Abstract
We report the development of a novel flow cytometry–based Ig capture assay (ICA) for the identification and sorting of individual Ab-secreting cells based on their Ag reactivity. The ICA represents a fast and versatile tool for single-cell sorting of peripheral plasmablasts, streamlining subsequent Ab analysis, and cloning. We demonstrate the utility of the assay by isolating Ag-reactive plasmablasts from cryopreserved PBMC obtained from volunteers vaccinated with a recombinant HIV envelope protein. To show the specificity of the ICA, we produced Ag-specific Abs from these cells and subsequently verified their Ag reactivity via ELISA. Furthermore, we used the ICA to track Ag-specific plasmablast responses in HIV-vaccine recipients over a period of 42 d and performed a head-to-head comparison with a conventional B cell ELISpot. Results were highly comparable, highlighting that this assay is a viable alternative for monitoring Ag-specific plasmablast responses at early time points after infection or vaccination. The ICA provides important added benefits in that phenotypic information can be obtained from the identified Ag-specific cells that can then be captured for downstream applications such as B cell sequencing and/or Ab cloning. We envisage the ICA as being a useful tool in Ab repertoire analysis for future clinical trials.
This article is featured in In This Issue, p.3927
Introduction
The development of a specific and sustained Ab response to a pathogen is a key feature for the induction of protective immunity. As such, vaccine candidates are often designed to induce highly specific Igs that can provide protection against future infections. Both the specificity and magnitude of the resulting Ab response are conventionally assessed via quantitative immunoassays such as serum ELISAs and B cell ELISpots (1). However, these assays do not allow for the phenotypic analysis of the individual B cells that produced the Abs nor the characterization of individual Abs themselves.
With advances in flow cytometry, FACS, and Ab cloning techniques, a number of methods have been established to produce Ag-specific recombinant Abs from individual memory B cells (MBC), plasmablasts, and plasma cells (2). Of these subsets, MBCs, comprising 40–50% of total B cells (3), have been most often used for Ab cloning due to their availability in peripheral blood even after infection has subsided, and their cell surface expression of Ig, facilitating their detection using fluorescent Ag probes (4). The most common type of reagent used as a probe is recombinant fluorescently tagged whole Ag, although groups have also used whole virions, peptide tetramers, Ag-coated beads, and even modified infected cells (5–12). However, these approaches are not directly applicable to plasmablasts, due to their low or complete lack of surface Ig expression. This makes the production of Ag-specific Abs from plasmablasts a difficult task, often requiring sorting of the entire plasmablast population, expression cloning of their Ig mRNA, followed by the postproduction screening of the recombinant Abs to confirm Ag reactivity (13).
Most plasmablasts are generated in germinal centers along with MBCs, but can also originate from the differentiation of reactivated MBCs or from early T-B cell interactions outside the germinal center (14). As such, peripheral blood plasmablasts are likely to be a mix of these, and may produce Abs containing variable degrees of somatic hypermutation and of different Ig classes. As the germinal center reaction and immune response progress, some B cells will differentiate into long-lived plasma cells and migrate to the bone marrow to provide sustained serum Ab titers (15). Therefore, plasmablasts represent an accessible source of highly matured Abs, despite their low numbers in the periphery after vaccination in comparison with other B cell subsets.
Although most healthy adult subjects exhibit low levels of circulating Ig-secreting plasmablasts [or Ab-secreting cells (ASC)] in the range of 1–4% at steady state, Ag-specific plasmablasts are significantly enriched in peripheral blood 5–10 d following infection or vaccination (16). The extent of this enrichment can be highly variable, ranging from a very small percentage to >95% of total plasmablasts being reactive to the immunogen, dependent on the nature of the infection or vaccine and individual donor (17). For highly immunogenic infections or vaccines, the common approach for production of Ag-specific Abs by the sorting and cloning of the entire population of isolated plasmablasts can be an efficient approach, with the large majority of Abs produced being specific to the Ag or pathogen (16, 18). However, experimental vaccines can often give rise to only a modest Ab response, which, in the absence of a method for screening Ab specificity prior to cell sorting, can result in a great deal of time and resources being spent producing nonspecific Abs to identify and isolate only a handful of Ag-specific clones (19, 20).
