Human monoclonal single-chain antibodies specific to dengue virus envelope protein.

UNLABELLED: Dengue virus (DENV) infection is an arthropod-borne disease with increasing prevalence worldwide. Attempts have been made to develop therapeutic molecules for treatment for DENV infection. However, most of potentially therapeutic DENV monoclonal antibody was originated from mouse, which could cause undesirable effects in human recipients. Thus, fully human antibody is preferable for therapeutic development. Human single-chain variable fragments (HuScFv) with inhibitory effect to DENV infection were generated in this study. HuScFv molecules were screened and selected from the human antibody phage display library by using purified recombinant DENV full-length envelope (FL-E) and its domain III (EDIII) proteins as target antigens for biopanning. HuScFv molecules were then tested for their bindings to DENV particles by indirect ELISA and immunofluorescent microscopy. EDIII-specific HuScFv exhibited neutralizing effect to DENV infection in Vero cells in a dose-dependent manner as determined by plaque formation and cell ELISA. Epitope mapping and molecular docking results concordantly revealed interaction of HuScFv to functional loop structure in EDIII of the DENV E protein. The neutralizing HuScFv molecule warrants further development as a therapeutic biomolecule for DENV infection. SIGNIFICANCE AND IMPACT OF THE STUDY: No approved vaccine and specific drug for dengue virus (DENV) infection are available; thus, their developments are urgently required. The human single-chain variable antibody fragments (HuScFv) specific to DENV envelope (E) protein are potential to be developed as therapeutic biomolecules. HuScFv that bound specifically to recombinant full-length DENV E (FL-E) and its domain III (EDIII) were generated and testified for its inhibitory effect in DENV infection. EDIII-specific HuScFv inhibited DENV infection in a dose-dependent manner and has potential to be further developed as a therapeutic biomolecule for DENV infection.

The severity of clinical symptoms in ragweed-allergic patients is related to the extent of ragweed-induced complement activation in their sera.

We have previously reported a correlation between the extent of ragweed allergen (RWA)-induced in vitro serum complement activation and the symptom scores registered daily during the ragweed (RW)-blooming season in RW-allergic patients. The present study was performed in 22 15-17-year-old RW-allergic adolescents. Serum samples were incubated with 100 micrograms/ml RWA, and the generation of different complement activation products was measured by ELISA or RIA. Symptom scores were registered for 4 weeks during the RW-blooming season. The patients were divided according to the extent (low or high) of the generation of complement activation products, and symptom scores registered in the two groups were compared by two-way ANOVA. Significantly higher symptom scores were obtained in the high than in the low complement activation group (P values: 0.049 for C1rC1sC1inh, 0.022 for C3bBbP, 0.015 for C5b-9, 0.0001 for C3a, and 0.0008 for C5a). Similar results were obtained at the measurement performed in the sera obtained from the same patients half a year before the season (P values: 0.022 for C3bBbP, and 0.005 for C5b-9). These findings indicate that complement activation induced by the allergen may enhance the clinical symptoms of RW allergy.

Clinical significance of longitudinal complement measurements in recipients of bone marrow transplant.

Overall activity of classical complement pathway, C3 and C4 levels, level of circulating immune complexes and concentration of serum immunoglobulins were measured in 38 patients transplanted with HLA-identical bone marrow before and after transplantation for at least 4 years. Changes of complement parameters and their association with acute and chronic GVHD and with infections were analysed. A strong association was found between the development of chronic GVHD and hypercomplementaemia measured in 16 long-term survivors. Low pre-transplantation C4 activity was found to predict the development of severe acute GVHD. These findings indicate that longitudinal complement measurements may have clinical value in BMT patients.

Effect of conditioned media of acute myeloid leukemia blast cells on complement synthesis by cultured human cells of monocyte and hepatocyte origin.

The effect of conditioned media of 3-day cultures of blast cells from peripheral blood of 5 patients with acute myeloid leukemia (CM-AML) was studied on the synthesis of C2, factor B (Bf) and C1 esterase inhibitor (C1-INH) by human monocyte-macrophage cultures and HepG2 hepatoma cell line. The level of C2 in the culture supernatants was measured by immune hemolysis, those of Bf and C1-INH by ELISA. CM-AML was added to the monocyte cultures on day 3 and replaced by culture fluid on day 6. Compared to the control cultures, CM-AML significantly increased C2 and Bf levels and slightly decreased C1-INH levels in the culture fluids on day 6. On day 9, Bf synthesis enhancement still could be observed but C2 and C1-INH levels did not significantly differ from those of the control. CM-AML significantly increased the synthesis of factor B by the HepG2 cells too. A strong correlation was found between the results of the Bf protein and RNA determinations, which means that the supernatants of AML blasts affect the gene expression of factor B at a pretranslational level. The selective complement synthesis modifying effect of CM-AML was not due to interferons (IFN) because neither IFN-alpha nor IFN-gamma could be detected in these conditioned media. The present findings indicate that the hypercomplementemia observed in AML patients can be due to unknown factor(s) produced by leukemic blast cells.

FN-C1q and C1 INH C1r-C1s complexes as indicators of complement activation in patients with chronic lymphocytic leukaemia.

We have previously found low levels of C1 and C4 INH in the sera of chronic lymphocytic leukaemia (CLL) patients. Hypocomplementaemia was supposed to be the consequence of a permanent activation of the classical pathway. We have compared the levels of C1 INH-C1rC1s and C1q-FN complexes in the sera of 95 CLL patients and 100 healthy controls, because these complexes are known to be formed in the early stage of classical pathway activation. A significant increase in the level of both types of complexes was found in sera of CLL patients as compared to the controls. These findings support the assumption that the classical complement pathway is activated in the patients with CLL.

Serum complement C2 levels in patients suffering from cystic fibrosis (CF).

Serum C2 complement levels were measured in 17 children suffering from CF, 17 with obstructive bronchitis, and 7 control children. No correlation was found between the C2 level and the clinical stage in Shwachman score, the HLA B7 or B18 antigens and the ventilation functional parameters. The mean serum C2 complement level did not differ in the three investigated groups, but in 5 of the 17 CF patients the serum C2 was diminished according to the possibility of C2 complement heterozygosity. The CF patients with diminished C2 complement levels exhibited HLA B7, B12 or B35 antigens. The serum C2 complement levels were significantly higher in the HLA B18 antigen-carrier CF homozygotes.