Conceived and designed the experiments: AHR SAJ JTD PJB LGV. Performed the experiments: AHR DS AWV XJ KD MG. Analyzed the data: AHR DS. Contributed reagents/materials/analysis tools: SAJ XJ PJB KD MG. Wrote the paper: AHR DS LGV. Revised manuscript: JTD.
The authors have declared that no competing interests exist.
Yellow fever vaccination (YF-17D) can cause serious adverse events (SAEs). The mechanism of these SAEs is poorly understood. Older age has been identified as a risk factor. We tested the hypothesis that the humoral immune response to yellow fever vaccine develops more slowly in elderly than in younger subjects.
We vaccinated young volunteers (18–28 yrs, N = 30) and elderly travelers (60–81 yrs, N = 28) with YF-17D and measured their neutralizing antibody titers and plasma YF-17D RNA copy numbers before vaccination and 3, 5, 10, 14 and 28 days after vaccination.
Ten days after vaccination seroprotection was attained by 77% (23/30) of the young participants and by 50% (14/28) of the elderly participants (p = 0.03). Accordingly, the Geometric Mean Titer of younger participants was higher than the GMT of the elderly participants. At day 10 the difference was +2.9 IU/ml (95% CI 1.8–4.7,
We found that elderly subjects had a delayed antibody response and higher viraemia levels after yellow fever primovaccination. We postulate that with older age, a weaker immune response to yellow fever vaccine allows the attenuated virus to cause higher viraemia levels which may increase the risk of developing SAEs. This may be one piece in the puzzle of the pathophysiology of YEL-AVD.
Trialregitser.nl
The live attenuated 17D yellow fever vaccine is regarded as one of the safest and most effective vaccines
The biological mechanism for the association between adverse events and older age has not yet been elucidated
The protocol for this trial and supporting checklist are available as supporting information; see
The protocol and consent forms were approved by the Dutch Central Committee of Human Research (CCMO) and by the Medical Ethical Committee of the Leiden University Medical Center (LUMC) in the Netherlands. The trial was registered under NTR1040 and ISRCTN42180653, (
This study was conducted to determine whether the adaptive immune response to yellow fever vaccine is slower to develop in persons of 60 years or older compared with persons aged 18 to 40 years. Primary outcomes were the humoral response to yellow fever vaccination, measured by Plaque Reduction Neutralization Test (PRNT), and Yellow Fever 17D (YF-17D) viraemia after vaccination, which was quantified by real time PCR (qRT-PCR). Secondary outcomes were adverse events.
In this prospective controlled cohort study, participants were recruited at the Travel Clinic of the Leiden University Medical Center (LUMC), and Municipal Health Centers of Leiden and The Hague, the Netherlands. Healthy volunteers aged between 18 and 40 years and eligible for inclusion into the control group were invited to participate. Participants in the control group were not necessarily planning to travel to a yellow fever endemic area. The study group consisted of healthy travelers aged 60 years or above, who had an indication for yellow fever vaccination based on their travel destination (National Coordination Center for Travelers' Health, LCR)
The live, attenuated, 17D vaccine used in this study was manufactured on embryonated chicken eggs according to WHO regulations and stored according to manufacturer's guidelines. All administered vaccines originated from the same vaccine lot (Stamaril, Lot no B5355, Sanofi Pasteur, France). The vaccine was administered subcutaneously in the deltoid region of the right arm.
At the time of inclusion, data on demographic characteristics of the participants were obtained. Blood samples for the determination of neutralizing antibodies (NA) and YF-17D viraemia were collected before (day 0), and 3, 5, 10, 14 and 28 days after vaccination. Participants were asked to document any injection site and systemic adverse events after vaccination in a three-week diary. Solicited symptoms were: erythema, pain and swelling at the site of injection, fever and myalgia. Non-solicited symptoms could also be reported.
