Antiviral Research 124 (2015) 61e68
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Antibody with an engineered Fc region as a therapeutic agent against dengue virus infection Ririn Ramadhany a, Itaru Hirai b, Tadahiro Sasaki a, 1, Ken-ichiro Ono c, Pongrama Ramasoota d, Kazuyoshi Ikuta a, 1, Takeshi Kurosu a, * a
Research Institute of Microbial Disease, Osaka University, Japan Faculty of Medicine, University of the Ryukyu, Okinawa, Japan Medical and Biological Laboratories Corporation Ltd., Japan d Center of Excellence of Antibody Research, Department of Social and Environment Medicine, Faculty of Tropical Medicine, Mahidol University, Thailand b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 December 2014 Received in revised form 28 August 2015 Accepted 10 October 2015 Available online 30 October 2015
Antibody-dependent enhancement (ADE) of dengue virus (DENV) infectivity is thought to play a crucial role in severe dengue disease. It occurs when pre-existing sub-neutralizing anti-DENV antibody (Ab) produced from a primary infection encounters a DENV serotype different from that of the initial infection and forms immune complexes, which enable the efficient infection of Fcg receptor-bearing cells. However, the exact role played by Abs during a secondary infection of patients remains unknown. We previously obtained a broadly cross-reactive neutralizing IgG1 human monoclonal anti-DENV envelope (E) Ab (HuMAb) D23-1G7C2-IgG1 from a DENV-infected patient; however, D23-1G7C2-IgG1 had ADE activity. With the aim of being able to reduce the ADE activity, we exchanged the Fc region of D23-1G7C2 to generate Abs bearing each of the three other IgG subclasses (IgG2e4). In addition, N297A, a mutation known to reduce the affinity of the IgG1 Fc region for Fcg receptors, was introduced into D23-1G7C2IgG1. Swapping D23-1G7C2-IgG1 to IgG2 or IgG4 subclasses reduced ADE activity in FcgRI and FcgRIIbearing THP-1 cells. By contrast, in FcgRII-bearing K562 cells, the change to IgG2 increased ADE activity. Introducing the N297A mutation into D23-1G7C2-IgG1 resulted in a marked reduction in ADE activity in both cell types. Compared to D23-1G7C2-IgG1, D23-1G7C2-IgG1-N297A was less protective in IFN-a/b/g receptor knockout mice infected with a lethal dose of recombinant chimeric DENV, carrying prME of DENV-2 in Japanese encephalitis virus (80% vs. 40% survival, respectively). These observations provide valuable information regarding the use of recombinant Abs as therapeutics. © 2015 Elsevier B.V. All rights reserved.
Keywords: Dengue Human monoclonal antibody Antibody-dependent enhancement IgG subclass Neutralization
1. Introduction DENV is classified into four serotypes (DENV 1e4) (Whitehorn and Simmons, 2011). Sero-epidemiologic studies suggest that antibody acquired from primary infection provides protective immunity against re-infection with the identical serotype of DENV, but provides only partial protection in cases of secondary infection with different DENV serotypes (Graham et al., 1999; Burke et al., 1988; Vaughn et al., 2000). The presence of a sub-neutralizing concentration of anti-DENV antibody acquired from the primary
* Corresponding author. E-mail address: [email protected] (T. Kurosu). 1 Present address: The Research Foundation for Microbial Disease of Osaka University, Japan. http://dx.doi.org/10.1016/j.antiviral.2015.10.012 0166-3542/© 2015 Elsevier B.V. All rights reserved.
infection can enhance a secondary infection by promoting the binding of antibody-virus complex to cells bearing Fcg receptors, so-called antibody-dependent enhancement (ADE) (Porterfield, 1982; Wahala, 2011; van der Schaar et al., 2009) . Dendritic cells and macrophages, target cells for DENV infection, express several types FcgRs (Boonak et al., 2008; Hotta et al., 1983; Guilliams et al., 2014). FcgRs are classified into three groups: FcgRI, FcgRII, and FcgRIII. Human FcgRIA and FcgRIIA have been demonstrated to enhance DENV infection through ADE (Ravetch and Bolland, 2001; Kontny et al., 1988; Chareonsirisuthigul et al., 2007; Boonnak et al., 2013; Mady et al., 1993; Moi et al., 2010; Rodrigo et al., 2006). We previously obtained an anti-DENV envelope (E) human monoclonal antibody (HuMAb), D23-1G7C2-IgG1, derived from patients in Thailand (Sasaki et al., 2013), which broadly neutralizes all serotypes of DENV. In this study, we have examined the effects of
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changing the IgG subclass of this HuMAb on its neutralizing and ADE abilities. Recombinant (r) D23-1G7C2-IgGs (IgG2-4) HuMAbs were constructed and then produced in the human embryonic kidney cell line, 293T. In addition, a single mutation N297A was introduced into D23-1G7C2-IgG1, which disrupts a glycosylation site, critical for binding the HuMAb to FcgRs (Lux et al., 2013). The ability of the HuMAbs bearing a rIgG Fc region to protect against DENV infection was further evaluated in a mouse model.
2. Materials and methods 2.1. Cells and viruses The African green monkey kidney cell line Vero was maintained in minimal essential medium with 10% fetal bovine serum (FBS) at 37 C. Mosquito C6/36 cells were cultured at 28 C in Leibovitz's L15 medium with 10% FBS and 0.3% tryptose phosphate broth. The human monocytic leukemia THP-1 and human erythroleukemia K562 cell lines were cultured in RPMI-1640 with 10% FBS at 37 C. DENV-1 Mochizuki, DENV-2 16681, DENV-3 H87, and DENV-4 H241 strains were propagated in C6/36 cells and stored at 80 C prior to use. Viral infectivity was determined using focus-forming unit (FFU) assays as described previously (Kurosu et al., submitted for publication).
2.3. Reactivity of rHuMAbs by immunofluorescence assay (IFA) and neutralization assay Vero cells infected with DENV-2 were fixed with PBS containing 4% paraformaldehyde (PFA), permeabilized with PBS containing 1% Triton X-100 for 5 min at room temperature, and then incubated with each HuMAb for 1 h. The cells were washed three times with PBS and then incubated with Alexa 488-goat anti-human IgG Ab (Invitrogen, Paisley, UK) for 30 min at room temperature. After washing, the cells were observed under a fluorescence microscope. HuMAb 5E4 (IgG1) reactive against influenza hemagglutinin (HA) (Yasugi et al., 2013) was used as an isotype control. For neutralization assay, each HuMAb or human normal IgG (50 mg/ml), was serially diluted (2-fold) and reacted with the DENV2 (150 ffu/well). The final concentration of MAb in each reaction was 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.10, 0.05, and 0.03 mg/ml. The following procedure was performed described previously (Sasaki et al., 2013). 2.4. Antibody-dependent enhancement assay in vitro Mixtures of serially diluted HuMAbs and DENV were incubated at 37 C for 30 min to allow the formation of immune complexes (Ab-DENV). THP-1 and K562 cells were infected with the Ab-DENV mixture at a multiplicity of infection of 0.1 and seeded in 48-well plates, and then incubated at 37 C for 72 h (Masrinoul et al., 2013; Sasaki et al., 2013). Viral RNA (vRNA) collected from cells was quantified as described previously (Masrinoul et al., 2013).
