Emerging chikungunya virus variants at the E1-E1 inter-glycoprotein spike interface impact virus attachment and Inflammation.

Chikungunya virus (CHIKV) is a re-emerging arthropod-borne alphavirus and a serious threat to human health. Therefore, efforts toward elucidating how this virus causes disease and the molecular mechanisms underlying steps of the viral replication cycle are crucial. Using an in vivo transmission system that allows intra-host evolution, we identified an emerging CHIKV variant carrying a mutation in the E1 glycoprotein (V156A) in the serum of mice and saliva of mosquitoes. E1 V156A has since emerged in humans during an outbreak in Brazil, co-occurring with a second mutation, E1 K211T, suggesting an important role for these residues in CHIKV biology. Given the emergence of these variants, we hypothesized that they function to promote CHIKV infectivity and subsequent disease. Here, we show that E1 V156A and E1 K211T modulate virus attachment and fusion and impact binding to heparin, a homolog of heparan sulfate, a key entry factor on host cells. These variants also exhibit differential neutralization by anti-glycoprotein monoclonal antibodies, suggesting structural impacts on the particle that may be responsible for altered interactions at the host membrane. Finally, E1 V156A and E1 K211T exhibit increased titers in an adult arthritic mouse model and induce increased foot-swelling at the site of injection. Taken together, this work has revealed new roles for E1 where discrete regions of the glycoprotein are able to modulate cell attachment and swelling within the host. IMPORTANCE Alphaviruses represent a growing threat to human health worldwide. The re-emerging alphavirus chikungunya virus (CHIKV) has rapidly spread to new geographic regions in the last several decades, causing overwhelming outbreaks of disease, yet there are no approved vaccines or therapeutics. The CHIKV glycoproteins are key determinants of CHIKV adaptation and virulence. In this study, we identify and characterize the emerging E1 glycoprotein variants, V156A and K211T, that have since emerged in nature. We demonstrate that E1 V156A and K211T function in virus attachment to cells, a role that until now has been only attributed to specific residues of the CHIKV E2 glycoprotein. We also demonstrate E1 V156A and K211T to increase foot-swelling of the ipsilateral foot in mice infected with these variants. Observing that these variants and other pathogenic variants occur at the E1-E1 inter-spike interface, we highlight this structurally important region as critical for multiple steps during CHIKV infection. Together, these studies further defines the function of E1 in CHIKV infection and can inform the development of therapeutic or preventative strategies.

A phase 1 trial of lipid-encapsulated mRNA encoding a monoclonal antibody with neutralizing activity against Chikungunya virus

Chikungunya virus (CHIKV) infection causes acute disease characterized by fever, rash and arthralgia, which progresses to severe and chronic arthritis in up to 50% of patients. Moreover, CHIKV infection can be fatal in infants or immunocompromised individuals and has no approved therapy or prevention. This phase 1, first-in-human, randomized, placebo-controlled, proof-of-concept trial conducted from January 2019 to June 2020 evaluated the safety and pharmacology of mRNA-1944, a lipid nanoparticle-encapsulated messenger RNA encoding the heavy and light chains of a CHIKV-specific monoclonal neutralizing antibody, CHKV-24 (NCT03829384). The primary outcome was to evaluate the safety and tolerability of escalating doses of mRNA-1944 administered via intravenous infusion in healthy participants aged 18–50 years. The secondary objectives included determination of the pharmacokinetics of mRNA encoding for CHKV-24 immunoglobulin heavy and light chains and ionizable amino lipid component and the pharmacodynamics of mRNA-1944 as assessed by serum concentrations of mRNA encoding for CHKV-24 immunoglobulin G (IgG), plasma concentrations of ionizable amino lipid and serum concentrations of CHKV-24 IgG. Here we report the results of a prespecified interim analysis of 38 healthy participants who received intravenous single doses of mRNA-1944 or placebo at 0.1, 0.3 and 0.6 mg kg−1, or two weekly doses at 0.3 mg kg−1. At 12, 24 and 48 h after single infusions, dose-dependent levels of CHKV-24 IgG with neutralizing activity were observed at titers predicted to be therapeutically relevant concentrations (≥1 µg ml−1) across doses that persisted for ≥16 weeks at 0.3 and 0.6 mg kg−1 (mean t1/2 approximately 69 d). A second 0.3 mg kg−1 dose 1 week after the first increased CHKV-24 IgG levels 1.8-fold. Adverse effects were mild to moderate in severity, did not worsen with a second mRNA-1944 dose and none were serious. To our knowledge, mRNA-1944 is the first mRNA-encoded monoclonal antibody showing in vivo expression and detectable ex vivo neutralizing activity in a clinical trial and may offer a treatment option for CHIKV infection. Further evaluation of the potential therapeutic use of mRNA-1944 in clinical trials for the treatment of CHIKV infection is warranted.

