|Liqun Zhang, PhD|
Viral infections of the human respiratory tract are the leading causes of morbidity and hospitalization worldwide and are a major trigger for acute exacerbations in cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and asthma. Many viruses are able to persist in human airways, yet the extent and impact of viral persistence is unknown. Viruses are efficient machines -- to overcome both the mechanical mucociliary clearance and immune-mediated anti-viral defenses in the respiratory airways, viruses have acquired, through evolution, specific mechanisms that efficiently counter host immunity to ensure their survival. I am interested in understanding the nature of the interactions between the respiratory viruses and the respiratory epithelium, specifically, the underlying mechanisms responsible for viral adaptation and propagation in the airways. The long-term goal of this research is to take advantage of viruses’ unique “talents” by developing virus-based gene delivery vehicles for therapeutic gene replacement and vaccine/immune-modulatory applications benefitting human lung diseases.
Figure 1. Replacement of CFTR protein to ciliated airway epithelial cells from CF patients. Isolated scrapes of human ciliated airway epithelium derived from a cystic fibrosis (CF) patient, after in vitro gene delivery of CFTR using a novel viral vector system that targets human ciliated cells. The restored CFTR protein expression (red) is found at the lumenal surface of the ciliated epithelial cells where it regulates ion and fluid transport in the airways. The virus vector transduced cells are identified in green with virus specific antibodies. Cell nuclei are in blue. The replacement of CFTR in CF ciliated epithelial cells are found to fully correct CF-related pathophysiology in vitro. (Zhang L. et al. PLoS Biol. 2009 Jul;7(7):e1000155).
Gene transfer for CF lung disease
The large surface area of the respiratory airways and the easy accessibility for inhaled agents make the lungs ideal organs for therapeutic interventions. CF, a hereditary monogenic disease, was chosen as a testing platform to investigate the feasibility of virus-based gene transfer. Since both RSV and PIV3 target ciliated cells that express CFTR in vivo, we have engineered recombinant viruses expressing CFTR as an extra gene. Upon inoculation to the CF HAE, RSV-CFTR, in which CFTR was placed after L gene, completely restored the forskolin-sensitive chloride transport to non-CF level. PIV3CFTR, in which CFTR was inserted between HN and L genes, fully corrected the basic bioelectric defects of chloride and sodium ion transport in CF HAE and restored airway surface liquid volume regulation and mucus transport to levels approaching those of non-CF. Studies aimed at reducing virus-induced cytopathology and at increasing the duration of transgene expression are underway.
CFTR gene delivery with adeno-associated virus (AAV) vectors
In collaboration with Jude Samulski of UNC Gene Therapy Center, we are developing ways to improve AAV’s transduction efficiency for the respiratory epithelium. DNA shuffling combined with in vitro evolution on HAE has generated several novel AAV variants with superior transduction than any known AAV serotypes. These AAV variants expressing luciferase have delivered sustained transgene expression for up to 10 months in mouse nose in vivo. The novel AAVs expressing CFTR will soon be tested in the nasal epithelia of CF knockout mouse model. The measurement of the nasal electrical potential differences (an indicator of CFTR activity) should help establish the efficacy and duration of AAV-mediated correction. Furthermore, we are performing computer-assisted three-dimensional capsid structural modeling based on the known structure of AAV2 capsid to map key amino acid residues responsible for the enhanced transduction, with the goal to assist a rational design of optimal AAV vectors balancing enhanced transduction with efficient virus packaging.
Analysis of the human respiratory viromes to uncover novel respiratory viruses
Approximately half of all respiratory tract infections are of unknown etiology, with a majority of these thought to be caused by viruses. We hypothesize that human lungs are a reservoir harboring a large number of viruses including both pathogenic and commensal viruses which have not been characterized. Since more than 90% of viruses are uncultivable in cultures, we aim to directly sequence the genomes of uncultured viruses in human respiratory specimens such as the bronchoalveolar lavage (BAL) from CF, CF transplant, COPD, and healthy subjects. By combining the state-of-the-art high-throughput sequencing technology with cutting-edge bioinformatics tools, we expect to reveal the makeup and diversity of viral species in human respiratory tract and potentially to uncover new viruses infecting the human lungs. If successful, these findings will transform our understanding of the respiratory virus ecology in diseases and health, ultimately leading to better treatment and prevention strategies for respiratory tract infections.
|Video 1. Three-dimensional (3D) models of human airway epithelia infected with parainfluenza virus type 3 (PIV3). The video shows a 360 rotation of 3D reconstructed images of HAE infected with PIV3 at 2 days post inoculation. Viral infected cells are shown in green (GFP), and cilia were labeled red with -tubulin IV antibody. The z-stacks of x-y confocal fluorescence images were first captured, followed by 3D volume rendering (Volocity software).
