Longitudinal dynamics of SARS-CoV-2-specific cellular and humoral immunity after natural infection or BNT162b2 vaccination.

The timing of the development of specific adaptive immunity after natural SARS-CoV-2 infection, and its relevance in clinical outcome, has not been characterized in depth. Description of the long-term maintenance of both cellular and humoral responses elicited by real-world anti-SARS-CoV-2 vaccination is still scarce. Here we aimed to understand the development of optimal protective responses after SARS-CoV-2 infection and vaccination. We performed an early, longitudinal study of S1-, M- and N-specific IFN-γ and IL-2 T cell immunity and anti-S total and neutralizing antibodies in 88 mild, moderate or severe acute COVID-19 patients. Moreover, SARS-CoV-2-specific adaptive immunity was also analysed in 234 COVID-19 recovered subjects, 28 uninfected BNT162b2-vaccinees and 30 uninfected healthy controls. Upon natural infection, cellular and humoral responses were early and coordinated in mild patients, while weak and inconsistent in severe patients. The S1-specific cellular response measured at hospital arrival was an independent predictive factor against severity. In COVID-19 recovered patients, four to seven months post-infection, cellular immunity was maintained but antibodies and neutralization capacity declined. Finally, a robust Th1-driven immune response was developed in uninfected BNT162b2-vaccinees. Three months post-vaccination, the cellular response was comparable, while the humoral response was consistently stronger, to that measured in COVID-19 recovered patients. Thus, measurement of both humoral and cellular responses provides information on prognosis and protection from infection, which may add value for individual and public health recommendations.

Hemodialysis-Associated Immune Dysregulation in SARS-CoV-2-Infected End-Stage Renal Disease Patients.

Patients on hemodialysis show dysregulated immunity, basal hyperinflammation and a marked vulnerability to COVID-19. We evaluated the immune profile in COVID-19 hemodialysis patients and the changes associated with clinical deterioration after the hemodialysis session. Recruited patients included eight hemodialysis subjects with active, PCR-confirmed SARS-CoV-2 infection, five uninfected hemodialysis patients and five healthy controls. In SARS-CoV-2-infected hemodialysis patients TNF-α, IL-6 and IL-8 were particularly increased. Lymphopenia was mostly due to reduction in CD4+ T, B and central memory CD8+ T cells. There was a predominance of classical and intermediate monocytes with reduced HLA-DR expression and enhanced production of pro-inflammatory molecules. Immune parameters were analysed pre- and post-hemodialysis in three patients with COVID-19 symptoms worsening after the hemodialysis session. There was a higher than 2.5-fold increase in GM-CSF, IFN-γ, IL-1ß, IL-2, IL-6, IL-17A and IL-21 in serum, and augmentation of monocytes-derived TNF-α, IL-1ß and IL-8 and CXCL10 (p < 0.05). In conclusion, COVID-19 in hemodialysis patients associates with alteration of lymphocyte subsets, increasing of pro-inflammatory cytokines and monocyte activation. The observed worsening during the hemodialysis session in some patients was accompanied by augmentation of particular inflammatory cytokines, which might suggest biomarkers and therapeutic targets to prevent or mitigate the hemodialysis-related deterioration during SARS-CoV-2 infection.

Immunogenicity of Anti-SARS-CoV-2 Vaccines in Common Variable Immunodeficiency.

Common variable immunodeficiency (CVID) is characterized by hypogammaglobulinemia and/or a defective antibody response to T-dependent and T-independent antigens. CVID response to immunization depends on the antigen type, the vaccine mechanism, and the specific patient immune defect. In CVID patients, humoral and cellular responses to the currently used COVID-19 vaccines remain unexplored. Eighteen CVID subjects receiving 2-dose anti-SARS-CoV-2 vaccines were prospectively studied. S1-antibodies and S1-specific IFN-γ T cell response were determined by ELISA and FluoroSpot, respectively. The immune response was measured before the administration and after each dose of the vaccine, and it was compared to the response of 50 healthy controls (HC). The development of humoral and cellular responses was slower in CVID patients compared with HC. After completing vaccination, 83% of CVID patients had S1-specific antibodies and 83% had S1-specific T cells compared with 100% and 98% of HC (p = 0.014 and p = 0.062, respectively), but neutralizing antibodies were detected only in 50% of the patients. The strength of both humoral and cellular responses was significantly lower in CVID compared with HC, after the first and second doses of the vaccine. Absent or discordant humoral and cellular responses were associated with previous history of autoimmunity and/or lymphoproliferation. Among the three patients lacking humoral response, two had received recent therapy with anti-B cell antibodies. Further studies are needed to understand if the response to COVID-19 vaccination in CVID patients is protective enough. The 2-dose vaccine schedule and possibly a third dose might be especially necessary to achieve full immune response in these patients.

