Expression profile of HERVs and inflammatory mediators detected in nasal mucosa as a predictive biomarker of COVID-19 severity.

Introduction: Our research group and others demonstrated the implication of the human endogenous retroviruses (HERVs) in SARS-CoV-2 infection and their association with disease progression, suggesting HERVs as contributing factors in COVID-19 immunopathology. To identify early predictive biomarkers of the COVID-19 severity, we analyzed the expression of HERVs and inflammatory mediators in SARS-CoV-2-positive and -negative nasopharyngeal/oropharyngeal swabs with respect to biochemical parameters and clinical outcome. Methods: Residuals of swab samples (20 SARS-CoV-2-negative and 43 SARS-CoV-2-positive) were collected during the first wave of the pandemic and expression levels of HERVs and inflammatory mediators were analyzed by qRT-Real time PCR. Results: The results obtained show that infection with SARS-CoV-2 resulted in a general increase in the expression of HERVs and mediators of the immune response. In particular, SARS-CoV-2 infection is associated with increased expression of HERV-K and HERV-W, IL-1ß, IL-6, IL-17, TNF-α, MCP-1, INF-γ, TLR-3, and TLR-7, while lower levels of IL-10, IFN-α, IFN-ß, and TLR-4 were found in individuals who underwent hospitalization. Moreover, higher expression of HERV-W, IL-1ß, IL-6, IFN-α, and IFN-ß reflected the respiratory outcome of patients during hospitalization. Interestingly, a machine learning model was able to classify hospitalized vs not hospitalized patients with good accuracy based on the expression levels of HERV-K, HERV-W, IL-6, TNF-a, TLR-3, TLR-7, and the N gene of SARS-CoV-2. These latest biomarkers also correlated with parameters of coagulation and inflammation. Discussion: Overall, the present results suggest HERVs as contributing elements in COVID-19 and early genomic biomarkers to predict COVID-19 severity and disease outcome.

T Lymphocyte Subset Counts and Interferon-Gamma Production in Adults and Children with COVID-19: A Narrative Review.

Adults and children exhibit a broad range of clinical outcomes from SARS-CoV-2 infection, with minimal to mild symptoms, especially in the pediatric age. However, some children present with a severe hyperinflammatory post-infectious complication named multisystem inflammatory syndrome in children (MIS-C), mainly affecting previously healthy subjects. Understanding these differences is still an ongoing challenge, that can lead to new therapeutic strategies and avoid unfavorable outcomes. In this review, we discuss the different roles of T lymphocyte subsets and interferon-γ (IFN-γ) in the immune responses of adults and children. Lymphopenia can influence these responses and represent a good predictor for the outcome, as reported by most authors. The increased IFN-γ response exhibited by children could be the starting point for the activation of a broad response that leads to MIS-C, with a significantly higher risk than in adults, although a single IFN signature has not been identified. Multicenter studies with large cohorts in both age groups are still needed to study SARS-CoV-2 pathogenesis with new tools and to understand how is possible to better modulate immune responses.

Novel evidence of Thymosin α1 immunomodulatory properties in SARS-CoV-2 infection: effect on innate inflammatory response in a peripheral blood mononuclear cell-based in vitro model

