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Emergence and evolution of enfuvirtide resistance following long-term therapy involves heptad repeat 2 mutations within gp41.

ABSTRACT: The objective of this study was to track the evolution of sequence changes in both the heptad region 1 (HR1) and HR2 domains of gp41 associated with resistance to enfuvirtide (ENF) in a patient cohort receiving long-term ENF treatment. We studied 17 highly antiretroviral agent-experienced patients receiving long-term ENF treatment with virological rebound or a lack of suppression. Sixty-two samples obtained after between 5 and 107 weeks of ENF therapy were analyzed. Baseline samples from 15 of these 17 patients were available for analysis. Viruses from five samples from four patients were also sequenced after the cessation of ENF therapy. Drug susceptibilities were assessed by a pseudotype virus reporter assay. We identified HR1 and HR2 sequence changes over time in relation to the baseline sequences. Mutations in HR1 (amino acids 36 to 45) were noted in all cases, including previously unreported changes N42Q/H and N43Q. In addition to a range of HR2 sequence changes at polymorphic sites, isolates from 6 of 17 (35%) patients developed an S138A substitution in the HR2 domain at least 8 weeks after the start of ENF treatment and also subsequent to the first emergence of HR1 mutations. In most, but not all, cases the S138A mutation accompanied HR1 mutations at position 43. Molecular modeling demonstrates the close proximity of S138A with amino acids 40 and 45 in HR1. Of note, isolates in samples available from four patients demonstrated the loss of both the HR1 and the S138A HR2 mutations following the cessation of therapy. We show that the S138A HR2 mutation increased the level of resistance by approximately threefold over that conferred by the HR1 mutation N43D. Continual evolution of HR1 in the domain from amino acids 36 to 45 was observed during long-term ENF therapy. We have identified, for the first time, an ENF resistance-associated HR2 mutation, S138A, which appeared in isolates from 6 of 17 patients with virological failure and demonstrated its potential to contribute to drug resistance. We propose that this represents a possible secondary and/or compensatory mutation, particularly when it coexists with mutations at position 43 in HR-1.

SUBMITTER: Xu L

PROVIDER: S-EPMC549241 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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Emergence and evolution of enfuvirtide resistance following long-term therapy involves heptad repeat 2 mutations within gp41.

Xu L L Pozniak A A Wildfire A A Stanfield-Oakley S A SA Mosier S M SM Ratcliffe D D Workman J J Joall A A Myers R R Smit E E Cane P A PA Greenberg M L ML Pillay D D

Antimicrobial agents and chemotherapy 20050301 3

The objective of this study was to track the evolution of sequence changes in both the heptad region 1 (HR1) and HR2 domains of gp41 associated with resistance to enfuvirtide (ENF) in a patient cohort receiving long-term ENF treatment. We studied 17 highly antiretroviral agent-experienced patients receiving long-term ENF treatment with virological rebound or a lack of suppression. Sixty-two samples obtained after between 5 and 107 weeks of ENF therapy were analyzed. Baseline samples from 15 of t ...[more]

PMID: 15728911

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Project description:Previous studies suggest that short peptides from the heptad repeat 2 (HR2) domain of gp41 expressed on the cell surface are more potent inhibitors of HIV-1 entry than soluble analogs. However, their therapeutic potential has only been examined using lentiviral vectors. Here, we aimed to develop CRISPR/Cas9-based fusion inhibitory peptide knock-in (KI) technology for the generation and selection of HIV-1-resistant T cells. First, we embedded a series of HIV-1 fusion inhibitory peptides in CD52, the shortest glycosylphosphatidylinositol (GPI)-anchored protein, which efficiently delivers epitope tags to the cell surface and maintains a sufficient level of KI. Among the seven peptides tested, MT-C34, HP-23L, and 2P23 exhibited significant activity against both cell-free and cell-to-cell HIV-1 infection. The shed variant of MT-C34 provided insufficient protection against HIV-1 due to its low concentration in the culture medium. Using Cas9 plasmids or ribonucleoprotein electroporation and peptide-specific antibodies, we sorted CEM/R5 cells with biallelic KI of MT-C34 and 2P23 peptides at the CXCR4 locus. In combination, these peptides provided a higher level of protection than individual KI. By extending homology arms and cloning donor DNA into a plasmid containing signals for nuclear localization, we achieved KI of MT-C34 into the CXCR4 locus and HIV-1 proviral DNA at levels of up to 35% in the T-cell line and up to 4 to 5% in primary CD4 lymphocytes. Compared to lentiviral delivery, KI resulted in the higher MT-C34 surface expression and stronger protection of lymphocytes from HIV-1. Thus, we demonstrate that KI is a viable strategy for peptide-based therapy of HIV infection. IMPORTANCE HIV is a human lentivirus that infects CD4-positive immune cells and, when left untreated, manifests in the fatal disease known as AIDS. Antiretroviral therapy (ART) does not lead to viral clearance, and HIV persists in the organism as a latent provirus. One way to control infection is to increase the population of HIV-resistant CD4 lymphocytes via entry molecule knockout or expression of different antiviral genes. Peptides from the heptad repeat (HR) domain of gp41 are potent inhibitors of HIV-1 fusion, especially when designed to express on the cell surface. Individual gp41 peptides encoded by therapeutic lentiviral vectors have been evaluated and some have entered clinical trials. However, a CRISPR/Cas9-based gp41 peptide delivery platform that operates through concomitant target gene modification has not yet been developed due to low knock-in (KI) rates in primary cells. Here, we systematically evaluated the antiviral activity of different HR2 peptides cloned into the shortest carrier molecule, CD52. The resulting small-size transgene constructs encoding selected peptides, in combination with improvements to enhance donor vector nuclear import, helped to overcome precise editing restrictions in CD4 lymphocytes. Using KI into CXCR4, we demonstrated different options for target gene modification, effectively protecting edited cells against HIV-1.

