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Arul SS, Balakrishnan B, Handanahal SS, Venkataraman S. Viral nanoparticles: Current advances in design and development. Biochimie 2024; 219:33-50. [PMID: 37573018 DOI: 10.1016/j.biochi.2023.08.006] [Citation(s) in RCA: 5] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2023] [Revised: 07/06/2023] [Accepted: 08/07/2023] [Indexed: 08/14/2023]
Abstract
Viral nanoparticles (VNPs) are self-assembling, adaptable delivery systems for vaccines and other therapeutic agents used in a variety of biomedical applications. The potential of viruses to invade and infect various hosts and cells renders them suitable as potential nanocarriers, possessing distinct functional characteristics, immunogenic properties, and improved biocompatibility and biodegradability. VNPs are frequently produced through precise genetic or chemical engineering, which involves adding diverse sequences or functional payloads to the capsid protein (CP). Several spherical and helical plant viruses, bacteriophages, and animal viruses are currently being used as VNPs, or non-infectious virus-like particles (VLPs). In addition to their broad use in cancer therapy, vaccine technology, diagnostics, and molecular imaging, VNPs have made important strides in the realms of tissue engineering, biosensing, and antimicrobial prophylaxis. They are also being used in energy storage cells due to their binding and piezoelectric properties. The large-scale production of VNPs for research, preclinical testing, and clinical use is fraught with difficulties, such as those relating to cost-effectiveness, scalability, and purity. Consequently, many plants- and microorganism-based platforms are being developed, and newer viruses are being explored. The goal of the current review is to provide an overview of these advances.
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Ghasemian K, Broer I, Schön J, Killisch R, Kolp N, Springer A, Huckauf J. Oral and Subcutaneous Immunization with a Plant-Produced Mouse-Specific Zona Pellucida 3 Peptide Presented on Hepatitis B Core Antigen Virus-like Particles. Vaccines (Basel) 2023; 11:vaccines11020462. [PMID: 36851339 PMCID: PMC9963689 DOI: 10.3390/vaccines11020462] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2023] [Revised: 02/14/2023] [Accepted: 02/16/2023] [Indexed: 02/19/2023] Open
Abstract
A short mouse-specific peptide from zona pellucida 3 (mZP3, amino acids 328-342) has been shown to be associated with antibody-mediated contraception. In this study, we investigated the production of mZP3 in the plant, as an orally applicable host, and examined the immunogenicity of this small peptide in the BALB/c mouse model. The mZP3 peptide was inserted into the major immunodominant region of the hepatitis B core antigen and was produced in Nicotiana benthamiana plants via Agrobacterium-mediated transient expression. Soluble HBcAg-mZP3 accumulated at levels up to 2.63 mg/g leaf dry weight (LDW) containing ~172 µg/mg LDW mZP3 peptide. Sucrose gradient analysis and electron microscopy indicated the assembly of the HBcAg-mZP3 virus-like particles (VLPs) in the soluble protein fraction. Subcutaneously administered mZP3 peptide displayed on HBcAg VLPs was immunogenic in BALB/c mice at a relatively low dosage (5.5 µg mZP3 per dose) and led to the generation of mZP3-specific antibodies that bound to the native zona pellucida of wild mice. Oral delivery of dried leaves expressing HBcAg-mZP3 also elicited mZP3-specific serum IgG and mucosal IgA that cross-reacted with the zona pellucida of wild mice. According to these results, it is worthwhile to investigate the efficiency of plants producing HBcAg-mZP3 VLPs as immunogenic edible baits in reducing the fertility of wild mice through inducing antibodies that cross-react to the zona pellucida.
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Affiliation(s)
- Khadijeh Ghasemian
- Department of Agrobiotechnology and Risk Assessment for Bio and Gene Technology, Faculty of Agricultural and Environmental Sciences, University of Rostock, 18059 Rostock, Germany
| | - Inge Broer
- Department of Agrobiotechnology and Risk Assessment for Bio and Gene Technology, Faculty of Agricultural and Environmental Sciences, University of Rostock, 18059 Rostock, Germany
| | - Jennifer Schön
- Department of Reproduction Biology, Leibniz Institute for Zoo and Wildlife Research (IZW), 10315 Berlin, Germany
| | - Richard Killisch
- BIOSERV, Analytik und Medizinprodukte GmbH, 18059 Rostock, Germany
| | - Nadine Kolp
- BIOSERV, Analytik und Medizinprodukte GmbH, 18059 Rostock, Germany
| | - Armin Springer
- Medical Biology and Electron Microscopy Center, Rostock University Medical Center, 18057 Rostock, Germany
| | - Jana Huckauf
- Department of Agrobiotechnology and Risk Assessment for Bio and Gene Technology, Faculty of Agricultural and Environmental Sciences, University of Rostock, 18059 Rostock, Germany
- Correspondence:
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Hepatitis B core-based virus-like particles: A platform for vaccine development in plants. ACTA ACUST UNITED AC 2021; 29:e00605. [PMID: 33732633 PMCID: PMC7937989 DOI: 10.1016/j.btre.2021.e00605] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2020] [Revised: 02/17/2021] [Accepted: 02/25/2021] [Indexed: 02/07/2023]
Abstract
Virus-like particles (VLPs) are a class of structures formed by the self-assembly of viral capsid protein subunits and contain no infective viral genetic material. The Hepatitis B core (HBc) antigen is capable of assembling into VLPs that can elicit strong immune responses and has been licensed as a commercial vaccine against Hepatitis B. The HBc VLPs have also been employed as a platform for the presentation of foreign epitopes to the immune system and have been used to develop vaccines against, for example, influenza A and Foot-and-mouth disease. Plant expression systems are rapid, scalable and safe, and are capable of providing correct post-translational modifications and reducing upstream production costs. The production of HBc-based virus-like particles in plants would thus greatly increase the efficiency of vaccine production. This review investigates the application of plant-based HBc VLP as a platform for vaccine production.
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Zahmanova G, Mazalovska M, Takova K, Toneva V, Minkov I, Peyret H, Lomonossoff G. Efficient Production of Chimeric Hepatitis B Virus-Like Particles Bearing an Epitope of Hepatitis E Virus Capsid by Transient Expression in Nicotiana benthamiana. Life (Basel) 2021; 11:life11010064. [PMID: 33477348 PMCID: PMC7830250 DOI: 10.3390/life11010064] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2020] [Revised: 01/13/2021] [Accepted: 01/14/2021] [Indexed: 12/12/2022] Open
Abstract
The core antigen of hepatitis B virus (HBcAg) is capable of self-assembly into virus-like particles (VLPs) when expressed in a number of heterologous systems. Such VLPs are potential carriers of foreign antigenic sequences for vaccine design. In this study, we evaluated the production of chimeric HBcAg VLPs presenting a foreign epitope on their surface, the 551–607 amino acids (aa) immunological epitope of the ORF2 capsid protein of hepatitis E virus. A chimeric construct was made by the insertion of 56 aa into the immunodominant loop of the HBcAg. The sequences encoding the chimera were inserted into the pEAQ-HT vector and infiltrated into Nicotiana benthamiana leaves. The plant-expressed chimeric HBcHEV ORF2 551–607 protein was recognized by an anti-HBcAg mAb and anti-HEV IgG positive swine serum. Electron microscopy showed that plant-produced chimeric protein spontaneously assembled into “knobbly” ~34 nm diameter VLPs. This study shows that HBcAg is a promising carrier platform for the neutralizing epitopes of hepatitis E virus (HEV) and the chimeric HBcAg/HEV VLPs could be a candidate for a bivalent vaccine.
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Affiliation(s)
- Gergana Zahmanova
- Department of Plant Physiology and Molecular Biology, University of Plovdiv, 4000 Plovdiv, Bulgaria; (M.M.); (K.T.); (V.T.)
- Center of Plant Systems Biology and Biotechnology, 4000 Plovdiv, Bulgaria;
- Correspondence: (G.Z.); (G.L.); Tel.: +359-32-261529 (G.Z.); +44-1603-450351 (G.L.)
| | - Milena Mazalovska
- Department of Plant Physiology and Molecular Biology, University of Plovdiv, 4000 Plovdiv, Bulgaria; (M.M.); (K.T.); (V.T.)
| | - Katerina Takova
- Department of Plant Physiology and Molecular Biology, University of Plovdiv, 4000 Plovdiv, Bulgaria; (M.M.); (K.T.); (V.T.)
| | - Valentina Toneva
- Department of Plant Physiology and Molecular Biology, University of Plovdiv, 4000 Plovdiv, Bulgaria; (M.M.); (K.T.); (V.T.)
- Institute of Molecular Biology and Biotechnologies, 4108 Markovo, Bulgaria
| | - Ivan Minkov
- Center of Plant Systems Biology and Biotechnology, 4000 Plovdiv, Bulgaria;
- Institute of Molecular Biology and Biotechnologies, 4108 Markovo, Bulgaria
| | - Hadrien Peyret
- Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney NR4 7UH, UK;
| | - George Lomonossoff
- Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney NR4 7UH, UK;
- Correspondence: (G.Z.); (G.L.); Tel.: +359-32-261529 (G.Z.); +44-1603-450351 (G.L.)