Graphical abstract The peculiar property of Thymosin alpha 1 (Tα1) to act as master regulator of immune homeostasis has been successfully defined in different physiological and pathological contexts ranging from cancer to infection. Interestingly, recent papers also demonstrated its mitigating effect on the "cytokine storm” as well as on the T-cell exhaustion/activation in SARS-CoV-2 infected individuals. Nevertheless, in spite of the increasing knowledge on Tα1-induced effects on T cell response confirming the distinctive features of this multifaceted peptide, little is known on its effects on innate immunity to SARS-CoV-2 infection. Here, we interrogated peripheral blood mononuclear cell (PBMC) cultures stimulated with SARS-CoV-2 to disclose Tα1 properties on the main cell players of early response to infection, namely monocytes and myeloid dendritic cells (mDC). Moving from ex vivo data showing an enhancement in the frequency of inflammatory monocytes and activated mDC in COVID-19 patients, a PBMC-based experimental setting reproduced in vitro a similar profile with an increased percentage of CD16+ inflammatory monocytes and mDC expressing CD86 and HLA-DR activation markers in response to SARS-CoV-2 stimulation. Interestingly, the treatment of SARS-CoV-2-stimulated PBMC with Tα1 dampened the inflammatory/activation status of both monocytes and mDC by reducing the release of pro-inflammatory mediators, including TNF-α, IL-6 and IL-8, while promoting the production of the anti-inflammatory cytokine IL-10. This study further clarifies the working hypothesis on Tα1 mitigating action on COVID-19 inflammatory condition. Moreover, these evidence shed light on inflammatory pathways and cell types involved in acute SARS-CoV-2 infection and likely targetable by newly immune-regulating therapeutic approaches.

Human Arboviral Infections in Italy: Past, Current, and Future Challenges.

Arboviruses represent a public health concern in many European countries, including Italy, mostly because they can infect humans, causing potentially severe emergent or re-emergent diseases, with epidemic outbreaks and the introduction of endemic circulation of new species previously confined to tropical and sub-tropical regions. In this review, we summarize the Italian epidemiology of arboviral infection over the past 10 years, describing both endemic and imported arboviral infections, vector distribution, and the influence of climate change on vector ecology. Strengthening surveillance systems at a national and international level is highly recommended to be prepared to face potential threats due to arbovirus diffusion.

Novel evidence of Thymosin α1 immunomodulatory properties in SARS-CoV-2 infection: Effect on innate inflammatory response in a peripheral blood mononuclear cell-based in vitro model.

The peculiar property of Thymosin alpha 1 (Tα1) to act as master regulator of immune homeostasis has been successfully defined in different physiological and pathological contexts ranging from cancer to infection. Interestingly, recent papers also demonstrated its mitigating effect on the "cytokine storm" as well as on the T-cell exhaustion/activation in SARS-CoV-2 infected individuals. Nevertheless, in spite of the increasing knowledge on Tα1-induced effects on T cell response confirming the distinctive features of this multifaceted peptide, little is known on its effects on innate immunity during SARS-CoV-2 infection. Here, we interrogated peripheral blood mononuclear cell (PBMC) cultures stimulated with SARS-CoV-2 to disclose Tα1 properties on the main cell players of early response to infection, namely monocytes and myeloid dendritic cells (mDC). Moving from ex vivo data showing an enhancement in the frequency of inflammatory monocytes and activated mDC in COVID-19 patients, a PBMC-based experimental setting reproduced in vitro a similar profile with an increased percentage of CD16+ inflammatory monocytes and mDC expressing CD86 and HLA-DR activation markers in response to SARS-CoV-2 stimulation. Interestingly, the treatment of SARS-CoV-2-stimulated PBMC with Tα1 dampened the inflammatory/activation status of both monocytes and mDC by reducing the release of pro-inflammatory mediators, including TNF-α, IL-6 and IL-8, while promoting the production of the anti-inflammatory cytokine IL-10. This study further clarifies the working hypothesis on Tα1 mitigating action on COVID-19 inflammatory condition. Moreover, these evidence shed light on inflammatory pathways and cell types involved in acute SARS-CoV-2 infection and likely targetable by newly immune-regulating therapeutic approaches.

Thymosin alpha 1 restores the immune homeostasis in lymphocytes during Post-Acute sequelae of SARS-CoV-2 infection.

