Heterologous SARS-CoV-2 booster vaccine for individuals with hematological malignancies after a primary SARS-CoV-2 mRNA vaccine series.

Heterologous COVID-19 vaccine boosters have not been evaluated for patients with hematological malignancies. A Novavax booster was administered for 56 individuals with hematological malignancies who had received a primary COVID-19 series and prior boosters with mRNA vaccines only. Blood specimens were obtained at baseline (pre-vaccine), 28 days, and 168 days after vaccination with the Novavax booster. The median fold change of anti-Spike IgG was 1.02 (IQR 0.79, 1.3) between baseline and Day 28. Circulating Spike protein-specific B cells increased 1.4-fold at Day 28 (p < 0.05). Increases in antibody and T cell responses were modest without significance, with a waning of humoral and cellular responses at 168 days after vaccination.

Impact of Donor and Recipient SARS-CoV-2 Vaccination or Infection on Immunity after Hematopoietic Cell Transplantation.

The role of donor and recipient Coronavirus disease 2019 (COVID-19) immunologic status pre-transplantation has not been fully investigated in allogeneic hematopoietic stem cell transplantation (HSCT) recipients. Given the poor immunogenicity to vaccines in this population and the serious outcomes of COVID-19, adoptive transfer of immunity may offer important insight into improving protection for this vulnerable population. In this study, we evaluated the role of adoptive transfer of immunity at 1 month post-transplantation and 6 months post-transplantation after vaccination of recipients, based on pre-transplantation severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination and infection exposures of both recipient and donor. Using banked specimens from related donor allogeneic HSCT recipients and clinical data from both donors and recipients, anti-Spike (S) IgG titers were analyzed at 1, 3, and 6 months post-transplantation according to prior SARS-CoV-2 immunologic exposures. Recipients were excluded if they had received SARS-CoV-2 monoclonal antibodies or had infection in the first 6 months post-transplantation. Of the 53 recipient-donor pairs, 29 donors and 24 recipients had prior SARS-CoV-2 immunologic exposure. Recipient-donor pairs with no prior SARS-CoV-2 exposure (D0R0) had significantly lower anti-S IgG titers at 1 month compared to those with prior exposures (D1R1) (D0R0: median, 2.43 [interquartile range (IQR), .41 to 3.77]; D1R1: median, 8.42; IQR, 5.58 to 12.20]; P = .008). At 6 months, anti-S IgG titers were higher in recipients who were vaccinated at 3 months post-transplantation in the D1R1 cohort (median IgG, 148.34; IQR, 92.36 to 204.33) compared with the D0R0 cohort (median IgG, 38.74; IQR, 8.93 to 119.71). Current strategies should be optimized to enhance SARS-CoV-2 protection for HSCT recipients, including augmentation of the immune response for both donors and recipients prior to transplantation.

Immunogenicity of a Three-Dose Primary Series of mRNA COVID-19 Vaccines in Patients With Lymphoid Malignancies.

Background: Patients with lymphoid malignancies are at risk for poor coronavirus disease 2019 (COVID-19)-related outcomes and have reduced vaccine-induced immune responses. Currently, a 3-dose primary regimen of mRNA vaccines is recommended in the United States for immunocompromised hosts. Methods: A prospective cohort study of healthy adults (n = 27) and patients with lymphoid malignancies (n = 94) was conducted, with longitudinal follow-up through completion of a 2- or 3-dose primary mRNA COVID vaccine series, respectively. Humoral responses were assessed in all participants, and cellular immunity was assessed in a subset of participants. Results: The rate of seroconversion (68.1% vs 100%) and the magnitude of peak anti-S immunoglobulin G (IgG) titer (median anti-S IgG = 32.4, IQR = 0.48-75.0 vs median anti-S IgG = 72.6, IQR 51.1-100.1; P = .0202) were both significantly lower in patients with lymphoid malignancies compared to the healthy cohort. However, peak titers of patients with lymphoid malignancies who responded to vaccination were similar to healthy cohort titers (median anti-S IgG = 64.3; IQR, 23.7-161.5; P = .7424). The third dose seroconverted 7 of 41 (17.1%) patients who were seronegative after the first 2 doses. Although most patients with lymphoid malignancies produced vaccine-induced T-cell responses in the subset studied, B-cell frequencies were low with minimal memory cell formation. Conclusions: A 3-dose primary mRNA series enhanced anti-S IgG responses to titers equivalent to healthy adults in patients with lymphoid malignancies who were seropositive after the first 2 doses and seroconverted 17.1% who were seronegative after the first 2 doses. T-cell responses were present, raising the possibility that the vaccines may confer some cell-based protection even if not measurable by anti-S IgG.

Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer.

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements. SIGNIFICANCE: Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1397.

Donor Clonal Hematopoiesis and Recipient Outcomes After Transplantation.

PURPOSE: Clonal hematopoiesis (CH) can be transmitted from a donor to a recipient during allogeneic hematopoietic cell transplantation. Exclusion of candidate donors with CH is controversial since its impact on recipient outcomes and graft alloimmune function is uncertain. PATIENTS AND METHODS: We performed targeted error-corrected sequencing on samples from 1,727 donors age 40 years or older and assessed the effect of donor CH on recipient clinical outcomes. We measured long-term engraftment of 102 donor clones and cytokine levels in 256 recipients at 3 and 12 months after transplant. RESULTS: CH was present in 22.5% of donors, with DNMT3A (14.6%) and TET2 (5.2%) mutations being most common; 85% of donor clones showed long-term engraftment in recipients after transplantation, including clones with a variant allele fraction < 0.01. DNMT3A-CH with a variant allele fraction ≥ 0.01, but not smaller clones, was associated with improved recipient overall (hazard ratio [HR], 0.79; P = .042) and progression-free survival (HR, 0.72; P = .003) after adjustment for significant clinical variables. In patients who received calcineurin-based graft-versus-host disease prophylaxis, donor DNMT3A-CH was associated with reduced relapse (subdistribution HR, 0.59; P = .014), increased chronic graft-versus-host disease (subdistribution HR, 1.36; P = .042), and higher interleukin-12p70 levels in recipients. No recipient of sole DNMT3A or TET2-CH developed donor cell leukemia (DCL). In seven of eight cases, DCL evolved from donor CH with rare TP53 or splicing factor mutations or from donors carrying germline DDX41 mutations. CONCLUSION: Donor CH is closely associated with clinical outcomes in transplant recipients, with differential impact on graft alloimmune function and potential for leukemic transformation related to mutated gene and somatic clonal abundance. Donor DNMT3A-CH is associated with improved recipient survival because of reduced relapse risk and with an augmented network of inflammatory cytokines in recipients. Risk of DCL in allogeneic hematopoietic cell transplantation is driven by somatic myelodysplastic syndrome-associated mutations or germline predisposition in donors.

Severe Acute Respiratory Syndrome Coronavirus 2 Messenger RNA Vaccines in Allogeneic Hematopoietic Stem Cell Transplant Recipients: Immunogenicity and Reactogenicity.

The severe acute respiratory syndrome coronavirus 2 messenger RNA vaccine-induced humoral response and reactogenicity profile are described in allogeneic hematopoietic stem cell transplant (HSCT) recipients. Findings showed that 75.0% (by Simoa assay) or 80.0% (by Roche assay) of the HSCT cohort had a positive antibody response on series completion, compared with 100% in the healthy cohort.

Single-Molecule Enzymology for Diagnostics: Profiling Alkaline Phosphatase Activity in Clinical Samples.

Enzymes can be used as biomarkers for a variety of diseases. However, profiling enzyme activity in clinical samples is challenging due to the heterogeneity in enzyme activity, and the low abundance of the target enzyme in biofluids. Single-molecule methods can overcome these challenges by providing information on the distribution of enzyme activities in a sample. Here, we describe the concept of using the single-molecule enzymology (SME) method to analyze enzymatic activity in clinical samples. We present recent work focused on measuring alkaline phosphatase isotypes in serum samples using SME. Future work will involve improving and simplifying this technology, and applying it to other enzymes for diagnostics.

Plasma IL-6 changes correlate to PD-1 inhibitor responses in NSCLC.

BACKGROUND: Blood-based biomarkers of anti-solid tumor immune checkpoint blockade (ICB) response are lacking. We hypothesized that changes in systemic cytokine levels with the initial doses of programmed cell death protein 1 (PD-1) pathway inhibitors would correlate with clinical responses. New ultrasensitive ELISA technology enables quantitation of plasma proteins in sub-picogram-per-milliliter concentrations. METHODS: We measured plasma cytokines by ultrasensitive single-molecule array assays in patients with non-small-cell lung carcinoma before and during treatment with anti-PD-1 therapy. Association with best overall response and progression-free survival (PFS) was assessed by Kruskall-Wallis test and Kaplan-Meier plots with log-rank test, respectively. RESULTS: A decrease in interleukin 6 (IL-6) levels was associated with improved PFS (n=47 patients, median PFS: 11 vs 4 months, HR 0.45 (95% CI 0.23 to 0.89), p=0.04). The extent of change in IL-6 differed between best overall response categories (p=0.01) and correlated with changes in C reactive protein levels. We also explored plasma cytokine levels in relation to immune-related adverse effects and observed some correlation. CONCLUSIONS: This study suggests the presence of a systemic, proteomic reflection of successful ICB outside the tumor microenvironment with plasma decreases in IL-6 and CRP.

