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1.
Int J Mol Sci ; 25(8)2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38673835

RESUMO

Virotherapy is one of the perspective technologies in the treatment of malignant neoplasms. Previously, we have developed oncolytic vaccinia virus VV-GMCSF-Lact and its high cytotoxic activity and antitumor efficacy against glioma was shown. In this work, using immortalized and patient-derived cells with different sensitivity to VV-GMCSF-Lact, we evaluated the cytotoxic effect of chemotherapy agents. Additionally, we studied the combination of VV-GMCSF-Lact with temozolomide which is the most preferred drug for glioma treatment. Experimental results indicate that first adding temozolomide and then the virus to the cells is inherently more efficient than dosing it in the reverse order. Testing these regimens in the U87 MG xenograft glioblastoma model confirmed this effect, as assessed by tumor growth inhibition index and histological analysis. Moreover, VV-GMCSF-Lact as monotherapy is more effective against U87 MG glioblastoma xenografts comparing temozolomide.


Assuntos
Glioma , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Terapia Viral Oncolítica , Vírus Oncolíticos , Temozolomida , Vaccinia virus , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Linhagem Celular Tumoral , Camundongos , Glioma/terapia , Glioma/tratamento farmacológico , Glioma/patologia , Vaccinia virus/genética , Vaccinia virus/fisiologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Camundongos Nus , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Glioblastoma/terapia , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Terapia Combinada
2.
Molecules ; 29(4)2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38398600

RESUMO

Aptamers are currently being investigated for their potential to improve virotherapy. They offer several advantages, including the ability to prevent the aggregation of viral particles, enhance target specificity, and protect against the neutralizing effects of antibodies. The purpose of this study was to comprehensively investigate an aptamer capable of enhancing virotherapy. This involved characterizing the previously selected aptamer for vaccinia virus (VACV), evaluating the aggregation and molecular interaction of the optimized aptamers with the recombinant oncolytic virus VV-GMCSF-Lact, and estimating their immunoshielding properties in the presence of human blood serum. We chose one optimized aptamer, NV14t_56, with the highest affinity to the virus from the pool of several truncated aptamers and built its 3D model. The NV14t_56 remained stable in human blood serum for 1 h and bound to VV-GMCSF-Lact in the micromolar range (Kd ≈ 0.35 µM). Based on dynamic light scattering data, it has been demonstrated that aptamers surround viral particles and inhibit aggregate formation. In the presence of serum, the hydrodynamic diameter (by intensity) of the aptamer-virus complex did not change. Microscale thermophoresis (MST) experiments showed that NV14t_56 binds with virus (EC50 = 1.487 × 109 PFU/mL). The analysis of the amplitudes of MST curves reveals that the components of the serum bind to the aptamer-virus complex without disrupting it. In vitro experiments demonstrated the efficacy of VV-GMCSF-Lact in conjunction with the aptamer when exposed to human blood serum in the absence of neutralizing antibodies (Nabs). Thus, NV14t_56 has the ability to inhibit virus aggregation, allowing VV-GMCSF-Lact to maintain its effectiveness throughout the storage period and subsequent use. When employing aptamers as protective agents for oncolytic viruses, the presence of neutralizing antibodies should be taken into account.


Assuntos
Aptâmeros de Nucleotídeos , Vírus Oncolíticos , Humanos , Vaccinia virus/genética , Aptâmeros de Nucleotídeos/metabolismo , Anticorpos Neutralizantes
3.
Cells ; 12(22)2023 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-37998351

