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While studying transgene expression after systemic administration of lentiviral vectors, we found that splenic B cells are robustly transduced, regardless of the types of pseudotyped envelope proteins. However, the administration of two different pseudotypes resulted in transduction of two distinct B cell populations, suggesting that each pseudotype uses unique and specific receptors for its attachment and entry into splenic B cells. Single-cell RNA sequencing analysis of the transduced cells demonstrated that different pseudotypes transduce distinct B cell subpopulations characterized by specific B cell receptor (BCR) genotypes. Functional analysis of the BCRs of the transduced cells demonstrated that BCRs specific to the pseudotyping envelope proteins mediate viral entry, enabling the vectors to selectively transduce the B cell populations that are capable of producing antibodies specific to their envelope proteins. Lentiviral vector entry via the BCR activated the transduced B cells and induced proliferation and differentiation into mature effectors, such as memory B and plasma cells. BCR-mediated viral entry into clonally specific B cell subpopulations raises new concepts for understanding the biodistribution of transgene expression after systemic administration of lentiviral vectors and offers new opportunities for BCR-targeted gene delivery by pseudotyped lentiviral vectors., Competing Interests: Declaration of interests K.M. holds 2 US patents (US patent numbers 8449875 and 9163248) related to this work., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, causes respiratory failure and damage to multiple organ systems. The emergence of viral variants poses a risk of vaccine failures and prolongation of the pandemic. However, our understanding of the molecular basis of SARS-CoV-2 infection and subsequent COVID-19 pathophysiology is limited. In this study, we have uncovered a critical role for the evolutionarily conserved Hippo signaling pathway in COVID-19 pathogenesis. Given the complexity of COVID-19-associated cell injury and immunopathogenesis processes, we investigated Hippo pathway dynamics in SARS-CoV-2 infection by utilizing COVID-19 lung samples and human cell models based on pluripotent stem cell-derived cardiomyocytes (PSC-CMs) and human primary lung air-liquid interface (ALI) cultures. SARS-CoV-2 infection caused activation of the Hippo signaling pathway in COVID-19 lung and in vitro cultures. Both parental and Delta variant of concern (VOC) strains induced Hippo pathway. The chemical inhibition and gene knockdown of upstream kinases MST1/2 and LATS1 resulted in significantly enhanced SARS-CoV-2 replication, indicating antiviral roles. Verteporfin, a pharmacological inhibitor of the Hippo pathway downstream transactivator, YAP, significantly reduced virus replication. These results delineate a direct antiviral role for Hippo signaling in SARS-CoV-2 infection and the potential for this pathway to be pharmacologically targeted to treat COVID-19., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Garcia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Zika virus (ZIKV), a mosquito-borne human pathogen, causes dire congenital brain developmental abnormalities in children of infected mothers. The global health crisis precipitated by this virus has led to a concerted effort to develop effective therapies and prophylactic measures although, unfortunately, not very successfully. The error-prone nature of RNA viral genome replication tends to promote evolution of novel viral strains, which could cause epidemics and pandemics. As such, our objective was to develop a safe and effective replication-deficient ZIKV vector-based vaccine candidate. We approached this by generating a ZIKV vector containing only the nonstructural (NS) 5'-untranslated (UTR)-NS-3' UTR sequences, with the structural proteins capsid (C), precursor membrane (prM), and envelope (E) (CprME) used as a packaging system. We efficiently packaged replication-deficient Zika vaccine particles in human producer cells and verified antigen expression in vitro . In vivo studies showed that, after inoculation in neonatal mice, the Zika vaccine candidate (ZVAX) was safe and did not produce any replication-competent revertant viruses. Immunization of adult, nonpregnant mice showed that ZVAX protected mice from lethal challenge by limiting viral replication. We then evaluated the safety and efficacy of ZVAX in pregnant mice, where it was shown to provide efficient maternal and fetal protection against Zika disease. Mass cytometry analysis showed that vaccinated pregnant animals had high levels of splenic CD8 + T cells and effector memory T cell responses with reduced proinflammatory cell responses, suggesting that endogenous expression of NS proteins by ZVAX induced cellular immunity against ZIKV NS proteins. We also investigated humoral immunity against ZIKV, which is potentially induced by viral proteins present in ZVAX virions. We found no significant difference in neutralizing antibody titer in vaccinated or unvaccinated challenged animals; therefore, it is likely that cellular immunity plays a major role in ZVAX-mediated protection against ZIKV infection. In conclusion, we demonstrated ZVAX as an effective inducer of protective immunity against ZIKV, which can be further evaluated for potential prophylactic application in humans. IMPORTANCE This research is important as it strives to address the critical need for effective prophylactic measures against the outbreak of Zika virus (ZIKV) and outlines an important vaccine technology that could potentially be used to induce immune responses against other pandemic-potential viruses.

Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR-Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V ( RhoV ) and WW domain-containing transcription regulator 1 ( WWTR1 ) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host-pathogen interactions.