Serological Responses in Patients Infected with Mayaro Virus and Evaluation of Cross-Protective Responses against Chikungunya Virus

Mayaro virus (MAYV) is an alphavirus endemic to both Latin America and the Caribbean. Recent reports have questioned the ability of MAYV and its close relative, Chikungunya virus (CHIKV), to generate cross-reactive, neutralizing antibodies to one another. Since CHIKV was introduced to South America in 2013, discerning whether individuals have cross-reactive antibodies or whether they have had exposures to both viruses previously has been difficult. Using samples obtained from people infected with MAYV prior to the introduction of CHIKV in the Americas, we performed neutralizing assays and observed no discernable neutralization of CHIKV by sera from patients previously infected with MAYV. These data suggest that a positive CHIKV neutralization test cannot be attributed to prior exposure to MAYV and that previous exposure to MAYV may not be protective against a subsequent CHIKV infection.

Model-based assessment of Chikungunya and O’nyong-nyong virus circulation in Mali in a serological cross-reactivity context

Serological surveys are essential to quantify immunity in a population but serological cross-reactivity often impairs estimates of the seroprevalence. Here, we show that modeling helps addressing this key challenge by considering the important cross-reactivity between Chikungunya (CHIKV) and O’nyong-nyong virus (ONNV) as a case study. We develop a statistical model to assess the epidemiology of these viruses in Mali. We additionally calibrate the model with paired virus neutralization titers in the French West Indies, a region with known CHIKV circulation but no ONNV. In Mali, the model estimate of ONNV and CHIKV prevalence is 30% and 13%, respectively, versus 27% and 2% in non-adjusted estimates. While a CHIKV infection induces an ONNV response in 80% of cases, an ONNV infection leads to a cross-reactive CHIKV response in only 22% of cases. Our study shows the importance of conducting serological assays on multiple cross-reactive pathogens to estimate levels of virus circulation.

MK2a inhibitor CMPD1 abrogates chikungunya virus infection by modulating actin remodeling pathway

Chikungunya virus (CHIKV) epidemics around the world have created public health concern with the unavailability of effective drugs and vaccines. This emphasizes the need for molecular understanding of host-virus interactions for developing effective targeted antivirals. Microarray analysis was carried out using CHIKV strain (Prototype and Indian) infected Vero cells and two host isozymes, MAPK activated protein kinase 2 (MK2) and MAPK activated protein kinase 3 (MK3) were selected for further analysis. The substrate spectrum of both enzymes is indistinguishable and covers proteins involved in cytokines production, endocytosis, reorganization of the cytoskeleton, cell migration, cell cycle control, chromatin remodeling and transcriptional regulation. Gene silencing and drug treatment were performed in vitro and in vivo to unravel the role of MK2/MK3 in CHIKV infection. Gene silencing of MK2 and MK3 abrogated around 58% CHIKV progeny release from the host cell and a MK2 activation inhibitor (CMPD1) treatment demonstrated 68% inhibition of viral infection suggesting a major role of MAPKAPKs during late CHIKV infection in vitro. Further, it was observed that the inhibition in viral infection is primarily due to the abrogation of lamellipodium formation through modulation of factors involved in the actin cytoskeleton remodeling pathway. Moreover, CHIKV-infected C57BL/6 mice demonstrated reduction in the viral copy number, lessened disease score and better survivability after CMPD1 treatment. In addition, reduction in expression of key pro-inflammatory mediators such as CXCL13, RAGE, FGF, MMP9 and increase in HGF (a CHIKV infection recovery marker) was observed indicating the effectiveness of the drug against CHIKV. Taken together it can be proposed that MK2 and MK3 are crucial host factors for CHIKV infection and can be considered as important target for developing effective anti-CHIKV strategies.

