CA2678775A1 - Protein markers useful to differentiate immunized and infected specimens - Google Patents
Protein markers useful to differentiate immunized and infected specimens Download PDFInfo
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- CA2678775A1 CA2678775A1 CA 2678775 CA2678775A CA2678775A1 CA 2678775 A1 CA2678775 A1 CA 2678775A1 CA 2678775 CA2678775 CA 2678775 CA 2678775 A CA2678775 A CA 2678775A CA 2678775 A1 CA2678775 A1 CA 2678775A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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Abstract
The current invention provides novel markers of protein origin added to veterinary vaccines used in immunization treatments, characterized by effectively differentiate immunized specimens from the infected ones. Markers generate specific antibodies in fish, they act at extremely low doses and the differentiation methods used are simple and cost effective.
Description
Protein Markers Useful To Differentiate Immunized And Infected Specimens DESCRIPTION
Vaccination continues being one of the most sustainable and used methods to control infection contagious diseases in veterinarian medicine, given their higher economic feasibility, and due to the problem caused by antibiotics residues in animal products for human consumption, such as milk, meat and eggs (Martinez, et al., 2008).
Nevertheless, few vaccines are able to gather ideal conditions. There are even microorganisms for which vaccines do not exist. For a vaccine to comply with such a condition it must be safe, specific, protective, with prolonged immunity, of unique dose, low cost, stable, it must prevent pathogen dispersion and be of easy administration (Wigdorovitz, 2007). Also, in the administration of such veterinarian compositions, the appearance of adverse effects must be avoided, such as intra-peritoneum adherences, which are directly related to the volume of the administered dose, as a function of a larger adjuvant amount administered with larger doses (Leal, 2003).
The first reports on the use of vaccines in fish did not result into a massive deployment of this kind of essay in the rest of laboratories, since in those years, after World War II, concerns were focused in the control of diseases with the use of recently discovered antibiotics. Only in the second half of the seventies, as a result of an increase in aquiculture centers, the issue of immunization was again considered as a viable option to prevent and control diseases in fish, thus greatly increasing the development of commercially available vaccinations (Evelyn, 1997).
According to figures generated by the World Bank, losses for the aquiculture industry originated by different pathologies amounted to US$3,000 million per year, at a worldwide level. In Chile, Piscirickettsia salmonis alone originates losses over US$100 million per year (www.aquahoy.com, 2007). Diseases originated by the appearance of new infections, having had a strong impact on this productive sector must be added to such losses.
Some figures show that the use of vaccinations in the domestic aquiculture industry has experienced a sustained growth during the last few years, thus allowing the control of several viral and bacterial diseases affecting normal development. The above is supported by an increase in the participation of vaccinated fish by three of the most grown species in Chile: 97.30% vaccinated of the total Atlantic Salmon spawn grown, corresponding to 161,190,000 spawn; 71.30% in the case of Rainbow Trout, corresponding to 57,520,000 spawn, and 77.70% in the case of Coho Salmon, corresponding to 35,050,000 spawn (Intervet, 2006).
Nevertheless, together with an increase in the development of veterinarian vaccinations to be administered in fish, new issues arose. One of them was the incapability of differentiating the pathogen agents detected in fish plasma, in the sense of knowing for certain if they had been originated by an infection (pathology), or if they had been administered with immunization purposes (vaccination). There is also no differentiation in the antibodies generated by them, consequently, this issue continues existing.
Aquiculture industry is a sector strongly monitored by the authorities, since it is pertinent to learn if fish are positive to a given pathogen as determined by a natural infection, or due to immunization policies designed to eliminate infectious sources. This is even more relevant when considering the restrictive measures by the health authorities against the allegedly infected culture centers, especially if quarantine zones delimitation and sanitary sacrifice of the cages are ordered by a decree (Chilean Government, SalmonChile, 2008), with the evident resulting economic consequences.
BACKGROUND OF THE INVENTION
Some proteins, antigens, enzymes, bacteria or genetically modified viruses, which genes code proteins, may be used as marker agents, useful in the diagnosis and treatment, as well as in the identification of treatments with vaccinations that are useful to differentiate the immunized individuals from the infected ones. All of them are well explained in the previous art, being its most pertinent documents as follows:
US 4673634: Patent protecting a specific antigen of non-A, non-B hepatitis, which is a serological marker in the diagnosis of this disease. Patent was granted in the United States, in June 1987, to the Health and Human Services Department of the United States Government.
US 5480774: Patent protecting a procedure to determine the sex of several salmon species, by means of the genomic sequence of a pseudo-gene associated to sexual determination in Y chromosome, which is used as marker for sex determination.
The patent was granted in the United States, in January 1996, to A/F Protein Inc.
US 5733748: Patent protecting a specific gene of human colon that codifies polypeptides used as markers in colon cancer and metastasis determination.
Patent was granted in the United States, in March 1998, to Human Genome Sciences Inc.
Vaccination continues being one of the most sustainable and used methods to control infection contagious diseases in veterinarian medicine, given their higher economic feasibility, and due to the problem caused by antibiotics residues in animal products for human consumption, such as milk, meat and eggs (Martinez, et al., 2008).
Nevertheless, few vaccines are able to gather ideal conditions. There are even microorganisms for which vaccines do not exist. For a vaccine to comply with such a condition it must be safe, specific, protective, with prolonged immunity, of unique dose, low cost, stable, it must prevent pathogen dispersion and be of easy administration (Wigdorovitz, 2007). Also, in the administration of such veterinarian compositions, the appearance of adverse effects must be avoided, such as intra-peritoneum adherences, which are directly related to the volume of the administered dose, as a function of a larger adjuvant amount administered with larger doses (Leal, 2003).
