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 set of specific oligonucleotide primers and hybridization testers with the support of TaqMan in real time is proposed to identify and differentiate such human pathogenic orthopoxviruses as smallpox virus, monkey smallpox virus, cowpox virus, variolovaccine. The analytical specificity of the set is determined upon the example of 20 strains of 6 types of human pathogenic orthopoxviruses and made up 100%. The sensibility is established using recombinant plasmids containing orthopoxviruses sequence. The minimal revealed amount of plasmid made up to 20 copies for smallpox and monkey smallpox viruses, 10 copies for cowpox and 60 copies for variolovaccine in reaction.

Efficacy of candidate DNA-vaccines based on the variola virus natural gene A30L and artificial gene A30Lopt with modified codon usage, optimized for expression in mammalian cells, was tested. The groups of mice were intracutaneously immunized three times with three-week intervals with candidate DNA-vaccines: pcDNA_A30L or pcDNA_A30Lopt, and in three weeks after the last immunization all mice in the groups were intraperitoneally infected by the ectromelia virus K1 strain in 10 LD50 dose for the estimation of protection. It was shown that the DNA-vaccines based on natural gene A30L and codon-optimized gene A30Lopt elicited virus, thereby neutralizing the antibody response and protected mice from lethal intraperitoneal challenge with the ectromelia virus with lack of statistically significant difference.