2.2. Construction of rHuMAbs The full-length coding sequence of D23-1G7C2-IgG1 was synthesized by reverse transcription polymerase chain reaction (RTPCR). The IgG heavy chains were amplified from the HuMAbs expressing IgG2, IgG3m or IgG4 (Setthapramote et al., 2012) with 25-9-9VH-F; 50 -AGCTCGGATCTGTACcATGAAACACCTGTGGTTCTTCCTC-30 and hIgGH4R; 50 -ATGTTAACGTACGATTCATTTACCCAGAGACAGGGAGAG-30 primers and cloned into the pIRESHyg vector (Clontech Laboratories, Mountain View, CA). The heavy chain was amplified with 25-19-32VK-F; 50 -CGAGCTCGGATCGATACCATGAGGCTCCCTGCTCAGCTC-30 and hIgGH4R primers and cloned into the pIRESpuro vector (Clontech Laboratories). To exchange the IgG1 Fc region, heavy chains were amplified with hIgGHF; 50 -CCTCCACCAAGGGCCCATCGG-30 and either hIgGH4R; 50 -ATGTTAACGTACGATTCATTTACCCAGAGACAGGGAGAG-30 or hIgGH1-3R; 50 -ATGTTAACGTACGATTCATTTACCCGGAGACAGGGAGAG-30 primers and cloned into pCR-BluntII (Life Technologies, Carlsbad, CA). The resultant IgG sequences corresponded to each reference (IgG2: BC062335, IgG3: AK097355, and IgG4: BX640824). After sequence confirmation, Fc constant regions of IgG2, IgG3, and IgG4 were amplified with corresponding primers (Fragment 1). Fab variable regions of D23-1G7C2-IgG1 were amplified with IgG constant change R; 50 -CCGATGGGCCCTTGGTGGAGG-30 and 25-99VH-F; 50 -AGCTCGGATCTGTACcATGAAACACCTGTGGTTCTTCCTC-3 (Fragment 2). Fragments 1 and 2 were cloned into pIREShyg2 using an In-Fusion Cloning Kit (Clontech) in accordance with the manufacturer's instructions. A point mutation, N297A, was introduced into the N-glycosylation site positioned at 297 using In-Fusion HD cloning kit. The light chain was amplified with hIgGH4R; 50 CGAGCTCGGATCGATACCATGAGGCTCCCTGCTCAGCTC-30 and IGLV1-47*02(57L)R2; 50 -CGTTACTAGTGGATCTATGAACATTCTGTAGGGGC-30 from D23-1G7C2-IgG1 and inserted into pIRESpuro. Plasmids containing heavy chain and light chain genes were transfected into 293T cells and selected in the presence of 1.5 mg/ml puromycin and 100e125 mg/ml hygromycin in Dulbecco's modified Eagle's medium with IgG depleted.10% FCS.
2.5. Protection of IFN-a/b/gR KO mice by D23-1G7C2-IgG or D231G7C2-IgG-N297A after lethal infection with chimeric DENV-2 All animal experiments were performed in accordance with the guidelines for the care and use of laboratory animals at the Research Institute for Microbial Diseases (RIMD), Osaka University. The study was approved by the Animal Experiment Committee of the RIMD (#H25-09-1). Five or six 6-week old interferon-a/b/g receptor knockout (IFN-a/b/gR KO) mice per group were infected intraperitoneally (i.p.) with 800 FFU of chimeric DENV-2, 20 h prior to HuMAb injection. HuMAbs D23-1G7C2-IgG1, D23-1G7C2-IgG1N297A or 5E4 (500 mg/mouse) were injected and the mice observed for 38 days. Viremia and antibody titer were monitored in blood samples collected from the tail vein at 2, 6, and 12 days p.i. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc). 2.6. Measurement of viral titer in mouse sera vRNA was extracted from mouse serum using a QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer's instructions. One-step real-time quantitative RT-PCR amplification was performed to measure vRNA using a CFX Connect Real-Time System (BIO-RAD) with a One-Step SYBR PrimeScript RT-PCR Kit II (TAKARA). The following primers were used: JEF; 50 -AGAGCGGGGAAAAAGGTCAT-30 , and JER#110; 50 -CTTCACGCTCTTCCTACAGT-30 (modified from Santhosh et al., 2007). The results were quantified by interpolation from a standard curve generated from 10-fold serial dilutions of in vitro-transcribed chimeric DENV RNA (made using a MEGAscript kit; Ambion). Data were analyzed using CFX Manager software version 1.6 (Bio-Rad). 2.7. Detection of persistent HuMAbs in IFN-a/b/gR KO mice To detect persistent HuMAb in mice blood, the reactivity of HuMAb D23-1G7C2-IgG1 and rHuMAb D23-1G7C2-IgG1-N297A
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from mouse sera to DENV were detected by IFA. Sera collected at 2, 6, 12, and 25 days p.i. were incubated with chimeric DENV-infected Vero cells. Infected cells were visualized with Alexa Fluor 488 goat anti-human IgG (Invitrogen). Collected sera were also subjected to neutralization assay against DENV.
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original HuMAb D23-1G7C2-IgG1 (FRNT50 (mg/ml): D23-1G7C2IgG1, 1.02; D23-1G7C2-IgG2, 1.64; D23-1G7C2-IgG3, 1.57; D231G7C2-IgG4, 1.18 and D23-1G7C2-IgG1-IgG1-N297A, 1.18; Fig. 1B and Table 1).
3. Results 3.1. Reactivity and neutralization activity of rHuMAbs against DENV To observe the effect on the reactivity to DENV of swapping the IgG subclass and of introducing the N279A mutation into the IgG1 Fc region, Vero cells infected with DENV-2 were stained with purified rHuMAbs and visualized by IFA. All the rHuMAbs reacted equally to DENV (Fig. 1A). The neutralizing activity (determined by the FRNT50) of each rHuMAb was found to be similar to that of the
Table 1 FRNT50 of rHuMAb IgG in neutralizing DENV. Antibody D23-1G7C2
FRNT50a (mg/ml)
IgG1 IgG2 IgG3 IgG4 IgG-N297A
1.02 1.64 1.57 1.18 1.18
a
50% reduction of focus reduction neutralization.
Fig. 1. Reactivity and neutralizing activity of HuMAbs. (A) DENV-2-infected Vero cells were fixed 20 h post-infection, and stained with rHuMAbs (D23-1G7C2-IgG1, -IgG2, -IgG3, -IgG4, or D23-1G7C2-IgG1-N297A) followed by Alexa Fluor 488-anti-human IgG heavy and light chains. Mock-infected cells were stained only with D23-1G7C2-IgG1. Anti-influenza HA HuMAb 5E4 was used as a negative control for staining. (B) Serially 2-fold diluted HuMAbs (50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.10, and 0.05 mg/ml) were incubated with 150 FFU of DENV-2. Vero cells were infected with each Ab-DENV mixture and visualized as described in the Materials and methods. Foci were counted, the neutralizing activity of the HuMAbs was calculated and expressed as percent inhibition. Data are shown as the means ± SD from 3 experiments.
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Fig. 2. Antibody-dependent enhancement activity in THP-1 and K562 cells. (A) DENV-1, (B) DENV-2, (C) DENV-3, or (D) DENV-4 were incubated with rHuMAbs (D23-1G7C2-IgG1, -IgG2, -IgG3, -IgG4, or D23-1G7C2-IgG1-N297A) for 30 min and then used to infect THP-1 cells. (E) DENV-2 was incubated with each rHuMAb for 30 min and then used to infect K562 cells. After 72 h incubation cells were collected and total RNA was extracted. vRNA was measured using quantitative RT-PCR and normalization to GAPDH. Enhancement was expressed as fold enhancement of virus titer from virus titer from cells infected with DENV without Ab. The x-axis scale numbers indicates concentration of Abs. The y-axis scale numbers indicates fold-enhancement of vRNA in cytoplasm. Data are shown as the means ± SD from 3 experiments.
3.2. Antibody-dependent enhancement in FcgRI- and FcgRIIbearing cells Next, the effect of swapping the IgG subclass and of modifying the IgG1 Fc region on the ADE activity of D23-1G7C2-IgG1 was examined. The ADE assay was performed in two types of cells, THP1 and K562. THP-1 cells bear both FcgRI and FcgRII (Fleit and Kobasiuk, 1991), while K562 cells bear only FcgRII (Littaua et al., 1990; Mady et al., 1993). THP-1 and K562 cells were each infected with mixtures containing serially diluted HuMAb and DENV After incubation for 72 h the vRNA in the cytoplasm was measured by quantitative RT-PCR. 3.2.1. FcgRI- and FcgRII-bearing THP-1 cells In THP-1 cells, D23-1G7C2-IgG1 and D23-1G7C2-IgG3 induced a
1024-fold enhancement of the production of DENV-1 vRNA in cells, while D23-1G7C2-IgG2 and D23-1G7C2-IgG4 induced a 64- and a 256-fold enhancement, respectively, suggesting that they had caused a reduction in ADE. D23-1G7C2-IgG1-N297A induced the least enhancement, with a 32-fold increase in production (Fig. 2A and Table 2). Production of DENV-2 was enhanced 256-fold by D23-1G7C2IgG1 and by 32-fold in the presence of D23-1G7C2-IgG3. By contrast, D23-1G7C2-IgG4 and D23-1G7C2-IgG2 induced less enhancement of viral production, at four- and eight-fold respectively. D23-1G7C2-IgG1-N297A induced a two-fold increase in DENV-2 production (Fig. 2B and Table 2). The pattern of the effect of the rHuMAbs on DENV-3 production was similar. D23-1G7C2-IgG1 induced the greatest enhancement (16-fold); D23-1G7C2-IgG3 and D23-1G7C2-IgG4 induced an
R. Ramadhany et al. / Antiviral Research 124 (2015) 61e68 Table 2 Summary of the maximum degree of enhancement reached for each HuMAb in an in vitro ADE assay. Virus
Antibody D23-1G7C2 IgG1
THP-1
K562
DENV-1 DENV-2 DENV-3 DENV-4 DENV-2
1024 256 16 32 64
a
IgG2
IgG3
IgG4
IgG-N297A
64 8 4 4 512
1024 32 8 128 128
256 4 8 16 32
32 2 3 4 16
a
The numbers indicate fold enhancement compared to cells that were not reacted with HuMAbs.
