Search Results (22)

Background/Objectives: Coronaviruses are a family of positive-sense RNA viruses that cause respiratory and gastrointestinal disease in mammals and birds. Their zoonotic nature and high mutability make them a pandemic threat. UNICOR-v is a pre-pandemic, pan-coronavirus vaccine composed of an adjuvanted mix of twelve synthetic peptides originating from conserved regions within Nsp12 and M coronavirus proteins containing clusters of predicted T-cell epitopes. Here, we evaluate the immunogenicity of UNICOR-v and its efficacy against Middle East Respiratory Syndrome-related coronavirus (MERS). Methods: Animals were vaccinated with an adjuvanted equimolar mix of UNICOR-v. Humoral and cellular immunogenicity were assessed 28 days later through ELISA and FLUOROSpot. Vaccine efficacy was assessed in a DPP4 knock-in (HDPP4-KI) mouse model where mice were challenged post-vaccination with a lethal or non-lethal dose of MERS-CoV-MA. Results: Vaccination with UNICOR-v induced high IgG titers in both mice and rabbits and cellular secretion of pro-inflammatory cytokines. Vaccination with UNICOR-v, or passive serum transfer, significantly reduced viral lung titers 4 days post-infection compared to placebo. Vaccination induced lower immune cell infiltration in the alveolar space and increased repair of the cells lining the major airways in vaccinated mice, translating to increased survival rate compared to placebo. Conclusions: These data demonstrate the ability of conserved T-cell epitopes to protect against MERS-CoV infection, supporting further characterization of the breadth of protection of UNICOR-v against other coronaviruses that affect humans and livestock, following a One Health approach to control this highly zoonotic family of viruses. Full article

Objective: We evaluate the performance and relevance of MRI to detect csPC in men with normal PSA. Methods: Out of our database of patients referred for prostate MRI, we selected men with PSA < 4 ng/mL for whom histopathology or at least 2 years of clinical follow-up data were available as standard of reference. Subgroup analyses were performed for the patients with PSA < 3 ng/mL, <2 ng/mL, and 2–3.9 ng/mL. The reasons for prostate MRI referral despite their normal PSA level were retrieved by exploring the patients’ files. The prostate MRIs were reported according to the Prostate Imaging and Reporting Data System (PI-RADS), and the overall assessment score was registered. For evaluation of the performance, PI-RADS ≥ 3 was set as a threshold for a positive exam. The patients without PC or only International Society of Urological Pathology (ISUP) grade group 1 PC (Gleason 3+3) were considered as one category having no csPC. The performance of prostate MRI was separately evaluated for detection of ISUP ≥ 2 and for ISUP ≥ 3 csPC. Results: A total of 148 men were included, with PSA ranging from 0.42 to 3.99 ng/mL (median 2.95, IQR 1.68–3.50) and age ranging from 36 to 84 years (median 58, IQR 52–66). A total of 74 men (50.0%) had a PSA level < 3 ng/mL, 42 (28.4%) had a PSA level < 2 ng/mL, and 106 (71.6%) had a PSA level of 2–3.9 ng/mL. They were referred for prostate MRI for a wide variety, and usually a combination of, reasons, such as younger age (<60 years in 55.4%, N = 82; <50 years in 17.6%, N = 26), abnormal digital rectal examination in 31.8% of cases (N = 47), suspicious PSA dynamics in 29.7% (N = 44), positive familial history in 27.0% (N = 40), clinical signs of prostatitis in 18.2% (N = 27), suspicious findings on Transrectal Ultrasound (TRUS) in 16.9% (N = 25), hematospermia in 7.4% (N = 11), hematuria in 4.1% (N = 6), incidental hot spot in the prostate on Fluoro-Deoxy-Glucose (FDG) Positron Emission Tomography (PET)–Computed Tomography (CT) in 4.1% (N = 6), lymphadenopathies on CT in 2.7% (N = 4), or severe patient anxiety in 3.4% (N = 5). Overall, ISUP ≥ 2 PC was present in 18.9% (N = 28) of cases, and MRI detected this with a sensitivity of 92.9%, a specificity of 66.7%, and a positive predictive value of 39.4%. ISUP ≥ 3 PC was present in 9.5% (N = 14) of cases, and prostate MRI detected this with a sensitivity of 100%, a specificity of 61.2%, and a positive predictive value of 21.2%. In patients with PSA < 2 ng/mL (N = 42), no csPC was found, but MRI generated false positives in 33.3%. Conclusions: Performing prostate MRI in men with normal PSA (<4 ng/mL) seems useful if there are other reasons that increase the clinical suspicion of csPC. In about one-fifth of these patients, csPC is present and MRI has high sensitivity for its detection. Prostate MRI has, however, low positive predictive value in this patient group, and clinicians should be aware of the risk of false-positive MRI. Below a PSA level of 2 ng/mL, no csPC was found and prostate MRI generated only false positives, suggesting limited value in this subgroup. Full article

