ABSTRACT
The large-scale production of a human diploid cell (HDC) vaccine (HDCV) for rabies is limited by several technical challenges. Kanghua Biological Products Co., Ltd., has successfully used microcarrier technology for the large-scale culture of HDCs in bioreactors to develop a lyophilized and purified HDCV. In this blinded, randomized, parallel-group study conducted between July and October 2014 in Mianzhu, Sichuan Province, China, we monitored the safety and immunogenicity of this vaccine in a healthy population vaccinated according to the Essen post-exposure immunization schedule. A hamster kidney cell vaccine was used as the control. Adverse reactions were monitored 0.5, 6, 24, 48, and 72 h post vaccination to assess safety. Neutralizing antibodies in venous blood were measured on day 7, 14, and 72 to evaluate the immunogenicity of the vaccine while follow-up monitoring continued for 1 month. No serious adverse reactions were observed in any volunteer. The incidence rates of systemic and local adverse reactions were, respectively, 10.6% and 2.9% in the test group and 20.0% and 13.6% in the control group. After the third injection, the positive conversion rates of antibodies in the test and control groups were 100% and 98.82%, respectively. In addition, the average antibody titers on day 7, 14, and 42, were respectively, 1.71, 2.72, and 1.29 times higher than those in the control group. These results indicate that HDCV had a better safety profile and higher immunogenicity than the hamster kidney cell rabies vaccine.
Trial registration number: 20130602.
KEYWORDS: immunogenicity, randomized, safety profile, rabies vaccine
Introduction
Rabies is a fatal infectious disease caused by the rabies virus that primarily attacks the central nervous system and it has a 100% mortality rate in humans.1 Presently, there is no effective treatment for rabies. Warm-blooded animals such as dogs, cats, and foxes are generally more susceptible to rabies infection.2 It is prevalent in more than 150 countries worldwide,3 and is responsible for more than 60,000 deaths annually.4,5 The majority of rabies cases occur in Africa and Asia, and approximately 40% occur in children aged< 15 years. In rabies endemic areas, more than 99% of cases in humans are caused by rabies virus transmitted by dogs.6 Vaccination of dogs, pre-exposure prophylaxis for humans, and post-exposure clinical treatment and prophylaxis are some of the steps taken to curb rabies incidence. The World Health Organization (WHO) also supports the development of vaccines,6 which are the most cost-effective means of preventing and controlling human infections. In China, chicken embryo cell vaccine,7 Vero cell vaccine,8,9 hamster kidney cell vaccine,10 and human diploid cell (HDC) rabies vaccine (HDCV) 11,12have been approved.
The WHO clearly states that HDCV is the “gold standard” rabies vaccine.13 Because HDCs are normal karyotype cells without carcinogenicity, the HDCV does not contain any foreign animal impurity or neurotoxicity factor. Furthermore, there are fewer injections required and mild adverse reactions and it is safe and efficacious; thus, it is recommended by the WHO as the “nearly ideal human vaccine.” As the gold standard rabies vaccine, HDCV has always been used as the standard control for developing new rabies vaccines. However, the difficulties encountered during the large-scale culture of HDCs have limited the production of rabies vaccines that require these cells as substrates.14 Prior to 2015, HDCV was primarily used in developed countries. China does not approve the import of foreign HDCV and in this regard, Kanghua Biological Products Co., Ltd., in China has overcome these technical difficulties to become the first worldwide to successfully utilize microcarrier technology for the large-scale culture of HDCs in bioreactors for developing a lyophilized and purified HDCV. This clinical study was conducted to assess the immunogenicity and safety of HDCV prepared under the conditions for large-scale production in a healthy population vaccinated according to the Essen post-exposure immunization schedule.
Results
Study population
A total of 232 participants were screened, consisting of 78 men and 154 women. Eighteen participants who were deemed ineligible were excluded from the study, including seven with drug allergies, four with food allergies, three who had received rabies vaccines 1 year ago, and one each for the use of immunosuppressive agents within 1 year, being prone to fainting during needle procedures, undergoing lymph node resection, and having a history of epilepsy. Finally, 214 participants (104 and 110 in the test and control groups, respectively) were included for safety monitoring. Among them, 65 were men and 149 were women, with a sex ratio of 0.44:1. The maximum and minimum age was 59.5 and 15.7 years, respectively, while the average was 45.6 years. The participants were asked to return their diary cards after vaccination for verification purposes, and the verification rate was 100%. A total of 194 participants, consisting of 91 and 103 in the test and control groups, respectively received all five rounds of vaccinations. The detailed results are presented in Table 1.
