|
1
|
Munetomo S, Uchiyama J, Takemura-Uchiyama I, Wanganuttara T, Yamamoto Y, Tsukui T, Hagiya H, Kanamaru S, Kanda H, Matsushita O. Examination of yield, bacteriolytic activity and cold storage of linker deletion mutants based on endolysin S6_ORF93 derived from Staphylococcus giant bacteriophage S6. PLoS One 2024; 19:e0310962. [PMID: 39441843 PMCID: PMC11498662 DOI: 10.1371/journal.pone.0310962] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2024] [Accepted: 09/10/2024] [Indexed: 10/25/2024] Open
Abstract
Methicillin-resistant Staphylococcus spp. present challenges in clinical and veterinary settings because effective antimicrobial agents are limited. Phage-encoded peptidoglycan-degrading enzyme, endolysin, is expected to be a novel antimicrobial agent. The enzymatic activity has recently been shown to be influenced by the linker between functional domains in the enzyme. S6_ORF93 (ORF93) is one of the endolysins derived from previously isolated Staphylococcus giant phage S6. The ORF93 was speculated to have a catalytic and peptidoglycan-binding domain with a long linker. In this study, we examined the influence of linker shortening on the characteristics of ORF93. We produce wild-type ORF93 and the linker deletion mutants using an Escherichia coli expression system. These mutants were designated as ORF93-Δ05, ORF93-Δ10, ORF93-Δ15, and ORF93-Δ20, from which 5, 10, 15, and 20 amino acids were removed from the linker, respectively. Except for the ORF93-Δ20, ORF93 and its mutants were expressed as soluble proteins. Moreover, ORF93-Δ15 showed the highest yield and bacteriolytic activity, while the antimicrobial spectrum was homologous. The cold storage experiment showed a slight effect by the linker deletion. According to our results and other studies, linker investigations are crucial in endolysin development.
Collapse
Affiliation(s)
- Sosuke Munetomo
- Department of Public Health, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University, Kita-ku, Okayama, Japan
| | - Jumpei Uchiyama
- Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University, Kita-ku, Okayama, Japan
| | - Iyo Takemura-Uchiyama
- Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University, Kita-ku, Okayama, Japan
| | - Thamonwan Wanganuttara
- Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University, Kita-ku, Okayama, Japan
| | - Yumiko Yamamoto
- Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University, Kita-ku, Okayama, Japan
| | | | - Hideharu Hagiya
- Department of Infectious Diseases, Okayama University Hospital, Kita-ku, Okayama, Japan
| | - Shuji Kanamaru
- School of Life Science and Technology, Tokyo Institute of Technology, Yokohama Kanagawa, Japan
| | - Hideyuki Kanda
- Department of Public Health, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University, Kita-ku, Okayama, Japan
| | - Osamu Matsushita
- Department of Bacteriology, Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama University, Kita-ku, Okayama, Japan
| |
Collapse
|
|
2
|
Messaabi A, Merindol N, Bohnenblust L, Fantino E, Meddeb-Mouelhi F, Desgagné-Penix I. In vivo thrombin activity in the diatom Phaeodactylum tricornutum: biotechnological insights. Appl Microbiol Biotechnol 2024; 108:481. [PMID: 39377797 PMCID: PMC11461642 DOI: 10.1007/s00253-024-13322-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2024] [Revised: 09/24/2024] [Accepted: 10/01/2024] [Indexed: 10/09/2024]
Abstract
Diatoms are responsible for 20% of global carbon dioxide fixation and have significant potential in various biotechnological and industrial applications. Recently, the pennate diatom Phaeodactylum tricornutum has emerged as a prominent platform organism for metabolic engineering and synthetic biology. The availability of its genome sequence has facilitated the development of new bioengineering tools. In this study, we used in silico analyses to identify sequences potentially encoding thrombin-like proteins, which are involved in recognizing and cleaving the thrombin sequence LVPRGS in P. tricornutum. Protein structure prediction and docking studies indicated a similar active site and ligand positioning compared to characterized human and bovine thrombin. The evidence and efficiency of the cleavage were determined in vivo using two fusion-protein constructs that included YFP to measure expression, protein accumulation, and cleavage. Western blot analysis revealed 50-100% cleavage between YFP and N-terminal fusion proteins. Our findings suggest the existence of a novel thrombin-like protease in P. tricornutum. This study advances the application of diatoms for the synthesis and production of complex proteins and enhances our understanding of the functional role of these putative thrombin sequences in diatom physiology. KEY POINTS: • Protein structure predictions reveal thrombin-like active sites in P. tricornutum. • Validated cleavage efficiency of thrombin-like protease on fusion proteins in vivo. • Study advances bioengineering tools for diatom-based biotechnological applications.
Collapse
Affiliation(s)
- Anis Messaabi
- Department of Chemistry, Biochemistry and Physics, Université du Québec À Trois-Rivières, Trois-Rivières, QC, Canada
| | - Natacha Merindol
- Department of Chemistry, Biochemistry and Physics, Université du Québec À Trois-Rivières, Trois-Rivières, QC, Canada
| | - Lea Bohnenblust
- Department of Chemistry, Biochemistry and Physics, Université du Québec À Trois-Rivières, Trois-Rivières, QC, Canada
| | - Elisa Fantino
- Department of Chemistry, Biochemistry and Physics, Université du Québec À Trois-Rivières, Trois-Rivières, QC, Canada
- Plant Biology Research Group, Université du Québec À Trois-Rivières, Trois-Rivières, QC, Canada
| | - Fatma Meddeb-Mouelhi
- Department of Chemistry, Biochemistry and Physics, Université du Québec À Trois-Rivières, Trois-Rivières, QC, Canada
- Plant Biology Research Group, Université du Québec À Trois-Rivières, Trois-Rivières, QC, Canada
| | - Isabel Desgagné-Penix
- Department of Chemistry, Biochemistry and Physics, Université du Québec À Trois-Rivières, Trois-Rivières, QC, Canada.
- Plant Biology Research Group, Université du Québec À Trois-Rivières, Trois-Rivières, QC, Canada.
| |
Collapse
|
|
3
|
Heidarnejad F, Namvar A, Sadat SM, Pordanjani PM, Rezaei F, Namdari H, Arjmand S, Bolhassani A. In silico designing of novel epitope-based peptide vaccines against HIV-1. Biotechnol Lett 2024; 46:315-354. [PMID: 38403788 DOI: 10.1007/s10529-023-03464-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2023] [Revised: 11/16/2023] [Accepted: 12/21/2023] [Indexed: 02/27/2024]
Abstract
The HIV-1 virus has been regarded as a catastrophe for human well-being. The global incidence of HIV-1-infected individuals is increasing. Hence, development of effective immunostimulatory molecules has recently attracted an increasing attention in the field of vaccine design against HIV-1 infection. In this study, we explored the impacts of CD40L and IFN-γ as immunostimulatory adjuvants for our candidate HIV-1 Nef vaccine in human and mouse using immunoinformatics analyses. Overall, 18 IFN-γ-based vaccine constructs (9 constructs in human and 9 constructs in mouse), and 18 CD40L-based vaccine constructs (9 constructs in human and 9 constructs in mouse) were designed. To find immunogenic epitopes, important characteristics of each component (e.g., MHC-I and MHC-II binding, and peptide-MHC-I/MHC-II molecular docking) were determined. Then, the selected epitopes were applied to create multiepitope constructs. Finally, the physicochemical properties, linear and discontinuous B cell epitopes, and molecular interaction between the 3D structure of each construct and CD40, IFN-γ receptor or toll-like receptors (TLRs) were predicted. Our data showed that the full-length CD40L and IFN-γ linked to the N-terminal region of Nef were capable of inducing more effective immune response than multiepitope vaccine constructs. Moreover, molecular docking of the non-allergenic full-length- and epitope-based CD40L and IFN-γ constructs to their cognate receptors, CD40 and IFN-γ receptors, and TLRs 4 and 5 in mouse were more potent than in human. Generally, these findings suggest that the full forms of these adjuvants could be more efficient for improvement of HIV-1 Nef vaccine candidate compared to the designed multiepitope-based constructs.
Collapse
Affiliation(s)
| | - Ali Namvar
- Iranian Comprehensive Hemophilia Care Center, Tehran, Iran
| | - Seyed Mehdi Sadat
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
| | | | - Fatemeh Rezaei
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
| | - Haideh Namdari
- Iranian Tissue Bank Research Center, Tehran University of Medical Sciences, Tehran, Iran
| | - Sina Arjmand
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran
| | - Azam Bolhassani
- Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran.
| |
Collapse
|
|
4
|
Aslam S, Zulfiqar F, Hameed W, Qureshi S, Zaroon, Bashir H. Fusion proteins development strategies and their role as cancer therapeutic agents. Biotechnol Appl Biochem 2024; 71:81-95. [PMID: 37822167 DOI: 10.1002/bab.2523] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2023] [Accepted: 10/01/2023] [Indexed: 10/13/2023]
Abstract
Cancer continues to be leading cause of morbidity and mortality despite decades of research and advancement in chemotherapy. Most tumors can be reduced via standard oncology treatments, such as chemotherapy, radiotherapy, and surgical resection, and they frequently recur. Significant progress has been made since targeted cancer therapy inception in creation of medications that exhibit improved tumor-selective action. Particularly in preclinical and clinical investigations, fusion proteins have shown strong activity and improved treatment outcomes for a number of human cancers. Synergistically combining many proteins into one complex allows the creation of synthetic fusion proteins with enhanced characteristics or new capabilities. Signal transduction pathways are important for onset, development, and spread of cancer. As result, signaling molecules are desirable targets for cancer therapies, and significant effort has been made into developing fusion proteins that would act as inhibitors of these pathways. A wide range of biotechnological and medicinal applications are made possible by fusion of protein domains that improves bioactivities or creates new functional combinations. Such proteins may function as immune effectors cell recruiters to tumors or as decoy receptors for various ligands. In this review article, we have outlined the standard methods for creating fusion proteins and covered the applications of fusion proteins in treatment of cancer. This article also highlights the role of fusion proteins in targeting the signaling pathways involved in cancer for effective treatment.
