Development of a Novel Chikungunya Virus-Like Replicon Particle for Rapid Quantification and Screening of Neutralizing Antibodies and Antivirals.

Chikungunya fever is a mosquito-transmitted infectious disease that induces rash, myalgia, and persistent incapacitating arthralgia. At present, no vaccines or antiviral therapies specific to Chikungunya virus (CHIKV) infection have been approved, and research is currently restricted to biosafety level 3 containment. CHIKV-like replicon particles (VRPs) are single-cycle infectious particles containing viral structure proteins, as well as a defective genome to provide a safe surrogate for living CHIKV to facilitate the testing of vaccines and antivirals. However, inefficient RNA transfection and the potential emergence of the competent virus through recombination in mammalian cells limit VRP usability. This study describes a transfection-free system for the safe packaging of CHIK VRP with all necessary components via transduction of mosquito cell lines using a single baculovirus vector. We observed the release of substantial quantities of mosquito cell-derived CHIK VRP (mos-CHIK VRP) from baculovirus-transduced mosquito cell lines. The VRPs were shown to recapitulate viral replication and subgenomic dual reporter expression (enhanced green fluorescent protein [eGFP] and luciferase) in infected host cells. Interestingly, the rapid expression kinetics of the VRP-expressing luciferase reporter (6 h) makes it possible to use mos-CHIK VRPs for the rapid quantification of VRP infection. Treatment with antivirals (suramin or 6-azauridine) or neutralizing antibodies (monoclonal antibodies [MAbs] or patient sera) was shown to inhibit mos-CHIK VRP infection in a dose-dependent manner. Ease of manufacture, safety, scalability, and high throughput make mos-CHIK VRPs a highly valuable vehicle for the study of CHIKV biology, the detection of neutralizing (NT) antibody activity, and the screening of antivirals against CHIKV. IMPORTANCE This study proposes a transfection-free system that enables the safe packaging of CHIK VRPs with all necessary components via baculovirus transduction. Those mosquito cell-derived CHIK VRP (mos-CHIK VRPs) were shown to recapitulate viral replication and subgenomic dual reporter (enhanced green fluorescent protein [eGFP] and luciferase) expression in infected host cells. Rapid expression kinetics of the VRP-expressing luciferase reporter (within hours) opens the door to using mos-CHIK VRPs for the rapid quantification of neutralizing antibody and antiviral activity against CHIKV. To the best of our knowledge, this is the first study to report a mosquito cell-derived alphavirus VRP system. Note that this system could also be applied to other arboviruses to model the earliest event in arboviral infection in vertebrates.

Antigenicity and immunogenicity of chikungunya virus-like particles from mosquito cells.

The spread of chikungunya virus (CHIKV) is reaching pandemic levels, and vaccines and antivirals to control CHIKV infection have yet to be approved. Virus-like particles (VLPs), a self-assembled native multi-subunit protein structure, could potentially be used as an antigen for serological detection and vaccine development. In the current study, we describe the production of novel CHIKV VLPs from mosquitoes using a Baculovirus/Mosquito (BacMos) system in a simple Biosafety Level-2 laboratory. Substantial envelope and capsid protein secretions were detected in culture medium. Co-fractionation of CHIKV E2, E1, and capsid proteins via sucrose gradient ultracentrifugation provided evidence of VLP formation. Transmission electron microscopy and dynamic light scattering analysis revealed the formation of VLPs in the form of spherical particles with a diameter of roughly 40 nm in transduced cells and culture medium. VLP-based IgM capture ELISA in CHIKV patient sera revealed native epitopes on the VLPs. These non-purified VLPs were shown to act as an antigen in CHIKV-specific IgM capture ELISA. The immunization of CHIKV-VLPs alone in mice induced a balance CHIKV-specific IgG2a/IgG1 antibodies and neutralized antibody responses. The study provides support for the hypothesis that mosquito cell-derived CHIKV VLPs could serve as a novel antigen for serological detection and the development of vaccines against CHIKV infection. KEY POINTS: • CHIKV VLPs secreted from BacMos-CHIKV 26S-transduced mosquito cell. • This CHIKV VLPs potentially serve as an alternative capture antigen for MAC-ELISA. • Unadjuvanted CHIK VLPs induce CHIKV-specific IgG and NT responses in mice.