Ultrasensitive Detection of Enzymatic Activity Using Single Molecule Arrays.

Enzyme assays are important for many applications including clinical diagnostics, functional proteomics, and drug discovery. Current methods for enzymatic activity measurement often suffer from low analytical sensitivity. We developed an ultrasensitive method for the detection of enzymatic activity using Single Molecule Arrays (eSimoa). The eSimoa assay is accomplished by conjugating substrates to paramagnetic beads and measuring the conversion of substrates to products using single molecule analysis. We demonstrated the eSimoa method for the detection of protein kinases, telomerase, histone H3 methyltransferase SET7/9, and polypeptide N-acetylgalactosaminyltransferase with unprecedented sensitivity. In addition, we tested enzyme inhibition and performed theoretical calculations for the binding of inhibitor to its target enzyme and show the need for an ultrasensitive enzymatic assay to evaluate the potency of tight binding inhibitors. The eSimoa assay was successfully used to determine inhibition constants of both bosutinib and dasatinib. Due to the ultrasensitivity of this method, we also were able to measure the kinase activities at the single cell level. We show that the eSimoa assay is a simple, fast, and highly sensitive approach, which can be easily extended to detect a variety of other enzymes, providing a promising platform for enzyme-related fundamental research and inhibitor screening.

Ultrasensitive Detection of Attomolar Protein Concentrations by Dropcast Single Molecule Assays.

Measurements of very low levels of biomolecules, including proteins and nucleic acids, remain a critical challenge in many clinical diagnostic applications due to insufficient sensitivity. While digital measurement methods such as Single Molecule Arrays (Simoa), or digital ELISA, have made significant advances in sensitivity, there are still many potential disease biomarkers that exist in accessible biofluids at levels below the detection limits of these techniques. To overcome this barrier, we have developed a simple strategy for single molecule counting, dropcast single molecule assays (dSimoa), that enables more target molecules to be counted through increased sampling efficiency and with a simpler workflow. In this approach, beads are simply dropcast onto a microscope slide and dried into a monolayer film for digital signal readout. The dSimoa platform achieves attomolar limits of detection, with an up to 25-fold improvement in sensitivity over Simoa, the current state of the art for ultrasensitive protein detection. Furthermore, due to its simple readout process and improved cost-effectiveness compared to existing digital bioassays, dSimoa increases amenability to integration into point-of-care platforms. As an illustration of the potential utility of dSimoa, we demonstrate its ability to measure previously undetectable levels of Brachyury, a tissue biomarker for chordoma, in plasma samples. With its significantly enhanced sensitivity and simplicity, dSimoa can pave the way toward the discovery of new biomarkers for early disease diagnosis and improved health outcomes.

Single Molecule Protein Detection with Attomolar Sensitivity Using Droplet Digital Enzyme-Linked Immunosorbent Assay.

Many proteins are present at low concentrations in biological samples, and therefore, techniques for ultrasensitive protein detection are necessary. To overcome challenges with sensitivity, the digital enzyme-linked immunosorbent assay (ELISA) was developed, which is 1000× more sensitive than conventional ELISA and allows sub-femtomolar protein detection. However, this sensitivity is still not sufficient to measure many proteins in various biological samples, thereby limiting our ability to detect and discover biomarkers. To overcome this limitation, we developed droplet digital ELISA (ddELISA), a simple approach for detecting low protein levels using digital ELISA and droplet microfluidics. ddELISA achieves maximal sensitivity by improving the sampling efficiency and counting more target molecules. ddELISA can detect proteins in the low attomolar range and is up to 25-fold more sensitive than digital ELISA using Single Molecule Arrays (Simoa), the current gold standard tool for ultrasensitive protein detection. Using ddELISA, we measured the LINE1/ORF1 protein, a potential cancer biomarker that has not been previously measured in serum. Additionally, due to the simplicity of our device design, ddELISA is promising for point-of-care applications. Thus, ddELISA will facilitate the discovery of biomarkers that have never been measured before for various clinical applications.

