scholarly journals Influenza A Virus Lacking M2 Protein as a Live Attenuated Vaccine

2009 ◽  
Vol 83 (11) ◽  
pp. 5947-5950 ◽  
Author(s):  
Shinji Watanabe ◽  
Tokiko Watanabe ◽  
Yoshihiro Kawaoka
Keyword(s):  
Influenza Virus ◽  
Immune Responses ◽  
Influenza A ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Lethal Challenge ◽  
Live Attenuated Vaccine ◽  
Attenuated Vaccine ◽  
Live Attenuated Influenza Vaccine

ABSTRACT Mutant influenza virus that lacks the transmembrane and cytoplasmic tail domains of M2 (M2 knockout [M2KO]) is attenuated in both cell culture and mice. Here, we examined the potency of M2KO influenza virus as a live attenuated influenza vaccine. M2KO virus grew as efficiently as the wild-type virus in cells stably expressing the wild-type M2, indicating the feasibility of efficient vaccine production. Mice intranasally vaccinated with M2KO virus developed protective immune responses and survived a lethal challenge with the wild-type virus, suggesting that the M2KO virus has potential as a live attenuated vaccine.

scholarly journals Immunogenicity and Cross Protection in Mice Afforded by Pandemic H1N1 Live Attenuated Influenza Vaccine Containing Wild-Type Nucleoprotein

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Andrey Rekstin ◽  
Irina Isakova-Sivak ◽  
Galina Petukhova ◽  
Daniil Korenkov ◽  
Igor Losev ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Immune Responses ◽  
Influenza Vaccine ◽  
Wild Type Virus ◽  
Pandemic H1n1 ◽  
Type Virus ◽  
Wild Type ◽  
Live Attenuated Influenza Vaccine ◽  
Np Gene

Since conserved viral proteins of influenza virus, such as nucleoprotein (NP) and matrix 1 protein, are the main targets for virus-specific CD8+ cytotoxic T-lymphocytes (CTLs), we hypothesized that introduction of the NP gene of wild-type virus into the genome of vaccine reassortants could lead to better immunogenicity and afford better protection. This paper describes in vitro and in vivo preclinical studies of two new reassortants of pandemic H1N1 live attenuated influenza vaccine (LAIV) candidates. One had the hemagglutinin (HA) and neuraminidase (NA) genes from A/South Africa/3626/2013 H1N1 wild-type virus on the A/Leningrad/134/17/57 master donor virus backbone (6 : 2 formulation) while the second had the HA, NA, and NP genes of the wild-type virus on the same backbone (5 : 3 formulation). Although both LAIVs induced similar antibody immune responses, the 5 : 3 LAIV provoked greater production of virus-specific CTLs than the 6 : 2 variant. Furthermore, the 5 : 3 LAIV-induced CTLs had higher in vivo cytotoxic activity, compared to 6 : 2 LAIV. Finally, the 5 : 3 LAIV candidate afforded greater protection against infection and severe illness than the 6 : 2 LAIV. Inclusion in LAIV of the NP gene from wild-type influenza virus is a new approach to inducing cross-reactive cell-mediated immune responses and cross protection against pandemic influenza.

scholarly journals Next generation methodology for updating HA vaccines against emerging human seasonal influenza A(H3N2) viruses

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
James D. Allen ◽  
Ted M. Ross
Keyword(s):  
Influenza Virus ◽  
Immune Responses ◽  
Influenza A ◽  
Seasonal Influenza ◽  
Influenza Viruses ◽  
Next Generation ◽  
Virus Infections ◽  
Wild Type ◽  
Influenza Hemagglutinin ◽  
H3n2 Influenza

AbstractWhile vaccines remain the best tool for preventing influenza virus infections, they have demonstrated low to moderate effectiveness in recent years. Seasonal influenza vaccines typically consist of wild-type influenza A and B viruses that are limited in their ability to elicit protective immune responses against co-circulating influenza virus variant strains. Improved influenza virus vaccines need to elicit protective immune responses against multiple influenza virus drift variants within each season. Broadly reactive vaccine candidates potentially provide a solution to this problem, but their efficacy may begin to wane as influenza viruses naturally mutate through processes that mediates drift. Thus, it is necessary to develop a method that commercial vaccine manufacturers can use to update broadly reactive vaccine antigens to better protect against future and currently circulating viral variants. Building upon the COBRA technology, nine next-generation H3N2 influenza hemagglutinin (HA) vaccines were designed using a next generation algorithm and design methodology. These next-generation broadly reactive COBRA H3 HA vaccines were superior to wild-type HA vaccines at eliciting antibodies with high HAI activity against a panel of historical and co-circulating H3N2 influenza viruses isolated over the last 15 years, as well as the ability to neutralize future emerging H3N2 isolates.

