Malaria Vaccines: TBV Antigens as Conjugates with Alternate Carriers

疟疾疫苗：TBV 抗原与替代载体的结合物

• 批准号: 10014128
• 负责人: Patrick Duffy
• 金额: $ 305万
• 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/10014128
• 关键词: Adjuvant; Agreement; Aluminum; Animals; Antibodies; Antibody Response; Antibody titer measurement; Antigens; Area; Atomic Force Microscopy; Biological Assay; Biological Models; Carrier Proteins; Chemicals; Clinical; Clinical Trials; Cold Chains; Collaborations; Complement Activation; Complex; Correlation Studies; Dimensions; Dose; Epitopes; Escherichia coli; Evaluation; Evolution; Formulation; Genomics; Germany; Goals; High Pressure Liquid Chromatography; IgG2; Immune response; Immunization; Immunoglobulin G; Indonesia; Injury; Japan; Liposomes; Malaria; Malaria Vaccines; Mammalian Cell; Manuscripts; Mass Immunization; Measurement; Measures; Medical; Membrane; Membrane Proteins; Messenger RNA; Methods; Modeling; Modification; Molecular Structure; Mus; National Institute of Allergy and Infectious Disease; Needles; Neisseria meningitidis; Nucleic Acids; Pain; Parasites; Performance; Pharmacologic Substance; Physical condensation; Plasmodium vivax; Polymers; Polysaccharides; Pregnancy; Progress Reports; Property; Proteins; Publications; Publishing; RNA vaccine; Reaction; Recombinant Proteins; Reporting; Salmonella typhi; Sampling; Scientist; Self Administration; Series; Serum; Sexual Development; Structure; Surface; Techniques; Technology; Testing; Tetanus Toxin; Tetanus Toxoid; Therapeutic; Thick; Time; Vaccine Antigen; Vaccines; Vesicle; Work; base; burden of illness; cost; cross reacting material 197; design; feeding; immunogenic; immunogenicity; innovation; light scattering; liquid chromatography mass spectroscopy; macromolecule; malaria transmission; malaria transmission-blocking vaccine; meetings; metrology; nanoGold; nanomaterials; nanoparticle; nonhuman primate; particle; pre-clinical; protein complex; research clinical testing; response; self assembly; transmission-blocking vaccine; unpublished works; uptake; vaccine candidate; vaccine delivery

In FY19, LMIV scientists contributed to 2 publications on conjugate or particle vaccines, and we describe progress reported in those manuscripts here: We have previously shown that chemical conjugation of poorly immunogenic TBV antigens to Exoprotein A (EPA) can enhance their immunogenicity. Here, we assessed Outer Membrane Protein Complex (OMPC), a membrane vesicle derived from Neisseria meningitidis, as a carrier for Pfs230 (Scaria PV, et al. npjVaccines). We prepared Pfs230-OMPC conjugates with varying levels of antigen load and examined immunogenicity in mice. Chemical conjugation of Pfs230 to OMPC enhanced immunogenicity and functional activity of the Pfs230 antigen, and OMPC conjugates achieved 2-fold to 20-fold higher antibody titers than Pfs230-EPA/AdjuPhos at different doses. OMPC conjugates were highly immunogenic even at low doses, indicating a dose-sparing effect. EPA conjugates induced an IgG subclass profile biased towards a Th2 response, whereas OMPC conjugates induced a strong Th1-biased immune response with high levels of IgG2, which can benefit Pfs230 antibody functional activity, which depends on complement activation. OMPC is a promising carrier for Pfs230 vaccines. Improvements in dimensional metrology and innovations in physical-chemical characterization of functionalized nanoparticles are critically important for the realization of enhanced performance and benefits of nanomaterials (Farkas N, Scaria PV, et al. Scientific Reports). Toward this goal, we propose a multi-technique measurement approach, in which correlated atomic force microscopy, dynamic light scattering, high performance liquid chromatography and mass spectroscopy measurements are used to assess molecular and structural properties of self-assembled polyplex nanoparticles with a core-shell structure. In this approach, measurement methods are first validated with a model system consisting of gold nanoparticles functionalized with synthetic polycationic branched polyethylenimine macromolecules. Shell thickness is measured by atomic force microscopy and dynamic light scattering, and the polyelectrolyte uptake determined by chromatographic separation and mass spectrometric analysis. Statistical correlation between size, structure and stability provide a basis for extending the methods to more complex self-assembly of nucleic acids and macromolecules via a condensation reaction. From these size and analytical chemical measurements, we obtain a comprehensive spatial description of these assemblies, obtain a detailed interpretation of the core-shell evolution, and identify regions of the parameter space where stable, discrete particle formation occurs. In unpublished work, we report below our progress on several ongoing projects: Further evaluation of OMPC as a delivery