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A r t i c l e s 1820 VOLUME 18 NUMBER 12 DECEMBER 2012 nAture medicine Of the approximately 335 emerging infectious diseases that were iden tified between 1940 and 2004 60 3 originated in wildlife 1 From past pandemics it is clear that highly pathogenic zoonoses are major threats to global human health economic stability and national secu rity 1 4 SARS CoV and swine influenza virus CA 04 09 H1N1 have caused substantial human morbidity and mortality in the 21st century Similar to influenza CoVs have a strong history of host shifting and cross species transmission 5 6 In addition to the emergence of SARS CoV in 2002 which caused 50 mortality in aged populations several other human CoVs such as HCoV NL63 HCoV OC43 and HCoV 229E probably emerged from animal reservoirs within the past 200 years 7 8 The sudden emergence of new respiratory viral pathogens from animals underscores the need for new broadly applicable vaccine strategies that rapidly and rationally attenuate emerging zoonoses especially to protect vulnerable populations in future outbreaks Vaccines have a long history of success in reducing viral disease bur dens Live attenuated viruses are ideal vaccine candidates as they elicit balanced innate and adaptive lifelong protective immune responses with low production and delivery costs 9 Unfortunately broadly appli cable strategies for the rational design of live attenuated virus vaccines have remained elusive and vaccines attenuated by chemical treatment or passage can revert to virulence resulting in disease outbreaks in unvaccinated and immunocompromised populations 9 Moreover the precise mechanism of attenuation often remains unclear thus the sta bility of the attenuation cannot be clearly evaluated or assured RNA viruses encode RNA dependent RNA polymerases that lack efficient proofreading capabilities the resulting high error rates which range from 10 3 to 10 5 mutations per site per round of rep lication render RNA viruses highly vulnerable to lethal mutagenesis using chemical agents 10 11 High mutation rates generate consider able genomic diversity allowing RNA viruses to rapidly adapt to changing environmental conditions and hosts 12 Increased replica tion fidelity has been shown to reduce the virulence of poliovirus and chikungunya virus 12 14 and has been proposed as a strategy for live attenuated virus design 15 CoVs encode the largest known RNA virus genomes 26 32 kb exceeding the theoretical limits of viable RNA genome size 11 Mutation rates are lower in CoVs than in other RNA viruses approaching 2 10 6 mutations per site per round of replication 16 Nsp14 encoded in the viral replicase gene contains a 3 5 exoRNase ExoN of the DEDDh exonu clease superfamily 17 In addition to the CoVs ExoN homologs are present in the members of the Nidovirales order whose genomes are 20 kb but are not present in the smaller arteriviruses with genomes of 12 16 kb suggesting that the ExoN had a crucial role in genome expansion 16 18 In vitro 3 5 exoRNase activity has been demonstrated for recombinant SARS CoV nsp14 ref 19 We have engineered and recovered viable ExoN inactivation mutants from mouse hepatitis virus MHV ExoN and SARS CoV Urbani background SARS ExoN Both MHV ExoN and SARS ExoN inactivations are maintained stably for more than ten passages 1 Department of Epidemiology University of North Carolina at Chapel Hill Chapel Hill North Carolina USA 2 Department of Pediatrics Vanderbilt University Nashville Tennessee USA 3 Department of Microbiology and Immunology University of North Carolina at Chapel Hill Chapel Hill North Carolina USA 4 Department of Pathology Microbiology and Immunology Vanderbilt University Nashville Tennessee USA Correspondence should be addressed to R S B rbaric email unc edu Received 9 August accepted 14 September published online 11 November 2012 doi 10 1038 nm 2972 A live impaired fidelity coronavirus vaccine protects in an aged immunocompromised mouse model of lethal