59 results on '"Boyington JC"'
Search Results
2. Transforming epitope-specific gp120 monomer-based probes into immunogens with N-linked glycan masking
- Author
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Chuang G, Boyington JC, Nabel GJ, Kwong PD, and Georgiev I
- Subjects
Immunologic diseases. Allergy ,RC581-607 - Published
- 2012
- Full Text
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3. Design of Nanoparticulate Group 2 Influenza Virus Hemagglutinin Stem Antigens That Activate Unmutated Ancestor B Cell Receptors of Broadly Neutralizing Antibody Lineages
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Subbarao, K, Corbett, KS, Moin, SM, Yassine, HM, Cagigi, A, Kanekiyo, M, Boyoglu-Barnum, S, Myers, SI, Tsybovsky, Y, Wheatley, AK, Schramm, CA, Gillespie, RA, Shi, W, Wang, L, Zhang, Y, Andrews, SF, Joyce, MG, Crank, MC, Douek, DC, McDermott, AB, Mascola, JR, Graham, BS, Boyington, JC, Subbarao, K, Corbett, KS, Moin, SM, Yassine, HM, Cagigi, A, Kanekiyo, M, Boyoglu-Barnum, S, Myers, SI, Tsybovsky, Y, Wheatley, AK, Schramm, CA, Gillespie, RA, Shi, W, Wang, L, Zhang, Y, Andrews, SF, Joyce, MG, Crank, MC, Douek, DC, McDermott, AB, Mascola, JR, Graham, BS, and Boyington, JC
- Abstract
Influenza vaccines targeting the highly conserved stem of the hemagglutinin (HA) surface glycoprotein have the potential to protect against pandemic and drifted seasonal influenza viruses not covered by current vaccines. While HA stem-based immunogens derived from group 1 influenza A viruses have been shown to induce intragroup heterosubtypic protection, HA stem-specific antibody lineages originating from group 2 may be more likely to possess broad cross-group reactivity. We report the structure-guided development of mammalian-cell-expressed candidate vaccine immunogens based on influenza A virus group 2 H3 and H7 HA stem trimers displayed on self-assembling ferritin nanoparticles using an iterative, multipronged approach involving helix stabilization, loop optimization, disulfide bond addition, and side-chain repacking. These immunogens were thermostable, formed uniform and symmetric nanoparticles, were recognized by cross-group-reactive broadly neutralizing antibodies (bNAbs) with nanomolar affinity, and elicited protective, homosubtypic antibodies in mice. Importantly, several immunogens were able to activate B cells expressing inferred unmutated common ancestor (UCA) versions of cross-group-reactive human bNAbs from two multidonor classes, suggesting they could initiate elicitation of these bNAbs in humans.IMPORTANCE Current influenza vaccines are primarily strain specific, requiring annual updates, and offer minimal protection against drifted seasonal or pandemic strains. The highly conserved stem region of hemagglutinin (HA) of group 2 influenza A virus subtypes is a promising target for vaccine elicitation of broad cross-group protection against divergent strains. We used structure-guided protein engineering employing multiple protein stabilization methods simultaneously to develop group 2 HA stem-based candidate influenza A virus immunogens displayed as trimers on self-assembling nanoparticles. Characterization of antigenicity, thermostability, and particle
- Published
- 2019
4. P13-01. Crystal structure and function of a monoclonal antibody against primate CD4 that blocks HIV/SIV infection
- Author
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Pegu, A, primary, Boyington, JC, additional, Kong, W, additional, Shi, W, additional, Kwong, PD, additional, and Nabel, GJ, additional
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- 2009
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5. Cleavage-intermediate Lassa virus trimer elicits neutralizing responses, identifies neutralizing nanobodies, and reveals an apex-situated site-of-vulnerability.
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Gorman J, Cheung CS, Duan Z, Ou L, Wang M, Chen X, Cheng C, Biju A, Sun Y, Wang P, Yang Y, Zhang B, Boyington JC, Bylund T, Charaf S, Chen SJ, Du H, Henry AR, Liu T, Sarfo EK, Schramm CA, Shen CH, Stephens T, Teng IT, Todd JP, Tsybovsky Y, Verardi R, Wang D, Wang S, Wang Z, Zheng CY, Zhou T, Douek DC, Mascola JR, Ho DD, Ho M, and Kwong PD
- Subjects
- Animals, Guinea Pigs, Lassa virus, Antibodies, Viral, Antibodies, Neutralizing, Single-Domain Antibodies, Lassa Fever
- Abstract
Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a pandemic biothreat. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into LASV neutralization, here we develop a prefusion-stabilized LASV glycoprotein trimer (GPC), pan it against phage libraries comprising single-domain antibodies (nanobodies) from shark and camel, and identify one, D5, which neutralizes LASV. Cryo-EM analyses reveal D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. The recognized site appears specific to GPC intermediates, with protomers lacking full cleavage between GP1 and GP2 subunits. Guinea pig immunizations with the prefusion-stabilized cleavage-intermediate LASV GPC, first as trimer and then as a nanoparticle, induce neutralizing responses, targeting multiple epitopes including that of D5; we identify a neutralizing antibody (GP23) from the immunized guinea pigs. Collectively, our findings define a prefusion-stabilized GPC trimer, reveal an apex-situated site-of-vulnerability, and demonstrate elicitation of LASV-neutralizing responses by a cleavage-intermediate LASV trimer., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)
- Published
- 2024
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6. Co-immunization with hemagglutinin stem immunogens elicits cross-group neutralizing antibodies and broad protection against influenza A viruses.
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Moin SM, Boyington JC, Boyoglu-Barnum S, Gillespie RA, Cerutti G, Cheung CS, Cagigi A, Gallagher JR, Brand J, Prabhakaran M, Tsybovsky Y, Stephens T, Fisher BE, Creanga A, Ataca S, Rawi R, Corbett KS, Crank MC, Karlsson Hedestam GB, Gorman J, McDermott AB, Harris AK, Zhou T, Kwong PD, Shapiro L, Mascola JR, Graham BS, and Kanekiyo M
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- Animals, Mice, Humans, Hemagglutinins, Broadly Neutralizing Antibodies, Hemagglutinin Glycoproteins, Influenza Virus, Antibodies, Viral, Ferrets, Antibodies, Neutralizing, Immunization, Influenza Vaccines, Influenza A Virus, H5N1 Subtype, Orthomyxoviridae Infections, Influenza A Virus, H7N9 Subtype, Influenza, Human
- Abstract
Current influenza vaccines predominantly induce immunity to the hypervariable hemagglutinin (HA) head, requiring frequent vaccine reformulation. Conversely, the immunosubdominant yet conserved HA stem harbors a supersite that is targeted by broadly neutralizing antibodies (bnAbs), representing a prime target for universal vaccines. Here, we showed that the co-immunization of two HA stem immunogens derived from group 1 and 2 influenza A viruses elicits cross-group protective immunity and neutralizing antibody responses in mice, ferrets, and nonhuman primates (NHPs). Immunized mice were protected from multiple group 1 and 2 viruses, and all animal models showed broad serum-neutralizing activity. A bnAb isolated from an immunized NHP broadly neutralized and protected against diverse viruses, including H5N1 and H7N9. Genetic and structural analyses revealed strong homology between macaque and human bnAbs, illustrating common biophysical constraints for acquiring cross-group specificity. Vaccine elicitation of stem-directed cross-group-protective immunity represents a step toward the development of broadly protective influenza vaccines., Competing Interests: Declaration of interests S.M.M., J.C.B., P.D.K., J.R.M., B.S.G., and M.K. are named inventors of US patents 9,441,019, 10,137,190, 10,363,301, and 11,338,033 on influenza HA nanoparticle vaccines and stabilized HA stem trimers and of several pending applications on related technologies filed by the US Department of Health and Human Services (NIH)., (Published by Elsevier Inc.)
- Published
- 2022
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7. A single residue in influenza virus H2 hemagglutinin enhances the breadth of the B cell response elicited by H2 vaccination.
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Andrews SF, Raab JE, Gorman J, Gillespie RA, Cheung CSF, Rawi R, Cominsky LY, Boyington JC, Creanga A, Shen CH, Harris DR, Olia AS, Nazzari AF, Zhou T, Houser KV, Chen GL, Mascola JR, Graham BS, Kanekiyo M, Ledgerwood JE, Kwong PD, and McDermott AB
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- Antibodies, Neutralizing, Antibodies, Viral, Child, Clinical Trials, Phase I as Topic, Epitopes, Hemagglutinin Glycoproteins, Influenza Virus, Hemagglutinins, Humans, Vaccination, Influenza A Virus, H5N1 Subtype, Influenza Vaccines, Influenza, Human
- Abstract
Conserved epitopes on the influenza hemagglutinin (HA) stem are an attractive target for universal vaccine strategies as they elicit broadly neutralizing antibodies. Such antibody responses to stem-specific epitopes have been extensively characterized for HA subtypes H1 and H5 in humans. H2N2 influenza virus circulated 50 years ago and represents a pandemic threat due to the lack of widespread immunity, but, unlike H1 and H5, the H2 HA stem contains Phe45
HA2 predicted to sterically clash with HA stem-binding antibodies characterized to date. To understand the effect of Phe45HA2 , we compared the HA stem-specific B cell response in post hoc analyses of two phase 1 clinical trials, one testing vaccination with an H2 ferritin nanoparticle immunogen ( NCT03186781 ) and one with an inactivated H5N1 vaccine ( NCT01086657 ). In H2-naive individuals, the magnitude of the B cell response was equivalent, but H2-elicited HA stem-binding B cells displayed greater cross-reactivity than those elicited by H5. However, in individuals with childhood H2 exposure, H5-elicited HA stem-binding B cells also displayed high cross-reactivity, suggesting recall of memory B cells formed 50 years ago. Overall, we propose that a one-residue difference on an HA immunogen can alter establishment and expansion of broadly neutralizing memory B cells. These data have implications for stem-based universal influenza vaccination strategies., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)- Published
- 2022
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8. Fusion peptide priming reduces immune responses to HIV-1 envelope trimer base.
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Corrigan AR, Duan H, Cheng C, Gonelli CA, Ou L, Xu K, DeMouth ME, Geng H, Narpala S, O'Connell S, Zhang B, Zhou T, Basappa M, Boyington JC, Chen SJ, O'Dell S, Pegu A, Stephens T, Tsybovsky Y, van Schooten J, Todd JP, Wang S, Doria-Rose NA, Foulds KE, Koup RA, McDermott AB, van Gils MJ, Kwong PD, and Mascola JR
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- Animals, Antibodies, Monoclonal immunology, Antibodies, Neutralizing immunology, Female, Humans, Immunization, Immunoglobulin Fab Fragments immunology, Macaca mulatta, Male, Models, Biological, Antibody Formation immunology, HIV-1 immunology, Peptides immunology, Protein Multimerization, Recombinant Fusion Proteins metabolism, env Gene Products, Human Immunodeficiency Virus metabolism
- Abstract
Soluble "SOSIP"-stabilized envelope (Env) trimers are promising HIV-vaccine immunogens. However, they induce high-titer responses against the glycan-free trimer base, which is occluded on native virions. To delineate the effect on base responses of priming with immunogens targeting the fusion peptide (FP) site of vulnerability, here, we quantify the prevalence of trimer-base antibody responses in 49 non-human primates immunized with various SOSIP-stabilized Env trimers and FP-carrier conjugates. Trimer-base responses account for ∼90% of the overall trimer response in animals immunized with trimer only, ∼70% in animals immunized with a cocktail of SOSIP trimer and FP conjugate, and ∼30% in animals primed with FP conjugates before trimer immunization. Notably, neutralization breadth in FP-conjugate-primed animals correlates inversely with trimer-base responses. Our data provide methods to quantify the prevalence of trimer-base responses and reveal that FP-conjugate priming, either alone or as part of a cocktail, can reduce the trimer-base response and improve the neutralization outcome., Competing Interests: Declaration of interests P.D.K., J.R.M., L.O., Y.T., K.X., and B.Z. are inventors on U.S. Patent Application 62/735,188 filed March 26, 2020, entitled “HIV-1 ENV fusion peptide immunogens and their use.” The other authors declare no competing interests., (Published by Elsevier Inc.)
- Published
- 2021
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9. Cryo-EM Structures of SARS-CoV-2 Spike without and with ACE2 Reveal a pH-Dependent Switch to Mediate Endosomal Positioning of Receptor-Binding Domains.
