Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology.

Analysis of the specificity and kinetics of neutralizing antibodies (nAbs) elicited by SARS-CoV-2 infection is crucial for understanding immune protection and identifying targets for vaccine design. In a cohort of 647 SARS-CoV-2-infected subjects, we found that both the magnitude of Ab responses to SARS-CoV-2 spike (S) and nucleoprotein and nAb titers correlate with clinical scores. The receptor-binding domain (RBD) is immunodominant and the target of 90% of the neutralizing activity present in SARS-CoV-2 immune sera. Whereas overall RBD-specific serum IgG titers waned with a half-life of 49 days, nAb titers and avidity increased over time for some individuals, consistent with affinity maturation. We structurally defined an RBD antigenic map and serologically quantified serum Abs specific for distinct RBD epitopes leading to the identification of two major receptor-binding motif antigenic sites. Our results explain the immunodominance of the receptor-binding motif and will guide the design of COVID-19 vaccines and therapeutics.

Antibody response to three-dose anti-SARS-CoV-2 mRNA-vaccination in treated solid cancer patients.

Solid cancer patients are at higher risk of SARS-CoV-2 infection and severe complications. Moreover, vaccine-induced antibody response is impaired in patients on anticancer treatment. In this retrospective, observational, hypothesis-generating, cohort study, we assessed the antibody response to the third dose of mRNA vaccine in a convenience sample of patients on anticancer treatment, comparing it to that of the primary two-dose cycle. Among 99 patients included, 62.6% were ≥60 years old, 32.3% males, 67.7% with advanced disease. Exactly 40.4% were receiving biological therapy, 16.2% chemotherapy only and 7.1% both treatments. After the third dose, seroconversion rate seems to increase significantly, especially in non-responders to two doses. Heterologous vaccine-type regimen (two-dose mRNA-1273 and subsequent tozinameran or vice versa) results in higher antibody levels. This explorative study suggests that repeated doses of mRNA-vaccines could be associated with a better antibody response in this population. Furthermore, heterologous vaccine-type three-dose vaccination seems more effective in this population. Since this is a hypothesis-generating study, adequately statistically powered studies should validate these results.

Early administration of nirmatrelvir/ritonavir leads to faster negative SARS-CoV-2 nasal swabs than monoclonal antibodies in COVID 19 patients at high-risk for severe disease.

PURPOSE: Besides the well-established efficacy in preventing severe COVID-19, the impact of early treatments, namely antivirals and monoclonal antibodies (mAbs), on the time length to negativization of SARS-CoV-2 nasal swabs is still unclear. The aim of this study was to compare the efficacy of different early treatments in reducing the SARS-CoV-2 viral shedding, identifying a single drug that might potentially lead to a more rapid negativization of SARS-CoV-2 nasal swab. METHODS: This was a single-centre, retrospective, observational study conducted at Ospedale Luigi Sacco in Milan. Data of high-risk COVID-19 patients who received early treatments between 23 December 2021 and March 2023 were extracted. The comparison across treatments was conducted using the Kruskall-Wallis test for continuous variables. Dunn's test with Bonferroni adjustment was performed for post-hoc comparisons of days to negativization. Secondly, a negative binomial regression adjusted for age, sex, number of comorbidities, immunosuppression, and SARS-CoV-2 vaccination status was implemented. RESULTS: Data from 428 patients receiving early treatments were collected. The majority were treated with Nirmatrelvir/Ritonavir and were affected by SARS-CoV-2 Omicron infection with BA.2 sublineage. The median length time to SARS-CoV-2 nasal swab negativization was 9 days [IQR 7-13 days]. We found that Nirmatrelvir/Ritonavir determined a significant decrease of the length time to SARS-CoV-2 nasal swab negativization compared to mAbs (p = 0.003), but not compared to Remdesivir (p = 0.147) and Molnupiravir (p = 0.156). CONCLUSION: Our findings highlight the importance of promptly treating high-risk COVID-19 patients with Nirmatrelvir/Ritonavir, as it also contributes to achieving a faster time to negative SARS-CoV-2 nasal swabs.