disease Rachel L Graham 1 Michelle M Becker 2 Lance D Eckerle 2 Meagan Bolles 3 Mark R Denison 2 4 increased fidelity is attenuating for some viruses Coronavirus CoV replication fidelity is approximately 20 fold greater than that of other RNA viruses and is mediated by a 3 5 exonuclease ExoN activity that probably functions in RNA proofreading In this study we demonstrate that engineered inactivation of severe acute respiratory syndrome SARS CoV ExoN activity results in a stable mutator phenotype with profoundly decreased fidelity in vivo and attenuation of pathogenesis in young aged and immunocompromised mice The ExoN inactivation genotype and mutator phenotype are stable and do not revert to virulence even after serial passage or long term persistent infection in vivo ExoN inactivation has potential for broad applications in the stable attenuation of CoVs and perhaps other RNA viruses npg 2012 Nature America Inc All rights reserved A r t i c l e s nAture medicine VOLUME 18 NUMBER 12 DECEMBER 2012 1821 in vitro and have 15 to 20 fold increased mutation frequencies compared to wild type MHV and SARS CoV 16 20 Thus ExoN has a crucial role in CoV RNA genome replication fidelity in vitro prob ably by directly mediating or stimulating proofreading a function previously unknown among RNA viruses 21 In this study we used the stable low fidelity mutator phenotype of the SARS CoV ExoN mutants to determine whether decreased replication fidelity could be used as a rational design strategy for a live attenuated vaccine with broad potential applications to other viruses 16 20 21 We evaluated i the impact of the inactiva tion of an RNA proofreading exonuclease and the resultant mutator phenotype on CoV replication fitness and pathogenesis ii virus stability after passage or persistence in vivo and iii the efficacy of using a decreased fidelity mutant as a vaccine Further we assessed the potential for generating stably attenuated reversion resistant immunogenic strains of known and newly identified CoVs to be used as vaccines in both immunocompetent and immunocompro mised populations RESULTS The mutator phenotype and decreased fitness of MA ExoN We engineered nsp14 ExoN inactivation mutations into the back ground of the virulent mouse adapted SARS CoV MAwt yielding MA ExoN Fig 1a b We compared MAwt which causes increased mortality and acute respiratory distress in young and aged mouse models 22 24 and MA ExoN in in vitro growth experiments multi plicity of infection MOI 0 1 PFU per cell MA ExoN showed a stable growth defect of less than 1 log Fig 1c When placed in direct competition MA ExoN was clearly less fit than MAwt over successive rounds of infection Supplementary Fig 1a b At 6 h after infection p i MA ExoN genome RNA levels were roughly equivalent to those of MAwt and were lower than those of MAwt at 12 h p i by 24 h p i MA ExoN genome RNA levels were approximately 10 of those of MAwt Supplementary Fig 1c Thus the data suggest that MA ExoN is able to initiate and establish replication efficiently through times of peak RNA synthesis 0 6 h but has impaired accumula tion which manifests late in one round of infection and is amplified over multiple rounds These results are consistent with accumulating defects resulting from a markedly increased mutation rate see the Discussion section We then sequenced RNA from multiple MAwt and MA ExoN plaques Both the MAwt background and engineered ExoN muta tions were present in all sequenced MA ExoN clones Additionally MA ExoN accumulated 14 fold more unique mutations and had a mean 11 5 fold greater mutation frequency compared to MAwt P 0 01 Fig 1d e These results confirm that the growth and repli cation fidelity impairments of the nsp14 ExoN mutator phenotype are present in MA ExoN and are indistinguishable from those in SARS ExoN during replication in culture MA ExoN is attenuated in vivo To assess MA ExoN virulence we infected young 10 week old and aged 14 month old female BALB c mice with MA ExoN or MAwt Fig 2 Young