A semi-quantitative serological method to assess the potency of inactivated rabies vaccine for veterinary use

Potency is one of the most important indexes of inactivated vaccines. A number of methods have been established to assay the potency, of which the NIH test and single-dose mouse protection test are the “prescribed methods”. Here, we report a method to semi-quantitatively assay the potency of an inactivated rabies vaccine, which uses fewer animals and takes less time to complete. Depending on the quality requirements of a vaccine (e.g. minimum potency), a rabies reference vaccine is, for example, diluted to the minimum potency, and 50 μL of the dilution is taken to inoculate 10 mice. The same amount of the test rabies vaccine is inoculated into another 10 mice. After two weeks, all mice are bled and serum samples are assayed for viral neutralizing antibody by the fluorescent antibody virus neutralization (FAVN) test. By comparing the median and interquartile range of antibody titers of the reference vaccine with those of the test vaccine, the test vaccine potency can be semi-quantitatively judged as to whether it is in accord with the required quality. The reliability of this method was also confirmed in dogs. The procedure can be recommended for batch potency testing during inactivated rabies vaccine production.     

A multi-dose serological assay suitable to quantify the potency of inactivated rabies vaccines for veterinary use

The mouse vaccination-challenge test, which is the most widely used method for determining the potency of inactivated rabies vaccines, is imprecise, time-consuming, and causes severe distress to the test animals. An alternative single-dose serological method has been implemented in the European Pharmacopoeia Monograph 0451 to replace the mouse challenge test for batch release. This single-dose limit method provides semi-quantitative results, but is not suitable for quantifying potency. We have now extended this serological method to a multi-dose format which allows a quantification of vaccine potency. In studies including all rabies vaccine strains relevant for Europe, we found dose-dependency for all vaccines and standard preparations. We have demonstrated that the multi-dose serological approach provides reliable quantitative potency results and is more precise than the mouse vaccination-challenge test. We have shown that adjuvanted vaccines can be calibrated against non-adjuvanted material, and that reference material can be calibrated against the International Standard. The method is therefore capable of assigning potency with the additional advantage of requiring fewer animals and reducing distress. Once the applicability of the method has been further verified in a collaborative study, it can complement the single-dose assay and eventually eliminate the need for the mouse challenge test.     

Standardization of an enzyme immunoassay for the in vitro potency assay of inactivated tissue culture rabies vaccines: determination of the rabies virus glycoprotein with polyclonal antisera

A non-competitive enzyme-linked immunoassay (ELISA) has been standardized to supplement the in vivo potency test used for the quality control of inactivated tissue culture vaccines against rabies. The essentials of the ELISA were: fixation of the virus in different dilutions of vaccine on the surface of microtitre plates; testing of the reference and up to six test vaccines on one plate; incubation with polyclonal antisera to rabies virus glycoprotein containing an excess of antibody; further incubation with a species-specific anti-IgG coupled to peroxidase; a final incubation with a substrate. The incubation periods were 1 h, 1 h and 30 min both at +37 degrees C. The relative potency determinations were made graphically or by a computer using a parallel line bioassay in which the potencies of the vaccines of unknown potency were tested against the reference preparation on a single microtitre plate. Under these conditions inactivated rabies vaccines of different types (virus strains, cell substrates, inactivation and concentration procedures) were tested for potency. Furthermore, it was possible with this in vitro method to assay adjuvanted vaccines, in process samples such as tissue culture supernatants with live or inactivated rabies virus, concentrates, and vaccines undergoing thermal stability tests. The rabies glycoprotein antigen-antibody reaction was highly specific according to the results and the glycoprotein content was measured quantitatively. The potency determined by the in vitro ELISA correlated with the in vivo NIH protection potency test. The lower limit of detection of the ELISA was 0.015 IU/ml. Quantitative antigen determination was possible with both homologous and heterologous antisera to rabies virus glycoprotein when vaccines of the same virus strain were tested. When the potencies of vaccines of different virus strain specificity were calculated, it was necessary to take into account the strain-specific antigenicity. Even so vaccines of high potency were found to give a stronger reaction with a heterologous serum than did weak vaccines with a homologous antiserum. Stability tests made on inactivated tissue culture vaccines such as vaccine from the human diploid cell strain (HDCS), from purified chicken embryo cell (PCEC) or from purified Vero cell rabies vaccine (PVRV), showed high stability of the glycoprotein antigen even after four months of storage at +37 degrees C or 24 h at +56 degrees C, provided that the vaccines were stored in a lyophilized state. The antigenicity of liquid vaccines was inactivated after a few hours at +56 degrees C. For tropical areas, therefore, only lyophilized vaccines should be considered.     

