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Efforts to minimize COVID-19 exposure during the current SARS-CoV-2 pandemic have led to limitations in access to medical care and testing. The Tasso-SST kit includes all of the components necessary for remote, capillary blood self-collection. In this study, we sought to investigate the accuracy and reliability of the Tasso-SST device as a self-collection device for measurement of SARS-CoV-2 IgG antibodies. Capillary blood was obtained via unsupervised and supervised application of the Tasso-SST device, and venous blood was collected by standard venipuncture. Unsupervised self-collected blood samples underwent either extreme summer or winter-simulated shipping conditions prior to testing. Sera obtained by all three methods were tested concurrently using the EuroImmun anti-SARS-CoV-2 S1 IgG assay in a CLIA-certified clinical laboratory. Successful Tasso-SST capillary blood collection by unsupervised and supervised administration was completed by 93.4% and 94.5% of participants, respectively. Sera from 56 participants, 55 with documented (PCR+) COVID-19, and 33 healthy controls were then tested for anti-SARS-CoV-2 IgG antibodies. Compared to venous blood results, Tasso-SST-collected (unstressed) and the summer- and winter-stressed blood samples demonstrated Deming regression slopes of 1.00 (95% CI: 0.99-1.02), 1.00 (95% CI: 0.98-1.01), and 0.99 (95% CI: 0.97-1.01), respectively, with an overall accuracy of 98.9%. Capillary blood self-collection using the Tasso-SST device had a high success rate. Moreover, excellent concordance was found for anti-SARS-CoV-2 IgG results between Tasso-SST capillary and standard venous blood-derived sera. The Tasso-SST device should enable widespread collection of capillary blood for testing without medical supervision, facilitating epidemiologic studies.

Silymarin, also known as milk thistle extract, inhibits hepatitis C virus (HCV) infection and also displays antioxidant, anti-inflammatory, and immunomodulatory actions that contribute to its hepatoprotective effects. In the current study, we evaluated the hepatoprotective actions of the seven major flavonolignans and one flavonoid that comprise silymarin. Activities tested included inhibition of: HCV cell culture infection, NS5B polymerase activity, TNF-α-induced NF-κB transcription, virus-induced oxidative stress, and T-cell proliferation. All compounds were well tolerated by Huh7 human hepatoma cells up to 80 μM, except for isosilybin B, which was toxic to cells above 10 μM. Select compounds had stronger hepatoprotective functions than silymarin in all assays tested except in T cell proliferation. Pure compounds inhibited JFH-1 NS5B polymerase but only at concentrations above 300 μM. Silymarin suppressed TNF-α activation of NF-κB dependent transcription, which involved partial inhibition of IκB and RelA/p65 serine phosphorylation, and p50 and p65 nuclear translocation, without affecting binding of p50 and p65 to DNA. All compounds blocked JFH-1 virus-induced oxidative stress, including compounds that lacked antiviral activity. The most potent compounds across multiple assays were taxifolin, isosilybin A, silybin A, silybin B, and silibinin, a mixture of silybin A and silybin B. The data suggest that silymarin- and silymarin-derived compounds may influence HCV disease course in some patients. Studies where standardized silymarin is dosed to identify specific clinical endpoints are urgently needed.

Background. Long-lasting protection resulting from hepatitis vaccine, despite loss of antibody against hepatitis virus (HBV) surface antigen (anti-HBs), is undetermined. Methods. We recruited persons from a cohort vaccinated with plasma-derived hepatitis vaccine in 1981 who have been followed periodically since. We performed serological testing for anti-HBs and microRNA-155 and assessed HBV-specific T-cell responses by enzyme-linked immunospot and cytometric bead array. Study subgroups were defined 32 years after vaccination as having an anti-HBs level of either ≥ 10 mlU/mL (group 1; n= 13) or <10 mlU/mL (group 2; n = 31). Results. All 44 participants, regardless of anti-HBs level, tested positive for tumor necrosis factor a, interleukin 10, or interleukin 6 production by HBV surface antigen-specific T cells. The frequency of natural killer T cells correlated with the level of anti-HBs (P = .008). The proportion of participants who demonstrated T-cell responses to HBV core antigen varied among the cytokines measured, suggesting some natural exposure to HBV in the study group. No participant had evidence of breakthrough HBV infection. Conclusions. Evidence of long-lasting cellular immunity, regardless of anti-HBs level, suggests that protection afforded by primary immunization with plasma-derived hepatitis vaccine during childhood and adulthood lasts at least 32 years.

