Small Molecule Inhibitors Targeting Chikungunya Virus.

Chikungunya virus (CHIKV) infection in humans is rarely fatal but is often associated with chronic joint and muscle pain. Chronic CHIKV disease is highly debilitating and is associated with viral persistence. To date, there are no approved vaccines or therapeutics to prevent or treat CHIKV infections once they are established. Current palliative treatments aim to reduce joint inflammation and pain associated with acute and chronic CHIKV disease. Development of novel therapeutics that reduces viral loads should positively impact virus inflammatory disease and improve patient outcomes following CHIKV infection. Therapies that target multiple aspects of CHIKV replication cycle should be developed since the virus is capable of rapidly mutating around any single therapeutic. This review summarizes the current status of small molecule inhibitor development against CHIKV.

Blocking the IL-1 receptor reduces cardiac transplant ischemia and reperfusion injury and mitigates CMV-accelerated chronic rejection.

Ischemia-reperfusion injury (IRI) is an important risk factor for accelerated cardiac allograft rejection and graft dysfunction . Utilizing a rat heart isogeneic transplant model, we identified inflammatory pathways involved in IRI in order to identify therapeutic targets involved in disease. Pathway analyses identified several relevant targets, including cytokine signaling by the IL-1 receptor (IL-1R) pathway and inflammasome activation. To investigate the role of IL-1R signaling pathways during IRI, we treated syngeneic cardiac transplant recipients at 1-hour posttransplant with Anakinra, a US Food and Drug Administration (FDA)-approved IL-1R antagonist; or parthenolide, a caspase-1 and nuclear factor kappa-light-chain-enhancer of activated B cells inhibitor that blocks IL-1ß maturation. Both Anakinra and parthenolide significantly reduced graft inflammation and cellular recruitment in the treated recipients relative to nontreated controls. Anakinra treatment administered at 1-hour posttransplant to recipients of cardiac allografts from CMV-infected donors significantly increased the time to rejection and reduced viral loads at rejection. Our results indicate that reducing IRI by blocking IL-1Rsignaling pathways with Anakinra or inflammasome activity with parthenolide provides a promising approach for extending survival of cardiac allografts from CMV-infected donors.

Identification of Quinolinones as Antivirals against Venezuelan Equine Encephalitis Virus.

Venezuelan equine encephalitis virus (VEEV) is a reemerging alphavirus that can cause encephalitis resulting in severe human morbidity and mortality. Using a high-throughput cell-based screen, we identified a quinolinone compound that protected against VEEV-induced cytopathic effects. Analysis of viral replication in cells identified several quinolinone compounds with potent inhibitory activity against vaccine and virulent strains of VEEV. These quinolinones also displayed inhibitory activity against additional alphaviruses, such as Mayaro virus and Ross River virus, although the potency was greatly reduced. Time-of-addition studies indicated that these compounds inhibit the early-to-mid stage of viral replication. Deep sequencing and reverse genetics studies identified two unique resistance mutations in the nsP2 gene (Y102S/C; stalk domain) that conferred VEEV resistance on this chemical series. Moreover, introduction of a K102Y mutation into the nsP2 gene enhanced the sensitivity of chikungunya virus (CHIKV) to this chemical series. Computational modeling of CHIKV and VEEV nsP2 identified a highly probable docking alignment for the quinolinone compounds that require a tyrosine residue at position 102 within the helicase stalk domain. These studies identified a class of compounds with antiviral activity against VEEV and other alphaviruses and provide further evidence that therapeutics targeting nsP2 may be useful against alphavirus infection.

Macrophage depletion of CMV latently infected donor hearts ameliorates recipient accelerated chronic rejection.