eight-fold enhancement, while D23-1G7C2-IgG2 increased production four-fold. D23-1G7C2-IgG1-N297A induced the least enhancement (three-fold; Fig. 2C and Table 2). In the presence of DENV-4, D23-1G7C2-IgG1 and D23-1G7C2IgG3 showed the greatest enhancement (both 128-fold), while D23-1G7C2-IgG2 and D23-1G7C2-IgG4 enhanced viral production four-fold and 14-fold, respectively. The D23-1G7C2-IgG1-N297A enhanced production four-fold (Fig. 2D and Table 2). While, compared to the other rHuMAbs, D23-1G7C2-IgG1 did induce the greatest degree of ADE on the infectivity of DENV-3, unlike its effect on the other DENV serotypes (1, 2, and 4), it was the lowest maximum enhancement observed, suggesting that ADE is also dependent on the virus serotype. Compared to the other rHuMAbs, in the presence of each DENV serotype D23-1G7C2IgG1-N297A consistently induced the least enhancement of infectivity, suggesting that the single mutation, N297A, in the Fc region was sufficient to disrupt ADE activity. 3.2.2. FcgRII-bearing K562 cells K562 cells express only type II FcgRs. Unlike ADE in THP-1 cells, in K562 cells the greatest enhancement was induced by D231G7C2-IgG2 (512-fold), while D23-1G7C2-IgG1, -IgG3, and -IgG4 induced enhancement 64-, 128-, and 32-fold, respectively. ADE activity of different IgG subclass depends on the type of FcgR and the target cell. Again, D23-1G7C2-IgG1-N297A induced the least enhancement (16-fold; Fig. 2E and Table 2), indicating that the introduction of a single mutation, N297A, into the Fc region of IgG1 disrupted ADE activity in both THP-1 and K562 cells. 3.3. D23-1G7C2-IgG1 protected mice from lethal infection with chimeric DENV, while D23-1G7C2-IgG1-N297A showed partial protection The protective ability of IgGs was examined in mice infected with virus. Because there was no other DENVs caused fatal infection in this mouse model, chimeric DENV carrying prME genome of DENV-2 and genome coding JEV NSs protein was used for challenge. All IgGs could equally neutralize chimeric DENV (Supplementary Fig. 1). IFN-a/b/gR KO mice were infected with a lethal dose of chimeric DENV, and 500 mg of rHuMAb was injected i.p. 20 h p.i. with 800 FFU of chimeric DENV. Mice that received the control 5E4 had all died by day 9 p.i. (Fig. 3A); by contrast, mice treated with D23-1G7C2-IgG1 did not start to die until 17 days p.i., and at the end of the 38-day study period had an 80% survival rate, whereas 40% of mice that received D23-1G7C2-IgG1-N297A survived (Fig. 3A). This result suggests that the introduction of the N297A mutation in the IgG1 Fc region reduced the protective activity of D23-1G7C2-IgG1. A similar tendency was found in IFN-a/b/ gR KO mice injected with 300 mg of HuMAb (Supplementary Fig. 2.). The viral titer in the sera of all the treated mice was determined on days 2, 6, and 12 p.i. At day-2 p.i., viremia was detected in mice
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injected with 5E4 (5.8 10 copies/ml) and at a higher level in mice injected with D23-1G7C2-IgG1-N297A (1.97 102 copies/ml; Fig. 3B). There was no detectable level of viremia in mice injected with D23-1G7C2-IgG1. At day-6 p.i., viremia had increased in 5E4and D23-1G7C2-IgG1-N297A-treated mice (9.3 103 and 2.4 103 copies/ml, respectively), while there was no detectable viremia in mice injected with D23-1G7C2-IgG1. At day-12 p.i., all the mice injected with D23-1G7C2-IgG1 and the surviving mice injected with D23-1G7C2-IgG1-N297A showed little or no viremia. These results raised the question of whether D23-1G7C2-IgG1N297A retains sufficient neutralizing activity in mouse because it may be instable in sera. Sera collected from 5E4-injected mice on day 2 p.i. showed no neutralizing, activity (Fig. 3B), confirming that normal mouse serum does not contain neutralizing antibodies; however, sera collected on day 6 p.i. showed moderate neutralizing activity (95.6%). This activity was probably due to newly-generated antibodies induced by the viral infection. Despite producing antisera, all mice injected with 5E4 died by day 9 (Fig. 3A). Sera from mice injected with D23-1G7C2-IgG1 collected on days 2, 6, and 12 p.i. efficiently neutralized DENV-2 (Fig. 3C). Meanwhile, sera from mice injected with D23-1G7C2-IgG1-N297A collected 2 and 6 days p.i. showed moderate neutralization activity (both 64% by undiluted serum), but reduced neutralizing activity (38.3% by undiluted serum) on day-12 p.i. (Fig. 3C). This suggests that D23-1G7C2-IgG1N297A rapidly loses neutralizing activity when injected into mice. To confirm that the HuMAbs were present in the sera, reactivity was examined using DENV-2-infected Vero cells. HuMAb-specific reactivity was detected by IFA with an Alexa Fluor 488 conjugated anti-human IgG antibody to distinguish it from that of newly produced DENV-reactive mouse Abs. D23-1G7C2-IgG1 was detected even at day-25 p.i. (Fig. 3D). On the other hand, D23-1G7C2IgG1-N297A was detected 2 days p.i., but the signal was reduced by day-6 p.i. and undetectable on day-12 p.i. D23-1G7C2-IgG1N297A was rapidly degraded. Thus, although D23-1G7C2-IgG1N297A showed neutralizing activity against chimeric DENV-2 similar to that of D23-1G7C2-IgG1 (Fig. 1B and Table 1), it partially lost its protective ability. Furthermore, ADE was examined by the introduction of lower concentrations of HuMAbs in mice. A maximum ADE was observed by introduction of 8 mg of mouse monoclonal anti-DENV E Ab 4G2 (Kurosu et al., submitted for publication). However, introduction of both D23-1G7C2-IgG1 and D23-1G7C2-IgG1-N297A did not increase lethality nor viral load by introduction of 2 or 8 mg of HuMAbs (Supplementary Fig. 3). 4. Discussion Passive immunization with MAbs, especially humanized MAbs or human-derived MAbs, represents an attractive therapeutic approach. Nevertheless, there is concern that introduction of MAbs may exacerbate the patient's condition by the induction of ADE. To reduce the risk of ADE, HuMAbs would have to be genetically modified (Hanley and Weaver, 2010). Replacing certain subclasses of IgG reduced ADE activity, although the ADE activity of these antibodies depended on the type of target cell (Fig. 2 and Table 2). In addition, HuMAb with the Fc region mutation, D23-1G7C2-IgG1-N297A, showed the least ADE activity (Fig. 2), without losing neutralizing activity (Fig. 1B). A single mutation at N297 residue of IgG has been shown to impair the binding to FcgR (Arnold et al., 2007; Sazinsky et al., 2008; Lux et al., 2013). D23-1G7C2-IgG1-N297A is an ideal therapeutic antibody; however, D23-1G7C2-IgG1-N297A partially lost its protective activity (Fig. 3A). The mutation at N297 of IgG possibly induce conformational change of the Fc region (Wright and Morrison, 1997; Siberil et al., 2006) and short half-life in mice. This may be
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Fig. 3. Protection of IFN-a/b/gR KO mice from chimeric DENV-2 by rHuMAbs. (A) Survival rate of IFN-a/b/gR KO mice. Five or six IFN-a/b/gR KO mice in each group were infected with 800 FFU of chimeric DENV-2 i.p. After 20 h mice were then injected i.p. with 500 mg of D23-1G7C2-IgG1, D23-1G7C2-IgG1-N297A, or 5E4, and observed until day-38 p.i. Asterisks indicate a statistically significant difference of P < 0.01. (B) Viral copy number in mouse sera collected from each group of mice at day-2, -6, and -12 p.i. was determined by quantitative RT-PCR as described in the Materials and methods. (C) Serially two-fold diluted sera collected from mice at day-2, -6, and -12, were mixed with 100 FFU of chimeric DENV-2 and inoculated into Vero cells. A neutralization assay was performed as described in the Materials and methods. (D) Vero infected with DENV were incubated with sera collected from chimeric DENV-2- and HuMAb-treated mice as described above for Fig. 3A, and visualized with Alexa Fluor 488- goat anti-human IgG by fluorescent microscopy.
