Novel E2 Glycoprotein Tetramer Detects Hepatitis C Virus–Specific Memory B Cells

Hepatitis C virus (HCV) infection remains a global public health problem. In the United States, infection rates have increased steadily over the past decade primarily due to injection drug use among adolescents and young adults (1). Although direct acting antivirals are generally safe and most persistent infections are cured within 2–3 mo of therapy (2), their high cost, limited availability, and the asymptomatic nature of most infections remain important challenges. Furthermore, successful treatment of chronic HCV infection with direct acting antivirals does not prevent reinfection, which is a recurrent problem for the high risk population (3). The development of a prophylactic vaccine to inhibit HCV transmission is still a major goal. An interesting candidate, currently in phase 2 clinical trials, is based on the prime-boost strategy with the first immunization using a chimpanzee adenovirus together with a boost using a modified vaccinia Ankara vector, both expressing the nonstructural region of HCV genome. In a phase 1 clinical study, this vaccine was well tolerated and induced a strong T cell response (4). However, an optimal vaccine would combine both T and B cell responses (5). It was shown that immunization with recombinant HCV E1E2 glycoproteins elicited a cross-reactive neutralizing Ab response in humans (6). In order to better evaluate the potential of a vaccine eliciting an effective humoral response, further insight into the development of a protective B cell response during acute HCV infection is required.

Seroconversion to HCV envelope glycoproteins 1 (E1) and 2 (E2) usually occurs several weeks postinfection, regardless of whether the virus is cleared or persists (7, 8). The neutralizing effect of anti-HCV Abs was demonstrated in both the chimpanzees (9, 10) and humanized mouse models of HCV infection (11, 12). In these studies, incubation of an HCV inoculum with anti-HCV Abs prevented infection, as did a passive transfer of the Abs before the challenge (9–12). In humans, broadly neutralizing Abs were shown to have developed more rapidly and to higher titers in individuals with an acute resolving infection when compared with those with persisting infection (13–17). In the context of reinfection, it was shown that a subsequent exposure to HCV led to the development of cross-reactive Abs, suggesting an improved humoral response (18). However, some reports suggest that the infection can be resolved in the absence of any detectable HCV-specific Ab responses in both chimpanzees and humans (19–21). In those studies, HCV viral loads were low, and HCV-specific T cell responses were detected suggesting that a very transient viremia might not be enough to prime HCV-specific Ab responses. Furthermore, examining immune responses in a cohort of women who spontaneously resolved a single source outbreak of HCV demonstrated that circulating HCV-specific Abs were undetectable in many subjects 18–20 y after recovery (22). These opposing results have led to confusion regarding the contribution of HCV-specific Abs to clearance of infection and highlight the need to obtain a better insight into the nature of humoral immune response during acute HCV infection.

Ag-specific IgG-secreting memory B cell frequencies can be evaluated by bulk B cell stimulation coupled with ELISPOT assays (23). Although informative, this method does not allow for direct characterization or recovery of the HCV-specific B cells for downstream analyses. Alternatively, identification of Ag-specific B cells is possible using tetramers generated from biotinylated Ags coupled with fluorescently labeled streptavidin. Such tetramers have been shown to specifically identify and isolate tetanus toxoid-specific B cells from the blood of vaccinated donors (24). Similarly, HIV gp41 tetramers have been used to characterize HIV-specific B cells and their Ab repertoire during different stages of HIV infection (25).

Here we report the development of an HCV-specific B cell tetramer reagent composed of the ectodomain of HCV envelope glycoprotein E2 (J6 strain, genotype 2a). The specificity of this tetramer was validated using an E2-specific hybridoma cell line and PBMCs from subjects persistently infected with HCV of different genotypes. To better understand the kinetics of Ag-specific B cell responses during HCV infection, we performed a longitudinal study to directly visualize and quantify the frequency of E2-specific B cells in the peripheral blood of subjects progressing from an acute to chronic infection. Finally, as a proof of concept, this novel HCV E2 tetramer enabled us to isolate E2-specific class-switched memory B cells and perform BCR deep sequencing on two persistently HCV-infected subjects. The dominant repertoire profiles, skewed CDR3 length distributions, and increased mutation frequencies all suggested that these cells were selected, expanded, and had undergone affinity maturation processes.

