Directed Evolution of Immune-Escaping Adeno-Associated Virus Vectors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3532-3532
Author(s):  
Stephan Maersch ◽  
Anke Huber ◽  
Michael Hallek ◽  
Hildegard Buening ◽  
Luca Perabo
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gene Transfer ◽  
Directed Evolution ◽  
Neutralizing Antibodies ◽  
Rational Design ◽  
Multiple Point ◽  
Repeated Treatment ◽  
Therapeutic Gene ◽  
Adeno Associated Virus

Abstract Efficiency of therapeutic gene transfer by adeno-associated virus of serotype 2 (AAV-2) vectors is hampered in patients with pre-existing immunity against the natural virus. Genetic engineering by rational design or directed evolution has been employed in the last 3 years to generate capsids that escape antibody neutralization and has led to identify several amino acid residues of the capsid proteins that can be mutated in order to decrease antibody recognition (Perabo et al., 2006; Maheshri et al, 2006; Lochrie et al., 2006). In this novel study, we aimed to exploit the comprehensive knowledge gathered so far by generating novel capsid variants that carried multiple point mutations at these previously identified sites. Capsid libraries were generated by codon randomization of several immunogenic residues and screened to isolate mutants that most efficiently infected human cells despite the presence of anti-AAV2 neutralizing antibodies. Besides testing novel combinations of concomitant mutations at these sites, this approach allowed for the first time an exhaustive scanning of combinations of all 20 natural amino acids at each position. We identified several novel capsid mutants that remain highly infectious even when incubated with serum concentrations that completely neutralize wild type AAV2. Our results demonstrate that combining mutations at several sites it is possible to improve the immune-escaping ability of the capsid. In addition, we show that escaping ability and other biological characteristics of these mutants are strongly dependent on the type of amino acid substituted, demonstrating that an exact choice of substituted amino acids is essential to maximize stealth properties and minimize loss of packaging ability, particle stability and transduction efficacy. These vectors can be used for therapeutic gene transfer to patients with pre-existing immunity, or for repeated treatment after antibodies are generated upon first application.

Structurally Mapping Antigenic Epitopes of Adeno-Associated Virus 9: Development of Antibody Escape Variants

2021 ◽  
Author(s):  
Shanan N. Emmanuel ◽  
J. Kennon Smith ◽  
Jane Hsi ◽  
Yu-Shan Tseng ◽  
Matias Kaplan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Gene Delivery ◽  
General Population ◽  
Neutralizing Antibodies ◽  
Rational Design ◽  
Transduction Efficiency ◽  
Viral Neutralization ◽  
Patient Cohort ◽  
Antigenic Epitopes

Adeno-associated viruses (AAV) serve as vectors for therapeutic gene delivery. AAV9 vectors have been FDA approved, as Zolgensma®, for the treatment of spinal muscular atrophy and is being evaluated in clinical trials for the treatment of neurotropic and musculotropic diseases. A major hurdle for AAV-mediated gene delivery is the presence of pre-existing neutralizing antibodies in 40 to 80% of the general population. These pre-existing antibodies can reduce therapeutic efficacy through viral neutralization, and the size of the patient cohort eligible for treatment. In this study, cryo-electron microscopy and image reconstruction was used to define the epitopes of five anti-AAV9 monoclonal antibodies (MAbs); ADK9, HL2368, HL2370, HL2372, and HL2374, on the capsid surface. Three of these, ADK9, HL2370, and HL2374, bound on or near the icosahedral 3-fold axes, HL2368 to the 2/5-fold wall, and HL2372 to the region surrounding the 5-fold axes. Pseudo-atomic modeling enabled the mapping and identification of antibody contact amino acids on the capsid, including S454 and P659. These epitopes overlap with previously defined parvovirus antigenic sites. Capsid amino acids critical for the interactions were confirmed by mutagenesis followed by biochemical assays testing recombinant AAV9 (rAAV9) variants capable of escaping recognition and neutralization by the parental MAbs. These variants retained parental tropism and had similar or improved transduction efficiency compared to AAV9. These engineered rAAV9 variants could expand the patient cohort eligible for AAV9-mediated gene delivery by avoiding pre-existing circulating neutralizing antibodies. IMPORTANCE The use of recombinant AAVs (rAAVs) as delivery vectors for therapeutic genes is becoming increasingly popular, especially following the FDA approval of Luxturna® and Zolgensma®, based on serotypes AAV2 and AAV9, respectively. However, high titer anti-AAV neutralizing antibodies in the general population, exempts patients from treatment. The goal of this study is to circumvent this issue by creating AAV variant vectors not recognized by pre-existing neutralizing antibodies. The mapping of the antigenic epitopes of five different monoclonal antibodies (MAbs) on AAV9, to recapitulate a polyclonal response, enabled the rational design of escape variants with minimal disruption to cell tropism and gene expression. This study, which included four newly developed and now commercially available MAbs, provides a platform for the engineering of rAAV9 vectors that can be used to deliver genes to patients with pre-exiting AAV antibodies.

