Role of IL-2 secreted by PADRE-specific CD4+ T cells in enhancing E7-specific CD8+ T-cell immune responses

CD4(+) T helper cells are known to play an integral role in the generation of CD8(+) T-cell immune responses. We have previously shown that co-administration of DNA vaccines containing E6 or E7 protein of human papillomavirus 16 (HPV-16) combined with DNA encoding invariant (Ii) chain in which class II-associated Ii peptide (CLIP) region is replaced with the CD4(+) T helper epitope, PADRE (Pan-DR-epitope) (Ii-PADRE DNA) enhanced HPV antigen-specific CD8(+) T-cell immune responses in vaccinated mice. In the current study, we investigated the enhancement of HPV E7-specific CD8(+) T-cell immune responses by PADRE-specific CD4(+) T cells. We showed that intradermal administration of Ii-PADRE DNA at the same location as E7-expressing DNA is necessary to generate strong E7-specific CD8(+) T-cell immune responses. We also showed that PADRE-specific CD4(+) T cells generated by Ii-PADRE DNA vaccination expressed Th1 cytokine profile. Furthermore, our in vitro study demonstrated that PADRE-specific CD4(+) T cells stimulated with PADRE-loaded dendritic cells secrete IL-2 that leads to the proliferation of E7-specific CD8(+) T cells. Thus, our data suggest that activated PADRE-specific CD4(+) T helper cells may be required at the vicinity of E7-specific CD8(+) T cells where they secrete IL-2, which enhances the E7-specific CD8(+) T-cell immune responses generated by DNA vaccination.     

TA-CIN, a vaccine incorporating a recombinant HPV fusion protein (HPV16 L2E6E7) for the potential treatment of HPV16-associated genital diseases

Commercially available prophylactic HPV vaccines for cervical cancer prevention have limited use in women with previous viral exposure. Therefore, a therapeutic HPV vaccine would benefit patients with HPV-associated genital diseases. Being developed by Cancer Research Technology Ltd, under license from Xenova Group plc, TA-CIN (Tissue Antigen - Cervical Intraepithelial Neoplasia) is a fusion protein vaccine comprising the HPV16 viral proteins L2, E6 and E7 for the treatment of HPV16-associated genital diseases. In mouse models, TA-CIN induced dose-dependent HPV16-specific CD4 and CD8 T-cell responses, which were enhanced when boosted with the vaccinia-based vector vaccine TA-HPV (Therapeutic Antigen - HPV). A phase I clinical trial of TA-CIN in healthy volunteers reported no serious adverse events and HPV16-specific cellular immune responses. Phase II trials in patients with anogenital and vulval intraepithelial neoplasia investigated heterologous prime/boost strategies with TA-CIN/TA-HPV and TA-HPV/TA-CIN, but neither of the regimens offered advantages over single-agent TA-HPV. A recent phase II trial investigating imiquimod/TA-CIN in patients with vulval intraepithelial neoplasia demonstrated significant infiltration of CD4 and CD8 T-cells in lesion responders and complete lesion regression in 63% of patients. More comprehensive case-controlled trials are needed to define responders to immunotherapy with TA-CIN and verify its prophylactic and therapeutic properties.     

