Human Herpesvirus-8 Glycoprotein B Interacts with Epstein–Barr Virus (EBV) Glycoprotein 110 but Fails to Complement the Infectivity of EBV Mutants

AbstractTo characterize human herpesvirus 8 (HHV-8) gB, the open reading frame was PCR amplified from the HHV-8-infected cell line BCBL-1 and cloned into an expression vector. To facilitate detection of expressed HHV-8 gB, the cytoplasmic tail of the glycoprotein was tagged with the influenza hemagglutinin (HA) epitope. Expression of tagged HHV-8 gB (gB-HA), as well as the untagged form, was readily detected in CHO-K1 cells and several lymphoblastoid cell lines (LCLs). HHV-8 gB-HA was sensitive to endoglycosidase H treatment, and immunofluorescence revealed that HHV-8 gB-HA was detectable in the perinuclear region of CHO-K1 cells. These observations suggest that HHV-8 gB is not processed in the Golgi and localizes to the endoplasmic reticulum or nuclear membrane. Because both HHV-8 and EBV are γ-herpesviruses, the ability of HHV-8 gB to interact with and functionally complement EBV gp110 was examined. HHV-8 gB-HA and EBV gp110 co-immunoprecipitated, indicating formation of hetero-oligomers. However, HHV-8 gB-HA and HHV-8 gB failed to restore the infectivity of gp110-negative EBV mutants. These findings indicate that although HHV-8 gB and EBV gp110 have similar patterns of intracellular localization and can interact, there is not sufficient functional homology to allow efficient complementation

Phase 2 Study of Anti-Human Cytomegalovirus Monoclonal Antibodies for Prophylaxis in Hematopoietic Cell Transplantation.

Human cytomegalovirus (HCMV) can cause significant disease in immunocompromised patients, and treatment options are limited by toxicities. CSJ148 is a combination of two anti-HCMV human monoclonal antibodies (LJP538 and LJP539) that bind to and inhibit the functions of viral HCMV glycoprotein B (gB) and the pentameric complex, consisting of glycoproteins gH, gL, UL128, UL130, and UL131. In this phase 2, randomized, placebo-controlled trial, we evaluated the safety and efficacy of CSJ148 for prophylaxis of HCMV in patients undergoing allogeneic hematopoietic stem cell transplantation. As would be expected in the study population, all the patients (100%) reported at least one treatment-emergent adverse event. There were 22 deaths during this study, and over 80% of the patients receiving placebo or CSJ148 developed at least one adverse event of grade 3 or higher severity. No subject who received antibody developed a hypersensitivity- or infusion-related reaction. CSJ148-treated patients showed trends toward decreased viral load, shorter median duration of preemptive therapy, and fewer courses of preemptive therapy. However, the estimated probability that CSJ148 decreases the need for preemptive therapy compared to placebo was 69%, with a risk ratio of 0.89 and a 90% credible interval of 0.61 to 1.31. The primary efficacy endpoint was therefore not met, indicating that CSJ148 did not prevent clinically significant HCMV reactivation in recipients of allogeneic hematopoietic cell transplants. (This study has been registered at ClinicalTrials.gov under identifier NCT02268526 and at EudraCT under number 2017-002047-15.)

Eradication of common pathogens at days 2, 3 and 4 of moxifloxacin therapy in patients with acute bacterial sinusitis

