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The polyepitope strategy is promising approach for successfully creating a broadly protective flu vaccine, which targets T-lymphocytes (both CD4+ and CD8+) to recognise the most conserved epitopes of viral proteins. In this study, we employed a computer-aided approach to develop several artificial antigens potentially capable of evoking immune responses to different virus subtypes. These antigens included conservative T-cell epitopes of different influenza A virus proteins. To design epitope-based antigens we used experimentally verified information regarding influenza virus T-cell epitopes from the Immune Epitope Database (IEDB) (http://www.iedb.org). We constructed two "human" and two "murine" variants of polyepitope antigens. Amino acid sequences of target polyepitope antigens were designed using our original TEpredict/PolyCTLDesigner software. Immunogenic and protective features of DNA constructs encoding "murine" target T-cell immunogens were studied in BALB/c mice. We showed that mice groups immunised with a combination of computer-generated "murine" DNA immunogens had a 37.5% survival rate after receiving a lethal dose of either A/California/4/2009 (H1N1) virus or A/Aichi/2/68 (H3N2) virus, while immunisation with live flu H1N1 and H3N2 vaccine strains provided protection against homologous viruses and failed to protect against heterologous viruses. These results demonstrate that mechanisms of cross-protective immunity may be associated with the stimulation of specific T-cell responses. This study demonstrates that our computer-aided approach may be successfully used for rational designing artificial polyepitope antigens capable of inducing virus-specific T-lymphocyte responses and providing partial protection against two different influenza virus subtypes.Communicated by Ramaswamy H. Sarma.

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.

Development of efficient antitumor modalities remains a priority of modern translation medicine. Adoptive transfer of autologous T cells expressing chimeric antigen receptors (CARs) specific for tumor-associated surface targets of B cells has demonstrated encouraging results in recent clinical trials. It is tempting therefore to adapt this approach to solid cancers and move it to universal/allogeneic formats. For this reason, it appears very attractive to use human NK cell lines as CAR carriers. Using lentiviral delivery, human NK cell line YT was modified to express a PSMA-specific CAR and to produce an anticancer peptide lactaptin. "Armored” CAR-YT-Lact cell line obtained shows cytotoxicity against PSMA-positive prostate adenocarcinoma cells. Accordingly, YT-Lact cells display a moderate and non-specific activity against PSMA-negative target cells, which may translate into a tighter control of tumor cell escape via antigen loss.

T and NK cells armed with chimeric antigen receptors (CAR) are promising tools for the specific elimination of cancer cells. In most CAR designs implemented to date, the recognition of target cells is mediated by single-chain variable fragments (scFvs) derived from murine monoclonal antibodies. This format, however, has a number of limitations, including its relatively large size and potential immunogenicity in humans. In this study, we explored the feasibility of using human fibronectin type III domains (Fn3) as the antigen recognition domain in CARs. Human Fn3 domains have lower predicted immunogenicity compared to mouse-derived sequences, and a reduced molecular weight compared to scFvs. We created a functional CAR using a VEGFR2-specific Fn3 module replacing the conventional scFv. The resulting FnCAR specifically potentiates the cytotoxic activity of human T cells and YT NK cells in the presence of VEGFR2-positive targets. These findings demonstrate that Fn3 domains can be used in CARs for antigen recognition.

Despite the multitude of anticancer cytostatic drugs available to oncologists today, most of such drugs have serious side effects that may preclude their use in some groups of patients. Hence, selective induction of apoptosis in cancer but not normal cells remains an attractive goal of molecular medicine. Lactaptin, a proteolytic fragment of the human milk kappa-casein, has been previously identified as a protein displaying potent killing of cancer cells in vitro. Its recombinant analog (RL2) produced in E. coli has been shown to delay solid tumor growth in vivo. Given that lactaptin is of human origin and is not immunogenic, it can be administered to patients multiple times without running the risk of immune response that could dampen the therapy efficacy. In the present study, we demonstrate that the combination of RL2 and cyclophosphamide treatments has an additive therapeutic effect against hepatoma tumor in immunocompetent mice. We asked whether production of lactaptin in human rather than bacterial cells would result in a protein with increased cytotoxic activity. Using lentiviral vector pCDH as a backbone, two constructs, pEL1 and pEL2, encoding secreted forms of lactaptin that differ in their signal sequences were created. Lactaptin expression in human cell lines was confirmed using Western-blot analysis, whereas ELISA was used for quantification of secreted lactaptin. Next, we measured the cytotoxic effects of the media conditioned by pEL1-transfected HEK293T cells, as assayed against the panel of three human cancer cell lines: MDA-MB-231 (adenocarcinoma), PC3 (prostate cancer), and T98G (glioblastoma). We show that EL1-derived lactaptin is at least 100-fold more cytotoxic than RL2. Taken together, our results provide an opportunity for developing armored immune cells as an “off-the-shelf” platform for targeted delivery of lactaptin to cancer cells.

