Patent Application
20250213674
The present disclosure provides viral vectors with therapeutic utility and a use in the generation and expression of recombinant proteins.
Virus can be modified for use as vectors to deliver therapeutic compounds to cells and other systems. Vectors based on PIV5 are useful as the virus is able to infect human cells but is not known to cause any disease. Moreover, the virus possesses a non-segmented negative strand RNA genome and does not move through a DNA phase when replicating in a cell—this avoids the possibility of viral genes being integrated into the host genome.
There is a need for improved vectors which can be used as adjuvants and which can be used in medicine to treat diseases like cancer.
The present disclosure provides a cohort of novel viral genome derived products for use:
The term ‘adjuvant’ may be applied to any product, molecule or compound which is able to augment or modulate an immune response to another product, molecule or compound. For example, adjuvants are often used to improve immune responses to antigens, wherein those antigens are, by themselves, poorly immunogenic or not capable of eliciting the correct immune response. An adjuvant may be administered together with the product (e.g. an antigen), molecule or compound which is to be subject to a modulated or augmented (for example improved) immune response. An adjuvant may be administered with a vaccine (as a ‘vaccine adjuvant’) to improve the immune response to the vaccine.
It should be noted that while this application refers to various viral genome derived products for use in the treatment and/or prevention of certain diseases, conditions and/or disorders, the disclosure extends to methods of treating those same diseases, conditions and/or disorders. Said methods may (generally) comprise steps in which subjects in need of treatment are administered any one or more of the viral genome derived products described herein. The subjects may be administered therapeutically effective amounts of any of the viral genome derived products described herein.
Moreover, throughout this specification, the term “comprising” is used to denote that the disclosed embodiments and teachings “comprise” the noted features and as such, may also include other features. However, the term “comprising” may also encompass embodiments and teachings which “consist essentially of” the relevant features or “consist of” the relevant features.
The disclosed viral genome derived products may be derived from members of the Paramyxoviridae Family. The Paramyxoviridae Family contains a wide variety of vertebrate viruses, including mumps, measles and human parainfluenza viruses.
In one teaching, the disclosed viral genome derived products are derived from the parainfluenza virus 5 (PIV5) genome. As such, the disclosed viral genome derived products are all PIV5 derived products.
PIV5 has a non-segmented, negative-sense RNA genome of 15,246 nucleotides (nt) containing seven tandemly arranged genes, that encode eight proteins, flanked by 3′-leader (Le) and 5′-trailer (Tr) sequences (at the genome ends). From the 3′-leader sequence, the genome encodes the nucleocapsid protein (NP), V protein (V), phosphoprotein (P), matrix protein (M), fusion protein (F), small hydrophobic protein (SH), haemagglutinin-neuraminidase (HN) and the large protein (L).
An exemplary PIV5 genome is shown in
The HN, F and SH proteins protrude through the virus envelope; the HN protein binds the virus to its target cell, and together with the F protein, facilitates virus entry into the cell to initiate the replication process. The matrix protein is located on the inner surface of the virus envelope and plays an essential role in virion assembly. The genomic RNA is encapsidated by NP, forming a flexible helical nucleocapsid complex that is associated with the viral RNA-dependent RNA polymerase complex (vRdRP) consisting of L and P (see
In view of the above, the disclosure provides a cohort of products derived from parainfluenza virus 5 (PIV5) and the genome thereof. All of the disclosed products may be for use as described above.
In one aspect, the viral genome derived product is a PIV5 vector. A PIV5 vector may comprise one or more wild-type PIV5 genes and a heterologous nucleic acid sequence for expression. Conversely, a PIV5 vector may (relative to a wild-type PIV5 genome) lack one or more wild-type PIV5 genes and comprise a heterologous nucleic acid sequence for expression.
The PIV5 vectors described herein may be used to generate recombinant proteins and/or to deliver therapeutic proteins.
The described PIV5 vectors are advantageous because:
By way of example, a PIV5 vector of this disclosure may lack, relative to a wild type PIV5 genome, one or more of the wild-type PIV5 gene(s). In one teaching a PIV5 vector of this disclosure may be characterised by, relative to a wild-type PIV5 genome, the full or partial deletion of any one or more of the wild-type genes.