RESUMO

Oncolytic virotherapy is a rapidly evolving approach that aims to selectively kill cancer cells. We designed a promising recombinant vaccinia virus, VV-GMCSF-Lact, for the treatment of solid tumors, including glioma. We assessed how VV-GMCSF-Lact affects human cells using immortalized and patient-derived glioma cultures and a non-malignant brain cell culture. Studying transcriptome changes in cells 12 h or 24 h after VV-GMCSF-Lact infection, we detected the common activation of histone genes. Additionally, genes associated with the interferon-gamma response, NF-kappa B signaling pathway, and inflammation mediated by chemokine and cytokine signaling pathways showed increased expression. By contrast, genes involved in cell cycle progression, including spindle organization, sister chromatid segregation, and the G2/M checkpoint, were downregulated following virus infection. The upregulation of genes responsible for Golgi vesicles, protein transport, and secretion correlated with reduced sensitivity to the cytotoxic effect of VV-GMCSF-Lact. Higher expression of genes encoding proteins, which participate in the maturation of pol II nuclear transcripts and mRNA splicing, was associated with an increased sensitivity to viral cytotoxicity. Genes whose expression correlates with the sensitivity of cells to the virus are important for increasing the effectiveness of cancer virotherapy. Overall, the results highlight molecular markers, biological pathways, and gene networks influencing the response of glioma cells to VV-GMCSF-Lact.


Assuntos
Glioma , Vírus Oncolíticos , Humanos , Vírus Oncolíticos/genética , Transcriptoma/genética , Replicação Viral/genética , Glioma/genética , Glioma/terapia , Glioma/patologia , Vaccinia virus/genética
4.
Viruses ; 15(1)2023 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-36680222

RESUMO

Human Adenovirus type 6 (HAdV-C6) is a promising candidate for the development of oncolytic vectors as it has low seroprevalence and the intrinsic ability to evade tissue macrophages. However, its further development as a therapeutic agent is hampered by the lack of convenient cloning methods. We have developed a novel technology when a shuttle plasmid carrying the distal genome parts with modified E1A and E3 regions is recombined in vitro with the truncated HAdV-C6 genome. Using this approach, we have constructed a novel Ad6-hT-GM vector controlled by the hTERT promoter and expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) instead of 6.7K and gp19K E3 proteins. We have demonstrated that control by the hTERT promoter may result in delayed viral replication, which nevertheless does not significantly change the cytotoxic ability of recombinant viruses. The insertion of the transgene by displacing the E3-6.7K/gp19K region does not drastically change the expression patterns of E3 genes; however, mild changes in expression from major late promoter were observed. Finally, we have demonstrated that the treatment of human breast cancer xenografts in murine models with Ad6-hT-GM significantly decreased the tumor volume and improved survival time compared to mock-treated mice.


Assuntos
Adenovírus Humanos , Neoplasias , Terapia Viral Oncolítica , Vírus Oncolíticos , Humanos , Camundongos , Animais , Adenovírus Humanos/genética , Estudos Soroepidemiológicos , Adenoviridae/genética , Adenoviridae/metabolismo , Transgenes , Regiões Promotoras Genéticas , Vetores Genéticos/genética , Terapia Viral Oncolítica/métodos , Linhagem Celular Tumoral , Replicação Viral , Vírus Oncolíticos/genética , Neoplasias/terapia , Neoplasias/genética
5.
Life (Basel) ; 11(10)2021 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-34685455

RESUMO

Glioblastoma is one of the most aggressive brain tumors. Given the poor prognosis of this disease, novel methods for glioblastoma treatment are needed. Virotherapy is one of the most actively developed approaches for cancer therapy today. VV-GMCSF-Lact is a recombinant vaccinia virus with deletions of the viral thymidine kinase and growth factor genes and insertions of the granulocyte-macrophage colony-stimulating factor and oncotoxic protein lactaptin genes. The virus has high cytotoxic activity against human cancer cells of various histogenesis and antitumor efficacy against breast cancer. In this work, we show VV-GMCSF-Lact to be a promising therapeutic agent for glioblastoma treatment. VV-GMCSF-Lact effectively decreases the viability of glioblastoma cells of both immortalized and patient-derived cultures in vitro, crosses the blood-brain barrier, selectively replicates into orthotopically transplanted human glioblastoma when intravenously injected, and inhibits glioblastoma xenograft and metastasis growth when injected intratumorally.

