scholarly journals Genetic signatures of human cytomegalovirus variants acquired by seronegative glycoprotein B vaccinees

2018 ◽  
Author(s):  
Cody S. Nelson ◽  
Diana Vera Cruz ◽  
Melody Su ◽  
Guanhua Xie ◽  
Nathan Vandergrift ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Rational Design ◽  
Hcmv Infection ◽  
Vaccine Trial ◽  
The United States ◽  
Glycoprotein B ◽  
Viral Population ◽  
The Impact ◽  
Placebo Recipients

AbstractHuman cytomegalovirus (HCMV) is the most common congenital infection worldwide, and a frequent cause of hearing loss or debilitating neurologic disease in newborn infants. Thus, a vaccine to prevent HCMV-associated congenital disease is a public health priority. One potential strategy is vaccination of women of child-bearing age to prevent maternal HCMV acquisition during pregnancy. The glycoprotein B (gB) + MF59 adjuvant subunit vaccine is the most efficacious tested clinically to date, demonstrating approximately 50% protection against HCMV infection of seronegative women in multiple phase 2 trials. Yet, the impact of gB/MF59-elicited immune responses on the population of viruses acquired by trial participants has not been assessed. In this analysis, we employed quantitative PCR as well as multiple sequencing methodologies to interrogate the magnitude and genetic composition of HCMV populations infecting gB/MF59 vaccinees and placebo recipients. We identified several differences between the viral dynamics of acutely-infected vaccinees and placebo recipients. First, there was reduced magnitude viral shedding in the saliva of gB vaccinees. Additionally, employing a panel of tests for genetic compartmentalization, we noted tissue-specific gB haplotypes in the majority of vaccinees though only in a single placebo recipient. Finally, we observed reduced acquisition of genetically-related gB1, gB2, and gB4 genotype “supergroup” HCMV variants among vaccine recipients, suggesting that the gB1 genotype vaccine construct may have elicited partial protection against HCMV viruses with antigenically-similar gB sequences. These findings indicate that gB immunization may have had a measurable impact on viral intrahost population dynamics and support future analysis of a larger cohort.Author SummaryThough not a household name like Zika virus, human cytomegalovirus (HCMV) causes permanent neurologic disability in one newborn child every hour in the United States - more than Down syndrome, fetal alcohol syndrome, and neural tube defects combined. There are currently no established effective preventative measures to inhibit congenital HCMV transmission following acute or chronic HCMV infection of a pregnant mother. However, the glycoprotein B (gB) vaccine is the most effective HCMV vaccine tried clinically to date. Here, we utilized high-throughput, next-generation sequencing of viral DNA isolated from patients enrolled in a gB vaccine trial, and identified several impacts that this vaccine had on the size, distribution, and composition of thein vivoviral population. These results have increased our understanding of why the gB/MF59 vaccine was partially efficacious and will inform future rational design of a vaccine to prevent congenital HCMV.

scholarly journals Intrahost Dynamics of Human Cytomegalovirus Variants Acquired by Seronegative Glycoprotein B Vaccinees

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Cody S. Nelson ◽  
Diana Vera Cruz ◽  
Melody Su ◽  
Guanhua Xie ◽  
Nathan Vandergrift ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Rational Design ◽  
Hcmv Infection ◽  
Vaccine Trial ◽  
The United States ◽  
Glycoprotein B ◽  
Viral Population ◽  
Vaginal Fluid ◽  
The Impact ◽  
Placebo Recipients