Nanoparticular CpG-adjuvanted SARS-CoV-2 S1 protein elicits broadly neutralizing and Th1-biased immunoreactivity in mice.

The spike (S) protein is a leading vaccine candidate against SARS-CoV-2 infection. The S1 domain of S protein, which contains a critical receptor-binding domain (RBD) antigen, potentially induces protective immunoreactivities against SARS-CoV-2. In this study, we presented preclinical evaluations of a novel insect cell-derived SARS-CoV-2 recombinant S1 (rS1) protein as a potent COVID-19 vaccine candidate. The native antigenicity of rS1 was characterized by enzyme-linked immunosorbent assay with a neutralizing monoclonal antibody targeting the RBD antigen. To improve its immunogenicity, rS1-adjuvanted with fucoidan/trimethylchitosan nanoparticles (FUC-TMC NPs) and cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODNs) were investigated using a mouse model. The S1-specific immunoglobulin G (IgG) titers, FluoroSpot assay, pseudovirus- and prototype SARS-CoV-2-based neutralization assays were assessed. The results showed that the rS1/CpG/ FUC-TMC NPs (rS1/CpG/NPs) formulation induced a broad-spectrum IgG response with potent, long-lasting, and cross-protective neutralizing activity against the emerging SARS-CoV-2 variant of concern, along with a Th1-biased cellular response. Thus, the rS1/CpG/NPs formulation presents a promising vaccination approach against COVID-19.

Facile method for delivering chikungunya viral replicons into mosquitoes and mammalian cells.

Reverse genetics is an important tool in the elucidation of viral replication and the development of countermeasures; however, these methods are impeded by laborious and inefficient replicon delivery methods. This paper demonstrates the use of a baculovirus to facilitate the efficient delivery of autonomous CHIKV replicons into mosquito and mammalian cells in vitro as well as adult mosquitoes in vivo. The efficacy of this approach was verified via co-localization among an eGFP reporter, nsP1, and dsRNA as well as through the inhibition of an RNA-dependent RNA polymerase (RdRp) null mutation (DDAA) in nsP4, or the treatment of a known antiviral compound (6-azauridine). We also investigated the correlation between CHIKV replicon-launched eGFP expression and the effectiveness of CHIKV replicon variants in inducing IFN-ß expression in human cell lines. This delivery method based on a single vector is applicable to mosquito and mammalian cells in seeking to decipher the mechanisms underlying CHIKV replication, elucidate virus-host interactions, and develop antivirals. This study presents an effective alternative to overcome many of the technological issues related to the study and utilization of autonomous arbovirus replicons.

Mosquito Cell-Derived Japanese Encephalitis Virus-Like Particles Induce Specific Humoral and Cellular Immune Responses in Mice.

The Japanese encephalitis virus (JEV) is the major cause of an acute encephalitis syndrome in many Asian countries, despite the fact that an effective vaccine has been developed. Virus-like particles (VLPs) are self-assembled multi-subunit protein structures which possess specific epitope antigenicities related to corresponding native viruses. These properties mean that VLPs are considered safe antigens that can be used in clinical applications. In this study, we developed a novel baculovirus/mosquito (BacMos) expression system which potentially enables the scalable production of JEV genotype III (GIII) VLPs (which are secreted from mosquito cells). The mosquito-cell-derived JEV VLPs comprised 30-nm spherical particles as well as precursor membrane protein (prM) and envelope (E) proteins with densities that ranged from 30% to 55% across a sucrose gradient. We used IgM antibody-capture enzyme-linked immunosorbent assays to assess the resemblance between VLPs and authentic virions and thereby characterized the epitope specific antigenicity of VLPs. VLP immunization was found to elicit a specific immune response toward a balanced IgG2a/IgG1 ratio. This response effectively neutralized both JEV GI and GIII and elicited a mixed Th1/Th2 response in mice. This study supports the development of mosquito cell-derived JEV VLPs to serve as candidate vaccines against JEV.

Baculovirus as an efficient vector for gene delivery into mosquitoes.

