Human cytomegalovirus blocks canonical TGFß signaling during lytic infection to limit induction of type I interferons.

Human cytomegalovirus (HCMV) microRNAs (miRNAs) significantly rewire host signaling pathways to support the viral lifecycle and regulate host cell responses. Here we show that SMAD3 expression is regulated by HCMV miR-UL22A and contributes to the IRF7-mediated induction of type I IFNs and IFN-stimulated genes (ISGs) in human fibroblasts. Addition of exogenous TGFß interferes with the replication of a miR-UL22A mutant virus in a SMAD3-dependent manner in wild type fibroblasts, but not in cells lacking IRF7, indicating that downregulation of SMAD3 expression to limit IFN induction is important for efficient lytic replication. These findings uncover a novel interplay between SMAD3 and innate immunity during HCMV infection and highlight the role of viral miRNAs in modulating these responses.

Altruism and hyperaltruism in children of three cultures.

Children's inclination to prioritize others' welfare over their own (hyperaltruism) was investigated from a developmental and cross-cultural perspective. The distribution of rewarding or aversive items to self and another child was probed in 3- to 14-year-old children (final sample: N = 158; 87 girls) from urban China (n = 51; Mage = 9.1 years, SD = 1.81; 27 girls), urban United States (n = 55; Mage = 8.89 years, SD = 2.18; 30 girls), and rural Samoa (n = 52; Mage = 8.6 years, SD = 2.61; 30 girls). In two counterbalanced conditions, the children were first asked to split either rewards (stickers or treats) or potentially harmful insects (biting bugs) with another child. In a third condition, children were asked to choose various levels of hypothetical pain to be inflicted on either themselves or another child in exchange for commensurate rewards. Results indicated that in conditions where children distributed rewards, older children were more egalitarian (i.e., gave an equal number of candies to self and other) compared with younger children. However, in conditions where they needed to allocate potential harm, older children displayed more altruism (taking more harm onto themselves so that others could be spared). These results varied by culture, where Chinese children showed more altruism, U.S. children were more egalitarian, and Samoan children showed no clear patterns of either egalitarianism or altruism. The results show that although there are some universal trends toward egalitarian and altruistic ways of sharing, significant group differences exist.

Emerging Alphaviruses Are Sensitive to Cellular States Induced by a Novel Small-Molecule Agonist of the STING Pathway.

The type I interferon (IFN) system represents an essential innate immune response that renders cells resistant to virus growth via the molecular actions of IFN-induced effector proteins. IFN-mediated cellular states inhibit growth of numerous and diverse virus types, including those of known pathogenicity as well as potentially emerging agents. As such, targeted pharmacologic activation of the IFN response may represent a novel therapeutic strategy to prevent infection or spread of clinically impactful viruses. In light of this, we employed a high-throughput screen to identify small molecules capable of permeating the cell and of activating IFN-dependent signaling processes. Here we report the identification and characterization of N-(methylcarbamoyl)-2-{[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-2-phenylacetamide (referred to as C11), a novel compound capable of inducing IFN secretion from human cells. Using reverse genetics-based loss-of-function assays, we show that C11 activates the type I IFN response in a manner that requires the adaptor protein STING but not the alternative adaptors MAVS and TRIF. Importantly, treatment of cells with C11 generated a cellular state that potently blocked replication of multiple emerging alphavirus types, including chikungunya, Ross River, Venezuelan equine encephalitis, Mayaro, and O'nyong-nyong viruses. The antiviral effects of C11 were subsequently abrogated in cells lacking STING or the type I IFN receptor, indicating that they are mediated, at least predominantly, by way of STING-mediated IFN secretion and subsequent autocrine/paracrine signaling. This work also allowed characterization of differential antiviral roles of innate immune signaling adaptors and IFN-mediated responses and identified MAVS as being crucial to cellular resistance to alphavirus infection.IMPORTANCE Due to the increase in emerging arthropod-borne viruses, such as chikungunya virus, that lack FDA-approved therapeutics and vaccines, it is important to better understand the signaling pathways that lead to clearance of virus. Here we show that C11 treatment makes human cells refractory to replication of a number of these viruses, which supports its value in increasing our understanding of the immune response and viral pathogenesis required to establish host infection. We also show that C11 depends on signaling through STING to produce antiviral type I interferon, which further supports its potential as a therapeutic drug or research tool.

