Patent Abstract:
methods of making vaccinia viruses for gene - directed prodrug therapy are disclosed and their use in the treatment of disease is provided . in particular , the anti - tumor effects of vaccinia viruses that are modified to express a prodrug - activating enzyme are disclosed .

Detailed Description:
gdept is a two - step treatment for tumours . foreign , prodrug - activating enzymes are delivered to , and expressed in , target cells where they can activate subsequently administered non - toxic prodrugs to form active drugs . fig2 shows a scheme of a gdept system in which , in a first step , a tumour - infecting virus is used to deliver the gene expressing the prodrug - activating enzyme . in the second step , a prodrug is administered that can be activated to form a toxic drug by the enzyme that has been expressed in the tumour . the prodrug - activating enzyme gene should be expressed exclusively , or with a relatively high ratio , in tumour cells compared with normal tissues and blood , and should achieve a sufficient concentration for clinical benefit . after gene delivery , prodrug administration should be delayed to permit protein expression in the targeted cells . the catalytic activity of the expressed enzyme should be sufficient for activation of the prodrug . since expression of the prodrug - activating enzymes will not occur in all cells of a targeted tumour in vivo , a bystander cytotoxic effect is beneficial , whereby the prodrug is cleaved to an active drug that kills not only tumour cells but also neighbouring non - expressing tumour cells . this means that expression in less than 100 % of tumour cells can still result in killing of all tumour cells . the prodrug - activating enzyme is usually expressed intracellularly , but it may be a membrane - tethered variant that is directed to the cell surface . a number of different prodrug - activating enzymes have been used in gdept therapy . for example , cytosine deaminase , hsv thymidine kinase and cytochrome p450 have been used in gdept ( hedley et . al ., nat . rev . can . 7 , 870 - 879 , 2007 ). any one of these enzymes can be used with vaccinia virus according to the present invention . the gdept system developed by the present inventors uses the enzyme carboxypeptidase g2 ( cpg2 ) from pseudomonas strain rs16 . peptidases are a class of enzymes which act upon a substrate to cleave a peptide ( amide ) bond ; carboxypeptidases cleave peptide bonds at the carboxy - terminal of a protein or peptide . in some embodiments of the present invention , the prodrug - activating enzyme is a carboxypeptidase , which converts the prodrug into an active drug by removing a protecting group from the prodrug . the prodrug - activating enzyme may be a glutamate carboxypeptidase , e . g ., cpg1 or cpg2 , which preferentially cleave glutamate and glutamate analogues from the prodrug . the preferred enzyme is truncated carboxypeptidase cpg2 from pseudomonas strain rs16 ( disclosed in wo88 / 07378 ), having the sequence shown in seq id no : 2 , although full - length cpg2 , membrane anchored cpg2 , other mutants , orthologues , homologues or variants of cpg2 may also be used . in the context of this application , a cpg2 homologue is an enzyme that shares a common ancestry with cpg2 , which is able to convert a prodrug to an active drug at substantially the same rate as the cpg2 of seq id no : 2 . in the context of this application , cpg2 mutants have an amino acid sequence consisting of at least 80 %, at least 90 %, at least 95 %, at least 98 %, or at least 99 % of seq id no : 2 and also optionally comprise a further amino acid sequence derived from another protein . variants may comprise an amino acid sequence which has at least 80 %, at least 90 %, at least 95 %, at least 98 %, or at least 99 % identity to seq id no : 2 . variants may be encoded by a nucleotide sequence which has at least 80 %, at least 90 %, at least 95 %, at least 98 %, or at least 99 % identity to seq id no : 1 . sequence comparison may be made over the full - length of the relevant sequence shown herein , or may more preferably be over a contiguous sequence of about or greater than about 20 , 25 , 30 , 33 , 40 , 50 , 67 , 133 , 167 , 200 , 233 , 267 , 300 , 333 , or more amino acids or nucleotide triplets , compared with the relevant amino acid sequence or nucleotide sequence as the case may be . alterations to the sequence will be such that the enzyme retains its ability to convert a prodrug to an active drug at substantially the same rate as the native enzyme . in this context , “ substantially the same rate ” will desirably be within 1 order of magnitude , and preferably from about 50 - fold