IL-6-based mortality prediction model for COVID-19: Validation and update in multicenter and second wave cohorts.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a highly variable condition. Validated tools to assist in the early detection of patients at high risk of mortality can help guide medical decisions. OBJECTIVE: We sought to validate externally, as well as in patients from the second pandemic wave in Europe, our previously developed mortality prediction model for hospitalized COVID-19 patients. METHODS: Three validation cohorts were generated: 2 external with 185 and 730 patients from the first wave and 1 internal with 119 patients from the second wave. The probability of death was calculated for all subjects using our prediction model, which includes peripheral blood oxygen saturation/fraction of inspired oxygen ratio, neutrophil-to-lymphocyte ratio, lactate dehydrogenase, IL-6, and age. Discrimination and calibration were evaluated in the validation cohorts. The prediction model was updated by reestimating individual risk factor effects in the overall cohort (N = 1477). RESULTS: The mortality prediction model showed good performance in the external validation cohorts 1 and 2, and in the second wave validation cohort 3 (area under the receiver-operating characteristic curve, 0.94, 0.86, and 0.86, respectively), with excellent calibration (calibration slope, 0.86, 0.94, and 0.79; intercept, 0.05, 0.03, and 0.10, respectively). The updated model accurately predicted mortality in the overall cohort (area under the receiver-operating characteristic curve, 0.91), which included patients from both the first and second COVID-19 waves. The updated model was also useful to predict fatal outcome in patients without respiratory distress at the time of evaluation. CONCLUSIONS: This is the first COVID-19 mortality prediction model validated in patients from the first and second pandemic waves. The COR+12 online calculator is freely available to facilitate its implementation (https://utrero-rico.shinyapps.io/COR12_Score/).

Circulatory follicular helper T lymphocytes associate with lower incidence of CMV infection in kidney transplant recipients.

Primary infection and/or reactivation of cytomegalovirus (CMV) in kidney transplant recipients (KTR) favor rejection and mortality. T follicular helper cells (TFH) could contribute to protection against CMV. Circulatory TFH (cTFH) were studied pretransplant and early posttransplant in 90 CMV seropositive KTR not receiving antithymocyte globulin or antiviral prophylaxis, followed-up for 1 year. Patients who presented CMV infection had significantly lower cTFH and activated cTFH pretransplant and early posttransplant. Pretransplant activated cTFH were also lower within patients who developed CMV disease. Pre- and 14 days posttransplant activated cTFH were an independent protective factor for CMV infection (HR 0.41, p = .01; and 0.52, p = .02, respectively). KTR with low cTFH 7 days posttransplant (<11.9%) had lower CMV infection-free survival than patients with high cTFH (28.2% vs. 67.6%, p = .002). cTFH were associated with CMV-specific neutralizing antibodies (Nabs). In addition, IL-21 increased interferon-γ secretion by CMV-specific CD8+ T cells in healthy controls. Thus, we show an association between cTFH and lower incidence of CMV infection, probably through their cooperation in CMV-specific Nab production and IL-21-mediated enhancement of CD8+ T cell activity. Moreover, monitoring cTFH pre- and early posttransplant could improve CMV risk stratification and help select KTR catalogued at low/intermediate risk who could benefit from prophylaxis.

T cell-mediated response to SARS-CoV-2 in liver transplant recipients with prior COVID-19.

