240 results
Search Results
2. Abstracts of Papers Presented at the Annual Meeting of the National Academy of Sciences (USA), Washington, D.C., 23-25 April 1973
- Author
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Breit, G., Tischler, M., Mukherjee, S., Pappas, G., Damon, Paul E., Hammel, H. T., Scholander, P. F., Knopoff, L., Bukowinski, M., Kristian, Jerome, Orowan, E., Sabin, Albert B., Tarro, Giulio, Schockley, W., and Szent-Gyorgyi, Albert
- Published
- 1973
3. Direct Immunoassay for Detecting Escherichia coli Colonies That Contain Polypeptides Encoded by Cloned DNA Segments
- Author
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Kemp, David J. and Cowman, Alan F.
- Published
- 1981
4. Anti-Immune Complex Antibodies Enhance Affinity and Specificity of Primary Antibodies
- Author
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Ullman, Edwin F., Milburn, Gary, Jelesko, John, Radika, Kesavan, Pirio, Marcel, Kempe, Thomas, and Skold, Carl
- Published
- 1993
5. Vaccines for Bacterial Sexually Transmitted Infections: A Realistic Goal?
- Author
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Sparling, P. Frederick, Elkins, Christopher, Wyrick, Priscilla B., and Cohen, Myron S.
- Published
- 1994
6. Impact of Malaria on Genetic Polymorphism and Genetic Diseases in Africans and African Americans
- Author
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Miller, Louis H.
- Published
- 1994
7. Molecular Genetics of Speciation and Human Origins
- Author
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Ayala, Francisco J., Escalante, Ananias, O'hUigin, Colm, and Klein, Jan
- Published
- 1994
8. A Genetic Approach to the Generation of Antibodies with Enhanced Catalytic Activities
- Author
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Lesley, Scott A., Patten, Phillip A., and Schultz, Peter G.
- Published
- 1993
9. Mechanism of the Enzymatic Synthesis of Cardiolipin in Escherichia coli
- Author
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Hirschberg, Carlos B. and Kennedy, Eugene P.
- Published
- 1972
10. A Comparison of the Tryptic Peptides Obtained from Immobilization Antigens of Paramecium aurelia
- Author
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Steers, Edward
- Published
- 1962
11. The Enzymatic Synthesis of Guanosine Diphosphate Colitose by a Mutant Strain of Escherichia coli
- Author
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Heath, Edward C. and Elbein, Alan D.
- Published
- 1962
12. The Capsular Polysaccharide of a Mucoid Variant of E. coli K12
- Author
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Sapelli, Rose V. and Goebel, Walther F.
- Published
- 1964
13. Antigenicity-defined conformations of an extremely neutralization-resistant HIV-1 envelope spike.
- Author
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Cai Y, Karaca-Griffin S, Chen J, Tian S, Fredette N, Linton CE, Rits-Volloch S, Lu J, Wagh K, Theiler J, Korber B, Seaman MS, Harrison SC, Carfi A, and Chen B
- Subjects
- Antibodies, Monoclonal, Antibodies, Neutralizing, Antigens chemistry, Epitopes, HEK293 Cells, HIV Antibodies immunology, HIV Envelope Protein gp120 immunology, HIV-1 genetics, Humans, Protein Conformation, Antigens metabolism, HIV Envelope Protein gp160 immunology, HIV Envelope Protein gp160 metabolism, HIV-1 metabolism
- Abstract
The extraordinary genetic diversity of the HIV-1 envelope spike [Env; trimeric (gp160)
3 , cleaved to (gp120/gp41)3 ] poses challenges for vaccine development. Envs of different clinical isolates exhibit different sensitivities to antibody-mediated neutralization. Envs of difficult-to-neutralize viruses are thought to be more stable and conformationally homogeneous trimers than those of easy-to-neutralize viruses, thereby providing more effective concealment of conserved, functionally critical sites. In this study we have characterized the antigenic properties of an Env derived from one of the most neutralization-resistant HIV-1 isolates, CH120.6. Sequence variation at neutralizing epitopes does not fully account for its exceptional resistance to antibodies. The full-length, membrane-bound CH120.6 Env is indeed stable and conformationally homogeneous. Its antigenicity correlates closely with its neutralization sensitivity, and major changes in antigenicity upon CD4 engagement appear to be restricted to the coreceptor site. The CH120.6 gp140 trimer, the soluble and uncleaved ectodomain of (gp160)3 , retains many antigenic properties of the intact Env, consistent with a conformation close to that of Env spikes on a virion, whereas its monomeric gp120 exposes many nonneutralizing or strain-specific epitopes. Thus, trimer organization and stability are important determinants not only for occluding many epitopes but also for conferring resistance to neutralization by all but a small set of antibodies. Env preparations derived from neutralization-resistant viruses may induce irrelevant antibody responses less frequently than do other Envs and may be excellent templates for developing soluble immunogens., Competing Interests: Conflict of interest statement: S.K-.G., S.T., C.E.L., and A.C. were employees of Novartis Vaccines and Diagnostics at the time of the study. Following the acquisition of Novartis Vaccines and Diagnostics by the GlaxoSmithKline (GSK) group of companies in March 2015, S.K.-G., S.T., C.E.L., and A.C. became employees of the GSK group of companies. A.C. reports ownership of GSK shares. M.S. and W. Weissenhorn (reviewer) are coauthors of several papers (the last in 2014). The role of M.S. in the work in those papers was to provide neutralization analyses, and he does not have an active research collaboration with W. Weissenhorn.- Published
- 2017
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14. Extraislet expression of islet antigen boosts T cell exhaustion to partially prevent autoimmune diabetes.
- Author
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Selck, Claudia, Jhala, Gaurang, De George, David J., Kwong, Chun-Ting J., Christensen, Marie K., Pappas, Evan G., Xin Liu, Tingting Ge, Trivedi, Prerak, Kallies, Axel, Thomas, Helen E., Kay, Thomas W. H., and Krishnamurthy, Balasubramanian
- Subjects
T-cell exhaustion ,ANTIGENS ,ISLANDS ,DIABETES ,T cells - Abstract
Persistent antigen exposure results in the differentiation of functionally impaired, also termed exhausted, T cells which are maintained by a distinct population of precursors of exhausted T (T
PEX ) cells. T cell exhaustion is well studied in the context of chronic viral infections and cancer, but it is unclear whether and how antigen-driven T cell exhaustion controls progression of autoimmune diabetes and whether this process can be harnessed to prevent diabetes. Using nonobese diabetic (NOD) mice, we show that some CD8+ T cells specific for the islet antigen, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) displayed terminal exhaustion characteristics within pancreatic islets but were maintained in the TPEX cell state in peripheral lymphoid organs (PLO). More IGRP-specific T cells resided in the PLO than in islets. To examine the impact of extraislet antigen exposure on T cell exhaustion in diabetes, we generated transgenic NOD mice with inducible IGRP expression in peripheral antigen-presenting cells. Antigen exposure in the extraislet environment induced severely exhausted IGRP-specific T cells with reduced ability to produce interferon (IFN)γ, which protected these mice from diabetes. Our data demonstrate that T cell exhaustion induced by delivery of antigen can be harnessed to prevent autoimmune diabetes. [ABSTRACT FROM AUTHOR]- Published
- 2024
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15. Antigen perception in T cells by long-term Erk and NFAT signaling dynamics.
- Author
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Wither, Matthew J., White, William L., Pendyala, Sriram, Leanza, Paul J., Fowler, Douglas M., and Hao Yuan Kueh
- Subjects
T cells ,T cell receptors ,MAJOR histocompatibility complex ,ANTIGENS ,GENETIC regulation - Abstract
Immune system threat detection hinges on T cells' ability to perceive varying peptide-major histocompatibility complex (pMHC) antigens. As the Erk and NFAT pathways link T cell receptor engagement to gene regulation, their signaling dynamics may convey information about pMHC inputs. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that, together, enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. Both pathways initially activate uniformly across various pMHC inputs but diverge only over longer (9+ h) timescales, enabling independent encoding of pMHC affinity and dose. These late signaling dynamics are decoded via multiple temporal and combinatorial mechanisms to generate pMHC-specific transcriptional responses. Our findings underscore the importance of long timescale signaling dynamics in antigen perception and establish a framework for understanding T cell responses under diverse contexts. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
