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The review contains a brief analysis of the results of investigations conducted during 40 years after smallpox eradication and directed to study genomic organization and evolution of variola virus (VARV) and development of modern diagnostics, vaccines and chemotherapies of smallpox and other zoonotic orthopoxviral infections of humans. Taking into account that smallpox vaccination in several cases had adverse side effects, WHO recommended ceasing this vaccination after 1980 in all countries of the world. The result of this decision is that the mankind lost the collective immunity not only to smallpox, but also to other zoonotic orthopoxvirus infections. The ever more frequently recorded human cases of zoonotic orthopoxvirus infections force to renew consideration of the problem of possible smallpox reemergence resulting from natural evolution of these viruses. Analysis of the available archive data on smallpox epidemics, the history of ancient civilizations, and the newest data on the evolutionary relationship of orthopoxviruses has allowed us to hypothesize that VARV could have repeatedly reemerged via evolutionary changes in a zoonotic ancestor virus and then disappeared because of insufficient population size of isolated ancient civilizations. Only the historically last smallpox pandemic continued for a long time and was contained and stopped in the 20th century thanks to the joint efforts of medics and scientists from many countries under the aegis of WHO. Thus, there is no fundamental prohibition on potential reemergence of smallpox or a similar human disease in future in the course of natural evolution of the currently existing zoonotic orthopoxviruses. Correspondingly, it is of the utmost importance to develop and widely adopt state-of-the-art methods for efficient and rapid species-specific diagnosis of all orthopoxvirus species pathogenic for humans, VARV included. It is also most important to develop new safe methods for prevention and therapy of human orthopoxvirus infections., Competing Interests: The authors declare no conflict of interest.

Studies of the primary cultures of granulocytes, mononuclear, and monocyte-macrophage cells derived from human blood were performed using variola virus (VARV) in the doses of 0.001-0.021 PFU/cell (plaques-forming units per cell). Positive dynamics of the virus accumulation was observed only in the monocyte-macrophages with maximum values of virus concentration (5.0-5.5 Ig PFU/ml) mainly within six days after the infection. The fact of VARV replication in the monocyte-macrophages was confirmed by the data of electron microscopy. At the same time, virus vaccines when tested in doses 3.3 and 4.2 Ig PFU/ml did not show the ability to reproduce in these human cells. The people sensitivity to VARV as assessed from the data obtained on human monocyte-macrophages corresponded to -1 PFU (taking into account the smooth interaction of the virus in the body to the cells of this type), which is consistent to previously found theoretical data on the virus sensitivity. The human susceptibility to VARV assessed experimentally can be used to predict the adequacy of developed smallpox models (in vivo) based on susceptible animals. This is necessary for reliable assessment of the efficiency of development of drugs for treatment and prophylaxis of the smallpox.

The review gives data on the history of smallpox vaccination and shows the high topicality of designing the current safe vaccines against orthopoxviruses. Four generations of live smallpox, protein subunit, and DNA vaccines are considered. Analysis of the data published leads to the conclusion that it is promising to use the up-to-date generations of safe smallpox subunit or DNA vaccines for mass primary immunization with possible further revaccination with classical live vaccine.

The seeding and working banks of a 4647-cell culture have been created. The 4647-cell culture in these banks has a high proliferative activity, as well as the morphology, typical of this line, and the karyotype and the enzymogram, which are characteristic for the cells of an African talapoin (Cercopithecus aethiops). The culture is not contaminated with bacteria, fungi, Mycoplasma, and viruses, including oncoviruses. The deposited 4647 cells have high viral productive properties for the accumulation of the recombinant virus strain b7,5S2-S vaccine and keep the stability of all biological properties during a long-term cultivation. The continuous 4647 cell line was tested at the L. A. Tarasevich State Institute of SK. The seeding and working banks of 4647-cell culture at passages 108 and 128 are recommended as a substrate for cultivation of the strain b7,5S2-S vaccinia, used to prepare a bivaccine against smallpox and hepatitis B.

