Expression of an immunocomplex consisting of Fc fragment fused with a consensus dengue envelope domain III in Saccharomyces cerevisiae.

OBJECTIVES: To explore Saccharomyces cerevisiae as an expression platform for dengue oral immune complex vaccine development. RESULTS: Molecular engineering was applied to create a fusion gene construct (scEDIII-PIGS) consisting of a yeast codon optimized sequence encoding for a synthetic consensus dengue envelope domain III (scEDIII) followed by a modified IgG Fc domain (PIGS). Northern blot showed transcription of the target gene, with a temporal expression pattern similar to those from previous work. Western blot showed assembly of various immune complexes from monomer to hexamer. Partial purification of scEDIII-PIGS was also attempted to demonstrate the feasibility of yeast system for immune complex vaccine development. Approximately 1 mg of scEDIII-PIGS can be produced from 1 l culture. CONCLUSION: This work demonstrated for the first time that various immunocomplex structures of our target protein could be efficiently produced in S. cerevisiae for future application in developing oral and injectable vaccines against various pathogens.

Variable selection and parameter estimation of viral amplification in vero cell cultures dedicated to the production of a dengue vaccine.

In this study, a dynamic model of a Vero cell culture-based dengue vaccine production process is developed. The approach consists in describing the process dynamics as functions of the whole living (uninfected and infected) biomass whereas previous works are based on population balance approaches. Based on the assumption that infected biomass evolves faster than other variable, the model can be simplified using a slow-fast approximation. The structural identifiability of the model is analysed using differential algebra as implemented in the software DAISY. The model parameters are inferred from experimental datasets collected from an actual vaccine production process and the model predictive capability is confirmed both in direct and cross-validation. The model prediction shows the impact of the metabolism on virus yield and confirms observations reported in previous studies. Multi-modality and sensitivity analysis complement the parameter estimation, and allow to obtain confidence intervals on both parameters and state estimates. Finally, the model is used to compute the maximum infectious virus yield that can be obtained for different combinations of multiplicity of infection (MOI) and time of infection (TOI). © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2687, 2019.

Human single-chain variable fragment antibody expressed in E. coli with optimal in vitro cross-neutralizing and no enhancing activity.

Single chain fragment variable (scFv) is a small molecule antibody comprising of only the variable region of heavy and light chain responsible for antigen binding. For dengue disease, the Fc region of antibody molecule was reported to be involved with dengue complication caused by Antibody-dependent enhancement (ADE). We attempted to produce small molecule scFv human monoclonal antibody (HuMAb), which lacking the Fc portion to eliminate the ADE effect of the IgG. This scFv antibody was produced in Escherichia coli. The biologically active form of scFv antibody was successfully generated. 23-1C2D2-scFv showed neutralizing activity similar to the IgG obtained from parental hybridoma, but lacked enhancing activity in all studied concentrations. This antibody was targeted to the 101WXN103 motif of dengue envelop protein domain II, studied by western blot analysis with truncated E protein and random peptide phage display. This scFv is verified as a candidate for further development as therapeutic candidate for DENV infection.

C5a receptor targeting of partial non-structural protein 3 of dengue virus promotes antigen-specific IFN-γ-producing T-cell responses in a mucosal dengue vaccine model.

Mucosal vaccination is an ideal strategy to induce protective immunity in both mucosal and parenteral areas. Successful induction of an antigen-specific immune response via mucosal administration essentially requires the effective delivery of antigen into a mucosal immune inductive site, which depends on antigen delivery into M cells. We previously reported that M cells specifically express C5aR, and antigen targeting to C5aR by using specific ligands, including Co1 peptide, promotes the antigen-specific immune response in both mucosal and systemic immune compartments. In this study, we found that application of the Co1 peptide to dengue virus antigen containing CD8 T cell epitopes effectively induced an antigen-specific IFN-γ-producing CD8+ T cell response after oral mucosal administration of antigen. Consequently, we suggest that Co1 peptide-mediated C5aR targeting of antigen into M cells can be used for the induction of an effective antigen-specific CD8+ T cell immune response in oral mucosal vaccine development.

