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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.

Two candidate reference rabies vaccines were tested in a collaborative study involving seven laboratories. Using four different testing procedures it was determined that both vaccine preparations were highly potent and demonstrated satisfactory stability in the thermal degradation test.

In a field trial 181 high risk persons of both sexes between ages of 2--71 years were prophylactically vaccinated against rabies with the beta-propiolactone inactivated rabies-vaccine (hdc) mrc-5. 1 ml of vaccine was administered intramuscularly on days 0, 7 and 21. Serum samples were collected on days 0, 7, 21 and 35. Neutralizing antibody titers against the rabies virus were determined in the rapid fluorescent focus inhibition test, 7 days after the first injection measurable titers of antibodies were found in all sera with one exception. On day 21 all persons showed a 100% seroconversion with a mean titer of 1:1000 (8,3 IU/ml). On day 35 the final mean titer measured 1:2400 (20 IU/ml). Major side effects during the prophylactic vaccination were not observed.

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.

126 patients underwent a post-exposure antirabies treatment using the beta-propiolactone inactivated (HDC) MRC-5 vaccine. The immunization schedule consisted of single doses of 1 ml of vaccine administered on days 0, 3, 7, 14, 30 and 90. Within the post-exposure group several patients suffered wounds from known rabid animals. Serum samples were collected on days 0, 14, 30, 90 and 104. Neutralizing antibodies against the rabies virus were determined in the rapid-fluorescent-focus-inhibition (RFFI) test. The rise in neutralizing antibodies was steeper than that in the prophylactic group whereas the mean titer at day 30 did not significantly exceed the values obtained with the preventive immunization scheme. A 1 ml booster injection on day 90 resulted in a three-fold increase of antibody titer within two weeks. Although no major side-effects were observed, an analysis of questionnaires filled out by the patients is presented.

Of the three major proteins associated with the rabies virus membrane, only the glycoprotein (G-protein) was found to be located on the external surface of the viral membrane. A minor glycoprotein (gp 50) detected by SDS-polyacrylamide gel electrophoresis (PAGE) of rabies virus appeared to be a breakdown product of the G-protein. Glycoprotein prepared by treatment of rabies virus with Triton X100 and purified by isoelectric focusing was found to be homogeneous with respect to size and isoelectric point. The apparent molecular weight of this component isolated under nondenaturing conditions is approximately 400,000 daltons. The same material analyzed by PAGE was found to be comprised solely of polypeptide chains of the G-protein (MW 80,000 daltons). Therefore, the Triton X100-released material represents homopolymers of the G-protein. The purified rabies virus glycoprotein (G) is only structural protein of the virus that induces the formation of virus-neutralizing antibodies and which confers immunity to animals. The total protective activity of the virus was recovered in the purified G protein preparation. The protective activity of G protein increased with purification : 9 ng of G protein was required to protect 50% of the mice as compared to 1.63 microgram of the virus. The number of oligosaccharide side chains on rabies virus glycoprotein was investigated. Analysis of glycopeptides obtained by protease digestion of desialated glycoprotein revealed three discrete glycopeptides. Comparison of the protease digestion products from desialated and from untreated glycoprotein indicated a heterogeneity among the glycopeptides in the sialic acid content. Two major tryptic glycopeptides were isolated from desialated rabies virus glycoprotein and were analyzed after protease digestion; one contained two oligosaccharide side chains and the other contained a single oligosaccharide side chain.