This review is focused on polyprenyl phosphatis (PPFs) (phosphorilated polyprenols), a unique class of natural compounds with a wide spectrum of biological activity. The main emphasis is put on their antiviral properties that were studied in numerous in vitro and in vivo experiments. The results formed the basis for the development of new drugs containing PPFs as the active substance. These drugs, due to their high efficiency and harmlessness, are widely used in veterinary practice in the treatment of viral diseases of both small domestic and agricultural animals. Also, the world’s first PPF-based drug is registered in Russia, and it can be used as part of a comprehensive therapy of chronic recurrent genital infection caused by the herpes simplex virus.

INTRODUCTION:Hemorrhagic fever with renal syndrome (HFRS) holds a leading place among natural focal human diseases in Russian Federation. There is no etiotropic therapy for the disease now. The vaccine prophylaxis is the most effective method to control this infection. The main criteria for inactivated vaccines evaluation are its immunogenicity and specific activity.The study purposes were to develop a sensitive and specific real-time PCR method for viral RNA quantification in the inactivated vaccine and to study the correlation between the viral RNA amount and vaccine immunogenicity.MATERIAL AND METHODS:L-segment fragments of the Puumala, Hantaan, and Sochi vaccine strains were selected as diagnostic targets for oligonucleotides and fluorescent probes. The immunogenicity of experimental vaccines was determined by the induction of neutralizing antibodies in BALB/c mice.RESULTS:A highly specific, sensitive and reproducible real-time PCR method has been developed. The analytical sensitivity was 1.24 ± 1.5 x 102 copies/ml for Puumala virus; 1.16 ± 1.4 * 102 copies/ml for Hantaan; 1.32 ± 1.8 * 102 copies/ ml for Sochi, with a virus content of 1.5 ± 0.5 lg FFU/ml; 1.8 ± 0.5 lg FFU/ml and 2.2 ± 0.5 lg FFU/ml, respectively. The viral RNA amount in experimental vaccine preparations inactivated with β-propiolactone was proportional to the neutralizing antibodies titer observed in mice following the immunization.DISCUSSION:It was found that different virus inactivators differently affects the detected viral RNA amount, but not the vaccine immunogenicity, which indicates the same degree of the immunogenic proteins damage. The direct relationship between the viral RNA copy number and vaccine immunogenicity makes it possible to use this criterion for vaccine dosage preparation.CONCLUSION:The developed method for viral RNA quantification is a promising tool for the specific activity control of the HFRS vaccine.

Introduction. Since the development of inactivated polio vaccines, different stages of the production process have been changed and improved. Current production of inactivated polio vaccines based on both wild and attenuated strains includes several technological stages, one of which is the concentration of the virus-containing liquid, which ensures poliovirus concentration, and purification of the virus-containing liquid from a significant part of the ballast components.Research objective is to compare the characteristics of ultrafiltration membranes and select the membranes that provide optimal value of purification and concentration of poliovirus type 1 (Sabin strain).Materials and methods. Laboratory ultrafiltration systems from two manufacturers with 50, 100, and 300 kDa membranes were used for the concentration. Results were evaluated by the content of total protein, which is the main stress for the subsequent purification stages, the value of infectious virus titer in the concentrate, and the content of D-antigen as the target product.Results and discussion. Obtained results demonstrated that the content of the target product (the highest D-antigen content) and purification from impurity proteins (the total protein content in the concentrate) were most optimal when a membrane with a cut-off of 300 kDa was used for concentration. The study also evaluated the real cut-off components by various membranes to determine the composition of the protein load on the target product.Conclusion. In terms of quality of the resulting target product and the manufacturability of the production process, the use of a 300 kDa membrane is the most appropriate when working out the technology for manufacturing inactivated polio vaccine based on Sabin strains of poliovirus and the Vero line as a producing culture.

Introduction . Since the development of inactivated polio vaccines, different stages of the production process have been changed and improved. Current production of inactivated polio vaccines based on both wild and attenuated strains includes several technological stages, one of which is the concentration of the virus-containing liquid, which ensures poliovirus concentration, and purification of the virus-containing liquid from a significant part of the ballast components. Research objective is to compare the characteristics of ultrafiltration membranes and select the membranes that provide optimal value of purification and concentration of poliovirus type 1 (Sabin strain). Materials and methods . Laboratory ultrafiltration systems from two manufacturers with 50, 100, and 300 kDa membranes were used for the concentration. Results were evaluated by the content of total protein, which is the main stress for the subsequent purification stages, the value of infectious virus titer in the concentrate, and the content of D-antigen as the target product. Results and discussion . Obtained results demonstrated that the content of the target product (the highest D-antigen content) and purification from impurity proteins (the total protein content in the concentrate) were most optimal when a membrane with a cut-off of 300 kDa was used for concentration. The study also evaluated the real cut-off components by various membranes to determine the composition of the protein load on the target product. Conclusion . In terms of quality of the resulting target product and the manufacturability of the production process, the use of a 300 kDa membrane is the most appropriate when working out the technology for manufacturing inactivated polio vaccine based on Sabin strains of poliovirus and the Vero line as a producing culture.

