[Preparation of an immunizing glycoprotein from inactivated rabies vaccine (author's transl)].
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Biomedical subjects
Publications and source records attributed to P Atanasiu.
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From a structural point of view an essential distinction between complete and defective rabies viruses is difference in size. In addition, isoelectric properties differ. The complete virus has an isoelectric point approaching neutrality, whereas the defective virus focuses between pH 3-4.7. The isoelectric points of the glycoprotein from complete and defective viruses differ in a corresponding fashion. The Pasteur virus cultivated on BHK21C13 cells, contains glycoproteins, the glycopeptides of which have a structure containing the following five monosaccharides: sialic acid, D-galactose, (N-acetyl)D-glucosamine, D-mannose and L-fucose. The glycosylation of the glycoproteins is different, at least in so far as the relative sialic acid/glucosamine ratio is concerned.
Immunological survey of 3 patients with proved rabies encephalitis shows three interesting facts. 1) An IgM local synthesis, sometimes proportionally higher than IgG local synthesis, is observed in 8 CSF (among 13 investigated) and detected early, during the first week of the disease for 2 patients. 2) A relative poor IgG response is noted; this response is absent in one and decreasing in the 2 others patients despite their progressive aggravation. 3) A very little elevation of blood nucleic antibodies is detected in 5 CSF only. This very poor synthesis of antinucleic IgG contrast with their common increase in viral encephalitis. Thus, a viral immunosuppression may be discussed in rabies encephalitis, which may contribute to the lethal prognosis.
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Techniques usually employed for the detection of rabies' antibodies are costly, time consuming, and sometimes fail to detect early antibodies. The introduction of immunoenzymatic techniques in the serology of viral disease represents a new and important advance. We therefore adapted this technique to the detection of rabies antibodies. We have found that the sera from rabies patients who had not received antirabies treatment do not show seroneutralizing antibodies until several days after the onset of symptoms. However, antibodies can be detected some days earlier by the immunoenzymatic method in the same samples. Furthermore, the immunoenzymatic test was applied to the detection of both the IgM or the IgG class of antirabies antibodies using an antihuman Ig-or antihuman IgG-peroxydase conjugate.
In vitro multiplication of rabies virus was inhibited by a condensed mineral ion, ammonium-5-tungsto-2-antimoniate (HPA 23). The inhibitory effect was evaluated by two different methods, plaque reduction and one step virus growth. Plaquing showed 50% inhibition with 4.5 microgram/ml of HPA 23 and complete inhibition with 12.5 microgram/ml. A reduction of two logs in virus yield was obtained in BHK21C13S cells in suspension treated with 50 microgram/ml of HPA 23. Inhibition also occurred when treatment with HPA 23 was started 18 to 24 h after infection in the plaque assay but no effect was seen when HPA 23 was added 48 h after virus inoculation. All these inhibitory effects of HPA 23 on rabies virus multiplication were observed at non cytotoxic doses. Therefore HPA 23 contrasts with other antiviral drugs which do not inhibit rabies virus multiplication without affecting the viability of cells.
Eleven laboratories from eight countries and four continents took part in a collaborative study to evaluate experimental procedures to be used in selecting the new standard reference rabies vaccine prepared in human diploid cell cultures. The following procedures were used : (a) the NIH potency test in mice, (b) the antibody binding technique (by either mouse inoculation or the tissue culture method), (c) virus-neutralizing antibody levels in mice used for the NIH test, and (d) antibody induction in human volunteers treated with vaccine alone and in combination with human rabies gamma globulin. The four methods used for determination of rabies antibodies were mouse inoculation, rapid fluorescent focus inhibition, plaque reduction and complement fixation techniques. All results were expressed in International Units as compared to the standard WHO serum and vaccine preparations. In general, a close correlation was noted in results from different laboratories, and it was recommended that the future rabies standard vaccine should be evaluated by three testing procedures, the NIH test, the antibody-binding technique, and antibody levels in mice used for the NIH test.
The comparative studies undertaken by 7 laboratories in 6 countries show that the calculation of I.U.'s did not, as anticipated, minimize but actually enhanced the variability of results of rabies antibody estimations in the sera of HDCS vaccinees. The high biological variance in the method(s) may not have been considered by individual laboratories and any neglect of fundamental biostatistical laws unfortunately diminishes the theoretical advantage of using the "International Standard (I.S.)" as a "tertium comparationis". Perhaps the intrinsic variability of the I.S. should be re-evaluated and it is conceivable that a pure IgG fraction of rabies antiserum would show less variability. Intralaboratory variation might be reduced by agreeing that only a geometric mean of the I.S., and not a single value obtained in an individual test, should be used for calculation of I.U.'s. Application of the principles of biochemical and pharmacological methods, such as test-to-test control of the I.S. and its analytical variances might well enhance the reproducibility of the results. MNT, RFFIT, PRT and CFT were unable to detect antibodies in HDCS vaccinees until 7 days after the first vaccination. The establishment of methods for detecting early antibody requires further investigation.
