Targeted Dendritic Cell Activation through Multi-Adjuvant Liposomes

通过多佐剂脂质体靶向激活树突状细胞

• 批准号: 8252011
• 负责人: Gretchen Jimenez
• 金额: $ 30万
• 依托单位: MOLECULAR EXPRESS, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8252011
• 关键词: Achievement; Adjuvant; Adult; Affect; Agonist; American; Amines; Amino Acids; Animals; Antibodies; Antigen Targeting; Antigen-Presenting Cells; Antigens; Biological; Biological Assay; Bioterrorism; Body Weight decreased; Buffers; CD40 Ligand; Caliber; Carbohydrates; Cause of Death; Cell Surface Receptors; Cells; Chimeric Proteins; Clinic; Clinical; Communicable Diseases; Coupled; Cyclic GMP; Dendritic Cells; Dendritic cell activation; Development; Disease; Drug Delivery Systems; Drug Formulations; Engineering; Ensure; Escherichia coli; Extracellular Matrix; Female; Glycoproteins; Goals; HDAC1 gene; HIV; Herpes Simplex Infections; Home environment; Human; Human Herpesvirus 2; Immune response; Immunization; Immunologist; Inbred BALB C Mice; Individual; Infection; Influenza; Influenza A virus; Interferons; Interleukin-12; Interleukin-4; Lead; Ligands; Lipid Bilayers; Lipids; Liposomes; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Market Research; Marketing; Membrane Glycoproteins; Methods; Modeling; Molecular; Monitor; Mothers; Mus; Myelogenous; Oligonucleotides; Pathway interactions; Peptides; Performance; Pharmacology and Toxicology; Phase; Precipitation; Predisposition; Prevalence; Production; Property; Proteins; Publishing; Recombinant Proteins; Reporting; Science; Sexually Transmitted Diseases; Small Business Innovation Research Grant; Solutions; Sulfhydryl Compounds; Surface; Surface Antigens; System; T-Lymphocyte; TLR4 gene; TLR9 gene; Technology; Testing; Time; Toll-like receptors; Update; Vaccine Research; Vaccines; Vesicle; Viral; Viral Antigens; Viral Load result; Virulent; Virus; Virus Diseases; Woman; analytical method; base; commercialization; cytokine; design; engineering design; flexibility; genital herpes; immunogenic; immunosuppressed; improved; in vivo; infectious disease model; innovation; meetings; mouse model; neonate; pandemic disease; pathogen; programs; receptor; receptor binding; research clinical testing; research study; response; statistics; transmission process; unilamellar vesicle; vaccine candidate; vaccine development; vaccine efficacy

DESCRIPTION (provided by applicant): The overall goal of this SBIR AT application is to develop a multi-adjuvant liposomal vaccine formulation that targets dendritic cells and can be applied to our current vaccine programs. The innovation is a dual adjuvant system where VesiVax(R) lipid vesicles are formulated with a TLR9 agonist (CpG) and CD40 Ligand fused to the Company's proprietary Hydrophobic Domain (HD) technology. The formulations, including testing of single adjuvants, will be assessed with influenza and Herpes Simplex 2 viral antigens in vivo in proven mouse models. We have shown that the TLR4 agonist, MPL has an immunostimulatory effect and hypothesize that other TLR agonists and receptor-binding ligands will influence the immune response elicited by target antigens. The ultimate goal of this program is to build an immunogenic liposome formulation that is broadly applicable to antigens, regardless of whether a predominantly Th1 or Th2 type of immune response is desired. The optimal formulation will be utilized to advance an influenza or Herpes Simplex Virus 2 (HSV2) vaccine candidate towards the clinic. Vaccines based on the VesiVax(R) system can be engineered using two formats: 1) a target antigen or adjuvant protein can be engineered to be expressed as a fusion protein with the HD which facilitates incorporation directly into the liposome or; 2) a target antigen or adjuvant in the form of a peptide or carbohydrate that can be attached via conjugation to the surface of the liposomes, or conjugatable adjuvant lipid vesicles (CALVs). The Company has assembled an internal team, as well as an external collaborator, with expertise in key core competencies in recombinant protein production, understanding structural and functional interactions of proteins and peptides with lipid bilayers in order to prepare liposomes with the appropriate properties, and to analyze the liposomes and lipid, protein, and adjuvant components to confirm the composition. Our collaborator for the proposed in vivo studies has been testing VesiVax(R)-TLR4 vaccines in established challenge models for influenza and HSV2. The approach to evaluating the proposed dual adjuvant system involves creating the CD40L and TLR9 adjuvanted