AU2022336620A1 - Bovine ephemeral fever and lumpy skin disease antigenic constructs - Google Patents
Bovine ephemeral fever and lumpy skin disease antigenic constructs Download PDFInfo
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Abstract
This invention relates to a recombinant LSDV vector encoding a consensus BEFV Gb protein and a BEFV M protein. The invention also relates to combinations of the BEFV Gb and M proteins and the LSDV vector, compositions containing the BEFV Gb protein, BEFV M protein, and the LSDV vector and vaccines containing the BEFV Gb and M proteins and the LSDV vector. The invention further relates to a dual vaccine containing the BEFV Gb and M proteins and a modified LSDV comprising a stabilised SOD-homolog (SODis) gene, methods of producing the BEFV Gb and M proteins and the recombinant LSDV and pharmaceutical compositions either comprising the recombinant LSDV vector encoding the BEFV Gb and BEFV M proteins. More specifically, the invention relates to a dual vaccine against lumpy skin disease virus and Bovine Ephemeral Fever virus.
Description
BOVINE EPHEMERAL FEVER AND LUMPY SKIN DISEASE ANTIGENIC CONSTRUCTS
BACKGROUND OF THE INVENTION
Lumpy skin disease (LSD) and bovine ephemeral fever (BEF) are two diseases of cattle of economic importance. There is an overlapping geographical distribution of LSD and BEF and a dual vaccine would be desirable to protect against the two diseases in one vaccine formulation.
Lumpy skin disease is caused by a poxvirus, lumpy skin disease virus (LSDV) and results in significant morbidity of cattle as well as economic losses due to damaged hides, decreased milk production, abortions and infertility. Bovine ephemeral fever, also known as three-day stiff-sickness, caused by the rhabdovirus, bovine ephemeral fever virus (BEFV), causes a sudden onset of fever, associated with an inability to move or swallow. Pregnant cows often abort and bulls temporarily lose fertility. Milk production declines dramatically. Currently these diseases are controlled by (separate) vaccines based on live attenuated virus or killed virus. There is presently no dual vaccine against LSDV and BEFV. (A vaccinia virus recombinant was made by Hertig et al (1995) where the BEFV G protein from an Australian strain of BEFV was expressed in vaccinia virus. This vaccine was designed to protect against BEFV, but has not been commercialized).
Poxviruses have been shown to be highly immunogenic vaccine vectors and the Neethling vaccine strain is a safe LSDV vaccine produced by Onderstepoort Biological Products (OBP). Our group has modified the Neethling strain to express a full-length SOD-homologue gene with the aim of improving the LSDV vaccine. This recombinant LSDV, nLSDVSODis-UCT, was used as a backbone for making a dual vaccine against both LSDV and BEFV.
Geographically, BEF and LSD show considerable overlap, notably in Africa. Both diseases are seasonal, with the causative viruses being spread by biting insects. Ideally, vector control could reduce the incidence of both viral infections, but the implementation of this intervention is impractical. Effective vaccines are available for LSD and BEF, but, due to the seasonal nature of the diseases, are not always considered necessary by animal owners. 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. In addition, concomitant vaccination against the two diseases will automatically reduce the incidence of BEF, which is largely under-reported, despite being of great economic importance.
SUMMARY OF THE INVENTION
This invention relates to a recombinant LSDV vector encoding a consensus BEFV Gb protein and a BEFV M protein. The invention also relates to combinations of the BEFV Gb and M proteins and the LSDV vector, compositions containing the BEFV Gb protein, BEFV M protein, and the LSDV vector and vaccines containing the BEFV Gb and M proteins and the LSDV vector. The invention further relates to a dual vaccine containing the BEFV Gb and M proteins and a modified LSDV comprising a stabilised SOD-homolog (SODis) gene, methods of producing the BEFV Gb and M proteins and the recombinant LSDV and pharmaceutical compositions either comprising the recombinant LSDV vector encoding the BEFV Gb and BEFV M proteins. More specifically, the invention relates to a dual vaccine against lumpy skin disease virus and Bovine Ephemeral Fever virus.
