scholarly journals A Mutated PB1 Residue 319 Synergizes with the PB2 N265S Mutation of the Live Attenuated Influenza Vaccine to Convey Temperature Sensitivity

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1246
Author(s):  
Andrew Cox ◽  
Jordana Schmierer ◽  
Josephine D’Angelo ◽  
Andrew Smith ◽  
Dustyn Levenson ◽  
...  
Keyword(s):  
Temperature Sensitivity ◽  
Genetic Background ◽  
Influenza A ◽  
Polymerase Activity ◽  
Influenza Vaccines ◽  
Potential Utility ◽  
Live Vaccines ◽  
Live Attenuated Influenza Vaccine ◽  
Complete Set ◽  
New Generation

Current influenza vaccines have modest efficacy. This is especially true for current live attenuated influenza vaccines (LAIV), which have been inferior to the inactivated versions in recent years. Therefore, a new generation of live vaccines may be needed. We previously showed that a mutation at PB1 residue 319 confers enhanced temperature sensitivity and attenuation in an LAIV constructed in the genetic background of the mouse-adapted Influenza A Virus (IAV) strain A/PR/8/34 (PR8). Here, we describe the origin/discovery of this unique mutation and demonstrate that, when combined with the PB2 N265S mutation of LAIV, it conveys an even greater level of temperature sensitivity and attenuation on PR8 than the complete set of attenuating mutations from LAIV. Furthermore, we show that the combined PB1 L319Q and PB2 N265S mutations confer temperature sensitivity on IAV polymerase activity in two different genetic backgrounds, PR8 and A/Cal/04/09. Collectively, these findings show that the PB2 LAIV mutation synergizes with a mutation in PB1 and may have potential utility for improving LAIVs.

scholarly journals Favipiravir-resistant influenza A virus shows potential for transmission

2020 ◽  
Author(s):  
Daniel H. Goldhill ◽  
Ada Yan ◽  
Rebecca Frise ◽  
Jie Zhou ◽  
Jennifer Shelley ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Genetic Background ◽  
Influenza A ◽  
Resistance Mutation ◽  
Polymerase Activity ◽  
Resistant Virus ◽  
Wild Type ◽  
Fitness Advantage

AbstractFavipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain.Author SummaryIn the event of a new influenza pandemic, drugs will be our first line of defence against the virus. However, drug resistance has proven to be particularly problematic to drugs against influenza. Favipiravir is a novel drug which might be used against influenza virus in the event of a new pandemic. Is resistance likely to be a problem for the use of favipiravir? Our previous work has shown that resistance to favipiravir can be generated in cell culture but we don’t know whether there will be a cost preventing the spread of resistance in whole organisms. Here, we used a mix of wild-type and resistant influenza viruses from early in the 2009 pandemic to test whether viruses resistant to favipiravir could transmit between ferrets. We found that the resistant viruses could transmit but that the resistance mutation was selected against within some ferrets. Using modelling and in vitro experiments, we found that the resistant mutation was selected against in the influenza strain from our experiment but not in more recently evolved strains. Our results show that favipiravir resistant viruses could spread if resistance is generated but the probability will depend on the genetic background of the virus.

scholarly journals Viral Subpopulation Screening Guides in Designing a High Interferon-Inducing Live Attenuated Influenza Vaccine by Targeting Rare Mutations in NS1 and PB2 Proteins

2020 ◽  
Vol 95 (2) ◽  
pp. e01722-20
Author(s):  
Amir Ghorbani ◽  
Michael C. Abundo ◽  
Hana Ji ◽  
Kara J. M. Taylor ◽  
John M. Ngunjiri ◽  
...  
Keyword(s):  
Cell Culture ◽  
Influenza Vaccine ◽  
Influenza Viruses ◽  
Translation Initiation Factor ◽  
Influenza Vaccines ◽  
Effective Control ◽  
Live Vaccines ◽  
Live Attenuated Influenza Vaccine ◽  
Nonstructural Protein 1 ◽  
Interferon Responses

