Enhancing the Immunogenicity of Vaccinia Virus.

The conventional live smallpox vaccine based on the vaccinia virus (VACV) cannot be widely used today because it is highly reactogenic. Therefore, there is a demand for designing VACV variants possessing enhanced immunogenicity, making it possible to reduce the vaccine dose and, therefore, significantly eliminate the pathogenic effect of the VACV on the body. In this study, we analyzed the development of the humoral and T cell-mediated immune responses elicited by immunizing mice with low-dose VACV variants carrying the mutant A34R gene (which increases production of extracellular virions) or the deleted A35R gene (whose protein product inhibits antigen presentation by the major histocompatibility complex class II). The VACV LIVP strain, which is used as a smallpox vaccine in Russia, and its recombinant variants LIVP-A34R*, LIVP-dA35R, and LIVP-A34R*-dA35R, were compared upon intradermal immunization of BALB/c mice at a dose of 104 pfu/animal. The strongest T cell-mediated immunity was detected in mice infected with the LIVP-A34R*-dA35R virus. The parental LIVP strain induced a significantly lower antibody level compared to the strains carrying the modified A34R and A35R genes. Simultaneous modification of the A34R gene and deletion of the A35R gene in VACV LIVP synergistically enhanced the immunogenic properties of the LIVP-A34R*-dA35R virus.

Adaptive Immune Response to Vaccinia Virus LIVP Infection of BALB/c Mice and Protection against Lethal Reinfection with Cowpox Virus.

Mass vaccination has played a critical role in the global eradication of smallpox. Various vaccinia virus (VACV) strains, whose origin has not been clearly documented in most cases, have been used as live vaccines in different countries. These VACV strains differed in pathogenicity towards various laboratory animals and in reactogenicity exhibited upon vaccination of humans. In this work, we studied the development of humoral and cellular immune responses in BALB/c mice inoculated intranasally (i.n.) or intradermally (i.d.) with the VACV LIVP strain at a dose of 105 PFU/mouse, which was used in Russia as the first generation smallpox vaccine. Active synthesis of VACV-specific IgM in the mice occurred on day 7 after inoculation, reached a maximum on day 14, and decreased by day 29. Synthesis of virus-specific IgG was detected only from day 14, and the level increased significantly by day 29 after infection of the mice. Immunization (i.n.) resulted in significantly higher production of VACV-specific antibodies compared to that upon i.d. inoculation of LIVP. There were no significant differences in the levels of the T cell response in mice after i.n. or i.d. VACV administration at any time point. The maximum level of VACV-specific T-cells was detected on day 14. By day 29 of the experiment, the level of VACV-specific T-lymphocytes in the spleen of mice significantly decreased for both immunization procedures. On day 30 after immunization with LIVP, mice were infected with the cowpox virus at a dose of 46 LD50. The i.n. immunized mice were resistant to this infection, while 33% of i.d. immunized mice died. Our findings indicate that the level of the humoral immune response to vaccination may play a decisive role in protection of animals from orthopoxvirus reinfection.

Enhancing the Protective Immune Response to Administration of a LIVP-GFP Live Attenuated Vaccinia Virus to Mice.

Following the WHO announcement of smallpox eradication, discontinuation of smallpox vaccination with vaccinia virus (VACV) was recommended. However, interest in VACV was soon renewed due to the opportunity of genetic engineering of the viral genome by directed insertion of foreign genes or introduction of mutations or deletions into selected viral genes. This genomic technology enabled production of stable attenuated VACV strains producing antigens of various infectious agents. Due to an increasing threat of human orthopoxvirus re-emergence, the development of safe highly immunogenic live orthopoxvirus vaccines using genetic engineering methods has been the challenge in recent years. In this study, we investigated an attenuated VACV LIVP-GFP (TK-) strain having an insertion of the green fluorescent protein gene into the viral thymidine kinase gene, which was generated on the basis of the LIVP (Lister-Institute for Viral Preparations) strain used in Russia as the first generation smallpox vaccine. We studied the effect of A34R gene modification and A35R gene deletion on the immunogenic and protective properties of the LIVP-GFP strain. The obtained data demonstrate that intradermal inoculation of the studied viruses induces higher production of VACV-specific antibodies compared to their levels after intranasal administration. Introduction of two point mutations into the A34R gene, which increase the yield of extracellular enveloped virions, and deletion of the A35R gene, the protein product of which inhibits presentation of antigens by MHC II, enhances protective potency of the created LIVP-TK--A34R*-dA35R virus against secondary lethal orthopoxvirus infection of BALB/c mice even at an intradermal dose as low as 103 plaque forming units (PFU)/mouse. This virus may be considered not only as a candidate attenuated live vaccine against smallpox and other human orthopoxvirus infections but also as a vector platform for development of safe multivalent live vaccines against other infectious diseases using genetic engineering methods.

