Journal: medRxiv

Article Title: DIFFERENTIAL SARS-COV-2 ANTIGEN SPECIFICITY OF THE HUMORAL RESPONSE IN INACTIVATED VIRUS-VACCINATED, CONVALESCENT, AND BREAKTHROUGH SUBJECTS

doi: 10.1101/2022.07.01.22277165

Figure Lengend Snippet: Five hundred nanograms (500 ng) of SARS-CoV-2 recombinant proteins (ORF1a, ORF3a, ORF8, NSP1, NSP8, NSP9, NSP10, NSP14, E (envelop), ME (membrane)) were immobilized in nitrocellulose membranes using 2 uL of proteins prepared in 8M Urea denaturant buffer. The membranes were blocked with 10% BSA and incubated overnight at 4°C with a sera pool obtained from control naïve individuals, convalescents (PCR (+) + 4w), CoronaVac-vaccinated with 2nd dose + 2 weeks, and breakthrough (PCR (+) + 2 weeks) subjects (dilution 1/250). As positive controls, all proteins (500 ng) were incubated with an anti-HisTag antibody conjugated with biotin. After incubation with sera, the membranes were treated with anti-human IgG-HRP. Finally, the membranes were incubated with an enhanced chemiluminescence Western blot detection system (Femto, ECL).

Article Snippet: In-house indirect ELISA assays were performed to detect humoral immunity against N-SARS-CoV-2 (R&D systems, #10474-CV), M (R&D systems, #10690-CV), E (Sino Biological, #40609-V10E3), ORF3a (LSBio, #LS-G145920) and NSP8 (R&D systems, #10633-CV).

Techniques: Recombinant, Membrane, Incubation, Western Blot

Journal: medRxiv

Article Title: DIFFERENTIAL SARS-COV-2 ANTIGEN SPECIFICITY OF THE HUMORAL RESPONSE IN INACTIVATED VIRUS-VACCINATED, CONVALESCENT, AND BREAKTHROUGH SUBJECTS

doi: 10.1101/2022.07.01.22277165

Figure Lengend Snippet: Serum samples from CoronaVac-vaccinated individuals were obtained at the moment of the first dose (pre-immune), two and four weeks after the second dose, and four weeks after a booster dose. D, E, and F. Convalescent individual serum sampling was performed at 1, 2, 4, and 8 weeks after recovering from SARS-CoV-2 infection. Nine convalescent individuals were evaluated without information about the time of sample collection (Unspecified). G, H, and I. Follow-up samples from CoronaVac breakthrough cases (right) were obtained two and four weeks after a positive PCR result. Serum from ten naïve individuals was added as controls. Reciprocal antibody titers elicited against structural SARS-CoV-2 proteins (N, M, and E) are shown in the upper panel. Reciprocal antibody titers elicited against non-structural SARS-CoV-2 proteins (NSP8 and ORF3a) are shown in the middle panel. The seropositivity rates were determined for each protein, and the respective time-points evaluated correspond to the lower panels. Bars show the Geometric Mean Titer (GMT), and the error bars indicate 95% CI. A two- way ANOVA test was used with Tukey’s multiple comparison post-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: In-house indirect ELISA assays were performed to detect humoral immunity against N-SARS-CoV-2 (R&D systems, #10474-CV), M (R&D systems, #10690-CV), E (Sino Biological, #40609-V10E3), ORF3a (LSBio, #LS-G145920) and NSP8 (R&D systems, #10633-CV).

Techniques: Sampling, Infection, Comparison

Journal: medRxiv

Article Title: DIFFERENTIAL SARS-COV-2 ANTIGEN SPECIFICITY OF THE HUMORAL RESPONSE IN INACTIVATED VIRUS-VACCINATED, CONVALESCENT, AND BREAKTHROUGH SUBJECTS

