storage and biochemical analyses of post - mortem human brain samples and transmission studies to mice were performed with written informed consent from patients with capacity to give consent .
where patients were unable to give informed consent , assent was obtained from their relatives in accordance with uk legislation and codes of practice .
samples were stored and used in accordance with the human tissue authority codes of practice and in line with the requirements of the human tissue authority licence held by ucl institute of neurology .
this study was performed with approval from the medical research advisory committee of the government of papua new guinea , the national hospital for neurology and neurosurgery and the ucl institute of neurology joint research ethics committee ( now national research ethics service committee , london  queen square ) - rec references : 03/n036 , 03/n038 and 03/n133 .
work with mice was performed under approval and licence granted by the uk home office ( animals ( scientific procedures ) act 1986 ) ; project licence number 70/6454 which conformed to university college london institutional and arrive guidelines ( www.nc3rs.org.uk/arrive/ ) .
the 759bp human prp orf was amplified by pcr with pfu polymerase from genomic dna prepared from the brain of a patient with the 127v polymorphism on the 129 m allele , using forward primer 5-gtcgaccagtcattatggcgaacctt-3 and reverse primer 5-ctcgagaagaccttcctcatcccact-3. restriction sites sal i and xho
i ( underlined ) were introduced in the forward and reverse primers respectively for cloning .
the blunt ended pcr fragment generated by pfu polymerase was subcloned into sma i digested psp72 vector and sequenced to ensure that no spurious alterations had been introduced by the pcr and to confirm the presence of valine-127 and methionine-129 polymorphisms matching the patient dna template .
the amplified human prp orf with the confirmed polymorphisms was then isolated by sal i and xho i digestion .
subsequent subcloning into the sal i site of the cosmid vector shacostt , packaging and preparation of high quality dna of the not i transgene insert was as previously reported .
microinjection of the purified not i dna fragment was carried out according to standard protocol into single cell eggs of prnp null mice which had been backcrossed onto an fvb / n genetic background .
genotyping was performed by pcr and prp expression levels estimated by western blot analysis as previously reported .
two homozygous lines were established for variant huprp vm described as tg(huprp vm / vm 
 prnp)-183 ( vm tg183 ) and tg(huprp vm / vm 
 prnp)-190 ( vm tg190 ) , with transgene expression levels of 2 and 1  respectively , that of pooled 10% ( w / v ) normal human brain homogenate ( table 1 ) .
fvb - congenic versions of tg35 mice homozygous for huprp gm 
  and tg152 mice homozygous for huprp gv 
  were used as wild type human prp - expressing controls , designated tg(huprp gm / gm 
 prnp)-35c ( gm tg35c ) and tg(huprp gv / gv 
 prnp)-152c ( gv tg152c ) respectively . the prnp codon 127 - 129 genotypes and relative expression levels of wild type and variant prp in the parental transgenic lines and in the f1 crosses are shown in table 1 .
inocula were prepared , using disposable equipment for each inoculum , in a microbiological containment level 3 laboratory and inoculations performed within a class 1 microbiological safety cabinet as described previously .
ten mice per group of control gm tg35c and gv tg152c lines and 15 per group of newly generated vm tg183 , vm tg190 lines and their respective crosses with tg35c and tg152c , were inoculated ( see below ) with human brain homogenates from neuropathologically confirmed patients comprising , four kuru cases , eight sporadic cjd cases , four iatrogenic cjd cases and two cases of vcjd .
the genotype of each mouse was confirmed by pcr of ear punch dna prior to inclusion and all mice were uniquely identified by sub - cutaneous transponders .
disposable cages were used and all cage lids and water bottles were also uniquely identified by transponder and remained with each cage of mice throughout the incubation period .
mice ( female , aged 6 - 8 weeks ) were randomly assigned to experimental groups and anaesthetised with a mixture of halothane and o2 , and intracerebrally inoculated into the right parietal lobe with 30 l of a 1% ( w / v ) brain homogenate prepared in dulbecco s phosphate buffered saline lacking ca or mg ions ( d - pbs ) .
all mice were thereafter examined daily for early indicators of clinical prion disease including piloerection , sustained erect ears , intermittent generalised tremor , unsustained hunched posture , rigid tail , mild loss of coordination , and clasping hind legs when lifted by the tail .
definite diagnosis of clinical prion disease ( triggering experimental end point ) was reached if mice exhibited any two early indicator signs in addition to one confirmatory sign , or any two confirmatory signs .