Rational design of a function-based selection method for genetically encoding acylated lysine derivatives

2019 ◽  
Vol 17 (25) ◽  
pp. 6127-6130
Author(s):  
Hui Miao ◽  
Chenguang Yu ◽  
Anzhi Yao ◽  
Weimin Xuan
Keyword(s):  
Amino Acids ◽  
Genetic Code ◽  
Directed Evolution ◽  
Rational Design ◽  
Selection Method ◽  
Genetic Code Expansion

Genetic code expansion depends on the directed evolution of aaRS to recognize non-canonical amino acids. Herein, we reported a function-based method that enables rapidly evolving aaRS for acylated lysine derivatives.

scholarly journals Engineering Improves Enzymatic Synthesis of L-Tryptophan by Tryptophan Synthase from Escherichia coli

2020 ◽  
Vol 8 (4) ◽  
pp. 519
Author(s):  
Lisheng Xu ◽  
Fangkai Han ◽  
Zeng Dong ◽  
Zhaojun Wei
Keyword(s):  
Thermal Stability ◽  
Amino Acid ◽  
Directed Evolution ◽  
Amino Acid Residue ◽  
Rational Design ◽  
Molecular Engineering ◽  
Mutant Enzyme ◽  
Tryptophan Synthase ◽  
Mutant Library ◽  
Thermal Stability Of

To improve the thermostability of tryptophan synthase, the molecular modification of tryptophan synthase was carried out by rational molecular engineering. First, B-FITTER software was used to analyze the temperature factor (B-factor) of each amino acid residue in the crystal structure of tryptophan synthase. A key amino acid residue, G395, which adversely affected the thermal stability of the enzyme, was identified, and then, a mutant library was constructed by site-specific saturation mutation. A mutant (G395S) enzyme with significantly improved thermal stability was screened from the saturated mutant library. Error-prone PCR was used to conduct a directed evolution of the mutant enzyme (G395S). Compared with the parent, the mutant enzyme (G395S /A191T) had a Km of 0.21 mM and a catalytic efficiency kcat/Km of 5.38 mM−1∙s−1, which was 4.8 times higher than that of the wild-type strain. The conditions for L-tryptophan synthesis by the mutated enzyme were a L-serine concentration of 50 mmol/L, a reaction temperature of 40 °C, pH of 8, a reaction time of 12 h, and an L-tryptophan yield of 81%. The thermal stability of the enzyme can be improved by using an appropriate rational design strategy to modify the correct site. The catalytic activity of tryptophan synthase was increased by directed evolution.

scholarly journals Single Amino Acid Substitutions in the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Determine Viral Entry and Immunogenicity of a Major Neutralizing Domain

2005 ◽  
Vol 79 (18) ◽  
pp. 11638-11646 ◽  
Author(s):  
Christopher E. Yi ◽  
Lei Ba ◽  
Linqi Zhang ◽  
David D. Ho ◽  
Zhiwei Chen
Keyword(s):  
Amino Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Rational Design ◽  
Antiviral Agents ◽  
Single Amino Acid ◽  
Major Mechanism ◽  
The Difference

ABSTRACT Neutralizing antibodies (NAbs) against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) spike (S) glycoprotein confer protection to animals experimentally infected with the pathogenic virus. We and others previously demonstrated that a major mechanism for neutralizing SARS-CoV was through blocking the interaction between the S glycoprotein and the cellular receptor angiotensin-converting enzyme 2 (ACE2). In this study, we used in vivo electroporation DNA immunization and a pseudovirus-based assay to functionally evaluate immunogenicity and viral entry. We characterized the neutralization and viral entry determinants within the ACE2-binding domain of the S glycoprotein. The deletion of a positively charged region SΔ(422-463) abolished the capacity of the S glycoprotein to induce NAbs in mice vaccinated by in vivo DNA electroporation. Moreover, the SΔ(422-463) pseudovirus was unable to infect HEK293T-ACE2 cells. To determine the specific residues that contribute to related phenotypes, we replaced eight basic amino acids with alanine. We found that a single amino acid substitution (R441A) in the full-length S DNA vaccine failed to induce NAbs and abolished viral entry when pseudoviruses were generated. However, another substitution (R453A) abolished viral entry while retaining the capacity for inducing NAbs. The difference between R441A and R453A suggests that the determinants for immunogenicity and viral entry may not be identical. Our findings provide direct evidence that these basic residues are essential for immunogenicity of the major neutralizing domain and for viral entry. Our data have implications for the rational design of vaccine and antiviral agents as well as for understanding viral tropism.

