《【病毒外文文献】2010 Intranasal immunization with plasmid DNA encoding spike protein of SARS-coronavirus_polyethylenimine nanoparticles》由会员分享，可在线阅读，更多相关《【病毒外文文献】2010 Intranasal immunization with plasmid DNA encoding spike protein of SARS-coronavirus_polyethylenimine nanoparticles（4页珍藏版）》请在金锄头文库上搜索。
1、RESEARCH ARTICLEOpen AccessIntranasal immunization with plasmid DNAencoding spike protein of SARS-coronavirus/polyethylenimine nanoparticles elicits antigen-specific humoral and cellular immune responsesByoung-Shik Shim1,3, Sung-Moo Park2,3, Ji-Shan Quan1,4, Dhananjay Jere1, Hyuk Chu5, Man Ki Song3,
2、Dong Wook Kim3, Yong-Suk Jang6, Moon-Sik Yang6, Seung Hyun Han3,7, Yong-Ho Park2, Chong-Su Cho1,Cheol-Heui Yun1,8*AbstractBackground: Immunization with the spike protein (S) of severe acute respiratory syndrome (SARS)-coronavirus(CoV) in mice is known to produce neutralizing antibodies and to preven
3、t the infection caused by SARS-CoV.Polyethylenimine 25K (PEI) is a cationic polymer which effectively delivers the plasmid DNA.Results: In the present study, the immune responses of BALB/c mice immunized via intranasal (i.n.) route withSARS DNA vaccine (pci-S) in a PEI/pci-S complex form have been e
4、xamined. The size of the PEI/pci-S nanoparticlesappeared to be around 194.7 99.3 nm, and the expression of the S mRNA and protein was confirmed in vitro.The mice immunized with i.n. PEI/pci-S nanoparticles produced significantly (P 0.05) higher S-specific IgG1 in thesera and mucosal secretory IgA in
5、 the lung wash than those in mice treated with pci-S alone. Compared to thosein mice challenged with pci-S alone, the number of B220+cells found in PEI/pci-S vaccinated mice was elevated.Co-stimulatory molecules (CD80 and CD86) and class II major histocompatibility complex molecules (I-Ad) wereincre
6、ased on CD11c+dendritic cells in cervical lymph node from the mice after PEI/pci-S vaccination. Thepercentage of IFN-g-, TNF-a- and IL-2-producing cells were higher in PEI/pci-S vaccinated mice than in controlmice.Conclusion: These results showed that intranasal immunization with PEI/pci-S nanoparti
7、cles induce antigen specifichumoral and cellular immune responses.BackgroundSevere acute respiratory syndrome (SARS) is an emer-ging infectious disease 1. In contrast to most othercoronaviruses, which cause mild infection, the newSARS-CoV has a high mortality rate. Because there-emergence of SARS is
8、 possible due to existence ofSARS-CoV like strains in animal reservoir, developmentof safe and effective vaccines is highly desired. TheSARS-CoV genome is composed of single positivestranded RNA and encodes four main structural pro-teins including spike protein (S), membrane protein(M), envelope pro
9、tein (E) and nucleocapsid protein (N)2. The S protein is involved in not only receptorrecognition but also in virus attachment and its entryinto target cells 3.In attempts to develop vaccines against various patho-gens, research on DNA vaccine has been widely carriedout. Using DNA vaccines, both hum
10、oral and cellularimmune responses are induced 4. A few studiesdemonstrated that DNA-based vaccines can induce pro-tective immune response against several viruses 5,6.However, one of the problems with DNA-based vaccinesis that they are incapable of inducing immune response* Correspondence: cyunsnu.ac
11、.kr Contributed equally1Department of Agricultural Biotechnology and Research Institute forAgriculture and Life Sciences, Seoul National University, Seoul 151-921,Republic of KoreaFull list of author information is available at the end of the articleShim et al. BMC Immunology 2010, 11:65http:/ 2010
12、Shim et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http:/creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is prope
13、rly cited.in mice when injected through the intranasal (i.n.) route7. In light of the fact that the entry of most respiratorydiseases is through the mucosal surface, it is obviousand ideal that a vaccine should induce both systemicand mucosal immune responses. Secretory IgA plays amajor role in medi
14、ating mucosal immunity 8. Mucosalimmune responses take an important role as a first lineof immune defense system against influenza virus infec-tion although parenteral immunization is not enough toprovoke protective immunity 9.Polyethylenimine (PEI) has been widely used as the non-viral vector in vi
