GB2624391A - Recombinant LSDV vectored bovine coronavirus antigen constructs - Google Patents
Recombinant LSDV vectored bovine coronavirus antigen constructs Download PDFInfo
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- GB2624391A GB2624391A GB2217048.4A GB202217048A GB2624391A GB 2624391 A GB2624391 A GB 2624391A GB 202217048 A GB202217048 A GB 202217048A GB 2624391 A GB2624391 A GB 2624391A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A recombinant lumpy skin disease virus (rLSDV) vector comprises a cassette comprising a nucleic acid encoding a bovine coronavirus (BCoV) spike protein and a nucleic acid encoding a BCoV nucleocapsid protein, wherein the BCoV spike protein comprises an S1 and S2 subunit, and wherein a native cleavage site between the S1 and S2 subunits of the spike protein are replaced by a linker. Preferably, the rLSDV comprises a stabilised SOD-homolog (SOD-is) gene and the native leader sequence of the BCoV spike protein has been replaced by a tissue plasminogen (TPA) leader sequence. More preferably, the S2 subunit of the BCoV spike protein comprises amino acid substitutions A1080P and L1081P and the nucleic acid encoding the BCoV spike protein is under the control of a mH5 pox virus promoter, whilst the nucleic acid encoding the BCoV nucleocapsid protein is under the control of a pLEO pox virus promoter. A composition comprising the vector and a vaccine for use in a method of inducing any immune response against BCoV preferably in cattle, pigs, sheep, goats, deer, antelope, giraffe, camels, or buffalo are also disclosed.
Description
RECOMBINANT LSDV VECTORED BOVINE CORONAVIRUS ANTIGEN
CONSTRUCTS
BACKGROUND OF THE INVENTION
Bovine coronaviruses (BCoV) and Lumpy skin disease (LSD) are highly contagious diseases among cattle and other farm animals with severe economic costs. Control strategies using inactivated vaccines against these two diseases have proven to be costly while providing limited efficacy. A live attenuated recombinant vaccine has the potential to improve immunogenic responses and coverage while reducing vaccine cost and the economic burden of these diseases. A dual vaccine against both BCoV and LSD would also be of economic benefit.
Bovine coronavirus is a member of the Betacoronavirus genus of the Coronaviridae family of viruses and is the causative agent of BCoV infection in cattle. Infection causes calf enteritis and contributes to the enzoofic pneumonia complex in calves. It can also cause winter dysentery in adult cattle. BCoV can infect domestic and wild ruminants and has a global distribution. Transmission is via the respiratory and oro-fecal routes. The virus is an enveloped, positive-sense, single-stranded RNA virus. BCoV has a surface protein called hemagglufinin esterase (HE) in addition to the four structural proteins shared by all other coronaviruses. These structural proteins include the spike protein, membrane protein, nucleocapsid protein, and envelope protein. Infection normally occurs in calves between the ages of one week and three months. Gastrointestinal signs include anorexia, dehydration, depression, profuse diarrhoea and reduced weight gain. Respiratory infection in the calf produces a serous to purulent nasal discharge. Clinical signs may worsen with secondary bacterial infection. Infection in adults is normally subclinical, the exception being with winter dysentery, which affects housed cattle over the winter months. In winter dysentery outbreaks the clinical signs include profuse diarrhoea and a drop in milk yield.
Lumpy skin disease virus (LSDV) is a member of the Capripoxvirus genus of the Poxviridae family of viruses and is the causative agent of lumpy skin disease (LSD) in cattle. Lactating cattle have been found to be the most susceptible to disease. The rate of morbidity is often higher during an outbreak and can rise to 85%. LSD in cattle ranges from subclinical to an acute infection and usually lasts for 2-5 weeks. The acute infection is characterised by fever and localised or disseminated nodules developing -2 -on the skin, where lesions are often found in the upper respiratory tract and a secondary bacterial infection often occurs. Nodules have been reported to occur on the skeletal muscles and the mucosa of the oral and upper respiratory tract. Systemic effects include pyrexia, anorexia, dysgalactia and pneumonia. Cows may lose reproductive ability and milk producing capacity for several months. Fever, anorexia and abortions are also common. The drop in milk production in lactating cows along with temporary or permanent infertility in cows and bulls and skin lesions caused by the virus all impact on the economic effects of the disease.
Eradication campaigns and imposed trade restrictions on live animals and animal products add to the financial toll of outbreaks. This has resulted in the World Organization for Animal Heath (01E) recognising LSD as a notifiable agricultural disease. Although both BCoV and LSD are agricultural diseases of great importance, there currently does not exist a dual vaccine that is effective against both diseases simultaneously. The development of a single vaccine for control of the two viral diseases would be attractive to both cattle owners as well as vaccine manufactures, due to the reduction in cost and number of vaccines administered
SUMMARY OF THE INVENTION
The present invention relates to a recombinant LSDV (rLSDV) vector comprising or consisting of a cassette encoding a modified bovine coronavirus (BCoV) spike protein and a BCoV nucleocapsid protein. The invention also relates to compositions containing the rLSDV vector and cassette and to a dual vaccine against LSDV and BCoV.
In a first aspect of the invention there is provided for a recombinant lumpy skin disease virus (rLSDV) vector that comprises or consists of a cassette comprising or consisting of a nucleic acid encoding a modified bovine coronavirus (BCoV) spike protein and a nucleic acid encoding a BCoV nucleocapsid protein. The modified BCoV spike protein comprises or consists of an Si and S2 subunit, separated by a linker. The linker between the Si and 52 subunits of the spike protein replace the native furin cleavage site.
In a first embodiment of the invention the rLSDV comprises or consists of a stabilised SOD-homolog (SOD-is) gene. Preferably, the stabilised SOD-is gene encodes an amino acid sequence of SEQ ID NO:24.
In a second embodiment of the invention the native leader sequence of the BCoV spike protein has been replaced by a tissue plasminogen (TPA) leader -3 -sequence. In a preferred embodiment the TPA leader sequence comprises or consists of the sequence of SEQ ID NO:10.
In a third embodiment of the invention the linker sequence comprises or consists of an amino acid sequence of SEQ ID NO:9. It will be appreciated that the linker sequence replaces the native furin cleavage site between the Si and S2 subunits of the spike protein.
