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Traditional Chinese medicine (TCM) has significant benefits in the prevention and treatment of diseases due to its unique theoretical system and research techniques. However, there are still key issues to be resolved in the full interpretation and use of TCM, such as vague active compounds and mechanism of action. Therefore, it is promising to promote the research on TCM through innovative strategies and advanced cutting-edge technologies. Microfluidic chips have provided controllable unique platforms for biomedical applications in TCM research with flexible composition and large-scale integration. In this review, the analysis and biomedical applications of microfluidics in the field of TCM are highlighted, including quality control of Chinese herbal medicines (CHMs), delivery of CHMs, evaluation of pharmacological activity as well as disease diagnosis. Finally, potential challenges and prospects of existing microfluidic technologies in the inheritance and innovation of TCM are discussed.Copyright © 2022 Elsevier B.V.
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Background: Current COVID-19 vaccines provide substantial protection against severe COVID-19, but they do not completely eliminate subsequent SARS-CoV-2 infections. We examined incidence of and immune differences against related but different common cold coronaviruses (ccCoV) as a proxy for response against a future emerging CoV among those with SARS-CoV-2 infection, COVID-19 vaccination, or neither exposure. Method(s): We assessed incidence of ccCoV (229E, HKU1, NL63, OC43) and rhinovirus/enterovirus infections among those with documented prior SARSCoV- 2 infection (n=493), prior COVID-19 vaccine, but no SARS-CoV-2 infection (n=1,568), or no prior SARS-CoV-2 infection or vaccination (n=2,874). We conducted a retrospective review of all individuals at Boston Medical Center that underwent a comprehensive respiratory panel polymerase chain reaction (CRP-PCR) test from November 30, 2020 to October 8, 2021 to estimate infection incidence. A subset within each group was assessed for coronavirus specific humoral and cellular immune responses, via pseudovirus neutralization and peptide stimulation T cell assays. Comparisons among the three groups were done using Chi-square and multi-variate Cox-proportional hazards models. Result(s): Incidence of symptomatic ccCoV was lower in those individuals with documented prior SARS-CoV-2 infection (1.0%) compared to those with COVID-19 vaccination (2.9%) or no prior SARS-CoV-2 exposure (1.8%, p = 0.01). Rhinovirus/enterovirus incidence was similar in all three groups (range 6.2 - 8.7%). Individuals with prior SARS-CoV-2 infection and those with previous COVID-19 vaccination had similar plasma neutralization against SARS-CoV-2, OC43, and 229E spike bearing pseudoviruses. SARS-CoV-2 (p = 0.01) and OC43 nucleocapsid (p = 0.02), but not spike specific peptides, yielded higher T cell responses in individuals with a prior SARS-CoV-2 infection as compared to those with COVID-19 vaccination or no prior SARS-CoV-2 exposure. Conclusion(s): Prior SARS-CoV-2 infection, but not COVID-19 vaccination, protects against subsequent related but different ccCoV symptomatic infection. This protection against symptomatic ccCoVs may be mediated by cellular responses to non-spike proteins. Future pan-coronavirus vaccines could be improved by including both spike and non-spike antigens.
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T cells, and especially cytotoxic T cells are at the forefront of the fight against viral infection. The killer cells are able not only to distinguish between self and foreign peptides, but also to engage in the fight to clear the viral infection by eliminating the infected cells. Our lab is focused on understanding how T cells engage with viral peptide antigens, that are presented by highly polymorphic HLA molecules. T cells have receptors on their surface called T cell receptors (TCRs) that allow them to recognize the composite surface of the peptide- HLA complex. Using x-ray crystallography we can understand at the atomic level both peptide antigens presentation and TCR recognition, both important to determine the quality of the subsequent immune response. We can then link that structural information with our cellular assay that determines the strength and magnitude of the anti-viral response, providing the basis for peptide modification to reach stronger response or an understanding of viral mutation that led to viral escape. Our current work compared the T cell response, at the antigen level against 32 single epitope derived from spike, between COVID-19 recovered and vaccinated donors. We have shown that the booster shot (3rd dose) increases the antigen-specific T cell response, increases the level of T cell cross-reactivity against variant of SARS-CoV-2, but also alters the phenotype of the T cell. Those results are important to future guide vaccination advise and better understand the immune response to SARS-CoV-2 infection. POSTER PRESENTATIONS Autoimmunity, Infection, Reproduction and Cancer.