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5
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Lacasta A, Mody KT, De Goeyse I, Yu C, Zhang J, Nyagwange J, Mwalimu S, Awino E, Saya R, Njoroge T, Muriuki R, Ndiwa N, Poole EJ, Zhang B, Cavallaro A, Mahony TJ, Steinaa L, Mitter N, Nene V. Synergistic Effect of Two Nanotechnologies Enhances the Protective Capacity of the Theileria parva Sporozoite p67C Antigen in Cattle. THE JOURNAL OF IMMUNOLOGY 2021; 206:686-699. [PMID: 33419770 PMCID: PMC7851744 DOI: 10.4049/jimmunol.2000442] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/21/2020] [Accepted: 12/03/2020] [Indexed: 11/19/2022]
Abstract
East Coast fever (ECF), caused by Theileria parva, is the most important tick-borne disease of cattle in sub-Saharan Africa. Practical disadvantages associated with the currently used live-parasite vaccine could be overcome by subunit vaccines. An 80-aa polypeptide derived from the C-terminal portion of p67, a sporozoite surface Ag and target of neutralizing Abs, was the focus of the efforts on subunit vaccines against ECF and subjected to several vaccine trials with very promising results. However, the vaccination regimen was far from optimized, involving three inoculations of 450 μg of soluble p67C (s-p67C) Ag formulated in the Seppic adjuvant Montanide ISA 206 VG. Hence, an improved formulation of this polypeptide Ag is needed. In this study, we report on two nanotechnologies that enhance the bovine immune responses to p67C. Individually, HBcAg-p67C (chimeric hepatitis B core Ag virus-like particles displaying p67C) and silica vesicle (SV)-p67C (s-p67C adsorbed to SV-140-C18, octadecyl-modified SVs) adjuvanted with ISA 206 VG primed strong Ab and T cell responses to p67C in cattle, respectively. Coimmunization of cattle (Bos taurus) with HBcAg-p67C and SV-p67C resulted in stimulation of both high Ab titers and CD4 T cell response to p67C, leading to the highest subunit vaccine efficacy we have achieved to date with the p67C immunogen. These results offer the much-needed research depth on the innovative platforms for developing effective novel protein-based bovine vaccines to further the advancement.
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Affiliation(s)
- Anna Lacasta
- Animal and Human Health Program, International Livestock Research Institute, Nairobi 00100, Kenya;
| | - Karishma T Mody
- Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, Queensland 4072, Australia
| | - Ine De Goeyse
- Enzootic, Vector-borne and Bee Diseases, Sciensano, 1180 Brussels, Belgium.,Department of Biomedical Sciences, Institute of Tropical Medicine, 2000 Antwerp, Belgium
| | - Chengzhong Yu
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia
| | - Jun Zhang
- Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia
| | - James Nyagwange
- Animal and Human Health Program, International Livestock Research Institute, Nairobi 00100, Kenya
| | - Stephen Mwalimu
- Animal and Human Health Program, International Livestock Research Institute, Nairobi 00100, Kenya
| | - Elias Awino
- Animal and Human Health Program, International Livestock Research Institute, Nairobi 00100, Kenya
| | - Rosemary Saya
- Animal and Human Health Program, International Livestock Research Institute, Nairobi 00100, Kenya
| | - Thomas Njoroge
- Animal and Human Health Program, International Livestock Research Institute, Nairobi 00100, Kenya
| | - Robert Muriuki
- Animal and Human Health Program, International Livestock Research Institute, Nairobi 00100, Kenya
| | - Nicholas Ndiwa
- Research Methods Group, International Livestock Research Institute, Nairobi 00100, Kenya; and
| | - Elisabeth Jane Poole
- Research Methods Group, International Livestock Research Institute, Nairobi 00100, Kenya; and
| | - Bing Zhang
- Department of Agriculture and Fisheries, Brisbane, Queensland 4102, Australia
| | - Antonino Cavallaro
- Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, Queensland 4072, Australia
| | - Timothy J Mahony
- Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, Queensland 4072, Australia
| | - Lucilla Steinaa
- Animal and Human Health Program, International Livestock Research Institute, Nairobi 00100, Kenya
| | - Neena Mitter
- Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, Queensland 4072, Australia
| | - Vishvanath Nene
- Animal and Human Health Program, International Livestock Research Institute, Nairobi 00100, Kenya
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Diamos AG, Larios D, Brown L, Kilbourne J, Kim HS, Saxena D, Palmer KE, Mason HS. Vaccine synergy with virus-like particle and immune complex platforms for delivery of human papillomavirus L2 antigen. Vaccine 2019; 37:137-144. [PMID: 30459071 PMCID: PMC6291209 DOI: 10.1016/j.vaccine.2018.11.021] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2018] [Revised: 11/09/2018] [Accepted: 11/10/2018] [Indexed: 01/18/2023]
Abstract
Diverse HPV subtypes are responsible for considerable disease burden worldwide, necessitating safe, cheap, and effective vaccines. The HPV minor capsid protein L2 is a promising candidate to create broadly protective HPV vaccines, though it is poorly immunogenic by itself. To create highly immunogenic and safe vaccine candidates targeting L2, we employed a plant-based recombinant protein expression system to produce two different vaccine candidates: L2 displayed on the surface of hepatitis B core (HBc) virus-like particles (VLPs) or L2 genetically fused to an immunoglobulin capable of forming recombinant immune complexes (RIC). Both vaccine candidates were potently immunogenic in mice, but were especially so when delivered together, generating very consistent and high antibody titers directed against HPV L2 (>1,000,000) that correlated with virus neutralization. These data indicate a novel immune response synergy upon co-delivery of VLP and RIC platforms, a strategy that can be adapted generally for many different antigens.
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Affiliation(s)
- Andrew G Diamos
- Center for Immunotherapy, Vaccines, & Virotherapy, Biodesign Institute at ASU; and School of Life Sciences, Arizona State University, Tempe, AZ 85287, United States
| | - Dalia Larios
- Center for Immunotherapy, Vaccines, & Virotherapy, Biodesign Institute at ASU; and School of Life Sciences, Arizona State University, Tempe, AZ 85287, United States
| | - Lauren Brown
- Center for Immunotherapy, Vaccines, & Virotherapy, Biodesign Institute at ASU; and School of Life Sciences, Arizona State University, Tempe, AZ 85287, United States
| | - Jacquelyn Kilbourne
- Center for Immunotherapy, Vaccines, & Virotherapy, Biodesign Institute at ASU; and School of Life Sciences, Arizona State University, Tempe, AZ 85287, United States
| | - Hyun Soon Kim
- Plant Systems Engineering Research Center, KRIBB, Gwahang-ro 125, Yuseong-gu, Daejeon 34141, Republic of Korea
| | - Divyasha Saxena
- Center for Predictive Medicine for Emerging Infectious Diseases and Department of Pharmacology and Toxicology, School of Medicine, University of Louisville, KY, United States, Center for Predictive Medicine, Louisville, KY 40202, United States
| | - Kenneth E Palmer
- Center for Predictive Medicine for Emerging Infectious Diseases and Department of Pharmacology and Toxicology, School of Medicine, University of Louisville, KY, United States, Center for Predictive Medicine, Louisville, KY 40202, United States
| | - Hugh S Mason
- Center for Immunotherapy, Vaccines, & Virotherapy, Biodesign Institute at ASU; and School of Life Sciences, Arizona State University, Tempe, AZ 85287, United States.
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7
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Yang M, Lai H, Sun H, Chen Q. Virus-like particles that display Zika virus envelope protein domain III induce potent neutralizing immune responses in mice. Sci Rep 2017; 7:7679. [PMID: 28794424 PMCID: PMC5550446 DOI: 10.1038/s41598-017-08247-9] [Citation(s) in RCA: 92] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2017] [Accepted: 07/07/2017] [Indexed: 11/10/2022] Open
Abstract
Several Zika virus (ZIKV) vaccine candidates have recently been described which use inactivated whole virus, DNA or RNA that express the virus' Envelope (E) glycoprotein as the antigen. These were successful in stimulating production of virus-targeted antibodies that protected animals against ZIKV challenges, but their use potentially will predispose vaccinated individuals to infection by the related Dengue virus (DENV). We have devised a virus like particle (VLP) carrier based on the hepatitis B core antigen (HBcAg) that displays the ZIKV E protein domain III (zDIII), and shown that it can be produced quickly and easily purified in large quantities from Nicotiana benthamiana plants. HBcAg-zDIII VLPs are shown to be highly immunogenic, as two doses elicited potent humoral and cellular responses in mice that exceed the threshold correlated with protective immunity against multiple strains of Zika virus. Notably, HBcAg-zDIII VLPs-elicited antibodies did not enhance the infection of DENV in Fc gamma receptor-expressing cells, offsetting the concern of ZIKV vaccines inducing cross-reactive antibodies and sensitizing people to subsequent DENV infection. Thus, our zDIII-based vaccine offers improved safety and lower cost production than other current alternatives, with equivalent effectiveness.