Collapse
Affiliation(s)
- Shakira Aslam
- Center for Applied Molecular Biology, University of the Punjab, Lahore, Pakistan
| | | | - Warda Hameed
- King Edward Medical University, Lahore, Pakistan
| | - Shahnila Qureshi
- Center for Applied Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Zaroon
- Center for Applied Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Hamid Bashir
- Center for Applied Molecular Biology, University of the Punjab, Lahore, Pakistan
| |
Collapse
|
|
5
|
Pagliari S, Dema B, Sanchez-Martinez A, Montalvo Zurbia-Flores G, Rollier CS. DNA Vaccines: History, Molecular Mechanisms and Future Perspectives. J Mol Biol 2023; 435:168297. [PMID: 37797831 DOI: 10.1016/j.jmb.2023.168297] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2023] [Revised: 09/22/2023] [Accepted: 09/28/2023] [Indexed: 10/07/2023]
Abstract
The history of DNA vaccine began as early as the 1960s with the discovery that naked DNA can transfect mammalian cells in vivo. In 1992, the evidence that such transfection could lead to the generation of antigen-specific antibody responses was obtained and supported the development of this technology as a novel vaccine platform. The technology then attracted immense interest and high hopes in vaccinology, as evidence of high immunogenicity and protection against virulent challenges accumulated from several animal models for several diseases. In particular, the capacity to induce T-cell responses was unprecedented in non-live vaccines. However, the technology suffered its major knock when the success in animals failed to translate to humans, where DNA vaccine candidates were shown to be safe but remained poorly immunogenic, or not associated with clinical benefit. Thanks to a thorough exploration of the molecular mechanisms of action of these vaccines, an impressive range of approaches have been and are currently being explored to overcome this major challenge. Despite limited success so far in humans as compared with later genetic vaccine technologies such as viral vectors and mRNA, DNA vaccines are not yet optimised for human use and may still realise their potential.
Collapse
Affiliation(s)
- Sthefany Pagliari
- School of Biosciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK; Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - Barbara Dema
- Pandemic Science Institute, Institute of Developmental and Regenerative Medicine (IDRM), University of Oxford, Oxford, UK
| | | | | | - Christine S Rollier
- School of Biosciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK.
| |
Collapse
|
|
6
|
Rahman T, Das A, Abir MH, Nafiz IH, Mahmud AR, Sarker MR, Emran TB, Hassan MM. Cytokines and their role as immunotherapeutics and vaccine Adjuvants: The emerging concepts. Cytokine 2023; 169:156268. [PMID: 37320965 DOI: 10.1016/j.cyto.2023.156268] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2023] [Revised: 06/02/2023] [Accepted: 06/06/2023] [Indexed: 06/17/2023]
Abstract
Cytokines are a protein family comprising interleukins, lymphokines, chemokines, monokines and interferons. They are significant constituents of the immune system, and they act in accordance with specific cytokine inhibiting compounds and receptors for the regulation of immune responses. Cytokine studies have resulted in the establishment of newer therapies which are being utilized for the treatment of several malignant diseases. The advancement of these therapies has occurred from two distinct strategies. The first strategy involves administrating the recombinant and purified cytokines, and the second strategy involves administrating the therapeutics which inhibits harmful effects of endogenous and overexpressed cytokines. Colony stimulating factors and interferons are two exemplary therapeutics of cytokines. An important effect of cytokine receptor antagonist is that they can serve as anti-inflammatory agents by altering the treatments of inflammation disorder, therefore inhibiting the effects of tumour necrosis factor. In this article, we have highlighted the research behind the establishment of cytokines as therapeutics and vaccine adjuvants, their role of immunotolerance, and their limitations.
Collapse
Affiliation(s)
- Tanjilur Rahman
- Department of Biochemistry and Molecular Biology, Faculty of Biological Sciences, University of Chittagong, Chattogram 4331, Bangladesh
| | - Ayan Das
- Department of Biochemistry and Molecular Biology, Faculty of Biological Sciences, University of Chittagong, Chattogram 4331, Bangladesh
| | - Mehedy Hasan Abir
- Faculty of Food Science and Technology, Chattogram Veterinary and Animal Sciences University, Chattogram 4225, Bangladesh
| | - Iqbal Hossain Nafiz
- Department of Biochemistry and Molecular Biology, Faculty of Biological Sciences, University of Chittagong, Chattogram 4331, Bangladesh
| | - Aar Rafi Mahmud
- Department of Biochemistry and Molecular Biology, Mawlana Bhashani Science and Technology University, Tangail 1902, Bangladesh
| | - Md Rifat Sarker
- Department of Biochemistry and Molecular Biology, Mawlana Bhashani Science and Technology University, Tangail 1902, Bangladesh
| | - Talha Bin Emran
- Department of Pharmacy, BGC Trust University Bangladesh, Chattogram 4381, Bangladesh; Department of Pharmacy, Faculty of Allied Health Sciences, Daffodil International University, Dhaka 1207, Bangladesh
| | - Mohammad Mahmudul Hassan
- Department of Physiology, Biochemistry and Pharmacology, Faculty of Veterinary Medicine, Chattogram Veterinary and Animal Sciences University, Chattogram 4225, Bangladesh; Queensland Alliance for One Health Sciences, School of Veterinary Science, The University of Queensland, Queensland 4343, Australia.
| |
Collapse
|
|
7
|
Bian Y, Walter DL, Zhang C. Efficiency of Interferon-γ in Activating Dendritic Cells and Its Potential Synergy with Toll-like Receptor Agonists. Viruses 2023; 15:v15051198. [PMID: 37243284 DOI: 10.3390/v15051198] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2023] [Revised: 05/14/2023] [Accepted: 05/18/2023] [Indexed: 05/28/2023] Open
Abstract
Interferon-γ (IFN-γ) is a cytokine that plays an important role in immune regulation, especially in the activation and differentiation of immune cells. Toll-like receptors (TLRs) are a family of pattern-recognition receptors that sense structural motifs related to pathogens and alert immune cells to the invasion. Both IFN-γ and TLR agonists have been used as immunoadjuvants to augment the efficacy of cancer immunotherapies and vaccines against infectious diseases or psychoactive compounds. In this study, we aimed to explore the potential of IFN-γ and TLR agonists being applied simultaneously to boost dendritic cell activation and the subsequent antigen presentation. In brief, murine dendritic cells were treated with IFN-γ and/or the TLR agonists, polyinosinic-polycytidylic acid (poly I:C), or resiquimod (R848). Next, the dendritic cells were stained for an activation marker, a cluster of differentiation 86 (CD86), and the percentage of CD86-positive cells was measured by flow cytometry. From the cytometric analysis, IFN-γ efficiently stimulated a considerable number of the dendritic cells, while the TLR agonists by themselves could merely activate a few compared to the control. The combination of IFN-γ with poly I:C or R848 triggered a higher amount of dendritic cell activation than IFN-γ alone. For instance, 10 ng/mL IFN-γ with 100 µg/mL poly I:C achieved 59.1% cell activation, which was significantly higher than the 33.4% CD86-positive cells obtained by 10 ng/mL IFN-γ. These results suggested that IFN-γ and TLR agonists could be applied as complementary systems to promote dendritic cell activation and antigen presentation. There might be a synergy between the two classes of molecules, but further investigation is warranted to ascertain the interaction of their promotive activities.
Collapse
Affiliation(s)
- Yuanzhi Bian
- Department of Biological Systems Engineering, College of Agriculture and Life Sciences & College of Engineering, Virginia Tech, Blacksburg, VA 24061, USA
| | - Debra L Walter
- Department of Biological Systems Engineering, College of Agriculture and Life Sciences & College of Engineering, Virginia Tech, Blacksburg, VA 24061, USA
| | - Chenming Zhang
- Department of Biological Systems Engineering, College of Agriculture and Life Sciences & College of Engineering, Virginia Tech, Blacksburg, VA 24061, USA
| |
Collapse
|
|
8
|
Jiang Y, Xia L, Gao S, Li N, Yu S, Zhou J. Engineering Saccharomyces cerevisiae for enhanced (-)-α-bisabolol production. Synth Syst Biotechnol 2023; 8:187-195. [PMID: 36824492 PMCID: PMC9941373 DOI: 10.1016/j.synbio.2023.01.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2022] [Revised: 01/15/2023] [Accepted: 01/17/2023] [Indexed: 01/21/2023] Open
Abstract
(-)-α-Bisabolol is naturally occurring in many plants and has great potential in health products and pharmaceuticals. However, the current extraction method from natural plants is unsustainable and cannot fulfil the increasing requirement. This study aimed to develop a sustainable strategy to enhance the biosynthesis of (-)-α-bisabolol by metabolic engineering. By introducing the heterologous gene MrBBS and weakening the competitive pathway gene ERG9, a de novo (-)-α-bisabolol biosynthesis strain was constructed that could produce 221.96 mg/L (-)-α-bisabolol. Two key genes for (-)-α-bisabolol biosynthesis, ERG20 and MrBBS, were fused by a flexible linker (GGGS)3 under the GAL7 promoter control, and the titer was increased by 2.9-fold. Optimization of the mevalonic acid pathway and multi-copy integration further increased (-)-α-bisabolol production. To promote product efflux, overexpression of PDR15 led to an increase in extracellular production. Combined with the optimal strategy, (-)-α-bisabolol production in a 5 L bioreactor reached 7.02 g/L, which is the highest titer reported in yeast to date. This work provides a reference for the efficient production of (-)-α-bisabolol in yeast.
