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Фундаментальные исследования

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A.V. Dubov

We are offering and developing a hypothesis on common and transbarrier (hystohematogenous barriers) cerebrospinal immunity in pantropic virus infections. Common humoral and cellular immunity does not make improbable the penetration of virulent neurotropic viruses, such as tick-borne encephalitis pathogen, through hematoencephalitic barrier and the development of acute virus infection or virus persistence with the formation of chronic or slow infection process. Makers of specific antivirus vaccines should take this possibility into account.

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Background

There is proof of pantropic character of tick-borne encephalitis (TBE) reproduction as well as similar diseases in inner organs and central nerve system (CNS) (1, 2).

The experience in vaccine prophylaxis against the diseases, caused by these viruses, testifies on high epidemic efficiency of TBE inactivated cultural vaccine, especially in western hotbeds of infection circulation area (3-7). Nevertheless vaccinated subjects show TBE slight clinical course, which is the evidence of virus penetration into CNS organs through hematoencephalitic barrier. We revealed chronic slow course TBE in some vaccinated subjects after aparalytic TBE or being healthy in half a year, a year or more after TBE inoculation. Poliomyelitic form, Kojewnikoff´s (cortical) epilepsy, amyotrophic lateral sclerosis syndrome are among these forms (2, 8-9).

We are offering and developing the conception of common, barrier (hystohematogenous barriers) and transbarrier (cerebrospinal) specific immunity in pantropic virus infections (10, 11). Common immunity is being formed under natural immunization or vaccination apart from CNS up to hystohematogenous (hematoencephalitic) barrier, but this does not protect target-organs. Cerebrospinal immunity starts to develop only after virus or virus antigen penetration into CNS organs and tissues.

The efficiency of TBE specific prophylaxis is based on the following: antigen and immune gen activity of specific antivirus vaccines; the ability of wild virus population to penetrate into CNS organs from introduction point; the intensity of reproduction in extraneural system; penetration through hematoencephalitic barrier; tropism to nerve tissue (12,13).

Materials and methods

In our tests we used BALB/C white mice weighing 8 to 10 gram from Rapolovo nursery (Leningradskaya oblast), adult Macacus rhesus, delivered by plane from India, who were in quarantine and passed adaptation in vivarium of virology laboratory for at least 30 days. Virus indication and titration was produced on mice in intracerebral infection. We used TBE highly virulent strains (Sofyin, Pan and Absettarov) and naturally slowed Elantzev strain (15 - 20/ 3 clone).

Immunization was performed by inactivated vaccine (producer - M.P.Chumakov Poliomyelitis and Virus Encephalitis Institute of Russian Academy of Medical Sciences) and/ or naturally slowed Elantzev strain (15 - 20/ 3 clone), were inoculated in 4.0 - 4.4 lg LD₅₀ concentration to macaques hypodermically or into head brain (left thalamus). In 1 to 2 months after the immunization the injections were repeated into left thalamus: virulent strains or Sofyin (5.7 - 7.8 lg LD₅₀) or Pan (6.7 - 7.7 lg LD₅₀) or Absettarov (3.9 - 8.0 lg LD₅₀).

Table 1. The results of many days monitoring for virus accumulation in different organs and tissues in white mice of BALB/ C line, inoculated hypodermically with Absettarov highly virulent strains of TBE virus.

Results

The tests for mice of BALB/ C line with high virulent strains of TBE virus (Sofyin, Pan and Absettarov and others) resulted in the same characteristics of the given strains (2). So, in 3 days after hypodermic inoculation, the number of virus in mice brain reached 3.5 lg LD₅₀/ 0.03 ml. During the first three days the number of the virus in inner organs was relatively higher, than in brain. The most concentration of the virus in inner organs blood was marked up to the moment of clinical symptoms development in mice (the 5^th day after inoculation). Before the death (the 7^th day after inoculation) there was considerable lowering of virus concentration in inner organs and blood. In head brain it increased greatly from 5.5 up to 7.2 lg LD₅₀/ 0.03 ml (Table 1). At all the stages viremia was tense. The curve of virus content in blood corresponded to the one in organs, rich with reticulo endothelium cells.

Table 2. The results of many days monitoring of virus accumulation in different organs and tissues in white mice, inoculated hypodermically with TBE virus Elanzev strain.

Dynamics of accumulation and distribution of naturally slowed Elantzev strain (15 - 20/ 3 clone) in hypodermically inoculated white mice of BALB/ C line had its own peculiarities.

In a day after the inoculation the virus was marked in all examined inner organs and tissues excluding head brain in white mice. The majority was isolated from organs, rich with reticulo endothelium elements: spleen, lymph nodes (Table 2).

In three days after inoculation the virus was marked in all the examined organs and tissues. It was few in head brain as compared to inner organs. Even in 5 days after inoculation the number of the virus in head brain was 100 times less in Elantzev strain than in Absettarov strain in the same period. From the 9^th to 12^th day in survived mice there was considerable lowering of virus concentration in both CNS and extraneural organs.

We studied the dynamics of prevalence and accumulation of TBE virus in BALB/C mice, inoculated with inactivated vaccine and naturally slowed Elantzev strain (15 - 20/ 3 clone) in different types of tests.

a) Twofold intraperitoneal immunization with inactivated vaccine with 7 to 8 days interval. Then hypodermic inoculations in 14 to 15 days with Sofyin, Pan, Absettarov and others virulent strains (Table 3).

The accumulation of virulent virus in the mice, inoculated by 2-times inactivated vaccine, differed greatly from non-immunized ones. Virulent virus penetrated into animal head brain, protractedly reproduced and persisted.

b) Twofold intraperitoneal immunization of mice of BALB/ C line with inactivated vaccine with 7 to 8 days interval. Then in 14 to 15 days hypodermic inoculation with 10 000 LD₅₀/ ml with naturally slowed Elantzev strain (Table 4).

