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* Department of Basic Medical Sciences,
Department of Microbiology and Immunology,
Department of Medical Technology, and
Department of Pediatrics, College of Medicine, National Cheng Kung University, Tainan, Taiwan; and
¶ Center for Disease Control, Department of Health, Taipei, Taiwan, Republic of China
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Abstract |
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RII pathway. Furthermore, anti-prM Ab also enhanced dengue virion binding and infection on cells lacking FcR, such as BHK-21 or A549 cells, by the mechanism of peptide (CPFLKQNEPEDIDCW)-specific binding. Anti-prM Ab cross-reacted with BHK-21 or A549 cells and recognized self-Ags such as heat shock protein 60. In summary, a novel mechanism of anti-prM Ab-mediated enhancement on dengue virus infection was found to be mediated by dual specific binding to dengue virion and to target cells, in addition to the traditional enhancement on FcR-bearing cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Cell lines bearing the FcR, such as murine macrophage P388D1, dendritic cell line DC2.4, human B cell line BJAB, murine myeloma FO, murine hybridoma A20, human macrophage cell line U937, human leukemia cell line K562, murine hybridoma H21F8-1, 206-35, and cell lines that do not have the FcR, such as human T cell line Jurkat T, murine fibroblast cell line NIH3T3, human epithelial carcinoma cell line A549, and baby hamster kidney cell line BHK-21 were maintained in DMEM supplemented with 10% FBS. All four dengue virus serotype strains (dengue serotype 1/766733A, serotype 2/PL046, serotype 3/739079A, and serotype 4/H-241) were obtained from the Center for Disease Control in Taiwan. Unless otherwise specified, the dengue virus serotype-2 strain PL046 was used for most experiments. Viruses were propagated in mosquito C6/36 cell line in Eagle’s MEM containing 2% heat-inactivated FBS at 28° C for 5 days and quantified by standard plaque assay as reported previously (7). Dengue virus-containing supernatant was first centrifuged at 10,000 rpm, and then ultracentrifuged at 100,000 x g at 4°C for 3 h to purify the dengue virion, which was then labeled using NHS-fluorescein (Pierce).
Production of anti-dengue mAb
Breeder mice of BALB/c strain were purchased from either The Jackson Laboratory or Charles River Japan. They were maintained on standard laboratory chow and water ad libitum in the animal facility of the Medical College, National Cheng Kung University (Tainan, Taiwan). The animals were raised and cared for following the guidelines set up by the National Science Council of the Republic of China. Groups of BALB/c adult mice were inoculated i.v. with dengue-2 virus PL046 as described previously (7). Hybridomas were generated by fusion of splenocytes with FO myeloma using 1% polyethylene glycol (8). Several methods, including cellular ELISA, immunohistochemical staining, and flow cytometric assay on dengue virus-infected BHK-21 cells, were used to screen the Abs. The isotypes of each mAb were identified using the mouse monoAb ID kit (Zymed Laboratories). The antigenicity of each clone was further characterized by Western blot or immunoprecipitation of dengue-infected C6/36 cell lysate. In most cases, the mAb was purified from hybridoma culture supernatant using the Montage Prosep-G kit (Millipore). However, for anti-E Ab and anti-NS1 Ab, when a large quantity of mAb was needed, the mouse ascites were used instead. The Abs were biotinylated by using NHS-LC-Biotin (Pierce), and immobilized pepsin (Pierce) was used to obtain the F(ab')2.
Infection of dengue virus and ADE
The infection was usually performed by coincubating cells with dengue viruses at a multiplicity of infection (m.o.i.) of 1 for 90 min at 37°C. In some cases, the m.o.i. could be decreased to 0.1. For cell lines that are not very sensitive to dengue virus infection such as myeloma FO, hybridoma 185-10, H21F8-1, 206-35, A20, and murine dendritic cell line DC2.4, human macrophage cell line U937, or human monocytes, a higher m.o.i. of 10 was used. In the Ab enhancement on FcR-bearing suspension cells, the virus-Ab complexes were prepared by mixing immune serum or mAb with viruses (m.o.i. = 1) for 30 min at 37°C before addition to the cell suspension (2 x 105/sample) for infection. In some experiments, cells were pretreated with anti-FcR mAb (anti-CD16 Ab or anti-CD32 Ab, 20 µg/ml; BD Biosciences Pharmingen) for 15 min at 37° C before incubation with the virus-Ab complexes. After infection, cells were washed twice and resuspended in complete medium for further culture. The cells were assayed for virus infection by flow cytometric analysis, and the supernatants were assayed by plaque assay. For Ab enhancement on non-FcR-bearing cells, BHK-21 cells (1 x 105/well) were plated in 24-well plates for 24 h and then subsequently coated with 5 µg of anti-prM (70-21) or anti-E (185-10) Ab for 30 min at 37°C. The Ab-coated cells were washed twice, then infected with dengue virus at the m.o.i. of 0.1 for 90 min at 37°C. The cells were washed again and cultured with complete growth medium for 24 h before flow cytometric analysis. For dengue virion binding, cells (2 x 105/well) were coated with 5 µg each of anti-prM (70-21) or anti-E (185-10) Ab for 30 min at 4°C. After thoroughly washing, the cells were then incubated with FITC-labeled virions (25 µl) for 30 min at 4°C. The cell-bound virions with green fluorescence were detected by flow cytometer. For the M3 region of the prM protein-blocking experiment, M3 peptide (CPFLKQNEPEDIDCW), control M1 (MAFHLTTRNGEPHMI), or M4 (GTCTTTGEHRREKRSVAL) peptides were added together with the virus (9).
