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Long-term immunogenicity and safety of inactivated Hantaan virus vaccine (HantavaxTM ) in healthy adults Joon Young Song a,1 , Heung Jeong Woo b,1 , Hee Jin Cheong a , Ji Yun Noh a , Luck Ju Baek c , Woo Joo Kim a,∗ a
Division of Infectious Diseases, Department of Internal Medicine, Korea University College of Medicine, Seoul, Republic of Korea Division of Infectious Diseases, Department of Internal Medicine, Hallym University College of Medicine, Seoul, Republic of Korea c Department of Microbiology, Institute for Viral Diseases, Korea University College of Medicine, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 12 November 2015 Received in revised form 29 December 2015 Accepted 15 January 2016 Available online xxx NCT02360514 Keywords: Hantaan virus vaccines Hantavirus Immunogenicity Safety
a b s t r a c t Background: Hemorrhagic fever with renal syndrome is a serious health problem in Eurasian countries, including Korea and China. This study evaluated the long-term immunogenicity and safety of formalininactivated Hantaan virus vaccine (HantavaxTM ). Methods: A phase III, multi-center clinical trial was undertaken to evaluate the immunogenicity and safety of HantavaxTM (three-dose schedule at 0, 1, and 13 months) among healthy adults. Immune response was assessed using the plaque reduction neutralizing antibody test (PRNT) and immunofluorescent antibody assay (IFA). Antibody levels were measured pre-vaccination and at 2, 13, 14, 25, 37, and 49 months after the initial vaccination. Systemic and local adverse events were assessed. Results: A total of 226 healthy subjects aged 19–75 years were enrolled. Following two primary doses of HantavaxTM , the seroconversion rate was 90.14% by IFA, but it was only 23.24% by PRNT50 . With booster administration, seropositive rates were 87.32% and 45.07% at one month post-vaccination according to IFA and PRNT50 , respectively. In young adults (19–39 years), the seropositive rate according to PRNT50 reached about 60% after booster vaccination. The mean duration of seropositive response was 735 days for PRNT50 and 845 days for IFA. Solicited local and systemic adverse events occurred in 47.79% and 25.22% of study subjects, respectively, and most were grade 1. Conclusion: HantavaxTM showed a booster effect and immunogenicity lasting two years with a three-dose schedule. The neutralizing antibody response was quite poor with two primary doses, so an early booster vaccination at 2–6 months might be warranted to provide timely protection to high-risk subjects. © 2016 Published by Elsevier Ltd.
1. Introduction Hemorrhagic fever with renal syndrome (HFRS) is a rodentborne zoonosis widely distributed over Eurasia [1]. Among more than 20 strains of Hantaviruses, the major causative agents are the Hantaan, Seoul, and Puumala viruses [1–3]. In the Republic of Korea (ROK), both the Hantaan and Seoul viruses are known as etiologic agents of HFRS [4,5]. These viruses establish chronic infections in certain species of rodents and are transmitted to humans primarily via aerosols or fomites from the feces, urine, and saliva of infected mice. Thus, the incidence of HFRS is particularly high among military persons and farmers during the harvest season. The overall prevalence of HFRS has been reported at 0.81
∗ Corresponding author. Tel.: +82 2 2626 3051; fax: +82 2 2626 1105. E-mail address: [email protected] (W.J. Kim). 1 JYS and HJW contributed equally to this study.
per 100,000 population, while that for military personnel was 40–64 per 100,000 in the ROK, with seasonal variation [6,7]. Since 1990, a commercialized vaccine (HantavaxTM ) has been marketed to prevent HFRS caused by the Hantaan and Seoul viruses [8,9]. Based on the immunogenicity data, HantavaxTM is recommended to be administered as a series of two primary doses administered one month apart and a booster administered 12 months later [9,10]. Although the incidence and severity of HFRS appeared to decrease in the ROK after the introduction of HantavaxTM , the efficacy of the vaccine has been questioned [11]. In addition to vaccination of high-risk groups, other factors should be considered to contribute to the secular reduction of disease burden, including improvements in the standard of living and housing in rural areas, ecological changes in rodent populations, or other natural factors [7]. In a former field trial, no cases of HFRS were identified in 1900 recipients of one-dose HantavaxTM , while 20 cases were confirmed among 2000 non-vaccinated controls (p < 0.01) [12]. However, the
http://dx.doi.org/10.1016/j.vaccine.2016.01.031 0264-410X/© 2016 Published by Elsevier Ltd.
Please cite this article in press as: Song JY, et al. Long-term immunogenicity and safety of inactivated Hantaan virus vaccine (HantavaxTM ) in healthy adults. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.01.031
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vaccine recipients and controls were not followed prospectively in that field trial; the medical records of patients with HFRS were used for comparative analysis. In addition, a case-control study conducted in the Korean army did not show statistically significant effectiveness even after the three-dose vaccination [13]. Interestingly, although statistically insignificant, vaccine effectiveness was dose-related, increasing from 25% for one dose, to 46% for two doses, to 75% for three doses. To ensure vaccine effectiveness, the long-term immunogenicity of the current vaccination series of HantavaxTM needs to be established.
2. Materials and methods 2.1. Study design A phase III, multi-center, open-labelled clinical trial was undertaken to evaluate the immunogenicity, immunogenic persistence, and safety of an inactivated hantavirus vaccine (HantavaxTM , Green Cross Corporation, Yongin, ROK) among healthy adults aged 19–75 years at two centers in the ROK (ClinicalTrials.gov Identifier: NCT02360514). Subjects were excluded for the following reasons: previous hantavirus infection, previous hantavirus vaccination, positive neutralizing antibody response at screening, allergy to vaccine components, history of seizure within the past year, pregnant or breastfeeding, febrile disorder, marked nutritional deficiency, uncontrolled medical condition (cardiovascular, renal, or hepatic disease), immunodeficiency or receipt of immunosuppressive therapy, administration of blood products or immunoglobulins within the past three months, or other previous vaccinations within two weeks prior to the study onset. After a baseline blood sample was collected, the hantavirus vaccine was administered to each eligible subject three times with respective one month and 12 month intervals (0-1-13 schedule), including two primary doses and one booster dose. To evaluate the immune responses, blood draws were performed at two months (one month after administration of the second primary dose), 13 months (one year after the two primary doses and before the booster dose), 14 months (one month after the booster dose), 25 months (one year after the booster dose), 37 months (two years after the booster dose), and 49 months (three years after the booster dose) from the initial vaccination. Each subject was followed-up until negative conversion of the neutralizing antibody was observed, up to three years after the three serial vaccinations. Subjects were required to report any solicited adverse events for seven days after each vaccination. At each visit, a brief survey was obtained from study subjects to track baseline demographics, occurrence of illnesses, and medication usage. The primary immunogenicity objective was to evaluate the seroconversion rate and sero-positive persistence of HantavaxTM among healthy adults after administration of two primary doses and a booster dose according to the plaque reduction neutralizing antibody test (PRNT). Secondary immunogenicity objective was to evaluate the seroconversion rate and sero-positive persistence of HantavaxTM using the immunofluorescent antibody assay (IFA). Antibody levels were serially assessed using both immunoassays. Safety objectives included the evaluation of solicited local and systemic adverse events (AEs), spontaneously reported unsolicited AEs, and serious adverse events (SAEs). This study was conducted in accordance with the Declaration of Helsinki and the standards of good clinical practice defined by the International Conference on Harmonization. The protocol and consent forms were approved by the institutional review board of each participating study site. Written informed consent was obtained from all participants following a detailed explanation of the schedules and study contents.