The first reports on the use of vaccines in fish did not result into a massive deployment of this kind of essay in the rest of laboratories, since in those years, after World War II, concerns were focused in the control of diseases with the use of recently discovered antibiotics. Only in the second half of the seventies, as a result of an increase in aquiculture centers, the issue of immunization was again considered as a viable option to prevent and control diseases in fish, thus greatly increasing the development of commercially available vaccinations (Evelyn, 1997).
According to figures generated by the World Bank, losses for the aquiculture industry originated by different pathologies amounted to US$3,000 million per year, at a worldwide level. In Chile, Piscirickettsia salmonis alone originates losses over US$100 million per year (www.aquahoy.com, 2007). Diseases originated by the appearance of new infections, having had a strong impact on this productive sector must be added to such losses.
Some figures show that the use of vaccinations in the domestic aquiculture industry has experienced a sustained growth during the last few years, thus allowing the control of several viral and bacterial diseases affecting normal development. The above is supported by an increase in the participation of vaccinated fish by three of the most grown species in Chile: 97.30% vaccinated of the total Atlantic Salmon spawn grown, corresponding to 161,190,000 spawn; 71.30% in the case of Rainbow Trout, corresponding to 57,520,000 spawn, and 77.70% in the case of Coho Salmon, corresponding to 35,050,000 spawn (Intervet, 2006).
Nevertheless, together with an increase in the development of veterinarian vaccinations to be administered in fish, new issues arose. One of them was the incapability of differentiating the pathogen agents detected in fish plasma, in the sense of knowing for certain if they had been originated by an infection (pathology), or if they had been administered with immunization purposes (vaccination). There is also no differentiation in the antibodies generated by them, consequently, this issue continues existing.
Aquiculture industry is a sector strongly monitored by the authorities, since it is pertinent to learn if fish are positive to a given pathogen as determined by a natural infection, or due to immunization policies designed to eliminate infectious sources. This is even more relevant when considering the restrictive measures by the health authorities against the allegedly infected culture centers, especially if quarantine zones delimitation and sanitary sacrifice of the cages are ordered by a decree (Chilean Government, SalmonChile, 2008), with the evident resulting economic consequences.
BACKGROUND OF THE INVENTION
Some proteins, antigens, enzymes, bacteria or genetically modified viruses, which genes code proteins, may be used as marker agents, useful in the diagnosis and treatment, as well as in the identification of treatments with vaccinations that are useful to differentiate the immunized individuals from the infected ones. All of them are well explained in the previous art, being its most pertinent documents as follows:
US 4673634: Patent protecting a specific antigen of non-A, non-B hepatitis, which is a serological marker in the diagnosis of this disease. Patent was granted in the United States, in June 1987, to the Health and Human Services Department of the United States Government.
US 5480774: Patent protecting a procedure to determine the sex of several salmon species, by means of the genomic sequence of a pseudo-gene associated to sexual determination in Y chromosome, which is used as marker for sex determination.
The patent was granted in the United States, in January 1996, to A/F Protein Inc.
US 5733748: Patent protecting a specific gene of human colon that codifies polypeptides used as markers in colon cancer and metastasis determination.
Patent was granted in the United States, in March 1998, to Human Genome Sciences Inc.
EP 19980202054: Patent application of a procedure to clone the Newcastle disease virus, useful in the manufacturing of vaccinations and diagnosis essays for this disease.
The procedure allows a modification of the virus antigenic composition, thus generating virus vaccinations that act as markers, serologically differentiating from the pathogenic stumps of the virus. This solution was presented in June, 1998, by the Dutch company Stichting Dienst Landbauwkundig Onderzoek, and as a reference, it was granted in the United States in April, 2004, under number US 6,719,979.
Mackay, D., et al., 1998: Research article showing the development of an ELISA
analysis methodology to differentiate an antibody type from the food-and-mouth disease, which is specifically caused by infected bovines.
US 19990240173: Patent application referred to a vaccination against bovine herpes virus, containing a recombinant virus that is suppressed in glycoprotein E
portion, thus allowing it to act as immunological marker, differentiating vaccinated from infected animals. This application was published in the United States, in January, 1999, the applicants is the University Kansas State.
EP 1318406: Patent referred to the human and veterinarian use of glycine N-acetyltransferase as a marker in the diagnosis, prognosis and monitoring of inflammatory and infectious diseases. The application was granted in June, 2003, to B.R.A.H.M.S. Aktiengesellschaft.
EP 1318407: Patent referred to human and veterinarian use of Aldose 1-epimerase as marker in the diagnosis, prognosis and monitoring of inflammatory and infectious diseases. The application was granted in June, 2003, to B.R.A.H.M.S.
Aktiengesellschaft.
The procedure allows a modification of the virus antigenic composition, thus generating virus vaccinations that act as markers, serologically differentiating from the pathogenic stumps of the virus. This solution was presented in June, 1998, by the Dutch company Stichting Dienst Landbauwkundig Onderzoek, and as a reference, it was granted in the United States in April, 2004, under number US 6,719,979.
Mackay, D., et al., 1998: Research article showing the development of an ELISA
analysis methodology to differentiate an antibody type from the food-and-mouth disease, which is specifically caused by infected bovines.
US 19990240173: Patent application referred to a vaccination against bovine herpes virus, containing a recombinant virus that is suppressed in glycoprotein E
portion, thus allowing it to act as immunological marker, differentiating vaccinated from infected animals. This application was published in the United States, in January, 1999, the applicants is the University Kansas State.