Whether immunosuppression impairs severe acute respiratory syndrome coronavirus 2-specific T cell-mediated immunity (SARS-CoV-2-CMI) after liver transplantation (LT) remains unknown. We included 31 LT recipients in whom SARS-CoV-2-CMI was assessed by intracellular cytokine staining (ICS) and interferon (IFN)-γ FluoroSpot assay after a median of 103 days from COVID-19 diagnosis. Serum SARS-CoV-2 IgG antibodies were measured by ELISA. A control group of nontransplant immunocompetent patients were matched (1:1 ratio) by age and time from diagnosis. Post-transplant SARS-CoV-2-CMI was detected by ICS in 90.3% (28/31) of recipients, with higher proportions for IFN-γ-producing CD4+ than CD8+ responses (93.5% versus 83.9%). Positive spike-specific and nucleoprotein-specific responses were found by FluoroSpot in 86.7% (26/30) of recipients each, whereas membrane protein-specific response was present in 83.3% (25/30). An inverse correlation was observed between the number of spike-specific IFN-γ-producing SFUs and time from diagnosis (Spearman's rho: -0.418; p value = .024). Two recipients (6.5%) failed to mount either T cell-mediated or IgG responses. There were no significant differences between LT recipients and nontransplant patients in the magnitude of responses by FluoroSpot to any of the antigens. Most LT recipients mount detectable-but declining over time-SARS-CoV-2-CMI after a median of 3 months from COVID-19, with no meaningful differences with immunocompetent patients.

Tocilizumab for the treatment of adult patients with severe COVID-19 pneumonia: A single-center cohort study.

Coronavirus disease 2019 (COVID-19) can lead to a massive cytokine release. The use of the anti-interleukin-6 receptor monoclonal antibody tocilizumab (TCZ) has been proposed in this hyperinflammatory phase, although supporting evidence is limited. We retrospectively analyzed 88 consecutive patients with COVID-19 pneumonia that received at least one dose of intravenous TCZ in our institution between 16 and 27 March 2020. Clinical status from day 0 (first TCZ dose) through day 14 was assessed by a 6-point ordinal scale. The primary outcome was clinical improvement (hospital discharge and/or a decrease of ≥2 points on the 6-point scale) by day 7. Secondary outcomes included clinical improvement by day 14 and dynamics of vital signs and laboratory values. Rates of clinical improvement by days 7 and 14 were 44.3% (39/88) and 73.9% (65/88). Previous or concomitant receipt of subcutaneous interferon-ß (adjusted odds ratio [aOR]: 0.23; 95% confidence interval [CI]: 0.06-0.94; P = .041) and serum lactate dehydrogenase more than 450 U/L at day 0 (aOR: 0.25; 95% CI: 0.06-0.99; P = .048) were negatively associated with clinical improvement by day 7. All-cause mortality was 6.8% (6/88). Body temperature and respiratory and cardiac rates significantly decreased by day 1 compared to day 0. Lymphocyte count and pulse oximetry oxygen saturation/FiO2 ratio increased by days 3 and 5, whereas C-reactive protein levels dropped by day 2. There were no TCZ-attributable adverse events. In this observational single-center study, TCZ appeared to be useful and safe as immunomodulatory therapy for severe COVID-19 pneumonia.

IL-6-based mortality risk model for hospitalized patients with COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has rapidly become a global pandemic. Because the severity of the disease is highly variable, predictive models to stratify patients according to their mortality risk are needed. OBJECTIVE: Our aim was to develop a model able to predict the risk of fatal outcome in patients with COVID-19 that could be used easily at the time of patients' arrival at the hospital. METHODS: We constructed a prospective cohort with 611 adult patients in whom COVID-19 was diagnosed between March 10 and April 12, 2020, in a tertiary hospital in Madrid, Spain. The analysis included 501 patients who had been discharged or had died by April 20, 2020. The capacity of several biomarkers, measured at the beginning of hospitalization, to predict mortality was assessed individually. Those biomarkers that independently contributed to improve mortality prediction were included in a multivariable risk model. RESULTS: High IL-6 level, C-reactive protein level, lactate dehydrogenase (LDH) level, ferritin level, d-dimer level, neutrophil count, and neutrophil-to-lymphocyte ratio were all predictive of mortality (area under the curve >0.70), as were low albumin level, lymphocyte count, monocyte count, and ratio of peripheral blood oxygen saturation to fraction of inspired oxygen (SpO2/FiO2). A multivariable mortality risk model including the SpO2/FiO2 ratio, neutrophil-to-lymphocyte ratio, LDH level, IL-6 level, and age was developed and showed high accuracy for the prediction of fatal outcome (area under the curve 0.94). The optimal cutoff reliably classified patients (including patients with no initial respiratory distress) as survivors and nonsurvivors with 0.88 sensitivity and 0.89 specificity. CONCLUSION: This mortality risk model allows early risk stratification of hospitalized patients with COVID-19 before the appearance of obvious signs of clinical deterioration, and it can be used as a tool to guide clinical decision making.