Flow cytometric assay
Cells were harvested and fixed with 2% paraformaldehyde in PBS for 20 min on ice. After being washed, the cells were stained with biotinylated anti-dengue mAb (anti-E, anti-prM, anti-NS1, and anti-Core Abs) in the permeabilization buffer (2% FBS, 0.1% saponin, 0.1% sodium azide) for 30 min on ice. After washing, these cells were then stained with streptavidin-FITC in the same buffer for 30 min. Finally, these cells were washed and resuspended in staining buffer (2% FBS and 0.1% sodium azide) for FACSCalibur analysis (BD Immunocytometry Systems), and analyzed using the software WinMDI, version 2.8.
Identification of autoantigens from A549 or BHK-21 cells
The membrane protein extracts of human epithelial carcinoma cell line A549 or baby hamster kidney cells BHK-21 were prepared with the Subcellular Proteome Extraction Kit (Calbiochem) and incubated with anti-prM or anti-Hsp60 Ab-coated Dynabeads M-450 GAM IgG (Dynal Biotech) for 1 h at 4°C. After washing with 1% Triton X-100/PBS, the immune-complexes were dissolved in native sample buffer and were subjected to 12.5% SDS-PAGE analysis. Western blot analysis was performed with anti-prM or anti-hsp60 Ab.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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To study the phenomena of Ab enhancement of dengue virus infection, a flow cytometric analysis can be used with anti-NS1 or anti-E Ab intracellular staining (Fig. 1A), in which both the percentage of dengue virus-infected cells and the intensity of dengue virus protein within the cells can be quantitated. Either Ab against dengue nonstructural protein NS1 or envelope protein can detect intracellular dengue virus-positive cells. The very low percentage of dengue virus-infected cells detected by the anti-core Ab was probably due to the low affinity of the anti-core Ab used. The isotype control Ab was used as a negative control. By testing on a sensitive human leukemia cell line K562, we could detect the degree of enhancement of dengue-2 virus cell infection in flow cytometry with a high dilution of heterologous dengue-3 immune serum at a m.o.i. from 0.01 to 10 (Fig. 1B). Furthermore, the mean fluorescent intensity of dengue virus protein per infected cell was also enhanced. The standard plaque assay was simultaneously tested for comparison. The traditional plaque assay detected the enhancement of dengue virion production at a m.o.i. of 0.01–1. Apparently, this flow cytometric method is comparable to the traditional plaque assay, but it has the advantages of being simple, fast, sensitive, time-saving, and target cell-based. It can be used on either cell lines or primary cells (Fig. 1, C and D). The dengue-2 virus-infected human B cell line BAJB was enhanced by dengue-3 immune serum, with a high dilution (1/6000) of serum enhancing more than that of 1/600. Furthermore, on human monocytes, taking the double staining of CD14+ and NS1/E+ monocytes to represent the dengue virus-infected monocytes, the addition of dengue-3 immune serum increased the percentage of the dengue-2-infected monocytes from 2.6 to 21.6%. The mean fluorescence intensity of dengue protein Ag also increased from 291 to 352, indicating that the dengue virus protein synthesis within the cells was increased as well. This suggests that heterologous dengue-3 immune sera not only increased the dengue-2 virus-infected cell percentage, but the dengue virus E and NS1 proteins recognized by anti-E and anti-NS1 Abs per cell also increased.
