An Overview of Current Accomplishments and Gaps of COVID-19 Vaccine Platforms and Considerations for Next Generation Vaccines.

Vaccines against SARS-CoV-2 have transformed the course of the COVID-19 pandemic with more than 30 authorizations. More than 2 billion people have been vaccinated with these vaccines developed on very different manufacturing platforms. We have reviewed the unprecedented work done in various aspects of the authorized vaccines and listed three potential improvements: 1) long-term stability at room-temperature conditions; 2) suitability for diverse populations such as infants, elderly, immune-compromised, and those with pre-existing or ongoing diseases; and 3) ability to act against different strains. In this article, we have discussed the current status of COVID-19 vaccines with respect to 1) diversity (strength and breadth) of initial immune responses and long-term immune memory; 2) prime-boost combinations that induce protection against variants; and 3) breakthrough infections. Further, we have listed host, product (critical quality attributes), and viral pathogenic factors that contribute to safety, efficacy, and effectiveness of vaccines. In addition, we have elaborated on the potential to (develop models and) determine the immune correlates that can predict long-term immune memory. The graphical representation of the abstract is provided as Fig. 1.

A new flow cytometry assay identifies recipient IgG subtype antibodies binding donor cells: increasing donor availability for highly sensitised patients.

Objectives: There are four immunoglobulin (IgG) subtypes that have varying complement-activating ability: strong (IgG3 and IgG1) and weak (IgG2 and IgG4). The standard flow cytometric crossmatch (FCM) assay does not distinguish between the various subtypes of the IgG molecule. This study outlines the development and use of a novel cell-based IgG subtype-specific FCM assay that is able to detect the presence of and quantitate the IgG subtypes bound to donor cells. Methods: A six-colour lyophilised reagent was designed that specifically detects the four IgG subtypes, as well as distinguishes between T cells and B cells in the lymphocyte population. To test the efficacy of this reagent, a retrospective evaluation of a group of highly sensitised patients awaiting heart and kidney transplant was carried out, who, because of positive standard FCM results, had been deemed incompatible with numerous prior potential donors. Results: Observations in this study demonstrate that the positive standard FCM results were mainly because of the presence of noncomplement-activating IgG2 or IgG4 antibodies. The results were supported by the absence of C3d-binding donor-specific antibodies (DSA) and a negative complement-dependent cytotoxicity crossmatch (CDC). Conclusion: Preliminary data presented in this study demonstrate the reliability of the novel IgG subtype assay to detect the presence of pretransplant, complement-activating antibodies bound to donor cells. The knowledge gained from the IgG subtype assay and the C3d-binding specificities of DSAs provides improved identification of donor suitability in pretransplant patients, potentially increasing the number of transplants.

Predicting the Severity of Disease Progression in COVID-19 at the Individual and Population Level: A Mathematical Model.

The impact of COVID-19 disease on health and economy has been global, and the magnitude of devastation is unparalleled in modern history. Any potential course of action to manage this complex disease requires the systematic and efficient analysis of data that can delineate the underlying pathogenesis. We have developed a mathematical model of disease progression to predict the clinical outcome, utilizing a set of causal factors known to contribute to COVID-19 pathology such as age, comorbidities, and certain viral and immunological parameters. Viral load and selected indicators of a dysfunctional immune response, such as cytokines IL-6 and IFNα which contribute to the cytokine storm and fever, parameters of inflammation D-Dimer and Ferritin, aberrations in lymphocyte number, lymphopenia, and neutralizing antibodies were included for the analysis. The model provides a framework to unravel the multi-factorial complexities of the immune response manifested in SARS-CoV-2 infected individuals. Further, this model can be valuable to predict clinical outcome at an individual level, and to develop strategies for allocating appropriate resources to manage severe cases at a population level.

Predicting the severity of disease progression in COVID-19 at the individual and population level: A mathematical model.