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Zhou T, Tsybovsky Y, Gorman J, Rapp M, Cerutti G, Chuang GY, Katsamba PS, Sampson JM, Schön A, Bimela J, Boyington JC, Nazzari A, Olia AS, Shi W, Sastry M, Stephens T, Stuckey J, Teng IT, Wang P, Wang S, Zhang B, Friesner RA, Ho DD, Mascola JR, Shapiro L, and Kwong PD
- Subjects
- Amino Acid Sequence genetics, Angiotensin-Converting Enzyme 2 ultrastructure, Antibodies, Neutralizing genetics, Antibodies, Neutralizing immunology, Binding Sites, COVID-19 pathology, COVID-19 virology, Cryoelectron Microscopy, Endosomes ultrastructure, Humans, Hydrogen-Ion Concentration, Protein Binding, Protein Domains, Receptors, Virus genetics, Receptors, Virus ultrastructure, SARS-CoV-2 genetics, SARS-CoV-2 ultrastructure, Spike Glycoprotein, Coronavirus genetics, Angiotensin-Converting Enzyme 2 genetics, COVID-19 genetics, Pandemics, Spike Glycoprotein, Coronavirus ultrastructure
- Abstract
The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the human ACE2 receptor and to facilitate virus entry, which can occur through low-pH-endosomal pathways. To understand how ACE2 binding and low pH affect spike conformation, we determined cryo-electron microscopy structures-at serological and endosomal pH-delineating spike recognition of up to three ACE2 molecules. RBDs freely adopted "up" conformations required for ACE2 interaction, primarily through RBD movement combined with smaller alterations in neighboring domains. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a solitary all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning through coordinated movements of the entire trimer apex. These structures provide a foundation for understanding prefusion-spike mechanics governing endosomal entry; we suggest that the low pH all-down conformation potentially facilitates immune evasion from RBD-up binding antibody., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)
- Published
- 2020
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10. Cryo-EM Structures Delineate a pH-Dependent Switch that Mediates Endosomal Positioning of SARS-CoV-2 Spike Receptor-Binding Domains.
- Author
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Zhou T, Tsybovsky Y, Olia AS, Gorman J, Rapp MA, Cerutti G, Chuang GY, Katsamba PS, Nazzari A, Sampson JM, Schon A, Wang PD, Bimela J, Shi W, Teng IT, Zhang B, Boyington JC, Sastry M, Stephens T, Stuckey J, Wang S, Friesner RA, Ho DD, Mascola JR, Shapiro L, and Kwong PD
- Abstract
The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the ACE2 receptor and to facilitate virus entry. Antibodies can engage RBD but some, such as CR3022, fail to inhibit entry despite nanomolar spike affinity. Here we show the SARS-CoV-2 spike to have low unfolding enthalpy at serological pH and up to 10-times more unfolding enthalpy at endosomal pH, where we observe significantly reduced CR3022 affinity. Cryo-EM structures -at serological and endosomal pH- delineated spike recognition of up to three ACE2 molecules, revealing RBD to freely adopt the 'up' conformation. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a locked all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning and spike shedding of antibodies like CR3022. An endosomal mechanism involving spike-conformational change can thus facilitate immune evasion from RBD-'up'-recognizing antibody.
- Published
- 2020
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11. Development of a 3Mut-Apex-Stabilized Envelope Trimer That Expands HIV-1 Neutralization Breadth When Used To Boost Fusion Peptide-Directed Vaccine-Elicited Responses.
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Chuang GY, Lai YT, Boyington JC, Cheng C, Geng H, Narpala S, Rawi R, Schmidt SD, Tsybovsky Y, Verardi R, Xu K, Yang Y, Zhang B, Chambers M, Changela A, Corrigan AR, Kong R, Olia AS, Ou L, Sarfo EK, Wang S, Wu W, Doria-Rose NA, McDermott AB, Mascola JR, and Kwong PD
- Subjects
- Animals, Antibodies, Neutralizing immunology, Female, Guinea Pigs, HEK293 Cells, HIV Antibodies immunology, HIV Seropositivity, HIV-1 immunology, Humans, Immunization, Secondary, Peptides, Vaccines, Subunit, AIDS Vaccines immunology, env Gene Products, Human Immunodeficiency Virus genetics, env Gene Products, Human Immunodeficiency Virus immunology
- Abstract
HIV-1 envelope (Env) trimers, stabilized in a prefusion-closed conformation, can elicit humoral responses capable of neutralizing HIV-1 strains closely matched in sequence to the immunizing strain. One strategy to increase elicited neutralization breadth involves vaccine priming of immune responses against a target site of vulnerability, followed by vaccine boosting of these responses with prefusion-closed Env trimers. This strategy has succeeded at the fusion peptide (FP) site of vulnerability in eliciting cross-clade neutralizing responses in standard vaccine-test animals. However, the breadth and potency of the elicited responses have been less than optimal. Here, we identify three mutations (3mut), Met302, Leu320, and Pro329, that stabilize the apex of the Env trimer in a prefusion-closed conformation and show antigenically, structurally, and immunogenically that combining 3mut with other approaches (e.g., repair and stabilize and glycine-helix breaking) yields well-behaved clade C-Env trimers capable of boosting the breadth of FP-directed responses. Crystal structures of these trimers confirmed prefusion-closed apexes stabilized by hydrophobic patches contributed by Met302 and Leu320, with Pro329 assuming canonically restricted dihedral angles. We substituted the N-terminal eight residues of FP (FP8, residues 512 to 519) of these trimers with the second most prevalent FP8 sequence (FP8v2, AVGLGAVF) and observed a 3mut-stabilized consensus clade C-Env trimer with FP8v2 to boost the breadth elicited in guinea pigs of FP-directed responses induced by immunogens containing the most prevalent FP8 sequence (FP8v1, AVGIGAVF). Overall, 3mut can stabilize the Env trimer apex, and the resultant apex-stabilized Env trimers can be used to expand the neutralization breadth elicited against the FP site of vulnerability. IMPORTANCE A major hurdle to the development of an effective HIV-1 vaccine is the elicitation of serum responses capable of neutralizing circulating strains of HIV, which are extraordinarily diverse in sequence and often highly neutralization resistant. Recently, we showed how sera with 20 to 30% neutralization breadth could, nevertheless, be elicited in standard vaccine test animals by priming with the most prevalent N-terminal 8 residues of the HIV-1 fusion peptide (FP8), followed by boosting with a stabilized BG505-envelope (Env) trimer. Here, we show that subsequent boosting with a 3mut-apex-stabilized consensus C-Env trimer, modified to have the second most prevalent FP8 sequence, elicits higher neutralization breadth than that induced by continued boosting with the stabilized BG505-Env trimer. With increased neutralizing breadth elicited by boosting with a heterologous trimer containing the second most prevalent FP8 sequence, the fusion peptide-directed immune-focusing approach moves a step closer toward realizing an effective HIV-1 vaccine regimen., (Copyright © 2020 American Society for Microbiology.)
- Published
- 2020
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12. Structure of Super-Potent Antibody CAP256-VRC26.25 in Complex with HIV-1 Envelope Reveals a Combined Mode of Trimer-Apex Recognition.
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Gorman J, Chuang GY, Lai YT, Shen CH, Boyington JC, Druz A, Geng H, Louder MK, McKee K, Rawi R, Verardi R, Yang Y, Zhang B, Doria-Rose NA, Lin B, Moore PL, Morris L, Shapiro L, Mascola JR, and Kwong PD
- Subjects
- Amino Acid Sequence, Antibodies, Neutralizing immunology, Cell Line, Cryoelectron Microscopy methods, HIV Antibodies metabolism, HIV Envelope Protein gp120 immunology, HIV Infections immunology, HIV-1 immunology, Humans, Immunoglobulin Fab Fragments metabolism, Protein Binding, Protein Multimerization, HIV Antibodies immunology, HIV Antibodies ultrastructure, env Gene Products, Human Immunodeficiency Virus immunology
- Abstract
Antibodies targeting the V1V2 apex of the HIV-1 envelope (Env) trimer comprise one of the most commonly elicited categories of broadly neutralizing antibodies. Structures of these antibodies indicate diverse modes of Env recognition typified by antibodies of the PG9 class and the PGT145 class. The mode of recognition, however, has been unclear for the most potent of the V1V2 apex-targeting antibodies, CAP256-VRC26.25 (named for donor-lineage.clone and referred to hereafter as VRC26.25). Here, we determine the cryoelectron microscopy structure at 3.7 Å resolution of the antigen-binding fragment of VRC26.25 in complex with the Env trimer thought to have initiated the lineage. The 36-residue protruding loop of VRC26.25 displays recognition incorporating both strand-C interactions similar to the PG9 class and V1V2 apex insertion similar to the PGT145 class. Structural elements of separate antibody classes can thus intermingle to form a "combined" class, which in this case yields an antibody of extraordinary potency., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)
- Published
- 2020
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13. Glycan repositioning of influenza hemagglutinin stem facilitates the elicitation of protective cross-group antibody responses.
- Author
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Boyoglu-Barnum S, Hutchinson GB, Boyington JC, Moin SM, Gillespie RA, Tsybovsky Y, Stephens T, Vaile JR, Lederhofer J, Corbett KS, Fisher BE, Yassine HM, Andrews SF, Crank MC, McDermott AB, Mascola JR, Graham BS, and Kanekiyo M
- Subjects
- Animals, Antibodies, Viral immunology, Antibody Specificity, Asparagine chemistry, Asparagine metabolism, Broadly Neutralizing Antibodies immunology, Cross Reactions, Epitopes immunology, Female, Glycosylation, Hemagglutinin Glycoproteins, Influenza Virus metabolism, Immunoglobulins immunology, Influenza A Virus, H7N9 Subtype pathogenicity, Mice, Inbred BALB C, Orthomyxoviridae Infections immunology, Orthomyxoviridae Infections prevention & control, Hemagglutinin Glycoproteins, Influenza Virus chemistry, Hemagglutinin Glycoproteins, Influenza Virus immunology, Influenza Vaccines immunology, Nanoparticles chemistry, Polysaccharides chemistry
- Abstract
The conserved hemagglutinin (HA) stem has been a focus of universal influenza vaccine efforts. Influenza A group 1 HA stem-nanoparticles have been demonstrated to confer heterosubtypic protection in animals; however, the protection does not extend to group 2 viruses, due in part to differences in glycosylation between group 1 and 2 stems. Here, we show that introducing the group 2 glycan at Asn38
HA1 to a group 1 stem-nanoparticle (gN38 variant) based on A/New Caledonia/20/99 (H1N1) broadens antibody responses to cross-react with group 2 HAs. Immunoglobulins elicited by the gN38 variant provide complete protection against group 2 H7N9 virus infection, while the variant loses protection against a group 1 H5N1 virus. The N38HA1 glycan thus is pivotal in directing antibody responses by controlling access to group-determining stem epitopes. Precise targeting of stem-directed antibody responses to the site of vulnerability by glycan repositioning may be a step towards achieving cross-group influenza protection.- Published
- 2020
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14. Activation Dynamics and Immunoglobulin Evolution of Pre-existing and Newly Generated Human Memory B cell Responses to Influenza Hemagglutinin.
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Andrews SF, Chambers MJ, Schramm CA, Plyler J, Raab JE, Kanekiyo M, Gillespie RA, Ransier A, Darko S, Hu J, Chen X, Yassine HM, Boyington JC, Crank MC, Chen GL, Coates E, Mascola JR, Douek DC, Graham BS, Ledgerwood JE, and McDermott AB
- Subjects
- Adult, Antibodies, Viral metabolism, Antibody Formation, Cells, Cultured, Epitopes immunology, Female, Hemagglutinin Glycoproteins, Influenza Virus immunology, Humans, Immunologic Memory, Lymphocyte Activation, Male, Middle Aged, Phenotype, Receptors, Antigen, B-Cell genetics, Single-Cell Analysis, Vaccination, Young Adult, B-Lymphocyte Subsets immunology, B-Lymphocytes immunology, Influenza A Virus, H7N9 Subtype physiology, Influenza Vaccines immunology, Influenza, Human immunology
- Abstract
Vaccine-induced memory B cell responses to evolving viruses like influenza A involve activation of pre-existing immunity and generation of new responses. To define the contribution of these two types of responses, we analyzed the response to H7N9 vaccination in H7N9-naive adults. We performed comprehensive comparisons at the single-cell level of the kinetics, Ig repertoire, and activation phenotype of established pre-existing memory B cells recognizing conserved epitopes and the newly generated memory B cells directed toward H7 strain-specific epitopes. The recall response to conserved epitopes on H7 HA involved a transient expansion of memory B cells with little observed adaptation. However, the B cell response to newly encountered epitopes was phenotypically distinct and generated a sustained memory population that evolved and affinity matured months after vaccination. These findings establish clear differences between newly generated and pre-existing memory B cells, highlighting the challenges in achieving long-lasting, broad protection against an ever-evolving virus., (Published by Elsevier Inc.)
