A booster regime of liposome-delivered live-attenuated CHIKV vaccine RNA genome protects against chikungunya virus disease in mice.

Mosquito-transmitted chikungunya virus (CHIKV) is the causal pathogen of CHIKV disease and is responsible for global epidemics of arthritic disease. CHIKV infection can lead to severe chronic and debilitating arthralgia, significantly impacting patient mobility and quality of life. Our previous studies have shown a live-attenuated CHIKV vaccine candidate, CHIKV-NoLS, to be effective in protecting against CHIKV disease in mice vaccinated with one dose. Further studies have demonstrated the value of a liposome RNA delivery system to deliver the RNA genome of CHIKV-NoLS directly in vivo, promoting de novo production of live-attenuated vaccine particles in vaccinated hosts. This system, designed to bypass live-attenuated vaccine production bottlenecks, uses CAF01 liposomes. However, one dose of CHIKV-NoLS CAF01 failed to provide systemic protection against CHIKV challenge in mice, with low levels of CHIKV-specific antibodies. Here we describe CHIKV-NoLS CAF01 booster vaccination regimes designed to increase vaccine efficacy. C57BL/6 mice were vaccinated with three doses of CHIKV-NoLS CAF01 either intramuscularly or subcutaneously. CHIKV-NoLS CAF01 vaccinated mice developed a systemic immune response against CHIKV that shared similarity to vaccination with CHIKV-NoLS, including high levels of CHIKV-specific neutralising antibodies in subcutaneously inoculated mice. CHIKV-NoLS CAF01 vaccinated mice were protected against disease signs and musculoskeletal inflammation when challenged with CHIKV. Mice given one dose of live-attenuated CHIKV-NoLS developed a long lasting protective immune response for up to 71 days. A clinically relevant CHIKV-NoLS CAF01 booster regime can overcome the challenges faced by our previous one dose strategy and provide systemic protection against CHIKV disease.

N-Linked Glycans Shape Skin Immune Responses during Arthritis and Myositis after Intradermal Infection with Ross River Virus.

Arthritogenic alphaviruses are mosquito-borne arboviruses that include several re-emerging human pathogens, including the chikungunya (CHIKV), Ross River (RRV), Mayaro (MAYV), and o'nyong-nyong (ONNV) virus. Arboviruses are transmitted via a mosquito bite to the skin. Herein, we describe intradermal RRV infection in a mouse model that replicates the arthritis and myositis seen in humans with Ross River virus disease (RRVD). We show that skin infection with RRV results in the recruitment of inflammatory monocytes and neutrophils, which together with dendritic cells migrate to draining lymph nodes (LN) of the skin. Neutrophils and monocytes are productively infected and traffic virus from the skin to LN. We show that viral envelope N-linked glycosylation is a key determinant of skin immune responses and disease severity. RRV grown in mammalian cells elicited robust early antiviral responses in the skin, while RRV grown in mosquito cells stimulated poorer early antiviral responses. We used glycan mass spectrometry to characterize the glycan profile of mosquito and mammalian cell-derived RRV, showing deglycosylation of the RRV E2 glycoprotein is associated with curtailed skin immune responses and reduced disease following intradermal infection. Altogether, our findings demonstrate skin infection with an arthritogenic alphavirus leads to musculoskeletal disease and envelope glycoprotein glycosylation shapes disease outcome. IMPORTANCE Arthritogenic alphaviruses are transmitted via mosquito bites through the skin, potentially causing debilitating diseases. Our understanding of how viral infection starts in the skin and how virus systemically disseminates to cause disease remains limited. Intradermal arbovirus infection described herein results in musculoskeletal pathology, which is dependent on viral envelope N-linked glycosylation. As such, intradermal infection route provides new insights into how arboviruses cause disease and could be extended to future investigations of skin immune responses following infection with other re-emerging arboviruses.

Interleukin-17 contributes to Ross River virus-induced arthritis and myositis.