Efficient gene delivery technologies play an essential role in the gene functional analyses that are necessary for basic and applied researches. Mosquitoes are ubiquitous insects, responsible for transmitting many deadly arboviruses causing millions of human deaths every year. The lack of efficient and flexible gene delivery strategies in mosquitoes are among the major hurdles for the study of mosquito biology and mosquito-pathogen interactions. We found that Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the type baculovirus species, can efficiently transduce mosquito cells without viral propagation, allowing high level gene expression upon inducement by suitable promoters without obvious negative effects on cell propagation and viability. AcMNPV transduces into several mosquito cell types, efficiently than in commonly used mammalian cell lines and classical plasmid DNA transfection approaches. We demonstrated the application of this system by expressing influenza virus neuraminidase (NA) into mosquito hosts. Moreover, AcMNPV can transduce both larvae and adults of essentially all blood-sucking mosquito genera, resulting in bright fluorescence in insect bodies with little or no tissue barriers. Our experiments establish baculovirus as a convenient and powerful gene delivery vector in vitro and in vivo that will greatly benefit research into mosquito gene regulation, development and the study of mosquito-borne viruses.

Antiviral activities of niclosamide and nitazoxanide against chikungunya virus entry and transmission.

Chikungunya disease results from an infection with the arbovirus, chikungunya virus (CHIKV). Symptoms of CHIKV include fever and persistent, severe arthritis. In recent years, several antiviral drugs have been evaluated in clinical trials; however, no registered antivirals have been approved for clinical therapy. In this study, we established a high-throughput screening (HTS) system based on CHIKV 26S mediated insect cell fusion inhibition assay. Our screening system was able to search potential anti-CHIKV drugs in vitro. Using this system, four compounds (niclosamide, nitazoxanide, niflumic acid, tolfenamic acid) were identified. These compounds were then further analyzed using a microneutralization assay. We determined that niclosamide and nitazoxanide exhibit ability to against CHIKV-induced CPE. The anti-CHIKV abilities of these compounds were further confirmed by RT-qPCR and IFA. Moreover, niclosamide and nitazoxanide were found to (1) limit virus entry, (2) inhibit both viral release and cell-to-cell transmission, and (3) possess broad anti-alphavius activities, including against two clinical CHIKV isolates and two alphaviruses: Sindbis virus (SINV) and Semliki forest virus (SFV). In conclusion, our findings suggested that niclosamide and nitazoxanide were able to inhibit CHIKV entry and transmission, which might provide a basis for the development of novel human drug therapies against CHIKV and other alphavirus infections.

Suramin treatment reduces chikungunya pathogenesis in mice.

The chikungunya virus (CHIKV), an arthritogenic alphavirus, has caused explosive epidemics involving millions of cases. Globally expanding pandemics involving CHIKV and post-CHIKV rheumatic disorders are increasing public health concerns. However, no antiviral interventions or vaccines to control CHIKV infection have yet been approved. Although suramin has been possess anti-CHIKV activity in vitro, whether suramin has anti-CHIKV activity in vivo remains unknown. This study aimed to determine whether suramin treatment could ameliorate CHIKV-induced arthritis in a C57BL/6 mice model. C57BL/6 mice were infected with CHIKVs to evaluate anti-CHIKV activities of suramin in terms of histopathology, viral burden and disease score. Not only did suramin treatment substantially decrease viral loads, but it also significantly ameliorated acute foot lesions in mice. In addition, suramin treatment markedly restores cartilage integrity and reduces the number of IHC positive chondrocyte in mice infected with CHIKV strains 0810bTw and 0706aTw. This in vivo study highlights the potential ability of suramin to treat CHIKV infection in clinical settings.

Topoisomerase II Inhibitors Can Enhance Baculovirus-Mediated Gene Expression in Mammalian Cells through the DNA Damage Response.

BacMam is an insect-derived recombinant baculovirus that can deliver genes into mammalian cells. BacMam vectors carrying target genes are able to enter a variety of cell lines by endocytosis, but the level of expression of the transgene depends on the cell line and the state of the transduced cells. In this study, we demonstrated that the DNA damage response (DDR) could act as an alternative pathway to boost the transgene(s) expression by BacMam and be comparable to the inhibitors of histone deacetylase. Topoisomerase II (Top II) inhibitor-induced DDR can enhance the CMV-IE/enhancer mediated gene expression up to 12-fold in BacMam-transduced U-2OS cells. The combination of a Top II inhibitor, VM-26, can also augment the killing efficiency of a p53-expressing BacMam vector in U-2OS osteosarcoma cells. These results open a new avenue to facilitate the application of BacMam for gene delivery and therapy.

