US20140348875A1 - Koi herpesvirus vaccine - Google Patents

Koi herpesvirus vaccine Download PDF

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US20140348875A1
US20140348875A1 US14/368,093 US201214368093A US2014348875A1 US 20140348875 A1 US20140348875 A1 US 20140348875A1 US 201214368093 A US201214368093 A US 201214368093A US 2014348875 A1 US2014348875 A1 US 2014348875A1
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Alain Vanderplasschen
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Universite de Liege ULG
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Definitions

  • the present invention relates to a recombinant Koi herpesvirus (KHV), methods for the production of such KHV, cells comprising such KHV and the use of such KHV as a vector and in vaccines for the prevention and/or therapeutic treatment of a disease in fish caused by Koi herpesvirus in carp such as Cyprinus carpio carpio or Cyprinus carpio koi.
  • KHV Koi herpesvirus
  • Common carp Cyprinus carpio carpio
  • Koi Cyprinus carpio koi subspecies
  • a virus causing a lethal disease in both common and Koi carp was detected in 1996 in the United Kingdom.
  • the virus was then rapidly identified as the cause of mass mortality among Koi and common carp in Israel, the USA, and Germany Intensive culture of common carp, Koi shows and international trading have contributed to the rapid global spread of this highly contagious and extremely virulent disease. Since its emergence, KHVD has caused severe financial and economic losses in both Koi and common carp culture industries worldwide.
  • the genome of the virus comprises linear double-stranded DNA (dsDNA) of ⁇ 295 kb similar to that of Cyprinid herpesvirus 1 (CyHV-1) but larger than those of Herpesviridae members generally ranging from 125 to 240 kb in size.
  • dsDNA linear double-stranded DNA
  • Cyprinid herpesvirus 1 Cyprinid herpesvirus 1
  • the sequence of KHV genome has been published quite recently (Aoki et al., J Virol, 81, pages 5058-5065 (2007)).
  • the KHV genome contains a significant number of DNA sequences without homology to any other known viral sequences. Moreover, it contains highly divergent DNA sequences encoding polypeptides, which resemble those of several dsDNA viruses, like herpesvirus, poxvirus, iridovirus and other large DNA viruses.
  • KHV Koi herpesvirus
  • CNGV carp interstitial nephritis and gill necrosis virus
  • CyHV-3 Cyprinid herpesvirus 3
  • KHV bears a genome of approximately 295 kb which represents the largest genome ever identified among Herpesvirales members. Although the first isolation of KHV dates from 1996, only little information is available about the role of individual genes in KHV pathogenesis and in the biology of the infection of the natural host.
  • HCMV Human Cytomegalovirus
  • a recombinant KHV in which the Open Reading Frame 57 (ORF57) is deficient shows a strongly reduced or no mortality at all, even in very young/small carp infected with this herpesvirus recombinant and provides immunity against wild-type Koi herpesvirus.
  • ORF57 Open Reading Frame 57
  • Such a recombinant KHV thus provides a safe and efficacious attenuated vaccine virus that can suitably be used in young and/or small carp.
  • a first embodiment of the present invention relates to a recombinant Koi herpesvirus in which ORF57 is deficient, resulting in a KHV which is attenuated and induces a mortality rate of 40% or less in carp, preferably Cyprinus carpio carpio or Cyprinus carpio koi , when infected with said herpesvirus.
  • a “deficient” ORF57 is an ORF57 that is no longer functional, i.e. no longer capable of encoding a functional protein.
  • a deficient ORF57 as used herein results in a KHV which is attenuated to the level that it induces a mortality rate of 40% or less in carp.
  • Such a deficiency can e.g. be obtained by mutation such as insertion or deletion of one or more nucleotides in the gene encoding ORF57, or in its promoter region.
  • Such a mutation can e.g. be a frame shift mutation at the 5′ site of the gene, or a deletion of (part of) the promoter region or (part of) the gene itself.
  • a putative promoter region is located at position 100212-100261 that may possibly be involved in the expression of the adjacent ORF58. For this reason, a mutation in ORF57 should preferably not extend into this region. Thus, it is preferred to introduce mutations in ORF57 in the region on the left hand of position 100212 or on the right hand of position 100261.
  • a preferred form of this embodiment relates to a recombinant Koi herpesvirus according to the invention which is deficient in at least one additional gene which contributes to virulence but is not essential for replication of the virus.
