Panel Of Monoclonal Antibodies Research Articles

Lysosomal Associated Membrane Protein Family Member 5 (LAMP5) As a Novel Mediator of, and Target in High Risk Multiple Myeloma (MM)

Background: Our recently published studies of single cell clonotypic and transcriptional evolution of multiple myeloma precursor disease (Dang et al. Cancer Cell, 2023), as well as related multi-omic studies of more advanced disease, identified a number of genes as potential contributors to progression. Among these is LAMP5, a gene on chromosome 20 that is markedly and specifically overexpressed in the malignant, monoclonal plasma cells in a high proportion of patients. As LAMP5 expression has also been associated with osteolytic bony disease in myeloma, we sought to delineate its potential contributions to the pathobiology of myeloma, which remain incompletely understood. Methods: The impact of LAMP5 knockdown and over-expression was studied using myeloma cell line models both in vitro and in vivo. Moreover, we generated a monoclonal antibody to LAMP5 after finding it was exposed on the myeloma cell plasma membrane, and converted this agent into an antibody drug conjugate (ADC) linked to monomethyl auristatin E (MMAE) or pyrrolobenzodiazepine ( PBD) dimers. Finally, we used the sequence of the single-chain variable fragment to generate LAMP5-targeted chimeric antigen receptor (CAR)-guided T-cells. Results: Analysis of the Multiple Myeloma Research Foundation's CoMMpass SM database indicated that high LAMP5 expression (defined as in the top quartile) in newly diagnosed myeloma patients was significantly associated with markers of increased disease burden, including a higher beta-2-microglobulin (p = 0.047), C-reactive protein (p = 0.0399), serum calcium (p < 0.0001), marrow plasmacytosis (p < 0.001), and a trend towards increased circulating plasma cells (p = 0.075). Moreover, high LAMP5 expression correlated with a shorter progression-free (p = 0.0006) as well as overall survival (p = 0.0006). Similarly, analysis of the University of Arkansas for Medical Sciences gene expression data indicated LAMP5 was increased in the hyperdiploid and proliferative groups that are associated with a poor prognosis. Inducible shRNA-mediated LAMP5 knockdown in myeloma cell lines conferred a survival disadvantage and activated apoptosis. RNA-sequencing analysis identified multiple dysregulated Hallmark pathways, including the G2M checkpoint, the mitotic spindle, apoptosis, mTORC1 signaling, and the unfolded protein response. Proteomic consequences of LAMP5 knockdown included Caspase-7, -8, and -3 cleavage, and increased abundance of Smac, Bim, and PUMA. By contrast, many G2M checkpoint proteins were down-regulated, including Aurora A, Checkpoint-1, Cell division cycle-6, and WEE1 kinases, and decreased levels of phospho-S807/S811 Rb protein were seen. Also, RNA-sequencing revealed mTORC1 pathway dysregulation, and proteomic studies showed decreased activity of AKT and AKT2, mTOR, and p70 S6 kinase. Notably, LAMP5 suppression was associated with reduced levels of expression of an RFP-LC3 fusion protein consistent with a role for LAMP5 in autophagy. In contrast, high LAMP5 expression was associated with improved colony formation in vitro ( A), and promoted disease progression in vivo. Since the LAMP5 promoter had consensus retinoic acid sites, we tested all-trans retinoic acid and found it suppressed LAMP5 protein expression, and reduced in vitro clonogenicity. Finally, since other LAMPs can under some circumstances be found at the plasma membrane, we performed flow and LAMP5 was indeed detected on the surface of unpermeabilized LAMP5-positive myeloma cells. We thus developed a panel of monoclonal antibodies that recognized the extracellular domains of LAMP5, and converted one of these into antibody drug conjugates (ADCs) linked to either MMAE or PBD. Both ADCs reduced the viability of LAMP5-positive myeloma cells in a time- and concentration-dependent manner ( B), activated immunogenic cell death in vitro, and also showed anti-tumor activity in vivo. Importantly, LAMP5-directed CAR T-cells similarly and specifically eliminated only LAMP5-positive myeloma cells with efficacy that was comparable to that of B-cell maturation antigen-targeted CAR T-cells. Conclusions: These pre-clinical in vitro and in vivo data support the possibility that LAMP5 represents a novel mediator of myeloma clonogenicity and high-risk behavior, and that it could serve as a rational therapeutic target leveraging immune therapies that recognize the extracellular domains of cell surface LAMP5.