EP 1318406: Patent referred to the human and veterinarian use of glycine N-acetyltransferase as a marker in the diagnosis, prognosis and monitoring of inflammatory and infectious diseases. The application was granted in June, 2003, to B.R.A.H.M.S. Aktiengesellschaft.
EP 1318407: Patent referred to human and veterinarian use of Aldose 1-epimerase as marker in the diagnosis, prognosis and monitoring of inflammatory and infectious diseases. The application was granted in June, 2003, to B.R.A.H.M.S.
Aktiengesellschaft.
DE 102005057643: Patent application referred to a vaccination against bacterial diseases in animals, with at least one bacterial agent of endogenous origin genetically modified, which genes codify a marking protein that, in turn, generates antibodies.
Patent granted in Germany, on May, 2007.
DE 102007010142: Patent application referred to a method to verify cholinergic neurons through a complex formed by specific cholinergic neurons marking protein (SLC1OA4) and limpet hemocyanin. The protein is detected with immunofluorescence, or with peroxidase application. This document was originally published in Germany, on September, 2007. DE200710010142.
Perez, E., 2008: Research article explaining the development of a recombinant virus in the diagnosis of porcine circovirus type 2 (PCV2). Such method also allows for differentiation of vaccinated from infected animals, since infected ones generate antibodies for structural and non structural proteins.
Detailed Description of the Invention The state-of-the-art shows a large variety of protein origin markers, nevertheless, they do not correspond to products that may be used in vaccines for fish. Also, there is no compound type able to act at low doses to generate an easily measurable immunogenic capability.
The current invention provides for novel protein markers that are added to veterinary vaccines to selectively differentiate the immunized from infected specimens, generating specific antibodies in fish. The compounds used as markers correspond to recombinant chimeric proteins with fusion at highly hydrophilic sequences, capable of generating specific immunologic response in fish, surprisingly at very low doses, significantly reducing the possibilities of generating adverse effects, such as intra-peritoneum adherences. Such markers are recognized by specific antibodies in immunized fish plasma, which may be detected by means of immunological essays techniques, such as ELISA or Western Blot. They both correspond to versatile, solid techniques, simple to carry out, efficient, that use cost-efficient reactives, thus translating into an advantage for markers identification.
Markers are highly stable, they are produced by cloning and expressed in totally exogen E. coli BL21 bacteria, induced by isopropyl-beta-D-thiogalactopyranoside (IPTG), and they generate a non pathogen immunogenic activity adequate to produce an easily measurable response to the immunological assay techniques detection limits.
Their immunogenic activity is independent from the immunogenic activity caused by the vaccine itself, so they are not related.
Protein markers have a histidine tag in the terminal amine, thus facilitating its eventual purification. The most relevant specifications are shown on Table 1; at the drawings section, figure 1 shows the hydrophobicity map of one of them.
Table 1. Specifications of protein markers.
Specification Values Range Molecular Weight 45000-65000 kDa Isoelectric point 8.0- 10.0 Extinction Coefficient at 280 cm" 1.0- 1.5 Absorbance at 280 nm (optical density) ABS 0.1 % 1.0- 1.5 Antigenic Dose 1 - 10 pg/ 0.1 mL
Protein markers Detection and Quantification Methods 1. ELISA
Material and equipment: This method uses ELISA Nunc Maxisorp, Greiner Bio One High Binding or similar plates, ELISA Nunc Immuno Wash 8 or similar plates washer, automatic ELISA plates washer, filter-calibrated ELISA plates reader, incubation stove, freezer, and timer.
Reactives: Capturing serum containing the monoclonal antibody (capture antibody), antigen, dissolution solution, ABTS- H202 (substrate), positive control serum, negative control serum and stop solution.
Sample: Immunized fish serum.
Procedure Summary: ELISA plates were sensitized by means of the capture antibody, and they were subject to cold incubation for 16 hours. Then the previously diluted antigen (exogen protein marker) was added to the plate, which was tied to the capture antibody and incubated at 37 C for 1 hour. Then immunized fish serum was added, which developed anti-protein marker antibodies, and was incubated for 1 hour at environment temperature. Successive dilutions were then carried out on the same plate with the dissolution solution from 1:2 to 1:256 and it was incubated at environment temperature for 2 hours. Then the ABTS-H202 substrate was added, which bonds with the antibodies developed by immunized fish and allows for revealing, in order to determine the antibody presence. The reaction was interrupted when the stop solution was added, and it was read at an ELISA automatic reader, which provided the necessary optical density data for processing with a calculation software that delivered the final results of the samples. Figure 2 of the drawings section shows an outlined summary of this procedure.
2. Western Blot Material and equipment: Electrophoresis and blotting equipment including glass plates, plates support, gel support, electrophoresis support, electrophoresis power source, transference cassette, transference support, nitrocellulose membrane for western blotting, orbital agitator, revealing cassette and timer.
Reactives: Protein marker (exogen antigen), 5X buffer sample, 1X buffer sample, polyacrylamide sodium dodecyl sulfate separating gel (PAGE-SDS), running buffer, transference buffer, Ponceau red solution, washing buffer, membrane blocking buffer, secondary antibody and ECL revealing kit, or revealing solution DAB-H2O2 (diaminobenzidine- hydrogen peroxide).
Sample: Immunized fish serum.
