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1、CLINICAL ANDVACCINEIMMUNOLOGY, July 2007, p. 894901Vol. 14, No. 71556-6811/07/$08.00?0doi:10.1128/CVI.00019-07Copyright 2007, American Society for Microbiology. All Rights Reserved.Induction of Specific Immune Responses by Severe Acute RespiratorySyndrome Coronavirus Spike DNA Vaccine with or withou
2、tInterleukin-2 Immunization Using DifferentVaccination Routes in Mice?Hui Hu,1,2Xinya Lu,1,2Ling Tao,1,2Bingke Bai,1,2Zhenfeng Zhang,1Yao Chen,1Fangliang Zheng,1Jianjun Chen,1Ze Chen,1and Hanzhong Wang1*State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Science, Wuhan
3、430071, Peoples Republic ofChina,1and Graduate University of Chinese Academy of Sciences, Beijing 100049, Peoples Republic of China2Received 26 December 2006/Returned for modification 16 March 2007/Accepted 25 April 2007DNA vaccines induce humoral and cellular immune responses in animal models and h
4、umans. To analyze theimmunogenicity of the severe acute respiratory syndrome (SARS) coronavirus (CoV), SARS-CoV, spike DNAvaccine and the immunoregulatory activity of interleukin-2 (IL-2), DNA vaccine plasmids pcDNA-S andpcDNAIL-2 were constructed and inoculated into BALB/c mice with or without pcDN
5、AIL-2 by using threedifferent immunization routes (the intramuscular route, electroporation, or the oral route with live attenuatedSalmonella enterica serovar Typhimurium). The cellular and humoral immune responses were assessed byenzyme-linked immunosorbent assays, lymphocyte proliferation assays,
6、enzyme-linked immunospot assays,and fluorescence-activated cell sorter analyses. The results showed that specific humoral and cellular immu-nities could be induced in mice by inoculating them with SARS-CoV spike DNA vaccine alone or by coinocu-lation with IL-2-expressing plasmids. In addition, the i
7、mmune response levels in the coinoculation groups weresignificantly higher than those in groups receiving the spike DNA vaccine alone. The comparison between thethree vaccination routes indicated that oral vaccination evoked a vigorous T-cell response and a weak responsepredominantly with subclass i
8、mmunoglobulin G2a (IgG2a) antibody. However, intramuscular immunizationevoked a vigorous antibody response and a weak T-cell response, and vaccination by electroporation evoked avigorous response with a predominant subclass IgG1 antibody response and a moderate T-cell response. Ourfindings show that
9、 the spike DNA vaccine has good immunogenicity and can induce specific humoral andcellular immunities in BALB/c mice, while IL-2 plays an immunoadjuvant role and enhances the humoral andcellular immune responses. Different vaccination routes also evoke distinct immune responses. This studyprovides b
10、asic information for the design of DNA vaccines against SARS-CoV.An international outbreak of severe acute respiratory syn-drome (SARS), an atypical form of pneumonia, caused about8,000 cases and 774 deaths across more than 30 countries,starting from its emergence in mid-November 2002 until 7August
11、2003. The etiology of SARS was identified as a newcoronavirus (CoV), named SARS-CoV (13, 22, 29, 37). TheSARS-CoV genome is approximately 29,000 bp and encodesfour main structure proteins, including the spike (S) protein,the membrane (M) protein, the envelope (E) protein, and thenucleocapsid (N) pro
12、tein (9, 22, 37). The S glycoprotein isresponsible for binding to receptors on host cells and plays animportant role in membrane fusion (6, 21, 30, 32, 33). More-over, antibodies to this protein not only neutralize the virus invitro but also protect against lethal SARS-CoV challenge (21,38, 46). It
13、has previously been shown that pseudotype lentiviralparticles bearing the SARS-CoV S protein could be inhibitedby sera from SARS patients (21). In addition, the CD8 T-cellepitopes identified in the SARS-CoV S protein have beenshown to elicit a T-cell response in SARS-CoV-infected pa-tients (39). Hen
14、ce, the S protein is an attractive target for boththerapeutics and vaccine development. Several SARS-CoV S-protein-based vaccines have been shown to generate antibod-ies and cellular immune responses (25, 42, 44). Therefore, ourstudy focused on the SARS-CoV S protein as a target antigenfor the devel
15、opment of a DNA vaccine.DNA vaccines induce both cellular and humoral immuneresponses to produce long-lasting immunity against infectiousdiseases (31). However, the low immunogenicity of DNA-based vaccines could compromise the application of such vac-cines (18, 28). In recent years, many efforts hav