15、tro and in vivo due to high transfectionefficiency and buffering capacity 10. It has been shownthat mucosal administration with PEI could function as apotent mucosal immunostimulator 11. It has beenrevealed that PEI is a very effective gene delivery vehiclefor lung transfection producing high antibo
16、dy titersagainst the encoded protein 12. In the present study, theimmune responses in BALB/c mice immunized withSARS DNA vaccine via i.n. route have been examined.ResultsCharacterization of PEI/pci-S complexesIt is well known that transfection efficiency of gene car-rier depends upon its ability to
17、condense DNA intonano-sized particles 13. As expected, PEI condensedDNA into nano-sized particles, suggesting their endocy-tosis potential (Figure 1A).The formation of PEI/pci-S nanoparticles was furtherconfirmed by morphology observation. Representativeenergy-filtering transmission electron microsc
18、opy(EF-TEM) images of the PEI/pci-S nanoparticles at N/Pratio 10 are shown in Figure 1B. The nanoparticles wereobserved as spherical shape with around 200 nm size,which are similar to those measured by dynamic lightscattering. It is notable that cytotoxicity of PEI was mea-sured in RAW 264.7 cells a
19、fter transfection with PEI/pci-S complexes by using MTT assay. The cell viabilitiesdecreased slightly when the N/P ratios of PEI/pci-Scomplexes increased. When the N/P ratio was 10, thecell viability was 87.5 7.3% (data not shown). In orderto confirm PEI/pci-S uptaken by RAW264.7 cells, rho-damine l
20、abeled pci-S DNA was used to form the nano-particles with the PEI and the complex was visualizedby confocal microscopy. As shown in Figure 1C, thelabeled nanoparticles can be seen in the cells, near tothe nucleus. RT-PCR analysis showed both pci-S DNAand PEI/pci-S nanoparticles can transfect the cel
21、ls. Infact, the PEI/pci-S nanoparticles induced much strongerS mRNA expression than that of naked DNA (Figure1D). Western blot analysis further confirmed that thecells treated with PEI/pci-S nanoparticles can induce theexpression of detectable S protein whereas no proteinwas detected when pci-S was
22、used alone (Figure 1E).SARS-CoV S-specific antibody responses and B cellproliferation capacityTo evaluate the influence of PEI on adaptive immunityto SARS-CoV S protein, specific antibody responseswere examined in mice immunized i.n. with SARS-CoVDNA vaccine. Immunization with PEI/pci-S complexeseli
23、cited high level of SARS-CoV S-specific-serum IgGantibody but not in mice immunized with SARS-CoV SDNA alone (Figure 2A). To access the balance of Th1/Th2 response, SARS S-specific IgG1 and IgG2 wereevaluated in immunized mice. SARS S-specific IgG1antibody was significantly (P 0.01) increased in mic
24、eimmunized with SARS-CoV S DNA vaccine plus PEIwhereas little increase was observed on SARS-specificIgG2a antibody production (Figure 2A), indicating Th2dominant response. To examine mucosal antibody pro-duction, lung wash, nasal wash, fecal extracts, saliva andvaginal wash of immunized mice were co
25、llected. Theresult showed that SARS S-specific IgA antibodyresponse was significantly (P 0.01) increased in lungwash from mice immunized with PEI/pci-S complexes(Figure 2B).To assess B lymphocyte proliferation against SARS-CoV spike protein, the notion that antibody responsesenhanced was further con
26、firmed by proliferation abilityof B220+cells at 1 week after the last vaccination. B220+cells from mice immunized with PEI/pci-S complexeswere highly proliferated after in vitro re-stimulationwith SARS-CoV S protein (Figure 2C).Expression of cell surface molecules and maturation ofdendritic cells (D
27、Cs) from the mice stimulated with PEI/pci-S complexesThe maturation of DCs is accompanied with enhancedexpression of surface markers, including co-stimulatoryand MHC class molecules. To examine the effect ofDNA vaccination on DC maturation in vivo, mice wereimmunized i.n. with PEI/pci-S complexes. T
28、he surfaceexpression of CD80 and CD86 co-stimulatory moleculeswere significantly (P 0.05) higher on DCs from micetreated with PEI/pci-S complexes than those fromSARS-CoV DNA S vaccine alone (Figure 3). MHC classII, I-Ad, expression was also up-regulated significantly (P 0.05) in PEI/pci-S complexes
29、group as compared withthat of SARS-CoV DNA alone (Figure 3).Improvement of SARS-CoV S-specific T cell responses byPEI/pci-S complexesTo examine T cell immunity to SARS-CoV S DNAvaccine, cytokine profiles were examined by using intra-cellular cytokine assays. The cells were harvested fromthe lung at
