Molecular engineering of PETase for efficient PET biodegradation.

The widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems. Compared with traditional thermomechanical or chemical PET cycling, the biodegradation of PET may offer a more feasible solution. Though the PETase from Ideonalla sakaiensis (IsPETase) displays interesting PET degrading performance under mild conditions; the relatively low thermal stability of IsPETase limits its practical application. In this study, enzyme-catalysed PET degradation was investigated with the promising IsPETase mutant HotPETase (HP). On this basis, a carbohydrate-binding module from Bacillus anthracis (BaCBM) was fused to the C-terminus of HP to construct the PETase mutant (HLCB) for increased PET degradation. Furthermore, to effectively improve PET accessibility and PET-degrading activity, the truncated outer membrane hybrid protein (FadL) was used to expose PETase and BaCBM on the surface of E. coli (BL21with) to develop regenerable whole-cell biocatalysts (D-HLCB). Results showed that, among the tested small-molecular weight ester compounds (p-nitrophenyl phosphate (pNPP), p-Nitrophenyl acetate (pNPA), 4-Nitrophenyl butyrate (pNPB)), PETase displayed the highest hydrolysing activity against pNPP. HP displayed the highest catalytic activity (1.94 µM(p-NP)/min) at 50 °C and increased longevity at 40 °C. The fused BaCBM could clearly improve the catalytic performance of PETase by increasing the optimal reaction temperature and improving the thermostability. When HLCB was used for PET degradation, the yield of monomeric products (255.7 µM) was ∼25.5 % greater than that obtained after 50 h of HP-catalysed PET degradation. Moreover, the highest yield of monomeric products from the D-HLCB-mediated system reached 1.03 mM. The whole-cell catalyst D-HLCB displayed good reusability and stability and could maintain more than 54.6 % of its initial activity for nine cycles. Finally, molecular docking simulations were utilized to investigate the binding mechanism and the reaction mechanism of HLCB, which may provide theoretical evidence to further increase the PET-degrading activities of PETases through rational design. The proposed strategy and developed variants show potential for achieving complete biodegradation of PET under mild conditions.

Combining Phage Display Technology with In Silico-Designed Epitope Vaccine to Elicit Robust Antibody Responses against Emerging Pathogen Tilapia Lake Virus.

Antigen epitope identification is a critical step in the vaccine development process and is a momentous cornerstone for the development of safe and efficient epitope vaccines. In particular, vaccine design is difficult when the function of the protein encoded by the pathogen is unknown. The genome of Tilapia lake virus (TiLV), an emerging virus from fish, encodes protein functions that have not been elucidated, resulting in a lag and uncertainty in vaccine development. Here, we propose a feasible strategy for emerging viral disease epitope vaccine development using TiLV. We determined the targets of specific antibodies in serum from a TiLV survivor by panning a Ph.D.-12 phage library, and we identified a mimotope, TYTTRMHITLPI, referred to as Pep3, which provided protection against TiLV after prime-boost vaccination; its immune protection rate was 57.6%. Based on amino acid sequence alignment and structure analysis of the target protein from TiLV, we further identified a protective antigenic site (399TYTTRNEDFLPT410) which is located on TiLV segment 1 (S1). The epitope vaccine with keyhole limpet hemocyanin (KLH-S1399-410) corresponding to the mimotope induced the tilapia to produce a durable and effective antibody response after immunization, and the antibody depletion test confirmed that the specific antibody against S1399-410 was necessary to neutralize TiLV. Surprisingly, the challenge studies in tilapia demonstrated that the epitope vaccine elicited a robust protective response against TiLV challenge, and the survival rate reached 81.8%. In conclusion, this study revealed a concept for screening antigen epitopes of emerging viral diseases, providing promising approaches for development and evaluation of protective epitope vaccines against viral diseases. IMPORTANCE Antigen epitope determination is an important cornerstone for developing efficient vaccines. In this study, we attempted to explore a novel approach for epitope discovery of TiLV, which is a new virus in fish. We investigated the immunogenicity and protective efficacy of all antigenic sites (mimotopes) identified in serum of primary TiLV survivors by using a Ph.D.-12 phage library. We also recognized and identified the natural epitope of TiLV by bioinformatics, evaluated the immunogenicity and protective effect of this antigenic site by immunization, and revealed 2 amino acid residues that play important roles in this epitope. Both Pep3 and S1399-410 (a natural epitope identified by Pep3) elicited antibody titers in tilapia, but S1399-410 was more prominent. Antibody depletion studies showed that anti-S1399-410-specific antibodies were essential for neutralizing TiLV. Our study demonstrated a model for combining experimental and computational screens to identify antigen epitopes, which is attractive for epitope-based vaccine development.

Biomimetic nanovaccine based on erythrocyte membrane enhances immune response and protection against tilapia lake virus.