16、e been made toenhance the immune responses elicited by DNA vaccines, in-cluding through the coexpression of cytokines, the use of het-erologous prime-boost regimens, and the use of the conven-tional route of delivery of DNA vaccines.Plasmid cytokine adjuvants can be used to augment DNAvaccine-elicit
17、ed humoral and cellular immune responses in an-imal models (2, 3, 7, 8). Interleukin-2 (IL-2) is a potent cyto-kine that can activate multiple compartments of the immunesystem. Several studies have reported that the immune re-sponses to DNA vaccines can be dramatically enhanced bycoadministration of
18、 plasmids encoding the IL-2 gene. Exam-ples of this phenomenon have been reported for DNA vaccinesagainst bovine herpes virus type 1 (26), hepatitis C virus (18),* Corresponding author. Mailing address: State Key Laboratory ofVirology, Wuhan Institute of Virology, Chinese Academy of Sciences,Wuhan 4
19、30071, Peoples Republic of China. Phone: 86-27-87199239.Fax: 86-27-87198072. E-mail: .?Published ahead of print on 9 May 2007.894hepatitis B virus (7, 8), bovine viral diarrhea virus (28), humanimmunodeficiency virus (27), foot-and-mouth disease virus(41), and measles virus (31). The IL-2 gene has n
20、ot been usedas a cytokine adjuvant in SARS-CoV DNA vaccines, andtherefore, its immune-modulating effects on the SARS-CoVS-protein DNA vaccine were investigated in the current study.In addition to DNA adjuvants, the vaccination route is an-other important factor that influences the efficiency of immu
21、-nization. A number of methods have been developed to in-crease the efficiency of plasmid delivery (15). Apart from theconventional intramuscular (i.m.) route of immunization, re-cent experiments have demonstrated that electroporation cangreatly enhance vaccination with plasmids and is associatedwit
22、h increased levels of gene expression. Additionally, electro-poration displays an adjuvant quality that increases gene ex-pression (48). Another convenient DNA vaccine delivery sys-tem is oral vaccination with live attenuated Salmonella entericaserovar Typhimurium (12, 35). The use of attenuated str
23、ains ofSalmonella as vehicles for the delivery plasmid DNA in vivo is aneffective method for the induction of strong cell-mediated andhumoral immune responses at mucosal sites (1, 4, 10, 16, 19).Thus, we were particularly interested in immunization byelectroporation and immunization by the oral rout
24、e with liveattenuated Salmonella enterica serovar Typhimurium in vivo.Few studies have directly compared the immune responsesgenerated by the use of different routes of vaccination forSARS-CoV DNA immunization. Therefore, in this study, theimmunogenicities of vaccinations with SARS-CoV S-proteinDNA
25、with or without an IL-2-expressing vector delivered bythe i.m. route, electroporation, and the oral route with liveattenuated S. enterica serovar Typhimurium were compared ina mouse model.MATERIALS AND METHODSPlasmids and bacterial strains. pcDNA3.1(?) (Invitrogen, Carlsbad, CA) wasused as the vecto
26、r for all DNA vaccines. The full-length S-protein gene ofSARS-CoV was amplified from pFastBacDULL-S (constructed by the State KeyLaboratory of Virology, Wuhan Institute of Virology). The mouse IL-2 gene waskindly provided by Cui Bo-an (Henan Agricultural University, Zhengzhou, Peo-ples Republic of C
27、hina). The S-protein and IL-2 genes were subcloned intopcDNA3.1 to construct recombinant plasmids pcDNA-S and pcDNAIL-2, re-spectively. The accuracies of the constructs were confirmed by restriction diges-tion and sequencing. DNA plasmids were purified with MegaPrep columns(QIAGEN), dissolved in end
28、otoxin-free phosphate-buffered saline (PBS) to afinal concentration of 2 ?g/?l, and stored at ?20C.The attenuated S. enterica serovar Typhimurium strain CS022 (ATCC 14028;phopc) was kindly provided by Guo Ai-zhen (Huazhong Agricultural University,School of Animal Medicine, Wuhan, Peoples Republic of
29、 China) and was usedas a carrier for oral genetic immunization.Animals and immunization. Six- to 8-week-old female BALB/c mice werepurchased from the Center of Experimental Animal of Hubei Medical Collegeand were randomly divided into groups (eight animals per group). The animalswere provided with p
30、athogen-free water and food. The mice were immunizedthree times at 2-week intervals by the i.m. route, electroporation, or the oralroute with S. enterica serovar Typhimurium. The immunization schedule is sum-marized in Table 1.The immunization dose was 200 ?g plasmid per animal and was injected into
31、the quadriceps muscles. For immunization by electroporation, the animals wereanesthetized and injected with 30 ?g plasmid pcDNA-S i.m. in the rear thigh.Two-needle-array electrodes were inserted into the muscles immediately afterthe injection of DNA by electroporation, and the array was inserted lon
