Exposure of Epitope Residues on the Outer Face of the Chikungunya Virus Envelope Trimer Determines Antibody Neutralizing Efficacy

ABSTRACT

Chikungunya virus (CHIKV) is a reemerging alphavirus that causes a debilitating arthritic disease and infects millions of people and for which no specific treatment is available. Like many alphaviruses, the structural targets on CHIKV that elicit a protective humoral immune response in humans are poorly defined. Here we used phage display against virus-like particles (VLPs) to isolate seven human monoclonal antibodies (MAbs) against the CHIKV envelope glycoproteins E2 and E1. One MAb, IM-CKV063, was highly neutralizing (50% inhibitory concentration, 7.4 ng/ml), demonstrated high-affinity binding (320 pM), and was capable of therapeutic and prophylactic protection in multiple animal models up to 24 h postexposure. Epitope mapping using a comprehensive shotgun mutagenesis library of 910 mutants with E2/E1 alanine mutations demonstrated that IM-CKV063 binds to an intersubunit conformational epitope on domain A, a functionally important region of E2. MAbs against the highly conserved fusion loop have not previously been reported but were also isolated in our studies. Fusion loop MAbs were broadly cross-reactive against diverse alphaviruses but were nonneutralizing. Fusion loop MAb reactivity was affected by temperature and reactivity conditions, suggesting that the fusion loop is hidden in infectious virions. Visualization of the binding sites of 15 different MAbs on the structure of E2/E1 revealed that all epitopes are located at the membrane-distal region of the E2/E1 spike. Interestingly, epitopes on the exposed topmost and outer surfaces of the E2/E1 trimer structure were neutralizing, whereas epitopes facing the interior of the trimer were not, providing a rationale for vaccine design and therapeutic MAb development using the intact CHIKV E2/E1 trimer.

IMPORTANCE CHIKV is the most important alphavirus affecting humans, resulting in a chronic arthritic condition that can persist for months or years. In recent years, millions of people have been infected globally, and the spread of CHIKV to the Americas is now beginning, with over 100,000 cases occurring in the Caribbean within 6 months of its arrival. Our study reports on seven human MAbs against the CHIKV envelope, including a highly protective MAb and rarely isolated fusion loop MAbs. Epitope mapping of these MAbs demonstrates how some E2/E1 epitopes are exposed or hidden from the human immune system and suggests a structural mechanism by which these MAbs protect (or fail to protect) against CHIKV infection. Our results suggest that the membrane-distal end of CHIKV E2/E1 is the primary target for the humoral immune response to CHIKV, and antibodies targeting the exposed topmost and outer surfaces of the E2/E1 trimer determine the neutralizing efficacy of this response.

INTRODUCTION

Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that was first isolated in Tanzania in 1952 (1). CHIKV is endemic in Africa, India, and Southeast Asia but also occurs in unpredictable outbreaks beyond these regions, infecting millions of people (2). A mutation in the CHIKV envelope glycoprotein 1 (E1-A226V) enabled viral transmission through Aedes albopictus mosquitoes, in addition to Aedes aegypti mosquitoes, and in 2005 resulted in widespread and severe epidemics in La Réunion, France (266,000 cases, representing a third of the population), India (at least 1.4 million cases), and Indonesia (>15,000 cases), with subsequent traveler-initiated outbreaks occurring in Italy (more than 200 cases), France, and China (3–7). Due to the extended geographic range of Aedes albopictus mosquitoes, Europe and the Americas are now at risk of CHIKV outbreaks (8, 9), and CHIKV infected over 100,000 people in the Caribbean in the first half of 2014 after arriving there only 6 months earlier (10).

CHIKV causes a debilitating rheumatic disease, characterized by arthritis and arthralgia, that lasts weeks to months and can remain unresolved for years postinfection (11, 12). Currently, there are no specific prophylactics or therapeutics for CHIKV, any other alphaviruses, or the long-term diseases that they cause, with current treatments primarily consisting of anti-inflammatory drugs and antiviral compounds with broad spectra of activity (13–15). Although vaccine candidates against CHIKV were first proposed 45 years ago and vaccines against CHIKV are in active development (16–21), many vaccine candidates tested to date have either failed to induce protective antibodies or demonstrated significant safety issues (22). As a result, many questions remain to be answered to understand the most protective human immune response to this virus.

Similar to the genomes of other alphaviruses, the CHIKV genome encodes two envelope glycoproteins, E2 and E1, which are derived from a larger polyprotein precursor (capsid/E3/E2/6K/E1) and are embedded in the viral membrane. E2 is believed to mediate receptor attachment, while E1 is a class II viral fusion protein responsible for membrane fusion (23–27). On the mature virus, heterodimers of E2 and E1 trimerize to form 80 spikes on the virion that constitute an icosahedral protein shell surrounding the viral membrane (28).

The majority of antibodies triggered by CHIKV infection appear to target the viral envelope glycoprotein E2 (29, 30). Prior studies have described four CHIKV envelope human monoclonal antibodies (MAbs; two isolated by us and two isolated by another group) from CHIKV-infected individuals (31, 32), as well as MAbs from mice (33–36), that neutralize CHIKV and show protection in CHIKV mouse and/or nonhuman primate models. Several neutralizing MAbs targeting other alphaviruses have also been identified (37–44), but protection in vivo has been demonstrated only for a few MAbs, and most require high doses or combinations of MAbs to achieve even prophylactic protection. Mechanisms proposed for MAb-mediated neutralization of CHIKV include blocking of viral attachment to target cells, prevention of the structural transitions necessary for membrane fusion, and inhibition of cell-to-cell viral transmission (33, 45). Limited efforts to identify small-molecule inhibitors of CHIKV have also been initiated (46–48), but most of the inhibitors described to date are weak, many give rise to resistant strains, and to date none have entered clinical trials (against CHIKV or any other alphavirus).

In the present study, we used phage display against structurally intact E2/E1 on virus-like particles (VLPs) to isolate a panel of seven human MAbs, including a highly neutralizing MAb (IM-CKV063) and several fusion loop-specific MAbs. IM-CKV063 neutralized virus and protected mice in two CHIKV animal models, while the fusion loop MAbs neither demonstrated neutralizing ability in vitro nor offered protection from lethality in mice, despite targeting a functionally critical region of the glycoprotein. Comparing the binding sites of MAbs from this study and the literature, we found that the epitope locations of 15 different MAbs on E2/E1 cluster around domains A and B, the fusion loop, and E1 domain II, but neutralizing and nonneutralizing epitope locations were spatially distinct. Cumulatively, these data suggest that epitopes on the topmost and outer surfaces of the E2/E1 trimer structure are neutralizing, whereas epitopes facing the interior of the trimer are not, providing a rationale for vaccine design and therapeutic MAb development.

MATERIALS AND METHODS

Immune phage antibody library construction.

A Fab phage display library was constructed from peripheral blood donated by three CHIKV-infected individuals, as described previously (32). All three individuals were infected in La Réunion, France, during the 2006 outbreak. Written informed consent was obtained from recovered CHIKV donors in La Réunion, and collection complied with the relevant human subject research protocols approved by the institutional review boards of Centre Hospitalier Universitaire. Briefly, peripheral blood samples were drawn 2 to 3 years after infection, and serum was analyzed for the presence of neutralizing antibodies using reporter HIV isolates pseudotyped with CHIKV E2/E1. Total RNA was prepared using the TRI Reagent (Sigma-Aldrich, St. Louis, MO). RNA was converted to cDNA using a SuperScript first-strand synthesis system for reverse transcription-PCR (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Construction of the phage library was performed by GenScript (Piscataway, NJ).

Screening of isolated Fab clones.

