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Previously we established a family of macrocyclic peptide triazoles (cPTs) that inactivate the Env protein complex of HIV-1, and identified the pharmacophore that engages Env’s receptor binding pocket. Here, we examined the hypothesis that the side chains of both components of the triazole Pro - Trp segment of cPT pharmacophore work in tandem to make intimate contacts with two proximal subsites of the overall CD4 binding site of gp120 to stabilize binding and function. Variations of the triazole Pro R group, which previously had been significantly optimized, led to identification of a variant MG-II-20 that contains a pyrazole substitution. MG-II-20 has improved functional properties over previously examined variants, with Kd for gp120 in the nM range. In contrast, new variants of the Trp indole side chain, with either methyl- or bromo- components appended, had disruptive effects on gp120 binding, reflecting the sensitivity of function to changes in this component of the encounter complex. Plausible in silico models of cPT:gp120 complex structures were obtained that are consistent with the overall hypothesisof occupancy by the triazole Pro and Trp side chains, respectively, into the β20/21 and Phe43 sub-cavities. The overall results strengthen the definition of the cPT-Env inactivator binding site and provide a new lead composition (MG-II-20) as well as structure-function findings to guide future HIV-1 Env inactivator design.

Host cell infection by SARS-CoV-2, similar to that by HIV-1, is driven by a conformationally metastable and highly glycosylated surface entry protein complex, and infection by these viruses has been shown to be inhibited by the mannose-specific lectins Cyanovirin-N (CV-N) and Griffithsin (GRFT). We discovered in this study that CV-N and His6-tagged GRFT not only inhibit SARS-CoV-2 infection but also lead to irreversibly inactivated pseudovirus particles. The irreversibility effect was revealed by the observation that pseudoviruses first treated with either CV-N or GRFT and then washed to remove all soluble lectin did not recover infectivity. Infection inhibition of SARS-CoV-2 pseudovirus mutants with single-site glycan mutations in Spike suggested that two glycan clusters in S1 are important for both CV-N and GRFT inhibition, one cluster associated with the RBD (receptor binding domain) and the second with the S1/S2 cleavage site. We observed lectin antiviral effects with several SARS-CoV-2 pseudovirus variants, including the recently emerged omicron, as well as a fully infectious coronavirus, therein reflecting the breadth of lectin antiviral function and the potential for pan-coronavirus inactivation. Mechanistically, observations made in this work indicate that multivalent lectin interaction with S1 glycans is likely a driver of the lectin infection inhibition and irreversible inactivation effect and suggest the possibility that lectin inactivation is caused by an irreversible conformational effect on Spike. Overall, lectins' irreversible inactivation of SARS-CoV-2, taken with their breadth of function, reflects the therapeutic potential of multivalent lectins targeting the vulnerable metastable Spike before host cell encounter.

Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have demonstrated strong immunogenicity and protection against severe disease, concerns about the duration and breadth of these responses remain. In this study, we show that codelivery of plasmid-encoded adenosine deaminase-1 (pADA) with SARS-CoV-2 spike glycoprotein DNA enhances immune memory and durability in vivo. Coimmunized mice displayed increased spike-specific IgG of higher affinity and neutralizing capacity as compared with plasmid-encoded spike-only–immunized animals. Importantly, pADA significantly improved the longevity of these enhanced responses in vivo. This coincided with durable increases in frequencies of plasmablasts, receptor-binding domain–specific memory B cells, and SARS-CoV-2–specific T follicular helper cells. Increased spike-specific T cell polyfunctionality was also observed. Notably, animals coimmunized with pADA had significantly reduced viral loads compared with their nonadjuvanted counterparts in a SARS-CoV-2 infection model. These data suggest that pADA enhances immune memory and durability and supports further translational studies.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease of 2019 (COVID-19), has infected millions of people causing a global pandemic. SARS-CoV-2 vaccines candidates have demonstrated acute immunogenicity and protection however, it has yet to be demonstrated whether natural or vaccine induced immunity against SARS-CoV-2 induces long term, protective immunity. In this study we sought to understand if adenosine deaminase (ADA), as a molecular adjuvant, can enhance immune memory and durability in the context of a SARS-CoV-2 DNA vaccine. Mice were immunized with a plasmid encoding for SARS-CoV-2 spike alone or in combination with plasmid encoded ADA. Subsequent B and T cell responses were measured until d60pi. In mice co-immunized with ADA, there were increased concentrations of spike receptor binding domain (RBD)-specific IgG in the sera which were found to bind RBD at an increased affinity as well as exhibit increased neutralization capability against SARS-CoV-2 pseudotyped viruses. Additionally, ADA co-immunized mice exhibited increased frequency of RBD specific memory B cells. In regard to T cell responses, mice co-immunized with ADA exhibited increased spike-specific IFN-γ, TNF-a and IL-2 as measured by flow cytometry and ELISpot. The ADA-enhanced anti-spike antibody durability over time was associated with increased frequencies of T follicular helper cells (TFH). Preliminary analysis supports that co-immunization with pADA impacts viral load in a SARS-CoV-2 infection model. These data suggest that ADA enhances immune memory and durability and supports further study with translational focus for enhancement of vaccines.

