Spatial distribution of Mycobacterium tuberculosis mRNA and secreted antigens in acid-fast negative human antemortem and resected tissue.

BACKGROUND: The ability to detect evidence of Mycobacterium tuberculosis (Mtb) infection within human tissues is critical to the study of Mtb physiology, tropism, and spatial distribution within TB lesions. The capacity of the widely-used Ziehl-Neelsen (ZN) staining method for identifying Mtb acid-fast bacilli (AFB) in tissue is highly variable, which can limit detection of Mtb bacilli for research and diagnostic purposes. Here, we sought to circumvent these limitations via detection of Mtb mRNA and secreted antigens in human tuberculous tissue. METHODS: We adapted RNAscope, an RNA in situ hybridisation (RISH) technique, to detect Mtb mRNA in ante- and postmortem human TB tissues and developed a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B). FINDINGS: We identified Mtb mRNA within intact and disintegrating bacilli as well as extrabacillary mRNA. Mtb mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found Mtb mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchiolar epithelium. Intriguingly, we observed accumulation of Mtb mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. Mtb mRNA was observed in ZN-negative antemortem lymph node biopsies. INTERPRETATION: RNAscope and dual ZN/immunohistochemistry staining are well-suited for identifying subsets of intact Mtb and/or bacillary remnants in human tissue. RNAscope can identify Mtb mRNA in ZN-negative tissues from patients with TB and may have diagnostic potential in complex TB cases. FUNDING: Wellcome Leap Delta Tissue Program, Wellcome Strategic Core Award, the National Institutes of Health (NIH, USA), the Mary Heersink Institute for Global Health at UAB, the UAB Heersink School of Medicine.

Detection of Mycobacterium tuberculosis in human tissue via RNA in situ hybridization.

Rationale: Accurate TB diagnosis is hampered by the variable efficacy of the widely-used Ziehl-Neelsen (ZN) staining method to identify Mycobacterium tuberculosis ( Mtb ) acid-fast bacilli (AFB). Here, we sought to circumvent this current limitation through direct detection of Mtb mRNA. Objectives: To employ RNAscope to determine the spatial distribution of Mtb mRNA within tuberculous human tissue, to appraise ZN-negative tissue from confirmed TB patients, and to provide proof-of-concept of RNAscope as a platform to inform TB diagnosis and Mtb biology. Methods: We examined ante- and postmortem human TB tissue using RNAscope to detect Mtb mRNA and a dual ZN/immunohistochemistry staining approach to identify AFB and bacilli producing antigen 85B (Ag85B). Measurements and main results: We adapted RNAscope for Mtb and identified intact and disintegrated Mtb bacilli and intra- and extracellular Mtb mRNA. Mtb mRNA was distributed zonally within necrotic and non-necrotic granulomas. We also found Mtb mRNA within, and adjacent to, necrotic granulomas in ZN-negative lung tissue and in Ag85B-positive bronchial epithelium. Intriguingly, we observed accumulation of Mtb mRNA and Ag85B in the cytoplasm of host cells. Notably, many AFB were negative for Ag85B staining. Mtb mRNA was observed in ZN-negative antemortem lymph node biopsies. Conclusions: RNAscope has diagnostic potential and can guide therapeutic intervention as it detects Mtb mRNA and morphology in ZN-negative tissues from TB patients, and Mtb mRNA in ZN-negative antemortem biopsies, respectively. Lastly, our data provide evidence that at least two phenotypically distinct populations of Mtb bacilli exist in vivo .

A high-resolution 3D atlas of the spectrum of tuberculous and COVID-19 lung lesions.

