Biomedical subjects
Publications and source records attributed to J R Stephenson.
A highly purified, inactivated tick-borne encephalitis (TBE) virus particle vaccine has been developed. In this study we report on the efficacy of this new vaccine to protect against TBE virus isolates from different geographical areas of Europe and the Asian part of the USSR.
Vaccines made by inactivating pathogenic microorganisms have been dramatically successful in controlling diseases in humans and animals. Despite their successes, they have a major disadvantage in that several inoculations are required for them to be effective. To overcome this problem, a commercial inactivated vaccine preparation against tickborne encephalitis was combined with human growth hormone (HGH). This formulation produced complete protection in a murine model with only one dose of vaccine, apparently by binding hormone and antigen to an insoluble matrix containing aluminium hydroxide. Thus it is postulated that when virus-specific lymphocytes are attracted to the site of injection, the hormone is at a high local concentration and stimulates the clonal expansion of antigen-specific T cells. The development of genetically engineered HGH now gives unlimited supplies of hormone, potentially resulting in an increase in efficacy of a wide variety of vaccines, especially those needing prolonged immunization schedules such as those being developed to combat human immunodeficiency virus infection.
We identified twenty patients maintained on continuous ambulatory peritoneal dialysis who suffered repeated episodes of peritonitis caused by coagulase-negative staphylococci. We documented hand and exist-site coagulase-negative staphylococcus-associated peritonitis over a total period of 32 months, and compared hand and exit-site strains with strains isolated from dialysate fluid using three typing methods: biotyping using the API Staph kit plus antibiograms, immunoblotting using sera raised in rabbits to three standard strains of coagulase-negative staphylococci, and 35S-methionine-labelled coagulase-negative staphylococcal profiles separated on sodium dodecylsulphate polyacrylamide gel electrophoresis and visualised by autoradiography (radioPAGE). In 5 of 84 episodes, strains isolated from skin were indistinguishable by all three typing methods from the dialysate strain. In a further two episodes, hand or exit-site isolates were indistinguishable by all three typing methods from the dialysate strain isolated in the subsequent, but not the same, episode. Thus in the majority of episodes, no inference of hand or exit-site origin of dialysate infection could be drawn.
A live attenuated vaccine virus variant of Rift Valley fever (RVF) virus was developed by passaging a human isolate in tissue culture under the influence of the mutagen 5-fluorouracil. This virus variant (MV P12) has been assessed in this study as to its suitability as a vaccine, by testing its pathogenicity in young lambs and measuring its ability to induce a protective immune response. Even high doses of the vaccine virus failed to induce any of the clinical or histopathologic changes associated with classical RVF virus infection. Although the vaccine induced mild pyrexia when given in high doses, viremia was not induced. Neutralizing antibody and a protective immune response was elicited with even low doses of vaccine virus. These data, along with data of other workers on the lack of abortigenicity of this virus variant, indicate that the MV P12 variant of RVF virus is an excellent candidate for a safe and effective vaccine against RVF.
Two derivatives of plasmid pAT153 which arose spontaneously in phosphate-limited continuous cultures of Escherichia coli HB101[pAT153] have been characterised. The smaller plasmid, pLAB-446, resulted from a deletion of 446 base pairs of DNA covering the translation start and codon sequence of the tetracycline resistance gene. The other plasmid, pLAB135, differed from pAT153 only by an A:T to G:C transition in the transcribed but untranslated leader region of the tet gene which decreased tet transcription by 84%, possibly due to the formation of a more stable stem-loop structure in the mRNA.
By employing the techniques of column chromatography and membrane filtration, we have succeeded in purifying flavivirus particles with low particle to infectivity ratios and free from contamination with cellular proteins. Virus particles purified by this method have an average diameter of 53 nm, a particle to infectivity ratio of less than 10, and a KD (partition coefficient) consistent with a molecular weight of 2.63 x 10(7). In addition it has been possible to purify the extracellular form of non-structural protein 1 (NS1), which in its native form appears to be a hexamer. It is also apparent from these studies that the slowly sedimenting haemagglutinin particle (or SHA) is an artifact of purification methods using gradient centrifugation. This technology should not only prove useful in the laboratory for studying the detailed structure of these viruses and the proteins encoded by them, but should also prove useful in industrial vaccine manufacture where large volumes of highly pathogenic material are handled.
