scholarly journals The Investigation of the Prospects for Using NS4A Antigen of Hepatitis C Virus for the Development of Recombinant Mosaic Vaccines with Self-Adjuvant Properties

2017 ◽  
Vol 16 (1) ◽  
pp. 69-74
Author(s):  
V. V. Koupriyanov ◽  
L. I. Nikolaeva ◽  
A. A. Zykova ◽  
P. I. Makhnovskiy
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Genetic Engineering ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Interleukin 2 ◽  
High Expression ◽  
Subsequent Work ◽  
Atomic Force ◽  
High Expression Level

The aim of this study was to design promising variants of recombinant proteins based on NS4A antigen of hepatitis C virus (HCV) for subsequent work on the creation of a mosaic recombinant vaccine against hepatitis C. Methods. The recombinant proteins, containing different fragments of NS4A (belong to HCV subtype 1b) and murine interleukin-2, were prepared by genetic engineering approaches, using vectors pQE30 and pQE60 for E. coli. The size of the recombinant protein particles were evaluated by atomic force microscopy. Immunogenicity of these recombinant proteins was tested for Balb/c mice. The murine sera were analyzed by enzyme immunoassay. The recombinant proteins were also tested by immunoblotting with human sera specific to HCV antigens. Results. Six variants of recombinant genetic engineering constructions based on NS4A antigen of hepatitis C virus were designed. In the first variant amino acid sequence of NS4A was inserted using vector pQE60 into the immunodominant loop of HBc protein (core protein of hepatitis B virus). However, further analysis of the product showed the absence of virus-like particles in it. The following three constructs (with glycine linker 19s), without it and N-truncated NS4A) were done using vector pQE30. Only N-truncated NS4а product had a high expression level. Then new protein, consisted of NS4A and N-truncated murine interleukin-2 (IL-2), was obtained to enhance immunogenicity. It is known that IL-2 has adjuvant property. The new product (NS4a-IL-2) is well expressed, but it is accumulated in inclusion bodies. It was extracted with 7M guanidine chloride, purified on a Ni-sorbent and dialyzed in PBS. A shortened version of NS4A (ANS4a-IL-2) was also obtained with a high expression level. Taking in account that increasing the repetition of antigenic regions in recombinant constructs can enhance their immunogenicity, we obtained a recombinant protein comprising three repeat of NS4A. But its efficiency of expression was low. The construction NS4a had very poor immunogenicity, but NS4a-IL-2 (which contains the full length NS4A) displayed the best one for Balb/c mice. As it was shown earlier the immunogenicity of the protein preparation is dependent on the presence of aggregates, so we investigated our recombinant proteins for the presence of protein aggregates by atomic force microscopy.The presence of the particles with size of 6 - 8 nm was revealed in solution of NS4a-IL-2. Conclution. Only ANS4a-IL-2 and ANS4a-IL-2 of the six constructs had high expression and antigenic properties. And only NS4a-IL-2 possessed the high immunogenic property. So, this construction can be used for subsequent work on the creation of a mosaic recombinant vaccine against hepatitis C.

scholarly journals IMMUNOGENIC PROPERTIES OF RECOMBINANT MOZAIC PROTEINS BASED ON ANTIGENS NS4A AND NS4B OF HEPATITIS C VIRUS

2018 ◽  
Vol 63 (3) ◽  
pp. 138-143
Author(s):  
V. V. Koupriyanov ◽  
L. I. Nikolaeva ◽  
A. A. Zykova ◽  
P. I. Makhnovskiy ◽  
R. Y. Kotlyarov ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Core Protein ◽  
Interleukin 2 ◽  
Gamma Interferon ◽  
Interferon Production ◽  
Helper Epitope ◽  
Immunogenic Properties