32、gitudi-nally relative to the direction of the muscle fibers. In vivo electroporation wasperformed with a model 820 square-wave generator (BTX, San Diego, CA), andthe parameters were as follows: 20-V/mm distance between the electrodes, 50-mspulse length, and six pulses with a reversal of polarity aft
33、er three pulses. For oralimmunization, attenuated S. enterica serovar Typhimurium strain CS022 cellsharboring the pcDNA-S or the pcDNAIL-2 DNA vaccine were cultured andgrown until they reached an optical density at 600 nm (OD600) of 1.0. The cellswere harvested by centrifugation and resuspended at t
34、he highest required densityin PBS (Sigma). The inoculum of S. enterica serovar Typhimurium was diluted tothe appropriate concentration with 0.1 ml 10% sodium bicarbonate buffer. Eachmouse was immunized by oral gavage with 5 ? 109CFU S. enterica serovarTyphimurium transformed with pcDNA-S.All of the
35、groups containing pcDNAIL-2 were immunized as follows: 50 ?gpcDNAIL-2 per mouse by the i.m. route, 8 ?g by electroporation, and 1 ? 109CFU by oral immunization. The DNA dosage and the numbers of CFU of theattenuated S. enterica serovar Typhimurium strain used to treat the mice wereoptimized by a ser
36、ies of preliminary experiments.Analysis of humoral immune response. Anti-SARS-CoV antibody levels inserum were assessed by enzyme-linked immunosorbent assay (ELISA). Chemi-cally killed SARS-CoV was purified and used as the detection antigen. Opti-mized concentrations (5 ?g/ml) of antigens were coate
37、d onto 96-well plates(Costar) overnight at 4C. The plates were washed and blocked with 1% bovineserum albumin buffered solution for 1 h at 37C prior to a 2-h incubation withmouse serum diluted 1:100 at 37C. Bound antibodies were detected with alka-line phosphatase-conjugated goat anti-mouse immunogl
38、obulin G (IgG; Sigma).The color was developed by adding para-nitrophenylphosphate substrate, andthe A405was read with a plate reader (Bio-Rad). The values obtained for serafrom the mice in the experimental groups were considered positive when they were?2.1 times the value for the control group. Valu
39、es of ?0.05 were not included.A similar ELISA protocol was followed to assess S-protein-specific IgG and itssubclasses (IgG1 and IgG2a). In this case, recombinant S protein expressed inEscherichia coli was purified and was used as the detection antigen. Horseradishperoxidase-conjugated goat anti-mou
40、se IgG, IgG1, and IgG2a (Sigma) were usedas secondary antibodies. The OD490(A490) was read.LPA. The antigen-specific lymphocyte proliferation assay (LPA) was per-formed as described previously (17). In brief, 10 days following the final injection,the mice were killed and single-cell suspensions were
41、 prepared from the spleensfor each group. Splenocytes (2 ? 105per well) in RPMI 1640 medium (Sigma)supplemented with 10% fetal bovine serum were seeded in 96-well plates intriplicate. The cultures were stimulated under the following various conditionsfor 60 h at 37C and 5% CO2: 5 ?g/ml concanavalin
42、A (ConA; positive control),5 ?g/ml purified S-protein antigen (specific antigen), 5 ?g/ml bovine serumalbumin (irrelevant antigen), or medium alone (negative control). The CellTiter96 Aqueous One solution reagent (20 ?l; Promega) was added to each well,according to the manufacturers protocols. Follo
43、wing a 4-h incubation at 37C,the A490was read. Proliferative activity was estimated by using the stimulationindex (SI), which was calculated from the mean OD490for the antigen-containingwells divided by the mean OD490for wells without the antigen.SARS-CoV S-protein-specific ELISPOTs. The cellular im
44、mune responses toSARS-CoV were assessed by gamma interferon (IFN-?) and IL-4 enzyme-linkedimmunospot assays (ELISPOTs) with mouse splenocytes. The assays were per-formed according to the instruction manual (U-CyTech, The Netherlands).Ninety-six-well plates were coated with 5 ?g/ml rat anti-mouse IFN
45、-? or IL-4(100 ?l/well) in PBS overnight. The plates were then washed three times withPBS containing 0.25% Tween 20 and blocked with PBS containing 5% fetalbovine serum for 2 h at 37C. After three additional washes with PBS containing0.25% Tween 20, 1 ? 105splenocytes in 100 ?l reaction buffer conta
46、ining 2 ?g/mlpurified S protein were added to each well. The plates were incubated for 16 hTABLE 1. Immunization schedule for the different forms of vaccinesGroupVaccineRoute ofadministrationDose per mouse1pcDNA-Si.m.200 ?g2pcDNA-S ? pcDNA-IL-2i.m.200 ? 50 ?g3pcDNA-Si.e.a30 ?g4pcDNA-S ? pcDNA-IL-2i.
