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1.
Acta Crystallogr D Struct Biol ; 78(Pt 5): 623-632, 2022 May 01.
Article in English | MEDLINE | ID: mdl-35503210

ABSTRACT

The structure of the antigen-binding fragment (Fab) of mouse monoclonal antibody 7H2.2 in complex with a 15-residue fragment from the metalloproteinase sperm acrosomal SLLP1 binding protein (SAS1B), which is a molecular and cellular candidate for both cancer therapy and female contraception, has been determined at 2.75 Šresolution by single-crystal X-ray diffraction. Although the crystallization conditions contained the final 148 C-terminal residues of SAS1B, the Fab was observed to crystallize in complex with a 15-residue fragment corresponding to one of only two elements of secondary structure that are predicted to be ordered within the C-terminal region of SAS1B. The antigen forms an amphipathic α-helix that binds the 7H2.2 combining site via hydrophilic residues in an epitope that spans the length of the antigen α-helix, with only two CH-π interactions observed along the edge of the interface between the antibody and antigen. Interestingly, the paratope contains two residues mutated away from the germline (YL32F and YH58R), as well as a ProH96-ThrH97-AspH98-AspH99 insertion within heavy chain CDR3. The intact 7H2.2 antibody exhibits high affinity for the SAS1B antigen, with 1:1 binding and nanomolar affinity for both the SAS1B C-terminal construct used for crystallization (3.38 ± 0.59 nM) and a 15-amino-acid synthetic peptide construct corresponding to the helical antigen observed within the crystal structure (1.60 ± 0.31 nM). The SAS1B-antibody structure provides the first structural insight into any portion of the subdomain architecture of the C-terminal region of the novel cancer-oocyte tumor surface neoantigen SAS1B and provides a basis for the targeted use of SAS1B.


Subject(s)
Antibodies, Monoclonal , Neoplasms , Animals , Antibodies, Monoclonal/chemistry , Binding Sites, Antibody , Crystallography, X-Ray , Female , Immunoglobulin Fab Fragments/chemistry , Mice , Oocytes/metabolism , Protein Conformation
2.
MAbs ; 14(1): 2031482, 2022.
Article in English | MEDLINE | ID: mdl-35377271

ABSTRACT

Generative machine learning (ML) has been postulated to become a major driver in the computational design of antigen-specific monoclonal antibodies (mAb). However, efforts to confirm this hypothesis have been hindered by the infeasibility of testing arbitrarily large numbers of antibody sequences for their most critical design parameters: paratope, epitope, affinity, and developability. To address this challenge, we leveraged a lattice-based antibody-antigen binding simulation framework, which incorporates a wide range of physiological antibody-binding parameters. The simulation framework enables the computation of synthetic antibody-antigen 3D-structures, and it functions as an oracle for unrestricted prospective evaluation and benchmarking of antibody design parameters of ML-generated antibody sequences. We found that a deep generative model, trained exclusively on antibody sequence (one dimensional: 1D) data can be used to design conformational (three dimensional: 3D) epitope-specific antibodies, matching, or exceeding the training dataset in affinity and developability parameter value variety. Furthermore, we established a lower threshold of sequence diversity necessary for high-accuracy generative antibody ML and demonstrated that this lower threshold also holds on experimental real-world data. Finally, we show that transfer learning enables the generation of high-affinity antibody sequences from low-N training data. Our work establishes a priori feasibility and the theoretical foundation of high-throughput ML-based mAb design.


Subject(s)
Antigen-Antibody Reactions , Machine Learning , Antibodies, Monoclonal/chemistry , Binding Sites, Antibody , Epitopes
3.
Viruses ; 14(2)2022 02 07.
Article in English | MEDLINE | ID: mdl-35215927

ABSTRACT

A new SARS-CoV-2 variant B.1.1.529 was named by the WHO as Omicron and classified as a Variant of Concern (VOC) on 26 November 2021. Because this variant has more than 50 mutations, including 30 mutations on the spike, it has generated a lot of concerns on the potential impacts of the VOC on COVID-19. Here through ELISA assays using the recombinant RBD proteins with sequences the same to that of SARS-CoV-2 WIV04 (lineage B.1), the Delta variant and the Omicron variant as the coating antigens, the binding capabilities between the RBDs and the antibodies in COVID-19 convalescent sera and vaccine sera after two doses of the inactivated vaccine produced by Sinopharm WIBP are compared with each other. The results showed that the Omicron variant may evade antibodies induced by the ancestral strain and by the inactivated vaccine, with significant reduction in the binding capability of its RBD much greater than that of the Delta variant.


