Classifications

• • 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
  • G01N33/56983—Viruses
• • 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
  • A61K39/12—Viral antigens
• • 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
  • A61K39/12—Viral antigens
  • A61K39/125—Picornaviridae, e.g. calicivirus
• • 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
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/525—Virus
  • A61K2039/5258—Virus-like particles
• • 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
• • 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
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/16011—Caliciviridae
  • C12N2770/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• the subjects of the present invention are a diagnostic kit for the examination of animal sera for the presence of anti-RHD antibodies and a diagnostic kit for detecting the RHD virus.
• the subjects of the present invention facilitate the application in ELISA tests of specific (monospecific) anti-sera obtained as a result of the immunization of laboratory animals with purified, recombinant RHD virus antigens (VLP RHDV). This yields high specificity, sensitivity and repeatability of the results obtained.
• VLP RHDV purified, recombinant RHD virus antigens
• Rabbit haemorrhagic disease often called rabbit plague in Tru [G ⁇ rski 1988] is a viral disease which occurs with an acute or hyper acute phase, and exhibits a high mortality, in some cases as high as 100%. The incubation period of the disease lasts from 24 to 72 hours. Post mortem analysis of rabbits shows edema of the lungs with clear signs of haemorrhagic inflammation. Swelling and distrophy of the liver are also observed, as well as enlargement of the spleen and bloodshot and pulpy kidneys.
• Histopathological analysis shows extensive haemorrhaging in the lungs, hyperemia and edema of lymph nodes, lysis and atrophy of hepatocyte nuclei, atrophy of spleen pulp, distrophy and hyperemia of the kidneys.
• the RHD virus was placed in the Caliciviridae family.
• the virus possesses the ability to agglutinate erythrocytes of blood type O [G ⁇ rski 1988, Liu
• the enzyme-linked immunosorbent assay is a widely known and useful diagnostic method used to detect various components present in bodily fluids such as serum or urine, or in the supernatants of tissue cultures and homogenates.
• ELISA test is a sensitive and specific method, while at the same time simple to perform.
• virus detection is performed using the hemagglutination index (HA), and the presence of specific antibodies in rabbit serum is detected using the hemagglutination inhibition index (HI).
• HA hemagglutination index
• HI hemagglutination inhibition index
• RHD diagnostics have made use of electron microscopy, Western blotting and immunofluorescence in addition to HA and HI. Detection of RHD using the above methods produces trustworthy results, but is also very expensive and is generally limited to scientific research. Much more frequently, RHD diagnostics make use of the ELISA test, used in a variety of modifications due to its sensitivity, specificity and simplicity.
• IZS kits make use of monoclonal antibodies against different RHDV epitopes, as well as polyclonal rabbit anti-sera and a negative RHDV antigen isolated from tissues of infected rabbits.
• a competitive ELISA test has been designed for the detection and quantification of neutralizing antibodies.
• an adsorbing plate is coated with a specific rabbit antiserum.
• a mixture of the studied serum and a known dose of RHD antigen is then applied to the plate.
• a virological diagnostics kit contains such reagents as: a mixture of several monoclonal antibodies facilitating the discovery of RHDV in the internal organs of infected rabbits as well as the detection of EBHSV in hares.
• a plate coated with a specific anti-RHDV serum is treated with the studied antigen, which is then detected using a conjugate of monoclonal antibodies with HRP.
• the presence of RHDV is visualized via a chromogenic reaction (positive sample) using a specific substrate.
• the intensity of the chromogenic reaction is measured spectrophotometrically and analyzed in relation to control samples, both positive and negative. The results are interpreted quantitatively.
• Literature on RHD also makes mention of using "direct" ELISA, in which the detection of antibodies is facilitated by absorbent plates which are coated directly with purified RHDV and then used to detect antibodies (Rodak 1990).
• an ELISA kit has been made available commercially from the Kalon company for the detection of anti-RHD antibodies. It uses a recombinant RHDV protein produced in a baculovirus system.
• the kit contains coated absorbent plates and positive and negative reference sera.
• the RHD virus genome is contained in an icosahedral, envelope-less capsid, and is a single, positive RNA strand (ss RNA+) some 7,5 kb long.
• the genome was cloned for the first time by Meyers et al. [Meyers 1991a, 1991b], and its full sequence was published by Rasschaert et al. showing 98% amino acid homology [Rasschaert 1994].
