GM-CSF和IL-4增强Aβ表位DNA疫苗的免疫原性研究

目的:探究和评价粒细胞-巨噬细胞集落刺激因子(GM-CSF)及白细胞介素4(IL-4)作为β淀粉样蛋白(Aβ)表位DNA疫苗的分子佐剂,增强DNA疫苗体液和细胞免疫反应的水平。方法:阿尔茨海默病DNA表位疫苗p VAX1-6Aβ15-T分别与重组DNA分子佐剂p VAX1-S-IL-4和p VAX1-S-GM-CSF联合免疫BALB/c小鼠,并检测其免疫原性。结果:分子佐剂IL-4组相比单独的DNA表位疫苗p VAX1-6Aβ15-T组抗体水平具有一定程度的提高;GM-CSF能明显提高DNA疫苗Aβ特异的抗体水平和泛DR辅助T细胞表位(PADRE)特异的细胞免疫反应,4次免疫后其抗体滴度提高了4倍。结论:GM-CSF佐剂能够有效地用于今后阿尔茨海默病DNA表位疫苗的研究中。     

IL-12对巨细胞病毒DNA疫苗免疫增强作用的研究

白介素12(interleukin 12,IL-12)主要和细胞免疫应答有关,是免疫过程中重要的调节因子。本研究探讨IL-12对编码巨细胞病毒(cytomegalovirus,CMV)即刻早期基因IE1的DNA疫苗的免疫增强作用。将CMVIE1质粒DNA单独或与编码IL-12的质粒DNA共同免疫小鼠,然后用致死量病毒攻击小鼠。通过检测小鼠体内诱导的细胞免疫应答、小鼠的存活率、体重丢失率、器官中的病毒滴度等来评价IL-12对疫苗免疫的佐剂效果。结果显示,与单独疫苗免疫组相比,IE1 DNA联合IL-12 DNA免疫组能够在小鼠体内诱导更高的细胞免疫应答水平,同时能够降低器官中的病毒滴度,显著提高保护率,从而更好地抵抗病毒攻击。实验证明,IL-12能够作为巨细胞病毒IE1 DNA疫苗的佐剂,提高免疫保护效果。     

含分子内佐剂的SARS-CoV-2 RBD域重组蛋白制备及免疫效果评价北大核心CSCD

制备含破伤风毒素肽(tetanus toxin,TT)、促吞噬肽(tuftsin)和新型冠状病毒刺突蛋白(spike,S蛋白)受体结合域(receptor-binding domain,RBD)的融合蛋白,探讨分子内佐剂对RBD蛋白体液免疫和细胞免疫效果的影响。将破伤风毒素肽、促吞噬肽与S蛋白RBD区域通过柔性多肽串联,密码子优化后构建重组载体,原核表达纯化制备重组S-TT-tuftsin蛋白,与铝佐剂混合后免疫BALB/c小鼠,对其体液及细胞免疫效果进行评价。重组S-TT-tuftsin蛋白以包涵体形式表达,离子交换层析纯化后采用梯度透析进行复性,复性蛋白经Dot blotting鉴定,可与新冠亚单位疫苗(安徽智飞公司)免疫后人血清发生反应。小鼠免疫实验结果表明,免疫35 d时抗体水平到达平台期,含分子内佐剂重组蛋白(铝佐剂)免疫小鼠后血清ELISA抗体效价高达1︰66240,显著高于S-RBD蛋白(铝佐剂)免疫小鼠抗体效价(P<0.05)。同时,含分子内佐剂重组蛋白刺激小鼠产生更强的淋巴细胞增殖能力,刺激指数可达4.71±0.15,相较于S-RBD蛋白的刺激指数1.83±0.09具有显著性差异(P<0.0001)。分子内佐剂破伤风毒素肽和促吞噬肽可显著增强新冠S蛋白RBD域的体液免疫和细胞免疫效果,可为新冠亚单位疫苗和其他病毒亚单位疫苗的研制提供理论基础和参考。     