  • the recombinant KHV is additionally deficient in at least the thymidine kinase gene or the putative thymidylate kinase gene.
  • the recombinant KHV according to the present invention is additionally deficient in the thymidine kinase gene and at least one further gene which contributes to virulence selected from the group consisting of ORF12: putative tumor necrosis factor (TNF) receptor gene; ORF16: putative G-protein coupled receptor (GPCR) gene; ORF134: putative Interleukin 10 homologue gene or ORF140: putative thymidylate kinase gene.
  • TNF tumor necrosis factor
  • GPCR G-protein coupled receptor
  • ORF134 putative Interleukin 10 homologue gene
  • ORF140 putative thymidylate kinase gene.
  • the recombinant KHV is additionally deficient in at least the thymidine kinase gene and the putative thymidylate kinase gene.
  • non-replicative form means that the recombinant Koi herpesvirus still has the capability to infect cells or fish individuals (e.g. Cyprinus carpio, Cyprinus carpio carpio or Cyprinus carpio koi ) but is not able to replicate to the extend that infective progeny virus is formed.
  • infect cells or fish individuals e.g. Cyprinus carpio, Cyprinus carpio carpio or Cyprinus carpio koi
  • Such a deleted virus is cultured on a permissive cell line stably expressing the deleted gene (trans-complementation).
  • any gene which contributes to replication may be made deficient in order to obtain a non-replicating recombinant Koi herpesvirus.
  • any gene of which the inactivation leads to a non-replicative recombinant Koi herpesvirus can be deleted.
  • a gene of the recombinant KHV according to the present invention that is deleted in order to provide a non-replicative form of the virus is selected from the group consisting of:
  • a recombinant Koi herpesvirus according to the present invention preferably comprises a bacterial artificial chromosome (BAC) vector sequence.
  • BAC bacterial artificial chromosome
  • herpesvirus genomes Since about one and a half decade, the manipulation of large herpesvirus genomes has been greatly facilitated by the use of such bacterial artificial chromosomes. These vectors allow the maintenance and the mutagenesis of the viral genome in Escherichia coli , followed by reconstitution of progeny virions by transfection of the BAC plasmid into permissive eukaryotic cells.
  • the sequences for the BAC vector are introduced into the herpesvirus genome by conventional homologous recombination in infected cells.
  • the linear double-stranded DNA genome of herpesviruses circularizes during replication. It suffices to isolate the circular replication intermediate of the BAC mutant and to shuttle it by DNA transformation into E. coli .
  • the herpesvirus BAC is then propagated and mutated in E. coli .
  • the homogenous, clonal herpesvirus BAC DNA is shuttled back into eukaryotic permissive cells only for virus reconstitution. As viral functions are not required, the virus genome remains sleeping while in E. coli , preserving the viral functions present at the time of cloning. This is important for viruses where in vitro culture procedures change the authentic properties of isolates.
  • sequence mediating homologous recombination refers to a sequence which causes homologous recombination which is dependent from a specific recombination protein, which is catalyzing, carrying out or assisting in homologous recombination. Such a recombination protein preferably acts specifically on a “sequence mediating homologous recombination” and does not act on other sequences.
  • BAC vector sequences are well-known in the art and their use in the construction of recombinant viruses such as herpesviruses has frequently been described in the art (Borst, E. M., Hahn, G., Koszinowski, U. H. & Messerle, M. (1999), J Virol 73, 8320-9. Costes, B., Fournier, G., Michel, B., Delforge, C., Raj, V. S., Dewals, B., Gillet, L., Drion, P., Body, A., Schynts, F., Lieffrig, F., Vanderplasschen, A., 2008. J Virol 82, 4955-4964.
  • the BAC vector sequence need not necessarily be inserted into ORF57. Alternatively it can be inserted in any other viral gene which contributes to virulence and/or any other viral gene which is or isn't essential for viral replication and/or any intergenic region.
  • the recombinant Koi herpesvirus comprises a BAC vector sequence which is inserted into ORF57.
  • Such insertion has the advantage that by inserting the BAC vector into ORF57, ORF57 becomes at the same time deficient, thus directly providing a recombinant KHV according to the invention.
  • An example of a recombinant KHV according to the present invention was achieved by cloning of the KHV genome by the insertion of a modified loxP-flanked BAC cassette into ORF55 (vide infra). This insertion led to a BAC recombinant virus whose genome was stably maintained in bacteria and was able to regenerate virions when transfected into permissive cells. (See: Costes, B., Fournier, G., Michel, B., Delforge, C., Raj, V.