• Capsid protein-coding sequences of the genome were reported by [Boga 1997] among others, for a strain isolated in Spain; Milton et al., for strains isolated in the Czech republic, and Austria, and by Guittre et al, who demonstrated 96 to 98% sequence identity among French strains.
• RHD virus genome exhibits significant homology to other caliciviruses ⁇ Feline c ⁇ licivirus -FCV, San Miguel sealion virus - SMSV, Vesicular exanthema virus of swine - VESV), but is organized differently, since it only possesses two open readfing frames (ORF), and not three as in the above examples.
• the main ORF of the RHD virus 2344 codons, encompasses virtually the entire genome from nucleotide 10 to 7042 and codes a 257 kDa polyprotein, which may be subdivided into non-structural proteins (nt. 1000 - 5000) and the structural capsid protein VP60 (5305 - 7044).
• the VP60-coding sequence is found near the 3 '-end of ORFl.
• ORF2 A smaller reading frame, ORF2 occurs beside ORFl (7025-7378) which contains 353 nucleotides and codes the VP12 protein, of as yet unknown function.
• subgenomic RNA has also been isolated (2.2 kb) which codes for the VP60 structural capsid protein shortened by two amino acids, and likely plays a part in replication [Meyers 1991a, 199Ib].
• RHDV viral cDNA obtained in vitro in rabbit reticulocyte lysate systems (RRL) and E. coli were used in the study of the mechanisms of formation and degradation of the large polyprotein (257 kDa) coded by ORF 1.
• RTL rabbit reticulocyte lysate systems
• E. coli E. coli
• RHDV structural capsid protein VP60 was achieved in the viral vaccinia (cow pox) system.
• RK13 cells were infected with the recombined virus, RecV-VP60, in order to enable it to proliferate.
• IF intermediate fluorescence
• anti-RHDV antibodies the 60 kDa protein product exhibited antigenic properties.
• the protein induced the production a high titre of specific antibodies. It also protected the animals during an attempt to infect them with the contagious RHD virus performed 15 days post-inoculation [Bertagnoli 1996].
• the gene coding the RHDV structural capsid protein was expressed using an E. coli system or using T7 RNA polymerase.
• the E. coli system produced a fusion protein, which only partially exhibited antigen similarity to native VP60 and did not produce immunity during infection trials using the contagious virus.
• the T7 RNA system used a recombinant protein which, following double immunization of rabbits (7 day interval) stimulated the production of specific antibodies and protection following infection with a lethal dose of RHDV 7 days following the administration of the second dose [Boga 1994]..
• the Saccharomyces cerevisiae yeast system was used to obtain a recombinant protein which protected against attempts to infect with the contagious RHD virus.
• the protein possessed antigenic properties typical for RHDV. Microscopic analysis showed the presence of VLP 's exhibiting a similar diameter.
• 4 rabbits were immunized subcutaneously with samples of total protein composed of an ultracentrifugation pellet at a dose of 400 ⁇ g (the relative amount of recombinant protein per sample was 10% by SDS-PAGE).
• Contagious virus infection at 10OxLD 50 was carried out 14 days post immunization with a single dose [Boga 1997].
• the RHD capsid protein was expressed in a baculovirual system in insect cells.
• Rabbits were immunized twice with intramuscular injections of 30 ⁇ g of purified VLP' s. White mice were immunized intraperitoneally with lO ⁇ g of the recombinant protein. The antigen was administered with the Titer Max adjuvant. A booster dose was given after 14 days. Four immunized rabbits were infected with the contagious virus at a dose of 1000xLD50. Immunized rabbits survived the infection, whereas 2 control rabbits died [Nagesha 1995].
• the RHD virus capsid protein was expressed in a baculovirus system in insect cells in two ways: 1) on the basis of genetic information encoded in an mRNA template analogous to the subgenomic RHD viral RNA, and 2) as fragments of a larger polyprotein encompassing nonstructural proteins (protease 3C and RNA polymerase) and the structural capsid protein, based on genetic information encoded in the genomic RNA. Both methods produce VLP' s antigenically and morphologically similar to purified native viral particles. Six rabbits not expressing specific antibodies were immunized once with the recombinant protein VLP' s in doses of 10 and lOO ⁇ g protein with an adjuvant (aluminum hydroxide gel).
