article: Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit infection of all three CHIKV genotypes. Four of 36 neutralizing MAbs (CHK-102, CHK-152, CHK-166, and CHK-263) provided complete protection against lethality as prophylaxis in highly susceptible immunocompromised mice lacking the type I IFN receptor (Ifnar−/−) and mapped to distinct epitopes on the E1 and E2 structural proteins. CHK-152, the most protective MAb, was humanized, shown to block viral fusion, and require Fc effector function for optimal activity in vivo. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs (CHK-102+CHK-152 or CHK-166+CHK-152) limited the development of resistance and protected immunocompromised mice against disease when given 24 to 36 hours before CHIKV-induced death. Selected pairs of highly neutralizing MAbs may be a promising treatment option for CHIKV in humans.
  Chikungunya virus (CHIKV) infection causes a severe febrile illness in humans that is characterized by a debilitating polyarthritis, which can persist for months and cause significant morbidity [1], [2]. There are three genotypes of CHIKV: Asian, East/Central/South African (ECSA), and West African [3]–[5], with 95. 2 to 99. 8% amino acid identity [4]. The CHIKV strains from the recent epidemics belong to the ECSA genotype and have affected millions in Africa and the Indian subcontinent [3], [6]. Imported cases in the United States and outbreaks in Europe highlight the threat of CHIKV to developed countries [7]. Currently, there are no approved vaccines or therapeutics for CHIKV [8]. CHIKV is an enveloped alphavirus of the Togaviridae family that enters cells via receptor-mediated internalization and a low pH-triggered type II membrane fusion event in early endosomes. The mature virion is comprised of three structural proteins: a nucleocapsid protein and two glycoproteins, E1 and E2, where E2 functions in attachment to cells and E1 participates in virus fusion. Each 700 Å CHIKV virion contains 240 copies of the envelope and capsid proteins, which are arranged in T = 4 quasi-icosahedral symmetry. E1-E2 heterodimers assemble into 80 trimeric spikes on the virus surface [9]. X-ray crystallographic structures of the precursor pE3-E2-E1, mature E2-E1, and E1 proteins [10]–[13] have elucidated the architecture of the glycoprotein shell. The E1 ectodomain consists of three domains. Domain I (DI) is located between DII and DIII, the latter of which adopts an immunoglobulin-like fold. The fusion peptide is located at the distal end of DII. E1 monomers lie at the base of the surface spikes and form a trimer around each of the icosahedral axes. E2 localizes to a long, thin leaf-like structure on the top of the spike. The mature E2 protein contains three domains with immunoglobulin-like folds: the N-terminal domain A, located at the center; domain B at the tip; and the C-terminal domain C, located proximal to the viral membrane. Mouse models have been developed for CHIKV infection. Newborn outbred and inbred mice are vulnerable to severe CHIKV infection with viral replication observed in muscle, joint, and skin [14], [15]. Adult mice with defects in type I interferon signaling (Ifnar−/− mice) develop lethal disease, with muscle, joint, and skin appearing as the primary sites of infection [15]. CHIKV infection of juvenile C57BL/6 mice by a subcutaneous route results in metatarsal foot swelling with histological evidence of arthritis, tenosynovitis and myositis [16], [17]. Passive transfer of MAbs or immune sera can protect animals against infection of alphaviruses including Sindbis (SINV), Semliki Forest (SFV), and Venezuelan equine encephalitis (VEEV) viruses [18]–[25]. Immune γ-globulin from human donors in the convalescent phase of CHIKV infection exhibited neutralizing activity in vitro and had partial therapeutic efficacy in Ifnar−/− and neonatal wild type mice when administered up to 24 hours after infection [26]. Although mouse and human MAbs that neutralize CHIKV infection have been reported [27], [28], their post-exposure efficacy against lethal infection in vivo has not been clearly established [29]. Here, we investigated the molecular basis of antibody-mediated neutralization of CHIKV using a panel of 230 newly generated, cloned MAbs. CHK-152 protected mice against CHIKV-induced mortality and disease. The inclusion of a second MAb (CHK-166 or CHK-102) prevented the emergence of viral resistance and extended the treatment window in Ifnar−/− mice up to 24 to 36 hours prior to death of the animals. Our results suggest that combination therapy with selected neutralizing MAbs has potential for treatment of CHIKV infection in humans.
