Role of aluminum-containing adjuvants in antigen internalization by dendritic cells in vitro

An important step in the induction of an immune response to vaccines is the internalization of antigens by antigen presenting cells, such as dendritic cells (DCs). Many current vaccines are formulated with antigens adsorbed to an aluminum-containing adjuvant. Following injection of the vaccine the antigens may either elute or stay adsorbed to the adjuvant surface. Antigens, which elute from the adjuvant surface, are internalized by dendritic cells through macropinocytosis while those that remain adsorbed are internalized with the adjuvant particle by phagocytosis. The relative efficiency of these two routes of internalization was studied. Alpha casein (AC) labeled with a green fluorescent dye was selected as the model antigen. In order to model vaccine antigens that elute from aluminum-containing adjuvants following administration, dendritic cells were incubated with a solution of fluorochrome-labeled alpha casein. To model vaccine antigens that do not elute from aluminum-containing adjuvants following administration, dendritic cells were exposed to fluorochrome-labeled alpha casein adsorbed to aluminum hydroxide adjuvant (AH). Alpha casein has eight phosphate groups and adsorbs to aluminum hydroxide adjuvant through ligand exchange. Alpha casein does not elute from aluminum hydroxide adjuvant upon exposure to cell culture media. The uptake of antigen by dendritic cells was determined at 0.5, 1, 2 and 3h by confocal microscopy and flow cytometry. Dendritic cells internalized both alpha casein in solution and alpha casein adsorbed to aluminum hydroxide adjuvant. However, the mean fluorescence intensity of dendritic cells incubated with adsorbed alpha casein was four times greater than dendritic cells incubated with alpha casein in solution. In addition, the internalization of alpha casein was enhanced when the mean aggregate diameter of the adjuvant in the cell culture media was reduced from 17 microm to 3 microm. It was concluded that antigen internalization by dendritic cells was enhanced when the antigen remained adsorbed to the aluminum-containing adjuvant following administration and the aggregate size of the adjuvant was smaller than dendritic cells which are approximately 10 microm in diameter.     

Enhanced antigen delivery via cell death induced by the vaccine adjuvants

The objective of this study was to examine the effect of cell death induced by the emulsion adjuvants on the in vitro delivery of antigens into the antigen-presenting cells (APCs). J774A.1 murine macrophage-like cells, serving as the APCs, were pulsed with various vaccine adjuvants, and incubated with fluorescein isothiocyanate-conjugated bovine serum albumin (BSA-FITC), with or without adjuvants-pretreated EL4 murine thymoma cells, followed by analysis by flow cytometry and fluorescence microscopy. To assess the potential oxidative burst in the macrophages after adjuvant treatment, cells were probed with 2',7'-dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE), and analyzed by flow cytometry. Treatment of macrophages with the emulsion adjuvants, followed by co-incubation with the adjuvant-induced dead EL4 cells, resulted in a substantial uptake of soluble antigens into the APCs and generation of a considerable amount of reactive oxygen species (ROS), including hydrogen peroxide and superoxide. Pre-treatment of J774A.1 cells with several endocytosis inhibitors, including amiloride, brefeldin A or cytochalasin B, on the other hand, reduced internalization of soluble antigens. It was concluded that co-treatment of macrophages with the emulsion adjuvants and the adjuvant-induced dead cells facilitated delivery of soluble antigens, via both phagocytosis and macropinocytosis, into the antigen-presenting cells.     

DC细胞在辐射损伤抗原递呈及T细胞活化中的作用

目的 利用体外细胞共培养技术模拟体内肺组织微环境,探索树突状细胞（DC）在辐射损伤细胞的抗原提呈作用。方法 60Co γ射线照射的小鼠肺上皮细胞（MLE-12）与骨髓来源DC和/或脾T淋巴细胞培养48 h,流式细胞术检测DC细胞共刺激分子CD80/86和抗原肽识别复合物MHCⅠ/Ⅱ表达水平,T细胞活化标志CD69/28/152表达水平以及CD4+和CD8+亚群细胞数。结果 60Co γ射线照射的MLE-12细胞凋亡率呈剂量依赖性增高,明显刺激DC细胞CD80/86和MHC II表达,但对T细胞无直接活化作用;6 Gy照射的MLE-12细胞与DC细胞和T淋巴细胞共培养48 h,T细胞CD69和CD28表达增加,CD4+和CD8+亚群细胞数均明显高于对照组,同时DC细胞出现CD86和MHCI特异性高表达。结论 辐射损伤细胞可刺激DC细胞抗原提呈功能,并对T细胞进行活化。     

