Development of a novel serological assay for the detection of mpox infection in vaccinated populations.

In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 104 nonendemic locations worldwide. Serologic detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.

Mpox vaccine and infection-driven human immune signatures: an immunological analysis of an observational study.

BACKGROUND: Monkeypox virus has recently infected more than 88 000 people, raising concerns about our preparedness against this emerging viral pathogen. Licensed and approved for mpox, the JYNNEOS vaccine has fewer side-effects than previous smallpox vaccines and has shown immunogenicity against monkeypox in animal models. This study aims to elucidate human immune responses to JYNNEOS vaccination compared with mpox-induced immunity. METHODS: Peripheral blood mononuclear cells and sera were obtained from ten individuals vaccinated with one or two doses of JYNNEOS and six individuals diagnosed with monkeypox virus infection. Samples were obtained from seven individuals before vaccination to serve as a baseline. We examined the polyclonal serum (ELISA) and single B-cell (heavy chain gene and transcriptome data) antibody repertoires and T-cell responses (activation-induced marker and intracellular cytokine staining assays) induced by the JYNNEOS vaccine versus monkeypox virus infection. FINDINGS: All participants were men between the ages of 21 and 60 years, except for one woman in the group of mpox-convalescent individuals, and none had previous orthopoxvirus exposure. All mpox cases were mild. Vaccinee samples were collected 6-33 days after the first dose and 5-40 days after the second dose. Mpox-convalescent samples were collected 20-102 days after infection. In vaccine recipients, gene-level plasmablast and antibody responses were negligible and sera displayed moderate binding to recombinant orthopoxviral proteins (A29L, A35R, E8L, A30L, A27L, A33R, B18R, and L1R) and native proteins from the 2022 monkeypox outbreak strain. By contrast, recent monkeypox virus infection (within 20-102 days) induced robust serum antibody responses to monkeypox virus proteins and to native monkeypox virus proteins from a viral isolate obtained during the 2022 outbreak. JYNNEOS vaccine recipients presented robust orthopoxviral CD4+ and CD8+ T-cell responses. INTERPRETATION: Infection with monkeypox virus resulted in robust B-cell and T-cell responses, whereas immunisation with JYNNEOS elicited more robust T-cell responses. These data can help to inform vaccine design and policies for preventing mpox in humans. FUNDING: National Cancer Institute (National Institutes of Health), National Institute of Allergy and Infectious Diseases (National Institutes of Health), and Icahn School of Medicine.

Development of a Novel Serological Assay for the Detection of Mpox Infection in Vaccinated Populations.

In 2022 the World Health Organization declared a Public Health Emergency for an outbreak of mpox, the zoonotic Orthopoxvirus (OPV) affecting at least 103 non-endemic locations world-wide. Serologic detection of mpox infection is problematic, however, due to considerable antigenic and serologic cross-reactivity among OPVs and smallpox-vaccinated individuals. In this report, we developed a high-throughput multiplex microsphere immunoassay (MIA) using a combination of mpox-specific peptides and cross-reactive OPV proteins that results in the specific serologic detection of mpox infection with 93% sensitivity and 98% specificity. The New York State Non-Vaccinia Orthopoxvirus Microsphere Immunoassay is an important diagnostic tool to detect subclinical mpox infection and understand the extent of mpox spread in the community through retrospective analysis.

Durable Immunity to Ricin Toxin Elicited by Intranasally Administered Monoclonal Antibody-Based Immune Complexes.

Inhalation of ricin toxin (RT) elicits profuse inflammation and cell death within the upper and lower airways, ultimately culminating in acute respiratory distress syndrome. We previously reported that the effects of pulmonary RT exposure in mice are nullified by intranasal administration of an mAb mixture consisting of PB10, directed against ricin's enzymatic subunit (RTA), and SylH3, directed against ricin's binding subunit (RTB). We now report that delivery of PB10 and SylH3 as an RT-mAb immune complex (RIC) to mice by the intranasal or i.p. routes stimulates the rapid onset of RT-specific serum IgG that persists for months. RIC administration also induced high-titer, toxin-neutralizing Abs. Moreover, RIC-treated mice were immune to a subsequent 5 × LD50 RT challenge on days 30 or 90. Intranasal RIC administration was more effective than i.p. delivery at rendering mice immune to intranasal RT exposure. Finally, we found that the onset of RT-specific serum IgG following RIC delivery was independent of FcγR engagement, as revealed through FcγR knockout mice and RICs generated with PB10/SylH3 LALA (leucine to alanine) derivatives. In conclusion, a single dose of RICs given intranasally to mice was sufficient to stimulate durable protective immunity to RT by an FcγR-independent pathway.

