Neutralization, effector function and immune imprinting of Omicron variants.

Currently circulating SARS-CoV-2 variants have acquired convergent mutations at hot spots in the receptor-binding domain1 (RBD) of the spike protein. The effects of these mutations on viral infection and transmission and the efficacy of vaccines and therapies remains poorly understood. Here we demonstrate that recently emerged BQ.1.1 and XBB.1.5 variants bind host ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1, XBB.1 and BN.1 RBDs bound to the fragment antigen-binding region of the S309 antibody (the parent antibody for sotrovimab) and human ACE2 explain the preservation of antibody binding through conformational selection, altered ACE2 recognition and immune evasion. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1 and hamsters challenged with XBB.1.5. Vaccine-elicited human plasma antibodies cross-react with and trigger effector functions against current Omicron variants, despite a reduced neutralizing activity, suggesting a mechanism of protection against disease, exemplified by S309. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring the role of persistent immune imprinting.

Autoantibodies against chemokines post-SARS-CoV-2 infection correlate with disease course.

Infection with severe acute respiratory syndrome coronavirus 2 associates with diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse coronavirus disease 2019 (COVID-19) outcomes. Here we discovered that antibodies against specific chemokines were omnipresent post-COVID-19, were associated with favorable disease outcome and negatively correlated with the development of long COVID at 1 yr post-infection. Chemokine antibodies were also present in HIV-1 infection and autoimmune disorders, but they targeted different chemokines compared with COVID-19. Monoclonal antibodies derived from COVID-19 convalescents that bound to the chemokine N-loop impaired cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising chemokine antibodies may modulate the inflammatory response and thus bear therapeutic potential.

Long-Term Evolution of Activities of Daily Life (ADLs) in Critically Ill COVID-19 Patients, a Case Series.

BACKGROUND: The most common long-term symptoms of critically ill COVID-19 patients are fatigue, dyspnea and mental confusion. Adequate monitoring of long-term morbidity, mainly analyzing the activities of daily life (ADLs), allows better patient management after hospital discharge. The aim was to report long-term ADL evolution in critically ill COVID-19 patients admitted to a COVID-19 center in Lugano (Switzerland). METHODS: A retrospective analysis on consecutive patients discharged alive from ICU with COVID-19 ARDS was performed based on a follow-up one year after hospital discharge; ADLs were assessed through the Barthel index (BI) and the Karnofsky Performance Status (KPS) scale. The primary objective was to assess differences in ADLs at hospital discharge (acute ADLs) and one-year follow-up (chronic ADLs). The secondary objective was to explore any correlations between ADLs and multiple measures at admission and during the ICU stay. RESULTS: A total of 38 consecutive patients were admitted to the ICU; a t-test analysis between acute and chronic ADLs through BI showed a significant improvement at one year post discharge (t = -5.211, p < 0.0001); similarly, every single task of BI showed the same results (p < 0.0001 for each task of BI). The mean KPS was 86.47 (SD 20.9) at hospital discharge and 99.6 at 1 year post discharge (p = 0.02). Thirteen (34%) patients deceased during the first 28 days in the ICU; no patient died after hospital discharge. CONCLUSIONS: Based on BI and KPS, patients reached complete functional recovery of ADLs one year after critical COVID-19.

Therapeutic and vaccine-induced cross-reactive antibodies with effector function against emerging Omicron variants.

Currently circulating SARS-CoV-2 variants acquired convergent mutations at receptor-binding domain (RBD) hot spots. Their impact on viral infection, transmission, and efficacy of vaccines and therapeutics remains poorly understood. Here, we demonstrate that recently emerged BQ.1.1. and XBB.1 variants bind ACE2 with high affinity and promote membrane fusion more efficiently than earlier Omicron variants. Structures of the BQ.1.1 and XBB.1 RBDs bound to human ACE2 and S309 Fab (sotrovimab parent) explain the altered ACE2 recognition and preserved antibody binding through conformational selection. We show that sotrovimab binds avidly to all Omicron variants, promotes Fc-dependent effector functions and protects mice challenged with BQ.1.1, the variant displaying the greatest loss of neutralization. Moreover, in several donors vaccine-elicited plasma antibodies cross-react with and trigger effector functions against Omicron variants despite reduced neutralizing activity. Cross-reactive RBD-directed human memory B cells remained dominant even after two exposures to Omicron spikes, underscoring persistent immune imprinting. Our findings suggest that this previously overlooked class of cross-reactive antibodies, exemplified by S309, may contribute to protection against disease caused by emerging variants through elicitation of effector functions.

