High Levels Of Viral Replication Research Articles

Elevated levels of cell-free NKG2D-ligands modulate NKG2D surface expression and compromise NK cell function in severe COVID-19 disease.

It is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2. To address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease. Although NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies. These observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.

Adipose Tissues from Human and Bat-Derived Cell Lines Support Ebola Virus Infection.

Ebola virus is a zoonotic pathogen with a geographic range covering diverse ecosystems that are home to many potential reservoir species. Although researchers have detected Ebola virus RNA and serological evidence of previous infection in different rodents and bats, the infectious virus has not been isolated. The field is missing critical knowledge about where the virus is maintained between outbreaks, either because the virus is rarely encountered, overlooked during sampling, and/or requires specific unknown conditions that regulate viral expression. This study assessed adipose tissue as a previously overlooked tissue capable of supporting Ebola virus infection. Adipose tissue is a dynamic endocrine organ helping to regulate and coordinate homeostasis, energy metabolism, and neuroendocrine and immune functions. Through in vitro infection of human and bat (Eptesicus fuscus) brown adipose tissue cultures using wild-type Ebola virus, this study showed high levels of viral replication for 28 days with no qualitative indicators of cytopathic effects. In addition, alterations in adipocyte metabolism following long-term infection were qualitatively observed through an increase in lipid droplet number while decreasing in size, a harbinger of lipolysis or adipocyte browning. The finding that bat and human adipocytes are susceptible to Ebola virus infection has important implications for potential tissue tropisms that have not yet been investigated. Additionally, the findings suggest how the metabolism of this tissue may play a role in pathogenesis, viral transmission, and/or zoonotic spillover events.

The Timing and Magnitude of the Type I Interferon Response Are Correlated with Disease Tolerance in Arbovirus Infection.

Infected hosts possess two alternative strategies to protect themselves against the negative impact of virus infections: resistance, used to abrogate virus replication, and disease tolerance, used to avoid tissue damage without controlling viral burden. The principles governing pathogen resistance are well understood, while less is known about those involved in disease tolerance. Here, we studied bluetongue virus (BTV), the cause of bluetongue disease of ruminants, as a model system to investigate the mechanisms of virus-host interactions correlating with disease tolerance. BTV induces clinical disease mainly in sheep, while cattle are considered reservoirs of infection, rarely exhibiting clinical symptoms despite sustained viremia. Using primary cells from multiple donors, we show that BTV consistently reaches higher titers in ovine cells than cells from cattle. The variable replication kinetics of BTV in sheep and cow cells were mostly abolished by abrogating the cell type I interferon (IFN) response. We identified restriction factors blocking BTV replication, but both the sheep and cow orthologues of these antiviral genes possess anti-BTV properties. Importantly, we demonstrate that BTV induces a faster host cell protein synthesis shutoff in primary sheep cells than cow cells, which results in an earlier downregulation of antiviral proteins. Moreover, by using RNA sequencing (RNA-seq), we also show a more pronounced expression of interferon-stimulated genes (ISGs) in BTV-infected cow cells than sheep cells. Our data provide a new perspective on how the type I IFN response in reservoir species can have overall positive effects on both virus and host evolution. IMPORTANCE The host immune response usually aims to inhibit virus replication in order to avoid cell damage and disease. In some cases, however, the infected host avoids the deleterious effects of infection despite high levels of viral replication. This strategy is known as disease tolerance, and it is used by animal reservoirs of some zoonotic viruses. Here, using a virus of ruminants (bluetongue virus [BTV]) as an experimental system, we dissected virus-host interactions in cells collected from species that are susceptible (sheep) or tolerant (cow) to disease. We show that (i) virus modulation of the host antiviral type I interferon (IFN) responses, (ii) viral replication kinetics, and (iii) virus-induced cell damage differ in tolerant and susceptible BTV-infected cells. Understanding the complex virus-host interactions in disease tolerance can allow us to disentangle the critical balance between protective and damaging host immune responses.

Use of Zebrafish Embryos To Reproduce Human Norovirus and To Evaluate Human Norovirus Infectivity Decay after UV Treatment.