The pCMJJ4 vector served as the backbone for expression of the HCV E2 ectodomain (J6 genotype 2a strain, aa 384–664). The E2 ectodomain sequence was linked to a BirA substrate peptide (GLNDIFEAQKIEWHE) (BSP85), followed by a PreScission protease-cleavable protein A (PA) tag (Fig. 1A). Lentiviruses were generated as described previously (26). Briefly, the E2 expression vector and accessory plasmids (psPAX2 and pMD2G) were introduced into HEK293T cells by calcium phosphate transfection. Supernatants containing lentiviruses were harvested 3 d later and were subsequently used to transduce HEK293T cells expressing the BirA enzyme, which biotinylates the BirA substrate peptide. Analysis of surface Thy1.1 expression was used to determine transduction efficiency. Cells expressing Thy1.1 were then inoculated into CELLine Flasks (Integra Biosciences, Hudson, NH) and supernatants were harvested every 7 d. The supernatants were clarified by centrifugation at 5000 × g for 30 min at 4°C and E2-biotin-protein A was purified by the use of an IgG fast flow affinity column (GE Healthcare, Atlanta, GA). The protein A tag was removed by cleavage with 50:1 ratio of protein to PreScission protease (GE Healthcare) overnight at 4°C. E2-biotin was subsequently purified with an IgG FF column (GE Healthcare) to remove uncleaved E2-protein A and the protein A tag. Finally, a GSTrap FF column was used to remove PreScission protease (GE Healthcare). As a control reagent, an HIV gp140 tetramer (clade C HIV-1, isolate DU422) was also generated using the same approach.

Biotinylated E2 monomers were incubated with either PE labeled ExtrAvidin (Sigma, St. Louis, MO) or allophycocyanin labeled streptavidin (Molecular Probes; Thermo Fisher Scientific, Rochester, NY) at a molar ratio of 4:1. Fluorescently labeled streptavidin reagent was added to the E2 monomer in six aliquots, each followed by an incubation of 10 min at room temperature (1 h total). Before hybridoma cell staining, tetramer preparations were centrifuged for 10 min at maximum speed to remove aggregates. Hybridoma cells were first stained with Ghost Dye Red 780 (Tonbo Biosciences, San Diego, CA) to exclude dead cells. Cells were washed and treated with BD FACS permeabilizing solution 2 for 10 min at room temperature. Cells were washed and incubated with 0.4 μg E2 tetramers for 30 min at 4°C. Cells were washed and fixed in 1% formaldehyde in PBS before FACS analysis.

Study subjects were recruited among people who inject drugs (PWID) participating in the Montreal Acute Hep C Cohort Study (HEPCO) as previously described (27) or presenting to the hepatology clinic of St-Luc Hospital as previously described (28). Acute infection was identified and followed as previously described (29). The estimated date of infection (EDI) was defined as the median point between the last negative and the first positive HCV test. Chronic HCV infection was defined as a positive HCV RNA test at 6 mo following the EDI. A total of 37 subjects were examined in a cross-sectional analysis: 31 chronic patients (HCV Ab +ve and HCV RNA +ve) and 6 spontaneous resolvers (HCV Ab +ve and HCV RNA −ve). In addition, seven PWIDs were analyzed longitudinally at three key time points before, during, and after acute HCV infection: baseline (negative both for HCV RNA and Abs), late acute phase (5 ± 2 mo post EDI) and chronic phase (>12 mo post EDI). Two control groups consisting of seven healthy donors and six HCV naive PWID were also included. Participant’s demographics and clinical characteristics are listed in Table I. This study was approved by the Institutional Ethics Committee of the Research Centre of the University of Montreal Hospital Centre (CRCHUM), protocols SL05.014 and SL05.025. All experiments were performed on cryopreserved PBMCs.

