The early-onset febrile reaction following vaccination and associated factors: An exploratory sub-study based on the Ebola vaccine clinical trial

ABSTRACT

A phase-1 clinical trial aimed to assess the safety and immunogenicity of the type-5 adenovirus vector based Ebola vaccine (Ad5-EBOV) was conducted in China. To provide more evidence for the safety evaluation and dose-selection, an exploratory sub-study using a wireless automatic temperature measuring platform was done based on the phase-1 clinical trial. The main aim of the sub-study was to obtain more information about the occurrence of fever and detect the potential associated factors, second was to assess the feasibility of the temperature measuring platform in vaccine clinical trials. Temperature data of 3 treatment groups all presented a rising tendency during the first 6 hours after vaccination, the incidence of elevated temperature and possible associated factors were analyzed. For the incidence of elevated temperature, no marked dose-response relationship was found in 6 hours with wireless thermometers; the information from mercury thermometers showed that the grade-1 fever proportion peaked at 6 hours and there was no difference between groups, while grade-2 fever proportion peaked at 24 hours and was significantly higher in high-dose group than those in the other 2 groups. Significant differences were found between sex groups (males vs. female, incidence rate ratios (IRR) = 2.93 and 7.62 for any-grade, grade-2 fever respectively, P<0.001); a decline in grade-2 fever incidence was found with the increasing age groups (IRR = 0.78, P = 0.003) and body mass index (BMI, IRR = 0.67, P<0.001) .Our findings show that the dose-dependent manner between fever and the dose of Ad5-EBOV in this study might emerge after 6 hours, and which is slight and transient. Wireless thermometers secured on the skin surface are not suitable for a long time (longer than 6 hours) measurement, new methods for temperature monitoring, like ear temperature measurement, should be tested in the further research.

KEYWORDS:

• Ebola vaccine
• Febrile reaction
• phase-1clinical trial
• Wireless automatic temperature measuring platform

Introduction

The 2014 Ebola epidemic in west Africa caused 23406 suspected, probable, and laboratory-confirmed cases of Ebola virus disease and 9457 deaths by Feb 20, 2015.¹ There was no licensed vaccine available to prevent Ebola virus so far.² Between the end of 2014 and early 2015, a phase I clinical trial designed to assess the safety and immunogenicity of a novel Ad5-EBOV (the first Ebola vaccine based on the 2014 Zaire Guinea epidemic strain) was implemented at one site in Taizhou County, Jiangsu Province, China.³ The primary safety end point was occurrence of solicited adverse reactions, including fever, within 7 d after vaccination, the primary immunogenicity end point was ELISA antigen-specific assays for antibody responses on day 0 and 28 and intracellular cytokine staining (ICS) assay for T cell responses on day 0 and 28.

Since it was the first Ebola vaccine clinical trial in China, the safety and tolerability of the vaccine were the dominating issues. A previous dose-finding clinical trial with Ebola vaccine showed that the fever counts was significant lower in low-dose group versus high-dose group,⁴ which suggested that the dose-dependent manner between the vaccine dosage and the fever counts may exist, so we did this exploratory sub-study which adopted both wireless automatic temperature measuring platform and traditional mercury thermometers to provide more evidence for the safety evaluation and dose-selection of the Ad5-EBOV. In this paper, we describe the occurrence of febrile reaction and figure out its associated factors. In addition, we also attempt to explore the application of the temperature measuring platform in vaccine clinical trials which monitoring the real-time body temperature of participants for 6 hours after injection, which has been rarely used in the vaccine evaluation process before.

