PMC8492986_01
SARS-CoV2 and non-CoV-2 antibody profiling using Luminex based assays and immunoglobulin subclass assays. (A) Reactivity of serum IgG antibody to CoV-2 proteins (Full Spike, S1, S2, RBD and NC) and non-SARS-CoV-2 proteins from community coronaviruses (CoV-229E-S1, HCoV-HKU1-S1, HCoV-NL63-S1, HCoV-OC43-S1), and novel coronaviruses (MERS-S1 and SARS-S1). X-axis represents days relative to first positive SARS-CoV-2 test in chronological serum dates. Y-axis represents the Mean Florescence Intensity (MFI) value of the reactivity. (B) Anti-SARS-CoV-2 IgG, IgA and IgM serum concentrations measured using the immunIQ assay (mg/L).
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No SARS-CoV-2 specific antibodies were detected in samples prior to day 49 by either assay (Figure 2A). Low levels of serum IgG antibodies (MFI <4,000) to community coronaviruses were detected prior to the first positive SARS-CoV-2 test, suggesting previous exposure to other coronaviruses (Figure 2A). IgM, IgG and IgA were below detectable limits in post-op day 5 samples. Increasing levels of all three isotypes were detected from day 49, with peak concentrations in day 68 samples (IgG 13.1, IgM 9.1, IgA 2.9 mg/L) (Figure 2B).
T cell repertoire sequencing of patient at study time points. (A) Differential abundance analysis comparing clonotypes that have significantly increased or decreased in frequency across baseline and post-infection Day 2, Day 5, and Day 111. This is based on a beta-binomial model where normal repertoire changes in healthy adults over time are accounted for while measuring clonal expansion. (B) Locations on the SARS-CoV-2 genome where TCR binding has likely occurring; different colours specify open reading frame, and size of dots indicate number of TCRbeta rearrangements associated with SARS-CoV-2 at a particular position. Screening was performed based on the ImmuneCODE database (Adaptive Biotechnologies). (C) Changes in frequencies of the 15 most frequently observed TCR clonotypes, relative to total TCR repertoire, across baseline and post-infection day 2, 5, and 111.
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By day 111 however, there were a number of public clonotypes which increased in frequency compared to baseline (Figure 3A). TCRs targeting different regions of the SARS-CoV-2 genome (Figure 3B). The evidence of such clonotypes in the baseline sample suggests previous exposure to community coronaviruses, with multiple targets mapping to the ORF1 and surface glycoprotein, both regions of which are conserved across various coronaviridae genomes (Figure 3B). The top 15 occurring clonotypes across all samples did not show demonstrable changes in the repertoire over the course of infection (Figure 3C).
Immunophenotyping of PBMCs of patient at study time points. (A) Absolute numbers of T cells. (B) CD4 and CD8 T cell frequencies. (C) Subpopulations frequencies of CD4 T cells. (D) Subpopulation frequencies of CD8 T cells. (E) CD38+ HLA-DR+ frequencies of CD8 non-naive T cells. (F) Absolute numbers of B cells. (G) B cell frequencies of lymphocytes. (H) Class switched -, non-class switched - and naive B cell frequencies of naive and memory B cells. (I) Plasmablast - and transitional B cell frequencies of total B cells. (J) Non-T- and Non-B-cell frequencies of lymphocytes. CM, central memory; EM, effector memory; SCM, stem cell memory; CS, class-switched; NCS, non class-switched; Non_T_B, non-T, and non-B cells.
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B cells. The patient was T cell lymphopenic post-transplant and during the early phase of infection as evident in the immunophenotypic profile on day 2 and 5 (0.37 and 0.43 x 109 cells/L) post-infection (Figure 4A). By day 49 (1.72 x 109 cells/L), T cell counts had returned to within normal range, however the patient was again lymphopenic by day 111 (0.26 x 109 cells/L) (Figure 4A). CD4 (50.5 - 58.4%) and CD8 T cell frequencies (33.7 - 38.8%) remained unchanged during the study period (Figure 4B) with only minor changes observed in CD4 T cell subsets (Figure 4C). T cells frequencies were decreased on day 2 and 5 (36.8 and 52.4% respectively), expanded on day 49 (61.1%), then contracted by day 111 (39.5%) (Figure 4D). The frequency of activated non-naive CD8 T cells (CD38+ HLA-DR+) was significantly increased on day 111 (62.7%), compared to the earlier time points (1.92 - 12.8%) (Figure 4E). T cell subsets, B cell absolute counts were generally low on day 2 and 5 (0.07 and 0.13 x 109 cells/L respectively), recovered to normal by day 49 (0.24 x 109 cells/L), then diminished again by day 111 (0.03 x 109 cells/L blood) (Figure 4F). The frequency of B cells, as a proportion of lymphocytes, on the other hand dropped steadily (12.1, 11.8, 9.3 and 3%) over the observation period (Figure 4G). The absolute counts of B cell subsets were low in the early phase of infection and the frequency of these subsets including naive, class-switched, and non-class switched memory B cells changed only slightly (Figure 4H). The frequency of plasmablasts was elevated on days 2 and 5 (1.41 and 1.65% respectively), then declined at day 49 (0.25%), and increased again by day 111 (0.72%) (Figure 4I). Subsequently it declined to 27.9 (day 49) before reaching 80% on day 111 (Figure 4J).