Collapse
Affiliation(s)
- Yinkun Jiang
- Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China,Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China
| | - Lu Xia
- Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China,Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China
| | - Song Gao
- Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China,Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China
| | - Ning Li
- Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China,Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China
| | - Shiqin Yu
- Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China,Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China
| | - Jingwen Zhou
- Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China,Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China,Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, China,Jiangsu Province Engineering Research Center of Food Synthetic Biotechnology, Jiangnan University, Wuxi, 214122, China,Corresponding author. Science Center for Future Foods, Jiangnan University, 1800 Lihu Rd, Wuxi, Jiangsu, 214122, China.
| |
Collapse
|
|
9
|
Designing a novel E2-IFN-γ fusion protein against CSFV by immunoinformatics and structural vaccinology approaches. Appl Microbiol Biotechnol 2022; 106:3611-3623. [PMID: 35524776 DOI: 10.1007/s00253-022-11919-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2022] [Revised: 04/01/2022] [Accepted: 04/08/2022] [Indexed: 11/02/2022]
Abstract
Subunit vaccines with high purity and safety are gradually becoming a main trend in vaccinology. However, adjuvants such as interferon-gamma (IFN-γ) are required to enhance immune responses of subunit vaccines due to their poor immunogenicity. The conjugation of antigen with adjuvant can induce more potent immune responses compared to the mixture of antigen and adjuvant. At the same time, the selection of linker, indispensable in the construction of the stable and bioactive fusion proteins, is complicated and time-consuming. The development of immunoinformatics and structural vaccinology approaches provides a means to address the abovementioned problem. Therefore, in this study, a E2-IFN-γ fusion protein with an optimal linker (E2-R2-PIFN) was designed by bioinformatics approaches to improve the immunogenicity of the classical swine fever virus (CSFV) E2 subunit vaccine. Moreover, the E2-R2-PIFN fusion protein was expressed in HEK293T cells and the biological effects of IFN-γ in E2-R2-PIFN were confirmed in vitro via Western blotting. Here, an alternative method is utilized to simplify the design and validation of the antigen-adjuvant fusion protein, providing a potential subunit vaccine candidate against CSFV. KEY POINTS: • An effective and simple workflow of antigen-adjuvant fusion protein design and validation was established by immunoinformatics and structural vaccinology. • A novel E2-IFN-γ fusion protein with an optimal linker was designed as a potential CSFV vaccine. • The bioactivity of the newly designed fusion protein was preliminarily validated through in vitro experiments.
Collapse
|
|
10
|
Gong W, Pan C, Cheng P, Wang J, Zhao G, Wu X. Peptide-Based Vaccines for Tuberculosis. Front Immunol 2022; 13:830497. [PMID: 35173740 PMCID: PMC8841753 DOI: 10.3389/fimmu.2022.830497] [Citation(s) in RCA: 34] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2021] [Accepted: 01/10/2022] [Indexed: 12/12/2022] Open
Abstract
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. As a result of the coronavirus disease 2019 (COVID-19) pandemic, the global TB mortality rate in 2020 is rising, making TB prevention and control more challenging. Vaccination has been considered the best approach to reduce the TB burden. Unfortunately, BCG, the only TB vaccine currently approved for use, offers some protection against childhood TB but is less effective in adults. Therefore, it is urgent to develop new TB vaccines that are more effective than BCG. Accumulating data indicated that peptides or epitopes play essential roles in bridging innate and adaptive immunity and triggering adaptive immunity. Furthermore, innovations in bioinformatics, immunoinformatics, synthetic technologies, new materials, and transgenic animal models have put wings on the research of peptide-based vaccines for TB. Hence, this review seeks to give an overview of current tools that can be used to design a peptide-based vaccine, the research status of peptide-based vaccines for TB, protein-based bacterial vaccine delivery systems, and animal models for the peptide-based vaccines. These explorations will provide approaches and strategies for developing safer and more effective peptide-based vaccines and contribute to achieving the WHO's End TB Strategy.
Collapse
Affiliation(s)
- Wenping Gong
- Tuberculosis Prevention and Control Key Laboratory/Beijing Key Laboratory of New Techniques of Tuberculosis Diagnosis and Treatment, Senior Department of Tuberculosis, The 8th Medical Center of PLA General Hospital, Beijing, China
| | - Chao Pan
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Biotechnology, Beijing, China
| | - Peng Cheng
- Tuberculosis Prevention and Control Key Laboratory/Beijing Key Laboratory of New Techniques of Tuberculosis Diagnosis and Treatment, Senior Department of Tuberculosis, The 8th Medical Center of PLA General Hospital, Beijing, China
- Hebei North University, Zhangjiakou City, China
| | - Jie Wang
- Tuberculosis Prevention and Control Key Laboratory/Beijing Key Laboratory of New Techniques of Tuberculosis Diagnosis and Treatment, Senior Department of Tuberculosis, The 8th Medical Center of PLA General Hospital, Beijing, China
| | - Guangyu Zhao
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China
| | - Xueqiong Wu
- Tuberculosis Prevention and Control Key Laboratory/Beijing Key Laboratory of New Techniques of Tuberculosis Diagnosis and Treatment, Senior Department of Tuberculosis, The 8th Medical Center of PLA General Hospital, Beijing, China
| |
Collapse
|
|
11
|
Effect and mechanism analysis of different linkers on efficient catalysis of subunit-fused nitrile hydratase. Int J Biol Macromol 2021; 181:444-451. [PMID: 33753198 DOI: 10.1016/j.ijbiomac.2021.03.103] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2021] [Revised: 03/16/2021] [Accepted: 03/17/2021] [Indexed: 11/21/2022]
Abstract
Protein fusion using a linker plays an important role for protein evolution. However, designing suitable linkers for protein evolution is yet challenging and under-explored. To further clarify the regular pattern of suitable type of linker for fusion proteins, one nitrile hydratase (NHase) was used as a target protein and subunit fusion strategy was carried out to improve its efficient catalysis. Subunit-fused variants with three different types of linkers were constructed and characterized. All variants exhibited higher stability than that of the wild type. The longer the linker was, the higher stability NHase showed, however, too long linker affected NHase activity and expression. Among the three types of linkers, the α-helical linker seemed more suitable for NHase than flexible or rigid linkers. Though it is not clear how the linkers affecting the activity, structure analysis indicated that the stability improvement is dependent on the additional salt bridge, H-bond, and the subunit interface area increasing due to the linker insertion, among which the additional salt bridge and interface area were more important factors. The results described here may be useful for redesigning other enzymes through subunit fusion.
Collapse
|
|
12
|
Knödler M, Buyel JF. Plant-made immunotoxin building blocks: A roadmap for producing therapeutic antibody-toxin fusions. Biotechnol Adv 2021; 47:107683. [PMID: 33373687 DOI: 10.1016/j.biotechadv.2020.107683] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Revised: 12/07/2020] [Accepted: 12/20/2020] [Indexed: 12/16/2022]
Abstract
Molecular farming in plants is an emerging platform for the production of pharmaceutical proteins, and host species such as tobacco are now becoming competitive with commercially established production hosts based on bacteria and mammalian cell lines. The range of recombinant therapeutic proteins produced in plants includes replacement enzymes, vaccines and monoclonal antibodies (mAbs). But plants can also be used to manufacture toxins, such as the mistletoe lectin viscumin, providing an opportunity to express active antibody-toxin fusion proteins, so-called recombinant immunotoxins (RITs). Mammalian production systems are currently used to produce antibody-drug conjugates (ADCs), which require the separate expression and purification of each component followed by a complex and hazardous coupling procedure. In contrast, RITs made in plants are expressed in a single step and could therefore reduce production and purification costs. The costs can be reduced further if subcellular compartments that accumulate large quantities of the stable protein are identified and optimal plant growth conditions are selected. In this review, we first provide an overview of the current state of RIT production in plants before discussing the three key components of RITs in detail. The specificity-defining domain (often an antibody) binds cancer cells, including solid tumors and hematological malignancies. The toxin provides the means to kill target cells. Toxins from different species with different modes of action can be used for this purpose. Finally, the linker spaces the two other components to ensure they adopt a stable, functional conformation, and may also promote toxin release inside the cell. Given the diversity of these components, we extract broad principles that can be used as recommendations for the development of effective RITs. Future research should focus on such proteins to exploit the advantages of plants as efficient production platforms for targeted anti-cancer therapeutics.
Collapse
Affiliation(s)
- M Knödler
- Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, Aachen 52074, Germany; Institute for Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany.
| | - J F Buyel
- Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, Aachen 52074, Germany; Institute for Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany.
| |
Collapse
|
|
13
|
Arai R. Design of helical linkers for fusion proteins and protein-based nanostructures. Methods Enzymol 2020; 647:209-230. [PMID: 33482989 DOI: 10.1016/bs.mie.2020.10.003] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The construction of recombinant fusion/chimeric proteins has been widely used for expression of soluble proteins and protein purification in a variety of fields of protein engineering and biotechnology. Fusion proteins are constructed by the linking of two protein domains with a peptide linker. The selection of a linker sequence is important for the construction of stable and bioactive fusion proteins. Empirically designed linkers are generally classified into two categories according to their structural features: flexible linkers and rigid linkers. Rigid linkers with the α-helix-forming sequences A(EAAAK)nA (n=2-5) were first designed about two decades ago to control the distance between two protein domains and to reduce their interference. Thereafter, the helical linkers have been applied to the construction of many fusion proteins to improve expression and bioactivity. In addition, the design of fusion proteins that self-assemble into supramolecular complexes is useful for nanobiotechnology and synthetic biology. A protein that forms a self-assembling oligomer was fused by a rigid helical linker to another protein that forms another self-assembling oligomer, and the fusion protein symmetrically self-assembled into a designed protein nanoparticle or nanomaterial. Moreover, to construct chain-like polymeric nanostructures, extender protein nanobuilding blocks were designed by tandemly fusing two dimeric de novo proteins with helical or flexible linkers. The linker design of fusion proteins can affect conformation and dynamics of self-assembling nanostructures. The present review and methods focus on useful helical linkers to construct bioactive fusion proteins and protein-based nanostructures.
Collapse
Affiliation(s)
- Ryoichi Arai
- Department of Biomolecular Innovation, Institute for Biomedical Sciences, Shinshu University, Ueda, Nagano, Japan; Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano, Japan.
| |
Collapse
|
|
14
|
The U3 and Env Proteins of Jaagsiekte Sheep Retrovirus and Enzootic Nasal Tumor Virus Both Contribute to Tissue Tropism. Viruses 2019; 11:v11111061. [PMID: 31739606 PMCID: PMC6893448 DOI: 10.3390/v11111061] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2019] [Revised: 11/12/2019] [Accepted: 11/12/2019] [Indexed: 12/11/2022] Open
Abstract
Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) are small-ruminant betaretroviruses that share high nucleotide and amino acid identity, utilize the same cellular receptor, hyaluronoglucosaminidase 2 (Hyal2) for entry, and transform tissues with their envelope (Env) glycoprotein; yet, they target discrete regions of the respiratory tract—the lung and nose, respectively. This distinct tissue selectivity makes them ideal tools with which to study the pathogenesis of betaretroviruses. To uncover the genetic determinants of tropism, we constructed JSRV–ENTV chimeric viruses and produced lentivectors pseudotyped with the Env proteins from JSRV (Jenv) and ENTV (Eenv). Through the transduction and infection of lung and nasal turbinate tissue slices, we observed that Hyal2 expression levels strongly influence ENTV entry, but that the long terminal repeat (LTR) promoters of these viruses are likely responsible for tissue-specificity. Furthermore, we show evidence of ENTV Env expression in chondrocytes within ENTV-infected nasal turbinate tissue, where Hyal2 is highly expressed. Our work suggests that the unique tissue tropism of JSRV and ENTV stems from the combined effort of the envelope glycoprotein-receptor interactions and the LTR and provides new insight into the pathogenesis of ENTV.
