Wendy W. L. Lee

Early neutralizing IgG response to Chikungunya virus in infected patients targets a dominant linear epitope on the E2 glycoprotein

Chikungunya virus (CHIKV) and related arboviruses have been responsible for large epidemic outbreaks with serious economic and social impact. The immune mechanisms, which control viral multiplication and dissemination, are not yet known. Here, we studied the antibody response against the CHIKV surface antigens in infected patients. With plasma samples obtained during the early convalescent phase, we showed that the naturally‐acquired IgG response is dominated by IgG3 antibodies specific mostly for a single linear epitope ‘E2EP3’. E2EP3 is located at the N‐terminus of the E2 glycoprotein and prominently exposed on the viral envelope. E2EP3‐specific antibodies are neutralizing and their removal from the plasma reduced the CHIKV‐specific antibody titer by up to 80%. Screening of E2EP3 across different patient cohorts and in non‐human primates demonstrated the value of this epitope as a good serology detection marker for CHIKV infection already at an early stage. Mice vaccinated by E2EP3 peptides were protected against CHIKV with reduced viremia and joint inflammation, providing a pre‐clinical basis for the design of effective vaccine against arthralgia‐inducing CHIKV and other alphaviruses.

An Essential Role of Antibodies in the Control of Chikungunya Virus Infection

In recent years, Chikungunya virus (CHIKV) was responsible for epidemic outbreaks in intertropical regions. Although acquired immunity has been shown to be crucial during CHIKV infection in both humans and mice, their exact role in the control of CHIKV infection remains unclear. In this study, wild-type (WT), CD4−/−, and B cell (μMT) knockout mice were infected with CHIKV. Sera were taken at different days postinfection and measured for anti-CHIKV Ab levels. Isotype and neutralizing capacity of these Abs were assessed in vitro, and specific linear epitopes were mapped. Viremia in CHIKV-infected μMT mice persisted for more than a year, indicating a direct role for B cells in mediating CHIKV clearance. These animals exhibited a more severe disease than WT mice during the acute phase. Characterization of CHIKV-specific Abs revealed that anti-CHIKV Abs were elicited early and targeted epitopes mainly at the C terminus of the virus E2 glycoprotein. Furthermore, CD4−/− mice could still control CHIKV infection despite having lower anti-CHIKV Ab levels with reduced neutralizing capacity. Lastly, pre-existing natural Abs in the sera of normal WT mice recognized CHIKV and were able to partially inhibit CHIKV. Taken together, natural and CHIKV infection–induced specific Abs are essential for controlling CHIKV infections.

Longitudinal Analysis of the Human Antibody Response to Chikungunya Virus Infection: Implications for Serodiagnosis and Vaccine Development

ABSTRACT Chikungunya virus (CHIKV) is an alphavirus which causes chronic and incapacitating arthralgia in humans. Although previous studies have shown that antibodies against the virus are produced during and after infection, the fine specificity of the antibody response against CHIKV is not known. Here, using plasma from patients at different times postinfection, we characterized the antibody response against various proteins of the virus. We have shown that the E2 and E3 glycoproteins and the capsid and nsP3 proteins are targets of the anti-CHIKV antibody response. Moreover, we have identified the different regions in these proteins which contain the linear epitopes recognized by the anti-CHIKV antibodies and determined their structural localization. Data also illustrated the effect of a single K252Q amino acid change at the E2 glycoprotein that was able to influence antibody binding and interaction between the antibodies and epitope because of the changes of epitope-antibody binding capacity. This study provides important knowledge that will not only aid in the understanding of the immune response to CHIKV infection but also provide new knowledge in the design of modern vaccine development. Furthermore, these pathogen-specific epitopes could be used for future seroepidemiological studies that will unravel the molecular mechanisms of human immunity and protection from CHIKV disease.

Chikungunya virus neutralization antigens and direct cell-to-cell transmission are revealed by human antibody-escape mutants.

Chikungunya virus (CHIKV) is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop “groove” as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis.

Caribbean and La Réunion Chikungunya Virus Isolates Differ in Their Capacity To Induce Proinflammatory Th1 and NK Cell Responses and Acute Joint Pathology