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Background. Benefit of COVID-19 vaccines is limited by need for freezing, high cost, requirement for multiple boosters, and waning immunity. An unmet need for safe and effective vaccines that can be quickly deployed worldwide during a pandemic exists. A barrier to developing scalable low-cost vaccines is the restricted access to vaccine- or adjuvant-formulations due to protection by intellectual property rights. This limits research to understand their mechanism of action and potential applicability towards vulnerable populations or emerging pathogens. Methods. We studied cellular and molecular mechanisms of action of adjuvant formulations developed for global open access in 4 distinct but complementary in vitro platforms that are human, age-specific, and enable the same individual to serve as control and test condition;generating data on adjuvant-induced responses in vitro that predict activity in vivo. Results. Whole blood assay that models magnitude of innate immune activation and identifies cell types activated showed that liposomal co-formulation of MPL +QS-21 activated monocytes and natural killer cells and induced cytokine production;tissue construct assay that models monocyte extravasation and autonomous differentiation into dendritic cells (DC) showed that only MPL+QS-21 co-formulation promoted CD14+ cells towards DC phenotype;monocyte-derived DC (MoDC) assay that interrogates immune activation type showed that MPL+QS-21 co-formulation in lipid nanoparticles promoted MoDC maturation by increasing CD40, CD86, CCR7 and HLA-DR expression and Th1-polarizing cytokine secretion;and dendritic cell-T cell interface assay that assesses potential of formulations to re-activate SARS-CoV-2 Spike antigen-specific T cells showed that only MPL+QS-21 coformulation enhanced activation of antigen-specific CD4+ and CD8+ T cells. Conclusion. Thus, human in vitro modeling provides new insight into the mechanism of action and synergistic effects of MPL+QS-21, and positions us to study them in vulnerable populations to assess potential age-specific application on vaccine development. Precision vaccinology coupled with global access may enable marked public health progress by accelerating, de-risking, and advancing affordable adjuvanted vaccines to the most vulnerable.
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Introduction: SARS-CoV-2 immune-response is mediated by both humoral and cellular immunity. However, since Ab levels wane faster than SARS-CoV-2 specific T cells immunity, cellular immunity represents an important factor for COVID-19 immune defence. Determining immunoreactivity of SARS-CoV-2 specific T cells is of clinical relevance in transplant recipients or patients treated with immunomodulant therapy. SARS-CoV-2 specific T cells assays are currently based on ELISA, whilst rapid tests are pivotal for real-time patients' evaluation. In this study, a novel direct real-time PCR (dRT-PCR) targeting mRNA of CXCL10 for measuring SARS-CoV-2 specific T cells, was tested and evaluated. Method(s): A total of 104 healthcare workers, with two or three doses of homologous (Pfizer/BioNTech, n = 82) or heterologous (Pfizer/BioNTech and Vaxzevria or Moderna, n = 22) vaccinations were asked to collect a blood (Li-He) sample. Blood was stimulated overnight with SARS-CoV-2 spike peptides (S-peptide) or treated with non-stimulating substance. Stimulated/treated samples were diluted in Buffer A, mixed with dqTACT MS then loaded into the cartridge. The analysis was performed using SCV2 T Activation kit, bCube and bApp (Hyris srl, Lodi, Italy), equipped by an automatic result interpretation based on artificial intelligence. For a subgroup of 49 samples, IFN-y releases to SARS-CoV-2 spike peptides were tested by Quant-T-Cell SARS-CoV-2 and ELISA (Euroimmune, Lubeck, Germany). Result(s): Seventy-nine (75.9%) and 25 (24.1%) were females and males, respectively. Twenty-nine subjects were previously infected by SARS-CoV-2. Overall mean age (+/- SD) was 45.9+/-13.3 years. At qualitative analyses, 97 subjects (93.2%) resulted reactive to S-peptides, 3 (2.8%) were borderline and 4 were negative (3.8%). These negatives had their third vaccinal dose in December/November 2021. Previous infected individuals presented reactivity to S-peptides, with the exception of one subject with resulted reactive also in the untreated sample. Samples tested with both dRT-PCR and ELISA perfectly agreed (100%) with both methods. At quantitative analyses, between-assay correlation was 0.32 (p<0.001). Conclusion(s): Hyris dRT-PCR appeared accurate for determining presence or absence of immunoreactivity of SARS-CoV-2 specific T cell, especially when rapid analyses are required, such as for organ transplantation.
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Early in the COVID-19 pandemic, it was recognized that infection with SARS-CoV-2 is associated with increased morbidity and mortality in patients with cancer; therefore, preventive vaccination in cancer survivors is expected to be particularly impactful. Heterogeneity in how a neoplastic disease diagnosis and treatment interferes with humoral and cellular immunity, however, poses a number of challenges in vaccination strategies. Herein, the available literature on the effectiveness of COVID-19 vaccines among patients with cancer is critically appraised under the lens of anti-neoplastic treatment optimization. The objective of this review is to highlight areas of uncertainty, where more research could inform future SARS-CoV-2 immunization programs and maximize benefits in the high-risk cancer survivor population, and also minimize cancer treatment deviations from standard practices.
