Cognate Antibodies Research Articles

Immunoanalytical Detection of Conserved Peptides: Refining the Universe of Biomarker Targets in Planetary Exploration.

Ancient peptides are remnants of early biochemistry that continue to play pivotal roles in current proteins. They are simple molecules yet complex enough to exhibit independent functions, being products of an evolved biochemistry at the interface of life and nonlife. Their adsorption to minerals may contribute to their stabilization and preservation over time. To investigate the feasibility of conserved peptide sequences and structures as target biomarkers for the search for life on Mars or other planetary bodies, we conducted a bioinformatics selection of well-conserved ancient peptides and produced polyclonal antibodies for their detection using fluorescence microarray immunoassays. Additionally, we explored how adsorbing peptides to Mars-representative minerals to form organomineral complexes could affect their immunological detection. The results demonstrated that the selected peptides exhibited autonomous folding, with some of them regaining their structure, even after denaturation. Furthermore, their cognate antibodies detected their conformational features regardless of amino acid sequences, thereby broadening the spectrum of target peptide sequences. While certain antibodies displayed unspecific binding to bare minerals, we validated that peptide-mineral complexes can be detected using sandwich immunoassays, as confirmed through desorption and competitive assays. Consequently, we conclude that the diversity of peptide sequences and structures suitable for use as target biomarkers in astrobiology can be constrained to a few well conserved sets, and they can be detected even if they are adsorbed in organomineral complexes.

Fast-Track Discovery of SARS-CoV-2-Neutralizing Antibodies from Human B Cells by Direct Functional Screening.

As the COVID-19 pandemic revealed, rapid development of vaccines and therapeutic antibodies are crucial to guarantee a quick return to the status quo of society. In early 2020, we deployed our droplet microfluidic single-cell-based platform DROPZYLLA® for the generation of cognate antibody repertoires of convalescent COVID-19 donors. Discovery of SARS-CoV-2-specific antibodies was performed upon display of antibodies on the surface of HEK293T cells by antigen-specific sorting using binding to the SARS-CoV-2 spike and absence of binding to huACE2 as the sort criteria. This efficiently yielded antibodies within 3-6 weeks, of which up to 100% were neutralizing. One of these, MTX-COVAB, displaying low picomolar neutralization IC50 of SARS-CoV-2 and with a neutralization potency on par with the Regeneron antibodies, was selected for GMP manufacturing and clinical development in June 2020. MTX-COVAB showed strong efficacy in vivo and neutralized all identified clinically relevant variants of SARS-CoV-2 at the time of its selection. MTX-COVAB completed GMP manufacturing by the end of 2020, but clinical development was stopped when the Omicron variant emerged, a variant that proved to be detrimental to all monoclonal antibodies already approved. The present study describes the capabilities of the DROPZYLLA® platform to identify antibodies of high virus-neutralizing capacity rapidly and directly.

Kelvin probe force microscopy on patterned large-area biofunctionalized surfaces: a reliable ultrasensitive platform for biomarker detection.

Kelvin probe force microscopy (KPFM) allows the detection of single binding events between immunoglobulins (IgM, IgG) and their cognate antibodies (anti-IgM, anti-IgG). Here an insight into the reliability and robustness of the methodology is provided. Our method is based on imaging the surface potential shift occurring on a dense layer of ∼5 × 107 antibodies physisorbed on a 50 μm × 90 μm area when assayed with increasing concentrations of antigens in phosphate buffer saline (PBS) standard solutions, in air and at a fixed scanning location. A comprehensive investigation of the influence of the main experimental parameters that may interfere with the outcomes of KPFM immune-assay is provided, showing the robustness and reliability of our approach. The data are supported also by a thorough polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS) analysis of the physisorbed biolayer, in the spectral region of the amide I, amide II and amide A bands. Our findings demonstrate that a 10 min incubation in 500 μL PBS encompassing ≈ 30 antigens (100 zM) triggers an extended surface potential shift that involves the whole investigated area. Such a shift quickly saturates at increasing ligand concentration, showing that the developed sensing platform works as an OFF/ON detector, capable of assessing the presence of a few specific biomarkers in a given assay volume. The reliability of the developed methodology KPFM is an important asset in single molecule detections at a wide electrode interface.

Production of Antibodies to Peptide Targets Using Hybridoma Technology.