  • BAC vector refers to a plasmid which is produced using F plasmid of E. coli and a vector which can stably maintain and grow a large size DNA fragment of about 300 kb or more in bacteria, such as E. coli and the like.
  • the BAC vector contains at least a BAC vector sequence essential for the replication of the BAC vector. Examples of such a region essential for replication include, but are not limited to, the origin of replication of F plasmid and variants thereof.
  • BAC vector sequence refers to a sequence comprising a sequence essential for the function of a BAC vector.
  • the BAC vector sequence may further comprise a “recombination protein-dependent recombinant sequence” and/or a “selectable marker”.
  • the BAC vector sequence is flanked by sequences mediating homologous recombination, preferably loxP.
  • the BAC vector sequence comprises a selectable marker (vide infra).
  • the selectable marker is a drug selectable marker (vide infra).
  • the genome of said recombinant herpesvirus is present in the form of a plasmid. This is achieved by isolating circular forms of the above mentioned recombinant Koi herpesvirus comprising a BAC vector sequence and introduction into bacterial cells.
  • the BAC (bacterial artificial chromosome) vector sequence is inserted into one or more of the viral genes which contribute to virulence or are necessary for replication, as long as one or more of the mentioned genes which contribute to virulence or are necessary for replication is/are made deficient by genetic engineering techniques.
  • the BAC vector sequence may be inserted into any region of the virus genome, provided that ORF57 and preferably one or more other viral genes which contribute to virulence are also deficient.
  • BAC vector mediated cloning techniques as described above can be used repeatedly: e.g. a first time to make ORF57 deficient and a second time to make an additional gene deficient.
  • the BAC vector sequence may in principle remain present in a recombinant KHV according to the invention in further applications without problems.
  • a Koi herpesvirus according to the invention in e.g. a vaccine, it is preferred that most of the BAC sequences are removed. This is e.g. the case for BAC sequences that comprise genes encoding the selectable markers and even more for resistance genes. The presence of such genes in a vaccine is not only considered unnecessary, but even undesirable.
  • At least a part e.g. a part that comprises a resistance gene or a selectable marker
  • more preferably most of the BAC vector sequence is excised from the herpesvirus genome, thereby preferably leaving behind a heterologous sequence at the excision site or former insertion site in the herpesvirus genome.
  • the heterologous sequence has a size of less than 200 nucleotides. The excision is achieved by introduction of the recombinant KHV into a permissive eukaryotic cell expressing the Cre recombinase which is excising the loxP-flanked BAC vector sequence.
  • a preferred form of this embodiment relates to a recombinant Koi herpesvirus according to the invention, characterised in that part of the BAC vector sequence is excised from the herpesvirus genome thereby leaving a heterologous sequence at the excision site or former insertion site, respectively, in the herpesvirus genome.
  • the part of the BAC vector sequence that is excised from the herpesvirus genome comprises at least one gene encoding a selectable marker and/or a resistance gene.
  • BAC cassette sequence by homologous recombination in eukaryotic cells using a DNA fragment of the wild type viral genome encompassing the site of insertion of the BAC cassette (e.g. ORF55 encoding TK). Selection of viral plaques that do not longer express EGFP (encoded by the BAC cassette) allows the selection of recombinants which have reverted the site of BAC insertion to wild type sequence.
  • the recombinant Koi herpesvirus according to the present invention in either form, the KHV BAC clone, and the above mentioned KHV construct where at least part of the BAC vector sequence is excised from the herpesvirus genome may be used for further manipulation involving for example genetic engineering techniques in order to make the genome deficient in further specific genes.
  • the deficiency of such further genes can equally be obtained using the BAC technique, as already said above.
  • a recombinant KHV according to the invention can be used for vaccine purposes as such (see below), merely in order to prevent fish, more specifically carp, even more specifically Cyprinus carpio carpio or Cyprinus carpio koi , from KHV disease, it can also be efficiently used as carrier virus for heterologous (i.e. non-KHV) DNA fragment.
  • the advantageous characteristics of the recombinant KHV according to the invention would be fully used and in addition the virus would e.g. gain additional properties such as marker properties, additional immunizing properties or adjuvating properties.