COVID-19 Serology Control Panel Using the Dried-Tube Specimen Method.

The dried-tube specimen (DTS) procedure was used to develop the COVID-19 serology control panel (CSCP). The DTS offers the benefit of shipping materials without a cold chain, allowing for greater access without deterioration of material integrity. Samples in the panel were sourced from COVID-19 convalescent persons from March to May 2020. The immunoglobulin subtypes (total Ig, IgM, and IgG) and their respective reactivity to severe acute respiratory syndrome coronavirus 2 nucleocapsid, spike, and receptor-binding domain antigens of the samples were delineated and compared with the WHO International Standard to elucidate the exact binding antibody units of each CSCP sample and ensure the CSCP provides adequate reactivity for different types of serological test platforms. We distribute the CSCP as a kit with five coded tubes to laboratories around the world to be used to compare test kits for external quality assurance, for harmonizing laboratory testing, and for use as training materials for laboratory workers.

Pharmacokinetics of convalescent plasma therapy in a COVID-19 patient with X-linked Agammaglobulinemia.

Convalescent plasma (CP) has been the first line of defense against numerous infectious diseases throughout history. The COVID-19 pandemic created a need for a quick, easily accessible, and effective treatment for severe disease and CP was able to meet that immediate need. The utility of CP warrants a better understanding of the pharmacokinetics of CP treatment. Here we present the case of a COVID-19 patient with a genetic deficiency in antibody production who received CP as a part of the treatment regimen. In depth serological analysis revealed a surprising lack of SARS-CoV-2 specific antibodies and reduced serum IgG following CP infusion. Our study highlights plasma dilution and accelerated antibody clearance as potential mechanisms for the variable efficacy of CP therapy.

Serological analysis reveals an imbalanced IgG subclass composition associated with COVID-19 disease severity.

Coronavirus disease 2019 (COVID-19) is associated with a wide spectrum of disease presentation, ranging from asymptomatic infection to acute respiratory distress syndrome (ARDS). Paradoxically, a direct relationship has been suggested between COVID-19 disease severity and the levels of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies, including virus-neutralizing titers. A serological analysis of 536 convalescent healthcare workers reveals that SARS-CoV-2-specific and virus-neutralizing antibody levels are elevated in individuals that experience severe disease. The severity-associated increase in SARS-CoV-2-specific antibody is dominated by immunoglobulin G (IgG), with an IgG subclass ratio skewed toward elevated receptor binding domain (RBD)- and S1-specific IgG3. In addition, individuals that experience severe disease show elevated SARS-CoV-2-specific antibody binding to the inflammatory receptor FcÉ£RIIIa. Based on these correlational studies, we propose that spike-specific IgG subclass utilization may contribute to COVID-19 disease severity through potent Fc-mediated effector functions. These results may have significant implications for SARS-CoV-2 vaccine design and convalescent plasma therapy.

Glycolipid-Containing Nanoparticle Vaccine Engages Invariant NKT Cells to Enhance Humoral Protection against Systemic Bacterial Infection but Abrogates T-Independent Vaccine Responses.