30、6 days after the immunization. It has beensuggested that T cells producing IFN-g, IL-2, IL-17, andTNF-a are especially effective in protective immunityShim et al. BMC Immunology 2010, 11:65http:/ 2 of 914. Amount of IFN-g-producing cells were increased inCD4+and CD8+T cells from mice immunized with
31、PEI/pci-S whereas IL-17-producing cells were increased onlyin CD4+, not CD8+T cells. It is notable that IFN-g+,IL-2+and IL-17+cells were not detected in CD8+T cells from the mice immunized with PEI/pci-Scomplexes (Figure 4A and 4B). Re-stimulation withSARS-CoV S peptide induced the activation of cyt
32、okine-producing CD4+and CD8+T cells with a predominantproduction of TNF-a as well as TNF-a and IL-2 doublecytokine-producing T cells in mice immunized withPEI/pci-S complexes (Figure 4A and 4B). Furthermore,PBSS geneGAPDHpci-S PEI/pci-Smockpci-SPEI/pci-SS protein-actinABAverage 194.7 ? 99.3CDEFigure
33、 1 Characterization of SARS-CoV S DNA vaccine (pci-S)-PEI complexes. (A) Transmission electron micrographic image of PEI/pci-Scomplexes at N/P ratio 10. Scale bar represents 0.5 m. (B) Size distribution of complexes prepared at N/P ratio 10. (C) The cell uptake of PEI/pci-S complexes was observed by
34、 confocal laser scanning microscope. Upper left, intracellular distribution of rhodamine-labeled PEI/pci-S (red).Upper right, cell nuclei by DAPI staining. Lower left, differential interference images of RAW 264.7 cells. Lower right, overlapping image of nucleiand rhodamine-labeled PEI/pci-S. (D) Ex
35、pression of S mRNA in RAW 264.7 cells transfected with PEI/pci-S complexes was detected by RT-PCR. (E)S protein in RAW 264.7 cells with PEI/pci-S complexes was detected by Western blot.Shim et al. BMC Immunology 2010, 11:65http:/ 3 of 9IFN-g and IL-17 double cytokine-producing cells werefound more i
36、n the PEI/pci-S complexes group while itwas not detectable in pci-S group. It is to note that IL-4-producing cells were detectable neither in the lungnor spleen (data not shown).DiscussionIn the twenty first century, SARS was the first emerginginfectious disease that has seriously threatened publich
37、ealth and the economy throughout the world 1. Over8,000 people from 26 countries were infected with SARS-coronavirus, resulting 774 deaths 15. It has been shownthat SARS-CoV spike protein (S) plays an important rolein receptor recognition, virus attachment and its entry16. It represented one of the
38、most important targets forthe development of SARS vaccine 6. To prevent and con-trol SARS outbreaks, several vaccine studies based on thespike protein of SARS have been done including S proteinvaccine, fragment DNA vaccine, full-length DNA vaccinesand receptor binding domain 17. DNA vaccine encoding
39、full-length S protein has shown to induce humoral, cellu-lar and protective immune responses against SARS-CoV6. In the current study, we evaluated the immunogenicityof a PEI/pci-S in mice through intranasal immunization.There have been several reports that PEI/DNA com-plexes enhance transfection eff
40、iciency in mammaliancells and augment immunogenicity 18. In the presentstudy, we have adapted this PEI/pci-S complex formucosal DNA vaccination. The size of PEI/pci-S com-plexes appeared to be about 200 nm. The effect of PEI/DNA complexes on transfection, and gene and proteinexpression in RAW 264.7
41、cells were evaluated bymeasuring the expression of the mRNA and protein,respectively. This result, thus, let us move ahead to invivo studies for mouse immunization via intranasalroute using PEI/pci-S complexes.A number of studies attempted to develop SARS DNAvaccines, exclusively via systemic routes
42、 includingVaginal washFecalSalivaNasal washLung washpci-Mockpci-Spci-S + PEIB*IgGIgG1IgG2aAbsorbance at 450nmpci-Mockpci-Spci-S + PEIA*pci-Mockpci-Spci-S+PEICFSECell NumberCell Number2.578.2319.7CFigure 2 SARS-CoV S protein-specific humoral and mucosal immune responses in BALB/c mice intranasally im
43、munized with PEI/pci-Scomplexes. The samples were collected on day 7 after the last immunization. Induction of (A) S-specific IgG subclasses in serum and (B) S-specific IgA in various mucosal samples were determined by ELISA. The results were expressed as means SEM for the group (n = 5 to 8).Signifi