Centralized MRD Assessment at Early Timepoints in Gimema AML1718 Trial, a Phase 2 Open-Label Study of Venetoclax, Fludarabine, Idarubicin and Cytarabine (V-FLAI) in the Induction Therapy of Non Low-Risk AML

Background: New therapeutic strategies are strongly needed to improve the prognosis of high risk Acute Myeloid Leukemia (AML) patients, such as combination of novel agents and conventional chemotherapy. The Italian GIMEMA AML1718 trial (NCT03455504) investigate the safety and efficacy of the BCL-2 inhibitor venetoclax (VEN) in combination with intensive fludarabine-based induction (FLAI) [fludarabine 30 mg/sqm from day 1 to day 5, cytarabine 2000 mg/sqm from day 1 to day 5, and idarubicin 8 mg/sqm on days 1, 3 and 5] as first-line therapy for newly diagnosed non low-risk ELN AML patients. Results from the Planned Interim Analysis of Safety Run-in and Part 1 (early expansion cohorts) were presented at the last 2022 ASH Meeting. Median overall survival (OS) was not reached; probability of 12-month OS was 76%. Median disease-free survival was not reached. With a median follow-up of 10.5 months, 28 patients (49%) received allogeneic stem cell transplantation (HSCT) in first complete remission (CR). Centralized multicolour flow cytometry minimal residual disease (MFC-MRD) assessment was planned during the phase 2, part 2 of the study (confirmatory cohort) where the lower effective dose of VEN (400 mg/day) was administered in association with FLAI. Here we report results from early time-points (TPs) MRD analysis with the aim of identify the most informative TPs for MRD assessment and prognostic correlations. Methods: Erythrocyte-lysed whole bone marrow (BM) samples obtained at diagnosis from patients enrolled in the confirmatory cohort were centralized and analysed with a broad panel of monoclonal antibodies to identify the leukemia-associated phenotype (LAIP) which was used to track residual leukemic cells during follow-up. Eight color flow cytometry analysis was performed at pre-defined TPs (TP1: post-induction I, TP2: post induction II/consolidation I, following TPs: post consolidation/pre-transplantation) (FACSCantoII; BD Facs Diva Software V6.1.3). A positive flow MRD was defined by the presence of no less than 10 clustered leukemic cells/10^4 total events. Enrollment closed on January 2023. Collection and analysis of later TPs is ongoing. Results: Sixty-seven patients from 11 centers were enrolled in the phase 2, part 2. Risk stratification according to ELN 2017 was intermediate in 46% of patients and high risk in 54%. Fifty-eight/67 patients (87%) obtained CR after induction I. In the centralized MRD analysis, 170 samples has been collected and analysed so far (60 baseline samples, 110 MRD samples). Seven patients lacking baseline sample for LAIP identification were excluded from the analysis. TP1 was available in 57/59 patient achieving CR (97%), and TP2 was available in 29 patients, so far. MFC-MRD negativity was obtained in 31/57 (54%) at TP1 (post V-FLAI, Fig. 1). An increase in MFC-MRD negativity rate was observed at TP2 (post Induction II or consolidation I) with 20 MFC-MRD negative patients/29 available samples (69%, Fig. 1). Thirteen/110 follow up samples (8%), albeit resulting MRD negative, were considered inadequate for the analysis due to haemodilution, 9 from TP1 and 4 from TP2 (Fig. 1). TP1 and TP2 MRD assessment were scheduled at day 28 of Induction I and Induction II/consolidation, respectively. However, in most cases, delayed haematological recovery was observed after V-FLAI, thus resulting in suboptimal samples for MRD evaluation. Conclusions: Preliminary results from centralized MRD analysis confirm that the combination therapy is able to induce high-quality remissions with a very high percentage of MFC-MRD negativity in a difficult cohort of patients, with a higher percentage of MFC-MRD-negative after the completion of the second course of therapy. Delayed haematological recovery may impact on reliability of MRD assessment due to hypocellular and regenerative marrow samples, suggesting that in those cases MRD analysis should be postponed. Correlation with survival will be performed as the data collection will be complete.

Identification of key residues of B cell epitopes in hemagglutinin of H6 influenza A virus.

