The effect of strain differences on the assay of rabies virus glycoprotein by single radial immunodiffusion

Antigenic differences between rabies virus strains used for vaccine manufacture can be demonstrated using monoclonal antibodies. We have shown that these differences are sufficiently large to affect the potency values of vaccines measured in single radial immunodiffusion (SRD) assays if the reference and test vaccines are antigenically heterologous. The production of reagents for use in SRD assays for each strain of rabies virus should be considered.     

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.     

A collaborative study on the use of single radial immunodiffusion for the assay of rabies virus glycoprotein

The single radial immunodiffusion (SRD) technique has been applied to the assay of the glycoprotein content of rabies vaccines produced in cell cultures. Fourteen laboratories in seven countries participated in a collaborative study to evaluate the reproducibility of the SRD technique; some laboratories also examined vaccines in the mouse protection (NIH) test and by enzyme immunoassay. Good agreement was found between potency estimates using the SRD technique: the geometric coefficients of variation for combined potency estimates of all laboratories were about 10%. SRD assays appear to have a role for the in vitro assay of antigen content of vaccine and could complement results obtained in in vivo assays which are subject to wide variability.     

Rabies vaccine standardization: International collaborative study for the characterization of the fifth international standard for rabies vaccine

A collaborative study was carried out to establish a replacement for the International Standard for Rabies Vaccine, the stocks of which are exhausted. Three rabies vaccines for human use derived from different rabies virus strains and prepared on different cell culture substrates were compared with the International Standard for Rabies Vaccine using in vivo and in vitro assay methods in a collaborative study involving 14 participants. The proposed fifth International Standard (PISRAV) which was derived from the same virus strain as the present international standard preparation, the Pitman Moore (PM) strain, was found to be approximately twice as potent relative to the International Standard in immunogenicity assays as in antigenicity assays. On the other hand another vaccine, derived from the LEP strain, was considerably more potent in antigenicity assays than in immunogenicity assays. The glycoprotein of the proposed replacement standard measured in antigenicity assays appeared to be stable at +37 degrees C for 245 days, whereas the immunogenicity of the proposed replacement vaccine was sensitive to this heat treatment and the vaccine lost 66% of its immunogenic potency. The results of this study indicate that the NIH protection test should continue to form the primary basis for potency assay of rabies vaccine as glycoprotein content does not appear to correlate with immunogenic potency for different types of vaccine. The vaccine coded PISRAV has been established as the fifth International Standard for Rabies Vaccine and a potency of 16 International Units of Rabies Vaccine (based on the immunogenicity assays) assigned to the contents of each ampoule. Each ampoule has also been assigned a unitage of 10 IU of PM Rabies Virus Glycoprotein and 135 IU of PM Rabies Virus Ribonucleoprotein.     

Use of a monoclonal antibody for quantitation of rabies vaccine glycoprotein by enzyme immunoassay

The use of a monoclonal antibody specific for the envelope glycoprotein of rabies virus has been used in a direct enzyme immunoassay to quantify the glycoprotein content of rabies vaccines produced in two kinds of cell culture. The results of this direct enzyme immunoassay are well correlated with the in vivo NIH potency test. This test may be a useful tool to rapidly and accurately control the antigenic value of a vaccine. It could also be used for the "in process' control of vaccine production before deciding whether a vaccine batch may reasonably be subjected to the NIH potency test.     

A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture.

Rabies is an endemic, fatal zoonotic disease in the developing countries. Prevention and post-exposure therapy require safe and efficacious vaccines. The vaccine potency depends on the amount of immunogenic rabies viral glycoprotein antigen in the vaccine preparation. In order to estimate the rabies viral glycoprotein antigen, a specific monoclonal antibody was developed and used in an immuno-capture ELISA (IC-ELISA). The monoclonal antibody binds a conformational epitope on the natively folded rabies viral glycoprotein as indicated by specific, membrane fluorescence on unfixed, rabies virus infected murine neuroblastoma (MNA) cells and glycoprotein gene encoding plasmid transfected COS cells. In addition, the monoclonal antibody competes with and blocks a glycoprotein antigen site III binding monoclonal antibody (mAb-D1, Institut Pasteur, Paris, France). The monoclonal antibody was used in an IC-ELISA using an in-house standard to quantify the rabies viral glycoprotein antigen in 12 vaccine preparations with potency values ranging from 4 to 18 IU. The results indicated a good correlation with the NIH mouse potency assay (r=0.83). The immuno-capture ELISA described in this study can be used to quantify the immunogenic rabies viral glycoprotein antigen in the inactivated rabies viral antigen preparation in a simple and rapid format, which enables better vaccine formulation.     

