scholarly journals Massively parallel profiling of HIV-1 resistance to the fusion inhibitor enfuvirtide

2018 ◽  
Author(s):  
Adam S. Dingens ◽  
Dana Arenz ◽  
Julie Overbaugh ◽  
Jesse D. Bloom
Keyword(s):  
Binding Site ◽  
Transmembrane Domain ◽  
Massively Parallel ◽  
Heptad Repeat ◽  
Entry Inhibitor ◽  
Resistance Mutations ◽  
Receptor Binding Site ◽  
Allosteric Sites ◽  
Additional Resistance ◽  
Hiv 1

AbstractResistance to enfuvirtide, the only clinically approved HIV-1 entry inhibitor, has primarily been mapped to the binding site in the N-terminal heptad repeat (NHR) of the Env transmembrane domain and a limited number of allosteric sites. To better delineate the genotypic determinants of resistance, we used deep mutational scanning to quantify how all mutations to HIV-1 Env affect enfuvirtide sensitivity. We identified numerous additional resistance mutations in the NHR and other regions of Env, including the co-receptor binding site. This complete map of resistance sheds light on the diverse mechanisms of enfuvirtide resistance and can inform clinical monitoring of patients.

scholarly journals Massively Parallel Profiling of HIV-1 Resistance to the Fusion Inhibitor Enfuvirtide

Viruses ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 439 ◽  
Author(s):  
Adam S. Dingens ◽  
Dana Arenz ◽  
Julie Overbaugh ◽  
Jesse D. Bloom
Keyword(s):  
Drug Resistance ◽  
Binding Site ◽  
Transmembrane Domain ◽  
Viral Evolution ◽  
Heptad Repeat ◽  
Single Amino Acid ◽  
Fusion Inhibitor ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Hiv 1

Identifying drug resistance mutations is important for the clinical use of antivirals and can help define both a drug’s mechanism of action and the mechanistic basis of resistance. Resistance mutations are often identified one-at-a-time by studying viral evolution within treated patients or during viral growth in the presence of a drug in cell culture. Such approaches have previously mapped resistance to enfuvirtide, the only clinically approved HIV-1 fusion inhibitor, to enfuvirtide’s binding site in the N-terminal heptad repeat (NHR) of the Envelope (Env) transmembrane domain as well as a limited number of allosteric sites. Here, we sought to better delineate the genotypic determinants of resistance throughout Env. We used deep mutational scanning to quantify the effect of all single-amino-acid mutations to the subtype A BG505 Env on resistance to enfuvirtide. We identified both previously characterized and numerous novel resistance mutations in the NHR. Additional resistance mutations clustered in other regions of Env conformational intermediates, suggesting they may act during different fusion steps by altering fusion kinetics and/or exposure of the enfuvirtide binding site. This complete map of resistance sheds light on the diverse mechanisms of enfuvirtide resistance and highlights the utility of using deep mutational scanning to comprehensively map potential drug resistance mutations.

scholarly journals Therapeutic Efficacy and Resistance Selection of a Lipopeptide Fusion Inhibitor in Simian Immunodeficiency Virus-Infected Rhesus Macaques