Subject(s)
Antibodies, Viral/metabolism , Binding Sites, Antibody/physiology , COVID-19 Vaccines/immunology , COVID-19/immunology , Convalescence , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/blood , Antibodies, Viral/immunology , Humans , Immune Evasion , Mutation , Neutralization Tests , Vaccines, Inactivated/immunology
4.
Viruses ; 14(2)2022 01 30.
Article in English | MEDLINE | ID: mdl-35215888

ABSTRACT

Currently, SARS-CoV-2 causing coronavirus disease 2019 (COVID-19) is responsible for one of the most deleterious pandemics of our time. The interaction between the ACE2 receptors at the surface of human cells and the viral Spike (S) protein triggers the infection, making the receptor-binding domain (RBD) of the SARS-CoV-2 S-protein a focal target for the neutralizing antibodies (Abs). Despite the recent progress in the development and deployment of vaccines, the emergence of novel variants of SARS-CoV-2 insensitive to Abs produced in response to the vaccine administration and/or monoclonal ones represent a potential danger. Here, we analyzed the diversity of neutralizing Ab epitopes and assessed the possible effects of single and multiple mutations in the RBD of SARS-CoV-2 S-protein on its binding affinity to various antibodies and the human ACE2 receptor using bioinformatics approaches. The RBD-Ab complexes with experimentally resolved structures were grouped into four clusters with distinct features at sequence and structure level. The performed computational analysis indicates that while single amino acid replacements in RBD may only cause partial impairment of the Abs binding, moreover, limited to specific epitopes, the variants of SARS-CoV-2 with multiple mutations, including some which were already detected in the population, may potentially result in a much broader antigenic escape. Further analysis of the existing RBD variants pointed to the trade-off between ACE2 binding and antigenic escape as a key limiting factor for the emergence of novel SAR-CoV-2 strains, as the naturally occurring mutations in RBD tend to reduce its binding affinity to Abs but not to ACE2. The results provide guidelines for further experimental studies aiming to identify high-risk RBD mutations that allow for an antigenic escape.


Subject(s)
Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Binding Sites, Antibody/genetics , Computational Biology/methods , Mutation , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Epitopes/metabolism , Host Microbial Interactions/genetics , Humans , Protein Binding , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology
5.
Viruses ; 14(2)2022 02 02.
Article in English | MEDLINE | ID: mdl-35215903

ABSTRACT

To investigate the dynamic association among binding and functional antibodies in health-care-workers receiving two doses of BNT162b2 mRNA COVID-19-vaccine, SARS-CoV-2 anti-RBD IgG, anti-Trimeric-S IgG, and neutralizing antibodies (Nabs) were measured in serum samples collected at 2 weeks, 3 months, and 6 months from full vaccination. Despite the high correlation, results for anti-RBD and anti-Trimeric S IgG were numerically different even after recalculation to BAU/mL following WHO standards indications. Moreover, after a peak response at 2 weeks, anti-RBD IgG levels showed a 4.5 and 13 fold decrease at 3 and 6 months, respectively, while the anti-Trimeric S IgG presented a less pronounced decay of 2.8 and 4.7 fold. Further different dynamics were observed for Nabs titers, resulting comparable at 3 and 6 months from vaccination. We also demonstrated that at NAbs titers ≥40, the area under the receiver operating characteristic curve and the optimal cutoff point decreased with time from vaccination for both anti-RBD and anti-Trimeric S IgG. The mutating relation among the anti-RBD IgG, anti-Trimeric S IgG, and neutralizing antibodies are indicative of antibody maturation upon vaccination. The lack of standardized laboratory procedures is one factor interfering with the definition of a correlate of protection from COVID-19.


