Evaluation of the Influenza A Replicon for Transient Expression of Recombinant Proteins in Mammalian Cells

Avian influenza A/H7N9 is a subtype of influenza viruses that has been detected in birds in the past. This subtype has not been previously seen in either animals or human until it was found in March 2013 in China. However, since then, infections of modified H7N9 in both human and birds have been observed such as H7N2, H7N3 and H7N5 subtypes. Hemagglutinin (HA) is the most important antigen for induction of immunity, and is a target to develop influenza vaccines. In this study, the gene encoding HA protein was sucessfully inserted into pRTRA vectors to generate 35S-HA-histag-cmyc-ELP-KDEL and 35S-HApII-histag-cmyc-ELP-KDEL cassettes in the absence and presence of trimer motif GCN4, respectively in order to enhance accumulation of recombinant proteins in plants. These cassettes were inserted into pCB301 binary vectors and transformed into Agrobacterium tumefaciens in order to carry out the transient expression in Nicotiana benthamiana leaves. Five days after infiltration, the total soluble protein was extracted and analysed in SDS-PAGE. The HA-ELP and (HApII-ELP)3 recombinant proteins were detected by Western blot using anti-cmyc monoclonal antibody. These results showed that HA protein fused ELP was observed in monomeric and trimeric forms. The plant extract containing hemagglutinin protein in monomeric and trimeric forms were characterized bio-function using hemagglutination assay. The plant extract containing hemagglutinin protein in trimeric form agglutinated chicken red blood cells at concentration of 94 µg protein/ml whereas the plant extract containing hemagglutinin protein in monomeric form did not agglutinate chicken red blood cells at the same of protein concentration.

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Faculty Opinions recommendation of Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1033203.375757 ◽

2006 ◽

Author(s):

David Waugh

Keyword(s):

Recombinant Proteins ◽

Mammalian Cells ◽

Highly Efficient

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Faculty Opinions recommendation of Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1148890.618063 ◽

2009 ◽

Author(s):

Dehua Pei

Keyword(s):

Chemical Modification ◽

Recombinant Proteins ◽

Mammalian Cells ◽

Specific Chemical ◽

Site Specific ◽

Specific Chemical Modification

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Dicer is involved in protection against influenza A virus infection

Journal of General Virology ◽

10.1099/vir.0.83103-0 ◽

2007 ◽

Vol 88 (10) ◽

pp. 2627-2635 ◽

Cited By ~ 80

Author(s):

Alexey A. Matskevich ◽

Karin Moelling

Keyword(s):

Virus Infection ◽

Influenza A Virus ◽

Mammalian Cells ◽

Influenza A ◽

Virus Production ◽

Vero Cells ◽

Antiviral Response ◽

Protein Levels ◽

Infected Cells ◽

Influenza A Virus Infection

In mammals the interferon (IFN) system is a central innate antiviral defence mechanism, while the involvement of RNA interference (RNAi) in antiviral response against RNA viruses is uncertain. Here, we tested whether RNAi is involved in the antiviral response in mammalian cells. To investigate the role of RNAi in influenza A virus-infected cells in the absence of IFN, we used Vero cells that lack IFN-α and IFN-β genes. Our results demonstrate that knockdown of a key RNAi component, Dicer, led to a modest increase of virus production and accelerated apoptosis of influenza A virus-infected cells. These effects were much weaker in the presence of IFN. The results also show that in both Vero cells and the IFN-producing alveolar epithelial A549 cell line influenza A virus targets Dicer at mRNA and protein levels. Thus, RNAi is involved in antiviral response, and Dicer is important for protection against influenza A virus infection.

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Host-cell dependent role of phosphorylated keratin 8 during influenza A/NWS/33 virus (H1N1) infection in mammalian cells

Virus Research ◽

10.1016/j.virusres.2021.198333 ◽

2021 ◽

Vol 295 ◽

pp. 198333

Author(s):

Flora De Conto ◽

Francesca Conversano ◽

Sergey V. Razin ◽

Silvana Belletti ◽

Maria Cristina Arcangeletti ◽

...