Our current understanding of the spectrum of TB and COVID-19 lesions in the human lung is limited by a reliance on low-resolution imaging platforms that cannot provide accurate 3D representations of lesion types within the context of the whole lung. To characterize TB and COVID-19 lesions in 3D, we applied micro/nanocomputed tomography to surgically resected, postmortem, and paraffin-embedded human lung tissue. We define a spectrum of TB pathologies, including cavitary lesions, calcium deposits outside and inside necrotic granulomas and mycetomas, and vascular rearrangement. We identified an unusual spatial arrangement of vasculature within an entire COVID-19 lobe, and 3D segmentation of blood vessels revealed microangiopathy associated with hemorrhage. Notably, segmentation of pathological anomalies reveals hidden pathological structures that might otherwise be disregarded, demonstrating a powerful method to visualize pathologies in 3D in TB lung tissue and whole COVID-19 lobes. These findings provide unexpected new insight into the spatial organization of the spectrum of TB and COVID-19 lesions within the framework of the entire lung.

Mycobacterium tuberculosis DosS binds H2S through its Fe3+ heme iron to regulate the DosR dormancy regulon.

Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we test the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KDapp = 5.30 µM) but not the ferrous (Fe2+) form. No interaction with DosT(Fe2+-O2) was detected. We found that the binding of sulfide can slowly reduce the DosS heme iron to the ferrous form. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce DosR regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS(Fe3+). These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase.

Mycobacterium tuberculosis H2S Functions as a Sink to Modulate Central Metabolism, Bioenergetics, and Drug Susceptibility.

H2S is a potent gasotransmitter in eukaryotes and bacteria. Host-derived H2S has been shown to profoundly alter M. tuberculosis (Mtb) energy metabolism and growth. However, compelling evidence for endogenous production of H2S and its role in Mtb physiology is lacking. We show that multidrug-resistant and drug-susceptible clinical Mtb strains produce H2S, whereas H2S production in non-pathogenic M. smegmatis is barely detectable. We identified Rv3684 (Cds1) as an H2S-producing enzyme in Mtb and show that cds1 disruption reduces, but does not eliminate, H2S production, suggesting the involvement of multiple genes in H2S production. We identified endogenous H2S to be an effector molecule that maintains bioenergetic homeostasis by stimulating respiration primarily via cytochrome bd. Importantly, H2S plays a key role in central metabolism by modulating the balance between oxidative phosphorylation and glycolysis, and it functions as a sink to recycle sulfur atoms back to cysteine to maintain sulfur homeostasis. Lastly, Mtb-generated H2S regulates redox homeostasis and susceptibility to anti-TB drugs clofazimine and rifampicin. These findings reveal previously unknown facets of Mtb physiology and have implications for routine laboratory culturing, understanding drug susceptibility, and improved diagnostics.

Micro-Computed Tomography Analysis of the Human Tuberculous Lung Reveals Remarkable Heterogeneity in Three-dimensional Granuloma Morphology.

Rationale: Our current understanding of tuberculosis (TB) pathophysiology is limited by a reliance on animal models, the paucity of human TB lung tissue, and traditional histopathological analysis, a destructive two-dimensional approach that provides limited spatial insight. Determining the three-dimensional (3D) structure of the necrotic granuloma, a characteristic feature of TB, will more accurately inform preventive TB strategies.Objectives: To ascertain the 3D shape of the human tuberculous granuloma and its spatial relationship with airways and vasculature within large lung tissues.Methods: We characterized the 3D microanatomical environment of human tuberculous lungs by using micro computed tomography, histopathology, and immunohistochemistry. By using 3D segmentation software, we accurately reconstructed TB granulomas, vasculature, and airways in three dimensions and confirmed our findings by using histopathology and immunohistochemistry.Measurements and Main Results: We observed marked heterogeneity in the morphology, volume, and number of TB granulomas in human lung sections. Unlike depictions of granulomas as simple spherical structures, human necrotic granulomas exhibit complex, cylindrical, branched morphologies that are connected to the airways and shaped by the bronchi. The use of 3D imaging of human TB lung sections provides unanticipated insight into the spatial organization of TB granulomas in relation to the airways and vasculature.Conclusions: Our findings highlight the likelihood that a single, structurally complex lesion could be mistakenly viewed as multiple independent lesions when evaluated in two dimensions. In addition, the lack of vascularization within obstructed bronchi establishes a paradigm for antimycobacterial drug tolerance. Lastly, our results suggest that bronchogenic spread of Mycobacterium tuberculosis reseeds the lung.

