BackgroundDengue disease is a leading cause of illness and death in the tropics and subtropics. Most severe cases occur among patients secondarily infected with a different dengue virus (DENV) serotype compared with that from the first infection, resulting in antibody-dependent enhancement activity (ADE). Our previous study generated the neutralizing human monoclonal antibody, D23-1B3B9 (B3B9), targeting the first domain II of E protein, which showed strong neutralizing activity (NT) against all four DENV serotypes. However, at sub-neutralizing concentrations, it showed ADE activity in vitro.MethodsIn this study, we constructed a new expression plasmid using the existing IgG heavy chain plasmid as a template for Fc modification at position N297Q by site-directed mutagenesis. The resulting plasmid was then co-transfected with a light chain plasmid to produce full recombinant IgG (rIgG) in mammalian cells (N297Q-B3B9). This rIgG was characterized for neutralizing and enhancing activity by using different FcγR bearing cells. To produce sufficient quantities of B3B9 rIgG for further characterization, CHO-K1 cells stably secreting N297Q-B3B9 rIgG were then established.ResultsThe generated N297Q-B3B9 rIgG which targets the conserved N-terminal fusion loop of DENV envelope protein showed the same cross-neutralizing activity to all four DENV serotypes as those of wild type rIgG. In both FcγRI- and RII-bearing THP-1 cells and FcγRII-bearing K562 cells, N297Q-B3B9 rIgG lacked ADE activity against all DENV serotypes at sub-neutralizing concentrations. Fortunately, the N297Q-B3B9 rIgG secreted from stable cells showed the same patterns of NT and ADE activities as those of the N297Q-B3B9 rIgG obtained from transient expression against DENV2. Thus, the CHO-K1 stably expressing N297Q-B3B9 HuMAb can be developed as high producer stable cells and used to produce sufficient amounts of antibody for further characterization as a promising dengue therapeutic candidate.DiscussionHuman monoclonal antibody, targeted to fusion loop of envelope domainII (EDII), was generated and showed cross-neutralizing activity to 4 serotypes of DENV, but did not cause any viral enhancement activity in vitro. This HuMAb could be further developed as therapeutic candidates.

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Background Dengue disease is a mosquito-borne infection caused by four dengue virus serotypes (DENV1-4). Secondary infections can produce flavivirus cross-reactive antibodies at sub-neutralizing levels. This phenomenon can significantly increase the severity of secondary infections via antibody-dependent enhancement (ADE). ADE activity is associated with a high risk of viral infection in immune effector cells, triggering cytokine cascade and activating the complement system, which lead to severe symptoms. Despite extensive studies, therapeutic antibodies, particularly fully human monoclonal antibodies, which can be an option for immune passive therapy, have not yet been discovered. Methodology/Principal Findings This study generated LALA-mutated human monoclonal antibody clone B3B9 (LALA-B3B9 HuMAb) that can neutralize all four DENV serotypes without enhancing viral activity. The number of infected cells obtained with the ADE assay was compared among wild-type antibody (B3B9), and modified Fc, LALA-B3B9 HuMAb, and N297Q (N297Q-B3B9), with or without complement proteins. Moreover, the therapeutic efficacy of these HuMAbs against ADE infection by competing with natural antibodies in patients with acute dengue was determined using the in vitro suppression-of-enhancement assay in K562 cells. The novel Fc-modified antibody LALA-B3B9 (Leu234Ala/Leu235Ala mutations) could have a therapeutic effect. Further, it exhibited neutralization properties against all dengue virus serotypes without triggering ADE activity at any antibody concentration. This outcome was similar to that of the previous Fc-modified N297Q-B3B9 antibody (N297Q mutation). Moreover, the effect of complement protein on enhancing and neutralizing activities was evaluated to assess unwanted inflammatory responses to these therapeutic antibodies. Results showed that the elimination of complement activity could reduce the severity of dengue. The activities of LALA-B3B9 and N297Q-B3B9 HuMAbs were complement-independent in all dengue virus serotypes. Conclusions LALA-B3B9 and N297Q-B3B9 HuMAbs can prevent the suppression-of-enhancement activity in K562 cells caused by human DENV2. Hence, they are promising candidates for dengue treatment.

Objective: To evaluate the expression of DNA plasmid-harboring modified antibody gene that produces neutralizing human monoclonal antibodies against four serotypes of dengue virus (DENV) without enhancing activity in BALB/c mice. Methods: We constructed pFUSE-based vectors (pFUSE_1G7C2_ hVH and pFUSE_1G7C2_hVL) containing genes encoding the variable domains of the heavy or light chain of the anti-dengue virus antibody 1G7C2, a human IgG1 that has been characterized for its neutralizing activity to DENV-1-4. Leucine (L) at positions 234 and 235 on the Fc CH2 domain in pFUSE_1G7C2_hVH was mutated to alanine (A) (LALA mutation) by site direct mutagenesis, and the new plasmid was termed pFUSE_1G7C2_hVH_LALA. An equal amount of pFUSE_1G7C2_hVL and 1G7C2_hG1-LALA plasmids were co-transfected into Chinese hamster ovary cells (CHO-K1) and a single dose of 100 μg 1G7C2_hG1-LALA plasmid was intramuscularly injected, followed by electroporation in BALB/c mice. The secreted 1G7C2_hG1-LALA antibodies in cell culture supernatant and mouse serum were examined for their biological functions, neutralization and enhancing activity. Results: The co-transfection of heavy- and light-chain 1G7C2_ hG1-LALA plasmids in CHO-K1 cells produced approximately 3 900 ng/mL human IgG and neutralized 90%-100% all four DENV, with no enhancing activity. Furthermore, the modified human IgG was produced more than 1 000 ng/mL in mouse serum on day 7 post plasmid injection and showed cross-neutralization to four DENV serotypes. Subsequently, antibody production and neutralization decreased rapidly. Nevertheless, the secreted neutralizing 1G7C2_ hG1-LALA in mouse serum demonstrated complete absence of enhancing activities to all DENV serotypes. Conclusions: These findings reveal that a new modified 1G7C2_ hG1-LALA expressing plasmid based on gene transfer is a possible therapeutic antibody candidate against DENV infection.