In a first aspect of the invention there is provided for a composition comprising (i) a recombinant Bovine Ephemeral Fever virus (BEFV) glycoprotein (Gb protein), (ii) a BEFV matrix protein (M protein), and (iii) a recombinant Lumpy Skin Disease virus (rLSDV) comprising a stabilised SOD-homolog (SODis) gene. Alternatively, there is provided for a combination of proteins comprising or consisting of (i) a recombinant Bovine Ephemeral Fever Virus (BEFV) glycoprotein (Gb protein), and (ii) a BEFV matrix protein (M protein).
In a first embodiment of the first aspect of the invention the recombinant BEFV Gb protein is a protein of SEQ ID NO:1 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1 .
In a second embodiment of the first aspect of the invention the BEFV M protein is a protein of SEQ ID NO:3 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3.
In a third embodiment of the first aspect of the invention the rLSDV comprises a stabilised SODis gene of SEQ ID NO:11 .
In a second aspect of the invention there is provided for a composition comprising or consisting of (i) a first nucleic acid sequence encoding a recombinant Bovine Ephemeral Fever Virus (BEFV) glycoprotein (Gb polypeptide), and (ii) a second nucleic acid encoding a BEFV matrix protein (M polypeptide), wherein the first nucleic
acid and second nucleic acid are contained in a vector, and wherein the vector is a rLSDV comprising a stabilised SOD-homolog (SODis) gene.
In a first embodiment of the second aspect of the invention the first nucleic acid sequence and the second nucleic acid sequence are contained in or inserted in the rLSDV between open reading frame 49 and 50.
In a second embodiment of the second aspect of the invention the recombinant BEFV Gb polypeptide is a polypeptide of SEQ ID NO:1 or a polypeptide having at least 90% sequence identity to SEQ ID NO:1 .
In a third embodiment of the second aspect of the invention the BEFV M polypeptide is a polypeptide of SEQ ID NO:3 or a polypeptide having at least 90% sequence identity to SEQ ID NO:3.
In a fourth embodiment of the second aspect of the invention the rLSDV comprises a stabilised SODis gene of SEQ ID NO:11 .
In a fifth embodiment of the second aspect of the invention the first nucleic acid and second nucleic acid are each operably linked to regulatory sequences that allow for the expression of the BEFV Gb and BEFV M polypeptides.
In a sixth embodiment of the second aspect of the invention the first nucleic acid and second nucleic acid are contained on an expression vector.
In a seventh embodiment of the second aspect of the invention the expression vector is a recombinant lumpy skin disease virus (LSDV) comprising or consisting of a stabilised SOD-homolog (SODis) gene.
In a third aspect of the invention there is provided for a vaccine comprising or consisting of the compositions described herein and a pharmaceutically acceptable carrier or adjuvant.
In a fourth aspect of the invention there is provided for the composition or the vaccine as described herein for use in a method of inducing an immune response against Bovine Ephemeral Fever virus in a subject, wherein the method comprises administering an immunogenically effective amount of the composition or the vaccine to the subject.
In one embodiment of the fourth aspect of the invention the composition or vaccine additionally induces an immunogenically effective response against Lumpy skin disease virus in the subject. Preferably, the subject is a mammal. Even more preferably the mammal is selected from cattle and buffalo.
In a fifth aspect of the invention there is provided for a method of inducing an immune response against Bovine Ephemeral fever in a subject, the method comprising
administering an immunogenically effective amount of the composition, or the vaccine to the subject.
In one embodiment of the fifth aspect of the invention the composition or vaccine additionally induces an immunogenically effective response against Lumpy Skin disease virus in the subject. Preferably the subject is a mammal. Even more preferably the subject is selected from cattle and buffalo.
In a sixth aspect of the invention there is provided for a recombinant LSDV variant vector comprising or consisting of a recombinant lumpy skin disease virus (LSDV) comprising or consisting of a stabilised SOD-homolog (SODis) gene and wherein the recombinant LSDV variant vector comprises (i) a nucleic acid encoding a recombinant Bovine Ephemeral Fever Virus (BEFV) glycoprotein (Gb protein); and (ii) a nucleic acid encoding a BEFV matrix protein (M protein).