- Published
- 2019
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15. Design of Nanoparticulate Group 2 Influenza Virus Hemagglutinin Stem Antigens That Activate Unmutated Ancestor B Cell Receptors of Broadly Neutralizing Antibody Lineages.
- Author
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Corbett KS, Moin SM, Yassine HM, Cagigi A, Kanekiyo M, Boyoglu-Barnum S, Myers SI, Tsybovsky Y, Wheatley AK, Schramm CA, Gillespie RA, Shi W, Wang L, Zhang Y, Andrews SF, Joyce MG, Crank MC, Douek DC, McDermott AB, Mascola JR, Graham BS, and Boyington JC
- Subjects
- Animals, Antigens, Viral genetics, Cross Reactions, Drug Carriers metabolism, Ferritins metabolism, Hemagglutinin Glycoproteins, Influenza Virus genetics, Immunity, Heterologous, Influenza Vaccines genetics, Influenza Vaccines isolation & purification, Mice, Protein Multimerization, Vaccines, Virus-Like Particle genetics, Vaccines, Virus-Like Particle isolation & purification, Antibodies, Neutralizing blood, Antibodies, Viral blood, Antigens, Viral immunology, B-Lymphocytes immunology, Hemagglutinin Glycoproteins, Influenza Virus immunology, Influenza Vaccines immunology, Vaccines, Virus-Like Particle immunology
- Abstract
Influenza vaccines targeting the highly conserved stem of the hemagglutinin (HA) surface glycoprotein have the potential to protect against pandemic and drifted seasonal influenza viruses not covered by current vaccines. While HA stem-based immunogens derived from group 1 influenza A viruses have been shown to induce intragroup heterosubtypic protection, HA stem-specific antibody lineages originating from group 2 may be more likely to possess broad cross-group reactivity. We report the structure-guided development of mammalian-cell-expressed candidate vaccine immunogens based on influenza A virus group 2 H3 and H7 HA stem trimers displayed on self-assembling ferritin nanoparticles using an iterative, multipronged approach involving helix stabilization, loop optimization, disulfide bond addition, and side-chain repacking. These immunogens were thermostable, formed uniform and symmetric nanoparticles, were recognized by cross-group-reactive broadly neutralizing antibodies (bNAbs) with nanomolar affinity, and elicited protective, homosubtypic antibodies in mice. Importantly, several immunogens were able to activate B cells expressing inferred unmutated common ancestor (UCA) versions of cross-group-reactive human bNAbs from two multidonor classes, suggesting they could initiate elicitation of these bNAbs in humans. IMPORTANCE Current influenza vaccines are primarily strain specific, requiring annual updates, and offer minimal protection against drifted seasonal or pandemic strains. The highly conserved stem region of hemagglutinin (HA) of group 2 influenza A virus subtypes is a promising target for vaccine elicitation of broad cross-group protection against divergent strains. We used structure-guided protein engineering employing multiple protein stabilization methods simultaneously to develop group 2 HA stem-based candidate influenza A virus immunogens displayed as trimers on self-assembling nanoparticles. Characterization of antigenicity, thermostability, and particle formation confirmed structural integrity. Group 2 HA stem antigen designs were identified that, when displayed on ferritin nanoparticles, activated B cells expressing inferred unmutated common ancestor (UCA) versions of human antibody lineages associated with cross-group-reactive, broadly neutralizing antibodies (bNAbs). Immunization of mice led to protection against a lethal homosubtypic influenza virus challenge. These candidate vaccines are now being manufactured for clinical evaluation.
- Published
- 2019
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16. Glycan Masking Focuses Immune Responses to the HIV-1 CD4-Binding Site and Enhances Elicitation of VRC01-Class Precursor Antibodies.
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Duan H, Chen X, Boyington JC, Cheng C, Zhang Y, Jafari AJ, Stephens T, Tsybovsky Y, Kalyuzhniy O, Zhao P, Menis S, Nason MC, Normandin E, Mukhamedova M, DeKosky BJ, Wells L, Schief WR, Tian M, Alt FW, Kwong PD, and Mascola JR
- Subjects
- AIDS Vaccines immunology, Animals, Antibodies, Monoclonal immunology, Broadly Neutralizing Antibodies, Cell Line, Female, Gene Knock-In Techniques, HIV Envelope Protein gp120 immunology, HIV Infections immunology, HIV Infections prevention & control, Humans, Immunoglobulin Heavy Chains immunology, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Polysaccharides chemistry, Antibodies, Neutralizing immunology, Binding Sites, Antibody immunology, CD4 Antigens immunology, HIV Antibodies immunology, HIV-1 immunology
- Abstract
An important class of HIV-1 broadly neutralizing antibodies, termed the VRC01 class, targets the conserved CD4-binding site (CD4bs) of the envelope glycoprotein (Env). An engineered Env outer domain (OD) eOD-GT8 60-mer nanoparticle has been developed as a priming immunogen for eliciting VRC01-class precursors and is planned for clinical trials. However, a substantial portion of eOD-GT8-elicited antibodies target non-CD4bs epitopes, potentially limiting its efficacy. We introduced N-linked glycans into non-CD4bs surfaces of eOD-GT8 to mask irrelevant epitopes and evaluated these mutants in a mouse model that expressed diverse immunoglobulin heavy chains containing human IGHV1-2
∗ 02, the germline VRC01 VH segment. Compared to the parental eOD-GT8, a mutant with five added glycans stimulated significantly higher proportions of CD4bs-specific serum responses and CD4bs-specific immunoglobulin G+ B cells including VRC01-class precursors. These results demonstrate that glycan masking can limit elicitation of off-target antibodies and focus immune responses to the CD4bs, a major target of HIV-1 vaccine design., (Published by Elsevier Inc.)- Published
- 2018
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17. Use of Hemagglutinin Stem Probes Demonstrate Prevalence of Broadly Reactive Group 1 Influenza Antibodies in Human Sera.
- Author
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Yassine HM, McTamney PM, Boyington JC, Ruckwardt TJ, Crank MC, Smatti MK, Ledgerwood JE, and Graham BS
- Subjects
- Adult, Age Factors, Cross Reactions, Cross-Sectional Studies, Female, Healthy Volunteers, Humans, Male, Middle Aged, Seroepidemiologic Studies, Young Adult, Antibodies, Neutralizing blood, Antibodies, Viral blood, Hemagglutinin Glycoproteins, Influenza Virus immunology, Influenza, Human immunology
- Abstract
A better understanding of the seroprevalence and specificity of influenza HA stem-directed broadly neutralizing antibodies (bNAbs) in the human population could significantly inform influenza vaccine design efforts. Here, we utilized probes comprising headless, HA stabilized stem (SS) to determine the prevalence, binding and neutralization breadth of antibodies directed to HA stem-epitope in a cross-sectional analysis of the general population. Five group-1 HA SS probes, representing five subtypes, were chosen for this analyses. Eighty-four percent of samples analyzed had specific reactivity to at least one probe, with approximately 60% of the samples reactive to H1 probes, and up to 45% reactive to each of the non-circulating subtypes. Thirty percent of analyzed sera had cross-reactivity to at least four of five probes and this reactivity could be blocked by competing with F10 bNAb. Binding cross-reactivity in sera samples significantly correlated with frequency of H1
+ H5+ cross-reactive B cells. Interestingly, only 33% of the cross-reactive sera neutralized both H1N1 and H5N1 pseudoviruses. Cross-reactive and neutralizing antibodies were more prevalent in individuals >50 years of age. Our data demonstrate the need to use multiple HA-stem probes to assess for broadly reactive antibodies. Further, a universal vaccine could be designed to boost pre-existing B-cells expressing stem-directed bNAbs.- Published
- 2018
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18. Preferential induction of cross-group influenza A hemagglutinin stem-specific memory B cells after H7N9 immunization in humans.
- Author
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Andrews SF, Joyce MG, Chambers MJ, Gillespie RA, Kanekiyo M, Leung K, Yang ES, Tsybovsky Y, Wheatley AK, Crank MC, Boyington JC, Prabhakaran MS, Narpala SR, Chen X, Bailer RT, Chen G, Coates E, Kwong PD, Koup RA, Mascola JR, Graham BS, Ledgerwood JE, and McDermott AB
- Abstract
Antigenic drift and shift of influenza strains underscore the need for broadly protective influenza vaccines. One strategy is to design immunogens that elicit B cell responses against conserved epitopes on the hemagglutinin (HA) stem. To better understand the elicitation of HA stem-targeted B cells to group 1 and group 2 influenza subtypes, we compared the memory B cell response to group 2 H7N9 and group 1 H5N1 vaccines in humans. Upon H7N9 vaccination, almost half of the HA stem-specific response recognized the group 1 and group 2 subtypes, whereas the response to H5N1 was largely group 1-specific. Immunoglobulin repertoire analysis of HA-specific B cells indicated that the H7N9 and H5N1 vaccines induced genetically similar cross-group HA stem-binding B cells, albeit at a much higher frequency upon H7N9 vaccination. These data suggest that a group 2-based stem immunogen could prove more effective than a group 1 immunogen at eliciting broad cross-group protection in humans., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)
- Published
- 2017
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19. Protection of calves by a prefusion-stabilized bovine RSV F vaccine.
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Zhang B, Chen L, Silacci C, Thom M, Boyington JC, Druz A, Joyce MG, Guzman E, Kong WP, Lai YT, Stewart-Jones GBE, Tsybovsky Y, Yang Y, Zhou T, Baxa U, Mascola JR, Corti D, Lanzavecchia A, Taylor G, and Kwong PD
- Abstract
Bovine respiratory syncytial virus, a major cause of respiratory disease in calves, is closely related to human RSV, a leading cause of respiratory disease in infants. Recently, promising human RSV-vaccine candidates have been engineered that stabilize the metastable fusion (F) glycoprotein in its prefusion state; however, the absence of a relevant animal model for human RSV has complicated assessment of these vaccine candidates. Here, we use a combination of structure-based design, antigenic characterization, and X-ray crystallography to translate human RSV F stabilization into the bovine context. A "DS2" version of bovine respiratory syncytial virus F with subunits covalently fused, fusion peptide removed, and pre-fusion conformation stabilized by cavity-filling mutations and intra- and inter-protomer disulfides was recognized by pre-fusion-specific antibodies, AM14, D25, and MPE8, and elicited bovine respiratory syncytial virus-neutralizing titers in calves >100-fold higher than those elicited by post-fusion F. When challenged with a heterologous bovine respiratory syncytial virus, virus was not detected in nasal secretions nor in respiratory tract samples of DS2-immunized calves; by contrast bovine respiratory syncytial virus was detected in all post-fusion- and placebo-immunized calves. Our results demonstrate proof-of-concept that DS2-stabilized RSV F immunogens can induce highly protective immunity from RSV in a native host with implications for the efficacy of prefusion-stabilized F vaccines in humans and for the prevention of bovine respiratory syncytial virus in calves., Competing Interests: Competing Interests The authors declare that an intellectual property application has been filed.
- Published
- 2017
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20. Vaccine-Induced Antibodies that Neutralize Group 1 and Group 2 Influenza A Viruses.
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Joyce MG, Wheatley AK, Thomas PV, Chuang GY, Soto C, Bailer RT, Druz A, Georgiev IS, Gillespie RA, Kanekiyo M, Kong WP, Leung K, Narpala SN, Prabhakaran MS, Yang ES, Zhang B, Zhang Y, Asokan M, Boyington JC, Bylund T, Darko S, Lees CR, Ransier A, Shen CH, Wang L, Whittle JR, Wu X, Yassine HM, Santos C, Matsuoka Y, Tsybovsky Y, Baxa U, Mullikin JC, Subbarao K, Douek DC, Graham BS, Koup RA, Ledgerwood JE, Roederer M, Shapiro L, Kwong PD, Mascola JR, and McDermott AB
- Subjects
- Adult, Amino Acid Sequence, Antibodies, Neutralizing chemistry, Antibodies, Neutralizing genetics, Antibodies, Viral chemistry, Antibodies, Viral genetics, B-Lymphocytes immunology, Epitopes, B-Lymphocyte, Female, Gene Rearrangement, B-Lymphocyte, Heavy Chain, Humans, Immunologic Memory, Influenza A Virus, H5N1 Subtype immunology, Male, Middle Aged, Models, Molecular, Protein Structure, Tertiary, Structure-Activity Relationship, Young Adult, Antibodies, Neutralizing immunology, Antibodies, Viral immunology, Influenza A virus immunology, Influenza Vaccines immunology
- Abstract
Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we sorted hemagglutinin cross-reactive memory B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and group 2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and-in half the subjects-multidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A immunization strategies., (Published by Elsevier Inc.)