44、cant differences compared with pci-S group were expressed as *P 0.05 and *P 0.01, respectively. (C) Proliferation activity of B220+cells from spleen of mice immunized with PEI/pci-S complexes. Bar and number in each panel present the percentage of proliferated cells.Shim et al. BMC Immunology 2010,
45、11:65http:/ 4 of 9intramuscular injection 19. As SARS is a respiratorypathogen, among the SARS vaccine candidates, targetingintranasal immunization was likely to be more effectiveto induce protective immune responses against infectionwhen compared with other delivery routes. Intranasalimmunization o
46、f PEI/pci-S complexes induced higherantigen-specific serum IgG responses than pci-S alone.Coincidently, antigen-specific IgG1 was also dramaticallyincreased when compared to IgG2a, suggesting Th2dominant response. Also the immunization enhancedantigen-specific IgA response in bronchoalveolar lavagef
47、luid and B cell proliferation after in vitro re-stimulationwith the spike protein. In Garzons study, antigen-speci-fic antibody and T cell responses have been dose-depen-dently increased in mice immunized with DNA vaccineup to 100 g 20. However, in our study each mousewas immunized with only 20 g of
48、 DNA. Despite thesmall amount of DNA, it has induced not only systemicbut also mucosal immune responses.DCs play a pivotal role for the effective induction ofantigen-specific immune responses. DCs are foundthroughout the body and are considered as one of thefirst-line sentinel cells 21. When DCs rec
49、ognizepathogen-associated molecular patterns from microor-ganisms, DCs became mature and acquired capacityfor the antigen presentation, concomitantly augmentedthe expression of MHC proteins 22, cytokines 23and number of co-stimulatory molecules includingCD80, CD83, and CD86 24. Thus, the maturation
50、ofDCs is essential for appropriate initiation of the subse-quent adaptive immune responses 22. In the presentstudy, we demonstrated that the PEI/pci-S complexesincrease the co-stimulatory and MHC class II mole-cules on DCs from cervical lymph nodes after intrana-sal immunization.The cellular immune
51、responses are mediated by bothCD4+and CD8+T cells. Functional study indicated thatantigen-specific T cells produce cytokines includingIFN-g, TNF-a, IL-2, and IL-17 after in vitro re-stimula-tion with SARS spike peptides. IFN-g is an effector cyto-kine, critical for activating macrophages and DCs and
52、inhibiting viral infection 25. TNF-a is a cytokine thatprobably regulates immune cells and inhibits viral repli-cation 26. IL-2 mediates the expansion of T cells andmaintains memory T cells 27. IL-17 mediates the pro-duction of antimicrobial peptide and immunoglobulinfor neutralizing viral infection
53、 28. In the current study,we have shown that antigen-specific CD4+and CD8+Tcells secreted IFN-g, TNF-a, IL-2, and IL-17 in non-lymphoid tissues such as lung. Furthermore multiple-cytokine producing cells are increased in mice immu-nized i.n. with PEI/pci-S. It is probable that these cellsare likely
54、responsible for the protection when host isinfected with SARS-CoV after the vaccination. In fact, ithas been suggested that multi-cytokine producing anti-gen-specific CD4+T cells are functionally superior onprotection to single-cytokine producing cells 29. Thereare several reports that multi-cytokin
55、e producing T cellshave shown to correlate with protection against Leish-mania major infection 30. In the current study, thePEI/pci-S complexes induced a high frequency of TNF-a+IL-2+CD4+T cells and IFN-g+IL17+CD4+T cells,and TNF-a+IL-2+CD8+T cells when assessed by SARSpeptide recall responses. Take
56、n together, our resultssupport the hypothesis that intranasal immunizationwith PEI/pci-S complexes induces ideal cellularresponses for the protection.ConclusionPEI is effective in delivering DNA onto the mucosal sur-face, in maturation of dendritic cells and in improvingthe immunogenicity of the DNA
57、 vaccine. Our resultsindicated that PEI can be used as a vector for the muco-sal delivery of DNA vaccine and play an important rolein B cell and T cell immunities.MethodsConstruction of plasmid expressing SARS-CoV S proteinThe gene encoding SARS-CoV spike (S) protein withouttransmembrane domain (ami
58、no acids 14-1154) wassynthesized. The sequence was codon optimized formammalian cell expression and the natural signalCD80CD83CD86CCR7I-AdMock44.0 ?3.438.9 ?2.349.9 ?3.435.4 ?2.51379.0 ?25.2pci-S35.2 ?0.119.0 ?0.551.4 ?0.137.3 ?5.41379.1 ?0.6pci-S+PEI61.3 ?3.7 *20.2 ?0.777.2 ?3.7 *41.3 ?3.81791.2 ?4
