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1、Long-term protection from SARS coronavirus infection conferred by asingle immunization with an attenuated VSV-based vaccineSagar U. Kapadiaa,b, John K. Rosea,*, Elaine Lamirandec, Leatrice Vogelc,Kanta Subbaraoc, Anjeanette RobertscaDepartment of Pathology, Yale University School of Medicine, 310 Ce
2、dar Street (LH 315), New Haven, CT 06510, USAbDepartment of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USAcLaboratory of Infectious Diseases, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, MD 20892, USAReceived 21 April 2005
3、; returned to author for revision 1 June 2005; accepted 10 June 2005Available online 25 July 2005AbstractAlthough the recent SARS coronavirus (SARS-CoV) that appeared in 2002 has now been contained, the possibility of re-emergence ofSARS-CoV remains. Due to the threat of re-emergence, the overall fa
4、tality rate of 10%, and the rapid dispersion of the virus viainternational travel, viable vaccine candidates providing protection from SARS are clearly needed. We developed an attenuated VSVrecombinant (VSV-S) expressing the SARS coronavirus (SARS-CoV) spike (S) protein. In cells infected with this
5、recombinant, S protein wassynthesized, glycosylated at approximately 17 Asn residues, and transported via the Golgi to the cell surface. Mice vaccinated with VSV-Sdeveloped SARS-neutralizing antibody and were able to control a challenge with SARS-CoV performed at 1 month or 4 months after a singleva
6、ccination. We also demonstrated, by passive antibody transfer, that the antibody response induced by the vaccine was sufficient forcontrolling SARS-CoV infection. A VSV-vectored SARS vaccine could have significant advantages over other SARS vaccine candidatesdescribed to date.D 2005 Published by Els
7、evier Inc.Keywords: Intranasal; Neutralizing antibodies; Protective immunity; SpikeIn the fall of 2002 an emerging human disease termedSARS (severe acute respiratory syndrome) drew globalattention. The disease was characterized as an atypicalpneumonia accompanied by high fever. According theWorld He
8、alth Organization, there were 8096 cases ofSARS, 21% of which were in health care workers.Approximately 10% of the infected individuals died as aresult of the illness, and the death rate exceeded 50% forthose over 60 years old (http:/www.who.int/csr/sars/en/WHOconsensus.pdf and Peiris et al., 2004).
9、 The causativeagent was identified as a coronavirus (Drosten et al., 2003;Ksiazek et al., 2003), a positive-stranded enveloped RNAvirus, and the 29.7-kb genome sequence was determinedrapidly. Like other coronaviruses, the SARS coronavirus(SARS-CoV) has 6 major open reading frames, twoencoding the po
10、lymerase, and four encoding the structuralproteins spike (S), membrane, envelope, and nucleocapsid(Marra et al., 2003; Rota et al., 2003). The SARS S proteinis a type I transmembrane glycoprotein. It is responsible forviral binding to host cellular receptors, identified asangiotensin converting enzy
11、me 2 (Li et al., 2003) andCD209L (Jeffers et al., 2004), followed by viral fusion tohost cells (Bosch et al., 2004; Liu et al., 2004; Yuan et al.,2004). Immunization with the S proteins of other corona-viruses (mouse hepatitis virus MHV, transmissible gastro-enteritis virus TGEV, and infectious bron
12、chitis virusIBV) generates protective immunity against these virusesin various animal models (De Diego et al., 1992; Ignjatovicand Galli, 1994; Wesseling et al., 1993). Expression of the Sprotein of SARS-CoV from either in vitro expression0042-6822/$ - see front matter D 2005 Published by Elsevier I
