scholarly journals Plasmid-only rescue of influenza A virus vaccine candidates

2001 ◽  
Vol 356 (1416) ◽  
pp. 1965-1973 ◽  
Author(s):  
J. H. Schickli ◽  
A. Flandorfer ◽  
T. Nakaya ◽  
L. Martinez-Sobrido ◽  
A. Garcia-Sastre ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Genetic Manipulation ◽  
Influenza Viruses ◽  
High Yield ◽  
Ns1 Protein ◽  
Potential Threat ◽  
Rescue System ◽  
Rapid Generation

The potential threat of another influenza virus pandemic stimulates discussion on how to prepare for such an event. The most reasonable prophylactic approach appears to be the use of effective vaccines. Since influenza and other negative–stranded RNA viruses are amenable to genetic manipulation using transfection by plasmids, it is possible to outline new reverse genetics–based approaches for vaccination against influenza viruses. We suggest three approaches. First, we use a plasmid–only rescue system that allows the rapid generation of high–yield recombinant vaccine strains. Second, we propose developing second–generation live influenza virus vaccines by constructing an attenuated master strain with deletions in the NS1 protein, which acts as an interferon antagonist. Third, we suggest the use of Newcastle disease virus recombinants expressing influenza virus haemagglutinin proteins of pandemic (epizootic) strains as novel vaccine vectors for use in animals and possibly humans.

scholarly journals Dual R108K and G189D Mutations in the NS1 Protein of A/H1N1 Influenza Virus Counteract Host Innate Immune Responses

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 905
Author(s):  
Meng-Ting Huang ◽  
Sen Zhang ◽  
Ya-Nan Wu ◽  
Wei Li ◽  
Yu-Chang Li ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Nonstructural Protein ◽  
H1n1 Influenza ◽  
Influenza Viruses ◽  
Innate Immune ◽  
Ns1 Protein ◽  
H1n1 Influenza Virus ◽  
Functional Sites ◽  
Nonstructural Protein 1

Influenza A viruses (IAV) modulate host antiviral responses to promote growth and pathogenicity. Here, we examined the multifunctional IAV nonstructural protein 1 (NS1) of influenza A virus to better understand factors that contribute to viral replication efficiency or pathogenicity. In 2009, a pandemic H1N1 IAV (A/California/07/2009 pH1N1) emerged in the human population from swine. Seasonal variants of this virus are still circulating in humans. Here, we compared the sequence of a seasonal variant of this H1N1 influenza virus (A/Urumqi/XJ49/2018(H1N1), first isolated in 2018) with the pandemic strain A/California/07/2009. The 2018 virus harbored amino acid mutations (I123V and N205S) in important functional sites; however, 108R and 189G were highly conserved between A/California/07/2009 and the 2018 variant. To better understand interactions between influenza viruses and the human innate immune system, we generated and rescued seasonal 2009 H1N1 IAV mutants expressing an NS1 protein harboring a dual mutation (R108K/G189D) at these conserved residues and then analyzed its biological characteristics. We found that the mutated NS1 protein exhibited systematic and selective inhibition of cytokine responses via a mechanism that may not involve binding to cleavage and polyadenylation specificity factor 30 (CPSF30). These results highlight the complexity underlying host–influenza NS1 protein interactions.

scholarly journals A Seven-Segmented Influenza A Virus Expressing the Influenza C Virus Glycoprotein HEF

2008 ◽  
Vol 82 (13) ◽  
pp. 6419-6426 ◽  
Author(s):  
Qinshan Gao ◽  
Edward W. A. Brydon ◽  
Peter Palese
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Chimeric Protein ◽  
Influenza Viruses ◽  
Viral Rna ◽  
Surface Glycoprotein ◽  
Influenza C Virus ◽  
Major Surface

