Immune response induced by standard and fractional doses of 17DD yellow fever vaccine | npj Vaccines – Nature.com

Study population

This is an observational cross-sectional investigation comparing two cohorts of vaccinees defined by vaccine dosage (fractional and standard doses) carried out in the metropolitan area of So Paulo (SP, Brazil) by the Collaborative Group for Studies of Yellow Fever Vaccine. The study protocol was submitted and approved by research ethics committees at Instituto Ren RachouFundao Oswaldo Cruz (CAAE: 82357718.5.0000.5091), Instituto Nacional de Infectologia Evandro Chagas/INIFundao Oswaldo Cruz (CAAE: 82357718.5.3001.5262), Secretaria Municipal da Sade de So Paulo - SMS/SP (CAAE: 82357718.5.3003.0086) and Instituto de Infectologia Emlio RibasIIER (CAAE: 82357718.5.3002.0061). The study population comprised a non-probabilistic convenience sampling including whole blood specimens (n=322 samples) collected from healthy subjects of both genders (Males=142 samples; Females=180 samples), with age ranging from 11 to 65years (Mean=4114), further categorized into two groups referred as Fractional Dose (FD) and Standard Dose (SD). Written informed consent was given from all participants that have joined this study. Only volunteers with negative results of YF-specific neutralizing antibodies prior to vaccination were enrolled in the present study.

The FD group was enrolled between January and July 2018 at primary care medical centers, during the large-scale vaccination campaign with the fractional dose (1/5 of SD) of the 17DD-YF vaccine in three cities at the metropolitan area of So Paulo (Diadema, Mau and Santo Andr). This group included 225 samples (Males=99; Females=126), with age ranging from 11 to 65years (Mean=4314).

Between April and August 2019, the SD group was selected during the routine 17DD-YF vaccination at primary care medical centers in So Paulo. This group included 97 samples (Males=44; Females=53), aged 1763years (Mean=3512).

Blood samples (10mL) were collected at baseline (D0) and at distinct timepoints after primary 17DD-YF vaccination (from D1 throughout D15 and at D30-45) by venipuncture using a gel separation vacuum system without anticoagulant. Aiming to assess the kinetics timeline of distinct parameters, categorical day intervals were defined by grouping D1 to D15 depending on the number of samples available.

Blood specimens were processed to obtain serum samples and peripheral blood mononuclear cells (PBMC) as previously described by Reis et al.30. Whole blood samples were submitted to centrifugation at 10002000g for 10min at room temperature to obtain serum samples, and aliquots stored at 80C at Laboratrio de Investigao Mdica from Universidade de So Paulo (USP) for further viremia quantification, YF-specific IgM, IgG and neutralizing antibodies detection, as well as analysis of soluble mediators. Clots were processed at Laboratrio de Biomarcadores from Instituto Ren Rachou (IRR, FIOCRUZ-Minas) for PBMC isolation. Clots were removed from the gel separation system, sliced into 12mm fragments, and minced with sterile syringe plungers through 100m filters attached to 50mL conical tubes containing RPMI-1640 medium and resuspended with RPMI supplemented with 10% Fetal Bovine Serum to obtain a final volume of 10mL. The suspension was slowly applied over 5mL Ficoll-Paque PLUS cushion (1077g/mL) and submitted to centrifugation at 410g for 40min at room temperature. PBMC were transferred to 50mL conical tubes and washed once with 15mL supplemented RPMI-1640 medium by centrifugation at 300g for 7min at 4C. Thereafter, the supernatant was discarded and the cell pellet was treated with ammonium chloride solution (155mM NH4Cl, 10mM KHCO3, 0.1mM EDTA, pH 7.2) for 10min at room temperature to lyse the remaining red blood cells. Following centrifugation at 300g for 7min at 4C, the PBMC pellet was washed once with RPMI-1640 and resuspended in 1mL. Aliquots of 10L were stained with Trypan Blue (0.4%) to estimate cell viability using the Countess Automated Cell Counter. PBMC suspensions were maintained at 4C for further use for immunophenotyping staining.

The quantification of YF viral copies in serum samples collected from D3 throughout D15 after primary immunization with SD or FD of 17DD-YF vaccine was performed through Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) at Laboratrio de Tecnologia Virolgica in Bio-Manguinhos (LATEV, FIOCRUZ), as previously described by Martins et al.15. The limit of detection (LOD) of the test was obtained through the validation process of the one step qPCR assay for yellow fever carried out in Bio-Manguinhos. LOD: 6.25 copies/L or 3.45 Log10 copies/mL. The results were expressed as viral copies/mL according to the standard curve included in each experimental batch.