59、0.9 *Apci-Mockpci-Spci-S+PEIBCell NumberI-AdFigure 3 Expression of cell surface molecules, CD80, CD83,CD86, CCR7 and MHC class II, on CD11c+cells in cervicallymph nodes from mice immunized with PEI/pci-S complexes.BALB/c mice were immunized with PBS, pci-mock, pci-S, or PEI/pci-Scomplexes and then c
60、ell surface molecules on CD11c+cells fromcervical lymph nodes were analyzed on day 3 after the lastimmunization. (A) The expressions of major cell surface molecules,CD80, CD83, CD86, CCR7, and MHC class II (I-Ad) on CD11c+DCwere determined by flow cytometry. Data were expressed as themean value of m
61、ean fluorescence intensity SD. Significantdifferences compared with pci-S group were expressed as *P 0.05.(B) Expression of MHC class II molecules was represented byhistogram.Shim et al. BMC Immunology 2010, 11:65http:/ 5 of 9sequence was replaced with the leader sequence ofhuman tissue plasminogen
62、activator (tPA). The tPA-Sgene and pci-neo (Promega, Madison, WI) were digestedwith Nhe I and Not I. Then, the plasmid expressingSARS-CoV S protein was generated by ligation.Particle size and morphology of the PEI/pci-SnanoparticlesPEI/pci-S nanoparticles were prepared by mixing poly-mer and pci-S D
63、NA in a solution form at N/P (PEI/pci-S) ratio of 10. The size of PEI/pci-S nanoparticles wasA% of cytokine producing cells / CD4+T cellsIL-17IFN-IL-17 + IFN-CD4+T cell*N.DTNF-IL-2TNF- + IL-2CD4+T cell*N.DN.DIL-17IFN-IL-17 + IFN-CD8+T cellB% of cytokine producingcells / CD8+T cellsN.DN.DN.DN.DN.DTNF
64、-IL-2TNF- + IL-2CD8+T cell*N.DN.DN.DN.DN.DNaivepci-mockpci-Spci-S + PEIpci-S+PEIIL-17IFN-CD4CD8CD4CD8IL-2TNF-Cpci-SIL 17IL 20.020.000.140.170.220.000.000.000.000.000.000.020.040.070.170.350.720.440.020.050.570.950.060.26Figure 4 Effector CD4+and CD8+T cell responses in BALB/c mice immunized with PEI
65、/pci-S complexes. Multi-intracellular cytokinestaining for IL-17, IFN-g, TNF-a and IL-2 was performed on (A) CD4+and (B) CD8+T cells after in vitro re-stimulation with SARS peptide. SARS S-specific CD4+T cells from lung were recovered on day 6 after the last immunization. ND indicates not detectable
66、. Data were expressed as themean value of mean fluorescence intensity SEM. Significant differences compared with pci-S group were expressed as *P 0.01. (C) Theresults showed representative example of flow cytometry analysis.Shim et al. BMC Immunology 2010, 11:65http:/ 6 of 9measured by an electropho
67、retic light scattering spectro-photometer (ELS8000, Otsuka Electronice, Osaka,Japan). Morphology of the PEI/pci-S nanoparticles wasobserved by EF-TEM (LIBRA 120, Carl Zeiss, Germany).Cell uptakeFor cell uptake observation, pci-S DNA was labeled withrhodamine by using Label ITTracker CX-Rhodaminekit
68、(Mirus, WI). RAW 264.7 cells were seeded in theplate. PEI/Rhodamine-labeled pci-S DNA nanoparticleswere incubated for 1 h and washed. Then, they weremounted using ProLongGold antifade reagent withDAPI (Invitrogen, Carlsbad, CA). The cell uptake imageswere observed by confocal laser scanning microsco
69、pe(Carl Zeiss-LSM510, Thornwood, NY).Expression of SARS-CoV S gene and proteinThe expression of SARS-CoV S was examined in RAW264.7 cells at both transcriptional and protein level. Thecells were transfected with naked pci-S DNA or PEI/pci-S nanoparticles at N/P ratio of 10. The cells were lysedwith
70、Trizol or cell lysis buffer 31.Reverse transcription (RT) were performed usingSuperscript III reverse transcriptase (Invitrogen). Theresulting cDNA was amplified by PCR. The sequence ofprimers were as following; for pci-S, forward (CGTCGT GAA AGG CGA TGA TG) and reverse (CGATGG TGT TGT TGC TGT AGG);
71、 for glyceraldehyde3-phosphate dehydrogenase (GAPDH), forward (ACCA-CAGTCCATGCCATCAC)andreverse(TCCAC-CACCCTGTTGCTGTA). The RT-PCR products wereanalyzed by electrophoresis.For Western blot assay, equal amount of lysates wasseparated by SDS-PAGE and subsequently transferredonto a nitrocellulose membr
72、ane (Amersham Biosciences,Piscataway, NY). Membranes were blocked with 5%non-fat milk. Spike protein primary antibody receivedfrom Chiron and horseradish peroxidase-conjugatedsecondary antibody (Santa Cruz Biotechnology, Inc.,Santa Cruz, CA) were incubated with the membrane, inturn. Antigen-antibody
73、 interaction was detected with anECL fluorescence system. b-actin was used as a control.Immunization of miceSix- to eight-week old female BALB/c mice (Orient,Korea) were anesthetized. Five mice per group wereimmunized i.n. with 20 g of pci-mock, pci-S, or PEI/pci-S complexes in total of 25 l ultrapu
