A collaborative study to establish an International Standard Rabies immunoglobulin of human origin

Because the supply of the International Standard for Anti-rabies Serum was very low, the WHO initiated a search for a replacement product. The US Food and Drug Administration agreed to undertake a collaborative study using a human rabies immunoglobulin previously purchased for use as a US standard. The potency of this product was determined, in International Units (IU) per millilitre using the rapid fluorescent focus inhibition test for measuring rabies antibody. The mean potency value was found to be 59 IU per ampoule. In June 1984 this preparation was accepted by WHO as the International Standard for Rabies Immunoglobulin.     

Evaluation of Fourth International Standard for Whole Cell Pertussis Vaccine

Whole cell pertussis vaccine is still widely used in many countries. An International Standard is needed for its potency control. The Third International Standard for Pertussis Vaccine was prepared about 40 years ago and its replacement was recommended by the Expert Committee for Biological Standardisation (ECBS) of the WHO. Material in ampoules coded 94/532 was prepared as a candidate replacement and has been evaluated in international collaborative studies which consisted of two parts. The first part, to assess the suitability of the candidate standard by comparing it with the Second International Standard for Pertussis Vaccine (IS2) involved 14 laboratories in 11 countries. The second part to compare the candidate standard with the Third International Standard for Pertussis Vaccine (IS3) involved 16 laboratories in 14 countries. Since 1995 various other studies have included the international standards and the results of these are also considered in assessing likely continuity of the IU for potency of whole cell pertussis vaccine. The preparation in ampoules coded 94/532 was adopted by the WHO ECBS in October 2006 as the 4th International Standard for whole cell pertussis vaccine and assigned an activity of 40 IU per ampoule on the basis of the studies reported here.     

A collaborative assay of the proposed third British Reference Preparation for Pertussis Vaccine and of the relative potencies of the second International Standard and the second British Reference Preparation for Pertussis Vaccine

A collaborative assay has been carried out to estimate the mouse protective potency of a freeze-dried preparation of Bordetella pertussis (88/522) intended to serve as the third British Reference Preparation for Pertussis Vaccine (third BRP). The opportunity was also taken of reassessing the relationship between the second International Standard for Pertussis Vaccine and the second British Reference Preparation for Pertussis Vaccine (second BRP). Workers in nine laboratories took part in the study and together completed 19 assays which were considered to be statistically valid. Based on the results of the study it is proposed that ampouled preparation code number 88/522 be established as the third BRP with an assigned potency of 50 IU per ampoule. The evidence of this study also suggests that the relationship between the second International Standard for Pertussis Vaccine and the second BRP has not changed significantly since they were originally established.     

Hepatitis A Immunoglobulin: An International Collaborative Study to Establish the Second International Standard

A collaborative study was carried out to assess the suitability of a candidate replacement material for the International Standard for hepatitis A immunoglobulin, which was found to be reactive for HCV RNA, and to calibrate it in International Units. The candidate standard, coded 97/646, was derived from a bulk of 16% immunoglobulin supplied by the Central Laboratory of the Netherlands Red Cross, Amsterdam, and diluted 1 in 2 in H2O resulting in a final immunoglobulin concentration of 8%. Sixteen laboratories from 11 countries participated in the study and contributed data from 64 assays performed using six commercial assay kits and four in-house methods. All assays were analysed as parallel line bioassays comparing assay response with log concentration. The overall mean potency of the candidate replacement immunoglobulin standard, 97/646, relative to the International Standard for hepatitis A immunoglobulin, was 98.6 IU/ml. A freeze-dried serum preparation, 97/648, was also calibrated in this study and had a potency of 22.64 IU/ml. The Second International Standard for hepatitis A immunoglobulin, human, was established by the World Health Organisation Expert Committee on Biological Standardisation in 1998 with a potency of 49 IU per ampoule when reconstituted in 0.5 ml.     

Collaborative study for the calibration of a replacement International Standard for Tetanus Toxoid Adsorbed

We present the results of a collaborative study for the establishment of a replacement International Standard (IS) for Tetanus Toxoid Adsorbed. Two candidate preparations were included in the study, one of which was established as the 4th IS for Tetanus Toxoid Adsorbed at the WHO Expert Committee on Biological Standardization meeting in October 2010. This preparation was found to have a unitage of 490 IU/ampoule, based on calibration in guinea pig challenge assays. Results from mouse challenge assays suggest that the relative performance of two candidate preparations may differ significantly between guinea pigs and mice. The authors note that the number of laboratories that performed guinea pig challenge assays, which are used to calibrate and assign IU, is much lower than in previous collaborative studies and this may have implications for calibration of replacement standards in the future. The issue of assigning separate units to the IS for guinea pig and mouse assays is discussed. The study also assessed performance of the replacement standard in serological assays which are used as alternative procedures to challenge assays for tetanus potency testing. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays.     

