Hydroxychloroquine is associated with lower seroconversion upon 17DD-Yellow fever primovaccination in patients with primary Sjögren's syndrome

Abstract

The present study aimed at investigating whether the hydroxychloroquine (HCQ) treatment would impact the neutralizing antibody production, viremia levels and the kinetics of serum soluble mediators upon planned 17DD-Yellow Fever (YF) primovaccination (Bio-Manguinhos-FIOCRUZ) of primary Sjögren's syndrome (pSS). A total of 34 pSS patients and 23 healthy controls (HC) were enrolled. The pSS group was further categorized according to the use of HCQ (HCQ and Non-HCQ). The YF-plaque reduction neutralization test (PRNT ≥1:50), YF viremia (RNAnemia) and serum biomarkers analyses were performed at baseline and subsequent time-points (Day0/Day3-4/Day5-6/Day7/Day14-D28). The pSS group showed PRNT titers and seropositivity rates similar to those observed for HC (GeoMean = 238 vs 440, p = .11; 82% vs 96%, p = .13). However, the HCQ subgroup exhibited lower seroconversion rates as compared to HC (GeoMean = 161 vs 440, p = .04; 69% vs 96%, p = .02) and Non-HQC (GeoMean = 161 vs 337, p = .582; 69% vs 94%, p = .049). No differences in YF viremia were observed amongst subgroups. Serum biomarkers analyses demonstrated that HCQ subgroup exhibited increased levels of CCL2, CXL10, IL-6, IFN-γ, IL1-Ra, IL-9, IL-10, and IL-2 at baseline and displayed a consistent increase of several biomarkers along the kinetics timeline up to D14-28. These results indicated that HCQ subgroup exhibited a deficiency in assembling YF-specific immune response elicited by 17DD-YF primovaccination as compared to Non-HCQ subgroup. Our findings suggested that hydroxychloroquine is associated with a decrease in the humoral immune response after 17DD-YF primovaccination.

Keywords: 17DD-YF vaccine; Primary Sjögren’s syndrome; humoral immunity; hydroxychloroquine; serum biomarkers.

Graphical abstract

Figure 1.

Neutralizing antibody titers and seropositivity rates in patients with primary Sjögren’s Syndrome upon 17DD-YF primovaccination. The YF-specific neutralizing antibodies were measured by plaque reduction neutralization test – PRNT [20] in serum samples from patients with primary Sjögren’s Syndrome (pSS, D0 = , D28 = , n = 34) and healthy controls (HC, D0 = ,D28 = , n = 23). Data are shown as scattering of individual values of reciprocal of serum dilution over column charts representing geometric mean titers (95%CI). The seropositivity rates are shown as frequency (%) of subjects with titers higher than 1:50 (dashed line).Comparative analysis of PRNT titers and seropositive rates (pSS vs HC) were assessed by Student t test and Chi-square test, respectively. In all cases, significant differences were considered at p < .05.

Figure 2.

Neutralizing antibody titers and seropositivity rates in patients with primary Sjögren’s Syndrome categorized according to the use of HCQ immunotherapy. The YF-specific neutralizing antibodies were measured by plaque reduction neutralization test – PRNT [20] in serum samples from patients with primary Sjögren’s Syndrome (pSS), categorized according to the use HCQ immunotherapy, referred as: Non-HCQ (D0 = , D28 = , n = 18) or HCQ (D0 = , D28 = , n = 16) and samples from healthy controls (HC, D0 = , D28 = , n = 23). Data are shown as scattering of individual values of reciprocal of serum dilution over column charts representing geometric mean titers (95% CI). The seropositivity rates are shown as frequency (%) of subjects with titers higher than 1:50 (dashed line). Comparative analysis of PRNT titers and seropositive rates (pSS subgroups vs HC) were assessed by Student t test and Chi-square test, respectively. In all cases, significant differences were considered at p < .05 and highlighted by bold format.

Figure 3.