As with MBCs, fluorescent Ag probes can be used to identify specific plasmablasts by binding to unsecreted Ab in the cytoplasm of the cell; however, this requires fixation and permeabilization of the cell, which precludes further culture and seriously affects mRNA integrity, making cloning extremely difficult on a single-cell level (4, 21). Alternatively, the secreted Ab can be captured on or around the cell, where it can be probed with a fluorescent Ag (22). Secretion assays are based upon forming an affinity matrix around the secreting cells, either by binding a capture reagent to the cell surface or by encapsulating single cells in a medium that prevents secreted product from leaving the vicinity of the cell. Early encapsulation methods used biotinylated agarose in combination with avidin and a biotinylated capture reagent, whereas more modern methods use water-in-oil droplets containing capture beads and fluorescent detection Abs (23, 24). However, these methods require specialist equipment for encapsulation, analysis, and isolation, and the process of microencapsulation in agarose in particular can be damaging to the cells.
Using a capture reagent is often more feasible. Initially, cell surface biotinylation was used to conjugate the capture reagent to the cell, whereas newer methods use reagents such as bispecific Abs, magnetic nanoparticles, or lipidated capture Abs (25–27). Although some bispecific capture Abs are available commercially as part of cytokine secretion assay kits, most other reagents must be produced by the experimenter, which takes both time and expertise and thus limits their widespread use.
In this paper, we describe a novel secretion assay for the detection and isolation of Ag-specific plasmablasts from both freshly isolated and frozen PBMC samples via FACS. We demonstrate the utility of the Ig capture assay (ICA) in identifying Ag-specific plasmablasts for three model vaccine Ags [HIV gp140, tetanus toxin C-fragment, and hepatitis B surface Ag (HBsAg)]. We also use the ICA to chart the kinetics of the plasmablast response to a model vaccine Ag (gp140) over time, with results comparable to B cell ELISpots. We demonstrate the additional benefit of ICA where the identified Ag-specific cells can be isolated and used in downstream assays such as Ab gene cloning to facilitate the generation of patient-derived monoclonal Abs. We believe the ICA provides a versatile new tool for isolation, characterization, and single-cell cloning of Ag-specific plasmablasts.
Materials and Methods
HIV clinical vaccine trial
To optimize and validate the ICA, we mainly used PBMC samples from a previous phase I HIV vaccine trial (X001), the results of which have been published (28). Briefly, the X001 study used a good manufacturing practice–grade recombinant uncleaved clade C HIV-1 envelope gp140 protein (CN54gp140) (Polymun Scientific, Vienna, Austria), which has previously been reported to be immunogenic in a number of preclinical and clinical trials. The vaccine Ag CN54gp140 was administered i.m. into the deltoid muscle of the upper arm at a dosage of 100 μg of CN54gp140 formulated with 5 μg GLA-AF (IDRI, Seattle, WA) in a total volume of 0.4 ml at weeks 0, 4, and 8 with a boost inoculation with the same material at either month 6 or 12 (28).
Hepatitis B and tetanus vaccination
For the validation of the HBsAg-GFP and tetanus toxoid (TTX)–GFP protein probes, healthy controls were vaccinated with the Engerix B hepatitis B vaccine (GlaxoSmithKline, U.K.) or the Revaxis combination diphtheria, polio, and tetanus vaccine (Sanofi Pasteur, U.K.), respectively. Revaxis, containing diphtheria, TTX, and inactivated poliomyelitis virus types 1–3 adsorbed to aluminum hydroxide, was administered i.m. into the deltoid muscle of the upper arm in a total volume of 0.5 ml. Engerix B, containing recombinant purified HBsAg adsorbed to aluminum hydroxide, was administered i.m. into the deltoid muscle in the upper arm in a total volume of 1 ml.
Protein production and purification
Protein probes were designed as fusion constructs of CN54gp140 linked by a short linker sequence (SSGR) with a superfolder GFP (sfGFP) (29Materials and Methods
ICA for the targeted isolation of Ag-specific plasmablasts by FACS
Cryopreserved PBMC samples were thawed at 37°C and washed twice in warm RPMI 1640 medium (Life Technologies) supplemented with 100 U/ml penicillin (Life Technologies), 100 μg/ml streptomycin (Life Technologies), and 2 mM l 2 Fragment Goat Anti-Human IgG-Fcγ (Jackson ImmunoResearch) at a 1:200 dilution for 20 min at 4°C (wet ice). Cells were washed and resuspended in 1 ml RPMI 1640 supplemented with 2 mM ll g for 2 min and then flash-frozen in dry ice, before storage at −80°C until RNA extraction.