Human neutralizing antibodies to cold linear epitopes and subdomain 1 of the SARS-CoV-2 spike glycoprotein.

Emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike glycoprotein that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization, and, similar to fp.006 and hr2.016, protects mice expressing human angiotensin-converting enzyme 2 against infection when present as a bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants.

Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape.

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity, and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month time frame. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both prefusion and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sublineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.

Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2.

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera , including the nine human coronaviruses, through recognition of a conserved motif that includes the S2' site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae , including SARS-CoV-2 variants. One sentence summary: Broadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery.

Maturation of SARS-CoV-2 Spike-specific memory B cells drives resilience to viral escape.

Memory B cells (MBCs) generate rapid antibody responses upon secondary encounter with a pathogen. Here, we investigated the kinetics, avidity and cross-reactivity of serum antibodies and MBCs in 155 SARS-CoV-2 infected and vaccinated individuals over a 16-month timeframe. SARS-CoV-2-specific MBCs and serum antibodies reached steady-state titers with comparable kinetics in infected and vaccinated individuals. Whereas MBCs of infected individuals targeted both pre- and postfusion Spike (S), most vaccine-elicited MBCs were specific for prefusion S, consistent with the use of prefusion-stabilized S in mRNA vaccines. Furthermore, a large fraction of MBCs recognizing postfusion S cross-reacted with human betacoronaviruses. The avidity of MBC-derived and serum antibodies increased over time resulting in enhanced resilience to viral escape by SARS-CoV-2 variants, including Omicron BA.1 and BA.2 sub-lineages, albeit only partially for BA.4 and BA.5 sublineages. Overall, the maturation of high-affinity and broadly-reactive MBCs provides the basis for effective recall responses to future SARS-CoV-2 variants.

Reply to Böning et al. Comment on "Ceruti et al. Temporal Changes in the Oxyhemoglobin Dissociation Curve of Critically Ill COVID-19 Patients. J. Clin. Med. 2022, 11, 788".

We would like to thank Böning et al. for all the important issues raised in the present commentary [...].

A Pilot, Prospective, Observational Study to Investigate the Value of NGS in Liquid Biopsies to Predict Tumor Response After Neoadjuvant Chemo-Radiotherapy in Patients With Locally Advanced Rectal Cancer: The LiBReCa Study.

Introduction: Circulating tumor DNA (ctDNA) correlates with the response to therapy in different types of cancer. However, in patients with locally advanced rectal cancer (LARC), little is known about how ctDNA levels change with neoadjuvant chemoradiation (Na-ChRT) and how they correlate with treatment response. This work aimed to explore the value of serial liquid biopsies in monitoring response after Na-ChRT with the hypothesis that this could become a reliable biomarker to identify patients with a complete response, candidates for non-operative management. Materials and Methods: Twenty-five consecutive LARC patients undergoing long-term Na-ChRT therapy were included. Applying next-generation sequencing (NGS), we characterized DNA extracted from formalin-fixed paraffin embedded diagnostic biopsy and resection tissue and plasma ctDNA collected at the following time points: the first and last days of radiotherapy (T0, Tend), at 4 (T4), 7 (T7) weeks after radiotherapy, on the day of surgery (Top), and 3-7 days after surgery (Tpost-op). On the day of surgery, a mesenteric vein sample was also collected (TIMV). The relationship between the ctDNA at those time-points and the tumor regression grade (TRG) of the surgical specimen was statistically explored. Results: We found no association between the disappearance of ctDNA mutations in plasma samples and pathological complete response (TRG1) as ctDNA was undetectable in the majority of patients from Tend on. However, we observed that the poor (TRG 4) response to Na-ChRT was significantly associated with a positive liquid biopsy at the Top. Conclusions: ctDNA evaluation by NGS technology may identify LARC patients with poor response to Na-ChRT. In contrast, this technique does not seem useful for identifying patients prone to developing a complete response.

