Patent Grant
11154611
The invention is in the medical field, especially in the veterinary field, and particularly pertains to vaccines, more particularly to vaccines against bovine leukemia virus. The invention more specifically relates to recombinant bovine leukemia viruses that have an attenuated phenotype, nucleic acids encoding such viruses, vectors comprising such nucleic acids, host cells comprising such nucleic acids or vectors, applications of these agents in medicine, particularly as vaccines, non-human animals vaccinated therewith, materials derived from such non-human animals, and downstream uses of such materials.
Although eradicated from Europe, bovine leukemia virus (BLV) is responsible for important economic losses worldwide. The great majority of BLV-infected animals are asymptomatic carriers of the virus. Approximately one-third of BLV-infected bovines develop a benign polyclonal proliferation of B cells called persistent lymphocytosis (PL), characterised by an increase in the absolute number of peripheral blood circulating B lymphocytes associated with an inversion of the B/T lymphocyte ratio. PL is usually stable for several years but can also progress to a tumour phase.
The most conspicuous clinical manifestation of BLV infection is the development of lymphoid tumours. Fatal lymphoma or lymphosarcoma (LS), characterised by mono- or oligo-clonal B cell expansion, occurs in less than 5-10% of infected animals, predominantly in adult cattle older than 4-5 years old. Local proliferation of B cells, called lymphosarcoma, can occur within different organs and tissues leading to a series of defects that are finally incompatible with the survival of the animal. In addition, transformed B cells can also induce the enlargement of lymph nodes and cause lymphoma. Besides an impact on survival, BLV infection also impairs the immune system leading to opportunistic infections.
Several attempts have been undertaken to develop vaccines against BLV, such as vaccines based on chemically inactivated BLV, vaccines based on lysates from, e.g., BLV-infected cells or BLV tumours, vaccines comprising BLV subunits, such as, e.g., the gp51 envelope glycoprotein. Other attempts used vaccinia virus as a vehicle and introduced BLV genes encoding, e.g., BLV envelope proteins, into its genome (i.e., recombinant vaccinia virus or RVV). Short peptides mimicking B and T cell epitopes of BLV proteins were also tested as immunogens. DNA vaccines comprising BLV genes, e.g., the env gene under the control of the cytomegalovirus promoter, were also developed. These ‘traditional’ vaccine candidates faced problems of inter alia efficacy (i.e., only an inadequately low fraction of vaccinated animals were protected), persistence (i.e., rapid decrease of immune protection in the vaccinated animals), cost (e.g., high cost of production of purified proteins), and/or safety (e.g., use of genetically modified hybrid viruses, such as RVV).
In an attempt to address the shortcomings of these earlier approaches, numerous attenuated BLV mutants were developed, e.g., by deleting genes dispensable for infectivity but required for efficient replication of the virus (Willems et al. 1993. J. Virol. 67: 4078-4085). Among these, an attenuated BLV provirus, pBLV6073, was obtained by introducing a mutation to an immunoreceptor tyrosine-based activation motif localised in the cytoplasmic tail of the transmembrane gp30 envelope glycoprotein (Willems et al. 1995. J. Virol. 69: 4137-4141). Another attenuated BLV provirus, pBLVDX, was constructed by deleting the R3 and G4 sequences (Willems et al. 1993. J. Virol. 67: 4078-4085). These BLV mutants (pBLV6073 and pBLVDX) were evaluated in Kerkhofs et al. 2000. J. Gen. Virol. 81: 957-963; Reichert et al. 2000. J. Gen. Virol. 81: 965-969; and Florins et al. 2007. J. Virol. 81: 10195-10200.
The present inventors have conducted extensive studies of existing attenuated BLV proviruses, and have confirmed that these BLV proviruses, including inter alia pBLV6073 and pBLVDX, do remain pathogenic at a level that may prevent their widespread use as vaccines in veterinary practice. For example, pathogenicity was observed in one sheep among 20 that have been infected with the pBLVDX provirus after a latency period of 7 years. Also, as summarised in Table 1 of Florins et al. 2007 supra, pathogenicity was observed in one sheep among 8 that have been infected with the pBLVDX provirus after a latency period of 7.5 years. Furthermore, the pBLV6073 provirus induced leukemia in 1 out of 4 sheep after 83 months of latency (also see Table 1 of Florins et al. 2007 supra).
Hence, the previously existing attenuated BLV proviruses are still at least weakly pathogenic.
Moreover, protection achieved by previously existing attenuated BLV proviruses has been reported as not effective enough and comparatively short-term. For example, one of two cows vaccinated using the pBLVDX provirus and evaluated in Kerkhofs et al. 2000 supra became infected by wild-type BLV 12 months after challenge. One of three sheep vaccinated using the pBLVDX provirus and evaluated in Reichert et al. 2000 supra became infected by BLV from a naturally infected cow. Further importantly, as shown in the experimental section, cow #269 vaccinated using the pBLV6073 provirus and evaluated in Kerkhofs et al. 2000 supra also became infected by wild-type BLV 24 months after challenge.
The present invention addresses one or more of such problems observed by the inventors.
As corroborated by the experimental section, which illustrates certain representative embodiments of the invention, the inventors have realised that by combining specific mutations in a BLV (pro)virus, greatly improved vaccines may be obtained. Hence, the inventors accomplished recombinant BLV proviruses which were infectious, but which replicated at desirably low levels in target animals, such as specifically in cows.
At least some embodiments of the present recombinant BLV proviruses display one or more further advantages improving their use as vaccines. For example, such recombinant BLV proviruses may display one or more or preferably all of the following advantages: they elicit a strong anti-BLV immune response comparable to an immune response to wild-type BLV; they do not spread to uninfected sentinels maintained for prolonged periods of time in the same herd (i.e., satisfactory biosafety as a vaccine); they lead to production of antibodies that are transmitted to the newborn calves via the maternal colostrum, whereby the anti-viral passive immunity persists during several months in the calves; they do not transmit from cows to calves; they cause the vaccinated animals to resist a challenge by a wild type BLV provirus.
In particular, vaccines provided for by the recombinant BLV proviruses in accordance with aspects and embodiments of the present invention are highly effective, preferably achieving long-term protection (e.g., protection for at least 18 months or for at least 24 months or for at least 36 months or for at least 48 months post-vaccination) of virtually all tested animals (e.g., at least 90%, preferably at least 95%, such as 98%, or 99%, or even 100%), more preferably of cattle, from infection by wild-type BLV. Hence, in contrast to previously existing vaccines, the present recombinant BLV proviruses are effective in bovids, such as more particularly in cows, rendering the present vaccines particularly advantageous for controlling BLV infections in cattle.
Accordingly, in an aspect the invention provides a recombinant attenuated bovine leukemia virus (BLV) characterised in that the virus comprises:
(i) at least one mutation selected from the group consisting of:
Further aspects of the invention provide:
These and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject-matter of the appended claims is hereby specifically incorporated in this specification.
As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.
The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of” and “consisting essentially of”, which enjoy well-established meanings in patent terminology.
The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.
The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/−10% or less, preferably +/−5% or less, more preferably +/−1% or less, and still more preferably +/−0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” refers is itself also specifically, and preferably, disclosed.
Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any ≥3, ≥4, ≥5, ≥6 or ≥7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.
The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.
Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.
Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined.
In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.
As noted, the inventors have realised that by combining certain mutations in the BLV genome an attenuated BLV may be obtained useful for the production of greatly improved vaccines. These attenuated BLV are infectious, thus facilitating their introduction into the to-be-vaccinated subjects, but replicate at desirably low levels in the vaccinated subjects.
Accordingly, in an aspect the invention provides a recombinant attenuated bovine leukemia virus (BLV) characterised in that the virus comprises:
Unexpectedly, the combinations of the mutations in the resulting recombinant BLV, rather than being deleterious for the recombinant BLV (e.g., completely destroying its infectivity in animals, particularly in cattle), preserves satisfactory levels of infectivity of the recombinant BLV and reduces or eliminates its pathogenicity, thereby achieving greatly improved attenuated vaccines in animals, particularly in cattle.
In certain preferred embodiments, the recombinant attenuated BLV may comprise:
In certain further preferred embodiments, the recombinant attenuated BLV may comprise:
In certain particularly preferred embodiments, the recombinant attenuated BLV may comprise: the mutation in the nucleic acid sequence encoding the most N-terminal YXXL signalling motif of the cytoplasmic domain of the transmembrane subunit (TM) of the envelope protein, said mutation disrupting the signal transduction activity of the motif, the mutation in G4 restricting the propagation of the BLV in vivo, and the mutation in R3 restricting the propagation of the BLV in vivo. Unexpectedly, whereas protection achieved by the previously existing attenuated BLV proviruses pBLVDX and pBLV6073 has been reported as not effective enough and comparatively short-term, the recombinant attenuated BLV in accordance with these embodiments, combining mutations in the N-terminal YXXL signalling motif of the cytoplasmic domain of TM of the envelope protein, in G4 and in R3, such as for example BLV6073DX described elsewhere in this specification, are highly effective and provide for long-term protection. Surprisingly, the combination of the mutations, rather than being deleterious for the recombinant BLV (e.g., completely destroying its infectivity, such as the infectivity of BLV6073DX in animals, particularly in cattle), preserves satisfactory levels of infectivity of the recombinant BLV and reduces or eliminates its pathogenicity, thereby achieving greatly improved attenuated vaccines in animals, particularly in cattle.
In certain further particularly preferred embodiments, the recombinant attenuated BLV may comprise: the mutation in the X region of the BLV nucleic acid sequence, said mutation abolishing the production of at least one or preferably all microRNA encoded by said X region, the mutation in G4 restricting the propagation of the BLV in vivo, and the mutation in R3 restricting the propagation of the BLV in vivo.
In certain further preferred embodiments, the recombinant attenuated BLV may comprise: the mutation in the nucleic acid sequence encoding the most N-terminal YXXL signalling motif of the cytoplasmic domain of the transmembrane subunit (TM) of the envelope protein, said mutation disrupting the signal transduction activity of the motif, the mutation in the X region of the BLV nucleic acid sequence, said mutation abolishing the production of at least one or preferably all microRNA encoded by said X region, and the mutation in G4 restricting the propagation of the BLV in vivo.
In certain further preferred embodiments, the recombinant attenuated BLV may comprise: the mutation in the nucleic acid sequence encoding the most N-terminal YXXL signalling motif of the cytoplasmic domain of the transmembrane subunit (TM) of the envelope protein, said mutation disrupting the signal transduction activity of the motif, the mutation in the X region of the BLV nucleic acid sequence, said mutation abolishing the production of at least one or preferably all microRNA encoded by said X region, and the mutation in R3 restricting the propagation of the BLV in vivo.
In yet further particularly preferred embodiments, the recombinant attenuated BLV may comprise: the mutation in the nucleic acid sequence encoding the most N-terminal YXXL signalling motif of the cytoplasmic domain of the transmembrane subunit (TM) of the envelope protein, said mutation disrupting the signal transduction activity of the motif, the mutation in the X region of the BLV nucleic acid sequence, said mutation abolishing the production of at least one or preferably all microRNA encoded by said X region, the mutation in G4 restricting the propagation of the BLV in vivo, and the mutation in R3 restricting the propagation of the BLV in vivo.
In various embodiments, the recombinant attenuated BLV may comprise combinations of mutations as individualised in Table 1.
For the purposes of Table 1, “mut TM” denotes the mutation in the nucleic acid sequence encoding the most N-terminal YXXL signalling motif of the cytoplasmic domain of the transmembrane subunit (TM) of the envelope protein, said mutation disrupting the signal transduction activity of the motif; “mut G4” denotes the mutation in G4 restricting the propagation of the BLV in vivo; “mut R3” denotes the mutation in R3 restricting the propagation of the BLV in vivo; and “mut microRNA” denotes the mutation in the X region of the BLV nucleic acid sequence, said mutation abolishing the production of at least one or preferably all microRNA encoded by said X region.
Preferred embodiments of those individualised in Table 1 may be embodiments #3 and #6 to #9, more preferred #3, #6 and #9, even more preferred #3 and #9.
The term “bovine leukemia virus” or “BLV” refers to a naturally occurring oncogenic, B-lymphotropic retrovirus that mainly infects cattle, preferably domestic cattle. It is a member of the Oncovirinae subfamily and belongs to the Deltaretrovirus genus, which also includes the human T-cell leukemia virus types 1 and 2 (HTLV-1 and-2). The term encompasses BLV of any and all geographical origins, such as without limitation BLV originating from (e.g., isolated or isolatable from cattle in) Argentina, Belgium, Brazil, Costa Rica, France, Iran, Japan, Russia, Ukraine, or USA. The term further encompasses any and all variants, clones, strains, isolates and genotypes of BLV. A useful but non-limiting overview of previously identified BLV isolates and genotypes, which may be useful in performing the present invention, is found inter alia in Rodriguez et al. 2009 (J Gen Virol. 90: 2788-97) and references cited therein.
Upon infecting a host cell, preferably a B lymphocyte, the viral +mRNA genome is reverse transcribed into DNA and integrated as a provirus into the genome of the BLV-infected host cell. The provirus can persist integrated into the host cell genome, thereby inducing a persistent or latent infection with diverse outcomes, ranging from asymptomatic to persistent lymphosis, lymphosarcoma and lymphoma. BLV can be transmitted through the transfer of BLV-infected cells (such as, e.g., B-lymphocytes and monocytes/macrophages) present in, e.g., blood or milk. Routes of transmission may include cattle management procedures involving transfer of infected blood such as dehorning, ear tattooing, rectal palpation, or the use of infected needles.
A non-limiting example of BLV is BLV clone 344 isolated as a provirus from a BLV-induced tumour (Van den Broeke et al. 1988, Proc. Natl. Acad. Sci. USA 85: 9263-9267). BLV 344 provirus is available inter alia cloned in the pSP64 plasmid, thereby yielding the plasmid pBLV344H as described in Willems et al. 1993 (J. Virol. 67: 4078-4085). The plasmid pBLV344H has been deposited under the Budapest Treaty with the Belgian
Coordinated Collections of Microorganisms BCCM/LMBP Collection under accession number LMBP 8165 on Feb. 5, 2013 (see Table 2A). The complete sequence of the BVL 344 provirus as sequenced from plasmid pBLV344H is shown in
In certain other embodiments, the BLV for performing the present invention may be the BLV isolate LS2 (complete proviral genome sequence annotated under GenBank acc. no. HE967302.1); BLV isolate LS3 (complete proviral genome sequence annotated under GenBank acc. no. HE967303.1); BLV isolate LS1 (complete proviral genome sequence annotated under GenBank acc. no. HE967301.1); BLV isolate of which the complete genome sequence is annotated under GenBank acc. no. NC_001414.1; BLV isolate of which the gag and pol genes sequence is annotated under GenBank acc. no. M10987.1; BLV isolate of which the env gene and post-env region sequence is annotated under GenBank acc. no. K02251.1; BLV isolate of which the complete genome sequence is annotated under GenBank acc. no. AF033818.1; BLV strain Arg41 (complete genome sequence annotated under GenBank acc. no. FJ914764.1); BLV isolate of which the complete genome sequence is annotated under GenBank acc. no. AF257515.1; BLV isolate of which the complete genome sequence is annotated under GenBank acc. no. K02120.1; BLV isolate of which the complete genome sequence is annotated under GenBank acc. no. D00647.1; or BLV isolate pBLV913 (complete proviral genome sequence annotated under GenBank acc. no. EF600696.1).