Immunogenicity of two doses of BNT162b2 and mRNA-1273 vaccines for solid cancer patients on treatment with or without a previous SARS-CoV-2 infection.

Previous studies on the immunogenicity of SARS-CoV-2 mRNA vaccines showed a reduced seroconversion in cancer patients. The aim of our study is to evaluate the immunogenicity of two doses of mRNA vaccines in solid cancer patients with or without a previous exposure to the virus. This is a single-institution, prospective, nonrandomized study. Patients in active treatment and a control cohort of healthy people received two doses of BNT162b2 (Comirnaty, BioNTech/Pfizer, The United States) or mRNA-1273 (Spikevax, Moderna). Vaccine was administered before starting anticancer therapy or on the first day of the treatment cycle. SARS-CoV-2 antibody levels against S1, RBD (to evaluate vaccine response) and N proteins (to evaluate previous infection) were measured in plasma before the first dose and 30 days after the second one. From January to June 2021, 195 consecutive cancer patients and 20 healthy controls were enrolled. Thirty-one cancer patients had a previous exposure to SARS-CoV-2. Cancer patients previously exposed to the virus had significantly higher median levels of anti-S1 and anti-RBD IgG, compared to healthy controls (P = .0349) and to cancer patients without a previous infection (P < .001). Vaccine type (anti-S1: P < .0001; anti-RBD: P = .0045), comorbidities (anti-S1: P = .0274; anti-RBD: P = .0048) and the use of G-CSF (anti-S1: P = .0151) negatively affected the antibody response. Conversely, previous exposure to SARS-CoV-2 significantly enhanced the response to vaccination (anti-S1: P < .0001; anti-RBD: P = .0026). Vaccine immunogenicity in cancer patients with a previous exposure to SARS-CoV-2 seems comparable to that of healthy subjects. On the other hand, clinical variables of immune frailty negatively affect humoral immune response to vaccination.

Genomic characterization and phylogenetic analysis of SARS-COV-2 in Italy.

This report describes the isolation, molecular characterization, and phylogenetic analysis of the first three complete genomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolated from three patients involved in the first outbreak of COVID-19 in Lombardy, Italy. Early molecular epidemiological tracing suggests that SARS-CoV-2 was present in Italy weeks before the first reported cases of infection.

Corrigendum: Definition of the immune parameters related to COVID-19 severity.

[This corrects the article DOI: 10.3389/fimmu.2022.850846.].

Clinical efficacy of the first two doses of anti-SARS-CoV-2 mRNA vaccines in solid cancer patients.

INTRODUCTION: Cancer patients are frail individuals, thus the prevention of SARS-CoV-2 infection is essential. To date, vaccination is the most effective tool to prevent COVID-19. In a previous study, we evaluated the immunogenicity of two doses of mRNA-based vaccines (BNT162b2 or mRNA-1273) in solid cancer patients. We found that seroconversion rate in cancer patients without a previous exposure to SARS-CoV-2 was lower than in healthy controls (66.7% vs. 95%, p = 0.0020). The present study aimed to evaluate the clinical efficacy of the vaccination in the same population. METHODS: This is a single-institution, prospective observational study. Data were collected through a predefined questionnaire through phone call in the period between the second and third vaccine dose. The primary objective was to describe the clinical efficacy of the vaccination, defined as the percentage of vaccinated subjects who did not develop symptomatic COVID-19 within 6 months after the second dose. The secondary objective was to describe the clinical features of patients who developed COVID-19. RESULTS: From January to June 2021, 195 cancer patients were enrolled. Considering that 7 (3.59%) patients tested positive for SARS-CoV-2 and 5 developed symptomatic disease, the clinical efficacy of the vaccination was 97.4%. COVID-19 disease in most patients was mild and managed at home; only one hospitalization was recorded and no patient required hospitalization in the intensive care unit. DISCUSSION: Our study suggests that increasing vaccination coverage, including booster doses, could improve the prevention of infection, hospitalization, serious illness, and death in the frail population of cancer patients.