H6 influenza A viruses are enzootic and genetically diverse in both domestic poultry and wild birds, occasionally causing spillovers to mammals, including humans, posing threat to public health. However, the antigenic sites of the hemagglutinin (HA) of H6 are poorly understood. In this study, a panel of murine monoclonal antibodies (MAbs) with different reactivity spectra against HA of H6 was generated and nine key residues of antigenic epitopes in H6 were identified through escape mutants of these MAbs. Of these, residues 139 and 140 belonged to antigenic Sa, residue 89 belonged to antigenic Cb, residue 149 belonged to Ca2, while residue 221 belonged to Ca1 regarding the H1 antigenic sites. However, the other four residues 69, 120, 124, and 246 were not located in the identified antigenic regions of H1. Although most HA variants of the escape mutants had the preference for avian-like receptor, whereas escape mutants m3B9 and m4C2 increased the binding affinity for both avian and human-like receptors, and m1A5 and m6D11 decreased the binding affinity for avian-like receptors. Moreover, a single dose of MAbs 4C2 or 6E3 could fully protect mice against lethal challenge of mouse-adapted A/Eurasian teal/Jiangxi/2018WB0417(H6N2) (MA E-Teal/417) in the prophylactic treatment and also could provide partial protection in the therapeutic treatment. Overall, the generated MAbs and the identified key residues of antigenic epitopes not only provide novel insights into the key markers for monitoring the antigenic variation of H6 but also demonstrate the potential for developing MAb-based passitive treatments for H6.IMPORTANCESince the escape immunity of influenza A viruses (IAVs) is mainly caused by the continuous antigenic variations in HA, the identification of key antigenic epitopes is crucial for better understanding of the escape immunity and vaccine development for IAVs. The antigenic sites of several HA subtypes, including H1, H3, H5, and H9, have been well characterized, whereas those of H6 subtype are poorly understood. Here, we mapped nine key residues of antigenic epitopes in H6 through escape mutants using a panel of MAbs. Moreover, MAbs 4C2 and 6E3, targeting 140 and 89 residues, respectively, could protect mice against lethal challenge of MA E-Teal/417. These key residues of antigenic epitopes identified here provide the molecular targets for further elucidating the antigenic evolution of H6 and better preparing the vaccine against H6 IAV.

Development and characterisation of a novel inhibitory anti-GH monoclonal antibody.

Excess growth hormone (GH) has been implicated in multiple cancer types and there is increasing interest in the development of therapeutic inhibitors targeting GH-GH receptor (GHR) signalling. Here we describe a panel of anti-GH monoclonal antibodies (mAbs) generated using a hybridoma approach and identify two novel inhibitory mAbs (1-8-2 and 1-46-3) that neutralised GH signalling. mAbs 1-8-2 and 1-46-3 exhibited strong inhibitory activity against GH-dependent cell growth in a Ba/F3-GHR cell viability assay, with EC50 values of 1.00 ± 0.27 and 0.5 ± 0.1 µg/mL, respectively. Cross-reactivity with the human placental hormones, placental lactogen (PL) and placental GH, was observed by ELISA, but neither antibody cross-reacted with mouse GH or human prolactin (PRL). mAb 1-8-2 had a binding affinity for GH of KD 0.62 ± 0.5 nM, while mAb 1-46-3 had a KD of 2.68 ± 0.53 nM, as determined by bio-layer interferometry. mAb 1-46-3 inhibited GH-dependent signal transduction in T-47D and LNCaP cancer cell lines and reduced GH-dependent cell growth and migration in the breast cancer cell line T-47D. mAb 1-46-3 inhibited T-47D cell viability more effectively than the GHR antagonist B2036. In conclusion, we describe two novel inhibitory anti-GH mAbs and provide in vitro evidence supporting development of these entities as anti-cancer therapeutics.

Blocking of ebolavirus spread through intercellular connections by an MPER-specific antibody depends on BST2/tetherin

Ebola virus (EBOV) and Bundibugyo virus (BDBV) belong to the family Filoviridae and cause a severe disease in humans. We previously isolated a large panel of monoclonal antibodies from B cells of human survivors from the 2007Uganda BDBV outbreak, 16 survivors from the 2014 EBOV outbreak in the Democratic Republic of the Congo, and one survivor from the West African 2013-2016 EBOV epidemic. Here, we demonstrate that EBOV and BDBV are capable of spreading to neighboring cells through intercellular connections in a process that depends upon actin and Tcell immunoglobulin and mucin 1 protein. We quantify spread through intercellular connections by immunofluorescence microscopy and flow cytometry. One of the antibodies, BDBV223, specific to the membrane-proximal external region, induces virus accumulation at the plasma membrane. The inhibiting activity of BDBV223 depends on BST2/tetherin.

The Use of CD200 in the Differential Diagnosis of B-Cell Lymphoproliferative Disorders.