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

mSphere ◽  
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.

scholarly journals Characterization of Human Influenza Virus Variants Selected In Vitro in the Presence of the Neuraminidase Inhibitor GS 4071

1998 ◽  
Vol 42 (12) ◽  
pp. 3234-3241 ◽  
Author(s):  
Chun Y. Tai ◽  
Paul A. Escarpe ◽  
Robert W. Sidwell ◽  
Matthew A. Williams ◽  
Willard Lew ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Neuraminidase Inhibitor ◽  
H3n2 Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Human Influenza ◽  
Wild Type ◽  
Viral Virulence ◽  
High Level

ABSTRACT An oral prodrug of GS 4071, a potent and selective inhibitor of influenza neuraminidases, is currently under clinical development for the treatment and prophylaxis of influenza virus infections in humans. To investigate the potential development of resistance during the clinical use of this compound, variants of the human influenza A/Victoria/3/75 (H3N2) virus with reduced susceptibility to the neuraminidase inhibitor GS 4071 were selected in vitro by passaging the virus in MDCK cells in the presence of inhibitor. After eight passages, variants containing two amino acid substitutions in the hemagglutinin (A28T in HA1 and R124M in HA2) but no changes in the neuraminidase were isolated. These variants exhibited a 10-fold reduction in susceptibility to GS 4071 and zanamivir (GG167) in an in vitro plaque reduction assay. After 12 passages, a second variant containing these hemagglutinin mutations and a Lys substitution for the conserved Arg292 of the neuraminidase was isolated. The mutant neuraminidase enzyme exhibited high-level (30,000-fold) resistance to GS 4071, but only moderate (30-fold) resistance to zanamivir and 4-amino-Neu5Ac2en, the amino analog of zanamivir. The mutant enzyme had weaker affinity for the fluorogenic substrate 2′-(4-methylumbelliferyl)-α-d- N -acetylneuraminic acid and lower enzymatic activity compared to the wild-type enzyme. The viral variant containing the mutant neuraminidase did not replicate as well as the wild-type virus in culture and was 10,000-fold less infectious than the wild-type virus in a mouse model. These results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.

scholarly journals The Influenza A Virus M2 Cytoplasmic Tail Is Required for Infectious Virus Production and Efficient Genome Packaging

2005 ◽  
Vol 79 (6) ◽  
pp. 3595-3605 ◽  
Author(s):  
Matthew F. McCown ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Infectious Virus ◽  
Virus Production ◽  
Cytoplasmic Tail ◽  
Host Cells ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Particles

ABSTRACT The M2 integral membrane protein encoded by influenza A virus possesses an ion channel activity that is required for efficient virus entry into host cells. The role of the M2 protein cytoplasmic tail in virus replication was examined by generating influenza A viruses encoding M2 proteins with truncated C termini. Deletion of 28 amino acids (M2Stop70) resulted in a virus that produced fourfold-fewer particles but >1,000-fold-fewer infectious particles than wild-type virus. Expression of the full-length M2 protein in trans restored the replication of the M2 truncated virus. Although the M2Stop70 virus particles were similar to wild-type virus in morphology, the M2Stop70 virions contained reduced amounts of viral nucleoprotein and genomic RNA, indicating a defect in vRNP packaging. The data presented indicate the M2 cytoplasmic tail plays a role in infectious virus production by coordinating the efficient packaging of genome segments into influenza virus particles.

scholarly journals Ubiquitination of the Cytoplasmic Domain of Influenza A Virus M2 Protein is Crucial for Production of Infectious Virus Particles

2017 ◽  
pp. JVI.01972-17 ◽  
Author(s):  
Wen-Chi Su ◽  
Wen-Ya Yu ◽  
Shih-Han Huang ◽  
Michael M.C. Lai
Keyword(s):  
Virus Replication ◽  
Influenza A ◽  
Functional Role ◽  
Infectious Virus ◽  
Virus Production ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus Particles