In a fourth embodiment of the invention the S2 subunit of the BCoV spike protein comprises or consists of two amino acid substitutions at positions 1080 and 1081 of the BCoV spike protein amino acid sequence. The aforementioned substitutions are A1080P and L1081P.
In a fifth embodiment of the invention the BCoV spike protein comprises or consists of a sequence of SEQ ID NO:2.
In a sixth embodiment of the invention the BCoV nucleocapsid protein comprises or consists of a sequence of SEQ ID NO:4.
In an eighth embodiment of the invention the cassette encoding the modified BCoV spike protein and the BCoV nucleocapsid protein is inserted into the LSDVSODis genome between ORF 49 and ORF 50.
In a seventh embodiment of the invention the nucleic acids encoding the BCoV spike and BCoV nucleocapsid proteins are operably linked to regulatory sequences which allow for the expression of the BCoV spike and the BCoV nucleocapsid proteins. It will be appreciated that the nucleic acids encoding the BCoV spike and/or the nucleic acid encoding the BCoV nucleocapsid may be under the control of any suitable promoter for expression of the fusion polypeptide. In one preferred embodiment the nucleic acid encoding the BCoV spike protein is under the control of a m H5 pox virus promoter which allows for the expression of the BCoV spike protein. In a second preferred embodiment the nucleic acid encoding the BCoV nucleocapsid protein is under the control of a pLE0 pox virus promoter which allows for the expression of the BCoV nucleocapsid protein. It will however be appreciated by those of skill in the art that any suitable promoter may be used for allowing expression of the modified BCoV spike protein and/or the BCoV nucleocapsid protein.
In a second aspect of the invention there is provided for a composition comprising or consisting of a rLSDV vector that comprises or consists of a cassette comprising or consisting of a nucleic acid encoding a modified BCoV spike protein and a nucleic acid encoding a BCoV nucleocapsid protein. The modified BCoV spike protein comprises or consists of an Si and S2 subunit, separated by a linker. The linker -4 -between the Si and 52 subunits of the spike protein replace the native furin cleavage site.
In a first embodiment of the second aspect of the invention the rLSDV vector comprises or consists of a stabilised SOD-homolog (SOD-is) gene. Preferably, the stabilised SOD-is gene encodes an amino acid sequence of SEQ ID NO:24.
In a second embodiment of the second aspect of the invention the native leader sequence of the BCoV spike protein has been replaced by a tissue plasminogen (TPA) leader sequence. In a preferred embodiment the TPA leader sequence comprises or consists of the sequence of SEQ ID NO:10.
In a third embodiment of the second aspect of the invention the linker comprises or consists of an amino acid sequence of SEQ ID NO:9. It will be appreciated that the linker sequence replaces the native furin cleavage site between the Si and S2 subunits of the spike protein.
In a fourth embodiment of the second aspect of the invention the S2 subunit of the BCoV spike protein comprises or consists of two amino acid substitutions at positions 1080 and 1081 of BCoV spike protein amino acid sequence. The aforementioned substitutions are Al 080P and L1081P.
In a fifth embodiment of the second aspect of the invention the BCoV spike protein comprises or consists of a sequence of SEQ ID NO:2.
In a sixth embodiment of the second aspect of the invention the BCoV nucleocapsid protein comprises or consists of a sequence of SEQ ID NO:4.
In a seventh embodiment of the second aspect of the invention the nucleic acids encoding the BCoV spike and BCoV nucleocapsid proteins are operably linked to regulatory sequences which allow for the expression of the BCoV spike and the BCoV nucleocapsid proteins. It will be appreciated that the nucleic acids encoding the BCoV spike and/or the nucleic acid encoding the BCoV nucleocapsid may be under the control of any suitable promoter for expression of the fusion polypepfide. In one preferred embodiment the nucleic acid encoding the BCoV spike protein is under the control of a mH5 pox virus promoter which allows for the expression of the BCoV spike protein. In a second preferred embodiment of the invention the nucleic acid encoding the BCoV nucleocapsid protein is under the control of a pLE0 pox virus promoter which allows for the expression of the BCoV nucleocapsid protein. It will however be appreciated by those of skill in the art that any suitable promoter may be used for allowing expression of the modified BCoV spike protein and/or the BCoV nucleocapsid protein. -5 -
In an eighth embodiment of the second aspect of the invention the cassette encoding the modified BCoV spike protein and the BCoV nucleocapsid protein is inserted into the LSDV-SODis genome between ORF 49 and ORF 50.
In a third aspect of the invention there is provided for a vaccine comprising or consisting of the rLSDV vector as described herein or the compositions as described herein and a pharmaceutically acceptable carrier or adjuvant.
In a fourth aspect of the invention there is provided for an rLSDV vector as described herein, a composition as described herein or a vaccine as described herein for use in a method of inducing any immune response against BCoV in a subject, the method comprising or consisting of administering an immunogenically effective amount of the vector, composition or vaccine to the subject.
In a first embodiment of the fourth aspect of the invention the rLSDV vector, composition or vaccine additionally induces an immunogenically effective response against BCoV and/or LSDV in the subject.
In a second embodiment of the fourth aspect of the invention it will be appreciated that the subject is or may be selected from cattle, pigs, sheep, goats, deer, antelope, giraffe, camels and buffalo.
In a fifth aspect of the invention there is provided for a method of inducing an immune response against BCoV in a subject, the method comprising or consisting of administering an immunogenically effective amount of the rLSDV vector as described herein, a composition as described herein or the vaccine as described herein to the subject.
In a first embodiment of the fifth aspect of the invention the rLSDV vector, composition or vaccine additionally induces an immunogenically effective response against BCoV and/or LSDV in the subject.
In a second embodiment of the fifth aspect of the invention it will be appreciated that the subject is or may be selected from cattle, pigs, sheep, goats, deer, antelope, giraffe, camels and buffalo.
