Protective efficiency of a combination of four recombinant Brucella abortus (B. abortus) proteins, namely, ribosomal protein L7/L12, outer membrane protein (OMP) 22, OMP25 and OMP31, was evaluated as a combined subunit vaccine (CSV) against B. abortus infection in RAW 264.7 cell line and murine model.
Four proteins were cloned, expressed and purified, and their immunocompetence was analysed.
BALB/c mice were immunized subcutaneously with single subunit vaccines (SSVs) or CSV.
Cellular and humoral immune responses were determined by ELISA.
Results of immunoreactivity showed that these four recombinant proteins reacted with Brucella-positive serum individually but not with Brucella-negative serum.
A massive production of IFN-γ and IL-2 but low degree of IL-10 was observed in mice immunized with SSVs or CSV.
In addition, the titres of IgG2a were heightened compared with IgG1 in SSV- or CSV-immunized mice, which indicated that SSVs and CSV induced a typical T-helper-1-dominated immune response in vivo.
Further investigation of the CSV showed a superior protective effect in mice against brucellosis.
The protection level induced by CSV was significantly higher than that induced by SSVs, which was not significantly different compared with a group immunized with RB51.
Collectively, these antigens of Brucella could be potential candidates to develop subunit vaccines, and the CSV used in this study could be a potential candidate therapy for the prevention of brucellosis.
Protein Biochemistry and Molecular Modeling of the Intra-Melanosomal Domain of Human Recombinant Tyrp2 Protein and OCA8-Related Mutant Variants
Tyrosinase-related protein 2 (Tyrp2) is involved in the melanogenesis pathway, catalyzing the tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA).
Recently, a new type of albinism was discovered with disease-causing mutations in the TYRP2 gene.
Here, for the first time, we characterized the intra-melanosomal protein domain of Tyrp2 (residues 1-474) and missense variants C40S and C61W, which mimic the alterations found in genetic studies. Know more about
Recombinant Proteins were produced in the Trichoplusia Ni (Ti. Ni) larvae, purified by a combination of immobilized metal affinity (IMAC) and gel-filtration (GF) chromatography, and biochemically characterized.
The mutants showed the protein expression in the lysates such as the wild type; however, undetectable protein yield after two steps of purification exhibited their misfolding and instability.
In addition, the misfolding effect of the mutations was confirmed computationally using homology modeling and molecular docking.
Together, experiments in vitro and computer simulations indicated the critical role of the Cys-rich domain in the Tyrp2 protein stability.
The results are consistent with molecular modeling, global computational mutagenesis, and clinical data, proving the significance of genetic alterations in cysteine residues, which could cause oculocutaneous albinism type 8.
An Image-Analysis-Based Method for the Prediction of Recombinant Protein Fiber Tensile Strength
Silk fibers derived from the cocoon of silk moths and the wide range of silks produced by spiders exhibit an array of features, such as extraordinary tensile strength, elasticity, and adhesive properties.
The functional features and mechanical properties can be derived from the structural composition and organization of the silk fibers.
Artificial recombinant protein fibers based on engineered spider silk proteins have been successfully made previously and represent a promising way towards the large-scale production of fibers with predesigned features.
However, for the production and use of protein fibers, there is a need for reliable objective quality control procedures that could be automated and that do not destroy the fibers in the process.
Furthermore, there is still a lack of understanding the specifics of how the structural composition and organization relate to the ultimate function of silk-like fibers.
In this study, we develop a new method for the categorization of protein fibers that enabled a highly accurate prediction of fiber tensile strength.
Based on the use of a common light microscope equipped with polarizers together with image analysis for the precise determination of fiber morphology and optical properties, this represents an easy-to-use, objective non-destructive quality control process for protein fiber manufacturing and provides further insights into the link between the supramolecular organization and mechanical functionality of protein fibers.
Safety and immunogenicity of two recombinant DNA COVID-19 vaccines containing the coding regions of the spike or spike and nucleocapsid proteins: an interim analysis of two open-label, non-randomised, phase 1 trials in healthy adults
We assessed the safety and immunogenicity of two recombinant DNA vaccines for COVID-19: GX-19 containing plasmid DNA encoding the SARS-CoV-2 spike protein, and GX-19N containing plasmid DNA encoding the SARS-CoV-2 receptor-binding domain (RBD) foldon, nucleocapsid protein, and plasmid DNA encoding the spike protein.
Two open-label non-randomised phase 1 trials, one of GX-19 and the other of GX-19N were done at two hospitals in South Korea.
We enrolled healthy adults aged 19-49 years for the GX-19 trial and healthy adults aged 19-54 years for the GX-19N trial. Participants who tested positive by serological testing for SARS-CoV-2 were excluded. At 4-week intervals, the GX-19 trial participants received two vaccine doses (either 1·5 mg or 3·0 mg), and the GX-19N trial participants received two 3·0 mg doses. The vaccines were delivered intramuscularly using an electroporator.
The participants were followed up for 52 weeks after first vaccination. Data collected up to day 57 after first vaccination were analyzed in this interim analysis.
The primary outcome was safety within 28 days after each vaccination measured in the intention-to-treat population.
The secondary outcome was vaccine immunogenicity using blood samples collected on day 43 or 57 after first vaccination measured in the intention-to-treat population. The GX-19 and GX-19N trials are registered with ClinicalTrials.gov.
Findings: Between June 17 and July 30, 2020, we screened 97 individuals, of whom 40 (41%) participants were enrolled in the GX-19 trial (20 [50%] in the 1·5 mg group and 20 [50%] in the 3·0 mg group).
Between Dec 28 and 31, 2020, we screened 23 participants, of whom 21 (91%) participants were enrolled on the GX-19N trial. 32 (52%) of 61 participants reported 80 treatment-emergent adverse events after vaccination.
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All solicited adverse events were mild except one (2%) case of moderate fatigue in the 1·5 mg GX-19 group; no serious vaccine-related adverse events were detected.
Binding antibody responses increased after second dose of vaccination in all groups (p=0·0002 in the 1·5 mg GX-19 group; p<0·0001 in the 3·0 mg GX-19; and p=0·0004 for the spike protein and p=0·0001 for the RBD in the 3·0 mg GX-19N group).
Interpretation:GX-19 and GX-19N are safe and well tolerated. GX-19N induces humoral and broad SARS-CoV-2-specific T-cell responses. GX-19N shows lower neutralising antibody responses and needs improvement to enhance immunogenicity.