scholarly journals Integration Stability of sHBsAg-Multi Expression Cassettes in Pichia pastoris GS115 during Methanol Induction

2020 ◽  
Vol 27 (4) ◽  
pp. 283
Author(s):  
Patricia Gita Naully ◽  
Neni Nurainy ◽  
Elvi Restiawaty ◽  
Dessy Natalia ◽  
Debbie Soefie Retnoningrum ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Hepatitis B ◽  
Vaccine Development ◽  
Expression Cassette ◽  
Foreign Gene ◽  
Pastoris Gs115 ◽  
Methanol Induction ◽  
Small Surface ◽  
Major Health Problem ◽  
Replacement Method

Hepatitis B is the major health problem worldwide including in Indonesia. Vaccination is the best prevention strategy for the disease. For the purpose of vaccine development and to decrease drug import, production of Hepatitis B Virus (HBV) small surface antigen (sHBsAg) from Indonesian HBV subtype is needed. The recombinant protein production can be conducted by integrating multi expression cassettes of sHBsAg gene in Pichia pastoris chromosome using gene replacement method. Such integration method turns out to allow loss of foreign gene from chromosome by excisional recombination-mediated looping out. This research was aimed to determine integration stability of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome inducted with 2% methanol in FM22 medium. The methanol induction was conducted twice at 63-h and 75-h. Integration stability determination was conducted qualitatively using PCR and quantitatively using qPCR absolute quantification. A band of 208 bp with similar intensity was observed after amplification of genomic DNA. All samples generated the same Ct value of around 22 with four copies of sHBsAg gene per genome. The result from this experiment shows that integration of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome is stable during methanol induction.

scholarly journals Specific Antibodies Induced by Immunization with Hepatitis B Virus-Like Particles Carrying Hepatitis C Virus Envelope Glycoprotein 2 Epitopes Show Differential Neutralization Efficiency

Vaccines ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 294
Author(s):  
Anna Czarnota ◽  
Anna Offersgaard ◽  
Anne Finne Pihl ◽  
Jannick Prentoe ◽  
Jens Bukh ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Neutralizing Antibodies ◽  
Expression System ◽  
Small Surface ◽  
Major Health Problem ◽  
Virus Like Particles ◽  
B Virus

Hepatitis C virus (HCV) infection with associated chronic liver diseases is a major health problem worldwide. Here, we designed hepatitis B virus (HBV) small surface antigen (sHBsAg) virus-like particles (VLPs) presenting different epitopes derived from the HCV E2 glycoprotein (residues 412–425, 434–446, 502–520, and 523–535 of isolate H77C). Epitopes were selected based on their amino acid sequence conservation and were previously reported as targets of HCV neutralizing antibodies. Chimeric VLPs obtained in the Leishmania tarentolae expression system, in combination with the adjuvant Addavax, were used to immunize mice. Although all VLPs induced strong humoral responses, only antibodies directed against HCV 412–425 and 523–535 epitopes were able to react with the native E1E2 glycoprotein complexes of different HCV genotypes in ELISA. Neutralization assays against genotype 1–6 cell culture infectious HCV (HCVcc), revealed that only VLPs carrying the 412–425 epitope induced efficient HCV cross-neutralizing antibodies, but with isolate specific variations in efficacy that could not necessarily be explained by differences in epitope sequences. In contrast, antibodies targeting 434–446, 502–520, and 523–535 epitopes were not neutralizing HCVcc, highlighting the importance of conformational antibodies for efficient virus neutralization. Thus, 412–425 remains the most promising linear E2 epitope for further bivalent, rationally designed vaccine research.

How Far we are towards Eradication of HBV Infection

2015 ◽  
Vol 24 (4) ◽  
pp. 473-479 ◽  
Author(s):  
Mihai Voiculescu
Keyword(s):  
Immune Response ◽  
Hepatitis B Virus ◽  
T Cell ◽  
Hepatitis B ◽  
T Cell Receptors ◽  
Hepatitis B Surface Antigen ◽  
Hbv Infection ◽  
Major Health Problem ◽  
Cell Receptors ◽  
B Virus