Envelope phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtr) have been shown to mediate binding of enveloped viruses. However, commonly used PtdSer binding molecules such as Annexin V cannot block PtdSer-mediated viral infection. Lack of reagents that can conceal envelope PtdSer and PtdEtr and subsequently inhibit infection hinders elucidation of the roles of the envelope phospholipids in viral infection. Here, we developed sTIM1dMLDR801, a reagent capable of blocking PtdSer- and PtdEtr-dependent infection of enveloped viruses. Using sTIM1dMLDR801, we found that envelope PtdSer and/or PtdEtr can support ZIKV infection of not only human but also mosquito cells. In a mouse model for ZIKV infection, sTIM1dMLDR801 reduced ZIKV load in serum and the spleen, indicating envelope PtdSer and/or PtdEtr support in viral infection in vivo. sTIM1dMLDR801 will enable elucidation of the roles of envelope PtdSer and PtdEtr in infection of various virus species, thereby facilitating identification of their receptors and transmission mechanisms., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The risk for breast cancer is significantly reduced in persons who engage in greater amounts of physical activity, and greater physical activity before or after diagnosis associates with reduced disease-specific mortality. Previous mechanistic studies indicate that components of innate immunity can mediate an inhibitory effect of physical activity on several types of tumor. However, in breast cancer specifically, the myeloid compartment of innate immunity is thought to exhibit high propensity for an immunosuppressive role that obstructs anti-tumor immunity. Thus, we tested the notion that greater physical activity alters mononuclear phagocytes in mammary tissue when inhibiting nascent tumor in a murine model of breast cancer. To model greater physical activity, we placed an angled running wheel in each mouse's home cage for two weeks before tumor engraftment with EO771 mammary cancer cells that express luciferase for bioluminescent detection. Fully immunocompetent mice and mice with compromised adaptive immunity showed significantly less mammary tumor signal when given access to running wheels, although the effect size was smaller in this latter group. To investigate the role of the myeloid compartment, mononuclear phagocytes were ablated by systemic injection of clodronate liposomes at 24 h before tumor engraftment and again at the time of tumor engraftment, and this treatment reversed the inhibition in wheel running mice. However, clodronate also inhibited mammary tumor signal in sedentary mice, in conjunction with an expected decrease in gene and protein expression of the myeloid antigen, F4/80 (Adgre1), in mammary tissue. Whole transcriptome digital cytometry with CIBERSORTx was used to analyze myeloid cell populations in mammary tissue following voluntary wheel running and clodronate treatment, and this approach found significant changes in macrophage and monocyte populations. In exploratory analyses, whole transcriptome composite scores for monocytic myeloid-derived suppressor cell (M-MDSC), macrophage lactate timer, and inflammation resolution gene expression programs were significantly altered. Altogether, the results support the hypothesis that physical activity inhibits nascent mammary tumor growth by enhancing the anti-tumor potential of mononuclear phagocytes in mammary tissue., Competing Interests: The authors declare there are no competing interests., (©2021 Lamkin et al.)

Coronavirus disease 2019 (COVID-19) is a pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19 is defined by respiratory symptoms, but cardiac complications including viral myocarditis are also prevalent. Although ischemic and inflammatory responses caused by COVID-19 can detrimentally affect cardiac function, the direct impact of SARS-CoV-2 infection on human cardiomyocytes is not well understood. Here, we utilize human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model to examine the mechanisms of cardiomyocyte-specific infection by SARS-CoV-2. Microscopy and RNA sequencing demonstrate that SARS-CoV-2 can enter hiPSC-CMs via ACE2. Viral replication and cytopathic effect induce hiPSC-CM apoptosis and cessation of beating after 72 h of infection. SARS-CoV-2 infection activates innate immune response and antiviral clearance gene pathways, while inhibiting metabolic pathways and suppressing ACE2 expression. These studies show that SARS-CoV-2 can infect hiPSC-CMs in vitro , establishing a model for elucidating infection mechanisms and potentially a cardiac-specific antiviral drug screening platform., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Zika virus (ZIKV) is a reemerging human pathogen that causes congenital abnormalities, including microcephaly and eye disease. The cellular/molecular basis of ZIKV and host interactions inducing ocular and neuronal pathogenesis are unclear. Herein, we noted that the Hippo/Salvador-Warts-Hippo signaling pathway, which controls organ size through progenitor cell proliferation and differentiation, is dysregulated after ZIKV infection. In human fetal retinal pigment epithelial cells, there is an early induction of transcriptional coactivator, Yes-associated protein (YAP), which is later degraded with a corresponding activation of the TANK binding kinase 1/interferon regulatory factor 3 type I interferon pathway. YAP/transcriptional co-activator with a PDZ-binding domain (TAZ) silencing results in reduced ZIKV replication, indicating a direct role of Hippo pathway in regulating ZIKV infection. Using an in vivo Ifnar1 -/- knockout mouse model, ZIKV infection was found to reduce YAP/TAZ protein levels while increasing phosphorylated YAP Ser127 in the retina and brain. Hippo pathway is activated in major cellular components of the blood-brain barrier, including endothelial cells and astrocytes. In addition, this result suggests AMP-activated protein kinase signaling pathway's role in regulating YAP/TAZ in ZIKV-infected cells. These data demonstrate that ZIKV infection might initiate a cross talk among AMP-activated protein kinase-Hippo-TBK1 pathways, which could regulate antiviral and energy stress responses during oculoneuronal inflammation., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Phosphatidylserine (PtdSer) is usually present only in the inner leaf of the lipid bilayers of the cell membrane, but is exposed on the outer leaf when cells are activated and/or die. Exposure of PtdSer has physiological functions. For example, the PtdSer exposed on dead cells can serve as "eat-me signals" for phagocytes to clear dead cells by phagocytosis, which prevents autoimmune reactions and inflammation. HIV-1 induces PtdSer exposure on infected and target cells and it also exposes PtdSer on its envelope. Recent studies showed that PtdSer exposed on the HIV-1 envelope and infected and target cells can facilitate or inhibit multiple steps of HIV-1 replication.At the virus binding and entry steps, interaction of the envelope PtdSer and the host's PtdSer-binding molecules can enhance HIV-1 infection of cells by facilitating virus attachment. At the virus budding step, HIV-1 can be trapped on the cell surface by one family of PtdSer-binding receptors, T-cell immunoglobulin mucin domain proteins (TIM)-1, 3, and 4 expressed on virus producer cells. Although this trapping can inhibit release of HIV-1, one of the HIV-1 accessory gene products, Negative Factor (Nef), can counteract virus trapping by TIM family receptors (TIMs) by inducing the internalization of these receptors. HIV-1 infection can induce exposure of PtdSer on infected cells by inducing cell death. A soluble PtdSer-binding protein in serum, protein S, bridges PtdSer exposed on HIV-1-infected cells and a receptor tyrosine kinase, Mer, expressed on macrophages and mediate phagocytic clearance of HIV-1 infected cells. HIV-1 can also induce exposure of PtdSer on target cells at the virus binding step. Binding of HIV-1 envelope proteins to its receptor (CD4) and co-receptors (CXCR4 or CCR5) elicit signals that induce PtdSer exposure on target cells by activating TMEM16F, a phospholipid scramblase. PtdSer exposed on target cells enhances HIV-1 infection by facilitating fusion between the viral envelope and target cell membrane. Because various other phospholipid channels mediating PtdSer exposure have recently been identified, it will be of interest to examine how HIV-1 actively interacts with these molecules to manipulate PtdSer exposure levels on cells and viral envelope to support its replication.

