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pre-clinical; protein complex; research clinical testing; response; self assembly; transmission-blocking vaccine; unpublished works; uptake; vaccine candidate; vaccine delivery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.

2019 财年，LMIV 科学家发表了 2 篇关于结合疫苗或颗粒疫苗的出版物，我们在此处描述了这些手稿中报告的进展： 我们之前已经证明，免疫原性差的 TBV 抗原与外蛋白 A (EPA) 的化学缀合可以增强其免疫原性。在这里，我们评估了外膜蛋白复合物 (OMPC)（一种源自脑膜炎奈瑟菌的膜囊泡）作为 Pfs230 的载体（Scaria PV 等人 npjVaccines）。我们制备了具有不同抗原负载水平的 Pfs230-OMPC 缀合物，并检查了小鼠的免疫原性。 Pfs230 与 OMPC 的化学缀合增强了 Pfs230 抗原的免疫原性和功能活性，并且 OMPC 缀合物在不同剂量下实现了比 Pfs230-EPA/AdjuPhos 高 2 倍至 20 倍的抗体滴度。 OMPC 缀合物即使在低剂量下也具有高度免疫原性，表明剂量节约效应。 EPA 缀合物诱导了偏向 Th2 反应的 IgG 亚类谱，而 OMPC 缀合物诱导了具有高水平 IgG2 的强烈偏向 Th1 的免疫反应，这有利于 Pfs230 抗体功能活性，而功能活性取决于补体激活。 OMPC 是 Pfs230 疫苗有前途的载体。 尺寸计量学的改进和功能化纳米粒子物理化学表征的创新对于实现纳米材料的增强性能和优势至关重要（Farkas N、Scaria PV 等人。科学报告）。为了实现这一目标，我们提出了一种多技术测量方法，其中使用相关原子力显微镜、动态光散射、高效液相色谱和质谱测量来评估具有核心的自组装聚合物纳米粒子的分子和结构特性外壳结构。在这种方法中，首先使用由合成聚阳离子支化聚乙烯亚胺大分子功能化的金纳米颗粒组成的模型系统来验证测量方法。通过原子力显微镜和动态光散射测量壳厚度，并通过色谱分离和质谱分析测定聚电解质的吸收。大小、结构和稳定性之间的统计相关性为将该方法扩展到通过缩合反应进行更复杂的核酸和大分子自组装提供了基础。从这些尺寸和分析化学测量中，我们获得了这些组件的全面空间描述，获得了核-壳演化的详细解释，并确定了参数空间中发生稳定、离散颗粒形成的区域。 在未发表的工作中，我们报告了几个正在进行的项目的进展情况： OMPC 作为阻断疫苗抗原递送平台的进一步评估： 2019财年，我们继续评估 OMPC 作为 TBV 抗原递送平台的能力。正如我们出版物中提到的，小鼠研究确定，与相同抗原的对照 EPA 缀合物相比，OMPC 缀合物具有更优异的免疫原性。与 EPA 缀合物的 Th2 反应相反，OMPC 缀合物观察到了性质不同的、偏向 Th1 的免疫反应。基于这些发现，我们开始在非人灵长类动物中评估 OMPC 缀合物，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结果于 2019 财年发表。对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（长达一年），通过标准膜补料测定法测定不同时间点的血清样本的抗体滴度和功能活性。 TBV 抗原替代蛋白载体的评估： 我们目前正在评估替代载体和佐剂。为此，我们通过合作协议从不同的商业实体采购了运营商。 2019财年，我们继续与Fina Biosolutions合作，获得了EcoCRM（大肠杆菌生产的CRM197）和TTHc（破伤风毒素重链），用于与Pfs230缀合。我们合成了一系列 Pfs230 与 EcoCRM、TTHC、CRM197（来自 Pfenex）和 TT（来自 SSI）的缀合物。这些缀合物在小鼠免疫原性研究中进行了测试，发现可以产生强烈的免疫反应。基于这些发现，我们与 Scarab Genomics 和 Biofarma（印度尼西亚）建立了额外的合作，分别获得 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缀合物；测试这些的小鼠研究正在进行中，初步结果已在 2018 年 10 月的 ASTMH 年会上公布。通过与 MVI/PATH 的合作，我们在这一领域的工作得到了加速，MVI/PATH 支持 Pfs230D1 和 Pfs230C1 的临床前缀合/免疫研究。 疟疾抗原mRNA技术评价： 2019财年，我们继续与德国CureVac合作，在CureVacs RNActive技术平台上测试LMIVs疟疾抗原的免疫原性。使用 mRNA 进行抗原递送作为一种可以快速生成用于临床测试的候选疫苗的技术，在疫苗领域引起了相当大的关注。该技术目前正在 CureVac 和 Moderna Therapeutics 的多项临床试验中进行测试；两家公司都拥有设计和制造疫苗有效 mRNA 的专有技术。我们正在与 CureVac 合作构建 TBV 和 PMV 抗原的 mRNA。在 2019 财年，CureVac 生成了一系列 LMIV 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。作为持续合作的一部分，将在 LMIV 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。 无针疫苗注射： 2019 财年，我们继续与日本武田药品工业株式会社建立合作，评估其专有的微针贴片递送技术，用于递送我们用于阻断传播的疫苗的结合免疫原。武田的溶解微针是一种疫苗输送技术，具有许多可用于疟疾疫苗的有吸引力的功能。微针贴片的施用不需要熟练的医疗专业人员，也可以自行施用。它通过消除意外针伤和针输送相关的疼痛来避免使用针。它还不需要冷链运输和储存，从而降低了大规模免疫活动的成本。我们目前正在与武田合作进行第一项涉及微针输送的动物研究。在 2019 财年，我们评估了用于微针制造的聚合物混合物中的缀合物抗原的兼容性，并发现它们是兼容的。我们还建立了定量微针贴片中抗原含量的测定方法。这使得武田能够微调微针制造。根据这些研究，武田生产了一系列用于动物研究的微针贴片。这些补丁正在 LMIV 的小鼠免疫原性研究中进行评估。 替代 TBV 抗原的缀合物 - Pfs47： 2019 财年，作为与 Carolina Barillas 集团 (NIAID) 建立的内部合作的一部分，以测试替代 TBV 抗原 Pfs47，我们合成了 Pfs47 的 EPA 结合物。该缀合物在小鼠免疫原性研究中进行了评估。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，目前正在寻求其他合成方案。