The immunomodulatory properties of immunobiological drugs Glutoxim and Phosprenyl we well as vesicular stomatitis virus and inactivated tick-borne encephalitis vaccine virus were studied using human diploid fibroblast cell line from the collection of M. P. Chumakov Federal Research Center for Research and Development of Immunobiological Products. All tested preparations exhibited immunomodulatory activity in human diploid fibroblast cell line. Glutoxim in doses of 0.1 and 0.25 μg/ml stimulated production of IL-6 and IL-10 during 24-48 h of culturing, but did not stimulate production of IL-1β. Phosprenyl, on the contrary, increased production of IL-1β and the levels of IL-6 and IL-10. Vesicular stomatitis virus stimulated the production of IL-1β, IL-6, and IL-10, while inactivated tick-borne encephalitis vaccine virus stimulated the production of cytokines IL-8 and IL-18. Immunomodulatory activity of inactivated tick-borne encephalitis vaccine virus was first demonstrated in the in vitro system.

Antiapoptosis activity of plant polyprenylphosphate against macrophage target cells infected with the murine encephalomyelitis virus. Infection caused by the Theiler’s murine encephalomyelitis virus (TMEV) is regarded as an experimental model of multiple sclerosis, since both of these diseases are characterized by similar pathology of the central nervous system tissues and involvement of the immune system in the development of the demielinization. The aim of the work was to study the effect of plant-derived polyprenylphosphate (PP) on the apoptosis of infected target cells. We showed that PP reduced apoptosis of macrophage target cells infected with TMEV. It is known that in the protocol of multiple sclerosis treatment some medicines possessing immunomodulatory, antiviral, anti-inflammatory and antioxidant activity are used. Since PPs of plant origin also have all these activities, the prospects of their use as therapeutic agents are discussed.

We studied the sensitivity of domestic proprietary human and animal cell lines from the collection of M. P. Chumakov Federal Scientific Center for Research and Development of Immuneand-Biological Products to infection with different enterovirus 71 strains. A cell system based on domestic proprietary permanent cell line 4647 was for the first time used for reproduction of four enterovirus 71 strains (BrCr, 42266, 42934, and 43374). It was shown that strain 4647 is the optimal cell substrate for enterovirus 71 reproduction. The titers of enterovirus 71 for all four strains considerably (by 2 lgTCID 50 /ml and more) increased during sequential passages in permanent cell line 4647. The prospects of using permanent cell line 4647 for creation of diagnostic and preventive preparations against 71 was demonstrated.

Рассмотрена роль первичных фибробластов и линий диплоидных клеток фибробластов человека в формировании противоинфекционного иммунитета. Обсуждается адекватность клеточной модели на основе фибробластов для изучения молекулярных механизмов иммунопатогенеза вирусных инфекций. Рассмотрена возможность использования фибробластов для изучения особенностей действия иммунотропных препаратов. Обсуждается перспективность клеточной модели на основе аттестованных линий диплоидных клеток фибробластов для изучения механизмов действия новых и известных иммуномодуляторов.

AIM:Study of antiviral activity of moraprenil phosphates (MPP) against herpes simplex type 1 virus (HSV1) in vitro and during experimental infection caused by HSV1 in mice.MATERIALS AND METHODS:Activity of MPP in vitro was tested by the ability to suppress formation of symplasts in VERO cells infected with HSV1, strain VR-3. A series of MPP that suppress virus-induced symplast-formation by 30 times was selected for in vivo experiments. Anti-viral activity of MPP in vivo was studied in HSV-1 infected mice after administration of either prophylaxis or therapy regimens.RESULTS:MPP at the dose of 20 microg/mice during s/c administration exhibited a pronounced prophylactic-therapeutic effect. Effectiveness of MPP during clinically evident herpes against the background of developing neurologic symptoms was demonstrated for the first time. Visual observation of the mice, that had received MPP as the first clinical symptoms of the disease appeared, has shown that against the background of preparation injection the clinical signs have ceased after 2 - 3 days and did not registered at least for the whole duration of the observation period (14 days).CONCLUSION:Active herpes infection is accompanied by the increase of FoxP3 expression in-thymus was shown. Possible mechanisms of anti-viral effect of MPP are discussed.

The attempts to decrease a toxic action of Freund complete adjuvant have led to development of new emulsion adjuvant compositions. More often they are based on metabolizable oil (squalene) with addition the Tween 80 (Polisorbate 80) as an emulsifier. The third component is an immunomodulator. Adjuvants, intended for prevention of virus infections, should include the immunomodulators polarizing the immune response to Th1. From this point of view adjuvant based on a colloidal solution prepared by means of the Tween 80 from polyprenyl phasphates of fir needles (Phosprenyl) can be rather perspective. The last one, as well as squalene, is constructed by isoprene links, but has them not 6, but 16 that essentially reduces its toxicity. The obtained data shows that Phosprenyl essentially enhances efficiency of vaccination in such virus infections as tick born encephalitis, bird flu, poliomyelitis and polarizes the immune response to Th1.