Conjugating certain types of lentiviral vectors with targeting ligands can redirect the vectors to specifically transduce desired cell types. However, extensive genetic and/or biochemical manipulations are required for conjugation, which hinders applications for targeting lentiviral vectors for broader research fields. We developed envelope proteins fused with biotin-binding molecules to conjugate the pseudotyped vectors with biotinylated targeting molecules by simply mixing them. The envelope proteins fused with the monomeric, but not tetrameric, biotin-binding molecules can pseudotype lentiviral vectors and be conjugated with biotinylated targeting ligands. The conjugation is stable enough to redirect lentiviral transduction in the presence of serum, indicating their potential in in vivo . When a signaling molecule is conjugated with the vector, the conjugation facilitates transduction and signaling in a receptor-specific manner. This simple method of ligand conjugation and ease of obtaining various types of biotinylated ligands will make targeted lentiviral transduction easily applicable to broad fields of research., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Efferocytosis, the phagocytic clearance of apoptotic cells, can provide host protection against certain types of viruses by mediating phagocytic clearance of infected cells undergoing apoptosis. It is known that HIV-1 induces apoptosis and HIV-1-infected cells are efferocytosed by macrophages, although its molecular mechanisms are unknown. To elucidate the roles that efferocytosis of HIV-1-infected cells play in clearance of infected cells, we sought to identify molecules that mediate these processes. We found that protein S, present in human serum, and its homologue, Gas6, can mediate phagocytosis of HIV-1-infected cells by bridging receptor tyrosine kinase Mer, expressed on macrophages, to phosphatidylserine exposed on infected cells. Efferocytosis of live infected cells was less efficient than dead infected cells; however, a significant fraction of live infected cells were phagocytosed over 12h. Our results suggest that efferocytosis not only removes dead cells, but may also contribute to macrophage removal of live virus producing cells., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: It is very difficult for dental professionals to objectively assess tooth brushing skill of patients, because an obvious index to assess the brushing motion of patients has not been established. The purpose of this study was to quantitatively evaluate toothbrush and arm-joint motion during tooth brushing., Materials and Methods: Tooth brushing motion, performed by dental hygienists for 15 s, was captured using a motion-capture system that continuously calculates the three-dimensional coordinates of object's motion relative to the floor. The dental hygienists performed the tooth brushing on the buccal and palatal sides of their right and left upper molars. The frequencies and power spectra of toothbrush motion and joint angles of the shoulder, elbow, and wrist were calculated and analyzed statistically., Results: The frequency of toothbrush motion was higher on the left side (both buccal and palatal areas) than on the right side. There were no significant differences among joint angle frequencies within each brushing area. The inter- and intra-individual variations of the power spectrum of the elbow flexion angle when brushing were smaller than for any of the other angles., Conclusions: This study quantitatively confirmed that dental hygienists have individual distinctive rhythms during tooth brushing. All arm joints moved synchronously during brushing, and tooth brushing motion was controlled by coordinated movement of the joints. The elbow generated an individual's frequency through a stabilizing movement., Clinical Relevance: The shoulder and wrist control the hand motion, and the elbow generates the cyclic rhythm during tooth brushing.

