ABSTRACT
Objective To observe the splenocytes immune response elicited by different concentrations of recombinant Toxo?plasma gondii profilin(rTgPRF)through the nasal route,and determine the optimal dose. Methods Fifty female BALB/c mice were randomly divided into 5 groups. The immunized groups were intranasally administered with 10,20,30μg or 40μg of rTgPRF that was separately dissolved in 20μl of phosphate?buffered saline(PBS)on days 0,14,and 21 respectively,while the control mice were given PBS solution instead. Two weeks after the last immunization,all mice were killed. Under asceptic conditions,the spleens from the immunized mice were dissected,and then the splenocyte proliferative responses in vitro were tested by CCK?8 kit. The levels of IFN?γ,IL?2,IL?4 and IL?10 of splenocyte culture supernatant were detected by ELISA. Re?sults Compared to the control group,the splenocytes from the 30μg and 40μg groups exhibited a significantly higher prolifer?ative response to rTgPRF(P<0.05),and SI from the 30μg rTgPRF group was higher than that from the 40μg group(P<0.05). The levels of IFN?γin all the immunized groups(P<0.05)and IL?2 in the 20,30μg and 40μg groups were significant?ly stronger than those in the control(P<0.05),and the 30μg group presented the highest concentrations of IFN?γ(P<0.01) and IL?2(P<0.01). There were no statistical differencesa mong the groups in the levels of IL?4 and IL?10. Conclusions The intranasal immunization with rTgPRF can induce the splenocyteproliferation and Th1?type mediated immunity. The best immu?nized dose is confirmed as 30μg.
ABSTRACT
Respiratory syncytial virus (RSV) and influenza virus are the most significant pathogens causing respiratory tract diseases. Composite vaccines are useful in reducing the number of vaccination and confer protection against multiple infectious agents. In this study, we generated fusion of RSV G protein core fragment (amino acid residues 131 to 230) and influenza HA1 globular head domain (amino acid residues 62 to 284) as a dual vaccine candidate. This fusion protein, Gcf-HA1, was bacterially expressed, purified by metal resin affinity chromatography, and refolded in PBS. BALB/c mice were intranasally immunized with Gcf-HA1 in combination with a mucosal adjuvant, cholera toxin (CT). Both serum IgG and mucosal IgA responses specific to Gcf and HA1 were significantly increased in Gcf-HA1/CT-vaccinated mice. To determine the protective efficacy of Gcf-HA1/CT vaccine, immunized mice were challenged with RSV (A2 strain) or influenza virus (A/PR/8/34). Neither detectable viral replication nor pathology was observed in the lungs of the immune mice. These results demonstrate that immunity induced by intranasal Gcf-HA1/CT immunization confers complete protection against both RSV and homologous influenza virus infection, suggesting our Gcf-HA1 vaccine candidate could be further developed as a dual subunit vaccine against RSV and influenza virus.
Subject(s)
Animals , Mice , Cholera Toxin , Chorionic Gonadotropin, beta Subunit, Human , Chromatography, Affinity , GTP-Binding Proteins , Head , Hemagglutinins , Immunization , Immunoglobulin A , Immunoglobulin G , Influenza, Human , Lung , Orthomyxoviridae , Peptide Fragments , Respiratory Syncytial Viruses , Respiratory Tract Diseases , Vaccination , VaccinesABSTRACT
Objective To determine the immune responses induced by recombinant Salmonella ty-phimurium expressing the secreting antigen ESAT-6 of Mycobacterium tuberculosis. Methods ESAT-6 cod-ing gene was cloned and identified by PCR and sequencing. Prokaryotic expression plasmid pYA33-esat car-rying the ESAT-6 coding sequence was constructed firstly and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria was named as X4550(33-esat). C57BL/6 mice were immunized intranasally (I. N) with 108 CFU recombinant bacteria at day 0 and 18. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patch (PP) were collected from mice after second immu-nization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected by ELISPOT assay u-sing ESAT-6 peptide as stimulus. Furthermore, CTL effects were in vivo evaluated by CFSE assay. Results The results showed that cellular immune responses specific for ESAT-6 could be detected by ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 were biased toward Th1 type, the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells. In splenocytes and MLN cells, the anti-gen specific immune responses acted as Thl and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. CFSE assay indicated that recombinant bacteria could induce the high level of CTL effects specific for ESAT-6 peptide. Conclusion These results suggested that recombinant Sal-monella typhimurium X4550(33-esat) not only can induce cellular immune responses, but also can elicit specific CTL responses after I. N immunization. It also provided the useful information for the control of infec-tious disease of tuberculosis.