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 : Expression of eGFP from different promoters in LSDV-infected BHK-21 cells. BHK-21 cells were infected with nLSDVSODis-UCT at an MOI of 0.5 and transfected with 2ug plasmid encoding the eGFP gene driven by poxvirus promoters pmH5, pmFP and pS. Images were taken using phase contrast (column 1), green fluorescence (column 2) and the two merged (column 3). pmH5 = modified vaccinia virus H5 promoter; pS = synthetic vaccinia virus promoter; pmFP = fowlpox virus promoter modified by Ruzaiq Omar.
Figure 2: Diagram of transfer vector, pBEFV_Gb_M_eGFP, used in construction of LSDV(SODis)BEFV-Gb-M.
Figure 3: Schematic diagram of LSDV(SODis)BEFV-Gb-M showing insertion into the LSDV 49-50 locus of the BEFV glycoprotein gene (Gb = SA consensus sequence), BEFV matrix protein gene (M) and the eGFP marker gene (for selection of the recombinant virus). All three foreign genes were under the control of poxvirus promoters - pmH5 (a widely used vaccinia virus promoter), pmFPV (a fowlpoxvirus promoter modified by our group) and pS (a widely used synthetic promoter) respectively.
Figure 4: PCR confirmation of recombinant LSDV(SODis)BEFV-Gb-M. Agarose gel electrophoresis showing the expected fragment size of 5,875bp for the PCR product amplified from recombinant LSDV(SODis)BEFV-Gb-M (lane 2). Lane 1 is a negative control of DNA isolated from uninfected cells and lane 3 shows the
amplified product from DNA isolated from cells infected with the parent nLSDVSODis- UCT. Refer to Figure 3 for expected sizes of PCR products generated from primers 38 for and 39 rev, which bind to LSDV outside of the flanking sequences of ORFs 49 and 50 respectively. M = molecular weight marker.
Figure 5: Detection of BEFV protein by immunofluorescence of live cells. LSDV(SODis)BEFV-Gb-M expresses eGFP, resulting in infected cells fluorescing green. Samples were treated with primary rabbit anti-B-Phemeral serum followed by secondary donkey anti-rabbit conjugated Cy3 antibody. BEFV protein expression is detected as red fluorescence.
Figure 6: SDS PAGE/western blot for LSDV(SODis)BEFV-Gb-M VLP isolation. MDBK cells in T75 flasks were infected with no virus (uninfected), nLSDVSODis-UCT (LSDV SODis) at MOI 0.5 and LSDV(SODis)BEFV-Gb-M (LSDV(SODis)-Gb-M) at MOI 0.1 . Clarified media and lysates (two freeze/thaws) were centrifuged through 12% OptiPrep cushions at 20 000 rpm for 1 hr. Media pellets were resuspended in TBS and subjected to polyacrylamide gel electrophoresis using a 12% resolving gel followed by Western blot analysis. Purified BEFV (BEFV (+)) was used as a positive control. Blots were probed with pre-absorbed anti-BEFV rabbit sera (1 :300) followed by a secondary anti-rabbit antibody labelled with alkaline phosphatase. The BEFV G protein is approximately 80kDa and the matrix protein approximately 26kDa as labelled G and M2 respectively.
Figure 7: Immunogenicity testing in rabbits. BEFV and LSDV neutralization titres were determined from rabbit sera isolated after three inoculations of different LSDV-BEFV vaccines. A. BEFV neutralization titres of rabbits inoculated with the parent virus, nLSDVASOD-UCT, and recombinants of this virus expressing BEFV Ga, Gb and Gb-M proteins. B. BEFV and LSDV neutralization titres for the same groups of rabbits as in A. C. Comparison of recombinants LSDV(ASOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb-M. nLSDVASOD-UCT = LSDV backbone lacking the SOD-homologue gene; LSDV(ASOD)BEFV-Ga = nLSDVASOD-UCT with the Australian BEFV G protein gene; LSDV(ASOD)BEFV-Gb = nLSDVASOD-UCT with the African consensus G protein gene, Gb; LSDV(ASOD)BEFV-Gb-M = nLSDVASOD- UCT + Gb + the published matrix (M) protein gene; and LSDV(SODis)BEFV-Gb-M = nLSDVSODis-UCT (the Neethling strain of LSDV containing the full-length SOD- homologue gene) + Gb + M. Titres are expressed as the reciprocal of the dilution required to neutralize BEFV or LSDV growth in 50% of infected wells.
Figure 8: Immunogenicity in two groups of cattle inoculated twice with LSDV(ASOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb-M respectively. A.