Keyword(s):

Host Cell ◽

Mammalian Cells ◽

Influenza A ◽

Virus H1n1 ◽

H1n1 Infection ◽

Keratin 8

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Combination of conserved recombinant proteins (NP & 3M2e) formulated with Alum protected BALB/c mice against influenza A/PR8/H1N1 virus challenge

Biotechnology Letters ◽

10.1007/s10529-021-03174-2 ◽

2021 ◽

Author(s):

Mehrnaz Forqani ◽

Seyed Masoud Hosseini ◽

Behrokh Farahmand ◽

Maryam Saleh ◽

Hadiseh Shokouhi ◽

...

Keyword(s):

Recombinant Proteins ◽

Influenza A ◽

H1n1 Virus ◽

Virus Challenge

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Comparison of Expression Vectors for Transient Expression of Recombinant Proteins in Plants

Plant Molecular Biology Reporter ◽

10.1007/s11105-013-0614-z ◽

2013 ◽

Vol 31 (6) ◽

pp. 1529-1538 ◽

Cited By ~ 22

Author(s):

Kausar Hussain Shah ◽

Bachar Almaghrabi ◽

Holger Bohlmann

Keyword(s):

Transient Expression ◽

Recombinant Proteins ◽

Expression Vectors

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Plasticity of the Influenza Virus H5 HA Protein

mBio ◽

10.1128/mbio.03324-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Huihui Kong ◽

David F. Burke ◽

Tiago Jose da Silva Lopes ◽

Kosuke Takada ◽

Masaki Imai ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Mammalian Cells ◽

Influenza A ◽

Viral Antigen ◽

Influenza Viruses ◽

Influenza A Viruses ◽

Highly Pathogenic ◽

Antigenic Properties ◽

Ha Protein

ABSTRACT Since the emergence of highly pathogenic avian influenza viruses of the H5 subtype, the major viral antigen, hemagglutinin (HA), has undergone constant evolution, resulting in numerous genetic and antigenic (sub)clades. To explore the consequences of amino acid changes at sites that may affect the antigenicity of H5 viruses, we simultaneously mutated 17 amino acid positions of an H5 HA by using a synthetic gene library that, theoretically, encodes all combinations of the 20 amino acids at the 17 positions. All 251 mutant viruses sequenced possessed ≥13 amino acid substitutions in HA, demonstrating that the targeted sites can accommodate a substantial number of mutations. Selection with ferret sera raised against H5 viruses of different clades resulted in the isolation of 39 genotypes. Further analysis of seven variants demonstrated that they were antigenically different from the parental virus and replicated efficiently in mammalian cells. Our data demonstrate the substantial plasticity of the influenza virus H5 HA protein, which may lead to novel antigenic variants. IMPORTANCE The HA protein of influenza A viruses is the major viral antigen. In this study, we simultaneously introduced mutations at 17 amino acid positions of an H5 HA expected to affect antigenicity. Viruses with ≥13 amino acid changes in HA were viable, and some had altered antigenic properties. H5 HA can therefore accommodate many mutations in regions that affect antigenicity. The substantial plasticity of H5 HA may facilitate the emergence of novel antigenic variants.

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Characterization of Subtype H6 Avian Influenza A Viruses Isolated From Wild Birds in Poyang Lake, China

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.685399 ◽

2021 ◽

Vol 8 ◽

Author(s):

Zhimin Wan ◽

Qiuqi Kan ◽

Zhehong Zhao ◽

Hongxia Shao ◽

Thomas J. Deliberto ◽

...

Keyword(s):

