Identification and characterization of meningococcal surface antigens with vaccine potential

Abstract

Neisseria meningitidis (menningococcus, MC) a capsulated Gram-negative bacterium, has become theleading cause of bacterial meningitis in children owing to the reduction in incidences of Streptococcuspneumoniae and Haemophilus influenzae type b infection achieved by the introduction of conjugatevaccines for the latter pathogens. Among the 13 serogroups of MC, groups A, B, C, W-135, and Y represent virtually all pathogenic isolates. Encouraging progress has been made in the development of CPS-conjugate vaccines against groups A, C, W135 and Y, but no effective vaccine against B is available. Recently, many potential vaccine candidates have been identified by reverse vaccinology based on genomics. However, still other candidates must have been unseen due to uncertain annotation or their peculiar properties. Therefore, we thought to explore this field by immuno-proteomic approach using monoclonal antibodies (mABs) obtained from mice immunized with intact MC. We have obtained six independent mAbs, each can recognize over 90% of the tested MC strains from different serogroups. Todate, the target protein (Ag473) for one of the bactericidal mAb, 4-7-3 (IgG3) has been identified. The vaccine potential of Ag473 has been demonstrated in adult mice model. With the identification of Ag473, a R & D project on meningococcal subunit vaccines has been initiated at the Vaccine Research and Development Center, NHRI. Ag473, undetectable by staining with Coomassie brilliant blue and UV absorbance, is a novel lipoprotein with no homologues of known function in the database. An ag473 mutant (MT) obtained by inserting a gentamicin cartridge in the coding region exhibits a reduced virulence suggesting that Ag473 is crucial for meningococcal pathogenesis. The two essential virulence factors for MC pathogenesis are type IV pili and CPS, with type IV pili being required for the attachment to the human cells and CPS for the survival in the bloodstream. Immunofluorescent microscopic analysis revealed that (1) MT do not form 3D-microcolonies on the surface of infected cells, a known trait associated with type IV pilus-mediated adherence of MC, (2) MT exhibit better efficiencies than it parental strain (WT) in adherence, and (3) the WT but not MT can form filamentous structures (presumably formed by type IV pili). Theseobservations indicate that the surface architecture of MT is strikingly different from that of WT. Based on its adherent properties, the MT apparently are nonpiliated and noncapsulated. One goal of this research is to understand the pathophysiological role Ag473 plays on meningococcal pathogenesis. The specific aims for this part of study are (1) to elucidate the role Ag473 plays in the type IV pilus biogenesisand maintaining the surface architecture by depicting the surface structures of in vitro grown MT and WT and determining the composition of the filamentous structures seen on WT after contact with NPC-076 cells, and (2) to elucidate the effect of Ag473 exerts on MC-host interactions, by determining (a) theexpression pattern of surface antigens, which are known to be modulated in the process of bacterial adherence after co-incubation with human epithelial cells, blood cells, or brain cells, and (b) the inflammatory cytokine expression profiles in human cells after co-incubation with WT, MT, or purifiedAg473. Since both type IV pili and CPS are critical for MC to invade meninges, defining the function of Ag473 can bring new insights into MC type IV pilus biogenesis. By examining the surface antigens on MC and the cytokine expression profiles of cells derived from nasopharyngeal, peripheral blood cells andbrain after MC-host cell interactions, the results may provide some clue towards understanding of the mechanism for MC to cross the brain-blood barrier. Another goal of this proposed research is to identify and characterize the target antigen for the remaining mAbs reactive to a broad spectrum of MCs. Among the five mAbs recognizing conserved epitopes on MC, identification of the antigen for mAb 24-03 will be set as the first priority because this antigen seems to be more accessible in the ag473 knock-out mutant. This implicates that it may be structurally orfunctionally associated with Ag473. For the remaining four, the priority of target finding will be set depending on the expression patterns of their antigen on MC when interacting with the human cells. The results of this study would benefit the on going R & D project in meningococcal B subunit vaccine.