Zhang L, Collins PL, Lamb RA, Pickles RJ. Comparison of differing cytopathic effects in human airway epithelium of parainfluenza virus 5 (W3A), parainfluenza virus type 3, and respiratory syncytial virus. Virology (accepted for publication).
Li W, Zhang L, Wu Z, Pickles RJ, Samulski RJ. AAV-6 mediated efficient transduction of mouse lower airways. Virology. 2011 Jul 11. [Epub ahead of print] PMID: 21752418.
Johnson JS, Gentzsch M, Zhang L, Ribeiro CMP, Kantor B, et al. (2011) AAV Exploits Subcellular Stress Associated with Inflammation, Endoplasmic Reticulum Expansion, and Misfolded Proteins in Models of Cystic Fibrosis. PLoS Pathog. 2011 May;7(5):e1002053. Epub 2011 May 19. PMID: 21625534; PMCID: PMC3098238.
Okada SF, Zhang L, Kreda SM, Abdullah LH, Davis CW, Pickles RJ, Lazarowski ER, Boucher RC. Coupled Nucleotide and Mucin Hypersecretion from Goblet Cell Metaplastic Human Airway Epithelium. Am J Respir Cell Mol Biol. 2011 Aug;45(2):253- 60. Epub 2010 Oct 8. PMID: 20935191; PMCID: PMC Journal – In Process.
Zhang L, Limberis M, Thompson C, Antunes MB, Luongo C, Wilson JM, Collins PL, Pickles RJ. Alpha-Fetoprotein Gene Delivery to the Nasal Epithelium of Nonhuman Primates by Human Parainfluenza Virus Vectors. Hum Gene Ther. 2010 Dec;21(12):1657-64. Epub 2010 Nov 3. PMID: 20735256; PMCID: PMC2999572. Feature Cover in Human Gene Therapy, December 2010, Volume 21, Number 12.
Kwilas AR, Yednak MA, Zhang L, Liesman R, Collins PL, Pickles RJ, Peeples ME. Respiratory Syncytial Virus Engineered to Express CFTR Corrects the Bioelectric Phenotype of Human Cystic Fibrosis Airway Epithelium In Vitro. J Virol. 2010 Aug;84(15):7770-81. Epub 2010 May 26. PMID: 20504917; PMCID: PMC2897634.
Feature Cover in Journal of Virology, November 2010, Volume 84, Issue 21.
Li W, Zhang L, Johnson JS, Wu Z, Grieger JC, Xiao P-J, Drouin LM, Agbandje- McKenna M, Pickles RJ, and Samulski RJ. Generation of novel AAV variants by
directed evolution for improved CFTR delivery to human ciliated airway epithelium. Mol Ther. 2009 Dec;17(12):2067-77. Epub 2009 Jul 14. PMID: 19603002; PMCID: PMC2801879.
Kwilas S, Liesman RM, Zhang L, Walsh E, Pickles RJ, and Peeples ME. Respiratory syncytial virus grown in vero cells contains a truncated attachment protein that alters its infectivity and dependence on glycosaminoglycans. J Virol. 2009 Oct;83(20):10710-8. Epub 2009 Aug 5. PMID: 19656891; PMCID: PMC2753119. SPOTLIGHT: Featured article of significant interest selected by the editors.
Zhang L, Button B, Gabriel SE, Burkett S, Yan Y, Skiadopoulos MH, Dang YL, Vogel LN, McKay T, Mengos A, Boucher RC, Collins PL, Pickles RJ. CFTR delivery to 25% of surface epithelial cells restores normal rates of mucus transport to human cystic fibrosis airway epithelium. PLoS Biol. 2009 Jul;7(7):e1000155. Epub 2009 Jul 21. PMID: 19621064; PMCID: PMC2705187. SYNOPSIS: Meadows R. Engineered common cold virus helps cultured cystic fibrosis tissues clear mucus. PLoS Biol. 2009 Jul;7(7):e1000155. PubMed PMID: 20076750; PMCID: PMC2704862. Feature Cover in PLoS Biology July 2009, Issue 7(7).
Limberis MP, Vandenberghe LH, Zhang L, Pickles RJ, and Wilson JM. Transduction efficiencies of novel AAV vectors in mouse airway epithelium In vivo and human ciliated airway epithelium in vitro. Mol Ther. 2009 Feb;17(2):294-301. Epub 2008 Dec 9. PMID: 19066597; PMCID: PMC2835069.
Bukreyev A, Marzi A, Feldmann F, Zhang L, Yang L, Ward JM, Dorward DW, Pickles RJ, Murphy BR, Feldmann H, Collins PL. Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge. Virology. 2009 Jan 20;383(2):348-61. Epub 2008 Nov 17. PMID: 19010509; PMCID: PMC2649782.
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