血小板对树突状细胞和肿瘤细胞抗原呈递活性的抑制作用

本研究探讨完整的血小板对骨髓来源的树突状细胞（BMDC）和肿瘤细胞的抗原呈递功能的影响。抗原呈递检测体系采用BMDC诱导的混合淋巴细胞反应或肿瘤细胞系EG7（表达卵白蛋白OVA）诱导的OVA特异性T细胞的活化,共培养体系中加入从小鼠外周血分离的血小板至不同浓度,培养一定时间后采用同位素掺入法检测淋巴细胞增殖,采用ELISA检测各种细胞因子的水平,采用流式细胞术检测细胞表面分子情况。结果发现,在反应体系中加入血小板时,淋巴细胞增殖和IFN,,／产生均明显受抑制。血小板还可以阻断细菌脂多糖或含CpG基序的寡核苷酸引起的BMDC表面B7之上调、细胞因子分泌及对同种异基因淋巴细胞的刺激反应。血小板也可抑制BMDC特异性T细胞呈递可溶性或细胞性抗原的能力,表现为淋巴细胞的增殖和细胞因子分泌受到抑制。血小板的这些抑制作用均依赖于加至培养体系中血小扳的浓度。流式细胞术分析表明,血小板结合于BMDC仅轻微增加后者的死亡比例。结论：在某些条件下,血小板可以通过抑制抗原呈递功能而对免疫反应起到负向调控作用,血小板对免疫系统的调节可能比原来预期的更加复杂。     

Enhanced antigen presentation and immunostimulation of dendritic cells using acid-degradable cationic nanoparticles.

Acid-degradable cationic nanoparticles encapsulating a model antigen (i.e., ovalbumin) were prepared by inverse microemulsion polymerization with acid-cleavable acetal cross-linkers. Incubation of these degradable nanoparticles with dendritic cells derived from bone marrow (BMDCs) resulted in the enhanced presentation of ovalbumin-derived peptides, as quantified by B3Z cells, a CD8+ T cell hybridoma. The cationic nature of the particles contributed to the increased surface endocytosis (or phagocytosis) observed with BMDCs, which is the first barrier to overcome for successful antigen delivery. The acid sensitivity of the particles served to direct more ovalbumin antigens to be processed into the appropriately trimmed peptide fragments and presented via the major histocompatibility complex (MHC) class I pathway following hydrolysis within the acidic lysosomes. It was also shown that adjuvant molecules such as unmethylated CpG oligonucleotides (CpG ODN) and anti-interleukin-10 oligonucleotides (AS10 ODN) could be co-delivered with the protein antigen for maximized cellular immune response.     

Immunization with analog peptide in combination with CpG and montanide expands tumor antigen-specific CD8+ T cells in melanoma patients

Analog peptides represent a promising tool to further optimize peptide-based vaccines in promoting the expansion of tumor antigen-specific cytotoxic T lymphocytes. Here, we report the results of a pilot trial designed to study the immunogenicity of the analog peptide NY-ESO-1 157-165V in combination with CpG 7909/PF3512676 and Montanide ISA 720 in patients with stage III/IV NY-ESO-1-expressing melanoma. Eight patients were immunized either with Montanide and CpG (arm 1, 3 patients); Montanide and peptide NY-ESO-1 157-165V (arm 2, 2 patients); or with Montanide, CpG, and peptide NY-ESO-1 157-165V (arm 3, 3 patients). Only the 3 patients immunized with Montanide, CpG, and peptide NY-ESO-1 157-165V in arm 3 developed a rapid increase of effector-memory NY-ESO-1-specific CD8+ T cells, detectable ex vivo. The majority of these cells exhibited an intermediate/late-stage differentiated phenotype (CD28-). Our study further demonstrated that our vaccine approach stimulated spontaneous tumor-reactive NY-ESO-1-specific CD8+ T cells in 2 patients with advanced disease, but failed to prime tumor-reactive NY-ESO-1-specific T cells in 1 patient with no spontaneously tumor-induced CD8+ T-cell responses to NY-ESO-1. Collectively, our data support the capability of the analog peptide NY-ESO-1 157-165V in combination with CpG and Montanide to promote the expansion of NY-ESO-1-specific CD8+ T cells in patients with advanced cancer. They also suggest that the presence of tumor-induced NY-ESO-1-specific T cells of well-defined clonotypes is critical for the expansion of tumor-reactive NY-ESO-1-specific CD8+ T cells after peptide-based vaccine strategies.     