Anti-chemokine antibodies after SARS-CoV-2 infection correlate with favorable disease course.

Infection by SARS-CoV-2 leads to diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse COVID-19 outcomes. Instead, we discovered that antibodies against specific chemokines are omnipresent after COVID-19, associated with favorable disease, and predictive of lack of long COVID symptoms at one year post infection. Anti-chemokine antibodies are present also in HIV-1 infection and autoimmune disorders, but they target different chemokines than those in COVID-19. Monoclonal antibodies derived from COVID- 19 convalescents that bind to the chemokine N-loop impair cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising anti-chemokine antibodies associated with favorable COVID-19 may be beneficial by modulating the inflammatory response and thus bear therapeutic potential. One-Sentence Summary: Naturally arising anti-chemokine antibodies associate with favorable COVID-19 and predict lack of long COVID.

Nasogastric tube in mechanical ventilated patients: ETCO2 and pH measuring to confirm correct placement. A pilot study.

INTRODUCTION: Nasogastric tube (NGT) placement is a procedure commonly performed in mechanically ventilated (MV) patients. Chest X-Ray is the diagnostic gold-standard to confirm its correct placement, with the downsides of requiring MV patients' mobilization and of intrinsic actinic risk. Other potential methods to confirm NGT placement have shown lower accuracy compared to chest X-ray; end-tidal CO2 (ETCO2) and pH analysis have already been singularly investigated as an alternative to the gold standard. Aim of this study was to determine threshold values in ETCO2 and pH measurement at which correct NGT positioning can be confirmed with the highest accuracy. MATERIALS & METHODS: This was a prospective, multicenter, observational trial; a continuous cohort of eligible patients was allocated with site into two arms. Patients underwent general anesthesia, orotracheal intubation and MV; in the first and second group we respectively assessed the difference between tracheal and esophageal ETCO2 and between esophageal and gastric pH values. RESULTS: From November 2020 to March 2021, 85 consecutive patients were enrolled: 40 in the ETCO2 group and 45 in the pH group. The ETCO2 ROC analysis for predicting NGT tracheal misplacement demonstrated an optimal ETCO2 cutoff value of 25.5 mmHg, with both sensitivity and specificity reaching 1.0 (AUC 1.0, p < 0.001). The pH ROC analysis for predicting NGT correct gastric placement resulted in an optimal pH cutoff value of 4.25, with mild diagnostic accuracy (AUC 0.79, p < 0.001). DISCUSSION: In patients receiving MV, ETCO2 and pH measurements respectively identified incorrect and correct NGT placement, allowing the identification of threshold values potentially able to improve correct NGT positioning. TRIAL REGISTRATION: NCT03934515 (www.clinicaltrials.gov).

Temporal Changes in the Oxyhemoglobin Dissociation Curve of Critically Ill COVID-19 Patients.

Critical COVID-19 is a life-threatening disease characterized by severe hypoxemia with complex pathophysiological mechanisms that are not yet completely understood. A pathological shift in the oxyhemoglobin curve (ODC) was previously described through the analysis of p50, intended as the oxygen tension at which hemoglobin is saturated by oxygen at 50%. The aim of this study was to analyze Hb-O2 affinity features over time in a cohort of critically ill COVID-19 patients, through the analysis of ODC p50 behavior. A retrospective analysis was performed; through multiple arterial blood gas (ABG) analyses, each p50 was calculated and normalized according to PaCO2, pH and temperature; patients' p50 evolution over time was reported, comparing the first 3 days (early p50s) with the last 3 days (late p50s) of ICU stay. A total of 3514 ABG analyses of 32 consecutive patients were analyzed. The majority of patients presented a left shift over time (p = 0.03). A difference between early p50s and late p50s was found (20.63 ± 2.1 vs. 18.68 ± 3.3 mmHg, p = 0.03); median p50 of deceased patients showed more right shifts than those of alive patients (24.1 vs. 18.45 mmHg, p = 0.01). One-way ANOVA revealed a p50 variance greater in the early p50s (σ2 = 8.6) than in the late p50s (σ2 = 3.84), associated with a reduction over time (p < 0.001). Comparing the Hb-O2 affinity in critically ill COVID-19 patients between ICU admission and ICU discharge, a temporal shift in the ODC was observed.