The term “recombinant” is generally used to indicate that the material (e.g., a virus, a nucleic acid, a genetic construct or a protein) has been altered by technical means (i.e., non-naturally) through human intervention. The term “recombinant nucleic acid” can commonly refer nucleic acids comprised of segments joined together using recombinant DNA technology. As used herein, the term may preferably denote material (e.g., a virus, a nucleic acid, a genetic construct or a protein) that has been altered by technical means of mutagenesis.
The term “attenuated” is well-known in the field of vaccination and when used in combination with a virus, preferably a bovine leukemia virus, denotes a virus variant or mutant which exhibits a substantially lower degree of virulence compared to a wild-type virus, preferably a virus variant or mutant exhibiting reduced propagation in the host (i.e., in vivo), e.g., due to slower growth rate and/or a reduced level of replication compared to a wild-type virus. Propagation of an attenuated virus in the host (i.e., in vivo) may be at least about 10 fold, e.g., at least about 25 fold, or at least about 50 fold, or at least about 75 fold, preferably at least about 100 fold, less than that of a wild-type virus.
Suitable methods for measuring the propagation of a virus, in particular attenuated BLV or wild-type BLV, in the host include without limitation determining the proviral loads in the challenged host. For example, the number of BLV proviral copies may be determined using a suitable methodology, e.g., quantitative PCR, per a given number, e.g., 100, of peripheral blood mononuclear cells at a given time or times, i.e., in function of time, following the challenge of the host, in particular cattle such as a cow, with the virus. See Example 3 for a specific, non-limiting application of this approach.
Typically, such attenuated virus will not induce symptoms of viral infection or will induce only mild symptoms upon infecting, preferably through vaccination, a subject, but severe symptoms of viral infection do not typically occur in the infected, preferably vaccinated, subject.
The terms “mutation” and “mutagenesis” and the like generally refer to changes in nucleic acid sequences. Such changes may naturally occur, e.g., due to errors that occur during nucleic acid replication, mitosis or meiosis, or due to insertion of transposons or viral sequences. They may also be artificially (i.e., non-naturally) introduced by technical means through human intervention, e.g., by chemicals, irradiation, or recombinant DNA technology. As used herein, the terms preferably refer to such ‘artificial’ mutations.
Mutations in general may either have no effect (e.g., silent mutations) or they may have an effect on a given transcription product and/or translation product, e.g., they may result in the production of no transcription and/or translation product, or may result in the production of a transcription and/or translation product that is substantially not functioning or not functioning properly (i.e., not as the wild-type product).
In the present specification, the term “mutation” may particularly refer to a sequence change in the nucleic acid of a BLV (i.e., mutated BLV, BLV mutant) compared to the nucleic acid of a BLV that has not been so-mutated, such as, preferably, compared to the nucleic acid of a wild-type BLV. “Wild-type” BLV as used herein may suitably refer to naturally occurring, pathogenic BLV found in or isolated from BLV-infected hosts. The term also includes wild-type BLV proviruses, isolated forms thereof and genetic constructs containing such.
Optionally, a BLV carrying the mutation(s) as taught by the present invention may also comprise one or more other mutations not specified herein, e.g., one or more other mutations vis-à-vis a wild-type BLV. Such one or more other mutations may be in any one of the BLV genes, for example, in any one or more of the gag, pol, env, microRNA, R3, G4, Tax and Rex genes. Preferably such one or more other mutations do not interfere with replication of the BLV, in particular such one or more other mutations do not restrict the propagation of the BLV in vivo.
Mutations affecting a given BLV polypeptide (e.g., the level of production and/or the amino acid sequence of the polypeptide) may reside in nucleic acid sequence(s) comprised in the open reading frame (ORF) coding for said polypeptide, and/or such mutations may reside in nucleic acid sequence(s) comprised in the non-coding portions (untranslated regions) of the messenger RNA (mRNA) encoding said polypeptide, and/or such mutations may reside in nucleic acid sequence(s) comprised in precursor RNA (pre-mRNA) encoding said polypeptide, but removed (spliced out) from the mature mRNA encoding said polypeptide.
An “open reading frame” or “ORF” as used herein refers to a succession of coding nucleotide triplets (codons) starting with a translation initiation codon and closing with a translation termination codon known per se, and not containing any internal in-frame translation termination codon, and potentially capable of encoding a protein or polypeptide. Reference to the “level” of a BLV polypeptide encompasses the quantity and/or the availability (e.g., availability for performing its biological function) of the BLV polypeptide, e.g., in a cell, tissue, organ or an organism.
By means of an example and without limitation, a mutation in G4 as intended herein, which may also be denoted as a mutation in G4 gene, which affects the G4 polypeptide (e.g., the level of production and/or the amino acid sequence of the G4 polypeptide) may reside in nucleic acid sequence(s) comprised in the ORF coding for the G4 polypeptide, and/or such mutation may reside in nucleic acid sequence(s) comprised in the non-coding portions of the mRNA encoding the G4 polypeptide, and/or such mutation may reside in nucleic acid sequence(s) comprised in pre-mRNA encoding the G4 polypeptide, but removed (spliced out) from the mature mRNA encoding the G4 polypeptide. Hence, “a mutation in G4” that may be denoted as “a mutation in G4 gene” may also be denoted as a mutation in the nucleic acid sequence encoding G4, in the sense that the mutation may be the nucleic acid sequence encoding G4 pre-mRNA, G4 mRNA and/or G4 ORF.
Similarly, by means of an example and without limitation, a mutation in R3 as intended herein, which may also be denoted as a mutation in R3 gene, which affects the R3 polypeptide (e.g., the level of production and/or the amino acid sequence of the R3 polypeptide) may reside in nucleic acid sequence(s) comprised in the ORF coding for the R3 polypeptide, and/or such mutation may reside in nucleic acid sequence(s) comprised in the non-coding portions of the mRNA encoding the R3 polypeptide, and/or such mutation may reside in nucleic acid sequence(s) comprised in pre-mRNA encoding the R3 polypeptide, but removed (spliced out) from the mature mRNA encoding the R3 polypeptide. Hence, “a mutation in R3” that may be denoted as “a mutation in R3 gene” may also be denoted as a mutation in the nucleic acid sequence encoding R3, in the sense that the mutation may be the nucleic acid sequence encoding R3 pre-mRNA, R3 mRNA and/or R3 ORF.
Any types of mutations achieving the intended effects, such as affecting a given BLV polypeptide (e.g., the level of production and/or the amino acid sequence of the polypeptide), are contemplated herein. For example, suitable mutations may include deletions, insertions, and/or substitutions, The term “deletion” refers to a mutation wherein one or more nucleotides, typically consecutive nucleotides, of a nucleic acid are removed, i.e., deleted, from the nucleic acid. The term “insertion” refers to a mutation wherein one or more nucleotides, typically consecutive nucleotides, are added, i.e., inserted, into a nucleic acid. The term “substitution” refers to a mutation wherein one or more nucleotides of a nucleic acid are each independently replaced, i.e., substituted, by another nucleotide.
In certain embodiments, a mutation may introduce a premature in-frame stop codon into the ORF coding for a given BLV polypeptide. Such premature stop codon may lead to production of a C-terminally truncated form of said polypeptide (this may preferably affect, such as diminish or abolish, some or all biological function(s) of the polypeptide) or, especially when the stop codon is introduced close to (e.g., about 20 or less, or about 10 or less amino acids downstream of) the translation initiation codon of the ORF, the stop codon may effectively abolish the production of the polypeptide. Various ways of introducing a premature in-frame stop codon in the ORF coding for the BLV polypeptide are apparent to a skilled person. For example but without limitation, a suitable insertion, deletion or substitution of one or more nucleotides in the ORF may introduce the premature in-frame stop codon.
In other embodiments, a mutation may introduce a frame shift (e.g., +1 or +2 frame shift) in the ORF coding for a given BLV polypeptide. Typically, such frame shift may lead to a previously out-of-frame stop codon downstream of the mutation becoming an in-frame stop codon. Hence, such frame shift may lead to production of a form of the polypeptide having an alternative C-terminal portion and/or a C-terminally truncated form of said polypeptide (this may preferably affect, such as diminish or abolish, some or all biological function(s) of the polypeptide) or, especially when the mutation is introduced close to (e.g., about 20 or less, or about 10 or less amino acids downstream of) the translation initiation codon of the ORF, the frame shift may effectively abolish the production of the polypeptide. Various ways of introducing a frame shift in the ORF coding for the BLV polypeptide are apparent to a skilled person. For example but without limitation, a suitable insertion or deletion of one or more (not multiple of 3) nucleotides in the ORF may lead to a frame shift.
In further embodiments, a mutation may delete at least a portion of the ORF coding for a given BLV polypeptide. Such deletion may lead to production of an N-terminally truncated form, a C-terminally truncated form and/or an internally deleted form of said polypeptide (this may preferably affect, such as diminish or abolish, some or all biological function(s) of the polypeptide). Preferably, the deletion may remove about 20% or more, or about 50% or more of the ORF's nucleotides. Especially when the deletion removes a sizeable portion of the ORF (e.g., about 50% or more, preferably about 60% or more, more preferably about 70% or more, even more preferably about 80% or more, still more preferably about 90% or more of the ORF's nucleotides) or when the deletion removes the entire ORF, the deletion may effectively abolish the production of the polypeptide. The skilled person can readily introduce such deletions.
In certain other embodiments, a mutation may be a substitution of one or more nucleotides in the ORF coding for a given BLV polypeptide resulting in substitution of one or more amino acids of said BLV polypeptide. Such mutation may typically preserve the production of the polypeptide, and may preferably affect, such as diminish or abolish, some or all biological function(s) of the polypeptide. The skilled person can readily introduce such substitutions.
In certain preferred embodiments, a mutation may abolish native splicing of a pre-mRNA encoding a given BLV polypeptide. In the absence of native splicing, the pre-mRNA may be degraded, or the pre-mRNA may be alternatively spliced, yielding mRNA(s) encoding other BLV polypeptide(s), or the pre-mRNA may be spliced improperly employing latent splice site(s) if available. Hence, such mutation may typically effectively abolish the production of the polypeptide's mRNA and thus the production of the polypeptide. Various ways of interfering with proper splicing are available to a skilled person, such as for example but without limitation, mutations which alter the sequence of one or more sequence elements required for splicing to render them inoperable, or mutations which comprise or consist of a deletion of one or more sequence elements required for splicing.
The terms “splicing”, “splicing of a gene”, “splicing of a pre-mRNA” and similar as used herein are synonymous and have their art-established meaning. By means of additional explanation, splicing denotes the process and means of removing intervening sequences (introns) from pre-mRNA in the process of producing mature mRNA. The reference to splicing particularly aims at native splicing such as occurs under normal physiological conditions. The terms “pre-mRNA” and “transcript” are used herein to denote RNA species that precede mature mRNA, such as in particular a primary RNA transcript and any partially processed forms thereof. Sequence elements required for splicing refer particularly to cis elements in the sequence of pre-mRNA which direct the cellular splicing machinery (spliceosome) towards correct and precise removal of introns from the pre-mRNA. Sequence elements involved in splicing are generally known per se and can be further determined by known techniques including inter alia mutation or deletion analysis. By means of further explanation, “splice donor site” or “5′ splice site” generally refer to a conserved sequence immediately adjacent to an exon-intron boundary at the 5′ end of an intron. Commonly, a splice donor site may contain a dinucleotide GU, and may involve a consensus sequence of about 8 bases at about positions +2 to −6. “Splice acceptor site” or “3′ splice site” generally refers to a conserved sequence immediately adjacent to an intron-exon boundary at the 3′ end of an intron. Commonly, a splice acceptor site may contain a dinucleotide AG, and may involve a consensus sequence of about 16 bases at about positions −14 to +2.
Reference herein to a mutation which abolishes splicing of a given gene, such as in particular splicing at a given exon-intron or intron-exon boundary, may in particular encompass a mutation involving the respective splice donor site or a mutation involving the respective splice acceptor site, whereby splicing at said splice donor site or splice acceptor site is abolished due to said mutation.
For example, a mutation involving a splice donor site may comprise or consist of a deletion, insertion and/or substitution of one or more nucleotides, thereby changing the sequence of the splice donor site. The change in the sequence the splice donor site may involve a change of any one or more nucleotides constituting the splice donor consensus sequence, more preferably may involve a change of any one or both of the 5′ most two nucleotides of an intron. For example, a deletion of a given splice donor site may refer to a deletion of any one or both of the 5′ most two nucleotides of an intron, e.g., a deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 10 5′ most nucleotides of an intron, and optionally an additional deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 10 3′ most nucleotides of the upstream exon.
For example, a mutation involving a splice acceptor site may comprise or consist of a deletion, insertion and/or substitution of one or more nucleotides, thereby changing the sequence of the splice acceptor site. The change in the sequence the splice acceptor site may involve a change of any one or more nucleotides constituting the splice acceptor consensus sequence, more preferably may involve a change of any one or both of the 3′ most two nucleotides of an intron. For example, a deletion of a given splice acceptor site may refer to a deletion of any one or both of the 3′ most two nucleotides of an intron, e.g., a deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 10 3′ most nucleotides of an intron, and optionally an additional deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 10 5′ most nucleotides of the downstream exon.
A skilled reader shall appreciate that various combinations of such exemplary types of mutations as mentioned above are foreseen herein.
The recombinant attenuated BLV and related aspects as disclosed herein comprise certain mutations as specified herein. The mutations are configured such as to not affect or not detrimentally affect BLV polypeptides (e.g., the level of production and/or the amino acid sequence of such BLV polypeptides) or other products, such as miRNA (e.g., the level of production and/or the nucleic acid sequence of such miRNA), which are not specified to be mutated.
Hence, for example, a mutation in the X region of the BLV nucleic acid sequence, said mutation abolishing the production of at least one or preferably all microRNA encoded by said X region, may be configured such as to not affect or not detrimentally affect BLV polypeptides encoded by gag, pol, env, R3, G4, Tax and Rex. Particular care when introducing a mutation in the miRNA region of BLV may need to be given to not affect or not detrimentally affect BLV polypeptides encoded by env and R3, which are adjacent to the miRNA region of BLV. In another example, a mutation in G4 restricting the propagation of the BLV in vivo may be configured such as to not affect or not detrimentally affect BLV polypeptides encoded by gag, pol, env, R3, Tax and Rex, and BLV miRNAs. Particular care when introducing a mutation in G4 may need to be given to not affect or not detrimentally affect BLV polypeptides encoded by R3, Tax and Rex, which are adjacent to/overlapping with G4. In a further example, a mutation in R3 restricting the propagation of the BLV in vivo may be configured such as to not affect or not detrimentally affect BLV polypeptides encoded by gag, pol, env, G4, Tax and Rex, and BLV miRNAs. Particular care when introducing a mutation in R3 may need to be given to not affect or not detrimentally affect BLV polypeptides encoded by G4, Tax and Rex, which are adjacent to/overlapping with R3.
