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

Abstract

We performed a retrospective immunological analysis of the antibody response in serum and in nasopharyngeal swabs (NPS) obtained from 46 individuals infected with ancestral SARS-CoV-2 Wuhan-Hu-1 strain during the first COVID-19 wave in Cagliari (Sardinia, Italy), with a 4-month follow-up after the hospital admission. We implemented a comprehensive antibody response in serum and in mucosal samples using assays established in our laboratories. In NPS we evaluated the viral load by real time PCR, presence and kinetics of anti-Spike IgG and IgA by ELISA as well as their anti-Wuhan neutralization activity, showing induction and persistence of anti-viral immunity at the mucosal level. Neutralizing antibodies were measured in serum and NPS using a safe pseudovirus-based assay validated after comparison with a standard neutralization test using live SARS-CoV-2. We evaluated cross-neutralizing antibodies against all the major early variants of concerns (VoC) in sera. Of note, we detected a remarkable reduction of neutralizing activity against BA.1 compared to BA.2 and BA.5 Omicron subvariants, which was confirmed in sera from an analogous cohort of patients at the San Raffaele hospital in Milan, a geographically distant region of Italy, infected with the ancestral virus during the same period of time.

Keywords: Mucosal antibodies; Neutralizing antibodies; SARS-CoV-2; Sardinia.

Fig. 1

Anti-SARS-CoV-2 IgM and IgG antibodies in serum. Serum samples were assayed by CLIA at the hospital. Anti-SARS-CoV-2 IgM and IgG levels expressed in log10 AU/ml of each patient are reported. The dotted lines indicate the cut off of the assay (10 AU/ml). (a) Anti-SARS-CoV-2 IgM and IgG levels in serum samples (N = 18) collected within 10 days from the hospital admission are shown. Kinetics of anti-SARS-CoV-2 IgM (b) and IgG (c) antibodies in all serum samples collected at different time points (days) after the admission.

Fig. 2

Comparison between neutralization assays. Selected serum samples (N = 19) were assayed using neutralization assays based on infectious SARS-CoV-2 (VNT) and on lentiviral vector pseudotyped with Spike (LV-Luc). (a) Correlation between the ID50 (log10) values obtained from the two assays. Dots correspond to individual measurements; the black line represents the regression line. (b) Comparison of ID50 (log10) values obtained using the two assays. A Wilcoxon matched-pairs signed rank test was used to compare the assays. The black line indicates the median ID50, the dotted line indicates the assay cut off (minimum serum dilution tested: 1:20 dilution).

Fig. 3

Analysis of anti-Wuhan nAbs in serum samples. (a) Serum samples collected within 10 days from the hospital admission (N = 18) were assayed by LV-Luc neutralization assay. Each dot represents a single patient. Data are expressed as ID50, ID75 and ID90. The black line indicates the median value. The dotted line indicates the assay cut off (minimum serum dilution tested: 1:40 dilution). (b) Kinetics of nAbs. Serum samples collected at different time points (days) after the admission were assayed for neutralization activity. Data are expressed as ID50. The red line represents the kinetics of median ID50.

Fig. 4

Kinetics of anti-Spike antibodies in NPS. Swabs collected at the hospital admission and during hospitalization were assayed by ELISA. (a) Kinetics of total IgG and IgA and anti-Spike (S) IgG and IgA (mean ± SEM). Data are expressed as ng/ml. (b) Kinetics of the percentage of anti-Spike IgG and IgA. Asterisks indicate a significant difference between IgG and IgA (*p < 0,05, **p < 0,01, ***p < 0,001, Wilcoxon matched-pairs signed rank test). (c) Evaluation of neutralizing activity in NPS. Selected patients (N = 19) with serum nAbs (ID50 > 100) were assayed for anti-Wuhan nAbs in NPS. The corresponding value in serum is also indicated. Each dot represents a single patient. Results are expressed as ID50. The dotted line indicates the assay cut off (minimum sample dilution tested: 1:10 dilution).

Fig. 5

Analysis of viral RNA content in NPS. Quantification of viral RNA in the available NPS by RT-PCR at different time points from the hospital admission (N = 28). Data are expressed as TCD50_eq/ml. Black circles indicate RNA content of a patient with a peculiar immune profile discussed in the text.

Fig. 6

Cross neutralization with VoC. NAbs against each VoC were measured in serum samples (N = 16) from Cagliari (a) and Milan cohort (b), using LV-Luc pseudotyped with Spike from the indicated VoC. Results are expressed as ID50. Each dot represents a single patient. The black line indicates the median ID50. The dotted line indicates the assay cut off (minimum serum dilution tested: 1:80 or 1:40 starting dilution). The asterisks indicate a statistically significant difference between ID50 Wuhan versus ID50 VoC, measured by mixed-effects analysis (*p < 0,05; ****p < 0,0001).