改组猪IL-2和重组IL-6C基因增强猪口蹄疫疫苗的免疫应答

以离子交联法制备壳聚糖纳米颗粒,包裹重组改组的猪IL-2基因表达质粒(VRIL2S)、重组IL-6和CpG(VPIL6C)质粒(CNP-VRIL2S-VPIL6C-CNP), 按0.5 mg/头肌肉注射壳聚糖包装质粒接种21日龄健康三元杂交猪,同时肌肉注射免疫灭活口蹄疫疫苗;在免疫后7、14、28、42和56 d,采血检测实验猪的免疫球蛋白、特异抗体和免疫细胞的动态变化.结果发现:接种后56 d,改组猪IL-2和重组IL-6C质粒接种猪血液中的特异性抗口蹄疫抗体、IgG、IgA和IgM含量均较对照空白疫苗免疫猪显著升高(P<0.05),其IL-2、IL-6水平和淋巴细胞、单核细胞的数量也相应较灭活疫苗对照组明显增加(P<0.05).这些证明猪IL-2和IL-6基因与CpG有良好的免疫协同增强效应,能有效地增强疫苗的特异体液和细胞免疫应答反应,可作为安全高效的新型免疫基因分子佐剂,促进猪对口蹄疫疫苗的免疫防御抗病力,抵御口蹄疫的感染.     

HEVAg-PLGA纳米颗粒疫苗小鼠体内免疫学研究

研究重组戊型肝炎抗原(HEVAg)-乳酸/乙醇酸共聚物(PLGA)纳米颗粒抗原能否在动物体内诱导产生免疫应答。制备HEVAg-PLGA纳米颗粒抗原后,通过皮下、滴鼻、口服途径接种Balb/c小鼠,每隔4周加强免疫两次,HEVAg与铝盐佐剂(铝佐剂疫苗Al_2O_3-Ag)为对照组,一定时间内检测抗体及细胞因子的应答水平。结果HEVAg-PLGA纳米颗粒抗原在小鼠体内诱导产生有效的体液免疫、细胞免疫。滴鼻、口服途径黏膜系统中诱导产生较高滴度的IgA抗体,ELISPOT结果显示鼻腔、唾液腺中IgA ASCs数量显著增加;皮下途径诱导产生较高滴度的IgG抗体;常规铝佐剂疫苗相比于HEVAg-PLGA纳米颗粒抗原诱导较强的IgG抗体水平,未诱导产生黏膜免疫应答;HEVAg-PLGA纳米颗粒抗原诱导产生较强细胞免疫应答,皮下接种途径IFN-γ、IL-4生成细胞数量显著高于其它免疫组。与铝佐剂疫苗相比,HEVAg-PLGA纳米颗粒抗原能有效诱导产生系统免疫及黏膜免疫应答,显示HEVAg-PLGA有潜力成为备选HEV黏膜疫苗抗原,同时展示PLGA颗粒作为黏膜系统抗原递送载体及黏膜佐剂的优越性。     

重组小鼠白细胞介素-33的原核表达制备及其粘膜免疫佐剂活性

目的:白细胞介素(IL)-33对树突状细胞、巨噬细胞及T细胞等免疫细胞具有重要调控作用。利用大肠杆菌制备重组小鼠的IL-33,并初步考察其作为粘膜免疫佐剂应用的潜能与特点。方法:以IPTG诱导硫氧还蛋白(Trx)/IL-33融合蛋白在大肠杆菌DH5α中的表达,并通过QSepharose离子交换和Ni~(++)金属螯合亲和层析纯化Trx/IL-33,进一步经肠激酶切割获得成熟形式的IL-33。重组HBcAg混合纯化的IL-33后经滴鼻免疫小鼠,考察HBcAg特异的IgA及IgG_1、IgG_(2a)的应答。结果:纯化的重组IL-33具有与标准品相当的促巨噬细胞RAW264.7表达TNF-α的体外细胞生物学活性。作为佐剂可显著增强滴鼻粘膜免疫激发的不同粘膜组织中HBcAg特异的IgA应答,以及血清与支气管肺泡灌洗液中特异IgG_1的应答水平,而抑制IgG_(2a)应答。结论:利用大肠杆菌可制备活性IL-33,其具有粘膜免疫佐剂的应用潜能。     