Hepatitis B virus (HBV) infection is a major health problem with an important biological and a significant socio-economic impact all over the world. There is a high pressure to come up with a new and more efficient strategy against HBV infection, especially after the recent success of HCV treatment. Preventing HBV infection through vaccine is currently the most efficient way to decrease HBV-related cirrhosis and liver cancer incidence, as well as the best way to suppress the HBV reservoir. The vaccine is safe and efficient in 80-95% of cases. One of its most important roles is to reduce materno-fetal transmission, by giving the first dose of vaccine in the first 24 hours after birth. Transmission of HBV infection early in life is still frequent, especially in countries with high endemicity.Successful HBV clearance by the host is immune-mediated, with a complex combined innate and adaptive cellular and humoral immune response. Different factors, such as the quantity and the sequence of HBV epitope during processing by dendritic cells and presenting by different HLA molecules or the polymorphism of T cell receptors (TOL) are part of a complex network which influences the final response. A new potential therapeutic strategy is to restore T-cell antiviral function and to improve innate and adaptive immune response by immunotherapeutic manipulation.It appears that HBV eradication is far from being completed in the next decades, and a new strategy against HBV infection must be considered. Abbreviations: ALT: alanine aminotransferase; APC: antigen presenting cells; cccDNA: covalently closed circular DNA; HBIG: hepatitis B immunoglobulin; HbsAg: hepatitis B surface antigen; HBV: hepatitis B virus; HCC: hepatocellular carcinoma; CTL: cytotoxic T lymphocyte; IFN: interferon; NUC: nucleos(t)ide analogues; pg RNA: pre genomic RNA; TLR: toll-like receptors; TOL: T cell receptors.

scholarly journals Covalent protein display on Hepatitis B core-like particles in plants through the in vivo use of the SpyTag/SpyCatcher system

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hadrien Peyret ◽  
Daniel Ponndorf ◽  
Yulia Meshcheriakova ◽  
Jake Richardson ◽  
George P. Lomonossoff
Keyword(s):  
Hepatitis B ◽  
Cost Saving ◽  
Capsid Protein ◽  
Recombinant Proteins ◽  
Nicotiana Benthamiana ◽  
Vaccine Development ◽  
Binding Partner ◽  
Gfp Fluorescence ◽  
Display Systems

Abstract Virus-like particles (VLPs) can be used as nano-carriers and antigen-display systems in vaccine development and therapeutic applications. Conjugation of peptides or whole proteins to VLPs can be achieved using different methods such as the SpyTag/SpyCatcher system. Here we investigate the conjugation of tandem Hepatitis B core (tHBcAg) VLPs and the model antigen GFP in vivo in Nicotiana benthamiana. We show that tHBcAg VLPs could be successfully conjugated with GFP in the cytosol and ER without altering VLP formation or GFP fluorescence. Conjugation in the cytosol was more efficient when SpyCatcher was displayed on tHBcAg VLPs instead of being fused to GFP. This effect was even more obvious in the ER, showing that it is optimal to display SpyCatcher on the tHBcAg VLPs and SpyTag on the binding partner. To test transferability of the GFP results to other antigens, we successfully conjugated tHBcAg VLPs to the HIV capsid protein P24 in the cytosol. This work presents an efficient strategy which can lead to time and cost saving post-translational, covalent conjugation of recombinant proteins in plants.

scholarly journals Hepatitis B core-based virus-like particles: A platform for vaccine development in plants

2021 ◽  
Vol 29 ◽  
pp. e00605
Author(s):  
Maryam Moradi Vahdat ◽  
Farshad Hemmati ◽  
Abozar Ghorbani ◽  
Daria Rutkowska ◽  
Alireza Afsharifar ◽  
...  
Keyword(s):  
Hepatitis B ◽  
Vaccine Development ◽  
Virus Like Particles

Methanol Induction Optimization for Recombinant Human Consensus Interferon Mutant Production in Pichia pastoris

2010 ◽  
Vol 150 ◽  
pp. 540-540
Author(s):  
Dan Wu ◽  
Ju Chu ◽  
Yu-You Hao ◽  
Yong-Hong Wang ◽  
Ying-Ping Zhuang ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methanol Induction ◽  
Consensus Interferon

Production of foreign proteins in the yeast Pichia pastoris

1995 ◽  
Vol 73 (S1) ◽  
pp. 891-897 ◽  
Author(s):  
James M. Cregg ◽  
David R. Higgins
Keyword(s):  
Gene Expression ◽  
Pichia Pastoris ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Methylotrophic Yeast ◽  
Culture Broth ◽  
Heterologous Gene ◽  
Foreign Gene ◽  
Heterologous Proteins ◽  
Yeast Pichia Pastoris

