|
1
|
Masavuli MG, Wijesundara DK, Torresi J, Gowans EJ, Grubor-Bauk B. Preclinical Development and Production of Virus-Like Particles As Vaccine Candidates for Hepatitis C. Front Microbiol 2017; 8:2413. [PMID: 29259601 PMCID: PMC5723323 DOI: 10.3389/fmicb.2017.02413] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2017] [Accepted: 11/22/2017] [Indexed: 12/16/2022] Open
Abstract
Hepatitis C Virus (HCV) infects 2% of the world’s population and is the leading cause of liver disease and liver transplantation. It poses a serious and growing worldwide public health problem that will only be partially addressed with the introduction of new antiviral therapies. However, these treatments will not prevent re-infection particularly in high risk populations. The introduction of a HCV vaccine has been predicted, using simulation models in a high risk population, to have a significant effect on reducing the incidence of HCV. A vaccine with 50 to 80% efficacy targeted to high-risk intravenous drug users could dramatically reduce HCV incidence in this population. Virus like particles (VLPs) are composed of viral structural proteins which self-assemble into non-infectious particles that lack genetic material and resemble native viruses. Thus, VLPs represent a safe and highly immunogenic vaccine delivery platform able to induce potent adaptive immune responses. Currently, many VLP-based vaccines have entered clinical trials, while licensed VLP vaccines for hepatitis B virus (HBV) and human papilloma virus (HPV) have been in use for many years. The HCV core, E1 and E2 proteins can self-assemble into immunogenic VLPs while inclusion of HCV antigens into heterogenous (chimeric) VLPs is also a promising approach. These VLPs are produced using different expression systems such as bacterial, yeast, mammalian, plant, or insect cells. Here, this paper will review HCV VLP-based vaccines and their immunogenicity in animal models as well as the different expression systems used in their production.
Collapse
Affiliation(s)
- Makutiro Ghislain Masavuli
- Virology Laboratory, Basil Hetzel Institute for Translational Medicine, Discipline of Surgery, University of Adelaide, Adelaide, SA, Australia
| | - Danushka K Wijesundara
- Virology Laboratory, Basil Hetzel Institute for Translational Medicine, Discipline of Surgery, University of Adelaide, Adelaide, SA, Australia
| | - Joseph Torresi
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, VIC, Australia
| | - Eric J Gowans
- Virology Laboratory, Basil Hetzel Institute for Translational Medicine, Discipline of Surgery, University of Adelaide, Adelaide, SA, Australia
| | - Branka Grubor-Bauk
- Virology Laboratory, Basil Hetzel Institute for Translational Medicine, Discipline of Surgery, University of Adelaide, Adelaide, SA, Australia
| |
Collapse
|
|
2
|
Ezzikouri S, Jadid FZ, Hamdi S, Wakrim L, Tsukiyama-Kohara K, Benjelloun S. Supplementing Conventional Treatment with Pycnogenol® May Improve Hepatitis C Virus-Associated Type 2 Diabetes: A Mini Review. J Clin Transl Hepatol 2016; 4:228-233. [PMID: 27777890 PMCID: PMC5075005 DOI: 10.14218/jcth.2016.00021] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/18/2016] [Revised: 06/20/2016] [Accepted: 07/07/2016] [Indexed: 12/20/2022] Open
Abstract
Hepatitis C virus (HCV) infection and type 2 diabetes mellitus (T2DM) present a significant health burden, with increasing complications and mortality rates worldwide. Pycnogenol® (PYC), a natural product, possesses antidiabetic and antiviral properties that may improve HCV-associated T2DM. In this review, we present previously published data on the effectiveness of PYC against HCV replication and T2DM. We believe that supplementing conventional treatment with PYC may improve the current HCV therapy, attenuate HCV-associated T2DM, and reduce the risk of complications such as cirrhosis or hepatocellular carcinoma and cardiovascular disease.
