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.

Phage display antibodies against ectromelia virus that neutralize variola virus: Selection and implementation for p35 neutralizing epitope mapping.

In this study, five phage display antibodies (pdAbs) against ectromelia virus (ECTV) were selected from vaccinia virus (VACV)-immune phage-display library of human single chain variable fragments (scFv). ELISA demonstrated that selected pdAbs could recognize ECTV, VACV, and cowpox virus (CPXV). Atomic force microscopy visualized binding of the pdAbs to VACV. Three of the selected pdAbs neutralized variola virus (VARV) in the plaque reduction neutralization test. Western blot analysis of ECTV, VARV, VACV, and CPXV proteins indicated that neutralizing pdAbs bound orthopoxvirus 35 kDa proteins, which are encoded by the open reading frames orthologous to the ORF H3L in VACV. The fully human antibody fh1A was constructed on the base of the VH and VL domains of pdAb, which demonstrated a dose-dependent inhibition of plaque formation after infection with VARV, VACV, and CPXV. To determine the p35 region responsible for binding to neutralizing pdAbs, a panel of truncated p35 proteins was designed and expressed in Escherichia coli cells, and a minimal p35 fragment recognized by selected neutralizing pdAbs was identified. In addition, peptide phage-display combinatorial libraries were applied to localize the epitope. The obtained data indicated that the epitope responsible for recognition by the neutralizing pdAbs is discontinuous and amino acid residues located within two p35 regions, 15-19 aa and 232-237 aa, are involved in binding with neutralizing anti-p35 antibodies.

New effective chemically synthesized anti-smallpox compound NIOCH-14.

Antiviral activity of the new chemically synthesized compound NIOCH-14 (a derivative of tricyclodicarboxylic acid) in comparison with ST-246 (the condensed derivative of pyrroledione) was observed in experiments in vitro and in vivo using orthopoxviruses including highly pathogenic ones. After oral administration of NIOCH-14 to outbred ICR mice infected intranasally with 100 % lethal dose of ectromelia virus, it was shown that 50 % effective doses of NIOCH-14 and ST-246 did not significantly differ. The 'therapeutic window' varied from 1 day before infection to 6 days post-infection (p.i.) to achieve 100-60 % survival rate. The administration of NIOCH-14 and ST-246 to mice resulted in a significant reduction of ectromelia virus titres in organs examined as compared with the control and also reduced pathological changes in the lungs 6 days p.i. Oral administration of NIOCH-14 and ST-246 to ICR mice and marmots challenged with monkeypox virus as compared with the control resulted in a significant reduction of virus production in the lungs and the proportion of infected mice 7 days p.i. as well as the absence of disease in marmots. Significantly lower proportions of infected mice and virus production levels in the lungs as compared with the control were demonstrated in experiments after oral administration of NIOCH-14 and ST-246 to ICR mice and immunodeficient SCID mice challenged with variola virus 3 and 4 days p.i., respectively. The results obtained suggest good prospects for further study of the chemical compound NIOCH-14 to create a new smallpox drug on its basis.

Using ICR and SCID mice as animal models for smallpox to assess antiviral drug efficacy.

The possibility of using immunocompetent ICR mice and immunodeficient SCID mice as model animals for smallpox to assess antiviral drug efficacy was investigated. Clinical signs of the disease did not appear following intranasal (i.n.) challenge of mice with strain Ind-3a of variola virus (VARV), even when using the highest possible dose of the virus (5.2 log10 p.f.u.). The 50 % infective doses (ID50) of VARV, estimated by the virus presence or absence in the lungs 3 and 4 days post-infection, were 2.7 ± 0.4 log10 p.f.u. for ICR mice and 3.5 ± 0.7 log10 p.f.u. for SCID mice. After i.n. challenge of ICR and SCID mice with VARV 30 and 50 ID50, respectively, steady reproduction of the virus occurred only in the respiratory tract (lungs and nose). Pathological inflammatory destructive changes were revealed in the respiratory tract and the primary target cells for VARV (macrophages and epithelial cells) in mice, similar to those in humans and cynomolgus macaques. The use of mice to assess antiviral efficacies of NIOCH-14 and ST-246 demonstrated the compliance of results with those described in scientific literature, which opens up the prospect of their use as an animal model for smallpox to develop anti-smallpox drugs intended for humans.

Intranasal immunization with inactivated tick-borne encephalitis virus and the antigenic peptide 89-119 protects mice against intraperitoneal challenge.