Hybridoma technology is a well-established and indispensable tool for generating high-quality monoclonal antibodies and has become one of the most common methods for monoclonal antibody production. In this process, antibody-producing B cells are isolated from mice following immunization of mice with a specific immunogen and fused with an immortal myeloma cell line to form antibody-producing hybridoma cell lines. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents but have also emerged as the most rapidly expanding class of therapeutic biologicals. In spite of the development of new high-throughput monoclonal antibody generation technologies, hybridoma technology still is applied for antibody production due to its ability to preserve innate functions of immune cells and to preserve natural cognate antibody paring information. In this chapter, an overview of hybridoma technology and the laboratory procedures used for hybridoma production and antibody screening of peptide-specific antibodies are presented.

Mapping immunological and host receptor binding determinants of SARS-CoV spike protein utilizing the Qubevirus platform

The motifs involved in tropism and immunological interactions of SARS-CoV spike (S) protein were investigated utilizing the Qubevirus platform. We showed that separately, 14 overlapping peptide fragments representing the S protein (F1-14 of 100 residues each) could be inserted into the C-terminus of A1 on recombinant Qubevirus without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F6 from S425-525, was found to contain the binding determinant of the recombinant human angiotensin converting enzyme 2 (rhACE2), with the shortest active binding motif situated between residues S437-492. Upstream, another fragment, F7, containing an overlapping portion of F6 would not bind to rhACE2, confirming that a contiguous stretch of residues could adopt the appropriate structural orientation of F6 as an insertion within the Qubevirus. The F6 (S441-460) and other inserts, including F7/F8 (S601-620) and F10 (S781-800), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes, improved substantially the recognition and binding to their cognate antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of subunit vaccines or other biologics against SARS-CoV.

Non-Genotoxic Conditioning for Hematopoietic Stem Cell Transplant through Engineered Stem Cell Antibody Paired Evasion (ESCAPE)

While ex vivo gene editing of CD34 + hematopoietic stem and progenitor cells (HSPCs) offers potentially curative therapies for patients with serious diseases, such as sickle cell anemia, significant autologous hematopoietic stem cell (HSC) transplant-related challenges remain, notably the requirement of myeloablative conditioning using chemotherapeutic agents such as busulfan. To address this challenge, we have developed a non-genotoxic conditioning strategy with our Engineered Stem Cell Antibody Paired Evasion (ESCAPE) approach where we aim to engineer the epitope of the HSC surface protein CD117 (cKIT) using base editors, in such a way that the engineered protein retains the normal CD117 receptor function, but escapes recognition by a cognate monoclonal antibody that recognizes the wildtype CD117 protein. The ESCAPE-CD117 edit can be combined with a therapeutic target edit to generate a multiplex-edited engineered HSC (eHSC). We previously reported that through large parallel antibody and antigen screening efforts, we could identify mAb: edit pairs in which the engineered epitope evades mAb-mediated blocking of the WT CD117 receptor-ligand interaction while maintaining normal CD117 receptor biology and HSPC function and differentiation potential in vitro and in vivo. To further characterize binding properties of our lead CD117 variant to the natural ligand SCF, we determined the 3-dimensional structure of the receptor-ligand complex using cryo-EM. Structural overlay between engineered CD117:SCF and WT CD117: SCF complexes showed high similarity, with root mean square deviation (RMSD) of 0.848-Å, indicating that our epitope engineering did not affect the structure of CD117 nor its binding to its ligand. We have previously demonstrated in vivo proof-of-concept data supporting selection of engrafted multiplex-edited eHSCs after a single dose of mAb in non-competitive and competitive transplant settings in multiple rodent xenotransplantation models. We next sought to examine whether mAb conditioning could achieve long-term, multilineage hematopoietic reconstitution of multiplex-edited eHSCs and potentially therapeutic mixed chimerism levels. To this end, we humanized an immunocompromised mouse model with unedited mobilized peripheral blood (mPB) hCD34 + cells to achieve stable human bone marrow chimerism and then administered a single dose of mAb followed by a second transplant from the same donor of eHSCs harboring both ESCAPE and therapeutic edits targeting the HBG1/2 promoter. Animals receiving CD117 mAb demonstrated significant engraftment by cells harboring our ESCAPE multiplex edits as detected by NGS of bulk BM (~50% multiplex editing) and within the sorted marrow CD34 + cell population (~60% multiplex editing), while the isotype control treated animals only showed 2-5% multiplex editing within the said compartments. We also isolated CD15 + myeloid cells and GlyA + erythroid cells from the bone marrow of the mAb treated animals, which also harbored >50% multiplex editing as detected by NGS. Furthermore, we could detect what is believed to be therapeutically relevant levels of HbF induction (>30%) in human GlyA + sorted bone marrow cells, suggesting that we could achieve therapeutic benefit with a single dose of mAb as the conditioning agent. In conclusion, our structural data showed a high level of structural similarity between our engineered CD117 epitope and the WT protein. Our in vitro assays and in vivo rodent xenotransplantation models showed enrichment of engineered HSCs upon mAb treatment. Altogether, our data suggest the feasibility of achieving therapeutically relevant levels of mixed bone marrow chimerism with our non-genotoxic conditioning ESCAPE platform. This approach may remove the need for patients to undergo systemic genotoxic conditioning prior to transplant and further unlock the promise of base editing therapies for the treatment of patients suffering from serious hematologic diseases.