In one teaching, a PIV5 vector of this disclosure may be characterised by the (functional) deletion of:
The disclosed PIV5 vectors may be classed as single cycle vectors. Thus, a PIV5 genome derived product of this disclosure may be a single cycle vector (i.e. a PIV5 single cycle vector).
In one teaching, a PIV5 single cycle vector lacks, as compared to the wild-type PIV5genome, a functional copy of one or more of the wild-type PIV5 genes.
Without wishing to be bound by theory, although a single cycle vector can infect cells it cannot make infectious virus particles unless the missing protein(s) (encoded by the (functionally) deleted genes) are provided in trans during its replication. To facilitate this, helper cell-lines capable of (inducibly) expressing the missing protein(s) can be used. Expression of the missing protein may be inducible; for example expression may be induced by contact with, or addition of, an inducing agent.
In one teaching, a PIV5 vector of this disclosure may, relative to a wild-type PIV5 genome, comprise a (functionally) deleted F gene. A PIV5 vector which lacks the F gene may be referred to as single cycle vector and may be designated PIV5ΔF. While a PIV5ΔF vector can infect cells, it cannot make infectious virus particles unless the missing protein(s) (encoded by the (functionally) deleted genes) are provided during its replication. To facilitate this, helper cell-lines capable of (inducibly) expressing the missing protein(s) can be used. The helper cells in effect provide the missing protein(s) (encoded by the (functionally) deleted genes) in trans during replication. Without wishing to be bound by theory, it is suggested that PIV5ΔF vectors may be particularly useful where a high viral titre is preferred—for example for use in raising immune responses, inducing immune responses and/or for application in vaccine relate methods.
In one teaching, a PIV5 vector of this disclosure may, relative to a wild-type PIV5 genome comprise a (functionally) deleted M and F genes (thereby rendering it unable to express the M and F). A PIV5 vector which lacks the M and F genes may be referred to as single cycle vector and may be designated PIV5ΔM/F. As stated, a PIV5ΔM/F vector may be rescued by a helper cell line that expresses the missing (or deleted/functionally deleted) wild-type PIV5 genes in trans.
In one teaching, a PIV5 vector of this disclosure may, relative to a wild-type PIV5 genome comprise a (functionally) deleted M gene, F gene and HN gene (thereby rendering it unable to express the M, F and HN proteins). A PIV5 vector which lacks the M gene, F gene and HN gene may be referred to as single cycle vector and may be designated PIV5ΔM/F/HN. As stated, a PIV5ΔM/F/HN vector may be rescued by a helper cell line that expresses the missing (or deleted/functionally deleted) wild-type PIV5 genes in trans. In another teaching, a PIV5 vector of this disclosure may, relative to a wild-type PIV5 genome comprise (functionally) deleted M gene, F gene, P gene, L gene and HN gene (thereby rendering it unable to express the M, F, P, L and HN proteins). A PIV5 vector which lacks the M, F, P, L and HN gene may be referred to as single cycle vector and may be designated PIV5ΔM/F/P/L/HN. As stated, a PIV5ΔM/F/P/L/HN vector may be rescued by a helper cell line that expresses the missing (or deleted/functionally deleted) wild-type PIV5 genes in trans.
In a further teaching, a PIV5 vector of this disclosure may comprise, consist of or consist essentially of, a functional NP gene. Where the PIV5 vector comprises, consist of or consists essentially of a functional copy of the NP gene, any of the other (missing, deleted and/or functionally deleted) wild type PIV5 genes may (as described below) be provided in trans—for example by a helper cell which expresses those missing wild-type PIV5 genes (see description below). Without wishing to be bound by theory, it is suggested that by retaining the NP gene within the vector, the NP concentration builds up inside the helper cell and this facilitates the switch from “transcription mode” to a “replication mode”.
Any of the PIV5 vectors/single cycle vectors described herein, including for example PIV5ΔF and any PIV5 vector which lacks the M, F and HN genes, may further comprise a heterologous sequence for expression. The heterologous sequence may encode a therapeutic protein or a recombinant protein.
As such, the disclosure provides a PIV5 vector or PIV5 single cycle vector as described herein, comprising a heterologous sequence for expression in a cell, wherein relative to the wild-type PIV5 genome, the vector lacks one or more (functional) copies of a wild-type gene.
