Immunization with adenovirus LIGHT-engineered dendritic cells induces potent T cell responses and therapeutic immunity in HBV transgenic mice

LIGHT, a TNF superfamily member (TNFSF14), is a type II transmembrane protein expressed on activated T cells and immature dendritic cells (DCs). However, the expression of LIGHT on mature DCs is down-regulated. Recent studies demonstrated that LIGHT provides potent costimulatory activity for T cells, enhancing proliferation and the production of Th1 cytokines independently of the B7-CD28 pathway. Here, we evaluated the effectiveness of peptide-pulsed DC-mediated antiviral immunity in HBV transgenic mice and the immunoadjuvant effect of LIGHT. The bone marrow-derived DCs were modified in vitro with an adenovirus (Ad) vector expressing mouse LIGHT (Ad-LIGHT), the expression of costimulatory molecules was up-regulated and the secretion of cytokines IL-12 and IFN-γ increased. LIGHT-modified DCs enhanced allostimulation for T cells in mixed lymphocyte reaction (MLR). HBV peptide-pulsed DCs elicited HBV specific CD8+ T cell response and reduced the level of HBsAg and HBV DNA in sera of HBV transgenic mice. Importantly, LIGHT-modified DCs could induce stronger antiviral immunity. These results support the concept that genetic modification of DCs with a recombinant LIGHT adenovirus vector may be a useful strategy for antiviral immunotherapy.     

负载gp96多肽复合物的树突状细胞抗耐药卵巢癌细胞株作用的实验研究

目的:探讨以卵巢癌细胞(SKOV3)热休克蛋白gp96多肽复合物作为抗原,刺激树突状细胞增强其抗原呈递功能,增加对卵巢癌细胞和耐药卵巢癌细胞在体外的杀伤作用,为临床卵巢癌的辅助免疫治疗提供实验依据。方法:从卵巢癌细胞中提纯出gp96多肽复合物,并对其进行鉴定;与脐血诱导培养的树突细胞(DC)结合,制备gp96-DC复合物;将DC和负载抗原的DC分别加入脐血有核细胞和人外周血T淋巴细胞中,研究二者对卵巢癌细胞和耐药卵巢癌细胞的体外CTL效应。结果:提取后的蛋白质经WesternBlot鉴定,表明该蛋白为热休克蛋白gp96;两种DC对脐血有核细胞和人外周血T淋巴细胞均有明显的活化作用,但gp96-DC对卵巢癌细胞和耐药卵巢癌细胞有更强的杀伤活性。结论:卵巢癌细胞提取的热休克蛋白gp96刺激的树突状细胞疫苗在体外对卵巢癌细胞和耐药卵巢癌细胞具有明显的杀伤功能,作为一种辅助治疗手段为卵巢癌患者的治疗带来希望。     

热休克蛋白-抗原肽复合物对移植黑色素瘤B16细胞小鼠的抑瘤效应

制备黑色素瘤B16细胞热休克蛋白-抗原肽复合物(HACs)及其粗提物(HAC-CEs),并进行其抑瘤效应的研究.结果表明应用凝胶过滤制备的HAC-CE3、HAC-CE4和HAC-CE5及应用亲和层析纯化的HAC60、HAC75和HAC97均不同程度地降低肿瘤发生率、延迟肿瘤发生时间和减少C57BL/6J小鼠死亡率.提示,60～97kD HACs具有抑瘤效应,而且为制备肿瘤疫苗提供了重要的实验依据.     

艾滋病合并感染现况及对CD4的影响研究

目的:研究艾滋病感染者和艾滋病人(HIV/AIDS)合并感染乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、梅毒(TP)的流行现况及对艾滋病抗病毒治疗的影响。方法:选取HIV/AIDS的病例采用ELISA法,分别对标本进行乙肝表面抗原、丙肝抗体、梅毒抗体检测,艾滋病抗病毒治疗后患者的CD4+T淋巴细胞的观察。结果:719例HIV/AIDS合并感染乙肝的有80例(11.13%),丙肝46例(6.40%),梅毒114例(15.86%)。接受艾滋病抗病毒治疗的326例患者中,单纯HIV感染的有232例,其中T淋巴细胞CD4<300/mm3者110例,占47.41%;合并多重感染的HIV患者有94例,其中T淋巴细胞CD4<300/mm3的有49例,占52.13%;两组比较差别无统计学意义(P>0.05)。结论:HIV合并感染TP、HCV和HBV率较高,易对T淋巴细胞CD4产生不同程度的影响。     