ABSTRACTHuman cytomegalovirus (HCMV) is the most common congenital infection worldwide and a frequent cause of hearing loss and debilitating neurologic disease in newborn infants. Thus, a vaccine to prevent HCMV-associated congenital disease is a public health priority. One potential strategy is vaccination of women of child bearing age to prevent maternal HCMV acquisition during pregnancy. The glycoprotein B (gB) plus MF59 adjuvant subunit vaccine is the most efficacious tested clinically to date, demonstrating 50% protection against primary HCMV infection in a phase 2 clinical trial. Yet, the impact of gB/MF59-elicited immune responses on the population of viruses acquired by trial participants has not been assessed. In this analysis, we employed quantitative PCR as well as multiple sequencing methodologies to interrogate the magnitude and genetic composition of HCMV populations infecting gB/MF59 vaccinees and placebo recipients. We identified several differences between the viral dynamics in acutely infected vaccinees and placebo recipients. First, viral load was reduced in the saliva of gB vaccinees, though not in whole blood, vaginal fluid, or urine. Additionally, we observed possible anatomic compartmentalization of gB variants in the majority of vaccinees compared to only a single placebo recipient. Finally, we observed reduced acquisition of genetically related gB1, gB2, and gB4 genotype “supergroup” HCMV variants among vaccine recipients, suggesting that the gB1 genotype vaccine construct may have elicited partial protection against HCMV viruses with antigenically similar gB sequences. These findings suggest that gB immunization had a measurable impact on viral intrahost population dynamics and support future analysis of a larger cohort.IMPORTANCEThough not a household name like Zika virus, human cytomegalovirus (HCMV) causes permanent neurologic disability in one newborn child every hour in the United States, which is more than that for Down syndrome, fetal alcohol syndrome, and neural tube defects combined. There are currently no established effective measures to prevent viral transmission to the infant following HCMV infection of a pregnant mother. However, the glycoprotein B (gB)/MF59 vaccine, which aims to prevent pregnant women from acquiring HCMV, is the most successful HCMV vaccine tested clinically to date. Here, we used viral DNA isolated from patients enrolled in a gB vaccine trial who acquired HCMV and identified several impacts that this vaccine had on the size, distribution, and composition of thein vivoviral population. These results have increased our understanding of why the gB/MF59 vaccine was partially efficacious, and such investigations will inform future rational design of a vaccine to prevent congenital HCMV.

scholarly journals Human Cytomegalovirus Selectively Suppresses the Megakaryo/Thrombopoiesis of PDGFR+ and αvβ3+ Megakaryocytes Via the TPO/c-Mpl Pathway after Allo-HSCT

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-25
Author(s):  
Feng-qi Liu ◽  
Fei-er Feng ◽  
Gao-chao Zhang ◽  
Yan Su ◽  
Xue-yan Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Conflicts Of Interest ◽  
Antiviral Treatment ◽  
Infection Model ◽  
Hematopoietic Stem ◽  
The Impact