This study reports an essential improvement of the method for replication of human norovirus (HNoV) with the use of zebrafish (Danio rerio) embryos. With three HNoV genotypes and P-types GII.2[P16], GII.4[P16], and GII.17[P31], we demonstrated that this tool had higher efficiency and robustness than the zebrafish larvae as reported previously. When zebrafish larvae were injected with virus (1.6 ± 0.3 log genome copies/10 larvae), a significant increase of virus genome copies was detected at 2 days postinfection (dpi; 4.4 ± 0.8 log genome copies/10 larvae, P < 0.05) and the viral loads started to decrease gradually from 3 dpi. In comparison, when the viruses were injected into the zebrafish embryos, significant virus replication was noticed from 1 dpi and lasted to 6 dpi (P < 0.05). The virus levels detected at 3 dpi had the highest mean value and the smallest variation (7.7 ± 0.2 log genome copies/10 larvae). The high levels of virus replication enabled continuous passaging for all three strains up to four passages. The zebrafish embryo-generated HNoVs showed clear patterns of binding to human histo-blood group antigens (HBGAs) in human saliva by a simple saliva-binding reverse transcription-quantitative PCR (RT-qPCR). Last, in a disinfection study, it was shown that a dose of 6 mJ/cm2 UV254 was able induce a >2-log reduction in HNoV infectivity for all three HNoV strains tested, suggesting that HNoVs were more UV susceptible than multiple enteric viruses and commonly used HNoV surrogates as tested before. IMPORTANCE HNoVs are a leading cause of gastroenteritis outbreaks worldwide. The zebrafish embryo tool as developed in this study serves as an efficient way to generate viruses with high titers and clean background and a straightforward platform to evaluate HNoV inactivation efficacies. It is expected that this tool will not only benefit epidemiological research on HNoV but also be used to generate HNoV inactivation parameters which are highly needed by the water treatment and food industries.

Mechanistic Contributions of lncRNAs to Cellular Signaling Pathways Crucial to the Lifecycle of Human Papillomaviruses.

Papillomaviruses are ubiquitous epitheliotropic viruses with double-stranded circular DNA genomes of approximately 8000 base pairs. The viral life cycle is somewhat unusual in that these viruses can establish persistent infections in the mitotically active basal epithelial cells that they initially infect. High-level viral genome replication ("genome amplification"), the expression of capsid proteins, and the formation of infectious progeny are restricted to terminally differentiated cells where genomes are synthesized at replication factories at sites of double-strand DNA breaks. To establish persistent infections, papillomaviruses need to retain the basal cell identity of the initially infected cells and restrain and delay their epithelial differentiation program. To enable high-level viral genome replication, papillomaviruses also need to hold the inherently growth-arrested terminally differentiated cells in a replication-competent state. To provide ample sites for viral genome synthesis, they target the DNA damage and repair machinery. Studies focusing on delineating cellular factors that are targeted by papillomaviruses may aid the development of antivirals. Whilst most of the current research efforts focus on protein targets, the majority of the human transcriptome consists of noncoding RNAs. This review focuses on one specific class of noncoding RNAs, long noncoding RNAs (lncRNAs), and summarizes work on lncRNAs that may regulate the cellular processes that are subverted by papillomavirus to enable persistent infections and progeny synthesis.

Accumulation of copy-back viral genomes during respiratory syncytial virus infection is preceded by diversification of the copy-back viral genome population followed by selection.

RNA viruses generate nonstandard viral genomes during their replication, including viral genomes of the copy-back (cbVGs) type that cannot replicate in the absence of a standard virus. cbVGs play a crucial role in shaping virus infection outcomes due to their ability to interfere with virus replication and induce strong immune responses. However, despite their critical role during infection, the principles that drive the selection and evolution of cbVGs within a virus population are poorly understood. As cbVGs are dependent on the virus replication machinery to be generated and replicated, we hypothesized that host factors that affect virus replication exert selective pressure on cbVGs and drive their evolution within a virus population. To test this hypothesis, we used respiratory syncytial virus (RSV) as a model and took an experimental evolution approach by serially passaging RSV in immune-competent human lung adenocarcinoma A549 control and immune-deficient A549 Signal transducer and activator of transcription 1 (STAT1) KO cells, which allow higher levels of virus replication. As predicted, we observed that virus populations accumulated higher amounts of cbVGs in the more permissive A549 STAT1 KO cells over time; however, unexpectedly, the predominant cbVG species after passages in the two conditions were different. While A549 STAT1 KO cells accumulated relatively short cbVGs, A549 control cells mainly contained cbVGs of much longer predicted size, which have not been described previously. These long cbVGs were predominant at first in both cell lines in vitro and the predominant ones observed in samples from RSV-infected patients. Although sustained high replication levels are associated with cbVG generation and accumulation, our data show that sustained high levels of virus replication are critical for cbVG population diversification, a process that precedes the generation of shorter cbVGs that selectively accumulate over time. Taken together, we show that selection and evolution of cbVGs within a virus population are shaped by how resistant or permissive a host is to RSV.

