Seasonal Betacoronavirus Antibodies' Expansion Post-BNT161b2 Vaccination Associates with Reduced SARS-CoV-2 VoC Neutralization.

SARS-CoV-2 vaccination is known to induce antibodies that recognize also variants of concerns (VoCs) of the virus. However, epidemiological and laboratory evidences indicate that these antibodies have a reduced neutralization ability against VoCs. We studied binding and neutralizing antibodies against the Spike protein domains and subunits of the Wuhan-Hu-1 virus and its alpha, beta, delta VoCs and of seasonal betacoronaviruses (HKU1 and OC43) in a cohort of 31 health care workers prospectively followed post-vaccination with BNT162b2-Comirnaty. The study of sequential samples collected up to 64 days post-vaccination showed that serological assays measuring IgG against Wuhan-Hu-1 antigens were a poor proxy for VoC neutralization. In addition, in subjects who had asymptomatic or mild COVID-19 prior to vaccination, the loss of nAbs following disease could be rapid and accompanied by post-vaccination antibody levels similar to those of naïve vaccinees. Interestingly, in health care workers naïve for SARS-CoV-2 infection, vaccination induced a rapid and transient reactivation of pre-existing seasonal coronaviruses IgG responses that was associated with a subsequent reduced ability to neutralize alpha and beta VoCs.

Mindfulness-Based Interventions for the Improvement of Well-Being in People With Multiple Sclerosis: A Systematic Review and Meta-Analysis.

OBJECTIVE: The study aims to meta-analytically review studies about the effects of mindfulness-based interventions (MBIs) on well-being of people with multiple sclerosis (MS). METHODS: Seven electronic databases were searched from June 2018 to September 2018. A systematic review and a meta-analysis were conducted. RESULTS: Twenty-one studies were included in qualitative synthesis, and 10 studies were included in meta-analysis. MBIs are effective with an overall moderate effect size (Hedges' g = 0.70) in improving well-being in people with MS, with lasting effects at the follow-up (g = 0.55). In particular, MBIs demonstrated to highly reduce stress (g = 1.07) and to improve depression and anxiety symptoms with a moderate to large effect at postintervention (g = 0.77 and g = 0.63, respectively). CONCLUSIONS: MBIs represent a valid and effective mind-body intervention to improve the well-being of patients with MS. Further studies should investigate which components of MBIs could be more beneficial for patients with progressive MS. PROSPERO REGISTRATION: CRD42018099704.

Adjustment to multiple sclerosis and identity satisfaction among newly diagnosed women: what role does motherhood play?

The present study aimed to describe the levels of depressive symptoms, affective well-being and identity satisfaction in a group of women recently diagnosed with multiple sclerosis (MS), accounting for differences in age, motherhood, and disease duration. Moreover, the role of identity satisfaction in depressive symptoms and affective well-being was evaluated, examining the moderating effect of motherhood. The study involved 74 women, aged between 19 and 57 years (Mean = 37.7 years, SD = 10.7 years). Thirty-two women (43.2%) had children, aged between 2 and 29 years. All women had relapsing-remitting multiple sclerosis (RRMS) and mild to moderate disability. Mothers experienced greater depressive symptoms than childless women. Moreover, motherhood moderated the effect of disease duration on adjustment, with mothers reporting greater depressive symptoms, less affective well-being and less identity satisfaction than childless women as time passed since the diagnosis. Finally, greater identity satisfaction was related to less depressive symptoms and greater affective well-being, with a moderating effect of motherhood. The results outline the relevance of the process of identity redefinition for women's adjustment to MS early in the illness. Moreover, the results underscore the need to take into account the additional burden of motherhood when promoting women's adjustment to MS.

Prevalence of Posttraumatic Stress Disorder in Patients With Multiple Sclerosis.