Collapse
|
|
15
|
Won Y, Pagar AD, Patil MD, Dawson PE, Yun H. Recent Advances in Enzyme Engineering through Incorporation of Unnatural Amino Acids. BIOTECHNOL BIOPROC E 2019. [DOI: 10.1007/s12257-019-0163-x] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
|
|
16
|
Development of heme protein based oxygen sensing indicators. Sci Rep 2018; 8:11849. [PMID: 30087408 PMCID: PMC6081431 DOI: 10.1038/s41598-018-30329-5] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2018] [Accepted: 07/27/2018] [Indexed: 12/16/2022] Open
Abstract
Oxygen is essential for aerobic life and is required for various oxygen-dependent biochemical reactions. In addition, oxygen plays important roles in multiple intracellular signaling pathways. Thus, to investigate oxygen homeostasis in living cells, we developed a genetically encoded oxygen sensor protein using the oxygen sensor domain of bacterial phosphodiesterase direct oxygen sensor protein (DosP), which was connected to yellow fluorescence protein (YFP) using an optimized antiparallel coiled-coil linker. The resulting ANA-Y (Anaerobic/aerobic sensing yellow fluorescence protein) was highly sensitive to oxygen and had a half saturation concentration of 18 μM. The ANA-Y reacts with dissolved oxygen within 10 s and the resulting increases in fluorescence are reversed with decreases in oxygen concentrations. This sensitivity of the ANA-Y enabled direct determinations of initial photosynthetic oxygen production by cyanobacteria. ANA-Y exhibits reversible fluorescence change of donor YFP following reversible absorbance change of acceptor DosH, and the operating mechanism of this ANA-Y could be used to develop various protein sensor probes for intracellular signaling molecules using natural sensor proteins.
Collapse
|
|
17
|
Liang MH, Zhou SS, Jiang JG. Construction, expression and characterization of a fusion protein HBscFv-IFNγ in Komagatella (Pichia) pastoris X33. Enzyme Microb Technol 2017; 102:74-81. [PMID: 28465064 DOI: 10.1016/j.enzmictec.2017.04.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2016] [Revised: 03/25/2017] [Accepted: 04/02/2017] [Indexed: 02/08/2023]
Abstract
HBscFv-IFNγ, a fusion protein constructed by fusing γ-interferon (IFNγ) with an antibody fragment HBscFv for the purpose of targeted delivery of the cytokine IFNγ, was designed in order to enhance its therapeutic efficacy through increasing its hepatoma localization. HBscFv and IFNγ were connected into HBscFv-IFNγ by the linker (Gly4Ser)3, and then the multicopy recombinant plasmids pPICZαA/(HBscFv-IFNγ)1,2,4 were constructed and transformed into Komagatella (Pichia) pastoris X33. The engineering strain X4, which had much higher copy number and could secretively express HBscFv-IFNγ, was screened from transformed X33 by qPCR. Results from SDS-PAGE, Western blotting and ELISA indicated that HBscFv-IFNγ displayed an excellent immunoreaction against HBsAg. The culture supernatant of X4 was purified by 14F7 affinity chromatography to obtain the fusion protein HBscFv-IFNγ in a purity of 95-98%. The HBscFv-IFNγ was able to bind 27.9% HBsAg in the serum of HBV transgenic mice, showing that the antibody of HBscFv-IFNγ has high binding affinity against HBsAg. The expressing of the recombinant HBscFv-IFNγ in P. pastoris provides a promising and inexpensive diagnostic reagent for preventing HBV infection.
Collapse
Affiliation(s)
- Ming-Hua Liang
- School of Biological Science & Engineering, South China University of Technology, Guangzhou, 510006, China; College of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China
| | - Shi-Shui Zhou
- School of Biological Science & Engineering, South China University of Technology, Guangzhou, 510006, China.
| | - Jian-Guo Jiang
- College of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China.
| |
Collapse
|
|
18
|
Cook JM, Charlesworth A. Insertion of inter-domain linkers improves expression and bioactivity of Zygote arrest (Zar) fusion proteins. Protein Eng Des Sel 2017; 30:313-319. [PMID: 28130327 DOI: 10.1093/protein/gzx002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2016] [Accepted: 01/10/2017] [Indexed: 11/12/2022] Open
Abstract
Developmentally important proteins that are crucial for fertilization and embryogenesis are synthesized through highly regulated translation of maternal mRNA. The Zygote arrest proteins, Zar1 and Zar2, are crucial for embryogenesis and have been implicated in binding mRNA and repressing mRNA translation. To investigate Zar1 and Zar2, the full-length proteins had been fused to glutathione-S-transferase (GST) or MS2 protein tags with minimal inter-domain linkers derived from multiple cloning sites; however, these fusion proteins expressed poorly and/or lacked robust function. Here, we tested the effect of inserting additional linkers between the fusion domains. Three linkers were tested, each 17 amino acids long with different physical and chemical properties: flexible hydrophilic, rigid extended or rigid helical. In the presence of any of the three linkers, GST-Zar1 and GST-Zar2 had fewer breakdown products. Moreover, in the presence of any of the linkers, MS2-Zar1 was expressed to higher levels, and in dual luciferase tethered assays, both MS2-Zar1 and MS2-Zar2 repressed luciferase translation to a greater extent. These data suggest that for Zar fusion proteins, increasing the length of linkers, regardless of their physical or chemical properties, improves stability, expression and bioactivity.
Collapse
Affiliation(s)
- Jonathan M Cook
- Department of Integrative Biology, University of Colorado Denver, Denver, CO 80204, USA
| | - Amanda Charlesworth
- Department of Integrative Biology, University of Colorado Denver, Denver, CO 80204, USA
| |
Collapse
|
|
19
|
Shafiee F, Rabbani M, Jahanian-Najafabadi A. Production and evaluation of cytotoxic effects of DT386-BR2 fusion protein as a novel anti-cancer agent. J Microbiol Methods 2016; 130:100-105. [DOI: 10.1016/j.mimet.2016.09.004] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2016] [Revised: 08/21/2016] [Accepted: 09/04/2016] [Indexed: 10/21/2022]
|
|
20
|
Designing cell-targeted therapeutic proteins reveals the interplay between domain connectivity and cell binding. Biophys J 2015; 107:2456-66. [PMID: 25418314 DOI: 10.1016/j.bpj.2014.10.007] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2014] [Revised: 08/11/2014] [Accepted: 10/03/2014] [Indexed: 01/22/2023] Open
Abstract
The therapeutic efficacy of cytokines is often hampered by severe side effects due to their undesired binding to healthy cells. One strategy for overcoming this obstacle is to tether cytokines to antibodies or antibody fragments for targeted cell delivery. However, how to modulate the geometric configuration and relative binding affinity of the two domains for optimal activity remains an outstanding question. As a result, many antibody-cytokine complexes do not achieve the desired level of cell-targeted binding and activity. Here, we address these design issues by developing a computational model to simulate the dynamics and binding kinetics of natural and engineered fusion proteins such as antibody-cytokine complexes. To verify the model, we developed a modular system in which an antibody fragment and a cytokine are conjugated via a DNA linker that allows for programmable linker geometry and protein spatial configuration. By assembling and testing several anti-CD20 antibody fragment-interferon ? complexes, we showed that varying the linker length and cytokine binding affinity controlled the magnitude of cell-targeted signaling activation in a manner that agreed with the model predictions, which were expressed as dose-signaling response curves. The simulation results also revealed that there is a range of cytokine binding affinities that would achieve optimal therapeutic efficacy. This rapid prototyping platform will facilitate the rational design of antibody-cytokine complexes for improved therapeutic outcomes.
Collapse
|
|
21
|
Wang Y, Lu Y, Liu D, Wei Y, Guo L, Wu H, Huang L, Liu J, Liu C. Enhanced Th1-biased immune efficacy of porcine circovirus type 2 Cap-protein-based subunit vaccine when coadministered with recombinant porcine IL-2 or GM-CSF in mice. Appl Microbiol Biotechnol 2014; 99:1155-63. [PMID: 25487886 DOI: 10.1007/s00253-014-6167-8] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2014] [Revised: 10/13/2014] [Accepted: 10/14/2014] [Indexed: 02/05/2023]
Abstract
Porcine circovirus type 2 (PCV2) capsid (Cap) protein is the primary protective antigen responsible for inducing PCV2-specific protective immunity, so it is a desirable target for the development of recombinant subunit vaccines to prevent PCV2-associated diseases. Interleukin 2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), used as immune adjuvants, have been shown to enhance the immunogenicity of certain antigens or vaccines in various experimental models. In this study, five different subunit vaccines (the PCV2-Cap, Cap-PoIL-2, PCV2-Cap + PoIL-2, Cap-PoGM-CSF, and PCV2-Cap + PoGM-CSF vaccines) were prepared based on baculovirus-expressed recombinant proteins. The immunogenicity of these vaccines was evaluated to identify the immunoenhancement by PoIL-2 and PoGM-CSF of the Cap-protein-based PCV2 subunit vaccine in mice. The PCV2-Cap + PoIL-2, Cap-PoGM-CSF, PCV2-Cap + PoGM-CSF, and PCV2-Cap vaccines induced significantly higher levels of PCV2-specific antibodies than the Cap-PoIL-2 vaccine, whereas there was no apparent difference between these four vaccines. Our results indicate that neither PoIL-2 nor PoGM-CSF had effect on the enhancement of the humoral immunity induced by the PCV2-Cap vaccine. Furthermore, the PCV2-Cap + PoIL-2, Cap-PoGM-CSF, and PCV2-Cap + PoGM-CSF vaccines elicited stronger lymphocyte proliferative responses and greater IL-2 and interferon gamma (IFN-γ) secretion. This suggests that PoIL-2 and PoGM-CSF substantially augmented the Th1-biased immune response to the PCV2-Cap vaccine. Following challenge, the viral loads in the lungs of the PCV2-Cap + PoIL-2-, Cap-PoGM-CSF-, and PCV2-Cap + PoGM-CSF-treated groups were dramatically lower than those in the Cap-PoIL-2- and PCV2-Cap-treated groups, indicating that the three vaccines induced stronger protective effects against challenge. These findings show that PoIL-2 and PoGM-CSF essentially enhanced the Th1-biased protective efficacy of the PCV2-Cap vaccine when coadministered with the protein or delivered as Cap-PoGM-CSF, and that the "antigen-cytokine"- or "antigen + cytokine"-based vaccines that we report here provide new basis for the development of safer and more effective vaccines.