钙调磷酸酶B亚单位(CnB)作为佐剂对新型乙型肝炎病毒疫苗免疫效果的影响北大核心CSCD

比较不同佐剂配伍的效果,探讨常规剂量(5μg)钙调磷酸酶B亚单位(Calcineurin subunit B,CnB)佐剂对含PreS1+S融合抗原的乙型肝炎病毒新型疫苗(HBSS1)免疫效果的影响。采用Al(OH)3、常规剂量(5μg)CnB及CnB+Al(OH)3等佐剂与HBV颗粒疫苗配伍初次免疫,重组腺病毒载体疫苗加强免疫的策略,在C57BL/6小鼠模型上研究不同佐剂对HBV颗粒疫苗肌肉注射后免疫应答的影响,主要包括抗体滴度、抗体亚型分类及特异性细胞免疫(γ-IFN ELISpot检测)。研究结果显示Al(OH)3佐剂存在明显免疫增强作用,而单独加入5μg CnB佐剂或CnB与Al(OH)3佐剂联合应用对Anti-PreS1抗体无明显的增强作用,但可显著降低anti-HBs抗体水平;各免疫组在重组腺病毒载体疫苗加强后,其抗体亚类包括IgG1、IgG2a和IgG2b;并可诱导高水平的细胞免疫应答反应。因而常规剂量(5μg)CnB单独或联合Al(OH)3佐剂对新型HBV疫苗无明显的免疫增强作用。     

钙调磷酸酶B亚单位(CnB)作为佐剂对新型乙型肝炎病毒疫苗免疫效果的影响

比较不同佐剂配伍的效果,探讨常规剂量(5μg)钙调磷酸酶B亚单位(Calcineurin subunit B,CnB)佐剂对含PreS1+S融合抗原的乙型肝炎病毒新型疫苗(HBSS1)免疫效果的影响。采用Al(OH)3、常规剂量(5μg)CnB及CnB+Al(OH)3等佐剂与HBV颗粒疫苗配伍初次免疫,重组腺病毒载体疫苗加强免疫的策略,在C57BL/6小鼠模型上研究不同佐剂对HBV颗粒疫苗肌肉注射后免疫应答的影响,主要包括抗体滴度、抗体亚型分类及特异性细胞免疫(γ-IFN ELISpot检测)。研究结果显示Al(OH)3佐剂存在明显免疫增强作用,而单独加入5μg CnB佐剂或CnB与Al(OH)3佐剂联合应用对Anti-PreS1抗体无明显的增强作用,但可显著降低anti-HBs抗体水平;各免疫组在重组腺病毒载体疫苗加强后,其抗体亚类包括IgG1、IgG2a和IgG2b;并可诱导高水平的细胞免疫应答反应。因而常规剂量(5μg)CnB单独或联合Al(OH)3佐剂对新型HBV疫苗无明显的免疫增强作用。     

DNA疫苗佐剂研究进展

DNA疫苗在免疫应答中能诱导机体产生持久的体液免疫和细胞免疫,然而DNA疫苗刺激机体免疫应答能力往往比常规疫苗引起的免疫反应弱。最近研究表明:使用DNA疫苗佐剂如细胞因子、CpGODN、补体C3d等有助于提高DNA疫苗的免疫效价。就DNA疫苗佐剂的研究进展做一综述。     