ABSTRACT Chikungunya virus (CHIKV) is a mosquito-borne arthralgic alphavirus that has garnered international attention as an important emerging pathogen since 2005. More recently, it invaded the Caribbean islands and the Western Hemisphere. Intriguingly, the current CHIKV outbreak in the Caribbean is caused by the Asian CHIKV genotype, which differs from the La Réunion LR2006 OPY1 isolate belonging to the Indian Ocean lineage. Here, we adopted a systematic and comparative approach against LR2006 OPY1 to characterize the pathogenicity of the Caribbean CNR20235 isolate and consequential host immune responses in mice. Ex vivo infection using primary mouse tail fibroblasts revealed a weaker replication efficiency by CNR20235 isolate. In the CHIKV mouse model, CNR20235 infection induced an enervated joint pathology characterized by moderate edema and swelling, independent of mononuclear cell infiltration. Based on systemic cytokine analysis, localized immunophenotyping, and gene expression profiles in the popliteal lymph node and inflamed joints, two pathogenic phases were defined for CHIKV infection: early acute (2 to 3 days postinfection [dpi]) and late acute (6 to 8 dpi). Reduced joint pathology during early acute phase of CNR20235 infection was associated with a weaker proinflammatory Th1 response and natural killer (NK) cell activity. The pathological role of NK cells was further demonstrated as depletion of NK cells reduced joint pathology in LR2006 OPY1. Taken together, this study provides evidence that the Caribbean CNR20235 isolate has an enfeebled replication and induces a less pathogenic response in the mammalian host. IMPORTANCE The introduction of CHIKV in the Americas has heightened the risk of large-scale outbreaks due to the close proximity between the United States and the Caribbean. The immunopathogenicity of the circulating Caribbean CHIKV isolate was explored, where it was demonstrated to exhibit reduced infectivity resulting in a weakened joint pathology. Analysis of serum cytokine levels, localized immunophenotyping, and gene expression profiles in the organs revealed that a limited Th1 response and reduced NK cells activity could underlie the reduced pathology in the host. Interestingly, higher asymptomatic infections were observed in the Caribbean compared to the La Réunion outbreaks in 2005 and 2006. This is the first study that showed an association between key proinflammatory factors and pathology-mediating leukocytes with a less severe pathological outcome in Caribbean CHIKV infection. Given the limited information regarding the sequela of Caribbean CHIKV infection, our study is timely and will aid the understanding of this increasingly important disease.

Loss of TLR3 aggravates CHIKV replication and pathology due to an altered virus‐specific neutralizing antibody response

RNA‐sensing toll‐like receptors (TLRs) mediate innate immunity and regulate anti‐viral response. We show here that TLR3 regulates host immunity and the loss of TLR3 aggravates pathology in Chikungunya virus (CHIKV) infection. Susceptibility to CHIKV infection is markedly increased in human and mouse fibroblasts with defective TLR3 signaling. Up to 100‐fold increase in CHIKV load was observed in Tlr3−/− mice, alongside increased virus dissemination and pro‐inflammatory myeloid cells infiltration. Infection in bone marrow chimeric mice showed that TLR3‐expressing hematopoietic cells are required for effective CHIKV clearance. CHIKV‐specific antibodies from Tlr3−/− mice exhibited significantly lower in vitro neutralization capacity, due to altered virus‐neutralizing epitope specificity. Finally, SNP genotyping analysis of CHIKF patients on TLR3 identified SNP rs6552950 to be associated with disease severity and CHIKV‐specific neutralizing antibody response. These results demonstrate a key role for TLR3‐mediated antibody response to CHIKV infection, virus replication and pathology, providing a basis for future development of immunotherapeutics in vaccine development.

Unique Epitopes Recognized by Antibodies Induced in Chikungunya Virus-Infected Non-Human Primates: Implications for the Study of Immunopathology and Vaccine Development

Chikungunya virus (CHIKV) is an Alphavirus that causes chronic and incapacitating arthralgia in humans. Although patient cohort studies have shown the production of CHIKV specific antibodies, the fine specificity of the antibody response against CHIKV is not completely defined. The macaque model of CHIKV infection was established due to limitations of clinical specimens. More importantly, its close relation to humans will allow the study of chronic infection and further identify important CHIKV targets. In this study, serum samples from CHIKV-infected macaques collected at different time-points post infection were used to characterize the antibody production pattern and kinetics. Results revealed that anti-CHIKV antibodies were neutralizing and the E2 glycoprotein, Capsid, nsP1, nsP3 and nsP4 proteins were targets of the anti-CHIKV antibody response in macaques. Furthermore, linear B-cell epitopes recognized by these anti-CHIKV antibodies were identified, and mapped to their structural localization. This characterizes the specificity of anti-CHIKV antibody response in macaques and further demonstrates the importance of the different regions in CHIKV-encoded proteins in the adaptive immune response. Information from this study provides critical knowledge that will aid in the understanding of CHIKV infection and immunity, vaccine design, and pre-clinical efficacy studies.