Introduction Virus-induced thrombocytopenia is a severe complication in immunocompromised hosts. Among patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT), human cytomegalovirus (HCMV) infection contributes to a variety of end-organ diseases and hematological complications, leading to increased mortality. Even with antiviral treatment, HCMV remains a potentially lethal infection due to the lack of understanding of the underlying mechanisms of host-virus interactions. The key to solving this problem is to identify the factors that predispose patients to HCMV infection and carry out targeted therapy. Here, we investigated the megakaryo/thrombopoiesis process, including the thrombopoietin (TPO)/c-Mpl pathway, after HCMV infection in vivo and in vitro, screened for susceptible subsets of megakaryocytes (MKs) and explored novel therapeutic targets for HCMV infection. Methods To test whether thrombocytopenia induced by HCMV results from an impaired megakaryo/thrombopoiesis process, we studied the impact of HCMV in an in vivo model of HCMV DNAemia patients following allo-HSCT and an in vitro model of bone marrow CD34+-derived MKs infected with serum from HCMV DNAemia patients. Forty patients who had received allo-HSCT were enrolled in this study, among whom 18 recipients had HCMV DNAemia and 22 were HCMV negative, and bone marrow-derived mononuclear cells (MNCs) from patients were tested for CD41, vWF, pp65, c-Mpl, PDGFR, αvβ3 and TLR2 using flow cytometry (FCM). Transmission electron microscopy (TEM) was used to detect HCMV capsids inside MKs. Cell apoptosis was measured by Annexin V. MK ploidy was determined by FCM for propidium iodide (PI) staining. Finally, inhibitors of PDGFR (IMC-3G3 and Gleevec), αvβ3 and TLR2 were cocultured with MKs. Results Our data showed that pp65+ cells accounted for 40.59±6.12% of total CD41+vWF+ MKs from HCMV DNAemia patients, and there was a significant increase in the expression of αvβ3, PDGFR and TLR2 in pp65+ MKs compared with that in control patients. Furthermore, the percentage of PDGFR+αvβ3+ MKs emerged as an independent factor associated with HCMV infection in multivariate analysis (p = 0.008). MKs in HCMV-infected patients showed increased apoptosis and necrosis and different patterns of MK ploidy distribution compared with those in HCMV-negative patients, with a decreased proportion from 16N to 64N and a peak at 8N. Meanwhile, the expression of TPO receptor c-Mpl was lower in pp65+ MKs from HCMV DNAemia patients (0.77±0.38% in pp65+ MKs from HCMV DNAemia patients, 1.75±0.40% in pp65- MKs from HCMV DNAemia patients, 1.97±0.67% in MKs from HCMV-negative patients, and 2.06±0.29% in MKs from healthy controls, p<0.01) while the TPO level in serum was increased compared with that in controls. Next, we established an in vitro HCMV infection model of CD34+-derived MKs with serum from HCMV DNAemia patients, and the laboratory HCMV strain Towne was used as a positive control. After 9 days of coculturing, the viral capsids of HCMV were observed in the nuclei of MKs (Figure 1A), and HCMV infection increased the apoptosis of MKs and shifted them to low ploidy, with a significant decrease in platelet release. As with the in vivo results, c-Mpl was downregulated in HCMV-infected MKs. The expression levels of PDGFR, TLR2 and αvβ3 on MKs were increased in coculture with HCMV DNAemia serum, and pp65-positive MKs were decreased compared with the control after treatment with inhibitors of PDGFR and αvβ3 (Figure 1B). However, neither Gleevec nor anti-TLR2 altered the HCMV infection rate. Conclusions Our study showed that HCMV could impair megakaryopoiesis throughout maturation, apoptosis, and platelet generation via the TPO/c-Mpl pathway both in vivo and in vitro. MKs with PDGFR+ and αvβ3+ phenotypes are susceptible to HCMV infection and we proposed PDGFR and αvβ3 inhibitors as potential therapeutic alternatives for allo-HSCT patients with HCMV infection. Disclosures No relevant conflicts of interest to declare.

scholarly journals HCMV glycoprotein B subunit vaccine efficacy was mediated by non-neutralizing antibody effector functions

2018 ◽  
Author(s):  
Cody S. Nelson ◽  
Tori Huffman ◽  
Eduardo Cisneros de la Rosa ◽  
Guanhua Xie ◽  
Nathan Vandergrift ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Vaccine Efficacy ◽  
Subunit Vaccine ◽  
Hcmv Infection ◽  
Neutralizing Antibody ◽  
The United States ◽  
Glycoprotein B ◽  
Vaccine Platform ◽  
Effector Functions ◽  
Epitope Specificity

AbstractHuman cytomegalovirus (HCMV) is the most common congenital infection worldwide, frequently causing hearing loss and brain damage in afflicted infants. A vaccine to prevent maternal acquisition of HCMV during pregnancy is necessary to reduce the incidence of infant disease. The glycoprotein B (gB) + MF59 adjuvant subunit vaccine platform is the most successful HCMV vaccine tested to-date, demonstrating approximately 50% efficacy in preventing HCMV acquisition in phase II trials. However, the mechanism of vaccine protection remains unknown. Plasma from 33 gB/MF59 vaccinees at peak immunogenicity was tested for gB epitope specificity as well as neutralizing and non-neutralizing anti-HCMV effector functions, and compared to an HCMV-seropositive cohort. gB/MF59 vaccination elicited IgG responses with gB-binding magnitude and avidity comparable to natural infection. Additionally, IgG subclass distribution was similar with predominant IgG1 and IgG3 responses induced by gB vaccination and HCMV infection. However, vaccine-elicited antibodies exhibited limited neutralization of the autologous virus, negligible neutralization of multiple heterologous strains, and limited binding responses against gB structural motifs targeted by neutralizing antibodies including AD-1, AD-2, and Domain I. Interestingly, vaccinees had high-magnitude IgG responses against AD-3 linear epitopes, demonstrating immunodominance against this non-neutralizing, cytosolic region. Finally, vaccine-elicited IgG robustly bound trimeric, membrane-associated gB on the surface of transfected or HCMV-infected cells and mediated virion phagocytosis, though were poor mediators of NK cell activation. Altogether, these data suggest that non-neutralizing antibody functions, including virion phagocytosis, likely played a role in the observed 50% vaccine-mediated protection against HCMV acquisition.SignificanceThe CDC estimates that every hour, a child is born in the United States with permanent neurologic disability resulting from human cytomegalovirus (HCMV) infection – more than is caused by Down syndrome, fetal alcohol syndrome, and neural tube defects combined. A maternal vaccine to block transmission of HCMV to the developing fetus is a necessary intervention to prevent these adverse outcomes. The gB/MF59 vaccine is the most successful tested clinically to-date, achieving 50% reduction in HCMV acquisition. This manuscript establishes the function and epitope specificity of the humoral response stimulated by this vaccine that may explain the partial vaccine efficacy. Understanding the mechanism of gB/MF59-elicited protective immune responses will guide rational design and evaluation of the next generation of HCMV vaccines.