流感新型黏膜疫苗的研究

目的评价PorA、PorB和Class4对流感裂解疫苗的免疫增强作用,从中挑选出最有效的流感黏膜佐剂,为发展流感黏膜疫苗提供理论基础。方法流感三价裂解抗原按比例与PorA、PorB和Class4非共价结合,滴鼻免疫Balb／c小鼠3次,采取间接ELISA检测血清特异性IgG抗体及抗体亚型,检测鼻咽、肺、小肠和阴道冲洗液中IgA效价,采用血凝抑制试验检测血清中HAI效价。结果PorB重组蛋白佐剂组较无佐剂的流感裂解抗原组在提高小鼠早期免疫应答的同时诱导较强的系统免疫应答和黏膜免疫应答;PorA组也有黏膜佐剂的功能,但和无佐剂的流感裂解抗原组相比,差异无统计学意义。结论在蛋白体的三分子中,以PorB为佐剂的流感黏膜疫苗不仅提高了抗原的系统免疫应答,而且诱导了较强的小鼠呼吸道、生殖道的局部黏膜免疫应答,为流感黏膜疫苗的研制奠定了理论基础。     

Mannosylated Mucin-Type Immunoglobulin Fusion Proteins Enhance Antigen-Specific Antibody and T Lymphocyte Responses

Targeting antigens to antigen-presenting cells (APC) improve their immunogenicity and capacity to induce Th1 responses and cytotoxic T lymphocytes (CTL). We have generated a mucin-type immunoglobulin fusion protein (PSGL-1/mIgG_2b), which upon expression in the yeast Pichia pastoris became multivalently substituted with O-linked oligomannose structures and bound the macrophage mannose receptor (MMR) and dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) with high affinity in vitro. Here, its effects on the humoral and cellular anti-ovalbumin (OVA) responses in C57BL/6 mice are presented.OVA antibody class and subclass responses were determined by ELISA, the generation of anti-OVA CTLs was assessed in ⁵¹Cr release assays using in vitro-stimulated immune spleen cells from the different groups of mice as effector cells and OVA peptide-fed RMA-S cells as targets, and evaluation of the type of Th cell response was done by IFN-γ, IL-2, IL-4 and IL-5 ELISpot assays.Immunizations with the OVA − mannosylated PSGL-1/mIgG_2b conjugate, especially when combined with the AbISCO®-100 adjuvant, lead to faster, stronger and broader (with regard to IgG subclass) OVA IgG responses, a stronger OVA-specific CTL response and stronger Th1 and Th2 responses than if OVA was used alone or together with AbISCO®-100. Also non-covalent mixing of mannosylated PSGL-1/mIgG_2b, OVA and AbISCO®-100 lead to relatively stronger humoral and cellular responses. The O-glycan oligomannoses were necessary because PSGL-1/mIgG_2b with mono- and disialyl core 1 structures did not have this effect.Mannosylated mucin-type fusion proteins can be used as versatile APC-targeting molecules for vaccines and as such enhance both humoral and cellular immune responses.     

A surfactin cyclopeptide of WH1fungin used as a novel adjuvant for intramuscular and subcutaneous immunization in mice

WH1fungin, a surfactin cyclopeptide from Bacillus amyloliquefaciens WH1, is firstly reported as a novel immunoadjuvant, which can markedly enhance the immune response when given in mixture with antigens. After intramuscular or subcutaneous immunization, WH1fungin can help to induce both of durable humoral and cellular immune response, even as strong as Freund's adjuvant. Both IgG1 and IgG2a antigen-specific antibodies were elicited from the immunizations indicating a mixed Th1/Th2 response. Splenocytes from mice intramuscularly immunized with OVA plus WH1fungin responded to OVA CTL peptide stimulation resulting in an increase in CD8⁺TNF-α⁺ and CD8⁺IFN-γ⁺ T cell populations, and also an increase in CD4⁺TNF-α⁺ T cells and CD4⁺IFN-γ⁺ T cell populations was found from mice subcutaneously immunized with OVA plus WH1fungin when responded to OVA Th peptide stimulation. These results further suggest that WH1fungin helps to elicit humoral and cellular responses to OVA. The potential mechanism of WH1fungin as an immunoadjuvant was investigated. In vitro assays showed that WH1fungin could enter into RAW 264.7 cells, induce ROS accumulation, and increase the expression of cell surface markers and cytokines in cells. Further investigation suggested that WH1fungin might exert its adjuvant activity by ligating with TLR-2 in antigen present cells such as RAW 264.7. Taken together, WH1fungin is very potent as a novel adjuvant for development of vaccines in the future.     