Objective: To evaluate morphological differences of the facial soft tissue surface between male Japanese adults and children., Design: 20 adult Japanese males (average age 28 years) and 20 Japanese boys (average age 5.5 years) with normal occlusion were selected for this study. The images of the subjects' facial surface were obtained with a 3-D laser scanner. To evaluate the three-dimensional morphological differences of the facial soft tissue, we transformed the coordinates of 16 facial landmarks to a new reference plane and compared the adults' and children's facial form drawn to the same scale in the same coordinate system., Results: The morphological difference ratio of the lower facial area was higher than in the upper facial area, and the nose and lower face changed more forward than downward. The morphological difference ratio of the mid face width was smaller than other areas., Conclusion: Our study suggests that the morphological facial soft tissue differences between Japanese adults and children are more forward and downward than laterally, manifesting in a facial form of adults that is deeper and narrow., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Unlabelled: We recently demonstrated that a soluble protein, Gas6, can facilitate viral entry by bridging viral envelope phosphatidylserine to Axl, a receptor tyrosine kinase expressed on target cells. The interaction between phosphatidylserine, Gas6, and Axl was originally shown to be a molecular mechanism through which phagocytes recognize phosphatidylserine exposed on dead cells. Since our initial report, several groups have confirmed that Axl/Gas6, as well as other phosphatidylserine receptors, facilitate entry of dengue, West Nile, and Ebola viruses. Virus binding by viral envelope phosphatidylserine is now a viral entry mechanism generalized to many families of viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most of the known human phosphatidylserine-recognizing molecules, including MFG-E8, TIM-1, -3, and -4, CD300a, BAI1, and stabilin-1 and -2, for their abilities to facilitate virus binding and infection. Using pseudotyped lentiviral vectors, we found that a soluble phosphatidylserine-binding protein, MFG-E8, enhances transduction. Cell surface receptors TIM-1 and -4 also enhance virus binding/transduction. The extent of enhancement by these molecules varies, depending on the type of pseudotyping envelope proteins. Mutated MFG-E8, which binds viral envelope phosphatidylserine without bridging virus to cells, but, surprisingly, not annexin V, which has been used to block phagocytosis of dead cells by concealing phosphatidylserine, efficiently blocks these phosphatidylserine-dependent viral entry mechanisms. These results provide insight into understanding the role of viral envelope phosphatidylserine in viral infection., Importance: Envelope phosphatidylserine has previously been shown to be important for replication of various envelope viruses, but details of this mechanism(s) were unclear. We were the first to report that a bifunctional serum protein, Gas6, bridges envelope phosphatidylserine to a cell surface receptor, Axl. Recent studies demonstrated that many envelope viruses, including vaccinia, dengue, West Nile, and Ebola viruses, utilize Axl/Gas6 to facilitate their entry, suggesting that the phosphatidylserine-mediated viral entry mechanism can be shared by various enveloped viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most human phosphatidylserine-recognizing molecules for their abilities to facilitate viral infection. The results provide insights into the role(s) of envelope phosphatidylserine in viral infection, which can be applicable to the development of novel antiviral reagents that block phosphatidylserine-mediated viral entry.

Background: We previously developed an antibody-avidin fusion protein (ch128.1Av) specific for the human transferrin receptor 1 (TfR1; CD71) to be used as a delivery vector for cancer therapy and showed that ch128.1Av delivers the biotinylated plant toxin saporin-6 into malignant B cells. However, as a result of widespread expression of TfR1, delivery of the toxin to normal cells is a concern. Therefore, we explored the potential of a dual targeted lentiviral-mediated gene therapy strategy to restrict gene expression to malignant B cells. Targeting occurs through the use of ch128.1Av or its parental antibody without avidin (ch128.1) and through transcriptional regulation using an immunoglobulin promoter., Methods: Flow cytometry was used to detect the expression of enhanced green fluorescent protein (EGFP) in a panel of cell lines. Cell viability after specific delivery of the therapeutic gene FCU1, a chimeric enzyme consisting of cytosine deaminase genetically fused to uracil phosphoribosyltransferse that converts the 5-fluorocytosine (5-FC) prodrug into toxic metabolites, was monitored using the MTS or WST-1 viability assay., Results: We found that EGFP was specifically expressed in a panel of human malignant B-cell lines, but not in human malignant T-cell lines. EGFP expression was observed in all cell lines when a ubiquitous promoter was used. Furthermore, we show the decrease of cell viability in malignant plasma cells in the presence of 5-FC and the FCU1 gene., Conclusions: The present study demonstrates that gene expression can be restricted to malignant B cells and suggests that this dual targeted gene therapy strategy may help to circumvent the potential side effects of certain TfR1-targeted protein delivery approaches., (Copyright © 2014 John Wiley & Sons, Ltd.)

Chronic active Epstein-Barr virus infection (CAEBV) is characterized by chronic or recurrent infectious mononucleosis-like symptoms, such as fever, extensive lymphadenopathy, and hepatosplenomegaly. A 44-year-old women visited our ENT clinic with a four-month history of fever and throat pain. She was diagnosed as having CAEBV based on the findings of fever, liver dysfunction, lymphadenopathy, pharyngeal ulcer, the titer for IgG to the EBV capsid and pathological findings. The whole-blood EBV DNA levels were high and above 3.7 x 10(3) copies/mL. After administration of intravenous predonine (1000 mg/day for 3 days) and oral predonine (1.5 mg/kg. 60 mg/day), the liver dysfunction and pharyngeal ulcer improved. Since the prognosis is poor in adult cases of CAEBV, chemotherapy is scheduled for this case.