The Pattern of Red Cell Antibodies in a Large Population of People with Sickle Cell Disease and Thalassemia

Background There is interest in strategies to match recipients to red blood cell (RBC) units for transfusion using antigens beyond ABO/Rh/K with the aim of reducing rates of alloantibody formation in heavily transfused people, for example sickle cell disease (SCD) and thalassemia (THAL). However, the efficacy of different strategies, often defined in national guidelines, is unknown. People with SCD/THAL in England are now offered free universal blood group typing using high-throughput, low-cost genotyping arrays, which will also be applied to 92,000 blood donors, extending the antigens available for matching. Key to an evaluation of the potential benefits of improved matching is an understanding of the patterns of alloimmunization, which have been reported to vary widely in SCD and THAL. We describe the distribution of alloantibodies in people with SCD/THAL, and evaluate the effect of transfusion exposure on the incidence of antibody formation alongside national guidelines. Methods In 2019 we performed a cross-sectional service evaluation of transfusion treatment for recipients with SCD and THAL at three comprehensive care centres in London, comparing practices against current British Society Haematology (BSH) guidelines, which require matching for compatibility with ABO/Rh/K antigens. We collated and curated data on red cell antigens and antibodies and transfusion treatment regimens from hospital records. Subsequently, at the largest centre, we collated additional longitudinal data on antigens, antibodies and treatment from hospital records retrospectively for the 5 years ending on 31st December 2022. The longitudinal cohort overlapped substantially with the recipients contributing to the cross-sectional service evaluation. Appropriate local and national (22/EM/0006) governance approvals were obtained. Results For the service evaluation, we collated data on 2,025 people: 1,331 SCD (of whom 199 regularly transfused); 453 THAL (of whom 257 regularly transfused). The prevalence of at least one antibody was 14% and 20% in SCD and THAL recipients respectively, and 26% and 24% in regularly transfused SCD and THAL recipients respectively. The commonest antibodies in SCD were anti-S (3.2% prevalence), anti-E (2.9% prevalence), anti-Kp(a) (2.3% prevalence) and anti-K (2.1% prevalence); and in THAL were anti-K (7.1% prevalence), anti-E (6.8% prevalence), anti-Kp(a) (6.6% prevalence) and anti-C (2.2% prevalence). We collated longitudinal data on 676 people treated at the largest centre, of whom 443 had a complete five-year transfusion history. Over five years, 317 SCD recipients received 45,707 RBC units of whom 46 developed new antibodies (Table 1); 126 THAL recipients received 15,251 RBC units and two developed new antibodies. In total, there were 57 alloimmunization events in SCD recipients and 3 in THAL recipients. The antibodies with the greatest frequency of incident formation were anti-Kp(a) (14 events) and anti-C (12 events), all in SCD. For each antibody, we calculated the number of antigen negative units required to eliminate the possibility of incident alloimmunization in the cohort, assuming transfusion was equally distributed across antigen types e.g. 93% of the 676 recipients are Fy(a) neg, so 0.93 x 45,707 units = 42,508 Fy(a) negative units would need to be supplied over 5 years to ensure that no Fy(a) negative recipients are exposed to Fy(a) positive blood. Conclusions The risk of new alloimmunization differs between THAL and SCD. In SCD, anti-Kp(a) and anti-C account for almost 50% of new alloimmunization. Most of the antibodies (anti C/E/K) prevalent in our longitudinal cohort are likely to have been formed before the introduction of the current matching strategies defined in national guidelines. Many people who are antigen negative never form cognate antibodies to the corresponding antigen, despite extensive transfusion exposure. Guidance around extended matching beyond ABO/Rh/K may not match the antibodies that are most commonly newly formed in SCD, but may not be indicated in THAL. Further studies are required to determine who will benefit from more precise RBC matching, given the relative paucity of antigen negative blood in donor populations, such as planned by novel algorithmic real-time approaches in the Haem-Match programme (www.haemmatch.org).