ABSTRACT Influenza viruses are classified into three types: A, B, and C. The genomes of A- and B-type influenza viruses consist of eight RNA segments, whereas influenza C viruses only have seven RNAs. Both A and B influenza viruses contain two major surface glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). Influenza C viruses have only one major surface glycoprotein, HEF (hemagglutinin-esterase fusion). By using reverse genetics, we generated two seven-segmented chimeric influenza viruses. Each possesses six RNA segments from influenza virus A/Puerto Rico/8/34 (PB2, PB1, PA, NP, M, and NS); the seventh RNA segment encodes either the influenza virus C/Johannesburg/1/66 HEF full-length protein or a chimeric protein HEF-Ecto, which consists of the HEF ectodomain and the HA transmembrane and cytoplasmic regions. To facilitate packaging of the heterologous segment, both the HEF and HEF-Ecto coding regions are flanked by HA packaging sequences. When introduced as an eighth segment with the NA packaging sequences, both viruses are able to stably express a green fluorescent protein (GFP) gene, indicating a potential use for these viruses as vaccine vectors to carry foreign antigens. Finally, we show that incorporation of a GFP RNA segment enhances the growth of seven-segmented viruses, indicating that efficient influenza A viral RNA packaging requires the presence of eight RNA segments. These results support a selective mechanism of viral RNA recruitment to the budding site.

scholarly journals Cloning of the Chicken RNA Polymerase I Promoter and Use for Reverse Genetics of Influenza A Viruses in Avian Cells

2005 ◽  
Vol 79 (21) ◽  
pp. 13811-13816 ◽  
Author(s):  
Pascale Massin ◽  
Pierre Rodrigues ◽  
Monica Marasescu ◽  
Sylvie van der Werf ◽  
Nadia Naffakh
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Promoter Sequence ◽  
Influenza Viruses ◽  
Rna Polymerase I ◽  
Influenza A Viruses ◽  
Virus Research ◽  
Influenza Vaccine Production ◽  
Polymerase I

ABSTRACT Reverse genetics techniques to rescue influenza viruses have thus far been based on the use of a human polymerase I (PolI) promoter to direct the synthesis of the eight viral RNAs. They can only be used on cells from primate origin due to the species specificity of the PolI promoter. Here we report the cloning of the chicken PolI promoter sequence and the generation of recombinant influenza virus upon transfection of bidirectional PolI/PolII plasmids in avian cells. Potential contributions of this new reverse genetics system in the fields of influenza virus research and influenza vaccine production are discussed.

scholarly journals Rescue of Influenza C Virus from Recombinant DNA

2007 ◽  
Vol 81 (20) ◽  
pp. 11282-11289 ◽  
Author(s):  
Bernadette Crescenzo-Chaigne ◽  
Sylvie van der Werf
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
Recombinant Dna ◽  
Helper Virus ◽  
Influenza Viruses ◽  
Expression Vectors ◽  
Efficient System ◽  
Human Embryonic Kidney Cells ◽  
Type C

ABSTRACT The rescue of influenza viruses by reverse genetics has been described only for the influenza A and B viruses. Based on a similar approach, we developed a reverse-genetics system that allows the production of influenza C viruses entirely from cloned cDNA. The complete sequences of the 3′ and 5′ noncoding regions of type C influenza virus C/Johannesburg/1/66 necessary for the cloning of the cDNA were determined for the seven genomic segments. Human embryonic kidney cells (293T) were transfected simultaneously with seven plasmids that direct the synthesis of each of the seven viral RNA segments of the C/JHB/1/66 virus under the control of the human RNA polymerase I promoter and with four plasmids encoding the viral nucleoprotein and the PB2, PB1, and P3 proteins of the viral polymerase complex. This strategy yielded between 103 and 104 PFU of virus per ml of supernatant at 8 to 10 days posttransfection. Additional viruses with substitutions introduced in the hemagglutinin-esterase-fusion protein were successfully produced by this method, and their growth phenotype was evaluated. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and for generation of expression vectors from type C influenza virus.

scholarly journals Generation of DelNS1 Influenza Viruses: a Strategy for Optimizing Live Attenuated Influenza Vaccines

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Pui Wang ◽  
Min Zheng ◽  
Siu-Ying Lau ◽  
Pin Chen ◽  
Bobo Wing-Yee Mok ◽  
...  
Keyword(s):  
Immune Response ◽  
Influenza Virus ◽  
T Cell ◽  
Protective Immunity ◽  
Influenza A ◽  
Influenza Viruses ◽  
Cross Protection ◽  
Influenza Vaccines ◽  
Influenza B ◽  
Ns1 Protein

