Alu dsRNAs as adjuvants for influenza vaccines

尽管医学取得了进步，但传染病仍然是全球主要的死亡原因之一。疫苗是预防传染病的最有效工具之一。免疫力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性扩张和分化。第二种是危险信号，它通过模式识别受体(PRRs)刺激天然免疫系统，模式识别受体识别真核细胞中不存在的保守的病原体相关分子模式(PAMP)。一般来说，最有效的病毒疫苗是减毒活病毒。灭活病毒疫苗也提供免疫力，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，由于免疫原性较差，用处较小。然而，亚单位疫苗在成本、生产的一致性、稳定性、控制产生的免疫类型的能力以及远远优越的安全性方面具有明显的优势，但亚单位疫苗需要有效的佐剂才能产生强大的免疫力。临床使用的例子包括矿物盐(明胶)、水包油乳剂和矿物盐-TLR4激动剂组合。其他作为佐剂成分的PRR激动剂正处于不同的临床试验阶段。使用PAMPS作为疫苗佐剂的一个限制是毒性。一般来说，通过PRRs的信号通路激活了两个主要的转录级联反应，即干扰素调节因子(IRF)和核因子-kB(NF-kB)信号通路，最终导致编码蛋白的基因表达，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)与核因子-kB信号通路的抑制剂结合起来，有可能保持PAMP的佐剂活性并减轻毒性。这一策略的局限性来自于系统性抑制NF-kB信号的内在严重的基于机制的毒性。自身免疫性疾病产生一种“危险信号”；被称为“干扰素信号”，在没有感染的情况下，干扰素诱导的许多基因在患者体内升高。我们发现多发性硬化症(MS)患者的“干扰素信号”是由于内源性双链Alu元件(Alu DsRNA)水平显著升高所致，并表明Alu dsRNA可刺激IRF和NF-kB的强烈激活。Alu dsRNAs与纳米粒子(AluJb/NP)复合后，在体内具有很强的抗肿瘤活性，显示了其免疫刺激活性。我们鉴定了Alu dsRNA元件，它们是IRF和NF-kB信号的强激活因子，以及那些只激活IRF信号而不是NF-kB信号的元件。因此，我们的假设是，我们可以设计和提供模拟内源性Alu元件的RNA佐剂，这些佐剂优先激活IRF反应，并最大限度地刺激NF-kB信号，以增强免疫反应和改善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.