Imbalance favoring follicular helper T cells over IL10+ regulatory B cells is detrimental for the kidney allograft.

A high frequency of regulatory B (Breg) cells, generally transitional B cells, has been associated with long-term kidney allograft survival and operational tolerance. However, circulating follicular helper T cells (cTfh) correlate with graft rejection. In order to better understand the interplay between these cell subsets and to determine their association with graft outcome we studied transitional and IL10+ Breg cells, as well as cTfh, pre- and post-transplantation in a prospective cohort of 200 kidney transplant recipients and in healthy volunteers. Patients with end-stage kidney disease had higher frequencies of transitional and IL10+ Breg cells compared to controls, and these subsets decreased during the one-year post-transplant follow-up. Higher frequencies of pre-transplant IL10+ Breg cells, and a larger reduction in these cells early post-transplantation, predicted acute rejection and graft failure. Moreover, IL10+ Breg cells correlated with cTfh pre-transplantation, and a post-transplant increase in the cTfh/IL10+Breg ratio preceded acute rejection. Thus, evaluation of pre-transplant IL10+ Breg cells and the regular monitoring of the cTfh/IL10+Breg ratio may be useful to assess post-transplant risk. Hence, our observations suggest the need to develop therapeutic strategies aimed at preserving regulatory B cells, and depleting Tfh, post-transplantation.

A PBX1 transcriptional network controls dopaminergic neuron development and is impaired in Parkinson's disease.

Pre-B-cell leukemia homeobox (PBX) transcription factors are known to regulate organogenesis, but their molecular targets and function in midbrain dopaminergic neurons (mDAn) as well as their role in neurodegenerative diseases are unknown. Here, we show that PBX1 controls a novel transcriptional network required for mDAn specification and survival, which is sufficient to generate mDAn from human stem cells. Mechanistically, PBX1 plays a dual role in transcription by directly repressing or activating genes, such as Onecut2 to inhibit lateral fates during embryogenesis, Pitx3 to promote mDAn development, and Nfe2l1 to protect from oxidative stress. Notably, PBX1 and NFE2L1 levels are severely reduced in dopaminergic neurons of the substantia nigra of Parkinson's disease (PD) patients and decreased NFE2L1 levels increases damage by oxidative stress in human midbrain cells. Thus, our results reveal novel roles for PBX1 and its transcriptional network in mDAn development and PD, opening the door for new therapeutic interventions.

Number and function of circulatory helper innate lymphoid cells are unaffected by immunosuppressive drugs used in solid organ recipients - a single centre cohort study.

In transplanted intestines, depletion of T cells together with long-term persistence of ILC is observed, suggesting ILC insensitivity to immunosuppressive drugs. To further analyze helper ILC (hILC) apparent resistance to therapy, cytotoxic ILC (NK cells), hILC subsets (ILC1, ILC2, and ILC precursors (ILCP)), and their signature cytokines (IFNγ, IL4 + IL13, and IL22) were analyzed in peripheral blood of kidney and liver transplant recipients. Early after transplantation (posTx), transplanted patients showed significantly lower Lin + and NK cells, whereas total hILC, ILC1, ILC2, and ILCP numbers were similar in patients and controls. Between paired pre- and posTx samples, Lin + cell and NK cell counts significantly decreased, whereas all three hILC counts and their cytokine production remained similar. ILC1, ILC2, and ILCP numbers were also similar in patients under thymoglobulin or basiliximab (BAS), patients without induction (only maintenance therapy) and controls. hILC showed lower TMG binding comparing to Lin + cells, reduced expression of CD25 (BAS target), and diminished calcineurin activity with undetectable calcineurin and FKBP12 (tacrolimus target). hILC counts were not related to delayed graft function or biopsy-proven acute rejection. Thus, hILC remain stable early after transplantation and seem unaffected by immunosuppressors, which may be related to reduced targets expression and low calcineurin activity.

Longitudinal profile of circulating T follicular helper lymphocytes parallels anti-HLA sensitization in renal transplant recipients.

