Development of a Bacteriophage Vaccine Vector System

描述(申请人提供)：本建议中研究的目标是进一步开发一种能够快速生产、纯化和应用表达甲型流感病毒样颗粒(Flu-VLP)的噬菌体载体系统(BVS)平台的方法。这一目标是有必要的，因为BVS是一种独特的载体，可以在细菌中快速生产，提供与基于RNA的载体相关的安全性，并具有作为体外生产亚单位疫苗的瞬时表达系统和作为可直接在体内给药的疫苗载体的潜力。BVS来源于双链RNA(DsRNA)囊状病毒噬菌体Phi-8。为了使这种噬菌体用于疫苗应用，PI和他的同事们对Phi-8的基因组片段--S(Gss)和基因组片段-M(Gsm)进行了基因改造，并在大肠杆菌宿主菌株中启动了BVS重组的Phi-8衣壳。在初步研究中，纯化的BVS衣壳在体外哺乳动物细胞中表达报告蛋白和疫苗抗原。这一背景经验和证据支持进一步开发BVS衣壳作为应急准备/生物防御应用平台的提议。PI证明，这一系统的成功开发将创造一种公共卫生工具和生物防御对策，它在正常情况下能够生产常规亚单位疫苗；然而，有人建议，在可能存在这种风险的紧急事件期间，BVS衣壳可以作为疫苗载体部署，从序列鉴定到疫苗释放在不到一个月的时间内。通过这种方式，该系统将细菌制造的优势和基于非复制RNA的核酸疫苗载体的实用性和安全性与快速反应时间表的需要结合在一起，以确保针对H5N1等新出现的流感病毒血清型的广泛应急准备。将在下面拟议的研究中检验的中心假设是，BVS衣壳有可能同时作为疫苗生产商和疫苗载体。为了解决这一假设，PI建议(I)开发一种快速生产纯化的、具有生物活性的表达Flu-VLP的BVS衣壳的方法，以及(Ii)在实验室动物中鉴定表达Flu-VLP的Flu-VLP和BVS衣壳的免疫原性。预计成功完成这些目标将建立一个有效的BVS衣壳和流感-VLP生产系统，该系统可以适应季节性流感和新出现的流感亚型的生产，并比其他疫苗模式部署得更快。此外，拟议的研究将提供与该系统的适应密切相关的基本信息，使其成为快速开发和部署公共卫生和生物防御疫苗的操作平台。与公共卫生的相关性：禽流感H5N1亚型可能引发灾难性的人类大流行，以及与其他生物制剂的生物恐怖主义威胁增加，促使人们重新认识到迫切需要制定快速开发、制造和分发疫苗的战略。这项提案中研究的目标是提供一个能够快速生产、提纯和管理疫苗的制造平台。此外，这里的研究将产生在控制季节性和大流行性流感方面立即有效的疫苗成分和制造程序。此外，预计该系统将成为快速开发和部署各种公共卫生和生物防御疫苗的业务平台。

DESCRIPTION (provided by applicant): The goal of the studies in this proposal is to further develop a process that enables rapid production, purification and application of a bacteriophage vector system (BVS) platform expressing influenza A virus-like particles (Flu-VLPs). This goal is warranted as BVS is a unique vector that can be rapidly produced in bacteria, affords the safety profile associated with RNA-based vectors and has the potential for dual application as a transient expression system to produce subunit vaccines in vitro and as a vaccine vector directly administrable to humans or animals in vivo. BVS is derived from the double- stranded RNA (dsRNA) Cystovirus bacteriophage phi-8. To render this bacteriophage for vaccine applications the PI and coworkers have genetically altered genomic segment-S (gsS) and genomic segment-M (gsM) of phi-8, and launched the BVS recombinant phi-8 capsids in Escherichia coli host strains. In preliminary studies purified BVS capsids have been shown to express reporter proteins and vaccine antigens in mammalian cells in vitro. This background experience and evidence supports the proposal to further develop BVS capsids as a platform for emergency preparedness/biodefense applications. The PI provides justification that successful development of this system will create a public health tool and biodefense countermeasure, which under normal circumstances is capable of producing conventional subunit vaccines; yet, it is proposed that BVS capsids can be deployed as a vaccine vector from sequence identification through to vaccine release in under a month during emergency events where such a risk may be warranted. In this manner, this system integrates the advantages of manufacturing in bacteria and the utility and safety of non-replicating RNA-based nucleic acid vaccine vectors with the need for rapid response timelines to ensure broad emergency preparedness against emerging influenza virus serotypes such as H5N1. The central hypothesis that will be tested in the proposed studies below is that BVS capsids have the potential to serve as both a vaccine producer and vaccine vector. To address this hypothesis the PI proposes to (i) To develop a procedure to rapidly produce purified and bioactive BVS capsids that express Flu-VLPs and (ii) To characterize the immunogenicity of Flu-VLPs and BVS capsids expressing Flu-VLPs in laboratory animals. It is envisaged that successful completion of these aims will create an effective BVS capsid and Flu-VLP production system that can be adapted for manufacturing of both seasonal influenza and emerging influenza subtypes, and deployed significantly more rapidly than other vaccine modes. In addition, the proposed studies will provide fundamental information germane to the adaptation of this system to become an operational platform for rapid development and deployment of public health and biodefense vaccines. PUBLIC HEALTH RELEVANCE: A renewed awareness of the urgent need to devise strategies for the rapid development, manufacture and distribution of vaccines was invoked by the threat of avian influenza subtype H5N1 to spawn a catastrophic human pandemic and the increased threat of bioterrorism with other biological agents. The goal of the studies in this proposal is to provide a manufacturing platform that enables rapid production, purification and administration of vaccines. Furthermore, the studies herein will generate vaccine components and manufacturing procedures that will have an immediate utility in the control of both seasonal and pandemic influenza. Additionally, it is envisaged that this system will serve as an operational platform for rapid development and deployment of a broad array of public health and biodefense vaccines.