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

• Main Authors: Wen-Yang Tsai, 蔡文揚
• Other Authors: Wei-Kung Wang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/07920709583242913187

碩士 === 國立臺灣大學 === 微生物學研究所 === 95 === 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.