Notwithstanding, it shall be understood that where mutations in two or more of miRNA region, R3, and G4 are specified, such as mutations in R3 and G4, or mutations in miRNA region and R3, or mutations in miRNA region, R3 and G4, a single mutation (e.g., a single deletion) may suitably affect (span) both or all three so-specified genes or regions.
The skilled reader is well aware how mutation(s) intended herein may be configured such as to not affect or not detrimentally affect BLV polypeptides or other products, such as miRNA, which are not specified to be mutated. Preferably, the mutation(s) may be located such as not to modify the transcription, splicing, translation and amino acid sequence of such non-mutated BLV polypeptides or not to modify the transcription and nucleic acid sequence of the non-mutated miRNA. For example, in order to not modify the amino acid sequence of the non-mutated BLV polypeptides, the mutation(s) may be located such as to avoid the ORFs of the non-mutated BLV polypeptides, or if present in the ORFs, to be silent, i.e., to not produce any amino acid change in the non-mutated BLV polypeptides. For example, in order to not modify the splicing of the pre-mRNA encoding the non-mutated BLV polypeptides, the mutation(s) may be located such as to avoid sequence elements required for splicing of the pre-mRNA encoding the non-mutated BLV polypeptides. For example, in order to not modify the nucleic acid sequence of the non-mutated miRNAs, the mutation(s) may be located such as to avoid the sequence(s) encoding the non-mutated miRNAs.
Techniques for introducing mutations into nucleic acids are well-known to the skilled person and include, for example, but without limitation site-directed mutagenesis by PCR, homologous recombination, restriction enzyme digestion and ligation, etc. Standard reference works setting forth the general principles of recombinant DNA technology include Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates) (“Ausubel et al. 1992”); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990.
The BLV genome comprises long terminal repeats (LTRs) bordering the genome at its 5′ terminus and its 3′ terminus (
The term “envelope” as used herein refers to the BLV envelope encoded by the env gene of the BLV genome. The BLV envelope is a multimeric complex comprising an extracellular subunit gp51 (SU) associated with a transmembrane protein gp30 (TM) through disulfide bonds. Both subunits are glycosylated polypeptides (glycoproteins). Nucleotide sequence of the envelope gene portion coding for the gp30 glycoprotein is located from position 5518 to position 6162 (stop codon at 6160-6162,
Note that as a suitable point of reference, numbering of nucleotides or amino acids throughout the present disclosure are according to the sequence described in Rice et al. 1987 (Sequence analysis of the Bovine Leukemia Virus Genome. In A. Burney and M. Mammerickx (ed.), Enzootic bovine leukosis and bovine leukemia virus. Martinus Nijhof, Leiden, The Netherlands, pp. 115-144): nucleotide 1 is the first at the 5′ end of the R region of the 5′ long terminal repeat (LTR). A certain portion of the “Rice” sequence (nucleotides 5790 to position 7409) that may particularly aid the perusal of the present specification is reproduced in
Understandably, due to natural sequence variation occurring between various BLV strains, variants, clones, isolates and genotypes, the sequence elements and features referred to herein may be located at different positions in such other BLV than they are in the BLV sequence published by Rice et al. 1987 supra. Hence, the “Rice” numbering adopted herein is not intended to be limiting, but rather is intended to aid the perusal of this specification. The skilled person can readily determine the actual positions of the sequence elements and features referred to herein in the respective sequences of such other BLV strains, variants, clones, isolates and genotypes.
The term “ITAM” or “immunoreceptor tyrosine-based activation motif” generally refers to a conserved YXXL sequence of amino acids, wherein X represents a variable residue, and is involved in signal transduction, in particular signal transduction in immune cells. As used herein, the term specifically refers to the YXXL motifs present in the cytoplasmic tail of the transmembrane envelope protein. The C-terminal cytoplasmic tail of gp30 contains three such YXXL motifs, which are involved in signal transduction (Willems et al. 1995. J. Virol. 69: 4137-4141). In the “Rice” sequence, nucleotide sequence encoding the most N-terminal YXXL motif of gp30 is located from position 6073 to position 6084 of (
The term “R3 polypeptide” refers herein to the accessory protein R3 which might have a regulatory function of viral expression, in particular by inhibiting the post-transcriptional regulator of viral expression Rex (Alexandersen et al. 1993. J. Virol. 67: 39-52). The R3 gene and R3 pre-mRNA contain 3 exons (herein consecutively numbered from 5′ as exon 1, 2, 3), which are present in R3 mRNA, and two intervening introns (herein consecutively numbered from 5′ as intron 1, 2), which are spliced out of R3 mRNA (
The terms “microRNA” or “miRNA” generally refer to short RNA molecules of 22 nucleotides on average. They are generally involved in post-transcriptional regulation of gene expression through binding to complementary sequences on target messenger RNA transcripts, usually resulting in translational repression or target degradation and gene silencing. They are often implicated in disease states, including cancer. As used herein, the term specifically refers to the miRNAs encoded by the BLV genome. The miRNA encoding region is located in the X region of the BLV genome (as noted previously, X region defines the region between the 3′ end of the env gene and the 3′ LTR), in particular between the 3′ end of the env gene and the start of the R3 ORF region located in the X region (Cullen, 2012. PNAS 109: 2695-2696). In the “Rice” sequence, the miRNA encoding region may be deemed as located from 6163 to position 6812.
By means of further guidance, Kincaid et al. 2012 (Proc Natl Acad Sci USA 109(8): 3077-82) has recently mapped eight BLV-encoded miRNA sequences—annotated as BLV-mir-B1-3p, BLV-mir-B2-5p, BLV-mir-B2-3p, BLV-mir-B3-5p, BLV-mir-B3-3p, BLV-mir-B4-3p, BLV-mir-B5-5p, BLV-mir-B5-3p—to the above-mentioned miRNA encoding region of the BLV nucleic acid sequence. Kincaid et al. 2012 proposed the following consensus sequences for these miRNA's:
Rosewick et al. 2013 (Proc Natl Acad Sci USA, PMID: 23345446) confirmed these findings and further identified BLV-mir-B1-5p and BLV-mir-B4-5p, with the following consensus sequences:
Rosewick et al. 2013 further determined that the BLV miRNAs resulted from the transcription of five independent transcriptional units encoding five hairpin structures in the BLV miRNA encoding region.
The terms “Tax polypeptide” and “Rex polypeptide” refer herein to the regulatory proteins Tax and Rex. Tax, the transactivating protein, stimulates the 5′ long terminal repeat to promote viral transcription and may be involved in tumorigenesis. Rex is involved in the transition from early expression of regulatory proteins to later expression of viral structural proteins. The Tax/Rex gene and Tax/Rex pre-mRNA contain 3 exons (herein consecutively numbered from 5′ as exon 1, 2, 3), which are present in Tax/Rex mRNA, and two intervening introns (herein consecutively numbered from 5′ as intron 1, 2), which are spliced out of Tax/Rex mRNA (
As noted, the recombinant attenuated BLV and related aspects as disclosed herein may comprise a mutation in the nucleic acid sequence encoding the most N-terminal YXXL signalling motif of the cytoplasmic domain of the transmembrane subunit (TM) of the envelope protein, said mutation disrupting the signal transduction activity of the motif.
To assess the signal transduction activity of the YXXL motif, calcium responses and cytokine production may be analysed in a lymphoid cell line, such as a B or T cell line, which has been stably transfected with a chimeric molecule comprising the extracellular and transmembrane portions of CD8 fused to the cytoplasmic tail of TM, in response to an anti-CD8 antibody as described in Beaufils et al. (1993. EMBO J. 12: 5105-5112).
In preferred embodiments, the mutation in the most N-terminal YXXL signalling motif of the cytoplasmic domain of the transmembrane subunit (TM) of the envelope protein disrupting the signal transduction activity of the motif is a substitution of the tyrosine residue of the motif, i.e., the tyrosine residue at position 186 of the BLV TM protein. The tyrosine residue may be substituted by any other amino acid residue, preferably by any other naturally occurring amino acid residue, more preferably wherein such residue does not comprise a hydroxyl moiety. Particularly suitable substitutions of the tyrosine residue include substitutions of the tyrosine residue with alanine or aspartic acid residues, preferably with aspartic acid residue (i.e., Y186D, resulting in the motif DXXL).
Accordingly, in preferred embodiments the mutation in the most N-terminal YXXL signalling motif of the cytoplasmic domain of the transmembrane subunit (TM) of the envelope protein as intended herein is a mutation of the TAT codon at positions 6073-6075 of the nucleic acid encoding BLV into a codon encoding an amino acid residue other than tyrosine, i.e., a codon other than TAT and TAC, preferably into a codon encoding alanine (GCT, GCC, GCA, or GCG) or aspartic acid (GAT or GAC) residues, preferably into a codon encoding aspartic acid residue (GAT or GAC). In certain embodiments, the mutation may be a missense point mutation (i.e., a mutation of a single nucleotide changing the amino acid encoding by the codon), in a particularly preferred example a point mutation of the T nucleotide at position 6073 of the nucleic acid encoding BLV, preferably BLV provirus, to a G nucleotide (i.e., TAT→GAT, resulting in Tyr→Asp).
As noted, the recombinant attenuated BLV and related aspects as disclosed herein may comprise a mutation in G4 restricting the propagation of the BLV in vivo.
As also noted, the recombinant attenuated BLV and related aspects as disclosed herein may comprise a mutation in R3 restricting the propagation of the BLV in vivo.
In these contexts, the phrase “restricting the propagation of the BLV in vivo” denotes that a BLV virus carrying the mutation in G4 or in R3, or in both G4 and R3, exhibits reduced propagation in a host, i.e., in vivo, compared to a reference BLV virus which is otherwise identical but does not comprise the mutation in G4 or in R3, or in both G4 and R3, respectively, preferably compared to a reference BLV virus which is otherwise identical but comprises wild-type G4 or R3, or both G4 and R3, respectively. The host may be as defined elsewhere in this specification, such as particularly cattle, such as more particularly a cow. The propagation of the virus in the host may be at least about 2 fold, e.g., at least about 5 fold, or preferably at least about 10 fold, e.g., at least about 20 fold, or more preferably at least about 50 fold, e.g., at least about 100 fold or less than that of the reference virus. Suitable methods for measuring the propagation of a virus, in particular BLV, in the host include without limitation determining the proviral loads in the challenged host. For example, the number of BLV proviral copies may be determined using a suitable methodology, e.g., quantitative PCR, per a given number, e.g., 100, of peripheral blood mononuclear cells at a given time or times, i.e., in function of time, following the challenge of the host. See Example 3 for a specific, non-limiting application of this approach.
In certain embodiments, the mutation in G4 restricting the propagation of the BLV in vivo may abolish the production of G4 polypeptide.
In certain other embodiments, the mutation in G4 restricting the propagation of the BLV in vivo may result in production of a C-terminally truncated G4 polypeptide lacking at least 20 C-terminal amino acids of G4 polypeptide (e.g., ≥21, ≥22, ≥23, ≥24, ≥25, ≥26, ≥27, ≥28, ≥29) or may result in production of a C-terminally truncated G4 polypeptide lacking at least 30 C-terminal amino acids of G4 polypeptide (e.g., ≥31, ≥32, ≥33, ≥34, ≥35, ≥36, ≥37, ≥38, ≥39), such as may result in production of a C-terminally truncated G4 polypeptide lacking between about 30 and about 40, e.g., between about 33 and about 47, e.g., about 35 C-terminal amino acids of G4 polypeptide.
In certain other embodiments, the mutation in G4 restricting the propagation of the BLV in vivo may inactivate G4 polypeptide such as to at least abolish the oncogenic potential of G4 polypeptide. The oncogenic potential of G4 may be assessed through testing its transforming potential in vitro. For example, tumour formation may be examined in immunocompromised mice, such as, e.g., thymus-less nude mice, injected with embryonic cells, such as, e.g., rat embryonic fibroblasts, that have been co-transfected with nucleic acid encoding BLV G4 and an expression vector comprising an oncogene, preferably Ha-ras (Kerkhofs et al. 1998. J. Virol. 72: 2554-2559).
Care when introducing a mutation in G4 may need to be given to not affect or not detrimentally affect R3 (where R3 mutation as taught herein is not specified), Tax and Rex, as explained elsewhere in this specification. Hence, the mutation is compatible with production of functional Tax and Rex proteins.
A suitable mutation in G4 restricting the propagation of the BLV in vivo may be located in any exon (e.g., exon 1 or 2) and/or intron 1 of G4. For example, the mutation may be located in any exon (e.g., exon 1 or 2) of G4. In a further example, the mutation may be located in G4 ORF, such as in the portion of G4 ORF present in exon 1 or in the portion of G4 ORF present in exon 2.
Without limitation, a suitable mutation in G4 restricting the propagation of the BLV in vivo may be located in exon 1 of G4, preferably in the portion of G4 ORF present in exon 1. For example, a premature in-frame stop codon or a frame shift mutation introduced in the portion of G4 ORF present in exon 1 would abolish production of G4. Such mutation does not affect or does not detrimentally affect the function of the 5′ LTR or the production of other BLV polypeptides or products.
Also without limitation, a suitable mutation in G4 restricting the propagation of the BLV in vivo may be located in the splice donor site of intron 1 of G4 and may abolish native splicing of G4 pre-mRNA and thereby abolish production of G4 polypeptide, e.g., a deletion comprising or consisting of a deletion of said splice donor site of intron 1 of G4.
Preferably, the mutation in G4 restricting the propagation of the BLV in vivo may be located in the X region of the BLV nucleic acid sequence. In particular, the portions of G4 present in the X region of the BLV nucleic acid sequence include a 3′ portion of intron 1 and exon 2.
Without limitation, a suitable mutation in G4 restricting the propagation of the BLV in vivo may be located in the splice acceptor site of intron 1 of G4 and may abolish native splicing of G4 pre-mRNA and thereby abolish production of G4 polypeptide, e.g., a deletion comprising or consisting of a deletion of said splice acceptor site of intron 1 of G4.
Also without limitation, a suitable mutation in G4 restricting the propagation of the BLV in vivo may be located in exon 2 of G4, preferably in the portion of G4 ORF present in exon 2. For example, a premature in-frame stop codon, a frame shift mutation, a deletion or a substitution introduced in the portion of G4 ORF present in exon 2 could produce C-terminally truncated G4 polypeptide or G4 polypeptide with altered amino acid sequence having diminished or abolished biological function(s), such as for example at least abolished oncogenic potential, or could abolish the production of G4.
Particular care especially when introducing a mutation in G4 in the X region of the BLV nucleic acid sequence may need to be given to not affect or not detrimentally affect the miRNA region (where miRNA mutation as taught herein is not specified), R3 (where R3 mutation as taught herein is not specified), Tax and Rex, as explained elsewhere in this specification. Hence, the mutation is compatible with production of functional Tax and Rex proteins.
