Determination of variable region sequences from hybridoma immunoglobulins that target Mycobacterium tuberculosis virulence factors.

Mycobacterium tuberculosis (Mtb) infects one-quarter of the world's population. Mtb and HIV coinfections enhance the comorbidity of tuberculosis (TB) and AIDS, accounting for one-third of all AIDS-associated mortalities. Humoral antibody to Mtb correlates with TB susceptibility, and engineering of Mtb antibodies may lead to new diagnostics and therapeutics. The characterization and validation of functional immunoglobulin (Ig) variable chain (IgV) sequences provide a necessary first step towards developing therapeutic antibodies against pathogens. The virulence-associated Mtb antigens SodA (Superoxide Dismutase), KatG (Catalase), PhoS1/PstS1 (regulatory factor), and GroES (heat shock protein) are potential therapeutic targets but lacked IgV sequence characterization. Putative IgV sequences were identified from the mRNA of hybridomas targeting these antigens and isotype-switched into a common immunoglobulin fragment crystallizable region (Fc region) backbone, subclass IgG2aκ. Antibodies were validated by demonstrating recombinant Ig assembly and secretion, followed by the determination of antigen-binding specificity using ELISA and immunoblot assay.

Introduction

Mycobacterium tuberculosis (Mtb) is the causative agent of the transmissible respiratory disease Tuberculosis (TB) and is considered one of the most insidious and intractable pathogens in history. Mtb can synergize destructively with other infective agents, including HIV [1, 2]. Mtb/HIV coinfections account for one-third of all AIDS-associated mortalities [3]. The World Health Organization and the National Institutes of Health consider TB a critical health priority [4, 5]. As with other bacterial pathogens under antibiotic pressure, the incidence of multi-drug and extensively drug-resistant Mtb is on the rise [6].

Diversified approaches are necessary for the detection and control of TB. Recent evidence points to humoral antibody (Ab) correlating well with TB susceptibility, suggesting recombinant Ab against Mtb are viable therapeutic strategies [7-14]. The complexity of Mtb infection and progression, however, hampers development of universal TB diagnostics or therapeutics. The pathogenesis of TB can vary from an active, open form of pulmonary TB to an asymptomatic, closed-form of latent TB [15, 16]. While host-pathogen interactions are diverse throughout infection and pathogenesis, Mtb remains replication-competent [17]. Therefore, a reasonable strategy to develop diagnostic and therapeutic antibodies would target the virulence-associated factors of Mtb.

Cellular components critical for development and pathogenesis help define virulence-associated factors. Mtb transmission occurs by aerosol and establishes infection in the host lung after macrophage infiltration [16, 18]. Mtb circumvents macrophage degradation from lysosomal hydrolases and reactive oxygen and nitrogen species by inhibiting phagosome-lysosome fusion or maturation [19-21] and escapes the acidic environment of the phagolysosome [19, 22]. Instead of clearing the mycobacterium bacilli, macrophages become the major Mtb reservoir [23, 24]. Thus, one of the hallmarks of infection with Mtb is its persistence within arrested, immature macrophage phagosomes [18, 25]. Virulence-associated factors are likely associated with Mtb infiltration or persistence.

The number of antibody sequences known for Mtb antigens, virulence-associated or not, is limited [26-28]. Two major groups of virulence-associated Mtb factors have been identified through gene inactivation in a validated TB model correlating to a measurable loss in Mtb macrophage residence or Mtb fitness [16, 18, 25, 29–33]. KatG, a catalase-peroxidase enzyme [16, 29], and SodA, a member of superoxide dismutase complex [16, 31], are key elements for bacterial redox homeostasis to counteract host macrophage reactive oxygen and nitrogen species [30]. PhoS1/PstS1 is a phosphate-binding factor within the ABC phosphate transporter system, important for nutrient import and drug export [32, 34]. PstS1 also acts as a mycobacterial cell adhesin, promoting macrophage phagocytosis [34, 35]. The heat shock proteins GroES and GroEL are conserved chaperone proteins [16, 36, 37], necessary for proper protein folding. Both PhoS1 and GroES stimulate host B cell and T cell immunity as immunodominant antigens and are considered TB vaccine candidates [38-41]. SodA and KatG antibodies could develop into diagnostic or therapeutic tools (e.g., inhibiting Mtb survival). All four targets offer some promise for further development.

The first step in such development is the identification of the sequence encoding the Immunoglobulin (Ig) variable region (IgV or Fab region). Structurally, the IgV region in antibodies is composed of portions of the heavy (IgVH) and light (IgVL) chains [42, 43]. The antigen-binding region within the IgV is constructed via three complementarity-determining regions (CDRs), flanked by four framework regions (FR 1–4) [42, 44]. CDRs are in the loop region of IgV, directly participating in antigen-recognition specificity and affinity, and are the key determinants of immune antigen-recognition diversity [45]. The remainder of the Ig, the constant or Fc region, determines the effector function within an Ab. While PCR can amplify Ig sequences for functional antibodies in hybridomas [46-49], a plethora of highly homologous but aberrant Ig transcripts confound interpretation [49-53].

In this study, we deciphered and validated sequences corresponding to the IgVH and IgVL chains of immunoglobulin expressed in hybridomas to Mtb-SodA, Mtb-KatG, Mtb-GroES, and Mtb-PstS1. Using 5’ Rapid Amplification of cDNA End (RACE) PCR, we amplified the CDR1-3 and FR 1–4 (IgV) regions. We sequenced the IgV amplicons through traditional cloning and Sanger sequencing and compared this approach with the deep sequencing ability of Next Generation Sequencing (NGS) to identify all potential IgV sequences. Aberrant IgV chains were systematically identified and eliminated using bioinformatics methods. Retention of paratope-determining sequences were confirmed by isotype-switching the putative IgV in an Fc-recombinant backbone. We validated each recombinant IgV for comparable antigen-binding activity to the target antigens as that from antibodies secreted by the parental hybridomas. We have deciphered sequences with this validation method corresponding to the IgVH and IgVL chains of immunoglobulin to Mtb-SodA, Mtb-KatG Mtb-GroES, and Mtb-PstS.

Results

Sequencing of the murine IgVH and IgVL regions of hybridoma transcripts

5’ RACE-PCR was employed to isolate IgV sequences expressed in hybridoma mRNAs. Since an isotype-specific antisense primer is required for the PCR assay, identification of hybridoma isotypes was integral prior to IgV sequencing. Isotypes of four hybridoma clones were determined by an anti-mouse, isotype-specific, lateral flow assay: IgG1κ for PhoS1/PstS1NRC-2410, IgG1κ for SodANRC-13810, IgMκ for KatGNRC-49680, and IgG2aκ for GroESNRC-2894 (). The isotype class enabled the design of the 3’ isotype-specific primer (3’ ISP) (Tables ).

High-quality RNA preparations were produced to ensure the isolation of contiguous IgV domains. Extracted RNA was evaluated for purity by absorbance at A260/A280 (>1.9), the integrity of 28S/18S rRNA (>2), and measurement of RNase contamination after 37°C treatment (retention of 28S band) (). RNAs satisfying all three criteria were used for 1st strand cDNA synthesis and subsequent IgV RACE-PCR amplification.

The diversity of antigen recognition encoded by IgV is one of the hallmarks of immunity and requires considerable sequence variability. IgV sequences were generated by 5’ RACE-PCR (Takara Inc., Mountain View, CA), using a 3’-antisense primer specific to conserved isotype-specific constant regions. The isotypes of the hybridoma clones included 4 different subclasses, IgG1, IgG2a, and IgM for Ig heavy chains and Igκ for Ig light chains. The respective 3’ isotype-specific primer sequences are highly conserved (>97% pairwise identity) (). Sequence alignment was performed for the corresponding CH1-constant region of heavy chains and the CH-constant region of light chains as downloaded from the IMGT/LIGM-DB database [54]. The IMGT/LIGM-DB database collectively contains immunoglobulin sequences deposited to INSDC by participating nucleotide databases [55]. The anticipated RACE-PCR-IgVH (~650 bp) and -IgVL (~550 bp) amplicons encompass an Ig leader sequence and full-length CDR1-3/FR1-4 (IgV) sequences (). The immunoglobulin heavy and light chain fragments of the 5’ RACE-PCR products were detected and isolated using agarose gel electrophoresis (. Though the amplicons of IgVH and IgVL appeared as a homogenous, single band on an agarose gel, direct Sanger sequencing of the eluted DNA fragments indicated a mix of Ig templates (), confirming the presence in the hybridomas of sequences encoding aberrant Ig chains.

5’ RACE-PCR immunoglobulin heavy (IgVH) and light (IgVL) chain products for each of the 4 hybridoma cell lines.

A. Illustration of the 5’ RACE-PCR amplicons of IgVH and IgVL used in this study. The 5’ RACE Universal Primer (5’ RACE UPM) and antisense, 3’ isotype-specific primer (3’ ISP) were marked. B. Agarose gel electrophoresis of the 5’ RACE-PCR amplicons of IgVH and IgVL for the 4 hybridoma cell lines. The amplicons were separated by 1.5% low melting agarose electrophoresis with ethidium bromide and were excised, extracted, and column purified. The corresponding amplicons of IgVH or IgVL were used either for generating an NGS library for MiSeq Illumina sequencing or for cloning into a TOPO-TA vector, followed by Sanger sequencing.