Protection against the allergic airway inflammation depends on the modulation of spleen dendritic cell function and induction of regulatory T cells in mice

Background

Allergen-induced imbalance of specific T regulatory (Treg) cells and T helper 2 cells plays a decisive role in the development of immune response against allergens.

Objective

To evaluate effects and potential mechanisms of DNA vaccine containing ovalbumin (OVA) and Fc fusion on allergic airway inflammation.

Methods

Bronchoalveolar lavage (BAL) levels of inflammatory mediators and leukocyte infiltration, expression of CD_11c ⁺CD₈₀ ⁺ and CD_11c ⁺CD₈₆ ⁺ co-stimulatory molecules in spleen dendritic cells (DCs), circulating CD₄ ⁺ and CD₈ ⁺ T cells, Foxp3⁺ in spleen CD₄ ⁺ T cells and spleen CD₄ ⁺ T cells were measured in OVA-sensitized and challenged animals pretreated with pcDNA, OVA-pcDNA, Fc-pcDNA, and OVA-Fc-pcDNA.

Results

OVA-Sensitized and challenged mice developed airway inflammation and Th2 responses, and decreased the proliferation of peripheral CD₄ ⁺and CD₈ ⁺ T cells and the number of spleen Foxp₃ ⁺ Treg. Those changes with increased INF-γ production and reduced OVA-specific IgE production were protected by the pretreatment with OVA-Fc-pcDNA.

Conclusion

DNA vaccine encoding both Fc and OVA showed more effective than DNA vaccine encoding Fc or OVA alone, through the balance of DCs and Treg.     

T cell precursor frequency differentially affects CTL responses under different immune conditions

Generation of effective CTL responses is the goal of many vaccination protocols. However, to what extant T cell precursor frequencies will generate a CD8⁺ CTL response has not been elucidated properly. In this study, we employed a model system, in which naive CD4⁺ and CD8⁺ T cells derived from ovalbumin (OVA)-specific TCR transgenic OT II and OT I mice were used for adoptive transfer into wild-type, Ia^b−/− gene knockout and transgenic RIP-mOVA mice, and assessed OVA-pulsed DC (DC_OVA)-stimulated CD8⁺ CTL responses in these mice. We demonstrated that (i) a critical threshold exists above which T cells precursor frequency cannot enhance the CTL responses in wild-type C57BL/6 mice, (ii) increasing CD8⁺ T cell precursors is required to generate CTL responses but with functional memory defect in absence of CD4⁺ T cell help, and (iii) increasing CD4⁺ and CD8⁺ T cell precursors overcomes immune suppression to DC_OVA-stimulated CD8⁺ CTL responses in transgenic RIP-mOVA mice with OVA-specific self immune tolerance. Taken together, these findings may have important implications for optimizing immunotherapy against cancer.     

Poly(I:C)/Alum Mixed Adjuvant Priming Enhances HBV Subunit Vaccine-Induced Immunity in Mice When Combined with Recombinant Adenoviral-Based HBV Vaccine Boosting

Background

Virus-specific cellular immune responses play a critical role in virus clearance during acute or chronic HBV infection. Currently, the commercially available HBV vaccine is combined with alum adjuvant, which stimulates mainly Th2 immune responses. Therefore, development of new therapeutic HBV vaccine adjuvants and immune strategies that also promote Th1 and CTL responses is urgently needed.

