Dengue Virus-like Particles Research Articles

Synergistic Role of the AuAg-Fe3O4 Nanoenzyme for Ultrasensitive Immunoassay of Dengue Virus.

A combination of magnetic and noble metal nanoparticles (NPs) has recently emerged as a potential substance for rapid and sensitive immunosorbent assays. However, to make the assay an alternative method for Enzyme-linked immunosorbent assay, the individual role of each nanoparticle must be explored properly. In this work, an immunoassay has been proposed using two antibody-conjugated iron oxide nanoparticles (Fe3O4NPs) and gold-silver bimetallic nanoparticles (AuAgNPs) to enhance the sensitivity of virus detection by colorimetric TMB/H2O2 signal amplification. A synergistic effect is monitored between Fe3O4NPs and AuAgNPs, which is explored for colorimetric virus detection. The sensor exploits the synergistic effect between the nanoparticles to successfully detect a wide range of dengue virus-like particle (DENV-LP) concentrations ranging from 10 to 100 pg/mL with a detection limit of up to 2.6 fg/mL. In the presence of a target DENV-LP, a sandwich-like structure is formed, which restricts the electron transfer and the associated synergistic effect between the nanoparticles, restricting the TMB oxidation process. Therefore, the synergistic effect is the key to the present work, which accounts for the enhanced rate of the enzymatic reaction on TMB and makes the current method of virus detection more sensitive and reliable compared to the others.

A tetravalent dengue virus-like particle vaccine induces high levels of neutralizing antibodies and reduces dengue replication in non-human primates.

Dengue virus (DENV) represents a significant global health burden, with 50% of the world's population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the virus but lack the viral genome, are an attractive approach. Here, we describe a dengue VLP (DENVLP) vaccine which generates a neutralizing antibody response against all four DENV serotypes in 100% of immunized non-human primates for up to 1 year. Additionally, DENVLP vaccination produced no ADE response against any of four DENV serotypes in vitro. DENVLP vaccination reduces viral replication in a non-human primate challenge model. We also show that transfer of purified IgG from immunized monkeys into immunodeficient mice protects against subsequent lethal DENV challenge, indicating a humoral mechanism of protection. These results indicate that this DENVLP vaccine is immunogenic and can be considered for clinical evaluation. Immunization of non-human primates with a tetravalent DENVLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes.IMPORTANCEDengue is a viral disease that infects nearly 400 million people worldwide and causes dengue hemorrhagic fever, which is responsible for 10,000 deaths each year. Currently, there is no therapeutic drug licensed to treat dengue infection, which makes the development of an effective vaccine essential. Virus-like particles (VLPs) are a safe and highly immunogenic platform that can be used in young children, immunocompromised individuals, as well as healthy adults. In this study, we describe the development of a dengue VLP vaccine and demonstrate that it induces a robust immune response against the dengue virus for over 1 year in monkeys. The immunity induced by this vaccine reduced live dengue infection in both murine and non-human primate models. These results indicate that our dengue VLP vaccine is a promising vaccine candidate.

Adaptation of single virus fusion assay to study fusion of dengue virus-like particles with model lipid membranes

Adaptation of single virus fusion assay to study fusion of dengue virus-like particles with model lipid membranes

An integrative and multiscale approach to study dengue virus-like particle dynamics.