Collapse
Affiliation(s)
- Yiping Wang
- Division of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150001, China
| | | | | | | | | | | | | | | | | |
Collapse
|
|
22
|
Isik G, Sliepen K, van Montfort T, Sanders RW. Enhanced immunogenicity of HIV-1 envelope gp140 proteins fused to APRIL. PLoS One 2014; 9:e107683. [PMID: 25247707 PMCID: PMC4172553 DOI: 10.1371/journal.pone.0107683] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2014] [Accepted: 08/17/2014] [Indexed: 11/24/2022] Open
Abstract
Current HIV-1 vaccines based on the HIV-1 envelope glycoprotein spike (Env), the only relevant target for broadly neutralizing antibodies, are unable to induce protective immunity. Env immunogenicity can be enhanced by fusion to costimulatory molecules involved in B cell activation, such as APRIL and CD40L. Here, we found that Env-APRIL signaled through the two receptors, BCMA and TACI. In rabbits, Env-APRIL induced significantly higher antibody responses against Env compared to unconjugated Env, while the antibody responses against the APRIL component were negligible. To extend this finding, we tested Env-APRIL in mice and found minimal antibody responses against APRIL. Furthermore, Env-CD40L did not induce significant anti-CD40L responses. Thus, in contrast to the 4-helix cytokines IL-21 and GM-CSF, the TNF-superfamily members CD40L and APRIL induced negligible autoantibodies. This study confirms and extends previous work and shows that fusion of Env-based immunogens to APRIL can improve Env immunogenicity and might help in designing HIV vaccines that induce protective humoral immunity.
Collapse
Affiliation(s)
- Gözde Isik
- Laboratory of Experimental Virology, Department of Medical Microbiology Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
| | - Kwinten Sliepen
- Laboratory of Experimental Virology, Department of Medical Microbiology Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
| | - Thijs van Montfort
- Laboratory of Experimental Virology, Department of Medical Microbiology Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
| | - Rogier W. Sanders
- Laboratory of Experimental Virology, Department of Medical Microbiology Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands
- Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, United States of America
- * E-mail:
| |
Collapse
|
|
23
|
Edwards W, Fung-Leung WP, Huang C, Chi E, Wu N, Liu Y, Maher MP, Bonesteel R, Connor J, Fellows R, Garcia E, Lee J, Lu L, Ngo K, Scott B, Zhou H, Swanson RV, Wickenden AD. Targeting the ion channel Kv1.3 with scorpion venom peptides engineered for potency, selectivity, and half-life. J Biol Chem 2014; 289:22704-22714. [PMID: 24939846 DOI: 10.1074/jbc.m114.568642] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Ion channels are an attractive class of drug targets, but progress in developing inhibitors for therapeutic use has been limited largely due to challenges in identifying subtype selective small molecules. Animal venoms provide an alternative source of ion channel modulators, and the venoms of several species, such as scorpions, spiders and snails, are known to be rich sources of ion channel modulating peptides. Importantly, these peptides often bind to hyper-variable extracellular loops, creating the potential for subtype selectivity rarely achieved with small molecules. We have engineered scorpion venom peptides and incorporated them in fusion proteins to generate highly potent and selective Kv1.3 inhibitors with long in vivo half-lives. Kv1.3 has been reported to play a role in human T cell activation, and therefore, these Kv1.3 inhibitor fusion proteins may have potential for the treatment of autoimmune diseases. Our results support an emerging approach to generating subtype selective therapeutic ion channel inhibitors.
Collapse
Affiliation(s)
- Wilson Edwards
- Janssen Research and Development, LLC, San Diego, California 92121.
| | | | - Chichi Huang
- Janssen Research and Development, LLC, San Diego, California 92121
| | - Ellen Chi
- Janssen Research and Development, LLC, San Diego, California 92121
| | - Nancy Wu
- Janssen Research and Development, LLC, San Diego, California 92121
| | - Yi Liu
- Janssen Research and Development, LLC, San Diego, California 92121
| | - Michael P Maher
- Janssen Research and Development, LLC, San Diego, California 92121
| | | | - Judith Connor
- Janssen Research and Development, LLC, San Diego, California 92121
| | - Ross Fellows
- Janssen Research and Development, LLC, San Diego, California 92121
| | - Elena Garcia
- Janssen Research and Development, LLC, San Diego, California 92121
| | - Jerry Lee
- Janssen Research and Development, LLC, San Diego, California 92121
| | - Lu Lu
- Janssen Research and Development, LLC, San Diego, California 92121
| | - Karen Ngo
- Janssen Research and Development, LLC, San Diego, California 92121
| | - Brian Scott
- Janssen Research and Development, LLC, San Diego, California 92121
| | - Hong Zhou
- Janssen Research and Development, LLC, San Diego, California 92121
| | - Ronald V Swanson
- Janssen Research and Development, LLC, San Diego, California 92121
| | - Alan D Wickenden
- Janssen Research and Development, LLC, San Diego, California 92121
| |
Collapse
|
|
24
|
Isik G, van Montfort T, Chung NPY, Moore JP, Sanders RW. Autoantibodies induced by chimeric cytokine-HIV envelope glycoprotein immunogens. THE JOURNAL OF IMMUNOLOGY 2014; 192:4628-35. [PMID: 24729614 DOI: 10.4049/jimmunol.1303401] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Cytokines are often used as adjuvants to increase the immunogenicity of vaccines because they can improve the immune response and/or direct it into a desired direction. As an alternative to codelivering Ags and cytokines separately, they can be fused into a composite protein, with the advantage that both moieties act on the same immune cells. The HIV-1 envelope glycoprotein (Env) spike, located on the outside of virus particles and the only relevant protein for the induction of neutralizing Abs, is poorly immunogenic. The induction of anti-Env Abs can be improved by coupling Env proteins to costimulatory molecules such as a proliferation inducing ligand (APRIL). In this study, we evaluated the immunogenicity of chimeric molecules containing uncleaved Env gp140 fused to the species-matched cytokines IL-21 or GM-CSF in rabbits and mice. Each cytokine was either fused to the C terminus of Env or embedded within Env at the position of the variable loops 1 and 2. The cytokine components of the chimeric Env-GM-CSF and Env-IL-21 molecules were functional in vitro, but none of the Env-cytokine fusion proteins resulted in improved Ab responses in vivo. Both the Env-GM-CSF and the Env-IL-21 molecules induced strong anticytokine Ab responses in both test species. These autoimmune responses were independent of the location of the cytokine in the chimeric Env molecules in that they were induced by cytokines inserted within the variable loops 1 and 2 of Env or fused to its C terminus. The induction of undesired autoimmune responses should be considered when using cytokines as costimulatory molecules in fusion proteins.
Collapse
Affiliation(s)
- Gözde Isik
- Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands
| | | | | | | | | |
Collapse
|
|
25
|
Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev 2013; 65:1357-69. [PMID: 23026637 DOI: 10.1016/j.addr.2012.09.039] [Citation(s) in RCA: 1309] [Impact Index Per Article: 109.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2012] [Revised: 09/05/2012] [Accepted: 09/20/2012] [Indexed: 01/21/2023]
Abstract
As an indispensable component of recombinant fusion proteins, linkers have shown increasing importance in the construction of stable, bioactive fusion proteins. This review covers the current knowledge of fusion protein linkers and summarizes examples for their design and application. The general properties of linkers derived from naturally-occurring multi-domain proteins can be considered as the foundation in linker design. Empirical linkers designed by researchers are generally classified into 3 categories according to their structures: flexible linkers, rigid linkers, and in vivo cleavable linkers. Besides the basic role in linking the functional domains together (as in flexible and rigid linkers) or releasing the free functional domain in vivo (as in in vivo cleavable linkers), linkers may offer many other advantages for the production of fusion proteins, such as improving biological activity, increasing expression yield, and achieving desirable pharmacokinetic profiles.
Collapse
Affiliation(s)
- Xiaoying Chen
- Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA
| | | | | |
Collapse
|
|
26
|
Isik G, van Montfort T, Boot M, Cobos Jiménez V, Kootstra NA, Sanders RW. Chimeric HIV-1 envelope glycoproteins with potent intrinsic granulocyte-macrophage colony-stimulating factor (GM-CSF) activity. PLoS One 2013; 8:e60126. [PMID: 23565193 PMCID: PMC3615126 DOI: 10.1371/journal.pone.0060126] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2013] [Accepted: 02/21/2013] [Indexed: 11/18/2022] Open
Abstract
HIV-1 acquisition can be prevented by broadly neutralizing antibodies (BrNAbs) that target the envelope glycoprotein complex (Env). An ideal vaccine should therefore be able to induce BrNAbs that can provide immunity over a prolonged period of time, but the low intrinsic immunogenicity of HIV-1 Env makes the elicitation of such BrNAbs challenging. Co-stimulatory molecules can increase the immunogenicity of Env and we have engineered a soluble chimeric Env trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF) domain. This chimeric molecule induced enhanced B and helper T cell responses in mice compared to Env without GM-CSF. We studied whether we could optimize the activity of the embedded GM-CSF as well as the antigenic structure of the Env component of the chimeric molecule. We assessed the effect of truncating GM-CSF, removing glycosylation-sites in GM-CSF, and adjusting the linker length between GM-CSF and Env. One of our designed Env(GM-CSF) chimeras improved GM-CSF-dependent cell proliferation by 6-fold, reaching the same activity as soluble recombinant GM-CSF. In addition, we incorporated GM-CSF into a cleavable Env trimer and found that insertion of GM-CSF did not compromise Env cleavage, while Env cleavage did not compromise GM-CSF activity. Importantly, these optimized Env(GM-CSF) proteins were able to differentiate human monocytes into cells with a macrophage-like phenotype. Chimeric Env(GM-CSF) should be useful for improving humoral immunity against HIV-1 and these studies should inform the design of other chimeric proteins.
