Serologic Testing of US Blood Donations to Identify Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-Reactive Antibodies: December 2019-January 2020.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), was first identified in Wuhan, China, in December 2019, with subsequent worldwide spread. The first US cases were identified in January 2020. METHODS: To determine if SARS-CoV-2-reactive antibodies were present in sera prior to the first identified case in the United States on 19 January 2020, residual archived samples from 7389 routine blood donations collected by the American Red Cross from 13 December 2019 to 17 January 2020 from donors resident in 9 states (California, Connecticut, Iowa, Massachusetts, Michigan, Oregon, Rhode Island, Washington, and Wisconsin) were tested at the Centers for Disease Control and Prevention for anti-SARS-CoV-2 antibodies. Specimens reactive by pan-immunoglobulin (pan-Ig) enzyme-linked immunosorbent assay (ELISA) against the full spike protein were tested by IgG and IgM ELISAs, microneutralization test, Ortho total Ig S1 ELISA, and receptor-binding domain/ACE2 blocking activity assay. RESULTS: Of the 7389 samples, 106 were reactive by pan-Ig. Of these 106 specimens, 90 were available for further testing. Eighty-four of 90 had neutralizing activity, 1 had S1 binding activity, and 1 had receptor-binding domain/ACE2 blocking activity >50%, suggesting the presence of anti-SARS-CoV-2-reactive antibodies. Donations with reactivity occurred in all 9 states. CONCLUSIONS: These findings suggest that SARS-CoV-2 may have been introduced into the United States prior to 19 January 2020.

Examination of SARS-CoV-2 serological test results from multiple commercial and laboratory platforms with an in-house serum panel.

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) is a novel human coronavirus that was identified in 2019. SARS-CoV-2 infection results in an acute, severe respiratory disease called coronavirus disease 2019 (COVID-19). The emergence and rapid spread of SARS-CoV-2 has led to a global public health crisis, which continues to affect populations across the globe. Real time reverse transcription polymerase chain reaction (rRT-PCR) is the reference standard test for COVID-19 diagnosis. Serological tests are valuable tools for serosurveillance programs and establishing correlates of protection from disease. This study evaluated the performance of one in-house enzyme linked immunosorbent assay (ELISA) utilizing the pre-fusion stabilized ectodomain of SARS-CoV-2 spike (S), two commercially available chemiluminescence assays Ortho VITROS Immunodiagnostic Products Anti-SARS-CoV-2 Total Reagent Pack and Abbott SARS-CoV-2 IgG assay and one commercially available Surrogate Virus Neutralization Test (sVNT), GenScript USA Inc., cPass SARS-CoV-2 Neutralization Antibody Detection Kit for the detection of SARS-CoV-2 specific antibodies. Using a panel of rRT-PCR confirmed COVID-19 patients' sera and a negative control group as a reference standard, all three immunoassays demonstrated high comparable positivity rates and low discordant rates. All three immunoassays were highly sensitive with estimated sensitivities ranging from 95.4-96.6 %. ROC curve analysis indicated that all three immunoassays had high diagnostic accuracies with area under the curve (AUC) values ranging from 0.9698 to 0.9807. High positive correlation was demonstrated among the conventional microneutralization test (MNT) titers and the sVNT inhibition percent values. Our study indicates that independent evaluations are necessary to optimize the overall utility and the interpretation of the results of serological tests. Overall, we demonstrate that all serological tests evaluated in this study are suitable for the detection of SARS-CoV-2 antibodies.

Flagellin, a TLR5 agonist, reduces graft-versus-host disease in allogeneic hematopoietic stem cell transplantation recipients while enhancing antiviral immunity.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in patients treated with allogeneic hematopoietic stem cell transplantation (HSCT). Posttransplant immunosuppressive drugs incompletely control GVHD and increase susceptibility to opportunistic infections. In this study, we used flagellin, a TLR5 agonist protein (∼50 kDa) extracted from bacterial flagella, as a novel experimental treatment strategy to reduce both acute and chronic GVHD in allogeneic HSCT recipients. On the basis of the radioprotective effects of flagellin, we hypothesized that flagellin could ameliorate GVHD in lethally irradiated murine models of allogeneic HSCT. Two doses of highly purified flagellin (administered 3 h before irradiation and 24 h after HSCT) reduced GVHD and led to better survival in both H-2(b) → CB6F1 and H-2(K) → B6 allogeneic HSCT models while preserving >99% donor T cell chimerism. Flagellin treatment preserved long-term posttransplant immune reconstitution characterized by more donor thymic-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells and significantly enhanced antiviral immunity after murine CMV infection. The proliferation index and activation status of donor spleen-derived T cells and serum concentration of proinflammatory cytokines in flagellin-treated recipients were reduced significantly within 4 d posttransplant compared with those of the PBS-treated control recipients. Allogeneic transplantation of radiation chimeras previously engrafted with TLR5 knockout hematopoietic cells showed that interactions between flagellin and TLR5 expressed on both donor hematopoietic and host nonhematopoietic cells were required to reduce GVHD. Thus, the peritransplant administration of flagellin is a novel therapeutic approach to control GVHD while preserving posttransplant donor immunity.

