Rationally Designed Influenza Virus Vaccines That Are Antigenically Stable during Growth in Eggs

Harding, Alfred T.; Heaton, Brook E.; Dumm, Rebekah E.; Heaton, Nicholas S.

doi:10.1128/mbio.00669-17

Cited by 43 publications

(71 citation statements)

References 41 publications

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“…We chose to use the H1N1 strain A/Puerto Rico/8/1934 (PR8) in this study for two major reasons. First, the virus grows robustly under laboratory conditions and we have many reporter strains available (including Cre expressing viruses) in this genetic background (Harding et al, 2017; Heaton et al, 2014; Heaton et al, 2013). Second, our strain of PR8 has been extensively passaged in embryonated chicken eggs, which causes the acquisition of an avian strain receptor preference (Gambaryan et al, 1999; Meng et al, 2010).…”

Section: Resultsmentioning

confidence: 99%

“…PR8 expressing Nanoluc luciferase was generated as previously described for our Gaussia luciferase virus (Heaton et al, 2013), simply replacing the reporter gene. PR8-mNeon was generated via insertion of the mNeon fluorescent gene (Shaner et al, 2013) into segment 4 of the virus (Harding et al, 2017). Dr. Peter Palese kindly provided wild-type influenza viruses A/Beijing/47/1992, A/Bayern/7/1995, and A/Wyoming/3/2005.…”

Section: Methodsmentioning

confidence: 99%

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A CRISPR Activation Screen Identifies a Pan-avian Influenza Virus Inhibitory Host Factor

et al. 2017

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Summary Influenza A virus (IAV) is a pathogen that poses significant risks to human health. It is therefore critical to develop strategies to prevent influenza disease. Many loss-of-function screens have been performed to identify the host proteins required for viral infection. However, there has been no systematic screen to identify the host factors that when over-expressed are sufficient to prevent infection. In this study, we utilized CRISPR/dCas9 activation technology to perform a genome-wide overexpression screen to identify IAV restriction factors. The major hit from our screen, B4GALNT2, showed inhibitory activity against influenza viruses with an α2,3 linked sialic acid receptor preference. In fact, B4GALNT2 overexpression prevented the infection of every avian influenza virus strain tested, including the H5, H9, and H7 subtypes, which have previously caused disease in humans. Thus, we have utilized CRISPR/dCas9 activation technology to identify a factor that can completely abolish infection by avian influenza viruses.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

A CRISPR Activation Screen Identifies a Pan-avian Influenza Virus Inhibitory Host Factor

et al. 2017

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“…Reporter-expressing, replication-competent IAV represents an excellent tool for basic and/or translational studies and has drastically improved our knowledge of viral replication and pathogenesis (2,(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). Various research groups, including ours, have previously described reporter-expressing recombinant IAV encoding a single fluorescent or luciferase reporter gene to study the biology of IAV and to evaluate the efficacy of new antiviral and/or vaccine approaches (2, 28-31, 33-40, 48, 54).…”

Section: Discussionmentioning

confidence: 99%

“…The modification of viral segments for the incorporation of reporter genes, such as fluorescent or luciferase proteins, in replication-competent IAV has been a crucial technological advance in the field. Genetically modified IAV expressing reporter genes is an excellent tool for the tracking of viral infection in vitro and in vivo, providing a robust quantitative readout of viral replication (2,(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). In addition, this readout is compatible with high-throughput screening (HTS) technologies and useful to assess viral infection in cultured cells and animal models without the use of laborious secondary approaches to identify the presence of the virus in infected cells and/or animals (2,(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41).…”

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confidence: 99%

A Novel Fluorescent and Bioluminescent Bireporter Influenza A Virus To Evaluate Viral Infections

