An Affinity-Enhanced Neutralizing Antibody against the Membrane-Proximal External Region of Human Immunodeficiency Virus Type 1 gp41 Recognizes an Epitope between Those of 2F5 and 4E10 (original) (raw)
Related papers
Archives of Virology, 2001
The envelope protein of human immunodeficiency virus type 1 (HIV-1) comprises the outer gp120 SU domain and the anchoring gp41 TM domain, and the conventional view is that it has a single transmembrane region with the following C-terminal sequence situated entirely within the virion. However, we have recently proposed that the gp41 C-terminal region comprises three transmembrane regions and an external loop structure. Part of this loop is the peptide 731 PRGPDRPEGIEEEGGERDRDRS 752 that carries three antibody epitopes, 734 PDRPEG 739 , 740 IEEE 743 , and 746 ERDRD 750. PDRPEG is not detected in virions but reacts with its cognate MAb (C8) in Western blots, IEEE is a linear and non-neutralizing epitope, and ERDRD is a conformational and neutralizing epitope. Here we show that escape mutants selected with neutralizing ERDRDspecific antibody had a single 732R→G substitution, 14 residues upstream of the cognate epitope, and no longer bound the selecting antibody. The same amino acid substitution altered epitope PDRPEG in the virion so that it now reacted with MAb C8, but left epitope IEEE unaffected. Introduction of 732R→G by site-specific mutagenesis into the gp41 of cloned HIV-1 NL4-3 virions allowed them to escape neutralization by ERDRD-specific IgG, and confirms that 732R
Virology, 1992
. In this study, we have mapped the regions and specific amino acid residues within NP involved in its anti-IFN activity. We identified a region spanning residues 382 to 386 as playing a critical role in the IFN-counteracting activity of NP. Alanine substitutions at several positions within this region resulted in NP mutants that lacked the IFN-counteracting activity but retained their functions in virus RNA synthesis and assembly of infectious particles. We used reverse genetics to rescue a recombinant LCMV strain carrying mutation D382A in its NP [rLCMV/ NP*(D382A)]. Compared to wild-type (WT) LCMV, rLCMV/NP*(D382A) exhibited a higher level of attenuation in IFN-competent than IFN-deficient cells. In addition, A549 cells infected with rLCMV/NP*(D382A), but not with WT LCMV, produced IFN and failed to rescue replication of the IFN-sensitive Newcastle disease virus.
Protein Expression and Purification, 1996
. Therapeutic trials were not successful (14-17), Hybrid molecules between MalE, the periplasmic partly due to a limitation in the amounts of available maltose binding protein of Escherichia coli, and CD4, materials and to the protein instability in vivo which the human T-lymphocyte receptor for the AIDS virus reduced the quantities effectively delivered to the tis-HIV, have been constructed and purified. We show that sues where high concentrations were needed, in particu-CD4 can be fused as multiple repeats to both ends of lar lymph nodes of seropositive patients, a site of persisa single MalE molecule. Hybrid proteins are exported tent high rate viral replication .
AIDS Research and Human Retroviruses, 2004
DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies. In this study, DNA vaccines were constructed to express the gp120 subunit of Env from the isolate HIV-1 R2 using both wild-type and codon-optimized gene sequences. Three copies of the murine C3d were added to the carboxyl terminus to enhance the immunogenicity of the expressed fusion protein.
Isolation of recombinant partial gag gene product p18 (HIV-1Bru) from Escherichia coli
Journal of Chromatography A, 1989
The membrane-associated structural protein, ~18, of the human immunodeticiency virus (HIV-l), has been expressed in Escherichiu coli. The recombinant protein was purified by cation-exchange chromatography on S Sepharose followed by cation-exchange high-performance liquid chromatography (HPLC) on Sulfoethyl Aspartamide. The isolation of 28.7 mg of recombinant ~18 from 16.7 1 of cell culture represents an overall yield of cu. 20%.