Individual Fab peripreparations were prepared from single colonies by induction with 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) at 28°C overnight, lysing of the cells in phosphate-buffered saline (PBS) by freeze-thaw, and then screening for CHIKV-specific binding in a Fab enzyme-linked immunosorbent assay (ELISA). Purified VLPs were directly coated on a microtiter plate overnight at 4°C using 0.25 μg of protein per well in 0.1 M sodium bicarbonate buffer, pH 8.6. The wells were washed with PBS and blocked with 4% PBS^−/− with 4% milk (PBSM) for 1 h. Fabs in 4% PBSM were added to each well, and the plate was incubated for 1 h at 37°C with gentle agitation. The Fab solution was discarded, and the plate was washed 3 times with PBS plus 0.01% Tween 20. To detect bound Fab, a 1:5,000 dilution of anti-human Fd horseradish peroxidase (HRP; Southern Biotech, Birmingham, AL) in 4% PBSM was added to the wells, and the plate was incubated at room temperature for 30 min with gentle agitation. The plate was washed 3 times with PBS plus 0.01% Tween 20 and developed according to the manufacturer's instructions (Super Signal West Pico; Thermo Scientific, Waltham, MA). Negative controls included a buffer blank (no antigen) and a nonspecific antigen.

Construction and transient production of recombinant IgG antibodies.

Candidate Fabs were converted to human IgG1 format for production in HEK293T cells. Briefly, phage-derived Fab variable domains were subcloned downstream of the human interleukin-2 signal sequence and upstream of either human IgG1 heavy-chain (γ1) or human IgG1 light-chain (κ1) constant domains in a mammalian expression vector. Heavy- and light-chain constructs were cotransfected into HEK293T cells at a ratio of 1:3 by calcium phosphate coprecipitation. Secreted IgG was purified from the culture medium at 48 to 72 h posttransfection by protein A chromatography, followed by concentration and buffer exchange against PBS. Quantification of the purified IgG was performed by a bicinchoninic acid assay (Thermo Scientific, Waltham, MA).

Purification of CHIKV VLPs.

CHIKV VLPs were produced by transfecting HEK293T cells with an expression plasmid encoding a codon-optimized chimera between O'Nyong-Nyong virus (ONNV) capsid (Igbo Ora strain, residues 1 to 260) and CHIKV E3/E2/E1 (S27 strain, residues 262 to 1248). After production for 2 to 3 days, the supernatant was filtered through a 0.22-μm-pore-size filter (catalog number 430517; Corning). Polyethylene glycol 8000 (37.5%; catalog number P4463; Sigma) was added 1:4 to the filtered supernatant, and the mixture was incubated overnight at 4°C to precipitate VLPs. The mixture was spun for 30 min at 7,000 rpm and 4°C using a JLA-8.1000 rotor. After the mixture was spun, the medium was aspirated and 25 ml HEPES buffered saline (HBS; 150 mM NaCl, pH 8.0) was added to resuspend the precipitated VLPs. The resuspension was added to a large Beckman Ultra-Clear centrifuge tube (25 by 89 mm; Beckman), and a 10-ml 20% sucrose cushion (10 mM HEPES, 100 mM NaCl, 1 mM EDTA, pH 8.0) was added. The sample was spun for 3 h at 31,000 rpm and 4°C using an SW-32 rotor. After the sample was spun, the suspension and sucrose layer were aspirated and the pellet was resuspended in 10 ml HBS. The resuspension was added to a small Beckman Ultra-Clear centrifuge tube (14 by 89 mm; Beckman), and a 1-ml 20% sucrose cushion was added. The sample was spun for 3 h at 31,000 rpm and 4°C using an SW-41 rotor. After the sample was spun, the suspension and sucrose layer were aspirated and the purified viral pellet was gently resuspended in 200 μl HBS. The sample was stored for 1 h at 4°C and thereafter at −80°C.

Construction of CHIKV E2/E1 mutation library.

A CHIKV envelope expression construct (S27 strain; UniProt accession number Q8JUX5) encoding a C-terminal V5 epitope tag was subjected to high-throughput alanine scanning mutagenesis to generate a comprehensive mutation library. Primers were designed to mutate each residue within the E2, 6K, and E1 regions of the envelope (residues Y326 to H1248) to alanine, while alanine codons were mutated to serine. In a few cases, other amino acid substitutions instead of alanine were generated. In total, 910 CHIKV envelope mutants were generated (98.5% coverage), the sequences were confirmed, and the mutants were arrayed into 384-well plates at one mutant per well.

Immunofluorescence assay.

The CHIKV mutation library, arrayed in 384-well microplates, was transfected into HEK293T cells and allowed to express for 22 h. For MAbs IM-CKV057, IM-CKV061, IM-CKV062, and IM-CKV067, cells were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS plus calcium and magnesium (PBS⁺⁺). Cells were stained with purified MAbs IM-CKV057 (0.25 μg/ml), IM-CKV061 (0.75 μg/ml), IM-CKV062 (0.5 μg/ml), IM-CKV065 (1.0 μg/ml), IM-CKV066 (1.0 μg/ml), and IM-CKV067 (1.0 μg/ml) and purified Fab IM-CKV063 (2.5 μg/ml) diluted in 10% normal goat serum (NGS; Sigma-Aldrich, St. Louis, MO). For IM-CKV063, it was necessary to employ a Fab for epitope mapping since the high-affinity MAb bound too tightly to identify individual point mutants that ablated MAb binding. Primary MAb concentrations were determined using an independent immunofluorescence titration curve against wild-type CHIKV E2/E1 to ensure that the signals were within the linear range of detection. MAbs were detected using 3.75 μg/ml Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) in 10% NGS. Cells were washed twice with PBS without calcium or magnesium (PBS^−/−) and resuspended in Cellstripper solution (Cellgro, Manassas, VA) with 0.1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO). Mean cellular fluorescence was detected using an Intellicyt high-throughput flow cytometer (HTFC; Intellicyt, Albuquerque, NM). MAb reactivities against each mutant E2/E1 clone relative to the reactivity against wild-type E2/E1 protein were calculated by subtracting the signal from mock-transfected controls and normalizing the signal to the signal from wild-type E2/E1-transfected controls.

Epitope identification.

Mutated residues within critical clones were identified to be critical to the MAb epitope if they did not support the reactivity of the test MAb but did support the reactivity of a reference CHIKV MAb and additional CHIKV MAbs. This counterscreen strategy facilitates the exclusion of E2/E1 mutants that are locally misfolded or that have an expression defect (49). The detailed algorithms used to interpret shotgun mutagenesis data are described elsewhere (50; U.S. patent application 61/938,894). For IM-CKV065, epitope residues were further refined on the basis of surface accessibility in the trimer structure. Residues constituting the MAb epitope were visualized on heterodimeric and trimeric CHIKV envelope crystal structures (E2/E1 heterodimer [PDB accession number 3N41] and E2/E1 trimer [PDB accession number 2XFC]).

Characterization of MAb binding kinetics using a biosensor.

All biosensor studies were performed in PBS buffer supplemented with 1 mg/ml BSA (PBS-B) at 25°C using a ForteBio Octet Red biosensor system (Pall-ForteBio, Inc., Menlo Park, CA). Purified CHIKV VLPs were loaded onto amine-reactive biosensor tips (AR2G) using a human MAb against CHIKV (E26D9; Dendritics, Lyon, France). Briefly, amine-reactive sensor tips were activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/sulfo-N-hydroxysulfosuccinimide (sulfo-NHS) (20 mM and 10 mM, respectively) for 5 min. E26D9 at 25 μg/ml in 10 mM sodium acetate (pH 5.5) was then immobilized on the sensor tips with a 10-min incubation. Following a 5-min deactivation in 1 M ethanolamine and a 10-min stabilization in PBS-B, CHIKV VLPs diluted to 20 μg/ml were loaded for 45 min, followed by another 10 min of stabilization. IM-CKV063 was prepared as a 2-fold serial dilution (starting at 20 μg/ml), and buffer blanks were prepared. Nonspecific binding was assessed using sensor tips without VLPs. Data analysis was performed using the Octet data analysis program (v6.4; ForteBio). Binding kinetics were analyzed using a standard 1:1 binding model.