  • Marker properties in this respect means that the heterologous DNA fragment allows, directly or indirectly, to discriminate between field virus infection or vaccine virus infection.
  • a direct way to discriminate between field virus infection and vaccine virus infection would e.g. comprise a PCR-reaction using primers that specifically reacts with a heterologous (i.e. non-KHV) DNA fragment in a recombinant KHV according to the invention, and not with DNA of a KHV field virus.
  • a heterologous (i.e. non-KHV) DNA fragment in a recombinant KHV according to the invention and not with DNA of a KHV field virus.
  • An indirect way to discriminate between field virus infection and vaccine virus infection would e.g. comprise an immunological reaction using an antibody that specifically reacts with an immunogenic protein encoded by a heterologous (i.e. non-KHV) DNA fragment in a recombinant KHV according to the invention, and not with any protein of a KHV field virus.
  • a heterologous (i.e. non-KHV) DNA fragment in a recombinant KHV according to the invention, and not with any protein of a KHV field virus.
  • another embodiment of the present invention relates to a recombinant KHV according to the invention that comprises a heterologous DNA fragment, e.g. a heterologous gene.
  • such a heterologous DNA fragment is a heterologous gene that encodes an immunogenic protein of another virus or microorganism that is pathogenic to fish, more specifically carp, even more specifically Cyprinus carpio carpio or Cyprinus carpio koi.
  • the heterologous gene is the G glycoprotein of rhabdovirus causing carp spring viraemia.
  • Such a construct when used in a vaccine, will not only protect carp against KHV but also against carp spring viraemia.
  • Suitable promoters for the expression of heterologous genes in eukaryotic cells are extensively known in the art.
  • An example of a suitable promoter for the expression of a heterologous gene, e.g. the G glycoprotein of rhabdovirus causing carp spring viraemia is the HCMV IE promoter.
  • the present invention further provides a method for the production of infectious particles of recombinant Koi herpesvirus (KHV), wherein said method comprises the steps of
  • the above mentioned recombinant Koi herpesviruses according to the invention and their DNA are very suitable for the immunization of fish, preferably Cyprinus carpio carpio or Cyprinus carpio koi individuals by injection or balneation or per os.
  • Still another embodiment of the present invention provides a recombinant Koi herpesvirus according to the invention and/or a KHV DNA comprising the genome of the recombinant Koi herpesvirus according to the invention for use in the prevention and/or therapeutic treatment of a disease in fish caused by Koi herpesvirus (KHV).
  • KHV Koi herpesvirus
  • Preventive use is a use that aims at preventing infection, or at least clinical manifestations of the disease
  • Therapeutic use is a use of said KHV or KHV DNA in fish that already suffer from the disease caused by KHV.
  • KHV Koi herpesvirus
  • Still another embodiment of the present invention provides a vaccine for the prevention and/or therapeutic treatment of a disease in fish caused by Koi herpesvirus (KHV), characterised in that said vaccine comprises a recombinant Koi herpesvirus according to the invention and/or a KHV DNA comprising the genome of the recombinant Koi herpesvirus according to the invention, and a pharmaceutically acceptable carrier.
  • KHV Koi herpesvirus
  • a recombinant KHV according to the invention carrying the gene encoding G glycoprotein of said rhabdovirus causing carp spring viraemia or a DNA sequence comprising the genome of said recombinant KHV, and a pharmaceutically acceptable carrier.
  • the term “vaccine” refers to a composition capable of prevention and/or therapeutic treatment of a host to a particular disease. Such a vaccine may produce prophylactic or therapeutic immunity.
  • the pharmaceutically acceptable carrier can be as simple as water or a buffer.
  • the pharmaceutically acceptable carrier may also comprise stabilizers. It can also comprise an adjuvant, or it can in itself be an adjuvant.
  • vaccines are prepared as liquid solutions, emulsions or suspensions for injection or delivery through immersion of fish in water.
  • a liquid emulsion or emulsifiable concentrate can be prepared in order to be added to a water tank or bath where the fish are held.
  • Solid (e.g. powder) forms suitable for dissolution in, or suspension in, liquid vehicles or for mixing with solid food, prior to administration may also be prepared.
  • the vaccine may be a lyophilized culture in a ready to use form for reconstitution with a sterile diluent.
  • lyophilized cells may be reconstituted in 0.9% saline (optionally provided as part of the packaged vaccine product).
  • a preferred formulation of injectable vaccine is an emulsion.
