Delivery of Experimental mRNA Vaccine Encoding the RBD of SARS-CoV-2 by Jet Injection.

We studied a needle-free jet injection delivery of an experimental mRNA vaccine encoding the receptor-binding domain of the SARS-CoV-2 S protein (mRNA-RBD). Immunization of BALB/c mice with mRNA-RBD by a needle-free jet injector induced high levels of antibodies with virus-neutralizing activity and a virus-specific T-cell response. The immune response was low in the group of mice that received intramuscular injection of mRNA-RBD. The effectiveness of this simple and safe method of mRNA delivering has been demonstrated. Thus, jet injection of mRNA vaccine can be a good alternative to lipid nanoparticles.

Removal of Double-Stranded RNA Contaminants During Template-Directed Synthesis of mRNA.

The removal of double-stranded RNA (dsRNA) contaminants during in vitro mRNA synthesis is one of the technological problems to be solved. Apparently, these contaminants are the result of the T7 RNA polymerase side activity. In this study, we used a modified method of mRNA purification based on the selective binding of dsRNA to cellulose in ethanol-containing buffer. It was shown both in vivo and in vitro that the cellulose-purified mRNA preparation leads neither to activation of the lymphocyte inflammatory marker CD69 nor to increased release of IFNα in mice, and does not contain impurities detectable by antibodies to dsRNA.

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.

DNA Vaccine Encoding the Artificial T-Cell Polyepitope Immunogen of Tick-Borne Encephalitis Virus.

A promising approach to the development of new means for preventing infection caused by tick-borne encephalitis virus can be DNA vaccines encoding polyepitope T-cell immunogens. A DNA vaccine pVAX-AG4-ub encoding an artificial polyepitope immunogen that includes cytotoxic and T-helper epitopes from the NS1, NS3, NS5, and E proteins of the tick-borne encephalitis virus has been obtained. The developed construct ensured the synthesis of the corresponding mRNAs in transfected eukaryotic cells. Immunization of mice with pVAX-AG4-ub induced the formation of a virus-specific T-cell response providing 50% protection from lethal infection with the virus.

Artificial COVID-19 T-Cell Immunogen.

An artificial T-cell immunogen consisting of conserved fragments of different proteins of the SARS-CoV-2 virus and its immunogenic properties were studied in BALB/c mice. To create a T-cell immunogen, we used an approach based on the design of artificial antigens that combine many epitopes from the main proteins of the SARS-CoV-2 virus in the one molecule. The gene of the engineered immunogen protein was cloned as part of the pVAX1 plasmid in two versions: with an N-terminal ubiquitin and without it. The obtained plasmids were analyzed for their ability to provide the synthesis of the immunogen protein in vitro and in vivo. It has been shown that protein product of the created artificial genes is actively processed in HEK293T cells and induces cellular immunity in mice.

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.

mRNA Vaccine Platform: mRNA Production and Delivery.

Vaccination is the most efficient way to prevent infectious diseases. mRNA-based vaccines is a new approach to vaccine development, which have several very useful advantages over other types of vaccines. Since mRNA encodes only the target antigen there is no potential risk of infection as in the case with attenuated or inactivated pathogens. The mode of action of mRNA-vaccines implies that their genetic information is expressed only in the cytosol, leaving very little possibility of mRNA integration into the host's genome. mRNA-vaccines can induce specific cellular and humoral immune responses, but do not induce the antivector immune response. The mRNA-vaccine platform allows for easy target gene replacement without the need to change the production technology, which is important to address the time lag between the epidemic onset and vaccine release. The present review discusses the history of mRNA vaccines, mRNA vaccine production technology, ways to increase mRNA stability, modifications of the cap, poly(A)-tail, coding and noncoding parts of mRNA, target mRNA vaccine purification from byproducts, and delivery methods.

Methods to Produce Monoclonal Antibodies for the Prevention and Treatment of Viral Infections.

