Ag/Au-incorporated trimethyl chitosan-shell hybrid particles as reinforcing and antioxidant fillers for trimethyl chitosan hydrogel.

N,N,N-Trimethyl chitosan (TMC) is a quaternized chitosan with versatile biological features. However, low mechanical strength limits its uses, for example, as hydrogels for tissue engineering applications. This study illustrates a viable synthesis of metal/polymer hybrid, core-shell colloidal particles and their use as reinforcing and antioxidant fillers for TMC hydrogels. The core-shell particles were initially synthesized by surfactant-free emulsion polymerization, induced by a photo-redox initiating system of riboflavin assisted by a 3° amine and 2° alcohol co-initiators. The synthesized core-shell particles were based on two polymeric shells: TMC and chitosan, and two polymeric cores: poly (hydroxypropyl methacrylate) (PHPMA) and poly(2-hydroxy ethyl methacrylate) (PHEMA). The presence of both 3° amine on TMC and 2° alcohol on HPMA monomer enhanced the photopolymerization performance. The TMC-based particles had sizes of 122-154 nm and zeta potentials of 10-35 mV, bringing the colloidal stability in the 4-10 pH range. Furthermore, due to the presence of TMC on the shell layer, the core-shell particles could be used as templates to grow the Ag/Au bimetallic nanoparticles with alloy and core-shell types through a thermal reduction. The prepared hybrid particles were incorporated in TMC hydrogels as a multifunctional filler, improving their mechanical and antioxidant properties.

Intranasal immunization with the bivalent SARS-CoV-2 vaccine effectively protects mice from nasal infection and completely inhibits disease development.

With the continuous emergence of coronavirus disease 2019 (COVID-19) waves, the scientific community has developed a vaccine that offers broad-spectrum protection at virus-targeted organs for inhibiting the transmission and protection of disease development. In the present study, a bivalent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine containing receptor-binding domain (RBD) protein of spike from Wuhan-1 and omicron BA.1 loaded in nanoparticles, bivalent RBD NPs, was developed. The immunogenicity and protective efficacy of this vaccine candidate were evaluated using an in vivo model. Results showed that mice that received intranasal cGAMP-adjuvanted bivalent RBD-NPs vaccine elicited robust and durable antibody responses. The stimulated antibody broadly neutralized the ancestral strain and variants of concerns (delta and omicron BA.1) in the upper and lower respiratory tracts. Furthermore, the immunized mice developed T-cell response in their lung tissue. Importantly, intranasal immunization with this vaccine candidate efficiently protected mice from nasal infection caused by both Wuhan-1 and BA.1 viruses. Immunized mice that remained susceptible to nasal infection did not develop any symptoms. This is because activated responses in the nasal cavity significantly suppressed virus production. Another word is this nasal vaccine completely protected the mice from disease development and mortality. Therefore, the bivalent RBD vaccine platform has potential to be developed into an anti-SARS-CoV-2 universal vaccine.

Responses of primary human nasal epithelial cells to COVID-19 vaccine candidate.

BACKGROUND: Upper respiratory tract is the primary target of SARS-CoV-2. Therefore, nasal immune responses act as the first line of defense against SARS-CoV-2 infection. OBJECTIVE: We aim to investigate the immune responses of human nasal epithelial cells (HNEpCs) upon stimulation with a COVID-19 vaccine candidate. This candidate named RBD-NPs is composed of SARS-CoV-2 receptor-binding domain (RBD) encapsulated within the N,N,N-trimethyl chitosan nanoparticles (TMC-NPs). METHODS: HNEpCs were stimulated with RBD-NPs, empty NPs, or soluble RBD at various concentrations. After 24 and 48 h of treatment, cells viability and delivery of the immunogens were assessed using XTT assay and flow cytometry. Levels of cytokines and chemokines in the supernatant were quantified with Bio-plex Human Cytokine Assay. Communication between RBD-NPs-stimulated HNEpCs and monocyte-derived dendritic cells (MoDCs) was assessed through differentiation of MoDCs into mature phenotype. RESULTS: RBD-NPs as high as 100 µg exerted no toxicity to HNEpCs and could effectively be delivered to HNEpCs. Treatment of HNEpCs with RBD-NPs strongly activated production of several pro-inflammatory cytokines, chemokines, Th1-related cytokines and the monocytes/macrophages growth factors. Interestingly, soluble mediators secreted from RBD-NPs treated HNEpCs significantly upregulated the expression of maturation markers (CD80, CD83, CD86 and HLA-DR) on the MoDCs. CONCLUSION: This study demonstrated that our COVID-19 vaccine candidate drove HNEpCs into immunologically competent cells that not only exerted anti-viral innate immune responses but also potently induced MoDCs maturation.

