Pitching cGAMP as a vaccine strategy One strategy to address the variable effectiveness of many influenza vaccines is to induce antiviral resident memory T cells, which can mediate cross-protection against multiple substrains (heterosubtypic immunity). Unfortunately, such vaccines typically use attenuated active viruses, which may be unsafe for certain populations. Wang et al. report a vaccine using an inactivated virus that effectively induced heterosubtypic immunity in both mice and ferrets (see the Perspective by Herold and Sander). They coadministered the virus with 2′,3′-cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), a potent activator of the innate immune system, encapsulated in pulmonary surfactant–biomimetic liposomes. This adjuvant was taken up by alveolar epithelial cells, whose activation resulted in effective antiviral T cell and humoral immune responses without accompanying immunopathology. Science, this issue p. eaau0810; see also p. 852 Biomimetic liposomes containing an innate immune adjuvant promote effective immunity using an inactivated influenza vaccine. INTRODUCTION Current influenza vaccines must be refreshed annually to address constant mutations of viral hemagglutinin (HA) and neuraminidase (NA) genes because the vaccines induce primarily neutralizing antibodies against these surface antigens. Even with annual updates, there have been years in which influenza vaccines were ineffective because of mismatched HA and/or NA antigenicity between the vaccine viral strains and strains in circulation. Thus, resources have been poured into developing “universal” influenza vaccines that can protect the population from divergent influenza viruses. However, none of these have passed human clinical trials thus far. Broad immunity can be evoked by natural viral infections or live vector–engineered and attenuated influenza vaccines, which all induce lung resident memory T cells (TRM cells) apart from humoral immunity. However, a delicate balance must be struck between safety and immunogenicity of these “replicating” vaccines. Moreover, these vaccines are suitable for only some populations. Thus, safe and potent mucosal adjuvants are urgently needed as part of nonreplicating vaccines in order to stimulate lung TRM cells and engender strong heterosubtypic immunity. RATIONALE Type I interferons (IFN-Is) are the chief immune mediators for protective immunity against viral infections and can be vigorously induced by influenza viral infection of alveolar epithelial cells (AECs) as well as immune cells. Thus, the activation of stimulator of interferon genes (STING) in these two cell types may recapitulate the immune responses provoked by viral infection or replicating vaccines. However, delivery of STING agonists into the cytosol of AECs without breaching the integrity of the pulmonary surfactant (PS) layer remains a substantial challenge because the PS layer forms a strong barrier to prevent nanoparticles and hydrophilic molecules from accessing them. To address this challenge, we encapsulated 2′,3′-cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), a natural and potent STING agonist, with PS-biomimetic liposomes (PS-GAMP) in an attempt to increase the breadth of nonreplicating influenza vaccines toward universality. RESULTS In mice, PS-GAMP entered alveolar macrophages (AMs) in concert with lung-specific surfactant protein–A (SP-A) and SP-D because of its resemblance to PS. Its cargo was released into the cytosol followed by a flux from AMs into AECs through gap junctions. Disguised as “self,” PS-GAMP escaped immune surveillance after intranasal immunization, activating the STING pathway in both AMs and AECs without breaching PS and alveolar epithelial barriers. Through this mechanism, PS-GAMP averted viral infection–provoked immunopathology while robustly augmenting the recruitment and differentiation of CD11b+ dendritic cells (DCs) and CD8+ T cell and humoral responses of influenza vaccines such as those induced by viral infection in terms of both timing and magnitude. The adjuvant in conjunction with inactivated H1N1 vaccine generated wide-spectrum cross-protection against distant H1N1 and heterosubtypic H3N2, H5N1, and H7N9 viruses as early as 2 days after a single immunization. This cross-protection lasted for at least 6 months, concurrent with durable lung CD8+ TRM cells in mice. The effectiveness of this vaccine approach was also demonstrated in a U.S. Food and Drug Administration–approved ferret model. PS-GAMP–mediated adjuvanticity was abrogated in vivo when AECs were deficient in Sting or when mice were administered gap junction inhibitors. CONCLUSION Nonreplicating influenza vaccines or conventional adjuvants primarily activate immune cells, but this approach appears to be inadequate to induce lung TRM cells, a key element of heterosubtypic immunity. By contrast, PS-GAMP activated immune cells as well as AECs without breaching PS and AEC barriers, effectively averting exaggerated inflammation in the lung. STING activation in both immune cells and AECs resulted in a broad spectrum of immune protection against heterosubtypic influenza viruses. The study sheds light on the pivotal role AECs play in generating broad cross-protection against various influenza viruses. Thus, PS-GAMP is a promising mucosal adjuvant for “universal” influenza vaccines. PS-GAMP–mediated adjuvanticity. In alveoli, PS-GAMP associates with SP-A or SP-D before entering AMs by means of SP-A– or SP-D–mediated endocytosis. cGAMP is subsequently released into the cytosol and fluxes into AECs by way of gap junctions. It then activates STING in these cells, resulting in the vigorous production of type 1 immune mediators. These mediators facilitate the recruitment and differentiation of CD11b+ DCs, which in turn direct robust antiviral CD8+ T cell and humoral immune responses. Current influenza vaccines only confer protection against homologous viruses. We synthesized pulmonary surfactant (PS)–biomimetic liposomes encapsulating 2′,3′-cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), an agonist of the interferon gene inducer STING (stimulator of interferon genes). The adjuvant (PS-GAMP) vigorously augmented influenza vaccine–induced humoral and CD8+ T cell immune responses in mice by simulating the early phase of viral infection without concomitant excess inflammation. Two days after intranasal immunization with PS-GAMP–adjuvanted H1N1 vaccine, strong cross-protection was elicited against distant H1N1 and heterosubtypic H3N2, H5N1, and H7N9 viruses for at least 6 months while maintaining lung-resident memory CD8+ T cells. Adjuvanticity was then validated in ferrets. When alveolar epithelial cells (AECs) lacked Sting or gap junctions were blocked, PS-GAMP–mediated adjuvanticity was substantially abrogated in vivo. Thus, AECs play a pivotal role in configuring heterosubtypic immunity.