Background: B-Cell Lymphoproliferative Disorders (B-LPDs) are a group of heterogenous disorders characterised by the accumulation of B-cells in peripheral blood, bone marrow, lymph nodes and spleen. They have a variable disease course and outcome and many share similar features making differential diagnosis challenging. Therefore, accurate diagnosis is fundamental in particular for determining treatment options. Immunophenotyping by flow cytometry plays a crucial role in the diagnosis of B-LPDs. However, overlapping immunophenotyping patterns exist and the use of novel monoclonal antibodies has become increasingly important in immunophenotyping analysis. More recently differential expression of CD200 has been reported in various B-LPDs and that CD200 may improve the differentiation between chronic lymphocytic leukaemia (CLL) and mantle cell lymphoma (MCL). In this study CD200 expression is evaluated in different B-LPDs. Methods: A total of 100 samples were collected and analysed by immunophenotyping flow cytometry over a period of 1year (2017-2018), by a panel of monoclonal antibodies including CD200. The percentage of CD200 and its expression intensity was evaluated and compared between different groups of B-LPDs. Results: All of the 50 cases of CLL expressed CD200 with moderate to bright intensity, 6 MCL cases lacked the expression of CD200. Furthermore, all 5 cases of hairy cell leukaemia (HCL) expressed CD200. Out of all B-LPDs evaluated, CD200 expression in HCL cases was noted to be the brightest. The other 39 cases were not found to be B-LPDs. Conclusion: CD200 has an important role in differentiating CLL from MCL, HCL has a consistent bright expression of CD200. By adding CD200 to the combinations of markers in routine testing panel, Immunophenotyping by flow cytometry can be an effective tool in the diagnosis of B-LPDs especially in cases with atypical immunophenotyping pattern. Our result support that CD200 can be added to routine testing panel as it is useful in differentiating them.

Costimulatory capacity of CD137 mAbs on T cells depends on elaborate CRD structures but not on blocking ligand-receptor binding.

CD137 is mainly a costimulatory receptor of CD8+ T cells. Two representative CD137 antibodies, utomilumab, and urelumab, show different costimulatory capacities in clinical trials. Balancing the antitumor effect and systemic toxicity of T cells activated by CD137 signaling is a challenge that requires clinical consideration. In this study, a panel of specific anti-human CD137 monoclonal antibodies (mAbs) were prepared and their affinities, isotypes, CD137-CRD (cysteine-rich domain) binding regions, cross-reactivity to mouse and rhesus CD137, inhibition of ligand-receptor binding and costimulatory activities were analyzed. The results showed that anti-human CD137 mAbs had high cross-reactivity with rhesus CD137. MAbs fell into three clusters according to their different binding regions of the CD137 extracellular domain. They bound to CRDI+CRDII, CRDIII or CRDIV+STP. CRDIII-binding mAbs had the strongest blocking activity. Highly costimulatory CD137 mAbs showed stronger abilities to promote CD8+ T-cell proliferation. However, the costimulatory capacity of mAbs on T cells was not closely related to their ability to block CD137L-CD137 binding and may be controlled by more elaborate CRD conformational structures. This study provides additional information for the development of next-generation CD137 mAbs to meet clinical needs.

Bone marrow composition features and possibilities of tumor involvement diagnosing in patients with classic Hodgkin’s lymphoma

Background. Classical Hodgkin’s lymphoma is a B-cell lymphoproliferative disease, the tumor substrate of which is Berezovsky–Reed–Sternberg cells, characterized by CD30, PAX-5, CD15 expression and the absence of CD3, CD45. In some cases, tumor cells express CD20. modern anticancer therapy has increased the survival probability for most patients, not only with early but also with advanced stages of classical Hodgkin’s lymphoma. Such successes are mainly due to the distribution of patients into prognostic groups and the choice of an appropriate treatment regimen. tumor infiltration of the bone marrow suggests assigning patients to the advanced stages group, followed by the choice of an intensive therapy program. Aim. To determine the bone marrow involvement frequency according to positron emission tomography combined with computed tomography (PET/CT), with 18F-fluorodeoxyglucose (FDG) and bone marrow trephine biopsy (bmtb), to compare the results obtained with primary tumor immunophenotype and bone marrow cellular composition, and to identify of prognostic risk factors. Materials and methods. The study included 107 patients with newly diagnosed classical Hodgkin’s lymphoma, who underwent a diagnostic examination at the moscow research institute of oncology named after P. A. Herzen – a branch of the National Medical Research Center for Radiology and the “Lapino” clinical Hospital from 2015 to 2022, followed by anticancer therapy and further follow-up. Morphology of the primary tumor biopsy specimen in all patients and immunohistochemical (IHC) study using a wide panel of monoclonal antibodies (CD15, CD30, CD3, CD45, CD20, PAX-5 anti-gens; in some cases epstein–barr virus proteins expression) in most cases were performed. All patients underwent a morphological and / or IHC study of BMTB and the majority underwent aspiration biopsy and PET/CT with 18F-FDG. Results. The most common histological variant of classical Hodgkin’s lymphoma was nodular sclerosis (86.9 %). The majority of patients (51.4 %) were assigned to the advanced stage prognostic group. bone marrow tumor infiltration was statistically significantly more frequently diagnosed during PET/CT with 18F-FDG compared with the results of the BMTB – in 27.1 % and 12.1 % of cases, respectively (p < 0.05). when comparing the results of both diagnostic methods, it was found that in 17.1 % of cases, bone marrow infiltration, detected during PET/CT, was not confirmed by IHC examination of the trephine biopsy. In addition, it was found that the majority of cases with CD20+/± and CD15+/± expression in the primary tumor were observed in the group of patients without bone marrow involvement. when assessing the cellular composition of bone marrow aspirates, it was revealed that in patients with bone marrow tumor infiltration, an increase in cellularity and megakaryocytes number along with a decrease in the plasma cells number is observed. Conclusion. The results suggest further study of bone marrow immunomorphological features in order to identify prognostic factors and search for new therapeutic targets. a more extended analysis of bone marrow aspirate immunomor-phological characteristics using new modern diagnostic methods, minimal residual disease status as a surrogate marker seems to be relevant and necessary to confirm the depth of the antitumor response achieved. Detection of CD20+/±- and cd15+/±-Berezovsky–Reed–Sternberg cells according to primary tumor IHC analysis indicates a low probability of bone marrow tumor infiltration, but further analysis is required on a large clinical and laboratory material.