AIDS Research and Human Retroviruses, 1997
The genetic subtypes of human immunodeficiency virus type 1 (HIV-1) display differences in immunologie reac-tivity1 and possibly transmissibility.2 In addition to the high replication rate of HIV-1 and the infidelity of its reverse transcriptase, interstrain recombination may increase the diversity of phenotypes this virus generates. The discoveries that more than 10% of the HIV-1 strains sequenced to date are intersubtype recombinant3 and that the subtype E viruses prevalent in Thailand are probably subtype A/E recombinant4,5 suggest that recombination between viruses belonging to distinct subtypes occurs to an appreciable extent and that the resulting recombinant progeny are transmissible. Cross-immunity between HIV-1 antigens with divergent primary sequences has been a desired goal of preventive and therapeutic vaccine design. Here we report our finding that the Zairian strain used to make an inactivated, gpl20-depleted, therapeutic HIV-1 immunogen6,7 is subtype G/subtype A recombinant. This strain, Z321, was isolated from a previously frozen serum sample taken in 1976 from a 26-year-old Zairian woman who died of an AIDS-like illness in 1978.8 The virus was propagated initially in peripheral blood lymphocytes (PBLs) and later in the transformed T cell line HUT 78 for the purpose of manufacturing the therapeutic immunogen. We amplified (by polymerase chain reaction [PCR]) short segments of the Z321
Journal of Virology, 2002
The epitopes of the V3 domain of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have complex structures consisting of linear and conformational antigenic determinants. Anti-V3 antibodies (Abs) recognize both types of elements, but Abs which preferentially react to the conformational aspect of the epitopes may have more potent neutralizing activity against HIV-1, as recently suggested. To test this hypothesis, human anti-V3 monoclonal Abs (MAbs) were selected using a V3 fusion protein (V3-FP) which retains the conformation of the third variable region. The V3-FP consists of the V3 JR-CSF sequence inserted into a truncated form of murine leukemia virus gp70. Six human MAbs which recognize epitopes at the crown of the V3 loop were selected with the V3-FP. They were found to react more strongly with molecules displaying conformationally intact V3 than with linear V3 peptides. In a virus capture assay, these MAbs showed cross-clade binding to native, intact virions of clades A, B, C, D, and F. No binding was found to isolates from subtype E. The neutralizing activity of MAbs against primary isolates was determined in three assays: the GHOST cell assay, a phytohemagglutinin-stimulated peripheral blood mononuclear cell assay, and a luciferase assay. While these new MAbs displayed various degrees of activity, the pattern of cross-clade neutralization of clades A, B, and F was most pronounced. The neutralization of clades C and D viruses was weak and sporadic, and neutralization of clade E by these MAbs was not detected. Analysis by linear regression showed a highly significant correlation (P < 0.0001) between the strength of binding of these anti-V3 MAbs to intact virions and the percent neutralization. These studies demonstrate that human MAbs to conformation-sensitive epitopes of V3 display cross-clade reactivity in both binding to native, intact virions and neutralization of primary isolates.
A native-like SOSIP.664 trimer based on a HIV-1 subtype B env gene
Journal of virology, 2015
33 34 X Present address for J-P. Julien: Program in Molecular Structure and Function, The Abstract word count: 197 39 Text word count: 8643 40 3 Abstract 41 Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) 42 envelope glycoprotein (Env) spike should expose as many epitopes as possible for 43 broadly neutralizing antibodies (bNAbs), but few if any for non-neutralizing antibodies 44 (non-NAbs). Soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A strain, 45 BG505, approach this ideal and are therefore plausible vaccine candidates. Here, we 46 report on the production and in vitro properties of a new SOSIP.664 trimer derived from 47 in practical amounts is via the introduction of specific sequence changes that confer 64 stability to the cleaved form of Env. The resulting trimers are known as SOSIP.664 65 gp140s, and the current paradigm is based on the BG505 subtype A env gene. Here, we 66 describe the production and characterization of a SOSIP.664 trimer derived from a 67 subtype B gene (B41), together with a simple, one-step method to purify native-like 68 trimers by affinity chromatography with a quaternary epitope-specific bNAb, PGT145. 69 The resulting trimers will be useful for structural and immunogenicity experiments aimed 70 at devising ways to make an effective HIV-1 vaccine. 71 72 It is now clear that the resulting uncleaved gp140 (gp140 UNC ) proteins, when purified by 90 size-exclusion chromatography (SEC), contain the right number of gp120 and gp41 ECTO 91 subunits (i.e., 3 of each). However, these various SEC-purified gp140 UNC protein 92 populations mostly (90-100%) adopt non-native configurations whether or not they 93 contain additional "trimerization motifs" at the C-terminus of gp41 ECTO (20-27). Electron 94 microscopy, other biophysical measurements, and glycan profiling have shown or 95 112 cryo-electron microscopy (cryo-EM) Env structures (24, 39-42). Their potential as 113 immunogens is currently under evaluation. 