Results

A total of 120 participants in the cohort (40 participants per group) were assessed in the Ad5-EBOV phase-1 clinical trial between Dec 28, 2014, and Jan 9, 2015.The sub-study based on the clinical trial was performed simultaneously. Baseline characteristics of the 120 participants were similar between groups (Table 1). We selected the maximum values of temperature data which measured by wireless thermometer from each participant in 6 hours following immunization, graded these values into corresponding fever levels and analyzed the proportion of fever in the participants. The febrile reactions were graded artificially according to the guidelines of CFDA.⁵ 92 of 120 participants (76.67%; 95%CI, 68.07%–83.90%) had a febrile reaction(any-grade fever, ≥ 37.1°C) within 6 hours after injection of the vaccine/placebo. The number of participants who had grade-1 fever (37.1–37.5°C) was 71(59.17%, 95%CI: 49.82%–68.05%), there was no significant statistical difference among treatment, sex, age and BMI groups. The number of participants who had grade-2 fever (37.6–39.0°C) was 21(17.50%, 95%CI: 11.17%–25.50%), there was a significant difference in occurrence of grade-2 fever among BMI groups (P = 0.007) and the result after Bonferroni correction indicated that the difference between 18.5–24.9 BMI group and 25–29.9 BMI group was significant (P = 0.012) (Table 2). No participant had grade-3 fever in this study.

Table 1. Baseline characteristics and fever proportion before vaccination.

	Placebo	Low-dose	High-dose
Variables	n = 40	n = 40	n = 40
Sex
Female	22(55.0)	20(50.0)	19(47.5)
Male	18(45.0)	20(50.0)	21(52.5)
Age
18-30	6(15.0)	4(10.0)	4(10.0)
31-40	11(27.5)	10(25.0)	10(25.0)
41-50	11(27.5)	16(40.0)	16(40.0)
51-60	12(30.0)	10(25.0)	10(25.0)
BMI
<18.5	0	1(2.5)	1(2.5)
18.5-24.9	26(65.0)	28(70.0)	24(60)
25-29.9	14(35.0)	10(25.0)	14(35)
≥30	0	1(2.5)	1(2.5)
Fever	0	0	0

Table 2. Incidence of fever in participants following vaccination (measured by wireless thermometers).

		Any fever (≥37.1℃)			Grade-1 fever (37.1-37.5℃)			Grade-2 fever (37.6-39.0℃)
Variables	N	n	%(95%CI)	P	n	%(95%CI)	P	n	%(95%CI)	P
Treatment-group				0.4127			0.7855			0.8410
Placebo	40	31	77.50(61.55-89.16)		24	60.00(43.33-75.14)		7	17.50(7.34-32.78)
Low-dose	40	28	70.00(53.47-83.44)		22	55.00(38.49-70.74)		6	15.00(5.71-29.84)
High-dose	40	33	82.50(67.22-92.66)		25	62.50(45.80-77.27)		8	20.00(9.05-35.65)
Sex				0.2323			0.4371			0.7456
Female	61	44	72.13(59.17-82.85)		34	55.74(42.45-68.45)		10	16.39(8.15-28.09)
Male	59	48	81.36(69.09-90.31)		37	62.71(49.15-74.96)		11	18.64(9.69-30.91)
Age				0.0505			0.7527			0.1839
18-30	14	10	71.43(41.90-91.61)		7	50.00(23.04-76.96)		3	21.43(4.66-50.80)
31-40	31	29	93.55(78.58-99.21)		20	64.52(45.37-80.77)		9	29.03(14.22-48.04)
41-50	43	30	69.77(53.87-82.82)		24	55.81(39.88-70.92)		6	13.95(5.30-27.93)
51-60	32	23	71.88(53.25-86.25)		20	62.50(43.69-78.90)		3	9.38(1.98-25.02)
BMI				0.1626			0.6633			0.0073
<18.5	2	1	50.00(1.26-98.74)		1	50.00(1.26-98.74)		0	0.00(0.00-84.19)
18.5-24.9	78	64	82.05(71.72-89.83)		44	56.41(44.70-67.61)		20	25.64(16.42-36.79)
25-29.9	38	25	65.79(48.65-80.37)		24	63.16(45.99-78.19)		1	2.63(0.07-13.81)
≥30	2	2	100.00(15.81-.)		2	100.00(15.81-.)		0	0.00(0.00-84.19)

^a P = 0.012 for 18.5-24.9 BMI group versus 25–29.9 BMI group.