The impact of COVID-19 disease on health and economy has been global, and the magnitude of devastation is unparalleled in modern history. Any potential course of action to manage this complex disease requires the systematic and efficient analysis of data that can delineate the underlying pathogenesis. We have developed a mathematical model of disease progression to predict the clinical outcome, utilizing a set of causal factors known to contribute to COVID-19 pathology such as age, comorbidities, and certain viral and immunological parameters. Viral load and selected indicators of a dysfunctional immune response, such as cytokines IL-6 and IFNα, which contribute to the cytokine storm and fever, parameters of inflammation d-dimer and ferritin, aberrations in lymphocyte number, lymphopenia, and neutralizing antibodies were included for the analysis. The model provides a framework to unravel the multi-factorial complexities of the immune response manifested in SARS-CoV-2 infected individuals. Further, this model can be valuable to predict clinical outcome at an individual level, and to develop strategies for allocating appropriate resources to mitigate severe cases at a population level.

Tumor Formation of Adult Stem Cell Transplants in Rodent Arthritic Joints.

PURPOSE: While imaging matrix-associated stem cell transplants aimed for cartilage repair in a rodent arthritis model, we noticed that some transplants formed locally destructive tumors. The purpose of this study was to determine the cause for this tumor formation in order to avoid this complication for future transplants. PROCEDURES: Adipose-derived stem cells (ADSC) isolated from subcutaneous adipose tissue were implanted into 24 osteochondral defects of the distal femur in ten athymic rats and two immunocompetent control rats. All transplants underwent serial magnetic resonance imaging (MRI) up to 6 weeks post-transplantation to monitor joint defect repair. Nine transplants showed an increasing size over time that caused local bone destruction (group 1), while 11 transplants in athymic rats (group 2) and 4 transplants in immunocompetent rats did not. We compared the ADSC implant size and growth rate on MR images, macroscopic features, histopathologic features, surface markers, and karyotypes of these presumed neoplastic transplants with non-neoplastic ADSC transplants. RESULTS: Implants in group 1 showed a significantly increased two-dimensional area at week 2 (p = 0.0092), 4 (p = 0.003), and 6 (p = 0.0205) compared to week 0, as determined by MRI. Histopathological correlations confirmed neoplastic features in group 1 with significantly increased size, cellularity, mitoses, and cytological atypia compared to group 2. Six transplants in group 1 were identified as malignant chondrosarcomas and three transplants as fibromyxoid sarcomas. Transplants in group 2 and immunocompetent controls exhibited normal cartilage features. Both groups showed a normal ADSC phenotype; however, neoplastic ADSC demonstrated a mixed population of diploid and tetraploid cells without genetic imbalance. CONCLUSIONS: ADSC transplants can form tumors in vivo. Preventive actions to avoid in vivo tumor formations may include karyotyping of culture-expanded ADSC before transplantation. In addition, serial imaging of ADSC transplants in vivo may enable early detection of abnormally proliferating cell transplants.

Stabilization of pre-optimized multicolor antibody cocktails for flow cytometry applications.

BACKGROUND: Flow cytometry has a multitude of applications in nearly all fields of biology. Newly described biological markers enable the creation of novel reagents which then aid in the elucidation of unique subsets of cells and their potential role in health and disease. In order to enable the simultaneous detection of an even greater number of parameters, the future progress of flow cytometry relies on advances in instrument engineering and the parallel development of new fluorophores. METHODS: In order to address the issues of reagent reliability, reproducibility, and work-flow optimization, we have used the freeze-dry technique to stabilize pre-mixed, pre-optimized, multicolor 'cocktails' of antibodies within 12 × 75 mm flow cytometry tubes (Lyotube). In this study we describe several lyophilized stabilized reagent combinations that are functional for extended periods of time (18 months and beyond), and can be stored at ambient temperature, eliminating cold-chain requirements during transportation and storage. This improves precision and reduces the redundant labor and error-potential associated with mixing antibodies to create "home-brew" cocktails. RESULTS: We have stained different types of samples including normal and leukemic whole blood, bone marrow, and PBMCs, as well as cell lines, directly with BD Lyotube reagents: The data show comparable and consistent performance of multiple batches of dehydrated, stabilized mixtures of antibodies and their liquid counterparts. CONCLUSIONS: The approach we describe here, the Lyotube, facilitates the improvement and implementation of standardization measures in clinical settings and in multi-site studies, a useful tool which can also be applied to determining the efficacy and safety of candidate therapeutics and vaccines. © 2016 International Clinical Cytometry Society.