More preferably, the mutation in G4 restricting the propagation of the BLV in vivo may be located in the region of the BLV nucleic acid sequence between the stop codon of R3 and the splice acceptor site of intron 2 of Tax/Rex. Advantageously, mutating this portion of G4 can ensure that no detrimental changes are introduced into the R3 (where R3 mutation as taught herein is not specified), Tax and Rex. This region corresponds to positions 6897 to 7039 according to the “Rice” sequence numbering, starting at the first nucleotide downstream of the R3 stop codon located at 6894-6896 and extending to nucleotide −3 of the intron 2-exon 3 boundary of Tax/Rex at position 7039, i.e., excluding the last two nucleotides of intron 2 of Tax/Rex at positions 7040-7041.
Without limitation, a premature in-frame stop codon, a frame shift mutation, a deletion or a substitution introduced in the region of the BLV nucleic acid sequence between the stop codon of R3 and the splice acceptor site of intron 2 of Tax/Rex could produce C-terminally truncated G4 polypeptide or G4 polypeptide with altered amino acid sequence having diminished or abolished biological function(s), such as for example at least abolished oncogenic potential, or could abolish the production of G4.
Also without limitation, a deletion in G4 may remove a sizeable portion of the region of the BLV nucleic acid sequence between the stop codon of R3 and the splice acceptor site of intron 2 of Tax/Rex, such as, e.g., about 50% or more, preferably about 60% or more, more preferably about 70% or more, even more preferably about 80% or more, still more preferably about 90% or more of the nucleotides constituting this region. Hence, without limitation, a deletion in G4 may remove a sizeable portion of the region of the BLV nucleic acid sequence between the stop codon of R3 and the splice acceptor site of intron 2 of Tax/Rex, such as, e.g., about 70 nucleotides or more, preferably about 90 nucleotides or more, more preferably about 110 nucleotides or more, even more preferably about 130 nucleotides or more, of the nucleotides constituting this region. This could produce C-terminally truncated or internally deleted G4 polypeptide having diminished or abolished biological function(s), such as for example at least abolished oncogenic potential, or could abolish the production of G4.
Preferably, the mutation in G4 restricting the propagation of the BLV in vivo may comprise or consist of an insertion of an in-frame stop codon in the G4 open reading frame. This can produce C-terminally truncated G4 polypeptide having diminished or abolished biological function(s), such as for example at least abolished oncogenic potential, or can abolish the production of G4.
Particularly preferably, the mutation in G4 restricting the propagation of the BLV in vivo may comprise or consist of an insertion of an in-frame stop codon in the G4 open reading frame in the region of the BLV nucleic acid sequence between the stop codon of R3 and the splice acceptor site of intron 2 of Tax/Rex. This can produce C-terminally truncated G4 polypeptide having diminished or abolished biological function(s), such as for example at least abolished oncogenic potential, or can abolish the production of G4.
In exemplary non-limiting embodiments, an in-frame stop codon may be introduced, with reference to the “Rice” sequence numbering, between positions 6947 and 7037, such as between positions 6957 and 7037, such as particularly between positions 6967 and 7027, such as more particularly between positions 6977 and 7017, such as even more particularly between positions 6987 and 7007, such as at about position 6997 of the BLV nucleic acid sequence.
In certain embodiments, the mutation in R3 restricting the propagation of the BLV in vivo may abolish the production of R3 polypeptide.
Care when introducing a mutation in R3 may need to be given to not affect or not detrimentally affect G4 (where G4 mutation as taught herein is not specified), Tax and Rex, as explained elsewhere in this specification. Hence, the mutation is compatible with production of functional Tax and Rex proteins.
A suitable mutation in R3 restricting the propagation of the BLV in vivo may be located in any exon (e.g., exon 1, 2 or 3) and/or any intron (e.g., intron 1 or 2) of R3. For example, the mutation may be located in any exon (e.g., exon 1, 2 or 3) of R3. In a further example, the mutation may be located in R3 ORF, such as in the portion of R3 ORF present in exon 2 or in the portion of R3 ORF present in exon 3.
Because exon 1 and 2 of R3 are common with the Tax/Rex mRNA, and the portion of R3 ORF present in exon 2 of R3 is identical to that of Rex, a suitable mutation in R3 restricting the propagation of the BLV may be advantageously located in the 3′ portion of intron 2 of R3 or in exon 3 of R3. However, as noted already, mutations in exon 1 or 2 of R3 and in intron 1 or in the 5′ portion of intron 2 of R3 that are compatible with production of functional Tax and Rex proteins are also possible and contemplated herein.
Preferably, the mutation in R3 restricting the propagation of the BLV in vivo may be located in the X region of the BLV nucleic acid sequence. In particular, the portions of R3 present in the X region of the BLV nucleic acid sequence include a 3′ portion of intron 2 and exon 3.
Without limitation, a suitable mutation in R3 restricting the propagation of the BLV in vivo may be located in the splice acceptor site of intron 2 of R3 and may abolish native splicing of R3 pre-mRNA and thereby abolish production of R3 polypeptide, e.g., a deletion comprising or consisting of a deletion of said splice acceptor site of intron 2 of R3.
Also without limitation, a suitable mutation in R3 restricting the propagation of the BLV in vivo may be located in exon 3 of R3, preferably in the portion of R3 ORF present in exon 3. For example, a premature in-frame stop codon, a frame shift mutation, a deletion or a substitution introduced in the portion of R3 ORF present in exon 3 could produce C-terminally truncated R3 polypeptide or R3 polypeptide with altered amino acid sequence having diminished or abolished biological function(s) or could abolish the production of R3.
Particular care especially when introducing a mutation in R3 in the X region of the BLV nucleic acid sequence may need to be given to not affect or not detrimentally affect the miRNA region (where miRNA mutation as taught herein is not specified), G4 (where G4 mutation as taught herein is not specified), Tax and Rex, as explained elsewhere in this specification. Hence, the mutation is compatible with production of functional Tax and Rex proteins.
For example, the mutation in R3 restricting the propagation of the BLV in vivo may be located in the region of the BLV nucleic acid sequence between the end of the miRNA encoding region and the splice acceptor site of intron 1 of G4, e.g., in the region of the BLV nucleic acid sequence between about 250 nucleotides upstream of the splice acceptor site of intron 2 of R3 and the splice acceptor site of intron 1 of G4).
Advantageously, mutating this portion of R3 can ensure that no detrimental changes are introduced into the miRNA region (where miRNA mutation as taught herein is not specified), G4 (where G4 mutation as taught herein is not specified), Tax and Rex.
For example, a premature in-frame stop codon, a frame shift mutation, a deletion or a substitution introduced in the portion of R3 ORF present in exon 3 upstream of the splice acceptor site of intron 1 of G4 (i.e., the region corresponding to positions 6813 to 6858 according to the “Rice” sequence numbering, starting at the first nucleotide of exon 3 of R3 stop codon located at 6813 and extending to nucleotide −3 of the intron 1-exon 2 boundary of G4 at 6858, i.e., excluding the last two nucleotides of intron 1 of G4 at positions 6859-6860) could produce C-terminally truncated R3 polypeptide or R3 polypeptide with altered amino acid sequence having diminished or abolished biological function(s) or could abolish the production of R3.
For example, a deletion in R3 may remove a sizeable portion of the portion of R3 ORF present in exon 3 upstream of the splice acceptor site of intron 1 of G4, i.e., the region corresponding to positions 6813 to 6858 according to the “Rice” sequence numbering. A sizeable portion of this region may be for example about 50% or more, preferably about 60% or more, more preferably about 70% or more, even more preferably about 80% or more, still more preferably about 90% or more of the nucleotides constituting this region. This could produce C-terminally truncated or internally deleted R3 polypeptide having diminished or abolished biological function(s) or could abolish the production of R3.
Preferably, the mutation in R3 restricting the propagation of the BLV in vivo may abolish splicing at the intron 2-exon 3 boundary of R3 pre-messenger RNA. Hereby, native splicing of R3 pre-mRNA and production of R3 polypeptide can be abolished. Any mutation involving the splice acceptor site of intron 2 of R3 is contemplated herein. Preferably, the mutation may comprise or consist of a deletion of the splice acceptor site of intron 2 of R3.
Particularly preferably, the mutation in R3 restricting the propagation of the BLV in vivo may be a deletion of at least a portion of the region of the BLV nucleic acid sequence between the end of the miRNA encoding region and the splice acceptor site of intron 1 of G4, more particularly between about 250 nucleotides upstream of the intron 2-exon 3 boundary of R3 and the splice acceptor site of intron 1 of G4, wherein the mutation abolishes splicing at the intron 2-exon 3 boundary of R3 pre-messenger RNA, as explained above. Advantageously, mutating this portion of R3 can ensure that no detrimental changes are introduced into the miRNA region (where miRNA mutation as taught herein is not specified), G4 (where G4 mutation as taught herein is not specified), Tax and Rex.
For example, the 5′ boundary of the deletion may be located between about 250 nucleotides upstream of the intron 2-exon 3 boundary of R3 and at the intron 2-exon 3 boundary of R3, or between about 250 and about 10 nucleotides upstream of the intron 2-exon 3 boundary of R3, or between about 250 and about 50 nucleotides upstream of the intron 2-exon 3 boundary of R3, or between about 250 and about 100 nucleotides upstream of the intron 2-exon 3 boundary of R3. For example, the 5′ boundary of the deletion may be located between about 249 and about 149, or between about 239 and about 159, or between about 229 and about 169, or between about 219 and about 179, or between about 209 and about 189, or at about 199 nucleotides upstream of the intron 2-exon 3 boundary of R3.
Any of such exemplary 5′ boundaries of the deletion listed in the previous paragraph may be combined with a 3′ boundary of the deletion located between about 45 and about 3 nucleotides upstream of the intron 1-exon 2 boundary of G4, or any of such exemplary 5′ boundaries of the deletion listed in the previous paragraph may be combined with a 3′ boundary of the deletion located between about 33 and about 3 nucleotides upstream of the intron 1-exon 2 boundary of G4, or any of such exemplary 5′ boundaries of the deletion listed in the previous paragraph may be combined with a 3′ boundary of the deletion located between about 23 and about 3 nucleotides upstream of the intron 1-exon 2 boundary of G4, or any of such exemplary 5′ boundaries of the deletion listed in the previous paragraph may be combined with a 3′ boundary of the deletion at about 13 nucleotides upstream of the intron 1-exon 2 boundary of G4.
For example, the 5′ boundary of the deletion may be located between about 249 and about 149, or preferably between about 239 and about 159, or more preferably between about 229 and about 169, or even more preferably between about 219 and about 179, or still more preferably between about 209 and about 189, or particularly preferably at about 199 nucleotides upstream of the intron 2-exon 3 boundary of R3, and the 3′ boundary of the deletion may be located between about 45 and about 3, or preferably between about 33 and about 3, or more preferably between about 23 and about 3, or still more preferably at about 13 nucleotides upstream of the intron 1-exon 2 boundary of G4.
In a particular example, the 5′ boundary of the deletion may be located between about 209 and about 189, e.g., at about 209, nucleotides upstream of the intron 2-exon 3 boundary of R3 and the 3′ boundary of the deletion may located between about 23 and about 3, e.g., at about 13, nucleotides upstream of the intron 1-exon 2 boundary of G4.
In further preferred embodiments, the 5′ boundary of the deletion may be located between positions 6564 and 6664, or preferably between positions 6574 and 6654, or more preferably between positions 6584 and 6644, or even more preferably between positions 6594 and 6634, or still more preferably between positions 6604 and 6624, or particularly preferably at about position 6614 of the BLV nucleic acid sequence according to “Rice” sequence numbering and the 3′ boundary of the deletion may be located between positions 6816 and 6858, or preferably between positions 6828 and 6858, or more preferably between positions 6838 and 6858, or still more preferably at about position 6828 of the BLV nucleic acid sequence according to “Rice” sequence numbering. In a particular example, the 5′ boundary of the deletion may be located between positions 6604 and 6624, e.g., at about position 6614, of the BLV nucleic acid sequence and the 3′ boundary of the deletion may be located between positions 6838 and 6858, e.g., at about position 6848, of the BLV nucleic acid sequence.
In certain embodiments, the recombinant BLV may comprise the mutation in both G4 and R3, said mutation restricting the propagation of the BLV in vivo.
In preferred embodiments, the mutation in both G4 and R3 may abolish the production of both G4 and R3 polypeptides. Preferably, the mutation may be located in the X region of the BLV nucleic acid sequence.
Care when introducing a mutation in both G4 and R3, particularly when introducing such mutation in the X region of the BLV nucleic acid sequence, may need to be given to not affect or not detrimentally affect the miRNA region (where miRNA mutation as taught herein is not specified), Tax and Rex, as explained elsewhere in this specification. Hence, the mutation is compatible with production of functional Tax and Rex proteins.
In certain embodiments, the recombinant BLV may comprise the mutation in G4 restricting the propagation of the BLV in vivo, and the mutation in R3 restricting the propagation of the BLV in vivo, wherein said mutations abolish splicing at the intron 2-exon 3 boundary of R3 pre-messenger RNA and at the intron 1-exon 2 boundary of G4 pre-messenger RNA. Hereby, native splicing of R3 pre-mRNA and production of R3 polypeptide and native splicing of G4 pre-mRNA and production of G4 polypeptide can be abolished. Any mutation involving the splice acceptor site of intron 2 of R3 and any mutation involving the splice acceptor site of intron 1 of G4 is contemplated herein. Preferably, the mutations may comprise or consist of a deletion of the splice acceptor site of intron 2 of R3 and a deletion of the splice acceptor site of intron 1 of G4.
Particularly preferably, said mutations in G4 and in R3 restricting the propagation of the BLV in vivo may be a deletion of at least a portion of the region of the BLV nucleic acid sequence between the end of the miRNA encoding region and the splice acceptor site of intron 2 of Tax/Rex, more particularly between about 250 nucleotides upstream of the intron 2-exon 3 boundary of R3 and the splice acceptor site of intron 2 of Tax/Rex, whereby the splice acceptor site of intron 2 of R3 and the splice acceptor site of intron 1 of G4 are deleted, such that the mutations abolish splicing at the intron 2-exon 3 boundary of R3 pre-messenger RNA and at the intron 1-exon 2 boundary of G4 pre-messenger RNA, as explained above.
For example, the 5′ boundary of the deletion may be located between about 250 nucleotides upstream of the intron 2-exon 3 boundary of R3 and at the intron 2-exon 3 boundary of R3, or between about 250 and about 10 nucleotides upstream of the intron 2-exon 3 boundary of R3, or between about 250 and about 50 nucleotides upstream of the intron 2-exon 3 boundary of R3, or between about 250 and about 100 nucleotides upstream of the intron 2-exon 3 boundary of R3. For example, the 5′ boundary of the deletion may be located between about 249 and about 149, or between about 239 and about 159, or between about 229 and about 169, or between about 219 and about 179, or between about 209 and about 189, or at about 199 nucleotides upstream of the intron 2-exon 3 boundary of R3.
Any of such exemplary 5′ boundaries of the deletion listed in the previous paragraph may be combined with a 3′ boundary of the deletion located between about 178 and about 3 nucleotides upstream of the intron 2-exon 3 boundary of Tax/Rex, or any of such exemplary 5′ boundaries of the deletion listed in the previous paragraph may be combined with a 3′ boundary of the deletion located between about 100 and about 3 nucleotides upstream of the intron 2-exon 3 boundary of Tax/Rex, or any of such exemplary 5′ boundaries of the deletion listed in the previous paragraph may be combined with a 3′ boundary of the deletion located between about 55 and about 35 nucleotides upstream of the intron 2-exon 3 boundary of Tax/Rex, or any of such exemplary 5′ boundaries of the deletion listed in the previous paragraph may be combined with a 3′ boundary of the deletion at about 45 nucleotides upstream of the intron 2-exon 3 boundary of Tax/Rex.