Expression and assembly of cholera toxin B subunit and domain III of dengue virus 2 envelope fusion protein in transgenic potatoes.

The rates of mosquito-transmitted dengue virus infection in humans have increased in tropical and sub-tropical areas. Domain III of dengue envelope protein (EDIII) is involved in cellular receptor binding and induces serotype-specific neutralizing antibodies. EDIII fused to the B subunit of Vibrio cholera (CTB-EDIII) was expressed in potatoes to develop a plant-based vaccine against dengue virus type 2. CTB-EDIII fused to an endoplasmic reticulum (ER) retention signal, SEKDEL, was introduced into potatoes by A. tumefaciens-mediated gene transformation. The integration of the CTB-EDIII fusion gene into the nuclear genome of transgenic plants was confirmed by genomic DNA polymerase chain reaction (PCR), and mRNA transcripts of CTB-EDIII were detected. CTB-EDIII fusion protein was expressed in potato tubers and assembled into a pentameric form capable of binding monosialotetrahexosylganglioside (GM1). The level of expression was determined to be ∼0.005% of total soluble protein in potato tubers. These results suggest that dengue virus antigen could be produced in potatoes, raising the possibility that edible plants are employed in mucosal vaccines for protection against dengue infection.

Immunogenic Properties of a BCG Adjuvanted Chitosan Nanoparticle-Based Dengue Vaccine in Human Dendritic Cells.

Dengue viruses (DENVs) are among the most rapidly and efficiently spreading arboviruses. WHO recently estimated that about half of the world's population is now at risk for DENV infection. There is no specific treatment or vaccine available to treat or prevent DENV infections. Here, we report the development of a novel dengue nanovaccine (DNV) composed of UV-inactivated DENV-2 (UVI-DENV) and Mycobacterium bovis Bacillus Calmette-Guerin cell wall components (BCG-CWCs) loaded into chitosan nanoparticles (CS-NPs). CS-NPs were prepared by an emulsion polymerization method prior to loading of the BCG-CWCs and UVI-DENV components. Using a scanning electron microscope and a zetasizer, DNV was determined to be of spherical shape with a diameter of 372.0 ± 11.2 nm in average and cationic surface properties. The loading efficacies of BCG-CWCs and UVI-DENV into the CS-NPs and BCG-CS-NPs were up to 97.2 and 98.4%, respectively. THP-1 cellular uptake of UVI-DENV present in the DNV was higher than soluble UVI-DENV alone. DNV stimulation of immature dendritic cells (iDCs) resulted in a significantly higher expression of DCs maturation markers (CD80, CD86 and HLA-DR) and induction of various cytokine and chemokine productions than in UVI-DENV-treated iDCs, suggesting a potential use of BCG- CS-NPs as adjuvant and delivery system for dengue vaccines.

Dengue-4 envelope domain III fused twice within the meningococcal P64k protein carrier induces partial protection in mice.

A vaccine against dengue virus must be able to induce an effective and equivalent immune response to the four viral serotypes; however, some studies have revealed that DEN4 (dengue-virus serotype 4) induces a weaker immune response than the others in quadrivalent (tetravalent') formulations. We have previously reported the protective capacity, in a viral encephalitis murine model, of fusion protein P64k-envelope domain III of DEN1, DEN2 and DEN3. We also reported that the P64k protein can be used as a carrier in two different positions: the insertion following the first 45 amino acids and the fusion at the C-terminus. Considering the low immunogenicity described for DEN4, in the present study we obtained a novel chimaeric protein by inserting two dengue-4 envelope domains III in both sites of P64k (PD24), and hence increasing the presence of the virus in the final construct. After expression in Escherichia coli and semipurification, the protein exhibited a pattern of high molecular mass and was well recognized by human and murine polyclonal antibodies. The protein was finally evaluated in mice, Al(OH)(3) being employed as the adjuvant. Even though the animals exhibited low levels of antiviral antibodies, the recombinant protein induced significant protection against lethal challenge with dengue-4 virus.