16. Identification of a second glycoform of the clinically prevalent O1 antigen from Klebsiella pneumoniae.
- Author
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Kelly, Steven D., Ovchinnikova, Olga G., Müller, Fabian, Steffen, Michael, Braun, Martin, Sweeney, Ryan P., Kowarik, Michael, Follador, Rainer, Lowary, Todd L., Serventi, Fabio, and Whitfield, Chris
- Subjects
KLEBSIELLA pneumoniae ,ANTIGENS ,WESTERN immunoblotting ,KLEBSIELLA infections ,ATP-binding cassette transporters ,HOSPITAL care of children - Abstract
Carbapenemase and extended β-lactamase-producing Klebsiella pneumoniae isolates represent a major health threat, stimulating increasing interest in immunotherapeutic approaches for combating Klebsiella infections. Lipopolysaccharide O antigen polysaccharides offer viable targets for immunotherapeutic development, and several studies have described protection with O-specific antibodies in animal models of infection. O1 antigen is produced by almost half of clinical Klebsiella isolates. The O1 polysaccharide backbone structure is known, but monoclonal antibodies raised against the O1 antigen showed varying reactivity against different isolates that could not be explained by the known structure. Reinvestigation of the structure by NMR spectroscopy revealed the presence of the reported polysaccharide backbone (glycoform O1a), as well as a previously unknown O1b glycoform composed of the O1a backbone modified with a terminal pyruvate group. The activity of the responsible pyruvyltransferase (WbbZ) was confirmed by western immunoblotting and in vitro chemoenzymatic synthesis of the O1b terminus. Bioinformatic data indicate that almost all O1 isolates possess genes required to produce both glycoforms. We describe the presence of O1ab-biosynthesis genes in other bacterial species and report a functional O1 locus on a bacteriophage genome. Homologs of wbbZ are widespread in genetic loci for the assembly of unrelated glycostructures in bacteria and yeast. In K. pneumoniae, simultaneous production of both O1 glycoforms is enabled by the lack of specificity of the ABC transporter that exports the nascent glycan, and the data reported here provide mechanistic understanding of the capacity for evolution of antigenic diversity within an important class of biomolecules produced by many bacteria. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
17. Fully synthetic platform to rapidly generate tetravalent bispecific nanobody-based immunoglobulins.
- Author
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Mindrebo, Laetitia Misson, Hejun Liu, Ozorowski, Gabriel, Quoc Tran, Woehl, Jordan, Khalek, Irene, Smith, Jessica M., Barman, Shawn, Fangzhu Zhao, Keating, Celina, Limbo, Oliver, Verma, Megan, Jingjia Liu, Stanfield, Robyn L., Xueyong Zhu, Turner, Hannah L., Sok, Devin, Po-Ssu Huang, Burton, Dennis R., and Ward, Andrew B.
- Subjects
IMMUNOGLOBULINS ,BISPECIFIC antibodies ,SARS-CoV-2 ,MODULAR design ,ANTIGENS - Abstract
Nanobodies bind a target antigen with a kinetic profile similar to a conventional antibody, but exist as a single heavy chain domain that can be readily multimerized to engage antigen via multiple interactions. Presently, most nanobodies are produced by immunizing camelids; however, platforms for animal-free production are growing in popularity. Here, we describe the development of a fully synthetic nanobody library based on an engineered human VH3-23 variable gene and a multispecific antibody-like format designed for biparatopic target engagement. To validate our library, we selected nanobodies against the SARS-CoV-2 receptor-binding domain and employed an on-yeast epitope binning strategy to rapidly map the specificities of the selected nanobodies. We then generated antibody-like molecules by replacing the VH and VL domains of a conventional antibody with two different nanobodies, designed as a molecular clamp to engage the receptor-binding domain biparatopically. The resulting bispecific tetra-nanobody immunoglobulins neutralized diverse SARS-CoV-2 variants with potencies similar to antibodies isolated from convalescent donors. Subsequent biochemical analyses confirmed the accuracy of the on-yeast epitope binning and structures of both individual nanobodies, and a tetra-nanobody immunoglobulin revealed that the intended mode of interaction had been achieved. This overall workflow is applicable to nearly any protein target and provides a blueprint for a modular workflow for the development of multispecific molecules. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
18. Prevention of Th2-mediated murine allergic airways disease by soluble antigen administration in the neonate.
- Author
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Hogan SP, Foster PS, Charlton B, and Slattery RM
- Subjects
- Animals, Animals, Newborn, Antibody Formation, Antigens pharmacology, Bronchial Hyperreactivity pathology, Cells, Cultured, Immunoglobulin G blood, Injections, Intraperitoneal, Interferon-gamma biosynthesis, Interleukin-4 biosynthesis, Interleukin-5 biosynthesis, Lung immunology, Lung pathology, Mice, Mice, Inbred BALB C, Ovalbumin administration & dosage, Ovalbumin pharmacology, Respiratory Hypersensitivity pathology, Antigens administration & dosage, Bronchial Hyperreactivity immunology, Bronchial Hyperreactivity prevention & control, Respiratory Hypersensitivity immunology, Respiratory Hypersensitivity prevention & control, Th2 Cells immunology
- Abstract
It has been demonstrated recently that neonatal antigen administration in the mouse can lead to priming for Th2-mediated immune responses. This observation has important implications for the development of vaccination strategies in humans, particularly for individuals who may be predisposed to atopy or asthma. In this paper it is shown that although i.p. administration of antigen (100 microg) in adjuvant to the neonate does indeed prime for Th2-mediated disease in mice [allergic airways disease (AAD)], when the same relatively low dose of antigen is given in soluble form no priming occurs. Further, administration of a larger dose of soluble antigen (1 mg) actually prevents the ability to prime for a Th2 response subsequently and so prevents the induction of AAD. Protection from disease was associated with evidence of functional inactivation of both Th1 and Th2 ovalbumin-specific T cells. In contrast, administration of a very low dose of antigen (10 microg) primed for a Th2 response in a similar fashion to antigen in adjuvant. We suggest that the adjuvant lowers the "effective" dose of antigen administered in the neonate and thereby primes for Th2-type immune responses. These findings demonstrate that neonatal antigen administration can inhibit Th2-mediated diseases, such as AAD, but the dose of antigen may be critical to avoid predisposition to disease.
- Published
- 1998
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19. Repertoire-scale measures of antigen binding.
- Author
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Arora, Rohit and Arnaout, Ramy
- Subjects
T cell receptors ,ANTIGENS ,INFLUENZA vaccines ,GIBBS' free energy - Abstract
Antibodies and T cell receptors (TCRs) are the fundamental building blocks of adaptive immunity. Repertoire-scale functionality derives from their epitope-binding properties, just as macroscopic properties like temperature derive from microscopic molecular properties. However, most approaches to repertoire-scale measurement, including sequence diversity and entropy, are not based on antibody or TCR function in this way. Thus, they potentially overlook key features of immunological function. Here we present a framework that describes repertoires in terms of the epitope-binding properties of their constituent antibodies and TCRs, based on analysis of thousands of antibody-antigen and TCR-peptide-major-histocompatibility-complex binding interactions and over 400 high-throughput repertoires. We show that repertoires consist of loose overlapping classes of antibodies and TCRs with similar binding properties. We demonstrate the potential of this framework to distinguish specific responses vs. bystander activation in influenza vaccines, stratify cytomegalovirus (CMV)-infected cohorts, and identify potential immunological "super-agers." Classes add a valuable dimension to the assessment of immune function. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
20. An unexplored angle: T cell antigen discoveries reveal a marginal contribution of proteasome splicing to the immunogenic MHC class I antigen pool.
- Author
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Verkerk, Tamara, Koomen, Sofie J. I., Fuchs, Kyra J., Griffioen, Marieke, and Spaapen, Robbert M.
- Subjects
T cells ,TUMOR antigens ,ANTIGENS ,PEPTIDES ,MASS spectrometry - Abstract
In the current era of T cell-based immunotherapies, it is crucial to understand which types of MHC-presented T cell antigens are produced by tumor cells. In addition to linear peptide antigens, chimeric peptides are generated through proteasome-catalyzed peptide splicing (PCPS). Whether such spliced peptides are abundantly presented by MHC is highly disputed because of disagreement in computational analyses of mass spectrometry data of MHC-eluted peptides. Moreover, such mass spectrometric analyses cannot elucidate how much spliced peptides contribute to the pool of immunogenic antigens. In this Perspective, we explain the significance of knowing the contribution of spliced peptides for accurate analyses of peptidomes on one hand, and to serve as a potential source of targetable tumor antigens on the other hand. Toward a strategy for mass spectrometry independent estimation of the contribution of PCPS to the immunopeptidome, we first reviewed methodologies to identify MHC-presented spliced peptide antigens expressed by tumors. Data from these identifications allowed us to compile three independent datasets containing 103, 74, and 83 confirmed T cell antigens from cancer patients. Only 3.9%, 1.4%, and between 0% and 7.2% of these truly immunogenic antigens are produced by PCPS, therefore providing a marginal contribution to the pool of immunogenic tumor antigens. We conclude that spliced peptides will not serve as a comprehensive source to expand the number of targetable antigens for immunotherapies. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
21. Identification of the Bartonella autotransporter CFA as a protective antigen and hypervariable target of neutralizing antibodies in mice.