Procedure Summary: 5 pL of a protein marker were taken, and 1 pL of 5X buffer sample was added, and then taken to 30 pL with 1 X buffer sample; it was boiled for 5 minutes and the plate was planted to start electrophoresis with PAGE-SDS gel, where the meal buffer was used for 130 minutes, under a constant flow of 45 miliamperes. Then the protein transfer was carried out from the plate to a nitrocellulose membrane included in the transfer cassette; the latter was assembled trying that all parts are covered by the transfer buffer. Then, the system was closed and the transfer support was placed. The conditions for the transfer were a constant voltage of 75 volts, for 90 minutes. The Ponceau red solution was added to check if proteins transfer was successful; the membrane color indicates that the transfer was satisfactorily made. The membrane was washed with washing buffer until colorant disappeared. The technique continued with membrane blocking, using the blocking buffer based on bovine serum albumin. Then the serum of immunized fish was added, which contained the protein anti-marker antibody (primary antibody) and it was incubated for 60 minutes at environment temperature. Four washings were carried out, and the secondary antibody was added, leaving it in incubation for 60 minutes more at environment temperature; the membrane was washed four times.
Revealing was carried out with the ECL kit, or with the substrate solution DAB-H202.
Safety Assessment The safety of protein markers was assessed through the measurement of intra-peritoneum adherences, an important parameter that indicates the application feasibility of injection compositions in fish. For this study Atlantic salmon species, at a sweet water stage were used, with weights between 20 and 30 g. Fish were selected among those that had not been vaccinated, that had not shown recent clinical states, and that had not been subject to treatment with any medication prior to the study. With the purpose of checking the health condition of the fish and to rule out the presence of any pathogen, a sanitary check-up was carried out during the first 7 days. Afterwards, 100 specimens were selected, which were moved under the requirements established for fish transfer, to a 350 liters tank (tank 1), under temperature conditions between 15 C and 18 C, with oxygen between 8 to 10 mg/L, maintaining a population density between 5 and 9 Kg/m3, with a water exchange rate of one tank/hour. The fish stayed there for 7 days more, to complete the adaptation process.
For this test, a protein marker that complies with the above described specifications was used, administering an injection dose of 0,1 mL sterile solution containing 5 pg of the protein marker per dose to the first group; such fish were marked with a cut on the adipose fin. The 50-fish second group (control group) was injected with 0.1 mL
of sterile physiological solution, and the fish were marked with a cut on the caudal fin.
Both groups were transferred to a tank with the same capacity of tank 1 (tank 2), maintaining the same conditions above, thus allowing both groups to live together until the end of the assay, eliminating external factors that could affect a given group.
Administration was carried out with intra-peritoneum injection on the ventral medium line, with fish anesthetized with 20% benzocaine to obtain a 40 ppm concentration in the tank. Then, fish were left in the tanks without being subject to any stressing handling until they completed 30 post-injection days. During this time the water temperature was monitored, and on the 30th day fish were sacrificed and sent to a laboratory for adherence assessment.
Feed during the study was based on a commercial diet, with a daily feeding rate of 2%, except for two days before and one day after the injection, when fish fasted.
With the purpose of avoiding fungi appearance, during the study 1 % salt, or Chloramine T baths were applied, with a 10 ppm concentration. Baths were applied for one hour, for 2 consecutive days, once a week.
Figure 3 shows a chronological summary of the main events during this study.
The assessment conditions and results are summarized on table 2. The acceptance criterion was established internally, based on fish vaccines safety studies, where a substance is safe in case less than 15% of the cases show an adherence level, and in any event no adherence higher than grade 3, according to Speilberg scale.
Table 2. Summary of safety assessment results.
Parameter Protein Marker Control Injected volumen 0.1 mL 0.1 mL
NO fish 50 50 Mark Adipose fin Caudal fin Adherences 6% 4%
Results showed that there exists a very low trend to intra-peritoneum adherences in the group of fish with application of protein marker, and the percentages as similar in the control group. Adherences shown in both groups were classified as grade 1 according to the Speilberg scale, which does not represent a significant toxicity. Also, no dead fish were recorded. From the above, we may conclude that injection administration of protein markers in fish is safe.
Compared Immunogenicity Study Immunogenicity of protein markers was assessed through a comparative study using a vaccine against Piscirickettsia salmonis. For this study, 100 specimens of Atlantic salmon species at sweet water stage were used, with weights between 20 and 30 g.
Sanitary check-up of fish, their selection, tanks transfer, their characteristics, adaptation procedure and the study term correspond to the same used for the safety study.
The total fish under study was divided in two groups of 50 fish each; the first group was injected with 0.1 mL of a vaccine against Piscirickettsia salmonis available in the market, and fish were marked with a cut on the adipose fin. The second group was injected with 0.1 mL of a solution prepared on the basis of the same commercial vaccine as group 1, but with the addition of a sufficient amount of protein marker, which complies with the above described specifications, to generate a 5 pg/ dose concentration. Such fish were marked with a cut on the caudal fin. Both groups were transferred, after the injection, to another tank with identical characteristics, so as to allow their living together until the end of the assay, eliminating external factors that could affect a given group. At the end of the living-together term, that is, after 30 days, fish were anesthetized and 6 specimens were selected at random from each group, a blood test was taken on the 6 specimens that received an injection of the commercial vaccine, 3 were analyzed with the ELISA immunological assay technique, and the other 3 with Western Blot. At the same time, the fish specimens that received injections of commercial vaccine with a protein marker were analyzed in the same manner, to detect the antibodies generated with the addition of the protein marker in the fish plasma. The comparative immunogenicity study results are summarized on table 3.
Table 3. Comparative immunogenicity study results.