Novel Potent Autophagy Inhibitor Ka-003 Inhibits Dengue Virus Replication.

Every year, dengue virus (DENV) affects millions of people. Currently, there are no approved drugs for the treatment of DENV infection. Autophagy is a conserved degradation process that was shown to be induced by DENV infection and required for optimal DENV replication. The modulation of autophagy is, therefore, considered an attractive target to treat DENV infection. This study carried out a high-content image screen analysis using Crispr-Cas9 GFP-LC3 knocked-in HeLa cells of a compound library synthesized from or inspired by natural products and their biocongener precursors to discover novel autophagy inhibitors. The screen identified Ka-003 as the most effective compound for decreasing the number of autophagic vacuoles inside cells upon autophagy induction. Ka-003 could inhibit autophagy in a dose-dependent manner at low micromolar concentrations. More importantly, Ka-003 demonstrated the concentration-dependent inhibition of DENV production in Crispr-Cas9 GFP-LC3 knocked-in THP-1 monocytes. The core structure of Ka-003, which is a methyl cyclohexene derivative, resembles those found in mulberry plants, and could be synthetically prepared in a bioinspired fashion. Taken together, data indicate that Ka-003 hampered autophagy and limited DENV replication. The low cytotoxicity of Ka-003 suggests its therapeutic potential, which warrants further studies for the lead optimization of the compound for dengue treatment.

The Adjuvant Activity of BCG Cell Wall Cytoskeleton on a Dengue Virus-2 Subunit Vaccine.

The uneven immunogenicity of the attenuated tetravalent dengue vaccine has made it difficult to achieve balanced protection against all four serotypes of the dengue virus (DENV). To overcome this problem, non-replicative vaccines have come into focus, as their immunogenicity is adjustable. This approach is excellent for multivalent vaccines but commonly faces the issue of low immunogenicity. In this present study, we developed a non-replicating dengue vaccine composed of UV-inactivated dengue virus-2 (UV-DENV-2) and DENV-2 NS1-279 protein encapsidated within nanoparticles. This vaccine candidate was administered in the presence of BCG cell wall cytoskeleton (BCG-CWS) as an adjuvant. We revealed, here, that encapsidated immunogens with BCG-CWS exerted potent activities on both B and T cells and elicited Th-1/Th-2 responses in mice. This was evidenced by BCG-CWS significantly augmenting antibody-mediated complement-fixing activity, strongly stimulating the antigen-specific polyfunctional T cell responses, and activating mixed Th-1/Th-2 responses specific to DENV-2- and NS1-279 antigens. In conclusion, BCG-CWS potently adjuvanted the inactivated DENV-2 and DENV subunit immunogens. The mechanism of adjuvanticity remains unclear. This study revealed the potential use of BCG-CWS in vaccine development.

A bivalent form of nanoparticle-based dengue vaccine stimulated responses that potently eliminate both DENV-2 particles and DENV-2-infected cells.

Dengue is the most prevalent mosquito-borne viral disease and continues to be a global public health concern. Although a licensed dengue vaccine is available, its efficacy and safety profile are not satisfactory. Hence, there remains a need for a safe and effective dengue vaccine. We are currently developing a bivalent dengue vaccine candidate. This vaccine candidate is composed of a C-terminus truncated non-structural protein 1 (NS11-279) and envelope domain III (EDIII) of DENV-2 encapsidated in the nanocarriers, N, N, N-trimethyl chitosan nanoparticles (TMC NPs). The immunogenicity of this bivalent vaccine candidate was investigated in the present study using BALB/c mice. In this work, we demonstrate that NS1 + EDIII TMC NP-immunized mice strongly elicited antigen-specific antibody responses (anti-NS1 and anti-EDIII IgG) and T-cell responses (NS1- and EDIII-specific-CD4+ and CD8+ T cells). Importantly, the antibody response induced by NS1 + EDIII TMC NPs provided antiviral activities against DENV-2, including serotype-specific neutralization and antibody-mediated complement-dependent cytotoxicity. Moreover, the significant upregulation of Th1- and Th2-associated cytokines, as well as the increased levels of antigen-specific IgG2a and IgG1, indicated a balanced Th1/Th2 response. Collectively, our findings suggest that NS1 + EDIII TMC NPs induced protective responses that can not only neutralize infectious DENV-2 but also eliminate DENV-2-infected cells.

