A potent, broadly neutralizing human monoclonal antibody that efficiently protects hACE2-transgenic mice from infection with the Wuhan, BA.5, and XBB.1.5 SARS-CoV-2 variants.

The COVID-19 pandemic has uncovered the high genetic variability of the SARS-CoV-2 virus and its ability to evade the immune responses that were induced by earlier viral variants. Only a few monoclonal antibodies that have been reported to date are capable of neutralizing a broad spectrum of SARS-CoV-2 variants. Here, we report the isolation of a new broadly neutralizing human monoclonal antibody, iC1. The antibody was identified through sorting the SARS-CoV-1 RBD-stained individual B cells that were isolated from the blood of a vaccinated donor following a breakthrough infection. In vitro, iC1 potently neutralizes pseudoviruses expressing a wide range of SARS-CoV-2 Spike variants, including those of the XBB sublineage. In an hACE2-transgenic mouse model, iC1 provided effective protection against the Wuhan strain of the virus as well as the BA.5 and XBB.1.5 variants. Therefore, iC1 can be considered as a potential component of the broadly neutralizing antibody cocktails resisting the SARS-CoV-2 mutation escape.

Serial Llama Immunization with Various SARS-CoV-2 RBD Variants Induces Broad Spectrum Virus-Neutralizing Nanobodies.

The emergence of SARS-CoV-2 mutant variants has posed a significant challenge to both the prevention and treatment of COVID-19 with anti-coronaviral neutralizing antibodies. The latest viral variants demonstrate pronounced resistance to the vast majority of human monoclonal antibodies raised against the ancestral Wuhan variant. Less is known about the susceptibility of the evolved virus to camelid nanobodies developed at the start of the pandemic. In this study, we compared nanobody repertoires raised in the same llama after immunization with Wuhan's RBD variant and after subsequent serial immunization with a variety of RBD variants, including that of SARS-CoV-1. We show that initial immunization induced highly potent nanobodies, which efficiently protected Syrian hamsters from infection with the ancestral Wuhan virus. These nanobodies, however, mostly lacked the activity against SARS-CoV-2 omicron-pseudotyped viruses. In contrast, serial immunization with different RBD variants resulted in the generation of nanobodies demonstrating a higher degree of somatic mutagenesis and a broad range of neutralization. Four nanobodies recognizing distinct epitopes were shown to potently neutralize a spectrum of omicron variants, including those of the XBB sublineage. Our data show that nanobodies broadly neutralizing SARS-CoV-2 variants may be readily induced by a serial variant RBD immunization.

Polymer-Stabilized Elemental Boron Nanoparticles for Boron Neutron Capture Therapy: Initial Irradiation Experiments.

Sufficient boron-10 isotope (10B) accumulation by tumor cells is one of the main requirements for successful boron neutron capture therapy (BNCT). The inability of the clinically registered 10B-containing borophenylalanine (BPA) to maintain a high boron tumor concentration during neutron irradiation after a single injection has been partially solved by its continuous infusion; however, its lack of persistence has driven the development of new compounds that overcome the imperfections of BPA. We propose using elemental boron nanoparticles (eBNPs) synthesized by cascade ultrasonic dispersion and destruction of elemental boron microparticles and stabilized with hydroxyethylcellulose (HEC) as a core component of a novel boron drug for BNCT. These HEC particles are stable in aqueous media and show no apparent influence on U251, U87, and T98G human glioma cell proliferation without neutron beam irradiation. In BNCT experiments, cells incubated with eBNPs or BPA at an equivalent concentration of 40 µg 10B/mL for 24 h or control cells without boron were irradiated at an accelerator-based neutron source with a total fluence of thermal and epithermal neutrons of 2.685, 5.370, or 8.055 × 1012/cm2. The eBNPs significantly reduced colony-forming capacity in all studied cells during BNCT compared to BPA, verified by cell-survival curves fit to the linear-quadratic model and calculated radiobiological parameters, though the effect of both compounds differed depending on the cell line. The results of our study warrant further tumor targeting-oriented modifications of synthesized nanoparticles and subsequent in vivo BNCT experiments.

Isolation of a panel of ultra-potent human antibodies neutralizing SARS-CoV-2 and viral variants of concern.