B cell ELISpot
Plasmablasts were tested for specificity and quantity using a standard ELISpot protocol. Sterile 96-well ELISpot plates (Millipore) were activated with 15 μl per well of 70% ethanol for 1 min. Following five washes with 200 μl per well sterile water, individual wells were coated with 100 μl per well sterile PBS containing either an anti-IgG Ab (MT91/145; Mabtech) at a concentration of 15 μg/ml to detect total IgG responses, or CN54gp140 (produced in house), HBsAg (Abcam), or tetanus toxin C-fragment (produced in house) at a concentration of 5 μg/ml to detect specific responses. Coated plates were incubated at 4°C overnight and then washed six times with 200 μl per well sterile PBS. Wells were blocked with 200 μl per well R-10 at room temperature for 1 h prior to the addition of cells, which were diluted in R-10. Once cells were plated, they were incubated at 37°C for 6 h (for PBMCs) or overnight (for sorted plasmablasts) before washing six times with 200 μl per well sterile PBS. Plates were then incubated with 100 μl per well sterile PBS containing 0.5% FBS (0.5% PBS/FBS) and 1 μg/ml biotin anti-IgG (MT78/145; Mabtech) and incubated overnight at 4°C (for PBMCs) or for 3 h at room temperature (sorted plasmablasts). Plates were washed as before and incubated for 1 h at room temperature in 100 μl per well 0.5% PBS/FBS containing streptavidin HRP (Mabtech) diluted 1:1000. Following a final wash as before, 100 μl per well prepared 3-amino-9-ethylcarbazole substrate (BD Biosciences) was added and incubated at room temperature for 5–10 min until spots developed. The reaction was stopped by adding tap water to the plates, which were then left to dry before imaging. Spots were imaged and counted using an automated AID ELISpot reader (Autoimmun Diagnostika).
Ab gene molecular cloning
The cloning method was adapted from a protocol published by Smith et al. (13). Briefly, 96-well PCR plates containing sorted plasmablasts were thawed and used directly for reverse transcription PCR by the addition of 2'-deoxynucleoside 5'-triphosphate, RT-PCR buffer, RNAase-free H2O, reverse transcriptase, and two primer pools containing forward and reverse primers for the H and L chains (RT-PCR reagents purchased from Qiagen, primers purchased from GeneArt, Invitrogen). These PCR products were used in three separate nested PCR reactions to amplify the heavy and light chains separately, and to introduce restriction sites to the ends of the PCR products. Next, the PCR products were digested using their respective restriction enzymes (AgeI-HF, SalI, XhoI, and BsiWI, all purchased from New England Biolabs) and ligated into human IgH, IgK, and IgL vectors containing a CMV promotor and ampicillin resistance (all expression vectors were kindly provided by Dr. J. Mongkolsapaya) using T4 DNA ligase (New England Biolabs). Vectors were expanded using TOP10 chemically competent Escherichia coli
Ab screening ELISA
Supernatants containing Ab secreted from cells transfected with H and L chain Ig plasmid DNA were assessed using a semiquantitative ELISA. Briefly, 96-well high binding MaxiSorp plates (Nunc) were coated with 50 μl per well recombinant CN54gp140 (Polymun Scientific) at 2.5 μg/ml in PBS overnight at 4°C. Purified Igs, which were captured with a combination of anti-human κ and λ L chain–specific mouse Abs, were used as standards. Capture Abs were coated onto the plates overnight at 4°C and coated plates were washed four times with PBS-T before blocking with the appropriate assay buffer. Following further washing, diluted samples were added to the Ag-coated wells and titrations of Ig standards were added to the κ/λ capture Ab-coated wells at 50 μl per well and incubated for 1 h at 37°C. Plates were washed four times prior to the addition of secondary Ab and incubated for 1 h at 37°C. Plates were washed four times and developed with 50 μl per well of KPL SureBlue TMB substrate (Insight Biotechnology, U.K.) (28).