Bimodal distribution pattern associated with the PCR cycle threshold (Ct) and implications in COVID-19 infections

SARS-CoV-2 is notable for its extremely high level of viral replication in respiratory epithelial cells, relative to other cell types. This may partially explain the high transmissibility and rapid global dissemination observed during the COVID-19 pandemic. Polymerase chain reaction (PCR) cycle threshold (Ct) number has been widely used as a proxy for viral load based on the inverse relationship between Ct number and amplifiable genome copies present in a sample. We examined two PCR platforms (Centers for Disease Control and Prevention 2019-nCoV Real-time RT-PCR, Integrated DNA Technologies; and TaqPath COVID-19 multi-plex combination kit, ThermoFisher Scientific) for their performance characteristics and Ct distribution patterns based on results generated from 208,947 clinical samples obtained between October 2020 and September 2021. From 14,231 positive tests, Ct values ranged from 8 to 39 and displayed a pronounced bimodal distribution. The bimodal distribution persisted when stratified by gender, age, and time period of sample collection during which different viral variants circulated. This finding may be a result of heterogeneity in disease progression or host response to infection irrespective of age, gender, or viral variants. Quantification of respiratory mucosal viral load may provide additional insight into transmission and clinical indicators helpful for infection control.

Japanese encephalitis virus induces vasodilation and severe lethality in adult and aged AG129 mice lacking alpha, beta and gamma interferon receptors

Japanese encephalitis virus (JEV) is a single-stranded positive-sense RNA virus belonging to the Flaviviridae family. The JEV is the leading cause of viral encephalitis in children and the elderly which is spread by mosquitoes. JEV infection has been established in different animal models such as mouse, hamster, guinea pig, swine, rat, monkey, rabbit by using the different routes of inoculations. Here, we have shown that the alpha/beta and gamma -receptor deficient AG129 mouse induces fatal encephalitis in both young and aged old mice, when challenged with high titer JEV Indian clinical isolate by both intraperitoneal and intradermal route. The JEV infected AG129 mouse have shown neurological symptoms, JEV-induced pathological features and supported high level viral replication. Additionally, administration of JEV in AG129 mice resulted in the induction of severe peripheral vascular permeability, which is a major hall mark of Dengue infection but not shown in JEV. Taken together, our results demonstrate interferon α/β and γ receptors knock out AG129 mouse does not need adaptation of JEV clinical isolates and could be is a promising JEV challenge mouse model by mimicking the natural intradermal route of administration for rapid screening of novel antivirals and vaccines.

Recapitulating infection, thermal sensitivity and antiviral treatment of seasonal coronaviruses in human airway organoids

SummaryBackgroundHuman seasonal coronaviruses usually cause mild upper-respiratory tract infection, but severe complications can occur in specific populations. Research into seasonal coronaviruses is limited and robust experimental models are largely lacking. This study aims to establish human airway organoids (hAOs)-based systems for seasonal coronavirus infection and to demonstrate their applications in studying virus-host interactions and therapeutic development.MethodsThe infections of seasonal coronaviruses 229E, OC43 and NL63 in 3D cultured hAOs with undifferentiated or differentiated phenotypes were tested. The kinetics of virus replication and production was profiled at 33 °C and 37 °C. Genome-wide transcriptome analysis by RNA sequencing was performed in hAOs under various conditions. The antiviral activity of molnupiravir and remdesivir, two approved medications for treating COVID19, was tested.FindingsHAOs efficiently support the replication and infectious virus production of seasonal coronaviruses 229E, OC43 and NL63. Interestingly, seasonal coronaviruses replicate much more efficiently at 33 °C compared to 37 °C, resulting in over 10-fold higher levels of viral replication. Genome-wide transcriptomic analyses revealed distinct patterns of infection-triggered host responses at 33 °C compared to 37 °C temperature. Treatment of molnupiravir and remdesivir dose-dependently inhibited the replication of 229E, OC43 and NL63 in hAOs.InterpretationHAOs are capable of modeling 229E, OC43 and NL63 infections. The intriguing finding that lower temperature resembling that in the upper respiratory tract favors viral replication may help to better understand the pathogenesis and transmissibility of seasonal coronaviruses. HAOs-based innovative models shall facilitate the research and therapeutic development against seasonal coronavirus infections.FundingThis research is supported by funding of a VIDI grant (No. 91719300) from the Netherlands Organization for Scientific Research and the Dutch Cancer Society Young Investigator Grant (10140) to Q.P., and the ZonMw COVID project (114025011) from the Netherlands Organization for Health Research and Development to R.R.

