DMAb optimization and in vitro characterization

The expression of human IgG antibodies from DNA-based vectors has briefly been explored in the past27 and resulted in low levels of serum-detectable antibodies in vivo. However, subsequent genetic optimizations to DNA plasmids and accompanying delivery systems, particularly EP, have resulted in increased expression of desired proteins28. With this in mind, we systematically optimized DMAb DNA through the creation of multiple iterations of two- and single-plasmid antibody-encoding DNA cassettes, with the aim of increasing human IgG production from DMAb in vivo (Supplementary Fig. 1). Dosage studies of two-plasmid DMAb delivery in C57BL/6 mice showed a dose-dependent increase in serum-detectable human IgG levels and a comparison between two- and single-plasmid DMAb constructs revealed a slight increase in expression from the single-plasmid condition (Supplementary Fig. 1). Ultimately, we designed and constructed highly optimized DNA plasmids encoding the heavy and light chains of the anti-DENV antibody DV87.1, a human IgG1 mAb that has been well characterized for its ability to neutralize DENV1–314. Two optimized plasmids were constructed: pDVSF-3 WT, which encodes for the heavy and light chains of DV87.1 and pDVSF-3 LALA, which encodes for an Fc region-modified version of DV87.1 with abrogated FcγR binding by way of two leucine-to-alanine (LALA) mutations in the CH2 region29 that have been shown to eliminate antibody-dependent enhancement14. In order to express a full-length antibody from a single open reading frame, the heavy and light chain genes were separated by a furin cleavage site and a P2A self-processing peptide. Each transgene was genetically optimized, synthesized and subcloned into a modified pVax1 mammalian expression vector (Fig. 1a). The plasmids were transfected into human embryonic kidney (HEK) 293T cells and secreted antibody levels in the supernatant were quantified after 48 hours by enzyme-linked immunosorbent assay (ELISA) (Fig. 1b). Both pDVSF-3 WT and pDVSF-3 LALA resulted in 600 ng/mL of human IgG, confirming that the plasmids can express human IgG and that the LALA mutation has no effect on antibody expression levels in vitro. To confirm proper antibody assembly, DVSF-3 and DVSF-3 LALA antibodies were collected from supernatants of transfected HEK293T cells and separated by SDS-PAGE gel for Western blot analysis (Fig. 1c). The heavy and light chain proteins were at their expected molecular weights, suggesting proper protein cleavage and antibody assembly.

Figure 1 In vitro expression of human anti-DENV neutralizing mAbs by DMAb. (a) Schematic illustration of DNA plasmid used for DMAb; antibody heavy and light chain sequences are separated by a combination of furin and 2A cleavage sites. (b) ELISA quantification analysis of human IgG in supernatants of pDVSF-3 WT- or LALA-transfected 293T cells. The data displayed are the mean of triplicate values +/− standard error of the mean (SEM) and are representative of three independent experiments. (c) Western blot analysis of pDVSF-3 WT-transfected 293T supernatants containing DVSF-3 WT. Antibodies were purified by Protein A spin columns and separated by SDS-PAGE under reducing (left) and non-reducing (right) conditions.(d) Vero cells were either uninfected (Mock) or infected by DENV1, 2, 3, or 4, then fixed, permeabilized and stained with supernatants of pDVSF-3 WT- or LALA-transfected 293T cells. The data displayed are representative of two independent experiments. Full size image

To assess the biological activity of the antibodies, we first performed a binding ELISA assay that measures whether the antibody-containing supernatant can bind to recombinant DENV1-3 E proteins. The supernatants of HEK293T cells that secreted either DVSF-3 WT or DVSF-3 LALA antibodies were able to recognize DENV1–3 E proteins, while DENV4 went unrecognized, as expected (Supplementary Fig. 2). Additionally, DVSF-3 WT- and DVSF-3 LALA-containing supernatants were able to stain Vero cells infected with DENV1–3, whereas Vero cells infected with DENV4 were not stained by the supernatants (Fig. 1d). Importantly, DVSF-3 WT enhanced DENV infection of FcγR-bearing human K562 cells, whereas DVSF-3 LALA had no such ADE activity in vitro (Supplementary Fig. 2).