Native PAGE and Western blotting
To visualize all proteins in the samples, gels were first fixed for 1 h in fixation buffer containing 40% methanol, 10% acetic acid, and 50% ultrapure water. Gels were then washed in ultrapure water overnight, with multiple changes of water. Following this, the gels were sensitized in a 0.02% solution of sodium thiosulfate for 1 min, before three 20 s washes of ultrapure water. Gels were incubated for 20 min in a chilled (4°C) solution of 0.1% silver nitrate and 0.02% formaldehyde. Gels were washed three times in ultrapure water for 1 min each, and then developed in a 0.05% solution of formaldehyde containing 3% sodium carbonate. Once protein bands had developed to a sufficient degree, the reaction was stopped by the addition of 5% acetic acid for 5 min, before washing in ultrapure water and dehydration for storage.
Statistical methods
All statistical analysis was carried out using Prism 7.0a (GraphPad, CA).
Ethics statement
The trials generating PBMC samples used in this study were conducted according to the U.K. Clinical Trials Regulations and Good Clinical Practice guidelines. The trial protocols, trial-specific information provided to volunteers, the consent forms, and substantial protocol amendments (if applicable) were reviewed by a recognized Research Ethics Committee and by the Medicines and Healthcare Products Regulatory Authority. All volunteers gave fully informed written consent.
Results
Design and validation of a flow cytometry–based assay for detection of Ag-specific plasmablasts
To directly detect Ag-specific plasmablasts, we designed a secretion assay based on the formation of an Ab capture matrix on the surface of plasmablasts. The capture matrix comprises a streptavidin-conjugated anti-CD45 Ab, followed by a biotinylated anti-human IgG F(ab′)2 fragment, specific for the Fc portion of the secreted Ab. The formation of this capture matrix on the surface of plasmablasts enables the localized capture and concentration of secreted IgG on the surface of the producing cell. The specificity of the captured Ab is then determined using an sfGFP-labeled Ag probe, which is further amplified using an anti-GFP Ab conjugated to Alexa Fluor 488 for enhanced signal detection and assay sensitivity (Fig. 1).
Diagram demonstrating the structure of the affinity matrix and the mechanism of the assay. Streptavidin-conjugated anti-CD45 is used as the base for the scaffold complex. Next, biotinylated F(ab′)2 anti-human IgG is added to capture IgG Abs secreted by the plasmablasts. Ag-specific human IgG is subsequently detected with sfGFP-conjugated Ag and the fluorescent signal is amplified by using fluorescently labeled anti-GFP.
We first tested this assay using a candidate antigenic probe: HIV-1 CN54gp140 conjugated to sfGFP by a flexible serine-glycine linker. This was selected based on the availability of clinical samples for downstream analysis. sfGFP has been shown to maintain its intrinsic fluorescence when expressed as a fusion protein, as well as allowing for efficient folding and trimerization of the recombinant gp140 envelope (Env) protein (29). Incorporation of this probe, which was validated by native PAGE analysis, into our secretion assay enabled us to identify CN54gp140-specific plasmablasts.
During sample acquisition and sorting, we used a gating strategy (Fig. 2A) to select CD3−CD14−CD19+CD20−CD27hiCD38hi plasmablasts, allowing us to exclude nonplasmablast cell types, such as CD4+ T cells or monocytes, or B cell subsets expressing high levels of surface IgG, which if Ag-specific would bind the gp140 probe. We also excluded any plasmablasts that were positive for IgA, IgD, or IgM to ensure a pure IgG+ plasmablast population. When this gating strategy was applied to samples of PBMCs from individuals vaccinated with recombinant CN54gp140, we were able to identify a range of responses (Fig. 2B). All control donor cells were acquired at baseline, whereas Ag-reactive cells were acquired 5–7 d after a booster vaccination.
Gating strategy for sorting of Ag-specific IgG+ plasmablasts. (A) PBMCs were first gated by forward (FSC) and side scatter (SSC), extending the normal lymphocyte gate beyond the standard limits into the monocytes, to account for the increased size and granularity of plasmablasts. These cells were then gated to exclude doublets by comparing the FSC area and height measurements. Single cells were gated on live cells shown by only level staining by Zombie NIR Fixable Viability Dye, followed by gating on CD3−CD14−CD19+ cells to exclude T cells and monocytes and to gate on CD19+ B cells. Within the B cell population, plasmablasts were identified as CD27hiCD38hi, followed by CD20−. Plasmablasts expressing IgM, IgD, or IgA were gated out because our cloning strategy only incorporated IgG primers. (B) The final IgA− population as shown in (A) was then interrogated for the level of anti-GFP binding, representing the Ag reactivity. Gates were set using either an unvaccinated sample or a fluorescence minus one (FMO) control. The donors shown in this study were gated using an unvaccinated control donor (left), and showed a range of CN54gp140-specific responses reflecting the time after vaccination after which these samples were taken. (C and D) This approach was also tested using two other recombinant GFP-tagged Ags; TTX C-fragment and HBsAg. All control donor cells were acquired at baseline, whereas Ag-reactive cells were acquired 5–7 d after booster vaccination.