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Background: Patients (pts) with indolent lymphomas are at increased risk of severe COVID-19 infection. We have shown limited seroconversion and live viral neutralisation (VN), but preserved COVID-specific T cell responses after 2 doses of mRNA COVID-19 vaccination in such pts. (Beaton, B ASH 2021, 149348). A 3rd vaccine dose to complete primary vaccination has since been recommended. Aims: To assess humoral & cellular immune responses to a 3rd COVID-19 (mRNA) vaccination in pts with follicular lymphoma (FL) & Waldenström Macroglobulinemia (WM), including assessment of response after pausing BTKi therapy in WM pts. Methods: Patients with WM, FL & healthy controls (HC) were enrolled in a prospective observational study to measure immune responses 21-28 days after a 3rd mRNA COVID-19 vaccine. Immune response was measured by mean fluorescence intensity (MFI) of anti-SARS-CoV-2 spike antibodies (ASAb) obtained using a high-sensitivity live cell assay, live VN to a panel of SARS-CoV-2 variants of concern, and CD4+ & CD8+ antigen-specific T cell responses. The associated TRIBECA (TReatment Interruption of BTKi to Enhance COVID-19 Antibody response) study sought to determine if a superior immune response could be gained by pause of BTKi therapy prior to and up to 4 weeks after 3rd vaccine dose. Patients were closely monitored during the BTKi pause with weekly clinical, full blood count and IgM assessments. WM pts receiving a 3rd dose while continuing on BTKi served as a control. Statistical analysis of medians between cohorts were compared by the non-parametric Mann-Whitney (Graphpad Prism). Comparison of medians between paired grouped data was assessed by 2-way ANOVA. Results: To date, 56 of 125 pts had their ASAb measured following 3rd vaccine dose administered between October 2021 and January 2022: 28 WM pts (including 6/9 WM pts on the BTKi pause sub-study), 24 FL pts and 4 HC. Median age was 68 years with 21 females and 35 males. Median follow up from 2nd dose was 140 days (range: 79- 170 days). In antibody responders, median MFI fell from 163042 (IQR 82663-249934) 28 days post 2nd dose to 52117 (IQR 19942-60973) (p<0.0001) immediately before the 3rd dose. Median MFI in all FL & WM pts pre- 3rd dose vaccine was 17111 (IQR 0-52650), rising significantly post 3rd dose to a median of 86730 (IQR 0-221937). Only 4/20 pts without measurable ASAb prior to the 3rd dose (2 WM, 2 FL) developed measurable ASAb following the 3rd dose: 16/20 patients (8 WM, 8FL, all treated) had no ASAb response. The median MFI in pts who underwent a BTKi treatment pause rose from 9151 (IQR 1671-21232) pre-3rd dose to 87720 (IQR 2785-152195) post 3rd dose, significantly higher than the median MFI in WM pts who did not pause their BTKi , which rose from 16769 (IQR 218-22447) pre- to 20252 (IQR 168-114262) post 3rd dose, (p = 0.016). Of the 5/125 with COVID infection in the study to date, only one patient (without measurable ASAb) in this 3-dose cohort had COVID, requiring intensive care support. Summary/Conclusion: Most WM & FL pts who responded to a 2nd dose COVID vaccine showed a decline in ASAb titre over time which increased following a 3rd mRNA vaccine. Only 20% of pts without detectable ASAb pre- 3rd dose showed improvement post 3rd dose, highlighting the importance of other COVID protection strategies in these pts. Although initial numbers are small, there may be a higher increment in ASAb when BTKi therapy is paused around the time of vaccination. Comprehensive immune analysis, including VN and T-cell response on the entire FL & WM cohort will be presented at the EHA congress.