中文翻译：

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肺表面活性剂-仿生纳米粒子增强异亚型流感免疫

将 cGAMP 用作疫苗策略解决许多流感疫苗的可变有效性的一种策略是诱导抗病毒驻留记忆 T 细胞，它可以介导针对多种亚株（异源亚型免疫）的交叉保护。不幸的是，此类疫苗通常使用减毒活性病毒，这对某些人群可能不安全。王等人。报道了一种使用灭活病毒的疫苗，可在小鼠和雪貂中有效诱导异亚型免疫（参见 Herold 和 Sander 的观点）。他们将病毒与 2',3'-环鸟苷单磷酸-腺苷单磷酸 (cGAMP) 共同给药，cGAMP 是一种先天免疫系统的有效激活剂，包裹在肺表面活性剂-仿生脂质体中。这种佐剂被肺泡上皮细胞吸收，其激活导致有效的抗病毒 T 细胞和体液免疫反应，而不伴随免疫病理学。科学，这个问题 p。eaau0810; 另见第 852 含有先天免疫佐剂的仿生脂质体使用灭活流感疫苗促进有效免疫。引言 当前的流感疫苗必须每年更新以解决病毒血凝素 (HA) 和神经氨酸酶 (NA) 基因的不断突变，因为疫苗主要诱导针对这些表面抗原的中和抗体。即使每年更新一次，由于疫苗病毒株和流通株之间的 HA 和/或 NA 抗原性不匹配，流感疫苗也有多年无效的情况。因此，已投入资源开发“通用”流感疫苗，以保护人群免受不同流感病毒的侵害。然而，到目前为止，这些都没有通过人体临床试验。天然病毒感染或活载体工程和减毒流感疫苗都可以引起广泛的免疫，除了体液免疫之外，它们都诱导肺常驻记忆 T 细胞（TRM 细胞）。然而，必须在这些“复制”疫苗的安全性和免疫原性之间取得微妙的平衡。此外，这些疫苗仅适用于某些人群。因此，迫切需要安全有效的粘膜佐剂作为非复制疫苗的一部分，以刺激肺 TRM 细胞并产生强大的异亚型免疫。基本原理 I 型干扰素 (IFN-Is) 是针对病毒感染的保护性免疫的主要免疫介质，可被流感病毒感染肺泡上皮细胞 (AEC) 和免疫细胞强烈诱导。因此，这两种细胞类型中干扰素基因刺激物 (STING) 的激活可能会重现由病毒感染或复制疫苗引起的免疫反应。然而，在不破坏肺表面活性剂 (PS) 层完整性的情况下将 STING 激动剂递送到 AEC 的细胞质中仍然是一个巨大的挑战，因为 PS 层形成了强大的屏障以防止纳米颗粒和亲水性分子进入它们。为了应对这一挑战，我们封装了 2',3'-环鸟苷单磷酸-腺苷单磷酸 (cGAMP)，一种天然且有效的 STING 激动剂，与 PS 仿生脂质体 (PS-GAMP) 一起尝试增加非复制流感疫苗的广度以实现通用性。结果 在小鼠中，PS-GAMP 与肺特异性表面活性蛋白-A (SP-A) 和 SP-D 一起进入肺泡巨噬细胞 (AMs)，因为它与 PS 相似。它的货物被释放到细胞质中，然后通过间隙连接从 AMs 流入 AECs。伪装成“自我”的 PS-GAMP 在鼻内免疫后逃避免疫监视，在不破坏 PS 和肺泡上皮屏障的情况下激活 AM 和 AEC 中的 STING 通路。通过这种机制，PS-GAMP 避免了病毒感染引起的免疫病理学，同时有力地增加了 CD11b+ 树突状细胞 (DC) 和 CD8+ T 细胞的募集和分化以及流感疫苗的体液反应，例如病毒感染在时间和强度方面引起的反应。佐剂与灭活 H1N1 疫苗相结合，早在单次免疫后 2 天就产生了针对远处 H1N1 和异亚型 H3N2、H5N1 和 H7N9 病毒的广谱交叉保护。