Collapse
Affiliation(s)
- Sayeh Ezzikouri
- Virology Unit, Viral Hepatitis Laboratory, Institut Pasteur du Maroc, Casablanca, Morocco
- *Correspondence to: Sayeh Ezzikouri, Viral Hepatitis Laboratory, Institut Pasteur du Maroc, 1 Place Louis Pasteur, Casablanca 20360, Morocco. Tel: +212-5-27016076, +212-5-22434450, Fax: +212-5-22260957, E-mail:
| | - Fatima Zahra Jadid
- Virology Unit, Viral Hepatitis Laboratory, Institut Pasteur du Maroc, Casablanca, Morocco
| | - Salsabil Hamdi
- Virology Unit, Viral Hepatitis Laboratory, Institut Pasteur du Maroc, Casablanca, Morocco
| | - Lahcen Wakrim
- Virology Unit, Immunovirology Laboratory, Institut Pasteur du Maroc, Casablanca, Morocco
| | - Kyoko Tsukiyama-Kohara
- Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
- Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, Japan
| | - Soumaya Benjelloun
- Virology Unit, Viral Hepatitis Laboratory, Institut Pasteur du Maroc, Casablanca, Morocco
| |
Collapse
|
|
3
|
Mehrlatifan S, Mirnurollahi SM, Motevalli F, Rahimi P, Soleymani S, Bolhassani A. The structural HCV genes delivered by MPG cell penetrating peptide are directed to enhance immune responses in mice model. Drug Deliv 2015; 23:2852-2859. [PMID: 26559939 DOI: 10.3109/10717544.2015.1108375] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
One of the significant problems in vaccination projects is the lack of an effective vaccine against hepatitis C virus (HCV). The goal of the current study is to evaluate and compare two DNA constructs encoding HCV core and coreE1E2 genes alone or complexed with MPG peptide as a delivery system for stimulation of antibody responses and IFN-γ secretion in Balb/c mice model. Indeed, MPG cell penetrating peptide was used to improve DNA immunization in mice. Our results demonstrated that MPG forms stable non-covalent nanoparticles with pcDNA-core and pcDNA-coreE1E2 at an N/P ratio of 10:1. The in vitro transfection efficiency of core or coreE1E2 DNA using MPG and TurboFect delivery systems was confirmed by western blot analysis. The results indicated the expression of the full-length core (∼21 kDa), and coreE1E2 (∼83 kDa) proteins using an anti-His monoclonal antibody. In addition, the expression of HCV core and coreE1E2 proteins was performed in bacteria and the purified recombinant proteins were injected to mice with Montanide 720 adjuvant. Our data showed that the immunized mice with HCV core and coreE1E2 proteins generated the mixture of sera IgG1 and IgG2a isotypes considerably higher than other groups. Furthermore, DNA constructs encoding core and coreE1E2 complexed with MPG could significantly induce IFN-γ secretion in lower concentrations than the naked core and coreE1E2 DNAs. Taken together, the DNA formulations as well as protein regimens used in this study triggered high-level IFN-γ production in mice, an important feature for the development of Th1 immune responses.