13、nc.doi:10.1016/j.virol.2005.06.016* Corresponding author.E-mail address: john.roseyale.edu (J.K. Rose).Virology 340 (2005) 174 or from live viral vectors has recently been reportedto generate protection against SARS-CoV infection inanimal models (Bisht et al., 2004; Bukreyev et al., 2004;Chen et al
14、., 2005; Gao et al., 2003; Yang et al., 2004).The spread of SARS was curtailed by public healthmeasures, and the outbreak was successfully contained inJuly 2003. Although several animals are susceptible toSARS-CoV infection, the source of the outbreak and hostreservoir are uncertain (Guan et al., 20
15、03). The possibility ofa re-emergence still exists, and in such an event, a SARSvaccine would be valuable, especially to protect the mostvulnerable groups, the elderly and health care workers.In this study, an experimental vesicular stomatitis virus(VSV)-based SARS vaccine was developed and tested.V
16、SV is a negative strand RNA virus with a non-segmentedgenome that encodes five structural proteins, nucleocapsid(N), phosphoprotein (P), matrix (M), glycoprotein (G), andan RNA-dependent RNA polymerase (L). VSV can elicitstrong humoral and cellular immune responses in a varietyof animals, and VSV-ba
17、sed vaccines have been shown toconfer immunity in animal models of respiratory syncytialvirus (Kahn et al., 2001), influenza virus (Roberts et al.,1998), SHIV/AIDS (Rose et al., 2001), measles virus(Schlereth et al., 2000), and human papilloma virus (Robertset al., 2004). VSV has significant advanta
18、ges over manyother vaccine vectors. It has a relatively small RNA genomebut can accommodate insertion of large foreign genes. Itreplicates exclusively in the cytoplasm via RNA intermedi-ates only and does not undergo recombination. VSVseropositivity is also extremely low in the general popula-tion (
19、Rose and Whitt, 2001). Recombinant VSV generatedby the plasmid DNA system employed here have beenshown to be less pathogenic (attenuated) when comparedwith naturally occurring strains of VSV (Roberts et al.,1998) and cause no disease symptoms in non-humanprimates (Rose et al., 2001) when given by in
20、tranasal, oral,or intramuscular routes. In the present study, we developed arecombinant, attenuated VSV expressing the SARS-CoV Sprotein and tested it as a SARS vaccine in a mouse model(Subbarao et al., 2004). In this model, SARS-CoV replicatesto high titers in the respiratory tract, allowing protec
21、tionfrom SARS infection to be assayed readily in vaccinatedanimals following SARS-CoV challenge.ResultsConstruction and characterization of a VSV recombinant(VSV-S) expressing SARS-CoV S proteinTo determine if the SARS-CoV S protein could beexpressed from a VSV recombinant, we first generated acDNA
22、clone of the SARS S gene by reverse transcriptionand PCR amplification of RNA purified from SARS-CoV-infected cells. We next constructed a plasmid with theSARS-CoV S coding sequence inserted between the VSV Gand VSV L genes (Fig. 1A). Appropriate VSV transcriptionstart and stop sites flanked the S g
23、ene. We recovered arecombinant VSV (designated VSV-S) from this plasmidusing published methods (Lawson et al., 1995). Todetermine if the recombinant virus expressed the SARS Sprotein, we infected BHK cells with the recovered virus orwith wt VSV and metabolically labeled cells for 1 h with35S-methion
24、ine. Lysates of radiolabeled cells were thenanalyzed by SDSPAGE. Since VSV shuts off host proteinsynthesis, wt VSV-infected cells express predominantly thefive viral proteins L, G, N, P, and M. VSV-S-infected cellsexpressed the five VSV proteins (Fig. 1B), and also aprotein of the expected size for
25、S (calculated as 197 kDaassuming N-glycosylation at all 23 potential sites). To verifythe identity of the S protein, S was immunoprecipitated fromthe cell lysates using a rabbit polyclonal antibody to the Scytoplasmic domain. The immunoprecipitated proteinFig. 1. VSV recombinant expressing the SARS-
