[Rapid immunochemical method for the detection of orthopoxviruses (Orthopoxvirus, Chordopoxvirinae, Poxviridae).]

INTRODUCTION: The abolition of smallpox vaccination has led to the disappearance of population immunity to pox viruses. However, the threat of infection by pathogenic orthopoxviruses persists and determines the need to develop sensitive and operational methods for indicating pathogens. OBJECTIVES: Development of a sensitive, fast and easy-to-use immunochemical test for the detection of orthopoxviruses in the «point of care¼ format. MATERIAL AND METHODS: We used preparations of cultural vaccinia virus (VV) with varying degrees of purification, polyclonal antibodies from hyperimmune rabbit serum, and equipment from a previously developed autonomous kit for dot-immunoassay on flat protein arrays. RESULTS AND DISCUSSION: It has been established that rabbit polyclonal antibodies can be used in a single-stage dotanalysis, both as a capture agent immobilized on a substrate and as a detection reagent bound with colloidal gold particles. It is shown that the effectiveness of the detection of VV is inversely related to the degree of purification of viruses from sub-viral structures. The sensitivity of the rapid detection of viruses in a crude preparation was about 30 times higher than in pure viral material. The increase in sensitivity, presumably, occurs due to binding to the capture antibodies of subviral structures, which form large aggregates of sensitized gold particles. The test does not detect cross-reactions with heterogeneous viruses (measles, rubella and chickenpox) that cause exantematous diseases. CONCLUSION: The one-stage variant of the dot-immunoassay reduces the analysis time to 40 minutes and improves the detection sensitivity of orthopoxviruses in crude viral preparations to the range of 105-104 PFU / ml. Full makeup, ease of analysis and the ability to visually accounting for results allow the test to be used outside of laboratories.

Establishment of a national network of validated and qualified laboratories for neutralizing anti-vaccinia antibodies titration.

A Proficiency Testing Study (PTS) was organized in France by the French Health Products Safety Agency (Afssaps) aiming at assessing the performance of laboratories in performing a neutralizing anti-vaccinia antibodies titration method by plaque reduction neutralization test (PRNT). The ultimate goal was to establish a national network of qualified and validated laboratories. Five laboratories were included in the PTS and four submitted their data. Three samples of human sera containing various immunoglobulin concentrations (a "high" serum: s-576, a "medium" serum: Ref-19584 and a "low" serum: s-483) were tested by PRNT as described in a procedure supplied by Afssaps and developed in each laboratory during preliminary assays. Data were sent to Afssaps which performed the statistical analysis. The overall performance of the four participating laboratories was satisfactory. This allowed the four participating laboratories to be validated and then to be qualified by the Ministry of Health. Finally a national network for anti-vaccinia immunoglobulins titration was established.

Standardization of a neutralizing anti-vaccinia antibodies titration method: an essential step for titration of vaccinia immunoglobulins and smallpox vaccines evaluation.

The possibility of mass population vaccination with smallpox vaccine implies the development of anti-vaccinia immunoglobulins for the treatment of severe side effects following vaccination. We have chosen to develop and validate the "gold standard method" (plaque reduction neutralization assay) to titrate neutralizing anti-vaccinia antibodies in two different French laboratories belonging to the Department of Defense (CRSSA) and to the French Health Products Safety Agency (Afssaps). The results of precision, linearity and accuracy of the method led to consider the method as validated. In parallel, we have prepared and lyophilized a pool of anti-vaccinia plasma samples issued from a unique donor and qualified this preparation versus the first British standard to use it as an in-house standard with a titer of 25 international units (IU). This work will allow to titrate, in IU, sera from vaccinated persons in order (i) to titrate purified anti-vaccinia immunoglobulin preparations for vaccine severe side effect treatments; (ii) to investigate the level of neutralizing antibodies in the general population; and (iii) to investigate clinical trials of new generation smallpox vaccines. In the future, this will allow comparability of studies on either smallpox vaccines or on the serological status of the population.

New generation of cell culture assay for smallpox vaccine potency.

The potency of smallpox vaccines produced in the 1970s was tested by titration onto chorioallantoic membranes of fertilized hen eggs (CAM assay). The potency specification commonly approved for these vaccines was a titer above 10(8) pock-forming units per milliliter. We developed and validated a cell culture titration assay to have a more reliable potency test. The cell titration assay and the CAM assay were tested in parallel on 34 first-generation smallpox vaccine lots. These allowed us to demonstrate that a correlation does exist between the two titration techniques and to determine a new in-house specification for the cell titration method. This in vitro potency assay will allow us to test first-generation smallpox vaccines produced on the skin of living animals but will also give a hint of the potency specification that should be assigned for new generations of cell-derived smallpox vaccines.

[Bacteriophages isolated from smallpox vaccine]. / Bakteriofagi, vydelennye iz ospennoi vaktsiny.

A number of lots of dermal and tissue culture smallpox vaccine was studied for the presence of bacteriophage. The presence of bacteriophage was established in lots of tissue culture vaccine but not in the dermal vaccine. Bacteriophage was detected by a modified method for phage isolation. The concentration and activity of the isolated bacteriophage were low. Its activity was increased by passages. The bacteriophage was stable and lysed the main serotypes of enteropathogenic E. coli as well as strains of Shigella sonnei and Salmonella of typhoid A.

A gas chromatographic method of measuring residual water in freeze-dried smallpox vaccine.

A new method for the accurate determination of residual water in freeze-dried vaccines is described. Tests may be carried out on very small samples in sealed ampoules. The method is based on the adsorption of water from the vaccine with benzene and its estimation by gas chromatography. It appears that the residual water and total nitrogen content are important factors in the stability of the vaccine.

Quantitation of residual host protein in chicken embryo-derived vaccines by radial immunodiffusion.

Quantitation of soluble residual host protein in chicken embryo-derived vaccines was performed rapidly, economically, and accurately by radial immunodiffusion.