Vaccine Adjuvant Program

疫苗佐剂计划

• 批准号: 8946548
• 负责人: Ronald N Germain
• 金额: $ 16.52万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8946548
• 关键词: Acute; Address; Adjuvant; Adjuvant Study; Affect; Affinity; Alhydrogel; Animals; Antibodies; Antibody Formation; Antigen Presentation; Antigens; Area; B-Lymphocytes; BCL1 Oncogene; BLR1 gene; Benchmarking; Biological Assay; Biological Models; Blocking Antibodies; Bone Marrow; CD4 Positive T Lymphocytes; CD80 Antigens; Carrier Proteins; Cell Communication; Cells; Cellular Immunity; Clinic; Complement; Conjugate Vaccines; Culicidae; Data; Dendritic Cells; Detection; Development; Dose; Double-Blind Method; Drug Formulations; Enzyme-Linked Immunosorbent Assay; Epitopes; Escherichia coli; Event; Exposure to; Fostering; Freund' s Adjuvant; Goals; Helper-Inducer T-Lymphocyte; Homing; Host Defense; Human; Image; Immune response; Immunoglobulin Class Switching; Immunoglobulin G; Immunoglobulin Somatic Hypermutation; Immunoglobulin-Secreting Cells; Inflammatory; Injection of therapeutic agent; Kinetics; Laboratories; Licensing; Life; Liposomes; Longevity; Lymphoid Tissue; MHC Class II Genes; Malaria; Malaria Vaccines; Mali; Measurement; Measures; Membrane; Memory B-Lymphocyte; Methods; Microscopy; Modeling; Molecular; Mus; Outcome; Peptides; Phase I Clinical Trials; Physiological; Pichia; Plasma Cells; Plasmablast; Population; Primates; Proliferating; Proteins; Protocols documentation; QS21; Reaction; Recombinant Proteins; Regimen; Regulation; Research Design; Rodent; Role; Serological; Serum; Signal Transduction; Spleen; Staging; Staining method; Stains; Stimulus; Structure of germinal center of lymph node; T cell response; T-Lymphocyte; Testing; Time; Toxoplasma; Transgenic Organisms; Vaccination; Vaccine Adjuvant; Vaccine Antigen; Vaccine Design; Vaccines; Variant; Work; base; experience; feeding; immune function; incomplete Freund' s adjuvant; insight; long bone; lymph nodes; migration; mycobacterial; novel; placebo controlled study; programs; prototype; research study; response; transmission process; vaccine candidate; vaccine development; volunteer

Nearly all licensed vaccines protect through antibodies rather than cell-mediated immunity. A critical aspect of vaccine-induced serological protection is the duration of antibody titer post-boost. Our basic understanding of T-cell dependent antibody responses involves presentation of antigens by dendritic cells to specific CD4+ T cells, which then proliferate and differentiate into helper T cells (Tfh) that engage antigen-engaged B cells that have moved to the T-B border of secondary lymphoid tissues (spleen or lymph node). These interactions can generate a population of short-lived, high-rate antibody secreting cells (the extrafollicluar antibody response), which contributes to acute host defense but not to long-lived protection. The latter involves migration of antigen-activated T cells and the associated antigen-specific B cells into the B cell follicle where they set up the germinal center reaction. Here continued T-B interaction leads to somatic hypermutation and isotype class switching, producing antibodies of higher affinity and different effector class, while generating both memory B cells and also plasmablasts that can become long-lived plasma cells if they reach the proper niche, which is mainly located in the bone marrow. While this general outline is well-established, the molecular signals that guide the engagement of T and B cells to generate a maximally productive response, the role of Tfh in determining the choice between memory B cells and long-lived plasma cells, and what determines how plasmablasts become long-lived plasma cells remain unclear. This project is a collaborative effort among laboratories with expertise in vaccine development, adjuvant function, and the cellular immune reactions that aims to examine how adjuvants affect each of these key steps in humoral immune responses and how variations in the quality and quantity of Tfh, stimuli for B cells, and niche space for plasma cells affects the peak titer and persistence of antibody responses post-vaccination. In FY14, progress has been made in systematically testing a variety of adjuvants with candidate antigens selected for a transmission-blocking malarial vaccine with respect to their capacity to produce durable antibody responses of adequate titer. Distinct adjuvants produced substantially different peak post-boost levels of Pfs25-specific antibodies, with a role also found the carrier protein. Surprisingly and in contrast to data from NHP studies performed previously, the decline in antibody titer over nearly a year showed a similar slope for all conditions. This raises the important question of whether this is a species-related difference or if it reflects the fact that the mice are clean in comparison to the NHP, influencing the availability of bone marrow niche space for long-lived plasma cells. This issue is being addressed