Third International Standard for pertussis vaccine: international confirmation study of activity of British Standard for pertussis vaccine, coded 66/303.

The first British Standard (BS) (Code 66/303) for pertussis whole cell vaccine was prepared from the same suspension of Bordetella pertussis as the now exhausted Second International Standard for Pertussis Vaccine (2ndIS). The BS and the 2ndIS were compared and calibrated in a previous international study. This report describes a small international study, which included the BS, the 1stIS and the 2ndIS so that the present relationship of these preparations to one another could be assessed. The results of this study show that the relationship of the BS to the 1stIS is consistent with its established unitage of 46 IU per ampoule. The results further show that the potency of the BS is broadly consistent with that of the 2ndIS and that the BS has not lost activity relative to the 2ndIS (from which it was previously found to be indistinguishable). Based on its original calibration and supported by the results of this present study, the BS has been established as the Third International Standard for Pertussis Vaccine, with an assigned unitage of 46 IU per ampoule.     

Collaborative study for the calibration of a replacement international standard for diphtheria toxoid adsorbed

We present the results of a collaborative study for the characterization of a preparation of diphtheria toxoid adsorbed, and its calibration in terms of the 3rd International Standard (IS) for Diphtheria Toxoid Adsorbed. Calibration was performed using established World Health Organization (WHO) and European Pharmacopoeia (Ph. Eur.) protection models. Two candidate toxoid preparations were included in the study, one of which was adopted as a replacement Ph. Eur. Biological Reference Preparation (BRP, batch 4) in February 2009. The second candidate preparation was found to have a unitage of 213 IU/ampoule based on the calibration by in vivo bioassay in 19 laboratories in 16 countries, and was established as the 4th IS for Diphtheria Toxoid Adsorbed by the WHO Expert Committee on Biological Standardization (ECBS) in October 2009.The study also assessed performance of the replacement standard in mouse and guinea pig serological assays which are used as alternative procedures for diphtheria potency testing. Participants tested both candidate preparations and potency was expressed in relative terms only. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays and that the Vero cell assay may be suitable for calibration of future replacement standards.     

Collaborative study to assess the suitability of a candidate International Standard for yellow fever vaccine

Yellow fever vaccines are routinely assayed by plaque assay. However, the results of these assays are then converted into mouse LD(50) using correlations/conversion factors which, in many cases, were established many years ago. The minimum required potency in WHO Recommendations is 10(3) LD(50)/dose. Thirteen participants from 8 countries participated in a collaborative study whose aim was to assess the suitability of two candidate preparations to serve as an International Standard for yellow fever vaccine. In addition, the study investigated the relationship between the mouse LD(50) test and plaque forming units with a view to updating the WHO recommendations. Plaque assays were more reproducible than mouse assays, as expected. Differences in sensitivities of plaque assays were observed between laboratories but these differences appear to be consistent within a laboratory for all samples and the expression of potency relative to the candidate standard vaccine improved the reproducibility of assays between laboratories. However, the use of potencies had little effect on the between laboratory variability in mouse LD(50) assays. There appears to be a consistent relationship between overall mean LD(50) and plaques titre for all study preparations other than sample E. The slope of the correlation curve is >1 and it would appear that 10(3) LD(50) is approximately equivalent to 10(4) plaque forming units (PFU), based on the overall means of all laboratory results. The First International Standard for yellow fever vaccine, NIBSC Code 99/616, has been established as the First International Standard for yellow fever vaccine by the Expert Committee of Biological Standards of the World Health Organisation. The International Standard has been arbitrarily assigned a potency of 10(4.5) International Units (IU) per ampoule. Manufacturers and National Control Laboratories are including the First International Standard for yellow fever vaccine in routine assays so that the minimum potency in IU of vaccines released for use and which meet the current minimum potency of 10(3) LD(50) in mouse assays, can be determined. These data will be analysed before a review of the WHO requirements, including the minimum potency per dose, is undertaken.     

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.     

The 1st International Standard for anti-measles serum

The 1st International Standard for anti-measles serum has been established on the basis of a collaborative study. There were four participating laboratories in four countries and three types of assay used. This standard has been assigned a potency of 5 IU anti-measles antibody per ampoule.     

Calibration of replacement international standard and European pharmacopoeia biological reference preparation for tetanus toxoid, adsorbed.

