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2021-22 Courses


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  • Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice. Science (New York, N.Y.) Cohen, A. A., Gnanapragasam, P. N., Lee, Y. E., Hoffman, P. R., Ou, S., Kakutani, L. M., Keeffe, J. R., Wu, H. J., Howarth, M., West, A. P., Barnes, C. O., Nussenzweig, M. C., Bjorkman, P. J. 2021; 371 (6530): 735-741

    Abstract

    Protection against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and SARS-related emergent zoonotic coronaviruses is urgently needed. We made homotypic nanoparticles displaying the receptor binding domain (RBD) of SARS-CoV-2 or co-displaying SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles with four to eight distinct RBDs). Mice immunized with RBD nanoparticles, but not soluble antigen, elicited cross-reactive binding and neutralization responses. Mosaic RBD nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs relative to sera from immunizations with homotypic SARS-CoV-2-RBD nanoparticles or COVID-19 convalescent human plasmas. Moreover, after priming, sera from mosaic RBD-immunized mice neutralized heterologous pseudotyped coronaviruses as well as or better than sera from homotypic SARS-CoV-2-RBD nanoparticle immunizations, demonstrating no loss of immunogenicity against particular RBDs resulting from co-display. A single immunization with mosaic RBD nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses.

    View details for DOI 10.1126/science.abf6840

    View details for PubMedID 33436524

    View details for PubMedCentralID PMC7928838

  • Naturally enhanced neutralizing breadth against SARS-CoV-2 one year after infection. Nature Wang, Z., Muecksch, F., Schaefer-Babajew, D., Finkin, S., Viant, C., Gaebler, C., Hoffmann, H. H., Barnes, C. O., Cipolla, M., Ramos, V., Oliveira, T. Y., Cho, A., Schmidt, F., da Silva, J., Bednarski, E., Aguado, L., Yee, J., Daga, M., Turroja, M., Millard, K. G., Jankovic, M., Gazumyan, A., Zhao, Z., Rice, C. M., Bieniasz, P. D., Caskey, M., Hatziioannou, T., Nussenzweig, M. C. 2021

    Abstract

    Over one year after its inception, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several excellent vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies1,2. Here we report on a cohort of 63 COVID-19-convalescent individuals assessed at 1.3, 6.2 and 12 months after infection, 41% of whom also received mRNA vaccines3,4. In the absence of vaccination antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable from 6 to 12 months. Vaccination increases all components of the humoral response, and as expected, results in serum neutralizing activities against variants of concern that are comparable to or greater than neutralizing activity against the original Wuhan Hu-1 achieved by vaccination of naive individuals2,5-8. The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover, and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in variants of concern4,9. In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand dramatically after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.

    View details for DOI 10.1038/s41586-021-03696-9

    View details for PubMedID 34126625

  • B cell genomics behind cross-neutralization of SARS-CoV-2 variants and SARS-CoV. Cell Scheid, J. F., Barnes, C. O., Eraslan, B., Hudak, A., Keeffe, J. R., Cosimi, L. A., Brown, E. M., Muecksch, F., Weisblum, Y., Zhang, S., Delorey, T., Woolley, A. E., Ghantous, F., Park, S. M., Phillips, D., Tusi, B., Huey-Tubman, K. E., Cohen, A. A., Gnanapragasam, P. N., Rzasa, K., Hatziioanno, T., Durney, M. A., Gu, X., Tada, T., Landau, N. R., West, A. P., Rozenblatt-Rosen, O., Seaman, M. S., Baden, L. R., Graham, D. B., Deguine, J., Bieniasz, P. D., Regev, A., Hung, D., Bjorkman, P. J., Xavier, R. J. 2021; 184 (12): 3205-3221.e24

    Abstract

    Monoclonal antibodies (mAbs) are a focus in vaccine and therapeutic design to counteract severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. Here, we combined B cell sorting with single-cell VDJ and RNA sequencing (RNA-seq) and mAb structures to characterize B cell responses against SARS-CoV-2. We show that the SARS-CoV-2-specific B cell repertoire consists of transcriptionally distinct B cell populations with cells producing potently neutralizing antibodies (nAbs) localized in two clusters that resemble memory and activated B cells. Cryo-electron microscopy structures of selected nAbs from these two clusters complexed with SARS-CoV-2 spike trimers show recognition of various receptor-binding domain (RBD) epitopes. One of these mAbs, BG10-19, locks the spike trimer in a closed conformation to potently neutralize SARS-CoV-2, the recently arising mutants B.1.1.7 and B.1.351, and SARS-CoV and cross-reacts with heterologous RBDs. Together, our results characterize transcriptional differences among SARS-CoV-2-specific B cells and uncover cross-neutralizing Ab targets that will inform immunogen and therapeutic design against coronaviruses.