双抗体夹心ELISA与NIH法在狂犬病疫苗抗原检测中的比较

通过用单克隆抗体制备双抗体夹心ELISA,快速检测狂犬病疫苗中狂犬病毒糖蛋白(G)的含量,将狂犬病疫 苗的检定时间由28天缩短至2个工作日,以此缩短疫苗库存待检时间,提高疫苗的生产和销售效率,并最终替代 NIH动物法。将待检的狂犬病疫苗样品在同一性别12～14g昆明鼠体内作效力检定试验(NIH),同时用双抗体夹 心ELISA检测狂犬病疫苗中狂犬病毒G蛋白含量,用Microsoft Office Excel做出标准品和各样品疫苗的线性关 系图并计算出疫苗效力值E-NIH。结果用双抗体夹心ELISA所得的E-NIH与对应的小鼠效力试验所得结果M- NIH之间呈正相关性;同一批狂犬病疫苗分次测得E-NIH值在2．41～5．85之间,而相应的M-NIH值在5．11～ 10．19之间。从而得出E-NIH与相应的M-NIH之间存在明显的线性关系;与NIH法比较,ELISA具有重复性好、 成本低、快速等优点;用双抗体夹心ELISA替代小鼠效力试验是可能并可行的。     

狂犬病毒糖蛋白DNA疫苗的研制及其免疫效果的观察

构建了含有狂犬病毒（RV）CVS株糖蛋白（GP）基因的重组质粒pCMVCVSRG,将其转染至鼠NIH3T3细胞中,用间接免疫荧光法和APAAP法均证实RVGP能在真核细胞中表达。分别将合RV不同毒株的GP基因的质粒（DNA疫苗）及空白载体质粒（对照组）免疫小鼠,仅DNA疫苗免疫的小鼠产生了中和抗体。以RV攻击后,DNA疫苗免疫组小鼠的存活率与对照组相比,差异有极显著性意义（P＜0．01）;不同的启动子（CMV或SV40）与不同GP基因（来源于CVS株或ERA株）对DNA疫苗的免疫效果无明显影响。在注射120d后．用PCR方法仍可检测出RVGP基因。结果表明：狂犬病DNA疫苗能够诱生低水平的中和抗体和记忆性B淋巴细胞,并能保护小鼠抵抗RV的攻击。该疫苗能在体内稳定存在。狂犬病DNA疫苗的研制为狂犬病免疫开辟了一条新途径,并可为防治其他疾病的DNA疫苗的研制奠定基础。     

Appraisal of rabies vaccine potency by determination of in vitro, specific interleukin-2 production

The potency of different rabies vaccines was measured via cell mediated immunity (CMI) assessed by the production of interleukin-2 (IL-2) by CD4+CD8- lymphocytes. IL-2 production by splenocytes from mice immunized with various vaccines was measured following in vitro stimulation with antigens from different rabies and rabies-related strains. IL-2 production was specific, reproducible and correlated with the vaccine protective activity as determined by the pre-exposure NIH test. Our results suggest that measurement of IL-2 production could be used for the appraisal of rabies vaccine potency.     

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.     

Effect of the contents and form of rabies glycoprotein on the potency of rabies vaccination in cattle

One of the methods used for controlling cattle rabies in Brazil consists of vaccination. Sometimes, however, rabies occurs in cattle supposedly protected. Since rabies vaccine batches are officially controlled by tests performed on laboratory animals, it is questionable whether the minimal mandatory requirements really correspond to immunogenicity in the target species. We have analyzed the association among potencies of rabies vaccines tested by the NIH test, the contents and form (free-soluble or virus-attached) of rabies glycoprotein (G) in the vaccine batches, and the virus-neutralizing antibodies (VNA) titers elicited in cattle. No correlation was found between G contents in the vaccine batches and the NIH values, whatever the presentation of G. There was no correlation either between NIH values and VNA titers elicited in cattle. There was, however, a positive correlation (r = 0.8681; p = 0.0001) between the amounts of virion-attached G present in the vaccine batches and VNA elicited in cattle. This was not observed when the same analysis was performed with total-glycoprotein or free-soluble glycoprotein. The study demonstrated that NIH values can not predict the effect of the immunogen in cattle. On the other hand, the quantification of virus-attached rabies glycoprotein has a strong correlation with VNA elicited in cattle.     