the confirmatory signs included ataxia , impairment of righting reflex , dragging of hind limbs , sustained hunched posture , or significant abnormal breathing .
mice were killed ( by co2 asphyxiation ) if they exhibited any signs of distress or once a diagnosis of prion disease was established . at post - mortem brains from inoculated mice
were removed , divided sagittally with half frozen and half fixed in 10% buffered formol saline .
fixed brain was immersed in 98% formic acid for 1 hour and paraffin wax embedded .
serial sections of 4 m thickness were pre - treated by boiling for 10 min in a low ionic strength buffer ( 2.1 mm tris , 1.3 mm edta , 1.1 mm sodium citrate , ph 7.8 ) before exposure to 98% formic acid for 5 min .
abnormal prp accumulation was examined using anti - prp monoclonal antibody icsm 35 ( d - gen ltd , london ) on a ventana automated immunohistochemical staining machine ( ventana medical systems inc . ,
tucson , arizona ) using proprietary secondary detection reagents ( ventana medical systems inc ) before development with 33 diaminobenzedine tetrachloride as the chromogen .
preparation of brain homogenates ( 10% ( w / v ) in d - pbs ) , proteinase k digestion ( 75 g / ml for 1 h at 37c ) and subsequent immunoblotting was performed as described previously .
blots were probed with anti - prp monoclonal antibody icsm 35 ( d - gen ltd , london ) in conjunction with an anti - mouse igg - alkaline phosphatase conjugate and development in chemiluminescent substrate ( cdp - star ; tropix inc ) .
storage and biochemical analyses of post - mortem human brain samples and transmission studies to mice were performed with written informed consent from patients with capacity to give consent .
where patients were unable to give informed consent , assent was obtained from their relatives in accordance with uk legislation and codes of practice .
samples were stored and used in accordance with the human tissue authority codes of practice and in line with the requirements of the human tissue authority licence held by ucl institute of neurology .
this study was performed with approval from the medical research advisory committee of the government of papua new guinea , the national hospital for neurology and neurosurgery and the ucl institute of neurology joint research ethics committee ( now national research ethics service committee , london  queen square ) - rec references : 03/n036 , 03/n038 and 03/n133 .
work with mice was performed under approval and licence granted by the uk home office ( animals ( scientific procedures ) act 1986 ) ; project licence number 70/6454 which conformed to university college london institutional and arrive guidelines ( www.nc3rs.org.uk/arrive/ ) .
the 759bp human prp orf was amplified by pcr with pfu polymerase from genomic dna prepared from the brain of a patient with the 127v polymorphism on the 129 m allele , using forward primer 5-gtcgaccagtcattatggcgaacctt-3 and reverse primer 5-ctcgagaagaccttcctcatcccact-3. restriction sites sal i and xho i ( underlined ) were introduced in the forward and reverse primers respectively for cloning .
the blunt ended pcr fragment generated by pfu polymerase was subcloned into sma i digested psp72 vector and sequenced to ensure that no spurious alterations had been introduced by the pcr and to confirm the presence of valine-127 and methionine-129 polymorphisms matching the patient dna template .
the amplified human prp orf with the confirmed polymorphisms was then isolated by sal i and xho i digestion .
subsequent subcloning into the sal i site of the cosmid vector shacostt , packaging and preparation of high quality dna of the not i transgene insert was as previously reported .
microinjection of the purified not i dna fragment was carried out according to standard protocol into single cell eggs of prnp null mice which had been backcrossed onto an fvb / n genetic background .
genotyping was performed by pcr and prp expression levels estimated by western blot analysis as previously reported .
two homozygous lines were established for variant huprp vm described as tg(huprp vm / vm 
 prnp)-183 ( vm tg183 ) and tg(huprp vm / vm 
 prnp)-190 ( vm tg190 ) , with transgene expression levels of 2 and 1  respectively , that of pooled 10% ( w / v ) normal human brain homogenate ( table 1 ) .
fvb - congenic versions of tg35 mice homozygous for huprp gm 
  and tg152 mice homozygous for huprp gv 
  were used as wild type human prp - expressing controls , designated tg(huprp gm / gm 
 prnp)-35c ( gm tg35c ) and tg(huprp gv / gv 
 prnp)-152c ( gv tg152c ) respectively .
the prnp codon 127 - 129 genotypes and relative expression levels of wild type and variant prp in the parental transgenic lines and in the f1 crosses are shown in table 1 .