Dengue virus is an enveloped virus which infects millions of people worldwide. The only available vaccine has low efficacy and is recommended only for individuals with prior dengue infection. Virus-like particles (VLPs) are promising platforms as vaccine candidates due to their non-infectious and highly immunogenic nature. These particles lack a genomic core and are highly unstable and heterogeneous in size. This motivates the characterization of the biophysical and structural properties of VLPs to facilitate the rational production of stable and homogenous particles. Dengue VLPs elicits neutralizing antibodies similar to infectious virions. Dengue VLPs are enveloped particles in which sixty envelope (E) and membrane (M) proteins are embedded in a lipid vesicle. Similarly, to the dengue virus, the VLPs undergo a maturation process, involving large conformational changes, leading to exposure of epitopes that subsequently determine the immune response. In this work, the structure and intrinsic dynamics of VLPs and their conformational transitions were investigated using integrative modeling and molecular simulations. To gain atomistic insights into the maturation process and to facilitate the development of stable enveloped VLPs, we employed an integrative approach combining multiscale modeling and simulations with experimental data from cryo-electron microscopy, lipidomics, and secretion assays. Coarse-grained simulations of mature and immature VLPs revealed the effective number of lipids that result in optimally stable and spherical particles. Furthermore, these models were used as a platform to simulate the transition between conformational states in order to understand the maturation process in unprecedented detail. Finally, atomistic MD simulations performed on native dengue and dengue-Japanese encephalitis virus chimeric E/M proteins and their mutants highlighted the role of protein-lipid interactions in governing VLP stability. This integrative approach aims towards the future development of stable and immunogenic next-generation vaccines.

Quality assessment of virus-like particle: A new transmission electron microscopy approach.

Transmission electron microscopy (TEM) is a gold standard analytical method for nanoparticle characterization and is playing a valuable role in virus-like particle (VLP) characterization extending to other biological entities such as viral vectors. A dedicated TEM facility is a challenge to both small and medium-sized enterprises (SMEs) and companies operating in low-and-middle income countries (LMICs) due to high start-up and running costs. A low-voltage TEM solution with assisted image acquisition and analysis such as the MiniTEM system, coupled with Vironova Imaging and Analysis Software (VIAS) could provide an affordable and practical alternative. The MiniTEM system has a small footprint and software that enables semi-automated data collection and image analysis workflows using built-in deep learning methods (convolutional neural networks) for automation in analysis, increasing speed of information processing and enabling scaling to larger datasets. In this perspective we outline the potential and challenges in the use of TEM as mainstream analytical tool in manufacturing settings. We highlight the rationale and preliminary findings from our proof-of-concept study aiming to develop a method to assess critical quality attributes (CQAs) of VLPs and facilitate adoption of TEM in manufacturing settings. In our study we explored all the steps, from sample preparation to data collection and analysis using synthetic VLPs as model systems. The applicability of the method in product development was verified at pilot-scale during the technology transfer of dengue VLPs development from a university setting to an LMIC- based vaccine manufacturing company, demonstrating the applicability of this analytical technique to VLP vaccine characterization.

Humoral immune response induced with dengue virus-like particles serotypes 1 and 4 produced in silkworm

Dengue is an arboviral disease, which threatens almost half the global population, and has emerged as the most significant of current global public health challenges. In this study, we prepared dengue virus-like particles (DENV-LPs) consisting of Capsid-premembrane-envelope (CprME) and premembrane-envelope (prME) polypeptides from serotype 1 and 4, which were expressed in the silkworms using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid. 1CprME, 1prME, 4CprME, and 4prME expressed proteins in hemolymph, and the molecular weight of the purified proteins was 55 kDa, respectively. The purified polypeptides formed spherical Dengue virus-like particles (DENV-LPs) with ~ 30–55 nm in diameter. The immunoelectron microscopy (IEM) images revealed antigens to the surface of a lipid bilayer of DENV-LPs. The heparin-binding assay shows a positive relationship between absorbance and E protein domain III (EDIII) quantity, which is supported by the isothermal titration calorimetry assay. This indicates a moderate binding affinity between heparin and DENV-LP. The high correlation between patient sera and DENV-LP reactivities revealed that these DENV-LPs shared similar epitopes with the natural dengue virus. IgG elicitation studies in mice have demonstrated that DENV-LPs/CPrMEs elicit a stronger immune response than DENV-LP/prMEs, which lends credence to this claim.