Effect of the Route of Administration of the Vaccinia Virus Strain LIVP to Mice on Its Virulence and Immunogenicity.

The mass smallpox vaccination campaign has played a crucial role in smallpox eradication. Various strains of the vaccinia virus (VACV) were used as a live smallpox vaccine in different countries, their origin being unknown in most cases. The VACV strains differ in terms of pathogenicity exhibited upon inoculation of laboratory animals and reactogenicity exhibited upon vaccination of humans. Therefore, each generated strain or clonal variant of VACV needs to be thoroughly studied in in vivo systems. The clonal variant 14 of LIVP strain (LIVP-14) was the study object in this work. A comparative analysis of the virulence and immunogenicity of LIVP-14 inoculated intranasally (i.n.), intradermally (i.d.), or subcutaneously (s.c.) to BALB/c mice at doses of 108, 107, and 106 pfu was carried out. Adult mice exhibited the highest sensitivity to the i.n. administered LIVP-14 strain, although the infection was not lethal. The i.n. inoculated LIVP-14 replicated efficiently in the lungs. Furthermore, this virus was accumulated in the brain at relatively high concentrations. Significantly lower levels of LIVP-14 were detected in the liver, kidneys, and spleen of experimental animals. No clinical manifestations of the disease were observed after i.d. or s.c. injection of LIVP-14 to mice. After s.c. inoculation, the virus was detected only at the injection site, while it could disseminate to the liver and lungs when delivered via i.d. administration. A comparative analysis of the production of virus-specific antibodies by ELISA and PRNT revealed that the highest level of antibodies was induced in i.n. inoculated mice; a lower level of antibodies was observed after i.d. administration of the virus and the lowest level after s.c. injection. Even at the lowest studied dose (106 pfu), i.n. or i.d. administered LIVP-14 completely protected mice against infection with the cowpox virus at the lethal dose. Our findings imply that, according to the ratio between such characteristics as pathogenicity/immunogenicity/protectivity, i.d. injection is the optimal method of inoculation with the VACV LIVP-14 strain to ensure the safe formation of immune defense after vaccination against orthopoxviral infections.

Rapid protocol of dot-immunnoassay for orthopoxviruses detection.

The aim of the work was to create a sensitive and fast immunochemical test for the detection of orthopoxviruses (OPXV) in the "point of care" format. This work presents the results of the comparative evaluation of a single-stage (rapid version) and two-stage protocol of dot-immunoassay based on plane protein array for detection of vaccinia virus (VACV), cowpoxvirus (CPXV) and ectromelia virus (ECTV) in viral culture materials with different degrees of purification. It has been established that rabbit polyclonal VACV-antibodies can be used in a one-stage dot-analysis, both as a capture agent immobilized on a substrate and as a detection reagent bound with colloidal gold particles. It is shown that the sensitivity of detection of OPXV is inversely related to the degree of purification of viruses. The one-stage variant of the dot-immunoassay allows reducing the analysis time to 39 min and increasing the detection sensitivity of all the studied orthopoxviruses in crude viral samples to a range of 104-103 PFU/mL. The increase in sensitivity in the rapid version of the analysis, presumably, occurs due to binding of capture antibodies to subviral structures that form large aggregates of gold particles. Ultrasonic treatment of culture virus reduces the detection sensitivity, presumably due to both the destruction of conformational epitopes located on the surface of subvirus structures, as well as the increase in the dispersion of cell debris, which limits diffusion and contacts of viral antigens with capture antibodies on the substrate. Both versions of the analysis are specific and do not detect interactions both with preparations of non-infected cell culture and with heterogeneous controls of the causative agents of erythematous infections. The rapid protocol of dot-immunnoassay described above can be used to detect, or help to exclude, the presence of threat viruses in samples and could be useful in a variety of biodefense applications. Ready-to-use setup, ease of analysis and the ability to visually accounting for results allow the test to be used outside of laboratories.