scholarly journals Lipophilicity and Click Reactivity Determine the Performance of Bioorthogonal Tetrazine Tools in Pretargeted in Vivo Chemistry

2020 ◽  
Author(s):  
Johanna Stéen ◽  
Jesper Tranekjær Jørgensen ◽  
Denk Christoph ◽  
Umberto Maria Battisti ◽  
Kamilla Nørregaard ◽  
...  
Keyword(s):  
Targeted Delivery ◽  
Rational Design ◽  
Structural Parameters ◽  
Early Time ◽  
High Rate ◽  
Drug Conjugates ◽  
Compound Libraries ◽  
Drug Concentrations ◽  
The Impact

<p>The development of highly selective and fast biocompatible reactions for ligation and cleavage has paved the way for new diagnostic and therapeutic applications of <i>in vivo</i> chemistry. The concept of bioorthogonal pretargeting has attracted considerable interest, in particular for the targeted delivery of radionuclides and drugs. In nuclear medicine, pretargeting can provide increased target-to-background ratios at early time-points compared to traditional approaches. This reduces the radiation burden to healthy tissue and, depending on the selected radionuclide, enables better imaging contrast or higher therapeutic efficiency. Moreover, bioorthogonally triggered cleavage of pretargeted antibody-drug conjugates represents an emerging strategy to achieve controlled release and locally increased drug concentrations. The toolbox of bioorthogonal reactions has significantly expanded in the past decade, with the tetrazine ligation being the fastest and one of the most versatile <i>in vivo</i> chemistries. Progress in the field, however, relies heavily on the development and evaluation of (radio)labeled compounds, preventing the use of compound libraries for systematic studies. The rational design of tetrazine probes and triggers has thus been impeded by the limited understanding of the impact of structural parameters on the <i>in vivo</i> ligation performance. In this work, we describe the development of a pretargeted blocking assay that allows for the investigation of the <i>in vivo</i> fate of a structurally diverse library of 45 unlabeled tetrazines and their capability to reach and react with pretargeted <i>trans</i>-cyclooctene (TCO)-tagged antibodies in tumor-bearing mice. This study enabled us to assess the correlation of click reactivity and lipophilicity of tetrazines with their <i>in vivo</i> performance. In particular, high rate constants (>50,000 M<sup>-1</sup>s<sup>-1</sup>) for the reaction with TCO and low calculated log<i>D</i><sub>7.4</sub> values (below -3) of the tetrazine were identified as strong indicators for successful pretargeted <i>in vivo</i> click chemistry. Click-radiolabeling gave access to a set of selected <sup>18</sup>F-labeled tetrazines, including highly reactive scaffolds, which were used in pretargeted PET imaging studies to confirm the results from the blocking study. These insights thus enable the rational design of tetrazine probes for <i>in vivo</i> application and will thereby assist the clinical translation of bioorthogonal pretargeting.</p>

scholarly journals In VitroSusceptibility of Canine Influenza A (H3N8) Virus to Nitazoxanide and Tizoxanide

2010 ◽  
Vol 2010 ◽  
pp. 1-5 ◽  
Author(s):  
Laura V. Ashton ◽  
Robert L. Callan ◽  
Sangeeta Rao ◽  
Gabriele A. Landolt
Keyword(s):  
Influenza Virus ◽  
Influenza A ◽  
The United States ◽  
Canine Influenza Virus ◽  
Valuable Adjunct ◽  
Canine Influenza ◽  
The Impact ◽  
H3n8 Virus

