A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein.

An Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 (isolate Wuhan-Hu-1) spike protein would significantly accelerate the search for anti-COVID-19 therapeutics because of its versatility and low cost. However, RBD contains four disulfide bonds and its expression in E. coli is limited by the formation of aberrant disulfide bonds resulting in inclusion bodies. Here, we show that a solubility-enhancing peptide (SEP) tag containing nine arginine residues (RBD-C9R) attached at the C-terminus can overcome this problem. The SEP-tag increased the expression in the soluble fraction and the final yield by five times (2 mg/L). The folding properties of the E. coli expressed RBD-C9R were demonstrated with biophysical characterization using RP-HPLC, circular dichroism, thermal denaturation, fluorescence, and light scattering. A quartz crystal microbalance (QCM) analysis confirmed the binding activity of RBD-C9R with ACE2, the host cell's receptor. In addition, RBD-C9R elicited a Th-2 immune response with a high IgG titer in Jcl: ICR mice. The RBD-C9R antisera interacted with both itself and the mammalian-cell expressed spike protein (S1), as demonstrated by ELISA, indicating that the E. coli expressed RBD-C9R harbors native-like epitopes. Overall, these results emphasize the potential of our SEP-tag for the E. coli production of active multi-disulfide-bonded RBD.

Protein and peptide delivery to lungs by using advanced targeted drug delivery.

The challenges and difficulties associated with conventional drug delivery systems have led to the emergence of novel, advanced targeted drug delivery systems. Therapeutic drug delivery of proteins and peptides to the lungs is complicated owing to the large size and polar characteristics of the latter. Nevertheless, the pulmonary route has attracted great interest today among formulation scientists, as it has evolved into one of the important targeted drug delivery platforms for the delivery of peptides, and related compounds effectively to the lungs, primarily for the management and treatment of chronic lung diseases. In this review, we have discussed and summarized the current scenario and recent developments in targeted delivery of proteins and peptide-based drugs to the lungs. Moreover, we have also highlighted the advantages of pulmonary drug delivery over conventional drug delivery approaches for peptide-based drugs, in terms of efficacy, retention time and other important pharmacokinetic parameters. The review also highlights the future perspectives and the impact of targeted drug delivery on peptide-based drugs in the coming decade.

A Global Review on Short Peptides: Frontiers and Perspectives.

Peptides are fragments of proteins that carry out biological functions. They act as signaling entities via all domains of life and interfere with protein-protein interactions, which are indispensable in bio-processes. Short peptides include fundamental molecular information for a prelude to the symphony of life. They have aroused considerable interest due to their unique features and great promise in innovative bio-therapies. This work focusing on the current state-of-the-art short peptide-based therapeutical developments is the first global review written by researchers from all continents, as a celebration of 100 years of peptide therapeutics since the commencement of insulin therapy in the 1920s. Peptide "drugs" initially played only the role of hormone analogs to balance disorders. Nowadays, they achieve numerous biomedical tasks, can cross membranes, or reach intracellular targets. The role of peptides in bio-processes can hardly be mimicked by other chemical substances. The article is divided into independent sections, which are related to either the progress in short peptide-based theranostics or the problems posing challenge to bio-medicine. In particular, the SWOT analysis of short peptides, their relevance in therapies of diverse diseases, improvements in (bio)synthesis platforms, advanced nano-supramolecular technologies, aptamers, altered peptide ligands and in silico methodologies to overcome peptide limitations, modern smart bio-functional materials, vaccines, and drug/gene-targeted delivery systems are discussed.

Oral inhalation for delivery of proteins and peptides to the lungs.

Oral inhalation is the preferred route for delivery of small molecules to the lungs, because high tissue levels can be achieved shortly after application. Biologics are mainly administered by intravenous injection but inhalation might be beneficial for the treatment of lung diseases (e.g. asthma). This review discusses biological and pharmaceutical challenges for delivery of biologics and describes promising candidates. Insufficient stability of the proteins during aerosolization and the biological environment of the lung are the main obstacles for pulmonary delivery of biologics. Novel nebulizers will improve delivery by inducing less shear stress and administration as dry powder appears suitable for delivery of biologics. Other promising strategies include pegylation and development of antibody fragments, while carrier-encapsulated systems currently play no major role in pulmonary delivery of biologics for lung disease. While development of various biologics has been halted or has shown little effects, AIR DNase, alpha1-proteinase inhibitor, recombinant neuraminidase, and heparin are currently being evaluated in phase III trials. Several biologics are being tested for the treatment of coronavirus disease (COVID)-19, and it is expected that these trials will lead to improvements in pulmonary delivery of biologics.

Biotin Functionalized Self-Assembled Peptide Nanofiber as an Adjuvant for Immunomodulatory Response.

Biotinylated peptide amphiphile (Biotin-PA) nanofibers, are designed as a noncovalent binding location for antigens, which are adjuvants to enhance, accelerate, and prolong the immune response triggered by antigens. Presenting antigens on synthetic Biotin-PA nanofibers generated a higher immune response than the free antigens delivered with a cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN) (TLR9 agonist) adjuvant. Antigen attached Biotin-PA nanofibers trigger splenocytes to produce high levels of cytokines (IFN-γ, IL-12, TNF-α, and IL-6) and to exhibit a superior cross-presentation of the antigen. Both Biotin-PA nanofibers and CpG ODN induce a Th-1-biased IgG subclass response; however, delivering the antigen with Biotin-PA nanofibers induce significantly greater production of total IgG and subclasses of IgG compared to delivering the antigen with CpG ODN. Contrary to CpG ODN, Biotin-PA nanofibers also enhance antigen-specific splenocyte proliferation and increase the proportion of the antigen-specific CD8(+) T cells. Given their biodegradability and biocompatibility, Biotin-PA nanofibers have a significant potential in immunoengineering applications as a biomaterial for the delivery of a diverse set of antigens derived from intracellular pathogens, emerging viral diseases such as COVID-19, or cancer cells to induce humoral and cellular immune responses against the antigens.