This report is concerned with the application of the enzyme immunoassay to measure the antibodies in humans vaccinated against rabies or presenting symptoms of rabies without having been vaccinated. By the same technique we identify the IgG and IgM classes of antibodies. The antigen (5 microgram/ml), purified virus, is readily adsorbed into polystyrene tube by passive adsorption. The use of only one dilution for each serum assay (1/200) is particularly suitable for epidemiological studies. Antibody response of subjects in the course of rabies vaccination was an obvious application. After 5 inoculations of tissue culture vaccine the IgM response was poor and late; it was even negative in two cases. The IgG response appeared early on the 7th day. In the same way we tried to follow antibody response in three cases of rabies in man. Seroneutralisation (SN) antibody were not detected at the beginning of the illness. In case 1 antibodies were found on the 12th day, in case 2 on the 7th day and in case 3 on the 8th day. When we assayed the serum samples for immunoenzymatic test, we found that the sera became positive some days earlier: on the 5th for case 1, already on the 1st day for the two others. In each of these three cases the positivity of the test corresponded to the presence of IgM class globulins since IgG detection remained negative as did the SN test. Our results could have some clinical interest concerning future rabies treatment and early diagnosis.
An inactivated human rabies vaccine prepared on bovine fetal kidney cells is concentrated and purified by zonal centrifugation. The peak of rabies particles is monitored by hemagglutination. Immunogenicity of the purified particles was evaluated by titration of specific antibodies from vaccinated animals. Protective activity of the vaccine was assayed on guinea pigs challenged with street rabies. Biological results were compared with those obtained with other tissue culture vaccines.
The comparative studies undertaken by 7 laboratories in 6 countries show that the calculation I.U.s did not as anticipated minimize but actually enhanced the variability of results of Rabies antibody estaminations in the sera of HDCS vaccines. The high biological variance in the method(s) may not have been considered by individual laboratories and any neglect of fundamental biostatistical laws, unfortunately, diminishes the theoretical advantage of using the "International Standard (I.S.)" as a "tertium comparationis". Perhaps the intrinsic variability of the I.S. should be re-evaluated and it is conceivable that a pure IgG fraction of Rabies antiserum would show less variability. Intralaboratory variation might be reduced by agreeing that only a geometric mean of the I.S., and not a single value obtained in an individual test, should be used for calculation of I.U.s. Application of the principles of biochemical and pharmacological methods, such as test-to-test control of the I.S. and its analytical variances might well enhance the reproducibility of the results. MNT, RFFIT, PRT and CFT were unable to detect antibodies in HDCS vaccinees until 7 days after the first vaccination. The establishment of methods for detecting early antibody requires further investigation.
The peripheral lymphocytes of vaccinated men were stimulated by homologous antigen or its subunits (nucleocapsid and glycoprotein) or by some parent viruses (Mokola and Lagos). All subjects produced a high level of antibody. A difference was noted between the two types of immunity. A lack of cell-mediated immunity could explain the very few failures in Pasteur antirabies treatment.
A glycoprotein was extracted with Triton X100 from rabies virus grown in primary foetal bovine kidney cells. This glycoprotein was further purified by iso electro focusing and showed a major peak at pH 7,0 and a smaller peak at pH 4,6. Purified fractions were migrated on polyacrylamide gels and assayed for immunogenicity.
Rabbit lymphocytes obtained from animals previously exposed to rabies virus undergo a specific lymphoblastic transformation when incubated in vitro in the presence of either the complete virus or a nucleocapsid fraction. This type of in vitro transformation was observed by the Pasteur, Lagos, Mokola, Obodhiang viruses as well as the virus HEP in cells originally exposed only to the virus HEP. In additions to the morphological changes of lymphoblastic transformation, 3H-thymidine incorporation was stimulated about 21 to 67 fold in homologous system and about 6 to 39 in heterologous systems and unrelated with other virus system like influenza. These results suggest an immunological relationship between the classic rabies strains (HEP, Pasteur) and subgroup rhabdovirus (Lagos, Mokola, Obodhiang).
The rabies virus and its glycoprotein are used as antigens in a microimmunoenzymatic assay (IEA). Human rabies antibodies are detected and titrated using anti-human antibodies and staphylococcus protein A conjugated to peroxidase. Several mammal antirabies sera are tested by this method with protein A. The results are compared to those obtained by seroneutralization in mice, which is the standard method for rabies antibody detection.
Rabies cell culture vaccines are able to induce circulating interferon in human sera. In 8/15 cases a low peak of interferon appears in the serum about 8 h after the vaccination. The inhibition has been considered as due to interferon because of the resistance to pH 2 and lack of activity on other animal species.
Lipid-free cells of Nocardia opaca and a cell extract (NWSM) are interferon inducers in syrian hamsters: peak interferon appears 2 h after injection as compared to 8 h in the case of interferon induced by inactivated NDV virus. The interferon induced by Nocardia product is inactivated by a treatment for 30 min at 56 degrees C or 24 h at pH 2: NDV-induced interferon is more stable under these conditions. Hamsters inoculated with NWSM before and after infection with rabies virus are partially protected: their survival time is slightly increased.