Single-Molecule Arrays for Ultrasensitive Detection of Blood-Based Biomarkers for Immunotherapy.

Single-molecule array (Simoa) technology enables ultrasensitive protein detection that is suited to the development of peripheral blood-based assays for assessing immuno-oncology responses. We adapted a panel of Simoa assays to measure systemic cytokine levels from plasma and characterized physiologic variation in healthy individuals and preanalytic variation arising from processing and handling of patient samples. Insights from these preclinical studies led us to a well-defined set of Simoa assay conditions, a specimen processing protocol, and a data processing approach that we describe here. Simoa enables accurate quantitation of soluble immune signaling molecules in an unprecedented femtomolar range, opening up the potential for liquid biopsy-type approaches in immuno-oncology. We are using the method described here to distinguish PD-1 inhibitor nonresponders as early as after one dose after therapy and envision applications in characterizing PD-1 inhibitor resistance and detection of immune-related adverse effects.

Evaluation of Antibody Biotinylation Approaches for Enhanced Sensitivity of Single Molecule Array (Simoa) Immunoassays.

In this study, we evaluated the performance of Single Molecule Array (Simoa) immunoassays based on various detection antibody biotinylation approaches. Simoa immunoassays, like other sandwich ELISAs, are highly dependent on the interaction of a biotinylated detection antibody with an enzyme conjugated to streptavidin. Thus, we sought to assess whether different biotinylation reagents can improve the performance and sensitivity of Simoa assays. We selected three proteins, GM-CSF, IFNγ, and IL-2, that are present at ultralow levels in many biological samples. We compared the performance of these Simoa assays by using five different biotinylation reagents and varying the amount of molar fold excess biotin during the biotinylation process. We found that the choice of biotinylation reagent and the molar fold excess biotin can highly affect the performance of the Simoa assays, with differences of up to an order of magnitude in sensitivity. We also tested the performance of bulk ELISAs using the different biotinylated detection antibodies and observe differences greater than an order of magnitude in sensitivity. We show that evaluating different strategies for detection antibody biotinylation is a simple approach for optimizing immunoassay performance for enhanced sensitivity.

Digital direct detection of microRNAs using single molecule arrays.

MicroRNAs (miRNAs) are involved in many biological pathways, and detecting miRNAs accurately is critical for diagnosing a variety of diseases including cancer. However, most current methods for miRNA detection require lengthy sample preparation and amplification steps that can bias the results. In addition, lack of specificity and reproducibility give rise to various challenges in detection of circulating miRNAs in biological samples. In this work, we applied the Single Molecule Array (Simoa) technique to develop an ultra-sensitive sandwich assay for direct detection of multiple miRNAs without pre-amplification. We successfully detected miRNAs at femtomolar concentrations (with limits of detection [LODs] ranging from 1 to 30 fM) and high specificity (distinguishing miRNAs with a single nucleotide mismatch). This method was effective against a range of diverse target sequences, suggesting a general approach for miRNA detection. To demonstrate the practical application of this technique, we detected miRNAs in a variety of sample types including human serum and total RNA. The high sensitivity and simple workflow of the Simoa method represent excellent advantages for miRNA-based diagnostics of human diseases.

Development of a Rapid Salivary Proteomic Platform for Oral Feeding Readiness in the Preterm Newborn.

Oral feeding competency is a major determinant of length of stay in the neonatal intensive care unit. An infant must be able to consistently demonstrate the ability to take all required enteral nutrition by mouth before discharge home. Most infants born prematurely (<37 weeks) will require days, if not weeks, to master this oral feeding competency skill. Inappropriately timed feeding attempts can lead to acute and long-term morbidities, prolonged hospitalizations, and increased health-care costs. Previously, a panel of five genes involved in essential developmental pathways including sensory integration (nephronophthisis 4, Plexin A1), hunger signaling [neuropeptide Y2 receptor (NPY2R), adenosine-monophosphate-activated protein kinase (AMPK)], and facial development (wingless-type MMTV integration site family, member 3) required for oral feeding success were identified in neonatal saliva. This study aimed to translate these five transcriptomic biomarkers into a rapid proteomic platform to provide objective, real-time assessment of oral feeding skills, to better inform care, and to improve neonatal outcomes. Total protein was extracted from saliva of 10 feeding-successful and 10 feeding-unsuccessful infants matched for age, sex, and post-conceptional age. Development of immunoassays was attempted for five oral feeding biomarkers and two reference biomarkers (GAPDH and YWHAZ) to normalize for starting protein concentrations. Normalized protein concentrations were correlated to both feeding status at time of sample collection and previously described gene expression profiles. Only the reference proteins and those involved in hunger signaling were detected in neonatal saliva at measurable levels. Expression patterns for NPY2R and AMPK correlated with the gene expression patterns previously seen between successful and unsuccessful feeders and predicted feeding outcome. Salivary proteins associated with hunger signaling are readily quantifiable in neonatal saliva and may be utilized to assess oral feeding readiness in the newborn. This study lays the foundation for the development of an informative, rapid, proteomic platform to assess neonatal oral feeding maturation.