Direct TOPO cloning and Sanger sequencing of murine IgVH and IgVL regions

We examined the Ig-transcript profile of IgVs by cloning and sequence analysis of the RACE-PCR amplicons. The in-gel IgV DNA amplicons were extracted and purified for making an IgV library for Illumina next generation sequencing (NGS) or for cloning into a TOPO-TA vector (Invitrogen Inc.) for Sanger sequencing. Amplicons were cloned, and multiple independent clones (7 to 10) from each hybridoma Ig-amplicon were sequenced using the Sanger method. The resulting Ig sequences were analyzed through multiple sequence alignment to determine homology, translated to assess open reading frames, and aligned to M. musculus VDJ genes with MiGMAP [56]. In addition to “productive” Ig transcripts (i.e., containing open reading frames), multiple “unproductive” or “aberrant” transcripts (ORFs disrupted with stop codons) were also found ().

Next generation sequencing and de novo transcript assembly of hybridoma IgVH and IgVL sequences

To enable a robust IgV sequencing approach, we compared cloning and Sanger sequencing results with deep sequencing directly from amplified Ig transcripts. MiSeq Illumina NGS coverage and high-fidelity sequences are limited to a continuous read length less than 300 bp, shorter than the IgV PCR amplicons (~500–600 bp), and assembly of reads into the complete IgV amplicon sequence is required. Selecting NGS contig assembly parameters, thresholds, or filtering schemes was challenging due to the highly homologous nature of aberrant transcripts but benefitted from the contiguous amplicon sequence information generated from Sanger sequencing. Sanger reference sequences guided Ig-NGS sequencing methodology with proper filters, thresholds, and metrics and provided cross-validation of the assembled transcript candidates.

Eight libraries from 5’ RACE-PCR IgV amplicons of the hybridoma heavy and light chains were sequenced on the MiSeq Illumina NGS platform using a V2 2X150 Nano flow cell. A read depth predicted to suffice for identifying immunoglobulin genes from hybridomas [48] [between 10,000 and 200,000 reads ()] was obtained. Using Trinity [57], reads were de novo assembled into contigs representing putative transcripts. These transcripts were filtered to detect the presence of a 3’-ISP sequence, then aligned against the IMGT Mus musculus database of mouse V, D, and J genes using MiGMAP [56] to ascertain identity as an Ig transcript of interest. In , contigs from the 8 NGS-hybridoma libraries contained their respective 3’-ISP sequence, demonstrating method fidelity to identify gene-specific, isotype-specific Igs. The final contigs, shown in , contained the signature of productive transcripts in all hybridoma clones. present the productive transcripts for each of the antibodies characterized in this study.

Hybridoma Ig sequencing using MiSeq Illumina NGS and de novo transcript assembly.

Profiles of Contig types among the 4 hybridomas. A. Summary of hybridoma sequence contig assemblies identified using isotype-specific ISPs. B. The Trinity de novo assembled Ig contigs were grouped and quantitated based on counts (Left) or expression levels (Right) into four functional categories: 1) Unique Productive—contigs without STOP codons in the CDRs and found in only one of the tested hybridomas, 2) Unproductive—contigs with one or more STOP codons in the CDRs, 3) Incomplete—only 1 or 2 CDRs identified, and 4) Unidentified—no CDRs identified.

Multiple homologous transcripts, however, were identified in the contigs generated from de novo assembly. To evaluate the fidelity of our bioinformatics approach, we examined if the NGS dataset included all cloned transcript sequences identified using the TOPO cloning/Sanger sequencing method. Using NRC-13810 as proof-of-concept, our approach identified identical productive Ig transcripts as well as additional unique, productive Ig contigs (e.g., for NRC-13810). For example, though Igκ-1 of NRC-13810 was identified using both methods, Igκ-2 of NRC-13810_Igκ was only identified using NGS (). summarizes IgV reads using TOPO cloning/Sanger sequencing and NGS/bioinformatics methods. PhoS1/PstS1NRC-2410, SodANRC-13810, and KatGNRC-49680 contained multiple Ig heavy and light chains, whereas GroESNRC-2894 featured a single pair of Ig heavy and light chains. For the hybridomas with multiple IgVs, validation of the correct combination of heavy and light chains encoding a functional Ab was necessary. IgV results that were subsequently validated are provided in the rightmost panel of to facilitate comparison of the methods for accuracy and efficiency of IgV identification. Most of the IgV sequences were identified by both methodologies. However, deep sequencing with NGS, identified putative Ig transcripts with greater sensitivity of detection than TOPO cloning/Sanger sequencing.

Functional validation of hybridoma IgVH and IgVL sequences

Each putative IgV sequence was cloned and isotype-switched into a common IgG2aκ–Fc vector for use in sequence validation (). Thus, the antigen-binding property of the secreted recombinant Ab (rAb) lies in the in-grafted IgV sequences () derived from Mtb Ig transcripts. Since glycosylation alters Ig recognition and binding efficiency to native antigens [58, 59], the IgVH and IgVL constructs were co-expressed in 293F cells to mimic B cell glycosylation of immunoglobulins (). The IgV were validated by demonstrating (1) the assembly and secretion of the recombinant isotype-switched Ig and (2) the secreted Ig elicits antigen-binding of the target Mtb antigen similar to that of the antibody from the parental hybridoma. Antigen-binding specificity and potency were determined through immunoblot and ELISA assays of purified Mtb full-length antigens (Ag). The native Mtb antigens for GroES and PstS1 were obtained from BEI Resources (GroES, NR-14861; PstS1, NR-14859). The recombinant Mtb antigens, KatG and SodA, were expressed and purified by affinity chromatography (Figs ).

Confirmation of the fidelity of recombinant validation platform.

A. Diagram depicts the Mtb antibody sequencing and validation workflow. Sequences of the putative IgV (red) were validated by a recombinant Ig platform in which the hybridoma Fc was isotype-switched to a standard mouse IgG2aκ-Fc vector (blue). The recombinant antibody (rAb) was expressed in a mammalian 293F cell line. B. Mtb antigens used in the validation study. Coomassie staining of 6 μg of Mtb native antigens (GroES [left panel] or PstS1[right panel]) demonstrated high purity antigens. Recombinant antigens (His-SodA [left panel] and His-KatG [right panel, 2 μg each) were partially purified. C. IgV sequence of GroESNRC-2894 hybridoma was validated. rAbNRC-2894 elicits a similar GroES-binding profile as its hybridoma parent, hyAbNRC-2894. The antigen-binding specificity is assessed by immunoblot against the whole cell extract of Mtb over Y. pestis, another human bacterial pathogen. D. The antigen-binding property is assessed by ELISA using equal amounts of purified rAbNRC-2894 or purified hyAbNRC-2894 to react with the purified GroES antigen. Relative Ig affinity was determined by ELISA endpoint titers normalized against that of an ELISA buffer control (negative control, NC). Data shown here were from two independent experiments performed in triplicate.

We first explored the fidelity of the in-grafting process by examining the antigen-binding properties after switching into the same isotype. hyAbNRC-2894 shares the same isotype as the common IgG2aκ–Fc cloning vector, implying antigen-binding prior to and following isotype-switching, if done correctly, should be similar in specificity and affinity. The supernatant of Day-6 post-transfection rAb NRC-2894 cells was collected. Both recombinant and hybridoma secreted antibodies were highly purified (Figs ). The rAbNRC-2894 displayed a similar GroES-recognition/binding profile by immunoblot analysis as the parental hybridoma hyAbNRC-2894 (). Immunoblot assay of Mtb whole-cell extracts indicates that both rAbNRC-2894 and hyAb NRC-2894 recognize Mtb_GroES as a monomer at ~10 kDa and as a multimeric complex at ~25 kDa (). The pair of GroES bands was also recognized by rAbNRC-2894 with purified GroES proteins (). Moreover, both rAbNRC-2894 and hyAbNRC-2894 recognize Mtb-GroES over cross-reacting protein in whole-cell extracts from Yersinia pestis (), indicating specificity for Mtb_GroES. Finally, the GroES-binding potency of hyAbNRC-2894 is indistinguishable from that of rAbNRC-2894 when using an ELISA assay with purified GroES antigen (). This data validates the IgV sequence identified from both NGS and TOPO cloning/Sanger sequencing strategies for hyAbNRC-2894, and the functional retention of antigen recognition following Fc engineering.

To pinpoint the correct Ig-pair within each of the PhoS1/PstS1NRC-2410, SodANRC-13810, or KatGNRC-49680 hybridomas, we followed the same in-grafting process to generate all possible Ig-pair constructs. Each construct pair was separately expressed in 293F cells. Ig culture supernatants were collected at Day-6 post-transfection and examined the presence of secreted Ig using a mouse isotype kit (). Supernatants with secreted Ig were purified using a series of stepwise acid wash cycles on a protein A column (). Spectrophotometric absorbance at A280 identified protein-containing fractions and immunoblotting confirmed the Ig-containing fractions using anti-IgG antibodies (). The Ig positive fractions were pooled, and the rAb was concentrated by centrifugation using a spin column concentrator with a 10K molecular weight cutoff. Sufficient purity for comparison against hyAb was confirmed using gel electrophoresis with Coomassie blue or Silver staining (). To validate rAb, the Ag-Ab binding potential of each IgVH/IgVL pair was compared to that of the corresponding hyAb. Equivalent amounts of either the rAb or hyAb were kept in immunoblot and ELISA assays to ensure equitable comparison. The validated rAb 1) recognized purified Mtb antigen in the immunoblot assay and 2) elicited affinity comparable to that of the hyAb in the ELISA assay. Each validated rAb had equal or greater antigen-specific binding activity towards its cognate Mtb antigen as that of the corresponding hyAb ().

IgV sequence validation of PstS1NRC-2410, SodANRC-13810, and KatGNRC-49680 hybridomas.

