# Inhibition of Ehrlichial Infection by Intracellular Nanobody

> **NIH NIH R21** · OHIO STATE UNIVERSITY · 2020 · $195,000

## Abstract

Rickettsia cause life-threatening vector-borne diseases, including Anaplasmosis and Ehrlichiosis, which are
greatly increasing in worldwide prevalence. There are two major barriers to progress for the development of
effective therapies and prophylactic measures for diseases caused by these obligatory intracellular bacteria: 1)
the important disease-associated bacterial and host molecules are intracellular, protected from direct antibody
(Ab) or drug attack, and 2) bacteria-specific lethal targets are difficult to identify because conventional
approaches, such as knockout mutants of essential genes for obligatory intracellular infection, are not feasible.
Ehrlichia chaffeensis (Ech) is a classic example. The Type IV Secretion System (T4SS) is conserved among all
Rickettsial organisms and is essential for host infection. The recent elucidation of the Ech T4SS effectors
Ehrlichial translocated factors 1 and 2 (Etf-1 and Etf-2) provide critical targets for innovative molecular
approaches. Nanobodies are the smallest intact antigen-binding fragments (VHH) derived from heavy chain-
only antibodies in camelids. Nanobodies are proteolytically stable and biologically active in reduced
intracellular environments; therefore, they have greater potential as therapeutic agents in Ech infection and
research tools than conventional antibodies. VHH can be cloned into a mammalian expression plasmid, and
expressed intracellularly at high levels in heterologous systems. Indeed, we showed transfection of VHH that
binds human heterogeneous nuclear ribonucleoprotein K can block Ech infection in human cells. The goal of
the proposed research is to carry out a pilot study to develop and characterize Ech infection-blocking
intracellular nanobodies for future therapeutic application. We will achieve this goal through two aims. Aim 1 is
to isolate and characterize Etf-1 and Etf-2-specific iAbs that inhibit Ech infection by: 1) cloning VHH cDNA from
Etf-1 and Etf-2-immunized llama lymphocyte mRNA into a phage display library and isolating Etf-1 and Etf-2
antigen-specific VHHs by panning; 2) cloning Etf-1 and Etf-2-specific VHHs into a mammalian expression
vector to produce iAbs by transfection; 3) identifying Ech infection-blocking iAbs and determining their amino
acid sequences; and 4) analyzing the mechanism(s) by which the iAbs inhibit Etf-1, -2 functions and block Ech
infection. Aim 2 is to deliver selected VHH peptides conjugated with cell penetrating peptide (VHH-CPP) to
block Ech infection by: 1) optimizing intracellular delivery of Ech infection-blocking VHH-CPP into human cells
in vitro; 2) testing the efficacy of selected VHH-CPP for inhibition of Ech infection in vitro; and 3) analyzing
VHH-CPP pharmacokinetics, toxicity, and efficacy for inhibiting Ech infection in mice. Our results will
demonstrate the novel use of nanobody technology and CPP to overcome current barriers to advance basic
and translational research on obligatory intracellular infection. Eventuall...

## Key facts

- **NIH application ID:** 9942364
- **Project number:** 5R21AI146736-02
- **Recipient organization:** OHIO STATE UNIVERSITY
- **Principal Investigator:** YASUKO RIKIHISA
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $195,000
- **Award type:** 5
- **Project period:** 2019-06-04 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9942364

## Citation

> US National Institutes of Health, RePORTER application 9942364, Inhibition of Ehrlichial Infection by Intracellular Nanobody (5R21AI146736-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9942364. Licensed CC0.

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