Seropositivity rates and neutralizing antibodies levels in patients with autoimmune disease upon planned primary 17DD-YF vaccination
The quantification of YF-specific neutralizing antibody was carried out using the standard protocol of plaque-reduction neutralization test (PRNT) and the results are shown in the Table 1. Data analysis demonstrated that the seropositivity rate was significant lower in AID patients (77%) as compared to HC (95%). Subsequent categorization of AID patients according to the type of autoimmune disease further demonstrated that SpA (73%) and SLE (71%) presented lower seropositivity rates, while RA (87%) and SS (77%) displayed similar seropositivity rates as compared to HC. The analysis of neutralizing antibodies levels (GeoMean) corroborate the seropositivity rates, showing lower levels of PRNT in AID (170), SpA (112) and SLE(133), but similar levels in RA (291) and SS (209) as compared to HC (448) (Table 1).
Viremia levels in patients with autoimmune disease upon planned primary 17DD-YF vaccination
The viremia level quantification (YF Viral RNAnemia) was performed by qRT-PCR assay and the data expressed as mean copies/mL at peak of viremia, presented in the Table 2. The results demonstrated that all groups exhibited the peak of viremia around Day5-6 after vaccination. The analysis of viremia levels at peak did not shown significant differences amongst AID subgroups as compared to HC (Table 2).
Timeline kinetics of serum biomarkers in patients with autoimmune disease following planned primary 17DD-YF vaccination
The quantification of serum chemokines, pro-inflammatory cytokines, regulatory cytokines and growth factors was carried out by high-performance microbeads array at baseline (Day0) and at four consecutive scheduled time points, including: Day3-4, Day5-6, Day7 and Day14-D28 after vaccination. The results are presented as median baseline fold changes in the Figure 2. Data analysis demonstrated that 17DD-YF vaccine triggered a more prominent serum biomarker response in AID as compared to HC, throughout the timeline kinetics. In general, prominent increase in baseline fold changes observed in AID patients occurred at Day3-4 (CXCL8, CCL11, CCL3, CCL4, CCL5, IL-1β, IL-6, TNF-α, IL-17, IL-9, FGF-basic, VEGF, G-CSF, IL-2), at Day5-6 (CXC10, IL-4, IL-5, IL-10, IL-13, IL-7) or at Day7 (IL1Ra). In a few situations along the timeline kinetics AID presented lower baseline fold changes as compared to HC, as observed at Day3-4 (IL-15, IL-10, PDGF, IL-7), at Day5-6 (CCL2, IFN-γ, IL1Ra) and at Day7 (CCL4, CCL2, IL-12, IL-15, PDGF, IL-7). Overall, the baseline fold changes were higher in AID at Day14-28 as compared to HC. Of note, was differential kinetic profile of IFN-γ and IL-10 observed for HC and AID. While HC showed high IFN-γ but low IL-10 at Day5-6, lower increase of IFN-γ concomitant with up-regulation of IL-10 was observed to AID at Day5-6 (Figure 2). These differences in baseline fold changes profiles of serum biomarkers detected along the timeline kinetic and particularly at the peak of viremia (Day5-6) may explain the distinct seroconversion profile observed in AID as compared to HC.
Timeline kinetics of serum biomarkers in patients with autoimmune disease according to the neutralizing antibody status after planned primary 17DD-YF vaccination
The timeline kinetics of changes in serum biomarkers were further evaluated in AID patients categorized according to the PRNT status after primary 17DD-YF vaccination as AID/PRNT(-) and AID/PRNT(+) and the results showed in the Figure 3. Data analysis demonstrated that, in general, more prominent serum biomarker response was observed in AID/PRNT(+) as compared to AID/PRNT(-), throughout the timeline kinetics. Prominent increase in baseline fold changes observed in AID/PRNT(+) patients occurred at Day3-4 (CXCL8, CCL11, CCL5, IL-1β, IL-6, TNF-α, IL-15, IL-17, FGF-basic, G-CSF, IL-2, IL-7), at Day5-6 (CCL2, CXC10, IFN-γ, IL-5, IL-10, IL-13, GM-CSF) or at Day7 (IL1Ra). Along the timeline kinetics, the AID/PRNT(+) presented lower baseline fold changes as compared to AID/PRNT(-), as observed at Day5-6 (CXCL8, CCL11) and at Day7 (CXCL8, CCL3, CCL2, CCL5, IL-6, TNF-α, IFN-γ, IL-15, IL-4, IL-9, PDGF, CM-CSF, IL-2, IL-7). The AID/PRNT(-) exhibited increased levels of serum biomarkers late at Day14-28 as observed for (CXCL8, CCL11, CCL3, CCL4, TNF-α, IL-17, IL-4, PDGF, VEGF, IL-2, IL-7). Of note, a synchronic pattern of high IFN-γ levels at the day of peak viremia (Day5-6) was observed in AID/PRNT(+) groups, whereas an asynchronous IFN-γ response was reported for AID/PRNT(-) later at Day7. However, both subgroups exhibited peak of IL-10 concomitant with the viremia peak at Day5-6 (Figure 3).