- Published
- 2016
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21. Structure-Based Design of Head-Only Fusion Glycoprotein Immunogens for Respiratory Syncytial Virus.
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Boyington JC, Joyce MG, Sastry M, Stewart-Jones GB, Chen M, Kong WP, Ngwuta JO, Thomas PV, Tsybovsky Y, Yang Y, Zhang B, Chen L, Druz A, Georgiev IS, Ko K, Zhou T, Mascola JR, Graham BS, and Kwong PD
- Subjects
- Animals, Antibodies, Viral immunology, Female, HEK293 Cells, Humans, Immunogenicity, Vaccine, Male, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Protein Conformation, Respiratory Syncytial Viruses genetics, Viral Fusion Proteins chemistry, Viral Fusion Proteins genetics, Viral Vaccines genetics, Respiratory Syncytial Viruses immunology, Viral Fusion Proteins immunology, Viral Vaccines immunology
- Abstract
Respiratory syncytial virus (RSV) is a significant cause of severe respiratory illness worldwide, particularly in infants, young children, and the elderly. Although no licensed vaccine is currently available, an engineered version of the metastable RSV fusion (F) surface glycoprotein-stabilized in the pre-fusion (pre-F) conformation by "DS-Cav1" mutations-elicits high titer RSV-neutralizing responses. Moreover, pre-F-specific antibodies, often against the neutralization-sensitive antigenic site Ø in the membrane-distal head region of trimeric F glycoprotein, comprise a substantial portion of the human response to natural RSV infection. To focus the vaccine-elicited response to antigenic site Ø, we designed a series of RSV F immunogens that comprised the membrane-distal head of the F glycoprotein in its pre-F conformation. These "head-only" immunogens formed monomers, dimers, and trimers. Antigenic analysis revealed that a majority of the 70 engineered head-only immunogens displayed reactivity to site Ø-targeting antibodies, which was similar to that of the parent RSV F DS-Cav1 trimers, often with increased thermostability. We evaluated four of these head-only immunogens in detail, probing their recognition by antibodies, their physical stability, structure, and immunogenicity. When tested in naïve mice, a head-only trimer, half the size of the parent RSV F trimer, induced RSV titers, which were statistically comparable to those induced by DS-Cav1. When used to boost DS-Cav1-primed mice, two head-only RSV F immunogens, a dimer and a trimer, boosted RSV-neutralizing titers to levels that were comparable to those boosted by DS-Cav1, although with higher site Ø-directed responses. Our results provide proof-of-concept for the ability of the smaller head-only RSV F immunogens to focus the vaccine-elicited response to antigenic site Ø. Decent primary immunogenicity, enhanced physical stability, potential ease of manufacture, and potent immunogenicity upon boosting suggest these head-only RSV F immunogens, engineered to retain the pre-fusion conformation, may have advantages as candidate RSV vaccines.
- Published
- 2016
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22. Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection.
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Yassine HM, Boyington JC, McTamney PM, Wei CJ, Kanekiyo M, Kong WP, Gallagher JR, Wang L, Zhang Y, Joyce MG, Lingwood D, Moin SM, Andersen H, Okuno Y, Rao SS, Harris AK, Kwong PD, Mascola JR, Nabel GJ, and Graham BS
- Subjects
- Animals, Antibodies, Viral blood, Female, Ferrets, Hemagglutinin Glycoproteins, Influenza Virus chemistry, Mice, Mice, Inbred BALB C, Nanoparticles, Vaccination, Hemagglutinin Glycoproteins, Influenza Virus immunology, Influenza A Virus, H5N1 Subtype immunology, Influenza Vaccines immunology
- Abstract
The antibody response to influenza is primarily focused on the head region of the hemagglutinin (HA) glycoprotein, which in turn undergoes antigenic drift, thus necessitating annual updates of influenza vaccines. In contrast, the immunogenically subdominant stem region of HA is highly conserved and recognized by antibodies capable of binding multiple HA subtypes. Here we report the structure-based development of an H1 HA stem-only immunogen that confers heterosubtypic protection in mice and ferrets. Six iterative cycles of structure-based design (Gen1-Gen6) yielded successive H1 HA stabilized-stem (HA-SS) immunogens that lack the immunodominant head domain. Antigenic characterization, determination of two HA-SS crystal structures in complex with stem-specific monoclonal antibodies and cryo-electron microscopy analysis of HA-SS on ferritin nanoparticles (H1-SS-np) confirmed the preservation of key structural elements. Vaccination of mice and ferrets with H1-SS-np elicited broadly cross-reactive antibodies that completely protected mice and partially protected ferrets against lethal heterosubtypic H5N1 influenza virus challenge despite the absence of detectable H5N1 neutralizing activity in vitro. Passive transfer of immunoglobulin from H1-SS-np-immunized mice to naive mice conferred protection against H5N1 challenge, indicating that vaccine-elicited HA stem-specific antibodies can protect against diverse group 1 influenza strains.
- Published
- 2015
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23. Enhanced potency of a broadly neutralizing HIV-1 antibody in vitro improves protection against lentiviral infection in vivo.
- Author
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Rudicell RS, Kwon YD, Ko SY, Pegu A, Louder MK, Georgiev IS, Wu X, Zhu J, Boyington JC, Chen X, Shi W, Yang ZY, Doria-Rose NA, McKee K, O'Dell S, Schmidt SD, Chuang GY, Druz A, Soto C, Yang Y, Zhang B, Zhou T, Todd JP, Lloyd KE, Eudailey J, Roberts KE, Donald BR, Bailer RT, Ledgerwood J, Mullikin JC, Shapiro L, Koup RA, Graham BS, Nason MC, Connors M, Haynes BF, Rao SS, Roederer M, Kwong PD, Mascola JR, and Nabel GJ
- Subjects
- Animals, Antibodies, Neutralizing administration & dosage, Antibodies, Neutralizing genetics, HIV Antibodies administration & dosage, HIV Antibodies genetics, HIV-1 genetics, Macaca mulatta, Male, Molecular Sequence Data, Sequence Analysis, DNA, Antibodies, Neutralizing immunology, HIV Antibodies immunology, HIV-1 immunology, Immunization, Passive methods, Simian Acquired Immunodeficiency Syndrome prevention & control
- Abstract
Unlabelled: Over the past 5 years, a new generation of highly potent and broadly neutralizing HIV-1 antibodies has been identified. These antibodies can protect against lentiviral infection in nonhuman primates (NHPs), suggesting that passive antibody transfer would prevent HIV-1 transmission in humans. To increase the protective efficacy of such monoclonal antibodies, we employed next-generation sequencing, computational bioinformatics, and structure-guided design to enhance the neutralization potency and breadth of VRC01, an antibody that targets the CD4 binding site of the HIV-1 envelope. One variant, VRC07-523, was 5- to 8-fold more potent than VRC01, neutralized 96% of viruses tested, and displayed minimal autoreactivity. To compare its protective efficacy to that of VRC01 in vivo, we performed a series of simian-human immunodeficiency virus (SHIV) challenge experiments in nonhuman primates and calculated the doses of VRC07-523 and VRC01 that provide 50% protection (EC50). VRC07-523 prevented infection in NHPs at a 5-fold lower concentration than VRC01. These results suggest that increased neutralization potency in vitro correlates with improved protection against infection in vivo, documenting the improved functional efficacy of VRC07-523 and its potential clinical relevance for protecting against HIV-1 infection in humans., Importance: In the absence of an effective HIV-1 vaccine, alternative strategies are needed to block HIV-1 transmission. Direct administration of HIV-1-neutralizing antibodies may be able to prevent HIV-1 infections in humans. This approach could be especially useful in individuals at high risk for contracting HIV-1 and could be used together with antiretroviral drugs to prevent infection. To optimize the chance of success, such antibodies can be modified to improve their potency, breadth, and in vivo half-life. Here, knowledge of the structure of a potent neutralizing antibody, VRC01, that targets the CD4-binding site of the HIV-1 envelope protein was used to engineer a next-generation antibody with 5- to 8-fold increased potency in vitro. When administered to nonhuman primates, this antibody conferred protection at a 5-fold lower concentration than the original antibody. Our studies demonstrate an important correlation between in vitro assays used to evaluate the therapeutic potential of antibodies and their in vivo effectiveness., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)
- Published
- 2014
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24. Neutralizing antibodies to HIV-1 envelope protect more effectively in vivo than those to the CD4 receptor.
- Author
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Pegu A, Yang ZY, Boyington JC, Wu L, Ko SY, Schmidt SD, McKee K, Kong WP, Shi W, Chen X, Todd JP, Letvin NL, Huang J, Nason MC, Hoxie JA, Kwong PD, Connors M, Rao SS, Mascola JR, and Nabel GJ
- Subjects
- Animals, Antibodies, Neutralizing immunology, CD4 Antigens immunology, Female, HIV Antibodies immunology, HIV Antibodies therapeutic use, Macaca mulatta, Male, Antibodies, Neutralizing therapeutic use, HIV Infections immunology, HIV Infections prevention & control, HIV-1 immunology, env Gene Products, Human Immunodeficiency Virus immunology
- Abstract
HIV-1 infection depends on effective viral entry mediated by the interaction of its envelope (Env) glycoprotein with specific cell surface receptors. Protective antiviral antibodies generated by passive or active immunization must prevent these interactions. Because the HIV-1 Env is highly variable, attention has also focused on blocking the HIV-1 primary cell receptor CD4. We therefore analyzed the in vivo protective efficacy of three potent neutralizing monoclonal antibodies (mAbs) to HIV-1 Env compared to an antibody against the CD4 receptor. Protection was assessed after mucosal challenge of rhesus macaques with simian/HIV (SHIV). Despite its comparable or greater neutralization potency in vitro, the anti-CD4 antibody did not provide effective protection in vivo, whereas the HIV-1-specific mAbs VRC01, 10E8, and PG9, targeting the CD4 binding site, membrane-proximal, and V1V2 glycan Env regions, respectively, conferred complete protection, albeit at different relative potencies. These findings demonstrate the protective efficacy of broadly neutralizing antibodies directed to the HIV-1 Env and suggest that targeting the HIV-1 Env is preferable to the cell surface receptor CD4 for the prevention of HIV-1 transmission., (Copyright © 2014, American Association for the Advancement of Science.)
- Published
- 2014
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25. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus.
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McLellan JS, Chen M, Joyce MG, Sastry M, Stewart-Jones GB, Yang Y, Zhang B, Chen L, Srivatsan S, Zheng A, Zhou T, Graepel KW, Kumar A, Moin S, Boyington JC, Chuang GY, Soto C, Baxa U, Bakker AQ, Spits H, Beaumont T, Zheng Z, Xia N, Ko SY, Todd JP, Rao S, Graham BS, and Kwong PD
- Subjects
- Animals, Antibodies, Neutralizing immunology, Antigens, Viral genetics, Antigens, Viral immunology, Crystallography, X-Ray, Cysteine chemistry, Cysteine genetics, Glycoproteins genetics, Glycoproteins immunology, Humans, Macaca, Mice, Protein Engineering, Protein Multimerization, Protein Stability, Protein Structure, Tertiary, Vaccination, Viral Fusion Proteins genetics, Viral Fusion Proteins immunology, Antigens, Viral chemistry, Glycoproteins chemistry, Respiratory Syncytial Virus Infections prevention & control, Respiratory Syncytial Virus Vaccines chemistry, Viral Fusion Proteins chemistry
- Abstract
Respiratory syncytial virus (RSV) is the leading cause of hospitalization for children under 5 years of age. We sought to engineer a viral antigen that provides greater protection than currently available vaccines and focused on antigenic site Ø, a metastable site specific to the prefusion state of the RSV fusion (F) glycoprotein, as this site is targeted by extremely potent RSV-neutralizing antibodies. Structure-based design yielded stabilized versions of RSV F that maintained antigenic site Ø when exposed to extremes of pH, osmolality, and temperature. Six RSV F crystal structures provided atomic-level data on how introduced cysteine residues and filled hydrophobic cavities improved stability. Immunization with site Ø-stabilized variants of RSV F in mice and macaques elicited levels of RSV-specific neutralizing activity many times the protective threshold.