ABSTRACTInfluenza A viruses continue to circulate among wild birds and poultry worldwide, posing constant pandemic threats to humans. Effective control of emerging influenza viruses requires new broadly protective vaccines. Live attenuated influenza vaccines with truncations in nonstructural protein 1 (NS1) have shown broad protective efficacies in birds and mammals, which correlate with the ability to induce elevated interferon responses in the vaccinated hosts. Given the extreme diversity of influenza virus populations, we asked if we could improve an NS1-truncated live attenuated influenza vaccine developed for poultry (PC4) by selecting viral subpopulations with enhanced interferon-inducing capacities. Here, we deconstructed a de novo population of PC4 through plaque isolation, created a large library of clones, and assessed their interferon-inducing phenotypes. While most of the clones displayed the parental interferon-inducing phenotype in cell culture, few clones showed enhanced interferon-inducing phenotypes in cell culture and chickens. The enhanced interferon-inducing phenotypes were linked to either a deletion in NS1 (NS1Δ76-86) or a substitution in polymerase basic 2 protein (PB2-D309N). The NS1Δ76-86 deletion disrupted the putative eukaryotic translation initiation factor 4GI-binding domain and promoted the synthesis of biologically active interferons. The PB2-D309N substitution enhanced the early transcription of interferon mRNA, revealing a novel role for the 309D residue in suppression of interferon responses. We combined these mutations to engineer a novel vaccine candidate that induced additive amounts of interferons and stimulated protective immunity in chickens. Therefore, viral subpopulation screening approaches can guide the design of live vaccines with strong immunostimulatory properties.IMPORTANCE Effectiveness of NS1-truncated live attenuated influenza vaccines relies heavily on their ability to induce elevated interferon responses in vaccinated hosts. Influenza viruses contain diverse particle subpopulations with distinct phenotypes. We show that live influenza vaccines can contain underappreciated subpopulations with enhanced interferon-inducing phenotypes. The genomic traits of such virus subpopulations can be used to further improve the efficacy of the current live vaccines.

scholarly journals A Live Attenuated Influenza Vaccine Elicits Enhanced Heterologous Protection When the Internal Genes of the Vaccine Are Matched to Those of the Challenge Virus

2019 ◽  
Vol 94 (4) ◽  
Author(s):  
Andrew Smith ◽  
Laura Rodriguez ◽  
Maya El Ghouayel ◽  
Aitor Nogales ◽  
Jeffrey M. Chamberlain ◽  
...  
Keyword(s):  
Influenza Vaccine ◽  
Influenza A ◽  
H1n1 Virus ◽  
Influenza Infection ◽  
Influenza Viruses ◽  
Influenza Vaccines ◽  
Temperature Sensitive ◽  
Live Attenuated Influenza Vaccine ◽  
Cell Immunity

