探討登革病人之抗體反應以及登革病毒前驅膜蛋白質之表現及其異類二聚體及重組次病毒顆粒之形成

Study of antibody responses in dengue patients and the expression of precursor membrane protein of dengue virus and its involvement in heterodimerization and recombinant subviral particles formation

蔡文揚(Wen-Yang Tsai)

指導教授 ： 王維恭

國立臺灣大學/醫學院/微生物學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.00595

摘要

登革病毒（Dengue virus, DENV）為黃質病毒科的一員，在將近80多種蟲媒病毒中，四種血清型的登革病毒造成最重要的人類蟲媒疾病，包括典型的登革熱（Degue fever）及嚴重的登革出血熱/登革休克症候群（Dengue hemorrhagic fever / Dengue shock syndrome）。被某一型的登革病毒感染則對該型有終生保護性，但是對其他型的登革病毒則否。登革病毒在非中和性抗體或低於最適濃度的中和性抗體作用下，會有所謂抗體決定促進現象，此現象可以解釋流行病學上觀察到二次感染者較一次感染者有更高的危險產生登革出血熱，此即免疫學說。傳統上對於登革病人抗體反應之研究，主要用血球凝集抑制法，酵素連結免疫吸附法或中和試驗，可以用於區別一次感染或二次感染，但是對於抗體所辨識之登革病毒抗原無法區別。雖有少數西方墨點法的報告，但是對各抗原的標定並不夠嚴謹，且對感染同型或不同型之登革病毒抗原無詳細分析。
本實驗第一個目標是利用西方墨點法來探討2001-2年高屏地區第二型登革病毒一次感染或二次感染之共69個病人，包括登革熱及登革出血熱，其血清中抗體對於同型以及不同型之四種抗原，包括外套膜（envelope, E），前驅膜蛋白（precursor membrane, PrM），殼（capsid, C）及非結構性蛋白質（nonstructural protein）NS1的辨識情形。此四種抗原均以已知的單株抗體標定位置。我們發現隨著發燒後天數增加，一次感染病人產生之抗體，首先可以辨識第二、第一及第三型E蛋白質以及第二型NS1蛋白質，能辨識第四型E蛋白質之抗體較晚出現。而二次感染病人之抗體首先可以辨識三到四型之E蛋白質，第二和第一型之NS1蛋白質以及三到四型之PrM蛋白質。發燒後七天血清中抗體則可以辨識四型登革病毒的E蛋白質、NS1蛋白質以及PrM蛋白質，相當於回復期時體內抗體之反應。比起一次感染的病人，二次感染之病人體內抗體反應來的更快更強，並且能夠辨識到更多病毒抗原。進一步分析發燒後大於7天之血清檢體，顯示二次感染之病人辨識四型NS1及四型PrM蛋白質的比率明顯高於一次感染之病人（P<0.005, chi-square test）。未來可以利用此種差異來區別一次及二次感染。不論是一次感染或二次感染，登革熱病人與登革出血熱病人血清中抗體對於不同抗原的辨識，並無明顯的不同。最後利用還原劑β-mercaptoethanol來破壞病毒抗原之構形觀察病人抗體之辨認情形，發現絕大多數病人血清中抗體無法辨識構形改變之E及PrM蛋白質，然而只有少數病人血清仍可以辨識構形改變之NS1蛋白質。
另一方面，表現登革病毒的E及PrM蛋白質可以交互作用形成異類二聚體（heterodimer），進一步形成重組次病毒顆粒（recombinant subviral particles, RSPs），此為研究PrM及E蛋白質功能的重要工具，也可用於血清診斷試劑及未來疫苗之用。過去對於PrM及E蛋白質異類二聚體及重組次病毒顆粒之研究，主要著重於E蛋白質，顯示其C端著柄區（stem）之α-螺旋結構（α-helix）（包括E-H1及E-H2）及嵌入區之α-螺旋結構（包括E-T1及E-T2）對於上述功能有重要的影響，但是對PrM蛋白質方面則較少研究。本研究的第二個目標主要以第四型登革病毒的PrM/E表現質體為材料來探討PrM蛋白質之表現以及其C端著柄區α-螺旋結構（M-H）對PrM及E蛋白質形成異類二聚體及重組次病毒顆粒之影響。我們根據螺旋構造預測軟體，在a、d位點上5個胺基酸進行點突變成丙胺酸或脯胺酸，發現所有的點突變對於PrM及E蛋白質形成異類二聚體並沒有太大的影響，但是M-H區域上的第120、123及127個胺基酸突變成脯胺酸就會破壞重組次病毒顆粒之形成，顯示M-H區域上α-螺旋結構對於重組次病毒顆粒形成是重要的。另外對於PrM蛋白質表現，我們以PrM與E蛋白質及系列C端截短質體進行研究。我們發現PrM蛋白質在有E蛋白質存在下才能有良好之表現，其C端截短質體不能單獨表現，顯示E蛋白質對於PrM蛋白質之表現及穩定有其角色。此外E蛋白質去除了E-T2、E-T1和E-H2區，PrM蛋白質之表現有下降的趨勢，顯示E蛋白質主要靠其C端之E-H2區來協助PrM蛋白質之表現及穩定。
在過去近30年來，全球各地登革疫情流行日趨嚴重，不但登革熱及登革出血熱之病例急速增加，有登革出血熱病例之國家數目也快速成長，顯示研究登革病毒之重要性。本研究第一部分以西方墨點法研究登革病人血清中之抗體反應顯示辨識PrM及NS1蛋白質的有無可以用來區分一次或二次感染。此外我們的研究發現絕大多數病人針對E及PrM蛋白質之抗體反應均是受抗原構形所影響，這對未來設計重組疫苗上提供重要訊息。本研究第二部分探討PrM蛋白質之表現與功能，顯示E蛋白質，特別是其C端部分對PrM蛋白質之表現有重大影響，這在黃質病毒屬的病毒中是特別的發現。而PrM蛋白質之C端M-H區之三個重要胺基酸對形成重組次病毒顆粒有重要影響，顯示此α-螺旋結構之重要功能，可以是未來發展抗病毒策略之標的之一。

