scholarly journals Different genetic barriers for resistance to HA stem antibodies in influenza H3 and H1 viruses

Science ◽  
2020 ◽  
Vol 368 (6497) ◽  
pp. 1335-1340 ◽  
Author(s):  
Nicholas C. Wu ◽  
Andrew J. Thompson ◽  
Juhye M. Lee ◽  
Wen Su ◽  
Britni M. Arlian ◽  
...  
Keyword(s):  
Influenza Vaccine ◽  
Viral Fitness ◽  
Resistance Mutations ◽  
Genetic Barrier ◽  
Influenza Hemagglutinin ◽  
Genetic Barriers ◽  
Universal Influenza Vaccine

The discovery and characterization of broadly neutralizing human antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have contributed to considerations of a universal influenza vaccine. However, the potential for resistance to stem bnAbs also needs to be more thoroughly evaluated. Using deep mutational scanning, with a focus on epitope residues, we found that the genetic barrier to resistance to stem bnAbs is low for the H3 subtype but substantially higher for the H1 subtype owing to structural differences in the HA stem. Several strong resistance mutations in H3 can be observed in naturally circulating strains and do not reduce in vitro viral fitness and in vivo pathogenicity. This study highlights a potential challenge for development of a truly universal influenza vaccine.

scholarly journals Influenza H3 and H1 hemagglutinins have different genetic barriers for resistance to broadly neutralizing stem antibodies

2019 ◽  
Author(s):  
Nicholas C. Wu ◽  
Andrew J. Thompson ◽  
Juhye M. Lee ◽  
Wen Su ◽  
Britni M. Arlian ◽  
...  
Keyword(s):  
Influenza Vaccine ◽  
Neutralizing Antibodies ◽  
Viral Fitness ◽  
Resistance Mutations ◽  
Genetic Barrier ◽  
Influenza Hemagglutinin ◽  
Genetic Barriers ◽  
Universal Influenza Vaccine

ABSTRACTIn the past decade, the discovery and characterization of broadly neutralizing antibodies (bnAbs) to the highly conserved stem region of influenza hemagglutinin (HA) have provided valuable insights for development of a universal influenza vaccine. However, the genetic barrier for resistance to stem bnAbs has not been thoroughly evaluated. Here, we performed a series of deep mutational scanning experiments to probe for resistance mutations. We found that the genetic barrier to resistance to stem bnAbs is generally very low for the H3 subtype but substantially higher for the H1 subtype. Several resistance mutations in H3 cannot be neutralized by stem bnAbs at the highest concentration tested, do not reduce in vitro viral fitness and in vivo pathogenicity, and are often present in circulating strains as minor variants. Thus, H3 HAs have a higher propensity than H1 HAs to escape major stem bnAbs and creates a potential challenge in the development of a bona fide universal influenza vaccine.ONE SENTENCE SUMMARYAcquisition of resistance by influenza virus to broadly neutralizing hemagglutinin stem antibodies varies tremendously depending on subtype.

scholarly journals Development and characterization of influenza M2 ectodomain and/or HA stalk-based DC-targeting vaccines for different influenza infections

2021 ◽  
Author(s):  
Xiaojian Yao ◽  
Titus Olukitibi ◽  
Zhujun Ao ◽  
Hiva Azizi ◽  
Mona Mahmoudi ◽  
...  
Keyword(s):  
Influenza Vaccine ◽  
Matrix Protein ◽  
Influenza Infection ◽  
Extracellular Matrix Protein ◽  
Influenza Hemagglutinin ◽  
Viral Particles ◽  
Vesicular Stomatitis ◽  
Universal Influenza Vaccine ◽  
Mouse Study

A universal influenza vaccine is required for broad protection against influenza infection. Here, we revealed the efficacy of novel influenza vaccine candidates based on Ebola glycoprotein (EboGP) DC-targeting domain (EΔM) fusion protein technology. We fused influenza hemagglutinin stalk (HAcs) and extracellular matrix protein (M2e) or four copies of M2e (referred to as tetra M2e (tM2e)) with EΔM to generate EΔM-HM2e or EΔM-tM2e, respectively, and revealed that EΔM facilitates DC/macrophage targeting in vitro. In a mouse study, EΔM-HM2e- or EΔM-tM2e-pseudotyped viral particles (PVPs) induced significantly higher titers of anti-HA and/or anti-M2e antibodies. We also developed recombinant vesicular stomatitis virus (rVSV)-EΔM-HM2e and rVSV-EΔM-tM2e vaccines that resulted in rapid and potent induction of HA and/or M2 antibodies in mouse sera and mucosa. Importantly, vaccination protects mice from influenza H1N1 and H3N2 challenges. Taken together, our study suggests that recombinant rVSV-EΔM-HM2e and rVSV-EΔM-tM2e are efficacious and protective universal vaccines against influenza.

In vitro and in vivo characterization of designed immunogens derived from the CD-helix of the stem of influenza hemagglutinin

2013 ◽  
Vol 81 (10) ◽  
pp. 1759-1775 ◽  
Author(s):  
V. Vamsee Aditya Mallajosyula ◽  
Michael Citron ◽  
Xianghan Lu ◽  
Jan ter Meulen ◽  
Raghavan Varadarajan ◽  
...  
Keyword(s):  
Influenza Hemagglutinin ◽  
In Vivo Characterization

scholarly journals In Vivo Validation of a Bioinformatics Based Tool to Identify Reduced Replication Capacity in HIV-1

2010 ◽  
Vol 4 (1) ◽  
pp. 225-232
Author(s):  
Christina M.R Kitchen ◽  
Paul Krogstad ◽  
Scott G Kitchen
Keyword(s):  
Drug Resistance ◽  
In Silico ◽  
Viral Fitness ◽  
Biological Assessment ◽  
Relative Fitness ◽  
Replication Capacity ◽  
Resistance Mutations ◽  
Hiv 1

Although antiretroviral drug resistance is common in treated HIV infected individuals, it is not a consistent indicator of HIV morbidity and mortality. To the contrary, HIV resistance-associated mutations may lead to changes in viral fitness that are beneficial to infected individuals. Using a bioinformatics-based model to assess the effects of numerous drug resistance mutations, we determined that the D30N mutation in HIV-1 protease had the largest decrease in replication capacity among known protease resistance mutations. To test thisin silicoresult in anin vivoenvironment, we constructed several drug-resistant mutant HIV-1 strains and compared their relative fitness utilizing the SCID-hu mouse model. We found HIV-1 containing the D30N mutation had a significant defectin vivo, showing impaired replication kinetics and a decreased ability to deplete CD4+ thymocytes, compared to the wild-type or virus without the D30N mutation. In comparison, virus containing the M184V mutation in reverse transcriptase, which shows decreased replication capacityin vitro, did not have an effect on viral fitnessin vivo. Thus, in this study we have verified anin silicobioinformatics result with a biological assessment to identify a unique mutation in HIV-1 that has a significant fitness defectin vivo.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

In vitro and in vivo characterization of graphene oxide coated porcine bone granules

Carbon ◽  
2016 ◽  
Vol 103 ◽  
pp. 291-298 ◽  
Author(s):  
Valeria Ettorre ◽  
Patrizia De Marco ◽  
Susi Zara ◽  
Vittoria Perrotti ◽  
Antonio Scarano ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
In Vivo Characterization

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

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