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Molecular Therapy Volume 9 Supplement 1 May 2004 Copyright The American Society of Gene Therapy S210 G49G4EG46 G45G43G54G49G4FG55G53G20G44G49G53G45G41G53G45G53G20G41G4EG44G20G56G41G43G43G49G4EG45G53 557 Co Administration of an Adenovirus Encoding the B Cell Stimulating Factor BAFF with Heat Inactivated Pseudomonas aeruginosa Leads to Increased Anti pseudomonal Humoral Immunity Christine Tertilt 1 Stefan Worgall 1 Michael C Rivara 1 Ronald G Crystal 1 1 Weill Medical College of Cornell University New York NY Pseudomonas aeruginosa is an important pathogen in conditions such as severe burns and cystic fibrosis causing significant morbidity and mortality Despite many efforts no clinically effective vaccine against P aeruginosa is available to date especially in CF patients In order to optimize an antipseudomonal vaccine and enhance mucosal immunity we focused our study on the enhancement of the B cell response Stimulating B cells through B cell activating molecules during immunization against P aeruginosa may increase the efficiency of a vaccine B cell activating factor BAFF a 34kD single chain TNF family member is produced by activated antigen presenting cells and stimulates B cells Similar to other members of the TNF family BAFF is secreted as a trimer by cleavage of the protein from the membrane Following binding to its receptors on B cells BAFF prolongs the life span of activated B cells by preventing apoptosis and induces T cell independent immunoglobulin class switch in vitro and is crucial for B cell development and for the generation of humoral immune responses in vivo Based on these findings we hypothesized that the strong B cell stimulatory properties of BAFF can be exploited for the induction of immunity against P aeruginosa and that the transient overexpression of BAFF during immunization will favor the development of a rapid strong humoral immune response To evaluate this concept AdmBAFF an E1 E3 adenovirus expressing full length murine BAFF under control of a CMV promotor was constructed After infection of A549 cells with AdmBAFF expression of full length BAFF could be seen in the cell lysate by Western analysis and a cleaved form of BAFF was detected in the supernatant demonstrating that the overexpressed BAFF was properly shed from the membrane To assess the potency of AdmBAFF to induce humoral immunity against P aeruginosa in vivo C57Bl 6 mice were injected subcutaneously with 7 5x10 10 particle units of AdmBAFF together with 10 5 cfu of heat inactivated P aeruginosa strain PAO1 Mice injected with AdNull or PBS plus PAO1 served as controls Serum binding antibodies against PAO1 were evaluated by ELISA 1 2 3 and 4 wk following immunization Mice injected with PAO1 AdmBAFF showed higher levels of PAO1 specific IgM titers 1 and 2 wk after immunization 1175 506 and 304 118 respectively compared to mice immunized with PAO1 AdNull 280 105 and 73 48 or PAO1 PBS 305 91 and 61 17 p 0 02 for both comparisons at both timepoints Similarly higher PAO1 specific total IgG levels were observed 2 3 and 4 wk following immunization with PAO1 AdmBAFF 373 206 158 35 and 164 99 respectively compared to mice immunized with PAO1 AdNull 79 70 47 45 and 38 32 or PAO1 PBS 63 41 30 9 and 40 15 p0 3 all pairwise comparisons At a dose of 10 11 pu anti SARS CoV neutralizing titers were also measurable 700 160 intravenous 130 20 intravenous 450 210 subcutaneous In contrast naive mice and mice immunized with AdNull an Ad with no transgene had no detectable neutralizing antibody titers at any time point Cellular immunity stimulated by immunization was evaluated in BALB c mice injected intravenously with AdnS or AdNull at a dose of 10 11 pu After 2 wk splenic CD8 T cells were isolated and exposed to syngeneic target cells stably expressing S Accumulation of intracellular IFN was observed in 8 of CD8 cells as opposed to 2 5 of cells exposed to syngeneic cells not expressing S Similar results were observed in C57Bl 6 mice with 11 5 of CD8 cells accumulating intracellular IFN in response to syngeneic cells expressing S as opposed to 3 3 of CD8 cells exposed to syngeneic cell lines not expressing S The AdNull vaccinated controls did not stimulate antigen specific IFN accumulation in the splenic CD8 cells To determine the location of the major cellular epitopes in the spike glycoprotein Ad vectors and target syngeneic cell lines expressing the N terminal domain S1 or the C terminal domain S2 of spike were constructed The specific accumulation of IFN in immunized BALB c mice at 2 wk post administration was more dependent on the S2 domain than the S1 domain 19 specific intracellular IFN accumulation using the S2 domain vs 6 3 with S1 We conclude that immunization with an Ad vaccine vector expressing the SARS CoV S elicits high titers of SARS CoV neutralizing antibodies and that the S2 domain of spike contains the immunodominant CD8 T cell epitopes