The quantification of Anti-YF IgM was performed at the Laboratrio de Tecnologia Imunolgica from Bio-Manguinhos (LATIM, FIOCRUZ) using a standardized in-house capture enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates were coated with 75L/well of anti-IgM antibody (10g/mL) in carbonate-bicarbonate buffer, pH 9.6, and incubated overnight at 4C in a humid chamber. Following incubation, the supernatant was discarded and plates were incubated with 200 L/well of blocking solution [BDS1, prepared as phosphate-buffered saline (PBS), pH 7.2, supplemented with 0.5% Tween-20 (PBS/T1) supplemented and 5% skimmed milk] for 30min in a humid chamber at room temperature (RT). After 5 times washing with PBS/T1, 50 L of pre-diluted serum samples and controls (1:200) in PBS/T1 were added together with 50L/well of 17DD-YF live-attenuated virus (2g/mL). The viral antigen concentration was previously determined during standardization steps using distinct protein concentrations measured by BCA protein assay kit (Pierce), according to the manufacturers instructions. The plate was incubated overnight at 4C in a humid chamber, following washing steps, and 50L/well of commercially available anti-Flavivirus antibody (6B6C-1) conjugated with Horseradish Peroxidase was added to each well for 1h at 37C. After washing steps, 75L/well of 3,3,5,5-tetramethylbenzidine substrate solution (TMB) was added to each well and plates were incubated for 10min before addition of 50 L/well of stop-solution (2M H2SO4). The endpoint measurements were performed at 450nm. The optical density (OD) values of each sample were subtracted from the negative control. The results were considered positive at OD>0.800, borderline at OD ranging from 0.800 to 0.667 and negative at OD<0.667.

The quantification of Anti-YF IgG was performed at the Laboratrio de Tecnologia Imunolgica from Bio-Manguinhos (LATIM, FIOCRUZ) using a standardized in-house enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates were coated with 50 L/well of 17DD-YF live-attenuated virus (2.5g/mL) in carbonate-bicarbonate buffer, pH 9.6. The viral antigen concentration was previously determined during standardization steps using distinct protein concentrations measured by BCA protein assay kit (Pierce), according to the manufacturers instructions. The microplates were incubated overnight at 4C in a humid chamber and were mechanically washed 5 times with 300 L/well of phosphate-buffered saline (PBS) pH 7.4, supplemented with 0.05% Tween-20 (PBS/T2). The plates were incubated with 100L/well of blocking solution [BDS2, prepared as PBS/T2 supplemented with 0.05% bovine serum albumin (BSA), 3% fetal bovine serum (FBS) and 5% skimmed milk], for 1h at 37C. Following, 50 L of two-fold serial dilution of serum samples (1:20 to 1:160) in BDS2 were incubated for 1h at RT. The standard curve was constructed using two-fold serial dilution (10.015 UI/mL) of commercially available anti-YF serum (YFNIBSC). After incubation, plates were washed and reincubated with 100 L/well of anti-Human IgG conjugated with Horseradish Peroxidase (HRP, BD Biosciences) diluted 1:3.000 in BDS2 for 1h at RT. Following washing procedures, 100L/well of TMB solution was added to each well and the plates were incubated for 15min before addition of 100L/well of stop-solution (2M H2SO4). The endpoint measurements were performed at 450nm. The absorbances of the serum sample dilutions were plotted and the standard curve was used to determine the antibody concentration according to the 4-parameter logistic regression, using the software SoftMax Pro. The results were expressed in IU/mL relative to the reference anti-YF serum standard curve.

The analysis of YF-specific neutralizing antibodies was performed at the Laboratrio de Tecnologia Virolgica at Bio-Manguinhos (LATEV, FIOCRUZ) using the micro plaque-reduction neutralizationHorseradish Peroxidase test (PRN-HRP), according to Simes M.67. Briefly, in 96-well plates, serial dilutions of serum samples (1:61:1458) were pre-incubated with 17D-213/77 vaccine virus (~100 PFU/well) for 2h at 37C, 5% CO2, and transferred to pre-formed Vero cells monolayer. Carboxymethylcellulose semisolid medium was overlaid on each well and plates were incubated for 48h at 37C, 5% CO2. After incubation, cell monolayers were washed and fixed with ethanol/methanol (1:1) solution and incubated with HRP-conjugated monoclonal antibody (clone 4G2) for 2h at 35C, 5% CO2, followed by the addition of True Blue Dye substrate. Thereafter, monolayers were washed, dried and photographed using the ScanLab microscope and images were used for the automated counts. The endpoint-neutralizing antibody titers were defined as the last serum dilution that reduced the number of plaques by 50% (PRN-HRP50) as compared to the virus control included in each assay. Seropositivity was defined considering the antibody titer 100 as the cut-off.