Enhancing DNA vaccine potency by combining a strategy to prolong dendritic cell life and intracellular targeting strategies with a strategy to boost CD4+ T cell

Intradermal administration of DNA vaccines, using a gene gun, represents an effective means of delivering DNA directly into professional antigen-presenting cells (APCs) in the skin and thus allows the application of strategies to modify the properties of APCs to enhance DNA vaccine potency. In the current study, we hypothesized that the potency of human papillomavirus (HPV) type 16 E7 DNA vaccines employing intracellular targeting strategies combined with a strategy to prolong the life of dendritic cells (DCs) could be further enhanced by the addition of a DNA vaccine capable of generating high numbers of pan-HLA-DR reactive epitope (PADRE)-specific CD4(+) T cells. We observed that the addition of PADRE DNA to E7 DNA vaccines employing intracellular targeting strategies with a strategy to prolong the life of DCs led to significant enhancement of E7-specific CD8(+) effector and memory T cells as well as significantly improved therapeutic effects against established E7-expressing tumors in tumor-challenged mice. Our data suggest that the potency of a DNA vaccine combining an intracellular targeting strategy as well as a strategy to prolong the life of DCs can be further enhanced by addition of DNA that is capable of generating high numbers of PADRE-specific CD4(+) T cells.     

Monitoring the trafficking of adoptively transferred antigen- specific CD8-positive T cells in vivo, using noninvasive luminescence imaging

Understanding of the trafficking of antigen-specific CD8(+) T cells in vivo will provide insight about how our immune system controls infectious diseases and cancers. In the current study we used a luciferase-expressing human papillomavirus type 16 (HPV-16) E7-specific CD8(+) T cell for adoptive transfer to control E7-expressing TC-1 tumor cells. We used noninvasive luminescence imaging to monitor the trafficking of E7-specific CD8(+) T cells over time. We also boosted the luciferase-expressing E7-specific CD8(+) T cells in vivo, using E7-expressing vaccinia. We found that injected E7-specific T cells preferentially migrated to the E7-expressing tumor site but not to the E7-negative control tumor site, and increased in number at the tumor site over time. In addition, vaccination with E7-expressing vaccinia led to a significant increase in the number of E7-specific CD8(+) T cells at the tumor site, resulting in a significant antitumor effect compared with vaccination with wild-type vaccinia. Thus, our data suggest that the antitumor effects generated by adoptive transfer of E7-specific CD8(+) T cells can be significantly enhanced by vaccination with E7-expressing vaccinia and that our system represents a plausible approach to investigate the trafficking and biology of antigen-specific T cells in vivo.     

Human papillomavirus 16 E6-specific CD45RA+ CCR7+ high avidity CD8+ T cells fail to control tumor growth despite interferon-gamma production in patients with cervical cancer

We defined the nature of the cellular immune response in 5 women with human papillomavirus (HPV) 16+cervical carcinoma at a single time point when surgery was performed for treatment. To monitor the differences in T-cell recognition, 2 approaches of tetramer-guided technology were employed: (i) the in situ localization of major histocompatibility complex class I peptide complexes in the tumor lesions and (ii) the ex vivo sorting of HLA-A*0201-restricted and HPV16 E6-reactive T cells. CD8 T cells from the periphery (peripheral blood lymphocytes), the tumor (tumor-infiltrating lymphocytes), and T cells harvested from draining lymph nodes (T-LN) were analyzed. HPV16 E6 tetramer-sorted lymphocytes from the different anatomic sites recognized an HLA-A*0201-restricted E6 peptide irrespective of the type of antigen-presenting cells used for stimulation as determined by interferon-gamma production: autologous tumor cells, HLA-A*0201 surrogate antigen-presenting cells pulsed with the nominal peptide, and an HLA-A*0201-matched human dendritic cell line transgenic for HPV16 E6. Further analysis showed that the HPV16 E6-reactive CD8 T cells were of high avidity defined by blocking with an anti-CD8-alpha specific monoclonal antibody. We found that HPV16 E6-reactive T cells reside preferentially within the CD45RA+ CCR7+ T-cell subpopulation of tumor-infiltrating lymphocyte, peripheral blood lymphocyte, and T-LN in cervical cancer patients, suggesting that successful immune surveillance of HPV16+ tumor cells in cervical cancer patients is impaired. The CD45RA+/CCR7+ phenotype of HPV antigen-reactive T cells may serve as an indicator of dysfunctional T cells, despite effective interferon-gamma production in response to HPV antigens.     