- Published
- 2013
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26. Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies.
- Author
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Kanekiyo M, Wei CJ, Yassine HM, McTamney PM, Boyington JC, Whittle JR, Rao SS, Kong WP, Wang L, and Nabel GJ
- Subjects
- Animals, Binding Sites, Cross Reactions immunology, Female, Ferrets immunology, Ferrets virology, Ferritins chemistry, Hemagglutination Inhibition Tests, Hemagglutinin Glycoproteins, Influenza Virus immunology, Influenza A Virus, H1N1 Subtype classification, Male, Mice, Mice, Inbred BALB C, Orthomyxoviridae Infections immunology, Orthomyxoviridae Infections prevention & control, Orthomyxoviridae Infections virology, Vaccines, Inactivated immunology, Antibodies, Neutralizing immunology, Antibodies, Viral immunology, Influenza A Virus, H1N1 Subtype immunology, Influenza Vaccines chemistry, Influenza Vaccines immunology, Nanoparticles chemistry
- Abstract
Influenza viruses pose a significant threat to the public and are a burden on global health systems. Each year, influenza vaccines must be rapidly produced to match circulating viruses, a process constrained by dated technology and vulnerable to unexpected strains emerging from humans and animal reservoirs. Here we use knowledge of protein structure to design self-assembling nanoparticles that elicit broader and more potent immunity than traditional influenza vaccines. The viral haemagglutinin was genetically fused to ferritin, a protein that naturally forms nanoparticles composed of 24 identical polypeptides. Haemagglutinin was inserted at the interface of adjacent subunits so that it spontaneously assembled and generated eight trimeric viral spikes on its surface. Immunization with this influenza nanoparticle vaccine elicited haemagglutination inhibition antibody titres more than tenfold higher than those from the licensed inactivated vaccine. Furthermore, it elicited neutralizing antibodies to two highly conserved vulnerable haemagglutinin structures that are targets of universal vaccines: the stem and the receptor binding site on the head. Antibodies elicited by a 1999 haemagglutinin-nanoparticle vaccine neutralized H1N1 viruses from 1934 to 2007 and protected ferrets from an unmatched 2007 H1N1 virus challenge. This structure-based, self-assembling synthetic nanoparticle vaccine improves the potency and breadth of influenza virus immunity, and it provides a foundation for building broader vaccine protection against emerging influenza viruses and other pathogens.
- Published
- 2013
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27. Outer domain of HIV-1 gp120: antigenic optimization, structural malleability, and crystal structure with antibody VRC-PG04.
- Author
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Joyce MG, Kanekiyo M, Xu L, Biertümpfel C, Boyington JC, Moquin S, Shi W, Wu X, Yang Y, Yang ZY, Zhang B, Zheng A, Zhou T, Zhu J, Mascola JR, Kwong PD, and Nabel GJ
- Subjects
- Amino Acid Sequence, Antibodies, Neutralizing chemistry, Antibodies, Neutralizing metabolism, Antigens, Viral chemistry, Antigens, Viral genetics, Antigens, Viral immunology, Crystallography, X-Ray, Epitopes chemistry, Epitopes genetics, Epitopes immunology, HIV Antibodies metabolism, HIV Envelope Protein gp120 genetics, HIV-1 genetics, Models, Molecular, Molecular Sequence Data, Protein Structure, Quaternary, Sequence Alignment, HIV Antibodies chemistry, HIV Envelope Protein gp120 chemistry, HIV Envelope Protein gp120 immunology, HIV-1 chemistry, HIV-1 immunology
- Abstract
The outer domain of the HIV-1 gp120 envelope glycoprotein contains the epitope for broadly neutralizing antibodies directed to the CD4-binding site, many of which are able to neutralize over 90% of circulating HIV-1 isolates. While the outer domain is conformationally more stable than other portions of the HIV-1 envelope, efforts to express the outer domain as an immunogen for eliciting broadly neutralizing antibodies have not been successful, potentially because natural outer domain variants do not bind strongly to antibodies such as VRC01. In this study, we optimized the antigenic properties of the HIV-1 Env outer domain to generate OD4.2.2, from the KER2018 strain of clade A HIV-1, enabling it to bind antibodies such as VRC01 with nanomolar affinity. The crystal structure of OD4.2.2 in complex with VRC-PG04 was solved at 3.0-Å resolution and compared to known crystal structures including (i) the structure of core gp120 bound by VRC-PG04 and (ii) a circularly permutated version of the outer domain in complex with antibody PGT128. Much of the VRC-PG04 epitope was preserved in the OD4.2.2 structure, though with altered N and C termini conformations. Overall, roughly one-third of the outer domain structure appeared to be fixed in conformation, independent of alterations in termini, clade, or ligand, while other portions of the outer domain displayed substantial structural malleability. The crystal structure of OD4.2.2 with VRC-PG04 provides atomic-level details for an HIV-1 domain recognized by broadly neutralizing antibodies and insights relevant to the rational design of an immunogen that could elicit such antibodies by vaccination.
- Published
- 2013
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28. Structural and genetic basis for development of broadly neutralizing influenza antibodies.
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Lingwood D, McTamney PM, Yassine HM, Whittle JR, Guo X, Boyington JC, Wei CJ, and Nabel GJ
- Subjects
- Amino Acid Sequence, Antibodies, Neutralizing genetics, Antibodies, Viral chemistry, Antibody Affinity immunology, Binding Sites, Antibody immunology, Complementarity Determining Regions chemistry, Complementarity Determining Regions immunology, Cross Reactions immunology, Humans, Immunoglobulin G chemistry, Immunoglobulin G immunology, Immunoglobulin Heavy Chains chemistry, Immunoglobulin Heavy Chains immunology, Immunoglobulin M chemistry, Immunoglobulin M immunology, Influenza Vaccines immunology, Models, Molecular, Molecular Sequence Data, Orthomyxoviridae chemistry, Protein Conformation, Receptors, Antigen, B-Cell chemistry, Receptors, Antigen, B-Cell immunology, Sequence Alignment, Antibodies, Neutralizing chemistry, Antibodies, Neutralizing immunology, Antibodies, Viral genetics, Antibodies, Viral immunology, Orthomyxoviridae classification, Orthomyxoviridae immunology
- Abstract
Influenza viruses take a yearly toll on human life despite efforts to contain them with seasonal vaccines. These viruses evade human immunity through the evolution of variants that resist neutralization. The identification of antibodies that recognize invariant structures on the influenza haemagglutinin (HA) protein have invigorated efforts to develop universal influenza vaccines. Specifically, antibodies to the highly conserved stem region of HA neutralize diverse viral subtypes. These antibodies largely derive from a specific antibody gene, heavy-chain variable region IGHV1-69, after limited affinity maturation from their germline ancestors, but how HA stimulates naive B cells to mature and induce protective immunity is unknown. To address this question, we analysed the structural and genetic basis for their engagement and maturation into broadly neutralizing antibodies. Here we show that the germline-encoded precursors of these antibodies act as functional B-cell antigen receptors (BCRs) that initiate subsequent affinity maturation. Neither the germline precursor of a prototypic antibody, CR6261 (ref. 3), nor those of two other natural human IGHV1-69 antibodies, bound HA as soluble immunoglobulin-G (IgG). However, all three IGHV1-69 precursors engaged HA when the antibody was expressed as cell surface IgM. HA triggered BCR-associated tyrosine kinase signalling by germline transmembrane IgM. Recognition and virus neutralization was dependent solely on the heavy chain, and affinity maturation of CR6261 required only seven amino acids in the complementarity-determining region (CDR) H1 and framework region 3 (FR3) to restore full activity. These findings provide insight into the initial events that lead to the generation of broadly neutralizing antibodies to influenza, informing the rational design of vaccines to elicit such antibodies and providing a model relevant to other infectious diseases, including human immunodeficiency virus/AIDS. The data further suggest that selected immunoglobulin genes recognize specific protein structural 'patterns' that provide a substrate for further affinity maturation.
- Published
- 2012
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29. Computational prediction of N-linked glycosylation incorporating structural properties and patterns.
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Chuang GY, Boyington JC, Joyce MG, Zhu J, Nabel GJ, Kwong PD, and Georgiev I
- Subjects
- Amino Acid Sequence, Amino Acids chemistry, Amino Acids genetics, Amino Acids metabolism, Glycoproteins metabolism, Glycosylation, Protein Structure, Secondary, Protein Structure, Tertiary, Proteins genetics, Proteins metabolism, Structure-Activity Relationship, Algorithms, Glycoproteins chemistry, Models, Biological, Proteins chemistry
- Abstract
Motivation: N-linked glycosylation occurs predominantly at the N-X-T/S motif, where X is any amino acid except proline. Not all N-X-T/S sequons are glycosylated, and a number of web servers for predicting N-linked glycan occupancy using sequence and/or residue pattern information have been developed. None of the currently available servers, however, utilizes protein structural information for the prediction of N-glycan occupancy., Results: Here, we describe a novel classifier algorithm, NGlycPred, for the prediction of glycan occupancy at the N-X-T/S sequons. The algorithm utilizes both structural as well as residue pattern information and was trained on a set of glycosylated protein structures using the Random Forest algorithm. The best predictor achieved a balanced accuracy of 0.687 under 10-fold cross-validation on a curated dataset of 479 N-X-T/S sequons and outperformed sequence-based predictors when evaluated on the same dataset. The incorporation of structural information, including local contact order, surface accessibility/composition and secondary structure thus improves the prediction accuracy of glycan occupancy at the N-X-T/S consensus sequon., Availability and Implementation: NGlycPred is freely available to non-commercial users as a web-based server at http://exon.niaid.nih.gov/nglycpred/.
- Published
- 2012
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30. Elicitation of broadly neutralizing influenza antibodies in animals with previous influenza exposure.
- Author
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Wei CJ, Yassine HM, McTamney PM, Gall JG, Whittle JR, Boyington JC, and Nabel GJ
- Subjects
- Animals, Antibody Formation immunology, Antibody Specificity immunology, Ferrets immunology, Ferrets virology, Hemagglutination Inhibition Tests, Hemagglutinin Glycoproteins, Influenza Virus immunology, Humans, Immune Sera, Immunity immunology, Immunization, Secondary, Influenza A Virus, H1N1 Subtype immunology, Influenza, Human prevention & control, Mice, Orthomyxoviridae Infections prevention & control, Orthomyxoviridae Infections virology, Vaccination, Antibodies, Neutralizing immunology, Antibodies, Viral immunology, Influenza, Human immunology, Orthomyxoviridae Infections immunology
- Abstract
The immune system responds to influenza infection by producing neutralizing antibodies to the viral surface protein, hemagglutinin (HA), which regularly changes its antigenic structure. Antibodies that target the highly conserved stem region of HA neutralize diverse influenza viruses and can be elicited through vaccination in animals and humans. Efforts to develop universal influenza vaccines have focused on strategies to elicit such antibodies; however, the concern has been raised that previous influenza immunity may abrogate the induction of such broadly protective antibodies. We show here that prime-boost immunization can induce broadly neutralizing antibody responses in influenza-immune mice and ferrets that were previously infected or vaccinated. HA stem-directed antibodies were elicited in mice primed with a DNA vaccine and boosted with inactivated vaccine from H1N1 A/New Caledonia/20/1999 (1999 NC) HA regardless of preexposure. Similarly, gene-based vaccination with replication-defective adenovirus 28 (rAd28) and 5 (rAd5) vectors encoding 1999 NC HA elicited stem-directed neutralizing antibodies and conferred protection against unmatched 1934 and 2007 H1N1 virus challenge in influenza-immune ferrets. Indeed, previous exposure to certain strains could enhance immunogenicity: The strongest HA stem-directed immune response was observed in ferrets previously infected with a divergent 1934 H1N1 virus. These findings suggest that broadly neutralizing antibodies against the conserved stem region of HA can be elicited through vaccination despite previous influenza exposure, which supports the feasibility of developing stem-directed universal influenza vaccines for humans.
- Published
- 2012
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31. DNA priming and influenza vaccine immunogenicity: two phase 1 open label randomised clinical trials.