Subject(s)
Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , COVID-19/immunology , Immunoglobulin G/metabolism , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Adult , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Binding Sites, Antibody , COVID-19/prevention & control , Cohort Studies , Female , Follow-Up Studies , Health Personnel/statistics & numerical data , Humans , Immunity, Humoral , Immunoglobulin G/blood , Immunoglobulin G/immunology , Kinetics , Longitudinal Studies , Male , Middle Aged , Vaccination
6.
Elife ; 112022 02 24.
Article in English | MEDLINE | ID: mdl-35200140

ABSTRACT

Antibody binding to cell surface proteins plays a crucial role in immunity, and the location of an epitope can altogether determine the immunological outcome of a host-target interaction. Techniques available today for epitope identification are costly, time-consuming, and unsuited for high-throughput analysis. Fast and efficient screening of epitope location can be useful for the development of therapeutic monoclonal antibodies and vaccines. Cellular morphology typically varies, and antibodies often bind heterogeneously across a cell surface, making traditional particle-averaging strategies challenging for accurate native antibody localization. In the present work, we have developed a method, SiteLoc, for imaging-based molecular localization on cellular surface proteins. Nanometer-scale resolution is achieved through localization in one dimension, namely, the distance from a bound ligand to a reference surface. This is done by using topological image averaging. Our results show that this method is well suited for antibody binding site measurements on native cell surface morphology and that it can be applied to other molecular distance estimations as well.


Antibodies play a key role in the immune system. These proteins stick to harmful substances, such as bacteria and other disease-causing pathogens, marking them for destruction or blocking their attack. Antibodies are highly selective, and this ability has been used to target particular molecules in research, diagnostics and therapies. Typically, antibodies need to stick to a particular segment, or 'epitope', on the surface of a cell in order to trigger an immune response. Knowing where these regions are can help explain how these immune proteins work and aid the development of more effective drugs and diagnostic tools. One way to identify these sites is to measure the nano-distance between antibodies and other features on the cell surface. To do this, researchers take multiple images of the cell the antibody is attached to using light microscopy. Various statistical methods are then applied to create an 'average image' that has a higher resolution and can therefore be used to measure the distance between these two points more accurately. While this approach works on fixed shapes, like a perfect circle, it cannot handle human cells and bacteria which are less uniform and have more complex surfaces. Here, Kumra Ahnlide et al. have developed a new method called SiteLoc which can overcome this barrier. The method involves two fluorescent probes: one attached to a specific site on the cell's surface, and the other to the antibody or another molecule of interest. These two probes emit different colours when imaged with a fluorescent microscope. To cope with objects that have uneven surfaces, such as cells and bacteria, the two signals are transformed to 'follow' the same geometrical shape. The relative distance between them is then measured using statistical methods. Using this approach, Kumra Ahnlide et al. were able to identify epitopes on a bacterium, and measure distances on the surface of human red blood cells. The SiteLoc system could make it easier to develop antibody-based treatments and diagnostic tools. Furthermore, it could also be beneficial to the wider research community who could use it to probe other questions that require measuring nanoscale distances.


Subject(s)
Antibodies, Monoclonal/metabolism , Binding Sites, Antibody , Cell Membrane/metabolism , Epitope Mapping/methods , Membrane Proteins/metabolism , Binding Sites , Cell Membrane/immunology , Epitopes/metabolism , Humans , Ligands , Membrane Proteins/immunology , Models, Molecular
7.
Methods Mol Biol ; 2446: 409-424, 2022.
Article in English | MEDLINE | ID: mdl-35157286

ABSTRACT

Nanobodies are single-domain antibody fragments that have found widespread use in basic research, therapy, and diagnostics. Like other antibody formats, nanobodies can be developed with high affinity and specificity for desired antigens. A photobody is a light-activatable nanobody, obtained by incorporating a photo-labile caging group into the paratope region. The caging group prevents antigen binding until removed with light (365 nm), thereby rendering the binding controllable with high temporal and spatial resolution. Thus far photocaged tyrosine residues have been used for this purpose, as tyrosine is a frequent residue at critical positions of nanobody paratopes. Nanobodies without a tyrosine residue at the antigen-binding interface may require a different strategy. In this chapter, we describe methods to design and prepare photobodies by recombinant expression in Escherichia coli in combination with genetic code expansion technology to incorporate ortho-nitrobenzyl-tyrosine residues. We use the conversion of the anti-green fluorescent protein enhancer nanobody into a photobody as an example. These protocols should be applicable to many other nanobodies.


