Problems of creating antibody phage libraries and their solutions.

Phage display has become an efficient, reliable and popular molecular technique for generating libraries encompassing millions or even billions of clones of divergent peptides or proteins. The method is based on the correspondence between phage genotype and phenotype, which ensures the presentation of recombinant proteins of known amino acid composition on the surface of phage particles. The use of affinity selection allows one to choose variants with affinity for different targets from phage libraries. The implementation of the antibody phage display technique has revolutionized the field of clinical immunology, both for developing tools to diagnose infectious diseases and for producing therapeutic agents. It has also become the basis for efficient and relatively inexpensive methods for studying protein-protein interactions, receptor binding sites, as well as epitope and mimotope identification. The antibody phage display technique involves a number of steps, and the final result depends on their successful implementation. The diversity, whether natural or obtained by combinatorial chemistry, is the basis of any library. The choice of molecular techniques is critical to ensure that this diversity is maintained during the phage library preparation step and during the transformation of E. coli cells. After a helper phage is added to the suspension of transformed E. coli cells, a bacteriophage library is formed, which is a working tool for performing the affinity selection procedure and searching for individual molecules. Despite the apparent simplicity of generating phage antibody libraries, a number of subtleties need to be taken into account. First, there are the features of phage vector preparation. Currently, a large number of phagemid vectors have been developed, and their selection is also of great importance. The key step is preparing competent E. coli cells and the technology of their transformation. The choice of a helper phage and the method used to generate it is also important. This article discusses the key challenges faced by researchers in constructing phage antibody libraries.

The Search for Single-Domain Antibodies Interacting with the Receptor-Binding Domain of SARS-CoV-2 Surface Protein.

We performed a search for nanoantibodies that specifically interact with the receptor-binding domain (RBD) of the SARS-CoV-2 surface protein. The specificity of single-domain antibodies from the blood sera of a llama immunized with RBD of SARS-CoV-2 surface protein S (variant B.1.1.7 (Alpha)) was analyzed by ELISA. Recombinant trimers of the SARS-CoV-2 spike protein were used as antigens. In this work, a set of single-domain antibodies was obtained that specifically bind to the RBD of the SARS-CoV-2 virus.

Immunogenicity of the DNA/Protein Combined Vaccine against COVID-19.

During the COVID-19 pandemic, the development of prophylactic vaccines, including those based on new platforms, became highly relevant. One such platform is the creation of vaccines combining DNA and protein components in one construct. For the creation of DNA vaccine, we chose the full-length spike protein (S) of the SARS-CoV-2 virus and used the recombinant receptor-binding domain (RBD) of the S protein produced in CHO-K1 cells as a protein component. The immunogenicity of the developed combined vaccine and its individual components was compared and the contribution of each component to the induction of the immune response was analyzed. The combined DNA/protein vaccine possesses the advantages of both underlying approaches and is capable of inducing both humoral (similar to subunit vaccines) and cellular (similar to DNA vaccines) immunity.

Immunogenic Properties of the DNA Construct Encoding the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein.

The development of preventive vaccines became the first order task in the COVID-19 pandemic caused by SARS-CoV-2. This paper reports the construction of the pVAX-RBD plasmid containing the Receptor-Binding Domain (RBD) of the S protein and a unique signal sequence 176 which promotes target protein secretion into the extracellular space thereby increasing the efficiency of humoral immune response activation. A polyglucine-spermidine conjugate (PGS) was used to deliver pVAX-RBD into the cells. The comparative immunogenicity study of the naked pVAX-RBD and pVAX-RBD enclosed in the PGS envelope showed that the latter was more efficient in inducing an immune response in the immunized mice. In particular, RBD-specific antibody titers were shown in ELISA to be no higher than 1 : 1000 in the animals from the pVAX-RBD group and 1 : 42 000, in the pVAX-RBD-PGS group. The pVAX-RBD‒PGS construct effectively induced cellular immune response. Using ELISpot, it has been demonstrated that splenocytes obtained from the immunized animals effectively produced INF-γ in response to stimulation with the S protein-derived peptide pool. The results suggest that the polyglucine-spermidine conjugate-enveloped pVAX-RBD construct may be considered as a promising DNA vaccine against COVID-19.

[Immunogenic Properties of the DNA Construct Encoding the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein].