e . g . about 2 - fold less to 2 , 5 or 10 fold more . cpg2 variants used according to the invention may be engineered to be directed to the cell membrane ( i . e . membrane - tethered cpg2 ), as described in wo 01 / 085960 , to enable prodrugs with poor intracellular availability to be activated in the extracellular space . alternatively , other bacterial carboxypeptidase enzymes may be used , e . g ., cpg2 enzymes from other pseudomonas species such as pseudomonas aeruginosa , pseudomonas cepacia , pseudomonas fluorescens , pseudomonas putida , pseudomonas syringae , pseudomonas savastanoi . preferred carboxypeptidases will have one or more of the following properties : immunological cross - reactivity with an antibody reactive to the polypeptide for which the sequence given in seq id no : 2 ; sharing an epitope with the polypeptide for which the amino acid sequence is shown in seq id no : 2 ( as determined for example by immunological cross - reactivity between the two polypeptides ); a biological activity which is inhibited by an antibody raised against the polypeptide whose sequence is shown in seq id no : 2 ; ability to release l - glutamic acid from benzoic acid mustard prodrugs . alteration of sequence may change the nature and / or level of activity and / or stability of the carboxypeptidase enzyme . a preferred substrate for cpg2 is an l - glutamic acid group , linked to an aromatic ring via an amidic , carbamic , or ureidic linkage . glutamic acid analogs are also acceptable substrates . for example , l - glutamic acid modified at the γ - carbon ( e . g ., with an amide , — conh 2 , instead of an acid , — cooh ) also serves as a suitable substrate for cpg2 . a range of prodrugs have been used in gdept therapy . for example , 5 - fluorocytosine has been used in cytosine deaminase gdept systems , ganciclovir has been used in hsv thymidine kinase gdept systems and cyclophosphamide has been used in cytochrome p450 gdept systems ( hedley et . al ., nat . rev . can . 7 , 870 - 879 , 2007 ). any one of these prodrug / enzyme systems can be used with vaccinia virus according to the present invention . the gdept system that the present inventors have developed uses the enzyme carboxypeptidase g2 ( cpg2 ) to activate prodrugs such as benzoic acid nitrogen mustard prodrugs , aniline nitrogen mustard prodrugs and phenol nitrogen mustard prodrugs such as zd2767p ( n -( 4 -[ bis ( 2 - iodoethyl ) amino ] phenoxycarbonyl )- l - glutamic acid ) and salts thereof . cpg2 cleaves the carbamate linkage of zd2767p to release l - glutamic acid , a carbon dioxide molecule and the dna alkylating nitrogen mustard n -( 4 -[ bis ( 2 - iodoethyl ) amino ] phenol , which is a potent cytotoxic agent . fig1 shows the cpg2 - catalysed conversion of zd2767p to produce n -( 4 -[ bis ( 2 - iodoethyl ) amino ] phenol . nitrogen mustards are related to sulfur mustard , ( clch 2 ch 2 ) 2 s , the “ mustard gas ” used during the first world war . nitrogen mustards have the general formula ( clch 2 ch 2 ) 2 nr . in vivo , each 2 - chloroethyl side - chain undergoes an intramolecular cyclisation with the release of a chloride ion . the resulting highly reactive ethylene immonium derivative can interact with dna and other molecules , for example , as an alkylating and / or crosslinking agent . nitrogen mustards are useful , for example , in the treatment of proliferative conditions , such as cancer . nitrogen mustard analogues , in which the chloro group is replaced by other groups , such as other halogens ( e . g ., bromo and iodo , as exemplified by zd2767p ) and other good leaving groups ( e . g ., sulfonates , such as mesyloxy , — oso 2 me ) are also known , and are included in the class denoted “ nitrogen mustards ”. nitrogen mustards may conveniently be grouped according to the group r . for example , two groups are phenolic nitrogen mustards and anilinic nitrogen mustards . in gdept systems , nitrogen mustards or other cytotoxic drugs are provided in the form of a prodrug , which has a protecting group linked to the cytotoxic moiety . in most cases , the protecting group will be cleaved as a whole from the prodrug . however , it is also possible for the enzyme to cleave or simply alter part of the protecting group , resulting in a partially cleaved or altered protecting group which is unstable , leading to spontaneous removal of the remainder of the group . a range of prodrugs which are suitable substrates for cpg2 have been described previously ( wo 94 / 002450 , wo 04 / 020400 ). a partial structure of typical substrates is ; - aromatic - ring - x — co — nh - glu , where x is — nh —, — o — or — ch2 , and glu is glutamic acid or a glutamic acid analogue . besides zd2767p , other nitrogen mustard prodrugs which can be used according to the present invention in cpg2 gdept systems include : wo 04 / 020400 describes “ self - immolative ” cpg2 substrates , which are defined as compounds that , following an activation process , generate an unstable intermediate that releases the active drug in a number of subsequent steps . hence , cpg2 is able to activate these self - immolative prodrugs by cleaving or simply altering part of the prodrug protecting group , resulting in a partially cleaved or altered protecting group which is unstable , leading to spontaneous removal of the remainder of the group . the skilled person would understand that any of the prodrugs described herein , or any other compounds that can be used with the prodrug - activating enzymes considered herein , can be used according to the invention without undue burden . vaccinia virus is a member of the poxviridae family of large dna viruses , which typically have a genome of between 130 - 300 kbp in size , which is encoded on a single length of linear double - stranded dna . to accommodate the relatively large quantity of genetic material , poxviruses have a large capsid having dimensions of around 200 nm by 300 nm . their large dna carrying capacity allows poxviruses to be engineered to deliver multiple transgenes and / or transgenes of a large size . poxvirus virions take different forms at different stages of their lifecycle . the extracellular enveloped virion is the infectious particle which binds and enters the cell . the extracellular enveloped virion ( eev ) has a viral core within a capsid particle , which is sheathed in an outer membrane . the outer membrane is removed upon cell entry , thus defining a first part of the two - part process of uncoating of the eev . in the second part of the uncoating process , the viral core containing the viral dna is released into the cytoplasm . vaccinia genes are expressed in the cytoplasm , and this process takes place in several stages including early gene expression , intermediate gene expression and late gene expression . genes which are necessary for genome replication , e . g . viral dna - dependent rna polymerase , are early phase genes . genes which modulate the host anti - viral response and promote virus replication are also predominantly expressed during the early phase of infection . in contrast , viral structural components are mainly expressed during the late phase , with a relatively small group of genes thought to be important in activating late gene expression being expressed in the intermediate phase . structural genes assemble in the cytosol to form the intracellular mature virion ( imv ), which becomes coated with two membranes by the golgi apparatus to form the intracellular enveloped virion ( iev ). the iev is translocated along the cytoskeletal microtubules to the cell periphery where it fuses with the cell membrane to form the cell - associated enveloped virion ( cev ), which is released as eev when the cell is lysed , causing cell death . vaccinia virus according to the present invention will predominantly be in the form of eev , however even the intracellular forms of the vaccinia virion are known to be infectious and are also considered to form a part of the invention . the vaccinia virus genome contains many virulence genes which function to modulate the host anti - viral response or disrupt the host cell cycle in order to promote intracellular conditions suitable for viral replication . for example , vaccinia viruses contain genes which express thymidine kinase , interferon - binding proteins , serine proteinase inhibitors and epidermal growth factor receptor ( egfr )- binding growth factors . mutations to members of each of these classes of vaccinia virulence genes in order to enhance vaccinia selectivity for infecting tumour cells have been previously reported ( kirn et al ., nat . rev . can ., 9 , 64 - 71 , 2009 ). vaccinia virus having a deletion of the type i ifn - binding binding protein selectively replicates in tumours with a loss of interferon response . deletion of the serine proteinase inhibitors spi - 1 and spi - 2 , which interfere with host antiviral defence , results in vaccinia that preferentially replicate in transformed cells . deletion of vaccinia thymidine kinase leads to dependence of the virus on cellular thymidine kinase expression , which is constitutively expressed in the majority of cancers , regardless of proliferation status ( kirn et al ., nat . rev . can ., 9 , 64 - 71 , 2009 ). vaccinia viruses have been shown to efficiently infect and kill a range of tumour types including lung cancer , cervix cancer and