The aim of the study was to investigate immunogenic properties of mosaic recombinant proteins constructed on the data of hepatitis C virus NS4A and NS4B antigens. Four mosaic recombinant proteins, containing the T and B epitopes of the NS4A and NS4B antigens, were created by genetic engineering methods in the E. coli system. To enhance the immune response they were linked in different variations to the nucleotide sequences of murine interleukin-2 (IL-2), the Neisseria meningiditis lipopeptide, and the T helper epitope of the core protein of hepatitis C virus. The immunogenic properties of these recombinant proteins were analyzed by immunoblotting, ELISA and ELISpot using sera from immunized mice and patients infected with hepatitis C virus. Recombinant proteins specifically reacted with the sera of immunized mice and infected patients in immunoblotting. According to the ELISA data, the predominant formation of antibodies to NS4B was observed when mice were immunized with the recombinant proteins containing both antigens. Analysis of gamma-interferon production by T-lymphocytes upon contact with activated dendritic cells showed in ELISpot that the maximum production of this cytokine was detected when adjuvant components were located at the N- and C-ends of the recombinant protein. The highest level of gamma-interferon production during stimulation with this drug was detected in lymphocytes from the bone marrow and lymph nodes. The recombinant protein containing the T and B epitopes of NS4A and NS4B, murine IL-2 and the lipopeptide Neisseria meningiditis had the greatest immunostimulate effect among the four constructions. This recombinant protein formed nanoparticles of 100-120 nm in size.

scholarly journals Hepatitis C Virus RNA-Dependent RNA Polymerase Is Regulated by Cysteine S-Glutathionylation

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Marina K. Kukhanova ◽  
Vera L. Tunitskaya ◽  
Olga A. Smirnova ◽  
Olga A. Khomich ◽  
Natalia F. Zakirova ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Recombinant Protein ◽  
Rna Polymerase ◽  
Reducing Agents ◽  
Cysteine Residues ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Redox Switches ◽  
Ns5b Protein

Hepatitis C virus (HCV) triggers massive production of reactive oxygen species (ROS) and affects expression of genes encoding ROS-scavenging enzymes. Multiple lines of evidence show that levels of ROS production contribute to the development of various virus-associated pathologies. However, investigation of HCV redox biology so far remained in the paradigm of oxidative stress, whereas no attention was given to the identification of redox switches among viral proteins. Here, we report that one of such redox switches is the NS5B protein that exhibits RNA-dependent RNA polymerase (RdRp) activity. Treatment of the recombinant protein with reducing agents significantly increases its enzymatic activity. Moreover, we show that the NS5B protein is subjected to S-glutathionylation that affects cysteine residues 89, 140, 170, 223, 274, 521, and either 279 or 295. Substitution of these cysteines except C89 and C223 with serine residues led to the reduction of the RdRp activity of the recombinant protein in a primer-dependent assay. The recombinant protein with a C279S mutation was almost inactive in vitro and could not be activated with reducing agents. In contrast, cysteine substitutions in the NS5B region in the context of a subgenomic replicon displayed opposite effects: most of the mutations enhanced HCV replication. This difference may be explained by the deleterious effect of oxidation of NS5B cysteine residues in liver cells and by the protective role of S-glutathionylation. Based on these data, redox-sensitive posttranslational modifications of HCV NS5B and other proteins merit a more detailed investigation and analysis of their role(s) in the virus life cycle and associated pathogenesis.

Combination of three adjuvants enhances the immunogenicity of a recombinant protein containing the CTL epitopes of non-structural proteins of hepatitis C virus

2020 ◽  
Vol 284 ◽  
pp. 197984 ◽  
Author(s):  
Victor V. Kuprianov ◽  
Liudmila I. Nikolaeva ◽  
Anna A. Zykova ◽  
Anna V. Dedova ◽  
Alexander E. Grishechkin ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Recombinant Protein ◽  
Structural Proteins ◽  
Ctl Epitopes

scholarly journals Detection of hepatitis C virus core protein in serum by atomic force microscopy combined with mass spectrometry

2015 ◽  
pp. 1597 ◽  
Author(s):  
Ivan Shumov ◽  
Yuri Ivanov ◽  
Anna Kaysheva ◽  
Pavel Frantsuzov ◽  
Tatyana Pleshakova ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Atomic Force Microscopy ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Force Microscopy ◽  
Virus Core ◽  
Atomic Force

scholarly journals Hepatitis C Virus-Related Lymphomagenesis in a Mouse Model

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Kyoko Tsukiyama-Kohara ◽  
Satoshi Sekiguchi ◽  
Yuri Kasama ◽  
Nagla Elwy Salem ◽  
Keigo Machida ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mouse Model ◽  
B Cell ◽  
Lymphoproliferative Disorder ◽  
Interleukin 2 ◽  
Elevated Serum ◽  
Hcv Infection ◽  
Α Subunit ◽  
Non Hodgkin Lymphoma

