Antibody and T-Cell Responses to COVID-19 Vaccination in Myeloproliferative Neoplasm Patients

The efficacy of COVID-19 vaccines in cancer populations remain unknown. Myeloproliferative neoplasms (MPNs), including chronic myeloid leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF) remain a vulnerable patient population and are immunocompromised due to impaired innate and adaptive immunity, heightened inflammation, and effects of ongoing treatment. We evaluate antibody and T-cell responses in MPN patients following completion of the BNT162b2 (Pfizer/BioNTech) and mRNA-1273 (Moderna) COVID-19 vaccine series. Patients with a known diagnosis of MPN presenting at Massachusetts General Hospital and eligible for COVID-19 vaccination were recruited. All participants gave informed consent and the study protocol was approved by the Institutional Review Board. 33 MPN patients were enrolled and 23 patients completed vaccination. Baseline and post-vaccination peripheral blood samples were collected and peripheral blood mononuclear cells (PBMCs) isolated. 26 vaccinated participants with no history of malignancy were included as healthy controls (PMID 33972942). Baseline characteristics are tabled below. Qualitative ELISA for human IgG/A/M against SARS-CoV-2 spike protein using donor serum was performed per manufacturer instructions. Seroconversion occurred in 22/23 (96%) of MPN patients and 25/26 (96%) of healthy controls (Figure). To measure SARS-CoV-2 T-cell immunity, an IFNγ ELISpot assay previously developed in convalescent and vaccinated healthy individuals was used. Freshly isolated PBMCs from patients were stimulated with commercially available overlapping 15mer peptide pools spanning the SARS-CoV-2 spike and nucleocapsid proteins. Given its size, the spike protein was split into two pools (Spike A or B). IFNγ-producing T-cells were quantified by counting the median spot forming units (SFU) per 2.5x10 5 PBMCs from duplicate wells. A positive threshold was defined as >6 SFUs per 2.5x10 5 PBMCs to either Spike A or B after subtraction of background, based on prior receiver operator curve (ROC) analysis of ELISpot responses (sensitivity 90% specificity 92%). Post-vaccination ELISpot responses occurred in 21/23 (91%) of MPN patients and 26/26 (100%) of healthy controls (p=0.99) (Figure). The median SFU to total spike protein (Spike A+B) increased after vaccination in both MPN patients (0 to 38, p=0.02) and healthy controls (6 to 134, p=0.002). MPN patients had significantly lower median SFU's on post-vaccination ELISpot compared to healthy controls (38 vs 134, p=0.044), although this was not significant after adjusting for age in multivariable logistic regression. MF patients had the lowest seroconversion and ELISpot response rates, and lowest median post-vaccination SFUs, although this was not significant. There were no other differences in post-vaccination SFUs with regards to gender, vaccine type, number of days post-vaccine, treatment, and absolute lymphocyte count. Whole-blood assay based on the in vitro diagnostic QuantiFERON TB Gold Plus assay was also used to assess T-cell response. Heparinized whole blood from donors was stimulated with S1 and S2 subdomains for the SARS-CoV-2 spike protein, with measurement of IFNγ released into plasma with the QuantiFERON ELISA. IFNγ release of >0.3 IU/mL was considered a positive threshold, based on prior ROC analysis (sensitivity and specificity 100%). MPN patients had significantly lower IFNγ response rates compared to healthy controls (57% versus 100%, p=0.003) (Figure). Our findings demonstrate robust antibody and T-cell responses to BNT162b2 and mRNA-1273 vaccination in MPN patients, with >90% serologic and ELISpot responder rates. We detected subtle differences in T-cell responses in MPN patients compared to healthy controls. MPN patients had lower median post-vaccination ELISpot SFUs and lower rates of T-cell responses on IFNγ-whole blood assay compared to healthy controls. As the whole blood assay uses whole protein antigen rather than peptide pools, differences from ELISpot testing may reflect deficiencies in antigen processing and presentation. It is unclear whether these subtle differences translate into less clinical protection from COVID-19, or to what extent our results are confounded by the older age of MPN patients. Further evaluation of B and T-cell responses to COVID-19 vaccination in a larger sample size of MPN patients is warranted. Figure 1 Figure 1. Disclosures Neuberg: Pharmacyclics: Research Funding; Madrigal Pharmaceuticals: Other: Stock ownership. Maus: Atara: Consultancy; Bayer: Consultancy; BMS: Consultancy; Cabaletta Bio (SAB): Consultancy; CRISPR therapeutics: Consultancy; In8bio (SAB): Consultancy; Intellia: Consultancy; GSK: Consultancy; Kite Pharma: Consultancy, Research Funding; Micromedicine: Consultancy, Current holder of stock options in a privately-held company; Novartis: Consultancy; Tmunity: Consultancy; Torque: Consultancy, Current holder of stock options in a privately-held company; WindMIL: Consultancy; AstraZeneca: Consultancy; Agenus: Consultancy; Arcellx: Consultancy; Astellas: Consultancy; Adaptimmune: Consultancy; tcr2: Consultancy, Divested equity in a private or publicly-traded company in the past 24 months; century: Current equity holder in publicly-traded company; ichnos biosciences: Consultancy, Current holder of stock options in a privately-held company. Hobbs: AbbVie.: Consultancy; Incyte Corporation: Research Funding; Novartis: Consultancy; Bayer: Research Funding; Merck: Research Funding; Constellation Pharmaceuticals: Consultancy, Research Funding; Celgene/Bristol Myers Squibb: Consultancy.