formulations and testing their effect formulated in vaccines containing the influenza M2e or HSV2 gD antigens. Using methods already developed by the Company for M2e-HD and gD-HD, we will prepare CD40L as an HD fusion expressed in E. coli, and we will prepare the selected CpG oligonucleotide sequence either synthesized coupled to a lipid or with a free thiol or free amine on the end to attach to lipid. We will use ou published female mouse model of intravaginal HSV2 infection and mouse influenza intranasal challenge model. These models will allow us to test each adjuvant in separate formulations as well as combined. Ultimately, we hypothesize that the combination of the two adjuvants will stimulate stronger protective immune responses, thus leading to an optimized commercial formulation for our vaccine programs. The first Milestone in this proposal is the production of liposomes formulated with CpG, CD40L-HD, and both CpG and CD40L-HD. The second Milestone is the choice of an optimal formulation by testing in the two infectious disease models. In Phase 2, we will develop commercial production and analytical methods and conduct nonclinical Pharmacology/Toxicology studies to support the filing of an IND. A secondary application of the new adjuvant system would be to make it available to vaccinologists and immunologists in a kit format. A Foresight Science & Technology market research report entitled "Liposomal Adjuvant for Vaccine Research" was commissioned by the Company. The report identified lipid-based adjuvants as having a "very broad range of potential applications and corresponding to more than 80% of the market for vaccines in development, which is estimated to reach more than $18 billion by 2015." Using this statistic, it is estimated that the total addressable market for lipid-based adjuvants may be over $14.4 billion by 2015. PUBLIC HEALTH RELEVANCE: With the continued emergence of new pathogens, the prevalence of certain viral infections, and the threat of bioterrorism, it is imperative to continu to optimize vaccine performance. Molecular Express is developing vaccines to two viral targets, influenza A virus (IAV) and Herpes Simplex Virus 2 (HSV2). Herpes genitalis caused by HSV2 is one of the most common, sexually transmitted diseases in humans, with as many as 1 out of 6 Americans having been infected. The disease affects both normal and immunosuppressed adults, and is associated with increased susceptibility to the human immunodeficiency virus, serious clinical disease in neonates following transmission of virus from their infected mothers, and increased likelihood of developing cervical cancer versus non-infected women. IAV infections have caused the deaths of millions of humans worldwide. While generally effective against individual viral subtypes, vaccines that protect against infection by IAV are difficult to manufacture rapidly and demonstrate limited activity against different subtypes due to antigenic shift and drift in the surface antigens of the virus. In addition, the recent emergence of highly virulent strains of IAV could result in a devastating human pandemic before effective vaccine candidates could be developed. Vaccines that provide broad protection against infection with emerging strains of IAV and are manufactured in a manner that allows the vaccine to be rapidly and effectively updated in response to new strains of IAV would be a major advance. Clearly there are critical needs to develop effective vaccines against these two diseases. There is unexplored potential to optimize our current influenza A virus (IAV) and Herpes Simplex Virus 2 (HSV2) vaccine candidates by targeting immunostimulation through Dendritic Cell (DC) activating pathways. We will track the immunostimulatory properties of vaccine formulations using established in vivo viral challenge models. We hypothesize that by combining a Toll-like receptor 9 agonist, CpG, with CD40 Ligand, DC cells will facilitate an augmented response to VesiVax(R) liposomal vaccines. Testing of the DC-targeted dual adjuvant approach will be conducted on two vaccines, IAV Matrix 2 protein ectodomain (L-M2e1-15-HD) and HSV2 surface glycoprotein (L-gD1-306), which generates predominantly Th2 and predominantly Th1 responses, respectively. The goal of this project is to understand and optimize vaccine formulations that are broadly applicable to our vaccine programs. Thus, successful execution of the studies proposed in this application could lead to new and/or improved vaccines for treatment of a variety of infectious diseases or cancers.