这种交叉保护持续了至少 6 个月，同时在小鼠中与持久的肺 CD8+ TRM 细胞同时存在。美国食品和药物管理局批准的雪貂模型也证明了这种疫苗方法的有效性。当 AEC 缺乏 Sting 或给小鼠施用间隙连接抑制剂时，PS-GAMP 介导的佐剂性在体内被废除。结论 非复制流感疫苗或常规佐剂主要激活免疫细胞，但这种方法似乎不足以诱导肺 TRM 细胞，这是异亚型免疫的关键因素。相比之下，PS-GAMP 在不破坏 PS 和 AEC 屏障的情况下激活免疫细胞和 AEC，有效地避免了肺部过度炎症。免疫细胞和 AEC 中的 STING 激活导致针对异亚型流感病毒的广谱免疫保护。该研究揭示了 AEC 在产生针对各种流感病毒的广泛交叉保护方面发挥的关键作用。因此，PS-GAMP 是一种很有前途的用于“通用”流感疫苗的粘膜佐剂。PS-GAMP 介导的佐剂性。在肺泡中，PS-GAMP 在通过 SP-A 或 SP-D 介导的内吞作用进入 AMs 之前与 SP-A 或 SP-D 结合。cGAMP 随后被释放到细胞质中，并通过间隙连接流入 AEC。然后激活这些细胞中的 STING，导致 1 型免疫介质的大量产生。这些介质促进 CD11b+ DC 的募集和分化，进而引导强大的抗病毒 CD8+ T 细胞和体液免疫反应。当前的流感疫苗仅提供针对同源病毒的保护。我们合成了肺表面活性剂 (PS)-仿生脂质体，包裹着 2',3'-环磷酸鸟苷-磷酸腺苷 (cGAMP)，干扰素基因诱导剂 STING（干扰素基因刺激剂）的激动剂。佐剂 (PS-GAMP) 通过模拟病毒感染的早期阶段而不会伴随过度炎症，有力地增强了流感疫苗诱导的小鼠体液和 CD8+ T 细胞免疫反应。用 PS-GAMP 佐剂的 H1N1 疫苗鼻内免疫两天后，在维持肺驻留记忆 CD8+ T 细胞的同时，对远处的 H1N1 和异亚型 H3N2、H5N1 和 H7N9 病毒产生了强烈的交叉保护至少 6 个月。然后在雪貂中验证了佐剂性。当肺泡上皮细胞 (AECs) 缺乏 Sting 或间隙连接被阻断时，PS-GAMP 介导的佐剂性在体内基本消失。因此，AEC 在配置异亚型免疫方面发挥着关键作用。佐剂 (PS-GAMP) 通过模拟病毒感染的早期阶段而不会伴随过度炎症，有力地增强了流感疫苗诱导的小鼠体液和 CD8+ T 细胞免疫反应。用 PS-GAMP 佐剂的 H1N1 疫苗鼻内免疫两天后，在维持肺驻留记忆 CD8+ T 细胞的同时，对远处的 H1N1 和异亚型 H3N2、H5N1 和 H7N9 病毒产生了强烈的交叉保护至少 6 个月。然后在雪貂中验证了佐剂性。当肺泡上皮细胞 (AECs) 缺乏 Sting 或间隙连接被阻断时，PS-GAMP 介导的佐剂性在体内基本消失。因此，AEC 在配置异亚型免疫方面发挥着关键作用。佐剂 (PS-GAMP) 通过模拟病毒感染的早期阶段而不会伴随过度炎症，有力地增强了流感疫苗诱导的小鼠体液和 CD8+ T 细胞免疫反应。用 PS-GAMP 佐剂的 H1N1 疫苗鼻内免疫两天后，在维持肺驻留记忆 CD8+ T 细胞的同时，对远处的 H1N1 和异亚型 H3N2、H5N1 和 H7N9 病毒产生了强烈的交叉保护至少 6 个月。然后在雪貂中验证了佐剂性。当肺泡上皮细胞 (AECs) 缺乏 Sting 或间隙连接被阻断时，PS-GAMP 介导的佐剂性在体内基本消失。因此，AEC 在配置异亚型免疫方面发挥着关键作用。