Clinical trials of oral live recombinant embryonic variola and hepatitis B bivaccine as tablets (Revax-BT) were performed. When volunteers were prevaccinated with oral variola vaccine first in a small dose and, 7, 14, 30, 90, and 180 days later, in a larger dose, a slight reactoginicity was sometimes observed after the first vaccination (with a small dose) whereas revaccination with a larger dose did not give rise to any clinical manifestations. A month after vaccination, a protective level of virus-neutralizing antibodies to vaccinia virus (VV) was observed in 90-100% of the volunteers twice immunized with the bivaccine (in a small dose and in a larger one at an administration intervals of 1-2 weeks under remote revaccination while 6-9 months following vaccination, this level was recorded in 80% of the volunteers. A month following vaccination, 50-55% seroconversion to VV was observed in the volunteers twice immunized with the bivaccine (at an interval of 1 or 3-6 months). Cellular immunity to VV was low (0-20%). Double immunization of volunteers with the oral bivaccine under remote vaccination failed to produce the significant levels of humoral and cellular immune responses to hepatitis B markers. Recombinant VV was not recorded in any blood, saliva, and urine samples taken in the volunteers twice immunized with the bivaccine.

Nowadays there are certain facts which evidence the necessity of return to smallpox vaccination in Russia, especially concerning people who will be engaged in the liquidation of the nidi of this infection, should such appear. Smallpox virus is kept in laboratories of some countries; there is a real threat of biological terrorism; population immunity to smallpox infection is virtually absent. Considering the present situation in development and production of smallpox vaccines and remedies against vaccine-related complications, the tactics of population vaccination in Russia may vary. The main problems concerning return to smallpox vaccination in Russia are the following: there are no modern schemes (schedules) of smallpox immunization; there are no currently available and safe vaccines for primary immunization of adults; there are no preparations for prevention and treatment of vaccine-related complications. Thus, the priority areas are: development of effective and safe oral adult vaccines for primary immunization; production of human and heterologous immune globulins and their storage.

The reactogenicity of the embryonic live recombinant variola and hepatitis B bivaccine as tablets (Revax-BT) as well as its safety and immunogenicity were evaluated in clinical trials made in volunteers who had previously immunized or not with variola vaccine. A preliminary conclusion was made on a lack of side effects and drug safety in primary vaccination and been revaccination with low and high doses. Primary immunization of volunteers and as bivaccination with high doses stimulated the most pronounced immune response to the vaccine virus versus such effect observed in immunization of volunteers with low vaccine doses. Humoral immune response to HBs was observed in 75% of volunteers of both groups after as bivaccination. Such response was most pronounced in examinees immunized with low vaccine doses versus those who received high bivaccine doses. At the same time, no protective levels of humoral immunity response to HBs Ag were observed in volunteers first vaccinated.

The results of fundamental and applied investigations on the development and trial of the oral administration of smallpox vaccine and live recombinant smallpox-hepatitis vaccine (Revax VT) in tablets are summarized. In comparative experiments on animals (rabbits, monkeys and guinea pigs) and human immunization the oral smallpox vaccine in tablets was shown to ensure equal effectiveness and greater safety in comparison with traditional smallpox vaccine for skin application. The study confirmed the natural and physiological character of oral immunization as a result of direct contact of immunogen with the mucous membrane of the digestive tract--an essential immunocompetent organ of the lymphoid system. The conclusion was made that oral immunization was the safe and most promising method of immunization against smallpox under modern conditions.

Experimentally, by stimulation of the activity of serum DNase and RNase direct evidence of their nonspecific protective role in herpes and influenza infections, respectively was obtained. Potentiation of the protective effect upon interaction of endogenous and exogenous nucleases with specific antibodies was demonstrated. Experiments in mice and observations in humans established a marked inhibition of serum RNase activity due to binding with the inhibitor, in mild and severe forms of influenza in nonimmune hosts and host immunized with live and inactivated vaccines. This is assumed to be one of the causes of insufficient effectiveness of artificial immunity against influenza.

The selective survey of the population of the Republic of Congo for the presence of antibodies to orthopoxviruses has been carried out with the use of the neutralization test, the hemagglutination inhibition (HAI) test and the ELISA. Despite a prolonged period (15 years) elapsed since the transmission of natural smallpox stopped in this country and despite the almost complete cessation of immunization against this infection since 1977, antibodies to orthopoxviruses can be detected in a considerable proportion of the population: 29%. This percentage grows as older age groups are examined, reaching 90.6% in the age groups of 16 years and over. Antibodies to orthopoxviruses have also been detected in children under 5 years of age, born after the eradication of smallpox and having no vaccination scars. The possible causes of this phenomenon are discussed. The comparison of the results obtained with the use of different tests has confirmed high sensitivity of ELISA. The HAI test is less sensitive, but this is compensated by its simplicity and its easy use for screening procedures. Besides, the positive results of this test indicate that the corresponding sera contain sufficiently high titers of virus-neutralizing antibodies detected by means of ELISA, which is of importance for their subsequent interspecific differentiation.