In addition to detecting CN54gp140-specific plasmablasts, we also intended for this assay to be easily adaptable to other studies involving the analysis of Ab responses to different Ags. Therefore, we tested whether other Ag probes could be easily substituted in the assay with minimal reoptimization. We produced two further recombinant Ags, tetanus toxin C-fragment and HBsAg, both also fused with sfGFP, and used them to identify specific plasmablasts from cryopreserved PBMC samples obtained from healthy volunteers 7 d postvaccination with either the Revaxis combination tetanus vaccine or Engerix B hepatitis B vaccine, respectively. The plasmablasts were gated using our standard strategy (Fig. 2A), and the specific plasmablasts were identified as GFP+ (Fig. 2C). Both tetanus and hepatitis B vaccination show a clear population of Ag-reactive plasmablasts (2.53 and 8.07% of total plasmablasts, respectively). Whereas vaccination with TTX often gives up to 95% enrichment for specific ASCs (17), vaccination with the combination vaccine as used in this study gives a reduced response. Vaccination with the hepatitis B vaccine, however, is known to produce only a small enrichment, with roughly 10% of total plasmablasts specific for HBsAg (17). This use of the assay required no adjustment of the concentration of reagents or incubation times, and titration of the two new Ags yielded the same values as for the CN54gp140-GFP probe, with 5 μg per assay being optimal for detection of specific plasmablasts.
The quantification of plasmablast response kinetics by ICA significantly correlates with measurements by traditional ELISpot methodology
The current gold standard for tracking humoral responses during vaccine trials are Ag-specific B cell ELISpots performed on PBMC samples and measurement of serum Ab titers by ELISA (16). Having established the ICA for identification of Ag-specific IgG plasmablasts, we wanted to investigate whether the assay could be adopted and used as an alternative to the traditional ELISpot format to accurately chart the kinetics of plasmablast generation following vaccination. Using frozen PBMCs collected from donors vaccinated with HIV CN54gp140 as part of the X001 clinical trial (28), we analyzed the Ag-specific plasmablast response for 11 donors over six to eight study visits, for which B cell ELISpots had been previously performed on isolated PBMCs. The ICA can be used to generate the equivalent data to the ELISpot spot-forming units per million PBMCs by simply recording the absolute number of Ag-reactive IgG plasmablasts and adjusting for the absolute number of live PBMCs acquired.
When the spot-forming unit (SFU) values from both the ICA and the B cell ELISpots are compared (Fig. 3, Supplemental Fig. 1), a strikingly similar pattern can be seen in the plasmablast kinetics, with a dramatic increase in the plasmablast response between 5 and 9 d after vaccination, peak levels occurring at 6 d postvaccination (ELISpot: 136 ± 31.6 SFU per million PBMC; ICA: 158 ± 56.6 SFU per million PBMC), then slowly reducing until returning to baseline.
Comparison of ICA and ELISpot data. The number of Ag-specific plasmablasts was measured using both the traditional ELISpot assay and the ICA methodology (n = 11). (A) The absolute numbers of plasmablasts (mean±SEM) following serial immunizations with CN54gp140 (indicated by numbers 1° and 2°) are compared, whereas (B) shows the Spearman correlation of the data from both assays, comparing all data points, demonstrating a strong correlation (r = 0.9272) between the results with a significance of p < 0.0001.
Comparison of the values for all donors and time points between the assays provided a highly significant correlation (p < 0.0001, r = 0.9272, Spearman). These data demonstrate that the ICA can be reliably used to monitor plasmablast responses to vaccination or infection, even when responses are low, as observed during the X001 study.
