without a national title.
Illinois (28). The Illini have won 39 NCAA games and advanced to five Final Fours but never cut down the nets. Closest call: They reached the championship game and lost a great game against North Carolina in 2005, but the better chance was in 1989. That team lost in the semifinals to eventual champion Michigan on a Sean Higgins putback in the final seconds. Illinois had beaten the Wolverines twice that season.
Oklahoma (29). The Sooners have 35 NCAA wins without a title. Closest call: the 1988 national title loss to Danny Manning and Kansas. Oklahoma was the No. 1 team going into the tournament and played like it for five games, all double-digit victories. But in the final, they lost by four points. As with Illinois above, Oklahoma had beaten the Jayhawks twice earlier in the season.
Purdue (30). The Boilermakers have a terrific history, with one glaring hole: no title, and nothing terribly close. They've won 34 NCAA games, but their only championship-game appearance ended in a blowout at the hands of Lew Alcindor and UCLA in 1969.
Texas (31). The Longhorns have 34 NCAA wins but only two Final Four appearances, and those were a mere 56 years apart. But if anyone has a more painful Final Four loss than the Longhorns, The Minutes wants to hear about it. In 1947, Texas led Oklahoma by a point in the final seconds when a Sooners sub threw in a shot from 40 feet to beat the Longhorns 55-54. Nothing in the Red River football rivalry can be as brutal a beat as that.
Temple (32). The Owls have won 32 NCAA games but haven't reached a Final Four since 1958. That run ended with a one-point loss to eventual champion Kentucky on a Vernon Hatton layup with 16 seconds left.
Kansas State (33). As with Temple, the Wildcats had their chances in the early days of the NCAA tourney but haven't been back since. They've won 32 NCAA games and made four Final Fours, most recently in 1964. Closest call: a 10-point loss to Kentucky in the championship game in 1951.
Memphis (34). The Tigers also have 32 NCAA victories but never, never on a Monday. The closest call in theory was the Mario Chalmers gut-punch in 2008. But since that Final Four appearance is vacated, the closest call is a 1973 championship-game loss to UCLA in which Bill Walton was 21-of-22 from the floor.
Perfection/imperfection update
We're down to three teams with a zero in either the left-hand or right-hand column. The update:
Binghamton (35). Now 0-19, the Bearcats gave it a run last Thursday at Maryland-Baltimore County, losing by six in a game that featured some late window-dressing by the losing team. Misery statistic: Binghamton hasn't had a second-half lead since Jan. 5. This week: at Albany on Wednesday and at Maine on Saturday. Ken Pomeroy gives Binghamton a 2 percent chance of winning the first game and a 5 percent chance of winning the second.
Towson (36). The Tigers are 0-21 after losing Monday night at Delaware. The Tigers were in it for about 31 minutes before submitting. Misery statistic: Towson has lost 40 in a row. Say that out loud if you dare. This week: The Tigers do get two more chances, and both at home. They play host to VCU on Wednesday and to UNC-Wilmington on Saturday. Pomeroy is predicting a 23-point rout at the hands of the Rams but only a 13-point loss to UNCW.
Murray State (37). And now for the happier side of 0: The Racers improved to 20-0 after road victories over Morehead State and SIU-Edwardsville. With every victory, Murray solidifies an at-large bid should it stumble in the Ohio Valley Conference tournament and seemingly improves its NCAA seeding. This week: Murray plays host to Eastern Illinois and Southeast Missouri State in what should be a pair of beatdowns.
The Minutes figures to be writing about all three teams again next week.
Coach who earned his comp car
Leonard Hamilton (38), Florida State. Has anyone had a better week this season than Hambo? His Seminoles mauled North Carolina at home and stunned Duke on the road, with a 14-point victory over Maryland squeezed in-between. That's good enough to make everyone forget about being 0-2 against the Ivy League (losses to Harvard and Princeton). The 'Noles suddenly are a live threat to break up the Duke-Carolina Tobacco Road cartel at the top of the ACC.
Coach who should take the bus to work
Jamie Dixon (39), Pittsburgh. The losing streak is now eight, stretching back to before Christmas. Included in that tailspin are a 23-point home loss to Rutgers, a 54-point effort against Wagner and just once scoring more than 63 points in that span. If Pitt doesn't win at home Wednesday against Providence, well, seriously, when?
Buzzer beater
With the Final Four in mind, The Minutes dished a New Orleans recommendation last week. But why stop at just one NOLA joint when there are so many to tout? When hungry for a deluxe dinner away from the madness of the French Quarter, The Minutes recommends a visit to Patois (40). The entire menu, from appetizer to dessert, is completely absurd. But the gumbo was the best. Ever. Order it and thank The Minutes later.
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