Clinical studies suggest that stress-related biobehavioral factors can accelerate the progression of hematopoietic cancers such as acute lymphoblastic leukemia (ALL), but it is unclear whether such effects are causal or what biological pathways mediate such effects. Given the network of sympathetic nervous system (SNS) fibers that innervates the bone marrow to regulate normal (non-leukemic) hematopoietic progenitor cells, we tested the possibility that stress-induced SNS signaling might also affect ALL progression. In an orthotopic mouse model, Nalm-6 human pre-B ALL cells were transduced with the luciferase gene for longitudinal bioluminescent imaging and injected i.v. into male SCID mice for bone marrow engraftment. Two weeks of daily restraint stress significantly enhanced ALL tumor burden and dissemination in comparison to controls, and this effect was blocked by the β-adrenergic antagonist, propranolol. Although Nalm-6 ALL cells expressed mRNA for β1- and β3-adrenergic receptors, they showed no evidence of cAMP signaling in response to norepinephrine, and norepinephrine failed to enhance Nalm-6 proliferation in vitro. These results show that chronic stress can accelerate the progression of human pre-B ALL tumor load via a β-adrenergic signaling pathway that likely involves indirect regulation of ALL biology via alterations in the function of other host cell types such as immune cells or the bone marrow microenvironment., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Envelope virus replication begins with receptor binding, followed by fusion of the viral envelope with the cell membrane. The binding and fusion steps are usually mediated by envelope proteins. The ability of envelope proteins of a particular virus to bind and fuse with target cells defines the host range of the virus, known as 'viral tropism'. The mechanism(s) of fusion by the viral envelope is largely categorized as either pH-dependent or pH-independent. By redirecting the binding specificities of envelope proteins to desired target molecules while maintaining fusion activity, it is possible to redirect the tropisms of virus and viral vectors, enabling specific killing and/or transduction of desired cells in vivo. Recently, a lipid, phosphatidylserine, was also shown to mediate binding of virus, which affects the tropisms of viruses and viral vectors.

Virus entry into cells is typically initiated by binding of virally encoded envelope proteins to specific cell surface receptors. Studying infectivity of lentivirus pseudotypes lacking envelope binding, we still observed high infectivity for some cell types. On further investigation, we discovered that this infectivity is conferred by the soluble bovine protein S in fetal calf serum, or Gas6, its human homolog. Gas6 enhances native infectivity of pseudotypes of multiple viral envelope proteins. Gas6 mediates binding of the virus to target cells by bridging virion envelope phosphatidylserine to Axl, a TAM receptor tyrosine kinase on target cells. Phagocytic clearance of apoptotic cells is known to involve bridging by Gas6. Replication of vaccinia virus, which was previously reported to use apoptotic mimicry to enter cells, is also enhanced by Gas6. These results reveal an alternative molecular mechanism of viral entry that can broaden host range and enhance infectivity of enveloped viruses., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Metastasis to distant tissues is the chief driver of breast cancer-related mortality, but little is known about the systemic physiologic dynamics that regulate this process. To investigate the role of neuroendocrine activation in cancer progression, we used in vivo bioluminescence imaging to track the development of metastasis in an orthotopic mouse model of breast cancer. Stress-induced neuroendocrine activation had a negligible effect on growth of the primary tumor but induced a 30-fold increase in metastasis to distant tissues including the lymph nodes and lung. These effects were mediated by β-adrenergic signaling, which increased the infiltration of CD11b(+)F4/80(+) macrophages into primary tumor parenchyma and thereby induced a prometastatic gene expression signature accompanied by indications of M2 macrophage differentiation. Pharmacologic activation of β-adrenergic signaling induced similar effects, and treatment of stressed animals with the β-antagonist propranolol reversed the stress-induced macrophage infiltration and inhibited tumor spread to distant tissues. The effects of stress on distant metastasis were also inhibited by in vivo macrophage suppression using the CSF-1 receptor kinase inhibitor GW2580. These findings identify activation of the sympathetic nervous system as a novel neural regulator of breast cancer metastasis and suggest new strategies for antimetastatic therapies that target the β-adrenergic induction of prometastatic gene expression in primary breast cancers., (©2010 AACR.)

Redirecting the tropism of viral vectors enables specific transduction of selected cells by direct administration of vectors. We previously developed targeting lentiviral vectors by pseudotyping with modified Sindbis virus envelope proteins. These modified Sindbis virus envelope proteins have mutations in their original receptor-binding regions to eliminate their natural tropisms, and they are conjugated with targeting proteins, including antibodies and peptides, to confer their tropisms on target cells. We investigated whether our targeting vectors interact with DC-SIGN, which traps many types of viruses and gene therapy vectors by binding to the N-glycans of their envelope proteins. We found that these vectors do not interact with DC-SIGN. When these vectors were produced in the presence of deoxymannojirimycin, which alters the structures of N-glycans from complex to high mannose, these vectors used DC-SIGN as their receptor. Genetic analysis demonstrated that the N-glycans at E2 amino acid (aa) 196 and E1 aa 139 mediate binding to DC-SIGN, which supports the results of a previous report of cryoelectron microscopy analysis. In addition, we investigated whether modification of the N-glycan structures could activate serum complement activity, possibly by the lectin pathway of complement activation. DC-SIGN-targeted transduction occurs in the presence of human serum complement, demonstrating that high-mannose structure N-glycans of the envelope proteins do not activate human serum complement. These results indicate that the strategy of redirecting viral vectors according to alterations of their N-glycan structures would enable the vectors to target specific cells types expressing particular types of lectins.

We recently developed a novel targeting Sindbis virus envelope pseudotyped lentiviral vector, 2.2ZZ, which acquires specific transduction capacity by antibody conjugation and binding with specific antigens on the surface of targeted cells. Here we characterize the virological properties of this vector by examining its targeting to CD4 antigen. Our results show that entry is dependent on CD4 cell surface density and occurs via the clathrin-mediated endocytic pathway. These findings provide insight into the mechanism of infection by a new viral vector with combined properties of Sindbis virus and lentiviruses and infectivity conferred by monoclonal antibody-ligand interactions.