BACKGROUND: Acute bacterial sinusitis (ABS) is a common infection in clinical practice. Data on time to bacteriologic eradication after antimicrobial therapy are lacking for most agents, but are necessary in order to optimize therapy. This was a prospective, single-arm, open-label, multicenter study to determine the time to bacteriologic eradication in ABS patients (maxillary sinusitis) treated with moxifloxacin. METHODS: Adult patients with radiologically and clinically confirmed ABS received once-daily moxifloxacin 400 mg for 10 days. Middle meatus secretion sampling was performed using nasal endoscopy pre-therapy, and repeated on 3 consecutive days during treatment. Target enrollment was 30 bacteriologically evaluable patients (pre-therapy culture positive for Streptococcus pneumoniae, Haemophilus influenzae or Moraxella catarrhalis and evaluable cultures for at least Day 2 and Day 3 during therapy visits), including at least 10 each with S. pneumoniae or H. influenzae. RESULTS: Of 192 patients enrolled, 42 were bacteriologically evaluable, with 48 pathogens isolated. Moxifloxacin was started on Day 1. Baseline bacteria were eradicated in 35/42 (83.3%) patients by day 2, 42/42 (100%) patients by day 3, and 41/42 (97.6%) patients by day 4. In terms of individual pathogens, 12/18 S. pneumoniae, 22/23 H. influenzae and 7/7 M. catarrhalis were eradicated by day 2 (total 41/48; 85.4%), and 18/18 S. pneumoniae and 23/23 H. influenzae were eradicated by day 3. On Day 4, S. pneumoniae was isolated from a patient who had negative cultures on Days 2 and 3. Thus, the Day 4 eradication rate was 47/48 (97.9%). Clinical success was achieved in 36/38 (94.7%) patients at the test of cure visit. CONCLUSION: In patients with ABS (maxillary sinusitis), moxifloxacin 400 mg once daily for 10 days resulted in eradication of baseline bacteria in 83.3% of patients by Day 2, 100% by Day 3 and 97.6% by Day 4

Cell-Cell Transmission Enables HIV-1 to Evade Inhibition by Potent CD4bs Directed Antibodies

HIV is known to spread efficiently both in a cell-free state and from cell to cell, however the relative importance of the cell-cell transmission mode in natural infection has not yet been resolved. Likewise to what extent cell-cell transmission is vulnerable to inhibition by neutralizing antibodies and entry inhibitors remains to be determined. Here we report on neutralizing antibody activity during cell-cell transmission using specifically tailored experimental strategies which enable unambiguous discrimination between the two transmission routes. We demonstrate that the activity of neutralizing monoclonal antibodies (mAbs) and entry inhibitors during cell-cell transmission varies depending on their mode of action. While gp41 directed agents remain active, CD4 binding site (CD4bs) directed inhibitors, including the potent neutralizing mAb VRC01, dramatically lose potency during cell-cell transmission. This implies that CD4bs mAbs act preferentially through blocking free virus transmission, while still allowing HIV to spread through cell-cell contacts. Thus providing a plausible explanation for how HIV maintains infectivity and rapidly escapes potent and broadly active CD4bs directed antibody responses in vivo

Human Herpesvirus 8 Glycoprotein B (gB), gH, and gL Can Mediate Cell Fusion

Herpesvirus entry into cells and herpesvirus-induced cell fusion are related processes in that virus penetration proceeds by fusion of the viral envelope and cell membrane. To characterize the human herpesvirus 8 (HHV-8) glycoproteins that can mediate cell fusion, a luciferase reporter gene activation assay was used. Chinese hamster ovary (CHO) cells expressing the HHV-8 glycoproteins of interest along with a luciferase reporter gene under the control of the T7 promoter were cocultivated with human cells transfected with T7 RNA polymerase. Because HHV-8 glycoprotein B (gB) expressed in CHO cells localizes to the perinuclear region, a truncated form of gB (designated gB(MUT)) that lacks putative endocytosis signals was constructed by deletion of the distal 58 amino acids of the cytoplasmic tail. HHV-8 gB(MUT) was expressed efficiently on the surface of CHO cells. HHV-8 gB, gH, and gL could mediate the fusion of CHO cells with two different human cell types, embryonic kidney cells and B lymphocytes. Substituting gB(MUT) for gB significantly enhanced the fusion of CHO cells with human embryonic kidney cells but not B lymphocytes. Thus, two human cell types known to be susceptible to HHV-8 entry were also suitable targets for cell fusion induced by HHV-8 gB, gH, and gL. For human embryonic kidney cells and B cells at least, optimal fusion was noted with the expression of all three HHV-8 glycoproteins

Cell Fusion Induced by Herpes Simplex Virus Glycoproteins gB, gD, and gH-gL Requires a gD Receptor but Not Necessarily Heparan Sulfate

AbstractTo characterize cellular factors required for herpes simplex virus type 1 (HSV-1)-induced cell fusion, we used an efficient and quantitative assay relying on expression of HSV-1 glycoproteins in transfected cells. We showed the following: (1) Cell fusion depended not only on expression of four viral glycoproteins (gB, gD, and gH-gL), as previously shown, but also on expression of cell surface entry receptors specific for gD. (2) Cell fusion required expression of all four glycoproteins in the same cell. (3) Heparan sulfate was not required for cell fusion. (4) Coexpression of receptor with the four glycoproteins in the same cell reduced fusion activity, indicating that interaction of gD and receptor can limit polykaryocyte formation. Overall, the viral and cellular determinants of HSV-1-induced cell fusion are similar to those for viral entry, except that HSV-1 entry is significantly enhanced by binding of virus to cell surface heparan sulfate

