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Vaccine 2008 26 797 808 available at journal homepage particles protein protect mice against Kumari G Lokugamage a 1 Naoko Yoshikawa Iwata a 1 Naoto Ito a Douglas M Watts b Philip R Wyde c Nan Wang a b Patrick Newman a b Chien Te Kent Tseng a C J Peters a b Shinji Makino a a Department of Microbiology and Immunology University of Texas Medical Branch Galveston TX 77555 United States b Department of Pathology University of Texas Medical Branch Galveston TX 77555 United States c Department of Molecular Virology and Microbiology Baylor College of Medicine Houston TX 77030 United States Received 1 October 2007 received in revised form 19 November 2007 accepted 29 November 2007 Available online 26 December 2007 KEYWORDS SARS coronavirus Virus like particles Neutralizing antibody Mouse Summary We tested the efficacy of coronavirus like particles VLPs for protecting mice against severe acute respiratory syndrome coronavirus SCoV infection Coexpression of SCoV S protein and E M and N proteins of mouse hepatitis virus in 293T or CHO cells resulted in the efficient production of chimeric VLPs carrying SCoV S protein Balb c mice inoculated with a mixture of chimeric VLPs and alum twice at an interval of four weeks were protected from SCoV challenge as indicated by the absence of infectious virus in the lungs The same groups of mice had high levels of SCoV specific neutralizing antibodies while mice in the negative control groups which were not immunized with chimeric VLPs failed to manifest neutralizing antibodies suggesting that SCoV specific neutralizing antibodies are important for the suppres sion of viral replication within the lungs Despite some differences in the cellular composition of inflammatory infiltrates we did not observe any overt lung pathology in the chimeric VLP treated mice when compared to the negative control mice Our results show that chimeric VLP can be an effective vaccine strategy against SCoV infection 2007 Elsevier Ltd All rights reserved Corresponding author Tel 1 409 772 2323 fax 1 409 772 5065 E mail address shmakino utmb edu S Makino 1 K G L and N Y I contributed equally to this study Introduction Severe acute respiratory syndrome SARS is a newly emerged disease caused by SARS coronavirus SCoV SARS originated in Southern China in 2002 and spread to five dif ferent continents causing 8000 infection and 700 deaths 0264 410X see front matter 2007 Elsevier Ltd All rights reserved doi 10 1016 j vaccine 2007 11 092 Chimeric coronavirus like acute respiratory syndrome carrying severe coronavirus SCoV S challenge with SCoV 798 K G Lokugamage et al before its apparent eradication as a human infection in 2004 1 Healthcare systems in affected areas were severely stressed and additional economic costs in trade and travel were very high It is not known if the virus will be reintroduced into the human population but ancestral coro naviruses are widely distributed in bats and are thought to have adapted to civets and then to humans in recent time periods 2 3 Because emerging viruses tend to reemerge as conditions change 4 it is highly desirable to develop safe and infections proteins proteins brane M Coronavirus susceptible tion and for and neutralizing surprising are protein of tects These ognize the tion virus cells sion production assembly particles Further aging 29 32 complex tein replicons to reported co transfected sion E SCoV 39 an protein MHV proteins present tralized in VLP tion Materials and methods Cells and virus Vero E6 cells 293T cells and CHO cells were grown in Dulbecco s modified minimum essential medium DMEM supplemented with penicillin 100units ml streptomycin 100H9262g ml 0 2 sodium bicarbonate and 10 fetal bovine serum FBS The Urbani strain of SCoV was obtained from T vention was virus cells tered Medium and Plasmid The pCAGGS MHV by E based was for of E M described Prepar For in 14 3 5 using tion 1550 to through a in EDT rotor dient the and sisting Purified 50 through pellets until 2 8 M cells were efficacious vaccines and or antivirals to prevent SCoV All coronaviruses including SCoV carry four structural nucleocapsid N protein and three envelope namely spike S protein a type I transmem glycoprotein envelope E protein and membrane protein which has three membrane spanning domains S protein is responsible for virus adsorption to cells through a specific virus receptor interac and induces membrane fusion between viral envelope host cell membrane 5 S protein is a main player determining coronavirus tissue tropism host specificity viral pathogenicity 6 12 Because most coronavirus antibodies recognize S protein 1 13 itisnot that most of the current SCoV vaccine candidates either the S protein subunit itself or those carrying S 14 19 Furthermore prophylactic administration monoclonal antibodies directed at the SCoV S protein pro animals against subsequent SCoV challenge 20 23 studies point out that neutralizing antibodies that rec SCoV S protein