Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine.

Despite success in vaccinating populations against SARS-CoV-2, concerns about immunity duration, continued efficacy against emerging variants, protection from infection and transmission, and worldwide vaccine availability remain. Molecular adjuvants targeting pattern recognition receptors (PRRs) on antigen-presenting cells (APCs) could improve and broaden the efficacy and durability of vaccine responses. Native SARS-CoV-2 infection stimulates various PRRs, including toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors. We hypothesized that targeting PRRs using molecular adjuvants on nanoparticles (NPs) along with a stabilized spike protein antigen could stimulate broad and efficient immune responses. Adjuvants targeting TLR4 (MPLA), TLR7/8 (R848), TLR9 (CpG), and RIG-I (PUUC) delivered on degradable polymer NPs were combined with the S1 subunit of spike protein and assessed in vitro with isogeneic mixed lymphocyte reactions (isoMLRs). For in vivo studies, the adjuvant-NPs were combined with stabilized spike protein or spike-conjugated NPs and assessed using a two-dose intranasal or intramuscular vaccination model in mice. Combination adjuvant-NPs simultaneously targeting TLR and RIG-I receptors (MPLA+PUUC, CpG+PUUC, and R848+PUUC) differentially induced T cell proliferation and increased proinflammatory cytokine secretion by APCs in vitro. When delivered intranasally, MPLA+PUUC NPs enhanced CD4+CD44+ activated memory T cell responses against spike protein in the lungs while MPLA NPs increased anti-spike IgA in the bronchoalveolar (BAL) fluid and IgG in the blood. Following intramuscular delivery, PUUC NPs induced strong humoral immune responses, characterized by increases in anti-spike IgG in the blood and germinal center B cell populations (GL7+ and BCL6+ B cells) in the draining lymph nodes (dLNs). MPLA+PUUC NPs further boosted spike protein-neutralizing antibody titers and T follicular helper cell populations in the dLNs. These results suggest that protein subunit vaccines with particle-delivered molecular adjuvants targeting TLR4 and RIG-I could lead to robust and unique route-specific adaptive immune responses against SARS-CoV-2.

Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine.

Despite recent success in vaccinating populations against SARS-CoV-2, concerns about immunity duration, continued efficacy against emerging variants, protection from infection and transmission, and worldwide vaccine availability, remain. Although mRNA, pDNA, and viral-vector based vaccines are being administered, no protein subunit-based SARS-CoV-2 vaccine is approved. Molecular adjuvants targeting pathogen-recognition receptors (PRRs) on antigen-presenting cells (APCs) could improve and broaden the efficacy and durability of vaccine responses. Native SARS-CoV-2 infection stimulate various PRRs, including toll-like receptors (TLRs) and retinoic-acid-inducible gene I-like receptors (RIG-I). We hypothesized that targeting the same PRRs using adjuvants on nanoparticles along with a stabilized spike (S) protein antigen could provide broad and efficient immune responses. Formulations targeting TLR4 (MPLA), TLR7/8 (R848), TLR9 (CpG), and RIG-I (PUUC) delivered on degradable polymer-nanoparticles (NPs) were combined with the S1 subunit of S protein and assessed in vitro with isogeneic mixed lymphocyte reactions (iso-MLRs). For in vivo studies, the adjuvanted nanoparticles were combined with stabilized S protein and assessed using intranasal and intramuscular prime-boost vaccination models in mice. Combination NP-adjuvants targeting both TLR and RIG-I (MPLA+PUUC, CpG+PUUC, or R848+PUUC) differentially increased proinflammatory cytokine secretion (IL-1ß, IL-12p70, IL-27, IFN-ß) by APCs cultured in vitro, and induced differential T cell proliferation. When delivered intranasally, MPLA+PUUC NPs enhanced local CD4+CD44+ activated memory T cell responses while MPLA NPs increased anti-S-protein-specific IgG and IgA in the lung. Following intramuscular delivery, PUUC-carrying NPs induced strong humoral immune responses, characterized by increases in anti-S-protein IgG and neutralizing antibody titers and germinal center B cell populations (GL7+ and BCL6+ B cells). MPLA+PUUC NPs further boosted S-protein-neutralizing antibody titers and T follicular helper cell populations in draining lymph nodes. These results suggest that SARS-CoV-2-mimicking adjuvants and subunit vaccines could lead to robust and unique route-specific adaptive immune responses and may provide additional tools against the pandemic.