2.2. Vaccines The study vaccine (HantavaxTM , Green Cross Corporation) was developed from suckling mouse brains infected with the ROK 84/105 strain and inactivated with 0.05% formalin. HantavaxTM is alum-adjuvanted and formulated in 0.5-mL pre-filled syringes containing 0.125 mL of virus. The vaccine was administered by intramuscular or subcutaneous routes as a series of two vaccinations spaced one month apart, with a booster administered 12 months later. 2.3. Immunogenicity assessment PRNT and IFA were performed at the Microbiology Laboratory of Korea University College of Medicine, as described previously [10]. Heat-inactivated (56 ◦ C, 30 min) serum samples were diluted in Eagle minimal essential medium with Earle’s salts (EMEM) and then combined with an equal volume (111 mL) of EMEM containing 75 PFU of virus and 10% guinea pig complement (catalog no. ACL4051; Accurate Chemical and Scientific Corp.). This mixture was incubated overnight at 4 ◦ C, and then a plaque assay was performed exactly as described previously, using 7day-old Vero E6 monolayers in six-well plates. The mixture was stained with neutral red (Gibco-BRL) after 1 week, and plaques were counted 2 days (37 ◦ C) after staining. The PRNT50 antibody titer was estimated as the highest serum dilution that reduced the standard viral dose (Hantaan virus strain 76–118) by 50%. Neutralizing titers of 1:10 or greater were regarded as positive. IFA was performed with Hantaan virus (strain 76–118)-infected Vero E6 cells. Cells were spread onto slides, air-dried, and fixed in acetone for 10 min at 4 ◦ C. Serum samples were serially diluted in 2-fold steps in phosphate-buffered saline, starting with the initial dilution of 1:2, then added to the cells. After incubation for 30 min at 37 ◦ C, slides were washed in phosphate-buffered saline and incubated with fluoroscein isothiocyanate-conjugated anti-guinea pig IgG (Sigma, St. Louis, MO, USA). The slides were then washed with phosphate buffered saline, and mounted with buffered alkaline glycerol. The results were read under the fluorescence microscope. IFA titers of 1:32 or greater were regarded as positive. 2.4. Safety assessment At the first visit, each enrolled subject was given a digital thermometer and a diary card containing a list of solicited AEs and their grades. For seven days after vaccination, subjects were requested to record the severity of solicited AEs and unsolicited AEs, axillary temperatures, and concomitant medications on the diary card. Subjects used a standard scale to grade adverse events. Solicited local AEs were pain, redness, and swelling, and solicited systemic AEs were fever, chill, headache, malaise, arthralgia, dizziness, and nausea. We collected reports of unsolicited AEs during the study. Subjects were required to report any SAEs within 24 h for up to six months after vaccination. 2.5. Statistical analysis Demographic data were analyzed for the subjects in the intention-to-treat (ITT) set. Immunogenicity analyses were performed in both per-protocol (PP) and ITT sets, while safety was analyzed for all subjects exposed to the study vaccines. The ITT analysis set comprised all participants who received at least one dose of vaccine, and the PP set included all enrolled subjects who received the correctly administered vaccine, provided evaluable serum samples at relevant time points, and had no major protocol deviations.
Please cite this article in press as: Song JY, et al. Long-term immunogenicity and safety of inactivated Hantaan virus vaccine (HantavaxTM ) in healthy adults. Vaccine (2016), http://dx.doi.org/10.1016/j.vaccine.2016.01.031
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Table 1 Demographic characteristics (intention-to-treat population). Parameters Age (years) Age group No. (%)
Sex, no. (%) BMI (kg/m2 )
Subjects (no. = 218) Mean ± SD Median 19–29 years 30–39 years 40–49 years 50–59 years 60–72 years Male Mean ± SD Median
43.26 ± 14.14 42.00 51 (23.39) 49 (22.48) 38 (17.43) 46 (21.10) 34 (15.60) 81 (37.16) 22.86 ± 2.96 22.60
SD, Standard deviation.
Among the 218 subjects in the ITT set, 142 were eligible for PP analysis. 3.2. Immunogenicity
Fig. 1. Flowchart of subjects through the study (PRNT, plaque reduction neutralization test; IFA, immunofluorescent antibody assay).
Sample sizes of 116 and 80 were estimated to provide sufficient power (80%) to examine the primary (≥80% seroconversion at one month after the booster dose) and secondary (persistent seroconversion up to two years after the booster dose in ≥50% of sero-responsive subjects) study objectives, respectively. Anticipating a 30% drop-out rate at the primary end point (one month after the booster dose) and an annual 25% drop-out rate up to the secondary end point (two years from the booster dose), we planned to enroll 226 subjects. All of the analyses were performed using the statistical software system SAS 9.3 (SAS Institute, Cary, NC, USA). The immunogenicity data were expressed in terms of seroconversion rate and geometric mean titer (GMT) with two-sided 95% confidence intervals (CIs). The two-sided 95% CIs for the GMTs were calculated using the normal approximation of log-transformed titers, and percentages were calculated with approximate or exact 95% CIs. The duration of the immune response (seroconversion) was estimated using the Kaplan–Meier method. Safety data were described as the proportion of study subjects reporting local and systemic adverse reactions. Student’s t-test (or the Wilcoxon rank-sum test) was used to compare continuous variables between the two groups, while the Chi-square test or Fisher’s exact test was conducted to analyze the categorical variables. Results were considered statistically significant if the associated p-value was less than 0.05. 3. Results 3.1. Study subjects Among 256 volunteers, a total of 226 healthy subjects aged 19–75 years were enrolled (Fig. 1). Thirty subjects were excluded because of violation of eligibility criteria (six subjects) or withdrawal of consent (24 subjects). Eight subjects were excluded from the ITT set for the analysis of baseline characteristics because a blood draw was not performed for immunogenicity analysis. The mean age of the participants was 43.3 ± 14.1 years (median age, 42 years), and a higher proportion of females (62.8%, or 137 subjects) was noted. The age distribution included 51 adults aged 19–29 years, 49 adults aged 30–39 years, 38 adults aged 40–49 years, 46 adults aged 50–59 years, and 34 adults aged ≥60 years (Table 1).
Table 2 shows the HantavaxTM -induced antibody responses among the per-protocol population, as assessed using PRNT50 and IFA at each time point after the two primary doses and a booster dose. At one month after the two primary doses, the proportion of subjects with positive responses (seroconversion) was 90.14% according to IFA, but it was only 23.24% according to PRNT50 . The GMTs and seropositive rates returned to pre-vaccination levels over one year, irrespective of the assay method (Table 2 and Fig. 2). With the booster administration of HantavaxTM , the seropositive rates increased to 45.07% and 87.32% at one month post-vaccination according to PRNT50 and IFA, respectively. The seropositive rate of PRNT50 was maintained above 40% at one year after the booster dose but decreased markedly after that. The decline in seropositive rate was rather slower after the administration of the booster dose than after the two primary doses (Fig. 2). When estimated using the Kaplan–Meier method, the mean duration of seropositive response (time from initial positive response to negative conversion) was 735 days (95% CI, 663–721; median, 712 days) and 845 days (95% CI, 713–721; median, 721 days) according to PRNT50 and IFA, respectively. Regarding PRNT50 response, there was no significant difference in age (43.1 ± 13.9 vs. 42.3 ± 13.2, p = 0.87), male sex (12.5% vs. 33.9%, p = 0.42) and body mass index (21.7 ± 3.3 vs. 23.3 ± 2.9, p = 0.13) between seropositive (N = 8) and seronegative (N = 56) subjects at three years after booster dose. However, postbooster GMT (at one month after booster dose) was significantly higher in seropositive subjects (33.6, 95% CI 18.5–61.3) compared to seronegative subjects (17.0, 95% CI 14.2–20.5) (p = 0.01). Age-stratified analysis showed that the seropositive rate of PRNT50 was highest in subjects aged 30–39 years (65.52%), followed by those aged 19–29 years (53.85%) at one month after the booster dose (Table 3). In the case of IFA, the seropositive rate at one month after the booster dose was around 90% in all age groups except for those aged ≥60 years (70.37%). Immune responses among the ITT set were consistent with the results of the PP set (Supplementary Tables 1 and 2). 3.3. Safety Overall, 154 (68.14%) of 226 subjects reported 929 AEs, most of which (84.0%, 780 of 929 events) were grade 1 (noticeable but did not interfere with daily activity). Solicited local and systemic AEs reported within seven days of vaccination are shown in Table 4. Solicited local and systemic AEs occurred in 108 (47.79%) and 57 (25.22%) of 226 subjects, respectively. Injection site pain (104 subjects, 46.02%) and myalgia (41 subjects, 18.14%) were the most frequently reported AEs. All subjects with unsolicited
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Table 2 Immune responses after administration of HantavaxTM among the per-protocol population.