Sero-Prevalence and Cross-Reactivity of Chikungunya Virus Specific Anti-E2EP3 Antibodies in Arbovirus-Infected Patients

Chikungunya virus (CHIKV) and clinically-related arboviruses cause large epidemics with serious economic and social impact. As clinical symptoms of CHIKV infections are similar to several flavivirus infections, good detection methods to identify CHIKV infection are desired for improved treatment and clinical management. The strength of anti-E2EP3 antibody responses was explored in a longitudinal study on 38 CHIKV-infected patients. We compared their anti-E2EP3 responses with those of patients infected with non-CHIKV alphaviruses, or flaviviruses. E2EP3 cross-reactive samples from patients infected with non-CHIKV viruses were further analyzed with an in vitro CHIKV neutralization assay. CHIKV-specific anti-E2EP3 antibody responses were detected in 72% to 100% of patients. Serum samples from patients infected with other non-CHIKV alphaviruses were cross-reactive to E2EP3. Interestingly, some of these antibodies demonstrated clearly in vitro CHIKV neutralizing activity. Contrastingly, serum samples from flaviviruses-infected patients showed a low level of cross-reactivity against E2EP3. Using CHIKV E2EP3 as a serology marker not only allows early detection of CHIKV specific antibodies, but would also allow the differentiation between CHIKV infections and flavivirus infections with 93% accuracy, thereby allowing precise acute febrile diagnosis and improving clinical management in regions newly suffering from CHIKV outbreaks including the Americas.

Expanding regulatory T cells alleviates chikungunya virus-induced pathology in mice.

ABSTRACT Chikungunya virus (CHIKV) infection is a reemerging pandemic human arboviral disease. CD4+ T cells were previously shown to contribute to joint inflammation in the course of CHIKV infection in mice. The JES6-1 anti-IL-2 antibody selectively expands mouse regulatory T cells (Tregs) by forming a complex with IL-2. In this study, we show that the IL-2 JES6-1-mediated expansion of Tregs ameliorates CHIKV-induced joint pathology. It does so by inhibiting the infiltration of CD4+ T cells due to the induction of anergy in CHIKV-specific CD4+ effector T cells. These findings suggest that activation of Tregs could also become an alternative approach to control CHIKV-mediated disease. IMPORTANCE Chikungunya virus (CHIKV) has reemerged as a pathogen of global significance. Patients infected with CHIKV suffer from incapacitating joint pain that severely affects their daily functioning. Despite the best efforts, treatment is still inadequate. While T cell-mediated immunopathology in CHIKV infections has been reported, the role of regulatory T cells (Tregs) has not been explored. The JES6-1 anti-interleukin 2 (IL-2) antibody has been demonstrated to selectively expand mouse Tregs by forming a complex with IL-2. We reveal here that IL-2 JES6-1-mediated expansion of Tregs ameliorates CHIKV-induced joint pathology in mice by neutralizing virus-specific CD4+ effector T (Teff) cells. We show that this treatment abrogates the infiltration of pathogenic CD4+ T cells through induction of anergy in CHIKV-specific CD4+ Teff cells. This is the first evidence where the role of Tregs is demonstrated in CHIKV pathogenesis, and its expansion could control virus-mediated immunopathology.

Fingolimod treatment abrogates chikungunya virus–induced arthralgia

Abrogation of pathogenic CD4+ T cells to the joints with fingolimod (FTY720) treatment prevents chikungunya virus–induced joint pathology. Taming T cells to ameliorate chikungunya arthritis Mosquito-borne chikungunya virus causes fever and joint pain; some patients suffer from arthritis for years with no treatment options. In this issue, two studies investigated targeting pathogenic CD4+ T cells to prevent arthritis symptoms in a mouse model of chikungunya virus infection. Teo et al. demonstrated that fingolimod, a drug that sequesters immune cells to lymphoid organs, was able to relieve arthritis symptoms without affecting viral replication. Miner et al. used a combination of abatacept, which blocks T cell costimulation, and a human chikungunya neutralizing antibody to reduce both viral replication and disease severity. Repurposing these clinically available therapies could provide treatment options for chikungunya patients. Chikungunya virus (CHIKV) is one of the many rheumatic arthropod-borne alphaviruses responsible for debilitating joint inflammation in humans. Despite the severity in many endemic regions, clinically approved intervention targeting the virus remains unavailable. CD4+ T cells have been shown to mediate CHIKV-induced joint inflammation in mice. We demonstrate here that transfer of splenic CD4+ T cells from virus-infected C57BL/6 mice into virus-infected T cell receptor–deficient (TCR−/−) mice recapitulated severe joint pathology including inflammation, vascular leakages, subcutaneous edema, and skeletal muscle necrosis. Proteome-wide screening identified dominant CD4+ T cell epitopes in nsP1 and E2 viral antigens. Transfer of nsP1- or E2-specific primary CD4+ T cell lines into CHIKV-infected TCR−/− recipients led to severe joint inflammation and vascular leakage. This pathogenic role of virus-specific CD4+ T cells in CHIKV infections led to the assessment of clinically approved T cell–suppressive drugs for disease intervention. Although drugs targeting interleukin-2 pathway were ineffective, treatment with fingolimod, an agonist of sphingosine 1-phosphate receptor, successfully abrogated joint pathology in CHIKV-infected animals by blocking the migration of CD4+ T cells into the joints without any effect on viral replication. These results set the stage for further clinical evaluation of fingolimod in the treatment of CHIKV-induced joint pathologies.