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  • 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 subject of the present invention is a method of manufacturing a recombinant vaccine against rabbit viral haemorrhagic disease, the vaccine and the application of a recombinant protein antigen in the manufacture of the vaccine.
• the subjects of the present invention facilitate the production of a recombinant capsid protein exhibiting hemagglutinin and antigenic properties, which is able to form virus-like particles (VLP) and to stimulate the production of specific antibodies in inoculated animals.
• VLP virus-like particles
• Rabbit haemorrhagic disease (RHD) often called rabbit plague in Poland [G ⁇ rski
• the RHDD virus was placed in the Caliciviridae family.
• the virus possesses the ability to agglutinate erythrocytes of blood type O [G ⁇ rski 1988, Liu 1984, Ohlinger 1990, Parra 1990].
• HA hemmaglutination index
• ELISA immunoenzymatic assay
• RHD virus genome exhibits significant homology to other caliciviruses (Feline calicivirus -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, nucleotides 10 through 7042, and codes for 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.
• ORFl 7025-73708
• subgenomic RNA exists (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].
• VLP-RHDV virus-like pariticle
• morphologically morphologically (diameter, appearance under electron microscopy) to a native RHD virus.
• VLP expression was achieved in a baculovirus system including hemagglutinizing RHD virus strains, as well as other caliciviruses for which in vitro proliferation was not possible, e.g the Norwalk virus, which causes bowel disease with diarrhea in humans, or the EBHS virus in rabbits.
• 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.
• T7 phage RNA polymerase system and the ⁇ 10 promoter along with the RBS region of the 10 gene of the T7 phage (which codes an envelope protein), using E. coli as a host.
• the T7 polymerase is an enzyme encoded by its 1 gene, and is a single polypeptide of 883 aa, with a molecular mass of 98,8 kDa.
• the T7 polymerase recognizes only its own terminators. Its activity rate is 8 times faster than the E. coli polymerase and the RBS region of the T7 phage 10 gene promoter increases translation efficiency 40-fold for most tested genes due to a series of translation signals.
• T7 RNA polymerase In order to use the T7 RNA polymerase, special vectors were constructed, in which the strong T7 phage ⁇ lO promoter was inserted along with the T ⁇ terminator. These vectors are considered to be derivatives of the pBR322 plasmid. These are the pT7-3 to ⁇ T7-7 vectors.
• 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].
• a 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 ultracentrifuge pellet at a dose of 400 ⁇ g (the relative amount of recombinant protein per sample was 10% by SDS-PAGE). Contagious virus infection at 10OxLDs 0 was carried out 14 days post immunization with a single dose [Boga 1997].
• the RHD virus structural capsid protein VP60 was produced in transgenic potato plants. Rabbits were immunized through an injection of leaf extract. Their serum exhibited high titres of specific anti-RHDV antibodies, and the rabbits were protected against experimental infection.
• the rabbits were infected with the contagion, HA titre l:16000/ml each. The rabbits survived the infection [Castanon 1999].
• 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 native viral particles.
• 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 RHD 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 proteins Nib and CP, as well as a cloning element.
• the antigen obtained is capable of stimulating immune reactions against RHDV, and may be used as a recombinant subunit vaccine against it. _ .
• Patent description EP0972840 (published 2000-01-19) describes a new attenuated recombined myxoma virus and its application in the production of mixed vaccines against myxomatosis and RHD.
• the new recombined myxoma virus has been deposited in the CNCM bank unde the accession number 1-1990. Its genome contains a nucleotide sequence capable of expressing the RHD antigen VP60.
• the strain is attenuated and exhibits a controlled transmission ability, both as a vaccine against myxomatosis and against RHDV.
• the patent also describes a method of producing the attenuated myxoma virus used in the manufacture of self-propagating combined vaccines against myxomatosis and other rabbit diseases.
• 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 RHDV 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 KR2002061139 (published 2002-07-23), HU53809 (published 1990-12-28), CN86100553 (published 1987-09-02), and CS8808644 (published 1989- 12-13) describe methods of producing inactivated vaccines against the viral haemorrhagic disease of rabbits (VHD).