In the absence of virus-targeting small-molecule drugs approved for the treatment and prevention of COVID-19, broadening the repertoire of potent SARS-CoV-2-neutralizing antibodies represents an important area of research in response to the ongoing pandemic. Systematic analysis of such antibodies and their combinations can be particularly instrumental for identification of candidates that may prove resistant to the emerging viral escape variants. Here, we isolated a panel of 23 RBD-specific human monoclonal antibodies from the B cells of convalescent patients. A surprisingly large proportion of such antibodies displayed potent virus-neutralizing activity both in vitro and in vivo. Four of the isolated nAbs can be categorized as ultrapotent with an apparent IC100 below 16 ng/mL. We show that individual nAbs as well as dual combinations thereof retain activity against currently circulating SARS-CoV-2 variants of concern (such as B.1.1.7, B.1.351, B.1.617, and C.37), as well as against other viral variants. When used as a prophylactics or therapeutics, these nAbs could potently suppress viral replication and prevent lung pathology in SARS-CoV-2-infected hamsters. Our data contribute to the rational development of oligoclonal therapeutic nAb cocktails mitigating the risk of SARS-CoV-2 escape.

Gold Nanoparticles Permit In Situ Absorbed Dose Evaluation in Boron Neutron Capture Therapy for Malignant Tumors.

Boron neutron capture therapy (BNCT) is an anticancer modality realized through 10B accumulation in tumor cells, neutron irradiation of the tumor, and decay of boron atoms with the release of alpha-particles and lithium nuclei that damage tumor cell DNA. As high-LET particle release takes place inside tumor cells absorbed dose calculations are difficult, since no essential extracellular energy is emitted. We placed gold nanoparticles inside tumor cells saturated with boron to more accurately measure the absorbed dose. T98G cells accumulated ~50 nm gold nanoparticles (AuNPs, 50 µg gold/mL) and boron-phenylalanine (BPA, 10, 20, 40 µg boron-10/mL), and were irradiated with a neutron flux of 3 × 108 cm-2s-1. Gamma-rays (411 keV) emitted by AuNPs in the cells were measured by a spectrometer and the absorbed dose was calculated using the formula D = (k × N × n)/m, where D was the absorbed dose (GyE), k-depth-related irradiation coefficient, N-number of activated gold atoms, n-boron concentration (ppm), and m-the mass of gold (g). Cell survival curves were fit to the linear-quadratic (LQ) model. We found no influence from the presence of the AuNPs on BNCT efficiency. Our approach will lead to further development of combined boron and high-Z element-containing compounds, and to further adaptation of isotope scanning for BNCT dosimetry.

Diversity of Immunoglobulin Light Chain Genes in Non-Teleost Ray-Finned Fish Uncovers IgL Subdivision into Five Ancient Isotypes.

The aim of this study was to fill important gaps in the evolutionary history of immunoglobulins by examining the structure and diversity of IgL genes in non-teleost ray-finned fish. First, based on the bioinformatic analysis of recent transcriptomic and genomic resources, we experimentally characterized the IgL genes in the chondrostean fish, Acipenser ruthenus (sterlet). We show that this species has three loci encoding IgL kappa-like chains with a translocon-type gene organization and a single VJC cluster, encoding homogeneous lambda-like light chain. In addition, sterlet possesses sigma-like VL and J-CL genes, which are transcribed separately and both encode protein products with cleavable leader peptides. The Acipenseriformes IgL dataset was extended by the sequences mined in the databases of species belonging to other non-teleost lineages of ray-finned fish: Holostei and Polypteriformes. Inclusion of these new data into phylogenetic analysis showed a clear subdivision of IgL chains into five groups. The isotype described previously as the teleostean IgL lambda turned out to be a kappa and lambda chain paralog that emerged before the radiation of ray-finned fish. We designate this isotype as lambda-2. The phylogeny also showed that sigma-2 IgL chains initially regarded as specific for cartilaginous fish are present in holosteans, polypterids, and even in turtles. We conclude that there were five ancient IgL isotypes, which evolved differentially in various lineages of jawed vertebrates.

B cell-specific protein FCRLA is expressed by plasmacytoid dendritic cells in humans.

