S V Gribencha’s research while affiliated with Ivanovsky Institute of Virology and other places

In experiments of curative vaccination, carried out with the use of an experimental model similar to the current practice of treatment with antirabies preparations, the advantages of using tissue-culture rabies vaccine with immunogenic potency equal to 1.3 international units (I. U.) were shown. In these experiments the vaccine was introduced into guinea pigs infected with fixed rabies virus, the course of vaccination consisting of 14 daily injections. No correlation between the induction of virus-neutralizing antibodies and the immunogenic potency of tissue-culture rabies vaccine was established: the use of the vaccine with immunogenic potency equal to 0,3 and 1,3 I.U. had no essential influence on the level of antibody formation in the animals.

Purified concentrates of rabies virus were prepared by both microfiltration chromatography and diafiltration methods. The diafiltration concentrate had a high level of protective activity and was not inferior to the microfiltration chromatographic one. The infectious activity was better retained after diafiltration, the chromatographic purity of both preparations was similar. It is concluded that diafiltration presents a good alternative to the currently used methods for purification and concentration of viruses.

The interaction of 7 monoclonal antibodies (MCA) to the nucleocapsid complex and 3 MCA to protein G of the vaccine virus Vnukovo-32 with 33 and 27 members of the rabies virus group, respectively, was studied. The indirect immunofluorescence test showed 7 MCA to the nucleocapsid complex to recognize 4 antigenic determinants (AD). Two MCA recognized the AD common for all the viruses under study. Individual ecological variants may be detected using other MCA. Three MCA to protein G had marked virus-neutralizing activity. MCA 1C5 neutralized all the rabies viruses under study. The other two MCA differed in the number of viruses they could neutralize. The viruses under study were divided into 4 groups depending on their interactions with certain MCA in neutralization test.

A series of hybridomas secreting monoclonal antibodies (MCA) to glycoprotein and nucleocapsid proteins of rabies virus strain Vnukovo-32 was selected as a result of fusion of splenocytes from immune BALB/c mice with cells of myeloma line Sp2/OAq14, screening and cloning by limiting dilution methods in semi-liquid agar. Four hybridomas secreted MCA to glycoprotein in high titres (5.0 x 10(5)-2.2 x 10(6)) and had marked virus-neutralizing and therapeutic properties. Eight hybridomas produced MCA to the nucleocapsid complex: five hybridomas secreted MCA of the G class in high titres (2.4 x 10(5)-1.6 x 10(6)) and three hybridomas secreted MCA of the M class in low titres.

In this work materials on the development of an experimental model for the study of rabies vaccines are presented. The comparative study of different immunization schedules for vaccines with different protective potency has been carried out. Guinea pigs infected with street rabies virus, strain k, were used as an experimental model. As shown in this investigation the optimum method of infecting the animals with strain k was intramuscular injection causing 50% mortality among the animals, the incubation period lasting 10-24 days. Only those tissue-culture rabies vaccines which had activity equal to 1.0-1.3 I. U. and, when injected into the animals, ensured survival rate ranging from 57% to 76%, depending on the immunization schedule, were shown to possess protective potency. It should be pointed out that survival rate among the animals receiving the preparation according to the reduced schedules recommended by WHO was higher than among those immunized daily for 14 days. In all groups immune response was observed. Still in the animals receiving the preparation according to the reduced schedules a higher level of virus-neutralizing antibodies was registered. Thus, an experimental model capable of being used for the evaluation of the quality of existing and newly developed antirabies preparations was obtained. Besides, we believe it to be expedient to carry out the field trial of rabies vaccines with activity equal to 1.0-1.3 I. U., using the reduced immunization schedules.

Experiments in CBA, mice immunized with inactivated unconcentrated cultural rabies vaccine (CRV) demonstrated that a clone of T-effectors of memory was formed within 30-37 days after primary immunization which reacted by the secondary type in response to inoculation of subthreshold doses of the rabies antigen. This clone of immunological memory seems to remain in the quiescent state for very long periods (the observation period 406 days). The immune splenocytes from the donors which had been given subthreshold doses of the antigen at intervals of 60, 103, 180 and 406 days, protected 2-4 times more animals than those after primary immunization with a threshold dose of the antigen. A continuous state of cell-mediated immunity--T-effectors processing the protective properties in vivo--may be maintained by repeated threshold doses of antigen given at 7-day intervals. The observed patterns of the induction of T-effectors possessing protective properties in vivo allowed some principles for construction of the optimal schedule of rabies vaccination to be formulated.

In the experiments on dogs inoculated intracerebrally with the "paralytic rabies" variant of the "Yak" strain of street rabies virus we observed spontaneous recovery of 1 out of 5 sick animals. Rabies in this dog was verified by three isolations of the virus from the saliva during the disease and by an increase of antibody titres in the time course of to 6776. In another experiment, among 7 dogs developing rabies after intracerebral inoculation with the "convulsive rabies" variant of the virus one animal showed recurrent form of rabies. The diagnosis of rabies was confirmed by isolation of the virus from the saliva in primary disease as well as by virus isolation from the salivary glands and the brain of the dog dying with recurrent rabies.

Street rabies virus strains can contain from one to three biological (clinical) variants as it has been estimated using random-bred white mice and dogs.

Daily administration of vaccine as well as combined use of antirabies gamma-globulin and vaccine were shown not to induce the production of immune T-killers possessing protective properties in adoptive transfer in mice pre-infected with street rabies virus in contrast to a single administration of a shock dose of antigen. Immune splenocytes protected 31-43% of mice infected with street rabies virus in a dose of 2-4 im LD50 and did not protect the animals infected with a dose of 12 im-LD50. After combined administration of immune splenocytes and an interferon inducer (double-stranded RNA) the additive protective effect was observed.