Investigation of the Presence of DNA of Highly Pathogenic Human Papillomaviruses in Water Bodies of the Lake Baikal Natural Territory.

Human papillomaviruses (HPVs) are extremely widespread throughout the world. There are more than 100 types of HPVs, of which at least 14 types represent high oncogenic risk viruses (World Health Organization, 2020). Numerous attempts were made to analyze various water sources in order to (i) reveal the presence of DNA of pathogenic human papillomaviruses in them and (ii) assess the potential risks of occurrence of epidemics caused by HPV. With time, the necessity to solve these important problems stimulated the formation of a new direction in the world medical and environmental investigations.This paper contains the investigation of the presence of DNA of highly dangerous types of human papillomaviruses (HPV6, HPV11, HPV16 and HPV18) in water bodies of the Baikal natural territory, in particular in the water reservoirs in and near the villages of Listvyanka, Bolshiye Koty, Kultuk and the cities of Baikalsk and Slyudyanka. In course of our work, the conditions good for the study of the biological material obtained from water samples by the PCR technique to reveal the presence of DNA of HPV6, HPV11, HPV16 and HPV18 papillomaviruses were chosen. PCR analysis was conducted with the aid of both the already well-known universal primers GP5 + /6 + and the primers developed by our team to be applied to the conservative domains of nucleotide sequences encoding the main capsid protein L1 of human papillomaviruses HPV6, HPV11 (these types of the virus contribute to the occurrence of anogenital condylomatosis and the development of respiratory papillomatosis) and HPV16, HPV16 (these types of virus contribute to the occurrence of cervical cancer).The analyzes conducted by our team have revealed the presence of DNA of the four types of HPVs (6, 11, 16 and 18) in the samples taken from various water sources of the Baikal natural territory.

A bioinformatics approach for identifying the probable cause of the cross-interaction of antibodies to the antigenic protein HPV16 L1 with the HPV6 L1 protein.

This paper describes an attempt to analyze, with the aid of bioinformatics resources (programs and databases), the probable cause of the cross-interaction of antibodies against HPV16 L1 with antigenic protein HPV6 L1, which has been revealed in the investigation of the candidate vaccine obtained on the base of a plant expression system (tomato plants). In our opinion, the most likely reason for the cross-interaction of antibodies with antigens of different pathogenic HPV types is the similarity of their antigenic determinants. In this work, the amino acid sequences of HPV16 L1 and HPV6 L1 used for the development of a binary vaccine against cervical cancer and anogenital papillomatosis have been analyzed. For the analysis of antigenic determinants, the programs BepiPred-2.0: Sequential B-Cell Epitope Predictor, DiscoTope 2.0 Server and SYFPEITHI have been used. As a result of the analysis of probable B-cell linear determinants (epitopes), it has been found that in both types of HPV the proteins have approximately the same location and size of linear antigenic determinants; the difference is observed only in the form of small shifts in the size of several amino acid residues. However, there are some differences in the amino acid composition of epitopes; therefore, the possibility for cross-interaction of the antibodies with the antigens due to the similarity of linear antigenic determinants for B-cells is very small. The analysis of potential threedimensional epitopes for B-cells has shown that due to little difference between them the HPV16 L1 and HPV6 L1 proteins have no prerequisites for cross-interaction of the antibodies with the antigens belonging to the two different pathogenic HPV types. The analysis of probable linear epitopes for T-cells has revealed a common antigenic determinant in the two protein sequences. According to the rank made with the SYFPEITHI program, the amino acid sequence AQL(I)FNKPYWL is the second most likely antigenic determinant for T-cells. Meanwhile, the amino acid sequences of this determinant in HPV16 L1 and HPV6 L1 are virtually identical. There is a difference in only one position, but it is not critical due to the similarity of the physicochemical properties of amino acids, for which there is a replacement in the amino acid sequence of antigenic determinants. Consequently, some moderate cross-interaction of the antibodies to HPV16 L1 with the antigens of HPV6 L1 may be expected.