Collapse
Affiliation(s)
- Gözde Isik
- Department of Medical Microbiology, Laboratory of Experimental Virology, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Thijs van Montfort
- Department of Medical Microbiology, Laboratory of Experimental Virology, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Maikel Boot
- Department of Medical Microbiology, Laboratory of Experimental Virology, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Viviana Cobos Jiménez
- Department of Experimental Immunology, Sanquin Research, Landsteiner Laboratory, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Neeltje A. Kootstra
- Department of Experimental Immunology, Sanquin Research, Landsteiner Laboratory, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
| | - Rogier W. Sanders
- Department of Medical Microbiology, Laboratory of Experimental Virology, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
- Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, United States of America
| |
Collapse
|
|
27
|
Wang YP, Liu D, Guo LJ, Tang QH, Wei YW, Wu HL, Liu JB, Li SB, Huang LP, Liu CM. Enhanced protective immune response to PCV2 subunit vaccine by co-administration of recombinant porcine IFN-γ in mice. Vaccine 2012; 31:833-8. [PMID: 23219694 DOI: 10.1016/j.vaccine.2012.11.062] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2012] [Revised: 11/15/2012] [Accepted: 11/21/2012] [Indexed: 11/24/2022]
Abstract
The capsid (Cap) protein of PCV2 is the major immunogenic protein that is crucial to induce PCV2-specific neutralizing antibodies and protective immunity; thus, it is a suitable target antigen for the research and development of genetically engineered vaccines against PCV2 infection. IFN-γ has exhibited potential efficacy as an immune adjuvant that enhances the immunogenicity of certain vaccines in experimental animal models. In this study, three recombinant proteins: PCV2-Cap protein, porcine IFN-γ (PoIFN-γ), and the fusion protein (Cap-PoIFN-γ) of PCV2-Cap protein and PoIFN-γ were respectively expressed in the baculovirus system, and analyzed by Western blot and indirect ELISA. Additionally, we evaluated the enhancement of the protective immune response to the Cap protein-based PCV2 subunit vaccine elicited by co-administration of PoIFN-γ in mice. Vaccination of mice with the PCV2-Cap+PoIFN-γ vaccine elicited significantly higher levels of PCV2-specific IPMA antibodies, neutralizing antibodies, and lymphocyte proliferative responses compared to the Cap-PoIFN-γ vaccine, the PCV2-Cap vaccine, and LG-strain. Following virulent PCV2 challenge, no viraemia was detected in all immunized groups, and the viral loads in lungs of the PCV2-Cap+PoIFN-γ group were significantly lower compared to the Cap-PoIFN-γ group, the LG-strain group, and the mock group, but slightly lower compared to the PCV2-Cap group. These findings suggested that PoIFN-γ substantially enhanced the protective immune response to the Cap protein-based PCV2 subunit vaccine, and that the PCV2-Cap+PoIFN-γ subunit vaccine potentially serves as an attractive candidate vaccine for the prevention and control of PCV2-associated diseases.
Collapse
Affiliation(s)
- Yi-Ping Wang
- Division of Swine Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
28
|
Tomchuck SL, Norton EB, Garry RF, Bunnell BA, Morris CA, Freytag LC, Clements JD. Mesenchymal stem cells as a novel vaccine platform. Front Cell Infect Microbiol 2012; 2:140. [PMID: 23162801 PMCID: PMC3499769 DOI: 10.3389/fcimb.2012.00140] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2012] [Accepted: 10/22/2012] [Indexed: 01/14/2023] Open
Abstract
Vaccines are the most efficient and cost-effective means of preventing infectious disease. However, traditional vaccine approaches have thus far failed to provide protection against human immunodeficiency virus (HIV), tuberculosis, malaria, and many other diseases. New approaches to vaccine development are needed to address some of these intractable problems. In this report, we review the literature identifying stimulatory effects of mesenchymal stem cells (MSC) on immune responses and explore the potential for MSC as a novel, universal vaccination platform. MSC are unique bone marrow-derived multipotent progenitor cells that are presently being exploited as gene therapy vectors for a variety of conditions, including cancer and autoimmune diseases. Although MSC are predominantly known for anti-inflammatory properties during allogeneic MSC transplant, there is evidence that MSC can actually promote adaptive immunity under certain settings. MSC have also demonstrated some success in anti-cancer therapeutic vaccines and anti-microbial prophylactic vaccines, as we report, for the first time, the ability of modified MSC to express and secrete a viral antigen that stimulates antigen-specific antibody production in vivo. We hypothesize that the unique properties of modified MSC may enable MSC to serve as an unconventional but innovative, vaccine platform. Such a platform would be capable of expressing hundreds of proteins, thereby generating a broad array of epitopes with correct post-translational processing, mimicking natural infection. By stimulating immunity to a combination of epitopes, it may be possible to develop prophylactic and even therapeutic vaccines to tackle major health problems including those of non-microbial and microbial origin, including cancer, or an infectious disease like HIV, where traditional vaccination approaches have failed.
Collapse
Affiliation(s)
- Suzanne L Tomchuck
- Department of Microbiology and Immunology, Tulane University School of Medicine New Orleans, LA, USA
| | | | | | | | | | | | | |
Collapse
|
|
29
|
Targeting HIV-1 envelope glycoprotein trimers to B cells by using APRIL improves antibody responses. J Virol 2011; 86:2488-500. [PMID: 22205734 DOI: 10.1128/jvi.06259-11] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
An HIV-1 vaccine remains elusive, in part because various factors limit the quantity and quality of the antibodies raised against the viral envelope glycoprotein complex (Env). We hypothesized that targeting Env vaccines directly to B cells, by fusing them to molecules that bind and activate these cells, would improve Env-specific antibody responses. Therefore, we fused trimeric Env gp140 to A PRoliferation-Inducing Ligand (APRIL), B-cell Activating Factor (BAFF), and CD40 Ligand (CD40L). The Env-APRIL, Env-BAFF, and Env-CD40L gp140 trimers all enhanced the expression of activation-induced cytidine deaminase (AID), the enzyme responsible for inducing somatic hypermutation, antibody affinity maturation, and antibody class switching. They also triggered IgM, IgG, and IgA secretion from human B cells in vitro. The Env-APRIL trimers induced higher anti-Env antibody responses in rabbits, including neutralizing antibodies against tier 1 viruses. The enhanced Env-specific responses were not associated with a general increase in total plasma antibody concentrations, indicating that the effect of APRIL was specific for Env. All the rabbit sera raised against gp140 trimers, irrespective of the presence of CD40L, BAFF, or APRIL, recognized trimeric Env efficiently, whereas sera raised against gp120 monomers did not. The levels of trimer-binding and virus-neutralizing antibodies were strongly correlated, suggesting that gp140 trimers are superior to gp120 monomers as immunogens. Targeting and activating B cells with a trimeric HIV-1 Env-APRIL fusion protein may therefore improve the induction of humoral immunity against HIV-1.
Collapse
|
|
30
|
Melchers M, Matthews K, de Vries RP, Eggink D, van Montfort T, Bontjer I, van de Sandt C, David K, Berkhout B, Moore JP, Sanders RW. A stabilized HIV-1 envelope glycoprotein trimer fused to CD40 ligand targets and activates dendritic cells. Retrovirology 2011; 8:48. [PMID: 21689404 PMCID: PMC3141652 DOI: 10.1186/1742-4690-8-48] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2010] [Accepted: 06/20/2011] [Indexed: 12/22/2022] Open
Abstract
Background One reason why subunit protein and DNA vaccines are often less immunogenic than live-attenuated and whole-inactivated virus vaccines is that they lack the co-stimulatory signals provided by various components of the more complex vaccines. The HIV-1 envelope glycoprotein complex (Env) is no exception to this rule. Other factors that limit the induction of neutralizing antibodies against HIV-1 lie in the structure and instability of Env. We have previously stabilized soluble trimeric mimics of Env by introducing a disulfide bond between gp120 and gp41 and adding a trimer stabilizing mutation in gp41 (SOSIP.R6 gp140). Results We further stabilized the SOSIP.R6 gp140 using a GCN4-based isoleucine zipper motif, creating SOSIP.R6-IZ gp140. In order to target SOSIP.R6-IZ to immune cells, including dendritic cells, while at the same time activating these cells, we fused SOSIP.R6-IZ to the active domain of CD40 ligand (CD40L), which may serve as a 'cis-adjuvant'. The Env component of the SOSIP.R6-IZ-CD40L fusion construct bound to CD4 and neutralizing antibodies, while the CD40L moiety interacted with CD40. Furthermore, the chimeric molecule was able to signal efficiently through CD40 and induce maturation of human dendritic cells. Dendritic cells secreted IL-6, IL-10 and IL-12 in response to stimulation by SOSIP.R6-IZ-CD40L and were able to activate naïve T cells. Conclusions Chimeric HIV-1 gp140 - CD40L trimers can target and activate dendritic cells. Targeting and activating immune cells using CD40L and other 'cis-adjuvants' may improve subunit protein vaccine immunogenicity for HIV-1 and other infectious diseases.