Before vaccination At one month after two primary doses At one year after two primary doses At one month after the booster dose At one year after the booster dose At two years after the booster dose At three years after the booster dose
Plaque reduction neutralization test (PRNT50 a )
Immunofluorescent antibody assay
No. of tested cases
Seropositive rate, (%) (95% CI)
GMTs (95% CI)
No. of tested cases
Seropositive rate, (%) (95% CI)
GMTs (95% CI)
142b 142b 142c 142d 64e 64f 64g
– 23.24 (16.29–30.19) 1.41 (0.00–3.35) 45.07 (36.89–53.25) 40.63 (28.59–52.66) 15.63 (6.73–24.52) 12.50 (4.40–20.60)
5.00 (5.00–5.00) 6.35 (5.86–6.89) 5.07 (4.97–5.18) 9.03 (7.89–10.33) 6.00 (5.64–6.37) 5.85 (5.31–6.44) 6.60 (5.59–7.78)
142a 142a 142b 142c 124d 124e 124f
– 90.14 (85.24–95.04) 10.56 (5.51–15.62) 87.32 (81.85–92.80) 34.68 (26.30–43.05) 17.74 (11.02–24.47) 10.48 (5.09–15.88)
16.08 (15.92–16.23) 57.76 (49.63–67.24) 17.73 (16.76–18.76) 87.90 (71.85–107.53) 23.91 (21.10–27.09) 27.00 (21.85–33.36) 34.78 (22.76–53.13)
GMTs, geometric mean titers. a The PRNT50 titer represents the reciprocal serum dilution that reduced virus plaque number by 50%. b Number of cases who took the antibody test before and one month after the two primary doses. c Number of cases who took the antibody test at one year after the two primary doses. d Number of cases who took the antibody test at one month after the booster dose. e Number of cases who took the antibody test at one month after the booster dose–number of drop-out cases up to one year after the booster dose. f Number of cases who took the antibody test at one month after the booster dose–number of drop-out cases up to two years after the booster dose. g Number of cases who took the antibody test at one month after the booster dose–number of drop-out cases up to three years after the booster dose. Table 3 Age-stratified immune responses (seropositive rates) after administration of HantavaxTM among the per-protocol population.
19–29 years
30–39 years
40–49 years
50–59 years
≥60 years
T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6
Plaque reduction neutralization test (PRNT50 a )
Immunofluorescent antibody assay
No.
Seropositive rate, (%) (95% confidence interval)
No.
Seropositive rate, (%) (95% confidence interval)
26 26 26 14 14 14 29 29 29 19 19 19 27 27 27 10 10 10 33 33 33 14 14 14 27 27 27 7 7 7
42.31 (23.32–61.30) 3.85 (0.00–11.24) 53.85 (34.68–73.01) 35.71 (10.61–60.81) 7.14 (0.00–20.63) 7.14 (0.00–20.63) 27.59 (11.32–43.85) 0.00 (0.00–0.00) 65.52 (48.22–82.82) 47.37 (24.92–69.82) 21.05 (2.72–39.38) 21.05 (2.72–39.38) 3.70 (0.00–10.83) 0.00 (0.00–0.00) 37.04 (18.82–55.25) 60.00 (29.64–90.36) 30.00 (1.60–58.40) 10.00 (0.00–28.59) 24.24 (9.62–38.86) 0.00 (0.00–0.00) 42.42 (25.56–59.29) 21.43 (0.00–42.92) 14.29 (0.00–32.62) 7.14 (0.00–20.63) 18.52 (3.87–33.17) 3.70 (0.00–10.83) 25.93 (9.40–42.46) 42.86 (6.20–79.52) 0.00 (0.00–0.00) 14.29 (0.00–40.21)
26 26 26 24 24 24 29 29 29 26 26 26 27 27 27 26 26 26 33 33 33 29 29 29 27 27 27 19 19 19
96.15 (88.76–100.00) 15.38 (1.52–29.25) 92.31 (82.06–100.00) 33.33 (14.47–52.19) 8.33 (0.00–19.39) 8.33 (0.00–19.39) 93.10 (83.88–100.00) 6.90 (0.00–16.12) 89.66 (78.57–100.00) 38.46 (19.76–57.16) 50.00 (30.78–69.22) 26.92 (9.87–43.97) 81.48 (66.83–96.13) 7.41 (0.00–17.29) 96.30 (89.17–100.00) 38.46 (19.76–57.16) 11.54 (0.00–23.82) 3.85 (0.00–11.24) 90.91 (81.10–100.00) 12.12 (0.99–23.26) 87.88 (76.74–99.01) 27.59 (11.32–43.85) 3.45 (0.00–10.09) 3.45 (0.00–10.09) 88.89 (77.03–100.00) 11.11 (0.00–22.97) 70.37 (53.15–87.59) 36.84 (15.15–58.53) 15.79 (0.00–32.19) 10.53 (0.00–24.33)
T1: one month after the two primary doses, T2: one year after the two primary doses, T3: one month after the booster dose, T4: one year after the booster dose, T5: two years after the booster dose, T6: three years after the booster dose. a The PRNT50 titer represents the reciprocal serum dilution that reduced virus plaque number by 50%.
AEs recovered without sequelae, and no vaccine-related SAE was reported up to six months post-vaccination. 4. Discussion This study showed that HantavaxTM was not usually immunogenic after the administration of two primary dose s, so a booster dose would be required. Despite vaccination with two primary doses, most of the vaccine recipients remained seronegative for one year before the booster dose administration. Thus, the vaccination schedule might need to be modified. An additional dosing
regimen at two to six months appears to be better, providing prompt protection against hantavirus infection among high-risk subjects. Vaccine-induced immunity (seropositive response) persisted for around two years in seroresponsive subjects. With respect to immunoassays, PRNT has been considered more specific to evaluate the protective immune responses against Hantaan virus compared to IFA [8,14]. IFA can detect antibodies against Hantaan virus nucleocapsid proteins, which are released from patients with HFRS during the acute stage. It is uncertain whether IFA antibody level is related to disease protection. In comparison, PRNT can detect neutralizing antibodies against G1 /G2 membrane
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Fig. 2. (A) Changes in seropositive rate after two primary doses and a booster dose over the study period (PRNT, plaque reduction neutralization test; IFA, immunofluorescent antibody assay). The PRNT50 titer represents the reciprocal serum dilution that reduced virus plaque number by 50%. (B) Age-stratified changes in seropositive rate (PRNT50 ) after two primary doses and a booster dose over the study period. (C) Age-stratified changes in seropositive rate (IFA) after two primary doses and a booster dose over the study period.
glycoproteins produced during the convalescent stage after Hantaan virus infection, providing protective immunity. Thus, PRNT better reflects the protective immune responses of HantavaxTM than IFA. In this study, the antibodies by IFA were detected at higher rates than neutralizing antibodies. The seroconversion rate was estimated to be ≥90% at one month after the two primary dose
vaccinations according to IFA. However, as reported previously [9,10], neutralizing antibodies were not sufficiently induced in the absence of booster vaccination in the present study. When measured using PRNT50 , the seroconversion rate was 23.24% at one month after the two primary dose vaccinations but the seropositive rate increased to 45.07% after the administration of a booster
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Table 4 Solicited local and systemic adverse events within seven days after HantavaxTM administration (N = 226).