26、CoV S protein. (A) Thediagram illustrates the genome of the VSV-S recombinant expressing the Sgene and shows the terminal coding and flanking VSV transcription startand stop signals. (B) BHK-21 cells were infected with wt VSV (lanes 1and 2) or VSV-S (lanes 3 and 4), labeled with 35S-methionine, andl
27、ysates were analyzed by SDSPAGE without (lanes 1 and 3) or withimmunoprecipitation with anti-S antibody (lanes 2 and 4). (C) Immuno-precipitated S was left undigested (lane 1), digested with PNGase F (lane 2),or Endo H (lane 3).S.U. Kapadia et al. / Virology 340 (2005) 174182175appeared as two bands
28、 (Fig. 1C, lane 1), potentiallyrepresenting two different glycosylation states of S. Toexamine this possibility we treated the protein with PNGaseF (Fig. 1C, lane 2), which cleaves all N-linked glycans.PNGase F-digestion yielded a single band of approximately156 kDa, confirming that variable glycosy
29、lation states of theS protein were responsible for the observed size hetero-geneity. Digestion of immunoprecipitated S protein withEndo H, an endoglycosidase that cleaves only unprocessedor partially processed glycans from proteins, yielded twobands (Fig. 1C, lane 3). The Endo-H-resistant band ofori
30、ginal size accounted for 15% of total S protein. Thelower band, representing protein which had not yet under-gone processing of its carbohydrate side chains, accountedfor 85% of total S protein. In a pulse-chase experimentwith a 20-min pulse labeling, we observed that 50% of Sexpressed by VSV was En
31、do H resistant within 30 min afterthe chase was begun (data not shown). From these experi-ments, we concluded that the S protein is transportedthrough the exocytic pathway at least as far as the Golgiapparatus where Endo H resistance is acquired.To determine if the S protein was transported from the
32、Golgi to the cell surface, we examined VSV-S-infected cellsusing indirect immunofluorescence microscopy. BHK-21cells were infected with VSV-S or wt VSV, fixed, and thenincubated with serum from a person who had recoveredfrom SARS-CoV infection. A secondary, Alexa Fluor 488-conjugated anti-human anti
33、body was used for visualizationwith fluorescence microscopy. We found that the SARS Sprotein was clearly expressed on the cell surface as indicatedby the strong fluorescent signal visible in VSV-S-but not wtVSV-infected cells (Fig. 2).Immunization with VSV-S elicits humoral immunityInitially we immu
34、nized four groups of eight mice witheither wt VSV, VSV-S, or SARS-CoV. Four weeks later,serum was collected from each mouse. To determine if theVSV and VSV-S inoculations were effective, we performedan assay to measure VSV-neutralizing antibodies. Pooledsera from mice infected with wt VSVor VSV-S ha
35、d a VSV-neutralizing antibody titer of 1:2560, while mice infectedwith SARS-CoV did not have any detectable VSV-neutralizing antibody.We then examined the individual serum samples forneutralizing antibodies to SARS-CoV (Table 1). Infection ofmice with VSV-S generated a stronger neutralizing antibody
36、response (average SARS-neutralizing titer of 1:32) toSARS-CoV than did infection with SARS-CoV (averageSARS-neutralizing titer of 1:12). Sera from control miceFig. 2. SARS-CoV S protein is expressed on the surface of VSV-S-infected cells. BHK-21 cells were infected with VSV-S (A and B) or wt VSV (C
37、and D).Cells were fixed and stained for SARS S as described in Materials and methods. The fluorescence (left) and DIC (right) images were generated with a NikonMicrophot-FX microscope using a 60? objective.S.U. Kapadia et al. / Virology 340 (2005) 174182176immunized with wt VSV did not have detectab