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The ability of passage in HeLa cells to attenuate flaviviruses was investigated for three different strains of the mosquito-borne West Nile (WN) virus and two tick-borne viruses, louping-ill and Langat. One strain of WN virus, Sarawak, was attenuated 4000-fold for adult mice by intraperitoneal or intranasal challenge after six HeLa passages. The HeLa-passaged virus was also found to be antigenically different and temperature-sensitive in its growth characteristics compared with the parent. After six HeLa cell passages the Egypt 101 and Smithburn strains of WN virus lost their ability to infect monkey kidney cells and no longer killed adult mice, although inoculated animals became sick for several days. In contrast, two tick-borne flaviviruses remained as virulent for mice after six HeLa passages as the parent non-HeLa-passaged virus. Neither of the tick-borne viruses exhibited characteristics associated with temperature sensitivity. The results, therefore, indicate that the mosquito-borne, but not tick-borne, flaviviruses can be attenuated by very few passages in HeLa cells. This observation may provide a model system with which to analyse the molecular basis of attenuation and/or virulence of mosquito-borne flaviviruses.
The effects of changing the composition of the growth medium, the dilution rate and the source of the bacterial host on maintenance of the plasmid pAT153 in Escherichia coli HB101 have been studied. In a medium supplemented with Casamino acids, the plasmid was maintained longer during phosphate-limited growth at a dilution rate of 0.3 h-1 than at 0.15 h-1. In contrast, phosphate-limited growth was not achieved when the Casamino acids were replaced by proline, leucine and thiamin to satisfy the auxotrophic requirements of the host. Although 100% of the bacteria were still ampicillin resistant after 72 generations of growth at a dilution rate of 0.15 h-1, the original plasmid had almost totally been replaced by a structurally modified plasmid which lacked a functional tet gene. Further experiments confirmed that neither the host nor the plasmid was retained unchanged in the minimal medium. The changes were highly reproducible and reflected periodic selection of sub-populations which were either plasmid-free or carried a structurally modified plasmid, which had reverted to Leu+ or Pro+, or had acquired other chromosomal mutations which gave them a selective advantage. We conclude that in complex media the plasmid is maintained longer by E. coli HB101 at a high than at a low growth rate and that different results reported from different laboratories are largely due to differences in analytical techniques and the growth medium rather than to differences in the bacterial host or the plasmid used. A fermenter-adapted strain was isolated which reproducibly maintained the plasmid longer during phosphate-limited continuous growth than the original strain which had been cultured on laboratory media.
We report an outbreak of serious infections with Serratia marcescens in patients on a neurosurgery ward. The epidemiological investigations undertaken are described. Features of outbreaks of infection with serratia and control measures are discussed.
The replication of flaviviruses results in the secretion of four virus-coded proteins into the extracellular environment. Three of these proteins, E, C and M (or pre-M), are found in purified virions. A fourth virus-specified extracellular protein which was not present in either the slowly sedimenting haemagglutinin particles or in virions is described. The relationship of this protein to the intracellular NS1 polypeptide was investigated along with its similarity to the soluble complement-fixing antigen (SCF) reported for mosquito-borne flaviviruses. The difference in the Mr of NS1 and SCF is the result of additional glycosylation of SCF, mostly by the addition of fucose molecules. The synthesis of E and NS1 is sequential but their secretion is simultaneous, suggesting a role for NS1 in virion protein transport or virion release.