Solicited local event Pain Redness Swelling Solicited systemic event Fever Chill Headache Malaise Myalgia Arthralgia Dizziness Nausea
Adverse reactions
Adverse drug reactions*
108 (47.79) 104 (46.02) 23 (10.18) 12 (5.31)
108 (47.79) 104 (46.02) 23 (10.18) 12 (5.31)
57 (25.22) 1 (0.44) 11 (4.87) 16 (7.08) 22 (9.73) 41 (18.14) 11 (4.87) 10 (4.42) 4 (1.77)
55 (24.34) 1 (0.44) 11 (4.87) 14 (6.19) 22 (9.73) 40 (17.70) 11 (4.87) 9 (3.98) 4 (1.77)
* Adverse reactions excluding the cases that were definitely not related or considered not related to the vaccine.
dose. In young adults aged 19–39 years, who are at greater risk since they are involved in more outdoor activities, the seropositive rate reached about 60% after booster vaccination. Considering the poor immunogenicity after the two primary dose vaccinations of HantavaxTM , the booster dosing schedule should be adjusted. An additional dosing regimen at two to six months might be better to achieve the protective antibody level timely among high-risk groups. In addition, post-booster titers were correlated with the longevity of response in this study. Thus, vaccination schedule should be optimized to achieve higher titer after the booster dose also. After the introduction of HantavaxTM in 1990, some studies reported the decreased incidence and severity of HFRS in the ROK [7,11]. Based on these descriptive epidemiological data, mass immunizations with HantavaxTM have been conducted at military units in high-risk areas. Annually, 230,000 to 620,000 doses of HantavaxTM have been administered in the last decade (sales data provided by the pharmaceutical company). However, the long-term immunogenicity and clinical efficacy were not thoroughly investigated, and these uncertainties were great obstacles to increasing the vaccine uptake rates, even among high-risk subjects. Although statistically insignificant, a case-control study demonstrated the dose-dependent protective effectiveness of HantavaxTM [13]. It is not clear whether HantavaxTM prevents or weakens HFRS, even at the sub-protective antibody level. Hantaviruses have a somewhat long incubation period [15]. After boosting is performed, efficient memory B cells are generated that can quickly respond to an infection with higher affinity antibodies. Even though low levels of antibody might be in circulation years after a boost, these B cells might be able to quickly respond by producing high affinity antibodies. Actually, among seroconverted participants, one subject aged ≥60 years was seronegative at two years, but seropositive at three years from the booster dose (Table 3). This subject might have a chance to be boosted by natural exposure in the field. In addition to antibody levels and memory B cell response, vaccineinduced cellular immunity might contribute to disease protection [16]. Thus, it is hopeful that a booster effect was demonstrated when administered at one year after the primary doses. Further studies are required to better clarify the memory B cell populations after each dose of HantavaxTM vaccine and their clinical significance. Rodent brain-derived inactivated hantavirus vaccines have been developed and tested in Korea and China [17]. Although HFRS is also widespread in Europe, rodent-derived hantavirus vaccines have not been developed there due to the concerns about autoimmune encephalitis caused by myelin basic protein contaminants in Western countries [16,17]. Due to these safety concerns, a variety of
techniques have been investigated to prevent hantavirus infection, especially cell culture-adapted vaccines and DNA vaccines [18–20]. However, in agreement with a previous report [15], HantavaxTM was safe and tolerable without vaccine-related SAEs in the subject included in the present study. Most AEs were mild and resolved within two to three days. In fact, a commercialized Hantaan virus vaccine (HantavaxTM ) has been on the market for more than 20 years, but no SAEs have ever been reported. This study has some limitations. First, vaccine-induced cellular immunity was not evaluated. Moreover, the absence of a suitable animal model and limited clinical efficacy data precluded the comprehensive understanding of the immunogenicity results. Second, diverse doses and schedules were not compared. Finally, crossreactive immunogenicity was not tested against other circulating hantaviruses in the ROK. Historically in the ROK, the Hantaan virus has been the most common (70%), followed by the Seoul virus (20%), Soochong virus, and Muju virus [21]. In summary, HantavaxTM showed a booster effect and immunogenicity lasting two years with three dose-scheduled vaccinations. The neutralizing antibody response was quite poor with two primary doses, so an early booster vaccination at two to six months might be warranted to provide timely protection to high-risk recipients. Financial support This work was supported by a Korea University Guro Hospital grant (no. KUGH0707) that was underwritten by the Green Cross Corporation of Yongin, Republic of Korea. Conflict of interest statement All authors have no conflict of interests. Acknowledgments The authors appreciate the HantavaxTM study team and participants for providing samples and data for this work. The authors also thank the laboratory staff at each of the participating institutions for assistance with sample management and testing. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2016.01. 031. References [1] Lee HW, van der Groen G. Hemorrhagic fever with renal syndrome. Prog Med Virol 1989;36:62–102. [2] LeDuc JW. Epidemiology of Hantaan and related viruses. Lab Anim Sci 1987;37(4):413–8. [3] Schmaljohn CS, Hasty SE, Dalrymple JM, LeDuc JW, Lee HW, von Bonsdorff CH, et al. Antigenic and genetic properties of viruses linked to hemorrhagic fever with renal syndrome. Science 1985;227(4690):1041–4. [4] Lee HW. Hemorrhagic fever with renal syndrome in Korea. Rev Infect Dis 1989;11(Suppl 4):S864–76. [5] Lee HW, Lee PW, Johnson KM. Isolation of the etiologic agent of Korean Hemorrhagic fever. J Infect Dis 1978;137(3):298–308. [6] Lee SH, Chung BH, Lee WC, Choi IS. Epidemiology of hemorrhagic fever with renal syndrome in Korea, 2001–2010. J Korean Med Sci 2013;28(10):1552–4. [7] Song JY, Chun BC, Kim SD, Baek LJ, Kim SH, Sohn JW, et al. Epidemiology of hemorrhagic fever with renal syndrome in endemic area of the Republic of Korea, 1995–1998. J Korean Med Sci 2006;21(4):614–20. [8] Cho HW, Howard CR, Lee HW. Review of an inactivated vaccine against hantaviruses. Intervirology 2002;45(4–6):328–33. [9] Sohn YM, Rho HO, Park MS, Kim JS, Summers PL. Primary humoral immune responses to formalin inactivated hemorrhagic fever with renal syndrome
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[15] Kim HY. Hemorrhagic fever with renal syndrome. Infect Chemother 2009;41(6):323–32. [16] Choi Y, Ahn CJ, Seong KM, Jung MY, Ahn BY. Inactivated Hantaan virus vaccine derived from suspension culture of Vero cells. Vaccine 2003;21(17–18):1867–73. [17] Schmaljohn C. Vaccines for hantaviruses. Vaccine 2009;27(Suppl 4):D61–4. [18] Maes P, Clement J, Van Ranst M. Recent approaches in hantavirus vaccine development. Expert Rev Vaccin 2009;8(1):67–76. [19] Schmaljohn CS, Spik KW, Hooper JW. DNA vaccines for HFRS: laboratory and clinical studies. Virus Res 2014;187:91–6. [20] Schmidt J, Jandrig B, Klempa B, Yoshimatsu K, Arikawa J, Meisel H, et al. Nucleocapsid protein of cell culture-adapted Seoul virus strain 80-39: analysis of its encoding sequence, expression in yeast and immuno-reactivity. Virus Genes 2005;30(1):37–48. [21] Noh JY, Cheong HJ, Song JY, Kim WJ, Song KJ, Klein TA, et al. Clinical and molecular epidemiological features of hemorrhagic fever with renal syndrome in Korea over a 10-year period. J Clin Virol 2013;58(1):11–7.