SARS-CoV-2 is responsible for a global pandemic claiming over 2 million lives and infecting over 100 million people. Elderly patients display increased COVID-19 morbidity and mortality. Vaccine candidates have been studied and deployed in the clinic. However, age-associated immune deficits are known to cause sub-optimal responses, and the longevity of vaccine-induced responses is under investigation. Germinal center follicular helper T cells (TFH) promote affinity maturation of B cell receptors and memory B cell differentiation and are defined by expression of adenosine deaminase-1 (ADA-1). We investigated the adjuvant properties of plasmid-encoded adenosine deaminase (pADA) in the context of a SARS-CoV-2 spike synDNA antigens. Young and aged mice were immunized with plasmid-encoded spike (pS) alone or co-immunized with pS and pADA and cellular and humoral responses were evaluated. When immunized with pS alone, aged mice had decreased spike-binding IgG and neutralization titers. However, young and aged animals co-immunized with pADA had similar humoral responses. We observed similar trends in serum antibody affinity as measured by SPR. pADA co-immunization also rescued age-associated decreases in spike-specific IFNy secretion in the spleens and lungs of co-immunized aged mice as measured by ELISpot. Finally, preliminary data analysis indicates that co-immunization with pADA significantly impacts viral load in a model of SARS-CoV-2 infection. These data suggest that pADA enhances antigen-specific cellular and humoral immunity in aged mice and supports further study of this molecule as an immunoadjuvant for vaccines targeting elderly populations.

In the human adaptation and optimization of a mouse anti-human respiratory syncytial virus neutralizing antibody, affinity assessment was crucial to distinguish among potential candidates and to evaluate whether this correlated with function in vitro and in vivo. This affinity assessment was complicated by the trimeric nature of the antigen target, respiratory syncytial virus F (RSV-F) glycoprotein. In the initial affinity screen, surface plasmon resonance was used to determine the intrinsic binding affinities of anti-RSV-F Fab and immunoglobulin G (IgG) to the extracellular domain of RSV-F. This assessment required minimal biotinylation of the RSV-F protein and design of a capture strategy to minimize avidity effects. Approximately 30 Fabs were selected from three optimization phage display libraries on the basis of an initial ELISA screen. Surface plasmon resonance analysis demonstrated the success of optimization with some candidates from the screened libraries having low picomolar dissociation constants, more than 700-fold tighter than the parental monoclonal antibody (B21M). The affinities of these antibodies were further evaluated by a kinetic exclusion assay, a solution binding technology. One IgG (monoclonal antibody 029) displayed a low picomolar K(D) comparable with that of motavizumab, an RSV antibody in clinical study. Kinetic exclusion assay showed that two other of the matured IgGs (011 and 019) had sub-picomolar dissociation constants that could not be resolved further. We discuss the relevance of these interaction analysis results in the light of recently published data on the mechanism of F-driven viral fusion during paramyxoviral infection and 101F epitope conservation revealed from the recent crystal structure of RSV-F in the post-fusion state.

