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Smallpox eradication and lack of adequate animal model for smallpox infection underlies a necessity to assess immunogenic and protective properties of genetic engineering-created live attenuated smallpox vaccines in several animal models of orthopoxviral infections. Here we compared immunogenic and protective properties of the recombinant vaccinia virus (VACV) LIVP-GFP intradermally (i.d.) inoculated to mice, guinea pigs and rabbits. LIVP-GFP immunization in all animal species was applied at dose of 2 × 104 or 2 × 106 PFU. Control animals were injected with saline. Blood sampling was performed on day 28 after virus LIVP-GFP or saline inoculation. Blood samples were taken intravitally from the retro-orbital venous sinus in mice, heart in guinea pigs or marginal ear vein in rabbits. Serum samples were isolated by precipitating blood cells via centrifugation. The serum anti-VACV IgG titers were determined by ELISA. On day 30 post-immunization animals were intranasally challenged with lethal dose of host specific orthopoxvirus species. Mice were infected by cowpox virus (CPXV) strain GRI-90 at dose 68 LD50, guinea pigs – by VACV GPA at dose 56 LD50, rabbits — by VACV HB-92 at dose 100 LD50. All animals in control group died afterwards, whereas all animals immunized by attenuated recombinant virus LIVP-GFP at dose 2 × 106 PFU survived. In case of the LIVP-GFP immunization at dose 2 × 104 PFU, 88% of mice, 67% of rabbits and 50% of guinea pigs survived after being challenged with species-specific CPXV, VACV HB-92, and VACV GPA. ELISA data for the blood serum samples revealed a correlation between level of VACV-specific antibodies and level of protection in animal species. Based on the data obtained, it could be concluded that all three “animal–orthopoxvirus” models allow to provide with a proper evaluation of immunogenicity and protectiveness for generated modern attenuated vaccines against smallpox and other orthopoxviral human infections. Upon that, it was shown that BALB/c mouse strain was the most convenient investigational host species.

Vaccinia virus had played a key role in the global smallpox eradication. However, in case of mass vaccination with various Vaccinia virus strains severe side effects were revealed sometimes ending up with lethal outcomes, especially in immunocompromised humans. Hence, in 1980 the World Health Organization recommended to cancel smallpox vaccination after declaring about smallpox eradication. Over the last 40 years, human population virtually lost immunity not only against smallpox, but also against other zoonotic orthopoxvirus infections, such as monkeypox, cowpox, buffalopox, and camelpox. All of them pose a represent increasing threat to human health and heighten a risk of emerging highly contagious viruses due to natural evolution of previous zoonotic orthopoxviruses. In order to prevent development of small outbreaks into spreading epidemics and, thus, to decrease a risk of emergence due to natural evolution of highly pathogenic for humans orthopoxviruses, efforts should be applied to develop safe new generation live vaccines based on Vaccinia virus with target virulence genes inactivation. These strains should be examined in laboratory animal models inoculated via different routes. Currently, Vaccinia virus often becomes attenuated to create live recombinant vaccines due to inserting target DNA sequences into the virus virulence genes resulting in their inactivation. Vaccinia virus strain LIVP used in the Russian Federation as smallpox vaccine as well as derivative attenuated variant LIVP-GFP created by using genetic engineering methods with inactivating its thymidine kinase gene were examined. Such viruses were intracerebrally inoculated into suckling mice at doses of 101 or 102 PFU/animal for neurovirulence assessment. Adult mice were infected intranasally, subcutaneously or intradermally at doses of 107 or 108 PFU/animal and clinical manifestations were analyzed for 14 days. On the 28th day after the onset, blood serum samples were collected from individual mice to measure virus specific antibody level by using ELISA. It was shown that recombinant Vaccinia virus strain LIVP-GFP displayed markedly lowered neurovirulence and pathogenicity for mice as compared to parental LIVP. Finally, intradermal route turned out to demonstrate the most safe and effective profile for immunization with both examined Vaccinia virus strains.

The lack of immunity to the variola virus in the population, increasingly more frequent cases of human orthopoxvirus infection, and increased risk of the use of the variola virus (VARV) as a bioterrorism agent call for the development of modern, safe vaccines against orthopoxvirus infections. We previously developed a polyvalent DNA vaccine based on five VARV antigens and an attenuated variant of the vaccinia virus (VACV) with targeted deletion of six genes (VAC6). Independent experiments demonstrated that triple immunization with a DNA vaccine and double immunization with VAC6 provide protection to mice against a lethal dose (10 LD50) of the ectromelia virus (ECTV), which is highly pathogenic for mice. The present work was aimed at comparing the immunity to smallpox generated by various immunization protocols using the DNA vaccine and VAC6. It has been established that immunization of mice with a polyvalent DNA vaccine, followed by boosting with recombinant VAC6, as well as double immunization with VAC6, induces production of VACV-neutralizing antibodies and provides protection to mice against a 150 LD50 dose of ECTV. The proposed immunization protocols can be used to develop safe vaccination strategies against smallpox and other human orthopoxvirus infections.

The LIVPΔ6 strain of vaccinia virus (VACV) was created by genetic engineering on the basis of previously obtained attenuated 1421ABJCN strain by target deletion of the A35R gene encoding an inhibitor of antigen presentation by the major histocompatibility complex class II. 1421ABJCN is the LIVP strain of VACV with five inactivated virulence genes encoding hemagglutinin (A56R), γ-interferon-binding protein (B8R), thymidine kinase (J2R), complement-binding protein (C3L), and Bcl2-like inhibitor of apoptosis (N1L). The highly immunogenic LIVPΔ6 strain could be an efficient fourth-generation attenuated vaccine against smallpox and other orthopoxvirus infections.