2020 ◽  
Vol 94 (15) ◽  
Author(s):  
Danwei Yu ◽  
Jing Xue ◽  
Huamian Wei ◽  
Zhe Cong ◽  
Ting Chen ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Therapeutic Efficacy ◽  
Rhesus Macaques ◽  
Heptad Repeat ◽  
Fusion Inhibitor ◽  
Resistance Mutations ◽  
Fusion Inhibitors ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We recently reported a group of lipopeptide-based membrane fusion inhibitors with potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). In this study, the in vivo therapeutic efficacy of such a lipopeptide, LP-52, was evaluated in rhesus macaques chronically infected with pathogenic SIVmac239. In a pilot study with one monkey, monotherapy with low-dose LP-52 rapidly reduced the plasma viral loads to below the limit of detection and maintained viral suppression during three rounds of structurally interrupted treatment. The therapeutic efficacy of LP-52 was further verified in four infected monkeys; however, three out of the monkeys had viral rebounds under the LP-52 therapy. We next focused on characterizing SIV mutants responsible for the in vivo resistance. Sequence analyses revealed that a V562A or V562M mutation in the N-terminal heptad repeat (NHR) and a E657G mutation in the C-terminal heptad repeat (CHR) of SIV gp41 conferred high resistance to LP-52 and cross-resistance to the peptide drug T20 and two newly designed lipopeptides (LP-80 and LP-83). Moreover, we showed that the resistance mutations greatly reduced the stability of diverse fusion inhibitors with the NHR site, and V562A or V562M in combination with E657G could significantly impair the functionality of viral envelopes (Envs) to mediate SIVmac239 infection and decrease the thermostability of viral six-helical bundle (6-HB) core structure. In conclusion, the present data have not only facilitated the development of novel anti-HIV drugs that target the membrane fusion step, but also help our understanding of the mechanism of viral evolution to develop drug resistance. IMPORTANCE The anti-HIV peptide drug T20 (enfuvirtide) is the only membrane fusion inhibitor available for treatment of viral infection; however, it exhibits relatively weak antiviral activity, short half-life, and a low genetic barrier to inducing drug resistance. Design of lipopeptide-based fusion inhibitors with extremely potent and broad antiviral activities against divergent HIV-1, HIV-2, and SIV isolates have provided drug candidates for clinical development. Here, we have verified a high therapeutic efficacy for the lipopeptide LP-52 in SIVmac239-infected rhesus monkeys. The resistance mutations selected in vivo have also been characterized, providing insights into the mechanism of action of newly designed fusion inhibitors with a membrane-anchoring property. For the first time, the data show that HIV-1 and SIV can share a similar genetic pathway to develop resistance, and that a lipopeptide fusion inhibitor could have a same resistance profile as its template peptide.

scholarly journals Broad Neutralization of Viral Pathogens

2014 ◽  
Vol 70 (a1) ◽  
pp. C245-C245
Author(s):  
Ian Wilson
Keyword(s):  
Hepatitis C ◽  
Binding Site ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Functional Characterization ◽  
Antigenic Site ◽  
Soluble Form ◽  
Receptor Binding Site ◽  
Fusion Domain ◽  
Hiv 1

Influenza, Hepatitis C, and HIV-1 continue to constitute significant threats to global health. We have structurally and functionally characterized several potent, broadly neutralizing antibodies (bnAbs) against HIV-1, influenza and hepatitis C viruses. The surface antigens of these viruses are the main target of neutralizing antibodies. However, most antibodies are strain-specific and protect only against highly related strains within the same subtype. Recently, a number of antibodies have been identified that are much broader and neutralize across multiple subtypes and types of these viruses through binding to functionally conserved sites, such as the receptor binding site or the fusion domain. For example, co-crystal structures of bnAbs with influenza hemagglutinin (HA) identified highly conserved sites in the fusion domain (stem) and in the receptor binding site (head) as target for broad neutralization[1]. HCV is also genetically diverse, but some antibodies have potent neutralizing activity across most genotypes of the virus. One family of these antibodies targets a conserved antigenic site on the HCV E2 envelope glycoprotein that overlaps with the CD81 receptor-binding site[2]. For HIV-1, structural and functional characterization of different families of bnAbs have led to identification of novel epitopes on HIV-1 Env, many of which involve glycans. These glycan-dependent Abs have unique features that enable them to penetrate the glycan shield and bind complex epitopes that consist of sugars and underlying protein segments on gp120 on HIV-1 Env. Recent x-ray[3] and EM structures of a soluble form of HIV-1 Env have revealed that the epitopes are more extensive and complex than previously appreciated. This structural information is now being used to aid in structure-assisted vaccine design for HIV-1, HCV and for a more universal flu vaccine. IAW is supported by NIH grants AI100663, AI082362, AI84817, AI099275 and GM094586 and the Crucell Vaccine Institute.