PBMCs from healthy controls and HCV-infected individuals were thawed and blocked with RPMI 1640 supplemented with 20% heat-inactivated human serum and 5 μl human Fc block/2 × 10⁶ cells (BD Biosciences, Mississauga, ON) for 15 min at 4°C. Cells were then washed with FACS buffer (PBS 1× [Wisent], 1% FBS [FBS; Sigma], 0.01% sodium azide [Thermo Fisher Scientific, Burlington, ON]) and stained with the tetramer for 30 min at room temperature. Cells were washed twice with FACS buffer and stained with either panel 1 (Figs. 3, 4) or panel 2 (cell sorting) for 30 min at 4°C. Cells were washed again twice and fixed with 1% formaldehyde in PBS before FACS analysis. The following conjugated anti-human mAbs were used in panel 1: CD3-Pacific Blue (clone UCHT1), CD14-V500 (clone M5E2), CD16-V500 (clone 3G8), CD19-Alexa Fluor 700 (clone HIB19), CD27-allophycocyanin-H7 (clone M-T271), IgM-BB515 (clone G20-127). Panel 2 contained all panel 1 Abs with the addition of CD10-BV605 (clone HI10a) and CD21-PE-Cy7 (clone B-ly4). All Abs were obtained from BD Biosciences. Live cells were identified using LIVE/DEAD fixable aqua dead cell stain kit (Molecular Probes; Thermo Fisher Scientific). Multiparameter flow cytometry was performed at the flow cytometry core of the CRCHUM using a BD LSRII instrument and cell sorting was completed using a BD Aria IIIu instrument, both equipped with violet (405 nm), blue (488 nm), yellow-green (561 nm) and red (633 nm) lasers and FACSDiva version 8.0.1 (BD Biosciences). FCS data files were analyzed using FlowJo version 10.0.8 for Mac (Tree Star, Ashland, OR). Fluorescence minus one controls were used to set the gate for CD27 and CD10. B cell gating included the selection of live cells that are also CD14⁻, CD16⁻ and CD3⁻ (Fig. 3A).

HCV E2 glycoprotein (30) (1 μg/ml in 0.1 M Na₂CO₃ buffer) was used to coat 96-well flat bottom immuno plates (Nalgene Nunc; Thermo Fisher Scientific) overnight at 4°C. Coated plates were washed twice with PBS plus 0.05% Tween 20 (PBS-T) and then blocked with 10% normal goat serum in PBS-T (Jackson ImmunoResearch, West Grove, PA) for 1 h at 37°C. Human plasma samples from HCV-1 to HCV-7 as well as three healthy controls were added to the plates (10-fold serial dilutions in binding buffer [0.1% normal goat serum in PBS-T]) for 90 min at room temperature and the plates were washed eight times with PBS-T. Then, 0.1 μg/ml biotinylated anti-human IgG mAb MT178/145 (Mabtech, Cincinnati, OH) diluted in binding buffer was added to each well for 90 min at room temperature and plates were washed eight times with PBS-T. Streptavidin-HRP (Mabtech) diluted 1:5000 in binding buffer was added to plates for 45 min at room temperature and plates were washed eight times with PBS-T. Tetramethylbenzidine substrate (BD, Franklin Lakes, NJ) was added to develop color according to the manufacturer. Absorbance (450 nm) was measured using a Versamax microplate reader and SoftMaxPro software (Molecular Devices, Sunnyvale, CA). Standard curves were done using serial dilutions of E2 mAb 2C1, goat anti-mouse IgG, biotin conjugate (Invitrogen, Thermo Fisher Scientific, Waltham, MA). The assay was quantified Elisaanalysis.com software. Three independent experiments were performed in duplicates.

B cell ELISPOT was performed with the human IgG ELISPOT kit (Mabtech) according to the manufacturer’s instructions and as described by Jahnmatz et al. (31). Briefly, PBMCs were thawed and rested for 1 h at 37°C, 5% CO₂ in R10 (RPMI 1640, supplemented with 10% FBS). Cell were stimulated with 1 μg/ml R848 and 10 ng/ml rhIL-2 in AIM-V supplemented with 10% FBS (AIM-V-FBS) in a 24 wells plate at 2 × 10⁶ cells/well or left unstimulated for 72 h at 37°C, 5% CO₂. Cells were washed three times and plated in duplicates in PVDF MSIPS4W10 ELISPOT plates (EMD Millipore, Etobicoke, ON) that were previously coated with anti-IgG (MT91/145; Mabtech) overnight at 4°C, washed and blocked with AIM-V-FBS for 1 h at 37°C, 5% CO₂. Plates were incubated 18 h at 37°C, 5% CO₂. Plates were washed nine times with PBS-T. Total IgG response was detected with anti-IgG-biotin (MT78/145) and HCV-specific Ab response was detected with J6-E2-biotin, for 2 h at room temperature. Plates were washed nine times and incubated with streptavidin-ALP (alkaline phosphatase, 1:1000; Mabtech) for 1 h at room temperature. Plates were washed seven times with PBS-T, three times with PBS, and one time with water. Spots were developed with the alkaline phosphatase conjugate substrate kit (BioRad, Montreal, QC) for 4 min in the dark, according to the manufacturer’s instructions. Plates were extensively washed with tap water, dried overnight and spots were counted with Immunospot plate reader (Cellular Technology, Shaker Heights, OH) and normalized to Ab-secreting cells (ASC)/1 × 10⁶ PBMC.

