Recombinant Human Anti-E protein Antibody (E60) (CAT#: FAMAB-0116CQ)

Figure 1 Anti-DENV MAbs are therapeutic following a virus-only or antibody-enhanced lethal infection.

A. Ribbon diagram of the DENV2 E protein homodimer (PDB ID code 1OAN). EDI is red, EDII is yellow and EDIII is blue. The epitopes targeted by MAbs include the fusion loop (green), dimer interface (white), C-C′ loop (orange) and A strand (magenta). B. AG129 mice were administered a lethal dose of DENV2 D2S10 and 24 hours later were treated with 20 µg of modified MAbs (n = 5 per group from 2 independent experiments). C. AG129 mice were administered an enhancing dose of polyvalent DENV1-immune mouse serum, infected with DENV2 D2S10, and 24 hours later treated with 20 µg of modified MAbs. (n = 3–19 per group from at least 2 independent experiments for each modified MAb). A Kaplan-Meier survival curve is shown (B–C), and log-rank analysis was used for statistical comparison.

Williams, K. L., Sukupolvi-Petty, S., Beltramello, M., Johnson, S., Sallusto, F., Lanzavecchia, A., ... & Harris, E. (2013). Therapeutic efficacy of antibodies lacking FcγR against lethal dengue virus infection is due to neutralizing potency and blocking of enhancing antibodies. PLoS pathogens, 9(2), e1003157.

Figure 2 Modified MAbs targeting the fusion loop epitope prevent antibody-enhanced lethal DENV infection in relation to their neutralizing potency.

A. AG129 mice were administered a lethal dose of DENV2 D2S10 and 24 hours later were treated with 20 µg of modified MAb targeting the fusion loop (n = 3–6 per group from 2 independent experiments). B. AG129 mice were administered an enhancing dose of polyvalent DENV1-immune mouse serum, infected with DENV2 D2S10, and 24 hours later treated with 20 µg of modified MAbs (n = 3–6 per group from 2 independent experiments). C. AG129 mice were administered an enhancing quantity (20 µg) of 4G2 MAb, infected with DENV2 D2S10, and 24 hours later treated with 20 µg of modified MAb (n = 3–9 per group from at least 2 independent experiments). A Kaplan-Meier survival curve is shown, and log-rank analysis was used for statistical comparison.

Williams, K. L., Sukupolvi-Petty, S., Beltramello, M., Johnson, S., Sallusto, F., Lanzavecchia, A., ... & Harris, E. (2013). Therapeutic efficacy of antibodies lacking FcγR against lethal dengue virus infection is due to neutralizing potency and blocking of enhancing antibodies. PLoS pathogens, 9(2), e1003157.

Figure 3 Fusion loop- and A strand-specific modified MAbs can compete for binding with fusion loop-specific MAbs of lesser neutralizing potency.

A. Fusion loop-specific mouse MAb 4G2 was incubated at 1 µg/mL with MAbs E60, 87.1 or E87 at 10, 1 or 0.1 µg/mL human MAb prior to addition to DENV2-virion coated plates (for each MAb concentration, data is represented as mean +/− SEM). Anti-mouse, Fc-specific secondary MAb was then added, followed by PNPP substrate. Optical density (OD) values are shown on the y-axis and were calculated after subtracting the average background (binding of mouse Fcγ-chain specific secondary antibody in the absence of 4G2) from the raw OD. Statistically significant differences in 4G2 binding across the different human MAb concentrations were calculated using a Kruskal-Wallis analysis from triplicate values within each experiment. This data shown is representative of seven independent experiments. B–D. MAb 4G2 was pre-mixed with MAb E60 N297Q, MAb 87.1 LALA or MAb E87 N297Q in ratios of 95% 4G2/5% modified MAb (B), 85% 4G2/15% modified MAb (C), or 75% 4G2/25% modified MAb (D). For each 4G2/modified MAb mixture, a Gaussian distribution was used to fit the enhancement curve. The area under the curve (AUC) was calculated for each curve, and relative infection was expressed by dividing the AUC in the presence of modified MAbs by the AUC measured with 4G2 (no modified MAb) only. The data displayed are the average of three to seven independent experiments +/− SEM. Comparisons between the MAb combinations E87 N297Q/4G2 and E60 N297Q/4G2 or 87.1 LALA/4G2 were performed using a Kruskal-Wallis test. E. AG129 mice (n = 3–6 per group from one or two experiments) were administered an enhancing quantity (20 µg) of 4G2 MAb, infected with DENV2 D2S10, and 24 hours later treated with 20 µg of modified MAb. A Kaplan-Meier survival curve is shown, and log-rank analysis was used for statistical comparison.