Can a Glove-Coach Technology Significantly Increase the Efficacy of Cardiopulmonary Resuscitation on Non-healthcare Professionals? A Controlled Trial.

Introduction: Cardiovascular accidents are the world's leading cause of death. A good quality cardiopulmonary resuscitation (CPR) can reduce cardiac arrest-associated mortality. This study aims to test the coaching system of a wearable glove, providing instructions during out-of-hospital CPR. Materials and Methods: We performed a single-blind, controlled trial to test non-healthcare professionals during a simulated CPR performed on an electronic mannequin. The no-glove group was the control. The primary outcome was to compare the accuracy of depth and frequency of two simulated CPR sessions. Secondary outcomes were to compare the decay of CPR performance and the percentage of the duration of accurate CPR. Results: About 130 volunteers were allocated to 1:1 ratio in both groups; mean age was 36 ± 15 years (min-max 21-64) and 62 (48%) were men; 600 chest compressions were performed, and 571 chest compressions were analyzed. The mean frequency in the glove group was 117.67 vs. 103.02 rpm in the control group (p < 0.001). The appropriate rate cycle was 92.4% in the glove group vs. 71% in the control group, with a difference of 21.4% (p < 0.001). Mean compression depth in the glove group was 52.11 vs. 55.17 mm in the control group (p < 0.001). A mean reduction of compression depth over time of 5.3 mm/min was observed in the control group vs. 0.83 mm/min of reduction in the glove group. Conclusion: Visual and acoustic feedbacks provided through the utilization of the glove's coaching system were useful for non-healthcare professionals' CPR performance.

Admission criteria in critically ill COVID-19 patients: A physiology-based approach.

INTRODUCTION: The COVID-19 pandemic required careful management of intensive care unit (ICU) admissions, to reduce ICU overload while facing limitations in resources. We implemented a standardized, physiology-based, ICU admission criteria and analyzed the mortality rate of patients refused from the ICU. MATERIALS AND METHODS: In this retrospective observational study, COVID-19 patients proposed for ICU admission were consecutively analyzed; Do-Not-Resuscitate patients were excluded. Patients presenting an oxygen peripheral saturation (SpO2) lower than 85% and/or dyspnea and/or mental confusion resulted eligible for ICU admission; patients not presenting these criteria remained in the ward with an intensive monitoring protocol. Primary outcome was both groups' survival rate. Secondary outcome was a sub analysis correlating SpO2 cutoff with ICU admission. RESULTS: From March 2020 to January 2021, 1623 patients were admitted to our Center; 208 DNR patients were excluded; 97 patients were evaluated. The ICU-admitted group (n = 63) mortality rate resulted 15.9% at 28 days and 27% at 40 days; the ICU-refused group (n = 34) mortality rate resulted 0% at both intervals (p < 0.001). With a SpO2 cut-off of 85%, a significant correlation was found (p = 0.009), but with a 92% a cut-off there was no correlation with ICU admission (p = 0.26). A similar correlation was also found with dyspnea (p = 0.0002). CONCLUSION: In COVID-19 patients, standardized ICU admission criteria appeared to safely reduce ICU overload. In the absence of dyspnea and/or confusion, a SpO2 cutoff up to 85% for ICU admission was not burdened by negative outcomes. In a pandemic context, the SpO2 cutoff of 92%, as a threshold for ICU admission, needs critical re-evaluation.