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BACKGROUND: Immune-modulating medications for inflammatory bowel diseases (IBD) have been associated with suboptimal vaccine responses. There is conflicting data with SARS-CoV-2 vaccination. METHODS: We measured SARS-CoV-2 vaccine immunogenicity at 2 weeks post 2nd mRNA vaccine in IBD patients as compared to normal healthy donors (NHD). We measured humoral immune responses to SARS-CoV-2: anti-spike Immunoglobulin G (IgG) and anti-receptor binding domain (RBD) IgG were measured by ELISA, and neutralizing antibody titers were measured using recombinant, reporter SARS-CoV-2. Antigen specific memory B cells were measured using recombinant SARS-CoV-2 proteins. Activation induced marker T cell (AIM) assays were performed using SARS-CoV-2 spike megapools. Immunophenotyping was performed by flow cytometry. RESULTS: We enrolled 29 patients with IBD (19 with Crohn's disease, 10 with ulcerative colitis) on infliximab (IFX) monotherapy (N=9), IFX combination therapy with a thiopurine (N=9), vedolizumab monotherapy (N= 11) as compared to matched NHD (N=12). At 2 weeks post vaccination, all subjects made detectable anti-spike IgG and anti-RBD IgG. There were no differences in anti-spike IgG titers among the different groups. IBD patients on IFX monotherapy, but not IBD patients on IFX combination therapy or vedolizumab monotherapy, had lower anti-RBD and neutralization titers as compared to NHD (p-value: 0.041 and 0.023, respectively) (Fig. 1). There were no significant differences in the percentage of spike-specific or RBD-specific memory B cells in IBD patients as compared to NHD (Fig. 1). There were no differences in the percentage of spike-specific CD4+ or CD8+ T cells in all IBD patients as compared to NHDs (Fig. 2). CONCLUSIONS: We demonstrate overall comparable and perserved cell-mediated immunity to SARS-CoV-2 vaccination in a small cohort of IBD patients treated with a range of different immune-modulating medications as compared to healthy controls. Larger numbers of patients are needed to validate these findings.
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Background: Knowledge about SARS-CoV2 infection in pregnancy and exposed newborns is deficient. We performed a longitudinal analysis of innate immune system status and determined soluble cytokines of women infected with SARS-CoV2 during pregnancy and their newborns Methods: Women with confirmed SARS-CoV2 infection (RT-PCR+ or SARS-CoV2 anti-IgM/IgG+) (COVID MOTHER group, CM n=29, median age of 31 years) and their SARS-CoV2 exposed uninfected newborns were recruited from Hospital Gregorio Marañón, Spain. Peripheral blood mononuclear cells (PBMCs), cord cells and plasma were collected at birth and 6 months later (n=15). The immunophenotyping of innate components (natural killer cells [NK] and monocytes) was studied on cryopreserved PBMCs and cord cells by multiparametric flow cytometry. Up to 4 soluble pro/anti-inflammatory cytokines were assessed in plasma and cord plasma by ELISA assay. CM was compared to a healthy non-SARS-CoV2 infected mothers' group matched by age (SARS-CoV2 PCR-and SARS-CoV2 anti-IgM/IgG-)(Uninfected Mothers, UM n=16) and their newborns (n=16) Results: On NK cell assays, CM show at baseline lower percentage of CD16++ subset, higher NKG2D and lower NKG2A expression on CD16++ and CD56++ subsets and reduced CD57 expression compared to UM;proportion of CD16++ subset and percentage of NKG2D reverted after 6 months(A). Regarding monocytes, CM show increased levels of CD62L and decreased CD49d expression on classical subset, elevated intermediate monocytes proportion and decreased CD40 expression on patrolling subset(B). No differences were found 6 months later. No newborn was infected by SARS-CoV2 and the phenotype analyzed on cord cells shows lower frequency of NK subsets compared with unexposed children and increased CD16++ subset after 6 months(C). In monocytes distribution, exposed children present lower frequency of total monocytes and its subsets than unexposed. Classical monocytes show significant changes at follow-up time-point(D). Increased TNFα and IL10 levels were found on CM compared to UM. Strong and direct correlations were observed between the age and IL6(E). No differences were observed in soluble cytokine levels comparing both groups of newborns Conclusion: SARS-CoV2 infection during pregnancy shows differences in activation, maturation and endothelial markers on innate immune system that could lead newborns clinical implications at birth. However, altered cell proportions and phenotypes found at SARS-CoV2 at birth time and on their exposed newborns is later reverted.