Here we report the characterisation of a preparation of tetanus toxoid, adsorbed, and its calibration by 27 laboratories in 19 countries in a joint international collaborative study co-sponsored by World Health Organization (WHO) Expert Committee of Biological Standardization (ECBS) and the European Biological Standardisation Programme of European Directorate for the Quality of Medicines (EDQM), Council of Europe. Calibration was in terms of the Second International Standard (I.S.) for Tetanus Toxoid, Adsorbed, by the established WHO/European Pharmacopoeia (Ph Eur) challenge methods. The replacement standard preparation was found to have a unitage of 469 IU/ampoule on the basis of its calibration in guinea-pigs and 496 IU/ampoule on the basis of its calibration in mice. Assessment, both within the collaborative study and as part of candidate characterisation, indicated satisfactory stability of the candidate preparation. This study also provided some information on the effect of mouse strain on potency testing of tetanus vaccines. A limited assessment of the impact of the replacement standard on testing of current production batches of vaccines was also carried out by four manufacturers. This study did not directly address the serological approaches to potency testing. However, one laboratory offered data from mouse serology assay, which gave comparable estimates to in vivo mouse bioassay.Based on the results of this study and with the agreement of participants, the candidate standard was established as the Third International Standard for Tetanus Toxoid, Adsorbed (coded 98/552) by the WHO Expert Committee of Biological Standardization (ECBS) in November 2000. The same preparation was also established as the second Ph Eur Biological Reference Preparation (Ph Eur BRP, batch no. 2) by the Steering Committee of the Biological Standardisation Programme of the EDQM and approved by the European Pharmacopoeia Commission.     

Rabies reference vaccine for use as a regional standard for Latin America and the Caribbean countries

A candidate rabies reference vaccine of suckling mouse brain (SMB) origin was prepared and standardized at the Pan American Zoonoses Center (PAHO/WHO) and evaluated in a collaborative study involving seven laboratories. On the basis of three different tests, its potency, immunogenicity, and stability were demonstrated to be satisfactory. The vaccine was proposed for consideration of the Latin American and Caribbean countries as a regional standard to determine the potency of SMB vaccines, the most widely used in the Region.     

The International Standard for Netilmicin

An International Standard for Netilmicin has been established on the basis of a collaborative assay. There were five participating laboratories in five countries. The activity of the contents of each ampoule of the International Standard for Netilmicin is defined as 4810 IU of netilmicin.     

Immunogenicity of multi-epitope-based vaccine candidates administered with the adjuvant Gp96 against rabies

Rabies, a zoonotic disease, causes 55,000 human deaths globally and results in at least 500 million dollars in losses every year. The currently available rabies vaccines are mainly inactivated and attenuated vaccines, which have been linked with clinical diseases in animals. Thus, a rabies vaccine with high safety and efficacy is urgently needed. Peptide vaccines are known for their low cost, simple production procedures and high safety. Therefore, in this study, we examined the efficacy of multi-epitope-based vaccine candidates against rabies virus. The ability of various peptides to induce epitope-specific responses was examined, and the two peptides that possessed the highest antigenicity and conservation, i.e., AR16 and h PAB, were coated with adjuvant canineGp96 and used to prepare vaccines. The peptides were prepared as an emulsion of oil in water(O/W) to create three batches of bivalent vaccine products. The vaccine candidates possessed high safety. Virus neutralizing antibodies were detected on the day 14 after the first immunization in mice and beagles, reaching 5–6 IU/m L in mice and 7–9 IU/m L in beagles by day 28. The protective efficacy of the vaccine candidates was about 70%–80% in mice challenged by a virulent strain of rabies virus. Thus, a novel multi-epitope-based rabies vaccine with Gp96 as an adjuvant was developed and validated in mice and dogs. Our results suggest that synthetic peptides hold promise for the development of novel vaccines against rabies.     

Preparation and Standardization of U.S. Standard Pertussis Vaccine, Lot No. 11

The Center for Biologics Evaluation and Research within the U.S. Food and Drug Administration has prepared a new U.S. Standard Pertussis Vaccine. Whole cell pertussis vaccine concentrate was diluted in 5% (w/v) lactose and lyophilized. The preparation was tested for toxicity, sterility heterogeneity and residual moisture. Based on data from an international sollaborative study involving 11 laboratories, the potency was estimated in relation to the U.S. Master Standard Pertussis Vaccine, Lot 4 and the International Standard for Pertussis Vaccine, Lot 2. The potency of the preparation was defined to be 90 units per ampoule. When reconstituted and stored according to instructions, no significant change in potency was observed in the 14 days following reconstitution. This material was shown to be suitable for a pertussis vaccine standard and accordingly it was designated as U.S. Standard Pertussis Vaccine, Lot 11 on March 22, 1994.     