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The occurrence of Aeromonas spp. in a hospital water supply, in patients and in the local community was investigated. In healthy persons outside the hospital the isolation rate was 3.6% and among hospital patients it was 6%. Seven per cent of water samples yielded Aeromonas strains. Isolates were typed by sodium dodecyl sulphate polyacrylamide gel electrophoresis of sulphur-35 methionine labelled proteins of Aeromonas isolates. No relationship between water and human isolates could be established, even when a strain of A. hydrophila producing Vero cell cytotoxin contaminated enteral feeds given to patients in the intensive care unit.
Diseases caused by flavivirus infection have been a scourge of mankind for over three centuries; with yellow fever, dengue fever and Russian spring-summer encephalitis causing epidemics resulting in thousands of fatalities. Due to the development of a safe and efficacious live-attenuated vaccine against yellow fever, this disease is no longer such a threat in countries where adequate vaccination is practised. A similarly safe and efficacious inactivated vaccine against central-European tick-borne encephalitis has also been developed and this has drastically reduced the incidence of this disease in many countries where it is endemic. In spite of these successes, the development of vaccines against other pathogenic flaviviruses, causing diseases such as dengue fever and Russian spring-summer encephalitis, have not been successful. This review attempts to summarize the development of flavivirus vaccines to date and identify areas for future improvements. Problems associated with designing flavivirus vaccines are discussed and the advantages and disadvantages of future strategies for vaccine development are considered.
Adult Balb/c mice were passively immunized with monoclonal antibodies (100 micrograms/mouse) raised against tick-borne encephalitis (TBE) virus then challenged 24 hours later s.c. with 10 LD50 of TBE virus (Nëudorfl isolate). None of the mice showed evidence of premature death although all except one of the monoclonal antibodies tested are capable of enhancing the infectivity of TBE virus in the Fc receptor-bearing mouse macrophage-like cell line P 388 D 1. The ability of monoclonal antibodies to neutralize TBE virus in vitro, and to fix complement was examined, and of these properties only a single monoclonal antibody, which was able to neutralize virus, was also able to protect mice against virus challenge.
Tick-borne encephalitis virus codes for two major immunogenic polypeptides, one of which is the major virion envelope protein E, and the other, NV3, does not have a designated function at present. The intracellular forms of both the E and NV3 polypeptides contain at least four types of sugar residues, i.e. galactose, glucosamine, fucose and mannose. The only glycoprotein in the extracellular virion particles is E. Experiments performed in the presence of tunicamycin have demonstrated that most of these sugars are N-linked. The kinetics of synthesis of E and NV3 have been studied and both show a distinct lag period between initiation of protein synthesis and the appearance of either protein. The kinetics of synthesis of these proteins are consistent with the hypothesis that initiation of protein synthesis starts at the 5' end of a polycistronic genome but the synthesis of the E and NV3 proteins only occurs after translocation of the polyribosome complex to specific areas in the infected cell. No precursors to either the E or NV3 glycoproteins were detected. Synthesis of both glycoproteins can be detected as early as 6 h after infection and rises to a maximum at 15 h after infection.
A method for typing species of Aeromonas by 35S-labelling and sodium-dodecyl sulphate polyacrylamide-gel electrophoresis (SDS-PAGE) of total soluble proteins is described. Radiolabelled-protein profiles from different strains were compared by use of the Dice coefficient. Typability was 100% and discrimination of pairs of differing strains was satisfactory with acceptable reproducibility. The results also yielded information on subgroups of phenotypic species.
Twenty-two displaced intra-articular fractures of the calcaneus (in twenty-two patients) were treated by open reduction and internal fixation with a screw and one or two staples using a lateral and, when needed, a medial approach, followed by early motion. Despite the early motion, a nearly normal anatomical reduction was achieved and maintained in nineteen feet. After follow-up that ranged in duration from sixteen to seventy-two months, the clinical and radiographic result was rated as good in seventeen patients and fair in one, and there were four failures. Twenty-one of the twenty-two patients returned to work within six months. The average subtalar motion at follow-up was 75 per cent of normal.