  • Liquid or reconstituted forms of the vaccine may be diluted in a small volume of water (e.g. 1 to 100 volumes) before addition to a pen, tank or bath.
  • said virus may be in the form of a tissue culture fluid.
  • Said fluid may be stored at the ambience, preferably at ⁇ 70° C., most preferably as a solution containing glycerol.
  • the tissue culture fluid contains 20% glycerol.
  • the recombinant KHV according to the invention is used as a vaccine component for oral administration (e.g. through dipping or balneation), there will usually be no need for the administration of an adjuvant.
  • An adjuvant is an immunostimulatory substance boosting the immune response of the host in a non-specific manner.
  • the adjuvant may be hydrophilic adjuvant, e.g., aluminum hydroxide or aluminum phosphate, or hydrophobic adjuvant, e.g. mineral oil based adjuvants.
  • Adjuvants such as muramyl dipeptides, avidine, aluminium hydroxide, aluminium phosphate, oils, oil emulsions, saponins, dextran sulphate, glucans, cytokines, block co-polymers, immunostimulatory oligonucleotides and others known in the art may be admixed with the recombinant KHV according to the invention.
  • adjuvants frequently used in fish farming are muramyldipeptides, lipopolysaccharides, several glucans and glycans and Carbopol® (a homopolymer).
  • Suitable adjuvants are e.g. water in oil (w/o) emulsions, o/w emulsions and w/o/w double-emulsions.
  • Oil adjuvants suitable for use in w/o emulsions are e g mineral oils or metabolisable oils. Mineral oils are e.g. Bayol®, Marcor and Drakeol®; metabolisable oils are e.g.
  • o/w emulsions are e.g. obtained starting from 5-50% w/w water phase and 95-50% w/w oil adjuvant, more preferably 20-50% w/w water phase and 80-50% w/w oil adjuvant are used. The amount of adjuvant added depends on the nature of the adjuvant itself, and information with respect to such amounts provided by the manufacturer.
  • the vaccine according to the invention additionally comprises a stabilizer.
  • a stabilizer can be added to a vaccine according to the invention e.g. to protect it from degradation, to enhance the shelf-life, or to improve freeze-drying efficiency.
  • Useful stabilizers are i.a. SPGA (Bovarnik et al., 1950, J. Bacteriology, vol. 59, p. 509), skimmed milk, gelatine, bovine serum albumin, carbohydrates e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, lactoses, proteins such as albumin or casein or degradation products thereof, and buffers, such as alkali metal phosphates.
  • Antibiotics such as neomycin and streptomycin may be added to prevent the potential growth of germs.
  • the vaccine may comprise one or more suitable surface-active compounds or emulsifiers, e.g. Span® or Tween®.
  • the vaccine may also comprise a so-called “vehicle”.
  • a vehicle is a compound to which the KHV virus (either in form of a virus particle or in form of DNA) according to the invention adheres, without being covalently bound to it.
  • Such vehicles are i.a. bio-microcapsules, micro-alginates, liposomes and macrosols, all known in the art.
  • a special form of such a vehicle is an Iscom.
  • the recombinant KHV when used in its dry form in a vaccine may further include a reconstitution fluid, preferably sterile water, saline or physiological solution. It may also contain small amounts of residual materials from the manufacturing process such as cell proteins, DNA, RNA, etc. While these materials are not additives per se, they may nonetheless be present in the vaccine formulation.
  • a reconstitution fluid preferably sterile water, saline or physiological solution. It may also contain small amounts of residual materials from the manufacturing process such as cell proteins, DNA, RNA, etc. While these materials are not additives per se, they may nonetheless be present in the vaccine formulation.
  • the vaccine may be administered to fish individually-orally, e.g. through their feed or by forced oral administration, or by injection (e.g. via the intramuscular or intraperitoneal route).
  • the vaccine may be administered simultaneously to the entire fish population contained in a body of water by spraying, dissolving and/or immersing the vaccine.
  • spraying, dissolving and/or immersing the vaccine are useful for vaccination of all kinds of fish, e.g., food and ornamental fish, and in various environments such as ponds, aquariums, natural habitat and fresh water reservoirs.
  • a further aspect of the invention relates to a DNA vaccine comprising the recombinant KHV according to the invention.
  • DNA vaccines according to the invention do not basically differ from vaccines comprising the recombinant KHV according to the invention, in the sense that they comprise the genome of a recombinant KHV according to the invention.