• Patent description CS8904066 (published 1991-02-12) describes an inactivated, oil- based vaccine against the rabbit haemorrhagic disease virus and the method of its preparation.
• Patent description RU2064304 (published 1996-07-27) describes a method of preparing a vaccine against myxomatosis and the rabbit haemorrhagic disease virus.
• Patent description RU2007452 (published 1994-02-15) describes a strain of Mus musculus hybrid cells producing monoclonal antibodies used in RHD diagnostics.
• Patent description CN1043739 (published 1990-07-11) describes a strongly contagious strain used against the rabbit haemorrhagic disease virus and method of producing the vaccine.
• Patent descriptions CS9005797 and CS8906486 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 FR2690838 (published 1993-11-12) describes a vaccine against the rabbit haemorrhagic disease virus containing inactivated YHD tissue cultures along with adjuvants, introduced into the organism without the use of a needle. Furthermore these cultures may be included in vaccines against myxomatosis.
• 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.
• Patent description BG52094 (published 1996-07-31) describes a method of producing a vaccine against RHD.
• the vaccine is produced from liver tissues infected by the virus, which is extracted, mechanically lysed in a sodium chloride solution containing 0.8- 0.2% saponin, and then heat inactivated at 67 - 69°C for 80 to 100 min and then lyophilized.
• the goal of the present invention is to produce the means which could be used to design a method of obtaining a recombinant structural capsid protein with antigenic and hemagglutinating properties, capable of forming virus-like particles (VLP) and stimulating the production of neutralizing antibodies in immunized animals.
• VLP virus-like particles
• the subject of the present invention is a method of producing a recombinant protein against Rabbit Haemorrhagic Disease (RHD), characterized in that it encompasses the infection of rabbits with the contagious SGM strain of RHDV, collection of the liver from infected rabbits, homogenization of the organs and isolation of genetic material through total RNA isolation, production of a DNA template (cDNA), amplification of the RHDV structural capsid protein (VP60) gene, 1.8kb long, cloning of the complete vp60 gene into the pGEM T-easy vector and the determination of the complete nucleotide and amino acid sequences of the RHDV vp60 gene and its product, cloning of the vp60 gene into a baculovirus genome, transfection of insect cells and expression of the recombinant RHDV VP60 structural protein, and the adsorption of the VP60 recombined protein produced onto an adjuvant (aluminum hydroxide gel).
• the present invention makes use of the Polish strain of the virus, SGM, which exhibits both antigenic and hemagglutinating propertiues, which is also proven to be contagious in rabbits.
• the rabbits are infected with the contagious SGM RHDV strain exhibiting hemagglutinating properties.
• liver tissues from infected rabbits are collected during the peak period of the viremia, between 36 and 72 hours post infection.
• the isolation of genetic material is performed by way of isolation of total
• the DNA template (cDNA strand) is synthesized by way of reverse transcription (RT).
• RT reverse transcription
• the amplification of the RHDV structural capsid protein gene vp60 1.8kb long is performed using enzymatic amplification, wherein the amplification via the polymerase chain reaction makes use of primers encompassing the full length of the vp60 gene (ca. 1.8 kb), containing the transcription start sequence atg at 5305-5307b and a stop codon at 7042-7044b.
• the recombined RHDV VP60 protein being the product of expression in a culture of Spodoptera frugiperdai insect cells, is collected from the medium following the fourth day of culturing and is then tested for the presence of hemagglutinating virus- like particles.
• the HA titre is ascertained via the hemagglutination assay and antigenic activity through the ELISA assay.
• the cloning of the vp60 gene into a baculovirus is performed via the pFast
• Bac vector which is a component of the Bac-to-Bac (Invitrogen) protein expression system.
• the first stage encompasses cloning into the pGEM T-easy vector (Promega).
• the second stage encompasses subcloning from pGEM T-easy into pFastBac (Invitrogen).
• the recombined VP60 protein antigen is adsorbed onto an adjuvant, the latter being 5-20% aluminium hydroxide gel.
• an adjuvant the latter being 5-20% aluminium hydroxide gel.
• the adsorption of the antigen onto the adjuvant is performed at a temperature of 2-10°C.