The correct IgV sequences of PstS1NRC-2410 (A), SodANRC-13810 (B) and KatGNRC-49680 (C) were identified from multiple IgVH and IgVL sequences (). Each heavy and light chain combination of the isotype-switched IgG2a and Igκ constructs was separately co-expressed in 293 F cells by transient transfection. The presence of an assembled, secreted rAb can be detected in the culture supernatant on Day-6 post-transfection using a mouse Ig Isotype kit (Isotyping label). Some combinations yield an undetectable Ig in the cellular supernatant. The cellular supernatant of each combination was collected and purified by protein A column chromatography. The Ig-equivalent elution fractions were pooled and concentrated. Coomassie or silver staining shows the purity of the rAbs. The Ag-Ab binding potential of each IgVH/IgVL pair was compared to that of the corresponding hyAb. Equivalent amounts of either the rAb or hyAb were kept in immunoblot and ELISA assays to ensure equitable comparison. Immunoblot assays detected recognition of their cognate purified antigen (Immunoblotting). The antigen-binding potency was quantified by ELISA using purified antigens. Relative Ig affinity was determined by ELISA endpoint titers normalized against that of an ELISA buffer control (negative control, NC). Data shown here are from two independent experiments performed in triplicate. P-value<0.001 indicated by the asterisks.

While isolation of the highest affinity rAb was achieved for these hybridomas, the data suggests additional complexity in the analysis. Multiple validated rAbs against PstS1 were identified. PstS1-rAb-DIV elicits higher affinity than its hyAb counterpart (, Immunoblotting and ELISA), while PstS1-rAb-S26 has affinity equivalent to the negative control. To explain the discrepancies between PstS1-hyAb and PstS1-rAb-DIV, either the Fc engineering enhanced PstS1-rAb-DIV binding activity or the hybridoma secreted a mixture of high- and low-affinity antibodies. Since both rAbs can be secreted but PstS1-rAb-S26 lacks significant activity, the later hypothesis is more plausible. To examine if PstS1-hyAb and PstS1-rAb-DIV share similar epitope-binding potential, a direct competition iELISA assay was employed (). Since SodA-hyAb, an IgG1κ isotype, has been purified to homogeneity () and shows no cross-recognition to PstS1 antigens (), it was selected as the isotype control to monitor non-specific mechanisms at high concentrations of antibody. As shown in , PstS1-hyAb inhibits PstS1-rAb binding in a dose-dependent manner, whereas its isotype control, SodA-hyAb, cannot, indicating that the inhibition is highly selective and supports that PstS1-hyAb and PstS1-rAb-DIV bind to identical or proximal epitopes. Multiple validated rAbs against SodA were also identified. SodA-rAb-Igκ1 and SodA-rAb-Igκ2 demonstrated similar antigen-binding potency, despite featuring different functional Ig chains (). In contrast, the rAbs for KatG exhibited properties of monoclonal antibody. KatG-rAb-KVS failed to secrete into the cellular supernatant and was a non-productive aberrant chain (). The rAb-IgM was validated, featuring binding activity equivalent to that of KatG-hyAb ( Taken together, the PstS1 and SodA rAb data support the recent finding that some hybridoma clones are polyclonal and express additional functional Igs with different variable regions [60].

Determination of CDRs and FRs from validated IgV sequences

The CDRs and FRs for each of the validated IgV sequences were defined using bioinformatics to facilitate potential downstream applications. We used two common Ig-CDR definition algorithms, KABAT [61, 62] and IMGT numbering schemes [63, 64]. The conventional KABAT Ig CDR search routine is based on antibody sequence and is used widely as the CDR delineation standard [61, 62]. The IMGT approach, currently accepted by the World Health Organization-International Union of Immunological Societies, identifies Ig-CDRs and FRs by integrating the KABAT Ig definition with antibody structural considerations [63-65]. The IMGT-Collier de Perles program provides a 2D view for the position of the amino acids in CDR/FR representation. In either program, the interpretation of CDR/FR assignments should be used with caution. Assigned CDR/FR stretches in one scheme can disagree with those from other schemes [66]. Using the bioinformatics search algorithms Ig-Blast [67] and IMGT/VQuest [68], we defined the Ig_FRs/CDRs, as presented in (IMGT), and Tables and (KABAT).

The approach described herein can be applied to the identification and sequencing of functional IgV pair sequences from any hybridoma with certain caveats. First, bioinformatics algorithms and filters should stringently and carefully screen for aberrant or nonfunctional IgV. Second, the potential functional IgV pairs should be validated in their native or recombinant form against the cognate antigen, especially when multiple possible chains are identified. Thus, we have successfully isolated and sequenced the IgV transcripts from Mtb-GroES, Mtb-PstS1, Mtb-SodA, and Mtb-KatG hybridomas. We identified the IgVH/IgVL pair that encodes the paratope-determining Ig and defined their CDR/FR regions for each hybridoma.

Discussion

Tuberculosis (TB) is considered a global infectious disease of urgent concern [4, 5, 69, 70]. Though more than a century has passed since the identification of the bacterial pathogen, no cures for TB exist. Mtb infects around 10 million people annually, resulting in a death rate of 1.4 million, highlighting the need for TB diagnostic and treatment strategies. Ideally, a uniform and robust immune response to infection would lead to a preventive vaccine against TB, but this currently appears unattainable [11, 71–73]. The heterogeneity of host immune responses to Mtb infection manifests in diverse clinical presentations, posing challenges for rapid diagnosis and treatment of TB [74, 75]. The development of antibody-based therapeutic approaches targeted to immunodominant antigens or virulence factors is an important strategy for combating infection.

As an initial step toward engineering candidate antibodies for potential therapeutic and diagnostic applications, the IgV sequences of monoclonal antibodies recognizing Mtb virulence factors were determined. The IgV sequences for cognate Abs from the hybridomas PhoS1/PstS1NRC-2410, SodANRC-13810, KatGNRC-49680, and GroESNRC-2894 () were determined using classical and contemporary sequencing methods, i.e., direct TOPO cloning/Sanger sequencing and next generation sequencing combined with transcript assembly. Since the abundance of transcripts determined through deep sequencing does not strongly correlate with the abundance of Ig protein expression [60], herein, greater than 50X coverage is performed to uncover even less abundant Ig-encoding transcripts. While Sanger sequencing is limited to only a single forward and a single reverse read length (2X coverage), the substantially longer Sanger reads provide a template for orthogonal verification of the transcript assembled from the NGS reads. Therefore, the combination of the two sequencing methods enabled a robust IgV sequencing approach in this study.

As is well-understood, hybridomas may lose expression of functional antibodies through passage [76]. Validation of IgV sequence preserves the functional utility of monoclonal antibodies by capturing the epitope recognition domain and enables generation of recombinant antibodies with equivalent properties. The determination of correct IgV/paratope sequence is a recognized challenge in hybridoma sequencing because of an abundance of aberrant Ig transcripts in hybridomas [49–53, 77]. Unproductive aberrant immunoglobulins (Igs) contain stop codons in the reading frame [51], while productive aberrant Igs achieve full-length transcripts without functional domains [53]. Since IgV sequence identification relies on eliminating these aberrant chains, we created a reference library of aberrant chain sequences for in silico subtraction. This library, provided as , includes the overlapping productive aberrant IgVs identified in SP2/0 hybridomas in this work and the earlier literature [49, 50, 53]. After eliminating aberrant IgVs, a minimal and highly refined set of sequence candidates, including all paratope-determining IgVs, were pinpointed. This library may be used whenever performing IgV sequencing in an SP2/0 myeloma background to arrive at, presumably, a similarly small set of sequence candidates for IgV validation.

Multiple IgV heavy or light chain sequences were identified among PhoS1/PstS1NRC-2410, SodANRC-13810, and KatGNRC-49680 hybridomas (). IgV sequence validation was needed to identify the functional IgV sequence pairs from each hybridoma. Fc isotype-switched recombinant antibodies were used to validate the paratope-determining IgVs. We showed that the Fc-isotype-switch leaves the IgV paratope unaffected in terms of binding-potency and specificity, as demonstrated by the representative comparison of GroESNRC-2894 hybridoma recombinant Ig activities (). The RACE-PCR amplification of this domain, therefore, captured the complete productive and functional IgV sequence. The Ig-pairs within PstS1NRC-2410, SodANRC-13810, and KatGNRC-49680 hybridomas were also identified using this isotype-switch validation method. The Fc isotype-switched rAbs elicited specific binding to their cognate Mtb antigens with at least the same potency as that of the corresponding hyAb. This approach works whether a single pair of Ig chains, such as in GroESNRC-2894, is present or when multiple potential IgVH/IgVL pairs are present. The recombinant Ig expression constructs generated from this study () will be available through NIAID’s BEI Resources (www.beiresources.org). The authenticated Mtb-IgV sequences were uploaded to the NCBI nucleotide database (), meaningfully diversifying the current Mtb IgV sequence collection.

The CDR/FR information provided herein may benefit antibody engineering by guiding the delineation of Mtb Ag-Ab interaction loops. CDRs are considered as the antigen-recognition loops representing the Ab-paratope [45]. Genetic mechanisms, such as VDJ/VJ recombination and somatic hypermutations, generate hypervariability within CDR1-3, and particularly CDR3 [42, 45] separated by conserved framework regions (FR1-4). However, some framework residues can contribute toward antigen reactivity [78, 79]. Hence, the currently assigned CDR/FRs provide a starting point for structural and mutational analyses to determine the key interacting residues in the Ag-Ab binding interface. Knowledge of the minimal but essential Ag-Ab epitope binding residues will enable successful CDR-grafting into humanized antibodies.