The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest. An industrial yeast selected for efficient growth on methanol for biomass generation, P. pastoris is readily grown on defined medium in continuous culture at high volume and density. A unique feature of the expression system is the promoter employed to drive heterologous gene expression, which is derived from the methanol-regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters known. The strength of the AOX1 promoter results in high expression levels in strains harboring only a single integrated copy of a foreign-gene expression cassette. Levels may often be further enhanced through the integration of multiple cassette copies into the P. pastoris genome and strategies to construct and select multicopy cassette strains have been devised. The system is particularly attractive for the secretion of foreign-gene products. Because P. pastoris endogenous protein secretion levels are low, foreign secreted proteins often appear to be virtually the only proteins in the culture broth, a major advantage in processing and purification. Key words: heterologous gene expression, methylotrophic yeast, Pichia pastoris, secretion, glycosylation.

scholarly journals Coronaviruses as Vectors: Stability of Foreign Gene Expression

2005 ◽  
Vol 79 (20) ◽  
pp. 12742-12751 ◽  
Author(s):  
Cornelis A. M. de Haan ◽  
Bert Jan Haijema ◽  
David Boss ◽  
Frank W. H. Heuts ◽  
Peter J. M. Rottier
Keyword(s):  
Genetic Stability ◽  
Vaccine Development ◽  
Hepatitis Virus ◽  
Firefly Luciferase ◽  
Foreign Gene ◽  
Rna Recombination ◽  
Large Deletions ◽  
Group 2 ◽  
Attenuated Viruses ◽  
Foreign Genes

ABSTRACT Coronaviruses are enveloped, positive-stranded RNA viruses considered to be promising vectors for vaccine development, as (i) genes can be deleted, resulting in attenuated viruses; (ii) their tropism can be modified by manipulation of their spike protein; and (iii) heterologous genes can be expressed by simply inserting them with appropriate coronaviral transcription signals into the genome. For any live vector, genetic stability is an essential requirement. However, little is known about the genetic stability of recombinant coronaviruses expressing foreign genes. In this study, the Renilla and the firefly luciferase genes were systematically analyzed for their stability after insertion at various genomic positions in the group 1 coronavirus feline infectious peritonitis virus and in the group 2 coronavirus mouse hepatitis virus. It appeared that the two genes exhibit intrinsic differences, the Renilla gene consistently being maintained more stably than the firefly gene. This difference was not caused by genome size restrictions, by different effects of the encoded proteins, or by different consequences of the synthesis of the additional subgenomic mRNAs. The loss of expression of the firefly luciferase was found to result from various, often large deletions of the gene, probably due to RNA recombination. The extent of this process appeared to depend strongly on the coronaviral genomic background, the luciferase gene being much more stable in the feline than in the mouse coronavirus genome. It also depended significantly on the particular genomic location at which the gene was inserted. The data indicate that foreign sequences are more stably maintained when replacing nonessential coronaviral genes.

scholarly journals Improved Production ofAspergillus usamiiendo-β-1,4-Xylanase inPichia pastorisvia Combined Strategies

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Jianrong Wang ◽  
Yangyuan Li ◽  
Danni Liu
Keyword(s):  
Pichia Pastoris ◽  
Cell Viability ◽  
Recombinant Proteins ◽  
Recombinant Strain ◽  
Xylanase Activity ◽  
Vitreoscilla Hemoglobin ◽  
Wild Type ◽  
Methanol Induction ◽  
Aspergillus Usamii ◽  
Induction Temperature

A series of strategies were applied to improve expression level of recombinant endo-β-1,4-xylanase fromAspergillus usamii(A. usamii) inPichia pastoris(P. pastoris). Firstly, the endo-β-1,4-xylanase (xynB) gene fromA. usamiiwas optimized forP. pastorisand expressed inP. pastoris. The maximum xylanase activity of optimized (xynB-opt) gene was 33500 U/mL after methanol induction for 144 h in 50 L bioreactor, which was 59% higher than that by wild-type (xynB) gene. To further increase the expression ofxynB-opt, theVitreoscilla hemoglobin(VHb) gene was transformed to the recombinant strain containingxynB-opt. The results showed that recombinant strain harboring thexynB-optandVHb(named X33/xynB-opt-VHb) displayed higher biomass, cell viability, and xylanase activity. The maximum xylanase activity of X33/xynB-opt-VHbin 50 L bioreactor was 45225 U/mL, which was 35% and 115% higher than that by optimized (xynB-opt) gene and wild-type (xynB) gene. Finally, the induction temperature of X33/xynB-opt-VHbwas optimized in 50 L bioreactor. The maximum xylanase activity of X33/xynB-opt-VHbreached 58792 U/mL when the induction temperature was 22°C. The results presented here will greatly contribute to improving the production of recombinant proteins inP. pastoris.

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