Virus-like particles with FLAG-tagged envelope protein as a tetravalent dengue vaccine candidate

The global incidence of dengue, which is caused by dengue virus (DENV) infection, has grown dramatically in recent decades and secondary infection with heterologous serotype of the virus may cause severe symptoms. Efficacious dengue vaccines should be able to provide long-lasting immunity against all four DENV serotypes simultaneously. In this study, we constructed a novel vaccine platform based on tetravalent dengue virus-like particles (DENV-LPs) in which envelope (E) protein carried a FLAG tag sequence at the position located not only in the exterior loop on the protruding domain but outside of dimerization interface of the protein. We demonstrated an effective strategy to produce the DENV-LPs by transient transfection with expression plasmids for pre-membrane and E proteins of DENV-1 to DENV-4 in mammalian cells and to concentrate and purify them with one-step affinity chromatography. Characteristic features of VLPs such as particle size, shape and density were comparable to flavivirus-like particles reported. The neutralizing activity against all four DENV serotypes was successfully induced by immunization with the purified tetravalent VLPs in mice. Simple, one-step purification systems for VLP vaccine platforms using epitope-tagging strategy should be advantageous for vaccine development not only for dengue but for emerging pandemics in the future.

Development of a Novel Assay to Assess the Avidity of Dengue Virus-Specific Antibodies Elicited in Response to a Tetravalent Dengue Vaccine.

Antibody affinity maturation is a critical step in development of functional antiviral immunity; however, accurate measurement of affinity maturation of polyclonal serum antibody responses to particulate antigens such as virions is challenging. We describe a novel avidity assay employing biolayer interferometry and dengue virus-like particles. After validation using anti-dengue monoclonal antibodies, the assay was used to assess avidity of antibody responses to a tetravalent dengue vaccine candidate (TAK-003) in children, adolescents, and adults during two phase 2 clinical trials conducted in dengue-endemic regions. Vaccination increased avidity index and avidity remained high through 1 year postvaccination. Neutralizing antibody titers and avidity index did not correlate overall; however, a correlation was observed between neutralizing antibody titer and avidity index in those subjects with the highest degree of antibody affinity maturation. Therefore, vaccination with TAK-003 stimulates polyclonal affinity maturation and functional antibody responses, including neutralizing antibodies.Clinical Trials RegistrationNCT01511250 and NCT02302066.

Production of dengue virus-like particles serotype-3 in silkworm larvae and their ability to elicit a humoral immune response in mice

To develop monovalent dengue virus-like particle for serotype 3 (DENV-LP/3), we prepared and expressed two structural polyprotein constructs using silkworm and Bm5 cells: DENV-3 Capsid-premembrane-envelope (DENV-3CprME) and premembrane-envelope (DENV-3prME). The expressed PA-tagged 3CprME and 3prME polypeptides were partially purified by PA-tag affinity chromatography and had molecular weights of 85 and 75 kDa, respectively. Expressed proteins were separately verified using the following primary antibodies: the anti-PA tag antibody, DENV premembrane polyclonal antibody, and DENV envelope polyclonal antibody. Transmission electron microscopy revealed that these DENV-3CprME and 3prME formed rough, spherical DENV-LPs (DENV-LP/3CprME and DENV-LP/3prME), respectively, with a diameter of 30–55 nm. The heparin-binding assay demonstrated that these DENV-LPs contained the envelope protein domain III on their surfaces. Both DENV-LPs showed an affinity to sera from human dengue patients and immunized mice. Immunization of mice with DENV-LP/3prME significantly induced the level of antibodies compared with DENV-LP/3CprME. These results indicate that DENV-LP/3prME is suitable as a vaccine candidate compared with DENV-LP/3CprME.

Production and Purification of Dengue Virus-like Particles from COS-1 Cells.

Non-infectious virus-like particles (VLPs) containing dengue virus (DENV) pre-membrane (prM) and envelope (E) proteins have been demonstrated to be highly immunogenic and can be used as a potential vaccine candidate as well as a tool for serodiagnostic assays. Successful application of VLPs requires abundant, and high-purity production methods. Here, we describe a robust protocol for producing DENV VLPs from transiently-transformed or stable COS-1 cells and further provide an easily adaptable antigen purification method by sucrose gradient centrifugation.

Epitope resurfacing on dengue virus-like particle vaccine preparation to induce broad neutralizing antibody.