BACKGROUND: Fc receptor-like A (FCRLA) is a unique member of a family of Fc receptor like-molecules that lacks a transmembrane region and is an ER-resident protein. In mice and humans, FCRLA has been known as a B cell specific protein. We report here that, in humans, FCRLA is also expressed in a subpopulation of plasmacytoid dendritic cells (pDCs). METHODS: Human peripheral blood mononuclear cells (PBMC), splenocytes, and tonsillar cells were stained for lineage markers followed by fixation/saponin permeabilization and intracellular staining for FCRLA, and then analyzed by flow cytometry with CD123 and CD303 used as pDC markers. RESULTS: We conducted an extensive flow cytometric analysis of a rare population of CD19-FCRLA+ cells found for the first time in human lymphoid tissues that we assigned to pDCs as they were lin-/CD123+/CD303+. FCRLA expression in human pDCs was further confirmed by the RT-PCR analysis of cDNA of pDCs isolated from the peripheral blood of a healthy donor. FCRLA-positive pDCs expressed a lower level of HLA-DR than their FCRLA-negative counterparts. CONCLUSIONS: FCRLA has long been viewed as a B cell specific protein, and this is the first time its expression has also been shown in human pDCs. © 2017 International Clinical Cytometry Society.

Radiobiological response of U251MG, CHO-K1 and V79 cell lines to accelerator-based boron neutron capture therapy.

In the current article, we provide in vitro efficacy evaluation of a unique accelerator-based neutron source, constructed at the Budker Institute of Nuclear Physics (Novosibirsk, Russian Federation), for boron neutron capture therapy (BNCT), which is particularly effective in the case of invasive cancers. U251MG, CHO-K1 and V79 cells were incubated and irradiated in various concentrations of boric acid with epithermal neutrons for 2-3 h in a plexiglass phantom, using 2.0 MeV proton energy and 1.5-3.0 mA proton current, resulting in a neutron fluence of 2.16 × 1012 cm-2. The survival curves of cells loaded with boron were normalized to those irradiated without boron (to exclude the influence of the fast neutron and gamma dose components) and fit to the linear-quadratic (LQ) model. Colony formation assays showed the following cell survival rates (means ± SDs): CHO-K1: 0.348 ± 0.069 (10 ppm), 0.058 ± 0.017 (20 ppm), 0.018 ± 0.005 (40 ppm); V79: 0.476 ± 0.160 (10 ppm), 0.346 ± 0.053 (20 ppm), 0.078 ± 0.015 (40 ppm); and U251MG: 0.311 ± 0.061 (10 ppm), 0.131 ± 0.022 (20 ppm), 0.020 ± 0.010 (40 ppm). The difference between treated cells and controls was significant in all cases (P < 0.01) and confirmed that the neutron source and irradiation regimen were sufficient for control over cell colony formation. We believe our study will serve as a model for ongoing in vitro experiments on neutron capture therapy to advance in this area for further development of accelerator-based BNCT into the clinical phase.

Peptide-MHC multimer-based monitoring of CD8 T-cells in HIV-1 infection and AIDS vaccine development.

The use of MHC multimers allows precise and direct detecting and analyzing of antigen-specific T-cell populations and provides new opportunities to characterize T-cell responses in humans and animals. MHC-multimers enable us to enumerate specific T-cells targeting to viral, tumor and vaccine antigens with exceptional sensitivity and specificity. In the field of HIV/SIV immunology, this technique provides valuable information about the frequencies of HIV- and SIV-specific CD8(+) cytotoxic T lymphocytes (CTLs) in different tissues and sites of infection, AIDS progression, and pathogenesis. Peptide-MHC multimer technology remains a very sensitive tool in detecting virus-specific T -cells for evaluation of the immunogenicity of vaccines against HIV-1 in preclinical trials. Moreover, it helps to understand how immune responses are formed following vaccination in the dynamics from priming point until T-cell memory is matured. Here we review a diversity of peptide-MHC class I multimer applications for fundamental immunological studies in different aspects of HIV/SIV infection and vaccine development.

Development and characterization of domain-specific monoclonal antibodies produced against human SLAMF9.