Subject(s)
Single-Domain Antibodies , Antibodies/chemistry , Antigens , Binding Sites, Antibody , Cloning, Molecular , Single-Domain Antibodies/chemistry
8.
Front Immunol ; 12: 801368, 2021.
Article in English | MEDLINE | ID: mdl-35087526

ABSTRACT

A subset of antibodies found in cattle comprises ultralong CDR-H3 regions of up to 70 amino acids. Interestingly, this type of immunoglobulin usually pairs with the single germline VL gene, V30 that is typically very conserved in sequence. In this work, we have engineered ultralong CDR-H3 common light chain bispecific antibodies targeting Epidermal Growth Factor Receptor (EGFR) on tumor cells as well as Natural Cytotoxicity Receptor NKp30 on Natural Killer (NK) cells. Antigen-specific common light chain antibodies were isolated by yeast surface display by means of pairing CDR-H3 diversities following immunization with a single V30 light chain. After selection, EGFR-targeting paratopes as well as NKp30-specific binders were combined into common light chain bispecific antibodies by exploiting the strand-exchange engineered domain (SEED) technology for heavy chain heterodimerization. Biochemical characterization of resulting bispecifics revealed highly specific binding to the respective antigens as well as simultaneous binding to both targets. Most importantly, engineered cattle-derived bispecific common light chain molecules elicited potent NK cell redirection and consequently tumor cell lysis of EGFR-overexpressing cells as well as robust release of proinflammatory cytokine interferon-γ. Taken together, this data is giving clear evidence that bovine bispecific ultralong CDR-H3 common light chain antibodies are versatile for biotechnological applications.


Subject(s)
Antibodies, Bispecific/immunology , Antibody Specificity , Binding Sites, Antibody/immunology , Complementarity Determining Regions/immunology , Immunoglobulin Light Chains/immunology , Animals , Antibodies, Bispecific/genetics , Antibody Specificity/immunology , Binding Sites, Antibody/genetics , Cattle , Complementarity Determining Regions/genetics , Cytokines/metabolism , Cytotoxicity, Immunologic , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin Light Chains/genetics , Inflammation Mediators/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Natural Cytotoxicity Triggering Receptor 3/antagonists & inhibitors , Protein Engineering , Recombinant Fusion Proteins
9.
Elife ; 112022 01 13.
Article in English | MEDLINE | ID: mdl-35023832

ABSTRACT

Malaria is a global health burden, with Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) responsible for the majority of infections worldwide. Circumsporozoite protein (CSP) is the most abundant protein on the surface of Plasmodium sporozoites, and antibodies targeting the central repeat region of CSP can prevent parasite infection. Although much has been uncovered about the molecular basis of antibody recognition of the PfCSP repeats, data remains scarce for PvCSP. Here, we performed molecular dynamics simulations for peptides comprising the PvCSP repeats from strains VK210 and VK247 to reveal how the PvCSP central repeats are highly disordered, with minor propensities to adopt turn conformations. Next, we solved eight crystal structures to unveil the interactions of two inhibitory monoclonal antibodies (mAbs), 2F2 and 2E10.E9, with PvCSP repeats. Both antibodies can accommodate subtle sequence variances in the repeat motifs and recognize largely coiled peptide conformations that also contain isolated turns. Our structural studies uncover various degrees of Fab-Fab homotypic interactions upon recognition of the PvCSP central repeats by these two inhibitory mAbs, similar to potent mAbs against PfCSP. These findings augment our understanding of host-Plasmodium interactions and contribute molecular details of Pv inhibition by mAbs to unlock structure-based engineering of PvCSP-based vaccines.


