Supplementary Table 1: Summary of Reactogenicity Events by Group and Time of Onset in Entebbe
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1 Supplementary Table 1: Summary of Reactogenicity Events by Group and Time of Onset in Group A (n=39) B (n=11) A (n=39) B (n=11) Onset Post-Vaccination Event 3 Minutes 3-14 days Pain 5 (13%) 1 (1%) 6 (15%) Headache 3 (8%) 8 (2%) 1 (1%) Malaise 3 (8%) 4 (1%) Nausea 2 (5%) 3 (8%) Induration 2 (5%) 2 (5%) Fatigue 1 (3%) 1 (1%) Fever 1 (1%) 3 (8%) Myalgia 2 (5%) Tenderness 2 (5%) Erythema 1 (3%) 1(1%) Total Events Reactogenicity Event Summary in Group A (First Vaccination) and Group B (Boost Vaccination) Overall, 27 of the 5 (54 %) vaccinated volunteers (23 from group A) reported 52 local and systemic reactogenicity events. Within 3 minutes post vaccination, 16 volunteers (32%) reported 18 events. Between 3 and 14 days post vaccination, 2 volunteers (4%) reported 34 events. All events were mild and all resolved within 8 days of onset. None of the reactogenicity events were unexpected based on the known safety profile of the YF-17D vaccine. None of the events interfered with daily activities. YF- 17D naïve volunteers (Group A) who were vaccinated during the study reported more reactogenicity events compared with those who were boosted (Group B).
2 Supplementary Table 2: Summary of Adverse Events by Group in Group A B AE n=39 n=11 Neutropenia (Grade 15 (38%) 3(3%) 1-3) URTI 5(13%) Conjunctivitis Tonsillitis 1(1%) Anaemia 2(5%) Increased ALT 1(3%) Chicken Pox 1(3%) Malaria 1(3%) Headache 1(3%) Thrombocytopenia 1(3%) Gastritis 1(3%) Total Events 28 4 Adverse Event (AE) Summary in Group A (First Vaccination) and Group B (Boost Vaccination) Overall, 31 of 5 volunteers reported at least one AE during the course of the study. Out of the 35 AEs reported, 13 were clinical events while 22 were laboratory events. All AEs due to laboratory abnormalities were not clinically significant and apart from anaemia were self-limiting. All AEs reported in this study resolved without sequelae.
3 Supplementary Table 3: Adverse events surveillance in study volunteers from Lausanne A total of 5 YF-17D immunized volunteers were reassessed at 4 visits: day 3, day 7, week 8 and ~ 1 year. Overall 43 (86%) were first-time vaccinees by contrast to 7 (14%) reimmunized subjects. In addition to YF-17D immunization, 19 (38%) individuals received one or more concomitant viral vaccines according to their trip destination needs and vaccination history. Frequency of other immunizations at day were as follow: Poliomyelitis / Poliorix or Revaxis = 12 Hepatitis A / Epaxal = 14 Hepatitis B / Engerix = 4 Combined Hepatitis A+B / Twinrix = 5 Mumps-Measles-Rubella / Priorix = 7 Varicella / Varilix = 1 African meningitis/ Mencevax ACWY= 5 Serious and none-mild adverse events were recorded in 3 volunteers (6%). Briefly, 2 presented mildmoderate adverse events and 1 volunteer was hospitalized for pneumonia treatment. All three subjects were first-time recipients of yellow fever vaccine. Adverse events reported in YF-vaccine recipients from Lausanne: 12 months follow-up. Volunteer Age Gender Adverse event Time of onset 5 29 M Flu-like symptoms Day 4 T :39 C 1 45 M Injection site Day 1 erythema and arm pain F Recurrent Week 16 guttate psoriasis F Community-acquired Day 4 pneumonia Concomitant vaccines none none Mencevax ACWY Malarone prophylaxis Poliorix Epaxal
4 Medical information concerning volunteers experiencing reported adverse events YF-5 No medical history of allergy or pre-existing condition. Following day 4 onset of headache, sore throat and abdominal pain he reported persistent fever (max.39 C) from day 7 onto day 11 regardless intake of antipyretics. After resolution of fever, convalescence due to fatigue was present until week 2. YF-1 Medical history of aspirin allergy and peptic ulcer disease. Injection site erythema and arm pain resolved by day 3 spontaneously. YF-38 History of chronic hepatitis B, on medical treatment for hypertension and diabetes type 2. According to the hospital records, community-acquired pneumonia was confirmed at day 4 and resolved at day 9 after 5 days of hospitalization.