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein VLP derived from DENV serotype-2 were engineered becoming highly matured (mD2VLP) and showed variable size distribution with diameter of ~31 nm forming the major population under cryo-electron microscopy examination. Furthermore, mD2VLP particles of 31 nm diameter possess a T = 1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Mice vaccinated with mD2VLP generated higher cross-reactive (CR) neutralization antibodies (NtAbs) and were fully protected against all 4 serotypes of DENV. Our results highlight the potential of 'epitope-resurfaced' mature-form D2VLPs in inducing quaternary structure-recognizing broad CR NtAbs to guide future dengue vaccine design.

An Envelope-Modified Tetravalent Dengue Virus-Like-Particle Vaccine Has Implications for Flavivirus Vaccine Design.

Dengue viruses (DENV) infect 50 to 100 million people each year. The spread of DENV-associated infections is one of the most serious public health problems worldwide, as there is no widely available vaccine or specific therapeutic for DENV infections. To address this, we developed a novel tetravalent dengue vaccine by utilizing virus-like particles (VLPs). We created recombinant DENV1 to -4 (DENV1-4) VLPs by coexpressing precursor membrane (prM) and envelope (E) proteins, with an F108A mutation in the fusion loop structure of E to increase the production of VLPs in mammalian cells. Immunization with DENV1-4 VLPs as individual, monovalent vaccines elicited strong neutralization activity against each DENV serotype in mice. For use as a tetravalent vaccine, DENV1-4 VLPs elicited high levels of neutralization activity against all four serotypes simultaneously. The neutralization antibody responses induced by the VLPs were significantly higher than those with DNA or recombinant E protein immunization. Moreover, antibody-dependent enhancement (ADE) was not observed against any serotype at a 1:10 serum dilution. We also demonstrated that the Zika virus (ZIKV) VLP production level was enhanced by introducing the same F108A mutation into the ZIKV envelope protein. Taken together, these results suggest that our strategy for DENV VLP production is applicable to other flavivirus VLP vaccine development, due to the similarity in viral structures, and they describe the promising development of an effective tetravalent vaccine against the prevalent flavivirus.IMPORTANCE Dengue virus poses one of the most serious public health problems worldwide, and the incidence of diseases caused by the virus has increased dramatically. Despite decades of effort, there is no effective treatment against dengue. A safe and potent vaccine against dengue is still needed. We developed a novel tetravalent dengue vaccine by using virus-like particles (VLPs), which are noninfectious because they lack the viral genome. Previous attempts of other groups to use dengue VLPs resulted in generally poor yields. We found that a critical amino acid mutation in the envelope protein enhances the production of VLPs. Our tetravalent vaccine elicited potent neutralizing antibody responses against all four DENV serotypes. Our findings can also be applied to vaccine development against other flaviviruses, such as Zika virus or West Nile virus.

Measuring Haitian children's exposure to chikungunya, dengue and malaria.

ObjectiveTo differentiate exposure to the newly introduced chikungunya virus from exposure to endemic dengue virus and other pathogens in Haiti.MethodsWe used a multiplex bead assay to detect immunoglobulin G (IgG) responses to a recombinant chikungunya virus antigen, two dengue virus-like particles and three recombinant Plasmodium falciparum antigens. Most (217) of the blood samples investigated were collected longitudinally, from each of 61 children, between 2011 and 2014 but another 127 were collected from a cross-sectional sample of children in 2014.FindingsOf the samples from the longitudinal cohort, none of the 153 collected between 2011 and 2013 but 78.7% (48/61) of those collected in 2014 were positive for IgG responses to the chikungunya virus antigen. In the cross-sectional sample, such responses were detected in 96 (75.6%) of the children and occurred at similar prevalence across all age groups. In the same sample, responses to malarial antigen were only detected in eight children (6.3%) but the prevalence of IgG responses to dengue virus antigens was 60.6% (77/127) overall and increased steadily with age. Spatial analysis indicated that the prevalence of IgG responses to the chikungunya virus and one of the dengue virus-like particles decreased as the sampling site moved away from the city of Léogâne and towards the ocean.ConclusionSerological evidence indicates that there had been a rapid and intense dissemination of chikungunya virus in Haiti. The multiplex bead assay appears to be an appropriate serological platform to monitor the seroprevalence of multiple pathogens simultaneously.

