Biomedical subjects
Publications and source records attributed to P D Minor.
Poliomyelitis has been largely controlled by the use of attenuated live vaccine strains. The molecular basis of attenuation is beginning to be understood, but the interactions between the virus and its human host remain mysterious.
The slow growth of hepatitis A virus (HAV) has made tissue culture assay for infectious virus difficult. Strains of the virus of greater cytopathogenicity have been selected for use in plaque cytopathic assays. However, in our hands, this assay has been difficult to reproduce consistently due to problems in maintaining intact cell monolayers over the long incubations involved. This report describes the development of a cytopathic TCID50 assay for HAV. From the results of repeated assay of one preparation of the virus, the coefficient of variation of the assay was calculated to be 4%. This assay has also been adapted to quantitate antibodies to HAV. Initial results of assaying the WHO standard immune serum globulin are comparable with the titre obtained by the radioimmunofocus inhibition test. Antibody titres in human and mouse sera could also be quantitated. The cytopathic TCID50 assay and the adapted inhibition assay described, may prove useful for the development and control of HAV vaccines and the validation of viral inactivation protocols.
The transmission of viral infections through the use of products derived from blood has emphasised the need for adequate validation of the production process, testing of materials used in production and quality control tests on the final product. Since the late 1980s, as part of its batch release procedures, NIBSC has tested for markers of viral infectivity plasma pools used in production of blood products used in the UK. As a result of testing over 9,000 pools, NIBSC has identified 9 pools contaminated with HBsAg and 2 pools containing antibodies to HIV-1. Since routine screening of plasma pools for anti-HCV was introduced in 1993, 8 pools out of the 4,000 tested have been found to contain antibodies to HCV. In addition, the release of 12 batches of blood products was withheld and it is known that further batches of material produced from the positive pools were not submitted for batch release. Studies involving assays of dilutions of known positive plasma samples indicated that there is considerable variation in the endpoint dilutions of antigen or antibody detected by test kits from different manufactures. The selection and validation of the kits used in such testing is therefore important. The usefulness of standardised low-level external controls in assays of plasma pools for markers of viral infection is discussed.
The relationships between different strains of mumps virus were established by determination of the sequence of the HN gene. They closely resemble those established from other portions of the genome, suggesting that the viruses are not recombinants over the areas examined. The relationships were consistent with those established by reaction with monoclonal antibodies raised against the Urabe strain, which has a similar antigenic structure to previously studied laboratory strains, and largely consistent with the specificity of the serological response of naturally infected or vaccinated human subjects.
Animal models were central to the development of poliovaccines and remain essential in some form in the routine quality control of both live and killed vaccines. The necessity of an animal model is illustrated by the examples of mumps and measles vaccines where the existing materials, while satisfactory, have a number of drawbacks and where changes in current practice raise concerns for safety and efficacy.
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Explore the source record for details and available documents.
The current understanding of the function of CD4+ T helper (Th) cells in immunity to infectious diseases is that Th1 cells, which secrete interleukin (IL)-2 and interferon-gamma, induce cellular immune responses, whereas Th2 cells, which secrete IL-4, IL-5, IL-6, and IL-10, provide helper function for humoral immunity. We have used a panel of poliovirus-specific murine CD4+ T cell clones and mice transgenic for the human poliovirus receptor to evaluate the role of Th cell subpopulations in protective immunity to poliovirus. The majority of T cell clones, as well as polyclonal T cells generated from mice infected or immunized with poliovirus, secreted IL-2 and interferon-gamma, but not IL-4, IL-5, or IL-10, a profile typical of Th1 cells. The Th1 clones displayed major histocompatibility complex class II-restricted cytotoxic T lymphocyte activity against specific poliovirus peptide-pulsed target cells, but also provided help for antipoliovirus neutralizing antibody production. To examine the mechanism of immunity in vivo, we have used poliovirus receptor-transgenic mice on a BALB/c (H-2d) background. These animals developed a poliomyelitis-like disease when challenged intravenously with a virulent wild-type strain of poliovirus, but not with an attenuated vaccine strain. Furthermore, mice immunized with the vaccine strain were protected against a subsequent challenge with wild-type virus. Using an adoptive transfer technique, we demonstrated that it was possible to confer protection with primed B cells in the presence of polyclonal poliovirus-specific T cells, but not when transgenic mice received either B cells or T cells alone. Furthermore, protection was observed when mice received primed B cells in the presence of a VP4-specific Th1 clone. The findings demonstrate that Th1 cells can mediate a protective immune response against poliovirus infection in vivo through helper activity for humoral immunity and that CD4+ T cells, specific for the internal poliovirus capsid protein, VP4, can provide effective help for a protective antibody response directed against surface capsid proteins.
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The French neurotropic vaccine, or FNV, was used extensively in Africa to control yellow fever (YF). Although efficacious, the vaccine caused an unacceptable rate of post-vaccinal complications in children and was subsequently replaced by the 17D vaccine. Here we report that the genomes of the wild-type YF virus French viscerotropic virus and its attenuated vaccine derivative, FNV virus from the Institut Pasteur, Paris, (FNV-IP) differ by 77 nucleotides encoding 35 amino acid substitutions. Comparison of FNV-IP and three other isolates of FNV with other YF vaccine strains (17D-204 and 17DD derived from wild-type strain Asibi) revealed that during the two attenuation processes two common nucleotide changes arose that encode two amino acid substitutions: one is in the membrane protein at amino acid 35 (M-35), the other in non-structural (NS) protein 4B at NS4B-95. These common substitutions may be important in the process of attenuation of viscerotropic disease for humans and monkeys, and/or may be involved in loss of mosquito competence of the vaccine viruses.
Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from its neurovirulent progenitor (P1/Mahoney), two have been strongly implicated by previous studies as determinants of the attenuation phenotype. A change of an A to a G at position 480, located within the 5' noncoding region, has been suggested to be the major attenuating mutation, analogous to the mutations at positions 481 and 472 in poliovirus types 2 and 3, respectively. In addition, the change of a U to a C at position 6203, resulting in an amino acid change in the polymerase protein 3D, has also been implicated as a determinant of attenuation, albeit to a lesser extent. To assess the contributions of these mutations to attenuation and temperature sensitivity, reciprocal changes were generated at these positions in infectious cDNA clones of Sabin 1 and P1/Mahoney. Assays in tissue culture and primates indicated that the two mutations make some contribution to the temperature sensitivity of the Sabin 1 strain but that neither is a strong determinant of attenuation.
CD55, or decay-accelerating factor (DAF), is a cell surface glycoprotein which regulates complement activity by accelerating the decay of C3/C5 convertases. Recently, we and others have established that this molecule acts as a cellular receptor for echovirus 7 and related viruses. DAF consists of five domains: four short consensus repeats (SCRs) and a serine/threonine-rich region, attached to the cell surface by a glycosylphosphatidyl inositol anchor. Chinese hamster ovary cells stably transfected with deletion mutants of DAF or DAF-membrane cofactor protein recombinants were analyzed for virus binding. The results indicate that the binding of echovirus 7 to DAF specifically requires SCR2, SCR3, and SCR4. There is also a nonspecific requirement for the S/T-rich region which probably functions to project the binding region away from the cell membrane. The three nonpeptide modifications of DAF, N-linked glycosylation, O-linked glycosylation, and the glycosylphosphatidyl inositol anchor, are not required for virus binding. The SCRs of membrane cofactor protein, the closest known relative of DAF, cannot substitute for those of DAF with retention of virus binding activity. The monoclonal antibody used to identify DAF as an echovirus receptor, and which inhibits binding of the virus (monoclonal antibody 854), binds to SCR3.
The Ad Hoc Working Party on Biotechnology and Pharmacy of the Committee for Proprietary Medicinal Products has developed a number of guidance notes for manufacturers intending to submit applications for market authorization in the European Union which includes one dealing specifically with the validation of production processes for the removal or inactivation of viruses. The strategies used to minimize the risk of viral transmission by biological products are: screening the source materials for viral contamination; examining the ability of the production process to remove or inactivate viruses; and examining the final product for evidence of viral contamination. It is concluded that validation studies provide a significant assurance of viral safety when properly carried out, using appropriate relevant and model viruses.
Reported are the results of a study to investigate the immunogenicity of oral poliovirus vaccine (OPV) when administered in mass campaigns compared with that following routine immunization programmes. For this purpose, paired sera were collected from a cohort of children before and after a mass vaccination with OPV in Morocco in 1987. Serum samples and information on vaccination status and other confounding factors that could influence antibody responses to OPV were collected. Neutralizing antibody titres to poliovirus types 1, 2 and 3 were determined using a standardized assay. OPV doses administered exclusively during the mass campaign were consistently associated with higher type-specific seroprevalence rates than the same number of doses administered in the routine programme. These findings could not be attributed to differences in confounding factors. Enhanced secondary spread of vaccine virus may have occurred but could not be demonstrated because of limitations in the study design. Mass campaigns appear to be highly effective in raising the dose-related poliovirus type-specific immunity of the population above that achieved by the routine immunization programme. Our findings support the continued use of mass campaigns as an adjunct to routine programmes in order to both enhance and catalyse current efforts to achieve the global eradication of poliomyelitis by the year 2000.
Using an anti-receptor mAb that blocks the attachment of echovirus 7 and related viruses (echoviruses 13, 21, 29 and 33), we have isolated a complementary DNA clone that encodes the human decay-accelerating factor (CD55). Mouse cells transfected with the CD55 clone bind echovirus 7, and this binding is blocked by the anti-receptor mAb. The method used (CELICS) allows rapid and direct cloning of genes encoding cell surface receptors. It is based on episomal replication and high efficiency expression of complementary DNA clones in the vector pCDM8 in COS or WOP cells, in conjunction with a sensitive immuno-focal screen that uses antibody probes linked to beta-galactosidase. Receptor positive cells were identified by a colour change and isolated individually using a micromanipulator. DNA extracted from a small number of cells was then cloned directly in Escherichia coli.
Viral protein synthesis in poliovirus infected cells was found to be influenced by mutations in part of the viral 5'-non-coding region (NCR) in a temperature dependent manner. At elevated temperatures these mutations resulted in virus titre reductions that allowed selection of revertant viruses. Some revertants were found to have retained the 5'-NCR mutations but had compensating mutations in the 2A protease gene that were responsible for the suppression of the temperature sensitive phenotypes. The mutations in 2A enhanced viral protein synthesis at a stage when cap dependent translation was already abolished, suggesting that the virally encoded protein 2A is directly involved in the process of cap independent translation in addition to its role in abolishing cap dependent translation.
The history of the use of biological products is littered with examples of the iatrogenic transmission of viruses. A major effort of the biotechnology industry is directed towards the elimination of any risk of infection from the products of cell culture. This article summarizes the overall strategy that has been followed, so far with great success, in that there have, as yet, been no reported cases of viral transmission by the products of biotechnology.