Development of an anti-SARS-CoV-2 monoclonal antibody panel and its applicability as a reagent in high-throughput fluorescence reduction neutralization and immunohistochemistry assays.

Since its emergence in late 2019, coronavirus disease 2019 (COVID-19) has caused millions of deaths and socioeconomic losses. Although vaccination significantly reduced disease mortality, it has been shown that protection wanes over time, and that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) may escape vaccine-derived immunity. Therefore, serological studies are necessary to assess protection in the population and guide vaccine regimens. A common measure of protective immunity is the presence of neutralizing antibodies (nAbs). However, the gold standard for measuring nAbs (plaque reduction neutralization test, or PRNT) is laborious and time-consuming, limiting its large-scale applicability. We developed a high-throughput fluorescence reduction neutralization assay (FRNA) to detect SARS-CoV-2 nAbs. Because the assay relies on immunostaining, we developed and characterized monoclonal antibodies (mAbs) to lower costs and reduce the assay's vulnerability to reagent shortages. Using samples of individuals vaccinated with COVID-19 and unvaccinated/pre-pandemic samples, we showed that FRNA results using commercial and in-house mAbs strongly correlated with those of the PRNT method while providing results in 70% less time. In addition to providing a fast, reliable, and high-throughput alternative for measuring nAbs, the FRNA can be easily customized to assess SARS-CoV-2 VOCs. Additionally, the mAb we produced was able to detect SARS-CoV-2 in pulmonary tissues by immunohistochemistry assays.

Development of monoclonal antibody-based blocking ELISA for detecting SARS-CoV-2 exposure in animals.

The global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a significant threat to public health. Besides humans, SARS-CoV-2 can infect several animal species. Highly sensitive and specific diagnostic reagents and assays are urgently needed for rapid detection and implementation of strategies for prevention and control of the infection in animals. In this study, we initially developed a panel of monoclonal antibodies (mAbs) against SARS-CoV-2 nucleocapsid protein. To detect SARS-CoV-2 antibodies in a broad spectrum of animal species, an mAb-based blocking enzyme-linked immunosorbent assay (bELISA) was developed. Test validation using a set of animal serum samples with known infection status obtained an optimal percentage of inhibition cut-off value of 17.6% with diagnostic sensitivity of 97.8% and diagnostic specificity of 98.9%. The assay demonstrates high repeatability as determined by a low coefficient of variation (7.23%, 4.89%, and 3.16%) between-runs, within-run, and within-plate, respectively. Testing of samples collected over time from experimentally infected cats showed that the bELISA was able to detect seroconversion as early as 7 days post-infection. Subsequently, the bELISA was applied for testing pet animals with coronavirus disease 2019 (COVID-19)-like symptoms and specific antibody responses were detected in two dogs. The panel of mAbs generated in this study provides a valuable tool for SARS-CoV-2 diagnostics and research. The mAb-based bELISA provides a serological test in aid of COVID-19 surveillance in animals. IMPORTANCE Antibody tests are commonly used as a diagnostic tool for detecting host immune response following infection. Serology (antibody) tests complement nucleic acid assays by providing a history of virus exposure, no matter symptoms developed from infection or the infection was asymptomatic. Serology tests for COVID-19 are in high demand, especially when the vaccines become available. They are important to determine the prevalence of the viral infection in a population and identify individuals who have been infected or vaccinated. ELISA is a simple and practically reliable serological test, which allows high-throughput implementation in surveillance studies. Several COVID-19 ELISA kits are available. However, they are mostly designed for human samples and species-specific secondary antibody is required for indirect ELISA format. This paper describes the development of an all species applicable monoclonal antibody (mAb)-based blocking ELISA to facilitate the detection and surveillance of COVID-19 in animals.

