Error-prone pcr-based mutagenesis strategy for rapidly generating high-yield influenza vaccine candidates

Ye, Jianqiang; Wen, Feng; Xu, Yifei; Zhao, Nan; Liang, Liping; Sun, Hailiang; Yang, Jialiang; Cooley, Jim; Pharr, G. T.; Webby, Richard J.; Wan, Xiu‐Feng

doi:10.1016/j.virol.2015.03.051

Cited by 16 publications

(15 citation statements)

References 43 publications

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“…Generation of mutants using an epPCR-based reverse-genetics strategy. The mutant library with random mutations in HA RBSs was generated by epPCR as previously described (48). In brief, the randomly mutated short sequences (about 200 nucleotides) were used as primers for site-directed mutagenesis with HA-pHW2000, leading to plasmids with random mutations in the HA RBSs.…”

Section: Methodsmentioning

confidence: 99%

“…In brief, the randomly mutated short sequences (about 200 nucleotides) were used as primers for site-directed mutagenesis with HA-pHW2000, leading to plasmids with random mutations in the HA RBSs. This strategy can avoid the need for a labor-intensive gene-cloning process, and HA-pHW2000 with mutations can be used directly for generating vaccine candidates (48). One day before transfection, 293T cells and MDCK cells were cocultured in 24-well plates, using a 20:1 ratio of 293T cells to MDCK cells.…”

Section: Methodsmentioning

confidence: 99%

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A Y161F Hemagglutinin Substitution Increases Thermostability and Improves Yields of 2009 H1N1 Influenza A Virus in Cells

Wen

Zhao

et al. 2018

J Virol

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Vaccination is the primary strategy for influenza prevention and control. However, egg-based vaccines, the predominant production platform, have several disadvantages, including the emergence of viral antigenic variants that can be induced during egg passage. These limitations have prompted the development of cell-based vaccines, which themselves are not without issue. Most importantly, vaccine seed viruses often do not grow efficiently in mammalian cell lines. Here we aimed to identify novel high-yield signatures for influenza viruses in continuous Madin-Darby canine kidney (MDCK) and Vero cells. Using influenza A(H1N1)pdm09 virus as the testing platform and an integrating error-prone PCR-based mutagenesis strategy, we identified a Y161F mutation in hemagglutinin (HA) that not only enhanced the infectivity of the resultant virus by more than 300-fold but also increased its thermostability without changing its original antigenic properties. The vaccine produced from the Y161F mutant fully protected mice against lethal challenge with wild-type A(H1N1)pdm09. Compared with A(H1N1)pdm09, the Y161F mutant had significantly higher avidity for avian-like and human-like receptor analogs. Of note, the introduction of the Y161F mutation into HA of seasonal H3N2 influenza A virus (IAV) and canine H3N8 IAV also increased yields and thermostability in MDCK cells and chicken embryotic eggs. Thus, residue F161 plays an important role in determining viral growth and thermostability, which could be harnessed to optimize IAV vaccine seed viruses. Although a promising complement to current egg-based influenza vaccines, cell-based vaccines have one large challenge: high-yield vaccine seeds for production. In this study, we identified a molecular signature, Y161F, in hemagglutinin (HA) that resulted in increased virus growth in Madin-Darby canine kidney and Vero cells, two cell lines commonly used for influenza vaccine manufacturing. This Y161F mutation not only increased HA thermostability but also enhanced its binding affinity for α2,6- and α2,3-linked Neu5Ac. These results suggest that a vaccine strain bearing the Y161F mutation in HA could potentially increase vaccine yields in mammalian cell culture systems.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A Y161F Hemagglutinin Substitution Increases Thermostability and Improves Yields of 2009 H1N1 Influenza A Virus in Cells

Wen

Zhao

et al. 2018

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…The fidelity of the reaction can be further reduced by altering the amount of bivalent cations Mn 2+ and Mg 2+ , introduction of biased concentrations of deoxyribonucleoside triphosphates (dNTPs) [210], using mutagenic dNTP analogues [211], or adjusting elongation time and the number of cycles [212]. Random mutations can also be induced by utilizing 3'-5' proofreading-deficient polymerases [213][214][215].…”

Section: Random Mutagenesismentioning

confidence: 99%

Evolving a Peptide: Library Platforms and Diversification Strategies

Bozovičar

Bratkovič

2019

IJMS

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Peptides are widely used in pharmaceutical industry as active pharmaceutical ingredients, versatile tools in drug discovery, and for drug delivery. They find themselves at the crossroads of small molecules and proteins, possessing favorable tissue penetration and the capability to engage into specific and high-affinity interactions with endogenous receptors. One of the commonly employed approaches in peptide discovery and design is to screen combinatorial libraries, comprising a myriad of peptide variants of either chemical or biological origin. In this review, we focus mainly on recombinant peptide libraries, discussing different platforms for their display or expression, and various diversification strategies for library design. We take a look at well-established technologies as well as new developments and future directions.

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“…By manipulating DNA amplification conditions, ideal mismatches can be created due to the nature of the polymerase that lacks 3′-5′ proofreading ability. A commercially available Taq polymerase, Mutazyme (Agilent Technologies), was engineered for error-prone PCR to reduce its mutational bias for prohibitive selection preference on certain nucleotides during amplification [131,132]. Meanwhile, MutaGen™ is an in vitro random mutagenesis approach that utilizes low-fidelity human DNA polymerase, known as mutases [133].…”

Section: In Vitro Affinity Maturation Strategiesmentioning

confidence: 99%

Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation

Lim

Choong

Lim

2019

IJMS

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Antibodies leverage on their unique architecture to bind with an array of antigens. The strength of interaction has a direct relation to the affinity of the antibodies towards the antigen. In vivo affinity maturation is performed through multiple rounds of somatic hypermutation and selection in the germinal centre. This unique process involves intricate sequence rearrangements at the gene level via molecular mechanisms. The emergence of in vitro display technologies, mainly phage display and recombinant DNA technology, has helped revolutionize the way antibody improvements are being carried out in the laboratory. The adaptation of molecular approaches in vitro to replicate the in vivo processes has allowed for improvements in the way recombinant antibodies are designed and tuned. Combinatorial libraries, consisting of a myriad of possible antibodies, are capable of replicating the diversity of the natural human antibody repertoire. The isolation of target-specific antibodies with specific affinity characteristics can also be accomplished through modification of stringent protocols. Despite the ability to screen and select for high-affinity binders, some ‘fine tuning’ may be required to enhance antibody binding in terms of its affinity. This review will provide a brief account of phage display technology used for antibody generation followed by a summary of different combinatorial library characteristics. The review will focus on available strategies, which include molecular approaches, next generation sequencing, and in silico approaches used for antibody affinity maturation in both therapeutic and diagnostic applications.

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Error-prone pcr-based mutagenesis strategy for rapidly generating high-yield influenza vaccine candidates

Cited by 16 publications

References 43 publications

A Y161F Hemagglutinin Substitution Increases Thermostability and Improves Yields of 2009 H1N1 Influenza A Virus in Cells

A Y161F Hemagglutinin Substitution Increases Thermostability and Improves Yields of 2009 H1N1 Influenza A Virus in Cells

Evolving a Peptide: Library Platforms and Diversification Strategies

Cognizance of Molecular Methods for the Generation of Mutagenic Phage Display Antibody Libraries for Affinity Maturation

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