Project description:HIV-1 infection is initiated by the viral glycoprotein Env, which, after interaction with cellular coreceptors, adopts a transient conformation known as the prehairpin intermediate (PHI). The N-heptad repeat (NHR) is a highly conserved region of gp41 exposed in the PHI; it is the target of the FDA-approved drug enfuvirtide and of neutralizing monoclonal antibodies (mAbs). However, to date, these mAbs have only been weakly effective against tier-1 HIV-1 strains, which are most sensitive to neutralizing antibodies. Here, we engineered and tested 11 IgG variants of D5, an anti-NHR mAb, by recombining previously described mutations in four of D5's six antibody complementarity-determining regions. One variant, D5_AR, demonstrated 6-fold enhancement in the 50% inhibitory dose (ID50) against lentivirus pseudotyped with HXB2 Env. D5_AR exhibited weak cross-clade neutralizing activity against a diverse set of tier-2 HIV-1 viruses, which are less sensitive to neutralizing antibodies than tier-1 viruses and are the target of current antibody-based vaccine efforts. In addition, the neutralization potency of D5_AR IgG was greatly enhanced in target cells expressing FcγRI, with ID50 values of <0.1 μg/ml; this immunoglobulin receptor is expressed on macrophages and dendritic cells, which are implicated in the early stages of HIV-1 infection of mucosal surfaces. D5 and D5_AR have equivalent neutralization potency in IgG, Fab, and single-chain variable-fragment (scFv) formats, indicating that neutralization is not impacted by steric hindrance. Taken together, these results provide support for vaccine strategies that target the PHI by eliciting antibodies against the gp41 NHR and support investigation of anti-NHR mAbs in nonhuman primate passive immunization studies. IMPORTANCE Despite advances in antiretroviral therapy, HIV remains a global epidemic and has claimed more than 32 million lives. Accordingly, developing an effective HIV vaccine remains an urgent public health need. The gp41 N-heptad repeat (NHR) of the HIV-1 prehairpin intermediate (PHI) is highly conserved (>90%) and is inhibited by the FDA-approved drug enfuvirtide, making it an attractive vaccine target. However, to date, anti-NHR antibodies have not been potent. Here, we engineered D5_AR, a more potent variant of the anti-NHR antibody D5, and established its ability to inhibit HIV-1 strains that are more difficult to neutralize and are more representative of circulating strains (tier-2 strains). The neutralizing activity of D5_AR was greatly potentiated in cells expressing FcγRI; FcγRI is expressed on cells that are implicated at the earliest stages of sexual HIV-1 transmission. Taken together, these results bolster efforts to target the gp41 NHR and the PHI for vaccine development.

Project description:BackgroundThe native pre-fusion structure of gp120/gp41 complex of human immunodeficiency virus type 1 was recently revealed. In the model, the helices of gp41 (α6, α7, α8, and α9) form a four-helix collar underneath trimeric gp120. Gp41 is a class I fusion protein and mediates membrane fusion by forming a post-fusion structure called the six-helix bundle (6HB). The comparison of the pre- and post-fusion structures revealed the large conformational changes in gp41 during the antiparallel packing of the N- and C-terminal heptad repeats (NHRs and CHRs) in membrane fusion. Several mutagenesis studies of gp41 performed in the past were interpreted based on 6HB, the only available structure at that time. To obtain an insight about the current pre-fusion structural model and conformational changes during membrane fusion, alanine insertion mutagenesis of the NHR, CHR and connecting loop regions of HXB2 gp41 was performed. The effects of mutations on biosynthesis and membrane fusion were analyzed by immunoblotting and fusion assays, respectively. The extent of membrane fusion was evaluated by split luciferase-based pore formation and syncytia formation assays, respectively.ResultsConsistent with the current structural model, drastic negative effects of mutations on biosynthesis and membrane fusion were observed for NHR, loop, and proximal regions of CHR (up to amino acid position 643). The insertions in α9 after it leaves the four-helix collar were tolerable for biosynthesis. These CHR mutants showed varying effects on membrane fusion. Insertion at position 644 or 645 resulted in poor pore and syncytia formation. Efficient pore and syncytia formation almost similar to that of the wild type was observed for insertion at position 647, 648 or 649. However, recovery of virus infectivity was only observed for the insertions beyond position 648.ConclusionsThe mutagenesis data for HXB2 gp41 is in agreement with the recent pre-fusion structure model. The virus infection data suggested that fusion pores sufficiently large enough for the release of the virus genome complex are formed after the completion of 6HB beyond position 648.

Dataset Information

Emergence and evolution of enfuvirtide resistance following long-term therapy involves heptad repeat 2 mutations within gp41.

Publications

Emergence and evolution of enfuvirtide resistance following long-term therapy involves heptad repeat 2 mutations within gp41.

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