Neutralization titres for BEFV (filled bars) and LSDV (unfilled bars). LSDV(ASOD)BEFV-Gb-M = nLSDVASOD-UCT with the African consensus G protein (Gb) as well as the published matrix (M) protein genes inserted between LSDV ORFs 49 and 50; LSDV(SODis)BEFV-Gb-M = nLSDVSODis-UCT with the African consensus G protein (Gb) as well as the M protein gene sequences inserted into nLSDVSODis- UCT (the Neethling strain of LSDV containing the full-length SOD-homologue gene) between LSDV ORFs 49 and 50. Titres are expressed as the reciprocal of the dilution required to neutralize BEFV or LSDV growth in 50% of infected wells. B. Comparison of BEFV neutralization titres of the two groups of cattle at 30 days post boost. The difference observed between the two groups, using the Mann Whitney test, was not significant (P>0.05).
Figure 9: Amino acid consensus sequence of the South African BEFV Gb polypeptide (SEQ ID NOU ).
Figure 10: Codon optimised nucleic acid sequence encoding the BEFV Gb polypeptide (SEQ ID NO:2).
Figure 11 : Amino acid sequence of the BEFV M polypeptide (SEQ ID NO:3).
Figure 12: Codon optimised nucleic acid sequence encoding the BEFV M polypeptide (SEQ ID NO:4).
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 NOU - Consensus amino acid sequence of the South African BEFV Gb polypeptide.
SEQ ID NO:2 - Consensus nucleic acid sequence encoding the BEFV Gb polypeptide.
SEQ ID NO:3 - Amino acid sequence of the BEFV M polypeptide.
SEQ ID NO:4 - Nucleic acid sequence encoding the BEFV M polypeptide. SEQ ID NO:5 - Amino acid sequence of eGFP protein.
SEQ ID NO:6 - Nucleic acid sequence encoding the eGFP protein.
SEQ ID NO:7 - Nucleic acid sequence for the transfer vector.
SEQ ID NO:8 - Nucleic acid sequence of the modified vaccinia virus promoter pmH5.
SEQ ID NO:9 - Nucleic acid sequence of the synthetic vaccinia virus promoter pS.
SEQ ID NQ:10 - Nucleic acid sequence of the modified fowl pox promoter pmFP.
SEQ ID NO:11 - Nucleic acid sequence of the SODis gene.
SEQ ID NO:12 - Amino acid sequence of the SODis gene.
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.
The present inventors sequenced the G protein gene from a number of BEFV isolates from South Africa (Omar et aL, 2020) and found them to be divergent from published sequences from Australia, Asia and Middle East. A consensus G protein gene sequence (Gb) was derived from the African strains of BEFV and this was commercially synthesized, together with the published BEFV M protein gene sequence, as well as an eGFP gene for construction of a transfer vector to generate recombinant LSDV expressing the BEFV Gb and M proteins. It was hypothesized that the Gb and M proteins would be expressed by LSDV and form virus like particles (VLPs), which would be secreted from infected cells. This dual vaccine is designed to protect against both LSD and BEF.
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 phosphorylation).
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.
As used herein the term “combination of proteins” refers to a combination of a recombinant Bovine Ephemeral Fever Virus (BEFV) glycoprotein (Gb protein) and a BEFV matrix protein (M protein), as described herein. It will be appreciated by those of skill in the art that the BEFV Gb and BEFV M proteins may be in the form of secreted proteins and/or may be in the form of a virus like particle (VLP), either as a small Gb- M particle, and/or as part of the larger LSDV virion.
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 antisense 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 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. Polypeptides, 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, 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. When 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 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 polynucleotide or gene sequence that is expressed from a vector, for example, the polynucleotide or gene sequences encoding the BEFV Gb and BEFV M polypeptides described herein. A cassette generally comprises a gene sequence 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 polypeptides 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 recombinant LSDV vector encoding BEFV Gb and BEFV M 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 nucleic acids encoding the BEFV Gb and BEFV M proteins are vectored in an LSDV vector.
