The Experts below are selected from a list of 157344 Experts worldwide ranked by ideXlab platform
Rogier W. Sanders - One of the best experts on this subject based on the ideXlab platform.
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Similarities and differences between native HIV-1 envelope glycoprotein Trimers and stabilized soluble Trimer mimetics
PLoS pathogens, 2019Co-Authors: Alba Torrents De La Peña, Kimmo Rantalainen, Christopher A. Cottrell, Joel D. Allen, Marit J. Van Gils, Jonathan L. Torres, Max Crispin, Rogier W. Sanders, Andrew B. WardAbstract:The HIV-1 envelope glycoprotein (Env) Trimer is located on the surface of the virus and is the target of broadly neutralizing antibodies (bNAbs). Recombinant native-like soluble Env Trimer mimetics, such as SOSIP Trimers, have taken a central role in HIV-1 vaccine research aimed at inducing bNAbs. We therefore performed a direct and thorough comparison of a full-length unmodified Env Trimer containing the transmembrane domain and the cytoplasmic tail, with the sequence matched soluble SOSIP Trimer, both based on an early Env sequence (AMC011) from an HIV+ individual that developed bNAbs. The structures of the full-length AMC011 Trimer bound to either bNAb PGT145 or PGT151 were very similar to the structures of SOSIP Trimers. Antigenically, the full-length and SOSIP Trimers were comparable, but in contrast to the full-length Trimer, the SOSIP Trimer did not bind at all to non-neutralizing antibodies, most likely as a consequence of the intrinsic stabilization of the SOSIP Trimer. Furthermore, the glycan composition of full-length and SOSIP Trimers was similar overall, but the SOSIP Trimer possessed slightly less complex and less extensively processed glycans, which may relate to the intrinsic stabilization as well as the absence of the membrane tether. These data provide insights into how to best use and improve membrane-associated full-length and soluble SOSIP HIV-1 Env Trimers as immunogens.
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Stabilizing HIV-1 envelope glycoprotein Trimers to induce neutralizing antibodies
Retrovirology, 2018Co-Authors: Alba Torrents De La Peña, Rogier W. SandersAbstract:An effective HIV-1 vaccine probably will need to be able to induce broadly neutralizing HIV-1 antibodies (bNAbs) in order to be efficacious. The many bNAbs that have been isolated from HIV-1 infected patients illustrate that the human immune system is able to elicit this type of antibodies. The elucidation of the structure of the HIV-1 envelope glycoprotein (Env) Trimer has further fueled the search for Env immunogens that induce bNAbs, but while native Env Trimer mimetics are often capable of inducing strain-specific neutralizing antibodies (NAbs) against the parental virus, they have not yet induced potent bNAb responses. To improve the performance of Env Trimer immunogens, researchers have studied the immune responses that Env Trimers have induced in animals; they have evaluated how to best use Env Trimers in various immunization regimens; and they have engineered increasingly stabilized Env Trimer variants. Here, we review the different approaches that have been used to increase the stability of HIV-1 Env Trimer immunogens with the aim of improving the induction of NAbs. In particular, we draw parallels between the various approaches to stabilize Env Trimers and ones that have been used by nature in extremophile microorganisms in order to survive in extreme environmental conditions.
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high throughput protein engineering improves the antigenicity and stability of soluble hiv 1 envelope glycoprotein sosip Trimers
Journal of Virology, 2017Co-Authors: Jonathan T Sullivan, Rogier W. Sanders, Andrew B. Ward, Gabriel Ozorowski, Anila Yasmeen, Chidananda Sulli, Alberto Nilo, P J Klasse, John P MooreAbstract:Soluble envelope glycoprotein (Env) Trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membrane-bound Env spike (gp160). Since engineering Trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying Trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151 and so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (MAbs), including PGT151. We identified 29 Trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 Trimer variants identified here may be useful for vaccine and structural studies.IMPORTANCE Soluble Env Trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble Trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble Trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine.