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Affiliation(s)
- Ming Yang
- The Biodesign Institute, Arizona State University, Tempe, AZ, 85287, USA
| | - Huafang Lai
- The Biodesign Institute, Arizona State University, Tempe, AZ, 85287, USA
| | - Haiyan Sun
- The Biodesign Institute, Arizona State University, Tempe, AZ, 85287, USA
| | - Qiang Chen
- The Biodesign Institute, Arizona State University, Tempe, AZ, 85287, USA.
- School of Life Sciences, Arizona State University, Tempe, AZ, 85287, USA.
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8
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Peyret H, Gehin A, Thuenemann EC, Blond D, El Turabi A, Beales L, Clarke D, Gilbert RJC, Fry EE, Stuart DI, Holmes K, Stonehouse NJ, Whelan M, Rosenberg W, Lomonossoff GP, Rowlands DJ. Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins. PLoS One 2015; 10:e0120751. [PMID: 25830365 PMCID: PMC4382129 DOI: 10.1371/journal.pone.0120751] [Citation(s) in RCA: 94] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2014] [Accepted: 01/26/2015] [Indexed: 01/03/2023] Open
Abstract
The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.
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Affiliation(s)
- Hadrien Peyret
- Department of Biological Chemistry, John Innes Centre, Norwich, United Kingdom
| | - Annick Gehin
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
| | - Eva C. Thuenemann
- Department of Biological Chemistry, John Innes Centre, Norwich, United Kingdom
| | - Donatienne Blond
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
| | - Aadil El Turabi
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
- iQur Ltd, London, United Kingdom
| | - Lucy Beales
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
- iQur Ltd, London, United Kingdom
| | - Dean Clarke
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
| | | | - Elizabeth E. Fry
- UK Division of Structural Biology, University of Oxford, Oxford, United Kingdom
| | - David I. Stuart
- UK Division of Structural Biology, University of Oxford, Oxford, United Kingdom
| | - Kris Holmes
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
| | - Nicola J. Stonehouse
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
| | | | | | | | - David J. Rowlands
- School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
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9
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Plasmid dimerization increases the production of hepatitis B core particles in E. coli. BIOTECHNOL BIOPROC E 2013. [DOI: 10.1007/s12257-013-0188-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
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10
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Roseman AM, Borschukova O, Berriman JA, Wynne SA, Pumpens P, Crowther RA. Structures of hepatitis B virus cores presenting a model epitope and their complexes with antibodies. J Mol Biol 2012; 423:63-78. [PMID: 22750730 PMCID: PMC3465560 DOI: 10.1016/j.jmb.2012.06.032] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2012] [Revised: 05/16/2012] [Accepted: 06/20/2012] [Indexed: 12/22/2022]
Abstract
The core shell of hepatitis B virus is a potent immune stimulator, giving a strong neutralizing immune response to foreign epitopes inserted at the immunodominant region, located at the tips of spikes on the exterior of the shell. Here, we analyze structures of core shells with a model epitope inserted at two alternative positions in the immunodominant region. Recombinantly expressed core protein assembles into T = 3 and T = 4 icosahedral shells, and atomic coordinates are available for the T = 4 shell. Since the modified protein assembles predominantly into T = 3 shells, a quasi-atomic model of the native T = 3 shell was made. The spikes in this T = 3 structure resemble those in T = 4 shells crystallized from expressed protein. However, the spikes in the modified shells exhibit an altered conformation, similar to the DNA containing shells in virions. Both constructs allow full access of antibodies to the foreign epitope, DPAFR from the preS1 region of hepatitis B virus surface antigen. However, one induces a 10-fold weaker immune response when injected into mice. In this construct, the epitope is less constrained by the flanking linker regions and is positioned so that the symmetry of the shell causes pairs of epitopes to come close enough to interfere with one another. In the other construct, the epitope mimics the native epitope conformation and position. The interaction of native core shells with an antibody specific to the immunodominant epitope is compared to the constructs with an antibody against the foreign epitope. Our findings have implications for the design of vaccines based on virus-like particles.
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Affiliation(s)
- A M Roseman
- MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
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11
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Whitacre DC, Lee BO, Milich DR. Use of hepadnavirus core proteins as vaccine platforms. Expert Rev Vaccines 2010; 8:1565-73. [PMID: 19863249 DOI: 10.1586/erv.09.121] [Citation(s) in RCA: 68] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
The first virus-like particle to be tested for use as a vaccine carrier was based on the hepatitis B virus nucleocapsid protein. This viral subunit, while not infectious on its own, is a 36-nm particle that is highly immunogenic during a natural infection. The self-assembly and high degree of immunogenicity is maintained when expressed as a recombinant protein and, moreover, can confer a high degree of immunogenicity on foreign antigens linked to the particle, either chemically or genetically. This review describes the current state of the hepadnaviral core protein as a vaccine carrier.
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Affiliation(s)
- David C Whitacre
- Vaccine Research Institute of San Diego, San Diego, CA 92121, USA.
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12
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Folding properties of the hepatitis B core as a carrier protein for vaccination research. Amino Acids 2009; 38:1617-26. [DOI: 10.1007/s00726-009-0365-1] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2009] [Accepted: 10/07/2009] [Indexed: 01/10/2023]
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13
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Yap WB, Tey BT, Ng MYT, Ong ST, Tan WS. N-terminally His-tagged hepatitis B core antigens: construction, expression, purification and antigenicity. J Virol Methods 2009; 160:125-31. [PMID: 19433111 DOI: 10.1016/j.jviromet.2009.04.038] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2008] [Revised: 04/28/2009] [Accepted: 04/30/2009] [Indexed: 11/16/2022]
Abstract
The core antigen of the hepatitis B virus (HBcAg) has been used widely as a diagnostic reagent for the identification of the viral infection. However, purification using the conventional sucrose density gradient ultracentrifugation is time consuming and costly. To overcome this, HBcAg particles displaying His-tag on their surface were constructed and produced in Escherichia coli. The recombinant His-tagged HBcAgs were purified using immobilized metal affinity chromatography. Transmission electron microscopy and enzyme-linked immunosorbent assay (ELISA) revealed that the displayed His-tag did not impair the formation of the core particles and the antigenicity of HBcAg.
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Affiliation(s)
- Wei Boon Yap
- Department of Microbiology, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
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14
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Neugebauer M, Walders B, Brinkman M, Ruehland C, Schumacher T, Bertling WM, Geuther E, Reiser COA, Reichel C, Strich S, Hess J. Development of a vaccine marker technology: Display of B cell epitopes on the surface of recombinant polyomavirus-like pentamers and capsoids induces peptide-specific antibodies in piglets after vaccination. Biotechnol J 2006; 1:1435-46. [PMID: 17109492 DOI: 10.1002/biot.200600149] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Highly immunogenic capsomers (pentamers) and virus-like particles (VLPs) were generated through insertion of foreign B cell epitopes into the surface-exposed loops of the VP1 protein of murine polyomavirus and via heterologous expression of the recombinant fusion proteins in E. coli. Usually, complex proteins like the keyhole limpet hemocyanin (KLH) act as standard carrier devices for the display of such immunogenic peptides after chemical linkage. Here, a comparative analysis revealed that antibody responses raised against the carrier entities, KLH or VP1 pentamers, did not significantly differ up to 18 weeks, demonstrating the highly immunogenic nature of VP1-based particulate structures. The carrier-specific antibody response was reproducibly detected in the meat juice after processing. More importantly, chimeric VP1 pentamers and VLPs carrying peptides of 12 and 14 amino acids in length, inserted into the BC2 loop, induced a strong and long-lasting humoral immune response against VP1 and the inserted foreign epitope. Remarkably, the epitope-specific antibody response was only moderately decreased when VP1 pentamers were used instead of VLPs. In conclusion, we identified polyomavirus VP1-based structures displaying surface-exposed immunodominant B cell epitopes as being an efficient carrier system for the induction of potent peptide-specific antibodies. The application of this approach in vaccine marker technology in livestock holding and the meat production chain is discussed.
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15
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Guo H, Aldrich CE, Saputelli J, Xu C, Mason WS. The insertion domain of the duck hepatitis B virus core protein plays a role in nucleocapsid assembly. Virology 2006; 353:443-50. [PMID: 16837020 DOI: 10.1016/j.virol.2006.06.004] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2006] [Revised: 05/11/2006] [Accepted: 06/06/2006] [Indexed: 01/08/2023]
Abstract
Synthesis of hepadnaviral DNA is dependent upon both the viral DNA polymerase and the viral core protein, the subunit of the nucleocapsids in which viral DNA synthesis takes place. In a study of natural isolates of duck hepatitis B virus (DHBV), we cloned full-length viral genomes from a puna teal. One of the clones failed to direct viral DNA replication in transfected cells, apparently as a result of a 3 nt inframe deletion of histidine 107 in the core protein. Histidine 107 is located in the center of a predicted helical region of the "insertion domain", a stretch of 45 amino acids which appears to be at the tip of a spike on the surface of the nucleocapsid. The mutation was introduced into a well-characterized strain of DHBV for further analysis. Core protein accumulated in cells transfected with the mutant DHBV but was partially degraded, suggesting that it was unstable. Assembled nucleocapsids were not detected by capsid gel electrophoresis. Interestingly, the mutant protein appeared to form chimeric nucleocapsids with wild-type core protein. The chimeric nucleocapsids supported viral DNA replication. These results suggest that the insertion domain of the spike may play a role either in assembly of stable nucleocapsids, possibly in formation of the dimer subunits, or in triggering nucleocapsid disintegration, required during initiation of new rounds of infection.