IL-6 in human cytomegalovirus secretome promotes angiogenesis and survival of endothelial cells through the stimulation of survivin.

Human cytomegalovirus (HCMV) is linked to the acceleration of vascular diseases such as atherosclerosis and transplant vasculopathy. One of the hallmarks of these diseases is angiogenesis (AG) and neovessel formation. Endothelial cells (ECs) are an integral part of AG and are sites of HCMV persistence. AG requires multiple synchronous processes that include EC proliferation, migration, and vessel stabilization. Virus-free supernatant (secretome) from HCMV-infected ECs induces AG. To identify factor(s) involved in this process, we performed a human cytokine array. Several cytokines were significantly induced in the HCMV secretomes including interleukin-6 (IL-6), granulocyte macrophage colony-stimulating factor, and IL-8/CXCL8. Using in vitro AG assays, neutralization of IL-6 significantly reduced neovessel formation. Addition of the HCMV secretome to preformed vessels extended neovessel survival, but this effect was blocked by neutralization of IL-6. In these cells, IL-6 prevented apoptosis by blocking caspase-3 and -7 activation through the induction of survivin. Neutralization of IL-6 receptor on ECs abolished the ability of HCMV secretome to increase survivin expression and activated effector caspases. Moreover, survivin shRNA expression induced rapid regression of tubule capillary networks in ECs stimulated with HCMV secretome and activated effector caspases. These observations may explain how CMV accelerates vascular disease despite limited infection in tissues.

Herpes simplex virus type 1 inflammasome activation in proinflammatory human macrophages is dependent on NLRP3, ASC, and caspase-1.

The proinflammatory cytokines interleukin (IL)-1ß and IL-18 are products of activation of the inflammasome, an innate sensing system, and important in the pathogenesis of herpes simplex virus type 1 (HSV-1). The release of IL-18 and IL-1ß from monocytes/macrophages is critical for protection from HSV-1 based on animal models of encephalitis and genital infection, yet if and how HSV-1 activates inflammasomes in human macrophages is unknown. To investigate this, we utilized both primary human monocyte derived macrophages and human monocytic cell lines (THP-1 cells) with various inflammasome components knocked-out. We found that HSV-1 activates inflammasome signaling in proinflammatory primary human macrophages, but not in resting macrophages. Additionally, HSV-1 inflammasome activation in THP-1 cells is dependent on nucleotide-binding domain and leucine-rich repeat-containing receptor 3 (NLRP3), apoptosis-associated speck-like molecule containing a caspase recruitment domain (ASC), and caspase-1, but not on absent in melanoma 2 (AIM2), or gamma interferon-inducible protein 16 (IFI16). In contrast, HSV-1 activates non-canonical inflammasome signaling in proinflammatory macrophages that results in IL-1ß, but not IL-18, release that is independent of NLRP3, ASC, and caspase-1. Ultraviolet irradiation of HSV-1 enhanced inflammasome activation, demonstrating that viral replication suppresses inflammasome activation. These results confirm that HSV-1 is capable of activating the inflammasome in human macrophages through an NLRP3 dependent process and that the virus has evolved an NLRP3 specific mechanism to inhibit inflammasome activation in macrophages.

Characterization of a Novel Compound That Stimulates STING-Mediated Innate Immune Activity in an Allele-Specific Manner.