- Author
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Siewert, Lena K., Korotaev, Aleksandr, Sedzicki, Jaroslaw, Fromm, Katja, Pinschewer, Daniel D., and Dehio, Christoph
- Subjects
BARTONELLA ,COMPLEMENT receptors ,FC receptors ,IMMUNOGLOBULINS ,ANTIGENS ,FIREPROOFING agents - Abstract
The bacterial genus Bartonella comprises numerous emerging pathogens that cause a broad spectrum of disease manifestations in humans. The targets and mechanisms of the anti-Bartonella immune defense are ill-defined and bacterial immune evasion strategies remain elusive. We found that experimentally infected mice resolved Bartonella infection by mounting antibody responses that neutralized the bacteria, preventing their attachment to erythrocytes and suppressing bacteremia independent of complement or Fc receptors. Bartonella-neutralizing antibody responses were rapidly induced and depended on CD40 signaling but not on affinity maturation. We cloned neutralizing monoclonal antibodies (mAbs) and by mass spectrometry identified the bacterial autotransporter CFA (CAMP-like factor autotransporter) as a neutralizing antibody target. Vaccination against CFA suppressed Bartonella bacteremia, validating CFA as a protective antigen. We mapped Bartonella-neutralizing mAb binding to a domain in CFA that we found is hypervariable in both human and mouse pathogenic strains, indicating mutational antibody evasion at the Bartonella subspecies level. These insights into Bartonella immunity and immune evasion provide a conceptual framework for vaccine development, identifying important challenges in this endeavor. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
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22. Peripheral tolerance by Treg via constraining OX40 signal in autoreactive T cells against desmoglein 3, a target antigen in pemphigus.
- Author
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Hisato Iriki, Hayato Takahashi, Naoko Wada, Hisashi Nomura, Miho Mukai, Aki Kamata, Hiromi Ito, Jun Yamagami, Takeshi Matsui, Yutaka Kurebayashi, Setsuko Mise-Omata, Hiroshi Nishimasu, Osamu Nureki, Akihiko Yoshimura, Shohei Hori, and Masayuki Amagai
- Subjects
T cells ,PEMPHIGUS ,REGULATORY T cells ,ANTIGENS ,BONE marrow ,COMMERCIAL products - Abstract
Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor-transgenic mice to thymus-transplanted mice, Dsg3-specific CD4+ T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg3 -/- mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3-mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3-mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40-dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40-dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
23. Antigen-binding specificities of antibodies are primarily determined by seven residues of VH.
- Author
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Ohno S, Mori N, and Matsunaga T
- Subjects
- Amino Acid Sequence, Animals, Antibody Specificity, Antigens immunology, Biological Evolution, Immunoglobulin Heavy Chains immunology, Immunoglobulin Variable Region immunology, Mice, Protein Conformation, p-Azobenzenearsonate immunology, Antigens genetics, Immunoglobulin Heavy Chains genetics, Immunoglobulin Variable Region genetics
- Abstract
Although the antigen-binding pocket of all antibodies consists of VL + VH dimers (where VL and VH represent immunoglobulin light and heavy chain regions, respectively), subgroups of their VH largely determine their antigen-binding specificities. This VH subgroup dependence automatically relegates subsidiary roles to VL as a whole and to the complementarity-determining region 3 (CDR-3) of VH encoded by independent diversity (D) and joining (J) coding segments in determining antigen-binding specificities of individual antibodies. As a sequel to our previous paper, which emphasized the role conserved residues in CDR-1 and CDR-2 of VH play in general shaping of the primordial antigen-binding cavity, here we propose that the three short clusters of amino acid sequences in CDR-1 and CDR-2 that are placed in the immediate vicinity of the tryptophan loop primarily determine subgroup-dependent antigen preference of individual VH, therefore, antibodies. The three clusters are the 31st to 35th positions of CDR-1 and the 50th to 52nd and 58th to 60th positions of CDR-2. Of those, the 32nd, 34th, 51st, and 59th positions tend to be occupied by tyrosine, methionine, isoleucine, and tyrosine, respectively. Nevertheless, free amino acid substitutions at the remaining seven sites can generate 20(7) or 1.28 X 10(9) varieties of amino acid sequence combinations. Some of these astronomically numerous sequence combinations no doubt contribute to the maintenance of the vast repertoire of antigen-combining diversity, which might be as large as 10(7), whereas others serve to vary binding affinities toward the same antigen. Ironically, but not surprisingly, a single nonconservative amino acid substitution at one of these sites often suffices to change the antigen preference of VH from one to another, whereas more substitutions affecting two or more clusters are apparently required to change the binding affinity toward the same antigen. In the case of mouse anti-p-azophenylarsonate antibodies, the principle of VH subgroup dependence is violated, their VH belonging to either subgroup 1 or 3. It appears that the mouse genome lacks anti-p-azophenylarsonate germ line VH, residues of CDR-3 derived from one particular JH coding segment coming to rescue to cope with this unnatural man-made antigen.
- Published
- 1985
- Full Text
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24. cDNA cloning of a human autoimmune nuclear ribonucleoprotein antigen.
- Author
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Wieben ED, Rohleder AM, Nenninger JM, and Pederson T
- Subjects
- Biological Evolution, Chromosome Mapping, Cloning, Molecular, DNA genetics, DNA Restriction Enzymes, Genes, Humans, RNA, Messenger genetics, Ribonucleoproteins immunology, Ribonucleoproteins, Small Nuclear, snRNP Core Proteins, Antigens genetics, Autoantigens genetics, Ribonucleoproteins genetics
- Abstract
Sera from patients with systemic lupus erythematosus and other autoimmune disorders contain antibodies against nuclear proteins. One such autoantibody system, known as Sm, reacts with antigens associated with small nuclear RNA molecules. In this paper we report the use of Sm autoantibodies to isolate a cDNA clone for the mRNA of one of these nuclear antigens. A HeLa cell cDNA library was screened by message selection followed by autoantibody reaction of cell-free translation products. This led to the identification of a cDNA clone, p281, containing sequences complementary to mRNA for an Sm autoantibody-reactive, 11,000 Mr protein. This cloned Sm antigen comigrated with the small nuclear RNA-associated protein known as "E" and reacted with four out of four Sm autoantibodies that precipitate E protein from total mRNA translation products. RNA gel blot hybridization with clone p281 DNA revealed a poly(A)+ mRNA of approximately equal to 600 nucleotides in human and marmoset (New World primate) cells. Southern blot hybridization of HeLa cell and human lymphocyte DNA indicated the presence of 6-10 copies of p281-homologous sequences. Similar copy numbers were observed with genomic DNA from baboon, cat, and mouse, indicating that the Sm antigen mRNA sequence represented in p281 is conserved across three classes of the Mammalia (primates, carnivores, and rodents). However, no cross-hybridization of p281 was observed with frog or Drosophila DNA. In light of existing evidence that the mammalian Sm antigen E is a weaker autoantigen than other small nuclear RNA-associated proteins, these results suggest a possible correlation between a protein's capacity to serve as an autoantigen during breakdown of the host's immunological tolerance and its extent of evolutionary conservation, whereas the inverse relationship applies to conventional immunity. We suspect, as have others, that this is a clue to the mechanism of autoimmunity.
- Published
- 1985
- Full Text
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25. Structural basis of P[II] rotavirus evolution and host ranges under selection of histo-blood group antigens.
- Author
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Shenyuan Xu, McGinnis, Kristen Rose, Yang Liu, Pengwei Huang, Ming Tan, Stuckert, Michael Robert, Burnside, Riley Erin, Jacob, Elsa Grace, Shuisong Ni, Xi Jiang, and Kennedy, Michael A.
- Subjects
ROTAVIRUSES ,ANTIGENS ,ANIMAL populations ,VACCINE development ,CRYSTAL structure - Abstract
Group A rotaviruses cause severe gastroenteritis in infants and young children worldwide, with P[II] genogroup rotaviruses (RVs) responsible for >90% of global cases. RVs have diverse host ranges in different human and animal populations determined by host histo-blood group antigen (HBGA) receptor polymorphism, but details governing diversity, host ranges, and species barriers remain elusive. In this study, crystal structures of complexes of the major P[II] genogroup P[4] and P[8] genotype RV VP8* receptor-binding domains together with Lewis epitope-containing LNDFH I glycans in combination with VP8* receptor-glycan ligand affinity measurements based on NMR titration experiments revealed the structural basis for RV genotype-specific switching between ββ and βα HBGA receptor-binding sites that determine RV host ranges. The data support the hypothesis that P[II] RV evolution progressed from animals to humans under the selection of type 1 HBGAs guided by stepwise host synthesis of type 1 ABH and Lewis HBGAs. The results help explain disease burden, species barriers, epidemiology, and limited efficacy of current RV vaccines in developing countries. The structural data has the potential to impact the design of future vaccine strategies against RV gastroenteritis. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
26. Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d.