Alzheimer's disease is a progressive neurodegenerative disease characterized by extracellular deposits of β-amyloid (Aβ) plaques. Aggregation of the Aβ42 peptide leading to plaque formation is believed to play a central role in Alzheimer's disease pathogenesis. Anti-Aβ monoclonal antibodies can reduce amyloid plaques and could possibly be used for immunotherapy. We have developed a monoclonal antibody C706, which recognizes the human Aβ peptide. Here we report the crystal structure of the antibody Fab fragment at 1.7 A resolution. The structure was determined in two crystal forms, P21 and C2. Although the Fab was crystallized in the presence of Aβ16, no peptide was observed in the crystals. The antigen-binding site is blocked by the hexahistidine tag of another Fab molecule in both crystal forms. The poly-His peptide in an extended conformation occupies a crevice between the light and heavy chains of the variable domain. Two consecutive histidines (His4–His5) stack against tryptophan residues in the central pocket of the antigen-binding surface. In addition, they form hydrogen bonds to the acidic residues at the bottom of the pocket. The mode of his-tag binding by C706 resembles the Aβ recognition by antibodies PFA1 and WO2. All three antibodies recognize the same immunodominant B-cell epitope of Aβ. By similarity, residues Phe–Arg–His of Aβ would be a major portion of the C706 epitope. Copyright © 2010 John Wiley & Sons, Ltd.

Protein aggregation is of great concern to pharmaceutical formulations and has been implicated in several diseases. We engineered an anti-IL-13 monoclonal antibody CNTO607 for improved solubility. Three structure-based engineering approaches were employed in this study: (i) modifying the isoelectric point (pI), (ii) decreasing the overall surface hydrophobicity and (iii) re-introducing an N-linked carbohydrate moiety within a complementarity-determining region (CDR) sequence. A mutant was identified with a modified pI that had a 2-fold improvement in solubility while retaining the binding affinity to IL-13. Several mutants with decreased overall surface hydrophobicity also showed moderately improved solubility while maintaining a similar antigen affinity. Structural studies combined with mutagenesis data identified an aggregation 'hot spot' in heavy-chain CDR3 (H-CDR3) that contains three residues ((99)FHW(100a)). The same residues, however, were found to be essential for high affinity binding to IL-13. On the basis of the spatial proximity and germline sequence, we reintroduced the consensus N-glycosylation site in H-CDR2 which was found in the original antibody, anticipating that the carbohydrate moiety would shield the aggregation 'hot spot' in H-CDR3 while not interfering with antigen binding. Peptide mapping and mass spectrometric analysis revealed that the N-glycosylation site was generally occupied. This variant showed greatly improved solubility and bound to IL-13 with affinity similar to CNTO607 without the N-linked carbohydrate. All three engineering approaches led to improved solubility and adding an N-linked carbohydrate to the CDR was the most effective route for enhancing the solubility of CNTO607.

Toll-like receptors are a family of pattern-recognition receptors that contribute to the innate immune response. Toll-like receptor 3 (TLR3) signals in response to foreign, endogenous and synthetic ligands including viral dsRNA, bacterial RNA, mitochondrial RNA, endogenous necrotic cell mRNA and the synthetic dsRNA analog, poly(I:C). We have generated a monoclonal antibody (mAb CNTO2424) that recognizes the extracellular domain (ECD) of human TLR3 in a conformation-dependent manner. CNTO2424 down-regulates poly(I:C)-induced production of IL-6, IL-8, MCP-1, RANTES, and IP-10 in human lung epithelial cells. In addition, mAb CNTO2424 was able to interfere with the known TLR3-dependent signaling pathways, namely NF-κB, IRF-3/ISRE, and p38 MAPK. The generation of this neutralizing anti-TLR3 mAb provides a unique tool to better understand TLR3 signaling and potential cross-talk between TLR3 and other molecules.

Granulocyte-macrophage colony stimulating factor (GM-CSF) activity has been linked to pro-inflammatory effects in autoimmune syndromes, such as rheumatoid arthritis. Thus GM-CSF mimetics with antagonist activity might play a therapeutic role in these diseases. The human GM-CSF core structure consists of a four alpha-helix bundle, and GM-CSF activity is controlled by its binding to a two-subunit receptor. A number of residues located on the B and C helices of GM-CSF are postulated to interact with the alpha chain of the GM-CSF receptor (GM-CSFR). Several approaches have been successfully utilized to develop peptide mimetics of this site, including peptides from the native sequence, a peptide derived from a recombinant antibody (rAb) light chain which mimicked GM-CSF receptor binding activity, and structurally guided de novo design. Analysis of the rAb light chain had suggested mimicry of GM-CSF with residues mostly contributed by the CDR I region. Key residues involved in CDR I peptide/GM-CSFR binding were identified by truncation and alteration of individual residues, while the structural elements required to antagonize the biological action of GM-CSF were separately tested in binding and inhibitory activity assays of multiple cyclic analogues. A peptide designed to retain the loop conformation of the CDR I region of the rAb light chain competed with GM-CSF for both antibody and receptor binding, but the role of specific residues in antibody versus receptor binding differed markedly. These studies suggest that structural analysis of peptide mimetics can reveal differences in receptor and antibody binding, perhaps including key interactions that impact binding kinetics. Peptide mimetics of other four-helix bundle cytokines are reviewed, including helical and reverse turn mimetics of helical structures. Use of peptide mimetics coupled with structural and kinetic analysis provides a powerful approach to identifying important receptor-ligand interactions, which implications for rational design of novel therapeutics.