the reason why, in our study, D23-1G7C2-IgG1-N297A was undetectable in mouse serum at day 12 p.i., while D23-1G7C2-IgG1 was detected even at day 25 p.i. (Fig. 3D). Another possible explanation for the short half-life of D23-1G7C2-IgG1-N297A is that the N297A mutation reduces its affinity for the neonatal Fc receptor (FcRn), which functions as a salvage receptor to rescue IgG from the lysosomal degradation pathway (Kobayashi et al., 2002). The recycling of Ab through FcRn contributes to their long half-life. To some extent, the N297A mutant Ab shows reduced binding affinity for FcRn (Shields et al., 2001). This may be the reason why the amount
of D23-1G7C2-IgG1-N297A in mouse serum decreased (Fig. 3D). In contrast to the present study, another study using a different model showed that a similar chimeric humanized N297Q mutant fully protected mice from lethal infection with DENV (Balsitis et al., 2010). These differing observations may be due to differences in the viruses studied. For example, chimeric DENV-2 replicates much faster in mice. Of note, a low amount (800 FFU) of chimeric DENV-2 was sufficient to cause 100% lethality (Fig. 3A), whereas a high dose of DENV was needed to cause lethal infection in the model studied by Balsitis et al. (2010). Furthermore, mice infected with 800 FFU of
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chimeric DENV-2 showed a serum titer greater than 106 FFU/ml at day 5 p.i. (Kurosu et al., submitted for publication). In the present model, mice did not survive unless the injected Ab reduced the virus tier to below at least 800 FFU. Human cases of infection begin after exposure to a small amount of DENV via a mosquito bite; the virus then actively replicates and spreads throughout the body, resulting in high levels of viremia. The mouse model used in the present study is similar to human cases of infection in terms of virus replication kinetics. Furthermore, mice injected with D231G7C2-IgG1-N297A did not generate a protective Ab response at the early stages of infection (Fig. 3C). This suggests that D231G7C2-IgG1-N297A interfered with the humoral immune response. We believe that this is an important observation with respect to the practical application of Ab therapy. This mouse model also revealed another possibility with respect to Ab therapy. Although D23-1G7C2-IgG1-N297A is safe in terms of ADE, it was rapidly cleared from mouse serum (Fig. 3D). By contrast, although D23-1G7C2-IgG1 efficiently protected mice (Fig. 3A), it showed higher ADE in vitro (Fig. 2 and Table 2). The half-life of an Ab is important with respect to therapy. Differences in IgG subclass do not affect the affinity for FcRn, and all subclasses have a similar half-life (West and Bjorkman, 2000). Other IgG subclass, such as IgG2 or IgG4, which showed lower ADE (Fig. 2 and Table 2), may be more secure than IgG1. Alternatively, changes in glycosylation may affect Ab affinity; for example, Ha et al. reported that IgG Ab carrying asymmetric glycosylations at N297 cannot bind FcgRs without losing affinity for FcRn. Although technical difficulties remain, it may be considered as a next candidate (Ha et al., 2011). This study raises the question of whether N297 mutant IgG has a short half-life in humans. Further study is needed to develop an effective and safe therapeutic HuMAb. Acknowledgments This work was supported by a grant-in-aid (21790444) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and a program of the Japan Initiative for Global Research Network on Infectious Diseases launched by a project commissioned by the MEXT. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.antiviral.2015.10.012. References Arnold, J.N., Wormald, M.R., Sim, R.B., Rudd, P.M., Dwek, R.A., 2007. The impact of glycosilation on the biological function and structure of human immunoglobulins. Annu. Rev. Immunol. 25, 21e50. Balsitis, S.J., Williams, K.L., Lachica, R., Flores, D., Kyle, J.L., Mehlhop, E., Johnson, S., Diamond, M.S., Beatty, P.R., Harris, E., 2010. Lethal antibody enhancement of Dengue disease in mice is prevented by Fc modification. PLoS Pathog. 6, e1000790. Boonak, K., Slike, B.M., Burgess, T.H., Mason, R.M., Wu, S., Sun, P., Porter, K., Rudiman, I.F., Yuwono, D., Puthavathana, P., Marovitch, M.A., 2008. Role of dendritic cells in antibody-dependent enhancement of Dengue virus infection. J. Virol. 82, 3939e3951. Boonnak, K., Slike, B.M., Donofrio, G.C., Marovich, M.A., 2013. Human FcgRII cytoplasmic domains differentially influence antibody-mediated dengue virus infection. J. Immunol. 190, 5659e5665. Burke, D.S., Nisalak, A., Johnson, D.E., Scott, R.M., 1988. A prospective study of dengue infections in Bangkok. Am. J. Trop. Med. Hyg. 38, 172e180. Chareonsirisuthigul, T., Kalayanarooj, S., Ubol, S., 2007. Dengue virus (DENV) antibody-dependent enhancement of infection upregulates the production of anti-inflammatory cytokines, but suppresses anti-DENV free radical and proinflammatory cytokine production, in THP-1 cells. J. Gen. Virol. 88, 365e375. Fleit, H.B., Kobasiuk, C.D., 1991. The human monocyte-like cell line THP-1 expresses FcgRI and FcgRII. J. Leukoc. Biol. 49, 556e565. Graham, R.R., Juffrie, M., Tan, R., Hayes, C.G., Laksono, I., Ma'roef, C., Erlin, Sutaryo,
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Porter, K.R., Halstead, S.B., 1999. A prospective seroepidemiologic study on dengue in children four to nine years of age in Yogyakarta, Indonesia I. studies in 1995-1996. Am. J. Trop. Med. Hyg. 61, 412e419. Guilliams, M., Bruhns, P., Saeys, Y., Hammad, H., Lambrecht, B.N., 2014. The function of Fcg receptors in dendritic cells and macrophages. Nat. Rev. Immunol. 14, 94e108. Ha, S., Ou, Y., Vlasak, J., Li, Y., Wang, S., Vo, K., Du, Y., Mach, A., Fang, Y., Zhang, N., 2011. Isolation and characterization of IgG1 with asymmetrical Fc glycosylation. Glycobiology 21, 1087e1096. Hanley, K.A., Weaver, S.C., 2010. Frontiers in Dengue Virus Research. Caister Academic Press, p. 266. Hotta, H., Hotta, S., Takada, H., Kotani, S., Tanaka, S., Ohki, M., 1983. Enhancement of Dengue virus type 2 replication in mouse macrophage cultures by bacterial cell walls, peptidoglycans, and a polymer of peptidoglycan subunits. Infect. Immun. 41, 462e469. Kobayashi, N., Suzuki, Y., Tsuge, T., Okumura, K., Ra, C., Tomino, Y., 2002. FcRnmediated transcytosis of immunoglobulin G in human renal proximal tubular epithelial cells. Am. J. Physiol. Ren. Physiol. 282, 358e365. Kontny, U., Kurane, I., Ennis, F.A., 1988. Interferon gamma augments Fcgreceptormediated dengue virus infection of human monocytic cells. J. Virol. 62, 3928e3933. Kurosu, T., Khamlert, C., Phanthanawiboon, S., Ikuta, K., Anantapreecha, S., 2010. Highly efficient rescue of dengue virus using a co-culture system with mosquito/mammalian cells. Biochem. Biophys. Res. Commun. 394, 398e404. Littaua, R., Kurane, I., Ennis, F.A., 1990. Human IgG Fc receptor II mediates antibodydependent enhancement of dengue virus infection. J. Immunol. 144, 3183e3186. Lux, A., Yu, X., Scanlan, C.N., Nimmerjahn, F., 2013. Impact of immune complex size and glycosylation on IgG binding to human FcgRs. J. Immunol. 190, 4315e4323. Mady, J.B., Kurane, I., Erbe, D.V., Fanger, M.W., Ennis, F.A., 1993. Neuraminidase augments Fcg receptor II-mediated antibody-dependent enhancement of dengue virus infection. J. Gen. Virol. 