Federico Pratesi

The production of antibodies against viral structural proteins such as the spike (S) and the nucleocapsid (N) is a hallmark of SARS-CoV-2 infection. The N protein contains several immunogenic regions. In this work we analysed epitope specificity and avidity of anti-N antibodies in COVID-19 patients. Eighty-nine COVID-19 patients were recruited. IgG, IgA, IgM antibodies to recombinant N protein and to 6 different peptides containing predicted B epitopes were measured by ELISA. Antibody avidity was evaluated by chaotropic ELISA. Anti-N IgG, IgA, and IgM were detected in 59%, 41% and 30% respectively; in 11% cases anti-N IgG are the only antibodies present in COVID patients. IgG and IgA anti-N antibodies levels correlate with levels of IL-6 and inversely with PaO₂/FiO₂ ratio (p < 0,005). Anti-N IgG displayed medium avidity in 51% and high avidity in 49% COVID patients; anti-N avidity was negatively correlated with PaO₂/FiO₂ (p < 0,05). Median anti-N antibody levels were similar in discharged or deceased patients and antibody avidity was not correlated with outcome. Among peptides, N_366-388 is the most frequently recognized by IgG, IgA and IgM antibodies followed by N_380-400, bound by patients IgG and IgA but anti-N_380-400 IgG display higher avidity than anti-N_366-388 IgG. These results indicate that anti-N antibodies are characterized by medium-high avidity and preferentially bind the COOH-terminus of the nucleoprotein. Anti-N antibodies, especially directed towards specific epitopes, might be relevant for the course of the infection, and their induction might improve current vaccination strategies.

Autoimmune diseases are conditions characterized by aberrant B‐cell and T‐cell reactivity against self‐antigens. Autoantibodies are serological biomarkers of autoimmune diseases, as such, autoantibody testing is a key step for diagnosing and classifying many autoimmune diseases, as well as monitoring disease activity and devising a treatment strategy. Considering the rising number of people affected by autoimmune diseases worldwide, it is even more important to have efficient techniques that combine high sensitivity and specificity with reduced sample processing times and an automated high‐throughput workflow. In this context, the identification and validation of new autoantigens and autoantibodies, together with the implementation of technological advancements, has led, in the last decades, to an improvement in patient diagnosis and stratification. Here, we review the major antigens of some of the most common autoimmune diseases, and the most widely used assays employed in diagnostic laboratories for the detection of their cognate antibody, confronting more traditional platforms with emerging ones in selected cases of study.

Objective: Rheumatoid arthritis (RA) associated interstitial lung disease (ILD) is the most common pulmonary manifestations of RA, with a progressive course and a poor survival. An early detection and better treatment is essential to improve outcome. We evaluated 16 analytes that could be relevant for the development of RA ILD. Method: In an inception cohort of 1118 early RA patients, pulmonary fibrosis (PF) were identified in 60 patients after a mean follow-up of 5.3 years using high resolution computer tomography (HRCT). As controls, 124 early RA patients without PF and 94 matched population controls without known rheumatic disease were studied. Analysis of antibodies against histones 3 and 4 derived citrullinated peptides (CitH3/H4), and cytokines/chemokines levels were performed in plasma samples collected at RA diagnosis using in-house ELISA and Luminex analysis. Result: Anti-CitH3(114–135) antibodies were the only antibody with increased frequency and levels in patients with PF versus without PF. The highest OR for PF development were found when combining positivity for anti-CitH3(114–135) and -CitH4(31–50) antibodies, OR 2.26. Levels of IL1α, IL1ß, TNFα, VEGFA and MIPα remained significantly elevated in patients with PF compared without PF, after adjustments and Bonferroni corrections. Several of the cytokines/chemokines correlated significantly with the histone antibodies in patients without PF. Partial least squares discriminant analysis including antibodies against citrullinated histon peptides and cytokines/chemokines identified significantly in PF in non-smokers. Conclusion: Antibodies against CitH3 peptides and several of the analysed cytokines/chemokines in samples collected at diagnosis were associated with subsequent delevopment of PF in patients with RA.