IgG3 subclass antibodies recognize antigenically drifted influenza viruses and SARS-CoV-2 variants through efficient bivalent binding

The constant domains of antibodies are important for effector functions, but less is known about how they can affect binding and neutralization of viruses. Here, we evaluated a panel of human influenza virus monoclonal antibodies (mAbs) expressed as IgG1, IgG2, or IgG3. We found that many influenza virus-specific mAbs have altered binding and neutralization capacity depending on the IgG subclass encoded and that these differences result from unique bivalency capacities of the subclasses. Importantly, subclass differences in antibody binding and neutralization were greatest when the affinity for the target antigen was reduced through antigenic mismatch. We found that antibodies expressed as IgG3 bound and neutralized antigenically drifted influenza viruses more effectively. We obtained similar results using a panel of SARS-CoV-2-specific mAbs and the antigenically advanced B.1.351 and BA.1 strains of SARS-CoV-2. We found that a licensed therapeutic mAb retained neutralization breadth against SARS-CoV-2 variants when expressed as IgG3, but not IgG1. These data highlight that IgG subclasses are not only important for fine-tuning effector functionality but also for binding and neutralization of antigenically drifted viruses.

Monovalent Omicron COVID-19 vaccine triggers superior neutralizing antibody responses against Omicron subvariants than Delta and Omicron bivalent vaccine

ABSTRACT The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses a challenge to determine the optimal updated composition of the coronavirus disease 2019 (COVID-19) vaccine. The present study aimed to investigate the immunogenicity of the Delta monovalent vaccine, the Omicron monovalent vaccine, and the Delta and Omicron BA.1 bivalent vaccine. Three COVID-19 vaccines were designed using the heterologous DNA prime-protein boost strategy, with each vaccine containing either Delta receptor-binding domain (RBD) of the spike protein, Omicron RBD, or both Delta and Omicron antigens. Temporal serum antibody binding titers and neutralizing antibody titers induced by the three vaccines in New Zealand White rabbits were analyzed. To further dissect the vaccine elicited antibodies (mAb) responses at the molecular level, a panel of rabbit monoclonal antibodies (RmAbs) was generated by a high-throughput single B cell sorting and discovery pipeline and further comprehensively characterized. The Omicron monovalent vaccine induced higher antibody binding titers and neutralization activities than the Delta and Omicron bivalent vaccine. Four RmAbs with robust neutralization capacity were isolated from rabbits immunized with the Omicron or Delta monovalent vaccine. Notably, 9E11 isolated from the Omicron monovalent vaccine group neutralized all the Omicron subvariants with an IC50 value ranging from 1.5 to 503.6 ng/mL; thus, this vaccine could serve as a prophylactic and therapeutic intervention. Given the increasing incidence of COVID-19 cases due to the Omicron variant, RBD from the Omicron strain could serve as a candidate immunogen that can induce higher neutralization activities against the SARS-CoV-2 Omicron sublineages.

Expression of β2 microglobulin on malignant multiple myeloma plasma cells

Background. β2‑microglobulin is a low molecular weight glycoprotein that is part of the major histocompatibility complex. The concentration of β2‑microglobulin in the blood serum of patients increases with various oncohematological pathologies, including multiple myeloma. One of the methods for detecting membrane β2‑microglobulin is flow cytometry. This article discusses the expression of β2‑microglobulin and key markers on the membrane of malignant cells in patients with multiple myeloma at the onset of the disease.Aim. To study the expression of β2‑microglobulin and markers CD45, CD56 and CD19 on the membrane of tumor plasma cells in the primary diagnosis of multiple myeloma.Materials and methods. The study of β2‑microglobulin and CD45, CD56 and CD19 was carried out in 37 patients with multiple myeloma by 8‑color flow cytometry using a panel of monoclonal antibodies for the diagnosis of plasma cell tumors (EuroFlow, 2012).Results. The expression of β2‑microglobulin in combination with markers CD45, CD56 and CD19 on tumor plasma cells was assessed, illustrations of the expression of these markers are presented. Out of 37 patients with multiple myeloma, hyperexpression of β2‑microglobulin was noted in 23 (62.2 %), in other cases there were normal or reduced levels of this protein in other cases.Conclusion. Tumor plasma cells overexpress β2‑microglobulin in a significant percentage of cases (62.2 %). This allows further assessment of the clinical significance of such aberrant expression and its relationship with serum levels of β2‑microglobulin.