Improved approach for chondrogenic differentiation of human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) have demonstrated great potential for hyaline cartilage regeneration. However, current approaches for chondrogenic differentiation of hiPSCs are complicated and inefficient primarily due to intermediate embryoid body formation, which is required to generate endodermal, ectodermal, and mesodermal cell lineages. We report a new, straightforward and highly efficient approach for chondrogenic differentiation of hiPSCs, which avoids embryoid body formation. We differentiated hiPSCs directly into mesenchymal stem /stromal cells (MSC) and chondrocytes. hiPSC-MSC-derived chondrocytes showed significantly increased Col2A1, GAG, and SOX9 gene expression compared to hiPSC-MSCs. Following transplantation of hiPSC-MSC and hiPSC-MSC-derived chondrocytes into osteochondral defects of arthritic joints of athymic rats, magnetic resonance imaging studies showed gradual engraftment, and histological correlations demonstrated hyaline cartilage matrix production. Results present an efficient and clinically translatable approach for cartilage tissue regeneration via patient-derived hiPSCs, which could improve cartilage regeneration outcomes in arthritic joints.

Toward development of a comprehensive external quality assurance program for polyfunctional intracellular cytokine staining assays.

The External Quality Assurance Program Oversight Laboratory (EQAPOL) Flow Cytometry Program assesses the proficiency of NIH/NIAID/DAIDS-supported and potentially other interested research laboratories in performing Intracellular Cytokine Staining (ICS) assays. The goal of the EQAPOL Flow Cytometry External Quality Assurance Program (EQAP) is to provide proficiency testing and remediation for participating sites. The program is not punitive; rather, EQAPOL aims to help sites identify areas for improvement. EQAPOL utilizes a highly standardized ICS assay to minimize variability and readily identify those sites experiencing technical difficulties with their assays. Here, we report the results of External Proficiency 3 (EP3) where participating sites performed a 7-color ICS assay. On average, sites perform well in the Flow Cytometry EQAP (median score is "Good"). The most common technical issues identified by the program involve protocol adherence and data analysis; these areas have been the focus of site remediation. The EQAPOL Flow Cytometry team is now in the process of expanding the program to 8-color ICS assays. Evaluating polyfunctional ICS responses would align the program with assays currently being performed in support of HIV immune monitoring assays.

Iron administration before stem cell harvest enables MR imaging tracking after transplantation.

PURPOSE: To determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem cells (MSCs) in vivo and can be used for tracking of stem cell transplants. MATERIALS AND METHODS: This study was approved by the institutional animal care and use committee. Sprague-Dawley rats (6-8 weeks old) were injected with ferumoxytol 48 hours prior to extraction of MSCs from bone marrow. Ferumoxytol uptake by these MSCs was evaluated with fluorescence, confocal, and electron microscopy and compared with results of traditional ex vivo-labeling procedures. The in vivo-labeled cells were subsequently transplanted in osteochondral defects of 14 knees of seven athymic rats and were evaluated with magnetic resonance (MR) imaging up to 4 weeks after transplantation. T2 relaxation times of in vivo-labeled MSC transplants and unlabeled control transplants were compared by using t tests. MR data were correlated with histopathologic results. RESULTS: In vivo-labeled MSCs demonstrated significantly higher ferumoxytol uptake compared with ex vivo-labeled cells. With electron microscopy, iron oxide nanoparticles were localized in secondary lysosomes. In vivo-labeled cells demonstrated significant T2 shortening effects in vitro and in vivo when they were compared with unlabeled control cells (T2 in vivo, 15.4 vs 24.4 msec; P < .05) and could be tracked in osteochondral defects for 4 weeks. Histologic examination confirmed the presence of iron in labeled transplants and defect remodeling. CONCLUSION: Intravenous ferumoxytol can be used to effectively label MSCs in vivo and can be used for tracking of stem cell transplants with MR imaging. This method eliminates risks of contamination and biologic alteration of MSCs associated with ex vivo-labeling procedures.

