第四型登革病毒單株抗體之特性探討

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(1)國立台灣師範大學生命科學系碩士論文. 第四型登革病毒單株抗體之特性探討 Characterization of Monoclonal Antibodies against Dengue Virus Type 4. 研究生：沈文凡 Wen-Fan Shen. 指導教授：吳漢忠博士 Han-Chung Wu, Ph.D. 童麗珠博士 Li-Chu Tung, Ph.D.. 中華民國九十八年七月. 1.

(2) 中文摘要登革病毒是盛行於熱帶及亞熱帶地區感染人類的病原體，主要的傳染途徑是經由帶有病毒的病媒蚊叮咬，後而易引起登革熱、登革出血熱和登革休克症候。登革病毒的套膜蛋白[envelope (E) protein]具有結合細胞表面受器及引發病毒與宿主間膜融合的功能，同時亦是能誘發人體免疫反應的重要抗原；套膜蛋白的膜外區域(E domain)可以再進一步區分為三個功能區塊：區塊一、區塊二與區塊三(EDI, II, III)。為了探究登革病毒中和性抗體之抗原決定位及未來研發登革的DNA 疫苗，本研究篩選了若干株由我們實驗室所生產的抗體，以釐清第四型登革病毒與中和性抗體之間的交互作用。作法上，我們先利用免疫光染色法與西方墨點法來確認單株抗體的專一性，並且找到若干株單株抗體能對抗病毒的套模蛋白或非結構性蛋白一 [Non-structural (NS)-1] 。我們更進一步利用蝕斑減少中和試驗 [Plaque reduction neutralization test (PRNT)]，與保護試驗檢測這些單株抗體對於登革第四型的中和能力。我們亦製造了兩個DNA載體，分別含有第四型登革病毒的功能區塊一、二及三，並將此載體利用基因槍施打於小鼠身上，希望能藉此比較功能區塊一、二及功能區塊三對於誘發免疫反應的能力。我們亦施打第四型登革病毒DNA疫苗，pCB8D4-2J，及我們製作的載體，進一步比較並評估該製造的載體是否有成為DNA疫苗的. 2.

(3) 潛力。在本實驗中我們t辨識出九株單株抗體能專一辨識第四型登革病毒與九株會變其他型登革病毒的單株抗體；其中有十四株抗體是辨識套模蛋白，兩株是辨識非結構性蛋白一。在辨識套模蛋白中，有七株單株抗體是辨識區塊一、二，另有兩株抗體能辨識區塊三。最後找到兩株能辨識登革第四型功能區塊一、二，並在細胞實驗上能阻止病毒感染，但只有一株抗體能在動物模式上提供保護。同時我們製造的 DNA疫苗，雖然能夠表現其蛋白質於細胞且被抗體所辨識，但是在誘發對抗登革病毒免疫的能力上，仍不及pCB8D4-2J；顯示該DNA疫苗需多次免疫方能有效誘導出對抗登革病毒的抗體。. 關鍵詞:登革病毒、套膜蛋白、單株抗體、抗原決定位、DNA疫苗. 3.

(4) Abstract Dengue virus (DENV), the human pathogen leading to dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), is epidemic in tropical and subtropical areas around the world. The envelope protein (E) of DENV, which could induce protective immunity, is critical for membrane fusion and mediates binding to cellular receptors. The ectodomain of the E monomer could further be divided into three domains assigned to domain I, domain II and domain III (EDI, EDII and EDIII). In order to confirm the target of neutralizing antibodies and develop the DNA vaccines of DENV, we generated a large panel of MAbs against DENV4 in our laboratory. In our study, we further these MAbs against DENV4. At first, we use the immunofluorescence and Western blotting to identify the specificity of those MAbs. Several MAbs could recognize the envelop protein (E protein) or non-structural protein 1 (NS-1). We further demonstrated their neutralizing activity of DENV4 by plaque reduction neutralization test (PRNT) and protection assay. We have identified 9 serotype-specific MAbs and 9 cross-reactive MAbs against DENV4, and 14 MAbs could recognize E protein and 2 MAbs recognized NS-1 protein. Among these MAb recognized E protein, seven MAbs could recognize EDI-II and 2 MAbs recognized EDIII. Finally, we identified 2 MAbs, which recognized EDI-II of DENV4, displayed neutralizing activity in vitro, but only one MAb could reveal protective activity in vivo. We also constructed two DNA vectors which contained the EDI-II and EDIII of DENV4, and immunized the mice for identification the neutralizing antibodies. We used the DNA vaccine of 4.

(5) DENV4, pCB8D4-2J, to immunize the mice to determine the neutralizing avtivity of EDI-II or EDIII DNA vaccine by PRNT and protection assay for imitating immune responses against DENV4 in vitro and in vivo. Although the two DNA vaccines, EDI-II and EDIII, could be detected the protein by IFA, their humoral immunity against DENV4 still weaker than pCB8D4-2J. It might result from less immunization, so we need to immunize mice more times. Keywords: dengue virus, envelope protein, monoclonal antibody (MAb), Epitopes, DNA vaccine. 5.

(6) 致謝在兩年研究所的生活中，最先要感謝吳漢忠老師。吳老師不僅願意指導在這個領域毫無背景的我，提供實驗的方向與想法更開拓了我的視野；除了實驗外，吳老師亦樂於與我們分享人生的歷程與經驗，使我受益匪淺。另外，非常感謝童麗珠老師從大學開始指導我實驗，讓我打下實驗的基礎。同時，也非常感謝金傳春老師能撥空擔任我的口試委員，並提供寶貴的建議。其次，我要感謝中研院細生所的各位: 小倩姊、顏博、美英、瑞旻、璧君、怡如、中道、德寬、東盈、國華、建勳、美櫻、逸平、慈文、慧宇、秉昌與中興趙老師，能在實驗及生活上提供幫助與意見。也要感謝同時進實驗室的凱文跟怡亘，一起分享研究時的喜怒哀樂；還有民珊跟育綾在平日的協助。此外，也感謝在師大的仁華與施兄在實驗與課業上所提供的掩護及各項的支援，也感謝惠如陪我走過碩士兩年七百多個日子。最後，我要感謝我的父母與弟弟，給予我經濟上的支持與鼓勵，讓我能無後顧之憂的完成學業。. 沈文凡 2009 年 7 月. 6.

(7) Contents 中文摘要..………………………………….……………………………2 Abstract……………………………………………………………….…4 致謝…………………………………………………………..…………6 Contents…………………………………………………………………7 Introduction…………………………………………………………..…8 Materials and Methods…………………………………………………22 Results…………………………………………………….………..…..28 Discuession……………………………………….………..…………..34 Figure……………………………….………..………….……………...39 Table………………………………….……………………..……….…55 Reference…………………………………………………………….…56. 7.

(8) Introduction Epidemiology of dengue Dengue viruses (DENV) which are mosquito-borne RNA virus are members of the Flavivirus genus of the Flaviviridae family. There are four serotypes (DENV1, DENV2, DENV3 and DENV4) and they causes from asymptomatic infection, dengue fever (DF) to dengue hemorrhagic fever (DHF) / dengue shock syndrome (DSS)(Henchal and Putnak, 1990; Kautner et al., 1997). The latter is fatal without treatment correctly (Gubler, 1998). There are about 50-100 million DENV infections each year, 1 % of which will develop DHF (Monath, 1994) and more than 2.5 billion people are at the risk of DENV infection. Especially, people live in tropical countries (Gubler, 2006; Halstead, 1990). In Asia, DENV infection is a leading factor of death among children and the major DHF cases in the world are from there (Gubler, 2002).. The structure of dengue virus The genome of DENV is a 10.8-kb, single, positive-strand RNA. The genomic RNA contains a cap at the 5’-end without a polyadenylate tail at the 3’-end. and it is has an open reading frame (ORF)(Chambers et al., 1990). It encodes 3 structural proteins, which is including capsid protein (C), membrance protein (M) and envelope proteins (E), and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). There are the 5’- and 3’-untranslated regions (UTRs) of around 100 and 400–600 nucleotides, which are crucial in the initiation and regulation of translation, replication, and virion assembly, ouside the 8.