Cytomegalovirus (CMV) infection is linked to acceleration of solid organ transplant vascular sclerosis (TVS) and chronic rejection (CR). Donor latent CMV infection increases cardiac-resident macrophages and T cells leading to increased inflammation, promoting allograft rejection. To investigate the role of cardiac-resident passenger macrophages in CMV-mediated TVS/CR, macrophages were depleted from latently ratCMV (RCMV)-infected donor allografts prior to transplantation. Latently RCMV-infected donor F344 rats were treated with clodronate, PBS, or control liposomes 3 days prior to cardiac transplant into RCMV-naïve Lewis recipients. Clodronate treatment significantly increased graft survival from post-operative day (POD)61 to POD84 and decreased TVS at rejection. To determine the kinetics of the effect of clodronate treatment's effect, a time study revealed that clodronate treatment significantly decreased macrophage infiltration into allograft tissues as early as POD14; altered allograft cytokine/chemokine protein levels, fibrosis development, and inflammatory gene expression profiles. These findings support our hypothesis that increased graft survival as a result of allograft passenger macrophage depletion was in part a result of altered immune response kinetics. Depletion of donor macrophages prior to transplant is a strategy to modulate allograft rejection and reduce TVS in the setting of CMV + donors transplanted into CMV naïve recipients.

Emerging Alphaviruses Are Sensitive to Cellular States Induced by a Novel Small-Molecule Agonist of the STING Pathway.

The type I interferon (IFN) system represents an essential innate immune response that renders cells resistant to virus growth via the molecular actions of IFN-induced effector proteins. IFN-mediated cellular states inhibit growth of numerous and diverse virus types, including those of known pathogenicity as well as potentially emerging agents. As such, targeted pharmacologic activation of the IFN response may represent a novel therapeutic strategy to prevent infection or spread of clinically impactful viruses. In light of this, we employed a high-throughput screen to identify small molecules capable of permeating the cell and of activating IFN-dependent signaling processes. Here we report the identification and characterization of N-(methylcarbamoyl)-2-{[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-2-phenylacetamide (referred to as C11), a novel compound capable of inducing IFN secretion from human cells. Using reverse genetics-based loss-of-function assays, we show that C11 activates the type I IFN response in a manner that requires the adaptor protein STING but not the alternative adaptors MAVS and TRIF. Importantly, treatment of cells with C11 generated a cellular state that potently blocked replication of multiple emerging alphavirus types, including chikungunya, Ross River, Venezuelan equine encephalitis, Mayaro, and O'nyong-nyong viruses. The antiviral effects of C11 were subsequently abrogated in cells lacking STING or the type I IFN receptor, indicating that they are mediated, at least predominantly, by way of STING-mediated IFN secretion and subsequent autocrine/paracrine signaling. This work also allowed characterization of differential antiviral roles of innate immune signaling adaptors and IFN-mediated responses and identified MAVS as being crucial to cellular resistance to alphavirus infection.IMPORTANCE Due to the increase in emerging arthropod-borne viruses, such as chikungunya virus, that lack FDA-approved therapeutics and vaccines, it is important to better understand the signaling pathways that lead to clearance of virus. Here we show that C11 treatment makes human cells refractory to replication of a number of these viruses, which supports its value in increasing our understanding of the immune response and viral pathogenesis required to establish host infection. We also show that C11 depends on signaling through STING to produce antiviral type I interferon, which further supports its potential as a therapeutic drug or research tool.

Correction: Zika Virus infection of rhesus macaques leads to viral persistence in multiple tissues.

[This corrects the article DOI: 10.1371/journal.ppat.1006219.].

Zika Virus infection of rhesus macaques leads to viral persistence in multiple tissues.

Zika virus (ZIKV), an emerging flavivirus, has recently spread explosively through the Western hemisphere. In addition to symptoms including fever, rash, arthralgia, and conjunctivitis, ZIKV infection of pregnant women can cause microcephaly and other developmental abnormalities in the fetus. We report herein the results of ZIKV infection of adult rhesus macaques. Following subcutaneous infection, animals developed transient plasma viremia and viruria from 1-7 days post infection (dpi) that was accompanied by the development of a rash, fever and conjunctivitis. Animals produced a robust adaptive immune response to ZIKV, although systemic cytokine response was minimal. At 7 dpi, virus was detected in peripheral nervous tissue, multiple lymphoid tissues, joints, and the uterus of the necropsied animals. Notably, viral RNA persisted in neuronal, lymphoid and joint/muscle tissues and the male and female reproductive tissues through 28 to 35 dpi. The tropism and persistence of ZIKV in the peripheral nerves and reproductive tract may provide a mechanism of subsequent neuropathogenesis and sexual transmission.

Animal Models of Chikungunya Virus Infection and Disease.