Genetic stability (in vivo) of the attenuated oral rabies virus vaccine SAD B19

The distribution of oral rabies vaccine baits containing replication-competent live viruses poses certain environmental safety risks; among others, the possibility of reversion to or an increase in virulence. Hence, the genetic stability of the complete genome of the most widely used oral rabies vaccine virus, SAD B19, was examined after four and 10 serial i.c. passages in foxes and mice, respectively. It was shown that the consensus strain of SAD B19 was extremely stable in vivo . After 10 consecutive passages in mice not a single mutation was observed. In foxes, seven single nucleotide exchanges were found between the first and fourth passage, of which only one resulted in an amino acid exchange at position 9240 of the L-gene. This mutation was not observed during the first three passages and, furthermore, it was shown that this mutation was not linked to enhanced virulence.     

Highly sensitive histamine-sensitization test for residual activity of pertussis toxin in acellular pertussis vaccine

Histamine-sensitization test method based on histamine-sensitizing death is widely used for controlling residual activity of pertussis toxin in acellular pertussis vaccines. The test method evaluates the residual activity according to the death of mice injected with a test vaccine after histamine challenge and the test result, therefore, depends on the sensitivity of mice. A highly sensitive test method based on change in rectal temperature of mice has been used in Japan for many years but has limited feasibility in other countries. We examined the possibility of a test method using dermal temperature measured by infrared thermometer to reduce animal suffering instead of rectal temperature. The dermal temperature method was shown to be as sensitive as the rectal temperature method. Furthermore, the dermal as well as rectal temperature methods can evaluate the activity of a test vaccine in relative to a reference preparation so as to allow direct comparison of the test results among different laboratories. The activity by means of the dermal temperature method was also found to be well consistent with that by the rectal temperature method.     

国产甲型肝炎灭活疫苗用于儿童免疫效果研究

为了探讨国产甲型肝炎灭活疫苗在儿童中应用的免疫效果,选择2～15岁抗-HAV阴性健康易感儿童91名作为接种对象,采用0、6程序接种国产甲型肝炎灭活疫苗250U／剂,观察免疫后的局部反应和全身反应,并于全程免疫后一个月检测抗-HAV阳转率和抗体GMT。结果91例观察对象在初免和加强免疫后均未见即时副反应,只在8～72小时内出现轻微的一过性局部和全身反应。全程免疫后一个月抗-HAV阳转率为100％。抗体GMT为14 407mIU／ml。国产甲肝灭活疫苗在儿童中应用具有良好的安全性和免疫原性,采用0、6个月程序可获得高滴度抗体。     

Specificity and detection limit of a dermal temperature histamine sensitization test for absence of residual pertussis toxin in vaccines

Currently, an assay based on fatal sensitization of mice to histamine challenge is widely used for testing absence of residual pertussis toxin in acellular pertussis containing vaccines. For replacement of this lethal end-point assay, an alternative method based on body temperature measurement in mice has been presented, and in this study the specificity and detection limit of a dermal temperature-based assay were assessed. Test preparations containing pertussis toxin were prepared in aluminum-adjuvanted pertussis toxoid vaccine and injected intraperitoneally in histamine sensitive mice. Later the mice were challenged with histamine and the pertussis toxin-induced decrease in dermal temperature recorded. By comparison of mice treated with pertussis toxoid vaccine spiked with pertussis toxin with mice treated with pertussis toxoid vaccine alone, the assay gave a response that specifically could detect presence of pertussis toxin. The acellular pertussis containing vaccine did not interfere with the pertussis toxin-induced temperature response recorded. In tests for presence of pertussis toxin in the pertussis vaccine preparation, the detection limit of the assay was estimated to approximately 5 ng pertussis toxin per human dose of pertussis toxoid. The dermal temperature-based assay was found to be a valid method to be applied in routine quality control of vaccines.     