- Author
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Ledgerwood JE, Wei CJ, Hu Z, Gordon IJ, Enama ME, Hendel CS, McTamney PM, Pearce MB, Yassine HM, Boyington JC, Bailer R, Tumpey TM, Koup RA, Mascola JR, Nabel GJ, and Graham BS
- Subjects
- Adolescent, Adult, Antibodies, Viral immunology, Cytokines immunology, Cytokines metabolism, Humans, Middle Aged, Vaccines, Inactivated immunology, Young Adult, Influenza A Virus, H5N1 Subtype genetics, Influenza A Virus, H5N1 Subtype immunology, Influenza Vaccines genetics, Influenza Vaccines immunology, Influenza, Human prevention & control, Vaccines, DNA immunology
- Abstract
Background: Because the general population is largely naive to H5N1 influenza, antibodies generated to H5 allow analysis of novel influenza vaccines independent of background immunity from previous infection. We assessed the safety and immunogenicity of DNA encoding H5 as a priming vaccine to improve antibody responses to inactivated influenza vaccination., Methods: In VRC 306 and VRC 310, two sequentially enrolled phase 1, open-label, randomised clinical trials, healthy adults (age 18-60 years) were randomly assigned to receive intramuscular H5 DNA (4 mg) at day 0 or twice, at day 0 and week 4, followed by H5N1 monovalent inactivated vaccine (MIV; 90 μg) at 4 or 24 weeks, and compared with a two-dose regimen of H5N1 MIV with either a 4 or 24 week interval. Antibody responses were assessed by haemagglutination inhibition (HAI), ELISA, neutralisation (ID(80)), and immunoassays for stem-directed antibodies. T cell responses were assessed by intracellular cytokine staining. After enrolment, investigators and individuals were not masked to group assignment. VRC 306 and VRC 310 are registered with ClinicalTrials.gov, numbers NCT00776711 and NCT01086657, respectively., Findings: In VRC 306, 60 individuals were randomly assigned to the four groups (15 in each) and 59 received the vaccinations. In VRC 310, of the 21 individuals enrolled, 20 received the vaccinations (nine received a two-dose regimen of H5N1 MIV and 11 received H5 DNA at day 0 followed by H5N1 MIV at week 24). H5 DNA priming was safe and enhanced H5-specific antibody titres following an H5N1 MIV boost, especially when the interval between DNA prime and MIV boost was extended to 24 weeks. In the two studies, DNA priming with a 24-week MIV boost interval induced protective HAI titres in 21 (81%) of 26 of individuals, with an increase in geometric mean titre (GMT) of more than four times that of individuals given the MIV-MIV regimen at 4 or 24 weeks (GMT 103-206 vs GMT 27-33). Additionally, neutralising antibodies directed to the conserved stem region of H5 were induced by this prime-boost regimen in several individuals. No vaccine-related serious adverse events were recorded., Interpretation: DNA priming 24 weeks in advance of influenza vaccine boosting increased the magnitude of protective antibody responses (HAI) and in some cases induced haemagglutinin-stem-specific neutralising antibodies. A DNA-MIV vaccine regimen could enhance the efficacy of H5 or other influenza vaccines and shows that anti-stem antibodies can be elicited by vaccination in man., Funding: National Institutes of Health., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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32. Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9.
- Author
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McLellan JS, Pancera M, Carrico C, Gorman J, Julien JP, Khayat R, Louder R, Pejchal R, Sastry M, Dai K, O'Dell S, Patel N, Shahzad-ul-Hussan S, Yang Y, Zhang B, Zhou T, Zhu J, Boyington JC, Chuang GY, Diwanji D, Georgiev I, Kwon YD, Lee D, Louder MK, Moquin S, Schmidt SD, Yang ZY, Bonsignori M, Crump JA, Kapiga SH, Sam NE, Haynes BF, Burton DR, Koff WC, Walker LM, Phogat S, Wyatt R, Orwenyo J, Wang LX, Arthos J, Bewley CA, Mascola JR, Nabel GJ, Schief WR, Ward AB, Wilson IA, and Kwong PD
- Subjects
- AIDS Vaccines chemistry, AIDS Vaccines immunology, Amino Acid Motifs, Amino Acid Sequence, Antibodies, Neutralizing chemistry, Antibody Affinity immunology, Antigen-Antibody Complex chemistry, Antigen-Antibody Complex immunology, Binding Sites, Antibody immunology, Conserved Sequence, Crystallography, X-Ray, Epitopes chemistry, Epitopes immunology, Glycopeptides chemistry, Glycopeptides immunology, Glycosylation, HIV Antibodies chemistry, Hydrogen Bonding, Immune Evasion, Models, Molecular, Molecular Sequence Data, Polysaccharides chemistry, Polysaccharides immunology, Protein Structure, Quaternary, Protein Structure, Tertiary, Antibodies, Neutralizing immunology, Antibody Specificity immunology, HIV Antibodies immunology, HIV Envelope Protein gp120 chemistry, HIV Envelope Protein gp120 immunology, HIV-1 chemistry, HIV-1 immunology
- Abstract
Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded β-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which-with PG9-involves a site of vulnerability comprising just two glycans and a strand.
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- 2011
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33. The 1.5 Å crystal structure of human receptor for advanced glycation endproducts (RAGE) ectodomains reveals unique features determining ligand binding.
- Author
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Park H, Adsit FG, and Boyington JC
- Subjects
- Crystallography, X-Ray, DNA chemistry, DNA genetics, DNA metabolism, Humans, Hydrophobic and Hydrophilic Interactions, Ligands, Nerve Growth Factors chemistry, Nerve Growth Factors genetics, Nerve Growth Factors metabolism, Protein Binding, Protein Structure, Tertiary, RNA, Double-Stranded chemistry, RNA, Double-Stranded genetics, RNA, Double-Stranded metabolism, Receptor for Advanced Glycation End Products genetics, Receptor for Advanced Glycation End Products metabolism, S100 Calcium Binding Protein beta Subunit, S100 Proteins chemistry, S100 Proteins genetics, S100 Proteins metabolism, Structure-Activity Relationship, Receptor for Advanced Glycation End Products chemistry
- Abstract
Interaction of the pattern recognition receptor, RAGE with key ligands such as advanced glycation end products (AGE), S100 proteins, amyloid β, and HMGB1 has been linked to diabetic complications, inflammatory and neurodegenerative disorders, and cancer. To help answer the question of how a single receptor can recognize and respond to a diverse set of ligands we have investigated the structure and binding properties of the first two extracellular domains of human RAGE, which are implicated in various ligand binding and subsequent signaling events. The 1.5-Å crystal structure reveals an elongated molecule with a large basic patch and a large hydrophobic patch, both highly conserved. Isothermal titration calorimetry (ITC) and deletion experiments indicate S100B recognition by RAGE is an entropically driven process involving hydrophobic interaction that is dependent on Ca(2+) and on residues in the C'D loop (residues 54-67) of domain 1. In contrast, competition experiments using gel shift assays suggest that RAGE interaction with AGE is driven by the recognition of negative charges on AGE-proteins. We also demonstrate that RAGE can bind to dsDNA and dsRNA. These findings reveal versatile structural features of RAGE that help explain its ability to recognize of multiple ligands.
- Published
- 2010
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34. Induction of broadly neutralizing H1N1 influenza antibodies by vaccination.
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Wei CJ, Boyington JC, McTamney PM, Kong WP, Pearce MB, Xu L, Andersen H, Rao S, Tumpey TM, Yang ZY, and Nabel GJ
- Subjects
- Animals, Antibodies, Neutralizing biosynthesis, Antibodies, Viral biosynthesis, Female, Ferrets, Genetic Vectors, Hemagglutinin Glycoproteins, Influenza Virus genetics, Humans, Immunization, Secondary, Influenza A Virus, H2N2 Subtype immunology, Influenza A Virus, H3N2 Subtype immunology, Influenza A Virus, H5N1 Subtype immunology, Influenza, Human immunology, Influenza, Human prevention & control, Macaca mulatta, Male, Mice, Mice, Inbred BALB C, Mutant Proteins immunology, Orthomyxoviridae Infections immunology, Orthomyxoviridae Infections prevention & control, Plasmids, Vaccination, Vaccines, DNA administration & dosage, Vaccines, DNA immunology, Antibodies, Neutralizing immunology, Antibodies, Viral immunology, Cross Protection, Hemagglutinin Glycoproteins, Influenza Virus immunology, Influenza A Virus, H1N1 Subtype immunology, Influenza Vaccines administration & dosage, Influenza Vaccines immunology
- Abstract
The rapid dissemination of the 2009 pandemic influenza virus underscores the need for universal influenza vaccines that elicit protective immunity to diverse viral strains. Here, we show that vaccination with plasmid DNA encoding H1N1 influenza hemagglutinin (HA) and boosting with seasonal vaccine or replication-defective adenovirus 5 vector encoding HA stimulated the production of broadly neutralizing influenza antibodies. This prime/boost combination increased the neutralization of diverse H1N1 strains dating from 1934 to 2007 as compared to either component alone and conferred protection against divergent H1N1 viruses in mice and ferrets. These antibodies were directed to the conserved stem region of HA and were also elicited in nonhuman primates. Cross-neutralization of H1N1 subtypes elicited by this approach provides a basis for the development of a universal influenza vaccine for humans.
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- 2010
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35. Cross-neutralization of 1918 and 2009 influenza viruses: role of glycans in viral evolution and vaccine design.
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Wei CJ, Boyington JC, Dai K, Houser KV, Pearce MB, Kong WP, Yang ZY, Tumpey TM, and Nabel GJ
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Disease Outbreaks prevention & control, Drug Design, Hemagglutinin Glycoproteins, Influenza Virus chemistry, History, 20th Century, History, 21st Century, Humans, Influenza, Human epidemiology, Influenza, Human immunology, Influenza, Human virology, Mice, Mice, Inbred BALB C, Models, Molecular, Molecular Sequence Data, Antibodies, Neutralizing immunology, Cross Protection immunology, Evolution, Molecular, Influenza A Virus, H1N1 Subtype immunology, Influenza Vaccines immunology, Neutralization Tests, Polysaccharides metabolism
- Abstract
New strains of H1N1 influenza virus have emerged episodically over the last century to cause human pandemics, notably in 1918 and recently in 2009. Pandemic viruses typically evolve into seasonal forms that develop resistance to antibody neutralization, and cross-protection between strains separated by more than 3 years is uncommon. Here, we define the structural basis for cross-neutralization between two temporally distant pandemic influenza viruses--from 1918 and 2009. Vaccination of mice with the 1918 strain protected against subsequent lethal infection by 2009 virus. Both were resistant to antibodies directed against a seasonal influenza, A/New Caledonia/20/1999 (1999 NC), which was insensitive to antisera to the pandemic strains. Pandemic strain-neutralizing antibodies were directed against a subregion of the hemagglutinin (HA) receptor binding domain that is highly conserved between the 1918 and the 2009 viruses. In seasonal strains, this region undergoes amino acid diversification but is shielded from antibody neutralization by two highly conserved glycosylation sites absent in the pandemic strains. Pandemic HA trimers modified by glycosylation at these positions were resistant to neutralizing antibodies to wild-type HA. Yet, antisera generated against the glycosylated HA mutant neutralized it, suggesting that the focus of the immune response can be selectively changed with this modification. Collectively, these findings define critical determinants of H1N1 viral evolution and have implications for vaccine design. Immunization directed to conserved receptor binding domain subregions of pandemic viruses could potentially protect against similar future pandemic viruses, and vaccination with glycosylated 2009 pandemic virus may limit its further spread and transformation into a seasonal influenza.
- Published
- 2010
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36. Biochemical and structural characterization of cathepsin L-processed Ebola virus glycoprotein: implications for viral entry and immunogenicity.