Chikungunya virus (CHIKV) is a reemerging alphavirus that causes acute febrile illness and severe joint pain in humans. Although acute symptoms often resolve within a few days, chronic joint and muscle pain can be long lasting. In the last decade, CHIKV has caused widespread outbreaks of unprecedented scale in the Americas, Asia, and the Indian Ocean island regions. Despite these outbreaks and the continued expansion of CHIKV into new areas, mechanisms of chikungunya pathogenesis and disease are not well understood. Experimental animal models are indispensable to the field of CHIKV research. The most commonly used experimental animal models of CHIKV infection are mice and nonhuman primates; each model has its advantages for studying different aspects of CHIKV disease. This review will provide an overview of animal models used to study CHIKV infection and disease and major advances in our understanding of chikungunya obtained from studies performed in these models.

Infection with chikungunya virus confers heterotypic cross-neutralizing antibodies and memory B-cells against other arthritogenic alphaviruses predominantly through the B domain of the E2 glycoprotein.

Infections with Chikungunya virus, a mosquito-borne alphavirus, cause an acute febrile syndrome often followed by chronic arthritis that persists for months to years post-infection. Neutralizing antibodies are the primary immune correlate of protection elicited by infection, and the major goal of vaccinations in development. Using convalescent blood samples collected from both endemic and non-endemic human subjects at multiple timepoints following suspected or confirmed chikungunya infection, we identified antibodies with broad neutralizing properties against other alphaviruses within the Semliki Forest complex. Cross-neutralization generally did not extend to the Venezuelan Equine Encephalitis virus (VEEV) complex, although some subjects had low levels of VEEV-neutralizing antibodies. This suggests that broadly neutralizing antibodies elicited following natural infection are largely complex restricted. In addition to serology, we also performed memory B-cell analysis, finding chikungunya-specific memory B-cells in all subjects in this study as remotely as 24 years post-infection. We functionally assessed the ability of memory B-cell derived antibodies to bind to chikungunya virus, and related Mayaro virus, as well as the highly conserved B domain of the E2 glycoprotein thought to contribute to cross-reactivity between related Old-World alphaviruses. To specifically assess the role of the E2 B domain in cross-neutralization, we depleted Mayaro and Chikungunya virus E2 B domain specific antibodies from convalescent sera, finding E2B depletion significantly decreases Mayaro virus specific cross-neutralizing antibody titers with no significant effect on chikungunya virus neutralization, indicating that the E2 B domain is a key target of cross-neutralizing and potentially cross-protective neutralizing antibodies.

Mayaro virus pathogenesis and immunity in rhesus macaques.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes debilitating and persistent arthritogenic disease. While MAYV was previously reported to infect non-human primates (NHP), characterization of MAYV pathogenesis is currently lacking. Therefore, in this study we characterized MAYV infection and immunity in rhesus macaques. To inform the selection of a viral strain for NHP experiments, we evaluated five MAYV strains in C57BL/6 mice and showed that MAYV strain BeAr505411 induced robust tissue dissemination and disease. Three male rhesus macaques were subcutaneously challenged with 105 plaque-forming units of this strain into the arms. Peak plasma viremia occurred at 2 days post-infection (dpi). NHPs were taken to necropsy at 10 dpi to assess viral dissemination, which included the muscles and joints, lymphoid tissues, major organs, male reproductive tissues, as well as peripheral and central nervous system tissues. Histological examination demonstrated that MAYV infection was associated with appendicular joint and muscle inflammation as well as presence of perivascular inflammation in a wide variety of tissues. One animal developed a maculopapular rash and two NHP had viral RNA detected in upper torso skin samples, which was associated with the presence of perivascular and perifollicular lymphocytic aggregation. Analysis of longitudinal peripheral blood samples indicated a robust innate and adaptive immune activation, including the presence of anti-MAYV neutralizing antibodies with activity against related Una virus and chikungunya virus. Inflammatory cytokines and monocyte activation also peaked coincident with viremia, which was well supported by our transcriptomic analysis highlighting enrichment of interferon signaling and other antiviral processes at 2 days post MAYV infection. The rhesus macaque model of MAYV infection recapitulates many of the aspects of human infection and is poised to facilitate the evaluation of novel therapies and vaccines targeting this re-emerging virus.