Without wishing to be bound by theory, it is suggested that the PIV5 vectors disclosed herein can establish persistent infections.
In order to make infectious particles, the vectors described herein, including the PIV5ΔF vectors and any PIV5ΔM/F/HN or PIV5ΔM/F/P/L/HN vectors, the missing PIV5 virus proteins may be provided in trans, for example through the use of helper cell lines. In the case of PIV5ΔF, only the F protein need be provided in trans. For PIV5ΔM/F/HN vectors the M, F and HN proteins need to be provided in trans. The present disclosure further provides helper cell lines which express the M and/or F and/or HN proteins (the exact helper cell PIV protein expression profile depending on the features of the corresponding PIV5 vector). These helper cell lines have been successfully used to rescue infectious particles that can in turn be used to infect cells in which the vector persists. Prior art methods of rescuing viral derived vectors may include methods which exploit the transient transfection of plasmids into cells. However, such methods result in a relatively low yield of “infectious” particles. In contrast, the yield infectious particles from a helper cell expressing the F protein is high (a high titre) similar to the wild type virus.
Any of the disclosed PIV5 vector(s)/PIV5 single cycle vector(s) may be used to deliver therapeutic proteins in vivo. Accordingly, disclosed is a method of administering a therapeutic protein, said method comprising administering a PIV5 vector/PIV5 single cycle vector according to this disclosure, which vector comprises a nucleic acid encoding the therapeutic protein for delivery. The method may be for in vivo and in vitro use. Where the method is for in vitro use, the administering step, may be replaced with a step in which a cell is contacted with a PIV5 vector or PIV5 single cycle vector of this disclosure.
The method may further require inducing the expression of the heterologous sequence.
In one teaching, PIV5 vectors of this disclosure and high titre PIV5 vectors, for example PIV5ΔF, can be grown in helper cell-lines. As stated, a helper cell line may (heterologously) express (via induction) the proteins which the PIV5 vector is unable to express. By way of example, where the vector is a PIV5ΔF vector, a helper cell may express the PIV5 F protein. This generates an infectious PIV5ΔF which can then be used to infect other cells (both in vitro and in vivo) to produce the required recombinant protein (for example an antibody). However, due to the lack of F protein expression, no infectious virus will be produced in these cells.
By way of a further example, where the vector lacks the M, F and HN genes, a helper cell may express the PIV5 M, F and HN proteins. This will generate an infectious PIV5 vector which can then be used to infect other cells (both in vitro and in vivo). However, due to the lack of M, F and HN protein expression, no infectious virus will be produced in these cells. In one teaching a vector of this disclosure (for example an Indel expression vector as described herein) expressing the relevant PIV5 viral protein (for example the F protein of PIV5) can be used as an alternative to a helper cell line.
A single cycle vector according to this disclosure, including, for example a PIV5ΔF vector is safe to use in any cell or system which does not (heterologously) express the necessary PIV proteins, for example (in the case of the PIV5ΔF vector), the PIV5 F protein. Indeed a single cycle vector according to this disclosure, including, for example a PIV5ΔF vector, is safe for use in patients with underlying health conditions and/or with cancer.
The single cycle vectors of this disclosure can be designed with an acute or persistent phenotype.
In another aspect, the invention provides a method of making a single cycle vector, said method comprising deleting or functionally deleting the F protein from the PIV genome.
This disclosure may provide helper cell lines. These helper cell lines may be engineered to express (potentially by induction) one or more of the PIV5 genes missing from the PIV vector. Thus the helper cell provides the PIV5 vector with the missing genes in trans.
A helper cell line may comprise any cell which is permissive to a PIV5 virus. For example, the helper cell may be a Vero cell.
A helper cell may express one or more PIV5 proteins. The PIV5 protein expression of the helper cell may correspond to the proteins that the PIV5 vector does not express. For example, a helper cell of this disclosure may be modified to express:
The disclosure provides a method of replicating a PIV5 vector of this disclosure, said method comprising culturing or replicating a PIV5 vector in a helper cell of this disclosure.
The disclosure provides a method of replicating a PIV5ΔF vector of this disclosure, said method comprising culturing or replicating a PIV5ΔF vector in a helper cell which expresses the PIV5 F protein.