Mice immunized with trimethyl chitosan nanoparticles containing DENV-2 envelope domain III elicit neutralizing antibodies with undetectable antibody-dependent enhancement activity.

Dengue is a disease that poses a significant global public health concern. Although a tetravalent live-attenuated dengue vaccine has been licensed, its efficacy is still debated due to evidence of vaccine breakthrough infection. To avoid this issue, dengue vaccines should stimulate a high degree of serotype-specific response. Thus, envelope domain III (EDIII), which contains serotype-specific neutralizing epitopes, is an attractive target for dengue vaccine development. In this study, we investigated how EDIII encapsidated in N, N, N-trimethyl chitosan chloride nanoparticles (TMC NPs) stimulates a serotype-specific response and whether this response exerts a potential in vitro breakthrough infection. The immune response to DENV-2 elicited by EDIII TMC NP-immunized mice was monitored. We demonstrated that immunization with EDIII TMC NPs resulted in a high level of anti-EDIII antibody production. These antibodies included IgG, IgG1, and IgG2a subtypes. Importantly, antibodies from the immunized mice exerted efficient neutralizing activity with undetectable antibody dependent enhancement (ADE) activity. We also found that EDIII TMC NPs activated functional EDIII-specific CD4+ and CD8+ T cell responses. In conclusion, EDIII TMC NPs stimulated humoral immunity with a strong neutralizing antibody response, as well as a cellular immune response against DENV-2.

The STING Ligand and Delivery System Synergistically Enhance the Immunogenicity of an Intranasal Spike SARS-CoV-2 Vaccine Candidate.

The respiratory organ serves as a primary target site for SARS-CoV-2. Thus, the vaccine-stimulating immune response of the respiratory tract is significant in controlling SARS-CoV-2 transmission and disease development. In this study, mucoadhesive nanoparticles were used to deliver SARS-CoV-2 spike proteins (S-NPs) into the nasal tracts of mice. The responses in the respiratory organ and the systemic responses were monitored. The administration of S-NPs along with cGAMP conferred a robust stimulation of antibody responses in the respiratory tract, as demonstrated by an increase of IgA and IgG antibodies toward the spike proteins in bronchoalveolar lavages (BALs) and the lungs. Interestingly, the elicited antibodies were able to neutralize both the wild-type and Delta variant strains of SARS-CoV-2. Significantly, the intranasal immunization also stimulated systemic responses. This is evidenced by the increased production of circulating IgG and IgA, which were able to neutralize and bind specifically to the SARS-CoV-2 virion and spike protein. Additionally, this intranasal administration potently activated a splenic T cell response and the production of Th-1 cytokines, suggesting that this vaccine may well activate a cellular response in the respiratory tract. The results demonstrate that STING agonist strongly acts as an adjuvant to the immunogenicity of S-NPs. This platform may be an ideal vaccine against SARS-CoV-2.

Peritoneal Administration of a Subunit Vaccine Encapsulated in a Nanodelivery System Not Only Augments Systemic Responses against SARS-CoV-2 but Also Stimulates Responses in the Respiratory Tract.