A relation between specific immune status indicators and activity of “lipid peroxidation — antioxidant defense” system in COVID-19 neonates

The 2019 coronavirus infection (COVID-19) has not been considered as a solved issue for public health. Pregnant women and newborns are specifically vulnerable to COVID-19 infection compared to older children and healthy young adults. Virtually no data on relation between diverse arms of immunity in patients in neonatal period and coronavirus infection are available. The obtained results can contribute to a better understanding of the pathogenetic mechanisms on reactivity of immune processes in young patients and corresponding formation of approaches for prevention and correction of such disorders. The aim of the study was to determine magnitude of specific altered parameters in immune system and their relation with lipid peroxidation parameters in COVID-19 newborns. Two groups of newborns (mean age 43.1 days) were examined: SARS-CoV-2-positive (COVID-19 patients, n = 44) and negative (control group, n = 80) PCR test of nasopharyngeal swab. All newborns were assessed for specific indicators of peripheral blood immune status and lipid peroxidation activity. The concentration of Th1-pro-inflammatory cytokines and Th2-anti-inflammatory interleukins was assessed by enzyme immunoassay method (a panel of monoclonal antibodies). Spectrophotometric, fluorometric and enzyme immunoassay methods to evaluate the lipid peroxidation system were used. According to our data, newborns with COVID-19 vs. healthy newborns had decreased CRP, pro-inflammatory cytokines TNF, IL-1, IL-6, IL-8, and anti-inflammatory factor (IL-4). Change in lipid peroxidation system in children with COVID-was 19 related to higher level of DC, KD and CT, TBARs, increased SOD activity and reduced GPO. Numerous intersystem dependencies in the group of newborns with COVID-19 (CRP Total AOA, IL-4 KD and CT, IL-4 TBARs, IL-4 Total AOA, IL-4 SOD, IL-8 SOD, IFN GSH) were noted. It can be concluded that in newborns with COVID-19, changes in the immune system are nonspecific and are accompanied by an increased intensity of lipid peroxidation reactions against the background of reduced values of pro- and anti-inflammatory cytokines. These results may contribute to a more accurate assessment of intensity and dynamics of emerging neonatal coronavirus infection, which should be an important arm in preventing subsequent complications.

Monoclonal Antibody Panel for Hepatitis B Virus Genotype and HBsAg Subtype Identification: Results Obtained for Blood-Serum Samples from Three Regions of Russia

Monoclonal Antibody Panel for Hepatitis B Virus Genotype and HBsAg Subtype Identification: Results Obtained for Blood-Serum Samples from Three Regions of Russia

A multiplexed assay for quantifying immunomodulatory proteins supports correlative studies in immunotherapy clinical trials.

Immunotherapy is an effective treatment for a subset of cancer patients, and expanding the benefits of immunotherapy to all cancer patients will require predictive biomarkers of response and immune-related adverse events (irAEs). To support correlative studies in immunotherapy clinical trials, we are developing highly validated assays for quantifying immunomodulatory proteins in human biospecimens. Here, we developed a panel of novel monoclonal antibodies and incorporated them into a novel, multiplexed, immuno-multiple reaction monitoring mass spectrometry (MRM-MS)-based proteomic assay targeting 49 proteotypic peptides representing 43 immunomodulatory proteins. The multiplex assay was validated in human tissue and plasma matrices, where the linearity of quantification was >3 orders of magnitude with median interday CVs of 8.7% (tissue) and 10.1% (plasma). Proof-of-principle demonstration of the assay was conducted in plasma samples collected in clinical trials from lymphoma patients receiving an immune checkpoint inhibitor. We provide the assays and novel monoclonal antibodies as a publicly available resource for the biomedical community.

A novel heterodimer composed of IL-12p40 and CD5L promotes a unique cytokine profile from T cells

Abstract The IL-12 family of cytokines (IL-12, IL-23, IL-27, and IL-35) are heterodimers composed of α and β subunits. Both IL-12 and IL-23 utilize IL-12p40 (p40) as their β subunit yet exert distinct proinflammatory outcomes on the T cell response. Upon immune stimulation, the p40 monomer is also secreted from innate immune cells without its binding partners p35 and p19. In order to examine if p40 functions by interacting with additional partners, we generated a library of p40 binders using immunoprecipitation and mass spectrometry. Of the 19 potential binding partners identified biochemically, CD5L, a protein characterized for its role in regulating macrophages was the top hit. We generated a panel of dimer-specific monoclonal antibodies recognizing the p40-CD5L heterodimer. Using this reagent, we found that p40CD5L was present in the serum of both C57BL/6 and BALB/c mice at baseline and increased in the BAL of mice after allergic sensitization. Adoptive transfer and restimulation of transgenic T cells with recombinant p40CD5L resulted in significantly increased production of IL-4 and IL-10 – a unique cytokine profile that has not been identified with other p40-containing cytokines. From these results, we suggest that p40CD5L may function as a novel innate heterodimeric cytokine potentially promoting IL-4 and IL-10 responses. Supported by grants from NIH (R21AI166330-02)

Neutralizing and protective murine monoclonal antibodies to the hemagglutinin of influenza H5 clades 2.3.2.1 and 2.3.4.4.