B cell non-Hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with hepatitis C virus (HCV) infection. The mechanism by which HCV infection leads to lymphoproliferative disorder remains unclear. Our group established HCV transgenic mice that expressed the full HCV genome in B cells (RzCD19Cre mice). We observed a 25.0% incidence of diffuse large B cell non-Hodgkin lymphomas (22.2% in male and 29.6% in female mice) within 600 days of birth. Interestingly, RzCD19Cre mice with substantially elevated serum-soluble interleukin-2 receptor α-subunit (sIL-2Rα) levels (>1000 pg/mL) developed B cell lymphomas. Another mouse model of lymphoproliferative disorder was established by persistent expression of HCV structural proteins through disruption of interferon regulatory factor-1 (irf-1_/_/CN2 mice). Irf-1_/_/CN2 mice showed extremely high incidences of lymphomas and lymphoproliferative disorders. Moreover, these mice showed increased levels of interleukin (IL)-2, IL-10, and Bcl-2 as well as increased Bcl-2 expression, which promoted oncogenic transformation of lymphocytes.

Natural Genetic Engineering of Hepatitis C Virus NS5A for Immune System Counterattack

2009 ◽  
Vol 1178 (1) ◽  
pp. 173-185 ◽  
Author(s):  
Mahmoud M. El Hefnawi ◽  
Wessam H. El Behaidy ◽  
Aliaa A. Youssif ◽  
Atek Z. Ghalwash ◽  
Lamya A. El Housseiny ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Immune System ◽  
Hepatitis C ◽  
Genetic Engineering

scholarly journals Control of Heterologous Hepatitis C Virus Infection in Chimpanzees Is Associated with the Quality of Vaccine-Induced Peripheral T-Helper Immune Response

2004 ◽  
Vol 78 (1) ◽  
pp. 187-196 ◽  
Author(s):  
C. Rollier ◽  
E. Depla ◽  
J. A. R. Drexhage ◽  
E. J. Verschoor ◽  
B. E. Verstrepen ◽  
...  
Keyword(s):  
Immune Response ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Interleukin 2 ◽  
Hcv Infection ◽  
T Helper ◽  
Vaccine Strategy ◽  
Ns3 Protein ◽  
Ifn Γ

ABSTRACT Prophylactic hepatitis C virus (HCV) vaccine trials with human volunteers are pending. There is an important need for immunological end points which correlate with vaccine efficacy and which do not involve invasive procedures, such as liver biopsies. By using a multicomponent DNA priming-protein boosting vaccine strategy, naïve chimpanzees were immunized against HCV structural proteins (core, E1, and E2) as well as a nonstructural (NS3) protein. Following immunization, exposure to the heterologous HCV 1b J4 subtype resulted in a peak of plasma viremia which was lower in both immunized animals. Compared to the naïve infection control and nine additional historical controls which became chronic, vaccinee 2 (Vac2) rapidly resolved the infection, while the other (Vac1) clearly controlled HCV infection. Immunization induced antibodies, peptide-specific gamma interferon (IFN-γ), protein-specific lymphoproliferative responses, IFN-γ, interleukin-2 (IL-2), and IL-4 T-helper responses in both vaccinees. However, the specificities were markedly different: Vac2 developed responses which were lower in magnitude than those of Vac1 but which were biased towards Th1-type cytokine responses for E1 and NS3. This proof-of-principle study in chimpanzees revealed that immunization with a combination of nonstructural and structural antigens elicited T-cell responses associated with an alteration of the course of infection. Our findings provide data to support the concept that the quality of the response to conserved epitopes and the specific nature of the peripheral T-helper immune response are likely pivotal factors influencing the control and clearance of HCV infection.

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