47、e.30 ? 8 ?g5pcDNA-SOralb5 ? 109CFU6pcDNA-S ? pcDNA-IL-2Oral5 ? 109CFU ? 1 ? 109CFU7pcDNA3.1i.m.200 ?g8pcDNAIL-2i.m.200 ?g9PBSi.m.100 ?l10pcDNA3.1Oral5 ? 109CFU11CS022Oral5 ? 109CFUai.e., electroporation.bOral immunization with attenuated S. enterica serovar Typhimurium CS022.VOL. 14, 2007SARS-CoV SP
48、IKE DNA VACCINE895at 37C in 5% CO2and then washed 10 times with PBS. Biotinylated anti-mouseIFN-? or IL-4 monoclonal antibody at a 1:500 dilution was subsequently added,and plates were incubated for 2 h at room temperature. After the plates werewashed, streptavidin-biotinylated horseradish peroxidas
49、e was added and theplates were incubated for an additional hour at room temperature. Following fivewashes with PBS, individual plates containing IFN-? or IL-4 were developed toobtain dark spots after a 10-min reaction with the peroxidase substrate 3-amino-9-ethylcarbazole. The reactions were stopped
50、 by rinsing the plates with demi-neralized water. The plates were air dried at room temperature, and the absor-bance was read by using an ELISPOT reader (Hitech Instruments). The numberof spot-forming cells per 106splenocytes was calculated. The medium back-grounds consistently had ?10 spot-forming
51、cells per 106splenocytes.Determinations of CD4?and CD8?cells in peripheral blood mononuclearcells. Flow cytometry was used to monitor the expression of T-cell surfacemarkers. Direct immunofluorescence was performed by standard techniques, asdescribed elsewhere (5). CD4?and CD8?cells were assessed 10
52、 days after thefinal boost with each vaccine. Lymphocytes were isolated from peripheral bloodmononuclear cells and stained with the following monoclonal antibodies: fluo-rescein isothiocyanate-labeled anti-mouse CD3 antibody, phycoerythrinCy-5-labeled anti-mouse CD4 antibody, phycoerythrin-labeled a
53、nti-mouse CD8 anti-body (BD PharMingen), or the corresponding isotype controls. One hundredthousand cells were acquired on a FACSCalibur flow cytometer, and the datawere analyzed with WinMDI software (Becton Dickinson, Lincoln Park, NJ).Statistical analysis. All data are presented as means for the i
54、mmunized micein each group ? standard deviations (SDs). SPSS 13.0 software for Windows wasused for statistical analysis. Differences in the humoral and the cellular immuneresponses between groups were assessed by using single-factor analysis of vari-ance. The least-significant-difference t test was
55、used for between-group compar-isons. P values of ?0.05 were considered statistically significant.RESULTSAntibody responses to SARS-CoV. In the present study, plas-mid pcDNA-S or pcDNA-S plus pcDNAIL-2 was used toimmunize mice by the i.m. route, electroporation, or the oralroute with attenuated S. en
56、terica serovar Typhimurium. Toexamine the humoral responses elicited by these vaccines, thelevels of anti-SARS-CoV antibodies in the immunized micewere determined by ELISA. Sera collected 10 days after thefinal boost with each vaccine were assayed, and the results areshown in Fig. 1. In all groups,
57、immunization induced signifi-cantly higher levels of anti-SARS-CoV IgG compared to thosein the control groups immunized with pcDNA3.1, pcDNAIL-2, or PBS (P ? 0.01). Immunization with pcDNA-S byelectroporation induced the highest level of antibody amongthose achieved by the three routes of vaccinatio
58、n, while immu-nization by the oral route evoked the lowest level of antibodyamong those achieved by the three routes of vaccination. Theantibody levels in the groups to which pcDNA and IL-2 werecoadministered were significantly higher than those in thegroups that received pcDNA-S alone (P ? 0.05). F
59、or thegroups immunized with pcDNA-S plus pcDNAIL-2, the an-tibody responses induced by i.m. injection were comparable tothose induced by immunization by the oral route (P ? 0.05)but were significantly lower than those induced by immuniza-tion by electroporation (P ? 0.01).S-protein-specific antibodi
60、es and antibody subclasses. Tendays after the final immunization, the levels of S-protein-spe-cific antibodies in mouse sera were determined by ELISA (Fig.2). All vaccinated mice developed substantial antibody re-sponses, whereas the animals in the control groups did notshow any detectable S-protein
61、-specific antibody response. Thetendency for the mice in the immunized groups to achieve anS-protein-specific IgG antibody response was consistent withthat for the mice to achieve an anti-SARS-CoV antibody re-sponse (Fig. 1).IgG1 and IgG2a levels were measured to determine thehumoral immune response
