Melissa A. Hickey

Lentivirus Vectors Pseudotyped with Filoviral Envelope Glycoproteins Transduce Airway Epithelia from the Apical Surface Independently of Folate Receptor Alpha

ABSTRACT The practical application of gene therapy as a treatment for cystic fibrosis is limited by poor gene transfer efficiency with vectors applied to the apical surface of airway epithelia. Recently, folate receptor alpha (FRα), a glycosylphosphatidylinositol-linked surface protein, was reported to be a cellular receptor for the filoviruses. We found that polarized human airway epithelia expressed abundant FRα on their apical surface. In an attempt to target these apical receptors, we pseudotyped feline immunodeficiency virus (FIV)-based vectors by using envelope glycoproteins (GPs) from the filoviruses Marburg virus and Ebola virus. Importantly, primary cultures of well-differentiated human airway epithelia were transduced when filovirus GP-pseudotyped FIV was applied to the apical surface. Furthermore, by deleting a heavily O-glycosylated extracellular domain of the Ebola GP, we improved the titer of concentrated vector severalfold. To investigate the folate receptor dependence of gene transfer with the filovirus pseudotypes, we compared gene transfer efficiency in immortalized airway epithelium cell lines and primary cultures. By utilizing phosphatidylinositol-specific phospholipase C (PI-PLC) treatment and FRα-blocking antibodies, we demonstrated FRα-dependent and -independent entry by filovirus glycoprotein-pseudotyped FIV-based vectors in airway epithelia. Of particular interest, entry independent of FRα was observed in primary cultures of human airway epithelia. Understanding viral vector binding and entry pathways is fundamental for developing cystic fibrosis gene therapy applications.

Persistent Gene Expression in Mouse Nasal Epithelia following Feline Immunodeficiency Virus-Based Vector Gene Transfer

ABSTRACT Gene transfer development for treatment or prevention of cystic fibrosis lung disease has been limited by the inability of vectors to efficiently and persistently transduce airway epithelia. Influenza A is an enveloped virus with natural lung tropism; however, pseudotyping feline immunodeficiency virus (FIV)-based lentiviral vector with the hemagglutinin envelope protein proved unsuccessful. Conversely, pseudotyping FIV with the envelope protein from influenza D (Thogoto virus GP75) resulted in titers of 106 transducing units (TU)/ml and conferred apical entry into well-differentiated human airway epithelial cells. Baculovirus GP64 envelope glycoproteins share sequence identity with influenza D GP75 envelope glycoproteins. Pseudotyping FIV with GP64 from three species of baculovirus resulted in titers of 107 to 109 TU/ml. Of note, GP64 from Autographa californica multicapsid nucleopolyhedrovirus resulted in high-titer FIV preparations (∼109 TU/ml) and conferred apical entry into polarized primary cultures of human airway epithelia. Using a luciferase reporter gene and bioluminescence imaging, we observed persistent gene expression from in vivo gene transfer in the mouse nose with A. californica GP64-pseudotyped FIV (AcGP64-FIV). Longitudinal bioluminescence analysis documented persistent expression in nasal epithelia for ∼1 year without significant decline. According to histological analysis using a LacZ reporter gene, olfactory and respiratory epithelial cells were transduced. In addition, methylcellulose-formulated AcGP64-FIV transduced mouse nasal epithelia with much greater efficiency than similarly formulated vesicular stomatitis virus glycoprotein-pseudotyped FIV. These data suggest that AcGP64-FIV efficiently transduces and persistently expresses a transgene in nasal epithelia in the absence of agents that disrupt the cellular tight junction integrity.

Lethal Infection of K18-hACE2 Mice Infected with Severe Acute Respiratory Syndrome Coronavirus

ABSTRACT The severe acute respiratory syndrome (SARS), caused by a novel coronavirus (SARS-CoV), resulted in substantial morbidity, mortality, and economic losses during the 2003 epidemic. While SARS-CoV infection has not recurred to a significant extent since 2003, it still remains a potential threat. Understanding of SARS and development of therapeutic approaches have been hampered by the absence of an animal model that mimics the human disease and is reproducible. Here we show that transgenic mice that express the SARS-CoV receptor (human angiotensin-converting enzyme 2 [hACE2]) in airway and other epithelia develop a rapidly lethal infection after intranasal inoculation with a human strain of the virus. Infection begins in airway epithelia, with subsequent alveolar involvement and extrapulmonary virus spread to the brain. Infection results in macrophage and lymphocyte infiltration in the lungs and upregulation of proinflammatory cytokines and chemokines in both the lung and the brain. This model of lethal infection with SARS-CoV should be useful for studies of pathogenesis and for the development of antiviral therapies.

