Viral and immune profiles during the first wave of the SARS-COV-2 infection in hospital patients in Sardinia, Italy,

Study population

The study comprised 46 patients (≥ 18 years) from the Trinità Hospital of Santissima (SS) in Cagliari (Sardinia, Italy) with a confirmed SARS-COV-2) from a nasal-pharngeal-dabper (NPS) and/or symptoms and radiological findings that indicate Covid 19-lens inflammation. All patients included in this study were hospitalized between March and April 2020 with indicators of serious illnesses, according to the guidelines of the World Health Organization (WHO)¹²And inscribed in the observation study “Covid-IMM: Anala della Risposta Immunologica Nei di Pazienti Covid-19”, checked and approved by the Institutional Review Board (RB) (protocol number 260/2020/CE).

During the recovery of the hospital, routine blood tests included a complete parameters with a differential, kidney and liver function tests, C reactive protein (CRP) and lactate dehydrogenase (LDH), serum ferris and D-Dimer. For each patient, aliquots of serums and NPs were collected at different times, stored at -80 ° C and other analyzes sent to the Istituto Superiore di Sanità (ISS). The patient samples were layered in accordance with the collection days from the day of the hospital stay.

Analysis of anti-Sars-Cov-2 IgG and IGM in the serum

Anti-Sars-COV-2-IGM and IGG antibodies that aim at the nucleoprotein, and the spike protein was measured in undistated serums in the hospital laboratory of the Cagliari by chemiluminesmen immunoassay (clia), according to the instructions from the manufacturer (yhlo Biotech Co. Ltd., Shenzen, China). The results are expressed in arbitrary units (AU)/ml with 10 Au/ml as a positive limit of the assays.

Analysis of anti-Spike-Sars-Cov-2 IGA and IGG in Nasopharyngeal flows

Nasopharnene copies were collected in the hospital using the swabs (floqswabs) in sterile tubes with transport medium (UTM universal transport medium, copan, Italy) and frozen at -80 ° C. The samples were then thawed, inactivated at 56 ° C for 30 minutes, centrifuged at 2800 × g for 10 minutes and the excess were aliquoted and stored at -80 ° C. Anti-Spike igg and IGA in Nasopharyngeal flows (NPS) were rated by Elisa using Sars-Cov-2-cleaned trimer spike¹³ as a coating protein. In short, 96-well plates (Greiner-Bio-Eins, Frickenhausen, Germany) were taken over at 4 ° C with 0.2 µg/vertical protein overnight. After 2 -hour washing and blocking with 200 µl PBS, which contained 1% BSA (SIGMA chemicals), two wrinkled serial dilements from NPS from 1:10 were added to double wells and 2 h incubated at room temperature. The panels were washed and biotin-conjugated goat-anti-Human-igg or IGA (Southern Biotech, Birmingham, AL, USA) added 2 hours at room temperature. The panels were washed again before the addition of horse -tingle peroxidase (HRP) conjugated streptavidine (Anaspec, Fremont, CA, USA) for 30 minutes at room temperature. The antigen antibody₂So₄. End pointers were determined as the subject of the highest dilution, which contained an absorption value at least the triple background (NPS of healthy individuals). To build the Elisa, various positive controls were used, including the entire IG, from the plasma of a Covid 19 convalescent patient, the monoclonal anti-spike¹³) Antibodies.

Total -IGG and IGA in NPs were rated by Sandwich Elisa using Bethyllabor's reagents (Montgomery, TX, USA). In short, the plates were coated with polyclonal goat-anti-human igg or IGA antibodies, and serial dilution of an internal standard (cleaned human serum igg or cleaned human IGA) and two folding seeds serial dilutions from NPS from 1: 100 Added fountains in double and 2 h incubated at room temperature. The next steps were the same as described above. The concentration of IG in the NPS was calculated using an interpolation curve by Graphpad Prism (Graphpad Software Inc., San Diego CA, USA). Since the concentration of total igg and IGA varies in each individual to compare the antigen-specific antibody isotype IgG and IGA in the NPs.

SARS-COV-2-neutralization casts

All serums were tested for the presence of neutralizing antibodies (NABS), which were directed to the spike derived from ancestral virus, using a pseudovirus-based assay developed in our laboratory, as described as described^11.14.15. The assay was validated by comparing the NAB titer in the serum of COVID-19-infected patients (n = 19) with a neutralization assay using live sars-cov-2 as described below. Statistical correlation tests were carried out to ensure comparability between the two assays.

