Pierre Sureau

Filoviridae: a taxonomic home for Marburg and Ebola viruses?

Filoviridae: a Taxonomic Home for Marburg and Ebola Viruses ? M.P. Kiley E.T.W. Bowen G.A. Eddy M. Isaäcson K.M. Johnson J.B. McCormick F.A. Murphy S.R. Pattyn D. Peters O.W. Prozesky R.L. Regnery D.I.H. Simpson W. Slenczka P. Sureau G. van der Groen P.A. Webb H. Wulff Center for Infectious Diseases, Centers for Disease Control, Atlanta, Ga., USA; PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, UK; US Army Medical Research Institute for Infectious Diseases, Ft. Detrick, Frederick, Md., USA; The South African Institute for Medical Research, Johannesburg, South Africa; Colorado State University, College of Veterinary Medicine and Biomedical Sciences, Ft. Collins, Col., USA;Prins Leopold Instituut voor Tropische Geneeskunde, Antwerpen, Belgium; Bernard-Nocht-Institut für Schiffsund Tropenkrankheiten, Abteilung für Virologie, Hamburg, FRG; National Institute for Virology, Sandringham, Transvaal, South Africa;Hygiene-Institut der Universität, Marburg, FRG; Institut Pasteur, Paris, France

Rabies immunosome (subunit vaccine) structure and immunogenicity. Pre- and post-exposure protection studies.

Rabies immunosomes (glycoprotein anchored on pre-formed liposomes) have been prepared in order to study their structural, biological and immunological properties. The glycoprotein molecules appear to have the same orientation on the immunosome as on the viral particle: (1) electron microscopy analysis shows particles of 40 to 70 nm with spikes protruding outward, (2) one particular epitope shows the same accessibility to a neutralizing monoclonal antibody as on the viral particle. When injected into animals, rabies immunosomes are cleared from the organism by a process different from that for the liposomes used to anchor the glycoprotein: a higher rate of transition through the spleen is observed with immunosomes than with purified glycoprotein or liposomes. Immunosomes induce high levels of neutralizing antibodies and protect animals against challenge with virulent strains. This protective activity is not altered after several months of storage at 4 degrees C. Furthermore, rabies immunosomes were shown to be efficient in post-exposure treatment of laboratory animals that had been experimentally infected with a lethal dose of a rabies wild strain.

Immunity against the European bat rabies (Duvenhage) virus induced by rabies vaccines: an experimental study in mice

Protection experiments were performed in mice with different inactivated vaccines prepared with the fixed rabies virus strains: PM (Pitman-Moore), PV4 (Pasteur virus) and LEP (Flury LEP) against an intracerebral challenge with a European bat virus (Duvenhage, strain Hamburg, DUV3). All vaccines protected mice against challenge with CVS (Challenge virus standard). Vaccines prepared with PV4 protected mice against a DUV3 challenge. On the contrary, PM or LEP vaccines did not protect mice against a DUV3 infection. The protection conferred by PV4 vaccines against Duvenhage could be due to the antigenic relationship which seems to exist between PV4 and European bat virus as revealed by serum-virus neutralization, absorption experiments and CTL crossreactivity. The four virus strains PAS, PV4, PM and CVS, originated from the Pasteur virus isolated in 1882 from the brain of a rabid cow, were classified in two groups on the basis of reactivity with neutralizing anti-glycoprotein monoclonal antibodies. One group contained the L. Pasteur (PAS) and the PV4 strains, the second contained PM and CVS strains. The divergence between the two virus groups possibly resulted from distinct passage histories.

The influence of the type of immunosorbent on rabies antibody EIA; advantages of purified glycoprotein over whole virus

Two types of in vitro assay (enzyme immunoassay and sero-neutralization test) for the titration of rabies antibodies were used to assay sera from mice and humans immunized with cell culture vaccines or neural tissue vaccines. Enzyme immunoassays (EIA) were performed in plates sensitized with whole virus, purified glycoprotein or purified nucleocapsid. Neutralizing antibody titres were determined by the rapid fluorescent focus inhibition test (REFIT) and by an in vitro seroneutralization test including a rapid enzyme immunotitration of intracellular antigens (REITICA). The results obtained with sera of immunized mice and humans showed that (1) cell culture vaccines mainly induced the synthesis of antiglycoprotein neutralizing antibodies; and (2) neural tissue vaccines induced a high synthesis of antinucleocapsid non-neutralizing antibodies and a more or less important synthesis of antiglycoprotein antibodies depending on the origin of the tissue used for their preparation. Consequently, it was emphasized that when using EIA, the antibody titration must be run in glycoprotein-coated plates rather than in whole virus-coated plates to appreciate correctly the immunizing potency of a rabies vaccine, especially neural tissue vaccine.

Interaction of rabies vaccine with human rabies immunoglobulin and reliability of a 2-1-1 schedule application for postexposure treatment

Five commercially available rabies vaccines (HDCV, FBKC vaccine, PCEC vaccine, PVRV and PDEV) applied alone or combined with human rabies immunoglobulin (HRIG) were administered, by random allocation, to 161 volunteer vaccinees, using the abbreviated 2-1-1 postexposure immunization schedule. Protective levels of rabies antibody were demonstrated in all vaccinees by day 14, and in all but one vaccinee from day 21 to day 90. Partial inhibition of the antibody response due to HRIG was observed for three vaccines (PCEC vaccine, PVRV and PDEV). In terms of economy, reliability and rapid antibody induction, the 2-1-1 schedule proves superior to the presently recommended regimen for postexposure rabies prophylaxis.