To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.

Aspartate 368 on human immunodeficiency virus type 1 (HIV-1) gp120 forms multiple contacts with CD4; in mutagenesis studies, its replacement by asparagine and corresponding changes in simian immunodeficiency virus SIVmac (D385N) reduced binding with CD4. Nevertheless, simian immunodeficiency virus envelopes with D385N were prevalent in several studies. Extending these observations, we also found D385N to be dominant among env clones from two rhesus macaques that progressed rapidly to simian AIDS. These envelopes showed a CD4-independent phenotype as well as reduced affinity to CD4. Moreover, an adjacent change, G383R, which was frequently coselected with D385N, further decreased binding. An optical biosensor study demonstrated that the SIVmac239 gp120 bound to CD4 with kinetics similar to those of HIV-1. However, the gp120s with D385N and G383R showed a 40-fold reduction in affinity, with a drastic increase in dissociation rate, indicating an inherently unstable complex. This finding showed that rapid progression to simian AIDS may be accompanied by the selection of CD4-independent gp120 variants with impaired CD4 binding ability.

The molecular mechanisms involved in the interaction of complement receptor 2 (CR2) with its natural ligands iC3b and C3d are still not well understood. In addition, studies regarding the binding site(s) of the receptor on C3 as well as the affinities of the C3 fragments for CR2 have produced contradictory results. In the present study, we have used surface plasmon resonance technology to study the interaction of CR2 with its ligands C3d, iC3b, and the EBV surface glycoprotein gp350/220. We measured the kinetics of binding of the receptor to its ligands, examined the influence of ionic contacts on these interactions, and assessed whether immobilized and soluble iC3b bound with similar kinetics to CR2. Our results indicate that 1) gp350 binding to CR2 follows a simple 1:1 interaction, whereas that of the C3 fragments is more complex and involves more than one intramolecular component; 2) kinetic differences exist between the binding of C3d and iC3b to CR2, which may be due to an additional binding site found on the C3c region of iC3b; and 3) iC3b binds to CR2 with different kinetics, depending on whether the iC3b is in solution or immobilized on the surface. These findings suggest that binding of CR2 to iC3b and C3d is more complex than previously thought.

Binding of the T-cell antigen CD4 to human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has been reported to induce conformational rearrangements in the envelope complex that facilitate recognition of the CCR5 coreceptor and consequent viral entry into cells. To better understand the mechanism of virus docking and cell fusion, we developed a three-component gp120-CD4-17b optical biosensor assay to visualize the CD4-induced conformational change of gp120 as seen through envelope binding to a neutralizing human antibody, 17b, which binds to epitopes overlapping the CCR5 binding site. The 17b Fab fragment was immobilized on a dextran sensor surface, and kinetics of gp120 binding were evaluated by both global and linear transformation analyses. Adding soluble CD4 (sCD4) increased the association rate of full-length JR-FL gp120 by 25-fold. This change is consistent with greater exposure of the 17b binding epitope on gp120 when CD4 is bound and correlates with CD4-induced conformational changes in gp120 leading to higher affinity binding to coreceptor. A smaller enhancement of 17b binding by sCD4 was observed with a mutant of gp120, DeltaJR-FL protein, which lacks V1 and V2 variable loops and N- and C-termini. Biosensor results for JR-FL and DeltaJR-FL argue that CD4-induced conformational changes in the equilibrium state of gp120 lead both to movement of V1/V2 loops and to conformational rearrangement in the gp120 core structure and that both of these lead to greater exposure of the coreceptor-binding epitope in gp120. A 17b binding enhancement effect on JR-FL also was observed with a 32-amino acid charybdotoxin miniprotein construct that contains an epitope predicted to mimic the Phe 43/Arg 59 region of CD4 and that competes with CD4 for gp120 binding. Results with this construct argue that CD4-mimicking molecules with surrogate structural elements for the Phe 43/Arg 59 components of CD4 are sufficient to elicit a similar gp120 conformational isomerization as expressed by CD4 itself.