Evolutionary Qβ Phage Displayed Nanotag Library and Peptides for Biosensing.

We selected a novel biotin-binding peptide for sensing biotin, biotinylated proteins, and nucleotides. From a 15-mer library displayed on the RNA coliphage Qβ, a 15-amino acid long peptide (HGHGWQIPVWPWGQG) hereby referred to as a nanotag was identified to selectively bind biotin. The target selection was achieved through panning with elution by infection. The selected peptide was tested as a transducer for an immunogenic epitope of the foot-and-mouth disease virus (FMDV) on Qβ phage platform separated by a linker. The biotin-tag showed no significant influence on the affinity of the epitope to its cognate antibody (SD6). The nanotag-bound biotin selectively fused either to the C- or N-terminus of the epitope. The epitope would not bind or recognize SD6 while positioned at the N-terminus of the nanotag. Additionally, the biotin competed linearly with the SD6 antibody in a competitive ELISA. Competition assays using the selected recombinant phage itself as a probe or transducer enable the operationalization of this technology as a biosensor toolkit to sense and quantify SD6 analyte. Herein, the published Strep II nanotag (DVEWLDERVPLVET) was used as a control and has similar functionalities to our proposed novel biotin-tag thereby providing a new platform for developing devices for diagnostic purposes.

A self-binding immune complex vaccine elicits strong neutralizing responses against herpes simplex virus in mice.

It has been known for over half a century that mixing an antigen with its cognate antibody in an immune complex (IC) can enhance antigen immunogenicity. However, many ICs produce inconsistent immune responses, and the use of ICs in the development new vaccines has been limited despite the otherwise widespread success of antibody-based therapeutics. To address this problem, we designed a self-binding recombinant immune complex (RIC) vaccine which mimics the larger ICs generated during natural infection. In this study, we created two novel vaccine candidates: 1) a traditional IC targeting herpes simplex virus 2 (HSV-2) by mixing glycoprotein D (gD) with a neutralizing antibody (gD-IC); and 2) an RIC consisting of gD fused to an immunoglobulin heavy chain and then tagged with its own binding site, allowing self-binding (gD-RIC). We characterized the complex size and immune receptor binding characteristics in vitro for each preparation. Then, the in vivo immunogenicity and virus neutralization of each vaccine were compared in mice. gD-RIC formed larger complexes which enhanced C1q receptor binding 25-fold compared to gD-IC. After immunization of mice, gD-RIC elicited up to 1,000-fold higher gD-specific antibody titers compared to traditional IC, reaching endpoint titers of 1:500,000 after two doses without adjuvant. The RIC construct also elicited stronger virus-specific neutralization against HSV-2, as well as stronger cross-neutralization against HSV-1, although the proportion of neutralizing antibodies to total antibodies was somewhat reduced in the RIC group. This work demonstrates that the RIC system overcomes many of the pitfalls of traditional IC, providing potent immune responses against HSV-2 gD. Based on these findings, further improvements to the RIC system are discussed. RIC have now been shown to be capable of inducing potent immune responses to a variety of viral antigens, underscoring their broad potential as a vaccine platform.

Germline-encoded amino acid-binding motifs drive immunodominant public antibody responses.

Despite the vast diversity of the antibody repertoire, infected individuals often mount antibody responses to precisely the same epitopes within antigens. The immunological mechanisms underpinning this phenomenon remain unknown. By mapping 376 immunodominant "public epitopes" at high resolution and characterizing several of their cognate antibodies, we concluded that germline-encoded sequences in antibodies drive recurrent recognition. Systematic analysis of antibody-antigen structures uncovered 18 human and 21 partially overlapping mouse germline-encoded amino acid-binding (GRAB) motifs within heavy and light V gene segments that in case studies proved critical for public epitope recognition. GRAB motifs represent a fundamental component of the immune system's architecture that promotes recognition of pathogens and leads to species-specific public antibody responses that can exert selective pressure on pathogens.

