Affinity of anti-spike antibodies to three major SARS-CoV-2 variants in recipients of three major vaccines.

Background: Measuring anti-viral antibody affinity in blood plasma or serum is a rational quantitative approach to assess humoral immune response and acquired protection. Three common vaccines against SARS-CoV-2-Comirnaty developed by Pfizer/BioNTech, Spikevax developed by Moderna/NIAID, and Jcovden (previously Janssen COVID-19 Vaccine) developed by Johnson & Johnson/Janssen (J&J)-induce antibodies to a variety of immunogenic epitopes including the epitopes located in the ACE2 receptor-binding domain (RBD) of the spike protein. Blocking RBD with antibodies interferes with the binding of the virus to ACE2 thus protecting against infection. Methods: We perform measurements in the serum of the recipients of Pfizer, Moderna, and J&J vaccines, and we compare the apparent affinities of vaccine-induced antibodies against the RBD of the ancestral SARS-CoV-2 virus and the Delta and Omicron variants. We use our recently published method to determine the apparent affinity of anti-spike protein antibodies directly in human serum. This involves probing antibody-antigen equilibria with a small number of antigen-coated magnetic microparticles and imaging them on a fluorescence microscope. Results: Recipients of two-dose Pfizer and Moderna vaccines, as well as recipients of the single-dose J&J vaccine, develop high-affinity antibodies toward RBD derived from ancestral SARS-CoV-2. Affinities of these antibodies to Delta-RBD are approximately 10 times weaker, and even more drastically reduced (∼1000-fold) toward Omicron-RBD. Conclusions: Vaccine-induced antibodies against ancestral SARS-CoV-2 RBD demonstrate ~10-fold and ~1000-fold weaker affinities toward Delta- and Omicron-RBD, respectively. Our approach offers a direct means for evaluating vaccine-induced adaptive immunity and can be helpful in designing or updating vaccines.

Affinity of anti-spike antibodies in SARS-CoV-2 patient plasma and its effect on COVID-19 antibody assays.

BACKGROUND: Measuring anti-spike protein antibodies in human plasma or serum is commonly used to determine prior exposure to SARS-CoV-2 infection and to assess the anti-viral protection capacity. According to the mass-action law, a lesser concentration of tightly binding antibody can produce the same quantity of antibody-antigen complexes as higher concentrations of lower affinity antibody. Thus, measurements of antibody levels reflect both affinity and concentration. These two fundamental parameters cannot be disentangled in clinical immunoassays, and so produce a bias which depends on the assay format. METHODS: To determine the apparent affinity of anti-spike protein antibodies, a small number of antigen-coated magnetic microparticles were imaged by fluorescence microscopy after probing antigen-antibody equilibria directly in patient plasma. Direct and indirect anti-SARS-CoV-2 immunoassays were used to measure antibody levels in the blood of infected and immunised individuals. FINDINGS: We observed affinity maturation of antibodies in convalescent and vaccinated individuals, showing that higher affinities are achieved much faster by vaccination. We demonstrate that direct and indirect immunoassays for measuring anti-spike protein antibodies depend differently on antibody affinity which, in turn, affects accurate interpretation of the results. INTERPRETATION: Direct immunoassays show substantial antibody affinity dependence. This makes them useful for identifying past SARS-CoV-2 exposure. Indirect immunoassays provide more accurate quantifications of anti-viral antibody levels. FUNDING: The authors are all full-time employees of Abbott Laboratories. Abbott Laboratories provided all operating funds. No external funding sources were used in this study.

Direct single-molecule imaging for diagnostic and blood screening assays.