Dengue DMAb delivery of DVSF-3 LALA protects against enhanced dengue disease in mice

In order to investigate antibody production kinetics in vivo, we determined the duration of DNA plasmid-encoded human IgG expression in nude mice, which would model antibody expression in an immune-accommodating host. The mice were injected intramuscularly with 100 μg of a DNA plasmid encoding another human IgG1 anti-DENV antibody, DVSF-1 WT (derived from DV82.11, a human IgG1 mAb that targets the DII fusion loop of the E protein and has been well characterized for its ability to neutralize DENV1–414), followed immediately by EP. Human IgG concentrations in the serum were detectable within 5 days of injection, with peak levels of ~1000 ng/mL at two weeks post-injection (Fig. 2a, left panel). Duration of human IgG expression lasted at least 19 weeks (Fig. 2a, right panel), showcasing the sustained expression levels attainable with DNA plasmids. Given that the mouse DENV challenge model uses mice from the 129/Sv background, we sought to determine whether the antibody-encoding DNA plasmid constructs could produce serum-detectable levels of DVSF-3 WT or LALA in this background strain. Serum from 129/Sv mice receiving either pDVSF-3 WT or pDVSF-3 LALA showed comparable human IgG levels (Fig. 2b) and stained Vero cells infected with DENV1–3 (Fig. 2c). Additionally, whereas naïve sera was unable to neutralize virus in a neutralization assay, both WT and LALA-containing serum were capable of neutralizing DENV1-3 (Fig. 2d).

Figure 2 DMAb results in long-term expression of neutralizing DENV antibodies in mouse serum. (a) Total serum-detectable levels of human IgG was measured by ELISA after a single intramuscular injection of DNA plasmid encoding the anti-DENV human IgG antibody DVSF-1 into Foxn1/NuJ immunodeficient mice. Human IgG levels between weeks 0–4 (left; data displayed are mean of duplicate values each animal +/−SEM) and at week 19 (right; error bars display the mean of duplicate values from five animals +/− SEM). Each line (left) or dot (right) represents an individual mouse (n = 5 mice). (b) Total human IgG in serum was measured by ELISA after intramuscular injection of pDVSF-3 WT or pDVSF-3 LALA plasmids in 129/Sv mice (n = 4–5 mice per group, data displayed are the mean +/− SEM of each group’s animals and are representative of two independent experiments). (c) Vero cells were either uninfected (Mock) or infected by DENV1, 2, 3, or 4, then fixed, permeabilized and stained with 129/Sv mouse serum taken at days 0 or 7 post-DNA injection of either pDVSF-3 WT, pDVSF-3 LALA or pVax empty vector (n = 5 mice per group, data representative of two independent experiments).(d) Neutralization was assessed by incubating DENV1, 2, 3, or 4 with serial dilutions of 129/Sv mouse serum taken at day 7 post-DNA injection of either pDVSF-3 WT or pDVSF-3 LALA (n = 5 mice per group) before addition to Vero cells. The percentage of infected cells is shown; error bars are the mean +/− SEM of each group’s animals). Full size image

To assess whether mice expressing DNA plasmid-encoded anti-DENV neutralizing mAbs would be protected from DENV challenge, we employed the AG129 mouse model, which lacks type I and type II interferon (IFN) receptors and, upon DENV infection, recapitulates many aspects of human disease30,31. Importantly, these mice have been shown to exhibit ADE, with low doses of serotype-specific as well as cross-reactive antibodies both enhancing infection30. For these studies, mice were infected with the mouse-adapted DENV2 strain S221, which, in the presence of sub-neutralizing amounts of the anti-DENV mAb 2H2, causes antibody-enhanced severe disease and acute lethality (4–6 days post-infection) in AG129 mice at sublethal doses30.

