Expression, purification and characterization of glycosylated influenza H5N1 hemagglutinin produced in Pichia pastoris.

Kopera, Edyta; Dwornyk, Angela; Kossoń, Piotr; Florys, Katarzyna; Sa̧czyńska, Violetta; Dębski, Janusz; Cecuda-Adamczewska, Violetta; Szewczyk, Bogusław; Zagórski-Ostoja, Włodzimierz; Grzelak, Krystyna

doi:10.18388/abp.2014_1882

Cited by 8 publications

(8 citation statements)

References 20 publications

(20 reference statements)

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“…Moreover, these viruses may acquire the ability of human-to-human transmission and thereby pose a pandemic threat. To control H5N1 HPAIVs, HA-based vaccines are being developed using predominantly eukaryotic expression systems (e.g., [ 8 – 10 ]). More recently, so-called bacterial HAs have become a promising alternative to vaccine antigens expressed in yeast, baculovirus-insect or mammalian cells.…”

Section: Discussionmentioning

confidence: 99%

“…The obvious candidate for the production of subunit vaccines against flu is HA, a key viral protein capable of eliciting potent neutralizing antibodies. To obtain HA antigens of various subtypes, including H5, yeast (e.g., [ 8 ]), baculovirus (e.g., [ 9 ]) and mammalian (e.g., [ 10 ]) expression systems, but not a bacterial expression system, have been used for years. The exploitation of bacteria enabling the relatively easy, low-cost and efficient production of the vaccine protein seems to have been hampered for a long time by the commonly accepted view that glycosylation determines the correct HA structure.…”

Section: Introductionmentioning

confidence: 99%

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A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies

et al. 2017

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The highly pathogenic (HP) H5N1 avian influenza viruses (AIVs) cause a mortality rate of up to 100% in infected chickens and pose a permanent pandemic threat. Attempts to obtain effective vaccines against H5N1 HPAIVs have focused on hemagglutinin (HA), an immunodominant viral antigen capable of eliciting neutralizing antibodies. The vast majority of vaccine projects have been performed using eukaryotic expression systems. In contrast, we used a bacterial expression system to produce vaccine HA protein (bacterial HA) according to our own design. The HA protein with the sequence of the H5N1 HPAIV strain was efficiently expressed in Escherichia coli, recovered in the form of inclusion bodies and refolded by dilution between two chromatographic purification steps. Antigenicity studies showed that the resulting antigen, referred to as rH5-E. coli, preserves conformational epitopes targeted by antibodies specific for H5-subtype HAs, inhibiting hemagglutination and/or neutralizing influenza viruses in vitro. The proper conformation of this protein and its ability to form functional oligomers were confirmed by a hemagglutination test. Consistent with the biochemical characteristics, prime-boost immunizations with adjuvanted rH5-E. coli protected 100% and 70% of specific pathogen-free, layer-type chickens against challenge with homologous and heterologous H5N1 HPAIVs, respectively. The observed protection was related to the positivity in the FluAC H5 test (IDVet) but not to hemagglutination-inhibiting antibody titers. Due to full protection, the effective contact transmission of the homologous challenge virus did not occur. Survivors from both challenges did not or only transiently shed the viruses, as established by viral RNA detection in oropharyngeal and cloacal swabs. Our results demonstrate that vaccination with rH5-E. coli could confer control of H5N1 HPAIV infection and transmission rates in chicken flocks, accompanied by reduced virus shedding. Moreover, the role of H5 subtype-specific neutralizing antibodies in anti-influenza immunity and a novel correlate of protection are indicated.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies

et al. 2017

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“…The second immunization experiment was similar to the previous one; however, a different adjuvant was used. Based on our results obtained from the immunization experiment with H5 antigen [8], the H1 vaccine formulation contained the Sigma Adjuvant System. The same doses of the H1 antigen were tested.…”