For example, the 5′ boundary of the deletion may be located between about 249 and about 149, or preferably between about 239 and about 159, or more preferably between about 229 and about 169, or even more preferably between about 219 and about 179, or still more preferably between about 209 and about 189, or particularly preferably at about 199 nucleotides upstream of the intron 2-exon 3 boundary of R3, and the 3′ boundary of the deletion may be located between about 178 and about 3, or preferably between about 100 and about 3, or more preferably between about 55 and about 35, or still more preferably at about 45 nucleotides upstream of the intron 2-exon 3 boundary of Tax/Rex.
In a particular example, the 5′ boundary of the deletion may be located between about 209 and about 189, e.g., at about 209, nucleotides upstream of the intron 2-exon 3 boundary of R3 and the 3′ boundary of the deletion may located between about 55 and about 35, e.g., at about 45, nucleotides upstream of the intron 2-exon 3 boundary of Tax/Rex.
In further preferred embodiments, the 5′ boundary of the deletion may be located between positions 6564 and 6664, or preferably between positions 6574 and 6654, or more preferably between positions 6584 and 6644, or even more preferably between positions 6594 and 6634, or still more preferably between positions 6604 and 6624, or particularly preferably at about position 6614 of the BLV nucleic acid sequence according to “Rice” sequence numbering and the 3′ boundary of the deletion may be located between positions 6957 and 7037, or preferably between positions 6967 and 7027, or more preferably between positions 6977 and 7017, or even more preferably between positions 6987 and 7007, or still more preferably at about position 6997 of the BLV nucleic acid sequence according to “Rice” sequence numbering. In a particular example, the 5′ boundary of the deletion may be located between positions 6604 and 6624, e.g., at about position 6614, of the BLV nucleic acid sequence and the 3′ boundary of the deletion may be located between positions 6987 and 7007, e.g., at about position 6997, of the BLV nucleic acid sequence.
As noted, the recombinant attenuated BLV and related aspects as disclosed herein may comprise a mutation in the X region of the BLV nucleic acid sequence abolishing the production of at least one or preferably all microRNA encoded by said X region. Any mutations, including deletions, insertions and/or substitutions, abolishing the production of at least one or preferably all microRNA encoded by said X region are contemplated herein.
By means of example and not limitation, the mutation in the X region of the BLV nucleic acid sequence abolishing the production of at least one or preferably all microRNA encoded by said X region may abolish the production of—in order of increasing preference—one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten BLV-encoded miRNA selected from the group consisting of BLV-mir-B1-5p, BLV-mir-B1-3p, BLV-mir-B2-5p, BLV-mir-B2-3p, BLV-mir-B3-5p, BLV-mir-B3-3p, BLV-mir-B4-5p, BLV-mir-B4-3p, BLV-mir-B5-5p, BLV-mir-B5-3p, as defined elsewhere in this specification.
Preferably, the mutation in the X region of the BLV nucleic acid sequence abolishing the production of at least one or preferably all microRNA encoded by said X region may be a deletion of at least a portion of the region of the BLV nucleic acid sequence between the stop codon of the transmembrane subunit (TM, gp30 glycoprotein) of the envelope protein and the splice acceptor site of intron 2 of R3. This region contains the nucleic acid encoding the miRNAs and may be suitably denoted as miRNA region or miRNA encoding region herein.
For example, the mutation in the X region of the BLV nucleic acid sequence abolishing the production of at least one or preferably all microRNA encoded by said X region may be a deletion of at least a portion of the region of the BLV nucleic acid sequence between the stop codon of TM and about 10 or about 20 or about 30 or about 40 or about 50 or about 60 or about 70 or about 80 or about 90 or about 100 or about 150 or about 200 nucleotides upstream of the intron 2-exon 3 boundary of R3.
For example, the 5′ boundary of the deletion may be located between about 1 and about 50, preferably between about 1 and about 40, more preferably between about 1 and about 30, even more preferably between about 1 and about 20, still more preferably between about 1 and about 11, such as at about 7, nucleotides downstream of the stop codon of TM and the 3′ boundary of the deletion may be located between about 200 and about 3, preferably between about 132 and about 32, more preferably between about 122 and about 42, even more preferably between about 112 and about 52, still more preferably between about 102 and about 62, yet more preferably between about 92 and about 72 nucleotides upstream of the intron 2-exon 3 boundary of R3.
In further preferred embodiments, the 5′ boundary of the deletion may be located between positions 6163 and 6213, or preferably between positions 6163 and 6203, or more preferably between positions 6163 and 6193, or even more preferably between positions 6163 and 6183, or still more preferably between positions 6163 and 6173, or particularly preferably at about position 6169 of the BLV nucleic acid sequence according to “Rice” sequence numbering and the 3′ boundary of the deletion may be located between positions 6681 and 6781, or preferably between positions 6691 and 6771, or more preferably between positions 6701 and 6761, or even more preferably between positions 6711 and 6751, or still more preferably between positions 6721 and 6741, or particularly preferably at about position 6731 of the BLV nucleic acid sequence according to “Rice” sequence numbering. In a particular example, the 5′ boundary of the deletion may be located between positions 6163 and 6173, e.g., at about position 6169, of the BLV nucleic acid sequence and the 3′ boundary of the deletion may be located between positions 6721 and 6741, e.g., at about position 6731, of the BLV nucleic acid sequence.
In certain embodiments, the recombinant BLV may comprise a mutation in both G4 and R3, said mutation restricting the propagation of the BLV in vivo, and a mutation in the X region of the BLV nucleic acid sequence abolishing the production of at least one or preferably all microRNA encoded by said X region. In preferred embodiments, the mutation in both G4 and R3 may abolish the production of both G4 and R3 polypeptides; preferably, the mutation may be located in the X region of the BLV nucleic acid sequence. Hence, in certain embodiments, the recombinant BLV may comprise a mutation in the X region of the BLV nucleic acid sequence abolishing the production of both G4 and R3 and abolishing the production of at least one or preferably all microRNA encoded by said X region.
Care when introducing a mutation in miRNA, G4 and R3, particularly when the mutation in both G4 and R3 is in the X region of the BLV nucleic acid sequence, may need to be given to not affect or not detrimentally affect Tax and Rex, as explained elsewhere in this specification. Hence, the mutation is compatible with production of functional Tax and Rex proteins.
In certain embodiments, the recombinant attenuated BLV may comprise the mutation in G4 restricting the propagation of the BLV in vivo, and the mutation in R3 restricting the propagation of the BLV in vivo, and the mutation in the X region of the BLV nucleic acid sequence abolishing the production of at least one or preferably all microRNA encoded by said X region, wherein said mutations are a deletion of at least a portion of the region of the BLV nucleic acid sequence between the stop codon of TM and the splice acceptor site of intron 2 of Tax/Rex, whereby the splice acceptor site of intron 2 of R3 and the splice acceptor site of intron 1 of G4 are deleted. In further embodiments, such recombinant attenuated BLV may further comprise the mutation in the nucleic acid sequence encoding the most N-terminal YXXL signalling motif of the cytoplasmic domain of the transmembrane subunit (TM) of the envelope protein, said mutation disrupting the signal transduction activity of the motif, as disclosed herein.
For example, the 5′ boundary of the deletion may be located between about 1 and about 50, preferably between about 1 and about 40, more preferably between about 1 and about 30, even more preferably between about 1 and about 20, still more preferably between about 1 and about 11, such as at about 7, nucleotides downstream of the stop codon of TM.
Any of such exemplary 5′ boundaries of the deletion listed in the previous paragraph may be combined with a 3′ boundary of the deletion located between about 178 and about 3 nucleotides upstream of the intron 2-exon 3 boundary of Tax/Rex, or any of such exemplary 5′ boundaries of the deletion listed in the previous paragraph may be combined with a 3′ boundary of the deletion located between about 100 and about 3 nucleotides upstream of the intron 2-exon 3 boundary of Tax/Rex, or any of such exemplary 5′ boundaries of the deletion listed in the previous paragraph may be combined with a 3′ boundary of the deletion located between about 55 and about 35 nucleotides upstream of the intron 2-exon 3 boundary of Tax/Rex, or any of such exemplary 5′ boundaries of the deletion listed in the previous paragraph may be combined with a 3′ boundary of the deletion at about 45 nucleotides upstream of the intron 2-exon 3 boundary of Tax/Rex.
For example, the 5′ boundary of the deletion may be located between about 1 and about 50, preferably between about 1 and about 40, more preferably between about 1 and about 30, even more preferably between about 1 and about 20, still more preferably between about 1 and about 11, such as at about 7, nucleotides downstream of the stop codon of TM, and the 3′ boundary of the deletion may be located between about 178 and about 3, or preferably between about 100 and about 3, or more preferably between about 55 and about 35, or still more preferably at about 45 nucleotides upstream of the intron 2-exon 3 boundary of Tax/Rex.
In a particular example, the 5′ boundary of the deletion may be located between about 1 and about 11, e.g., at about 7, nucleotides downstream of the stop codon of TM and the 3′ boundary of the deletion may located between about 55 and about 35, e.g., at about 45, nucleotides upstream of the intron 2-exon 3 boundary of Tax/Rex.
In further preferred embodiments, the 5′ boundary of the deletion may be located between positions 6163 and 6213, or preferably between positions 6163 and 6203, or more preferably between positions 6163 and 6193, or even more preferably between positions 6163 and 6183, or still more preferably between positions 6163 and 6173, or particularly preferably at about position 6169 of the BLV nucleic acid sequence according to “Rice” sequence numbering and the 3′ boundary of the deletion may be located between positions 6957 and 7037, or preferably between positions 6967 and 7027, or more preferably between positions 6977 and 7017, or even more preferably between positions 6987 and 7007, or still more preferably at about position 6997 of the BLV nucleic acid sequence according to “Rice” sequence numbering.
In a particular example, the 5′ boundary of the deletion may be located between positions 6163 and 6173, e.g., at about position 6169, of the BLV nucleic acid sequence and the 3′ boundary of the deletion may be located between positions 6987 and 7007, e.g., at about position 6997, of the BLV nucleic acid sequence.
In certain embodiments, the invention provides the recombinant attenuated BLV as taught herein, preferably wherein the BLV is BLV isolate 344, and wherein one of the following applies:
As already noted, aspect of the invention provides the recombinant attenuated BLV encoded by the plasmid as deposited under the Budapest Treaty with the Belgian Coordinated Collections of Microorganisms BCCM™/LMBP Collection under accession number LMBP 8166 on Feb. 5, 2013 (see Table 2B). This encodes the BLV6073DX provirus as described in the experimental section.
As already noted, aspect of the invention provides the recombinant attenuated BLV encoded by the plasmid as deposited under the Budapest Treaty with the Belgian Coordinated Collections of Microorganisms BCCM/LMBP Collection under accession number LMBP 8166 on Feb. 5, 2013 (see Table 2B). This encodes the BLV6073DX provirus as described in the experimental section.
As also already noted, aspect of the invention provides the recombinant attenuated BLV encoded by the plasmid as deposited under the Budapest Treaty with the Belgian Coordinated Collections of Microorganisms BCCM/LMBP Collection under accession number LMBP 8167 on Feb. 5, 2013 (see Table 2C). This encodes the BLVGPDX provirus as described in the experimental section.
As also already noted, aspect of the invention provides the recombinant attenuated BLV encoded by the plasmid as deposited under the Budapest Treaty with the Belgian Coordinated Collections of Microorganisms BCCM/LMBP Collection under accession number LMBP 8713 on Oct. 25, 2013 (see Table 2D). This encodes the BLV6073GPDX provirus as described in the experimental section.
A further aspect provides a recombinant nucleic acid encoding the recombinant attenuated BLV as disclosed herein.
By “nucleic acid” is meant oligomers and polymers of any length composed essentially of nucleotides, e.g., deoxyribonucleotides and/or ribonucleotides. Nucleic acids can comprise purine and/or pyrimidine bases and/or other natural (e.g., xanthine, inosine, hypoxanthine), chemically or biochemically modified (e.g., methylated), non-natural, or derivatised nucleotide bases. The backbone of nucleic acids can comprise sugars and phosphate groups, as can typically be found in RNA or DNA, and/or one or more modified or substituted sugars and/or one or more modified or substituted phosphate groups.
Modifications of phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. A “nucleic acid” can be for example double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear. The term “nucleic acid” as used herein preferably encompasses DNA and RNA, specifically including RNA, genomic RNA, cDNA, DNA, provirus, pre-mRNA and mRNA.
The term “oligonucleotide” as used herein refers to a nucleic acid oligomer or polymer as defined herein. Preferably, an oligonucleotide is (substantially) single-stranded. Oligonucleotides as intended herein may be preferably between about 10 and about 100 nucleoside units (i.e., nucleotides) in length, preferably between about 15 and about 50, more preferably between about 15 and about 40, also preferably between about 20 and about 30.
With the term “provirus” is meant herein the reverse transcribed genome of a virus, in particular a retrovirus, that is integrated into the DNA genome of a host cell. The term also includes isolated forms of proviruses and genetic constructs containing such.
In preferred embodiments, the recombinant nucleic acid encoding the recombinant attenuated BLV disclosed herein is recombinant DNA. By means of example, said DNA may comprise, consist essentially of or consist of isolated provirus.
In a further aspect, the invention provides a vector comprising the recombinant nucleic acid disclosed herein.
The term “vector” encompasses nucleic acid molecules, typically DNA, to which nucleic acid fragments, preferably the recombinant nucleic acid disclosed herein, may be inserted and cloned, i.e., propagated. Hence, a vector will typically contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible. A vector may also preferably contain a selection marker, such as e.g. an antibiotic resistance gene, to allow selection of recipient cells that contain the vector. Vectors may include, without limitation, plasmids, phagemids, bacteriophages, bacteriophage-derived vectors, PAC, BAC, linear nucleic acids, e.g., linear DNA, etc., as appropriate (see, e.g., Sambrook et al., 1989; Ausubel 1992).
Factors of importance in selecting a particular vector include inter alia: choice of recipient host cell, ease with which recipient cells that contain the vector may be recognised and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in particular recipient cells; whether it is desired for the vector to integrate into the chromosome or to remain extra-chromosomal in the recipient cells; and whether it is desirable to be able to “shuttle” the vector between recipient cells of different species.
Preferred vectors comprise a selection marker. Preferably, the selection marker is not an ampicillin resistance gene, which helps to avoid issues of subject sensitivity to beta-lactams. A suitable selection marker may include, for example, but without limitation, a kanamycin resistance gene. Another suitable selection marker may include an auxotrophic selection marker for use with auxotrophic recipient cells as known per se. The auxotrophic growth-based selection system is based on the restoration of growth of auxotrophic recipient cells (i.e., recipient cells that lack a functional essential gene for growth) upon introducing a plasmid that allows expression of the functional gene product (i.e., a plasmid comprising an auxotrophic selection marker). The recipient cells are first modified, e.g., by introducing a deletion or a nonsense point mutation into an essential or conditionally essential chromosomal gene, resulting in auxotrophy, and the plasmid comprises e.g., the deleted gene or encodes a suppressor tRNA which allows a complete translation of the truncated gene product.