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Long-term immunogenicity and safety of inactivated Hantaan virus vaccine (HantavaxTM ) in healthy adults Joon Young Song a,1 , Heung Jeong Woo b,1 , Hee Jin Cheong a , Ji Yun Noh a , Luck Ju Baek c , Woo Joo Kim a,∗ a
Division of Infectious Diseases, Department of Internal Medicine, Korea University College of Medicine, Seoul, Republic of Korea Division of Infectious Diseases, Department of Internal Medicine, Hallym University College of Medicine, Seoul, Republic of Korea c Department of Microbiology, Institute for Viral Diseases, Korea University College of Medicine, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 12 November 2015 Received in revised form 29 December 2015 Accepted 15 January 2016 Available online xxx NCT02360514 Keywords: Hantaan virus vaccines Hantavirus Immunogenicity Safety
a b s t r a c t Background: Hemorrhagic fever with renal syndrome is a serious health problem in Eurasian countries, including Korea and China. This study evaluated the long-term immunogenicity and safety of formalininactivated Hantaan virus vaccine (HantavaxTM ). Methods: A phase III, multi-center clinical trial was undertaken to evaluate the immunogenicity and safety of HantavaxTM (three-dose schedule at 0, 1, and 13 months) among healthy adults. Immune response was assessed using the plaque reduction neutralizing antibody test (PRNT) and immunofluorescent antibody assay (IFA). Antibody levels were measured pre-vaccination and at 2, 13, 14, 25, 37, and 49 months after the initial vaccination. Systemic and local adverse events were assessed. Results: A total of 226 healthy subjects aged 19–75 years were enrolled. Following two primary doses of HantavaxTM , the seroconversion rate was 90.14% by IFA, but it was only 23.24% by PRNT50 . With booster administration, seropositive rates were 87.32% and 45.07% at one month post-vaccination according to IFA and PRNT50 , respectively. In young adults (19–39 years), the seropositive rate according to PRNT50 reached about 60% after booster vaccination. The mean duration of seropositive response was 735 days for PRNT50 and 845 days for IFA. Solicited local and systemic adverse events occurred in 47.79% and 25.22% of study subjects, respectively, and most were grade 1. Conclusion: HantavaxTM showed a booster effect and immunogenicity lasting two years with a three-dose schedule. The neutralizing antibody response was quite poor with two primary doses, so an early booster vaccination at 2–6 months might be warranted to provide timely protection to high-risk subjects. © 2016 Published by Elsevier Ltd.
1. Introduction Hemorrhagic fever with renal syndrome (HFRS) is a rodentborne zoonosis widely distributed over Eurasia [1]. Among more than 20 strains of Hantaviruses, the major causative agents are the Hantaan, Seoul, and Puumala viruses [1–3]. In the Republic of Korea (ROK), both the Hantaan and Seoul viruses are known as etiologic agents of HFRS [4,5]. These viruses establish chronic infections in certain species of rodents and are transmitted to humans primarily via aerosols or fomites from the feces, urine, and saliva of infected mice. Thus, the incidence of HFRS is particularly high among military persons and farmers during the harvest season. The overall prevalence of HFRS has been reported at 0.81
∗ Corresponding author. Tel.: +82 2 2626 3051; fax: +82 2 2626 1105. E-mail address: [email protected] (W.J. Kim). 1 JYS and HJW contributed equally to this study.
per 100,000 population, while that for military personnel was 40–64 per 100,000 in the ROK, with seasonal variation [6,7]. Since 1990, a commercialized vaccine (HantavaxTM ) has been marketed to prevent HFRS caused by the Hantaan and Seoul viruses [8,9]. Based on the immunogenicity data, HantavaxTM is recommended to be administered as a series of two primary doses administered one month apart and a booster administered 12 months later [9,10]. Although the incidence and severity of HFRS appeared to decrease in the ROK after the introduction of HantavaxTM , the efficacy of the vaccine has been questioned [11]. In addition to vaccination of high-risk groups, other factors should be considered to contribute to the secular reduction of disease burden, including improvements in the standard of living and housing in rural areas, ecological changes in rodent populations, or other natural factors [7]. In a former field trial, no cases of HFRS were identified in 1900 recipients of one-dose HantavaxTM , while 20 cases were confirmed among 2000 non-vaccinated controls (p < 0.01) [12]. However, the
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vaccine recipients and controls were not followed prospectively in that field trial; the medical records of patients with HFRS were used for comparative analysis. In addition, a case-control study conducted in the Korean army did not show statistically significant effectiveness even after the three-dose vaccination [13]. Interestingly, although statistically insignificant, vaccine effectiveness was dose-related, increasing from 25% for one dose, to 46% for two doses, to 75% for three doses. To ensure vaccine effectiveness, the long-term immunogenicity of the current vaccination series of HantavaxTM needs to be established.
2. Materials and methods 2.1. Study design A phase III, multi-center, open-labelled clinical trial was undertaken to evaluate the immunogenicity, immunogenic persistence, and safety of an inactivated hantavirus vaccine (HantavaxTM , Green Cross Corporation, Yongin, ROK) among healthy adults aged 19–75 years at two centers in the ROK (ClinicalTrials.gov Identifier: NCT02360514). Subjects were excluded for the following reasons: previous hantavirus infection, previous hantavirus vaccination, positive neutralizing antibody response at screening, allergy to vaccine components, history of seizure within the past year, pregnant or breastfeeding, febrile disorder, marked nutritional deficiency, uncontrolled medical condition (cardiovascular, renal, or hepatic disease), immunodeficiency or receipt of immunosuppressive therapy, administration of blood products or immunoglobulins within the past three months, or other previous vaccinations within two weeks prior to the study onset. After a baseline blood sample was collected, the hantavirus vaccine was administered to each eligible subject three times with respective one month and 12 month intervals (0-1-13 schedule), including two primary doses and one booster dose. To evaluate the immune responses, blood draws were performed at two months (one month after administration of the second primary dose), 13 months (one year after the two primary doses and before the booster dose), 14 months (one month after the booster dose), 25 months (one year after the booster dose), 37 months (two years after the booster dose), and 49 months (three years after the booster dose) from the initial vaccination. Each subject was followed-up until negative conversion of the neutralizing antibody was observed, up to three years after the three serial vaccinations. Subjects were required to report any solicited adverse events for seven days after each vaccination. At each visit, a brief survey was obtained from study subjects to track baseline demographics, occurrence of illnesses, and medication usage. The primary immunogenicity objective was to evaluate the seroconversion rate and sero-positive persistence of HantavaxTM among healthy adults after administration of two primary doses and a booster dose according to the plaque reduction neutralizing antibody test (PRNT). Secondary immunogenicity objective was to evaluate the seroconversion rate and sero-positive persistence of HantavaxTM using the immunofluorescent antibody assay (IFA). Antibody levels were serially assessed using both immunoassays. Safety objectives included the evaluation of solicited local and systemic adverse events (AEs), spontaneously reported unsolicited AEs, and serious adverse events (SAEs). This study was conducted in accordance with the Declaration of Helsinki and the standards of good clinical practice defined by the International Conference on Harmonization. The protocol and consent forms were approved by the institutional review board of each participating study site. Written informed consent was obtained from all participants following a detailed explanation of the schedules and study contents.