Plasmatic Sample ELISA Western Blot Commercial vaccine fish Negative (n=3) Negative (n=3) Commercial vaccine fish + marker Positive (n=3) Positive (n=3) The results showed that in no event the commercial vaccine produces antibodies detected with the above described methodologies for immunological assay test, Elisa and Western Blot, that is to say, the antibodies generated by the vaccine and the markers are different and they may be differentiated.
It may be concluded from the above that protein markers generate an independent and specific immunogenicity, entirely different from the one produced by a given vaccine, eliminating the possibility of crossed detections between the antibodies produced by a vaccine and those produced by the protein markers.
Also, the current invention refers to the use of protein markers in veterinary vaccines to differentiate fish with an antigenic activity due to immunization, and fish with an antigenic activity due to infection.
Examples The following examples describe the invention in detail.
Example 1. Aminoacidic sequence showing the primary structure of a 408-aminoacid protein marker.
MHHHHHHSSGLVPRGSGMKQTAAAKFGRQHMNSPDLGTNNNNKAMADISIPSQKSVL
YFLIEKGQHEAAIEFFEGMVHDSIKEELRPLIQQTSFVKRAFKRLKENFEIVALCLTLLANI
VIMIRETHKRQKMVDDAVNEYIEKANITTDDQTLDEAEKNPLETSGASTVGFRERTLPG
QKARDDVNSEPAQPTEEQPQAEGPYAGPLERQRPLKVRAKLPRQEGPYAGPMERQK
PLKVKARAPVVKEGPYEGPVKKPVALKVKAKNLIVTESGAPPTDLQKMVMGNTKPVELI
LDGKTVAICCAVINNADVGRLIFSGEALTYKDIWCMDGDTMPGLFAYKAATKAGYCGG
AVLAKDGADTFIVGTHSAGGNGVGYCSCVSRSMLLKMKAHI DPEPHH EGLI.
Example 2. Aminoacidic sequence showing the primary structure of a 420-aminoacid protein marker.
MHHHHHHSQTAAAKFGRQHMNSPDLGTNNEAAIEFFEGMVHDSIKEELRPLIQQTSFV
KRAFKRLKENFIMIRETHKRQKMVDDAVNEYIEKANITTDDQTLDEAENNVLYFLIEKGQ
HKNPLETAHIDPEPHHEGLISGASTVGFRERTLPGQKARDDVNSEPAQPTEEQPQKAM
ADISIPSQKSAEGPYAGPLERQRPLKVRSGLVPRGSGMKAKLPRQEGPYAGPMERQK
PLKVKARAPWKEGPYEGPVKKPVALKVKAKNLIVTESGAPPTDLQKMVMGNTKPVELI
LDDEKRLNQGAVGFATGKTVAICCAVINNADVGRLIFSGEALTYKDIWCMDGDTMPGL
FAYKAATKAGYCGGAVLEIVALCLTLLANIVAKDGADTFIVGTHSAGGNGVGYCSCVSR
SMLLKMK.
Example 3. Aminoacidic sequence showing the primary structure of a 427-aminoacid protein marker.
THHHHHHSSGLVPRGSGMKQTAAAKFGRQHMNSPDLGTNNNNKAMADISIPEFFVAIC
CAVINNADEGMVHDSIKEELFKRLKENFEIVALCLTLLANIQKMVDDAVNEYIEKANITTD
DQTLDSQKSVLYFLIEKGQHEAAIEAEKNPLETSGASTVGFRERTLPGQKARDDVNSEP
AQPTEEQPQAEGPYAGPLAKLPRQEGPRPLIQQTSFVKRAYAGPMERQKHIDPEPHH
EGLPLKVKARAPWKEGPYEGPVKKPVALKVKAKNLIVTESGAPPTDLERQRPLKVRQ
KMVMGNTKPVELILDGKSTCVLIQDLIMPFHVAGGGICRPYQDKTVGRLIFSGEALTVIMI
RETHKRYKDIWCMDGDTMPGLFAYKAATKAGYCGGAVLAKDGADTFIVGTHSAGGN
GVGYCSCVSRSMLLKMKAI.
The above indicated protein markers correspond to examples of the invention, and they do not limit the scope of its protection, since they are only considered as an orientation on the general characteristics of the invention.
Patent granted in Germany, on May, 2007.
DE 102007010142: Patent application referred to a method to verify cholinergic neurons through a complex formed by specific cholinergic neurons marking protein (SLC1OA4) and limpet hemocyanin. The protein is detected with immunofluorescence, or with peroxidase application. This document was originally published in Germany, on September, 2007. DE200710010142.
Perez, E., 2008: Research article explaining the development of a recombinant virus in the diagnosis of porcine circovirus type 2 (PCV2). Such method also allows for differentiation of vaccinated from infected animals, since infected ones generate antibodies for structural and non structural proteins.
Detailed Description of the Invention The state-of-the-art shows a large variety of protein origin markers, nevertheless, they do not correspond to products that may be used in vaccines for fish. Also, there is no compound type able to act at low doses to generate an easily measurable immunogenic capability.
The current invention provides for novel protein markers that are added to veterinary vaccines to selectively differentiate the immunized from infected specimens, generating specific antibodies in fish. The compounds used as markers correspond to recombinant chimeric proteins with fusion at highly hydrophilic sequences, capable of generating specific immunologic response in fish, surprisingly at very low doses, significantly reducing the possibilities of generating adverse effects, such as intra-peritoneum adherences. Such markers are recognized by specific antibodies in immunized fish plasma, which may be detected by means of immunological essays techniques, such as ELISA or Western Blot. They both correspond to versatile, solid techniques, simple to carry out, efficient, that use cost-efficient reactives, thus translating into an advantage for markers identification.