Tetravalent recombinant dengue virus-like particles as potential vaccine candidates: immunological properties.

BackgroundCurrently, a licensed vaccine for Dengue Virus (DENV) is not yet available. Virus-like particles (VLP) have shown considerable promise for use as vaccines and have many advantages compared to many other types of viral vaccines. VLPs have been found to have high immunogenic potencies, providing protection against various pathogens.ResultsIn the current study, four DENV-VLP serotypes were successfully expressed in Pichia pastoris, based on co-expression of the prM and E proteins. The effects of a tetravalent VLP vaccine were also examined. Immunization with purified, recombinant, tetravalent DENV1-4 VLPs induced specific antibodies against all DENV1-4 antigens in mice. The antibody titers were higher after immunization with the tetravalent VLP vaccine compared to titers after immunization with any of the dengue serotype VLPs alone. Indirect immunofluorescence assay (IFA) results indicated that sera from VLP immunized mice recognized the native viral antigens. TNF-α and IL-10 were significantly higher in mice immunized with tetravalent DENV-VLP compared to those mice received PBS. The tetravalent VLP appeared to stimulate neutralizing antibodies against each viral serotype, as shown by PRNT50 analysis (1:32 against DENV1 and 2, and 1:16 against DENV3 and 4). The highest titers with the tetravalent VLP vaccine were still a little lower than the monovalent VLP against the corresponding serotype. The protection rates of tetravalent DENV-VLP immune sera against challenges with DENV1 to 4 serotypes in suckling mice were 77, 92, 100, and 100%, respectively, indicating greater protective efficacy compared with monovalent immune sera.ConclusionsOur results provide an important basis for the development of the dengue VLP as a promising non-infectious candidate vaccine for dengue infection.

An optimized expression vector for improving the yield of dengue virus-like particles from transfected insect cells

Recombinant virus-like particles (rVLPs) of flaviviruses are non-infectious particles released from cells expressing the envelope glycoproteins prM and E. Dengue virus rVLPs are recognized as a potential vaccine candidate, but large scale production of these particles is hindered by low yields and the occurrence of cytopathic effects. In an approach to improve the yield of rVLPs from transfected insect cells, several components of a dengue serotype 2 virus prM+E expression cassette were modified and the effect of these modifications was assessed during transient expression. Enhancement of extracellular rVLP levels by simultaneous substitutions of the prM signal peptide and the stem-anchor region of E with homologous cellular and viral counterparts, respectively, was further augmented by codon optimization. Extensive formation of multinucleated cells following transfection with the codon-optimized expression cassette was abrogated by introducing an E fusion loop mutation. This mutation also helped restore the extracellular E levels affected negatively by alteration of a charged residue at the pr-M junction, which was intended to promote maturation of rVLPs during export. Optimized expression cassettes generated in this multiple add-on modification approach should be useful in the generation of stably expressing clones and production of dengue virus rVLPs for immunogenicity studies.

Dengue virus-like particles: construction and application

Virus-like particles (VLPs) are shell-like viruses that lack virus-specific genetic materials. Many viral-structured proteins can assemble into VLPs, which mimic the overall structure of virus particles and can elicit strong immune responses in a host. Dengue viruses (DENVs), from the genus Flavivirus, are transmitted to humans through the bites of an infected Aedes mosquito. DENVs cause several diseases that prevailed mainly in tropical and subtropical areas. However, effective treatment measures and preventive strategies for dengue diseases are still lacking. The present minireview summarized the assembly and maturation of DENVs, the strategies and effective factors for dengue VLP construction, and the application of DENV VLPs.