Therapeutic and prophylactic treatment with a virus-specific antibody is highly effective in rodent models of Chikungunya infection and disease.

Chikungunya virus (CHIKV) has re-emerged as a significant human pathogen in the 21st century, causing periodic, and sometimes widespread, outbreaks over the past 15 years. Although mortality is very rare, a debilitating arthralgia is very common and may persist for months or years. There are no antivirals that are approved for the treatment of CHIKV infection, and current treatment options consist of supportive care only. Herein, we demonstrate the efficacy of a CHIKV-specific antibody in the prophylactic and therapeutic treatment of CHIKV in mouse models of disease. The fully human anti-CHIKV monoclonal Ab SVIR023 demonstrated broad in vitro activity against representative strains from the three major CHIKV clades. Therapeutic treatment with SVIR023 administered 1- or 3-days post-infection resulted in reduced virus in various tissues in a dose- and time-dependent manner. Prophylactic treatment up to 4 weeks prior to virus challenge was also effective in preventing disease in mice. Mice treated with SVIR023 and infected with CHIKV were resistant to secondary challenge and no evidence of antibody enhancement of disease was observed. Treatment with SVIR023 was effective in mouse models of CHIKV infection and disease and further evaluation towards clinical development is warranted.

Nonhuman Primate Models of Zika Virus Infection and Disease during Pregnancy.

Since the explosive outbreak of Zika virus in Brazil and South/Central America in 2015-2016, the frequency of infections has subsided, but Zika virus remains present in this region as well as other tropical and sub-tropical areas of the globe. The most alarming aspect of Zika virus infection is its association with severe birth defects when infection occurs in pregnant women. Understanding the mechanism of Zika virus pathogenesis, which comprises features unique to Zika virus as well as shared with other teratogenic pathogens, is key to future prophylactic or therapeutic interventions. Nonhuman primate-based research has played a significant role in advancing our knowledge of Zika virus pathogenesis, especially with regard to fetal infection. This review summarizes what we have learned from these models and potential future research directions.

Non-replicating adenovirus based Mayaro virus vaccine elicits protective immune responses and cross protects against other alphaviruses.

Mayaro virus (MAYV) is an alphavirus endemic to South and Central America associated with sporadic outbreaks in humans. MAYV infection causes severe joint and muscle pain that can persist for weeks to months. Currently, there are no approved vaccines or therapeutics to prevent MAYV infection or treat the debilitating musculoskeletal inflammatory disease. In the current study, a prophylactic MAYV vaccine expressing the complete viral structural polyprotein was developed based on a non-replicating human adenovirus V (AdV) platform. Vaccination with AdV-MAYV elicited potent neutralizing antibodies that protected WT mice against MAYV challenge by preventing viremia, reducing viral dissemination to tissues and mitigating viral disease. The vaccine also prevented viral-mediated demise in IFN⍺R1-/- mice. Passive transfer of immune serum from vaccinated animals similarly prevented infection and disease in WT mice as well as virus-induced demise of IFN⍺R1-/- mice, indicating that antiviral antibodies are protective. Immunization with AdV-MAYV also generated cross-neutralizing antibodies against two related arthritogenic alphaviruses-chikungunya and Una viruses. These cross-neutralizing antibodies were protective against lethal infection in IFN⍺R1-/- mice following challenge with these heterotypic alphaviruses. These results indicate AdV-MAYV elicits protective immune responses with substantial cross-reactivity and protective efficacy against other arthritogenic alphaviruses. Our findings also highlight the potential for development of a multi-virus targeting vaccine against alphaviruses with endemic and epidemic potential in the Americas.

Pyrimidone inhibitors targeting Chikungunya Virus nsP3 macrodomain by fragment-based drug design.

