Recombinant Endopeptidases IdeS and IdeZ and Their Potential Application.

Endopeptidases IdeS and IdeZ (streptococcal virulence factors that specifically cleave IgG heavy chains) are of particular interest because of their potential use in biotechnology, medicine, and veterinary. Genes encoding these enzymes were cloned and expressed in Escherichia coli heterologous expression system (ideS was cloned from a Streptococcus pyogenes collection strain; ideZ from Streptococcus zooepidemicus was synthesized). The 6His-tag was introduced into the amino acid sequence of each endopeptidase, and IdeS and IdeZ were purified by metal affinity chromatography to an apparent homogeneity (according to polyacrylamide gel electrophoresis). Purified enzymes were active against human and animal IgGs; their specificity toward human IgGs was confirmed by polyacrylamide gel electrophoresis. Recombinant IdeZ was used for immunological analysis of equine strangles infection (diagnostics and determination of the titer of specific antibodies in blood). Hence, IdeZ can be used in veterinary and sanitary microbiology to diagnose infections caused by Streptococcus equi and S. zooepidemicus in addition to its application in medicine and biotechnology.

Hybrid Proteins with Short Conformational Epitopes of the Receptor-Binding Domain of SARS-CoV-2 Spike Protein Promote Production of Virus-Neutralizing Antibodies When Used for Immunization.

Based on the previously developed approach, hybrid recombinant proteins containing short conformational epitopes (a.a. 144-153, 337-346, 414-425, 496-507) of the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) were synthesized in Escherichia coli cells as potential components of epitope vaccines. Selected epitopes are involved in protein-protein interactions in the S protein complexes with neutralizing antibodies and ACE2 (angiotensin-converting enzyme 2). The recombinant proteins were used for immunization of mice (three doses with 2-week intervals), and the immunogenicity of protein antigens and ability of the resulting sera to interact with inactivated SARS-CoV-2 and RBD produced in eukaryotic cells were examined. All recombinant proteins showed high immunogenicity; the highest titer in the RBD binding assay was demonstrated by the serum obtained after immunization with the protein containing epitope 414-425. At the same time, the titers of sera obtained against other proteins in the RBD and inactivated virus binding assays were significantly lower than the titers of sera obtained with the previously produced four proteins containing the loop-like epitopes 452-494 and 470-491, the conformation of which was fixed with a disulfide bond. We also studied activation of cell-mediated immunity by the recombinant proteins that was monitored as changes in the levels of cytokines in the splenocytes of immunized mice. The most pronounced increase in the cytokine synthesis was observed in response to the proteins containing epitopes with disulfide bonds (452-494, 470-491), as well as epitopes 414-425 and 496-507. For some recombinant proteins with short conformational epitopes, adjuvant optimization allowed to obtained mouse sera displaying virus-neutralizing activity in the microneutralization assay with live SARS-CoV-2 (hCoV-19/Russia/StPetersburg-3524/2020 EPI_ISL_415710 GISAID). The results obtained can be used to develop epitope vaccines for prevention of COVID-19 and other viral infections.

Development of a Platform for Producing Recombinant Protein Components of Epitope Vaccines for the Prevention of COVID-19.

A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS-CoV-2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS-CoV-2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS-CoV-2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS-CoV-2 virus and the Spike protein receptor-binding domain at high titers.

Fusion of Lysostaphin to an Albumin Binding Domain Prolongs Its Half-Life and Bactericidal Activity in the Systemic Circulation.

Antibacterial lysins are promising proteins that are active against both antibiotic-susceptible and antibiotic-resistant bacterial strains. However, a major limitation of antibacterial lysins is their fast elimination from systemic circulation. PEGylation increases the plasma half-life of lysins but renders them inactive. Here we report the construction of a fusion protein of lysostaphin, a potent anti-staphylococcal lysin, and an albumin-binding domain from streptococcal protein G. The resulting fusion protein was less active than the parent enzyme lysostaphin, but it still retained significant antibacterial activity even when bound to serum albumin. The terminal half-life of the fusion protein in rats was five-fold greater than that of lysostaphin (7.4 vs. 1.5 h), and the area under the curve increased more than 115 times. Most importantly, this increase in systemic circulation time compensated for the decrease in activity. The plasma from rats that received an injection of the fusion protein retained bactericidal activity for up to 7 h, while plasma from rats that received plain lysostaphin lacked any detectable activity after 4 h. To the best of our knowledge, this is the first report of an antibacterial lysin with both improved pharmacokinetic parameters and prolonged bactericidal activity in the systemic circulation.

The Influence of Dimerization on the Pharmacokinetics and Activity of an Antibacterial Enzyme Lysostaphin.

The increasing prevalence of antibiotic-resistant strains of pathogenic bacteria is a major healthcare problem. Antibacterial lysins are enzymes that cleave the peptidoglycan of the bacterial cell wall. These proteins hold potential as a supplement or an alternative to traditional antibiotics since they are active against antibiotic resistant strains. However, antibacterial lysins are rapidly eliminated from the systemic circulation, which limits their application. Dimerization of an anti-pneumococcal lysin Cpl-1 has been demonstrated to decrease the clearance rate of this protein in mice. In the present work, we constructed a dimer of an anti-staphylococcal lysin lysostaphin by fusing it with an anti-parallel α-helical dimerization domain. Lysostaphin dimer had a more favorable pharmacokinetic profile with increased terminal half-life and area under the curve (AUC) values compared to monomeric lysostaphin. However, the staphylolytic activity of dimerized lysostaphin was decreased. This decrease in activity was likely caused by the dimerization; since the catalytic efficacy of lysostaphin dimer towards pentaglycine peptide was unaltered. Our results demonstrate that, although dimerization is indeed beneficial for the pharmacokinetics of antibacterial lysins, this approach might not be suitable for all lysins, as it can negatively affect the lysin activity.

Recombinant domains III of Tick-Borne Encephalitis Virus envelope protein in combination with dextran and CpGs induce immune response and partial protectiveness against TBE virus infection in mice.

BACKGROUND: E protein of tick-borne encephalitis virus (TBEV) and other flaviviruses is located on the surface of the viral particle. Domain III of this protein seems to be a promising component of subunit vaccines for prophylaxis of TBE and kits for diagnostics of TBEV. METHODS: Three variants of recombinant TBEV E protein domain III of European, Siberian and Far Eastern subtypes fused with dextran-binding domain of Leuconostoc citreum KM20 were expressed in E. coli and purified. The native structure of domain III was confirmed by ELISA antibody kit and sera of patients with tick-borne encephalitis. Immunogenic and protective properties of the preparation comprising these recombinant proteins immobilized on a dextran carrier with CpG oligonucleotides as an adjuvant were investigated on the mice model. RESULTS: All 3 variants of recombinant proteins immobilized on dextran demonstrate specific interaction with antibodies from the sera of TBE patients. Thus, constructed recombinant proteins seem to be promising for TBE diagnostics. The formulation comprising the 3 variants of recombinant antigens immobilized on dextran and CpG oligonucleotides, induces the production of neutralizing antibodies against TBEV of different subtypes and demonstrates partial protectivity against TBEV infection. CONCLUSIONS: Studied proteins interact with the sera of TBE patients, and, in combination with dextran and CPGs, demonstrate immunogenicity and limited protectivity on mice compared with reference "Tick-E-Vac" vaccine.