Alignment of the amino acid sequences containing the fusion loop domain from E proteins of DENV 1–4, WNV, JEV, YFV, TBEV and the four point mutations of the QUAD proteins.

<p>Amino acids numbering: 1 is start of the E protein.</p

A: Expression and purification of DENV-2 Ewt and Equad from Drosophila S2 culture supernatants; supernatant before induction (b.ind.), after 7 days of expression culture (7 d expr.), concentrated via tangential flow (Conc.) and the two step purification with immobilized imidazole affinity (IMAC) and size exclusion chromatography (SEC) were separated on a 10% SDS-PAGE gel under reducing conditions. B: 6 μg of purified DENV1-4 (D1-D4) Equad and DENV-2 Ewt proteins were analyzed with SDS-PAGE. Proteins were stained with Coomassie blue. Size of molecular weight markers in kilo Daltons is indicated on the left.

<p>A: Expression and purification of DENV-2 Ewt and Equad from Drosophila S2 culture supernatants; supernatant before induction (b.ind.), after 7 days of expression culture (7 d expr.), concentrated via tangential flow (Conc.) and the two step purification with immobilized imidazole affinity (IMAC) and size exclusion chromatography (SEC) were separated on a 10% SDS-PAGE gel under reducing conditions. B: 6 μg of purified DENV1-4 (D1-D4) Equad and DENV-2 Ewt proteins were analyzed with SDS-PAGE. Proteins were stained with Coomassie blue. Size of molecular weight markers in kilo Daltons is indicated on the left.</p

Antigen titration using the indicated amounts of DENV-1 (A), -2 (B), -3 (C) and -4 (D) Equad and DENV-2 Ewt (E) proteins with three different DENV, WNV and negative (NEG) human sera.

<p>Measurements were performed in duplicates in at least two independent experiments.</p

Comparison of different antigens for the detection of DENV IgG.

<p>Sera positive for IgG against DENV, WNV, TBEV or negative control sera were analyzed with the Panbio Indirect IgG ELISA (black), the DENV-2 Ewt protein (light gray, lined) or the DENV1-4 Equad mix (dark grey, lined). The absolute absorbance is indicated. Cut-Off values for the Panbio test were obtained by calculation of the internal standard of the manufacturer; these are indicated at the right and only refer to this test (horizontal bars: DENV-positive results with an OD-value higher than 1.1*cut-off, equivocal results having an OD-value between 1.1*cut-off and 0.9*cut-off, negative results with an OD-value lower than 0.9*cut-off).</p

IgM-ELISA on 300 ng of DENV-2 Ewt (A) and DENV 1–4 Equad mixture (B) with different DENV, WNV and negative (NEG) sera (n = number of individuals).

<p>Bottom and top of the boxes are the first and third quartiles. The median signal is depicted as a line inside the box. Whiskers represent the 9^th and the 91^st percentile. Measurements were performed in duplicates in at least two independent experiments.</p

300 ng per well of DENV-2 Ewt (A) and Equad (B) and 160 ng per well of a DENV 1–4 Equad mixture (C) were tested with DENV- WNV- and TBEV- infected and YFV-vaccinated sera compared to negative (NEG) samples in an IgG-ELISA (n = number of individuals).

<p>Bottom and top of the boxes are the first and third quartiles. The median signal is depicted as a line inside the box. Whiskers represent the 9^th and the 91^st percentile. Outliers in B and C are marked with numbers (1–5). Measurements were performed in duplicates in at least two independent experiments.</p

Image_1_Neutralizing Antibody Responses to SARS-CoV-2 in Recovered COVID-19 Patients Are Variable and Correlate With Disease Severity and Receptor-Binding Domain Recognition.jpeg

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) caused outbreaks of the pandemic starting from the end of 2019 and, despite ongoing vaccination campaigns, still influences health services and economic factors globally. Understanding immune protection elicited by natural infection is of critical importance for public health policy. This knowledge is instrumental to set scientific parameters for the release of “immunity pass” adopted with different criteria across Europe and other countries and to provide guidelines for the vaccination of COVID-19 recovered patients. Here, we characterized the humoral response triggered by SARS-CoV-2 natural infection by analyzing serum samples from 94 COVID-19 convalescent patients with three serological platforms, including live virus neutralization, pseudovirus neutralization, and ELISA. We found that neutralization potency varies greatly across individuals, is significantly higher in severe patients compared with mild ones, and correlates with both Spike and receptor-binding domain (RBD) recognition. We also show that RBD-targeting antibodies consistently represent only a modest proportion of Spike-specific IgG, suggesting broad specificity of the humoral response in naturally infected individuals. Collectively, this study contributes to the characterization of the humoral immune response in the context of natural SARS-CoV-2 infection, highlighting its variability in terms of neutralization activity, with implications for immune protection in COVID-19 recovered patients.</p

Distribution of HAV strains in the 8 Italian Regions for which enough sequences were available.

Distribution of HAV strains in the 8 Italian Regions for which enough sequences were available.</p

Summary of the 15 sequences showing one only nucleotide difference <i>vs</i>. the reference outbreak sequence (Accession Number KF182323).

Summary of the 15 sequences showing one only nucleotide difference vs. the reference outbreak sequence (Accession Number KF182323).</p

Additional file 1 of Neutralising reactivity against SARS-CoV-2 Delta and Omicron variants by vaccination and infection history

Additional file 1: Figure S1. Anti-N antibody titres and dynamics in vaccinated and unvaccinated subjects; Figure S2. Anti-N antibody titres of vaccinated and unvaccinated individuals previously exposed to SARS-CoV-2 infection differ according to age; Figure S3. Correlation among DiaSorin S1/S2, DiaSorin trimeric tests and neutralisation assay; Figure S4. Features of the cohorts analysed in this study. Table S1. Concordance between S1/S2 and trimericS DiaSorin assays