The disclosure provides a method of replicating a PIV5ΔM/F/HN vector of this disclosure, said method comprising culturing or replicating a PIV5ΔM/F/HN vector in a helper cell which expresses the PIV5 M, F and HN protein.
In a further aspect the viral genome derived product is disabled helper paramyxovirus.
The viral genome derived product is disabled a defective interfering (DI) particle. DIs are subgenomic and often contain extensive deletions that render the DIs unable to complete a full replication cycle in the absence of a coinfecting, non-defective “helper” virus.
In one teaching, the viral genome derived product is a defective interfering (DI) particle derived from:
DIs derived from any of the above sources may be referred to as “copyback” DIs or “internal deletion” (Indel) DIs.
As such, this disclosure provides PIV5 derived copyback DIs.
The disclosure further provides PIV5 derived internal deletion (Indel) DIs.
In a copyback DI, the 3′ genomic promoter has been replaced by a sequence complementary to the 5′ antigenomic promoter; this is due to template switching from the antigenome to the nascent strand during synthesis of genomic RNA; as a consequence, the copyback DI cannot be transcribed. The termini of copyback DIs are thus complementary and form a dsRNA stem-loop structure when SDS treatment is used to dissociate the RNA genomes from encapsidating NP protein.
A PIV5 derived copyback DI of this disclosure may comprise the 3′ replication promoter of the PIV5 genome, duplicated and in the opposite orientation together with some of the L gene sequence.
A DI according to this disclosure may be obtainable by:
The term ‘relevant helper F cell’ may comprise any cell which expresses the necessary missing PIV5 wild type genes. For a PIV5ΔM vector a helper cell may express the PIV5 M gene in trans (to provide the PIV5 M protein); for a PIV5ΔF/M vector, a helper cell may express the PIV5 F and M genes in trans (to provide the PIV5 F and M proteins) and for a PIV5ΔF vector, a helper cell may express the PIV5 F gene (to provide the PIV5 F protein) in trans.
In one teaching, the passaging may be done at a high multiplicity of infection.
Additionally or alternatively (and again without being bound by theory), it has been noted that a copyback DI may be best or most efficiently produced using a PIV5 virus/vector (including any vector of this disclosure-for example a single cycle vector; a PIV5ΔM, a PIV5ΔF/M or a PIV5ΔF vector) having an acute but not persistent phenotype. It is noted that a switch between a virus/vector with a persistent to acute phenotype may be achieved through the use of a single nucleotide change (resulting in single or point amino acid changes). For example, the inventors note a serine to phenylalanine change at position 157 in the PIV5 P protein (as encoded by the PIV5 P gene) can change a PIV5 virus/vector from having a persistent phenotype to having an acute/lytic phenotype (a limited number of other changes can achieve the same switch). This is in contrast to methods or uses for the preparation of cell lines which constitutively produce recombinant proteins, those cell lines may comprise vectors (of this disclosure) with a persistent phenotype.
Accordingly, a DI according to this disclosure may be obtainable by:
DIs obtainable by the above-described passaging method, may comprise copyback DIs.
A PIV5 derived copyback DI obtainable by the disclosed method may comprise the 3′ replication promoter of the PIV5 genome, duplicated and in the opposite orientation together with some of the L gene sequence.
A copy back DI of this disclosure may act as a potent inducer of: innate immune responses, aspects and features of the innate immune system and, for example, IFN.
A copyback DI of this disclosure may be:
The disclosure further provides a method of modulating, improving or augmenting the immune response to an antigen or vaccine, said method comprising immunising a subject with the vaccine or antigen and a DI of this disclosure.
Moreover, the disclosure provides a method of treating or preventing:
The term ‘subject’ may refer to any human or animal subject having or suspected of having any relevant disease, condition, infection or cancer. The term ‘subject’ may further embrace any human or animal subject predisposed or susceptible to any relevant disease, condition, infection or cancer.
It should be noted that Copyback DIs do not express any viral proteins (and cannot currently be used as expression vectors); they are generated by mistakes during virus replication. Therefore, and without wishing to be bound by theory, it is suggested that PIV5 copy-back DIs are safe to use in vivo, including in immunosuppressed individuals.