In addition, we took advantage of all the additional data acquired by flow cytometry during the analysis of the PBMC samples and were able to analyze phenotypic data for both plasmablast and MBC populations (Fig. 4). Using the Abs targeted against IgA, IgD, and IgM, we tracked the relative percentages of plasmablasts expressing these isotypes, and gated on the IgA/D/M negative population as the IgG+ plasmablast population. We also compared these variables against the percentages of plasmablasts within the B cell pool, B cells within the PBMC pool, CN54gp140-reactive plasmablasts within the total and IgG+ plasmablast pools, and CN54gp140-reactive MBCs.
Correlation matrix of recorded parameters by ICA. Comparison between the SFU values obtained by B cell ELISpot and by ICA representing the number of Ag-reactive plasmablasts per 106 PBMCs, the percentages of Env-reactive total plasmablasts, Env-reactive IgG+ plasmablasts, Env-reactive MBCs, plasmablasts within the B cell pool, B cells within the PBMC pool, and IgM+, IgD+, IgA+, and IgG+ (IgM/D/A−) plasmablasts within the total plasmablast pool. Values were plotted against one another as shown in the scatter plots in the bottom left segment of the matrix, and the Spearman coefficient (rs) was calculated by Spearman correlation and shown in the top right segment of the matrix. The box colors reflect degree of correlation. The significance of the correlations is represented by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). The density of points on the scatter plots is shown by the density plots in the center of the matrix.
By comparing all measured parameters obtained by flow cytometry (Fig. 4), we were able to note several significant correlations between plasmablast subsets. Further validating the previous data comparing B cell ELISpot and ICA absolute cell numbers, the percentages of both Ag-specific IgG+ and total plasmablasts correlated significantly (***p < 0.001) with both the ELISpot and the calculated ICA SFU values. The percentage of total IgG+ plasmablasts also correlated with both the size of the plasmablast population relative to the total B cell pool (**p < 0.01) and the ELISpot and ICA SFU values (***p < 0.001), suggesting that the expansion of the plasmablast pool following vaccination is mainly due to an expansion of Ag-specific IgG+ plasmablasts. The percentage of total IgA+ plasmablasts also decreased significantly as the IgG+ plasmablast pool expanded (***p < 0.001), whereas the IgM+ plasmablast pool was not significantly different.
This gave us a more complete picture of the B cell response to the vaccine, as well as demonstrating a major advantage of the ICA over the B cell ELISpot in that multiple parameters can be analyzed simultaneously, saving both time and samples.
Ab cloning, production, and screening validates the specificity of the ICA
To both further validate the Ag specificity of identified plasmablasts and to investigate the Ab repertoire of CN54gp140-vaccinated volunteers, we then produced a library of individual Abs against the vaccine Ag. Using frozen PBMC samples collected from donors vaccinated with CN54gp140 as part of the X001 trial, we single-cell sorted Ag-reactive plasmablasts into 96-well PCR plates containing a lysis and RNA-preservation buffer. The timepoints selected for sorting were 6 d after a third and fourth vaccination.
Following sorting, the IgG mRNA from the individual cells was amplified and extracted using a modified version of the protocol published by Smith et al. (13). The efficiency of this step remained at 48.3% throughout the X001 trial, which is in agreement with similar studies that report a range of 30–60% PCR efficiency (30). Where a paired H and L chain was obtained, both chains were cloned into suitable vectors, and successful plasmid products were then transfected as a paired Ab (both an H chain plasmid and a κ or λ L chain plasmid) into HEK293T/17 cells for expression.
To demonstrate their specificity, Abs were collected from the supernatant of cultured cells and screened for binding to a recombinant clade C HIV CN54gp140 protein (the vaccinating Ag) by ELISA. We also investigated the Abs for cross-clade reactivity by screening for binding to clade A and D Env proteins (Fig. 5A, 5E). The Ab genes were also sequenced and analyzed for Ig gene family usage and mutation frequency and type (Fig. 5B–D, Supplemental Fig. 2).