Rapid and strong human CD8+ T cell responses to vaccination with peptide, IFA, and CpG oligodeoxynucleotide 7909

The induction of potent CD8+ T cell responses by vaccines to fight microbes or tumors remains a major challenge, as many candidates for human vaccines have proved to be poorly immunogenic. Deoxycytidyl-deoxyguanosin oligodeoxynucleotides (CpG ODNs) trigger Toll-like receptor 9, resulting in dendritic cell maturation that can enhance immunogenicity of peptide-based vaccines in mice. We tested whether a synthetic ODN, CpG 7909, could improve human tumor antigen-specific CD8+ T cell responses. Eight HLA-A2+ melanoma patients received 4 monthly vaccinations of low-dose CpG 7909 mixed with melanoma antigen A (Melan-A; identical to MART-1) analog peptide and incomplete Freund's adjuvant. All patients exhibited rapid and strong antigen-specific T cell responses: the frequency of Melan-A-specific T cells reached over 3% of circulating CD8+ T cells. This was one order of magnitude higher than the frequency seen in 8 control patients treated similarly but without CpG and 1-3 orders of magnitude higher than that seen in previous studies with synthetic vaccines. The enhanced T cell populations consisted primarily of effector memory cells, which in part secreted IFN- and expressed granzyme B and perforin ex vivo. In vitro, T cell clones recognized and killed melanoma cells in an antigen-specific manner. Thus, CpG 7909 is an efficient vaccine adjuvant that promotes strong antigen-specific CD8+ T cell responses in humans.     

Vaccination with a DNA vaccine encoding herpes simplex virus type 1 VP22 linked to antigen generates long-term antigen-specific CD8-positive memory T cells and protective immunity

Intradermal vaccination with DNA encoding herpes simplex virus type 1 (HSV-1) VP22 linked to antigen leads to spread of antigen within the epithelium and results in enhanced antigen-specific CD8+ T cell immune responses in vaccinated mice. In this study, we characterized the number of antigen-expressing dendritic cells (DCs) in the draining lymph nodes of vaccinated mice and determined whether the linkage of VP22 to antigen would influence the ability of antigen-expressing DCs to activate antigen-specific CD8+ T cells in vivo. Vaccination with DNA encoding HSV-1 VP22 linked to human papillomavirus type 16 E7 antigen generated more antigen-expressing DCs in the draining lymph nodes of vaccinated mice than E7 alone. In addition, the linkage of VP22 to E7 improved the MHC class I presentation of E7 in transfected DCs and led to enhanced activation of E7-specific CD8+ T cells. We also observed that vaccination with DNA encoding VP22 linked to E7 generated more E7-specific CD8+ memory T cells, and enhanced long-term protective antitumor immunity against an E7-expressing tumor in vaccinated mice compared with vaccination with DNA encoding E7 alone. Thus, administration of DNA encoding VP22 linked to antigen represents a plausible approach for the development of protective DNA vaccines.     

Intramuscular administration of E7-transfected dendritic cells generates the most potent E7-specific anti-tumor immunity

Dendritic cells (DCs) are highly efficient antigen-presenting cells capable of priming both cytotoxic and helper T cells in vivo. Recent studies have demonstrated the potential use of DCs that are modified to carry tumor-specific antigens in cancer vaccines. However, the optimal administration route of DC-based vaccines to generate the greatest anti-tumor effect remains to be determined. This study is aimed at comparing the levels of immune responses and anti-tumor effect generated through different administration routes of DC-based vaccination. We chose the E7 gene product of human papillomavirus (HPV) as the model antigen and generated a stable DC line (designated as DC-E7) that constitutively expresses the E7 gene. Among the three different routes of DC-E7 vaccine administration in a murine model, we found that intramuscular administration generated the greatest anti-tumor immunity compared with subcutaneous and intravenous routes of administration. Furthermore, intramuscular administration of DC-E7 elicited the highest levels of E7-specific antibody and greatest numbers of E7-specific CD4+ T helper and CD8+ T cell precursors. Our results indicate that the potency of DC-based vaccines depends on the specific route of administration and that intramuscular administration of E7-transfected DCs generates the most potent E7-specific anti-tumor immunity.     