(9) ORF (Alvarez et al., 2005; Chiu et al., 2005). The C protein which is composed of ~120 amino acids has 4 helices(α1-α4) and form dimers in solution (Jones et al., 2003). Its dimer structure is involved in forming the nucleocapsid (NC) core and packaging of the viral genome (Kiermayr et al., 2004; Zhang et al., 2004). The pre-membrane (prM ~165 amino acids), the precursor to M, and E (~495 amino acids) are glycoprotein, each of which has two transmembrane helices. Additionally, before the prM protein is cleaved during particle maturation, it seems to function as a chaperone for the assembly of the E protein (Lorenz et al., 2002). The diameter of mature DENV is 500-Å approximately. The outer of mature DENV is envelope which consists of 50-Å thick lipid bilayer, 180 M and E proteins (Kuhn et al., 2002).. Envelope protein of Dengue virus By X-ray crystallographics, it showed that the E proteins form 90 homodimers on the surface of the mature virion. The length of E protein is approximately 500 amino acids and its N-terminal 400 amino acids form the ectodomain. In addition, the ectodomains of the E monomer could be divided into three domains assigned to domain I, domain II, and domain III (EDI, EDII, and EDIII) (Modis et al., 2003). Of the E protein monomer, the central structural domain is EDI and it is linked the EDII and EDIII together by four and single polypeptide chains. Another domain, EDII, is an elongated dimerization domain which contains the fusion peptide. The fusion peptide is important to trigger membrane fusion with endosomal membrane in the low-pH conditions, and then, 9.

(10) release the viral RNA into cytoplasm host cell (Allison et al., 2001). The function of fusion peptide is not only above but also it is buried between EDI and EDIII of the adjacent monomer within a dimer (Rey et al., 1995; Zhang et al., 2004). Moreover, one of the crystal structures of E protein of DENV2 revealed a molecule of N-octyl-β-D-glucoside to be positioned at the EDI-II interface (Modis et al., 2003), which this flexible structure might be necessary for the large conformational alterations during maturation and fusion.The other domain, EDIII, is an immunoglobulin (Ig)-like domain which is regarded as the theoretical binding site with the receptor of the target cell (Chen et al., 1997; Chiu and Yang, 2003; Hung et al., 1999). This supposition result from monoclonal antibodies (MAbs) that bind to EDIII are blockers of virus adsorption (Crill and Roehrig, 2001), and the dominant and type-specific neutralization sites for MAbs are localized in here (Roehrig, 2003).. The candidate receptors for Dengue virus The principal vectors of DENV transmission are Aedes aegypti and Aedes albopictus mosquitoes. These viruses evolved in subhuman primates from a common ancestor and were separately introduced into the urban cycle some 500 years ago (Wang et al., 2000). When the mosquitoes that infected DENV bite the human, the DENV can be transmit to the host. Then, the DENV infect the target cell via the binding of applicable receptor. Some receptors may be potential receptor for DENV, including: heparin sulfate, Fc receptor, heat shock protein 70 (Hsp70) and Hsp90, 37-kDa/67-kDa high affinity laminin receptor, 10.

(11) glucose-regulating protein 78 (GRP78/BiP), CD14-associated molecules, and dendritic-cell-specific ICAM-grabbing non-integrin (DC-SIGN) (Chen et al., 1999; Jindadamrongwech et al., 2004; Littaua et al., 1990; Navarro-Sanchez et al., 2003; Reyes-Del Valle et al., 2005; Thepparit and Smith, 2004). In the above receptors, Fc receptors and the DC-SIGN are related with the immune system principally. The Fc receptors which contain FcγRI and FcγRII play the major roles in the antibody-dependent enhancement (ADE) (Halstead and O'Rourke, 1977; Halstead et al., 1977). The DC-SIGN (CD209), which is a mannose-specific lectin, is suggested that it has specific interaction with the a few carbohydrate residues on the E protein of DENV (Navarro-Sanchez et al., 2003). In addition, DC-SIGN could make four serotypes DENV infect and produce ectopic. expression. of. on. normally. non-permissive. cell. lines. (Navarro-Sanchez et al., 2003; Tassaneetrithep et al., 2003). In 2005, it is observed that endocytosis-defective DC-SIGN molecules could let efficient DENV replication and N-terminal internalization signals of DC-SIGN are dispensable for the viral uptake (Lozach et al., 2005). In terms of that, it has been presented that low-affinity DC-SIGN might increase the local concentration of DENV at the cell surface at first. Afterward, an unidentified receptor, which is less-common and high-affinity, is drafted to mediate DENV internalization and low-pH triggered fusion into dendritic cells (DCs). Moreover, the binding site on virion for such an unknown factor is left vacant upon binding to DC-SIGN (Pokidysheva et al., 2006).. 11.

(12) The hypothesis of DENV life cycle The DENV enter the host cell by receptor-mediated endocytosis. It triggers the irreversible class II fusion by acidic environment in endosome (Allison et al., 1995). After the internalization and acidification of endosomes, the NC is released into the cytoplasm. Subsequently, the viral RNA and capsid protein dissociate, and then replication of the RNA genome and particle assembly is initiated (Brinton, 2002; Lindenbach and Rice, 2003). At first, the immature particles, which contain E and prM proteins, lipid membrane and NC, are formed in the lumen of the endoplasmic reticulum (ER). However, those immature particles could not induce host-cell fusion and make them non-infectious (Guirakhoo et al., 1992; Guirakhoo et al., 1991). These immature particles are transported into Golgi and the prM is cleaved by furin in the trans-Golgi network. The cleavage of prM could make the viral particles mature and infectious (Elshuber et al., 2003; Stadler et al., 1997). In addition, the process also creats the subviral particles. Subviral particles are also produced in the ER, but only contain the glycoproteins and membrane, and lack capsid protein and genomic RNA. These structure make these particles non-infectious (Schalich et al., 1996). Finally, Mature virus and subviral particles are released from the host cell by exocytosis.. Pathogenesis of dengue Up to the present, a lot of factors, viral or host, have been reported which might relate with the pathogenesis of dengue. Some factors is 12.

(13) notable, such as viral serotypes (Balmaseda et al., 2006; Gubler, 1998) and genotypes (Messer et al., 2003; Rico-Hesse et al., 1997), secondary infection (Sangkawibha et al., 1984; Thein et al., 1997), ADE (Halstead, 1983, 2003; Kliks et al., 1989), and interferons (IFNs) (Diamond et al., 2000; Kurane et al., 1991; Laoprasopwattana et al., 2005). During the process of secondary infection, Hoskins effect, also called “original antigenic sin”, plays a part in DHF/DSS (Halstead, 2003; Halstead et al., 1983; Mongkolsapaya et al., 2006). In this hypothesis, it is supposed that the immune system preferentially capitalizes on memory based on the primary infection ward off the other primary infection of the pathogen which is extremely similar. However, it might cause that the low-affinity antibodies from memory B-cell populations and inefficient cytotoxic effects exerted by pre-existing T cells were produced in this situation. These low-affinity or non-neutralizing antibodies could restrain native B-cell from producing more effective antibodies. Moreover, it might result in the worse viral clearance that the extension of memory cross-reactive T-cell for the similar virus than the current viral infection (Cummings et al., 2005; Kliks et al., 1989; Laoprasopwattana et al., 2005). Those low-affinity or non-neutralizing antibodies do not only make the slower viral clearance, but also induce the ADE. The assumption of ADE describes that the infection of cells which bear Fcγ receptors or complement receptors increase when the presence of sub-neutralizing concentrations of antibody or immune sera. Therefore, even thought the antibodies could neutralize the viral virulence, they might cause ADE while neutralizing antibodies below their functional levels (Halstead, 13.

(14) 1983, 2003; Kliks et al., 1989). For DENV, the antibody-virion complexes are entered the monocytes or macrophages by Fcγ receptors on them. It will increases the risk of DHF/DSS in secondary infections (Chareonsirisuthigul et al., 2007; Huang et al., 2006; Littaua et al., 1990; Mady et al., 1991; Mady et al., 1993). Except the relevant mechanisms of antibodies, IFNs and the other cytokines may be another critical role in the immune responses to viral infection. For DENV, it has been reported that IFN-α/β and IFN-γ could render protection from virus in vitro, but the host need treating with IFN-α/β and IFN-γ before the viral infection (Diamond et al., 2000; Ho et al., 2005). However, IFN-α/β and IFN-γ are not only good for protection, but also good for viral infection. In Thailand, some children with DF and DHF/DSS are detected the higher concentration of IFN-α/β and IFN-γ than the health ones. The same circumstances are observed between DHF/DSS patients and DF patients, too (Kurane et al., 1993; Kurane et al., 1991). Relatively, pro-inflammatory cytokines are significantly proliferated during the DHF phase. Such as interleukin 6 (IL-6) and interleukin 10 (IL-10), both signal transference and activator of transcription 1 (STAT-1) dependent and STAT-1 independent pathways are implied to be related to IFN-induced responses to dengue infection. In a recent study, the level of type-I IFN mRNA transcripts in peripheral-blood mononuclear cells (PBMCs) from eight non-shock DHF patients was higher than that from six DSS patients (Simmons et al., 2007). The nature killer cells (NK cells) participate in innate immune responses by secreting IFN-γ which could activate the other members of 14.