Background/Objectives: Despite clonal expansion during a primary immune response, or after subsequent antigen encounters, the frequency of memory B cells (B_mem) specific for an antigen remains low, making their detection difficult. However, unlike serum antibodies, which have a short half-life in vivo and thus require continuous replenishment to maintain stable titers, circulating B_mem are long-lived; they preserve immunological preparedness through their ability to rapidly engage in recall responses and differentiate into antibody-secreting cells (ASCs) upon antigen encounter. To this end, development of assays suited for the reliable detection of rare antigen-specific B_mem is critical and can provide insights into an individual’s antigen exposure history and immune status beyond that offered by traditional serum antibody measurements alone. Methods: ImmunoSpot^® has emerged as a suitable technique for the detection of individual antigen-specific B cells through visualizing their antibody-derived secretory footprints. Here, we report the theoretical and practical foundations for detecting rare antigen-specific B_mem in human peripheral blood mononuclear cells (PBMC). Leveraging the unique availability of verifiably naïve vs. antigen-experienced human samples, we used SARS-CoV-2 Spike (S-) and Nucleocapsid (NCAP) antigens to interrogate the presence of B_mem with these respective specificities. Results: While 100% diagnostic accuracy was achieved for both antigens, detection of NCAP-specific B_mem required reducing the lower detection limit of the standard assay. Specifically, this was achieved by testing a total of 2 million PBMC across multiple replicate assay wells and assessing the cumulative number of secretory footprints detected. Conclusion: The protocols described here should facilitate the reliable detection of ASCs present at varying precursor frequencies and serve as guidance for routine immune monitoring of rare B_mem with specificity for any antigen. Full article

Background: Memory B cells (B_mem) facilitate the generation of renewed and rapid antigen-specific antibody responses long after the initial antigen exposure, at a time when circulating serum antibodies may have declined. As the generation and/or recruitment of B_mem is at the core of most vaccination strategies, the assessment of antigen-specific B_mem is highly informative for forecasting and profiling the elicited B cell immune response. Methods: The two prevalent techniques used to detect antigen-specific B_mem cells at single-cell resolution are probe-based flow cytometry and B cell ImmunoSpot, while the measurement of B cell-derived antibodies in culture supernatants of stimulated B cells offers a semi-quantitative alternative. To the best of our knowledge, a direct side-by-side comparison of these assay systems has not yet been reported using the same starting PBMC material in a blinded fashion to test all three assays simultaneously. Results: These three assay systems were run in parallel to detect SARS-CoV-2 Wuhan-1 strain Spike-specific IgG⁺ B_mem in peripheral blood mononuclear cell (PBMC) samples obtained from well-defined cohorts comprising pre-COVID-19 era “naïve” individuals (negative controls), individuals shortly after recovery from a PCR-verified SARS-CoV-2 infection (positive controls), and a cohort of donor PBMCs isolated in 2024 (the experimental group). Each assay was able to discern Spike-exposed individuals from naïve , with ImmunoSpot suggesting superior sensitivity and specificity. ImmunoSpot and flow cytometry results were closely correlated. Conclusions: The study demonstrates that all three assays are suited for the detection of specific B_mem in antigen-primed individuals when such B_mem occur in the mid- to high-frequency range, and that they broadly concur. Strengths and weaknesses of the three test systems are discussed. Full article