Hydrogen sulfide stimulates Mycobacterium tuberculosis respiration, growth and pathogenesis.

Hydrogen sulfide (H2S) is involved in numerous pathophysiological processes and shares overlapping functions with CO and •NO. However, the importance of host-derived H2S in microbial pathogenesis is unknown. Here we show that Mtb-infected mice deficient in the H2S-producing enzyme cystathionine ß-synthase (CBS) survive longer with reduced organ burden, and that pharmacological inhibition of CBS reduces Mtb bacillary load in mice. High-resolution respirometry, transcriptomics and mass spectrometry establish that H2S stimulates Mtb respiration and bioenergetics predominantly via cytochrome bd oxidase, and that H2S reverses •NO-mediated inhibition of Mtb respiration. Further, exposure of Mtb to H2S regulates genes involved in sulfur and copper metabolism and the Dos regulon. Our results indicate that Mtb exploits host-derived H2S to promote growth and disease, and suggest that host-directed therapies targeting H2S production may be potentially useful for the management of tuberculosis and other microbial infections.

Microanatomic Distribution of Myeloid Heme Oxygenase-1 Protects against Free Radical-Mediated Immunopathology in Human Tuberculosis.

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that controls inflammatory responses and redox homeostasis; however, its role during pulmonary tuberculosis (TB) remains unclear. Using freshly resected human TB lung tissue, we examined the role of HO-1 within the cellular and pathological spectrum of TB. Flow cytometry and histopathological analysis of human TB lung tissues showed that HO-1 is expressed primarily in myeloid cells and that HO-1 levels in these cells were directly proportional to cytoprotection. HO-1 mitigates TB pathophysiology by diminishing myeloid cell-mediated oxidative damage caused by reactive oxygen and/or nitrogen intermediates, which control granulocytic karyorrhexis to generate a zonal HO-1 response. Using whole-body or myeloid-specific HO-1-deficient mice, we demonstrate that HO-1 is required to control myeloid cell infiltration and inflammation to protect against TB progression. Overall, this study reveals that zonation of HO-1 in myeloid cells modulates free-radical-mediated stress, which regulates human TB immunopathology.

Ergothioneine Maintains Redox and Bioenergetic Homeostasis Essential for Drug Susceptibility and Virulence of Mycobacterium tuberculosis.

The mechanisms by which Mycobacterium tuberculosis (Mtb) maintains metabolic equilibrium to survive during infection and upon exposure to antimycobacterial drugs are poorly characterized. Ergothioneine (EGT) and mycothiol (MSH) are the major redox buffers present in Mtb, but the contribution of EGT to Mtb redox homeostasis and virulence remains unknown. We report that Mtb WhiB3, a 4Fe-4S redox sensor protein, regulates EGT production and maintains bioenergetic homeostasis. We show that central carbon metabolism and lipid precursors regulate EGT production and that EGT modulates drug sensitivity. Notably, EGT and MSH are both essential for redox and bioenergetic homeostasis. Transcriptomic analyses of EGT and MSH mutants indicate overlapping but distinct functions of EGT and MSH. Last, we show that EGT is critical for Mtb survival in both macrophages and mice. This study has uncovered a dynamic balance between Mtb redox and bioenergetic homeostasis, which critically influences Mtb drug susceptibility and pathogenicity.

Fiber-modified adenovirus for central nervous system Parkinson's disease gene therapy.