SLAMF9 is a member of the signaling lymphocyte-activating molecule (SLAM) immunoreceptor family. The SLAM family receptors are expressed in a broad range of immune cells and play an important role in immunity. To date, SLAMF9 is the least studied member of this family. Its ligand, signaling properties, and cells on whose surface it is expressed are unknown. We generated hybridoma clones 6E11 and 7G5 secreting monoclonal antibodies specific to human SLAMF9. BALB/c mice were immunized with Escherichia coli-expressed purified SLAMF9 protein; splenocytes from these mice were fused with mouse myeloma cell line NS-1. Based on isotyping of the MAbs, clone 6E11 was referred to the IgG1 subclass, while 7G5 to IgG2b. The specificity of these MAbs was assessed by ELISA, immunoblotting, immunohistochemistry, and flow cytometry. According to the results of epitope analysis, clone 6E11 reacts with the C2-like domain, whereas 7G5 is specific to the V-like domain of the SLAMF9 molecule. The generated MAbs were demonstrated to be applicable in various immunochemical analyses. They may be useful tools in studies clarifying the expression and function of human SLAMF9.

Characterization of human FCRLA isoforms.

FCRLA is an ER-resident B-cell specific protein. The exact function of this protein remains unclear although human FCRLA has been recently shown to interact with IgM, IgG and IgA. The retention of FCRLA in ER is mediated by the N-terminal domain. The major human FCRLA isoform is encoded by five exons, of which one encodes a short signal peptide (SSP) and the others code four protein domains. Here we show that human tissues also produce transcripts which contain an additional exon and encode proteins with signal peptide that is six residues longer (LSP). Transfection experiments demonstrated that the extension of the signal peptide had no visible effect on the topology and molecular mass of the processed four-domain FCRLA isoform. However, the length of the signal peptide was found to affect processing of two-domain FCRLA isoforms composed of the third and fourth domains (FCRLAd2). The signal peptide was not cleaved in the SSP-FCRLAd2 and this isoform was found to accumulate in the ER. In contrast, the LSP-containing FCRLAd2 isoform was processed, O-glycosylated and secreted. The secreted FCRLAd2 isoform did not interact with IgG- or IgM-immunosorbents.

Differential expression of FCRLA in naïve and activated mouse B cells.

FCRLA is an intracellular B cell protein that belongs to the FcR-like family. Using newly generated FCRLA-specific antibodies, we studied the constitutive expression pattern of mouse FCRLA and monitored changes during an immune response and following in vitro B cell activation. All B cell subpopulations examined expressed FCRLA. However, the level of FCRLA expression is determined by the stage of B cell differentiation. Low expression of FCRLA is characteristic of naïve follicular and marginal zone B cells. High expression was detected in a small fraction of activated B cells scattered along migratory pathways in the lymphoid tissues. FCRLA-bright cells could be subdivided into two subpopulations, with high and low/undetectable level of intracellular immunoglobulins, which phenotypically resemble either plasma or memory B cells. High expression of FCRLA in subset(s) of terminally differentiated B-cells suggests that, being an ER protein, FCRLA may participate in the regulation of immunoglobulin assembly and secretion.

FCRLA is a resident endoplasmic reticulum protein that associates with intracellular Igs, IgM, IgG and IgA.

Fc receptor-like A (FCRLA) is an unusual member of the extended Fc receptor family. FCRLA has homology to receptors for the Fc portion of Ig (FCR) and to other FCRL proteins. However, unlike these other family representatives, which are typically transmembrane receptors with extracellular ligand-binding domains, FCRLA has no predicted transmembrane domain or N-linked glycosylation sites and is an intracellular protein. We show by confocal microscopy and biochemical assays that FCRLA is a soluble resident endoplasmic reticulum (ER) protein, but it does not possess the amino acid sequence KDEL as an ER retention motif in its C-terminus. Using a series of deletion mutants, we found that its ER retention is most likely mediated by the amino terminal partial Ig-like domain. We have identified ER-localized Ig as the FCRLA ligand. FCRLA is unique among the large family of Fc receptors, in that it is capable of associating with multiple Ig isotypes, IgM, IgG and IgA. Among hemopoietic cells, FCRLA expression is restricted to the B lineage and is most abundant in germinal center B lymphocytes. The studies reported here demonstrate that FCRLA is more broadly expressed among human B lineage cells than originally reported; it is found at significant levels in resting blood B cells and at varying levels in all B-cell subsets in tonsil.

