Binding affinity landscapes constrain the evolution of broadly neutralizing anti-influenza antibodies

Over the past two decades, several broadly neutralizing antibodies (bnAbs) that confer protection against diverse influenza strains have been isolated. Structural and biochemical characterization of these bnAbs has provided molecular insight into how they bind distinct antigens. However, our understanding of the evolutionary pathways leading to bnAbs, and thus how best to elicit them, remains limited. Here, we measure equilibrium dissociation constants of combinatorially complete mutational libraries for two naturally isolated influenza bnAbs (CR-9114, 16 mutations; CR-6261, 11 mutations), reconstructing all possible intermediates back to the unmutated germline sequences. We find that these two libraries exhibit strikingly different patterns of breadth: while many variants of CR-6261 display moderate affinity to diverse antigens, those of CR-9114 display appreciable affinity only in specific, nested combinations. By examining the extensive pairwise and higher-order epistasis between mutations, we find key sites with strong synergistic interactions that are highly similar across antigens for CR-6261 and different for CR-9114. Together, these features of the binding affinity landscapes strongly favor sequential acquisition of affinity to diverse antigens for CR-9114, while the acquisition of breadth to more similar antigens for CR-6261 is less constrained. These results, if generalizable to other bnAbs, may explain the molecular basis for the widespread observation that sequential exposure favors greater breadth, and such mechanistic insight will be essential for predicting and eliciting broadly protective immune responses.

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Binding affinity landscapes constrain the evolution of broadly neutralizing anti-influenza antibodies

eLife ◽

10.7554/elife.71393 ◽

2021 ◽

Vol 10 ◽

Author(s):

Angela M Phillips ◽

Katherine R Lawrence ◽

Alief Moulana ◽

Thomas Dupic ◽

Jeffrey Chang ◽

...

Keyword(s):

Binding Affinity ◽

Heavy Chain ◽

Neutralizing Antibodies ◽

Biochemical Characterization ◽

Dissociation Constants ◽

Synergistic Interactions ◽

Key Sites ◽

Equilibrium Dissociation Constants ◽

Equilibrium Dissociation

Over the past two decades, several broadly neutralizing antibodies (bnAbs) that confer protection against diverse influenza strains have been isolated. Structural and biochemical characterization of these bnAbs has provided molecular insight into how they bind distinct antigens. However, our understanding of the evolutionary pathways leading to bnAbs, and thus how best to elicit them, remains limited. Here, we measure equilibrium dissociation constants of combinatorially complete mutational libraries for two naturally isolated influenza bnAbs (CR9114, 16 heavy-chain mutations; CR6261, 11 heavy-chain mutations), reconstructing all possible evolutionary intermediates back to the unmutated germline sequences. We find that these two libraries exhibit strikingly different patterns of breadth: while many variants of CR6261 display moderate affinity to diverse antigens, those of CR9114 display appreciable affinity only in specific, nested combinations. By examining the extensive pairwise and higher-order epistasis between mutations, we find key sites with strong synergistic interactions that are highly similar across antigens for CR6261 and different for CR9114. Together, these features of the binding affinity landscapes strongly favor sequential acquisition of affinity to diverse antigens for CR9114, while the acquisition of breadth to more similar antigens for CR6261 is less constrained. These results, if generalizable to other bnAbs, may explain the molecular basis for the widespread observation that sequential exposure favors greater breadth, and such mechanistic insight will be essential for predicting and eliciting broadly protective immune responses.

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Preparation and Characterization of Paclitaxel Imprinted Silica Nanoparticles

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.399-401.1894 ◽

2011 ◽

Vol 399-401 ◽

pp. 1894-1897

Author(s):

Jian Hua Li ◽

Zong Jian Zheng ◽

Shao Ping Fu ◽

Jing Bo Zhu

Keyword(s):

Silica Nanoparticles ◽

Dissociation Constants ◽

Sol Gel ◽

Scatchard Analysis ◽

Binding Properties ◽

Imprinted Polymers ◽

Sol Gel Process ◽

Equilibrium Dissociation Constants ◽

Equilibrium Dissociation

Highly selective molecularly imprinted layer-coated silica nanoparticles for paclitaxel were synthesized by molecular imprinting technique with a sol–gel process on the supporter of silica nanoparticles. The morphology of the obtained polymers was characterized by scanning electron microscopy (SEM). The binding properties of the imprinted polymers were evaluated through the equilibrium rebinding experiments. Scatchard analysis revealed that two classes of binding sites were formed in the imprinted polymers with equilibrium dissociation constants of 0.0509 g•L-1and 0.0094 g•L-1, respectively. Paclitaxel and its analogue were employed for selectivity tests. The results indicated that the imprinted polymers exhibited good selectivity and specificity toward paclitaxel.