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

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

Switching to unboosted atazanavir reduces bilirubin and triglycerides without compromising treatment efficacy in UGT1A1*28 polymorphism carriers.

OBJECTIVES: Hyperbilirubinaemia is a frequent complication of atazanavir-containing antiretroviral therapy and its severity is related to UDP-glucuronosyl transferase (UGT) 1A1*28 polymorphism. The aim of this study was to evaluate the safety and outcome of unboosted atazanavir-containing regimens based on the genetic constitution. METHODS: Fifty-one HIV-1-infected patients on boosted atazanavir were prospectively enrolled in the study. Twenty-five patients with a UGT1A1*28 allele switched to 400 mg of unboosted atazanavir. RESULTS: At baseline, UGT1A1 heterozygous and homozygous patients had significantly higher bilirubin levels than wild-type (P = 0.012 and P < 0.001, respectively). After ritonavir removal, a reduction was observed in total bilirubin (from 4.09 to 1.82 mg/dL; P < 0.001), γ-glutamyl transpeptidase (P = 0.015), triglycerides (P = 0.03) and total cholesterol (P = 0.05). No significant changes in CD4 T cell count and no increases in viral load were observed 12 months after unboosting. Plasma drug monitoring after ritonavir removal revealed the presence of therapeutic atazanavir concentrations in all patients except one with poor therapy adherence. CONCLUSIONS: UGT1A1*28 is significantly related to hyperbilirubinaemia in HIV-1 patients receiving atazanavir. Genotyping before the initiation of antiretroviral therapy can reduce the emergence of severe hyperbilirubinaemia. Unboosted atazanavir-containing therapy is safe and efficacious in patients with an undetectable viral load with a UGT1A1*28 polymorphism, allowing the use of atazanavir in patients otherwise likely unable to receive it.

Definition of the Immune Parameters Related to COVID-19 Severity.

A relevant portion of patients with disease caused by the severe acute respiratory syndrome coronavirus 2 (COVID-19) experience negative outcome, and several laboratory tests have been proposed to predict disease severity. Among others, dramatic changes in peripheral blood cells have been described. We developed and validated a laboratory score solely based on blood cell parameters to predict survival in hospitalized COVID-19 patients. We retrospectively analyzed 1,619 blood cell count from 226 consecutively hospitalized COVID-19 patients to select parameters for inclusion in a laboratory score predicting severity of disease and survival. The score was derived from lymphocyte- and granulocyte-associated parameters and validated on a separate cohort of 140 consecutive COVID-19 patients. Using ROC curve analysis, a best cutoff for score of 30.6 was derived, which was associated to an overall 82.0% sensitivity (95% CI: 78-84) and 82.5% specificity (95% CI: 80-84) for detecting outcome. The scoring trend effectively separated survivor and non-survivor groups, starting 2 weeks before the end of the hospitalization period. Patients' score time points were also classified into mild, moderate, severe, and critical according to the symptomatic oxygen therapy administered. Fluctuations of the score should be recorded to highlight a favorable or unfortunate trend of the disease. The predictive score was found to reflect and anticipate the disease gravity, defined by the type of the oxygen support used, giving a proof of its clinical relevance. It offers a fast and reliable tool for supporting clinical decisions and, most important, triage in terms of not only prioritization but also allocation of limited medical resources, especially in the period when therapies are still symptomatic and many are under development. In fact, a prolonged and progressive increase of the score can suggest impaired chances of survival and/or an urgent need for intensive care unit admission.

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

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

Anti-tumour necrosis factor α antibodies and circulating lymphocyte phenotypes in inflammatory bowel disease.

