Pharmacokinetics and Safety of the Nucleoside Analog Antiviral Drug Galidesivir Administered to Healthy Adult Subjects.

Galidesivir (BCX4430) is an adenosine nucleoside analog broadly active in cell culture against multiple RNA virus families, and active in animal models of viral diseases associated with Ebola, Marburg, yellow fever, Zika, and Rift Valley fever. Current studies demonstrated the pharmacokinetics and safety of the first-in-human evaluations of galidesivir as intramuscular (IM) and intravenous (IV) formulations. Two double-blind, placebo-controlled, dose-ranging studies were conducted enrolling 126 healthy subjects. Study 1 evaluated the safety and tolerability of IM galidesivir over single day dosing, single day dosing ± lidocaine, and 7-day dosing with lidocaine. Study 2 evaluated the safety and tolerability of single ascending doses of IV galidesivir. Safety and tolerability were evaluated via clinical and laboratory monitoring. The plasma concentration-time profile of galidesivir at doses 0.3 to 10 mg/kg IM was characterized by rapid absorption, an initial rapid distribution and clearance phase, and an extended terminal elimination phase. The initial rapid distribution and extended terminal elimination were mimicked in the profile of galidesivir at doses 5 to 20 mg/kg IV. No fatal events or related serious adverse events were reported. No clinically significant dose-related trends in laboratory values, vital signs, electrocardiograms, or echocardiograms were noted. Galidesivir was safe and generally well tolerated.

An update on the progress of galidesivir (BCX4430), a broad-spectrum antiviral.

Galidesivir (BCX4430) is an adenosine nucleoside analog that is broadly active in cell culture against several RNA viruses of various families. This activity has also been shown in animal models of viral disease associated with Ebola, Marburg, yellow fever, Zika, and Rift Valley fever viruses. In many cases, the compound is more efficacious in animal models than cell culture activity would predict. Based on favorable data from in vivo animal studies, galidesivir has recently undergone evaluation in several phase I clinical trials, including against severe acute respiratory syndrome coronavirus 2, and as a medical countermeasure for the treatment of Marburg virus disease.

Activity of Galidesivir in a Hamster Model of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) has claimed the lives of millions of people worldwide since it first emerged. The impact of the COVID-19 pandemic on public health and the global economy has highlighted the medical need for the development of broadly acting interventions against emerging viral threats. Galidesivir is a broad-spectrum antiviral compound with demonstrated in vitro and in vivo efficacy against several RNA viruses of public health concern, including those causing yellow fever, Ebola, Marburg, and Rift Valley fever. In vitro studies have shown that the antiviral activity of galidesivir also extends to coronaviruses. Herein, we describe the efficacy of galidesivir in the Syrian golden hamster model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Treatment with galidesivir reduced lung pathology in infected animals compared with untreated controls when treatment was initiated 24 h prior to infection. These results add to the evidence of the applicability of galidesivir as a potential medical intervention for a range of acute viral illnesses, including coronaviruses.

Latent and lytic Epstein-Barr virus gene expression in the peripheral blood of astronauts.

Epstein-Barr virus (EBV) latent and replicative gene transcription was analyzed in peripheral blood B-lymphocytes from astronauts who flew on short-duration (∼11 days) Shuttle missions and long-duration (∼180 days) International Space Station (ISS) missions. Latent, immediate-early, and early gene replicative viral transcripts were detected in samples from six astronauts who flew on short-duration Shuttle missions, whereas viral gene transcription was mostly absent in samples from 24 healthy donors. Samples from six astronauts who flew on long-duration ISS missions were characterized by expanded expression of latent, immediate-early, and early gene transcripts and new onset expression of late replicative transcription upon return to Earth. These data indicate that EBV-infected cells are no longer expressing the restricted set of viral genes that characterize latency but are expressing latent and lytic gene transcripts. These data also suggest the possibility of EBV-related complications in future long-duration missions, in particular interplanetary travel.

Chronic herpesvirus reactivation occurs in aging.