  We generated a panel of neutralizing MAbs against CHIKV as a first step towards a possible therapy in humans. We infected adult C57BL/6 mice deficient for interferon regulatory factor 7 (Irf7−/−) with 104 PFU of the La Reunion 2006 OPY-1 strain of CHIKV (CHIKV-LR); these mice were boosted with CHIK virus-like particles [30], soluble recombinant CHIKV E2 protein, or live CHIKV-LR. We immunized Irf7−/− rather than wild type (WT) mice, as CHIKV replicated to higher titers, induced stronger neutralizing antibody responses, yet did not cause lethal infection in these innate immune-deficient animals ([31], and data not shown). We screened four independent myeloma cell-splenocyte fusions for binding of hybridoma supernatants to CHIKV-LR infected cells (Fig. S1) and cloned 230 CHIKV-specific MAbs for further analysis (Table S1 in Text S1). Using a single endpoint neutralization assay, we identified 36 MAbs with inhibitory activity against infection of CHIKV-LR in BHK21-15 cells (data not shown). To assess the inhibitory potential of our anti-CHIKV MAbs against the homologous CHIKV-LR and representative strains from the Asian and West African genotypes (RSU1 and IbH35 respectively), we performed focus reduction neutralization tests (FRNTs) on Vero cells. We determined the concentration of MAb that reduced the number of foci of infection by 50 or 90% (EC50 and EC90 values, Fig. 1A and B, and Table 1). CHK-152 was the most strongly neutralizing MAb we identified; 3 and 15 ng/ml of this MAb prevented 50 and 90% of CHIKV infection against all three CHIKV genotypes (Fig. 1C). Ten other MAbs inhibited CHIKV infection with EC50 values of <10 ng/ml against all three genotypes, and many others inhibited all three strains similarly, with a few exceptions. For example, CHK-9 failed to neutralize the Asian strain to the same extent as the West African or La Reunion (ECSA genotype) strains (Fig. 1D), whereas CHK-151 inhibited infection of the Asian strain better than the others (Table 1). Also, for reasons that are unclear, some neutralizing MAbs (e. g. , CHK-143, CHK-264, and CHK-269) were incapable of inhibiting all viruses (EC90>10,000 ng/ml) in this assay, even at high MAb concentrations. We speculated that some MAbs might show cell type-dependent neutralization if they blocked attachment to cell type-specific factors. To test this hypothesis, we assessed MAb neutralization of CHIKV-LR infection in cells of another species, NIH 3T3 mouse fibroblasts (Table 1). For most MAbs, the EC50 values were comparable to those achieved with Vero cells. However, two MAbs (CHK-96 and CHK-176) showed a 12 to 250-fold reduction (P<0. 05) in neutralizing activity on NIH 3T3 compared to Vero cells; although further study is warranted, these MAbs may block a step in the entry pathway that varies among different cell types. To evaluate whether neutralizing MAbs protect against CHIKV infection in vivo, we initially used a stringent test model: prevention of lethal infection in immunodeficient Ifnar−/− C57BL/6 mice. One hundred micrograms of 14 different MAbs with strong, modest, or poor neutralizing activity were administered to Ifnar−/− mice one day prior to CHIKV-LR infection. As seen previously [15], all Ifnar−/− mice died by day 4 after infection when treated with saline or a negative control MAb (Fig. 2A, and data not shown). Strongly neutralizing (e. g. , CHK-102, CHK-152, and CHK-263) and one moderately inhibitory (CHK-166) MAb protected 100% of mice from lethal infection (P<0. 0001). In comparison, and somewhat surprisingly, CHK-95, a potently neutralizing MAb of the same IgG2c isotype, protected only 12% of mice from death. The other MAbs tested conferred intermediate levels of protection (Fig. 2A). Thus, although several strongly neutralizing MAbs prevented against lethal CHIKV infection in Ifnar−/− mice, in vitro neutralization activity per se did not directly correlate with protection. To define the relative potency of the four MAbs that completely prevented lethal disease, we administered a lower (10 µg) dose. Whereas CHK-152 and CHK-263 still protected most mice from lethal infection, CHK-102 and CHK-166 protected to a lesser degree or only prolonged survival (Fig. 2B). Consistent with their ability to protect against lethal infection, passive transfer of CHK-102, CHK-152, CHK-166, and CHK-263 MAbs all markedly reduced viral loads in serum, spleen, liver, muscle, and brain at 48 hours after infection relative to a non-binding isotype control (DENV1-E98) MAb (Fig. 2C–G). The level of protection afforded by CHK-102, CHK-152, CHK-166, and CHK-263 MAbs, however, did not correlate directly with their binding strength to CHIKV surface glycoproteins (Fig. S2). Although a stringent test of MAb protection, CHIKV-infected Ifnar−/− mice do not develop the arthritis observed in humans. To evaluate this, we utilized a WT C57BL/6 mouse model in which inoculation of CHIKV into the footpad results in localized swelling and induction of arthritis and fasciitis within the foot and ankle [16], [17], although infection does not cause lethality. Pretreatment of mice with either 