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Background <p>Rickettsiosis is among the deadliest vector-borne infectious diseases worldwide, in part because rickettsiae replicate within human cells, where antibodies and most drugs cannot effectively reach this obligatory intracellular pathogen. <i>Ehrlichia chaffeensis</i>, an emerging rickettsia, is the causative agent of human monocytic ehrlichiosis. We therefore aim to generate intrabodies (IBs), the variable domain of heavy chain of heavy-chain-only antibodies (VHHs) that bind intracellular bacterial proteins to inhibit <i>E. chaffeensis</i> infection.</p> Methods <p><i>E. chaffeensis</i> replicates in membrane-bound vacuoles resembling early endosomes in human monocytes/macrophages. The type IV secretion system effector <i>Ehrlichia</i> translocated factor-2 (Etf-2) directly binds to RAB5-GTP on <i>E. chaffeensis-</i>containing vacuoles. Consequently, Etf-2 hinders the engagement of RAB5 GTPase-activating protein with RAB5-GTP, delays maturation of <i>Ehrlichia</i> vacuoles to late endosomes, thus facilitates infection. As C-terminal half of Etf-2 (Etf-2C) binds RAB5-GTP, a random synthetic library of VHHs was screened for binding to Etf-2C, and for inhibition of Etf-2 binding to RAB5 in human cells when expressed intracellularly (IBs). Positive IBs were tested for inhibition of Etf-2 functions and <i>E. chaffeensis</i> infection, and lipid nanoparticles-encapsulated mRNAs (mRNAs-LNP) platform was used to deliver IBs in vitro and in mice.</p> Results <p>We have identified two distinct IBs that inhibit Etf-2 binding to RAB5 and Etf-2 functions in vitro. Synthesized mRNA-LNP encoding anti-Etf-2 IBs significantly inhibited <i>E. chaffeensis</i> infection in cell cultures and in a mouse model.</p> Conclusions <p>The results demonstrate the feasibility of mRNA-LNP encoding IBs as intracellular probes and a precision therapy addressing underlying cause of obligatory intracellular infection.</p>

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Development of mRNA–lipid nanoparticle intrabodies against rickettsial infection

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年8月12日

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  • Qi Yan,
  • Nan Duan,
  • Mingqun Lin,
  • Wenqing Zhang,
  • Stephen Denton,
  • Yichen Zhong,
  • Yizhou Dong,
  • Yasuko Rikihisa

摘要

Background

Rickettsiosis is among the deadliest vector-borne infectious diseases worldwide, in part because rickettsiae replicate within human cells, where antibodies and most drugs cannot effectively reach this obligatory intracellular pathogen. Ehrlichia chaffeensis, an emerging rickettsia, is the causative agent of human monocytic ehrlichiosis. We therefore aim to generate intrabodies (IBs), the variable domain of heavy chain of heavy-chain-only antibodies (VHHs) that bind intracellular bacterial proteins to inhibit E. chaffeensis infection.

Methods

E. chaffeensis replicates in membrane-bound vacuoles resembling early endosomes in human monocytes/macrophages. The type IV secretion system effector Ehrlichia translocated factor-2 (Etf-2) directly binds to RAB5-GTP on E. chaffeensis-containing vacuoles. Consequently, Etf-2 hinders the engagement of RAB5 GTPase-activating protein with RAB5-GTP, delays maturation of Ehrlichia vacuoles to late endosomes, thus facilitates infection. As C-terminal half of Etf-2 (Etf-2C) binds RAB5-GTP, a random synthetic library of VHHs was screened for binding to Etf-2C, and for inhibition of Etf-2 binding to RAB5 in human cells when expressed intracellularly (IBs). Positive IBs were tested for inhibition of Etf-2 functions and E. chaffeensis infection, and lipid nanoparticles-encapsulated mRNAs (mRNAs-LNP) platform was used to deliver IBs in vitro and in mice.

Results

We have identified two distinct IBs that inhibit Etf-2 binding to RAB5 and Etf-2 functions in vitro. Synthesized mRNA-LNP encoding anti-Etf-2 IBs significantly inhibited E. chaffeensis infection in cell cultures and in a mouse model.

Conclusions

The results demonstrate the feasibility of mRNA-LNP encoding IBs as intracellular probes and a precision therapy addressing underlying cause of obligatory intracellular infection.