Enhanced Influenza Virus-Like Particle Vaccination with a Structurally Optimized RIG-I Agonist as Adjuvant

Abstract

The molecular interaction between viral RNA and the cytosolic sensor RIG-I represents the initial trigger in the development of an effective immune response against infection with RNA viruses, resulting in innate immune activation and subsequent induction of adaptive responses. In the present study, the adjuvant properties of a sequence-optimized 5=-triphosphate-containing RNA (5=pppRNA) RIG-I agonist (termed M8) were examined in combination with influenza virus-like particles (VLP) (M8-VLP) expressing H5N1 influenza virus hemagglutinin (HA) and neuraminidase (NA) as immunogens. In combination with VLP, M8 increased the antibody response to VLP immunization, provided VLP antigen sparing, and protected mice from a lethal challenge with H5N1 influenza virus. M8-VLP immunization also led to long-term protective responses against influenza virus infection in mice. M8 adjuvantation of VLP increased endpoint and antibody titers and inhibited influenza virus replication in lungs compared with approved or experimental adjuvants alum, AddaVax, and poly(I·C). Uniquely, immunization with M8-VLP stimulated a T H 1-biased CD4 T cell response, as determined by increased T H 1 cytokine levels in CD4 T cells and increased IgG2 levels in sera. Collectively, these data demonstrate that a sequence-optimized, RIG-I-specific agonist is a potent adjuvant that can be utilized to increase the efficacy of influenza VLP vaccination and dramatically improve humoral and cellular mediated protective responses against influenza virus challenge. IMPORTANCEThe development of novel adjuvants to increase vaccine immunogenicity is an important goal that seeks to improve vaccine efficacy and ultimately prevent infections that endanger human health. This proof-of-principle study investigated the adjuvant properties of a sequence-optimized 5=pppRNA agonist (M8) with enhanced capacity to stimulate antiviral and inflammatory gene networks using influenza virus-like particles (VLP) expressing HA and NA as immunogens. Vaccination with VLP in combination with M8 increased anti-influenza virus antibody titers and protected animals from lethal influenza virus challenge, highlighting the potential clinical use of M8 as an adjuvant in vaccine development. Altogether, the results describe a novel immunostimulatory agonist targeted to the cytosolic RIG-I sensor as an attractive vaccine adjuvant candidate that can be used to increase vaccine efficacy, a pressing issue in children and the elderly population.

Crosstalk between Cytoplasmic RIG-I and STING Sensing Pathways

“…A recently characterized sequence-optimized immunostimulatory RNA (termed M8) that maintains specificity for RIG-I engagement was identified as a potent antiviral agent that blocked influenza, chikungunya and dengue infection in vitro and in vivo in animal models of viral pathogenesis [14, 15]. At picomolar concentrations, M8 stimulated in MoDCs greater IFN and inflammatory responses compared to other RLR binding sequences; this immune modulation required RIG-I, but was not affected by knockdown of other RNA receptors, a clear indication of M8 selectivity for RIG-I.…”

“…In combination with VLP, M8 increased the antibody response to VLP immunization, provided VLP antigen sparing, and protected mice from a lethal challenge with influenza H5N1. M8-VLP immunization also led to long term protective responses against influenza infection in mice [14, 15]. Collectively, these data argue that stimulation of the conserved RIG-I pathway may be an additional target for antiviral and vaccine development.…”

“…RIG-I agonists have also been successfully used as vaccine adjuvants: M8 and 5’-pppRNA, in combination with influenza hemagglutinin as antigen, protected mice against challenge with a lethal inoculation of influenza, an effect dependent on antibody production and cytotoxic T lymphocyte activation [14, 91]. With their ability to induce tumor cell death and lymphocyte cross-priming, RIG-I ligands are among the most promising molecules for the development of new immune-stimulatory adjuvants in cancer vaccines [69].…”

“…Activated DC drive adaptive immune responses. Activated DC engage CD4 + T cells and stimulate a Th1 skewed T cell response that includes IFNγ release, maturation of B lymphocytes to antibody producing plasma cells and the production of antibodies directed against viral or tumor antigens (14,15). Abbreviations used: RIG-I, retinoic acid inducible gene-I; MAVS, mitochondrial antiviral-signaling protein; IFN, interferon; cGAS, cyclic GMP-AMP synthase; cGAMP, 2’3’ guanosine-adenosine monophosphate; STING, stimulator of interferon genes…”

Harnessing Nanoparticles for Immunomodulation and Vaccines

“…One study showed STING activation by membrane disturbance caused by the entry of viral vectors, VLPs and liposomes [63]. The use of 5′-triphosphate–containing RNA, a RIG-I agonist, combined with an influenza-derived VLP was capable of increasing antibody titers and protecting mice against a lethal challenge with H5N1 [64]. Another study suggested an involvement of endogenous retrovirus-derived nucleic acids that would activate RIG-I and cGAS [65] upon VLP recognition by B cells.…”