Every year, over 100 million units of donated blood undergo mandatory screening for HIV, hepatitis B, hepatitis C, and syphilis worldwide. Often, donated blood is also screened for human T cell leukemia-lymphoma virus, Chagas, dengue, Babesia, cytomegalovirus, malaria, and other infections. Several billion diagnostic tests are performed annually around the world to measure more than 400 biomarkers for cardiac, cancer, infectious, and other diseases. Considering such volumes, every improvement in assay performance and/or throughput has a major impact. Here, we show that medically relevant assay sensitivities and specificities can be fundamentally improved by direct single-molecule imaging using regular epifluorescence microscopes. In current microparticle-based assays, an ensemble of bound signal-generating molecules is measured as a whole. By contrast, we acquire intensity profiles to identify and then count individual fluorescent complexes bound to targets on antibody-coated microparticles. This increases the signal-to-noise ratio and provides better discrimination over nonspecific effects. It brings the detection sensitivity down to the attomolar (10-18 M) for model assay systems and to the low femtomolar (10-16 M) for measuring analyte in human plasma. Transitioning from counting single-molecule peaks to averaging pixel intensities at higher analyte concentrations enables a continuous linear response from 10-18 to 10-5 M. Additionally, our assays are insensitive to microparticle number and volume variations during the binding reaction, eliminating the main source of uncertainties in standard assays. Altogether, these features allow for increased assay sensitivity, wide linear detection ranges, shorter incubation times, simpler assay protocols, and minimal reagent consumption.

Acridone and acridinium constructs with red-shifted emission.

Acridinium 9-carboxylic acid derivatives have been extensively used as chemiluminescent labels in diagnostic assays. Triggering acridinium with basic hydrogen peroxide produces a highly strained dioxetanone intermediate, which converts into an acridone in an electronically excited state and emits light at 420-440 nm. Here, we introduce a novel acridinium-fluorescein construct emitting at 530 nm, in which fluorescein is covalently attached to the acridinium N-10 nitrogen via a propyl sulfonamide linker. To characterize the spectral properties of the acridinium-fluorescein chemiluminophores, we synthesized the analogous acridone-fluorescein constructs. Both acridinium and acridone were linked to either 5- or 6-carboxyfluorescein and independently synthesized as individual structural isomers. Using fluorescent acridone-fluorophore tandems, we investigated and optimized the diluent composition to prevent dye aggregation. As monomolecular species, the acridone isomers demonstrated similar absorption, excitation, and emission spectra, as well as the expected fluorescence lifetimes and molecular brightness. Chemical triggering of acridinium-fluorescein tandems, as well as direct excitation of their acridone-fluorescein analogs, resulted in a nearly complete energy transfer from acridone to fluorescein. Acridone-based dyes can be studied with steady-state spectroscopy. Thus, they will serve as useful tools for structure and solvent optimizations, as well as for studying chemiluminescent energy transfer mechanisms in related acridinium-fluorophore tandems. Direct investigations of the light-emitting molecules generated in the acridinium chemiluminescent reaction empower further development of chemiluminescent labels with red-shifted emission. As illustrated by the two-color HIV model immunoassay, such labels can find immediate applications for multicolor detection in clinical diagnostic assays.

A Single Common Protocol for the Expression and Purification of Soluble Mammalian DSPs from Escherichia coli.

Mammalian DSPs have been historically isolated either from native tissue sources or from transfected insect cell cultures via time-consuming and cumbersome protocols often yielding protein of variable quality and quantity. A facile and highly reproducible alternative methodology involving the heterologous expression and purification of soluble mammalian DSPs from E. coli, which yields highly active and functional protein of a uniform quality and quantity, free of spurious posttranslational modifications inherent to mammalian and insect cell expression systems, is described in this chapter.

Direct single-molecule counting for immunoassay applications.