Background: Targeted gene transduction in vivo is the ultimate preferred method for gene delivery. We previously developed targeting lentiviral vectors that specifically recognize cell surface molecules with conjugated antibodies and mediate targeted gene transduction both in vitro and in vivo. Although effective in some experimental settings, the conjugation of virus with antibodies is mediated by the interaction between protein A and the Fc region of antibodies, which is not as stable as covalent conjugation. We have now developed a more stable conjugation strategy utilizing the interaction between avidin and biotin., Methods: We inserted the biotin-adaptor-peptide, which was biotinylated by secretory biotin ligase at specific sites, into our targeting envelope proteins, enabling conjugation of the pseudotyped virus with avidin, streptavidin or neutravidin., Results: When conjugated with avidin-antibody fusion proteins or the complex of avidin and biotinylated targeting molecules, the vectors could mediate specific transduction to targeted cells recognized by the targeting molecules. When conjugated with streptavidin-coated magnetic beads, transduction by the vectors was targeted to the locations of magnets., Conclusions: This targeting vector system can be used for broad applications of targeted gene transduction using biotinylated targeting molecules or targeting molecules fused with avidin.

Background: Targeting gene therapy vectors that can home in on desired cell and tissue types in vivo comprise the ultimate gene delivery system. We have previously developed targeting lentiviral vectors by pseudotyping vectors with modified Sindbis virus envelope proteins. The envelope protein contains the Fc-binding region of protein A (ZZ domain), so the virus can be conjugated with antibodies. The conjugated antibody mediates specific transduction of the cells and tissues expressing the target antigens, both in vitro and in vivo. However, more stable conjugation of targeting molecules would be optimal for use in immunocompetent animals, as well as in humans., Methods: We inserted integrin-targeting peptides into two sites of the targeting envelope proteins and determined whether the peptides serve as receptor-binding regions of the envelope proteins and redirect the pseudotyped viruses., Results: The integrin-targeting peptides can mediate binding to cells via the interaction with integrins on target cells and transduction. Peptides with a higher binding affinity increase titers of pseudotyped virus. We found two regions on the envelope protein that can accommodate insertion and serve as receptor-binding regions. Combining the peptides in two distinct regions increased the titers of the virus., Conclusions: Successful incorporation of targeting molecules into the envelope protein will broaden the application of targeting vectors for a wide variety of experimental and clinical settings., (2009 John Wiley & Sons, Ltd.)

Background: Conventional gene-therapy applications of hematopoietic stem cells (HSCs) involve purification of CD34+ progenitor cells from the mobilized peripheral blood, ex vivo transduction of the gene of interest into them, and reinfusion of the transduced CD34+ progenitor cells into patients. Eliminating the process of purification would save labor, time and money, while enhancing HSCs viability, transplantability and pluripotency. Lentiviral vectors have been widely used in gene therapy because they infect both dividing and nondividing cells and provide sustained transgene expression. One of the exceptions to this rule is quiescent primary lymphocytes, in which reverse transcription of viral DNA is not completed., Methods: In the present study, we tested the possibility of targeting CD34+ progenitor cells within nonpurified human mobilized peripheral blood mononuclear cells (mPBMCs) utilizing vesicular stomatitis virus G (VSV-G) pseudotyped lentiviral vectors, based on the assumption that the CD34+ progenitor cells would be preferentially transduced. To further enhance the specificity of vector transduction, we also examined utilizing a modified Sindbis virus envelope (2.2) pseudotyped lentiviral vector, developed in our laboratory, that allows targeted transduction to specific cell receptors via antibody recognition., Results: Both the VSV-G and 2.2 pseudotyped vectors achieved measurable results when they were used to target CD34+ progenitor cells in nonpurified mPBMCs., Conclusions: Overall, the data obtained demonstrate the potential of ex vivo targeting of CD34+ progenitor cells without purification.

Angiogenesis is a rate-limiting factor for numerous human diseases. Angiogenic vessels and also the endothelium of certain organs such as the lung display molecular addresses that can be exploited for the selective delivery of gene therapeutics. Lentiviral vectors (LVs) are powerful tools for stable gene delivery but their integration and expression in undesired cell types poses a serious safety concern. We have developed a dual-targeted LV that can specifically target primary endothelial cells (ECs). Cell selectivity is achieved during entry, using a modified Sindbis virus envelope, and during transcription, with an EC-specific promoter. We evaluated four surface markers for EC targeting and seven promoter sequences from genes preferentially expressed in ECs. The efficiency and specificity of the double targeted vector were assayed in a panel of human primary cultures and tumor cell lines. A vector targeted to CD146, an endothelial adhesion molecule, and carrying a derivative of the EC tyrosine kinase Tie2 promoter, increased specificity of transduction up to 50 times and was also effective at selectively transducing ECs in a mixed coculture with human fibroblasts. The vector presented here is a potentially powerful tool that could be used in a variety of human diseases., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Prostate cancer dissemination is difficult to detect in the clinic, and few treatment options exist for patients with advanced-stage disease. Our aim was to investigate the role of tumor lymphangiogenesis during metastasis. Further, we implemented a noninvasive molecular imaging technique to facilitate the assessment of the metastatic process. The metastatic potentials of several human prostate cancer xenograft models, LAPC-4, LAPC-9, PC3 and CWR22Rv-1 were compared. The cells were labeled with luciferase, a bioluminescence imaging reporter gene, to enable optical imaging. After tumor implantation the animals were examined weekly during several months for the appearance of metastases. Metastatic lesions were confirmed by immunohistochemistry. Additionally, the angiogenic and lymphangiogenic profiles of the tumors were characterized. To confirm the role of lymphangiogenesis in mediating metastasis, the low-metastatic LAPC-9 tumor cells were engineered to overexpress VEGF-C, and the development of metastases was evaluated. Our results show CWR22Rv-1 and PC3 tumor cell lines to be more metastatic than LAPC-4, which in turn disseminates more readily than LAPC-9. The difference in metastatic potential correlated with the endogenous production levels of lymphangiogenic growth factor VEGF-C and the presence of tumor lymphatics. In agreement, induced overexpression of VEGF-C in LAPC-9 enhanced tumor lymphangiogenesis leading to the development of metastatic lesions. Taken together, our studies, based on a molecular imaging approach for semiquantitative detection of micrometastases, point to an important role of tumor lymphatics in the metastatic process of human prostate cancer. In particular, VEGF-C seems to play a key role in prostate cancer metastasis., ((c) 2007 Wiley-Liss, Inc.)