74, 839e844. Masrinoul, P., Omokoko, M.D., Pambudi, S., Ikuta, K., Kurosu, T., 2013. Serotypespecific anti-dengue virus NS1 mouse antibodies cross-react with prM and are potentially involved in virus production. Viral Immunol. 26, 250e258. Moi, M.L., Lim, C.K., Takasaki, T., Kurane, I., 2010. Involvement of the Fcg receptor IIA cytoplasmic domain in antibody-dependent enhancement of dengue virus infection. J. Gen. Virol. 91, 103e111. Porterfield, J.S., 1982. Immunological enhancement and the pathogenesis of dengue haemorrhagic fever. J. Hyg. Lond. 89, 355e364. Ravetch, J.V., Bolland, S., 2001. IgG fc receptors. Annu. Rev. Immunol. 19, 275e290. Rodrigo, W.W., Jin, X., Blackley, S.D., Rose, R.C., Schlesinger, J.J., 2006. Differential enhancement of dengue virus immune complex infectivity mediated by signaling-competent and signaling-incompetent human FcgRIA (CD64) or FcgRIIA (CD32). J. Virol. 80, 128e138. Santhosh, S.R., Parida, M.M., Dash, P.K., Pateriya, A., Pattnaik, B., Pradhan, H.K., Tripathi, N.K., Ambuj, S., Gupta, N., Saxena, P., Lakshmana Rao, P.V., 2007. Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantitation of Japanese encephalitis virus. J. Virol. Methods 143, 73e80. Sasaki, T., Setthapramote, C., Kurosu, T., Nishimura, M., Asai, A., Omokoko, M.D., Pipattanaboon, C., Pitaksajjakul, P., Limkittikkul, K., Subchareon, A., Chaichana, P., Okabayashi, T., Hirai, I., Leaungwutisong, P., Misaki, R., Fujiyama, K., Ono, K., Okuno, Y., Ramasoota, P., Ikuta, K., 2013. Dengue virus neutralization and antibody-dependent enhancement activities of human monoclonal antibodies derived from dengue patients at acute phase of secondary infection. 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Antibody with an engineered Fc region as a therapeutic agent against dengue virus infection Ririn Ramadhany a, Itaru Hirai b, Tadahiro Sasaki a, 1, Ken-ichiro Ono c, Pongrama Ramasoota d, Kazuyoshi Ikuta a, 1, Takeshi Kurosu a, * a
Research Institute of Microbial Disease, Osaka University, Japan Faculty of Medicine, University of the Ryukyu, Okinawa, Japan Medical and Biological Laboratories Corporation Ltd., Japan d Center of Excellence of Antibody Research, Department of Social and Environment Medicine, Faculty of Tropical Medicine, Mahidol University, Thailand b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 December 2014 Received in revised form 28 August 2015 Accepted 10 October 2015 Available online 30 October 2015
Antibody-dependent enhancement (ADE) of dengue virus (DENV) infectivity is thought to play a crucial role in severe dengue disease. It occurs when pre-existing sub-neutralizing anti-DENV antibody (Ab) produced from a primary infection encounters a DENV serotype different from that of the initial infection and forms immune complexes, which enable the efficient infection of Fcg receptor-bearing cells. However, the exact role played by Abs during a secondary infection of patients remains unknown. We previously obtained a broadly cross-reactive neutralizing IgG1 human monoclonal anti-DENV envelope (E) Ab (HuMAb) D23-1G7C2-IgG1 from a DENV-infected patient; however, D23-1G7C2-IgG1 had ADE activity. With the aim of being able to reduce the ADE activity, we exchanged the Fc region of D23-1G7C2 to generate Abs bearing each of the three other IgG subclasses (IgG2e4). In addition, N297A, a mutation known to reduce the affinity of the IgG1 Fc region for Fcg receptors, was introduced into D23-1G7C2IgG1. Swapping D23-1G7C2-IgG1 to IgG2 or IgG4 subclasses reduced ADE activity in FcgRI and FcgRIIbearing THP-1 cells. By contrast, in FcgRII-bearing K562 cells, the change to IgG2 increased ADE activity. Introducing the N297A mutation into D23-1G7C2-IgG1 resulted in a marked reduction in ADE activity in both cell types. Compared to D23-1G7C2-IgG1, D23-1G7C2-IgG1-N297A was less protective in IFN-a/b/g receptor knockout mice infected with a lethal dose of recombinant chimeric DENV, carrying prME of DENV-2 in Japanese encephalitis virus (80% vs. 40% survival, respectively). These observations provide valuable information regarding the use of recombinant Abs as therapeutics. © 2015 Elsevier B.V. All rights reserved.
Keywords: Dengue Human monoclonal antibody Antibody-dependent enhancement IgG subclass Neutralization
1. Introduction DENV is classified into four serotypes (DENV 1e4) (Whitehorn and Simmons, 2011). Sero-epidemiologic studies suggest that antibody acquired from primary infection provides protective immunity against re-infection with the identical serotype of DENV, but provides only partial protection in cases of secondary infection with different DENV serotypes (Graham et al., 1999; Burke et al., 1988; Vaughn et al., 2000). The presence of a sub-neutralizing concentration of anti-DENV antibody acquired from the primary
* Corresponding author. E-mail address: [email protected] (T. Kurosu). 1 Present address: The Research Foundation for Microbial Disease of Osaka University, Japan. http://dx.doi.org/10.1016/j.antiviral.2015.10.012 0166-3542/© 2015 Elsevier B.V. All rights reserved.
infection can enhance a secondary infection by promoting the binding of antibody-virus complex to cells bearing Fcg receptors, so-called antibody-dependent enhancement (ADE) (Porterfield, 1982; Wahala, 2011; van der Schaar et al., 2009) . Dendritic cells and macrophages, target cells for DENV infection, express several types FcgRs (Boonak et al., 2008; Hotta et al., 1983; Guilliams et al., 2014). FcgRs are classified into three groups: FcgRI, FcgRII, and FcgRIII. Human FcgRIA and FcgRIIA have been demonstrated to enhance DENV infection through ADE (Ravetch and Bolland, 2001; Kontny et al., 1988; Chareonsirisuthigul et al., 2007; Boonnak et al., 2013; Mady et al., 1993; Moi et al., 2010; Rodrigo et al., 2006). We previously obtained an anti-DENV envelope (E) human monoclonal antibody (HuMAb), D23-1G7C2-IgG1, derived from patients in Thailand (Sasaki et al., 2013), which broadly neutralizes all serotypes of DENV. In this study, we have examined the effects of
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changing the IgG subclass of this HuMAb on its neutralizing and ADE abilities. Recombinant (r) D23-1G7C2-IgGs (IgG2-4) HuMAbs were constructed and then produced in the human embryonic kidney cell line, 293T. In addition, a single mutation N297A was introduced into D23-1G7C2-IgG1, which disrupts a glycosylation site, critical for binding the HuMAb to FcgRs (Lux et al., 2013). The ability of the HuMAbs bearing a rIgG Fc region to protect against DENV infection was further evaluated in a mouse model.
2. Materials and methods 2.1. Cells and viruses The African green monkey kidney cell line Vero was maintained in minimal essential medium with 10% fetal bovine serum (FBS) at 37 C. Mosquito C6/36 cells were cultured at 28 C in Leibovitz's L15 medium with 10% FBS and 0.3% tryptose phosphate broth. The human monocytic leukemia THP-1 and human erythroleukemia K562 cell lines were cultured in RPMI-1640 with 10% FBS at 37 C. DENV-1 Mochizuki, DENV-2 16681, DENV-3 H87, and DENV-4 H241 strains were propagated in C6/36 cells and stored at 80 C prior to use. Viral infectivity was determined using focus-forming unit (FFU) assays as described previously (Kurosu et al., submitted for publication).
