Publications by authors named "Helen J Kim"

14 Publications

  • Page 1 of 1

Chemical Cross-Linking Stabilizes Native-Like HIV-1 Envelope Glycoprotein Trimer Antigens.

J Virol 2016 01 28;90(2):813-28. Epub 2015 Oct 28.

The Sir William Dunn School of Pathology, The University of Oxford, Oxford, United Kingdom

Unlabelled: Major neutralizing antibody immune evasion strategies of the HIV-1 envelope glycoprotein (Env) trimer include conformational and structural instability. Stabilized soluble trimers such as BG505 SOSIP.664 mimic the structure of virion-associated Env but nevertheless sample different conformational states. Here we demonstrate that treating BG505 SOSIP.664 trimers with glutaraldehyde or a heterobifunctional cross-linker introduces additional stability with relatively modest effects on antigenicity. Thus, most broadly neutralizing antibody (bNAb) epitopes were preserved after cross-linking, whereas the binding of most weakly or nonneutralizing antibodies (non-NAb) was reduced. Cross-linking stabilized all Env conformers present within a mixed population, and individual conformers could be isolated by bNAb affinity chromatography. Both positive selection of cross-linked conformers using the quaternary epitope-specific bNAbs PGT145, PGT151, and 3BC315 and negative selection with non-NAbs against the V3 region enriched for trimer populations with improved antigenicity for bNAbs. Similar results were obtained using the clade B B41 SOSIP.664 trimer. The cross-linking method may, therefore, be useful for countering the natural conformational heterogeneity of some HIV-1 Env proteins and, by extrapolation, also vaccine immunogens from other pathogens.

Importance: The development of a vaccine to induce protective antibodies against HIV-1 is of primary public health importance. Recent advances in immunogen design have provided soluble recombinant envelope glycoprotein trimers with near-native morphology and antigenicity. However, these trimers are conformationally flexible, potentially reducing B-cell recognition of neutralizing antibody epitopes. Here we show that chemical cross-linking increases trimer stability, reducing binding of nonneutralizing antibodies while largely maintaining neutralizing antibody binding. Cross-linking followed by positive or negative antibody affinity selection of individual stable conformational variants further improved the antigenic and morphological characteristics of the trimers. This approach may be generally applicable to HIV-1 Env and also to other conformationally flexible pathogen antigens.
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http://dx.doi.org/10.1128/JVI.01942-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702668PMC
January 2016

Impaired PIEZO1 function in patients with a novel autosomal recessive congenital lymphatic dysplasia.

Nat Commun 2015 Sep 21;6:8329. Epub 2015 Sep 21.

ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, Utah 84108, USA.

Piezo1 ion channels are mediators of mechanotransduction in several cell types including the vascular endothelium, renal tubular cells and erythrocytes. Gain-of-function mutations in PIEZO1 cause an autosomal dominant haemolytic anaemia in humans called dehydrated hereditary stomatocytosis. However, the phenotypic consequence of PIEZO1 loss of function in humans has not previously been documented. Here we discover a novel role of this channel in the lymphatic system. Through whole-exome sequencing, we identify biallelic mutations in PIEZO1 (a splicing variant leading to early truncation and a non-synonymous missense variant) in a pair of siblings affected with persistent lymphoedema caused by congenital lymphatic dysplasia. Analysis of patients' erythrocytes as well as studies in a heterologous system reveal greatly attenuated PIEZO1 function in affected alleles. Our results delineate a novel clinical category of PIEZO1-associated hereditary lymphoedema.
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http://dx.doi.org/10.1038/ncomms9329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4578306PMC
September 2015

HIV-1 VACCINES. HIV-1 neutralizing antibodies induced by native-like envelope trimers.

Science 2015 Jul 18;349(6244):aac4223. Epub 2015 Jun 18.

Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10065, USA.

A challenge for HIV-1 immunogen design is the difficulty of inducing neutralizing antibodies (NAbs) against neutralization-resistant (tier 2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation, BG505 SOSIP.664, induced NAbs potently against the sequence-matched tier 2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (tier 1) viruses. Tier 2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas tier 1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous tier 2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for the development of HIV-1 vaccines aimed at inducing bNAbs.
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http://dx.doi.org/10.1126/science.aac4223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498988PMC
July 2015

Structural Constraints Determine the Glycosylation of HIV-1 Envelope Trimers.