74、re water ondays 0, 14, 28, and 42. All studies were approved byIACUC at International Vaccine Institute (Seoul, Korea).Sample collectionSera and mucosal samples were collected on day 6 or 7after the last immunization. Blood was collected fromthe retro-orbital plexus. Fecal extracts were dissolved in
75、phosphate-buffered saline (PBS) containing 0.02%sodium azide. For other samples, mice were anesthetizedand vaginal washes were collected by pipetting withPBS. Lung washes were performed by repeated flushingand aspiration with PBS into the lungs. Nasal washeswere collected twice by flushing with PBS
76、through thenasal cavity.Enzyme-linked immunosorbent assay (ELISA)Microtiter plates (Nunc, Denmark) were coated with 2g/ml of S protein (Protein Sciences Corporation, Meri-den, CT). Plates were blocked with 5% skim milk.Serum (1:20) or mucosal samples (1:2) except lung wash(no dilution) were diluted
77、in the blocking buffer. Each100 l samples were applied into separate wells. Goat-anti-mouse IgG, IgG1, IgG2a or IgA conjugated withhorseradish peroxidase (Santa Cruz Biotechnology, Inc)in the blocking buffer was added to each well. Colorwas developed with TMB solution (Sigma) in dark. Thereaction wa
78、s stopped by 0.5 N HCl. The absorbance at450 nm was measured in a microplate reader (MolecularDevices Corp., Menlo Park, CA).B cell proliferationMice were sacrificed on day 7 after the last vaccinationand spleens were collected. Splenocytes were labeledwith Carboxyfluorescein succinimidyl ester (CFS
79、E) (Invi-trogen) and stimulated with 2 g/ml SARS-S protein for5 days. B cells were stained with anti-B220-PerCP (BDBiosciences). The degree of proliferation was detectedusing flow cytometry, FACSCalibur (BD Biosciences).All cytometric data were analyzed by using FlowJo soft-ware (Tree Star, San Carl
80、os, CA).DC maturation in vivoCervical lymph nodes (CLN) were removed on day 3after the last vaccination. Single cell suspension wasstained with following antibodies: CD11c-APC andCD80-PE, CD83-PE, CD86-biotin, or I-Ad-biotin (BDBiosciences). The degree of expression was detectedusing flow cytometry,
81、 FACSCalibur. All cytometric datawere expressed as MFI (mean fluorescence intensity)and analyzed by using FlowJo software.Intracellular cytokine stainingLungs, removed from mice on day 6 after the last vacci-nation, were disrupted into single-cell suspensions. Thecells were seeded onto 96-well plate
82、 at 2 105cells perwell and re-stimulated with SARS peptide (Peptron) at 5g/ml for 12 hrs. Intracellular cytokine staining assaywas followed by the manufacturers instruction. Thecells were stained with anti-CD4-PerCP and anti-CD8-FITC together with anti-IFN-g-APC and -IL-17-PE, orShim et al. BMC Immu
83、nology 2010, 11:65http:/ 7 of 9anti-TNF-a-APCand-IL-2-PE(allfromBDBiosciences). The percentage of cells with associatedfluorescence was determined by using a flow cytometry,FACSCalibur. All cytometric data were analyzed byusing FlowJo software.Statistical analysisStatistical tests were performed by
84、using Students t test.P value less than 0.05 was considered significant.AbbreviationsSARS: severe acute respiratory syndrome; CoV: coronavirus; PEI:polyethylenimine 25K; S: spike protein; M: membrane protein; E: envelopeprotein; N: nucleocapsid protein; i.n.: Intranasal; pci-S: SARS DNA Vaccine; Ag:
85、antigen; DCs: Dendritic cells; PAMPs: pathogen-associated molecularpatterns; tPA: tissue plasmnogen activator; EF-TEM: energy-filteringtransmission electron microscopy; CLN: cervical lymph nodesAcknowledgementsThis work was supported by National Research Foundation (NRF) grants(2010-0003291 and 2010
86、-0027222) funded through the Ministry of Education,Science and Technology, and was conducted as a part of the TechnologyDevelopment Program for Agriculture (Agriculture Research Centerprogram), supported by the Ministry for Food, Agriculture, Forestry andFisheries, Republic of Korea. We thank Ho-Hyu
87、n Song, Dong-Hyun Joo, andKyung-Min Cho for supporting animal experiments.Author details1Department of Agricultural Biotechnology and Research Institute forAgriculture and Life Sciences, Seoul National University, Seoul 151-921,Republic of Korea.2Department of Microbiology, College of Veterinarymedi