Many serologic tests are now available for measuring severe acute respiratory syndrome coronavirus 2 antibodies to evaluate potential protective immunity and for seroprevalence studies. We describe an approach to standardizing positivity thresholds and quantitative values for different assays that uses z-scores to enable rapid and efficient comparison of serologic test performance.

VTX-2337 (USAN: motolimod) is a selective toll-like receptor 8 (TLR8) agonist, which is in clinical development as an immunotherapy for multiple oncology indications, including squamous cell carcinoma of the head and neck (SCCHN). Activation of TLR8 enhances natural killer cell activation, increases antibody-dependent cell-mediated cytotoxicity, and induces Th1 polarizing cytokines. Here, we show that VTX-2337 stimulates the release of mature IL-1β and IL-18 from monocytic cells through coordinated actions on both TLR8 and the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome complex. In vitro, VTX-2337 primed monocytic cells to produce pro-IL-1β, pro-IL-18, and caspase-1, and also activated the NLRP3 inflammasome, thereby mediating the release of mature IL-1β family cytokines. Inhibition of caspase-1 blocked VTX-2337-mediated NLRP3 inflammasome activation, but had little impact on production of other TLR8-induced mediators such as TNFα. IL-18 activated natural killer cells and complemented other stimulatory pathways, including FcγRIII and NKG2D, resulting in IFNγ production and expression of CD107a. NLRP3 activation in vivo was confirmed by a dose-related increase in plasma IL-1β and IL-18 levels in cynomolgus monkeys administered VTX-2337. These results are highly relevant to clinical studies of combination VTX-2337/cetuximab treatment. Cetuximab, a clinically approved, epidermal growth factor receptor-specific monoclonal antibody, activates NK cells through interactions with FcγRIII and facilitates ADCC of tumor cells. Our preliminary findings from a Phase I open-label, dose-escalation, trial that enrolled 13 patients with recurrent or metastatic SCCHN show that patient NK cells become more responsive to stimulation by NKG2D or FcγRIII following VTX-2337 treatment. Together, these results indicate that TLR8 stimulation and inflammasome activation by VTX-2337 can complement FcγRIII engagement and may augment clinical responses in SCCHN patients treated with cetuximab. ClinicalTrials.gov NCT01334177.