Infection of dogs with canine influenza virus (CIV) is considered widespread throughout the United States following the first isolation of CIV in 2004. While vaccination against influenza A infection is a common and important practice for disease control, antiviral therapy can serve as a valuable adjunct in controlling the impact of the disease. In this study, we examined the antiviral activity of nitazoxanide (NTZ) and tizoxanide (TIZ) against three CIV isolatesin vitro. NTZ and TIZ inhibited virus replication of all CIVs with 50% and 90% inhibitory concentrations ranging from 0.17 to 0.21 μMand from 0.60 to 0.76 μM, respectively. These results suggest that NTZ and TIZ are effective against CIV and may be useful for treatment of canine influenza in dogs but further investigation of thein vivoefficacy against CIV as well as the drug's potential for toxicity in dogs is needed.

In vitro infection of megakaryocytes and their precursors by human cytomegalovirus

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 487-493 ◽  
Author(s):  
Kirsten Crapnell ◽  
Esmail D. Zanjani ◽  
Aniruddho Chaudhuri ◽  
Joao L. Ascensao ◽  
Stephen St. Jeor ◽  
...  
Keyword(s):  
Life Cycle ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Allogeneic Bone Marrow Transplantation ◽  
Laboratory Strain ◽  
Allogeneic Bone ◽  
Cd34 Cells ◽  
Hcmv Disease

Apart from congenital human cytomegalovirus (HCMV) infection, manifest HCMV disease occurs primarily in immunocompromised patients. In allogeneic bone marrow transplantation, HCMV is frequently associated with graft failure and cytopenias involving all hematopoietic lineages, but thrombocytopenia is the most commonly reported hematologic complication. The authors hypothesized that megakaryocytes (MK) may be a specific target for HCMV. Although the susceptibility of immature hematopoietic progenitors cells to HCMV has been established, a productive viral life cycle has only been linked to myelomonocytic maturation. The authors investigated whether HCMV can also infect MK and impair their function. They demonstrated that HCMV did not affect the thrombopoietin (TPO)-driven proliferation of CD34+ cells until MK maturation occurred. MK challenged with HCMV showed a 50% more rapid loss of viability than mock-infected cells. MK and their early precursors were clearly shown to be susceptible to HCMV in vitro, as evidenced by the presence of HCMV in magnetic column-purified CD42+ MK and 2-color fluorescent staining with antibodies directed against CD42a and HCMV pp65 antigen. These findings were confirmed by the infection of MK with a laboratory strain of HCMV containing the β-galactosidase (β-gal) gene. Using chromogenic β-gal substrates, HCMV was detected during MK differentiation of infected CD34+ cells and after infection of fully differentiated MK. Production of infectious virus was observed in cultures infected MK, suggesting that HCMV can complete its life cycle. These results demonstrate that MK are susceptible to HCMV infection and that direct infection of these cells in vivo may contribute to the thrombocytopenia observed in patients infected with HCMV.

scholarly journals Diagnosis and Management of Human Cytomegalovirus Infection in the Mother, Fetus, and Newborn Infant

2002 ◽  
Vol 15 (4) ◽  
pp. 680-715 ◽  
Author(s):  
Maria Grazia Revello ◽  
Giuseppe Gerna
Keyword(s):  
Prenatal Diagnosis ◽  
Human Cytomegalovirus ◽  
Primary Infection ◽  
Hcmv Infection ◽  
Prognostic Markers ◽  
Recurrent Infection ◽  
Congenital Infection ◽  
Immunoglobulin M ◽  
Hcmv Disease ◽  
The Impact