Baseline profile of serum biomarkers in patients with autoimmune disease according to the neutralizing antibody status after planned primary 17DD-YF vaccination
Aiming at investigating whether the baseline profile of serum biomarker was associated with the seroconversion status achieved after planned primary 17DD-YF vaccination, the AID patients were categorized according to the PRNT status after primary 17DD-YF vaccination as AID/PRNT(-) and AID/PRNT(+), and the biomarker levels prior planned primary 17DD-YF vaccination compared between AID subgroups. The results are presented in the Figure 4. Data analysis demonstrated that AID/PRNT(-) exhibited higher levels of several biomarkers at baseline (CXCL8, CCL11, CXCL10, TNF-α, IL-15, IL-17, IL-10, G-CSF, GM-CSF) as compared to AID/PRNT(+), suggesting that an inflammatory status prior vaccination may orchestrated the lack of antibody response (Figure 4).
Serum biomarker signature in patients with autoimmune diseaseafter planned primary 17DD-YF vaccination
The serum biomarker signature of patients with autoimmune diseases were assessed at distinct time points after planned primary 17DD-YF vaccination and the results presented in Figure 5. Data analysis demonstrated that, in general, AID patients exhibited a more prominent increased in serum biomarkers levels as compared to health controls. In fact, a massive up-regulation of several soluble mediators was observed in AID patients at Day3-4 and Day5-6 with subsequent decrease at Day7 towards Day14-28 (Figure 5A). Noteworthy, was that while HC presented a synchronic increase of IFN-γ at the peak of viremia (Day5-6) with late increase of IL-10 at Day7, AID patients displayed a simultaneous and persistent increase of IFN-γ and IL-10 at the peak of viremia (Day5-6) up to Day7 after vaccination (Figure 5A). Heatmap analysis further illustrated these synchronic/asynchronous phenomena observed for the IFN-γ and IL-10 rhythm nearby the viremia peak (Figure 5B).
Serum biomarker signatures after planned primary 17DD-YF vaccination according to the type of autoimmune disease
Serum biomarker signatures were further assembled for AID patients according to the type of autoimmune disease (Figure 6). The results demonstrated that SpA and SLE patients presented early increase in serum biomarkers upon planned primary 17DD-YF vaccination, more prominent at Day3-4 with subsequent shift towards unaltered baseline fold changes in most soluble mediators at Day14-28 (Figure 6). Conversely, RA and SS subgroups exhibited a robust and persistent increase in serum biomarkers upon planned primary 17DD-YF vaccination. While the RA subgroup still presented a decrease in several biomarkers later at Day14-28, the SS subgroup maintained most serum biomarkers elevated throughout the kinetic timeline after planned primary 17DD-YF vaccination (Figure 6). Of note, was the finding that IFN-γ levels increase at the peak of viremia (Day5-6) in most AID subgroups, except for the SLE subgroup which presented IFN-γ up-regulation early at Day 3-4 (Supplementary Figure 1).
Serum biomarker signatures in patients with autoimmune diseaseaccording to the neutralizing antibody status after planned primary 17DD-YF vaccination
In order to further characterize the changes in serum biomarkers observed after planned primary 17DD-YF vaccination and its association with the post vaccination PRNT status, the AID patients were categorized as AID/PRNT(-) and AID/PRNT(+) and the fold changes in biomarker levels compared between AID subgroups (Figure 7). Data analysis that AID/PRNT(+) exhibited an earlier serum biomarker response with higher fold change values at Day3-4 and Day5-6 with following decrease in a range of soluble mediators at Day7 towards Day14-28 (Figure 7A). On the other hand, AID/PRNT(-) presented higher fold change in serum biomarkers at late time-points with a progressive increase towards Day7, maintaining a sustained up-regulation of serum biomarkers up to Day14-28 (Figure 7A). These findings suggest the occurrence of putative immunomodulatory effect of live attenuated 17DD-YF vaccine associated with the seroconversion status observed in AID (Figure 7A).
Heatmap analysis further corroborates these findings (Figure 7B). AID/PRNT(-) exhibited an increase of IL-10 concomitant with the peak of viremia (Day5-6) and a late increase of IFN-γ at Day7. Conversely, AID/PRNT(+) patients displayed an early and persistent increase of IL-10 at Day3-4, Day5-6 and Day7 around the peak of viremia, maintaining the IFN-γ up-regulation concomitant with the peak of viremia (Day5-6) (Figure 7B).