38、le neutraliz-ing antibody titers to SARS-CoV.Immunization with VSV-S protects against SARS-CoVinfectionMice inoculated intranasally with SARS-CoV do notshow any clinical symptoms of infection; however, the virusreplicates to high titers in the lungs and nasal turbinates(NTs). SARS-CoV replication in
39、 respiratory tissues peaksby 2 days post-infection and virus is cleared within a week(Subbarao et al., 2004). We examined SARS-CoV repli-cation in vaccinated and control animals to evaluate theeffectiveness of the VSV-S vaccine in protecting againstSARS-CoV infection.Three groups of four mice each w
40、ere immunized witheither wt VSV, VSV-S, or SARS-CoV. Four weeks later themice were challenged with SARS-CoV, and 2 days after thechallenge, the lungs and nasal turbinates were collected,and the corresponding viral titers were determined (Fig.3A). Control mice, immunized with wt VSV, had highSARS vir
41、al titers in both lungs and nasal turbinatesfollowing SARS-CoV challenge. In contrast, mice immu-nized with VSV-S or SARS-CoV were completely pro-tected and controlled the SARS-CoV challenge as indicatedby SARS-CoV titers at or below the detection limits in boththe lungs and nasal turbinates. These
42、data indicate thatvaccination with VSV-S is as effective as a primaryinfection with SARS-CoV in preventing subsequentSARS-CoV infections.To determine if VSV-S vaccination could providesustained protection from SARS-CoV infection, a similarexperiment was carried out with a 4-month interval betweenimm
43、unizations and challenge (Fig. 3B). Two days afterchallenge, SARS-CoV titers in the lungs of VSV-S immu-nized mice were at the limit of detection (1 of 4 mice) orFig. 3. Protection from SARS-CoVinfection in immunized mice. Balb/c mice were inoculated intranasally with wt VSV, VSV-S, or SARS-CoV. The
44、 mice werechallenged 4 weeks later with 105TCID50SARS-CoV. Two days later, lungs and nasal turbinates were collected, and the viral titers were determined (A).Additional mice were challenged 4 months after immunization with wt VSV, VSV-SARS S, and SARS-CoV. Two days after the challenge, lungs and na
45、salturbinates were collected and viral titers were determined (B). Limits of detection were 1.5 log10TCID50/g tissue in 10% lung homogenates and 1.8log10TCID50/g tissue in 5% nasal turbinate homogenates.Table 1Neutralizing antibody titers to SARS-CoVPrimary immunogenVSVVSV-SSARSIndividualSARS-CoV-ne
46、utralizingantibody titer1:161:131:81:81:251:201:81:201:101:81:321:251:41:811:111:41:161:111:81:401:81:8Average1:321:12S.U. Kapadia et al. / Virology 340 (2005) 174182177undetectable (3 of 4 mice), while the titer in the lungs ofSARS-CoV immunized mice was slightly above the limit ofdetection (1 of 4
47、 mice) or undetectable (3 of 4 mice). Viraltiters in the nasal turbinates were slightly above the limit ofdetection for both VSV-S (3 of 4 mice)- and SARS-CoV(2 of 4 mice)-immunized mice. The mean SARS viraltiters in nasal turbinates of both VSV-S- and SARS-CoV-inoculated mice were nearly 10,000-fol
48、d lower than theviral titers in nasal turbinates from wt VSV-immunizedmice. These findings demonstrate that VSV-S vaccinationprovides sustained protection from SARS-CoV challengeand may provide protective immunity equal to or betterthan primary SARS-CoV infection.Serum from mice immunized with VSV-S
49、 can confer passiveprotectionVSV infection can elicit strong humoral and cellularimmune responses. To determine if antibody alone couldprovide protection against SARS-CoV infection, serumfrom wt VSV-, VSV-S-, or SARS-CoV-infected mice wasadministered intraperitoneally into na ve mice (Fig. 4). Micew
50、ere bled 24 h after administration of antisera and SARS-CoV-neutralizing antibody titers were measured. Mice weresubsequently challenged with SARS-CoV, and 2 days laterlungs were collected and SARS-CoV titers were deter-mined. SARS-CoV-neutralizing antibody titers were detect-able in the groups of m