In one embodiment of the invention the LSDV vector including the nucleic acids encoding the BEFV Gb and BEFV M proteins 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 genes encoding the BEFV Gb and BEFV M polypeptides to subjects either with Bovine Ephemeral Fever or Lumpy Skin Disease or to subjects which are
presymptomatic for a condition associated with Bovine Ephemeral Fever 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 nucleic acids encoding BEFV Gb and BEFV M, alternatively a composition of the invention may contain a recombinant BEFV Gb and BEFV M virus like particle.
For vaccine formulations and pharmaceutical compositions, an effective amount of a composition comprising an LSDV vector comprising nucleic acids encoding BEFV Gb and BEFV M, alternatively a composition of the invention may contain a chimaeric BEFV virus like particle 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 nucleic acids encoding BEFV Gb and BEFV M, alternatively a recombinant BEFV VLP of the invention 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 nucleic acids encoding BEFV Gb and BEFV M, alternatively a recombinant BEFV VLP comprising the BEFV Gb of SEQ ID NO:1 and the BEFV M of SEQ ID NO:3 that prime and/or boost an immune response to BEF and/or LSDV.
In one embodiment, the BEFV Gb and BEFV M polypeptides are capable of “priming” an immune response to Bovine Ephemeral Fever and/or the LSDV vector is capable of “priming an immune response to lumpy skin disease. Examples of such priming compositions include the compositions and/or VLPs of the invention.
It will further be appreciated that a “boost” composition may include a composition comprising the LSDV vector comprising nucleic acids encoding BEFV Gb and BEFV M, alternatively a recombinant BEFV VLP which is administered to the subject in two or more doses after the initial priming inoculation. 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 nucleic acids encoding BEFV Gb and BEFV M, alternatively a recombinant BEFV VLP.
In some embodiments, the LSDV vector comprising nucleic acids encoding BEFV Gb and BEFV M, alternatively a recombinant BEFV VLP 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 nucleic acids encoding BEFV Gb and BEFV M, alternatively the recombinant BEFV VLP according to the invention 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 Bovine Ephemeral Fever 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 any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.
The dosage of the LSDV vector comprising nucleic acids encoding BEFV Gb and BEFV M, alternatively the recombinant BEFV VLP 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 Bovine Ephemeral Fever 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 nucleic acids encoding BEFV Gb and BEFV M, alternatively the recombinant BEFV VLP 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 Bovine Ephemeral Fever 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 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 nucleic acids encoding BEFV Gb and BEFV M, alternatively the recombinant BEFV VLPs 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 LD5o and the ED5o. 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 within a range of circulating concentrations that include the ED5o 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
Construction of LSDV(SODis)BEFV-Gb-M
As a vector backbone, and LSDV vaccine, the SOD knock-in virus nLSDVSODis-UCT was used (Douglass et al. 2020). nLSDVSODis was created by replacing the native SOD gene with a stabilised SOD gene of SEQ ID NO:11. Two BEFV genes were inserted into this backbone between the convergent LSDV open reading frames ORFs 49 and 50. In addition a marker gene, eGFP (SEQ ID NO:6), was inserted for purification of the recombinant vaccine. The BEFV genes used encoded the membrane glycoprotein (Gb protein) (SEQ ID NO:1 ) as well as the matrix
protein (SEQ ID NO:3). The glycoprotein sequence was a consensus sequence derived from sequences of African strains of BEFV, generated by our group (Omar et al. 2020) and the matrix protein gene sequence (SEQ ID NO:4) was that of the published Walker strain. The three foreign genes were placed downstream of poxvirus promoters which were shown to be expressed by LSDV (Figure 1 ). pmH5 is a modified vaccinia promoter (SEQ ID NO:8), pmFP is a modified fowlpox virus promoter (SEQ ID NQ:10) designed by Ruzaiq Omar and pS is a synthetic vaccinia virus promoter (SEQ ID NO:9). The transfer vector (SEQ ID NO:7) for construction of this recombinant was designed by our group (RO) and ordered from Genscript (Figure 2). Using this transfer vector, recombinant LSDV(SODis)BEFV-Gb-M was generated by insertion of the foreign gene cassette at the LSDV 49-50 locus by homologous recombination (Figure 3).