DNA疫苗抗原提呈机制研究进展

DNA疫苗是基因治疗研究中衍生并发展起来的一个新的研究领域。虽然该疫苗的免疫效应在动物实验及部分临床工作中得到验证,但其免疫效果仍不够理想,免疫效价低于现有疫苗。深入研究DNA疫苗免疫后抗原提呈机制成为近年研究热点内容,此文就近年来这方面的研究进展综述如下。     

Bovine immune response to leptospira antigen in different novel adjuvants and vaccine delivery platforms

Leptospirosis is a global zoonosis causing significant economic losses for cattle production. Current cattle vaccines against leptospirosis need improvement to provide efficacy against multiple serovars, reduce shedding in urine, and to induce earlier and more robust immune responses. In this study, Leptospira borgpetersenii serovar Hardjo strain 203 antigen was combined with novel adjuvants (a biodegradable polyanhydride compressed rod implant (VPEAR), poly(diaminosulfide) microparticles, a water-oil-water emulsion adjuvant, and aluminum hydroxide) to develop novel vaccines. Cattle were immunized twice, at a 4 week interval, with inoculums containing adjuvants alone or leptospira antigens and immune responses were compared to responses of cattle receiving a commercial monovalent leptospirosis vaccine (Spirovac). All animals were inoculated with a single dose of Spirovac at 20 weeks to assess antigen recall responses. Serum antibody responses were increased (P > 0.05) at 8 and 20 weeks after vaccination in cattle receiving inoculums containing leptospira antigens combined with water-oil-emulsion, poly(diaminosulfide) microparticles (PNSN-MP), or aluminum hydroxide and in cattle vaccinated with Spirovac. Humoral responses were predominantly IgG1 isotypes. Antigen-specific proliferative responses were detected after initial vaccination in cattle vaccinated with Spirovac, PNSN-MP and water-oil-water treatments. Most proliferative responses occurring within CD4+ and gamma delta T cell populations expressing CD45RO and CD25 markers, a response consistent with an effector memory phenotype. Antigen-specific immune responses were not detected in cattle vaccinated with VPEAR after initial inoculation, but were detected in the antigen recall responses. PBMCs from cattle vaccinated with Spirovac, oil-water-oil, or PNSN-MP treatments had increased (P < 0.05) IL-17A release after in vitro stimulation with leptospirosis antigens, whereas all groups produced IFN-γ and IL-17A after in vitro stimulation during the antigen recall response. Our data demonstrates that combining leptospirosis antigens with these adjuvants enhances immunogenicity in cattle.Interpretative Summary: Vaccination of livestock is a key mechanism for minimizing transmission of leptospirosis, a zoonotic disease. Leptospirosis vaccines for cattle need to be improved to provide greater levels of protection from kidney colonization, better immune responses, and protection against multiple serovars. This could be accomplished using new vaccine adjuvants. In this study, several novel adjuvants were evaluated for their ability to induce effective immune responses in cattle to leptospira antigens as compared to currently available vaccines. Data suggested that vaccines containing biodegradable polymer microparticles and oil-emulsion adjuvants induced similar or greater immune responses as compared to a commercial vaccine. Our data suggest these new vaccine formulations warrant further investigation as new vaccine formulations for cattle and other livestock.     

