复合辅助性T淋巴细胞表位高效及亚显性HB疫苗研究

There are more than 300 millions of HBsAg carriers all over the world. Chronic hepatitis B virus infection may cause significant disease morbidity, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HB vaccine inoculation is the important measure for prevention of HBV infection. Considerable variability exists, however, in the vaccine response to hepatitis B with 5-10% of healthy young adults demonstrating no or inadequate responses following a standard vaccination schedule. The causes of the weak or non response to hepatitis B might be genetic specificity or HBV infection of the individual. For genetic specificity of the individual, we studied on the effect of exogenous epitope of helper T lymphocyte (HTL) on humoral immunity of HBV S gene DNA immunity. Two universal HTL epitopes, amino acid residue (aa) 830-843 of the tetanus toxoid (TTE) and artificial epitope (PADRE), and 3 unique epitopes, aa1-20 of tubercle bacteria hot shock protein 65 (TBE), aa54-65 of rubella protein E2-4 (ME) and aa35-48 of trachoma hot shock protein 60 (CE) were chosen. Eukaryotic expression vectors were constructed by inserting single or multiple exogenous epitopes in HBV S gene just after the initial code of translation. BALB/c mice were inoculated with 100mg of recombinant DNA per mouse, and given boost inoculation for 3 times with 3-week interval. Mouse blood were collected one month just after the third boost inoculation. Anti-HBs was detected using Abbott test kits. HBV S eukaryotic gene expression vectors, pHB and 6 exogenous HTL epitope HBV S gene vectors, pHB-TBE, pHB-PADRE, pHB-TTE, pHB-MTE2, pHB-MTE3 and pHB-MTE5 were constructed successfully with anti-HBs level (IU/L) of 10.38 ± 4.54, 5.44 ± 4.69, 48.67 ± 6.95, 28.69 ± 6.41, 16.20 ± 8.13, 23.12 ± 7.46 and 27.54 ± 7.68 respectively. Among 3 single epitopes, TTE and PADRE had obviously effect on promoting the anti-HBs response of HBV S gene, while TBE had no promoting effect. All of the 3 multiple epitopes were shown the effect of immune promoting. Some exogenous HTL epitopes had obviously effect on promoting the anti-HBs response of HBV S gene. Multiple epitopes also had humoral immunity promoting effect, but there was no synergic effect among their own HTL epitopes. PADRE might be an important candidate for new efficient HB vaccine. The multiple epitope cluster consisted form 5 exogenous epitopes might be an important candidate for the reinoculating HB vaccine or therapy HB vaccine.Large portion of these non-responders may have got HBV infection in low level or with infection of HBV variants before inoculation. Immunologic tolerance is the main mechanism for those non-responders of HB vaccine and patients with chronic HBV infection. In addition to immunologic tolerance, immune escape is also the important mechanism for for those non-responders of HB vaccine and patients with chronic HBV infection. It is difficult for routine HB vaccine to obtain response in those individuals. Fortunately, many recent researches suggested that weak epitopes, such as subdominant epitopes, might not be tolerant and could be responded through modifying its structure in immunologic tolerance individuals, or in patients with variant infection since there was no mutation in area of weak epitopes. For HBsAg, the main component of HB vaccine, has more than four epitopes of B lymphocyte. The so called a determinant is the strongest one among those epitopes. a determinant is prone to take place escape mutants. The antigenicity of HBsAg with escape mutant was much weaker since it can not react to antiserum raised by HB vaccine. Epitope of helper T lymphocyte is one of the reasons for the weak antigenicity. For these reasons, recombinant vectors of HBsAg with deletion of a determinant and adding exogenous epitopes of helper T lymphocyte were constructed in order to let subdominant epitopes be responded by hosts. Their antigenicity was evaluated by DNA immunization in BALB/c mice.

采用基因工程技术将多个通用和特异Th细胞表位插入中分子HBsAg中，获得新型重组疫苗，又中∈笃兰燮淇朔﨟－2抗原多态性对疫苗应答的不利影响的效果。构建亚显表位真核表达柿＃谧蛐∈竽Ｐ蜕掀兰燮淇朔庖吣褪芎兔庖咛颖苣芰ΑＬ教秩死嗫朔﨟LA多态性及低水平HBV感染造成HB疫苗不应答的新途径，为新型高效疫苗研究提供理论基础和新技术。