Formulation of LbL microparticle vaccine in microneedle array for intradermal del

摘要 免疫系统识别并启动对外来颗粒的免疫应答的能力， 细菌和病毒引起了对微粒疫苗技术的开发的兴趣。我们 开发一种创新的方法，通过逐层（LbL）制造生产颗粒疫苗， 合成微粒（MP）。我们已经表明，LbL-MP装载有来自呼吸道合胞体的表位， 病毒（RSV）或恶性疟原虫（Pf）引起有效和平衡的体液和细胞免疫应答 保护免疫宿主免受病原体攻击后的感染和疾病。LbL-MP 疫苗在水悬浮液中以低剂量（1-10 μ g）给药时具有免疫原性和有效性 不含任何外源性佐剂，因此提供了优于竞争疫苗技术的安全性优势。在 在第一阶段的可行性研究中，我们将通过将MP装载到 微针阵列设计用于将有效载荷直接输送到皮肤中，皮肤是一个丰富的免疫储存库， 细胞，包括朗格汉斯细胞和真皮树突细胞。我们将使用两个LbL-MP，我们已经 广泛表征的，一个装载有来自RSV的两个表位，一个装载有来自Pf的三个表位， 人类疟疾的病原体。每个MP将被加载到微针贴片使用我们的标准 包含海藻糖的贴剂制剂用于疫苗稳定性。将监测MP加载的效率 通过将贴片溶解在盐水缓冲液中并进行氨基酸分析以确定疫苗抗原的量 回收的疫苗抗原，而回收的疫苗抗原的完整性将通过ELISA使用单克隆抗体进行监测。 靶抗原特异性抗体。我们将使用小鼠模型来比较MP的免疫原性 在微针贴剂中配制，与在水性溶液中使用相同MP的肠胃外免疫相比 无佐剂悬浮液。在RSV模型中，我们将通过以下方式检查微针贴片的功效： 用活RSV攻击免疫的小鼠，并在攻击后监测肺病毒负荷。我们预计 MP的微针贴片递送将导致更大的免疫效力（例如，较高抗体滴度 引发的或所需的较低剂量），更有利的免疫表型（例如，同种型转换和Th 1- 偏倚的细胞应答）和上级疗效（攻毒后病毒负荷降低）， 与肠胃外注射相同的LbL-MP相比。LbL-MP疫苗平台的这一进展 应导致改进的疫苗效力，更容易管理，更大的病人接受，由于其 微创性。

Abstract The ability of the immune system to recognize and initiate an immune response to foreign particles such as bacteria and viruses has generated interest in development of particulate vaccine technologies. We are developing an innovative approach to produce particulate vaccines via layer-by-layer (LbL) fabrication of synthetic microparticles (MP). We have shown that LbL-MP loaded with epitopes from respiratory syncytial virus (RSV) or Plasmodium falciparum (Pf) elicit potent and balanced humoral and cellular immune responses that protect the immunized host from infection and disease following pathogen challenge. The LbL-MP vaccines are immunogenic and efficacious when administered at low doses (1-10 ¿g) in aqueous suspension without any exogenous adjuvant, thus offering safety advantages over competing vaccine technologies. In this Phase I feasibility study, we will improve the LbL-MP technology by loading the MP into microneedle arrays designed to deliver the payload directly into the skin, a rich reservoir of immune cells including Langerhans cells and dermal dendritic cells. We will use two LbL-MP that we have already extensively characterized, one loaded with two epitopes from RSV and one loaded with three epitopes from Pf, the causative agent of human malaria. Each MP will be loaded into microneedle patches using our standard patch formulation which includes trehalose for vaccine stability. The efficiency of MP loading will be monitored by dissolution of the patch in saline buffer and amino acid analysis to determine the amount of vaccine antigen recovered, while integrity of the recovered vaccine antigen will be monitored by ELISA using monoclonal antibody specific for the target antigen. We will use mouse models to compare the immunogenicity of MP formulated in microneedle patches against that of parenteral immunization with the same MP in aqueous suspension without adjuvant. In the RSV model, we will examine efficacy of the microneedle patches by challenging the immunized mice with live RSV and monitoring lung viral burden post-challenge. We anticipate that microneedle patch delivery of MP will result in greater immune potency (e.g., higher antibody titer elicited or lower dose required), more favorable immune phenotype (e.g., isotype switching and Th1- biased cellular response), and superior efficacy (reduction in viral burden post-challenge) when compared to parenteral injection of the same LbL-MP. This advancement in the LbL-MP vaccine platform should lead to improved vaccine potency, easier administration, and greater patient acceptance due to its minimally invasive nature.