Preferred vectors are plasmids, more preferably bacterial plasmids or yeast shuttle vectors.
Non-limiting examples of suitable bacterial plasmids are those capable of replication in E. coli, such as, for example, pSP64.
With the term “yeast shuttle vector” is meant herein a plasmid capable of cloning in yeast, preferably Saccharomyces cerevisiae, but also capable of replication in a bacterial host, preferably E. coli. Such shuttle vectors typically comprise a genetic element, preferably an origin of replication, which enable the plasmid to be propagated in a bacterial host, preferably E. coli, a selectable marker for the bacterial host, a selectable marker for the yeast, and a multiple cloning site.
Preferred yeast shuttle vectors are yeast integrative plasmids, yeast episomal plasmids, or yeast centromeric plasmids.
With the term “yeast integrative plasmid” is meant herein a yeast plasmid which by homologous recombination is integrated into the host genome. A non-limiting example of a yeast integrative plasmid is pRS306.
With “yeast episomal plasmids” are meant herein yeast plasmids which maintain as episomes in the host. Such episomal plasmids typically comprise part of the 2μ plasmid DNA sequence necessary for autonomous replication. A non-limiting example of a yeast episomal plasmid is pRS426.
The term “yeast centromeric plasmid” denotes a yeast plasmid which replicates autonomously and controlled in a way that the copy number of the self-replicated plasmid is just one. A yeast centromeric plasmid may typically comprise a yeast origin of replication (ARS sequence) and a centromeric sequence which guarantees stable mitotic segregation. A non-limiting example of a yeast centromeric plasmid is pRS316.
In preferred embodiments, the vector disclosed herein may be selected from the group comprising or consisting of: a bacterial plasmid, a yeast integrative plasmid, a yeast episomal plasmid, and a yeast centromeric plasmid.
A further aspect provides the plasmid as deposited under the Budapest Treaty with the Belgian Coordinated Collections of Microorganisms BCCM/LMBP Collection under accession number LMBP 8166 on Feb. 5, 2013 (see Table 2B). The plasmid corresponds to the pBLV6073DX plasmid as described in the experimental section.
A further aspect provides the plasmid as deposited under the Budapest Treaty with the Belgian Coordinated Collections of Microorganisms BCCM/LMBP Collection under accession number LMBP 8167 on Feb. 5, 2013 (see Table 2C). The plasmid corresponds to the pBLVGPDX plasmid as described in the experimental section.
A further aspect provides the plasmid as deposited under the Budapest Treaty with the Belgian Coordinated Collections of Microorganisms BCCM/LMBP Collection under accession number LMBP 8713 on Oct. 25, 2013 (see Table 2D). The plasmid corresponds to the pBLV6073GPDX plasmid as described in the experimental section.
Further aspects provide a recombinant nucleic acid encoding a recombinant attenuated BLV, wherein the recombinant nucleic acid comprises, consists essentially of or consists of the insert of the plasmid as deposited under the Budapest Treaty with the Belgian Coordinated Collections of Microorganisms BCCM/LMBP Collection under accession number LMBP 8166 on Feb. 5, 2013 (see Table 2B), and a vector comprising the recombinant nucleic acid.
Further aspects provide a recombinant nucleic acid encoding a recombinant attenuated BLV, wherein the recombinant nucleic acid comprises, consists essentially of or consists of the insert of the plasmid as deposited under the Budapest Treaty with the Belgian Coordinated Collections of Microorganisms BCCM/LMBP Collection under accession number LMBP 8167 on Feb. 5, 2013 (see Table 2C), and a vector comprising the recombinant nucleic acid.
Further aspects provide a recombinant nucleic acid encoding a recombinant attenuated BLV, wherein the recombinant nucleic acid comprises, consists essentially of or consists of the insert of the plasmid as deposited under the Budapest Treaty with the Belgian Coordinated Collections of Microorganisms BCCM/LMBP Collection under accession number LMBP 8713 on Oct. 25, 2013 (see Table 2D), and a vector comprising the recombinant nucleic acid.
The invention further provides a host cell comprising the recombinant attenuated BLV, the recombinant nucleic acid, the vector, or the plasmid as taught herein. The terms “host cell” and may suitably refer to cells encompassing both prokaryotic cells, such as bacteria, and eukaryotic cells, such as yeast, fungi, protozoan, plant and animal cells. A host cell may particularly refer to an isolated host cell, e.g., a host cell maintained and/or propagated in laboratory conditions, e.g., in microbiological culture or in cell or tissue culture.
In preferred embodiments, the host cell may be a bacterial cell, a yeast cell, an animal cell, or a mammalian cell.
Non-limiting examples of suitable bacterial cells include Escherichia coli, such as, e.g., E. coli strain STBL2™ (competent cells) (Invitrogen; genotype and background: [F-mcrA Δ(mcrBC-hsdRMSmrr) recA1 endA1 gyrA96 thi supE44 relA1λ-Δ(lac-proAB)]) or SURE (Stratagene; genotype and background: e14-(McrA-) Δ(mcrCB-hsdSMR-mrr)171 endA1 gyrA96 thi-1 supE44 relA1 lac recB recJ sbcC umuC:Tn5 (Kanr) uvrC [F′ proAB lacIqZΔM15 Tn10 (Tetr)]); Yersinia enterocolitica; or Brucella sp., such as, e.g., Brucella abortus strain S19 or strain RB51. Other non-limiting examples of suitable bacterial cells include, e.g., Salmonella tymphimurium, Serratia marcescens, or Bacillus subtilis. Preferably, such bacteria, e.g., E. coli, may carry at least the recA, in order to reduce or prevent recombination of direct repeats in the BLV provirus plasmid.
A non-limiting example of a suitable yeast cell includes yeast of the genera Saccharomyces, Schizosaccharomyces, or Pichia, e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Pichia pastoris.
Non-limiting examples of suitable animal cells may include human and non-human animal cells, such as vertebrate animal cells, mammalian cells, primate cells, human cells or insect cells.
Animal cells, such as mammalian cells, such as human or non-human mammalian cells, may include primary cells, secondary, tertiary etc. cells, or may include immortalised cell lines, including clonal cell lines. Preferred animal cells can be readily maintained and transformed in tissue culture.
Preferred but non-limiting example of human cells include the human HeLa (cervical cancer) cell line. Other human cell lines common in tissue culture practice include inter alia DU145 (prostate cancer), Lncap (prostate cancer), MCF-7 (breast cancer), MDA-MB-438 (breast cancer), PC3 (prostate cancer), T47D (breast cancer), THP-1 (acute myeloid leukemia), U87 (glioblastoma), SHSY5Y (neuroblastoma), or Saos-2 cells (bone cancer).
A non-limiting example of primate cells are Vero (African green monkey Chlorocebus kidney epithelial cell line) cells.
Non-limiting examples of rodent cells are rat GH3 (pituitary tumor) or PC12 (pheochromocytoma) cell lines, or mouse MC3T3 (embryonic calvarium) cell line.
Non-limiting examples of insect cells include cells derived from Drosophila melanogaster such as Schneider 2 cells, cell lines derived from the army worm Spodoptera frugiperda, such as Sf9 and Sf21 cells, or cells derived from the cabbage looper Trichoplusia ni, such as High Five cells.
Methods for introducing nucleic acids, including vectors, into a host cell (i.e., transfection or transformation) are known to the person skilled in the art, and may include calcium phosphate co-precipitation, electroporation, micro-injection, lipofection, transfection employing polyamine transfection reagents, bombardment of cells by nucleic acid-coated tungsten micro projectiles, etc.
The host cells as taught herein may be live or may be inactivated (i.e., dead) by a suitable cell inactivation procedure, e.g. by freeze-drying, sonication or irradiation.
The recombinant attenuated BLV, the recombinant nucleic acid, the vector, the plasmid or the host cell as taught herein may be formulated in a pharmaceutical composition with a pharmaceutically acceptable excipient, i.e., one or more pharmaceutically acceptable carrier substances and/or additives, e.g., buffers, carriers, excipients, stabilisers, etc. The term “pharmaceutically acceptable” as used herein is consistent with the art and means compatible with the other ingredients of the pharmaceutical composition and not deleterious to the recipient thereof.
Accordingly, in a further aspect the invention provides a pharmaceutical composition comprising the recombinant attenuated BLV, the recombinant nucleic acid, the vector, the plasmid, or the host cell as disclosed herein.
In a related aspect the invention provides the recombinant attenuated BLV, the recombinant nucleic acid, the vector, the plasmid, or the host cell as disclosed herein for use in medicine.
Also disclosed herein is the use of the recombinant attenuated BLV, the recombinant nucleic acid, the vector, the plasmid, or the host cell as taught herein, for the production of a medicament.
For example, host cells as disclosed herein may be allowed to produce and optionally secrete recombinant attenuated BLV particles, which require expression of the proviral sequence, packaging of the viral genome into a capsid, a complex formation of envelope proteins with cell membranes and budding of the virion similar to natural infection. The harvested viral particles may optionally after further purification and/or processing steps such as lyophilisation, suspended in a medium suitable for administration. The recombinant attenuated BLV particles may be lyophilized in the presence of common excipients such as lactose, other sugars, alkaline and/or alkali earth stearate, carbonate and/or sulphate (for example, magnesium stearate, sodium carbonate and sodium sulphate), kaolin, silica, flavourants and aromas.
In another example, the vectors disclosed herein, preferably the proviral plasmids disclosed herein (i.e., plasmids comprising recombinant BLV provirus), may be allowed to propagate into an appropriate host cell, after which the purified vectors may be formulated with cationic liposomes, such as, e.g., N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP), in a medium suitable for administration.
In yet another example, the host cell disclosed herein may be cultured, upon which the harvested cells, optionally after further purification and/or processing steps, such as freeze-drying, sonication or irradiation to inactivate them, can be used to prepare the formulation. Such formulations include but are not limited to live or inactivated (i.e., dead), e.g., by freeze-drying, sonication or irradiation, bacteria, yeast or mammalian cells, such as, e.g., HeLa cells, comprising a proviral DNA plasmid or episome or proviral DNA integrated in their genome and a medium suitable for administration.
In preferred embodiments, the pharmaceutical composition may be a vaccine.
In a related aspect the invention provides the recombinant attenuated BLV, the recombinant nucleic acid, the vector, the plasmid, the host cell, or the pharmaceutical composition as disclosed herein, for use as a vaccine, in particular for use as a vaccine against a BLV-associated disease, more in particular for use as a prophylactic vaccine against a BLV-associated disease. As discussed elsewhere in this specification, such vaccine may advantageously be intended for long-term protection (e.g., protection for at least 18 months or for at least 24 months or for at least 36 months or for at least 48 months post-vaccination) of animals, preferably bovids, more preferably cattle, from infection by wild-type BLV (which may be heterologous to the vaccine). Such vaccine may so-protect virtually all animals, preferably bovids, more preferably cattle, e.g., at least 90%, preferably at least 95%, such as 98%, or 99%, or even 100% of the vaccinated animals.
Also disclosed herein is the use of the recombinant attenuated BLV, the recombinant nucleic acid, the vector, the plasmid or the host cell as taught herein, for the production of such vaccine.
A vaccine may typically comprise an immunologically effective amount of an immunogenic substance or composition.
The term “immunologically effective amount” refers to an amount of an immunogenic substance or composition effective to enhance the immune response of a subject against a subsequent exposure to the immunogen. Levels of induced immunity can be determined, e.g. by measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assay.
By means of example, an immunologically effective amount of the recombinant nucleic acid, the vector, or the plasmid as taught herein may comprise at least about 25 ng nucleic acid, or at least at least about 50 ng nucleic acid, or at least about 100 ng nucleic acid, or at least at least about 250 ng nucleic acid, or at least at least about 500 ng nucleic acid, or at least at least about 750 ng nucleic acid, or at least at least about 1 μg nucleic acid, or at least at least about 2 μg nucleic acid, or at least at least about 5 μg nucleic acid, or at least at least about 10 μg nucleic acid, or at least at least about 50 μg nucleic acid, or at least at least about 100 μg nucleic acid, e.g., in a single or repeated dose. Dosages of the nucleic acid for administration will vary depending upon any number of factors including the type of BLV mutant, the subject, the route of administration to be used, prevalence of the disease to be treated, etc. Thus, precise dosages cannot be defined for each and every embodiment of the invention, but will be readily apparent to those skilled in the art once armed with the present invention.
By means of example, an immunologically effective amount of a vaccine comprising host cells, e.g., bacteria, comprising proviral plasmid may comprise at least 104 bacteria, or at least 105 bacteria, or at least 106 bacteria, or at least 107 bacteria, or at least 108 bacteria, or at least 109, or at least 1010, or at least 1011, or at least 1012, or at least 1013, or at least 1014, or at least 1015, or more bacteria, e.g., in a single or repeated dose. Dosages of host cells for administration will vary depending upon any number of factors including the type of host cell, expression levels, the route of administration to be used, prevalence of the disease to be treated, etc. Thus, precise dosages cannot be defined for each and every embodiment of the invention, but will be readily apparent to those skilled in the art once armed with the present invention.
The vaccine may further comprise one or more adjuvants for enhancing the immune response. Suitable adjuvants include, for example, but without limitation, saponin, mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, bacilli Calmette-Guerin (BCG), Corynebacterium parvum, and the synthetic adjuvant QS-21.
Optionally, the vaccine may further comprise one or more immunostimulatory molecules. Non-limiting examples of immunostimulatory molecules include various cytokines, lymphokines and chemokines with immunostimulatory, immunopotentiating, and pro-inflammatory activities, such as interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc.
In preferred embodiments the vaccine as taught herein may comprise one or more further immunogenic substances or compositions.
Any substance or composition capable of eliciting an immune response may be added to the vaccine. By means of example, but without limitation, such immunogenic substance may be a recombinant nucleic acid, e.g. a plasmid, comprising coding sequences for epitopes or antigens; or live attenuated viruses, e.g. in the form of a proviral plasmid; or live attenuated bacteria. For example, vaccines comprising host cells, e.g. bacteria, as disclosed herein may comprise a BLV proviral plasmid and one or more plasmids comprising coding sequences for antigens or epitopes.
Such combination vaccines may be aimed at preventing several diseases or one disease caused by different variants of the same organism causing the disease.
Suitable immunogenic substances for use in a combination with the vaccine disclosed herein are without limitation live attenuated bovine herpesvirus, such as e.g. live attenuated bovine herpesvirus type I, or live attenuated Clostridium sp.
The invention further provides the recombinant attenuated BLV, the recombinant nucleic acid, the vector, the plasmid, the host cell, or the pharmaceutical composition as disclosed herein, for use in treatment of a BLV-associated disease, in particular for use in prevention (i.e., preventative treatment, prophylactic treatment, prophylaxis) of a BLV-associated disease.
The term “BLV-associated disease” as intended herein generally encompasses any disease and disorder caused by BLV infection.