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To further characterize the ADE on dengue virus infection, a panel of anti-E, prM, and NS-1 or core mAb was generated from dengue 2-virus-infected mice, the specificities of these mAbs determined by flow cytometry, immunofluorescent staining, Western blot, or immunoprecipitation assay as shown in Fig. 2, and summarized in Table I. On FcR-bearing cells of the murine macrophage cell line P388D1 or murine dendritic-like cell line DC2.4, the ADE effect of different monoclonal anti-dengue Abs was shown in Fig. 3A. Anti-prM Abs and some anti-E Abs (185-10 and 113-19) enhanced dengue virus infection, but another anti-E Ab, 137-22, was found to be inhibitory. Meanwhile, the anti-NS1 or anti-core Abs did not have any effect. To further characterize this finding, the enhancing anti-prM Ab (70-21), anti-E Ab (185-10), and the neutralizing anti-E Ab (137-22 and 50-21) were studied with different concentrations. It was found that the ADE effect was concentration-dependent (Fig. 3B). Anti-prM Ab enhanced the dengue virus infection at doses of 0.1 
10 µg/ml, but not at 100 µg/ml, and the anti-E Ab 185-10 enhanced the dengue virus infection at doses of 1 µg/ml 100 µg/ml. In contrast, the anti-E Ab 137-22 acted as a neutralizing Ab at doses higher than 1 µg/ml, whereas it had an enhancing activity at a dose of 0.1 µg/ml. Likewise, anti-E Ab 50-21 neutralized at doses higher than 10 µg/ml, but enhanced at 0.1 µg/ml. Furthermore, the ADE effect was independent of the four serotypes of dengue virus. Although these anti-dengue mAbs were derived from dengue 2-virus-infected mice, most of them could recognize the four serotypes of dengue virus as shown by immunohistochemical staining of dengue virus-infected cells (Table I). As shown in Fig. 3C, anti-prM Ab 70-21 or anti-E Ab 185-10 enhanced the dengue virus infection of all four serotypes, whereas anti-E Ab 137-22, which was dengue 2-specific, neutralized the dengue-2 virus infection but had no effect on the other three serotypes. The ADE effect is known to require the Fc portion of the enhancing Ab and the presence of FcR on target cells, and we also observed this. Pepsin digestion of the Fc portion of the anti-prM Ab resulted in the loss of its enhancing activity. The addition of anti-FcR Abs (anti-CD16/CD32 Ab) to the target cells blocked the anti-prM Ab-mediated enhancement (Fig. 3D). We titrated the concentration of anti-FcR Ab and found that 20 µg/ml is optimal to completely inhibit the enhancement. The involvement of the Fc portion and FcR on the target cells was also applied to the anti-E Ab (185-10)-mediated enhancement. In this experiment, Protein A was used to block the interaction of the Fc portion with the FcR. This suggests that both anti-prM and anti-E Ab mediate the enhancement of dengue virus infection on FcR-bearing cells by the Fc-FcR pathway. If the anti-E or anti-prM Ab, once bound to the dengue virion, does not inhibit the infection, then the Ab-virus complex can subsequently enter the target cells more efficiently through the FcR of the cells, thus leading to infection.
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Anti-prM Ab could enhance the dengue virus infection on all FcR+ cell lines tested (murine myeloma FO, hybridoma 185-10 and H21F8-1, A20, murine macrophage P388D1, DC2.4, and human B cell line BJAB, macrophage cell line U937) (Fig. 4A). Surprisingly, it also enhanced the dengue virus infection on non-FcR-bearing cells such as baby hamster kidney cell BHK-21, human epithelial carcinoma cell line A549, mosquito cell line C6/36, and murine fibroblast cell line NIH3T3 (Fig. 4B). How the anti-prM Ab enhanced the dengue virus infection was therefore further investigated. Using FITC-labeled dengue virus, the dengue virion binding to the FcR-bearing K562 cells was enhanced by either the anti-prM Ab (70-21) or anti-E Ab (185-10), both of these being Fc and FcRII-dependent, because anti-CD32 (FcRII) Ab, and not anti-CD16 (FcRIII) Ab, blocked the binding (Fig. 5A). The anti-E Ab (137-22), although acting as a neutralizing Ab in Fig. 3A, also enhanced the dengue virion binding. We have mapped the epitope of the anti-prM Ab recognized as corresponding to the M3 region (CPFLKQNEPEDIDCW) of the prM (data not shown). The enhanced infection by anti-prM Ab on K562 cells was blocked by M3 peptide dose-dependently (Fig. 5B), suggesting that the anti-prM Ab-mediated enhancement of dengue virus infection is M3 Ag-binding dependent. Furthermore, for the target cells without FcR like BHK-21 cells, the anti-prM Ab could also enhance the dengue virion binding (Fig. 5C), which was also blocked by M3 peptide dose-dependently (Fig. 5D). The enhanced dengue virion binding would subsequently lead to the enhancement of dengue virus infection in BHK-21 cells (Fig. 5E). The effect of anti-prM Ab on the enhancement of dengue virion binding and infection was also observed for A549 cells (data not shown).