A viral threat can arise suddenly and quickly turn into a major epidemic or pandemic. In such a case, it is necessary to develop effective means of therapy and prevention in a short time. Vaccine development takes decades, and the use of antiviral compounds is often ineffective and unsafe. A quick response may be the use of convalescent plasma, but a number of difficulties associated with it forced researchers to switch to the development of safer and more effective drugs based on monoclonal antibodies (mAbs). In order to provide protection, such drugs must have a key characteristic-neutralizing properties, i.e., the ability to block viral infection. Currently, there are several approaches to produce mAbs in the researchers' toolkit, however, none of them may serve as a gold standard. Each approach has its own advantages and disadvantages. The choice of the method depends both on the characteristics of the virus and on time constraints and technical challenges. This review provides a comparative analysis of modern methods to produce neutralizing mAbs and describes current trends in the design of antibodies for therapy and prevention of viral diseases.

Model systems of human immunodef iciency virus (HIV-1) for in vitro eff icacy assessment of candidate vaccines and drugs against HIV-1.

HIV infection still remains a major challenge for healthcare systems of the world. There are several aspects on counteracting the HIV/AIDS epidemic. The f irst aspect covers preventive measures including educational campaigns on HIV/AIDS and promotion of a healthy lifestyle, protected sex, and pre-exposure prophylaxis of vulnerable groups. The second aspect is timely HIV testing and the use of antiretroviral therapy when test results come back positive. The third aspect is the scientif ic research associated with discovering new pharmaceutical agents and developing HIV-1 vaccines. Selecting an adequate tool for quick and accurate in vitro eff icacy assessment is the key aspect for eff icacy assessment of vaccines and chemotherapy drugs. The classical method of virology, which makes it possible to evaluate the neutralizing activity of the sera of animals immunized with experimental vaccines and the eff icacy of chemotherapy agents is the method of neutralization using viral isolates and infectious molecular clones, i. e. infectious viral particles obtained via cell transfection with a plasmid vector including the full-length HIV-1 genome coding structural, regulatory, and accessory proteins of the virus required for the cultivation of replication-competent viral particles in cell culture. However, neutralization assessment using viral isolates and infectious molecular clones is demanding in terms of time, effort, and biosafety measures. An alternative eliminating these disadvantages and allowing for rapid screening is the use of pseudoviruses, which are recombinant viral particles, for the analysis of neutralizing activity. Pseudotyped viruses have defective genomes restricting their replication to a single cycle, which renders them harmless compared to infectious viruses. The present review focuses on describing viral model systems for in vitro eff icacy assessment of vaccines and drugs against HIV-1, which include primary HIV-1 isolates, laboratoryadapted strains, infectious molecular clones, and env-pseudoviruses. A brief comparison of the listed models is presented. The HIV-1 env-pseudoviruses approach is described in more detail.

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.

Characterization of the Altai Maral Chymosin Gene, Production of a Chymosin Recombinant Analog in the Prokaryotic Expression System, and Analysis of Its Several Biochemical Properties.

For the first time, the chymosin gene (CYM) of a maral was characterized. Its exon/intron organization was established using comparative analysis of the nucleotide sequence. The CYM mRNA sequence encoding a maral preprochymosin was reconstructed. Nucleotide sequence of the CYM maral mRNA allowed developing an expression vector to ensure production of a recombinant enzyme. Recombinant maral prochymosin was obtained in the expression system of Escherichia coli [strain BL21 (DE3)]. Total milk-coagulation activity (MCA) of the recombinant maral chymosin was 2330 AU/ml. The recombinant maral prochymosin relative activity was 52955 AU/mg. The recombinant maral chymosin showed 100-81% MCA in the temperature range 30-50°C, thermal stability (TS) threshold was 50°C, and the enzyme was completely inactivated at 70°C. Preparations of the recombinant chymosin of a single-humped camel and recombinant bovine chymosin were used as reference samples. Michaelis-Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) of the recombinant maral chymosin, were 1.18 ± 0.1 µM, 2.68 ± 0.08 s-1 and 2.27± 0.10 µm M-1·s-1, respectively.

mRNA technology as one of the promising platforms for the SARS-CoV-2 vaccine development.