Human microglial models to study HIV infection and neuropathogenesis: a literature overview and comparative analyses.

HIV persistence in the CNS despite antiretroviral therapy may cause neurological disorders and poses a critical challenge for HIV cure. Understanding the pathobiology of HIV-infected microglia, the main viral CNS reservoir, is imperative. Here, we provide a comprehensive comparison of human microglial culture models: cultured primary microglia (pMG), microglial cell lines, monocyte-derived microglia (MDMi), stem cell–derived microglia (iPSC-MG), and microglia grown in 3D cerebral organoids (oMG) as potential model systems to advance HIV research on microglia. Functional characterization revealed phagocytic capabilities and responsiveness to LPS across all models. Microglial transcriptome profiles of uncultured pMG showed the highest similarity to cultured pMG and oMG, followed by iPSC-MG and then MDMi. Direct comparison of HIV infection showed a striking difference, with high levels of viral replication in cultured pMG and MDMi and relatively low levels in oMG resembling HIV infection observed in post-mortem biopsies, while the SV40 and HMC3 cell lines did not support HIV infection. Altogether, based on transcriptional similarities to uncultured pMG and susceptibility to HIV infection, MDMi may serve as a first screening tool, whereas oMG, cultured pMG, and iPSC-MG provide more representative microglial culture models for HIV research. The use of current human microglial cell lines (SV40, HMC3) is not recommended.Supplementary informationThe online version contains supplementary material available at 10.1007/s13365-021-01049-w.

Use of Micro-Computed Tomography to Visualize and Quantify COVID-19 Vaccine Efficiency in Free-Breathing Hamsters.

The SARS-CoV-2 pandemic has impacted the health of humanity after the outbreak in Hubei, China in late December 2019. Ever since, it has taken unprecedented proportions and rapidity causing over a million fatal cases. Recently, a robust Syrian golden hamster model recapitulating COVID-19 was developed in search for effective therapeutics and vaccine candidates. However, overt clinical disease symptoms were largely absent despite high levels of virus replication and associated pathology in the respiratory tract. Therefore, we used micro-computed tomography (μCT) to longitudinally visualize lung pathology and to preclinically assess candidate vaccines. μCT proved to be crucial to quantify and noninvasively monitor disease progression, to evaluate candidate vaccine efficacy, and to improve screening efforts by allowing longitudinal data without harming live animals. Here, we give a comprehensive guide on how to use low-dose high-resolution μCT to follow-up SARS-CoV-2-induced disease and test the efficacy of COVID-19 vaccine candidates in hamsters. Our approach can likewise be applied for the preclinical assessment of antiviral and anti-inflammatory drug treatments in vivo.

Assessing the infiltration of immune cells in the upper trachea mucosa after infectious laryngotracheitis virus (ILTV) vaccination and challenge