are sufficient to prevent or decrease morbidity and mortality associated with SCoV infec by primarily suppressing replication of the challenge Coronavirus like particles VLPs are produced from the coexpressing the S M and E proteins 24 expres of the latter two proteins are sufficient for VLP 24 M protein plays a central role in virus while S protein is assembled into coronavirus through S protein M protein interaction 25 28 interactions of the M protein with the RNA pack signal of the viral RNA 29 and with N protein drive incorporation of the helical nucleocapsid which consists of the viral genome and N pro into virus particles Vaccinia virus and or alphavirus have been used to express coronavirus proteins enable generation of VLPs 33 35 while we have production of SCoV VLPs from 293T cells that are with four eukaryotic pCAGGS based expres plasmids each of which encodes SCoV S M N and proteins 36 Others have also reported production of VLP from insect cells 37 38 and mammalian cells During our studies of coronavirus assembly we found efficient production of chimeric VLPs carrying SCoV S and murine coronavirus mouse hepatitis virus or M N and E proteins from cells coexpressing those In mice immunized with the chimeric VLPs the study describes elicitation of antibodies that neu SCoV and suppressed challenged SCoV replication the lungs These findings suggest that the use of chimeric is an effective vaccine strategy against SCoV infec G Ksiazek at the Centers for Disease Control and Pre Atlanta GA and a working stock of this virus prepared by serially passaging a portion of the seed twice in Vero E6 cells The culture fluid from infected was clarified by low speed centrifugation and was fil using a 0 45H9262m filter portioned and frozen at 80 C from uninfected Vero E6 cells was similarly treated processed construction plasmids pCAGGS MHV A59 S pCAGGS MHV A59 E A59 M and pCAGGS A59 N were constructed inserting the entire region of MHV structural proteins S M and N respectively into a chicken beta actin promoter expression plasmid pCAGGS MCS plasmid pMH54 40 obtained from Paul Masters and used as a template PCR amplification of MHV structural genes Construction plasmids pCAGGS S expressing SCoV S protein pCAGGS expressing SCoV E protein pCAGGS M expressing SCoV protein and pCAGGS N expressing SCoV N protein was previously 36 ation and purification of VLPs preparation of MHV VLPs subconfluent 293T cell cultures 100 mm tissue culture dishes were cotransfected with H9262g of pCAGGS MHV A59 S 0 4H9262g of pCAGGS MHV A59 E H9262g of pCAGGS MHV A59 M and 14H9262g of pCAGGS A59 N TransIT 293 reagent Mirus At 3 days posttransfec the culture media were collected centrifuged at g for 10min and filtered through a 0 45 H9262m filter remove cell debris The MHV VLPs were pelleted down 20 sucrose cushion at 26 000rpm for 3h by using Beckman SW 28 rotor After the pellets were suspended NTE buffer 100mM NaCl 10mM Tris HCl pH 7 0 1mM A the VLPs were centrifuged using a Beckman SW 28 at 26 000rpm for 3h on a discontinuous sucrose gra consisting of 60 50 30 and 20 sucrose The VLPs at interface of 30 and 50 sucrose were collected diluted further purified on a discontinuous sucrose gradient con of 60 50 30 and 20 sucrose at 26 000rpm for 18h and concentrated VLPs at the interface between and 30 sucrose were collected diluted and pelleted a 20 sucrose cushion at 26 000rpm for 2h The were suspended in NTE buffer and kept at 80 C further use For preparation of SCoV VLP a mixture of H9262g of pCAGGS S 13 2H9262g of pCAGGS E 1 4H9262g of pCAGGS and 2 8H9262g of pCAGGS N was cotransfected into 293T grown on a 100 mm tissue culture plate Chimeric VLPs generated by transfecting 293T cells or CHO cells in a Chimeric coronavirus like particles protect mice against challenge with SCoV 799 100 mm tissue culture plate with 14H9262g of pCAGGS S 0 4H9262g of pCAGGS MHV A59 E 3 5H9262g of pCAGGS MHV A59 M and 14H9262g of pCAGGS A59 N SCoV VLPs and chimeric VLPs were purified using the same procedure as described for the MHV VLP purification Quantitation of VLP amounts The total protein concentration of VLPs was quantitated by Bio Rad DC protein assay according to the manufacturer s Procedures for immunization and SCoV challenge MHV VLPs chimeric VLPs or influenza virus vaccine were adjusted to twice their desired final concentration H9262g 0 1ml and then mixed with an equal volume of phos phate buffered saline PBS or alum adjuvant Imject alum Pierce cat no 77161 At day 0 they were administered intramuscularly i m using 1 ml tuberculin syringes Norm Ject Tuttingen Germany and 26 g 3 8 in beveled needles Becton were ture were indicated given directly thetized Chicago of undergo immunized ulation euthanized inoculated at reports confirmed T Mice the 56 specific 2 fold of culture diluted 120 incubated of ferred in virus the assessed induced expressed completely Collection immunohistochemistry T their dle removed quent IHC remaining instructions BIO RAD CA Western blot