Markers are highly stable, they are produced by cloning and expressed in totally exogen E. coli BL21 bacteria, induced by isopropyl-beta-D-thiogalactopyranoside (IPTG), and they generate a non pathogen immunogenic activity adequate to produce an easily measurable response to the immunological assay techniques detection limits.
Their immunogenic activity is independent from the immunogenic activity caused by the vaccine itself, so they are not related.
Protein markers have a histidine tag in the terminal amine, thus facilitating its eventual purification. The most relevant specifications are shown on Table 1; at the drawings section, figure 1 shows the hydrophobicity map of one of them.
Table 1. Specifications of protein markers.
Specification Values Range Molecular Weight 45000-65000 kDa Isoelectric point 8.0- 10.0 Extinction Coefficient at 280 cm" 1.0- 1.5 Absorbance at 280 nm (optical density) ABS 0.1 % 1.0- 1.5 Antigenic Dose 1 - 10 pg/ 0.1 mL
Protein markers Detection and Quantification Methods 1. ELISA
Material and equipment: This method uses ELISA Nunc Maxisorp, Greiner Bio One High Binding or similar plates, ELISA Nunc Immuno Wash 8 or similar plates washer, automatic ELISA plates washer, filter-calibrated ELISA plates reader, incubation stove, freezer, and timer.
Reactives: Capturing serum containing the monoclonal antibody (capture antibody), antigen, dissolution solution, ABTS- H202 (substrate), positive control serum, negative control serum and stop solution.
Sample: Immunized fish serum.
Procedure Summary: ELISA plates were sensitized by means of the capture antibody, and they were subject to cold incubation for 16 hours. Then the previously diluted antigen (exogen protein marker) was added to the plate, which was tied to the capture antibody and incubated at 37 C for 1 hour. Then immunized fish serum was added, which developed anti-protein marker antibodies, and was incubated for 1 hour at environment temperature. Successive dilutions were then carried out on the same plate with the dissolution solution from 1:2 to 1:256 and it was incubated at environment temperature for 2 hours. Then the ABTS-H202 substrate was added, which bonds with the antibodies developed by immunized fish and allows for revealing, in order to determine the antibody presence. The reaction was interrupted when the stop solution was added, and it was read at an ELISA automatic reader, which provided the necessary optical density data for processing with a calculation software that delivered the final results of the samples. Figure 2 of the drawings section shows an outlined summary of this procedure.
2. Western Blot Material and equipment: Electrophoresis and blotting equipment including glass plates, plates support, gel support, electrophoresis support, electrophoresis power source, transference cassette, transference support, nitrocellulose membrane for western blotting, orbital agitator, revealing cassette and timer.
Reactives: Protein marker (exogen antigen), 5X buffer sample, 1X buffer sample, polyacrylamide sodium dodecyl sulfate separating gel (PAGE-SDS), running buffer, transference buffer, Ponceau red solution, washing buffer, membrane blocking buffer, secondary antibody and ECL revealing kit, or revealing solution DAB-H2O2 (diaminobenzidine- hydrogen peroxide).
Sample: Immunized fish serum.
Procedure Summary: 5 pL of a protein marker were taken, and 1 pL of 5X buffer sample was added, and then taken to 30 pL with 1 X buffer sample; it was boiled for 5 minutes and the plate was planted to start electrophoresis with PAGE-SDS gel, where the meal buffer was used for 130 minutes, under a constant flow of 45 miliamperes. Then the protein transfer was carried out from the plate to a nitrocellulose membrane included in the transfer cassette; the latter was assembled trying that all parts are covered by the transfer buffer. Then, the system was closed and the transfer support was placed. The conditions for the transfer were a constant voltage of 75 volts, for 90 minutes. The Ponceau red solution was added to check if proteins transfer was successful; the membrane color indicates that the transfer was satisfactorily made. The membrane was washed with washing buffer until colorant disappeared. The technique continued with membrane blocking, using the blocking buffer based on bovine serum albumin. Then the serum of immunized fish was added, which contained the protein anti-marker antibody (primary antibody) and it was incubated for 60 minutes at environment temperature. Four washings were carried out, and the secondary antibody was added, leaving it in incubation for 60 minutes more at environment temperature; the membrane was washed four times.
Revealing was carried out with the ECL kit, or with the substrate solution DAB-H202.
Safety Assessment The safety of protein markers was assessed through the measurement of intra-peritoneum adherences, an important parameter that indicates the application feasibility of injection compositions in fish. For this study Atlantic salmon species, at a sweet water stage were used, with weights between 20 and 30 g. Fish were selected among those that had not been vaccinated, that had not shown recent clinical states, and that had not been subject to treatment with any medication prior to the study. With the purpose of checking the health condition of the fish and to rule out the presence of any pathogen, a sanitary check-up was carried out during the first 7 days. Afterwards, 100 specimens were selected, which were moved under the requirements established for fish transfer, to a 350 liters tank (tank 1), under temperature conditions between 15 C and 18 C, with oxygen between 8 to 10 mg/L, maintaining a population density between 5 and 9 Kg/m3, with a water exchange rate of one tank/hour. The fish stayed there for 7 days more, to complete the adaptation process.
For this test, a protein marker that complies with the above described specifications was used, administering an injection dose of 0,1 mL sterile solution containing 5 pg of the protein marker per dose to the first group; such fish were marked with a cut on the adipose fin. The 50-fish second group (control group) was injected with 0.1 mL
of sterile physiological solution, and the fish were marked with a cut on the caudal fin.
Both groups were transferred to a tank with the same capacity of tank 1 (tank 2), maintaining the same conditions above, thus allowing both groups to live together until the end of the assay, eliminating external factors that could affect a given group.