A PIV5 copy-back DI of this disclosure may be for use in the induction of an innate immune response both in vitro and in vivo. Without wishing to be bound by theory, the engagement of a PIV5 derived copy-back DI with pathogen recognition receptors (PRRs) activates a number of cellular kinases and transcription factors (e.g., IRF3, NF-κB) that regulate the expression of several cytokines, including, for example, IFNs, tumour necrosis factor (TNF), and interleukin 6 (IL-6), and can stimulate DC maturation and enhance antigen specific immunity to pathogen associated antigens.
In another aspect the viral genome derived product is an Indel vector (this may also be referred to as an Indel DI).
Indel vectors do not express any viral (PIV5) proteins but retain the 3′ and 5′ ends of the virus genome (with the Le and Tr sequences) and therefore possess at least the transcription and replication signals essential for virus transcription and replication.
Transcription and replication of the disclosed Indel vectors is dependent on additional viral proteins being expressed either in trans, for example in helper cell-lines, or by co-infection with helper viruses including, for example helper virus products derived from PIV5.
A potential advantage of such vectors is that they have a very high coding capacity. For example, the coding capacity of any of the PIV5 vectors described herein may include heterologous sequences in the region of 16-20 kb. One of skill will appreciate that the size of a heterologous insert may depend on the number and size of the PIV5 genes retained.
The disclosed ‘Indel’ vectors may be to express heterologous sequences (e.g. genes, or combination of genes) at sizes up to, for example 16-20 kb.
Thus in one teaching, the viral genome derived product is an Indel vector derived from a Paramyxovirus, for example, PIV5.
PIV5 derived Indel vector of this disclosure may comprise a nucleic acid sequence which, relative to a wild-type PIV5 genome, lacks all of the NP, P/V, M, F, SH, HN and/or L genes. As stated, a PIV5 Indel vector may retain (or comprise) the 3′ Le and 5′ Tr sequences of PIV5 viral genome (i.e. the ends comprising the Le and Tr sequences).
A PIV5 Indel vector may further comprise one or more heterologous nucleic acid sequences. The heterologous sequence(s) may encode recombinant and/or therapeutic proteins. The heterologous sequence(s) may be for expression in a cell. The heterologous sequence(s) may be inducibly expressible. A PIV5 derived Indel vector of this disclosure may comprise, or further comprise a nucleic acid sequence encoding a heterologous protein. It should be understood that the Indel vectors of this disclosure can be modified to receive almost any heterologous sequence encoding almost any sort of heterologous protein. For example, the Indel vectors of this disclosure may comprise, for example, heterologous nucleic acid sequences which encode any one or more of the following categories of protein:
By way of example only, a PIV5 derived Indel vector of this disclosure may comprise a nucleic acid sequence encoding a SRAS CoV-2 antigen for example the SRAS-CoV-2 spike protein.
In a further example, the PIV5 derived Indel vector of this disclosure may comprise a nucleic acid sequence encoding interferon (IFN).
In a further teaching, a PIV5 derived Indel vector of this disclosure may comprise a nucleic acid sequence encoding all or part of an antibody. The PIV5 derived Indel vector may be modified to express the heavy and/or light chain of an antibody or any fragment(s) thereof. In one teaching, the PIV5 derived Indel vector may be modified to include nucleic acid encoding the heavy and/or the light chain(s) of the humanised anti-V5 mAb.
A PIV5 derived Indel vector of this disclosure may comprise or further comprise an internal ribosome entry site (IRES).
The PIV5 derived Indel vector of this disclosure may comprise or further comprise a nucleic acid sequence encoding a heterologous reporter moiety, for example an optically detectable reporter moiety such as a fluorescent protein. Examples may include nucleic acid sequences which encode fluorescence proteins such as mCherry and/or GFP.
A PIV5 derived Indel vector of this disclosure may comprise, or further comprise a nucleic acid sequence for use in a method of selection or enrichment. For example, A PIV5 derived Indel vector of this disclosure may comprise, or further comprise an antibiotic resistance gene. Examples may include genes which encode an enzyme which inactivates or neutralises an antibiotic. A PIV5 derived Indel vector of this disclosure may comprise, or further comprise a nucleic acid sequence encoding enzymes which provide blasticidine and/or puromycin resistance.
