Alu dsRNAs as adjuvants for influenza vaccines

尽管医学取得了进步，但传染病仍然是全世界死亡的主要原因之一。疫苗是预防传染病最有效的工具之一。免疫的产生有两个组成部分。首先是T和B细胞受体识别的抗原的独特化学结构，允许适应性免疫系统细胞的克隆扩增和分化。第二种是“危险信号”，它通过模式识别受体（PRRs）刺激先天免疫系统，这种受体识别真核细胞中缺失的保守病原体相关分子模式（PAMPS）。一般来说，最有效的病毒疫苗是减毒活病毒。灭活病毒疫苗也具有免疫力，但提供的保护较少，需要多次免疫。亚单位疫苗，如病毒蛋白抗原，由于免疫原性差，用处不大。然而，亚单位疫苗在成本、生产的统一性、稳定性、控制产生的免疫类型的能力以及更优越的安全性方面有明显的优势，但亚单位疫苗需要有效的佐剂来产生强免疫力。临床应用的例子包括无机盐（明矾）、水包油乳剂和无机盐- tlr4激动剂组合。其他作为佐剂的PRR激动剂正处于临床试验的不同阶段。使用PAMPS作为疫苗佐剂的一个限制是毒性。一般来说，通过PRRs发出的信号激活了两个主要的转录级联，干扰素调节因子（IRF）和核因子κ B （NF-kB）信号，最终表达编码蛋白的基因，从而抑制病原体复制并强烈激活免疫系统。最近的证据表明，通过将PAMP （CpG）与NF-kB信号通路抑制剂结合，可能保留PAMP的佐剂活性并减轻毒性。这一策略的局限性源于固有的基于机制的系统性抑制NF-kB信号的毒性。自身免疫性疾病会产生一种“危险信号”；被称为“干扰素特征”，干扰素诱导的许多基因在明显没有感染的患者中升高。我们发现多发性硬化症（MS）中的“干扰素特征”是由内源性双链Alu元件（Alu dsRNA）水平显著升高引起的，并表明Alu dsRNA刺激强烈的IRF和NF-kB激活。Alu dsRNAs与纳米颗粒（AluJb/NP）复合物在体内具有强大的抗肿瘤活性，表明其具有免疫刺激活性。我们发现Alu dsRNA元件是IRF和NF-kB信号的强激活因子，以及那些仅是IRF信号而不是NF-kB信号的强激活因子。因此，我们要验证的假设是，我们可以设计和递送RNA佐剂，模拟内源性Alu元素，优先激活IRF反应，最小程度地刺激NF-kB信号，以增强免疫反应，提高流感亚单位疫苗的耐受性。

Despite medical progress, infectious diseases remain one of the leading causes of death worldwide. Vaccines are one of the most effective tools to prevent infectious diseases. Generation of immunity has two components. The first is the unique chemical structure of the antigen recognized by T and B cell receptors allowing clonal expansion and differentiation of cells of the adaptive immune system. The second is a ‘danger signal’ that stimulates the innate immune system via pattern recognition receptors (PRRs) that recognize conserved pathogen-associated molecular patterns (PAMPS) absent from eukaryotic cells. In general, the most effective viral vaccines are live attenuated viruses. Inactivated virus vaccines also confer immunity but offer less protection and require multiple immunizations. Subunit vaccines, such as viral protein antigens, are less useful due to poor immunogenicity. However, there are clear advantages to subunit vaccines in terms of cost, uniformity of production, stability, ability to control the type of immunity that is generated, and far superior safety profiles, but subunit vaccines require effective adjuvants to generate strong immunity. Examples in clinical use include mineral salts (Alum), oil-in-water emulsions, and a mineral salt-TLR4 agonist combination. Other PRR agonists as components of adjuvants are in various stages of clinical trials. A limitation to use of PAMPS as vaccine adjuvants is toxicity. In general terms, signaling through PRRs activates two major transcriptional cascades, interferon regulatory factor (IRF) and nuclear factor-kappa B (NF-kB) signaling culminating in expression of genes encoding proteins to both inhibit pathogen replication and strongly activate the immune system. Recent evidence argues that it may be possible to retain adjuvant activity of a PAMP and alleviate toxicity by combining a PAMP (CpG) with an inhibitor of the NF-kB signaling path. A limitation to this strategy derives from inherent serious mechanism-based toxicity of systemic inhibition of NF-kB signaling. Autoimmune disease generates a kind of ‘danger signal’; referred to as an ‘interferon signature’ in which many genes induced by interferons are elevated in patients in apparent absence of infection. We found that the ‘interferon signature’ in multiple sclerosis (MS) results from markedly increased levels of endogenous double-stranded Alu elements (Alu dsRNA) and showed that Alu dsRNAs stimulate strong IRF and NF-kB activation. Alu dsRNAs complexed with nanoparticles (AluJb/NP) have potent anti-tumor activity, in vivo, demonstrating their immunostimulatory activity. We identified Alu dsRNA elements that are strong activators of both IRF and NF-kB signaling and those that are strong activators of only IRF signaling but not NF-kB. Thus, our hypothesis to test is that we can design and deliver RNA adjuvants that mimic endogenous Alu elements that preferentially activate IRF responses and minimally agonize NF-kB signaling to enhance immune responses and improve tolerability of influenza subunit vaccines.