BRIEF DESCRIPTION OF THE FIGURES
Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures: Figure 1: Schematic diagram showing the modifications made to the BCoV spike protein (S). The native leader sequence (SS) was replaced with the tissue plasminogen leader (TPA), the cleavage site (X) was replaced with a linker sequence, -6 -GGGGSGGGS (L) (SEQ ID NO:9) and two stabilising proline mutations were introduced (PP), A1080 to P and L1081 to P. Figure 2: Schematic diagram showing the design of the recombinant LSDV expressing the BCoV spike (S) and nucleocapsid (N) proteins.
Figure 3: PCR amplification of the region between open reading frames 49 and 50 of LSDV. Schematic diagram showing the location of PCR primer binding sites and expected DNA fragment sizes.
Figure 4: PCR amplification of the region between open reading frames 49 and 50 of LSDV. PCR 1 using primers #57 (SEQ ID NO:5) and #35 (SEQ ID NO:6). 1 kb DNA ladder (ThermoScientific) lane 1; water only control lane 2; DNA from uninfected MBDK cells lane 3; DNA from MBDK cells infected with LSDVSODis p67HA BLV Gag lane 4 (control); MBDK cells infected with cell lysate from passage 5 of recombinant LSDV-BCoV-K1L; 1 kb DNA ladder.
Figure 5: PCR amplification of the region between open reading frames 49 and 50 of LSDV. PCR 2 using primers #34 (SEQ ID NO:7) and #99 (SEQ ID NO:8). 1 kb DNA ladder (ThermoScienfific) lane 1; water only control lane 2; DNA from uninfected MBDK cells lane 3; DNA from MBDK cells infected with LSDVSODi5p67HA-BLV-Gag lane 4 (control); MBDK cells infected with cell lysate from passage 5 of recombinant LSDV-BCoV-K1L; 1 kb DNA ladder.
Figure 6: Confirmation of spike and nucleocapsid expression from MDBKs infected with LSDV-BCoV-K1L by SDS PAGE and western blot analysis. Western blot: Lane 1 -MBDK cells infected with nLSDVSODis-UCT negative control. Lane 2-MBDK cells infected with LSDV-BCoV-K1L. The blot was probed with antiBCoV antibody (GTX40364, GeneTex).
Figure 7: Immunisation schedule for animal study.
Figure 8: IFN-Y ELISPOT responses of vaccinated mice to BCoV spike and nucleocapsid proteins. IFN-Y ELISPOT assay. Cryopreserved murine splenocytes were revived and plated at 5 x 105 cells/ well. The ELISPOT was performed using the murine ELISPOT kit (ab64029, Abcam, UK) and cells were stimulated with 5 pg/ml of BCoV S, BCoV N or p67 proteins and 0.5 pg/ml Con A (data not shown). The spots were developed for 5 min. Two-way ANOVA with a post-hoc Bonferroni test was conducted; p <0.05 -", ns -nonsignificant.
Figure 9: Evaluation of BCoV nucleocapsid binding antibodies. Mice were inoculated according to the schedule shown in Figure 7. Serum was collected at day 4 (pre-bleed) and day 42. The end-point titres were defined as the reciprocal of the last 7 -dilution to give a signal above the ELISA signal from appropriate pre-bleed groups at a 1:10 dilution. Each character represents an individual mouse's endpoint titre, in the form 1/logio. Horizontal lines indicate mean. A one-way ANOVA with a post-hoc Bonferroni test was conducted. p <0.0001 -*. ;Figure 10: Evaluation of BCoV spike binding antibodies. Mice were inoculated according to the schedule shown in Figure 7. Serum was collected at day 4 (pre-bleed) and day 42. The end-point titres were defined as the reciprocal of the last dilution to give a signal above the ELISA signal from appropriate pre-bleed groups at a 1:10 dilution. Each character represents an individual mouse's endpoint titre, in the form 1/logio. Horizontal lines indicate mean. A one-way ANOVA with a post-hoc Bonferroni test was conducted. p <0.0001 -"*.
Figure 11: Codon optimised nucleotide sequence encoding BCoV spike protein (SEQ ID NO:1).
Figure 12: Consensus amino acid sequence of the BCoV spike protein (SEQ ID NO:2).
Figure 13: Codon optimised nucleotide sequence encoding BCoV nucleocapsid protein (SEQ ID NO:3).
Figure 14: Consensus amino acid sequence of the bovine coronavirus nucleocapsid protein (SEQ ID NO:4).
Figure 15: Full length nucleic acid sequence of the BCoV S+N cassette (mH5 promoter-spike-pLE0 promoter-nucleocapsid-mFP promoter-mCherry) (SEQ ID NO:20).
Figure 16: Full length nucleic acid sequence of the BCoV S+N K1L cassette (mH5 promoter-spike-pLE0 promoter-nucleocapsid-mFP promoter-mCherryK1L promoter-K1L) (SEQ ID NO:21).
SEQUENCE LISTING
The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and the standard three letter abbreviations for amino acids. It will be understood by those of skill in the art that only one strand of each nucleic acid sequence is shown, but that the complementary strand is included by any reference to the displayed strand. In the accompanying sequence listing: SEQ ID NO:1 -Codon optimised nucleotide sequence encoding BCoV spike protein. -8 -
SEQ ID NO:2 -Consensus amino acid sequence of the BCoV spike protein.
SEQ ID NO:3 -Codon optimised nucleotide sequence encoding BCoV nucleocapsid protein.
SEQ ID NO:4 -Consensus amino acid sequence of the BCoV nucleocapsid protein.
SEQ ID NO:5 -Forward primer for amplification of spike protein inserted into LSDVSODis (#57).
SEQ ID NO:6 -Reverse primer for amplification of spike protein inserted into LSDVSODis (#35).
SEQ ID NO:7 -Forward primer for amplification of nucleocapsid protein inserted into LSDVSODis (#34).
SEQ ID NO:8 -Reverse primer for amplification of nucleocapsid protein inserted into LSDVSODis (#99).
SEQ ID NO:9 -Linker sequence.
SEQ ID NO:10 -Nucleic acid sequence encoding the tissue plasminogen leader sequence SEQ ID NO:11 -Amino acid sequence of the tissue plasminogen leader sequence.
SEQ ID NO:12 -Nucleic acid sequence encoding the K1L polypeptide.