Methodology
Lamb testes cells were split and infected the following day at an MOI of 1 . After 2 hours the cells were transfected with 6 pg of pBEFV Gb_M_eGFP and 2 pl of Xtreme Gene HP transfection reagent (ratio 3:1 ). After overnight incubation with the transfection mix, 1 ml of cDMEM was added and cells left for another 48 hrs, by which time there were many GFP positive cells. Cells were lysed and used to infect MDBK cells (P1 ). After more than 72 hours of incubation, 9 foci were identified and picked. These foci were suspended in 150 pl DMEM and lysed. 10 pl was used to infect MDBK cells (P2). The plates were observed 97 hours post infection. 17 fluorescing foci were marked and picked after P2. Using the same procedure used above, the P2 virus was then used to infect MDBK cells to generate P3 virus. At P3, 33 fluorescing foci were observed after 144 hours. Most of the fluorescing foci were picked and the same procedure repeated to generate P4 virus. Some wells of the 12 well plate containing P4 virus had no parental virus. These wells had media removed, washed with PBS and 500 pl DMEM added. The plate was frozen and thawed three times. Dilutions were made of the lysate and 100 pl of these dilutions were added to each well of a 96 well plate, with the aim of isolating a single fluorescing focus in a single well for clonal expansion. Such a focus was identified by P6 after two series of passaging on 96 well plates. 10 pl and 30 pl were used to infect MDBK cells in a 12 well plate for passaging and PCR respectively.
Confirmation of recombinant LSDV(SODis)BEFV-Gb-M
The recombinant was confirmed to be correct by PCR, using conditions of 30 sec denaturation at 94 °C, 30 sec of annealing at 55 °C and 6 min of extension at 68 °C. After 30 cycles a final extension was performed at 68 °C for 5 min. The PCR products were separated by agarose gel electrophoresis. PCR generated the expected fragment sizes of 5,857 bp for the recombinant and 1 ,088 bp for the parent virus (Figures 3 and 4). Additional PCR reactions were performed to generate products for sequencing. The construct was confirmed to be correct by DNA sequencing.
Confirmation of expression of BEFV protein from LSDV(SODis)BEFV-Gb-M
Expression of BEFV protein was shown by immunofluorescence (Figure 5) and western blot analysis (Figure 6). Live cell staining indicated that the BEFV protein was located on the surface of cells (Figure 5). The western blot analysis showed that BEFV proteins were secreted into the medium of MDBK cells infected with LSDV(SODis)BEFV-Gb-M (Figure 6).
Immunogenicity Testing of different LSDV-BEFV vaccines in a rabbit model
LSDV(SODis)BEFV-Gb-M was tested for immunogenicity both in a rabbit model as well as in cattle. A number of different vaccines were tested in rabbits to determine which vaccines to carry forward into cattle. Vaccines were made in two different LSDV backbones, one without the SOD-homologue (nLSDVASOD-UCT) and one with a stabilized full-length SOD-homologue (nLSDVSODis-UCT). Rabbits were inoculated three times with 106 ffu vaccine i.m. at 4 week intervals. The final bleed was taken two weeks post final inoculation.
The published Walker G protein gene sequence (Ga) was compared to the African consensus G protein sequence (Gb) and Gb gave higher BEFV neutralization titres in rabbits inoculated three times with 106 ffu vaccine (LSDV(ASOD)BEFV-Ga vs LSDV(ASOD)BEFV-Gb (Figure 7A). The vaccine with both Gb and M genes (LSDV(ASOD)BEFV-Gb-M) was also tested in this same experiment and 2 out of the 5 rabbits gave superior neutralization responses (Figure 7A). All rabbits produced neutralizing responses to LSDV (Figure 7B, open bars), indicating that the superior BEFV neutralization response was specifically due to the Gb insert and not to any experimental error.
A comparison was made of the two vector backbones expressing BEFV Gb and M genes in the same way in a separate experiment. All rabbits generated neutralizing responses to both BEFV and LSDV (Figure 7C). There was no clear
indication which backbone was superior, so both of these vaccines were tested in cattle.