scholarly journals Elicitation of Cluster A and Co-Receptor Binding Site Antibodies Are Required to Eliminate HIV-1 Infected Cells

2020 ◽  
Vol 8 (5) ◽  
pp. 710 ◽  
Author(s):  
Guillaume Beaudoin-Bussières ◽  
Jérémie Prévost ◽  
Gabrielle Gendron-Lepage ◽  
Bruno Melillo ◽  
Junhua Chen ◽  
...  
Keyword(s):  
Binding Site ◽  
Envelope Glycoprotein ◽  
Antibody Dependent Cellular Cytotoxicity ◽  
Receptor Binding Site ◽  
Coreceptor Binding ◽  
Infected Cells ◽  
Cluster A ◽  
Hiv 1 ◽  
Coreceptor Binding Site

HIV-1-infected individuals raise a polyclonal antibody response targeting multiple envelope glycoprotein (Env) epitopes. Interestingly, two classes of non-neutralizing CD4-induced (CD4i) antibodies, present in the majority of HIV-1-infected individuals have been described to mediate antibody-dependent cellular cytotoxicity (ADCC) in the presence of small CD4 mimetic compounds (CD4mc). These antibodies recognize the coreceptor binding site (CoRBS) and the constant region one and two (C1C2 or inner domain cluster A) of the gp120. In combination with CD4mc they have been shown to stabilize an antibody-vulnerable Env conformation, known as State 2A. Here we evaluated the importance of these two families of Abs in ADCC responses by immunizing guinea pigs with gp120 immunogens that have been modified to elicit or not these types of antibodies. Underlying the importance of anti-CoRBS and anti-cluster A Abs in stabilizing State 2A, ADCC responses were only observed in the presence of these two types of CD4i antibodies. Altogether, our results suggest that these two families of CD4i antibodies must be taken into account when considering future strategies relying on the use of CD4mc to eliminate HIV-1-infected cells in vivo.

scholarly journals Topological analysis of the gp41 MPER on lipid bilayers relevant to the metastable HIV-1 envelope prefusion state

2019 ◽  
Vol 116 (45) ◽  
pp. 22556-22566 ◽  
Author(s):  
Yi Wang ◽  
Pavanjeet Kaur ◽  
Zhen-Yu J. Sun ◽  
Mostafa A. Elbahnasawy ◽  
Zahra Hayati ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Neutralizing Antibodies ◽  
Structural Changes ◽  
Transmembrane Domain ◽  
Topological Analysis ◽  
Heptad Repeat ◽  
Structural Topology ◽  
Paramagnetic Resonance ◽  
Viral Membrane ◽  
Hiv 1

The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.

scholarly journals MiniCD4 protein resistance mutations affect binding to the HIV-1 gp120 CD4 binding site and decrease entry efficiency

Retrovirology ◽  
2012 ◽  
Vol 9 (1) ◽  
pp. 36 ◽  
Author(s):  
Katrijn Grupping ◽  
Philippe Selhorst ◽  
Johan Michiels ◽  
Katleen Vereecken ◽  
Leo Heyndrickx ◽  
...  
Keyword(s):  
Binding Site ◽  
Resistance Mutations ◽  
Protein Resistance ◽  
Cd4 Binding Site ◽  
Hiv 1 ◽  
Entry Efficiency

scholarly journals Structure-Based Stabilization of HIV-1 gp120 Enhances Humoral Immune Responses to the Induced Co-Receptor Binding Site