in mouse experiments employing a new method for testing the bone marrow homing capacity of recently generated plasmablasts and their longevity in that compartment. Studies are now under way in conventionally reared animals and in those given strong infectious challenges to fill up the bone marrow plasma cell niche. We have also initiated studies examining the role of inflammatory signals in mobilizing bone marrow resident plasma cells and allowing their replacement with newly generated plasmablasts derived from vaccination. Additional experiments are examining whether the physiological state of the plasmablasts also controls the duration of the serological response, with preliminary studies showing that we can detect differential rates of titer decline in mice given plasmablasts from different priming regimens. To complement these functional studies and directly examine the plasma cell niche in bone marrow, we have developed new methods for long bone whole mount microscopy that allows detection of antigen specific and well as IgG producing plasma cells and the detailed characterization of the niche in which they reside. In prior work, we used a model system in which mice were immunized with a peptide epitope from Toxoplasma emulsified in Complete Freunds Adjuvant and then measured T cell responses using a specific Class II MHC tetramer at day 14 post-injection. With this protocol we found that approximately 7% of the CD4+, Foxp3- cells in draining lymph nodes from immunized mice were Tfh as defined by co-staining with CXCR5 and BCL-6; of these roughly 15% were tetramer positive. A more stringent analysis using a combination of 4 Tfh canonical markers revealed that approximately 1% of the CD4+, Foxp3- cells in immunized mice were CXCR5+, BCL-6 +, ICOShi, PD1hi versus 0.1 % in sham immunized mice. These responses were substantially reduced in immunized mice deficient in the TLR/IL-1R adaptor MyD88. This work is currently being extended with kinetic analyses and to studies with Incomplete Freunds adjuvant to evaluate the contribution of the mycobacterial component in CFA to Tfh induction. Additional studies are underway using a new set of Pfs25-protein conjugates to allow more definitive tracking of Tfh responses using tetramers. Pfs25 has been conjugated with the mycobacterial antigen Ag85b, for which the specific peptide determinant is well characterized. This will also allow imaging studies of the dynamics of T-DC and T-B interactions during development of the humoral response using TCR transgenic P25 T cells specific for Ag85b. This will permit direct and quantitative analysis of Tfh formation via dendritic cell and antigen-specific B cell interactions, assessment of B cell activation by antigen and innate stimuli, examination of the outcome of Tfh-B cell interactions in germinal centers, and determination of the proportion of memory B cells vs. plasmablasts formed by this latter response. The data generated by these rodent experiments will be used to plan NHP studies focused on determining if the top candidate adjuvants produce the same types of responses in these primates at the macro (i.e., antibody titer) level and with respect to the cellular events involved, such as bone marrow niche regulation. A key aim of this project is the identification of vaccine formulations that will extend the duration of the antibody response against malaria vaccine candidates. Our prototype target malaria antigen is Pfs25, which is undergoing Phase 1 trials in humans as a Pichia-expressed recombinant protein conjugated to the carrier protein ExoProtein A (EPA) expressed in E. coli with a molar ratio of 3:1, and formulated with the commercially available adjuvant Alhydrogel: Pfs25-EPA/Alhydrogel. Pfs25-EPA/Alhydrogel is undergoing Phase 1 trials in malaria-nave volunteers in the US (dose-escalating trial) and malaria-experienced volunteers in Mali (dose-escalating; double-blinded; placebo-controlled trial). Sera collected from volunteers in the US and Mali are being assessed for seroreactivity by standardized ELISA, and transmission-blocking antibody activity is measured in membrane feeding assays. Serum antibody levels in either assay are being measured before and after each vaccine dose, and then periodically thereafter to assess the duration of the antibody response, including the functional antibody response. In Mali, mosquitoes are fed directly on vaccinees to determine their infectivity/malaria transmission potential, and this will be related to the antibody measurements. The Pfs25-EPA/Alhydrogel product will be a benchmark against which we will compare novel Pfs25 products and formulations in our animal studies. Our animal studies of adjuvants to date support our plan to test Pfs25-based conjugate vaccines using alternative adjuvants in humans, and our initial focus is on the commercial product AS01 from GSK and a similar product (GLA + QS21 in liposomal formulation) from IDRI that has not yet been in the clinic.