88、cine, Seoul National University, Seoul 151-921, Republic of Korea.3Laboratory Science Division, International Vaccine Institute, Seoul 151-818,Republic of Korea.4College of Pharmacy, Yanbian University, Jilin Province133000, PR China.5Division of Zoonoses, Center for Immunology &Pathology, National
89、Institute of Health, Korea Center for Disease Control &Prevention, Seoul 122-701, Republic of Korea.6Division of Biological Sciencesand The Institute for Molecular Biology and Genetics, Chonbuk NationalUniversity, Jeonju 561-756, Republic of Korea.7Department of OralMicrobiology & Immunology, Dental
90、 Research Institute, and BK21 Program,School of Dentistry, Seoul National University, Seoul 110-749, Republic ofKorea.8Center for Agricultural Biomaterials, Seoul National University, Seoul151-921, Republic of Korea.Authors contributionsBSS, SMP and JSQ performed most of experiments, analyzed data,
91、andcarried out some statistical analysis. They participated in the design of thestudy and helped with draft of the manuscript. DJ carried out the assay,measuring the size of PEI/pci-S nanoparticles. HC performed statisticalanalysis. MKS, DWK, YSJ, MSY, SHH and YHP advised with analysing the dataand
92、drafting the manuscript. CSC and CHY conceived and designed thestudy and finalized the manuscript. All authors have read and approved thefinal manuscript.Competing interestsThe authors declare that they have no competing interests.Received: 29 March 2010 Accepted: 31 December 2010Published: 31 Decem
93、ber 2010References1.Zhong N, Zheng B, Li Y, Poon L, Xie Z, Chan K, Li P, Tan S, Chang Q, Xie J:Epidemiology and cause of severe acute respiratory syndrome (SARS) inGuangdong, Peoples Republic of China. February Lancet 2003,362:1353-1358.2.Rota P, Oberste M, Monroe S, Nix W, Campagnoli R, Icenogle J,
94、Penaranda S, Bankamp B, Maher K, Chen M: Characterization of a novelcoronavirus associated with severe acute respiratory syndrome. Science2003, 300:1394-1399.3.Li W, Moore M, Vasilieva N, Sui J, Wong S, Berne M, Somasundaran M,Sullivan J, Luzuriaga K, Greenough T: Angiotensin-converting enzyme 2 isa
95、 functional receptor for the SARS coronavirus. Nature 2003, 426:450-454.4.Rice J, Ottensmeier C, Stevenson F: DNA vaccines: precision tools foractivating effective immunity against cancer. Nature Reviews Cancer 2008,8:108-120.5.Wang B, Ugen K, Srikantan V, Agadjanyan M, Dang K, Refaeli Y, Sato A,Boy
96、er J, Williams W, Weiner D: Gene inoculation generates immuneresponses against human immunodeficiency virus type 1. Proceedings ofthe National Academy of Sciences 1993, 90:4156-4160.6.Yang Z, Kong W, Huang Y, Roberts A, Murphy B, Subbarao K, Nabel G: ADNA vaccine induces SARS coronavirus neutralizat
97、ion and protectiveimmunity in mice. Nature 2004, 428:561-564.7.Wang D, Christopher M, Nagata L, Zabielski M, Li H, Wong J, Samuel J:Intranasal immunization with liposome-encapsulated plasmid DNAencoding influenza virus hemagglutinin elicits mucosal, cellular andhumoral immune responses. Journal of C
98、linical Virology 2004, 31:99-106.8.Fagarasan S, Honjo T: Intestinal IgA synthesis: regulation of front-linebody defences. Nature Reviews Immunology 2003, 3:63-72.9.Tumpey T, Renshaw M, Clements J, Katz J: Mucosal delivery of inactivatedinfluenza vaccine induces B-cell-dependent heterosubtypic cross-
99、protection against lethal influenza A H5N1 virus infection. Journal ofvirology 2001, 75:5141-5150.10.Boussif O, Lezoualch F, Zanta M, Mergny M, Scherman D, Demeneix B,Behr J: A versatile vector for gene and oligonucleotide transfer into cellsin culture and in vivo: polyethylenimine. Proceedings of t
100、he NationalAcademy of Sciences 1995, 92:7297-7301.11.Regnstrm K, Ragnarsson E, ping-H gg rd MK, Torstensson E, Nyblom H,Artursson P: PEI a potent, but not harmless, mucosal immuno-stimulatorof mixed T-helper cell response and FasL-mediated cell death in mice.Gene Therapy 2003, 10:1575-1583.12.Densmo
101、re C, Orson F, Xu B, Kinsey B, Waldrep J, Hua P, Bhogal B, Knight V:Aerosol delivery of robust polyethyleneimine-DNA complexes for genetherapy and genetic immunization. Molecular Therapy 2000, 1:180-188.13.Arote R, Kim T, Kim Y, Hwang S, Jiang H, Song H, Nah J, Cho M, Cho C: Abiodegradable poly (est
102、er amine) based on polycaprolactone andpolyethylenimine as a gene carrier. Biomaterials 2007, 28:735-744.14.Seder R, Darrah P, Roederer M: T-cell quality in memory and protection:implications for vaccine design. Nature Reviews Immunology 2008,8:247-258.15.Skowronski D, Astell C, Brunham R, Low D, Pe
