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RESEARCH ARTICLE Open Access Intranasal immunization with plasmid DNA encoding spike protein of SARS coronavirus polyethylenimine nanoparticles elicits antigen specific humoral and cellular immune responses Byoung Shik Shim 1 3 Sung Moo Park 2 3 Ji Shan Quan 1 4 Dhananjay Jere 1 Hyuk Chu 5 Man Ki Song 3 Dong Wook Kim 3 Yong Suk Jang 6 Moon Sik Yang 6 Seung Hyun Han 3 7 Yong Ho Park 2 Chong Su Cho 1 Cheol Heui Yun 1 8 Abstract Background Immunization with the spike protein S of severe acute respiratory syndrome SARS coronavirus CoV in mice is known to produce neutralizing antibodies and to prevent 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 with SARS DNA vaccine pci S in a PEI pci S complex form have been examined The size of the PEI pci S nanoparticles appeared 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 the sera and mucosal secretory IgA in the lung wash than those in mice treated with pci S alone Compared to those in 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 A d were increased on CD11c dendritic cells in cervical lymph node from the mice after PEI pci S vaccination The percentage of IFN g TNF a and IL 2 producing cells were higher in PEI pci S vaccinated mice than in control mice Conclusion These results showed that intranasal immunization with PEI pci S nanoparticles induce antigen specific humoral and cellular immune responses Background Severe acute respiratory syndrome SARS is an emer ging infectious disease 1 In contrast to most other coronaviruses which cause mild infection the new SARS CoV has a high mortality rate Because the re emergence of SARS is possible due to existence of SARS CoV like strains in animal reservoir development of safe and effective vaccines is highly desired The SARS CoV genome is composed of single positive stranded RNA and encodes four main structural pro teins including spike protein S membrane protein M envelope protein E and nucleocapsid protein N 2 The S protein is involved in not only receptor recognition but also in virus attachment and its entry into target cells 3 In attempts to develop vaccines against various patho gens research on DNA vaccine has been widely carried out Using DNA vaccines both humoral and cellular immune responses are induced 4 A few studies demonstrated that DNA based vaccines can induce pro tective immune response against several viruses 5 6 However one of the problems with DNA based vaccines is that they are incapable of inducing immune response Correspondence cyun snu ac kr Contributed equally 1 Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences Seoul National University Seoul 151 921 Republic of Korea Full list of author information is available at the end of the article Shim et al BMC Immunology 2010 11 65 2010 Shim et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License http creativecommons org licenses by 2 0 which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited in mice when injected through the intranasal i n route 7 In light of the fact that the entry of most respiratory diseases is through the mucosal surface it is obvious and ideal that a vaccine should induce both systemic and mucosal immune responses Secretory IgA plays a major role in mediating mucosal immunity 8 Mucosal immune responses take an important role as a first line of immune defense system against influenza virus infec tion although parenteral immunization is not enough to provoke protective immunity 9 Polyethylenimine PEI has been widely used as the non viral vector in vitro and in vivo due to high transfection efficiency and buffering capacity 10 It has been shown that mucosal administration with PEI could function as a potent mucosal immunostimulator 11 It has been revealed that PEI is a very effective gene delivery vehicle for lung transfection producing high antibody titers against the encoded protein 12 In the present study the immune responses in BALB c mice immunized with SARS DNA vaccine via i n route have been examined Results Characterization of PEI pci S complexes It is well known that transfection efficiency of gene car rier depends upon its ability to condense DNA into nano sized particles 13 As expected PEI condensed DNA into nano sized particles suggesting their endocy tosis potential Figure 1A The formation of PEI pci S nanoparticles was further confirmed by morphology observation Representative energy filtering transmission electron microscopy