ABSTRACT Influenza A virus (IAV) causes significant morbidity and mortality, despite the availability of viral vaccines. The efficacy of live attenuated influenza vaccines (LAIVs) has been especially poor in recent years. One potential reason is that the master donor virus (MDV), on which all LAIVs are based, contains either the internal genes of the 1960 A/Ann Arbor/6/60 or the 1957 A/Leningrad/17/57 H2N2 viruses (i.e., they diverge considerably from currently circulating strains). We previously showed that introduction of the temperature-sensitive (ts) residue signature of the AA/60 MDV into a 2009 pandemic A/California/04/09 H1N1 virus (Cal/09) results in only 10-fold in vivo attenuation in mice. We have previously shown that the ts residue signature of the Russian A/Leningrad/17/57 H2N2 LAIV (Len LAIV) more robustly attenuates the prototypical A/Puerto Rico/8/1934 (PR8) H1N1 virus. In this work, we therefore introduced the ts signature from Len LAIV into Cal/09. This new Cal/09 LAIV is ts in vitro, highly attenuated (att) in mice, and protects from a lethal homologous challenge. In addition, when our Cal/09 LAIV with PR8 hemagglutinin and neuraminidase was used to vaccinate mice, it provided enhanced protection against a wild-type Cal/09 challenge relative to a PR8 LAIV with the same attenuating mutations. These findings suggest it may be possible to improve the efficacy of LAIVs by better matching the sequence of the MDV to currently circulating strains. IMPORTANCE Seasonal influenza infection remains a major cause of disease and death, underscoring the need for improved vaccines. Among current influenza vaccines, the live attenuated influenza vaccine (LAIV) is unique in its ability to elicit T-cell immunity to the conserved internal proteins of the virus. Despite this, LAIV has shown limited efficacy in recent years. One possible reason is that the conserved, internal genes of all current LAIVs derive from virus strains that were isolated between 1957 and 1960 and that, as a result, do not resemble currently circulating influenza viruses. We have therefore developed and tested a new LAIV, based on a currently circulating pandemic strain of influenza. Our results show that this new LAIV elicits improved protective immunity compared to a more conventional LAIV.

scholarly journals Intersection of Sex and Frailty in Humoral Immune Responses to Influenza Vaccine in Community-Dwelling Older Adults

2020 ◽  
Vol 4 (Supplement_1) ◽  
pp. 271-272
Author(s):  
Janna Shapiro ◽  
Helen Kuo ◽  
Rosemary Morgan ◽  
Huifen Li ◽  
Sabra Klein ◽  
...  
Keyword(s):  
Older Adults ◽  
Immune Responses ◽  
Influenza A ◽  
Community Dwelling ◽  
Influenza Vaccines ◽  
Health Study ◽  
Cardiovascular Health Study ◽  
High Dose ◽  
Humoral Immune ◽  
Humoral Immune Responses

Abstract Older adults bear the highest burden of severe disease and complications associated with seasonal influenza, with annual vaccination serving as the best option for protection. Variability in vaccine efficacy exists, yet the host factors that affect immune responses to inactivated influenza vaccines (IIV) are incompletely understood. We hypothesized that sex and frailty interact to affect vaccine-induced humoral responses among older adults. To test this hypothesis, community-dwelling adults above 75 years of age were recruited yearly, assessed for frailty (as defined by the Cardiovascular Health Study criteria), and vaccinated with the high-dose trivalent IIV. Humoral immune responses were evaluated via hemagglutination inhibition titers. The study began during the 2014-2015 influenza season, with yearly cohorts ranging from 76-163 individuals. A total of 617 vaccinations were delivered from 2014-2019. In preliminary analyses, the outcome of interest was seroconversion, defined as ≥ 4-fold rise in titers. Crude odds ratios suggest that females are more likely to seroconvert to influenza A strains (H1N1: OR = 1.39, (0.98-1.96) ; H3N2: 1.17 (0.85 – 1.62)), while males are more likely to seroconvert to the B strain (OR = 0.85 (0.60 – 1.22)). Furthermore, this sex difference was modified by frailty – for example, the odds of seroconversion to H1N1 were 65% higher for females than males among those who were nonfrail, and only 30% higher among females who were frail. Together, these results suggest that sex and frailty interact to impact immune responses to influenza vaccines. These findings may be leveraged to better protect vulnerable populations.

scholarly journals Structural and Thermodynamic Analysis of the Resistance Development to Pimodivir (VX-787), the Clinical Inhibitor of Cap Binding to PB2 Subunit of Influenza A Polymerase

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 1007
Author(s):  
Jiří Gregor ◽  
Kateřina Radilová ◽  
Jiří Brynda ◽  
Jindřich Fanfrlík ◽  
Jan Konvalinka ◽  
...  
Keyword(s):  
Thermodynamic Analysis ◽  
Influenza A ◽  
Polymerase Activity ◽  
Second Phase ◽  
Missense Mutations ◽  
Wild Type ◽  
Resistance Development ◽  
Third Phase ◽  
Major Mutation ◽  
Control And Prevention

Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.

scholarly journals Human Cytomegalovirus Inhibitor AL18 Also Possesses Activity against Influenza A and B Viruses

2012 ◽  
Vol 56 (11) ◽  
pp. 6009-6013 ◽  
Author(s):  
Giulia Muratore ◽  
Beatrice Mercorelli ◽  
Laura Goracci ◽  
Gabriele Cruciani ◽  
Paul Digard ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Influenza A ◽  
Molecular Model ◽  
Polymerase Activity

ABSTRACTAL18, an inhibitor of human cytomegalovirus DNA polymerase, was serendipitously found to also block the interaction between the PB1 and PA polymerase subunits of influenza A virus. Furthermore, AL18 effectively inhibited influenza A virus polymerase activity and the overall replication of influenza A and B viruses. A molecular model to explain the binding of AL18 to both cytomegalovirus and influenza targets is proposed. Thus, AL18 represents an interesting lead for the development of new antivirals.

scholarly journals Multiple polymerase acidic (PA) I38X substitutions in influenza A(H1N1)pdm09 virus permit polymerase activity and cause reduced baloxavir inhibition

2020 ◽  
Author(s):  
Jeremy C Jones ◽  
Philippe N Q Pascua ◽  
Walter N Harrington ◽  
Richard J Webby ◽  
Elena A Govorkova
Keyword(s):  
Inhibitory Activity ◽  
Influenza A ◽  
Antiviral Drug ◽  
Polymerase Activity ◽  
Resistance Marker ◽  
Complex Activity ◽  
Replication Complex ◽  
Virus Rescue ◽  
Influenza A H1n1 ◽  
Targeted Approach

Abstract Background Baloxavir marboxil is an antiviral drug that targets the endonuclease activity of the influenza virus polymerase acidic (PA) protein. PA I38T/M/F substitutions reduce its antiviral efficacy. Objectives To understand the effects of the 19 possible amino acid (AA) substitutions at PA 38 on influenza A(H1N1)pdm09 polymerase activity and inhibition by baloxavir acid, the active metabolite of baloxavir marboxil. Methods Influenza A(H1N1)pdm09 viral polymerase complexes containing all 19 I38X AA substitutions were reconstituted in HEK293T cells in a mini-replicon assay. Polymerase complex activity and baloxavir inhibitory activity were measured in the presence or absence of 50 nM baloxavir acid. Results Only three substitutions (R, K, P) reduced polymerase activity to <79% of I38-WT. When compared with the prototypical baloxavir marboxil resistance marker T38, 5 substitutions conferred 10%–35% reductions in baloxavir acid inhibitory activity (M, L, F, Y, C) and 11 substitutions conferred >50% reductions (R, K, S, N, G, W, A, Q, E, D, H), while two substitutions (V, P) maintained baloxavir acid inhibitory activity. Conclusions Most PA 38 substitutions permit a functional replication complex retaining some drug resistance in the mini-replicon assay. This study provides a targeted approach for virus rescue and analysis of novel baloxavir marboxil reduced-susceptibility markers, supports the consideration of a broader range of these markers during antiviral surveillance and adds to the growing knowledge of baloxavir marboxil resistance profiles.

scholarly journals Effectiveness of seasonal influenza vaccine for adults and children in preventing laboratory-confirmed influenza in primary care in the United Kingdom: 2015/16 end-of-season results

2016 ◽  
Vol 21 (38) ◽  
Author(s):  
Richard Pebody ◽  
Fiona Warburton ◽  
Joanna Ellis ◽  
Nick Andrews ◽  
Alison Potts ◽  
...  
Keyword(s):  
Primary Care ◽  
United Kingdom ◽  
Influenza Vaccine ◽  
Influenza A ◽  
Immunisation Programme ◽  
Influenza B ◽  
Live Attenuated Influenza Vaccine ◽  
The United Kingdom ◽  
Influenza A H1n1 ◽  
The Uk

