RHEUMATOID ARTHRITIS: LABORATORY MODELS OF THE DISEASE

The  establishment and  application of animal  models  represent effective  tools  for  research  in rheumatoid arthritis (RA) pathogenesis. Animal models that replicate various mechanisms reflecting all aspects of RA, including early RA pathology, have provided important insights into studying etiology and pathogenetic mechanisms of RA in humans. This review article was compiled in order to give an introduction to the current state of RA models.  Application of these  experimental disorders  for testing  potential therapeutic approaches will help to make better predictions for drug efficiency in human R

Orthopoxvirus Infections: Epidemiology, Clinical Picture, and Diagnostics (Scientific Review)

Lack of immunity among the population against pathogenic orthopoxviruses and an increased number of these infections human cases testify to the need of development of the rapid high-sensitive methods for species-specific orthopoxvirus diagnostics. The review presents current epidemiological situation on human orthopoxvirus infections. Addressed are clinical aspects of the disease, caused by small pox virus (SPV), Monkeypox virus, cowpox virus, and vaccinia virus. Represented is a historical survey of the conventional methods for diagnostics of these particular viruses. Reconsidered are the benefits of researches into the sphere of state-of-the-art molecular-diagnostic techniques taking into view both genus-specific and species-specific detection of agents, causing orthopoxvirus infections in humans. Demonstrated is the urgency of new-generation typing in view of occurrence of a novel SPV-like virus emerged as a result of natural evolution of existing zoonotic orthopoxviruses or SPV application as a biological terroristic agent

Recombinant short TNF-BD protein from smallpox virus is pharmacologically active in an experimental septic shock model

Tumor necrosis factor (TNF) is one among the key cytokines that mediate the immune system to protect humans against viral infections. Throughout evolution, anthropogenic Variola virus (VARV) has developed effective mechanisms to overcome human defense reactions. The viral genome encodes soluble proteins imitating the structure of cellular cytokine receptors. These proteins compete with cellular receptors for cytokine binding, thus blocking the antiviral immune response. In particular, the G2R gene of VARV encodes the TNF decoy receptor, VARV-CrmB protein. This protein consists of N-ended TNF-biding (TNF-BD) and C-ended chemokine binding (Ch-BD) domains. Recombinant VARV-CrmB protein has been produced in insect cells using molecular cloning methods and its TNF neutralizing activity has been shown in vitro and in vivo. To decrease the immunogenicity of this protein, a recombinant plasmid coding for shortened TNF-BD protein of VARV in Escherichia coli cells has been constructed. Using the method of immobilized metal affinity chromatography, recombinant TNF-BD protein corresponding to the TNF-biding domain of VARV-CrmB protein was purified from E. coli cells. The therapeutic potential of TNF-BD was studied using an experimental model of LPS-induced septic shock. After septic shock induction, several doses of recombinant TNF-BD were injected and the mortality of experimental animals was observed during 7 days. All mice not injected with TNF-BD had been dead by day 3 of the experiment, but 30, 40 and 60 % of the experimental animals, who received different TNF-BD doses, survived in a dose-dependent manner. Data obtained demonstrate that recombinant TNF-BD protein is pharmacologically active in the experimental model of LPS-induced septic shock