Collapse
Affiliation(s)
- Saloume Mehrlatifan
- a Department of Hepatitis and AIDS , Pasteur Institute of Iran , Tehran , Iran.,b Department of Biotechnology , Faculty of Advanced Sciences and Technology, Pharmaceutical Sciences Branch, Islamic Azad University , Tehran , Iran , and
| | | | - Fatemeh Motevalli
- a Department of Hepatitis and AIDS , Pasteur Institute of Iran , Tehran , Iran
| | - Pooneh Rahimi
- a Department of Hepatitis and AIDS , Pasteur Institute of Iran , Tehran , Iran
| | - Sepehr Soleymani
- a Department of Hepatitis and AIDS , Pasteur Institute of Iran , Tehran , Iran
| | - Azam Bolhassani
- a Department of Hepatitis and AIDS , Pasteur Institute of Iran , Tehran , Iran
| |
Collapse
|
|
4
|
Mirnurollahi SM, Bolhassani A, Irani S, Davoudi N. Expression and Purification of HCV Core and Core-E1E2 Proteins in Different Bacterial Strains. IRANIAN JOURNAL OF BIOTECHNOLOGY 2015; 13:57-62. [PMID: 28959300 DOI: 10.15171/ijb.1249] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
BACKGROUND Hepatitis C virus (HCV) is a main public health problem causing chronic liver infection and subsequently liver cirrhosis and lethal hepatocellular carcinoma (HCC). Vaccination based on HCV capsid proteins has attracted a special interest for prevention of viral infections. The core protein is a basic and evolutionary most conserved protein, which regulates the cellular processes related to viral replication and pathogenesis. The envelope E1 and E2 proteins involve in generation of the infectious particles, viral entry by binding to a host cell receptor, and modulation of the immune responses. OBJECTIVES In current study, the efficient generation of recombinant core and core-E1E2 proteins was developed in bacterial expression systems. MATERIALS AND METHODS The expression of HCV core and core-E1E2 proteins was performed using prokaryotic pET-28a and pQE-30 expression systems in BL21/ Rosetta, and M15 strains, respectively. The recombinant proteins were purified using affinity chromatography under native conditions and also reverse staining method. Finally, the levels of recombinant proteins were assessed by BCA kit and spectrophotometer. RESULTS The data showed a clear band of ~573 bp for HCV core and ~2238 bp for core-E1E2 genes in agarose gel. Moreover, a ~21 kDa band of core protein and a ~83 kDa band of core-E1E2 protein were revealed in SDS-PAGE. The affinity chromatography could not purify the core and core-E1E2 proteins completely, because of low affinity to Ni-NTA bead in comparison with reverse staining method. CONCLUSIONS This study is the first report for purification of HCV core and core-E1E2 proteins using the reverse staining procedure with no need of any chromatography columns. The BL21 strain was more potent than Rosetta strain for HCV core protein in pET 28a expression system. Furthermore, M15 strain was suitable for expression of coreE1E2 in pQE-30 bacterial system.
Collapse
Affiliation(s)
| | - Azam Bolhassani
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
| | - Shiva Irani
- Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
| | - Noushin Davoudi
- Biotechnology Research Center, Department of Medical Biotechnology, Pasteur Institute of Iran, Tehran, Iran
| |
Collapse
|
|
5
|
Liu F, Wu X, Zhao Y, Li L, Wang Z. Budding of peste des petits ruminants virus-like particles from insect cell membrane based on intracellular co-expression of peste des petits ruminants virus M, H and N proteins by recombinant baculoviruses. J Virol Methods 2014; 207:78-85. [PMID: 24992672 DOI: 10.1016/j.jviromet.2014.06.021] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2014] [Revised: 06/01/2014] [Accepted: 06/24/2014] [Indexed: 01/13/2023]
Abstract
Peste des petits ruminants virus (PPRV), an etiological agent of peste des petits ruminants (PPR), is classified into the genus Morbillivirus in the family Paramyxovirida. In this study, two full-length open reading frames (ORF) corresponding to the PPRV matrix (M) and haemagglutinin (H) genes underwent a codon-optimization based on insect cells, respectively. Two codon-optimized ORFs along with one native nucleocapsid (N) ORF were used to construct recombinant baculoviruses co-expressing the PPRV M, H and N proteins in insect cells. Analysis of Western blot, immunofluorescence, confocal microscopy and flow cytometry demonstrated co-expression of the three proteins but at different levels in insect cells, and PPR virus-like particles (VLPs) budded further from cell membrane based on self-assembly of the three proteins by viewing of ultrathin section with a transmission electron microscope (TEM). Subsequently, a small number of VLPs were purified by sucrose density gradient centrifugation for TEM viewing. The PPR VLPs, either purified by sucrose density gradient centrifugation or budding from insect cell membrane on ultrathin section, morphologically resembled authentic PPRVs but were smaller in diameter by the TEM examination.