• the next subject of the present invention is the application of the recombined VP60 protein antigen (obtained via infection of rabbits with the contagious, hemagglutinating SGM strain of RHDV, collection of livers from infected rabbits, homogenization of tissues and isolation of genetic material by way of isolating total RNA, production of a DNA template (cDNA), amplification of the RHDV structural capsid protein gene vp60 (1,8 kb long), subcloning of the full vp60 gene into the pGEM T-easy vector and determination of the nucleotide and amino acid sequence of the RHDV vp60 gene and its product, cloning of the vp60 gene into the baculo virus genome, transfection of insect cells and expression of the RHDV recombinant structural protein VP60 and the adsorption of the produced recombined VP60 protein onto an adjuvant in the form of aluminium hydroxide gel, to produce a vaccine against Rabbit
• the amplification of the RHDV structural capsid protein gene vp60 1.8kb long is performed using enzymatic amplification, wherein the amplification via the polymerase chain reaction makes use of primers encompassing the full length of the vp60 gene (ca. 1.8 kb), containing the transcription start sequence atg at 5305-5307b and a stop codon at 7042-7044b.
• the recombined RHDV VP60 protein being the product of expression in a culture of Spodoptera frugiperdai insect cells, is collected from the medium following the fourth day of culturing and is then tested for the presence of hemagglutinating virus- like particles.
• the cloning of the vp60 gene into a baculovirus is performed via the pFast Bac vector, which is a component of the Bac-to-Bac (Invitrogen) protein expression system.
• the first stage encompasses cloning into the pGEM T-easy vector (Promega).
• the second stage encompasses subcloning from pGEM T-easy into pFastBac (Invitrogen).
• the recombined VP60 protein antigen is adsorbed onto an adjuvant, the latter being 5-20% aluminium hydroxide gel.
• the next subject of the present invention jest a vaccine against Rabbit Haemorrhagic Disease (RHD), characterized in that it contains the recombined VP60 protein antigen obtained via infection of rabbits with the contagious, hemagglutinating SGM strain of RHDV, collection of livers from infected rabbits, homogenization of tissues and isolation of genetic material by way of isolating total RNA, production of a DNA template (cDNA), amplification of the RHDV structural capsid protein gene vp60 (1,8 kb long), subcloning of the full vp60 gene into the pGEM T-easy vector and determination of the nucleotide and amino acid sequence of the RHDV vp60 gene and its product, cloning of the vp60 gene into the baculovirus genome, transfection of insect cells and expression of the RHDV recombinant structural protein VP60 and the adsorption of the produced recombined VP60 protein onto an adju
• the amplification of the RHDV structural capsid protein gene vp60 1.8kb long is performed using enzymatic amplification, wherein the amplification via the polymerase chain reaction makes use of primers encompassing the full length of the vp60 gene (ca. 1.8 kb), containing the transcription start sequence atg at 5305-5307b and a stop codon at 7042-7044b.
• the recombined RHDV VP60 protein being the product of expression in a culture of Spodoptera frugiperdai insect cells, is collected from the medium following the fourth day of culturing and is then tested for the presence of hemagglutinating virus- like particles.
• the cloning of the vp60 gene into a baculovirus is performed via the pFast Bac vector, which is a component of the Bac-to-Bac (Invitrogen) protein expression system.
• the first stage encompasses cloning into the pGEM T-easy vector (Promega).
• the second stage encompasses subcloning from pGEM T-easy into pFastBac (Invitrogen).
• Example I 3 rabbits were experimentally infected with RHDV at a HA titre of 10240. These animals died between 36 and 56 bhrs. post infection. The lungs, heart, spleen, liver and kidneys were collected during the dissection. The organs of each animal were separately tagged and frozen at -18°C for 24 h. Next, the liver from one rabbit was defrosted, and a 2 g fragment was excised, cut into pieces with scissors and homogenized in a mortar, this was supplemented with 8ml of chilled 0,85% NaCl and the extraction was performed at 2-8 0 C for 1 hour.