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native like env Trimers as a platform for hiv 1 vaccine design
Immunological Reviews, 2017Co-Authors: Rogier W. Sanders, John P MooreAbstract:Summary We describe the development and potential use of various designs of recombinant HIV-1 envelope glycoprotein Trimers that mimic the structure of the virion-associated spike, which is the target for neutralizing antibodies. The goal of Trimer development programs is to induce broadly neutralizing antibodies with the potential to intervene against multiple circulating HIV-1 strains. Among the topics we address are the designs of various constructs; how native-like Trimers can be produced and purified; the properties of such Trimers in vitro and their immunogenicity in various animals; and the immunization strategies that may lead to the eventual elicitation of broadly neutralizing antibodies. In summary, native-like Trimers are a now a platform for structure- and immunology-based design improvements that could eventually yield immunogens of practical value for solving the long-standing HIV-1 vaccine problem.
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hiv 1 envelope Trimer design and immunization strategies to induce broadly neutralizing antibodies
Trends in Immunology, 2016Co-Authors: Steven W De Taeye, Rogier W. Sanders, John P MooreAbstract:The identification of multiple broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) Trimer has facilitated its structural characterization and guided Env immunogen design. Several recent studies constitute progress in utilizing this knowledge for the development of an HIV-1 vaccine that induces bNAbs. Native-like Env Trimers can induce autologous NAb responses against resistant (Tier-2) viruses in several animal models. Here we review recent studies aimed at addressing the challenge of driving the strong but narrowly focused NAb responses to Env Trimers towards ones with much greater breadth. Among strategies that merit pursuing are using multiple Trimers as sequential or simultaneous immunogens, targeting the germline precursors of bNAbs, delivering sequential lineages of Trimers derived from infected individuals who developed bNAbs, and presenting Trimers as particulate antigens.
Gabriel Ozorowski - One of the best experts on this subject based on the ideXlab platform.
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rational design of dna expressed stabilized native like hiv 1 envelope Trimers
Cell Reports, 2018Co-Authors: Yoann Aldon, Joel D. Allen, Gabriel Ozorowski, Linling He, Paul F Mckay, R Levai, Monica Tolazzi, Paul Rogers, Katalin Fabian, Gabriella ScarlattiAbstract:The HIV-1-envelope glycoprotein (Env) is the main target of antigen design for antibody-based prophylactic vaccines. The generation of broadly neutralizing antibodies (bNAb) likely requires the appropriate presentation of stabilized Trimers preventing exposure of non-neutralizing antibody (nNAb) epitopes. We designed a series of membrane-bound Envs with increased Trimer stability through the introduction of key stabilization mutations. We derived a stabilized HIV-1 Trimer, ConSOSL.UFO.750, which displays a dramatic reduction in nNAb binding while maintaining high quaternary and MPER-specific bNAb binding. Its soluble counterpart, ConSOSL.UFO.664, displays similar antigenicity, and its native-like Env structure is confirmed by negative stain-EM and glycosylation profiling of the soluble ConSOSL.UFO.664 Trimer. A rabbit immunization study demonstrated that the ConSOSL.UFO.664 can induce autologous tier 2 neutralization. We have successfully designed a stabilized native-like Env Trimer amenable to nucleic acid or viral vector-based vaccination strategies.