    View details for DOI 10.1016/j.cell.2021.04.032

    View details for PubMedID 34015271

    View details for PubMedCentralID PMC8064835

  • mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants. Nature Wang, Z., Schmidt, F., Weisblum, Y., Muecksch, F., Barnes, C. O., Finkin, S., Schaefer-Babajew, D., Cipolla, M., Gaebler, C., Lieberman, J. A., Oliveira, T. Y., Yang, Z., Abernathy, M. E., Huey-Tubman, K. E., Hurley, A., Turroja, M., West, K. A., Gordon, K., Millard, K. G., Ramos, V., Da Silva, J., Xu, J., Colbert, R. A., Patel, R., Dizon, J., Unson-O'Brien, C., Shimeliovich, I., Gazumyan, A., Caskey, M., Bjorkman, P. J., Casellas, R., Hatziioannou, T., Bieniasz, P. D., Nussenzweig, M. C. 2021; 592 (7855): 616-622

    Abstract

    Here we report on the antibody and memory B cell responses of a cohort of 20 volunteers who received the Moderna (mRNA-1273) or Pfizer-BioNTech (BNT162b2) vaccine against SARS-CoV-21-4. Eight weeks after the second injection of vaccine, volunteers showed high levels of IgM and IgG anti-SARS-CoV-2 spike protein (S) and receptor-binding-domain (RBD) binding titre. Moreover, the plasma neutralizing activity and relative numbers of RBD-specific memory B cells of vaccinated volunteers were equivalent to those of individuals who had recovered from natural infection5,6. However, activity against SARS-CoV-2 variants that encode E484K-, N501Y- or K417N/E484K/N501-mutant S was reduced by a small-but significant-margin. The monoclonal antibodies elicited by the vaccines potently neutralize SARS-CoV-2, and target a number of different RBD epitopes in common with monoclonal antibodies isolated from infected donors5-8. However, neutralization by 14 of the 17 most-potent monoclonal antibodies that we tested was reduced or abolished by the K417N, E484K or N501Y mutation. Notably, these mutations were selected when we cultured recombinant vesicular stomatitis virus expressing SARS-CoV-2 S in the presence of the monoclonal antibodies elicited by the vaccines. Together, these results suggest that the monoclonal antibodies in clinical use should be tested against newly arising variants, and that mRNA vaccines may need to be updated periodically to avoid a potential loss of clinical efficacy.

    View details for DOI 10.1038/s41586-021-03324-6

    View details for PubMedID 33567448

  • Convergent antibody responses to SARS-CoV-2 in convalescent individuals. Nature Robbiani, D. F., Gaebler, C., Muecksch, F., Lorenzi, J. C., Wang, Z., Cho, A., Agudelo, M., Barnes, C. O., Gazumyan, A., Finkin, S., Hägglöf, T., Oliveira, T. Y., Viant, C., Hurley, A., Hoffmann, H. H., Millard, K. G., Kost, R. G., Cipolla, M., Gordon, K., Bianchini, F., Chen, S. T., Ramos, V., Patel, R., Dizon, J., Shimeliovich, I., Mendoza, P., Hartweger, H., Nogueira, L., Pack, M., Horowitz, J., Schmidt, F., Weisblum, Y., Michailidis, E., Ashbrook, A. W., Waltari, E., Pak, J. E., Huey-Tubman, K. E., Koranda, N., Hoffman, P. R., West, A. P., Rice, C. M., Hatziioannou, T., Bjorkman, P. J., Bieniasz, P. D., Caskey, M., Nussenzweig, M. C. 2020; 584 (7821): 437-442

    Abstract

    During the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has led to the infection of millions of people and has claimed hundreds of thousands of lives. The entry of the virus into cells depends on the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Although there is currently no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-21-5. Here we report on 149 COVID-19-convalescent individuals. Plasma samples collected an average of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titres; titres were less than 50 in 33% of samples, below 1,000 in 79% of samples and only 1% of samples had titres above 5,000. Antibody sequencing revealed the expansion of clones of RBD-specific memory B cells that expressed closely related antibodies in different individuals. Despite low plasma titres, antibodies to three distinct epitopes on the RBD neutralized the virus with half-maximal inhibitory concentrations (IC50 values) as low as 2 ng ml-1. In conclusion, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.