Expansion of the in vitro assay for Leptospira potency testing to other serovars: Case study with Leptospira Hardjo

Evaluation of leptospiral vaccines for potency against Leptospira interrogans serovars Pomona, Icterohaemorrhagiae, Canicola, and Grippotyphosa is accomplished using the hamster potency test method described in 9 CFR 113.101-104. Applicability of this method to evaluation of bacterins developed for immunization against infection with L. interrogans serovar Hardjo or Leptospira borgpetersenii serovar Hardjo is complicated by several issues. Information from research on target host animal efficacy studies and evaluation of the immune response elicited using effective whole-cell bacterin formulations have revealed problems in relating these studies to either hamster-based or other potency testing methods. Future work on serovar Hardjo vaccines employing recombinant proteins will require preliminary testing methods in models other than the host animal. These models may also prove applicable to evaluation of potency for protein-based vaccines. Both an acute lethal infection model and a chronic infection model have been developed using two different strains of serovar Hardjo and will be described.     

丙型肝炎病毒疫苗的研究进展

丙型肝炎是由丙型肝炎病毒(HCV)感染引起的重要传染病,有效的治疗性和预防性疫苗目前均尚未研制成功.HCV的高变异性及多种免疫逃避途径是疫苗研制的主要障碍.目前,已有多种类型的HCV候选疫苗进入临床试验或临床前期试验阶段,但其有效性、安全性等还不能令人满意.随着对HCV相关免疫应答机制的深入了解,有望研制出相应的治疗性和预防性疫苗.     

基于纳米颗粒的赭曲霉毒素A高灵敏酶联免疫检测方法的建立及应用

赭曲霉毒素(ochratoxins)主要是由青霉菌Penicillium和曲霉菌Aspergillus产生的有毒次级代谢产物,常见于发霉或发酵的农产品中,其中赭曲霉毒素A(ochratoxin A,OTA)毒性最强且最为普遍。OTA是粮食作物和饲料的重要污染物,在加工、储存或运输过程中均可产生,具有肾毒性和免疫毒性,可通过蓄积作用发挥毒性效应,对人类和动物健康造成严重威胁。本研究通过将OTA单克隆抗体包被于纳米磁珠(magnetic nanoparticles,MNPs)表面,获得具有免疫活性的磁珠抗体复合物(MNPs-Anti OTA),并制备生物素标记的偶联抗原OTA-BSA-Bio,后续采用链酶亲和素标记的纳米金颗粒(Strep-HRP-AuNPs)催化底物进行信号检测,最终建立了OTA高灵敏检测方法(MNPs-bs-AuNPs-ELISA)。在最优条件下,经计算该方法检测下限(IC10)为0.01ng/mL,检测区间(IC20-IC80)为0.02-0.73ng/mL,半数抑制率(IC50)为0.13ng/mL。与OTA类似物OTB、OTC交叉反应性为4.3%和8.1%,对其他常见真菌毒素AFB1、ZEN、FB1、DON、CIT和PAT均无交叉反应。玉米、面粉和大豆样本中的加标回收率可达85.6%-115.7%,对天然样本中OTA含量的检测结果表明,该方法与LC-MS/MS相关性良好。本研究建立的MNPs-bs-AuNPs-ELISA可满足谷物及饲料样本中OTA的快速、高灵敏度定量检测,成本较低,具有很好的应用前景。     

The role of the Badger (Meles meles) in rabies epizootiology and the implications for Great Britain