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hiv 1 vaccine design through minimizing envelope metastability
bioRxiv, 2018Co-Authors: Linling He, Joel D. Allen, Sonu Kumar, Deli Huang, Colin J Mann, Karen L Sayefrancisco, Jeffrey Copps, Anita Sarkar, Gabrielle S Blizard, Gabriel OzorowskiAbstract:Overcoming envelope metastability is crucial to Trimer-based HIV-1 vaccine design. Here, we present a coherent vaccine strategy by minimizing metastability. For ten strains across five clades, we demonstrate that gp41 ectodomain (gp41ECTO) is the main source of envelope metastability by replacing wild-type gp41ECTO with BG505 gp41ECTO of the uncleaved prefusion-optimized (UFO) design. These gp41ECTO-swapped Trimers can be produced in CHO cells with high yield and high purity. Crystal structure of a gp41ECTO-swapped Trimer elucidates how a neutralization-resistant tier 3 virus evades antibody recognition of the V2 apex. UFO Trimers of transmitted/founder (T/F) viruses and UFO Trimers containing a consensus-based ancestral gp41ECTO suggest an evolutionary root of the metastability. Gp41ECTO-stabilized Trimers can be readily displayed on 24- and 60-meric nanoparticles, with incorporation of additional T cell help illustrated for a hyperstable 60-mer. In mice and rabbits, gp140 nanoparticles induced more effective tier 2 neutralizing antibody response than Trimers with statistical significance.
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structure based design of native like hiv 1 envelope Trimers to silence non neutralizing epitopes and eliminate cd4 binding
Nature Communications, 2017Co-Authors: Daniel W Kulp, Matthias Pauthner, Christopher A. Cottrell, Jon M Steichen, Xiaozhen Hu, Torben Schiffner, Alessia Liguori, Colin Havenardaughton, Gabriel OzorowskiAbstract:Elicitation of broadly neutralizing antibodies (bnAbs) is a primary HIV vaccine goal. Native-like Trimers mimicking virion-associated spikes present nearly all bnAb epitopes and are therefore promising vaccine antigens. However, first generation native-like Trimers expose epitopes for non-neutralizing antibodies (non-nAbs), which may hinder bnAb induction. We here employ computational and structure-guided design to develop improved native-like Trimers that reduce exposure of non-nAb epitopes in the V3-loop and Trimer base, minimize both CD4 reactivity and CD4-induced non-nAb epitope exposure, and increase thermal stability while maintaining bnAb antigenicity. In rabbit immunizations with native-like Trimers of the 327c isolate, improved Trimers suppress elicitation of V3-directed and tier-1 neutralizing antibodies and induce robust autologous tier-2 neutralization, unlike a first-generation Trimer. The improved native-like Trimers from diverse HIV isolates, and the design methods, have promise to assist in the development of a HIV vaccine.
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high throughput protein engineering improves the antigenicity and stability of soluble hiv 1 envelope glycoprotein sosip Trimers
Journal of Virology, 2017Co-Authors: Jonathan T Sullivan, Rogier W. Sanders, Andrew B. Ward, Gabriel Ozorowski, Anila Yasmeen, Chidananda Sulli, Alberto Nilo, P J Klasse, John P MooreAbstract:Soluble envelope glycoprotein (Env) Trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membrane-bound Env spike (gp160). Since engineering Trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying Trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151 and so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (MAbs), including PGT151. We identified 29 Trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 Trimer variants identified here may be useful for vaccine and structural studies.IMPORTANCE Soluble Env Trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble Trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble Trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine.