SEQ ID NO:13 -Amino acid sequence of the KlL polypeptide.
SEQ ID NO:14 -Nucleic acid sequence encoding the mCherry polypeptide.
SEQ ID NO:15 -Amino acid sequence of the mCherry polypeptide.
SEQ ID NO:16 -Nucleic acid sequence of the mH5 promoter.
SEQ ID NO:17 -Nucleic acid sequence of the pLE0 promoter.
SEQ ID NO:18 -Nucleic acid sequence of the mFP promoter.
SEQ ID NO:19 -Nucleic acid sequence of the KlL promoter.
SEQ ID NO:20 -Full length nucleic acid sequence of the BCoV S+N cassette.
SEQ ID NO:21 -Full length nucleic acid sequence of the BCoV S+N K1L cassette.
SEQ ID NO:22 -Nucleic acid sequence encoding the linker sequence used to replace the furin cleavage site between spike subunits Si and 52.
SEQ ID NO:23 -Nucleic acid encoding the stabilised SOD-is polypeptide.
SEQ ID NO:24 -Amino acid sequence of the stabilised SOD-is polypeptide. -9 -
DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.
The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
As used throughout this specification and in the claims which follow, the singular forms "a", "an" and "the" include the plural form, unless the context clearly indicates otherwise.
The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms "comprising", "containing", "having" and "including" and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items.
Use of a lumpy skin disease virus (LSDV) vector backbone for a coronavirus vaccine is different from other vaccine technologies that are available for coronaviruses, which are based on inactivated virus, protein (subunit or nanoparticles), recombinant adenoviruses or mRNA. The advantages and improvements of this technology lies in the safety and stability of the vector, which has been used for decades in the cattle industry. Like other poxviruses, LSDV can accommodate large insertions, meaning both the spike and nucleocapsid coronavirus genes could be inserted into the same vector. In addition, LSDV replicates in cattle and thus a lower dose of the vaccine can be used as compared to non-replicating viral vectors. This saves costs in manufacturing and therefore for the farmer too.
Although technologies exist for constructing recombinant LSDV, the present invention differs in the site of insertion of the BCoV genes; this site is intergenic, between LSDV ORFs 49 and 50 and does not inactivate any LSDV genes. Other recombinant LSDV candidate vaccines have used the thymidine kinase or ribonucleotide reductase genes for insertion of foreign genes. Insertion of foreign genes at these sites causes further attenuation of LSDV.
The present LSDV backbone (nLSDVSODis-UCT) is also unique and elicits strong humoral and T cell responses.
In the case of BCoV, LSDV is an attenuated replicating vector and serves as a dual vaccine against both LSDV and BCoV. This means cattle can be immunized -10 -against two diseases using one vaccine. The manufacturing saving further translates into savings for cattle farmers. In addition, the cattle require fewer inoculations.
The present vaccine expresses both the spike (S) and nucleocapsid (N) genes. Although T cell responses were higher to nucleocapsid (N), spike-specific T cell responses were also elicited. High titres of binding antibodies to the spike protein were elicited and some mice developed low levels of binding antibodies to the nucleocapsid protein.
The inventors have produced consensus amino acid spike (SEQ ID NO:2) and nucleocapsid (SEQ ID NO:4) sequences from 38 different spike protein and 24 different nucleocapsid BCoV proteins from the 13 different genotypes. The genes encoding the spike (SEQ ID NO:1) and nucleocapsid sequences (SEQ ID NO:3) were codon optimised for expression in cattle. Additionally, the spike sequence was modified by replacing the native leader sequence with a tissue plasminogen leader (TPA) sequence (SEQ ID NO:11), the cleavage site between the Si and S2 subunits was replaced with a linker sequence (SEQ ID NO:9) and two stabilising proline mutations, namely A1080P and L1081P, were introduced.
As used herein, the term "LSDV vector' refers to a LSDV vector backbone containing a stabilised SOD-homologue gene, as disclosed in international publication number WO 2019/220403.
A "protein," "peptide" or "polypeptide" is any chain of two or more amino acids, including naturally occurring or non-naturally occurring amino acids or amino acid analogues, irrespective of post-translational modification (e.g., glycosylation or phosphorylafion).
An "antigen" is a compound, composition, or substance that can stimulate the production of antibodies and/or a CD4+ or CD8+ T cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens.
The terms "nucleic acid", "nucleic acid molecule" and "polynucleotide" are used herein interchangeably and encompass both ribonucleotides (RNA) and deoxyribonucleotides (DNA), including cDNA, genomic DNA, and synthetic DNA. The nucleic acid may be double-stranded or single-stranded. Where the nucleic acid is single-stranded, the nucleic acid may be the sense strand or the anfisense strand. A nucleic acid molecule may be any chain of two or more covalently bonded nucleotides, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives. By "RNA" is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides. The term "DNA" refers to a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides. By "cDNA" is meant a complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase).
The term "isolated", is used herein and means having been removed from its natural environment.
The term "purified", relates to the isolation of a molecule or compound in a form that is substantially free of contamination or contaminants. Contaminants are normally associated with the molecule or compound in a natural environment, purified thus means having an increase in purity as a result of being separated from the other components of an original composition. The term "purified nucleic acid" describes a nucleic acid sequence that has been separated from other compounds including, but not limited to polypeptides, lipids and carbohydrates which it is ordinarily associated with in its natural state.
The term "complementary" refers to two nucleic acids molecules, e.g., DNA or RNA, which are capable of forming Watson-Crick base pairs to produce a region of double-strandedness between the two nucleic acid molecules. It will be appreciated by those of skill in the art that each nucleotide in a nucleic acid molecule need not form a matched Watson-Crick base pair with a nucleotide in an opposing complementary strand to form a duplex. One nucleic acid molecule is thus "complementary" to a second nucleic acid molecule if it hybridizes, under conditions of high stringency, with the second nucleic acid molecule. A nucleic acid molecule according to the invention includes both complementary molecules.
