Inhibition Of Anti-prM-related Antibody-dependent Enhancement Of Dengue Virus By Fc Gamma Rllb

Background and ObjectiveDengue fever is prevalent in tropical and subtropical regions and the incidence increased significantly in recent decades in the world. More than 40% of the world population (about 2.5 billion people) are at risk of suffering from dengue fever. WHO estimates that there may be 50 million to 100 million cases of dengue virus infection in the world which is a harmful insect-borne viral disease with the most widely distributed and the largest number of cases. Health and Family Planning Commission of Guang Dong reported about 45,000 cases of dengue fever including many cases of severe dengue in December,2014. Because of no effective drugs and vaccines,we only have to treat symptomatically, resulting in a high mortality rate.Therefore, dengue fever has become a global public health problem which we must control the infection of dengue vrius without delay.The pathogenesis of severe dengue has not yet been elucidated. But antibody-dependent enhancement of infection (ADE) dominate in the pathogenesis of dengue virus, immunology and vaccinology studies.This theory holds that the cell of mononuclear phagocyte system with the Fc receptors on the surface are the main target of dengue virus. Dengue virus and its specific antibody combine to form a complex, and then through the Conjugate of the Fc receptor of mononuclear phagocyte and the Fc segments of antibody bind, it gets into cells and replicate, resulting in viral load addition.Dengue virus can simultaneously produce mature and immature particles during its proliferation. ADE mediated by prM antibody related primarily immature particles. Electron microscopy and functional studies show that the projections on immature particles surface are composed of prM-E protein heterodimer, and the prM protein closes the fusion peptide of E protein in space which results the immature particles can not start the process of membrane fusion. After the cleavage by protein furin of the host, prM protein can release its N-terminal pr-peptide and start the structure rearrangement of immature particle,and then induces the prM-E heterodimer depolymerization to promote the formation of E protein homodimers tile end to end in a sleek surface of the particles. Finally complete the process of maturation of viral particles.Therefore, under normal conditions only mature particles are infectious and immature particles are non-infectious. In the presence of prM antibodies, prM protein-specific antibody can bind with the prM protein of immature particle on the surface.Then mediated by Fc receptors, or heat shock proteins,vrius can infect host cells and make immature particles acquired their infection capacity, resulting the performance of the ADE. The discovery of prM antibody and immature particles’ interaction greatly deepened the understanding of the ADE’mechanisms of dengue virus.Most target cells infected by dengue virus including monocytes, macrophages, dendritic cells can express activated receptors and inhibitory receptors. Recent studies show that activated receptors like FcyR Ⅱ and FcyR I play a catalytic role for ADE and there are little studies on FcyRⅡb. FcyRIIb as an inhibitory receptors can mediate a variety of negative feedback regulation reaction for immune cells. In its intracellular region contain an immunoreceptortyrosine-based inhibition motif (ITIM) which mainly mediated negative feedback regulation effect.When FcyRⅡb crosslinks immunoreceptor tyrosine -based activation motif (ITAM), it can maintain the balance of peripheral tolerance by mainly relying ITIM way to signal transduction. So FcyRIIb is the only FcyR inhibitory receptors of several white blood cells on the surface, which is being a growing concern for its cellular immune function.Fc receptor activation type FcyR Ⅰ and FcyR Ⅱ a has been shown to promote thedengue virus in the infected macrophages, but the influence of inhibitory receptor FcyRIIb has little effect on the ADE, Fc receptors ADE mechanism is not clear, especially the role of inhibitory receptors on ADE,and co-expression upon activation