Rapid development of replication-controlled vaccinia virus vectors for vaccines and therapeutics with single or double safety features

项目摘要 该应用程序响应PA-15-313，促进疫苗安全的研究（R21）。痘苗病毒 （VACV）被用作天花的活疫苗，天花是由天花病毒引起的疾病。VACV也被 成功地用作开发有效的人类和动物疫苗的活病毒载体，以及 免疫疗法和溶瘤病毒疗法。然而，VACV可在患有以下疾病的个体中引起并发症： 病症，如特应性皮炎、心脏病和免疫抑制。因此，个人与 这些条件或与具有这些条件的接触者的接触禁忌用于接种复制型 VACV载体。我们最近生成了具有内置安全机制的VACV载体，该机制仅在 存在四环素（TC）抗生素（复制诱导型VACV）。在这个系统中，VACV基因是必需的， 用于复制的基因（例如D 6 R）由TC使用泰特操纵子的元件诱导表达。给药时 作为疫苗（在不存在抗生素的情况下），载体不复制但保留其免疫原性，以及 因此对人类使用更安全。方便地，载体可以在细胞培养物中以高滴度在培养基中增殖。 这与其他基于复制缺陷型VACV的载体如MVA不同。我们还开发 VACV载体在不存在抗生素的情况下正常复制，但在存在抗生素的情况下复制缺陷。 复制抑制型VACV（Replication-Repressive VACV）当作为疫苗施用时（在不存在抗生素的情况下）， 载体与传统VACV载体一样具有复制能力（因此具有高度免疫原性）， 任何不良反应的治疗都像TC抗生素治疗一样简单。我们可以进一步加强 通过将必需基因（D 6 R）的表达与干扰素-γ联系起来的故障安全功能， (IFN-γ），通过内部核糖体进入位点（IRES）。我们发现VACV表达IFN-γ， 组成性地或诱导性地，导致体内VACV的完全衰减，即使当以非常低的水平表达时也是如此。 程度.令人惊讶的是，该病毒仍然能够在体外生长到野生型水平。因此，这两种策略都可以 组合以开发可诱导或可抑制地表达D 6 R的复制缺陷型VACV（在其天然表达下）。 启动子）和IFN-γ（在小鼠IRES下）。所得到的载体应该能够生长到高滴度， 在存在TC（诱导型载体）或不存在TC（抑制型载体）的情况下， 向量）。由于D 6 R的表达将与IFN-γ的表达相关联，因此任何潜在的具有复制能力的IFN-γ表达都可能与IFN-γ的表达相关联。 可能在体外繁殖或体内给药期间产生的VACV将是无复制能力的 在体内由于IFN-γ的伴随表达。在目标1中，我们将确定复制诱导型 和复制抑制型VACV载体的体内试验。在目标2中，我们将开发， 表征并确定具有故障安全功能的复制缺陷VACV载体的安全性。我们计划 利用VACV疫苗平台快速开发寨卡等传染病的安全疫苗 病毒、免疫疗法（例如，个性化癌症疫苗）和溶瘤病毒疗法。

PROJECT SUMMARY This application is responsive to PA-15-313, Research to Advance Vaccine Safety (R21). Vaccinia virus (VACV) was used as a live vaccine for smallpox, a disease caused by variola virus. VACV has also been successfully used as a live viral vector for the development of effective human and animal vaccines, as well as immunotherapies and oncolytic virotherapies. However, VACV can cause complications in individuals with conditions such as atopic dermatitis, cardiac disease, and immunosuppression. Consequently, individuals with such conditions or with contacts that have these conditions are contraindicated for vaccination with replicating VACV vectors. We recently generated VACV vectors with a built-in safety mechanism that replicate only in the presence of tetracycline (TC) antibiotics (replication-inducible VACVs). In this system, a VACV gene essential for replication (eg, D6R) is inducibly expressed by TCs using elements of the tet operon. When administered as a vaccine (in the absence of antibiotics), the vector does not replicate but retains its immunogenicity, and therefore is safer for human use. Conveniently, the vector can be propagated in cell culture at high titers in the presence of TCs, unlike other replication-defective VACV-based vectors such as MVA. We also developed VACV vectors that replicate normally in the absence of antibiotics, but are replication-defective in the presence of TCs (replication-repressible VACVs). When administered as a vaccine (in the absence of antibiotics), the vector is replication competent like traditional VACV vectors (and therefore highly immunogenic), and treatment of any adverse reactions would be as simple as TC antibiotic therapy. We can further enhance the safety of our vectors by a fail-safe feature that links expression of the essential gene (D6R) with interferon-γ (IFN-γ), via an internal ribosome entry site (IRES). We showed that expression of IFN-γ by VACV, either constitutively or inducibly, leads to complete attenuation of VACV in vivo, even when expressed at very low levels. Surprisingly, the virus is still able to grow to wild-type levels in vitro. Thus, both strategies can be combined to develop replication-defective VACVs inducibly or repressibly expressing D6R (under its natural promoter) and IFN-γ (under a small murine IRES). The resulting vectors should be able to grow to high titers in vitro (thus allowing propagation) in the presence of TCs (inducible vectors) or absence of TCs (repressible vectors). Since expression of D6R will be linked to IFN-γ expression, any potential replication-competent VACV that may originate during in vitro propagation or in vivo administration would be replication-incompetent in vivo due to concomitant expression of IFN-γ. In Aim 1 we will determine the safety of replication-inducible and replication-repressible VACV vectors in vivo using immunodeficient SCID mice. In Aim 2, we will develop, characterize, and determine the safety of replication-defective VACV vectors with a fail-safe feature. We plan to use this VACV vaccine platform for rapid development of safe vaccines for infectious diseases such as Zika virus, immunotherapies (eg, personalized cancer vaccines), and oncolytic virotherapies.