62、 profiles. As shown in Fig. 2, theanti-SARS-CoV IgG subtype profile revealed that both IgG1and IgG2a were induced by all immunization regimens. Theantibody levels in the group vaccinated with the combinationvaccine showed greater increases than those in the groupsvaccinated with pcDNA-S, although th
63、e antibody subclass pro-files were similar in both groups. The antibodies produced inthe animals vaccinated i.m. were mainly of the IgG2a subclass.The level of the IgG1 subclass that was induced in the groupvaccinated by the mucosal (oral) route was appreciably higherthan the level of the IgG2a subc
64、lass that was induced. Ofinterest, in the groups immunized by electroporation, IgG2awas predominant when the mice were immunized withpcDNA-S alone, but IgG1 was the major subclass in the ani-mals vaccinated with pcDNAIL-2 plus pcDNA-S. In mostcases, coadministration of pcDNAIL-2 and pcDNA-S mainlyen
65、hanced the antibody response, but it did not change theantibody subclass types.S-protein-specific T-cell proliferation. The activation andthe proliferation of lymphocytes play a critical role in both theFIG. 1. Antibody responses to SARS-CoV induced by vaccinationswith or without pcDNAIL-2 by differ
66、ent immunization routes in mice.Serum samples (eight per group) were taken 10 days after the finalimmunization. Data are presented as means ? SDs. Values of ?2.1were considered positive by taking into account the absolute ratio ofthe responses in postimmunization serum/na ve serum.FIG. 2. Detection
67、of SARS-CoV S-protein-specific IgG and othersubclasses in vaccinated mice. Mouse sera (eight per group) werecollected 10 days after the final immunization and were assayed forIgG1 and IgG2a antibodies against the S protein of SARS-CoV. Dataare presented as means ? SDs.896HU ET AL.CLIN. VACCINEIMMUNO
68、L.humoral and the cellular immune responses induced by vacci-nation. Therefore, we next evaluated whether vaccination withpcDNA-S in the presence or the absence of immunization withpcDNAIL-2 by the different routes could influence antigen-specific T-cell proliferation. As shown in Fig. 3, higher lev
69、els oflymphocytes stimulated by the S protein were observed in miceimmunized with pcDNA-S alone or with pcDNA-S pluspcDNAIL-2 than in the controls (P ? 0.01). The level of cellproliferation in animals coimmunized with pcDNAIL-2 wasappreciably higher than that in animals immunized withpcDNA-S alone (
70、P ? 0.05). The level of splenocyte prolifera-tion in response to oral immunization with pcDNA-S orpcDNA-S plus pcDNAIL-2 was consistently and significantlyhigher than that in response to immunization by the other tworoutes (P ? 0.05). Immunization by the i.m. route induced astronger T-cell response
71、than immunization by electropora-tion. These results indicate that immunization with pcDNA-Sand pcDNA-S plus pcDNAIL-2 elicits recognizable levels ofantigen-specific T-cell responses and that immunization by theoral routes evokes the strongest immune response.S-protein-specific Th1- and Th2-type res
72、ponses. ELISPOTwas used to assess the magnitudes of the S-protein-specificIFN-? (Th1) and IL-4 (Th2) T-cell responses after the micewere vaccinated with pcDNA-S DNA or pcDNA-S pluspcDNAIL-2. The protein dosage used for stimulation and thenumber of splenocytes were optimized to induce IFN-? andIL-4 T
73、-cell responses (data not shown). Splenocytes from vac-cinated mice were harvested 10 days after the final vaccination,and S-protein-specific IFN-? and IL-4 levels were enumeratedby ELISPOTs. As shown in Fig. 4A and B, only small numbersof nonspecific IFN-?- and IL-4-secreting cells were detected in
74、the control groups by ELISPOTs (?10 spots/106cells). ByELISPOT, the background counts in wells containing spleno-cytes in the absence of mitogens or nominal antigens wereabout the same as those for the control groups. Significantnumbers of S-protein-specific IFN-? and IL-4 spots were de-tected for a
75、ll the immunized groups by ELISPOTs (P ? 0.01).Compared to immunization with pcDNA-S alone, a two- orthreefold increase in antigen-specific IFN-?-secreting cellnumbers was detected in mice coimmunized with pcDNA andIL-2 by all three immunization routes (P ? 0.05). However, theIL-4-secreting cell num
76、ber was not as high as the IFN-?-se-creting cell number in the groups to which pcDNA and IL-2were coadministered and was appreciably higher only than thatin the group immunized with pcDNA-S alone.Immunization of the mice by the oral route, which inducedthe lowest IgG level and appreciably more of th