ABSTRACT The types of immune cells that populate the trachea after ILTV vaccination and infection have not been assessed. The objective of this study was to quantify CD4+, CD8α+, CD8β+, TCRγδ+, and MRC1LB+ cells that infiltrate the trachea after vaccination with chicken embryo origin (CEO), tissue culture origin (TCO), and recombinant herpesvirus of turkey-laryngotracheitis (rHVT-LT) vaccines, and after challenge of vaccinated and non-vaccinated chickens with a virulent ILTV strain. Eye-drop vaccination with CEO, or TCO, or in ovo vaccination with rHVT-LT did not alter the number of CD4+, CD8α+, CD8β+, TCRγδ+, and MRC1LB+ cells in the trachea. After challenge, the CEO vaccinated group of chickens showed swift clearance of the challenge virus, the mucosa epithelium of the trachea remained intact, and a limited number of CD4+, CD8α+, and CD8β+ cells were detected in the upper trachea mucosa. The TCO and rHVT-LT vaccinated groups of chickens showed narrow viral clearance with moderate disruption of the trachea epithelial integrity, and a significant increase in CD4+, CD8α+, CD8β+, and TCRγδ+ cells infiltrated the upper trachea mucosa. Non-vaccinated challenged chickens showed high levels of viral replication, the epithelial organization of the upper trachea mucosa was heavily disrupted, and the predominant infiltrates were CD4+, TCRγδ+, and MRC1LB+ cells. Hence, the very robust protection provided by CEO vaccination was characterized by minimal immune cell infiltration to the trachea mucosa. In contrast, partial protection induced by the TCO and rHVT-LT vaccines requires a prolonged period of T cell expansion to overcome the established infection in the trachea mucosa.

Protective efficacy of Ad26.COV2.S against SARS-CoV-2 B.1.351 in macaques

The emergence of SARS-CoV-2 variants that partially evade neutralizing antibodies poses a threat to the efficacy of current COVID-19 vaccines1,2. The Ad26.COV2.S vaccine expresses a stabilized spike protein from the WA1/2020 strain of SARS-CoV-2, and has recently demonstrated protective efficacy against symptomatic COVID-19 in humans in several geographical regions—including in South Africa, where 95% of sequenced viruses in cases of COVID-19 were the B.1.351 variant3. Here we show that Ad26.COV2.S elicits humoral and cellular immune responses that cross-react with the B.1.351 variant and protects against B.1.351 challenge in rhesus macaques. Ad26.COV2.S induced lower binding and neutralizing antibodies against B.1.351 as compared to WA1/2020, but elicited comparable CD8 and CD4 T cell responses against the WA1/2020, B.1.351, B.1.1.7, P.1 and CAL.20C variants. B.1.351 infection of control rhesus macaques resulted in higher levels of virus replication in bronchoalveolar lavage and nasal swabs than did WA1/2020 infection. Ad26.COV2.S provided robust protection against both WA1/2020 and B.1.351, although we observed higher levels of virus in vaccinated macaques after B.1.351 challenge. These data demonstrate that Ad26.COV2.S provided robust protection against B.1.351 challenge in rhesus macaques. Our findings have important implications for vaccine control of SARS-CoV-2 variants of concern.

Application of the woodchuck animal model for the treatment of hepatitis B virus-induced liver cancer.

This review describes woodchucks chronically infected with the woodchuck hepatitis virus (WHV) as an animal model for hepatocarcinogenesis and treatment of primary liver cancer or hepatocellular carcinoma (HCC) induced by the hepatitis B virus (HBV). Since laboratory animal models susceptible to HBV infection are limited, woodchucks experimentally infected with WHV, a hepatitis virus closely related to HBV, are increasingly used to enhance our understanding of virus-host interactions, immune response, and liver disease progression. A correlation of severe liver pathogenesis with high-level viral replication and deficient antiviral immunity has been established, which are present during chronic infection after WHV inoculation of neonatal woodchucks for modeling vertical HBV transmission in humans. HCC in chronic carrier woodchucks develops 17 to 36 mo after neonatal WHV infection and involves liver tumors that are comparable in size, morphology, and molecular gene signature to those of HBV-infected patients. Accordingly, woodchucks with WHV-induced liver tumors have been used for the improvement of imaging and ablation techniques of human HCC. In addition, drug efficacy studies in woodchucks with chronic WHV infection have revealed that prolonged treatment with nucleos(t)ide analogs, alone or in combination with other compounds, minimizes the risk of liver disease progression to HCC. More recently, woodchucks have been utilized in the delineation of mechanisms involved in innate and adaptive immune responses against WHV during acute, self-limited and chronic infections. Therapeutic interventions based on modulating the deficient host antiviral immunity have been explored in woodchucks for inducing functional cure in HBV-infected patients and for reducing or even delaying associated liver disease sequelae, including the onset of HCC. Therefore, woodchucks with chronic WHV infection constitute a well-characterized, fully immunocompetent animal model for HBV-induced liver cancer and for preclinical evaluation of the safety and efficacy of new modalities, which are based on chemo, gene, and immune therapy, for the prevention and treatment of HCC in patients for which current treatment options are dismal.