Characteristics of routine coagulationtesting and thromboelastometry in polytrauma patients at admission

Background: polytrauma is one of the emergency surgeries with a high mortality rate. One of the leading causes of death is coagulopathy that is not detected early and treated promptly. Thromboelastometry (ROTEM) is a whole blood assay that evaluates the viscoelastic properties during clot formation and clot lysis. This method can detect coagulopathy rapidly and accurately, thereby improving the management of bleeding after trauma. Objectives: describing and evaluating the correlation between ROTEM parameters and routine coagulation tests in polytrauma patients at admission. Method: 110 patients admitted to the Emergency Department, Viet Duc University Hospital from May 2021 to July 2021 were diagnosed with polytrauma. All patients underwent routine coagulation testing and ROTEM parameters at admission. Result: the average age of patients is 41.4±14.7 years old, men accounts for 77.3%, average ISS score is 24.5±6.3. The proportion of the polytrauma patients with coagulopathy by routine coagulation testing was 50.9%. A significant correlation was found between routine coagulation parameters and ROTEM: between APTT with CFT-INTEM (r=0.65; p<0.01); between PT and CFT-EXTEM (r=0.64; p<0.01); between platelet count and MCF-INTEM (r=0.56; p=0.00) and MCF-EXTEM (r=0.57; p=0.00); between fibrinogen level and MCF-INTEM (r=0.71; p=0.00), MCF-EXTEM (r=0.71; p=0.00), and MCF-FIBTEM (r=0.91; p=0.00). Conclusion: proportion of the polytrauma patients with coagulopathy by routine coagulation testing was 50.9%. A significant correlation was found between routine coagulation parameters and ROTEM

Development of an Improved Whole Blood Assay for Diagnosis of Latent and Active Tuberculosis Cases

Background: Latent TB infection (LTBI) is an infection where the presence of disease causing organism M. tuberculosis is there without any sign and symptoms of the disease hence mostly remains undiagnosed, though Tuberculin skin test (TST) and Interferon Gamma Release Assay (IGRA) were used to diagnose the LTBI. They have their limitations, TST gives major cross-reactivity with BCG vaccine and gives inaccurate results in individuals who have taken BCG and IGRA are very costly and variable sensitivity is repeated in various populations hence the modifications are needed in the IGRA for proper diagnosis of LTBI. Objectives: In the proposed study we aimed to develop an improved whole blood assay towards a diagnosis of latent and active TB infection as an alternative to the Quantiferon QFT assay Methodology: Synthetic antigenic peptides against latency specific antigens will be designed and synthesized. Designed peptides will be screened for LTBI specific cytokine by in-vitro experiments. Development & production of Whole assay using selected peptides evaluation of developed assay through ELISA in clinical samples. Expected Results: Latent specific peptides will be identified and peptide-based whole blood assay for detection and diagnosis of tuberculosis will be developed as an indigenous alternative for the existing QFT assay. Conclusion: An improved whole blood assay will be developed for screening of LTBI in the Indian population.

Ex Vivo Antioxidant Capacities of Fruit and Vegetable Juices. Potential In Vivo Extrapolation

Background: In support of claims that their products have antioxidant properties, the food industry and dietary supplement manufacturers rely solely on the in vitro determination of the ORAC (oxygen radical antioxidant capacity) value, despite its acknowledged lack of any in vivo relevance. It thus appears necessary to use tests exploiting biological materials (blood, white blood cells) capable of producing physiological free radicals, in order to evaluate more adequately the antioxidant capacities of foods such as fruit and vegetable juices. Materials: Two approaches to assessing the antioxidant capacities of 21 commercial fruit and vegetable juices were compared: the ORAC assay and the “PMA–whole blood assay,” which uses whole blood stimulated by phorbol myristate acetate to produce the superoxide anion. We described in another paper the total polyphenol contents (TPCs) and individual phenolic compound contents of all the juices investigated here (Matute et al. Antioxidants 2020, 9, 1–18). Results: Ranking of the juices from highest to lowest antioxidant capacity differed considerably according to the test used, so there was no correlation (r = 0.33, p = 0.13) between the two assays when considering all juices. Although the results of the ORAC assay correlated positively with TPC (r = 0.50, p = 0.02), a much stronger correlation (r = 0.70, p = 0.004) emerged between TPC and % superoxide anion inhibition. In the PMA–whole blood assay, peonidin-3-O-glucoside, epigallocatechin gallate, catechin, and quercetin present in juices were found to inhibit superoxide anion production at concentrations below 1 µM, with a strong positive correlation. Conclusions: Associated with the determination of total and individual phenolic compounds contained in fruit and vegetable juices, the PMA–whole blood assay appears better than the ORAC assay for evaluating juice antioxidant capacity.