- Author
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Almeida, Catarina F., Smith, Dylan G. M., Tan-Yun Cheng, Harpur, Chris M., Batleska, Elena, Nguyen-Robertson, Catriona V., Nguyen, Tram, Thelemann, Tamara, Reddiex, Scott J. J., Shihan Li, Eckle, Sidonia B. G., Van Rhijn, Ildiko, Rossjohn, Jamie, Uldrich, Adam P., Moody, D. Branch, Williams, Spencer J., Pellicci, Daniel G., and Godfrey, Dale I.
- Subjects
SMALL molecules ,T cell receptors ,BENZOFURAN ,DRUG allergy ,ANTIGENS ,COMMERCIAL products ,CURCUMIN - Abstract
Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant aß T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug-like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αβTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1-TRAJ6) that was conserved across donors. By trapping a CD1d-type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
27. An in vivo selection-derived D-peptide for engineering erythrocyte-binding antigens that promote immune tolerance.
- Author
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Loftis, Alexander R., Genwei Zhang, Backlund, Coralie, Quartararo, Anthony J., Pishesha, Novalia, Hanna, Cameron C., Schissel, Carly K., Garafola, Daniel, Loas, Andrei, Collier, R. John, Ploegh, Hidde, Irvine, Darrell J., and Pentelute, Bradley L.
- Subjects
IMMUNOLOGICAL tolerance ,BACTERIAL proteins ,ANTIGENS ,BLOOD cells ,MASS spectrometry ,COMMERCIAL products - Abstract
When displayed on erythrocytes, peptides and proteins can drive antigen-specific immune tolerance. Here, we investigated a straightforward approach based on erythrocyte binding to promote antigen-specific tolerance to both peptides and proteins. We first identified a robust erythrocyte-binding ligand. A pool of one million fully D-chiral peptides was injected into mice, blood cells were isolated, and ligands enriched on these cells were identified using nano-liquid chromatography-tandem mass spectrometry. One round of selection yielded a murine erythrocyte-binding ligand with an 80 nM apparent dissociation constant, Kd. We modified an 83-kDa bacterial protein and a peptide antigen derived from ovalbumin (OVA) with the identified erythrocyte-binding ligand. An administration of the engineered bacterial protein led to decreased protein-specific antibodies in mice. Similarly, mice given the engineered OVA-derived peptide had decreased inflammatory anti-OVA CD8+ T cell responses. These findings suggest that our tolerance-induction strategy is applicable to both peptide and protein antigens and that our in vivo selection strategy can be used for de novo discovery of robust erythrocyte-binding ligands. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
28. Inactivation of common airborne antigens by perfluoroalkyl chemicals modulates early life allergic asthma.
- Author
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Mengjing Wang, Qianqian Li, Meifang Hou, Chan, Louisa L. Y., Meng Liu, Ter, Soo Kai, Ting Dong, Yun Xia, Chotirmall, Sanjay H., and Mingliang Fang
- Subjects
ASTHMA ,PEDIATRIC respiratory diseases ,HOUSE dust mites ,COMMERCIAL products ,POLLUTANTS ,ANTIGENS ,ALKYLBENZENE sulfonates - Abstract
Allergic asthma, driven by T helper 2 cell-mediated immune responses to common environmental antigens, remains the most common respiratory disease in children. Perfluorinated chemicals (PFCs) are environmental contaminants of great concern, because of their wide application, persistence in the environment, and bioaccumulation. PFCs associate with immunological disorders including asthma and attenuate immune responses to vaccines. The influence of PFCs on the immunological response to allergens during childhood is unknown. We report here that a major PFC, perfluorooctane sulfonate (PFOS), inactivates house dust mite (HDM) to dampen 5-wk-old, early weaned mice from developing HDMinduced allergic asthma. PFOS further attenuates the asthma protective effect of the microbial product lipopolysaccharide (LPS). We demonstrate that PFOS prevents desensitization of lung epithelia by LPS, thus abolishing the latter’s protective effect. A close mechanistic study reveals that PFOS specifically binds the major HDM allergen Der p1 with high affinity as well as the lipid A moiety of LPS, leading to the inactivation of both antigens. Moreover, PFOS at physiological human (nanomolar) concentrations inactivates Der p1 from HDM and LPS in vitro, although higher doses did not cause further inactivation because of possible formation of PFOS aggregates. This PFOS-induced neutralization of LPS has been further validated in primary human cell models and extended to an in vivo bacterial infection mouse model. This study demonstrates that early life exposure of mice to a PFC blunts airway antigen bioactivity to modulate pulmonary inflammatory responses, which may adversely affect early pulmonary health. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
29. Nanoparticle-enabled innate immune stimulation activates endogenous tumor-infiltrating T cells with broad antigen specificities.
- Author
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Qian Yin, Wong Yu, Grzeskowiak, Caitlin L., Jing Li, Huang Huang, Jing Guo, Liang Chen, Feng Wang, Fan Zhao, von Boehmer, Lotta, Metzner, Thomas J., Leppert, John T., Chien, Yueh-hsiu, Kuo, Calvin J., and Davis, Mark M.
- Subjects
REGULATORY T cells ,T cells ,LABORATORY mice ,IMMUNOLOGIC memory ,ANTIGENS ,TUMOR-infiltrating immune cells - Abstract
Tumors are often infiltrated by T lymphocytes recognizing either self- or mutated antigens but are generally inactive, although they often show signs of prior clonal expansion. Hypothesizing that this may be due to peripheral tolerance, we formulated nanoparticles containing innate immune stimulants that we found were sufficient to activate self-specific CD8
+ T cells and injected them into two different mouse tumor models, B16F10 and MC38. These nanoparticles robustly activated and/or expanded antigen-specific CD8+ tumor-infiltrating T cells, along with a decrease in regulatory CD4+ T cells and an increase in Interleukin-17 producers, resulting in significant tumor growth retardation or elimination and the establishment of immune memory in surviving mice. Furthermore, nanoparticles with modification of stimulating human T cells enabled the robust activation of endogenous T cells in patientderived tumor organoids. These results indicate that breaking peripheral tolerance without regard to the antigen specificity creates a promising pathway for cancer immunotherapy. [ABSTRACT FROM AUTHOR]- Published
- 2021
- Full Text
- View/download PDF
30. Optofluidic multiplex detection of single SARS-CoV-2 and influenza A antigens using a novel bright fluorescent probe assay.
- Author
-
Stambaugh, Alexandra, Parks, Joshua W., Stott, Matthew A., Meena, Gopikrishnan G., Hawkins, Aaron R., and Schmidt, Holger
- Subjects
FLUORESCENT probes ,SARS-CoV-2 ,COVID-19 pandemic ,INFLUENZA ,ANTIGENS ,COMMERCIAL products - Abstract
The urgency for the development of a sensitive, specific, and rapid point-of-care diagnostic test has deepened during the ongoing COVID-19 pandemic. Here, we introduce an ultrasensitive chipbased antigen test with single protein biomarker sensitivity for the differentiated detection of both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A antigens in nasopharyngeal swab samples at diagnostically relevant concentrations. The single-antigen assay is enabled by synthesizing a brightly fluorescent reporter probe, which is incorporated into a bead-based solid-phase extraction assay centered on an antibody sandwich protocol for the capture of target antigens. After optimization of the probe release for detection using ultraviolet light, the full assay is validated with both SARS-CoV-2 and influenza A antigens from clinical nasopharyngeal swab samples (PCR-negative spiked with target antigens). Spectrally multiplexed detection of both targets is implemented by multispot excitation on amultimode interferencewaveguide platform, and detection at 30 ng/mL with single-antigen sensitivity is reported. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
31. Homeostatic regulation of T follicular helper and antibody response to particle antigens by IL-1Ra of medullary sinus macrophage origin.
- Author
-
Xinwen Lin, Trix Twelkmeyer, Danming Zhu, Li Zhang, Yang Zhao, Chao Zhang, Yoichiro Iwakura, Guangxun Meng, Zhaolin Hua, Bingyu Yan, Liu, William J., Zhongguang Luo, Sitang Gong, Hairong Chen, Shuran Li, Baidong Hou, and Hong Tang
- Subjects
ANTIBODY formation ,HEPATITIS associated antigen ,ANTIGENS ,CELL surface antigens ,MACROPHAGES ,MILIEU therapy - Abstract
Hepatitis B virus (HBV) vaccines are composed of surface antigen HBsAg that spontaneously assembles into subviral particles. Factors that impede its humoral immunity in 5% to 10% of vaccinees remain elusive. Here, we showed that the low-level interleukin-1 receptor antagonist (IL-1Ra) can predict antibody protection both in mice and humans. Mechanistically, murine IL-1Ra-inhibited T follicular helper (Tfh) cell expansion and subsequent germinal center (GC)-dependent humoral immunity, resulting in significantly weakened protection against the HBV challenge. Compared to soluble antigens, HBsAg particle antigen displayed a unique capture/uptake and innate immune activation, including IL-1Ra expression, preferably of medullary sinus macrophages. In humans, a unique polymorphism in the RelA/p65 binding site of IL-1Ra enhancer associated IL-1Ra levels with ethnicity-dependent vaccination outcome. Therefore, the differential IL-1Ra response to particle antigens probably creates a suppressive milieu for Tfh/GC development, and neutralization of IL-1Ra would resurrect antibody response in HBV vaccine nonresponders. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
32. Orthogonal immunoassays for IgG antibodies to SARS-CoV-2 antigens reveal that immune response lasts beyond 4 mo post illness onset.