Oncolytic Vaccinia Virus Expressing White-Spotted Charr Lectin Regulates Antiviral Response in Tumor Cells and Inhibits Tumor Growth In Vitro and In Vivo.

Oncolytic vaccina virus (oncoVV) used for cancer therapy has progressed in recent years. Here, a gene encoding white-spotted charr lectin (WCL) was inserted into an oncoVV vector to form an oncoVV-WCL recombinant virus. OncoVV-WCL induced higher levels of apoptosis and cytotoxicity, and replicated faster than control virus in cancer cells. OncoVV-WCL promoted IRF-3 transcriptional activity to induce higher levels of type I interferons (IFNs) and blocked the IFN-induced antiviral response by inhibiting the activity of IFN-stimulated responsive element (ISRE) and the expression of interferon-stimulated genes (ISGs). The higher levels of viral replication and antitumor activity of oncoVV-WCL were further demonstrated in a mouse xenograft tumor model. Therefore, the engineered oncoVV expressing WCL might provide a new avenue for anticancer gene therapy.

Clinical significance of quantitative e antigen in a cohort of hepatitis B virus-infected children and adults in North America.

In chronic hepatitis B (CHB) viral infection, e antigen positivity (HBeAg+) is associated with high levels of viral replication and infectivity. Furthermore, HBeAg-positive CHB is associated with a liver disease spectrum ranging from none to severe. To assess whether the level of circulating HBeAg is associated with different clinical presentations of HBeAg-positive CHB. A cross-sectional analysis was conducted among HBV mono-infected participants enrolled in Hepatitis B Research Network (HBRN) cohorts to explore clinical and virological associations with quantitative HBeAg (qHBeAg). Among 763 HBeAg+ participants (56% female; 85% Asian; median age 26years), multivariable median regression modelling significantly associated qHBeAg with liver injury (inverse qHBeAg association with ALT p<.001 and APRI p<.001), and with both race and age (p=0.01). Among Asians, qHBeAg was inversely related to age; a relationship less clear among Blacks and Whites. Among Asians also, median qHBeAg levels were higher among those infected with HBV genotype C versus B (p<0.001), suggesting causal virologic differences. Across all races, median qHBeAg was higher in women (p=.01). Independent of sex, age, race and HBV genotype, qHBeAg was higher in participants with predominant wild-type versus basal core promoter and/or precore 'stop' viral variants (p<0.001). Lower qHBeAg was observed among HBRN participants with the greatest degree of liver injury independent of demographics and HBV genotype. These data support longitudinal studies to examine the role of qHBeAg in modulating the host immune response and predicting the outcomes of chronic HBV infection.

Abstract PS12-08: A window-of-opportunity study with atezolizumab and the oncolytic virus pelareorep in early breast cancer (REO-027, AWARE-1)