ABSTRACT Nonstructural protein 1 (NS1) of influenza virus is a key virulence element with multifunctional roles in virus replication and a potent antagonist of host immune response. Deletion of NS1 (DelNS1) would create a safer and more extensively immunogenic live attenuated influenza virus (LAIV) vaccine. However, DelNS1 viruses are very difficult to grow in regular vaccine-producing systems, which has hampered the application of DelNS1 LAIV vaccines in humans. We have developed two master backbones of deleted-NS1 (DelNS1) viral genomes from influenza A or B viruses which contain novel adaptive mutations to support DelNS1-LAIV replication. These DelNS1-LAIVs are highly attenuated in human cells in vitro and nonpathogenic in mice but replicate well in vaccine-producing cells. Both influenza A and influenza B DelNS1 LAIVs grow better at 33°C than at 37 to 39°C. Vaccination with DelNS1 LAIV performed once is enough to provide potent protection against lethal challenge with homologous virus and strong long-lasting cross protection against heterosubtypic or antigenically distantly related influenza viruses in mice. Mechanistic investigations revealed that DelNS1-LAIVs induce cross protective neutralizing antibody and CD8+ and CD4+ T cell immunities. Importantly, it has been shown that DelNS1-LAIV can be used to enhance specific anti-influenza immunity through expression of additional antigens from the deleted-NS1 site. Generation of DelNS1 viruses which are nonpathogenic and able to grow in vaccine-producing systems is an important strategy for making highly immunogenic LAIV vaccines that induce broad cross protective immunity against seasonal and emerging influenza. IMPORTANCE Current seasonal influenza vaccines are suboptimal and low in immunogenicity and do not provide long-lasting immunity and cross protection against influenza virus strains that have antigenically drifted. More-effective influenza vaccines which can induce both humoral immunity and T cell immunity are needed. The NS1 protein of influenza virus is a virulence element and the critical factor for regulation of the host immune response during virus infection. Deletion of the NS1 protein is a strategy to make an optimal LAIV vaccine. However, DelNS1 viruses are very difficult to grow in regular vaccine-producing systems, hampering the application of DelNS1 LAIV vaccines in humans. We have generated a panel of both influenza A and influenza B DelNS1 LAIVs which are able to grow in regular vaccine-producing cells. These DelNS1 LAIV vaccines are completely nonpathogenic, exhibit potent and long-lasting immunity, and can be used to express extra viral antigen to induce cross protective immunity against seasonal and emerging influenza.

scholarly journals Novel Approaches for The Development of Live Attenuated Influenza Vaccines

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 190 ◽  
Author(s):  
Pilar Blanco-Lobo ◽  
Aitor Nogales ◽  
Laura Rodríguez ◽  
Luis Martínez-Sobrido
Keyword(s):  
Influenza A ◽  
Reverse Genetics ◽  
Seasonal Influenza ◽  
Influenza Viruses ◽  
Antigenic Drift ◽  
Influenza Vaccines ◽  
Global Public Health ◽  
Protection Efficacy ◽  
Seasonal Epidemics ◽  
Rapid Generation

Influenza virus still represents a considerable threat to global public health, despite the advances in the development and wide use of influenza vaccines. Vaccination with traditional inactivate influenza vaccines (IIV) or live-attenuated influenza vaccines (LAIV) remains the main strategy in the control of annual seasonal epidemics, but it does not offer protection against new influenza viruses with pandemic potential, those that have shifted. Moreover, the continual antigenic drift of seasonal circulating influenza viruses, causing an antigenic mismatch that requires yearly reformulation of seasonal influenza vaccines, seriously compromises vaccine efficacy. Therefore, the quick optimization of vaccine production for seasonal influenza and the development of new vaccine approaches for pandemic viruses is still a challenge for the prevention of influenza infections. Moreover, recent reports have questioned the effectiveness of the current LAIV because of limited protection, mainly against the influenza A virus (IAV) component of the vaccine. Although the reasons for the poor protection efficacy of the LAIV have not yet been elucidated, researchers are encouraged to develop new vaccination approaches that overcome the limitations that are associated with the current LAIV. The discovery and implementation of plasmid-based reverse genetics has been a key advance in the rapid generation of recombinant attenuated influenza viruses that can be used for the development of new and most effective LAIV. In this review, we provide an update regarding the progress that has been made during the last five years in the development of new LAIV and the innovative ways that are being explored as alternatives to the currently licensed LAIV. The safety, immunogenicity, and protection efficacy profile of these new LAIVs reveal their possible implementation in combating influenza infections. However, efforts by vaccine companies and government agencies will be needed for controlled testing and approving, respectively, these new vaccine methodologies for the control of influenza infections.