5 Supplementary Table 4: Comparison of YF-17D neutralizing antibody titers within subgroups in Group Comparisons P value P value Male/Female.71 NS* Neutropenia Yes/No.69 NS Viral Load Yes/No.78 NS *NS= not significant Naïve vaccinees in did not show significant differences in neutralizing antibody titers when comparing gender, neutropenia status, or detectable viremia. Therefore, these parameters were not impacting the response to the YF-17D vaccine. Supplementary Table 5: Correlation between hematological data at baseline in and YF- 17D neutralizing antibody titers Basophils Eosinophils Hematocrit Hemoglobin Lymphocyte Spearman r P value P value NS* NS NS NS NS Monocytes Neutrophils Platelets RBC WBC Spearman r P value P value Significant NS NS NS NS *NS= not significant
6 Supplementary Table 6: TCR Vbeta usage of the A2/NS4B-specific CD8 T cells at two time points in Lausanne and.!non%persis+ng!clonotype! Persis+ng!clonotype! Lausanne Boosted SUBJECT VISIT TRBV4-1 TRBV5-5 TRBV28 TRBV3 TRBV14 TRBV19 TRBV5-1 TRBV18 TRBV3 TRBV6-5 TRBV6-6 TRBV12 TRBV5-6 TRBV1 TRBV2 TRBV9 TRBV11-2 TRBV13 TRBV2 TRBV25 TRBV27 TRBV4-3 TRBV6-2-3 TRBV29 1 W Y W Y W Y W Y W Y W Y W Y W Y V V V V V V V V V V V V V V V
7 Supplementary Figure 1: No association between YF-17D viral load and reactogenicity reported either 3 minutes or in the follow up 3-14 days after vaccination in the cohort. YF-17D Viral Load (copies/ml) No Reactogenicity Reported Reactogenicity
8 Supplementary Figure 2: Normalized neutralizing activity against DENV-2 of the sera of vaccinees from at baseline and sera from controls at dilutions of 1:1 (A) and 1:1 (B). A B DENV Controls DENV Controls
9 Supplementary Figure 3: Gating strategy for the NK cell populations analysis and monocytes by flow cytometry. 25K 2K 15K 1K 5K 49.7 FSC-H 25K 2K 15K 1K 5K 99.2 FSC-H 25K 2K 15K 1K 5K 86.1 Dump%& <AmCyan-A>: DUMP CD3%& <PE-Cy7 Green-A>: CD Nkp46+&CD56+& 31 <PE-Green-A>: NKP CD16%& CD16+& K 1K 15K 2K 25K FSC-A Ungated YF146 V2_Unstimulated.fcs Event Count: 7 5K 1K 15K 2K 25K FSC-A Scatter YF146 V2_Unstimulated.fcs Event Count: <AmCyan-A>: DUMP Singlets YF146 V2_Unstimulated.fcs Event Count: <Qdot 655-A>: CD3 Live cells YF146 V2_Unstimulated.fcs Event Count: <PE-Green-A>: NKP46 CD3 Neg YF146 V2_Unstimulated.fcs Event Count: <Pacific Blue-A>: CD16 V45 CD56+Nkp46+ YF146 V2_Unstimulated.fcs Event Count: K 25K 25K 25K 2K 2K 2K 2K 15K 1K 15K 1K K 1K K 1K.448 5K 5K 5K 5K <Alexa Fluor 7-A>: IFN G CD56+Nkp46+CD16+ YF146 V2_Unstimulated.fcs Event Count: <FITC-A>: KI67 CD56+Nkp46+CD16+ YF146 V2_Unstimulated.fcs Event Count: <PerCP-Cy5-5-A>: HLA DR CD56+Nkp46+CD16+ YF146 V2_Unstimulated.fcs Event Count: <APC-Cy7-A>: CD17A CD56+Nkp46+CD16+ YF146 V2_Unstimulated.fcs Event Count: K 25K 25K 25K 2K 2K 2K 2K 15K 1K K 1K K 1K K 1K K 5K 5K 5K <Alexa Fluor 7-A>: IFN G CD56+Nkp46+CD16- YF146 V2_Unstimulated.fcs Event Count: <FITC-A>: KI67 CD56+Nkp46+CD16- YF146 V2_Unstimulated.fcs Event Count: <PerCP-Cy5-5-A>: HLA DR CD56+Nkp46+CD16- YF146 V2_Unstimulated.fcs Event Count: <APC-Cy7-A>: CD17A CD56+Nkp46+CD16- YF146 V2_Unstimulated.fcs Event Count: K 25K 2K 2K 15K K.391 1K 1K 5K 5K <Alexa Fluor 7-A>: IFN G CD56+Nkp46+CD16- YF146 V2_Unstimulated.fcs Event Count: <Alexa Fluor 7-A>: IFN G CD56+Nkp46+CD16- YF146 V2_Stimulated.fcs Event Count: 162 Monocyte(subsets( Vivid( CD3CD19( CD14+(