The occurrence of a wildlife rabies epizootic in Britain remains a very unlikely event, but it is important to examine all the possible consequences of such an event. Here, I examine the possible role of the European Badger (Meles meles) in such an epizootic. The population density of Badgers in Britain is much higher than that in Europe, and appears to have increased substantially over the last decade or so. The population parameters and epizootiology of rabies in the Badger are reviewed in comparison with the Fox (Vulpes vulpes) and other species. Mustelids appear to be very susceptible to rabies, with the smaller mustelids becoming aggressive, although Badgers do not appear to show heightened aggression when infected. Badger populations on the continent become severely reduced when rabies arrives in the area, and circumstantial evidence strongly suggests that Badgers can easily transmit the virus. Preliminary models support the idea that the Badger could be a very significant secondary host, especially in the initial rabies outbreak. The population recovery rate of the Badger suggests that it is unlikely to become a primary host, although short‐term epizootics in the Badger population are likely. The potential for controlling rabies in the Badger is also examined.     

Using phylogeographic approaches to analyse the dispersal history,velocity and direction of viral lineages — Application to rabies virus spread in Iran

Recent years have seen the extensive use of phylogeographic approaches to unveil the dispersal history of virus epidemics. Spatially explicit reconstructions of viral spread represent valuable sources of lineage movement data that can be exploited to investigate the impact of underlying environmental layers on the dispersal of pathogens. Here, we performed phylogeographic inference and applied different post hoc approaches to analyse a new and comprehensive data set of viral genomes to elucidate the dispersal history and dynamics of rabies virus (RABV) in Iran, which have remained largely unknown. We first analysed the association between environmental factors and variations in dispersal velocity among lineages. Second, we present, test and apply a new approach to study the link between environmental conditions and the dispersal direction of lineages. The statistical performance (power of detection, false‐positive rate) of this new method was assessed using simulations. We performed phylogeographic analyses of RABV genomes, allowing us to describe the large diversity of RABV in Iran and to confirm the cocirculation of several clades in the country. Overall, we estimate a relatively high lineage dispersal velocity, similar to previous estimates for dog rabies virus spread in northern Africa. Finally, we highlight a tendency for RABV lineages to spread in accessible areas associated with high human population density. Our analytical workflow illustrates how phylogeographic approaches can be used to investigate the impact of environmental factors on several aspects of viral dispersal dynamics.     

直接免疫荧光法检测狂犬病毒滴度的可行性研究

实验研究中建立用于定量检测狂犬病毒滴度的直接免疫荧光法,并将检测结果与传统小鼠脑内滴定法进行了比较,对照两种试验方法的灵敏度、特异性和重复性。结果显示,两种检测方法特异性基本一致;相同样品经多次检测的病毒滴度相近,重复性好、灵敏度高。直接免疫荧光法具有特异、灵敏、快速、操作简单、无需使用动物等优点,可应用于狂犬病毒滴度的定量检测。     

A comparison of an enzyme-linked immunosorbent assay and counter current electrophoresis for the detection of bovine serum albumin in virus vaccines

A monoclonal antibody directed against bovine serum albumin (BSA) has been developed and used in an enzyme-linked immunosorbent assay (ELISA) system for the detection of BSA in virus vaccines. The results correlated well with those obtained with a counter current electrophoresis system which has been employed routinely for this purpose. The ELISA was slightly more sensitive and more readily applicable to the screening of large numbers of samples but could not be used in the presence of certain stabilizers.     

Results of an international transferability study of the BINACLE (binding and cleavage) assay for in vitro detection of tetanus toxicity

Tetanus vaccines contain detoxified tetanus neurotoxin. In order to check for residual toxicity, the detoxified material (toxoid) has to be tested in guinea pigs. These tests are time-consuming and raise animal welfare issues. In line with the “3R” principles of replacing, reducing and refining animal tests, the “binding and cleavage” (BINACLE) assay for detection of active tetanus neurotoxin has been developed as a potential alternative to toxicity testing in animals. This in vitro test system can discriminate well between toxic and detoxified toxin molecules based on their receptor-binding and proteolytic characteristics.Here we describe an international study to assess the transferability of the BINACLE assay. We show that all participating laboratories were able to successfully perform the assay. Generally, assay variability was within an acceptable range. A toxin concentration-dependent increase of assay signals was observed in all tests. Furthermore, participants were able to detect low tetanus neurotoxin concentrations close to the estimated in vivo detection limit.In conclusion, the data from this study indicate that the methodology of the BINACLE assay seems to be robust, reproducible and easily transferable between laboratories. These findings substantiate our notion that the method can be suitable for the routine testing of tetanus toxoids.     