Increasing protectivity of the smallpox vaccine

At the present time, vast majority of human population lacks immunity against orthopoxvirus infections caused by variola (smallpox), monkeypox, cowpox, or buffalopox viruses. More and more mass outbreaks of orthopoxvirus infections are yearly registered among humans on different continents. To prevent transition of these outbreaks to widespread epidemics, we should develop appropriate immunoprophylaxis strategies. Currently, massive usage of the classic live vaccine based on vaccinia virus is not acceptable, due to its high reactogenicity. Therefore, it is necessary to develop the variants of vaccinia virus with reduced virulence and increased immunogenicity/protectivity. The aim of this work was to study protective effects against a lethal orthopoxvirus infection occuring after low-dose immunization of mice with vaccinia virus variants, i.e., carrying mutant A34R gene causing increased production of extracellular virions, or a A35R gene deletion encoding protein product inhibiting antigen presentation by the major histocompatibility complex class II. The LIVP viral strain used in Russia as a smallpox vaccine, and its recombinant variants (LIVP-A34R*, LIVP-dA35R and LIVP-A34R*-dA35R) were compared with intranasal or intradermal immunization of BALB/c mice at the doses of 105 or 103 PFU. 28 days following administration of viral preparations (experimental groups) or saline (control groups), the mice underwent intravital blood sampling from retroorbital venous sinus. The levels of virion-specific antibodies were determined in individual serum samples by enzyme immunoassay. On the day 30 of experiment, the mice were infected with cowpox virus at a dose of 32 LD50, which caused total death of control mice on days 6-10. In the groups immunized with the studied viruses at a dose of 105 PFU, all the animals survived, regardless of strain, or immunization method. Upon intradermal immunization (103 PFU) of mice immunized with the original LIVP virus, 83% of the animals survived, whereas all mutant strains of the vaccinia virus provided 100% protection of the mice from subsequent cowpox virus infection. Intranasal immunization of mice at a dose of 103 PFU with LIVP strain protected only 33% of animals from lethal infection with cowpox virus, while the mutant strains LIVP-A34R* and LIVP-A34R*-dA35R provided 67% protection, and the LIVP-dA35R strain has resqued 75% of the mice. The studied mutant vaccinia viruses can be considered not only new candidate vaccines against smallpox and other human orthopoxvirus infections, but also as vector platforms for creating live multivalent vaccines against other infectious diseases

Candidate antirheumatic genotherapeutic plasmid constructions have low immunogenicity

Rheumatoid arthritis (RA) is a serious systemic disease of connective tissue, mainly affecting joints but also with different extra-articular manifestations. In the course of RA the degenerative changes occur in cartilage surfaces of affected joints and also in subchondral bone tissue, joints get deformed and lose their mobility. RA affects about 1 % of the global human population. Biological therapy with recombinant protein inhibitors of inflammatory cytokines is an effective and well-accepted treatment of RA. TNF inhibitors such as recombinant receptors or monoclonal antibodies are the most widely used biotherapeutics in clinical practice. However, this treatment has some serious side effects. The patients treated with TNF inhibitors are more susceptible to infection diseases, they are also at higher risk of developing neoplastic or autoimmune disorders. Biotherapeutics become less effective or even lose their efficiency with evoking specific antidrug antibodies. These drawbacks are in general associated with repeated systemic injections of large amounts of recombinant protein required to achieve the therapeutic efficacy. Genetic therapy might provide a good and effective solution. Viral genes coding for immunomodulatory factors could be used to create new gene therapy products to treat RA and other human disease. Poxviruses, as compared to other viral families, have an unprecedentedly rich set of such immunomodulatory genes. In particular, they have genes encoding TNF-binding proteins. Previously in a variety of laboratory models we have shown that recombinant TNF-binding protein CrmB can effectively block TNF. In this work we demonstrated that candidate antirheumatic genotherapeutic plasmid constructions encoding poxviral TNF-binding proteins have low immunogenicity