• Sera were collected after 2 weeks and contagious virus infections were performed, with doses of 10 3 XLD 50 .
• the rabbits exhibited specific antibodies with titres ranging from 1/60 to 1/400. Immunized rabbits survived without exhibiting any symptoms of disease, whereas control animals died within 52 hours of infection [Sibilia 1995].
• Patent description PL183976 (published 1998-12-07) describes a method of utilizing an ELISA assay to test rabbit sera for RHD.
• the ELISA test is based on microplates coated with a specific chicken anti-serum against RHDV, RHD viral antigen, conserved with glycerol, specific guinea pig antiserum against RHDV, antiglobulin conjugate, enzymatic substrate - H2O2/OPD and a reference system consisting of VHD negative rabbit serum, highly positive serum and a threshold serum.
• Patent description PL183983 (published 1998-12-07) describes a method of utilizing an ELISA assay to test rabbit sera for RHD.
• the ELISA test is based on microplates coated with a specific chicken anti-serum against RHDV, specific guinea pig antiserum against RHDV, antiglobulin conjugate, enzymatic substrate - H2O2/OPD and a reference system: a positive, hemagglutinating reference and a negative, non- hemagglutinating reference.
• Patent description PL160369 (published 1991-03-11) describes a method of producing a vaccine against RHD.
• Patent description US2003186431 (published 2003-10-02) describes a vaccine against RHDV and the antigen.
• the structural RHDV antigen VP60 or a fragment thereof may be obtained from plants through the use of a viral vector based on the pox virus (PPV), containing a promoter, a recombined DNA sequence consisting of cDNA fused to the PPV genome, full length, and a DNA sequence coding for the RHDV protein VP60 or its fragment introduced between the nucleotide sequences coding the
• PPV pox virus
• the antigen obtained is capable of stimulating immune reactions against RHDV, and may be used as a recombinant subunit vaccine against it.
• Patent description WO9639177 (published 1996-12-12) describes a composition of a recombined virus, poxvirus-calicivirus (RHDV) and the application thereof. It describes attenuated, recombined viruses containing DNA coding for calicivirus antigens such as the REODV antigen, namely the capsid gene, as well as methods and compositions containing said viruses, the expression of their products and antibodies produced through the activity of said viruses and gene products with numerous prophylactic, therapeutic and diagnostic applications.
• the viral DNA may be used both in probes and in primers.
• Patent description CNl 134461 (published 1996-10-30) describes recombined capsids and proteins of RHDV, diagnostic kits and vaccines containing said capsids and p[roteins.
• Recombined capsids and recombined RHDV proteins were produced in a recombinant baculovirus expression system in an insect cell culture.
• the capsids and proteins produced are immunogenic and antigenically analogous to the native VP60 RHDV protein, and are thus suitable for the production of vaccines used against RHD as well as for the preparation of diagnostic tests capable of detecting antibodies against RHDV and the presence of RHDV in biological samples collected from rabbits.
• Patent description EP0704529 (published 1996-04-03) describes a vaccine against the rabbit haemorrhagic disease virus, production of the structural antigen (VP60) of the RHD virus and its application in a vaccine.
• Antigen production and immunization encompasses the following steps: a) isolation of the gene coding the RHD virus structural protein VP60; b) cloning of said gene in the baculovirus transfer vector pAcYMl; c) production and isolation of the recombined baculovirus, AcVP60; d) infection of SJ9 insect cells with the recombined AcVP60 baculovirus; e) production and isolation of the VP60 protein expressed by the recombined baculovirus AcVP60; f) inoculation of rabbits using the antigen.
• Patent descriptions CS9005797 (published 1992-03-18) and CS 8906486 (published 1992-03-18) describe lymphocytes produced in hybrydoma cell lines of mice or related rodents (RHDV-02 and RHDV-01, for descriptions CS9005797 and CS8906486 respectively) which produce monoclonal antibodies against the rabbit haemorrhagic disease virus.
• Patent description AU6247000 (published 2000-12-14) describes the composition of a recombinant poxvirus-calicivirus virion and its application.
• patent description US5989561 (published 1999-11-23) describes the composition of a recombinant poxvirus-calicivirus RHD virion and its application.
• This solution presents atenuated recombined viruses containing DNA coding for a calicivirus antigen such as the RHDV antigen (capsid gene) as well as methods and compositions pertaining to the virus, products of their expression and antibodies produced by said virus or expression products.