Abstract Background: A previous phase 2 study in metastatic breast cancer compared treatment with intravenously delivered oncolytic reovirus, pelareorep (pela), in combination with paclitaxel (PTX) versus PTX alone. This study demonstrated a statistically significant improvement in overall survival (OS), without differences in objective response or progression-free survival. We hypothesized that the OS benefit from pela + PTX may be attributed to an adaptive immune response triggered by pela. To test this hypothesis, and examine if pela can mediate the priming of an anti-tumor immune response, we designed a study called AWARE-1 (A window-of-opportunity study of pela in Early Breast Cancer), which is currently enrolling and for which initial translational research results are presented. Methods: AWARE-1 is evaluating the safety and effect of pela ± atezolizumab on the tumor microenvironment (TME) in 38 women with early breast cancer. Patients are treated with pela on days 1, 2, 8, and 9, while atezolizumab is administered on day 3. Tumor biopsies are collected at diagnosis, day 3, and day ~21. Five cohorts will be examined: Cohort 1: Hormone Receptor-positive/HER2-negative (HR+/HER2-neg) (10 patients), pelareorep + letrozole. Cohort 2: HR+/HER2-neg (10 patients), pelareorep + letrozole + atezolizumab. Cohort 3: Triple Negative Breast Cancer (TNBC) (6 patients), pelareorep + atezolizumab. Cohort 4: Hormone Receptor-positive/HER2-positive (HR+/HER2+) (6 patients), pelareorep + trastuzumab + atezolizumab. Cohort 5: Hormone Receptor-negative/HER2-positive (HR-/HER2+) (6 patients), pelareorep + trastuzumab + atezolizumab. The primary endpoint of the study is CelTIL score, a metric for quantifying the changes in tumor cellularity and infiltration of TILs, where an increase in CelTIL is associated with a favorable response to treatment. Tumor tissue was examined for pela replication, and changes to the TME were assessed by imaging mass cytometry (IMC), immunohistochemistry, and T cell receptor sequencing (TCR-seq). Peripheral blood was also examined by TCR-seq. Results: Detailed translational research results will be presented from patients in cohort 1, who received just pelareorep and letrozole. CelTIL score increased in 5/10 patients at day 3 biopsies and 6/10 patients at day 21 biopsies. Preliminary results show high levels of viral replication (&amp;gt;50% of tumor cells) while immunohistochemistry and IMC analysis revealed changes to the TME, with increases in CD8+ T cells and upregulation of PD-L1 at both day 3 and day 21 biopsies. Overall, preliminary data from cohort 1 of AWARE-1 demonstrate pela-mediated priming of an adaptive immune response. (NCT04102618) Citation Format: Luis Manso, Patricia Villagrasa, Nuria Chic, Begoña Bermejo, Juan Miguel Cejalvo, Yann Izarzugaza, Blanca Cantos, Salvador Blanch, Mireia Margeli, Jose Luis Alonso, Alejandro Martínez, Rafael Villanueva, Juan Antonio Guerra, Raquel Andrés, Pilar Zamora, Esteban Nogales, Manel Juan, Blanca González, Rita Laeufle, Gerard Nuovo, Grey Wilkinson, Matt Coffey, Azucena González, Débora Martínez, Laia Paré, Fernando Salvador, Xavier Gonzalez, Aleix Prat, Joaquín Gavilá. A window-of-opportunity study with atezolizumab and the oncolytic virus pelareorep in early breast cancer (REO-027, AWARE-1) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS12-08.

Abstract OT-13-02: Bracelet-1 (pre0113): A study to assess overall response rate by inducing an inflammatory phenotype in metastatic breast cancer with the oncolytic reovirus pelareorep in combination with anti-PD-L1 avelumab and paclitaxel

Abstract Background: A randomized phase 2 study with the intravenously delivered oncolytic virus, pelareorep, in combination with paclitaxel (PTX) demonstrated a statistically significant improvement in overall survival (OS) from 10.4 months with PTX alone to 17.4 months with pelareorep + PTX (HR 0.65, 80% CI 0.46-0.91, P = 0.1) in metastatic breast cancer (mBC) patients. The greatest benefit in OS was seen in patients with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) disease (Bernstein et al, 2018). However, pelareorep + PTX did not improve progression-free survival or objective response relative to PTX alone, suggesting a late-onset adaptive immune response. A subsequent window of opportunity study in early breast cancer has shown that pelareorep can indeed promote an adaptive immune response in breast cancer tissue, enhancing CD8+ T cell infiltration and upregulating PD-L1 expression, correlating with high levels of viral replication in HR+/HER2- tumor tissue (Manso et al, 2020). Moreover, high levels of peripheral T cell clonality (PTCC) have been identified as a candidate blood-based on-treatment biomarker for pelareorep therapy, further highlighting the role of an adaptive immune response in driving pelareorep mediated efficacy (Mahalingam et al, 2020; Manso et al, 2020). Thus, BRACELET-1 will test the hypothesis that pelareorep mediated priming of an adaptive immune response will be synergistic with checkpoint blockade therapy in HR+/HER2- mBC. Moreover, BRACELET-1 will further assess PTCC as an on-treatment biomarker. The overall goal of this study is to expand the number of mBC patients who can benefit from better immunotherapy. Study Design: This is an open-label randomized phase 2, three-cohort study in HR+/HER2- mBC. Patients must be refractory to endocrine therapy and have received prior treatment with a CDK4/6 inhibitor. Study cohorts include: Cohort 1, a control group receiving PTX (n = 15); Cohort 2, treatment with pelareorep added to PTX (n = 15); Cohort 3, treatment with pelareorep, PTX, and avelumab (n = 18). The study includes a three patient safety run-in for Cohort 3. Specific aims: (1) Evaluation of efficacy in terms of overall response rate (ORR) at week 16, according to RECIST v1.1; (2) Examination of the safety of the study treatments; and (3) Assessment of key biomarkers, such as PTCC which will be correlated to treatment efficacy. Present accrual and target accrual: The study is currently enrolling and is registered on clinicaltrials.gov: NCT04215146. Current enrollment = 2; Target enrollment = 48. This study is conducted through PrECOG, LLC and Oncolytics Biotech, Inc. Study contact information: PrE0113@precogllc.org. Citation Format: Kathy Miller, Fengmin Zhao, Amy Clark, Grey Wilkinson, Rita Laeufle, Antonio Wolff. Bracelet-1 (pre0113): A study to assess overall response rate by inducing an inflammatory phenotype in metastatic breast cancer with the oncolytic reovirus pelareorep in combination with anti-PD-L1 avelumab and paclitaxel [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-13-02.