几乎所有有执照的疫苗通过抗体保护而不是细胞介导的免疫力保护。疫苗诱导的血清学保护的一个关键方面是抗体效率后的持续时间。我们对T细胞依赖性抗体反应的基本理解涉及树突状细胞对特定CD4+ T细胞的抗原，然后将其扩增并分化为辅助抗原引入的B细胞的辅助T细胞（TFH），这些B细胞已转移到抗原引入的B细胞中，这些B细胞已移至二级淋巴结组织的T-B边界（Spleen或淋巴结型）。这些相互作用可以产生一系列短寿命的高速抗体分泌细胞（外向抗体反应），这有助于急性宿主防御，但不能有助于长期保护。后者涉及抗原激活的T细胞和相关的抗原特异性B细胞迁移到B细胞卵泡中，在那里它们建立了生发中心反应。在这里，持续的t-b相互作用会导致体细胞过度和同种型类切换，从而产生较高亲和力和不同效应子类的抗体，同时同时产生记忆B细胞，以及如果它们达到适当的颗粒（主要位于骨术中），则可以成为长期寿命的血浆细胞。 尽管该一般轮廓是良好的，但指导T和B细胞参与以产生最大生产力的分子​​信号，TFH在确定记忆B细胞和长期寿命的血浆细胞之间的选择中的作用，以及确定静脉曲张如何确定长期植物的血浆细胞的作用仍然不清楚。 This project is a collaborative effort among laboratories with expertise in vaccine development, adjuvant function, and the cellular immune reactions that aims to examine how adjuvants affect each of these key steps in humoral immune responses and how variations in the quality and quantity of Tfh, stimuli for B cells, and niche space for plasma cells affects the peak titer and persistence of antibody responses post-vaccination. 在2014财年，在系统地测试各种佐剂的候选抗原方面已取得了进展，该抗原因其产生适当滴答的持久抗体反应的能力而选择了传输阻断疟疾疫苗。不同的佐剂产生了PFS25特异性抗体的峰值后峰值水平，其作用也发现了载体蛋白。出乎意料的是，与先前进行的NHP研究的数据相反，近一年来抗体滴度的下降显示出所有条件的斜率相似。这就提出了一个重要的问题，即这是否是物种相关的差异，还是反映了与NHP相比的小鼠清洁的事实，影响了长期寿命的等离子体细胞的骨骨髓利基空间的可用性。在小鼠实验中正在解决此问题，该方法采用了一种新方法来测试最近产生的浆膜的骨髓归巢能力及其在该隔室中的寿命。现在，在传统饲养的动物以及对填充骨髓浆细胞小众群体填充的强烈感染挑战的研究中正在进行研究。我们还开始了研究，研究了炎症信号在动员骨髓驻留的浆细胞中的作用，并允许其替换为疫苗接种的新产生的浆膜。其他实验正在研究浆膜的生理状态是否还控制着血清学反应的持续时间，初步研究表明，我们可以检测到给定不同启动方案的浆膜的小鼠滴度下降的差异率。