Browsing by Author "Waranyoo Phoolcharoen"

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    Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2
    (2021-01-07) Konlavat Siriwattananon; Suwimon Manopwisedjaroen; Phongthon Kanjanasirirat; Priyo Budi Purwono; Kaewta Rattanapisit; Balamurugan Shanmugaraj; Duncan R. Smith; Suparerk Borwornpinyo; Arunee Thitithanyanont; Waranyoo Phoolcharoen; Universitas Airlangga; Chulalongkorn University; Mahidol University
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease (COVID-19) which has recently emerged as a potential threat to global public health. SARS-CoV-2 is the third known human coronavirus that has huge impact on the human population after SARS-CoV and MERS-CoV. Although some vaccines and therapeutic drugs are currently in clinical trials, none of them are approved for commercial use yet. As with SARS-CoV, SARS-CoV-2 utilizes angiotensin-converting enzyme 2 (ACE2) as the cell entry receptor to enter into the host cell. In this study, we have transiently produced human ACE2 fused with the Fc region of human IgG1 in Nicotiana benthamiana and the in vitro neutralization efficacy of the plant-produced ACE2-Fc fusion protein was assessed. The recombinant ACE2-Fc fusion protein was expressed in N. benthamiana at 100 μg/g leaf fresh weight on day 6 post-infiltration. The recombinant fusion protein showed potent binding to receptor binding domain (RBD) of SARS-CoV-2. Importantly, the plant-produced fusion protein exhibited potent anti-SARS-CoV-2 activity in vitro. Treatment with ACE2-Fc fusion protein after viral infection dramatically inhibit SARS-CoV-2 infectivity in Vero cells with an IC50 value of 0.84 μg/ml. Moreover, treatment with ACE2-Fc fusion protein at the pre-entry stage suppressed SARS-CoV-2 infection with an IC50 of 94.66 μg/ml. These findings put a spotlight on the plant-produced ACE2-Fc fusion protein as a potential therapeutic candidate against SARS-CoV-2.
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    Immunodominant linear B cell epitopes in the spike and membrane proteins of SARS-CoV-2 identified by immunoinformatics prediction and immunoassay
    (2021-12-01) Kanokporn Polyiam; Waranyoo Phoolcharoen; Namphueng Butkhot; Chanya Srisaowakarn; Arunee Thitithanyanont; Prasert Auewarakul; Tawatchai Hoonsuwan; Marasri Ruengjitchatchawalya; Phenjun Mekvichitsaeng; Yaowaluck Maprang Roshorm; Siriraj Hospital; Chulalongkorn University; Mahidol University; King Mongkut's University of Technology Thonburi; B.F. Feed Company Limited
    SARS-CoV-2 continues to infect an ever-expanding number of people, resulting in an increase in the number of deaths globally. With the emergence of new variants, there is a corresponding decrease in the currently available vaccine efficacy, highlighting the need for greater insights into the viral epitope profile for both vaccine design and assessment. In this study, three immunodominant linear B cell epitopes in the SARS-CoV-2 spike receptor-binding domain (RBD) were identified by immunoinformatics prediction, and confirmed by ELISA with sera from Macaca fascicularis vaccinated with a SARS-CoV-2 RBD subunit vaccine. Further immunoinformatics analyses of these three epitopes gave rise to a method of linear B cell epitope prediction and selection. B cell epitopes in the spike (S), membrane (M), and envelope (E) proteins were subsequently predicted and confirmed using convalescent sera from COVID-19 infected patients. Immunodominant epitopes were identified in three regions of the S2 domain, one region at the S1/S2 cleavage site and one region at the C-terminus of the M protein. Epitope mapping revealed that most of the amino acid changes found in variants of concern are located within B cell epitopes in the NTD, RBD, and S1/S2 cleavage site. This work provides insights into B cell epitopes of SARS-CoV-2 as well as immunoinformatics methods for B cell epitope prediction, which will improve and enhance SARS-CoV-2 vaccine development against emergent variants.
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    Immunogenicity studies of plant-produced sars-cov-2 receptor binding domain-based subunit vaccine candidate with different adjuvant formulations
    (2021-07-01) Konlavat Siriwattananon; Suwimon Manopwisedjaroen; Balamurugan Shanmugaraj; Eakachai Prompetchara; Chutitorn Ketloy; Supranee Buranapraditkun; Kittipan Tharakhet; Papatsara Kaewpang; Kiat Ruxrungtham; Arunee Thitithanyanont; Waranyoo Phoolcharoen; Chulalongkorn University; Mahidol University; Faculty of Medicine, Chulalongkorn University; Ltd.