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Introduction Lymphoid malignancies are a risk factor for severe COVID-19. Vaccination with BNT162b2 protects the general population from severe disease, but recent studies have shown limited seroconversion after vaccination in patients with lymphoid malignancy. This reduced response is likely related to disease and treatment factors altering both humoral and cellular immunity. Assessing response in patients with the indolent lymphomas, Waldenström's Macroglobulinaemia (WM) and Follicular Lymphoma (FL), including cohorts on differing treatment regimens, may help elucidate some of these factors. Australia has had low prevalence of SARS-CoV-2 infection to date, affording a unique opportunity to assess efficacy to vaccination without the confounding impact of endemic infection. Methods Patients with WM and FL and controls were enrolled in a prospective study of immune response after two doses of BNT162b2 administered 21 days apart. The study had Human Research Ethics Committee approval and all patients gave informed consent prior to participation. Recruitment was targeted to obtain comparable proportions of controls to treatment cohorts. PBMC and sera were collected from participants immediately prior to the first dose (T1), at day 21 immediately prior to the second dose (T2), and day 49 (+/-7d) (T3). Immune response was measured by: flow cytometric detection of anti-SARS-CoV-2 spike antibodies (ASAb), performed using our recently validated flow cytometric live cell assay (Tea et.al. PLoS Medicine 2021) with increased sensitivity compared to currently available commercial ELISAs;live virus neutralisation to a panel of SARS-CoV-2 variants of concern;and CD4+and CD8+ antigenspecific T cell responses. Statistical analysis of medians between cohorts were compared by the Mann-Whitney non-parametric test using Graphpad Prism. Initial ASAb IgG data for T1 and T2 is presented here. Complete immune response data at all time points will be available for the ASH meeting. Results Eighty-five participants received their first dose of BNT162b2 from 18 May 2021 to 7 June 2021 with a second dose 21 days later: 72 lymphoma patients (WM and FL) and 13 age-matched healthy volunteers (controls). Of 37 with WM [19 (51.3%) female, median 71 years (IQR 63-74)] 9 were treatment naïve (WMN), 15 had received rituximab-chemotherapy (WMT), and 13 were currently treated with a Bruton Tyrosine Kinase inhibitor (BTKi): 5 ibrutinib, 8 zanubrutinib (WMB). Of 35 patients with FL [16 (45.7%) female, median 65 years (IQR 54-71)], 11 were treatment naïve (FLN), and 24 had received rituximab-chemotherapy (FLT). Of the 13 controls 8 (61.5%) were female, median age 72 years (IQR 57-74)]. No participants had detectable ASAb at T1, confirming no prior SARS-CoV-2 exposure. Figure 1 shows ASAb results at T2. The median mean fluorescence intensity (MFI) of healthy controls: 60802 (IQR 17565 -78443), is higher than all WM: 0 (IQR 0-15010) p<0.0001, and all FL patients: 1687 (IQR 0-25421) p=0.002, Fig 1A. The median MFI of controls was higher than WMN (p=0.036), but not higher than FLN (p=0.28). The median MFI of WMN: 20074 (IQR 5421-35695), is higher than WMB: 0 (IQR 0-4217) p=0.018, but not significantly higher than WMT: 0 (IQR 0-14356) p=0.13, Fig 1B. Median MFI of FLN: 31476 (IQR 19351-51317), is higher than FLT: 0 (IQR 0-32849) p=0.01, Fig 1B. Conclusion These early serological data show measurable ASAb in all healthy controls 21 days post first dose of BNT162b2 vaccination. Treatment naïve patients had a better response than treated patients, and this did not differ significantly to healthy controls in the FL cohort. In WM, patients on BTKi had a significantly reduced response compared to treatment-naïve patients. This same reduction was not observed in the chemotherapy-rituximab cohort, but the characteristics of early responders versus non-responders, including time since last therapy, is being analysed. FL patients treated with chemotherapy-rituximab had a significantly reduced response compared to the treatmentnaïve cohort. Time constraints before the submission deadline prevented reporting of all mature vaccination response data. Measurement of ASAb one month after second vaccination, live virus neutralisation to a panel of SARS-CoV-2 variants of concern, and CD4+ and CD8+ antigen-specific T cell responses at T1, T2 and T3, to fully characterise the immune response to BNT162b2, will be reported at the ASH meeting.
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Background. Our understanding of the SARS-CoV-2 immune response has critical gaps that are inadequately addressed with available tools. We report the clinical performance of T-Detect COVID, the first T-cell assay to identify prior SARS-CoV-2 infection using T-cell receptor (TCR) sequencing and repertoire profiling from whole blood samples. Methods. The T-Detect COVID assay combines high-throughput immunosequencing of the TCRß gene from blood samples with a statistical classifier demonstrating 99.8% specificity for identifying prior SARS-CoV-2 infection. The assay was employed in several retrospective and prospective cohorts to assess primary and secondary Positive Percent Agreement (PPA) with SARS-CoV-2 RT-PCR (N=205;N=77);primary and secondary Negative Percent Agreement (NPA;N=87;N=79);PPA compared to SARS-CoV-2 serology (N=55);and pathogen cross-reactivity (N=38). The real-world performance of the test was also evaluated in a retrospective review of test ordering (N=69) at a single primary care clinic in Park City, Utah. Results. In validation studies, T-Detect COVID demonstrated high PPA (97.1% ≥15 days from diagnosis) in subjects with prior PCR-confirmed SARSCoV-2 infection;high NPA (~100%) in SARS-CoV-2 negative cases;equivalent or higher PPA with RT-PCR compared to two commercial EUA antibody tests;and no evidence of pathogen cross-reactivity. Review of assay use in a single clinic showed 100% PPA with RT-PCR in individuals with past confirmed SARS-CoV-2 vs. 85.7% for antibody testing, 100% agreement with positive antibody results, and positive results in 2/4 convalescent subjects with seroreversion to a negative antibody. In addition, 12/69 (17.3%) individuals with absent or negative RT-PCR tested positive by T-Detect COVID, nearly all of whom had compatible symptoms and/or exposure. TCR positivity was observed up to 12+ months (median 118 days) from the date of positive RT-PCR. Conclusion. A T-cell immunosequencing assay shows high clinical performance for identifying past SARS-CoV-2 infection from whole blood samples. This assay can provide additional insights on the SARS-CoV-2 immune response, with practical implications for clinical management, risk stratification, surveillance, assessing vaccine immunity, and understanding long-term sequelae.