Methodology/Principal findings

To improve the immunity induced by the novel HBSS1 HBV vaccine, we evaluated the ability of adjuvants, including alum, CpG and polyriboinosinic polyribocytidylic acid [poly(I:C)], to enhance the response when boosted with the recombinant adenoviral vector vaccine rAdSS1. The immune responses to different adjuvant combinations were assessed in C57BL/6 mice by enzyme-linked immunosorbent assay (ELISA), ELISpot and cytokine release assays. Among the combinations tested, a HBV protein particle vaccine with CpG/alum and poly(I:C)/alum priming combinations accelerated specific seroconversion and produced high antibody (anti-PreS1, anti-S antibody) titres with a Th1 bias. After boosting with recombinant adenoviral vector vaccine rAdSS1, both groups produced a strong multi-antigen (S and PreS1)-specific cellular immune response. HBSS1 immunisation with poly(I:C)/alum priming also generated high-level CD4⁺ and CD8⁺ T cell responses in terms of Th1 cytokines (IFN-γand IL-2).

Conclusions

The protein-vaccine HBSS1 with mixed poly(I:C)/alum adjuvant priming, followed by a rAdSS1 vaccine boost, maximises specific antibody and Th1-biased cellular immune responses. This regime might prove useful in the development of HBV therapeutic vaccines. Furthermore, this promising strategy might be applied to vaccines against other persistent infections, such as human immunodeficiency virus and tuberculosis.     

Improvement of adaptive immunity by antigen-carrying biodegradable nanoparticles

One of the most important aspects in vaccine development is to induce potent antigen-specific immune responses. In this study, we examined the immunological activities of antigen-carrying biodegradable poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) in mice. The immunization with ovalbumin (OVA)-carrying γ-PGA NPs (OVA-NPs) could induce significant expansion of antigen-specific CD8⁺ T cells. Unlike complete Freund’s adjuvant, subcutaneous (s.c.) inoculation of OVA-NPs to footpad did not generate injection site swelling. Although OVA-NPs could induce both antigen-specific cellular and humoral immune responses, the dominant induction of either cellular or humoral immunity was found to depend on their administration routes. Strong antibody production was observed by s.c. immunization, yet no antibody was identified by intranasal immunization. Thus, γ-PGA NPs are a safe and efficient antigen carrier with unique immunological properties.     

An mRNA vaccine encoding Chikungunya virus E2-E1 protein elicits robust neutralizing antibody responses and CTL immune responses

Arthropod-borne chikungunya virus (CHIKV) infection can cause a debilitating arthritic disease in human. However, there are no specific antiviral drugs and effective licensed vaccines against CHIKV available for clinical use. Here, we developed an mRNA-lipid nanoparticle (mRNA-LNP) vaccine expressing CHIKV E2-E1 antigen, and compared its immunogenicity with soluble recombinant protein sE2-E1 antigen expressed in S2 cells. For comparison, we first showed that recombinant protein antigens mixed with aluminum adjuvant elicit strong antigen-specific humoral immune response and a moderate cellular immune response in C57BL/6 mice. Moreover, sE2-E1 vaccine stimulated 12-23 folds more neutralizing antibodies than sE1 vaccine and sE2 vaccine. Significantly, when E2-E1 gene was delivered by an mRNA-LNP vaccine, not only the better magnitude of neutralizing antibody responses was induced, but also greater cellular immune responses were generated, especially for CD8⁺ T cell responses. Moreover, E2-E1-LNP induced CD8⁺ T cells can perform cytotoxic effect in vivo. Considering its better immunogenicity and convenience of preparation, we suggest that more attention should be placed to develop CHIKV E2-E1-LNP mRNA vaccine.     