An infectious disease emerged in recent years, Tilapia Lake Virus Disease (TiLVD), has severely restricted the development of global tilapia industry. Vaccination has proved potential strategy to prevent its causative agent Tilapia Lake Virus (TiLV) infectious. However, the response intensity of subunit vaccine is limited by its low immunogenicity, thus inclusion of adjuvants is required. Thus, we prepared a biomimetic nano-system (Cs-S2@M-M) with a particle size of ∼100 nm and an encapsulation efficiency of about 79.15% based on erythrocyte membrane. The immune response was detected after intramuscular injection to assess the effectiveness of the vaccine. The biomimetic system significantly up-regulates the expression of immune genes, enhances the activity of non-specific immune-related enzymes (P < 0.05) and improved relative percentage survival by 17.4%-26.1% in TiLV challenge. The biomimetic nano-system based on erythrocyte membrane induced significant immune response in tilapia and enhanced protection against TiLV, promising as a model for fish vaccines.

Optimization of immunization procedure for SWCNTs-based subunit vaccine with mannose modification against spring viraemia of carp virus in common carp.

Immersion vaccination of single-walled carbon nanotubes loaded with mannose-modified glycoprotein (SWCNTs-MG) vaccine has been proved to be effective in preventing spring viraemia of carp virus (SVCV). Immunization procedure has immense consequence on the immune effect of the immersion vaccine. However, immunization procedure optimization for SWCNTs-MG vaccine against SVCV has not been reported. In this study, accordingly, a full-factor experiment was designed to optimize the immunization procedure of SWCNTs-MG vaccine by three aspects of vaccine dose (30 mg/L, 40 mg/L and 50 mg/L), immunization density (8 fish L-1 , 24 fish L-1 and 48 fish L-1 ) and immunization time (6, 12 and 24 hr). Furthermore, we used the immunization group (A1B2C1, 30 mg/L, 24 fish L-1 and 6 hr) in the previous study as a positive control (PC) to evaluate the immunization effect optimized conditions from the expression of immune-related genes and relative percentage survival (RPS). At 28 days post-vaccination (DPV), common carps were intraperitoneal injected SVCV challenged test indicated that the A1B2C2 group (30 mg/L, 24 fish L-1 , 12 hr) displayed superiority of protective efficacy compare with other groups and the RPS with 77.9%, which was 15.6% higher than the PC group of RPS with 62.3%. Moreover, the expression of immune-related genes such as IL-10, CD4 and MHC-II was also significantly higher than PC group. The specific experimental flow chart is shown in Figure 1. Conclusively, these results demonstrated that vaccine dose, immunization density and immunization time are 30 mg/L, 24 fish L-1 and 12 hr, which is the more appropriate immunization programme with juvenile carp for SWCNTs-MG vaccine. This study provides a profitable reference for improving the immune efficiency of aquatic immersion vaccine. [Figure: see text].

Preliminary screening and immunogenicity analysis of antigenic epitopes of spring viremia of carp virus.

Glycoprotein (G) is the most common gene used in SVCV vaccine constructions. To identify the major immunogenicity determinant region of SVCV G gene, herein we truncated G gene to 4 parts (G-1, G-2, G-3 and G-4). Bioinformatics and the enzyme linked immunosorbent assay (ELISA) were used to identify the antigenicity of these 4 truncated G proteins. Immunological assays (serum antibody production, enzyme activity, immune genes expression and challenge test) were carried out to further identify the immunogenicity of the screened G protein in common carp. Moreover, to further verify the immune response of the screened G protein-based subunit vaccine, its protective effects on common carp against SVCV infection using single-walled carbon nanotubes (SWCNTs) as a carrier were evaluated. Results showed that G-3 protein could induce higher antibody titer than other truncated G proteins. Furthermore, carps vaccinated with G-3 and G (positive control) showed significant enhancement of immune response (serum antibody production, enzyme activity and immune related genes expression) when compared with control groups. Meanwhile, as a promising vaccine carrier, SWCNTs could significantly enhance the immune effect of naked subunit vaccine (G-3 and G). Notably, after SVCV challenge, there was no significant difference in immune protection between G-3 and G, nor between SWCNTs-G-3 and SWCNTs-G. These results so far suggest G-3 might be the potential antigen epitope of SVCV. This study lays a foundation for developing vaccine and immunodiagnostic techniques.

Evaluation of immune response and protection against spring viremia of carp virus induced by a single-walled carbon nanotubes-based immersion DNA vaccine.

Spring viremia of carp virus (SVCV) has caused mass mortality in cyprinids, with case fatality rates of young fish up to 90%, resulting in enormous economic losses in the aquaculture industry. Immersion vaccination is considered as the most effective method for juvenile fish to combating disease, due to its convenience for mass vaccination and stress-free administration. However, immune responses following immersion vaccination are generally less robust and of shorter duration as those induced through intraperitoneal injection. Herein, to enhance the efficient of immersion vaccine, functionalized single-walled carbon nanotubes (SWCNTs) as carrier were used to manufacture immersion DNA vaccine system (SWCNTs-pEGFP-M) with chemical modification. Results showed that SWCNTs-pEGFP-M could enter into fish body via immersion administration and express antigen proteins in fish kidney and spleen. Moreover, stronger and longer duration immune responses (including serum antibody production and immune genes expression) can be induced in fish vaccinated with SWCNTs-pEGFP-M in comparison with those vaccinated with pEGFP-M alone. Notably, SWCNTs can increase the immune protective effect of naked DNA vaccine by ca. 23.8%. Altogether, this study demonstrates that SWCNTs as a promising DNA vaccine carrier might be used to vaccinate large-scale juvenile fish by bath administration approach, which can provide an outlook for future vaccination strategies against SVCV.