用Vero细胞制备马抗狂犬病血清抗原

以Vero细胞为基质制备马抗狂犬病血清用抗原,以期建立有效、经济、简便的抗原制备方法。用含10％马血清营养液对Vero细胞作适应性培养,接种狂犬病毒,以含1％～3％马血清营养液作维持液培养病毒,于第5,8天收获病毒液,经灭活、浓缩、离心等制成抗原。第5,8天收获病毒滴度可稳定在7．010gLD50／mL以上,灭活抗原具良好的抗原性,用NIH法测定效价达6．0IU／mL以上,可用作抗原生产抗狂犬病血清。     

A collaborative study to establish the International Reference Reagent for hepatitis B vaccine containing plasma-derived hepatitis B surface antigen

A collaborative study was conducted to establish a suitable international reference reagent for hepatitis B vaccine for use in immunogenicity assays. The limiting dilution required to induce antibodies in 50% of the test animals was determined for the proposed international reference reagent and three other plasma-derived hepatitis B vaccines. The minimum antigenic dose of these preparations varied widely (100-fold range) between laboratories. However, the expression of potencies of vaccines relative to the proposed International Reference Reagent reduced the variation between laboratories to within a 10-fold range. The reference reagent is intended for use in assays of hepatitis B vaccines in mouse (or guinea-pig) immunogenicity studies. For products made by different procedures, clinical trials in humans are necessary to establish a correlation between the immunogenic potency in animals and man.     

Calibration and commutability assessment of the 1st International Standard for Diphtheria Antitoxin Human

The 1st International Standard for Diphtheria Antitoxin Human (coded 10/262) was established by the World Health Organization Expert Committee on Biological Standardization in 2012. This paper describes the production, characterization and calibration of the new standard which is intended for use in the standardization of assays used to measure diphtheria antibody responses in human serum. The new standard was calibrated in terms of the International Standard for Diphtheria Antitoxin Equine in an international collaborative study. A total of 8 participants from 8 different countries performed in vivo and/or in vitro toxin neutralization tests and returned data that was used to assign units to the proposed new standard. The new standard has a diphtheria antitoxin potency of 2 IU/ampoule and is predicted to be stable. A follow up study was performed to assess commutability of the new standard. The follow up study was an existing external quality assessment, modified to include the new standard. Results obtained suggest that the new standard is commutable, showing comparable behaviour to native human serum samples in the majority of the assays compared, and is therefore suitable for use as a reference preparation in assays used to measure the level of anti-diphtheria antibodies in human serum.     

The efficacy of a cultured rabies vaccine studied in guinea pigs preliminarily infected with a fixed rabies virus]

In experiments of curative vaccination, carried out with the use of an experimental model similar to the current practice of treatment with antirabies preparations, the advantages of using tissue-culture rabies vaccine with immunogenic potency equal to 1.3 international units (I. U.) were shown. In these experiments the vaccine was introduced into guinea pigs infected with fixed rabies virus, the course of vaccination consisting of 14 daily injections. No correlation between the induction of virus-neutralizing antibodies and the immunogenic potency of tissue-culture rabies vaccine was established: the use of the vaccine with immunogenic potency equal to 0,3 and 1,3 I.U. had no essential influence on the level of antibody formation in the animals.     

Report on the international workshop on alternative methods for human and veterinary rabies vaccine testing: State of the science and planning the way forward

Potency testing of most human and veterinary rabies vaccines requires vaccination of mice followed by a challenge test using an intracerebral injection of live rabies virus. NICEATM, ICCVAM, and their international partners organized a workshop to review the availability and validation status of alternative methods that might reduce, refine, or replace the use of animals for rabies vaccine potency testing, and to identify research and development efforts to further advance alternative methods. Workshop participants agreed that general anesthesia should be used for intracerebral virus injections and that humane endpoints should be used routinely as the basis for euthanizing animals when conducting the mouse rabies challenge test. Workshop participants recommended as a near-term priority replacement of the mouse challenge with a test validated to ensure potency, such as the mouse antibody serum neutralization test for adjuvanted veterinary rabies vaccines for which an international collaborative study was recently completed. The workshop recommended that an in vitro antigen quantification test should be a high priority for product-specific validation of human and non-adjuvanted veterinary rabies vaccines. Finally, workshop participants recommended greater international cooperation to expedite development, validation, regulatory acceptance, and implementation of alternative test methods for rabies vaccine potency testing.