As used herein a "substantially identical" sequence is an amino acid or nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, or by one or more non-conservative substitutions, deletions, or insertions located at positions of the sequence that do not destroy or substantially reduce the antigenicity of one or more of the expressed polypeptides or of the polypeptides encoded by the nucleic acid molecules. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the knowledge of those with skill in the art. These include using, for instance, computer software such as ALIGN, Megalign (DNASTAR), CLUSTALW or BLAST software. Those skilled in the art can readily determine appropriate parameters for measuring -12 -alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. In one embodiment of the invention there is provided for a polypeptide or polynucleotide sequence that has at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the sequences described herein.
Alternatively, or additionally, two nucleic acid sequences may be "substantially identical" if they hybridize under high stringency conditions. The "stringency" of a hybridisation reaction is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation which depends upon probe length, washing temperature, and salt concentration. In general, longer probes required higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridisation generally depends on the ability of denatured DNA to re-anneal when complementary strands are present in an environment below their melting temperature. A typical example of such "stringent" hybridisation conditions would be hybridisation carried out for 18 hours at 65°C with gentle shaking, a first wash for 12 min at 65°C in Wash Buffer A (0.5% SDS; 2XSSC), and a second wash for 10 min at 65°C in Wash Buffer B (0.1% SDS; 0.5% SSC).
Those skilled in the art will appreciate that polypeptides, peptides or peptide analogues can be synthesised using standard chemical techniques, for instance, by automated synthesis using solution or solid phase synthesis methodology. Automated peptide synthesisers are commercially available and use techniques known in the art. Polypepfides, peptides and peptide analogues can also be prepared from their corresponding nucleic acid molecules using recombinant DNA technology.
As used herein, the term "gene" refers to a nucleic acid that encodes a functional product, for instance an RNA, polypeptide or protein. A gene may include regulatory sequences upstream or downstream of the sequence encoding the functional product.
As used herein, the term "coding sequence" refers to a nucleic acid sequence that encodes a specific amino acid sequence. On the other hand, a "regulatory sequence" refers to a nucleotide sequence located either upstream, downstream or within a coding sequence. Generally regulatory sequences influence the transcription, RNA processing or stability, or translation of an associated coding sequence. Regulatory sequences include but are not limited to: effector binding sites, enhancers, -13 -introns, polyadenylation recognition sequences, promoters, RNA processing sites, stem-loop structures, translation leader sequences.
In some embodiments, the genes used in the method of the invention may be operably linked to other sequences. By "operably linked" is meant that the nucleic acid molecules encoding the recombinant polypeptides of the invention and regulatory sequences are connected in such a way as to permit expression of the proteins when the appropriate molecules are bound to the regulatory sequences. Such operably linked sequences may be contained in vectors or expression constructs which can be transformed or transfected into host cells for expression. It will be appreciated that any vector or vectors can be used for the purposes of expressing the recombinant antigenic polypeptides of the invention.
The term "promoter" refers to a DNA sequence that is capable of controlling the expression of a nucleic acid coding sequence or functional RNA. A promoter may be based entirely on a native gene or it may be comprised of different elements from different promoters found in nature. Different promoters are capable of directing the expression of a gene in different cell types, or at different stages of development, or in response to different environmental or physiological conditions. A "constitutive promoter" is a promoter that direct the expression of a gene of interest in most host cell types most of the time.
The term "recombinant" means that something has been recombined. VVhen used with reference to a nucleic acid construct the term refers to a molecule that comprises nucleic acid sequences that are joined together or produced by means of molecular biological techniques. The term "recombinant" when used in reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed from a recombinant nucleic acid construct created by means of molecular biological techniques. Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Accordingly, a recombinant nucleic acid construct indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention. Recombinant nucleic acid constructs may be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species.
As used herein, the term "chimeric", means that a sequence comprises of sequences that have been "recombined". By way of example sequences are -14 -recombined and are not found together in nature. The term "recombine" or "recombination" refers to any method of joining two or more polynucleotides. The term includes end to end joining, and insertion of one sequence into another. The term is intended to include physical joining techniques, for instance, sticky-end ligation and blunt-end ligation. Sequences may also be artificially synthesized to contain a recombined sequence. The term may also encompass the integration of one sequence into a second sequence by way of, for example, homologous recombination.
The term "vector" refers to a means by which polynucleotides or gene sequences can be introduced into a cell. There are various types of vectors known in the art including plasmids, viruses, bacteriophages and cosmids. Generally polynucleotides or gene sequences are introduced into a vector by means of a cassette. The term "cassette" refers to a gene sequence or gene sequences inserted into a vector, which in some embodiments, provides regulatory sequences for expressing the polynucleotide or gene sequences. In other embodiments, the vector provides the regulatory sequences for the expression of the polypepfides of the invention. In further embodiments, the vector provides some regulatory sequences and the nucleotide or gene sequence provides other regulatory sequences. "Regulatory sequences" include but are not limited to promoters, transcription termination sequences, enhancers, splice acceptors, donor sequences, introns, ribosome binding sequences, poly(A) addition sequences, and/or origins of replication.
The cassette of the present invention comprises a codon optimised gene encoding a BCoV spike protein consensus sequence (SEQ ID NO:2), a codon optimised gene encoding a BCoV nucleocapsid consensus sequence (SEQ ID NO:4) and a mCherry polypeptide (SEQ ID NO:15) under control of the pox virus promoters mH5 (SEQ ID NO:16), pLE0 (SEQ ID NO:17) and mFP (SEQ ID NO:18), respectively. The expression cassettes are inserted between ORF's 49 and 50 in the LSDV genome. Optionally, the mCherry nucleic acid sequence (SEQ ID NO:14) may be followed by a nucleic acid sequence encoding a K1L polypepfide (SEQ ID NO:13) under the control of the native K1L promoter (SEQ ID NO:19). The BCoV spike protein consensus sequence contains additional modifications including replacement of the native leader sequence (SS) with a tissue plasminogen leader (TPA), the cleavage site between the Si and S2 subunits of the spike protein has been replaced with a linker of SEQ ID NO:9 and two stabilising proline mutations have been introduced at the positions A1080P and L1081P. The cassette is hereinafter referred to as the "BCoV cassette".