The Mtb IgV sequence library and approach described herein will particularly benefit future TB research using epitope binding domains for detection, prevention, and treatment. Antibody engineering can further enhance the epitope-binding affinity of the IgV [80-82]. Increasing evidence points to a protective role for antibodies targeting Mtb virulence-associated factors against TB susceptibility, particularly with IgA in the mucosa [8, 14, 83, 84]. Zimmermann et al. recently demonstrated that an anti-HBHA domain in an IgA isotype is protective, whereas, in an IgG1 isotype background, anti-HBHA exasperates Mtb infection [8]. To further decipher Mtb antibody roles targeting other Mtb virulence-associated factors, antibody engineering that includes Fc-engineering is required. By altering the Fc backbone, changes in Ab effector function are possible, potentially leading to enhanced pharmacokinetics or other biological outcomes such as prolonged Ab half-life in circulation without loss of potency or specificity [80-82]. A simple extension of the robust IgV sequencing and validation workflow described herein may be used to determine and alter the functional IgV sequences of Mtb antibodies targeting Mtb-LAM, Mtb-HBHA, Mtb-Ag85A/B/C complex, and other antigens with therapeutic and diagnostic potential.

Materials and methods

Cell cultures and isotyping

The Mtb hybridomas PhoS1/PstS1NRC-2410, SodANRC-1381, KatGNRC-49680 and GroESNRC-2894, were obtained from BEI Resources. Hybridomas were generated by chemical fusion of murine SP2/0-Ag14 (SP2/0) myeloma cell lines [85] with the harvested splenocytes from the immunized mice challenged with one of the Mtb antigens PhoS1/PstS1, SodA, KatG, and GroES. Hybridoma clones were grown and expanded in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum at 37°C with 5% CO2. Cellular supernatants were collected to determine their antibody isotypes using a mouse antibody isotyping kit (cassette method, ThermoFisher Scientific, Waltham, MA; IsoStrip method, Sigma-Aldrich, St. Louis, MO). Cells were grown in Hybridoma-Serum Free Media (SFM) (ThermoFisher Scientific, Waltham, MA) to generate hybridoma antibodies suited for protein G purification.

RNA extractions

Total RNA from each hybridoma was extracted using a NucleoSpin RNA Plus kit (Takara Inc., Mountain View, CA) following the manufacturer’s protocol. RNA concentration and integrity were determined using UV absorbance and RNA gel electrophoresis (Lonza™ Reliant™ Precast RNA Gels, Fisher Scientific Inc.).

5’ RACE-PCR

mRNA was reverse-transcribed to synthesize first-strand cDNA using a SMARTer RACE 5’/3’ Kit (Takara Inc., Mountain View, CA). 5’ RACE-PCR was performed according to manufacturer’s protocol (Takara Inc., Mountain View, CA) using 3’-primers specific to highly conserved antibody isotype-specific constant regions [isotype specific primers (ISP); referred to as gene-specific primers (GSP) by the manufacturer (Takara Inc., Mountain View, CA)].

DNA preparation for TOPO cloning

Variable heavy and light chains of the 4 hybridomas were PCR amplified and isolated using a gel extraction kit (Qiagen Inc., Germantown, MD). IgV RACE-PCR amplicons were cloned into a TOPO sequencing vector (Invitrogen Inc., Carlsbad, CA). Seven to ten transformants were randomly selected for Sanger sequencing by M13 forward and reverse primers (Invitrogen Inc., Carlsbad, CA).

MiSeq Illumina NGS

Library preparation was performed using ~1 μg of IgV RACE-PCR amplicons and MiSeq® Reagent Micro Kit v2 (Illumina Inc. San Diego, CA). Pairwise-sequencing was performed on an Illumina MiSeq Instrument using a V2 2X150 Nano flow cell (Illumina Inc. San Diego, CA). At least 10,000 reads were obtained per sample ().

Bioinformatic analysis of the Ig sequence resulting from direct cloning or NGS-high throughput sequencing

The quality of all input sequence data was confirmed using FastQC, a standard tool for assessing next generation sequencing quality metrics [86]. Reads were then assembled into contigs representing individual transcripts using the RNASeq de novo assembler, Trinity v2.6.6 [57], followed by quantification of transcript expression levels using a pseudoalignment approach with Salmon v0.10.0 [87]. To ensure the identification of contigs with a well-defined V(D)J region, we implemented MiGMAP v1.0.3 [56], a wrapper for the NCBI IgBLAST tool v.1.4.0 that adds user convenience functions to the base IgBLAST tool, to align all contigs again the IMGT Mus musculus database of mouse V, D, and J genes. Based on these approaches, we grouped these contigs into four categories:

Productive contigs—the V(D)J region present without frameshifts or stop codons;

Unproductive contigs—the V(D)J region present with one or more frameshifts or stop codons;

Incomplete contigs—only a partial V(D)J region present without frameshifts or stop codons;

Unidentified contigs—no V(D)J region found.

De novo assembled contigs were further searched via an alignment approach using BlastN [88] for the existence of specific ISP primer sequences in the putative constant Ig regions. Finally, a custom R script () was applied to both the assembled NGS contigs and TOPO cloning/Sanger sequencing datasets to identify the aberrant sequences shared among the hybridoma cell clones examined, including the four hybridomas described in this study and ten additional Mtb hybridomas with an SP2/0 background. After eliminating common aberrant Ig chains, the sequences of the productive contigs were examined and shared little identity with the sequences of well-known aberrant Igs in the literature ([49, 50, 53]. Genbank Accession numbers have been assigned as follows: AF220155, AF220156, AF220157, D14170, D14171, D14173, D50398, S65377, AF089740, AF019945, AF230099, DQ355823, X80944, X80954, AF089742, AF039853, X80944, X80954, AF039853 and M183140.

Mouse IgG2a Isotype-switched antibody

Synthesis of the mouse IgG2a Isotype-switched IgV chimeras was provided by GenScript Inc. Gene blocks of the putative IgV sequences (FR1/CDR1 to FR4/CDR4, ) were cloned into an intermediate cloning vector, pUC57 and subsequently into GenScript IgG2a/Igκ Fc-expression vectors. Note that the leader sequences of all the recombinant antibodies are MGWSCIILFLVATATGVHS. This leader peptide has been successfully employed in antibody production with 293F suspension cell systems (GenScript Inc. Piscataway, NJ).

Recombinant antibody production

FreeStyle 293F cells (Invitrogen Inc., Carlsbad, CA) were grown in suspension on a platform shaker (ThermoFisher Scientific, Waltham, MA) in a humidified 37°C, 8% CO2 incubator with rotation at around 150 rpm. Cultures were maintained for 5–10 passages prior to performing transfections to ensure stable growth patterns. Polyethylenimine (PEI) (25 kDa linear PEI, Polysciences Inc., Warrington, PA) was prepared as a stock solution at a concentration of 1 mg/ml in a buffer containing 25 mM HEPES and 150 mM NaCl (pH 7.5). For best transfection efficiency, cells had a viability of >95% at the time of transfection. Twenty-four hours prior to transfection, cells were split to a density of ~1 x 106 cells/ml and cultured overnight in the CO2 incubator with shaking at 37°C. Cell density was ~2 x 106/ml at the time of transfection. For transfection, the Ig expression vector plasmid DNAs (1:1 of Ig heavy:light in μg) were added to the cells at a final concentration of 3 μg/ml and PEI at a final concentration of 3 μg/ml of transfection volume (at 1:1, DNA: PEI ratio). After 24 hr, the cells were diluted 1:1 with pre-warmed Freestyle™ 293 Medium supplemented with valproic acid (VPA) (Sigma-Aldrich, St. Louis, MO) to a final concentration of 2.2 mM. Ig enriched cellular supernatants were harvested at day 4–6 post-transfection with viability slightly greater than 55%.

Recombinant and hybridoma antibody purification

Purification employed a protein A column for recombinant antibodies and a protein G column for hybridoma antibodies (ThermoFisher Scientific, Waltham, MA). Cellular supernatants were added at 1:10 v/v of the binding buffer appropriate for the column (ThermoFisher Scientific, Waltham, MA) to ensure proper ionic and pH conditions for later affinity matrix-binding. The insoluble precipitates were removed by centrifugation at 9,500 rpm for 10 mins at 4°C. The appropriate column was utilized according to the manufacturer’s recommendations for equilibration and sample loading to purify the Ig. The column was washed with 5 column volumes (CVs) of binding buffer prior to stepwise acid washes (2 CVs in each pH buffer, 8.5, 6.5, 5.5) followed by Elution buffer (ThermoFisher Scientific, Waltham, MA) and neutralized using 1/10 (v/v) of 1M Tris/HCl, pH 8.0 (VWR, Radnor, PA). Protein dye staining and gel electrophoresis were employed to identify Ig within collected fractions. The final Ig-yield was determined by BCA protein assay (ThermoFisher Scientific, Waltham, MA)

Induction of recombinant Mtb antigens expressed in BL21/DE3, pLysS or BL21/DE3

Bacterial expression plasmids encoding the six-Histidine tagged SodA (rSodA) (BEI Resources, Manassas, VA) were transformed into BL21/DE3, pLysS competent cells (Invitrogen Inc., Carlsbad, CA). Bacterial expression plasmids encoding the six-Histidine tagged KatG (rKatG) (BEI Resources, Manassas, VA) were transformed into BL21/DE3 competent cells (Invitrogen Inc., Carlsbad, CA). Once bacterial density reached an A600 of 0.4 to 0.6, 0.2 mM to 1 mM IPTG (ThermoFisher Scientific, Waltham, MA) was added to induce recombinant protein expression. The culture was harvested after overnight incubation at 37°C with shaking at 200 rpm.