Development of a genotype‐by‐sequencing immunogenetic assay as exemplified by screening for variation in red fox with and without endemic rabies exposure

Pathogens are recognized as major drivers of local adaptation in wildlife systems. By determining which gene variants are favored in local interactions among populations with and without disease, spatially explicit adaptive responses to pathogens can be elucidated. Much of our current understanding of host responses to disease comes from a small number of genes associated with an immune response. High‐throughput sequencing (HTS) technologies, such as genotype‐by‐sequencing (GBS), facilitate expanded explorations of genomic variation among populations. Hybridization‐based GBS techniques can be leveraged in systems not well characterized for specific variants associated with disease outcome to “capture” specific genes and regulatory regions known to influence expression and disease outcome. We developed a multiplexed, sequence capture assay for red foxes to simultaneously assess ~300‐kbp of genomic sequence from 116 adaptive, intrinsic, and innate immunity genes of predicted adaptive significance and their putative upstream regulatory regions along with 23 neutral microsatellite regions to control for demographic effects. The assay was applied to 45 fox DNA samples from Alaska, where three arctic rabies strains are geographically restricted and endemic to coastal tundra regions, yet absent from the boreal interior. The assay provided 61.5% on‐target enrichment with relatively even sequence coverage across all targeted loci and samples (mean = 50×), which allowed us to elucidate genetic variation across introns, exons, and potential regulatory regions (4,819 SNPs). Challenges remained in accurately describing microsatellite variation using this technique; however, longer‐read HTS technologies should overcome these issues. We used these data to conduct preliminary analyses and detected genetic structure in a subset of red fox immune‐related genes between regions with and without endemic arctic rabies. This assay provides a template to assess immunogenetic variation in wildlife disease systems.     

A comparison of complete genome sequences of the attenuated RC-HL strain of rabies virus used for production of animal vaccine in Japan, and the parental Nishigahara strain

In order to establish the molecular basis of the pathogenicity of the attenuated RC-HL strain of rabies virus used for the production of animal vaccine in Japan, the complete genome sequence of this strain was determined and compared with that of the parental Nishigahara strain which is virulent for adult mice. The viral genome of both strains was composed of 11,926 nucleotides. The nucleotide sequences of the two genomes showed a high homology of 98.9%. The homology of the G gene was lower than those of N, P, M and L genes at both nucleotide and deduced amino acid levels, and the percentage of radical amino acid substitutions on the G protein was the highest among the five proteins. These findings raise the possibility that the structure of the G protein is the most variable among the five proteins of the two strains. Furthermore, we found two clusters of amino acid substitutions on the G and L proteins. The relevance of these clusters to the difference in the pathogenicity between the two strains is discussed.     

Development of reverse transcription loop‐mediated isothermal amplification assay for sensitive and rapid detection of Hosta virus X

The one‐step real‐time turbidity loop‐mediated isothermal amplification assay (RealAmp) was developed to detect Hosta virus X (HVX), the most devastating threat to hosta industry. The reaction was performed in a single tube at 63°C for 15 min, and real‐time turbidimetry was used to monitor the amplification results. Specificity and sensitivity analyses demonstrated that this RealAmp method was sensitive as real‐time TaqMan RT‐PCR and about 100‐fold higher than conventional RT‐PCR with no cross‐reaction with other viral pathogens. Field samples detection showed that HVX could be identified effectively with this method. Overall, this RealAmp assay for HVX detection was simple, specific, sensitive, convenient and time‐saving and could assist in the quarantine measures for prevention and control of the disease caused by HVX.