The aged immune system is characterized by clonal expansions of CD8+ T cells of which a substantial portion are directed against Epstein-Barr virus (EBV) and cytomegalovirus (CMV). It is unknown if these expansions represent increased viral reactivation or simply reflect an accumulation over time. We investigated herpesvirus reactivation in young and old subjects co-infected with CMV and EBV. Using molecular and serological techniques, we found significant increases in both the frequency and magnitude of EBV and CMV reactivation in elderly subjects. CMV DNA was frequently detected in the urine of elderly subjects; EBV load in peripheral blood was also significantly increased. Notably, EBV DNA in plasma was detected in a majority of the elderly subjects which was supported by frequent transcription of late structural genes. Furthermore, CD8+ T cells specific for EBV structural antigens were detected in samples from the elderly. Samples from our younger control group were negative for EBV DNA in plasma, CMV DNA in urine, expression of structural transcripts, and lacked CD8+ T cells specific for EBV structural antigens. These findings indicate that the aged immune system is no longer able to control EBV and CMV reactivation that could now be characterized as chronic instead of latent.

Epstein-Barr virus infection of Langerhans cell precursors as a mechanism of oral epithelial entry, persistence, and reactivation.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with many malignant and nonmalignant human diseases. Life-long latent EBV persistence occurs in blood-borne B lymphocytes, while EBV intermittently productively replicates in mucosal epithelia. Although several models have previously been proposed, the mechanism of EBV transition between these two reservoirs of infection has not been determined. In this study, we present the first evidence demonstrating that EBV latently infects a unique subset of blood-borne mononuclear cells that are direct precursors to Langerhans cells and that EBV both latently and productively infects oral epithelium-resident cells that are likely Langerhans cells. These data form the basis of a proposed new model of EBV transition from blood to oral epithelium in which EBV-infected Langerhans cell precursors serve to transport EBV to the oral epithelium as they migrate and differentiate into oral Langerhans cells. This new model contributes fresh insight into the natural history of EBV infection and the pathogenesis of EBV-associated epithelial disease.

Oncogenic activity of Epstein-Barr virus latent membrane protein 1 (LMP-1) is down-regulated by lytic LMP-1.

The Epstein-Barr virus (EBV) is an oncogenic human herpesvirus. EBV latent membrane protein 1 (LMP-1) is a viral oncogene that manifests its oncogenic phenotype through activation of cellular signaling pathways involved in cell growth, survival, differentiation, and transformation. Lytic LMP-1 (lyLMP-1) is a related EBV gene without oncogenic properties. The lyLMP-1 gene is found in 60% of the EBV strains circulating in nature, but it is not found in EBV strains associated with nasopharyngeal carcinoma. We recently demonstrated that lyLMP-1 down-regulates the half-life of LMP-1 in epithelial cells. Therefore in this study, we tested the hypothesis that lyLMP-1 concomitantly down-regulates LMP-1 oncogenic activity. The results demonstrated that lyLMP-1 inhibits LMP-1-mediated intracellular signaling activation, epithelial cell growth and survival, and fibroblast cell transformation in a dose-dependent manner. Lytic LMP-1 manifested this effect through the promotion of LMP-1 degradation and a reduction in the expressed quantity of LMP-1. Thus, lyLMP-1 functions as a posttranslational negative regulator of LMP-1 oncogenesis. These results support a model of EBV-associated epithelial oncogenesis in which lyLMP-1 may act in vivo to reduce the risk of LMP-1-mediated transformation and is therefore subjected to negative selection in nasopharyngeal carcinoma pathogenesis.

Epstein-Barr virus latent membrane protein 1 (LMP-1) half-life in epithelial cells is down-regulated by lytic LMP-1.

This study examined the effect of naturally occurring Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) gene sequence variation on the LMP-1 half-life in epithelial cells. The LMP-1 half-life was not influenced by sequence variation in amino acids 250 to 307 or amino acids 343 to 352. The LMP-1 half-life was short when the amino acid encoded at position 129 was methionine, the initiation codon product of lytic LMP-1 (lyLMP-1). The mutation of amino acid 129 to isoleucine greatly increased the LMP-1 half-life. Expression of lyLMP-1 in trans down-regulated the LMP-1 half-life in a dose-dependent manner and restored a short-half-life phenotype to the mutated LMP-1 construct lacking the cis ability to express lyLMP-1. This observed dominant negative effect of lyLMP-1 expression on the LMP-1 half-life in epithelial cells in vitro may have implications for EBV epithelial oncogenesis in vivo.

Molecular markers of clonality and identity in Epstein-Barr virus-associated B-cell lymphoproliferative disease.