    Due to the rapid transmission of the coronavirus disease 2019 (COVID-19) causing serious public health problems and economic burden, the development of effective vaccines is a high priority for controlling the virus spread. Our group has previously demonstrated that the plant-produced receptor-binding domain (RBD) of SARS-CoV-2 fused with Fc of human IgG was capable of eliciting potent neutralizing antibody and cellular immune responses in animal studies, and the immunogenicity could be improved by the addition of an alum adjuvant. Here, we performed a head-to-head comparison of different commercially available adjuvants, including aluminum hydroxide gel (alum), AddaVax (MF59), monophosphoryl lipid A from Salmonella minnesota R595 (mPLA-SM), and polyinosinic-polycytidylic acid (poly(I:C)), in mice by combining them with plant-produced RBD-Fc, and the differences in the immunogenicity of RBD-Fc with different adjuvants were evaluated. The specific antibody responses in terms of total IgG, IgG1, and IgG2a subtypes and neutralizing antibodies, as well as vaccine-specific T-lymphocyte responses, induced by the different tested adjuvants were compared. We observed that all adjuvants tested here induced a high level of total IgG and neutralizing antibodies, but mPLA-SM and poly (I:C) showed the induction of a balanced IgG1 and IgG2a (Th2/Th1) immune response. Further, poly (I:C) significantly increased the frequency of IFN-γ-expressing cells compared with control, whereas no significant difference was observed between the adjuvanted groups. This data revealed the adjuvants’ role in enhancing the immune response of RBD-Fc vaccination and the immune profiles elicited by different adjuvants, which could prove helpful for the rational development of next-generation SARS-CoV-2 RBD-Fc subunit vaccines. However, additional research is essential to further investigate the efficacy and safety of this vaccine formulation before clinical trials.
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    Internalization and propagation of the dengue virus in human hepatoma (HepG2) cells
    (2004-07-07) Chutima Thepparit; Waranyoo Phoolcharoen; Lukkana Suksanpaisan; Duncan R. Smith; Mahidol University
    Objectives: This study sought to undertake a comparative analysis of the internalization and propagation of all four dengue serotypes in a single cell line of human liver origin, HepG2. Methods: Virus production after infection was determined by the plaque assay technique. Internalization profiles were determined by incubating virus and cells on ice and then raising the temperature for various times. The contribution of extracellular matrix components to internalization was determined by pretreatment of cells with either trypsin or heparinase III. Results: HepG2 cells were able to support the propagation of all four serotypes with mature viruses being produced by 12 h for dengue serotype 4 and by 17-18 h for the remaining serotypes. Virus internalization showed a plateau for serotypes 1, 2 and 4 entry while serotype 3 showed a constant increase in internalization for up to 5 h. Pretreatment of HepG2 cells with heparinase III or trypsin both resulted in a reduction in viral production, with the smallest effect being noted for dengue serotype 3. Conclusion: These results suggest that the interaction between the dengue virus and liver cells is a complex one that requires both protein and nonprotein elements, and has a significant serotype/strain element. Copyright © 2004 S. Karger AG, Basel.
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    Internalization of the dengue virus is cell cycle modulated in HepG2, but not Vero cells
    (2004-11-01) Waranyoo Phoolcharoen; Duncan R. Smith; Mahidol University
    While many studies have investigated the relationship between cell type and dengue virus infection, no study to date has examined the effect of cell physiology on permissiveness to infection. Unsynchronized and artificially synchronized cell populations at different stages of the cell cycle of two cell types (Vero and HepG2) were examined for permissiveness to infection by two dengue virus serotypes (serotypes 2 and 3) by determining both the levels of virus produced as well as the percentage of cells infected. Vero cells showed no significant differences between either viral production or percentage of cells infected as compared to unsynchronized cells for any of the phases investigated, although production of virus (for both serotypes 2 and 3) was somewhat lower for cells infected during S phase. In contrast, HepG2 cells were significantly more permissive for both infection and virus production in the G2 phase as compared to other phases examined and serotype differences in permissiveness to infection were noted with cells in the M phase of the cell cycle. These results suggest that the cell cycle may be a mediator of cell permissiveness in some cell types. © 2004 Wiley-Liss, Inc.