The COVID-19 pandemic has currently created an unprecedented threat to human society and global health. A rapid mass vaccination to create herd immunity against SARS-CoV-2 is a crucial measure to ease the spread of this disease. Here, we investigated the immunogenicity of a SARS-CoV-2 subunit vaccine candidate, a SARS-CoV-2 spike glycoprotein encapsulated in N,N,N-trimethyl chitosan particles or S-TMC NPs. Upon intraperitoneal immunization, S-TMC NP-immunized mice elicited a stronger systemic antibody response, with neutralizing capacity against SARS-CoV-2, than mice receiving the soluble form of S-glycoprotein. S-TMC NPs were able to stimulate the circulating IgG and IgA as found in SARS-CoV-2-infected patients. In addition, spike-specific T cell responses were drastically activated in S-TMC NP-immunized mice. Surprisingly, administration of S-TMC NPs via the intraperitoneal route also stimulated SARS-CoV-2-specific immune responses in the respiratory tract, which were demonstrated by the presence of high levels of SARS-CoV-2-specific IgG and IgA in the lung homogenates and bronchoalveolar lavages of the immunized mice. We found that peritoneal immunization with spike nanospheres stimulates both systemic and respiratory mucosal immunity.

Intranasal Administration of RBD Nanoparticles Confers Induction of Mucosal and Systemic Immunity against SARS-CoV-2.

Mucosal immunity plays a significant role in host defense against viruses in the respiratory tract. Because the upper respiratory airway is a primary site of SARS-CoV-2 entry, immunization at the mucosa via the intranasal route could potentially lead to induction of local sterilizing immunity that protects against SARS-CoV-2 infection. In this study, we evaluated the immunogenicity of a receptor-binding domain (RBD) of SARS-CoV-2 spike glycoprotein loaded into N,N,N-trimethyl chitosan nanoparticles (RBD-TMC NPs). We showed that intranasal delivery of RBD-TMC NPs into mice induced robust local mucosal immunity, as evidenced by the presence of IgG and IgA responses in BALs and the lungs of immunized mice. Furthermore, mice intranasally administered with this platform of immunogens developed robust systemic antibody responses including serum IgG, IgG1, IgG2a, IgA and neutralizing antibodies. In addition, these immunized mice had significantly higher levels of activated splenic CD4+ and CD8+ cells compared with those that were administered with soluble RBD immunogen. Collectively, these findings shed light on an alternative route of vaccination that mimics the natural route of SARS-CoV-2 infection. This route of administration stimulated not only local mucosal responses but also the systemic compartment of the immune system.

Whole inactivated dengue virus-loaded trimethyl chitosan nanoparticle-based vaccine: immunogenic properties in ex vivo and in vivo models.

Dengue virus (DENV) is a mosquito-borne virus that poses an incomparable public health problem, particularly in tropical and subtropical areas. Vaccination remains the most rational measure for controlling DENV infection. In this study, an ultraviolet irradiation (UV)-inactivated DENV-2 carried by N,N,N-trimethyl chitosan nanoparticles (UV-inactivated DENV2 TMC NPs) was investigated as a potential non-replicating dengue vaccine candidate. Using a human ex vivo model, the human monocyte-derived dendritic cells (MoDCs), we showed that TMC served as both a vaccine vehicle and a potent adjuvant. TMC NPs not only efficiently enhanced UV-inactivated DENV2 internalization into MoDCs but also greatly increased the breadth of UV-inactivated DENV2 immunogenicity to drive the maturation of MoDCs. Moreover, UV-inactivated DENV2 TMC NPs were highly immunogenic in mice, inducing greater levels of antibodies (total IgG, IgG1, IgG2a and neutralizing antibodies) and T cells (activated CD4⁺ and CD8⁺ T cells) against DENV-2 compared to soluble DENV-2 immunogens. Notably, the neutralizing activity of sera from mice immunized with UV-inactivated DENV2 TMC NPs was significantly augmented in the presence of complement activation, leading to the strong elimination of both DENV-2 particles and infected cells. We further showed that the immunogenicity of an inactivated dengue-based vaccine was significantly improved in a concentration-dependent manner. These positive results warrant further investigations of this platform of vaccine delivery for tetravalent vaccines or monovalent vaccines in sequential immunizations.

Nanodelivery system enhances the immunogenicity of dengue-2 nonstructural protein 1, DENV-2 NS1.