The complex alterations of the immune system and the immune-mediated multiorgan injury plays a key role in host response to SARS-CoV-2 infection and in the pathogenesis of COVID-19, being also associated with adverse outcomes. Thymosin alpha 1 (Tα1) is one of the molecules used in the treatment of COVID-19, as it is known to restore the homeostasis of the immune system during infections and cancer. The use of Tα1 in COVID-19 patients had been widely used in China and in COVID-19 patients, it has been shown to decrease hospitalization rate, especially in those with greater disease severity, and reduce mortality by restoring lymphocytopenia and more specifically, depleted T cells. Persistent dysregulation with depletion of naive B and T cell subpopulations and expansion of memory T cells suggest a chronic stimulation of the immune response in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC). Our data obtained from an ex vivo study, showed that in PASC individuals with a chronically altered immune response, Tα1 improve the restoration of an appropriate response, most evident in those with more severe illness and who need respiratory support during acute phase, and in those with specific systemic and psychiatric symptoms of PASC, confirming Tα1 treatment being more effective in compromised patients. The results obtained, along with promising reports on recent trials on Tα1 administration in patients with COVID-19, offer new insights into intervention also for those patients with long-lasting inflammation with post-infectious symptoms, some of which have a delayed onset.

Sarilumab plus standard of care vs standard of care for the treatment of severe COVID-19: a phase 3, randomized, open-labeled, multi-center study (ESCAPE study).

Background: Among interleukin-6 inhibitors suggested for use in COVID-19, there are few robust evidences for the efficacy of sarilumab. Herein, we evaluated the efficacy and safety of sarilumab in severe COVID-19. Methods: In this phase 3, open-labeled, randomized clinical trial, conducted at 5 Italian hospitals, adults with severe COVID-19 pneumonia (excluding mechanically ventilated) were randomized 2:1 to receive intravenous sarilumab (400 mg, repeatable after 12 h) plus standard of care (SOC) (arm A) or to continue SOC (arm B). Randomization was web-based. As post-hoc analyses, the participants were stratified according to baseline inflammatory parameters. The primary endpoint was analysed on the modified Intention-To-Treat population, including all the randomized patients who received any study treatment (sarilumab or SOC). It was time to clinical improvement of 2 points on a 7-points ordinal scale, from baseline to day 30. We used Kaplan Meier method and log-rank test to compare the primary outcome between two arms, and Cox regression stratified by clinical center and adjusted for severity of illness, to estimate the hazard ratio (HR). The trial was registered with EudraCT (2020-001390-76). Findings: Between May 2020 and May 2021, 191 patients were assessed for eligibility, of whom, excluding nine dropouts, 176 were assigned to arm A (121) and B (55). At day 30, no significant differences in the primary endpoint were found (88% [95% CI 81-94] in arm A vs 85% [74-93], HR 1.07 [0.8-1.5] in arm B; log-rank p = 0.50). After stratifying for inflammatory parameters, arm A showed higher probability of improvement than B without statistical significance in the strata with C reactive protein (CRP) < 7 mg/dL (88% [77-96] vs 79% [63-91], HR 1.55 [0.9-2.6]; log-rank p = 0.049) and in the strata with lymphocytes <870/mmc (90% [79-96]) vs (73% [55-89], HR 1.53 [0.9-2.7]; log-rank p = 0.058). Overall, 39/121 (32%) AEs were reported in arm A and 14/55 (23%) in B (p = 0.195), while serious AEs were 22/121 (18%) and 7/55 (11%), respectively (p = 0.244). There were no treatment-related deaths. Interpretation: The efficacy of sarilumab in severe COVID-19 was not demonstrated both in the overall and in the stratified for severity analysis population. Exploratory analyses suggested that subsets of patients with lower CRP values or lower lymphocyte counts might have had benefit with sarilumab treatment, but this finding would require replication in other studies. The relatively low rate of concomitant corticosteroid use, could partially explain our results. Funding: This study was supported by INMI "Lazzaro Spallanzani" Ricerca Corrente Linea 1 on emerging and reemerging infections, funded by Italian Ministry of Health.

Admission eGFR predicts in-hospital mortality independently of admission glycemia and C-peptide in patients with type 2 diabetes mellitus and COVID-19.