OBJECTIVE: Circulating lymphocyte subtypes are not fully explored parameters for monitoring chronic T cell activation during inflammatory bowel disease (IBD). Tumor necrosis factor α (TNFα), one of the main mediators of IBD related inflammation induces expression of CD70 on T cells. CD70 limits T cell expansion and controls CD27 receptor on activated B lymphocytes. Aim of this study was to assess the number and the frequency of CD70+ T cells and CD27+ B cells in IBD patients during inactive phase of the disease under or without anti-TNFα treatment. DESIGN: We studied 91 patients with inactive IBD, 31 untreated, 29 treated with infliximab (IFX), and 31 treated with adalimumab (ADA). Lymphocyte phenotypes were assessed by flow cytometry using anti-CD45, CD19, CD27, CD3, and CD70 monoclonal antibodies. IFX and ADA actual capacity of TNFα neutralization in serum was estimated by the recoveryELISA technique. RESULTS: Whereas CD3+ T cells were increased in treated compared to untreated patients, the percentage of the CD70+ T cells was significantly lower in treated patients indicating a 'cooling' effect of the biological therapy. This effect differs between samples according to the therapeutic range of the circulating drug. Although the CD19+ B-cell percentage tended to be lower in treated patients, CD19+27+ memory B cells did not show significant differences between groups. CONCLUSIONS: Frequency of peripheral blood CD70+ T cells was significantly reduced by treatment with anti-TNFα antibodies. Monitoring of this parameter of T cells can give better insight to the disease progression and therapy application in IBD patients.

H3K4me3 Histone ChIP-Seq Analysis Reveals Molecular Mechanisms Responsible for Neutrophil Dysfunction in HIV-Infected Individuals.

Peripheral neutrophils in HIV-infected individuals are characterized by impairment of chemotaxis, phagocytosis, bactericidal activity, and oxidative burst ability regardless of whether patients are receiving antiretroviral therapy or not. Neutrophil dysfunction leads not only to increased susceptibility to opportunistic infections but also to tissue damage through the release of reactive oxygen species (ROS), proteases, and other potentially harmful effector molecules contributing to AIDS progression. In this study, we demonstrated high levels of histone H3 lysine K4 trimethylated (H3K4me3) and dysregulation of DNA transcription in circulating neutrophils of HIV-infected subjects. This dysregulation was accompanied by a deficient response of neutrophils to LPS, impaired cytokine/chemokine/growth factor synthesis, and increased apoptosis. Chromatin immunoprecipitation sequencing (ChIPseq) H3K4me3 histone analysis revealed that the most spectacular abnormalities were observed in the exons, introns, and promoter-TSS regions. Bioinformatic analysis of Gene Ontology, including biological processes, molecular function, and cellular components, demonstrated that the main changes were related to the genes responsible for cell activation, cytokine production, adhesive molecule expression, histone remodeling via upregulation of methyltransferase process, and downregulation of NF-κB transcription factor in canonical pathways. Abnormalities within H3K4me3 implicated LPS-mediated NF-κB canonical activation pathway that was a result of low amounts of κB DNA sites within histone H3K4me3, low NF-κB (p65 RelA) and TLR4 mRNA expression, and reduced free NF-κB (p65 RelA) accumulation in the nucleus. Genome-wide survey of H3K4me3 provided evidence that chromatin modifications lead to an impairment within the canonical NF-κB cell activation pathway causing the neutrophil dysfunction observed in HIV-infected individuals.

Anti-SARS-CoV-2 Immunoglobulin Isotypes, and Neutralization Activity Against Viral Variants, According to BNT162b2-Vaccination and Infection History.