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reducing v3 antigenicity and immunogenicity on soluble native like hiv 1 env sosip Trimers
Journal of Virology, 2017Co-Authors: Rajesh Ringe, Kimmo Rantalainen, Christopher A. Cottrell, Jonathan L. Torres, Gabriel Ozorowski, Anila Yasmeen, Thomas A Ketas, Weston B Struwe, Katie Matthews, Celia C LabrancheAbstract:Native-like Trimers of the SOSIP design are being developed as immunogens in human immunodeficiency virus type 1 (HIV-1) vaccine development programs. These Trimers display the epitopes for multiple broadly neutralizing antibodies (bNAbs) but can also expose binding sites for some types of nonneutralizing antibodies (non-NAbs). Among the latter are epitopes in the gp120 V3 region that are highly immunogenic when SOSIP Trimers are evaluated in animal models. It is presently uncertain whether antibodies against V3 can interfere with the induction of NAbs, but there are good arguments in favor of suppressing such "off-target" immune responses. Accordingly, we have assessed how to minimize the exposure of V3 non-NAb epitopes and thereby reduce their immunogenicity by introducing N-glycans within the V3 region of BG505 SOSIP Trimers. We found that inserting glycans at positions 306 and 314 (termed M1 and M7) markedly reduced V3 antigenicity while improving the presentation of Trimer apex bNAb epitopes. Both added glycans were shown to be predominantly of the Man6GlcNAc2 form. The additional introduction of the E64K ground-state stabilizing substitution markedly reduced or ablated soluble CD4 (sCD4) induction of non-NAb epitopes in V3 and/or associated with the coreceptor binding site. When a V3 glycan- and E64K-modified Trimer variant, BG505 SOSIP.664-E64K.M1M7, was tested in rabbits, V3 immunogenicity was eliminated while the autologous NAb response was unchanged.IMPORTANCE Trimeric proteins are being developed for future HIV-1 vaccine trials in humans, with the goal of eliciting broadly active neutralizing antibodies (NAbs) that are active against a wide variety of circulating strains. In animal models, the present generation of native-like Trimer immunogens, exemplified by the BG505 SOSIP.664 construct, induces narrow-specificity antibodies against the neutralization-resistant (tier-2), sequence-matched virus and more broadly active antibodies against sequence-divergent atypically neutralization-sensitive (tier-1) viruses. A concern in the Trimer immunogen design field has been whether the latter off-target antibodies might interfere with the induction of the more desired responses to tier-2 epitopes. Here, we have inserted two glycans into the dominant site for tier-1 NAbs, the gp120 V3 region, to block the induction of off-target antibodies. We characterized the new Trimers, tested them as immunogens in rabbits, and found that the blocking glycans eliminated the induction of tier-1 NAbs to V3-epitopes.
Anila Yasmeen - One of the best experts on this subject based on the ideXlab platform.
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structural and functional evaluation of de novo designed two component nanoparticle carriers for hiv env Trimer immunogens
bioRxiv, 2020Co-Authors: Aleksandar Antanasijevic, Christopher A. Cottrell, Joel D. Allen, Anila Yasmeen, Deli Huang, Jeffrey Copps, George Ueda, Philip J M Brouwer, Leigh M Sewall, Ilja BontjerAbstract:Abstract Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Nanoparticles of different sizes and geometries can be designed and combined with appropriate antigens to fit the requirements of different immunization strategies. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying Trimeric HIV envelope glycoprotein (Env) ectodomains. Env Trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to the underlying Trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env Trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. Comparing the humoral responses elicited by ConM-SOSIP Trimers presented on a two-component tetrahedral nanoparticle to the corresponding soluble protein revealed that multivalent presentation increased the proportion of the overall antibody response directed against autologous neutralizing Ab epitopes present on the ConM-SOSIP Trimers. Author Summary Protein constructs based on soluble ectodomains of HIV glycoprotein (Env) Trimers are the basis of many current HIV vaccine platforms. Multivalent antigen display is one strategy applied to improve the immunogenicity of different subunit vaccine candidates. Here, we describe and comprehensively evaluate a library of de novo designed, protein nanoparticles of different geometries for their ability to present Trimeric Env antigens. We found three nanoparticle candidates that can stably incorporate model Env Trimer on their surface while maintaining its structure and antigenicity. Immunogenicity of the designed nanoparticles is assessed in vitro and in vivo. In addition to introducing a novel set of reagents for multivalent display of Env Trimers, this work provides both guiding principles and a detailed experimental roadmap for the generation, characterization, and optimization of Env-presenting, self-assembling nanoparticle immunogens.