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Affiliation(s)
- Haitao Guo
- Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA
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16
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Peng M, Dai CB, Chen YD. Expression and immunoreactivity of an epitope of HCV in a foreign epitope presenting system. World J Gastroenterol 2005; 11:3363-7. [PMID: 15948240 PMCID: PMC4315989 DOI: 10.3748/wjg.v11.i22.3363] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To construct and highly express an epitope of hepatitis C virus (HCV) in a foreign epitope presenting vector based on an insect virus, and to study the antigenicity of the epitope.
METHODS: The HCV epitope sequence (amino acid residues 315 to 328: EGHRMAWDMMMNWS) of the E1 region was constructed at different positions of a foreign epitope presenting vector based on an insect virus, flock house virus (FHV) capsid protein encoding gene as a vector, and expressed in E. coli cells. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant proteins.
RESULTS: The gene encoding of the concerned B-cell epitope of HCV E1 envelope protein was expressed on FHV capsid carrier protein at positions I1 (aa 106), I2 (aa 153) and I3 (aa 305), respectively, on the surface of FHV capsid protein. The recombinant proteins in this system could be highly expressed in more than 40% of total cell protein of E. coli BL21. All the expressed recombinant proteins were in inclusion body form, and showed obvious immunoreactivity by Western blotting. Further purified recombinant proteins were detected by indirect ELISA as coating antigen respectively. All recombinant proteins could still show immunoreactivity.
CONCLUSION: The epitope of HCV E1 envelope protein can be highly expressed in FHV carrier system as a chimeric protein with high immunoreactivity. This system has multiple entry sites conferring many possible conformations closer to the native one for a given sequence.
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Affiliation(s)
- Mei Peng
- Department of Molecular Biology, Institute of Medical Biology, Chinese Academy of Medical Sciences/Peking Union Medical College, 379 Jiaoling Road, Kunming 650118, Yunnang Province, China.
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17
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Aguilar JC, Lobaina Y, Muzio V, García D, Pentón E, Iglesias E, Pichardo D, Urquiza D, Rodríguez D, Silva D, Petrovsky N, Guillén G. Development of a nasal vaccine for chronic hepatitis B infection that uses the ability of hepatitis B core antigen to stimulate a strong Th1 response against hepatitis B surface antigen. Immunol Cell Biol 2005; 82:539-46. [PMID: 15479440 DOI: 10.1111/j.0818-9641.2004.01278.x] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
There are estimated to be 350 million chronic carriers of hepatitis B infection worldwide. Patients with chronic hepatitis B are at risk of liver cirrhosis with associated mortality because of hepatocellular carcinoma and other complications. An important goal, therefore, is the development of an effective therapeutic vaccine against chronic hepatitis B virus (HBV). A major barrier to the development of such a vaccine is the impaired immune response to HBV antigens observed in the T cells of affected patients. One strategy to overcome these barriers is to activate mucosal T cells through the use of nasal vaccination because this may overcome the systemic immune downregulation that results from HBV infection. In addition, it may be beneficial to present additional HBV epitopes beyond those contained in the traditional hepatitis B surface antigen (HbsAg) vaccine, for example, by using the hepatitis B core antigen (HBcAg). This is advantageous because HBcAg has a unique ability to act as a potent Th1 adjuvant to HbsAg, while also serving as an immunogenic target. In this study we describe the effect of coadministration of HBsAg and HBcAg as part of a strategy to develop a more potent and effective HBV therapeutic vaccine.
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Affiliation(s)
- J C Aguilar
- Biomedical Branch, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
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18
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Kazaks A, Lachmann S, Koletzki D, Petrovskis I, Dislers A, Ose V, Skrastina D, Gelderblom HR, Lundkvist A, Meisel H, Borisova G, Krüger DH, Pumpens P, Ulrich R. Stop codon insertion restores the particle formation ability of hepatitis B virus core-hantavirus nucleocapsid protein fusions. Intervirology 2003; 45:340-9. [PMID: 12602354 DOI: 10.1159/000067927] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
In recent years, epitopes of various origin have been inserted into the core protein of hepatitis B virus (HBc), allowing the formation of chimeric HBc particles. Although the C-terminus of a C-terminally truncated HBc (HBc) tolerates the insertion of extended foreign sequences, the insertion capacity is still a limiting factor for the construction of multivalent vaccines. Previously, we described a new system to generate HBc mosaic particles based on a read-through mechanism in an Escherichia coli suppressor strain [J Gen Virol 1997;78:2049-2053]. Those mosaic particles allowed the insertion of a 114-amino acid (aa)-long segment of a Puumala hantavirus (PUUV) nucleocapsid (N) protein. To study the value and the potential limitations of the mosaic approach in more detail, we investigated the assembly capacity of 'non-mosaic' HBc fusion proteins and the corresponding mosaic constructs carrying 94, 213 and 433 aa of the hantaviral N protein. Whereas the fusion proteins carrying 94, 114, 213 or 433 aa were not assembled into HBc particles, or only at a low yield, the insertion of a stop codon-bearing linker restored the ability to form particles with 94, 114 and 213 foreign aa. The mosaic particles formed exhibited PUUV-N protein antigenicity. Immunization of BALB/c mice with these mosaic particles carrying PUUV-N protein aa 1-114, aa 1-213 and aa 340-433, respectively, induced HBc-specific antibodies, whereas PUUV-N protein-specific antibodies were detected only in mice immunized with particles carrying N-terminal aa 1-114 or aa 1-213 of the N protein. Both the anti-HBc and anti-PUUV antibody responses were IgG1 dominated. In conclusion, stop codon suppression allows the formation of mosaic core particles carrying large-sized and 'problematic', e.g. hydrophobic, hantavirus sequences.
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Affiliation(s)
- Andris Kazaks
- Biomedical Research and Study Centre, University of Latvia, Riga, Latvia
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19
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Lobaina Y, García D, Abreu N, Muzio V, Aguilar JC. Mucosal immunogenicity of the hepatitis B core antigen. Biochem Biophys Res Commun 2003; 300:745-50. [PMID: 12507513 DOI: 10.1016/s0006-291x(02)02897-8] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The hepatitis B virus (HBV) core antigen (HBcAg) is a potent immunogen in animal models and humans and has been used as a carrier for several antigens, however, the mucosal immunogenicity of HBcAg or chimeric HBcAg proteins has been poorly studied and only using the truncated variant of the HBcAg. In this study we explored the mucosal immunogenicity in mice of the recombinant complete nucleocapside of HBcAg. The antigen was administered by different mucosal and parenteral routes. The antibody response in sera was evaluated after each immunization and mucosal lavages were tested with the final extraction. To characterize the immune response, the serum IgG antibody response was tested during six months and also the ratio IgG2a to IgG1 was determined. The results obtained evidenced that the mucosal immunogenicity of HBcAg depended on the administration route, being the intranasal (i.n.) route the one that generated the higher IgG responses in sera, similar in intensity and duration to parenteral administrations. The IgA response in mucosal washes was superior for nasally immunized mice compared to the rest of mucosal and parenteral groups. The nasal route also induced the higher IgG2a to IgG1 ratio, evidencing a Th1-like Ab subclass pattern. In addition to the high Ab responses, preliminary results of the cellular response induced by nasal administration evidenced the induction of strong lymphoproliferative responses in spleen cells.
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Affiliation(s)
- Y Lobaina
- Division of Vaccines, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, C.P. 10600, C. Habana, Cuba
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20
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Abstract
In the middle 80s, recombinant hepatitis B virus cores (HBc) gave onset to icosahedral virus-like particles (VLPs) as a basic class of non-infectious carriers of foreign immunological epitopes. The recombinant HBc particles were used to display immunodominant epitopes of hepatitis B, C, and E virus, human rhinovirus, papillomavirus, hantavirus, and influenza virus, human and simian immunodeficiency virus, bovine and feline leukemia virus, foot-and-mouth disease virus, murine cytomegalovirus and poliovirus, and other virus proteins, as well as of some bacterial and protozoan protein epitopes. Practical applicability of the HBc particles as carriers was enabled by their ability to high level synthesis and correct self-assembly in heterologous expression systems. The interest in the HBc VLPs was reinforced by the resolution of their fine structure by electron cryomicroscopy and X-ray crystallography, which revealed an unusual alpha-helical organization of dimeric units of HBc shells, alternative packing into icosahedrons with T = 3 and T = 4 symmetry, and the existence of long protruding spikes. The tips of the latter seem to be the optimal targets for the display of foreign sequences up to 238 amino acid residues in length. Combination of numerous experimental data on epitope display with the precise structural information enables a knowledge-based design of diagnostic, and vaccine and gene therapy tools on the basis of the HBc particles.
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Affiliation(s)
- P Pumpens
- Biomedical Research and Study Center, University of Latvia, Riga, Latvia.