關鍵字

登革病毒 ； 異類二聚體 ； 次病毒

並列摘要

Dengue viruses（DENV）are members of the family Flaviviridae. Among the 80 or so arthropod-borne flaviviruses, the four serotypes of DENV（DENV1, DENV2, DENV3, DENV4）cause the most important arboviral diseases in human, including the classic dengue fever（DF）and severe dengue hemorrhagic fever/dengue shock syndrome（DHF/DSS）. Infection by one serotype of DENV can provide life-long protection against that serotype, but not against other serotypes. Infection of DENV in mononuclear cells has been shown to be enhanced by the presence of non-neutralizing antibody or suboptimal concentration of neutralizing antibody. This is the so called antibody-dependent enhancement, which may explain the epidemiological observation that people with secondary infection have a significant higher risk of developing DHF than people with primary infection. Previous studies of antibody responses against DENV by hemagglutination inhibition, ELISA and neutralization tests can distinguish between primary and secondary infection, but not different DENV antigens recognized. Although some studies have used Western blot analysis to examine antibody responses in DENV patients, the antibody responses against various DENV antigens of homologous or heterologous serotypes in DENV patients remain unclear.
The first specific aim of this study is to use Western blot analysis to investigate the antibody responses against four DENV proteins, envelope（E）, precursor membrane（PrM）, capsid（C）, and nonstructural protein NS1 of 4 serotypes of DENV in 69 patients with DF or DHF and with primary or secondary infection during the DENV2 outbreak in southern Taiwan in 2001-2. Several known monoclonal antibodies against these 4 DENV proteins were used to identify the DENV proteins recognized. With the increase in fever days, sera in patients with primary infection can first recognize E protein of DENV2, DENV1 and DENV3, as well as NS1 of DENV2. Anti-DENV4 E responses appeared later. In patients with secondary infection, sera can first recognize E and PrM proteins of three to four serotypes and NS1 of DENV1 and DENV2. After 7 days from the fever onset, the responses reached to the level and extent similar to that of convalescent stage, in which sera can recognize E, PrM and NS1 proteins of all 4 serotypes. Compared with those with primary infection, antibody responses in patients with secondary infection developed quicker and recognized more antigens at higher levels. Statistical analysis reveals that the proportion of patients with secondary infection developed anti-NS1 and anti-PrM antibodies ≧7 days from fever onset were significantly higher than those with primary infection（P<0.005, chi-square test）. There was no difference in the antibody responses against the four DENV proteins tested between patients with DF and DHF. After treating the antigens with reducing agent, β-mercaptoethanol, antibodies responses to E, PrM and NS1 proteins disappeared in the majority of cases, with anti-NS1 responses left in few cases.
On the other hand, expression of E and PrM proteins of DENV can form heterodimer, which is important for the production of recombinant subviral particles（RSPs）. RSPs have been shown to be a useful tool to study the function of PrM/E proteins as well as a potential serodiagnostic antigen and vaccine candidate. Previous studies of the heterodimerization and RSPs of PrM/E proteins focused primarily on the E protein, of which the two α-helices in the stem region（E-H1 and E-H2）and the two α-helices in the anchor region（E-T1 and E-T2）at the C-terminal have been shown to play an important role. Few studies examined the involvement of PrM protein in the process. The second specific aim of this study is to study the expression of PrM protein and the involvement of the C-terminal α-helix（M-H）of the stem region of PrM protein in the heterodimerization with E protein and RSPs production by using DENV4 based expression constructs. Amino acid substitutions of the 5 amino acid residues located at a,d position of the M-H in PrM protein with Alanine or Proline revealed no effect on heterodimerization. In contrast, amino acid substitutions of 3 amino acid residues at position 120, 123 and 127 with Proline resulted in severe impairment in RSPs formation. A serious of C-terminal truncation constructs of PrM and E proteins were also generated. In the absence of E protein, PrM protein can not express well, especially its C-terminal truncation mutants. Moreover, in the presence of C-terminal truncated E protein（without E-H2, E-T1 and E-T2）, the expression of PrM protein significantly decreased. These findings suggested that E protein, especially its C-terminal domain E-H2, play a critical role in the expression of PrM protein.
During the past three decades, there was a significant increase in the numbers of DHF/DSS cases and the numbers of countries reported to have DHF/DSS, indicating the importance and need for dengue research. The first part of this study suggested that reactivities to PrM and NS1 proteins recognized by Western blot analysis can be used to distinguish primary and secondary infection. Moreover, the majority of anti-E and anti-PrM antibody responses in DENV patients were conformational sensitive. These findings may provide useful information for future design of subunit vaccines. The second part of this study revealed that the E protein, especially its C-terminal domain, was important for expression of PrM protein. In addition, three critical amino acid residues at the M-H region of PrM protein were critical for the formation of RSPs, suggesting that this α-helix has an important function and may represent potential target for future antiviral strategies.

並列關鍵字

dengue virus ； heterodimer ； subviral particles

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