of the receptor and inhibitory receptors, the relationship of the two receptors for ADE action remains to be elucidated. Something has recently been reported in the literature, FcyRⅡb plays an inhibitory effect on the ADE, however, its exact mechanism of inhibitory effect is worth exploring. For example, whether the immune tyrosine-based inhibitory motif (ITIM) of FcyRIIb directly inhibited immune tyrosine-based activation motif (ITAM) of the activation receptor, resulting in an inhibitory effect? Or after immune complexes binding to the receptor, induce various cellular pathways? Or FcyRⅡb and FcyRIIa competed or limited immune complexes? Immune complexes by FcyRIIb and FcyRIIa signal transduction, causing an inhibitory or activating effect, to explore the mechanism of ADE provides a new way of thinking.Furthermore, in addition to antibodies and dengue virus by inhibiting viral binding to target cells or fusion of two mechanisms, but recent studies have indicated that there is a larger and mechanism of antigen-antibody complex by FcyRIIb receptor crosslinking inhibition of monocyte-macrophage immune complexes. The author believes FcyRIIB inhibitory effect can be used to explain the DV infection after four serotypes brief cross-protection. In the short term after DV infection, the presence of a large number of antibodies in vivo, at this time if the re-infection of DV, larger volumes can be formed immune complex, bulky and easier to immune complexes FcyRIIB crosslinking, thereby inhibiting endocytosis virus. The body gradually reduced antibody DV over time, and then re-infection DV, it is difficult to form a larger volume of immune complexes, and relatively easy to exert inhibitory FcyRIIB crosslinking to other Fc receptor-mediated cross-linking ADE effect.However, most surface receptors of the dengue virus target cell inhibited the activation of the receptor and the role of both crosslinked only apply to explore the ADE and the mechanisms and effects.The study was prepared by I-type dengue virus prM antibodies, DV2 virus combine to form immune complexes, respectively infection express FcyRIIa, FcyRIIb and BHK-21 cells co-expressing FcyRIIa and FcyRIIb model to explore the influence of FcyRⅡb on ADE.Methods1.Preparation and Purincation of Premembrane Protein of Dengue Type I Virus(1) The total RNA was extracted and prM gene(498bp) was amplified from genomic cDNA of DV1 by RT-PCR.(2) The amplified prM gene was inserted into PMD-18T and PGEX-4T-2 vetor and prM-gene-inserted vetor was transfered into BL-21(DE3).(3) Isopropyl thiogalactoside(with final concentration of 0.5mmol/L) was used to derivate BL-21 cell over night at 18℃, leading protein expression of BL-21 cell.(4) The expression product was purincation by glutathione peptide agarose gel chromatography and purity identification was applied by SDS-PAGE electrophoresis. The purified protein can be used as envelope antigen.2.Preparation and Identification of Polyclonal Antibody of prM ProteinNew Zealand White rabbits was immuned by purified prM protein. Indirect ELISA was used to detect Serum samples of immuned rabbits and titer of depurated polyclonal antibody. After ELISA detecting, specificity was detected by Western-blot.3.Preparation and Identification of Monoclonal Antibody of prM Protein(1) BALB/c mice was immuned by purified prM protein and immuned mice was uesd to isolated high antibody level spleen cells. Then isolated spleen cells was fuse with myeloma cell and was cultured by HT and HAT selective culture-medium. Single hybrid tumor cell line was isolated by limiting dilution.(2)Positive single hybrid tumor cell was applied to expanding cultivation. Then the hybridoma cells were injected into mice abdominal cavity to prepare ascites and the collected monoclonal antibody was purified.(3) Indirect ELISA was used to detect ascites of immuned mice and titer of depurated monoclonal antibody. After ELISA detecting, specificity of monoclonal antibody was detected by Western-blot.4.Construction and Expression of FcyRIIa and FcyRIIb Eukaryotic Vectors(1) FcyRIIa and FcyRIIb gene were amplified by PCR and double-enzyme cleavage with Not I/EcoR I.