2.3. Reactivity of rHuMAbs by immunofluorescence assay (IFA) and neutralization assay Vero cells infected with DENV-2 were fixed with PBS containing 4% paraformaldehyde (PFA), permeabilized with PBS containing 1% Triton X-100 for 5 min at room temperature, and then incubated with each HuMAb for 1 h. The cells were washed three times with PBS and then incubated with Alexa 488-goat anti-human IgG Ab (Invitrogen, Paisley, UK) for 30 min at room temperature. After washing, the cells were observed under a fluorescence microscope. HuMAb 5E4 (IgG1) reactive against influenza hemagglutinin (HA) (Yasugi et al., 2013) was used as an isotype control. For neutralization assay, each HuMAb or human normal IgG (50 mg/ml), was serially diluted (2-fold) and reacted with the DENV2 (150 ffu/well). The final concentration of MAb in each reaction was 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.10, 0.05, and 0.03 mg/ml. The following procedure was performed described previously (Sasaki et al., 2013). 2.4. Antibody-dependent enhancement assay in vitro Mixtures of serially diluted HuMAbs and DENV were incubated at 37 C for 30 min to allow the formation of immune complexes (Ab-DENV). THP-1 and K562 cells were infected with the Ab-DENV mixture at a multiplicity of infection of 0.1 and seeded in 48-well plates, and then incubated at 37 C for 72 h (Masrinoul et al., 2013; Sasaki et al., 2013). Viral RNA (vRNA) collected from cells was quantified as described previously (Masrinoul et al., 2013).
2.2. Construction of rHuMAbs The full-length coding sequence of D23-1G7C2-IgG1 was synthesized by reverse transcription polymerase chain reaction (RTPCR). The IgG heavy chains were amplified from the HuMAbs expressing IgG2, IgG3m or IgG4 (Setthapramote et al., 2012) with 25-9-9VH-F; 50 -AGCTCGGATCTGTACcATGAAACACCTGTGGTTCTTCCTC-30 and hIgGH4R; 50 -ATGTTAACGTACGATTCATTTACCCAGAGACAGGGAGAG-30 primers and cloned into the pIRESHyg vector (Clontech Laboratories, Mountain View, CA). The heavy chain was amplified with 25-19-32VK-F; 50 -CGAGCTCGGATCGATACCATGAGGCTCCCTGCTCAGCTC-30 and hIgGH4R primers and cloned into the pIRESpuro vector (Clontech Laboratories). To exchange the IgG1 Fc region, heavy chains were amplified with hIgGHF; 50 -CCTCCACCAAGGGCCCATCGG-30 and either hIgGH4R; 50 -ATGTTAACGTACGATTCATTTACCCAGAGACAGGGAGAG-30 or hIgGH1-3R; 50 -ATGTTAACGTACGATTCATTTACCCGGAGACAGGGAGAG-30 primers and cloned into pCR-BluntII (Life Technologies, Carlsbad, CA). The resultant IgG sequences corresponded to each reference (IgG2: BC062335, IgG3: AK097355, and IgG4: BX640824). After sequence confirmation, Fc constant regions of IgG2, IgG3, and IgG4 were amplified with corresponding primers (Fragment 1). Fab variable regions of D23-1G7C2-IgG1 were amplified with IgG constant change R; 50 -CCGATGGGCCCTTGGTGGAGG-30 and 25-99VH-F; 50 -AGCTCGGATCTGTACcATGAAACACCTGTGGTTCTTCCTC-3 (Fragment 2). Fragments 1 and 2 were cloned into pIREShyg2 using an In-Fusion Cloning Kit (Clontech) in accordance with the manufacturer's instructions. A point mutation, N297A, was introduced into the N-glycosylation site positioned at 297 using In-Fusion HD cloning kit. The light chain was amplified with hIgGH4R; 50 CGAGCTCGGATCGATACCATGAGGCTCCCTGCTCAGCTC-30 and IGLV1-47*02(57L)R2; 50 -CGTTACTAGTGGATCTATGAACATTCTGTAGGGGC-30 from D23-1G7C2-IgG1 and inserted into pIRESpuro. Plasmids containing heavy chain and light chain genes were transfected into 293T cells and selected in the presence of 1.5 mg/ml puromycin and 100e125 mg/ml hygromycin in Dulbecco's modified Eagle's medium with IgG depleted.10% FCS.
2.5. Protection of IFN-a/b/gR KO mice by D23-1G7C2-IgG or D231G7C2-IgG-N297A after lethal infection with chimeric DENV-2 All animal experiments were performed in accordance with the guidelines for the care and use of laboratory animals at the Research Institute for Microbial Diseases (RIMD), Osaka University. The study was approved by the Animal Experiment Committee of the RIMD (#H25-09-1). Five or six 6-week old interferon-a/b/g receptor knockout (IFN-a/b/gR KO) mice per group were infected intraperitoneally (i.p.) with 800 FFU of chimeric DENV-2, 20 h prior to HuMAb injection. HuMAbs D23-1G7C2-IgG1, D23-1G7C2-IgG1N297A or 5E4 (500 mg/mouse) were injected and the mice observed for 38 days. Viremia and antibody titer were monitored in blood samples collected from the tail vein at 2, 6, and 12 days p.i. Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software, Inc). 2.6. Measurement of viral titer in mouse sera vRNA was extracted from mouse serum using a QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer's instructions. One-step real-time quantitative RT-PCR amplification was performed to measure vRNA using a CFX Connect Real-Time System (BIO-RAD) with a One-Step SYBR PrimeScript RT-PCR Kit II (TAKARA). The following primers were used: JEF; 50 -AGAGCGGGGAAAAAGGTCAT-30 , and JER#110; 50 -CTTCACGCTCTTCCTACAGT-30 (modified from Santhosh et al., 2007). The results were quantified by interpolation from a standard curve generated from 10-fold serial dilutions of in vitro-transcribed chimeric DENV RNA (made using a MEGAscript kit; Ambion). Data were analyzed using CFX Manager software version 1.6 (Bio-Rad). 2.7. Detection of persistent HuMAbs in IFN-a/b/gR KO mice To detect persistent HuMAb in mice blood, the reactivity of HuMAb D23-1G7C2-IgG1 and rHuMAb D23-1G7C2-IgG1-N297A
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from mouse sera to DENV were detected by IFA. Sera collected at 2, 6, 12, and 25 days p.i. were incubated with chimeric DENV-infected Vero cells. Infected cells were visualized with Alexa Fluor 488 goat anti-human IgG (Invitrogen). Collected sera were also subjected to neutralization assay against DENV.
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original HuMAb D23-1G7C2-IgG1 (FRNT50 (mg/ml): D23-1G7C2IgG1, 1.02; D23-1G7C2-IgG2, 1.64; D23-1G7C2-IgG3, 1.57; D231G7C2-IgG4, 1.18 and D23-1G7C2-IgG1-IgG1-N297A, 1.18; Fig. 1B and Table 1).
3. Results 3.1. Reactivity and neutralization activity of rHuMAbs against DENV To observe the effect on the reactivity to DENV of swapping the IgG subclass and of introducing the N279A mutation into the IgG1 Fc region, Vero cells infected with DENV-2 were stained with purified rHuMAbs and visualized by IFA. All the rHuMAbs reacted equally to DENV (Fig. 1A). The neutralizing activity (determined by the FRNT50) of each rHuMAb was found to be similar to that of the
Table 1 FRNT50 of rHuMAb IgG in neutralizing DENV. Antibody D23-1G7C2
FRNT50a (mg/ml)
IgG1 IgG2 IgG3 IgG4 IgG-N297A
1.02 1.64 1.57 1.18 1.18
a
50% reduction of focus reduction neutralization.
Fig. 1. Reactivity and neutralizing activity of HuMAbs. (A) DENV-2-infected Vero cells were fixed 20 h post-infection, and stained with rHuMAbs (D23-1G7C2-IgG1, -IgG2, -IgG3, -IgG4, or D23-1G7C2-IgG1-N297A) followed by Alexa Fluor 488-anti-human IgG heavy and light chains. Mock-infected cells were stained only with D23-1G7C2-IgG1. Anti-influenza HA HuMAb 5E4 was used as a negative control for staining. (B) Serially 2-fold diluted HuMAbs (50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.10, and 0.05 mg/ml) were incubated with 150 FFU of DENV-2. Vero cells were infected with each Ab-DENV mixture and visualized as described in the Materials and methods. Foci were counted, the neutralizing activity of the HuMAbs was calculated and expressed as percent inhibition. Data are shown as the means ± SD from 3 experiments.