Neisseria meningitidis 俗稱腦膜炎球菌(MC)是一個具有莢膜的革蘭氏陰性菌。自從有了鏈球菌與嗜血桿菌的結合疫苗(conjugate vaccine)之後，MC已成為兒童細菌性腦膜炎的主要病因。MC雖可分為13群，致病菌主要為血清群A、B、C、Y及W135。在血清群A、C、Y及W135的疫苗研發上已有莢膜多醣結合疫苗上市，但目前尚未有針對血清群B的有效疫苗。近來在MC的基因體解碼後，利用反向疫苗學，已有許多具有疫苗潛力之抗原被發現。雖然如此，有些目標抗原卻因為基因註解不確實或是因其性質特殊而未被發現。有鑑於此，我們曾以MC全細胞免疫小鼠，製備單株抗體，獲得六株分別能辨識屬於不同血清群的MC (140株)之中至少90%菌株之抗體株。目前已完成具有殺菌力的抗體4-7-3之抗原鑑定，此抗原稱為Ag473。小鼠實驗顯示Ag473具有成為疫苗之可能性。此抗原的發現，是國衛院疫苗研發中心開始「B型腦膜炎重組次單元疫苗研發計畫」的 主因。 Ag473是一個功能未知、無法以Coomassie brilliant blue染色，也無法以280 nm的吸光值偵測的膜連脂蛋白，而且在目前的資料庫中也查不到任何功能已知的類似蛋白。以gentamicin抗藥基因插入ag473基因得到的MC 突變株(MT)，致病力明顯降低，顯示Ag473蛋白對MC致病力的重要性。已知第四型纖毛以及多醣莢膜(CPS)是MC致病過程中必要的兩個因子，藉著第四型纖毛，MC才能黏附在人類細胞上，而CPS則是MC在血液中能存活的主要關鍵。以螢光顯微鏡檢視發現：(1) MT在感染的細胞表面不會形成如第四型纖毛黏附所引發的3D-微菌落，(2)MT與人類細胞的黏附能力比野生型(WT)好，(3)只有WT具有可能係由type IV 纖毛所形成的絲狀構造。這些觀察結果顯示MT的表面結構與WT差異極大，而由其顯示的外觀推測，MT可能不具有第四型纖毛，而且也沒有莢膜多醣。本研究的第一個目的是要(1)藉由分析試管中生長的MT與WT之表面構造以及分析WT與NPC-076細胞接觸後所形成的絲狀構造之成分，了解Ag473在第四型纖毛生成上以及在維持表面構造正常上所扮演的角色; 以及(2)藉由以下兩項分析，了解Ag473對MC-宿主細胞互相作用的影響：(a)分析表面抗原的表現態樣(該等物質之表現在MC與人類上皮細胞、血液、腦細胞等接觸之後，其表現會受到調控)，以及(b)分析人類細胞與WT、MT或純化的A473共同培養之後，其發炎相關細胞素 的表現態樣。由於第四型纖毛與CPS 均為MC入侵腦膜所需，對Ag473功能的了解將對MC第四型纖毛的生合成有所了解。藉由上述諸項研究，我們將能獲得線索，供了解MC穿越腦與血之間的藩籬之機轉。本計劃的另一目標為，對之前的研究中獲得的其餘單株抗體之目標抗原，加以鑑定及特性描述。在該五株單株抗體(均各能辨認MC的保守epitopes)之中，24-03將列為第一優先，因為此一抗原在ag473突變株似乎比較容易被接觸到，表示其與Ag473在構造與功能上有關連。至於其餘的四個，對其目標抗原尋找工作之優先順序，將依它們與人類細胞接觸之後，在MC表面所表現的態樣來決定。這些研究結果將對目前進行中的腦膜炎雙球菌B群次單元疫苗之R&D計畫有實質的助益。