103、tric M, Roper R, Talbot P,Tam T, Babiuk L: Severe acute respiratory syndrome (SARS): a year inreview. Annual Review of Medicine 2005, 56:357-381.16.Sainz B, Rausch J, Gallaher W, Garry R, Wimley W: Identification andcharacterization of the putative fusion peptide of the severe acuterespiratory syndr
104、ome-associated coronavirus spike protein. Journal ofVirology 2005, 79:7195-7206.17.Wang Z, Yuan Z, Matsumoto M, Hengge U, Chang Y: Immune responseswith DNA vaccines encoded different gene fragments of severe acuterespiratory syndrome coronavirus in BALB/c mice. Biochemical andBiophysical Research Co
105、mmunications 2005, 327:130-135.18.Huang X, Xu J, Qiu C, Ren L, Liu L, Wan Y, Zhang N, Peng H, Shao Y:Mucosal priming with PEI/DNA complex and systemic boosting withrecombinant TianTan vaccinia stimulate vigorous mucosal and systemicimmune responses. Vaccine 2007, 25:2620-2629.19.Jin H, Xiao C, Chen
106、Z, Kang Y, Ma Y, Zhu K, Xie Q, Tu Y, Yu Y, Wang B:Induction of Th1 type response by DNA vaccinations with N, M, and Egenes against SARS-CoV in mice. Biochemical and Biophysical ResearchCommunications 2005, 328:979-986.20.Garzon Rodrigo M, Berraondo P, Crettaz J, Ochoa L, Vera M, Lasarte J,Vales A, V
107、an Rooijen N, Ruiz J, Prieto J: Induction of gp120-specificprotective immune responses by genetic vaccination with linearpolyethylenimine-plasmid complex. Vaccine 2005, 23:1384-1392.21.Cochran A, Morton D, Stern S, Lana A, Essner R, Wen D: Sentinel lymphnodes show profound downregulation of antigen-
108、presenting cells of theparacortex: implications for tumor biology and treatment. ModernPathology 2001, 14:604-608.22.Medzhitov R: Recognition of microorganisms and activation of theimmune response. Nature 2007, 449:819-826.Shim et al. BMC Immunology 2010, 11:65http:/ 8 of 923.Perry A, Gang C, Zheng
109、D, Hong T, Cheng G: The host type I interferonresponse to viral and bacterial infections. Cell Research 2005, 15:407-422.24.Son Y, Ahn S, Jang M, Moon Y, Kim S, Cho K, Han S, Yun C:Immunomodulatory effect of resistin in human dendritic cells stimulatedwith lipoteichoic acid from Staphylococcus aureu
110、s. Biochemical andBiophysical Research Communications 2008, 376:599-604.25.Wu C, Kirman J, Rotte M, Davey D, Perfetto S, Rhee E, Freidag B, Hill B,Douek D, Seder R: Distinct lineages of Th1 cells have differentialcapacities for memory cell generation in vivo. Nature immunology 2002,3:852-858.26.Agga
111、rwal B: Signalling pathways of the TNF superfamily: a double-edgedsword. Nature Reviews Immunology 2003, 3:745-756.27.Kim H, Jo E, Park J, Kim J: The enhancing IL-2R alpha mRNA expressioninduces a marked T cell proliferation with interleukin-2 and anti-CD3mAb. Korean Journal of Immunology 1998, 20:4
112、27-434.28.Kolls J, McCray P, Chan Y: Cytokine-mediated regulation of antimicrobialproteins. Nature Reviews Immunology 2008, 8:829-835.29.Kannanganat S, Ibegbu C, Chennareddi L, Robinson H, Amara R: Multiple-cytokine-producing antiviral CD4 T cells are functionally superior tosingle-cytokine-producin
113、g cells. Journal of Virology 2007, 81:8468-8476.30.Darrah P, Patel D, De Luca P, Lindsay R, Davey D, Flynn B, Hoff S,Andersen P, Reed S, Morris S: Multifunctional Th1 cells define a correlateof vaccine-mediated protection against Leishmania major. NatureMedicine 2007, 13:843-850.31.Beloor J, Kang H,
114、 Yun C, Kim S, Moon Y: Low lysine treatment increasesadipogenic potential of bovine intramuscular preadipocytes. Asian-Australasian Journal of Animal Sciences 2009, 22:721-726.doi:10.1186/1471-2172-11-65Cite this article as: Shim et al.: Intranasal immunization with plasmidDNA encoding spike protein
115、 of SARS-coronavirus/polyethyleniminenanoparticles elicits antigen-specific humoral and cellular immuneresponses. BMC Immunology 2010 11:65.Submit your next manuscript to BioMed Centraland take full advantage of: Convenient online submission Thorough peer review No space constraints or color figure
116、charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistributionSubmit your manuscript at et al. BMC Immunology 2010, 11:65http:/ 9 of 9BioMed Central publishes under the Creative Commons Attribution License (CCAL). Under the CCAL, authorsretain copyright to the article but users are allowed to download, reprint, distribute and /or copy articles inBioMed Central journals, as long as the original work is properly cited.