(15) innate immune system, such as macrophages and DCs, and stimulate the adaptive immune response toward Th1-type immunity. It results in the immune system recognizes and kills host somatic cells that are coated with antibody or fail to express the appropriate MHC proteins. NK cells could also secret inflammatory cytokines during antiviral activity (Degli-Esposti and Smyth, 2005; Hilleman, 2004; van Den Broek et al., 2000). It is observed that the NK cells is not only involved in immune response, but also related to milder DF in patients (Azeredo et al., 2006). The nitric oxide (NO) is a multifunctional molecule which’s property is not only cytotoxic but also cytoprotective. It could be produced from L-arginine by NO synthase (NOS). The many pathological effects, such as vasodilatation, inflammation, thrombosis, immune response and neurotransmission, could influence the production of NO. The NO might either induce or suppress apoptosis in different cells. In fact, the IFN-γ, form NK cells, would activate macrophages and then macrophage would inhibit the DENV infection via NO. In the spleen cells, NO is induced by CF / CF2 in the Ca+-dependent manner, which may be a mechanism of target cell killing (Mukerjee et al., 1996). Moreover, it is reported that the inducible nitric oxide synthase (iNOS) is activated by the infection of DENV both in vitro and in vivo (Neves-Souza et al., 2005). However, the surfeit of NO could result in the apoptosis of endothelial in blood vessels and cells are damaged by cross-reactive antibodies via the NO- dependent pathway (Lin et al., 2004; Takhampunya et al., 2006). In addition to IFNs, the other cytokines are also involved in the dengue pathogenesis. Cytokines are reported that it could cause more severe DHF/DSS in patients. This phenomenon is called “cytokine tsunami” 15.

(16) (Chaturvedi et al., 2007). The most cytokine secretion profiles distinguish helper T (Th) 1 and Th2 cells. The former secretes IFN-γ, interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α). Those cytokines are related with cell-mediated inflammatory reactions, delayed type hypersensitivity, tissue injury in infections and autoimmune diseases. The latter secretes IL-4, IL-5, IL-6, IL-10, IL-13and TNF-β. They are associated with support the antibody production. The cytokines play a important role in the process of plasma leakage, which is the major pathogenesis of DHF. Anderson et al. reported that monocytes would secrete TNF-α with enhancing antibodies in the infection of DENV, and TNF-α would triggers plasma leakage in vitro (Anderson et al., 1997). So far, more and more studies prove that increase of TNF-α would result in the severe disease, and reducing TNF-α level by antibodies would mitigate the symptoms of DHF (Braga et al., 2001; Chakravarti and Kumaria, 2006; Hober et al., 1996). The TNF-β is also reported the similar situation to TNF-α. It would cause the more severe disease and longer duration of illness in the DHF patients with higher levels of TNF-β (Agarwal et al., 1999). In addition to IFNs and TNFs, various interleukins, such as IL-6, IL-8 and IL-10, have been consider which are involved in DHF/DSS. IL-10 is a human cytokine synthesis inhibitory factor (CSIF), which is related with feedback loop of down-regulates inflammatory responses. It is reported that the level of IL-10 is higher in DHF/DSS patients than in DF (Chen et al., 2006; Perez et al., 2004). IL-8 is synthesized by macrophages or and epithelial cells and the level of IL-8 would be elevated by viral infection, like DENV (Raghupathy et al., 1998). It 16.

(17) might draft neutrophils and activate monocytes from infected DCs in vitro (Medin et al., 2005). However, it is observed the severity of the disease is proportional to the levels of IL-8 in the serum (Raghupathy et al., 1998). In fact, Talavera et al. show that endothelial cell monolayers, DEN-infected, release IL-8 and increased permeability and this condition could be alleviated by treatment with antibodies against IL-8 (Talavera et al., 2004). The levels of IL-6, which is from T cells and macrophages, would be increased and indirectly lead to hyperfibrinolysis and enhanced permeability which result from induced tissue plasminogen activators by IL-6 (Huang and Shi, 2001). On the whole, dengue is a multi-factorial disease. Its recovery percentage and the severity of disease would rely on protective immune responses, serotypic virulence, autoimmune activities, and host cells. The vaccine of dengue The first dengue vaccine could trace back to 1945 by Sabin and Schlesinger. It is the “Hawaiian” strain, serotype DENV1, live attenuated vaccines, which is attenuated by serial passage in mouse brain. However, these vaccines of single serotype could induce long-term protection to re-infection with the homologous serotype for years, but immunity resist heterotypic serotypes only for several months (Sabin, 1952). In addition, those vaccines might induce the ADE with the other serotype infections. The tetravalent dengue vaccines (TDVs) should be a better way to solve it. TDVs might induce immunity against all serotypes. So far, the unknown pathogenesis of DHF/DSS and the lack of suitable animal model for dengue disease are the major problems for dengue studies. Now, the 17.

(18) development of dengue vaccines would focus mainly on live attenuated virus vaccines, inactivated virus vaccines, recombinant subunit virus vaccines and DNA vaccines. The live attenuated virus vaccines could be made by passage in tissue culture cell (Eckels et al., 1984). The Walter Reed Army Institute of Research (WRAIR) attenuates virus through serial passage in primary dog kidney (PDK) cells, and terminal passages in fetal rhesus lung cells. However, these PDK-derived vaccines only the DENV1 monovalent candidate would cause 40% of volunteers developing fever and generalized rash, and the other serotypes monovalent candidates cause mildly reactogenicity (Sun et al., 2003). It was reported that D2 16681-PDK53 was safe and induced both neutralizing antibodies in vivo and cytokine gene expression in vitro (Rabablert and Yoksan, 2009). The other way for making the tetravalent live attenuated virus vaccines is using the “Chimeric virus”. The recombinant DNA technology comes into use in the chimeric virus. It is possible to create chimeric viruses which contain the structural protein genes for the target antigens of a virus are replaced by the corresponding genes of another virus. The ChimeriVax platform has been used to make chimeric vaccine candidates, ChimeriVax-DENV, which could express the pr-M and E from DENV in the yellow fever (YF) virus genetic background, YF17D (van Der Most et al., 2000). Guirakhoo et al. created a ChimeriVax-DENV representing DENV serotypes 1 to 4 by electroporation of Vero cells, in which RNA transcripts prepared from viral cDNA (Guirakhoo et al., 2001; Guirakhoo et al., 2002). It has been proved that this ChimeriVax-DENV have non-infection for Ae. aegypti and low infectious rates for Ae. Albopictus 18.

(19) (Higgs et al., 2006). It is also reported that the monovalent ChimeriVax-DENV2 could offer great resistance to DENV for human (Guirakhoo et al., 2006). Inactivated virus vaccines are using the inactive virus to induce immune response against DENV. These vaccine candidates could induce no immunity to the non-structural protein, because these vaccines only have structural protein. Moreover, those vaccine candidates require adjuvants to increase immune response for seronegative inoculators. There are both advantages and disadvantages. These vaccines are much safer than live attenuated virus vaccines, as it is not possible for inactivated vaccines to return to a pathogenic phenotype. Furthermore, these multivalent vaccine candidates could induce the equally immunogenic response for the four serotypes. It is reported that the DENV2 inactive virus vaccine could result in effective protection in the mouse and rhesus monkeys (Putnak et al., 1996a; Putnak et al., 1996b). In recent studies, the more effective adjuvants are reported in the primate model (Robert Putnak et al., 2005). The recombinant DNA technology is applied to recombinant subunit virus vaccines, too. These vaccine candidates contain the recombinant E protein, so the recombinant subunit virus vaccines might have safer than live attenuated virus vaccines. They also could induce the immunity against viral non-structure proteins with recombinant non-structure protein in the same vector. So far, there is no report about recombinant subunit virus vaccines testing for human. Nevertheless, it is proved that the recombinant ED-III of DENV2 could protect culture cells from DENV2 infection by blocking the virus from binding to host cells 19.