The United Kingdom (UK) is in the third season of introducing universal paediatric influenza vaccination with a quadrivalent live attenuated influenza vaccine (LAIV). The 2015/16 season in the UK was initially dominated by influenza A(H1N1)pdm09 and then influenza of B/Victoria lineage, not contained in that season’s adult trivalent inactivated influenza vaccine (IIV). Overall adjusted end-of-season vaccine effectiveness (VE) was 52.4% (95% confidence interval (CI): 41.0–61.6) against influenza-confirmed primary care consultation, 54.5% (95% CI: 41.6–64.5) against influenza A(H1N1)pdm09 and 54.2% (95% CI: 33.1–68.6) against influenza B. In 2–17 year-olds, adjusted VE for LAIV was 57.6% (95% CI: 25.1 to 76.0) against any influenza, 81.4% (95% CI: 39.6–94.3) against influenza B and 41.5% (95% CI: −8.5 to 68.5) against influenza A(H1N1)pdm09. These estimates demonstrate moderate to good levels of protection, particularly against influenza B in children, but relatively less against influenza A(H1N1)pdm09. Despite lineage mismatch in the trivalent IIV, adults younger than 65 years were still protected against influenza B. These results provide reassurance for the UK to continue its influenza immunisation programme planned for 2016/17.

scholarly journals HDAC6 Restricts Influenza A Virus by Deacetylation of the RNA Polymerase PA Subunit

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Huan Chen ◽  
Yingjuan Qian ◽  
Xin Chen ◽  
Zhiyang Ruan ◽  
Yuetian Ye ◽  
...  
Keyword(s):  
Life Cycle ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Molecular Mechanisms ◽  
Polymerase Activity ◽  
Negative Regulator ◽  
Host Protein ◽  
Spectrometric Analysis ◽  
Rna Polymerase Activity

ABSTRACT The life cycle of influenza A virus (IAV) is modulated by various cellular host factors. Although previous studies indicated that IAV infection is controlled by HDAC6, the deacetylase involved in the regulation of PA remained unknown. Here, we demonstrate that HDAC6 acts as a negative regulator of IAV infection by destabilizing PA. HDAC6 binds to and deacetylates PA, thereby promoting the proteasomal degradation of PA. Based on mass spectrometric analysis, Lys(664) of PA can be deacetylated by HDAC6, and the residue is crucial for PA protein stability. The deacetylase activity of HDAC6 is required for anti-IAV activity, because IAV infection was enhanced due to elevated IAV RNA polymerase activity upon HDAC6 depletion and an HDAC6 deacetylase dead mutant (HDAC6-DM; H216A, H611A). Finally, we also demonstrate that overexpression of HDAC6 suppresses IAV RNA polymerase activity, but HDAC6-DM does not. Taken together, our findings provide initial evidence that HDAC6 plays a negative role in IAV RNA polymerase activity by deacetylating PA and thus restricts IAV RNA transcription and replication. IMPORTANCE Influenza A virus (IAV) continues to threaten global public health due to drug resistance and the emergence of frequently mutated strains. Thus, it is critical to find new strategies to control IAV infection. Here, we discover one host protein, HDAC6, that can inhibit viral RNA polymerase activity by deacetylating PA and thus suppresses virus RNA replication and transcription. Previously, it was reported that IAV can utilize the HDAC6-dependent aggresome formation mechanism to promote virus uncoating, but HDAC6-mediated deacetylation of α-tubulin inhibits viral protein trafficking at late stages of the virus life cycle. These findings together will contribute to a better understanding of the role of HDAC6 in regulating IAV infection. Understanding the molecular mechanisms of HDAC6 at various periods of viral infection may illuminate novel strategies for developing antiviral drugs.

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