scholarly journals The NS1 Protein of a Human Influenza Virus Inhibits Type I Interferon Production and the Induction of Antiviral Responses in Primary Human Dendritic and Respiratory Epithelial Cells

2009 ◽  
Vol 83 (13) ◽  
pp. 6849-6862 ◽  
Author(s):  
Kester Haye ◽  
Svetlana Burmakina ◽  
Thomas Moran ◽  
Adolfo García-Sastre ◽  
Ana Fernandez-Sesma
Keyword(s):  
Influenza Virus ◽  
Epithelial Cells ◽  
Viral Replication ◽  
Influenza A ◽  
Type I Interferon ◽  
Influenza Viruses ◽  
Type I ◽  
Ns1 Protein ◽  
Human Influenza ◽  
Human Influenza Virus

ABSTRACT The NS1 protein of the influenza A virus is a potent virulence factor that inhibits type I interferon (IFN) synthesis, allowing the virus to overcome host defenses and replicate efficiently. However, limited studies have been conducted on NS1 function using human virus strains and primary human cells. We used NS1 truncated mutant influenza viruses derived from the human isolate influenza A/TX/91 (TX WT, where WT is wild type) to study the functions of NS1 in infected primary cells. Infection of primary differentiated human tracheo-bronchial epithelial cells with an NS1 truncated mutant demonstrated limited viral replication and enhanced type I IFN induction. Additionally, human dendritic cells (DCs) infected with human NS1 mutant viruses showed higher levels of activation and stimulated naïve T-cells better than TX WT virus-infected DCs. We also compared infections of DCs with TX WT and our previously characterized laboratory strain A/PR/8/34 (PR8) and its NS1 knockout strain, deltaNS1. TX WT-infected DCs displayed higher viral replication than PR8 but had decreased antiviral gene expression at late time points and reduced naïve T-cell stimulation compared to PR8 infections, suggesting an augmented inhibition of IFN production and human DC activation. Our findings show that human-derived influenza A viruses have a high capacity to inhibit the antiviral state in a human system, and here we have evaluated the possible mechanism of this inhibition. Lastly, C-terminal truncations in the NS1 protein of human influenza virus are sufficient to make the virus attenuated and more immunogenic, supporting its use as a live attenuated influenza vaccine in humans.

scholarly journals Reverse genetics approach towards understanding pathogenesis of H5N1 Hong Kong influenza A virus infection

2001 ◽  
Vol 356 (1416) ◽  
pp. 1841-1843 ◽  
Author(s):  
Masato Hatta ◽  
Gabriele Neumann ◽  
Yoshihiro Kawaoka
Keyword(s):  
Influenza Virus ◽  
Hong Kong ◽  
Influenza A ◽  
Reverse Genetics ◽  
Gene Segment ◽  
Influenza Viruses ◽  
H5n1 Influenza ◽  
Influenza A Virus Infection ◽  
Cloned Cdna ◽  
First Time

In 1990, Palese and colleagues established a method (reverse genetics) that allowed one to generate influenza virus containing a gene segment derived from cloned cDNA. Although this method contributed tremendously to our understanding of influenza pathogenesis, the requirement of helper viruses limited its use in many experimental settings. Recently, we and others established systems for the generation of influenza viruses entirely from cloned cDNAs. These systems require only DNA cloning and transfection techniques, and can therefore be easily implemented by laboratories working in the fields of molecular biology and virology. Thus, for the first time, a system is now available that allows highly efficient generation of influenza virus without technical limitations. Using this technology, we generated the same strain of H5N1 influenza viruses that caused an outbreak in Hong Kong in 1997, killing six people.