The innate immune response to cytosolic DNA involves transcriptional activation of type I interferons (IFN-I) and proinflammatory cytokines. This represents the culmination of intracellular signaling pathways that are initiated by pattern recognition receptors that engage DNA and require the adaptor protein Stimulator of Interferon Genes (STING). These responses lead to the generation of cellular and tissue states that impair microbial replication and facilitate the establishment of long-lived, antigen-specific adaptive immunity. Ultimately this can lead to immune-mediated protection from infection but also to the cytotoxic T cell-mediated clearance of tumor cells. Intriguingly, pharmacologic activation of STING-dependent phenotypes is known to enhance both vaccine-associated immunogenicity and immune-based anti-tumor therapies. Unfortunately, the STING protein exists as multiple variant forms in the human population that exhibit differences in their reactivity to chemical stimuli and in the intensity of molecular signaling they induce. In light of this, STING-targeting drug discovery efforts require an accounting of protein variant-specific activity. Herein we describe a small molecule termed M04 that behaves as a novel agonist of human STING. Importantly, we find that the molecule exhibits a differential ability to activate STING based on the allelic variant examined. Furthermore, while M04 is inactive in mice, expression of human STING in mouse cells rescues reactivity to the compound. Using primary human cells in ex vivo assays we were also able to show that M04 is capable of simulating innate responses important for adaptive immune activation such as cytokine secretion, dendritic cell maturation, and T cell cross-priming. Collectively, this work demonstrates the conceivable utility of a novel agonist of human STING both as a research tool for exploring STING biology and as an immune potentiating molecule.

What role for human rhinoviruses in the lower respiratory tract?

Human rhinoviruses (HRV) usually cause upper airway infections. However, viral replication in the tracheobronchial tree has been disclosed, although its clinical role is poorly known. We evaluated the prevalence of HRV in 159 bronchoalveolar lavages from 88 patients and describe a lung transplant recipient with a high HRV load in association with acute rejection. HRV was detected in 22/88 patients (25.0%): 7/18 lung transplant recipients, 11/41 immunocompetent, and 4/29 immunocompromised (p = n.s.). No lung disease was significantly associated with HRV positivity. It should be recommended to include HRV in the virological diagnostic work-up of lower respiratory specimens to elucidate their role.

Detection of PARV4, genotypes 1 and 2, in healthy and pathological clinical specimens.

The molecular epidemiology and tissue distribution of Human Parvovirus 4 (PARV4) and its variant PARV5 (Parvoviridae family) are poorly known. The aim of this study was to investigate the epidemiological role and prevalence of PARV4/5 by a nested-PCR on different clinical specimens, including blood samples from healthy donors, healthy and pathological skin samples, and bronchoalveolar lavages (BAL). Among blood specimens, 2/53 were positive; 3/37 and 23/105 of healthy and pathological skin specimens resulted positive, respectively, whereas no BAL was positive. PARV4/5 may be present in different healthy and pathological samples, suggesting the need for further investigating its tissue distribution.

Human Cytomegalovirus Immediate Early 86-kDa Protein Blocks Transcription and Induces Degradation of the Immature Interleukin-1ß Protein during Virion-Mediated Activation of the AIM2 Inflammasome.

Secretion of interleukin-1ß (IL-1ß) represents a fundamental innate immune response to microbial infection that, at the molecular level, occurs following activation of proteolytic caspases that cleave the immature protein into a secretable form. Human cytomegalovirus (HCMV) is the archetypal betaherpesvirus that is invariably capable of lifelong infection through the activity of numerous virally encoded immune evasion phenotypes. Innate immune pathways responsive to cytoplasmic double-stranded DNA (dsDNA) are known to be activated in response to contact between HCMV and host cells. Here, we used clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) genome editing to demonstrate that the dsDNA receptor absent in melanoma 2 (AIM2) is required for secretion of IL-1ß following HCMV infection. Furthermore, dsDNA-responsive innate signaling induced by HCMV infection that leads to activation of the type I interferon response is also shown, unexpectedly, to play a contributory role in IL-1ß secretion. Importantly, we also show that rendering virus particles inactive by UV exposure leads to substantially increased IL-1ß processing and secretion and that live HCMV can inhibit this, suggesting the virus encodes factors that confer an inhibitory effect on this response. Further examination revealed that ectopic expression of the immediate early (IE) 86-kDa protein (IE86) is actually associated with a block in transcription of the pro-IL-1ß gene and, independently, diminishment of the immature protein. Overall, these results reveal two new and distinct phenotypes conferred by the HCMV IE86 protein, as well as an unusual circumstance in which a single herpesviral protein exhibits inhibitory effects on multiple molecular processes within the same innate immune response.IMPORTANCE Persistent infection with HCMV is associated with the operation of diverse evasion phenotypes directed at antiviral immunity. Obstruction of intrinsic and innate immune responses is typically conferred by viral proteins either associated with the viral particle or expressed immediately after entry. In line with this, numerous phenotypes are attributed to the HCMV IE86 protein that involve interference with innate immune processes via transcriptional and protein-directed mechanisms. We describe novel IE86-mediated phenotypes aimed at virus-induced secretion of IL-1ß. Intriguingly, while many viruses target the function of the molecular scaffold required for IL-1ß maturation to prevent this response, we find that HCMV and IE86 target the IL-1ß protein specifically. Moreover, we show that IE86 impairs both the synthesis of the IL-1ß transcript and the stability of the immature protein. This indicates an unusual phenomenon in which a single viral protein exhibits two molecularly separate evasion phenotypes directed at a single innate cytokine.

