Trained laboratory personnel performed the anesthesia of mice via i.p. a significant reduction of viral load compared to isotype control (p?P?=?0.11) compared to 3G9-original, the significance was observed at 3 dpi (P?=?0.041), demonstrating faster viral clearance. These results indicated that this reduced ADE shown by these altered antibodies promotes their effectiveness in vivo and suggests that recombinant 3G9 antibodies are promising therapeutic agents. Competition assay using ELISA and ADE assays Our animal experiments were contamination models without ADE. Thus, it remained unclear whether the Fc-modified 3G9 could suppress contamination with ADE in vivo. An antibody highly competitive to 4G2 (low-avidity FLE antibody) has been reported to protect mice from lethal DENV contamination with ADE58. Since we had not established an in vivo ADE contamination model, competition assays were conducted. Competitive ELISA showed that 3G9 strongly competed with 4G2 but not with other mouse monoclonal antibodies (7F4 and 15C12, targeting the Tamibarotene central a part of domain name II and the A strand of domain name III, respectively) (Fig.?6A)20. 3G9 inhibited 4G2 binding by more than 50% when both were applied to the assay at the same concentration. Open in a separate windows Physique 6 Competition ELISA and ADE assay. (A) Competition ELISA. Mouse monoclonal antibodies 4G2, 7F4, or 15C12 at 1?g/ml were mixed with serially diluted 3G9 and incubated in a DENV-coated ELISA plate. The OD relative to that of the no competition well (without 3G9) is usually shown. Each data represents the average of two impartial experiments. (B) Competition ADE. 100?ng/ml of 4G2 and 1:640 diluted mouse serum, which showed the peak level of enhancement, were mixed with DENV-2 NGC and serially diluted 3G9-N297A. The number of infected cells relative to that of the Slit1 no competition wells is usually shown. Each data represents the average of two impartial experiments. It has been reported that this suppression of ADE caused by pre-existing antibodies in vitro could be an indicator of in vivo protection efficacy58. Thus, a competitive ADE assay was performed using 4G2 and DENV2-immunized mouse serum, which showed peak levels of ADE at 100?ng/ml and a 1:640 dilution, respectively (Fig.?3, Fig. S2). These dilutions Tamibarotene were used, therefore, for the subsequent competitive ADE assay. 3G9-N297A suppressed the peak ADE caused by 4G2 and D2-immunized mouse serum by 50% at only 30?ng/ml (Fig.?6B). A previous study indicated that an antibody that strongly reduced ADE contamination (>?50%) at 1000?ng/ml offered good therapeutic efficacy in an in vivo ADE model58. The competition intensities found in the current Tamibarotene study were higher than the threshold for in vivo protection quoted in the previous report, although direct comparison is impossible. Immunogenetic analysis Even though 3G9 is an FLE antibody, it showed high potency in terms of NT50 values and in vivo protection. Therefore, we analyzed the sequences of the 3G9 VH and VL regions using the IMGT tool to identify the closest VH and V germline genes. The results indicated that this VH gene was derived from IGHV3-23*02 and the V gene from IGLV7-46*01 (Table ?(Table5).5). Somatic hyper-mutation (SHM) rates of the VH and V genes were 14.3% and 7.1%, respectively. 3G9 showed distinct VH germline gene usage and the highest SHM rate of the VH regions than the HuMAbs generated in this study (Table S1). The high SHM rate of the VH regions indicated the levels of affinity-maturation during the secondary contamination50. The sequences of HuMAbs have been added in Supplementary Table S2. Table 5 Immunogenetic analysis of 3G9.
Heavy chainIGHV3-23*02IGHD3-16*01IGHJ4*0214.3% (14/98) AKLFGVGDSDGYLight chainIGLV7-46*01CIGLJ3*027.1% (7/98) LLSYGGGRPV Open in a separate windows The closest germline genes were determined using the IMGT tool. Discussion To date, no specific therapeutic agent against flavivirus contamination has been made available. Considering the current success of antibody therapy against respiratory syncytial computer virus, Ebola computer virus, and severe acute respiratory syndrome coronavirus 2 infections, antibody therapy for dengue is usually a promising target. DENV-neutralizing HuMAbs.