mice infected with MAwt showed dose dependent weight loss and recovery Fig 2a though they had no observable dose dependent differences in lung titers or clearance after day 4 p i Fig 2b In contrast young mice infected with MA ExoN showed no signs of clinical disease and had high but not dose dependent lung titers that were rapidly cleared by day 4 p i Fig 2a b We then compared MA ExoN and MAwt infec tion in aged immunosenescent mice 25 Mice infected with either virus experienced dose dependent weight loss Fig 2c however although lung titers were equivalent across all doses of MA ExoN and MAwt on day 2 p i mice infected with MA ExoN cleared the virus independ ent of inoculation dose whereas mice infected with MAwt had begun to clear the virus from higher titer infections more efficiently at day 4 p i than from lower titer infections Fig 2d Additionally whereas aged mice infected with MA ExoN had no mortality MAwt infected mice had dose dependent mortality Fig 2e As described previously 23 we found little if any virus in other organs These experiments demonstrate that MA ExoN is attenuated in both young and aged diseased mice compared to virulent MAwt and that disease symptoms when present are less pronounced in MA ExoN infections than MAwt infections A potential concern with live attenuated vaccines is the chance that they could revert to virulence in vivo particularly in immunocom promised individuals Therefore we assessed whether MA ExoN was nsp14 nsp14 nsp14 S EM N Motif I MAwt MAwt n 5 clones MA ExoN n 5 clones ORF1a ba b c d e Ala90 Ala92 MA ExoN Motif II Motif III 9 8 7 6 5 4 Virus titer log 10 PFU ml 1 3 2 1 0 1 4 8 12 16 20 SARS CoV S ExoN MA ExoN MAwt Time h p i 24 30 36 48 12 11 5 MAwt n 5 MA ExoN n 5 9 Mutation frequency 6 3 0 Figure 1 The nsp14 ExoN mutator virus in a virulent mouse adapted SARS CoV isogenic background a Genome organization with the locations of the nsp14 coding sequence black rectangle and the mouse adapted mutations triangles shown ORF1a b ORF1a and ORF1b Structural proteins are labeled as follows S spike E envelope M membrane N nucleocapsid b Nsp14 ExoN motifs DEDD domain residues underlined and alanine substitutions D90A and E92A in motif I recovered in wild type SARS CoV and MAwt backgrounds c Growth analysis MOI 0 1 PFU per cell of wild type SARS CoV S ExoN MAwt and MA ExoN on Vero cells Error bars s d d Mutation frequency from complete genome sequencing of plaque isolates of MAwt and MA ExoN n 5 for both at passage 3 The increase in mean mutation frequency horizontal lines in MA ExoN compared to MAwt 11 5 is indicated P 0 01 Mann Whitney nonparametric test for independent samples e The mutations identified with complete genome sequencing across five clones from each group Filled circles nonsynonymous mutations open circles synonymous mutations black noncoding mutations red mutations present in more than one clone blue mutations present in only one clone Mouse adapted mutations are shown as triangles on the genome schematic and were present in all sequenced genomes npg 2012 Nature America Inc All rights reserved A r t i c l e s 1822 VOLUME 18 NUMBER 12 DECEMBER 2012 nAture medicine attenuated in immunocompromised mice We used MAwt and MA ExoN to infect young Rag recombination activating gene severe combined immunodeficiency SCID and Stat1 signal transducer and activator of transcription 1 mice as well as background con trols C57BL 6 BALB c and 129 mice respectively All MA ExoN infected mice had significantly less weight loss than MAwt infected mice P 0 05 Fig 3a c and Supplementary Table 1 Only Stat1 mice had any notable weight loss 15 as a result of MA ExoN infection however these mice did not pass experimental morbidity thresholds Fig 3c In contrast all MAwt infected Stat1 mice died or were moribund by day 9 p i but MAwt infection was not lethal in C57BL 6 or 129 control mice Fig 3a c as has been previously reported 22 26 Rag and SCID mice maintained detectable amounts of MAwt and MA ExoN virus for 14 d Rag or 60 