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21
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Aggarwal N, Barnett PV. Antigenic sites of foot-and-mouth disease virus (FMDV): an analysis of the specificities of anti-FMDV antibodies after vaccination of naturally susceptible host species. J Gen Virol 2002; 83:775-782. [PMID: 11907326 DOI: 10.1099/0022-1317-83-4-775] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Of the known neutralizing antigenic sites of foot-and-mouth disease virus (FMDV), site 1 or A, formed in part by the G-H loop of VP1, has historically been considered immunodominant because of evidence implicating its importance in the induction of a protective immune response. However, no systematic study has been done to determine the relative importance of the various specificities of antibodies against the known neutralizing antigenic sites of FMDV in the polyclonal immune response of a natural host after vaccination. In this report, we have adopted a monoclonal antibody-based competition ELISA and used antibodies specific to sites 1, 2 and 3 to provide some insight into this issue. Following vaccination of the three main target species, cattle, pigs and sheep, with an O1 serotype strain, results indicate that none of these three antigenic sites can be considered immunodominant in a polyclonal serum. Interestingly, pigs did not respond to epitopes on the carboxy terminus end of VP1 as efficiently as the ruminant species. In addition to the known sites, other as yet undefined sites might also be important in the induction of a protective immune response. Possible implications for the design of new vaccine strategies for foot-and-mouth disease are discussed.
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Affiliation(s)
- N Aggarwal
- Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK1
| | - P V Barnett
- Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK1
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22
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Arnon R, Tarrab-Hazdai R, Ben-Yedidia T. Peptide-based synthetic recombinant vaccines with anti-viral efficacy. Biologicals 2001; 29:237-42. [PMID: 11851322 DOI: 10.1006/biol.2001.0303] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Synthetic recombinant vaccines are constructs in which a synthetic oligonucleotide coding for a protective epitope is inserted into an adequate gene for expression of the epitope. We report the results obtained using recombinant flagella of Salmonella vaccine strain expressing epitopes of influenza virus or of the parasite Schistosoma mansoni. In the case of influenza virus, three conserved epitopes of the haemagglutinin and the nucleoprotein of the virus inducing B- and T-cell immune response, were expressed and the flagella were used for intranasal immunization without any adjuvant. Both humoral and cellular immune responses specific to the virus induced in mice cross-strain long-term protection against challenge infection. Aged mice were also able to resist infection. For the design of a human influenza vaccine, epitopes recognized by the HLAs prevalent in Caucasian populations were used, and the resulting vaccine was evaluated in human/mouse radiation chimaera in which human PBMC are functionally engrafted. The vaccinated mice demonstrated efficient clearance of the virus after challenge and resistance to lethal infection. In the case of the parasitic disease schistosomiasis, a 14-residue peptide denoted 9B peptide 1 was expressed in the flagella. Intranasal vaccination of mice with this construct, without the use of adjuvant, resulted in 40% protection against challenge infection.
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Affiliation(s)
- R Arnon
- Department of Immunology, Weizmann Institute of Science, Rehovot, Israel 76100.
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23
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Koletzki D, Lundkvist A, Sjölander KB, Gelderblom HR, Niedrig M, Meisel H, Krüger DH, Ulrich R. Puumala (PUU) hantavirus strain differences and insertion positions in the hepatitis B virus core antigen influence B-cell immunogenicity and protective potential of core-derived particles. Virology 2000; 276:364-75. [PMID: 11040127 DOI: 10.1006/viro.2000.0540] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Hepatitis B virus (HBV) core-derived chimeric particles carrying a Puumala (PUU) hantavirus (strain Vranica/Hällnäs) nucleocapsid (N) protein sequence (aa 1-45), alternatively inserted at three distinct positions (N-, C-terminus, or the internal region), and mosaic particles consisting of HBV core as well as core/PUU (Vranica/Hällnäs) N (aa 1-45) readthrough protein were generated. Chimeric particles carrying the insert at the N-terminus or the internal region of core induced some protective immune response in bank voles (Clethrionomys glareolus) against a subsequent PUU virus (strain Kazan) challenge; 40-50% of the animals showed markers of protection. In contrast, internal insertion of PUU strain CG18-20 N (aa 1-45) into the HBV core caused a highly protective immune response in the bank vole model. Immunizations with particles carrying aa 75-119 of PUU (CG18-20) N at the C-terminus of core verified the presence of a second, minor protective region in the N protein. A strong PUU N-specific antibody response was detected not only in bank voles immunized with chimeric particles containing internal and N-terminal fusions of PUU N protein but also in animals immunized with the corresponding mosaic particles. Except for the exclusive occurrence of antibodies directed against aa 231-240 of N in non-protected animals post virus challenge, there was no additional obvious difference in the epitope-specificity of N-specific antibodies from immunized animals prior and post virus challenge.
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Affiliation(s)
- D Koletzki
- Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden
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24
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Ulrich R, Koletzki D, Lachmann S, Lundkvist A, Zankl A, Kazaks A, Kurth A, Gelderblom HR, Borisova G, Meisel H, Krüger DH. New chimaeric hepatitis B virus core particles carrying hantavirus (serotype Puumala) epitopes: immunogenicity and protection against virus challenge. J Biotechnol 1999; 73:141-53. [PMID: 10486924 DOI: 10.1016/s0168-1656(99)00117-0] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
Virus-like particles generated by the heterologous expression of virus structural proteins are able to potentiate the immunogenicity of foreign epitopes presented on their surface. In recent years epitopes of various origin have been inserted into the core antigen of hepatitis B virus (HBV) allowing the formation of chimaeric HBV core particles. Chimaeric core particles carrying the 45 N-terminal amino acids of the Puumala hantavirus nucleocapsid protein induced protective immunity in bank voles, the natural host of this hantavirus. Particles applied in the absence of adjuvant are still immunogenic and partially protective in bank voles. Although a C-terminally truncated core antigen of HBV (HBcAg delta) tolerates the insertion of extended foreign sequences, for the construction of multivalent vaccines the limited insertion capacity is still a critical factor. Recently, we have described a new system for generating HBV 'mosaic particles' in an Escherichia coli suppressor strain based on a readthrough mechanism on a stop linker located in front of the insert. Those mosaic particles are built up by both HBcAg delta and the HBcAg delta/Puumala nucleocapsid readthrough protein. The particles formed presented the 114 amino acid (aa) long hantavirus sequence, at least in part, on their surface and induced antibodies against the hantavirus sequence in bank voles. Variants of the stop linker still allowed the formation of mosaic particles demonstrating that stop codon suppression alone is sufficient for the packaging of longer foreign sequences in mosaic particles. Another approach to increase the insertion capacity is based on the simultaneous insertion of different Puumala nucleocapsid protein sequences (aa 1-45 and aa 75-119) into two different positions (aa 78 and behind aa 144) of a single HBcAg molecule. The data presented are of high relevance for the generation of multivalent vaccines requiring a high insertion capacity for foreign sequences.
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Affiliation(s)
- R Ulrich
- Institute of Virology, Humboldt University, Charité Medical School, Berlin, Germany
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25
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Lachmann S, Meisel H, Muselmann C, Koletzki D, Gelderblom HR, Borisova G, Krüger DH, Pumpens P, Ulrich R. Characterization of potential insertion sites in the core antigen of hepatitis B virus by the use of a short-sized model epitope. Intervirology 1999; 42:51-6. [PMID: 10393504 DOI: 10.1159/000024960] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Core particles of hepatitis B virus (HBV) are able to improve the immunogenicity of foreign sequences exposed on the particle surface. The insertion site in the core antigen of HBV (HBcAg) determines the surface presentation and thus the immunogenicity of the foreign sequence. For direct comparison of the value of potential insertion sites in the core antigen, we constructed vectors allowing insertions of a model marker epitope DPAFR. This epitope was inserted at the N-terminus, the c/e1 loop, behind amino acid (aa) 144 and behind aa 183 (DPAF only). In addition, we generated a mosaic construct allowing the co-expression of HBcAg and a HBcAg/DPAFR fusion protein due to a suppressor tRNA-mediated readthrough mechanism. All 6 constructs allowed the formation of chimaeric or mosaic core-like particles. Western blot analyses and a direct ELISA demonstrated the presence of the DPAFR sequence in the chimaeric and mosaic particles. Competitive ELISA and immune electron-microscopic data suggested the c/e1 loop as the insertion site of choice for presenting foreign sequences on the surface of chimaeric HBV core particles. However, the N-terminal fusion also allowed partial surface exposure of the DPAFR motif. In contrast, in particles of constructs carrying the DPAFR insert at aa position 144 or 183, respectively, the epitope seemed not to be surface accessible.