77、e IgG1 isotype,elicited significantly higher levels of IFN-? and IL-4 than im-munization by the i.m. route (P ? 0.01) or electroporation(P ? 0.05). Moreover, in all groups, IFN-? was induced to amuch higher level than IL-4. These results suggest that thisvaccine formulation is more immunogenic and l
78、ikely induces astronger Th1 bias.CD8?- and CD4?-lymphocyte responses. Since activatedCD4?and CD8?T lymphocytes are among the most crucialcomponents of antiviral effectors, the responses to these lym-phocytes were assessed in the vaccinated mice (Fig. 5). Flowcytometric analysis of unstimulated cells
79、 was used to standard-ize the background responses, and there was little variation inthe responses among nonimmunized mice. The vaccinationswith pcDNA-S and pcDNA-S plus pcDNAIL-2 significantlyincreased the percentages of activated CD4?and CD8?cellsFIG. 3. S-protein-specific LPA. Pooled splenocytes
80、were obtainedfrom mice (three mice per group) immunized with the DNA vaccineon day 10 postimmunization with pcDNAIL-2. Splenocytes werestimulated in vitro with S protein (test groups), ConA (positive con-trols), and bovine serum albumin (irrelevant antigen controls). Spleno-cytesfromthecontrolgroups
81、(immunizedwithpcDNAIL-2,pcDNA3.1, or PBS) were stimulated with the S protein and served asnegative controls and sham controls. The SI was calculated by use ofthe following formula: (mean OD of ConA- or antigen-stimulatedproliferation)/(mean OD of nonstimulated proliferation). Each barrepresents the
82、mean SI ? SD for three mice.FIG. 4. SARS-CoV S protein-specific IFN-? (A) and IL-4 (B) ELISPOT results. The numbers of IFN-?-secreting cells in the spleens of miceharvested 10 days after the final immunization and stimulated in vitro with the S protein are shown. The results represent the averages f
83、or triplicatewells for three mice and are expressed as means ? SDs.VOL. 14, 2007SARS-CoV SPIKE DNA VACCINE897compared to the percentages for the control groups (P ? 0.01).The numbers of activated CD4?and CD8?cells increased inall immunized groups. The CD8?/CD4?ratio in the groupsimmunized with pcDNA
84、-S plus pcDNAIL-2 was higher thanthat in the groups immunized with pcDNA-S for all threeimmunization routes, but the difference was not statisticallysignificant (P ? 0.05). Moreover, immunization by the oralroute induced a substantial CD8?response, while immuniza-tion by electroporation induced a ne
85、gligible CD8?response.This result paralleled the Ig response of the group immunizedby electroporation, which induced the highest level of CD4?and the lowest level of CD8?. These results further reinforcethe fact that the S-protein DNA vaccine can elicit a T-cellresponse in mice by use of the three i
86、mmunization routes andthat IL-2 expression can enhance the T-lymphocyte activityinduced by the S-protein DNA vaccine.DISCUSSIONIn the present study, a new DNA vaccination approach wasinvestigated by coadministering plasmid pcDNA-S with apcDNAIL-2 plasmid encoding murine IL-2 cDNA by the useof three
87、immunization routes. We clearly showed that vacci-nation with SARS-CoV S-protein DNA elicited SARS-CoVS-protein-specific humoral and cellular immune responses andthat these responses were significantly enhanced by the coad-ministration of an IL-2-expressing vector. The animal trial alsoshowed that t
88、he administration of these DNA vaccine candi-dates by different immunization routes could induce a qualita-tively different immune response profile. Our findings providebasic information for the design of DNA vaccines targetingother antigenic proteins of SARS-CoV.From our results on the anti-SARS-Co
89、V and anti-S-protein-specific antibodies and T-cell proliferation, activated CD4?and CD8?cells were shown to be successfully evoked afterS-protein DNA vaccination. We also found that antigen-spe-cific T cells were capable of secreting high levels of the Th1cytokine IFN-? and moderate levels of the T
90、h2 cytokine IL-4upon in vitro stimulation with the SARS-CoV S protein. Theseresults indicate that the S-protein DNA vaccine activates boththe Th1 and the Th2 subsets, and the level of activation of theTh1 subset was much higher, which correlated with the ten-dency for the IgG2a antibody levels to be