(20) (Jaiswal et al., 2004). Moreover, these monovalent vaccine candidates of DENV2 and DENV4 could make effective immune response in rhesus monkeys (Guzman et al., 2003; Robert Putnak et al., 2005). The DNA vaccines would induce the immunity by the live vectors, which are inserted the viral antigens of DENV. Therefore, these vaccines candidates are easier production of tetravalent vaccines and more stable in room temperature. However, in order to inducing higher immunity, these DNA vaccines are required multiple doses, appropriate adjuvants, special immunostimulatory motifs or specialized injection equipment. The first DNA vaccines of DENV are made by Kochel et al, and their vectors are inserted the prM and E proteins of DENV2 (Kochel et al., 1997). Afterward, it is proved that the prM might play an important role in these vaccines (Ocazionez Jimenez and Lopes da Fonseca, 2000). Those DNA monovalent vaccine candidates of DENV1 and DENV2 could induce effective antibodies in rhesus and Aotus monkeys (Raviprakash et al., 2001; Raviprakash et al., 2000). Furthermore, Raviprakash et al suggest that co-immunization with GM-CSF gene and injection by the needle-free Biojector system could increase the antibody titer and effective term (Raviprakash et al., 2003). Recently, De Paula et al., reported a DENV3 DNA vaccine, pVAC3DEN3, which could expressed prM and E protein of DENV3. In their study, the survival rate of pVACDEN3 injected mice was 80% after challenged with 50 LD50 (De Paula et al., 2008).. Our study In order to confirm the target of neutralizing antibodies and develop 20.

(21) the DNA vaccines of DENV, we screened a large panel of MAbs generated in our laboratory and characterized the MAbs against DENV4 by immunofluorescence, Western blotting, PRNT and protection assay. We also constructed two DNA vectors which contained the EDI-II and EDIII of DENV4 and immunized the mice for identification the neutralizing antibodies. We used the DNA vaccine of DENV4, pCB8D4-2J, to immunize the mice to determine the neutralizing activity of EDI-II or EDIII DNA vaccine by PRNT and protection assay for imitating immune responses against DENV4 in vitro and in vivo.. 21.

(22) Materials and Methods Cells and viruses DENV1 strain 766733 is a Taiwanese strain isolated from patients with DF. Four prototype dengue viruses, DENV1 (Hawaii), DENV2 (New Guinea C), DENV3 (H87), and DENV4 (H241), were provided by Duane J. Gubler from the Centers for Disease Control and Prevention, Fort Collins, CO. All viral strains were used to infect mosquito C6/36 cells with growth medium containing 50 % Mitsumashi and Maramorsch insect medium (Sigma) plus 50 % Dulbecco’s modified Eagle’s minimal essential medium (DMEM; GIBCO). The DENV-infected C6/36 cells were incubated at 28°C for 7 to 9 days. The viruses were harvested from the supernatants and titrated in a baby hamster kidney fibroblast cell line, BHK-21 cells, by plaque assay, and aliquots were stored at -80°C. BHK-21 cells were grown in minimal essential medium (MEM) containing 10% heat-inactivated fetal bovine serum (FBS).. Preparation of DENV antigens C6/36 cells were infected with DENV4. Infected cells were then lysed in lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1% Nonidet P-40) containing protease inhibitors. The supernatant was collected by centrifugation at 3000g for 10 minutes at 4°C, and then the protein concentration was quantified by the Bradford assay.. 22.

(23) DNA vaccine and Gene-gun immunization DNA vaccines were used pCB8D4-2J, which had the similar construct. to pCB8D2-2J-9-1 (Chang et al., 2003). The difference between them was one with the prM and E proteins of DENV2 and the other with DENV4. This DNA vaccine was coated onto 1.6nm gold particles with the concentration of 2 μg DNA per mg gold. The complexes of DNA-gold were coated onto a plastic tube, and then the tube coated gold was cut to 35~40 shots (1μg DNA per shot, approximately). These procedure were followed the manufacturer’s instructions (Bio-Rad). BALB/c mice were immunized in their abdominal skin by hand-held gene-gun, and the golds in the tubes were driven with compressed helium (400 p.s.i). Mice might be immunized twice at interval of 3 weeks, and then waited 3~4 weeks before further investigation.. Enzyme-linked immunosorbent assay (ELISA) Serially diluted sera of immunized mice were added to the plates of DENV4-infected C6/36 cells and incubated at room temperature for 1 hour. Then the plates were washed four times with phosphate-buffered saline containing 0.1 % (wt/vol) Tween 20 (PBST0.1) and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG; Jackson ImmunoResearch Laboratories, West Grove, PA). The plates were washed five times with PBST0.1 and incubated with the peroxidase substrate o-phenylenediamine dihydrochloride (OPD; Sigma). The reaction was stopped with 3 N HCl and then the plates were read using a microplate reader at 490 nm. 23.

(24) Western blot analysis Proteins or antigens were mixed with an equal volume of the native sample buffer (50 mM Tris-HCl, pH 6.8, 0.1 % bromophenol blue, 10 % glycerol), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane (Hybond-C Super; Amersham, Little Chalfont, United Kingdom). Nonspecific antibody-binding sites were blocked with 5% skimmed milk in phosphate-buffered saline (PBS), and then membranes were incubated with primary antibodies and HRP-conjugated goat anti-mouse IgG (Jackson Immuno-Research Laboratories, West Grove, PA) and were developed with chemiluminescence reagents (ECL; Amersham).. Expression of preparation recombinant EDI-IIs and EDIIIs of DENV4 The pET-21a/rEDI-II and pET-21a/rEDIII plasmids were transformed into Escherichia coli BL21 (DE3). Five ml BL21 cultures were grown in LB medium containing ampicillin at 37 °C overnight. Then, the cultures were diluted in fresh 50 ml LB medium containing ampicillin in the ratio of 1:50 and incubated to an OD600nm of approximately 0.6-0.8 and induced with 1 mM IPTG at 37 °C for 4 h. Bacterial cells were pelleted and stored at -20 °C overnight. The following day cells were lysed in 5 ml of lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1% Nonidet P-40) containing protease inhibitors. Then, sonicate on ice and centrifuge lysate at 10000×g , at 4°C for 30 minutes. Resuspend the pellet 24.

(25) in 5 ml of buffer B (100mM NaH2PO4, 10mM Tris-HCl, 8M urea and pH 8.0). The mixture were stired at room temperature for 1 hour, and then centrifuge lysate at 1000×g, at 4°C for 30 minutes. Afterwards, the collected supernatant would be added to five-fold volume with lysis buffer (containing 0.5M arginine, 1.5mM reduced form glutathione and 1mM PMSF) and stir them at room temperature for 10 minutes. After stir the mixture containing 1mM oxidized form glutathione at 4°C overnight, the total proteins were concentrated by 3kDa Amicon at 7000×g. In the end, the concentrated proteins were dialyzed with lysis buffer and used for Western blot analysis with diluted MAbs.. Construction of plasmid cDNA clones expressing recombinant EDI-IIs and EDIIIs of DENV4 The eukaryotic expression vector, pCR3.1-Flag, was used as a template DNA for rEDI-II and rEDIII of DENV4. The DNA fragments of DENV4 rEDI-II and rEDIII was from pET-21a/rEDI-II and pET-21a/rEDIII by PCR, and then, the PCR product was extracted and purified by Qiagen plasmid purification kit (Qiagen). The purified PCR products encoding EDI-II and EDIII were amplified with NheI and XhoI sites added at the 5’ and 3’ ends, respectively. Those PCR products were digested and ligated into the pCR3.1-Flag vector according to the manufacturer’s manual and transformed into Escherichia coli (DH5αF’). Selection of E. coli. colonies. containing. the. recombinant. plasmid,. pCR3.1/D4-EDI-II/Flag or pCR3.1/D4-EDIII/Flag, was performed by colony polymerase chain reaction and restriction enzyme digestion. The pCR3.1/D4-EDI-II/Flag and pCR3.1/D4-EDIII/Flag were extracted by 25.

