Transgenic potato plants expressing the gene of hepatitis B surface antigen (HBsAg) under the control of the double promoter of 35S RNA of cauliflower mosaic virus (CaMV 35SS) and the promoter of patatin gene of potato tubers have been obtained. Biochemical analysis of the plants was performed. The amount of HBsAg in leaves, microtubers, and tubers of transgenic potatoes growing in vitro and in vivo was 0.005-0.035% of the total soluble protein. HBsAg content reached 1 microg/g in potato tubers and was maximal in plants expressing the HBsAg gene under the control of CaMV 35SS promoter. In transgenic plants expressing HBsAg gene under the control of tuber-specific patatin promoter, HBsAg was found only in microtubers and tubers and was absent in leaves. Western blot analysis of HBsAg eluted from immunoaffinity protein A-Sepharose matrix has been performed. The molecular weight of HBsAg peptide was approximately 24 kD, which is in agreement with the size of the major protein of the envelope of hepatitis B virus. Using gel filtration, it was determined that the product of HBsAg gene expression in potato plants is converted into high-molecular-weight multimeric particles. Therefore, as well as in recombinant HBsAg-yeast cells, assembling of HBsAg monomers into immunogenic aggregates takes place in HBsAg-transgenic potato, which can be used as a source of recombinant vaccine against hepatitis B virus.

Background: One of the important reasons for spreading of hepatitis B virus (HBV) under conditions of vaccine pressure is emergence of escape mutations. Prevalent G145R mutation in S-gene leads to the most expressed changes of serological properties of HBV. Consequently, HBsAg is modified so thoroughly that it cannot be recognized by the majority of anti-HBs. Mutant G145R also differs from a wild type HBsAg by its immunogenic properties. At present, the relevance of enhancement of hepatitis B vaccine in view of mutant virus variants has been recognized. Objectives: a comparative study of antigenic and immunogenic properties of native and recombinant G145R mutants and an estimation of possibility for developing antigenic component of hepatitis B vaccine with G145R mutation in HBsAg. Methods: antigenic properties of recombinant HBsAg with G145R mutation were compared with each other and with native mutants by serological fingerprinting method. Then, BALB/c mice and sheep were immunized with selected recombinant antigen under different protocols. Titers of antibodies specific to wild type or mutant G145R type of HBsAg in sera of immunized animals were measured. Results: it was found that not all the recombinant HBsAg variants with G145R substitution have the same antigenic properties as native HBsAg with similar mutation. Recombinant HBsAg selected according to the principle of antigenic similarity possesses immunogenicity in mice and sheep causing the production of antibodies reacting with native wild and mutant type HBsAg. It was shown that mutant antigen is less immunogenic, requires larger doses and more time for the development of immune response; however, it is capable of causing an antibody level comparable with wild type antigen. Conclusion: preliminary selection of recombinant HBsAg containing G145R mutation with antigenic and immunogenic properties similar to the native analogue creates the basis for development of a specific component of hepatitis B vaccine with escape mutation G145R in HBsAg.

Immune-escape hepatitis B virus (HBV) mutants play an important role in HBV spread. Recently, the multivalent vaccine Bubo®-Unigep has been developed to protect against both wild-type HBV and the most significant G145R mutant. Here, we compared the effects of recombinant HBsAg antigens, wild-type and mutated at G145R, both included in the new vaccine, on activation of a human high-density culture of peripheral blood mononuclear cells (PBMC) in vitro. The antigens were used either alone or in combination with phytohemagglutinin (PHA). None of the antigens alone affected the expression of CD40, HLA-DR or CD279. Wild-type HBsAg enhanced CD86 and CD69 expression, and induced TNF-α, IL-10, and IFN-γ, regardless of the anti-HBsAg status of donor. In the presence of PHA, wild-type HBsAg had no effect on either of the tested surface markers, but increased IFN-γ and IL-10 and inhibited IL-2. In contrast, the G145R mutant alone did not affect CD86 expression, it induced less CD69, and stimulated IL-2 along with lowering levels of TNF-α, IL-10, and IFN-γ. The G145R mutant also suppressed PHA-induced activation of CD69. The dramatic differences in the immune responses elicited by wild-type HBsAg and the G145R mutant HBsAg suggest distinct adaptive capabilities of the G145R mutant HBV.

Background. In terms of serological properties and immunization, the wild type of HBsAg HBV and its G145R mutant behave as different antigens. This testifies to serious structural changes, which presumably could have a significant impact on the morphogenesis of virions and subviral particles. Nevertheless, morphological and ultrastructural investigations of HBV with G145R mutation have not been carried yet. Objectives. Research of structural and morphological organization of HBV in the presence of the G145R escape mutation. Methods. Studies of sera, purified viruses and recombinant HBsAg were carried out by transmission electron microscopy by the method of negative staining and indirect reaction of immunelabeling using monoclonal antibodies of different specificity. Specimens of wild type HBV and HBV with S143L mutation obtained in an identical manner were used as the control. Results. The presence of typical virus particles of HBV was shown in the specimens of wild strain and HBV with S143L mutation. Specimens of HBV with G145R mutation were characterized by expressed morphological heterogeneity. In the initial serum and in the specimen of purified virus containing G145R mutant, large oval particles 60-70 nm and up to 200 nm in size, respectively, were found. The presence of antigen structures of HBV in all heterogeneous forms was confirmed. It was shown that forming of subviral particles in the process of expression of the recombinant HBsAg with G145R mutation depends on conditions of expression and purification of the protein. They can vary from well-formed circular and oval particles to practically unstructured fine-grained masses. Conclusion. Direct data on the impact of G145R escape-mutation in S-gene, in contrast to S143L mutation, on the morphogenesis of virions and subviral particles of HBV were obtained.

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