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[Formula presented] Background: Patients with B-cell lymphoma have poor clinical outcomes to SARS-CoV-2 infection (COVID-19) and are also more likely to have suboptimal responses to immunization. An understanding of which lymphoma patients are at greatest risk of poor COVID-19 vaccine response and what aspects of immunity are most impaired is critical for developing strategies to protect these patients from a potentially fatal infection. Methods: We enrolled 149 participants, including 129 with lymphoma and 20 age-matched controls, who received a complete COVID-19 vaccination series to assess how B- and T-cell vaccine responses vary among clinically relevant subgroups and over time. This cohort included 99 patients with prior anti-CD20 treatment, ranging from 1 week to 17 years prior, allowing us to assess relationships between timing and intensity of anti-CD20 exposure and vaccine response. The cohort also included 18 patients who began treatment with an anti-CD20-containing regimen after being fully vaccinated, in whom we are assessing the potential efficacy of a pre-therapy vaccination strategy. Peripheral blood samples were taken on average 28 days and 4 months after last vaccine dose. B-cell responses are being profiled by measuring anti-Spike serum IgG, RBD-ACE2 blocking activity, and spike-specific memory B cells. T cell assessments include quantitation of spike-specific activation and cytokine production via interferon-gamma ELISPOTs and multiparameter flow cytometry. Results: The 129 participants with lymphoma had a median age of 68, were 59% male, and 70% had either diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma (FL). Thirteen percent had no therapy prior to vaccination, half had not been treated in the past six months and 36% had been treated recently or were currently being treated. Ninety-three percent received an mRNA COVID-19 vaccine. Although we did not detect a statistically significant difference in vaccine response between previously untreated lymphoma patients and controls, 3/17 were negative for blocking antibodies post-vaccine, compared to 0/20 controls (Fishers exact test p=0.09, Figure 1A). Lymphoma patients with any history of treatment had impaired serologic responses to the vaccine compared to previously untreated patients (p=0.01). Responses did not vary by specific lymphoma histology. Blocking antibodies were evident in only 45% and 3% of patients recently/currently treated with a BTK inhibitor or an anti-CD20 antibody, respectively (Figure 1A). Closely evaluating all patients who had ever received anti-CD20 antibody therapy, we found a strong linear correlation between time since last anti-CD20 treatment and RBD-ACE2 binding inhibition (p<0.0001, Figure 1B-C). T cell and memory B cell assays are ongoing as is analysis of response persistence and of the pre-therapy vaccination cohort, and results from these efforts will be included in the final presentation. Conclusions: Treatment with anti-CD20 antibodies significantly impaired COVID-19 vaccine-induced humoral responses in patients with lymphoma in a manner dependent on the time elapsed since last anti-CD20 treatment. Vaccination at least six months after anti-CD20 treatment, likely co-incident with recovery of the B-cell compartment, was associated with positive blocking antibody titers. These data suggest that booster vaccination strategies are more likely to succeed in lymphoma patients who have not received anti-CD20 treatment for at least 6 months and that those patients with recent anti-CD20 treatment may benefit most from passive immunization strategies. Forthcoming results from the pre-therapy vaccination cohort will also help inform sequencing of additional vaccine doses, passive immunization, and anti-cancer treatments. [Formula presented] Disclosures: Shree: Gilead: Other: Spouse's employment. Beygi: Kite/Gilead: Current Employment. Advani: Astellas/Agensys: Research Funding;AstraZeneca: Membership on an entity's Board of Directors or advisory committees;Bayer: Membership on an entity's Board of Directors or dvisory committees;Bristol Myer Squibb: Membership on an entity's Board of Directors or advisory committees;Cell Medica: Membership on an entity's Board of Directors or advisory committees;Forty Seven: Membership on an entity's Board of Directors or advisory committees, Research Funding;Genetech Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding;Gilead: Membership on an entity's Board of Directors or advisory committees;Janssen Pharmaceutical: Research Funding;Juno: Membership on an entity's Board of Directors or advisory committees;Kite Pharma: Membership on an entity's Board of Directors or advisory committees;Kura: Research Funding;Kyowa: Membership on an entity's Board of Directors or advisory committees;Merck: Research