(26) Qiagen plasmid purification kit (Qiagen), and determine vectors expression by IFA.. Immunofluorescence assay BHK-21 was cultured on the slides in the 12-well tissue culture plate overnight. Then cells were infected by DENV4 or transfected vectors, which were contained rNS-1, prM-E proteins, rEDI-II or rEDIII by using LipofectamineTM. 2000. reagent. (Invitrogen). according. to. the. manufacturer’s protocols. Then, BHK-21 cells were incubated at 37°C and 5 % CO2 for 48 hour. The cells on the slides were fixed with 3 % paraformaldehyde in PBS at room temperature for 30 minutes. The slides were washed once with PBS and perforated by 0.5% triton X-100 in PBS for 10 minutes. Afterward, the slides were blocked with 3 % bovine serum albumin (BSA) in PBS at room temperature for 30 minutes and then incubated with MAbs in 3 % BSA in PBS at room temperature for 1 hour.. The. slides. were. stained. by. fluorescent-isothiocyanate. (FITC)-labeled goat anti-mouse IgG antibody (Jackson Immuno Research, Pennsylvania,. USA). and. 4’,6-diamidino-2-phenylindole. (DAPI,. invitrogen) in 3 % BSA in PBS at room temperature for 1 hour, after washed PBS three times. the slides were washed PBS four times and cover slip was mounted for examination under a fluorescent microscope.. Plaque reduction neutralization test (PRNT) Plaque reduction neutralization test protocol was modified from Morens et al (Morens et al., 1985). BHK-21 was seeded 2×105 per well in 26.

(27) 12-well tissue culture plate and incubated at 37°C and 5% carbon dioxide (CO2) for 48 hour. For screening neutralizing activity, MAbs and normal mouse IgG (NMIgG) were diluted ten-fold with serum-free MEM from 10 μg/ml to 0.001 μg/ml. The sera of immunized mice was also diluted ten-fold to one hundred thousand-fold with serum-free MEM. Subsequently, we mixed 150 μl media containing diluted MAbs or sera with 150 μl media containing 40~60 plaque-forming units (PFU) of virus, and then, incubated the mixture at 4°C for 1 hour. Later, we add 150 μl the mixture in each well which included 300μl MEM and incubated at 37°C and 5 % CO2 for 1 hour. Finally, cells would be incubated with overlay media, which consisted 1% methyl-cellulose, MEM, and 2 % FBS, at 37°C and 5 % CO2 for 5~7 days. At the end, we fixed the cell with 10 % formaldehyde for 1hour and removed the mixture, then stained with 1 % crystal violet solution for 1 hour. After, washing with tap water, the plaques were counted. The DENV2-AB 43 and NMIgG were used as positive and negative control.. Protection assay The diluated MAbs or immunized mice sera would be mixed with DENV4 and incubated 4°C for 30 minutes. The virus titer of mixture was from 50 PFU to 1000 PFU. Then, each newborn, 2-day-old ICR mice were intracerebrally injected 20 μl mixture of DENV4-MAbs or DENV4-NMIgG. The NMIgG was used as negative control. The morbidity was recorded daily and observed 3 weeks.. 27.

(28) Result Identification of MAbs against DENV4 The specificity of MAbs was identified by immunofluorescent assay using BHK21 cells infected by DENV2 and DENV4 (Table 1 and Fig. 1). It was showed that all MAbs could recognize, and most MAbs could not recognize DENV2-infected cell. The MAbs, such as DENV4-Ab20 and DENV4-Ab17, recognized both DENV2-infected BHK-21 cells and DENV4-infected cells (Fig. 2A). DENV4-Ab5 could recognize BHK-21 cell infected with DENV1 and DENV4 (Fig. 2B). DENV4-Ab19 recognized BHK-21 cell infected with DENV1, DENV2 and DENV4 (Fig. 2C). The DENV4-Ab9 and DENV4-Ab10 could recognize BHK-21 cell infected with DENV3 and DENV4 (Fig. 3). In addition, DENV4-Ab7, DENV4-Ab13 and DENV4-Ab18 could recognize the all serotypes (Fig. 4).. Identification of the specificity of MAbs against DENV We wanted to understand the target protein of these MAbs, thus we utilized non-reducing SDS-PAGE for Western blot analysis. We found eight MAbs (DENV4-Ab1, DENV4-Ab3, DENV4-Ab7, DENV4-Ab8, DENV4-Ab9,. DENV4-Ab13,. DENV4-Ab14. and. DENV4-Ab16). specifically recognized the E protein of DENV4 without cross-reactivity to other serotype of DENV, and four MAbs (DENV4-Ab2, DENV4-Ab5, DENV4-Ab11 and DENV4-Ab17) could recognize the NS-1 proteins of DENV (Fig. 5). Among these four MAbs, only the DENV4-Ab2 specifically recognized the NS-1 proteins of DENV4 and DENV4-Ab5 28.

(29) specifically recognized the NS-1 proteins of DENV1 (Fig. 5). DENV4-Ab11 was showed it could recognize the NS-1 proteins of DENV1 and DENV4 (Fig. 5). In addition, DENV4-Ab17 also showed cross-reactivity to NS-1 protein of DENV2 and DENV4 (Fig. 5). Furthermore, we also tested these MAbs with the reducing SDS-PAGE for Western blot analysis. All MAbs did not recognize E or NS-1 proteins (data not shown). It is suggested that these MAbs were recognized conformational epitopes of DENV4. To further identify the target antigens of these conformational antibodies, we used prM-E or NS-1 transfected BHK-21 cells to test their reactivity by IFA. The results revealed that DENV4-Ab5, DENV4-Ab11 and DENV4-Ab12 were recognized the E protein, not NS-1 proteins (Fig. 6).. Identification of the domains of E proteins recognized by anti-E MAbs In the previous experiment, the target antigens of these antibodies were identified. We were interested that the binding domains of anti-E MAbs. Therefore, we identified the binding domains of anti-E MAbs by Western blot analysis. We found seven MAbs (DENV4-Ab1, DENV4-Ab8, DENV4-Ab9,. DENV4-Ab11,. DENV4-Ab3,. DENV4-Ab15. and. DENV4-Ab16) could recognize rEDI-II of DENV4, and two MAbs (DENV4-Ab12 and DENV4-Ab14) bound to rEDIII of DENV4 (Fig. 7). We also observed that most anti-E MAbs recognized EDI-II of DENV4. In addition, we also used the rEDI-II and rEDIII to test those other 29.

(30) MAbs with the reducing SDS-PAGE for Western blot analysis, but those MAbs conld not recognize rEDI-II or rEDIII again (data not shown). It was reinforced that these anti-E MAbs were recognized conformational epitopes of DENV4.. Determination of the neutralizing activities of MAbs by PRNT To estimate the neutralizing effects of individual MAbs on the infection of DENV4 in vitro, we infected BHK-21 cells with the mixture of individual MAbs and DENV4. Among all MAbs against DENV4, the DENV4-Ab11 showed the best neutralizing activity for DENV4 (50 % reduction of plaque formation at 2 μg/ml) (Fig. 8B and Fig. 9). DENV4-AB 13 also showed the lower neutralizing activity for DENV4 (50 % reduction of plaque formation at 20 μg/ml) than DENV4-Ab11 (Fig. 8C and Fig. 9). In addition to DENV4-Ab11 and DENV4-Ab13, we also find some MAbs, such as DENV4-Ab7, DENV4-Ab12, DENV4-AB 14 and DENV4-Ab15, which could neutralize DENV4, but their PRNT50 was higher than 50 μg/ml (Fig. 9).. Determination of the 50% lethal dose (LD50) by challenging with DENV4 Before we proceed to protection assay, we need to determine the LD50 (Lethal Dose, 50%) of DENV4. We injected the DENV4 into brain of suckling mice. The result showed that the all of suckling mice died after 30.

(31) nine days by challenging with the 100 PFU of DENV4. 50% of the suckling mice died after two weeks by challenging with 50 PFU of DENV4 (Fig. 10).. Determination of the neutralizing activities of MAbs by protection assay We found two MAbs, DENV4-Ab11 and DENV4-Ab13, could neutralize the DENV4 in vitro by PRNT. We also estimate the protection activity of individual MAbs on the infection of DENV in vivo, MAbs or NMIgG were incubated with 1000 PFU DENV4 (twenty-fold of LD50) and injected into brain of suckling mice. In Figure 11, it is observed that the suckling mice, treated with NMIgG and DENV4, were dead after seven days. The suckling mice which were treated with DENV4-Ab13 were similar to those treated with NMIgG. The DENV4-Ab13 could delay mice death until tenth-day after challenge (Fig. 11). By contrast, the protection. activity. of. DENV4-Ab11. was. more. powerful. than. DENV4-Ab13. The suckling mice treated with 100 μg/ml and 50 μg/ml DENV4-Ab11 were still lived more than 80 % after two weeks (Fig. 11). These result suggested that the DENV4-Ab11 could not neutralize DENV4 only in vitro, but in vivo.. Expression of rEDI-II and rEDIII of DENV4 in BHK-21 cells In our previous experiments and other reports, we were interested that 31.