VLP ELISAs.

Ninety-six-well white, flat-bottom microtiter plates were coated with retroviral VLPs (lipoparticles) containing CHIKV E2/E1 at 5.0 μg/well or CHIKV VLPs at 0.5 μg/ml and incubated overnight at 4°C. For IM-CKV057, IM-CKV061, IM-CKV062, IM-CKV063, and IM-CKV067, the particles were fixed in 4% paraformaldehyde. The plates were blocked with 3% BSA (Sigma) for 15 min at room temperature. For the comparative retroviral/CHIKV VLP ELISA, primary MAb was diluted to a previously optimized concentration (see “Immunofluorescence assay” above for the methodology), added to the plates, and allowed to incubate for 1 h at room temperature (22°C). For the temperature-dependent ELISA, primary MAb was diluted to 2 μg/ml in blocking buffer, and the diluted MAb was added to the plates and allowed to incubate for 1 h at room temperature (22°C), 37°C, or 45°C. The plates were washed 3 times with PBS^−/−, and then HRP-conjugated rabbit antihuman secondary antibody diluted 1:5,000 in blocking buffer was added for 1 h at room temperature. The plates were washed 3 times with PBS^−/−, and reactivity was detected using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Waltham, MA).

CHIKV pseudovirus neutralization assay.

Lentiviral reporter viruses pseudotyped with CHIKV E2/E1 were produced essentially as described previously (51, 52) by cotransfecting a plasmid carrying CHIKV E2/E1 with plasmids carrying the genes for HIV core proteins (gag-pol, based on previous work [53]) and luciferase (pNL-luc, based on pNL4-3-R-E- [54]). Cells were incubated at 37°C in 5% CO₂ to allow transfection and pseudovirus production. At 24 to 72 h posttransfection, the supernatants were harvested, filtered, and stored at −80°C. Target HEK293T cells were plated at 0.4 × 10⁶ cells/well in Dulbecco modified Eagle medium (DMEM; Thermo Scientific, Waltham, MA) containing additives and incubated at 37°C in 5% CO₂ overnight. On the following day, serial dilutions of MAb and virus preincubated for 45 min were added to the HEK293T cells. A spinoculation was performed at 2,000 rpm for 60 min at 20°C, and cells were then incubated at 37°C. At 24 h postinfection, 100 μl of fresh medium was added to each well. Infected target cells were lysed at 48 h postinfection, and lysates were assayed for luciferase activity (Promega, Madison, WI).

Wild-type CHIKV production and plaque assay.

Replication-competent CHIKV S27 (ATCC vr-64), a strain originally isolated in 1953 from a patient in East Africa, was grown in Vero cells. Vero cells (0.5 × 10⁵) were plated in a 6-well (Costar) plate and grown overnight. Serially diluted MAbs were mixed with S27 diluted to 400 PFU/ml, and S27 was preincubated with the MAbs for an hour at 37°C. Following this, 250 μl of the MAb-virus mixture was added to the confluent Vero cell monolayer for an additional hour. Subsequently, the virus was removed, an overlay of 4% agarose in DMEM supplemented with 2% fetal bovine serum was added, and the cells were incubated at 37°C for 72 h. The plaques were stained and counted as described previously (32). The plaque reduction neutralization test (PRNT) titer was calculated as the MAb concentration that resulted in a 50% reduction in the number of plaques compared to the number for the negative control in the presence of medium and no MAbs.

CHIKV neonatal mouse model.

The ability of MAbs to protect against lethal CHIKV infection was evaluated in a murine model with 9-day-old mice as previously described (32). All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee at ISIS Services, LLC (San Carlos, CA). C57BL/6 mice were purchased from The Jackson Laboratory (Sacramento, CA). Breeder pairs were housed under specific-pathogen-free conditions in microisolator cages (Innovive Inc., San Diego, CA). The mice were checked daily, and the date when litters were first observed was considered day 0. On day 9, litters with their mothers were transferred to static disposable cages (Innovive, Inc., San Diego, CA) and transferred into a biosafety level 3 facility for infection and treatment. Neonatal C57BL/6J mice were infected with 5 × 10⁵ PFU of CHIKV (strain S27; the strain was expanded and the titer was determined in Vero cells) intradermally in the ventral thorax. Each mouse was intraperitoneally injected with test MAb or control human IgG in 0.2 ml PBS immediately prior to CHIKV infection. Purified IgG from human serum was used as a control (Sigma-Aldrich, St. Louis, MO). Mice were then observed daily for up to 20 days. Results were analyzed using Kaplan-Meier survival curves and GraphPad Prism (version 5) software (GraphPad, CA).

CHIKV arthritis mouse model.

The ability of MAbs to protect against the arthralgia induced by CHIKV infection was evaluated in an arthritic mouse model as previously described (55). Briefly, female C57BL/6 mice (8 weeks old) were inoculated with CHIKV (LR2006-OPY-1 strain; 10⁴ log₁₀ 50% cell culture infectivity doses [CCID₅₀s]) or heat-inactivated CHIKV in 20 μl DMEM (supplemented with 2% fetal calf serum). Mice were inoculated by a shallow subcutaneous injection into the top, toward the lateral side, of each hind foot in the metatarsal region, injecting toward the ankle. CHIKV-infected mice (n = 5 mice per group) were injected with 100 μg purified MAb IM-CKV063 by the intraperitoneal route, and this was considered treatment day 0. Mice not treated with IM-CKV063 did not receive intraperitoneal injections. In order to avoid stimulating nonspecific immune responses that may interfere with CHIKV infection of adult mice (55), IM-CKV063 with endotoxin levels below 10 endotoxin units/mg were used. Arthritis was monitored by measuring the height and width of the metatarsal area of both hind feet using digital calipers (55).

Statistical analysis.

All data were analyzed using Prism software (GraphPad, San Diego, CA). Kaplan-Meier survival curves were analyzed by the log rank chi-square test. Log 50% effective concentrations (EC₅₀s) were compared using Prism's extra sum-of-squares F test. Other values were compared using Student's t test when comparing the results for two samples or a one-way or two-way analysis of variance when comparing the results for more than two samples.

RESULTS

Isolation of human CHIKV MAbs by phage display.

To better understand the types of antibodies that are elicited in response to natural human infection with CHIKV, we isolated MAbs from infected patients. Serum samples collected 2 to 3 years postinfection were screened from individuals who were infected during the 2006 CHIKV outbreak in La Réunion. Virus neutralization assays were used to identify samples containing highly neutralizing antibodies (Fig. 1A), and B cells from the most reactive patients were used to construct a Fab library for phage display. The phage library was panned using virus-like particles (VLPs) with retroviral cores (from murine leukemia virus Gag) that present CHIKV envelope proteins E2 and E1 (Fig. 1B) or using CHIKV VLPs (based on native alphavirus capsid) that are structurally equivalent to native CHIKV. Two different VLP types and slightly different panning protocols were used to increase the chances of isolating diverse MAbs. A number of E2/E1-reactive antibodies were derived from both panning strategies and bound to CHIKV E2/E1 with a signal-to-background noise ratio of >10:1 (Fig. 1C). Genes encoding the MAb heavy and light chains were sequenced and cloned into full-length human IgG1 vectors for MAb production and evaluation.

FIG 1.