Antibody-mediated rejection is responsible for 30%-50% of renal graft failures. Differentiation of B cells into antibody-producing plasmablasts depends on the collaboration of follicular helper T cells (Tfh). We analyzed circulating Tfh (cTfh) in kidney recipients and studied cTfh relationship with anti-HLA antibody production and graft outcome. cTfh were longitudinally analyzed in a prospective cohort of patients (n = 206), pre- and posttransplantation. Clinical data, HLA sensitization, and cTfh function were recorded. Both pretransplant and 6-month posttransplant cTfh were able to derive IgG-producing plasmablasts. Pretransplant cTfh was decreased in patients, especially in those who received dialysis. However, these cells were increased in patients with previous allograft or transfusions and in HLA-sensitized recipients. After transplantation cTfh expanded, significantly more in patients who developed de novo anti-HLA antibodies than in patients who remained unsensitized. Augmented pretransplant cTfh positively correlated with higher intensity of pretransplant anti-HLA class I and with de novo anti-HLA class I and anti-HLA class II antibodies. Consistently, pretransplantation cTfh were higher in patients who experienced acute rejection (HR = 1.14 [1.04-1.25]). Thus, we show a role for Tfh in anti-HLA sensitization and rejection. Multicenter studies with additional patient cohorts are needed to validate these results. Immunosuppressive drugs targeting Tfh could be useful to improve outcomes.

Complement C3F allotype synthesized by liver recipient modifies transplantation outcome independently from donor hepatic C3.

Complement component 3 (C3) presents both slow (C3S) and fast (C3F) variants, which can be locally produced and activated by immune system cells. We studied C3 recipient variants in 483 liver transplant patients by RT-PCR-HRM to determine their effect on graft outcome during the first year post-transplantation. Allograft survival was significantly decreased in C3FF recipients (C3SS 95% vs C3FS 91% vs C3FF 83%; P=.01) or C3F allele carriers (C3F absence 95% vs C3F presence 90%, P=.02). C3FF genotype or presence of C3F allele independently increased risk for allograft loss (OR: 2.38, P=.005 and OR: 2.66, P=.02, respectively). C3FF genotype was more frequent among patients whose first infection was of viral etiology (C3SS 13% vs C3FS 18% vs C3FF 32%; P=.04) and independently increased risk for post-transplant viral infections (OR: 3.60, P=.008). On the other hand, C3FF and C3F protected from rejection events (OR: 0.54, P=.03 and OR: 0.63, P=.047, respectively). Differences were not observed in hepatitis C virus recurrence or patient survival. In conclusion, we show that, independently from C3 variants produced by donor liver, C3F variant from recipient diminishes allograft survival, increases susceptibility to viral infections, and protects from rejection after transplantation. C3 genotyping of liver recipients may be useful to stratify risk.

Early renal graft function deterioration in recipients with preformed anti-MICA antibodies: partial contribution of complement-dependent cytotoxicity.

BACKGROUND: We previously reported that preformed anti-MHC class I-related chain A (MICA) antibodies increase the risk for renal graft rejection and enhance the deleterious effect of PRA(+) status early after transplantation. METHODS: We studied 727 kidney recipients. Days to reach optimal serum creatinine level, estimated glomerular filtration rate (eGFR) at Month 3 and chronic kidney disease (CKD) stages were recorded. Anti-MICA specificities and C1q binding were tested by solid-phase assay. Complement-dependent cytotoxicity (CDC) and flow cytometry (FC) cross-matches with HeLa and PMA/CD28-T-blasts were performed. RESULTS: PRA(+)MICA(+) recipients exhibited longer time to reach optimal serum creatinine level after transplantation (P = 0.005) and had the lowest eGFR at Month 3 (P = 0.006). PRA(+)MICA(+) status independently increased the risk for CKDT stage 5 at Month 3 [hazard ratio (HR) 4.92, P = 0.030]. Pre-transplant anti-MICA antibodies were polyspecific and showed stronger reactions when coexisting with anti-HLA antibodies (mean standard fluorescent intensity 112 157 ± 44 426 in HLA(+)MICA(+) sera versus 49 680 ± 33 116 in HLA(-)MICA(+) sera, P = 0.0006). Anti-AYVE supereplet reactivity was significantly higher in HLA(+)MICA(+) versus HLA(-)MICA(+) patients (P < 0.001) and significantly superior than anti-CMGWS supereplet within HLA(+)MICA(+) patients (P = 0.001). Three of 13 anti-MICA(+) pre-transplant sera were positive for the C1q binding assay; one of them (serum 3) exclusively recognized AYVE supereplet with a strong reactivity against MICA*027 antigen (same as MICA*008). Anti-MICA antibodies in anti-HLA-absorbed serum 3 bound native MICA molecules in MICA*008(+) HeLa and PMA/CD28-T-blasts and mediated cell death by activating complement. CONCLUSION: Preformed anti-MICA antibodies may occasionally be cytotoxic by fixing and activating complement. This way they might contribute to worse early kidney graft function.

