Introduction:

Hepatitis B virus is a hepadnavirus that is transmitted sexually and from mother to child. Chronic hepatitis may lead to liver disease such as cirrosis or in some cases liver cancer. The link between cirrhosis, liver cancer and chronic hepatitis B is the main reason why there is a need for early and effective treatment of infected populations as well as the development of vaccines that will hopefully lead to the eradication of the disease.

One of the methods of immunization that is currently being studied is to stimulate an immune response to a protein expressed in the form of a gene introduced directly in the form of pure plasmid DNA (Geissler et al., 1997). Other methods of this type of immunization have also been studied. Co-immunizations of cytokine and hepatitis B virus DNA (Geissler et al., 1998) and immunization using a plasmid DNA in combination with antigen and antibody complex (Qu et al. 1998) are two of these methods. Experimental results with the two methods show that DNA immunization will be very effective in the treatment of hepatitis B and possibly other diseases as well. Methods

Mice were the animal used in the experiments and were immunized with plasmid vectors encoding the large (LHB) or middle (MHB) envelope proteins of the hepatitis B virus (Geissler et al., 1997; Chow et al, 1997; Geissler et al, 1998; Qu et al., 1998). The reason for this is to compare the immune response with vectors encoding the middle or large envelope of the hepatitis B virus (Chow et al., 1997). The cells that were transfected with the vectors were from mice, and other cell lines such as the mastocytoma cell line P815, the myeloma cell line SP2/0 and cells that were grown in the laboratory; HEPG2, HuH-7 etc. (Geissler et al.,1997). These cell cells consists of one copy of the HBV genome with the core gene deleted (Geissler et al., 1997). Different cell lines were used to determine if the Hbvirus elicit a response over a range of cells that have different characteristics. For the co-immunization with Interlukin-2; a vector encoding IL-2 and a bicistronic vector separately encoding IL-2 and the middle envelope was also constructed for comparison (Chow et al., 1997). The efficiency of immune responses was measured through antibody and T-cell proliferation assays (Chow et al.,1997). Discussion of Results

The result of these experiments revealed that immunization of hepatitis B virus wit enhances the immune response to the hepatitis B antigen (Geissler et al.,1997). HuH-7 cells generated in the laboratory, cells generated from different cell lines and mice were injected with plasmid encoding the HBV middle or large envelope (Geissler et al., 1997). Levels of hepatitis B antigens and antibody and T-cell proliferation were measured and compared. Immunoblotting was performed to detect level and expression of HB antigen revealed large levels of HBV antigens in the serum revealing that the cell is in effect generating its own vaccine (Geissler et al., 1997). It was also found that the protein encoding the large envelope is non-secreted while the one encoding the middle envelope is secreted and retained on cell membrane (Geissler et al.,1997; Chow et al., 1997). This could account for the slight differences in immune responses induced by the two vectors. Mice immunized with LHB plasmid vectors had a weaker immune response than mice with MHB plasmid vectors (Geissler et al., 1997; Chow et al., 1997; Geissler et al.,1998). There is also suggestion that the large envelope of the hepatitis B virus has some inhibitory effect of antigen presenting cells (Geissler et al.,1998). It could also mean that proteins encoding the middle or large envelope of the HBV elicit a different immune response. Geissler et al., 1997 also found the level of anti Hbs antibody titres were maintained for over six months without boosting. Geissler et al., 1997 found the levels of HB antigens decreased as level of Hb antibodies increased.

The majority of proliferating T cells derived from all groups of immunigized mouse was CD4+ Th1 cells which in turn activates other factors that induce CD8+cytotoxic T-cell proliferation such as IL-2 leading to the destruction of the foreign antigen (Geissler et al., 1998). Cytotoxic CD8+ T-cells recognizes antigen peptides on surface of MHC class II cells and kills it. The need for IL-2 in immune response to Hb virus may be a reason why co-immunization with the by plasmids expressing Hb antigen and IL-2 induces a stronger immune response (Chow, et al., 1997). Antibody analysis was done with cells transfected with plasmid vectors encoding large envelope, vectors encoding middle envelope, a vector encoding the middle envelope fused with IL-2 (pS2-S-IL-2) and a bicistronic vector encoding the middle envelope with IL-2 (Chow et al., 1997). Analysis was done over a period of two months and it revealed that anti HBsAgs levels were over 100 times higher in mice immunized with the bicistronic vector and pS2-S-IL-2 vector (Chow et al., 1998). Antibody analysis also revealed that level of anti HbsAgs is in correlation with the dose of vaccine being administered (Chow et al., 1997; Geissler et al., 1998). The IgM isotype was the first immunoglobulin detected and was shifted to the IgG isotype which indicates a secondary immune response are stimulated by DNA inoculation (Chow et al., 1998). Geissler et al., 1998 also revealed that co-immunization with the LHB and Il-2 did not enhance humoral immune response. The reasons are not clear, but the aforementioned inhibitory effect of the large envelope protein may play a role.The results are promising, but further studies should be done in case of any harmful side effects that may be present.

Immunization with a combination of Hepatitis B surface antigen (HBsAg) complexed with anti-HBs and plasmid DNA encoding the HBV envelope produced an even stronger immunogenic response (Qu et al., 1998). Levels of anti Hbs were enhanced with this method of immunization as well as levels of Interleukin-2 (Qu et al., 1998). This method is also being considered for treatment of hepatitis C (Qu et al., 1998). Further experimentation is needed to determine whether or not this method of DNA immunization is applicable, but the initial results are promising.

Future Prospects The experiments have proved that DNA immunization can be a powerful tool in the battle against Hepatitis B. Each of these experiments was based on direct gene transfer using plasmid DNA into one or more tissues. The expression of specific HB proteins induces the production of HB antigens in the cell and the production of anti HBV. It is therefore clear that DNA immunization induces the body to make its own vaccine and the possibility also exists for a broad range of immune responses with DNA immunization.

Many issues still need to be addressed before this can be applied to humans. Safety of this treatment is the main concern. Mutations in the individual's cell DNA could occur if integration of plasmid DNA with the cell's DNA occur is one concern and further studies is necessary.


REFERENCES Chow, Y-H., Huang, W-L., Chi, Y-D, and Tao, M-H. 1997. Improvement of Hepatitis B Virus DNA Vaccines by Plasmids Coexpressing Hepatitis B Surface Antigen and Interleukin-2. Journal of Virology, Jan. 1997: 169-178. Geissler, M., Tokushige, K., Chante, Charles C., Zurawski, Vincent R. Jr., and Wands, Jack R., 1997. Cellular and Humoral Immune Response to Hepatitis B Virus Structural Proteins in Mice After DNA-Based Immunization. Gastroenterology 1997; 112:1307-1321. Geissler, M., Schirmbeck, R., Reimann, J., Hubert, E. Blum, and Wands, Jack R., 1998. Cytokine and Hepatitis B Virus DNA Co-immunizations Enhance Cellular and Humoral Immune Responses to the Middle But Not to the Large Hepatitis B Surface Antigen in Mice. Hepatology 1998;28:202-210. Qu, D., Yuan, ZH., Yang, L., Li, GD, and Wen, YM. 1998. Effect of Plasmid DNA on Immunogenicity of Hbs Antigen-anti-Hbs Complex. Viral Immunology 1998, 11(2):65-72.


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