Safe, effective, and inexpensive topical microbicides are urgently needed to curb the global human immunodeficiency virus type-1 (HIV-1) epidemic. Actinohivin (AH) is an actinomycete-derived lectin. This lectin specifically binds to high-mannose clusters uniquely found on the HIV-1 envelope (Env), thereby eliciting nanomolar antiviral activity against multiple HIV strains. Preliminary analyses revealed that AH has a high safety profile in human peripheral blood mononuclear cells (PBMCs) and in the rabbit vaginal irritation assay. Meanwhile, a translational AH-AH fusion protein (recombinant dimer [rd] AH) was suggested to have stronger and broader anti-HIV-1 activity than the original monomer. Given these high potentials, we hypothesize that rAH and/or rdAH (r/rdAH) are excellent HIV-1 microbicide candidates. This project's goal is to reveal the feasibilities of r/rdAH in terms of manufacture, antiviral efficacy, and safety upon use as a vaginal microbicide. In the R21 phase, we will initially focus on developing a highly efficient, scalable production system for r/rdAH that allows for extensive efficacy and safety studies and possible global use. We will utilize recombinant plant virus-based expression systems and various molecular biological approaches for rapid and high-level expression of high-quality r/rdAH. Upon obtaining bulk r/rdAH active pharmaceutical ingredients with high purity standards, we will analyze HIV-1 neutralization effects against selected R5-type viruses in two in vitro HIV neutralization assays based on Env-pseudotyped virus-reporter gene expression and primary isolate- PBMC infection systems. Next, r/rdAH'cytotoxic, mitogenic, and inflammatory potentials will be tested in PBMCs and/or human cervicovaginal (CV) epithelial cell lines to establish the minimal safety profile. Our success criteria in the R21 phase are: (1) establishing the bulk preparation procedure;(2) demonstrating cross- clade antiviral effects to R5 viruses;and (3) demonstrating no apparent in vitro cytotoxicity, mitogenic activity, or inflammatory potential at >100 times above an average anti-HIV IC50, for plant-made r/rdAH. Upon approval of our transition to the R33 phase, we will comprehensively analyze anti-HIV-1 efficacy of r/rdAH for various modes of HIV-1 infection and transmission, using various in vitro assay systems. In addition, we will investigate potential overlap, complementation, synergy, and antagonism of anti-HIV activities between r/rdAH and other inhibitors toward potential microbicide combination strategies. Finally, we will perform extensive evaluations of r/rdAH upon vaginal application in rabbit and mouse models. We will thoroughly evaluate r/rdAH'vaginal toxicity, inflammatory potential, and stability. Upon determining the maximal tolerated dose of r/rdAH, we will examine their potential immunogenicity and toxicity after a long-term exposure. Potential toxicity to the symbiotic vaginal commensal bacteria, the Lactobacillus species, will be examined. In summary, the proposed studies should answer the question of whether r/rdAH is justified for advanced next-stage preclinical studies. . The proposed studies will analyze the feasibilities of the novel HIV-1-binding lectin Actinohivin and its derivative recombinant dimer, as a candidate vaginal HIV-1 microbicide. The proposed studies should generate a comprehensive data set that will reveal their large-scale producibility, anti-HIV-1 efficacy, and broad toxicity profile upon vaginal application, thereby providing criteria of whether Actinohivin and its derivative are justified for further extensive preclinical and clinical studies.

Public Health Relevance

. The proposed studies will analyze the feasibilities of the novel HIV-1-binding lectin Actinohivin and its derivative recombinant dimer, as a candidate vaginal HIV-1 microbicide. The proposed studies should generate a comprehensive data set that will reveal their large-scale producibility, anti-HIV-1 efficacy, and broad toxicity profile upon vaginal application, thereby providing criteria of whether Actinohivin and its derivative are justified for further extensive preclinical and clinical studies.

Agency
National Institute of Health (NIH)
Type
Exploratory/Developmental Grants Phase II (R33)
Project #
5R33AI088585-05
Application #
8685097
Study Section
Special Emphasis Panel (ZAI1)
Program Officer
Turpin, Jim A
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Louisville
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
Louisville
State
KY
Country
United States
Zip Code
40202