Background: The COVID-19 pandemic provided a unique opportunity to evaluate how the human immune system responded to a novel pathogen and to determine whether immune responses initiated through natural infection differ from those elicited by vaccination against the same antigen. Here, we provide a comprehensive analysis of SARS-CoV-2 Spike (S-antigen)-reactive memory B cells (B_mem) elicited in previously immunologically naïve subjects following their first infection with the original Wuhan-Hu-1 (WH1)-like strain or their initial COVID-19 mRNA prime-boost regimen encoding the same WH1-S-antigen. In particular, we tested the hypothesis that the primary encounter of SARS-CoV-2 S-antigen in lung mucosal tissues during infection vs. intramuscular COVID-19 mRNA injection would elicit different B_mem responses. Methods: Cryopreserved peripheral blood mononuclear cell (PBMC) samples collected following primary infection with the WH1 strain or completion of the initial prime-boost vaccination regimen were tested in ImmunoSpot^® assays to assess the frequency, Ig class/subclass usage, and cross-reactivity of the S-antigen-reactive B_mem compartment; pre-pandemic blood draws served as naïve controls. Results: The B_mem repertoires generated post-infection vs. post-vaccination were found to be quite similar but with some subtle differences. In both cases, the prevalent induction of IgG1-expressing B_mem in similar frequencies was seen, ~30% of which targeted the receptor binding domain (RBD) of the WH1-S-antigen. Also, the extent of cross-reactivity with the future Omicron (BA.1) RBD was found to be similar for both cohorts. However, IgA⁺ B_mem were preferentially induced after infection, while IgG4⁺ B_mem were detected only after vaccination. Conclusions: B_mem elicited in naïve human subjects following SARS-CoV-2 infection or after WH1-S encoding mRNA vaccination were only subtly different, although the relevance of these differences as it relates to immune protection warrants further investigation. Our findings serve to illustrate the usefulness and feasibility of performing comprehensive monitoring of antigen-specific B cell memory in larger cohorts using the ImmunoSpot^® technique. Full article

Background: Measuring frequencies of antigen-specific memory B cells (B_mem), their immunoglobulin (Ig) class and subclass usage, cross-reactivity, and affinity can provide insights into the efficacy of future antibody responses in case of antigen re-encounter. B cell ImmunoSpot^® assays can provide such information; however, like most cell-based tests, they require considerable amounts of blood to be drawn from the donor and this has hindered their inclusion in clinical trials and routine immune diagnostics. Methods: We introduce strategies for reducing the cell numbers required to 2–3 million peripheral blood mononuclear cells (PBMCs) per antigen, obtainable from 2–3 mL of blood from healthy adult donors. Results: Except when B_mem frequencies were very low, we found that testing PBMCs in singlet wells, but in serial dilution, enables as reliable B_mem frequency assessments as when testing replicate wells at a single fixed cell number. Additionally, B cell ImmunoSpot^® assays can be multiplexed for detecting four Ig classes, or IgG subclasses, simultaneously and without loss of sensitivity. The requirement for low cell numbers and the retention of B cell functionality by cryopreserved PBMCs equivalent to freshly isolated material implies that fewer than the standard 10 million PBMCs per vial can be frozen. This would reduce the number of individuals who could not be tested for B_mem due to insufficient availability of PBMCs, a common problem with such assays. Conclusions: The predictable need for and recovery of cryopreserved PBMCs facilitates planning of and optimal cell utilization in B cell ImmunoSpot^® assays and increases the practical feasibility of extensive B_mem characterization in larger cohorts. Full article

Determining an individual’s humoral immune reactivity to a pathogen, autoantigen, or environmental agent is traditionally accomplished through the assessment of specific antibody levels in blood. However, in many instances, titers of specific antibodies decline over time and thus do not faithfully reveal prior antigen exposure or establishment of immunological memory. To estimate an individual’s humoral immune competence, it is therefore necessary to assess functional B cell memory. Here, we describe novel B cell ELISPOT and FluoroSpot assays (collectively referred to as ImmunoSpot) that can be rapidly developed and validated to characterize the memory B cell (B_mem) repertoire specific for any desired antigen ex vivo and at single-cell resolution. Moreover, multiplexed variants of the B cell FluoroSpot assay enable high-throughput testing of antigen-specific B cells secreting distinct antibody classes and/or IgG subclasses, with minimal cell material requirements. B cell ImmunoSpot assays also enable measurement of affinity distributions within the antigen-specific B_mem compartment and permit cross-reactivity measurements that can provide insights into B_mem established against future pathogen variants. Collectively, the ImmunoSpot^® system presented here is highly reproducible, and can be readily validated for regulated tests. The newly gained ability to monitor the antigen-specific B_mem compartment should catalyze a more comprehensive understanding of humoral immunity in health and disease. Full article