    View details for DOI 10.1038/s41586-020-2456-9

    View details for PubMedID 32555388

    View details for PubMedCentralID PMC7442695

  • De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2. Science (New York, N.Y.) Linsky, T. W., Vergara, R., Codina, N., Nelson, J. W., Walker, M. J., Su, W., Barnes, C. O., Hsiang, T. Y., Esser-Nobis, K., Yu, K., Reneer, Z. B., Hou, Y. J., Priya, T., Mitsumoto, M., Pong, A., Lau, U. Y., Mason, M. L., Chen, J., Chen, A., Berrocal, T., Peng, H., Clairmont, N. S., Castellanos, J., Lin, Y. R., Josephson-Day, A., Baric, R. S., Fuller, D. H., Walkey, C. D., Ross, T. M., Swanson, R., Bjorkman, P. J., Gale, M., Blancas-Mejia, L. M., Yen, H. L., Silva, D. A. 2020; 370 (6521): 1208-1214

    Abstract

    We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo human angiotensin-converting enzyme 2 (hACE2) decoys to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The best monovalent decoy, CTC-445.2, bound with low nanomolar affinity and high specificity to the receptor-binding domain (RBD) of the spike protein. Cryo-electron microscopy (cryo-EM) showed that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, showed ~10-fold improvement in binding. CTC-445.2d potently neutralized SARS-CoV-2 infection of cells in vitro, and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge.

    View details for DOI 10.1126/science.abe0075

    View details for PubMedID 33154107

    View details for PubMedCentralID PMC7920261

  • A broadly neutralizing macaque monoclonal antibody against the HIV-1 V3-Glycan patch. eLife Wang, Z., Barnes, C. O., Gautam, R., Cetrulo Lorenzi, J. C., Mayer, C. T., Oliveira, T. Y., Ramos, V., Cipolla, M., Gordon, K. M., Gristick, H. B., West, A. P., Nishimura, Y., Raina, H., Seaman, M. S., Gazumyan, A., Martin, M., Bjorkman, P. J., Nussenzweig, M. C., Escolano, A. 2020; 9

    Abstract

    A small fraction of HIV-1- infected humans develop broadly neutralizing antibodies (bNAbs) against HIV-1 that protect macaques from simian immunodeficiency HIV chimeric virus (SHIV). Similarly, a small number of macaques infected with SHIVs develop broadly neutralizing serologic activity, but less is known about the nature of simian antibodies. Here, we report on a monoclonal antibody, Ab1485, isolated from a macaque infected with SHIVAD8 that developed broadly neutralizing serologic activity targeting the V3-glycan region of HIV-1 Env. Ab1485 neutralizes 38.1% of HIV-1 isolates in a 42-pseudovirus panel with a geometric mean IC50 of 0.055 µg/mLl and SHIVAD8 with an IC50 of 0.028 µg/mLl. Ab1485 binds the V3-glycan epitope in a glycan-dependent manner. A 3.5 Å cryo-electron microscopy structure of Ab1485 in complex with a native-like SOSIP Env trimer showed conserved contacts with the N332gp120 glycan and gp120 GDIR peptide motif, but in a distinct Env-binding orientation relative to human V3/N332gp120 glycan-targeting bNAbs. Intravenous infusion of Ab1485 protected macaques from a high dose challenge with SHIVAD8. We conclude that macaques can develop bNAbs against the V3-glycan patch that resemble human V3-glycan bNAbs.

    View details for DOI 10.7554/eLife.61991

    View details for PubMedID 33084569

    View details for PubMedCentralID PMC7577740

  • SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies. Nature Barnes, C. O., Jette, C. A., Abernathy, M. E., Dam, K. A., Esswein, S. R., Gristick, H. B., Malyutin, A. G., Sharaf, N. G., Huey-Tubman, K. E., Lee, Y. E., Robbiani, D. F., Nussenzweig, M. C., West, A. P., Bjorkman, P. J. 2020; 588 (7839): 682-687