6.
Pharmaceuticals (Basel) ; 14(5)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068113

RESUMO

In this study, two strains of the yeast P. pastoris were constructed, one of which produced authentic recombinant human granulocyte-macrophage colony-stimulating factor (ryGM-CSF), and the other was a chimera consisting of ryGM-CSF genetically fused with mature human apolipoprotein A-I (ApoA-I) (ryGM-CSF-ApoA-I). Both forms of the cytokine were secreted into the culture medium. The proteins' yield during cultivation in flasks was 100 and 60 mg/L for ryGM-CSF and ryGM-CSF-ApoA-I, respectively. Both forms of recombinant GM-CSF stimulated the proliferation of human TF-1 erythroleukemia cells; however, the amount of chimera required was 10-fold that of authentic GM-CSF to induce a similar proliferative effect. RyGM-CSF exhibited a 2-fold proliferative effect on BFU-E (burst-forming units-erythroid) at a concentration 1.7 fold less than non-glycosylated E. coli-derived GM-CSF. The chimera together with authentic ryGM-CSF increased the number of both erythroid precursors and BMC granulocytes after 48 h of incubation of human bone marrow cells (BMCs). In addition, the chimeric form of ryGM-CSF was more effective at increasing the viability of the total amount of BMCs, decreasing apoptosis compared to the authentic form. ryGM-CSF-ApoA-I normalized the proliferation, maturation, and segmentation of neutrophils within the physiological norm, preserving the pool of blast cells under conditions of impaired granulopoiesis. The chimera form of GM-CSF exhibited the properties of a multilinear growth factor, modulating the activity of GM-CSF and, perhaps, it may be more suitable for the normalization of granulopoiesis.

7.
Eur Urol ; 77(3): 299-308, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31471138

RESUMO

CONTEXT: Progress achieved in the treatment of prostate cancer (PCa) with surgical, radiation, and hormonal therapies has drastically reduced mortality from this disease. Yet, patients with advanced PCa have few, if any, curative options. Recent success in treating patients with hematological malignancies of B-cell origin using T cells engineered to express chimeric antigen receptors (CARs) has inspired multiple groups worldwide to adapt this approach to the problem of late-stage PCa. OBJECTIVE: To summarize the available clinical results for CAR T-cell therapy of PCa and discuss future technological advancements in the CAR T-cell field that may help patients with metastatic PCa. EVIDENCE ACQUISITION: A literature review was conducted of clinical trial data, abstracts presented at recent oncology conferences, as well as reports highlighting critical bottlenecks of CAR T-cell therapy that became apparent from preclinical and clinical studies. EVIDENCE SYNTHESIS: Current understanding of why CAR T-cell therapy may fail, particularly in the context of solid cancers, is as follows. First, a CAR design that provides potent activity and persistence of engineered T cells in the hostile tumor microenvironment is a must. The choice of the targetable epitope(s) is critical to counteract tumor antigen escape. Preclinical and clinical evidence indicates that the efficacy of CAR T-cell therapy can be enhanced significantly in combination with other therapeutic approaches. We propose that several improvements to CAR design and patient conditioning, such as unbiased identification of novel PCa-specific CAR targets, use of next-generation (multispecific, resistant to the tumor microenvironment, and with prolonged persistence) CAR T-cell products, and combination therapies may translate into improved patient outcomes and more durable responses. CONCLUSIONS: Although significant preclinical experience of testing CAR T cells in solid cancer models has identified important technological and biological bottlenecks, information from clinical trials, particularly those focusing on the PCa, will be instrumental to the rational design of advanced CAR T therapies that will be both safe and effective in patients with advanced PCa. PATIENT SUMMARY: So far, chimeric antigen receptor (CAR) T-cell therapy has not shown significant activity in patients with metastatic prostate cancer (PCa). CAR T-cell products used for such trials represent one of the pioneering efforts to adapt this technology to the problem of metastatic PCa. In retrospect, both CAR design and cell composition appear to have been suboptimal to expect strong patient responses. Given the impressive results of CAR-based approaches observed in preclinical models of solid cancers, emerging CAR T-cell products are expected to be more successful in the clinic. Here, we discuss the challenges that need to be overcome to boost the efficacy of PCa-targeted CAR T-cell therapy and call for dialogue between clinicians and cell biologists to address these challenges.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Neoplasias da Próstata/patologia , Neoplasias da Próstata/terapia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Humanos , Masculino , Metástase Neoplásica , Neoplasias da Próstata/imunologia
8.
Anticancer Res ; 39(11): 6073-6086, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31704835