Enhancing therapeutic HPV DNA vaccine potency through depletion of CD4CD25 T regulatory cells

Therapeutic human papillomavirus (HPV) vaccines targeting E6 and/or E7 antigens represent an opportunity to control HPV-associated lesions. We have previously generated several therapeutic DNA vaccines targeting HPV-16 E7 antigen and generated significant antitumor effects. Since regulatory T cells (Tregs) play an important role in suppressing immune responses against tumors by immunotherapy, such as DNA vaccines, we tested if the therapeutic effects of a DNA vaccine encoding E7 linked to heat shock protein 70 (Hsp70) can be improved by a strategy to deplete Tregs using a anti-CD25 monoclonal antibody (PC61) in vaccinated mice. We found that administration of PC61 prior to vaccination with E7/Hsp70 DNA was capable of generating higher levels of E7-specific CD8⁺ T cells compared to the control antibody, leading to significantly improved therapeutic and long-term protective antitumor effects against an E7-expressing tumor, TC-1. Thus, a strategy to deplete CD4⁺CD25⁺ Tregs in conjunction with therapeutic tumor antigen-specific DNA vaccine may represent a potentially promising approach to control tumor. The clinical implications of our study are discussed.     

Mushroom lectin enhanced immunogenicity of HBV DNA vaccine in C57BL/6 and HBsAg-transgenic mice

DNA vaccination is a promising strategy for activating immune responses against hepatitis B virus (HBV) infection. However, the accumulated data have shown that DNA vaccination alone generates weak immune responses. To enhance the immunogenicity of HBV DNA vaccine, lectin purified from pleurotus ostreatus (POL) was used as adjuvant of HBV DNA vaccine for C57BL/6 and HBV surface antigen transgenic (HBVsAg-Tg) mice. Our data demonstrate that low dose of POL (1 μg/mouse) in conjunction with HBV DNA vaccine stimulated stronger HBV-specific delayed-type hypersensitivity (DTH) responses and higher HBV-specific IgG level than that in high dose of POL groups (5 μg/mouse and 10 μg/mouse). POL activated strong Th2 and Tc1 cell responses in immunized C57BL/6 and HBVsAg-Tg mice. POL as adjuvant of HBV DNA vaccine effectively enhanced HBV surface protein antibody (HBVsAb) and decreased HBVsAg level for HBV Tg mice treatment. Furthermore, POL infiltrated more lymphocytes excluding Th1, Th2 and Tc1 cell subtypes to liver of HBVsAg-Tg mice. Together, these results suggest that POL as adjuvant enhanced immunogenicity of HBV DNA vaccination and effectively stimulated immune reaponse for HBsAg-Tg mice treatment. Our findings implicate the potential of mushroom lectin as adjuvant of HBV DNA vaccine.     

A multi-trimeric fusion of CD40L and gp100 tumor antigen activates dendritic cells and enhances survival in a B16-F10 melanoma DNA vaccine model

Vaccination with tumor-associated antigens can induce cancer-specific CD8+ T cells. A recent improvement has been the targeting of antigen to dendritic cells (DC) using antibodies that bind DC surface molecules. This study explored the use of multi-trimers of CD40L to target the gp100 melanoma tumor antigen to DC. The spontaneously-multimerizing gene Surfactant Protein D (SPD) was used to fuse gp100 tumor antigen and CD40L, creating the recombinant protein SPD-gp100-CD40L. This “third generation” DC-targeting vaccine was designed to both target antigen to DC and optimally activate dendritic cells by aggregating CD40 trimers on the DC membrane surface. SPD-gp100-CD40L expressed as a 110 kDa protein. Analytical light scattering analysis gave elution data corresponding to 4-trimer and multi-trimer SPD-gp100-CD40L oligomers. The protein was biologically active on dendritic cells and induced CD40-mediated NF-κB signaling. DNA vaccination with SPD-gp100-CD40L plasmid, together with plasmids encoding IL-12p70 and GM-CSF, significantly enhanced survival and inhibited tumor growth in a B16-F10 melanoma model. Expression of gp100 and SPD-CD40L as separate molecules did not enhance survival, highlighting the requirement to encode gp100 within SPD-CD40L for optimal vaccine activity. These data support a model where DNA vaccination with SPD-gp100-CD40L targets gp100 to DC in situ, induces activation of these DC, and generates a protective anti-tumor response when given in combination with IL-12p70 and GM-CSF plasmids.