scholarly journals Amino acids 484 and 494 of SARS-CoV-2 spike are hotspots of immune evasion affecting antibody but not ACE2 binding

2021 ◽  
Author(s):  
Marta Alenquer ◽  
Filipe Ferreira ◽  
Diana Lousa ◽  
Mariana Valério ◽  
Mónica Medina-Lopes ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Angiotensin Converting Enzyme ◽  
Immune Evasion ◽  
Neutralizing Antibodies ◽  
Receptor Binding Domain ◽  
Converting Enzyme ◽  
Antibody Neutralization ◽  
Angiotensin Converting Enzyme 2 ◽  
The Core

AbstractUnderstanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike and, predominantly, of its receptor-binding-domain (RBD). Mutations in spike impacting antibody or ACE2 binding are known, but the effect of mutation synergy is less explored. We engineered 22 spike-pseudotyped lentiviruses containing individual and combined mutations, and confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent sera. This amino acid emerges as an additional hotspot for immune evasion and a target for therapies, vaccines and diagnostics.One-Sentence SummaryAmino acids in SARS-CoV-2 spike protein implicated in immune evasion are biased for binding to neutralizing antibodies but dispensable for binding the host receptor angiotensin-converting enzyme 2.

scholarly journals Assessment of gene and protein similarities of Beijing-1 vaccine producing strain with Japanese encephalitis virus strains circulating in Vietnam

2021 ◽  
Vol 18 (4) ◽  
pp. 663-670
Author(s):  
Nguyen Thi Ly ◽  
Huynh Thi Phuong Lien ◽  
Do Tuan Dat ◽  
Nguyen Kim Bach ◽  
Nguyen Hoang Tung ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Japanese Encephalitis ◽  
Neutralizing Antibodies ◽  
Vero Cells ◽  
Japanese Encephalitis Vaccine ◽  
Protective Antibodies ◽  
Encoding Gene ◽  
Genomic Regions ◽  
Virus Strains

Since 2006, the inactivated Japanese encephalitis vaccine has been studied and produced by the VABIOTECH company from the Beịing-1 strain on Vero cells. Vaccines produced from the materseed and working seed virus have been evaluated at laboratory and clinical scale in humans. The results showed that the vaccine was safe and created 100% protective antibodies after the booster dose. To officially put this vaccine into production and mass use, the master seed virus BV-MSV-0210 and working seed virus BV-WSV-0310 with the reference standard strain JEV Beijing-Kanonji has been tested for genetic stability. By the method of Sanger sequencing and genetic analysis software, we have evaluated the similarity of nucleotide and proteinsequencesof the E antigen-encoding gene. The results showed that the seed virus similarity of amino acids and nucleotides is 100% compared with the reference strain. Thus, it can be concluded, the seed virus has antigen stability. Nucleotide and amino acid gene sequences of E genomic regions of the two seed lots were compared with virus strains isolated from human, pig and mosquito in Vietnam. The results showed that the nucleotide similarity of seed virus compared with the JEV strains isolated from humans ranged from 86.67 to 97.54%; from pigs is 87.47 to 88.33%, and from mosquitoes is 86.05 to 99%. Meanwhile, the amino acid similarity of the seed virus with the JEV strains isolated from humans ranged from 96.73 to 99.02%; from pigs is 98.00 to98.40% and from mosquitoes 94.55 to 98.40%. The sequence of amino acids in the epitope producing neutralizing antibodies of the seed virus did not differ from that of the JEV strain circulating in humans isolated in 2014.

scholarly journals Mechanism and site of action of big dynorphin on ASIC1a

2019 ◽  
Author(s):  
C.B. Borg ◽  
N. Braun ◽  
S.A. Heusser ◽  
Y. Bay ◽  
D. Weis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ischemic Stroke ◽  
Amino Acid ◽  
Binding Site ◽  
Neuronal Death ◽  
Electrostatic Interactions ◽  
Rational Design ◽  
Site Of Action ◽  
Canonical Amino Acid

AbstractAcid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to neurotransmission, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is upregulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs and non-canonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding results in an ASIC1a closed resting conformation that is distinct from open and desensitized states induced by protons. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is mediated through electrostatic interactions of basic amino acids in the BigDyn N-terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the non-canonical amino acid azido-phenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics.Significance StatementNeuropeptides such as big dynorphin (BigDyn) play important roles in the slow modulation of fast neurotransmission, which is mediated by membrane-embedded receptors. In fact, BigDyn is the most potent known endogenous modulator of one such receptor, the acid-sensing ion channel (ASIC), but the mode of action remains unknown. In this work, we employ a broad array of technologies to unravel the details of where big dynorphin binds to ASIC and how it modulates its activity. As both BigDyn and ASIC are implicated in pain pathways, this work might pave the way towards future analgesics.

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