Williams, K. L., Sukupolvi-Petty, S., Beltramello, M., Johnson, S., Sallusto, F., Lanzavecchia, A., ... & Harris, E. (2013). Therapeutic efficacy of antibodies lacking FcγR against lethal dengue virus infection is due to neutralizing potency and blocking of enhancing antibodies. PLoS pathogens, 9(2), e1003157.

Figure 4 A high ratio of modified to non-modified MAb is necessary to prevent enhancement in vitro and in vivo.

A–B. Non-modified and modified MAbs E60/E60 N297Q (A) and 87.1/87.1 LALA (B) were pre-mixed at ratios of 100% intact MAb, 75% intact:25% modified MAb, 50% intact:50% modified MAb, 25% intact:75% modified MAb and 10% intact:90% modified MAb. The data is plotted as the average of duplicate values where the absolute percent infection of K562 cells is shown on the y-axis. This data is representative of two or three independent experiments. C. AG129 mice (n = 3 per experimental group and n = 5 for non-treated control group) were administered a polyvalent DENV1-immune enhancing mouse serum, infected with DENV2 D2S10, and 24 hours later treated with a total of 20 µg of E60/E60 N297Q MAbs in the same combinations tested in vitro in (A). A Kaplan-Meier survival curve is shown, and log-rank analysis was used for statistical comparison.

Williams, K. L., Sukupolvi-Petty, S., Beltramello, M., Johnson, S., Sallusto, F., Lanzavecchia, A., ... & Harris, E. (2013). Therapeutic efficacy of antibodies lacking FcγR against lethal dengue virus infection is due to neutralizing potency and blocking of enhancing antibodies. PLoS pathogens, 9(2), e1003157.

Figure 5 In vitro suppression-of-enhancement assay predicts therapeutic efficacy of MAbs in vivo with enhancing polyvalent DENV-immune serum from mice.

A. The peak enhancing titer (PENT = 1∶180) for DENV1-immune mouse serum was determined in K562 cells. B. DENV1-immune mouse serum was diluted 1∶180 (PENT) and incubated with modified MAbs at six 2-fold dilutions beginning at 2,000 ng/ml. Relative infection was calculated by dividing the percent infection in the presence of modified MAbs by the percent infection measured with mouse DENV1-immune serum alone. The data displayed are the average of duplicate values and are representative of four independent experiments. A † indicates modified MAbs that are statistically therapeutic in vivo following mouse DENV1-enhanced, lethal DENV2 infection. C. The average infection across four experiments at 1,000 ng/ml of modified MAb (mean +/− SEM shown for each MAb). P<0.04 was obtained when comparing the average relative infection values for therapeutic to non-therapeutic MAbs using a Wilcoxon rank-sum analysis. The solid line indicates relative infection of 0.5 (50% infection).

Williams, K. L., Sukupolvi-Petty, S., Beltramello, M., Johnson, S., Sallusto, F., Lanzavecchia, A., ... & Harris, E. (2013). Therapeutic efficacy of antibodies lacking FcγR against lethal dengue virus infection is due to neutralizing potency and blocking of enhancing antibodies. PLoS pathogens, 9(2), e1003157.

Figure 6 In vitro suppression-of-enhancement assay correlates with therapeutic efficacy of MAbs in vivo with enhancing polyvalent DENV-immune serum from humans.

A. The PENT (1∶540) for DENV4-immune human serum was determined in K562 cells. B. DENV4-immune human serum diluted 1∶540 was incubated with modified MAbs at 1,000 ng/ml. Relative infection was calculated. The data displayed are combined from five independent experiments, and the mean +/− SEM is displayed for each MAb. A sign rank test was used to determine whether relative infection with each modified MAb was significantly lower than relative infection of 0.5 (50% infection), * P<0.05, ** P<0.08. C. AG129 mice (n = 3 per experimental group and n = 6 for non-treated control group) were administered an enhancing dose of DENV4-immune human serum, infected with DENV2 D2S10, and 24 hours later treated with 20 µg of modified MAbs. A Kaplan-Meier survival curve is shown, and log-rank analysis was used for statistical comparison.

Williams, K. L., Sukupolvi-Petty, S., Beltramello, M., Johnson, S., Sallusto, F., Lanzavecchia, A., ... & Harris, E. (2013). Therapeutic efficacy of antibodies lacking FcγR against lethal dengue virus infection is due to neutralizing potency and blocking of enhancing antibodies. PLoS pathogens, 9(2), e1003157.