Collapse
Affiliation(s)
- Mark Melchers
- Laboratory of Experimental Virology, Department of Medical Microbiology Center for Infection and Immunity Amsterdam, Netherlands
| | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
31
|
Feng X, Su X, Wang F, Wei J, Wang F, Cao R, Zhou B, Mao X, Zheng Q, Chen P. Isolation and potential immunological characterization of TPSGLVY, a novel bursal septpeptide isolated from the bursa of Fabricius. Peptides 2010; 31:1562-8. [PMID: 20493914 DOI: 10.1016/j.peptides.2010.05.007] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/28/2010] [Revised: 05/12/2010] [Accepted: 05/12/2010] [Indexed: 11/21/2022]
Abstract
The bursa of Fabricius is central immune organ unique to birds, and the extract is immunocompetent in stimulating B cell differentiation and enhancing antibody production. However, except for bursin, the active peptides from the bursa of Fabricius are little reported. In the paper, a novel bursal septpeptide (BSP-II) with the amino acids sequence of TPSGLVY was identified and similar to the MGC53864 protein of Gallus gallus. We investigated the effects of BSP-II on the immune response in terms of the antibodies titers (IgG1 and IgG2alpha), the levels of interferon-gamma and interleukin-4 cytokines, spleen cell lymphocyte proliferation, and the T-lymphocyte subtype composition. It was noteworthy that BSP-II potentiates the Th1 and Th2-type immune responses in dose-dependent manner. BSP-II had specific enhancing effects on the hybridoma SP2/0 cell proliferation at two different serum concentrations (20% and 5%), but had no connection with the dose of BSP-II. The antibody secreting level of hybridoma SP2/0 cells rose in 5% and 20% serum when the concentrations of BSP-II increased. Also, BSP-II had effect on the viabilities of tumor cells (Hela and SP2/0). All the results indicated that BSP-II was able to significantly induce various immune responses and involved in the cell viability of different tumor cell lines. Our observations implied that BSP-II might be a novel biological active factor from the bursa of Fabricius with immunomodulatory activities.
Collapse
Affiliation(s)
- Xiuli Feng
- Division of Key Lab of Animal Disease Diagnosis and Immunology of China's Department of Agriculture, Nanjing Agricultural University, Nanjing, 210095, China
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
32
|
Abstract
The activity of several potent adjuvants, including incomplete Freund's adjuvant, CpG oligodeoxynucleotides, and alum, has been shown to be due at least in part to the induction of cytokines, including type I interferons (IFNs), IFN-gamma, interleukin-2 (IL-2), and IL-12, that play key roles in the regulation of innate and adaptive immunity. The relatively short half-life of recombinant homologues of cytokines has limited their use as vaccine adjuvants. These difficulties have been overcome by encapsulation into liposomes and the use of cytokine expression vectors co-administered with DNA vaccines. Although a number of cytokines including IFN-alpha, IFN-gamma, IL-2, IL-12, IL-15, IL-18, IL-21, GM-CSF, and Flt-3 ligand have been shown to potentiate the immune response to vaccination in various experimental models, the full potential of cytokines as vaccine adjuvants remains to be established.
Collapse
|
|
33
|
Larsen M, Jensen KB, Christensen PA, Suarez E, Paris D, Sanz L, Ravn P, Sauce D, Saas P, Goletz S, Alvarez-Vallina L, Kristensen P. Functionally fused antibodies--a novel adjuvant fusion system. J Immunol Methods 2008; 339:220-7. [PMID: 18854189 DOI: 10.1016/j.jim.2008.09.015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2008] [Revised: 09/19/2008] [Accepted: 09/22/2008] [Indexed: 11/18/2022]
Abstract
Antibodies capable of recognizing key molecular targets isolated e.g. by phage display technology have been used in the pursuit of new and improved therapies for prevalent human diseases. These approaches often take advantage of non-immunogenic antibody fragments to achieve specific toxin-, radioactivity- or effector-domain delivery. There is now a growing interest in using anti-idiotypic antibodies or other antigen mimics to induce potent immune responses against antigen structures in question. We have earlier reported on the functional rescue of antibodies that are active when fused to the phage, but inactive as soluble protein [Jensen, K.B., Larsen, M., Pedersen, J.S., Christensen, P.A., Alvarez-Vallina, L., Goletz, S., Clark, B.F. and Kristensen, P. (2002) Functional improvement of antibody fragments using a novel phage coat protein III fusion system. Biochem. Biophys. Res. Commun. 298, 566-73.]. The rescue was accomplished by maintaining the fusion between the antibody fragment and portions of the filamentous bacteriophage coat protein 3, as present in the original antibody-displaying phage. In the present study, we have applied this system in an attempt to improve immunogenicity of anti-idiotypic antibodies isolated by phage display. Here we demonstrate that by preserving linkage between phage antibody and the N-terminal domain of phage coat protein 3, we induce multimerization of the antibody fragments, and improve their immunogenicity. This immunization approach allows induction of anti-idiotypic antibodies in mice, and facilitates the use of antibodies that are non-functional as non-fused soluble protein.
Collapse
Affiliation(s)
- Martin Larsen
- Department of Molecular Biology, University of Aarhus, Denmark
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
34
|
Abstract
Interferons, IFNs, are among the most widely studied and clinically used biopharmaceuticals. Despite their invaluable therapeutic roles, the widespread use of IFNs suffers from some inherent limitations, mainly their relatively short circulation lifespan and their unwanted effects on some non-target tissues. Therefore, both these constraints have become the central focus points for the research efforts on the development of a variety of novel delivery systems for these therapeutic agents with the ultimate goal of improving their therapeutic end-points. Generally, the delivery systems currently under investigation for IFNs can be classified as particulate delivery systems, including micro- and nano-particles, liposomes, minipellets, cellular carriers, and non-particulate delivery systems, including PEGylated IFNs, other chemically conjugated IFNs, immunoconjugated IFNs, and genetically conjugated IFNs. All these strategies and techniques have their own possibilities and limitations, which should be taken into account when considering their clinical application. In this article, currently studied delivery systems/techniques for IFN delivery have been reviewed extensively, with the main focus on the pharmacokinetic consequences of each procedure.
Collapse
Affiliation(s)
- Mehrdad Hamidi
- Department of Pharmaceutics, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.
| | | | | |
Collapse
|
|
35
|
Zhao HL, Yao XQ, Xue C, Wang Y, Xiong XH, Liu ZM. Increasing the homogeneity, stability and activity of human serum albumin and interferon-alpha2b fusion protein by linker engineering. Protein Expr Purif 2008; 61:73-7. [PMID: 18541441 DOI: 10.1016/j.pep.2008.04.013] [Citation(s) in RCA: 125] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2008] [Revised: 04/21/2008] [Accepted: 04/22/2008] [Indexed: 11/15/2022]
Abstract
Previous studies in our laboratory have shown that when the N-terminus of interferon-alpha2b (IFN-alpha2b) was directly fused of to the C-terminus of human serum albumin (HSA), the resultant fusion protein (HSA-IFN-alpha2b) was heterogeneous (migrated as doublets on non-reducing SDS-PAGE) and unstable (prone to form covalent aggregates). The heterogeneity and instability of HSA-IFN-alpha2b was ascribed to the structural disturbance between HSA and IFN-alpha2b. To alleviate such structural disturbance, linkers with different lengths (1, 2, 5, 10 amino acid residues) or different conformation (flexible linker (FL, GGGGS), rigid linker (RL, PAPAP) or helix-forming linker (HL, AEAAAKEAAAKA)) were inserted between HSA and IFN-alpha2b. It was demonstrated that linker with 5 amino acid residues was sufficient to separated HSA and IFN-alpha2b effectively, as fusion protein with this linker migrated as single band on non-reducing SDS-PAGE. The fusion proteins with FL, RL and HL linkers were purified to homogeneity with yields of 20%, while the recovery rate of HSA-IFN-alpha2b was only 10%. Accelerated thermal stress tests showed that in contrast to HSA-IFN-alpha2b, fusion proteins with FL, RL and HL linkers were free of aggregates after stored at 37 degrees C for 10 days. Stability tests also revealed that fusion proteins with FL, RL and HL linkers had different susceptibility to hydrolysis, with HSA-RL-IFN-alpha2b being the least susceptible to hydrolysis at pH 6 and 7. Activity assay revealed that the insertion of FL, RL and HL linkers increased the anti-viral activity of fusion protein by 39%, 68% and 115%, respectively.
Collapse
Affiliation(s)
- Hong Liang Zhao
- Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, People's Republic of China
| | | | | | | | | | | |
Collapse
|
|
36
|
Nimal S, Thomas MS, Heath AW. Fusion of antigen to Fas-ligand in a DNA vaccine enhances immunogenicity. Vaccine 2007; 25:2306-15. [PMID: 17239500 DOI: 10.1016/j.vaccine.2006.11.059] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2006] [Revised: 11/20/2006] [Accepted: 11/28/2006] [Indexed: 11/21/2022]
Abstract
DNA vaccines have considerable potential for the prophylaxis and therapy of a range of diseases, but their potential has not been realised largely due to poor immunogenicity. Fas ligand is a pro-apoptotic molecule, able to induce death of Fas expressing cells. We describe the construction of a DNA vaccine encoding a chimeric fusion between Fas ligand and a truncated version of HIV gp120 as a model antigen. The fusion DNA was used as a priming vaccine, along with boosting with recombinant gp120 protein. Priming with fusion protein DNA resulted in a powerful enhancement of immune responses to the protein boost, and, in the presence of aluminum phosphate, to a strong enhancement in T helper 2 type responses. Fas ligand delivered in a separate plasmid also had an adjuvant effect, although it was weaker than that delivered by the fusion protein.