Neutrophil extracellular traps (NETs) are web-like structures composed of chromatin and proteins from neutrophil granules. Several studies highlight the heterogeneity of NETs, underscoring the challenges associated with their detection. In patients with COVID-19, high levels of NET fragments, called NET remnants, are detected in the circulation but also in alveoli and bronchioles. NET remnants are usually measured as complexes of DNA and myeloperoxidase (DNA−MPO). Taking advantage of proteomic data on NET composition, we developed new solid-phase assays to detect NET remnants, measuring complexes of DNA with alpha enolase (DNA−eno) or calprotectin (DNA−cal). The two assays were compared with the DNA−MPO test for the detection of in vitro-generated NET and serum NET remnants; all of them showed similar sensitivity in the detection of in vitro-generated NET. In an analysis of 40 patients with severe COVID-19 and 25 healthy subjects, the results of the three assays were highly correlated, and all detected significantly higher levels of NET remnants in patient sera. Moreover, the level of NET remnants correlated with impaired gas exchange and increased with the progressive decline of pulmonary function. The proposed assays thus represent a novel tool with which to evaluate NETosis; using antibodies to different NET constituents may allow their fingerprinting in different disorders.

Hidradenitis suppurativa (HS) is a chronic skin disease, characterized by clinical inflammation of the hair follicle with the recurrence of abscesses, nodules, and tunnels. Recently, several studies suggested a role of IL‐1 family (IL‐1F) cytokines in eliciting and sustaining the disease. The aim of this work is to perform a comprehensive analysis of IL‐1F cytokines, soluble inhibitors and receptors in a cohort of HS patients not treated with biological agents. Sixteen patients affected by HS and 16 healthy controls were recruited; clinical data were collected and disease severity evaluated by means of the International HS Severity Score System (IHS4). Serum levels of IL‐1F cytokines, inhibitors and receptors were measured using a Multiplex Assays. IL‐18 and free IL‐18 levels were significantly higher in patients vs controls. Among soluble inhibitors, IL‐1Ra, IL‐1R2 and ST2/IL‐1R4 were significantly increased. IL‐18, free IL‐18 and IL‐33 levels are strongly correlated with IHS4. Also the inhibitors IL‐1Ra and IL‐18BP show a correlation with IHS4. The data obtained in this study confirm the involvement of IL‐1F cytokines in mediating the disease and determining its severity and suggest a possible role for IL‐18 as novel serum biomarker of active disease.

The delivery of drugs through the skin barrier at a predetermined rate is the aim of transdermal drug delivery systems (TDDSs). However, so far, TDDS has not fully attained its potential as an alternative to hypodermic injections and oral delivery. In this study, we presented a proof of concept of a dual drug-loaded patch made of nanoparticles (NPs) and ultrafine fibers fabricated by using one equipment, i.e., the electrospinning apparatus. Such NP/fiber systems can be useful to release drugs locally through the skin and the tympanic membrane. Briefly, dexamethasone (DEX)-loaded poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) fiber meshes were decorated with rhodamine (RHO)-loaded poly(lactic-co-glycolic acid) (PLGA) NPs, with RHO representing as a second drug model. By properly tuning the working parameters of electrospinning, DEX-loaded PHBHV fibers (i.e., by electrospinning mode) and RHO-loaded PLGA NPs (i.e., by electrospray mode) were successfully prepared and straightforwardly assembled to form a TDDS patch, which was characterized via Fourier transform infrared spectroscopy and dynamometry. The patch was then tested in vitro using human dermal fibroblasts (HDFs). The incorporation of DEX significantly reduced the fiber mesh stiffness. In vitro tests showed that HDFs were viable for 8 days in contact with drug-loaded samples, and significant signs of cytotoxicity were not highlighted. Finally, thanks to a beaded structure of the fibers, a controlled release of DEX from the electrospun patch was obtained over 4 weeks, which may accomplish the therapeutic objective of a local, sustained and prolonged anti-inflammatory action of a TDDS, as is requested in chronic inflammatory conditions, and other pathological conditions, such as in sudden sensorineural hearing loss treatment.