The article reports the development of a collection of lentiviral vector constructs enabling time-efficient production and testing of different variants of chimeric antigen receptors (CAR). These artificial surface proteins make it possible to redirect the activity of immune cytotoxic T-cells towards cancer cells. Chimeric antigen receptors usually encompass four functional modules, namely, antigen recognition, flexible linker, transmembrane, and signal modules. The use of modules with different properties allows modulating the affinity and specificity of CAR interaction with target antigens, as well as intensity and quality of activation signaling, which determines the cytotoxic properties of CAR T-cells, as well as their proliferation rate and time of persistence in the organism. The proposed vector system make it possible to easily test various combinations of CAR modules while its being open to distribution allows the direct comparison of the results obtained by different scientific groups.

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.

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.

Summary This study is focusing on elucidation of the capacity of attenuated Salmonella enteritidis E23 (cya, crp )t o serve as a vehicle for the rectal delivery of the DNA vaccine. Earlier for creation HIV-1 candidate DNA vaccine we have designed the polyepitope protein TCI (T-cell immunogen), which comprises over 80 CTL epitopes from subtype A, B and C HIV-1 proteins. The gene coding for TCI protein was used to construct the eukaryotic expression plasmid pcDNA-TCI. The attenuated S. enteritidis E23 was transformed by electroporation with recombinant plasmid pcDNA-TCI and the expression of the TCI gene was determined in vitro andinvivo.BALB/cmicewererectallyimmunizedwith S. enteritidis E23/pcDNA-TCI (10 8 cfu) twice at 4 week interval. Bacteria were not pathogenic for mice and spontaneously eliminated from mice spleen and liver to 60 days post the immunization. Detectable antibodies were generated in 2 weeks after immunization and their level increased after second immunization. The results of INF-g ELISpot show that mice immunized with S. enteritidis E23/pcDNA-TCI elicited HIV-specific cellular immune response. This study demonstrates that attenuated S. enteritidis E23 is an effective live vector for rectal delivery of the DNA vaccine pcDNATCI to generate humoral and T-cellular responses against HIV-1.

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.

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.

A novel conserved member of the leukocyte Fc receptor (FcR) family was identified in human and mouse. The presumably secreted protein, designated FCRL (FcR-like) is comprised of four domains. The three N-terminal domains are related to the extracellular region of FcγRI, with the second (35–37% residue identity) and the third (46–52%) domains showing highest similarity. The C-terminaldomain is a unique sequence enriched with proline residues. In humans, alternative transcripts for six FCRL isoforms were revealed. Spleen and tonsils were found to be the major sources of FCRL mRNA in human tissues. Western blotting of tonsil cell lysate using FCRL-specific antibodies recognized a 44-kDa protein produced as a monomer containing free sulfhydryl groups. The monomer, however, was able to form disulfide-linked homo-oligomer upon oxidation. In COS-7 cells transiently transfected with two human FCRL isoforms, both resided intracellularly. Immunohistochemical staining of tonsil sections demonstrated the FCRL expression in germinal centers, suggesting that the protein may be implicated in germinal center-specific stages of B cell development. The phylogenetic analysis of the FCRL relationships with the leukocyte FcR supports a view that the three-domain structure was primordial in the evolution of the family.