CpG ODN1826刺激人外周血树突状细胞对胃癌细胞杀伤效应的研究

目的探讨CpGODN1826在体外增强树突状细胞（dendriticcells,DC）抗胃癌作用的影响。方法分离正常人外周血DC,用GM-CSF和IL-4培养至第5天分组：GM-CSF＋IL-4组、GM-CSF＋IL-4＋TNF-α组、GM-CSF＋IL-4＋LPS组和GM-CSF＋IL-4＋CpGODN组。自外周血单个核细胞（PBMC）诱导分离DC,流式细胞仪检测其表面标志,MTT法测定其刺激同种异体淋巴细胞增殖的影响,检测其活性及对胃癌细胞的杀伤效应,ELISA检测各组分泌IL-12情况。结果体外培养第10天,CpGODN组出现大量典型的DC形态;流式细胞仪检测CpGODN组显著高表达CD40、CD1a、CD80、CD86、MHC-Ⅱ和分泌IL-12;在体外能强烈刺激同种异体混合淋巴细胞的增殖,显著增强DC杀伤MKN-45细胞的活性。结论 CpGODN可显著地诱导人外周血DC分化成熟,增强DC对胃癌细胞的杀伤作用。     

Control of cervicovaginal HPV-16 E7-expressing tumors by the combination of therapeutic HPV vaccination and vascular disrupting agents

Abstract Antigen-specific immunotherapy and vascular disrupting agents, such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA), have emerged as attractive approaches for the treatment of cancers. In the current study, we tested the combination of DMXAA treatment with therapeutic human papillomavirus type 16 (HPV-16) E7 peptide-based vaccination for their ability to generate E7-specific CD8+ T-cell immune responses, as well as their ability to control E7-expressing tumors in a subcutaneous and a cervicovaginal tumor model. We found that the combination of DMXAA treatment with E7 long peptide (amino acids 43-62) vaccination mixed with polyriboinosinic:polyribocytidylic generated significantly stronger E7-specific CD8+ T-cell immune responses and antitumor effects compared with treatment with DMXAA alone or HPV peptide vaccination alone in the subcutaneous model. Additionally, we found that the DMXAA-mediated enhancement of E7-specific CD8+ T-cell immune responses generated by the therapeutic HPV peptide-based vaccine was dependent on the timing of administration of DMXAA. Treatment with DMXAA in tumor-bearing mice was also shown to lead to increased dendritic cell maturation and increased production of inflammatory cytokines in the tumor. Furthermore, we observed that the combination of DMXAA with HPV-16 E7 peptide vaccination generated a significant enhancement in the antitumor effects in the cervicovaginal TC-1 tumor growth model, which closely resembles the tumor microenvironment of cervical cancer. Taken together, our data demonstrated that administration of the vascular disrupting agent, DMXAA, enhances therapeutic HPV vaccine-induced cytotoxic T-lymphocyte responses and antitumor effects against E7-expressing tumors in two different locations. Our study has significant implications for future clinical translation.     

Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against leishmania major infection

CpG oligodeoxynucleotides (ODN) have potent effects on innate and adaptive cellular immune responses. In this report, the ability of CpG ODN to confer long-term immunity and protection when used as a vaccine adjuvant with a clinical grade of leishmanial antigen, autoclaved Leishmania major (ALM), or a recombinant leishmanial protein was studied. In two different mouse models of L. major infection, vaccination with ALM plus CpG ODN was able to control infection and markedly reduce lesion development in susceptible BALB/c and resistant C57BL/6 (B6) mice, respectively, up to 12 wk after immunization. Moreover, B6 mice immunized with ALM plus CpG ODNs were still protected against infectious challenge even 6 mo after vaccination. In terms of immune correlates of protection, ALM plus CpG ODN-vaccinated mice displayed L. major-specific T helper cell 1 and CD8+ responses. In addition, complete protection was markedly abrogated in mice depleted of CD8+ T cells at the time of vaccination. Similarly, mice vaccinated with a recombinant leishmanial protein plus CpG ODN also had long-term protection that was dependent on CD8+ T cells in vivo. Together, these data demonstrate that CpG ODN, when used as a vaccine adjuvant with either a recombinant protein or heat-killed leishmanial antigen, can induce long-term protection against an intracellular infection in a CD8-dependent manner.     

Innovative DNA vaccine for human papillomavirus (HPV)-associated head and neck cancer

Human papillomavirus (HPV), particularly type 16, has been associated with a subset of head and neck cancers. The viral-encoded oncogenic proteins E6 and E7 represent ideal targets for immunotherapy against HPV-associated head and neck cancers. DNA vaccines have emerged as attractive approaches for immunotherapy due to its simplicity, safety and ease of preparation. Intradermal administration of DNA vaccine by means of gene gun represents an efficient method to deliver DNA directly into dendritic cells for priming antigen-specific T cells. We have previously shown that a DNA vaccine encoding an invariant chain (Ii), in which the class II-associated Ii peptide (CLIP) region has been replaced by a Pan-DR-epitope (PADRE) sequence to form Ii-PADRE, is capable of generating PADRE-specific CD4+ T cells in vaccinated mice. In the current study, we hypothesize that a DNA vaccine encoding Ii-PADRE linked to E6 (Ii-PADRE-E6) will further enhance E6-specific CD8+ T cell immune responses through PADRE-specific CD4+ T-helper cells. We found that mice vaccinated with Ii-PADRE-E6 DNA generated comparable levels of PADRE-specific CD4+ T-cell immune responses, as well as significantly stronger E6-specific CD8+ T-cell immune responses and antitumor effects against the lethal challenge of E6-expressing tumor compared with mice vaccinated with Ii-E6 DNA. Taken together, our data indicate that vaccination with Ii-E6 DNA with PADRE replacing the CLIP region is capable of enhancing the E6-specific CD8+ T-cell immune response generated by the Ii-E6 DNA. Thus, Ii-PADRE-E6 represents a novel DNA vaccine for the treatment of HPV-associated head and neck cancer and other HPV-associated malignancies.     

Comparison of HPV DNA vaccines employing intracellular targeting strategies

Intradermal vaccination via gene gun efficiently delivers DNA vaccines into dendritic cells (DCs) of the skin, resulting in the activation and priming of antigen-specific T cells in vivo. In the context of DNA vaccines, we previously used the gene gun approach to test several intracellular targeting strategies that are able to route a model antigen, such as the human papillomavirus type-16 (HPV-16) E7, to desired subcellular compartments in order to enhance antigen processing and presentation to T cells. These strategies include the use of the sorting signal of lysosome-associated membrane protein (LAMP-1), Mycobacterium tuberculosis heat-shock protein 70 (HSP70), calreticulin (CRT) and the translocation domain (dII) of Pseudomonas aeruginosa exotoxin A (ETA). Vaccination with DNA vaccines encoding E7 antigen linked to any of these molecules all led to a significant enhancement of E7-specific CD8(+) T-cell immune responses and strong antitumor effects against an E7-expressing tumor, TC-1. However, we were interested in identifying the most potent DNA vaccine for our future clinical trials. Thus, we performed a series of experiments to directly compare the potency of the various DNA vaccines. Among the DNA vaccines we tested, we found that vaccination with pcDNA3-CRT/E7 generated the highest number of E7-specific CD8(+) T cells and potent long-term protection and treatment effects against E7-expressing tumors in mice. Interestingly, we observed that pcDNA3-CRT/E7 is also capable of protecting against an E7-expressing tumor with downregulated MHC class I expression, a common feature associated with most HPV-associated cervical cancers. Our data suggest that the DNA vaccine linking CRT to E7 (CRT/E7) may be a suitable candidate for human trials for the control of HPV infections and HPV-associated lesions.     