Nonstructural protein 1 (NS1) of dengue virus (DENV) is currently recognized as a dengue vaccine candidate. Unfortunately, most of non-replicating immunogens typically stimulate unsatisfactory immune responses, thus, the additional adjuvant is required. In this study, C-terminal truncated DENV-2 NS1 loaded in N,N,N, trimethyl chitosan nanoparticles (NS11-279TMC NPs) was prepared through the ionic gelation method. The immunogenicity of NS11-279TMC NPs was investigated using human ex vivo as well as the murine model. Through a human ex vivo model, it was demonstrated in this study that not only can TMC particles effectively deliver NS11-279 protein into monocyte-derived dendritic cells (MoDCs), but also potently stimulate those cells, resulting in increased expression of maturation marker (CD83), costimulating molecules (CD80, CD86 and HLA-DR) and markedly secreted various types of innate immune cytokines/chemokines. Moreover, mice administered with NS11-279TMC NPs strongly elicited both antibody and T cell responses, produced higher levels of IgG, IgG1, IgG2a and potently activated CD8+ T cells, as compared to mice administered with soluble NS11-279. Importantly, we further demonstrated that anti-NS11-279 antibody induced by this platform of NS11-279 effectively eliminated DENV-2 infected cells through antibody dependent complement-mediated cytotoxicity. Significantly, anti-DENV2 NS11-279 antibody exerted cross-antiviral activity against DENV-1 and -4 but not against DENV-3 infected cells. These findings demonstrate that TMC exerts a desirable adjuvant for enhancing delivery and antigenicity of NS1 based dengue vaccine.

Development of an adjuvanted nanoparticle vaccine against influenza virus, an in vitro study.

Influenza is an infectious respiratory illness caused by influenza viruses. Despite yearly updates, the efficacy of influenza vaccines is significantly curtailed by the virus antigenic drift and antigenic shift. These constant changes to the influenza virus make-up also challenge the development of a universal flu vaccine, which requires conserved antigenic regions shared by influenza viruses of different subtypes. We propose that it is possible to bypass these challenges by the development of an influenza vaccine based on conserved proteins delivered in an adjuvanted nanoparticle system. In this study, we generated influenza nanoparticle constructs using trimethyl chitosan nanoparticles (TMC nPs) as the carrier of recombinant influenza hemagglutinin subunit 2 (HA2) and nucleoprotein (NP). The purified HA2 and NP recombinant proteins were encapsulated into TMC nPs to form HA2-TMC nPs and NP-TMC nPs, respectively. Primary human intranasal epithelium cells (HNEpCs) were used as an in vitro model to measure immunity responses. HA2-TMC nPs, NP-TMC nPs, and HA2-NP-TMC nPs (influenza nanoparticle constructs) showed no toxicity in HNEpCs. The loading efficiency of HA2 and NP into the TMC nPs was 97.9% and 98.5%, respectively. HA2-TMC nPs and NP-TMC nPs more efficiently delivered HA2 and NP proteins to HNEpCs than soluble HA2 and NP proteins alone. The induction of various cytokines and chemokines was more evident in influenza nanoparticle construct-treated HNEpCs than in soluble protein-treated HNEpCs. In addition, soluble factors secreted by influenza nanoparticle construct-treated HNEpCs significantly induced MoDCs maturation markers (CD80, CD83, CD86 and HLA-DR), as compared to soluble factors secreted by protein-treated HNEpCs. HNEpCs treated with the influenza nanoparticle constructs significantly reduced influenza virus replication in an in vitro challenge assay. The results indicate that TMC nPs can be used as influenza vaccine adjuvants and carriers capable of delivering HA2 and NP proteins to HNEpCs.

Proteomic analysis of CHIKV-infected human fibroblast-like synoviocytes: Identification of host factors potentially associated with CHIKV replication and cellular pathogenesis.

Chikungunya virus (CHIKV) is a mosquito-borne virus that causes arthralgic fever. Fibroblast-like synoviocytes play a key role in joint damage in inflammatory arthritides and can additionally serve as target cells for CHIKV infection. To gain a better understanding of CHIKV-induced arthralgia, the interaction between CHIKV and synoviocytes was investigated at the protein level. A gel-enhanced liquid chromatography-mass spectrometry (GeLC-MS/MS) approach was used to examine protein expression from primary human fibroblast-like synoviocytes (HFLS) infected with clinical isolates of CHIKV at 12 and 24 hr post infection. Our analysis identified 259 and 241 proteins of known function that were differentially expressed (>1.5 or <-1.5 fold change) following CHIKV infection at 12 and 24 hpi, respectively. These proteins are involved in cellular homeostasis, including cellular trafficking, cytoskeletal organization, immune response, metabolic process, and protein modification. Some of these proteins have previously been reported to participate in arthralgia/arthritis and the death of infected cells. Our results provide information on the CHIKV-induced modulation of cellular proteins of HFLS at an early stage of infection, as well as highlighting biological processes associated with CHIKV infection in the main target cells of the joint.