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enhancing and shaping the immunogenicity of native like hiv 1 envelope Trimers with a two component protein nanoparticle
Nature Communications, 2019Co-Authors: Philip J M Brouwer, Zachary Berndsen, Anila Yasmeen, Aleksandar Antanasijevic, Brooke Fiala, Tom P L Bijl, Ilja Bontjer, Jacob B Bale, William Sheffler, Joel D. AllenAbstract:The development of native-like HIV-1 envelope (Env) Trimer antigens has enabled the induction of neutralizing antibody (NAb) responses against neutralization-resistant HIV-1 strains in animal models. However, NAb responses are relatively weak and narrow in specificity. Displaying antigens in a multivalent fashion on nanoparticles (NPs) is an established strategy to increase their immunogenicity. Here we present the design and characterization of two-component protein NPs displaying 20 stabilized SOSIP Trimers from various HIV-1 strains. The two-component nature permits the incorporation of exclusively well-folded, native-like Env Trimers into NPs that self-assemble in vitro with high efficiency. Immunization studies show that the NPs are particularly efficacious as priming immunogens, improve the quality of the Ab response over a conventional one-component nanoparticle system, and are most effective when SOSIP Trimers with an apex-proximate neutralizing epitope are displayed. Their ability to enhance and shape the immunogenicity of SOSIP Trimers make these NPs a promising immunogen platform.
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high throughput protein engineering improves the antigenicity and stability of soluble hiv 1 envelope glycoprotein sosip Trimers
Journal of Virology, 2017Co-Authors: Jonathan T Sullivan, Rogier W. Sanders, Andrew B. Ward, Gabriel Ozorowski, Anila Yasmeen, Chidananda Sulli, Alberto Nilo, P J Klasse, John P MooreAbstract:Soluble envelope glycoprotein (Env) Trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membrane-bound Env spike (gp160). Since engineering Trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying Trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151 and so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (MAbs), including PGT151. We identified 29 Trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 Trimer variants identified here may be useful for vaccine and structural studies.IMPORTANCE Soluble Env Trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble Trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble Trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine.
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reducing v3 antigenicity and immunogenicity on soluble native like hiv 1 env sosip Trimers
Journal of Virology, 2017Co-Authors: Rajesh Ringe, Kimmo Rantalainen, Christopher A. Cottrell, Jonathan L. Torres, Gabriel Ozorowski, Anila Yasmeen, Thomas A Ketas, Weston B Struwe, Katie Matthews, Celia C LabrancheAbstract:Native-like Trimers of the SOSIP design are being developed as immunogens in human immunodeficiency virus type 1 (HIV-1) vaccine development programs. These Trimers display the epitopes for multiple broadly neutralizing antibodies (bNAbs) but can also expose binding sites for some types of nonneutralizing antibodies (non-NAbs). Among the latter are epitopes in the gp120 V3 region that are highly immunogenic when SOSIP Trimers are evaluated in animal models. It is presently uncertain whether antibodies against V3 can interfere with the induction of NAbs, but there are good arguments in favor of suppressing such "off-target" immune responses. Accordingly, we have assessed how to minimize the exposure of V3 non-NAb epitopes and thereby reduce their immunogenicity by introducing N-glycans within the V3 region of BG505 SOSIP Trimers. We found that inserting glycans at positions 306 and 314 (termed M1 and M7) markedly reduced V3 antigenicity while improving the presentation of Trimer apex bNAb epitopes. Both added glycans were shown to be predominantly of the Man6GlcNAc2 form. The additional introduction of the E64K ground-state stabilizing substitution markedly reduced or ablated soluble CD4 (sCD4) induction of non-NAb epitopes in V3 and/or associated with the coreceptor binding site. When a V3 glycan- and E64K-modified Trimer variant, BG505 SOSIP.664-E64K.M1M7, was tested in rabbits, V3 immunogenicity was eliminated while the autologous NAb response was unchanged.IMPORTANCE Trimeric proteins are being developed for future HIV-1 vaccine trials in humans, with the goal of eliciting broadly active neutralizing antibodies (NAbs) that are active against a wide variety of circulating strains. In animal models, the present generation of native-like Trimer immunogens, exemplified by the BG505 SOSIP.664 construct, induces narrow-specificity antibodies against the neutralization-resistant (tier-2), sequence-matched virus and more broadly active antibodies against sequence-divergent atypically neutralization-sensitive (tier-1) viruses. A concern in the Trimer immunogen design field has been whether the latter off-target antibodies might interfere with the induction of the more desired responses to tier-2 epitopes. Here, we have inserted two glycans into the dominant site for tier-1 NAbs, the gp120 V3 region, to block the induction of off-target antibodies. We characterized the new Trimers, tested them as immunogens in rabbits, and found that the blocking glycans eliminated the induction of tier-1 NAbs to V3-epitopes.