Recombinant antigen purification

Recombinant KatG

After IPTG induction, rKatG-BL21/DE3 bacterial pellets were collected by centrifugation at 9,500 rpm for 10 mins. The rKatG pellets were resuspended in a lysis buffer containing 20 mM Tris, pH 8.0, 500mM NaCl, 30 mM imidazole, and protease inhibitors (Calbiochem, Burlington, MA) at 1:5 ratio (lysis buffer: bacterial culture, v/v) to prepare rKatG lysates. rKatG was extracted by briefly sonicating at an output setting of 10, for 3 to 5 times at 10–20 second intervals in ice to prevent overheating. Insoluble cell debris was then removed by centrifugation at 13,000 rpm for 10 min. The rKatG lysates were incubated with Ni++-NTA beads (GE Healthcare, Chicago, IL) overnight at 4°C with gentle mixing to ensure sufficient reaction time. The beads were washed three times with the lysis buffer containing 30 mM imidazole prior to 0.5M imidazole incubation to release rKatG from the beads. The purity of the released rKatG was shown by Coomassie Blue stain on SDS-PAGE gels.

Recombinant Sod (rSodA)

After IPTG induction, rSodA-BL21/DE3 bacterial pellets were collected by centrifugation at 9,500 rpm for 10 mins. To prepare SodA lysates, the rSodA pellets were resuspended in lysis buffer containing 20 mM Tris, pH 8.0, 500 mM NaCl, 60 mM imidazole, 8 M urea and protease inhibitors (Calbiochem, Burlington, MA) at 1:5 ratio (lysis buffer:bacterial culture, v/v). rSodA was extracted by mixing with lysis buffer at room temperature for 2 hours. Insoluble cell debris was then removed by centrifugation at 13,000 rpm for 10 min. The rSodA lysates were incubated with Ni++-NTA beads (GE Healthcare, Chicago, IL) overnight at room temperature with gentle mixing to ensure sufficient reaction time. The beads were washed three times with the lysis buffer containing 60 mM imidazole, 8 M urea prior to 0.5 M imidazole with urea incubation to release rSodA from the beads. The purity of the released rSodA was shown by Coomassie Blue stain on SDS-PAGE gels.

Immunological assays

Western blot or immunoblot

Protein samples were treated with sample buffer containing reducing agents (DTT or β-ME) and boiled at 70°C to 100°C for 10 mins prior to protein gel electrophoresis. The resolving gel was then transferred onto a polyvinylidene fluoride membrane for immunological detection by reacting with specific antibodies. To compare the Ag-Ab binding potential between rAb and hyAb, equivalent amounts of either the rAb or hyAb at 1 μg/ml were incubated with cognate Mtb antigen at 1 μg/lane in an immunoblot overnight at 4οC. Secondary HRP-conjugated antibody [anti-mouse IgG H+L chains (ab6789, Abcam, Waltham, MA) for GroES, PstS1, and SodA; anti-mouse IgM (ab97230, Abcam, Waltham, MA) for KatG] was added, washed and developed using ECL reagents (Cytiva, Marlborough, MA). Positive signals were detected using Imager c600 (Azure Biosystems, Dublin, CA).

Enzyme-linked immunosorbent assay (ELISA)

An indirect ELISA (iELISA) method was employed to detect the level of antigen-binding of hybridoma and recombinant antibodies. Plates were coated with 2.0 μg/ml of cognate, purified antigens overnight at 4°C, washed with PBS containing 0.1%, v/v, Tween 20 (PBS-Tween), and blocked in 5% non-fat milk (VWR, Radnor, PA) for one hour at room temperature. Then, plates were incubated with serial dilutions of the purified recombinant or hybridoma antibodies, washed in PBS-Tween, and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (Abcam, Cambridge, United Kingdom) prior to the addition of tetramethylbenzidine substrate (TMB, ThermoFisher Scientific, Waltham, MA). Spectral absorbance of plates at A650 was determined by microplate reader after addition of TMB blueStop (SeraCare Life Science, Gaithersburg, MD).

A directly competitive iELISA method was employed to measure the relative epitope-binding potential between rAb-Pst1 and hyAb-PstS1. Plates were coated with 1.0 μg/ml of PstS1, purified antigen overnight at 4°C, washed with PBS containing 0.1%, v/v, Tween 20 (PBS-Tween), and blocked in 5% non-fat milk (VWR, Radnor, PA) for one hour at room temperature. The rAb-Pst1, an IgG2a subclass, was kept at a constant concentration of 100 ng/ml, while the concentration of hyAb-PstS1, an IgG1 subclass, was varied from 0 to 1000 ng/ml. The rAb-PstS1 was selectively recognized by the HRP-conjugated anti-IgG2a antibody (A10685, Invitrogen, Carlsbad, CA). Total binding of either IgG1 or IgG2a was measured by the HRP-conjugated anti-IgG heavy and light chain antibody (ab6789, Abcam, Waltham, MA) in the parallel replicate.

Characterization of the isotypes and quality assessment of the extracted RNA from GroESNRC-2894, PstS1NRC-2410, SodANRC-13810 and KatGNRC-49680.

The extracted RNA from the 4 clones yielded high quality RNA with minimal RNase contamination. A. Secreted immunoglobulins from the 4 hybridoma clones and their isotypes were confirmed through isotype cassette by examining their culture supernatants prior to RNA extraction. B. RNA purity was determined by a NanoDrop™ 2000 Spectrophotometer. The ratio of A260/A280 and A260/A230 is greater than 1.9 and 2.0 respectively, indicating high RNA purity. C. RNA integrity was assessed by the rRNA ratio of 28S:18S larger than 2-fold after 1.25% formaldehyde, RNA gel electrophoresis. D. The degree of RNase contamination was measured by the differential amount of 28S present after (+) and before (-) incubation of the extracted RNA at 37°C for 2 hours. Minimal RNase contamination was observed.

(TIF)

Click here for additional data file.

Homology alignment to characterize the IgV found from direct cloning—Sanger methodology using a representative of the 4 hybridoma clones, SodANRC-13810.

A. Homology alignment of the NRC-13810-Ig chains, RACE-IgG1, and Igκ of TOPO TA clones: IgVH (Left) and IgVL (Right). B. Nucleic acid alignment of the sequence reads against each other. Positions #131- and #111-mark initiation codons of clone #1124 and #1267 PCR-IgV amplicons, respectively. If an IgV transcript contains a STOP codon within its FR/CDR domains, it was classified as an unproductive chain and eliminated. CLC Sequence Viewer v8 was used for Ig alignment.

(TIF)

Click here for additional data file.

Purified hybridoma antibody from PstS1NRC-2410 (A), GroESNRC-2894 (B), and SodANRC-13810 (C). Cellular supernatants were collected and incubated with protein G beads overnight at 4°C. The Ig-bound beads were then gently spun down at low speed and packed into a column. The column was washed with 5 column volumes (CVs) of binding buffer prior to stepwise acid washes (2 CVs in each pH buffer) followed by acid elution (E1-6). The presence of Ig heavy and light chains was examined by gel electrophoresis followed by immunoblot against a polyclonal anti-mouse IgG antibody (top gel). The remaining proteins left on the transferred gels were stained by Silver staining to confirm Ig presence (bottom gel).

(TIF)

Click here for additional data file.

Purified recombinant immunoglobulin IgG2aκ from NRC-2410 (A), NRC-2894 (B), NRC-13810 (C), and NRC-49680 (D). Cellular supernatants of the recombinant antibodies were collected and incubated with protein A beads overnight at 4°C with mixing. The Ig-bound beads were then gently spun down at low speed and packed into a column. The column was washed with 5 column volumes (CVs) of binding buffer prior to stepwise acid washes (2 CVs in each pH buffer) followed by acid elution (E1-9 fractions). The presence of Ig heavy and light chains was examined by A280 and gel electrophoresis followed by immunoblot against a polyclonal anti-mouse IgG antibody (top gel). The remaining proteins left on the transferred gels were stained by Coomassie or Silver staining to confirm Ig presence (bottom gel).

(TIF)

Click here for additional data file.

Mtb Recombinant SodA (A) and KatG (B) were purified by affinity chromatography. Bacterial expression plasmids bearing 6X His-tagged SodA or 6X His-tagged KatG were transformed into BL21, a T7/IPTG expression system. The presence of recombinant proteins was examined by gel electrophoresis followed by immunoblot against an anti-His antibody (top gel). The remaining proteins left on the transferred gels were stained by Coomassie staining to confirm recombinant protein presence (bottom gel). The arrow marks the respective recombinant protein. A. 8M urea was employed to extract rSodA-His from insoluble inclusion bodies. B. Brief sonication of rKatG bacteria pellets in the mild lysis buffer was used to extract rKatG-His.

(TIF)

Click here for additional data file.

Complementarity determining regions (CDRs) and framework regions (FRs) of IgVH and IgVL sequences through IMGT analysis.

Collier de Perles displays of validated Ig heavy (Left) and Ig light (Right) chains of GroESNRC-2894 (A), PstS1NRC-2410 (B), SodANRC-13810 (C), and KatGNRC-49680(D) using IMGT/V-QUEST (http://www.imgt.org/IMGT_vquest/vquest) for CDR analysis.

(TIF)

Click here for additional data file.

RACE-PCR amplicon features as a mix of templates.

NRC-13806, an Mtb hybridoma, was run in parallel with the hybridoma clones from this study and was selected as a representative for the Sanger sequence analysis. Chromatogram (A) or summary (B) reports confirmed the presence of a mixture of DNA template composites within a single RACE-PCR amplicon. Sanger sequencing was performed by ATCC’s sequencing facility.

(TIF)

Click here for additional data file.

Competitive iELISA assay of hyAb-PstS1 against rAb-Igk-DIV.

A. PstS1-rAb-lgk-DIV, an IgG2a subclass, was kept at a constant concentration of 100 ng/ml; while concentrations of PstS1-hyAb (solid lines), an IgG1 subclass, or its isotype control, SodA-hyAb (dash lines), were increased from 0 to 1000 ng/ml in a directly competitive iELISA assay for PstS1 antigen binding. B. The specificity of antibody reagents was examined using an iELISA assay with increasing concentrations from 0 to 1000 ng/ml of PstS1-hyAb or SodA-hyAb for PstS1 antigen binding. The PstS1-rAb is selectively recognized using the HRP-conjugated secondary isotype-specific antibody, anti-IgG2a (Red). Total binding of either IgG1 or IgG2a was measured by HRP-conjugated anti-IgG heavy and light chain antibody (Blue) in the parallel replicate. Data shown here from two independent experiments in duplicate.