Background Myeloid-derived suppressor cells (MDSC) have been found to play an important role in limiting immune responses in cancer. Higher circulating MDSC levels have been associated with greater tumor burden, poorer response to immunotherapy, and poorer survival. Optimal measurement of MDSC levels could provide clinicians with a useful prognostic and/or management tool. Methods A whole blood (WB) nine color, 11 parameter flow cytometric assay was designed, utilizing fluorescently-labeled antibodies against CD45, CD3, CD19, CD20, CD56, CD16, HLA-DR, CD33, CD11b, CD14 and CD15, and BD Trucount beads for quantitation. Total MDSC were defined as CD45 + CD3 − CD19 − CD20 − CD56 − CD16 − HLA-DR − CD33 + CD11b + cells, while the monocytic (M-MDSC) and polymorphonuclear subsets were defined as CD14+ or CD15+, respectively. Results A novel gating strategy was devised to eliminate granulocytes and improve consistency in gating. Several pre-analytical variables were found to significantly affect MDSC quantitation, including collection tube type and time elapsed between blood collection and testing. Total and M-MDSC levels were a mean of 63% and 73% greater, respectively, with K 2 EDTA compared to Na + heparin collection tubes ( N  = 5). In addition, time elapsed at room temperature prior to cell labeling affected MDSC quantitation; by 24 h after blood collection, total and M-MDSC levels were a mean of 26% and 57% lower compared to testing as soon as possible after collection ( N  = 6). Refrigeration of samples at 4 °C ameliorated time-dependent effects at both 4 and 8 h, but not 24 h after blood collection. To establish normal ranges for this assay, MDSC levels were quantified in 67 healthy subjects (30 male, 37 female) ages 20–93. No significant differences in total or M-MDSC levels were detected for ages ≤60 compared to > 60 ( p  = 0.5 and p  = 0.8, respectively). Finally, assay results demonstrated significantly higher MDSC levels among patients with hepatocellular carcinoma ( N  = 55) compared to age-matched healthy controls ( N  = 27) for total and M-MDSC ( p  = 0.006 and 0.004, respectively). Conclusions MDSC are a heterogenous group of cells, and their quantitation in WB can be affected by a number of pre-analytical variables. Consideration of these factors, and measurement using a material type that has not been manipulated, such as whole blood, is likely to yield the most accurate results.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder that causes debilitating swelling and destruction of the joints. People with RA are treated with drugs that actively suppress one or more parts of their immune system, and these may alter the response to vaccination against SARS-CoV-2. In this study, we analyzed blood samples from a cohort of patients with RA after receiving a 2-dose mRNA COVID-19 vaccine regimen. Our data show that individuals on the cytotoxic T lymphocyte antigen 4-Ig therapy abatacept had reduced levels of SARS-CoV-2-neutralizing antibodies after vaccination. At the cellular level, these patients showed reduced activation and class switching of SARS-CoV-2-specific B cells, as well as reduced numbers and impaired helper cytokine production by SARS-CoV-2-specific CD4+ T cells. Individuals on methotrexate showed similar but less severe defects in vaccine response, whereas individuals on the B cell-depleting therapy rituximab had a near-total loss of antibody production after vaccination. These data define a specific cellular phenotype associated with impaired response to SARS-CoV-2 vaccination in patients with RA on different immune-modifying therapies and help inform efforts to improve vaccination strategies in this vulnerable population.

BACKGROUNDMeasuring the immune response to SARS-CoV-2 enables assessment of past infection and protective immunity. SARS-CoV-2 infection induces humoral and T cell responses, but these responses vary with disease severity and individual characteristics.METHODSA T cell receptor (TCR) immunosequencing assay was conducted using small-volume blood samples from 302 individuals recovered from COVID-19. Correlations between the magnitude of the T cell response and neutralizing antibody (nAb) titers or indicators of disease severity were evaluated. Sensitivity of T cell testing was assessed and compared with serologic testing.RESULTSSARS-CoV-2-specific T cell responses were significantly correlated with nAb titers and clinical indicators of disease severity, including hospitalization, fever, and difficulty breathing. Despite modest declines in depth and breadth of T cell responses during convalescence, high sensitivity was observed until at least 6 months after infection, with overall sensitivity ~5% greater than serology tests for identifying prior SARS-CoV-2 infection. Improved performance of T cell testing was most apparent in recovered, nonhospitalized individuals sampled > 150 days after initial illness, suggesting greater sensitivity than serology at later time points and in individuals with less severe disease. T cell testing identified SARS-CoV-2 infection in 68% (55 of 81) of samples with undetectable nAb titers (<1:40) and in 37% (13 of 35) of samples classified as negative by 3 antibody assays.CONCLUSIONThese results support TCR-based testing as a scalable, reliable measure of past SARS-CoV-2 infection with clinical value beyond serology.TRIAL REGISTRATIONSpecimens were accrued under trial NCT04338360 accessible at clinicaltrials.gov.FUNDINGThis work was funded by Adaptive Biotechnologies, Frederick National Laboratory for Cancer Research, NIAID, Fred Hutchinson Joel Meyers Endowment, Fast Grants, and American Society for Transplantation and Cell Therapy.