BLV-associated diseases include inter alia and preferably enzootic bovine leukosis, which may include bovine persistent lymphosis, bovine lymphosarcoma and bovine lymphoma.
As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures. The terms “treatment”, “treating”, and the like, as used herein include amelioration or elimination of a developed disease or condition once it has been established or alleviation of the characteristic symptoms of such disease or condition. As used herein these terms preferably encompass, depending on the condition of the subject, preventing the onset of a disease or condition or of symptoms associated with a disease or condition, including reducing the severity of a disease or condition or symptoms associated therewith prior to affliction with said disease or condition. Such prevention or reduction prior to affliction refers to administration of the compound or composition of the invention to a patient that is not at the time of administration afflicted with the disease or condition. “Preventing” also encompasses preventing the recurrence or relapse-prevention of a disease or condition or of symptoms associated therewith, for instance after a period of improvement.
In preferred embodiments, the treatment is prophylactic treatment, such as preferably prophylactic vaccination, whereby (super)infection with another BLV virus may be prevented.
The recombinant attenuated BLV, the recombinant nucleic acid, the vector, the plasmid, the host cell, or the pharmaceutical composition such as vaccine as disclosed herein may in certain embodiments benefit from more than one administration to a subject. Hence, in such embodiments, following an initial administration of the recombinant attenuated BLV, the recombinant nucleic acid, the vector, the plasmid, the host cell, or the pharmaceutical composition such as vaccine as disclosed herein to a subject (such initial administration may be denoted as primary antigen stimulation or “priming”), one or more subsequent administrations (such subsequent administration(s) may be denoted as “boosting”) of the recombinant attenuated BLV, the recombinant nucleic acid, the vector, the plasmid, the host cell, or the pharmaceutical composition such as vaccine as disclosed herein to the subject may be advantageous to sustain or preferably increase the anti-viral immune response in the subject.
In certain embodiments, such “boosting” may involve repeated administrations of the recombinant attenuated BLV, the recombinant nucleic acid, the vector, the plasmid, the host cell, or the pharmaceutical composition such as vaccine as disclosed herein at regular intervals following the initial administration, e.g., at a regular interval of about 0.5 year, or about 1.0 year, or about 1.5 year or about 2.0 years or about 2.5 years, or about 3 years, or about 4 years, or about 5 years, preferably at an interval of about 1.0 year or about 2.0 years, even more preferably at an interval of about 1.0 year. In this context, such “repeated” administrations may mean for example two or more administrations, three or more administrations, four or more administrations or five or more administrations following the initial administration. In an example, “repeated” administrations may mean that such administrations are repeated throughout the entire lifetime of a subject.
The term “subject” as used herein particularly refers to animals, more particularly to mammals, such as non-human mammals, and most particularly to bovides. Bovides are cloven-hoofed ruminant mammals belonging to the Bovidae family. Members include, for example, but without limitation bison, African buffalo, water buffalo, antelopes, gazelles, sheep, goats, muskoxen, and domestic cattle. Preferred bovides are bovines, such as, e.g., buffalos, zebus and domestic cattle, particularly animals belonging to the genus Bos, more particularly Bos primigenius, including cattle (cows), more preferably cattle (cows). Hence, in preferred embodiments, the subject is a bovid, more preferably a bovine, even more preferably cattle, such as a cow.
The invention further pertains to a non-human animal, preferably a non-human mammal, more preferably a bovid, even more preferably a bovine, such as cattle, to which the recombinant attenuated BLV, the recombinant nucleic acid, the vector, the plasmid, the host cell, or the pharmaceutical composition as disclosed herein has been administered. Examples of non-human animals useful in this context include without limitation cows, pigs, donkeys, horses, rabbits, goats, sheep, guinea pigs, rats, mice, and the like.
Such non-human animal may harbour in the genome of at least some of its cells, preferably in the genome of at least some of its peripheral blood mononuclear cells (PBMC), a provirus encoding the recombinant attenuated bovine leukemia virus as taught herein.
The invention thus also provides methods for preparing a non-human animal-derived material, comprising obtaining material from the non-human animal as taught here above (e.g., by milking, or by slaughtering the animal), and optionally further processing said material into a non-human animal-derived product (e.g., portioning, treating with preservatives, packaging, etc.).
The invention further provides a non-human animal-derived material or a non-human animal-derived product obtainable or directly obtained from said non-human animal, or obtainable or directly obtained by said method.
In preferred embodiments, the non-human animal-derived material or the non-human animal-derived product may comprise, consist of or may be isolated from a secretion of the mammary gland of the non-human animal or a part thereof, in particular wherein the material or product comprises, consists of or is isolated from milk or colostrum.
In other preferred embodiments, the non-human animal-derived material or the non-human animal-derived product may comprise, consist of or may be isolated from whole blood of the non-human animal, in particular wherein the material or product comprises, consists of or is whole blood, or fraction of whole blood, such as plasma or serum.
The invention further pertains to said non-human animal-derived material or said non-human animal-derived product for use as a vaccine, in particular for use as a vaccine against a BLV-associated disease, more in particular for use as a prophylactic vaccine against a BLV-associated disease.
It shall be appreciated that such non-human animal-derived material or said non-human animal-derived product may thus contain the attenuated BLV virus, or the BLV provirus or cells containing such, or may contain antibodies that have been raised against the attenuated BLV virus in the subject from which the material or product is obtained (i.e., passive immunisation).
Deposits of Biological Material
The following Tables 2A, 2B, 2C and 2D summarise the requisite indications relating to deposited microorganisms or other biological material referred to throughout this specification.
E. coli STBL2 ™ (competent cells) (Invitrogen) or SURE (Stratagene)
E. coli STBL2 ™ (competent cells) (Invitrogen) or SURE (Stratagene)
E. coli STBL2 ™ (competent cells) (Invitrogen) or SURE (Stratagene)
E. coli STBL2 ™ (competent cells) (Invitrogen) or SURE (Stratagene)
The recombinant bovine leukemia virus (BLV) provirus plasmid pBLV6073DX is derived from the plasmid pBLV344H described in Willems et al. 1993 (J. Virol. 67: 4078-4085), specifically incorporated by reference herein. pBLV344H comprises complete wild-type BLV provirus derived from infected tissues of the sheep animal 344 experimentally infected with a Belgian variant of BLV, as described by Van den Broeke et al. 1988 (Proc. Natl. Acad. Sci. USA 85: 9263-9267), specifically incorporated by reference herein. The plasmid pBLV344H has been deposited under the Budapest Treaty with the Belgian Coordinated Collections of Microorganisms BCCM/LMBP Collection under accession number LMBP 8165 on Feb. 5, 2013 (see Table 2A).
The recombinant BLV provirus plasmid pBLV6073DX was constructed using standard molecular cloning techniques. Schematically, the KpnI-XbaI fragment of pBLV6073 (positions 2111-6614; size 4.5 Kbp) was ligated to the XbaI-KpnI fragment of pBLVDX (position 6997-2111 (XbaI site at adjacent to position 6997 in pBLVDX was introduced through cloning, see below); size 4.7 Kbp). Nucleotide positions of BLV proviruses are numbered in this specification according to the sequence as described in Rice et al. 1987 supra, a certain portion of which is reproduced in
The pBLV6073 recombinant BLV provirus plasmid is derived from the plasmid pBLV344H using PCR-based site-directed mutagenesis procedure as described in Willems et al. 1995 (J. Virol. 69: 4137-4141), specifically incorporated by reference herein. pBLV6073 carries a substitution of a T residue with a G residue at position 6073 in an immunoreceptor tyrosine-based activation motif (ITAM) located in the transmembrane protein gp30 of the envelope (
The pBLVDX recombinant BLV provirus plasmid is derived from the plasmid pBLV344H by cloning a double oligonucleotide segment composed of two hybridised oligonucleotides with the sequences 5′-CTAGAAAGCTTG-3′ (SEQ ID NO: 1) and 5′-GATCCAAGCTTT-3′ (SEQ ID NO: 2) into the XbaI and BamHI restriction sites (positions 6614 and 6997) of pBLV344H, as described in Willems et al. 1993 (J. Virol. 67: 4078-4085), specifically incorporated by reference herein. pBLVDX carries deletions in the R3 and G4 open reading frames (ORFs) (
A protocol was developed based on transient transfection of HeLa cells with proviral plasmids and subsequent subcutaneous or intradermal injection. In particular, two 150 cm2 Petri dishes of subconfluent HeLa cell monolayers were transfected with pBLV6073DX (35 μg per plate) complexed (ratio 1:5) with transfection reagent (TransIT®, Mirus Bio LCC or FuGENE®, Roche). After 2 days of culture (37° C. in a 95%-5% air-CO2 humidified atmosphere) in complete (i.e., supplemented with 10% foetal calf serum (FCS), 2 mM L-glutamine, 100 U of penicillin, 100 μg of streptomycin per ml) Dulbecco's Modified Eagle Medium (DMEM, Invitrogen), transfected cells were trypsinised, washed in phosphate-buffered saline (PBS) and injected subcutaneously.
This delivery protocol provides an alternative to more familiar protocols, such as infection by BLV or injection of purified proviral DNA, and offers certain advantages over such protocols. For example, natural infection by BLV requires expression of the viral RNA genome, its packaging into a capsid, a complex formation of envelope proteins with cell membranes and budding of the virion. However, in this extracellular form, the viral particle is comparatively unstable. This can also be avoided, for instance, by infecting animals by injection of purified proviral DNA. For example, packaging proviral DNA into cationic liposomes and intradermal injection permits viral infection. However, this technique requires production and purification of large quantities of plasmid DNA (100-500 micrograms per animal), and infection through direct DNA injection tends to be comparatively less efficient, potentially necessitating a second injection and/or extending the latency period before seroconversion.
We also developed another strategy based on intradermal injection of the E. coli strain STBL2™ (competent cells) ([F-mcrA Δ(mcrBC-hsdRMSmrr) recA1 endA1 gyrA96 thi supE44 relA1λ-A(lac-proAB)]; Trinh et al. (1994, FOCUS 16: 78)); available from Invitrogen carrying the proviral plasmid. STBL2™ (competent cells) cells transformed with pBLV6073DX were cultured overnight in 5 ml of Luria-Bertani (LB) broth medium (Invitrogen) containing 50 μg/ml of ampicillin at 28° C. Bacteria were then centrifuged, washed, resuspended in 2 ml of PBS and injected subcutaneously or intradermally.
This straightforward and cost-effective technique of injecting STBL2™ (competent cells) cells carrying the proviral plasmid very reproducibly transmitted infection to naïve hosts after a single injection. No side or toxic effects of STBL2™ (competent cells) cell injection were recorded, in agreement with its safety data sheet. Advantageously, using bacterial cells instead of mammalian cells such as the human HeLa cells, avoids the risk of inadvertently introducing other pathogens, such as HPV, and reduces the complexity of vaccine formulations based on cells (e.g., bacterial cells need not be preserved in liquid nitrogen when transported).
Experiments detailed below were performed using subcutaneous injection of HeLa cells carrying the proviral plasmids. Comparable results are obtained using subcutaneous injection of E. coli strain STBL2™ (competent cells) carrying the proviral plasmids.
Experiments detailed below were performed using subcutaneous injection of HeLa cells carrying the proviral plasmids. Comparable results are obtained using subcutaneous injection of E. coli strain STBL2™ carrying the proviral plasmids.
A preliminary trial performed under restricted conditions demonstrated that the recombinant BLV6073DX provirus is safe because of the: (i) absence of pathology or toxicity in vaccinated cows and in the highly susceptible ovine experimental model, (ii) lack of transmission of the recombinant BLV6073DX provirus to uninfected sentinels over a 3 year period, (iii) absence of detectable levels of plasmid DNA (including the β-lactamase gene) as revealed by nested PCR (data not shown).
A large scale experimental setting was designed. Ten cows were infected with recombinant BLV6073DX provirus (i.e., vaccinated) and 5 others were infected with wild-type BLV provirus (WT). All cows were then kept in a herd of 74-82 animals (depending on the year) among which about 15-30% were naturally infected with Argentinean BLV strain (ArgWT). Besides vaccine efficacy, this design also allows an evaluation of safety under real farm conditions (i.e., transmission from cow to calf and infection of sentinels).
As revealed by a competitive ELISA test (ELISA Bovine Leukosis Serum blocking test, Institut Pourquier), injection of pBLV6073DX elicited an antiviral antibody response with kinetics similar to wild-type infection (
Proviral loads were measured by qPCR. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated by Percoll density gradient centrifugation (GE Healthcare) and washed twice with phosphate-buffered saline (PBS)/0.075% EDTA and at least three times with PBS alone. DNA was isolated using DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer instructions. One hundred nanograms of genomic DNA were used for real-time PCR amplification of BLV proviral sequences. A segment corresponding to the pol gene was amplified using primers 5′-GAAACTCCAGAGCAATGGCATAA-3′ (SEQ ID NO: 5) and 5′-GGTTCGGCCATCGAGACA-3′ (SEQ ID NO: 6) and MESA GREEN mPCR MasterMix (Eurogentec) on a light cycler (Roche) following manufacturer instructions. A standard curve was generated after amplification of defined proviral copy numbers (from 102 to 106 of plasmid pBLV344) diluted in 100 ng of control genomic DNA. To correct for differences in DNA concentrations the actin DNA was quantified in parallel using primers 5′-TCCCTGGAGAAGAGCTACGA-3′ (SEQ ID NO: 7) and 5′-GGCAGACTTAGCCTCCAGTG-3′ (SEQ ID NO: 8). Thermal conditions: 95° C. 5 min; (95° C. 15 sec, 60° C. 20 sec, 72° C. 40 sec, 45 times). Proviral load was calculated from the number of proviral copies divided by half of the number of actin copies and expressed as number of proviral copies per 100 of PBMCs.
The proviral loads (PVL) were significantly reduced in vaccinated animals (
Importantly, no pathogenicity of BLV6073DX was observed in any of the 10 vaccinated cows in a period of almost 3.5 years post-inoculation (vaccination on 21 Oct. 2010). Similarly, none of the 3 cows used in the preliminary trial displayed any pathogenicity of BLV6073DX in a period of almost 5.5 years post-inoculation (vaccination on Oct. 10, 2008). Also, two sheep used in earlier trial studies and vaccinated with BLV6073DX have not shown any pathogenicity of BLV6073DX in a period of about 4 and 5 years post-inoculation. The absence of detectable pathogenicity in these experiments corroborates the suitability of BLV6073DX as a safe vaccine, emphasising its superiority over BLV6073, which caused pathogenicity in 1 of 4 sheep, and BLVDX, which caused pathogenicity in 1 of 8 sheep (Florins et al. 2007 supra).
Additionally, sequencing studies performed on the vaccinated animals have confirmed that BLV6073DX was not subject to mutations in the inoculated animals, thereby further corroborating the vaccine's stability and safety.
Preliminary experiments also supported the conclusion that BLV6073DX advantageously induces a cytotoxic immune response in vaccinated cows.