- Author
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Sasisekharan, Varun, Pentakota, Niharika, Jayaraman, Akila, Tharakaraman, Kannan, Wogan, Gerald N., and Narayanasami, Uma
- Subjects
SARS-CoV-2 ,MIDDLE East respiratory syndrome ,COVID-19 ,IMMUNE response ,IMMUNOASSAY - Abstract
Immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection during the current pandemic remains a field of immense interest and active research worldwide. Although the severity of acute infection may depend on the intensity of innate and adaptive immunity, leading to higher morbidity and mortality, the longevity of IgG antibodies, including neutralizing activity to SARS-CoV-2, is viewed as a key correlate of immune protection. Amid reports and concern that there is a rapid decay of IgG antibody levels within 1 mo to 2 mo after acute infection, we set out to study the pattern and duration of IgG antibody response to various SARS-CoV-2 antigens in asymptomatic and symptomatic patients in a community setting. Herein, we show the correlation of IgG anti-spike protein S1 subunit, receptor binding domain, nucleocapsid, and virus neutralizing antibody titers with each other and with clinical features such as length and severity of COVID-19 illness. More importantly, using orthogonal measurements, we found the IgG titers to persist for more than 4 mo post symptom onset, implying that long-lasting immunity to COVID-19 from infection or vaccination might be observed, as seen with other coronaviruses such as SARS and Middle East respiratory syndrome. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
33. Cryo-EM structure of the full-length WzmWzt ABC transporter required for lipid-linked O antigen transport.
- Author
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Caffalette, Christopher A. and Zimmer, Jochen
- Subjects
ATP-binding cassette transporters ,CHEMICAL processes ,CELL membranes ,ANTIGENS ,MEMBRANE lipids - Abstract
O antigens are important cell surface polysaccharides in gramnegative bacteria where they extend core lipopolysaccharides in the extracellular leaflet of the outer membrane. O antigen structures are serotype specific and form extended cell surface barriers endowing many pathogens with survival benefits. In the ABC transporter-dependent biosynthesis pathway, O antigens are assembled on the cytosolic side of the inner membrane on a lipid anchor and reoriented to the periplasmic leaflet by the channelforming WzmWzt ABC transporter for ligation to the core lipopolysaccharides. In many cases, this process depends on the chemical modification of the O antigen's nonreducing terminus, sensed by WzmWzt via a carbohydrate-binding domain (CBD) that extends its nucleotide-binding domain (NBD). Here, we provide the cryo-electron microscopy structure of the full-length WzmWzt transporter from Aquifex aeolicus bound to adenosine triphosphate (ATP) and in a lipid environment, revealing a highly asymmetric transporter organization. The CBDs dimerize and associate with only one NBD. Conserved loops at the CBD dimer interface straddle a conserved peripheral NBD helix. The CBD dimer is oriented perpendicularly to the NBDs and its putative ligand-binding sites face the transporter to likely modulate ATPase activity upon O antigen binding. Further, our structure reveals a closed WzmWzt conformation in which an aromatic belt near the periplasmic channel exit seals the transporter in a resting, ATP-bound state. The sealed transmembrane channel is asymmetric, with one open and one closed cytosolic and periplasmic portal. The structure provides important insights into O antigen recruitment to and translocation by WzmWzt and related ABC transporters. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
34. Plasmacytoid dendritic cells cross-prime naive CD8 T cells by transferring antigen to conventional dendritic cells through exosomes.
- Author
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Chunmei Fu, Peng Peng, Loschko, Jakob, Li Feng, Phuong Pham, Weiguo Cui, Lee, Kelvin P., Krug, Anne B., and Aimin Jiang
- Subjects
DENDRITIC cells ,T cells ,ANTIGENS ,ANTIGEN presentation ,EXOSOMES - Abstract
Although plasmacytoid dendritic cells (pDCs) have been shown to play a critical role in generating viral immunity and promoting tolerance to suppress antitumor immunity, whether and how pDCs cross-prime CD8 T cells in vivo remain controversial. Using a pDC-targeted vaccine model to deliver antigens specifically to pDCs, we have demonstrated that pDC-targeted vaccination led to strong cross-priming and durable CD8 T cell immunity. Surprisingly, cross-presenting pDCs required conventional DCs (cDCs) to achieve cross-priming in vivo by transferring antigens to cDCs. Taking advantage of an in vitro system where only pDCs had access to antigens, we further demonstrated that cross-presenting pDCs were unable to efficiently prime CD8 T cells by themselves, but conferred antigennaive cDCs the capability of cross-priming CD8 T cells by transferring antigens to cDCs. Although both cDC1s and cDC2s exhibited similar efficiency in acquiring antigens from pDCs, cDC1s but not cDC2s were required for cross-priming upon pDC-targeted vaccination, suggesting that cDC1s played a critical role in pDC-mediated cross-priming independent of their function in antigen presentation. Antigen transfer from pDCs to cDCs was mediated by previously unreported pDC-derived exosomes (pDCexos), that were also produced by pDCs under various conditions. Importantly, all these pDCexos primed naive antigen-specific CD8 T cells only in the presence of bystander cDCs, similarly to cross-presenting pDCs, thus identifying pDCexo-mediated antigen transfer to cDCs as a mechanism for pDCs to achieve cross-priming. In summary, our data suggest that pDCs employ a unique mechanism of pDCexo-mediated antigen transfer to cDCs for cross-priming. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
35. Autoantibodies against central nervous system antigens in a subset of B cell-dominant multiple sclerosis patients.
- Author
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Kuerten, Stefanie, Lanz, Tobias V., Lingampalli, Nithya, Lahey, Lauren J., Kleinschnitz, Christoph, Mäurer, Mathias, Schroeter, Michael, Braune, Stefan, Ziemssen, Tjalf, Ho, Peggy P., Robinson, William H., and Steinman, Lawrence
- Subjects
CENTRAL nervous system ,MULTIPLE sclerosis ,AUTOANTIBODIES ,B cells ,ANTIGENS - Abstract
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), with characteristic inflammatory lesions and demyelination. The clinical benefit of cell-depleting therapies targeting CD20 has emphasized the role of B cells and autoantibodies in MS pathogenesis. We previously introduced an enzyme-linked immunospot spot (ELISpot)-based assay to measure CNS antigen-specific B cells in the blood of MS patients and demonstrated its usefulness as a predictive biomarker for disease activity in measuring the successful outcome of disease-modifying therapies (DMTs). Here we used a planar protein array to investigate CNS-reactive antibodies in the serum of MS patients as well as in B cell culture supernatants after polyclonal stimulation. Anti- CNS antibody reactivity was evident in the sera of the MS cohort, and the antibodies bound a heterogeneous set of molecules, including myelin, axonal cytoskeleton, and ion channel antigens, in individual patients. Immunoglobulin reactivity in supernatants of stimulated B cells was directed against a broad range of CNS antigens. A group of MS patients with a highly active B cell component was identified by the ELISpot assay. Those antibody reactivities remained stable over time. These assays with protein arrays identify MS patients with a highly active B cell population with antibodies directed against a swathe of CNS proteins. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
36. Plasmodium vivax spleen-dependent genes encode antigens associated with cytoadhesion and clinical protection.
- Author
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Fernandez-Becerra, Carmen, Bernabeu, Maria, Castellanos, Angélica, Correa, Bruna R., Obadia, Thomas, Ramirez, Miriam, Rui, Edmilson, Hentzschel, Franziska, López-Montañés, Maria, Ayllon-Hermida, Alberto, Martin-Jaular, Lorena, Elizalde-Torrent, Aleix, Siba, Peter, Vêncio, Ricardo Z., Arevalo-Herrera, Myriam, Herrera, Sócrates, Alonso, Pedro L., Mueller, Ivo, and del Portillo, Hernando A.
- Subjects
PLASMODIUM vivax ,RECOMBINANT proteins ,MEMBRANE transport proteins ,ANTIGENS ,PLASMODIUM falciparum - Abstract
Plasmodium vivax, the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi, another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
37. CD5 signalosome coordinates antagonist TCR signals to control the generation of Treg cells induced by foreign antigens.