Gene-based therapies for neurological diseases continue to develop briskly. As disease mechanisms are elucidated, flexible gene delivery platforms incorporating transcriptional regulatory elements, therapeutic genes and targeted delivery are required for the safety and efficacy of these approaches. Adenovirus serotype 5 (Ad5)-based vectors can carry large genetic payloads to provide this flexibility, but do not transduce neuronal cells efficiently. To address this, we have developed a tropism-modified Ad5 vector with neuron-selective targeting properties for evaluation in models of Parkinson disease therapy. A panel of tropism-modified Ad5 vectors was screened for enhanced gene delivery in a neuroblastoma cell line model system. We used these observations to design and construct an unbiased Ad vector platform, consisting of an unmodified Ad5 and a tropism-modified Ad5 vector containing the fiber knob domain from canine Ad serotype 2 (Ad5-CGW-CK2). Delivery to the substantia nigra or striatum showed that this vector produced a neuronally-restricted pattern of gene expression. Many of the transduced neurons were from regions with afferent projections to the injection site, implicating that the vector binds the presynaptic terminal resulting in presynaptic transduction. We show that Ad5-CGW-CK2 can selectively transduce neurons in the brain and hypothesize that this modular platform is potentially adaptable to clinical use.

An adenovirus vector incorporating carbohydrate binding domains utilizes glycans for gene transfer.

BACKGROUND: Vectors based on human adenovirus serotype 5 (HAdV-5) continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting. METHODOLOGY/PRINCIPAL FINDINGS: As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4). This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells. CONCLUSIONS/SIGNIFICANCE: These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.

Iron sulfur cluster proteins and microbial regulation: implications for understanding tuberculosis.

All pathogenic and nonpathogenic microbes are continuously exposed to environmental or endogenous reactive oxygen and nitrogen species, which can critically effect survival and disease. Iron-sulfur [Fe-S] cluster containing prosthetic groups provide the microbial cell with a unique capacity to sense and transcriptionally respond to diatomic gases (e.g. NO and O2) and redox-cycling agents. Recent advances in our understanding of the mechanisms for how the FNR and SoxR [Fe-S] cluster proteins respond to NO and O2 have provided new insights into the biochemical mechanism of action of the Mycobacterium tuberculosis (Mtb) family of WhiB [Fe-S] cluster proteins. These insights have provided the basis for establishing a unifying paradigm for the Mtb WhiB family of proteins. Mtb is the etiological agent for tuberculosis (TB), a disease that affects nearly one-third of the world's population.

Transduction of brain dopamine neurons by adenoviral vectors is modulated by CAR expression: rationale for tropism modified vectors in PD gene therapy.

BACKGROUND: Gene-based therapy is a new paradigm for the treatment of Parkinson disease (PD) and offers considerable promise for precise targeting and flexibility to impact multiple pathobiological processes for which small molecule agents are not available. Some success has been achieved utilizing adeno-associated virus for this approach, but it is likely that the characteristics of this vector system will ultimately create barriers to progress in clinical therapy. Adenovirus (Ad) vector overcomes limitations in payload size and targeting. The cellular tropism of Ad serotype 5 (Ad5)-based vectors is regulated by the Ad attachment protein binding to its primary cellular receptor, the coxsackie and adenovirus receptor (CAR). Many clinically relevant tissues are refractory to Ad5 infection due to negligible CAR levels but can be targeted by tropism-modified, CAR-independent forms of Ad. Our objective was to evaluate the role of CAR protein in transduction of dopamine (DA) neurons in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Ad5 was delivered to the substantia nigra (SN) in wild type (wt) and CAR transgenic animals. Cellular tropism was assessed by immunohistochemistry (IHC) in the SN and striatal terminals. CAR expression was assessed by western blot and IHC. We found in wt animals, Ad5 results in robust transgene expression in astrocytes and other non-neuronal cells but poor infection of DA neurons. In contrast, in transgenic animals, Ad5 infects SNc neurons resulting in expression of transduced protein in their striatal terminals. Western blot showed low CAR expression in the ventral midbrain of wt animals compared to transgenic animals. Interestingly, hCAR protein localizes with markers of post-synaptic structures, suggesting synapses are the point of entry into dopaminergic neurons in transgenic animals. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate that CAR deficiency limits infection of wild type DA neurons by Ad5 and provide a rationale for the development of tropism-modified, CAR-independent Ad-vectors for use in gene therapy of human PD.

An adenoviral vector expressing human adenovirus 5 and 3 fiber proteins for targeting heterogeneous cell populations.