platform for Transmission Blocking Vaccine antigens: In FY2019, we continued the evaluation of OMPC as a delivery platform for TBV antigens. As noted above in our publication, mouse studies determined that OMPC conjugates provided superior immunogenic properties compared to control EPA conjugates of the same antigens. A qualitatively different, Th1-biased immune response was observed for OMPC conjugates as opposed to a Th2 response of EPA conjugates. Based on these findings, we have initiated evaluation of OMPC conjugates in nonhuman primates to determine their efficacy in this model and to evaluate the duration of immune response. Findings from the mouse immunogenicity studies were published during FY2019. Studies in nonhuman primates will evaluate the level of antibody response and different time points, up to a period of one year, assaying serum samples at different times points for their antibody titer and functional activity by Standard Membrane Feed assay. Evaluation of Alternate protein carriers for TBV antigens: We are currently evaluating alternate carriers and adjuvants. For this effort, we procured carriers from different commercial entities through collaborative agreements. In FY2019, we continued our collaboration with Fina Biosolutions to obtain EcoCRM (E. Coli produced CRM197) and TTHc (Tetanus Toxin heavy chain) for conjugation with Pfs230. We synthesized a series of conjugates of Pfs230 with EcoCRM, TTHC, CRM197 (from Pfenex) and TT (from SSI). These conjugates were tested in mouse immunogenicity studies and were found to generate strong immune responses. Based on these findings, additional collaborations were established with Scarab Genomics and Biofarma (Indonesia) to obtain clinically viable versions of CRM197 and TT respectively. These materials were obtained and conjugates were synthesized using these carriers; mouse studies to test these are ongoing, and preliminary results were presented at the ASTMH annual meeting in Oct 2018. Our work in this area has been accelerated through a partnership with MVI/PATH who is supporting preclinical conjugation/immunization studies of Pfs230D1 and Pfs230C1. Evaluation of mRNA technology for malaria antigens: In FY2019, we continued the collaboration with CureVac, Germany to test the immunogenicity of LMIVs malaria antigens in CureVacs RNActive technology platform. Antigen delivery using mRNA has generated considerable excitement in the vaccine field as a technology that can rapidly generate vaccine candidates for clinical testing. This technology is now being tested in a number of clinical trials by CureVac and Moderna Therapeutics; both have their proprietary technologies for designing and manufacturing potent mRNAs for vaccine. We are working with CureVac to construct mRNA for our TBV and PMV antigens. In FY2019, CureVac generated a series of mRNA constructs for LMIVs TBV and pregnancy malaria antigens and tested their expression in mammalian cells. As part of this continuing collaboration, mouse immunogenicity studies will be performed at LMIV to test the immunogenicity and functional activity of these mRNA constructs. Needle-free vaccine delivery: In FY2019, we continued the collaboration established with Takeda Pharmaceuticals, Japan to evaluate their proprietary Microneedle Patch delivery technology for delivery of our conjugate immunogens for transmission blocking vaccine. Takedas dissolving microneedle is a technology for vaccine delivery that has a number of attractive features useful for malaria vaccines. Administration of microneedle patches do not require a skilled medical professional or can be self-administered. It avoids needle use by eliminating accidental needle injuries and pain associated with needle delivery. It also does not require cold-chain transport and storage, thereby reducing the cost of mass immunization campaigns. We are currently working with Takeda to perform the first animal study involving microneedle delivery. In FY2019, we evaluated the compatibility of our conjugate antigens in polymer mix used for microneedle fabrication and found them compatible. We also established assays to quantify the antigen content in microneedle patches. This allows Takeda to fine tune microneedle fabrication. Based on these studies, Takeda generated a series of microneedle patches for animal studies. These patches are being evaluated in mouse immunogenicity studies at LMIV. Conjugates of Alternate TBV antigens - Pfs47: In FY2019 as part of the established intramural collaboration with Carolina Barillas group (NIAID) to test an alternate TBV antigen, Pfs47, we synthesized the EPA conjugate of Pfs47. This conjugate was evaluated in mouse immunogenicity studies. These studies indicated that chemical modification may alter the epitope important for functional activity. Based on these studies, other synthetic options are currently being pursued.