Creation of DNA vaccine vector based on codon-optimized gene of rabies virus glycoprotein (G protein) with consensus amino acid sequence

An optimized design of the rabies virus glycoprotein (G protein) for use within DNA vaccines has been suggested. The design represents a territorially adapted antigen constructed taking into account glycoprotein amino acid sequences of the rabies viruses registered in the Russian Federation and the vaccine Vnukovo-32 strain. Based on the created consensus amino acid sequence, the nucleotide codon-optimized sequence of this modified glycoprotein was obtained and cloned into the pVAX1 plasmid (a vector of the last generation used in the creation of DNA vaccines). A twofold increase in this gene expression compared to the expression of the Vnukovo-32 strain viral glycoprotein gene in a similar vector was registered in the transfected cell culture. It has been demonstrated that the accumulation of modified G protein exceeds the number of the control protein synthesized using the plasmid with the Vnukovo-32 strain viral glycoprotein gene by 20 times. Thus, the obtained modified rabies virus glycoprotein can be considered to be a promising DNA vaccine antigen.     

The influence of homologous vs. heterologous challenge virus strains on the serological test results of rabies virus neutralizing assays

The effect that the relatedness of the viral seed strain used to produce rabies vaccines has to the strain of challenge virus used to measure rabies virus neutralizing antibodies after vaccination was evaluated. Serum samples from 173 subjects vaccinated with either purified Vero cell rabies vaccine (PVRV), produced from the Pittman Moore (PM) seed strain of rabies virus, or purified chick embryo cell rabies vaccine (PCECV), produced from the Flury low egg passage (Flury-LEP) seed strain of rabies virus, were tested in parallel assays by RFFIT using a homologous and a heterologous testing system. In the homologous system, CVS-11 was used as the challenge virus in the assay to evaluate the humoral immune response in subjects vaccinated with PVRV and Flury-LEP was used for subjects vaccinated with PCECV. In the heterologous system, CVS-11 was used as the challenge virus in the assay to evaluate subjects vaccinated with PCECV and Flury-LEP was used for subjects vaccinated with PVRV. Although the difference in G protein homology between the CVS-11 and Flury-LEP rabies virus strains has been reported to be only 5.8%, the use of a homologous testing system resulted in approximately 30% higher titers for nearly two-thirds of the samples from both vaccine groups compared to a heterologous testing system. The evaluation of equivalence of the immune response after vaccination with the two different vaccines was dependent upon the type of testing system, homologous or heterologous, used to evaluate the level of rabies virus neutralizing antibodies. Equivalence between the vaccines was achieved when a homologous testing system was used but not when a heterologous testing system was used. The results of this study indicate that the strain of virus used in the biological assays to measure the level of rabies virus neutralizing antibodies after vaccination could profoundly influence the evaluation of rabies vaccines.     

Inactivated rabies vaccine control and release: use of an ELISA method.

Quality control of human rabies vaccines performed by National Control Laboratories (NCLs) prior to marketing vaccines batches requires in vivo and in vitro potency assays as requested by the relevant European Pharmacopoeia monographs, OMCLs guidelines and WHO technical recommendations.The aim of the present study was to check the suitability of an enzyme-linked immunosorbent assay (ELISA) using a virus neutralizing monoclonal antibody, directed to the rabies virus glycoprotein, to monitor the consistency of the lot to lot rabies vaccines production. Furthermore, this work was implemented to establish in house specifications for the glycoprotein content.     

The influence of the type of immunosorbent on rabies antibody EIA; advantages of purified glycoprotein over whole virus

Two types of in vitro assay (enzyme immunoassay and sero-neutralization test) for the titration of rabies antibodies were used to assay sera from mice and humans immunized with cell culture vaccines or neural tissue vaccines. Enzyme immunoassays (EIA) were performed in plates sensitized with whole virus, purified glycoprotein or purified nucleocapsid. Neutralizing antibody titres were determined by the rapid fluorescent focus inhibition test (REFIT) and by an in vitro seroneutralization test including a rapid enzyme immunotitration of intracellular antigens (REITICA). The results obtained with sera of immunized mice and humans showed that (1) cell culture vaccines mainly induced the synthesis of antiglycoprotein neutralizing antibodies; and (2) neural tissue vaccines induced a high synthesis of antinucleocapsid non-neutralizing antibodies and a more or less important synthesis of antiglycoprotein antibodies depending on the origin of the tissue used for their preparation. Consequently, it was emphasized that when using EIA, the antibody titration must be run in glycoprotein-coated plates rather than in whole virus-coated plates to appreciate correctly the immunizing potency of a rabies vaccine, especially neural tissue vaccine.     

In vitro rabies vaccine potency appraisal by ELISA: advantages of the immunocapture method with a neutralizing anti-glycoprotein monoclonal antibody

The replacement of the in vivo potency test (NIH test) for rabies vaccine evaluation by in vitro methods is at present discussed in many reports and also by WHO expert working groups. For this purpose, in vitro glycoprotein titration has been proposed. Among the different glycoprotein assays, we have studied two ELISA methods (immunocapture and direct plate coating with the antigen to be tested) using neutralizing mono- and polyclonal antibodies. In our view, the immunocapture method based on the use of a neutralizing monoclonal anti-glycoprotein antibody seems to be a convenient tool for the determination of the in vitro potency of rabies vaccine and of the products corresponding to the different steps of their production process.     