2009 ◽  
Vol 5 (5) ◽  
pp. e1000445 ◽  
Author(s):  
Barna Dey ◽  
Krisha Svehla ◽  
Ling Xu ◽  
Dianne Wycuff ◽  
Tongqing Zhou ◽  
...  
Keyword(s):  
Immune Responses ◽  
Binding Site ◽  
Receptor Binding ◽  
Receptor Binding Site ◽  
Humoral Immune ◽  
Humoral Immune Responses ◽  
Hiv 1

scholarly journals Proviral DNA as a Target for HIV-1 Resistance Analysis

Intervirology ◽  
2015 ◽  
Vol 58 (3) ◽  
pp. 184-189 ◽  
Author(s):  
Nadine Lübke ◽  
Veronica Di Cristanziano ◽  
Saleta Sierra ◽  
Elena Knops ◽  
Eugen Schülter ◽  
...  
Keyword(s):  
Resistance Testing ◽  
Detectable Viral Load ◽  
Resistance Mutations ◽  
Proviral Dna ◽  
Drug Resistance Mutations ◽  
Resistance Analysis ◽  
Additional Resistance ◽  
Plasma Rna ◽  
Hiv 1 ◽  
Rna And Dna

Background: Resistance analysis from viral RNA is restricted to detectable viral load. Therefore, analysis from proviral DNA could help in cases with low-level or suppressed viremia. Methods: Viral plasma RNA and the corresponding cellular proviral DNA of 78 EDTA samples from 48 therapy-naïve (TN) and 30 therapy-experienced (TE) HIV-1-infected patients were isolated and analyzed for their resistance profiles in the protease and reverse transcriptase genes. Results: Overall, 175 drug-resistance mutations (DRMs) were detected in 25/30 TE (83.3%) and 5/48 TN (10.4%) samples. The TE patients displayed a mean number of 6.68 DRMs in RNA and 5.20 in DNA. In the TN patients, a mean of 0.8 DRMs was found in RNA and 1.0 in DNA; 75% of the DRMs were detected in RNA and DNA simultaneously. In the TE samples, 76% of the DRMs were detected simultaneously in RNA and DNA, 23% exclusively in RNA and 1% in DNA only. The TN samples revealed a significantly higher frequency of DRMs in DNA than in RNA. Conclusions: Proviral DNA resistance testing provides additional resistance information for TN patients. It is also a reliable alternative for TE patients with unsuccessful RNA testing and can provide valuable information when no records are available.

scholarly journals Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer

2021 ◽  
Author(s):  
Zhi Yang ◽  
Kim-Marie A. Dam ◽  
Michael D. Bridges ◽  
Magnus A.G. Hoffmann ◽  
Andrew T. DeLaitsch ◽  
...  
Keyword(s):  
Binding Site ◽  
Neutralizing Antibodies ◽  
Resonance Spectroscopy ◽  
Broadly Neutralizing Antibodies ◽  
Receptor Binding Site ◽  
Double Electron ◽  
Immunogen Design ◽  
Cd4 Binding Site ◽  
Double Electron Electron Resonance ◽  
Hiv 1

Broadly-neutralizing antibodies (bNAbs) against HIV-1 Env can protect from infection. We characterized Ab1303 and Ab1573, neutralizing CD4-binding site (CD4bs) antibodies, isolated from sequentially-immunized macaques. Ab1303/Ab1573 binding was observed only when Env trimers were not constrained in the closed, prefusion conformation. Fab-Env cryo-EM structures showed that both antibodies recognized the CD4bs on Env trimer with an occluded-open conformation between closed, as targeted by bNAbs, and fully-open, as recognized by CD4. The occluded-open Env trimer conformation included outwardly-rotated gp120 subunits, but unlike CD4-bound Envs, did not exhibit V1V2 displacement, co-receptor binding site exposure, or a 4-stranded gp120 bridging sheet. Inter-protomer distances within trimers measured by double electron-electron resonance spectroscopy suggested an equilibrium between occluded-open and closed Env conformations, consistent with Ab1303/Ab1573 binding stabilizing an existing conformation. Studies of Ab1303/Ab1573 demonstrate that CD4bs neutralizing antibodies that bind open Env trimers can be raised by immunization, thereby informing immunogen design and antibody therapeutic efforts.

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