Isolation of patient-derived MAbs against CHIKV, using viral particles containing native E2/E1. (A) Serum samples from patients (patients C1 to C5) infected with CHIKV during the 2006 outbreak in La Réunion were screened by neutralization assays. All neutralization results shown are normalized to the results for the maximum (uninhibited) infection achieved after background subtraction with a noninfected control. An immune phage Fab library was created from the B cells of the most highly reactive patient samples (red symbols; patients C2, C3, and C4). (B) The phage Fab library created from the B cells of CHIKV-infected patients was used to pan against retroviral (Gag core) and CHIKV (alphavirus capsid core) VLPs displaying the native form of CHIKV E2/E1. (C) Fabs isolated from phage display were further tested for E2/E1 target selectivity in ELISAs using CHIKV VLPs for specificity and dengue virus serotype 1 virions (64) as a negative control. The majority of the clones tested displayed at least a 10:1 selectivity for the E2/E1 target. (D) ClustalW alignment of VH chain amino acid sequences followed by neighbor-joining analysis was used to generate the unrooted dendrogram (MacVector, version 10). Tree distances represent the number of residue differences between sequences. Bar, 5 amino acid (aa) residues. IM-CKV057, IM-CKV061, IM-CKV062, and IM-CKV067 have identical heavy chains but different light chains.

Seven MAbs with unique heavy chain and/or light chain sequences were selected for further characterization (Fig. 1D). Four of the most similar MAbs (IM-CKV057, IM-CKV061, IM-CKV062, and IM-CKV067) were derived from panning campaigns using different types of VLPs (Table 1), suggesting that CHIKV VLPs and retroviral VLPs displayed similar conformations of E2/E1. Each MAb was also shown to be immunoreactive against CHIKV E2/E1 expressed on the surface of HEK293T cells (Fig. 2A). To characterize the cross-reactivity of these MAbs, each was screened against the envelopes of a panel of related alphaviruses (Semliki Forest virus [SFV], Ross River virus [RRV], and Sindbis virus [SINV]) expressed on HEK293T cells. Four of the seven MAbs were found to be broadly cross-reactive, having high immunoreactivity to all other alphavirus envelopes that were screened, suggesting that their epitopes are broadly conserved among alphavirus envelopes. Two MAbs were selectively reactive with CHIKV E2/E1 alone. Our previously described neutralizing MAb, C9 (32), was selectively reactive with CHIKV and SFV envelopes, as was the newly isolated MAb IM-CKV063, suggesting that the epitopes are relatively conserved between these two alphaviruses, at least when expressed in cells.

TABLE 1.

Residues critical for CHIKV MAb binding^a

^aImmunoreactivities are expressed as a percentage of the immunoreactivity of the wild type, with ranges (maximum minus minimum values) given in parentheses. Values shaded in gray are for critical residues. For IM-CKV063, immunoreactivity for the Fab is shown. At least two replicate values were obtained for each experiment. Either CHIKV VLPs (the S27 strain with or without an A226V mutation) or retroviral VLPs (lipoparticles, made with murine leukemia virus Gag) were used for phage panning to isolate MAbs. Surface expression was determined by immunoreactivity with a rabbit polyclonal antibody (rPAb; a gift from IBT Bioservices). Percent sequence identity at individual residues was determined by comparing the sequences of 14 different alphaviruses (28). Data in the surface expression column represent the percentage of rabbit polyclonal antibody immunoreactivity for each mutant relative to that of the wild type. Data in the infectivity column represent the functional activity of mutant CHIKV E2/E1 reporter virus relative to that of the wild type. ND, not done.

FIG 2.

CHIKV MAb reactivity with alphavirus envelopes, VLP surfaces, and cell surface E2/E1. (A) MAbs against CHIKV E2/E1 that were isolated were tested by flow cytometry for immunoreactivity with envelope proteins of alphaviruses CHIKV, SFV, RRV, and SINV expressed on HEK293T cells. Cells were either fixed with 4% paraformaldehyde to test immunoreactivity with select MAbs (IM-CKV057, IM-CKV061, IM-CKV062, IM-CKV067, and V5) or left unfixed (for MAbs IM-CKV063, IM-CKV065, IM-CKV066, and C9). As a control, cells were permeabilized for immunodetection of the V5 epitope tag engineered onto the C terminus of each envelope. Cells transfected with the pUC19 empty vector were used as a negative control (No Env). The data shown represent the mean and standard deviation of four data points, and data are representative of those from at least two independent experiments. (B) MAb reactivities against E2/E1 were tested with retroviral VLPs, CHIKV VLPs, and HEK293T cells expressing CHIKV E2/E1. VLP reactivity was detected using ELISA, while cell surface reactivity was detected by flow cytometry. Samples were either fixed with 4% paraformaldehyde or left unfixed, as described in the legend to panel A. Bars represent the mean and standard deviation of three data points for VLP reactivity and four data points for cell surface reactivity. All data are representative of those from at least two independent experiments. MAbs showed negligible reactivity with controls that included dengue virus VLPs (Control VLPs) and mock-transfected HEK293T cells (Control Cells). RFU, relative fluorescence units; RLU, relative light units.

MAbs were also characterized for their ability to recognize CHIKV E2/E1 epitopes that were presented in different contexts, including on retroviral VLPs and CHIKV VLPs that were used for MAb isolation and on HEK293T cells that were used for alphavirus cross-reactivity and epitope mapping studies (Fig. 2B). For all MAbs, with the exception of IM-CKV065, the pattern of reactivity was similar between VLPs and cells, suggesting that the E2/E1 epitopes for these MAbs are similarly presented on retroviral VLPs, CHIKV VLPs, and the cell surface. IM-CKV065 demonstrated high reactivity with E2/E1 presented on cells but low reactivity when E2/E1 was presented in the context of retroviral and CHIKV VLPs, despite being isolated using retroviral VLPs. This apparent discrepancy could reflect a difference in epitope presentation on the different surfaces, but it could also be due to a number of experimental factors, including the different assays used for cell versus VLP detection and the very fast off rate of IM-CKV065 (see below). Nevertheless, the signal-to-background noise ratio of IM-CKV065 binding to VLPs was 4:1 to 8:1, suggesting that this MAb still recognizes E2/E1 on the virus surface.

IM-CKV063 is a highly potent CHIKV-neutralizing human MAb.

To determine if the seven CHIKV-reactive MAbs were capable of inhibiting viral infectivity, they were tested in cellular neutralization assays, initially using reporter viruses pseudotyped with CHIKV E2/E1 (Fig. 3A). The MAbs showed a range of activity, from nonneutralizing (IM-CKV057, IM-CKV061, IM-CKV062, IM-CKV066, and IM-CKV067) to moderately neutralizing (IM-CKV065; average 50% inhibitory concentration [IC₅₀] = 170 ng/ml) and strongly neutralizing (IM-CKV063). All MAbs were presented in an identical IgG1 format, so antibody isotype was not a factor in neutralization ability. MAb IM-CKV063 potently neutralized CHIKV pseudoviruses with an average IC₅₀ of 7.4 ng/ml, comparable to or better than the neutralization activity of the recently published anti-CHIKV MAb C9 (data for which are shown for comparison; IC₅₀ = 51 ng/ml) that we previously demonstrated could inhibit CHIKV in cell culture and animal models of viral pathogenesis (32).

FIG 3.

MAb IM-CKV063 strongly neutralizes CHIKV. Anti-CHIKV MAbs were tested for the ability to neutralize the infectivity of reporter HIV isolates pseudotyped with CHIKV S27 E2/E1 (A) or the VSV envelope (B). Viruses were preincubated with MAbs, as described in the Materials and Methods section, and infection of HEK293T target cells was detected by the expression of Renilla luciferase. Each data point is the mean of two replicates, and data are representative of those from at least two independent experiments. (C) IM-CKV063 was tested for neutralization of additional alphavirus envelope proteins pseudotyped onto reporter HIV isolates. Data points represent the mean of three replicates, and data are representative of those from two independent experiments. (D) Live CHIKV was preincubated with MAbs before addition to Vero cells. The S27 strain of CHIKV was used in all experiments. Strain 37997 was also tested against IM-CKV065. Infectivity was assessed after 72 h using a PRNT. Data points represent the mean and standard deviation of two to three replicates and are representative of those from at least two individual experiments. Neut, neutralization.