FCRL6 receptor: expression and associated proteins.

FCRL6 receptor is a more recently identified representative of the FCRL family. We generated a panel of mouse mAbs to baculovirus-derived recombinant FCRL6 protein. The clone 7B2 was found to specifically recognize a 63kDa protein expressed preferentially on the surface of CD8 T and CD56 NK cells in human peripheral blood and spleen. The clone 7B2 reacts with FCRL6 in Western blotting, FACS, and immunohistochemistry. In the T cell lineage, FCRL6 functions in antigen-experienced cells. Mitogenic stimulation of PB leukocytes in vitro resulted in an abrogation of the FCRL6 gene expression. We found a significant decrease in the FCRL6 gene expression in peripheral T cells of patients with certain autoimmune and blood diseases, and its upregulation at the late stages of HIV infection. Study of the FCRL6 association with signaling molecules showed its ability to recruit SHP-1, SHP-2, SHIP-1, and SHIP-2 phosphatases, and also adaptor protein Grb2 through phosphorylated cytoplasmic tyrosines. The current results demonstrate inhibitory potential of FCRL6 and suggest its possible involvement in modulation of CTL effector functions in various immune disorders.

The amphibians Xenopus laevis and Silurana tropicalis possess a family of activating KIR-related Immunoglobulin-like receptors.

In this study, we searched the amphibian species Xenopus laevis and Silurana (Xenopus) tropicalis for the presence of genes homologous to mammalian KIRs and avian CHIRs (KRIR family). By experimental and computational procedures, we identified four related ILR (Ig-like Receptors) genes in S. tropicalis and three in X. laevis. ILRs encode type I transmembrane receptors with 3-4 Ig-like extracellular domains. All predicted ILR proteins appear to be activating receptors. ILRs have a broad expression pattern, the gene transcripts were found in both lymphoid and non-lymphoid tissues. Phylogenetic analysis shows that the amphibian KRIR family receptors evolved independently from their mammalian and avian counterparts. The only conserved structural element of tetrapod KRIRs is the NxxR motif-containing transmembrane domain that facilitates association with FcRgamma subunit. Our findings suggest that if KRIRs of various vertebrates have any common function at all, such a function is activating rather than inhibitory.

The Xenopus FcR family demonstrates continually high diversification of paired receptors in vertebrate evolution.

BACKGROUND: Recent studies have revealed an unexpected diversity of domain architecture among FcR-like receptors that presumably fulfill regulatory functions in the immune system. Different species of mammals, as well as chicken and catfish have been found to possess strikingly different sets of these receptors. To better understand the evolutionary history of paired receptors, we extended the study of FcR-like genes in amphibian representatives Xenopus tropicalis and Xenopus laevis. RESULTS: The diploid genome of X. tropicalis contains at least 75 genes encoding paired FcR-related receptors designated XFLs. The allotetraploid X. laevis displays many similar genes primarily expressed in lymphoid tissues. Up to 35 domain architectures generated by combinatorial joining of six Ig-domain subtypes and two subtypes of the transmembrane regions were found in XFLs. None of these variants are shared by FcR-related proteins from other studied species. Putative activating XFLs associate with the FcRgamma subunit, and their transmembrane domains are highly similar to those of activating mammalian KIR-related receptors. This argues in favor of a common origin for the FcR and the KIR families. Phylogenetic analysis shows that the entire repertoires of the Xenopus and mammalian FcR-related proteins have emerged after the amphibian-amniotes split. CONCLUSION: FcR- and KIR-related receptors evolved through continual species-specific diversification, most likely by extensive domain shuffling and birth-and-death processes. This mode of evolution raises the possibility that the ancestral function of these paired receptors was a direct interaction with pathogens and that many physiological functions found in the mammalian receptors were secondary acquisitions or specializations.

A direct antigen-binding assay for detection of antibodies against native epitopes using alkaline phosphatase-tagged proteins.

