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

Hui-Chen Chang Foreman,Andrew Frank,Timothy T. Stedman,Frederick Quinn

doi:10.1371/journal.pone.0256079

Hui-Chen Chang Foreman, Andrew Frank + Show 2 more

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https://doi.org/10.1371/journal.pone.0256079

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Journal: PloS one	Publication Date: Aug 20, 2021
Citations: 1	License type: CC BY 4.0

Affiliation: American Type Culture Collection

Abstract

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.

Highlights

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
5’ Rapid Amplification of cDNA End (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

Summary

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]. As with other bacterial pathogens under antibiotic pressure, the incidence of multi-drug and extensively drug-resistant Mtb is on the rise [6]. Recent evidence points to humoral antibody (Ab) correlating well with TB susceptibility, suggesting recombinant Ab against Mtb are viable therapeutic strategies [7,8,9,10,11,12,13,14].

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