    Abstract

    The coronavirus disease 2019 (COVID-19) pandemic presents an urgent health crisis. Human neutralizing antibodies that target the host ACE2 receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein1-5 show promise therapeutically and are being evaluated clinically6-8. Here, to identify the structural correlates of SARS-CoV-2 neutralization, we solved eight new structures of distinct COVID-19 human neutralizing antibodies5 in complex with the SARS-CoV-2 spike trimer or RBD. Structural comparisons allowed us to classify the antibodies into categories: (1) neutralizing antibodies encoded by the VH3-53 gene segment with short CDRH3 loops that block ACE2 and bind only to 'up' RBDs; (2) ACE2-blocking neutralizing antibodies that bind both up and 'down' RBDs and can contact adjacent RBDs; (3) neutralizing antibodies that bind outside the ACE2 site and recognize both up and down RBDs; and (4) previously described antibodies that do not block ACE2 and bind only to up RBDs9. Class 2 contained four neutralizing antibodies with epitopes that bridged RBDs, including a VH3-53 antibody that used a long CDRH3 with a hydrophobic tip to bridge between adjacent down RBDs, thereby locking the spike into a closed conformation. Epitope and paratope mapping revealed few interactions with host-derived N-glycans and minor contributions of antibody somatic hypermutations to epitope contacts. Affinity measurements and mapping of naturally occurring and in vitro-selected spike mutants in 3D provided insight into the potential for SARS-CoV-2 to escape from antibodies elicited during infection or delivered therapeutically. These classifications and structural analyses provide rules for assigning current and future human RBD-targeting antibodies into classes, evaluating avidity effects and suggesting combinations for clinical use, and provide insight into immune responses against SARS-CoV-2.

    View details for DOI 10.1038/s41586-020-2852-1

    View details for PubMedID 33045718

    View details for PubMedCentralID PMC8092461

  • Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies. Cell Barnes, C. O., West, A. P., Huey-Tubman, K. E., Hoffmann, M. A., Sharaf, N. G., Hoffman, P. R., Koranda, N., Gristick, H. B., Gaebler, C., Muecksch, F., Lorenzi, J. C., Finkin, S., Hägglöf, T., Hurley, A., Millard, K. G., Weisblum, Y., Schmidt, F., Hatziioannou, T., Bieniasz, P. D., Caskey, M., Robbiani, D. F., Nussenzweig, M. C., Bjorkman, P. J. 2020; 182 (4): 828-842.e16

    Abstract

    Neutralizing antibody responses to coronaviruses mainly target the receptor-binding domain (RBD) of the trimeric spike. Here, we characterized polyclonal immunoglobulin Gs (IgGs) and Fabs from COVID-19 convalescent individuals for recognition of coronavirus spikes. Plasma IgGs differed in their focus on RBD epitopes, recognition of alpha- and beta-coronaviruses, and contributions of avidity to increased binding/neutralization of IgGs over Fabs. Using electron microscopy, we examined specificities of polyclonal plasma Fabs, revealing recognition of both S1A and RBD epitopes on SARS-CoV-2 spike. Moreover, a 3.4 Å cryo-electron microscopy (cryo-EM) structure of a neutralizing monoclonal Fab-spike complex revealed an epitope that blocks ACE2 receptor binding. Modeling based on these structures suggested different potentials for inter-spike crosslinking by IgGs on viruses, and characterized IgGs would not be affected by identified SARS-CoV-2 spike mutations. Overall, our studies structurally define a recurrent anti-SARS-CoV-2 antibody class derived from VH3-53/VH3-66 and similarity to a SARS-CoV VH3-30 antibody, providing criteria for evaluating vaccine-elicited antibodies.

    View details for DOI 10.1016/j.cell.2020.06.025

    View details for PubMedID 32645326

    View details for PubMedCentralID PMC7311918

  • The crystal structure of dGTPase reveals the molecular basis of dGTP selectivity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Barnes, C. O., Wu, Y., Song, J., Lin, G., Baxter, E. L., Brewster, A. S., Nagarajan, V., Holmes, A., Soltis, S., Sauter, N. K., Ahn, J., Cohen, A. E., Calero, G. 2019; 116 (19): 9333-9339

    Abstract

    Deoxynucleotide triphosphohydrolases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools. While the vast majority of these enzymes display broad activity toward canonical dNTPs, such as the dNTPase SAMHD1 that blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric Ec-dGTPase, including an X-ray free-electron laser structure of the free Ec-dGTPase enzyme to 3.2 Å. To obtain this structure, we developed a method that applied UV-fluorescence microscopy, video analysis, and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly located on fixed target holders, resulting in the highest indexing rates observed for a serial femtosecond crystallography experiment. Our structures show a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo- form. Moreover, despite no sequence homology, Ec-dGTPase and SAMHD1 share similar active-site and HD motif architectures; however, Ec-dGTPase residues at the end of the substrate-binding pocket mimic Watson-Crick interactions providing guanine base specificity, while a 7-Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures shed light on the mechanism by which long distance binding (25 Å) of single-stranded DNA in an allosteric site primes the active site by conformationally "opening" a tyrosine gate allowing enhanced substrate binding.