CD4+ T cells enable the critical B cell humoral immune protection afforded by most effective vaccines. We and others have recently identified an alternative source of help for B cells in mice, invariant NK T (iNKT) cells. iNKT cells are innate glycolipid-specific T cells restricted to the nonpolymorphic Ag-presenting molecule CD1d. As such, iNKT cells respond to glycolipids equally well in all people, making them an appealing adjuvant for universal vaccines. We tested the potential for the iNKT glycolipid agonist, α-galactosylceramide (αGC), to serve as an adjuvant for a known human protective epitope by creating a nanoparticle that delivers αGC plus antigenic polysaccharides from Streptococcus pneumoniae αGC-embedded nanoparticles activate murine iNKT cells and B cells in vitro and in vivo, facilitate significant dose sparing, and avoid iNKT anergy. Nanoparticles containing αGC plus S. pneumoniae polysaccharides elicits robust IgM and IgG in vivo and protect mice against lethal systemic S. pneumoniae However, codelivery of αGC via nanoparticles actually eliminated Ab protection elicited by a T-independent S. pneumoniae vaccine. This is consistent with previous studies demonstrating iNKT cell help for B cells following acute activation, but negative regulation of B cells during chronic inflammation. αGC-containing nanoparticles represent a viable platform for broadly efficacious vaccines against deadly human pathogens, but their potential for eliminating B cells under certain conditions suggests further clarity on iNKT cell interactions with B cells is warranted.

Potency determination of ricin toxin using a monoclonal antibody-based competition assay.

Mouse challenge studies with death as an endpoint remain the gold standard in assessing the potency of ricin toxin, a Category B biothreat agent derived from the castor bean (Ricinus communis). However, animal studies are expensive, time consuming and ethically concerning. In an effort to reduce reliance on animals in vaccine development, we developed a monoclonal antibody (MAb)-based ricin competition ELISA (RiCoE) that indicates conformation integrity of ricin toxin. In forced degradation (heat-denaturation) experiments with native ricin holotoxin, we demonstrate a correlation between the decline in MAb reactivity in RiCoE and a corresponding loss of toxin potency in Vero cells (IC50) and mice (LD50). The RiCoE assay was applied to differentially sourced commercial lots of ricin toxin derived from R. communis blends and compared to toxin potency in mice. There was near perfect congruence between RiCoE values with two different MAbs (PB10, SyH7) and ricin potency in the mouse model using morbidity as an endpoint. In conclusion, we propose that RiCoE can serve as a rapid and sensitive substitute to mouse lethal dose challenge studies as a means to determine ricin toxin potency and will be valuable at various stages of vaccine development.

Endpoint and epitope-specific antibody responses as correlates of vaccine-mediated protection of mice against ricin toxin.

The successful licensure of vaccines for biodefense is contingent upon the availability of well-established correlates of protection (CoP) in at least two animal species that can be applied to humans, without the need to assess efficacy in the clinic. In this report we describe a multivariate model that combines pre-challenge serum antibody endpoint titers (EPT) and values derived from an epitope profiling immune-competition capture (EPICC) assay as a predictor in mice of vaccine-mediated immunity against ricin toxin (RT), a Category B biothreat. EPICC is a modified competition ELISA in which serum samples from vaccinated mice were assessed for their ability to inhibit the capture of soluble, biotinylated (b)-RT by a panel of immobilized monoclonal antibodies (mAbs) directed against four immunodominant toxin-neutralizing regions on the enzymatic A chain (RTA) of RT. In a test cohort of mice (n = 40) vaccinated with suboptimal doses of the RTA subunit vaccine, RiVax®, we identified two mAbs, PB10 and SyH7, which had EPICC inhibition values in pre-challenge serum samples that correlated with survival following a challenge with 5 × LD50 of RT administered by intraperitoneal (IP) injection. Analysis of a larger cohort of mice (n = 645) revealed that a multivariate model combining endpoint titers and EPICC values for PB10 and SyH7 as predictive variables had significantly higher statistical power than any one of the independent variables alone. Establishing the correlates of vaccine-mediated protection in mice represents an important steppingstone in the development of RiVax® as a medical countermeasure under the United States Food and Drug Administration's "Animal Rule."

Thermal stability and epitope integrity of a lyophilized ricin toxin subunit vaccine.

Biodefense vaccine are destined to be stockpiled for periods of time and deployed in the event of a public health emergency. In this report, we compared the potency of liquid and lyophilized (thermostabilized) formulations of a candidate ricin toxin subunit vaccine, RiVax, adsorbed to aluminum salts adjuvant, over a 12-month period. The liquid and lyophilized formulations were stored at stressed (40 °C) and unstressed (4 °C) conditions and evaluated at 3, 6 and 12-month time points for potency in a mouse model of lethal dose ricin challenge. At the same time points, the vaccine formulations were interrogated in vitro by competition ELISA for conformational integrity using a panel of three monoclonal antibodies (mAbs), PB10, WECB2, and SyH7, directed against known immunodominant toxin-neutralizing epitopes on RiVax. We found that the liquid vaccine under stress conditions declined precipitously within the first three months, as evidenced by a reduction in in vivo potency and concomitant loss of mAb recognition in vitro. In contrast, the lyophilized RiVax vaccine retained in vivo potency and conformational integrity for up to one year at 4 °C and 40 °C. We discuss the utility of monitoring the integrity of one or more toxin-neutralizing epitopes on RiVax as a possible supplement to animal studies to assess vaccine potency.