The Use of the Antigenic Protein HPV6 L1 to Induce the Synthesis of Interferon, CD4 and CD8 T Lymphocytes, and Granzyme B in Blood and Splenocytes of Mice in Order to Develop a Broad Vaccine against Anogenital Papillomatoses.

The antigenic protein HPV6 L1 was synthesized in the plant expression system based on transgenic tomato fruit during the development of an oral vaccine against anogenital papillomaviruses. In experiments on mice immunization, new data on the induction of the T-cell immune response were obtained, which were recorded by the results of activation of the synthesis of interferon, CD4 and CD8 T lymphocytes, and granzyme B secreted by them in peripheral mononuclear blood cells and splenocytes of mice that were previously vaccinated with the vaccine material of tomato fruit with the HPV6 L1 gene.

The Synthesis of Main Capsid Protein of Anogenital Type HPV6 L1 in Plant Expression System on the Basis of Tomato Fruits.

The anogenital type HPV6 L1 major capsid protein was synthesized in a plant expression system on the basis of tomato fruits. The content of HPV6 L1 reached 380 µg per 1 mg of total soluble protein of raw fruit mass, which was represented as a single band with a molecular mass of 56 kDa on the SDS electrophoregram. When orally administrated to mice, the vaccine material from the tomato fruit transgenic for HPV6 L1 induced highly effective antibody immune response with a high titer. The cross-reactivity during the interaction of the antibody to the HPV6 L1 protein from peripheral blood serum of mice vaccinated with HPV6 L1 with the antigenic proteins HPV16 L1, HPV18 L1, HPV31 L1, and HPV45 L1 was found. This is promising for creating a vaccine with a broad reactivity against dangerous anogenital papillomatoses and cervical cancer.

The Antiproliferative Effect of the "Early" Protein E2 of Papillomavirus HPV16 on Testis Tumors of Mice Induced by the Injection of HeLa Cells.

The antiproliferative effect of the "early" protein E2 of the high-risk oncogenic human papillomavirus HPV16 on mouse testis tumors, which were induced by the intramuscular injection of HeLa cells, was discovered. The regression of tumors was maximum in the first 2 days after the oral vaccination with HPV16 E2 (500 mg per mouse) and then gradually decreased to the control variant. A typical monolayer of HeLa cells on the cultivation flask bottom, of which only 18% were functionally active, appeared after seeding testis tissue cells on DMEM medium.

The Induction of the Synthesis of Interferon, CD4 and CD8 T Lymphocytes in Blood and Spleen of Mice after Oral Vaccination with the "Early" Protein HPV16 E2.

High contents of interferon and CD4 and CD8 T lymphocytes were found in blood and spleen cells of mice after their oral vaccination with the "early" protein of HPV16 E2 by using ELISPOT, Western blot hybridization, and enzyme immunoassay technique. It was concluded that the "early" protein HPV16 E2 is involved in the immune system activation during control of pathogenesis and neoplasia.

The New Plant Expression System for the Development of Vaccines against Papillomaviruses.

To enhance the synthesis of antigenic envelope proteins L1 of high-grade papillomavirus types HPV16, HPV18, HPV31, and HPV45, the sequence of the gene encoding the cucumber mosaic virus replicase (RdRP CMV) was inserted into the genetic construct. This made it possible to increase the production of these antigenic proteins to 25-27 µg/mg total soluble protein.

Synthesis of Proteins Encoded by the Early Genes E2, E6, and E7 of Papillomavirus of Type 16 in the Plant Expression System.

An expression system for the synthesis of early antigenic proteins HPV16 E2, HPV16 E6, and HPV16 E7 of high-risk papillomavirus based on transgenic tomato fruits was developed. It is planned to use the early antigenic proteins synthesized to create a therapeutic vaccine against cervical cancer.

Calcium as a Modulator of the Adenylyl Cyclase Activity of Potato Cells in Bacterial Pathogenesis.