    View details for DOI 10.1073/pnas.1814999116

    View details for Web of Science ID 000467226400032

    View details for PubMedID 31019074

    View details for PubMedCentralID PMC6511015

  • Broad and Potent Neutralizing Antibodies Recognize the Silent Face of the HIV Envelope. Immunity Schoofs, T., Barnes, C. O., Suh-Toma, N., Golijanin, J., Schommers, P., Gruell, H., West, A. P., Bach, F., Lee, Y. E., Nogueira, L., Georgiev, I. S., Bailer, R. T., Czartoski, J., Mascola, J. R., Seaman, M. S., McElrath, M. J., Doria-Rose, N. A., Klein, F., Nussenzweig, M. C., Bjorkman, P. J. 2019; 50 (6): 1513-1529.e9

    Abstract

    Broadly neutralizing antibodies (bNAbs) against HIV-1 envelope (Env) inform vaccine design and are potential therapeutic agents. We identified SF12 and related bNAbs with up to 62% neutralization breadth from an HIV-infected donor. SF12 recognized a glycan-dominated epitope on Env's silent face and was potent against clade AE viruses, which are poorly covered by V3-glycan bNAbs. A 3.3Å cryo-EM structure of a SF12-Env trimer complex showed additional contacts to Env protein residues by SF12 compared with VRC-PG05, the only other known donor-derived silentface antibody, explaining SF12's increased neutralization breadth, potency, and resistance to Env mutation routes. Asymmetric binding of SF12 was associated with distinct N-glycan conformations across Env protomers, demonstrating intra-Env glycan heterogeneity. Administrating SF12 to HIV-1-infected humanized mice suppressed viremia and selected for viruses lacking the N448gp120 glycan. Effective bNAbs can therefore be raised against HIV-1 Env's silent face, suggesting their potential for HIV-1 prevention, therapy, and vaccine development.

    View details for DOI 10.1016/j.immuni.2019.04.014

    View details for PubMedID 31126879

    View details for PubMedCentralID PMC6591006

  • Immunization expands B cells specific to HIV-1 V3 glycan in mice and macaques. Nature Escolano, A., Gristick, H. B., Abernathy, M. E., Merkenschlager, J., Gautam, R., Oliveira, T. Y., Pai, J., West, A. P., Barnes, C. O., Cohen, A. A., Wang, H., Golijanin, J., Yost, D., Keeffe, J. R., Wang, Z., Zhao, P., Yao, K. H., Bauer, J., Nogueira, L., Gao, H., Voll, A. V., Montefiori, D. C., Seaman, M. S., Gazumyan, A., Silva, M., McGuire, A. T., Stamatatos, L., Irvine, D. J., Wells, L., Martin, M. A., Bjorkman, P. J., Nussenzweig, M. C. 2019; 570 (7762): 468-473

    Abstract

    Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires.

    View details for DOI 10.1038/s41586-019-1250-z

    View details for PubMedID 31142836

    View details for PubMedCentralID PMC6657810

  • Structural characterization of a highly-potent V3-glycan broadly neutralizing antibody bound to natively-glycosylated HIV-1 envelope NATURE COMMUNICATIONS Barnes, C. O., Gristick, H. B., Freund, N. T., Escolano, A., Lyubimov, A. Y., Hartweger, H., West, A. P., Cohen, A. E., Nussenzweig, M. C., Bjorkman, P. J. 2018; 9: 1251