Section: Resultsmentioning

confidence: 99%

“…The aim of this study was to test an H1N1pdm09 influenza virus HA produced in a yeast expression system as a potential vaccine antigen. Our previous study showed that the H5 antigen produced in the P. pastoris cells is capable of inducing a specific immune response in mice [8, 10] and providing full protection in chicken [9]. Ease of preparation, low cost of production, and high immunogenicity of the yeast-derived antigen prompted us to test an H1N1pdm09 influenza virus antigen.…”

Section: Introductionmentioning

confidence: 99%

High-Titre Neutralizing Antibodies to H1N1 Influenza Virus after Mouse Immunization with Yeast Expressed H1 Antigen: A Promising Influenza Vaccine Candidate

Kopera

Zdanowski

Uranowska

et al. 2019

Journal of Immunology Research

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H1N1 influenza virus is still regarded as a serious pandemic threat. The most effective method of protection against influenza virus and the way to reduce the risk of epidemic or pandemic spread is vaccination. Influenza vaccine manufactured in a traditional way, though well developed, has some drawbacks and limitations which have stimulated interest in developing alternative approaches. In this study, we demonstrate that the recombinant H1 vaccine based on the hydrophilic haemagglutinin (HA) domain and produced in the yeast system elicited high titres of serum haemagglutination-inhibiting antibodies in mice. Transmission electron microscopy showed that H1 antigen oligomerizes into functional higher molecular forms similar to rosette-like structures. Analysis of the N-linked glycans using mass spectrometry revealed that the H1 protein is glycosylated at the same sites as the native HA. The recombinant antigen was secreted into a culture medium reaching approximately 10 mg/l. These results suggest that H1 produced in Pichia pastoris can be considered as the vaccine candidate against H1N1 virus.

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“…Similarly, A/swan/Poland/305-135V08/2006 (H5N1) HA gene was expressed in P. pastoris and the protein was purified for mouse immunization. This recombinant HA was found to be effective in eliciting a high immune response in mice [125]. As P. pastoris is used mainly for large-scale protein production and antibody screening, it will not be further discussed here.…”

Section: The Yeast Saccharomyces Cerevisiae: a Powerful System For Idmentioning

confidence: 99%

Alternative Experimental Models for Studying Influenza Proteins, Host–Virus Interactions and Anti-Influenza Drugs

Chua

Engelberg

et al. 2019

Pharmaceuticals

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Ninety years after the discovery of the virus causing the influenza disease, this malady remains one of the biggest public health threats to mankind. Currently available drugs and vaccines only partially reduce deaths and hospitalizations. Some of the reasons for this disturbing situation stem from the sophistication of the viral machinery, but another reason is the lack of a complete understanding of the molecular and physiological basis of viral infections and host-pathogen interactions. Even the functions of the influenza proteins, their mechanisms of action and interaction with host proteins have not been fully revealed. These questions have traditionally been studied in mammalian animal models, mainly ferrets and mice (as well as pigs and non-human primates) and in cell lines. Although obviously relevant as models to humans, these experimental systems are very complex and are not conveniently accessible to various genetic, molecular and biochemical approaches. The fact that influenza remains an unsolved problem, in combination with the limitations of the conventional experimental models, motivated increasing attempts to use the power of other models, such as low eukaryotes, including invertebrate, and primary cell cultures. In this review, we summarized the efforts to study influenza in yeast, Drosophila, zebrafish and primary human tissue cultures and the major contributions these studies have made toward a better understanding of the disease. We feel that these models are still under-utilized and we highlight the unique potential each model has for better comprehending virus-host interactions and viral protein function.

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Expression, purification and characterization of glycosylated influenza H5N1 hemagglutinin produced in Pichia pastoris.

Cited by 8 publications

References 20 publications

A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies

A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies

High-Titre Neutralizing Antibodies to H1N1 Influenza Virus after Mouse Immunization with Yeast Expressed H1 Antigen: A Promising Influenza Vaccine Candidate

Alternative Experimental Models for Studying Influenza Proteins, Host–Virus Interactions and Anti-Influenza Drugs

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