analysis Western blotting was performed as described previously 36 For detection of MHV S M and N proteins we used anti MHV serum which was produced by immunizing rabbits with a purified JHM strain of MHV kindly provided by Susan Baker Loyola University of Chicago For detection of SCoV S protein a mixture of purified rabbit polyclonal anti SCoV S protein antibody ABGENT cat no AP6000a and polyclonal rabbit anti SCoV S protein antibody IMGENEX cat no IMG 541 was used SCoV N protein was detected by using rabbit polyclonal anti SCoV N protein antibody IMGENEX cat no IMG 548 and SCoV M protein was detected by using a mix ture of SCoV PUPM antibody N terminal ABGENT cat no AP6008a SCoV PUPM antibody C terminal ABGENT cat no AP6008b and anti SCoV M antibodies ProSci cat no 3527P and cat no 3529P Colloidal Coomassie blue staining After washing the gel with water proteins in the gel were stained by soaking the gel with Bio Safe Coomassie Stain BIO RAD with gentle agitation for 1h The gel was rinsed extensively with water overnight Electron microscopic analysis of chimeric VLP Carbon coated 200 mesh copper grids were floated on drops of chimeric VLP for 10min After washing the grids with water three times negative staining was performed using 2 phosphotungstic acid pH 7 0 for 1min After air drying of the grids the sample was examined under a Philips 201 transmission electron microscope and pictures were taken at 60kV Animals Six to 8 week old female Balb c mice Charles River labo ratory Wilmington MA were housed in cages covered with barrier filters in an approval biosafety level 3 animal facil ity maintained by the University of Texas Medical Branch at Galveston Texas All of the mouse experiments were performed using experimental protocols approved by the University of Texas Medical Branch Investigational Animal Care and Use Committee all of the experiments were car ried out following National Institutes of Health and United States Department of Agriculture guidelines Dickinson cat no 305110 In control groups mice inoculated i m with Vero E6 cell culture fluid a mix of influenza vaccine and alum or alum alone or they left untreated Mice were inoculated on day 0 and as in the body of the paper a second injection was on day 28 in most experiments SCoV was inoculated into the nares of mice 40H9262l that were lightly anes with isoflurane IsoFlo Abbott Laboratories North IL Mice that were inoculated with 1 10 6 TCID 50 SCoV at day 0 to provide post infection immunity did not a second inoculation In most of our experiments mice were challenged by intranasal i n inoc of 1 10 6 TCID 50 of SCoV at day 56 and they were at day 58 while in some experiments mice were with SCoV at day 28 and they were euthanized day 30 Day 2 post inoculation was chosen because of that peak titer occurs at that time 23 and this was in our laboratory itration of SCoV specific neutralizing antibodies were anesthetized with isoflurane and then bled from retro orbital sinus plexus After heat inactivation at C for 30min sera were stored at 4 C The assay for virus neutralizing antibodies was performed on serial diluted samples of each serum beginning at a dilution 1 5 using 2 FBS DMEM as the diluent in 96 well tissue plates Falcon 3072 the final volume of the serially samples in each well was 60H9262l After addition of TCID 50 of SCoV in 60H9262l into each well the samples were for 45 60min at room temperature Then 100H9262l these mixtures containing 100 TCID 50 of SCoV were trans into duplicate wells of confluent Vero E6 cells grown 96 well microtiter plates After 72h incubation when the control wells exhibited advanced virus induced CPE neutralizing capacity of individual serum samples was by determining the presence or absence of virus CPE SCoV specific neutralizing antibody titers were as the reciprocal of the last dilution of serum that inhibited virus induced CPE of lungs histology and virus titration wo days post SCoV challenge mice were euthanized and lungs were removed Lung lobes including right mid lobe right lower lobe accessory lobe and left lobe were and placed in 10 buffered formalin for subse histological examination and immunohistochemistry as described previously 41 For virus titration the lobes of the lungs were weighed and frozen at 80 C before being homogenized in PBS 10 FBS solution 800 K G Lokugamage et al using the TissueLyser Qiagen Retsch Haan Germany The homogenates were then centrifuged and SCoV titers in the clarified fluid were determined in a TCID 50 assay in quadru plicate wells of Vero E6 cells in 96 well plates Titers of virus in lung homogenates were expressed as TCID 50 gram of the lungs log 10 and the minimal detectable level of virus was 2 3 log 10 TCID 50 g Formalin fixed and paraffin embedded tissue sections were subjected to the standard hematoxylin and eosin and IHC SCoV Statistical By pad Kruskal W test tralizing assigned 2 3 titers Results Production W SCoV based proteins expression of ate cells analysis SCoV based proteins