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<p>Increase in tuberculosis (TB) mortality worldwide necessitates the development of more efficient new vaccines than the only approved TB vaccine, Bacillus Calmette Guérin (BCG), which is not effective in preventing adult pulmonary and latent TB. In this study, an optimal hairy root induction system was established in Indian cucumber cultivars. It was used to synthesize efficiently, for the first time, a novel tuberculosis subunit oral vaccine comprising two <i>Mycobacterium tuberculosis-</i>specific antigens, antigen 85B (Ag85B) and culture filtrate protein (CFP10) (absent in BCG), linked to the cholera toxin non-toxic B subunit (CTB) expressed as a single polypeptide. All the explants developed transgenic roots (100%), and 58.4% of them showed the presence of co-transformed desired transgenes by polymerase chain reaction (PCR), their stable integration and copy number by Southern blot analysis, transgene transcript levels by reverse transcriptase-PCR and quantitative real-time PCR, and the recombinant fusion protein expression by western blot analysis and quantification by enzyme-linked immunosorbent assay. The highest amount of a single polypeptide (CTB.CFP10.Ag85B) vaccine in transgenic roots was 692&#xa0;µg g<sup>-1</sup> fresh weight (0.34% of total soluble protein), which is substantially higher than that expressed previously in microbial and transgenic cucumber whole-plant systems (&lt; 0.2% total soluble protein). Oral immunization of Wistar rats with transgenic roots triggered a robust mucosal IgA response, surpassing that induced by BCG alone. Notably, a heterologous prime-boost strategy (BCG followed by an oral booster containing the synthesized vaccine) further enhanced systemic IgG and mucosal IgA immune responses. This study established the cucumber hairy root system as an efficient platform for synthesizing an oral, self-adjuvanted tuberculosis subunit vaccine that elicited enhanced humoral and systemic immune responses in vivo.</p>

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Cucumber hairy root cultures as an efficient platform for producing a self-adjuvanted Mycobacterium tuberculosis subunit vaccine

  • 期刊论文

  • 发表日期：

    2026年4月25日

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  • Supriya Phogat,
  • Yachna K. Jaiwal,
  • Darshna Chaudhary,
  • Ranjana Jaiwal,
  • Pawan K. Jaiwal

摘要

Increase in tuberculosis (TB) mortality worldwide necessitates the development of more efficient new vaccines than the only approved TB vaccine, Bacillus Calmette Guérin (BCG), which is not effective in preventing adult pulmonary and latent TB. In this study, an optimal hairy root induction system was established in Indian cucumber cultivars. It was used to synthesize efficiently, for the first time, a novel tuberculosis subunit oral vaccine comprising two Mycobacterium tuberculosis-specific antigens, antigen 85B (Ag85B) and culture filtrate protein (CFP10) (absent in BCG), linked to the cholera toxin non-toxic B subunit (CTB) expressed as a single polypeptide. All the explants developed transgenic roots (100%), and 58.4% of them showed the presence of co-transformed desired transgenes by polymerase chain reaction (PCR), their stable integration and copy number by Southern blot analysis, transgene transcript levels by reverse transcriptase-PCR and quantitative real-time PCR, and the recombinant fusion protein expression by western blot analysis and quantification by enzyme-linked immunosorbent assay. The highest amount of a single polypeptide (CTB.CFP10.Ag85B) vaccine in transgenic roots was 692 µg g-1 fresh weight (0.34% of total soluble protein), which is substantially higher than that expressed previously in microbial and transgenic cucumber whole-plant systems (< 0.2% total soluble protein). Oral immunization of Wistar rats with transgenic roots triggered a robust mucosal IgA response, surpassing that induced by BCG alone. Notably, a heterologous prime-boost strategy (BCG followed by an oral booster containing the synthesized vaccine) further enhanced systemic IgG and mucosal IgA immune responses. This study established the cucumber hairy root system as an efficient platform for synthesizing an oral, self-adjuvanted tuberculosis subunit vaccine that elicited enhanced humoral and systemic immune responses in vivo.