Virus replication is mediated by interactions between virus and host. Here, we demonstrate that influenza A virus membrane protein 2 (M2) can be ubiquitinated. The lysine residue at position 78, which is located in the cytoplasmic domain of M2, is essential for M2 ubiquitination. An M2-K78R (Lys78→Arg78) mutant, which produces ubiquitination-deficient M2, showed a severe defect in production of infectious virus particles. M2-K78R mutant progeny contained more HA proteins, less viral RNAs and less internal viral proteins, including M1 and NP, than the wild-type virus. Furthermore, most of the M2-K78R mutant viral particles lacked viral ribonucleoproteins upon examination under electron microscopy and exhibited slightly lower densities. We also found that mutant M2 colocalized with M1 protein to a lesser extent than for wild-type virus. These findings may account for the reduced incorporation of viral ribonucleoprotein into virions. By blocking the second round of virus infection, we showed that the M2 ubiquitination-defective mutant exhibited normal level of virus replication during the first round of infection, thereby proving that M2 ubiquitination is involved in the virus production step. Finally, we found that M2-K78R mutant virus induced autophagy and apoptosis earlier than wild-type virus. Collectively, these results suggest that M2 ubiquitination plays an important role in infectious virus production by coordinating efficient packaging of the viral genome into virus particles and timing of viral-induced cell death.IMPORTANCEAnnual epidemics and recurring pandemics of influenza viruses represent a very high global health and economic burden. Influenza virus M2 protein has been extensively studied for its important roles in virus replication, particularly in viral entry and release. Rimantadine, one of the most commonly used antiviral drugs, binds to the channel lumen near the N-terminus of M2 proteins. However, viruses resistant to Rimantadine have emerged. M2 undergoes several posttranslational modifications, such as phosphorylation and palmitoylation. Here, we reveal that ubiquitination mediates the functional role of M2. A ubiquitination-deficient M2 mutant predominately produced virus particles either lacking viral ribonucleoproteins or containing smaller amounts of internal viral components, resulting in lower infectivity. Our findings offer insights into the mechanism of influenza virus morphogenesis, particularly the functional role of M1-M2 interactions in viral particle assembly, and can be applied to the development of new influenza therapies.

scholarly journals Cell culture (Vero) derived whole virus (H5N1) vaccine based on wild-type virus strain induces cross-protective immune responses

Vaccine ◽  
2007 ◽  
Vol 25 (32) ◽  
pp. 6028-6036 ◽  
Author(s):  
Otfried Kistner ◽  
M. Keith Howard ◽  
Martin Spruth ◽  
Walter Wodal ◽  
Peter Brühl ◽  
...  
Keyword(s):  
Cell Culture ◽  
Immune Responses ◽  
Virus Strain ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virus H5n1 ◽  
H5n1 Vaccine

scholarly journals Codon Deletions in the Influenza A Virus PA Gene Generate Temperature-Sensitive Viruses

2016 ◽  
Vol 90 (7) ◽  
pp. 3684-3693 ◽  
Author(s):  
Léa Meyer ◽  
Alix Sausset ◽  
Laura Sedano ◽  
Bruno Da Costa ◽  
Ronan Le Goffic ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Wild Type Virus ◽  
Deletion Mutants ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type

ABSTRACTThe influenza virus RNA-dependent RNA polymerase, which is composed of three subunits, PB1, PB2, and PA, catalyzes genome replication and transcription within the cell nucleus. The PA linker (residues 197 to 256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts), attenuated mutants that display a defect in the transport of the PA–PB1 complex to the nucleus at a restrictive temperature. In this study, we investigated the ability of the PA linker to tolerate deletion mutations for furtherin vitroandin vivocharacterization. Four viable mutants with single-codon deletions were generated; all of them exhibited atsphenotype that was associated with the reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using fluorescently tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at a restrictive temperature (39.5°C). Mouse infections showed that the four mutants were attenuated and induced antibodies that were able to protect mice from challenge with a lethal homologous wild-type virus. Serialin vitropassages of two deletion mutants at 39.5°C and 37°C did not allow the restoration of a wild-type phenotype among virus progeny. Thus, our results identify codons that can be deleted in the PA gene to engineer genetically stabletsmutants that could be used to design novel attenuated vaccines.IMPORTANCEIn order to generate genetically stable live influenza A virus vaccines, we constructed viruses with single-codon deletions in a discrete domain of the RNA polymerase PA gene. The four rescued viruses exhibited a temperature-sensitive phenotype that we found was associated with a defect in the transport of the PA–PB1 dimer to the nucleus, where viral replication occurs. Thesetsdeletion mutants were shown to be attenuated and to be able to produce antibodies in mice and to protect them from a lethal challenge. Assays to select revertants that were able to grow efficiently at a restrictive temperature failed, showing that these deletion mutants are genetically more stable than conventional substitution mutants. These results are of interest for the design of genetically stable live influenza virus vaccines.

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