Isolation of a nanobody specific to the PstS-1 protein and evaluation of its immunoreactivity with structural components of Mycobacterium tuberculosis granuloma

Yogesh P Dhekale^1,2Kumarasamy Jothivel¹Samar Kumar Ghorui³Gagan Deep Gupta¹Shweta Singh¹Nawab Singh Baghel¹Savita Kulkarni^1,2*Pramod Kumar Gupta^1,2*

• ¹Bhabha Atomic Research Centre (BARC), Mumbai, India
• ²Homi Bhabha National Institute, Mumbai, India
• ³ICAR-National Research Centre on Camel, Bikaner, India

Mycobacterium tuberculosis (Mtb) causes infectious granulomatous disease tuberculosis (TB), and existing in vitro TB-diagnosis is insensitive for extrapulmonary TB (EPTB) as well as paucibacillary TB due to low bacillary load, therefore, alternative non-invasive molecular imaging-based diagnostic tools are urgently required. Within TB granulomas, foci of Mtb secreted antigens anchored on the surface of either bacilli or host cells may serve as targetable biomarkers for antibody based molecular imaging of TB. Nanobody is better suited over conventional antibody or fragment derivatives for molecular imaging due to its quick localization in target tissue and rapid clearance from off-target organs. Here, we report the production of a high affinity nanobody against PstS-1 protein of Mtb which helps bacilli in phosphate uptake as well as host cell adhesion. C8 nanobody (C8Nb) was isolated from a phage displayed nanobody library which was constructed from a camel immunized with secreted proteins of Mtb. C8Nb was characterized in vitro and in vivo for immunoreactivity against PstS-1 protein. The ability of C8Nb to bind the PstS-1 protein, associated with the surface of Mtb bacilli or adhered on the macrophages, and its localization around BCG cells injected intramuscularly into mice, demonstrate its potential in the development of molecular imaging-based diagnostic tools for TB.