Chronic and life-threatening illnesses, such as multiple sclerosis (MS), have been identified as significant stressors potentially triggering posttraumatic stress disorder (PTSD). The study aims to investigate the prevalence of PTSD according to Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, Text Revision (DSM-IV-TR) criteria in a large sample of patients with MS. A total of 988 patients with MS were screened with the Impact of Event Scale-Revised, and then assessed with the PTSD module of the Structured Clinical Interview for DSM-IV and with the Clinician-Administered PTSD Scale to confirm PTSD diagnosis. Posttraumatic symptoms were reported by 25.5% of the sample. A confirmed diagnosis of PTSD was found in 5.7% of patients, but prevalence could reach 8.5%, including also dropout patients. Further studies are needed to evaluate if adjustment disorder could better encompass the frequently encountered subthreshold posttraumatic stress symptoms and how clinicians can deal with these symptoms with appropriate interventions.

Coping strategies and adjustment to multiple sclerosis among recently diagnosed patients: the mediating role of sense of coherence.

OBJECTIVE: To examine the relationship between coping strategies (problem solving, emotional release, and avoidance) and adjustment (health-related quality of life, depression, and affective well-being) in a group of recently diagnosed multiple sclerosis patients (up to three years since diagnosis), and to explore the mediating role of sense of coherence between coping strategies and adjustment. DESIGN: Cross-sectional. SETTING: Multiple Sclerosis Clinic Centre. SUBJECTS: A total of 102 patients (61.8% women; age (years): M = 35.8, SD = 11.9; 95% with a relapsing-remitting form of multiple sclerosis; Expanded Disability Status Scale score, between 1 and 4). INTERVENTIONS: Not applicable. MAIN MEASURES: Coping with multiple sclerosis (problem solving, emotional release, and avoidance), sense of coherence, health-related quality of life (SF-12), depression (CES-D), and affective well-being (PANAS). RESULTS: Problem solving was linked to higher mental health ( ß = 0.28) and higher affective well-being ( ß = 0.36), emotional release was related to lower depression ( ß = -0.22); avoidance was associated to higher mental health ( ß = 0.25), higher affective well-being ( ß = 0.24), and lower depression ( ß = -0.29 ) (all betas were significant at p < 0.05). Sense of coherence mediated the relationship between emotional release and depression (Sobel z-value = -2.00; p < 0.05) and the relationship between avoidance and all the indicators of adjustment (mental health: Sobel z-value = 1.97; depression: Sobel z-value = -2.02; affective well-being: Sobel z-value= 2.05; p < 0.05). CONCLUSIONS: Emotional and avoidant coping strategies seem to be adaptive among recently diagnosed multiple sclerosis patients. A mediating role between coping strategies and adjustment is played by sense of coherence.

The efficacy of a Mindfulness Based Intervention for depressive symptoms in patients with Multiple Sclerosis and their caregivers: study protocol for a randomized controlled clinical trial.

BACKGROUND: Multiple Sclerosis has a great impact on psychological functioning of patients and can be associated with various mental health disorders and symptoms. The most prevalent one is depression, which ranges from 15 to 47%. Mindfulness Based Interventions are a relatively brief and cost-effective program that has been studied in patients with several chronic diseases and recently also in patients with Multiple Sclerosis. Mindfulness Based Interventions are based on the assumption that a non-judgmental awareness and acceptance of one's moment-to-moment experience can have a positive effect on the adaptation to the disease, reducing the psychological burden and improving patients' quality of life. Several studies concluded that Mindfulness Based Interventions can be beneficial in terms of improving both psychological and psychical aspects of Multiple Sclerosis, but none of them compared the intervention with an active control group. The primary objective of the study is to evaluate the efficacy of a group-based Mindfulness Based Intervention on depressive symptoms in patients with Multiple Sclerosis, as compared with an active control group. METHODS: The study design is a randomized controlled clinical trial. Eighty-eight patients with Multiple Sclerosis and depressive symptoms will be recruited and randomized to either Mindfulness Based Intervention or an active control group. The latter is designed to control for non-specific elements of the intervention and it comprises psycho-education and relaxation techniques. The primary outcome is the reduction of depressive symptoms as measured via the Beck Depressive Inventory-II. Secondary outcome measures are level of quality of life, anxiety, perceived stress, illness perception, fatigue and quality of interpersonal relationship. Outcomes will be assessed at baseline, after treatment and 6 months after the end of the treatment. Caregivers will participate in groups together with patients. DISCUSSION: As far as we know this trial will be the first randomized controlled trial testing the efficacy of group-based Mindfulness Based Intervention for patients with Multiple Sclerosis with a comparison with an active control group with a specific focus on depressive symptoms. TRIAL REGISTRATION: NCT02611401.