The development of preventive vaccines became the first order task in the COVID-19 pandemic caused by SARS-CoV-2. This paper reports the construction of the pVAX-RBD plasmid containing the Receptor-Binding Domain (RBD) of the S protein and a unique signal sequence 176 which promotes target protein secretion into the extracellular space thereby increasing the efficiency of humoral immune response activation. A polyglucine-spermidine conjugate (PGS) was used to deliver pVAX-RBD into the cells. The comparative immunogenicity study of the naked pVAX-RBD and pVAX-RBD enclosed in the PGS envelope showed that the latter was more efficient in inducing an immune response in the immunized mice. In particular, RBD-specific antibody titers were shown in ELISA to be no higher than 1 : 1000 in the animals from the pVAX-RBD group and 1 : 42000, in the pVAX-RBD-PGS group. The pVAX-RBD-PGS construct effectively induced cellular immune response. Using ELISpot, it has been demonstrated that splenocytes obtained from the immunized animals effectively produced INF-y in response to stimulation with the S protein-derived peptide pool. The results suggest that the polyglucine-spermidine conjugate-enveloped pVAX-RBD construct may be considered as a promising DNA vaccine against COVID-19.

Prototype of a DNA Vaccine against Marburg Virus.

The preparation and study of the biological properties of the pVAKS-GPVM DNA immunogen containing a gene encoding Marburgvirus glycoprotein are described. The specificity of blood serum antibodies of guinea pigs immunized with DNA immunogen was analyzed by ELISA. Inactivated viral preparation, recombinant glycoprotein (GP) obtained in the prokaryotic system and virus-like particles based on the recombinant vesicular stomatitis virus exhibiting Marburgvirus GP were used as the antigens. The neutralizing activity of antibodies of immunized animals was tested in vitro using a pseudovirus system. It was demonstrated that the developed immunogen administered to guinea pigs induced the production of specific antibodies that neutralize virus-like particles and Marburgvirus in cultured Vero cells.

[Nd:YAG laser goniopuncture as a mandatory adjuvant procedure after non-penetrating deep sclerectomy (long-term observation results)]. / Nd:YAG-lazernaia goniopunktura kak obiazatel'naia ad''iuvantnaia protsedura posle nepronikaiushchei glubokoi skleréktomii (rezul'taty dolgosrochnogo nabliudeniia).

Today, Nd:YAG laser goniopuncture (LGP) is considered a mandatory non-penetrating deep sclerectomy adjuvant procedure. However, its indications and timing remain debatable. PURPOSE: To evaluate the effect of Nd:YAG laser goniopuncture on the long-term hypotensive effectiveness of non-penetrating deep sclerectomy. MATERIAL AND METHODS: The study included 114 patients after non-penetrating deep sclerectomy (NPDS). In the control group (n=58), Nd:YAG laser goniopuncture was performed within 3.4±1.9 (1.5-6.7) months, and in the main group (n=56) - within 1.12±0.08 (0.9-1.5) months after the surgery. Ultrasound biomicroscopy (UBM) was used to evaluate the semiotics of trabecular-Descemet's membrane (TDM), intrascleral canal (ISC) and filtering bleb. The follow-up period was 5 years. RESULTS: According to UBM data, the thickness (0.10±0.009 mm) and density (50±6%) of TDM (p=0.0001) increased before LGP in the control group, the height of ISC decreased to 0.49±0.19 (0.20-0.40) (p=0.03), the height of UBM scan - to 1.49±0.05 (1.41-2.9) (p=0.0001); IOP (P0) was 18.48±4.7 (11.2-22.9) mmHg (p=0.001). In the main group before LGP, TDM thickness was 0.08±0.006 mm, density was 40±5%, and IOP (P0) was 15.7±4.1 (9.1-18.5) mm Hg. Complete hypotensive success was achieved in 83.6% of cases in the control group and 96.2% in the main group in 6 months; 68.07% and 92.59% in 12 months; 41.3% and 75.8% in 24 months; 15.25% and 48.93% in 36; 15% and 34.8% in 60 months after the surgery, respectively (p=0.0001, 95% confidence interval). CONCLUSION: TDM is an additional level of retention of aqueous humor and plays key role in the formation of outflow pathways after NPDS. Performing LGP in the early postoperative period is an effective and safe adjuvant option, which excludes the influence of TDM on the formation of aqueous humor outflow pathways and significantly increases the long-term hypotensive efficacy of non-penetrating deep sclerectomy.

Novel method for facial rejuvenation using Er:YAG laser equipped with a spatially modulated ablation module: An open prospective uncontrolled cohort study.

Facial aging is a complex biological process that affects the skin and superficial musculoaponeurotic system (SMAS). A new technology (RecoSMA) for skin rejuvenation based on acoustic-interference method using Er:YAG laser (2936 nm) equipped with a special module SMA that targets both the dermis and SMAS was evaluated in an open-label prospective cohort study of 100 female patients treated for facial rejuvenation. Measure of clinical improvement included investigator-rated clinical photography using the Modified Fitzpatrick Wrinkle Scale, and ultrasound measurements in the dermis a week, 30 days and six months post treatment. All patients completed the study and no complications were noted. Improvements in skin tone and texture were noted in all participants and significant decrease in wrinkle depth was demonstrated at the six-month follow-up that was confirmed by ultrasound skin measurements. Data presented herein confirm the safety and efficacy of RecoSMA treatment for facial rejuvenation.