Ag-specific Abs originating from plasmablasts that were single-cell sorted using the ICA. (A) The specificity of the recombinant Abs generated from 34 single-sorted plasmablasts was tested by semiquantitative ELISA. Cognate IgH and IgL gene pairs were transiently transfected into HEK293T cells, and Abs specific for CN54gp140 were detected in 32 of 34 (94%) supernatants. Shown are mean values from technical duplicates. The HIV Ab 5F3 was used as a positive control (green line) and a nonspecific Ab from clinical trial participants was used as a negative control (red line). In total, 27 of 32 CN54gp140-specific Abs were successfully upscaled for further functional assays. (B and C) Histogram representation of mutations in VH genes and plot showing CDRH3 length distribution (amino acids) as a composite of 27 recombinant vaccine-induced Abs that were tested for cross-reactivity against HIV clades C, A, and D. (D) Pie charts showing clonal IGHV families from 27 recombinant vaccine-induced Abs. (E) ELISA test to detect cross-reactivity of 27 Abs derived after sequential CN54gp140 immunizations (C, C.CN54gp140; A, A.UG37gp140; and D, D.UG21gp140 HIV protein). Strong binding is coded in red (>1.8 OD450), intermediate in orange (1.0–1.8 OD450), weak in yellow (0.39–1.0 OD450), and negative in light gray (<0.39 OD450). The EC50 values of 27 CN54gp140-specific Abs were extracted from the fitting of the raw data to a sigmoidal five-parameter logistic curve.
This approach, using the ICA to pre-enrich for Ag-reactive IgG+ plasmablasts, led us to produce a total of 34 functional Abs, of which 32, or 94%, were shown to be specific to CN54gp140 by ELISA (Fig. 6, Supplemental Table I). To demonstrate that the ICA was responsible for this enrichment of Ag-specific cells, we single-cell sorted the entire plasmablast population of several participants without the use of the ICA and proceeded to produce recombinant Abs from the cloned Ig mRNA. Using this approach, we needed to produce a total of 73 functional Abs to isolate a single CN54gp140-specific Ab. This represents just 1.37% of the total, whereas the application of ICA resulted in 94% enrichment of Ag-specific plasmablasts, and clearly shows the benefits of the assay over conventional approaches for plasmablast-derived Ab isolation of low frequency responses.
ICA significantly increases yield of Ag-specific Abs. Shown are the yields of Ag-specific Abs using ICA-sorted plasmablasts (left side) and total plasmablast sorts (right side). PBMC samples from the X001 study (28) were used to isolate individual plasmablasts and re-engineer Abs. Env-specific Abs recognize the HIV-1 CN54gp140 used for vaccinations, whereas nonspecific Abs recognize a different and unknown target. Shown are percentages of Env-specific and nonspecific Abs for the respective Ab platforms. The outermost rings are colored based on the participant. The gray and green inner rings represent the OD450 values of total IgG and Env-specific ELISAs. The pie chart in the center shows the percentages of produced Abs specific for Env.
Discussion
In this paper, we demonstrate the potential utility of a novel flow cytometry–based ICA for the detection of Ag-specific ASCs. Using this assay, we were able to enrich the low frequency of vaccine-induced gp140 Ag-specific plasmablasts from 1.37 to 94%. Furthermore, our assay was sensitive enough to detect Ag-reactive plasmablasts from cryopreserved PBMC samples. Having tested this assay on multiple unvaccinated controls during development, we established early on that our combination of a carefully selected flow cytometry panel and precise titration of the individual components of the affinity matrix gave very little background once nonplasmablasts had been gated out. Any residual background was eliminated by gating out plasmablasts expressing other Ig isotypes, predominantly IgM plasmablasts, which appear to be more promiscuous in their binding. With these cells removed from the analysis, a gating strategy such as that shown in Fig. 2 allows for even low-affinity Ag binding plasmablasts to be identified and sorted. Ensuring that the identified cells are Ag specific enables a much higher throughput of Ab repertoire analysis, facilitating either individual single-cell or total Ag-specific population analysis of produced Abs by conventional ELISA or gene somatic mutation and family usage by next-generation sequencing.