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Hood CL, Abraham J, Boyington JC, Leung K, Kwong PD, and Nabel GJ
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Neutralizing immunology, Cell Line, Female, Humans, Mice, Mice, Inbred BALB C, Models, Molecular, Molecular Sequence Data, Protein Multimerization, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Alignment, Viral Envelope Proteins genetics, Cathepsin L metabolism, Ebolavirus immunology, Ebolavirus physiology, Viral Envelope Proteins chemistry, Viral Envelope Proteins metabolism, Virus Internalization
- Abstract
Ebola virus (EBOV) cellular attachment and entry is initiated by the envelope glycoprotein (GP) on the virion surface. Entry of this virus is pH dependent and associated with the cleavage of GP by proteases, including cathepsin L (CatL) and/or CatB, in the endosome or cell membrane. Here, we characterize the product of CatL cleavage of Zaire EBOV GP (ZEBOV-GP) and evaluate its relevance to entry. A stabilized recombinant form of the EBOV GP trimer was generated using a trimerization domain linked to a cleavable histidine tag. This trimer was purified to homogeneity and cleaved with CatL. Characterization of the trimeric product by N-terminal sequencing and mass spectrometry revealed three cleavage fragments, with masses of 23, 19, and 4 kDa. Structure-assisted modeling of the cathepsin L-cleaved ZEBOV-GP revealed that cleavage removes a glycosylated glycan cap and mucin-like domain (MUC domain) and exposes the conserved core residues implicated in receptor binding. The CatL-cleaved ZEBOV-GP intermediate bound with high affinity to a neutralizing antibody, KZ52, and also elicited neutralizing antibodies, supporting the notion that the processed intermediate is required for viral entry. Together, these data suggest that CatL cleavage of EBOV GP exposes its receptor-binding domain, thereby facilitating access to a putative cellular receptor in steps that lead to membrane fusion.
- Published
- 2010
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37. The 1.4 angstrom crystal structure of the human oxidized low density lipoprotein receptor lox-1.
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Park H, Adsit FG, and Boyington JC
- Subjects
- Amino Acid Sequence, Animals, Crystallography, X-Ray, Dimerization, Humans, Lipoproteins, LDL metabolism, Models, Molecular, Molecular Sequence Data, Receptors, LDL genetics, Receptors, LDL metabolism, Receptors, Oxidized LDL, Scavenger Receptors, Class E, Sequence Alignment, Protein Structure, Quaternary, Receptors, LDL chemistry
- Abstract
The lectin-like oxidized low density lipoprotein receptor-1 (Lox-1) mediates the recognition and internalization of oxidatively modified low density lipoprotein by vascular endothelial cells. This interaction results in a number of pro-atherogenic cellular responses that probably play a significant role in the pathology of atherosclerosis. The 1.4 angstrom crystal structure of the extracellular C-type lectin-like domain of human Lox-1 reveals a heart-shaped homodimer with a ridge of six basic amino acids extending diagonally across the apolar top of Lox-1, a central hydrophobic tunnel that extends through the entire molecule, and an electrostatically neutral patch of 12 charged residues that resides next to the tunnel at each opening. Based on the arrangement of critical binding residues on the Lox-1 structure, we propose a binding mode for the recognition of modified low density lipoprotein and other Lox-1 ligands.
- Published
- 2005
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38. Overview of protein structural and functional folds.
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Sun PD, Foster CE, and Boyington JC
- Subjects
- Animals, Computational Biology, Cytoskeleton metabolism, DNA metabolism, Databases, Protein, Electron Transport, Humans, Immune System physiology, Models, Molecular, Muscles metabolism, RNA metabolism, Receptors, Cell Surface, Signal Transduction physiology, Software, Protein Conformation, Protein Folding, Proteins chemistry, Proteins genetics, Proteins metabolism
- Abstract
This overview provides an illustrated, comprehensive survey of some commonly observed protein-fold families and structural motifs, chosen for their functional significance. It opens with descriptions and definitions of the various elements of protein structure and associated terminology. Following is an introduction into web-based structural bioinformatics that includes surveys of interactive web servers for protein fold or domain annotation, protein-structure databases, protein-structure-classification databases, structural alignments of proteins, and molecular graphics programs available for personal computers. The rest of the overview describes selected families of protein folds in terms of their secondary, tertiary, and quaternary structural arrangements, including ribbon-diagram examples, tables of representative structures with references, and brief explanations pointing out their respective biological and functional significance.
- Published
- 2004
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39. A structural perspective on MHC class I recognition by killer cell immunoglobulin-like receptors.
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Boyington JC and Sun PD
- Subjects
- Amino Acid Sequence, Animals, H-2 Antigens chemistry, H-2 Antigens immunology, HLA-C Antigens immunology, Histocompatibility Antigen H-2D, Histocompatibility Antigens Class I immunology, Humans, Immunoglobulin Allotypes immunology, Lectins, C-Type, Membrane Glycoproteins chemistry, Membrane Glycoproteins immunology, Models, Molecular, Molecular Sequence Data, Protein Structure, Tertiary, Receptors, Immunologic immunology, Receptors, KIR, Receptors, KIR2DL1, Receptors, KIR2DL2, Receptors, NK Cell Lectin-Like, Antigens, Ly, HLA-C Antigens chemistry, Histocompatibility Antigens Class I chemistry, Killer Cells, Natural immunology, Receptors, Immunologic chemistry
- Abstract
Killer cell immunoglobulin-like receptors (KIR) play a critical role in the regulation of natural killer (NK) cell activity through their recognition of class I MHC molecules expressed on target cells. KIR recognition provides vital information to NK cells about whether a target cell should be lysed or spared. Understanding the molecular mechanism of this recognition has remained a strong focus of investigation. This has resulted in the crystal structures of several members of the KIR family and more recently the determinations of the three dimensional structures of KIR2DL2 and KIR2DL1 complexed with their respective ligands, HLA-Cw3 and HLA-Cw4. A strong structural conservation has been revealed both in the receptor design and in the overall mode of KIR binding to class I molecules. Nevertheless, distinct differences in the receptor binding sites allow for high specificity between ligands. Furthermore, unexpected similarities with T-cell receptor (TCR) recognition of MHC molecules are also observed. The detailed interactions between KIR and HLA-C molecules and their functional implications will be reviewed here.
- Published
- 2002
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40. Overview of protein folds in the immune system.
- Author
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Sun PD and Boyington JC
- Subjects
- Animals, Crystallography, X-Ray, Humans, Immune System, Immunologic Factors immunology, Models, Molecular, Molecular Structure, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Protein Folding, Proteins immunology, Immunologic Factors chemistry, Proteins chemistry
- Abstract
The rapid advancement of X-ray crystallography and nuclear magnetic resonance techniques in recent years has resulted in the solution of macromolecular structures at an unprecedented rate. This review aims at providing a comprehensive description of structures and folds related to the function of the immune system. Focus is placed on immunologically relevant proteins such as immunoreceptors and major histocompatibility complexes. Information is also provided regarding protein structure data banks.
- Published
- 2001
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41. Structure of killer cell immunoglobulin-like receptors and their recognition of the class I MHC molecules.
- Author
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Boyington JC, Brooks AG, and Sun PD
- Subjects
- Amino Acid Sequence, Animals, Binding Sites genetics, Carrier Proteins chemistry, Carrier Proteins genetics, Carrier Proteins metabolism, HLA-C Antigens chemistry, HLA-C Antigens genetics, HLA-C Antigens metabolism, Histocompatibility Antigens Class I chemistry, Histocompatibility Antigens Class I genetics, Humans, Hydrogen Bonding, Lectins, C-Type, Ligands, Macromolecular Substances, Membrane Proteins chemistry, Membrane Proteins genetics, Membrane Proteins metabolism, Mice, Models, Molecular, Molecular Sequence Data, Molecular Structure, Receptors, Immunologic genetics, Receptors, KIR, Receptors, KIR2DL2, Receptors, NK Cell Lectin-Like, Sequence Homology, Amino Acid, Antigens, Ly, Histocompatibility Antigens Class I metabolism, Receptors, Immunologic chemistry, Receptors, Immunologic metabolism
- Abstract
The recognition of class I MHC molecules by killer cell immunoglobulin-like receptors (KIR) constitutes an integral part of immune surveillance by the innate immune system. To understand the molecular basis of this recognition, the structures of several members of this superfamily have been determined. Despite their functional diversity, members of this superfamily share many conserved structural features. A central question is how these receptors recognize their ligands. The recent determination of the crystal structure of KIR2DL2 in complex with HLA-Cw3 has revealed the molecular mechanisms underpinning this interaction, which ultimately modulates the cytolytic activity of natural killer cells. While the recognition of MHC molecules by KIR is characterized by a number of unique features, some unexpected similarities with T-cell receptor recognition of MHC molecules are also observed. The detailed interactions between KIR2DL2 and HLA-Cw3 and their functional implications will be reviewed here.
- Published
- 2001
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42. Crystal structure of an NK cell immunoglobulin-like receptor in complex with its class I MHC ligand.
- Author
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Boyington JC, Motyka SA, Schuck P, Brooks AG, and Sun PD
- Subjects
- Amino Acid Sequence, Crystallography, X-Ray, Electrochemistry, Escherichia coli, HLA-C Antigens immunology, Humans, Killer Cells, Natural immunology, Ligands, Major Histocompatibility Complex, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Conformation, Receptor Aggregation, Receptors, Antigen, T-Cell immunology, Receptors, Immunologic immunology, Receptors, KIR, Receptors, KIR2DL2, Recombinant Proteins chemistry, Recombinant Proteins immunology, HLA-C Antigens chemistry, Killer Cells, Natural chemistry, Receptors, Immunologic chemistry
- Abstract
Target cell lysis is regulated by natural killer (NK) cell receptors that recognize class I MHC molecules. Here we report the crystal structure of the human immunoglobulin-like NK cell receptor KIR2DL2 in complex with its class I ligand HLA-Cw3 and peptide. KIR binds in a nearly orthogonal orientation across the alpha1 and alpha2 helices of Cw3 and directly contacts positions 7 and 8 of the peptide. No significant conformational changes in KIR occur on complex formation. The receptor footprint on HLA overlaps with but is distinct from that of the T-cell receptor. Charge complementarity dominates the KIR/HLA interface and mutations that disrupt interface salt bridges substantially diminish binding. Most contacts in the complex are between KIR and conserved HLA-C residues, but a hydrogen bond between Lys 44 of KIR2DL2 and Asn 80 of Cw3 confers the allotype specificity. KIR contact requires position 8 of the peptide to be a residue smaller than valine. A second KIR/HLA interface produced an ordered receptor-ligand aggregation in the crystal which may resemble receptor clustering during immune synapse formation.
- Published
- 2000
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43. Reconstitution of bacterial expressed human CD94: the importance of the stem region for dimer formation.
- Author
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Boyington JC, Raiz AN, Brooks AG, Patamawenu A, and Sun PD
- Subjects
- Amino Acid Sequence, Dimerization, Disulfides chemistry, Electrophoresis, Polyacrylamide Gel, Escherichia coli chemistry, Escherichia coli metabolism, Gas Chromatography-Mass Spectrometry, Humans, Inclusion Bodies chemistry, Inclusion Bodies metabolism, Molecular Sequence Data, NK Cell Lectin-Like Receptor Subfamily C, NK Cell Lectin-Like Receptor Subfamily D, Protein Folding, Protein Renaturation, Protein Structure, Tertiary, Receptors, Natural Killer Cell, Sequence Analysis, Protein, Antigens, CD chemistry, Lectins, C-Type, Membrane Glycoproteins chemistry, Receptors, Immunologic chemistry, Receptors, Mitogen chemistry
- Abstract
Human CD94 is a subunit of the disulfide-linked, heterodimeric natural killer (NK) cell surface receptor CD94/NKG2. This receptor, a member of the C-type lectin superfamily, participates in regulating NK cell directed lysis through interaction with the major histocompatibility antigen HLA-E. Two forms of CD94 were expressed using a bacterial expression system and refolded in vitro. One form, residues 34-179, designated S34, corresponds to the entire extracellular region of the receptor, including a 23-residue stem region, and the other, residues 51-179, designated E51, corresponds only to the putative carbohydrate recognition domain of the receptor. The refolded full-length S34 protein existed as a noncovalent dimer initially but formed an interchain disulfide bond upon storage for several months. In contrast, the stemless construct, E51, existed largely as a monomeric form. The stem region of S34, residues 34-56, is sensitive to proteolysis and its absence results in dissociation of the dimer. This suggests that the residues in the stem region of CD94 help to stabilize the dimeric conformation., (Copyright 2000 Academic Press.)
- Published
- 2000
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44. Natural killer cell recognition of HLA class I molecules.