(2) Double-enzyme cleavaged product was separately inserted into pcDNA3.1(-) vector and in order inserted into pIRES. Then monoclone was selected and characterize its properties.(3) After sequencing result was shown to be correct, single-expressed FcyRIIa and FcyRIIb eukaryotic expression plasmid and double-expressed FcyRⅡa and FcyRIIb eukaryotic expression plasmid was collected.5.Preparation and Identification of Differential Fc Receptor-Expressed-BHK-21 cells.3 types of recombinant expressing plasmid was transfered into BL-21 cells using lip-2000(named in three group:BHK-CD32a, BHK-CD32b and BHK-CD32a+b cell). 48 hours later flow cytometry was used to detect Fc receptor expression of 3 types of plasmid-transfered-cell.6. Dengue Virus Reacting with prM Antibody and Infecting Differental Fc-Receptor-Expressed-BHK-21 Cells.(1) Depurated polyclonal antibody of prM protein was quantified by BCA method and titre of degue type II virus was detected by Plaque experiment.(2) Polyclonal antibody (with concentration of 100ug/ml) was mixed with DENV-2 (MOI=1) and was incubated for 2 hours at 37℃.(3) Immune complex was used to infect differental Fc-receptor-expressed BHK-21 cells. After 16 hours, Virus infection rate was detected by flow cytometry.Results1.prM recombinant expressing plasmid was successfully constructed using the PGEX-4T-2 prokaryotic expression vector. IPTG was successfully derivated BHK-21 cells and rich soluble proteins was collected. New Zealand white rabbits was immuned by purified prM protein and the indirect ELISA result showed that the titer of serum polyclonal antibody is 1:100000 and the titer of depurated polyclonal antibody is 1:200000. The result of western-blot showed well specificity of polyclonal antibody.2.BALB/c mice was immuned by purified prM protein and the collect monoclonal antibody was purified. Indirect ELISA result showed that the titer of ascites of immuned mice was 1:800000 and titer of depurated monoclonal antibody was 1:400000. The result of western-blot showed well specificity of monoclonal antibody and SDS-PAGE electrophoresis showed good purification effects.3. Three types of recombinant eukaryotic expression plasmid (pcDNA3.1 (-)-CD32a with single-expressed FcyRIIa, pcDNA3.1 (-)-CD32b with single-expressed FcyRIIb and IRES-CD32a+b eukaryotic expression plasmid with double-expressed FcyRIIa and FcyRIIb) was successfully constructed.4.Three types of recombinant expressing plasmid was transfered into BL-21 cells using lip-2000(named in three group:BHK-CD32a, BHK-CD32b and BHK-CD32a+b cell). Flow cytometry result showed 50% Expression rate of all 3 types of cells. We unified expression quantity of Receptor and lay the foundations of evaluating the effect of FcR in ADE effect.5.Titre of degue type II virus was detected by plaque experiment and result was 2x107PFU/ml. Depurated polyclonal antibody of prM protein was quantified by BCA method and result was 10mg/ml. Flow cytometry was used to detect virus infection rate and result showed high level ADE effect in positive-antibody-cell but low level in negative-antibody-cell. The peak of the infection rate of positive-antibody-cell is 8.95%.6.1mmune complex was used to infect differental Fc-receptor-expressed-BHK-21 cells. Flow cytometry result showed the same viral quantity(24.5%and 23.1%) in BHK-CD32a and BHK-CD32b cells. Viral quantity of BHK-CD32a+b(18.1%) was a little lower with statistical difference.Conclusion1. Polyclonal antibodies and a monoclonal antibody against prM with high antibody titers and specificity were obtained. After combination of DENV-2, the immune complex showed significant infection enhancement after infected THP-1 cells.2. The recombinant plasmid which contained Fc gama RⅡa and Fc gama RⅡb were constructed and transfected to BHK-21 cells to get three different kinds of BHK-21 cells expressed FcR.After infected by immune complex,we found that BHK-21 cells coexpressed with Fc gama RⅡa and Fc gama RⅡb had a litter lower infection rate than BHK-21 cells expressed Fc gama RⅡa or Fc gama RⅡb alone.As a result,it may be explained by the crosslinking of Fc gama RⅡa and Fc gama RⅡb to inhibit the infection.