All neutralizing MAbs targeted E2/E1 and not other viral components since they did not neutralize reporter virus pseudotyped with the vesicular stomatitis virus (VSV) envelope (Fig. 3B). Neutralization by IM-CKV063 was selective for CHIKV E2/E1; this MAb did not neutralize viruses bearing envelopes of other alphaviruses (SFV, RRV, SINV) (Fig. 3C), despite being moderately cross-reactive with SFV (Fig. 2A). It is possible that its lower level of reactivity with SFV is not sufficient for neutralization (56) or that residues critical for SFV infectivity are not inhibited by IM-CKV063 binding.

Select MAbs were also tested for antiviral activity using native replication-competent CHIKV isolates. In a plaque reduction neutralization test (PRNT), IM-CKV063 demonstrated a dose-dependent inhibitory effect on the infectivity of live virus (concentration of neutralizing antibody required to achieve a 50% reduction in plaque counts [PRNT₅₀], 11 ng/ml) and had potency similar to that of C9 (for which the PRNT₅₀ was 12 ng/ml in the same assays; Fig. 3D). MAb IM-CKV065 did not neutralize S27 live virus using PRNTs but did neutralize CHIKV strain 37997, with a PRNT₅₀ value of 81 ng/ml.

To further characterize the activities of neutralizing MAbs, we used biosensor analyses to assess the kinetics of MAb binding to E2/E1 presented in its native state on the CHIKV virion surface. Noninfectious CHIKV VLPs were immobilized onto biosensor tips, and MAb binding was assessed using biolayer interferometry (Fig. 4). IM-CKV063 showed a specific association with immobilized E2/E1 and did not demonstrate nonspecific binding in the absence of CHIKV VLPs. Titration experiments revealed IM-CKV063's strong binding affinity to E2/E1, characterized by rapid association (association rate constant [k_a] = 2.9e5 M⁻¹ s⁻¹), slow dissociation (dissociation rate constant [k_d] = 9.4e−5 s⁻¹), and a strong affinity (apparent equilibrium dissociation constant [K_D] = 0.32 nM) that is close to the published affinity of MAb C9 (apparent K_D, 1.2 nM) (32). In contrast, moderately neutralizing MAb IM-CKV065 had a much weaker affinity (apparent K_D, 9.3 nM) for CHIKV S27 E2/E1. Although the association rate for this MAb was relatively fast (k_a = 1.6e5 M⁻¹s⁻¹), its rapid dissociation (k_d = 1.5e−3 s⁻¹) led to a decreased affinity. The fast dissociation of IM-CKV065 may also explain its relatively weak binding to VLPs under the experimental conditions used for detection (Fig. 2B). Taken together, the distinct binding characteristics of these MAbs may help explain their relative neutralizing potencies.

FIG 4.

Kinetic analysis of MAb binding to intact CHIKV VLPs. (A) Direct binding of IM-CKV063 to CHIKV VLPs was detected using a ForteBio Octet Red biosensor, with VLPs immobilized on biosensor tips via the CHIKV E2/E1 capture MAb E26D9 (Dendritics). MAb IM-CKV063 at 10 μg/ml was applied to the captured VLPs or an unconjugated surface at 100 s to monitor MAb association, and dissociation was measured starting at 400 s. IM-CKV063 bound specifically to CHIKV VLPs. (B) The kinetics of IM-CKV063 binding to CHIKV E2/E1 were assessed by fitting the data to a 1:1 binding model to determine the rate constants. An apparent binding affinity of 320 pM was calculated for IM-CKV063. Raw data curves for MAb association and dissociation from captured antigen are shown in black, and fitted curves are in red. (C) Binding kinetics of MAb IM-CKV065. An apparent binding affinity of 9.3 nM was calculated for IM-CKV065.

IM-CKV063 is protective in animal models of CHIKV infection.

We next investigated whether the virus-neutralizing effects of IM-CKV063 in cell culture would translate to in vivo efficacy in animal models of CHIKV disease. First, we employed a pathogenic neonatal mouse model of CHIKV-induced lethality (57), which evaluates absolute protection from lethality upon treatment. Here, mice were concurrently inoculated with live virus and test MAbs. Complete survival was observed with 100-μg, 20-μg, and 10-μg doses of IM-CKV063, and 90% survival was observed with a 4-μg dose (P < 0.02 for doses of 4 μg and above relative to the results for the IgG control) (Fig. 5A, blue curves). Control mice that received 100 μg (approximately 25 mg/kg) of human IgG or a nonspecific (anti-HCV) control MAb succumbed to infection within 4 to 5 days. The level of protection afforded by MAb IM-CKV063 was similar to that afforded by previously described MAb C9 at doses of 1 μg and 4 μg (P > 0.05). In contrast, MAbs that were nonneutralizing in cellular assays (IM-CKV057 and IM-CKV066) provided little or no protection relative to that provided by IgG or nonspecific MAb controls, even at high doses of 100 μg, and all mice died within 9 days of infection (Fig. 5A). Importantly, IM-CKV063 also provided significant protection from lethality when administered postexposure in a more therapeutically relevant scenario. A 10-μg dosage of IM-CKV063 was injected at 12 or 24 h after infection and led to the survival of at least 50% of mice (P < 0.03 relative to the results for IgG) (Fig. 5B).

FIG 5.

MAb IM-CKV063 provides therapeutic protection in mouse models of CHIKV pathogenesis. (A) The ability of MAb IM-CKV063 to protect neonatal mice from death due to viral infection was determined by injection of MAb concurrently with infection by live CHIKV. Survival of >90% was observed at doses of >4 μg of IM-CKV063 (blue curves; P < 0.02 relative to the results for IgG). IM-CKV057, IM-CKV066, and nonspecific human IgG and human anti-HCV antibody controls (all at 100 μg) were also tested, and MAb C9 was included for comparison. Groups of 4 to 9 mice were used for each condition, depending on the litter size available. (B) MAb IM-CKV063 was tested for the ability to protect mice after virus exposure. Groups of 6 to 7 mice were used for each condition. Ten micrograms of MAb provided complete protection when administered simultaneously with virus and protected a subgroup of the animals when administered 12 or 24 h following virus exposure (P < 0.03 relative to the results for IgG). (C) IM-CKV063 was tested for the ability to protect against foot swelling in an adult mouse model. Mice were injected intraperitoneally with 100 μg of MAb concurrently with the injection of CHIKV (LR2006-OPY-1). Footpad dimensions were measured according to foot width and breadth in the metatarsal region. Five mice were tested under each condition, and measurements on both feet were considered to be replicates. Error bars represent standard errors of the means. Statistically significant differences in foot dimensions between IM-CKV063-treated and untreated (no MAb treatment) mice are indicated (*, P < 0.05). Heat Inact. CHIKV, heat-inactivated CHIKV.

We also assessed IM-CKV063 in a second in vivo animal model of CHIKV infectivity to assess a different parameter of protection, the ability to protect adult mice from a CHIKV-induced arthritic phenotype. In this model, mice received a 100-μg intraperitoneal injection of purified IM-CKV063 (approximately 5 mg/kg of body weight) concurrently with the administration of CHIKV. Infected mice were monitored for foot swelling as described previously (55). Mice injected with CHIKV alone experienced foot swelling at 5 to 6 days, as expected (32, 55). In contrast, mice injected with IM-CKV063 showed no detectable foot swelling at any point during the experiment (Fig. 5C) (P < 0.05 between the results for treated and untreated mice). Taken together, these results demonstrate the ability of IM-CKV063 to neutralize CHIKV effectively in vivo.

Epitope mapping of CHIKV MAbs.

