Combination Adjuvants to Program Durable Immunity to Respiratory Viral and Fungal Pathogens

摘要：呼吸道病毒和真菌感染是全球主要的公共卫生问题。 除流感病毒外，没有获得许可的针对病毒或真菌的疫苗。人们普遍认为 诱导T细胞记忆对于防御呼吸道中的病毒和真菌至关重要。我们和其他人 已经表明，诱导组织驻留记忆(TRM)CD8和CD4T细胞和系统性迁移记忆 CD4T细胞对于预防甲型流感病毒(IAV)和吸入性真菌分别是必不可少的。然而， TRM细胞和系统记忆CD4T细胞都会发生磨损，导致短暂的免疫，这是 对于Tc1/TH1细胞尤其如此。因此，诱导持久的T细胞免疫对 疫苗专家。与TH1细胞相比，TH17细胞显示出干细胞特性、耐久性和 功能可塑性。我们建议量身定做组合佐剂以利用T17编程并诱导耐用性 保护肺TRM细胞和移行记忆CD4T细胞免受病毒和真菌的侵袭。 我们发现，当与TLR4激动剂葡萄糖吡喃葡萄糖联合使用时，纳米乳佐剂Adjuplex 脂质A(GLA)，在肺内激发抗原特异性的CD8和CD4T细胞反应：(I)持久和 多方面(Tc1/TC17/TH1/TH17)，和(Ii)提供对IAV的异型免疫，持续400天。 我们进一步发现，将这些佐剂与真菌CLR配体Blstomyces endoganase 2(Bl-Eng2； Dectin-2激动剂)和b-葡聚糖颗粒(Dectin-1激动剂)增强抗病毒TC17/TH17/Tc1/TH1并诱导 移行记忆T细胞，可预防真菌性肺炎。通过单细胞RNAseq，我们发现我们的 联合佐剂诱导表达ICOS的记忆、抗病毒和抗真菌CD8和CD4T细胞群 (可诱导T细胞共刺激因子)，转录因子c-Maf，以及促进组织驻留的转录组， 干细胞特性和非致病性T17编程。环二核苷酸也促进T17编程 肺部。这一点，我们假设，编程干细胞样的，功能可塑性的，非致病的TC17/TH17 使用我们的组合佐剂(参与TLR-4、Dectin-1/2和STING通路)的记忆细胞将促进 对肺部的病毒和真菌病原体具有持久的保护性免疫。我们的具体目标将考验三个 假设：目标1：联合佐剂可激发TC17/TH17干细胞样功能可塑性TRM或 系统性迁移记忆将产生对呼吸道病毒和真菌病原体的持久免疫力；目标2： TC17/TH17记忆的功能可塑性对病毒和真菌的保护性免疫很重要；目标3： ICOS/c-maf通路对于建立和/或维持疫苗诱导的持久保护性是不可或缺的 对呼吸道病毒和真菌感染具有免疫力。拟议的工作意义重大，影响很大，因为 它有可能创造一种易于处理的佐剂系统/工具包，以促进疫苗的配制：(1) 以粘膜或非肠道给药为目标的；(Ii)旨在诱导TRM或系统T细胞记忆的；以及(Iii) 量身定做，以诱导CD8和CD4T细胞，以保护免受各种病原体，如病毒和真菌。

ABSTRACT: Respiratory infections with viruses and fungi constitute major public health problems globally. Except for influenza virus, there are no licensed vaccines against viruses or fungi. It is generally agreed that induction of T-cell memory is critical for defense against viruses and fungi in the respiratory tract. We and others have shown that induction of tissue-resident memory (TRM) CD8 and CD4 T cells and systemic migratory memory CD4 T cells are essential for protection against influenza A virus (IAV) and inhaled fungi, respectively. However, both TRM cells and systemic memory CD4 T cells undergo attrition, leading to short-lived immunity, which is especially true for TC1/TH1 cells. Therefore, induction of durable T-cell immunity poses major challenges for vaccinologists. As compared to TH1 cells, TH17 cells display sought-after attributes of stem-ness, durability and functional plasticity. We propose to tailor combination adjuvants to harness T17 programming and induce durable and protective lung TRM cells and migratory memory CD4 T cells against viruses and fungi. We find that Adjuplex, a nano-emulsion adjuvant, when combined with the TLR4 agonist glucopyranosyl lipid A (GLA), evokes antigen-specific CD8 and CD4 T-cell responses in the lung that are: (i) durable and multifaceted (TC1/TC17/TH1/TH17), and (ii) confer heterosubtypic immunity against IAV that persists >400 days. We further find that combining those adjuvants with fungal CLR ligands Blastomyces endoglucanase 2 (Bl-Eng2; Dectin-2 agonist) and b-glucan particles (Dectin-1 agonist) augments antiviral TC17/TH17/TC1/TH1 and elicits migratory memory T cells that protect against fungal pneumonia. By single-cell RNAseq, we found that our combined adjuvants induce memory antiviral and antifungal CD8 and CD4 T-cell clusters that express ICOS (Inducible T Cell Co-stimulator), the transcription factor c-Maf, and a transcriptome that fosters tissue residency, stem cell-ness and non-pathogenic T17 programming. Cyclic dinucleotides also promote T17 programming in lungs. This, we postulate that programming stem cell-like, functionally plastic, non-pathogenic TC17/TH17 memory cells with our combination adjuvants (that engage TLR-4, Dectin-1/2 and STING pathways) will foster durable protective immunity to viral and fungal pathogens in the lung. Our specific aims will test three hypotheses: Aim 1: Combination adjuvants that evoke TC17/TH17 stem cell-like functionally-plastic TRM or systemic migratory memory will engender durable immunity to respiratory viral and fungal pathogens; Aim 2: Functional plasticity of TC17/TH17 memory is important for protective immunity to viruses and fungi; Aim 3: The ICOS/c-Maf pathway is integral to establishment and/or maintenance of durable vaccine-induced protective immunity to respiratory viral and fungal infections. The proposed work is significant and of high impact because it has the potential to create a tractable adjuvant system/tool kit that will advance the formulation of vaccines: (i) targeted for mucosal or parenteral administration; (ii) designed to induce TRM or systemic T-cell memory; and (iii) tailored to elicit CD8 and CD4 T cells that protect against diverse pathogens such as viruses and fungi.