Nogales

Ávila‐Pérez

Rangel-Moreno

et al. 2019

J Virol

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Studying influenza A virus (IAV) requires the use of secondary approaches to detect the presence of virus in infected cells. To overcome this problem, we and others have generated recombinant IAV expressing fluorescent or luciferase reporter genes. These foreign reporter genes can be used as valid surrogates to track the presence of virus. However, the limited capacity for incorporating foreign sequences in the viral genome forced researchers to select a fluorescent or a luciferase reporter gene, depending on the type of study. To circumvent this limitation, we engineered a novel recombinant replication-competent bireporter IAV (BIRFLU) expressing both fluorescent and luciferase reporter genes. In cultured cells, BIRFLU displayed growth kinetics comparable to those of wild-type (WT) virus and was used to screen neutralizing antibodies or compounds with antiviral activity. The expression of two reporter genes allows monitoring of viral inhibition by fluorescence or bioluminescence, overcoming the limitations associated with the use of one reporter gene as a readout. In vivo, BIRFLU effectively infected mice, and both reporter genes were detected using in vivo imaging systems (IVIS). The ability to generate recombinant IAV harboring multiple foreign genes opens unique possibilities for studying virus-host interactions and for using IAV in high-throughput screenings (HTS) to identify novel antivirals that can be incorporated into the therapeutic armamentarium to control IAV infections. Moreover, the ability to genetically manipulate the viral genome to express two foreign genes offers the possibility of developing novel influenza vaccines and the feasibility for using recombinant IAV as vaccine vectors to treat other pathogen infections. IMPORTANCE Influenza A virus (IAV) causes a human respiratory disease that is associated with significant health and economic consequences. In recent years, the use of replication-competent IAV expressing an easily traceable fluorescent or luciferase reporter protein has significantly contributed to progress in influenza research. However, researchers have been forced to select a fluorescent or a luciferase reporter gene due to the restricted capacity of the influenza viral genome for including foreign sequences. To overcome this limitation, we generated, for the first time, a recombinant replication-competent bireporter IAV (BIRFLU) that stably expresses two reporter genes (one fluorescent and one luciferase) to track IAV infections in vitro and in vivo. The combination of cutting-edge techniques from molecular biology, animal research, and imaging technologies brings researchers the unique opportunity to use this new generation of reporter-expressing IAV to study viral infection dynamics in both cultured cells and animal models of viral infection. RESULTSGeneration of a BIRFLU expressing NLuc and Venus. To generate replicationcompetent bireporter IAV (BIRFLU) (Fig. 1), the sequence of NLuc and the porcine teschovirus 1 (PTV-1) 2A autoproteolytic cleavage site were cloned in front ...

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“…To do this, we transfected a pool of 6 crRNAs targeting a given IAV genome segment. Two days after crRNA transfection, the Cas13d A549 cells were challenged with PR8-mNeon, a strain of H1N1 IAV (A/Puerto Rico/8/1934) engineered to express the mNeonGreen gene, a fluorescent reporter protein (hereafter referred to as PR8-mNeon) at an MOI of 2.5 or 5.0 [26][27][28] . At approximately 18 hours post-challenge, the cells were analyzed for IAV infection through flow cytometry and microscopy.…”

Section: Crispr Pac-man Is Able To Inhibit Iav Infection In Human Lunmentioning

confidence: 99%

Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza

Abbott

Dhamdhere

Liu

et al. 2020

Preprint

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The outbreak of the coronavirus disease 2019 , caused by the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), has infected more than 100,000 people worldwide with over 3,000 deaths since December 2019. There is no cure for COVID-19 and the vaccine development is estimated to require 12-18 months. Here we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (Prophylactic Antiviral CRISPR in huMAN cells), for viral inhibition that can effectively degrade SARS-CoV-2 sequences and live influenza A virus (IAV) genome in human lung epithelial cells. We designed and screened a group of CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs for cleaving SARS-CoV-2. The approach is effective in reducing respiratory cell viral replication for H1N1 IAV. Our bioinformatic analysis showed a group of only six crRNAs can target more than 90% of all coronaviruses. The PAC-MAN approach is potentially a rapidly implementable pan-coronavirus strategy to deal with emerging pandemic strains.

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Rationally Designed Influenza Virus Vaccines That Are Antigenically Stable during Growth in Eggs

Cited by 43 publications

References 41 publications

A CRISPR Activation Screen Identifies a Pan-avian Influenza Virus Inhibitory Host Factor

A CRISPR Activation Screen Identifies a Pan-avian Influenza Virus Inhibitory Host Factor

A Novel Fluorescent and Bioluminescent Bireporter Influenza A Virus To Evaluate Viral Infections

Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza

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