91、 elevated. Thesefindings suggest that the S-protein DNA vaccine is effective inactivating both B and T cells to generate anti-S-protein anti-bodies and cellular immune (mainly Th1) responses in mice.Our results are consistent with those observed in other SARS-CoV DNA vaccine studies (22, 25, 42, 44,
92、 45).Several studies have indicated that the codelivery of vectorsencoding cytokines, such as IL-2, IL-12, IFN-?, or granulocyte-macrophage colony-stimulating factor, is able to direct thenature of the resulting immune response by augmenting theefficacy of DNA vaccines (18, 24, 28, 36). IL-2 is a cy
93、tokine thatcan potently activate multiple compartments of the immunesystem, including T-helper cells, cytotoxic T cells, B cells, mac-rophages, and NK cells (14, 23, 40). Some evidence has sug-gested that the coadministration of plasmids encoding IL-2and a given antigen results in the enhancement of
94、 both hu-moral and cell-mediated immune responses to that antigen andmostly favors Th1 cell differentiation (14, 34, 40, 41). In thecurrent study, the immune responses in mice immunized withthe SARS-CoV S-protein DNA vaccine alone were comparedto those in mice coimmunized with a plasmid encoding IL-
95、2 byanalyzing antibodies, T-cell proliferation, T-helper-cell re-sponses, and CD8?T-cell responses. The mice which receivedpcDNAIL-2 adjuvant rapidly generated IgG antibodies (datanot shown), and the antibody levels were much higher thanthose in mice given the antigen-encoding plasmid alone. BothIgG
96、1 and IgG2a antibody levels increased in all groups immu-nized with the combination, and the dominant isotype did notchange by coinjection of the IL-2-expressing vector. A similarpattern was observed in the T-cell immune responses mea-sured by LPA, ELISPOT, and fluorescence-activated cellsorter anal
97、ysis. Our results showed that immunization withpcDNA-S plus pcDNAIL-2 elicited recognizably higher levelsof T-cell responses compared to those in the groups immu-nized with pcDNA-S alone. Taken together, our results pro-vide evidence that strategies that include IL-2 as an adjuvantcan be used to enh
98、ance the protective immunity of candidateSARS-CoV vaccines. These results are consistent with previ-ous findings obtained with animal models of chronic viral in-fection, which showed that the administration of IL-2 enhancesviral antigen-specific Th1 immune responses and improvesclinical outcomes (2,
99、 8, 23, 41).It has been documented that the route and the method ofimmunization are important modulators of vaccination withDNA vaccines. A DNA vaccine can be delivered by the i.m.route through a needle, by the intradermal route, by the sub-cutaneous route with a gene gun, by electroporation, or by
100、theoral route with live attenuated bacteria. In general, immuni-zation by injection induces both humoral and cellular immuneresponses. Recently, it has been found that the application ofan electric field to tissues in vivo significantly increases thelevels of DNA uptake and gene expression (48). How
101、ever, oralimmunization by the use of Salmonella vaccines which bearforeign antigens can induce strong protective immune re-FIG. 5. Analysis of CD8?and CD4?lymphocytes by flow cytometry.Peripheral blood mononuclear cells were isolated from vaccinated mice(n ? 5) 10 days after the final immunization.
102、CD4?and CD8?T cellsfrom healthy and immunized BALB/c mice were counted. Values areexpressed as means of the ratio of CD8?/CD4? SDs.898HU ET AL.CLIN. VACCINEIMMUNOL.sponses against a wide variety of infectious diseases in animalmodels (10, 11, 16, 43), and such oral vaccines can influencethe immune p
103、rofiles by augmenting the mucosal and cellularimmunities compared to those achieved by administration byinjection. For the SARS-CoV vaccine, it was previously de-scribed that immunization with Salmonella-carrying S-proteinpeptides induced protective antibodies (42). Therefore, we in-vestigated wheth
104、er different vaccination routes could changethe immune types, especially when an IL-2 gene adjuvant iscombined with the SARS-CoV S DNA vaccine.To facilitate DNA vaccine delivery in a mouse model, wechose three different immunization routes, the i.m. route, elec-troporation, and the oral route by the
105、 use of live attenuated S.enterica serovar Typhimurium. The vaccine with or withoutpcDNAIL-2 described here was administered by these immu-nization routes. After the evaluation and a comparison study ofthe efficiencies of these immunization schedules, our resultsshowed that immunization by the i.m.