Flow-based combinatorial antibody profiling: an integrated approach to cell characterization.

BD FACS™ CAP (CAP = combinatorial antibody profile) is a screening tool for rapid characterization of human cell surface protein expression profiles using semi-automated high-throughput flow cytometry. The current configuration consists of 229 directly conjugated antibodies arrayed in a 96-well plate as three-color cocktails, which enables the characterization of each of the 229 individual surface markers. Each individual cell type of interest is analyzed on the 96-well screening plates and the data are acquired on a flow cytometer equipped with a high-throughput sampler. The expression level of each marker for each cell type is then calculated using semiautomated custom flow cytometry software. The process of characterizing these surface markers in a highly efficient manner using BD FACS™ CAP is enabled by automated liquid handling for staining, automated flow cytometry for data acquisition, and standardized algorithms for automated data analysis.

Regulation of autoimmune arthritis by the pro-inflammatory cytokine interferon-gamma.

The pathogenesis of T cell-mediated diseases like rheumatoid arthritis (RA) has typically been explained in the context of the Th1-Th2 paradigm: the initiation/propagation by pro-inflammatory cytokines, and downregulation by Th2 cytokines. However, in our study based on the adjuvant-induced arthritis (AA) model of RA, we observed that Lewis (LEW) (RT.1(l)) rats at the recovery phase of AA showed the highest level of IFN-gamma in recall response to mycobacterial heat-shock protein 65 (Bhsp65), whereas AA-resistant Wistar-Kyoto (WKY) (RT.1(l)) rats secreted high levels of IFN-gamma much earlier following disease induction. However, no significant secretion of IL-10 or TGF-beta was observed in either strain. Furthermore, pre-treatment of LEW rats with a peptide of self (rat) hsp65 (R465), which induced T cells secreting predominantly IFN-gamma, afforded protection against AA and decreased IL-17 expression by the arthritogenic epitope-restimulated T cells. These results provide a novel perspective on the pathogenesis of autoimmune arthritis.

Identifying the estrogen receptor coactivator PELP1 in autophagosomes.

Resveratrol, a well-established phytoestrogen and chemopreventive agent, has gained much attention among oncologists because it can act as both estrogen receptor agonist and antagonist, depending on dosage and cell context. It is increasingly accepted that steroidal receptor coregulators may also function in the cytoplasmic compartment. Deregulation and altered localization of these coregulators could influence target gene expression and participate in the development of hormone-responsive cancers. Proline-, glutamic acid-, and leucine-rich protein-1 (PELP1), a novel estrogen receptor (ER) coactivator, plays an important role in the genomic and nongenomic actions of ER. Furthermore, recent studies have shown that differential compartmentalization of PELP1 could be crucial in modulating sensitivity to tamoxifen. In this study, we investigated the role of PELP1 in resveratrol-induced autophagy in lung cancer and salivary gland adenocarcinoma cell lines. Resveratrol reversibly inhibited the growth of these cancer cell lines and induced autophagy, as evidenced by microtubule-associated protein 1 light chain 3 (LC3) up-regulation in a time-dependent and 3-methyladenine-sensitive manner. Confocal microscopic analysis showed that resveratrol induced PELP1 accumulation in autophagosomes with green fluorescent protein-LC3. The intermediary molecule involved in PELP1 accumulation in resveratrol-induced autophagosomes is hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a trafficking molecule that binds to PELP1. These results identify PELP1 for the first time in autophagosomes, implying that both PELP1 and HRS reallocate to autophagosomes in response to resveratrol treatment, which might be important in the process of autophagy in the cancer cells.

Feasibility of a multiplex flow cytometric bead immunoassay for detection of anti-epoetin alfa antibodies.