描述（由申请人提供）：该SBIR在申请中的总体目标是开发一种靶向树突状细胞的多辅助脂质体疫苗配方，并可以应用于我们当前的疫苗程序。该创新是一种双重辅助系统，使用TLR9激动剂（CPG）和CD40配体配制了Vesivax（R）脂质囊泡，并融合了该公司专有疏水域（HD）技术。在经过验证的小鼠模型中，将用流感和单纯疱疹2病毒抗原评估包括单个佐剂的测试，包括对单个佐剂的测试。我们已经表明，TLR4激动剂，MPL具有免疫刺激作用，并假设其他TLR激动剂和受体结合配体将影响靶抗原引起的免疫反应。该程序的最终目标是建立一种免疫原性的脂质体配方，该配方通常适用于抗原，而不管是否需要主要的TH1或TH2类型的免疫反应。最佳配方将用于促进流感或单纯疱疹病毒2（HSV2）候选疫苗降落到诊所。 基于Vesivax（R）系统的疫苗可以使用两种格式进行设计：1）靶抗原或辅助蛋白可以设计为具有HD的融合蛋白，该蛋白具有HD，从而促进直接掺入脂质体或； 2）靶抗原或佐剂的形式，可以通过偶联到脂质体表面或可结合的辅助脂质囊泡（CALVS）的形式（CALVS）。该公司已经组建了一个内部团队以及外部合作者，在重组蛋白质生产方面的关键核心能力方面具有专业知识，了解蛋白质和肽与脂质双层的结构和功能相互作用，以便与适当的特性准备脂质体，并确认脂质体和脂质体和脂质体，蛋白质和杂质的组合，以供脂质体和脂质体组合。我们在拟议的体内研究的合作者一直在为型流感和HSV2的既定挑战模型中测试Vesivax（R）-TLR4疫苗。 评估所提出的双重辅助系统的方法涉及创建CD40L和TLR9辅助配方，并测试其在含有流感M2E或HSV2 GD抗原的疫苗中制定的效果。使用公司已经为M2E-HD和GD-HD开发的方法，我们将准备CD40L作为大肠杆菌表达的HD融合，我们将准备所选的CpG寡核苷酸序列合成的纤维或硫醇或自由氨基的合成，或者在脂质的末端与脂肪固定。我们将使用OU发表的雌性小鼠模型的阴道内HSV2感染和小鼠流感鼻内挑战模型。这些模型将使我们能够以单独的配方和组合测试每个佐剂。最终，我们假设两个佐剂的组合将刺激更强的保护性免疫反应，从而为我们的疫苗计划提供了优化的商业配方。 该提案中的第一个里程碑是用CPG，CD40L-HD以及CPG和CPG和CD40L-HD制成的脂质体的产生。第二个里程碑是通过在两个传染病模型中测试最佳配方的选择。在第2阶段，我们将开发商业生产和分析方法，并进行非临床药理学/毒理学研究，以支持IND提交。 新辅助系统的次要应用将是使疫苗学家和免疫学家以套件格式使用。该公司委托一份题为“疫苗研究的脂质体辅助”的前瞻性科学与技术市场研究报告。该报告确定基于脂质的佐剂具有“广泛的潜在应用，并且对应于开发疫苗的80％以上，估计到2015年，该疫苗的开发量超过180亿美元。”使用此统计数据，据估计，到2015年，基于脂质的佐剂的总可寻址市场可能超过144亿美元。 公共卫生相关性：随着新病原体的持续出现，某些病毒感染的流行以及生物恐怖主义的威胁，必须继续优化疫苗性能。 Molecular Express正在向两个病毒靶标开发疫苗，即流感病毒（IAV）和单纯疱疹病毒2（HSV2）。 HSV2引起的疱疹生殖器是人类最常见的性传播疾病之一，其中有6名美国人中有多达1种被感染。该疾病会影响正常和免疫抑制的成年人，并且与人类免疫缺陷病毒的易感性增加有关，在感染母亲传播病毒后，新生儿的严重临床疾病，以及宫颈癌与未感染的妇女的可能性增加。 IAV感染导致了全球数百万人类的死亡。虽然通常对单个病毒亚型有效，但防止IAV感染的疫苗很难迅速生产，并且由于抗原移位和病毒表面抗原的漂移而对不同亚型的活性有限。此外，在开发有效的候选疫苗之前，最近高度毒性的IAV菌株的出现可能导致毁灭性的人类大流行。通过IAV的新兴菌株提供广泛保护的疫苗，并以使疫苗能够迅速有效地对IAV的新菌株进行迅速有效地更新的方式生产，这将是一个重大进展。显然，针对这两种疾病开发有效的疫苗有至关重要的需要。 通过通过树突状细胞（DC）激活途径靶向免疫刺激，可以优化我们当前的流感病毒（IAV）和单纯疱疹病毒2（HSV2）疫苗的潜力。我们将使用已建立的体内病毒挑战模型来跟踪疫苗制剂的免疫刺激性能。我们假设，通过将Toll样受体9激动剂CPG与CD40配体组合，DC细胞将促进对Vesivax（R）脂质体疫苗的增强反应。将在两种疫苗IAV基质2蛋白质e骨（L-M2E1-15-HD）和HSV2表面糖蛋白（L-GD1-306）上进行两种疫苗（L-GD1-306），对两种疫苗进行了对DC靶向双重辅助方法的测试，该疫苗将分别产生，主要产生TH2，并主要产生Th2，并分别产生TH1反应。该项目的目的是了解和优化广泛适用于我们的疫苗计划的疫苗配方。因此，在本应用中提出的研究的成功执行可能会导致新的和/或改进的疫苗，以治疗各种传染病或癌症。