51、ice that received VSV-S antisera andSARS-CoV antisera (diluted 1:10). Only these mice wereable to protect against SARS-CoV infection upon challenge.Mice receiving wt VSV antisera or normal mouse sera didnot have measurable neutralizing antibodies to SARS-CoV,nor were they protected from SARS-CoV inf
52、ection uponchallenge. These results demonstrate that VSV-S can elicit astrong antibody response that is sufficient for controllingSARS-CoV infection.DiscussionIn this study we used an attenuated VSV vector toexpress the SARS-CoV S protein gene. Because bothSARS-CoV and VSV replicate in the cytoplasm
53、 of infectedcells, we anticipated efficient expression of SARS-CoV Sprotein by a recombinant VSV without problems associatedwith nuclear transcription, including mRNA modificationand mRNA export from the nucleus. The level of S proteinexpression obtained from the VSV-S recombinant wasclearly suffici
54、ent to generate long-term protective immunityto SARS-CoV in this animal model.The levels of SARS-CoV-neutralizing antibody titersfollowing VSV-S immunization were equal to or better thanthose elicited following primary infection with SARS-CoV.Although passive transfer of immune sera demonstrated tha
55、tantibody induced by either VSV-S or SARS-CoV inocu-lations is sufficient to prevent SARS-CoV infection, cellularimmune responses may also contribute to protection.Similarly, it has been shown with other coronaviruses, suchas mouse hepatitis virus type 3, that antibody-mediatedimmunity is sufficient
56、 for protection (Pope et al., 1996).Furthermore, the complete protection from SARS viralreplication in the lower respiratory tissues of VSV-S-immu-nized mice observed when challenge was administered 4months post-immunization suggests that prolonged protec-tion may be provided from a single immunizat
57、ion withVSV-S. Ours is the first study to show such long-termprotection after immunization with an experimental SARS-CoV vaccine. InpreviousstudiesonvaccinationofmicewithVSV expressing an influenza hemagglutinin, we found thatprotective influenza-neutralizing antibody titers were verylong-lived,decl
58、iningonly2-to4-foldoverthecourseofmorethan one year (Roberts and Rose, unpublished data).Our VSV-based SARS vaccine could have significantadvantages over previously described SARS vaccines (BishtFig. 4. Inhibition of SARS-CoV replication by transfer of serum from immunized mice. Sera from uninfected
59、 mice or mice immunized with VSV-S, SARS-CoV (diluted 1:10 in PBS), or wt VSV was injected into na ve mice. Serum was then collected from mice to measure SARS-CoV-neutralizing antibody titer.(The limit of detection was a reciprocal dilution of 8.) The mice were then infected with SARS-CoV, and 2 day
60、s later the lungs were harvested to measureSARS-CoV titer. The limit of detection was 1.5 log10TCID50/g.S.U. Kapadia et al. / Virology 340 (2005) 174182178et al., 2004; Bukreyev et al., 2004; Chen et al., 2005; Gao etal., 2003; Yang et al., 2004). Pre-existing immunity to VSVis rare in the human pop
61、ulation. Effectiveness of othervaccine candidates based on adenovirus or parainfluenzavirus could be limited by a high prevalence of pre-existingimmunity to the vectors in the human population. Vaccinesbased on modified vaccinia Ankara (MVA) could face thesame problem because the parental vaccinia v
62、irus has beenused worldwide as the smallpox vaccine, and there isextensive immunity to vaccinia, especially in the olderpopulation most susceptible to SARS (Crotty et al., 2003).DNA vaccines have been effective, especially in smallanimal models; however, efficacy in humans has yet to bedemonstrated.