Immunogenicity profiles of recombinant therapeutic proteins are important to understand because antibodies raised against these molecules may have important clinical sequelae. The purpose of the present study was to demonstrate that a flow cytometric bead array could be used to detect clinically relevant antibodies with specificity to such therapeutics. We chose to evaluate well-characterized specimens from persons treated with epoetin alfa that developed antibody-mediated pure red blood cell aplasia as a means to demonstrate the utility of this platform. Our data show that this assay is capable of detecting anti-epoetin alfa antibodies with a relative antibody concentration of 50 ng/ml, where 25 of 25 sera spiked with antibodies at this concentration scored positive. Moreover, the assay was designed to include positive and negative control beads for each specimen that is processed to ensure the specificity of the signal when detected. Measurement of interassay precision supports quantitative estimates of relative antibody concentrations in the range of 313 to 5,000 ng/ml, where the percent coefficient of variation did not exceed 20%. With respect to clinical specimens, antibodies with specificity for epoetin alfa could be easily detected in a set of specimens from persons with pure red blood cell aplasia that had prior exposure to the EPREX brand of recombinant epoetin alfa. Further development and validation of this approach may facilitate successful widespread application of the method for detection of anti-epoetin alfa antibodies, as well as antibodies directed against other recombinant therapeutic proteins.

Cytometric bead array: a multiplexed assay platform with applications in various areas of biology.

The introduction of flow cytometric bead-based technology has added a new approach for investigators to simultaneously measure multiple analytes in biological and environmental samples. This new technology allows for (1) evaluation of multiple analytes in a single sample; (2) utilization of minimal sample volumes to glean data; (3) reproducibility and results comparative with previous experiments; (4) direct comparison with existing assays; and (5) a more rapid evaluation of multiple samples in a single platform. The cytometric bead array (CBA) system enables simultaneous measurement of multiple analytes in sample volumes too small for traditional immunoassays. Results have been presented for the analysis of a variety of human cytokines. In addition, the technology allows for the design and creation of assays to measure a variety of analytes including inflammatory mediators, chemokines, immunoglobulin isotypes, intracellular signaling molecules, apoptotic mediators, adhesion molecules, and antibodies. New initiatives put forward by the Human Genome Project and the FDA require the development and use of assays for the rapid simultaneous quantitation of multiple analytes. The CBA technology provides the ability to quantify multiple proteins within a given sample, with precision and consistency.

Immunological characterization and therapeutic activity of an altered-peptide ligand, NBI-6024, based on the immunodominant type 1 diabetes autoantigen insulin B-chain (9-23) peptide.

The nonobese diabetic (NOD) mouse is a good model for human type 1 diabetes, which is characterized by autoreactive T-cell-mediated destruction of insulin-producing islet beta-cells of the pancreas. The 9-23 amino acid region of the insulin B-chain [B((9-23))] is an immunodominant T-cell target antigen in the NOD mouse that plays a critical role in the disease process. By testing a series of B((9-23)) peptide analogs with single or double alanine substitutions, we identified a set of altered peptide ligands (APLs) capable of inhibiting B((9-23))-induced proliferative responses of NOD pathogenic T-cell clones. These APLs were unable to induce proliferation of these clones. However, vaccinations with the APLs induced strong cellular responses, as measured by in vitro lymphocyte proliferation and Th2 cytokine production (i.e., interleukin [IL]-4 and IL-10, but not gamma-interferon [IFN-gamma]). These responses were cross-reactive with the native antigen, B((9-23)), suggesting that the APL-induced Th2 responses may provide protection by controlling endogenous B((9-23))-specific Th1 (i.e., IFN-gamma-producing) pathogenic responses. One of these APLs that contained alanine substitutions at residues 16 and 19 (16Y-->A, 19C-->A; NBI-6024) was further characterized for its therapeutic activity because it consistently induced T-cell responses (e.g., T-cell lines and clones) that were of the Th2 type and that were cross-reactive with B((9-23)). Subcutaneous injections of NBI-6024 to NOD mice administered either before or after the onset of disease substantially delayed the onset and reduced the incidence of diabetes. This study is the first to report therapeutic activity of an APL derived from an islet beta-cell-specific antigen in type 1 diabetes.