The macrodomain of nsP3 (nsP3MD) is highly conserved among the alphaviruses and ADP-ribosylhydrolase activity of Chikungunya Virus (CHIKV) nsP3MD is critical for CHIKV viral replication and virulence. No small molecule drugs targeting CHIKV nsP3 have been identified to date. Here we report small fragments that bind to nsP3MD which were discovered by virtually screening a fragment library and X-ray crystallography. These identified fragments share a similar scaffold, 2-pyrimidone-4-carboxylic acid, and are specifically bound to the ADP-ribose binding site of nsP3MD. Among the fragments, 2-oxo-5,6-benzopyrimidine-4-carboxylic acid showed anti-CHIKV activity with an IC50 of 23 µM. Our fragment-based drug discovery approach provides valuable information to further develop a specific and potent nsP3 inhibitor of CHIKV viral replication based on the 2-pyrimidone-4-carboxylic acid scaffold. In silico studies suggest this pyrimidone scaffold could also bind to the macrodomains of other alphaviruses and coronaviruses and thus, have potential pan-antiviral activity.

Targeting Chikungunya Virus Replication by Benzoannulene Inhibitors.

A benzo[6]annulene, 4-(tert-butyl)-N-(3-methoxy-5,6,7,8-tetrahydronaphthalen-2-yl) benzamide (1a), was identified as an inhibitor against Chikungunya virus (CHIKV) with antiviral activity EC90 = 1.45 µM and viral titer reduction (VTR) of 2.5 log at 10 µM with no observed cytotoxicity (CC50 = 169 µM) in normal human dermal fibroblast cells. Chemistry efforts to improve potency, efficacy, and drug-like properties of 1a resulted in a novel lead compound 8q, which possessed excellent cellular antiviral activity (EC90 = 270 nM and VTR of 4.5 log at 10 µM) and improved liver microsomal stability. CHIKV resistance to an analog of 1a, compound 1c, tracked to a mutation in the nsP3 macrodomain. Further mechanism of action studies showed compounds working through inhibition of human dihydroorotate dehydrogenase in addition to CHIKV nsP3 macrodomain. Moderate efficacy was observed in an in vivo CHIKV challenge mouse model for compound 8q as viral replication was rescued from the pyrimidine salvage pathway.

Isolation and Detection of Zika Virus-Infected Rhesus Macaques Lymph Node Cells and Splenocytes.

Zika virus (ZIKV) is a mosquito-borne viral infection that is shed in biological fluids promoting vertical and sexual transmission. Recent outbreaks of ZIKV have been associated with an increase in adult and fetal infection-related diseases. ZIKV infection in rhesus macaques is considered a robust animal model for studying Zika viral infection dynamics and fetal disease. A compelling feature of ZIKV is its ability to persist for long periods of time in immunocompetent hosts and during pregnancy, which may be linked to adverse infection outcomes. One consistent site of viral persistence is lymph node tissues. Utilizing this feature of ZIKV infection could be useful to diagnose viral persistence and to improve efficacy evaluation of antiviral vaccines and therapeutics, as well as for diagnostic and prognostic assessments in humans. We have developed a protocol to isolate lymph node cells using cell type-specific antibody-magnetic bead techniques followed by a one-step qRT-PCR detection of Zika virus RNA. This method fostered the identification of dendritic cells, macrophages, and B cells from the lymph node and spleen as harboring persistent ZIKV RNA.

Rat Cytomegalovirus Virion-Associated Proteins R131 and R129 Are Necessary for Infection of Macrophages and Dendritic Cells.

Cytomegalovirus (CMV) establishes persistent, latent infection in hosts, causing diseases in immunocompromised patients, transplant recipients, and neonates. CMV infection modifies the host chemokine axis by modulating chemokine and chemokine receptor expression and by encoding putative chemokine and chemokine receptor homologues. The viral proteins have roles in cellular signaling, migration, and transformation, as well as viral dissemination, tropism, latency and reactivation. Herein, we review the contribution of CMV-encoded chemokines and chemokine receptors to these processes, and further elucidate the viral tropism role of rat CMV (RCMV) R129 and R131. These homologues of the human CMV (HCMV)-encoded chemokines UL128 and UL130 are of particular interest because of their dual role as chemokines and members of the pentameric entry complex, which is required for entry into cell types that are essential for viral transmission and dissemination. The contributions of UL128 and UL130 to acceleration of solid organ transplant chronic rejection are poorly understood, and are in need of an effective in vivo model system to elucidate the phenomenon. We demonstrated similar molecular entry requirements for R129 and R131 in the rat cells, as observed for HCMV, and provided evidence that R129 and R131 are part of the viral entry complex required for entry into macrophages, dendritic cells, and bone marrow cells.