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How the anti-prM Ab enhances the dengue virion binding and infection by its M3 specificity was further investigated. The anti-prM Ab could cross-react with BHK-21 or A549 cells as shown by flow cytometric and immunofluorescent staining (Fig. 6A). The anti-prM Ab binding to BHK-21 or A549 cells was dose-dependent (data now shown), and can be inhibited by M3 peptide (Fig. 6B). The proteins on cell membrane recognized by the anti-prM Ab were further identified. Anti-prM Ab can bind to the membrane protein extracts from baby hamster kidney cell BHK-21 or human epithelial carcinoma cell line A549 on Western blot. One of the proteins has a m.w. equivalent to HSP60. The membrane protein immunoprecipitated by the anti-hsp60 Ab was also recognized by anti-prM Ab on Western blot. The HSP60 is one of the autoantigens recognized by the cross-reactive anti-prM Ab (Fig. 6C). Based on the above data, we conclude that in addition to the anti-E Ab-mediated enhancement of dengue virus infection on FcR-bearing cells, the anti-prM Ab is also an enhancing Ab for cells with or without FcR, its enhancement being mediated by dual specific binding to dengue virion and to target cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Dengue disease has a broad spectrum of clinical symptoms. Most dengue infections are subclinical or self-limited, but there is a risk of progressive development to DHF or DSS. Viral replication plays an important role in the disease pathogenesis. Severe DHF is associated with a higher virus load than mild dengue fever (11). ADE is a central hypothesis to explain the increased dengue virus-infected cell mass (4). Newborn babies <1 yr of age with maternal anti-dengue IgG Abs are known to be susceptible to DHF/DSS after primary infection (12, 13). The enhancing Ab was presumed to act against the dengue virus envelope protein. We reported in this study that both anti-E Ab and anti-prM Ab could work as the enhancing Abs. Although monoclonal anti-prM and anti-E Ab were used in this study, anti-prM Ab has indeed been found in dengue patient sera, which can also enhance dengue virus infection (our unpublished observation). In our study, the anti-E 137- 22 and 50-21 are neutralizing Abs, whereas the anti-E 185-10 and all the anti-prM are non-neutralizing ones according to plaque assay on P388D1 cells (our unpublished observation), but their enhancement or neutralization effect seems to be dependent on the concentration of the Ab, and is also dengue serotype-independent. Both of the anti-E Ab, 137-22 and 185-10, enhanced the dengue virion binding to K562 cells (Fig. 5A), but the former neutralized while the latter enhanced the dengue virus infection. This suggests that dengue virion binding to the putative receptor is a necessary but not a sufficient condition for virus infection.
From in vitro observation of ADE, it is known that the non-neutralizing Ab or subneutralization amount of Ab would help the virus to enter the cells through the FcR. In this study, we extended this phenomenon to cells lacking FcR such as BHK-21 or A549, and it was found that the anti-prM Ab could enhance the dengue virus infection of these cells. Takada and Kawaoka (14) have mentioned that ADE can involve the complement C3 receptor, C1q and its receptor C1qR, in addition to the FcR. Mature dengue virion is icosahedral, 500 Å in diameter (15, 16, 17). It has a smooth surface, which is different from the immature virion that has prM-containing spikes. Our anti-prM Ab could bind to the dengue virion produced in the mosquito C6/36 cell line. The possibility that anti-prM Ab, when it binds to the prM-containing particle, induces a conformational change that increases the exposure of fusion peptide on E domain II, thereby becoming fusogenic to target cells and hence increasing the infection, is not excluded. For dengue virus infection on FcR-bearing macrophages, ADE is mediated primarily by the FcR pathway because anti-FcR Abs block the dengue virus binding and infection. Two reasons are proposed for this complete inhibition by anti-FcR Ab blocking. One is the assay protocol used for the enhancing study, in which the dengue virus is mixed with the Ab first, so the Ab-virus complex would bind to the cells via the FcR pathway. Alternatively, the affinity of FcR is higher than the putative dengue virus receptor, so the FcR dominates the receptor binding. In contrast, complement receptors were not involved because we used heat-inactivated FCS to culture the cells. The anti-prM Ab can cross-react to BHK-21 and A549 cells, and by this process, the dual specific binding on dengue virion and target cells would bridge the dengue virion to its receptor. Mady et al. (18) have reported that the artificially bispecific Ab, contrived by conjugating anti-dengue E/prM Ab with anti-cell Ab, could enhance the dengue virus infection. Our study demonstrated that the anti-prM Ab could indeed have dual specificities, one binding to dengue virion with another to target cross-reactive HSP60 protein. It is possible that the enhancement of dengue virus infection is caused by the enhanced entry of the virus into more cells, thereby increasing both the virus-infected cell percentage and the rate of virion entry into each cell, and thus increasing the degree of virus replication within the cell. However, it should be noted that viral replication via ADE entry to suppress cellular antiviral response was reported for Ross River virus (19). The possibility of an antiviral effect by IL-10 is not excluded because the immune complex binding to FcRII was reported to enhance IL-10 production (20).