We describe a simple and efficient method to detect antibodies against native epitopes following immunization with denatured proteins and peptides. With this method, soluble antigens genetically fused with placental alkaline phosphatase (AP) are used as probes to detect antibodies immobilized on nitrocellulose membranes. The AP-tagged proteins can be produced in sufficient amounts using transient transfection of eukaryotic cells with an appropriate cDNA fragment in a commercial AP-tag vector. The intrinsic thermo-stable phosphatase activity of a tagged protein obviates the need for its purification. To evaluate the method, three recently identified proteins of the FcR family, FCRLA, FCRL1, and FCRL4, were fused with AP and tested in a reaction with various polyclonal and monoclonal antibodies raised by immunization with bacterially produced antigens and peptide conjugates. All the three probes demonstrated high specificity in analysis of immune sera and hybridoma supernatants. Sensitivity of the assay varied depending on antibody tested and, in some cases, was in the subnanogram range. The results obtained show that AP-tagged proteins are useful tools for discrimination of antibodies against native epitopes when production of antigen in its native conformation is laborious and expensive.

Natural human gene correction by small extracellular genomic DNA fragments.

Classical gene targeting employs natural homologous recombination for a gene correction using a specially designed and artificially delivered DNA construct but the method is very inefficient. On the other hand, small DNA fragments in the form of tiny chromatin-like particles naturally present in blood plasma can spontaneously penetrate into human cells and cell nuclei. We hypothesized that these natural DNA nanoparticles with recombinagenic free ends might be effective agents for gene replacement therapy. We demonstrate that a mixture of small fragments of total human chromatin from non-mutant cells added to a culture medium without transfection agents efficiently repaired a 47 base pair deletion in the CASP3 gene in 30% of treated human MCF7 breast cancer cells, as shown by restoration of caspase-3 apoptotic function and CASP3 DNA and mRNA structure. Such an innate gene replacement mechanism might function naturally in an organism using its own apoptotic DNA fragments. This mechanism might enable human cancer cell phenotype normalization in the presence of excess normal cells.

Generation and characterization of monoclonal antibodies specific for human FCRLA.

FCRLA is a recently identified intracellular protein structurally related to the classic Fc receptors and expressed primarily in the germinal centers of B cells. We generated six monoclonal antibodies (MAbs) specific to the human protein. The MAbs recognize three different epitopes, which were shown to be localized on the D3 domain of the FCRLA molecule. The clones M101 and M616 were demonstrated to be applicable in various immunochemical analyses, such as immunoblotting, immunohistochemistry, and immunoprecipitation. In addition, this pair of antibodies was used for development of a sandwich version of ELISA to quantitatively detect FCRLA in cell lysates. Using these MAbs, we studied FCRLA expression in a panel of human B cell lines, such as Raji, Daudi, Bjab, BL-2, RPMI 1788, RPMI 8226, IM-9, and SKW6.4. It was found that all these lines, except RPMI 8226, produce FCRLA but may vary in the proportion of FCRLA-positive cells. The MAbs we established can be a useful tool to investigate the functional role of FCRLA and its applicability as a B cell development and malignant transformation marker.

Expression pattern of FCRL (FREB, FcRX) in normal and neoplastic human B cells.

FCRL (also known as FREB and FcRX) is a recently described member of the family of Fc receptors for immunoglobulin G (IgG). In the present study we analysed its expression in normal and neoplastic lymphoid tissue using immunohistochemical techniques. FCRL was preferentially expressed in a proportion of germinal centre cells and, more weakly, in mantle zone B cells. In addition, strong labelling was observed in marginal zone B cells in the spleen, representing one of the few markers for this cell type. The majority of cases of small B-cell lymphoma, diffuse large B-cell lymphoma and lymphocyte predominance Hodgkin's disease were positive for FCRL. However, the number of positive cells varied widely, and in consequence we could not define a cut-off that distinguished subsets of diffuse large B-cell lymphoma. Our results also showed that FCRL tended to be negative in T-cell-rich B-cell lymphoma and in classical Hodgkin's disease. FCRL may therefore represent a novel marker for normal B cells (e.g. splenic marginal zone cells) and may also be useful as a potential marker of B-cell neoplasms.