Using Bicistronic Baculovirus Expression Vector System to Screen the Compounds That Interfere with the Infection of Chikungunya Virus.

Chikungunya virus (CHIKV) is the etiologic agent of Chikungunya fever and has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. A bicistronic baculovirus expression system was utilized to co-express CHIKV structural proteins C (capsid), E2 and E1 and the enhanced green fluorescence protein (EGFP) in Spodoptera frugiperda insect cells (Sf21). The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form a syncytium is mediated by the CHIKV E1 allowing it to identify chemicals that can prevent syncytium formation. The compounds characterized by this method could be anti-CHIKV drugs.

Suramin Inhibits Chikungunya Virus Entry and Transmission.

The mosquito-borne Chikungunya virus (CHIKV) is a profound global threat due to its high rate of contagion and the lack of vaccine or effective treatment. Suramin is a symmetric polyanionic naphthylurea that is widely used in the clinical treatment of parasite infections. Numerous studies have reported the broad antiviral activities of suramin; however, inhibition effects against CHIKV have not yet been demonstrated. The aim of this study was thus to investigate the antiviral effect of suramin on CHIKV infection and to elucidate the molecular mechanism underlying inhibition using plaque reduction assay, RT-qPCR, western blot analysis, and plaque assay. Microneutralization assay was used to determine the EC50 of suramin in the CHIKV-S27 strain as well as in three other clinical strains (0611aTw, 0810bTw and 0706aTw). Time-of-addition was used to reveal the anti-CHIKV mechanism of suramin. We also evaluated anti-CHIKV activity with regard to viral entry, virus release, and cell-to-cell transmission. Cytopathic effect, viral RNA, viral protein, and the virus yield of CHIKV infection were shown to diminish in the presence of suramin in a dose-dependent manner. Suramin was also shown the inhibitory activities of the three clinical isolates. Suramin inhibited the early progression of CHIKV infection, due perhaps to interference with virus fusion and binding, which subsequently prevented viral entry. Results of a molecular docking simulation indicate that suramin may embed within the cavity of the E1/E2 heterodimer to interfere with their function. Suramin was also shown to reduce viral release and cell-to-cell transmission of CHIKV. In conclusion, Suramin shows considerable potential as a novel anti-CHIKV agent targeting viral entry, extracellular transmission, and cell-to-cell transmission.

Cell-based analysis of Chikungunya virus E1 protein in membrane fusion.

BACKGROUND: Chikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV). E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored. METHODS: A bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein) were employed to investigate the roles of four E1 amino acid residues (G91, V178, A226, and H230) in membrane fusion activity. RESULTS: Western blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only) was greater than that of cells bearing 26S-based constructs (expressing all structural proteins), the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds. Cells bearing the V178A mutation exhibited a slight decrease in cholesterol-dependence and a higher-pH threshold for fusion. CONCLUSIONS: Cells expressing amino acid substitutions of conserved protein E1 residues of E1-G91 and E1-H230 lost most of the CHIKV E1-mediated membrane fusion activity. Cells expressing mutations of less-conserved amino acids, E1-V178A and E1-A226V, retained membrane fusion activity to levels similar to those expressing wild type E1, but their fusion properties of pH threshold and cholesterol dependence were slightly altered.

Cell-based analysis of Chikungunya virus membrane fusion using baculovirus-expression vectors.

Chikungunya virus infection has emerged in many countries over the past decade. There are no effective drugs for controlling the disease. To develop cell-based system for screening anti-virus drugs, a bi-cistronic baculovirus expression system was utilized to co-express viral structural proteins C (capsid), E2 and E1 and the enhanced green fluorescence protein (EGFP) in Spodoptera frugiperda insect cells (Sf21). The EGFP-positive Sf21 cells fused with each other and with uninfected cells to form a syncytium, allowing characterization of cholesterol and low pH requirements for syncytium formation. Western blot analysis showed three structural proteins were expressed in baculovirus infected cells. The structural proteins of Chikungunya virus that is required for cell fusion was determined with various recombinant baculoviruses bearing different lengths of the viral structural protein genes. Protein E1 was required for cell fusion and indicating that Chikungunya viral membrane fusion was a class II membrane fusion. It was also demonstrated that the heterologous expression of alphavirus monomeric E1 can induce insect cell fusions. Furthermore, this cell-based system provides a model for studying class II viral membrane fusion.