To understand the structural basis by which each MAb binds to CHIKV E2/E1 and how neutralizing and nonneutralizing epitopes differ, the residues required for binding of each MAb were next determined. To accomplish this, we used comprehensive alanine scanning, where MAb binding was assessed against a shotgun mutagenesis mutation library of CHIKV E2/E1 variants (32, 49, 50, 58). Nearly every residue of CHIKV E2/E1 was mutated, generating a library of 910 alanine mutants with 98.5% sequence coverage. The entire mutation library was transfected into human HEK293T cells in a 384-well array format (one clone per well) and assessed for immunoreactivity using high-throughput flow cytometry (Fig. 6A).

FIG 6.

Critical residues for MAb IM-CKV063 binding. (A) A shotgun mutagenesis mutation library for CHIKV envelope protein encompassing 910 E2/E1 mutations, where each amino acid was individually mutated to alanine, was constructed. Each well of each mutation array plate contained one mutant with a defined substitution. Reactivity results for a representative 384-well plate are shown. Eight positive (wild-type E2/E1) and eight negative (mock-transfected) control wells were included on each plate. (B) Human HEK293T cells expressing the CHIKV envelope mutation library were tested for immunoreactivity with MAb IM-CKV063, which was measured using an Intellicyt high-throughput flow cytometer. Using algorithms described elsewhere (50; U.S. patent application 61/938,894), clones with reactivity of <20% relative to that of wild-type CHIKV E2/E1 yet >70% reactivity for a different CHIKV E2/E1 MAb were initially identified to be critical for IM-CKV063 binding. (C) Mutation of six individual residues reduced IM-CKV063 binding (red bars) but did not greatly affect the binding of other conformation-dependent MAbs (gray bars). Bars represent the mean and range of at least two replicate data points.

Prior to testing of the MAbs against the entire CHIKV mutation library, the immunoreactivity of each MAb was optimized by testing reactivity with fixed and unfixed cells and by testing a range of MAb concentrations that resulted in good signal-to-background noise ratios of >5:1. Once optimized, each MAb was screened against the CHIKV mutation library, and residues critical for MAb binding were initially identified as those where E2/E1 mutations resulted in less than 20% reactivity for the MAb of interest (relative to that of wild-type CHIKV E2/E1), yet greater than 70% wild-type binding by a reference MAb (Fig. 6B). Residues were further validated to be critical by comparing their reactivity across a panel of MAbs to verify that the mutation did not globally disrupt the binding of diverse MAbs (Fig. 6C), using algorithms described elsewhere (50; U.S. patent application 61/938,894). Using this approach, we systematically mapped the detailed epitopes of all seven MAbs (Table 1).

For IM-CKV063, we identified six critical residues whose mutation greatly impaired MAb binding to levels of <20% of that to wild-type E2/E1. These residues all localized on E2 domain A in close proximity to each other (Fig. 7A). Interestingly, visualization of the epitope on the trimeric structure of E2/E1 (28) suggests that these residues form a conformational epitope that lies at the interface of two different E2 subunits (Fig. 7B), constituting a unique intersubunit epitope. This model suggests that residues E24 and I121 on one E2 subunit form a single MAb binding site together with G55, W64, K66, and R80 on the adjacent E2 subunit within the trimer. The distance between the epitope residues on different heterodimers in the trimer is predicted to be approximately 13 Å, consistent with the size of a MAb binding site. This epitope is predicted to be solvent exposed at neutral pH and easily accessible for binding on the native trimeric structure of the envelope, which is consistent with the ability of IM-CKV063 to bind and neutralize infectious virions.

FIG 7.

MAb IM-CKV063 epitope. The critical residues comprising the epitope for IM-CKV063 are visualized on the CHIKV envelope crystal structure, showing E2 (red), E1 (yellow), and the fusion loop (cyan). The epitope (green spheres) is depicted on the neutral pH heterodimeric structure (PDB accession number 3N41) (A) and trimeric structure (PDB accession number 2XFC) (B) of E2/E1 (28). The IM-CKV063 epitope appears to span two E2 subunits in the trimer, on the basis of structural proximity. A single epitope crossing two E2 subunits (one gray and one blue) is shown with multicolored residues and in the expanded view. Subunit I, E2-E24 (orange) and E2-I121 (green); subunit II, E2-G55 (yellow), E2-W64 (red), E2-K66 (purple), and E2-R80 (black). Residues comprising the other two IM-CKV063 epitopes on the trimer are indicated in cyan. The distance between residue I121 (shown in green) and residue W64 (shown in red) on two different E2 subunits of the trimer is 13 Å.

We also mapped the epitopes of the nonneutralizing MAbs IM-CKV057, IM-CKV061, IM-CKV062, IM-CKV066, and IM-CKV067, as well as moderately neutralizing MAb IM-CKV065. Interestingly, all nonneutralizing MAbs bound to epitopes that encompassed the fusion loop region of E1 (residues 83 through 100). Only a single MAb known to bind the fusion loop of any alphavirus has been previously reported (59, 60). The fusion loop is highly conserved among alphaviruses, explaining the broad reactivity of the fusion loop MAbs that we isolated with other alphavirus envelope proteins (from RRV, SINV, and SFV) (Fig. 2A). Detailed mapping studies showed that MAbs IM-CKV057, IM-CKV061, IM-CKV062, and IM-CKV067 bind to identical or overlapping epitopes directly on the fusion loop (Fig. 8A and B), thus also explaining their similarities in cross-reactivity and neutralization. The epitopes of MAbs IM-CKV061 and IM-CKV062 also included the seemingly distant residue E2-M267, which is in fact proximal to fusion loop epitope residues on adjacent heterodimers in the trimeric structure of the protein, thus contributing to an intersubunit epitope. The epitope for IM-CKV066 encompassed a different set of highly conserved residues in the fusion loop (G83, Y85, F87, and D97), as well as additional residues on E1 domain II (T228 and V229) and E2 arch 1 (Q146). Together, these residues constitute a discontinuous, intersubunit epitope in the envelope trimer (Fig. 8C). The E1 domain II residues are less well conserved than fusion loop residues, so the involvement of these residues in MAb binding may explain the lack of reactivity of IM-CKV066 with other alphaviruses (Fig. 2A). The moderately neutralizing MAb IM-CKV065 was mapped to an area of E2 encompassing domains A and B and arches 1 and 2 (Fig. 8D). The larger footprint of this MAb may be related to its relatively low affinity, with each individual residue presumably making only minor contributions to the total energetic landscape of the protein-protein interaction.

FIG 8.

Epitopes of nonneutralizing and moderately neutralizing anti-CHIKV MAbs. Critical residues for nonneutralizing CHIKV MAbs IM-CKV057 and IM-CKV067 (A), IM-CKV061 and IM-CKV062 (B), and IM-CKV066 (C) and moderately neutralizing MAb IM-CKV065 (D) are visualized. All epitopes are visualized on the heterodimeric and top-down trimeric forms of the CHIKV envelope crystal structure (PDB accession numbers 3N41 and 2XFC [28]). E1 is shown in yellow, E2 is shown in red, the fusion loop is shown in cyan, and epitope residues are shown as green spheres.

Interestingly, when all seven epitopes mapped in this study are visualized together with the eight epitope-mapped human and murine MAbs described in the literature (31, 33, 45), the epitopes reveal an immunogenic region at the tip of the E2/E1 heterodimer encompassing E2 domains A and B, the fusion loop, and E1 domain II (Fig. 9A). When the mapped epitopes are visualized on the CHIKV envelope trimer structure, a distinct spatial difference can be observed for the location of neutralizing versus nonneutralizing epitopes. The epitopes for our neutralizing MAbs (IM-CKV063, IM-CKV065, and C9) and other neutralizing MAbs described in the literature (CHK-152, CHK-263, CHK-102, CHK-166, and 5F10) are largely found on the accessible outer face and top of the trimer spike (Fig. 9B, orange residues), consistent with their ability to bind infectious virus and prevent infection. In contrast, the epitopes for nonneutralizing MAbs localize to the inner regions of the E2/E1 subunits (i.e., facing the interior of the trimer spike and not readily accessible) (Fig. 9B, green residues). The poor accessibility of these epitopes on the native infectious virion likely explains the lack of MAb neutralizing activity. Cumulatively, these results suggest that the membrane-distal domains of E2/E1 are the most highly immunogenic region of the protein and that epitopes exposed on the topmost and outer surfaces of the trimer face are neutralizing, whereas epitopes facing the interior of the trimer are not.

