Changes in nonstructural protein 3 are associated with attenuation in avian coronavirus infectious bronchitis virus.

Full-length genome sequencing of pathogenic and attenuated (for chickens) avian coronavirus infectious bronchitis virus (IBV) strains of the same serotype was conducted to identify genetic differences between the pathotypes. Analysis of the consensus full-length genome for three different IBV serotypes (Ark, GA98, and Mass41) showed that passage in embryonated eggs, to attenuate the viruses for chickens, resulted in 34.75-43.66% of all the amino acid changes occurring in nsp 3 within a virus type, whereas changes in the spike glycoprotein, thought to be the most variable protein in IBV, ranged from 5.8 to 13.4% of all changes. The attenuated viruses did not cause any clinical signs of disease and had lower replication rates than the pathogenic viruses of the same serotype in chickens. However, both attenuated and pathogenic viruses of the same serotype replicated similarly in embryonated eggs, suggesting that mutations in nsp 3, which is involved in replication of the virus, might play an important role in the reduced replication observed in chickens leading to the attenuated phenotype.

Bovine serum--regulatory issues.

For many years users of bovine serum in the manufacture of human and veterinary biological medicinal products have relied upon USDA 9CFR to ensure the viral safety of their serum. Recently, EU regulators have formalised their position by issuing guidelines on the use of bovine serum during manufacture. Additionally, the European Pharmacopoeia has drafted a monograph on bovine serum. There is good harmonisation among the recommendations and requirements although the EU CHMP guideline calls for greater attention to be paid to the potential presence of infectious bovine polyoma virus. The EU guidelines also call for various tests to assess the effect of BVDV antibodies in the detection of BVDV. However, in response to criticisms from serum suppliers and users, the stringency of these recommendations is being relaxed. The overall viral safety of bovine serum should be subject to a risk analysis as infectious virus will invariably be present in some batches of serum but remain undetected. Other factors such as the geographical source of the serum, the efficacy of viral inactivation/removal steps and the ability of specific viruses to grow in the production cells being used, should be taken into consideration.

Changes in biological source material.

A change in source (or raw) material can be radical, e.g. changing the derivation of a therapeutic protein from human plasma to recombinant DNA manufacture, or the change may be more subtle, e.g. the change from a non-inactivated bovine serum growth supplement to an inactivated serum. The former type of change is usually driven by manufacturing strategy and has vast consequences for the regulation of the product concerned. The latter type of change is usually driven by a need to increase the assurance of viral safety and the regulatory implications for the product are significantly less severe. In this latter example, inadvertent alterations to the product may result from changes in cell metabolism brought about by the change in its growth conditions and these need to be addressed in comparability studies. Ultimately, the implication of any slight change in the fine structure of a biotech medicinal product on its efficacy and/or immunogenicity will have to be dealt with on a case-by-case basis.

Preparation of vaccines against H5N1 influenza.

In response to the pandemic warning provided by the highly pathogenic H5N1 influenza virus infections in Hong Kong, there were world-wide attempts to develop vaccines. Three strategies were followed and although each was associated with some success, there were also some problems. Pre-clinical vaccine efficacy results are presented from one such strategy, that of using an apathogenic H5N3 avian strain for vaccine production.

[Differences in receptor specificity between the influenza A viruses isolated from the duck, chicken, and human]. / Sravnenie retseptornoi spetsifichnosti virusov grippa A, vydelennykh ot utok, kur i cheloveka.

The affinity of the duck, chicken, and human influenza viruses to the host cell sialosides was determined, and considerable distinctions between duck and chicken viruses were found. Duck viruses bind to a wide range of sialosides, including the short-stem gangliosides. Most of the chicken viruses, like human ones, lose the ability to bind these gangliosides, which strictly correlates with the appearance of carbohydrate at position 158-160. The affinity of the chicken viruses to sialoglycoconjugates of chicken intestine as well as chicken, monkey, and human respiratory epithelial cells exceeds that of the duck viruses. The human influenza viruses have high affinity to the same cells but do not bind at all to the duck epithelial cell. This testifies to the absence of 6'-sialylgalactose residues from the duck cells, in contrast to chicken and monkey cells. The alteration of the receptor specificity of chicken viruses in comparison with duck ones results in the similarity of the patterns of accessible cells for chicken and human influenza viruses. This may be the cause of the appearance of the line of H9N2 viruses from Hong Kong live bird markets with receptor specificity similar to that of H3N2 human viruses, and of the ability of H5N1 and H9N2 chicken influenza viruses to infect humans.

Assuring the quality, safety, and efficacy of DNA vaccines.

Scientists in academia whose research is aimed at the development of a novel vaccine or approach to vaccination may not always be fully aware of the regulatory process by which a candidate vaccine becomes a licensed product. It is useful for such scientists to be aware of these processes as the development of a novel vaccine could be problematic owing to the starting material often being developed in a research laboratory under ill-defined conditions. This paper examines the regulatory process with respect to the development of a DNA vaccine. DNA vaccines present unusual safety considerations that must be addressed during preclinical safety studies, including adverse immunopathology, genotoxicity through integration into a vaccinees chromosomes, and the potential for the formation of anti-DNA antibodies.