Efficient expansion and dopaminergic differentiation of human fetal ventral midbrain neural stem cells by midbrain morphogens.

Human fetal midbrain tissue grafting has provided proof-of-concept for dopamine cell replacement therapy (CRT) in Parkinson's disease (PD). However, limited tissue availability has hindered the development and widespread use of this experimental therapy. Here we present a method for generating large numbers of midbrain dopaminergic (DA) neurons based on expanding and differentiating neural stem/progenitor cells present in the human ventral midbrain (hVM) tissue. Our results show that hVM neurospheres (hVMN) with low cell numbers, unlike their rodent counterparts, expand the total number of cells 3-fold, whilst retaining their capacity to differentiate into midbrain DA neurons. Moreover, Wnt5a promoted DA differentiation of expanded cells resulting in improved morphological maturation, midbrain DA marker expression, DA release and electrophysiological properties. This method results in cell preparations that, after expansion and differentiation, can contain 6-fold more midbrain DA neurons than the starting VM preparation. Thus, our results provide evidence that by improving expansion and differentiation of progenitors present in the hVM it is possible to greatly enrich cell preparations for DA neurons. This method could substantially reduce the amount of human fetal midbrain tissue necessary for CRT in patients with PD, which could have major implications for the widespread adoption of this approach.

Defending against SARS-CoV-2: The T cell perspective.

SARS-CoV-2-specific T cell response has been proven essential for viral clearance, COVID-19 outcome and long-term memory. Impaired early T cell-driven immunity leads to a severe form of the disease associated with lymphopenia, hyperinflammation and imbalanced humoral response. Analyses of acute SARS-CoV-2 infection have revealed that mild COVID-19 course is characterized by an early induction of specific T cells within the first 7 days of symptoms, coordinately followed by antibody production for an effective control of viral infection. In contrast, patients who do not develop an early specific cellular response and initiate a humoral immune response with subsequent production of high levels of antibodies, develop severe symptoms. Yet, delayed and persistent bystander CD8+ T cell activation has been also reported in hospitalized patients and could be a driver of lung pathology. Literature supports that long-term maintenance of T cell response appears more stable than antibody titters. Up to date, virus-specific T cell memory has been detected 22 months post-symptom onset, with a predominant IL-2 memory response compared to IFN-γ. Furthermore, T cell responses are conserved against the emerging variants of concern (VoCs) while these variants are mostly able to evade humoral responses. This could be partly explained by the high HLA polymorphism whereby the viral epitope repertoire recognized could differ among individuals, greatly decreasing the likelihood of immune escape. Current COVID-19-vaccination has been shown to elicit Th1-driven spike-specific T cell response, as does natural infection, which provides substantial protection against severe COVID-19 and death. In addition, mucosal vaccination has been reported to induce strong adaptive responses both locally and systemically and to protect against VoCs in animal models. The optimization of vaccine formulations by including a variety of viral regions, innovative adjuvants or diverse administration routes could result in a desirable enhanced cellular response and memory, and help to prevent breakthrough infections. In summary, the increasing evidence highlights the relevance of monitoring SARS-CoV-2-specific cellular immune response, and not only antibody levels, as a correlate for protection after infection and/or vaccination. Moreover, it may help to better identify target populations that could benefit most from booster doses and to personalize vaccination strategies.

Decreased breadth of the antibody response to the spike protein of SARS-CoV-2 after repeated vaccination.