Purpose: To compare SARS-CoV-2 antigen-specific antibody production and plasma neutralizing capacity against B.1 wild-type-like strain, and Gamma/P.1 and Delta/B.1.617.2 variants-of-concern, in subjects with different Covid-19 disease and vaccination histories. Methods: Adult subjects were: 1) Unvaccinated/hospitalized for Covid-19; 2) Covid-19-recovered followed by one BNT162b2 vaccine dose; and 3) Covid-19-naïve/2-dose BNT162b2 vaccinated. Multiplex Luminex® immunoassays measured IgG, IgA, and IgM plasma levels against SARS-CoV-2 receptor-binding domain (RBD), spike-1 (S), and nucleocapsid proteins. Neutralizing activity was determined in Vero E6 cytopathic assays. Results: Maximum anti-RBD IgG levels were similar in Covid-19­recovered individuals 8‒10 days after single-dose vaccination and in Covid-19-naïve subjects 7 days after 2nd vaccine dosing; both groups had ≈2­fold higher anti-RBD IgG levels than Unvaccinated/Covid-19 subjects tracked through 2 weeks post-symptom onset. Anti-S IgG expression patterns were similar to RBD within each group, but with lower signal strengths. Viral antigen-specific IgA and IgM levels were more variable than IgG patterns. Anti-nucleocapsid immunoglobulins were not detected in Covid-19-naïve subjects. Neutralizing activity against the B.1 strain, and Gamma/P.1 and Delta/B.1.617.2 variants, was highest in Covid­19-recovered/single-dose vaccinated subjects; although neutralization against the Delta variant in this group was only 26% compared to B.1 neutralization, absolute anti-Delta titers suggested maintained protection. Neutralizing titers against the Gamma and Delta variants were 33‒77% and 26‒67%, respectively, versus neutralization against the B.1 strain (100%) in the three groups. Conclusion: These findings support SARS-CoV-2 mRNA vaccine usefulness regardless of Covid-19 history, and confirm remarkable protection provided by a single vaccine dose in people who have recovered from Covid-19.

SARS-CoV-2 seroprevalence trends in healthy blood donors during the COVID-19 outbreak in Milan.

BACKGROUND: The Milan metropolitan area in Northern Italy was among the most severely hit by the SARS-CoV-2 outbreak. The aim of this study was to examine the seroprevalence trends of SARS-CoV-2 in healthy asymptomatic adults, and the risk factors and laboratory correlates of positive tests. MATERIALS AND METHODS: We conducted a cross-sectional study in a random sample of blood donors, who were asymptomatic at the time of evaluation, at the beginning of the first phase (February 24th to April 8th 2020; n=789). Presence of IgM/IgG antibodies against the SARS-CoV-2-Nucleocapsid protein was assessed by a lateral flow immunoassay. RESULTS: The test had a 100/98.3 sensitivity/specificity (n=32/120 positive/negative controls, respectively), and the IgG test was validated in a subset by an independent ELISA against the Spike protein (n=34, p<0.001). At the start of the outbreak, the overall adjusted seroprevalence of SARS-CoV-2 was 2.7% (95% CI: 0.3-6%; p<0.0001 vs 120 historical controls). During the study period, characterised by a gradual implementation of social distancing measures, there was a progressive increase in the adjusted seroprevalence to 5.2% (95% CI: 2.4-9.0; 4.5%, 95% CI: 0.9-9.2% according to a Bayesian estimate) due to a rise in IgG reactivity to 5% (95% CI: 2.8-8.2; p=0.004 for trend), but there was no increase in IgM+ (p=not significant). At multivariate logistic regression analysis, IgG reactivity was more frequent in younger individuals (p=0.043), while IgM reactivity was more frequent in individuals aged >45 years (p=0.002). DISCUSSION: SARS-CoV-2 infection was already circulating in Milan at the start of the outbreak. The pattern of IgM/IgG reactivity was influenced by age: IgM was more frequently detected in participants aged >45 years. By the end of April, 2.4-9.0% of healthy adults had evidence of seroconversion.