In vitro rabies vaccine potency appraisal by ELISA: Advantages of the immunocapture method with a neutralizing anti-glycoprotein monoclonal antibody

The replacement of the in vivo potency test (NIH test) for rabies vaccine evaluation by in vitro methods is at present discussed in many reports and also by WHO expert working groups. For this purpose, in vitro glycoprotein titration has been proposed. Among the different glycoprotein assays, we have studied two ELISA methods (immunocapture and direct plate coating with the antigen to be tested) using neutralizing mono- and polyclonal antibodies. In our view, the immunocapture method based on the use of a neutralizing monoclonal anti-glycoprotein antibody seems to be a convenient tool for the determination of the in vitro potency of rabies vaccine and of the products corresponding to the different steps of their production process.     

两个NIH小鼠种群对不同种类的乙肝疫苗免疫应答效应的比较

目的比较两种不同的NIH小鼠种群基因组DNA中H-2q单倍型的百分比,对重组基因乙型肝炎疫苗（酿酒酵母）、重组基因乙型肝炎疫苗（CHO）、重组基因乙型肝炎疫苗（汉逊酵母）和治疗性乙型肝炎疫苗（乙克）免疫应答的影响。方法检测乙肝疫苗免疫效力用酶标法,测定乙肝疫苗的ED50值,再通过微量细胞毒法和PCR方法检测小鼠的H-2单倍型,计算不同品系小鼠H-2q的百分数,得出乙肝疫苗与不同动物种群的相关性。结果经验证,经1号NIH小鼠检测结果证实,重组基因乙型肝炎免疫效力依次为CHO〉汉逊〉酿酒,疗性乙型肝炎疫苗（乙克）效力检测合格,但经2号NIH小鼠检测结果显示除CHO疫苗合格外,其他三种疫苗检测结果均为不合格。基因检测结果为,1号种群NIH鼠含有H-2q型基因的百分比是96%,而2号种群NIH小鼠含有H-2q型基因基因百分比为30%,因此不同的q型基因百分比导致了不同的检测结果。结论根据本研究结果表明虽然NIH鼠的H-2单倍型中有q型基因,但不同来源的小鼠种群之间会存在差异,经不同的环境饲养后,小鼠的基因会发生不同的变化,因此在进行生物制品检定时,要注意不同品系小鼠及同一品系小鼠不同种群之间的差异对试验本身的影响。建立一种＂动物免疫＂的概念。     

A modification of the single radial immunodiffusion potency test (SRD) for rabies vaccines

In 1983 a WHO collaborative study showed that the single radial immunodiffusion test provided a reliable system for the assay of the glycoprotein content of inactivated rabies vaccines. We have modified the method used in the collaborative study by using round plastic plates with wells arranged in a circle. Potency estimates were made by the multiple slope ratio method and not by single slope estimation procedures adjusted for a common intercept. We carried out a statistical evaluation of the accuracy and reliability of the precipitation area calculation. We confirmed that for the purpose of the potency estimation the area calculations were sufficiently precise in most instances. By analysing the sources of variation for round and for glass plates and by testing the variability of these methods we found that the round plates were as good as the glass plates. The advantage of the round plates was mainly in the ease of their handling: the agarose solution was poured in the plates without the need for a separate mould; an integral moist chamber with these plates was readily available; the precipitation zones were measured easily using a viewer which gave fourfold enlargements and the method required less antiglycoprotein.     

Spatio-temporal Use of Oral Rabies Vaccines in Fox Rabies Elimination Programmes in Europe

In Europe, the elimination of wildlife rabies using oral rabies vaccination [ORV] of foxes for more than 30 years has been a success story. Since a comprehensive review on the scope of the different oral rabies vaccine baits distributed across Europe has not been available yet, we evaluated the use of different vaccine baits over the entire period of ORV [1978–2014]. Our findings provide valuable insights into the complexity of ORV programs in terms of vaccine related issues. More than 10 oral vaccines against rabies were used over the past four decades. Depending on many factors, the extent to which oral rabies virus vaccines were used varied considerably resulting in huge differences in the number of vaccine doses disseminated in ORV campaigns as well as in large spatial and temporal overlaps. Although vaccine virus strains derived from the SAD rabies virus isolate were the most widely used, the success of ORV campaigns in Europe cannot be assigned to a single oral rabies virus vaccine alone. Rather, the successful elimination of fox rabies is the result of an interaction of different key components of ORV campaigns, i.e. vaccine strain, vaccine bait and strategy of distribution.