Genetic Diversity ◽

Avian Influenza ◽

Mammalian Cells ◽

Influenza A ◽

Poyang Lake ◽

Wild Birds ◽

Mouse Lung ◽

Influenza A Viruses ◽

Domestic Poultry ◽

History Of

Subtype H6 avian influenza A viruses (IAVs) are enzootic and genetically diverse in both domestic poultry and wild waterfowl and may cause spillovers in both pigs and humans. Thus, it is important to understand the genetic diversity of H6 IAVs in birds and their zoonotic potential. Compared with that in domestic poultry, the genetic diversity of H6 viruses in wild birds in China has not been well-understood. In this study, five H6 viruses were isolated from wild birds in Poyang Lake, China, and genetic analyses showed that these isolates are clustered into four genotypes associated with reassortments among avian IAVs from domestic poultry and wild birds in China and those from Eurasia and North America and that these viruses exhibited distinct phenotypes in growth kinetics analyses with avian and mammalian cells lines and in mouse challenge experiments. Of interest is that two H6 isolates from the Eurasian teal replicated effectively in the mouse lung without prior adaptation, whereas the other three did not. Our study suggested that there are variations in the mammalian viral replication efficiency phenotypic among genetically diverse H6 IAVs in wild birds and that both intra- and inter-continental movements of IAVs through wild bird migration may facilitate the emergence of novel H6 IAV reassortants with the potential for replicating in mammals, including humans. Continued surveillance to monitor the diversity of H6 IAVs in wild birds is necessary to increase our understanding of the natural history of IAVs.

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Broadly Reactive H2 Hemagglutinin Vaccines Elicit Cross-Reactive Antibodies in Ferrets Preimmune to Seasonal Influenza A Viruses

mSphere ◽

10.1128/msphere.00052-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Z. Beau Reneer ◽

Amanda L. Skarlupka ◽

Parker J. Jamieson ◽

Ted M. Ross

Keyword(s):

Influenza Virus ◽

Immune Responses ◽

Recombinant Proteins ◽

Human Population ◽

Influenza A ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Wild Type ◽

Influenza A Viruses ◽

Global Pandemic

ABSTRACT Influenza vaccines have traditionally been tested in naive mice and ferrets. However, humans are first exposed to influenza viruses within the first few years of their lives. Therefore, there is a pressing need to test influenza virus vaccines in animal models that have been previously exposed to influenza viruses before being vaccinated. In this study, previously described H2 computationally optimized broadly reactive antigen (COBRA) hemagglutinin (HA) vaccines (Z1 and Z5) were tested in influenza virus “preimmune” ferret models. Ferrets were infected with historical, seasonal influenza viruses to establish preimmunity. These preimmune ferrets were then vaccinated with either COBRA H2 HA recombinant proteins or wild-type H2 HA recombinant proteins in a prime-boost regimen. A set of naive preimmune or nonpreimmune ferrets were also vaccinated to control for the effects of the multiple different preimmunities. All of the ferrets were then challenged with a swine H2N3 influenza virus. Ferrets with preexisting immune responses influenced recombinant H2 HA-elicited antibodies following vaccination, as measured by hemagglutination inhibition (HAI) and classical neutralization assays. Having both H3N2 and H1N1 immunological memory regardless of the order of exposure significantly decreased viral nasal wash titers and completely protected all ferrets from both morbidity and mortality, including the mock-vaccinated ferrets in the group. While the vast majority of the preimmune ferrets were protected from both morbidity and mortality across all of the different preimmunities, the Z1 COBRA HA-vaccinated ferrets had significantly higher antibody titers and recognized the highest number of H2 influenza viruses in a classical neutralization assay compared to the other H2 HA vaccines. IMPORTANCE H1N1 and H3N2 influenza viruses have cocirculated in the human population since 1977. Nearly every human alive today has antibodies and memory B and T cells against these two subtypes of influenza viruses. H2N2 influenza viruses caused the 1957 global pandemic and people born after 1968 have never been exposed to H2 influenza viruses. It is quite likely that a future H2 influenza virus could transmit within the human population and start a new global pandemic, since the majority of people alive today are immunologically naive to viruses of this subtype. Therefore, an effective vaccine for H2 influenza viruses should be tested in an animal model with previous exposure to influenza viruses that have circulated in humans. Ferrets were infected with historical influenza A viruses to more accurately mimic the immune responses in people who have preexisting immune responses to seasonal influenza viruses. In this study, preimmune ferrets were vaccinated with wild-type (WT) and COBRA H2 recombinant HA proteins in order to examine the effects that preexisting immunity to seasonal human influenza viruses have on the elicitation of broadly cross-reactive antibodies from heterologous vaccination.

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