• the homogenate was supplemented with 2 ml of analytically pure chloroform, mixed and left to extract for 1 h at 2-8°C, and then centrifuged for 10-15 min at 5000 rpm. About 9 ml of clear supernatant was obtained. This constituted the viral suspension. The viral hemagglutination titre was determined as was the antigenic activity using ELISA.
• Viral genetic material was isolated from the suspension using the guanidine thyocyanate - phenol-chloroforrn-isoamyl alcohol method and ethanol precipitation (after Chomczy ⁇ skiego i Sacchi) [Chomczy ⁇ ski 1978] and/or using the rapid RNA isolation kit from Qiagen (RNeasy mini kit).
• RT RNA isolation kit from Qiagen
• AMV reverse transcriptase and the VP2 antisense primer 7051-7071
• 5' GCACCTGCAAGTCCCAATCCG from the 3' end of the genome
• universal dtl5 primer facilitating DNA strand synthesis from the polyA end according to previously described methods (Fitzner A. 1999).
• the complete RHD vp60 gene was amplified using PCR, using the oligonucleotide primer VPl(5302-5321) containing the atg start codon: 5' ATGGAGGGCAAAGCCCG.
• the vp60 gene was cloned into a pGEM T-easy vector (Promega) according to the manufacturer's specifications. This vector is meant for cloning products destined for subsequent sequencing.
• vp60 was subcloned into the pFast Bacl vector and introduced into a baculovirus genome using the Bac-to-Bac procedure (Invitrogen, Life
• S ⁇ Spodoptera frugiperda insect cells were maintained on a synthetic medium, HyQ SFX-Insect MP (powder) with the addition of antibiotics in a monolayer culture or liquid culture at a density of 2xlO 6 /ml.
• the cells were infected with the recombined baculovirus bearing the RHDV vp60 gene (after Summers M.D., Smith E., 1987 (A manual of methods for baculovirus vectors and insect Wells culture proedures. Bulletin no 1555. teras Experimental Stadion, College stadion TX, USA) and cultured over a period from 48 to 120 hrs., preferentially 96 hrs.
• the cells were serparated from the medium.
• the suspension was separated into a solid phase and supernatant, the soluble portion of the medium containing the recombined VP60 protein, which was used as an antigen.
• IPMA IPMA
• Western blotting electron microscopy
• hemagglutination index ELISA assay
• the medium containing the recombined RHDV protein in the form of VLP's at a HA index of 5120-10240 was combined with an adjuvant, 3% aluminium hydroxide gel at a rate of 10% ( 27 ml medium and 3 ml hydroxide gel in the form of the sterile product Alhydrogel).
• the components were mixed and maintained at 2-8 °C for 1 hour. The mixture was used as an experimental vaccine.
• the remaining two rabbits comprised the unimmunized control group. Following 8 days, blood was sampled from 4 rabbits (peripheral vein of the ear) in order to determine the levels of specific antibodies. On Day 12 following the immunization, all 11 rabbits were blood-sampled again and the specific antibody levels were determined.
• the experimental infection was performed through subcutaneous injection. The rabbits were given 1,5 ml of a suspension of the contagious RHD virus at a dose of 100xLD50/ml and a HA titre of HA 10240.
• the immunized rabbits Nos. 1-7) all survived the infection and were bled following 10 days.
• the control rabbits NOS. 8k-llk
• Table 1 presents the results of assays for anti-RHDV antibodies and the determination of titres in the immunized rabbits. Increasing antibody levels were noted in sera sampled during the consecutive days following immunization and infection. Specific antibody titres in 6 immunized rabbits on day 12 following inoculation ranged from 1:60 to 1:320, and in one animal they were at the threshold of detection. Following infection, a large increase in the antibody titres was noted in all rabbits.
• VLP' s present in the SJ9 insect cell culture were purified by centrifugation against a saccharose gradient (60%- 10%) at 82000 x g and 4°C foir 1.3 h. Selected gradient fractions were used in the immunization which contained VLP' s with the highest antigenic activity (as determined by ELISA, Western blotting and HA).
• 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. Blood was sampled from immunized animals prior to immunization, and following 14 and 21 (or 28) days after the first immunization, and 14 days after the antigen booster dose.

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