Introduction: The rapid development of vaccines to prevent COVID-19 has raised the need to compare the capacity of different vaccines in terms of developing a protective humoral response. Previous studies have shown inconsistent results in this area, highlighting the importance of further research to evaluate the efficacy of different vaccines. Methods: This study utilized a highly sensitive and reliable flow cytometry method to measure the titers of IgG1 isotype antibodies in the blood of healthy volunteers after receiving one or two doses of various vaccines administered in Spain. The method was also used to simultaneously measure the reactivity of antibodies to the S protein of the original Wuhan strain and variants B.1.1.7 (Alpha), B.1.617.2 (Delta), and B.1.617.1 (Kappa). Results: Significant differences were observed in the titer of anti-S antibodies produced after a first dose of the vaccines ChAdOx1 nCov-19/AstraZeneca, mRNA-1273/Moderna, BNT162b2/Pfizer-BioNTech, and Ad26.COV.S/Janssen. Furthermore, a relative reduction in the reactivity of the sera with the Alpha, Delta, and Kappa variants, compared to the Wuhan strain, was observed after the second boosting immunization. Discussion: The findings of this study provide a comparison of different vaccines in terms of anti-S antibody generation and cast doubts on the convenience of repeated immunization with the same S protein sequence. The multiplexed capacity of the flow cytometry method utilized in this study allowed for a comprehensive evaluation of the efficacy of various vaccines in generating a protective humoral response. Future research could focus on the implications of these findings for the development of effective COVID-19 vaccination strategies.

Cellular and humoral immune responses and breakthrough infections after three SARS-CoV-2 mRNA vaccine doses.

Background: SARS-CoV-2 vaccination has proven the most effective measure to control the COVID-19 pandemic. Booster doses are being administered with limited knowledge on their need and effect on immunity. Objective: To determine the duration of specific T cells, antibodies and neutralization after 2-dose vaccination, to assess the effect of a third dose on adaptive immunity and to explore correlates of protection against breakthrough infection. Methods: 12-month longitudinal assessment of SARS-CoV-2-specific T cells, IgG and neutralizing antibodies triggered by 2 BNT162b2 doses followed by a third mRNA-1273 dose in a cohort of 77 healthcare workers: 17 with SARS-CoV-2 infection prior to vaccination (recovered) and 60 naïve. Results: Peak levels of cellular and humoral response were achieved 2 weeks after the second dose. Antibodies declined thereafter while T cells reached a plateau 3 months after vaccination. The decline in neutralization was specially marked in naïve individuals and it was this group who benefited most from the third dose, which resulted in a 20.9-fold increase in neutralization. Overall, recovered individuals maintained higher levels of T cells, antibodies and neutralization 1 to 6 months post-vaccination than naïve. Seventeen asymptomatic or mild SARS-CoV-2 breakthrough infections were reported during follow-up, only in naïve individuals. This viral exposure boosted adaptive immunity. High peak levels of T cells and neutralizing antibodies 15 days post-vaccination associated with protection from breakthrough infections. Conclusion: Booster vaccination in naïve individuals and the inclusion of viral antigens other than spike in future vaccine formulations could be useful strategies to prevent SARS-CoV-2 breakthrough infections.

Analysis of the factors predicting clinical response to tocilizumab therapy in patients with severe COVID-19.

BACKGROUND: Controversy remains about the efficacy of tocilizumab (TCZ) for the treatment of severe COVID-19. We aimed to analyze the profile of TCZ-respondent patients. METHODS: We retrospectively analyzed a cohort of patients with severe COVID-19 who received off-label TCZ after recommendation by a local committee and were admitted to the University Hospital "12 de Octubre" until May 2020. The primary end point was a significant clinical improvement (SCI) on day 14 after administration of TCZ. Factors independently related to SCI were analyzed by multivariate logistic regression models. RESULTS: Of 428 (63.3%) patients treated with TCZ, 271 (63.3%) experienced SCI. After adjustment for factors related to unfavorable outcomes, TCZ administration within the first 48 hours from admission (odds ratio [OR]: 1.98, 95% confidence Interval [95% CI]: 1.1-3.55; P = 0.02) and ALT levels >100 UI/L at day 0 (OR: 3.28; 95% CI: 1.3-8.1; P = 0.01) were independently related to SCI. The rate of SCI significantly decreased according to the time of TCZ administration: 70.2% in the first 48 hours from admission, 58.5% on days 3-7, and 45.1% after day 7 (P = 0.03 and P = 0.001, respectively). CONCLUSION: TCZ improves the prognosis of patients with COVID-19 the most if treatment starts within the first 48 hours after admission.