PBMCs were thawed and total B cells were purified by negative selection using the MACS Pan B cell Isolation Kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s protocol. Tetramer staining and cell surface staining for CD3, CD10, CD14, CD16, CD19, CD21, CD27, and IgM were performed as described above. Tetramer positive class-switched memory B cells (CD19⁺CD27⁺IgM⁻) and naive B cells (CD19⁺CD27⁻CD10⁻CD21^hi) were FACS sorted with an Aria IIIu flow cytometer (BD Biosciences).

Sorted cells were frozen and shipped to Adaptive Biotechnologies (Seattle, WA) for genomic DNA extraction and BCR H chain (IGH) deep sequencing. Lists of unique CDR3 sequences (clonotypes) and their frequency within the repertoire were obtained for each sample. The raw data can be accessed at https://clients.adaptivebiotech.com/pub/boisvert-2016-JI. Data were filtered to remove out-of-frame sequences and sequences with stop codons within the CDR3 region. Clonotypes with sequence counts equivalent to or less than the average count per cell were also removed from the analysis. Data were analyzed using the ImmunoSEQ analysis platform. To study the dominance profile, clonotypes were classified into four groups according to their frequency within the repertoire. The first group was composed of dominant clonotypes that were each present at a frequency >1% of the repertoire. The second group comprised subdominant clonotypes, with frequencies between 0.1 and 1% of the repertoire. The third group was made of low abundance clonotypes with frequencies between 0.05 and 0.1% of the repertoire. Finally, the fourth group contained clonotypes of lowest abundance with frequencies of <0.05% of the repertoire. The CDR3 region length in amino acids (aa) was determined and the distribution of the frequency of each length was analyzed. Very short (<7 aa) and very long (>31 aa) CDR3 lengths were rare and omitted from the analysis. V gene mutation frequency of each clonotype sequence was calculated as a percentage and represents the number of substitutions/100 bp compared with germline sequence according to the ImMunoGeneTics (IMGT) database (32).

Differences between groups were analyzed using the Mann–Whitney U test. Correlations were analyzed by the Spearman test using GraphPad Prism version 5.0 (La Jolla, CA).

There are currently no reagents available to directly examine HCV-specific B cells. Thus, we used the ectodomain of HCV envelope glycoprotein E2 (aa 384–664) derived from genotype 2a (J6 strain) to develop a novel B cell tetramer capable of identifying HCV-specific B cells from the peripheral blood of infected individuals. The J6 genotype 2a strain was chosen because its ectodomain was previously successfully expressed and crystalized to resolve the E2 protein structure diffracted to 2.4 Å resolution (30). This protein was also shown to be properly folded when expressed in HEK293T cells (33). A cartoon diagram for the generation of the E2 expression vector is shown in Fig. 1A. The insertion of the biotinylation site BSP85 sequence enabled site-specific monobiotinylation, whereas the addition of a protein A tag allowed for affinity purification. Biotinylated E2 monomers were produced in lentivirus-transduced HEK293T cells as described in Materials and Methods. Monomer purity and size were confirmed by SDS-PAGE (Fig. 1B). Purified E2-PA (90 kDa; lane 4), was cleaved by PreScission protease (PP, 46 kDa) to remove the protein A tag (PA, 30 kDa; lane 5), and E2 monomer (expected size 60 kDa) was purified by passage over IgG then GST columns (lanes 6–8). Since the purified E2 monomers corresponded to the expected size, they were used to generate tetramers by incubation with fluorophore-conjugated streptavidin or ExtrAvidin at a molar ratio of 4:1.

