Mannose binding lectin is required for alphavirus-induced arthritis/myositis

Mosquito-borne alphaviruses such as chikungunya virus and Ross River virus (RRV) are emerging pathogens capable of causing large-scale epidemics of virus-induced arthritis and myositis. The pathology of RRV-induced disease in both humans and mice is associated with induction of the host inflammatory response within the muscle and joints, and prior studies have demonstrated that the host complement system contributes to development of disease. In this study, we have used a mouse model of RRV-induced disease to identify and characterize which complement activation pathways mediate disease progression after infection, and we have identified the mannose binding lectin (MBL) pathway, but not the classical or alternative complement activation pathways, as essential for development of RRV-induced disease. MBL deposition was enhanced in RRV infected muscle tissue from wild type mice and RRV infected MBL deficient mice exhibited reduced disease, tissue damage, and complement deposition compared to wild-type mice. In contrast, mice deficient for key components of the classical or alternative complement activation pathways still developed severe RRV-induced disease. Further characterization of MBL deficient mice demonstrated that similar to C3(-/-) mice, viral replication and inflammatory cell recruitment were equivalent to wild type animals, suggesting that RRV-mediated induction of complement dependent immune pathology is largely MBL dependent. Consistent with these findings, human patients diagnosed with RRV disease had elevated serum MBL levels compared to healthy controls, and MBL levels in the serum and synovial fluid correlated with severity of disease. These findings demonstrate a role for MBL in promoting RRV-induced disease in both mice and humans and suggest that the MBL pathway of complement activation may be an effective target for therapeutic intervention for humans suffering from RRV-induced arthritis and myositis.This work was supported by NIH/NIAMS R01 AR 047190 awarded to MTH

The contribution of type I interferon signaling to immunity induced by alphavirus replicon vaccines

The type I interferon (IFN) system is critical for protecting the mammalian host from numerous virus infections and plays a key role in shaping the anti-viral adaptive immune response. In this report, the importance of type I IFN signaling was assessed in a mouse model of alphavirus-induced humoral immune induction. Venezuelan equine encephalitis virus replicon particles (VRP) expressing the hemagglutinin (HA) gene from influenza virus (HA-VRP) were used to vaccinate both wildtype (wt) and IFN α/β receptor knockout (RKO) mice. HA-VRP vaccination induced equivalent levels of flu-specific systemic IgG, mucosal IgG, and systemic IgA antibodies in both wt and IFN RKO mice. In contrast, HA-VRP vaccination of IFN RKO mice failed to induce significant levels of flu-specific mucosal IgA antibodies at multiple mucosal surfaces. In the VRP adjuvant system, co-delivery of null VRP with ovalbumin (OVA) protein significantly increased the levels of OVA-specific serum IgG, fecal IgG, and fecal IgA antibodies in both wt and RKO mice, suggesting that type I IFN signaling plays a less significant role in the VRP adjuvant effect. Taken together, these results suggest that, 1) at least in regard to IFN signaling, the mechanisms which regulate VRP-induced immunity differ when VRP are utilized as expression vectors as opposed to adjuvants, and 2) type I IFN signaling is required for the induction of mucosal IgA antibodies directed against VRP-expressed antigen. These results potentially shed new light on the regulatory networks which promote immune induction, and specifically mucosal immune induction, with alphavirus vaccine vectors

Alphavirus replicon particles acting as adjuvants promote CD8+ T cell responses to co-delivered antigen

Alphavirus replicon particles induce strong antibody and CD8+ T cell responses to expressed antigens in numerous experimental systems. We have recently demonstrated that Venezuelan equine encephalitis virus replicon particles (VRP) possess adjuvant activity for systemic and mucosal antibody responses. In this report, we demonstrate that VRP induced an increased and balanced serum IgG subtype response to co-delivered antigen, with simultaneous induction of antigen-specific IgG1 and IgG2a antibodies, and increased both systemic and mucosal antigen-specific CD8+ T cell responses, as measured by an IFN-γ ELISPOT assay. Additionally, VRP further increased antigen-specific T cell immunity in an additive fashion following co-delivery with the TLR ligand, CpG DNA. VRP infection led to recruitment of CD8+ T cells into the mucosal compartment, possibly utilizing the mucosal homing receptor, as this integrin was upregulated on CD8+ T cells in the draining lymph node of VRP-infected animals, where VRP-infected dendritic cells reside. This newly recognized ability of VRP to mediate increased T cell response towards co-delivered antigen provides the potential to both define the molecular basis of alphavirus-induced immunity, and improve alphavirus-based vaccines