Transmission potential of vaccinated and unvaccinated persons infected with the SARS-CoV-2 Delta variant in a federal prison, July-August 2021.

BACKGROUND: The extent to which vaccinated persons who become infected with SARS-CoV-2 contribute to transmission is unclear. During a SARS-CoV-2 Delta variant outbreak among incarcerated persons with high vaccination rates in a federal prison, we assessed markers of viral shedding in vaccinated and unvaccinated persons. METHODS: Consenting incarcerated persons with confirmed SARS-CoV-2 infection provided mid-turbinate nasal specimens daily for 10 consecutive days and reported symptom data via questionnaire. Real-time reverse transcription-polymerase chain reaction (RT-PCR), viral whole genome sequencing, and viral culture was performed on these nasal specimens. Duration of RT-PCR positivity and viral culture positivity was assessed using survival analysis. RESULTS: A total of 957 specimens were provided by 93 participants, of whom 78 (84 %) were vaccinated and 17 (16 %) were unvaccinated. No significant differences were detected in duration of RT-PCR positivity among vaccinated participants (median: 13 days) versus those unvaccinated (median: 13 days; p = 0.50), or in duration of culture positivity (medians: 5 days and 5 days; p = 0.29). Among vaccinated participants, overall duration of culture positivity was shorter among Moderna vaccine recipients versus Pfizer (p = 0.048) or Janssen (p = 0.003) vaccine recipients. In post-hoc analyses, Moderna vaccine recipients demonstrated significantly shorter duration of culture positivity compared to unvaccinated participants (p = 0.02). When restricted to participants without reported prior infection, the difference between Moderna vaccine recipients and unvaccinated participants was more pronounced (medians: 3 days and 6 days, p = 0.002). CONCLUSIONS: Infectious periods for vaccinated and unvaccinated persons who become infected with SARS-CoV-2 are similar and can be highly variable, though some vaccinated persons are likely infectious for shorter durations. These findings are critically important, especially in congregate settings where viral transmission can lead to large outbreaks. In such settings, clinicians and public health practitioners should consider vaccinated, infected persons to be no less infectious than unvaccinated, infected persons.

Tipifarnib sensitizes cells to proteasome inhibition by blocking degradation of bortezomib-induced aggresomes.

In this report, we investigated the mechanism responsible for synergistic induction of myeloma cell apoptosis induced by the combination of tipifarnib and bortezomib. Immunofluorescence studies revealed that bortezomib alone resulted in an accumulation of puncta of ubiquitinated proteins that was further enhanced by the addition of tipifarnib. These data suggest inhibition of the degradation of bortezomib-induced aggresomes; and consistent with this possibility, we also observed an increase in p62SQSTM1 in cells treated with the combination. However, autophagy in these cells appears to be normal as LC3BII is present, and autophagic flux appears to be unaffected as demonstrated by the addition of bafilomycin A1. Together, these data demonstrate that tipifarnib synergizes with bortezomib by inducing protein accumulation as a result of the uncoupling of the aggresome and autophagy pathways.

Perifosine synergistically enhances TRAIL-induced myeloma cell apoptosis via up-regulation of death receptors.