Subject(s)
Antibodies, Protozoan/metabolism , Binding Sites, Antibody , Molecular Dynamics Simulation , Plasmodium vivax/immunology , Plasmodium vivax/metabolism , Protozoan Proteins/immunology , Protozoan Proteins/metabolism , Animals , Antibodies, Protozoan/chemistry , Cell Line , Crystallization , HEK293 Cells , Humans , Mice , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Sporozoites/metabolism
10.
Front Immunol ; 12: 670561, 2021.
Article in English | MEDLINE | ID: mdl-35003053

ABSTRACT

Broadly neutralizing antibodies (bNAbs), known to mediate immune control of HIV-1 infection, only develop in a small subset of HIV-1 infected individuals. Despite being traditionally associated with patients with high viral loads, bNAbs have also been observed in therapy naïve HIV-1+ patients naturally controlling virus replication [Virus Controllers (VCs)]. Thus, dissecting the bNAb response in VCs will provide key information about what constitutes an effective humoral response to natural HIV-1 infection. In this study, we identified a polyclonal bNAb response to natural HIV-1 infection targeting CD4 binding site (CD4bs), V3-glycan, gp120-gp41 interface and membrane-proximal external region (MPER) epitopes on the HIV-1 envelope (Env). The polyclonal antiviral antibody (Ab) response also included antibody-dependent cellular phagocytosis of clade AE, B and C viruses, consistent with both the Fv and Fc domain contributing to function. Sequence analysis of envs from one of the VCs revealed features consistent with potential immune pressure and virus escape from V3-glycan targeting bNAbs. Epitope mapping of the polyclonal bNAb response in VCs with bNAb activity highlighted the presence of gp120-gp41 interface and CD4bs antibody classes with similar binding profiles to known potent bNAbs. Thus, these findings reveal the induction of a broad and polyfunctional humoral response in VCs in response to natural HIV-1 infection.


Subject(s)
Broadly Neutralizing Antibodies/immunology , CD4 Antigens/immunology , Epitopes/immunology , HIV Antibodies/immunology , HIV Envelope Protein gp120/immunology , HIV Infections/immunology , HIV-1/immunology , Peptide Fragments/immunology , Survivors , Viremia/immunology , Antibody Specificity , Binding Sites, Antibody , CD4 Antigens/metabolism , CD4 Lymphocyte Count , Epitope Mapping , Female , Genes, env , HLA-B Antigens/immunology , Humans , Immune Evasion , Immunoglobulin Fc Fragments/immunology , Immunoglobulin Fragments/immunology , Male , Models, Molecular , Phagocytosis , Protein Domains , Recombinant Proteins/immunology , Viral Load
11.
Front Immunol ; 12: 779100, 2021.
Article in English | MEDLINE | ID: mdl-35003094

ABSTRACT

Interleukin-1 (IL-1) family cytokines are potent mediators of inflammation, acting to coordinate local and systemic immune responses to a wide range of stimuli. Aberrant signaling by IL-1 family cytokine members, however, is linked to myriad inflammatory syndromes, autoimmune conditions and cancers. As such, blocking the inflammatory signals inherent to IL-1 family signaling is an established and expanding therapeutic strategy. While several FDA-approved IL-1 inhibitors exist, including an Fc fusion protein, a neutralizing antibody, and an antagonist cytokine, none specifically targets the co-receptor IL-1 receptor accessory protein (IL-1RAcP). Most IL-1 family cytokines form productive signaling complexes by binding first to their cognate receptors - IL-1RI for IL-1α and IL-1ß; ST2 for IL-33; and IL-36R for IL-36α, IL-36ß and IL-36γ - after which they recruit the shared secondary receptor IL-1RAcP to form a ternary cytokine/receptor/co-receptor complex. Recently, IL-1RAcP was identified as a biomarker for both AML and CML. IL-1RAcP has also been implicated in tumor progression in solid tumors and an anti-IL1RAP antibody (nadunolimab, CAN04) is in phase II clinical studies in pancreatic cancer and non-small cell lung cancer (NCT03267316). As IL-1RAcP is common to all of the abovementioned IL-1 family cytokines, targeting this co-receptor raises the possibility of selective signaling inhibition for different IL-1 family cytokines. Indeed, previous studies of IL-1ß and IL-33 signaling complexes have revealed that these cytokines employ distinct mechanisms of IL-1RAcP recruitment even though their overall cytokine/receptor/co-receptor complexes are structurally similar. Here, using functional, biophysical, and structural analyses, we show that antibodies specific for IL-1RAcP can differentially block signaling by IL-1 family cytokines depending on the distinct IL-1RAcP epitopes that they engage. Our results indicate that targeting a shared cytokine receptor is a viable therapeutic strategy for selective cytokine signaling inhibition.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Antibodies/pharmacology , Epitopes , Interleukin-1 Receptor Accessory Protein/antagonists & inhibitors , Interleukin-1beta/metabolism , Interleukin-33/metabolism , Anti-Inflammatory Agents/immunology , Anti-Inflammatory Agents/metabolism , Antibodies/immunology , Antibodies/metabolism , Antibody Affinity , Antibody Specificity , Binding Sites, Antibody , HEK293 Cells , Humans , Interleukin-1 Receptor Accessory Protein/immunology , Interleukin-1 Receptor Accessory Protein/metabolism , Molecular Docking Simulation , Molecular Targeted Therapy , Protein Binding , Signal Transduction
12.
MAbs ; 14(1): 2002236, 2022.
Article in English | MEDLINE | ID: mdl-34967699