• the recombined viruses may be recombined NYVAC or ALVAC viruses.
• Recombined viruses and gene products obtained therefrom as well as antibodies produced by the virus and gene products may have numerous applications in prophylaxis, therapeutics and diagnostics. DNA from the viruses may be used in probes and primers.
• kits for the examination of rabbit (and other species') sera for the presence of anti-REID antibodies and to detect the presence of REDDV antigens in animals.
• kits encompass a purified, antigenically stable preparations of recombined VP60 protein in the form of VLP' s, which facilitate the use of monospecific antisera in ELISA tests, obtained as a result of the immunization of laboratory animals with purified, recombined RHD antigen (VLP RHDV).
• VLP RHDV purified, recombined RHD antigen
• the subject of the present invention is a serological diagnostic kit used for the examination of animal sera for the presence of anti-RHD antibodies based on the ELISA test, characterized in that the diagnostic kit encompasses the coating serum - rabbit monospecific antiserum against VLP RHDV, the specific antigen - VLP-RHDV, a detecting serum - monospecific guinea pig antiserum against VLP-RHDV VP60, an anti-globulin conjugate tagged with an enzyme, an enzymatic substrate, as well as control rabbit sera: negative, highly positive and threshold containing specific anti-RHD antibodies.
• the active components of the kit were obtained through the immunization of laboratory animals with a recombinant structural RHDV protein VP60, purified through ultracentrifugation in a saccharose desnity gradient.
• absorbent plates are coated with a polyvalent, monospecific antiserum (1° antibody) from a rabbit immunized with a fraction of RHD VLP, a recombinant structural protein with a mass of 60 kDa (VLP RHDV VP60) obtained in a baculovirus system as a result of the expression of the full vp60 gene (1800 bp) from the hemagglutinating strain SGM of RHDV.
• the antigen used in the liquid phase of the test consists of the medium from cultures of Spodoptera frugiperda insect cells transfected with the recombinant baculovirus (SGMl 800 BacV) at m.o.i 2,0, collected 4 days post infection of the cells, with glycerol as a preservative.
• SGMl 800 BacV recombinant baculovirus
• the VLP 's are in the form of a medium from an insect cell culture containing the recombinant RHDV structural capsid protein (VLP RHDV VP60).
• the anti-globulin conjugate consists of a serum containing anti-guinea pig IgG antibodies conjugated with HRP and the substrate is the chromogen o- phenylenediamine dihydrochloride, OPD, activated by hydrogen peroxide, H 2 O 2 .
• the negative reference serum directed against RHD is obtained through the combination of equal parts of many portions of sera from clinically healthy rabbits, never immunized, which have never had contact with RHD.
• the highly positive reference serum directed against RHD is composed of rabbit serum obtained through the immunization of rabbits with inactivated, native RHDV and then challenged with the contagious virus.
• the highly positive refrence serum exhibits an HI titre no smaller than 1280.
• the threshold positive reference serum consists of serum from rabbits immunized with an anti-RHD vaccine, collected between 10 and 14 days post inoculation, with a HI titre of 1/40 - 1/80.
• the next subject of the present invention is a diagnostic kit used for the examination of the presence of RHDV, based on the' ELISA test, characterized in that the diagnostic kit encompasses rabbit antiserum against VLP RHDV VP60, guinea pig antiserum against VLP-RHDV VP60, an anti-globulin conjugate tagged with an enzyme, an enzymatic substrate, as well as control viral samples: negative and positive.
• the active components of the kit were obtained through the immunization of laboratory animals with a recombinant structural RHDV protein VP60, purified through ultracentrifugation in a saccharose density gradient.
• the RHD virus antigen contained in a studied sample binds to the coating serum, a rabbit antiserum against VLP RHDV VP60 and is available to anti-VLP RHDV VP60 immunoglobulins (IgG) present in the guinea pig serum.
• the tested samples whose OD 492 is at least twofold higher than that of the negative sample, are considered positive.
• the polyvalent, monospecific rabbit and guinea pig antisera were obtained as a result of the active immunization of animals, following a sequence of two inoculations via subcutaneous injection at 21-28 day intervals.