Human adenovirus serotype 5 (HAdV-5) attaches to its primary receptor, the coxsackie and adenovirus receptor (CAR) as the first step of infection. However, CAR expression decreases as tumors progress, thereby diminishing the utility of HAdV-5-based vectors for cancer therapy. In contrast, many aggressive tumor cells highly express CD46, a cellular receptor for HAdV-3. We hypothesized that a mosaic HAdV vector, containing two kinds of fiber proteins, would provide extensive transduction in a heterogeneous population of tumor cells with varying expression levels of HAdV receptors. We therefore generated a fiber-mosaic HAdV vector displaying both a chimeric HAdV-3 fiber and the HAdV-5 fiber protein. We verified the structural integrity of purified viral particles and confirmed that the fiber-mosaic HAdV vector has expanded tropism. We conclude that the use of fiber-mosaic HAdV vectors is a promising approach for transducing a heterogeneous cell population with different expression levels of adenovirus receptors.

Crystallographic structure of porcine adenovirus type 4 fiber head and galectin domains.

Adenovirus isolate NADC-1, a strain of porcine adenovirus type 4, has a fiber containing an N-terminal virus attachment region, shaft and head domains, and a C-terminal galectin domain connected to the head by an RGD-containing sequence. The crystal structure of the head domain is similar to previously solved adenovirus fiber head domains, but specific residues for binding the coxsackievirus and adenovirus receptor (CAR), CD46, or sialic acid are not conserved. The structure of the galectin domain reveals an interaction interface between its two carbohydrate recognition domains, locating both sugar binding sites face to face. Sequence evidence suggests other tandem-repeat galectins have the same arrangement. We show that the galectin domain binds carbohydrates containing lactose and N-acetyl-lactosamine units, and we present structures of the galectin domain with lactose, N-acetyl-lactosamine, 3-aminopropyl-lacto-N-neotetraose, and 2-aminoethyl-tri(N-acetyl-lactosamine), confirming the domain as a bona fide galectin domain.

A strategy for adenovirus vector targeting with a secreted single chain antibody.

BACKGROUND: Successful gene therapy will require targeted delivery vectors capable of self-directed localization. In this regard, the use of antibodies or single chain antibody fragments (scFv) in conjunction with adenovirus (Ad) vectors remains an attractive means to achieve cell-specific targeting. However, a longstanding barrier to the development of Ad vectors with genetically incorporated scFvs has been the biosynthetic incompatibility between Ad capsid proteins and antibody-derived species. Specifically, scFv require posttranslational modifications not available to Ad capsid proteins due to their cytoplasmic routing during protein synthesis and virion assembly. METHODOLOGY/PRINCIPAL FINDINGS: We have therefore sought to develop scFv-targeted Ad vectors using a secreted scFv that undergoes the requisite posttranslational modifications and is trafficked for secretion. Formation of the scFv-targeted Ad vector is achieved via highly specific association of the Ad virion and a targeting scFv employing synthetic leucine zipper-like dimerization domains (zippers) that have been optimized for structural compatibility with the Ad capsid and for association with the secreted scFv. Our results show that zipper-containing Ad fiber molecules trimerize and incorporate into mature virions and that zippers can be genetically fused to scFv without ablating target recognition. Most importantly, we show that zipper-tagged virions and scFv provide target-specific gene transfer. CONCLUSIONS/SIGNIFICANCE: This work describes a new approach to produce targeted Ad vectors using a secreted scFv molecule, thereby avoiding the problem of structural and biosynthetic incompatibility between Ad and a complex targeting ligand. This approach may facilitate Ad targeting using a wide variety of targeting ligands directed towards a variety of cellular receptors.

Crystallization of the head and galectin-like domains of porcine adenovirus isolate NADC-1 fibre.

The porcine adenovirus NADC-1 isolate, a strain of porcine adenovirus type 4, has a fibre with an atypical architecture. In addition to a classical virus attachment region, shaft and head domains, it contains an additional galectin like domain C-terminal to the head domain and connected to the head domain by a long RGD-containing loop. The galectin-like domain contains two putative carbohydrate-recognition domains. The head and galectin-like domains have been independently crystallized. Diffraction data have been obtained to 3.2 angstrom resolution from crystals of the head domain and to 1.9 angstrom resolution from galectin-like domain crystals.