100 µg of CHK-102 or CHK-152 completely protected against CHIKV-induced swelling, compared to control animals, which developed clinically apparent swelling (data not shown). While CHIKV infected control animals developed inflammatory arthritis in the ankle and foot, CHK-102 or CHK-152 MAb treated animals had normal appearing joint tissues (Fig. 2H). Antibody neutralization of enveloped viruses can occur by inhibiting attachment, internalization, and/or fusion [32], [33]. To determine how many of our most protective MAbs inhibited infection in cell culture, we performed pre- and post-attachment neutralization assays [34], [35]. Anti-CHK MAbs were incubated with CHIKV before or after virus binding to cells, and infection was measured. As expected, all MAbs efficiently neutralized infection when pre-mixed with virus (Fig. 3A). While CHK-102, CHK-152, CHK-166, and CHK-263 also inhibited CHIKV infection when added after virus adsorption to the cell surface, suggesting that at least part of their blocking activity was at a post-attachment step, differences in the extent of neutralization were noted in this context for several MAbs. CHK-152 completely neutralized all CHIKV virions without a resistant fraction when added post-attachment. When studies were repeated with eight other neutralizing MAbs that showed pre-exposure protection in vivo, no other MAb inhibited infection completely when added after virus adsorption to the cell. As expected, an isotype control MAb (DENV1-E98) and a non-neutralizing anti-CHK MAb (CHK-84) had no inhibitory effects in this assay (Fig. S3). Since CHK-152 neutralized infection efficiently at a post-attachment step, we investigated whether it blocked fusion using a viral fusion from without (FFWO) assay [36]. CHIKV was adsorbed to Vero cell monolayers on ice and then treated with MAbs. Fusion at the plasma membrane was triggered after a brief exposure to low pH buffered medium at 37°C. Subsequently, cells were incubated in the presence of 20 mM NH4Cl to prevent CHIKV fusion via canonical endosomal pathways. As expected, at 14 hours after initial treatment, CHIKV infection was not observed when adsorbed virus was incubated at neutral pH (Fig. 3B). In comparison, in the absence of MAb or in the presence of a control MAb, a short exposure of cell surface-adsorbed virus to acidic pH resulted in infection and CHIKV-antigen positive cells. Notably, CHK-152 completely inhibited (P<0. 0001) plasma membrane fusion and infection, whereas other anti-CHIKV neutralizing MAbs showed significant yet incomplete inhibition in this assay (Fig. 3B and C). These studies suggest that CHK-152 efficiently neutralizes infection by preventing the structural changes on the virion necessary for viral fusion with host cell membranes. We utilized a model liposome fusion assay with pyrene-labeled virus [37], [38] to confirm these results. Pyrene-labeled CHIKV was pre-incubated with different concentrations of MAb, mixed with liposomes at 37°C, and fusion was triggered by addition of a low-pH buffer [37]. In the absence of MAb or in the presence of 10 nM (1. 5 µg/ml) of a non-binding control MAb, fusion was complete within seconds of acidification. In contrast, pre-incubation of virus with increasing doses of CHK-152 inhibited fusion (Fig. 3D and E). Thus, CHK-152 can block low-pH-induced fusion of virus with liposomes. To define additional mechanisms by which our most strongly protective MAb (CHK-152) conferred protection in vivo, we generated a chimeric mouse-human CHK-152 (ch-CHK-152) as well as an aglycosyl variant (ch-CHK-152 N297Q) that lacks the ability to engage C1q or Fc-γ receptors; this mutation does not affect the ability to bind the neonatal Fc receptor (FcRn) or half-life of antibody in mouse serum [39]. The affinity of ch-CHK-152 and ch-CHK-152 N297Q binding to purified pE2-E1 was measured by surface plasmon resonance (SPR) and compared to the parent murine MAb. Notably, ch-CHK-152, ch-CHK-152 N297Q, and the murine CHK-152 all had similar affinity (KD of 3 to 4 nM) (Fig. 4A and data not shown) and neutralizing activity in cell culture (Fig. 4B). As expected, ch-CHK-152 N297Q failed to bind efficiently to soluble Fc-γ receptors or C1q (Fig. 4C). We transferred ch-CHK-152 and ch-CHK152 N297Q to Ifnar−/− mice prior to infection. Although high doses (100 µg) of ch-CHK-152 and ch-CHK-152 N297Q provided similar protection against CHIKV infection (data not shown), lower doses (10 µg) of the aglycosyl variant were less protective; whereas 62% of the mice receiving ch-CHK152 N297Q survived, all Ifnar−/− mice given ch-CHK-152 MAb remained alive (Fig. 4D, P<0. 05). When parallel studies were performed with WT C57BL/6 mice and MAb was administered 18 hours after infection, ch-CHK-152 N297Q also provided less protection against arthritis compared to ch-CHK-152 (Fig. 4E). These data suggest that the Fc effector interactions contribute to the potency of CHK-152 in mice. We humanized CHK-152 as a first step towards a MAb therapeutic (see Text S1). The affinity for pE2-E1 and neutralizing activity of the hu-CHK-152 were similar to mouse CHK-152 (Fig. S4A and B). Hu-CHK-152 also protected Ifnar−/− mice (P>0. 