Complement-mediated killing of Mycoplasma bovis does not play a role in the protection of animals against an experimental challenge.

Despite numerous efforts, developing recombinant vaccines for the control of M. bovis infections has not been successful. Many factors are contributing to the lack of success including the identification of protective antigens, use of effective adjuvants, and relatively limited information on the quality of immune responses needed for protection. Experimental trials using vaccination with many M. bovis proteins resulted in significant humoral immune responses before and after the challenges, however these responses were not enough to confer protection. We explored the role of complement-fixing antibodies in the killing of M. bovis in-vitro and whether animals vaccinated with proteins that elicit antibodies capable of complement-fixing would be protected against an experimental challenge. We found that antibodies against some of these proteins fixed complement and killed M. bovis in-vitro. Vaccination and challenge experiments with proteins whose cognate antibodies either fixed complement or not resulted in lack of protection against a M. bovis experimental challenge suggesting that complement fixation does not play a role in protection.

Chemically modified aptamers for improving binding affinity to the target proteins via enhanced non-covalent bonding.

Nucleic acid aptamers are ssDNA or ssRNA fragments that specifically recognize targets. However, the pharmacodynamic properties of natural aptamers consisting of 4 naturally occurring nucleosides (A, G, C, T/U) are generally restricted for inferior binding affinity than the cognate antibodies. The development of high-affinity modification strategies has attracted extensive attention in aptamer applications. Chemically modified aptamers with stable three-dimensional shapes can tightly interact with the target proteins via enhanced non-covalent bonding, possibly resulting in hundreds of affinity enhancements. This review overviewed high-affinity modification strategies used in aptamers, including nucleobase modifications, fluorine modifications (2'-fluoro nucleic acid, 2'-fluoro arabino nucleic acid, 2',2'-difluoro nucleic acid), structural alteration modifications (locked nucleic acid, unlocked nucleic acid), phosphate modifications (phosphorothioates, phosphorodithioates), and extended alphabets. The review emphasized how these high-affinity modifications function in effect as the interactions with target proteins, thereby refining the pharmacodynamic properties of aptamers.

Production of two SARS-CoV-2 neutralizing antibodies with different potencies in Nicotiana benthamiana

Monoclonal antibodies are considered to be highly effective therapeutic tools for the treatment of mild to moderate COVID-19 patients. In the present work, we describe the production of two SARS-CoV-2 human IgG1 monoclonal antibodies recognizing the spike protein receptor-binding domain (RBD) and endowed with neutralizing activity (nAbs) in plants. The first one, mAbJ08-MUT, was previously isolated from a COVID-19 convalescent patient and Fc-engineered to prolong the half-life and reduce the risk of antibody-dependent enhancement. This nAb produced in mammalian cells, delivered in a single intramuscular administration during a Phase I clinical study, was shown to (i) be safe and effectively protect against major variants of concern, and (ii) have some neutralizing activity against the recently emerged omicron variant in a cytopathic-effect-based microneutralization assay (100% inhibitory concentration, IC100 of 15 μg/mL). The second antibody, mAb675, previously isolated from a vaccinated individual, showed an intermediate neutralization activity against SARS-CoV-2 variants. Different accumulation levels of mAbJ08-MUT and mAb675 were observed after transient agroinfiltration in Nicotiana benthamiana plants knocked-out for xylosil and fucosil transferases, leading to yields of ~35 and 150 mg/kg of fresh leaf mass, respectively. After purification, as a result of the proteolytic events affecting the hinge-CH2 region, a higher degradation of mAb675 was observed, compared to mAbJ08-MUT (~18% vs. ~1%, respectively). Both nAbs showed a human-like glycosylation profile, and were able to specifically bind to RBD and compete with angiotensin-converting enzyme 2 binding in vitro. SARS-CoV-2 neutralization assay against the original virus isolated in Wuhan demonstrated the high neutralization potency of the plant-produced mAbJ08-MUT, with levels (IC100 < 17 ng/mL) comparable to those of the cognate antibody produced in a Chinese hamster ovary cell line; conversely, mAb675 exhibited a medium neutralization potency (IC100 ~ 200 ng/mL). All these data confirm that plant expression platforms may represent a convenient and rapid production system of potent nAbs to be used both in therapy and diagnostics in pandemic emergencies.