Collapse
Affiliation(s)
- Fuxiao Liu
- National Research Center for Exotic Animal Diseases, China Animal Health and Epidemiology Center, No. 369 Nanjing Road, Qingdao 266032, Shandong, China
| | - Xiaodong Wu
- National Research Center for Exotic Animal Diseases, China Animal Health and Epidemiology Center, No. 369 Nanjing Road, Qingdao 266032, Shandong, China
| | - Yonggang Zhao
- National Research Center for Exotic Animal Diseases, China Animal Health and Epidemiology Center, No. 369 Nanjing Road, Qingdao 266032, Shandong, China
| | - Lin Li
- National Research Center for Exotic Animal Diseases, China Animal Health and Epidemiology Center, No. 369 Nanjing Road, Qingdao 266032, Shandong, China
| | - Zhiliang Wang
- National Research Center for Exotic Animal Diseases, China Animal Health and Epidemiology Center, No. 369 Nanjing Road, Qingdao 266032, Shandong, China.
| |
Collapse
|
|
6
|
Liu F, Wu X, Li L, Liu Z, Wang Z. Formation of peste des petits ruminants spikeless virus-like particles by co-expression of M and N proteins in insect cells. Res Vet Sci 2014; 96:213-6. [DOI: 10.1016/j.rvsc.2013.10.012] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2013] [Revised: 09/22/2013] [Accepted: 10/26/2013] [Indexed: 10/26/2022]
|
|
7
|
Liu F, Wu X, Li L, Liu Z, Wang Z. Use of baculovirus expression system for generation of virus-like particles: successes and challenges. Protein Expr Purif 2013; 90:104-16. [PMID: 23742819 PMCID: PMC7128112 DOI: 10.1016/j.pep.2013.05.009] [Citation(s) in RCA: 88] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2013] [Revised: 05/13/2013] [Accepted: 05/15/2013] [Indexed: 11/10/2022]
Abstract
A brief overview of principles and applications of BES. Generation of VLPs using BES. Major properties of BES: promoting generation of VLPs. Bioprocess considerations for generation of VLPs. The baculovirus expression system (BES) has been one of the versatile platforms for the production of recombinant proteins requiring multiple post-translational modifications, such as folding, oligomerization, phosphorylation, glycosylation, acylation, disulfide bond formation and proteolytic cleavage. Advances in recombinant DNA technology have facilitated application of the BES, and made it possible to express multiple proteins simultaneously in a single infection and to produce multimeric proteins sharing functional similarity with their natural analogs. Therefore, the BES has been used for the production of recombinant proteins and the construction of virus-like particles (VLPs), as well as for the development of subunit vaccines, including VLP-based vaccines. The VLP, which consists of one or more structural proteins but no viral genome, resembles the authentic virion but cannot replicate in cells. The high-quality recombinant protein expression and post-translational modifications obtained with the BES, along with its capacity to produce multiple proteins, imply that it is ideally suited to VLP production. In this article, we critically review the pros and cons of using the BES as a platform to produce both enveloped and non-enveloped VLPs.
Collapse
Affiliation(s)
- Fuxiao Liu
- National Research Center for Exotic Animal Diseases, China Animal Health and Epidemiology Center, Qingdao, Shandong 266032, China
| | | | | | | | | |
Collapse
|
|
8
|
Belzhelarskaya SN, Koroleva NN, Popenko VV, Drutza VL, Orlova OV, Rubtzov PM, Kochetkov SN. Hepatitis C virus structural proteins and virus-like particles produced in recombinant baculovirus-infected insect cells. Mol Biol 2010. [DOI: 10.1134/s0026893310010139] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
|
|
9
|
Cellular models for the screening and development of anti-hepatitis C virus agents. Pharmacol Ther 2009; 124:1-22. [PMID: 19555718 DOI: 10.1016/j.pharmthera.2009.05.010] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2009] [Accepted: 05/19/2009] [Indexed: 12/24/2022]
Abstract
Investigations on the biology of hepatitis C virus (HCV) have been hampered by the lack of small animal models. Efforts have therefore been directed to designing practical and robust cellular models of human origin able to support HCV replication and production in a reproducible, reliable and consistent manner. Many different models based on different forms of virions and hepatoma or other cell types have been described including virus-like particles, pseudotyped particles, subgenomic and full length replicons, virion productive replicons, immortalised hepatocytes, fetal and adult primary human hepatocytes. This review focuses on these different cellular models, their advantages and disadvantages at the biological and experimental levels, and their respective use for evaluating the effect of antiviral molecules on different steps of HCV biology including virus entry, replication, particles generation and excretion, as well as on the modulation by the virus of the host cell response to infection.