Detailed studies of the pathogenesis of certain neuroviral infections allow for a better understanding of the special role of the olfactory neuroepithelial cells in the invasion of viruses into the CNS. Several studies using animal models demonstrated that neurotropic viruses belonging to various families invade the brain via the olfactory tract after parenteral infection. We suppose that intranasal (i.n.) immunization inducing mucosal and systemic immunity will block neurotropic virus propagation into the brain via the olfactory pathway and neutralize virus multiplication in visceral organs. Subject of the present study was the efficacy of i.n. immunization of Balb/c mice with both killed tick-borne encephalitis (TBE) virus and antigenic peptide 89-119 from the envelope protein E of TBE virus in a nanoemulsion formulation. Intranasal immunization with nanoemulsion containing inactivated TBE virus particles induced specific both neutralizing and HAI antibodies. TBE virus specific IgA antibodies were detected in lung and nasal lavages of mice. The fourth i.n. immunization resulted in a drastic titer increase of the specific antibodies to 1:12,800, which was protective in all i.n. immunized mice against intraperitoneal (i.p.) challenge with 100 LD(50) of TBE virus. The ratio of specific IgG2a to IgG1 indicated the occurrence of a Th2 type immune response. We observed a similar balance of TBE virus-specific IgG2a/IgG1 after s.c. immunization of mice with the commercial FSME-Immun Inject vaccine against TBE virus. Thus, the experimental data obtained for the first time demonstrates the feasibility of using nanoparticles containing inactivated TBE virus for effective protection of i.n. immunized mice against the usually lethal infection. We analyzed a number of fragments of E protein of the TBE virus for antigenic similarity with human proteins by computer analysis. Local antigenic similarity of the fragments of E protein of TBE virus and human proteins allows for the identification of the epitopes, which may induce an autoimmune response if applied in a vaccine construct. One of the candidates, peptide 89-119 of E protein of the TBE virus does not contain any epitopes with local similarities to human protein epitopes. This peptide was synthesized and applied intranasally in nanoparticulate formulation. It induced TBE virus-specific antibodies and led to protection in the case of i.p. challenge with TBE virus.

A study of systems for delivering antigens and plasmid DNA for intranasal immunization against tick-borne encephalitis virus.

Our previous studies indicated the possibility for some neurotropic viruses to spread into the brain of immune animals through the olfactory pathway. Thus, nasal mucosa in the olfactory region is likely to be a promising target for mucosal immunization to protect the CNS from neurotropic viral infections. THE MAIN IDEA OF THE RESEARCH: Intranasal immunization inducing mucosal and systemic immune responses blocks the propagation of neurotropic virus into the brain via the olfactory pathway and neutralizes the multiplication of virus in visceral organs, allowing more effective protection against neurotropic infections transmitted by bloodsucking arthropods to be achieved. Thus, study of the efficiency of delivery systems for intranasal immunization against tick-borne encephalitis (TBE) virus is an urgent task in the development of anti-TBE mucosal vaccine. To study intranasal immunization against TBE virus, we have chosen four delivery systems (DSs), namely, (i) biodegradable microparticles, (ii) cationic liposomes, and live attenuated (iii) bacterial and (iv) viral vectors. The gene of TBE virus protein E was inserted into the pcDNA3 plasmid (designated as pcDNA3/E-TBE). Three types of delivery system for plasmid DNA were developed and studied in vitro. The first system, artificial virus-like microparticles (VLP), consists of polyglycan-spermidine complexes that cover pcDNA3/E-TBE DNA. The second system is cationic liposomes with DNA of the plasmid pcDNA3/E-TBE. The third system is an attenuated Salmonella strain containing pcDNA3/E-TBE. The fourth system is a recombinant vaccinia strain with inserted genes of TBE virus proteins C, prM, E, NS1, NS2a, NS2b, and NS3. The DSs were tested in COS-7 and CV-1 cell lines and macrophages by ELISA of cell lysate. The results obtained showed the expression of the E gene in transfected cells, thereby demonstrating that these DSs are suitable for mucosal immunization. High levels of immune response shifted to the Th1 type were detected in BALB/c mice following intranasal immunization with recombinant vaccinia-TBE strain and VLP-pcDNA3/E-TBE. The mice immunized intranasally with recombinant vaccinia-TBE strains were completely protected against intraperitoneal challenge with TBE virus strain Sofjin, whereas intranasal immunization with killed TBE vaccine failed to induce a significant level of protection.