Arthritogenic alphaviruses are mosquito-borne viruses that are a major cause of infectious arthropathies worldwide, and recent outbreaks of chikungunya virus and Ross River virus (RRV) infections highlight the need for robust intervention strategies. Alphaviral arthritis can persist for months after the initial acute disease, and is mediated by cellular immune responses. A common strategy to limit inflammation and pathology is to dampen the overwhelming inflammatory responses by modulating proinflammatory cytokine pathways. Here, we investigate the contribution of interleukin-17 (IL-17), a cytokine involved in arthropathies such as rheumatoid arthritis, in the development RRV-induced arthritis and myositis. IL-17 was quantified in serum from RRV-infected patients, and mice were infected with RRV and joints and muscle tissues collected to analyse cellular infiltrates, tissue mRNA, cytokine expression, and joint and muscle histopathology. IL-17 expression was increased in musculoskeletal tissues and serum of RRV-infected mice and humans, respectively. IL-17-producing T cells and neutrophils contributed to the cellular infiltrate in the joint and muscle tissue during acute RRV disease in mice. Blockade of IL-17A/F using a monoclonal antibody (mAb) reduced disease severity in RRV-infected mice and led to decreased proinflammatory proteins, cellular infiltration in synovial tissues and cartilage damage, without affecting viral titers in inflamed tissues. IL-17A/F blockade triggered a shift in transcriptional profile of both leukocyte infiltrates and musculoskeletal stromal cells by downregulating proinflammatory genes. This study highlights a previously uncharacterized role for an effector cytokine in alphaviral pathology and points towards potential therapeutic benefit in targeting IL-17 to treat patients presenting with RRV-induced arthropathy.

Liposomal Delivery of the RNA Genome of a Live-Attenuated Chikungunya Virus Vaccine Candidate Provides Local, but Not Systemic Protection After One Dose.

Chikungunya virus (CHIKV) is the causative pathogen of chikungunya fever, a mosquito-borne viral disease causing highly debilitating arthralgia that can persist for months and progress to chronic arthritis. Our previous studies have identified the CHIKV live-attenuated vaccine candidate CHIKV-NoLS. Like most live-attenuated vaccines, attenuated replication of CHIKV-NoLS has the potential to limit scalable production. To overcome production limits, as well as other drawbacks of live-attenuated vaccines, we developed an in vivo liposome RNA delivery system to deliver the self-replicating RNA genome of CHIKV-NoLS directly into mice, allowing the recipients' body to produce the live-attenuated vaccine particles. CAF01 liposomes were able to deliver replication-competent CHIKV-NoLS RNA in vitro. Immunodeficient AG129 mice inoculated with liposome-delivered CHIKV-NoLS RNA developed viremia and disease signs representative of this lethal model of CHIKV infection, demonstrating de novo vaccine particle production in vivo. In immunocompetent C57BL/6 mice, liposome-delivered CHIKV-NoLS RNA inoculation was associated with reduced IgM and IgG levels with low antibody CHIKV-neutralizing capacity, compared to vaccination with the original live-attenuated vaccine CHIKV-NoLS. One dose of liposome-delivered CHIKV-NoLS RNA did not provide systemic protection from CHIKV wild-type (WT) challenge but was found to promote an early onset of severe CHIKV-induced footpad swelling. Liposome-delivered CHIKV-NoLS RNA inoculation did, however, provide local protection from CHIKV-WT challenge in the ipsilateral foot after one dose. Results suggest that in the presence of low CHIKV-specific neutralizing antibody levels, local inflammatory responses, likely brought on by liposome adjuvants, have a role in the protection of CHIKV-induced footpad swelling in the ipsilateral foot of mice inoculated with liposome-delivered CHIKV-NoLS RNA. Low IgG and CHIKV-specific neutralizing antibody levels may be responsible for early onset of severe swelling in the feet of CHIKV-WT-challenged mice. These results support previous studies that suggest CHIKV is vulnerable to antibody-mediated enhancement of disease. Further studies using booster regimes aim to demonstrate the potential for liposomes to deliver the self-replicating RNA genome of live-attenuated vaccines and offer a novel immunization strategy.

Modulation of Monocyte-Driven Myositis in Alphavirus Infection Reveals a Role for CX3CR1+ Macrophages in Tissue Repair.