A detailed characterisation of the distribution and presentation of DNA vaccine encoded antigen

The association between plasmid DNA distribution, the amount of Ag produced, Ag persistence and the identity and localisation of cells presenting DNA-encoded Ag all have important consequences for both quantitative and qualitative aspects of T cell responses induced by DNA vaccines. Using a variety of approaches to detect and quantify the uptake of injected DNA, and the production and presentation of DNA-encoded antigen, we report that injected DNA vaccines rapidly enter the peripheral blood from the injection site and also reach muscle-draining lymph nodes directly as free DNA. 24 h after plasmid injection, MHCII⁺CD11b⁺B220⁻CD11c^low/− cells in the draining and distal LNs and spleen contain pDNA. Interestingly, we also observed pDNA⁺MHCII^low/−CD11b⁺ within the bone marrow. Concomitantly, we detected Ag-containing/expressing cells at both the injection site and in draining lymph nodes. Three days after plasmid injection we detected rare pMHC⁺CD11c⁺ cells within secondary lymphoid tissue and simultaneously observed Ag-specific CD4⁺ T cell accumulation and blastogenesis in these tissues. Our results show that the events that determine the induction of DNA vaccine immune responses occur within days of DNA injection and that the response becomes systemic very rapidly, possibly with involvement from resident BM cells.     

In vivo antigen loading and activation of dendritic cells via a liposomal peptide vaccine mediates protective antiviral and anti-tumour immunity

Initiation of antiviral and anti-tumour T cell responses is probably achieved mainly by dendritic cells (DC) transporting antigen from the periphery into organised lymphoid tissues. To develop T cell vaccines it is, therefore, important to understand the accessibility of the antigen to DC in vivo and whether DC are activated by vaccination. Here we have evaluated the immunogenicity of a liposomal vaccine formulation with antigenic peptides derived from the glycoprotein of the lymphocytic choriomeningitis virus. Liposome-encapsulated peptides were highly immunogenic when administered intradermally and elicited protective antiviral immunity. After intradermal injection, liposomes formed antigen depots which facilitated long-lasting in vivo antigen loading of dendritic cells almost exclusively in the local draining lymph nodes. The immunogenicity of the liposomal peptide vaccine was further enhanced by incorporation of immunostimulatory oligonucleotides leading to activation of DC. This optimised liposomal peptide vaccine elicited also anti-tumour immunity and induced CTL responses comparable to adoptively transferred, peptide-presenting DC. Thus, our data show that liposomal formulations of peptide vaccines are highly effective at direct in vivo antigen loading and activation of DC leading to protective antiviral and anti-tumour immune responses.     

树突状细胞肿瘤疫苗的研究进展

树突状细胞（DendriticCellsDCs）是人体内最重要的抗原呈递细胞，其特殊的迁移功能以及活化静息T细胞的功能，使其在抗肿瘤免疫方面处于极其重要的地位。目前多种形式以DC为基础的肿瘤疫苗已在动物模型中取得了较好的抗肿瘤效果，且部分疫苗已应用于临床试验。现就DC疫苗近几年的研究进展和存在的问题及展望作一综述。     

泛素化及自噬参与蛋白降解及抗原提呈的研究进展

细胞内所有的蛋白质都在不断的被降解,并被新合成的蛋白质所取代。真核细胞内主要有两条蛋白降解途径,分别为泛素一蛋白酶体系统途径和白噬途径,两条途径都在维持细胞内稳态方面起着重要的作用。泛素-蛋白酶体系统是由泛素介导的一种高度复杂的蛋白降解机制,它通过一种特定的方式降解并再循环利用细胞内的蛋白,而自噬是一种自我降解过程,它通过溶酶体介导的方式降解蛋白。被降解的蛋白可以被提呈至MHCⅡ类分子或MHCⅠ类分子,从而激发有效的T细胞反应。此文就这两个途径参与蛋白降解及抗原提呈的相关研究进展进行综述。     

Surfactants in vaccine adjuvants: description and perspectives

Amphiphilic molecules, called surfactants, are made of a lipophilic part linked to a hydrophilic part. These substances have physicochemical properties of self-aggregation, solubilisation or emulsification. Their ability to be attracted at the same time by polar and non-polar compounds forces them to be at the interface. Surfactants most frequently used in vaccines can be of natural or synthetic origin, non-ionic, cationic or amphoteric with a formula weight between 600 and 4000 g/mol. They can be used as adjuvants, solubilisers or stabilisers of emulsions. Physicochemical properties are defined through solubility parameters or solution behaviours and hydrophilic lipophilic balance (HLB). Application properties are assessed through placebo formulations simulating the final use. Synthetic products are preferred to products of natural origin for quality reasons. Development of a new generation of surfactant with precise chemical definition and consistency may generate new candidates for potential vaccine adjuvants.     