0001) when a single dose (10 or 100 µg) was administered one day before infection (Fig. S4C). To define the therapeutic potential of our most protective MAbs, a single dose (100 µg) was administered to Ifnar−/− mice 24 hours after CHIKV infection (Fig. 5A). Whereas CHK-152 and 166 protected 58% and 63% of mice from death, respectively (P<0. 0001), CHK-263 and CHK-102 had less activity although both MAbs increased the median survival time (7 days versus 4 days with the control DENV1-E98 MAb, P<0. 0006). Administration of CHK-152 at 12 or 18 hours post infection also protected WT mice from CHIKV-induced swelling and arthritis (Fig. 5B and Fig. 4E). We next tested the activity of combinations of the most protective neutralizing MAbs in Ifnar−/− mice. Remarkably, administration of 50 µg each (100 µg total dose) of CHK-102+CHK-152, CHK-263+CHK-152, or CHK-166+CHK-152 at 24 hours post infection completely prevented mortality in all animals (Fig. 5A, P<0. 0001 for MAb combinations). This observation was not true for all MAb combinations, as administration of 50 µg each of CHK-102+CHK-263 provided substantially less protection with a 14% survival rate. We then performed a more stringent test in which 100 µg each (200 µg total) of our most protective combinations was delivered as a single dose at 48 hours post-infection (Fig. 5C). Treatment with CHK-102+CHK-152 or CHK-166+CHK-152 protected 62% of the Ifnar−/− mice (P<0. 003) and the combination of CHK-263+CHK-152 functioned almost as well, with 50% of animals surviving (P<0. 03). To define the limits of protection in Ifnar−/− mice, which all succumb to CHIKV between days 3 and 4, therapy was initiated at 60 and 72 hours after infection. At 60 hours after infection, Ifnar−/− mice receiving 250 µg each of CHK-102+CHK-152 or CHK-166+CHK-152 had survival rates of 28 and 71%, respectively (Fig. 5D, P = 0. 03 and P = 0. 004). Nonetheless, when combination therapy was given at 72 hours after infection, a time when overt disease was present, no survival benefit was conferred. Thus, combination MAb therapy is superior to monotherapy in protecting against lethal CHIKV infection in highly immunocompromised mice. To begin to understand the basis for enhanced in vivo activity, we assessed whether CHK-152 and selected MAbs could bind simultaneously to the CHIKV virion. We developed a competition ELISA in which virions were captured by a mouse MAb (CHK-65), and then incubated with increasing concentrations of CHK-102, CHK-152, CHK-166, or CHK-263 mouse MAbs. After washing, hu-CHK-152 MAb was added, and binding was assessed. While pre-bound mouse CHK-152 competed against hu-CHK-152 binding as expected, CHK-102, CHK-166, and CHK-263 minimally competed hu-CHK-152 binding (Fig. S5A), suggesting their epitopes largely were distinct. However, addition of CHK-102, CHK-166, or CHK-263 failed to augment the inhibitory activity of CHK-152 when neutralization was measured in cell culture (Fig. S5B), as no synergy was observed. To identify epitopes targeted by the therapeutic MAbs, we generated escape mutants in cell culture. After sequential virus passage under CHK-102, CHK-152, CHK-166, or CHK-263 selection, CHIKV became resistant to neutralization by these MAbs (Fig. 6A–D). We assessed whether the escape variants generated in the presence of one MAb remained sensitive to neutralization by the other MAbs. The CHK-152 escape variant was neutralized efficiently by CHK-102, CHK-166, and CHK-263 (Fig. 6B, Table S2 in Text S1, and data not shown), and analogously the CHK-166 escape variant was inhibited by CHK-102, CHK-152, and CHK-263 (Fig. 6C, and data not shown). In contrast, CHK-102 and CHK-263 escape variants reciprocally were resistant, suggesting their epitopes were the same or overlapping (Fig. 6A and D); however, CHK-102 and CHK-263 escape variants remained sensitive to neutralization by CHK-152 and CHK-166. Notably, selection with combinations of MAbs (e. g. , CHK-102+CHK-152) failed to produce escape variants despite several independent attempts (data not shown). To identify the mutations that conferred resistance, we sequenced plaque-purified escape variants (Table 2, top). Six of eight sequences from CHK-102 escape variants contained an L210P mutation in the E2 protein; the remaining two sequences had a G209E mutation in E2. For CHK-152 resistant variants, all sequences (9 of 9) contained a D59N mutation in E2 and two contained a second A89E substitution in E2. For CHK-263,3 of 4 escape variants had a K215E change in E2, whereas 1 of 4 had mutations in E2 at G209E. All escape variants (14 of 14) of CHK-166 had a single K61T mutation in the E1 protein. To verify the amino acid changes that conferred MAb resistance in vitro, we introduced several of these substitutions into a chimeric SFV-GFP-CHIKV cDNA comprised of SFV non-structural genes, a GFP reporter gene, and the CHIKV structural genes (T. Lin, K. Dowd, and T. Pierson, unpublished results). Parental and SFV-GFP-CHIKV with single amino acid mutations were analyzed for neutralization by