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Fig. 2. Antibody-dependent enhancement activity in THP-1 and K562 cells. (A) DENV-1, (B) DENV-2, (C) DENV-3, or (D) DENV-4 were incubated with rHuMAbs (D23-1G7C2-IgG1, -IgG2, -IgG3, -IgG4, or D23-1G7C2-IgG1-N297A) for 30 min and then used to infect THP-1 cells. (E) DENV-2 was incubated with each rHuMAb for 30 min and then used to infect K562 cells. After 72 h incubation cells were collected and total RNA was extracted. vRNA was measured using quantitative RT-PCR and normalization to GAPDH. Enhancement was expressed as fold enhancement of virus titer from virus titer from cells infected with DENV without Ab. The x-axis scale numbers indicates concentration of Abs. The y-axis scale numbers indicates fold-enhancement of vRNA in cytoplasm. Data are shown as the means ± SD from 3 experiments.
3.2. Antibody-dependent enhancement in FcgRI- and FcgRIIbearing cells Next, the effect of swapping the IgG subclass and of modifying the IgG1 Fc region on the ADE activity of D23-1G7C2-IgG1 was examined. The ADE assay was performed in two types of cells, THP1 and K562. THP-1 cells bear both FcgRI and FcgRII (Fleit and Kobasiuk, 1991), while K562 cells bear only FcgRII (Littaua et al., 1990; Mady et al., 1993). THP-1 and K562 cells were each infected with mixtures containing serially diluted HuMAb and DENV After incubation for 72 h the vRNA in the cytoplasm was measured by quantitative RT-PCR. 3.2.1. FcgRI- and FcgRII-bearing THP-1 cells In THP-1 cells, D23-1G7C2-IgG1 and D23-1G7C2-IgG3 induced a
1024-fold enhancement of the production of DENV-1 vRNA in cells, while D23-1G7C2-IgG2 and D23-1G7C2-IgG4 induced a 64- and a 256-fold enhancement, respectively, suggesting that they had caused a reduction in ADE. D23-1G7C2-IgG1-N297A induced the least enhancement, with a 32-fold increase in production (Fig. 2A and Table 2). Production of DENV-2 was enhanced 256-fold by D23-1G7C2IgG1 and by 32-fold in the presence of D23-1G7C2-IgG3. By contrast, D23-1G7C2-IgG4 and D23-1G7C2-IgG2 induced less enhancement of viral production, at four- and eight-fold respectively. D23-1G7C2-IgG1-N297A induced a two-fold increase in DENV-2 production (Fig. 2B and Table 2). The pattern of the effect of the rHuMAbs on DENV-3 production was similar. D23-1G7C2-IgG1 induced the greatest enhancement (16-fold); D23-1G7C2-IgG3 and D23-1G7C2-IgG4 induced an
R. Ramadhany et al. / Antiviral Research 124 (2015) 61e68 Table 2 Summary of the maximum degree of enhancement reached for each HuMAb in an in vitro ADE assay. Virus
Antibody D23-1G7C2 IgG1
THP-1
K562
DENV-1 DENV-2 DENV-3 DENV-4 DENV-2
1024 256 16 32 64
a
IgG2
IgG3
IgG4
IgG-N297A
64 8 4 4 512
1024 32 8 128 128
256 4 8 16 32
32 2 3 4 16
a
The numbers indicate fold enhancement compared to cells that were not reacted with HuMAbs.
eight-fold enhancement, while D23-1G7C2-IgG2 increased production four-fold. D23-1G7C2-IgG1-N297A induced the least enhancement (three-fold; Fig. 2C and Table 2). In the presence of DENV-4, D23-1G7C2-IgG1 and D23-1G7C2IgG3 showed the greatest enhancement (both 128-fold), while D23-1G7C2-IgG2 and D23-1G7C2-IgG4 enhanced viral production four-fold and 14-fold, respectively. The D23-1G7C2-IgG1-N297A enhanced production four-fold (Fig. 2D and Table 2). While, compared to the other rHuMAbs, D23-1G7C2-IgG1 did induce the greatest degree of ADE on the infectivity of DENV-3, unlike its effect on the other DENV serotypes (1, 2, and 4), it was the lowest maximum enhancement observed, suggesting that ADE is also dependent on the virus serotype. Compared to the other rHuMAbs, in the presence of each DENV serotype D23-1G7C2IgG1-N297A consistently induced the least enhancement of infectivity, suggesting that the single mutation, N297A, in the Fc region was sufficient to disrupt ADE activity. 3.2.2. FcgRII-bearing K562 cells K562 cells express only type II FcgRs. Unlike ADE in THP-1 cells, in K562 cells the greatest enhancement was induced by D231G7C2-IgG2 (512-fold), while D23-1G7C2-IgG1, -IgG3, and -IgG4 induced enhancement 64-, 128-, and 32-fold, respectively. ADE activity of different IgG subclass depends on the type of FcgR and the target cell. Again, D23-1G7C2-IgG1-N297A induced the least enhancement (16-fold; Fig. 2E and Table 2), indicating that the introduction of a single mutation, N297A, into the Fc region of IgG1 disrupted ADE activity in both THP-1 and K562 cells. 3.3. D23-1G7C2-IgG1 protected mice from lethal infection with chimeric DENV, while D23-1G7C2-IgG1-N297A showed partial protection The protective ability of IgGs was examined in mice infected with virus. Because there was no other DENVs caused fatal infection in this mouse model, chimeric DENV carrying prME genome of DENV-2 and genome coding JEV NSs protein was used for challenge. All IgGs could equally neutralize chimeric DENV (Supplementary Fig. 1). IFN-a/b/gR KO mice were infected with a lethal dose of chimeric DENV, and 500 mg of rHuMAb was injected i.p. 20 h p.i. with 800 FFU of chimeric DENV. Mice that received the control 5E4 had all died by day 9 p.i. (Fig. 3A); by contrast, mice treated with D23-1G7C2-IgG1 did not start to die until 17 days p.i., and at the end of the 38-day study period had an 80% survival rate, whereas 40% of mice that received D23-1G7C2-IgG1-N297A survived (Fig. 3A). This result suggests that the introduction of the N297A mutation in the IgG1 Fc region reduced the protective activity of D23-1G7C2-IgG1. A similar tendency was found in IFN-a/b/ gR KO mice injected with 300 mg of HuMAb (Supplementary Fig. 2.). The viral titer in the sera of all the treated mice was determined on days 2, 6, and 12 p.i. At day-2 p.i., viremia was detected in mice
65
injected with 5E4 (5.8 10 copies/ml) and at a higher level in mice injected with D23-1G7C2-IgG1-N297A (1.97 102 copies/ml; Fig. 3B). There was no detectable level of viremia in mice injected with D23-1G7C2-IgG1. At day-6 p.i., viremia had increased in 5E4and D23-1G7C2-IgG1-N297A-treated mice (9.3 103 and 2.4 103 copies/ml, respectively), while there was no detectable viremia in mice injected with D23-1G7C2-IgG1. At day-12 p.i., all the mice injected with D23-1G7C2-IgG1 and the surviving mice injected with D23-1G7C2-IgG1-N297A showed little or no viremia. These results raised the question of whether D23-1G7C2-IgG1N297A retains sufficient neutralizing activity in mouse because it may be instable in sera. Sera collected from 5E4-injected mice on day 2 p.i. showed no neutralizing, activity (Fig. 3B), confirming that normal mouse serum does not contain neutralizing antibodies; however, sera collected on day 6 p.i. showed moderate neutralizing activity (95.6%). This activity was probably due to newly-generated antibodies induced by the viral infection. Despite producing antisera, all mice injected with 5E4 died by day 9 (Fig. 3A). Sera from mice injected with D23-1G7C2-IgG1 collected on days 2, 6, and 12 p.i. efficiently neutralized DENV-2 (Fig. 3C). Meanwhile, sera from mice injected with D23-1G7C2-IgG1-N297A collected 2 and 6 days p.i. showed moderate neutralization activity (both 64% by undiluted serum), but reduced neutralizing activity (38.3% by undiluted serum) on day-12 p.i. (Fig. 3C). This suggests that D23-1G7C2-IgG1N297A rapidly loses neutralizing activity when injected into mice. To confirm that the HuMAbs were present in the sera, reactivity was examined using DENV-2-infected Vero cells. HuMAb-specific reactivity was detected by IFA with an Alexa Fluor 488 conjugated anti-human IgG antibody to distinguish it from that of newly produced DENV-reactive mouse Abs. D23-1G7C2-IgG1 was detected even at day-25 p.i. (Fig. 3D). On the other hand, D23-1G7C2IgG1-N297A was detected 2 days p.i., but the signal was reduced by day-6 p.i. and undetectable on day-12 p.i. D23-1G7C2-IgG1N297A was rapidly degraded. Thus, although D23-1G7C2-IgG1N297A showed neutralizing activity against chimeric DENV-2 similar to that of D23-1G7C2-IgG1 (Fig. 1B and Table 1), it partially lost its protective ability. Furthermore, ADE was examined by the introduction of lower concentrations of HuMAbs in mice. A maximum ADE was observed by introduction of 8 mg of mouse monoclonal anti-DENV E Ab 4G2 (Kurosu et al., submitted for publication). However, introduction of both D23-1G7C2-IgG1 and D23-1G7C2-IgG1-N297A did not increase lethality nor viral load by introduction of 2 or 8 mg of HuMAbs (Supplementary Fig. 3). 