ABSTRACT

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Cell Surface Display Techniques , Immunoglobulin G/immunology , Peptide Library , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/metabolism , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/genetics , Antibodies, Viral/metabolism , Antibody Specificity , Binding Sites, Antibody , COVID-19/metabolism , COVID-19/prevention & control , COVID-19/virology , Chlorocebus aethiops , Disease Models, Animal , Epitopes , Female , Host-Pathogen Interactions , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Immunoglobulin G/pharmacology , Mesocricetus , SARS-CoV-2/pathogenicity , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Single-Domain Antibodies/pharmacology , Vero Cells
13.
Toxins (Basel) ; 13(12)2021 12 10.
Article in English | MEDLINE | ID: mdl-34941720

ABSTRACT

Immunochemical methods for mycotoxin analysis require antigens with well-defined structures and antibodies with outstanding binding properties. Immunoreagents for the mycotoxins alternariol and/or alternariol monomethyl ether have typically been obtained with chemically uncharacterized haptens, and antigen conjugates have most likely been prepared with mixtures of functionalized molecules. For the first time, total synthesis was performed, in the present study, to obtain two haptens with opposite linker attachment locations. The functionalized synthetic haptens were purified and deeply characterized by different spectrometric methods, allowing the preparation of bioconjugates with unequivocal structures. Direct and indirect competitive enzyme-linked immunosorbent assays, using homologous and heterologous conjugates, were employed to extensively evaluate the generated immunoreagents. Antibodies with high affinity were raised from conjugates of both haptens, and a structure-activity relationship between the synthetic haptens and the specificity of the generated antibodies could be established. These results pave the way for the development of novel highly sensitive immunoassays selective of one or two of these Alternaria mycotoxins.


Subject(s)
Antibody Formation/drug effects , Binding Sites, Antibody/drug effects , Haptens/chemistry , Haptens/immunology , Lactones/chemistry , Lactones/immunology , Mycotoxins/chemistry , Mycotoxins/immunology , Immunoassay/methods , Immunologic Tests , Molecular Structure
14.
Elife ; 102021 12 23.
Article in English | MEDLINE | ID: mdl-34939934

ABSTRACT

HAP2 is a transmembrane gamete fusogen found in multiple eukaryotic kingdoms and is structurally homologous to viral class II fusogens. Studies in Plasmodium have suggested that HAP2 is an attractive target for vaccines that block transmission of malaria. HAP2 has three extracellular domains, arranged in the order D2, D1, and D3. Here, we report monoclonal antibodies against the D3 fragment of Plasmodium berghei HAP2 and crystal structures of D3 in complex with Fab fragments of two of these antibodies, one of which blocks fertilization of Plasmodium berghei in vitro and transmission of malaria in mosquitoes. We also show how this Fab binds the complete HAP2 ectodomain with electron microscopy. The two antibodies cross-react with HAP2 among multiple plasmodial species. Our characterization of the Plasmodium D3 structure, HAP2 ectodomain architecture, and mechanism of inhibition provide insights for the development of a vaccine to block malaria transmission.