The anti-prM Ab cross-reactivity with HSP60 is interesting. Schett et al. (21) reported that the autoantibody acting against HSP60 could mediate endothelial cytotoxicity. A homology shared between CMV-derived protein UL122/US28 and HSP60, Ab against these viral peptides also caused endothelial cell apoptosis (22). We have reported the generation of autoantibody to platelet and endothelial cells that were cross-reactive with the dengue virus protein (5, 6, 7, 23). The cross-reactive anti-prM Ab reported in this study also enhanced the dengue virus infection with the dual specificities on dengue virion binding and the target cells. A molecular mimicry between prM and HSP60 suggests an additional pathway for dengue virion binding and infection via the enhancing Ab.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by Grant NSC92-3112-B006-017 from the National Science Council, Taiwan. 
2 Address correspondence and reprint requests to Dr. Huan-Yao Lei, Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan, Republic of China. E-mail address: hylei{at}mail.ncku.edu.tw
3 Abbreviations used in this paper: DHF, hemorrhagic fever; DSS, dengue shock syndrome; ADE, Ab-dependent enhancement; dengue; HSP60, heat shock protein 60; m.o.i., multiplicity of infection.
Received for publication September 26, 2005. Accepted for publication December 7, 2005.
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References |
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receptors. J. Immunol. 147: 3139-3144. [Abstract] receptors. J. Immunol. 166: 6861-6868. This article has been cited by other articles:
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  H.-J. Cheng, C.-F. Lin, H.-Y. Lei, H.-S. Liu, T.-M. Yeh, Y.-H. Luo, and Y.-S. Lin Proteomic Analysis of Endothelial Cell Autoantigens Recognized by Anti-Dengue Virus Nonstructural Protein 1 Antibodies Experimental Biology and Medicine, January 1, 2009; 234(1): 63 - 73. [Abstract] [Full Text] [PDF]
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 I. A. Zybert, H. van der Ende-Metselaar, J. Wilschut, and J. M. Smit Functional importance of dengue virus maturation: infectious properties of immature virions J. Gen. Virol., December 1, 2008; 89(12): 3047 - 3051. [Abstract] [Full Text] [PDF]
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C.-Y. Lai, W.-Y. Tsai, S.-R. Lin, C.-L. Kao, H.-P. Hu, C.-C. King, H.-C. Wu, G.-J. Chang, and W.-K. Wang Antibodies to Envelope Glycoprotein of Dengue Virus during the Natural Course of Infection Are Predominantly Cross-Reactive and Recognize Epitopes Containing Highly Conserved Residues at the Fusion Loop of Domain II J. Virol., July 1, 2008; 82(13): 6631 - 6643. [Abstract] [Full Text] [PDF]
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K. Boonnak, B. M. Slike, T. H. Burgess, R. M. Mason, S.-J. Wu, P. Sun, K. Porter, I. F. Rudiman, D. Yuwono, P. Puthavathana, et al. Role of Dendritic Cells in Antibody-Dependent Enhancement of Dengue Virus Infection J. Virol., April 15, 2008; 82(8): 3939 - 3951. [Abstract] [Full Text] [PDF]
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H. M. van der Schaar, M. J. Rust, B.-L. Waarts, H. van der Ende-Metselaar, R. J. Kuhn, J. Wilschut, X. Zhuang, and J. M. Smit Characterization of the Early Events in Dengue Virus Cell Entry by Biochemical Assays and Single-Virus Tracking J. Virol., November 1, 2007; 81(21): 12019 - 12028. [Abstract] [Full Text] [PDF]
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T. Oliphant, G. E. Nybakken, M. Engle, Q. Xu, C. A. Nelson, S. Sukupolvi-Petty, A. Marri, B.-E. Lachmi, U. Olshevsky, D. H. Fremont, et al. Antibody Recognition and Neutralization Determinants on Domains I and II of West Nile Virus Envelope Protein J. Virol., December 15, 2006; 80(24): 12149 - 12159. [Abstract] [Full Text] [PDF]
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