为了补充这些功能研究并直接检查骨髓中的浆细胞壁ni，我们开发了长骨整个固定显微镜的新方法，可以检测抗原特异性和IgG产生血浆细胞以及它们所居住的小生物的详细表征。 在先前的工作中，我们使用了一个模型系统，在该模型系统中，用完全弗朗德斯佐剂中的毒性乳腺癌对小鼠进行免疫，然后在注射后第14天使用特定的II类MHC Tetramer测量T细胞反应。使用该方案，我们发现来自免疫小鼠的排水淋巴结中约7％的CD4+FOXP3细胞是通过与CXCR5和BCL-6共染色的TFH的。在这些大约15％的四聚体阳性。使用4 TFH规范标记的组合进行更严格的分析表明，免疫小鼠中约有1％的CD4+FOXP3-细胞为CXCR5+，Bcl-6+，ICOSHI，PD1HI，PD1HI，pD1HI，而在假免疫小鼠中为0.1％。 在TLR/IL-1R适配器MyD88中缺乏免疫小鼠中，这些反应大大降低了。 目前，这项工作正在扩展到动力学分析，并使用不完整的Freunds辅助研究来评估CFA中分枝杆菌成分对TFH诱导的贡献。使用一组新的PFS25-蛋白结合物正在进行其他研究，以便使用四聚体对TFH反应进行更确定的跟踪。 PFS25已与分枝杆菌抗原AG85B结合，特异性肽的决定因素的表征很好。这还将允许对使用特异性AG85B的TCR转基因P25 T细胞进行体液反应过程中T-DC和T-B相互作用的动力学进行成像研究。这将允许通过树突状细胞和抗原特异性B细胞相互作用进行直接和定量分析，通过抗原和先天刺激评估B细胞激活，检查生发中心中TFH-B细胞相互作用的结果以及确定记忆B细胞的比例与该层面响应形成的静态BLABLS的比例。这些啮齿动物实验产生的数据将用于计划NHP研究，重点是确定顶级候选佐剂是否在宏（即抗体滴度）水平以及涉及的细胞事件（例如骨髓小裂元法）中产生相同类型的反应。 该项目的主要目的是鉴定疫苗制剂，该制剂将扩大针对疟疾疫苗候选物的抗体反应的持续时间。我们的原型靶标疟疾抗原是PFS25，它正在人类中进行1期试验，作为一种斑岩表达的重组蛋白，该蛋白结合在大肠杆菌中以3：1表示的大肠杆菌表达，并以3：1表示，并用商业上可用的苏格氏素添加Alhyhyhyhyhyhyhydrog-epa-pfa-pfa-pfa-pfa-pfa- pfa。 PFS25-EPA/Alhydrogel正在美国（剂量升级试验）和马里疟疾经验的志愿者（剂量 - 标准化； Double-Blinded;安慰剂控制的试验）中，正在接受美国疟疾北极志愿者的1期试验。从美国和马里志愿者那里收集的血清正在通过标准化ELISA评估血清反应性，并且在膜进食测定中测量了传播阻断抗体活性。每种疫苗剂量之前和之后，都在测量两种测定中的血清抗体水平，然后定期评估抗体反应的持续时间，包括功能抗体反应。在马里，蚊子直接以疫苗为食，以确定其感染性/疟疾传播潜力，这将与抗体测量有关。 PFS25-EPA/Alhydrogel产品将是一个基准，我们将在动物研究中比较新颖的PFS25产品和制剂。我们迄今为止对佐剂的动物研究支持我们的计划，该计划使用人类中的替代佐剂测试基于PFS25的共轭疫苗，我们最初的重点是GSK的商业产品AS01和类似产品（GLA + QS21（脂质体配方中的GLA + QS21）），IDRI尚未在诊所中。