Tissue factor (TF) has been implicated in several important biologic processes, including fibrin formation, atherogenesis, angiogenesis, and tumor cell migration. In that plasminogen activators have been implicated in the same processes, the potential for interactions between TF and the plasminogen activator system was examined. Plasminogen was found to bind directly to the extracellular domain of TF apoprotein (amino acids 1-219) as determined by optical biosensor interaction analysis. A fragment of plasminogen containing kringles 1 through 3 also bound to TF apoprotein, whereas isolated kringle 4 and miniplasminogen did not. Expression of TF on the surface of a stably transfected Chinese hamster ovary (CHO) cell line stimulated plasminogen binding to the cells by 70% more than to control cells. Plasminogen bound to a site on the TF apoprotein that appears to be distinct from the binding site for factors VII and VIIa as judged by a combination of biosensor and cell assays. TF enhanced two-chain urokinase (tcuPA) activation of Glu-plasminogen, but not of miniplasminogen, in a dose-dependent, saturable manner (half maximal stimulation at 59 pmol/L). TF apoprotein induced an effect similar to that of relipidated TF, but a relatively higher concentration of the apoprotein was required (half maximal stimulation at 3.8 nmol/L). The stimulatory effect of TF on plasminogen activation was confirmed when plasmin formation was examined directly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In accord with this, TF inhibited fibrinolysis by approximately 74% at a concentration of 14 nmol/L and almost totally inhibited the binding of equimolar concentrations of plasminogen to human umbilical vein endothelial cells and human trophoblasts. Further, CHO cells expressing TF inhibited uPA-mediated fibrinolysis relative to a wild-type control. TF apoprotein and plasminogen were found to colocalize in atherosclerotic plaque. These data suggest that plasminogen localization and activation may be modulated at extravascular sites through a high-affinity interaction between kringles 1 through 3 of plasminogen and the extracellular domain of TF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) activity is mediated by a cellular receptor (GM-CSFR) that is comprised of an alpha-chain (GM-CSFRalpha), which specifically binds GM-CSF, and a beta-chain (betac), shared with the interleukin-3 and interleukin-5 receptors. GM-CSFRalpha exists in both a transmembrane (tmGM-CSFRalpha) and a soluble form (sGM-CSFRalpha). We designed an sGM-CSFRalpha-Fc fusion protein to study GM-CSF interactions with the GM-CSFRalpha. The construct was prepared by fusing the coding region of the sGM-CSFRalpha with the CH2-CH3 regions of murine IgG2a. Purified sGM-CSFRalpha-Fc ran as a monomer of 60 kDa on reducing SDS-polyacrylamide gel electrophoresis but formed a trimer of 160-200 kDa under nonreducing conditions. The sGM-CSFRalpha-Fc bound specifically to GM-CSF as demonstrated by standard and competitive immunoassays, as well as by radioligand assay with 125I-GM-CSF. The sGM-CSFRalpha-Fc also inhibited GM-CSF-dependent cell growth and therein is a functional antagonist. Kinetics of sGM-CSFRalpha-Fc binding to GM-CSF were evaluated using an IAsys biosensor (Affinity Sensors, Paramus, NJ) with two assay systems. In the first, the sGM-CSFRalpha-Fc was bound to immobilized staphylococcal protein A on the biosensor surface, and binding kinetics of GM-CSF in solution were determined. This revealed a rapid koff of 2.43 x 10(-2)/s. A second set of experiments was performed with GM-CSF immobilized to the sensor surface and the sGM-CSFRalpha-Fc in solution. The dissociation rate constant (koff) for the sGM-CSFRalpha-Fc trimer from GM-CSF was 1.57 x 10(-3)/s, attributable to the higher avidity of binding in this assay. These data indicate rapid dissociation of GM-CSF from the sGM-CSFRalpha-Fc and suggest that in vivo, sGM-CSFRalpha may need to be present in the local environment of a responsive cell to exert its antagonist activity.

