Successful clearance of persistent SARS-CoV-2 asymptomatic infection following a single dose of Ad5-nCoV vaccine

Abstract

Persistent asymptomatic (PA) SARS-CoV-2 infections have been identified. The immune responses in these patients are unclear, and the development of effective treatments for these patients is needed. Here, we report a cohort of 23 PA cases carrying viral RNA for up to 191 days. PA cases displayed low levels of inflammatory and interferon response, weak antibody response, diminished circulating follicular helper T cells (cTfh), and inadequate specific CD4⁺ and CD8⁺ T-cell responses during infection, which is distinct from symptomatic infections and resembling impaired immune activation. Administration of a single dose of Ad5-nCoV vaccine to 10 of these PA cases elicited rapid and robust antibody responses as well as coordinated B-cell and cTfh responses, resulting in successful viral clearance. Vaccine-induced antibodies were able to neutralize various variants of concern and persisted for over 6 months, indicating long-term protection. Therefore, our study provides an insight into the immune status of PA infections and highlights vaccination as a potential treatment for prolonged SARS-CoV-2 infections.

Fig. 1

Persistent asymptomatic SARS-CoV-2-infected cases exhibited unique immunological features. a Viral shedding times are displayed. Comparisons between NA, Mild, and Severe groups with the PA group, respectively, were performed using Mann–Whitney test. b–h Plasma levels of IL-6 (b), IL-1β (c), IL-10 (d), nAb against SARS-CoV-2 (WT) (e), and frequencies of cTfh (f), specific CD4⁺ T (g), and specific CD8⁺ T (h) in PBMCs at various time points post-onset in each group are presented. wpo, weeks post-onset. Wilcoxon matched-pairs signed-rank test was used to compare the data of subsequent time points with the first time point in the same group. Mann–Whitney test was used to compare the data of the first time point in different groups with Naive or PA groups, as indicated. ns: P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. i–l Spearman correlation analysis was performed to analyze the correlation between viral shedding time and initial values of nAb (i), cTfh (j), specific CD4⁺ (k), and CD8⁺(l) T-cell frequencies, and the data of the four groups are presented. Data collected at the first time point for each patient was defined as the initial values. Each circle indicates an individual case, and the horizontal black lines indicate the mean ± SEM or geometric mean ± geometric SD (e) values. Naive, SARS-CoV-2-naive participants; PA, persistent asymptomatic SARS-CoV-2-infected cases; NA, non-persistent asymptomatic SARS-CoV-2-infected cases; Mild, patients with mild COVID-19; Severe, patients with severe COVID-19

Fig. 2

A single dose of Ad5-nCoV vaccine elicited robust antibody responses and viral clearance. a Vaccination scheme indicating that 10 of the PA cases received one dose of Ad5-nCoV vaccine (Convidecia) (9 via inhalation and 1 via intramuscular injection) at 18 wpo when they were still SARS-CoV-2 viral RNA-positive. Samples were collected at 4 consecutive time points (T1, T2, T3, and T4). b Viral loads in oropharyngeal swab were detected at given time points using q-RT-PCR targeting on ORF1ab (left) and N (right) genes. The dashed lines represent the threshold, and a Ct value of <40 indicates the presence of SARS-CoV-2 nucleic acid in the sample. c Dynamics of nAb titers against SARS-CoV-2 (WT) in PA cases post vaccination are presented. The dashed line represents the limit of detection. d Dynamics of IgG and IgA levels against multiple structural proteins (RBD, S, S1, and S2) in plasma are presented. Plasma samples from six healthy donors collected in 2017 − 2018 were used to define the thresholds (mean + 3 SD), indicated as dashed lines. b–d Wilcoxon matched-pairs signed-rank test was performed to compare data between T1 and other time points, respectively (*P < 0.05; **P < 0.01). e–j Spearman correlation analysis was performed to analyze the correlation between Ct values of ORF1ab or N genes and the values of neutralizing antibodies (e) or various binding antibodies (g–j), which are summarized in a heatmap (f)

Fig. 3

Ad5-nCoV vaccination enhanced B-cell and cTfh responses in PA cases; these responses were positively correlated with nAbs. a–c Frequencies of RBD⁺ B cells (a), ASCs (b), and cTfhs (c) in PBMCs of PA cases post vaccination are presented. Representative flow plots displaying the gating of RBD⁺ B cells (a), ASCs (b) and cTfhs (c) are presented on the left, and summary panels are presented on the right. d–h Spearman correlation analysis was performed to analyze the correlations among nAbs, RBD-IgG, RBD⁺ B cells, ASCs and cTfhs. i, j Frequencies of specific CD4⁺ (i) and CD8⁺ (j) T cells in PA cases post vaccination are presented. k PCA plot showing the distribution of patients in the PA group at different time points. Each dot represents a case. l PCA plot displaying corresponding trajectories of key markers affecting separation between groups. a–c, i, j Wilcoxon matched-pairs signed-rank test was conducted to compare data between T1 and other time points, respectively

Fig. 4

A single-vaccination dose induced durable and broad nAbs against various SARS-CoV-2 VOCs in PA cases. a nAb titers against five SARS-CoV-2 VOCs (WT, Beta, Delta, Omicron BA.1, and Omicron BA.5) at 0 wpv (before vaccination), 2 wpv (peak response) and 25 wpv are presented. The fold changes of nAb titers are presented as the geometric mean (min-max). b–d Frequencies of RBD⁺ B cells (b), ASCs (c), and cTfhs (d) at 0, 2, and 25 wpv are presented. Data were analyzed using Wilcoxon matched-pair signed-rank test

Fig. 5

Ad5-nCoV vaccination induced distinct immune profiles in PA and HD groups. a–g Comparisons of nAbs against SARS-CoV-2 (WT) (a), RBD⁺ B cells (b), ASCs (c), cTfhs (d), specific CD4⁺ T cells (f), and specific CD8⁺ T cells (g) at 0, 2, and 6 wpv between PA and HD groups (SARS-CoV-2-naive participants who had completed two doses of inactivated vaccine) are presented. Representative flow plots of specific CD4⁺ and CD8⁺ T cells are presented (e). f, g The presented values were obtained with the formula: (unstimulated cells − (peptide-stimulated cells)). h, i Representative flow plots (h) and summary (i) of memory subsets of specific CD4⁺ T cells at 0, 2, and 6 wpv in the two groups are presented. Wilcoxon matched-pairs signed-rank test was performed to compare the data between different time points in the same group. j, k Spearman correlation test was conducted to analyze the correlation between specific CD4⁺ TEM (0 wpv) and specific CD4⁺ T (2 wpv) (j), and between specific CD4⁺ T before (wpv0) and after (wpv2) vaccination (k). nAb titers are presented as geometric mean ± geometric SD. Other data are presented as mean ± SEM