This is the first study to detect the effect of calcium ions on the activity of transmembrane adenylyl cyclase (tmAC), the key enzyme of the adenylyl cyclase signaling system, under normal conditions and after a short-term exposure to exopolysaccharides (EPS) of the bacterial ring rot pathogen Clavibacter michiganensis ssp. sepedonicus (Cms). After the treatment of the roots of plants with the Cms EPS, the response to Ca2+ changed: the activity of the tmAC of plants of the resistant cultivar significantly increased, whereas in the cells of the susceptible cultivar it remained unchanged.

Detection of lipid-protein microdomains (rafts) and investigation of their functional role in the chloroplast membranes of halophytes.

To study the characteristics of the structure and the functional role of chloroplast membranes of halophytes with different salt tolerance strategy, raft structures were isolated. Lipid analysis data provided evidence for the presence of rafts in the chloroplast membranes. The discovered significant differences in the content of raft-forming lipids in different halophytes suggest a relationship between the functions of rafts and the mechanisms of salt tolerance in plants.

The study of immunogenicity of the antigenic protein of high risk oncogenic type of the human papillomavirus HPV16 L1 produced in the plant expression system on the base of transgenic tomato.

The immunogenicity of plant-made peroral vaccine against cervical cancer was studied in mice during 342 days after vaccination with the material of tomato fruits genetically transformed with HPV16 L1 Ώ 5'UTR TMV. The immune response was found on day 4 after vaccination in blood serum of vaccinated mice. On days 90-100, the rise to maximum value of the level of antibodies to HPV16 L1 was in the range of 500 ng of the standard antibodies to HPV16 L1 (Santa Cruz Biotechnology, United States). This level of antibodies was retained until the end of the study.

Cross-reactivity of antigens and antibodies belonging to different pathogenic types of human papillomaviruses.

The analysis of the properties of a quadrivalent peroral vaccine against the cervical cancer, which was created in a plant expression system on the base of transgenic tomato fruits, by immunoassay and Western blot hybridization showed that the antibodies against human papilloma virus 16 L1 (HPV16 L1) actively interacted not only with the antigenic proteins HPV18 L1, HPV31 L1, and HPV45 L1, but also with the antigenic protein HPV6 L1, which belongs to another HPV family. Thus, new data on the possibility of crossreactivity between antibodies and antigens belonging to remote HPV families were obtained.

Using the omega leader sequence of tobacco mosaic virus to transform tomato fruits with the papillomavirus hpv16 L1 gene to enhance production of the antigenic protein HPV16 L11.

To enhance translation of the L1 protein antigen of the oncogenic human papillomavirus type HPV16 L1, the Ω sequence of the 5'-untranslated region of tobacco mosaic virus was inserted into a genetic construct as an enhancer. To transform plants, A. tumefaciens EHA105 cells were transfected with this construct. After the genetic transformation, the HPV16 L1 protein antigen was detected in tomato fruit in amounts of 287-2330 ng per 1 mg total soluble protein, which significantly exceeds the amount of the protein antigen obtained in our previous studies without using the omega leader sequence.

[PECULIAR PROPERTIES OF SOME COMPONENTS OF MORPHOLOGICAL STRUCTURE IN A PLANT CELL VACUOLE REVEALED BY CONFOCAL MICROSCOPY].

Results of investigations of peculiar properties related to the structure of plant cell vacuolar membranes are discussed. The study was carried out using confocal microscopy, which allowed us in the process of scanning to identify membrane tubes and vesicules in the preparations of isolated vacuoles. Such membrane tubes were found both inside and outside the vacuoles, and, in the case of scanning intermittently at equal time intervals, transition of vesicles with the membrane tube was observed. Furthermore, scanning of isolated vacuoles was conducted at various distances from the glass substrate. Each time, in the upper area of the isolated vacuole lying on the substrate, we observed a large segment of vacuolar membrane and registered the effect of highly intensive fluorescing of some of membrane segments. The distributions of laurdan fluorescence generalized polarization (GP) values for the vacuolar membrane on the whole and for the intensively fluorescing membrane segments have been obtained. We have found that the microviscosity of the intensively fluorescing membrane segments essentially differs from that of the rest part of the membrane.