  • a preferred amount of a recombinant KHV DNA according to the invention, in a pharmaceutical composition according to the invention is in the range between 10 pg and 1000 ⁇ g. Preferably, amounts in the range between 0.1 and 100 ⁇ g are used.
  • fish can be immersed in solutions comprising e.g. between 10 pg and 1000 ⁇ g/ml of the DNA to be administered. All these techniques and routes of administration are well-known in the art.
  • the vaccine according to the invention is formulated in a form suitable for injection or for immersion vaccination, such as a suspension, solution, dispersion, emulsion, and the like.
  • the dosing scheme for the application of a vaccine according to the invention to the target organism can be the application of single or multiple doses, which may be given at the same time or sequentially, in a manner compatible with the dosage and formulation and in such an amount as will be immunologically effective. It is well within the capacity of the skilled person to determine whether a treatment is “immunologically effective”, for instance by administering an experimental challenge infection to vaccinated animals, and next determining a target animals' clinical signs of disease, serological parameters, or by measuring re-isolation of the pathogen.
  • What constitutes a “pharmaceutically effective amount” for a vaccine according to the invention that is based upon a recombinant KHV or a recombinant KHV DNA according to the invention, is dependent on the desired effect and on the target organism. Determination of the effective amount is well within the skills of the routine practitioner.
  • a preferred amount of a recombinant KHV DNA according to the invention, comprised in a pharmaceutical composition according to the invention, has been described above.
  • a preferred amount of a live vaccine comprising recombinant KHV virus strain according to the invention is expressed for instance as plaque forming units (pfu).
  • a dose range between 1 and 10 10 plaque forming units (pfu) per animal dose may advantageously be used; preferably a range between 10 2 and 10 6 pfu/dose.
  • a vaccine comprises a non-replicative form of the recombinant KHV according to the invention
  • the dose would be expressed as the number of non-replicative virus particles to be administered. Then dose would usually be somewhat higher when compared to the administration of live virus particles, because live virus particles replicate to a certain extent in the target animal, before they are removed by the immune system.
  • an amount of virus particles in the range of about 10 4 to 10 9 particles would usually be suitable.
  • FIG. 2 , 3 flowchart of stages performed to produce FL BAC galK recombinant plasmids deleted for ORF57 ( FIG. 2 ) or ORF56 ( FIG. 3 ), and to demonstrate the reconstitution of infectious virus from the produced plasmids.
  • the regions of ORF57 or ORF56, as identified in FIG. 1 were replaced by a galK expression cassette using homologous recombination in E. coli .
  • TK thymidine kinase locus
  • the recombinant plasmids were co-transfected in permissive CCB cells with pGEMT-TK plasmid.
  • To reconstitute infectious virus with a truncated form of TK FL BAC excised strains
  • the recombinant plasmids were transfected in CCB cells expressing Cre recombinase.
  • FIG. 4 flowchart of stages performed to produce FL BAC recombinant plasmids deleted for ORF57 and ORF56 (ORF56-57), and to demonstrate the reconstitution of infectious virus from the produced plasmids.
  • the region of ORF56-57, as identified in FIG. 1 was replaced by a galK expression cassette using homologous recombination in E. coli .
  • the galK expression cassette was then removed by homologous recombination with a synthetic DNA sequence corresponding to KHV genome regions flanking the galK expression cassette (ORF56-57 Del cassette).
  • FIG. 6 safety of ORF56 single deleted recombinants.
  • FIG. 7 safety (A-C) and vaccination/challenge (D-G) tests of the FL BAC excised ORF56-57 Del strain.
  • the FL BAC excised strain (A) and mock-infection (C) were used as positive and negative controls, respectively. Mock-infection was performed on duplicate groups. Percentages of surviving carp are expressed according to days post-infection taking day 0 as the reference. Vaccination/challenge (D-G) tests.
  • FIG. 8 safety (A-C) and vaccination/challenge (D-G) tests of the FL BAC revertant ORF56-57 Del strain.
  • the FL BAC revertant strain (A) and mock-infection (C) were used as positive and negative controls, respectively. Mock-infection was performed on duplicate groups. Percentages of surviving carp are expressed according to days post-infection taking day 0 as the reference. Vaccination/challenge (D-G) tests.