Stimulated Immune Response by TruCulture® Whole Blood Assay in Patients With European Lyme Neuroborreliosis: A Prospective Cohort Study

IntroductionBorrelia burgdorferi sensu lato complex (B. burgdorferi) can cause a variety of clinical manifestations including Lyme neuroborreliosis. Following the tick-borne transmission, B. burgdorferi initially evade immune responses, later symptomatic infection is associated with occurrence of specific antibody responses. We hypothesized that B. burgdorferi induce immune hyporesponsiveness or immune suppression and aimed to investigate patients with Lyme neuroborreliosis ability to respond to immune stimulation.MethodsAn observational cohort study investigating the stimulated immune response by standardized whole blood assay (TruCulture®) in adult patients with Lyme neuroborreliosis included at time of diagnosis from 01.09.2018-31.07.2020. Reference intervals were based on a 5-95% range of cytokine concentrations from healthy individuals (n = 32). Patients with Lyme neuroborreliosis and references were compared using Mann-Whitney U test. Heatmaps of cytokine responses were generated using the webtool Clustvis.ResultsIn total, 22 patients with Lyme neuroborreliosis (19 definite, 3 probable) were included. In the unstimulated samples, the concentrations of cytokines in patients with Lyme neuroborreliosis were comparable with references, except interferon (IFN)-α, interleukin (IL)-17A, IL-1β and IL-8, which were all significantly below the references. Patients with Lyme neuroborreliosis had similar concentrations of most cytokines in all stimulations compared with references. IFN-α, IFN-γ, IL-12 and IL-17A were lower than references in multiple stimulations.ConclusionIn this exploratory cohort study, we found lower or similar concentrations of circulating cytokines in blood from patients with Lyme neuroborreliosis at time of diagnosis compared with references. The stimulated cytokine release in blood from patients with Lyme neuroborreliosis was in general slightly lower than in the references. Specific patterns of low IL-12 and IFN-γ indicated low Th1-response and low concentrations of IL-17A did not support a strong Th17 response. Our results suggest that patients with Lyme neuroborreliosis elicit a slightly suppressed or impaired immune response for the investigated stimulations, however, whether the response normalizes remains unanswered.

A novel whole-blood stimulation assay to detect and quantify memory T-cells in COVID-19 patients

SARS-CoV-2 specific T-cells responses are essential for virus clearance. We present a novel and simple whole-blood assay allowing the detection of interferon-gamma-producing antiviral T-cells following peptide stimulation. We show that unlike neutralizing antibodies, antiviral memory T-cells persist at least 6 months in convalescent Covid-19 individuals.

Rapid, simplified whole blood-based multiparameter assay to quantify and phenotype SARS-CoV-2 specific T cells

ABSTRACTRapid tests to evaluate SARS-CoV-2-specific T cell responses are urgently needed to decipher protective immunity and aid monitoring vaccine-induced immunity. Using a rapid whole blood assay requiring minimal amount of blood, we measured qualitatively and quantitatively SARS-CoV-2-specific CD4 T cell responses in 31 healthcare workers, using flow cytometry. 100% of COVID-19 convalescent participants displayed a detectable SARS-CoV-2-specific CD4 T cell response. SARS-CoV-2-responding cells were also detected in 40.9% of participants with no COVID-19-associated symptoms or who tested PCR negative. Phenotypic assessment indicated that, in COVID-19 convalescent participants, SARS-CoV-2 CD4 responses displayed an early differentiated memory phenotype with limited capacity to produce IFNγ. Conversely, in participants with no reported symptoms, SARS-CoV-2 CD4 responses were enriched in late differentiated cells, co-expressing IFNγ and TNFα and also Granzyme B. This proof of concept study presents a scalable alternative to PBMC-based assays to enumerate and phenotype SARS-CoV-2-responding T cells, thus representing a practical tool to monitor adaptive immunity in vaccine trials.SummaryIn this proof of concept study, we show that SARS-CoV-2 T cell responses are easily detectable using a rapid whole blood assay requiring minimal blood volume. Such assay could represent a suitable tool to monitor adaptive immunity in vaccine trials.