63、 VSV has the additional advantages ofgrowing to very high titers in cell lines such as Vero thatare approved for human vaccine production. VSV is alsoeffective at very low doses (Roberts et al., 1998, 1999) andis effective as an intranasal vaccine (Egan et al., 2004).Inactivated SARS-CoV itself has
64、also been shown toelicit an immune response (He et al., 2004; Qu et al., 2005;Takasuka et al., 2004; Tang et al., 2004; Xiong et al., 2004;Zhang et al., 2004); however, the production of such avaccine would carry the inherent risks of exposure to SARS-CoV from handling large volumes of infectious ma
65、terial thatcould result in accidental infection and secondary spread.Additionally, there might be concerns of proper andcomplete inactivation of the virus.The SARS-CoV S protein expressed by VSV wasestimated from gel mobility to be approximately 200 kDaand was converted to about 156 kDa upon the rem
66、oval of allN-linked glycans. Based on this calculation, we estimatethat at least 17 of the 23 potential N-linked glycosylationsites are used (assuming 2.5 kDa per N-linked glycan). Wealso demonstrated that S expressed from the VSV recombi-nant moves to the Golgi apparatus where it acquires Endo Hres
67、istance. About 15% of S expressed during a 1-h labelingperiod contained Endo-H-resistant glycans. In a pulse-chaseexperiment with a 20-min pulse labeling, we observed that50% of S expressed by VSV was Endo H resistant within 30min after the chase was begun (data not shown). Finally, wereadily observ
68、ed S protein on the cell surface. Our resultsindicate that S protein moves efficiently through theexocytic pathway when it is expressed in the absence ofother SARS virus proteins. Exposure of S protein on the cellsurface of infected cells is likely to be important in theinduction of a neutralizing a
69、ntibody response.Other coronavirus vaccines, including those for MHV,IBV, and TGEV, have been effective in controlling infection.In contrast, vaccine against feline infectious peritonitis virususing the S protein of that virus as an antigen enhanceddisease in cats (Vennema et al., 1990). No such enh
70、ancementof infection was observed in the present mouse model in thisand previous studies. However, since SARS-CoV does notcause a disease in mice, this model may not reveal sucheffects. Mild to moderate hepatic lesions have been observedin a single set of experiments where ferrets were vaccinatedwit
71、h MVA, MVA-SAR-S, or MVA-SARS-N and challengedwith SARS-CoV (Czub et al., 2005; Weingartl et al., 2004).The significance of these lesions is unclear since they occurin MVAvaccinated, SARS-CoV-challenged animals, as wellas unvaccinated (PBS controls) SARS-CoV challengedanimals. Although two of three
72、ferrets receiving MVA-SAR-S vaccine have slightly larger lesions following SARS-CoV challenge, it is difficult to ascertain whether hepaticdisease is enhanced or just variable. Furthermore, SARS-CoVdiseaseisprimarilyarespiratorydisease,andthelesionsin the liver may have little or no relevance to SAR
73、S-CoVdisease. These observations may therefore be isolated to theuse of this vector or this animal model or the specificcombination. The potential for SARS-CoV-enhanced diseasein previously vaccinated or SARS-exposed animals requiresfurther studies in models that more closely mimic the humandisease.
74、Materials and methodsPlasmid construction and recombinant VSV recoveryTheSARS-CoVSgenewasamplifiedfromRNAofvirus-infected cells (provided by Dr. William Bellini, CDC) byreverse transcription-PCR using Superscript One-Step RT-PCR for Long Templates (Invitrogen, Carlsbad, CA). Thefollowing primers wer
75、e used in the RT-PCR: 5V-GATC-GATCCTCGAGAACATGTTTATTTTCTTAATTATTTC-3Vand 5V-CGATCCCCCCGGGCTAGCTTATGTGTAATG-TAATTTGACACCC. The PCR product was digested withXhoI and NheI and ligated into pVSVXN2 (Schnell et al.,1996), also digested with the same enzymes. The resultingclone, pVSV-SARS S, was sequenced
76、. The S gene sequencematched the published sequence (GenBank accession no.AY278741) with the exception of a silent mutation atnucleotide 1173 which was G instead of A.Recombinant virus was recovered as previously described(Lawson et al., 1995). Briefly, BHK-21 cells were infectedwith vTF7-3 (Fuerst
77、et al., 1986) for 1 h and then transfectedwith pVSV-SARS S and support plasmids (pBS-N, pBS-M,pBS-G, and pBS-L) for 3 h. Two days after the transfection,the media were transferred onto fresh BHK-21 cells. Themedia were collected after 2 days. A stock of VSV-S from asingle plaque was grown on BHK-21.