Severe haematological diseases and lymphoid malignancies require bone marrow (BM)-suppressive treatments. Knowledge regarding the impact of BM-suppressive treatments on children’s memory T cells is very limited. Memory T cells play a crucial role in defending against herpesviruses, which is particularly relevant in paediatric cancer care. We studied 53 children in total; 34 with cancer and 2 with severe haematological disorders, with some receiving BM-suppressive treatment with or without allogeneic–haematopoietic stem cell transplantation (allo-HSCT), alongside 17 healthy controls. We focused on peripheral blood proportions of memory T-cell subsets using flow cytometry and analysed cytokine-secreting T cells with a four-parameter FluoroSpot assay in response to T-cell mitogen and varicella zoster virus (VZV) peptides. Patients on BM-suppressive treatment showed increased clusters of differentiation (CD)4⁺ and CD8⁺ effector memory (TEM)/terminally differentiated effector (TEFF) T cells compared to the healthy controls. They also exhibited, amongst other things, when compared to the healthy controls, a reduced total number of cytokine-secreting cells, by means of interferon (IFN)-γ, interleukin (IL)-17A, IL-10, and IL-22, following mitogen activation. A diminished IFN-γ response among the children with BM-suppressive treatment was observed upon VZV-peptide stimulation, compared to the healthy children. Collectively, the findings herein indicate that the children who are undergoing or have finished BM-suppressive treatment display qualitative differences in their T-cell memory compartment, potentially increasing their susceptibility to severe viral infections and impacting their immunotherapy, which relies on the functional ability of autologous T cells. Full article

Cannabis use is continuously increasing in Canada, raising concerns about its potential impact on immunity. The current study assessed the impact of a cannabinoid mixture (CM) on B cells and the mechanisms by which the CM exerts its potential anti-inflammatory properties. Peripheral blood mononuclear cells (PBMCs) were treated with different concentrations of the CM to evaluate cytotoxicity. In addition, flow cytometry was used to evaluate oxidative stress, antioxidant levels, mitochondrial membrane potential, apoptosis, caspase activation, and the activation of key signaling pathways (ERK1/2, NF-κB, STAT5, and p38). The number of IgM- and IgG-expressing cells was assessed using FluoroSpot, and the cytokine production profile of the B cells was explored using a cytokine array. Our results reveal that the CM induced B-cell cytotoxicity in a dose-dependent manner, which was mediated by apoptosis. The levels of ROS and those of the activated caspases, mitochondrial membrane potential, and DNA damage increased following exposure to the CM (3 µg/mL). In addition, the activation of MAP Kinase, STATs, and the NF-κB pathway and the number of IgM- and IgG-expressing cells were reduced following exposure to the CM. Furthermore, the exposure to the CM significantly altered the cytokine profile of the B cells. Our results suggest that cannabinoids have a detrimental effect on B cells, inducing caspase-mediated apoptosis. Full article

Immunogenicity, defined as the ability to provoke an immune response, can be either wanted (i.e., vaccines) or unwanted. The latter refers to an immune response to protein or peptide therapeutics, characterized by the production of anti-drug antibodies, which may affect the efficacy and/or the safety profiles of these drugs. Consequently, evaluation of the risk of immunogenicity early in the development of biotherapeutics is of critical importance for defining their efficacy and safety profiles. Here, we describe and validate a fit-for-purpose FluoroSpot-based in vitro assay for the evaluation of drug-specific T cell responses. A panel of 24 biotherapeutics with a wide range of clinical anti-drug antibody response rates were tested in this assay. We demonstrated that using suitable cutoffs and donor cohort sizes, this assay could identify most of the compounds with high clinical immunogenicity rates (71% and 78% for sensitivity and specificity, respectively) while we characterized the main sources of assay variability. Overall, these data indicate that the dendritic cell and CD4+ T cell restimulation assay published herein could be a valuable tool to assess the risk of drug-specific T cell responses and contribute to the selection of clinical candidates in early development. Full article