Alpha-galactosylceramide (αGalCer) enhances vaccine-induced protection in a model of ricin intoxication.

Alpha-galactosylceramide (αGalCer) is a glycolipid derived from a marine sponge that is a potent activator of both mouse and human invariant natural killer T (iNKT) cells. For that reason, αGalCer is a promising vaccine adjuvant that has been shown to improve both humoral and cellular immunity when co-administered with various vaccines, including candidate vaccines for biodefense. In the current study, we tested the effectiveness of αGalCer as an adjuvant for the clinically-relevant ricin toxin subunit vaccine, RiVax. αGalCer had a potent adjuvant effect, as shown by a rapid onset of anti-ricin IgG titers, accelerated development of serum toxin-neutralizing activity, and enhanced protection from lethal ricin challenge in a mouse model. These results underscore the potential of αGalCer to augment the protective immune response to a vaccine designed to counteract ricin toxin, a fast-acting biothreat agent.

Early derivation of IgM memory cells and bone marrow plasmablasts.

IgM memory cells are recognized as an important component of B cell memory in mice and humans. Our studies of B cells elicited in response to ehrlichial infection identified a population of CD11c-positive IgM memory cells, and an IgM bone marrow antibody-secreting cell population. The origin of these cells was unknown, although an early T-independent spleen CD11c- and T-bet-positive IgM plasmablast population precedes both, suggesting a linear relationship. A majority of the IgM memory cells detected after day 30 post-infection, also T-bet-positive, had undergone somatic hypermutation, indicating they expressed activation-induced cytidine deaminase (AID). Therefore, to identify early AID-expressing precursor B cells, we infected an AID-regulated tamoxifen-inducible Cre-recombinase-EYFP reporter strain. Tamoxifen administration led to the labeling of both IgM memory cells and bone marrow ASCs on day 30 and later post-infection. High frequencies of labeled cells were identified on day 30 post-infection, following tamoxifen administration on day 10 post-infection, although IgM memory cells were marked when tamoxifen was administered as early as day 4 post-infection. Transcription of Aicda in the early plasmablasts was not detected in the absence of CD4 T cells, but occurred independently of TLR signaling. Unlike the IgM memory cells, the bone marrow IgM ASCs were elicited independent of T cell help. Moreover, Aicda was constitutively expressed in IgM memory cells, but not in bone marrow ASCs. These studies demonstrate that two distinct long-term IgM-positive B cell populations are generated early in response to infection, but are maintained via separate mechanisms.

Neutrophils license iNKT cells to regulate self-reactive mouse B cell responses.

The innate responsiveness of the immune system is important not only for quick responses to pathogens but also for the initiation and shaping of the subsequent adaptive immune response. Activation via the cytokine IL-18, a product of inflammasomes, gives rise to a rapid response that includes the production of self-reactive antibodies. As increased concentrations of this cytokine are found in inflammatory diseases, we investigated the origin of the B cell response and its regulation. We identified an accumulation of B cell-helper neutrophils in the spleen that interacted with innate-type invariant natural killer T cells (iNKT cells) to regulate B cell responses. We found that neutrophil-dependent expression of the death-receptor ligand FasL by iNKT cells was needed to restrict autoantibody production. Neutrophils can thus license iNKT cells to regulate potentially harmful autoreactive B cell responses during inflammasome-driven inflammation.

Identification of secreted and membrane-bound bat immunoglobulin using a Microchiropteran-specific mouse monoclonal antibody.