EF TEM images of the PEI pci S nanoparticles at N P ratio 10 are shown in Figure 1B The nanoparticles were observed as spherical shape with around 200 nm size which are similar to those measured by dynamic light scattering It is notable that cytotoxicity of PEI was mea sured in RAW 264 7 cells after transfection with PEI pci S complexes by using MTT assay The cell viabilities decreased slightly when the N P ratios of PEI pci S complexes increased When the N P ratio was 10 the cell viability was 87 5 7 3 data not shown In order to confirm PEI pci S uptaken by RAW264 7 cells rho damine labeled pci S DNA was used to form the nano particles with the PEI and the complex was visualized by confocal microscopy As shown in Figure 1C the labeled nanoparticles can be seen in the cells near to the nucleus RT PCR analysis showed both pci S DNA and PEI pci S nanoparticles can transfect the cells In fact the PEI pci S nanoparticles induced much stronger SmRNAexpressionthanthatofnakedDNA Figure 1D Western blot analysis further confirmed that the cells treated with PEI pci S nanoparticles can induce the expression of detectable S protein whereas no protein was detected when pci S was used alone Figure 1E SARS CoV S specific antibody responses and B cell proliferation capacity To evaluate the influence of PEI on adaptive immunity to SARS CoV S protein specific antibody responses were examined in mice immunized i n with SARS CoV DNA vaccine Immunization with PEI pci S complexes elicited high level of SARS CoV S specific serum IgG antibody but not in mice immunized with SARS CoV S DNA alone Figure 2A To access the balance of Th1 Th2 response SARS S specific IgG1 and IgG2 were evaluated in immunized mice SARS S specific IgG1 antibody was significantly P 0 01 increased in mice immunized with SARS CoV S DNA vaccine plus PEI whereas little increase was observed on SARS specific IgG2a antibody production Figure 2A indicating Th2 dominant response To examine mucosal antibody pro duction lung wash nasal wash fecal extracts saliva and vaginal wash of immunized mice were collected The result showed that SARS S specific IgA antibody response was significantly P 0 01 increasedinlung wash from mice immunized with PEI pci S complexes Figure 2B To assess B lymphocyte proliferation against SARS CoV spike protein the notion that antibody responses enhanced was further confirmed by proliferation ability of B220 cells at 1 week after the last vaccination B220 cells from mice immunized with PEI pci S complexes were highly proliferated after in vitro re stimulation with SARS CoV S protein Figure 2C Expression of cell surface molecules and maturation of dendritic cells DCs from the mice stimulated with PEI pci S complexes The maturation of DCs is accompanied with enhanced expression of surface markers including co stimulatory and MHC class molecules To examine the effect of DNA vaccination on DC maturation in vivo micewere immunized i n with PEI pci S complexes The surface expression of CD80 and CD86 co stimulatory molecules were significantly P 0 05 higher on DCs from mice treated with PEI pci S complexes than those from SARS CoV DNA S vaccine alone Figure 3 MHC class II I A d expression was also up regulated significantly P 0 05 in PEI pci S complexes group as compared with that of SARS CoV DNA alone Figure 3 Improvement of SARS CoV S specific T cell responses by PEI pci S complexes To examine T cell immunity to SARS CoV S DNA vaccine cytokine profiles were examined by using intra cellular cytokine assays The cells were harvested from the lung at 6 days after the immunization It has been suggested that T cells producing IFN g IL 2 IL 17 and TNF a are especially effective in protective immunity Shim et al BMC Immunology 2010 11 65 Page 2 of 9 14 Amount of IFN g producing cells were increased in CD4 and CD8 T cells from mice immunized with PEI pci S whereas IL 17 producing cells were increased only in CD4 notCD8 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 S complexes Figure 4A and 4B Re stimulation with SARS CoV S peptide induced the activation of cytokine producing CD4 and CD8 T cells with a predominant production of TNF a as well as TNF a and IL 2 double cytokine producing T cells in mice immunized with PEI pci S complexes Figure 4A and 4B Furthermore PBS S gene GAPDH pci S PEI pci S mock pci S PEI pci S S protein actin AB Average 194 7 99 3 C D E Figure 1 Characterization of SARS CoV S DNA vaccine pci S PEI complexes A Transmission