Table 1.
Demographics and baseline characteristics of participants in the test and control groups.
| Category | Test group (n = 104) | Control group (n = 110) |
|---|---|---|
| Male | 39 | 26 |
| Female | 65 | 84 |
| Male/female ratio | 0.60 | 0.31 |
| Mean age (years) | 44.9 | 46.3 |
| SD | 9.57 | 8.41 |
Exclusion criteria: recipients with fever or chronic diseases such as autoimmune diseases, diabetes, hypertensive, liver and kidney diseases; history of rabies vaccination; contraindication to vaccination; and poor tolerance for blood collection.
Participant compliance
The dropout rates in the test and control groups were 12.5 and 6.4%, respectively. The collection rates of valid blood samples in the test and control groups were 88.5 and 90.9%, respectively. The vaccination rates of participants who received all five injections in the test and control groups were 88 and 94%, respectively and the detailed results are presented in Table 2.
Table 2.
Compliance analysis of participants in the test and control groups.
| Group | Number of enrolled participants | Number of participants who received all vaccinations | Number of participants from whom blood was collected | Full vaccination rate% | Vaccination compliance rate% |
|---|---|---|---|---|---|
| Test group | 104 | 91 | 92 | 88 | 89 |
| Control group | 110 | 103 | 100 | 94 | 91 |
There was no significant difference in vaccination compliance rates and full-vaccination rates between the 2 groups, Χ2 = 0.35, P = 0.56; Χ2 = 2.4, P = 0.12.
No significant differences in vaccination compliance and full-vaccination rates between the two groups were found, Chi-squared test (Χ2) = 0.35, P = 0.56; Χ2 = 2.4, P = 0.12.
Safety
The incidence rates of systemic and local adverse reactions were 10.6% and 2.9% in the test group and 20.0% and 13.6% in the control group, respectively. The systemic and local reactions in the test and control groups were mainly mild reactions (e.g., pain at the injection site) and the symptoms disappeared within 48 h in both groups (Table 3). A significant difference was observed in the incidence rates of local reactions between the two groups (Χ2 = 8.0, P = 0.0046), whereas the difference in incidence rates of systemic reactions was not significant (Χ2 = 3.6, P = 0.056). The difference in overall incidence rates of adverse reactions between the two groups was significant (Χ2 = 12.0, P = 0.0005). No serious adverse reactions were observed during the study.
Table 3.
Adverse reaction analysis in the test and control groups.
| Overall incidence |
Incidence of moderate or more serious adverse reactions |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Type | Group | Number of vaccinations | No. | % | x2 | P | No. | % | x2 | P |
| Systemic reactions | Test group | 104 | 11 | 10.6 | 3.6 | 0.056 | 2 | 1.9 | 0.003 | 0.95 |
| Control group | 110 | 22 | 20.0 | 1 | 0.9 | |||||
| Local reactions | Test group | 104 | 3 | 2.9 | 8 | 0.046 | 0 | 0 | ||
| Control group | 110 | 15 | 13.6 | 0 | 0 | |||||
| Overall reactions | Test group | 104 | 14 | 13.5 | 12 | 0.0005 | 2 | 1.9 | 0.003 | 0.95 |
| Control group | 110 | 37 | 33.6 | 1 | 0.9 | |||||
Immunogenicity
A post-vaccination antibody titer not less than 0.5 IU/mL was considered positive. A total of 192 serum samples were collected, including 15 that were positive for antibodies prior to vaccination and two that showed abnormal values during antibody testing. A total of 175 cases, including 85 and 90 in the test and control groups, respectively, were included in the analysis. The positive conversion rates of serum antibody post vaccination are presented in Table 4. Blood samples were collected on day 7, 14, and 42 after the first injection from the test and control groups.
Table 4.
Positive conversion rates of antibody in the test and control groups.