Targeted gene transduction to organs and tissues of interest is the ultimate goal of therapeutic gene delivery. Lentiviral vectors (LVs) are powerful tools for stable gene delivery but their integration into undesired cell types poses a serious safety concern for their use in the clinic. Here we report the development of a new dual-targeted LV that can preferentially home to and express in prostate cancer bone metastases in vivo after systemic delivery. Transductional targeting is mediated by a modified Sindbis virus envelope that interacts with the prostate stem cell antigen (PSCA) expressed by prostate cancer cells, and transcriptional targeting is mediated by a prostate cell specific promoter. Homing to prostate tumors was achieved in 70% of the animals. Importantly, tumors could be detected in some cases by molecular imaging prior to X-ray detection. The dual-targeted vector presents enhanced specificity with respect to individual transcriptional or transductional targeted vectors. Transgene expression in the liver was 190 times lower than the expression associated with solely transductionally targeted vectors, and there was 12 times less vector DNA than the amount present with solely transcriptionally targeted vectors. The LV presented here is a powerful tool for obtaining stable and site-specific gene expression and can be easily modified for its use in other diseases.

Efficient transduction of primary hematopoietic cell types by oncoretroviral vectors and lentiviral vectors with a variety of different envelope pseudotypes has proven to be difficult. We recently developed a lentiviral vector based upon a modified Sindbis virus envelope that allows targeted transduction via antibody recognition to specific cells in unfractionated cell populations. However, similar to other envelope pseudotypes, the utility of this vector for some primary hematopoietic cells was limited by low transduction efficiencies. Here, we report that transient treatment of cells with low pH culture medium immediately following infection results in marked enhancements in transduction efficiency for primary hematopoietic cells. In combination with antibody directed targeting, this simple technique expands the utility of targeting transduction to specific cells in mixed populations of primary cells.

Unlabelled: Noninvasive imaging of a reporter gene is a new and promising technique to quantify transgene expression after gene therapy. This study was performed to demonstrate visualization of lentiviral-marked cells by PET., Methods: We transduced nonhuman primate CD34+ hematopoietic cells with a lentiviral vector expressing a PET reporter gene, the mutant viral herpes simplex virus type 1-thymidine kinase (HSV1-sr39tk) gene. 1-(2-Fluoro-2-deoxy-beta-D-arabinofuranosyl)-76Br-5-bromouracil (76Br-FBAU) was used as the substrate for the viral tk enzyme. Upon phosphorylation, 76Br-FBAU was retained by cells and imaged by PET. The long half-life of 76Br, 16.2 h, permitted us to perform extended pharmacokinetic and imaging studies., Results: 76Br-FBAU was retained in vascular tissues of the animals with transplanted tk lentiviral vector-transduced CD34+ cells. Elimination of 76Br-FBAU was through renal and hepatic excretion., Conclusion: Noninvasive molecular imaging using PET will help us, in the future, to define the contribution and distribution of cells and their progeny to tissue repair and development.

Specifically and effectively directing a therapeutic gene to its intended site of action is a critical issue for translation of basic genomics to clinical gene therapy. Delivering gene therapy vectors to specific cells or tissues through intravenous injection is the most desirable method for this purpose. In 2001, we reported successful targeted gene transduction in vitro utilizing both oncoretroviral and lentiviral vectors pseudotyped with a chimeric Sindbis virus envelope (ZZ SINDBIS). However, these pseudotypes mediated nonspecific gene transduction to liver and spleen in vivo. To address this problem we generated the modified ZZ SINDBIS (termed m168) with significantly less nonspecific infectivity. To investigate the ability of m168 pseudotyped lentiviral vector to mediate targeted gene transduction in vivo, we utilized a metastatic tumor model by using mouse melanoma cells engineered to express human P-glycoprotein. We administered the m 168 pseudotyped vector conjugated with anti-P-glycoprotein antibody into the mice intravenously to target metastatic melanoma. The m168 pseudotyped vector selectively infected metastatic melanoma cells demonstrating successful targeted gene transduction in vivo. Targeting technology based upon m168 can be further modified for application not only to cancer but also potentially to genetic, neurologic, infectious and immune diseases, thereby expanding the future application of gene therapy.

Targeted gene transduction to specific tissues and organs through intravenous injection would be the ultimate preferred method of gene delivery. Here, we report successful targeting in a living animal through intravenous injection of a lentiviral vector pseudotyped with a modified chimeric Sindbis virus envelope (termed m168). m168 pseudotypes have high titer and high targeting specificity and, unlike other retroviral pseudotypes, have low nonspecific infectivity in liver and spleen. A mouse cancer model of metastatic melanoma was used to test intravenous targeting with m168. Human P-glycoprotein was ectopically expressed on the surface of melanoma cells and targeted by the m168 pseudotyped lentiviral vector conjugated with antibody specific for P-glycoprotein. m168 pseudotypes successfully targeted metastatic melanoma cells growing in the lung after systemic administration by tail vein injection. Further development of this targeting technology should result in applications not only for cancers but also for genetic, infectious and immune diseases.