We first sought to confirm that the E2 tetramer reagent could specifically recognize Abs targeting the E2 glycoprotein ectodomain. E2 tetramer reactivity was validated by two approaches. First, anti-mouse Ig BD flow cytometry compensation beads (CompBeads) were incubated with either 2C1 mAb that recognizes HCV E2 glycoprotein (30) or 1D6 mAb that recognizes HIV gp120 (34). Next, the Ab-coated beads were incubated with E2 or gp140 tetramers, and the tetramer allophycocyanin fluorescence intensity was analyzed by flow cytometry. The E2 tetramer was able to recognize the CompBeads that were coated with the E2-specific mAb 2C1 (Fig. 2A, left). This interaction was specific, as the E2 tetramer could not recognize the HIV Ab 1D6 and the HIV-specific gp140 tetramer did not recognize the beads coated with the anti-E2 2C1 Ab. The gp140 tetramer did, however, recognize the beads containing the gp120-specific Ab 1D6 (Fig. 2A, right). We next asked whether the HCV-E2 tetramer could directly recognize hybridoma cells that produce anti-HCV E2 mAbs. To test this, the E2 tetramer was used in an intracellular staining of the hybridoma cell line (2C1) that produces a mAb to J6-E2 protein (Fig. 2B, top). The E2 tetramer stained the 2C1 hybridoma, but did not recognize the hybridoma that produces the HIV envelope protein gp140-specific Ab (1G12) (Fig. 2B, bottom). Together these data suggest that the E2 tetramer could specifically recognize Abs targeting the HCV E2 glycoprotein.

Next, we evaluated the capacity of the E2 tetramer to detect HCV-specific B cells in HCV-infected individuals (Table I). Tetramer and surface staining were performed on PBMC to identify HCV E2-specific class-switched memory B cells (CD19⁺CD27⁺IgM⁻) in subjects with established chronic HCV infection (HCV RNA +ve for >1 y, genotypes 1, 2, or 3; n = 21, 5 and 5 respectively). A representative gating strategy is presented in Fig. 3A. The tetramer positive gate was set relative to the background staining observed on CD19⁻ cells. Tetramer staining was evaluated at three temperatures: 4°C, room temperature, and 37°C. The staining was most efficient at room temperature, enabling detection of more HCV-specific B cells (Fig. 3B), with minimal background on control samples as compared with 37°C (data not shown). Within the total class-switched memory B cell compartment, the average frequency of HCV-specific B cells was 0.40% (range 0.05–2.03%) (Fig. 3C). The mean background binding from healthy donors (n = 7) and HCV negative PWID (n = 6) was 0.04%, which when combined ranged from 0 to 0.11%. The threshold of detection was set at 0.095% (mean frequency + 2 SD of healthy donors and naive PWID) and HCV E2-specific tetramer-positive populations were detected in 28 out of 31 chronic HCV participants.

Sensitivity of the tetramer detection was compared with the detection of HCV-specific B cells using an IgG ELISPOT assay. As shown in Fig. 3D, there was a significant correlation between the number of E2-specific ASC and the number of tetramer positive cells (Spearman r = 0.86; p = 0.0003). In addition, the ELISPOT assay showed that in the three samples for which we could not detect a tetramer positive population, E2-specific ASC were not detected or were barely detectable (gray dots, Fig. 3D).

Analyses of other B cell subsets, unswitched memory B cells (CD27⁺IgM⁺), and naive/atypical B cells (CD27⁻IgM⁺) in genotype 1 samples showed that the tetramer positive cells were detectable in both populations (Fig. 3E). However, in the majority of cases (15 out of 21, p < 0.01) we detected more HCV-specific cells in the class-switched memory B cell subset. The remaining six samples had an equivalent frequency of tetramer positive cells in the unswitched memory population. There were also five samples where the frequency of naive/atypical B cells was equivalent or higher than the frequency of class-switched memory B cells, but the overall frequency of tetramer positive B cells was quite low and did not allow for a more detailed comparison of these two subsets. None of the three samples that were below the threshold of detection in class-switched memory B cells had a significant tetramer positive population in the unswitched or naive/atypical memory B cells. Collectively, these results suggest that the J6-E2 tetramer can be used to successfully identify HCV-specific B cells in infected participants and that HCV E2-specific B cells are present in the majority of individuals with chronic infection.