A genetically engineered adenovirus vector targeted to CD40 mediates transduction of canine dendritic cells and promotes antigen-specific immune responses in vivo.

Targeting viral vectors encoding tumor-associated antigens to dendritic cells (DCs) in vivo is likely to enhance the effectiveness of immunotherapeutic cancer vaccines. We have previously shown that genetic modification of adenovirus (Ad) 5 to incorporate CD40 ligand (CD40L) rather than native fiber allows selective transduction and activation of DCs in vitro. Here, we examine the capacity of this targeted vector to induce immune responses to the tumor antigen CEA in a stringent in vivo canine model. CD40-targeted Ad5 transduced canine DCs via the CD40-CD40L pathway in vitro, and following vaccination of healthy dogs, CD40-targeted Ad5 induced strong anti-CEA cellular and humoral responses. These data validate the canine model for future translational studies and suggest targeting of Ad5 vectors to CD40 for in vivo delivery of tumor antigens to DCs is a feasible approach for successful cancer therapy.

Localization of the N-terminus of minor coat protein IIIa in the adenovirus capsid.

Minor coat protein IIIa is conserved in all adenoviruses (Ads) and is required for correct viral assembly, but its precise function in capsid organization is unknown. The latest Ad capsid model proposes that IIIa is located underneath the vertex region. To obtain experimental evidence on the location of IIIa and to further define its role, we engineered the IIIa gene to encode heterologous N-terminal peptide extensions. Recombinant Ad variants with IIIa encoding six-histidine (6His) tag, 6His, and FLAG peptides, or with 6His linked to FLAG with a (Gly(4)Ser)(3) linker were rescued and analyzed for virus yield, capsid incorporation of heterologous peptides, and capsid stability. Longer extensions could not be rescued. Western blot analysis confirmed that the modified IIIa proteins were expressed in infected cells and incorporated into virions. In the Ad encoding the 6His-linker-FLAG-IIIa gene, the 6His tag was present in light particles, but not in mature virions. Immunoelectron microscopy of this virus showed that the FLAG epitope is not accessible to antibodies on the viral particles. Three-dimensional electron microscopy and difference mapping located the IIIa N-terminal extension beneath the vertex complex, wedged at the interface between the penton base and peripentonal hexons, therefore supporting the latest proposed model. The position of the IIIa N-terminus and its low tolerance for modification provide new clues for understanding the role of this minor coat protein in Ad capsid assembly and disassembly.

Enhanced Gene Delivery to Human Primary Endothelial Cells Using Tropism-Modified Adenovirus Vectors.

Endothelial cells have been noted to have relatively low expression of the native receptor for adenovirus serotype 5 (Ad5), coxsackie and adenovirus receptor (CAR), and are thus refractory to Ad5 infection. In this study, we hypothesize that increases in the infectivity of Ad5 in primary human pulmonary artery (HPAEC), coronary artery (HCAEC) and umbilical vein endothelial cells (HUVEC) can be achieved through genetic capsid modification of Ad5 to bypass CAR-dependent infection. The modifications tested in this study include incorporation of an integrin-binding RGD peptide motif (Ad5.RGD), a poly-lysine motif (Ad5.pK7), a combination of both of these peptide domains (Ad5.RGD.pK7), an adenovirus serotype 3 knob domain (Ad5/3Luc1) and canine adenovirus serotype 1 or 2 knob domains (Ad5Luc1-CK1 and Ad5Luc1-CK2). In HPAEC and HCAEC, the greatest infectivity enhancements were achieved using Ad5/3Luc1 (26-fold and 30-fold respectively). HUVEC was most readily infected by Ad5Luc1-CK1 (213-fold). These results demonstrate that gains in Ad5 infectivity in endothelial cells can be accomplished with genetic capsid modifications.