- Author
-
Blaize, Gaëtan, Daniels-Treffandier, Hélène, Aloulou, Meryem, Rouquié, Nelly, Cui Yang, Marcellin, Marlène, Gador, Mylène, Benamar, Mehdi, Ducatez, Mariette, Ki-duk Song, Burlet-Schiltz, Odile, Saoudi, Abdelhadi, Love, Paul E., Fazilleau, Nicolas, Gonzalez de Peredo, Anne, and Lesourne, Renaud
- Subjects
SUPPRESSOR cells ,T cell receptors ,T cells ,SIGNAL recognition particle receptor ,ANTIGENS - Abstract
CD5 is characterized as an inhibitory coreceptor with an important regulatory role during T cell development. The molecular mechanism by which CD5 operates has been puzzling and its function in mature T cells suggests promoting rather than repressing effects on immune responses. Here, we combined quantitative mass spectrometry and genetic studies to analyze the components and the activity of the CD5 signaling machinery in primary T cells. We found that T cell receptor (TCR) engagement induces the selective phosphorylation of CD5 tyrosine 429, which serves as a docking site for proteins with adaptor functions (c-Cbl, CIN85, CRKL), connecting CD5 to positive (PI3K) and negative (UBASH3A, SHIP1) regulators of TCR signaling. c-CBL acts as a coordinator in this complex enabling CD5 to synchronize positive and negative feedbacks on TCR signaling through the other components. Disruption of CD5 signalosome in mutant mice reveals that it modulates TCR signal outputs to selectively repress the transactivation of Foxp3 and limit the inopportune induction of peripherally induced regulatory T cells during immune responses against foreign antigen. Our findings bring insights into the paradigm of coreceptor signaling, suggesting that, in addition to providing dualistic enhancing or dampening inputs, coreceptors can engage concomitant stimulatory and inhibitory signaling events, which act together to promote specific functional outcomes. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
38. Molecular determinants of chaperone interactions on MHC-I for folding and antigen repertoire selection.
- Author
-
McShan, Andrew C., Devlin, Christine A., Overall, Sarah A., Jihye Park, Toor, Jugmohit S., Danai Moschidi, Flores-Solis, David, Choi, Hannah, Tripathi, Sarvind, Procko, Erik, and Sgourakis, Nikolaos G.
- Subjects
MOLECULAR chaperones ,CELL surface antigens ,MOLECULAR shapes ,ANTIGENS ,T cells - Abstract
The interplay between a highly polymorphic set of MHC-I alleles and molecular chaperones shapes the repertoire of peptide antigens displayed on the cell surface for T cell surveillance. Here, we demonstrate that the molecular chaperone TAP-binding protein related (TAPBPR) associates with a broad range of partially folded MHC-I species inside the cell. Bimolecular fluorescence complementation and deep mutational scanning reveal that TAPBPR recognition is polarized toward the α
2 domain of the peptide-binding groove, and depends on the formation of a conserved MHC-I disulfide epitope in the α2 domain. Conversely, thermodynamic measurements of TAPBPR binding for a representative set of properly conformed, peptide-loaded molecules suggest a narrower MHC-I specificity range. Using solution NMR, we find that the extent of dynamics at "hotspot" surfaces confers TAPBPR recognition of a sparsely populated MHC-I state attained through a global conformational change. Consistently, restriction of MHC-I groove plasticity through the introduction of a disulfide bond between the α1/α2 helices abrogates TAPBPR binding, both in solution and on a cellular membrane, while intracellular binding is tolerant of many destabilizing MHC-I substitutions. Our data support parallel TAPBPR functions of 1) chaperoning unstable MHC-I molecules with broad allele-specificity at early stages of their folding process, and 2) editing the peptide cargo of properly conformed MHC-I molecules en route to the surface, which demonstrates a narrower specificity. Our results suggest that TAPBPR exploits localized structural adaptations, both near and distant to the peptide-binding groove, to selectively recognize discrete conformational states sampled by MHC-I alleles, toward editing the repertoire of displayed antigens. [ABSTRACT FROM AUTHOR]- Published
- 2019
- Full Text
- View/download PDF
39. Developmental competence and antigen switch frequency can be uncoupled in Trypanosoma brucei.
- Author
-
McWilliam, Kirsty R., Ivens, Alasdair, Morrison, Liam J., Mugnier, Monica R., and Matthews, Keith R.
- Subjects
TRYPANOSOMA brucei ,ANTIGENIC variation ,MEMBRANE glycoproteins ,ANTIGENS ,QUORUM sensing - Abstract
African trypanosomes use an extreme form of antigenic variation to evade host immunity, involving the switching of expressed variant surface glycoproteins by a stochastic and parasite-intrinsic process. Parasite development in the mammalian host is another feature of the infection dynamic, with trypanosomes undergoing quorum sensing (QS)-dependent differentiation between proliferative slender forms and arrested, transmissible, stumpy forms. Longstanding experimental studies have suggested that the frequency of antigenic variation and transmissibility may be linked, antigen switching being higher in developmentally competent, fly-transmissible, parasites ("pleomorphs") than in serially passaged "monomorphic" lines that cannot transmit through flies. Here, we have directly tested this tenet of the infection dynamic by using 2 experimental systems to reduce pleomorphism. Firstly, lines were generated that inducibly lose developmental capacity through RNAi-mediated silencing of the QS signaling machinery ("inducible monomorphs"). Secondly, de novo lines were derived that have lost the capacity for stumpy formation by serial passage ("selected monomorphs") and analyzed for their antigenic variation in comparison to isogenic preselected populations. Analysis of both inducible and selected monomorphs has established that antigen switch frequency and developmental capacity are independently selected traits. This generates the potential for diverse infection dynamics in different parasite populations where the rate of antigenic switching and transmission competence are uncoupled. Further, this may support the evolution, maintenance, and spread of important trypanosome variants such as Trypanosoma brucei evansi that exploit mechanical transmission. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
40. Bacterial outer membrane vesicles engineered with lipidated antigens as a platform for Staphylococcus aureus vaccine.
- Author
-
Irene, Carmela, Fantappiè, Laura, Caproni, Elena, Zerbini, Francesca, Anesi, Andrea, Tomasi, Michele, Zanella, Ilaria, Stupia, Simone, Prete, Stefano, Valensin, Silvia, König, Enrico, Frattini, Luca, Gagliardi, Assunta, Isaac, Samine J., Grandi, Alberto, Guella, Graziano, and Grandi, Guido
- Subjects
BACTERIAL cell walls ,STAPHYLOCOCCUS aureus ,ANTIGENS ,LIPOPOLYSACCHARIDE structure ,VACCINES - Abstract
Bacterial outer membrane vesicles (OMVs) represent an interesting vaccine platform for their built-in adjuvanticity and simplicity of production process. Moreover, OMVs can be decorated with foreign antigens using different synthetic biology approaches. However, the optimal OMV engineering strategy, which should guarantee the OMV compartmentalization of most heterologous antigens in quantities high enough to elicit protective immune responses, remains to be validated. In this work we exploited the lipoprotein transport pathway to engineer OMVs with foreign proteins. Using 5 Staphylococcus aureus protective antigens expressed in Escherichia coli as fusions to a lipoprotein leader sequence, we demonstrated that all 5 antigens accumulated in the vesicular compartment at a concentration ranging from 5 to 20% of total OMV proteins, suggesting that antigen lipidation could be a universal approach for OMV manipulation. Engineered OMVs elicited high, saturating antigenspecific antibody titers when administered to mice in quantities as low as 0.2 μg/dose. Moreover, the expression of lipidated antigens in E. coli BL21(DE3)ΔompAΔmsbBΔpagP was shown to affect the lipopolysaccharide structure, with the result that the TLR4 agonist activity of OMVs was markedly reduced. These results, together with the potent protective activity of engineered OMVs observed in mice challenged with S. aureus Newman strain, makes the 5-combo-OMVs a promising vaccine candidate to be tested in clinics. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
41. Rickettsia conorii O antigen is the target of bactericidal Weil-Felix antibodies.
- Author
-
Hwan Keun Kim, Premaratna, Ranjan, Missiakas, Dominique M., and Schneewind, Olaf
- Subjects
RICKETTSIAL diseases ,IMMUNOGLOBULINS ,ANTIGENS ,RICKETTSIA ,SEROLOGY - Abstract
Rickettsial diseases have long been diagnosed with serum antibodies cross-reactive against Proteus vulgaris (Weil-Felix reaction). Although Weil-Felix antibodies are associated with the development of immunity, their rickettsial target and contribution to disease pathogenesis are not established. Here, we developed a transposon for insertional mutagenesis of Rickettsia conorii, isolating variants defective for replication in cultured cells and in spotted fever pathogenesis. Mutations in the polysaccharide synthesis operon (pso) abolish lipopolysaccharide O-antigen synthesis and Weil-Felix serology and alter outer-membrane protein assembly. Unlike wild-type R. conorii, pso mutants cannot elicit bactericidal antibodies that bind O antigen. The pso operon is conserved among rickettsial pathogens, suggesting that bactericidal antibodies targeting O antigen may generate universal immunity that could be exploited to develop vaccines against rickettsial diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