    Abstract

    Broadly neutralizing antibodies (bNAbs) isolated from HIV-1-infected individuals inform HIV-1 vaccine design efforts. Developing bNAbs with increased efficacy requires understanding how antibodies interact with the native oligomannose and complex-type N-glycan shield that hides most protein epitopes on HIV-1 envelope (Env). Here we present crystal structures, including a 3.8-Å X-ray free electron laser dataset, of natively glycosylated Env trimers complexed with BG18, the most potent V3/N332gp120 glycan-targeting bNAb reported to date. Our structures show conserved contacts mediated by common D gene-encoded residues with the N332gp120 glycan and the gp120 GDIR peptide motif, but a distinct Env-binding orientation relative to PGT121/10-1074 bNAbs. BG18's binding orientation provides additional contacts with N392gp120 and N386gp120 glycans near the V3-loop base and engages protein components of the V1-loop. The BG18-natively-glycosylated Env structures facilitate understanding of bNAb-glycan interactions critical for using V3/N332gp120 bNAbs therapeutically and targeting their epitope for immunogen design.

    View details for DOI 10.1038/s41467-018-03632-y

    View details for Web of Science ID 000428520600006

    View details for PubMedID 29593217

    View details for PubMedCentralID PMC5871869

  • Partially Open HIV-1 Envelope Structures Exhibit Conformational Changes Relevant for Coreceptor Binding and Fusion. Cell host & microbe Wang, H., Barnes, C. O., Yang, Z., Nussenzweig, M. C., Bjorkman, P. J. 2018; 24 (4): 579-592.e4

    Abstract

    HIV-1 Env, a trimer of gp120-gp41 heterodimers, mediates membrane fusion after binding host receptor CD4. Receptor binding displaces V1V2 loops from Env's apex, allowing coreceptor binding and opening Env to enable gp41-mediated fusion. We present 3.54 Å and 4.06 Å cryoelectron microscopy structures of partially open soluble native-like Env trimers (SOSIPs) bound to CD4. One structure, a complex with a coreceptor-mimicking antibody that binds both CD4 and gp120, stabilizes the displaced V1V2 and reveals its structure. Comparing partially and fully open Envs with closed Envs shows that gp41 rearrangements are independent of the CD4-induced rearrangements that result in V1V2 displacement and formation of a 4-stranded bridging sheet. These findings suggest ordered conformational changes before coreceptor binding: (1) gp120 opening inducing side-chain rearrangements and a compact gp41 central helix conformation, and (2) 4-stranded bridging-sheet formation and V1V2 displacement. These analyses illuminate potential receptor-induced Env changes and inform design of therapeutics disrupting viral entry.

    View details for DOI 10.1016/j.chom.2018.09.003

    View details for PubMedID 30308160

    View details for PubMedCentralID PMC6185872

  • The DDB1-DCAF1-Vpr-UNG2 crystal structure reveals how HIV-1 Vpr steers human UNG2 toward destruction NATURE STRUCTURAL & MOLECULAR BIOLOGY Wu, Y., Zhou, X., Barnes, C. O., DeLucia, M., Cohen, A. E., Gronenborn, A. M., Ahn, J., Calero, G. 2016; 23 (10): 933-940

    Abstract

    The HIV-1 accessory protein Vpr is required for efficient viral infection of macrophages and promotion of viral replication in T cells. Vpr's biological activities are closely linked to the interaction with human DCAF1, a cellular substrate receptor of the Cullin4-RING E3 ubiquitin ligase (CRL4) of the host ubiquitin-proteasome-mediated protein degradation pathway. The molecular details of how Vpr usurps the protein degradation pathway have not been delineated. Here we present the crystal structure of the DDB1-DCAF1-HIV-1-Vpr-uracil-DNA glycosylase (UNG2) complex. The structure reveals how Vpr engages with DCAF1, creating a binding interface for UNG2 recruitment in a manner distinct from the recruitment of SAMHD1 by Vpx proteins. Vpr and Vpx use similar N-terminal and helical regions to bind the substrate receptor, whereas different regions target the specific cellular substrates. Furthermore, Vpr uses molecular mimicry of DNA by a variable loop for specific recruitment of the UNG2 substrate.