inocula were prepared , using disposable equipment for each inoculum , in a microbiological containment level 3 laboratory and inoculations performed within a class 1 microbiological safety cabinet as described previously .
ten mice per group of control gm tg35c and gv tg152c lines and 15 per group of newly generated vm tg183 , vm tg190 lines and their respective crosses with tg35c and tg152c , were inoculated ( see below ) with human brain homogenates from neuropathologically confirmed patients comprising , four kuru cases , eight sporadic cjd cases , four iatrogenic cjd cases and two cases of vcjd .
the genotype of each mouse was confirmed by pcr of ear punch dna prior to inclusion and all mice were uniquely identified by sub - cutaneous transponders .
disposable cages were used and all cage lids and water bottles were also uniquely identified by transponder and remained with each cage of mice throughout the incubation period .
mice ( female , aged 6 - 8 weeks ) were randomly assigned to experimental groups and anaesthetised with a mixture of halothane and o2 , and intracerebrally inoculated into the right parietal lobe with 30 l of a 1% ( w / v ) brain homogenate prepared in dulbecco s phosphate buffered saline lacking ca or mg ions ( d - pbs ) .
all mice were thereafter examined daily for early indicators of clinical prion disease including piloerection , sustained erect ears , intermittent generalised tremor , unsustained hunched posture , rigid tail , mild loss of coordination , and clasping hind legs when lifted by the tail .
definite diagnosis of clinical prion disease ( triggering experimental end point ) was reached if mice exhibited any two early indicator signs in addition to one confirmatory sign , or any two confirmatory signs .
the confirmatory signs included ataxia , impairment of righting reflex , dragging of hind limbs , sustained hunched posture , or significant abnormal breathing .
mice were killed ( by co2 asphyxiation ) if they exhibited any signs of distress or once a diagnosis of prion disease was established . at post - mortem brains from inoculated mice
were removed , divided sagittally with half frozen and half fixed in 10% buffered formol saline .
fixed brain was immersed in 98% formic acid for 1 hour and paraffin wax embedded .
serial sections of 4 m thickness were pre - treated by boiling for 10 min in a low ionic strength buffer ( 2.1 mm tris , 1.3 mm edta , 1.1 mm sodium citrate , ph 7.8 ) before exposure to 98% formic acid for 5 min .
abnormal prp accumulation was examined using anti - prp monoclonal antibody icsm 35 ( d - gen ltd , london ) on a ventana automated immunohistochemical staining machine ( ventana medical systems inc . ,
tucson , arizona ) using proprietary secondary detection reagents ( ventana medical systems inc ) before development with 33 diaminobenzedine tetrachloride as the chromogen .
preparation of brain homogenates ( 10% ( w / v ) in d - pbs ) , proteinase k digestion ( 75 g / ml for 1 h at 37c ) and subsequent immunoblotting was performed as described previously .
blots were probed with anti - prp monoclonal antibody icsm 35 ( d - gen ltd , london ) in conjunction with an anti - mouse igg - alkaline phosphatase conjugate and development in chemiluminescent substrate ( cdp - star ; tropix inc ) .
the provenance of each brain sample is designated above each lane and molecular markers are indicated on the left .
the prnp codon 127 ( g , glycine , v , valine ) or codon 129 ( m , methionine , v , valine ) genotypes of the transgenic mice are designated below .
transgenic mouse brains were analysed by enhanced chemiluminescence without proteinase - k digestion and equal amounts of total protein loaded in each well and probed with anti - prp monoclonal antibody icsm 35 .
wild type human prp expression levels of gm / gm tg35c and gv / gv tg152c mice are 2- and 6-fold higher respectively , than seen in 10 % ( w / v ) pooled human brain homogenate .
homozygous vm tg183 and tg190 mice have 2-fold higher or equivalent prp expression levels respectively compared to 10 % ( w / v ) pooled human brain homogenate .
all mice were intracerebrally challenged with the same vcjd prion isolates ( i342 and i7042 ) .
abnormal prp deposition in fixed post - mortem brain from affected mice was detected using anti - prp monoclonal antibody icsm 35 .