Single-molecule methods offer specificity in studying complex systems and dynamics, but they also offer high sensitivity for basic enumeration. We apply single-molecule TIRF to immunoassays by counting the number of target molecules captured on a streptavidin surface. We demonstrate the utility of using single-molecule counting on eluted detection conjugate, following the capture and sandwich formation portions of the assay having been completed on microparticles. This approach is simple and effective, and creates the opportunity for a universal detection platform that can be used to perform a variety of diagnostic and blood screening assays. We take advantage of the low volume requirements of single-molecule detection and apply a sample reloading approach to concentrate sample onto the detection surface. Due to the high affinity of the streptavidin-biotin reaction, concentration through reloading is both quick and robust. These findings are demonstrated on a model system and in an HIV p24 antigen assay. Single-molecule detection techniques do not need to be complex to exhibit power and flexibility, and so can become valuable in the field of immunoassay diagnostics.

Steric interference from intrinsically disordered regions controls dynamin-related protein 1 self-assembly during mitochondrial fission.

The self-assembling, mechanoenzymatic dynamin superfamily GTPase, dynamin-related protein 1 (Drp1), catalyzes mitochondrial and peroxisomal fission. Distinct intrinsically disordered regions (IDRs) in Drp1 substitute for the canonical pleckstrin homology (PH) domain and proline-rich domain (PRD) of prototypical dynamin, which cooperatively regulate endocytic vesicle scission. Whether the Drp1 IDRs function analogously to the corresponding dynamin domains however remains unknown. We show that an IDR unique to the Drp1 GTPase (G) domain, the 'extended 80-loop', albeit dissimilar in location, structure, and mechanism, functions akin to the dynamin PRD by enabling stable Drp1 mitochondrial recruitment and by suppressing Drp1 cooperative GTPase activity in the absence of specific partner-protein interactions. Correspondingly, we find that another IDR, the Drp1 variable domain (VD), in conjunction with the conserved stalk L1N loop, functions akin to the dynamin PH domain; first, in an 'auto-inhibitory' capacity that restricts Drp1 activity through a long-range steric inhibition of helical inter-rung G-domain dimerization, and second, as a 'fulcrum' for Drp1 self-assembly in the proper helical register. We show that the Drp1 VD is necessary and sufficient for specific Drp1-phospholipid interactions. We further demonstrate that the membrane-dependent VD conformational rearrangement essential for the alleviation of Drp1 auto-inhibition is contingent upon the basal GTP hydrolysis-dependent generation of Drp1 dimers from oligomers in solution. IDRs thus conformationally couple the enzymatic and membrane activities of Drp1 toward membrane fission.

Rhodamine-Derived Fluorescent Dye with Inherent Blinking Behavior for Super-Resolution Imaging.

Super-resolution microscopy enables imaging of structures smaller than the diffraction limit. Single-molecule localization microscopy methods, such as photoactivation localization microscopy and stochastic optical reconstruction microscopy, reconstruct images by plotting the centroids of fluorescent point sources from a series of frames in which only a few molecules are fluorescing at a time. These approaches require simpler instrumentation than methods that depend on structured illumination and thus are becoming widespread. The functionalized rhodamine derivative reported in this paper spontaneously converts between a bright and dark state due to pH-dependent cyclization. At pH 7, less than 0.5% of the dye molecules are fluorescent at any given time. Blinking occurs on time scales of seconds to minutes and can therefore be used for single-molecule localization microscopy without sample treatment or activation. The dye is bright and straightforward to use, and it is easy to synthesize and functionalize. Thus, it has potential to become a new and powerful addition to the toolset for super-resolution imaging.

Distinct Splice Variants of Dynamin-related Protein 1 Differentially Utilize Mitochondrial Fission Factor as an Effector of Cooperative GTPase Activity.