This high level of precision also facilitates the quantitative and phenotypic analysis of Ag-reactive cell numbers in comparison with ELISpot data. The ICA provides several advantages over the current gold standard of the B cell ELISpot when measuring the B cell response to an Ag. For example, cells used for an ELISpot cannot be recovered for downstream analysis, whereas flow cytometric identification of Ag-reactive cells means that they can be sorted and further analyzed using downstream methods such as RNA-Seq or in vitro culture experiments. Index sorting, a feature of many modern sorters, enables sorted single cells to be identified individually in the recorded data. This allows for the exclusion of potentially nonspecific cells that may have been sorted accidentally and can be identified in the postsort data with the benefit of comparison with the entire sample population. Additional markers can easily be incorporated into the Ab flow panel, so that Ag-reactive cells can be interrogated for their expression of activation or homing markers for example. Ag-reactive cells with a specific phenotype can then be identified in postsort analysis, and either prioritized or excluded for sequencing or Ab production, or even used for analysis alongside sequencing or Ab affinity data. An immune response can also be profiled in more detail by identifying and tracking changes in other immune subsets as we demonstrate with IgA/D/M plasmablast populations. The high throughput nature of flow cytometry also enables far more samples to be processed at once, which for large clinical trials reduces both the time spent processing samples and the variability between samples, because all samples can be prepared and analyzed in a single run. Our testing of the ICA on cryopreserved PBMC samples from the X001 clinical trial in comparison with B cell ELISpots performed on freshly isolated PBMCs (Fig. 3) demonstrates that highly comparable data can be obtained using the ICA. Therefore, the use of the ICA can eliminate the need for B cell ELISpots, which for a large trial can be both time consuming and prone to interassay variability (31), due to the number of samples that can feasibly be run concurrently by a single operator. With the ICA, samples can be cryopreserved and the plasmablast responses analyzed in bulk, allowing multiple samples from the same participant, time point, or group to be analyzed in a single experiment.
Due to its modular design, the assay can be easily and rapidly adapted to study different infectious diseases and/or vaccine candidates. By substituting the capture reagents, the assay can be used to probe different isotypes of secreted Ab or plasmablasts from other species. The Ag probe can also be replaced, with minimal reoptimization, to detect plasmablasts specific to other Ags, as we demonstrate by detecting plasmablasts specific against HIV-1 CN54gp140, TTX, and HBsAg. Furthermore, use of different fluorochrome or tags would allow different Ags to be multiplexed within the same assay, facilitating measurement of Ab breadth and/or cross-reactivity.
In conclusion, we have demonstrated the utility of a novel and highly customizable flow cytometry–based ICA that is able to probe the specificity of secreted Ab from plasmablasts following vaccination. Using the ICA, we were able to isolate Ag-reactive plasmablasts from weakly responding vaccine clinical trial participants and express 34 recombinant Abs of which 94% displayed specificity to the vaccinating Ag. We also tracked the course of the Ag-specific plasmablast response in a separate vaccine clinical trial with accuracy comparable to B cell ELISpots that had been previously used to monitor this response. We believe the ICA will allow for much greater and focused analysis of the plasmablast Ab repertoire than has been previously possible, facilitating the identification and characterization of Ag-specific populations beyond the MBC pool.
Disclosures
The authors have no financial conflicts of interest.
Acknowledgments
We thank the St. Mary’s National Heart and Lung Institute FACS core facility and staff for support and instrumentation. We thank volunteers and clinical teams involved in the trials that generated samples for analysis. We thank Dormeur Investment Service for providing funds to purchase equipment used in these studies.
Footnotes
The X001 study was funded by the International AIDS Vaccine Initiative. Provision of samples from individuals vaccinated with Engerix B hepatitis B vaccine (GlaxoSmithKline, U.K.) or the Revaxis vaccine was supported by the European AIDS Vaccine Initiative 2020 funding through the European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement 681137. The conduct of both clinical studies was supported by the National Institute for Health Research at Imperial College Healthcare National Health Service Trust. Provision of CN54 gp140 and GLA-AF was supported through core funding from the Wellcome Trust via the UK HIV Vaccine Consortium (083844/Z/07/Z). Work by C.L.P. in developing the assay was supported by funding from the Bill and Melinda Gates Foundation (OPP1084580). The work of L.M. was supported by a grant from the Wellcome Trust (102413/Z/13/Z). The work of P.F.M. was supported by funding from the European Community’s European 7th Framework Program Advanced Immunization Technologies (HEALTH-F4-2011-18 280873).
The online version of this article contains supplemental material.
Abbreviations used in this article:
- ASC
- Ab-secreting cell
- CN54gp140
- clade C HIV-1 envelope gp140 protein
- Env
- envelope
- HBsAg
- hepatitis B surface Ag
- ICA
- Ig capture assay
- MBC
- memory B cell
- sfGFP
- superfolder GFP
- SFU
- spot-forming unit
- TTX
- tetanus toxoid.
- Received September 1, 2017.
- Accepted October 10, 2017.
- Copyright © 2017 by The American Association of Immunologists, Inc.