A therapy modality using recombinant IL-12 adenovirus plus E7 protein in a human papillomavirus 16 E6/E7-associated cervical cancer animal model

Interleukin (IL)-12 has been reported to induce cellular immune responses for protection against tumor formation. Here we investigate the utility of adenoviral delivery of IL-12 as an adjuvant for a human papillomavirus E7 subunit vaccine in a mouse tumor challenge model. Direct intratumoral injection of AdIL-12 resulted in a significant suppression of tumor growth compared to the control group. Injection of E7 protein into either a tumor site or the distance site along with AdIL-12 further enhanced antitumor effects significantly higher than either AdIL-12 or E7 injection alone. This combined injection resulted in complete regression of 9-mm-sized tumor in 40% of animals as well as lasting antitumor immunity against tumor recurrence. We also evaluated immune responses induced by these injections. AdIL-12 plus E7 enhanced E7-specific antibody responses significantly higher than AdIL-12 or E7 injection. In particular, the production level of interferon (IFN)-gamma from E7-specific CD4(+) T cells was similar between AdIL-12 group and AdIL-12 + E7 group. However, IFN-gamma production from E7-specific CD8(+) T cells was the most significant when injected with AdIL-12 + E7. This was consistent with intracellular IFN-gamma staining levels of CD8(+) T cells, suggesting that AdIL-12 + E7 injection enhances antitumor immunity in the human papillomavirus (HPV) 16 tumor model through increased expansion of the cytotoxic T-lymphocyte (CTL) subset. This enhanced protection appeared to be mediated by CD8(+) T cells, as determined by in vivo T-cell subset deletion. Thus, these studies demonstrate that E7 vaccines can induce CTL responses responsible for antitumor effects in the presence of IL-12 delivered via adenovirus vectors. This likely provides one additional approach for immune therapy against cervical cancers.     

CpG oligonucleotide therapy cures subcutaneous and orthotopic tumors and evokes protective immunity in murine bladder cancer

Bacillus Calmette-Guerin (BCG) instillation is standard immunotherapy for superficial bladder carcinoma. However, many patients become refractory to BCG, giving impetus to the development of alternative therapies. CpG oligodeoxynucleotide (ODN) therapy has been shown to promote T(H)1-oriented antitumor responses in various tumor models. To investigate its therapeutic effect in bladder cancer, we used different CpG ODNs to treat C57BL/6 mice bearing the subcutaneous murine bladder tumor MB49. CpG type B ODN 1668 was superior at inhibiting tumor growth, leading to complete regression of large tumors. More importantly, CpG ODN 1668 also regressed orthotopically growing MB49 tumors for the first time. Rechallenge of CpG ODN-cured mice with MB49 showed that a majority of the mice were protected long term, demonstrating that CpG ODN therapy evokes a memory response. Adenoviral vectors (Ad) encoding CD40L, tumor necrosis factor-related activation-induced cytokine, lymphotactin, interleukin (IL) 2, and IL-15 were also investigated. AdCD40L and AdIL-15 transduction could abolish MB49 tumorigenicity, and these vectors were combined with CpG ODN 1668 to investigate any enhanced effects. No such effects were seen. All groups of mice treated with CpG ODNs, alone or in combination with adenoviral vector, exhibited increased serum concentrations of IL-12, indicative of a T(H)1 response. Our results show that CpG ODN therapy cures established subcutaneous and orthotopic bladder cancer via a T(H)1-mediated response and provides long-lasting protective immunity.     