78、 Recombinant wild-type (wt) VSV (Lawson et al., 1995) was also grown onBHK-21.SARS S antibodyIn order to detect SARS-CoV S protein, antibodiesdirected to the c-terminal domain were produced. Rabbitswere immunized and boosted with the peptide KFDEDD-SEPVLKGVKLHYT coupled to KLH. Serum was collectedan
79、d affinity purified (Pocono Rabbit Farm and LaboratoryInc., Canadensis, PA).S.U. Kapadia et al. / Virology 340 (2005) 174182179Metabolic labeling, immunoprecipitation, and SDSPAGEBHK-21 cells were infected with either VSV-SARS S orwt VSVat a multiplicity of infection (MOI) of 10. After 5 h,the cells
80、 were washed twice with methionine-free Dulbec-cos modified Eagles medium (DMEM) and incubated withDMEM containing 100 ACi of 35S-methionine for 1 h.Cells were washed with phosphate-buffered saline (PBS)and then lysed with a detergent solution (1% Nonidet P-40,0.4% deoxycholate, 50 mM TrisHCl pH 8,
81、62.5 mMEDTA). Protein samples were analyzed by SDS10%PAGE.SARS S was immunoprecipitated from lysate with the S-tail antiserum at a dilution of 1:100 at 4 -C overnight.Protein A-Sepharose (Zymed Laboratories Inc., San Fran-cisco, CA) was added and allowed to incubate for 1 h atroom temperature. The S
82、epharose was washed with HNTGbuffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mMEGTA, 10% glycerol, 0.1% Triton X-100).The immunoprecipitates were treated with Endoglycosi-dase (Endo) H and peptide N-Glycosidase (PNGase) F(New England Biolabs, Beverly, MA) according to manu-facturers instructions.Immu
83、nofluorescence microscopyBHK-21 cells seeded onto glass coverslips were infectedwith either VSV-S or wt VSV at an MOI of 10. 6 h afterinfection, the cells were washed with PBS and fixed with 3%paraformaldehyde. The cells were then washed with PBSglycine (10 mM). Coverslips were incubated with a 1:20
84、0dilution serum from a person who had recovered from SARS(provided by Dr. William Bellini, CDC). The coverslips werewashed with PBSglycine and incubated with a 1:500dilution of Alexa Fluor 488 goat anti human IgG (MolecularProbes, Eugene, OR). Coverslips were washed and mountedon slides using glycer
85、ol containing 0.1 M n-propylgallate.Cells were observed with Nikon Microphot-FX epi-fluores-cence microscope using a 60? objective.Animal challenge protocolThree groups of eight BABL/c mice were inoculatedintranasally (i.n.) with either 1.6 ? 104pfu of wt VSV,1.4 ? 104pfu of VSV-SARS S, or 105TCID50
86、of SARS-CoV. Twenty-eight days after the immunization, serumwas collected from all mice to measure neutralizingantibody titers. Four mice from each group were thenchallenged i.n. with 104TCID50SARS-CoV as previouslydescribed (Subbarao et al., 2004). Two days later, lungsand nasal turbinates of the m
87、ice were collected todetermine viral titers (Subbarao et al., 2004). Theremaining four mice were challenged 4 months post-immunization. Lungs and nasal turbinates were collected2daysafterthechallenge,andviraltitersweredetermined (Subbarao et al., 2004).Neutralizing antibody titersVSV-neutralizing an
88、tibody titers were measured bydetermining the highest dilution of serum that couldprevent killing of a monolayer of BHK-21 cells by VSV(Rose et al., 2001). SARS-CoV-neutralizing antibody titerswere determined by evaluating the serum dilution at whichthe cytopathic effects (CPE) of SARS-CoV were inhi
89、bitedin half of the wells containing Vero E6 monolayer(Subbarao et al., 2004).Passive protection studySera from mice immunized with wt VSV, VSV-S, andSARS-CoV were collected and heat inactivated. Two groupsof six mice were injected intraperitoneally (i.p.) with 500 Alof wt VSVor VSV-S immune sera. T
90、wo additional groups ofsix mice were also injected i.p. with 500 Al of SARSimmune sera diluted 1:10 in PBS or sera of na ve mice.Serum was collected from each mouse to determineneutralizing antibody titers against SARS-CoV (Subbaraoet al., 2004). Mice were then inoculated i.n. with 105TCID50of SARS-
91、CoV. Two days later, the lungs werecollected from each mouse, and viral titer was determined asdescribed above.AcknowledgmentsWe thank Dr. William Bellini for providing RNA fromSARS-CoV infected cells and for serum samples. This workwas supported by NIH grant AI057158.ReferencesBisht, H., Roberts, A
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