RESUMO

BACKGROUND/AIM: Oncolytic adenoviruses are promising therapeutic agents against both the bulk of tumor cells and cancer stem cells. The present study intended to test the oncolytic capability of adenovirus serotype 6 (Ad6), which has a lower seroprevalence and hepatotoxicity relatively to adenovirus 5 (Ad5), against the glioblastoma and its cancer stem cells. MATERIALS AND METHODS: Oncolytic efficacy of Ad6 was compared to widespread Ad5 both in vitro and in vivo, using the U87 and U251 human glioblastoma cell lines and subcutaneously transplanted U87 cells in SCID mice, respectively. RESULTS: Ad6 had a dose-dependent cytotoxicity toward glioblastoma cells in vitro and its intratumoral injections lead to a significant (p<0.05) decrease in volume of U87 xenografts, similarly to Ad5. Based on the innate capability of glioblastoma cancer stem cells to internalize a fluorescent-labeled double-stranded DNA probe, the spatial localization of these cells was estimated and it was shown that the number of cancer stem cells tended to decrease under adenovirus therapy as compared to the control group. CONCLUSION: Ad6 was shown to be a promising agent for treating glioblastomas.


Assuntos
Adenovírus Humanos/genética , Glioblastoma/terapia , Células-Tronco Neoplásicas/metabolismo , Terapia Viral Oncolítica , Replicação Viral , Adenovírus Humanos/classificação , Animais , Apoptose , Proliferação de Células , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/virologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Oncolytic Virother ; 8: 9-26, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31372363

RESUMO

Viruses have some characteristics in common with cell-based life. They can evolve and adapt to environmental conditions. Directed evolution can be used by researchers to produce viral strains with desirable phenotypes. Through bioselection, improved strains of oncolytic viruses can be obtained that have better safety profiles, increased specificity for malignant cells, and more efficient spread among tumor cells. It is also possible to select strains capable of killing a broader spectrum of cancer cell variants, so as to achieve a higher frequency of therapeutic responses. This review describes and analyses virus adaptation studies performed with members of four RNA virus families that are used for viral oncolysis: reoviruses, paramyxoviruses, enteroviruses, and rhabdoviruses.

10.
Microbes Infect ; 20(3): 212-216, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29247709

RESUMO

Previously, we studied an association of two IL28B gene single nucleotide polymorphisms (SNPs) and three IL10 gene SNPs with predisposition to tick-borne encephalitis in a Russian population. In this study, a possible involvement of these SNPs in the development of predisposition to chronic hepatitis C (caused by structurally similar, related virus from the Flaviviridae family) was investigated in the same population. Only the IL10 promoter rs1800872 SNP was associated with predisposition to chronic hepatitis C. This SNP seems to be a common genetic marker of predisposition to two diseases caused by hepatitis C and tick-borne encephalitis viruses in Russian population.


Assuntos
Predisposição Genética para Doença , Hepatite C Crônica/genética , Interleucina-10/genética , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Idoso , Alelos , Feminino , Genótipo , Hepatite C Crônica/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Federação Russa/epidemiologia , Adulto Jovem
11.
Biomed Res Int ; 2017: 3620510, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28951871

RESUMO

Genetic modifications of the oncolytic vaccinia virus (VV) improve selective tumor cell infection and death, as well as activation of antitumor immunity. We have engineered a double recombinant VV, coding human GM-CSF, and apoptosis-inducing protein apoptin (VV-GMCSF-Apo) for comparing with the earlier constructed double recombinant VV-GMCSF-Lact, coding another apoptosis-inducing protein, lactaptin, which activated different cell death pathways than apoptin. We showed that both these recombinant VVs more considerably activated a set of critical apoptosis markers in infected cells than the recombinant VV coding GM-CSF alone (VV-GMCSF-dGF): these were phosphatidylserine externalization, caspase-3 and caspase-7 activation, DNA fragmentation, and upregulation of proapoptotic protein BAX. However, only VV-GMCSF-Lact efficiently decreased the mitochondrial membrane potential of infected cancer cells. Investigating immunogenic cell death markers in cancer cells infected with recombinant VVs, we demonstrated that all tested recombinant VVs were efficient in calreticulin and HSP70 externalization, decrease of cellular HMGB1, and ATP secretion. The comparison of antitumor activity against advanced MDA-MB-231 tumor revealed that both recombinants VV-GMCSF-Lact and VV-GMCSF-Apo efficiently delay tumor growth. Our results demonstrate that the composition of GM-CSF and apoptosis-inducing proteins in the VV genome is very efficient tool for specific killing of cancer cells and for activation of antitumor immunity.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , Transgenes/genética , Vaccinia virus/genética , Animais , Antineoplásicos , Biomarcadores Tumorais/genética , Caspase 3/genética , Caspase 7/genética , Morte Celular/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Fragmentação do DNA , Feminino , Vetores Genéticos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Proteínas de Choque Térmico HSP70/genética , Humanos , Potencial da Membrana Mitocondrial/genética , Camundongos , Camundongos SCID , Neoplasias/genética , Vírus Oncolíticos/genética , Fosfatidilserinas/genética , Regulação para Cima/genética , Replicação Viral/genética , Proteína X Associada a bcl-2/genética
12.
Oncotarget ; 7(45): 74171-74188, 2016 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-27708236