ACE2 Receptor Expression and Severe Acute Respiratory Syndrome Coronavirus Infection Depend on Differentiation of Human Airway Epithelia

ABSTRACT Studies of patients with severe acute respiratory syndrome (SARS) demonstrate that the respiratory tract is a major site of SARS-coronavirus (CoV) infection and disease morbidity. We studied host-pathogen interactions using native lung tissue and a model of well-differentiated cultures of primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2), the receptor for both the SARS-CoV and the related human respiratory coronavirus NL63, was expressed in human airway epithelia as well as lung parenchyma. As assessed by immunofluorescence staining and membrane biotinylation, ACE2 protein was more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia. Interestingly, ACE2 expression positively correlated with the differentiation state of epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected. Expression of ACE2 in poorly differentiated epithelia facilitated SARS spike (S) protein-pseudotyped virus entry. Consistent with the expression pattern of ACE2, the entry of SARS-CoV or a lentivirus pseudotyped with SARS-CoV S protein in differentiated epithelia was more efficient when applied to the apical surface. Furthermore, SARS-CoV replicated in polarized epithelia and preferentially exited via the apical surface. The results indicate that infection of human airway epithelia by SARS coronavirus correlates with the state of cell differentiation and ACE2 expression and localization. These findings have implications for understanding disease pathogenesis associated with SARS-CoV and NL63 infections.

Apical barriers to airway epithelial cell gene transfer with amphotropic retroviral vectors

Gene transfer to airway epithelia with amphotropic pseudotyped retroviral vectors is inefficient following apical vector application. To better understand this inefficiency, we localized the expression of Pit2, the amphotropic receptor, in polarized human airway epithelia. Pit2 was expressed on both the apical and basolateral surfaces of the cells, suggesting that factors other than receptor abundance may limit apical gene transfer efficiency. Binding studies performed with radiolabeled amphotropic MuLV suggested that the apically applied virus binds to Pit2. Hypothetical barriers to retroviral gene transfer include the apical glycocalyx and other secreted products of epithelia. In this study, we demonstrated that sialic acid, keratan sulfate and collagen type V are present on the apical surface of well-differentiated human airway epithelia. While enzyme treatment reduced the abundance of these components, the treatment also decreased the transepithelial resistance to ~35% of the controls, suggesting that the epithelial integrity was impaired. To attain an airway epithelial culture with a modified apical surface and intact epithelial integrity, we utilized 100 mM 2-deoxy-D-glucose, a glycosylation inhibitor, to prevent the glycocalyx from reforming following enzyme treatment. This approach allowed the resistance, but not the apical glycocalyx to recover. Despite this physical modification of the cell surface, the amphotropic retroviral vector failed to transduce airway epithelia following apical application. These results suggest that factors other than apical receptor abundance and the glycocalyx inhibit amphotropic retroviral gene transfer in human airway epithelia.

Infection of Human Airway Epithelia by Sars Coronavirus is Associated with ACE2 Expression and Localization

Severe acute respiratory syndrome (SARS) emerged as a regional and global health threat in 2002–2003 resulting in approximately 800 1 deaths. An intense, cooperative worldwide effort rapidly led to the identification of the disease causing agent as a novel coronavirus (SARS-CoV) and the subsequent complete sequencing of the viral genome. Although limited human pathological studies demonstrate that the respiratory tract is a major site of SARS-CoV infection and morbidity, little is known regarding the initial steps in SARSCoV-host cell interactions in the respiratory tract, such as the cell types in which primary viral infection and replication occur. Angiotensin converting enzyme 2 (ACE2) was identified as a receptor for both SARS-CoV and NL63. ACE2 is a membrane-associated aminopeptidase expressed in vascular endothelia, renal and cardiovascular tissues, and epithelia of the small intestine, and testes. and lysine 353 and proximal residues of the N-terminus of β-sheet 5 interacts with high affinity to the receptor binding domain of the SARS-CoV S glycoprotein. Several unanswered questions remain regarding ACE2 expression in human respiratory epithelia and its role as a receptor for SARS-CoV, including identification of the specific epithelial cell types expressing ACE2, the polarity of ACE2 expression, and whether SARS-CoV infection of respiratory epithelia is ACE2-dependent.