OBJECTIVE: Type 2 diabetes mellitus (T2DM) and impaired kidney function are associated with a higher risk of poor outcomes of coronavirus disease 2019 (COVID-19). We conducted a retrospective study in hospitalized T2DM patients with COVID-19 to assess the association between in-hospital mortality and admission values of different hematological/biochemical parameters, including estimated glomerular filtration rate (eGFR), plasma glucose and C-peptide (the latter serving as a marker of beta-cell function). METHODS: The study included T2DM patients with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection who were consecutively admitted to our Institution between 1 October 2020 and 1 April 2021. RESULTS: Patients (n = 74) were categorized into survivors (n = 55) and non-survivors (n = 19). Non-survivors exhibited significantly higher median white blood cell (WBC) count, D-dimer, neutrophil-to-lymphocyte ratio, high-sensitivity C-reactive protein (hsCRP), and procalcitonin levels, as well as significantly lower median serum 25-hydroxyvitamin D [25(OH)D] levels compared to survivors. Non-survivors exhibited significantly higher median admission plasma glucose (APG) values compared to survivors (210 vs. 166 mg/dL; p = .026). There was no statistically significant difference in median values of (random) plasma C-peptide between non-survivors and survivors (3.55 vs. 3.24 ng/mL; p = .906). A significantly higher percentage of patients with an eGFR < 60 mL/min/1.73 m2 was observed in the non-survivor group as compared to the survivor group (57.9% vs. 23.6%; p = .006). A multivariate analysis performed by a logistic regression model after adjusting for major confounders (age, sex, body mass index, major comorbidities) showed a significant inverse association between admission eGFR values and risk of in-hospital mortality (OR, 0.956; 95% CI, 0.931-0.983; p = .001). We also found a significant positive association between admission WBC count and risk of in-hospital mortality (OR, 1.210; 95% CI, 1.043-1.404; p = .011). CONCLUSIONS: Admission eGFR and WBC count predict in-hospital COVID-19 mortality among T2DM patients, independently of traditional risk factors, APG and random plasma C-peptide. Hospitalized patients with COVID-19 and comorbid T2DM associated with impaired kidney function at admission should be considered at high risk for adverse outcomes and death.

Humoral Response after Two Doses of BNT162b2 mRNA Vaccine Has a Role in Predicting Response after Three Doses That Is Related to Plasma HIV Viremia and Nadir CD4+ Cell Count in HIV-Positive Patients.

We investigated the spike IgG levels of HIV+ patients on antiretroviral therapy six months after they received their second dose (T2) and six months after the third dose (T3) of the BNT162b2 mRNA vaccine, as well as the influence of different levels of plasma HIV viremia of overall CD4+ cell count and nadir value on the humoral time course. One hundred eighty-four patients were enrolled. The median age was 55 years, the median CD4+ cell count was 639 cells/mm3 and the median nadir value was 258 cells/mm3. On the basis of all tests performed during the study period, persistently undetectable plasma HIV RNA (PUD) was found in 66 patients, low-level viremia (LLV) in 57 and ongoing viremia (OV) in 61. Serum levels of IgG antibodies against a trimeric S-protein antigen were tested with DiaSorin Liaison SARS-CoV-2 TrimericS IgG and the response was classified as optimal (>75th percentile), intermediate (50th−25th percentile) and low (<25th percentile). The frequencies of the three different patterns of plasma HIV viremia (PUD, LLV and OV) were comparable in patients with low, intermediate and optimal IgG response evaluated at T2, with no difference in overall CD4+ cell count or nadir count. At T3, 92.9% of patients achieved an optimal response: T2 response proved to be the most important factor in predicting T3 optimal response in patients with LLV and OV.A nadir value ≤ 330 cells/mm3 had 100% sensitivity in predicting a non-optimal response. In conclusion, we demonstrated the persistence of anti-spike IgG, with high serum levels occurring in most patients six months after the third dose of the BNT162b2 mRNA vaccine and a predictive role of humoral response at T2 in subjects with detectable plasma HIV viremia. Immunological alterations related to past immunodeficiency may persist despite immune reconstitution, and the nadir value could be a useful tool for elaborating personalized vaccine schedules.