(TIF)

Click here for additional data file.

Summary of the Isotype Specific Primers (ISP) conservation scores in alignments of Ig-heavy and -light chains.

(TIF)

Click here for additional data file.

Number of reads per NGS library.

(TIF)

Click here for additional data file.

Summary of all the Ig nucleic acid sequence (CDR/FR domains defined via a KABAT-based algorithm).

Red asterisks mark the validated IgVH and IgVL sequences.

(TIF)

Click here for additional data file.

Summary of the deduced Ig amino acid sequence (CDR/FR domains defined via a KABAT-based algorithm).

Red asterisks mark the validated IgVH and IgVL sequences.

(TIF)

Click here for additional data file.

The recombinant constructs generated from this study.

Sequences are available in the BEI-product information sheet.

(TIF)

Click here for additional data file.

The Ig-VDJ gene family used to define the CDR/FR domains in Table 3.

(TIF)

Click here for additional data file.

Aberrant chain library used in this study.

(ZIP)

Click here for additional data file.

Validated, FASTA IgV sequence for Mtb hybridomas.

(ZIP)

Click here for additional data file.

Custom R-script to identify the aberrant sequences shared among most hybridoma cell clones in this study.

(ZIP)

Click here for additional data file.

(PDF)

Click here for additional data file.

2 Jun 2021

PONE-D-21-15036

Determination of variable region sequences from hybridoma immunoglobulins that target Mycobacterium tuberculosis virulence factors

PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: In this paper authors have described their work on the determination of variable region sequences of hybridoma immunoglobulins (hyAbs) targeting four Mtb proteins (termed as “virulence factors”): PstS1, GroES (both provided by BEI), SodA and KatG (both plasmids provided by BEI. Corresponding hybridomas were also provided by BEI). Putative IgV CDR and FR regions of each hybridoma were sequenced by Sanger and NGS methods to identify all potential IgV (H and L) sequences. Sequences encoding aberrant Ig chains were eliminated through bioinformatics. Retention of paratope sequences were confirmed by isotype-switching of the putative IgV in a common Fc backbone (this work was outsourced). Each H and L chain combination of the isotype-switched constructs was co-expressed and each secreted recIgV was validated (using antibody from the parental hybridoma as reference) for binding to the target antigens. Authors conclude to have successfully isolated and sequenced the IgV transcripts from the 4 hybridomas, and also to have identified the CDR and FR regions in the IgV H/L pairs. The work is interesting since, as the authors have stated, extension of this workflow may help determine/alter the IgV sequences of Mtb antibodies targeting other antigens with therapeutic or diagnostic potential. Nonetheless, readers will be benefitted if following concerns could be addressed.

1. It is stated (with the help of Figures 3 and 4) that the epitope binding potency and specificity of IgV paratopes were unaffected by Fc-isotype switch. Even so, the concern remains whether both antibodies (rAb and hyAb) are binding to the same epitope. One way to address this is to allow one of them to compete with the other during immunoblotting and ELISA.

2. As the authors have stated, glycosylation at specific sites plays a very important role in overall function of the antibody. Hence, they have coexpressed the IgVH and IgVL constructs in 293F cells. However, have they also checked whether the glycosylation actually happened in the rAb and whether it was equal or equivalent to that seen in hyAb?

3. The CDRs and FRs for each validated IgV sequence were defined by bioinformatics, using KABAT/IMGT algorithms. However, not all the positions in the traditionally defined CDRs are important for binding. Many positions that contribute critically to the binding energy may reside outside of the CDRs. Moreover, different CDR identification methods may often identify radically different stretches. This and other limitations of the work may be stated in a paragraph under Discussion.

4. It will be helpful if all bioinformatic tools used in this study are briefly described in the ‘Materials and Methods’ section.

5. More details on Immunoblotting will be helpful for the reader.

Reviewer #2: The manuscript by Chang Foreman et al entitled, “Determination of variable region sequences from hybridoma immunoglobulins that target Mycobacterium tuberculosis virulence factors” describes the process to identify the specific variable domains responsible for binding against SodA (Superoxide Dismutase), KatG (Catalase), PhoS1/PstS1 (regulatory factor), and GroES (heat shock protein) from the hybridomas. This is a good study as hybridoma antibodies have been reported to present multiple antibody sequences in some cases. The authors applied RACE-PCR to amplify the antibody domains after identifying the isotypes and was subsequently sequenced. Sequence analysis using both NGS and Sanger sequencing were compared. The report highlights the depth and advantage of NGS method to identify the key domains from a hybridoma for conversion to functional recombinant scFv antibodies. Overall, the study is technically sound with sufficient validation to support the conclusion of the manuscript. I only have some comments to the authors which are listed below.

Comments:

Table 3: The domain sequences would read clearer if the CDR and FR sequences are expressed in amino acid sequence rather than gene sequence. The gene family for the VDJ used should also be detailed.

Line 341: please rephrase ready detection.

As the authors mentioned that deep sequencing capability is preferred to uncover even less abundant Ig-encoding transcripts, was gene analysis or VDJ gene analysis done to identify the clonality of the hybridoma?

Line 450 to 452: Finally, a custom R script was applied to both the assembled NGS contigs and TOPO cloning/Sanger sequencing datasets to identify the aberrant sequences shared among most hybridoma cell clones. Is the script provided?

Line 363, Since IgV sequence identification relies on eliminating these aberrant chains, we created a reference library of aberrant chain sequences for in silico subtraction. How was this library characterized and validated? There is no mention of the details to the reference library in terms of the selection process, characterization, and validation.

There is the added point that the sequence information provided would also help to improve structural studies especially those on antibody-antigen interactions which should be added in the discussion to highlight the importance of the study.

In addition, please provide a percentage of coverage when using Sanger vs NGS. Also discuss the potential reasons/complications that resulted in a lower coverage using Sanger.

The apparent V,D,J segments used and their combination should be mentioned and discussed. Also, are the combinations commonly used in TB.

There are some spelling and grammatical errors which would benefit from another round of editing.

Overall, the paper shows an interesting concept of generating scFv from hybridomas with a specific interest on MTb targets. The message that could be highlighted additionally is the flexibility and coverage provided by NGS as well as the potential identification of multiple sequences from a hybridoma which indicates the presence of multiple clonality.

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Reviewer #1: Yes: Sudhir Sinha

Reviewer #2: No

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11 Jul 2021

PONE-D-21-15036

Determination of variable region sequences from hybridoma immunoglobulins that target Mycobacterium tuberculosis virulence factors

PLOS ONE

> We want to express our thanks to both reviewers and to the editor for the encouraging feedback and the opportunity to revise our manuscript. We are grateful for the thorough and careful review. We have addressed the concerns in a point-by-point response below. This thoughtful review process has improved the revised manuscript, and we hope you find it worthy for publication. Please note that all manuscript line number changes addressed below refer to text visible when “No Markup” is selected in the “Track changes” tab of Microsoft Word.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf

and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

> Our manuscript meets PLOS ONE’s style requirements, including those for file naming.

2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

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> The original blot/gel image data are now provided in Supporting Information.

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> The statement “Recombinant KatG was unstable in this strain as a His-tag antibody detected multiple degradation products (data not shown)” has been removed.

Reply to the Reviewer #1 (Dr. Sinha)

1. It is stated (with the help of Figures 3 and 4) that the epitope binding potency and specificity of IgV paratopes were unaffected by Fc-isotype switch. Even so, the concern remains whether both antibodies (rAb and hyAb) are binding to the same epitope. One way to address this is to allow one of them to compete with the other during immunoblotting and ELISA.

> Although rAb and hyAb exhibit similar affinity and specificity in the antigen recognition, direct analysis of the interface between paratope and epitope has not been performed. Therefore, we changed the wording of “epitope-binding” to “antigen-binding” throughout the text. We agree that Figure 3 and 4 only demonstrate the antigen-binding potency of rAb and hyAb. When biochemical features such as affinity and specificity are similar (Figs 3C-3D and Figs 4B-C), the underlying molecular mechanisms (e.g. paratope:epitope interaction), are often conserved.

However, to address Dr. Sinha’s question on measuring the epitope-binding potential between rAb and hyAb, we chose PstS1-hyAb and its rAb as an exemplar. Among the 4 hybridoma clones, the PstS1 clone elicited the most notable, differential affinity between rAb and hyAb. We examined if rAb and hyAb of PstS1 can compete for PstS1 in a direct competitive iELISA assay (Fig 8s). With PstS1 antigen as the limiting reagent (Fig 8s, blue), PstS1-hyAb, a lower affinity antibody than rAb, can still outcompete PstS1-rAb in PstS1-binding, eliminating almost all PstS1-rAb binding, clearly indicating that both PstS1-rAb and -hyAb recognize co-localized or identical PstS1 epitopes. This result supports our hypothesis that PstS1 hybridoma is polyclonal, secreting a mixture of antibodies with varied affinities.

We have added the antibody competition result in the Result section from line 302 to line 305 in the edited text, and description of the method in the Materials and Methods section from line #573 to line #579 of the edited text.

2. As the authors have stated, glycosylation at specific sites plays a very important role in overall function of the antibody. Hence, they have coexpressed the IgVH and IgVL constructs in 293F cells. However, have they also checked whether the glycosylation actually happened in the rAb and whether it was equal or equivalent to that seen in hyAb?