Administration was carried out with intra-peritoneum injection on the ventral medium line, with fish anesthetized with 20% benzocaine to obtain a 40 ppm concentration in the tank. Then, fish were left in the tanks without being subject to any stressing handling until they completed 30 post-injection days. During this time the water temperature was monitored, and on the 30th day fish were sacrificed and sent to a laboratory for adherence assessment.
Feed during the study was based on a commercial diet, with a daily feeding rate of 2%, except for two days before and one day after the injection, when fish fasted.
With the purpose of avoiding fungi appearance, during the study 1 % salt, or Chloramine T baths were applied, with a 10 ppm concentration. Baths were applied for one hour, for 2 consecutive days, once a week.
Figure 3 shows a chronological summary of the main events during this study.
The assessment conditions and results are summarized on table 2. The acceptance criterion was established internally, based on fish vaccines safety studies, where a substance is safe in case less than 15% of the cases show an adherence level, and in any event no adherence higher than grade 3, according to Speilberg scale.
Table 2. Summary of safety assessment results.
Parameter Protein Marker Control Injected volumen 0.1 mL 0.1 mL
NO fish 50 50 Mark Adipose fin Caudal fin Adherences 6% 4%
Results showed that there exists a very low trend to intra-peritoneum adherences in the group of fish with application of protein marker, and the percentages as similar in the control group. Adherences shown in both groups were classified as grade 1 according to the Speilberg scale, which does not represent a significant toxicity. Also, no dead fish were recorded. From the above, we may conclude that injection administration of protein markers in fish is safe.
Compared Immunogenicity Study Immunogenicity of protein markers was assessed through a comparative study using a vaccine against Piscirickettsia salmonis. For this study, 100 specimens of Atlantic salmon species at sweet water stage were used, with weights between 20 and 30 g.
Sanitary check-up of fish, their selection, tanks transfer, their characteristics, adaptation procedure and the study term correspond to the same used for the safety study.
The total fish under study was divided in two groups of 50 fish each; the first group was injected with 0.1 mL of a vaccine against Piscirickettsia salmonis available in the market, and fish were marked with a cut on the adipose fin. The second group was injected with 0.1 mL of a solution prepared on the basis of the same commercial vaccine as group 1, but with the addition of a sufficient amount of protein marker, which complies with the above described specifications, to generate a 5 pg/ dose concentration. Such fish were marked with a cut on the caudal fin. Both groups were transferred, after the injection, to another tank with identical characteristics, so as to allow their living together until the end of the assay, eliminating external factors that could affect a given group. At the end of the living-together term, that is, after 30 days, fish were anesthetized and 6 specimens were selected at random from each group, a blood test was taken on the 6 specimens that received an injection of the commercial vaccine, 3 were analyzed with the ELISA immunological assay technique, and the other 3 with Western Blot. At the same time, the fish specimens that received injections of commercial vaccine with a protein marker were analyzed in the same manner, to detect the antibodies generated with the addition of the protein marker in the fish plasma. The comparative immunogenicity study results are summarized on table 3.
Table 3. Comparative immunogenicity study results.
Plasmatic Sample ELISA Western Blot Commercial vaccine fish Negative (n=3) Negative (n=3) Commercial vaccine fish + marker Positive (n=3) Positive (n=3) The results showed that in no event the commercial vaccine produces antibodies detected with the above described methodologies for immunological assay test, Elisa and Western Blot, that is to say, the antibodies generated by the vaccine and the markers are different and they may be differentiated.
It may be concluded from the above that protein markers generate an independent and specific immunogenicity, entirely different from the one produced by a given vaccine, eliminating the possibility of crossed detections between the antibodies produced by a vaccine and those produced by the protein markers.
Also, the current invention refers to the use of protein markers in veterinary vaccines to differentiate fish with an antigenic activity due to immunization, and fish with an antigenic activity due to infection.
Examples The following examples describe the invention in detail.
Example 1. Aminoacidic sequence showing the primary structure of a 408-aminoacid protein marker.
MHHHHHHSSGLVPRGSGMKQTAAAKFGRQHMNSPDLGTNNNNKAMADISIPSQKSVL
YFLIEKGQHEAAIEFFEGMVHDSIKEELRPLIQQTSFVKRAFKRLKENFEIVALCLTLLANI
VIMIRETHKRQKMVDDAVNEYIEKANITTDDQTLDEAEKNPLETSGASTVGFRERTLPG
QKARDDVNSEPAQPTEEQPQAEGPYAGPLERQRPLKVRAKLPRQEGPYAGPMERQK
PLKVKARAPVVKEGPYEGPVKKPVALKVKAKNLIVTESGAPPTDLQKMVMGNTKPVELI
LDGKTVAICCAVINNADVGRLIFSGEALTYKDIWCMDGDTMPGLFAYKAATKAGYCGG
AVLAKDGADTFIVGTHSAGGNGVGYCSCVSRSMLLKMKAHI DPEPHH EGLI.
Example 2. Aminoacidic sequence showing the primary structure of a 420-aminoacid protein marker.