42. Immunogenicity of a rheumatoid arthritis protective sequence when acquired through microchimerism.
- Author
-
Kanaan, Sami B., Sensoy, Oyku, Zhen Yan, Gadi, Vijayakrishna K., Richardson, Michael L., and Nelson, J. Lee
- Subjects
RHEUMATOID arthritis ,T cells ,CELL lines ,ALLELES ,ANTIGENS - Abstract
HLA class II genes provide the strongest genetic contribution to rheumatoid arthritis (RA). HLA-DRB1 alleles encoding the sequence DERAA are RA-protective. Paradoxically, RA risk is increased in women with DERAA
+ children born prior to onset. We developed a sensitive qPCR assay specific for DERAA, and found 53% of DERAA-/- women with RA had microchimerism (Mc; pregnancyderived allogeneic cells) carrying DERAA (DERAA-Mc) vs. 6% of healthy women. DERAA-Mc quantities correlated with an RA-risk genetic background including DERAA-binding HLA-DQ alleles, early RA onset, and aspects of RA severity. CD4+ T cells showed stronger response against DERAA+ vs. DERAA- allogeneic cell lines in vitro, in line with an immunogenic role of allogeneic DERAA. Results indicate a model where DERAA-Mc activates DERAA-directed T cells that are naturally present in DERAA-/- individuals and can have crossreactivity against joint antigens. Moreover, we provide an explanation for the enigmatic observation that the same HLA sequence differentially affects RA risk through Mendelian inheritance vs. microchimeric cell acquisition. [ABSTRACT FROM AUTHOR]- Published
- 2019
- Full Text
- View/download PDF
43. Molecular mimicry between Anoctamin 2 and Epstein-Barr virus nuclear antigen 1 associates with multiple sclerosis risk.
- Author
-
Tengvall, Katarina, Huang, Jesse, Hellström, Cecilia, Kammer, Patrick, Biström, Martin, Ayoglu, Burcu, Bomfim, Izaura Lima, Stridh, Pernilla, Butt, Julia, Brenner, Nicole, Michel, Angelika, Lundberg, Karin, Padyukov, Leonid, Lundberg, Ingrid E., Svenungsson, Elisabet, Ernberg, Ingemar, Olafsson, Sigurgeir, Dilthey, Alexander T., Hillert, Jan, and Alfredsson, Lars
- Subjects
MOLECULAR mimicry ,EPSTEIN-Barr virus ,MULTIPLE sclerosis ,CENTRAL nervous system diseases ,ANTIGENS - Abstract
Multiple sclerosis (MS) is a chronic inflammatory, likely autoimmune disease of the central nervous system with a combination of genetic and environmental risk factors, among which Epstein-Barr virus (EBV) infection is a strong suspect. We have previously identified increased autoantibody levels toward the chloride-channel protein Anoctamin 2 (ANO2) in MS. Here, IgG antibody reactivity toward ANO2 and EBV nuclear antigen 1 (EBNA1) was measured using bead-based multiplex serology in plasma samples from 8,746 MS cases and 7,228 controls. We detected increased anti-ANO2 antibody levels in MS (P = 3.5 × 10
−36 ) with 14.6% of cases and 7.8% of controls being ANO2 seropositive (odds ratio [OR] = 1.6; 95% confidence intervals [95%CI]: 1.5 to 1.8). The MS risk increase in ANO2-seropositive individuals was dramatic when also exposed to 3 known risk factors for MS: HLA-DRB1*15:01 carriage, absence of HLA-A*02:01, and high anti-EBNA1 antibody levels (OR = 24.9; 95%CI: 17.9 to 34.8). Reciprocal blocking experiments with ANO2 and EBNA1 peptides demonstrated antibody cross-reactivity, mapping to ANO2 [aa 140 to 149] and EBNA1 [aa 431 to 440]. HLA gene region was associated with anti-ANO2 antibody levels and HLA-DRB1*04:01 haplotype was negatively associated with ANO2 seropositivity (OR = 0.6; 95%CI: 0.5 to 0.7). Anti-ANO2 antibody levels were not increased in patients from 3 other inflammatory disease cohorts. The HLA influence and the fact that specific IgG production usually needs T cell help provides indirect evidence for a T cell ANO2 autoreactivity in MS. We propose a hypothesis where immune reactivity toward EBNA1 through molecular mimicry with ANO2 contributes to the etiopathogenesis of MS. [ABSTRACT FROM AUTHOR]- Published
- 2019
- Full Text
- View/download PDF
44. Antigen structure affects cellular routing through DC-SIGN.
- Author
-
Jarvis, Cassie M., Zwick, Daniel B., Grim, Joseph C., Alam, Mohammad Murshid, Prost, Lynne R., Gardiner, Jaye C., Soyeong Park, Zimdars, Laraine L., Sherer, Nathan M., and Kiessling, Laura L.
- Subjects
CELL anatomy ,ANTIGENS ,MAJOR histocompatibility complex ,POLYMER structure ,RING-opening polymerization - Abstract
Dendritic cell (DC) lectins mediate the recognition, uptake, and processing of antigens, but they can also be coopted by pathogens for infection. These distinct activities depend upon the routing of antigens within the cell. Antigens directed to endosomal compartments are degraded, and the peptides are presented on major histocompatibility complex class II molecules, thereby promoting immunity. Alternatively, HIV-1 can avoid degradation, as virus engagement with C-type lectin receptors (CLRs), such as DC-SIGN (DC-specific ICAM-3-grabbing nonintegrin) results in trafficking to surface-accessible invaginated pockets. This process appears to enable infection of T cells in trans. We sought to explore whether antigen fate upon CLR-mediated internalization was affected by antigen physical properties. To this end, we employed the ring-opening metathesis polymerization to generate glycopolymers that each display multiple copies of mannoside ligand for DC-SIGN, yet differ in length and size. The rate and extent of glycopolymer internalization depended upon polymer structure--longer polymers were internalized more rapidly and more efficiently than were shorter polymers. The trafficking, however, did not differ, and both short and longer polymers colocalized with transferrin-labeled early endosomes. To explore how DC-SIGN directs larger particles, such as pathogens, we induced aggregation of the polymers to access particulate antigens. Strikingly, these particulate antigens were diverted to the invaginated pockets that harbor HIV-1. Thus, antigen structure has a dramatic effect on DC-SIGN-mediated uptake and trafficking. These findings have consequences for the design of synthetic vaccines. Additionally, the results suggest strategies for targeting DC reservoirs that harbor viral pathogens. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
45. Borrelia burgdorferi peptidoglycan is a persistent antigen in patients with Lyme arthritis.
- Author
-
Jutras, Brandon L., Lochhead, Robert B., Kloos, Zachary A., Biboy, Jacob, Strle, Klemen, Booth, Carmen J., Govers, Sander K., Gray, Joe, Schumann, Peter, Vollmer, Waldemar, Bockenstedt, Linda K., Steere, Allen C., and Jacobs-Wagner, Christine
- Subjects
LYME disease ,BORRELIA burgdorferi ,SYNOVIAL fluid ,THERAPEUTICS ,DISEASE complications ,IMMUNOGLOBULIN G ,ANTIGENS - Abstract
Lyme disease is a multisystem disorder caused by the spirochete Borrelia burgdorferi. A common late-stage complication of this disease is oligoarticular arthritis, often involving the knee. In ~10% of cases, arthritis persists after appropriate antibiotic treatment, leading to a proliferative synovitis typical of chronic inflammatory arthritides. Here, we provide evidence that peptidoglycan (PG), a major component of the B. burgdorferi cell envelope, may contribute to the development and persistence of Lyme arthritis (LA). We show that B. burgdorferi has a chemically atypical PG (PG
Bb ) that is not recycled during cell-wall turnover. Instead, this pathogen sheds PGBb fragments into its environment during growth. Patients with LA mount a specific immunoglobulin G response against PGBb , which is significantly higher in the synovial fluid than in the serum of the same patient. We also detect PGBb in 94% of synovial fluid samples (32 of 34) from patients with LA, many of whom had undergone oral and intravenous antibiotic treatment. These same synovial fluid samples contain proinflammatory cytokines, similar to those produced by human peripheral blood mononuclear cells stimulated with PGBb . In addition, systemic administration of PGBb in BALB/c mice elicits acute arthritis. Altogether, our study identifies PGBb as a likely contributor to inflammatory responses in LA. Persistence of this antigen in the joint may contribute to synovitis after antibiotics eradicate the pathogen. Furthermore, our finding that B. burgdorferi sheds immunogenic PGBb fragments during growth suggests a potential role for PGBb in the immunopathogenesis of other Lyme disease manifestations. [ABSTRACT FROM AUTHOR]- Published
- 2019
- Full Text
- View/download PDF
46. Select sequencing of clonally expanded CD8+ T cells reveals limits to clonal expansion.
- Author
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Huang Huang, Sikora, Michael J., Islam, Saiful, Chowdhury, Roshni Roy, Yueh-hsiu Chien, Scriba, Thomas J., Davis, Mark M., and Steinmetz, Lars M.