(32) the efficacy of DNA vaccines contained rEDI-II or rEDIII of DENV4. For this purpose, we constructed two vectors, pCR3.1/D4-EDI-II/Flag (Fig. 12A) and pCR3.1/D4-EDIII/Flag (Fig. 12B). To confirm the expression of rEDI-II and rEDIII of DENV4, we transferred the pCR3.1/D4-EDI-II/Flag or pCR3.1/D4-EDIII/Flag to the BHK-21 cell by LipofectamineTM 2000. The result showed that the two vectors could express in BHK-21 cells and their recombinant protein could be detected by antibodies against Flag (Fig. 12C).. Determination of polyclone antibodies from immunized mice sera by ELISA In order to test the efficacy of pCR3.1/D4-EDI-II/Flag and pCR3.1/D4-EDIII/Flag in vivo, BALB/c mice were immunized with the pCB8D4-2J, pCR3.1/D4-EDI-II/Flag or pCR3.1/D4-EDIII/Flag by gene gun. After boost of immunization three weeks, hyper-immune mice sera were tested by ELISA with DENV4-infected C6/36 cells. ELISA data showed that sera of three immunized pCB8D4-2J mice were contained high titer of polyclone antibodies against DENV4 (Fig. 13). However, the antibody. titer. of. immunized. pCR3.1/D4-EDI-II/Flag-. or. pCR3.1/D4-EDIII/Flag-immunized mice was not markedly increased (Fig. 14 and Fig. 15).. Determination of the neutralizing activities of immunized mice sera by PRNT 32.

(33) We have observed that the antibody titer was increased in mice immunized with DNA vaccine. We want to test the neutralizing activity of these antibodies. The neutralizing activity of pCB8D4-2J-immunized mice was estimated by PRNT. We infected BHK-21 cells with DENV4 treated with serial dilute mice sera. In Figure 16, it is showed that the neutralizing activity PRNT50 (50 % reduction of plaque formation) of immunized mice sera was 1: 400.. 33.

(34) Discussion The adaptive immune response plays an elaborate role for DENV infection. It could provide protection against DENV infection or re-infection, but it also could make the much severer symptom in DENV patient, such as DHF or DSS. The immunization against dengue is supposed to address the issues of protective immunity and the postulated immunopathogenic factor of antibodies in patients. The E proteins of DENV is proved that it is the major target for neutralizing antibodies against DENV infection, and it could induce those antibodies to protective level. The E proteins are composed of there domains (domain I ~ III). The most neutralizing antibodies could bind to the domain III of E proteins, especially. In consequence, it is important that identification of viral B-cell epitopes for development of subunit vaccines and virus-specific diagnostics. So far, it is reported utilizing the generation of escape mutations to localize the epitopes of B-cell (Goncalvez et al., 2004; Lin et al., 1994; Serafin and Aaskov, 2001), recombinant protein fragment (Megret et al., 1992), recombinant constructs containing specific amino acid substitutions (Hiramatsu et al., 1996; Stiasny et al., 2006; Trainor et al., 2007), or synthetic peptide sequences to binding studies (Falconar, 1999; Kanai et al., 2006). The similarity of genome between the all four serotypes of DENV was much high. Presumably, the similarity of amino acid sequences between the all four serotypes of DENV was high, too (Aaskov et al., 1989; Palmer et al., 1999). In our study, we want to characterize the MAbs against DENV4, so we screened all MAbs against DENV4 generated in our laboratory and found 34.

(35) most anti-E MAbs were recognized the EDI-II of DENV4 (Fig. 6). Two MAbs exerted notable neutralizing effects on the entry of DENV4 by PRNT (Fig. 8B, Fig. 8C and Fig. 9). Especially, the DENV4-Ab11 had the greatest neutralizing effects with DENV4 among the screened MAbs by PRNT (Fig. 8B and Fig. 9). It was also observed that DENV4-Ab11 protected the suckling mice from DENV4 challenge in protection assay (Fig. 11). The DENV4-Ab13 also has some the neutralizing activity against DENV4 in vitro (Fig. 8C and Fig. 9), but it could not protect the suckling mice in protection assay from DENV4 challenge (Fig. 11). It is much surprised that both DENV4-Ab11 and DENV4-Ab13 were binding to rEDI-II of DENV4 (Fig. 7), since EDIIs of DENV were reported that it was the major target of most cross-reactive antibodies. In the studies with west nile virus (WNV), B-cell repertoire analysis of WNV patients was showed that only 8% of WNV-specific antibodies clones were specific to EDIII, and approximately half WNV-specific antibodies were binding to EDII, particularly the fusion loop (Throsby et al., 2006). However, Oliphant et al., proved that the MAbs against EDI-II of WNV could provide neutralization and protection both in vitro and in vivo. They assumed that the neutralizing mechanism of MAbs against EDI-II of WNV might block attachment (Oliphant et al., 2006). Moreover, the EDII of DENV played a critical role that it could be triggered membrane fusion by low-pH for virus infection. We suppose that DENV4-Ab11 may prevent the membrane fusion from virus infection or block DENV attachment, because the E proteins of DENV are similar to WNV. In fact, it was reported that those MAbs, the binding epitopes within EDIIIs, had more powerful neutralizing activity against DENV2 (Crill 35.

(36) and Roehrig, 2001), and the location of epitopes was at either end of EDIII (Hiramatsu et al., 1996; Lin et al., 1994). Additionally, we also found two MAbs (DENV4-Ab12 and DENV4-Ab14) could recognize rEDIII of DENV4 (Fig. 5), but neutralizing activity against DENV4 was weaker than DENV4-Ab11 and DENV4-Ab13 (Fig. 7). Many of the most potent neutralizing antibodies characterized to date recognize the upper lateral surface of DIII that protrudes off the surface of the virion and many neutralizing eptiopes were at the EDIII (Sukupolvi-Petty et al., 2007). Even though the MAbs could recognize the EDIII, the MAbs might have lower neutralizing activity when the binding eptiopes of MAbs were not neutralizing eptiopes. Neutralization would occur when the number of antibodies bound to an individual virion exceeds a required threshold (Pierson et al., 2007). Focus on this point; two biochemical factors, antibody affinity and the accessibility of epitopes on the virus particle, play a crucial role to determining neutralizing activity of antibodies in their concentration over the stoichiometric requirements. The “affinity” was the interactive strength between antibodies and antigen. If the antibodies have the more high-affinity, it means that the antibodies could more achieve threshold easily. It is no surprise that the most potent neutralizing antibodies, against flaviviruses, neutralization appears to occur at a relatively low occupancy (Gromowski and Barrett, 2007; Pierson et al., 2007). However, for some antibodies, even high-affinity and complete occupancy of epitopes on the virion is not sufficient to exceed the threshold for neutralization (Pierson et al., 2007). The pseudo-icosahedral arrangement of E proteins on the virion displays the E protein in three distinct chemical environments defined by 36.

(37) proximity to the two-, three- or fivefold axes of symmetry (Kuhn et al., 2002; Mukhopadhyay et al., 2003). In this regard, because of steric constraints imposed by adjacent E proteins on the virus particle from the perspective of the antibody, epitopes in each of these environments may be differentially accessible for antibody binding. The highly exposed determinants might be bound with antibodies easily and those antibodies may achieve the stoichiometric threshold for neutralization by binding the virion at relatively low occupancy. By contrast, it was predicted that the antibodies, which bind poorly exposed epitopes, may require nearly complete occupancy to achieve threshold requirements for neutralization (Pierson et al., 2007; Stiasny et al., 2006). Therefore, we could make conjecture that the target eptiopes of DENV4-Ab12 and DENV4-Ab14 was at lowly exposed place, their affinity was too weak to achieve threshold or not the neutralizing eptiopes. The immunized pCBD2-2J-2-9-1 mice sera were reported that ninety percent neutralization titers ranging from 1:40 to >1:1000 were observed in seven of nine serum specimens. In passive protection experiment, the half 2-day-old neonatal mice, born from immunized female mice, could survived with the challenge of DENV2 (New Guinea C)(Chang et al., 2003). In our study, the immunized pCB8D4-2J mice sera also showed neutralizing activity against DENV4 by PRNT (Fig. 16). However, the antibodies. titer. of. injected. pCR3.1/D4-EDI-II/Flag. or. pCR3.1/D4-EDIII/Flag mice was not rise (Fig.14 and Fig.15). It might result from less immunization, so we need to immunize mice more times. In conclusion, we found a MAb, DENV4-Ab11, which had neutralizing activity in vitro and in vivo. Additionally, we also found 37.