-15 -The recombinant LSDV vector comprising the BCoV cassette or compositions of the invention can be provided either alone or in combination with other compounds (for example, nucleic acid molecules, small molecules, peptides, or peptide analogues), in the presence of a liposome, an adjuvant, or any carrier, such as a pharmaceutically acceptable carrier and in a form suitable for administration to mammals, for example, humans, cattle, sheep, etc. Preferably, the BCoV cassette is vectored in an LSDV vector.
In one embodiment of the invention the LSDV vector including the BCoV cassette is formulated for immunization together with an adjuvant. Adjuvants are well known to those of skill in the art of vaccine development and are not limited to the adjuvants specifically exemplified herein.
As used herein a "pharmaceutically acceptable carrier' or "excipient" includes any and all antibacterial and antifungal agents, coatings, dispersion media, solvents, isotonic and absorption delaying agents, and the like that are physiologically compatible. A "pharmaceutically acceptable carrier" may include a solid or liquid filler, diluent or encapsulating substance which may be safely used for the administration of the recombinant antigen or vaccine composition to a subject. The pharmaceutically acceptable carrier can be suitable for intramuscular, intradermal, intravenous, intraperitoneal subcutaneous, oral or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, dispersions and sterile powders for the preparation of sterile solutions. The use of media and agents for the preparation of pharmaceutically active substances is well known in the art. Where any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is not contemplated. Supplementary active compounds can also be incorporated into the compositions.
Suitable formulations or compositions to administer the LSDV vector comprising the BCoV cassette to subjects either with BCoV infection or Lumpy Skin Disease or to subjects which are presymptomafic for a condition associated with BCoV or Lumpy Skin Disease also fall within the scope of the invention. Any appropriate route of administration may be employed, such as, parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracistemal, intraperitoneal, intranasal, aerosol, topical, or oral administration. It will be appreciated that a composition of the invention may include an LSDV vector comprising the BCoV cassette.
-16 -For vaccine formulations and pharmaceutical compositions, an effective amount of a composition comprising the BCoV cassette and can be provided, either alone or in combination with other compounds, with immunological adjuvants, for example, aluminium hydroxide dimethyldioctadecyl-ammonium hydroxide or Freund's incomplete adjuvant. The LSDV vector comprising the BCoV cassette may also be linked with suitable carriers and/or other molecules, such as bovine serum albumin or keyhole limpet haemocyanin in order to enhance immunogenicity. Vaccine formulations and compositions that are useful in the present invention include the LSDV vector comprising the BCoV cassette that primes and/or boosts an immune response to BCoV and/or LSDV.
In one embodiment, the BCoV cassette is capable of "priming" an immune response to BCoV and/or the LSDV vector is capable of producing polypepfides and/or virus like particles for "priming an immune response to lumpy skin disease.
It will further be appreciated that a "boost" composition may include a composition comprising the LSDV vector comprising the BCoV cassette. The boosting composition may include at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten subsequent inoculations with the LSDV vector comprising the BCoV cassette.
In some embodiments, the LSDV vector comprising the BCoV cassette according to the invention may be provided in a kit, optionally with a carrier and/or an adjuvant, together with instructions for use.
An "effective amount" of the LSDV vector comprising the BCoV cassette includes a therapeutically effective amount, immunologically effective amount, or a prophylactically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as treatment of an infection or a condition associated with such infection. The outcome of the treatment may for example be measured by a decrease in viraemia, inhibition of viral gene expression, delay in development of a pathology associated with BCoV and/or lumpy skin disease infection, stimulation of the immune system, or any other method of determining a therapeutic benefit. A therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which -17 -any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.
The dosage of the LSDV vector comprising the BCoV cassette or compositions of the present invention will vary depending on the symptoms, age and body weight of the subject, the nature and severity of the disorder to be treated or prevented, the route of administration, and the form of the composition. Any of the compositions of the invention may be administered in a single dose or in multiple doses. The dosages of the compositions of the invention may be readily determined by techniques known to those of skill in the art or as taught herein.
By "immunogenically effective amount" is meant an amount effective, at dosages and for periods of time necessary, to achieve a desired immune response. The desired immune response may include stimulation or elicitation of an immune response, for instance a T-cell response.
A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result, such as prevention of onset of a condition associated with either BCoV and/or lumpy skin disease infection. Typically, a prophylactic dose is used in a subject prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.
Dosage values may vary and be adjusted over time according to the individual need and the judgment of the person administering or supervising the administration of the LSDV vector comprising the BCoV cassette of the invention. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected. The amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single dose may be administered, or multiple doses may be administered over time. It may be advantageous to formulate the compositions in dosage unit forms for ease of administration and uniformity of dosage.
The vaccination protocol for eliciting an immune response against BCoV infection and/or lumpy skin disease in a subject as defined herein typically comprises a series of single doses of the antigens or compositions described herein. A single dose or dosage, as used herein, refers to the priming dose (i.e. initial first or second dose with the same antigens), and any subsequent dose, respectively, which are preferably administered in order to "boost" the immune reaction. In this context, each -18 -single dosage comprises the administration of one of the antigens or compositions according to the invention, wherein the interval between the administration of two single dosages can vary from at least one week, preferably 2, 3, 4, 5, 6, 7, 8,9, 10, 11 or 12 weeks apart. Most preferably, the antigens or compositions of the invention are administered at intervals of either 4 or 8 weeks apart. It will be appreciated that the intervals between single dosages may be constant or vary over the course of the immunization protocol, e.g. the intervals may be shorter in the beginning (such as 4 weeks apart) and longer towards the end of the protocol (such as 8 weeks apart). Additionally, depending on the total number of single dosages and the interval between single dosages, the immunization protocol may extend over a period of time, which preferably lasts at least one week, more preferably several weeks, even more preferably several months (e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 months). Each single dosage encompasses the administration of one of the LSDV vector comprising the BCoV cassette described herein.
The term "preventing", when used in relation to an infectious disease, or other medical disease or condition, is well understood in the art, and includes administration of a composition which reduces the frequency of or delays the onset of symptoms of a condition in a subject relative to a subject which does not receive the composition. Prevention of a disease includes, for example, reducing the number of diagnoses of the infection in a treated population versus an untreated control population, and/or delaying the onset of symptoms of the infection in a treated population versus an untreated control population.