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immunogenicity of stabilized hiv 1 envelope Trimers with reduced exposure of non neutralizing epitopes
Cell, 2015Co-Authors: Steven W De Taeye, Alba Torrents De La Peña, Gabriel Ozorowski, Miklos Guttman, Jeanphilippe Julien, Tom L G M Van Den Kerkhof, Judith A Burger, Laura K Pritchard, Pavel Pugach, Anila YasmeenAbstract:The envelope glycoprotein Trimer mediates HIV-1 entry into cells. The Trimer is flexible, fluctuating between closed and more open conformations and sometimes sampling the fully open, CD4-bound form. We hypothesized that conformational flexibility and transient exposure of non-neutralizing, immunodominant epitopes could hinder the induction of broadly neutralizing antibodies (bNAbs). We therefore modified soluble Env Trimers to stabilize their closed, ground states. The Trimer variants were indeed stabilized in the closed conformation, with a reduced ability to undergo receptor-induced conformational changes and a decreased exposure of non-neutralizing V3-directed antibody epitopes. In rabbits, the stabilized Trimers induced similar autologous Tier-1B or Tier-2 NAb titers to those elicited by the corresponding wild-type Trimers but lower levels of V3-directed Tier-1A NAbs. Stabilized, closed Trimers might therefore be useful components of vaccines aimed at inducing bNAbs.
Andrew B. Ward - One of the best experts on this subject based on the ideXlab platform.
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Similarities and differences between native HIV-1 envelope glycoprotein Trimers and stabilized soluble Trimer mimetics
PLoS pathogens, 2019Co-Authors: Alba Torrents De La Peña, Kimmo Rantalainen, Christopher A. Cottrell, Joel D. Allen, Marit J. Van Gils, Jonathan L. Torres, Max Crispin, Rogier W. Sanders, Andrew B. WardAbstract:The HIV-1 envelope glycoprotein (Env) Trimer is located on the surface of the virus and is the target of broadly neutralizing antibodies (bNAbs). Recombinant native-like soluble Env Trimer mimetics, such as SOSIP Trimers, have taken a central role in HIV-1 vaccine research aimed at inducing bNAbs. We therefore performed a direct and thorough comparison of a full-length unmodified Env Trimer containing the transmembrane domain and the cytoplasmic tail, with the sequence matched soluble SOSIP Trimer, both based on an early Env sequence (AMC011) from an HIV+ individual that developed bNAbs. The structures of the full-length AMC011 Trimer bound to either bNAb PGT145 or PGT151 were very similar to the structures of SOSIP Trimers. Antigenically, the full-length and SOSIP Trimers were comparable, but in contrast to the full-length Trimer, the SOSIP Trimer did not bind at all to non-neutralizing antibodies, most likely as a consequence of the intrinsic stabilization of the SOSIP Trimer. Furthermore, the glycan composition of full-length and SOSIP Trimers was similar overall, but the SOSIP Trimer possessed slightly less complex and less extensively processed glycans, which may relate to the intrinsic stabilization as well as the absence of the membrane tether. These data provide insights into how to best use and improve membrane-associated full-length and soluble SOSIP HIV-1 Env Trimers as immunogens.