Background: Cancer patients are more vulnerable to COVID-19 and are thus given high priority in vaccination campaigns. In solid cancer patients treated with checkpoint inhibitors, we evaluated the amount of anti-RBD and neutralizing antibodies and antibody avidity after two or three doses of the vaccine. Methods: Thirty-eight solid cancer patients, 15 untreated hematological patients and 21 healthy subjects were enrolled in the study. Blood was collected before the first dose (T0), 21 days after the second (T2) and in 18 solid cancer patients also 15 days after the third dose of vaccine (T3). IgG, IgM and IgA anti-RBD antibodies were detected by ELISA. Neutralizing antibodies were measured testing the inhibition of RBD binding to ACE2. Antibody avidity was evaluated in 18 patients by a urea avidity ELISA. Results: IgG anti-RBD antibodies were produced in 65.8% of the cancer patients at T2, and in 60% of hematological patients at levels lower than healthy controls. IgM and IgA anti-RBD antibodies were also produced in 5.3% and 21% cancer patients, respectively. At T3, a significant increase in anti-RBD IgG levels was observed. Neutralizing antibodies were produced in 68.4% of cancer patients as compared with 93% of untreated hematological patients and 100% of controls, at titers lower than in healthy subjects. At T3, neutralizing antibodies and avidity of IgG anti-RBD increased; 6/18 patients negative at T2 developed neutralizing antibodies at T3. Conclusion: The data indicate that in cancer patients mRNA vaccine induces high avidity anti-RBD antibodies and neutralizing antibodies that increase after the third dose. The process of induction and selection of high-affinity antibodies is apparently unaffected by the treatment with anti-PD-1 or anti-PD-L1 antibodies.

Introduction: Immediate and delayed hypersensitivity reactions (HSR) to COVID-19 vaccines are rare adverse events that need to be prevented, diagnosed, and managed in order to guarantee adherence to the vaccination campaign. The aims of our study were to stratify the risk of HSR to COVID-19 vaccines and propose alternative strategies to complete the vaccination. Methods: 1,640 subjects were screened for vaccinal eligibility, according to national and international recommendations. Among them, we enrolled for allergy workup 152 subjects, 43 with HSR to COVID-19 vaccines and 109 at high risk of HSR to the first dose. In vivo skin tests with drugs and/or vaccines containing PEG/polysorbates were performed in all of them, using skin prick test and, when negative, intradermal tests. In a subgroup of patients resulted negative to the in vivo skin tests, the programmed dose of COVID-19 vaccine (Pfizer/BioNTech) was administered in graded doses regimen, and detection of neutralizing anti-spike antibodies was performed in these patients after 4 weeks from the vaccination, using the SPIA method. Results: Skin tests for PEG/polysorbates resulted positive in only 3% (5/152) of patients, including 2 with previous HSR to COVID-19 vaccines and 3 at high risk of HSR to the first dose. Among the 147 patients with negative skin tests, 97% (143/147) were eligible for vaccination and 87% (124/143) of them received safely the programmed COVID-19 vaccine dose. Administration of graded doses of Pfizer/BioNTech vaccine were well tolerated in 17 out of 18 patients evaluated; only 1 developed an HSR during the vaccination, less severe than the previous one, and all developed neutralizing anti-spike antibodies after 4 weeks with values comparable to those subjects who received the vaccine in unfractionated dose. Conclusion: On the whole, the usefulness of the skin tests for PEG/polysorbates seems limited in the diagnosis of HSR to COVID-19 vaccines. Graded doses regimen (Pfizer/BioNTech) is a safe and effective alternative strategy to complete the vaccinal course.

Objectives In patients with systemic autoimmune rheumatic disorders (SARDs), vaccination with SARS-CoV-2 mRNA vaccines has been proposed. The aim of this study is to evaluate the immune response elicited by vaccination with mRNA vaccine, testing IgM, IgA and IgG antibodies to SARS-CoV-2 receptor-binding domain (RBD) and measuring neutralising antibodies. Methods IgG, IgM and IgA anti-RBD antibodies were measured in 101 patients with SARDs. Antibodies inhibiting the interaction between RBD and ACE2 were evaluated. Antibody avidity was tested in a chaotropic ELISA using urea. Twenty-one healthcare workers vaccinated with mRNA vaccine served as control group. Results Anti-RBD IgG and IgA were produced after the first dose (69% and 64% of the patients) and after the boost (93% and 83%). Antibodies inhibiting the interaction of RBD with ACE2 were detectable in 40% of the patients after the first dose and 87% after boost, compared with 100% in healthy controls (pConclusions These data show that double-dose vaccination induced in patients with SARDs anti-RBD IgG and IgA antibodies in amounts not significantly different from controls, and, most interestingly, characterised by high avidity and endowed with neutralising activity.