The term "prophylactic or therapeutic" treatment is well known to those of skill in the art and includes administration to a subject of one or more of the compositions of the invention. If the composition is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the subject) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilise the existing unwanted condition or side effects thereof).
Toxicity and therapeutic efficacy of compositions of the invention may be determined by standard pharmaceutical procedures in cell culture or using experimental animals, such as by determining the LD50 and the ED50. Data obtained from the cell cultures and/or animal studies may be used to formulate a dosage range for use in a subject. The dosage of any composition of the invention lies preferably -19 -within a range of circulating concentrations that include the ED50 but which has little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilised. For compositions of the present invention, the therapeutically effective dose may be estimated initially from cell culture assays.
The following example is offered by way of illustration and not by way of limitation.
EXAMPLE
Source of genes The BCoV spike (S) and nucleocapsid (N) genes utilised in this vaccine are consensus sequences derived from the alignment of 38 different spike and 24 different nucleocapsid BCoV proteins from the 13 different genotypes. The genes were codon optimised for expression in cattle and some modifications were made to the spike protein as shown in Figure 1. The native leader sequence of the spike protein was replaced with that of the tissue plasminogen activator leader sequence to facilitate entry of the protein into the secretory pathway. The cleavage site between the Si and 52 subunits was replaced with a flexible linker sequence to stabilise the linkage of the two proteins and two proline mutations were introduced to stabilise the spike protein in a pre-fusion conformation to expose the correct epitopes for eliciting neutralising antibodies.
Construction of transfer vectors A transfer vector has been constructed as shown below. A LSDV recombinant was constructed containing the BCoV spike gene under the control of the mH5 promoter, the BCoV nucleocapsid gene under the control of the pLE0 promoter, the mCherry fluorescent marker gene (SEQ ID NO:14) under the control of a modified fowlpox virus promoter and the KlL gene under the control of the native ML promoter (Figure 2). This expression cassette was inserted between open reading frames 49 and 50 in the LSDV genome.
Primary lamb testes (LT) cells were infected with LSDV(SODis)BEFV-Gb (green fluorescence) and transfected with the transfer vector containing the genes encoding the BCoV spike and nucleocapsid, mCherry and ML gene. After two days the cells were frozen and thawed twice and the cell lysate was passaged on RK13 cells. Cell lysate was passaged repeatedly on RK13 cells until no green fluorescent -20 -parent virus and only red fluorescent recombinant virus could be detected. Before high titre stocks were prepared the recombinant virus was diluted and passaged in 96-well plates such that, for each recombinant, a single focus in a single well was purified and expanded.
Confirmation of LSDV-BCoV-K1L by PCR and sequencing MBDK cells were infected with LSDV-BCoV-K1L and DNA was isolated and used for PCR. The insertion of the foreign gene cassette between LSDV ORFs 49 and 50 was confirmed by polymerase chain reaction (PCR) followed by agarose gel electrophoresis and Sanger DNA sequencing of the amplicon (Figure 3, 4 and 5).
Expression of spike and nucleocapsid from LSDV-BCoV-K1L Expression of the BCoV spike and nucleocapsid antigens in MDBK cells infected with the recombinant vaccine LSDV-BCoV-K1L was confirmed by western blot analysis (Figure 6).
Immunopenicity of LSDV expressing the BCoV spike and nucleocapsid proteins (LSDV-BC0V-K1L) in mice Mice were immunised with 106 ffu LSDVSODi5-BC0V-K1L, 106 ffu nLSDVSODis-UCT or PBS, as indicated in the immunisation schedule (Figure 7). IFNY ELISPOT assays were performed to evaluate the magnitude of IFN-Y secreting T cells, following stimulation with purified BCoV S and N proteins. The number of BCoV spike-specific cells in the LSDV-BCoV-K1L-immunised group was not significantly (p > 0.05) greater than those in the nLSDVSODis-UCT group (Figure 8), whereas the number of BCoV nucleocapsid-specific cells in the LSDV-BCoV-K1L-immunised group was significantly (p < 0.005) greater than those in the nLSDVSODis-UCT group (Figure 8), indicating that there was a higher response in IFN-Y secreting T cells, after stimulation with purified BCoV nucleocapsid protein. Although the background responses in the stimulated cells were high, there was still a significantly higher response in the LSDV-BCoV-K1L immunized group compared to the nLSDVSODisUCT and PBS immunised groups.
Evaluation of BCoV N and S binding antibodies ELISAS were conducted to evaluate the levels of BCoV nucleocapsid and spike binding antibodies, following immunisation with LSDV-BCoV-K1L.The end-point titres -21 -were defined as the reciprocal of the last dilution to give a signal above the ELISA signal from appropriate pre-bleed groups, at a 1:10 dilution. The animals immunized with LSDV-BCoV-K1L, developed very low titres of antibodies against the nucleocapsid protein (Figure 9). Some of the mice from the LSDV-BCoV-K1L group had detectable titres of nucleocapsid-specific antibodies, while others had titres that were not significantly (p > 0.05) different to the titres detected in the nLSDVSODisUCT and PBS groups (data not shown). All three groups had low levels of antibodies against the nucleocapsid protein, as a result all the titres below or equal to the PBS group titres were set to zero (Figure 9).
The animals immunized with LSDV-BC0V-K1L developed high titres of antibodies against the spike protein. The animals inoculated with nLSDVSODis-UCT, developed low titres of what is probably background antibodies, specific to cells and cell media in which the spike protein was prepped. The animals inoculated with PBS did not develop any detectable antibodies (Figure 10) The differences between the LSDV-BCoV-K1L-vaccinated animals and the control animals were statistically significant (p value < 0.0001).
Claims (27)
- -22 -CLAIMS1. A recombinant lumpy skin disease virus (rLSDV) vector that comprises a cassette comprising a nucleic acid encoding a bovine coronavirus (BCoV) spike protein and a nucleic acid encoding a BCoV nucleocapsid protein, wherein the BCoV spike protein comprises an Si and S2 subunit, and wherein a native cleavage site between the Si and S2 subunits of the spike protein are replaced by a linker.
- 2. The rLSDV vector of claim 1, wherein the rLSDV comprises a stabilised SOD-homolog (SOD-is) gene.