The tests were carried out in 6-well plates (Corning Inc., USA) using a slightly modified technique described originally by De Madrid and Porterfield
Viral RNA was isolated from 200 µl plasma using a MagNa Pure LC Total Nucleic Acid Isolation Kit (Roche Molecular Diagnostics, Penzberg, Germany). cDNA was synthesized with 10 µl elute (200 µl total) in a professional ThermoCycler (Biometra, Germany), and quantitative reverse transcription-PCR (qRT-PCR) of YFV RNA was performed in a BioRad i-cycler IQ™ real-time PCR detection system (BioRad, Veenendaal, The Netherlands). The following YFV specific primers and probe were used
YFV-1 (forward)
YFV-2 (reverse)
YFV-P (probe) FAM-
FAM (6-carboxyfluorescein) was used as 5′-reporter dye and BHQ (Black Hole Quencher) as 3′-quencher dye. In order to quantify YFV RNA, log10 dilutions of in vitro transcribed RNA standards were included as standard curves. RNA virus levels were calculated with standard curves from Cycle threshold (Ct) values to compare viraemia in both groups quantitatively, and were expressed as IU/ml.
Power calculations were based on an expected 80% virus neutralization of 95% in the control group and 66% in the elderly group at day 14, based on previous observations at the Travel Clinic (unpublished data). With an α of 0·05 and β of 0·2, 26 participants per group were needed to confirm a significant difference under these assumed conditions. To take into account a possible attrition rate of 15%, 30 participants were included per group. We analyzed the between group difference in GMT over the four time points (day 5, 10, 14, 28) using a mixed linear model. This model takes into account that each subject had repeated measurements of the antibody titer over time. More specifically, a unique identification number for each subject was entered as a random effect in the model and separate variables for all time points and for the groups (elderly versus young) were entered as fixed effects. Antibody titers below the detection threshold were assigned an arbitrary value of 0.05 IU/ml, which is twofold lower than the lowest detectable titer (i.e. 0.2 IU/ml). Where appropriate, Chi-square tests were used, and Wilcoxon's test for non-parametrical distributed numerical data. Statistical analysis was performed using a computer-assisted software package (SPSS version 16.0, SPSS Inc., Chicago, IL).
We enrolled 60 participants, none of whom withdrew prematurely. In 2 elderly participants, 17D-YF neutralizing antibodies were already present at day 0. In retrospect, these participants remembered that they were vaccinated against yellow fever many years ago. These two individuals were excluded from further analysis. In both groups 70% were female and 30% had visited flavivirus endemic countries in the past. The median age of the younger participants was 21 years (interquartile range 20–22.5) and of the elderly was 66 years (interquartile range 65–69). Although we invited persons of 18 to 40 years of age for the control group, the oldest participant in this group was 28 years old. Therefore the control group is defined as age 18–30 years. We recorded the incidence of previous travel to countries that are endemic for flaviviruses because past infections with other flaviviruses can cause cross-neutralization in the YF PRNT.
At day 3 and 5 after vaccination, no neutralizing antibodies were found in any of the participants. Ten days after vaccination seroprotection was attained by 77% (23/30) of the young participants and by 50% (14/28) of the elderly participants (p = 0.03, Chi-square test) (
Reverse cumulative distribution curves of yellow fever neutralizing antibody titers at 5, 10, 14 and 28 days after vaccination in 30 young and 28 elderly participants. Antibody titers were determined with Plaque Reduction Neutralization Tests and reflect the serum dilution at which 80% of virus was neutralized.
YF-17D viraemia was measured by qRT-PCR at day 0, 3, 5, 10 and 14 (
YF-17D viraemia | Young N = 30 | Elderly N = 28 | p-value | |
Day 0 | Number positive (%) | 0 (0) | 0 (0) | - |
Day 3 | Number positive (%) | 6 (20) | 11 (39) | 0.1 |
IU/ml (95% CI) | 1.4 (0.9–1.9) | 2.9 (2.1–4.4) | 0.04 | |
Day 5 | Number positive (%) | 16 (53) | 23 (82) | 0.02 |
IU/ml (95% CI) | 4.8 (0–10.7) | 20.8 (10.2–31.5) | 0.07 | |
Day 10 | Number positive (%) | 0 (0) | 2 (7) | 0.2 |
IU/ml (95% CI) | - | 1.00 (0.8–1.2) | - | |
Day 14 | Number positive | 0 (0) | 0 (0) | - |
1 time point positive (%) | 14 (78) | 12 (50) | 0.02 | |
2 sequential time points positive (%) | 4 (22) | 12 (50) |
YF-17D RNA virus levels were calculated with standard curves from Cycle threshold (Ct) values and were expressed as IU/ml. Comparison of number of participants positive for viraemia was calculated by Fisher's Exact test. Comparison of quantitative viraemia (only of participants who had measurable viraemia) was calculated with Student's t-test. IU = International Units, 95% CI = 95% Confidence Interval.