Sensitivity to the evaluation of others emerges by 24 months.

Although the human proclivity to engage in impression management and care for reputation is ubiquitous, the question of its developmental outset remains open. In 4 studies, we demonstrate that the sensitivity to the evaluation of others (i.e., evaluative audience perception) is manifest by 24 months. In a first study, 14- to 24-month-old children (N = 49) were tested in situations in which the attention of an audience was systematically manipulated. Results showed that when the experimenter was inattentive, as opposed to attentive, children were more likely to explore an attractive toy. A second study (N = 31) explored whether same-aged children would consider not only the attention of the experimenter but also the values the experimenter expressed for two different outcomes when exploring a toy. We found that children reproduced outcomes that were positively valued by the experimenter significantly more when the experimenter was attentive but were more likely to reproduce negatively valued outcomes when the experimenter was inattentive. A third control study (N = 30) showed that the significant effect of Study 2 disappeared in the absence of different values. Lastly, Study 4 (N = 34) replicated and extended the phenomenon by showing toddler's propensity to modify their behavior in the presence of 2 different experimenters, depending on both the experimenter's evaluation of an outcome and their attention. Overall, these data provide the first convergent demonstration of evaluative audience perception in young children that precedes the full-fledged normative, mentalizing, and strong conformity psychology documented in 4- to 5-year-old children. (PsycINFO Database Record

The Heme Metabolite Carbon Monoxide Facilitates KSHV Infection by Inhibiting TLR4 Signaling in Endothelial Cells.

Kaposi sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi sarcoma (KS) and certain rare B cell lymphoproliferative disorders. KSHV infection of endothelial cells (EC) in vitro increases expression of the inducible host-encoded enzyme heme oxygenase-1 (HO-1), which is also strongly expressed in KS tumors. HO-1 catalyzes the rate-limiting step in the conversion of heme into iron, biliverdin and the gasotransmitter carbon monoxide (CO), all of which share anti-apoptotic, anti-inflammatory, pro-survival, and tumorigenic activities. Our previous work has shown that HO-1 expression in KSHV-infected EC is characterized by a rapid yet transient induction at early times post-infection, followed by a sustained upregulation co-incident with establishment of viral latency. These two phases of expression are independently regulated, suggesting distinct roles for HO-1 in the virus life cycle. Here, we investigated the role of HO-1 during acute infection, prior to the onset of viral gene expression. The early infection phase involves a series of events that culminate in delivery of the viral genome to the nucleus. Primary infection also leads to activation of host innate immune effectors, including the pattern recognition receptor TLR4, to induce an antiviral response. It has been shown that TLR4-deficient EC are more susceptible to KSHV infection than wild-type controls, suggesting an important inhibitory role for TLR4 in the KSHV life cycle. TLR4 signaling is in turn subject to regulation by several virus-encoded immune evasion factors. In this report we identify HO-1 as a host protein co-opted by KSHV as part of a rapid immune evasion strategy. Specifically, we show that early HO-1 induction by KSHV results in increased levels of endogenous CO, which functions as a TLR4 signaling inhibitor. In addition, we show that CO-mediated inhibition of TLR4 signaling leads to reduced expression of TLR4-induced antiviral genes, thus dampening the host antiviral response and facilitating KSHV infection. Conversely, inhibition of HO-1 activity decreases intracellular CO, enhances the host antiviral response and inhibits KSHV infection. In conclusion, this study identifies HO-1 as a novel innate immune evasion factor in the context of KSHV infection and supports HO-1 inhibition as a viable therapeutic strategy for KS.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus.