Summary This study is focusing on elucidation of the capacity of attenuated Salmonella enteritidis E23 (cya, crp) to serve as a vehicle for the rectal delivery of the DNA vaccine. Earlier for creation HIV‐1 candidate DNA vaccine we have designed the polyepitope protein TCI (T‐cell immunogen), which comprises over 80 CTL epitopes from subtype A, B and C HIV‐1 proteins. The gene coding for TCI protein was used to construct the eukaryotic expression plasmid pcDNA‐TCI. The attenuated S. enteritidis E23 was transformed by electroporation with recombinant plasmid pcDNA‐TCI and the expression of the TCI gene was determined in vitro and in vivo. BALB/c mice were rectally immunized with S. enteritidis E23/pcDNA‐TCI (108 cfu) twice at 4 week interval. Bacteria were not pathogenic for mice and spontaneously eliminated from mice spleen and liver to 60 days post the immunization. Detectable antibodies were generated in 2 weeks after immunization and their level increased after second immunization. The results of INF‐γ ELISpot show that mice immunized with S. enteritidis E23/pcDNA‐TCI elicited HIV‐specific cellular immune response. This study demonstrates that attenuated S. enteritidis E23 is an effective live vector for rectal delivery of the DNA vaccine pcDNA‐TCI to generate humoral and T‐cellular responses against HIV‐1.

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.

We report cloning and characterization of FCRL2, a novel human gene that belongs to the FcR family. The gene is closely linked and structurally similar to the recently identified FCRL/FREB/FcRX gene. The encoded protein is composed of three Ig-like domains and a C-terminal mucin-like domain containing a conserved alpha-helical motif with dileucine signals. Intraexonic splicing may generate two alternative transcripts, coding for isoforms with the third and fourth domains replaced by entirely different amino acid sequences. Like FCRL, the full-length isoform of FCRL2 is expressed intracellularly in transfected 293T cells. Expression analysis revealed FCRL2 mRNA only in placenta. The gene transcripts were not detected in lymphoid tissues or in the main leukocyte subsets isolated from peripheral blood. However, we found that FCRL2 is differentially expressed by transformed B cell lines. Of interest is also the finding that the gene expression may be up-regulated in the progression of melanocytic tumors.

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.

A novel conserved member of the leukocyte Fc receptor (FcR) family was identified in human and mouse. The presumably secreted protein, designated FCRL (FcR-like) is comprised of four domains. The three N-terminal domains are related to the extracellular region of FcgammaRI, with the second (35-37% residue identity) and the third (46-52%) domains showing highest similarity. The C-terminal domain is a unique sequence enriched with proline residues. In humans, alternative transcripts for six FCRL isoforms were revealed. Spleen and tonsils were found to be the major sources of FCRL mRNA in human tissues. Western blotting of tonsil cell lysate using FCRL-specific antibodies recognized a 44-kDa protein produced as a monomer containing free sulfhydryl groups. The monomer, however, was able to form disulfide-linked homo-oligomer upon oxidation. In COS-7 cells transiently transfected with two human FCRL isoforms, both resided intracellularly. Immunohistochemical staining of tonsil sections demonstrated the FCRL expression in germinal centers, suggesting that the protein may be implicated in germinal center-specific stages of B cell development. The phylogenetic analysis of the FCRL relationships with the leukocyte FcR supports a view that the three-domain structure was primordial in the evolution of the family.

A group of genes encoding proteins structurally related to the leukocyte Fc receptors (FcRs) and termed the IFGP family was identified in human and mouse. Sequences of four human and two mouse cDNAs predict proteins differing by domain composition. One of the mouse cDNAs encodes a secreted protein, which, in addition to four immunoglobulin (Ig)-like domains, contains a scavenger receptor superfamily-related domain at the C-terminus. The other cDNAs code for the type I transmembrane proteins with the extracellular parts comprised of one to six Ig-like domains. Five homologous types of the Ig-like domains were defined and each protein was found to have a unique combination of the domain types. The cytoplasmic tails of the transmembrane proteins show different patterns of the tyrosine-based signal motifs. While the human IFGP members appear to be B-cell antigens, the mouse genes have a broader tissue distribution with predominant expression in brain. Sequence comparisons revealed that the IFGP family may be regarded as a phylogenetic link joining the leukocyte FcRs with the rat NK cell-specific gp42 antigen and platelet endothelial cell adhesion molecule-1 (PECAM-1), two mammalian leukocyte receptors whose close relatives were not found previously. It is suggested that FcRs, the IFGP proteins and gp42 have arisen by a series of duplications from a common ancestor receptor comprised of five Ig-like domains. The organization of the human genes shows that the IFGP family evolved through differential gain and loss of exons due to recombination and/or mutation accumulation in the duplicated copies.