Funding;Millenium: Research Funding;Pharmacyclics: Consultancy, Research Funding;Portola Pharmaceuticals: Consultancy;Regeneron: Research Funding;Roche: Membership on an entity's Board of Directors or advisory committees;Sanofi: Membership on an entity's Board of Directors or advisory committees;Seattle Genetics: Research Funding;Takeda: Membership on an entity's Board of Directors or advisory committees. Khodadoust: CRISPR Therapeutics, Nutcracker Therapeutics: Research Funding;Myeloid Therapeutics: Membership on an entity's Board of Directors or advisory committees;Alexion, AstraZeneca Rare Disease: Other: Study investigator. Kurtz: Roche: Consultancy;Foresight Diagnostics: Consultancy, Current holder of stock options in a privately-held company;Genentech: Consultancy. Alizadeh: Bristol Myers Squibb: Research Funding;Gilead: Consultancy;Celgene: Consultancy, Research Funding;Janssen Oncology: Honoraria;Roche: Consultancy, Honoraria;Foresight Diagnostics: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company;Forty Seven: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company;CAPP Medical: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company;Cibermed: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Levy: GigaGen: Membership on an entity's Board of Directors or advisory committees;Teneobio: Membership on an entity's Board of Directors or advisory committees;Nurix: Membership on an entity's Board of Directors or advisory committees;Dragonfly: Membership on an entity's Board of Directors or advisory committees;Apexigen: Membership on an entity's Board of Directors or advisory committees;Viracta: Membership on an entity's Board of Directors or advisory committees;Spotlight: Membership on an entity's Board of Directors or advisory committees;Immunocore: Membership on an entity's Board of Directors or advisory committees;Walking Fish: Membership on an entity's Board of Directors or advisory committees;Kira: Membership on an entity's Board of Directors or advisory committees;Abintus Bio: Membership on an entity's Board of Directors or advisory committees;Khloris: Membership on an entity's Board of Directors or advisory committees;Virsti: Membership on an entity's Board of Directors or advisory committees;BiolineRx: Membership on an entity's Board of Directors or advisory committees;BeiGene: Membership on an entity's Board of Directors or advisory committees;Quadriga: Membership on an entity's Board of Directors or advisory committees.
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It is not exactly clear yet which type of immune response prevails to accomplish viral clearance in coronavirus disease 2019 (COVID-19). Studying a patient with chronic lymphocytic leukemia and hypogammaglobulinemia who suffered from COVID-19 provided insight in the immunological responses after treatment with COVID-19 convalescent plasma (CCP). Treatment consisted of oxygen, repeated glucocorticosteroids and multiple dosages of CCP guided by antibody levels. Retrospectively performed humoral and cellular immunity analysis made clear that not every serological test for COVID-19 is appropriate for follow-up of sufficient neutralizing antibodies after CCP. In retrospect, we think that CCP merely bought time for this patient to develop an adequate cellular immune response which led to viral clearance and ultimately clinical recovery.
Subject(s)
COVID-19 , Clinical Laboratory Techniques , Humans , Laboratories , SARS-CoV-2 , Sensitivity and Specificity , T-LymphocytesABSTRACT
Vaccination is the main public health measure to reduce SARS-CoV-2 transmission and hospitalization, and a massive worldwide scientific effort resulted in the rapid development of effective vaccines. This work aimed to define the dynamics of humoral and cell-mediated immune response in a cohort of health care workers (HCWs) who received a two-dose BNT162b2-mRNA vaccination. The serological response was evaluated by quantifying the anti-RBD and neutralizing antibodies. The cell-mediated response was performed by a whole blood test quantifying Th1 cytokines (IFN-γ, TNF-α, IL-2), produced in response to spike peptides. The BNT162b2-mRNA vaccine induced both humoral and cell-mediated immune responses against spike peptides in virtually all HCWs without previous SARS-CoV-2 infection, with a moderate inverse relation with age in the anti-RBD response. Spike-specific T cells produced several Th1 cytokines (IFN-γ, TNF-α, and IL-2), which correlated with the specific-serological response. Overall, our study describes the ability of the BNT162b2 mRNA vaccine to elicit a coordinated neutralizing humoral and spike-specific T cell response in HCWs. Assessing the dynamics of these parameters by an easy immune monitoring protocol can allow for the evaluation of the persistence of the vaccine response in order to define the optimal vaccination strategy.