Epstein-Barr virus (EBV)-associated B-cell lymphoproliferative disease may be polyclonal, oligoclonal, or monoclonal. The degree of tumor clonality reflects the disease pathogenesis and may have implications for disease diagnosis, prognosis, and treatment. In this study, specimens of EBV-associated B-cell lymphoproliferative disease obtained from immunocompromised hosts were analyzed for molecular markers of cellular and virologic clonality and virologic identity. Each tumor specimen was assessed for immunoglobulin gene JH region rearrangement, the structure of the EBV genome termini, and the EBV genotype(s) present using a new EBV genotyping assay based upon LMP-1 gene sequence variation. The results of the JH rearrangement and EBV termini assays were generally concordant in their assessment of tumor specimen clonality, and both assays contributed to establishing clonal identity between different tumor specimens. The EBV genotyping assay did not significantly contribute to the assessment of tumor clonality but did established clear virologic identity between different tumor specimens obtained from the same individual. In one individual, these three assays together characterized a multi-focal, monoclonal tumor that may have arisen through clonal selection after sequential infections with two different EBV genotypes. In summary, the JH rearrangement and EBV termini assays each provided different but complementary information on tumor clonality, while the EBV genotyping assay proved most useful for establishing virologic identity among tumors. Utilization of these three assays together may provide new insight into the pathogenesis of EBV-associated B-cell lymphoproliferative disease.

Persistence and transition of Epstein-Barr virus genotypes in the pathogenesis of oral hairy leukoplakia.

This prospective study examined the persistence and transition of Epstein-Barr virus (EBV) in human immunodeficiency virus (HIV)-seropositive subjects with and without oral hairy leukoplakia, a replicative EBV-associated epithelial disease. The intrahost molecular epidemiology of EBV infection was characterized in subjects treated with valacyclovir to suppress EBV replication. Tongue epithelial tissues of HIV-seropositive subjects were found to support not only EBV replication but also persistent, nonproductive EBV infection. EBV appeared to enter the tongue from the blood reservoir of infection and, possibly, from exogenous sources as well. EBV transition from the blood to the tongue appeared to occur even during valacyclovir-mediated suppression of EBV replication, suggesting EBV entry into tongue epithelial tissue as a cell-associated latent infection. In conclusion, these results describe the persistence and transition of EBV as a dynamic interaction between the blood and epithelial reservoirs of EBV infection and suggest a role for entry, persistence, and reactivation of oral epithelial EBV in the pathogenesis of oral hairy leukoplakia.

Expression of Epstein-Barr virus latent genes in oral epithelium: determinants of the pathogenesis of oral hairy leukoplakia.

This retrospective study examined expression of Epstein-Barr virus (EBV) latent genes in oral epithelium from human immunodeficiency virus-seropositive subjects, to identify genes associated with the pathogenesis of oral hairy leukoplakia (HLP). Transcription of EBV latent genes was detected in tissues with productive EBV replication and, also, in normal oral epithelial tissues without EBV replication. Expression of the EBV EBNA-2 open-reading frame in oral epithelium was identified as an important cofactor associated with the pathogenesis of HLP. In vitro experiments suggested that a recombinant variant of the EBNA-2 gene may play a role in the pathogenesis of HLP, through modulation of EBNA-2 protein function.

Effect of Epstein-Barr virus replication on Langerhans cells in pathogenesis of oral hairy leukoplakia.

Epstein-Barr virus (EBV) replicates productively in oral hairy leukoplakia (HLP). One characteristic of human immunodeficiency virus (HIV)-associated HLP is a decreased oral epithelial Langerhans cell count. This prospective study tested the hypothesis that oral epithelial EBV replication decreases oral Langerhans cell counts. EBV replication in HLP was highly correlated with decreased oral Langerhans cell counts. Inhibition of EBV replication restored oral Langerhans cell counts to normal control levels, and the return of EBV replication after treatment resulted in a recurrent decline in oral Langerhans cell counts. Decreased oral Langerhans cell counts occurred independently of HIV infection, as demonstrated in HLP of otherwise healthy HIV-seronegative individuals. These results support the tested hypothesis and suggest that EBV manipulates and evades the mucosal immune response in oral epithelial infection. This novel EBV strategy for eliminating oral Langerhans cells may facilitate the persistence of oral epithelial EBV and may contribute to the pathogenesis of HLP.

Epstein-Barr virus replication in oral hairy leukoplakia: response, persistence, and resistance to treatment with valacyclovir.