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Affiliation(s)
- S Lachmann
- Institute of Virology, Humboldt University, Charité Medical School, Berlin, Germany
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26
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Borisova G, Borschukova O, Skrastina D, Dislers A, Ose V, Pumpens P, Grens E. Behavior of a short preS1 epitope on the surface of hepatitis B core particles. Biol Chem 1999; 380:315-24. [PMID: 10223334 DOI: 10.1515/bc.1999.043] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/11/2023]
Abstract
The major immunodominant region of hepatitis B core particles is widely recognized as the most prospective target for the insertion of foreign epitopes, ensuring their maximal antigenicity and immunogenicity. This region was mapped around amino acid residues 79-81, which were shown by electron cryo-microscopy to be located on the tips of the spikes protruding from the surface of hepatitis B core shells. Here we tried to expose a model sequence, the short immunodominant hepatitis B preS1 epitope 31-DPAFR-35, onto the tip of the spike, with simultaneous deletion of varying stretches from the major immunodominant region of the HBc molecule. Accessibility to the monoclonal anti-preS1 antibody MA18/7 and specific immunogenicity of the preS1 epitope depended on the location and length of the deletion. While chimeras with deletions within the stretch 79-88 presented the preS1 epitope on their surface and demonstrated remarkable preS1 immunogenicity, the corresponding chimeras without any deletion or with a more prolonged deletion (79-93) were unable to provide such presentation and possessed a lower specific preS1 immunogenicity. Deletion of the stretch 79-81 was sufficient to avoid the intrinsic HBc immunogenicity of the core particles, although chimeras with deleted major immunodominant region retained their property to be recognized by human polyclonal or hyperimmune anti-HBc antibodies.
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Affiliation(s)
- G Borisova
- Biomedical Research and Study Centre, University of Latvia, Riga
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27
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Koletzki D, Biel SS, Meisel H, Nugel E, Gelderblom HR, Krüger DH, Ulrich R. HBV core particles allow the insertion and surface exposure of the entire potentially protective region of Puumala hantavirus nucleocapsid protein. Biol Chem 1999; 380:325-33. [PMID: 10223335 DOI: 10.1515/bc.1999.044] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Core particles of the hepatitis B virus (HBV) potentiate the immune response against foreign epitopes presented on their surface. Potential insertion sites in the monomeric subunit of the HBV core protein were previously identified at the N- and C-terminus and in the immunodominant c/e1 region. In a C-terminally truncated core protein these sites were used to introduce the entire 120 amino acid (aa)-long potentially immunoprotective region of the hantavirus (serotype Puumala) nucleocapsid protein. The N- and C-terminal fusion products were unable to form core-like particles in detectable amounts. However, a suppressable stop codon located between the HBV core and the C-terminally fused hantavirus sequence restored the ability to form particles ('mosaic particles'); in contrast to the C-terminal fusion product the mosaic construct allowed the formation of particles built up by the core protein itself and the HBV core-Puumala nucleocapsid-readthrough protein. The mosaic particles exposed the 120 aa region of the PUU nucleocapsid protein on their surface as demonstrated by ELISA and immuno electron microscopy applying different monoclonal antibodies. Insertion of the hantaviral sequence into the c/e1 region not only allowed the formation of chimeric particles, but again the surface accessibility of the sequence. HBV core antigenicity itself was, however, reduced in the particles carrying insertions in the c/e1 region, probably due to a masking effect of the 120 aa long insert.
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Affiliation(s)
- D Koletzki
- Institute of Virology, Charité Medical School, Humboldt University, Berlin, Germany
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28
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Lohnas GL, Roberts SF, Pilon A, Tramontano A. Epitope-Specific Antibody and Suppression of Autoantibody Responses Against a Hybrid Self Protein. THE JOURNAL OF IMMUNOLOGY 1998. [DOI: 10.4049/jimmunol.161.12.6518] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Abstract
This study addresses the relationship of epitope-specific Ab responses and alternative autoantibody responses in a model system in which an antigenized self protein serves as the carrier for a defined heterologous B cell epitope. Ubiquitin, a nonimmunogenic self protein, was engineered to present heterologous B and T cell epitopes in the recombinant molecule. Fusion to the C terminus introduced a universal T cell epitope from a Mycobacterium tuberculosis Ag. The B cell epitope was created by inserting a 12-residue loop sequence of HIV-1 gp120 at a surface-exposed position of ubiquitin. These modifications preserved the ubiquitin fold, allowing a new conformational epitope to be presented among native self epitopes. Mice immunized with the hybrid protein bearing only the mycobacterial T cell epitope elicited a strong autoantibody response to native ubiquitin. In contrast, antisera elicited against hybrid ubiquitin presenting the HIV B cell epitope reacted specifically with the foreign epitope but not with native ubiquitin. Absence of autoantibody in the response was attributed to poor competition of autoreactive B cells for limiting T cell help. Both types of responses were associated with Th responses to defined epitopes of the ubiquitin hybrid protein. These results may have implications for a tolerance mechanism dependent on B-T cell cooperation.
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29
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Ulrich R, Nassal M, Meisel H, Krüger DH. Core particles of hepatitis B virus as carrier for foreign epitopes. Adv Virus Res 1998; 50:141-82. [PMID: 9520999 DOI: 10.1016/s0065-3527(08)60808-8] [Citation(s) in RCA: 115] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
To be effective as vaccines, most monomeric proteins and peptides either require chemical coupling to high molecular weight carriers or application together with adjuvants. More recently, recombinant DNA techniques have been used to insert foreign epitopes into proteins with inherent multimerization capacity, such as particle-forming viral capsid or envelope proteins. The core protein of hepatitis B virus (HBcAg), because of its unique structural and immunological properties, has gained widespread interest as a potential antigen carrier. Foreign sequences of up to approximately 40 amino acid residues at the N terminus, 50 or 100 amino acids in the central immunodominant c/e 1 epitope region of HBcAg, and up to 100 or even more residues at the C terminus, did not interfere with particle formation. The humoral immunogenicity of inserted epitopes is determined by the immunogenicity of the peptide itself and its surface exposure, and is influenced by the route of application. The probably flexible and surface-exposed c/e1 region emerged as the most promising insertion site. When applied together with adjuvants approved for human and veterinary use, or even without adjuvants, such chimeric particles induced B and T cell immune responses against the inserted epitopes. In some cases neutralizing antibodies, cytotoxic T cells and protection against challenge with the intact pathogen were demonstrated. Major factors for the potentiated immune response against the foreign epitopes are the multimeric structure of chimeric HBcAg that results in a high epitope density per particle, and the provision of T cell help by the carrier moiety. Beyond its use as subunit vaccine, chimeric HBcAg produced in attenuated Salmonella strains may be applicable as live vaccine.
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Affiliation(s)
- R Ulrich
- Charité Medical School, Humboldt University, Berlin, Germany
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30
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Böttcher B, Wynne SA, Crowther RA. Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy. Nature 1997; 386:88-91. [PMID: 9052786 DOI: 10.1038/386088a0] [Citation(s) in RCA: 599] [Impact Index Per Article: 21.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Hepatitis B virus, a major human pathogen with an estimated 300 million carriers worldwide, can lead to cirrhosis and liver cancer in cases of chronic infection. The virus consists of an inner nucleocapsid or core, surrounded by a lipid envelope containing virally encoded surface proteins. The core protein, when expressed in bacteria, assembles into core shell particles, closely resembling the native core of the virus. Here we use electron cryomicroscopy to solve the structure of the core protein to 7.4 A resolution. Images of about 6,400 individual particles from 34 micrographs at different levels of defocus were combined, imposing icosahedral symmetry. The three-dimensional map reveals the complete fold of the polypeptide chain, which is quite unlike previously solved viral capsid proteins and is largely alpha-helical. The dimer clustering of subunits produces spikes on the surface of the shell, which consist of radial bundles of four long alpha-helices. Our model implies that the sequence corresponding to the immunodominant region of the core protein lies at the tip of the spike and also explains other properties of the core protein.
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Affiliation(s)
- B Böttcher
- Medical Research Council Laboratory of Molecular Biology, Cambridge, UK
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31
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Buratti E, Tisminetzky SG, Scodeller ES, Baralle FE. Conformational display of two neutralizing epitopes of HIV-1 gp41 on the Flock House virus capsid protein. J Immunol Methods 1996; 197:7-18. [PMID: 8890890 DOI: 10.1016/0022-1759(96)00097-x] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
We have developed an antigen presenting system based on the capsid protein of the Flock House virus (FHV) and used it to display, in different positions on its external surface, two neutralizing epitopes found at residues 735-752 of HIV-1 gp160. We have compared the immunoreactivity of these FHV chimeric proteins and of the corresponding synthetic peptide using a panel of neutralizing mouse monoclonal antibodies (mAbs) directed against two distinct sequences (IEEE and ERDRD) contained in this epitope of the gp41 region. We have observed that both the FHV chimeric protein and the synthetic peptide are clearly detected in ELISA procedures by the mAbs recognizing the sequence IEEE. The denaturation of these recombinant proteins had little effect on the recognition pattern of this group of monoclonals, suggesting minor conformational requirements for the display of this epitope. The FHV chimeric proteins were also recognized by the mAbs directed against the ERDRD epitope, whereas the corresponding synthetic peptide was not recognized. In this case, denaturation of these recombinant proteins completely abolished the reactivity of the second group of mAbs, arguing for the existence of strong conformational constraints. Additionally, we have investigated whether an isolated loop structure from the FHV protein was sufficient to provide the conformational requirements for the presentation of these epitopes. These experiments have shown that the stabilized loop structure, although improving the presentation of both epitopes, is not as efficient as the native loop in the intact FHV protein. The data obtained with these mAbs support the recently observed limitations in the use of synthetic peptides for the screening of the immune response against conformational epitopes. The establishment of appropriate tools able to present epitope sequences in a structure resembling the native conformation will be useful for accurate epidemiological studies and for the design of new epitope-specific vaccines.