Proton pump inhibitors while belonging to the same family of generic drugs show different anti-tumor effect.

CONTEXT: Tumor acidity represents a major cause of chemoresistance. Proton pump inhibitors (PPIs) can neutralize tumor acidity, sensitizing cancer cells to chemotherapy. OBJECTIVE: To compare the anti-tumor efficacy of different PPIs in vitro and in vivo. MATERIALS AND METHODS: In vitro experiments PPIs anti-tumor efficacy in terms of cell proliferation and cell death/apoptosis/necrosis evaluation were performed. In vivo PPIs efficacy experiments were carried out using melanoma xenograft model in SCID mice. RESULTS: Lansoprazole showed higher anti-tumor effect when compared to the other PPIs. The lansoprazole effect lasted even upon drug removal from the cell culture medium and it was independent from the lipophilicity of the PPIs formulation. DISCUSSION: These PPIs have shown different anti-tumoral efficacy, and the most effective at low dose was lansoprazole. CONCLUSION: The possibility to contrast tumor acidity by off-label using PPIs opens a new field of oncology investigation.

The effects of a group-based cognitive behavioral therapy on people with multiple sclerosis: a randomized controlled trial.

OBJECTIVE: To evaluate the effectiveness of a cognitive behavioral group-based intervention aimed at reducing depression and fostering quality of life and psychological well-being of multiple sclerosis patients through the promotion of identity redefinition, sense of coherence, and self-efficacy. DESIGN: A randomized controlled trial. SETTING: Non-medical setting, external to the Multiple Sclerosis Clinic Centre. SUBJECTS: Eighty-two patients: 64% women; mean age 40.5, SD = 9.4; 95% with relapsing-remitting multiple sclerosis; Expanded Disability Status Scale (EDSS) between 1 and 5.5 were included in the study. INTERVENTIONS: Patients were randomly assigned to an intervention group (five cognitive behavioral group-based sessions, n = 41) or to a control group (three informative sessions, n = 41). MAIN MEASURES: Depression (CES-D), Quality of life (MSQOL revised), Psychological well-being (PANAS), Identity Motives Scale, Sense of Coherence (SOC), and Self Efficacy in Multiple Sclerosis. RESULTS: Quality of life increased in the intervention group compared with the control at 6-months follow-up (mean change 0.72 vs. -1.76, p < 0.05). Well-being in the intervention group increased for males and slightly decreased for females at 6-months follow-up (mean change 6.58 vs. -0.82, p < 0.05). Contrasts revealed an increase in self-efficacy in the intervention group at posttreatment compared with the control (mean change 2.95 vs. -0.11, p < 0.05). Depression tended to lower, while identity and coherence increased in the intervention group compared with the control, though the differences were not significant. CONCLUSIONS: Preliminary evidence suggests that intervention promotes patients' quality of life and has an effect on psychological well-being and self-efficacy.

Prevalence and significant determinants of post-traumatic stress disorder in a large sample of patients with multiple sclerosis.

Chronic and life-threatening neurodegenerative diseases may be associated with post-traumatic stress disorder (PTSD). Therefore, the current study was an investigation of the prevalence of PTSD in multiple sclerosis (MS) patients, and identification of significant determinants of PTSD. Two hundred thirty-two MS patients were consecutively recruited and screened for the presence of PTSD with the Impact of Event Scale-Revised, corroborated by the Structured Clinical Interview for DSM-IV. Furthermore, participants were administered the Hospital Anxiety and Depression Scale and the Fatigue Severity Scale. Twelve patients (12/232, i.e. 5.17 %) were diagnosed as suffering from PTSD. Levels of education, anxiety and depression were significant determinants of the presence of PTSD. The role played by the levels of education, anxiety and depression in determining the presence of PTSD has been discussed. Further research on the psychological features of neurodegenerative diseases is urgently needed in order to plan appropriate treatments and improve patients' quality of life.