- 3. The rLSDV vector of claim 1 or 2, wherein the native leader sequence of the BCoV spike protein has been replaced by a tissue plasminogen (TPA) leader sequence.
- 4. The rLSDV vector of any one of claims 1 to 3, wherein the linker comprises an amino acid sequence of SEQ ID NO:9.
- 5. The rLSDV vector of any one of claims 1 to 4, wherein the S2 subunit of the BCoV spike protein comprises two amino acid substitutions at positions 1080 and 1081 of the amino acid sequence, and where in the substitutions are A1080P and L1081 P.
- 6. The rLSDV vector of any one of claims 1 to 5, wherein the BCoV spike protein comprises a sequence of SEQ ID NO:2.
- 7. The rLSDV vector of any one of claims 1 to 6, wherein the BCoV nucleocapsid protein comprises a sequence of SEQ ID NO:4.
- 8. The rLSDV vector of any one of claims 1 to 7, wherein the nucleic acids encoding the BCoV spike and BCoV nucleocapsid proteins are operably linked to regulatory sequences which allow for the expression of the BCoV spike and the BCoV nucleocapsid proteins.-23 -
- 9. The rLSDV vector of claim 8, wherein the nucleic acid encoding the BCoV spike protein is under the control of a mH5 pox virus promoter which allows for the expression of the BCoV spike protein.
- 10. The rLSDV vector of claim 8, wherein the nucleic acid encoding the BCoV nucleocapsid protein is under the control of a pLE0 pox virus promoter which allows for the expression of the BCoV nucleocapsid protein.
- 11. A composition comprising a rLSDV vector that comprises a cassette comprising a nucleic acid encoding a BCoV spike protein and a nucleic acid encoding a BCoV nucleocapsid protein, wherein the BCoV spike protein comprises a Si and a 32 subunit, and wherein a native cleavage site between the Si and S2 subunits of the spike protein are replaced by a linker.
- 12. The composition of claim 11, wherein the rLSDV vector comprises a stabilised SOD-homolog (SOD-is) gene.
- 13. The composition of claim 11 or 12, wherein the native leader sequence of the BCoV spike protein has been replaced by a tissue plasminogen (TPA) leader sequence.
- 14. The composition of any one of claims 11 to 13, wherein the linker comprises an amino acid sequence of SEQ ID NO:9.
- 15. The composition of any one of claims 11 to 14, wherein the 52 subunit of the BCoV spike protein comprises two amino acid substitutions at positions 1080 and 1081 of the amino acid sequence, and where in the substitutions are A1080P and L1081P.
- 16. The composition of any one of claims 11 to 15, wherein the BCoV spike protein comprises a sequence of SEQ ID NO:2.
- 17. The composition of any one of claims 11 to 16, wherein the BCoV nucleocapsid protein comprises a sequence of SEQ ID NO:4.-24 -
- 18. The composition of any one of claims 11 to 17, wherein the nucleic acids encoding the BCoV spike and BCoV nucleocapsid proteins are operably linked to regulatory sequences which allow for the expression of the BCoV spike and the BCoV nucleocapsid proteins.
- 19. The composition of claim 18, wherein the nucleic acid encoding the BCoV spike protein is under the control of a mH5 pox virus promoter which allows for the expression of the BCoV spike protein.
- 20. The composition of claim 18, wherein the nucleic acid encoding the BCoV nucleocapsid protein is under the control of a pLE0 pox virus promoter which allows for the expression of the BCoV nucleocapsid protein.
- 21. A vaccine comprising the rLSDV vector of any one of claims 1 to 10 or the composition of any one of claims 11 to 20 and a pharmaceutically acceptable carrier or adjuvant.
- 22. The rLSDV vector of any one of claims 1 to 10, the composition of any one of claims 11 to 20 or the vaccine of claim 21 for use in a method of inducing any immune response against BCoV in a subject, the method comprising administering an immunogenically effective amount of the vector, composition or vaccine to the subject.
- 23. The rLSDV vector, composition or vaccine for use of claim 22, wherein the rLSDV vector, composition or vaccine additionally induces an immunogenically effective response against BCoV in the subject.
- 24. The rLSDV vector, composition or vaccine for use of claim 22 or 23, wherein the subject is selected from cattle, pigs, sheep, goats, deer, antelope, giraffe, camels and buffalo.
- 25. A method of inducing an immune response against BCoV in a subject, the method comprising administering an immunogenically effective amount of the rLSDV vector of any one of claims 1 to 10, the composition of any one of claims 11 to 20 or the vaccine of claim 21 to the subject.-25 -
- 26. The method of claim 25, wherein the rLSDV vector, composition or vaccine additionally induces an immunogenically effective response against BCoV in the subject.
- 27. The rLSDV vector, composition or vaccine for use of claim 25 or 26, wherein the subject is selected from cattle, pigs, sheep, goats, deer, antelope, giraffe, camels and buffalo.
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| PCT/IB2023/061442 WO2024105539A1 (en) | 2022-11-15 | 2023-11-13 | Recombinant lsdv vectored bovine coronavirus antigen constructs |
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| EP4103231A4 (en) * | 2020-02-14 | 2024-03-27 | Geovax, Inc. | Vaccines and uses thereof to induce an immune response to sars-cov2 |
| TW202146428A (en) * | 2020-02-26 | 2021-12-16 | 美商托尼克斯醫藥控股公司 | Recombinant poxvirus based vaccine against sars-cov-2 virus |
| GB202006376D0 (en) * | 2020-04-30 | 2020-06-17 | Univ Cape Town | Recombinant sars-cov-2 polypeptides and uses |
| US20230293670A1 (en) * | 2020-08-27 | 2023-09-21 | Cellid Co., Ltd. | Novel coronavirus recombinant spike protein, polynucleotide encoding same, vector comprising polynucleotide, and vaccine for preventing or treating coronavirus infection, comprising vector |
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| Journal of infection and public health, vol. 14, 2020, Dawood Reham M et al., "Bioinformatics prediction of B and T cell epitopes within the spike and nucleocapsid proteins of SARS-CoV2.", p. 169-178. * |
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