(38) some MAbs, such as DENV4-Ab13, that could block DENV4 entry into cell. In future, it is important to search B-cell epitopes of these MAbs. The identification of neutralizing MAbs and epitopes of DENV4 are valuable for developing DENV vaccine, diagnostic regents and studying pathogenesis of DENV.. 38.

(39) DENV4-Ab1 DD1-4. 4G2 DENV4. DENV2. DENV4. DENV2. DENV4. Merge. DAPI. FITC. DENV2. DENV4-Ab3 DD4-3. DENV4-Ab6 DD9-4 DENV4. DENV2. DENV4. DENV4-Ab12 DD17-4 DENV2. DENV4. Merge. DAPI. FITC. DENV2. DENV4-Ab8 DD13-4. DENV4-Ab14 DD27-8 DENV4. DENV2. DENV4. DENV4-Ab16 DD31-3 DENV2. DENV4. Merge. DAPI. FITC. DENV2. DENV4-Ab15 DD30-4. Figure 1. Identification of Serotype-specific MAbs against DENV4 by IFA. 39.

(40) A.. DENV4-Ab20 DD12-8. 4G2 DENV4. DENV2. DENV4. DENV2. DENV4. Merge. DAPI. FITC. DENV2. DENV4-Ab17 DD33-2. B.. DENV4-Ab5 DD7-8 DENV2. DENV3. DENV4. Merge. DAPI. FITC. DENV1. C.. DENV4-Ab19 DD19-1 DENV2. DENV3. DENV4. Merge. DAPI. FITC. DENV1. Figure 2. Identification of cross-reactive MAbs against (A) DENV2 and DENV4 (B) DENV1 and DENV4 (C) DENV1, DENV2 and DENV4 by IFA. 40.

(41) 4G2 DENV2. DENV3. DENV4. Merge. DAPI. FITC. DENV1. DENV4-Ab9 DD14-1 DENV2. DENV3. DENV4. Merge. DAPI. FITC. DENV1. DENV4-Ab10 DD15-2 DENV2. DENV3. DENV4. Merge. DAPI. FITC. DENV1. Figure 3. Identification of cross-reactive MAbs against DENV3 and DENV4 by IFA. 41.

(42) DENV4-Ab7 DD11-4 DENV2. DENV3. DENV4. Merge. DAPI. FITC. DENV1. DENV4-Ab13 DD18-5 DENV2. DENV3. DENV4. Merge. DAPI. FITC. DENV1. DENV4-Ab18 DD23-8 DENV2. DENV3. DENV4. Merge. DAPI. FITC. DENV1. Figure 4. Identification of cross-reactive MAbs against DENV1, DENV2, DENV3 and DENV4 by IFA. 42.

(43) 4G2. DENV2-Ab29. DENV4-Ab1. DENV4-Ab2. DENV4-Ab3. D1 D2 D3 D4. D1 D2 D3 D4. D1 D2 D3 D4. D1 D2 D3 D4. D1 D2 D3 D4. 2NS-1 E protein. DENV4-Ab5. DENV4-Ab7. DENV4-Ab8. DENV4-Ab9. DENV4-Ab11. D1 D2 D3 D4. D1 D2 D3 D4. D1 D2 D3 D4. D1 D2 D3 D4. D1 D2 D3 D4. DENV4-Ab12. DENV4-Ab13. DENV4-Ab14. DENV4-Ab16. DENV4-Ab17. D1 D2 D3 D4. D1 D2 D3 D4. D1 D2 D3 D4. D1 D2 D3 D4. D1 D2 D3 D4. Figure 5. Identification of MAbs against DENV4 by Western blots analysis. Four serotypes of DENV antigens (DENV1 to DENV4) from infected C6/36 cell lysates were size fractionated in non-reducing polyacrylamide gels. It was showed 4G2, DENV2-Ab29, DENV4-Ab1, DENV4-Ab2, DENV4-Ab3, DENV4-Ab5, DENV4-Ab7, DENV4-Ab8, DENV4-Ab9, DENV4-Ab11, DENV4-Ab12, DENV4-Ab13, DENV4-Ab14, DENV4-Ab16 and DENV4-Ab17. Those 4G2 and DENV2-Ab29 were positive control for E (55 kDa) and dimer-form NS-1 (75 kDa) protein.. 43.

(44) 4G2 NS-1. prM-E. NS-1. DENV4-Ab1 prM-E. NS-1. Merge. DAPI. FITC. prM-E. DENV2-Ab29. DENV4-Ab9 NS-1. prM-E. NS-1. DENV4-Ab11 prM-E. NS-1. Merge. DAPI. FITC. prM-E. DENV4-Ab10. DENV4-Ab12 NS-1. prM-E. NS-1. DENV4-Ab14 prM-E. NS-1. Merge. DAPI. FITC. prM-E. DENV4-Ab13. Figure 6. Identification of MAbs against prM-E or NS-1 protein of DENV4 by IFA.. 44.

(45) D4 -r E D4 DI-I I -r E DI II. D4 -r E D4 DI-I I -rE DI II. DENV4-Ab8 DENV4-Ab9 DENV4-Ab11 D4 -rE D 4 D I- I I -r E DI II. DENV4-Ab1 D4 -r E D 4 DI-I I -r E DI II. D4 -. D4 -r. ED I-I I rE DI II. Anti-His. rEDI-II. 34 26. rEDIII. 17 DENV4-Ab16 D4 -r E D4 DI-I I -rE DI II. D4 -rE D4 DI-I I -r E DI II. D4 -rE D4 DI-I I -rE DI II. D4 -rE D 4 D I -I -r E I DI II. D4 -rE D4 DI-I I -rE DI II. DENV4-Ab12 DENV4-Ab13 DENV4-Ab14 DENV4-Ab15. 34 26 17. Figure 7. Characterization of MAbs against rEDI-II or rEDIII of DENV4 E protein. Lysates which contained rEDI-II (36 kDa) and rEDIII (17 kDa) of DENV4 were used for Western blot analysis. It is showed that DENV4-Ab1, DENV4-Ab8, DENV4-Ab9, DENV4-Ab11, DENV4-Ab13, DENV4-Ab15 and DENV4-Ab16recognized EDI-II of DENV4; DENV4-Ab 12 and DENV4-Ab 14 recognized EDIII of DENV4.. 45.

(46) Inhibition Percentage (%). Number of Plaque. A. DENV2-Ab43. 40 30 20 10 0 20. 10. 5. 2.5. 1. 0. DENV2-Ab43 100 75 50 25 0 20. 10. Concentration (μg/ml). 5. 2.5. 1. 0. Concentration (μg/ml). Inhibition Percentage (%). Number of Plaque. B. DENV4-Ab11. 40 30 20 10 0 8. 4. 2. 1. 0.5. 0. DENV4-Ab11. 100 75 50 25 0 8. Concentration (μg/ml). 4. 2. 1. 0.5. 0. Concentration (μg/ml). Inhibition Percentage (%). Number of Plaque. C. DENV4-Ab13. 30 20 10 0 10. 1. 0.1. 0.01. 0.001. 0. DENV4-Ab-13. 100 75 50 25 0 10. Concentration (μg/ml). 1. 0.1. 0.01. 0.001. 0. Concentration (μg/ml). NMIgG. Inhibition Percentage (%). Number of Plaque. D. 40 30 20 10 0 10. 1. 0.1. 0.01. 0.001. 0. Concentration (μg/ml). NMIgG. 100 75 50 25 0 10. 1. 0.1. 0.01. 0.001. 0. Concentration (μg/ml). Figure 8. Determination of the neutralizing activity of MAbs. The neutralizing activity of MAbs, (A) DENV2-Ab43 (B) DENV4-Ab11 (C) DENV4-Ab13 (D) NMIgG, were test by PRNT. DENV4-Ab11 and DENV4-Ab13 were shown to neutralize DENV4 infection. The DENV2-Ab 43 and NMIgG were used for positive and negative control.. 46.