Different configurations of SARS-CoV-2 spike protein delivered by integrase-defective lentiviral vectors induce persistent functional immune responses, characterized by distinct immunogenicity profiles.

Several COVID-19 vaccine strategies utilizing new formulations for the induction of neutralizing antibodies (nAbs) and T cell immunity are still under evaluation in preclinical and clinical studies. Here we used Simian Immunodeficiency Virus (SIV)-based integrase defective lentiviral vector (IDLV) delivering different conformations of membrane-tethered Spike protein in the mouse immunogenicity model, with the aim of inducing persistent nAbs against multiple SARS-CoV-2 variants of concern (VoC). Spike modifications included prefusion-stabilizing double proline (2P) substitutions, mutations at the furin cleavage site (FCS), D614G mutation and truncation of the cytoplasmic tail (delta21) of ancestral and Beta (B.1.351) Spike, the latter mutation to markedly improve IDLV membrane-tethering. BALB/c mice were injected once with IDLV delivering the different forms of Spike or the recombinant trimeric Spike protein with 2P substitutions and FCS mutations in association with a squalene-based adjuvant. Anti-receptor binding domain (RBD) binding Abs, nAbs and T cell responses were detected up to six months from a single immunization with escalating doses of vaccines in all mice, but with different levels and kinetics. Results indicated that IDLV delivering the Spike protein with all the combined modifications, outperformed the other candidates in terms of T cell immunity and level of both binding Abs and nAbs soon after the single immunization and persistence over time, showing the best capacity to neutralize all formerly circulating VoC Alpha, Beta, Gamma and Delta. Although present, the lowest response was detected against Omicron variants (BA.1, BA.2 and BA.4/5), suggesting that the magnitude of immune evasion may be related to the higher genetic distance of Omicron as indicated by increased number of amino acid substitutions in Spike acquired during virus evolution.

New Monoclonal Antibodies Specific for Different Epitopes of the Spike Protein of SARS-CoV-2 and Its Major Variants: Additional Tools for a More Specific COVID-19 Diagnosis.

The emergence of the new pathogen SARS-CoV-2 determined a rapid need for monoclonal antibodies (mAbs) to detect the virus in biological fluids as a rapid tool to identify infected individuals to be treated or quarantined. The majority of commercially available antigenic tests for SARS-CoV-2 rely on the detection of N antigen in biologic fluid using anti-N antibodies, and their capacity to specifically identify subjects infected by SARS-CoV-2 is questionable due to several structural analogies among the N proteins of different coronaviruses. In order to produce new specific antibodies, BALB/c mice were immunized three times at 20-day intervals with a recombinant spike (S) protein. The procedure used was highly efficient, and 40 different specific mAbs were isolated, purified and characterized, with 13 ultimately being selected for their specificity and lack of cross reactivity with other human coronaviruses. The specific epitopes recognized by the selected mAbs were identified through a peptide library and/or by recombinant fragments of the S protein. In particular, the selected mAbs recognized different linear epitopes along the S1, excluding the receptor binding domain, and along the S2 subunits of the S protein of SARS-CoV-2 and its major variants of concern. We identified combinations of anti-S mAbs suitable for use in ELISA or rapid diagnostic tests, with the highest sensitivity and specificity coming from proof-of-concept tests using recombinant antigens, SARS-CoV-2 or biological fluids from infected individuals, that represent important additional tools for the diagnosis of COVID-19.

Persistent immunogenicity of integrase defective lentiviral vectors delivering membrane-tethered native-like HIV-1 envelope trimers.