Nineteen cases of human immunodeficiency virus (HIV)-associated oral hairy leukoplakia (HLP) and Epstein-Barr virus (EBV) replication were treated with high-dose oral valacyclovir to inhibit productive EBV replication. The clinical, histopathological, and molecular viral responses to treatment were assessed in surgical biopsy specimens obtained before, during, and after treatment. In the majority of treated cases, HLP was resolved, and EBV replication was terminated. In many cases, the initial response to inhibition of replication was a persistent, nonproductive, EBV infection of the oral mucosa, characterized by limited expression of replicative EBV genes, especially BZLF1. In some cases, productive EBV replication recurred after discontinuation of treatment with valacyclovir. In a few treated cases, treatment failed, and productive EBV replication persisted, possibly because of the evolution of acyclovir-resistant EBV. In summary, safe treatment of HLP and of EBV replication, with valacyclovir, provides new insight into the mechanisms of EBV persistence in oral mucosa.

Multiple Epstein-Barr virus infections in healthy individuals.

We employed a newly developed genotyping technique with direct representational detection of LMP-1 gene sequences to study the molecular epidemiology of Epstein-Barr virus (EBV) infection in healthy individuals. Infections with up to five different EBV genotypes were found in two of nine individuals studied. These results support the hypothesis that multiple EBV infections of healthy individuals are common. The implications for the development of an EBV vaccine are discussed.

A non-invasive technique for studying oral epithelial Epstein-Barr virus infection and disease.

Oral Epstein-Barr virus (EBV) infection is associated with hairy leukoplakia and possibly other oral diseases. Many studies of oral EBV infection utilize surgical specimens. This study tested a non-invasive brush biopsy technique as an alternative to surgical biopsy to study oral EBV infection and disease. Paired, same-site, samples of tongue epithelium were obtained from research subjects, first by brush and then by surgical biopsy. Brush cells and surgical specimens were fixed and prepared for histologic sectioning and/or processed for nucleic acid extraction. Brush cell pellet sections proved equivalent to surgical specimen tissue sections for hairy leukoplakia diagnosis by routine histologic staining and EBV immunohistochemistry or in situ hybridization. Amplification of EBV sequences demonstrated superiority of the brush cells over surgical specimens for both sensitivity (90% vs. 73%) and negative predictive value (93% vs. 82%). This non-invasive brush biopsy technique should facilitate larger, prospective studies of oral EBV infection and pathogenesis.

Unexpected absence of the Epstein-Barr virus (EBV) lyLMP-1 open reading frame in tumor virus isolates: lack of correlation between Met129 status and EBV strain identity.

The lytic cycle-associated lytic latent membrane protein-1 (lyLMP-1) of Epstein-Barr virus (EBV) is an amino-terminally truncated form of the oncogenic LMP-1. Although lyLMP-1 shares none of LMP-1's transforming and signal transducing activities, we recently reported that lyLMP-1 can negatively regulate LMP-1-stimulated NF-kappaB activation. The lyLMP-1 protein encoded by the B95-8 strain of EBV initiates from methionine 129 (Met129) of the LMP-1 open reading frame (ORF). The recent report that Met129 in the B95-8 LMP-1 ORF is not conserved in the Akata strain of EBV prompted us to screen a panel of EBV-positive cell lines for conservation of Met129 and lyLMP-1 expression. We found that 15 out of 16 tumor-associated virus isolates sequenced encoded an ATT or ACC codon in place of ATG in the LMP-1 ORF at position 129, and tumor cell lines harboring isolates lacking an ATG at codon 129 did not express the lyLMP-1 protein. In contrast, we found that EBV DNA from 22 out of 37 healthy seropositive donors retained the Met129 codon. Finally, the lyLMP-1 initiator occurs variably within distinct EBV strains and its presence cannot be predicted by EBV strain identity. Thus, Met129 is not peculiar to the B95-8 strain of EBV, but rather can be found in the background of several evolutionarily distinct EBV strains. Its absence from EBV isolates from tumors raises the possibility of selective pressure on Met129 in EBV-dependent tumors.

Primary Nocardia osteomyelitis as a presentation of AIDS.

Human infection with Nocardia species presents as a wide range of clinical syndromes. Nocardia is an important opportunistic pathogen in immunocom-promised patients. We report a case of primary Nocardia asteroides osteomyelitis as the initial clinical presentation of AIDS. The infection was successfully treated with a prolonged course of trimethoprim-sulfamethoxazole in conjunction with HAART. Nocardia osteomyelitis should be recognized as an unusual but important and treatable opportunistic infection in patients living with HIV infection.