Ross River virus envelope glycans contribute to disease through activation of the host complement system

Mannose binding lectin (MBL) generally plays a protective role during viral infection, yet MBL-mediated complement activation promotes Ross River virus (RRV)-induced inflammatory tissue destruction, contributing to arthritis and myositis. As MBL binds to carbohydrates, we hypothesized that N-linked glycans on the RRV envelope glycoproteins act as ligands for MBL. Using a panel of RRV mutants lacking the envelope N-linked glycans, we found that MBL deposition onto infected cells was dependent on the E2 glycans. Moreover, the glycan-deficient viruses exhibited reduced disease and tissue damage in a mouse model of RRV-induced myositis compared to wild-type RRV, despite similar viral load and inflammatory infiltrates within the skeletal muscle. Instead, the reduced disease induced by glycan-deficient viruses was linked to decreased MBL deposition and complement activation within inflamed tissues. These results demonstrate that the viral N-linked glycans promote MBL deposition and complement activation onto RRV-infected cells, contributing to the development of RRV-induced myositis

Nonmucosal Alphavirus Vaccination Stimulates a Mucosal Inductive Environment in the Peripheral Draining Lymph Node

The strongest mucosal immune responses are induced following mucosal Ag delivery and processing in the mucosal lymphoid tissues, and much is known regarding the immunological parameters which regulate immune induction via this pathway. Recently, experimental systems have been identified in which mucosal immune responses are induced following nonmucosal Ag delivery. One such system, footpad delivery of Venezuelan equine encephalitis virus replicon particles (VRP), led to the local production of IgA Abs directed against both expressed and codelivered Ags at multiple mucosal surfaces in mice. In contrast to the mucosal delivery pathway, little is known regarding the lymphoid structures and immunological components that are responsible for mucosal immune induction following nonmucosal delivery. In this study, we have used footpad delivery of VRP to probe the constituents of this alternative pathway for mucosal immune induction. Following nonmucosal VRP delivery, J chain-containing, polymeric IgA Abs were detected in the peripheral draining lymph node (DLN), at a time before IgA detection at mucosal surfaces. Further analysis of the VRP DLN revealed up-regulated α4β7 integrin expression on DLN B cells, expression of mucosal addressin cell adhesion molecule 1 on the DLN high endothelia venules, and production of IL-6 and CC chemokines, all characteristics of mucosal lymphoid tissues. Taken together, these results implicate the peripheral DLN as an integral component of an alternative pathway for mucosal immune induction. A further understanding of the critical immunological and viral components of this pathway may significantly improve both our knowledge of viral-induced immunity and the efficacy of viral-based vaccines

Disruption of the Opal Stop Codon Attenuates Chikungunya Virus-Induced Arthritis and Pathology

ABSTRACT Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for several significant outbreaks of debilitating acute and chronic arthritis and arthralgia over the past decade. These include a recent outbreak in the Caribbean islands and the Americas that caused more than 1 million cases of viral arthralgia. Despite the major impact of CHIKV on global health, viral determinants that promote CHIKV-induced disease are incompletely understood. Most CHIKV strains contain a conserved opal stop codon at the end of the viral nsP3 gene. However, CHIKV strains that encode an arginine codon in place of the opal stop codon have been described, and deep-sequencing analysis of a CHIKV isolate from the Caribbean identified both arginine and opal variants within this strain. Therefore, we hypothesized that the introduction of the arginine mutation in place of the opal termination codon may influence CHIKV virulence. We tested this by introducing the arginine mutation into a well-characterized infectious clone of a CHIKV strain from Sri Lanka and designated this virus Opal524R. This mutation did not impair viral replication kinetics in vitro or in vivo . Despite this, the Opal524R virus induced significantly less swelling, inflammation, and damage within the feet and ankles of infected mice. Further, we observed delayed induction of proinflammatory cytokines and chemokines, as well as reduced CD4 + T cell and NK cell recruitment compared to those in the parental strain. Therefore, the opal termination codon plays an important role in CHIKV pathogenesis, independently of effects on viral replication. IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes significant outbreaks of viral arthralgia. Studies with CHIKV and other alphaviruses demonstrated that the opal termination codon within nsP3 is highly conserved. However, some strains of CHIKV and other alphaviruses contain mutations in the opal termination codon. These mutations alter the virulence of related alphaviruses in mammalian and mosquito hosts. Here, we report that a clinical isolate of a CHIKV strain from the recent outbreak in the Caribbean islands contains a mixture of viruses encoding either the opal termination codon or an arginine mutation. Mutating the opal stop codon to an arginine residue attenuates CHIKV-induced disease in a mouse model. Compared to infection with the opal-containing parental virus, infection with the arginine mutant causes limited swelling and inflammation, as well as dampened recruitment of immune mediators of pathology, including CD4 + T cells and NK cells. We propose that the opal termination codon plays an essential role in the induction of severe CHIKV disease