The random effects Poisson regression model was then used to analyze the incidence of fever and its associated factors. The change of body temperature is relatively a slow and smooth process, we assumed that grade-1 fever was more like a ‘process’ rather than a ‘state’ for participants of which highest temperature had reached to grade-2 fever, the practical meaning of focusing exclusively on the grade-1 fever of these participants was not significant, so we analyzed the risk factors associated with any-grade fever and grade-2 fever below. It is particularly worth mentioning that during the process of temperature measurement with wireless thermometers, some participants showed varying degrees of erythema and itch on the skin pasted with adhesive tapes, it urged the investigators to suspend the measurement with wireless thermometers to evaluate if it would cause adverse impact on the safety of the subjects and the accuracy of the temperature data. During the pulse time, there were 7 participants enrolled in the clinical trial and the temperature data were completely missing. However, to follow the intention-to-treat principle, the 7 missing observations which merely having basic demographic data were also involved in the regression model, therefore, temperature data from 120 participants, accounting for 20307 records, were obtained effectively in the first 6 hours after vaccination with wireless thermometers. The output of the regression model indicated that, for the incidence rate of any-grade fever, the high-dose group was 0.67 times of the placebo group (P<0.001), while the low-dose group was 0.71 times that of the placebo group (P<0.001), with other variables being held constant. A single-degree-of-freedom chi-square test was used to compare the incidence rate of fever between high-dose group and low-dose group and the results showed no statistical significant difference (P = 0.607). For the incidence rate of grade-2 fever, there was no statistical significant difference between high-dose group, low-dose group and the placebo group (Table 3).

Table 3. Random effects Poisson regression model outputs for analysis of risk factors of fever.

	Any (≥37.1℃)		Grade-2 (37.6-39.0℃)
Variables	IRR (95%CI)	P	IRR (95%CI)	P
Group2 (low-dose VS placebo)	0.713(0.575-0.884)	0.002	0.996(0.615-1.612)	0.986
Group3 (high-dose VS placebo)	0.673(0.551-0.822)	<0.001a	1.442(0.966-2.152)	0.073b
Sex (male VS female)	2.932(2.427-3.541)	<0.001	7.624(4.686-12.404)	<0.001
Agegroup	1.071(0.984-1.166)	0.113	0.783(0.668-0.919)	0.003
BMI	1.034(0.999-1.070)	0.054	0.667(0.613-0.726)	<0.001
Base-temperature	2.177(1.635-2.900)	<0.001	2.842(1.581-5.107)	<0.001
Time	1.003(1.003-1.003)	<0.001	1.006(1.004-1.008)	<0.001

^a P = 0.607 for high-dose group versus low-dose group.

^b P = 0.089 for high-dose group versus low-dose group.

Male gender was a significant risk factor for fever compared with female gender (adjusted incidence rate ratios (IRRs) of 2.93 and 7.62 for any-grade fever or grade-2 fever respectively, P<0.001). The incidence rate of grade-2 fever decreased with the increasing of age, the expected decline of percentage for a 10-years increase in age was 22%. The percentage change in the incidence rate of any-grade fever was an increase of about 3%, while that of grade-2 fever was a decrease of about 23% for every unit increase in BMI. A positive correlation of fever rate with base temperature before immunization and time process after vaccination was also found (Table 3).

Mercury thermometer was used to measure the axillary temperature simultaneously at 30 minutes, 6 hours and fixed daily time points in 7 d after vaccination for each participant. the proportions of participants with fever were low and similar between groups in 30 minutes, but rose from 6 hours to 24 hours, and decreased slowly from day2 to day 7. Overall, 21 participants had fever, 20 of them were grade-1(n = 8 in the placebo group, n = 3 in the low-dose group, and n = 9 in the high-dose group) at the time point of 6 hours. Whereas, there were 8 participants had fever with 6 of them were grade-2(all the 6 participants were from high-dose group, P = 0.003) at 24 hours (Table 4).

Table 4. Temperature data from mercury thermometers in 7 days after vaccination.

