Prophylactic efficacy of orally administered Bacillus poly-\u03b3-glutamic acid, a non-LPS TLR4 ligand, against norovirus infection in mice

Wooseong Lee,Hae-Gwang Jung,Jong-Won Oh,Seung-Hoon Lee,Minwoo Kim

doi:10.1038/s41598-018-26935-y

Abstract

Poly-gamma-glutamic acid (γ-PGA), an extracellular biopolymer produced by Bacillus sp., is a non-canonical toll-like receptor 4 (TLR4) agonist. Here we show its antiviral efficacy against noroviruses. γ-PGA with a molecular mass of 2,000-kDa limited murine norovirus (MNV) replication in the macrophage cell line RAW264.7 by inducing interferon (IFN)-β and conferred resistance to viral infection-induced cell death. Additionally, γ-PGA interfered with viral entry into cells. The potent antiviral state mounted by γ-PGA was not attributed to the upregulation of TLR4 or TLR3, a sensor known to recognize norovirus RNA. γ-PGA sensing by TLR4 required the two TLR4-associated accessory factors MD2 and CD14. In ex vivo cultures of mouse ileum, γ-PGA selectively increased the expression of IFN-β in villi. In contrast, IFN-β induction was negligible in the ileal Peyer’s patches (PPs) where its expression was primarily induced by the replication of MNV. Oral administration of γ-PGA, which increased serum IFN-β levels without inducing proinflammatory cytokines, reduced MNV loads in the ileum with PPs and mesenteric lymph nodes in mice. Our results disclose a γ-PGA-mediated non-conventional TLR4 signaling in the ileum, highlighting the potential use of γ-PGA as a prophylactic antiviral agent against noroviruses.

Highlights

Toll-like receptor (TLR) 4 is a pattern-recognition receptor (PRR) that recognizes lipopolysaccharide (LPS) from Gram-negative bacteria as a pathogen-associated molecular pattern (PAMP)
We first determined the range of multiplicity of infection (MOI) of murine norovirus (MNV) in which viral infection-induced apoptosis is triggered during the course of viral infection
We investigated the ability of orally administered γ-PGA to induce an innate immune response and to mount an antiviral state in the ileum

Summary

Introduction

Toll-like receptor (TLR) 4 is a pattern-recognition receptor (PRR) that recognizes lipopolysaccharide (LPS) from Gram-negative bacteria as a pathogen-associated molecular pattern (PAMP). Human norovirus (HuNoV) is transmitted via the fecal-oral route and propagates in the gastrointestinal (GI) tract[11] It is the most common cause of non-bacterial acute outbreaks of gastroenteritis worldwide across all age groups[12]. In norovirus-infected cells, IFN-α/β production is initiated by the sensing of viral RNA genome via the cytoplasmic PRR MDA5 (melanoma differentiation-associated gene 5) and TLR3, followed by phosphorylation of IFN regulatory factor 3 (IRF-3) through two independent pathways[24]. This type I IFN signaling plays a major role in clearing mouse norovirus[24,25,26] and HuNoV27,28

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Conclusion