在195财年，LMIV科学家为两家关于共轭或颗粒疫苗的出版物做出了贡献，我们在这里描述了这些手稿中报道的进度： 我们先前已经表明，免疫原性TBV抗原与脱甲蛋白A（EPA）的化学结合可以增强其免疫原性。在这里，我们评估了源自奈瑟氏菌的膜囊泡的外膜蛋白复合物（OMPC），作为PFS230的载体（Scaria PV等人，NPJVACCINE）。我们准备了具有不同水平的抗原载荷的PFS230-CompC结合物，并检查了小鼠的免疫原性。 PFS230与OMPC的化学共轭增强了PFS230抗原的免疫原性和功能活性，而OMPC偶联物的化学性能达到了比PFS230-EPA/Adjuphos在不同剂量下的抗体​​滴度高2至20倍。即使在低剂量下，OMPC结合物也具有高度免疫原性，表明剂量的效果。 EPA偶联物诱导了偏向Th2响应的IgG亚类谱，而OMPC偶联物诱导了强大的Th1偏置免疫反应和高水平的IgG2，这可以使PFS230抗体功能活性受益，这取决于补体激活。 OMPC是PFS230疫苗的有前途的载体。 维度计量学的改进和功能化纳米颗粒的物理化学表征的创新对于实现增强纳米材料的性能和益处至关重要（Farkas N，Scaria PV等人。为了实现这一目标，我们提出了一种多技术测量方法，其中使用了相关的原子力显微镜，动态光散射，高性能液相色谱和质谱测量值，用于评估具有核心壳结构的自组装polyplex纳米颗粒的分子和结构特性。在这种方法中，首先使用由金纳米颗粒组成的模型系统验证测量方法，该模型系统由合成的聚卵枝聚乙二胺大分子官能化。通过原子力显微镜和动态光散射来测量壳厚度，以及通过色谱分离和质谱分析确定的聚电解质摄取。大小，结构和稳定性之间的统计相关性为通过凝结反应扩展到更复杂的核酸和大分子的更复杂自组装的方法提供了基础。从这些大小和分析化学测量值中，我们获得了这些组件的全面空间描述，获得对核心壳演化的详细解释，并确定发生稳定的离散粒子形成的参数空间区域。 在未发表的工作中，我们在下面的几个正在进行的项目上报告了下面的进展： 进一步评估OMPC作为传输阻断疫苗抗原的输送平台： 在2019财年，我们继续评估OMPC作为TBV抗原的输送平台。如上所述，小鼠研究确定OMPC结合物与同一抗原的对照EPA结合物相比提供了优异的免疫原性。与EPA偶联物的Th2响应相反，观察到OMPC偶联物的质量不同，Th1偏置的免疫反应。基于这些发现，我们已经开始评估非人类灵长类动物中的OMPC结合物，以确定其在该模型中的功效并评估免疫反应的持续时间。小鼠免疫原性研究的发现发表了2019财年。非人类灵长类动物的研究将评估抗体反应水平和不同时间点的水平，直到一年内，在不同时间点测定其抗体滴度的血清样品，并通过标准的膜饲料饲料测定法进行功能活性。 评估TBV抗原的替代蛋白载体： 我们目前正在评估替代载体和佐剂。为此，我们通过协作协议从不同商业实体采购了承运人。在2019财年，我们继续与Fina Biosolutions合作，以获取EcoCRM（大肠杆菌生产CRM197）和TTHC（Tetanus Toxin重链），以与PFS230结合。我们与EcoCRM，TTHC，CRM197（来自Pfenex）和TT（来自SSI）合成了一系列PFS230的共轭物。在小鼠免疫原性研究中测试了这些结合物，并被发现会产生强大的免疫反应。基于这些发现，与圣甲虫基因组学和生物武器（印度尼西亚）建立了其他合作，分别获得了CRM197和TT的临床可行版本。获得这些材料，并使用这些载体合成结合物。对这些测试的小鼠研究正在进行中，并在2018年10月的ASTMH年度会议上提出了初步结果。我们在该领域的工作通过与MVI/Path的合作伙伴关系加速了，他支持临床前共轭/免疫研究PFS230D1和PFS230C1。 评估疟疾抗原的mRNA技术： 在2019财年，我们继续与德国Curevac进行合作，以测试Curevacs rnactive技术平台中LMIVS疟疾抗原的免疫原性。使用mRNA的抗原递送已经在疫苗场中引起了极大的兴奋，它可以迅速生成候选疫苗进行临床测试的技术。现在，Curevac和Moderna Therapeutics在许多临床试验中对该技术进行了测试。两者都有专有技术，用于设计和制造疫苗的有效mRNA。我们正在与CUREVAC合作为TBV和PMV抗原构建mRNA。在2019年，Curevac生成了一系列用于LMIVS TBV和妊娠疟疾抗原的mRNA构建体，并测试了它们在哺乳动物细胞中的表达。作为这种持续合作的一部分，将在LMIV上进行小鼠免疫原性研究，以测试这些mRNA构建体的免疫原性和功能活性。 无针疫苗输送： 在2019财年，我们继续与日本武田制药公司（Takeda Pharmaceuticals）建立的合作，评估其专有的微针贴片输送技术，用于递送我们的共轭免疫原，用于传输疫苗。溶解微针的人是一种用于疫苗输送的技术，具有许多有吸引力的特征，可用于疟疾疫苗。微针贴剂的管理不需要熟练的医疗专业人员，也不需要自我管理。它通过消除意外针损伤和针头递送疼痛来避免使用针头。它还不需要冷链运输和存储，从而降低了大规模免疫运动的成本。我们目前正在与武田合作进行涉及微针交付的第一个动物研究。在2019财年，我们评估了用于微针制造的聚合物混合物中的共轭抗原的兼容性，并发现它们兼容。我们还建立了测定方法，以量化微针斑块中的抗原含量。这使得田可以微调微针制造。基于这些研究，武田为动物研究生成了一系列微针斑块。这些斑块正在LMIV的小鼠免疫原性研究中评估。 替代TBV抗原的偶联物-PFS47： 在2019财年，作为与Carolina Barillas Group（NIAID）建立的室内合作的一部分，以测试替代TBV抗原，PFS47，我们合成了PFS47的EPA结合物。在小鼠免疫原性研究中评估了这种结合物。这些研究表明，化学修饰可能会改变对功能活性重要的表位。基于这些研究，目前正在追求其他合成选择。