Adjuvant IL-15 does not enhance the efficacy of tumor cell lysate-pulsed dendritic cell vaccines for active immunotherapy of T cell lymphoma

There has been a recent interest in using IL-15 to enhance antitumor activity in several models because of its ability to stimulate CD8⁺ T cell expansion, inhibit apoptosis and promote memory T cell survival and maintenance. Previously, we reported that C6VL tumor lysate-pulsed dendritic cell vaccines significantly enhanced the survival of tumor-bearing mice by stimulating a potent tumor-specific CD8⁺ T cell response. In this study, we determined whether IL-15 used as immunologic adjuvant would augment vaccine-primed CD8⁺ T cell immunity against C6VL and further improve the survival of tumor-bearing mice. We report that IL-15 given after C6VL lysate-pulsed dendritic cell vaccines stimulated local and systemic expansion of NK, NKT and CD8⁺ CD44^hi T cells. IL-15 did not, however, augment innate or cellular responses against the tumor. T cells from mice infused with IL-15 following vaccination did not secrete increased levels of tumor-specific TNF-α or IFN-γ or have enhanced C6VL-specific CTL activity compared to T cells from recipients of the vaccine alone. Lastly, IL-15 did not enhance the survival of tumor-bearing vaccinated mice. Thus, while activated- and memory-phenotype CD8⁺ T cells were dramatically expanded by IL-15 infusion, vaccine-primed CD8⁺ T cell specific for C6VL were not significantly expanded. This is the first account of using IL-15 as an adjuvant in a therapeutic model of active immunotherapy where there was not a preexisting pool of tumor-specific CD8⁺ T cells. Our results contrast the recent studies where IL-15 was successfully used to augment tumor-reactivity of adoptively transferred transgenic CD8⁺ T cells. This suggests that the adjuvant potential of IL-15 may be greatest in settings where it can augment the number and activity of preexisting tumor-specific CD8⁺ T cells.     

Astragalus polysaccharides enhance the humoral and cellular immune responses of hepatitis B surface antigen vaccination through inhibiting the expression of transforming growth factor β and the frequency of regulatory T cells

Astragalus polysaccharides (APS), extracted from the root of Astragalus membranaceus, a traditional Chinese medicinal herb, have extensive pharmacological and strong immunomodulatory effects. In this study, the potential adjuvant effect of APS on humoral and cellular immune responses to hepatitis B subunit vaccine was investigated. Coadministration of APS with recombinant hepatitis B surface antigen significantly increased antigen-specific antibody production, T-cell proliferation and CTL (cytotoxic T lymphocyte) activity. Production of interferon-γ (IFN-γ), interleukin-2 (IL-2) and IL-4 in CD4(+) T cells and of IFN-γ in CD8(+) T cells were dramatically increased. Furthermore, expression of the genes PFP, GraB, Fas L and Fas were up-regulated; interestingly, expression of transforming growth factor β (TGF-β) and the frequency of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg cells) were down-regulated. Expression of Toll-like receptor 4 (TLR4) was significantly increased by administration of APS. Together, these results suggest that APS is a potent adjuvant for the hepatitis B subunit vaccine and can enhance both humoral and cellular immune responses via activating the TLR4 signaling pathway and inhibit the expression of TGF-β and frequency of Treg cells.     

Cimetidine enhances immune response of HBV DNA vaccination via impairment of the regulatory function of regulatory T cells

Cimetidine (CIM), a histamine 2-receptor antagonist, is postulated to enhance immune responses owing to its inhibitory effects on suppressor T cells. In this report, we evaluated effects of cimetidine on the potency of antigen-specific immunity generated by DNA vaccine encoding hepatitis B surface antigen (HBsAg, pcD-S2). Our data demonstrate that CIM as adjuvant significantly increased HBsAg-specific cell-mediated and humoral immunities that were characterized by higher Ig2a/IgG1 ratio. In addition, CIM significantly promotes an elevated level of IL-4 and IFN-γ in antigen-specific CD4⁺ T cells and a robust antigen-specific cytotoxic response in the animals immunized with pcD-S2 plus CIM. Further, CIM induces pro-inflammatory cytokine expression such as the IL-12 and down-regulates anti-inflammatory cytokine expression such as IL-10 and TGF-β, which may lead to an impairment of CD4⁺CD25⁺ Treg cell-mediated suppression. Collectively these findings suggest that CIM enhances the immune responses of HBV DNA vaccine through the stimulation of pro-inflammatory and inhibition of anti-inflammatory cytokine expression patterns.