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Title: A microscopic examination of the interaction between antibodies, dengue virus and monocytes
Keywords: dengue virus, Alexa Fluor, antibodies, monocytes, neutralization
Issue Date: 29-Sep-2010
Citation: ZHANG LIXIN (2010-09-29). A microscopic examination of the interaction between antibodies, dengue virus and monocytes. ScholarBank@NUS Repository.
Abstract: Dengue is a significant disease globally. An estimated 50 to 100 million dengue infections occur annually, and more are at risk of being infected with 2.5 billion people living in dengue endemic countries. Although vector reduction programmes may limit dengue virus (DENV) transmission, it has not been carried out at a scale sufficient to control the disease globally. A tetravalent dengue vaccine is therefore needed to halt this worldwide escalation in disease incidence. Serotype-specific antibodies generated in a course of infection are thought to confer lifelong immunity to the same serotype of DENV; whereas cross-reactive antibodies are more frequently associated with antibodymediated enhancement of infection, leading to more severe disease. Despite the fact that antibody-DENV interactions can lead to immunity or immunopathogenesis, the factors governing such outcomes of infection have not been well defined. This has thus led to long delays in the development of a safe and effective vaccine. In this thesis, we sought to understand the immunity end of the spectrum through early antibody-DENV interactions with monocytes (the primary targets of dengue infection) that lead to neutralization of the virus, using confocal microscopy. A simplified method of labelling DENV with a fluorescent Alexa Fluor dye with minimal modification to viral viability was developed in this study and subsequently used to visualize the early cellular processes taking place when monocytes encounter antibody- DENV complexes. Using two human-mouse chimeric antibodies, h3H5 and h4G2, as our model for serotype-specific and cross-reactive antibodies, respectively, we observed significantly different sub-cellular trafficking characteristics in human monocytes. At the minimal antibody concentration to fully neutralize 10 multiplicity of infection (MOI) of DENV, immune complexes with 3?g/ml h3H5 were rapidly internalized through the activatory Fc?RI and transported to LAMP-1 positive compartments within 30min, while that with 100?g/ml h4G2 bound to both Fc?RI and Fc?RII but internalization was delayed. This delay in internalization appeared to be antibody concentration dependent as increasing h3H5 concentration to 100 and 400?g/ml showed similar blockade of uptake. These observations were also verified in primary monocyte cultures. One possible explanation would be that larger viral aggregates were formed at higher antibody concentrations and that inhibited efficient Fc receptor-mediated uptake by the monocytes. Using a combination of sucrose gradient to separate the viral aggregates by size and dynamic light scattering to estimate their diameter, the data indicates that viral aggregates with average diameter of 192nm were formed with 100?g/ml of antibody, which is significantly larger than virus only (49.1nm) or Fab only controls (57.7nm). Taken collectively, increasing concentrations of antibody result in the formation of DENV aggregates of different sizes, which appeared to inhibit internalization. The mechanism for this is not through competition for FcR by free and unbound antibody. Instead the data suggests that larger viral aggregates may enable antibodies to cross-link FcR that are normally expressed at lower density. Lowering the antibody concentration allowed for efficient internalization, followed rapidly by trafficking of the immune complex to the late endosome. However, at these concentrations, viral replication was only prevented with serotype-specific but not cross-reactive antibody.
Appears in Collections:Master's Theses (Open)

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