Natural products and the search for novel vaccine adjuvants

Vaccines that protect against intracellular infections such as malaria, Leishmania and Chlamydia require strong cellular responses based on CD4⁺ T cells and CD8⁺ T cells in addition to antibodies. Such cell-mediated responses can be potentiated with adjuvants. However, very few adjuvants have been licensed for use in humans; thus there is an urgent need for the discovery of new non-toxic adjuvants in order to produce more efficacious vaccines. Until recently, the mechanisms of how adjuvants worked remained largely unknown, but, it is becoming clearer that many function via host germline-encoded pattern recognition receptors (PRRs) expressed by most immune and non-immune cells. Most PRRs sense infection and transmit a series of signals that ultimately lead to the development of immunity. PRR mediated signalling can be harnessed to search for new vaccine adjuvants. Dendritic cells (DCs) express many PRRs and are remarkably effective at directing T cell immunity. Natural products (NPs) have been the basis of many drugs and are a rich source of immune activators and/or regulators of the immune response. Here we review PRRs in the context of NPs and propose the use of DCs as biological probes to help identify novel immune type molecules and adjuvants within collections of NPs.     

Overview of vaccine adjuvants: present and future

The history of infectious diseases together with the evidence of emerging and reemerging infections underscores the need for new vaccines and the obstacles to producing them. Vaccine adjuvants are usually thought of as agents that increase the intensity of immune responses and influence the balance between antibody and cell-mediated immunity at the cost of toxicity. This review uses copolymer adjuvants as examples to encourage a more sophisticated approach to the study of adjuvants as agents that can influence many parameters of immune responses including the specificity, titer, duration, memory, class, isotype, and avidity of antibody as well as the type of cell-mediated immunity and the incidence of genetic non-responders. Much research will be needed for design of effective vaccines against emerging and reemerging infections and for improvement of existing vaccines.     

The immunogenicity-enhancing effect of emulsion vaccine adjuvants is independent of the dispersion type and antigen release rate--a revisit of the role of the hydrophile-lipophile balance (HLB) value

Effective antigen delivery is one of the most important issues in vaccine development. It has been suggested that adjuvant action results from a depot effect by prolonging the duration of the interaction between antigen and cells, and thus is related to the antigen-releasing properties of emulsion adjuvants. The objective of this study was to investigate the effect of the dispersion properties of emulsion-type vaccine adjuvants on the immune response with the aim of optimizing vaccine adjuvant formulation. Emulsion-type adjuvants with various dispersion properties of either the oil-in-water or water-in-oil type were prepared using emulsifiers with various hydrophilic-hydrophobic balance (HLB) values. The physicochemical properties of the emulsions, including the conductivity and viscosity, and antigen release rates were then determined. Cell death induced by the vaccine adjuvants was examined in EL4 cells by Annexin V/propidium iodide (PI) staining and flow cytometric analysis. Mice were immunized with or without the adjuvants and the immunogenicity-enhancing effect of the adjuvants determined by measuring antibody production using an enzyme linked immunosorbent assay. The conductivity, viscosity, and antigen release rates varied widely among emulsions containing emulsifiers with different HLB values. However, the magnitude of the antigen-specific antibody response was similar in most emulsions adjuvants containing Spans or Tweens. L121-adjuvant, the control adjuvant inducing the strongest apoptosis in vitro, was shown to stimulate the highest antibody response in vivo. The results obtained in this study indicate that the immunogenicity-enhancing effect of emulsion adjuvants is independent of the dispersion type and the antigen release rate of the vaccine delivery system.     