Highly pathogenic avian H5 influenza viruses have spread and diversified genetically and antigenically into multiple clades and subclades. Most isolates of currently circulating H5 viruses are in clade 2.3.2.1 or 2.3.4.4. Panels of murine monoclonal antibodies (mAbs) were generated to the influenza hemagglutinin (HA) of H5 viruses from the clade 2.3.2.1 H5N1 vaccine virus A/duck/Bangladesh/19097/2013 and the clade 2.3.4.4 H5N8 vaccine virus A/gyrfalcon/Washington/41088-6/2014. Antibodies were selected and characterized for binding, neutralization, epitope recognition, cross-reactivity with other H5 viruses, and the ability to provide protection in passive transfer experiments. All mAbs bound homologous HA in an ELISA format; mAbs 5C2 and 6H6 were broadly binding for other H5 HAs. Potently neutralizing mAbs were identified in each panel, and all neutralizing mAbs provided protection in passive transfer experiments in mice challenged with a homologous clade influenza virus. Cross-reacting mAb 5C2 neutralized a wide variety of clade 2.3.2.1 viruses, as well as H5 viruses from other clades, and also provided protection against heterologous H5 clade influenza virus challenge. Epitope analysis indicated that the majority of mAbs recognized epitopes in the globular head of the HA. The mAb 5C2 appeared to recognize an epitope below the globular head but above the stalk region of HA. The results suggested that these H5 mAbs would be useful for virus and vaccine characterization. The results confirmed the functional cross-reactivity of mAb 5C2, which appears to bind a novel epitope, and suggest the therapeutic potential for H5 infections in humans with further development.

Universal Candidacell-surface-specific monoclonal antibodies protect mice against multidrug-resistant Candida aurisbloodstream infections

Abstract Candidaspecies are the leading cause of life-threatening fungal infections in humans, especially among immunocompromised and hospitalized individuals. Of utmost concern is the emerging “superbug” Candida auriswhich has the potential to disseminate into bloodstream infections with up to 60% mortality in susceptible populations. Importantly, C. aurisis the first inherently multidrug-resistant fungal pathogen, which contributes to a delay in effective treatment and increased mortality rates. Consequently, C. aurisis the first fungal pathogen to cause a serious global public health threat. Our lab has identified and isolated a panel of universal monoclonal antibodies (mAbs) specific to Candidacell wall epitopes that share high homology among various human pathogenic Candidaspecies. Using an established murine model of disseminated infection, we showed that passive transfer of mAb C3.1 (anti-β-1,2-mannotriose) significantly extended survival and reduced fungal burdens and inflammation during early infection. We also showed that mAbs C3.1 and 9F2 (anti-phosphoglycerate kinase 1) significantly enhanced phagocytosis of C. aurisyeast by macrophages and inhibited the proliferation of multiple C. aurisclinical isolates in vitro. Since there are currently no approved antifungal vaccines or antibodies and current treatments are inadequate, critically needed antifungal antibodies can provide long-awaited effective therapies against this deadly pathogen. Our future directions include developing humanized mAbs to evaluate their protective efficacy in genetic mouse models of human immunodeficiency. We will then utilize Fc engineering to derive human IgG1 variants for enhanced complement-based effector efficacy. Supported by grants from NIH /NIAID (R21AI168929) and the Department of Defense (PR171482)

Development of swine immune reagents for swine immunity and biomedical research

Abstract The USDA-NIFA Swine Immune Toolkit Initiative has been involved in generating priority immune reagents for swine immunity and biomedical research and pipelining them for marketing. With the help of commercial partners we expressed each target immune molecule as a soluble protein using yeast expression systems and then produced panels of monoclonal antibodies (mAbs) against each target. Most recently we generated panels of mAbs reactive with porcine IL-5, IL-6, IL-13, IL-21, IL-28B, CXCL10, and BAFF and screened their reactivity in multiple immune assays. We determined mAb reactivity with orthologous proteins for most panels of mAbs. A sensitive sandwich ELISA is now available for IL-13 and CXCL10; other targets are being screened for best mAb pairs. Reactivity tests of labeled α-CXCL10, α-IL-6 and α-IL-13 mAbs for intracellular staining of porcine immune cells using flow cytometry assays are underway using different cell stimulation conditions. Immunohistochemistry analyses for binding of α-CXCL10 and other mAbs on formalin fixed pig lymph nodes and spleen tissues are in progress. Further, analysis of a large panel of commercial α-human CD antigen mAbs for cross-reactivity with porcine cells is ongoing. For each target, our goal is to provide the veterinary community with new commercial reagents and standardized assay techniques for their research efforts. Finally, efforts are proceeding to develop SLA tetramers for antigen presentation studies. Tools and reagents generated by this project will undoubtedly advance our understanding of swine immune responses to disease and vaccine and use for biomedical research efforts. Supported by USDA-NIFA AFRI grant # 2019-67015-29815 and USDA ARS project 8042-32000-117.