cns cancer ( a ascierto et al ., bmc cancer , 11 : 451 , 2011 ) and melanoma , ovarian cancer , kidney cancer , breast cancer , leukemia , non - small cell lung carcinoma , colon cancer , brain tumour , prostate cancer ( parato et al ., mol . ther ., 20 ( 4 ), 749 - 758 , 2012 ). the tumour selectivity of a virus can be measured or quantified by using standard techniques . for example , viral dna copy number in tumour samples can be compared with viral dna copy number in non - tumour samples by performing the polymerase chain reaction ( pcr ) on each sample , thereby allowing the skilled person to quantitatively assess the degree of tumour selectivity of a virus . the skilled person would understand that alternative techniques such as viral protein - specific enzyme - linked immunosorbent assay ( elisa ) may also be used to determine and compare viral load in in tumour and non - tumour samples . the present inventors have found that vaccinia viruses which are modified to express cpg2 are capable of eliciting a potent anti - tumour response in vivo when used in gdept protocols together with a suitable prodrug . dna expressing cpg2 under the control of the vaccinia p7 . 5 promoter was inserted into the vaccinia thymidine kinase ( tk ) gene , resulting in vaccinia virus which has inactivated thymidine kinase . the vaccinia growth factor ( vgf ) gene , which is homologous to epidermal growth factor , was also deleted . furthermore , dna expressing luciferase under the control of the vaccinia p syn ( e / l ) promoter was also inserted to allow infected cells to be conveniently identified . fig3 shows the orientation of luciferase and cpg2 in the tk gene of vv - cpg2 . authenticated fadu , sw620 and wm266 . 4 were obtained from american type culture collection ( lgc promochem ). fadu cells are a telomerase - positive human carcinoma cell line . the cell lines used for virus production and characterization , cv1 cells ( from american type culture collection ), 143b tk - ( authenticated by str profiling ) and the parental virus , vsc20 . vgf - and the shuttle vector , psc65 . lacz . luc , based on psc65 ( chakrabarti et al ., 1997 ), were supplied by jennerex inc . construction of the plasmid containing the cpg2 sequence and adv - htert have been previously described ( marais et al ., 1996 ) ( schepelmann et al , 2005 , 2007 ). fig2 shows a schematic of the molecular biology steps in the cloning of the vv - cpg2 plasmid . psc65 . lacz . luc was digested with xhoi and bamhi to remove the lacz sequence and religated with the polylinker sequence tcgagacgcgtgatatcatgcatacatgtcacgtggaattcactagtg ( top ) and gatccactagtgaattccacgtgacatgtatgcatgatatcacgcgtc ( bottom ) to produce psc65 . polylinker . luc , which was digested with mlui and spei . pef . cpg2 was modified to contain the adaptor - kozak sequence acgcgtgccgccacc ( top ) and ggtggcggcacgcgt ( bottom ) immediately upstream of the cpg2 atg start codon , giving pef . adaptor . cpg2 , which was digested with mlui and xbai . the linearized shuttle plasmid , psc65 . polylinker . luc , was religated with the excised cpg2 sequence to give psc65 . cpg2 . luc , in which cpg2 and luc are downstream of the vaccinia promoters p7 . 5 and psyn ( e / l ), respectively . recombination of psc65 . cpg2 . luc with vsc20 . vgf - and plaque purification of recombinants was performed according to standard protocols ( earl et al ., 1998 ). briefly , cv1 cells were infected with vsc20 . vgf - and subsequently transfected with psc65 . cpg2 . luc using lipofectamine ( invitrogen ). bioluminescence indicated cellular colocalisation of vaccinia transcription factors and the luciferase gene . recombinants were selected by three rounds of plaque purification in 143b tk - cells in the presence of bromodeoxyuridine , and validated by transgene region sequencing , bioluminescence and cpg2 activity assay ( stribbling et al ., 1997 ). recombinant virus was expanded to working stocks on cv1 cells , purified by sucrose gradient centrifugation and quantified by plaque titration . the engineered virus containing deletions of tk and vgf and insertions of cpg2 and luc was designated vv - cpg2 . insert fidelity was confirmed by dye - terminator cycle sequencing . the following cell lines were tested for susceptibility to transduction with vv - cpg2 ; sw620 and ht29 : colorectal cancer cell lines a549 : lung cancer fadu : head and neck cancer wm266 . 4 : melanoma mda - mb - 231 : breast cancer each cell tumour cell line tested was found to express cpg2 following infection with vv - cpg2 . for example , a549 cells , mda - mb - 231 cells and ht29 cells each transduced with vv - cpg2 at a multiplicity of infection ( moi ) of 0 . 