Sequence Determination of Hybridoma Antibody Transcripts Targeting Virulent or Immunogenic Factors of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), a causative agent for tuberculosis (TB), remains one of the most challenging pathogens to control. Mtb infects nearly a quarter of the world’s population and sinisterly synergizes with HIV to claim the lives of around 1/3 of all AIDS patients. While the WHO has considered this epidemic a global emergency since 1994, TB control has been hampered by lack of protective vaccines and a rapid, effective diagnostic tool. Only in recent years has TB antibody development offered new potential to control TB infections. The humoral antibody functions of TB immunity have been discovered and distinguishable antibody patterns in active/chronic stages of TB have been uncovered. Thus, knowledge of the paratope sequences of Mtb antibodies enables engineering diagnostic and therapeutic tools. Currently, few validated TB antibody sequences are available. Here, we identified the sequence of functional variable immunoglobulins (IgVs) expressed in 14 hybridomas encoding antibodies to Mtb targets with potential therapeutic/diagnostic value: (1) Mpt64, a mycobacterial diagnostic peptide; (2) Ag85 complex, the most immunogenic Mtb protein to date; (3) the Mtb bacterial surface components (A) glycolipid LAM, (B) lipoprotein LprG, and (C) HBHA, an epithelial cell adhesion factor; (4) the Mtb enzymes (A) Superoxide Dismutase SodA and (B) Catalase KatG, crucial for Mtb survival within the hostile macrophage phagosome; (5) the Mtb regulatory factors PhoS1/PstS1, factors within the ABC transporter system; and (6) the Mtb Heat Shock Proteins HspX, DnaK, and GroES. We isolated the paratope-determining CDR 1-3 regions of the heavy and light chains of these IgVs using a 5’RACE-PCR amplification from the cDNA of each hybridoma via an isotype (gene)-specific primer (GSP) for each of the light chains of Igk/Igl_1, 2, and 3 and heavy chains of IgG1, IgG2a, IgG3, and IgM. Using an Illumina^® NGS-MiSeq™, 2X150bp, pair-wise sequencing platform, we generated 28 IgV libraries. Most libraries contained sufficient reads and coverage for de novo assembly of Ig chains via a bioinformatics algorithm workflow for analysis of Ig sequences. Thirty-three (33) putative TB IgV sequences were identified. Validation of their antigen-binding capability via recombinant antibody techniques is in progress.