Generation and in vitro characterization of engineered cold-adapted influenza A strains with modified NS gene

2021 ◽  
Vol 21 (3) ◽  
pp. 153-158
Author(s):  
Anna K. Chistyakova ◽  
Polina I. Prokopenko ◽  
Elena V. Krutikova ◽  
Ekaterina A. Stepanova ◽  
Irina N. Isakova-Sivak ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza Vaccine ◽  
Influenza A ◽  
Influenza Viruses ◽  
T Cell Epitopes ◽  
Ns1 Protein ◽  
Cold Adapted ◽  
Ns Gene ◽  
Wide Range

BACKGROUND: The high variability of influenza strains and the emergence of new variants of viruses lead to the need for constant updating of the composition of influenza vaccines. One of the options for solving this problem is the development of vaccines with enhanced cross-protection against a wide range of influenza strains. Genetically engineered preparations based on live influenza vaccine can be used for targeted stimulation of the cellular immune response. It has been experimentally established that CTL epitopes inserted into the NS gene of the live influenza vaccine strain cause the activation of lymphocytes and the formation of a pool of resident memory T-cells in the lungs of model animals. It is optimal to use experimentally confirmed immunogenic regions for insertion. AIM: The aim of this study was to rescue a panel of experimental cold-adapted live attenuated influenza vaccine strains with a modified NS gene using A/Leningrad/134/17/57 backbone and recent influenza strains of H1N1, H3N2 and H7N9 subtypes, and evaluate their properties in vitro. MATERIALS AND METHODS: A cassette encoding immunogenic, conserved among a wide range of influenza strains T-cell epitopes of the influenza virus PB1 protein restricted by common HLA-allotypes was inserted into the gene encoding the NS1 protein. The modified NS gene was cloned into the pCIPolISapIT influenza virus reverse genetics vector. Chimeric influenza viruses were rescued by transfection of Vero cells by electroporation using a standard 8-plasmid system. The growth characteristics of viruses were assessed in developing chicken embryos. Results: Three strains were successfully obtained based on the live influenza vaccine master donor virus A/Leningrad/ 134/17/57 with a modified NS gene and influenza viruses of the H1N1, H3N2, H7N9 subtypes. Thus, modification of NS gene by insertion of immunogenic PB1 epitopes did not affect the viability and replicative activity of the rescued chimeric live influenza vaccine strains, regardless of the composition of the surface proteins. The strains replicated well at an optimal temperature, had temperature-sensitive phenotype and were able to grow at low temperature. CONCLUSIONS: The strains will be further studied as candidates for influenza prophylaxis as an experimental universal influenza vaccine.

scholarly journals Virulence Determinants of Avian H5N1 Influenza A Virus in Mammalian and Avian Hosts: Role of the C-Terminal ESEV Motif in the Viral NS1 Protein

2010 ◽  
Vol 84 (20) ◽  
pp. 10708-10718 ◽  
Author(s):  
Florian Zielecki ◽  
Ilia Semmler ◽  
Donata Kalthoff ◽  
Daniel Voss ◽  
Susanne Mauel ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Viral Replication ◽  
Influenza A ◽  
Pdz Domain ◽  
Influenza Viruses ◽  
Dependent Manner ◽  
Ns1 Protein ◽  
H5n1 Influenza

ABSTRACT We assessed the prediction that access of the viral NS1 protein to cellular PDZ domain protein networks enhances the virulence of highly pathogenic avian influenza A viruses. The NS1 proteins of most avian influenza viruses bear the C-terminal ligand sequence Glu-Ser-Glu-Val (ESEV) for PDZ domains present in multiple host proteins, whereas no such motif is found in the NS1 homologues of seasonal human virus strains. Previous analysis showed that a C-terminal ESEV motif increases viral virulence when introduced into the NS1 protein of mouse-adapted H1N1 influenza virus. To examine the role of the PDZ domain ligand motif in avian influenza virus virulence, we generated three recombinants, derived from the prototypic H5N1 influenza A/Vietnam/1203/04 virus, expressing NS1 proteins that either have the C-terminal ESEV motif or the human influenza virus RSKV consensus or bear a natural truncation of this motif, respectively. Cell biological analyses showed strong control of NS1 nuclear migration in infected mammalian and avian cells, with only minor differences between the three variants. The ESEV sequence attenuated viral replication on cultured human, murine, and duck cells but not on chicken fibroblasts. However, all three viruses caused highly lethal infections in mice and chickens, with little difference in viral titers in organs, mean lethal dose, or intravenous pathogenicity index. These findings demonstrate that a PDZ domain ligand sequence in NS1 contributes little to the virulence of H5N1 viruses in these hosts, and they indicate that this motif modulates viral replication in a strain- and host-dependent manner.

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