2.2. Vaccines The study vaccine (HantavaxTM , Green Cross Corporation) was developed from suckling mouse brains infected with the ROK 84/105 strain and inactivated with 0.05% formalin. HantavaxTM is alum-adjuvanted and formulated in 0.5-mL pre-filled syringes containing 0.125 mL of virus. The vaccine was administered by intramuscular or subcutaneous routes as a series of two vaccinations spaced one month apart, with a booster administered 12 months later. 2.3. Immunogenicity assessment PRNT and IFA were performed at the Microbiology Laboratory of Korea University College of Medicine, as described previously [10]. Heat-inactivated (56 ◦ C, 30 min) serum samples were diluted in Eagle minimal essential medium with Earle’s salts (EMEM) and then combined with an equal volume (111 mL) of EMEM containing 75 PFU of virus and 10% guinea pig complement (catalog no. ACL4051; Accurate Chemical and Scientific Corp.). This mixture was incubated overnight at 4 ◦ C, and then a plaque assay was performed exactly as described previously, using 7day-old Vero E6 monolayers in six-well plates. The mixture was stained with neutral red (Gibco-BRL) after 1 week, and plaques were counted 2 days (37 ◦ C) after staining. The PRNT50 antibody titer was estimated as the highest serum dilution that reduced the standard viral dose (Hantaan virus strain 76–118) by 50%. Neutralizing titers of 1:10 or greater were regarded as positive. IFA was performed with Hantaan virus (strain 76–118)-infected Vero E6 cells. Cells were spread onto slides, air-dried, and fixed in acetone for 10 min at 4 ◦ C. Serum samples were serially diluted in 2-fold steps in phosphate-buffered saline, starting with the initial dilution of 1:2, then added to the cells. After incubation for 30 min at 37 ◦ C, slides were washed in phosphate-buffered saline and incubated with fluoroscein isothiocyanate-conjugated anti-guinea pig IgG (Sigma, St. Louis, MO, USA). The slides were then washed with phosphate buffered saline, and mounted with buffered alkaline glycerol. The results were read under the fluorescence microscope. IFA titers of 1:32 or greater were regarded as positive. 2.4. Safety assessment At the first visit, each enrolled subject was given a digital thermometer and a diary card containing a list of solicited AEs and their grades. For seven days after vaccination, subjects were requested to record the severity of solicited AEs and unsolicited AEs, axillary temperatures, and concomitant medications on the diary card. Subjects used a standard scale to grade adverse events. Solicited local AEs were pain, redness, and swelling, and solicited systemic AEs were fever, chill, headache, malaise, arthralgia, dizziness, and nausea. We collected reports of unsolicited AEs during the study. Subjects were required to report any SAEs within 24 h for up to six months after vaccination. 2.5. Statistical analysis Demographic data were analyzed for the subjects in the intention-to-treat (ITT) set. Immunogenicity analyses were performed in both per-protocol (PP) and ITT sets, while safety was analyzed for all subjects exposed to the study vaccines. The ITT analysis set comprised all participants who received at least one dose of vaccine, and the PP set included all enrolled subjects who received the correctly administered vaccine, provided evaluable serum samples at relevant time points, and had no major protocol deviations.
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Table 1 Demographic characteristics (intention-to-treat population). Parameters Age (years) Age group No. (%)
Sex, no. (%) BMI (kg/m2 )
Subjects (no. = 218) Mean ± SD Median 19–29 years 30–39 years 40–49 years 50–59 years 60–72 years Male Mean ± SD Median
43.26 ± 14.14 42.00 51 (23.39) 49 (22.48) 38 (17.43) 46 (21.10) 34 (15.60) 81 (37.16) 22.86 ± 2.96 22.60
SD, Standard deviation.
Among the 218 subjects in the ITT set, 142 were eligible for PP analysis. 3.2. Immunogenicity
Fig. 1. Flowchart of subjects through the study (PRNT, plaque reduction neutralization test; IFA, immunofluorescent antibody assay).
Sample sizes of 116 and 80 were estimated to provide sufficient power (80%) to examine the primary (≥80% seroconversion at one month after the booster dose) and secondary (persistent seroconversion up to two years after the booster dose in ≥50% of sero-responsive subjects) study objectives, respectively. Anticipating a 30% drop-out rate at the primary end point (one month after the booster dose) and an annual 25% drop-out rate up to the secondary end point (two years from the booster dose), we planned to enroll 226 subjects. All of the analyses were performed using the statistical software system SAS 9.3 (SAS Institute, Cary, NC, USA). The immunogenicity data were expressed in terms of seroconversion rate and geometric mean titer (GMT) with two-sided 95% confidence intervals (CIs). The two-sided 95% CIs for the GMTs were calculated using the normal approximation of log-transformed titers, and percentages were calculated with approximate or exact 95% CIs. The duration of the immune response (seroconversion) was estimated using the Kaplan–Meier method. Safety data were described as the proportion of study subjects reporting local and systemic adverse reactions. Student’s t-test (or the Wilcoxon rank-sum test) was used to compare continuous variables between the two groups, while the Chi-square test or Fisher’s exact test was conducted to analyze the categorical variables. Results were considered statistically significant if the associated p-value was less than 0.05. 3. Results 3.1. Study subjects Among 256 volunteers, a total of 226 healthy subjects aged 19–75 years were enrolled (Fig. 1). Thirty subjects were excluded because of violation of eligibility criteria (six subjects) or withdrawal of consent (24 subjects). Eight subjects were excluded from the ITT set for the analysis of baseline characteristics because a blood draw was not performed for immunogenicity analysis. The mean age of the participants was 43.3 ± 14.1 years (median age, 42 years), and a higher proportion of females (62.8%, or 137 subjects) was noted. The age distribution included 51 adults aged 19–29 years, 49 adults aged 30–39 years, 38 adults aged 40–49 years, 46 adults aged 50–59 years, and 34 adults aged ≥60 years (Table 1).
Table 2 shows the HantavaxTM -induced antibody responses among the per-protocol population, as assessed using PRNT50 and IFA at each time point after the two primary doses and a booster dose. At one month after the two primary doses, the proportion of subjects with positive responses (seroconversion) was 90.14% according to IFA, but it was only 23.24% according to PRNT50 . The GMTs and seropositive rates returned to pre-vaccination levels over one year, irrespective of the assay method (Table 2 and Fig. 2). With the booster administration of HantavaxTM , the seropositive rates increased to 45.07% and 87.32% at one month post-vaccination according to PRNT50 and IFA, respectively. The seropositive rate of PRNT50 was maintained above 40% at one year after the booster dose but decreased markedly after that. The decline in seropositive rate was rather slower after the administration of the booster dose than after the two primary doses (Fig. 2). When estimated using the Kaplan–Meier method, the mean duration of seropositive response (time from initial positive response to negative conversion) was 735 days (95% CI, 663–721; median, 712 days) and 845 days (95% CI, 713–721; median, 721 days) according to PRNT50 and IFA, respectively. Regarding PRNT50 response, there was no significant difference in age (43.1 ± 13.9 vs. 42.3 ± 13.2, p = 0.87), male sex (12.5% vs. 33.9%, p = 0.42) and body mass index (21.7 ± 3.3 vs. 23.3 ± 2.9, p = 0.13) between seropositive (N = 8) and seronegative (N = 56) subjects at three years after booster dose. However, postbooster GMT (at one month after booster dose) was significantly higher in seropositive subjects (33.6, 95% CI 18.5–61.3) compared to seronegative subjects (17.0, 95% CI 14.2–20.5) (p = 0.01). Age-stratified analysis showed that the seropositive rate of PRNT50 was highest in subjects aged 30–39 years (65.52%), followed by those aged 19–29 years (53.85%) at one month after the booster dose (Table 3). In the case of IFA, the seropositive rate at one month after the booster dose was around 90% in all age groups except for those aged ≥60 years (70.37%). Immune responses among the ITT set were consistent with the results of the PP set (Supplementary Tables 1 and 2). 3.3. Safety Overall, 154 (68.14%) of 226 subjects reported 929 AEs, most of which (84.0%, 780 of 929 events) were grade 1 (noticeable but did not interfere with daily activity). Solicited local and systemic AEs reported within seven days of vaccination are shown in Table 4. Solicited local and systemic AEs occurred in 108 (47.79%) and 57 (25.22%) of 226 subjects, respectively. Injection site pain (104 subjects, 46.02%) and myalgia (41 subjects, 18.14%) were the most frequently reported AEs. All subjects with unsolicited
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Table 2 Immune responses after administration of HantavaxTM among the per-protocol population.