Dysphagic disorder in a cohort of COVID-19 patients: Evaluation and evolution.

BACKGROUND: COVID-19 is a multisystem disease complicated by respiratory failure requiring sustanined mechanical ventilation (MV). Prolongued oro-tracheal intubation is associated to an increased risk of dysphagia and bronchial aspiration. Purpose of this study was to investigate swallowing disorders in critically ill COVID-19 patients. MATERIAL AND METHODS: This was a retrospective study analysing a consecutive cohort of COVID-19 patients admitted to the Intensive Care Unit (ICU) of our hospital. Data concerning dysphagia were collected according to the Gugging Swallowing Screen (GUSS) and related to demographic characteristics, clinical data, ICU Length-Of-Stay (LOS) and MV parameters. RESULTS: From March 2 to April 30, 2020, 31 consecutive critically ill COVID-19 patients admitted to ICU were evaluated by speech and language therapists (SLT). Twenty-five of them were on MV (61% through endotracheal tube and 19% through tracheostomy); median MV length was 11 days. Seventeen (54.8%) patients presented dysphagia; a correlation was found between first GUSS severity stratification and MV days (p < 0.001), ICU LOS (p < 0.001), age (p = 0.03) and tracheostomy (p = 0.042). No other correlations were found. At 16 days, 90% of patients had fully recovered; a significant improvement was registered especially during the first week (p < 0.001). CONCLUSION: Compared to non-COVID-19 patiens, a higher rate of dysphagia was reported in COVID-19 patients, with a more rapid and complete recovery. A systematic early SLT evaluation of COVID-19 patients on MV may thus be useful to prevent dysphagia-related complications.

Multidisciplinary team approach in critically ill COVID-19 patients reduced pronation-related complications rate: A retrospective cohort study.

BACKGROUND: In the pandemic scenario, critically ill COVID-19 patients' management presented an increased workload for Intensive Care Unit (ICU) nursing staff, particularly during pronation maneuvers, with high risk of complications. In this contest, some authors described an increase in complications incidence after pronation. An ICU Pronation Team (IPT) was implemented to support this maneuver. MATERIAL AND METHODS: Retrospective analysis was conducted on consecutive critically ill COVID-19 patients in COVID-19 Center in southern Switzerland, between March and April 2020. Aim of the study was to determine rates and characteristics of pronation-related complications managed by IPT according to standard protocols. RESULTS: Forty-two patients undergoing mechanical ventilation (MV) were enrolled; 296 prone/supine positioning were performed, with 3.52 cycles/patient. All patients were equipped with arterial line, central venous catheter, urinary catheter, 28 (66%) endotracheal tube, 8 (19%), tracheostomy, 6 (14%) dialysis catheter, 3 (7%) abdominal drainage and 8 (19%) femoral thermodilution catheter; mean BMI was 28.3 kg/m2. One (0.3%) major complication was observed, while fourteen (33.3%) patients developed minor complications (pressure injuries). ICU length-of-stay and MV days correlated with both incidence (p = 0.029 and p = 0.015 respectively) and number (p = 0.001 and p = 0.001 respectively) of pressure sores (n = 27). Propensity matching score analysis did not show any protective factor of pronation regarding pressure injuries (p = 0.448). No other significant correlation was found. CONCLUSION: Multidisciplinary healthcare professional management can reduce most severe complication related to pronation in critical care setting. Rather than from pronation, the persistent high rate of minor complications appeared to be related to disease severity.

Low PEEP Mechanical Ventilation and PaO2/FiO2 Ratio Evolution in COVID-19 Patients.