CHK-102, CHK-152, CHK-166, and CHK-263 (Fig. 6E–H). Consistent with our sequencing results, viruses encoding mutations in E2-G209 and E2-L210 were resistant to CHK-102, changes in E2-D59 conferred resistance to CHK-152, substitutions in E1-K61 resulted in resistance to CHK-166, and mutation of E2-G209 and E2-K215 caused resistance to CHK-263. However, introduction of E2-A89E (which was present in 2 of 9 clones) failed to affect the neutralizing activity of CHK-152. In addition to selecting escape variants in cell culture, we harvested organs from the few mice that became ill after infection despite single MAb treatment (Table 3, bottom). In these moribund Ifnar−/− mice, CHIKV was present in the brain and muscle but absent from the spleen or liver (data not shown). This in vivo-derived virus was tested for MAb resistance and sequenced. For mice receiving a 10 µg dose of CHK-102 as prophylaxis, resistant variants with a L210P mutation in E2 were obtained. For mice receiving CHK-263 or CHK-102 at 24 hours post infection, resistant viruses with a G209E mutation in E2 were identified. None of the animals that were pre-treated with 10 µg of CHK-166 developed escape mutants, as the virus harvested from all 3 mice tested retained sensitivity to CHK-166 (data not shown). However, in one animal receiving CHK-166 at 24 hours post infection, a single resistant virus with a G64S substitution in the E1 gene was recovered (Fig. S6). For mice receiving a 10 µg dose of hu-CHK-152 as prophylaxis, partially resistant viruses with N231D and K233E mutations in E2 were isolated and confirmed by reverse genetics using the chimeric SFV-GFP-CHIKV infectious clone (Fig. S7). In comparison, when CHK-152 was given as a therapeutic, a single mutation at D59N in E2 was obtained in 4 of the 5 mice tested, with a K233T mutation in virus from the remaining animal. For animals treated at 48 hours with combination MAb therapy, all recovered viruses remained sensitive to CHK-152 yet showed partial resistance to CHK-102 or CHK-166 (Fig. S8). Mutations in E2 (N332I, CHK-166+CHK-152) were identified. Comparison of 140 available E1 and E2 sequences from historical and circulating CHIKV strains in a public database (http: //www. viprbrc. org/) revealed nearly complete conservation of the residues in which escape mutants were selected: E1-K61,100%; E1-G64,100%; E2-D59,100%; E2-G209,100%; E2-L210,99. 3%; E2-K215,100%; E2-N231,100%; and E2-K233,99. 3%. To define spatially the location of the amino acids that conferred resistance to our highly protective MAbs, these residues were mapped onto the existing CHIKV protein crystal structures [10] (Fig. 6I, left). Amino acids that conferred neutralization escape to CHK-102 and CHK-263 were located in the B domain of E2. The residues that modulated CHK-152 neutralization mapped to the A domain of E2. In contrast, CHK-166 recognized amino acids on DII of E1, adjacent to the fusion loop. All amino acids that conferred neutralization escape appear solvent accessible and highly exposed when docked onto the E2-E1 spike (Fig. 6I, right).
  We set out to identify MAbs with the greatest therapeutic activity against CHIKV in mice as a first step toward generating an immunotherapy for humans. Thirty-six MAbs with neutralizing activity against CHIKV-LR were identified, the majority of which also inhibited infection of strains corresponding to the two heterologous CHIKV genotypes. Although all fourteen of the selected anti-CHIKV MAbs improved outcome in vulnerable Ifnar−/− mice, only four of these (CHK-102, CHK-152, CHK-166, and CHK-263) completely prevented lethality when administered as prophylaxis. CHK-152 provided the greatest benefit as post-exposure therapy, although by itself, the window of treatment activity was limited in the Ifnar−/− mouse model. While addition of a second MAb (CHK-102, CHK-166, or CHK-263) failed to enhance CHK-152 neutralization in vitro, it limited the development of viral resistance in vitro and in vivo. Remarkably, combinations of CHK-102+CHK-152 or CHK-166+CHK-152 protected Ifnar−/− mice against mortality even when a single dose was administered 24 to 36 hours prior to the death of untreated or isotype control MAb-treated animals. In comparison to the highly therapeutic activity of 0. 5 mg of CHK-152+CHK-166, a single 25 mg dose of immune IgG purified from a convalescent human subject protected only 50% of Ifnar−/− mice when administered 24 hours after CHIKV infection [26]. The administered dose of neutralizing antibody likely is critical to post-exposure treatment of CHIKV infection because of the high viral burden [14], [16], [17], [40]. A high viral load impacts therapeutic activity of antibodies as it (a) increases the chance for pre-existing or selected resistant variants to emerge through quasispecies [28], [41]; and (b) results in a low relative fractional occupancy of binding to any individual virion, which allows antibodies recognizing key epitopes to fall below their stoichiometric threshold of neutralization [42]. Although there is extensive literature on the protective efficacy of MAbs or immune sera against alphavirus infection [18]–[25], no prior study has demonstrated reduced CHIKV-induced mortality with MAbs. Although a recent study showed that combination post-exposure therapy with two human anti-CHIKV MAbs (5F10 and 8B10,250 µg each at +8 h) prolonged survival of AG129 (Ifnar−/−×Ifngr−/−) mice by ten days, they failed to prevent lethal infection [29]; the basis of this treatment failure remains unclear but could reflect the lower neutralizing potency of the MAbs (compared to CHK-152), rapid emergence of resistant mutants, or the relative susceptibility of the immunocompromised mouse host. In comparison, a neutralizing MAb (UM 5. 