Telmisartan restricts Chikungunya virus infection in vitro and in vivo through the AT1/PPAR-γ/MAPKs pathways

Chikungunya virus (CHIKV) has re-emerged as a global public health threat. The inflammatory pathways of RAS and PPAR-γ are usually involved in viral infections. Thus, Telmisartan (TM) with known capacity to block AT1 receptor and activate PPAR-γ, was investigated against CHIKV. The anti-CHIKV effect of TM was investigated in vitro (Vero, RAW 264.7 cells and hPBMCs) and in vivo (C57BL/6 mice). TM was found to abrogate CHIKV infection efficiently (IC50 of 15.34-20.89μM in the Vero and RAW 264.7 cells respectively). Viral RNA and proteins were reduced remarkably. Additionally, TM interfered in the early and late stages of CHIKV life cycle with efficacy in both pre and post-treatment assay. Moreover, the agonist of AT1 receptor and antagonist of PPAR-γ increased CHIKV infection suggesting TM’s anti-viral potential by modulating host factors. Besides, reduced activation of all major MAPKs, NF-κB (p65) and cytokines by TM through the inflammatory axis supported the fact that the anti-CHIKV efficacy of TM is partly mediated through the AT1/PPAR-γ/MAPKs pathways. Interestingly, at the human equivalent dose, TM abrogated CHIKV infection and inflammation significantly leading to reduced clinical score and complete survival of C57BL/6 mice. Additionally, TM reduced infection in hPBMC derived monocyte-macrophage populations in vitro . Hence, TM was found to reduce CHIKV infection by targeting both viral and host factors. Considering its safety and in vivo efficacy, it can be a suitable candidate in future for repurposing against CHIKV.

Neonatal Chikungunya Presented as Apnea

Neonatal Chikungunya virus (CHIKV) infection is sporadic, and the prevalence of the entity has been described only recently. Neurological complications in adults have been reported, but there is a lack of data in this regard in neonates. In this retrospective case series done during the outbreak of Chikungunya, we observed 7 neonates who presented with fever, irritability, excessive cry, and rash, which was confirmed by polymerase chain reaction. Out of 7, 5 neonates presented with encephalopathy with apnea and seizures (80%), which were the most common presenting symptoms. Identifying this entity based on clinical and epidemiological background helps in management and aids in prognostication of the affected neonate.

Overview on Chikungunya Virus Infection: From Epidemiology to State-of-the-Art Experimental Models

Chikungunya virus (CHIKV) is currently one of the most relevant arboviruses to public health. It is a member of the Togaviridae family and alphavirus genus and causes an arthritogenic disease known as chikungunya fever (CHIKF). It is characterized by a multifaceted disease, which is distinguished from other arbovirus infections by the intense and debilitating arthralgia that can last for months or years in some individuals. Despite the great social and economic burden caused by CHIKV infection, there is no vaccine or specific antiviral drugs currently available. Recent outbreaks have shown a change in the severity profile of the disease in which atypical and severe manifestation lead to hundreds of deaths, reinforcing the necessity to understand the replication and pathogenesis processes. CHIKF is a complex disease resultant from the infection of a plethora of cell types. Although there are several in vivo models for studying CHIKV infection, none of them reproduces integrally the disease signature observed in humans, which is a challenge for vaccine and drug development. Therefore, understanding the potentials and limitations of the state-of-the-art experimental models is imperative to advance in the field. In this context, the present review outlines the present knowledge on CHIKV epidemiology, replication, pathogenesis, and immunity and also brings a critical perspective on the current in vitro and in vivo state-of-the-art experimental models of CHIKF.

Acute-Phase Levels of CXCL8 as Risk Factor for Chronic Arthralgia Following Chikungunya Virus Infection

The immunopathogenesis of chikungunya virus (CHIKV) infection and the role of acute-phase immune response on joint pain persistence is not fully understood. We investigated the profile of serum chemokine and cytokine in CHIKV-infected patients with acute disease, compared the levels of these biomarkers to those of patients with other acute febrile diseases (OAFD) and healthy controls (HC), and evaluated their role as predictors of chronic arthralgia development. Chemokines and cytokines were measured by flow Cytometric Bead Array. Patients with CHIKV infection were further categorized according to duration of arthralgia (≤ 3 months vs >3 months), presence of anti-CHIKV IgM at acute-phase sample, and number of days of symptoms at sample collection (1 vs 2-3 vs ≥4). Patients with acute CHIKV infection had significantly higher levels of CXCL8, CCL2, CXCL9, CCL5, CXCL10, IL-1β, IL-6, IL-12, and IL-10 as compared to HC. CCL2, CCL5, and CXCL10 levels were also significantly higher in patients with CHIKV infection compared to patients with OAFD. Patients whose arthralgia lasted > 3 months had increased CXCL8 levels compared to patients whose arthralgia did not (p<0.05). Multivariable analyses further indicated that high levels of CXCL8 and female sex were associated with arthralgia lasting >3 months. Patients with chikungunya and OAFD had similar cytokine kinetics for IL-1β, IL-12, TNF, IFN-γ, IL-2, and IL-4, although the levels were lower for CHIKV patients. This study suggests that chemokines may have an important role in the immunopathogenesis of chronic chikungunya-related arthralgia.