Vaccination with dengue virus-like particles induces humoral and cellular immune responses in mice

BackgroundThe incidence of dengue, an infectious disease caused by dengue virus (DENV), has dramatically increased around the world in recent decades and is becoming a severe public health threat. However, there is currently no specific treatment for dengue fever, and licensed vaccine against dengue is not available. Vaccination with virus-like particles (VLPs) has shown considerable promise for many viral diseases, but the effect of DENV VLPs to induce specific immune responses has not been adequately investigated.ResultsBy optimizing the expression plasmids, recombinant VLPs of four antigenically different DENV serotypes DENV1-4 were successfully produced in 293T cells. The vaccination effect of dengue VLPs in mice showed that monovalent VLPs of each serotype stimulated specific IgG responses and potent neutralizing antibodies against homotypic virus. Tetravalent VLPs efficiently enhanced specific IgG and neutralizing antibodies against all four serotypes of DENV. Moreover, vaccination with monovalent or tetravalent VLPs resulted in the induction of specific cytotoxic T cell responses.ConclusionsMammalian cell expressed dengue VLPs are capable to induce VLP-specific humoral and cellular immune responses in mice, and being a promising subunit vaccine candidate for prevention of dengue virus infection.

A high-throughput assay using dengue-1 virus-like particles for drug discovery

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for 50–100 million human infections each year. The development of DENV chemotherapy requires high-throughput screening (HTS) assays. A dengue virus-like particle (VLP) has been constructed using viral structural proteins to package a Renilla luciferase reporter replicon. VLP could be produced by either the sequential electroporation of the replicon RNAs and the structural gene RNAs or by electroporating replicon RNA into a stable cell line expressing the structural proteins. In both approaches, the key to produce high titer VLP (3 × 10 6 foci-forming unit/ml) is to use low temperature (30 °C) in the packaging step. In addition, exogenous expression of host protease furin increased VLP infectivity. The infection could be blocked by antibodies against viral envelope protein and by an inhibitor of viral NS5 polymerase, but not by an inhibitor of host alpha-glucosidase (castanospermine). The VLP infection assay was optimized for HTS in a 384-well format with consistent and robust signal, providing a simple and rapid cell-based assay for screening inhibitors against DENV entry, translation, and replication in an HTS format.

Secretion of noninfectious dengue virus-like particles and identification of amino acids in the stem region involved in intracellular retention of envelope protein

DNA plasmids that express flavivirus premembrane/membrane (prM/M) and envelope (E) proteins in the form of virus-like particles (VLPs) have an excellent potential as DNA vaccine candidates against virus infection. The plasmid-expressed VLPs are also useful as safe, noninfectious antigens in serodiagnostic assays. We have constructed plasmids containing the prM/M and E gene regions for DENV-1, -3, and -4 that express and secrete VLPs when electroporated into Chinese hamster ovary cells. Constructs containing the full-length DENV-1 E protein gene did not secrete VLPs into tissue culture fluid effectively. However, a 16-fold increase in ELISA titers of DENV-1 VLPs was achieved after replacing the carboxy-terminal 20% region of DENV-1 E protein gene with the corresponding sequence of Japanese encephalitis virus (JEV). DENV-3 plasmids containing either the full-length DENV-3 E protein gene or the 20% JEV sequence replacement secreted VLPs to similarly high levels. Whereas DENV-4 VLPs were secreted to high levels by plasmids containing the full-length DENV-4 E protein gene but not by the chimeric plasmid containing 20% JEV E replacement. Domain substitutions by replacing prM/M protein stem-anchor region with the corresponding prM/M stem-anchor region of JEV or DENV-2 in the chimeric DENV-4 construct failed to promote the secretion of DENV-4 VLPs. Using the DENV-2 chimeric plasmid with carboxy-terminal 10% of JEV E gene, the sequence responsible for intracellular localization of E protein was mapped onto the E-H1 α-helix domain of DENV-2 E protein. Substitution of three amino acids from the DENV-2 sequence to the corresponding amino acids in the JEV sequence (I398L, M401A, and M412L) in the E-H1 was sufficient to promote extracellular secretion and resulted in detectable titers of DENV-2 VLP secretion.