Retroviral aspects of the characterisation of avian and mammalian cell substrates.

There have been no reported incidents of adventitious retroviral infection of cell lines used for the production of vaccines and other biologicals. However, due to the unique molecular biology of retroviruses, cell lines may contain endogenous retroviral genomes and these can give rise to defective retroviral particles, e.g. murine hybridomas and CHO cells. Recently, a similar situation was reported for a cell substrate in use for decades for vaccine production, namely chick embryo fibroblasts, and this raised some concern regarding the safety of these vaccines.

European Union guidance on the quality, safety and efficacy of DNA vaccines and regulatory requirements.

A European Union note for guidance on gene transfer medicinal products is being developed by the Biotechnology Working Party (BWP) of the Committee for Proprietary Medicinal Products (CPMP) which will include guidance for DNA vaccines. The 'Note for Guidance on the Quality, Preclinical and Clinical Aspects of Gene Transfer Medicinal Products' outlines the information required to assure the quality of the plasmid DNA intended to be used as a vaccine. It also provides guidance on preclinical safety evaluation and on clinical efficacy and safety evaluation with regard to obtaining marketing authorisation according to Council Regulation No. (EEC) 2309/93. Before initiating a clinical trial, it is necessary to obtain an appraisal from the relevant central and/or local ethics committees, and the competent authority within the member state(s) concerned has to authorise, or be notified of, the clinical trial. Harmonisation of gene therapy and DNA vaccine regulations as well as co-operation between relevant ethics committees and authorities in Europe is currently being improved.

Assuring the quality, safety, and efficacy of DNA vaccines.

Scientists in academia whose research is aimed at the development of a novel vaccine or approach to vaccination may not always be fully aware of the regulatory process by which a candidate vaccine becomes a licensed product. This chapter will provide an overview of the regulatory process and will discuss in more detail the quality and pre-clinical safety issues of plasmid DNA vaccines intended for human use. It is useful for research scientists to be aware of these processes as the development of a novel vaccine could be problematic due to the starting material often being developed in a research laboratory under ill-defined conditions.

An overview of host cell selection.

A virus with a new phenotype can arise from an apparently homogeneous population after a single passage by the application of a strong selection pressure. With less pressure, a variant population may arise gradually during repeated passage of the virus. Strong host selection has been observed when naturally occurring influenza virus is grown in embryonated hens' eggs whereas the natural virus grows apparently unrestricted in MDCK cells. However, repeated passage in MDCK cells can select for a virus more suited to replication in these cells and variants which resemble those selected in eggs have been observed in such passaged virus.

The influence of the host cell on standardisation of influenza vaccine potency.

Conventional influenza vaccines are standardised using the single-radial-immunodiffusion (SRD) test where reagents are produced from egg-grown viruses. It is important to ensure homology between SRD antigen reagents and test vaccines. There was concern that cell-grown vaccines may differ antigenically from corresponding egg-grown vaccines, which may in turn affect vaccine standardisation. In an examination of five cell-grown vaccines from two companies, only one vaccine was affected by the specificity of the SRD test. Options for standardisation of cell-grown vaccines are considered and recommendations are made for further studies.

Effects of egg-adaptation on the receptor-binding properties of human influenza A and B viruses.

Propagation of human influenza viruses in embryonated chicken eggs (CE) results in the selection of variants with amino acid substitutions near the receptor-binding site of the hemagglutinin (HA) molecule. To evaluate the mechanisms by which these substitutions enable human virus growth in CE, we studied the binding of 10 human influenza A (H1N1, H3N2) and B strains, isolated and propagated solely in MDCK cells, and of their egg-adapted counterparts to preparations of cellular membranes, gangliosides, sialylglycoproteins, and sialyloligosaccharides. All egg-adapted variants differed from nonadapted strains by increased binding to the plasma membranes of chorio-allantoic (CAM) cells of CE and by the ability to bind to CAM gangliosides. In addition, there was no decrease in affinity for inhibitors within allantoic fluid. These findings indicate that growth of human influenza viruses in CE is restricted because of their inefficient binding to receptors on CAM cells and that gangliosides can play an important role in virus binding and/or penetration. The effects of the egg-adaptation substitutions on the receptor-binding properties of the viruses include (i) enhancement of virus binding to the terminal Sia(alpha2-3)Gal determinant (substitutions in HA positions 190, 225 of H1N1 strains and in position 186 of H3N2 strains); (ii) a decrease of steric interference with more distant parts of the Sia(alpha2-3Gal)-containing receptors (a loss of glycosylation sites in positions 163 of H1 HA and 187 of type B HA); and (iii) enhanced ionic interactions with the negatively charged molecules due to charged substitutions at the tip of the HA [187, 189, 190 (H1), and 145, 156 (H3)]. Concomitantly with enhanced binding to Sia(alpha2-3)Gal-terminated receptors, all egg-adapted variants decreased their affinity for equine macroglobulin, a glycoprotein bearing terminal 6'-sialyl(N-acetyllactosamine)-moieties.