| Test group |
Control group |
Analysis |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Blood collection time | Number of participants | Number of participants with antibody conversion | Positive conversion rate (%) | GMT | Number of participants | Number of participants with antibody conversion | Positive conversion rate (%) | GMT | x2 | P |
| Day 0 | 85 | 90 | 0.193 | 0.66 | ||||||
| Day 7 | 34 | 6 | 17.65 | 1.11 | 36 | 4 | 11.11 | 0.65 | ||
| Day 14 | 81 | 81 | 100.00 | 19.54 | 85 | 84 | 98.82 | 7.19 | ||
| Day 42 | 85 | 85 | 100.00 | 11.72 | 90 | 90 | 100.00 | 9.06 | ||
The antibody titers were determined using the rapid fluorescent focus inhibition test (RFFIT), and the average geometric mean titer (GMT) of the antibodies was calculated. The GMTs were, respectively, 1.11 IU/mL (95% confidence interval [CI]: 0.19 to 6.98), 19.54 IU/mL (95% CI: 13.34 to 25.76), and 11.71 IU/mL (95% CI: 1.95 to 150.33) in the test group and 0.65 IU/mL (95% CI: 0.1 to 21.38), 7.46 IU/mL (95% CI: 1.35 to 13.59), and 9.13 IU/mL (95% CI: 4.38 to 13.89) in the control group. No significant difference was observed in the GMT of serum antibodies between the two groups on day 42 post vaccination (t = 1.57, P = 0.12). The serum GMT on day 7 and 14 in the test group was significantly higher than that in the control group (t ≥ 2.21, P ≤ 0.03). The antibody titers are shown in Table 5 and Figure 1.
Table 5.
GMT analysis of antibodies in the test and control groups.
| Blood collection time | Group | Number of cases | GMT | 95CI | t value | P value |
|---|---|---|---|---|---|---|
| Blood collection on day 0 | Test group | 84 | ||||
| Control group | 89 | |||||
| Blood collection on day 7 | Test group | 33 | 1.11 | 0.19–6.98 | −2.21 | 0.03 |
| Control group | 34 | 0.65 | 0.1–21.38 | |||
| Blood collection on day 14 | Test group | 81 | 19.54 | 13.34–25.76 | −5.4 | 0 |
| Control group | 84 | 7.46 | 1.35–13.59 | |||
| Blood collection on day 42 | Test group | 84 | 11.71 | 1.95–150.33 | −1.57 | 0.12 |
| Control group | 89 | 9.13 | 4.38–13.89 |
Figure 1.
Antibody titers at different time points after first vaccination.
The positive conversion rates of serum antibody after two injections in the test and control groups were 17.6% and 11.1%, respectively and the corresponding rates after three injections, in the test group and control groups were 100% and 98.82%, respectively. After completing the entire vaccination course, the positive conversion rates of antibody in both the groups were 100%. No significant difference in the positive conversion rates of antibody was observed between the test and the control groups for each vaccination. In the first blood collection, 15 participants had serum antibody titers >0.5 IU/mL, two had abnormal serum antibody levels, and 17 excluded. The detailed results are presented in Table 6. A total of 14 out of 15 participants who tested positive for antibodies during the initial testing were vaccinated. One participant did not receive the vaccination (Table 7).
Table 6.
Participants excluded from the serum antibody titer analysis for immunogenicity (IU/mL).
| Serial number | Day 0 | Day 7 | Day 14 | Day 42 |
|---|---|---|---|---|
| 1 | 1.90 | / | 121.71 | 41.26 |
| 2 | 0.80 | 53.46 | 505.95 | 327.15 |
| 3 | 4.39 | 16.37 | 192.60 | 650.37 |
| 4 | 1.03 | / | 52.26 | 100.82 |
| 5 | 1.92 | 16.37 | / | 41.48 |
| 6 | 0.70 | / | 39.49 | 24.09 |
| 7 | 1.02 | / | 81.46 | 38.64 |
| 8 | 7.60 | 18.29 | 121.71 | 25.63 |
| 9 | 0.60 | / | 46.00 | 15.52 |
| 10 | 0.70 | 5.05 | 109.05 | 40.57 |
| 11 | 0.87 | / | 70.54 | 51.97 |
| 12 | 5.77 | / | 23.51 | 17.32 |
| 13 | 4.39 | 5.46 | 32.69 | 24.99 |
| 14 | 0.50 | / | 118.47 | 577.8 |
| 15 | 0.92 | / | 35.77 | 46.00 |
| 16 | 0.35 | 1990.47 | 1566.23 | 561.75 |
| 17 | 0.25 | 251.37 | 522.08 | 577.80 |
Table 7.