- Author
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Brooks AG, Boyington JC, and Sun PD
- Subjects
- Amino Acid Sequence, Antigens, CD immunology, Binding Sites, Cytotoxicity, Immunologic, Dimerization, HLA Antigens chemistry, HLA Antigens immunology, HLA-C Antigens chemistry, HLA-C Antigens immunology, Histocompatibility Antigens Class I chemistry, Humans, Hydrogen Bonding, Leukocyte Immunoglobulin-like Receptor B1, Macromolecular Substances, Membrane Glycoproteins immunology, Models, Molecular, Molecular Sequence Data, Multigene Family, NK Cell Lectin-Like Receptor Subfamily C, NK Cell Lectin-Like Receptor Subfamily D, Peptide Fragments immunology, Peptide Fragments metabolism, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Receptors, Immunologic chemistry, Receptors, Immunologic genetics, Receptors, KIR, Receptors, KIR2DL2, Receptors, Natural Killer Cell, Sequence Alignment, Sequence Homology, Amino Acid, Structure-Activity Relationship, Zinc physiology, HLA-E Antigens, Histocompatibility Antigens Class I immunology, Lectins, C-Type, Receptors, Immunologic immunology
- Abstract
Human NK cells express multiple receptors that interact with HLA class I molecules. These receptors belong to one of two major protein superfamilies, the immunoglobulin superfamily or the C type lectin superfamily. The killer cell immunoglobulin-like receptor (KIR) family predominantly recognise classical HLA class I molecules and different family members interact with discrete HLA class I allotypes. The solution of the crystal structure of KIR2DL2 in complex with its ligand, HLA-Cw3 has provided the molecular details of a KIR/class I interaction. The interaction site spans both the alpha1 and alpha2 helices of class I and the KIR makes direct contact with peptide residues 7 and 8. The allotype specificity of KIR2DL2 for HLA-Cw3 is the result of a single hydrogen bond from Lys44 of the KIR to Asn80 of HLA-C as all other HLA-C residues that contact KIR are conserved. The lectin-like CD94/NKG2 receptors specifically interact with the non-classical class I molecule, HLA-E. Cell surface expression of HLA-E is dependent on the expression of other class I molecules as they are the major source of HLA-E binding peptides in normal cells. Consequently recognition of HLA-E by the CD94/NKG2 receptors allows NK cells to indirectly monitor the expression of a broad array of class I molecules. While the molecular interactions underlying ligand recognition by both KIR and CD94/NKG2 receptors are likely to be distinct, recognition of class I by both families of receptors appears peptide dependent. This suggest that cells that lack class I and also those that are impaired in their ability to load class I molecules with peptide will become targets for NK-mediated destruction.
- Published
- 2000
45. Stoichiometric arginine binding in the oxygenase domain of inducible nitric oxide synthase requires a single molecule of tetrahydrobiopterin per dimer.
- Author
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Rafferty SP, Boyington JC, Kulansky R, Sun PD, and Malech HL
- Subjects
- Animals, Binding Sites drug effects, Biopterins analysis, Biopterins metabolism, Biopterins pharmacology, Carbon Monoxide pharmacology, Chromatography, Gel, Conserved Sequence, Dimerization, Dithionite pharmacology, Ferrous Compounds metabolism, Heme metabolism, Mice, Nitric Oxide Synthase chemistry, Nitric Oxide Synthase Type II, Oxygenases chemistry, Protein Binding drug effects, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Spectrophotometry, Ultraviolet, Titrimetry, Arginine metabolism, Biopterins analogs & derivatives, Nitric Oxide Synthase metabolism, Oxygenases metabolism
- Abstract
In addition to its catalytic roles, the nitric oxide synthase (NOS) cofactor tetrahydrobiopterin (H4B) is required for substrate binding and for stabilization of the dimeric structure. We expressed and purified the core of the iNOS oxygenase domain consisting of residues 75-500 (CODiNOS) in the presence (H4B+) and absence (H4B-) of this cofactor. Both forms bound stoichiometric amounts of heme (>0.9 heme per protein subunit). H4B- CODiNOS was unable to bind arginine, gave an unstable ferrous carbonyl adduct, and was a mixture of monomer and dimer. H4B+ CODiNOS bound arginine, gave a stable ferrous carbonyl adduct, and was exclusively dimeric. The H4B cofactor content of this species was only one per dimer yet this was sufficient to form two competent arginine binding sites as determined by optical stoichiometric titrations., (Copyright 1999 Academic Press.)
- Published
- 1999
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46. Structure of CD94 reveals a novel C-type lectin fold: implications for the NK cell-associated CD94/NKG2 receptors.
- Author
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Boyington JC, Riaz AN, Patamawenu A, Coligan JE, Brooks AG, and Sun PD
- Subjects
- Amino Acid Sequence, Antigens, CD metabolism, Binding Sites, Crystallography, X-Ray, Dimerization, HLA Antigens metabolism, Histocompatibility Antigens Class I metabolism, Humans, Lectins metabolism, Membrane Glycoproteins metabolism, Models, Molecular, Molecular Sequence Data, NK Cell Lectin-Like Receptor Subfamily C, NK Cell Lectin-Like Receptor Subfamily D, Receptors, Immunologic metabolism, Receptors, Mitogen metabolism, Receptors, Natural Killer Cell, Sequence Homology, Amino Acid, HLA-E Antigens, Antigens, CD chemistry, Killer Cells, Natural metabolism, Lectins chemistry, Lectins, C-Type, Membrane Glycoproteins chemistry, Protein Folding, Receptors, Immunologic chemistry, Receptors, Mitogen chemistry
- Abstract
The crystal structure of the extracellular domain of CD94, a component of the CD94/NKG2 NK cell receptor, has been determined to 2.6 A resolution, revealing a unique variation of the C-type lectin fold. In this variation, the second alpha helix, corresponding to residues 102-112, is replaced by a loop, the putative carbohydrate-binding site is significantly altered, and the Ca2+-binding site appears nonfunctional. This structure may serve as a prototype for other NK cell receptors such as Ly-49, NKR-P1, and CD69. The CD94 dimer observed in the crystal has an extensive hydrophobic interface that stabilizes the loop conformation of residues 102-112. The formation of this dimer reveals a putative ligand-binding region for HLA-E and suggests how NKG2 interacts with CD94.
- Published
- 1999
- Full Text
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47. Structure and mechanism of lipoxygenases.
- Author
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Prigge ST, Boyington JC, Faig M, Doctor KS, Gaffney BJ, and Amzel LM
- Subjects
- Animals, Humans, Models, Molecular, Lipoxygenase chemistry, Lipoxygenase metabolism, Protein Conformation
- Abstract
In mammals, lipoxygenases catalyze the formation of hydroperoxides as the first step in the biosynthesis of several inflammatory mediators. The substrate of this reaction, arachidonic acid, is the key precursor of two families of potent physiological effectors. It is the branch point between two central pathways: one, involving the enzyme cyclooxygenase, leads to the synthesis of prostaglandins and thromboxanes; the other, involving lipoxygenases, leads to the synthesis of leukotrienes and lipoxins, compounds that regulate important cellular responses in inflammation and immunity. While aspirin and other non-steroidal anti-inflammatory compounds are potent inhibitors of cyclooxygenase, no effective pharmacological inhibitor of lipoxygenase is presently available. Lipoxygenases are large non-heme, iron-containing enzymes that use molecular oxygen for the diooxygenation of arachidonic acid to form hydroperoxides, the first step in the biosynthetic pathways leading to leukotrienes and lipoxins. Because of the importance of these compounds, lipoxygenases have been the subject of extensive study: from detailed kinetic measurements to cloning, expression, and site-directed mutagenesis. The sequences of over 50 lipoxygenases have been reported. In addition, the structure of soybean lipoxygenase-1, determined by X-ray diffraction methods, has recently been reported. The structure revealed that the 839 amino acids in the protein are organized in two domains: a beta-sheet N-terminal domain and a large, mostly helical C-terminal domain. The iron is present in the C-terminal domain facing two internal cavities that are probably the conduits through which the fatty acid and molecular oxygen gain access to the metal. Models of the mammalian lipoxygenases based on the soybean structure provide clues about the structural determinants of the positional specificity of the enzyme, and can be used as targets for the design of more effective inhibitors.
- Published
- 1997
- Full Text
- View/download PDF
48. Crystal structure of formate dehydrogenase H: catalysis involving Mo, molybdopterin, selenocysteine, and an Fe4S4 cluster.
- Author
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Boyington JC, Gladyshev VN, Khangulov SV, Stadtman TC, and Sun PD
- Subjects
- Binding Sites, Carbon Dioxide metabolism, Catalysis, Crystallography, X-Ray, Electron Transport, Escherichia coli enzymology, Formate Dehydrogenases metabolism, Guanine Nucleotides chemistry, Guanine Nucleotides metabolism, Hydrogen Bonding, Hydrogenase metabolism, Ligands, Models, Molecular, Molecular Sequence Data, Molybdenum chemistry, Molybdenum metabolism, Multienzyme Complexes metabolism, Nitrites chemistry, Oxidation-Reduction, Protein Structure, Secondary, Protein Structure, Tertiary, Protons, Pterins chemistry, Pterins metabolism, Selenocysteine chemistry, Selenocysteine metabolism, Ferrous Compounds chemistry, Formate Dehydrogenases chemistry, Formates metabolism, Hydrogenase chemistry, Multienzyme Complexes chemistry, Protein Conformation
- Abstract
Formate dehydrogenase H from Escherichia coli contains selenocysteine (SeCys), molybdenum, two molybdopterin guanine dinucleotide (MGD) cofactors, and an Fe4S4 cluster at the active site and catalyzes the two-electron oxidation of formate to carbon dioxide. The crystal structures of the oxidized [Mo(VI), Fe4S4(ox)] form of formate dehydrogenase H (with and without bound inhibitor) and the reduced [Mo(IV), Fe4S4(red)] form have been determined, revealing a four-domain alphabeta structure with the molybdenum directly coordinated to selenium and both MGD cofactors. These structures suggest a reaction mechanism that directly involves SeCys140 and His141 in proton abstraction and the molybdenum, molybdopterin, Lys44, and the Fe4S4 cluster in electron transfer.
- Published
- 1997
- Full Text
- View/download PDF
49. The three-dimensional structure of soybean lipoxygenase-1: an arachidonic acid 15-lipoxygenase.
- Author
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Boyington JC, Gaffney BJ, and Amzel LM
- Subjects
- Amino Acid Sequence, Animals, Crystallography, X-Ray methods, Iron analysis, Mammals, Models, Molecular, Protein Structure, Secondary, Arachidonate 15-Lipoxygenase chemistry, Lipoxygenase chemistry, Protein Conformation, Glycine max enzymology
- Published
- 1997
- Full Text
- View/download PDF
50. Characterization of crystalline formate dehydrogenase H from Escherichia coli. Stabilization, EPR spectroscopy, and preliminary crystallographic analysis.
- Author
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Gladyshev VN, Boyington JC, Khangulov SV, Grahame DA, Stadtman TC, and Sun PD
- Subjects
- Crystallography, X-Ray, Electron Spin Resonance Spectroscopy, Escherichia coli enzymology, Formate Dehydrogenases ultrastructure, Freezing, Humans, Hydrogen-Ion Concentration, Metalloproteins chemistry, Metalloproteins ultrastructure, Molybdenum, Oxidation-Reduction, Selenium, Spectrum Analysis, Formate Dehydrogenases chemistry
- Abstract
The selenocysteine-containing formate dehydrogenase H (FDH) is an 80-kDa component of the Escherichia coli formate-hydrogen lyase complex. The molybdenum-coordinated selenocysteine is essential for catalytic activity of the native enzyme. FDH in dilute solutions (30 microg/ml) was rapidly inactivated at basic pH or in the presence of formate under anaerobic conditions, but at higher enzyme concentrations (>/=3 mg/ml) the enzyme was relatively stable. The formate-reduced enzyme was extremely sensitive to air inactivation under all conditions examined. Active formate-reduced FDH was crystallized under anaerobic conditions in the presence of ammonium sulfate and PEG 400. The crystals diffract to 2.6 A resolution and belong to a space group of P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions a = b = 146.1 A and c = 82.7 A. There is one monomer of FDH per crystallographic asymmetric unit. Similar diffraction quality crystals of oxidized FDH could be obtained by oxidation of crystals of formate-reduced enzyme with benzyl viologen. By EPR spectroscopy, a signal of a single reduced FeS cluster was found in a crystal of reduced FDH, but not in a crystal of oxidized enzyme, whereas Mo(V) signal was not detected in either form of crystalline FDH. This suggests that Mo(IV)- and the reduced FeS cluster-containing form of the enzyme was crystallized and this could be converted into Mo(VI)- and oxidized FeS cluster form upon oxidation. A procedure that combines anaerobic and cryocrystallography has been developed that is generally applicable to crystallographic studies of oxygen-sensitive enzymes. These data provide the first example of crystallization of a substrate-reduced form of a Se- and Mo-containing enzyme.
- Published
- 1996
- Full Text
- View/download PDF
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