The authors present the results of studying the PHAT sensitivity in comparison with the neutralization test and the hemagglutination inhibition test in examination of 254 sera of revaccinated and 95 sera of primarily vaccinated children. It appeared that PHAT was characterized by a sufficiently high sensitivity, and reproducibility, in case the same batch of erythrocytic diagnostic agent was used; at the same time the test was simple, accessible, economic, and gave rapid results. This test can be used for assessment of the immunological efficacy of smallpox revaccination.

The correlation between the reactogenicity and immunogenicity characteristics of different smallpox vaccines, determined under the conditions of a strictly controlled field trial, has been studied by a number of laboratory tests. The most informative and reproducible laboratory methods for evaluating the provisory antigenic and immunogenic potency of smallpox vaccines are the evaluation of the character of lesions developing in rabbits as a result of the intradermal injection of the virus and the determination of the capacity of the virus for multiplication in the skin of a rabbit (reproduction). The most informative and reproducible method for evaluating the provisory reactogenicity of smallpox vaccines for humans is the determination of the capacity of the virus for multiplication at elevated temperature.

The authors sum up the results of decade of investigations on the elaboration and trial of live oral smallpox vaccine in tablet form. A conclusion was made that due to mild reactogenic properties, satisfactory immunogenicity and weak allergy producing properties, the mentioned preparation, along with a possibility of express and mass use, can posses future prospects under conditions of final smallpox eradication.

Smallpox vaccine injection to 2-month-old C57BL/6j female mice caused during the first days significant rise in the number of precursor cells of rosette-forming lymphocytes, sensitive to the differentiating thymus extract effect. The number of these cells returned to the normal level by the 10th day after vaccination.

Monkeypox virus causing in M, rhesus upon aerogenic infection a disease similar to human varioloid was used to evaluate the intensity of immunity against smallpox in immunized M. rhesus monkeys. Postvaccination immunity was solid in all the animals vaccinated intradermally or orally. In 2 out of 14 monkeys immunized orally, however, the immunity was partially overcome. Neutralizing antibody titers in these two monkeys were 1:5 and 1:25, respectively. This agreed with the observations made in Pakistan indicating the possibility that some humans having serum neutralizing antibody titers up to 1:32 could contract smallpox and develop the disease.

Of 20 suckling rabbits, 4-5-days old, inoculated with live smallpox vaccine intradermally 6 displayed symptoms of generalized pox virus and neuroparalysis complications. Intensive accumulation of specific antigen in the brain, lungs, spleen, and the lymph glands was revealed by immunofluorescent method. The smallpox vaccine virus was isolated from these organs. Prolonged persistance of the attenuated smallpox virus was observed in the brain, spinal cord, lungs, spleen, and the lymph glands of 14 suckling rabbits showing no signs of any disease; specific antigen was revealed by immunofluorescent test. Vascular disturbances and slight cell changes were observed in the brain tissue of the inoculated animals. These changes were more severe in the sick animals.

In this work the multiple puncture method (in 76 cases) was compared with the scarification method--2 clinear cuts (in 70 cases). "Vaccine take" of the vaccines demonstrated a statistically significant difference: by the first method the percentage of personal "vaccine take" was 98.7, and by the second--86.4. The multiple puncture method produced a less pronounced local and general reaction. Immunological efficacy of the multiple puncture method (evaluated by hemagglutination inhibition test and neutralization test in the tissue culture) was quite satisfactory and failed to differ from such in vaccination by scarification.

As a result of observations carried out on children the authors present immunological characteristics of two-stage smallpox vaccination at different intervals (1 to 60 days) between the injection of inactivated and live vaccine. There proved to be acceleration and intensification of antibody formation after two-stage immunization in comparison with the rutine vaccination. A seven-day interval between the injection of the inactivated and live preparations was recommended on the basis of the data obtained.

Dynamics of accumulation and preservation of antibodies detectable in the PHAT (PHAT-AT) was studied on rabbits and guinea pigs with the use of various doses of the living inactivated virus and their combination in comparison with the virus-neutralizing antibodies, antihemagglutinins and precipitins. Accumulation of the virus-neutralizing antibodies did not coincide in time with the curve of the PHAT-AT accumulation; the titres of the virus-neutralizing antibodies were higher than the PHAT-AT titres. At the same time the percentage of seroconversions determined by PHAT was equal to 100 and the PHAT-AT level directly depended on the immunizing dose, the time of administration and the type of the antigen. On the basis of the data obtained PHAT could be recommended as a test for the assessment of the immunological efficacy of the smallpox vaccinations.