		Any		Grade-1		Grade-2
time point	group	n(%)	P	n(%)	P	n(%)	P
30min	Placebo(n = 40)	1(2.50)	0.772	1(2.50)	0.772	0	NA
	Low-dose(n = 40)	2(5.00)		2(5.00)		0
	High-dose(n = 40)	0		0		0
6h	Placebo(n = 40)	8(20.00)	0.098	8(20.00)	0.161	0	1.000
	Low-dose(n = 40)	3(7.50)		3(7.50)		0
	High-dose(n = 40)	10(25.00)		9(22.50)		1(2.50)
day1(24h)	Placebo(n = 40)	1(2.50)	0.006	1(2.50)	1.000	0	0.003
	Low-dose(n = 40)	0		0		0
	High-dose(n = 40)	7(17.50)		1(2.50)		6(15.00)
day2	Placebo(n = 40)	1(2.50)	0.617	1(2.50)	1.000	0	0.328
	Low-dose(n = 40)	1(2.50)		1(2.50)		0
	High-dose(n = 40)	3(7.50)		1(2.50)		2(5.00)
day3	Placebo(n = 40)	2(5.00)	0.544	2(5.00)	0.544	0	NA
	Low-dose(n = 40)	2(5.00)		2(5.00)		0
	High-dose(n = 40)	0		0		0
day4	Placebo(n = 40)	1(2.50)	1.000	1(2.50)	1.000	0	NA
	Low-dose(n = 40)	1(2.50)		1(2.50)		0
	High-dose(n = 40)	0		0		0
day5	Placebo(n = 40)	1(2.50)	1.000	1(2.50)	1.000	0	NA
	Low-dose(n = 40)	1(2.50)		1(2.50)		0
	High-dose(n = 40)	1(2.50)		1(2.50)		0
day6	total	0	NA	0	NA	0	NA
day7	total	0	NA	0	NA	0	NA

Discussion

We carefully analyzed the correlation between the proportions of fever (measured by wireless thermometers) and different dose groups. The results were similar with our previous report.³ Temperature data of 3 treatment groups all presented a rising tendency in 6 hours after vaccination, but there were no significant differences in the proportion of participants with fever between groups. Random effects Poisson regression model indicated that the low-dose and high-dose groups showed lower incidence rate of any-grade fever than the placebo group; for incidence rate of grade-2 fever, there was no statistical significant difference between high-dose group, low-dose group and the placebo group. The information from mercury thermometers showed that the grade-1 fever proportion peaked at 6 hours and there was no difference between groups, while grade-2 fever proportion peaked at 24 hours and was significantly higher in high-dose group than those in the other 2 groups. After 24 hours, the fever proportion slowly fell down to 0 in 7 d. These results from wireless thermometers combined with those from mercury thermometers, suggested that the dose-dependent manner between fever and the dose of Ad5-EBOV in this study might emerge after 6 hours, and which was slight and transient.

The sex distribution of participants with fever was comparable, but there was a remarkable higher incidence rate in males comparing to females, 2.93-fold for the any-grade fever and 7.62-fold for the grade-2 fever. Similar sex differences in clinically significant adverse reactions were reported with rubella⁶ and yellow fever⁷ vaccines. There are several works describing the sex differences in responses to vaccines and to explain mechanisms of sex differences in vaccine responses^8,9: Cook pointed out that the sex difference is not related entirely to gonadal hormones but probably reflects an antigen specific interaction with the immune system via mechanism as yet to be defined⁸; Klein et al.⁹ summarized the sex differences of immune responses to viral vaccines and discussed the possible mechanisms, in their review, they concluded that the hormones, genes, humoral immunity and the activity of immune cells are crucial in influencing immune responses to vaccines.

The incidence rate of grade-2 fever reduced with the increasing of age and BMI, similar with the report from Broder et al.¹⁰ and Krams et al.¹¹ which reported that fever (> 37.8°C) is more common in younger children than in older children following influenza immunization.¹² The age and BMI differences of grade-2 fever, together with the sex difference, encouraged further in-depth research should be done to reveal the relationship between immune response (humoral and cell-mediated) and demographical features such as sex, age and BMI.