d SCID beyond the background controls Fig 3d e but showed no signs of illness over the course of the experiment despite a lack of viral clearance expanding earlier reports from our laboratory that MAwt does not clear from Rag mice 26 The rapid clearance of MA ExoN infection from Stat1 mice Fig 3f further supports the hypothesis that clear ance of SARS CoV infection is dependent on both B and T cells 27 Mutation accumulation during persistent in vivo infection Infection with both MAwt and MA ExoN persisted for at least 60 d in SCID mice Fig 3e potentially allowing for the most longitudinal 110 105 100 95 90 85 80 75 70 Percentage initial mass Time d p i 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 a 10 Titer log 10 PFU per lung MA ExoN 2 log 3 log 4 log 2 log 3 log 4 log 2 4 15 2 4 15 2 4 15 2 4 15 2 4 15 2 4 15 MAwt 9 8 7 6 5 4 3 2 1 0 b 110 105 100 95 90 85 80 75 70 65 Percentage initial mass Time d p i 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 c Survival Time d p i 100 90 80 70 60 50 40 30 20 10 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 e Titer log 10 PFU per lung 2 4 8 14 2 4 8 14 2 4 8 14 2 4 8 14 2 42 4 7 MA ExoN MAwt 10 9 8 7 6 5 4 3 2 1 0 d 2 log 3 log 4 log 2 log 3 log 4 log Figure 2 Weight loss and lung titer in BALB c mice a e MA ExoN and MAwt infections of 10 week old a b and 12 to 14 month old c e female BALB c mice a c Weight loss Dark shapes and solid lines MA ExoN white shapes and dashed lines MAwt diamonds 10 2 PFU squares 10 3 PFU triangles 10 4 PFU Error bars indicate s d b d Lung titers The titer for each mouse lung is indicated by an open circle the mean titer of all mice at each time point is indicated by a horizontal bar Days p i are indicated on the x axes 2log 3log and 4log indicate the MOIs of each virus 10 2 10 3 or 10 4 PFU respectively e Survival within aged mouse groups calculated as the percentage of surviving mice compared to the total number of mice remaining on each day of the experiment Line weights and symbols are as in a and c d 8 7 6 5 4 3 2 Titer log 10 PFU per lung 1 4 9 15 30 4 9 30 4 9 4 9 MA ExoN MA ExoN MAwt C57BL 6Rag MAwt 0 e MA ExoN MAwt BALB cSCID MA ExoN MAwt 10 9 8 7 6 5 4 3 2 Titer log 10 PFU per lung 1 0 2 4 8 15 30 60 2 4 8 15 30 60 2 4 15 42 15 f Stat1 MA ExoN MA ExoN MAwt MAwt 9 8 7 6 5 4 3 2 Titer log 10 PFU per lung 1 0 4 9 15 30 129 4 9 4 9 4 9 115 a 110 105 100 95 90 Percentage initial mass 85 Time d p i 80 75 70 65 0 2 4 6 8 10 12 14 16 18 20 Rag MA ExoN Rag MAwt BL6 MAwt BL6 MA ExoN c 110 105 100 95 90 Percentage initial mass 85 Time d p i 80 75 70 65 0 2 4 6 8 10 12 14 16 18 20 Stat1 MA ExoN Stat1 MAwt 129 MAwt 129 MA ExoN b 110 105 100 95 90 Percentage initial mass 85 Time d p i 80 75 70 65 0 2 4 6 8 10 12 14 16 18 20 SCID MA ExoN SCID MAwt BALB c MAwt BALB c MA ExoN Figure 3 Weight loss and lung titer in young immunocompromised mice a c MA ExoN and MAwt infections of 10 week old Rag a SCID b and Stat1 c mice C57BL 6 a BALB c b and 129 c mice were included as background controls Weight loss is shown Dark shapes and solid lines immunocompromised mice white shapes and dashed lines background control mice circles MA ExoN squares MAwt Error bars indicate s d d f Lung titers for Rag d SCID e and Stat1 f mice and background controls The titer of each mouse lung is indicated by an open circle the mean of all titers at each time point is indicated by a horizontal bar Days p i are indicated on the x axes npg 2012 Nature America Inc All rights reserved A r t i c l e s nAture medicine VOLUME 18 NUMBER 12 DECEMBER 2012 1823 cycles of replication and lowest immune barriers to the emergence of mutations conferring increased fitness reversion to virulence and fidelity compensating changes of any of the groups examined To test this we sequenced viral genomes from viral plaques grown from SCID mouse lung homogenates at 30 d p i Fig 4 and Supplementary Table 2 We identified a total of 14 consensus mutations 100 000 nt for MAwt with 3 mutations shared in two or three genomes