> As the reviewer mentioned, glycosylation is very important for antibody function. The majority of glycan studies center on the glycosylation impact on Fc-mediated antibody effector functions; however, a few studies mention glycan impact on Ab stability and recognition. Though our focus lies in the paratope determining IgV sequences, and not the Fc-region, expression of rAbs in 293F with post-translation modifications mimicking that of mammalian B cells avoids possible issues such as Ab aggregation, secretability, and/or alterations in antigen-binding. As demonstrated in Figs 3c and 3d, rAbGroES expressed in 293F cells featured comparable affinity and specificity toward Mtb-GroES as hyAbGroES.

We did not compare the glycan profiles between the rAb and hyAb, since the validation of rAbGroES supports that our expression system preserves enough antibody features for interrogating IgV-determining paratope properties. However, we agree that knowledge of the glycan profiles between rAb and hyAb could benefit future Ab production for commercialization. However, glycan profiling will require more than simple sugar staining. Genetic approaches coupled with Mass Spectromery analysis may be necessary and lie outside the scope of this current paper.

3. The CDRs and FRs for each validated IgV sequence were defined by bioinformatics, using KABAT/IMGT algorithms. However, not all the positions in the traditionally defined CDRs are important for binding. Many positions that contribute critically to the binding energy may reside outside of the CDRs. Moreover, different CDR identification methods may often identify radically different stretches. This and other limitations of the work may be stated in a paragraph under Discussion.

> The reviewer’s points are well-taken. We have added caution statements about interpreting CDR/FR stretches in line #318 to #323 of the Result section under “Determination of CDRs and FRs from validated IgV sequences”. The application of CDR/FR information was added in line #396 to #404 of the Discussion section.

4. It will be helpful if all bioinformatic tools used in this study are briefly described in the ‘Materials and Methods’ section.

> We have provided additional description of the tools and modified the Materials and Methods, lines #455-462, as well as included our custom R-scripts in Appendix 3s.

5. More details on Immunoblotting will be helpful for the reader.

> We have added immunoblotting details in the Materials and Methods, line #555 to line #561.

Reply to the Reviewer #2

1. Table 3: The domain sequences would read clearer if the CDR and FR sequences are expressed in amino acid sequence rather than gene sequence. The gene family for the VDJ used should also be detailed.

> We have correspondingly changed Table 3 and added Table 6s to annotate the gene family-associated Kabat alignments to define CDR/FR stretches.

2. The expression in Line 341: please rephrase ready detection.

> We have rephrased the statement to “The heterogeneity of host immune responses to Mtb infection manifests in diverse clinical presentations, posing challenges for rapid diagnosis and treatment of TB.”

3. As the authors mentioned that deep sequencing capability is preferred to uncover even less abundant Ig-encoding transcripts, was gene analysis or VDJ gene analysis done to identify the clonality of the hybridoma?

> Thank you for pointing out that the NGS workflow could be extended to investigate hybridoma clonality. NGS offers high throughput, high fidelity and deep sequencing capability, perfect for sequence annotation of VDJ. Deciphering the molecular mechanisms driving genome heterogeneity, of course, is very interesting and could be beneficial for the study of B-cell derived tumors. However, we focus on deciphering the hybridoma’s IgV-transcript sequences responsible for encoding the functional, antigen-binding antibody. The source of aberrant chains and/or the mechanisms giving rise to aberrant chains or multi-clonality lies outside the scope of the current effort.

4. Line 450 to 452: Finally, a custom R script was applied to both the assembled NGS contigs and TOPO cloning/Sanger sequencing datasets to identify the aberrant sequences shared among most hybridoma cell clones. Is the script provided?

> The script is now provided in Appendix 3s.

5. Line 363, Since IgV sequence identification relies on eliminating these aberrant chains, we created a reference library of aberrant chain sequences for in silico subtraction. How was this library characterized and validated? There is no mention of the details to the reference library in terms of the selection process, characterization, and validation.

> The reference library of aberrrant chain sequences was built from a large IgV- Mtb hybridoma sequencing project. All hybridoma clones were made from fusion of SP2/0 myeloma cells. The 4 clones described in this report were members of that project. All hybridoma cell lines were sequenced using both TOPO and NGS/Bioinformatic methods. The shared IgV contigs between hybridoma clones were collected as common aberrant chain sequences, since each hybridoma clone was known to secrete antibodies recognizing distinct Mtb antigens. The known IgV aberrant chains collected from the published literature were used to populate the the aberrant chain reference library, described in lines #476-479. After filtration, none of the reported IgV sequences share sequence identity with the members of our aberrant library. We have added additional details to describe how we generated the aberrant library in lines #473-474.

6. There is the added point that the sequence information provided would also help to improve structural studies especially those on antibody-antigen interactions which should be added in the discussion to highlight the importance of the study.

> Thank you for the suggestions. We have added these points in Discussion lines #396-404.

7. In addition, please provide a percentage of coverage when using Sanger vs NGS. Also discuss the potential reasons/complications that resulted in a lower coverage using Sanger.

> The definition of coverage, also called depth of sequencing, is the number of reads aligned to a particular base in a target sequence, typically averaged across all bases in a given target sequence. The necessary coverage for NGS to uncover a TRUE IgV is hard to predict, since it depends on the expression level of the target transcript and the variable size of the IgV transcriptome arising from a mixture of true and aberrant chains. In other studies, coverages from 10X to 30X is sufficient to study human mutations or SNPs. Our sequencing depth reached greater than 50X. The paratope-determining IgV in each clone was successfully identified in this report, indicating that 50X coverage is sufficient depth for hybridoma IgV sequencing.

> The sequencing principles of Sanger and NGS are different. NGS employs a parallel sequencing methodology, whereas Sanger sequencing is a clonal sequencing method. Once a sequencing clone is selected, Sanger sequencing is capable of producing one forward read and one reverse read, which depending on read overlap, can only produce between 1-2X coverage (considering potential strand read discrepancies). The advantage of the Sanger method resides in its longer, continuous reads, as noted in lines #166-170, which typically can reach up to 1kb. In our IgV amplicon, averaging around 500-600 bp, the IgV sequence was determined using a forward and a reverse primer. Thus, all IgVs reported here using the Sanger method have 2X coverage. Detection sensitivity of TRUE IgV lies in the initial selection of TOPO clones. Herein, we randomly selected 7-10 clones in each TOPO-IgV cloning. As shown in Table 2, many failed to uncover a true IgV sequence. Clearly, NGS provided a much more robust and greater sensitivity of detection, as previously described in lines #205-207. We have modified/added lines #354-361 in Discussion to emphasize the superiority of the NGS method in uncovering TRUE IgV.

8. The apparent V,D,J segments used and their combination should be mentioned and discussed. Also, are the combinations commonly used in TB

> As described in line #122 and Fig 1A, our PCR approach to isolate an IgV amplicon employed a 5’-universial prime and a gene (isotype) specific primer (ISP). The ISP aligned with the conserved sequence of CH, Constant region of Ig chain (Table 1 & 1s) so that multiple primers aligned with V-D-J gene segments were unnecessary for amplification. We avoid a multiplex PCR method which frequently gives rise to a distorted representation of immune repertoire due to primer bias. No segments or combinations were used, so no discussion is possible.

> Mycobacterial antigens trigger a diverse array of B cells-VDJ recombination. Heavy chains of IgM, IgA, IgE, IgG1, IgG2, IgG3 isotypes/subclasses in combination with either light chains of kappa or lambda can be found in human TB patients. In mice, Mtb-antigens induce similar humoral profiles consisting of IgM, IgA, IgG1, IgG2a, IgG3, kappa and lambda. Nevertheless, we cannot comment on combinations in the manuscript since we did not employ them.

9. There are some spelling and grammatical errors which would benefit from another round of editing.

> Thank you. We have made corrections in Tables (fonts) and in the text throughout using tracked changes in Word. We also further confirm our format to PLOS requirements.

> Please note we have also rearranged the order of presentation of Figures 4A to 4C in the revised Results section.

> We have made corrections in the following Tables/Figures

Table 3: We have changed the data from nucleic acid to amino acid presentation and added a footnote indicating the source of gene family used for the alignment (Table 6s)

Fig 1A. Correction on IgL-mRNA label: change VH to VL

Fig 4B. Correction on Immunoblotting/Mw Labels: 50- to 40- & alignments

Table 6s: Added Table.

Fig 1s-B. Reformatted the A260/A280 label

Fig 4s-B. Correction on bottom label: Coomassie to Sliver Stain

Fig 6s. Reformatted the NRC-2894_Ig��label

Fig 8s. Added Figure.

Appendix 3s: Added Appendix

Submitted filename: Rebuttal_PONE-D-21-15036.docx

Click here for additional data file.

16 Jul 2021

PONE-D-21-15036R1

Determination of variable region sequences from hybridoma immunoglobulins that target Mycobacterium tuberculosis virulence factors

PLOS ONE

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Reviewer #1: I am satisfied with authors’ response to my queries/comments but it would be nice if they could address one lingering concern about the data shown as Fig 8S. A large amount of PstS1 hyAb was needed to inhibit the binding of rAb, which raises concern about the specificity of this competitive binding. Could it have arisen from a non-specific mechanism such as steric hindrance? Is it possible to address this doubt with the use of an ‘isotype control’ (same IgG subclass but with no or irrelevant antibody activity)?

Reviewer #2: (No Response)

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23 Jul 2021

1. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

> We have reviewed our reference for completeness and accuracy.

1. In manuscript line #340, we removed the reference #71 and replaced it with references #4 & #5. (Reference #71 was not the correct reference)

2. We also check if our references meet PLOS journal format recommendations (https://journals.plos.org/plosone/s/submission-guidelines). We have made the following changes.