• absorbent plates are coated with a polyvalent, monospecific antiserum (1° antibody) from a rabbit immunized with a fraction of RHD VLP, a recombinant structural protein with a mass of 60 kDa (VLP RHDV VP60) obtained in a baculovirus system as a result of the expression of the full vp60 gene (1800 bp) from the hemagglutinating strain SGM of RHDV.
• the detecting serum (2° antibody) is a polyvalent, monospecific antiserum from a guinea pig immunized with a fraction of RHD VLP, a recombinant structural protein with a mass of 60 kDa (VLP RHDV VP60) obtained in a baculovirus system as a result of the expression of the full vp60 gene (1800 bp) from the hemagglutinating strain SGM of RHDV.
• the anti-globulin conjugate consists of a serum containing anti-guinea pig IgG antibodies conjugated with HRP and the substrate is the chromogen o- phenylenediamine dihydrochloride, OPD, activated by hydrogen peroxide, H 2 O 2 .
• the RHDV antigen positive and negative control samples consist of purified tissue homogenates.
• the positive viral antigen (+)RHDV consists of a 20% liver suspension from rabbits infected with RHDV exhibiting hemagglutinating properties, inactivated and purified through centrifugation, chloroform and conserved 1:1 with glycerol.
• the negative viral antigen (-)RHDV consists of a 20% liver suspension from rabbits unexposed to RHDV, purified with chloroform and conserved 1:1 with glycerol.
• the reagents comprise a complete diagnostic set for the detection of the presence of RHD virus (antigen) and for the standardization of reagents necessary for the serological ELISA test for anti-RHD antibodies.
• Example I Seroconversion was evaluated in the sera of a laboratory animals: rabbits and guinea pigs immunized with a double purified, recombined SGMl 800 VP60 protein at a concentration of ca. 100 ⁇ g/ml, with an addition of Freund's complete adjuvant (CFA) and incomplete (ICFA).
• CFA complete adjuvant
• ICFA incomplete
• the animals were injected cutaneously and subcutaneously at 21 day intervals (guinea pigs) or 28 days (rabbits).
• the preparation of recombined SGMl 800 VP60 protein (VLP' s present in the Sf9 insect cell culture) were purified by centrifugation against a saccharose gradient (60%-10%) at 82000 x g and 4°C for 1.3 h.
• the fifth, unimmunized rabbit constituted the control.
• guinea pigs were immunized twice with a composition consisting of equal parts insect cell culture medium containing VP60 and either CFA or ICFA adjuvants. Each guinea pig was given 0.5 ml of the mixture.
• Example II Serum titration or point study To perform an ELISA in order to examine the rabbit sera flat-bottomed ELISA microplates with high absorbency for IgG (maxisorb, Nunc) were used, as well as round-bottomed plates.
• the ELISA plates are coated with a monospecific rabbit antiserum against VLP RHDV at an optimal dilution of 1/4000 in carbonate buffer, pH 9.6, and incubated for 18-24h at 4-8°C.
• the studied samples are placed along with the controls (columns 11 and 12) at a dilution of 1/10 or in serial, two-fold dilutions, meaning 1/10, 1/20, 1/40 and so on in PBS + 0.05% Tween 20 (PBST).
• 50 ⁇ l of the recombinant antigen is added to each well (VLP RHDV) at a dilution of 1/1500.
• Each serum in the point study is applied twice (schematic of the distribution of studied samples and controls: Schematic No. 1).
• the plates are incubated over 18-24h at a temperature of 4-8 0 C.
• the ELISA plates are washed 5 times with PBST and 50 ⁇ l of the antigen/serum mixture from the round-bottomed plate are placed in appropriate wells. Following Ih incubation at 37°C, the plate is washed as above and 50 ⁇ l of monospecific guinea pig antiserum against VLP RHDV diluted 1/8000 in PBST with 1-2% bovine serum albumin (PBST- BSA) are added. The plate is incubated for a period of Ih at 37°C and following washing as above, 50 ⁇ l of the conjugate of specific IgG directed against guinea pig IgG and HRP is added to each well at a 1/1000 dilution in PBST +BSA.
• PBST- BSA bovine serum albumin
• the chromogenic substrate OPD (50 ⁇ l/well) activated by H 2 O 2 , is added in citrate buffer (pH 5.0) in order to carry out the chromogenic reaction. After 10-15 minutes, the reaction is stopped through the addition of 50 ⁇ l IM H 2 SO 4 .

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