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

Kaposi Sarcoma Herpesvirus Induces HO-1 during De Novo Infection of Endothelial Cells via Viral miRNA-Dependent and -Independent Mechanisms.

UNLABELLED: Kaposi sarcoma (KS) herpesvirus (KSHV) infection of endothelial cells (EC) is associated with strong induction of heme oxygenase-1 (HO-1), a stress-inducible host gene that encodes the rate-limiting enzyme responsible for heme catabolism. KS is an angioproliferative tumor characterized by the proliferation of KSHV-infected spindle cells, and HO-1 is highly expressed in such cells. HO-1 converts the pro-oxidant, proinflammatory heme molecule into metabolites with antioxidant, anti-inflammatory, and proliferative activities. Previously published work has shown that KSHV-infected EC in vitro proliferate in response to free heme in a HO-1-dependent manner, thus implicating virus-enhanced HO-1 activity in KS tumorigenesis. The present study investigated the molecular mechanisms underlying KSHV induction of HO-1 in lymphatic EC (LEC), which are the likely spindle cell precursors. In a time course analysis of KSHV-infected cells, HO-1 expression displays biphasic kinetics characterized by an early transient induction that is followed by a more sustained upregulation coincident with the establishment of viral latency. A viral microRNA miR-K12-11 deletion mutant of KSHV was found to be defective for induction of HO-1 during latency. A potential mechanism for this phenotype was provided by BACH1, a cellular HO-1 transcriptional repressor targeted by miR-K12-11. In fact, in KSHV-infected LEC, the BACH1 message level is reduced, BACH1 subcellular localization is altered, and miR-K12-11 mediates the inverse regulation of HO-1 and BACH1 during viral latency. Interestingly, the data indicate that neither miR-K12-11 nor de novo KSHV gene expression is required for the burst of HO-1 expression observed at early times postinfection, which suggests that additional virion components promote this phenotype. IMPORTANCE: While the mechanisms underlying KSHV induction of HO-1 remain unknown, the cellular mechanisms that regulate HO-1 expression have been extensively investigated in the context of basal and pathophysiological states. The detoxifying action of HO-1 is critical for the protection of cells exposed to high heme levels. KS spindle cells are erythrophagocytic and contain erythrocyte ghosts. Erythrocyte degeneration leads to the localized release of heme, creating oxidative stress that may be further exacerbated by environmental or other cofactors. Our previous work showed that KSHV-infected cells proliferate in response to heme and that this occurs in a HO-1-dependent manner. We therefore hypothesize that KSHV induction of HO-1 contributes to KS tumor development via heme metabolism and propose that HO-1 be evaluated as a therapeutic target for KS. Our present work, which aimed to understand the mechanisms whereby KSHV induces HO-1, will be important for the design and implementation of such a strategy.

A cytomegalovirus-based vaccine provides long-lasting protection against lethal Ebola virus challenge after a single dose.

Ebola virus (Zaire ebolavirus; EBOV) is a highly lethal hemorrhagic disease virus that most recently was responsible for two independent 2014 outbreaks in multiple countries in Western Africa, and the Democratic Republic of the Congo, respectively. Herein, we show that a cytomegalovirus (CMV)-based vaccine provides durable protective immunity from Ebola virus following a single vaccine dose. This study has implications for human vaccination against ebolaviruses, as well as for development of a 'disseminating' vaccine to target these viruses in wild African great apes.

Human cytomegalovirus-encoded pUL7 is a novel CEACAM1-like molecule responsible for promotion of angiogenesis.