Integrase Defective Lentiviral Vectors (IDLVs) represent an attractive vaccine platform for delivering HIV-1 antigens, given their ability to induce specific and persistent immune responses in both mice and non-human primates (NHPs). Recent advances in HIV-1 immunogen design demonstrated that native-like HIV-1 Envelope (Env) trimers that mimic the structure of virion-associated Env induce neutralization breadth in rabbits and macaques. Here, we describe the development of an IDLV-based HIV-1 vaccine expressing either soluble ConSOSL.UFO.664 or membrane-tethered ConSOSL.UFO.750 native-like Env immunogens with enhanced bNAb epitopes exposure. We show that IDLV can be pseudotyped with properly folded membrane-tethered native-like UFO.750 trimers. After a single IDLV injection in BALB/c mice, IDLV-UFO.750 induced a faster humoral kinetic as well as higher levels of anti-Env IgG compared to IDLV-UFO.664. IDLV-UFO.750 vaccinated cynomolgus macaques developed unusually long-lasting anti-Env IgG antibodies, as underlined by their remarkable half-life both after priming and boost with IDLV. After boosting with recombinant ConM SOSIP.v7 protein, two animals developed neutralization activity against the autologous tier 1B ConS virus mediated by V1/V2 and V3 glycan sites responses. By combining the possibility to display stabilized trimeric Env on the vector particles with the ability to induce sustained humoral responses, IDLVs represent an appropriate strategy for delivering rationally designed antigens to progress towards an effective HIV-1 vaccine.

Long-term Cognitive Outcomes and Socioprofessional Attainment in People With Multiple Sclerosis With Childhood Onset.

BACKGROUND AND OBJECTIVES: Patients with pediatric-onset multiple sclerosis (MS) can be especially vulnerable to cognitive impairment (CI) due to the onset of MS during a critical period for CNS development and maturation. The objective of this longitudinal study was to assess long-term cognitive functioning and socioprofessional attainment in the Italian pediatric MS cohort, previously assessed at baseline and 2 and 5 years. METHODS: The 48 patients evaluated at the 5-year assessment were screened for inclusion. All participants were assessed with a cognitive test battery exploring 4 different cognitive abilities. Depression, fatigue, and socioprofessional attainment were also assessed. Mean cognitive z scores were calculated for the whole cohort, and their evolution over time was analyzed with an analysis of variance for repeated measurements test. Predictors of cognitive worsening or improvement were assessed with a linear mixed-model analysis. RESULTS: Thirty-three participants were included (mean follow-up 12.8 ± 0.8 years). The global cognitive performance worsened at year 2 and improved at year 5, although the z score remained significantly lower than at baseline (-0.9 ± 1.2 vs -0.3 ± 0.9, p = 0.002). There was no significant variation between years 5 and 12 (-0.7 ± 1.1, p = 0.452). Higher IQ (>90) at baseline (effect 0.3, 95% CI 0.1-0.5, p = 0.017) and lower number of relapses in the 2 years before baseline (effect -0.1, 95% CI -0.1 to 0.1, p = 0.025) predicted better cognitive performances. Eighteen (54.5%) patients failed at least 2 tests compared with healthy controls and were defined as cognitively impaired. The presence of CI predicted worse socioprofessional attainment (ß = 4.8, 95% CI 1.4-8.2, p = 0.008). DISCUSSION: The longitudinal cognitive trajectory in pediatric-onset MS has a heterogeneous course over time, with a decline in the first years followed by a partial recovery over the long term. However, at the last follow-up evaluation, the proportion of impaired patients was more than double compared with baseline, with a negative impact on the individual's socioprofessional attainment in adulthood. This study underscores how cognitive reserve may partially mitigate the negative effects of brain damage, highlighting the critical importance of intellectual enrichment early during the disease course.

The human homologue of Dictyostelium discoideum phg1A is expressed by human metastatic melanoma cells.

Tumour cannibalism is a characteristic of malignancy and metastatic behaviour. This atypical phagocytic activity is a crucial survival option for tumours in conditions of low nutrient supply, and has some similarities to the phagocytic activity of unicellular microorganisms. In fact, Dictyostelium discoideum has been used widely as a model to study phagocytosis. Recently, phg1A has been described as a protein that is primarily involved in the phagocytic process of this microorganism. The closest human homologue to phg1A is transmembrane 9 superfamily protein member 4 (TM9SF4). Here, we report that TM9SF4 is highly expressed in human malignant melanoma cells deriving from metastatic lesions, whereas it is undetectable in healthy human tissues and cells. TM9SF4 is predominantly expressed in acidic vesicles of melanoma cells, in which it co-localizes with the early endosome antigens Rab5 and early endosome antigen 1. TM9SF4 silencing induced marked inhibition of cannibal activity, which is consistent with a derangement of intracellular pH gradients, with alkalinization of acidic vesicles and acidification of the cell cytosol. We propose TM9SF4 as a new marker of malignancy, representing a potential new target for anti-tumour strategies with a specific role in tumour cannibalism and in the establishment of a metastatic phenotype.