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ATP induces two cholecystokinin binding affinity states in permeabilized rat pancreatic acini

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1992.263.1.g44 ◽

1992 ◽

Vol 263 (1) ◽

pp. G44-G51

Author(s):

G. T. Blevins ◽

J. A. Williams

Keyword(s):

Binding Affinity ◽

Competitive Inhibition ◽

Dissociation Constants ◽

Specific Binding ◽

Pancreatic Acini ◽

Streptolysin O ◽

Equilibrium Dissociation Constants ◽

Equilibrium Dissociation ◽

Gamma S ◽

Single Binding Site

The influence of adenine and guanine nucleotides on cholecystokinin (CCK) receptor binding was examined in streptolysin O-permeabilized rat pancreatic acini. Specific binding of tracer to intact acini was 12.1 +/- 0.4% per milligram protein, while permeabilized acini bound 34.6 +/- 2.9% (n = 7). The increase in binding was also seen when normalized to DNA. Binding to permeabilized acini was reduced by the presence of 1 mM ATP to 23.0 +/- 1.3%. Analysis of competitive inhibition of tracer binding by unlabeled CCK-8 was consistent with binding to two affinity states on intact acini, with the equilibrium dissociation constants for the high (KdH)- and low (KdL)-affinity states equal to 41 +/- 5 pM and 5.2 +/- 0.4 nM, respectively; permeabilized acini displayed a single binding site with Kd = 598 +/- 40 pM. In the presence of 1 mM ATP, two states were seen on permeabilized acini with KdH = 85 +/- 11 pM and KdL = 2.7 +/- 0.6 nM. ATP, ATP gamma S, GTP, and GTP gamma S all inhibited binding, with half-maximal inhibition occurring at greater than 1 mM, 21 microM, 5 microM, and 0.4 microM, respectively. GTP gamma S (1 microM) also induced two affinity states with KdH = 112 +/- 7 pM and KdL = 1.5 +/- 0.2 nM (n = 3). Binding of CCK to pancreatic membranes was also decreased by ATP, and a similar regeneration of two binding affinity states was observed. ATP also decreased binding of [125I-Tyr4]bombesin to permeabilized acini, but in contrast did not generate two measurable binding affinity states.(ABSTRACT TRUNCATED AT 250 WORDS)

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Structural and Biochemical Characterization of Cysteinylation in Broadly Neutralizing Antibodies to HIV-1

Journal of Molecular Biology ◽

10.1016/j.jmb.2021.167303 ◽

2021 ◽

pp. 167303

Author(s):

Oluwarotimi Omorodion ◽

Ian A. Wilson

Keyword(s):

Neutralizing Antibodies ◽

Biochemical Characterization ◽

Broadly Neutralizing Antibodies ◽

Hiv 1

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Asprosin Neutralizing Antibodies as a Treatment for Metabolic Syndrome

10.1101/2020.09.15.298489 ◽

2020 ◽

Author(s):

Ila Mishra ◽

Clemens Duerrschmid ◽

Zhiqiang Ku ◽

Wei Xie ◽

Elizabeth Sabath Silva ◽

...

Keyword(s):

Metabolic Syndrome ◽

Blood Glucose ◽

Neutralizing Antibodies ◽

Dissociation Constants ◽

Drug Application ◽

Distinct Species ◽

Pharmacologic Therapy ◽

Equilibrium Dissociation Constants ◽

Equilibrium Dissociation

AbstractRecently, we discovered a new glucogenic and centrally-acting orexigenic hormone – asprosin. Asprosin is elevated in metabolic syndrome (MS) patients, and importantly, its genetic loss results in reduced appetite, leanness and robust insulin sensitivity, leading to protection from MS. Here we demonstrate that anti-asprosin monoclonal antibodies (mAbs) are a dual-effect pharmacologic therapy that targets the two key pillars of MS – over-nutrition and the blood glucose burden. Anti-asprosin mAbs from three distinct species lowered appetite and body weight, and improved blood glucose in a dose-dependent and epitope-agnostic fashion in three independent MS mouse models, with an IC50 of ∼1.5 mg/kg. In addition, mAb treatment ameliorated MS associated dyslipidemia and hepatic dysfunction. The mAbs displayed half-life of over 3 days in vivo, with equilibrium dissociation-constants in picomolar to low nanomolar range. This evidence paves the way for further development towards an investigational new drug application and subsequent human trials for treatment of MS, a defining physical ailment of our time.