10 Supplementary Figure 4: (A) Frequency of Ki67+ NK cells at day, 3 and 7 after vaccination in Lausanne and. (B) IFNg concentration in the supernatant of PBMCs from Lausanne and at day, 3 and 7 after vaccination incubated without any stimulation overnight. (C) Expression of PD-L1 and HLA-DR on monocytes CD14+ CD16- and CD14+ CD16+. A P=.4 Percentage of Ki67+ NK cells ns P=.4 D D3 D7 D D3 D7 Lausanne B P=.2 P=.7 P=.5 IFNg Concentration (pg/ml) D D D3 D3 D7 D7 Lausanne C %"PD%L1+" P"<".1" HLA%DR"MFI" P"<".1"
11 Supplementary Figure 5: Gating strategy for the T cell population analysis by flow cytometry. PD1$on$CD4$Naive$cells$ PD11$on$CD4$Memory$cells$ CD8+$CD38+$ HLA1DR+$Cells$ T$cell$memory$subsets$ Memory'CD4+ICOS+'T'cells' CD4+' CD45RA6' CD4+%Treg%cells% CD4+% CD45RA%
12 Supplementary Figure 6: Gating strategy for the B cell population analysis by flow cytometry. B"cell"subsets" Vivid." CD19+" CD21+CD27+" Plasma&Cells/Plasmablasts& Vivid& CD19+&
13 Supplementary Figure 7: No difference of neutralizing antibody titers in individuals receiving YF-17D alone or concomitant with other vaccines in the Lausanne cohort. YF-17D Neutralizing Antibodies (Log1) Single Vaccination NS Multiple Vaccination
14 Supplementary Figure 8: Negative associations between baseline immune activation and YF-17D vaccine response. (A) Negative correlation between the frequency of CD38+ CD21+ CD27- B cells at baseline and the YF-17D NAb titers in the Lausanne and cohorts. (B) Negative correlation between the frequency of tissue-like memory B cells at baseline and the YF-17D NAb titers in the Lausanne and cohorts. (C) Negative correlation between the expression level of PD-1 on memory CD8 T cells at baseline and the YF-17D NAb titers in the Lausanne and cohorts. A Percentage of CD38+ CD21+ CD27- B cells Lausanne (Log1) -4.5 Percentage of CD38+ CD21+ CD27- B cells ρ=.54 P=.1-4. (Log1) -4.5 B Percentage of Tissue-like Memory B cells Lausanne (Log1) -4.5 Percentage of Tissue-like Memory B cells (Log1) -4.5 Lausanne C Percentage of PD1+ Memory CD8+ T cells P= Percentage of PD1+ Memory CD8+ T cells P= (Log1) (Log1)
15 Supplementary Figure 9: Adaptive response to YF-17D vaccination in boosted vaccinees from. Boosting of the A2/NS4B-specific CD8 T cell responses was observed in the two HLA-A2*21 subjects receiving a second YF-17D vaccination (A). Neutralizing antibody boost against YF-17D was detected both in subjects with undetectable (B) and detectable (C) neutralizing antibody titers at baseline. A Frequency of A2-NS4B Tetramer+ CD8+ T cells (Log2) D D28 D84 B D D7 D1 D14 D28 D84 C D D7 D1 D14 D28 D84
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