1) administered two days after SFV infection completely protected immunocompetent BALB/c mice [43]. Why were some combinations of two MAbs effective in vivo? (a) Pairs of MAbs may show neutral, additive, or synergistic effects on neutralization. Positive antiviral effects could occur through cooperative binding or by trapping CHIKV in conformations that makes it less competent to bind a receptor or fuse with host membranes. Nonetheless, when we added increasing concentrations of CHK-102, CHK-166, or CHK-263 to CHK-152, we failed to observe synergy. (b) Certain MAb combinations could prevent the emergence of resistance due to the low frequency of two escape mutations occurring simultaneously in a single replication cycle. Although we could readily select for neutralization escape against a single MAb in vitro and in vivo, we failed to isolate resistant mutants against CHK-152 when two MAbs (e. g. , CHK-102+CHK-152) were combined. However, some viruses from moribund animals treated with combination MAb therapy showed reduced sensitivity (up to 200-fold) to the other MAb (e. g. , CHK-102) in the pair. In comparison, when mice were treated with a combination of 50 µg each of CHK-102+CHK-263, we failed to observe the same survival benefit that was conferred by the combinations of CHK-102, CHK-166, or CHK-263 with CHK-152. Since CHK-102 and CHK-263 appear to share overlapping footprints on domain B of E2, this particular MAb combination may fail to prevent the rapid emergence of escape mutants relative to others targeting distinct epitopes on E1 and E2 proteins. (c) Combinations of MAbs could select for resistant viruses that have reduced fitness [44], and thus are less pathogenic in vivo. Virulence studies with CHIKV encoding selected single and double mutations are planned to evaluate this possibility. We localized the epitopes of our four highly protective MAbs using neutralization escape selection, sequencing, and reverse genetics. CHK-152, which blocked viral fusion, mapped to the wings of the A domain on E2, a result that we recently confirmed by cryo-electron microscopic analysis of CHK-152 Fab-virus particle complexes [45]. This epitope also was identified as a recognition site for neutralizing MAbs against VEEV [46] and SINV [47]. CHK-166, which was the least neutralizing (EC50 of ∼100 ng/ml) of our highly protective MAbs mapped to an epitope in domain II of the E1 protein, adjacent to the highly conserved fusion loop. While anti-E1 MAbs against SINV and VEEV that protect or neutralize infection have been described [46], [48], [49], none have been characterized against CHIKV. A neutralizing human MAb (8B10) against CHIKV was reported with possible reactivity against E1, although further analysis revealed that it bound to the E1/E2 heterodimer [27], [28]. CHK-102 and CHK-263 mapped to residues within the B domain on E2. A related epitope also was identified in mapping studies of strongly neutralizing antibodies against Ross River virus [50], SINV [51], [52], VEEV [46], [53], [54], and CHIKV [10], [28]. The B domain on E2 comprises an important antigenic domain that is under selective pressure for antibody neutralization [41]. It serves as a cap to the fusion loop on E1 and because of its location at the tip of the heterodimeric spike [10], [11] may contribute to attachment of cellular receptors. In summary, we identified combinations of MAb pairs that were highly effective as post-exposure therapeutic agents. These findings are consistent with recent studies showing enhanced post-exposure efficacy of MAb combinations against Ebola [55], influenza A [56] and rabies [57] viruses. Our most promising pair of MAbs mapped to distinct epitopes, limited the generation of resistance, blocked multiple stages of the viral entry pathway, and protected Ifnar−/− mice against mortality even when administered 60 hours after infection. CHK-152 was humanized as a first step towards a possible therapeutic for humans and demonstrated similar efficacy compared to the parent murine MAb. Tailored combinations of potently neutralizing MAbs show promise to prevent or treat infection by CHIKV, and likely other pathogenic alphaviruses in humans. Ultimately, a more detailed kinetic analysis of CHIKV infection in humans and determination of a treatment window relative to symptom onset is warranted to establish whether combination MAb therapy can prevent or mitigate acute or chronic and persistent infection and joint disease.
