Neutralization of antibody-enhanced dengue infection by VIS513, a pan serotype reactive monoclonal antibody targeting domain III of the dengue E protein

<div><p>Dengue virus (DENV) infection imposes enormous health and economic burden worldwide with no approved treatment. Several small molecules, including lovastatin, celgosivir, balapiravir and chloroquine have been tested for potential anti-dengue activity in clinical trials; none of these have demonstrated a protective effect. Recently, based on identification and characterization of cross-serotype neutralizing antibodies, there is increasing attention on the potential for dengue immunotherapy. Here, we tested the ability of VIS513, an engineered cross-neutralizing humanized antibody targeting the DENV E protein domain III, to overcome antibody-enhanced infection and high but brief viremia, which are commonly encountered in dengue patients, in various <i>in vitro</i> and <i>in vivo</i> models. We observed that VIS513 efficiently neutralizes DENV at clinically relevant viral loads or in the presence of enhancing levels of DENV immune sera. Single therapeutic administration of VIS513 in mouse models of primary infection or lethal secondary antibody-enhanced infection, reduces DENV titers and protects from lethal infection. Finally, VIS513 administration does not readily lead to resistance, either in cell culture systems or in animal models of dengue infection. The findings suggest that rapid viral reduction during acute DENV infection with a monoclonal antibody is feasible.</p></div

Identification of VIS513 escape mutants.

<p>A. Neutralization profile of a titration of 14c10 with DENV1 population passaged in presence of 14c10 at 100 μg/ml, 10 μg/ml, or trastuzumab at 150 μg/ml for 5 passages. B-C. Neutralization profile of a titration of VIS513 with DENV3 (passage 7) (B) or DENV4 (passage 5) (C) passaged in the presence of indicated concentrations of VIS513 or trastuzumab. For DENV4, passaging in the presence of 150 μg/ml VIS513 yielded insufficient virus for subsequent neutralization assays and hence could not be assayed. The solid lines and error bars represent mean ± SEM of experiment performed in triplicate.</p

Summary of escape mutations observed from cell culture studies.

<p>Summary of escape mutations observed from cell culture studies.</p

Structural analysis of escape mutations.

<p>A. Molecular interactions of E-DENV1 protein (shown in rainbow color cartoon) are shown with the 14c10 antibody (shown in transparent surface and cartoon diagram with heavy and light chains shown in green and cyan color, respectively). In the complex, the Lys136 residue of the E-DENV1 protein is found in the vicinity of the HCDR3 loop of the mAb. Mutation of Lys136 to a Glu is expected to cause loss of mAb binding due to the change in charge of the residue. B. Molecular interactions of Lys308/310 of EDIII-DENV3/4 protein (shown in magenta cartoon) are shown with VIS513 (shown in transparent surface and cartoon diagram with heavy and light chains shown in green and cyan color, respectively). The Lys308 of E-DENV3 or Lys310 of E-DENV4 residues are the same and it is found to form hydrogen bonding and salt bridges with the VIS513 heavy chain residues Asp52 and Glu54 (left panel). The right panel shows the modeling of mutation Lys308Arg. C. Modeling of position E311 of E-DENV4 region found to be mutated in virus from mouse samples. A hydrogen bond with the antibody molecule is shown with dotted line.</p

PRNT₅₀ (ng/mL) values for anti-DENV antibodies.

<p>PRNT₅₀ (ng/mL) values for anti-DENV antibodies.</p

Amino acid alterations identified in H77 HCV E1/E2 envelope glycoproteins after initial viral infection in untreated (#1) and treated (#2) chimpanzees.

<p>Amino acid alterations identified in H77 HCV E1/E2 envelope glycoproteins after initial viral infection in untreated (#1) and treated (#2) chimpanzees.</p

Amino acid alterations in E1/E2 envelope glycoproteins following viral rebound in treated chimpanzee B.

<p>Amino acid alterations in E1/E2 envelope glycoproteins following viral rebound in treated chimpanzee B.</p

HCV1 does not bind mutant E2 envelope glycoprotein produced in serum-containing medium.

<p>A vector encoding wild-type E2₆₆₀ was transfected into CHO cells grown in defined media (<b>A</b>) or serum-containing medium (<b>B</b>). In addition, a vector encoding E2₆₆₀–N415K was also transfected into CHO cells grown in defined media (<b>C</b>) or serum-containing media (<b>D</b>). All proteins were purified from culture supernatant using nickel-affinity chromatography, quantified, assessed for purity and coated on ELISA plates. Varying dilutions of HCV1 (red circles) was applied to each protein and binding assessed. In addition, varying dilutions of 96-2 (epitope II specific, blue squares), irrelevant antibody (blue triangles) and a mouse antibody specific to the (His)₆ tag (blue crosses) at the C-terminus of E2₆₆₀ were assayed to control for protein coating and non-specific binding. For HCV1, 96-2 and irrelevant mAb, bound antibody was detected employing goat anti–human secondary antibody conjugated to alkaline phosphatase, followed by development with PNPP substrate, and the absorbance at 405 nm for each dilution was plotted. For the mouse anti-(His)₆ antibody, goat anti-mouse secondary antibody was used.</p

Amino acid alterations in E1/E2 envelope glycoproteins following viral rebound in treated chimpanzee #1.

<p>Amino acid alterations in E1/E2 envelope glycoproteins following viral rebound in treated chimpanzee #1.</p

Serum from poorly-responding chimpanzees inhibits HCV1 neutralization of H77–HCVpp.

<p><b>A</b>. Serum from the three chronically-infected chimpanzees, collected prior to HCV1 infusion, and serum from an uninfected chimpanzee were mixed at varying dilutions (1∶16 to 1∶1024) with a constant concentration (100 nM) of HCV1 or irrelevant human antibody. The mixture was incubated with H77 genotype 1a HCVpp and applied to Hep3B cells for 72 hours. Bright-Glo luciferase detection reagent was used to quantitate infection of the luciferase containing HCVpp. Light output was quantified and the counts per second (CPS) for the HCV1 sample was divided by the CPS for the irrelevant antibody sample for each dilution of the serum. The CPS quotient as related to each serum dilution was plotted. A value of 1 would represent no neutralization of HCVpp with HCV1 and a value approaching 0 would signify that HCV1 neutralized 100% of the HCVpp. <b>B</b>. E2₆₆₀ envelope glycoprotein ELISA was performed on varying dilutions of chronically-infected chimpanzee serum collected prior to HCV1 infusion. Bound antibody was detected employing goat anti-human secondary antibody conjugated to alkaline phosphatase, followed by development with PNPP substrate, and the absorbance at 405 nm for each dilution was plotted.</p