CD40配体-受体载体介导的CpG ODN靶向脐血B淋巴细胞的特异传递及其免疫效应

背景:CpG寡核苷酸是一种高效低毒的免疫佐剂,在许多疾病的基因治疗中具有广阔的应用价值.但存在种属和细胞特异性,细胞摄取率低,易被酶降解,成为临床应用的障碍.目的:拟验证CpG寡核苷酸通过CD40配体-受体载体系统对脐血B淋巴细胞特异靶向传递及其免疫效应的增强作用.设计、时间及地点:观察对比实验,于2004-0412007-10在广州暨南大学附属第一医院血液科、儿科完成.材料:取健康新生儿的肝素抗凝新鲜脐血,事先征得父母知情同意并获医院伦理委员会审核批准.方法:制备CD40配体-碳二亚胺-多聚左旋赖氨酸-CpG寡核苷酸交联复合物,与脐血单个核细胞共培养.分别于24,48,72,96 h应用流式细胞仪检测单个核细胞对FAM标记CpG寡核苷酸的摄取率及平均摄入强度;培养96 h后用直接免疫荧光标记法标记CD19,CD20,CD22单抗,以流式细胞仪检测阳性表达率;细胞悬液加入96孔板,3组分别加入CD40配体-碳二亚胺-多聚左旋赖氨酸-CpG寡核苷酸、单纯CpG寡核苷酸及ddH2O,培养92 h,以四甲基偶氮唑盐比色法测细胞增殖能力;以酶联免疫吸附法测以上3组细胞培养上清IgG水平.主要观察指标:单个核细胞对CpG寡核苷酸的摄取率及平均摄入强度,淋巴细胞亚群的表达,细胞增殖能力,培养上清IgG水平.结果:与单纯CpG寡核苷酸未交联组相比,单个核细胞对交联复合物摄取率更高(98%),达摄取峰值时间吏早.共培养后CD19+,CD22+,CD20+细胞表达升高,细胞增殖量及上清IgG水平均高于对照组(P<0.05).结论:CD40配体-受体载体系统有助于CpG寡核苷酸特异高效的B细胞靶向投递,并增强其免疫效应.     

Potent antigen-specific immune responses stimulated by codelivery of CpG ODN and antigens in degradable microparticles

CpG ODN stimulates a TH1 response through its receptor Toll-like receptor 9 (TLR9). TLR9 is a receptor that is found intracellularly. Microparticles are efficiently internalized by dendritic cells (DCs) and macrophages and would thus be an ideal delivery vehicle for CpG ODN to reach its target site thereby enhancing the TH1 response to an antigen also encapsulated in the microparticle. Here, we show that careful control over fabrication parameters can produce biodegradable microparticles with predictable size distributions, surface morphology, and shape. Entrapment efficiencies of the model antigen OVA ranged from 19% to 23% with an average loading of 10 microg/mg of microparticles. For CpG ODN, these values were 33% to 35%, which corresponded to an average loading of 8.5 microg/mg of microparticles. The microparticles release CpG ODN and OVA in a burst followed by sustained release profile. At the highest concentration of microparticles incubated with a pure DC cell line, 92% of DCs had internalized microparticles by 16 hours, confirming that DCs efficiently take up the microparticles. Microparticles are capable of inducing DC maturation as determined by up-regulation of CD80 and CD86 markers. Although the presence of CpG ODN in the microparticles did not impact on the phenotype of the DCs, it was necessary for DCs to induce activation of antigen-specific T cells as indicated by interferon-gamma production. Microparticles entrapping both antigen and CpG ODN induced significantly higher amounts of anti-OVA antibody production than other preparations such as the soluble OVA and CpG ODN (P<0.01) and stimulated stronger IgG2a production than delivery of microparticles entrapping antigen alone. We conclude that co-encapsulating immunostimulatory CpG ODN and antigen in degradable microparticles is an effective approach to enhancing development of a TH1 immune response.