RESUMO

Vaccinia virus (VACV) oncolytic therapy has been successful in a number of tumor models. In this study our goal was to generate a double recombinant vaccinia virus (VV-GMCSF-Lact) with enhanced antitumor activity that expresses exogenous proteins: the antitumor protein lactaptin and human granulocyte-macrophage colony-stimulating factor (GM-CSF). Lactaptin has previously been demonstrated to act as a tumor suppressor in mouse hepatoma as well as MDA-MB-231 human adenocarcinoma cells grafted into SCID mice. VV-GMCSF-Lact was engineered from Lister strain (L-IVP) vaccinia virus and has deletions of the viral thymidine kinase and vaccinia growth factor genes. Cell culture experiments revealed that engineered VV-GMCSF-Lact induced the death of cultured cancer cells more efficiently than recombinant VACV coding only GM-CSF (VV-GMCSF-dGF). Normal human MCF-10A cells were resistant to both recombinants up to 10 PFU/cell. The selectivity index for breast cancer cells measured in pair cultures MCF-7/MCF-10A was 200 for recombinant VV-GMCSF-Lact coding lactaptin and 100 for VV-GMCSF-dGF. Using flow cytometry we demonstrated that both recombinants induced apoptosis in treated cells but that the rate in the cells with active caspase-3 and -7 was higher after treatment with VV-GMCSF-Lact than with VV-GMCSF-dGF. Tumor growth inhibition and survival outcomes after VV-GMCSF-Lact treatment were estimated using immunodeficient and immunocompetent mice models. We observed that VV-GMCSF-Lact efficiently delays the growth of sensitive and chemoresistant tumors. These results demonstrate that recombinant VACVs coding an apoptosis-inducing protein have good therapeutic potential against chemoresistant tumors. Our data will also stimulate further investigation of coding lactaptin double recombinant VACV in clinical settings.


Assuntos
Engenharia Genética/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Vaccinia virus/fisiologia , Células A549 , Animais , Apoptose , Caseínas/biossíntese , Caseínas/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Humanos , Células MCF-7 , Camundongos , Camundongos SCID , Neoplasias/virologia , Vaccinia virus/genética , Vaccinia virus/metabolismo
13.
Genome Announc ; 4(4)2016 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-27516510

RESUMO

We report here the complete genome sequence of Sendai virus Moscow strain. Anecdotal evidence for the efficacy of oncolytic virotherapy exists for this strain. The RNA genome of the Moscow strain is 15,384 nucleotides in length and differs from the nearest strain, BB1, by 18 nucleotides and 11 amino acids.

14.
Genome Announc ; 4(3)2016 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-27174282

RESUMO

Most of the live vaccine doses of vaccinia virus donated to the Intensified Smallpox Eradication Programme after 1971 were prepared using the L-IVP strain. A mixture of three clones of the L-IVP strain was sequenced using MySEQ. Consensus sequence similarity with the vaccinia virus Lister strain is 99.5%.