Subject(s)
Antibodies, Monoclonal/metabolism , Germ Cells/immunology , Malaria/prevention & control , Malaria/transmission , Plasmodium berghei/immunology , Protozoan Proteins/immunology , Protozoan Proteins/metabolism , Animals , Binding Sites, Antibody , Biophysical Phenomena , Culicidae/parasitology , Germ Cells/physiology , Malaria/immunology , Membrane Fusion , Protein Binding , Protozoan Proteins/chemistry
15.
PLoS Pathog ; 17(12): e1010175, 2021 12.
Article in English | MEDLINE | ID: mdl-34929007

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.


Subject(s)
Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/metabolism , Protein Domains , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , Binding Sites , Binding Sites, Antibody , COVID-19/prevention & control , Chlorocebus aethiops , Female , HEK293 Cells , Humans , Immunoglobulin Fc Fragments/therapeutic use , Immunoglobulin G/therapeutic use , Mice, Transgenic , Neutralization Tests , Protein Binding , Recombinant Fusion Proteins/therapeutic use , SARS-CoV-2/drug effects , Vero Cells
16.
Front Immunol ; 12: 793953, 2021.
Article in English | MEDLINE | ID: mdl-34899762

ABSTRACT

Durability of SARS-CoV-2 Spike antibody responses after infection provides information relevant to understanding protection against COVID-19 in humans. We report the results of a sequential evaluation of anti-SARS-CoV-2 antibodies in convalescent patients with a median follow-up of 14 months (range 12.4-15.4) post first symptom onset. We report persistence of antibodies for all four specificities tested [Spike, Spike Receptor Binding Domain (Spike-RBD), Nucleocapsid, Nucleocapsid RNA Binding Domain (N-RBD)]. Anti-Spike antibodies persist better than anti-Nucleocapsid antibodies. The durability analysis supports a bi-phasic antibody decay with longer half-lives of antibodies after 6 months and antibody persistence for up to 14 months. Patients infected with the Wuhan (WA1) strain maintained strong cross-reactive recognition of Alpha and Delta Spike-RBD but significantly reduced binding to Beta and Mu Spike-RBD. Sixty percent of convalescent patients with detectable WA1-specific NAb also showed strong neutralization of the Delta variant, the prevalent strain of the present pandemic. These data show that convalescent patients maintain functional antibody responses for more than one year after infection, suggesting a strong long-lasting response after symptomatic disease that may offer a prolonged protection against re-infection. One patient from this cohort showed strong increase of both Spike and Nucleocapsid antibodies at 14 months post-infection indicating SARS-CoV-2 re-exposure. These antibodies showed stronger cross-reactivity to a panel of Spike-RBD including Beta, Delta and Mu and neutralization of a panel of Spike variants including Beta and Gamma. This patient provides an example of strong anti-Spike recall immunity able to control infection at an asymptomatic level. Together, the antibodies from SARS-CoV-2 convalescent patients persist over 14 months and continue to maintain cross-reactivity to the current variants of concern and show strong functional properties.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Binding Sites, Antibody/immunology , COVID-19/virology , Cohort Studies , Cross Reactions/immunology , Female , Humans , Male , Middle Aged , Neutralization Tests/methods , Nucleocapsid/immunology , Nucleocapsid/metabolism , Protein Binding/immunology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/metabolism , Time Factors
17.
Front Immunol ; 12: 732938, 2021.
Article in English | MEDLINE | ID: mdl-34887850

ABSTRACT

Staphylococcal enterotoxin B (SEB) simultaneously crosslinks MHC class II antigen and TCR, promoting proliferation of T cells and releasing a large number of toxic cytokines. In this report, we computationally examined the possibility of using a single-chain biparatopic bispecific antibody to target SEB and prevent TCR binding. The design was inspired by the observation that mixing two anti-SEB antibodies 14G8 and 6D3 can block SEB-TCR activation, and we used 14G8-6D3-SEB tertiary crystal structure as a template. Twelve simulation systems were constructed to systematically examine the effects of the designed bispecific scFV MB102a, including isolated SEB, MB102a with different linkers, MB102a-SEB complex, MB102a-SEB-TCRß complex, MB102a-SEB-TCR-MHC II complex, and MB102a-SEB-MHC II. Our all atom molecular dynamics simulations (total 18,900 ns) confirmed that the designed single-chain bispecific antibody may allosterically prevent SEB-TCRß chain binding and inhibit SEB-TCR-MHC II formation. Subsequent analysis indicated that the binding of scFV to SEB correlates with SEB-TCR binding site motion and weakens SEB-TCR interactions.