Before vaccination At one month after two primary doses At one year after two primary doses At one month after the booster dose At one year after the booster dose At two years after the booster dose At three years after the booster dose
Plaque reduction neutralization test (PRNT50 a )
Immunofluorescent antibody assay
No. of tested cases
Seropositive rate, (%) (95% CI)
GMTs (95% CI)
No. of tested cases
Seropositive rate, (%) (95% CI)
GMTs (95% CI)
142b 142b 142c 142d 64e 64f 64g
– 23.24 (16.29–30.19) 1.41 (0.00–3.35) 45.07 (36.89–53.25) 40.63 (28.59–52.66) 15.63 (6.73–24.52) 12.50 (4.40–20.60)
5.00 (5.00–5.00) 6.35 (5.86–6.89) 5.07 (4.97–5.18) 9.03 (7.89–10.33) 6.00 (5.64–6.37) 5.85 (5.31–6.44) 6.60 (5.59–7.78)
142a 142a 142b 142c 124d 124e 124f
– 90.14 (85.24–95.04) 10.56 (5.51–15.62) 87.32 (81.85–92.80) 34.68 (26.30–43.05) 17.74 (11.02–24.47) 10.48 (5.09–15.88)
16.08 (15.92–16.23) 57.76 (49.63–67.24) 17.73 (16.76–18.76) 87.90 (71.85–107.53) 23.91 (21.10–27.09) 27.00 (21.85–33.36) 34.78 (22.76–53.13)
GMTs, geometric mean titers. a The PRNT50 titer represents the reciprocal serum dilution that reduced virus plaque number by 50%. b Number of cases who took the antibody test before and one month after the two primary doses. c Number of cases who took the antibody test at one year after the two primary doses. d Number of cases who took the antibody test at one month after the booster dose. e Number of cases who took the antibody test at one month after the booster dose–number of drop-out cases up to one year after the booster dose. f Number of cases who took the antibody test at one month after the booster dose–number of drop-out cases up to two years after the booster dose. g Number of cases who took the antibody test at one month after the booster dose–number of drop-out cases up to three years after the booster dose. Table 3 Age-stratified immune responses (seropositive rates) after administration of HantavaxTM among the per-protocol population.
19–29 years
30–39 years
40–49 years
50–59 years
≥60 years
T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6 T1 T2 T3 T4 T5 T6
Plaque reduction neutralization test (PRNT50 a )
Immunofluorescent antibody assay
No.
Seropositive rate, (%) (95% confidence interval)
No.
Seropositive rate, (%) (95% confidence interval)
26 26 26 14 14 14 29 29 29 19 19 19 27 27 27 10 10 10 33 33 33 14 14 14 27 27 27 7 7 7
42.31 (23.32–61.30) 3.85 (0.00–11.24) 53.85 (34.68–73.01) 35.71 (10.61–60.81) 7.14 (0.00–20.63) 7.14 (0.00–20.63) 27.59 (11.32–43.85) 0.00 (0.00–0.00) 65.52 (48.22–82.82) 47.37 (24.92–69.82) 21.05 (2.72–39.38) 21.05 (2.72–39.38) 3.70 (0.00–10.83) 0.00 (0.00–0.00) 37.04 (18.82–55.25) 60.00 (29.64–90.36) 30.00 (1.60–58.40) 10.00 (0.00–28.59) 24.24 (9.62–38.86) 0.00 (0.00–0.00) 42.42 (25.56–59.29) 21.43 (0.00–42.92) 14.29 (0.00–32.62) 7.14 (0.00–20.63) 18.52 (3.87–33.17) 3.70 (0.00–10.83) 25.93 (9.40–42.46) 42.86 (6.20–79.52) 0.00 (0.00–0.00) 14.29 (0.00–40.21)
26 26 26 24 24 24 29 29 29 26 26 26 27 27 27 26 26 26 33 33 33 29 29 29 27 27 27 19 19 19
96.15 (88.76–100.00) 15.38 (1.52–29.25) 92.31 (82.06–100.00) 33.33 (14.47–52.19) 8.33 (0.00–19.39) 8.33 (0.00–19.39) 93.10 (83.88–100.00) 6.90 (0.00–16.12) 89.66 (78.57–100.00) 38.46 (19.76–57.16) 50.00 (30.78–69.22) 26.92 (9.87–43.97) 81.48 (66.83–96.13) 7.41 (0.00–17.29) 96.30 (89.17–100.00) 38.46 (19.76–57.16) 11.54 (0.00–23.82) 3.85 (0.00–11.24) 90.91 (81.10–100.00) 12.12 (0.99–23.26) 87.88 (76.74–99.01) 27.59 (11.32–43.85) 3.45 (0.00–10.09) 3.45 (0.00–10.09) 88.89 (77.03–100.00) 11.11 (0.00–22.97) 70.37 (53.15–87.59) 36.84 (15.15–58.53) 15.79 (0.00–32.19) 10.53 (0.00–24.33)
T1: one month after the two primary doses, T2: one year after the two primary doses, T3: one month after the booster dose, T4: one year after the booster dose, T5: two years after the booster dose, T6: three years after the booster dose. a The PRNT50 titer represents the reciprocal serum dilution that reduced virus plaque number by 50%.
AEs recovered without sequelae, and no vaccine-related SAE was reported up to six months post-vaccination. 4. Discussion This study showed that HantavaxTM was not usually immunogenic after the administration of two primary dose s, so a booster dose would be required. Despite vaccination with two primary doses, most of the vaccine recipients remained seronegative for one year before the booster dose administration. Thus, the vaccination schedule might need to be modified. An additional dosing
regimen at two to six months appears to be better, providing prompt protection against hantavirus infection among high-risk subjects. Vaccine-induced immunity (seropositive response) persisted for around two years in seroresponsive subjects. With respect to immunoassays, PRNT has been considered more specific to evaluate the protective immune responses against Hantaan virus compared to IFA [8,14]. IFA can detect antibodies against Hantaan virus nucleocapsid proteins, which are released from patients with HFRS during the acute stage. It is uncertain whether IFA antibody level is related to disease protection. In comparison, PRNT can detect neutralizing antibodies against G1 /G2 membrane
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Fig. 2. (A) Changes in seropositive rate after two primary doses and a booster dose over the study period (PRNT, plaque reduction neutralization test; IFA, immunofluorescent antibody assay). The PRNT50 titer represents the reciprocal serum dilution that reduced virus plaque number by 50%. (B) Age-stratified changes in seropositive rate (PRNT50 ) after two primary doses and a booster dose over the study period. (C) Age-stratified changes in seropositive rate (IFA) after two primary doses and a booster dose over the study period.
glycoproteins produced during the convalescent stage after Hantaan virus infection, providing protective immunity. Thus, PRNT better reflects the protective immune responses of HantavaxTM than IFA. In this study, the antibodies by IFA were detected at higher rates than neutralizing antibodies. The seroconversion rate was estimated to be ≥90% at one month after the two primary dose
vaccinations according to IFA. However, as reported previously [9,10], neutralizing antibodies were not sufficiently induced in the absence of booster vaccination in the present study. When measured using PRNT50 , the seroconversion rate was 23.24% at one month after the two primary dose vaccinations but the seropositive rate increased to 45.07% after the administration of a booster
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Table 4 Solicited local and systemic adverse events within seven days after HantavaxTM administration (N = 226).