  Vero, Vero76 (ATCC), BHK21-15, and NIH 3T3 mouse fibroblast cells were cultured in Dulbecco' s modified Eagle medium (DMEM) supplemented with 5% or 15% (for 3T3 cells) fetal bovine serum (FBS) (Omega Scientific). C6/36 Aedes albopictus cells were grown in Leibovitz-15 medium supplemented with 10% FBS at 27°C. The infectious clones of CHIKV La Reunion 2006 OPY-1 (strain 142, CHIKV-LR) and CHIKV-GFP (strain 145) were gifts from S. Higgs (Manhattan, KS) [58]. CHIKV-RSU1 and CHIKV-IbH35 were gifts of R. Tesh, (Galveston, TX). Infection studies of WT mice used the SL15649 strain of CHIKV, which was generated from an infectious clone [17]. The S27 African prototype CHIKV strain was a gift from Dr. S. Günther (Bernhard-Nocht-Institute for Tropical Medicine, Germany) and isolated from a patient in Tanzania in 1953. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at the Washington University School of Medicine (Assurance Number: A3381-01) and the University of North Carolina (A3410-04). Dissections and footpad injections were performed under anesthesia that was induced and maintained with ketamine hydrochloride and xylazine, and all efforts were made to minimize suffering. Chimeric SFV-CHIKV virus was generated by complementation of a double sub-genomic DNA-launched SFV replicon “backbone” plasmid (pSFV-GFP-BB) with the structural genes of CHIKV as described recently for WNV [59]. The vectors and methods will be described in detail elsewhere (TY Lin, K. Dowd, and T. Pierson, in preparation). To generate SFV-CHIKV, a DNA fragment encoding WT or mutant CHIKV structural genes was ligated into the pSFV-GFP-BB plasmid and transfected directly into HEK-293T cells using Lipofectamine LTX. The source of CHIKV structural genes was a sub-cloning vector pCHIKV-struct: mutations were introduced into this vector using site-directed mutagenesis and fully sequenced. Virus was harvested at 48,72, or 96 hours after transfection, filtered, and stored at −80°C. The CHIKV E2 ectodomain (residues S1-E361) and pE2-E1 (E3-E2-E1: residues S1-R64 of E3, S1-E161 of E2, and Y1-Q411 of E1 including a (GGGS) 4 polylinker between E2 and E1) of the CHIKV-LR strain were amplified from the infectious cDNA clone using high-fidelity Phusion PCR (Thermo Scientific). The E2 ectodomain was cloned into pET21a, expressed in E. coli, and purified using an oxidative refolding protocol followed by size-exclusion column purification using fast protein liquid chromatography [60]. pE2-E1 was cloned into the mammalian expression vector pHLsec (Invitrogen) with a C-terminal octa-histidine tag and modified to express the Epstein–Barr virus EBNA-1 protein for enhanced protein expression. pE2-E1 was expressed in serum-free HEK-293F suspension cells and purified by Ni-NTA agarose affinity (Qiagen) and Superdex 200 gel filtration chromatography. Irf7−/− mice were infected and boosted with 104 PFU of CHIKV-LR and, depending on the experiment, given a final intravenous (i. v.) boost with CHIKV virus-like particles [30], 25 µg of E2 protein, or 2×105 PFU of CHIKV-LR three days prior to fusion with myeloma cells. Hybridomas secreting antibodies that reacted with CHIKV-GFP-infected BHK21-15 cells were identified by flow cytometry and cloned by limiting dilution. MAbs were isotyped by ELISA (Pierce), adapted for growth under serum-free conditions, and purified by protein G affinity and size exclusion chromatography. All MAbs were screened initially with a single endpoint neutralization assay using neat hybridoma supernatant (∼10 µg/ml), which was incubated with 100 FFU of CHIKV-LR for one hour at 37°C. MAb-virus complexes were added to BHK21-15 cell monolayers in 6-well plates. After 90 minutes, cells were overlaid with 1% (w/v) agarose in Modified Eagle Media (MEM) supplemented with 4% FBS. Plates were fixed with 10% formaldehyde in PBS 48 hours later, stained with crystal violet, and plaques were counted. The VH and VL sequence of neutralizing MAbs CHK-102, CHK-152, CHK-166, and CHK-263 were amplified from hybridoma cell RNA by a 5′ RACE procedure Table S3 in Text S1). The generation of a chimeric mouse-human CHK-9 and CHK-152 with mouse VH and VL and human IgG1 constant regions was performed as described previously [60]. A point mutation that abolishes FcγR and C1q binding (N297Q) was introduced by QuikChange mutagenesis (Stratagene). Recombinant antibodies were produced after transfection of HEK-293T cells, harvesting of supernatant, and purification by protein A affinity chromatography. Serial dilutions of MAb were incubated with 100 FFU of CHIKV for one hour at 37°C. MAb-virus complexes were added to cells in 96-well plates. After 90 minutes, cells were overlaid with 1% (w/v) methylcellulose in Modified Eagle Media (MEM) supplemented with 4% FBS. Plates were harvested 18 to 24 hours later, and fixed with 1% PFA in PBS. The plates were incubated sequentially with 500 ng/ml of ch-CHK-9 and horseradish peroxidase (HRP) -conjugated goat anti-human IgG in PBS supplemented with 0. 1% saponin and 0. 1% BSA. CHIKV-infected foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot 5. 0. 37 macroanalyzer (Cellular Technologies Ltd). Non-linear regression analysis was performed, and EC50 values were calculated after comparison to wells infected with CHIKV in the absence of antibody. 