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Affiliation(s)
- E Buratti
- International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
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32
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Chambers MA, Dougan G, Newman J, Brown F, Crowther J, Mould AP, Humphries MJ, Francis MJ, Clarke B, Brown AL, Rowlands D. Chimeric hepatitis B virus core particles as probes for studying peptide-integrin interactions. J Virol 1996; 70:4045-52. [PMID: 8648742 PMCID: PMC190284 DOI: 10.1128/jvi.70.6.4045-4052.1996] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
Abstract
An RGD-containing epitope from the foot-and-mouth disease virus (FMDV) VP1 protein was inserted into the e1 loop of the hepatitis B virus core (HBc) protein. This chimeric protein was expressed at high levels in Escherichia coli and spontaneously assembled into virus-like particles which could be readily purified. These fusion particles elicited high levels of both enzyme-linked immunosorbent assay- and FMDV-neutralizing antibodies in guinea pigs. The chimeric particles bound specifically to cultured eukaryotic cells. Mutant particles carrying the tripeptide sequence RGE in place of RGD and the use of a competitive peptide, GRGDS, confirmed the critical involvement of the RGD sequence in this binding. The chimeric particles also bound to purified integrins, and inhibition by chain-specific anti-integrin monoclonal antibodies implicated alpha 5 beta 1 as a candidate cell receptor for both the chimeric particle and FMDV. Some serotypes of FMDV bound to beta 1 integrins in solid- phase assays, and the chimeric particles competed with FMDV for binding to susceptible eukaryotic cells. Thus, HBc particles may provide a simple, general system for exploring the interactions of specific peptide sequences with cellular receptors.
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Affiliation(s)
- M A Chambers
- Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, United Kingdom
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33
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McLain L, Durrani Z, Wisniewski LA, Porta C, Lomonossoff GP, Dimmock NJ. Stimulation of neutralizing antibodies to human immunodeficiency virus type 1 in three strains of mice immunized with a 22 amino acid peptide of gp41 expressed on the surface of a plant virus. Vaccine 1996; 14:799-810. [PMID: 8817828 DOI: 10.1016/0264-410x(95)00229-t] [Citation(s) in RCA: 77] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
A plant virus, cowpea mosaic virus, expressing a 22 amino acid peptide 731-752 of the gp41 glycoprotein of human immunodeficency virus type 1 (HIV-1 IIIB), was shown previously to stimulate HIV-1 cross reactive neutralizing antibodies in adult C57/BL6 mice. Here some parameters concerning the stimulation of HIV-1-specific neutralizing and ELISA antibody have been determined in adult C57/BL6, C3H/He-mg and BALB/c mice. Two injections per mouse of all CPMV-HIV/1 doses tested (100, 10 and 1 microgram chimera which contained, respectively, 1700, 170 and 17 ng HIV peptide per injection) stimulated a strong serum neutralizing antibody response in all mice. One hundred micrograms or 10 micrograms CPMV-HIV/1 per injection gave 99% neutralization of HIV-1 IIIB in C8166 cells at a serum dilution of 1/200, whereas sera from mice immunized with 1 microgram per injection neutralized virus to 97%, 79% and 63% at a 1/200 dilution of serum from C3H/He-mg, C57/BL6 and BALB/c mice, respectively. Restimulation of these mice with the same immunogen dose marginally increased the neutralization titres. The longevity of the neutralizing antibody response increased as the immunogen dose decreased, and was dependent on the strain of mouse, in the order C57/BL6C3H/He-mg BALB/c. Re-immunization with a third injection improved the longevity of the antibody response. All mice immunized with 100 micrograms CPMV-HIV/1 responded with ELISA antibody to the gp41 peptide bound in solid phase. Ten micrograms stimulated ELISA antibody in some but not all mice, while mice immunized with 1 microgram had no detectable ELISA antibody. This synthesis of ELISA antibody decreased > or = 230-fold over the range of immunogen doses tested but, in the same mice, the neutralizing antibody response decreased only twofold, showing an unusual bias to production of the latter. Neutralizing antibodies were thus stimulated at a lower immunogen dose than ELISA antibodies. Antibody which was affinity purified using the free gp41 peptide gave a good ELISA titre but did not neutralize HIV-1, suggesting that the neutralizing antibody is recognizing a conformational epitope on the gp41 oligomer.
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Affiliation(s)
- L McLain
- Department of Biological Sciences, University of Warwick, Coventry, UK
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34
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Lomonossoff GP, Johnson JE. Use of macromolecular assemblies as expression systems for peptides and synthetic vaccines. Curr Opin Struct Biol 1996; 6:176-82. [PMID: 8728650 PMCID: PMC7133382 DOI: 10.1016/s0959-440x(96)80072-8] [Citation(s) in RCA: 61] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
The past decade has witnessed the development of numerous systems for the presentation of antigens on the surface of self-assembling macromolecules. Although the sites for insertion were initially chosen empirically, the determination of the three-dimensional structures of a number of carrier macromolecules has enabled structure-based insertional mutagenesis to be used increasingly. Furthermore, it is now feasible to determine the structure of an inserted sequence as presented in a heterologous environment, making it possible to correlate the detailed structure of a peptide with its immunological properties.
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Affiliation(s)
- G P Lomonossoff
- Department of Virus Research, John Innes Centre, Norwich, UK.
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35
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Londoño LP, Chatfield S, Tindle RW, Herd K, Gao XM, Frazer I, Dougan G. Immunisation of mice using Salmonella typhimurium expressing human papillomavirus type 16 E7 epitopes inserted into hepatitis B virus core antigen. Vaccine 1996; 14:545-52. [PMID: 8782354 DOI: 10.1016/0264-410x(95)00216-n] [Citation(s) in RCA: 56] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Live vaccines based on BRD509, an attenuated S. typhimurium (aroA, aroD) strain, were constructed that directed the expression of hepatitis B core antigen particles (HBcAg) (BRD969) or HBcAg harbouring human papillomavirus type 16 E7 protein sequences (BRD974), under the control of the in vivo inducible nirB promoter. These strains were used to orally or intravenously immunise different inbred mouse strains and humoral, secretory and cellular anti-E7 and anti-HBcAg responses were monitored. Both BRD969 and BRD974 induced anti-HBcAg humoral IgG responses following oral or intravenous immunisation of B10 mice, although responses were higher in BRD969 immunised animals. IgG subclass analysis revealed a predominantly IgG2a response in these animals. BRD974, but not BRD969, induced anti-E7 humoral IgG responses. Anti-HBcAg (BRD969 and BRD974) and anti-E7 (BRD974) IgA responses were detected in the intestines of orally immunised mice. Anti-Salmonella but not anti-HBcAg or anti-E7 T helper cell responses were detected in mice immunised with BRD509, BRD969 and BRD974. Thus Salmonella vaccine strains can be used to efficiently deliver HBcAg and E7 epitopes to the mucosal and systemic immune systems.
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Affiliation(s)
- L P Londoño
- Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK
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36
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Boulter NR, Glass EJ, Knight PA, Bell-Sakyi L, Brown CG, Hall R. Theileria annulata sporozoite antigen fused to hepatitis B core antigen used in a vaccination trial. Vaccine 1995; 13:1152-60. [PMID: 8578798 DOI: 10.1016/0264-410x(95)00026-w] [Citation(s) in RCA: 43] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
A C terminal fragment (SR1) of SPAG-1, a sporozoite surface antigen of Theileria annulata, has been expressed as a fusion protein in the e1 loop of hepatitis B core antigen (HBcAg). This recombinant antigen (HBcAg-SR1) is produced in the form of self-assembling polyhedral particles which have been visualised under the electron microscope. Cattle immunised with HBcAg-SR1 produced high titres of neutralising antibodies. A significant T cell response to both the HBcAg and SR1 determinants was observed but evidence of a T suppressor determinant in SR1 was also revealed. Immunised cattle showed some evidence of protection to sporozoite challenge as assessed by severity of the disease. The significance of these findings for the development of a sub-unit vaccine against T. annulata is discussed.
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Affiliation(s)
- N R Boulter
- Department of Biology, University of York, UK
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37
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Scodeller EA, Tisminetzky SG, Porro F, Schiappacassi M, De Rossi A, Chiecco-Bianchi L, Baralle FE. A new epitope presenting system displays a HIV-1 V3 loop sequence and induces neutralizing antibodies. Vaccine 1995; 13:1233-9. [PMID: 8578809 DOI: 10.1016/0264-410x(95)00058-9] [Citation(s) in RCA: 23] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The principal neutralizing domain, IGPGRAF sequence, from the V3-loop of HIV-1 was inserted in two positions on the surface of the protein that makes up the capside shell of the insect Flock House Virus. The hybrid proteins were expressed in insect cells via recombinant baculoviruses. Three different hybrids were used as immunogens: two with a single copy of the insert in different positions of the carrier protein and a third with two copies of the insert at the same positions as before. All hybrid proteins induced strong and broad specific immune response in guinea pigs against different V3-loop sequences. However, only one of the hybrid proteins was able to induce a strong neutralizing response against MN and IIIB HIV-1 isolates. Our results demonstrate that a very short peptide sequence of HIV-1 can constitute a valuable immunogen able to induce a neutralizing response if presented to the immune system in the context of the FHV capsomer structure.