Identification of HCV-specific B cells during acute HCV infection would enable the characterization of early changes in that population leading to the development of the Ab response. We thus examined the longitudinal frequency of HCV E2-specific B cells in acutely infected participants who went on to develop persistent HCV infection (n = 7). During the late acute phase of infection (5 ± 2 mo, EDI), E2 tetramer positive cells were detected in approximately half of the samples (3 out of 7) within both the total CD19⁺ B cell population (Fig. 4A) and the class-switched memory B cell population (CD19⁺CD27⁺IgM⁻) (Fig. 4B). All participants tested (7 out of 7) developed a tetramer positive population within the class-switched memory B cell population at their latest follow-up chronic time point (>12 mo). These results suggest that at least in some participants the development of the HCV-specific B cell response was significantly delayed. ELISA assay was performed with the plasma samples from the same participants during both the late acute and chronic time points. At the late acute time point, we detected E2-specific Abs only in the three samples with a tetramer positive population (Fig. 4C). All samples showed seroconversion at the chronic time point (Fig. 4D). For the majority of the samples, there was an observable correlation between the E2 Ab titers and the frequency of tetramer positive class-switched memory B cells (Fig. 4E). The only exception was sample HCV-4 where we detected a high frequency of tetramer positive cells but very low anti-E2 Ab titers (gray dot in Fig. 4E).

As a proof of concept, we sequenced the BCR of naive and HCV-specific B cells of two subjects to analyze the characteristics of B cell antigenic selection and maturation process within the repertoire. The selection and amplification of B cell clonotypes led to a focusing of the BCR repertoire with fewer clones detected and a dominant profile emerging. J6-E2 tetramer positive class-switched memory B cells (CD19⁺CD27⁺IgM⁻) from subjects HCV-8 and HCV-9 were sorted at their latest follow-up time point (≥5 y) and deep sequenced for the BCR IgH (IGH). Naive B cells (CD19⁺CD27⁻CD10⁻CD21^hi) were also sorted and used as a control for the unselected BCR repertoire. Sample information, including the number of sorted cells and the number of sequencing reads, is listed in Table II. The resulting list of unique clonotypes were divided into four groups based on their frequency within the repertoire: 1) dominant clonotypes each representing >1% of the repertoire, 2) subdominant clonotypes representing 0.1–1% of the repertoire, 3) low abundance clonotypes representing 0.05–0.1% of the repertoire, and 4) lowest abundance clonotypes, with frequencies of <0.05% of the repertoire. As expected, the BCR repertoire of naive B cells showed no selection of dominant clonotypes and very few subdominant clonotypes (Fig. 5, left pie charts). For both subjects, most of the naive repertoire (>90%) was composed of the lowest abundance clonotypes. In sharp contrast, the repertoire of HCV E2-specific tetramer positive class-switched memory B cells was focused in both subjects where most of the clonotypes identified (90–99%) were dominant (Fig. 5, middle pie charts). Participant HCV-9 possessed 16 clonotypes totaling 99% of the B cell repertoire, whereas the repertoire of participant HCV-8 was composed of 37 clonotypes, totaling 90% of the total repertoire. The most dominant clonotype for participant HCV-9 (IGHV03-D05-J06-01) represented 42% of the repertoire, whereas the most dominant clonotype for participant HCV-8 (IGHV04-D02-02J05-01) represented only 6% of the repertoire (Fig. 5, right pie charts). Together, these results demonstrate that the J6-E2 tetramer can be used to identify, select, and sort HCV-specific B cells for downstream analyses. Further, the focused repertoire from both HCV-infected subjects suggest Ag-specific selection and/or expansion of this B cell population.

In a naive B cell repertoire, it is estimated that CDR3 lengths have a normal Gaussian-like distribution with no particular selection or amplification of any clonotype (35). However, in an Ag-specific population particular clonotypes are selected and go through the process of affinity maturation leading to the enrichment and dominance of certain CDR3 lengths (35). We analyzed the distribution of CDR3 lengths for both the naive and E2-specific sorted samples. As demonstrated in Fig. 6A and 6C, the CDR3 lengths within the naive samples showed a normal bell shape distribution in both subjects (R² > 0.94). In contrast, the CDR3 length distributions from E2-specific B cells were skewed in both subjects (Fig. 6B, 6D; R² < 0.75). In participant HCV-8, lengths of 14, 18 and 19 aa were highly enriched (Fig. 6B). Likewise, CDR3 lengths of 16 and 18–21 aa were enriched in participant HCV-9 (Fig. 6D). These results provide additional evidence that E2-tetramer positive cells are indeed Ag-specific and have undergone specific selection and expansion during HCV infection.