Multiple isoforms of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) arise from the alternative splicing of its single gene-encoded pre-mRNA transcript. Among these, the longer Drp1 isoforms, expressed selectively in neurons, bear unique polypeptide sequences within their GTPase and variable domains, known as the A-insert and the B-insert, respectively. Their functions remain unresolved. A comparison of the various biochemical and biophysical properties of the neuronally expressed isoforms with that of the ubiquitously expressed, and shortest, Drp1 isoform (Drp1-short) has revealed the effect of these inserts on Drp1 function. Utilizing various biochemical, biophysical, and cellular approaches, we find that the A- and B-inserts distinctly alter the oligomerization propensity of Drp1 in solution as well as the preferred curvature of helical Drp1 self-assembly on membranes. Consequently, these sequences also suppress Drp1 cooperative GTPase activity. Mitochondrial fission factor (Mff), a tail-anchored membrane protein of the mitochondrial outer membrane that recruits Drp1 to sites of ensuing fission, differentially stimulates the disparate Drp1 isoforms and alleviates the autoinhibitory effect imposed by these sequences on Drp1 function. Moreover, the differential stimulatory effects of Mff on Drp1 isoforms are dependent on the mitochondrial lipid, cardiolipin (CL). Although Mff stimulation of the intrinsically cooperative Drp1-short isoform is relatively modest, CL-independent, and even counter-productive at high CL concentrations, Mff stimulation of the much less cooperative longest Drp1 isoform (Drp1-long) is robust and occurs synergistically with increasing CL content. Thus, membrane-anchored Mff differentially regulates various Drp1 isoforms by functioning as an allosteric effector of cooperative GTPase activity.

Cardiolipin's propensity for phase transition and its reorganization by dynamin-related protein 1 form a basis for mitochondrial membrane fission.

Cardiolipin (CL) is an atypical, dimeric phospholipid essential for mitochondrial dynamics in eukaryotic cells. Dynamin-related protein 1 (Drp1), a cytosolic member of the dynamin superfamily of large GTPases, interacts with CL and functions to sustain the balance of mitochondrial division and fusion by catalyzing mitochondrial fission. Although recent studies have indicated a role for CL in stimulating Drp1 self-assembly and GTPase activity at the membrane surface, the mechanism by which CL functions in membrane fission, if at all, remains unclear. Here, using a variety of fluorescence spectroscopic and imaging approaches together with model membranes, we demonstrate that Drp1 and CL function cooperatively in effecting membrane constriction toward fission in three distinct steps. These involve 1) the preferential association of Drp1 with CL localized at a high spatial density in the membrane bilayer, 2) the reorganization of unconstrained, fluid-phase CL molecules in concert with Drp1 self-assembly, and 3) the increased propensity of CL to transition from a lamellar, bilayer arrangement to an inverted hexagonal, nonbilayer configuration in the presence of Drp1 and GTP, resulting in the creation of localized membrane constrictions that are primed for fission. Thus we propose that Drp1 and CL function in concert to catalyze mitochondrial division.

Quantifying protein-protein interactions of peripheral membrane proteins by fluorescence brightness analysis.

Fluorescently labeled proteins that are found both in the cytoplasm and at the plasma membrane, such as peripheral membrane proteins, create stratified fluorescent layers that present a challenging environment for brightness studies with fluorescence fluctuation spectroscopy. The geometry of each layer along with fluorescence and brightness contributions from adjacent layers generates a convoluted raw brightness that conceals the underlying brightness of each individual layer. Because the brightness at a layer establishes the oligomeric state of the fluorescently labeled protein at said layer, we developed a method that connects the experimental raw brightness with the physical brightness at each layered compartment. The technique determines the oligomerization in each compartment from an axial intensity scan through the sample, followed by a fluorescence fluctuation spectroscopy measurement at each layer. We experimentally verify the technique with H-Ras-EGFP as a model system and determine its oligomeric state at both the plasma membrane and in the cytoplasm. Furthermore, we study the oligomerization of the Gag matrix domain of Human T-lymphotropic virus Type 1. The matrix domain targets the Gag polyprotein to the plasma membrane where, subsequently, viral assembly occurs. We determine the oligomerization of matrix in the cytoplasm and observe the onset of protein-protein interactions at the membrane. These observations shed light on the early assembly steps of the retrovirus.

Quantitative measurement of brightness from living cells in the presence of photodepletion.