Remdesivir Influence on SARS-CoV-2 RNA Viral Load Kinetics in Nasopharyngeal Swab Specimens of COVID-19 Hospitalized Patients: A Real-Life Experience.

There are still conflicting data on the virological effects of the SARS-CoV-2 direct antivirals used in clinical practice, in spite of the documented clinical efficacy. The aim of this monocentric retrospective study was to compare virologic and laboratory data of patients admitted due to SARS-CoV-2 infection from March to December 2020 treated with either remdesivir (R), a protease inhibitor (lopinavir or darunavir/ritonavir (PI)) or no direct antiviral drugs (NT). Viral load variation was indirectly assessed through PCR cycle threshold (Ct) values on the nasopharyngeal swab, analyzing the results from swabs obtained at ward admission and 7 (±2) days later. Overall, 253 patients were included: patients in the R group were significantly older, more frequently males with a significantly higher percentage of severe COVID-19, requiring more often intensive care admission, compared to the other groups. Ct variation over time did not differ amongst the three treatment groups and did not seem to be influenced by corticosteroid use, even after normalization of the treatment groups for disease severity. Non-survivors had lower Ct on admission and showed a significantly slower viral clearance compared to survivors. CD4 T-lymphocytes absolute count assessed at ward admission correlated with a reduced Ct variation over time. In conclusion, viral clearance appears to be slower in COVID-19 non-survivors, while it seems not to be influenced by the antiviral treatment received.

B- and T-Cell Responses After SARS-CoV-2 Vaccination in Patients With Multiple Sclerosis Receiving Disease Modifying Therapies: Immunological Patterns and Clinical Implications.

Background: Vaccination campaign to contrast the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has raised the issue of vaccine immunogenicity in special populations such as people with multiple sclerosis (PwMS) on highly effective disease modifying treatments (DMTs). While humoral responses to SARS-CoV-2 mRNA vaccines have been well characterized in the general population and in PwMS, very little is known about cell-mediated responses in conferring protection from SARS-CoV-2 infection and severe coronavirus disease-2019 (COVID-19). Methods: PwMS on ocrelizumab, fingolimod or natalizumab, vaccinated with two doses of mRNABNT162b2 (Comirnaty®) vaccine were enrolled. Anti-Spike (S) and anti-Nucleoprotein (N) antibody titers, IFN-gamma production upon S and N peptide libraries stimulation, peripheral blood lymphocyte absolute counts were assessed after at least 1 month and within 4 months from vaccine second dose administration. A group of age and sex matched healthy donors (HD) were included as reference group. Statistical analysis was performed using GraphPad Prism 8.2.1. Results: Thirty PwMS and 9 HDs were enrolled. All the patients were negative for anti-N antibody detection, nor reported previous symptoms of COVID-19. Peripheral blood lymphocyte counts were assessed in PwMS showing: (i) reduction of circulating B-lymphocytes in PwMS on ocrelizumab; (ii) reduction of peripheral blood B- and T-lymphocyte absolute counts in PwMS on fingolimod and (iii) normal B- and T-lymphocyte absolute counts with an increase in circulating CD16+CD56+ NK-cells in PwMS on natalizumab. Three patterns of immunological responses were identified in PwMS. In patients on ocrelizumab, anti-S antibody were lacking or reduced, while T-cell responses were normal. In patients on fingolimod both anti-S titers and T-cell mediated responses were impaired. In patients on natalizumab both anti-S titers and T-cell responses were present and comparable to those observed in HD. Conclusions: The evaluation of T-cell responses, anti-S titers and peripheral blood lymphocyte absolute count in PwMS on DMTs can help to better characterize the immunological response after SARS-CoV-2 vaccination. The evaluation of T-cell responses in longitudinal cohorts of PwMS will help to clarify their protective role in preventing SARS-CoV-2 infection and severe COVID-19. The correlation between DMT treatment and immunological responses to SARS-CoV-2 vaccines could help to better evaluate vaccination strategies in PwMS.

