Enhanced Influenza Virus-Like Particle Vaccination with a Structurally Optimized RIG-I Agonist as Adjuvant - PubMed (original) (raw)

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

Vladimir Beljanski et al. J Virol. 2015 Oct.

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 TH1-biased CD4 T cell response, as determined by increased TH1 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.

Importance: The 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.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Figures

FIG 1

FIG 1

Transfecting monocyte-derived dendritic cells (MDDC) with WT, M5, or M8 5′pppRNA or poly(I·C) increases expression of activation and differentiation markers and their mRNA levels. MDDC were isolated from peripheral blood mononuclear cells (n = 4), differentiated, and transfected with 10 ng WT, M5, M8, or poly(I·C) using HiPerFect transfection reagent for 24 h. (A) Gene expression analysis using the Fluidigm BioMark platform for several genes (indicated on the right) in MDDC transfected with 20 fmol of WT, M5, M8, or poly(I·C) for 24 h. (B) MDDC surface expression of activation and differentiation markers as assessed by flow cytometry (mean plus standard error of the mean [SEM] [error bar]). Values that are significantly different are indicated by bars and asterisks as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.

FIG 2

FIG 2

Protective efficacy of the VLP vaccine adjuvanted with M5, M8, or poly(I·C). Mice (n = 5) were immunized intramuscularly with 2 μg of VLP alone or combined with 5 μg M5, M8, or poly(I·C) as a 50-μl injection, and 3 weeks later, they were challenged with 5,000 PFU of H5N1 influenza virus. (A) Hemagglutination inhibition (HAI) antibody titers in immunized mice prior to infection were determined by hemagglutination inhibition assay using horse red blood cells. (B) Assessment of viral replication in lungs of infected animals 3 days postinfection by a plaque assay. (C) TUNEL-positive (apoptotic) lung cells in infected mice were quantified by a TUNEL assay as described in Materials and Methods. All values in panels A to C are expressed as means plus SEMs. Values that are significantly different are indicated by bars and asterisks as follows: *, P ≤ 0.05; ***, P ≤ 0.005. (D) H&E staining of paraffin-embedded lung cross sections from mice 3 days after challenge. The yellow arrows indicate the airways of mice that were vaccinated with M8 only (top) or M8-VLP (bottom).

FIG 3

FIG 3

Dose-response assessment of VLP and M8. (A and B) Mice (n = 5) were immunized with decreasing doses of VLP (2 μg to 0.5 μg) in combination with 5 μg of M8; 3 weeks later, they were challenged with H5N1 influenza virus, and lungs from infected animals were harvested 3 days postchallenge. HAI antibody titers in immunized mice prior to infection were determined by HAI assay (A), and viral replication in lungs was assessed by plaque assay (B). (C to F) Mice were immunized with 0.5 μg of VLP with 0.1 to 5 μg of M8. HAI antibody titers were determined by HAI assay (C), and viral replication was determined by plaque assay (D). Weight loss (E) and survival (F) were determined as well. Values are expressed as means plus SEMs. Values that are significantly different are indicated by bars and asterisks as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.

FIG 4

FIG 4

Adjuvant comparison strategy and antibody immune responses for M8, alum, AddaVax, and poly(I·C)-adjuvanted VLP vaccine. (A) Strategy for adjuvant comparison. 3d, 3 days. (B to D) Mice were immunized with 0.5 μg of VLP in combination with 5 μg of M8 or poly(I·C) or in combination with 50% volume of alum or AddaVax, and 5 days and 3 weeks after immunization, sera were collected. HA-specific IgG antibodies (B) and influenza virus HAI antibody titers (C) were determined 3 weeks after immunization by ELISA and HAI assay, respectively. (D) HA-specific IgM antibodies were determined 5 days after immunization by ELISA. Values are expressed as means plus SEMs. Values that are significantly different are indicated by bars and asterisks as follows: *, P ≤ 0.05; **, P ≤ 0.01. O.D., optical density.

FIG 5

FIG 5

Protective efficacy of 0.5 μg of VLP in combination with 5 μg of M8 or poly(I·C) or in combination with 50% volume of alum or AddaVax. (A to C) Three weeks after vaccination, mice (n = 8) were challenged with the lethal dose of H5N1 (5,000 PFU), and their survival (A), weight (B), and sickness score (C) were monitored. (D) Viral replication in lungs was assessed by plaque assay in a separate group of immunized animals (n = 5) 3 days postinfection. Values are expressed as means ± SEMs. Values that are significantly different (P ≤ 0.005) are indicated by bars and asterisks (***).

FIG 6

FIG 6

Long-term protective responses in mice immunized with 0.5 μg of VLP in combination with 5 μg of M8 or poly(I·C) or in combination with 50% volume of alum or AddaVax. Mouse sera (n = 8) were collected 4 weeks (white bars) and 16 weeks (black bars) postvaccination to determine HA-specific IgG antibodies (ELISA) (A) and HAI antibody titers (B). Upon challenge with a lethal H5N1 dose (5,000 PFU) weight (C), survival (D), and sickness score (E) were monitored for 3 weeks. Values are expressed as the means ± SEMs.

FIG 7

FIG 7

Quantification of germinal center (GC) B cells from spleens of i.m. immunized mice (n = 5) by flow cytometry. (A) Gating strategy for quantification of GC B cells; (B) percentage of GC B cells in B220hi splenocytes; (C) quantification of IgG1+ GC B cells. Values are expressed as means plus SEMs. Values that are significantly different are indicated by bars and asterisks as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.

FIG 8

FIG 8

Quantification of IgG subclasses from i.m. vaccinated animals (n = 8) and intracellular cytokine levels in T cells isolated from the spleens of i.p. vaccinated animals (n = 5) after 24 h of VLP stimulation. (A) IgG subclasses, IgG1, IgG2a, IgG2b, and IgG3, from sera of vaccinated animals (as indicated below the bars) were determined by ELISA using HA-coated plates. (B to F) The percentages of cytokine-secreting cells were obtained by subtracting the numbers of cytokine-secreting cells that were not stimulated with VLP. The percentages of IFN-γ+ CD8hi cells (B), IL-2+ CD4hi cells (C), TNF-α+ CD4hi cells (D), IFN-γ+ CD4hi cells (E), and IL-10+ CD4hi cells (F) are shown. All values are expressed as means plus SEMs. Values that are significantly different are indicated by bars and asterisks as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.

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