Lymph Node Follicle‐Targeting STING Agonist Nanoshells Enable Single‐Shot M2e Vaccination for Broad and Durable Influenza Protection

Abstract

The highly conserved matrix protein 2 ectodomain (M2e) of influenza viruses presents a compelling vaccine antigen candidate for stemming the pandemic threat of the mutation-prone pathogen, yet the low immunogenicity of the diminutive M2e peptide renders vaccine development challenging. A highly potent M2e nanoshell vaccine that confers broad and durable influenza protectivity under a single vaccination is shown. Prepared via asymmetric ionic stabilization for nanoscopic curvature formation, polymeric nanoshells co-encapsulating high densities of M2e peptides and stimulator of interferon genes (STING) agonists are prepared. Robust and long-lasting protectivity against heterotypic influenza viruses is achieved with a single administration of the M2e nanoshells in mice. Mechanistically, molecular adjuvancy by the STING agonist and nanoshell-mediated prolongation of M2e antigen exposure in the lymph node follicles synergistically contribute to the heightened anti-M2e humoral responses. STING agonist-triggered T cell helper functions and extended residence of M2e peptides in the follicular dendritic cell network provide a favorable microenvironment that induces Th1-biased antibody production against the diminutive antigen. These findings highlight a versatile nanoparticulate design that leverages innate immune pathways for enhancing the immunogenicity of weak immunogens. The single-shot nanovaccine further provides a translationally viable platform for pandemic preparedness.

Single-Component Multilayered Self-Assembling Protein Nanoparticles Displaying Extracellular Domains of Matrix Protein 2 as a Pan-influenza A Vaccine

“…Importantly, M2ex3 I3-01v9a and BG505 Env I3-01v9 SApNPs showed significantly longer follicular retention than the SARS-CoV-2 spike-presenting I3-01v9 SApNP (∼8 weeks vs ∼2 weeks), , suggesting a correlation between SApNP retention and antigen thermostability (a T m value of 65 °C or greater for M2ex3 and BG505 Env vs 48 °C for SARS-CoV-2 spike). Of note, a shorter follicular retention (<2 weeks) was reported for a recently developed M2e vaccine, in which M2e peptides were encapsulated within a poly­( d,l -lactide- co -glycolide), or PLGA, polymer matrix, possibly due to polymer degradation and/or M2e peptide release. Next, we examined the accumulation and retention patterns of these three M2ex3 immunogens at 2 and 5 weeks using a prime-boost regimen (injected into 4 footpads at weeks 0 and 3, 10 μg/footpad) (Figure G).…”

“…In our current study, the trafficking and retention of M2ex3 I3-01v9a SApNP were quantified via immunostaining of tandem M2e antigens displayed on SApNPs using an M2e-specific antibody (mAb148) at individual time points. In comparison, a previous study conjugated fluorescent dyes to the M2e peptide, which may have influenced the experimental readouts due to signal degradation in vivo over time.…”

“…In this study, a tandem M2e antigen derived from IAVs of human, avian, and swine hosts was presented on our recently developed SApNP platforms. − The multivalent display allows M2e epitopes to directly engage and cross-link BCRs to stimulate a robust B cell response. In recent studies, M2e peptides were encapsulated within a liposome or a polymer matrix − as nanovaccines against influenza, for which the degradation rates of the biomaterials used, the release kinetics of the encapsulated M2e peptides, and how M2e peptides interact with B cells in vivo can be difficult to quantify. Furthermore, different methods have been used to analyze intralymph node transport for M2e-based nanovaccines.…”

“…Although M2e is small (∼23 aa) and poorly immunogenic, it can be conjugated to large molecular carriers to elicit antibody responses that effectively reduce viral replication . Unlike HA and NA, M2e-specific antibodies protect via FcγR-dependent mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), rather than direct virus neutralization. ,− Various carriers have been used to increase the immunogenicity of M2e vaccines, including hepatitis B core protein (HBc), tobacco mosaic virus (TMV) coat protein, keyhole limpet hemocyanin (KLH), rotavirus NSP4, GCN4, bacterial flagellin, liposomes, polymers, − and gold nanoparticles (NPs). , Early human trials confirmed the immunogenicity and tolerance of M2e vaccines but also revealed several weaknesses. For example, an adjuvanted M2e-HBc fusion vaccine induced a short-lived anti-M2e antibody response, and an M2e-flagellin fusion vaccine caused undesirable side effects at higher doses.…”

Single-component multilayered self-assembling protein nanoparticles displaying extracellular domains of matrix protein 2 as a pan-influenza A vaccine

“…Unlike HA and NA, M2e-specific antibodies protect via FcγR-dependent mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), rather than direct neutralization ( 39, 41–43 ). Various carriers have been used to increase the immunogenicity of M2e vaccines, including hepatitis B core protein (HBc) ( 41 ), tobacco mosaic virus (TMV) coat protein ( 44 ), keyhole limpet hemocyanin (KLH) ( 40 ), rotavirus NSP4 ( 45 ), GCN4 ( 46 ), bacterial flagellin ( 47 ), liposomes ( 48 ), polymers ( 49–51 ), and gold nanoparticles (NPs) ( 52, 53 ). Early human trials confirmed the immunogenicity and tolerance of M2e vaccines but also revealed several weaknesses.…”