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high throughput protein engineering improves the antigenicity and stability of soluble hiv 1 envelope glycoprotein sosip Trimers
Journal of Virology, 2017Co-Authors: Jonathan T Sullivan, Rogier W. Sanders, Andrew B. Ward, Gabriel Ozorowski, Anila Yasmeen, Chidananda Sulli, Alberto Nilo, P J Klasse, John P MooreAbstract:Soluble envelope glycoprotein (Env) Trimers (SOSIP.664 gp140) are attractive HIV-1 vaccine candidates, with structures that mimic the native membrane-bound Env spike (gp160). Since engineering Trimers can be limited by the difficulty of rationally predicting beneficial mutations, here we used a more comprehensive mutagenesis approach with the goal of identifying Trimer variants with improved antigenic and stability properties. We created 341 cysteine pairs at predicted points of stabilization throughout gp140, 149 proline residue substitutions at every residue of the gp41 ectodomain, and 362 space-filling residue substitutions at every hydrophobic and aromatic residue in gp140. The parental protein target, the clade B strain B41 SOSIP.664 gp140, does not bind the broadly neutralizing antibody PGT151 and so was used here to identify improved variants that also provide insight into the structural basis for Env antigenicity. Each of the 852 mutants was expressed in human cells and screened for antigenicity using four different monoclonal antibodies (MAbs), including PGT151. We identified 29 Trimer variants with antigenic improvements derived from each of the three mutagenesis strategies. We selected four variants (Q203F, T538F, I548F, and M629P) for more comprehensive biochemical, structural, and antigenicity analyses. The T538F substitution had the most beneficial effect overall, including restoration of the PGT151 epitope. The improved B41 SOSIP.664 Trimer variants identified here may be useful for vaccine and structural studies.IMPORTANCE Soluble Env Trimers have become attractive HIV-1 vaccine candidates, but the prototype designs are capable of further improvement through protein engineering. Using a high-throughput screening technology (shotgun mutagenesis) to create and evaluate 852 variants, we were able to identify sequence changes that were beneficial to the antigenicity and stability of soluble Trimers based on the clade B B41 env gene. The strategies described here may be useful for identifying a wider range of antigenically and structurally improved soluble Trimers based on multiple genotypes for use in programs intended to create a broadly protective HIV-1 vaccine.
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the hiv 1 envelope glycoprotein structure nailing down a moving target
Immunological Reviews, 2017Co-Authors: Andrew B. Ward, Ian A WilsonAbstract:Structure determination of the HIV-1 envelope glycoprotein (Env) presented a number of challenges, but several high-resolution structures have now become available. In 2013, cryo-EM and x-ray structures of soluble, cleaved SOSIP Env Trimers from the clade A BG505 strain provided the first glimpses into the Env Trimer fold as well as more the variable regions. A recent cryo-EM structure of a native full-length Trimer without any stabilizing mutations had the same core structure, but revealed new insights and features. A more comprehensive and higher resolution understanding of the glycan shield has also emerged, enabling a more complete representation of the Env glycoprotein structure. Complexes of Env Trimers with broadly neutralizing antibodies have surprisingly illustrated that most of the Env surface can be targeted in natural infection and that the neutralizing epitopes are almost all composed of both peptide and glycan components. These structures have also provided further evidence of the inherent plasticity of Env and how antibodies can exploit this flexibility by perturbing or even stabilizing the Trimer to facilitate neutralization. These breakthroughs have stimulated further design and stabilization of Env Trimers as well as other platforms to generate Trimers that now span multiple subtypes. These Env Trimers when used as immunogens, have led to the first vaccine-induced neutralizing antibodies for structural and functional analyses.