Arterial antibodies promote atherosclerotic plaque development in mice

Abstract Atherosclerosis is characterized by the progressive buildup of cholesterol-rich plaques within the arterial wall. Plaques activate the immune system, of which B cells play a major role. Antibodies bind autoantigens released from necrotic cells and form immune complexes to increase inflammation. We isolated aorta-associated B cells from atherosclerotic ApoE-deficient mice on a high-fat diet and performed single cell sequencing of the B cell receptors to obtain antibody pairs. 32 over-represented antibodies were identified and then expressed in Expi293F cells. Epitope determination of these antibodies was performed against a human proteome array, and 15 potential autoantigens bound the monoclonal antibodies with significant affinity and specificity. The 12 most-promising antigens were scrutinized for their location in plaque tissue by immunohistochemistry using the cognate antibodies. Nine of these relevant antigens were then successfully synthesized using the pET bacterial expression system, and purified proteins were used to interrogate antibodies in human and mouse sera by ELISA. Mice were then immunized with the 5 best antigens to confirm their immunogenicity. The relevant antigens and antibodies are now being tested to see if they will protect or promote atherosclerosis development, and if they can be used as biomarkers in human disease. This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging.

Boosting the analysis of protein interfaces with multiple interface string alignments: Illustration on the spikes of coronaviruses.

We introduce multiple interface string alignment (MISA), a visualization tool to display coherently various sequence and structure based statistics at protein-protein interfaces (SSE elements, buried surface area, , B factor values, etc). The amino acids supporting these annotations are obtained from Voronoi interface models. The benefit of MISA is to collate annotated sequences of (homologous) chains found in different biological contexts, that is, bound with different partners or unbound. The aggregated views MISA/SSE, MISA/BSA, MISA/ΔASA, and so forth, make it trivial to identify commonalities and differences between chains, to infer key interface residues, and to understand where conformational changes occur upon binding. As such, they should prove of key relevance for knowledge-based annotations of protein databases such as the Protein Data Bank. Illustrations are provided on the receptor binding domain of coronaviruses, in complex with their cognate partner or (neutralizing) antibodies. MISA computed with a minimal number of structures complement and enrich findings previously reported. The corresponding package is available from the Structural Bioinformatics Library (http://sbl.inria.frand https://sbl.inria.fr/doc/Multiple_interface_string_alignment-user-manual.html).

HSymM-guided engineering of the immunodominant p53 transactivation domain putative peptide antigen for improved binding to its anti-p53 monoclonal antibody

A novel engineering strategy to improve autoantibody detection with peptide fragments derived from the parent antigen is presented. The model system studied was the binding of the putative p53 TAD peptide antigen (residues 46–55) to its cognate anti-p53 antibody, ab28. Each engineered peptide contained the full decapeptide epitope and differed only in the flanking regions. Since minimal structural information was available to guide the design, a simple epitope:paratope binding model was applied. The Hidden Symmetry Model, which we recently reported, was used to guide peptide design and estimate per-residue contributions to interaction free energy as a function of added C- and N-terminal flanking peptides. Twenty-four peptide constructs were designed, synthesized, and assessed for binding affinity to ab28 by surface plasmon resonance, and a subset of these peptides were evaluated in a simulated immunoassay for limit of detection. Many peptides exhibited over 200-fold enhancements in binding affinity and improved limits of detection. The epitope was reevaluated and is proposed to be the undecapeptide corresponding to residues 45–55. HSymM calculated binding free energy and experimental data were found to be in good agreement (R2 > 0.75).

Assessment of humoral and cellular immunity induced by the BNT162b2 SARS-CoV-2 vaccine in healthcare workers, elderly people, and immunosuppressed patients with autoimmune disease.