Safety and efficiency modifications of SIV-based integrase-defective lentiviral vectors for immunization.

Integrase-defective lentiviral vectors (IDLVs) represent an attractive platform for vaccine development as a result of the ability to induce persistent humoral- and cellular-mediated immune responses against the encoded transgene. Compared with the parental integrating vector, the main advantages for using IDLV are the reduced hazard of insertional mutagenesis and the decreased risk for vector mobilization by wild-type viruses. Here we report on the development and use in the mouse immunogenicity model of simian immunodeficiency virus (SIV)-based IDLV containing a long deletion in the U3 region and with the 3' polypurine tract (PPT) removed from the transfer vector for improving safety and/or efficacy. Results show that a safer extended deletion of U3 sequences did not modify integrase-mediated or -independent integration efficiency. Interestingly, 3' PPT deletion impaired integrase-mediated integration but did not reduce illegitimate, integrase-independent integration efficiency, contrary to what was previously reported in the HIV system. Importantly, although the extended deletion in the U3 did not affect expression or immunogenicity from IDLV, deletion of 3' PPT considerably reduced both expression and immunogenicity of IDLV.

Integrase-Defective Lentiviral Vector Is an Efficient Vaccine Platform for Cancer Immunotherapy.

Integrase-defective lentiviral vectors (IDLVs) have been used as a safe and efficient delivery system in several immunization protocols in murine and non-human primate preclinical models as well as in recent clinical trials. In this work, we validated in preclinical murine models our vaccine platform based on IDLVs as delivery system for cancer immunotherapy. To evaluate the anti-tumor activity of our vaccine strategy we generated IDLV delivering ovalbumin (OVA) as a non-self-model antigen and TRP2 as a self-tumor associated antigen (TAA) of melanoma. Results demonstrated the ability of IDLVs to eradicate and/or controlling tumor growth after a single immunization in preventive and therapeutic approaches, using lymphoma and melanoma expressing OVA. Importantly, LV-TRP2 but not IDLV-TRP2 was able to break tolerance efficiently and prevent tumor growth of B16F10 melanoma cells. In order to improve the IDLV efficacy, the human homologue of murine TRP2 was used, showing the ability to break tolerance and control the tumor growth. These results validate the use of IDLV for cancer therapy.

Robust Neutralizing Antibodies to SARS-CoV-2 Develop and Persist in Subjects with Diabetes and COVID-19 Pneumonia.

CONTEXT: Demonstrating the ability to mount a neutralizing antibody response to SARS-CoV-2 in the presence of diabetes is crucial to understand COVID-19 pathogenesis, reinfection potential, and vaccine development. OBJECTIVE: The aim of this study was to characterize the kinetics and durability of neutralizing antibody (Nab) response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the presence of hyperglycemia. METHODS: Using a lentiviral vector-based SARS-CoV-2 neutralization assay to measure Nabs, we characterized 150 patients randomly selected from a cohort of 509 patients with confirmed COVID-19 pneumonia. We analyzed Nab response according to the presence of diabetes or hyperglycemia, at the time of hospitalization and during the postdischarge follow-up: 1-, 3-, and 6-month outpatient visits. RESULTS: Among 150 randomly selected patients 40 (26.6%) had diabetes. Diabetes (hazard ratio [HR] 8.9, P < .001), glucose levels (HR 1.25 × 1.1 mmol/L, P < .001), and glucose variability (HR 1.17 × 0.6 mmol/L, P < .001) were independently associated with an increased risk of mortality. The neutralizing activity of SARS-CoV-2 antibodies in patients with diabetes was superimposable, as for kinetics and extent, to that of patients without diabetes. It was similar across glucose levels and correlated with the humoral response against the SARS-CoV-2 spike protein. Positivity for Nabs at the time of hospital admission conferred protection on mortality, both in the presence (HR 0.28, P = .046) or absence of diabetes (HR 0.26, P = .030). The longevity of the Nab response was not affected by diabetes. CONCLUSION: Diabetes and hyperglycemia do not affect the kinetics and durability of the neutralizing antibody response to SARS-CoV-2. These findings provide the rational to include patients with diabetes in the early phase of the vaccination campaign against SARS-CoV-2.