Perinatal exposure of mice to TCDD decreases allergic sensitisation through inhibition of IL-4 production rather than T regulatory cell-mediated suppression.

OBJECTIVE: The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a widespread, man-made, persistent organic pollutant with high immunotoxic potentials. It suppresses cell-mediated and humoral immune responses through mechanisms dependent on aryl-hydrocarbon receptor expression and immunosuppressive activity of the cells. Most sensitive to TCDD are organisms during fetal and infant life, mostly due to the developmental stage of many biological systems of the host, including immune system. Recent data show that T regulatory cells that have the potential to suppress immune reactions and which develop after TCDD exposure are also responsible for protection from allergy development. Our goal was to investigate if perinatal exposure to TCDD can affect allergic sensitisation and if T reg cells participate in this phenomenon. MATERIALS AND METHODS: Mice, Balb/c, were perinatally exposed to TCDD or to the carrier. Six weeks old control or exposed mice were sensitised with ovalbumin. Spleen cells of the animals were used to assess the content of T reg cells by means of flow cytometry. Levels of cytokines were assessed by ELISA technique in supernatants of the cells stimulated with anti-CD3 antibody. As a measure of sensitisation, total IgE and anti-OVA IgE were measured in serum of mice by ELISA method. To assess the function of T reg cells isolated from OVA-sensitised control or TCDD exposed animals we performed transfer studies. RESULTS: Here we show that perinatal exposure to TCDD decreases allergic sensitisation and that this process is related to inhibition of IL-4 synthesis rather than suppression mediated by T regulatory cells. CONCLUSION: We hypothesise that dioxin exposure can be an important environmental modulator of immunological responses that participate in allergic reactions.

Multiple Sclerosis CD49d+CD154+ As Myelin-Specific Lymphocytes Induced During Remyelination.

Multiple sclerosis (MS) is a demyelinating autoimmune disease of the central nervous system (CNS) mediated by autoreactive lymphocytes. The role of autoreactive lymphocytes in the CNS demyelination is well described, whereas very little is known about their role in remyelination during MS remission. In this study, we identified a new subpopulation of myelin-specific CD49d+CD154+ lymphocytes presented in the peripheral blood of MS patients during remission, that proliferated in vitro in response to myelin peptides. These lymphocytes possessed the unique ability to migrate towards maturing oligodendrocyte precursor cells (OPCs) and synthetize proinflammatory chemokines/cytokines. The co-culture of maturing OPCs with myelin-specific CD49d+CD154+ lymphocytes was characterized by the increase in proinflammatory chemokine/cytokine secretion that was not only a result of their cumulative effect of what OPCs and CD49d+CD154+ lymphocytes produced alone. Moreover, maturing OPCs exposed to exogenous myelin peptides managed to induce CD40-CD154-dependent CD49d+CD154+ lymphocyte proliferation. We confirmed, in vivo, the presence of CD49d+CD154+ cells close to maturating OPCs and remyelinating plaque during disease remission in the MS mouse model (C57Bl/6 mice immunized with MOG35-55) by immunohistochemistry. Three weeks after an acute phase of experimental autoimmune encephalomyelitis, CD49d+/CD154+ cells were found to be co-localized with O4+ cells (oligodendrocyte progenitors) in the areas of remyelination identified by myelin basic protein (MBP) labelling. These data suggested that myelin-specific CD49d+CD154+ lymphocytes present in the brain can interfere with remyelination mediated by oligodendrocytes probably as a result of establishing proinflammatory environment.

MS CD49d+CD154+ Lymphocytes Reprogram Oligodendrocytes into Immune Reactive Cells Affecting CNS Regeneration.