Mutations in the <i>A34R</i> gene increase the immunogenicity of vaccinia virus

Vaccination is the most simple and reliable approach of protection to virus infections. The most effective agents are live vaccines, usually low-virulence organisms for humans and closely related to pathogenic viruses or attenuated as a result of mutations/deletions in the genome of pathogenic virus. Smallpox vaccination with live vaccinia virus (VACV) closely related to smallpox virus played a key role in the success of the global smallpox eradication program carried out under the World Health Organization auspices. As a result of the WHO decision as of 1980 to stop smallpox vaccination, humankind has lost immunity not only to smallpox, but also to other zoonotic, orthopoxviruscaused human infections. This new situation allows orthopoxviruses to circulate in the human population and, as a consequence, to alter several established concepts of the ecology and range of sensitive hosts for various orthopoxvirus species. Classic VACV-based live vaccine for vaccination against orthopoxvirus infections is out of the question, because it can cause severe side effects. Therefore, the development of new safe vaccines against orthopoxviral infections of humans and animals is an important problem. VACV attenuation by modern approaches carried out by targeted inactivation of certain virus genes and usually leads to a decrease in the effectiveness of VACV in vivo propagation. As a result, it can cause a diminishing of the immune response after administration of attenuated virus to patients at standard doses. The gene for thymidine kinase is frequently used for insertion/inactivation of foreign genes and it causes virus attenuation. In this research, the effect of the introduction of two point mutations into the A34R gene of attenuated strain LIVP-GFP (ТК–), which increase the yield of extracellular enveloped virions (EEV), on the pathogenicity and immunogenicity of VACV LIVP-GFP-A34R administered intranasally to laboratory mice were studied. It was shown that increase in EEV production by recombinant strain VACV LIVP-GFP-A34R does not change the attenuated phenotype characteristic of the parental strain LIVP-GFP, but causes a significantly larger production of VACV-specific antibodies

Route-coupled pathogenicity and immunogenicity of vaccinia virus variant inoculated mice

Vaccinia virus had played a key role in the global smallpox eradication. However, in case of mass vaccination with various Vaccinia virus strains severe side effects were revealed sometimes ending up with lethal outcomes, especially in immunocompromised humans. Hence, in 1980 the World Health Organization recommended to cancel smallpox vaccination after declaring about smallpox eradication. Over the last 40 years, human population virtually lost immunity not only against smallpox, but also against other zoonotic orthopoxvirus infections, such as monkeypox, cowpox, buffalopox, and camelpox. All of them pose a represent increasing threat to human health and heighten a risk of emerging highly contagious viruses due to natural evolution of previous zoonotic orthopoxviruses. In order to prevent development of small outbreaks into spreading epidemics and, thus, to decrease a risk of emergence due to natural evolution of highly pathogenic for humans orthopoxviruses, efforts should be applied to develop safe new generation live vaccines based on Vaccinia virus with target virulence genes inactivation. These strains should be examined in laboratory animal models inoculated via different routes. Currently, Vaccinia virus often becomes attenuated to create live recombinant vaccines due to inserting target DNA sequences into the virus virulence genes resulting in their inactivation. Vaccinia virus strain LIVP used in the Russian Federation as smallpox vaccine as well as derivative attenuated variant LIVP-GFP created by using genetic engineering methods with inactivating its thymidine kinase gene were examined. Such viruses were intracerebrally inoculated into suckling mice at doses of 101 or 102 PFU/animal for neurovirulence assessment. Adult mice were infected intranasally, subcutaneously or intradermally at doses of 107 or 108 PFU/animal and clinical manifestations were analyzed for 14 days. On the 28th day after the onset, blood serum samples were collected from individual mice to measure virus specific antibody level by using ELISA. It was shown that recombinant Vaccinia virus strain LIVP-GFP displayed markedly lowered neurovirulence and pathogenicity for mice as compared to parental LIVP. Finally, intradermal route turned out to demonstrate the most safe and effective profile for immunization with both examined Vaccinia virus strains