Table 3. The results of many days monitoring for virus accumulation in different organs and tissues in BALB/C white mice, inoculated twice with inactivated vaccine and injected hypodermically with 10 000 LD₅₀/ ml Absettarov strain virulent virus.

During multiple tests we revealed very rarely the penetration of naturally slowed Elantzev strain into mice brain, mainly in 9 days after hypodermic inoculation. The reproduction of virus was not marked during 30 - days monitoring.

In tests for Macacus rhesus after extraneural inoculation (hypodermic, intravenous) with both naturally slowed Elantzev strain and highly virulent Sofyin and Pan and Absettarov we didn´t registered the disease. Highly virulent strains reproduced actively in inner organs, penetrating into CNS, where we marked their protracted persistence  (Table 5).

We didn´t found virus penetration into CNS organs in macaques, inoculated extraneurally with high concentrations of naturally slowed Elantzev strain (4.0 - 7.7 lg LD₅₀/ ml) (Table 6).

Table 4. The results of many day monitoring for virus accumulation in different organs and tissues in BALB/ C white mice, inoculated twice with inactivated vaccine and injected hypodermically with naturally slowed Elantzev strain

Table 5. The results of many days monitoring of virus accumulation in macaques, inoculated intravenously with TBE virus Sofyin strain (in lg LD₅₀ )

Macaque extraneural inoculation (immunization) with both TBE virus high virulent and naturally slowed strains lead to intensive production of specific virus neutralizing and antihemagglutinating antibodies. But repeated inoculation for macaques head brain with VE virus high virulent strains didn´t protect against mortal encephalitis in 7 to 10 days after inoculation.

After initial inoculation to the left thalamus with Absettarov, Pan or Sofyin strain, severe panencephalitis was developed with macaques death on the 7^th to 10^th day with intensive virus reproduction in different CNS organs (Table 7).

In macaques, dissected on the 3^rd, 8^th, 15^th and 30^th day after intracerebral inoculation with Elantzev strain, we marked its weak reproduction in CNS organs (Table 8).

All macaques, momentary immunized with naturally slowed Elantzev strain into the left thalamus and then in 1 to 2 months inoculated into head brain with large concentrations of TBE highly virulent viruses, were not ill during 30 days of monitoring and farther.

Table 6. The results of many days monitoring of virus accumulation in macaques, inoculated intravenously with TBE virus Elantzev strain (in lg LD₅₀ )

Table 7. The number of virus in lg LD₅₀/ml on the level of clinical signs on the 7^th day after inoculation of macaques into left thalamus with (M+m) strains.

In animals, immunized into head brain with Elantzev strain and then repeatedly inoculated by Sofyin, Pan or Absettarov strains on the 8^th, 15^th and 30^th day after virulent strain inoculation, there were neither slowed, nor virulent TBE viruses being determined in CNS and inner organs.

In Macacus rhesus, immunized into head brain (left thalamus) with inactivated vaccine against tick-borne encephalitis or naturally slowed Elantzev strain, there was pericyte proliferation around capillaries and precapillaries with the formation of perivascular ferrule. Cells are located as lines, forming the gathering, clearly confined from the rest of brain tissue. They show clear mitosis pictures. In the structure of perivascular ferrule there are large cells of reticular type with friable cytoplasm, nuclear and pirocytophilic neucleolus as well as the cells of lymph histocytic type, plasmoblasts and "ripe" plasma cells.

Table 8. The results of many day monitoring for virus accumulation in macaques, injected into left thalamus with Elantzev strain (clone 15-20/ 3) TBE virus(in lg LD₅₀ )

Rather similar morphological formations were marked in lymph nodes during the first ten days after extra neural immunization in macaques. We found plasmatic cells of transition type in medullated cords. They are large polygonal cells with pyrononinophilic cytoplasm, oval-shape light nucleus with basophilic neucleolus, appeared as a result of reticular cells transformation. Later monitoring (30 to 54 days after TBE virus inoculation) showed ripe plasmatic cells in cortical and cerebral layers of lymph nodes on the level of plasmoblasts. In some cases the last were bedded abundantly in medullated cords, replacing lymphoid tissue. Free phagocytes were often revealed in follicle sinuses and reactive centers.

So, we showed that immunity morphology in peripheral lymph nodes is similar to one in CNS as a result of perivascular ferrules formation in infection process. This is the basis of the assumption, that perivascular ferrules in CNS play the role of morphofunctional lymph nodes.

Conclusion

We revealed two independent types of immunity formation in neurotropic virus infection: common (extraneural) and off - hematoencephallic barrier (cerebral spinal). Both immunities develop independently only after the penetration of virus or virus antigen into target organs. Common immunity leads to the lowering of reproduction activity of virus virulent populations, but it does not provide its complete elimination in CNS organs. This forms the conditions for virus persistence, leading in some cases to chronic or slow forms of the disease.

Extraneural common immunity, being induced by inactivated vaccine against TBE does not provide reliable blockade against virus virulent populations in extraneural system and does not stop its penetration through hematoencephalitic barrier, which leads to virus persistence in CNS organs and tissues and slows TBE in some cases.

At the same time the immunity, induced with inactivated vaccine, is enough for the blockade of naturally slowed viruses penetration from extraneural system to CNS organs and tissues. On grundimmunity background, created by inactivated vaccine with further introduction of live vaccine, the reliable protection against TBE virus virulent populations is provided. In this connection the necessity to turn to combinatory vaccination appears again, as well as the creation of grundimmunity by inactivated vaccine with final immunization by safe but highly immunogenic live vaccine against TBE, which surely protects from western and eastern genotypes of TBE virus, and TBE complex agents.

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