Subject(s)
Antibodies, Bacterial/immunology , Antibodies, Bispecific/chemistry , Antibodies, Bispecific/immunology , Enterotoxins/immunology , Animals , Antibodies, Bacterial/chemistry , Antibodies, Bacterial/genetics , Antibodies, Bispecific/genetics , Binding Sites, Antibody , Computer Simulation , Crystallography, X-Ray , Drug Design , Enterotoxins/chemistry , Humans , Mice , Molecular Dynamics Simulation , Protein Conformation , Protein Engineering , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/immunology , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology , Staphylococcus aureus/immunology , T-Lymphocytes/immunology , T-Lymphocytes/microbiology
18.
Immunol Lett ; 241: 55-57, 2022 01.
Article in English | MEDLINE | ID: mdl-34785254

ABSTRACT

Gene as the basic functional unit of DNA encodes information about the product such as protein. The majority of proteins realize function through protein-protein interactions involving short protein motifs. However, some proteins such as antibodies are established by the rearrangement of several (V-D-J) gene segments with the potential addition of nontemplated nucleotides that may change information encoded by the respective gene segment used. Antibody VH domain sequence analysis by ISM bioinformatics approach that is based on amino acids physicochemical features, enable to distinguish the contribution of the information encoded by VH gene or generated during VDJ gene recombination for antibody-antigen interaction. The data presented in this report revealed the significance of CDRH3 for the interaction of antibody specific for immunogenic molecules while CDRH3 contribution is minor for antibody interaction with nonimmunogenic molecules such as haptens and native mammalian dsDNA. Thus, paratopes might be located in the CDRH3 or VH regions.


Subject(s)
Antigen-Antibody Complex/genetics , Binding Sites, Antibody/genetics , Computational Biology/methods , Epitope Mapping/methods , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Protein Domains/genetics , Amino Acid Sequence , Amino Acids/chemistry , Amino Acids/genetics , Antigen-Antibody Complex/metabolism , Gene Rearrangement , Humans , Protein Interaction Maps , Sequence Analysis
19.
Front Immunol ; 12: 750386, 2021.
Article in English | MEDLINE | ID: mdl-34764961

ABSTRACT

Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , Epitopes/immunology , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/genetics , Animals , Binding Sites, Antibody/immunology , COVID-19/drug therapy , Cell Line, Tumor , Chlorocebus aethiops , Female , Glycosylation , HEK293 Cells , Humans , Mice, Inbred BALB C , Neutralization Tests , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Vero Cells
20.
Proteins ; 90(3): 824-834, 2022 03.
Article in English | MEDLINE | ID: mdl-34761442

ABSTRACT

The coronavirus disease 2019 (COVID-19) has affected the lives of millions of people around the world. In an effort to develop therapeutic interventions and control the pandemic, scientists have isolated several neutralizing antibodies against SARS-CoV-2 from the vaccinated and convalescent individuals. These antibodies can be explored further to understand SARS-CoV-2 specific antigen-antibody interactions and biophysical parameters related to binding affinity, which can be utilized to engineer more potent antibodies for current and emerging SARS-CoV-2 variants. In the present study, we have analyzed the interface between spike protein of SARS-CoV-2 and neutralizing antibodies in terms of amino acid residue propensity, pair preference, and atomic interaction energy. We observed that Tyr residues containing contacts are highly preferred and energetically favorable at the interface of spike protein-antibody complexes. We have also developed a regression model to relate the experimental binding affinity for antibodies using structural features, which showed a correlation of 0.93. Moreover, several mutations at the spike protein-antibody interface were identified, which may lead to immune escape (epitope residues) and improved affinity (paratope residues) in current/emerging variants. Overall, the work provides insights into spike protein-antibody interactions, structural parameters related to binding affinity and mutational effects on binding affinity change, which can be helpful to develop better therapeutics against COVID-19.


Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Neutralizing/chemistry , Binding Sites, Antibody , Epitopes/chemistry , Epitopes/immunology , Humans , Molecular Docking Simulation , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry
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