UNLABELLED: Persistent human cytomegalovirus (HCMV) infection has been linked to several diseases, including atherosclerosis, transplant vascular sclerosis (TVS), restenosis, and glioblastoma. We have previously shown that factors secreted from HCMV-infected cells induce angiogenesis and that this process is due, at least in part, to increased secretion of interleukin-6 (IL-6). In order to identify the HCMV gene(s) responsible for angiogenesis promotion, we constructed a large panel of replication-competent HCMV recombinants. One HCMV recombinant deleted for UL1 to UL10 was unable to induce secretion of factors necessary for angiogenesis. Fine mapping using additional HCMV recombinants identified UL7 as a viral gene required for production of angiogenic factors from HCMV-infected cells. Transient expression of pUL7 induced phosphorylation of STAT3 and ERK1/2 MAP kinases and production of proangiogenic factors, including IL-6. Addition of recombinant pUL7 to cells was sufficient for angiogenesis and was again associated with increased IL-6 expression. Analysis of the UL7 structure revealed a conserved domain similar to the immunoglobulin superfamily domain and related to the N-terminal V-like domain of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Our report therefore identifies UL7 as a novel HCMV-encoded molecule that is both structurally and functionally related to cellular CEACAM1, a proangiogenic factor highly expressed during vasculogenesis. IMPORTANCE: A hallmark of cytomegalovirus (CMV) infection is its ability to modulate the host cellular machinery, resulting in the secretion of factors associated with long-term diseases such as vascular disorders and cancer. We previously demonstrated that HCMV infection alters the types and quantities of bioactive proteins released from cells (designated the HCMV secretome) that are involved in the promotion of angiogenesis and wound healing. A key proangiogenic and antiapoptotic factor identified from a proteomic-based approach was IL-6. In the present report, we show for the first time that HCMV UL7 encodes a soluble molecule that is a structural and functional homologue of the CEACAM1 proangiogenic cellular factor. This report thereby identifies a critical component of the HCMV secretome that may be responsible, at least in part, for the vascular dysregulation associated with persistent HCMV infection.

Aberrant proliferation in CXCR7+ endothelial cells via degradation of the retinoblastoma protein.

Angiogenesis is a critical factor in the growth and dissemination of solid tumors. Indeed, tumor vasculature is abnormal and contributes to the development and spread of malignancies by creating a hostile microenvironment. The alternative SDF-1/CXCL12 receptor, CXCR7, is frequently and specifically expressed in tumor-associated vessels. In this study, we examine the role of endothelium-expressed CXCR7 in tumor vascular dysfunction by specifically examining the contribution of CXCR7 to endothelial cell (EC) proliferation. We demonstrate that CXCR7 expression is sufficient to drive post-confluent growth in EC cultures. Further, we provide a novel mechanism for CXCR7-mediated proliferation via proteasomal degradation of the tumor suppressor protein Rb. These findings identify a heretofore unappreciated role for CXCR7 in vascular dysfunction and confirm this receptor as a plausible target for anti-tumor therapy.

A cytomegalovirus-based vaccine expressing a single tumor-specific CD8+ T-cell epitope delays tumor growth in a murine model of prostate cancer.

Cytomegalovirus (CMV) is a highly immunogenic virus that results in a persistent, life-long infection in the host typically with no ill effects. Certain unique features of CMV, including its capacity to actively replicate in the presence of strong host CMV-specific immunity, may give CMV an advantage compared with other virus-based vaccine delivery platforms. In the present study, we tested the utility of mouse CMV (mCMV)-based vaccines expressing human prostate-specific antigen (PSA) for prostate cancer immunotherapy in double-transgenic mice expressing PSA and HLA-DRB1*1501 (DR2bxPSA F1 mice). We assessed the capacity of 2 mCMV-based vectors to induce PSA-specific CD8 T-cell responses and affect the growth of PSA-expressing Transgenic Adenocarcinoma of the Mouse Prostate tumors (TRAMP-PSA). In the absence of tumor challenge, immunization with mCMV vectors expressing either a H2-D(b)-restricted epitope PSA(65-73) (mCMV/PSA(65-73)) or the full-length gene for PSA (mCMV/PSA(FL)) induced comparable levels of CD8 T-cell responses that increased (inflated) with time. Upon challenge with TRAMP-PSA tumor cells, animals immunized with mCMV/PSA(65-73) had delay of tumor growth and increased PSA-specific CD8 T-cell responses, whereas animals immunized with mCMV/PSA(FL) showed progressive tumor growth and no increase in number of splenic PSA(65-73)-specific T cells. The data show that a prototype CMV-based prostate cancer vaccine can induce an effective antitumor immune response in a "humanized" double-transgenic mouse model. The observation that mCMV/PSA(FL) is not effective against TRAMP-PSA is consistent with our previous findings that HLA-DRB1*1501-restricted immune responses to PSA are associated with suppression of effective CD8 T-cell responses to TRAMP-PSA tumors.