Validation of the T-Lymphocyte Subset Index (TLSI) as a Score to Predict Mortality in Unvaccinated Hospitalized COVID-19 Patients.

Lymphopenia has been consistently reported as associated with severe coronavirus disease 2019 (COVID-19). Several studies have described a profound decline in all T-cell subtypes in hospitalized patients with severe and critical COVID-19. The aim of this study was to assess the role of T-lymphocyte subset absolute counts measured at ward admission in predicting 30-day mortality in COVID-19 hospitalized patients, validating a new prognostic score, the T-Lymphocyte Subset Index (TLSI, range 0-2), based on the number of T-cell subset (CD4+ and CD8+) absolute counts that are below prespecified cutoffs. These cutoff values derive from a previously published work of our research group at Policlinico Tor Vergata, Rome, Italy: CD3+CD4+ < 369 cells/µL, CD3+CD8+ < 194 cells/µL. In the present single-center retrospective study, T-cell subsets were assessed on admission to the infectious diseases ward. Statistical analysis was performed using JASP (Version 0.16.2. JASP Team, 2022, Amsterdam, The Netherlands) and Prism8 (version 8.2.1. GraphPad Software, San Diego, CA, USA). Clinical and laboratory parameters of 296 adult patients hospitalized because of COVID-19 were analyzed. The overall mortality rate was 22.3% (66/296). Survivors (S) had a statistically significant lower TLSI score compared to non-survivors (NS) (p < 0.001). Patients with increasing TLSI scores had proportionally higher rates of 30-day mortality (p < 0.0001). In the multivariable logistic analysis, the TLSI was an independent predictor of in-hospital 30-day mortality (OR: 1.893, p = 0.003). Survival analysis showed that patients with a TLSI > 0 had an increased risk of death compared to patients with a TLSI = 0 (hazard ratio: 2.83, p < 0.0001). The TLSI was confirmed as an early and independent predictor of COVID-19 in-hospital 30-day mortality.

Neuro-Axonal Damage and Alteration of Blood-Brain Barrier Integrity in COVID-19 Patients.

Neurofilament light chain (NfL) is a specific biomarker of neuro-axonal damage. Matrix metalloproteinases (MMPs) are zinc-dependent enzymes involved in blood-brain barrier (BBB) integrity. We explored neuro-axonal damage, alteration of BBB integrity and SARS-CoV-2 RNA presence in COVID-19 patients with severe neurological symptoms (neuro-COVID) as well as neuro-axonal damage in COVID-19 patients without severe neurological symptoms according to disease severity and after recovery, comparing the obtained findings with healthy donors (HD). Overall, COVID-19 patients (n = 55) showed higher plasma NfL levels compared to HD (n = 31) (p < 0.0001), especially those who developed ARDS (n = 28) (p = 0.0005). After recovery, plasma NfL levels were still higher in ARDS patients compared to HD (p = 0.0037). In neuro-COVID patients (n = 12), higher CSF and plasma NfL, and CSF MMP-2 levels in ARDS than non-ARDS group were observed (p = 0.0357, p = 0.0346 and p = 0.0303, respectively). SARS-CoV-2 RNA was detected in four CSF and two plasma samples. SARS-CoV-2 RNA detection was not associated to increased CSF NfL and MMP levels. During COVID-19, ARDS could be associated to CNS damage and alteration of BBB integrity in the absence of SARS-CoV-2 RNA detection in CSF or blood. CNS damage was still detectable after discharge in blood of COVID-19 patients who developed ARDS during hospitalization.

Increased Mild Vaccine-Related Side Effects and Higher Specific Antibody Titers in Health Care Workers with Previous SARS-CoV-2 Infection after the mRNA BNT162b2 Vaccine.