Viremia kinetic timeline in patients with Sjögren’s Syndrome upon 17DD-YF primovaccination.The viremia levels were measured by qRT-PCR assay in serum samples from patients with Sjögren’sSyndrome (pSS), categorized according to the use of HCQ immunotherapy, referred as: Non-HCQ (, n = 18) or HCQ (, n = 16) and healthy controls (HC, , n = 23) at distinct time points upon 17DD-YF primovaccination (D3,D4, D5, D6, D7, and D14). Data are shown as GeoMean of copies/mL (95%CI) along the kinetics timeline. Dashed line represents the limit of detection (6.25 copies/µL). Undetectable levels were computed as 0.001 copies/mL. Viremia kinetic curves are shown as GeoMean of copies/mL in overlayed line charts. Comparative analysis of viremia levels at the day of viremia peak (gray rectangles) was carried out by ANOVA followed by Tukey’s post-test and the viremia kinetic curves compared by Kolmogorov-Smirnov (KS) test. In all cases, significant differences at were considered at p < .05.

Figure 4.

Serum soluble mediators in patients with Sjögren’sSyndrome categorized according to the use of HCQ immunotherapy. The levels of serum soluble mediators were measured by high-throughput microbeads array in serum samples from patients with Sjögren’s Syndrome (pSS), categorized according to the use of immunotherapy, referred as: Non-HCQ (D0 = , n = 18) or HCQ (D0 = , n = 16) prior 17DD-YF primovaccination. The levels of chemokines(CXCL8, CCL11, CCL3, CCL4, CCL2, CCL5, CXCL10), pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, IL-12, IFN-γ, IL-15, IL-17),regulatory cytokines (IL-1Ra, IL-4, IL-5, IL-9, IL-10, IL-13) and growth factors (FGF-basic, PDGF, VEGF, G-CSF, GM-CSF, IL-2 and IL-7) are shown as violin plot distribution (min and max) of serum concentrations (pg/mL) underscoring the median (continuous line) and 25^th and 75^th interquartile range (dashedlines). Significant differences at p < .05 are identified by connecting line and underscored by gray background.

Figure 5.

Serum biomarker signatures in patients with Sjögren’s Syndrome upon 17DD-YF primovaccination. The levels of serum soluble mediators were measured by high-throughput microbeads array in serum samples from patients with Sjögren’s Syndrome (pSS), categorized according to the use of HCQ immunotherapy, referred as: Non-HCQ (D0 = , D5-6 = , D14-28 = , n = 18) or HCQ (D0 = , D5-6 = , D14-28 = , n = 16) prior 17DD-YF primovaccination. Serum biomarker signatures were assembled converting the biomarker levels originally express as continuous values (pg/mL) into categorial data, and the results reported as proportion of subjects (%) with biomarker levels above the global median cutoff defined for each serum soluble mediator. Signature curves were assembled to identify the biomarkers with proportion of subjects above the 50^th percentile (dashed line). Heatmaps were constructed considering the proportion of subjects with biomarker levels above the global median cutoff at each time point (D0, D5-6 and D14-28). Color key underscores the proportion of subjects with increased biomarkers levels in percentiles: 10^th (green), 50^th (black) and 90^th (red). Line charts show the total number of biomarkers above the global median cutoff.

Figure 6.

Serum biomarker integrative networks in patients with Sjögren’s Syndrome upon 17DD-YF primovaccination. The levels of serum soluble mediators were measured by high-throughput microbeads array in serum samples from patients with Sjögren’s Syndrome (pSS), categorized according to the use of HCQ immunotherapy, referred as: Non-HCQ (D0 = , D5-6 = , D14-28 = , n = 18) or HCQ (D0 = , D5-6 = , D14-28 = , n=16) prior 17DD-YF primovaccination. The integrative networks were assembled based on Pearson and Spearman “r” scores between chemokines ( = CXCL8, CCL11, CCL3, CCL4, CCL2, CCL5, CXCL10), pro-inflammatory cytokines ( = IL-1β, IL-6, TNF-α, IL-12, IFN-γ, IL-15, IL-17), regulatory cytokines ( = IL-1Ra, IL-4, IL-5, IL-9, IL-10, IL-13) and growth factors ( = FGF-basic, PDGF, VEGF, G-CSF, GM-CSF, IL-2 and IL-7). The networks were created considering only significant correlations (p < .05). Connecting edges show weak/moderate (0.1 to 0.67, thin lines) and strong “r” scores (≥ 0.67, bold lines) between pairs of attributes. Line charts show the total number of connections between the biomarkers.