A replicating cytomegalovirus-based vaccine encoding a single Ebola virus nucleoprotein CTL epitope confers protection against Ebola virus.

BACKGROUND: Human outbreaks of Ebola virus (EBOV) are a serious human health concern in Central Africa. Great apes (gorillas/chimpanzees) are an important source of EBOV transmission to humans due to increased hunting of wildlife including the 'bush-meat' trade. Cytomegalovirus (CMV) is an highly immunogenic virus that has shown recent utility as a vaccine platform. CMV-based vaccines also have the unique potential to re-infect and disseminate through target populations regardless of prior CMV immunity, which may be ideal for achieving high vaccine coverage in inaccessible populations such as great apes. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesize that a vaccine strategy using CMV-based vectors expressing EBOV antigens may be ideally suited for use in inaccessible wildlife populations. To establish a 'proof-of-concept' for CMV-based vaccines against EBOV, we constructed a mouse CMV (MCMV) vector expressing a CD8+ T cell epitope from the nucleoprotein (NP) of Zaire ebolavirus (ZEBOV) (MCMV/ZEBOV-NP(CTL)). MCMV/ZEBOV-NP(CTL) induced high levels of long-lasting (>8 months) CD8+ T cells against ZEBOV NP in mice. Importantly, all vaccinated animals were protected against lethal ZEBOV challenge. Low levels of anti-ZEBOV antibodies were only sporadically detected in vaccinated animals prior to ZEBOV challenge suggesting a role, at least in part, for T cells in protection. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the ability of a CMV-based vaccine approach to protect against an highly virulent human pathogen, and supports the potential for 'disseminating' CMV-based EBOV vaccines to prevent EBOV transmission in wildlife populations.

Quantitative detection of Epstein-Barr virus in bronchoalveolar lavage from transplant and nontransplant patients.

BACKGROUND: The lower respiratory tract is a latency site of Epstein-Barr virus (EBV); however, its pathogenic role is poorly known, particularly in transplant patients. The aim of this study was to evaluate the prevalence and role of EBV in bronchoalveolar lavages (BAL) from transplant recipients (TR) in comparison with nontransplant (NT) patients. METHODS: Real-time quantitative polymerase chain reaction for EBV, human herpesvirus-6 (HHV-6), and HHV-7 and rapid shell-vial culture for human cytomegalovirus (HCMV) were performed on 272 consecutive BAL from 194 patients (107 from 59 TR and 165 from 143 NT). RESULTS: EBV-DNA was positive in 65 specimens (23.9%) from 57 patients (29.4%): 24 of 59 (40.7%) TR and 33 of 143 (23.1%) NT (P<0.05). There was no significant difference of EBV positivity considering the type of transplanted organ. Viral load did not significantly differ comparing specimens of TR versus NT, specimens of solid organ transplant versus bone marrow transplant recipients. EBV was frequently positive in patients with a diagnosis of pneumonia (28.6%), respiratory insufficiency (24.5%), and exacerbation of underlying bronchopneumopathies (30.8%); however, there was no difference comparing TR and NT. EBV was mostly detected in concomitance with other infectious pathogens. Mortality within 28 days of BAL sampling was not related to EBV-DNA positivity and load. CONCLUSIONS: EBV is frequently detected in BAL from TR and NT; however, its pathogenic role in lower respiratory tract remains poorly known, also because of the frequent detection of concomitant infectious pathogens. Further studies are needed to better elucidate this issue and the underlying local conditions favoring viral replication.