The development of vaccines to prevent SARS-CoV-2 infection has mainly relied on the induction of neutralizing antibodies (nAbs) to the Spike protein of SARS-CoV-2, but there is growing evidence that T cell immune response can contribute to protection as well. In this study, an anti-receptor binding domain (RBD) antibody assay and an INFγ-release assay (IGRA) were used to detect humoral and cellular responses to the Pfizer-BioNTech BNT162b2 vaccine in three separate cohorts of COVID-19-naïve patients: 108 healthcare workers (HCWs), 15 elderly people, and 5 autoimmune patients treated with immunosuppressive agents. After the second dose of vaccine, the mean values of anti-RBD antibodies (Abs) and INFγ were 123.33 U/mL (range 27.55–464) and 1513 mIU/mL (range 145–2500) in HCWs and 210.7 U/mL (range 3–500) and 1167 mIU/mL (range 83–2500) in elderly people. No correlations between age and immune status were observed. On the contrary, a weak but significant positive correlation was found between INFγ and anti-RBD Abs values (rho = 0.354, p = 0.003). As to the autoimmune cohort, anti-RBD Abs were not detected in the two patients with absent peripheral CD19+B cells, despite high INFγ levels being observed in all 5 patients after vaccination. Even though the clinical relevance of T cell response has not yet been established as a correlate of vaccine-induced protection, IGRA testing has showed optimal sensitivity and specificity to define vaccine responders, even in patients lacking a cognate antibody response to the vaccine.

Soluble α-synuclein–antibody complexes activate the NLRP3 inflammasome in hiPSC-derived microglia

Parkinson's disease is characterized by accumulation of α-synuclein (αSyn). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal death in part via microglial activation. Heretofore, it remained unknown if oligomeric/fibrillar αSyn could activate the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome in human microglia and whether anti-αSyn antibodies could prevent this effect. Here, we show that αSyn activates the NLRP3 inflammasome in human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) via dual stimulation involving Toll-like receptor 2 (TLR2) engagement and mitochondrial damage. In vitro, hiMG can be activated by mutant (A53T) αSyn secreted from hiPSC-derived A9-dopaminergic neurons. Surprisingly, αSyn-antibody complexes enhanced rather than suppressed inflammasome-mediated interleukin-1β (IL-1β) secretion, indicating these complexes are neuroinflammatory in a human context. A further increase in inflammation was observed with addition of oligomerized amyloid-β peptide (Aβ) and its cognate antibody. In vivo, engraftment of hiMG with αSyn in humanized mouse brain resulted in caspase-1 activation and neurotoxicity, which was exacerbated by αSyn antibody. These findings may have important implications for antibody therapies aimed at depleting misfolded/aggregated proteins from the human brain, as they may paradoxically trigger inflammation in human microglia.

Morphology and Composition of Immunodiffusion Precipitin Complexes Evaluated via Microscopy and Proteomics.

New approaches to rapid, simple, in vitro diagnostic immunoassays that do not rely on centralized laboratory facilities are urgently needed for disease diagnosis and to inform treatment strategies. The recent and ongoing COVID-19 pandemic has emphasized that rapid diagnostics are needed to help guide government policies on quarantines, social distancing measures, and community lockdowns. A common approach to developing new immunoassays is to modify existing platforms (e.g., automated ELISA and lateral flow assays) for the new analyte, even though this does not address the drawbacks of existing platforms. An alternate approach is to search for robust assays that have been superseded but could in fact solve important challenges using modern technologies. Immunodiffusion is one such platform based on unique "precipitin ring" patterns formed in gels or paper following interactions between proteins and cognate antibodies in diffusion/reaction systems. Herein, we investigate the microstructure of these precipitin rings using a combination of fluorescence and electron microscopy and also perform a mass spectrometry investigation to determine the proteomic composition of the rings. We observed that the rings were composed of microparticles, which we termed "precipitin complexes", and that these complexes were composed of at least 19 key proteins, including immunoglobulins and complement factors along with a range of plasma proteins, possibly related to immune complexes and/or high-density lipoprotein particles. This information will be useful in developing new in vitro diagnostics using reaction/diffusion systems-techniques that require a single assay step and that only require calibrated length measurements for target protein quantification.

Synthetic chemical ligands and cognate antibodies for biorthogonal drug targeting and cell engineering.

A vast range of biomedical applications relies on the specificity of interactions between an antigen and its cognate receptor or antibody. This specificity can be highest when said antigen is a non-natural (synthetic) molecule introduced into a biological setting as a bio-orthogonal ligand. This review aims to present the development of this methodology from the early discovery of haptens a century ago to the recent clinical trials. We discuss such methodologies as antibody recruitment, artificial internalizing receptors and chemically induced dimerization, present the use of chimeric receptors and/or bispecific antibodies to achieve drug targeting and transcytosis, and illustrate how these platforms most impressively found use in the engineering of therapeutic cells such as the chimeric antigen receptor cells. This review aims to be of interest to a broad scientific audience and to spur the development of synthetic artificial ligands for biomedical applications.