Live virus-based neutralization assay

The virus neutralization test (VNN) was used using the Sars-Cov-2-Isolate (Genbank: MT066156.1; CAT: NR-52284; SARS-based Coronavirus 2, Isoly Italy-Inmi1; near-resources, Manassas, VA, USA). . The virus spread and the neutralization assay were carried out as already described¹⁴. In short, after a heatingness for 30 minutes at 56 ° C, serum samples from 1:10 dilution with the same volume of 50 TCID50 SARS-COV-2 virus solution were mixed and 5% CO2 incubated at 37 ° C at 37 ° C in one moistened atmosphere. Then became a 100 & mgr; L Virus serum mixture to a 96-well plate with a 70% confluent Vero-E6 cell monochtight (Cercopithecus Aethiops derived, the epithelial kidney, ATCC C1008) derived. After 4 days of incubation at 37 ° C and 5% CO2 in a moistened atmosphere, the plates were inspected by an inverse optical microscope on the presence/absence of a cytopathic effect (CPE). The highest serum thinning, which protected more than 50% of CPE cells, was assumed as a neutralization titer. The VNT title was expressed as the subject of the highest serum dilution, which showed protection against virus infections and CPE.

Pseudotyped virus-based neutralization assay

Lentiviral Vector-BASED Pseudoviruses Expressing Luciferase (LV-LUC) Pseudotyped with Spike (LV-Luc/Spike) Were Obained by Transient Transfection of 293 T Lenti-X Cells on 10 cm Petri Dishes (Corning Incorporated-Life Sciences, OneTonta Ny, USA) using the PGAE-Luc lentiviral transfer vector plasmid, which expresses the luciferase coding sequence, the packaging plasmid-pad-Siv3 + and each of the pseudotyping plasmid that express the spike proteins, as described^11.14. Forty-eight hours after the transfection, the excesses contained the LV-LUC Pseudoviren (Perkinelmer, Groningen, Netherlands) with a density of 2.2 × 10⁴ Cells/good. After 48 h, the Luciferase expression was determined by the Britelite Plus Reporter -Say -System (Perkinelmer) and measured in a Varioskan luminometer (Thermo Fisher Scientific, Waltham, MA, USA). Dilutions that deliver 2 × 10⁵ Relative luminescent units (RLU) were used in the neutralization test. In short, the serial double dilutions of the sample from 1:40 or 1:80 for serum and 1:10 for NPs were in double LV-LUC/spike for 30 minutes at 37 ° C in 96-Tiep borehole plates (Resnova, Roma , Italy) and then added to Vero E6 cells, which in a 96-well isoplate (perkinelmer) at one Density of 2.2 were sown × 10⁴ Cells/good. Only virus and cell checks were included. After 48 hours, the Luciferase expression was determined by the Britelite Plus Reporter -Sassay system. RLU data points were converted into a percentage neutralization value, which was only calculated in relative controls by viruses. The results are expressed as an inhibition concentration (ID) 90, 75 and 50, which correspond to the sample dilution, which corresponds to 90%, 75% and 50% of the infection inhibition compared to the control deepening. ID values ​​were calculated using a linear interpolation method^11.14.

Neutralization of the activity against SARS-COV-2 variants

In order to determine the cross-neutralization activity against variants of concerns (VOC), various LV-Luc/Spike Ba.2, Ba.4/5) were used using the methodology described above and described above¹⁴. The pseudoviruses were used to compare the cross-neutralization activity with omicronic sub-variants in selected Sardinian patients and in a cohort of Covid-19 convalescent patients from the IRCCS San Raffaele Hospital in Milan, a geographically distant Italian population. Serum samples were preserved by patients (adapted to age, gender relationship and anti-Wuhan neutralization institution), which are characterized in an earlier study (COVID-19-patient, biobank, treatment reaction and the predictor [COVID-BioB]EC protocol number 34/Int/2020, Clinicaltrials.gov NCT04318366)¹⁶.

Quantification of Sars-Cov-2-RNA in NPS

The viral RNA of NPs was extracted using the DSP virus/pathogen -Midi kits with automated qiasymphony (Qiagen). Viral copies were evaluated by real -time -PCR how the protocol from the US centers for the control and prevention of diseases (CDC) is displayed.¹⁷. In short, the real-time PCR was on CFX 96 Biorad with a mixing reaction of 20 & MGR; l carried out, the 5 & mgr; L Extracted RNA contained 5 µl 4 × Taqpath ™ 1-step-RT-QPCR-Master mix (Thermo Fisher Scientific Inc.), and Primer and probes for the N2 gene; The human ribonuclease -P gene (RP constitutive gene) was amplified as an internal control. SARS-COV-2-TITER, expressed as TCD50/ML equivalent (TCD50_Glow/ml), was through crossing point⁶ TCD50/ml.

Statistical analysis

The data was analyzed with Graphpad Prism 9.5.1 (Graphpad software Inc.) and expressed as a mean ± standard error of the average (SEM) or median with 95% CI. The correlation was assessed with several comparisons by Spearman correlation analysis, the comparison of the Wilcoxon Matched Pairs Rank test and the mixed effect analysis. P Values