PURPOSE: The aim of this study is to investigate the efficacy of a novel Akt inhibitor, perifosine, in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human myeloma cells and primary patient samples. EXPERIMENTAL DESIGN: The activity of perifosine in combination with TRAIL was evaluated with experiments testing the effect of perifosine on DR4/DR5 expression by the use of chimeric blocking antibodies, as well as siRNA. RESULTS: DR4 and DR5 expression was induced by exposure to single-agent perifosine. After exposure of human myeloma cell lines or primary patient samples to increasing doses of perifosine with exogenous TRAIL, we identified synergistically enhanced apoptosis when compared with the perifosine alone, which was achieved with levels well below clinically achievable concentrations for both agents. Transfection with siRNA against DR4, and DR5 reduced the level of apoptosis induced by the combination but did not result in total abrogation of the combination effect. Overexpression of activated Akt, the proposed target for perifosine, did not inhibit the combination effect. Anti-DR4 and DR5 chimeric proteins blocked the cytotoxicity induced by the combination, and the use of c-FLICE-like interleukin protein (FLIP) siRNA enhanced the efficacy at the combination, further supporting the importance of the DR4/DR5 axis in the effect of perifosine. CONCLUSION: Our observation seems to be independent of the effects of perifosine on Akt signaling, and may represent an additional mechanism of action for this agent, and supports future clinical trials combining these two agents.

Regulation of alloimmune responses by dendritic cell subsets.

OBJECTIVE: Dendritic cells (DCs) are powerful mediators of immune responses. We have demonstrated that the content of plasmacytoid (type 2) dendritic cells (DC2) within allogeneic bone marrow grafts impacts survival and graft-vs-host disease following transplantation. In order to better understand the effect of DC subsets on regulation of immunity, we tested the effect of DC subsets on T cells in a model of indirect antigen presentation to mimic presentation of host-type alloantigen by donor-type DC. MATERIALS AND METHODS: Volunteers underwent apheresis without cytokine priming, and DC1, DC2, naïve, and memory T cells were purified by immunomagnetic bead and fluorescein-activated cell sorting. Purified DC1 and DC2 cells were cultured with third-party irradiated blood mononuclear cells and either naïve or memory homologous T cells in mixed lymphocyte reactions. RESULTS: Myeloid (type 1) dendritic cells (DC1) induced significant proliferation of homologous T cells and were more effective in priming naïve T-cell responses than memory T cells responding to alloantigen. DC2 cells induced minimal T-cell proliferation regardless of the T-cell subset used as the responding fraction. Secondary mixed lymphocyte reaction studies demonstrated that DC2 primed T cells remained hyporesponsive even when challenged with a third-party alloantigen. The immunostimulatory effect of DC1 required DC-to-T-cell contact, and induced interleukin-12 secretion, while DC2 cells induced interferon-gamma secretion. Polymerase chain reaction analysis of DC2-primed T cells demonstrated a significant increase in Foxp3 expression, supporting induction of a regulatory T-cell population. CONCLUSION: DC1 and DC2 cells induced divergent T-cell responses using homologous cells. Better understanding of DC2-mediated T-cell suppression may yield strategies that overcome tumor-specific immune tolerance and regulate graft-vs-host disease.

The combination of the farnesyl transferase inhibitor lonafarnib and the proteasome inhibitor bortezomib induces synergistic apoptosis in human myeloma cells that is associated with down-regulation of p-AKT.

The identification of signaling pathways critical to myeloma growth and progression has yielded an array of novel agents with clinical activity. Multiple myeloma (MM) growth is IL-6 dependent, and IL-6 is secreted in an autocrine/paracrine fashion with signaling via the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway. We hypothesized that combining a Ras pathway inhibitor (lonafarnib, SCH66336) with a proteasome inhibitor (bortezomib, Velcade, PS-341) would enhance myeloma-cell killing. MM cell lines and primary human cells were used to test either single agent bortezomib, lonafarnib, or the combination on MM signaling and apoptosis. Combination therapy induced synergistic tumor-cell death in MM cell lines and primary MM plasma cells. Cell death was rapid and associated with increased caspase 3, 8, and 9 cleavage and concomitant down-regulation of p-AKT. Down-regulation of p-AKT was seen only in combination therapy and not seen with either single agent. Cells transfected with constitutively active p-AKT, wild-type AKT, or Bcl-2 continued to demonstrate synergistic cell death in response to the combination. The order of addition was critically important, supporting bortezomib followed by lonafarnib as the optimal schedule. The combination of a proteasome inhibitor and farnesyl transferase inhibitor demonstrates synergistic myeloma-cell death and warrants further preclinical and clinical studies.