Development of a broadly accessible Venezuelan equine encephalitis virus replicon particle vaccine platform

Zoonotic viruses circulate as swarms in animal reservoirs and can emerge into human populations, causing epidemics that adversely affect public health. Portable, safe, and effective vaccine platforms are needed in the context of these outbreak and emergence situations. In this work, we report the generation and characterization of an alphavirus replicon vaccine platform based on a non-select agent, attenuated Venezuelan equine encephalitis (VEE) virus vaccine, strain 3526 (VRP 3526). Using both noroviruses and coronaviruses as model systems, we demonstrate the utility of the VRP 3526 platform in the generation of recombinant proteins, production of virus-like particles, and in vivo efficacy as a vaccine against emergent viruses. Importantly, packaging under biosafety level 2 (BSL2) conditions distinguishes VRP 3526 from previously reported alphavirus platforms and makes this approach accessible to the majority of laboratories around the world. In addition, improved outcomes in the vulnerable aged models as well as against heterologous challenge suggest improved efficacy compared to that of previously attenuated VRP approaches. Taking these results together, the VRP 3526 platform represents a safe and highly portable system that can be rapidly deployed under BSL2 conditions for generation of candidate vaccines against emerging microbial pathogens.IMPORTANCE While VEE virus replicon particles provide a robust, established platform for antigen expression and vaccination, its utility has been limited by the requirement for high-containment-level facilities for production and packaging. In this work, we utilize an attenuated vaccine strain capable of use at lower biocontainment level but retaining the capacity of the wild-type replicon particle. Importantly, the new replicon platform provides equal protection for aged mice and following heterologous challenge, which distinguishes it from other attenuated replicon platforms. Together, the new system represents a highly portable, safe system for use in the context of disease emergence

γδ T cells play a protective role in Chikungunya virus-induced disease

Chikungunya virus (CHIKV) is an alphavirus responsible for causing epidemic outbreaks of polyarthralgia in humans. Because CHIKV is initially introduced via the skin, where γδ T cells are prevalent, we evaluated the response of these cells to CHIKV infection. CHIKV infection led to a significant increase in γδ T cells in the infected foot and draining lymph node that was associated with the production of proinflammatory cytokines and chemokines in C57BL/6J mice. γδ T cell-/- mice demonstrated exacerbated CHIKV disease characterized by less weight gain and greater foot swelling than occurred in wild-type mice, as well as a transient increase in monocytes and altered cytokine/chemokine expression in the foot. Histologically, γδ T cell-/- mice had increased inflammation-mediated oxidative damage in the ipsilateral foot and ankle joint compared to wild-type mice which was independent of differences in CHIKV replication. These results suggest that γδ T cells play a protective role in limiting the CHIKV-induced inflammatory response and subsequent tissue and joint damage

Gamma-delta T cells play a protective role in chikungunya virus-induced disease.

Chikungunya virus (CHIKV) is an alphavirus responsible for causing epidemic outbreaks of polyarthralgia in humans. As CHIKV is initially introduced into the skin where γδ T cells are prevalent, we evaluated their response to CHIKV infection. CHIKV infection led to a significant increase in γδ T cells in the infected foot and draining lymph node associated with the production of pro-inflammatory cytokines and chemokines in C57BL/6J mice. γδ T cell-/- mice demonstrated exacerbated CHIKV disease characterized by less weight gain and greater foot swelling compared to wild-type mice as well as a transient increase in monocytes and altered cytokine/chemokine expression in the foot. Histologically, γδ T cell-/- mice had increased inflammation-mediated oxidative damage in the ipsilateral foot and ankle joint of versus wild-type mice which was independent of differences in CHIKV replication. These results suggest that γδ T cells play a protective role in limiting the CHIKV-induced inflammatory response and subsequent tissue and joint damage. Recent epidemics including the 2004-2007 outbreak and the spread of CHIKV to naïve populations in the Caribbean, Central and South America with resultant cases imported into the U.S highlighted the capacity of CHIKV to cause explosive epidemics where the virus can spread to millions of people and rapidly move into new areas. These studies identify γδ T cells as being important to both recruitment of key inflammatory cell populations and dampening the tissue injury due to oxidative stress. Given the importance of these cells in the early response to CHIKV, this information may inform the development of CHIKV vaccines and therapeutics