15.
Viruses ; 8(1)2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26771631

RESUMO

Oncolytic abilities of vaccinia virus (VACV) served as a basis for the development of various recombinants for treating cancer; however, "natural" oncolytic properties of the virus are not examined in detail. Our study was conducted to know how the genetically unmodified L-IVP strain of VACV produces its antitumor effect. Human A431 carcinoma xenografts in nude mice and murine Ehrlich carcinoma in C57Bl mice were used as targets for VACV, which was injected intratumorally. A set of virological methods, immunohistochemistry, light and electron microscopy was used in the study. We found that in mice bearing A431 carcinoma, the L-IVP strain was observed in visceral organs within two weeks, but rapidly disappeared from the blood. The L-IVP strain caused decrease of sizes in both tumors, however, in different ways. Direct cell destruction by replicating virus plays a main role in regression of A431 carcinoma xenografts, while in Ehrlich carcinoma, which poorly supported VACV replication, the virus induced decrease of mitoses by pushing tumor cells into S-phase of cell cycle. Our study showed that genetically unmodified VACV possesses at least two mechanisms of antitumor effect: direct destruction of tumor cells and suppression of mitoses in tumor cells.


Assuntos
Carcinoma de Ehrlich/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/fisiologia , Vaccinia virus/fisiologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Vírus Oncolíticos/genética , Vaccinia virus/genética , Replicação Viral
16.
Oncotarget ; 5(22): 11269-82, 2014 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-25358248

RESUMO

A recombinant vaccinia virus VVdGF-ApoS24/2 expressing apoptin selectively kills human cancer cells in vitro [Kochneva et al., 2013]. We compared the oncolytic activity of this recombinant with that of the parental strain L-IVP using a model of human A431 carcinoma xenografts in nude mice. Single intratumoral injections (2×10^7 PFU/mouse) of the viruses produced a dramatic decrease in tumor volumes, which was higher after injection of apoptin-producing virus. The tumor dried out after the injection of recombinant while injection of L-IVP strain resulted in formation of cavities filled with cell debris and liquid. Both viruses rapidly spread in xenografts and replicate exclusively in tumor cells causing their destruction within 8 days. Both viruses induced insignificant level of apoptosis in tumors. Unlike the previously described nuclear localization of apoptin in cancer cells the apoptin produced by recombinant virus was localized to the cytoplasm. The apoptin did not induce a typical apoptosis, but it rather influenced pathway of cell death and thereby caused tumor shrinkage. The replacement of destroyed cells by filamentous material is the main feature of tumor regression caused by the VVdGF-ApoS24/2 virus. The study points the presence of complicated mechanisms of apoptin effects at the background of vaccinia virus replication.


Assuntos
Carcinoma de Células Escamosas/terapia , Terapia Viral Oncolítica/métodos , Vaccinia virus/fisiologia , Animais , Apoptose/fisiologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/virologia , Linhagem Celular Tumoral , Feminino , Haplorrinos , Humanos , Camundongos , Camundongos Nus , Vaccinia virus/metabolismo , Replicação Viral , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Microbes Infect ; 16(5): 445-9, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24594345

RESUMO

Chronic hepatitis C is a severe liver disease caused by positive-strand RNA virus. Previously, we reported an association between seven single nucleotide polymorphisms (SNPs) in four innate immunity genes (OAS2, OAS3, CD209, and TLR3) and human predisposition to tick-borne encephalitis, caused by a virus from the same Flaviviridae family, in a Russian population. Currently, genotype and allele frequencies for these SNPs were analyzed in 75 chronic hepatitis C patients and compared with the population control (269 Novosibirsk citizens). Data obtained suggest that the OAS2 rs1293762 and CD209 rs2287886 SNPs are associated with predisposition to chronic hepatitis C in Russian population.


