Immunological basis for protection in a murine model of tick-borne encephalitis by a recombinant adenovirus carrying the gene encoding the NS1 non-structural protein.

The humoral immune response to flaviviruses is mainly directed to the major envelope protein, E, and a glycosylated non-structural protein, NS1. Cell-mediated immune responses, however, appear to be directed mainly against non-structural proteins. Experiments described here show that a defective recombinant adenovirus (Rad51) containing the gene encoding the NS1 protein of tick-borne encephalitis virus can induce a strong protective immune response against several pathogenic tick-borne flaviviruses in an experimental animal model, and can enhance the efficacy of conventional vaccine preparations. A protective immune response against a lethal virus challenge can also be induced by the passive transfer of antibodies, B cells or T cells from animals vaccinated with Rad51. Raised levels of non-neutralizing antibodies and cytokines associated with a T helper cell-type 1 immune response are also observed. These data demonstrate the importance of non-structural viral proteins in the protective immune response against flaviviruses and support the use of non-structural viral proteins as vaccine components.

[A recombinant adenovirus expressing the NS1 nonstructural protein of tick-borne encephalitis virus: some characteristics of the immunologic basis of antiviral action]. / Rekombinantnyi adenovirus, ékspressiruiushchii nestrukturnyi belok virusa kleshchevogo éntsefalita NS1: nekotorye kharakteristiki immunologicheskikh osnov protivovirusnogo deistviia.

Recombinant adenovirus expressing NS1 nonstructural protein of trick-borne encephalitis (TBE) virus (Rad 51) protected mice from many strains of TBE and Omsk hemorhagic fever (OHF) viruses, but virtually did not protect them from Negishi virus. During combined use of whole-virion inactivated TBE vaccine and Rad 51 the recombinant adenovirus notably potentiated the protective effect of the traditional vaccine. The results of adaptive transfer of immunological material from mice infected with Rad 51 showed that both the vaccinated animals' sera and the pool of T and B cells partially protected the recipient mice from lethal TBE infection. NS1 protein expressed by adenovirus increased the level of the key interleukins (IL) interferon, tumor necrosis factor, IL-1 beta, IL-2, and, probably, IL-4. Vaccination of mice with Rad 51 resulted in the appearance of antibodies to NS1 protein in rather high titers. The prospects of using Rad 51 as a vaccine against TBE are discussed.

Rapid vaccination protocols for commercial vaccines against tick-borne encephalitis.

Although inactivated viral vaccines have been dramatically successful in controlling many of the world's most devastating diseases, they frequently need several injections to ensure high levels of protection, and thus their efficacy is reduced in many situations. We have developed several rapid vaccination protocols for two commercial vaccine preparations against tick-borne encephalitis virus and studied their efficacy in an experimental murine model. Vaccination protocols as brief as two doses given over two days elicit efficient protection against challenge with potentially fatal doses of virus and this protection is afforded as soon as 5 or as long as 100 days after the first vaccination. The very rapid induction of protection and the poor antibody responses observed would suggest that cell-mediated immune responses are the most important mechanisms for the protection elicited by conventional inactivated vaccines against tick-borne encephalitis.

Immunogenicity of inactivated purified tissue culture vaccine against hepatitis A (HepAvac) assessed in laboratory rodents.

Immune response of laboratory rodents (guinea-pigs, CBA and Balb/c mice, Wistar and August rats) to inactivated hepatitis A vaccine was quantitatively assessed. Under comparable conditions of experiment, the mice showed the highest antibody titres and were capable of reacting to the lower doses of immunogen; meanwhile their individual variations in immune response were more pronounced; white rats were the least susceptible to the vaccine, demonstrating the minimal antibody formation; guinea-pigs produced antibody at intermediate levels but the antibody titres were the most homogeneous. The enhancing effect of aluminium hydroxide was observed in guinea-pigs examined at the late postimmunization stage. Differences in immunogenicity of three vaccine lots were essentially similar when these lots were tested as undiluted preparations in guinea-pigs and mice for mean antibody titres and in mice for 50% immune response using serial dilutions of vaccine. All three tests could be routinely employed for vaccine immunogenicity control.

Immunogenicity trial of inactivated hepatitis A virus vaccine in human volunteers.

An inactivated hepatitis A virus (HAV) vaccine was tested on a group of human adult volunteers. The vaccine was administered subcutaneously, and a control group received a placebo (aluminium hydroxide). The vaccine was found to be relatively well tolerated and non-reactogenic, and levels of anti-HAV were comparable to those in other studies.