Cell Rep 2015 Jun 4;11(10):1604-13. Epub 2015 Jun 4.

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address:

A highly glycosylated, trimeric envelope glycoprotein (Env) mediates HIV-1 cell entry. The high density and heterogeneity of the glycans shield Env from recognition by the immune system, but paradoxically, many potent broadly neutralizing antibodies (bNAbs) recognize epitopes involving this glycan shield. To better understand Env glycosylation and its role in bNAb recognition, we characterized a soluble, cleaved recombinant trimer (BG505 SOSIP.664) that is a close structural and antigenic mimic of native Env. Large, unprocessed oligomannose-type structures (Man8-9GlcNAc2) are notably prevalent on the gp120 components of the trimer, irrespective of the mammalian cell expression system or the bNAb used for affinity purification. In contrast, gp41 subunits carry more highly processed glycans. The glycans on uncleaved, non-native oligomeric gp140 proteins are also highly processed. A homogeneous, oligomannose-dominated glycan profile is therefore a hallmark of a native Env conformation and a potential Achilles' heel that can be exploited for bNAb recognition and vaccine design.
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http://dx.doi.org/10.1016/j.celrep.2015.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4555872PMC
June 2015

A native-like SOSIP.664 trimer based on an HIV-1 subtype B env gene.

J Virol 2015 Mar 14;89(6):3380-95. Epub 2015 Jan 14.

Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, USA

Unlabelled: Recombinant trimeric mimics of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) spike should expose as many epitopes as possible for broadly neutralizing antibodies (bNAbs) but few, if any, for nonneutralizing antibodies (non-NAbs). Soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A strain BG505 approach this ideal and are therefore plausible vaccine candidates. Here, we report on the production and in vitro properties of a new SOSIP.664 trimer derived from a subtype B env gene, B41, including how to make this protein in low-serum media without proteolytic damage (clipping) to the V3 region. We also show that nonclipped trimers can be purified successfully via a positive-selection affinity column using the bNAb PGT145, which recognizes a quaternary structure-dependent epitope at the trimer apex. Negative-stain electron microscopy imaging shows that the purified, nonclipped, native-like B41 SOSIP.664 trimers contain two subpopulations, which we propose represent an equilibrium between the fully closed and a more open conformation. The latter is different from the fully open, CD4 receptor-bound conformation and may represent an intermediate state of the trimer. This new subtype B trimer adds to the repertoire of native-like Env proteins that are suitable for immunogenicity and structural studies.

Importance: The cleaved, trimeric envelope protein complex is the only neutralizing antibody target on the HIV-1 surface. Many vaccine strategies are based on inducing neutralizing antibodies. For HIV-1, one approach involves using recombinant, soluble protein mimics of the native trimer. At present, the only reliable way to make native-like, soluble trimers in practical amounts is via the introduction of specific sequence changes that confer stability on the cleaved form of Env. The resulting proteins are known as SOSIP.664 gp140 trimers, and the current paradigm is based on the BG505 subtype A env gene. Here, we describe the production and characterization of a SOSIP.664 protein derived from a subtype B gene (B41), together with a simple, one-step method to purify native-like trimers by affinity chromatography with a trimer-specific bNAb, PGT145. The resulting trimers will be useful for structural and immunogenicity experiments aimed at devising ways to make an effective HIV-1 vaccine.
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http://dx.doi.org/10.1128/JVI.03473-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4337520PMC
March 2015

Structural evolution of glycan recognition by a family of potent HIV antibodies.

Cell 2014 Sep;159(1):69-79

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA; International AIDS Vaccine Initiative Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA; Scripps Center for HIV/AIDS Vaccine Immunology & Immunogen Discovery, The Scripps Research Institute, La Jolla, CA 92037, USA; Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. Electronic address:

The HIV envelope glycoprotein (Env) is densely covered with self-glycans that should help shield it from recognition by the human immune system. Here, we examine how a particularly potent family of broadly neutralizing antibodies (Abs) has evolved common and distinct structural features to counter the glycan shield and interact with both glycan and protein components of HIV Env. The inferred germline antibody already harbors potential binding pockets for a glycan and a short protein segment. Affinity maturation then leads to divergent evolutionary branches that either focus on a single glycan and protein segment (e.g., Ab PGT124) or engage multiple glycans (e.g., Abs PGT121-123). Furthermore, other surrounding glycans are avoided by selecting an appropriate initial antibody shape that prevents steric hindrance. Such molecular recognition lessons are important for engineering proteins that can recognize or accommodate glycans.
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http://dx.doi.org/10.1016/j.cell.2014.09.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4278586PMC
September 2014

Stable 293 T and CHO cell lines expressing cleaved, stable HIV-1 envelope glycoprotein trimers for structural and vaccine studies.