1 had cpg2 activity of 1 unit / mg ( u / mg ), 1 . 2 u / mg or 1 . 1 u / mg at 72 hours post - transduction , respectively . the reduction of cell viability in wm226 . 4 cells , fadu cells , sw620 and a549 cells is shown in fig1 to 14 . in each case , cell viability was measured by mts assay . a reduction of the ec50 of zd2767p in cells transduced with vv - cpg2 at moi of 0 . 1 is : 114 - fold ( wm226 . 4 cells ); 119 - fold ( sw620 cells ); and 103 - fold ( fadu cells ). hence , in each case , transduction with vv - cpg2 at moi = 0 . 1 results in over 100 - fold increase in sensitivity to zd2767p . all animal work was compliant with personal and project uk home office license restrictions and ukcccr guidelines ( workman , 1998 ). cd1 nu / nu athymic female mice were obtained from charles river at 5 - 7 weeks of age , and allowed to acclimatize for one week prior to study . a specific pathogen - free environment was maintained . animals had unlimited access to food and water and were housed at no more than five per cage . to establish tumour xenografts , cells in exponential growth phase were injected into the subcutaneous compartment of the right flank . fadu cells were injected at 10 6 cells per animal in pbs . interventions were initiated at 14 or 15 days after cell instillation in the presence of established tumours . a549 cells were injected at 10 7 cells per animal in pbs and matrigel , in a 1 : 1 ratio . interventions were initiated when tumours were approximately 100 mm 3 . sw620 cells were injected at 10 6 cells per animal in pbs . animals were sacrificed by cervical dislocation when the mean tumour diameter exceeded 15 mm or any single diameter reached 17 mm , or in the presence of progressive tumour ulceration , as directed by project license conditions . for intratumoural studies adv - htert was administered at 4 × 10 8 plaque forming units / kg ( pfu / kg ) in 0 . 2 ml / 20 g bodyweight , while vv - cpg2 was administered 4 × 10 7 pfu / kg by intratumoral injection in a total volume of 20 μl vehicle , using a systematic method adapted from published precedent ( bazan - peregrino et al ., 2008 ). for intravenous studies , adv - htert was administered at 4 × 10 9 pfu / kg in 0 . 2 ml / 20 g bodyweight by tail vein injection . vv - cpg2 was also administered at 4 × 10 9 pfu / kg in 0 . 2 ml / 20 g bodyweight by tail vein injection . zd2767p was administered by intraperitoneal ( ip ) injection at 150 - 300 mg / kg in three divided doses at one hour intervals . four to six prodrug administrations were performed over 4 - 8 weeks , guided by toxicity monitoring . in control groups , substance administrations were substituted by appropriate vehicles . tumour caliper measurements were taken twice weekly and volumes calculated using the formula length × width × height × π / 6 . tissue cpg2 activities were quantified by an indirect endpoint assay as previously described ( stribbling et al ., 1997 ). briefly , samples were homogenized and incubated with methotrexate for 30 minutes when the reaction was terminated . the concentration of the degradation product , dampa , was measured by high performance liquid chromatography . the end product concentration was converted to cpg2 activity by reference to standard curves , generated for each analysis using an alternative kinetic assay . irradiation was performed using a gammacell 40 exactor ( best theratronics , ottawa , canada ), adapted to deliver unilateral localised tumor irradiation from a single caesium - 137 source at 662 kv . offline dosimetry was performed using gafchromic ebt film ( international specialty products , vertec scientific , uk ), giving a current dose rate of 0 . 6 gy per minute . in control groups , substance administrations were substituted by appropriate vehicles , and mock - irradiation was performed by sedation and placement of animals in the treatment chamber for similar duration . fig4 shows concentration of the cpg2 enzyme in tumours following intratumoural ( it ) or intravenous ( iv ) injection of adv - htert or vv - cpg2 in the fadu human tumour xenograft model . cpg2 enzyme levels in the fadu human tumour xenograft model , measured in units per gram of tumour tissue ( cpg2 u / g ), were determined via an activity endpoint assay , as described below . fig4 shows that similar levels of cpg2 activity are achieved following iv injection of vv - cpg2 in the fadu human tumour xenograft model compared with adv - htert administered at the same dose of 4 × 10 9 plaque forming units / kg . for it administration in the fadu human tumour xenograft model , a 4 × 10 7 pfu / kg vv - cpg2 is sufficient to achieve a similar level of cpg2 activity to that achieved