100. Development of an Integrase Deficient FIV Vector for Transient Gene Expression

Lentiviral vectors mediate long term transgene expression through integration into host genomic DNA. However, integration comes with potential risks of insertional mutagenesis. In cases where transient gene expression is desired, an integrase (IN) deficient retroviral vector might be a useful alternative. Furthermore, IN deficient vectors may provide some advantages over alternative methods of transient gene expression due to the ability to pseudotype these vectors with heterologous envelopes that target specific cell types. Class I IN mutants have point mutations in the catalytic triad amino acids (DX39-58 DX35 E) of IN that disable this protein but leave other viral proteins functional. Transient gene expression from IN deficient vectors is mediated from episomal forms of vector DNA. 1-LTR and 2-LTR circles form through homologous recombination and non-homologous end joining, respectively, of the linear form of the reverse transcribed viral DNA. Although 1-LTR and 2-LTR circles are unable to efficiently integrate in the presence of functional IN protein, they can mediate transgene expression. It was previously reported that IN deficient HIV and FIV vectors could mediate transient gene transfer. In growth arrested cells, transgene expression was maintained. However, in rapidly dividing cells transgene expression was lost, and duration of expression was related to the rate of cell division. Because some gene transfer targets are mitotically quiescent and slowly dividing cells, IN deficient mutants may promote long term, though ultimately transient, gene expression. We hypothesized that IN deficient FIV vectors can be used to maintain transient gene expression in slowly dividing cell types, such as airway epithelia. We developed IN deficient FIV vectors with single (D66V, D118A, E154G), double (D66V/D118A, D66V/E154G, D118A/E154G), and triple (D66V/D118A/E154G) mutations that disrupt the catalytic triad of the IN protein. Preliminary data indicate that the FIV D66V IN mutant has titers similar to wild type IN vectors. Furthermore, we documented the production of 1-LTR and 2-LTR circles in human cell line cultures transduced with FIV D66V IN deficient vector. Ongoing studies are comparing the persistence of transgene expression in IN deficient versus wild type IN FIV in dividing and non-dividing cells. Furthermore, to ensure that transgene expression seen in IN deficient vectors represents episomal and not integrated vector DNA, Southern blotting will be used. IN deficient lentiviral vectors may have applications for transient gene expression, long term expression in slowly dividing cells, and can be used as controls in studies with integration competent vectors.

688. Pseudotyping FIV-based lentiviral vectors with baculovirus GP64 confers apical entry into airway epithelia

Top of pageAbstract The use of gene transfer to treat or prevent cystic fibrosis lung disease has been limited in part by the inability of vectors to efficiently transduce airway epithelia from the apical surface. Using a feline immunodeficiency virus (FIV)-based lentiviral vector system, we recently observed that the envelope glycoprotein GP64 from baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) confers apical entry into polarized primary cultures of human airway epithelia. High titer FIV-vector (>10e9 TU/ml) was achieved by pseudotyping with baculovirus GP64 (GP64-FIV) and these titers meet or exceed those obtained with the VSV-G envelope. We tested fusion (F) proteins from four other baculoviruses for their ability to pseudotype FIV, and obtained very low titers (less than or equal to 10e2). Interestingly, AcMNPV GP64 shares sequence identity with influenza D envelope GPs such as Thogotovirus. Pseudotyping FIV with Thogoto GP resulted in titers of ~10e6 TU/ml and also conferred apical entry into polarized human airway epithelial cell cultures. These data lend support to the notion of horizontal transfer of GPs during the evolution of AcMNPV and Influenza D viruses. The receptor for GP64 is currently unknown; however, we investigated the entry of GP64-FIV into epithelia. Pretreating A549 cells with the ionophores monensin or NH4Cl significantly decreased the transduction by GP64-FIV as measured by beta-Galactosidase expression. These data suggest that similar to VSV-G, GP64-FIV transduction requires a low pH endosome pathway. These findings identify a pseudotyped, integrating viral vector with the capacity to infect from the apical surface for the convenient delivery of transgenes to airway epithelia.