For challenge studies, AG129 mice were given a single intramuscular injection of pDVSF-3 WT or pDVSF-3 LALA followed immediately by EP. Negative controls received a single intramuscular injection of pVax1 empty vector followed by EP. Five days later, the mice were challenged with a sub-lethal dose (1 × 109 GE) of DENV2 S221 in the presence (ADE) or absence (virus-only infection) of exogenous anti-DENV mAb 2H2. Mice in the pDVSF-3 WT, pDVSF-3 LALA and pVax1 cohorts had mean human IgG concentrations of 750 ng/mL, 1139 ng/mL and undetectable levels, respectively, one day before challenge (Supplementary Fig. 3; p ≤ 0.0930 for comparison between pDVSF-3 WT and pDVSF-3 LALA). Under virus-only infection conditions, we expect pDVSF-3 WT-treated mice to experience ADE and acute lethality, as immune complexes formed by DVSF-3 WT antibodies with DENV should lead to increased infection14. Conversely, we expect pVax1- and pDVSF-3 LALA-treated mice to survive, being unable to enhance disease. Indeed, five of six pDVSF-3 LALA-treated mice and all five pVax1 mice showed no lethal disease enhancement; all pDVSF-3 WT-treated mice succumbed to disease by day 5 (Fig. 3a; p ≤ 0.0084 for comparison between pDVSF-3 LALA and pDVSF-3 WT), demonstrating the non-enhancing functionality of pDVSF-3 LALA against virus-only infection. Under ADE conditions, we expect both pDVSF-3 WT- and pVax1-treated mice to experience acute lethality due to enhanced infection, whereas pDVSF-3 LALA-treated mice should be protected from severe disease. All five mice receiving pDVSF-3 LALA survived under ADE conditions, while those receiving either pDVSF-3 WT or pVax1 empty vector succumbed to acute, antibody-enhanced disease within 4–5 days (Fig. 3b; p ≤ 0.0072 for comparison between pDVSF-3 LALA and pDVSF-3 WT). Taken together, these data show that injection of pDVSF-3 LALA does not cause lethal enhancement after virus-only infection and protects against severe disease in ADE conditions, supporting the concept of muscle correctly processing and expressing functional antibodies from this platform.

Figure 3 DMAb protects against virus-only and antibody-enhanced disease. (a) Virus-only challenge: AG129 mice received an intramuscular injection of either pDVSF-3 WT, pDVSF-3 LALA, or pVax empty vector five days prior to challenge with a sublethal dose of DENV2 S221 (n = 5–6 mice per group; p ≤ 0.0084 for comparison between pDVSF-3 LALA and pDVSF-3 WT).(b) Antibody-dependent enhancement challenge: AG129 mice received an intramuscular injection of either pDVSF-3 WT, pDVSF-3 LALA, or pVax empty vector five days prior to administration of an enhancing dose of the non-neutralizing anti-DENV mAb 2H2. Thirty minutes later, mice were challenged with a sublethal dose of DENV2 S221 (n = 5–6 mice per group; p ≤ 0.0072 for comparison between pDVSF-3 LALA and pDVSF-3 WT). A Kaplan-Meier survival curve is shown (a–b). Full size image

DMAb delivery of multiple antibodies increases human IgG concentration and breadth of viral coverage in mice

Given that DENV serotypes have been shown to escape neutralization15, it is likely that an antibody cocktail targeting multiple epitopes on the DENV virion would produce an ideal prophylactic strategy. DNA plasmids have been shown in numerous experiments to be delivered in multi-plasmid formulations32,33, suggesting that delivery of multiple antibody-encoding plasmids is feasible. To test this concept, we injected 129/Sv mice with pDVSF-3 WT (anti-DENV1–3) in one leg and pDVSF-1 WT (anti-DENV1–4) in the other. Mice injected with both plasmids had significantly higher serum human antibody levels at day 7 compared to mice receiving a single plasmid (Supplementary Fig. 4a; p ≤ 0.0088 for comparison between pDVSF-1 WT and pDVSF-1 + 3; p ≤ 0.0240 for comparison between pDVSF-3 WT and pDVSF-1 + 3). Furthermore, Sera from mice injected with both plasmids stained Vero cells infected with all four DENV serotypes (Supplementary Fig. 4b). We extended this analysis to their LALA variants, also including a plasmid encoding an additional anti-DENV4 neutralizing antibody, DVSF-2 (derived from DV22.3, a human IgG1 mAb that targets DI/DII of the E protein and has been well characterized for its ability to neutralize DENV414). Mice injected with pDVSF-2 and pDVSF-3 were able to bind all four serotypes and mice injected with all three antibodies had even greater binding against all four serotypes (Supplementary Figure 4c). These data suggest that DMAb can ultimately increase breadth of protection against infectious diseases.