Arthritogenic alphaviruses such as Ross River and Chikungunya viruses cause debilitating muscle and joint pain and pose significant challenges in the light of recent outbreaks. How host immune responses are orchestrated after alphaviral infections and lead to musculoskeletal inflammation remains poorly understood. Here, we show that myositis induced by Ross River virus (RRV) infection is driven by CD11bhi Ly6Chi inflammatory monocytes and followed by the establishment of a CD11bhi Ly6Clo CX3CR1+ macrophage population in the muscle upon recovery. Selective modulation of CD11bhi Ly6Chi monocyte migration to infected muscle using immune-modifying microparticles (IMP) reduced disease score, tissue damage, and inflammation and promoted the accumulation of CX3CR1+ macrophages, enhancing recovery and resolution. Here, we detail the role of immune pathology, describing a poorly characterized muscle macrophage subset as part of the dynamics of alphavirus-induced myositis and tissue recovery and identify IMP as an effective immunomodulatory approach. Given the lack of specific treatments available for alphavirus-induced pathologies, this study highlights a therapeutic potential for simple immune modulation by IMP in infected individuals in the event of large alphavirus outbreaks.IMPORTANCE Arthritogenic alphaviruses cause debilitating inflammatory disease, and current therapies are restricted to palliative approaches. Here, we show that following monocyte-driven muscle inflammation, tissue recovery is associated with the accumulation of CX3CR1+ macrophages in the muscle. Modulating inflammatory monocyte infiltration using immune-modifying microparticles (IMP) reduced tissue damage and inflammation and enhanced the formation of tissue repair-associated CX3CR1+ macrophages in the muscle. This shows that modulating key effectors of viral inflammation using microparticles can alter the outcome of disease by facilitating the accumulation of macrophage subsets associated with tissue repair.

Chikungunya: vaccines and therapeutics.

Chikungunya virus (CHIKV) has come to prominence as a global, re-emerging pathogen over the last two decades, progressing from sporadic, remote outbreaks to worldwide explosive epidemics. From contained, though considerable, outbreaks in the southern Indian Ocean, parts of South America and the Caribbean, CHIKV continues to be a significant pathogen in Southeast Asia and India. CHIKV circulates during epidemics through an urban mosquito-to-human transmission cycle, and with no available treatments or licensed vaccines to specifically target CHIKV disease, limiting transmission relies on vector control, which poses significant challenges, especially in developing countries. This review summarizes the current findings and progress in the development of safe, effective and affordable therapeutics and vaccines for CHIKV disease.

Specific inhibition of NLRP3 in chikungunya disease reveals a role for inflammasomes in alphavirus-induced inflammation.

Mosquito-borne viruses can cause severe inflammatory diseases and there are limited therapeutic solutions targeted specifically at virus-induced inflammation. Chikungunya virus (CHIKV), a re-emerging alphavirus responsible for several outbreaks worldwide in the past decade, causes debilitating joint inflammation and severe pain. Here, we show that CHIKV infection activates the NLRP3 inflammasome in humans and mice. Peripheral blood mononuclear cells isolated from CHIKV-infected patients showed elevated NLRP3, caspase-1 and interleukin-18 messenger RNA expression and, using a mouse model of CHIKV infection, we found that high NLRP3 expression was associated with peak inflammatory symptoms. Inhibition of NLRP3 activation using the small-molecule inhibitor MCC950 resulted in reduced CHIKV-induced inflammation and abrogated osteoclastogenic bone loss and myositis, but did not affect in vivo viral replication. Mice treated with MCC950 displayed lower expression levels of the cytokines interleukin-6, chemokine ligand 2 and tumour necrosis factor in joint tissue. Interestingly, MCC950 treatment abrogated disease signs in mice infected with a related arthritogenic alphavirus, Ross River virus, but not in mice infected with West Nile virus-a flavivirus. Here, using mouse models of alphavirus-induced musculoskeletal disease, we demonstrate that NLRP3 inhibition in vivo can reduce inflammatory pathology and that further development of therapeutic solutions targeting inflammasome function could help treat arboviral diseases.

Ross River virus disease clinical presentation, pathogenesis and current therapeutic strategies.

Ross River virus (RRV) is an arthitogenic alphavirus capable of causing outbreaks of debilitating musculoskeletal inflammatory disease in humans. RRV is the most common mosquito-borne disease in Australia, with outbreaks of RRV generally occurring during seasonal wet and warm conditions. Patients with Ross River virus disease (RRVD) typically present with fever, polyarthralgia, myalgia and a maculopapular erythematous rash. Treatment of the disease is usually palliative with no licensed vaccines or antiviral therapies currently available. In an effort to better inform therapeutic design, much progress has been made to understand the pathogenesis of RRVD. Progress has been largely driven by clinical evaluations supported by research using established murine models of RRVD, able to accurately replicate human disease. In this review we describe RRVD pathogenesis and the role of the host immune response, with particular focus on insights from studying animal models. We also discuss prospects for effective vaccines, preclinical development of therapeutic strategies and raise important questions for future RRV research.