4. Discussion Passive immunization with MAbs, especially humanized MAbs or human-derived MAbs, represents an attractive therapeutic approach. Nevertheless, there is concern that introduction of MAbs may exacerbate the patient's condition by the induction of ADE. To reduce the risk of ADE, HuMAbs would have to be genetically modified (Hanley and Weaver, 2010). Replacing certain subclasses of IgG reduced ADE activity, although the ADE activity of these antibodies depended on the type of target cell (Fig. 2 and Table 2). In addition, HuMAb with the Fc region mutation, D23-1G7C2-IgG1-N297A, showed the least ADE activity (Fig. 2), without losing neutralizing activity (Fig. 1B). A single mutation at N297 residue of IgG has been shown to impair the binding to FcgR (Arnold et al., 2007; Sazinsky et al., 2008; Lux et al., 2013). D23-1G7C2-IgG1-N297A is an ideal therapeutic antibody; however, D23-1G7C2-IgG1-N297A partially lost its protective activity (Fig. 3A). The mutation at N297 of IgG possibly induce conformational change of the Fc region (Wright and Morrison, 1997; Siberil et al., 2006) and short half-life in mice. This may be
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Fig. 3. Protection of IFN-a/b/gR KO mice from chimeric DENV-2 by rHuMAbs. (A) Survival rate of IFN-a/b/gR KO mice. Five or six IFN-a/b/gR KO mice in each group were infected with 800 FFU of chimeric DENV-2 i.p. After 20 h mice were then injected i.p. with 500 mg of D23-1G7C2-IgG1, D23-1G7C2-IgG1-N297A, or 5E4, and observed until day-38 p.i. Asterisks indicate a statistically significant difference of P < 0.01. (B) Viral copy number in mouse sera collected from each group of mice at day-2, -6, and -12 p.i. was determined by quantitative RT-PCR as described in the Materials and methods. (C) Serially two-fold diluted sera collected from mice at day-2, -6, and -12, were mixed with 100 FFU of chimeric DENV-2 and inoculated into Vero cells. A neutralization assay was performed as described in the Materials and methods. (D) Vero infected with DENV were incubated with sera collected from chimeric DENV-2- and HuMAb-treated mice as described above for Fig. 3A, and visualized with Alexa Fluor 488- goat anti-human IgG by fluorescent microscopy.
the reason why, in our study, D23-1G7C2-IgG1-N297A was undetectable in mouse serum at day 12 p.i., while D23-1G7C2-IgG1 was detected even at day 25 p.i. (Fig. 3D). Another possible explanation for the short half-life of D23-1G7C2-IgG1-N297A is that the N297A mutation reduces its affinity for the neonatal Fc receptor (FcRn), which functions as a salvage receptor to rescue IgG from the lysosomal degradation pathway (Kobayashi et al., 2002). The recycling of Ab through FcRn contributes to their long half-life. To some extent, the N297A mutant Ab shows reduced binding affinity for FcRn (Shields et al., 2001). This may be the reason why the amount
of D23-1G7C2-IgG1-N297A in mouse serum decreased (Fig. 3D). In contrast to the present study, another study using a different model showed that a similar chimeric humanized N297Q mutant fully protected mice from lethal infection with DENV (Balsitis et al., 2010). These differing observations may be due to differences in the viruses studied. For example, chimeric DENV-2 replicates much faster in mice. Of note, a low amount (800 FFU) of chimeric DENV-2 was sufficient to cause 100% lethality (Fig. 3A), whereas a high dose of DENV was needed to cause lethal infection in the model studied by Balsitis et al. (2010). Furthermore, mice infected with 800 FFU of
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chimeric DENV-2 showed a serum titer greater than 106 FFU/ml at day 5 p.i. (Kurosu et al., submitted for publication). In the present model, mice did not survive unless the injected Ab reduced the virus tier to below at least 800 FFU. Human cases of infection begin after exposure to a small amount of DENV via a mosquito bite; the virus then actively replicates and spreads throughout the body, resulting in high levels of viremia. The mouse model used in the present study is similar to human cases of infection in terms of virus replication kinetics. Furthermore, mice injected with D231G7C2-IgG1-N297A did not generate a protective Ab response at the early stages of infection (Fig. 3C). This suggests that D231G7C2-IgG1-N297A interfered with the humoral immune response. We believe that this is an important observation with respect to the practical application of Ab therapy. This mouse model also revealed another possibility with respect to Ab therapy. Although D23-1G7C2-IgG1-N297A is safe in terms of ADE, it was rapidly cleared from mouse serum (Fig. 3D). By contrast, although D23-1G7C2-IgG1 efficiently protected mice (Fig. 3A), it showed higher ADE in vitro (Fig. 2 and Table 2). The half-life of an Ab is important with respect to therapy. Differences in IgG subclass do not affect the affinity for FcRn, and all subclasses have a similar half-life (West and Bjorkman, 2000). Other IgG subclass, such as IgG2 or IgG4, which showed lower ADE (Fig. 2 and Table 2), may be more secure than IgG1. Alternatively, changes in glycosylation may affect Ab affinity; for example, Ha et al. reported that IgG Ab carrying asymmetric glycosylations at N297 cannot bind FcgRs without losing affinity for FcRn. Although technical difficulties remain, it may be considered as a next candidate (Ha et al., 2011). This study raises the question of whether N297 mutant IgG has a short half-life in humans. Further study is needed to develop an effective and safe therapeutic HuMAb. Acknowledgments This work was supported by a grant-in-aid (21790444) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and a program of the Japan Initiative for Global Research Network on Infectious Diseases launched by a project commissioned by the MEXT. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.antiviral.2015.10.012. References Arnold, J.N., Wormald, M.R., Sim, R.B., Rudd, P.M., Dwek, R.A., 2007. The impact of glycosilation on the biological function and structure of human immunoglobulins. Annu. Rev. Immunol. 25, 21e50. Balsitis, S.J., Williams, K.L., Lachica, R., Flores, D., Kyle, J.L., Mehlhop, E., Johnson, S., Diamond, M.S., Beatty, P.R., Harris, E., 2010. Lethal antibody enhancement of Dengue disease in mice is prevented by Fc modification. PLoS Pathog. 6, e1000790. Boonak, K., Slike, B.M., Burgess, T.H., Mason, R.M., Wu, S., Sun, P., Porter, K., Rudiman, I.F., Yuwono, D., Puthavathana, P., Marovitch, M.A., 2008. Role of dendritic cells in antibody-dependent enhancement of Dengue virus infection. J. Virol. 82, 3939e3951. Boonnak, K., Slike, B.M., Donofrio, G.C., Marovich, M.A., 2013. Human FcgRII cytoplasmic domains differentially influence antibody-mediated dengue virus infection. J. Immunol. 190, 5659e5665. Burke, D.S., Nisalak, A., Johnson, D.E., Scott, R.M., 1988. A prospective study of dengue infections in Bangkok. Am. J. Trop. Med. Hyg. 38, 172e180. Chareonsirisuthigul, T., Kalayanarooj, S., Ubol, S., 2007. Dengue virus (DENV) antibody-dependent enhancement of infection upregulates the production of anti-inflammatory cytokines, but suppresses anti-DENV free radical and proinflammatory cytokine production, in THP-1 cells. J. Gen. Virol. 88, 365e375. Fleit, H.B., Kobasiuk, C.D., 1991. The human monocyte-like cell line THP-1 expresses FcgRI and FcgRII. J. Leukoc. Biol. 49, 556e565. Graham, R.R., Juffrie, M., Tan, R., Hayes, C.G., Laksono, I., Ma'roef, C., Erlin, Sutaryo,
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