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Affiliation(s)
- E A Scodeller
- International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
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38
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Kingsman AJ, Burns NR, Layton GT, Adams SE. Yeast retrotransposon particles as antigen delivery systems. Ann N Y Acad Sci 1995; 754:202-13. [PMID: 7625653 DOI: 10.1111/j.1749-6632.1995.tb44452.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
The development of technologies to produce recombinant proteins for use in the pharmaceutical industry has made substantial advances, in particular in the area of generating antigens containing multiple copies of important immunological regions. One such antigen-carrier system is based on the ability of a protein encoded by the yeast retrotransposon, Ty, to self-assemble into virus-like particles. Ty-fusion proteins retain this ability to form particles, and a range of hybrid VLPs carrying a variety of heterologous antigens have been produced and shown to induce potent immune responses. In particular, hybrid VLPs carrying the core protein p24 of HIV (p24-VLPs) have been shown to induce antibody and T-cell proliferative responses in both experimental animals and human volunteers, and immunization of rabbits with VLPs carrying the principal neutralizing determinant of HIV (V3-VLPs) resulted in the induction of neutralizing antibody responses and T-cell proliferation. Further studies with V3-VLPs have shown that this particulate antigen stimulates enhanced V3-specific lymphoproliferative responses as compared to whole recombinant gp120 or to V3 peptide conjugated to albumin. The V3-VLPs also induce potent CTL responses following immunization of mice in the absence of adjuvant. These responses are MHC class I restricted and are mediated by CD8-positive cells. These observations therefore demonstrate that hybrid Ty-VLPs induce both humoral and cellular immune responses against HIV and suggest that these immunogens may be important in combatting AIDS and other infections.
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Affiliation(s)
- A J Kingsman
- British Bio-technology Ltd., Oxford, United Kingdom
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39
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Milich DR, Peterson DL, Zheng J, Hughes JL, Wirtz R, Schödel F. The hepatitis nucleocapsid as a vaccine carrier moiety. Ann N Y Acad Sci 1995; 754:187-201. [PMID: 7542855 DOI: 10.1111/j.1749-6632.1995.tb44451.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
The "carrier effect," defined as the provision of T cell recognition sites physically linked to B cell epitopes in order to provide Th cell function for antibody synthesis, is well known. Peptides, proteins, and more recently particulate protein antigens have been used for this purpose. The hepatitis B core antigen represents a highly immunogenic antigen in humans as well as in experimental animal models. Studies in mice have provided insight into this enhanced immunogenicity. For example, HBcAg directly activates B cells (i.e., T cell independence), HBcAg elicits strong T cell responses, and HBcAg is efficiently processed and presented by antigen presenting cells (APCs). These characteristics suggested that HBcAg may be an ideal carrier moiety for B cell epitopes requiring additional Th cell function. Therefore, a number of HBV and non-HBV B cell epitopes have been chemically linked or fused by recombinant methods to HBcAg as a method to increase immunogenicity with significant success. We have designed bacterial expression vectors that allow insertion of heterologous B cell epitopes at various positions within HBcAg particles and permit efficient purification of hybrid HBcAg particles. Studies of positional effects have demonstrated that an internal insertion into a dominant HBcAg-specific B cell site represents a superior location for enhanced antibody production. Immunogenicity studies have been extended to protection against experimental challenge in several systems. For example, a malaria CS repeat sequence derived from P. berghei was inserted into HBcAg at the internal site, and purified hybrid HBcAg/CS particles were highly immunogenic and protected 100% of experimentally challenged BALB/c mice. This system has also been exploited for purposes of oral vaccination by expressing genes coding for hybrid HBcAg particles in live, avirulent vaccine strains of Salmonella species.
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Affiliation(s)
- D R Milich
- Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037, USA
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40
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Francis MJ. Peptide Vaccines; New Approaches to Immunopotentiation. Vaccines (Basel) 1995. [DOI: 10.1007/978-1-4613-0357-2_14] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
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41
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Katzer F, Carrington M, Knight P, Williamson S, Tait A, Morrison IW, Hall R. Polymorphism of SPAG-1, a candidate antigen for inclusion in a sub-unit vaccine against Theileria annulata. Mol Biochem Parasitol 1994; 67:1-10. [PMID: 7838169 DOI: 10.1016/0166-6851(94)90090-6] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
SPAG-1, a Theileria annulata sporozoite surface antigen, is a vaccine candidate. Data is presented, based on the clonal segregation of SPAG-1 associated RFLPs, showing that this antigen is encoded by a single copy gene. We have cloned and sequenced a full-length genomic copy of the SPAG-1 gene and a comparison of this with a previously published SPAG-1 cDNA sequence demonstrates a high degree of polymorphism. We infer that these sequences represent two distinct allelic SPAG-1 variants. The deduced polypeptides show an overall identity of 92% with the most variable stretch (60% identity) occurring towards the middle of the molecule. The N and C termini are more conserved with identities of 92% and 97% respectively. The elastin receptor ligand, VGVAPG, present 3 times in the protein sequence derived from the cDNA is not found in that deduced from the genomic copy. Evidence for 2 further SPAG-1 alleles was obtained from PCR based sequences using macroschizont clones containing different SPAG-1 associated RFLPs. In summary we have shown the existence of at least 4 highly polymorphic SPAG-1 alleles. The implications of such polymorphism between and within distinct geographical isolates for the development of a SPAG-1 based subunit vaccine is discussed.
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Affiliation(s)
- F Katzer
- Department of Biology, University of York, UK
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42
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Brown F. The Leeuwenhoek Lecture, 1993. Peptide vaccines: dream or reality? Philos Trans R Soc Lond B Biol Sci 1994; 344:213-9. [PMID: 7521966 DOI: 10.1098/rstb.1994.0062] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Small fragments of micro-organisms which elicit protective immune responses have now been identified for several disease-causing agents. This major advance has made it possible to envisage the chemical synthesis of vaccines which could replace those in current use and may also furnish products which cannot be made by traditional methods. In my lecture I will illustrate the principles involved by describing the advances made with synthetic vaccines for foot-and-mouth disease, hepatitis B and malaria.
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Affiliation(s)
- F Brown
- U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, New York 11944-0848
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43
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Schödel F, Peterson D, Hughes J, Milich D. Hepatitis B virus core particles as a vaccine carrier moiety. Int Rev Immunol 1994; 11:153-65. [PMID: 7519230 DOI: 10.3109/08830189409061723] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Affiliation(s)
- F Schödel
- Institut National de la Santé et de la Recherche Médicale, Hôpital Edouard Herriot, Lyon, France
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44
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Yoshikawa A, Tanaka T, Hoshi Y, Kato N, Tachibana K, Iizuka H, Machida A, Okamoto H, Yamasaki M, Miyakawa Y. Chimeric hepatitis B virus core particles with parts or copies of the hepatitis C virus core protein. J Virol 1993; 67:6064-70. [PMID: 8396669 PMCID: PMC238027 DOI: 10.1128/jvi.67.10.6064-6070.1993] [Citation(s) in RCA: 27] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
Either parts or multiple copies of the core gene of hepatitis C virus (HCV) were fused to the 3' terminus of the hepatitis B virus (HBV) core gene with 34 codons removed. As many as four copies of HCV core protein (720 amino acids) were fused to the carboxy terminus of truncated HBV core protein (149 amino acids) without preventing the assembly of HBV core particles. Chimeric core particles were sandwiched between monoclonal antibody to HBV core and that to HCV core, thereby indicating that antigenic determinants of both HBV and HCV cores were accessible on them. Proteolytic digestion deprived chimeric core particles of the antigenicity for the HCV core without affecting that of the HBV core, confirming the surface exposure of HCV core determinants. The density of HCV core determinants on chimeric core particles increased as copies of fused HCV core protein were increased. Hybrid core particles with multiple HCV core determinants would be instrumental as an antigen probe for detecting class-specific antibodies to the HCV core in patients with acute and chronic hepatitis C and for simultaneous detection of antibodies to HBV core and those to HCV core in donated blood.
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45
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46
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47
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Brown CS, Jensen T, Meloen RH, Puijk W, Sugamura K, Sato H, Spaan WJ. Localization of an immunodominant domain on baculovirus-produced parvovirus B19 capsids: correlation to a major surface region on the native virus particle. J Virol 1992; 66:6989-96. [PMID: 1433504 PMCID: PMC240344 DOI: 10.1128/jvi.66.12.6989-6996.1992] [Citation(s) in RCA: 26] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
An immunodominant region on baculovirus-produced parvovirus B19 VP2 capsids was localized between amino acids 259 and 426 by mapping the binding sites of a panel of monoclonal antibodies which recognize determinants on the particles. The binding sites of three monoclonal antibodies were fine-mapped within this antigenic domain. Six VP2-specific monoclonal antibodies recognized determinants common to both the empty capsids and native parvovirus. The defined antigenic region is most probably exposed on the native B19 virion and corresponds to part of the threefold spike on the surface of canine parvovirus particles.
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Affiliation(s)
- C S Brown
- Department of Virology, Faculty of Medicine, Leiden University, The Netherlands
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