Abstract
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.
Highlights
Preventive vaccination is the most reliable method to protect against viral infections
Modification or deletion of some Vaccinia virus (VACV) genes is known to increase the immunogenicity of this virus [1,10]
One of these examples is the VACV A34R gene that controls the release of enveloped virions (EEVs) from infected cells [25,27,42]
Summary
Preventive vaccination is the most reliable method to protect against viral infections. At an infective dose of 105 PFU for VACV variants, all mice, regardless of the route of viral administration, were completely protected against subsequent lethal CPXV infection, while all animals of the positive control group died on days 4–8 In this case, mice i.n. immunized with the LIVP-GFP virus had the highest temporary weight loss after infection with the CPXV virus. I.n. immunization with VACV variants at a dose of 103 PFU provided partial protection of mice against subsequent CPXV infection only in the case of LIVP (protection of 67% of animals) and LIVP-GFP-A34R*-dA35R (83%) strains (Figure 5A) In these groups, survived mice developed CPXV infection, but the animals’ condition normalized by day 8 (Figure 3B). Data are given for groups of 6 animals immunized with appropriate viruses as well as for non-immunized and not inoculated (Negative control) or inoculated with CPXV GRI-90 (Positive control) groups. (B) Curves for negative control and LIVP, LIVP-GFP-A34R*, and LIVP-GFP-A34R*-dA35R groups overlap
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