Assuntos
2',5'-Oligoadenilato Sintetase/genética , Moléculas de Adesão Celular/genética , Predisposição Genética para Doença , Hepatite C Crônica/epidemiologia , Lectinas Tipo C/genética , Polimorfismo de Nucleotídeo Único , Receptores de Superfície Celular/genética , Adolescente , Adulto , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Federação Russa , Adulto Jovem
18.
Vaccine ; 30(1): 69-77, 2011 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-22041300

RESUMO

One of the greatest challenges to HCV vaccine development is the induction of effective immune responses using recombinant proteins or vectors. In order to better understand which vaccine-induced antibodies contribute to neutralization of HCV the quality of polyclonal anti-E1E2 antibody responses in immunized mice and chimpanzees was assessed at the level of epitope recognition using peptide scanning and neutralization of chimeric 1a/2a, 1b/2a and 2a HCVcc after blocking or affinity elution of specific antibodies. Mice and chimpanzees were immunized with genotype 1a (H77) HCV gpE1E2; all samples contained cross-neutralizing antibody against HCVcc. By functionally dissecting the polyclonal immune responses we identified three new regions important for neutralization within E1 (aa264-318) and E2 (aa448-483 and aa496-515) of the HCV glycoproteins, the third of which (aa496-515) is highly conserved (85-95%) amongst genotypes. Antibodies to aa496-515 were isolated by affinity binding and elution from the serum of a vaccinated chimpanzee and found to specifically neutralize chimeric 1a/2a, 1b/2a and 2a HCVcc. IC50 titres (IgG ng/mL) for the aa496-515 eluate were calculated as 142.1, 239.37 and 487.62 against 1a/2a, 1b/2a and 2a HCVcc, respectively. Further analysis demonstrated that although antibody to this new, conserved neutralization epitope is efficiently induced with recombinant proteins in mice and chimpanzees; it is poorly induced during natural infection in patients and chimpanzees (7 out of 68 samples positive) suggesting the epitope is poorly presented to the immune system in the context of the viral particle. These findings have important implications for the development of HCV vaccines and strategies designed to protect against heterologous viruses. The data also suggest that recombinant or synthetic antigens may be more efficient at inducing neutralizing antibodies to certain epitopes and that screening virally infected patients may not be the best approach for finding new cross-reactive epitopes.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Mapeamento de Epitopos , Epitopos/imunologia , Hepacivirus/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Camundongos , Testes de Neutralização , Pan troglodytes
19.
Biochim Biophys Acta ; 1764(11): 1710-8, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17070121

RESUMO

Tumor necrosis factor (TNF), a potent proinflammatory and antiviral cytokine, is a critical extracellular immune regulator targeted by poxviruses through the activity of virus-encoded family of TNF-binding proteins (CrmB, CrmC, CrmD, and CrmE). The only TNF-binding protein from variola virus (VARV), the causative agent of smallpox, infecting exclusively humans, is CrmB. Here we have aligned the amino acid sequences of CrmB proteins from 10 VARV, 14 cowpox virus (CPXV), and 22 monkeypox virus (MPXV) strains. Sequence analyses demonstrated a high homology of these proteins. The regions homologous to cd00185 domain of the TNF receptor family, determining the specificity of ligand-receptor binding, were found in the sequences of CrmB proteins. In addition, a comparative analysis of the C-terminal SECRET domain sequences of CrmB proteins was performed. The differences in the amino acid sequences of these domains characteristic of each particular orthopoxvirus species were detected. It was assumed that the species-specific distinctions between the CrmB proteins might underlie the differences in these physicochemical and biological properties. The individual recombinant proteins VARV-CrmB, MPXV-CrmB, and CPXV-CrmB were synthesized in a baculovirus expression system in insect cells and isolated. Purified VARV-CrmB was detectable as a dimer with a molecular weight of 90 kDa, while MPXV- and CPXV-CrmBs, as monomers when fractioned by non-reducing SDS-PAGE. The CrmB proteins of VARV, MPXV, and CPXV differed in the efficiencies of inhibition of the cytotoxic effects of human, mouse, or rabbit TNFs in L929 mouse fibroblast cell line. Testing of CrmBs in the experimental model of LPS-induced shock using SPF BALB/c mice detected a pronounced protective effect of VARV-CrmB. Thus, our data demonstrated the difference in anti-TNF activities of VARV-, MPXV-, and CPXV-CrmBs and efficiency of VARV-CrmB rather than CPXV- or MPXV-CrmBs against LPS-induced mortality in mice.


Assuntos
Vírus da Varíola Bovina/metabolismo , Monkeypox virus/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Vírus da Varíola/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Camundongos , Dados de Sequência Molecular , Receptores do Fator de Necrose Tumoral/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Spodoptera , Proteínas Virais/química
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