Retrovirology 2014 Apr 25;11:33. Epub 2014 Apr 25.

Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY, USA.

Background: Recombinant soluble, cleaved HIV-1 envelope glycoprotein SOSIP.664 gp140 trimers based on the subtype A BG505 sequence are being studied structurally and tested as immunogens in animals. For these trimers to become a vaccine candidate for human trials, they would need to be made in appropriate amounts at an acceptable quality. Accomplishing such tasks by transient transfection is likely to be challenging. The traditional way to express recombinant proteins in large amounts is via a permanent cell line, usually of mammalian origin. Making cell lines that produce BG505 SOSIP.664 trimers requires the co-expression of the Furin protease to ensure that the cleavage site between the gp120 and gp41 subunits is fully utilized.

Results: We designed a vector capable of expressing Env and Furin, and used it to create Stable 293 T and CHO Flp-In™ cell lines through site-specific recombination. Both lines produce high quality, cleaved trimers at yields of up to 12-15 mg per 1 × 109 cells. Trimer expression at such levels was maintained for up to 30 days (10 passages) after initial seeding and was consistently superior to what could be achieved by transient transfection. Electron microscopy studies confirm that the purified trimers have the same native-like appearance as those derived by transient transfection and used to generate high-resolution structures. They also have appropriate antigenic properties, including the presentation of the quaternary epitope for the broadly neutralizing antibody PGT145.

Conclusions: The BG505 SOSIP.664 trimer-expressing cell lines yield proteins of an appropriate quality for structural studies and animal immunogenicity experiments. The methodology is suitable for making similar lines under Good Manufacturing Practice conditions, to produce trimers for human clinical trials. Moreover, any env gene can be incorporated into this vector system, allowing the manufacture of SOSIP trimers from multiple genotypes, either by transient transfection or from stable cell lines.
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http://dx.doi.org/10.1186/1742-4690-11-33DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032163PMC
April 2014

Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies.

Nature 2014 May 2;509(7498):55-62. Epub 2014 Mar 2.

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development.
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http://dx.doi.org/10.1038/nature13036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395007PMC
May 2014

A structurally distinct human mycoplasma protein that generically blocks antigen-antibody union.

Science 2014 Feb;343(6171):656-661

Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

We report the discovery of a broadly reactive antibody-binding protein (Protein M) from human mycoplasma. The crystal structure of the ectodomain of transmembrane Protein M differs from other known protein structures, as does its mechanism of antibody binding. Protein M binds with high affinity to all types of human and nonhuman immunoglobulin G, predominantly through attachment to the conserved portions of the variable region of the κ and λ light chains. Protein M blocks antibody-antigen union, likely because of its large C-terminal domain extending over the antibody-combining site, blocking entry to large antigens. Similar to the other immunoglobulin-binding proteins such as Protein A, Protein M as well as its orthologs in other Mycoplasma species could become invaluable reagents in the antibody field.
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http://dx.doi.org/10.1126/science.1246135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3987992PMC
February 2014

Structural basis for enhanced HIV-1 neutralization by a dimeric immunoglobulin G form of the glycan-recognizing antibody 2G12.

Cell Rep 2013 Dec 5;5(5):1443-55. Epub 2013 Dec 5.

Division of Biology and Biological Engineering 114-96, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA; Howard Hughes Medical Institute, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA. Electronic address:

The human immunoglobulin G (IgG) 2G12 recognizes high-mannose carbohydrates on the HIV type 1 (HIV-1) envelope glycoprotein gp120. Its two antigen-binding fragments (Fabs) are intramolecularly domain exchanged, resulting in a rigid (Fab)2 unit including a third antigen-binding interface not found in antibodies with flexible Fab arms. We determined crystal structures of dimeric 2G12 IgG created by intermolecular domain exchange, which exhibits increased breadth and >50-fold increased neutralization potency compared with monomeric 2G12. The four Fab and two fragment crystalline (Fc) regions of dimeric 2G12 were localized at low resolution in two independent structures, revealing IgG dimers with two (Fab)2 arms analogous to the Fabs of conventional monomeric IgGs. Structures revealed three conformationally distinct dimers, demonstrating flexibility of the (Fab)2-Fc connections that was confirmed by electron microscopy, small-angle X-ray scattering, and binding studies. We conclude that intermolecular domain exchange, flexibility, and bivalent binding to allow avidity effects are responsible for the increased potency and breadth of dimeric 2G12.
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http://dx.doi.org/10.1016/j.celrep.2013.11.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919625PMC
December 2013

Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation.

Proc Natl Acad Sci U S A 2013 Nov 21;110(45):18256-61. Epub 2013 Oct 21.

Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065.

We compare the antigenicity and conformation of soluble, cleaved vs. uncleaved envelope glycoprotein (Env gp)140 trimers from the subtype A HIV type 1 (HIV-1) strain BG505. The impact of gp120-gp41 cleavage on trimer structure, in the presence or absence of trimer-stabilizing modifications (i.e., a gp120-gp41 disulfide bond and an I559P gp41 change, together designated SOSIP), was assessed. Without SOSIP changes, cleaved trimers disintegrate into their gp120 and gp41-ectodomain (gp41ECTO) components; when only the disulfide bond is present, they dissociate into gp140 monomers. Uncleaved gp140s remain trimeric whether SOSIP substitutions are present or not. However, negative-stain electron microscopy reveals that only cleaved trimers form homogeneous structures resembling native Env spikes on virus particles. In contrast, uncleaved trimers are highly heterogeneous, adopting a variety of irregular shapes, many of which appear to be gp120 subunits dangling from a central core that is presumably a trimeric form of gp41ECTO. Antigenicity studies with neutralizing and nonneutralizing antibodies are consistent with the EM images; cleaved, SOSIP-stabilized trimers express quaternary structure-dependent epitopes, whereas uncleaved trimers expose nonneutralizing gp120 and gp41ECTO epitopes that are occluded on cleaved trimers. These findings have adverse implications for using soluble, uncleaved trimers for structural studies, and the rationale for testing uncleaved trimers as vaccine candidates also needs to be reevaluated.
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http://dx.doi.org/10.1073/pnas.1314351110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831437PMC
November 2013

A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies.

PLoS Pathog 2013 Sep 19;9(9):e1003618. Epub 2013 Sep 19.

Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York, United States of America ; Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1) vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs). One approach to the problem is to create trimeric mimics of the native envelope glycoprotein (Env) spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs). Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM). We used several techniques, including ELISA and surface plasmon resonance (SPR), to determine the relationship between the ability of monoclonal antibodies (MAbs) to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145). Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits). Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogens.
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http://dx.doi.org/10.1371/journal.ppat.1003618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3777863PMC
September 2013

Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis.

Proc Natl Acad Sci U S A 2013 Apr 18;110(14):E1263-72. Epub 2013 Mar 18.

Howard Hughes Medical Institute, Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.

Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify the molecular determinants within E3 ligases that modulate activity, we scored each member of a library of nearly 100,000 protein variants of the murine ubiquitination factor E4B (Ube4b) U-box domain for auto-ubiquitination activity in the presence of the E2 UbcH5c. This assay identified mutations that enhance activity both in vitro and in cellular p53 degradation assays. The activity-enhancing mutations fall into two distinct mechanistic classes: One increases the U-box:E2-binding affinity, and the other allosterically stimulates the formation of catalytically active conformations of the E2∼Ub conjugate. The same mutations enhance E3 activity in the presence of another E2, Ube2w, implying a common allosteric mechanism, and therefore the general applicability of our observations to other E3s. A comparison of the E3 activity with the two different E2s identified an additional variant that exhibits E3:E2 specificity. Our results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity.
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http://dx.doi.org/10.1073/pnas.1303309110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3619334PMC
April 2013

Clinical challenges: 18 year old male with bloody diarrhea.

J Pediatr 2005 Aug;147(2):267-70

Department of Pediatrics, Division of Gastroenterology, University of Pittsburgh School of Medicine and Children's Hospital of Pittsburgh, PA 15213, USA.

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http://dx.doi.org/10.1016/j.jpeds.2005.04.018DOI Listing
August 2005