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    Plant-made antibody against miroestrol: a new platform for expression of full-length immunoglobulin G against small-molecule targets in immunoassays
    (2021-04-01) Kaewta Rattanapisit; Tharita Kitisripanya; Atthaphon Konyanee; Worapol Sae-Foo; Apisit Burapapiruin; Waraporn Putalun; Seiichi Sakamoto; Waranyoo Phoolcharoen; Gorawit Yusakul; Chulalongkorn University; Walailak University; Khon Kaen University; Mahidol University; Kyushu University
    Key message: Plant expression platform is the new source of immunoglobulin G (IgG) toward small low-molecular-weight targets. The plant-made monoclonal antibody-based immunoassay exhibits comparable analytical performance with hybridoma antibody. Abstract: Immunoassays for small molecules are efficiently applied for monitoring of serum therapeutic drug concentration, food toxins, environmental contamination, etc. Immunoglobulin G (IgG) is usually produced using hybridoma cells, which requires complicated procedures and expensive equipment. Plants can act as alternative and economic hosts for IgG production. However, the production of free hapten (low-molecular-weight target)-recognizing IgG from plants has not been successfully developed yet. The current study aimed at creating a plant platform as an affordable source of IgG for use in immunoassays and diagnostic tools. The functional IgG was expressed in Nicotiana benthamiana leaves infiltrated with Agrobacterium tumefaciens strain GV3101 with recombinant geminiviral vectors (pBY3R) occupying chimeric anti-miroestrol IgG genes. The appropriate assembly between heavy and light chains was achieved, and the yield of expression was 0.57 µg/g fresh N. benthamiana leaves. The binding characteristics of the IgG to miroestrol and binding specificity to related compounds, such as isomiroestrol and deoxymiroestrol, were similar to those of hybridoma-produced IgG (monoclonal antibody, mAb). The plant-based mAbs exhibited high sensitivity for miroestrol (IC50, 23.2 ± 2.1 ng/mL), precision (relative standard deviation ≤ 5.01%), and accuracy (97.8–103% recovery), as determined using quantitative enzyme-linked immunosorbent assay. The validated enzyme-linked immunosorbent assay was applicable to determine miroestrol in plant samples. Overall, the plant-produced functional IgG conserved the binding activity and specificity of the parent IgG derived from mammalian cells. Therefore, the plant expression system may be an efficient and affordable platform for the production of antibodies against low-molecular-weight targets in immunoassays.
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    Plant-Produced Receptor-Binding Domain of SARS-CoV-2 Elicits Potent Neutralizing Responses in Mice and Non-human Primates
    (2021-05-13) Konlavat Siriwattananon; Suwimon Manopwisedjaroen; Balamurugan Shanmugaraj; Kaewta Rattanapisit; Supaporn Phumiamorn; Sompong Sapsutthipas; Sakalin Trisiriwanich; Eakachai Prompetchara; Chutitorn Ketloy; Supranee Buranapraditkun; Wassana Wijagkanalan; Kittipan Tharakhet; Papatsara Kaewpang; Kantinan Leetanasaksakul; Taratorn Kemthong; Nutchanat Suttisan; Suchinda Malaivijitnond; Kiat Ruxrungtham; Arunee Thitithanyanont; Waranyoo Phoolcharoen; Chulalongkorn University; Thailand Ministry of Public Health; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; Faculty of Medicine, Chulalongkorn University; Ltd.
    The emergence of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected global public health and economy. Despite the substantial efforts, only few vaccines are currently approved and some are in the different stages of clinical trials. As the disease rapidly spreads, an affordable and effective vaccine is urgently needed. In this study, we investigated the immunogenicity of plant-produced receptor-binding domain (RBD) of SARS-CoV-2 in order to use as a subunit vaccine. In this regard, RBD of SARS-CoV-2 was fused with Fc fragment of human IgG1 and transiently expressed in Nicotiana benthamiana by agroinfiltration. The plant-produced RBD-Fc fusion protein was purified from the crude extract by using protein A affinity column chromatography. Two intramuscular administration of plant-produced RBD-Fc protein formulated with alum as an adjuvant have elicited high neutralization titers in immunized mice and cynomolgus monkeys. Further it has induced a mixed Th1/Th2 immune responses and vaccine-specific T-lymphocyte responses which was confirmed by interferon-gamma (IFN-γ) enzyme-linked immunospot assay. Altogether, our results demonstrated that the plant-produced SARS-CoV-2 RBD has the potential to be used as an effective vaccine candidate against SARS-CoV-2. To our knowledge, this is the first report demonstrating the immunogenicity of plant-produced SARS-CoV-2 RBD protein in mice and non-human primates.