SUMMARY Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection and mental retardation. HCMV infection, while causing asymptomatic infections in most immunocompetent subjects, can be transmitted during pregnancy from the mother with primary (and also recurrent) infection to the fetus. Hence, careful diagnosis of primary infection is required in the pregnant woman based on the most sensitive serologic assays (immunoglobulin M [IgM] and IgG avidity assays) and conventional virologic and molecular procedures for virus detection in blood. Maternal prognostic markers of fetal infection are still under investigation. If primary infection is diagnosed in a timely manner, prenatal diagnosis can be offered, including the search for virus and virus components in fetal blood and amniotic fluid, with fetal prognostic markers of HCMV disease still to be defined. However, the final step for definite diagnosis of congenital HCMV infection is detection of virus in the blood or urine in the first 1 to 2 weeks of life. To date, treatment of congenital infection with antiviral drugs is only palliative both prior to and after birth, whereas the only efficacious preventive measure seems to be the development of a safe and immunogenic vaccine, including recombinant, subunit, DNA, and peptide-based vaccines now under investigation. The following controversial issues are discussed in the light of the most recent advances in the field: the actual perception of the problem; universal serologic screening before pregnancy; the impact of correct counseling on decision making by the couple involved; the role of prenatal diagnosis in ascertaining transmission of virus to the fetus; the impact of preconceptional and periconceptional infections on the prevalence of congenital infection; and the prevalence of congenitally infected babies born to mothers who were immune prior to pregnancy compared to the number born to mothers undergoing primary infection during pregnancy.

scholarly journals Modelling co-translational dimerisation for programmable nonlinearity in synthetic biology

2020 ◽  
Author(s):  
Ruud Stoof ◽  
Ángel Goñi-Moreno
Keyword(s):  
Nonlinear Dynamics ◽  
Biological Networks ◽  
Rational Design ◽  
Fine Tuning ◽  
Genetic Circuits ◽  
On Demand ◽  
Regulatory Circuits ◽  
Protein Dimers ◽  
The Impact

AbstractNonlinearity plays a fundamental role in the performance of both natural and synthetic biological networks. Key functional motifs in living microbial systems, such as the emergence of bistability or oscillations, rely on nonlinear molecular dynamics. Despite its core importance, the rational design of nonlinearity remains an unmet challenge. This is largely due to a lack of mathematical modelling that accounts for the mechanistic basics of nonlinearity. We introduce a model for gene regulatory circuits that explicitly simulates protein dimerization—a well-known source of nonlinear dynamics. Specifically, our approach focusses on modelling co-translational dimerization: the formation of protein dimers during—and not after—translation. This is in contrast to the prevailing assumption that dimer generation is only viable between freely diffusing monomers (i.e., post-translational dimerization). We provide a method for fine-tuning nonlinearity on demand by balancing the impact of co- versus post-translational dimerization. Furthermore, we suggest design rules, such as protein length or physical separation between genes, that may be used to adjust dimerization dynamics in-vivo. The design, build and test of genetic circuits with on-demand nonlinear dynamics will greatly improve the programmability of synthetic biological systems.

scholarly journals Complex Interplay of theUL136Isoforms Balances Cytomegalovirus Replication and Latency

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Katie Caviness ◽  
Farah Bughio ◽  
Lindsey B. Crawford ◽  
Daniel N. Streblow ◽  
Jay A. Nelson ◽  
...  
Keyword(s):  
Virus Replication ◽  
Human Cytomegalovirus ◽  
Secretory Pathway ◽  
Hcmv Infection ◽  
Genetic Locus ◽  
Protein Isoforms ◽  
Hematopoietic Progenitor Cell ◽  
Transplant Patients