Insect anionic septapeptides suppress DENV replication by activating antiviral cytokines and miRNAs in primary human monocytes.

Dengue viruses (DENVs) have threatened 2/3 of the world population for decades. Thus, combating DENV infection with either antiviral therapy or protective vaccination is an urgent goal. In the present study, we investigated the anti-DENV activity of insect cell-derived anionic septapeptides from C6/36 mosquito cell cultures persistently infected with DENV. These molecules were previously shown to protect C6/36 and Vero cells against DENV infection. We found that treatment with these septapeptides strongly and rapidly downregulated the multiplication of DENV-1 16007, DENV-3 16562, and DENV-4 1036 but not that of DENV-2 16681 in primary human monocytes. This inhibitory effect was likely mediated through various routes including the increased production of antiviral cytokines (IFN-I), activation of mononuclear cell migration, and upregulation of the expression of antiviral miRNAs (has-miR-30e*, has-miR-133a, and has-miR-223) and inflammation-related miRNAs (has-miR-146a and has-miR-147). In conclusion, anionic septapeptides exerted anti-DENV activity in human monocytes through the upregulation of innate immune responses and the activation of several previously reported antiviral and inflammation-related miRNAs.

In vitro antiviral activity of spirotetronate compounds against dengue virus serotype 2.

Spirotetronate compounds are polyketide secondary metabolites with diverse biological functions, such as antibacterial, antitumor and antiviral activities. Three pure spirotetronate compounds (2EPS-A, -B, -C) isolated from Actinomadura strain 2EPS showed inhibitory activity against dengue virus serotype 2 (DENV-2). 2EPS-A, -B and -C demonstrated the LC50 values of 11.6, 27.5 and 12.0 µg/ml, respectively, in a test of cytotoxicity to Vero cells. The least cytotoxic, 2EPS-B, was further analyzed for its impact on viral propagation in a cell-based replication assay. At a concentration of 6.25 µg/ml, it could reduce the DENV-2 infection in Vero cells by about 94% when cells infected with DENV-2 were exposed to 2EPS-B, whereas direct treatment of DENV-2 with 2EPS-B at the same concentration prior to subsequent infection to Vero cell yielded no inhibition. 2EPS-A, -B an -C showed strong DENV-2 NS2B-NS3 protease inhibition in an in vitro assay, with IC50 values of 1.94 ± 0.18, 1.47 ± 0.15 and 2.51 ± 0.21 µg/ml, respectively. Therefore, the spirotetronate compounds appear to prevent viral replication and viral assembly by inhibition of the viral protease.

Heterogeneity of clinical isolates of chikungunya virus and its impact on the responses of primary human fibroblast-like synoviocytes.

Low-passage clinical isolates of chikungunya virus (CHIKV) were found to be a mixture of large- and small-plaque viruses, with small-plaque viruses being the predominant species. To investigate the contribution of plaque variants to the pathology of the joint, primary human fibroblast-like synoviocytes (HFLS) were used. Large- and small-plaque viruses were purified from two clinical isolates, CHIKV-031C and CHIKV-033C, and were designated CHIKV-031L and CHIKV-031S and CHIKV-033L and CHIKV-033S, respectively. The replication efficiencies of these viruses in HFLSs were compared and it was found that CHIKV-031S and CHIKV-033S replicated with the highest efficiency, while the parental clinical isolates had the lowest efficiency. Interestingly, the cytopathic effects (CPE) induced by these viruses correlated with neither the efficiency of replication nor the plaque size. The small-plaque viruses and the clinical isolates induced cell death rapidly, while large-plaque viruses induced slow CPE in which only 50 % of the cells in infected cultures were rounded up and detached on day 5 of infection. The production of proinflammatory cytokines and chemokines from infected HFLSs was evaluated. The results showed that the large-plaque viruses and the clinical isolates, but not small-plaque variants, were potent inducers of IL-6, IL-8 and MCP-1, and were able to migrate monocytes/macrophages efficiently. Sequencing data revealed a number of differences in amino acid sequences between the small- and large-plaque viruses. The results suggest that it is common for clinical isolates of CHIKV to be heterogeneous, while the variants may have distinct roles in the pathology of the joint.