Fever is a frequent systemic adverse event following immunization¹³ and commonly defined as ‘a body temperature greater than…’, however, normal body temperature is not a single value but a range of values affected by both diurnal variation and the site of measurement.^13,14 According to the recommendation of the Brighton Collaboration Fever Working Group, fever is defined as the endogenous elevation of at least one measured body temperature to be 38.0°C or more, regardless of measurement device, anatomic site, age or environmental conditions.¹⁵ In this paper, we graded fever according to the scale issued by the CFDA⁵ for 3 reasons: first, there were very few participants (2, 1.77%) had a fever with temperature higher than 38.0°C in this study; second, the phase-1 clinical trial was conducted in China and we followed local regulations and guidelines; finally, we used the same scale in previous study and report,³ to maintain the consistency and comparability, the former standard was still adopted.

This sub-study also explored the application of wearable wireless medical devices in vaccine clinical trials. With the advent of sensor and mobile technologies, smart monitoring systems are widely used in medical activities¹⁶: Fensli et al.¹⁷ evaluated a novel wireless electrocardiogram (ECG) recorder with regard to its ability to perform arrhythmia diagnostics; Boano et al.¹⁸ described several applications of accurate remote temperature monitoring for medical research. However, there is almost no literature about such application in vaccine clinical trials, most vaccine studies focus only on general adverse events and immune responses after vaccination, and the body temperature after vaccination is usually measured via oral cavity, axilla or rectum with standardized mercury or digital thermometers in vaccine clinical trials. In this sub-study, we designed a wireless automatic temperature measuring platform to continuously measure the body temperature in real-time in a non-intrusive manner after vaccination. The real-time feedback presented on the screen helped researchers to grasp the dynamic information and give suitably early warning when real problems were to emerge. Furthermore, the continuous body temperature monitoring gives a direct and complete picture of the body temperature after vaccination, which helps the researchers to get the peep into the age-, sex-, and BMI-specific changes in the kinetics of the immune responses.

There were also some potential limitations in this study should be acknowledged. Firstly, as mentioned above, the sub-study was subjected to the ‘Ad5-EBOV phase-1 clinical trial’, the sample size was constrained by the main research. Secondly, we measured the post-immunization temperature with wireless thermometers for only 6 hours, while other studies usually measured for at least 72 hours.^19-26 We initially planned to record temperature for 72 hours after vaccination, but some participants showed varying degrees of erythema and itch on the skin pasted with adhesive tapes, which urged the investigators to suspend the measurement with wireless thermometers to evaluate if the measurement would cause adverse impact on the safety of the subjects and the accuracy of temperature data. After evaluation, the monitoring time interval was reduced to 6 hours. So, the conclusions of this study were all derived from the observed results in 6 hours following vaccination, we cannot make the extrapolation beyond 6 hours. However, even if the measure time was reduced, we still get the information of early-onset febrile reactions and find the differences among sex, age and BMI. Furthermore, due to subject intolerance of the adhesive tapes used to secure the monitors in the exploratory study, the authors come to a conclusion that wireless thermometers secured on the skin surface are not suitable for a long time (longer than 6 hours) measurement, and new methods for temperature monitoring should be tested in the further research. For example, a wireless thermometer measuring the tympanic temperature, which is noninvasive and don't need the adhesive tapes, may be tested in subsequent vaccine clinical trials.

Participants and methods

Ethics statement

The study was approved by the institutional review board of Jiangsu Provincial Center for Disease Control and Prevention. Each participant signed an informed consent. The research protocol was performed in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines.

Study design and participants

This was an exploratory sub-study based on the randomized, double blinded, placebo-controlled Ad5-EBOV phase-1 clinical trial. Eligible participants were healthy adults (aged 18–60 years) who had no HIV infection or hepatorenal dysfunction, as confirmed by physical examination and laboratory tests at the time of screening. Exclusion criteria were family history of seizure or mental disease, allergy to any component of the vaccine formulation, acute febrile disease on the day of enrolment, urticaria in last one year, receipt of any blood products in the past 4 months, receipt of any research drugs or vaccine in the past month, and non-compliance with the study schedule. The protocol outlines further details of the inclusion and exclusion criteria.^{3, 27}