A multi-center, randomized clinical trial to compare the safety and efficacy of LFF571 and vancomycin for Clostridium difficile infections

Background: Clostridium difficile cause serious diarrheal disease with potentially fatal complications. Several drugs are available for the treatment of C. difficile, including vancomycin, metronidazole, and fidaxomicin. Recurrences after successful therapy, however, remain a significant problem. LFF571 is a semi-synthetic thiopeptide antibacterial that is potent against C. difficile in vitro and in animal models of infection. Here, we assess the safety and efficacy of LFF571 in patients with primary or first recurrent C. difficile infections. Methods: This was a multi-center, randomized, evaluator-blind, active-controlled study to compare the safety and efficacy of LFF571 to those of vancomycin. Adults aged 18-90 with primary episodes or first recurrences of moderate C. difficile infection were eligible to enroll. Patients were randomized to receive 200 mg LFF571 or 125 mg vancomycin four times daily for 10 days. The primary endpoint was the proportion of clinical cures at the end of therapy, with the primary analysis comparing cure rates in the per-protocol population. Secondary endpoints included time to resolution of diarrhea and the recurrence rate 30 days following completion of treatment. Results: Seventy-two patients were enrolled and randomized, of which 46 were assigned to receive LFF571. Based on the protocol-specified definition, the rates of clinical cure for LFF571 (90.6%) were non-inferior to those of vancomycin (78.3%). The 30-day sustained cure rates for LFF571- and vancomycin- treated patients were 56.7% and 65.0%, respectively, in the per-protocol population and were 58.7% and 60.0%, respectively, in the modified intent-to-treat population. Analysis of recurrence using only toxin-confirmed cases, however, indicated that recurrence rates were lower for LFF571 (19% compared to 25% for vancomycin in the per-protocol population). The incidence of adverse events was higher in the LFF571 group (76.1%) compared to vancomycin (69.2%), although more adverse events in the vancomycin group were suspected to be related to study drug (38.5% versus 32.6% for LFF571). One patient receiving LFF571 discontinued due to an adverse event. Conclusion: LFF571 was non-inferior to vancomycin for the clinical cure of primary or first recurrent moderate C. difficile infections. The sustained cure rates at the end-of-study were slightly lower for LFF571, although toxic-confirmed recurrences were reduced compared to vancomycin. LFF571 was generally safe and well-tolerated in patients with moderate C. difficile infections

A first-in-human, randomized, double-blind, placebo-controlled, time-lagged, single- and multiple-ascending oral dose study to assess the safety and tolerability of LFF571 in healthy volunteers

Clostridium difficile is the leading cause of hospital-acquired infectious diarrhea. LFF571 is a novel inhibitor of the prokaryotic translation elongation factor Tu (EF-Tu) and is active against a range of bacterial species, including C. difficile. This first-in-human study investigated the safety and pharmacokinetics of single and multiple ascending oral doses of LFF571 in healthy subjects. The study was randomized, double-blind, placebo-controlled, and time-lagged. Except for one cohort, LFF571 was given with a high fat meal in all single dose cohorts (25 mg, 100 mg, 400 mg, 1000 mg). In the multiple dose cohorts (25 mg, 100 mg, 200 mg, every 6 hours for 10 days), LFF571 was given without regard to food. A total of 56 subjects completed the study, with 32 and 25 receiving single and multiple doses, respectively. There were no deaths, no serious adverse events, and no subject discontinued due to an adverse event. The most common adverse event was diarrhea; gastrointestinal pain or distension was also noted. Diarrhea did not develop more frequently among subjects who received LFF571 compared to placebo. LFF571 had limited systemic exposure and high steady-state fecal concentrations. The highest serum concentration of LFF571 (3.2 ng/mL) was observed after the last dose in a subject receiving 200 mg every 6 hours. LFF571 was generally safe and well-tolerated after single and multiple oral doses in healthy subjects. The minimal serum and high fecal concentrations support the further development of LFF571 for the treatment of C. difficile infections