Fig 1.
Clinical cohort characteristics.
(A) Cohort participant’s antibody responses to EBV were measured by standard diagnostic ELISA over the sampling period of one year. Assay signals were normalized to the measurable signal range of the assay and mean values for all samples at a given time point are shown. (VCA = viral capsid antigen, EA = early antigen). (B) SOI was self-assessed by cohort participants at the time points of sample collection. Each individual line represents the SOI score of a single individual over the observation period. The height of grey bars reflects the mean SOI for a given time point. (C) A qPCR for BamHI-W repeats was run on 4x106 B cells of cohort participants derived from PBMC samples collected at the indicated time points. Dots represent the results for all available samples. Different colors indicate individual cohort participants. Grey bar graphs indicate the median.
Fig 2.
EBV proteins induce distinct IgG subclass responses.
(A) EBV antigen-specific titers for IgM were determined by class-/subclass-specific direct quantitative ELISAs using a reference Ab of known concentration as standard. Dots represent the median Ab concentration for the indicated time point. (B) EBV antigen-specific titers for IgG1 and IgG3 were determined using a similar approach as for IgM. (C) The peak median concentration for EBV antigen-specific IgM was calculated. Potential differences between all groups were determined by one-way ANOVA using Tukey`s correction for multiple comparisons. Only statistically significant differences are plotted. (D) Peak median concentration for EBV antigen-specific IgG1 and IgG3 were calculated as for IgM. (E) Total median IgG1 and IgG3 concentrations over all time points were calculated and accumulative concentrations set as 100% of the respective pie charts.
Fig 3.
ADCD during acute EBV infection is exclusively mediated by Abs against p18.
(A, B) The ability of EBV p18, gp350/220, p47/54, EBNA-1- (A) and influenza HA1-specific antibodies (B) present in cohort samples to mediate ADCD was determined by flow cytometry. Every dot represents the area under the curve of the average measured signal of a given control (grey) or sample. Colors indicate the different cohort subjects and black lines represent the grand median. (C) An ADCD assay was performed after depletion of p18-specific IgM or IgG in serum samples of 0 or 1 wpe. Significant differences in the levels of ADCD activity before and after depletion of IgM or IgG were determined by two-tailed T-test.
Fig 4.
EBV proteins induce Ab dependent phagocytosis inconsistently at low or minimal levels during the first year of infection, except for p18.
(A, B, D, E) The induction of ADNP (A, B) or ADCP (D, E) by antibodies against EBV p18, gp350/220, p47/54, EBNA-1 and influenza HA1 was analyzed by flow cytometry. Every dot represents the average measured signal of a given control (grey) or sample. Colors indicate the different cohort subjects and black horizontal lines represent the median. (C) An ADNP assay was performed after depletion of p18-specific IgM or IgG in serum samples of 0 or 1 wpe. Significant differences in the levels of ADNP activity before and after depletion of IgM or IgG were determined by two-tailed T-test.
Fig 5.
EBV-specific antibodies, including gp350/220 RBD-specific Abs, are inefficient at stimulating NK cell degranulation during acute infection.
(A, B) EBV antigens p18, p47/54, gp350/220 or EBNA-1 or influenza HA1 as a control were coated to ELISA plates and incubated with serum of EBV infected cohort participants collected at the indicated time points. After an incubation period NK cell degranulation induced by antigen-specific antibodies or PMA/I as an unspecific control was assessed by determining the percentage of CD107a (A) or MIP-1beta (B) positive NK cells by flow cytometry. Every dot represents the average measured signal of a given control (grey) or sample. Colors indicate the different cohort subjects and black horizontal lines represent the median.
Fig 6.
EBV p18-specific Abs bind highly efficiently to FcgRIIIb.
The ability of antigen-specific EBV and influenza antibodies to bind to FcgRs was measured by luminex. To account for titer dependent influences on FcgR binding signals, for each sample the ratio between FcgR binding signal and the sum of IgG1 and IgG3 titers totaling to above 0.1μg/ml was calculated. Lines in violin plots represent the divisions between quartiles, with the black line representing the grand median. A one-way ANOVA with Tukey’s correction for multiple comparisons was applied to determine statistical differences between all groups. Only statistically significant differences are plotted.
Fig 7.
The breadth of EBV-specific Ab responses expands from acute to chronic infection.
IgG recognition of EBV-specific peptides was determined in 18 individuals at an early (<4 wpe) and a late (>24 wpe) time point of EBV infection by the phage-based VirScan assay. The heatmap shows antibody responses to EBV proteins when antibody binding was detectable in at least two individuals.
Fig 8.
Acute EBV infection fails to induce a persistent Fc-functional Ab profile compared to acute HIV infection.
Radar plots show the average value of the Z-scored functional data for EBV and HIV antigens (in the respective cohort) at the 4 or 52-week time point.