BACKGROUND: to evaluate whether prior SARS-CoV-2 infection affects side effects and specific antibody production after vaccination with BNT162b2. METHODS: We included 1106 health care workers vaccinated with BNT162b2. We assessed whether prior SARS-CoV-2 infection affects the number and type of side effects and performed a nested case-control analysis comparing plasma levels of specific IgG titers between SARS-CoV-2-naïve and previously infected subjects after the first and the second vaccine doses. RESULTS: After the first dose, SARS-CoV-2-naïve subjects experienced side effects more often than SARS-CoV-2 naïve subjects. Individuals with prior SARS-CoV-2 infection more often reported pain at the injection site, weakness, and fever than SARS-CoV-2-naïve subjects. After the second dose, the frequency of side effects was similar in the two groups. All subjects with prior SARS-CoV-2 infection developed either a high (>100 AU/mL) or intermediate (10-100 AU/mL) antibody titer. Among SARS-CoV-2-naïve subjects, the majority developed an intermediate titer. After the second dose, a high (>2000 AU/mL) antibody titer was more common among subjects with prior SARS-CoV-2 infection. CONCLUSIONS: vaccine-related side effects and a higher anti-SARS-CoV-2-RBD IgG titer were more common in subjects with previous infection than in SARS-CoV-2-naïve after the first, but not after the second dose of the BNT162b2 vaccine.

Long-Term Longitudinal Analysis of Neutralizing Antibody Response to Three Vaccine Doses in a Real-Life Setting of Previously SARS-CoV-2 Infected Healthcare Workers: A Model for Predicting Response to Further Vaccine Doses.

We report the time course of neutralizing antibody (NtAb) response, as measured by authentic virus neutralization, in healthcare workers (HCWs) with a mild or asymptomatic SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection diagnosed at the onset of the pandemic, with no reinfection throughout and after a three-dose schedule of the BNT162b2 mRNA vaccine with an overall follow-up of almost two years since infection. Forty-eight HCWs (median age 47 years, all immunocompetent) were evaluated: 29 (60.4%) were asymptomatic. NtAb serum was titrated at eight subsequent time points: T1 and T2 were after natural infection, T3 on the day of the first vaccine dose, T4 on the day of the second dose, T5, T6, and T7 were between the second and third dose, and T8 followed the third dose by a median of 34 days. NtAb titers at all postvaccination time points (T4 to T8) were significantly higher than all those at prevaccination time points (T1 to T3). The highest NtAb increase was following the first vaccine dose while subsequent doses did not further boost NtAb titers. However, the third vaccine dose appeared to revive waning immunity. NtAb levels were positively correlated at most time points suggesting an important role for immunogenetics.

Early and Polyantigenic CD4 T Cell Responses Correlate with Mild Disease in Acute COVID-19 Donors.

We assessed SARS-CoV-2-specific CD4+ and CD8+ T cell responses in samples from 89 acute COVID-19 patients, utilizing blood samples collected during the first wave of COVID-19 in Italy. The goal of the study was to examine correlations between SARS-CoV-2-specific T cell responses in the early phase comparing mild, moderate, or severe COVID-19 disease outcomes. T cell responses to the spike (S) and non-S proteins were measured in a combined activation-induced marker (AIM) and intracellular cytokine staining (ICS) assay. Early CD4+ T cell responses to SARS-CoV-2 S correlated with milder disease by both AIM and IFNγ ICS readouts. The correlation of S-specific CD4+ T cell responses with milder disease severity was most striking within the first two weeks of symptom onset compared to later time points. Furthermore, donors with milder disease were associated with polyantigenic CD4+ T cell responses that recognized more prominently non-S proteins in addition to S, while severe acute COVID-19 was characterized by lower magnitudes of CD4+ T cell responses and a narrower repertoire. In conclusion, this study highlights that both the magnitude and breadth of early SARS-CoV-2-specific CD4+ T cell responses correlated with milder disease outcomes in acute COVID-19 patients.

In Patients with Severe COVID-19, the Profound Decrease in the Peripheral Blood T-Cell Subsets Is Correlated with an Increase of QuantiFERON-TB Gold Plus Indeterminate Rates and Reflecting a Reduced Interferon-Gamma Production.