The synergistic effect of nsP2-L618, nsP3-R117, and E2-K187 on the large plaque phenotype of chikungunya virus.

Chikungunya virus (CHIKV), a mosquito-borne Alphavirus, is the etiological agent of chikungunya fever. CHIKV re-emerged from 2004 onwards, and subsequently caused major outbreaks in many parts of the world including the Indian Ocean islands, Asia, and the Americas. In this study, a large plaque variant of CHIKV isolated from patient in Thailand was subjected to repeated cycles of plaque-purification in Vero cells. The resulting virus produced homogenous large plaques and showed a more pathogenic phenotype than the parental wild-type CHIKV. Whole genome analysis of the large plaque virus in comparison to parental isolate revealed a number of mutations, leading to the following amino acid changes: nsP2 (P618→L), nsP3 (G117→R), and E2 (N187→K). Eight recombinant CHIKVs were constructed to determine which amino acids mediated the large plaque phenotype. The results showed the recombinant virus which contains all three mutations, rCHK-L, produced significantly larger plaques than the other recombinant viruses (p < 0.01). Moreover, the plaque size of the other recombinant virus tended to be smaller if they contained only one or two of the large plaque associated mutations in the viral genome. In conclusion, the combination of all three residues (nsP2-L618, nsP3-R117, and E2-K187) is required to produce the large plaque phenotype of CHIKV.

Ubiquitin-Conjugating Enzyme E2 L3 is Downregulated by the Chikungunya Virus nsP2 Protease.

PURPOSE: Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus that causes chikungunya fever in humans. The CHIKV non-structural protein 2 (nsP2) is a multifunctional protein that additionally modulates the host cell to dampen the innate immune response and inhibit other cellular processes. EXPERIMENTAL DESIGN: To further investigate the interactions of nsP2 with host cells, the protease domain of CHIKV nsP2 (nsP2-pro) is transfected into Hela cells, and differential protein expression is detected by 2D polyacrylamide gel electrophoresis. RESULTS: A total of 21 differentially regulated (six upregulated, 15 downregulated) spots are observed, of which five are identified by mass spectrometry. The downregulation of one of the identified proteins, ubiquitin-conjugating enzyme E2 L3 (UBE2L3) is confirmed by western blotting of both nsP2-pro transfection and CHIKV natural infection, and the downregulation of UBE2L3 is additionally shown to require an enzymatically active nsP2 protease domain. Transfection of full length UBE2L3 into HEK293T/17 cells prior to CHIKV infection reduce levels of infection and E protein expression but do not alter RNA genome levels. CONCLUSION: These results suggest that UBE2L3 is a cellular target of the CHIKV nsP2 protease, and this possibly mediates the pathogenesis of chikungunya fever.

Small plaque size variant of chikungunya primary isolate showed reduced virulence in mice.

BACKGROUND: Plaque size is a common feature of viral characterization. Small plaque size is used as a marker of attenuation for live-attenuated vaccine development. OBJECTIVE: To investigate whether the naturally occurring plaque size variation reflects virulence of the variants of chikungunya virus (CHIKV). METHODS: We selected and purified a variant with small plaque size from the primary isolate. The viral variant was tested for the plaque morphology, in vitro growth kinetics and mouse neurovirulence in comparison with the parental wild type. RESULTS: The small plaque size variant showed stable homogenous small plaques after 4 plaque purifications. The small plaque virus grew slower and to the lower titer when compared with wild type virus. After 21 days of infection, mice that received small plaque virus showed 98% survival rate while 74% of mice survived after infected with wild type virus. CONCLUSION: The small plaque size variant of CHIKV can be obtained by plaque purification and the virus displays decreased virulence.