Genome stability of the vaccine strain VAC∆6

Due to cessation of mass smallpox vaccination in 1980, the collective immunity of humans against orthopoxvirus infections has virtually been lost. Therefore, the risk of spreading zoonotic human orthopoxvirus infections caused by monkeypox and cowpox viruses has increased in the world. First-generation smallpox vaccines based on Vaccinia virus (VAC) are reactogenic and therefore not suitable for mass vaccination under current conditions. This necessitates the development of modern safe live vaccines based on VAC using genetic engineering. We created the VACΔ6 strain by transient dominant selection. In the VACΔ6 genome, five virulence genes were intentionally deleted, and one gene was inactivated by inserting a synthetic DNA fragment. The virus was passaged 71 times in CV-1 cells to obtain the VACΔ6 strain from the VAC LIVP clonal variant. Such a long passage history might have led to additional off-target mutations in VACΔ6 compared to the original LIVP variant. To prevent this, we performed a genome-wide sequencing of VAC LIVP, VACΔ6, and five intermediate viral strains to assess possible off-target mutations. A comparative analysis of complete viral genomes showed that, in addition to target mutations, only two nucleotide substitutions occurred spontaneously when obtaining VACΔ4 from the VACΔ3 strain; the mutations persisting in the VACΔ5 and VACΔ6 genomes. Both nucleotide substitutions are located in intergenic regions (positions 1431 and 189738 relative to LIVP), which indicates an extremely rare occurrence of off-target mutations when using transient dominant selection to obtain recombinant VAC variants with multiple insertions/deletions. To assess the genome stability of the resulting attenuated vaccine strain, 15 consecutive cycles of cultivation of the industrial VACΔ6 strain were performed in 4647 cells certified for vaccine production in accordance with the “Guidelines for Clinical Trials of Medicinal Products”. PCR and sequencing analysis of six DNA fragments corresponding to the regions of disrupted genes in VACΔ6 showed that all viral DNA sequences remained unchanged after 15 passages in 4647 cells

A comparative study of replicative properties of antitumor recombinant vaccinia viruses on human glioblastoma cell culture U87 and monkey kidney cell culture CV-1

In the world of today, virotherapy is one of the rapidly developing areas in the treatment of cancer, and its advantage is selective destruction of cancer cells with minimizing the destructive effect on normal cells of the body. A promising basis for the creation of oncolytic drugs is orthopoxviruses, which have a number of advantages over other viral vectors, and one of these advantages is a large capacity of the genome, which allows genes encoding proteins with antitumor properties to be cloned into their genome. In this study, we compared the replicative properties of ten variants of vaccinia virus (the strain LIVP of VACV) using human glioblastoma cell culture; some of these viruses have additional genes, such as the gene encoding granulocyte-macrophage colony stimulating factor, gene encoding apoptosis-inducing protein TRAIL and gene encoding green fluorescent protein. Furthermore, the virus with five virulence genes deleted (genes encoding hemagglutinin, γ-interferonbinding protein, thymidine kinase, complementbinding protein and Bcl2-like inhibitor of apoptosis), which has significantly lower reactogenicity and neurovirulence compared to the original strain LIVP of VACV, was studied. These data suggest that variants of vaccinia virus with a defective gene encoding thymidine kinase most actively replicate in glioblastoma cell culture

Obtainingvaccinia virus with increased production of extracellular enveloped virions and directing GM-CSF synthesis as a promising basis for development of antitumor drug

The problems of oncological disease treatment are considered relevant and timely issues of the current research programs. Since monotherapy is increasingly clear to be less effective than combination therapy, the novel studies seek for advancement of current treatments and development of new ones employing oncolytic immunotherapy being among the most rapidly evolving approaches. Modern genetic engineering techniques enable new applications of oncolytic viruses in the frames of combined cancer therapy. These applications are feasible, due to the abilities of oncolytic viruses to destruct tumor cells, like as by changing susceptibility of cancer cells to anti-tumor drug, and upon the whole body, thus overcoming the mechanisms conferring immunoresistance of tumor cells. In the present work, we have developed a recombinant vaccinia virus which is a promising platform for designing the antitumor drugs. The following modifications of viral genome were made by means of genetic engineering: gene encoding granulocyte-macrophage colony-stimulating factor was inserted into the region of viral thymidine kinase gene; viral A34R gene encoding a membrane glycoprotein, was replaced by A34R gene with two nucleotide substitutions resulting into D110N and K151E mutations which cause increased proportion of extracellular enveloped virions during the virus reproduction. Some properties of the recombinant virus were studied in vitro. The virus was shown to produce granulocyte-macrophage colony stimulating factor, and high numbers of extracellular enveloped virions. The genome modifications had no effect upon viral replication