- Subjects
T cells ,CLONING ,T cell receptors ,ANTIGENS ,IMMUNE response ,MYCOBACTERIUM tuberculosis ,KILLER cells ,IMMUNOLOGY - Abstract
To permit the recognition of antigens, T cells generate a vast diversity of T cell receptor (TCR) sequences. Upon binding of the TCR to an antigen-MHC complex, T cells clonally expand to establish an immune response. To study antigen-specific T cell clonality, we have developed a method that allows selection of rare cells, based on RNA expression, before in-depth scRNA-seq (named SELECT-seq). We applied SELECT-seq to collect both TCR sequences and then transcriptomes from single cells of peripheral blood lymphocytes activated by a Mycobacterium tuberculosis (Mtb) lysate. TCR sequence analysis allowed us to preferentially select expanded conventional CD8
+ T cells as well as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells. The iNKT and MAIT cells have a highly similar transcriptional pattern, indicating that they carry out similar immunological functions and differ considerably from conventional CD8+ T cells. While there is no relationship between expression profiles and clonal expansion in iNKT or MAIT cells, highly expanded conventional CD8+ T cells down-regulate the interleukin 2 (IL-2) receptor alpha (IL2RA, or CD25) protein and show signs of senescence. This suggests inherent limits to clonal expansion that act to diversify the T cell response repertoire. [ABSTRACT FROM AUTHOR]- Published
- 2019
- Full Text
- View/download PDF
47. Successive crystal structure snapshots suggest the basis for MHC class I peptide loading and editing by tapasin.
- Author
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Hafstrand, Ida, Sayitoglu, Ece Canan, Apavaloaei, Anca, Josey, Benjamin John, Renhua Sun, Xiao Han, Pellegrino, Sara, Ozkazanc, Didem, Potens, Renée, Janssen, Linda, Nilvebrant, Johan, Nygren, Per-Åke, Sandalova, Tatyana, Springer, Sebastian, Georgoudaki, Anna-Maria, Duru, Adil Doganay, and Achour, Adnane
- Subjects
EPITOPES ,ANTIGENS ,MAJOR histocompatibility complex ,PEPTIDES ,CRYSTAL structure ,PROTEINS - Abstract
MHC-I epitope presentation to CD8
+ T cells is directly dependent on peptide loading and selection during antigen processing. However, the exact molecular bases underlying peptide selection and binding by MHC-I remain largely unknown. Within the peptideloading complex, the peptide editor tapasin is key to the selection of MHC-I-bound peptides. Here, we have determined an ensemble of crystal structures of MHC-I in complex with the peptide exchange-associated dipeptide GL, as well as the tapasin-associated scoop loop, alone or in combination with candidate epitopes. These results combined with mutation analyses allow us to propose a molecular model underlying MHC-I peptide selection by tapasin. The N termini of bound peptides most probably bind first in the N-terminal and middle region of the MHC-I peptide binding cleft, upon which the peptide C termini are tested for their capacity to dislodge the tapasin scoop loop from the F pocket of the MHC-I cleft. Our results also indicate important differences in peptide selection between different MHC-I alleles. [ABSTRACT FROM AUTHOR]- Published
- 2019
- Full Text
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48. Breaking tolerance with engineered class I antigen-presenting molecules.
- Author
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Parks, Christopher A., Henning, Kalli R., Pavelko, Kevin D., Hansen, Michael J., Van Keulen, Virginia P., Reed, Brendan K., Stone, Jennifer D., Schrum, Adam G., Gil, Diana, Kranz, David M., Bordner, Andrew J., Barry, Michael A., and Peas, Larry R.
- Subjects
ANTIGENS ,T cells ,PEPTIDES ,CANCER immunotherapy ,MAJOR histocompatibility complex - Abstract
Successful efforts to activate T cells capable of recognizing weak cancer-associated self-antigens have employed altered peptide antigens to activate T cell responses capable of cross-reacting on native tumor-associated self. A limitation of this approach is the requirement for detailed knowledge about the altered selfpeptide ligands used in these vaccines. In the current study we considered allorecognition as an approach for activating CTL capable of recognizing weak or self-antigens in the context of self-MHC. Nonself antigen-presenting molecules typically contain polymorphisms that influence interactions with the bound peptide and TCR interface. Recognition of these nonself structures results in peptide-dependent alloimmunity. Alloreactive T cells target their inducing alloantigens as well as third-party alloantigens but generally fail to target self-antigens. Certain residues located on the alpha-1/2 domains of class I antigen-presenting molecules primarily interface with TCR. These residues are more conserved within and across species than are residues that determine peptide antigen binding properties. Class I variants designed with amino acid substitutions at key positions within the conserved helical structures are shown to provide strong activating signals to alloreactive CD8 T cells while avoiding changes in naturally bound peptide ligands. Importantly, CTL activated in this manner can break self-tolerance by reacting to self-peptides presented by native MHC. The ability to activate self-tolerant T cells capable of cross-reacting on self-peptide-MHC in vivo represents an approach for inducing autoimmunity, with possible application in cancer vaccines. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
49. Molecular basis for AU-rich element recognition and dimerization by the HuR C-terminal RRM.
- Author
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Ripin, Nina, Boudet, Julien, Duszczyk, Malgorzata M., Hinniger, Alexandra, Faller, Michael, Krepl, Miroslav, Gadi, Abhilash, Schneider, Robert J., Šponer, Jiří, Meisner-Kober, Nicole C., and Allain, Frédéric H.- T.
- Subjects
ANTIGENS ,MESSENGER RNA ,ADENYLATE cyclase ,NUCLEAR magnetic resonance spectroscopy ,RNA-binding proteins - Abstract
Human antigen R (HuR) is a key regulator of cellular mRNAs containing adenylate/uridylate-rich elements (AU-rich elements; AREs). These are a major class of cis elements within 3' untranslated regions, targeting these mRNAs for rapid degradation. HuR contains three RNA recognition motifs (RRMs): a tandem RRM1 and 2, followed by a flexible linker and a C-terminal RRM3. While RRM1 and 2 are structurally characterized, little is known about RRM3. Here we present a 1.9-Å-resolution crystal structure of RRM3 bound to different ARE motifs. This structure together with biophysical methods and cellculture assays revealed the mechanism of RRM3 ARE recognition and dimerization.While multiple RNA motifs can be bound, recognition of the canonical AUUUA pentameric motif is possible by binding to two registers. Additionally, RRM3 forms homodimers to increase its RNA binding affinity. Finally, although HuR stabilizes ARE-containing RNAs, we found that RRM3 counteracts this effect, as shown in a cell-based ARE reporter assay and by qPCR with native HuRmRNA targets containing multiple AUUUA motifs, possibly by competing with RRM12. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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50. Multistate design of influenza antibodies improves affinity and breadth against seasonal viruses.
- Author
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Sevy, Alexander M., Wu, Nicholas C., Gilchuk, Iuliia M., Parrish, Erica H., Burger, Sebastian, Yousif, Dina, Nagel, Marcus B. M., Schey, Kevin L., Wilson, Ian A., Crowe Jr., James E., and Meiler, Jens
- Subjects
INFLUENZA ,IMMUNOGLOBULINS ,PUBLIC health ,ANTIGENS ,PROTEINS - Abstract
Influenza is a yearly threat to global public health. Rapid changes in influenza surface proteins resulting from antigenic drift and shift events make it difficult to readily identify antibodies with broadly neutralizing activity against different influenza subtypes with high frequency, specifically antibodies targeting the receptor binding domain (RBD) on influenza HA protein. We developed an optimized computational designmethod that is able to optimize an antibody for recognition of large panels of antigens. To demonstrate the utility of this multistate design method, we used it to redesign an antiinfluenza antibody against a large panel of more than 500 seasonal HA antigens of the H1 subtype. As a proof of concept, we tested this method on a variety of known antiinfluenza antibodies and identified those that could be improved computationally. We generated redesigned variants of antibody C05 to the HA RBD and experimentally characterized variants that exhibited improved breadth and affinity against our panel. C05 mutants exhibited improved affinity for three of the subtypes used in design by stabilizing the CDRH3 loop and creating favorable electrostatic interactions with the antigen. These mutants possess increased breadth and affinity of binding while maintaining high-affinity binding to existing targets, surpassing a major limitation up to this point. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
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