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thermostability of well ordered hiv spikes correlates with the elicitation of autologous tier 2 neutralizing antibodies
PLOS Pathogens, 2016Co-Authors: Yi Feng, Karen Tran, Shridhar Bale, Shailendra Kumar, Javier Guenaga, Richard K Wilson, Heather Arendt, Joanne Destefano, Andrew B. WardAbstract:In the context of HIV vaccine design and development, HIV-1 spike mimetics displaying a range of stabilities were evaluated to determine whether more stable, well-ordered Trimers would more efficiently elicit neutralizing antibodies. To begin, in vitro analysis of Trimers derived from the cysteine-stabilized SOSIP platform or the uncleaved, covalently linked NFL platform were evaluated. These native-like Trimers, derived from HIV subtypes A, B, and C, displayed a range of thermostabilities, and were “stress-tested” at varying temperatures as a prelude to in vivo immunogenicity. Analysis was performed both in the absence and in the presence of two different adjuvants. Since partial Trimer degradation was detected at 37°C before or after formulation with adjuvant, we sought to remedy such an undesirable outcome. Cross-linking (fixing) of the well-ordered Trimers with glutaraldehyde increased overall thermostability, maintenance of well-ordered Trimer integrity without or with adjuvant, and increased resistance to solid phase-associated Trimer unfolding. Immunization of unfixed and fixed well-ordered Trimers into animals revealed that the elicited tier 2 autologous neutralizing activity correlated with overall Trimer thermostability, or melting temperature (Tm). Glutaraldehyde fixation also led to higher tier 2 autologous neutralization titers. These results link retention of Trimer quaternary packing with elicitation of tier 2 autologous neutralizing activity, providing important insights for HIV-1 vaccine design.
Karin Stiasny - One of the best experts on this subject based on the ideXlab platform.
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Extensive flavivirus E Trimer breathing accompanies stem zippering of the post‐fusion hairpin
EMBO Reports, 2020Co-Authors: Iris Medits, Marie‐christine Vaney, Alexander Rouvinski, Martial Rey, Julia Chamot‐rooke, Felix Rey, Franz Heinz, Karin StiasnyAbstract:Flaviviruses enter cells by fusion with endosomal membranes through a rearrangement of the envelope protein E, a class II membrane fusion protein, into fusogenic Trimers. The rod-like E subunits bend into "hairpins" to bring the fusion loops next to the C-terminal transmembrane (TM) anchors, with the TM-proximal "stem" element zippering the E Trimer to force apposition of the membranes. The structure of the complete class II Trimeric hairpin is known for phleboviruses but not for flaviviruses, for which the stem is only partially resolved. Here, we performed comparative analyses of E-protein Trimers from the tick-borne encephalitis fla-vivirus with sequential stem truncations. Our thermostability and antibody-binding data suggest that the stem "zipper" ends at a characteristic flavivirus conserved sequence (CS) that cloaks the fusion loops, with the downstream segment not contributing to Trimer stability. We further identified a highly dynamic behavior of E Trimers C-terminally truncated upstream the CS, which, unlike fully stem-zippered Trimers, undergo rapid deuterium exchange at the Trimer interface. These results thus identify important "breathing" intermediates in the E-protein-driven membrane fusion process.
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Characterization of a membrane-associated Trimeric low-pH-induced form of the class II viral fusion protein E from tick-borne encephalitis virus and its crystallization
Journal of Virology, 2004Co-Authors: Karin Stiasny, Stéphane Bressanelli, Jean Lepault, Felix A. Rey, Franz X. HeinzAbstract:The interaction of a dimeric membrane anchor-free form of the envelope protein E (sE dimer) from tick-borne encephalitis virus with liposomes at acidic pH levels leads to its conversion into membrane-inserted sE Trimers. Electron microscopy shows that these Trimers have their long dimensions along the threefold molecular axis, which is oriented perpendicularly to the plane of the membrane, where the protein inserts via the internal fusion peptide. Liposomes containing sE at their surface display paracrystalline arrays of protein in a closely packing arrangement in which each Trimer is surrounded by six others, suggesting cooperativity in the insertion process. sE Trimers, solubilized with nonionic detergents, yielded three-dimensional crystals suitable for X-ray diffraction analysis.