A neonatal nonhuman primate model of gestational Zika virus infection with evidence of microencephaly, seizures and cardiomyopathy.

Zika virus infection during pregnancy is associated with miscarriage and with a broad spectrum of fetal and neonatal developmental abnormalities collectively known as congenital Zika syndrome (CZS). Symptomology of CZS includes malformations of the brain and skull, neurodevelopmental delay, seizures, joint contractures, hearing loss and visual impairment. Previous studies of Zika virus in pregnant rhesus macaques (Macaca mulatta) have described injury to the developing fetus and pregnancy loss, but neonatal outcomes following fetal Zika virus exposure have yet to be characterized in nonhuman primates. Herein we describe the presentation of rhesus macaque neonates with a spectrum of clinical outcomes, including one infant with CZS-like symptoms including cardiomyopathy, motor delay and seizure activity following maternal infection with Zika virus during the first trimester of pregnancy. Further characterization of this neonatal nonhuman primate model of gestational Zika virus infection will provide opportunities to evaluate the efficacy of pre- and postnatal therapeutics for gestational Zika virus infection and CZS.

Vaccine-Induced Skewing of T Cell Responses Protects Against Chikungunya Virus Disease.

Chikungunya virus (CHIKV) infections can cause severe and debilitating joint and muscular pain that can be long lasting. Current CHIKV vaccines under development rely on the generation of neutralizing antibodies for protection; however, the role of T cells in controlling CHIKV infection and disease is still unclear. Using an overlapping peptide library, we identified the CHIKV-specific T cell receptor epitopes recognized in C57BL/6 infected mice at 7 and 14 days post-infection. A fusion protein containing peptides 451, 416, a small region of nsP4, peptide 47, and an HA tag (CHKVf5) was expressed using adenovirus and cytomegalovirus-vectored vaccines. Mice vaccinated with CHKVf5 elicited robust T cell responses to higher levels than normally observed following CHIKV infection, but the vaccine vectors did not elicit neutralizing antibodies. CHKVf5-vaccinated mice had significantly reduced infectious viral load when challenged by intramuscular CHIKV injection. Depletion of both CD4+ and CD8+ T cells in vaccinated mice rendered them fully susceptible to intramuscular CHIKV challenge. Depletion of CD8+ T cells alone reduced vaccine efficacy, albeit to a lesser extent, but depletion of only CD4+ T cells did not reverse the protective phenotype. These data demonstrated a protective role for CD8+ T cells in CHIKV infection. However, CHKVf5-vaccinated mice that were challenged by footpad inoculation demonstrated equal viral loads and increased footpad swelling at 3 dpi, which we attributed to the presence of CD4 T cell receptor epitopes present in the vaccine. Indeed, vaccination of mice with vectors expressing only CHIKV-specific CD8+ T cell epitopes followed by CHIKV challenge in the footpad prevented footpad swelling and reduced proinflammatory cytokine and chemokines associated with disease, indicating that CHIKV-specific CD8+ T cells prevent CHIKV disease. These results also indicate that a T cell-biased prophylactic vaccination approach is effective against CHIKV challenge and reduces CHIKV-induced disease in mice.

Studies on Dibenzylamines as Inhibitors of Venezuelan Equine Encephalitis Virus.

Alphaviruses are arthropod-transmitted members of the Togaviridae family that can cause severe disease in humans, including debilitating arthralgia and severe neurological complications. Currently, there are no approved vaccines or antiviral therapies directed against the alphaviruses, and care is limited to treating disease symptoms. A phenotypic cell-based high-throughput screen was performed to identify small molecules that inhibit the replication of Venezuelan Equine Encephalitis Virus (VEEV). The compound, 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-(3-fluoro-4-methoxybenzyl)ethan-1-amine (1), was identified as a highly active, potent inhibitor of VEEV with an effective concentration for 90% inhibition of virus (EC90) of 0.89 µM and 7.49 log reduction in virus titers at 10 µM concentration. These data suggest that further investigation of compound 1 as an antiviral therapeutic against VEEV, and perhaps other alphaviruses, is warranted. Experiments suggested that the antiviral activity of compound 1 is directed at an early step in the VEEV replication cycle by blocking viral RNA and protein synthesis.