In Vivo Enhances The Immune Response Against HBV Following Gene Immunization

DNA vaccines are an attractive approach for the development of vaccines forHBV because they are stable and affordable and therefore potentially practical tobe used in the developing world. Following up on reports that direct injection ofplasmid DNA resulted in gene expression, several groups pursued the possibilitythat direct injection of plasmid DNA could be exploited as a vaccine strategy. Thefirst peer-reviewed report of protective immunity and cytotoxic T lymphocyte(CTL) induction in mice after i.m. injection of a DNA plasmid appeared in 1993.Subsequently, the use of DNA vaccines in preclinical studies has become wellestablished, with reports of protective immunity in many different independentstudies. In recent studies, both antibody and CTL responses were induced innonhuman primates, although 1-2 mg of DNA was immunized on multipleoccasions in these studies. Antibody and CTL responses also have been induced inhuman volunteers, but again, high doses of DNA were used. For example, in onestudy in naive subjects, optimal CTL responses were induced with 2.5 mg of DNAfrom Plasmodium falciparum. Nevertheless, DNA vaccines have proven veryeffective in small animal models and are also effective in larger animals, includingcattle, horses, and swine. However, although the use of DNA vaccines atmilligram doses is feasible, it would impose serious limitations on the number ofconstructs that could be included in a vaccine. In addition, the use of very highdoses of DNA is less favorable from a process economics standpoint. This lowerimmunogenicity in primates may be due to cellular barriers, nuclease degradationin vivo, and the fact that pDNA has an overall net negative electrostatic charge.Therefore, there is a clear need to induce effective immunity in humans withlower and fewer doses of DNA, as well as to increase the magnitude of theimmune responses obtained.There are a number of strategies available that have the potential to improvethe potency of DNA vaccines. These strategies include:(i) vector modification toenhance antigen expression, which may involve targeting of the expressed proteinto a particular cellular location, the inclusion of immunostimulatory sequences, orthe elimination of inhibitory sequences in the plasmid;(ii) improvements in DNAdelivery;or (iii) the inclusion of adjuvants, either as a gene or as a coadministeredagent. Our work focused predominantly on the use of DNA delivery systems toenhance the response to DNA vaccines.To achieve this, HBV-S2 S coding sequence was introduced into theeukaryotic expression vector pVAX1 and identified by PCR and DNA sequencinganalysis, and can be over-expressed in COS-7 cell line. Female BALB/c micewere primed by i.m. gene immunization with recombinant plasmid on day 0, thengiven electroporation in vivo. The level of anti-HBs-IgG antibodies and thekillrate of CTL were evaluated to identify the superiority of electroporation invivo.The experiment indicated that HBsAg specific antibody response waselicited in groups primed with plasmid pVAX1MS at 5ug dosage and the level ofanti-HBs-IgG antibodies was homologous to the control group (withoutelectroporation in vivo at 100ug dosage). What's more, in contrast to nakedpDNA, the group with electroporation in vivo induced potent cytotoxic Tlymphocyte (CTL) responses at a low dose. In addition, the routes (Intradermal,intramuscular and hypodermal) of DNA electroporation in vivo vaccination wereevaluated and the the route of intradermal is superior to the others. These resultsindicated electroporation in vivo could enhance humoral and cellular immuneresponse against HBV induced by gene immunization, which proved the fact thatthe immunogenicity of DNA vaccine was decided by transfection efficiency andantigen presention.To ulteriorly evaluate the immune response and safety of the therapeuticHBV DNA vaccine against the hepatitis B virus in established HBV Balb/c micemodel primed by intramuscular injection of therapeutic DNA vaccine and boostwith MVA (modified Vaccine Angala), HBV-s gene sequence was introduced intoboth the eukaryotic expression vector pV1012 and MVA. HBV DNA vaccine wasadministered to established HBV Balb/c mice model by repeated intramuscularinjections three times at 0,2,4 week and boost with MVA at the 6 week. It wasfound that immunization with the therapeutic DNA vaccine and MVA againsthepatitis B virus by means of priming-boost induced a strong and specific immuneresponse in established HBV Balb/c mice model. The anti-HBs antibody level, theHBs antigen concentration in sera, HBV DNA titer, change at ALT andpathological slice-up were used to synthetically evaluate the validity and safety ofDNA-priming/MVA-boost regimen. The experiment indicated the anti-HBsantibody level, the HBs antigen concentration in sera, HBV DNA titer of DNA-priming/MVA-boost group were straightened up,and CTL is crucial at anti-HBVimmunotherapy. What's more, no further evidences were observed expect theslight rise at ALT. These results suggested that DNA-priming/MVA-boostregimen can be use to antivirus immunotherapy.