( a - b ) homozygous tg35c mice ( expressing gm / gm wild type prp only ) show intense and widespread prp plaque deposits .
magnified areas show ( ai ) hippocampus , ( bi ) frontal cortex and ( aii and bii ) thalamus .
in contrast , heterozygous gm / vm tg35c / tg183 mice expressing equivalent levels of gm and vm prp ( c - d ) show only weak prp deposition in the corpus callosum ( ci and di ) with no abnormal prp deposition detected in other brain areas , for example , in the thalamus ( cii and dii ) .
heterozygous gm / vm tg35c / tg190 mice which express a lower level of vm prp relative to wild type gm prp ( e - f ) show greater levels of prp deposition than seen in tg35c / tg183 mice following challenge with the same vcjd prion isolates , ( ei , eii and fi ) corpus callosum ; ( fii ) pons .
scale bar , upper panels ( a - f ) 2 mm ; magnified panels , 100 m .
mice were intracerebrally challenged with kuru , classical and variant cjd prions . following prolonged ( > 600 days ) post inoculation periods , abnormal prp deposition in fixed post - mortem brain
( a - d ) vm / vm tg183 mice with 2-fold overexpression of vm prp . ( e - h ) vm / vm tg190 mice expressing endogenous levels of vm prp .
red square boxes in the main panels ( a - h ) define the area of magnified images of hippocampus ( i ) and thalamus ( ii ) shown in the lower panels .
scale bar , a - h , 2 mm ; magnified panels i and ii , 100 m .
the lack of detection of abnormal prp deposition in brain indicates that the mice are not subclinically infected with prions .
gm / gm tg35c mice or gv / gv tg152c mice are homozygous for wild type human prp alleles and are fully susceptible to kuru and classical cjd prions .
vm / vm tg183 or vm / vm tg190 transgenic mice are homozygous for the variant vm allele found only in humans from the kuru - exposed population of papua new guinea and are entirely resistant to infection with kuru and classical cjd prions .
the levels of prp expression in the brain of these homozygous transgenic mice relative to a pooled human brain homogenate are 1 ( tg190 mice ) 2 ( tg35c and tg183 mice ) and 6 ( tg152c mice ) .
generation of f1 mice through inter - breeding the various homozygous lines produces different combinations of the various human prp alleles leading to differences in the relative expression levels of the various prion proteins in brain .
the prp expression ratios from the two prp alleles in the crosses are shown in parentheses above the cartoon mice .
full , intermediate or low susceptibility of the mice to infection with kuru and classical cjd prions is indicated by three , two or one diagonal red bar , respectively , drawn across the mice .
mice with no red bar are entirely resistant to infection with kuru and classical cjd prions .
mice were intracerebrally challenged with kuru and classical cjd prions and abnormal prp deposition in fixed post - mortem brain from affected mice was examined using anti - prp monoclonal antibody icsm 35 .
red square boxes labelled i and ii in panels a - f mark brain areas that are magnified and displayed below ; ( i ) cortex and ( ii ) thalamus .
( a - b ) wild type gv / gv tg152c mice . ( c - d ) heterozygous gv / vm tg152c / tg183 mice .
( e - f ) heterozygous gv / vm tg152c/190 mice scale bar , a - f , 2 mm ; magnified panels i and ii , 100 m .
the detection of abnormal prp deposition in brain indicates that the mice are infected with prions .
 mammalian prions , transmissible agents causing lethal neurodegenerative diseases , are composed of assemblies of misfolded cellular prion protein ( prp ) 1 . a novel prp variant , g127v , was under positive evolutionary selection during the epidemic of kuru , an acquired prion disease epidemic of the fore population in papua new guinea , and appeared to provide strong protection against disease in the heterozygous state2 . 
 we have now investigated the protective role of this variant and its interaction with the common worldwide m129v prp polymorphism ; v127 was seen exclusively on a m129 
 prnp allele . here 
 we demonstrate that transgenic mice expressing both variant and wild type human prp are completely resistant to both kuru and classical cjd prions ( which are closely similar ) but can be infected with variant cjd prions , a human prion strain resulting from exposure to bse prions to which the fore were not exposed . 
 remarkably however , mice expressing only prp v127 were completely resistant to all prion strains demonstrating a different molecular mechanism to m129v , which provides its relative protection against classical cjd and kuru in the heterozygous state . 
 indeed this single amino acid substitution ( gv ) at a residue invariant in vertebrate evolution is as protective as deletion of the protein . 
 further study in transgenic mice expressing different ratios of variant and wild type prp indicates that not only is prp v127 completely refractory to prion conversion , but acts as a potent dose - dependent inhibitor of wild type prion propagation . 
Methods
Ethics Statement
Generation of transgenic mice
Transmission Studies
Neuropathology and Immunohistochemistry
Immunoblotting
Extended Data