optimization MHV efficiency than teins S 25 27 28 42 teins M and cient concentrations interacts duction result production cotransfected S tein blot of Figure and were and P WB are indicate P with ative coronavirus mization was chimeric of from the ous concentrations ciencies about 10 times lower than those from 293T cells data not shown We found that when we used TransIT 293 the pro duction of chimeric VLPs from CHO cells was about one third of those from 293T cells in repeated experiments data not shown Prolonged incubation of transfected 293T cells and CHO cells for more than 4 days posttransfection did not improve the accumulation of chimeric VLPs in the super natant data not shown Serum neutralizing antibody titers and lung SCoV titers in the mice immunized once with chimeric VLPs For assessing the usefulness of chimeric VLPs as a SCoV vac cine candidate we first tested the effects of immunization of mice with chimeric VLPs on serum neutralizing antibody production and inhibition of SCoV replication in the lung Six to 8 week old female Balb c mice were inoculated i m once with a mixture of 2H9262g of chimeric VLPs prepared from 293T staining for evaluating histopathology and detecting antigen respectively 41 analysis using a statistical program Instat version 3 Graph Software Inc San Diego CA we performed the allis non parametric analysis of variance ANOVA to compare arithmetic mean SCoV lung titers and neu antibody titers undetectable virus titers were a value of 1 8 the minimal detection limit being log 10 g lung and undetectable virus specific neutralizing were assigned a value of 10 and characterization of chimeric VLPs e have previously reported the successful production of VLPs from 293T cells cotransfected with four pCAGGS plasmids each of which encodes SCoV S M N or E 36 without using exogenous viruses for protein In the study reported here we varied the amount each plasmid for cotransfection and were able to gener approximately 1 3H9262g of SCoV VLPs from 2 10 7 293T Colloidal Coomassie blue staining and Western blot identified SCoV S N and M proteins in the purified VLPs Fig 1A Likewise transfection of four pCAGGS plasmids each of which encoded MHV S E M or N resulted in production of MHV VLPs Fig 1A After we obtained approximately 22 3H9262g of purified VLPs from 2 10 7 293T cells demonstrating that the of MHV VLP production was substantially better that of SCoV VLP production Because M and E pro drive VLP assembly and release 24 and coronavirus protein is incorporated into VLPs through M S interaction these data indicated that MHV M and E pro resulted in more efficient VLP production than did SCoV and E proteins We next tested whether coexpression of SCoV S protein MHV E N and M protein could result in the effi production of chimeric VLPs which would contain high of the SCoV S protein If SCoV S protein with MHV M protein then the robust VLP pro machinery driven by MHV M and E proteins could in efficient chimeric VLP production Chimeric VLP indeed occurred from 293T cells that were with plasmids each of which expressed SCoV protein MHV N protein MHV M protein or MHV E pro Fig 1A Colloidal Coomassie blue staining and Western analysis of purified chimeric VLP showed the presence SCoV S MHV N and MHV M proteins in chimeric VLPs 1 Characterization of VLPs A SCoV VLPs MHV VLPs chimeric VLPs all of which were produced from 293T cells independently purified by sucrose gradient centrifugation 5H9262g of purified VLPs were applied to each lane of SDS AGE Colloidal Coomassie blue staining CCB and Western blot analysis of purified SCoV VLPs MHV VLPs and chimeric VLPs shown B Negative staining of a chimeric VLP Arrowheads peplomers Bars 100nm Courtesy of Dr Vsevolod L opov only a low level of host protein contamination Neg staining of chimeric VLP revealed a typical spherical structure with peplomers Fig 1B After opti approximately 8 7H9262g of purified chimeric VLPs produced from 2 10 7 293T cells the efficiency of VLP production was about 8 times better than that SCoV VLPs We next tested efficiencies of chimeric VLP production Vero and CHO cells both of which have been used for preparation of human vaccines 43 47 By using vari DNA transfection reagents and combinations of different of each plasmid for transfection the effi of chimeric VLP production from Vero cells were Chimeric coronavirus like particles protect mice against challenge with SCoV 801 than those of the mice immunized with a mixture of chimeric VLPs and PBS and the placebo group After challenge the mice immunized with Vero E6 cell culture fluid placebo or with a mixture of 2H9262g chimeric VLP and PBS had high mean virus titers of 6 5log 10 lung and 5 2log 10 lung respectively In contrast no virus was detectable in the lungs of the mice previously administered either live SCoV or 2H9262gof chimeric VLP mixed with alum The SCoV titers in the lungs of these mice two days post virus challenge were inversely proportional titers Effects antibody replica