Participants with pre-vaccination serum levels of neutralizing antibody greater than 0.5 IU/mL.
| Serial number | Group | First blood collection (IU/mL) | Vaccination time |
|---|---|---|---|
| 1 | Test group | 1.9 | 2013 |
| 2 | Test group | 4.4 | 2007 |
| 3 | Test group | 0.7 | Not vaccinated |
| 4 | Test group | 1.0 | 2009 |
| 5 | Test group | 7.6 | 2011 |
| 6 | Test group | 0.7 | 2011 |
| 7 | Test group | 0.5 | 2008 |
| 8 | Control group | 0.8 | 2012 |
| 9 | Control group | 1.0 | 2006 |
| 10 | Control group | 1.9 | 2008 |
| 11 | Control group | 0.6 | 2011 |
| 12 | Control group | 0.9 | 2011 |
| 13 | Control group | 5.8 | 2014 |
| 14 | Control group | 4.4 | 2007 |
| 15 | Control group | 0.9 | 2012 |
Discussion
In this blinded, randomized, parallel-group study, we monitored the safety and immunogenicity of lyophilized and purified HDCV in a healthy population vaccinated according to the Essen post-exposure immunization schedule. We found that HDCV had a better safety profile and higher immunogenicity than the hamster kidney cell rabies vaccine.
The HDCV currently available on the market in China uses the WHO-recommended Pitman-Moore (PM) strain of fixed rabies virus for vaccine production, which has also been approved by the China National Drug Administration [formerly the China Food and Drug Administration (CFDA)].15 The cell substrate used for the HDCV is the MRC-5 cell line, which is approved and recommended by the WHO. This cell line is not carcinogenic when xenotransplanted and is not contaminated by exogenous factors. In addition, it is well tolerated and suitable for the production of various viral vaccines.16 MRC-5 cells have been used by many vaccine manufacturers worldwide to develop various vaccines.17,18 Regarding medical expenses, foreign HDCV in developed countries such as Europe and America cost US$1800 for the entire post-exposure prophylaxis process. In contrast, the HDCV used in this study has benefited more patients and costs only US$234 for the entire process of post-exposure prophylaxis, which greatly reduces the cost of medical care and the medical expenses of patients.
Vero cell rabies vaccine has great advantages in large-scale production. Scientists worldwide are attempting to produce low-cost vaccines using Vero cells and expect the safety and efficacy to meet or exceed that of HDCV. In 1984, Merieux successfully developed purified Vero cell rabies vaccine (PVRV) using microcarrier suspension culture and, subsequently, it was approved in Europe and many developing countries. Due to the high productivity, low cost and good immune effect of rabies virus cultured in Vero cells, the WHO provided a production procedure for purified inactivated rabies vaccine for human use cultured in Vero cells in 1987, and recommended the use of rabies vaccines produced using Vero cells. Currently, the Vero cell rabies vaccine is used worldwide, including in China. The development of the China hamster kidney cell rabies vaccine was led by Wuhan Institute of Biological Products, in cooperation with the China National Institute for the Control of Pharmaceutical and Biological Products. Since 1965, after years of research and development aG strain was finally produced in 1979. The primary hamster kidney cell vaccine, which has high safety and immunogenicity is also widely used in China.19
In 2009, a phase III clinical trial of the HDCV was conducted. A total of 1200 potentially infected subjects aged 10 to 60 years infected with rabies were included and randomized into groups. Furthermore, 600 were vaccinated with HDCV according to the post-exposure immunization schedule while 600 in the control group were vaccinated with the Vero vaccine. The results of the clinical trial showed that the positive conversion rate of antibody was 100% on day 14 after the first vaccination while the incidence rate of local and systemic adverse reaction was 7.67% and 5% respectively. All local and systemic reactions were mild, resolved spontaneously within 3 days without medical intervention, and no grade III or higher adverse reactions occurred.20 In a previous study, the immunogenicity of HDCV as a booster injection after 8 years of the initial vaccination was investigated. Sixty subjects who participated in phase III clinical trials and completed the full course of vaccination with HDCV were selected. The subjects received a single-dose booster injection after 8 years of the initial vaccination. The results showed that the GMT of rabies virus neutralizing antibodies (RVNA) before and after the booster injections was 1.42 and 30.61 IU/mL, respectively.21
In the present study, we evaluated data from phase IV clinical trials and found that the adverse effects of rabies vaccines using animal-derived cell substrates were significantly worse than those of HDCV. This observation proved that there was a significant difference between the two types of vaccines. This might be related to the cell substrates used by the vaccines as well as the purity of the vaccines. No serious reactions or serious adverse events were observed in this study, and most adverse reactions in the two groups were mild. The incidence rates of local, systemic and overall adverse reactions were lower in the test group than in the control group. Except for systemic reactions, which were not significantly different, the incidence rate of local reactions was significantly different between the two groups, suggesting that the purified vaccine from diploid cells might have a superior safety profile. The symptoms of local adverse reactions, which mainly manifested as pain at the injection site, disappeared within 48 h. The Chinese Pharmacopoeia has clearly stated that when introducing vaccines produced from animal-derived cell substrates, the amount of DNA residues must be strictly controlled to ensure the safety of subjects.