Increased rates of indeterminate QuantiFERON-TB Gold Plus Assay (QFT-Plus) were demonstrated in patients hospitalized with Coronavirus Disease (COVID)-19. We aimed to define the prevalence and characteristics of hospitalized COVID-19 patients with indeterminate QFT-Plus. A retrospective study was performed including hospitalized COVID-19 patients, stratified in survivors and non-survivors, non-severe and severe according to the maximal oxygen supply required. Statistical analysis was performed using JASP ver0.14.1 and GraphPad Prism ver8.2.1. A total of 420 patients were included, median age: 65 years, males: 66.4%. The QFT-Plus was indeterminate in 22.1% of patients. Increased rate of indeterminate QFT-Plus was found in non-survivors (p = 0.013) and in severe COVID-19 patients (p < 0.001). Considering the Mitogen-Nil condition of the QFT-Plus, an impaired production of interferon-gamma (IFN-γ) was found in non-survivors (p < 0.001) and in severe COVID-19 patients (p < 0.001). A positive correlation between IFN-γ levels in the Mitogen-Nil condition and the absolute counts of CD3+ (p < 0.001), CD4+ (p < 0.001), and CD8+ (p < 0.001) T-lymphocytes was found. At the multivariable analysis, CD3+ T-cell absolute counts and CD4/CD8 ratio were confirmed as independent predictors of indeterminate results at the QFT-Plus. Our study confirmed the increased rate of indeterminate QFT-Plus in COVID-19 patients, mainly depending on the peripheral blood T-lymphocyte depletion found in the most severe cases.

B- and T-Cell Responses After SARS-CoV-2 Vaccination in Patients With Multiple Sclerosis Receiving Disease Modifying Therapies: Immunological Patterns and Clinical Implications

Background Vaccination campaign to contrast the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has raised the issue of vaccine immunogenicity in special populations such as people with multiple sclerosis (PwMS) on highly effective disease modifying treatments (DMTs). While humoral responses to SARS-CoV-2 mRNA vaccines have been well characterized in the general population and in PwMS, very little is known about cell-mediated responses in conferring protection from SARS-CoV-2 infection and severe coronavirus disease-2019 (COVID-19). Methods PwMS on ocrelizumab, fingolimod or natalizumab, vaccinated with two doses of mRNABNT162b2 (Comirnaty®) vaccine were enrolled. Anti-Spike (S) and anti-Nucleoprotein (N) antibody titers, IFN-gamma production upon S and N peptide libraries stimulation, peripheral blood lymphocyte absolute counts were assessed after at least 1 month and within 4 months from vaccine second dose administration. A group of age and sex matched healthy donors (HD) were included as reference group. Statistical analysis was performed using GraphPad Prism 8.2.1. Results Thirty PwMS and 9 HDs were enrolled. All the patients were negative for anti-N antibody detection, nor reported previous symptoms of COVID-19. Peripheral blood lymphocyte counts were assessed in PwMS showing: (i) reduction of circulating B-lymphocytes in PwMS on ocrelizumab;(ii) reduction of peripheral blood B- and T-lymphocyte absolute counts in PwMS on fingolimod and (iii) normal B- and T-lymphocyte absolute counts with an increase in circulating CD16+CD56+ NK-cells in PwMS on natalizumab. Three patterns of immunological responses were identified in PwMS. In patients on ocrelizumab, anti-S antibody were lacking or reduced, while T-cell responses were normal. In patients on fingolimod both anti-S titers and T-cell mediated responses were impaired. In patients on natalizumab both anti-S titers and T-cell responses were present and comparable to those observed in HD. Conclusions The evaluation of T-cell responses, anti-S titers and peripheral blood lymphocyte absolute count in PwMS on DMTs can help to better characterize the immunological response after SARS-CoV-2 vaccination. The evaluation of T-cell responses in longitudinal cohorts of PwMS will help to clarify their protective role in preventing SARS-CoV-2 infection and severe COVID-19. The correlation between DMT treatment and immunological responses to SARS-CoV-2 vaccines could help to better evaluate vaccination strategies in PwMS.