106、route induced a moder-ate T-cell response and an antibody response, predominantlycomprising an IgG2a response, and that these responses werebetter than those obtained by oral immunization. Vaccinationby electroporation resulted in the highest antibody responseamong the three routes of immunization a
107、nd a midlevel cellu-lar response. In contrast, when the same DNA vaccine wasdelivered orally with live attenuated Salmonella, vigorous T-cell responses but weak antibody production dominated by theIgG2a subclass was evoked. IgG1 subclass antibody responseswere evoked by immunization by all three rou
108、tes, further sug-gesting that the processed antigens were recognized by Th2cells in association with IL-4. In addition, for all three vacci-nation routes enhanced immune responses were observed ingroups coimmunized with IL-2 DNA, including increased an-tigen-specific proliferative responses, higher
109、levels of IFN-?production, and increased CD8?T-cell numbers. These resultssuggest that those distinct types of immune responses gener-ated were due to the different immunization routes but not tocoimmunization with IL-2 DNA. On the basis of our observa-tion that vaccination with DNA by the oral rout
110、e induced astrong T-cell-mediated immune response, whereas immuniza-tion by the i.m. route and electroporation induced moderateT-cell responses but vigorous antibody responses, further stud-ies should be carried out by using a combination of both oraland injection immunizations to stimulate higher c
111、ellular andhumoral immune responses. In addition, our results also showthat the delivery of DNA vaccines by electroporation andorally by using live attenuated Salmonella in vivo is an effectivemethod for increasing the level of antigen expression in muscletissues, leading to marked improvements in i
112、mmune responses.It should be noted that delivery of the same DNA by differ-ent routes induced different immune response profiles. Thismight be due to the different ways of presentation of theS-protein antigen by professional antigen-presenting cellswhen it is delivered by the i.m. route, electropora
113、tion, or theoral route. Live attenuated S. enterica serovar Typhimuriumcould selectively infect M cells. If the DNA encoding the Sprotein was selectively carried into mucosa-associated lym-phoid tissue cells, termed M cells, most of the S peptides withinthe M cells might be cleaved and presented thr
114、ough the majorhistocompatibility complex (MHC) class I pathway, giving riseto a strong cellular response. On the other hand, only a verysmall amount of the S-protein peptides generated in the Mcells might be secreted and presented by B cells through theMHC class II pathway, resulting in a poor antib
115、ody response.In our study, the MHC class I and class II pathways weresimultaneously evoked to some extent by both the i.m. andthe electroporation immunization routes. This is supported bythe findings from a study of the hepatitis B virus DNA vaccinepresented by live attenuated S. enterica serovar Ty
116、phimurium(43). Similar results were also reported by Zheng et al. (47),who showed that live oral vaccination of mice with S. entericaserovar Typhimurium delivering DNA-HBsAg (oral DNA vac-cine) evoked a vigorous T-cell response and a weak antibodyresponse predominantly of the IgG2a subclass. Thus, d
117、ifferentformulations of the same plasmid DNA vaccine can inducedistinct immune responses.Apart from the different ways of antigen presentation byantigen-presenting cells, the reason why the different routes ofvaccine administration evoked distinct immune responses tothe SARS-CoV S protein may be the
118、 different amount of DNAused for the different routes, although the dose of DNA re-quired to stimulate immunity was optimized for the differentimmunization routes. Further refinements of the immunizationconditions, especially the DNA immune dose, are required toensure the maximal expansion of SARS-C
119、oV S-protein DNAvaccine effectors.DNA immunization has been well modeled in mice for theassessment of the optimal parameters for immunization andthe types of immune responses produced. DNA vaccines alsohold promise for use in humans. However, the effects in micemay be more dramatic than those in hum
120、ans, and the currenttechnologies have significant limitations that prevent the fulleffectiveness of DNA vaccines in larger animals. Many aspectsstill remain to be considered prior to the development of aDNA vaccine against SARS-CoV in humans. In the presentreport, we have demonstrated that the SARS-
121、CoV S-proteinDNA vaccine coadministered with an IL-2-expressing plas-mid induces specific immune responses in mice. However,we did not run tests with any other animal models; so it isstill unknown whether this approach could be applied toother animal models, and its immunogenicity in humansremains t
122、o be established. Therefore, it is very important toevaluate the efficacy of this SARS-CoV DNA vaccine insome highly relevant translational models to demonstratethe responsiveness of humans, and further studies should beconducted to validate whether this type of vaccination canbe extended to humans.
123、ACKNOWLEDGMENTSThis work was supported in part by a national 973 Project grant(2005CB523000) and an 863 Project grant (2005AA219070) from theMinistry of Science and Technology, Peoples Republic of China, andthe Chinese Academy of Sciences (KSCX1-YW-R-07).We thank Qinxue Hu for scientific editing of
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