electron micrographic image of PEI pci S complexes 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 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 nuclei and rhodamine labeled PEI pci S D Expression 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 65 Page 3 of 9 IFN g and IL 17 double cytokine producing cells were found more in the PEI pci S complexes group while it was not detectable in pci S group It is to note that IL 4 producing cells were detectable neither in the lung nor spleen data not shown Discussion In the twenty first century SARS was the first emerging infectious disease that has seriously threatened public health and the economy throughout the world 1 Over 8 000 people from 26 countries were infected with SARS coronavirus resulting 774 deaths 15 It has been shown that SARS CoV spike protein S plays an important role in receptor recognition virus attachment and its entry 16 It represented one of the most important targets for the development of SARS vaccine 6 To prevent and con trol SARS outbreaks several vaccine studies based on the spike protein of SARS have been done including S protein vaccine fragment DNA vaccine full length DNA vaccines and receptor binding domain 17 DNA vaccine encoding full length S protein has shown to induce humoral cellu lar and protective immune responses against SARS CoV 6 In the current study we evaluated the immunogenicity of a PEI pci S in mice through intranasal immunization There have been several reports that PEI DNA com plexes enhance transfection efficiency in mammalian cells and augment immunogenicity 18 In the present study we have adapted this PEI pci S complex for mucosal 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 protein expression in RAW 264 7 cells were evaluated by measuring the expression of the mRNA and protein respectively This result thus let us move ahead to in vivo studies for mouse immunization via intranasal route using PEI pci S complexes A number of studies attempted to develop SARS DNA vaccines exclusively via systemic routes including Vaginal wash Fecal Saliva Nasal wash Lung wash pci Mock pci S pci S PEI B IgG IgG1 IgG2a A b sorbance at 450nm pci Mock pci S pci S PEI A pci Mock pci S pci S PEI CFSE Cell NumberCell Number 2 57 8 23 19 7 C Figure 2 SARS CoV S protein specific humoral and mucosal immune responses in BALB c mice intranasally immunized with PEI pci S complexes 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 Significant 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 11 65 Page 4 of 9 intramuscular injection 19 As SARS is a respiratory pathogen among the SARS vaccine candidates targeting intranasal immunization was likely to be more effective to induce protective immune responses against infection when compared with other delivery routes Intranasal immunization of PEI pci S complexes induced higher antigen specific serum IgG responses than pci S alone Coincidently antigen specific IgG1 was also dramatically increased when compared to IgG2a suggesting Th2 dominant response Also the immunization enhanced antigen specific IgA response in bronchoalveolar lavage fluid and B cell proliferation after in vitro re stimulation with the spike protein In Garzon s study antigen speci fic antibody and T cell responses have been dose depen dently increased in mice immunized with DNA vaccine up to 100 g 20 However in our study each mouse was immunized with only 20 g of DNA Despite the small amount of DNA it has induced not only systemic but also mucosal immune responses DCs play a pivotal role for the effective induction of antigen specific immune responses DCs are found throughout the body and are considered as one of the first line sentinel cells 21 When DCs recognize pathogen associated molecular patterns from microor ganisms DCs became mature and acquired capacity for the antigen presentation concomitantly augmented the expression of MHC proteins 22 cytokines 23 and number of co stimulatory molecules including CD80 CD83 and CD86 24 Thus the maturation of DCs is essential for appropriate initiation of the subse quent adaptive immune responses 22 In the present study we demonstrated that the PEI pci S complexes increase the co stimulatory and MHC class II mole cules on DCs from cervical lymph nodes after intrana sal immunization The cellular immune responses are mediated by both CD4 and CD8 T cells Functional study indicated that antigen specific T cells produce cytokines including IFN 