During the process of affinity maturation, the V region of the BCR undergoes somatic hypermutation, followed by selection of clones with the highest affinity to the Ag (36). There was a statistically significant increase in the V gene mutation frequency from HCV E2-specific samples compared with the corresponding naive B cell samples in both subjects (Fig. 6E). There were no differences in the average mutation frequencies between the HCV E2-specific HCV-8 and HCV-9 samples. This suggests that HCV E2-specific class-switched memory B cells underwent affinity maturation and accumulated a significant number of mutations that were specific for each participant.

The importance of B cell-mediated Ab responses in spontaneous clearance of primary HCV infection is controversial. In chimpanzees, no correlation could be established between the Ab response and spontaneous viral clearance (37). In humans, a study from a cohort of a single-source outbreak of HCV infection showed that the rapid development of neutralizing Abs correlated with spontaneous clearance (15). However, other reports described cases where HCV infection was spontaneously resolved without the detection of HCV-specific Abs in humans (19–22). The development of new methods enabling characterization of HCV-specific B cells in acutely infected patients is needed to establish better understanding of the correlates of the humoral immune response associated with spontaneous resolutions of HCV infection. Generation of B cell tetramer is an important strategy because it enables deeper characterization of the B cell subpopulation that is involved, as well as its activation level or exhaustion status. Previously, a gp41 B cell tetramer was successfully used to study the development of the Ab responses in the context of HIV infection (25). In the current study, we used the ectodomain of HCV glycoprotein E2 from a genotype 2a strain (J6) to develop a biotinylated monomer that could be used to generate a tetramer specifically recognizing HCV E2-specific B cells (Fig. 1). Our reagent was first validated using E2-specific Abs and hybridoma cells producing E2-specific Abs (Fig. 2).

Next, using this novel E2 tetramer, we successfully identified Ag-specific B cells in HCV-infected subjects (Figs. 3–4). The most frequent genotype in the Montreal Acute Hep C Cohort Study is genotype 1. Despite high variability of sequences between genotypes (identity <70%), especially in the hypervariable regions of E2 (HVR1-3), exposed conserved regions are present and have been shown to be important for the binding of E2 protein with the coreceptor CD81 protein on the cell surface and subsequent viral entry (38, 39). Cross-reactive human Abs have been described that bind various E2 proteins from multiple HCV genotypes (40–42). We tested two different E2 tetramers that were derived from genotype 1 (H77 strain) and genotype 2 (J6 strain) with PBMC samples obtained from genotype 1, 2, and 3 infected participants. The staining of Ag-specific B cell with the J6-E2 tetramer was far more specific as compared with the H77-E2 tetramer (data not shown). The H77-E2 tetramer staining had increased background levels and there was less intensity in signal. As a result, all experiments were done using the J6-E2 tetramer. With this tetramer, E2-specific class-switched memory B cells were detected in the majority of chronically infected individuals (18 out of 21 for genotype 1 and 5 out of 5 for both genotypes 2 and genotype 3, Fig. 3C). Therefore, the usage of a different E2 genotype (J6, 2a) did not affect binding of Ag-specific B cells from patients with different infection genotypes. Furthermore, the sensitivity of the tetramer was comparable to the B cell ELISPOT assay as shown in Fig. 3D.

However, in a longitudinal analysis, tetramer positive populations could only be detected in approximately half of the tested individuals during the late acute HCV infection (Fig. 4A, 4B). This was in agreement with the anti-E2 titer, measured by ELISA where only the samples with a positive tetramer population exhibited a detectable anti-E2 titer (Fig. 4C). This is consistent with previous reports showing a delayed Ab response during acute infection. It suggests that at least in some patients, the development and/or expansion of HCV E2-specific B cells is hindered, which may be a factor that contributes to the establishment of a chronic infection. It is also possible that early HCV-specific B cells are of lower affinity and thus are unable to bind the E2 tetramer efficiently. To expand this analysis, in a preliminary experiment, we were able to detect HCV-specific B cells in three out of six subjects after spontaneous resolution of HCV infection with a mean frequency of 0.3% (range: 0.15–0.49%) of class-switched memory B cells (1 y post EDI, data not shown). This is also consistent with a previous report that showed Ab responses during HCV infection are usually of low titer, and decline rapidly after spontaneous resolution (7). Analysis of the E2 tetramer positive population frequency and function longitudinally during earlier time points in a larger cohort of participants with different infection outcomes may provide more detailed insight into the role of these Ag-specific B cells in determining the course and outcome of HCV infection. We focused our analysis on class-switched memory B cells for this pilot project. This new tool however, will also allow us to characterize the phenotype of HCV-specific B cells in greater detail. In the genotype 1 chronically infected subjects, we could also detect HCV-specific unswitched memory B cells (CD27⁺IgM⁺), and to a lower extent naive/atypical B cells (CD27⁻IgM⁺), but the frequency of HCV-specific class-switched memory B cells was certainly higher in the majority of samples analyzed (15 out of 21, Fig. 3E). Future studies will be needed to address this question in more detail, as well as to characterize the activation and/or exhaustion profiles of HCV-specific B cells.