The review is dedicated to immunologic adjuvants--various natural and synthetics substances that are added to vaccines for stimulation of specific immune response, but they do not induce specific response themselves. Critically important is the selection of the correct adjuvants, for which mechanisms of effect on immune system are studied the most. The majority of these mechanisms as well as physical-chemical and biological features of modern adjuvants are analyzed in the review. The problem of safety of adjuvants, types of immune response induced by adjuvants of various nature, excipients that are being verified or already in use in modern medicine and veterinary are also examined.

Исследована транскрипционная активность генов некоторых цитокинов (ЦТ)в головном мозге мышей при экспериментальной инфекции, вызвынной вирусом клещевого энцефалита (ВКЭ) после интрацеребрального заражения. Животные были разделены на группы с учетом выраженности признаков ВКЭ-инфекции. Показано, что ВКЭ-инфекция, сопровождающаяся неврологической симптоматикой, ассоциирована с высокими уровнями вирусной РНК в головном мозге. При этом регистрировались высокие уровни экспрессии генов провоспалительных ЦТ, что свидетельствует о выраженной воспалительной реакции, повреждающей головной мозг. Пероральное введение преднизолона достоверно снижало летальность инфицированных мышей. При и.ц. введении препарата на основе полипренилфосфата натрия (ППФ) наблюдалось снижение летальности с 67 % в группе животных, инфицированных ВКЭ, до 9 % в группе мышей, которым одновременно вводили ППФ и ВКЭ. При этом обнаружено, что и.ц. введение ППФ вызывает увеличение экспрессии гена IL6 в головном мозге в 5,4 раз по сравнению с контролем через 6 часов после введения, что, по-видимому, обусловливает активацию механизмов естественной резистентности и раннее подавление репродукции вируса. Эксперименты с антителами (АТ) к ВКЭ показали, что защитные свойства АТ проявляются лишь при условии их применения до или на ранних сроках после инфицирования. Введение АТ через 3–4 суток после интраперитонеального заражения мышей было неэффективно. Использование инъекций ППФ на поздних сроках относительно инфицирования также малоэффективно. По-видимому, это связано со значительным иммунопатогенным действием ВКЭ в этот период времени. В то же время введение ППФ с АТ оказывало защитное действие.

Shark cartilage extracts were examined for induction of cytokines and chemokines in human peripheral blood leukocytes. Primary leukocyte cultures were exposed to a variety of aqueous and organic extracts prepared from several commercial brands of shark cartilage. From all commercial sources of shark cartilage tested the acid extracts induced higher levels of TNFα than other extracts. Different commercial brands of shark cartilage varied significantly in cytokine-inducing activity. TNFα induction was seen as early as 4 h and IFNγ at detectable levels for up to four days. Shark cartilage extracts did not induce physiologically significant levels of IL-4. Results suggest that shark cartilage, preferentially, induces Th1 type inflammatory cytokines. When compared to bovine cartilage extract, collagen, and chondroitin sulfate, shark cartilage induced significantly higher levels of TNFα. Treatment with digestive proteases (trypsin and chymotrypsin) reduced the cytokine induction response by 80%, suggesting that the active component(s) in cartilage extracts is proteinaceous. The induction of Th1 type cytokine response in leukocytes is a significant finding since shark cartilage, taken as a dietary supplement for a variety of chronic degenerative diseases, would be contraindicated in cases where the underlying pathology of the chronic condition is caused by inflammation.

AIM:Study of macrophage migration inhibiting factor (MIF) effect after intracerebral administration on the course of experimental infection induced in mice by tick borne encephalitis virus (TEV), and study of sodium polyprenyl phosphate (PPP) and/or antibodies against MIF on the course of this infection against the background of MIF administration.MATERIALS AND METHODS:Phosprenil preparation was used as a source of PPP. PPP was administered intracerebrally. MIF--human recombinant (R&D, USA), mice--Balb/c line.RESULTS:In the sera of mice infected with TEV, MIF production stimulation was detected at days 8 through 10 after the infection--against the background of clinical signs presentation of tick borne encephalitis (TE). Administration of PPP to infected mice, on the contrary, resulted in MIF production suppression at the specified period. After administration of 20 ng of MIF to mice, lethality increased by 40% and average life span decreased by 2.3 days. Thus, MIF at high doses caused an increase of infection course severity, induced by TEV in mice, and administration of 60 microg of PPP resulted in the protection from infection in 100% of cases. Intracerebral administrationto mice of antibodies against MIF resulted in a decrease of lethality indicator up to 26% as compared with control and an increase of averagelife span by 5.5 days. During simultaneous administration into the brain of infected mice of MIF, PPP and antibodies against MIF, prevention of MIF-induced increase of TE course severity was registered.CONCLUSION:The data obtained allow to conclude that MIF may serve as an indicator of TE course severity, and possible prognostic indicator of meningo-encephalitic form development in humans.