An evaluation of second generation tissue culture rabies vaccines for use in man: a four-vaccine comparative immunogenicity study using a pre-exposure vaccination schedule and an abbreviated 2-1-1 postexposure schedule.

In a double-blind comparative trial the immunogenicity of three new tissue culture rabies vaccines was evaluated, using a commercial human diploid cell vaccine (HDCV) lot as the reference. Two different vaccination regimens, a pre-exposure schedule, and an abbreviated 2-1-1 postexposure schedule (two doses of the vaccine applied bilaterally on day 0, with subsequent single doses given on days 7 and 21) were employed. In both, two of the new vaccines, purified chick embryo cell vaccine and purified Vero rabies vaccine, induced an antibody response equivalent to that of HDCV, while the geometric mean titres for the fetal bovine kidney cell vaccine were somewhat lower. The 2-1-1 regimen, a candidate regimen for economical rabies postexposure treatment, evoked a rapid and high titre antibody response with all four vaccines, peaking on day 14.

Étude de l'adsorption et de la pénétration du virus rabique: Interactions avec les cellules BHK21 et des membranes artificielles

Summary The binding of rabies virus and its entry into susceptible cells was studied using BHK21 cells and a liposomal membrane model of cellular constituents. When BHK21 cells were incubated with radiolabelled virus or purified glycoprotein, binding of viral material to cell surfaces was observed. Saturation of cellular membrane sites by unlabelled rabies glycoprotein did not inhibit binding of radiolabelled virus. This result suggested that the sites and/or the mechanism of binding of the glycoprotein molecules were independant, depending on whether glycoprotein was free in the incubating solution or anchored to the viral membrane. Binding of the virus to the cell membrane and entry of viral material were enhanced by incubating virus-cell mixtures in acidic conditions: the amount of virus radioactivity bound to cells was 10-fold higher at pH 5.4 than at pH 7.2, but the viral infectivity was the same. Furthermore, these two phenomenons required energy because lower amounts of viral radioactivity were detected when virus and cells were incubated at 4°C (instead of 20°C) with sodium azide. Attempts to saturate cell sites with unlabelled virus led to two types of results: (1) inhibition of binding of labelled virus by low concentrations. Rabies virus also bound to liposomes prepared with either cellular lipids alone or lipids and 0.01% Triton-X100 cellular extracts. This binding was specific only at pH 7.2 and only when virus was incubated with liposomes containing detergent extract. Under these conditions, liposomes could be partially saturated when incubated with unlabelled virus before contact with labelled virus. This observation suggests that virus bind to a detergent-soluble receptor. All these results led us to propose that the attachment of rabies virus to the BHK21-cell surface requires a specific receptor and that entry of viral material mainly involves a fusion of the viral membrane and the cell plasma membrane.

In vitro rabies vaccine potency appraisal by ELISA: Advantages of the immunocapture method with a neutralizing anti-glycoprotein monoclonal antibody

The replacement of the in vivo potency test (NIH test) for rabies vaccine evaluation by in vitro methods is at present discussed in many reports and also by WHO expert working groups. For this purpose, in vitro glycoprotein titration has been proposed. Among the different glycoprotein assays, we have studied two ELISA methods (immunocapture and direct plate coating with the antigen to be tested) using neutralizing mono- and polyclonal antibodies. In our view, the immunocapture method based on the use of a neutralizing monoclonal anti-glycoprotein antibody seems to be a convenient tool for the determination of the in vitro potency of rabies vaccine and of the products corresponding to the different steps of their production process.

Interleukin 2 increases protection against experimental rabies.

Vaccination with either whole inactivated rabies virus or immunosome (rabies glycoprotein anchored on liposomes) induces a high level of interleukin 2 (IL 2) production after in vitro specific stimulation of splenocytes from primed mice (9). On the contrary, infection with a live rabies virus does not specifically induce the production of IL 2: splenocytes from ill mice previously infected with wild rabies virus cannot be specifically stimulated by rabies antigens, whereas they can be non-specifically stimulated by a mitogen (Concanavalin A (Con A]. When injected in mice, exogenous IL 2 (purified rat IL 2 or human recombinant IL 2) exhibits an adjuvant effect on rabies virus vaccine or subunit vaccine tested in a pre-exposure potency test (NIH test). When injected in hamsters, according to a post-exposure potency test (infection with a wild rabies virus followed by vaccination), IL 2 has no adjuvant effect on the rabies vaccine. Nevertheless, when injected alone, IL 2 protects thirty to fifty percent of the infected animals treated (1 hour, 3 and 7 days post-infection) with 10 international units of human recombinant IL 2.

A rapid rabies enzyme immuno-diagnosis (RREID): a useful and simple technique for the routine diagnosis of rabies

A Rapid Rabies Enzyme Immuno-Diagnosis (RREID) technique has been developed. This technique for the diagnosis of rabies was performed in microplates which had been previously sensitized with IgG to purified antinucleocapsids. Suspensions of homogenized material were incubated in the plate and the specific binding of rabies antigen was revealed by the use of the same IgG conjugated with peroxidase. With the RREID technique it was possible to detect rabies antigens in brain specimens with the same specificity and sensitivity as that of the direct immunofluorescence test or the neuroblastoma cell inoculation technique regardless of the species of animal from which the specimen was derived. Moreover, RREID was performed with fox salivary gland specimens with the same results as were obtained with brain specimens. RREID does not require an UV light microscope and a photometer is not essential. It is a useful and simple technique for the routine laboratory diagnosis of rabies.