114 We now seek to increase the repertoire of native-like soluble trimers available for 115 structural and immunogenicity studies. Here, we describe the B41 SOSIP.664 trimer 116 based on a subtype B env gene. These trimers are fully native-like when viewed by 117 negative stain electron microscopy (NS-EM) and have comparable antigenicity properties 118 157 of gp120 subunits of some of the B41 SOSIP.664 trimers, and of the B41 gp120 158 monomers, damaging the proteins (see Results). To overcome this problem we used the 159 293Fectin lipofection system to transiently transfect 293F suspension cells with Env and 160 Furin expression plasmids (4:1 ratio), and cultured the transfected cells in serum-free 161 medium, following the manufacturer's (Invitrogen) recommendations (see Results). 162 Env protein production from stable cell lines 163 PGV04 complex with the corresponding BG505 trimer (EMDB 5779; correlation 595 coefficient 0.95) (Fig.8A). Hence, like their BG505 counterparts, the B41 trimers have a 596 high structural integrity. 597 777 donating antibodies and reagents directly or through the AIDS reagents reference 778 program. We are grateful to David Montefiori and Celia Labranche for information on 779 the Tier-2 neutralization profile of the B41 Env-pseudotyped virus. The reconstruction 780 data reported in this paper has been deposited in the Electron Microscopy Data Bank, 781 809 Travis B, Kuhmann S, Burton DR, Hu SL, Olson WC, Moore JP. 2002. 810 Enhancing the proteolytic maturation of human immunodeficiency virus type 1 811 envelope glycoproteins. J. Virol. 76:2606-2616. 812 6. Sanders RW, Vesanen M, Schuelke N, Master A, Schiffner L, Kalyanaraman 813 R, Paluch M, Berkhout B, Maddon PJ, Olson WC, Lu M, Moore JP. 2002. 814 guinea pigs. J. Virol. 84:3270-3279.
Virology, 2008
The native, functional HIV-1 envelope glycoprotein (Env) complex is a trimer of two non-covalently associated subunits: the gp120 surface glycoprotein and the gp41 transmembrane glycoprotein. However, various non-functional forms of Env are present on virus particles and HIV-1-infected cells, some of which probably arise as the native complex decays. The aberrant forms include gp120-gp41 monomers and oligomers, as well as gp41 subunits from which gp120 has dissociated. The presence of non-functional Env creates binding sites for antibodies that do not recognize native Env complexes and that are, therefore, non-neutralizing. Non-native Env forms (monomers, dimers, tetramers and aggregates) can also arise when soluble gp140 proteins, lacking the cytoplasmic and transmembrane domains of gp41, are expressed for vaccine studies. We recently identified 5 amino acids in the gp41 N-terminal region (I535, Q543, S553, K567 and R588) that promote gp140 trimerization. We have now studied their influence on the function and antigenic properties of JR-FL Env expressed on the surfaces of pseudoviruses and Envtransfected cells. The 5 substitutions in gp41 reduce the expression of non-trimeric gp160s, without affecting trimer levels. Pseudovirions bearing the mutant Env are fully infectious with similar kinetics of Env-mediated fusion. Various non-neutralizing antibodies bind less strongly to the Env mutant, but neutralizing antibody binding is unaffected. Hence the gp41 substitutions do not adversely affect Env structure, supporting their use for making new Env-based vaccines. The mutant Env might also help in studies intended to correlate antibody binding to virus neutralization. Of note is that the 5 residues are much more frequent, individually or collectively, in viruses from subtypes other than B.
Construction of a Prokaryotic Expression Vector harboring Two HIV-1 Accessory Genes
Medical Laboratory Journal, 2021
Background and objectives: HIV-1 Nef and Vpr antigens have been described as suitable candidates for therapeutic HIV vaccine development. The aim of this study was to generate Nef-Vpr fusion gene construct and to clone the construct into pET-23a (+), a prokaryotic expression vector. Methods: HIV-1 Nef and Vpr genes were PCR-amplified from the pNL4-3 plasmid using specific primers and Pfu DNA polymerase. Results of PCR amplification were visualized by electrophoresis on 0.8% agarose gel. At first, the amplified Nef fragment was cloned into NheI and BamHI restriction sites of pET-23a expression vector. Next, cloning of Vpr gene was performed into BamHI and HindIII restriction sites of the pET-23a-Nef vector. Finally, purity of the recombinant pET-23-Nef-Vpr construct was determined by NanoDrop spectrophotometry. Results: PCR amplification of Nef and Vpr genes was confirmed by detection of 620 bp and 291 bp bands, respectively. Cloning of the Nef-Vpr construct into the vector was confirmed by detection of a 911 bp fragment following enzymatic digestion with NheI and HindIII and sequencing. Conclusion: The successful construction of recombinant fusion plasmid encoding a chimeric Nef-Vpr gene was performed in a prokaryotic expression vector for development of HIV-1 recombinant protein vaccine in near future.