The brightness of fluorescently labeled proteins provides an excellent marker for identifying protein interactions in living cells. Quantitative interpretation of brightness, however, hinges on a detailed understanding of the processes that affect the signal fluctuation of the fluorescent label. Here, we focus on the cumulative influence of photobleaching on brightness measurements in cells. Photobleaching within the finite volume of the cell leads to a depletion of the population of fluorescently labeled proteins with time. The process of photodepletion reduces the fluorescence signal which biases the analysis of brightness data. Our data show that even small reductions in the signal can introduce significant bias into the analysis of the data. We develop a model that quantifies the bias and introduce an analysis method that accurately determines brightness in the presence of photodepletion as verified by experiments with mammalian and yeast cells. In addition, photodepletion experiments with the fluorescent protein EGFP reveal the presence of a photoconversion process, which leads to a marked decrease in the brightness of the EGFP protein. We also identify conditions where the effect of EGFP's photoconversion on brightness experiments can be safely ignored.

A dimeric equilibrium intermediate nucleates Drp1 reassembly on mitochondrial membranes for fission.

The GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial division, but the mechanisms remain poorly understood. Much of what is attributed to Drp1's mechanism of action in mitochondrial membrane fission parallels that of prototypical dynamin in endocytic vesicle scission. Unlike the case for dynamin, however, no lipid target for Drp1 activation at the mitochondria has been identified. In addition, the oligomerization properties of Drp1 have not been well established. We show that the mitochondria-specific lipid cardiolipin is a potent stimulator of Drp1 GTPase activity, as well as of membrane tubulation. We establish further that under physiological conditions, Drp1 coexists as two morphologically distinct polymeric species, one nucleotide bound in solution and the other membrane associated, which equilibrate via a dimeric assembly intermediate. With two mutations, C300A and C505A, that shift Drp1 polymerization equilibria in opposite directions, we demonstrate that dimers, and not multimers, potentiate the reassembly and reorganization of Drp1 for mitochondrial membrane remodeling both in vitro and in vivo.

Brightness experiments.

This chapter presents an overview of quantitative fluorescence brightness experiments with special emphasis on single-color measurements of protein homo-interactions inside living cells. We discuss practical considerations in the choice of the fluorescent labels and the calibration measurements necessary for quantitative interpretation of brightness experiments. Methods to identify and avoid common pitfalls, such as bleaching and saturation, are addressed. We examine the interpretation of brightness data with moment analysis. In particular, we focus on how to avoid or correct for undersampling, as well as how to characterize and adjust for photon detector effects. We conclude by describing brightness titration experiments which determine the binding curve and stoichiometry of a protein from apparent brightness data.

APOBEC3 multimerization correlates with HIV-1 packaging and restriction activity in living cells.

APOBEC3G belongs to a family of DNA cytosine deaminases that are involved in the restriction of a broad number of retroviruses including human immunodeficiency virus type 1 (HIV-1). Prior studies have identified two distinct mechanistic steps in Vif-deficient HIV-1 restriction: packaging into virions and deaminating viral cDNA. APOBEC3A, for example, although highly active, is not packaged and is therefore not restrictive. APOBEC3G, on the other hand, although having weaker enzymatic activity, is packaged into virions and is strongly restrictive. Although a number of studies have described the propensity for APOBEC3 oligomerization, its relevance to HIV-1 restriction remains unclear. Here, we address this problem by examining APOBEC3 oligomerization in living cells using molecular brightness analysis. We find that APOBEC3G forms high-order multimers as a function of protein concentration. In contrast, APOBEC3A, APOBEC3C and APOBEC2 are monomers at all tested concentrations. Among other members of the APOBEC3 family, we show that the multimerization propensities of APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3H (haplotype II) bear more resemblance to APOBEC3G than to APOBEC3A/3C/2. Prior studies have shown that all of these multimerizing APOBEC3 proteins, but not the monomeric family members, have the capacity to package into HIV-1 particles and restrict viral infectivity. This correlation between oligomerization and restriction is further evidenced by two different APOBEC3G mutants, which are each compromised for multimerization, packaging and HIV-1 restriction. Overall, our results imply that multimerization of APOBEC3 proteins may be related to the packaging mechanism and ultimately to virus restriction.

Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system.

Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37°C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction.

Characterization of cytoplasmic Gag-gag interactions by dual-color z-scan fluorescence fluctuation spectroscopy.

Fluorescence fluctuation spectroscopy (FFS) quantifies the interactions of fluorescently-labeled proteins inside living cells by brightness analysis. However, the study of cytoplasmic proteins that interact with the plasma membrane is challenging with FFS. If the cytoplasmic section is thinner than the axial size of the observation volume, cytoplasmic and membrane-bound proteins are coexcited, which leads to brightness artifacts. This brightness bias, if not recognized, leads to erroneous interpretation of the data. We have overcome this challenge by introducing dual-color z-scan FFS and the addition of a distinctly colored reference protein. Here, we apply this technique to study the cytoplasmic interactions of the Gag proteins from human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type 1 (HTLV-1). The Gag protein plays a crucial role in the assembly of retroviruses and is found in both membrane and cytoplasm. Dual-color z-scans demonstrate that brightness artifacts are caused by a dim nonpunctate membrane-bound fraction of Gag. We perform an unbiased brightness characterization of cytoplasmic Gag by avoiding the membrane-bound fraction and reveal previously unknown differences in the behavior of the two retroviral Gag species. HIV-1 Gag exhibits concentration-dependent oligomerization in the cytoplasm, whereas HTLV-1 Gag lacks significant cytoplasmic Gag-Gag interactions.

Brightness analysis by Z-scan fluorescence fluctuation spectroscopy for the study of protein interactions within living cells.

Fluorescence fluctuation spectroscopy (FFS) quantifies interactions of fluorescently labeled proteins inside living cells by brightness analysis. Conventional FFS implicitly requires that the sample thickness exceeds the size of the observation volume. This condition is not always fulfilled when measuring cells. Cytoplasmic sections, especially, can be thinner than the axial size of the observation volume. The finite sample thickness introduces a brightness bias which, if not recognized, leads to an erroneous interpretation of the data. To avoid this artifact, we introduce z-scan FFS which consists of a fluorescence intensity z scan through the sample followed by an FFS measurement. To model the experimental z-scan data, a new PSF model had to be introduced. We use the intensity z scan together with the PSF model to determine the geometry of the sample and then extract the brightness from the FFS data. Cells expressing EGFP serve as a model system for testing the experimental approach. We demonstrate that z-scan FFS abolishes the brightness artifact and use the method to determine the oligomerization of cytoplasmic nuclear transport factor 2.

Probing nucleocytoplasmic transport by two-photon activation of PA-GFP.

Two-photon activation of photoactivatable green fluorescent protein (PA-GFP) provides a unique tool for probing cellular transport processes, because activation is strictly limited to the sub-femtoliter optical volume of the two-photon spot. We demonstrate two-photon activation of PA-GFP immobilized in a gel and freely diffusing within cells and recover a quadratic power dependence. Illumination at 820 nm allows simultaneous activation and fluorescence monitoring by two-photon excitation. Alternatively, we activate PA-GFP using two-photon excitation and monitor the fluorescence of the photoconverted product with one-photon excitation. We probe nucleocytoplasmic transport through the nuclear pore complex of COS-1 cells, by observing the time-dependent fluorescence at various locations within the cell after two-photon activation of PA-GFP in the nucleus and in the cytoplasm. Two-photon activation of a tandem construct of two PA-GFPs showed a markedly slower rate of crossing through the nuclear pore. Analysis based on a restricted diffusion model yields a nuclear pore radius of 4.5 nm, which is in good agreement with previously reported values. This application demonstrates the attractive features of two-photon photoactivation over traditional techniques, such as photobleaching, for studying transport processes in cells.