(47) Inhibition Percentage (%). Concentration (50 μg/ml) 125 100 75 50 25 0 D2-Ab43. D4-Ab7. D4-Ab11. D4-Ab12 D4-Ab13. D4-Ab14. D4-Ab15 NMIgG. Inhibition Percentage (%). Concentration (10 μg/ml) 100 75 50 25 0. Inhibition Percentage (%). D2-Ab43. D4-Ab7. D4-Ab11 D4-Ab12. D4-Ab13 D4-Ab14 D4-Ab15. NMIgG. Inhibition activity of DENV4. 100 75 50 25 0. D2-Ab43 (20 μg/ml). D2-Ab43 (10 μg/ml). D4-Ab11 (2 μg/ml). D4-Ab11 (1 μg/ml). D4-Ab13 (20μg/ml). D4-Ab13 (10μg/ml). NMIgG (20μg/ml). NMIgG (10μg/ml). Figure 9. Determination of the neutralizing activity of MAbs against DENV4 by PRNT. We examined the neutralizing activity of MAbs, DENV2-Ab43, DENV4-Ab7, DENV4-Ab11, DENV4-Ab12, DENV4-Ab13, DENV4-Ab14 and DENV4-Ab15, at different concentration of MAbs. The DENV2-Ab43 and NMIgG were used for positive and negative control. 47.

(48) LD50 of DENV4. Figure 10. Determination of the LD50 of DENV4 using ICR suckling mice. The 50 and 100PFU of DENV4 were injected into mice brain.. 48.

(49) Figure 11. Determination of the neutralizing activity of DENV4-Ab11 and DENV4-Ab13 against DENV4 by protection assay. Different concentration of DENV4-AB 11 was incubated with twenty-fold of LD50 of DENV4 and injected into ICR suckling mice. DENV4-Ab11 protected the mice from twenty times (1000 PFU) of DENV4 challenge.. 49.

(50) A.. B. f1 ori. PCMV. f1 ori PCMV. T7 promter/priming site. T7 promter/priming site NheI (671). Nhe I (671). Ampicillin. DENV4 EDI-II. Ampicillin. D4-DIII #1 XhoI (989). pCR3.1-DENV4-EDI-II-Flag. pCR3.1-DENV4-EDIII-Flag. XhoI (1565). 6062 bp. Psv40/ori. 5486 bp. Flag. Flag pIRES pCR3.1 reverse priming site. pIRES pCR3.1 reverse priming site Psv40/ori ColE1 origin. Neomycin/Kanamycin ColE1 origin. Neomycin/Kanamycin. TKpA. TKpA. C. Cell only. Anti-Flag DENV4-EDI-II. DENV4-EDIII. Figure 12. Construction and expression of DENV4-EDI-II and DENV4-EDIII in BHK-21 cells. (A) pCR3.1/DENV4-EDI-II/Flag and (B) pCR3.1/DENV4-EDIII/Flag were constructed using pCR3.1-Flag vector. (C) Determine the expression of these recombinant proteins by IFA with antibody against Flag proteins. The “Cell only” was negative control.. 50.

(51) D 4 p rM -E L1. 0.8. DENV 4. A490. 0.6. cell only. 0.4 0.2 0 500. 1000. 2000. 4000. 8000. 16000. 32000 64000. D ilution fo ld. D4 prM -E R1. 0.8. DE N V4. A490. 0.6. ce ll o nly. 0.4 0.2 0 500. 1000. 2000. 4000. 8000. 16000. 32000 64000. D ilu tio n fo ld D 4 prM -E Lx. 0.8. D EN V4. A490. 0.6. cell only 0.4 0.2 0 500. 1000. 2000. 4000. 8000. 16000. 32000. 64000. D ilution fo ld. Figure 13. Determination of the titer of DENV4 prM-E hyper-immune sera against DENV4 by ELISA assay. The mice sera were incubated with DENV4 H241 infected C6/36 cell plates. The absorbance values were read at O.D.490 nm as described under Materials and Methods. A490, optical density at O.D.490 nm.. 51.

(52) D 4 E DI-II L1. 0.8. DE N V4. A490. 0.6. ce ll o nly. 0.4 0.2 0 500. 1000. 2000. 4000. 8000. 16000 32000 64000. D ilution fold D4 EDI-II L2. 0.8. DE N V4. A490. 0.6. cell on ly. 0.4 0.2 0 500. 1000. 2000. 4000. 8000. 16000 32000 64000. Dilution fold D 4 E DI-II Lx. 0.8. D EN V4. A490. 0.6. ce ll o nly 0.4 0.2 0 500. 1000. 2000. 4000. 8000. 16000 32000 64000. Dilution fold. Figure 14. Determination of the titer of DENV4 EDI-II hyper-immune sera against DENV4 by ELISA assay. The mice sera were incubated with DENV4 H241 infected C6/36 cell plates. The absorbance values were read at O.D.490 nm as described under Materials and Methods. A490, optical density at O.D. 490 nm.. 52.

(53) D 4 E DIII L1. 0.8. D EN V4. 0.6. A490. cell only 0.4 0.2 0 500. 1000. 2000. 4000. 8000. 16000. 32000 64000. D ilution fold D4 EDIII L2. 0.8. D EN V4. 0.6. A490. cell only 0.4 0.2 0 500. 1000. 2000. 4000. 8000. 16000 32000 64000. Dilutio n fold D4 EDIII Lx. 0.8. D EN V4. 0.6. A490. cell o nly 0.4 0.2 0 500. 1000. 2000. 4000. 8000. 16000. 32000 64000. Dilution fold. Figure 15. Determination of the titer of DENV4 EDIII hyper-immune sera against DENV4 by ELISA assay. The mice sera were incubated with DENV4 H241 infected C6/36 cell plates. The absorbance values were read at O.D.490 nm as described under Materials and Methods. A490, optical density at O.D.490 nm.. 53.

(54) Inhibition Percentage (%). Number of Plaque. A. NMS. 25 20 15 10 5 0 50. 100. 200. 400. 800. 0. NMS. 100 75 50 25 0 50. 100. 200. 400. 800. 0. 800. 0. 800. 0. 800. 0. Dilution fold. Dilution fold. Inhibition Percentage (%). Number of Plaque. B. D4 prM-E L1 sera. 25 20 15 10 5 0 50. 100. 200. 400. 800. 0. D4 prM-E L1 sera. 100 75 50 25 0 50. 100. D4 prM-E L2 sera. 25 20 15 10 5 0 50. 100. 200. 400. 800. 0. 75 50 25 0 50. 100. Inhibition Percentage (%). Number of Plaque. 20 15 10 5 0 100. 200. 400. 200. 400. Dilution fold. D4 prM-E Lx sera. 50. 400. D4 prM-E L2 sera. 100. Dilution fold. 25. 200. Dilution fold. Inhibition Percentage (%). Number of Plaque. Dilution fold. 800. 0. Dilution fold. D4 prM-E Lx sera. 100 75 50 25 0 50. 100. 200. 400. Dilution fold. Figure 16. Determination of the neutralizing activity of DENV4 prM-E immunized mice sera against DENV4 by PRNT. It was showed that neutralizing activity of (A) NMS and (B) immunized mice sera. The NMS was used for negative control.. 54.

(55) Table 1. Characterization of MAbs against DENV4 specificity a. MAb IFA. PRNT50. Western Blot Analysis DENV1. DENV2. DENV3. DENV4. DENV4-Ab1. E. -. -. -. EDI-II. DENV4-Ab2. NS-1. -. -. -. NS-1. DENV4-Ab3. E. -. -. -. E. DENV4-Ab4. E. -. -. -. -. DENV4-Ab5. E. NS-1. -. -. -. DENV4-Ab 6. E. -. -. -. E. DENV4-Ab 7. E. -. -. +. EDI-II. DENV4-Ab 8. E. -. -. -. EDI-II. DENV4-Ab9. E. -. -. -. EDI-II. DENV4-Ab10. E. -. -. -. -. DENV4-Ab11. E. -. -. -. EDI-II. DENV4-Ab12. E. -. -. -. EDIII. DENV4-Ab13. E. -. -. +. EDI-II. DENV4-Ab14. E. -. -. -. E. DENV4-Ab15. E. -. -. -. EDI-II. DENV4-Ab16. E. -. -. -. EDI-II. DENV4-Ab17. NS-1. -. +. -. NS-1. a. < 2 μg/ml. < 20 μg/ml. E, envelope proteins; NS1, nonstructural protein 1, EDI-II, domain I and domain II of E protein, EDIII, domain III of E protein.. 55.

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