Immunogenicity of LSDV(SODis)BEFV-Gb-M in Cattle
Two of our vaccines, LSDV(ASOD)BEFV-Gb-M and LSDV(SODis)BEFV-Gb- M, were tested in cattle, the natural host for LSDV and BEFV. Two vaccinations of 105 and 5 x 104 ffu respectively were given with a 32-day time interval between vaccinations. The cattle serum was tested for BEFV and LSDV neutralization 30 days following the second vaccination. All animals generated neutralization responses to both BEFV and LSDV (Figure 8A). The LSDV responses were variable, showing no clear distinction between the two vaccines. Figure 8B shows that animals inoculated with LSDV(SODis)BEFV-Gb-M generated higher neutralization titres than those inoculated with LSDV(ASOD)BEFV-Gb-M; however, this difference was not statistically significant. LSDV(SODis)BEFV-Gb-M has been chosen to develop further as a dual vaccine against both LSDV and BEFV.
REFERENCES
Douglass, N., et aL, Influence of the Viral Superoxide Dismutase (SOD) Homologue on Lumpy Skin Disease Virus (LSDV) Growth, Histopathology and Pathogenicity. Vaccines (Basel), 2020. 8(4).
Omar, R., et aL, South African bovine ephemeral fever virus glycoprotein sequences are phylogenetically distinct from those from the rest of the world. Arch Virol, 2020. 165(5): p. 1207-1210.
Claims (19)
1. A composition comprising:
(i) a recombinant Bovine Ephemeral Fever virus (BEFV) glycoprotein (Gb protein);
(ii) a BEFV matrix protein (M protein); and
(iii) a recombinant Lumpy Skin Disease virus (rLSDV) comprising a stabilised SOD-homolog (SODis) gene.
2. The composition of claim 1 , wherein the recombinant BEFV Gb protein is a protein of SEQ ID NO:1 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:1 .
3. The composition of claim 1 or 2, wherein the BEFV M protein is a protein of SEQ ID NO:3 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:3.
4. The composition of any one of claims 1 to 3, wherein the rLSDV comprises a stabilised SODis gene of SEQ ID NO:11 .
5. A composition comprising:
(i) a first nucleic acid sequence encoding a recombinant Bovine Ephemeral Fever virus (BEFV) glycoprotein (Gb polypeptide); and
(ii) a second nucleic acid encoding a BEFV matrix protein (M polypeptide), wherein the first nucleic acid and second nucleic acid are contained in a vector, and wherein the vector is a rLSDV comprising a stabilised SOD- homolog (SODis) gene.
6. The composition of claim 5, wherein the first nucleic acid sequence and the second nucleic acid sequence are contained in the rLSDV between open reading frame 49 and 50.
7. The composition of claim 5 or 6, wherein the recombinant BEFV Gb polypeptide is a polypeptide of SEQ ID NO:1 or a polypeptide having at least 90% sequence identity to SEQ ID NO:1 .
8. The composition of any one of claims 5 to 7, wherein the BEFV M polypeptide is a polypeptide of SEQ ID NO:3 or a polypeptide having at least 90% sequence identity to SEQ ID NO:3.
9. The composition of any one of claim 5 to 8, wherein the stabilised SODis gene has a sequence of SEQ ID NO:11 .
10. The composition of any one of claims 5 to 9, wherein the first nucleic acid and second nucleic acid are each operably linked to regulatory sequences that allow for the expression of the BEFV Gb and BEFV M polypeptides.
11. A vaccine comprising the composition of any one of claims 1 to 10 and a pharmaceutically acceptable carrier or adjuvant.
12. The composition of any one of claims 1 to 10 or the vaccine of claim 11 for use in a method of inducing an immune response against Bovine Ephemeral Fever virus in a subject, the method comprising administering an immunogenically effective amount of the composition or the vaccine to the subject.
13. The composition or vaccine for use of claim 12, wherein the composition or vaccine additionally induces an immunogenically effective response against Lumpy Skin Disease virus in the subject.
14. The composition or vaccine for use of claim 12 or 13, wherein the subject is a mammal.
15. The composition, or vaccine for use of any one of claims 12 to 14, wherein the subject is selected from cattle and buffalo.
16. A method of inducing an immune response against Bovine Ephemeral Fever virus in a subject, the method comprising administering an immunogenically
effective amount of the composition of any one of claims 1 to 10, or the vaccine of claim 11 to the subject.
17. The method of claim 16, wherein the composition or vaccine additionally induces an immunogenically effective response against Lumpy Skin Disease virus in the subject.
18. The method of claim 16 or 17, wherein the subject is a mammal.
19. The method of any one of claims 16 to 18, wherein the subject is selected from cattle and buffalo.
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