The glycan shield of the human immunodeficiency virus (HIV) envelope protein presents many potential epitopes for vaccine development. To augment immune responses to HIV, type 1 (HIV-1), envelope-associated carbohydrate antigens, we are defining peptide mimics of HIV-associated carbohydrate antigens that function as antigen mimotopes that upon immunization will induce antibodies cross-reactive with carbohydrate antigens. We have previously defined peptides with a putative sequence tract RYRY that mimic concanavalin A-binding glycans. To imitate the multivalent binding of carbohydrates, we compared the avidity of a linear (911) and cyclic peptide (D002) reactive with concanavalin A presented in a multiple antigen peptide (MAP) format. The affinity of the MAP-D002 peptide was higher than that of the peptide MAP-911, whereas the avidity of D002 peptide was lower than that of 911. Serum from mice immunized with MAP-911 had lower titer for oligomannose-9 than those elicited by MAP-D002 under the same conditions, but both immunogens elicited antibodies that can block the binding of GP120 to dendritic cells. Antibodies that bind to the studied MAPs were found in a preparation of normal human immunoglobulin for intravenous use. Those that were purified on 911 bound back to 911 and D002, whereas anti-D002 antibodies were specific only for D002. Human antibodies reactive with both mimotopes and with a mannosyl preparation were observed to bind to envelope protein. These results suggested the potential to fine-tune the antibody response to carbohydrate antigens by modifying structural features of peptide mimotope-based immunogens.

Peptide mimotopes provide a strategy to augment human immunodeficiency virus 1 (HIV-1) specific carbohydrate reactive immune responses. Their antigenic and immunological properties will depend on the optimization of motif clustering and multimerization. We observe that structural variants of the same mimetic motif, linear versus cyclic, can be used to tune the properties of the antibodies elicited. The expansion of the database of mimotope sequence motifs can be increased by analyzing structures that bind to HIV directed monoclonal antibody 2G12 and the lectin Concanavalin A (Con A), fostering new mimotope designs. Such analysis indicates that these reagents bind to subsets of mannosyl antigens on the envelope (env) protein.

Experimental and data analysis protocols were developed to screen antibodies from hybridoma culture supernatants using Biacore surface plasmon resonance biosensor platforms. The screening methods involved capturing antibodies from crude supernatants using Fc-specific antibody surfaces and monitoring antigen binding at a single concentration. After normalizing the antigen responses for the amount of antibody present, a simple interaction model was fit to all of the binding responses simultaneously. As a result, the kinetic rate constants (k(a) and k(d)) and affinity (K(D)) could be determined for each antibody interaction under identical conditions. Higher-resolution studies involving multiple concentrations of antigen were performed to validate the reliability of single-concentration measurements. The screening protocols can be used to characterize antigen binding kinetics to approximately 200 antibody supernatants per day using automated Biacore 2000 and 3000 instruments.

The HIV envelope (Env) protein mediates entry into cells by binding CD4 and an appropriate coreceptor, which triggers structural changes in Env that lead to fusion between the viral and cellular membranes. The major HIV-1 coreceptors are the seven transmembrane domain chemokine receptors CCR5 and CXCR4. The type of coreceptor used by a virus strain is an important determinant of viral tropism and pathogenesis, and virus-receptor interactions can be therapeutic targets. However, Envs from many virus strains interact with CXCR4 and CCR5 with low affinity such that direct study of this important interaction is difficult if not impossible using standard cell-surface binding techniques. We have developed an approach that makes it possible to study ligand binding to membrane proteins, including Env–coreceptor interactions, using an optical biosensor. CCR5, CXCR4, and other membrane proteins were incorporated into retrovirus particles, which were purified and attached to the biosensor surface. Binding of conformationally sensitive antibodies as well as Env to these receptors was readily detected. The equilibrium dissociation constant for the interaction between an Env derived from the prototype HIV-1 strain IIIB for CXCR4 was approximately 500 nM, explaining the difficulty in measuring this interaction using standard equilibrium binding techniques. Retroviral pseudotypes represent easily produced, stable, homogenous structures that can be used to present a wide array of single and multiple membrane-spanning proteins in a native lipid environment for biosensor studies, thus avoiding the need for detergent solubilization, purification, and reconstitution. The approach should have general applicability and can be used to correlate Env–receptor binding constants to viral tropism and pathogenesis.