The scope of immune monitoring is to define the existence, magnitude, and quality of immune mechanisms operational in a host. In clinical trials and praxis, the assessment of humoral immunity is commonly confined to measurements of serum antibody reactivity without accounting for the memory B cell potential. Relying on fundamentally different mechanisms, however, passive immunity conveyed by pre-existing antibodies needs to be distinguished from active B cell memory. Here, we tested whether, in healthy human individuals, the antibody titers to SARS-CoV-2, seasonal influenza, or Epstein–Barr virus antigens correlated with the frequency of recirculating memory B cells reactive with the respective antigens. Weak correlations were found. The data suggest that the assessment of humoral immunity by measurement of antibody levels does not reflect on memory B cell frequencies and thus an individual’s potential to engage in an anamnestic antibody response against the same or an antigenically related virus. Direct monitoring of the antigen-reactive memory B cell compartment is both required and feasible towards that goal. Full article

Notwithstanding the current SARS-CoV-2 pandemic, influenza virus infection still represents a global health concern in terms of hospitalizations and possible pandemic threats. The objective of next-generation influenza vaccines is not only to increase the breadth of response but also to improve the elicitation of an effective and robust immune response, especially in high-risk populations. To achieve this second objective, the administration of adjuvanted influenza vaccines has been considered. In this regard, the monitoring and characterization of the antibody response associated with the administration of adjuvanted vaccines has been evaluated in this study in order to shed light on the kinetic, magnitude and subclass usage of antibody secreting cells (ASCs) as well as of circulating antigen-specific serum antibodies. Specifically, we utilized the DBA/2J mouse model to assess the kinetic, magnitude and IgG subclass usage of the antibody response following an intramuscular (IM) or intraperitoneal (IP) immunization regimen with AddaVax-adjuvanted bivalent H1N1 and H3N2 computationally optimized broadly reactive antigen (COBRA) influenza recombinant hemagglutinins (rHAs). While the serological evaluation revealed a homogeneous kinetic of the antibody response, the detection of the ASCs through a FluoroSpot platform revealed a different magnitude, subclass usage and kinetic of the antigen-specific IgG secreting cells peaking at day 5 and day 9 following the IP and IM immunization, respectively. Full article

The progression of the COVID-19 pandemic has led to the emergence of variants of concern (VOC), which may compromise the efficacy of the currently administered vaccines. Antigenic drift can potentially bring about reduced protective T cell immunity and, consequently, more severe disease manifestations. To assess this possibility, the T cell responses to the wild-type Wuhan-1 SARS-CoV-2 ancestral spike protein and the Omicron B.1.1.529 spike protein were compared. Accordingly, peripheral blood mononuclear cells (PBMC) were collected from eight healthy volunteers 4–5 months following a third vaccination with BNT162b2, and stimulated with overlapping peptide libraries representing the spike of either the ancestral or the Omicron SARS-CoV-2 virus variants. Quantification of the specific T cells was carried out by a fluorescent ELISPOT assay, monitoring cells secreting interferon-gamma (IFNg), interleukin-10 (IL-10) and interleukin-4 (IL-4). For all the examined individuals, comparable levels of reactivity to both forms of spike protein were determined. In addition, a dominant Th1 response was observed, manifested mainly by IFNg-secreting cells and only limited numbers of IL-10- and IL-4-secreting cells. The data demonstrate stable T cell activity in response to the emerging Omicron variant in the tested individuals; therefore, the protective immunity to the variant following BNT162b2 vaccination is not significantly affected. Full article

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally suited for antigen-specific B-cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen-coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high-affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen-coating approach streamlines characterization of the memory B-cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune-monitoring efforts of large donor cohorts in general. Full article

Monitoring antigen-specific T cell immunity relies on functional tests that require T cells and antigen presenting cells to be uncompromised. Drawing of blood, its storage and shipment from the clinical site to the test laboratory, and the subsequent isolation, cryopreservation and thawing of peripheral blood mononuclear cells (PBMCs) before the actual test is performed can introduce numerous variables that may jeopardize the results. Therefore, no T cell test is valid without assessing the functional fitness of the PBMC being utilized. This can only be accomplished through the inclusion of positive controls that actually evaluate the performance of the antigen-specific T cell and antigen presenting cell (APC) compartments. For Caucasians, CEF peptides have been commonly used to this extent. Moreover, CEF peptides only measure CD8 cell functionality. We introduce here universal CD8+ T cell positive controls without any racial bias, as well as positive controls for the CD4+ T cell and APC compartments. In summary, we offer new tools and strategies for the assessment of PBMC functional fitness required for reliable T cell immune monitoring. Full article