FIG 9.

Epitope mapping reveals highly immunogenic and neutralizing regions of CHIKV E2/E1. (A) The epitopes of nonneutralizing (IM-CKV057, IM-CKV061, IM-CKV062, IM-CKV066, IM-CKV067) and neutralizing (IM-CKV063, IM-CKV065) MAbs along with additional CHIKV epitopes reported in the literature (33, 45) are mapped onto the trimeric crystal structures of E2/E1 (PDB accession number 2XFC [28]). The names of the MAbs described in this study are boxed. All of the published epitopes map to the membrane-distal domains of E2/E1. Each individual E2/E1 heterodimeric subunit is shown in a different color for clarity. Epitopes on the outer-facing surfaces of the trimer (visible in the blue subunit) and residues on the top surface of the trimer correlate with MAbs that are neutralizing (residues in orange), whereas epitopes on the inner regions of the E2/E1 subunits facing the interior of the trimer spike (visible on the dark gray subunit) are correlated with MAbs that do not neutralize virus infectivity (colored green). (B) The CHIKV trimer spike is rotated 180 degrees relative to the orientation in panel A to illustrate that nonneutralizing and fusion loop residues are poorly accessible on the inner face of the virus trimer. A cutaway view with two subunits made transparent reveals nonneutralizing and fusion loop residues that face the interior of the trimer. Residue E2-E24 is located on the inner face in this view but is part of the cross-subunit epitope formed by neutralizing MAb IM-CKV063.

Fusion loop epitope residues are poorly exposed on native virions.

Our findings suggest that a major portion of E2/E1 is immunogenic in patients but does not elicit neutralizing MAbs. The localization of these nonneutralizing epitopes to an unexposed face of the envelope trimer further suggests that these nonneutralizing epitopes may be hidden from the immune system in the native infectious virion (but exposed under other circumstances). Interestingly, in the course of our studies, we observed that the immunoreactivity of some nonneutralizing MAbs was highly dependent on the immunofluorescence assay conditions employed and the format used for E2/E1 presentation. For example, the nonneutralizing fusion loop MAbs IM-CKV057, IM-CKV061, IM-CKV062, and IM-CKV067 were essentially nonreactive on cells expressing E2/E1 under native unfixed conditions at room temperature but were highly reactive following fixation with paraformaldehyde (Fig. 10). A similar trend was observed when CHIKV E2/E1 was presented on the surface of retroviral VLPs, where low levels of MAb reactivity were increased by fixation (Fig. 11A to D, blue versus green data points). These data are consistent with a model where epitopes near or within the fusion loop are not well exposed on the native virus.

FIG 10.

CHIKV MAb reactivity is influenced by the fixation state of cells expressing E2/E1. CHIKV MAbs were tested for immunoreactivity on HEK293T cells expressing CHIKV E2/E1 or a vector control (pUC19). Cells were either fixed with 4% paraformaldehyde for 5 min or left unfixed prior to the addition of primary MAb. Immunoreactivity was determined by flow cytometry. Bars represent the mean of eight replicate data points, and data are representative of those from at least two independent experiments. *, statistically significant differences (P < 0.01) for a given MAb using fixed and unfixed conditions.

FIG 11.

Temperature-dependent MAb reactivity. The immunoreactivity of MAbs IM-CKV057 (A), IM-CKV061 (B), IM-CKV062 (C), IM-CKV067 (D), IM-CKV066 (E), IM-CKV063 (F), IM-CKV065 (G), and C9 (H) with CHIKV E2/E1 displayed on the surface of retroviral VLPs was determined at different temperatures and under different fixation conditions by ELISA. Retroviral VLPs were either fixed using 4% paraformaldehyde at 22°C or left unfixed. Primary MAb was added at the temperatures indicated for 60 min. Data points represent the mean and standard deviation of three replicates. Green asterisks, MAb reactivities on unfixed cells at elevated temperatures that are different (P < 0.05) from their respective reactivity at 22°C; blue plus signs, MAb reactivities at a specific temperature that are different (P < 0.05) due to fixation. The experiment was performed twice, and data for one representative experiment are shown. IM-CKV065 and IM-CKV066 were not tested under fixed conditions since fixation renders these MAbs poorly reactive.

Because of these observations, we investigated if fusion loop MAbs IM-CKV057, IM-CKV061, IM-CKV062, and IM-CKV067 might also display increased reactivity under more physiologically relevant temperatures. In the native unfixed state (Fig. 11A to D, green data points), these MAbs demonstrated increased immunoreactivity at temperatures of 37°C and 45°C compared with that at room temperature, and the MAb reactivity at elevated temperatures was comparable to the MAb reactivity under fixed conditions. These results suggest that fusion loop epitopes are shielded at room temperature in their native state but that both elevated temperature and fixation can increase fusion loop epitope exposure. Interestingly, these fusion loop MAbs were still nonneutralizing even after incubation with virus at elevated temperatures (Fig. 12), suggesting that the fusion loop MAbs may be recognizing a subpopulation of E2/E1 with exposed fusion loop structures that no longer contribute to infectivity.

FIG 12.

Temperature-dependent effects of CHIKV MAb neutralization. The effect of temperature on MAb neutralization is shown for a representative nonneutralizing MAb (IM-CKV061) (A), a moderately neutralizing MAb (IM-CKV065) (B), and a strongly neutralizing MAb (IM-CKV063) (C). Reporter HIV isolates pseudotyped with CHIKV S27 E2/E1 were preincubated with MAbs at the indicated temperatures, and infection of HEK293T target cells was detected by the expression of Renilla luciferase. Each data point represents the mean and range of two replicates within an experiment. Data shown are representative of those from two independent experiments. For IM-CKV065 and IM-CKV063, there were no statistically significant differences in log EC₅₀ at different temperatures (P > 0.05). IM-CKV061 did not reach 50% neutralization irrespective of temperature.

The conformational MAb IM-CKV066 was also sensitive to temperature and fixation; however, it showed a trend opposite of that for the MAbs described above, where increased temperature and protein fixation led to diminished reactivity (Fig. 10 and 11E), suggesting that these conditions lead to protein conformational changes that disrupt this discontinuous epitope. Moderately neutralizing MAb IM-CKV065 has a discontinuous epitope that appears to be sensitive to conformational changes resulting from fixation but not temperature (Fig. 10 and 11G). It is possible that the Lys233 residue of this epitope is cross-linked to paraformaldehyde during fixation, decreasing its ability to interact with IM-CKV065. Interestingly, all neutralizing MAbs (regardless of potency) were highly reactive under native unfixed conditions, and temperature did not have a pronounced effect on their ability to bind their epitopes (Fig. 11F to H). This is consistent with these neutralizing MAbs targeting a well-exposed epitope on the native infectious virus that contributes to the human immune response against CHIKV.

DISCUSSION

In this study, we isolated and characterized a panel of human MAbs against CHIKV that were derived from naturally infected patients. To correlate the structural targets of CHIKV MAbs with their protective efficacy, we determined the in vitro neutralization abilities of the MAbs and identified their epitopes using a comprehensive shotgun mutagenesis strategy. Notably, we isolated a potent neutralizing MAb, IM-CKV063, which prevents CHIKV disease in vivo when administered up to 24 h postinfection. The results for other MAbs isolated in our studies suggest that functionally important structures that include the fusion loop are largely hidden from immune recognition in the infectious form of the virus.

