Searching for tumor-associated antigens and development of non-capsular meningococcal vaccine.

Abstract

In the field of tumor immunology, discover of tumor-associated antigens is not only beneficial to understand how tumors escape from the immune responses, but also can be the basis for diagnosis and immunotherapy development. In this study, rabbit anti-esophageal carcinoma tissue serum (anti-ESO-C) was to identify potential tumor associated antigens using the affinity selection assays against phage-displayed random peptide libraries－Ph.D.-12 and Ph.D.-C7C. Using the plaque filter immunoscreening, 7 anti-ESO-C phage clones were identified. The amino acid sequences of the peptide epitopes (phagotopes) were revealed by nucleotide sequencing. To identify the corresponding natural antigen(s) of each phagotope, the bioinformation Blast Search was performed to identify the putative targets and conformed by RT-PCR analysis. A brain-specific phosphatidic acid phosphatase-like protein, PHP1, was identified to be one of the putative targets. RT-PCR analysis revealed that this gene was expressed in esophageal, lung, and some liver tumor tissues implying that PHP1 may be classified as a tumor-associated antigen. In addition, immunochemical techniques were applied directly to identify the natural antigen. To generate the peptide specific antibody, phage particles displaying the desired peptide were produced to immunize mice. The resultant antiserum exhibited little or no specificity to the displayed peptide. To improve immunization efficacy, oligopeptides containing multiple repeats were used as the immunogens. A phagotope derived from the outer membrane PorA protein of N. meningitidis was used as an example, in which a DNA fragment encoding 4.5 repeats of the PorA phagotope (PorA-F1) was obtained by template-repeated polymerase chain reaction (TR-PCR) and cloned into expression vectors to be expressed either alone or fused to the C-terminus of Ag473, an lipoprotein of N. meningitidis. No specific antibody was detected in the mice immunized with PorA-F1. Although anti-PorA antibody can be detected in the antiserum against Ag473-PorA-F1 by Western blotting, it varied significantly among immunized mice. To evaluate whether Ag473 and Ag473-PorA-F1 can serve as vaccine components, in vitro bactericidal assay and in vivo protection assay were performed. Although neither of the antisera showed bactericidal activity, significant protection against N. meningitidis Nm22209 was obtained in immunized mice, suggesting that opsonophagocytic activity may be the mechanism of protection. When the whole cells of E. coli expressing Ag473 or Ag473-PorA-F1 were used as immunogens, significant bactericidal activity was observed. The results present in this thesis clearly demonstrate the potential of using Ag473 as a meningococcal vaccine component and also provide a preliminary basis for vaccine formulation.

在腫瘤免疫學的研究領域中，找出腫瘤相關抗原有助於了解癌細胞逃避免疫系統的機轉，且可作為診斷之用和免疫療法的基礎。本研究以兔子抗食道癌組織血清 (anti-ESO-C) 對Ph.D.-12及Ph.D.-C7C噬菌體胜肽展示庫因進行親和選擇，再以噬菌斑濾膜免疫篩選分離得到7個呈陽性反應之噬菌斑。核酸定序顯示此七個重組噬菌體各展示不同的胜肽。為了找出各序列所模擬之原始抗原，首先利用 BLAST 搜尋可能之目標抗原，再以 RT-PCR 分析此基因在 mRNA 層次之表現概況，初步分析結果發現一腦部專一性表現蛋白 (PHP1) 於食道癌、肺癌及部分肝癌組織皆有表現，但表現量不一，推測可能為腫瘤相關性抗原。另一方面嘗試製備展示胜肽專一抗體，直接找尋原始抗原。為達此目的，首先以重組噬菌體進行免疫注射，結果效果不彰。為了提高免疫原的效力，進而提升抗體專一性，嘗試以胜肽片段重複之形式作為免疫原。為了評估此策略之可行性，以腦膜炎雙球菌的外膜蛋白 PorA 之抗原表位模擬胜肽片段作為實驗材料，觀察所得之抗血清是否可如預期辨識原始抗原 PorA。 首先以 template-repeated polymerase chain reaction (TR-PCR) 技術合成一段主導4.5個重複序列的 DNA 片段，將此片段單獨表現得到重複胜肽 PorA-F1，或與另一腦膜炎雙球菌的脂蛋白 Ag473 以融合方式 (Ag473-PorA-F1) 表現，並進行免疫力分析。結果發現 PorA-F1 無法引起有效之抗體反應；以Western blot分析各Ag473-PorA-F1免疫小鼠之血清對 PorA 之辨識情形，結果發現小鼠間之差異極大。為了測試 Ag473 以及 Ag473-PorA-F1 是否具有疫苗潛力，分別測試抗血清之殺菌力以及腦膜炎雙球菌菌株 Nm22209 對免疫以及未接受免疫小鼠之影響。結果發現雖然in vitro分析 抗血清不具有殺菌活性，但無論是Ag473或Ag473-PorA-F1免疫之小鼠對 Nm22209 之感染都有顯著的保護力。此結果顯示，此兩種形式之免疫原引發之抗體主要透過調理吞噬能力達到殺菌效果。進一歨以表現 Ag473 或 Ag473-PorA-FI 之 E.coli 全菌進行免疫注射，結果發現所得之抗血清具有明顯的殺菌活性。本論文研究結果不但證明 Ag473 具有疫苗成分之可行性，同時也提供了後續疫苗配方研發之初步基礎。