MHHHHHHSQTAAAKFGRQHMNSPDLGTNNEAAIEFFEGMVHDSIKEELRPLIQQTSFV
KRAFKRLKENFIMIRETHKRQKMVDDAVNEYIEKANITTDDQTLDEAENNVLYFLIEKGQ
HKNPLETAHIDPEPHHEGLISGASTVGFRERTLPGQKARDDVNSEPAQPTEEQPQKAM
ADISIPSQKSAEGPYAGPLERQRPLKVRSGLVPRGSGMKAKLPRQEGPYAGPMERQK
PLKVKARAPWKEGPYEGPVKKPVALKVKAKNLIVTESGAPPTDLQKMVMGNTKPVELI
LDDEKRLNQGAVGFATGKTVAICCAVINNADVGRLIFSGEALTYKDIWCMDGDTMPGL
FAYKAATKAGYCGGAVLEIVALCLTLLANIVAKDGADTFIVGTHSAGGNGVGYCSCVSR
SMLLKMK.
Example 3. Aminoacidic sequence showing the primary structure of a 427-aminoacid protein marker.
THHHHHHSSGLVPRGSGMKQTAAAKFGRQHMNSPDLGTNNNNKAMADISIPEFFVAIC
CAVINNADEGMVHDSIKEELFKRLKENFEIVALCLTLLANIQKMVDDAVNEYIEKANITTD
DQTLDSQKSVLYFLIEKGQHEAAIEAEKNPLETSGASTVGFRERTLPGQKARDDVNSEP
AQPTEEQPQAEGPYAGPLAKLPRQEGPRPLIQQTSFVKRAYAGPMERQKHIDPEPHH
EGLPLKVKARAPWKEGPYEGPVKKPVALKVKAKNLIVTESGAPPTDLERQRPLKVRQ
KMVMGNTKPVELILDGKSTCVLIQDLIMPFHVAGGGICRPYQDKTVGRLIFSGEALTVIMI
RETHKRYKDIWCMDGDTMPGLFAYKAATKAGYCGGAVLAKDGADTFIVGTHSAGGN
GVGYCSCVSRSMLLKMKAI.
The above indicated protein markers correspond to examples of the invention, and they do not limit the scope of its protection, since they are only considered as an orientation on the general characteristics of the invention.
Claims (10)
1. Markers of protein origin CHARACTERIZED for being added to veterinary vaccines to effectively differentiate the immunized specimens from the infected ones.
2. Markers of protein origin CHARACTERIZED in that, according to claim 1, by generating specific antibodies in fish.
3. Markers of protein origin CHARACTERIZED in that, according to claims 1 to 2, by generating a high non-pathogen immunogenic activity on the specimens to which it is administered, independently from the immunogenic activity caused by the vaccine itself.
4. Markers of protein origin CHARACTERIZED in that, according to claims 1 to 3, by the fact that the antibodies generated by the specimens they are applied to are easily detectable and quantifiable with simple immunological essay techniques.
5. Markers of protein origin CHARACTERIZED in that, according to claims 1 to 4, by the fact that they are of safe administration as injections, without causing significant adverse effects (intra-peritoneum adherences).
6. Markers of protein origin CHARACTERIZED in that, according to claims 1 to 5, by a molecular weight within the range of 45000 to 65000 kDa
7. Markers of protein origin CHARACTERIZED in that, according to claims 1 to 6, by an isoelectric point within the range of 8.0 to 10Ø
8. Markers of protein origin CHARACTERIZED in that, according to claims 1 to 7, by an extinction coefficient at 280 nm within the range of 1.0 to 1.5.
9. Markers of protein origin CHARACTERIZED in that, according to claims 1 to 8, by an absorbance at 280 nm ABS 0.1 % within the range of 1.0 to 1.5.
10. Use of the markers of protein origin CHARACTERIZED in that, according to claims 1 to 9, because they may be added to veterinarian vaccines to differentiate fish with an antigenic activity due to immunization from fish with an antigenic activity due to infection.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CL2009001197A CL2009001197A1 (en) | 2009-05-15 | 2009-05-15 | Veterinary pharmaceutical composition containing the Chilean variety of infectious salmon anemia virus, protein markers and pharmaceutically acceptable excipients; procedure for obtaining the virus; use of the composition to prepare a vaccine for the prevention of the isa virus. |
| CL1197-2009 | 2009-05-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2678775A1 true CA2678775A1 (en) | 2010-11-15 |
Family
ID=41202926
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2678775 Abandoned CA2678775A1 (en) | 2009-05-15 | 2009-09-15 | Protein markers useful to differentiate immunized and infected specimens |
Country Status (4)
| Country | Link |
|---|---|
| CA (1) | CA2678775A1 (en) |
| CL (1) | CL2009001197A1 (en) |
| GB (1) | GB2470238A (en) |
| NO (1) | NO20092716L (en) |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI350310B (en) * | 2002-12-13 | 2011-10-11 | Novartis Ag | Immunization of fish with plant-expressed recombinant proteins |
| EP1685848A1 (en) * | 2005-01-26 | 2006-08-02 | Plant Research International B.V. | Oral vaccines for fish |
| CN101848995A (en) * | 2007-06-25 | 2010-09-29 | 南达科他州立大学 | Recombinant north american type 1 porcine reproductive and respiratory syndrome virus and methods of use |
| US20090110698A1 (en) * | 2007-10-31 | 2009-04-30 | Newport Laboratories, Inc. | Method of determining vaccine compliance |
-
2009
- 2009-05-15 CL CL2009001197A patent/CL2009001197A1/en unknown
- 2009-07-20 NO NO20092716A patent/NO20092716L/en not_active Application Discontinuation
- 2009-08-28 GB GB0915140A patent/GB2470238A/en not_active Withdrawn
- 2009-09-15 CA CA 2678775 patent/CA2678775A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| CL2009001197A1 (en) | 2009-08-21 |
| NO20092716L (en) | 2010-11-16 |
| GB0915140D0 (en) | 2009-10-07 |
| GB2470238A (en) | 2010-11-17 |
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