  • Cyprinus carpio brain cells (CCB) (Neukirch et al., 1999) were cultured in minimum essential medium (MEM, Invitrogen) containing 4.5 g/l glucose (D-glucose monohydrate, Merck) and 10% fetal calf serum (FCS). Cells were cultured at 25° C. in a humid atmosphere containing 5% CO 2 .
  • MEM minimum essential medium
  • FCS fetal calf serum
  • Electroporated cells were plated on solid M63 minimal medium supplemented with 20% galactose and chloramphenicol (17 ⁇ g/ml) to select bacteria in which homologous recombination occurred. Finally, colonies obtained were streaked onto MacConkey indicator plates as described elsewhere to confirm the production of galK positive clones.
  • Recombinant BAC molecules were amplified and purified (QIAGEN Large-Construct Kit), and their molecular structure was controlled using a combined restriction endonuclease-Southern blot approach, PCR and sequencing.
  • CyHV-3 BAC plasmids were transfected (Lipofectamine Plus, Invitrogen) into permissive CCB.
  • CyHV-3 BAC plasmids were co-transfected in CCB cells together with the pGEMT-TK vector (molecular ratio 1:75).
  • viral plaques negative for EGFP expression (the BAC cassette encodes an EGFP expression cassette) were picked and enriched by three successive rounds of plaque purification.
  • Two CyHV-3 FL BAC recombinant plasmids with deletion in the ORF56 locus were produced using a galK positive selection in bacteria as previously described (Warming et al., 2005) ( FIG. 3 ).
  • the recombination fragment consisted of a galactokinase (galK) gene (1231 bp) flanked by 50 bp sequences homologous to the regions of the CyHV-3 genome flanking the sequence to be deleted ( FIG. 1 ). These fragments were produced by PCR using the pgalK vector as template.
  • Electroporated cells were plated on solid M63 minimal medium supplemented with 20% galactose and chloramphenicol (17 ⁇ g/ml) to select bacteria in which homologous recombination occurred. Finally, colonies obtained were streaked onto MacConkey indicator plates as described elsewhere to confirm the production of galK positive clones.
  • Recombinant BAC molecules were amplified and purified (QIAGEN Large-Construct Kit), and their molecular structure was controlled using a combined restriction endonuclease-Southern blot approach, PCR and sequencing.
  • CyHV-3 BAC plasmids were transfected (Lipofectamine Plus, Invitrogen) into permissive CCB.
  • CyHV-3 BAC plasmids were co-transfected in CCB cells together with the pGEMT-TK vector (molecular ratio 1:75).
  • viral plaques negative for EGFP expression (the BAC cassette encodes an EGFP expression cassette) were picked and enriched by three successive rounds of plaque purification.
  • BAC plasmids were co-transfected in CCB cells together with the pEFIN3-NLS-Cre vector encoding Cre recombinase fused to a nuclear localization signal (Costes et al; 2008 JVI) (molecular ratio: 1:70).
  • CyHV-3 FL BAC recombinant plasmids with deletion in the ORF56 and ORF57 loci were produced using galK positive and negative selections in bacteria as previously described (Warming et al., 2005) ( FIG. 4 ).
  • the first recombination process was to replace the identified sequence of ORF56 and ORF57 by the galactokinase (galK) gene (1231 bp).
  • the recombination fragment consisted of the galK gene flanked by 50 bp sequences homologous to the regions of the CyHV-3 genome flanking the sequence to be deleted ( FIG. 1 ) (ORF56-57 Del galK, FIG. 4 ).
  • This fragment was produced by PCR using the primers ORF56-ORF57 Del fw and ORF56-ORF57 Del rev (Table 3) and the pgalK vector as template.
  • the amplification product was purified (QlAquick Gel Extraction Kit).
  • electrocompetent SW102 cells containing the CyHV-3 FL BAC plasmid were electroporated with 50 ng of the PCR product described above. Electroporated cells were plated on solid M63 minimal medium supplemented with 20% galactose and chloramphenicol (17 ⁇ g/ml) to select bacteria in which homologous recombination occurred. Finally, colonies obtained were streaked onto MacConkey indicator plates as described elsewhere to confirm the production of galK positive clones.
  • Recombinant BAC molecules were amplified and purified (QIAGEN Large-Construct Kit), and their molecular structure was controlled using a combined restriction endonuclease-Southern blot approach, PCR and sequencing.
  • the second recombination process was to remove the galK cassette from the FL BAC ORF56-57 Del galK plasmid.