Silymarin prevents liver disease in many experimental rodent models, and is the most popular botanical medicine consumed by patients with hepatitis C. Silibinin is a major component of silymarin, consisting of the flavonolignans silybin A and silybin B, which are insoluble in aqueous solution. A chemically modified and soluble version of silibinin, SIL, has been shown to potently reduce hepatitis C virus (HCV) RNA levels in vivo when administered intravenously. Silymarin and silibinin inhibit HCV infection in cell culture by targeting multiple steps in the virus lifecycle. We tested the hepatoprotective profiles of SIL and silibinin in assays that measure antiviral and anti-inflammatory functions. Both mixtures inhibited fusion of HCV pseudoparticles (HCVpp) with fluorescent liposomes in a dose-dependent fashion. SIL inhibited 5 clinical genotype 1b isolates of NS5B RNA dependent RNA polymerase (RdRp) activity better than silibinin, with IC50 values of 40-85 µM. The enhanced activity of SIL may have been in part due to inhibition of NS5B binding to RNA templates. However, inhibition of the RdRps by both mixtures plateaued at 43-73%, suggesting that the products are poor overall inhibitors of RdRp. Silibinin did not inhibit HCV replication in subgenomic genotype 1b or 2a replicon cell lines, but it did inhibit JFH-1 infection. In contrast, SIL inhibited 1b but not 2a subgenomic replicons and also inhibited JFH-1 infection. Both mixtures inhibited production of progeny virus particles. Silibinin but not SIL inhibited NF-κB- and IFN-B-dependent transcription in Huh7 cells. However, both mixtures inhibited T cell proliferation to similar degrees. These data underscore the differences and similarities between the intravenous and oral formulations of silibinin, which could influence the clinical effects of this mixture on patients with chronic liver diseases.

BackgroundSipuleucel-T (Provenge) is the first therapeutic vaccination approved by the FDA so far, indicated for advanced metastatic prostate cancer patients. Despite an improvement of the overall survival, the benefits of the therapy are still short-term so increasing the duration of the efficacy is necessary. Specifically, T-cell anergy is one of the challenges that we need to overcome to improve the overall efficacy. IL-7 is known to promote the naive T cell activation and to increase the proliferation and activation of the T cell memory subsets. Therefore, in this phase II clinical trial, we tested the therapeutic potential of a human recombinant glycosylated IL-7 after completion of the Provenge therapy on asymptomatic advanced prostate cancer patients.MethodsTo get a comprehensive analysis of the immune landscape in these patients, we performed CyTOF analysis on PBMC samples obtained at week 1 (baseline) and week 6 after the beginning of the IL-7 therapy. After stimulation with PMA/Ionomycin, we proceeded to surface and intracellular cytokine staining before acquisition on the CyTOF. The data were then analyzed by expert gating on Cytobank.ResultsAt 6 weeks post therapy, our data showed an increase in the number of circulating T lymphocytes in the IL-7 cohort, especially CD8 T cells, in accordance with previous literature. Even though of the frequency of CD4 T cells did not increase, the cells showed greater functionalities, with increased expression of IL-2, TNFα and IL-6 upon stimulation by PMA-Ionomycine. Cytotoxic subsets were also positively affected, with increased expression of IFNγ in CD8 T cells, TNFα in NK cells and IL-2 in γδ T cells. Moreover, PD-1 expression was decreased on CD4, CD8 and γδ T cells while CD137 increased on CD4, CD8 and NK cells. In addition, despite a reduction in the pool of circulating monocytes, we observed higher TNFα expression in these cells.ConclusionsAltogether, our data revealed multiple effects of IL-7 in these patients, highlighting a complex set of in vivo mechanisms. In the future, knowledge of these effects may help in choosing the best agents to use in combination with IL-7 and/or the best patients to benefit from IL-7 as part of their therapeutic approach.Trial RegistrationNCT01881867Ethics ApprovalThe study was last approved by Fred Hutchinson Cancer Research Center Institutional Review Board, IR file 8037, on January 23, 2020