MIRD pamphlet no. 16: Techniques for quantitative radiopharmaceutical biodistribution data acquisition and analysis for use in human radiation dose estimates.

This report describes recommended techniques for radiopharmaceutical biodistribution data acquisition and analysis in human subjects to estimate radiation absorbed dose using the Medical Internal Radiation Dose (MIRD) schema. The document has been prepared in a format to address two audiences: individuals with a primary interest in designing clinical trials who are not experts in dosimetry and individuals with extensive experience with dosimetry-based protocols and calculational methodology. For the first group, the general concepts involved in biodistribution data acquisition are presented, with guidance provided for the number of measurements (data points) required. For those with expertise in dosimetry, highlighted sections, examples and appendices have been included to provide calculational details, as well as references, for the techniques involved. This document is intended also to serve as a guide for the investigator in choosing the appropriate methodologies when acquiring and preparing product data for review by national regulatory agencies. The emphasis is on planar imaging techniques commonly available in most nuclear medicine departments and laboratories. The measurement of the biodistribution of radiopharmaceuticals is an important aspect in calculating absorbed dose from internally deposited radionuclides. Three phases are presented: data collection, data analysis and data processing. In the first phase, data collection, the identification of source regions, the determination of their appropriate temporal sampling and the acquisition of data are discussed. In the second phase, quantitative measurement techniques involving imaging by planar scintillation camera, SPECT and PET for the calculation of activity in source regions as a function of time are discussed. In addition, nonimaging measurement techniques, including external radiation monitoring, tissue-sample counting (blood and biopsy) and excreta counting are also considered. The third phase, data processing, involves curve-fitting techniques to integrate the source time-activity curves (determining the area under these curves). For some applications, compartmental modeling procedures may be used. Last, appendices are included that provide a table of symbols and definitions, a checklist for study protocol design, example formats for quantitative imaging protocols, temporal sampling error analysis techniques and selected calculational examples. The utilization of the presented approach should aid in the standardization of protocol design for collecting kinetic data and in the calculation of absorbed dose estimates.

The MIRD perspective 1999. Medical Internal Radiation Dose Committee.

The MIRD schema is a general approach for medical internal radiation dosimetry. Although the schema has traditionally been used for organ dosimetry, it is also applicable to dosimetry at the suborgan, voxel, multicellular and cellular levels. The MIRD pamphlets that follow in this issue and in coming issues, as well as the recent monograph on cellular dosimetry, demonstrate the flexibility of this approach. Furthermore, these pamphlets provide new tools for radionuclide dosimetry applications, including the dynamic bladder model, S values for small structures within the brain (i.e., suborgan dosimetry), voxel S values for constructing three-dimensional dose distributions and dose-volume histograms and techniques for acquiring quantitative distribution and pharmacokinetic data.

MIRD pamphlet No. 17: the dosimetry of nonuniform activity distributions--radionuclide S values at the voxel level. Medical Internal Radiation Dose Committee.

The availability of quantitative three-dimensional in vivo data on radionuclide distributions within the body makes it possible to calculate the corresponding nonuniform distribution of radiation absorbed dose in body organs and tissues. This pamphlet emphasizes the utility of the MIRD schema for such calculations through the use of radionuclide S values defined at the voxel level. The use of both dose point-kernels and Monte Carlo simulation methods is also discussed. PET and SPECT imaging can provide quantitative activity data in voxels of several millimeters on edge. For smaller voxel sizes, accurate data cannot be obtained using present imaging technology. For submillimeter dimensions, autoradiographic methods may be used when tissues are obtained through biopsy or autopsy. Sample S value tabulations for five radionuclides within cubical voxels of 3 mm and 6 mm on edge are given in the appendices to this pamphlet. These S values may be used to construct three-dimensional dose profiles for nonuniform distributions of radioactivity encountered in therapeutic and diagnostic nuclear medicine. Data are also tabulated for 131I in 0.1-mm voxels for use in autoradiography. Two examples illustrating the use of voxel S values are given, followed by a discussion of the use of three-dimensional dose distributions in understanding and predicting biologic response.

International standardization of gene amplification technology.

The WHO international Working Group on the standardization of Gene Amplification Techniques for the Virological Safety Testing of Blood and Blood Products (SoGAT) was established in 1995 as a technical discussion group. It is the only international forum for the exchange of information on scientific aspects of the technology, evaluation of the technology for the testing of products and the organisation of international collaborative studies for the development, evaluation and provision of reference materials and working standards. Materials suitable as working reagents in hepatitis C virus (HCV) gene amplification are in widespread use and plans for a collaborative study for the establishment on an HCV RNA International Standard are well underway as are studies aimed at standardizing the technology for other viruses.