树突状细胞疫苗在宫颈癌治疗中的研究进展

树突状细胞是至今发现的功能最强的专职抗原提呈细胞 ,人们能通过各种刺激提高其抗原提呈能力和诱导T细胞应答能力 ,本文简单介绍了树突状细胞疫苗在宫颈癌的免疫治疗方面的新进展。     

Activation of dendritic cells and induction of CD4(+) T cell differentiation by aluminum-containing adjuvants

Aluminum-containing adjuvants are widely used in licensed human and veterinary vaccines. However, the mechanism by which these adjuvants enhance the immune response and predominantly stimulate a T(H)2 humoral immune response is not well understood. In this study, the effects of aluminum hydroxide and aluminum phosphate adjuvants on antigen presentation, expression of costimulatory molecules and cytokines by mouse dendritic cells (DCs) and the ability of DCs to induce T helper cell differentiation were investigated. Dendritic cells pulsed with ovalbumin (OVA) adsorbed to aluminum-containing adjuvants activated antigen-specific T cells more effectively than DCs pulsed with OVA alone. Aluminum hydroxide adjuvant had a significantly stronger effect than aluminum phosphate adjuvant. Both aluminum-containing adjuvants significantly increased the expression of CD86 on DCs but only aluminum hydroxide adjuvant also induced moderate expression of CD80. Aluminum-containing adjuvants stimulated the release of IL-1beta and IL-18 from DCs via caspase-1 activation. DCs incubated with LPS and OVA induced T(H)1 differentiation of na?ve CD4(+) T cells. In contrast, DCs incubated with aluminum/OVA activated CD4(+) T cells to secrete IL-4 and IL-5 as well as IFN-gamma. Addition of neutralizing anti-IL-1beta antibodies decreased IL-5 production and addition of anti-IL-18 antibodies decreased both IL-4 and IL-5 production. Inhibition of IL-1beta and IL-18 secretion by DCs via inhibition of caspase-1 also led to a marked decrease of IL-4 and IL-5 by CD4(+) T cells. These results indicate that aluminum-containing adjuvants activate DCs and influence their ability to direct T(H)1 and T(H)2 responses through the secretion of IL-1beta and IL-18.     

Effect of cigarette smoke on mucosal vaccine response with activation of plasmacytoid dendritic cells: The outcomes of in vivo and in vitro experiments

Mucosal vaccines offer several advantages over transdermal vaccines, including the ability to acquire systemic and mucosal immunities. Smoking is a huge public health threat and major risk factor for various diseases that exacerbate or prolong respiratory symptoms and conditions. However, its impact on the efficacy of mucosal vaccines remains partially explored. Thus, this study investigates the effects of smoking on mucosal vaccine reactivity by assessing the induction of Th1 immunity, a vital response in infection defense. Cigarette smoke condensate was prepared as a substitute for mainstream smoke. We intranasally administered diphtheria toxoid as an antigen and natural CpG oligonucleotide G9.1, which enhances the Th1-type antibody (Ab) response in a plasmacytoid dendritic cells (pDCs) dependent manner, as an adjuvant to mice to assess the effect of cigarette smoke condensate on Ab responses. The mechanism of its effect was evaluated using human peripheral blood mononuclear cells and their pDC-rich fraction cultured with or without G9.1. In mice, cigarette smoke condensate tended to decrease diphtheria toxoid-specific Ab response, with a higher reduction in Th1-type IgG2 Ab response than in Th2-type IgG1 Ab response. In human peripheral blood mononuclear cells, cigarette smoke condensate significantly reduced the induction of IFN-α production by G9.1. Moreover, G9.1-induced increases in the CD83 expression in pDCs and the CD80 expression in DCs were suppressed via treatment with cigarette smoke condensate. Among the mechanisms suggested were decreased expression of toll-like receptor 9 mRNA, decreased expression of mRNA for IFN regulatory factor 7, and increased CpG methylation of its promoter region. The analysis of Tbet and GATA3 expressions revealed that cigarette smoke condensate exhibits Th1-directed immunostimulatory activity at a steady state but becomes more Th2-directed under G9.1 stimulation. In conclusion, smoking could reduce mucosal vaccine responses by decreasing pDC activation and, consequently, Th1-dominant immunity.     

