Neutralizing and non-neutralizing monoclonal antibodies against dengue virus E protein derived from a naturally infected patient

Abstract

Background: Antibodies produced in response to infection with any of the four serotypes of dengue virus generally provide homotypic immunity. However, prior infection or circulating maternal antibodies can also mediate a non-protective antibody response that can enhance the course of disease in a subsequent heterotypic infection. Naturally occurring human monoclonal antibodies can help us understand the protective and pathogenic roles of the humoral immune system in dengue virus infection.

Results: Epstein-Barr Virus (EBV) transformation of B cells isolated from the peripheral blood of a human subject with previous dengue infection was performed. B cell cultures were screened by ELISA for antibodies to dengue (DENV) envelope (E) protein. ELISA positive cultures were cloned by limiting dilution. Three IgG1 human monoclonal antibodies (HMAbs) were purified and their binding specificity to E protein was verified by ELISA and biolayer interferometry. Neutralization and enhancement assays were conducted in epithelial and macrophage-like cell lines, respectively. All three HMAbs bound to E from at least two of the four DENV serotypes, one of the HMAbs was neutralizing, and all were able to enhance DENV infection.

Conclusions: HMAbs against DENV can be successfully generated by EBV transformation of B cells from patients at least two years after naturally acquired DENV infections. These antibodies show different patterns of cross-reactivity, neutralizing, and enhancement activity.

Figure 1

Validation of the ConA ELISA. DENV E protein from serotypes 1-4 derived from DENV infected LLC-MK-2 cells was immobilized by ConA. Reactivity with patient serum and dengue negative human serum diluted in PBS/Tween as well as MMAbs 4G2 and 3H5 was measured by detection with HRP anti-human and anti-mouse IgG, respectively. MMAb 4G2 were tested at 5 ug/ml. Mouse MAb 3H5 was tested as undiluted culture fluid. Negative controls of uninfected LLC-MK-2 culture fluid and Vaccinia ADA were also tested.

Figure 2

Serial five fold dilutions of each HMAb. Starting at 10,000 ng/ml, Human and Mouse MAbs were tested by ConA ELISA against each dengue virus serotype. A HMAb 2.3D. B 3.6D. C 4.8A. D. 4G2 (dilutions were started at 50,000 ng/ml). E. 3H5 (tested as dilutions of culture fluid).

Figure 3

Cross-competition between HMAbs. A cross-competition assay was performed to determine whether the three HMAbs and MMAb 4G2 recognized overlapping or non-overlapping sties on DENV-1 E protein. Purified MMAb 4G2 and HMAbs were incubated with biotinylated HMAbs, washed, and the presence of biotinylated antibodies was detected using streptavidin.

Figure 4

Virus neutralization by patient serum. Approximately 100 FFU of virus were incubated with serial dilutions of heat inactivated patient serum and then allowed to infect LLC-MK-2 cells. Following an incubation period, virus foci were detected using specific mouse anti-DENV E protein MMAb E60.

Figure 5

Virus neutralization by HMAbs. Approximately 100 FFU of virus were incubated with serial dilutions of purified monoclonal antibody. Virus mixtures were allowed to infect LLC-MK-2 cells and then incubated at 37C. Virus foci were detected using anti-DENV E protein MMAb E60. Results are expressed as pooled data from two independent experiments with three replicates each. A HMAb 2.3D. B 3.6D. C 4.8A. D 4G2. E 3H5.

Figure 6

Enhanced DENV uptake by K562 cells in the presence of HMAbs. Human K562 cells were infected with DENV-1 in the presence of increasing concentrations of the antibodies. RNA was extracted from cell lysates and quantitative RT-PCR was performed with a DENV-1 specific primer pair specific for the NS1 region as a measure of infectious equivalents. A HMAb 2.3D. B 3.6D. C 4.8A.

Figure 7

HMAb measured by biolayer interferometry. Label free, real time binding analysis between the HMAbs 2.3D, 3.6D and 4.8A and purified, recombinant E protein from each DENV serotype to measure binding affinity was performed. The affinities of 2.3D and 3.6D for DENV 3 or 4 E proteins could not be measured due to Ab aggregation at the higher concentrations needed to detect lower affinity binding. A: Association rate constants (kon) for Ab:E protein interactions. B: Dissociation rate constants (koff) for Ab:E protein interactions. C: Equilibrium dissociation constants (Kd) for HMAb:E protein complexes.