After written informed consent were provided, a total of 120 participants were screened according to the protocol, sequentially enrolled and then randomly assigned into different treatment groups in a 2-step manner, with participants in group 1 receiving the low-dose vaccine (containing 4.0× 10¹⁰ viral particles per vial) or placebo (containing the vaccine excipients only, with no viral particles) and participants in group 2 receiving the high-dose vaccine (containing 1.6× 10¹¹ viral particles per vial) or placebo, then randomly assigned by computer-generated block randomization to receive the low-dose vaccine, the high-dose vaccine, or the placebo at the same ratio of 2:1.³

Before blood donating and vaccination, wireless thermometers were secured with adhesive tapes at the intersection of left mid-axillary line and 3rd intercostal space. The wireless automatic temperature measurement platform recorded the temperature data for at least 6 hours after vaccination. Meanwhile, mercury thermometers were used to screen the volunteers, measure the axillary temperature immediately before and 30 minutes, 6 hours after vaccination, in addition with daily measurement for the following 7 d.

Wireless automatic temperature measuring platform

The wireless automatic temperature measuring platform used in this study was developed by TOSHONG Technologies (Nanjing, China). This platform was composed of wireless thermometers, temperature measuring stations, server and temperature measuring platform software.

The wireless thermometer had an infrared sensor to measure the temperature of energy radiating in the armpit continuously, which transmitted the temperature data signal every 2 minutes. The measurement range was from 36.0°C to 42.0°C with an accuracy of 0.2°C. The wireless transmitting distance of this equipment was about 30 m, and power supply of the built-in battery could last 30 d or more.

The temperature measuring station was used to accept the temperature data signal transmitted by one or more wireless thermometers, and then transmitted the data to the server through the network. Two types of temperature measuring stations were used in this study: the stationary type (one-to-many pattern), which was placed at clinical trial site, could hold out data from a maximum of 1000 wireless thermometers; the portable type (one-to-one pattern) was carried by the participant with each station corresponding to an unique wireless thermometer.

The wireless automatic temperature measuring platform software was used to realize the function of processing, real-time displaying, managing and early warning of the temperature data.

The definition of fever

Fever (axillary temperature) was graded into 3 levels according to the scale issued by CFDA⁵: grade-1 (37.1–37.5°C), grade-2 (37.6–39.0°C) and grade-3 (greater than 39.0°C).

Statistical analysis

As this sub-study was conducted based on the Ad5-EBOV phase-1 clinical trial, the sample size was constrained by the main study scheme. Considering the minimum sample size for a phase-1 clinical trial is 20 participants per group according to requirements of the Chinese Food and Drug Administration (CFDA), the phase-1 clinical trial recruited 40 participants per group.

χ² test or Fisher exact test was adopted to analyze the differences in the proportion of participants with elevated temperature following vaccination among treatment groups, age groups, etc. Multiple comparisons based on Bonferroni adjustment were used if a statistical significant difference was found.

Random effects Poisson regression model was used to evaluate the incidence rate ratios (IRRs) of associated factors for elevated temperature. The temperature data from the same participant were likely to be correlated, but the data from different participants were assumed in general to be independent.²⁸ Therefore, random effects model was used for repeated measurement, the repeated count of elevated temperature was referred as response variable which followed Poisson distribution. Log link was used in the modeling process, generalized estimated equation (GEE) method was used for parameter estimation, the intra-participant correlation structure was assumed to be exchangeable.

Analysis was by intension-to-treat, the last observation carry-forward (LOCF) method was used to impute the missing temperature data. Statistical analyses were done with SAS (version 9.4).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Author contributions

Q.L, F.Z and J.L designed the trial and the study protocol, F.Z contributed to the critical review and revision of the report. Q.L, Y.H, L.H, F.M,H.Z, K.C, X.H, R.T, W.W, J.H, Z.L, S.Y led and participated in the site work, including the recruitment, follow-up. TOSHONG Technology Co., Ltd provided the wireless automatic temperature measuring platform. Q.D and Q.L contributed to the data collection, data management, statistical analysis and wrote the paper. H.H contributed to the English language revisions.

Funding

This study was funded by The National Science and Technology Major Project (2013ZX10004001), Beijing Institute of Biotechnology and Tianjin CanSino Biotechnology Inc.