Solicited local event Pain Redness Swelling Solicited systemic event Fever Chill Headache Malaise Myalgia Arthralgia Dizziness Nausea
Adverse reactions
Adverse drug reactions*
108 (47.79) 104 (46.02) 23 (10.18) 12 (5.31)
108 (47.79) 104 (46.02) 23 (10.18) 12 (5.31)
57 (25.22) 1 (0.44) 11 (4.87) 16 (7.08) 22 (9.73) 41 (18.14) 11 (4.87) 10 (4.42) 4 (1.77)
55 (24.34) 1 (0.44) 11 (4.87) 14 (6.19) 22 (9.73) 40 (17.70) 11 (4.87) 9 (3.98) 4 (1.77)
* Adverse reactions excluding the cases that were definitely not related or considered not related to the vaccine.
dose. In young adults aged 19–39 years, who are at greater risk since they are involved in more outdoor activities, the seropositive rate reached about 60% after booster vaccination. Considering the poor immunogenicity after the two primary dose vaccinations of HantavaxTM , the booster dosing schedule should be adjusted. An additional dosing regimen at two to six months might be better to achieve the protective antibody level timely among high-risk groups. In addition, post-booster titers were correlated with the longevity of response in this study. Thus, vaccination schedule should be optimized to achieve higher titer after the booster dose also. After the introduction of HantavaxTM in 1990, some studies reported the decreased incidence and severity of HFRS in the ROK [7,11]. Based on these descriptive epidemiological data, mass immunizations with HantavaxTM have been conducted at military units in high-risk areas. Annually, 230,000 to 620,000 doses of HantavaxTM have been administered in the last decade (sales data provided by the pharmaceutical company). However, the long-term immunogenicity and clinical efficacy were not thoroughly investigated, and these uncertainties were great obstacles to increasing the vaccine uptake rates, even among high-risk subjects. Although statistically insignificant, a case-control study demonstrated the dose-dependent protective effectiveness of HantavaxTM [13]. It is not clear whether HantavaxTM prevents or weakens HFRS, even at the sub-protective antibody level. Hantaviruses have a somewhat long incubation period [15]. After boosting is performed, efficient memory B cells are generated that can quickly respond to an infection with higher affinity antibodies. Even though low levels of antibody might be in circulation years after a boost, these B cells might be able to quickly respond by producing high affinity antibodies. Actually, among seroconverted participants, one subject aged ≥60 years was seronegative at two years, but seropositive at three years from the booster dose (Table 3). This subject might have a chance to be boosted by natural exposure in the field. In addition to antibody levels and memory B cell response, vaccineinduced cellular immunity might contribute to disease protection [16]. Thus, it is hopeful that a booster effect was demonstrated when administered at one year after the primary doses. Further studies are required to better clarify the memory B cell populations after each dose of HantavaxTM vaccine and their clinical significance. Rodent brain-derived inactivated hantavirus vaccines have been developed and tested in Korea and China [17]. Although HFRS is also widespread in Europe, rodent-derived hantavirus vaccines have not been developed there due to the concerns about autoimmune encephalitis caused by myelin basic protein contaminants in Western countries [16,17]. Due to these safety concerns, a variety of
techniques have been investigated to prevent hantavirus infection, especially cell culture-adapted vaccines and DNA vaccines [18–20]. However, in agreement with a previous report [15], HantavaxTM was safe and tolerable without vaccine-related SAEs in the subject included in the present study. Most AEs were mild and resolved within two to three days. In fact, a commercialized Hantaan virus vaccine (HantavaxTM ) has been on the market for more than 20 years, but no SAEs have ever been reported. This study has some limitations. First, vaccine-induced cellular immunity was not evaluated. Moreover, the absence of a suitable animal model and limited clinical efficacy data precluded the comprehensive understanding of the immunogenicity results. Second, diverse doses and schedules were not compared. Finally, crossreactive immunogenicity was not tested against other circulating hantaviruses in the ROK. Historically in the ROK, the Hantaan virus has been the most common (70%), followed by the Seoul virus (20%), Soochong virus, and Muju virus [21]. In summary, HantavaxTM showed a booster effect and immunogenicity lasting two years with three dose-scheduled vaccinations. The neutralizing antibody response was quite poor with two primary doses, so an early booster vaccination at two to six months might be warranted to provide timely protection to high-risk recipients. Financial support This work was supported by a Korea University Guro Hospital grant (no. KUGH0707) that was underwritten by the Green Cross Corporation of Yongin, Republic of Korea. Conflict of interest statement All authors have no conflict of interests. Acknowledgments The authors appreciate the HantavaxTM study team and participants for providing samples and data for this work. The authors also thank the laboratory staff at each of the participating institutions for assistance with sample management and testing. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2016.01. 031. References [1] Lee HW, van der Groen G. Hemorrhagic fever with renal syndrome. Prog Med Virol 1989;36:62–102. [2] LeDuc JW. Epidemiology of Hantaan and related viruses. Lab Anim Sci 1987;37(4):413–8. [3] Schmaljohn CS, Hasty SE, Dalrymple JM, LeDuc JW, Lee HW, von Bonsdorff CH, et al. Antigenic and genetic properties of viruses linked to hemorrhagic fever with renal syndrome. Science 1985;227(4690):1041–4. [4] Lee HW. Hemorrhagic fever with renal syndrome in Korea. Rev Infect Dis 1989;11(Suppl 4):S864–76. [5] Lee HW, Lee PW, Johnson KM. Isolation of the etiologic agent of Korean Hemorrhagic fever. J Infect Dis 1978;137(3):298–308. [6] Lee SH, Chung BH, Lee WC, Choi IS. Epidemiology of hemorrhagic fever with renal syndrome in Korea, 2001–2010. J Korean Med Sci 2013;28(10):1552–4. [7] Song JY, Chun BC, Kim SD, Baek LJ, Kim SH, Sohn JW, et al. Epidemiology of hemorrhagic fever with renal syndrome in endemic area of the Republic of Korea, 1995–1998. J Korean Med Sci 2006;21(4):614–20. [8] Cho HW, Howard CR, Lee HW. Review of an inactivated vaccine against hantaviruses. Intervirology 2002;45(4–6):328–33. [9] Sohn YM, Rho HO, Park MS, Kim JS, Summers PL. Primary humoral immune responses to formalin inactivated hemorrhagic fever with renal syndrome
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[15] Kim HY. Hemorrhagic fever with renal syndrome. Infect Chemother 2009;41(6):323–32. [16] Choi Y, Ahn CJ, Seong KM, Jung MY, Ahn BY. Inactivated Hantaan virus vaccine derived from suspension culture of Vero cells. Vaccine 2003;21(17–18):1867–73. [17] Schmaljohn C. Vaccines for hantaviruses. Vaccine 2009;27(Suppl 4):D61–4. [18] Maes P, Clement J, Van Ranst M. Recent approaches in hantavirus vaccine development. Expert Rev Vaccin 2009;8(1):67–76. [19] Schmaljohn CS, Spik KW, Hooper JW. DNA vaccines for HFRS: laboratory and clinical studies. Virus Res 2014;187:91–6. [20] Schmidt J, Jandrig B, Klempa B, Yoshimatsu K, Arikawa J, Meisel H, et al. Nucleocapsid protein of cell culture-adapted Seoul virus strain 80-39: analysis of its encoding sequence, expression in yeast and immuno-reactivity. Virus Genes 2005;30(1):37–48. [21] Noh JY, Cheong HJ, Song JY, Kim WJ, Song KJ, Klein TA, et al. Clinical and molecular epidemiological features of hemorrhagic fever with renal syndrome in Korea over a 10-year period. J Clin Virol 2013;58(1):11–7.
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