RNA interference is an evolutionarily conserved process of gene silencing that in plants serves as a natural defense mechanism against exogenous viral agents. RNA interference is becoming an important tool for the study of biological processes through reverse genetics and has potential for therapeutic applications in humans; however, effective delivery is still a major issue. Small interfering RNA (siRNA) and short hairpin RNA (shRNA) have been introduced into cells by transfection of chemically synthesized and RNA expression via plasmid cassettes utilizing RNA polymerase III transcription. The employment of siRNA/shRNA for gene knockout requires an efficient stable transfection or transduction process. Here, we report the successful construction of lentiviral vectors to express shRNA stably in human cells. We demonstrate that lentiviral vectors expressing siRNA directed to the reporter gene luciferase, when stably transduced into human cells without drug selection, are capable of protecting the cells from infection by a lentiviral vector encoding humanized firefly luciferase as a reporter gene. We observed 16- to 43-fold reduction of gene expression in infected cells transduced with shRNA vectors relative to cells transduced with control vectors. This model system demonstrates the utility of lentiviral vectors to stably express shRNA as both a cellular gene knockout tool and as a means to inhibit exogenous infectious agents such as viruses in human cells.

We have characterized a population of mesenchymal progenitor cells from adipose tissue, termed processed lipoaspirate (PLA) cells, which have multilineage potential similar to bone marrow-derived mesenchymal stem cells and are also easily expanded in culture. The primary benefit of using adipose tissue as a source of multilineage progenitor cells is its relative abundance and ease of procurement. We examined the infection of PLA cells with adenoviral, oncoretroviral, and lentiviral vectors. We demonstrate that PLA cells can be transduced with lentiviral vectors at high efficiency. PLA cells maintain transgene expression after differentiation into adipogenic and osteogenic lineages after lentiviral transduction. Therefore, PLA cells and lentiviral vectors may be an efficient combination for use as a therapeutic gene delivery vehicle.

Our laboratory has recently characterized a population of cells from adipose tissue, termed processed lipoaspirate (PLA) cells, which have multi-lineage potential similar to bone-marrow-derived mesenchymal stem cells (MSCs). This study is the first comparison of PLA cells and MSCs isolated from the same patient. No significant differences were observed for yield of adherent stromal cells, growth kinetics, cell senescence, multi-lineage differentiation capacity, and gene transduction efficiency. Adipose tissue is an abundant and easily procured source of PLA cells, which have a potential like MSCs for use in tissue-engineering applications and as gene delivery vehicles., (Copyright 2003 S. Karger AG, Basel)

Targeted stable transduction of specific cells is a highly desirable goal for gene therapy applications. We report an efficient and broadly applicable approach for targeting retroviral vectors to specific cells. We find that the envelope of the alphavirus Sindbis virus can pseudotype human immunodeficiency virus type 1- and murine leukemia virus-based retroviral vectors. When modified to contain the Fc-binding domain of protein A, this envelope gives a significant enhancement in specificity in combination with antibodies specific for HLA and CD4 relative to that without antibody. Unlike previous targeting strategies for retroviral transduction, the virus titers are relatively high and stable and can be further increased by ultracentrifugation. This study provides proof of principle for a targeting strategy that would be generally useful for many gene therapy applications.

Recently, gene therapy vectors based upon the human immunodeficiency virus type 1 (HIV-1) genome have been developed. Here, we create an HIV-1 vector which is defective for all HIV-1 genes, but which maintains cis-acting elements required for efficient packaging, infection, and expression. In T cells transduced by this vector, vector expression is low but efficiently induced following HIV-1 infection. Remarkably, although the HIV-1 vector does not contain specific anti-HIV-1 therapeutic genes, the presence of the vector alone is sufficient to inhibit the spread of HIV-1 infection. The mechanism of inhibition is likely to be at the level of competition for limiting substrates required for either efficient packaging or reverse transcription, thereby selecting against propagation of wild-type HIV-1. These results provide proof of a concept for potential application of a novel HIV-1 vector in HIV-1 disease.

In order to clarify the transmission process of human immunodeficiency virus type 1 (HIV-1) through the epithelial cell barrier, HeLa cells susceptible and non-susceptible to HIV-1 were cloned and designated as P6 HeLa and N7 HeLa cells, respectively. P6 HeLa cells could be infected with the LAI strain of HIV-1 and mediated HIV-1 transcytosis. In contrast, N7 HeLa cells exhibited neither HIV-1 infection nor transcytosis. CD4 and galactosylceramide as the receptors for HIV-1 were not detected on P6 HeLa cells, although an anti-CD4 monoclonal antibody (mAb) blocked HIV-1 infection. Since HIV-1-infected P6 HeLa cells exhibited no fusion and survived, we speculated that the P6 HeLa cells expressed molecules other than CD4 which facilitated HIV-1 infection. Two mAbs (A-14 ITK and C57 a9-9) which inhibited the HIV-1 infection of P6 HeLa cells were generated. Each mAb recognized distinct molecule(s) as shown by Western blotting. Transcytosis by the P6 HeLa cells was inhibited by C57 a9-9 but not by A-14 ITK or anti-CD4 mAb. Both infection and transcytosis may be responsible for HIV-1 transmission through epithelial cells in a complex manner. Although infection and transcytosis occurred via different mechanisms, the molecule(s) recognized by C57 a9-9 mAb may be associated with both processes.