96-well tissue culture plates were coated with 100 µl of poly-L lysine and seeded with 3×104 Vero cells/well overnight. For pre-attachment assays, dilutions of MAb were prepared at 4°C in DMEM with 2% FBS and pre-incubated with 100 FFU of CHIKV-LR for one hour at 4°C. MAb-virus complexes were added to pre-chilled Vero cells for one hour at 4°C. Non-adsorbed virus was removed with three washes of DMEM and adsorbed virus was allowed to internalize during a 37°C incubation for 15 minutes. Cells were overlaid with 1% (w/v) methylcellulose in MEM supplemented with 4% FBS. The post-attachment assay was performed similarly, except that an equivalent amount of CHIKV was adsorbed first onto Vero cells for one hour at 4°C. After removing free virus, dilutions of MAb were added to the virus-adsorbed cells for one hour at 4°C. Virus was allowed to internalize and cells were overlaid with methylcellulose as described above. Nineteen hours later, the plates were harvested and analyzed for antigen-specific foci as described above. The binding of human FcγR and C1q to ch-CHK-152 and ch-CHK-152 (N297Q) was analyzed by SPR using a BIAcore 3000 biosensor (GE Healthcare Life Sciences). MAbs were captured (∼900 RU) after flowing over immobilized F (ab) ′2 fragments of goat anti-human F (ab) ′2 specific IgG on a CM-5 sensor chip. Binding experiments were performed in HBS-EP buffer (10 mM Hepes, pH 7. 4,150 mM NaCl, 3 mM EDTA, and 0. 005% P20 surfactant). Binding of CD16A and CD64 (as monomeric soluble FcγR), CD32A (as dimeric soluble FcγR-aglycosylated Fc fusion), and C1q (Sigma-Aldrich) was analyzed at a single concentration. The FcγR and C1q were injected for 60 sec at a flow rate of 30 µl/min then allowed to dissociate over 2 minutes. Affinity measurements of CHK-152 MAbs for pE2-E1 were performed by SPR in HBS-EP buffer. Ch-CHK-152, ch-CHK-152 N297Q, hu-CHK-152 and mouse CHK-152 were captured (∼300 RU) after flowing over immobilized F (ab) ′2 fragments of goat anti-human or anti-mouse Fc specific IgG. Purified pE2-E1 was injected at concentrations of 0,6. 25,12. 5,25,50, and 100 nM, at a flow rate of 30 µl/min for 120 sec, and then allowed to dissociate over 2 minutes. Regeneration of capture surfaces was performed by pulse injection of 10 mM glycine pH 1. 5. Binding curve at the zero concentration of pE2-E1 was subtracted from each experimental curve as a blank. Data were analyzed using BIAevaluation 4. 1 software. Kinetic constants, ka and kd, were estimated by global fitting analysis of the association/dissociation curves to the 1∶1 Langmuir interaction model. CHIKV-LR (1. 2×105 FFU) was incubated with 25 µg/ml of MAbs for one hour at 37°C. Virus-MAb complexes were added to Vero cells and infection proceeded for 24 hours. At each passage, half of the supernatant was mixed (1∶1) with 50 µg/ml of the selection MAb for one hour at 37°C. These complexes were added to a new monolayer of Vero cells for 2 hours, and the procedure was repeated from 3 to 6 times depending on the selection MAb. Individual MAb-resistant viral plaques were picked and virus was grown in Vero cells in the presence of 10 µg/ml of MAb for 24 hours. RNA was isolated from cells using an RNeasy kit (Qiagen) and cDNA was made with random hexamers using the Superscript III Reverse Transcriptase kit (Invitrogen) and amplified by PCR with primers flanking the structural genes (Table S4). The PCR product was sequenced using ten overlapping primer sets (Table S4). Figures were prepared using the atomic coordinates of CHIKV pE2-E1 (RCSB accession number 3N44) using the program CCP4MG [61]. For survival analysis, Kaplan-Meier survival curves were analyzed by the log-rank test. For growth kinetics and neutralization an unpaired T-test or analysis of variance was used to determine significance. These analyses were assessed using Prism software (GraphPad software). The protective effects of ch-CHK-152 versus ch-CHK-152 N297Q in wild type C57BL/6 mice were analyzed by the Kruskal-Wallace test with Bonferroni correction using the agricolae package of R (R Development Core Team, 2010. Foundation for Statistical Computing, Vienna, Austria).
summary: Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes outbreaks of polyarthritis in humans, and is currently a threat to spread to the United States due to the presence of its mosquito vector, Aedes albopictus. At present, there is no licensed human vaccine or therapeutic available to protect against CHIKV infection. The primary goal of this study was to develop an antibody-based therapeutic agent against CHIKV. To do this, we developed a panel of 230 new mouse anti-CHIKV MAbs and tested them for their ability to neutralize infection of different CHIKV strains in cell culture. We identified 36 MAbs with broad neutralizing activity, and then tested several of these for their ability to protect immunocompromised Ifnar−/− mice against lethal CHIKV infection. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs limited the development of resistance and protected Ifnar−/− mice against disease even when given just 24 to 36 hours before CHIKV-induced death. Analogous protection against CHIKV-induced arthritis was seen in a disease model in wild type mice. Our data suggest that pairs of highly neutralizing MAbs may be a therapeutic option against CHIKV infection.
keywords: humoral immunity
  emerging viral diseases
  immunology
  microbiology
  viral structure
  emerging infectious diseases
  immune defense
  immunotherapy
  biology
  pathogenesis
  viral envelope
  immunity
  virology
  antivirals
  immunoglobulins
title: Development of a Highly Protective Combination Monoclonal Antibody Therapy against Chikungunya Virus