ABSTRACTHuman cytomegalovirus (HCMV), a betaherpesvirus, persists indefinitely in the human host through poorly understood mechanisms. TheUL136gene is carried within a genetic locus important to HCMV latency termed theUL133/8locus, which also carriesUL133,UL135, andUL138. Previously, we demonstrated thatUL136is expressed as five protein isoforms ranging from 33-kDa to 19-kDa, arising from alternative transcription and, likely, translation initiation mechanisms. We previously showed that theUL136isoforms are largely dispensable for virus infection in fibroblasts, a model for productive virus replication. In our current work,UL136has emerged as a complex regulator of HCMV infection in multiple contexts of infection relevant to HCMV persistence: in an endothelial cell (EC) model of chronic infection, in a CD34+hematopoietic progenitor cell (HPC) model of latency, and in anin vivoNOD-scidIL2Rγcnullhumanized (huNSG) mouse model for latency. The 33- and 26-kDa isoforms promote replication, while the 23- and 19-kDa isoforms suppress replication in ECs, in CD34+HPCs, and in huNSG mice. The role of the 25-kDa isoform is context dependent and influences the activity of the other isoforms. These isoforms localize throughout the secretory pathway, and loss of the 33- and 26-kDaUL136isoforms results in virus maturation defects in ECs. This work reveals an intriguing functional interplay between protein isoforms that impacts virus replication, latency, and dissemination, contributing to the overall role of theUL133/8locus in HCMV infection.IMPORTANCEThe persistence of DNA viruses, and particularly of herpesviruses, remains an enigma because we have not completely defined the viral and host factors important to persistence. Human cytomegalovirus, a herpesvirus, persists in the absence of disease in immunocompetent individuals but poses a serious disease threat to transplant patients and the developing fetus. There is no vaccine, and current therapies do not target latent reservoirs. In an effort to define the viral factors important to persistence, we have studied viral genes with no known viral replication function in contexts important to HCMV persistence. Using models relevant to viral persistence, we demonstrate opposing roles of protein isoforms encoded by theUL136gene in regulating latent and replicative states of infection. Our findings reveal an intriguing interplay betweenUL136protein isoforms and defineUL136as an important regulator of HCMV persistence.

scholarly journals 1591. Updated Fluoroquinolone MIC Breakpoints: Impact on Susceptibility Rates in the United States

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S580-S581
Author(s):  
Robert K Flamm ◽  
Michael A Pfaller ◽  
Paul G Ambrose ◽  
David Andes ◽  
John S Bradley ◽  
...  
Keyword(s):  
United States ◽  
Pseudomonas Aeruginosa ◽  
Ad Hoc ◽  
The United States ◽  
Broth Microdilution ◽  
Successful Model ◽  
Consultative Process ◽  
The Impact ◽  
Comparison Data

Abstract Background In 2015 USCAST, the National Advisory Committee for the United States (US) to EUCAST, produced a report (Version 1.0) on their website (www.uscast.org) re-evaluating fluoroquinolone (FQ) breakpoint interpretive criteria (IC) based on analysis of current microbiology and pharmacokinetic/pharmacodynamic (PK/PD) data. EUCAST initiated a consultative process using USCAST analyses in an effort to update FQ IC, released in 2017. CLSI formed an ad-hoc working group in late 2015 to review the USCAST FQ document and formulate questions about content. In 2018, USCAST released V1.3 of the FQ document and CLSI subsequently published updated FQ MIC IC in the M100-S29 (2019) document. This study evaluated the impact on susceptibility (S) rates for US surveillance data that these IC changes created. Methods Clinical isolates (reference broth microdilution MIC) from 2016–2018 US SENTRY Program were analyzed for S based on current and previous IC. FQ results for ciprofloxacin (CIP), levofloxacin (LEV), and moxifloxacin (MOX) were evaluated. Benchmark S comparison data for meropenem, cefepime, piperacillin–tazobactam and delafloxacin (new FQ) were also included. Results S rates for Enterobacteriaceae (ENT;Figure) were reduced by 3.8/3.7% for CIP/LEV (CLSI) and 2.3/2.5% (EUCAST). MOX-S rate vs. ENT declined 5.7% (EUCAST). Although reductions in S occurred for most organism groups, K. pneumoniae (6.0/5.5% for CIP/LEV [CLSI] and 4.0/4.2% [EUCAST]) and S. marcescens (7.4/4.1% for CIP/LEV [CLSI] and 4.1/5.0% [EUCAST]) reductions were among the largest changes. For Pseudomonas aeruginosa (PSA), CIP-S decreased 6.8% and LEV-S 10.1% (CLSI); but potential for false-S results remain using CLSI IC (5 pathogens). Conclusion USCAST’s comprehensive analyses of FQ IC in 2015 led to revised breakpoints for most organism/drug combinations among ENT and PSA compared with those being used before. USCAST analysis was most influenced by PK/PD in vivo data as current clinical outcomes data by MIC was limited. Awareness and interactions (both formal and informal) among breakpoint setting organizations has modified FQ ICs which are lower than previously recommended, and although not perfectly harmonized in time and detail, this represents a successful model. Disclosures All authors: No reported disclosures.

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