Participants reported the occurrence and duration of adverse events after yellow fever vaccination in a 3-week diary (
Adverse event (AE) | Young N = 30 | Elderly N = 28 | p-value | ||
Injection site AE | Any | Yes (%) | 9 (30) | 4 (14) | 0.15 |
Days to onset (range) | 0 (0-2) | 0.5 (0-6) | 0.6 | ||
Erythema | Yes (%) | 8 (27) | 2 (7) | 0.05 | |
Days duration (range) | 2.5 (1-8) | 2 (1-3) | 0.4 | ||
Swelling | Yes (%) | 3 (10) | 1 (4) | 0.3 | |
Days duration (range) | 2 (1-5) | 2 (-) | 1.0 | ||
Pain | Yes (%) | 3 (10) | 2 (7) | 0.7 | |
Days duration (range) | 1 (1-3) | 2 (2-2) | 0.5 | ||
Systemic AE | Any | Yes (%) | 12 (40) | 8 (29) | 0.4 |
Days to onset (range) | 0.5 (0-4) | 5 (1-6) | 0.002 | ||
Myalgia | Yes (%) | 12 (40) | 6 (21) | 0.4 | |
Days to onset (range) | 1 (0-6) | 5 (1-6) | 0.12 | ||
Fever | Yes (%) | 3 (10) | 4 (14) | 0.6 | |
Days to onset (range) | 0 (0-4) | 5 (5-6) | 0.03 |
Safety of vaccination expressed in various parameters. Numbers of days are medians. Fever was defined as self-measured temperature above 38 degrees Celsius. P-values based on Chi-square test and Wilcoxon's test. AE = Adverse event.
The main finding of this study was that after primary vaccination with 17D YF vaccine, elderly persons (≥60 years) were slower to develop an antibody response and had higher viraemia levels than younger persons. Only half of the elderly vaccinees had protective antibody levels 10 days after vaccination compared with over three quarters of younger vaccinees. In addition, GMT of neutralizing antibodies were significantly lower at 10 and 14 days after vaccination. The difference was less pronounced and no longer statistically significant 28 days after vaccination. Besides showing higher levels of viraemia in elderly subjects, our data also suggest that the duration of viraemia is prolonged in these subjects as two elderly participants and none of the younger participants had detectable viraemia at day 10.
These results provide insight into the etiology of the increased susceptibility to YEL-AVD after yellow fever vaccination in old age. Immunosenescence leading to an impaired ability to clear the vaccine virus has been put forth as a possible explanation for the increased risk of YEL-AVD in elderly people
Beside immunosenescence in elderly subjects, other factors contributing to YEL-AVD have been postulated. For example, it has previously been suggested that the vaccine virus reverts or mutates to a more virulent form during replication in a vaccinated individual, but extensive genetic analyses of the viral strains extracted from patients with YEL-AVD do not provide evidence to support this hypothesis
Although occurrence of YEL-AVD is very rare, fear of this adverse event could reduce utilization of yellow fever vaccine. An “International Laboratory Network for Yellow Fever Vaccine-Associated Adverse Events” has been established in 2008, to complement the USA and the European Yellow Fever Vaccine Safety Working Groups
The findings of our study can have the following practical implication: in travelers of 60 years and older, it would be prudent to vaccinate against yellow fever at least 14 days instead of 10 days before departure to guarantee that all vaccinees have obtained protective antibody levels.
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