doi: 10.1101/2022.07.01.22277165

Figure Lengend Snippet: Serum samples from CoronaVac-vaccinated individuals were obtained at the first dose (pre-immune), two and four weeks after the second dose, and four weeks after a booster dose for evaluating A. N-specific IgG antibodies, B. M-specific IgG antibodies, C. NSP8-specific IgG antibodies. Serum sampling of convalescent individuals was performed at 1, 2, 4, and 8 weeks after recovery from SARS-CoV-2 infection. Nine convalescent individuals without information regarding the time of sample collection (Unspecified) were included for evaluating, D. N-specific IgG antibodies, E. M-specific IgG antibodies, F. NSP8-specific IgG antibodies. Follow-up samples from breakthrough cases were obtained two and four weeks after a positive PCR result for evaluating G. N-specific IgG antibodies, H. M-specific IgG antibodies, and I. NSP8-specific IgG antibodies. Sera from ten naïve individuals were added as controls. Bars show the Geometric Mean Units (GMU), and the error bars indicate 95% CI. A Kruskal-Wallis test was used with Dunn’s multiple comparison post-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: In-house indirect ELISA assays were performed to detect humoral immunity against N-SARS-CoV-2 (R&D systems, #10474-CV), M (R&D systems, #10690-CV), E (Sino Biological, #40609-V10E3), ORF3a (LSBio, #LS-G145920) and NSP8 (R&D systems, #10633-CV).

Techniques: Sampling, Infection, Comparison

Journal: medRxiv

Article Title: DIFFERENTIAL SARS-COV-2 ANTIGEN SPECIFICITY OF THE HUMORAL RESPONSE IN INACTIVATED VIRUS-VACCINATED, CONVALESCENT, AND BREAKTHROUGH SUBJECTS

doi: 10.1101/2022.07.01.22277165

Figure Lengend Snippet: Specific IgG antibodies titers in samples collected at four weeks after the second or booster dose in individuals belonging to the vaccinated group and four weeks after the diagnosis of SARS-CoV-2 infection by positive PCR result. Individuals from the breakthrough group were compared with those from the convalescent group (samples obtained four weeks after the recovery from SARS-CoV-2 infection and individuals for whom there is no data regarding when the samples were collected). A, B, and C . Reciprocal antibody titers against N, M, and E- specific IgG. D and E. Reciprocal IgG specific antibody titers against NSP-8 and ORF3a. The numbers above the groups show the Geometric Mean Titer (GMT), and the error bars indicate the 95% CI. The numbers in red indicate the fold decrease. The blue text indicates fold increases. A Kruskal-Wallis test was used with Dunn’s multiple comparison post-test, *p < 0.05, **p < 0.01, ***p < 0.001. F and G. ROC analyses of IgG responses to the N (violet), M (yellow), E (blue), NSP-8 (green), and ORF3a (pink) proteins to compare vaccinated individuals after the administration of two or three doses of CoronaVac and convalescent subjects, respectively. Ara under the curve (AUC) values are indicated in parenthesis.

Article Snippet: In-house indirect ELISA assays were performed to detect humoral immunity against N-SARS-CoV-2 (R&D systems, #10474-CV), M (R&D systems, #10690-CV), E (Sino Biological, #40609-V10E3), ORF3a (LSBio, #LS-G145920) and NSP8 (R&D systems, #10633-CV).

Techniques: Infection, Comparison

Journal: medRxiv

Article Title: DIFFERENTIAL SARS-COV-2 ANTIGEN SPECIFICITY OF THE HUMORAL RESPONSE IN INACTIVATED VIRUS-VACCINATED, CONVALESCENT, AND BREAKTHROUGH SUBJECTS

doi: 10.1101/2022.07.01.22277165

Figure Lengend Snippet: ROC curves were constructed to identify SARS-CoV-2 proteins that can differentiate breakthrough cases from A. individuals that received two doses of CoronaVac. B. individuals receiving three doses (two in the primary schedule, plus one booster) of CoronaVac C. Convalescent individuals. Sera collected four weeks after recovery from infection, vaccination, and breakthrough cases were analyzed. For the convalescent group, nine individuals with an unspecified time of sample collection were included. D, E, and F. AUC values, negative predictive power, and positive predictive power for the combinations of M and N antibodies titers to differentiate the breakthrough cases from D. individuals that received two doses of CoronaVac. E. individuals that received three doses of CoronaVac F. Convalescent individuals. G, H and I . Scatter plots distributions along with cutoff values for IgG responses based on optimal Youden values related to ROC curves: convalescent individuals (light blue), individuals vaccinated with two (dark blue), or three (green) doses, and breakthrough cases (orange).

Article Snippet: In-house indirect ELISA assays were performed to detect humoral immunity against N-SARS-CoV-2 (R&D systems, #10474-CV), M (R&D systems, #10690-CV), E (Sino Biological, #40609-V10E3), ORF3a (LSBio, #LS-G145920) and NSP8 (R&D systems, #10633-CV).

Techniques: Construct, Infection