After the genome sequence of SARS-CoV-2 (Severe acute respiratory syndrome-related coronavirus 2) was published and the number of infected people began to increase rapidly, many global companies began to develop a vaccine. Almost all known approaches to vaccine design were applied for this purpose, including inactivated viruses, mRNA and DNA-vaccines, vaccines based on various viral vectors, synthetically generated peptides and recombinant proteins produced in cells of insects and mammals. This review considers one of the promising vaccine platforms based on messenger RNA. Until recent years, mRNA-vaccination was out of practical implementation due to high sensitivity to nuclease degradation and consequent instability of drugs based on mRNA. Latest technological advances significantly mitigated the problems of low immunogenicity, instability, and difficulties in RNA-vaccine delivery. It is worth noting that mRNA-vaccines can efficiently activate both components of the immune system, i. e. T-cell and humoral responses. The essential advantage of mRNAvaccines includes fast, inexpensive, scalable and uniform production providing a large output of desirable products in vitro. Synthesis and purification processes significantly simplify the process technology of mRNA drugs with injectable purity. Thus, mRNA production via in vitro transcription is more advantageous as compared with DNA-vaccines since it is a chemical process without the use of cells. mRNA techniques make it possible to pass all the phases of vaccine development much faster in comparison with the production of vaccines based on inactivated viruses or recombinant proteins. This property is critically important when designing vaccines against viral pathogens as the main problem of disease control includes a time gap between an epidemic and vaccine development. This paper discusses studies on the development of vaccines against coronaviruses including SARS-CoV-2 with special attention to the mRNA technique.

The Structural and Immunological Properties of Chimeric Proteins Containing HIV-1 MPER Sites.

The human immunodeficiency virus (HIV-1) poses a serious risk to global public health. The development of a safe and effective vaccine could stop the HIV/AIDS pandemic. Much of the research focused on HIV-1 prevention through vaccination is aimed at developing immunogens and immunization strategies to induce the formation of antibodies with neutralizing activity against a broad range of HIV-1 isolates (bNAbs). The objective of this study was to develop immunogens capable of targeting an immune response to MPER, one of the regions of bNAb binding in Env. Two immunogens carrying MPER fragments on their scaffolds (protein YkuJ Bacillus subtilis and artificial polypeptide TBI) were constructed. Circular dichroism spectroscopy was used to show that the secondary structure of the immunogens was consistent with their theoretical models. The antigenic structure of the MPER-TBI and YkuJ-MPER proteins was characterized using bNAbs that recognize HIV-1 MPER (2F5, 4E10, and 10E8). The rabbit model made it possible to show the immunogenicity of the constructed recombinant proteins. The resulting serum was found to be cross-reactive with immunogens carrying MPER. The constructs designed and characterized in this study can be used for targeting the humoral immune response to MPER, which is known to be one of the sites of HIV-1 vulnerability.

Design of Artificial Immunogens Containing Melanoma-associated T-cell Epitopes.

OBJECTIVE: Immunotherapy based on induction of T-cell responses is a promising approach to cancer treatment. The study aims to design artificial epitope-based immunogens, DNA vaccine candidates against melanoma and evaluate their ability to stimulate tumor cytotoxicity of ex vivo generated T-cells. METHODS: The original computational methods were used for predicting T-cell epitopes and designing polyepitope melanoma antigens. Artificial genes encoding the target antigens were cloned into DNA vaccine plasmid vector. Target gene expression was confirmed both at transcriptional and translational level in HEK-293T cells transfected with DNA-vaccine constructs. Dendritic cells were generated from adherent peripheral blood mononuclear cells of HLA-A*02:01+ donors. Cytotoxic activity of effector lymphocytes stimulated in co-culture with autologous antigen-presenting dendritic cells towards melanoma Mel Is cells was assessed with lactate dehydrogenase release assay. The proportion of granzyme B producing CD8+ T-cells was estimated using intracellular cytokine staining and flow cytometry. RESULTS: Two DNA vaccine constructions were created - pMEL-TCI and pMEL-A0201 - encoding polypeptides containing T-cell epitopes of six immunodominant melanoma antigens (NY-ESO-1, MART1, MAGE-A1, MAGE-A11, MAGE-A3, and MAGE-C1). Dendritic cells transfected with DNA vaccine constructs were found to stimulate both tumor cytotoxicity mediated by autologous lymphocytes and granzyme B production by CD8+ T-cells, and pMEL-A0201 was found to be the most efficient. CONCLUSION: The described approach may become a common platform for designing immunotherapeutic vaccines against oncological diseases.