A timely, full-course vaccination with lyophilized rabies vaccine administered for human use according to the immunization schedule can produce the ideal antibody. The positive conversion rate of antibody after the third injection was 100% in the HDCV group. Although the positive conversion rate was not significantly different from that of the control group (98.82%), the average antibody titer of the test group was higher. The antibody titers of the test group on day 7, 14, and 42 were respectively 1.71, 2.72, and 1.29 times the antibody titers of the control group, these differences were significant. The average age of test group and control group was 44.9 years old and 46.3 years old, respectively, the conversion rate was 98.82% in control group at day 14, this result may be related to the higher average age.
This study showed that the test group achieved 100% positive conversion of antibody on day 14, and the antibody titer was higher on day 14 than day 42. These findings suggested that the vaccine rapidly produces sufficient antibodies to protect the body from rabies virus, thereby providing superior protective effects.
Patients and methods
Study design and population
We selected no less than 200 healthy volunteers aged 10 to 60 years using a blinded randomized parallel-group method. And then all the volunteers were randomly assigned to two groups in a 1:1 ratio. Both groups were expected to have no less than 100 subjects vaccinated with the test and control vaccines to study the immunogenicity and clinical adverse reactions of HDCV. Numbers were randomly generated within 400 using the Microsoft Word EXCEL software, and used to code the subjects. Volunteers with odd and even numbers were assigned to the test and control groups, respectively.
This blinded, randomized, parallel-group study included no less than 200 healthy volunteers according to the following inclusion criteria: healthy individuals aged 10–60 years, no history of rabies vaccination within 3 years of the study, no contraindications for rabies vaccination, and free from any severe acute or chronic diseases. Monitoring of participants who received immunosuppressive agents during the monitoring period of the vaccinations was required to be discontinued. In case of serious adverse events, participant monitoring was required to be discontinued after the assessment by investigators. Participants who failed to complete the follow-ups during the monitoring period were deemed to have withdrawn (participants could withdraw voluntarily from the study at any time). Informed consent was obtained from the participants or their guardians.
Test and control vaccines
The test vaccine was lyophilized HDCV manufactured by Chengdu Kanghua Biological Products Co., Ltd. The potency of rabies vaccine was not less than 2.5 IU [batch number: 20130602, specification: 1 mL/dose]. The hamster kidney cell vaccine for human use used as the control vaccine was manufactured by Henan Yuanda Biopharmaceutical Co., Ltd. The potency of rabies vaccine was not less than 2.5 IU [batch number: 20141352, specification: 1 mL/bottle].
Interventions and outcomes
Included volunteers were randomly assigned to the two groups in a 1:1 ratio and vaccinated with 1 mL of the test or control vaccines in the deltoid muscle of the upper arm according to the “Essen” immunization schedule. Venous blood was collected four times before the vaccination and on day 7, 14, and 42 after injecting the first dose. All participants who were injected with one or more doses were included in the safety evaluation. Adverse reactions were monitored 0.5, 6, 24, 48, and 72 h post vaccination to determine the tolerance and safety of the vaccine. The types and grades of adverse reactions including systemic and local adverse reactions were determined based on the Management approaches for monitoring and reporting of adverse drug reactions. Guidelines for the grading of clinical adverse reactions of prophylactic vaccines, and other criteria specified in the relevant manuals. Follow-up monitoring continued for 1 month. Blood samples were collected from > 50% of the enrolled study population. The entrusted inspection was conducted by the China National Institute for the Control of Pharmaceutical and Biological Products through the use of FITC Anti-Rabies Monoclonal Globulin 5.0 mL/bottle and national standard rabies immunoglobulin 1.0 mL/bottle. Neutralizing antibodies were measured using the RFFIT to evaluate the immunogenicity of the vaccine. A post-vaccination RVNA titer no less than 0.5 IU/mL was considered a positive immunogenicity response.
Statistical analysis
The data were sorted using the Microsoft (MS) Excel 2010 program and analyzed using Chi-square tests and t-tests with the statistical package for the social sciences (SPSS) version 18.0. Differences with a P < 0.05 were deemed statistically significant.
Funding Statement
This study was supported by the Chengdu Municipal Key New Products Project in China: Enterprise Capacity Enhancement Project (2015-cp03-01010-GX)", which was held by Science and Technology Bureau of Chengdu City, aimed to encourage the R&D of enterprises.
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
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