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 inhibiting viral infection 25 TNF a is a cytokine that probably regulates immune cells and inhibits viral repli cation 26 IL 2 mediates the expansion of T cells and maintains memory T cells 27 IL 17 mediates the pro duction of antimicrobial peptide and immunoglobulin for neutralizing viral infection 28 In the current study we have shown that antigen specific CD4 and CD8 T cells 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 cells are likely responsible for the protection when host is infected with SARS CoV after the vaccination In fact it has been suggested that multi cytokine producing anti gen specific CD4 T cells are functionally superior on protection to single cytokine producing cells 29 There are several reports that multi cytokine producing T cells have shown to correlate with protection against Leish mania major infection 30 In the current study the PEI pci S complexes induced a high frequency of TNF a IL 2 CD4 T cells and IFN g IL17 CD4 Tcells and TNF a IL 2 CD8 T cells when assessed by SARS peptide recall responses Taken together our results support the hypothesis that intranasal immunization with PEI pci S complexes induces ideal cellular responses for the protection Conclusion PEI is effective in delivering DNA onto the mucosal sur face in maturation of dendritic cells and in improving the immunogenicity of the DNA vaccine Our results indicated that PEI can be used as a vector for the muco sal delivery of DNA vaccine and play an important role in B cell and T cell immunities Methods Construction of plasmid expressing SARS CoV S protein The gene encoding SARS CoV spike S protein without transmembrane domain amino acids 14 1154 was synthesized The sequence was codon optimized for mammalian cell expression and the natural signal CD80 CD83 CD86 CCR7 I A d Mock 44 0 3 4 38 9 2 3 49 9 3 4 35 4 2 5 1379 0 25 2 pci S 35 2 0 1 19 0 0 5 51 4 0 1 37 3 5 4 1379 1 0 6 pci S PEI 61 3 3 7 20 2 0 7 77 2 3 7 41 3 3 8 1791 2 40 9 A pci Mock pci S pci S PEIB Cell Number I A d Figure 3 Expression of cell surface molecules CD80 CD83 CD86 CCR7 and MHC class II on CD11c cells in cervical lymph nodes from mice immunized with PEI pci S complexes BALB c mice were immunized with PBS pci mock pci S or PEI pci S complexes and then cell surface molecules on CD11c cells from cervical lymph nodes were analyzed on day 3 after the last immunization A The expressions of major cell surface molecules CD80 CD83 CD86 CCR7 and MHC class II I A d on CD11c DC were determined by flow cytometry Data were expressed as the mean value of mean fluorescence intensity SD Significant differences compared with pci S group were expressed as P 0 05 B Expression of MHC class II molecules was represented by histogram Shim et al BMC Immunology 2010 11 65 Page 5 of 9 sequence was replaced with the leader sequence of human tissue plasminogen activator tPA The tPA S gene and pci neo Promega Madison WI were digested with Nhe I and Not I Then the plasmid expressing SARS CoV S protein was generated by ligation Particle size and morphology of the PEI pci S nanoparticles PEI pci S nanoparticles were prepared by mixing poly mer and pci S DNA in a solution form at N P PEI pci S ratio of 10 The size of PEI pci S nanoparticles was A of c y tok i ne producing ce l l s C D 4 T c e l l s IL 17 IFN IL 17 IFN CD4 T cell N D TNF IL 2 TNF IL 2 CD4 T cell N D N D IL 17 IFN IL 17 IFN CD8 T cell B of c y tok i ne producing ce l l s C D 8 T c e l l s N D N D N D N D N D TNF IL 2 TNF IL 2 CD8 T cell N D N D N D N D N D Naive pci mock pci S pci S PEI pci S PEI IL 17 IFN CD4 CD8 CD4 CD8 IL 2 TNF C pci S 0 02 0 00 0 14 0 17 0 22 0 00 0 00 0 00 0 00 0 00 0 00 0 02 0 04 0 07 0 170 350 72 0 44 0 020 05 0 570 95 0 06 0 26 Figure 4 Effector CD4 and CD8 T cell responses in BALB c mice immunized with PEI pci S complexes Multi intracellular cytokine staining 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 Data were expressed as the mean value of mean fluorescence intensity SEM Significant differences compared with pci S group were expressed as P 0 01 C The results showed representative example of flow cytometry analysis Shim et al BMC Immunology 2010 11 65 Page 6 of 9 measured by an electrophoretic light scattering spectro photometer ELS8000 Otsuka Electronice Osaka Japan Morphology of the PEI pci S nanoparticles was observed by EF TEM LIBRA 120 Carl Zeiss Germany Cell uptake For cell uptake observation pci S DNA was labeled with rh