Protein Counting in Single Cancer Cells.

The cell is the basic unit of biology and protein expression drives cellular function. Tracking protein expression in single cells enables the study of cellular pathways and behavior but requires methodologies sensitive enough to detect low numbers of protein molecules with a wide dynamic range to distinguish unique cells and quantify population distributions. This study presents an ultrasensitive and automated approach for quantifying phenotypic responses with single cell resolution using single molecule array (SiMoA) technology. We demonstrate how prostate specific antigen (PSA) expression varies over several orders of magnitude between single prostate cancer cells and how PSA expression shifts with genetic drift. Single cell SiMoA introduces a straightforward process that is capable of detecting both high and low protein expression levels. This technique could be useful for understanding fundamental biology and may eventually enable both earlier disease detection and targeted therapy.

Correlations of salivary biomarkers with clinical assessments in patients with cystic fibrosis.

RATIONALE: Monitoring clinical disease status in cystic fibrosis frequently requires invasive collection of clinical samples. Due to its noninvasive collection process and direct anatomic relationship with the lower airway, saliva shows great potential as a biological fluid for cystic fibrosis monitoring. OBJECTIVES: To measure the levels of multiple protein markers in human saliva supernatants and investigate the possibility of utilizing them to provide a more quantitative measure of disease state for use in research and monitoring of patients with cystic fibrosis clinically. METHODS: Whole saliva samples were collected and processed from cystic fibrosis patients at two distinct time points (2010 and 2013) and measured by two separate platforms. In this cross sectional study, a convenience sample of 71 participants were recruited with samples measured by multiplexed fluorescence microarray (fiber microarray) and another 117 participant samples were measured by an automated, point-of-care, analyzer (SDReader) using a microsphere-based array via fluorescence sandwich immunoassay. For comparison, saliva from 56 and 50 healthy subjects were collected, respectively. The levels of six target proteins were quantified. Various demographic and clinical data, including spirometry, medical history, and clinicians' assessments were also collected from patients with cystic fibrosis on the day of saliva collection. MEASUREMENTS AND MAIN RESULTS: Similar trends were observed with both platforms and compared with healthy subjects, cystic fibrosis patients had significantly elevated levels of VEGF, IP-10, IL-8, and EGF as well as lower levels of MMP-9 (P ≤ 0.005) using fiber microarray and significantly elevated levels of IP-10, IL-8 with lower levels of MMP-9 and IL-1ß (P ≤ 0.02) using the SDReader. The levels of the six proteins correlated with each other significantly, and in some cases, biomarker levels could be used to differentiate between subgroups of patients with different clinical presentations. For example, IP-10 levels significantly correlated with FEV1 and disease severity (as evaluated by clinicians) with both platforms (P < 0.05). CONCLUSIONS: Significant variations of the levels of six proteins in saliva supernatants, and the correlations of these levels with clinical assessments, demonstrated the potential of saliva for cystic fibrosis research and monitoring.

Ultra-sensitive protein detection via Single Molecule Arrays towards early stage cancer monitoring.

The early diagnosis of cancers and continued monitoring of tumor growth would be greatly facilitated by the development of a blood-based, non-invasive, screening technique for early cancer detection. Current technologies for cancer screening and detection typically rely on imaging techniques or blood tests that are not accurate or sensitive enough to definitively diagnose cancer at its earliest stages or predict biologic outcomes. By utilizing Single Molecule Arrays (SiMoA), an ultra-sensitive enzyme-linked immunosorbent assay (ELISA) technique, we were able to measure increasing levels of prostate specific antigen (PSA) within murine serum over time, which we attribute to tumor development. The measured concentrations of PSA were well below the detectable limits of both a leading clinical diagnostic PSA ELISA assay as well as a commercial ultra-sensitive PSA assay. Our work benchmarks the role of SiMoA as a vital tool in monitoring previously non-detectable protein biomarkers in serum for early cancer detection and offers significant potential as a non-invasive platform for the monitoring of early stage cancer.