Collapse
Affiliation(s)
- Sonali Nimal
- Unit of Infection and Immunity, University of Sheffield Medical School, Sheffield S10 2RX, UK
| | | | | |
Collapse
|
|
37
|
Gupta S, Leutenegger CM, Dean GA, Steckbeck JD, Cole KS, Sparger EE. Vaccination of cats with attenuated feline immunodeficiency virus proviral DNA vaccine expressing gamma interferon. J Virol 2006; 81:465-73. [PMID: 17079309 PMCID: PMC1797444 DOI: 10.1128/jvi.00815-06] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
A feline immunodeficiency virus (FIV) provirus with a vif gene deletion (FIVDelta vifATGgamma) that coexpresses feline gamma interferon (IFN-gamma) was tested as a proviral DNA vaccine to extend previous studies showing efficacy with an FIV-pPPRDelta vif DNA vaccine. Cats were vaccinated with either FIVDelta vifATGgamma or FIV-pPPRDelta vif proviral plasmid DNA or with both FIV-pPPRDelta vif DNA and a feline IFN-gamma expression plasmid (pCDNA-IFNgamma). A higher frequency of FIV-specific T-cell proliferation responses was observed in cats immunized with either FIVDelta vifATGgamma or FIV-pPPRDelta vif plus pCDNA-IFNgamma, while virus-specific cytotoxic-T-lymphocyte responses were comparable between vaccine groups. Antiviral antibodies were not observed postvaccination. Virus-specific cellular and humoral responses were similar between vaccine groups after challenge with a biological FIV isolate (FIV-PPR) at 13 weeks postimmunization. All vaccinated and unvaccinated cats were infected after FIV-PPR challenge and exhibited similar plasma virus loads. Accordingly, inclusion of plasmids containing IFN-gamma did not enhance the efficacy of FIV-pPPRDelta vif DNA immunization. Interestingly, the lack of protection associated with FIV-pPPRDelta vif DNA immunization contrasted with findings from a previous study and suggested that multiple factors, including timing of FIV-pPPRDelta vif inoculations and challenge, as well as route of challenge virus delivery, may significantly impact vaccine efficacy.
Collapse
Affiliation(s)
- Soumi Gupta
- Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA
| | | | | | | | | | | |
Collapse
|
|
38
|
Nimal S, McCormick AL, Thomas MS, Heath AW. An interferon gamma-gp120 fusion delivered as a DNA vaccine induces enhanced priming. Vaccine 2005; 23:3984-90. [PMID: 15917120 DOI: 10.1016/j.vaccine.2005.01.160] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2004] [Accepted: 01/18/2005] [Indexed: 10/25/2022]
Abstract
Nucleic acid vaccination has many potential advantages over traditional methods, but suffers from the fact that DNA vaccines tend to be relatively poorly immunogenic. Attempts to enhance DNA vaccine immunogenicity have included the addition of cytokine-encoding plasmids into the formulation, as well as the use of heterologous prime-boost regimes and the addition of conventional adjuvants, such as alum. We have previously shown that interferon gamma fusions have enhanced immunogenicity as recombinant protein vaccines. We have assessed here the immunogenicity of an interferon gamma-gp120 fusion delivered as a DNA vaccine, in the context of a prime-boost strategy and in the presence of absence of aluminium phosphate. Fusion of gp120 DNA to interferon gamma-encoding DNA resulted in strongly enhanced priming, especially of Th1 responses, including IgG2a responses to a protein boost.
Collapse
Affiliation(s)
- Sonali Nimal
- Infection and Immunity Department, F floor, Division of Genomic Medicine, University of Sheffield Medical School, Beech Hill Rd, Sheffield S10 2RX, UK
| | | | | | | |
Collapse
|
|
39
|
Yang YF, Yuan HY, Liu NS, Chen XL, Gao BY, Lu H, Li YY. Construction, expression and characterization of human interferon α2b-(G4S) n-thymosin α1 fusion proteins in Pichia pastoris. World J Gastroenterol 2005; 11:2597-602. [PMID: 15849818 PMCID: PMC4305750 DOI: 10.3748/wjg.v11.i17.2597] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [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: Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNα2b and Tα1 linked by different lengths of (G4S)n (n = 1-3) were constructed and expressed in Pichia pastoris.
METHODS: Using PCR and molecular clone techniques, the fusion genes of IFNα2b-(G4S)n-Tα1 (n = 1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S)n-Tα1 (n = 1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex™ 75 gel filtration and analyzed by SDS-PAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins.
RESULTS: DNA sequencing confirmed that the fusion genes of IFNα2b-(G4S)n-Tα1 (n = 1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1 (n = 1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex™ 75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFNα2b monoclonal antibody and Tα1 polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay.
CONCLUSION: The recombinant IFNα2b-(G4S)n-Tα1 (n = 1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNα2b and immunomodulatory activity of Tα1 in vitro. These results will be the basis for further evaluation of the fusion proteins’ function in vivo.
Collapse
Affiliation(s)
- You-Feng Yang
- State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China
| | | | | | | | | | | | | |
Collapse
|
|
40
|
Abstract
The conventional, currently available vaccines, though quite successful, suffer from a few shortcomings which hamper future vaccine development. We present herewith some of the new approaches that are presently being pursued, including (1) the development of recombinant, or genetically engineered, vaccines which are based either on the expression of the relevant protective antigen and its formulation into vaccine, or the production of live vaccines, where an appropriate live vector (virus or bacterium) presents the foreign antigen. (2) The development of naked DNA vaccines that include the gene(s) coding for the relevant protective antigen(s). (3) Peptide vaccines that include defined B cell and T cell epitopes, either in a chemically synthesized molecule or in a synthetic recombinant construct. The efficacy of such vaccines is usually dependent on adequate presentation and delivery, namely, carrier/adjuvant technology. (4) Therapeutic vaccines, based on all of the above approaches, may be applied for chronic or long-term infections, or for noninfectious diseases including autoimmune diseases, various neurological disorders, allergy and cancer.
Collapse
Affiliation(s)
- Ruth Arnon
- Department of Immunology, The Weizmann Institute, PO Box 26, 76100 Rehovot, Israel.
| | | |
Collapse
|
|
41
|
Hackney K, Cavanagh D, Kaiser P, Britton P. In vitro and in ovo expression of chicken gamma interferon by a defective RNA of avian coronavirus infectious bronchitis virus. J Virol 2003; 77:5694-702. [PMID: 12719562 PMCID: PMC154032 DOI: 10.1128/jvi.77.10.5694-5702.2003] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Coronavirus defective RNAs (D-RNAs) have been used for site-directed mutagenesis of coronavirus genomes and for expression of heterologous genes. D-RNA CD-61 derived from the avian coronavirus infectious bronchitis virus (IBV) was used as an RNA vector for the expression of chicken gamma interferon (chIFN-gamma). D-RNAs expressing chIFN-gamma were shown to be capable of rescue, replication, and packaging into virions in a helper virus-dependent system following electroporation of in vitro-derived T7 RNA transcripts into IBV-infected cells. Secreted chIFN-gamma, under the control of an IBV transcription-associated sequence derived from gene 5 of the Beaudette strain, was expressed from two different positions within CD-61 and shown to be biologically active. In addition, following infection of 10-day-old chicken embryos with IBV containing D-RNAs expressing chIFN-gamma, the allantoic fluid was shown to contain biologically active chIFN-gamma, demonstrating that IBV D-RNAs can express heterologous genes in vivo.
Collapse
Affiliation(s)
- Karen Hackney
- Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, United Kingdom
| | | | | | | |
Collapse
|
|
42
|
Faulkner L, Buchan G, Slobbe L, Lockhart E, Wales J, Wilson M, Baird M. Influenza hemagglutinin peptides fused to interferon gamma and encapsulated in liposomes protects mice against influenza infection. Vaccine 2003; 21:932-9. [PMID: 12547605 DOI: 10.1016/s0264-410x(02)00543-1] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
The immunogenicity of a peptide vaccine may be improved by fusing antigen to a cytokine and administering this chimeric protein in a particulate delivery system. We have investigated this using a vaccine comprising an immunodominant T cell epitope and a B cell epitope from influenza haemagglutinin (HATB) fused to interferon gamma and encapsulated in liposomes (HATB/IFN-gamma/lipo). Controls comprised groups receiving HATB/IFN-gamma mixed with liposomes, HATB incorporated in liposomes or heat inactivated PR8 influenza virus (HI PR8). IFN-gamma production in mice treated with HATB/IFN-gamma/lipo was significantly higher than in mice inoculated with either HATB/IFN-gamma mixed with liposomes or HATB incorporated in liposomes but less than HI PR8. Lung viral titres were significantly lower in mice treated with HATB/IFN-gamma/lipo compared with those treated with HATB/IFN-gamma mixed with liposomes. HI PR8-treated mice recorded a nil viral titre. There was no correlation between the level of antibody production and clearance of virus from the lungs. These data suggest that particulate delivery systems may be useful adjuncts to improve immune responses to chimeric proteins and to induce protection against disease.
Collapse
Affiliation(s)
- L Faulkner
- Department of Microbiology, University of Otago, PO Box 56, Dunedin, New Zealand
| | | | | | | | | | | | | |
Collapse
|
|
43
|
Gomes-Keller MA, Nunez R, Schroff M, Oswald D, Willett BJ, Lutz H. Up-regulation by feline interleukin-4 and down-regulation by feline interferon-gamma of major histocompatibility complex class II on cat B-lymphocytes. Vet Immunol Immunopathol 2002; 88:197-208. [PMID: 12127417 DOI: 10.1016/s0165-2427(02)00171-x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Interleukin-4 (IL-4) exhibits numerous biological and immunoregulatory functions on B- and T-lymphocytes, monocytes, and dendritic cells in both mice and humans. In the present study, we show that IL-4 also has a regulatory function in the cat species. Cells transfected with IL-4 DNA expressed a biologically active protein as demonstrated by the up-regulation of MHC class II molecules on B-lymphocytes (CD21(+)) in a flow cytometric assay. Increased levels of MHC class II expression on CD21(+) cells were seen in 11 out of 12 cats (p<0.05). In addition, 12 out of 12 cats showed up-regulation of MHC class II on CD21(-) cells, mainly consisting of T-lymphocytes (p<0.05). In contrast, concanavalin A (ConA)-induced culture supernatant from peripheral blood mononuclear cells (PBMCs) containing high levels of interferon-gamma (IFN-gamma) transcripts induced down-regulation of MHC class II molecules on CD21(+) cells of all samples (p<0.05). Variable results were observed for CD21(-) cells incubated with ConA-conditioned medium (p=0.71). The nature of the cytokine(s) responsible for these effects remains to be determined. However, the fact that down-regulation of MHC class II molecules on B cells occurred in all cats tested suggests that IFN-gamma may be involved. These data provide further insight into the mechanism by which MHC class II expression is regulated in feline lymphocytes, and suggest that the Th1/Th2 paradigm is also present in the cat.
Collapse
Affiliation(s)
- M A Gomes-Keller
- Clinical Laboratory, Faculty of Veterinary Medicine, University of Zurich, 8057 Zurich, Switzerland.
| | | | | | | | | | | |
Collapse
|