Use of protamine sulphate for elimination of substrate DNA in poliovaccines produced on continuous cell lines.

Cell substrate DNA was shown to be an abundant contaminant in the clarified preparations of the Sabin type 1, 2 and 3 poliovaccines produced on a continuous cell line (4647). The size of the DNA, as evaluated for the Sabin type 1 poliovaccine, was highly heterogeneous, ranging from 100 to 20,000 base pairs. In view of potential oncogenicity of this DNA a simple and efficient procedure for its elimination is proposed. The method is based on use of protamine sulphate which at the concentration of 2.0 mg ml-1 precipitated cell DNA almost completely without affecting the virus titres.

pH-dependent fusion of tick-borne encephalitis virus with artificial membranes.

pH-dependent fusion of TBE virus with artificial membranes was effective at slightly acidic pH with maximum at 6.4. The influence of various changes in E protein conformation on fusion process was studied.

Purification of tick-borne encephalitis virus in tris-modified porous glasses.

Macroporous glasses, chemically modified by tris-hydroxymethyl-aminomethane, were used for gel-permeation chromatography of tick-borne encephalitis (TBE) virus suspension. The highest yield of purified virus was observed in the carriers containing not less than 0.01 micrograms-equivalent/m2 surface-attached tris-hydroxymethyl-aminomethane groups at pH of the eluent higher than 7.6. The obtained modified porous carriers appeared to be suitable for the purification of both infectious and inactivated preparations of TBE virus.

Comparative study of infectious and formaldehyde-inactivated tick-borne encephalitis virus particles.

The structure and properties of infectious and formaldehyde-treated particles of tick-borne encephalitis virus, concentrated and purified by chromatography on macroporous glass, were studied. In addition to complete virions, such preparations contain some incomplete forms that differ in density, morphology and protein composition (incomplete forms do not contain nucleocapsid protein). The physico-chemical analysis of complete virions showed that formaldehyde treatment causes a) the formation of glycoprotein dimers and b) a portion of nucleocapsid protein to become tightly cross-linked with viral RNA. Formaldehyde treatment of incomplete forms resulted only in the formation of a small amount of glycoprotein dimers. Incomplete forms and glycoprotein extracted from inactivated preparations had protective and antigenic activity.

A study of candidate rabies vaccines for a national reference preparation for the evaluation of antigenic potency.

An unconcentrated and a concentrated lot of rabies vaccine prepared in Syrian hamster kidney cell culture from the Vnukovo-32 strain have been examined for their suitability as national reference preparations for rabies vaccine. The antigenic potencies of the preparations were assessed by the NIH test using both fixed and street strains of rabies virus and by the method of antibody induction. The candidate vaccines were calibrated in comparative assays with the WHO 3rd International Reference Preparation of Rabies Vaccine (IRP3). The correlation between the level of virus-neutralizing antibody and the resistance of mice to intracerebral challenge with a fixed rabies virus strain was studied. The candidate vaccines were also examined by the thermal degradation test. It was found that both vaccines had appropriate antigenic potency and thermostability to be used as a national reference preparation of rabies vaccine.

Virus-induced blast transformation of lymphocytes and its inhibition by F(ab1)2 fragments of human IgG.

Blast transformation of lymphocytes from persons immunized by vaccines against tick-borne Japanese encephalitides, in response to stimulation by homologous viral antigens was studied. 3H-Thymidine incorporation into lymphocytes was completely inhibited by F(ab1)2 fragments of normal human IgG containing no antibody to the viruses examined. A correlation of the inhibitory action of F(ab1)2 fragments on lymphocyte transformation induced by viruses and phytohaemagglutinin was observed.

Reevaluation of the proteins in rabies virus particles.

Protein content and localization of individual proteins of rabies virus have been studied. Four major proteins (estimated molecular weights, about 65,000, 54,000, 37,000 and 21,000), one minor component (molecular weight, about 200,000), and one intermediate (as regards its molar concentration) component (molecular weight, about 43,000) were revealed in rabies virus particles. In subviral particles accumulating in virus-infected cells, the 200,000-, 54,000-, and 37,000-dalton components were revealed. Some properties of the subviral particles allow them to be considered as viral nucleocapsids and the proteins composing them as analogs of L, N, and NS proteins of other rhabdoviruses. Thus, the protein composition of the rabies virus strain studied does not differ from that of other rhabdoviruses.