result ing in 11 distinct mutations 4 synonymous and 7 nonsynonymous For MA ExoN the engineered inactivation mutations were main tained In contrast to MAwt MA ExoN plaques contained a total of 91 mutations 89 distinct 32 synonymous and 57 nonsynonymous corresponding to a 9 6 fold higher total mutation accumulation compared to MAwt We compared mutation accumulations across two separate regions open reading frame 1a ORF1a nucleotides 493 8603 and ORF1b nucleotides 12915 16520 Supplementary Figs 2 and 3 and Supplementary Table 2 for statistical determinations Mutation accumulations were significantly higher in MA ExoN infected mice for both regions P 0 01 Additionally there was a mean 18 3 fold increased accumulation for MA ExoN across the ORF1a region When we normalized the accumulations of mutations per 10 kb MA ExoN mutation accumulations in the ORF1a compared to the ORF1b region were not significantly different P 0 340 but remained significantly increased compared to the accumulation in MAwt P 0 001 for both ORF1a and ORF1b We identified no new ORF1 MAwt 1 1 MAwt 2 1 MAwt 3 1 MA ExoN 1 1 MA ExoN 2 1 MA ExoN 3 1 Synonymous unique Nonsynonymous unique Synonymous present in three sequences Nonsynonymous present in two sequences ExoN inactivation mutations 20 nt deletion Creates stop codon 1 2 3 4 5 6 78 910 12 ExoN inactivation D90A E92A ORF2 ORF9 RBD 5 M 3b 2 S 3a 4 E 6 7b 9b 8b 8a 9a N 7a 13 14 15 16 Figure 4 Mutation accumulation in infected SCID mice at 30 d p i The SARS CoV genome is depicted at the top The nsp14 ExoN coding region is denoted by a purple box with the inactivating amino acid changes indicated above the schematic The receptor binding domain RBD is denoted by a black box Individual SCID mouse genome sequences are represented by black horizontal lines Dashed lines separate the nonstructural protein sequences in ORF1 and downstream ORFs Mutations are indicated by lollipop shapes colored as follows blue synonymous unique to one sequence light blue synonymous present in three sequences red nonsynonymous unique to one sequence green synonymous present in two sequences purple nsp14 ExoN inactivation mutations Mutations that alter the size of an ORF are indicated by a red deletion or a red S stop codon Genome sizes ORF and nonstructural protein boundaries and mutation marker placements are approximate Time d p i Percentage initial mass 105 100 95 90 85 80 0 1 2 3 MA ExoN P1 MA ExoN P2 MA ExoN P3 MAwt P1 MAwt P2 c Time d p i Percentage initial mass 0 1 2 3 4 5 6 7 105 110 100 95 90 85 70 65 75 80 MAwt pop 2 MAwt pop 1 MAwt pop 3 PBS MA ExoN pop 2 MA ExoN pop 1 MA ExoN pop 3 e Time d p i Percentage initial mass 105 110 100 95 90 85 0 1 2 3 4 5 6 7 SCID30 MA ExoN 2 1 SCID30 MA ExoN 3 1 f Time d p i Percentage initial mass 105 110 100 95 90 85 70 65 75 80 0 1 2 3 4 5 6 7 24 h MA ExoN 24 h MAwt 72 h MA ExoN 72 h MAwt d 10 9 8 7 6 5 4 3 2 1 0 MA ExoN P1 P2 P3 P4 P5 P6 P7 P8 P1 P2 P3 P4 P5 P6 P7 P8 MAwt Titer log 10 PFU per lung a 7 6 5 4 3 2 1 0 MA ExoN P1 P2 P3 P1 P2 P3 MAwt Titer log 1 0 PFU per lung b Figure 5 Virulence of passaged MA ExoN and MAwt viruses a c MA ExoN and MAwt serial infections of 12 month old female BALB c mice Passages P are indicated on the x axes a Serial passage after 24 h b c Serial passage after 72 h a b Lung titers are shown with average titers indicated by bars In the 72 h passage MAwt infected mice died by day 3 of passage 2 thus the viruses were not passaged further c Weight loss in the mice infected in the 72 h passage is shown Error bars indicate s d d MA ExoN 24 h passage 8 and 72 h passage 3 and MAwt passage 1 infections of 12 month old female BALB c mice Weight loss is shown Error bars indicate s d e Infections of 10 week old female BALB c mice with MA ExoN and MAwt population viruses isolated from SCID mice at 30 d p i weight loss is shown Dashed line mock PBS inoculation dark shapes and solid lines MA ExoN white shapes and solid lines MAwt For each virus lungs from three separate mice were harvested mou