A PIV5 derived Indel vector of this disclosure may comprise or further comprise a nucleic acid encoding a reporter gene, for example an optically detectable moiety, a fluorescent protein. Examples may include nucleic acid sequences which encode fluorescence proteins such as mCherry and/or GFP.
A PIV5 derived Indel vector of this disclosure may comprise or further comprise an internal ribosome entry site (IRES).
An Indel DI of this disclosure may find application as an expression vector for in vitro and in vivo use. An Indel DI of this disclosure may be used in the production of recombinant/therapeutic proteins.
Without wishing to be bound by theory, Indel DIs do not encode any viral proteins and are thus unable to replicate or be packaged into infectious particles without the co-expression of the appropriate viral replication and/or structural proteins. These co-expressed and essential proteins can be provided by:
Helper cell-lines which inducibly co-express, for example, any of the M, F, NP, V, P, L and/or HN PIV5 proteins may facilitate the replication of any of the disclosed Indel DIs—in turn this will facilitate the expression of heterologous proteins in the absence of co-infecting virus.
The disclosure further provides helper cell-lines which inducibly co-express all the PIV5 viral proteins. Cell lines of this type may facilitate the replication and packaging of DIs in the absence of co-infecting virus.
The present disclosure provides the disclosed copyback DIs for use in (directly or indirectly) modulating gene expression. The expression of one or more of the following genes 49 genes (see table 1) may be modulated by a copyback DI of this disclosure. The modulation may be direct modulation or indirect modulation occurring via the action of multiple pathways and/or cytokines in response to the presence of a copyback vector of this disclosure.
The modulated gene expression may occur in a cell, for example a cancer cell. The modulated gene expression may occur in an adenocarcinomec human alveolar basal epithelial cell, for example an A549 cell.
The present disclosure further provides a method of modulating the expression of any one or more of the above disclosed genes, said method comprising contacting the gene the expression of which is to be modulated, with a disclosed copyback DI.
The present disclosure further provides a (in vitro or in vivo) method of modulating the expression of any one or more of the above disclosed genes in a cell (for example a cancer cell, an adenocarcinoma human alveolar basal epithelial cell or an A549 cell), said method comprising contacting the cell with a disclosed copyback DI.
The term modulation may embrace any up or down regulation of the expression of any of the genes listed above. For example, the disclosed copyback DIs may be used to upregulate the expression of any one or more of the above listed genes.
Without wishing to be bound by theory, the disclosed copyback DIs may initiate innate signalling through the RIG-I/mda5 pathway; this may lead to the transcriptional activation of many cellular immune genes in addition to IFN genes.
The present invention will now be disclosed by reference to the following Figures which show:
This disclosure provides:
To further study the molecular biology of PIV5 and its interaction with cells we have generated a number of recombinant viruses that express mCherry facilitating the rapid and easy identification of infected cells (
Paramyxoviruses, including PIV5, spontaneously generate defective interfering virus genomes (DIs) due to errors during replication. These DIs are subgenomic and contain deletions (often extensive) that render the DIs unable to complete a full replication cycle in the absence of a coinfecting, non-defective “helper” virus. Paramyxovirus DIs may be “internal deletion” or “copyback” and these two types of DIs differ considerably in their genome structures (
Internal deletion DIs retain the Le and Tr sequences of the genome and therefore possess transcription and replication signals and have been shown to generate viral translation products. In contrast, the 3′ genomic promoter in trailer copyback DI [DI(TrCB)] genomes has been replaced by a sequence complementary to the 5′ antigenomic promoter (and therefore cannot be transcribed) due to template switching from the antigenome to the nascent strand during synthesis of genomic RNA; the termini of DI(TrCB)s are thus complementary and form a dsRNA stem-loop structure when SDS treatment is used to dissociate the RNA genomes from encapsidating NP protein. This structure is thought to be responsible for the ability of DI(TrCB)s to act as potent inducers of IFN.
Paramyxoviruses are poor activators of early innate immunity for two main reasons. First, they encode interferon (IFN) antagonists that can both inhibit the activation of the IFN-induction cascade and can block IFN signalling. In the case of PIV5, its IFN antagonist, the V protein, interacts with, and blocks the activity of melanoma differentiation-associated protein 5 (MDA5), as well as binding to the protein called laboratory of genetics and physiology 2 (LGP2) to negatively regulate retinoic acid-inducible gene I (RIG-I). In addition, PIV5 V targets STAT1 for proteasome-mediated degradation to block IFN-signalling.