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Altered plasma catecholamines and numbers of α- and β-adrenergic receptors in platelets and leucocytes in hyperthyroid patients normalized under antithyroid treatment

Acta Endocrinologica ◽

10.1530/acta.0.1100075 ◽

1985 ◽

Vol 110 (1) ◽

pp. 75-82 ◽

Cited By ~ 7

Author(s):

Dieter Ratge ◽

Sabine Hansel-Bessey ◽

Hermann Wisser

Keyword(s):

Dissociation Constants ◽

Plasma Catecholamines ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Plasma Norepinephrine ◽

Thyroid State ◽

Antithyroid Treatment ◽

Equilibrium Dissociation Constants ◽

Equilibrium Dissociation ◽

Hyperthyroid Patients

Abstract. We measured plasma catecholamines, α- and β-adrenoreceptor numbers and the accumulation of cyclic adenosine monophosphate (cAMP) in the unstimulated state and in response to 10 μmol/l (-) isoproterenol in blood cells from 29 euthyroid controls and from 18 patients with spontaneous hyperthyroidism. In the thyrotoxic patients plasma norepinephrine (1.14 ± 0.5 nmol/l) and epinephrine (0.3 ±0.14 nmol/l) were significantly decreased compared with plasma norepinephrine (1.87 ± 0.7 nmol) and epinephrine (0.41 ± 0.19 nmol/l) in the controls (P < 0.01 and P < 0.05, respectively) and the values obtained in subjects rendered euthyroid by antithyroid treatment (P < 0.001, respectively). α-adrenoceptor density in platelet membranes obtained from patients in the hyperthyroid state (114 ± 38 sites per cell) was significantly decreased when compared with controls (159 ± 48 sites per cell, P < 0.01) and the values from patients under effective antithyroid treatment (136 ± 35 sites per cell, P < 0.01). On the contrary, a significant increase in β-adrenoceptor density in mononuclear leucocyte (MNL) membranes was found in hyperthyroid patients (1751 ± 237 sites/cell) when compared with controls (1510 ± 351 sites/cell, P < 0.05) and the same patients following antithyroid treatment (1455 ± 260 sites/cell, P < 0.001). The equilibrium dissociation constants (KD) did not change in hyperthyroidism. Basal cAMP concentrations in MNL were higher in untreated thyrotoxicosis (45 ± 18 pmol/106 cells/10 min) than in patients in the euthyroid state (35 ± 9 pmol/106 cells/10 min, P < 0.05). Our data support the hypothesis that the balance of α- and β-adrenoceptors depends on the thyroid state. However, before the reputed catecholamine supersensitivity in hyperthyroid man can be accepted, the relationship between alterations in adrenoceptors and the biological responsiveness to catecholamines has to be demonstrated in different human tissues.

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A comprehensive influenza reporter virus panel for high-throughput deep profiling of neutralizing antibodies

Nature Communications ◽

10.1038/s41467-021-21954-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Adrian Creanga ◽

Rebecca A. Gillespie ◽

Brian E. Fisher ◽

Sarah F. Andrews ◽

Julia Lederhofer ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Depth Profiling ◽

Influenza Viruses ◽

Effective Vaccine ◽

Viral Gene ◽

Broadly Neutralizing Antibodies ◽

Level 2 ◽

Biosafety Level ◽

Major Target

AbstractBroadly neutralizing antibodies (bnAbs) have been developed as potential countermeasures for seasonal and pandemic influenza. Deep characterization of these bnAbs and polyclonal sera provides pivotal understanding for influenza immunity and informs effective vaccine design. However, conventional virus neutralization assays require high-containment laboratories and are difficult to standardize and roboticize. Here, we build a panel of engineered influenza viruses carrying a reporter gene to replace an essential viral gene, and develop an assay using the panel for in-depth profiling of neutralizing antibodies. Replication of these viruses is restricted to cells expressing the missing viral gene, allowing it to be manipulated in a biosafety level 2 environment. We generate the neutralization profile of 24 bnAbs using a 55-virus panel encompassing the near-complete diversity of human H1N1 and H3N2, as well as pandemic subtype viruses. Our system offers in-depth profiling of influenza immunity, including the antibodies against the hemagglutinin stem, a major target of universal influenza vaccines.

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