ABSTRACT
BACKGROUND: The overall clinical outcome of inflammatory conditions is the result of the balance between pro-inflammatory and anti-inflammatory mediators. Because nuclear factor kappa B (NF-ĸB) is at the bottom of many inflammatory conditions, methods to evaluate the net effect of inflammation modulators on this master regulator have been conceptualized for years. METHODS: Using an ex vivo NF-ĸB reporter cell line-based assay, plasma samples of patients with rheumatoid arthritis (n = 27), psoriasis (n = 15), or severe coronavirus disease-19 (COVID-19) (n = 21) were investigated for NF-ĸB activation compared to plasma samples from 9 healthy volunteers. RESULTS: When separated by C-reactive protein (CRP) threshold levels, samples of patients exhibiting increased CRP levels (≥5 mg/l) activated NF-ĸB more efficiently than samples from patients with levels below 5 mg/l (P = 0.0001) or healthy controls (P = 0.04). Overall, there was a moderate association of CRP levels with NF-ĸB activation (Spearman r = 0.66; p < 0.0001). Plasma from COVID-19 patients activated NF-ĸB more efficiently (mean 2.4-fold compared to untreated reporter cells) than samples from any other condition (healthy controls, 1.8-fold, P = 0.0025; rheumatoid arthritis, 1.7-fold, P < 0.0001; psoriasis, 1.7-fold, P < 0.0001). In contrast, effects of rheumatoid arthritis, psoriasis, or healthy volunteer samples did not differ. CONCLUSION: This study shows that a NF-ĸB reporter cell line can be used to evaluate the net inflammatory effect of clinical plasma samples. Patients with chronic but stable rheumatoid arthritis or psoriasis do not exhibit increased plasma levels of NF-ĸB-activating compounds as opposed to COVID-19 patients with high inflammatory burden.
Subject(s)
Arthritis, Rheumatoid/pathology , COVID-19/pathology , NF-kappa B/blood , NF-kappa B/metabolism , Psoriasis/pathology , Arthritis, Rheumatoid/blood , C-Reactive Protein/analysis , Cell Line , Enzyme Activation/physiology , Female , HEK293 Cells , Humans , Inflammation/pathology , Inflammation Mediators/metabolism , Male , Middle Aged , Psoriasis/blood , SARS-CoV-2/immunologyABSTRACT
BACKGROUND: The ongoing COVID-19 pandemic has created an urgency to identify novel vaccine targets for protective immunity against SARS-CoV-2. Early reports identify protective roles for both humoral and cell-mediated immunity for SARS-CoV-2. METHODS: We leveraged our bioinformatics binding prediction tools for human leukocyte antigen (HLA)-I and HLA-II alleles that were developed using mass spectrometry-based profiling of individual HLA-I and HLA-II alleles to predict peptide binding to diverse allele sets. We applied these binding predictors to viral genomes from the Coronaviridae family and specifically focused on T cell epitopes from SARS-CoV-2 proteins. We assayed a subset of these epitopes in a T cell induction assay for their ability to elicit CD8+ T cell responses. RESULTS: We first validated HLA-I and HLA-II predictions on Coronaviridae family epitopes deposited in the Virus Pathogen Database and Analysis Resource (ViPR) database. We then utilized our HLA-I and HLA-II predictors to identify 11,897 HLA-I and 8046 HLA-II candidate peptides which were highly ranked for binding across 13 open reading frames (ORFs) of SARS-CoV-2. These peptides are predicted to provide over 99% allele coverage for the US, European, and Asian populations. From our SARS-CoV-2-predicted peptide-HLA-I allele pairs, 374 pairs identically matched what was previously reported in the ViPR database, originating from other coronaviruses with identical sequences. Of these pairs, 333 (89%) had a positive HLA binding assay result, reinforcing the validity of our predictions. We then demonstrated that a subset of these highly predicted epitopes were immunogenic based on their recognition by specific CD8+ T cells in healthy human donor peripheral blood mononuclear cells (PBMCs). Finally, we characterized the expression of SARS-CoV-2 proteins in virally infected cells to prioritize those which could be potential targets for T cell immunity. CONCLUSIONS: Using our bioinformatics platform, we identify multiple putative epitopes that are potential targets for CD4+ and CD8+ T cells, whose HLA binding properties cover nearly the entire population. We also confirm that our binding predictors can predict epitopes eliciting CD8+ T cell responses from multiple SARS-CoV-2 proteins. Protein expression and population HLA allele coverage, combined with the ability to identify T cell epitopes, should be considered in SARS-CoV-2 vaccine design strategies and immune monitoring.