Invasive mechanical ventilation (IMV) is the standard treatment in critically ill COVID-19 patients with acute severe respiratory distress syndrome (ARDS). When IMV setting is extremely aggressive, especially through the application of high positive-end-expiratory respiration (PEEP) values, lung damage can occur. Until today, in COVID-19 patients, two types of ARDS were identified (L- and H-type); for the L-type, a lower PEEP strategy was supposed to be preferred, but data are still missing. The aim of this study was to evaluate if a clinical management with lower PEEP values in critically ill L-type COVID-19 patients was safe and efficient in comparison to usual standard of care. A retrospective analysis was conducted on consecutive patients with COVID-19 ARDS admitted to the ICU and treated with IMV. Patients were treated with a lower PEEP strategy adapted to BMI: PEEP 10 cmH2O if BMI < 30 kg m-2, PEEP 12 cmH2O if BMI 30-50 kg m-2, PEEP 15 cmH2O if BMI > 50 kg m-2. Primary endpoint was the PaO2/FiO2 ratio evolution during the first 3 IMV days; secondary endpoints were to analyze ICU length of stay (LOS) and IMV length. From March 2 to January 15, 2021, 79 patients underwent IMV. Average applied PEEP was 11 ± 2.9 cmH2O for BMI < 30 kg m-2 and 16 ± 3.18 cmH2O for BMI > 30 kg m-2. During the first 24 h of IMV, patients' PaO2/FiO2 ratio presented an improvement (p<0.001; CI 99%) that continued daily up to 72 h (p<0.001; CI 99%). Median ICU LOS was 15 days (10-28); median duration of IMV was 12 days (8-26). The ICU mortality rate was 31.6%. Lower PEEP strategy treatment in L-type COVID-19 ARDS resulted in a PaO2/FiO2 ratio persistent daily improvement during the first 72 h of IMV. A lower PEEP strategy could be beneficial in the first phase of ARDS in critically ill COVID-19 patients.

Circulating immune cell populations related to primary breast cancer, surgical removal, and radiotherapy revealed by flow cytometry analysis.

BACKGROUND: Advanced breast cancer (BC) impact immune cells in the blood but whether such effects may reflect the presence of early BC and its therapeutic management remains elusive. METHODS: To address this question, we used multiparametric flow cytometry to analyze circulating leukocytes in patients with early BC (n = 13) at the time of diagnosis, after surgery, and after adjuvant radiotherapy, compared to healthy individuals. Data were analyzed using a minimally supervised approach based on FlowSOM algorithm and validated manually. RESULTS: At the time of diagnosis, BC patients have an increased frequency of CD117+CD11b+ granulocytes, which was significantly reduced after tumor removal. Adjuvant radiotherapy increased the frequency of CD45RO+ memory CD4+ T cells and CD4+ regulatory T cells. FlowSOM algorithm analysis revealed several unanticipated populations, including cells negative for all markers tested, CD11b+CD15low, CD3+CD4-CD8-, CD3+CD4+CD8+, and CD3+CD8+CD127+CD45RO+ cells, associated with BC or radiotherapy. CONCLUSIONS: This study revealed changes in blood leukocytes associated with primary BC, surgical removal, and adjuvant radiotherapy. Specifically, it identified increased levels of CD117+ granulocytes, memory, and regulatory CD4+ T cells as potential biomarkers of BC and radiotherapy, respectively. Importantly, the study demonstrates the value of unsupervised analysis of complex flow cytometry data to unravel new cell populations of potential clinical relevance.

Clonal analysis of immunodominance and cross-reactivity of the CD4 T cell response to SARS-CoV-2.

The identification of CD4+ T cell epitopes is instrumental for the design of subunit vaccines for broad protection against coronaviruses. Here, we demonstrate in COVID-19-recovered individuals a robust CD4+ T cell response to naturally processed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein and nucleoprotein (N), including effector, helper, and memory T cells. By characterizing 2943 S-reactive T cell clones from 34 individuals, we found that the receptor-binding domain (RBD) is highly immunogenic and that 33% of RBD-reactive clones and 94% of individuals recognized a conserved immunodominant S346-S365 region comprising nested human leukocyte antigen DR (HLA-DR)- and HLA-DP-restricted epitopes. Using pre- and post-COVID-19 samples and S proteins from endemic coronaviruses, we identified cross-reactive T cells targeting multiple S protein sites. The immunodominant and cross-reactive epitopes identified can inform vaccination strategies to counteract emerging SARS-CoV-2 variants.