Neutralizing antibody responses to SARS-CoV-2 in symptomatic COVID-19 is persistent and critical for survival.

Understanding how antibody responses to SARS-CoV-2 evolve during infection may provide important insight into therapeutic approaches and vaccination for COVID-19. Here we profile the antibody responses of 162 COVID-19 symptomatic patients in the COVID-BioB cohort followed longitudinally for up to eight months from symptom onset to find SARS-CoV-2 neutralization, as well as antibodies either recognizing SARS-CoV-2 spike antigens and nucleoprotein, or specific for S2 antigen of seasonal beta-coronaviruses and hemagglutinin of the H1N1 flu virus. The presence of neutralizing antibodies within the first weeks from symptoms onset correlates with time to a negative swab result (p = 0.002), while the lack of neutralizing capacity correlates with an increased risk of a fatal outcome (p = 0.008). Neutralizing antibody titers progressively drop after 5-8 weeks but are still detectable up to 8 months in the majority of recovered patients regardless of age or co-morbidities, with IgG to spike antigens providing the best correlate of neutralization. Antibody responses to seasonal coronaviruses are temporarily boosted, and parallel those to SARS-CoV-2 without dampening the specific response or worsening disease progression. Our results thus suggest compromised immune responses to the SARS-CoV-2 spike to be a major trait of COVID-19 patients with critical conditions, and thereby inform on the planning of COVID-19 patient care and therapy prioritization.

Isolation and Characterization of Mouse Monoclonal Antibodies That Neutralize SARS-CoV-2 and Its Variants of Concern Alpha, Beta, Gamma and Delta by Binding Conformational Epitopes of Glycosylated RBD With High Potency.

Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in Escherichia coli, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.

Transient depletion of CD4(+) T cells augments IL-21-based immunotherapy of disseminated neuroblastoma in syngeneic mice.

IL-21 is a member of the IL-2 cytokine family, produced by CD4+ T cells. We previously showed that immunotherapy (IT) with IL-21-transduced neuroblastoma cells (Neuro2a/IL-21) cured 33% of syngeneic mice bearing systemic NB. Here, we studied whether the removal of Treg cells could potentiate the therapeutic efficacy of Neuro2a/IL-21 vaccine. The administration of anti-CD25 mAb, which targets Treg cells, slightly potentiated the effect of vaccine IT (50% cure rate), but anti-CD4 mAb had a more potent effect leading to 80% cure rate. Anti-CD25 mAb, indeed, only partially depleted CD4+CD25+FoxP3+ Treg cells, whereas anti-CD4 mAb was more effective in this respect, leading to 90% depletion of Treg cells. In mice receiving vaccine+anti-CD4 mAb, which developed systemic immunity to NB, CD4+ T cells counts completely recovered in 90 days. Depletion of CD8+ T cells abrogated the effect of the combined IT, indicating a predominant role of these cells in driving the immune response. In addition, CD8+ T cells from cured mice coinjected with Neuro2a/parental cells (pc) in NOD-SCID mice completely inhibited tumor growth. Spleen cells from mice receiving Neuro2a/IL-21 vaccination showed increased expression of IFN-alpha2, -beta1 and -gamma mRNA. Moreover, mice receiving vaccine therapy alone or vaccine+anti-CD4 mAb showed increased IFN-gamma serum levels and IFN-gamma-producing CD8+ T cells were found in spleen cells. In conclusion, anti-CD4 mAb potentiated IL-21-based IT by removing Treg cells and/or their precursors and other potentially immune-suppressive CD4+ cell subsets, thus allowing the development of an IL-21-driven CD8+ T cell response, which mediates NB rejection.