HCV E2-specific class-switched memory B cells were sorted using the E2 tetramer for BCR deep sequencing. In two independent subjects, the HCV-specific BCR repertoire was focused, suggestive of Ag driven selection. Moreover, we have shown that the normal Gaussian-like distribution of CDR3 lengths was skewed in both participants analyzed, suggesting an amplification and/or selection of specific clonotypes (35, 43). It is also postulated that the affinity maturation leads to a shorter CDR3 length in Ag-specific B cells (44). However, we did not observe any differences between the average CDR3 length of naive B cells and HCV E2-specific cells in the two subjects analyzed thus far (data not shown). Also, the accumulation of mutations in the CDR3 sequence is indicative of affinity maturation process (36). Our analysis showed an increased mutation frequency in HCV-specific BCRs compared with naive receptors. Together these results suggest that sorted E2-specific class-switched memory B cells were indeed Ag-specific, selected, expanded, and accumulated mutations during the affinity maturation process.

The BCR repertoire of bulk memory and naive B cells was previously investigated during HCV infection (45). However, in this study total memory B cells as opposed to Ag-specific cells were analyzed. It was shown that the gene usage was distinct between those who spontaneously resolved the infection versus those who were chronically infected. Also, the clonality of the repertoire was greater in resolving infection compared with chronically infected individuals. Phylogenetic analysis demonstrated tight clustering of a limited number of related B cell clonotypes in resolvers compared with a more dispersed pattern in chronically infected individuals, suggestive of an increased clonal selection in the resolvers. Finally, in the same report, the CDR3 length distribution was particularly skewed in BCRs of subjects who resolved the infection with a single CDR3 length being the dominant clone compared with BCRs obtained from persistent infection where the deviation from the Gaussian-like distribution was less apparent. Utilizing HCV E2-specific tetramers will now enable us to investigate the evolution of the repertoire in HCV-specific B cells, in particular the association of distinct BCR gene family usage among individuals who spontaneously clear versus those that progress to chronicity. One limitation of this approach is the low frequency of HCV-specific B cells. Nevertheless, given the advent of sensitive technologies, we are hopeful that the longitudinal comparisons of the BCR repertoire at the single cell level from subjects with different infection outcomes will be feasible in the near future, as this important tool will allow for a better characterization of HCV-specific B cells.

B cell disorders such as mixed cryoglobulinemia and non-Hodgkin lymphomas are complications associated with chronic HCV infection (46). It has been demonstrated that clonal B cell expansion in the liver is associated with these extrahepatic manifestations (47). Furthermore, E2-specific B cells isolated from an asymptomatic HCV patient used the VH01-69 gene (48) that is associated with B cell lymphomas in chronic HCV (49). B cells were also implicated in liver fibrosis, where they have been shown to be activated, produce inflammatory cytokines, and constitutively secrete IgG (50). Isolation and characterization of HCV-specific B cells in individuals with such phenotypes will provide a better insight into the role of B cells in these aberrant manifestations and may provide better B cell–based immunotherapies. Little is known about the interaction between B cells and CD4 helper T cells, specifically the follicular helper T cells (Tfh) during an acute and chronic HCV infection. A recent report demonstrated that HCV-specific Tfh cells had an activated phenotype during the acute phase of HCV infection, with an increased expression of ICOS, which correlated with Ab production (51). Future studies combining HCV-specific B cell visualization (together with subset identification, activation, and exhaustion markers) with detailed analyses of the neutralizing Ab repertoire and Tfh development and function should elucidate the importance of this interaction and how it influences the generation of virus-specific neutralizing Abs and infection outcome prognoses.

We thank all of the donors who participated in this study. We thank Dominique Gauchat of the flow cytometry core of the CRCHUM for technical help with cell sorting experiments.

The authors have no financial conflicts of interest.