Collapse
|
|
10
|
Tsitoura P, Georgopoulou U, Pêtres S, Varaklioti A, Karafoulidou A, Vagena D, Politis C, Mavromara P. Evidence for cellular uptake of recombinant hepatitis C virus non-enveloped capsid-like particles. FEBS Lett 2007; 581:4049-57. [PMID: 17678898 DOI: 10.1016/j.febslet.2007.07.032] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2007] [Accepted: 07/16/2007] [Indexed: 12/17/2022]
Abstract
Although the hepatitis C virus (HCV) is an enveloped virus, naked nucleocapsids have been reported in the serum of infected patients, and most recently novel HCV subgenomes with deletions of the envelope proteins have been identified. However the significance of these findings remains unclear. In this study, we used the baculovirus expression system to generate recombinant HCV capsid-like particles, and investigated their possible interactions with cells. We show that expression of HCV core in insect cells can sufficiently direct the formation of capsid-like particles in the absence of the HCV envelope glycoproteins and of the 5' untranslated region. By confocal microscopy analysis, we provide evidence that the naked capsid-like particles could be uptaken by human hepatoma cells. Moreover, our findings suggest that they have the potential to produce cell-signaling effects.
Collapse
Affiliation(s)
- Panagiota Tsitoura
- Molecular Virology Laboratory, Hellenic Pasteur Institute, 127, Vas. Sofias Ave, Athens 11521, Greece
| | | | | | | | | | | | | | | |
Collapse
|
|
11
|
Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotechnol 2005; 23:567-75. [PMID: 15877075 PMCID: PMC3610534 DOI: 10.1038/nbt1095] [Citation(s) in RCA: 700] [Impact Index Per Article: 35.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.
Collapse
|
|
12
|
Choi SH, Park KJ, Kim SY, Choi DH, Park JM, Hwang SB. C-terminal domain of hepatitis C virus core protein is essential for secretion. World J Gastroenterol 2005; 11:3887-92. [PMID: 15991288 PMCID: PMC4504891 DOI: 10.3748/wjg.v11.i25.3887] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/12/2004] [Revised: 01/02/2004] [Accepted: 01/05/2004] [Indexed: 02/06/2023] Open
Abstract
AIM We have previously demonstrated that hepatitis C virus (HCV) core protein is efficiently released into the culture medium in insect cells. The objective of this study is to characterize the HCV core secretion in insect cells. METHODS We constructed recombinant baculoviruses expressing various-length of mutant core proteins, expressed these proteins in insect cells, and examined core protein secretion in insect cells. RESULTS Only wild type core was efficiently released into the culture medium, although the protein expression level of wild type core was lower than those of other mutant core proteins. We found that the shorter form of the core construct expressed the higher level of protein. However, if more than 18 amino acids of the core were truncated at the C-terminus, core proteins were no longer secreted into the culture medium. Membrane flotation data show that the secreted core proteins are associated with the cellular membrane protein, indicating that HCV core is secreted as a membrane complex. CONCLUSION The C-terminal 18 amino acids of HCV core were crucial for core secretion into the culture media. Since HCV replication occurs on lipid raft membrane structure, these results suggest that HCV may utilize a unique core release mechanism to escape immune surveillance, thereby potentially representing the feature of HCV morphogenesis.
Collapse
Affiliation(s)
- Soo-Ho Choi
- Ilsong Institute of Life Science, Hallym University, 1 Ockcheon-dong, Chuncheon 200-702, South Korea
| | | | | | | | | | | |
Collapse
|