2.1. Ref #4: added a Web link

2.2. Ref #5: corrected author information and conformed to journal citation format

2.3. Ref #18: font size adjustment

2.4. Ref #55: conformed to journal citation format

2.5. Ref #67: conformed to journal citation format

2.6. Ref #68: conformed to journal citation format

2.7. Ref #70: conformed to journal citation format

2.8. Ref #71: removed. It is an incorrect reference.

Review Comments to the Author

Reviewer #1: I am satisfied with authors’ response to my queries/comments but it would be nice if they could address one lingering concern about the data shown as Fig 8S. A large amount of PstS1 hyAb was needed to inhibit the binding of rAb, which raises concern about the specificity of this competitive binding. Could it have arisen from a non-specific mechanism such as steric hindrance? Is it possible to address this doubt with the use of an ‘isotype control’ (same IgG subclass but with no or irrelevant antibody activity)?

> Thank you for the suggestion. As we have mentioned in our 1st Rebuttal letter, we have changed the wording from epitope-binding to antigen-binding throughout the text since we did not yet provide a direct analysis on the interface between paratope and epitope in Figs 3 & 4. We performed a competitive assay with isotype controls to address your concern over non-specific competition. In Fig 8S, the competitive assay, we tested whether rAb and hyAb of the same antigen can compete for their recognition epitopes. PstS1 group is of particular interest since the hyAb seems to be around 80% as potent as its rAb. In the previous Fig 8S, we did a quick diagnosis, and it was very promising. To provide a more stringent and quantitative analysis, we have revised our Fig 8S and made the modifications listed below.

1. We decreased the amount of PstS1 antigen from 2 to 1 ug/ml in the overnight coating to the ELISA plate. The reduced number of epitopes should create more selective binding competition between rAb and hyAb, avoiding/reducing the amount of non-specific binding or Ab absorption.

2. All antibody and antigen reagents used herein are purified or purchased in purified form, thereby reducing the likelihood of impurities interfering with the readouts.

3. In addition, we also cross-examined all the antibody reagents used herein for their recognition specificity (Fig 8sB).

4. We also included an hAb’s isotype control in parallel to control for non-specific mechanisms at high concentrations of antibody. hyAb-SodA, an IgG1� isotype, has been purified to homogeneity and is highly selective for its binding antigen (Fig8sB, blue dash). As shown in Fig 8sA, hyAb-PstS1 inhibits rAb-PstS1 binding in a dose-dependent manner, whereas its isotype control, hyAb-SodA, cannot, indicating that the assay is highly selective and depends on specific antibody recognition of the antigenic sites (epitopes).

5. Quantitatively, our assay is in good agreement with the relative affinities between rAb and hyAb-PstS1 (Fig 4A, similar, within 20% difference). Shown below is the Ag-Ab binding equation:

• When Ka1 is relatively close to Ka2 (less than 1 log difference; in our scenario, less than 20% difference), Ag-Ab complexes comprise roughly a 1:1 ratio of Ab1- and Ab2-components when Ab1 and Ab2 have equimolar starting concentrations. We observed that the concentration of hyAb-PstS1 antibody required to achieve 50% inhibition is between 64 and 128 ng/ml, quite comparable to the concentration of rAb-PstS1 in the reaction (100 ng/ml).

• Only by increasing the relative concentration of one antibody over the other (significantly) can one achieve “complete” inhibition of the other’s binding when both Ka1 and Ka2 are similar. 1 log (herein 1ug/ml) higher concentration of hyAb over that of rAb should begin to strongly favor hyAb binding, consistent with our data.

> We modified lines #302-309 and #577-585 to reflect this result.

Submitted filename: 2nd_Rebuttal letter.docx

Click here for additional data file.

30 Jul 2021

Determination of variable region sequences from hybridoma immunoglobulins that target Mycobacterium tuberculosis virulence factors

PONE-D-21-15036R2

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12 Aug 2021

PONE-D-21-15036R2

Determination of variable region sequences from hybridoma immunoglobulins that target Mycobacterium tuberculosis virulence factors

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Table 1

List of the 3’ isotype-specific primer (ISP) sequences in this study.

Name of 3’ISP*s	Isotype Targets (Mus musculus)	5’ to 3’ nucleic acid sequence
mIgG1	IgG1	CCGCTGGACAGGGATCCAGAGTTCCAGG
mIgG2a	IgG2a	CCACTGGACAGGGATCCAGAGTTCCAGG
mIgκ	Igκ	GGATACAGTTGGTGCAGCATCAGCCCG
mIgM	IgM	CAGGTGAAGGAAATGGTGCTGGGCAGG

*: 3’-isotype-specific primer.

Table 2

IgV sequences identified by NGS/bioinformatics and Sanger sequencing methods.

Items	Ig Subtypes	transcript tpm*	transcript read count	transcript avg coverage	TOPO-Sanger finds	IgV validated by recombinant ab: antigen-binding assay
SodANRC-13810	heavy, IgG1	963725.1311	88897.61877	10633.68646	Yes	True
	light, Igκ-1	39232.47749	3635.001071	883.7117677	Yes	True
	light, Igκ-2	304067.109	21428.2634	6339.72289	No	True
	light, Igκ-3	38357.25488	2533.438146	783.5375709	No	False No/little Secretion
	light, Igκ-4	13688.0569	849.329898	273.9773865	No	False No/little Secretion
PstS1NRC-2410	heavy, IgG1	968826.8725	40043.93523	9073.399221	No	True
	light, Igκ-DIV	843113.5427	20217.92773	6151.499309	Yes	True
	light, Igκ-S26	3807.603	74.34237	25.69437	No	False
KatGNRC-49680	heavy, IgM	610640.4988	17956.89359	4519.352413	No	True
	heavy, IgM_KVS	378156.7621	11071.18364	2795.753444	Yes	False No/little Secretion
	light, Igκ	453166.48	9730.178	2884.44	Yes	True
GroESNRC-2894	heavy, IgG2a	974745.1045	6680.858709	1397.669186	Yes	True
	light, Igκ	47876.02964	245.414241	74.97380071	Yes	True

*: transcripts per million transcripts.

Table 3

Summary of the deduced Ig amino acid sequence of the validated IgVH and IgVL sequences.

CDR/FR domains were defined via a KABAT-based algorithm*.

Hybridoma	Ig (GenBank#)	FR1	CDR1	FR2	CDR2	FR3	CDR3	FR4	Antigen
RC-2410	IgG1 (MW812375)	QVQLQQSGAELMKPGASVKISCKATGYTFS	GYWVE	WVKQRPGHGLEWIG	EILPGRVSTNYNEKFKA	KATFTADTSSNTAYMQLSSLTSEDSAVYYCAR	FKNYYGSSYNYFDY	WGQGTTLTVSS	PhoS1/PstS1
	Igκ-DIV (MW812376)	DIVLTQAAPSVPVTPGESLSISC	RSSKSLLHSNGNTYLY	WFLQRPGQSPQLLIY	RMSNLAS	GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYC	MQHLEYPYT	FGGGTKLEIK
NRC-13810	IgG1 (MW812377)	EVRLEESGGGLVLPGGSMKLSCVASGFTFN	NYWMN	WVRQSPEKGLEWVA	EIRLKSNNYATHYAESVKG	RFTISRDDSKGGVYLQMNNLRAEDTGIYYCTR	EANRGFAY	WGQGTLVTVSA	SodA
	Igκ-1 (MW812378)	KIVLTQSPASLAVSLRQRATISC	RASESVDSYGKSFMH	WYQQKSGQPPKLLIY	RASNLES	GVPARFSGSGSRTDFTLTIDPVEADDAATYYC	QQNYEAPRT	FGGGTKLEIK
	Igκ-2 (MW812379)	DIVLTQSPASLAVSLRQRATISC	RASESVDSYGKSFMH	WYQQKSGQPPKLLIY	RASNLES	GVPARFSGSGSRTDFTLTIDPVEADDAATYYC	QQNYEAPRT	FGGGTKLEIK
NRC-49680	IgM (MW812380)	QVQLKESGPGLVAPSQSLSITCTVSGFSLT	DYGVS	WIRQPPGKGLEWLG	VIWGGGSTYYNSALKS	RLSISKDNSKSQVFLKMNSLQTDDTAMYYCAK	HGNFAY	WGQGTLVTVSA	KatG
	Igκ (MW812381)	QIVLTQSPAIMSASLGERVTMTCTAS	SSVSSSY	LHWYQQKPGSSPKLWIY	STSNLAS	GVPARFSGSGSGTSYSLTISSMEAEDAATYYC	HQYHRSPWT	FGGGTKLEIK
NRC-2894	IgG2a (MW812373)	EVQLVESGGGLVQPKGSLKLSCAASGFTFK	TYAMN	WVRHTPGKGLEWVA	RIRSKSNNFATYYADSVKD	RFTISRDDSQSMLYLQMNNLKTEDTAMYYCVK	LTNGYFDS	WGQGTTLTVSS	GroES
	Igκ (MW812374)	DIQMTQSPSSLSASLGGKVTITC	KASQDINNYIA	WYQHKPGKGPRLLIH	DTSTLQP	GIPSRFSGSGSGRDYSFSISNLEPEDIATYYC	LQYDNLRT	FGGGTKVEIK

*S6 Table lists the CDR/FR gene family used for alignment.

Table 4

List and features of BEI resources M. tuberculosis hybridomas in this study.

BEI Resources Item No.	Clone #	Isotype	Antigen	Target gene	PMID
NRC-13810	CS-18	IgG1κ	SodA	Rv03846	24586151
NRC-49680	clone A	IgMκ	KatG	Rv1908c	n/a
NRC-2894	IT-3 (SA-12)	IgG2aκ	GroES	Rv3418c	n/a
NRC-2410	IT-15 (TB72)	IgG1κ	PhoS1/PstS1	Rv0934	n/a