A comparison of non-toxin vaccine adjuvants for their ability to enhance the immunogenicity of nasally-administered anthrax recombinant protective antigen

Development of nasal immunization for human use is hindered by the lack of acceptable adjuvants. Although CT is an effective adjuvant, its toxicity will likely prevent its use in nasal vaccines. This study compared non-toxin adjuvants to CT for their ability to induce protective antibody responses with nasal immunization. C3H/HeN and C57BL/6 mice were immunized with rPA formulated with the following adjuvants: CT, IL-1α, LPS, CpG, Pam3CSK4, 3M-019, resiquimod/R848 or c48/80. Serum and nasal wash cytokine concentrations were monitored 6 h post-vaccination as biomarkers for acute activation of the innate immune system. Not all of the adjuvants induced significant changes in innate serum or nasal wash cytokines, but when changes were observed, the cytokine signatures were unique for each adjuvant. All adjuvants except Pam3CSK4 induced significantly increased anti-rPA serum IgG titers in both strains of mice, while only IL-1α, c48/80 and CpG enhanced mucosal anti-rPA IgA. Pam3CSK4 was the only adjuvant unable to enhance the induction of serum LeTx-neutralizing antibodies in C3H/HeN mice while c48/80 was the only adjuvant to induce increased serum LeTx-neutralizing antibodies in C57BL/6 mice. Only CT enhanced total serum IgE in C3H/HeN mice while IL-1α enhanced total serum IgE in C57BL/6 mice. The adjuvant influenced antigen-specific serum IgG subclass and T cell cytokine profiles, but these responses did not correlate with the induction of LeTx-neutralizing activity. Our results demonstrate the induction of diverse innate and adaptive immune responses by non-toxin nasal vaccine adjuvants that lead to protective humoral immunity comparable to CT and that these responses may be influenced by the host strain.     

MUC1体外转染脐带血DC疫苗抗乳腺肿瘤的免疫效应研究

目的:研究人粘蛋白核心肽-连续重复序列(MUC1-VNTR)基因转染的脐血DC疫苗抗乳腺肿瘤的免疫效应。方法:提取人脐带血淋巴细胞,培养脐血树突状细胞(DCs),采用Lipofectamine2000阳离子脂质体法将MUC1-VNTR转染入脐血DC,用Western印迹法检测MUC1-VNTR基因的表达;与自体T细胞共培养,用MTT法检测转染MUC1基因的脐血DC所致敏的细胞毒性淋巴细胞对乳腺癌MCF-7的杀伤活性;ELISA法检测其刺激T细胞分泌IFN-γ的能力。结果:Western印迹法检测到1条MUC1-VNTR基因表达的特殊条带,转染MUC1-VNTR基因的脐血DC对MUC1表达阳性的乳腺癌细胞系MCF-7的杀伤活性显著高于单纯脐血DC组(P<0.05),而且其刺激同种T细胞分泌高水平的IFN-γ,显著高于未转染的脐血DC(P<0.05)。结论:MUC1-VNTR基因转染的人脐血树突状细胞可诱导特异性CTL,产生有效的针对MUC1阳性乳腺癌细胞MCF-7的杀伤效应,并诱导特异性抗乳腺癌免疫应答。     

Replacing the measles ten-dose vaccine presentation with the single-dose presentation in Thailand

Introduced to minimize open vial wastage, single-dose vaccine vials require more storage space and therefore may affect vaccine supply chains (i.e., the series of steps and processes involved in distributing vaccines from manufacturers to patients). We developed a computational model of Thailand's Trang province vaccine supply chain to analyze the effects of switching from a ten-dose measles vaccine presentation to each of the following: a single-dose measles-mumps-rubella vaccine (which Thailand is currently considering) or a single-dose measles vaccine. While the Trang province vaccine supply chain would generally have enough storage and transport capacity to accommodate the switches, the added volume could push some locations’ storage and transport space utilization close to their limits. Single-dose vaccines would allow for more precise ordering and decrease open vial waste, but decrease reserves for unanticipated demand. Moreover, the added disposal and administration costs could far outweigh the costs saved from preventing open vial wastage.