台灣是B型肝炎病毒 (HBV) 的高度流行區域，因此政府在1984年全面實施B型肝炎疫苗接種，在接種疫苗的10到15年之後，B型肝炎帶原者的比例及肝細胞癌的發生率都有下降的現象，疫苗施用成效顯著。但是初次接種20年之後，有50%的人其抗B型肝炎病毒表面抗原抗體 (anti-HBs) 效價逐漸消失，在之前針對目前抗B型肝炎病毒表面抗原抗體低下的人評估是否需要追加注射的研究中，發現多數的人再經過一次的追加注射都能產生保護性抗體，更重要的是均具有B型肝炎病毒表面抗原 (HBsAg) 特異記憶性B細胞，即使在抗體低下之時，也能因為抗原的刺激發揮其抗體再生性的特性。由於抗體低下的人其記憶性B細胞頻率與高抗體的人並無差異，因此我們想要進一步去了解在B型肝炎疫苗接種後的記憶性免疫反應中，記憶性T細胞是否與抗體的產生與維持有相關性。本研究利用具有高敏感度、便利等優點的酵素結合免疫斑點分析法 (ELISPOT) 來偵測這些疫苗接種者體內的記憶性T細胞的變化。經過不同的細胞濃度、抗原濃度與反應時間等各種測試後，調整出適於檢測分泌干擾素-γ (IFN-γ) 的B型肝炎病毒表面抗原特異記憶性T細胞的條件，10 μg/ml HBsAg刺激2×105 cells/well 的周邊血液單核細胞 (PBMC) 反應36小時後，能夠得到最佳的免疫斑點產生。在我們所收集的抗B型肝炎病毒表面抗原低抗體者的個案中，經過一劑疫苗追加注射後，年齡平均為22 ± 2歲的醫學院學生，85.7%的人能因此而獲得高效價抗體 (>100 mIU/ml)，而且記憶性T細胞頻率由追加注射前的3.32 ± 0.74 SFC/2×105 cells增加為追加注射後的10.89 ± 2.75 SFC/2×105 cells (P < 0.05, n=35)；年齡平均為45 ± 8歲且多數曾經自然感染過的醫院員工，只有37.5%的人能因此而獲得保護性抗體，記憶性T細胞頻率在追加注射前後並沒有顯著差異 (0.90 ± 0.52 SFC/2×105 cells vs.1.14 ± 0.67 SFC/2×105 cells, P=0.782, n=8)。在這些族群中有少數是即使經過追加注射也不產生抗體的疫苗不反應者 (non responder)，其記憶性T細胞頻率在追加注射前與追加注射一劑或是完成3劑疫苗注射後都沒有顯著差異 (1 ± 0.89 SFC/2×105 cells vs. 2 ± 1.24 SFC/2×105 cells vs. 2.5 ± 1.22 SFC/2×105 cells, P> 0.05, n=4)。而多數經過追加疫苗注射之後獲得保護性抗體的人也均具有HBsAg特異記憶性B細胞的存在 (1.97 ± 0.53% vs. 3.40 ± 0.67%, P < 0.05, n=17)。本研究發現多數具有HBsAg特異記憶性T細胞的人在經過追加注射後，其記憶性T細胞的頻率會增加，並且可以獲得高效價的抗體，而經過3劑追加仍不能產生抗體的人，雖然也具有HBsAg特異記憶性B細胞，但是其HBsAg特異記憶性T細胞並沒有任何變化。由此可知，無法產生記憶性T細胞的確影響抗體的產生與維持，未來也許可以藉此判斷疫苗追加必要性的依據。

A universal vaccination program to hepatitis B virus (HBV) infection has been launched in Taiwan since 1984. It significantly reduced HBV carrier rate, fulminant hepatitis, and hepatocellular carcinoma (HCC). Fifteen years after initiation of vaccination, it is not completely understood whether vaccinees with low anti-HBs titer (< 10 mIU/ml) can against HBV infection. Our previous study showed that it is unnecessary to boost the low anti-HBs titer population, due to the expression frequency of HBsAg-specific memory B cells in low anti-HBs titer population was similar to that of high anti-HBs titer ones. Further, we used the antigen-specific enzyme-linked immunospot (ELISPOT) to examine the interrelationship between the production of protective antibody and HBsAg-specific memory T cells. For optimizing immunospot formation, the following conditions for testing the IFN--secreting memory T cells production were established: 2105 peripheral blood mononuclear cells (PBMCs) per well with 10 g/ml HBsAg for 36 hours. Frozen and fresh PBMC demonstrated the similar results of spot formation. In the assay, the production of non-specific immunospot was not stimulated by Keyhole limpet hemocyanin (KLH). Individuals with anti-HBs titer below 100 mIU/ml before booster, 85.7% of them had high anti-HBs titer (> 100 mIU/ml) after one dose vaccine booster. In the college students (age, 22 ± 2 years), HBsAg-specific memory T cells were significantly increased after booster, in contrast to pre-booster (10.89 ± 2.75 SFC/2×105 cells vs. 3.32 ± 0.74 SFC/2×105 cells, P < 0.05, n=35). However, there is no significant difference of HBsAg-specific memory T cells among the hospital employees (age, 45 ± 8 years) after booster. The frequency of HBsAg-specific memory B cells was increased in the most subjects after booster (1.97 ± 0.53% vs. 3.40 ± 0.67%, P < 0.05, n=17). Some individuals didn’t have protective antibody after booster, even received three doses. We assumed that individuals without protective antibody were non responder for HBV vaccine. The HBsAg-specific memory T cells were increased in individuals with high anti-HBs after booster, however, in the non responders there were no differences after booster. In conclusion, the HBsAg-specific memory T cells were increased in individuals who had protective antibody after booster. But for HBV vaccine non responders did not change in the HBsAg-specific memory T cells even though they had increasing HBsAg-specific memory B cells. These resultes suggested that HBsAg-specific memory T cells play an important role in the maintenance and production of anti-HBs antibodies.

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