To generate IgG 1A5 D, containing the 9-aa deletion, two DraIII sites at positions 1053 and 1080 were 1st introduced to remove the intervening 27 nucleotides, and then the original splice site sequence was restored having a QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Sequence Analysis of mRNA. recognized chimpanzeeChuman chimeric IgG1 mAbs capable of neutralizing or binding to one or more DENV serotypes (14, 15). Cross-reactive IgG 1A5 neutralizes DENV-1 and DENV-2 more efficiently than DENV-3 and DENV-4, and type-specific IgG 5H2 neutralizes DENV-4 at a high titer (14, 15). Analysis of antigenic variants offers localized the IgG 1A5 binding site to the conserved fusion peptide in E (11). Therefore, IgG 1A5 shares many characteristics with the cross-reactive antibodies recognized in flavivirus infections. We investigated the ability of IgG 1A5 to mediate enhancement of DENV replication in monocyte-derived cell lines and in juvenile rhesus monkeys after passive transfer. We also explored strategies to reduce ADE by mutational analysis of the key constructions in the Fc of IgG JI051 1A5. A 9-aa deletion in the N terminus of Fc was identified as responsible for total abrogation of DENV ADE but recognized from the viral yield. IgG 1A5-mediated enhancement of DENV-4 illness in main monocytes from juvenile rhesus monkeys was also analyzed. At a MOI of 1 1 or 10 and in the presence of dengue-negative human being serum, 1% of the monocytes were infected with DENV-4. The number of infected cells recognized by circulation cytometry reached 31 1.2%, when IgG 1A5 was added at 5 g/ml (Fig. 2shows the result of common DENV-4 viremia titers from days 2C10 for each group of monkeys. The viremia titers on these days were not significantly different between the monkey group that received 18 mg/kg of IgG 1A5 and the monkey group that received PBS. By comparison, a significant difference in the viremia titer in all monkey organizations was observed for days 3C6 after challenge ( 0.05; KruskalCWallis test). Based on the analysis of these four days, quantitative PCR recognized a mean maximum viremia titer of 0.76 log10 FFU/ml in the control group. The mean viremia titer improved from 0.58 to 2.76 log10 FFU/ml in the organizations, as antibody concentration decreased from 18 to 0.22 mg/kg (Table 1). The viremia titer improved 15- and 8-fold in the monkey organizations that received 6 and 2 mg/kg IgG 1A5, respectively, compared with that observed in the control group ( 0.05; MannCWhitney test). The monkey organizations given 0.67 and 0.22 mg/kg IgG 1A5 had nearly 56- and 100-fold raises in viral titers, respectively, a highly significant increase compared with that observed in the control group ( 0.001; MannCWhitney test). Open in a separate windows Fig. 3. ADE of DENV-4 illness in juvenile rhesus monkeys passively given JI051 with IgG 1A5. ( 0.05 (MannCWhitney test). , 0.001 (MannCWhitney 0.05; KruskalCWallis test). ?Mean peak viremia titer was calculated on days 4 and 5 after infection ( 0.05). The viremia titers of infected monkeys were also determined by FFU assay. Viremia was recognized on days 3C8 after challenge in the control group but not in the monkey organizations that received 18 and 6 mg/kg of IgG 1A5 (Fig. 3 0.05; KruskalCWallis test). The mean viremia titer in the monkey organizations that received 0.67 and 0.22 mg/kg of antibody increased 36- and 165-fold, respectively ( 0.05; MannCWhitney test) (Table 1). The time of peak viremia was delayed 2C3 days in the monkey group that received the highest dose of IgG 1A5 compared JI051 with the monkey organizations that received lower doses of antibody or PBS. The high antibody concentration might have reduced DENV-4 replication and selected for escape variants in these monkeys. The latter probability was ruled out by sequencing the E-specific DNA amplified from viremic samples on days 7, Rabbit Polyclonal to RhoH 8, and 9 after challenge, because no mutation was found in the sequence. DENV-4 illness in monkeys was also confirmed by sero-analysis 6 weeks after challenge. Semiquantitative analysis by radio-immunoprecipitation exposed the levels of anti-NS1 antibody, an indirect measure of the degree of DENV replication, were significantly higher in the monkey organizations that received antibody, except for the group that received 18 mg/kg, compared with that of the control group (= 0.049) (data not shown). Therefore, analysis JI051 of anti-NS1 antibodies also supported IgG 1A5-mediated enhancement of DENV-4.