Visualization of the binding sites for our and others' MAbs suggests that most epitopes are located in highly immunogenic membrane-distal domains of E2/E1 and that epitopes on the exposed topmost and outer surfaces of the E2/E1 trimer structure are neutralizing, whereas epitopes facing the interior of the trimer are not. These results suggest that the neutralizing efficacy of CHIKV MAbs and, possibly, MAbs against other alphaviruses can be predicted, at least in part, on the basis of their epitope location. Specifically, our data suggest that whether the MAbs are neutralizing or nonneutralizing is dependent on the epitope's exposure on the native trimer, with neutralizing MAbs being elicited to the exposed external faces and top of the trimer and nonneutralizing MAbs being elicited to the occluded inner regions of the E2/E1 subunits facing the interior of the trimer spike. This hypothesis provides a rationale for vaccine design and the development of a therapeutic MAb to target the regions of E2/E1 with the greatest neutralization potential. For example, protein subunit vaccines may induce both nonneutralizing and neutralizing MAbs, while virus particle-based vaccines that are locked in their native configuration may preferentially induce neutralizing MAbs against the exposed surfaces of the E2/E1 trimer, helping to explain the efficacy of a CHIKV VLP-based vaccine currently being tested (16).

IM-CKV063 is one of the most potent CHIKV MAbs reported to date (IC₅₀, 7.4 ng/ml; PRNT₅₀, 11 ng/ml) and has potency comparable to that of the most potent human MAb, C9 (IC₅₀, 51 ng/ml; PRNT₅₀, 12 ng/ml [32]), and murine MAb, CHK-152 (PRNT₅₀, 2 ng/ml [33]), described previously. MAb IM-CKV063 was protective in vivo in two distinct animal models, protecting neonatal mice from death when administered up to 24 h after virus exposure. Postexposure experiments testing therapeutic efficacy are particularly relevant to human clinical use, as treatment after infection is often more feasible than prophylaxis. In a separate adult mouse model of CHIKV arthritic disease that more closely recapitulates human symptoms, we found that IM-CKV063 effectively prevented the arthritis caused by CHIKV. Taken together, the results from these two animal models demonstrate the potential clinical utility of IM-CKV063 for prophylactic and therapeutic protection against CHIKV-induced disease in humans.

To date, few other reported CHIKV MAbs have been evaluated in a therapeutic (postinfection) context. MAb CHK-152 also provided protection when administered at 24 h postinfection, although a 10-fold higher dose (100 μg of CHK-152 versus 10 μg of IM-CKV063 in the present study) and a different animal model (type I IFN receptor-knockout mice) were used (33). MAb IM-CKV063 appears to be greatly superior to previously reported human MAbs 5F10 and 8B10, which prolonged survival but did not protect animals from lethality even at much higher MAb doses of 250 μg (45, 61). IM-CKV063 thus offers the opportunity to design both therapeutic and passive immunization strategies for treating infected patients or protecting those at risk prior to exposure (e.g., travelers, military personnel). The fact that IM-CKV063 was originally derived from a patient immune response further suggests that this epitope may be successfully targeted by the human immune system in response to the right vaccine.

MAb IM-CKV063 binds to an E2 epitope not previously described in the literature. The specific residues involved in IM-CKV063 binding mapped to the surface-exposed regions of domain A known as the N flap and wings, which are possible sites of interaction with a cellular receptor in mammals (28, 42). Thus, a possible mechanism of action of IM-CKV063 neutralization is via blocking viral attachment to cells. MAb IM-CKV063 reactivity was relatively unaffected by changes in temperature or fixation conditions, likely reflecting the favorable surface exposure of its epitope in the native state of the virion. Furthermore, our data suggest that IM-CKV063 binds to a conformational epitope that spans two E2 subunits. Thus, this MAb may effectively cross-link different E2 domains on the E2/E1 trimer, preventing conformational changes that expose the E1 fusion peptide. Residues in this region of E2 have been shown to be important for conformational changes in E2/E1 that occur during membrane fusion (28, 62), consistent with this proposed neutralization mechanism. A mutation at residue E2-I121, part of the IM-CKV063 epitope, has previously been shown to enhance viral infectivity in Aedes aegypti mosquitoes (63) and allow MAb escape for Venezuelan equine encephalitis virus (39), so IM-CKV063 could also block other functionalities of E2/E1.

Previous studies have described other neutralizing MAbs that protect against CHIKV in animal models (32–36, 61) and also showed that some combinations of MAbs can extend the window for effective treatment after CHIKV exposure and reduce the development of viral resistance (33). IM-CKV063 potently targets an exposed epitope on E2 domain A in a location distinct from the locations targeted by other neutralizing MAbs, such as C9 or CHK-152. Thus, IM-CKV063 may be even more highly effective as part of an optimized MAb combination therapy for CHIKV, while its use may also minimize the possibility of emergence of resistant mutants.

The nonneutralizing MAbs that we isolated (IM-CKV057, IM-CKV061, IM-CKV062, IM-CKV066, and IM-CKV067) bound overlapping epitopes encompassing the fusion loop and neighboring residues. The fusion loop is a highly conserved structure among alphaviruses and is of crucial functional importance in viral infection (28). Consistent with the conserved sequence of this region, MAbs targeting the CHIKV fusion loop demonstrated cross-reactive binding to SFV, RRV, and SINV. However, the fusion loop MAbs were not capable of neutralizing CHIKV in our assays, even after incubation at elevated temperatures. The lack of neutralization was further evident in animal studies, where fusion loop MAbs IM-CKV057 and IM-CKV066 did not protect mice from CHIKV mortality. We hypothesize that this lack of neutralizing ability is because the fusion loop is hidden in the prefusion state of infectious virus, preventing these MAbs from binding and inhibiting infectivity. The increased reactivity of these MAbs with E2/E1 under altered reactivity conditions (elevated temperatures, fixation) is consistent with such a hidden epitope. The presence of fusion loop MAbs in patient samples may be due to an immune response against defective virions or E2/E1 proteins triggered during a natural infection. It is also possible that the method of MAb isolation used in our study, phage display, could influence the types of MAbs recovered.

Given the apparent immunogenicity of the fusion loop, it is interesting to note that only one other MAb against the fusion loop of any alphavirus has been previously reported (59, 60). In the related alphavirus SFV, fusion loop MAb E1f (which is also nonneutralizing) similarly demonstrated little binding to virus particles under native conditions, binding the fusion loop only when dissociation of the E2/E1 dimer was triggered by low pH (59, 60). It is likely that nonneutralizing fusion loop MAbs have been isolated by others but have been either not characterized as binding the fusion loop or not reported due to their apparent lack of therapeutic potential. For example, we found only two of seven MAbs isolated (29%) to be neutralizing, and Pal et al. similarly identified a relatively small subset (16%) that was neutralizing (33). The present study mapped epitopes irrespective of MAb neutralization status, a strategy that allows a broader representation of the overall human antibody response against CHIKV. In addition, the method of epitope mapping used here, shotgun mutagenesis, does not require MAb neutralization (as neutralization escape mapping methodologies do), nor does it require the maintenance of viral fitness during the acquisition of neutralization escape mutants, so it offers a comprehensive epitope mapping approach for conformational and linear MAbs across the entire envelope protein.

In conclusion, the MAbs isolated and characterized in this study demonstrate that the human immune system can generate highly potent, neutralizing MAbs against CHIKV but that nonneutralizing (e.g., fusion loop-specific) MAbs against epitopes that are not normally exposed on the native virion are also generated in a natural infection. Comparison of the binding sites of neutralizing and nonneutralizing MAbs suggests that vaccination with structurally intact trimers of E2/E1, as they exist on the native virion, may result in the most highly protective immune response.