The critical aspect in multiple sclerosis (MS) progression involves insufficient regeneration of CNS resulting from deficient myelin synthesis by newly generated oligodendrocytes (OLs). Although many studies have focused on the role of autoreactive lymphocytes in the inflammatory-induced axonal loss, the problem of insufficient remyelination and disease progression is still unsolved. To determine the effect of myelin-specific lymphocytes on OL function in MS patients and in a mouse model of MS, we cultured myelin induced MS CD49d+CD154+ circulating lymphocytes as well as Experimental Autoimmune Encephalomyelitis (EAE) mouse brain-derived T and memory B cells with maturing oligodendrocyte precursor cells (OPCs). We found that myelin-specific CD49d+CD154+ lymphocytes affected OPC maturation toward formation of immune reactive OLs. Newly generated OLs were characterized by imbalanced myelin basic protein (MBP) and proteolipid protein (PLP) production as well as proinflammatory chemokine/cytokine synthesis. The analysis of cellular pathways responsible for OL reprogramming revealed that CD49d+CD154+ lymphocytes affected miRNA synthesis by dysregulation of polymerase II activity. miR-665 and ELL3 turned out to be the main targets of MS myelin-specific lymphocytes. Neutralization of high intracellular miR-665 concentration restored miRNA and MBP/PLP synthesis. Together, these data point to new targets for therapeutic intervention promoting CNS remyelination.

Structural Basis for Broad HIV-1 Neutralization by the MPER-Specific Human Broadly Neutralizing Antibody LN01.

Potent and broadly neutralizing antibodies (bnAbs) are the hallmark of HIV-1 protection by vaccination. The membrane-proximal external region (MPER) of the HIV-1 gp41 fusion protein is targeted by the most broadly reactive HIV-1 neutralizing antibodies. Here, we examine the structural and molecular mechansims of neutralization by anti-MPER bnAb, LN01, which was isolated from lymph-node-derived germinal center B cells of an elite controller and exhibits broad neutralization breadth. LN01 engages both MPER and the transmembrane (TM) region, which together form a continuous helix in complex with LN01. The tilted TM orientation allows LN01 to interact simultaneously with the peptidic component of the MPER epitope and membrane via two specific lipid binding sites of the antibody paratope. Although LN01 carries a high load of somatic mutations, most key residues interacting with the MPER epitope and lipids are germline encoded, lending support for the LN01 epitope as a candidate for lineage-based vaccine development.

Dysfunction of CD4+CD25high T regulatory cells in patients with recurrent aphthous stomatitis.

BACKGROUND: Recurrent aphthous stomatitis (RAS) is a chronic inflammatory disease of unknown etiology characterized by recurring formation of painful oral ulcers. RAS may result from oral epithelium damage caused by T-cell-mediated immune response. CD4(+)CD25(+) T regulatory (Treg) cells suppress proliferation and effector functions of other immune cells, and therefore are crucial in regulating the immune response. METHODS: We tested the function of peripheral CD4(+)CD25(high) Treg cells in active RAS through their ability to inhibit proliferation and cytokine production of conventional CD4(+) T cells. We also attempted to detect the presence of FOXP3 and indoleamine 2,3-dioxygenase (IDO) mRNA in the lesional and non-lesional oral mucosa of RAS patients and healthy individuals using real-time PCR assay. RESULTS: Treg cells derived from RAS patients were less efficient in the suppression of cytokine production of CD4(+) T effector cells than Treg cells from healthy individuals. Moreover, in RAS, Treg cells were nearly twice less potent in the inhibition of CD4(+)CD25(-) T cell proliferation than in healthy donors. Furthermore, we have demonstrated the decreased proportion of CD4(+)CD25(+)FOXP3(+) Treg cells in peripheral blood of RAS patients compared with controls. We failed to detect FOXP3 mRNA, while IDO mRNA expression was decreased in non-lesional mucosa biopsies from RAS patients compared with ulcer biopsies or normal mucosa from healthy donors. CONCLUSIONS: These findings suggest that CD4(+)CD25(high) Treg cells are both functionally and quantitatively compromised in RAS and that decreased constitutive expression of IDO in oral mucosa in RAS may lead to the loss of local immune tolerance.