Bat immunity has received increasing attention because some bat species are being decimated by the fungal disease, White Nose Syndrome, while other species are potential reservoirs of zoonotic viruses. Identifying specific immune processes requires new specific tools and reagents. In this study, we describe a new mouse monoclonal antibody (mAb) reactive with Eptesicus fuscus immunoglobulins. The epitope recognized by mAb BT1-4F10 was localized to immunoglobulin light (lambda) chains; hence, the mAb recognized serum immunoglobulins and B lymphocytes. The BT1-4F10 epitope appeared to be restricted to Microchiropteran immunoglobulins and absent from Megachiropteran immunoglobulins. Analyses of sera and other E. fuscus fluids showed that most, if not all, secreted immunoglobulins utilized lambda light chains. Finally, mAb BT1-4F10 permitted the identification of B cell follicles in splenic white pulp. This Microchiropteran-specific mAb has potential utility in seroassays; hence, this reagent may have both basic and practical applications for studying immune process.

T cell-dependent IgM memory B cells generated during bacterial infection are required for IgG responses to antigen challenge.

Immunological memory has long considered to be harbored in B cells that express high-affinity class-switched IgG. IgM-positive memory B cells can also be generated following immunization, although their physiological role has been unclear. In this study, we show that bacterial infection elicited a relatively large population of IgM memory B cells that were uniquely identified by their surface expression of CD11c, CD73, and programmed death-ligand 2. The cells lacked expression of cell surface markers typically expressed by germinal center B cells, were CD138 negative, and did not secrete Ab ex vivo. The population was also largely quiescent and accumulated somatic mutations. The IgM memory B cells were located in the region of the splenic marginal zone and were not detected in blood or other secondary lymphoid organs. Generation of the memory cells was CD4 T cell dependent and required IL-21R signaling. In vivo depletion of the IgM memory B cells abrogated the IgG recall responses to specific Ag challenge, demonstrating that the cell population was required for humoral memory, and underwent class-switch recombination following Ag encounter. Our findings demonstrate that T cell-dependent IgM memory B cells can be elicited at high frequency and can play an important role in maintaining long-term immunity during bacterial infection.

SSR-marker analysis of the intracultivar phenotypic variation discovered within 3 soybean cultivars.

Genetic variation within homogeneous gene pools in various crops is assumed to be very limited. One objective of this study was to use 144 simple sequence repeat (SSR) markers to determine if the single-plant lines selected at ultra-low plant density in honeycomb designs within the soybean cultivars Benning, Haskell, and Cook had unique SSR genetic fingerprints. Another objective was to investigate if the variation found was the result of residual genetic heterozygosity that could be detected in the original gene pool where selection initiated. Our results showed that the phenotypic variation for seed protein content and seed weight has a genotypic component identified by the SSR band variation. The 7 lines from Haskell had a total of 63 variant alleles, the 5 lines from Benning had 34 variant alleles, and the 7 lines from Cook had 34 variant alleles, therefore, possessing unique genetic fingerprints. Most of the intracultivar SSR band variation discovered was the result of residual heterozygosity in the initial plant selected to become the cultivar. More specifically, 82% of the SSR variant alleles were traced in the Benning Foundation seed source, 93% in the Haskell seed source, and 82% in the Cook seed source. The remaining variant bands (18% for Benning, 7% for Haskell, and 18% for Cook) could not be detected in the Foundation seed source and were likely the result of mutation or some other mechanism generating de novo variation. These results provide evidence that genetic variation among individual plants is present even in homogeneous gene pools and can be further utilized in breeding programs.

Collaborative learning in radiologic science education.

CONTEXT: Radiologic science is a complex health profession, requiring the competent use of technology as well as the ability to function as part of a team, think critically, exercise independent judgment, solve problems creatively and communicate effectively. OBJECTIVE: This article presents a review of literature in support of the relevance of collaborative learning to radiologic science education. In addition, strategies for effective design, facilitation and authentic assessment of activities are provided for educators wishing to incorporate collaborative techniques into their program curriculum. RESULTS: The connection between the benefits of collaborative learning and necessary workplace skills, particularly in the areas of critical thinking, creative problem solving and communication skills, suggests that collaborative learning techniques may be particularly useful in the education of future radiologic technologists. SUMMARY: This article summarizes research identifying the benefits of collaborative learning for adult education and identifying the link between these benefits and the necessary characteristics of medical imaging technologists.