Research Article

Pulmonary surfactant–biomimetic nanoparticles potentiate heterosubtypic influenza immunity

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Science  21 Feb 2020:
Vol. 367, Issue 6480, eaau0810
DOI: 10.1126/science.aau0810

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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

Structured Abstract


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.


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.


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.


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.

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