For traceability, a protocol was designed to identify vaccinated animals based on nested PCR using primers that flank the deletion in the R3 and G4 ORFs of pBLV6073DX (
The protocol effectively identified the 10 vaccinated cows, i.e., the cows infected with the recombinant BLV6073DX provirus, as demonstrated by the amplification of the small fragment (
Since all animals were kept in the same herd, these data also show that the wild-type provirus does not transmit to vaccinated cows, suggesting that the recombinant BLV6073DX provirus efficiently protects against superinfection. Of note, the pBLV6073DX plasmid originates from wild-type BLV strain 344, which is different from Argentinean BLV variants. This observation thus indicates that infection with recombinant BLV6073DX provirus (i.e., vaccination) protects against infection of heterologous BLV viruses.
The observation that all 10 vaccinated cattle remained free of wild-type BLV virus for almost 3.5 years post-vaccination corroborates the advantages of BLV6073DX as a vaccine with a comparatively long-term protective effect, e.g., protective effect of at least 18 months, preferably at least 24 months, more preferably at least 36 months, even more preferably at least 48 months or even longer post-vaccination, in animals, especially in cattle. In contrast, one of two cows vaccinated using the previously existing pBLVDX provirus became infected by wild-type BLV 12 months after challenge (Kerkhofs et al. 2000 supra), and one cow (#269) vaccinated using the previously existing pBLV6073 provirus became infected by wild-type BLV 24 months after challenge (Kerkhofs et al. 2000 supra, and Example 10).
We designed a trial to evaluate the ability of vaccinated (i.e., infected with recombinant BLV6073DX provirus) animals to resist challenge with wild-type BLV provirus. Briefly, 60 μg of pBLVWT plasmid DNA (corresponding to 6×1012 wild-type proviral copies) were transfected into HeLa cells (two 15 cm diameter subconfluent Petri dishes) and, after 48 hours, the transfected HeLa cells were injected subcutaneously into the back of 3 vaccinated animals and 3 uninfected controls.
Infection with wild-type BLV provirus was assessed by a competitive ELISA to determine seropositivity and anti-BLV antibody titres as described in Example 3 and nested PCR according to the protocol described in Example 4 to detect the presence of WT BLV provirus.
Two months post-injection, the 3 controls (#77, #83 and #85) became infected with the wild-type provirus as demonstrated by nested PCR and ELISA (Table 3,
The data clearly demonstrate that vaccinated animals resist challenge by wild-type BLV.
Transmission of the recombinant BLV6073DX provirus from the cows to their calves was analyzed by inseminating animals and analyzing infection in the calves using nested PCR as described in Example 4.
Among four calves from wild-type infected cows, one (#100) became infected (Table 4). This pattern is consistent with previous observations describing intrauterine or perinatal transmission of BLV infection.
In contrast, proviral sequences could not be amplified in any of the 6 calves born from vaccinated cows, indicating that the pBLV6073DX provirus plasmid was not transmitted (Table 4). Importantly, these calves contained anti-BLV antibodies in their serum revealing passive immunity. The antibody titres persisted a few months and then slowly decreased with time further supporting lack of infection of vaccinated cows progeny. It should also be mentioned that we did observe neither signs of abortion in vaccinated cows nor side effects in their calves (e.g., weight, abnormalities, disease, . . . ).
In summary, cows vaccinated with pBLV6073DX transmit anti-BLV passive immunity but not viral infection to their calves.
Examples 1 to 6 set forth above demonstrate that by combining a mutation at residue 6073 and a deletion of the R3/G4 genes in an embodiment of the invention, a BLV strain has been achieved that (i) is infectious in cows but transmits neither to their offspring nor to sentinels, (ii) replicates at low levels compared to wild type but lacks pathogenicity, (iii) elicits a strong immune response and protects from wild type challenge, and (iv) is readily traceable by PCR. This attenuated strain can therefore be used as a protective vaccine against BLV infection.
A recombinant BLV provirus plasmid pBLVGPX carrying a deletion of the microRNAs ORFs, in particular harbouring a deletion between positions 6169 and 6731 (numbering as described in Rice et al. 1987 supra) in the X region between the env gene and the Tax/Rex sequences has been described in Willems et al. (2000, AIDS Res Hum Retroviruses. 16: 1787-95), specifically incorporated by reference herein.
pBLVGPX was derived from the plasmid pBLV344H (see Example 1). Schematically, 5′ proviral sequences were PCR-amplified using the upstream primer 5′-TGACAACATATAACCAAGA-3′ (SEQ ID NO: 17) (Rice positions 4751-4769) and the downstream primer 5′-TCTAGAGGGGGTGTCAAGGGCAGGGT-3′ (SEQ ID NO: 13). Nucleotides 7-26 of this downstream primer are complementary to BLV positions 6169-6150, and nucleotides 1-6 of the primer introduce an XbaI restriction site (underlined) at the 3′ end of the resulting PCR product. Thermal conditions for PCR: 95° C. 5 min; (95° C. 30 sec, 57° C. 30 sec, 72° C. 60 sec, 36 times); 72° C. 5 min. The amplicon was cloned into plasmid pCRII (Invitrogen) yielding pCREA. To construct pBLVGPX, 4 fragments were ligated: a 68 bp BglII-XbaI fragment of pCREA (BglII at positions 6101-6106 of BLV), and 3 fragments from pBLV344H(XbaI-KpnI 8 kb, KpnI-NcoI 2.8 kb and NcoI-BglII 1.2 kb). pBLV344H was described in Willems et al. 1993 (J. Virol. 67: 4078-4085).
Proviral loads were measured by qPCR, as described in Example 3. The recombinant BLVGPX provirus is infectious in vivo in cows (
Recombinant BLV provirus plasmids pBLVGPDX and pBLV6073GPDX are constructed using standard molecular cloning techniques.
pBLVGPDX was derived from the plasmid pBLV344H (see Example 1). Schematically, 5′ proviral sequences were PCR-amplified using the upstream primer 5′-TGACAACATATAACCAAGA-3′ (SEQ ID NO: 17) (Rice positions 4751-4769) and the downstream primer 5′-TCTAGAGGGGGTGTCAAGGGCAGGGT-3′ (SEQ ID NO: 13). Nucleotides 7-26 of this downstream primer are complementary to BLV positions 6169-6150, and nucleotides 1-6 of the primer introduce an XbaI restriction site (underlined) at the 3′ end of the resulting PCR product. Thermal conditions for PCR: 95° C. 5 min; (95° C. 30 sec, 57° C. 30 sec, 72° C. 60 sec, 36 times); 72° C. 5 min. The amplicon was cloned into plasmid pCRII (Invitrogen) yielding pCREA. To construct pBLVGPDX, 4 fragments were ligated: a 68 bp BglII-XbaI fragment of pCREA (BglII at positions 6101-6106 of BLV), and 3 fragments from pBLV344H(BamHI-KpnI 8.3 kb, KpnI-NcoI 2.8 kb and NcoI-BglII 1.2 kb). A double oligonucleotide linker segment composed of two hybridised oligonucleotides with the sequences 5′-CTAGAAAGCTTG-3′ (SEQ ID NO: 1) and 5′-GATCCAAGCTTT-3′ (SEQ ID NO: 2) was used to connect the XbaI and BamHI overhangs. As a result, the nucleic acid sequence 5′-TCTAGAAAGCTT-3′ (SEQ ID NO: 4) replaces the nucleic acid sequence between position 6170 and position 6996 of the BLV nucleic acid sequence (numbering as described in Rice et al. 1987 supra). pBLVGPDX has been deposited under the Budapest Treaty with the Belgian Coordinated Collections of Microorganisms BCCM/LMBP Collection under accession number LMBP 8167 on Feb. 5, 2013 (see Table 2C).
Subsequently, PCR-based site-directed mutagenesis was performed with QuikChange XL Site-Directed Mutagenesis Kit (Agilent) using the primers 6073S: 5′-GATTCTGATGATCAGGCCT-3′ (SEQ ID NO: 14) and 6073C: 5′-AGGCCTGATCATCAGAATC-3′ (SEQ ID NO: 15) to introduce the 6073 mutation.
The recombinant BLV6073GPDX provirus (
pBLV6073GPDX has been deposited under the Budapest Treaty with the Belgian Coordinated Collections of Microorganisms BCCM/LMBP Collection under accession number LMBP 8713 on Oct. 25, 2013 (see Table 2D).
The recombinant BLVGPDX provirus and the recombinant BLV6073GPDX provirus are each expected to provide a particularly advantageous BLV strain displaying at least some and preferably all of the following properties: (i) it is infectious in cows but transmits neither to their offspring nor to sentinels, (ii) it replicates at low levels compared to wild type but lacks pathogenicity, (iii) it elicits a strong immune response and protects from wild type challenge, and (iv) it is readily traceable by PCR. This attenuated strain can therefore be used as a protective vaccine against BLV infection.
One sheep (#2187) was infected with provirus pBLV6073GPDX using the following protocol. Two 15 cm-diameter dishes containing subconfluent Hela cells were transfected with 10 micrograms of plasmid pBLV6073GPDX, recovered in 5 ml PBS at day 3 and injected subcutaneously into sheep 2187. Infection was confirmed by competitive ELISA revealing the presence of anti-BLV antibodies and by PCR-sequencing demonstrating the presence of the mutations. No pathogenicity has been observed in sheep 2187 in almost 6 months (vaccination on 18 Sep. 2013).
In another trial, 50 calves are vaccinated with pBLV6073GPDX. pBLV6073GPDX will display long-term protection (e.g., at least 18 months, preferably at least 24 months, more preferably at least 36 months, even more preferably at least 48 months post-vaccination) of virtually all calves (e.g., at least 90% (45 calves or more), preferably at least 95% (48 calves or more), such as 98% (49 calves), or 99%, or even 100% (50 calves)) from infection by wild-type BLV. pBLV6073GPDX will not cause pathogenicity in the calves over extended time periods (e.g., 3 years, 4 years, 5 years, 6 years, or 7 years or more).
About 500 cows are included in another large-scale vaccination trial in dairy herds with about 80% BLV prevalence in Argentina. Calves (about 40 births per month) are vaccinated with pBLV6073GPDX on day 0 and day 60-90 after birth. Vaccinated heifers are mated at about 17-20 months, giving birth at about 27-30 months, and are followed-up to the age of at least 40 months. BLV6073GPDX will display long-term protection (e.g., at least 18 months, preferably at least 24 months, more preferably at least 36 months, even more preferably at least 48 months post-vaccination) of virtually all cows (e.g., at least 90%, preferably at least 95%, such as 98%, or 99%, or even 100%) from infection by wild-type BLV. BLV6073GPDX will not cause pathogenicity in the cows over the period of the trial. BLV6073DX will not transmit to the offspring of the cows nor to sentinels.
In another trial, 10 calves are vaccinated with pBLVGPDX. pBLVGPDX will display long-term protection (e.g., at least 18 months, preferably at least 24 months, more preferably at least 36 months, even more preferably at least 48 months post-vaccination) of virtually all calves (e.g., at least 90% (9 calves), preferably 100% (10 calves)) from infection by wild-type BLV. pBLVGPDX will not cause pathogenicity in the calves over extended time periods (e.g., 3 years, 4 years, 5 years, 6 years, or 7 years or more).
By combining specific mutations as described throughout Examples 1-8, various useful embodiments of pBLV344H-derived attenuated recombinant BLV proviruses illustrating the present invention were or are constructed using standard molecular cloning techniques, as listed in Table 5.
For the purposes of Table 5:
Cows vaccinated in trial studies (for example including about 10 animals or about 50 animals) with attenuated recombinant BLV proviruses exemplified in Table 5 will display long-term protection (e.g., at least 18 months, preferably at least 24 months, more preferably at least 36 months, even more preferably at least 48 months post-vaccination) of virtually all cows (e.g., at least 90%, preferably at least 95%, such as 98%, or 99%, or even 100%) from infection by wild-type BLV. The attenuated recombinant BLV proviruses exemplified in Table 5 will not cause pathogenicity in the cows over extended time periods (e.g., 3 years, 4 years, 5 years, 6 years, or 7 years or more).
As mentioned previously, the present inventors have demonstrated that cow #269 vaccinated using the pBLV6073 provirus and evaluated in Kerkhofs et al. 2000 supra in fact became infected by wild-type BLV 24 months after challenge with wild-type BLV, evidencing that BLV6073 provides for only comparatively short-term protection.
In particular, blood was collected by jugular venipuncture at 18 and 24 months post-challenge of pBLV6073-vaccinated cow #269 (Kerkhofs et al. 2000 supra). After nucleic acid extraction, DNA was amplified by PCR using 3 different pairs of primers: 6073S+7049R (lane 1 in
Sequencing of the virus infecting cow #269 confirmed infection by wild-type BLV. In particular, blood was collected by jugular venipuncture at 18 and 24 months post-challenge of pBLV6073-vaccinated cow #269 (Kerkhofs et al. 2000 supra). After nucleic acid extraction, DNA was amplified by PCR using the primer pair 5719S+7000R. The amplification product was sequenced using the primer 5719S. As control, the same experiment was performed with an env gene from a wild-type BLV virus. As shown in
To introduce AmyE and Lys-A genes of Bacillus subtilis strain 168 into the pBLV6073GPDX plasmid, a double recombination strategy is used (see
The resulting construct is transformed in Bacillus subtilis 168 amyE+ lysA−. Selection for amyE− lysA+ leads to isolation of a Bacillus having integrated the pBLV6073GPDX by homologous recombination.
Sequence Listing Free Text
| Number | Date | Country | Kind |
|---|---|---|---|
| 13156921 | Feb 2013 | EP | regional |
This application is a continuation of U.S. patent application Ser. No. 14/832,863, filed Aug. 21, 2015, now U.S. Pat. No. 10,029,006, which is a continuation of International Patent Application No. PCT/EP2014/053855, filed Feb. 27, 2014, which claims priority to U.S. Provisional Patent Application Ser. No. 61/769,971, filed Feb. 27, 2013, and European Patent Application No. 13156921.2, filed Feb. 27, 2013, the entire disclosures of which are hereby incorporated herein by reference in their entirety.
| Number | Date | Country |
|---|---|---|
| 2396978 | Aug 2010 | RU |
| Entry |
|---|
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| GenBank (2016): D00647. |
| GenBank (2014): AB934283. |
| GenBank (2016): FJ914764. |
| GenBank (2017): AF257515. |
| GenBank (2015): LC164085. |
| GenBank (2012): HE967303. |
| GenBank (2016): K02120. |
| GenBank (2017): AP018023. |
| GenBank (2017): AP018030. |
| GenBank (2015): LC005616. |
| GenBank (2017): AB987702. |
| U.S. Appl. No. 16/006,472. |
| U.S. Appl. No. 14/832,863 / 2016/0045593 / U.S. Pat. No. 10,029,006, filed Aug. 21, 2015 / Feb. 18, 2016 / Jul. 24, 2018, Luc Willems. |
| PCT/EP2014/05855 / 2014/131844, Feb. 27, 2014 Sep. 4, 2014, Luc Willems. |
| Number | Date | Country | |
|---|---|---|---|
| 20190000963 A1 | Jan 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61769971 | Feb 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14832863 | Aug 2015 | US |
| Child | 16006472 | US | |
| Parent | PCT/EP2014/053855 | Feb 2014 | US |
| Child | 14832863 | US |