    View details for DOI 10.1038/nsmb.3284

    View details for Web of Science ID 000384964500011

    View details for PubMedID 27571178

    View details for PubMedCentralID PMC5385928

  • Assessment of microcrystal quality by transmission electron microscopy for efficient serial femtosecond crystallography ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Barnes, C. O., Kovaleva, E. G., Fu, X., Stevenson, H. P., Brewster, A. S., Deponte, D. P., Baxter, E. L., Cohen, A. E., Calero, G. 2016; 602: 61-68

    Abstract

    Serial femtosecond crystallography (SFX) employing high-intensity X-ray free-electron laser (XFEL) sources has enabled structural studies on microcrystalline protein samples at non-cryogenic temperatures. However, the identification and optimization of conditions that produce well diffracting microcrystals remains an experimental challenge. Here, we report parallel SFX and transmission electron microscopy (TEM) experiments using fragmented microcrystals of wild type (WT) homoprotocatechuate 2,3-dioxygenase (HPCD) and an active site variant (H200Q). Despite identical crystallization conditions and morphology, as well as similar crystal size and density, the indexing efficiency of the diffraction data collected using the H200Q variant sample was over 7-fold higher compared to the diffraction results obtained using the WT sample. TEM analysis revealed an abundance of protein aggregates, crystal conglomerates and a smaller population of highly ordered lattices in the WT sample as compared to the H200Q variant sample. While not reported herein, the 1.75 Å resolution structure of the H200Q variant was determined from ∼16 min of beam time, demonstrating the utility of TEM analysis in evaluating sample monodispersity and lattice quality, parameters critical to the efficiency of SFX experiments.

    View details for DOI 10.1016/j.abb.2016.02.011

    View details for Web of Science ID 000378364900007

    View details for PubMedID 26944553

  • Transmission electron microscopy for the evaluation and optimization of crystal growth ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY Stevenson, H. P., Lin, G., Barnes, C. O., Sutkeviciute, I., Krzysiak, T., Weiss, S. C., Reynolds, S., Wu, Y., Nagarajan, V., Makhov, A. M., Lawrence, R., Lamm, E., Clark, L., Gardella, T. J., Hogue, B. G., Ogata, C. M., Ahn, J., Gronenborn, A. M., Conway, J. F., Vilardaga, J., Cohen, A. E., Calero, G. 2016; 72: 603-615

    Abstract

    The crystallization of protein samples remains the most significant challenge in structure determination by X-ray crystallography. Here, the effectiveness of transmission electron microscopy (TEM) analysis to aid in the crystallization of biological macromolecules is demonstrated. It was found that the presence of well ordered lattices with higher order Bragg spots, revealed by Fourier analysis of TEM images, is a good predictor of diffraction-quality crystals. Moreover, the use of TEM allowed (i) comparison of lattice quality among crystals from different conditions in crystallization screens; (ii) the detection of crystal pathologies that could contribute to poor X-ray diffraction, including crystal lattice defects, anisotropic diffraction and crystal contamination by heavy protein aggregates and nanocrystal nuclei; (iii) the qualitative estimation of crystal solvent content to explore the effect of lattice dehydration on diffraction and (iv) the selection of high-quality crystal fragments for microseeding experiments to generate reproducibly larger sized crystals. Applications to X-ray free-electron laser (XFEL) and micro-electron diffraction (microED) experiments are also discussed.

    View details for DOI 10.1107/S2059798316001546

    View details for Web of Science ID 000375851700002

    View details for PubMedID 27139624

  • High-density grids for efficient data collection from multiple crystals. Acta crystallographica. Section D, Structural biology Baxter, E. L., Aguila, L., Alonso-Mori, R., Barnes, C. O., Bonagura, C. A., Brehmer, W., Brunger, A. T., Calero, G., Caradoc-Davies, T. T., Chatterjee, R., DeGrado, W. F., Fraser, J. S., Ibrahim, M., Kern, J., Kobilka, B. K., Kruse, A. C., Larsson, K. M., Lemke, H. T., Lyubimov, A. Y., Manglik, A., McPhillips, S. E., Norgren, E., Pang, S. S., Soltis, S. M., Song, J., Thomaston, J., Tsai, Y., Weis, W. I., Woldeyes, R. A., Yachandra, V., Yano, J., Zouni, A., Cohen, A. E. 2016; 72: 2-11