Design of Polyepitope DNA Vaccine against Breast Carcinoma Cells and Analysis of Its Expression in Dendritic Cells.

Polyepitope DNA vaccine inducing T-cell-mediated immune response against cancer-specific antigens is a promising tool for selective elimination of tumor cells. Breast cancer-specific polyepitope DNA vaccine was designed using TEpredict and PolyCTLDesigner software on the basis of immunogenic peptides of HER2 and Mammaglobin-1 (Mam) tumor antigens. LPS-free preparations of plasmid DNA encoding polyepitope T-cell antigen and full-length copies of HER2 and Mam antigens were obtained. TaqMan-PCR systems for evaluation of the expression of immunogens in cells were created. The protocol of vaccine DNA delivery into dendritic cells was optimized. Expression of the target immunogens in dendritic cells derived from human peripheral blood mononuclear fraction after transfection with plasmid DNA preparations is demonstrated.

Broadly Neutralizing Antibodies against HIV-1 As a Novel Aspect of the Immune Response.

The human immunodeficiency virus-1 (HIV-1) has the ability to evade the adaptive immune response due to high mutation rates. Soon after the discovery of HIV-1, it was originally proposed that neutralizing of antibodies to the virus occurs rarely or cannot be elicited at all. In the 1990s, there appeared reports that sera of select HIV-1-infected individuals contained antibodies capable of neutralizing different virus subtypes. Such antibodies were named broadly neutralizing antibodies (bNAbs). Since 2009, the development of new cell technologies has intensified research efforts directed at identifying new bNAbs with a neutralization potency of over 90% of primary HIV-1 isolates. These antibodies have unique characteristics which include high levels of somatic mutations and unusually long variable loops that penetrate through the glycan shield of HIV-1 Env to contact the protein surface. In this review, we will attempt to summarize the latest data on bNAbs against HIV-1 in terms of their interactions with the sites of vulnerability on HIV-1 glycoproteins.

Rational design based synthetic polyepitope DNA vaccine for eliciting HIV-specific CD8+ T cell responses.

Advances in defining HIV-1 CD8+ T cell epitopes and understanding endogenous MHC class I antigen processing enable the rational design of polyepitope vaccines for eliciting broadly targeted CD8+ T cell responses to HIV-1. Here we describe the construction and comparison of experimental DNA vaccines consisting of ten selected HLA-A2 epitopes from the major HIV-1 antigens Env, Gag, Pol, Nef, and Vpr. The immunogenicity of designed gene constructs was assessed after double DNA prime, single vaccinia virus boost immunization of HLA-A2 transgenic mice. We compared a number of parameters including different strategies for fusing ubiquitin to the polyepitope and including spacer sequences between epitopes to optimize proteasome liberation and TAP transport. It was demonstrated that the vaccine construct that induced in vitro the largest number of [peptide-MHC class I] complexes was also the most immunogenic in the animal experiments. This most immunogenic vaccine construct contained the N-terminal ubiquitin for targeting the polyepitope to the proteasome and included both proteasome liberation and TAP transport optimized spacer sequences that flanked the epitopes within the polyepitope construct. The immunogenicity of determinants was strictly related to their affinities for HLA-A2. Our finding supports the concept of rational vaccine design based on detailed knowledge of antigen processing.