by a dosage of 4 × 10 8 of adv - htert in the fadu human tumour xenograft model . fig5 and 6 show , in the fadu human tumour xenograft model , the effect of gdept on tumour volume and animal survival following it administration of the adv - htert vector or the vv - cpg2 vector , respectively . fig5 a shows that adv - htert - mediated gdept does not effect a reduction in tumour volume . the tumour volume of adv - htert - gdept treated animals is not significantly different from the tumour volume of control animals or animals treated with the adv - htert vector alone ( without subsequent prodrug administration ). fig5 b shows that animal survival following adv - htert - mediated gdept is not extended ; if anything , survival time is shortened . in contrast , in the fadu human tumour xenograft model , fig6 a shows that vv - cpg2 - mediated gdept results in a reduction in tumour volume which is significant and persistent over time , with tumour volume tending to zero in surviving animals . fig6 b shows that animal survival following vv - cpg2 - mediated gdept is extended , with approximately 35 % of vv - cpg2 - gdept animals surviving for over 100 days . fig7 and 8 show , in the fadu human tumour xenograft model , the effect of gdept on tumour volume and animal survival following iv administration of the adv - htert vector and the vv - cpg2 vector , respectively . fig7 a shows that adv - htert - mediated gdept does not effect a reduction in tumour volume . the tumour volume of adv - htert - gdept treated animals is not significantly different from the tumour volume of control animals or animals treated with the adv - htert vector alone ( without subsequent prodrug administration ). fig7 b shows that animal survival following adv - htert - mediated gdept is only slightly extended . fig8 a , in the fadu human tumour xenograft model , shows that vv - cpg2 - mediated gdept results in a reduction in tumour volume over time . fig8 b shows that animal survival following vv - cpg2 - mediated gdept is extended , with approximately 50 % vv - cpg2 - gdept animals surviving for over 100 days . fig9 shows the combination efficacy between vv - cpg2 - mediated gdept and radiotherapy ( rt ) in the fadu human tumour xenograft model . vv - cpg2 was delivered intratumouraly . all animals , except controls , received a 20 gy radiation dose . fig9 a shows that rt in combination with the vector alone results in a stabilisation of tumour volume over time and that rt in combination with vv - cpg2 - mediated gdept results in reduced tumour volumes , which tend to zero . fig9 b shows that over 75 % of animals that received vv - cpg2 - mediated gdept in combination with radiotherapy survived for over 150 days . fig1 shows that vv - cpg2 - mediated gdept in combination with radiotherapy ( rt ) results in stable tumour volume when vv - cpg2 is delivered intravenously . all animals , except controls , received a 10 gy radiation dose . other combinations of anticancer therapy may be envisaged as forming a part of the invention . for example , cell checkpoint inhibitors such as chk1 , known to sensitise tumours to cytotoxic agents , may be used alongside the gdept systems described herein for cancer therapies . fig1 shows concentration of the cpg2 enzyme in ( a ) lung a549 tumours and ( b ) colorectal sw620 tumours following intravenous ( iv ) injection of vv - cpg2 . cpg2 enzyme levels , measured in units per gram of tumour tissue ( cpg2 u / g ), were determined via an activity endpoint assay . fig1 shows that similar levels of cpg2 activity are achieved in lung cancer models as with the head & amp ; neck cancer model shown in fig4 . fig1 illustrates the excellent tumour specificity of the vv - cpg2 vaccinia vector for head & amp ; neck fadu tumours ( a ), colorectal sw620 tumours ( b ), and lung cancer a549 tumours ( c ). fig1 a shows the effect of gdept on tumour volume and animal survival following iv administration of the vv - cpg2 to animals with lung cancer a549 tumours . the tumour volume of control animals or animals treated with the vv - cpg2 vector alone ( without subsequent prodrug administration ) was only slightly reduced ( central plot ). this antitumoural effect is enhanced when the prodrug is also administered ( lower plot ). the zd2767p prodrug was administered starting 4 days after vv - cpg2 injection by intraperitoneal ( ip ) injection at 150 mg / kg in three divided doses at one hour intervals . six prodrug administrations were performed over 7 weeks . in control groups , substance administrations were substituted by appropriate vehicles . fig1 b shows that animal survival following vv - 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