  • a synthetic 499 bp DNA fragment (ORF56-57 Del cassette, vide infra) was used to reach this goal.
  • Electroporated cells were plated on solid minimal medium supplemented with 2-deoxy-galactose to select bacteria in which homologous recombination occurred (digestion of 2-deoxy-galactose by galK produce toxic products).
  • Recombinant BAC molecules were amplified and purified (QIAGEN Large-Construct Kit), and their molecular structure was controlled using a combined restriction endonuclease-Southern blot approach, PCR and sequencing.
  • CyHV-3 BAC plasmids were transfected (Lipofectamine Plus, Invitrogen) into permissive CCB.
  • CyHV-3 BAC plasmids were co-transfected in CCB cells together with the pGEMT-TK vector (molecular ratio 1:75).
  • viral plaques negative for EGFP expression (the BAC cassette encodes an EGFP expression cassette) were picked and enriched by three successive rounds of plaque purification.
  • BAC plasmids were co-transfected in CCB cells together with the pEFIN3-NLS-Cre vector encoding Cre recombinase fused to a nuclear localization signal (Costes et al; 2008 JVI) (molecular ratio: 1:70).
  • carp were acclimatized in 60-liter tanks at 24° C. for 10 days.
  • carp biomass of 50 g of fish/l
  • water containing 4, 40 or 400 PFU/ml of the KHV strain to be tested At the end of the incubation period, the fish were returned to the larger tank.
  • fish were challenged with virulent KHV by co-habitation with na ⁇ ve fish infected just before their release in the tank of vaccinated fish.
  • These fish were inoculated by immersion in water containing 300 PFU/ml of the virulent parental FL strain for 2 h. Two infected fish were added to each tank containing vaccinated fish.
  • FIGS. 5E and F Six weeks post-infection with the ORF57 single deleted recombinants ( FIGS. 5E and F), fish were challenged as described in the examples (vaccination/challenge). Mock-infected fish were used as controls ( FIG. 5G ). Percentages of surviving carp are expressed according to days post-infection taking day 42 as the reference.
  • a KHV ORF57 deletion mutant according to the invention is very suitable as an efficacious vaccine, especially when administered in a dose of 40 pfu/ml or higher.
  • a KHV carrying a deletion in both ORF57 and ORF56 shows a safety and efficacy profile that is comparable to that of KHV carrying a single ORF57 deletion.

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CN110101854A (zh) * 2019-05-14 2019-08-09 四川农业大学 一种鲤疱疹病毒ⅲ型疫苗及其制备方法
CN110452926A (zh) * 2019-08-07 2019-11-15 西北农林科技大学 一种展示CyHV-2膜蛋白的重组杆状病毒及其制备方法和应用
CN112279898A (zh) * 2018-11-05 2021-01-29 共鳞实业(深圳)有限公司 预防或治疗鱼类感染CyHV-2的试剂及其应用
CN113679832A (zh) * 2021-05-24 2021-11-23 苏州大学 一种利用冷冻干燥制备杆状病毒载鲤疱疹病毒ii型dna疫苗的方法
CN115960903A (zh) * 2022-12-23 2023-04-14 中国水产科学研究院淡水渔业研究中心 一种抑制CyHV-2病毒增殖的反义RNA组、重组载体和纳米粒递送系统及应用

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Publication number Priority date Publication date Assignee Title
WO2017177071A1 (en) * 2016-04-08 2017-10-12 William Marsh Rice University Improved galactose utilization
CN112279898A (zh) * 2018-11-05 2021-01-29 共鳞实业(深圳)有限公司 预防或治疗鱼类感染CyHV-2的试剂及其应用
CN110101854A (zh) * 2019-05-14 2019-08-09 四川农业大学 一种鲤疱疹病毒ⅲ型疫苗及其制备方法
CN110452926A (zh) * 2019-08-07 2019-11-15 西北农林科技大学 一种展示CyHV-2膜蛋白的重组杆状病毒及其制备方法和应用
CN113679832A (zh) * 2021-05-24 2021-11-23 苏州大学 一种利用冷冻干燥制备杆状病毒载鲤疱疹病毒ii型dna疫苗的方法
CN115960903A (zh) * 2022-12-23 2023-04-14 中国水产科学研究院淡水渔业研究中心 一种抑制CyHV-2病毒增殖的反义RNA组、重组载体和纳米粒递送系统及应用

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