A Mouse Monoclonal Antibody against Dengue Virus Type 1 Mochizuki Strain Targeting Envelope Protein Domain II and Displaying Strongly Neutralizing but Not Enhancing Activity

Abstract

Dengue fever and its more severe form, dengue hemorrhagic fever, are major global concerns. Infection-enhancing antibodies are major factors hypothetically contributing to increased disease severity. In this study, we generated 26 monoclonal antibodies (MAbs) against the dengue virus type 1 Mochizuki strain. We selected this strain because a relatively large number of unique and rare amino acids were found on its envelope protein.

Expression of enhancing-activity-free neutralizing antibody against dengue type 1 virus in plasmid-inoculated mice

“…Specifically, 50% plaque reduction was achieved at approximately 10 ng/ml 7F4 in a conventional neutralization test using Vero cells, whereas no enhancing activity was seen, even at low concentrations (0.01-1 ng/ml) in our enhancing antibody assay system using Fc␥R-bearing cells, such as K562, U937, or HL-60 cells [16]. The target epitope of 7F4 is located in domain II of the envelope (E) protein of DENV-1 [27].…”

“…Briefly, microplates sensitized with rabbit hyperimmune serum against DENV-1 were incubated sequentially with the corresponding DENV-1 antigen, mouse serum samples diluted 1:30, alkalinephosphatase-conjugated anti-mouse IgG, and p-nitrophenyl phosphate. H (pFUSE-CHIg-mG2b or pFUSE-CHIg-mG3 for producing IgG2b or IgG3 subclasses, respectively) or L chains (pFUSECLIg-mK for the chain) were constructed previously [27]. Mixtures of equal amounts of 7F4-gene-containing plasmids based on pFUSE-CHIg-mG2b and pFUSE-CLIg-mK (p7F4G2b) or pFUSE-CHIg-mG3 and pFUSE-CLIg-mK (p7F4G3) were used for transfection.…”

“…Antibody expression vector (pFUSE)-based plasmids containing the gene encoding the variable region of MAb 7F4 and the constant region of the H chain (IgG2b or IgG3) or L chain () were constructed previously [27] (Fig. 1A).…”

“…We previously generated various monoclonal antibodies (MAbs) in mice immunized with the Mochizuki strain of DENV-1 [27]. Among these, the 7F4 antibody, an IgG3 subclass antibody, showed high neutralizing activity but no detectable enhancing activity.…”

Dengue-Immune Humans Have Higher Levels of Complement-Independent Enhancing Antibody than Complement-Dependent Neutralizing Antibody

“…Before the antibody assays, the sera were heat-inactivated at 56 C for 30 min. Mouse monoclonal antibodies (MAbs) against DENV-1 (D1-IV-7F4, D1-V-3H12) have been reported (14). Another MAb against DENV-1 (D1-IV-1C8) was obtained when the MAbs described above were generated.…”