    Abstract

    Higher throughput methods to mount and collect data from multiple small and radiation-sensitive crystals are important to support challenging structural investigations using microfocus synchrotron beamlines. Furthermore, efficient sample-delivery methods are essential to carry out productive femtosecond crystallography experiments at X-ray free-electron laser (XFEL) sources such as the Linac Coherent Light Source (LCLS). To address these needs, a high-density sample grid useful as a scaffold for both crystal growth and diffraction data collection has been developed and utilized for efficient goniometer-based sample delivery at synchrotron and XFEL sources. A single grid contains 75 mounting ports and fits inside an SSRL cassette or uni-puck storage container. The use of grids with an SSRL cassette expands the cassette capacity up to 7200 samples. Grids may also be covered with a polymer film or sleeve for efficient room-temperature data collection from multiple samples. New automated routines have been incorporated into the Blu-Ice/DCSS experimental control system to support grids, including semi-automated grid alignment, fully automated positioning of grid ports, rastering and automated data collection. Specialized tools have been developed to support crystallization experiments on grids, including a universal adaptor, which allows grids to be filled by commercial liquid-handling robots, as well as incubation chambers, which support vapor-diffusion and lipidic cubic phase crystallization experiments. Experiments in which crystals were loaded into grids or grown on grids using liquid-handling robots and incubation chambers are described. Crystals were screened at LCLS-XPP and SSRL BL12-2 at room temperature and cryogenic temperatures.

    View details for DOI 10.1107/S2059798315020847

    View details for PubMedID 26894529

    View details for PubMedCentralID PMC4756618

  • Crystal Structure of a Transcribing RNA Polymerase II Complex Reveals a Complete Transcription Bubble MOLECULAR CELL Barnes, C. O., Calero, M., Malik, I., Graham, B. W., Spahr, H., Lin, G., Cohen, A. E., Brown, I. S., Zhang, Q., Pullara, F., Trakselis, M. A., Kaplan, C. D., Calero, G. 2015; 59 (2): 258–69

    Abstract

    Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF-stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop 1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the trigger loop (TL), allowing visualization of its open state. Overall, our observations suggest that "open/closed" conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation.

    View details for PubMedID 26186291

  • Goniometer-based femtosecond crystallography with X-ray free electron lasers PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cohen, A. E., Soltis, S. M., Gonzalez, A., Aguila, L., Alonso-Mori, R., Barnes, C. O., Baxter, E. L., Brehmer, W., Brewster, A. S., Brunger, A. T., Calero, G., Chang, J. F., Chollet, M., Ehrensberger, P., Eriksson, T. L., Feng, Y., Hattne, J., Hedman, B., Hollenbeck, M., Holton, J. M., Keable, S., Kobilka, B. K., Kovaleva, E. G., Kruse, A. C., Lemke, H. T., Lin, G., Lyubimov, A. Y., Manglik, A., Mathews, I. I., McPhillips, S. E., Nelson, S., Peters, J. W., Sauter, N. K., Smith, C. A., Song, J., Stevenson, H. P., Tsai, Y., Uervirojnangkoorn, M., Vinetsky, V., Wakatsuki, S., Weis, W. I., Zadvornyy, O. A., Zeldin, O. B., Zhu, D., Hodgson, K. O. 2014; 111 (48): 17122-17127

    Abstract

    The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.

    View details for DOI 10.1073/pnas.1418733111

    View details for Web of Science ID 000345920800042

    View details for PubMedID 25362050

    View details for PubMedCentralID PMC4260607

  • Use of transmission electron microscopy to identify nanocrystals of challenging protein targets PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Stevenson, H. P., Makhov, A. M., Calero, M., Edwards, A. L., Zeldin, O. B., Mathews, I. I., Lin, G., Barnes, C. O., Santamaria, H., Ross, T. M., Soltis, S. M., Khosla, C., Nagarajan, V., Conway, J. F., Cohen, A. E., Calero, G. 2014; 111 (23): 8470-8475

    Abstract

    The current practice for identifying crystal hits for X-ray crystallography relies on optical microscopy techniques that are limited to detecting crystals no smaller than 5 μm. Because of these limitations, nanometer-sized protein crystals cannot be distinguished from common amorphous precipitates, and therefore go unnoticed during screening. These crystals would be ideal candidates for further optimization or for femtosecond X-ray protein nanocrystallography. The latter technique offers the possibility to solve high-resolution structures using submicron crystals. Transmission electron microscopy (TEM) was used to visualize nanocrystals (NCs) found in crystallization drops that would classically not be considered as "hits." We found that protein NCs were readily detected in all samples tested, including multiprotein complexes and membrane proteins. NC quality was evaluated by TEM visualization of lattices, and diffraction quality was validated by experiments in an X-ray free electron laser.

    View details for DOI 10.1073/pnas.1400240111

    View details for Web of Science ID 000336976000051

    View details for PubMedID 24872454

    View details for PubMedCentralID PMC4060711