Decrease of Clone Diversity in IgM Repertoires of HBV Chronically Infected Individuals With High Level of Viral Replication.

High-throughput antibody sequencing allows in-depth insights into human antibody repertoires. To investigate the characteristics of antibody repertoires in patients with chronic HBV infection, we performed Illumina sequencing and IMGT/HighV-QUEST analysis of B lymphocytes from healthy adults and the HBV carriers with high or low level of viral replication. The comparative study revealed high levels of similarity between the IgM and IgG repertoires of the HBV carriers and the healthy adults, including the somatic mutations in V regions, the average CDR3 length, and the occurrence of junctional modifications. Nevertheless, the diversity of the unique clones decreased and some clusters of unique clones expanded in the IgM repertoire of chronic HBV carriers (CHB) compared with healthy adults (HH) and inactive HBV carriers (IHB). Such difference in clone diversity and expansion was not observed in the IgG repertoires of the three populations. More shared antibody clones were found between the IgM repertoires of IHB and HH than that found between CHB and HH (7079 clones vs. 2304 clones). Besides, the biased used IGHD genes were IGHD2-2 and IGHD3-3 in CHB library but were IGHD3-10 and IGHD3-22 in IHB and HH library. In contrast, for IgG repertories, the preferred used VDJ genes were similar in all the three populations. These results indicated that low level of serum HBV might not induce significant changes in BCR repertoires, and high level of HBV replication could have more impacts on IgM repertories than IgG repertoires. Taken together, our findings provide a better understanding of the antibody repertoires of HBV chronically infected individuals.

HCV RdRp, sofosbuvir and beyond.

The therapeutic targeting of the nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase (RdRp) of the Hepatitis C Virus (HCV) with nucleotide analogs led to a deep understanding of this enzymes structure, function and substrate specificity. Unlike previously studied DNA polymerases including the reverse transcriptase of Human Immunodeficiency Virus, development of biochemical assays for HCV RdRp proved challenging due to low solubility of the full-length protein and inefficient acceptance of exogenous primer/templates. Despite the poor apparent specific activity, HCV RdRp was found to support rapid and processive transcription once elongation is initiated in vitro consistent with its high level of viral replication in the livers of patients. Understanding of the substrate specificity of HCV RdRp led to the discovery of the active triphosphate of sofosbuvir as a nonobligate chain-terminator of viral RNA transcripts. The ternary crystal structure of HCV RdRp, primer/template, and incoming nucleotide showed the interaction between the nucleotide analog and the 2'-hydroxyl binding pocket and how an unfit mutation of serine 282 to threonine results in resistance by interacting with the uracil base and modified 2'-position of the analog. Host polymerases mediate off-target toxicity of nucleotide analogs and the active metabolite of sofosbuvir was found to not be efficiently incorporated by host polymerases including the mitochondrial RNA polymerase (POLRMT). Knowledge from studying inhibitors of HCV RdRp serves to advance antiviral drug discovery for other emerging RNA viruses including the discovery of remdesivir as an inhibitor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), the virus that causes COVID-19.