Peptides may substitute for carbohydrate antigens in carbohydrate-specific immunological reactions. Using the recognition properties of an anti-Lewis Y (LeY) antibody, BR55-2, as a model system, we establish a molecular perspective for peptide mimicry by comparing the three-dimensional basis of BR55-2 binding to LeY with the binding of the same antibody to peptides. The peptides compete with LeY, as demonstrated by enzyme-linked immunosorbent assay and Biacore analysis. The computer program LUDI was used to epitope map the antibody-combining site, correlating peptide reactivity patterns. This approach identified amino acids interacting with the same BR55-2 functional residue groups that recognize the Fucalpha(1-3) moiety of LeY. Molecular modeling indicates that the peptides adopt an extended turn conformation within the BR55-2 combining site, serving to overlap the peptides with the LeY spatial position. Peptide binding is associated with only minor changes in BR55-2, relative to the BR55-2-LeY complex. Anti-peptide serum distinguishes the Fucalpha(1-3) from the Fucalpha(1-4) linkage, therefore differentiating difucosylated neolactoseries antigens. These results further confirm that peptides and carbohydrates can bind to the same antibody-binding site and that peptides can structurally and functionally mimic salient features of carbohydrate epitopes.

Understanding the basic forces that determine molecular recognition helps to elucidate mechanisms of biological processes and facilitates discovery of innovative biotechnological methods and materials for therapeutics, diagnostics, and separation science. The ability to measure interaction properties of biological macromolecules quantitatively across a wide range of affinity, size, and purity is a growing need of studies aimed at characterizing biomolecular interactions and the structural elements that drive them. Optical biosensors have provided an increasingly impactful technology for such biomolecular interaction analyses. These biosensors record the binding and dissociation of macromolecules in real time by transducing the accumulation of mass of an analyte molecule at the sensor surface coated with ligand molecule into an optical signal. Interactions of analytes and ligands can be analyzed at a microscale and without the need to label either interactant. Sensors enable the detection of bimolecular interaction as well as multimolecular assembly. Most notably, the method is quantitative and kinetic, enabling determination of both steady-state and dynamic parameters of interaction. This article describes the basic methodology of optical biosensors and presents several examples of its use to investigate such biomolecular systems as cytokine growth factor–receptor recognition, coagulation factor assembly, and virus–cell docking.

We report the functional phage display of single chain human interleukin-5 (scIL-5) and its use for receptor-binding epitope randomization. Enzyme-linked immunosorbent assays and optical biosensor analyses verified expression of scIL-5 on the phage surface and binding of scIL-5 phage to interleukin-5 receptor alpha chain. Furthermore, an asymmetrically disabled but functional scIL-5 mutant, (wt/A5)scIL-5, was displayed on phage. (wt/A5)scIL-5 was constructed from an N-terminal half containing the original five charged residues (88EERRR92) in the CD loop, including the Glu89 and Arg91 believed key in the alpha chain recognition site, combined with a C-terminal half containing a disabled CD loop sequence (88AAAAA92) missing the key recognition residues. This asymmetric variant was used as a starting point to generate an scIL-5 library in which the intact 88-92 N-terminal CD loop was randomized. From this epitope library, a receptor-binding variant of IL-5 was detected, (SLRGG/A5)scIL-5, in which the only charged residue in the CD loop is an Arg at position 90. Characterization of this variant expressed as a soluble protein in E. coli shows that the IL-5 pharmacophore for receptor alpha chain binding can function with a single positive charge in the CD loop. Charge-depleted CD loop mimetics of IL-5 suggest the importance of charge distribution in functional IL-5 receptor recruitment.

A chimeric antibody was human adapted and then affinity matured. Biological activity studies revealed that the affinity matured antibody is 10-fold more potent that the chimeric antibody. To determine the correlation between affinity and activity of these antibodies, the binding profile to their antigen was analyzed by Biacore and Kinexa. The studies showed the two mAbs have different thermodynamic profiles. These differences, particularly the equilibrium dissociation constant, KD, revealed a positive correlation with potency (biological activity). The data showed that the affinity of the chimeric antibody is picomolar, whereas the affinity of the human adapted antibody is femtomolar. Molecular modeling studies showed that several of the mutations introduced in the CDRs during the affinity maturation process were hydrophobic replacements of hydrophilic amino acids. The replacements are mostly located at the periphery of the antigen binding-site but are potentially in contact with the antigen, thus encircling the center of the epitope recognized by these antibodies. These hydrophobic contacts may account for more than 15- and 10-fold increase in affinity and potency, respectively.