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Objective To evaluate the protective effectiveness of intranasal immunizations with recombinated-pneumococcal autolysin(Re-LytA), which protects mice against local and systemic Streptococ- cus pneumoniae(Sp) infection. Methods Testing group (group A): CpG as an adjuvant, the mice were intranasally immunized with purified Re-LytA, obtained by affinity chromatograph. The negative control group(group B) were intranasally immunized with sterile saline. And the positive control group (group C) were received 23-valent polysaccharide commercial vaccine through intramuscular injection. All the samples were collected 2 weeks post the last immunization. The levels of antibody was determined by ELISA. Then the mice were challenged intraperitoneally and intranasally with Sp, respectively. The infection and coloniza- tion was followed by monitoring colony-forming units of Sp in the blood, homogenized lung, and nasopharyn- geal lavage fluid 4 days post intranasal immunization. The mice were observed daily to note the livability of each group. Results The level of the LytA antibody (IgG, IgA, slgA) in group A were higher than that in group B and C (P < 0.05). Neither the LytA nor polysaccharide antibody could be detected in group B. Polysaccharide antibody could be detected in group C. After challenged intraperitoneally there was no signifi- cant difference in survival rates between group A and group C (P > 0.05), which was significant higher than that in group B (P <0.05). After challenged intranasally, compared with the group A, the geometric mean colony-forming units washed from the nasopharyngeal lavage fluid of the group B and group C were signifi- cantly higher (P <0.05). Conclusion lntranasal immunizations with Re-LytA can protect mice against lo- cal and systemic pneumococcal infection, and the protective immunity may be related to sIgA.
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Objective:To observe the effect on different mucosal sites and system immune sites after intranasal immunization with bivalent Shigella vaccines Methods:BALB/c mice were divided into three groups at random , 10 mice per group Mice were intranasally immunized respectively with FSM 2117or FS 5416 (4?10 7CFU) three doses with an interval of two weeks The NALT, NP, spleen, PP, MLN, lymphocytes were isolated on the seventh day after the last immunization to assay the change of the cell phenotype with FACS The nasal ?lung?intestine?genital tract lavage fluid and serum were taken to assay the specific IgA or IgG against F2a or Sonni LPS with ELISA Results:The specific IgA and IgG in different mucosal sites and serum increased significantly after intranasal immunization with two Shigella vaccines compared with the control (P
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Objective To study the mucosal and systemic immune response after intranasal immunization with mucosal complex vaccine for Toxoplasma gondii,and to observe the protective effect on mice. Methods The mucosal complex vaccine was made of soluble tachyzoite antigen (STAg) and cholera toxin (CT),which were mixed and dissolved in PBS (1 ml PBS containing 1 mg STAg and 50 ?g CT). Fifty-two BALB/c mice were randomly divided into two groups: immunized group and control. Mice were intranasally immunized with 20 ?l mucosal complex vaccine (20 ?g STAg and 1?g CT) per mouse twice at an interval of two weeks,while the control mice were given PBS solution instead. Six mice of each group were killed by dislocation of cervical vertebra on day 14 after the last immunization. The specific IgG antibodies in serum and IgA in feces were detected by ELISA. Lymphocytes in spleen,Peyer's patches (PP) and intestinal intraepithelial lymphocyte(IEL) were isolated and counted. Percentage of CD4+ and CD8+ T cells was determined by immunocytochemistry. Other mice were challenged intragastrically each with 4?104 tachyzoites of RH strain Toxoplasma gondii on day 14 after the last immunization. Their health condition was observed and the number of tachyzoites in liver and brain was determined microscopically on the 30 th day after challenge. Results IgG antibodies in serum and IgA antibodies in feces of immunized mice were higher than the control (P
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Objective: To observe the distribution and expression of recombinant Mycobacterium smegmatis carrying the plasmid encoding human granulysin(GLS) and murine IL-12 by intranasal immunization in mice.Methods: BALB/C mice were intranasally immunized with recombinant M.smegmatis carrying the plasmid encoding green fluorescent protein(GFP).The distribution of GFP and recombinant Mycobacterium smegmatis in the lungs and spleens was detected 14 days after the first immunization.BALB/C mice were intranasally immunized three times with recombinant M.smegmatis carrying the plasmid encoding GLS and IL-12.28 days after the first immunization,the expression of GLS in tissue,levels of IL-12 in serum and SIgA in BALF(bronchoalveolar lavage fluid)were detected with immunohistochemistry and ELISA respectively.Results: The distribution of GFP and recombinant Mycobacterium smegmatis in lung and spleen was detected.The expression of GLS,the increased of IL-12 in serum and the specific SIgA in BALF were found.Conclusion: The distribution of recombinant Mycobacterium smegmatis carrying the plasmid encoding of GFP in lung and spleen is detected.The specific anti-bacterium SIgA is generated.GLS and IL-12 are expressed in BALB/C mice after intranasal immunization with recombinant Mycobacterium smegmatis carrying the plasmid encoding GLS and IL-12.The results lay the foundation for new vaccine study.