High-performance microbead array (Bio-Plex ProTM Human Assay) was used to quantify serum soluble mediators, comprising: chemokines (CCL11, CXCL8, CCL2, CCL3, CCL4, CCL5 and CXCL10), cytokines (IL-1, IL-6, TNF, IL-12, IFN-, IL-17, IL-1Ra, IL- 4, IL-5, IL-9, IL-10 and IL-13) and growth factors (FGF, PDGF, VEGF, G-CSF, IL-2 and IL-7). The assays were performed according to the manufacturers instructions. The results were expressed in pg/mL according to the standard curve of each soluble factor.

Immunophenotyping of T and B-cell subpopulations was performed in 96-well plates by incubating 5 105 live PBMC with two panels of monoclonal antibodies. The T-cell panel included anti-CD3-Qdot655 (Invitrogen, clone S4.1, dilution: 0.12:100, Cat #: Q10012), anti-CD4-BV605 (BD, clone RPA-T4, dilution: 1:100, Cat #: 562658), anti-CD8-Alexa Fluor 700 (eBioscience, clone RPA-T8, dilution: 5:100, Cat #: 56-0088-42), anti-CD45RO-BV421 (BD, clone UCHL1, dilution: 1:100, Cat #: 562641), anti-CD27-APC-H7 (BD, clone M-T271, dilution: 5:100, Cat #: 560222), anti-CXCR5-Alexa Fluor 488 (BD, clone RF8B2, dilution: 5:100, Cat #: 558112), anti-CXCR3-PE (BD, clone 1C6, dilution: 5:100, Cat #: 557185), anti-CCR6-PerCP-Cy5.5 (BD, clone 11A9, dilution: 5:100, Cat #: 560467), anti-ICOS-PE-Cy7 (Invitrogen, clone ISA-3, dilution: 5:100, Cat #: 25-9948-42) and anti-PD-1-APC (BioLegend, clone EH12.2H7, dilution: 5:100, Cat #: 329908). The B-cell panel comprised anti-CD19-PE-Cy7 (eBioscience, clone: SJ25C1, dilution: 0.20:100, Cat #: 25-0198-42), anti-CD20-BV650 (BD, clone 2H7, dilution: 2:100, Cat #: 563780), anti-CD21-FITC (eBioscience, clone HB5, dilution: 1.25:100, Cat #: 11-0219-42), anti-CD38- PE (BD, clone HIT2, dilution: 2.5:100, Cat #: 555460), anti-CD27-APC-H7 (BD, clone M-T271, dilution: 2.5:100, Cat #: 560222), and anti-IgD-Alexa Fluor 700 (BD, clone IA6-2, dilution: 2.5:100, Cat #: 561302). Cells were incubated for 20min at room temperature, washed and resuspended in PBS prior to acquisition on a LSR Fortessa Flow Cytometer (BD Biosciences). The FlowJo V10.8.1 (BD Bioscience) software was employed for data analysis using distinct gating strategies. Lymphocytes were selected based on the size and granularity properties within gated single cells. Thereafter, CD4+ and CD8+ T-cell subsets selected amongst CD3+ T cells were further characterized by additional phenotypic features. B-cell were identified as CD19+ cells within CD3- followed by further additional phenotypic analysis of cell subsets.

The GraphPad Prism V8.0 (GraphPad-Software) was used for statistical analyses and graphical arts. Data normality distribution was assessed by the Shapiro-Wilk test. MannWhitney test or Student t-test were used for comparison between two groups. KruskalWallis or ANOVA variance analysis followed by Dunns or Tukey post-test were employed for multiple comparisons. In all cases, significance was considered at p<0.05.

The baseline fold change indices were calculated as the ratio between the results observed for individual samples at distinct timepoints after primary 17DD-YF vaccination divided by the baseline values obtained for the same volunteer before vaccination. Chi-square test was used for the comparison of baseline fold changes categorized as decreased (<1), unaltered (=1) or increased (>1). Significance was considered at p<0.05. Venn Diagram analysis, available at (http://bioinformatics.psb.ugent.be/webtools/Venn/), was employed to assess the common and selective serum soluble mediators with increased (>1.5) or decreased (<0.5) baseline fold-change profiles at distinct timepoints.

Correlation analyses were carried out using Pearson and Spearman rank tests. The r scores of significant correlations (p<0.05) were employed to build correlation matrices and to assemble networks, using the corrplot package of the R software (Project for Statistical Computing Version 3.0.1) and the open-source Cytoscape software platform (available at https://cytoscape.org), respectively. Networks were compiled to arrange clusters of antibodies, chemokines, cytokines, growth factors, T and B-cell subsets. Nodes were used to represent each parameter and connecting lines were employed to identify positive (continuous line) and negative (dashed line) correlations. The node sizes are proportional to the number of correlations between parameters. Line thickness illustrates the correlation strength, ranging from weak/moderate (r scores from 0.1 to 0.5 or 0.5 to 0.1, thin lines) to strong correlations (r scores from 0.5 or0.5, thick lines).

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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Immune response induced by standard and fractional doses of 17DD yellow fever vaccine | npj Vaccines - Nature.com