Paramyxoviruses also tightly control virus transcription and replication, thereby limiting the production of pathogen-associated molecular patterns (PAMPs) that active pathogen recognition receptors (PRRs) and the IFN response. However, during replication paramyxoviruses make mistakes, including the generation of copyback DVGs. Copyback DVGs are powerful inducers of innate immune responses both in vitro and in vivo. DVG engagement of PRRs activates a number of cellular kinases and transcription factors (e.g., IRF3, NF-κB) that regulate (directly or indirectly) the expression of several cytokines, including IFNs, tumour necrosis factor (TNF), and interleukin 6 (IL-6), and can stimulate DC maturation and enhance antigen specific immunity to pathogen associated antigens.
To visualise the ability of viruses to induce innate immune responses we generated a cell-line that expresses GFP following activation of a number of innate intracellular responses, including the IFN pathway. Using this cell-line we showed that the IFN-induction cascade was only activated in a very few cells infected with preparations PIV5 that had few copyback DIs. In contrast, preparations of PIV5 rich in copyback DIs activate the IFN-response in most infected cells (
Given the ability of paramyxovirus copyback DIs to induce powerful innate immune responses in vivo, it has been suggested that they may be used therapeutically in a variety of clinical settings, for example:
A disabled PIV5 vector of this disclosure may have a range of therapeutic purposes; this includes generation of copyback DIs for use in cancer patients. The fusion (F) protein of PIV5 is essential to initiate entry of the virus into cells for replication to occur. To develop a safe PIV5 vector that could be used in patients with underlying health issues, such as cancer patients, the F gene was deleted from PIV5, generating a vector termed PIV5ΔF (
Whilst PIV5ΔF can infect cells it cannot make infectious virus particles unless the F protein is provided in trans during its replication. To facilitate this, helper cell-lines are provided. In these cell-lines, the expression of F is induced by the addition of Dox to the tissue culture medium (
Infectious PIV5ΔF can be readily grown in these F-helper cell-lines (
Generation of Copyback DIs with wt PIV5 and PIV5ΔF
By passaging wt PIV5 in tissue culture cells, and PIV5ΔF in helper F-expressing tissue culture cells, at high multiplicities of infection, copyback DIs are generated that are powerful inducers of innate immune responses (
The disclosed PIV5 vectors able to establish persistent infections for the production of recombinant proteins in vitro and may be used to delivering therapeutic proteins in vivo.
There are a number of advantages over other (prior art) systems
PIV5, unlike most other RNA viruses, can readily and immediately establish persistent infections in tissue culture cells. Persistently infected tissue culture cells can be readily passaged. Cell-lines can be readily made that persistently express heterologous proteins following infection with a variety of recombinant PIV5 viruses, including wt PIV5 and PIV5ΔF, exemplified by expression of mCherry, respectively (see
Internal deletion (Indel) DIs retain the Le and Tr sequences of the genome and therefore possess transcription and replication signals and can therefore be developed as novel expression vectors for in vitro and in vivo production of recombinant/therapeutic proteins. Indel DIs do not encode any viral proteins and are thus unable to replicate or be packaged into infectious particles without the co-expression of the appropriate viral replication and structural proteins. These proteins can be provided by:
An Indel expression vector that express the F protein of PIV5 can be used to rescue PIV5ΔF.mCherry and may be an alternative method to using helper cell-lines for the rescue PIV5ΔF vectors for the production of recombinant/therapeutic proteins in vitro and in vivo.
Indel Vectors Can be Replicated in Cells Co-Infected with PIV5 Min-Replicons That Express the NP, P and L Proteins.
Mini replicons can replicate independently of helper virus as they express the replication proteins (NP, P and L) but they cannot be packaged into infectious virus particles as they do not express the structural M, F and HN proteins. However, mini-replicons can be rescued and packaged into infectious particles by transient expression of the M, F and HN proteins. Alternatively, disclosed are cell-lines which inducibly express the M, F and HN proteins (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2209861.0 | Jul 2022 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2023/051766 | 7/5/2023 | WO |
