Susan E. Sloan

Treatment with Monoclonal Antibodies against Clostridium difficile Toxins

BACKGROUND New therapies are needed to manage the increasing incidence, severity, and high rate of recurrence of Clostridium difficile infection. METHODS We performed a randomized, double-blind, placebo-controlled study of two neutralizing, fully human monoclonal antibodies against C. difficile toxins A (CDA1) and B (CDB1). The antibodies were administered together as a single infusion, each at a dose of 10 mg per kilogram of body weight, in patients with symptomatic C. difficile infection who were receiving either metronidazole or vancomycin. The primary outcome was laboratory-documented recurrence of infection during the 84 days after the administration of monoclonal antibodies or placebo. RESULTS Among the 200 patients who were enrolled (101 in the antibody group and 99 in the placebo group), the rate of recurrence of C. difficile infection was lower among patients treated with monoclonal antibodies (7% vs. 25%; 95% confidence interval, 7 to 29; P<0.001). The recurrence rates among patients with the epidemic BI/NAP1/027 strain were 8% for the antibody group and 32% for the placebo group (P=0.06); among patients with more than one previous episode of C. difficile infection, recurrence rates were 7% and 38%, respectively (P=0.006). The mean duration of the initial hospitalization for inpatients did not differ significantly between the antibody and placebo groups (9.5 and 9.4 days, respectively). At least one serious adverse event was reported by 18 patients in the antibody group and by 28 patients in the placebo group (P=0.09). CONCLUSIONS The addition of monoclonal antibodies against C. difficile toxins to antibiotic agents significantly reduced the recurrence of C. difficile infection. (ClinicalTrials.gov number, NCT00350298.)

Identification and Characterization of Broadly Neutralizing Human Monoclonal Antibodies Directed against the E2 Envelope Glycoprotein of Hepatitis C Virus

ABSTRACT Nearly all livers transplanted into hepatitis C virus (HCV)-positive patients become infected with HCV, and 10 to 25% of reinfected livers develop cirrhosis within 5 years. Neutralizing monoclonal antibody could be an effective therapy for the prevention of infection in a transplant setting. To pursue this treatment modality, we developed human monoclonal antibodies (HuMAbs) directed against the HCV E2 envelope glycoprotein and assessed the capacity of these HuMAbs to neutralize a broad panel of HCV genotypes. HuMAb antibodies were generated by immunizing transgenic mice containing human antibody genes (HuMAb mice; Medarex Inc.) with soluble E2 envelope glycoprotein derived from a genotype 1a virus (H77). Two HuMAbs, HCV1 and 95-2, were selected for further study based on initial cross-reactivity with soluble E2 glycoproteins derived from genotypes 1a and 1b, as well as neutralization of lentivirus pseudotyped with HCV 1a and 1b envelope glycoproteins. Additionally, HuMAbs HCV1 and 95-2 potently neutralized pseudoviruses from all genotypes tested (1a, 1b, 2b, 3a, and 4a). Epitope mapping with mammalian and bacterially expressed proteins, as well as synthetic peptides, revealed that HuMAbs HCV1 and 95-2 recognize a highly conserved linear epitope spanning amino acids 412 to 423 of the E2 glycoprotein. The capacity to recognize and neutralize a broad range of genotypes, the highly conserved E2 epitope, and the fully human nature of the antibodies make HuMAbs HCV1 and 95-2 excellent candidates for treatment of HCV-positive individuals undergoing liver transplantation.

A broadly neutralizing human monoclonal antibody is effective against H7N9

Significance Emerging influenza subtypes, such as the recently identified H7N9 strains, are of considerable public health concern. Although vaccines are an important countermeasure, influenza vaccines tend to be subtype- and strain-specific, such that they may not be widely available in the event of the human adaptation and spread of an unanticipated strain or subtype. Additionally, influenza strains have demonstrated the ability to develop resistance to existing antivirals, including oseltamivir. As such, there is a need for novel interventions that can treat and/or prevent serious influenza infection. We demonstrate that VIS410, a human mAb, neutralizes a wide range of influenza A viruses and effectively manages H7N9 infection. Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (−24 h) or double-dose (−12 h, +48 h) administration (P < 0.001). A single dose of VIS410 at 50 mg/kg (−12 h) combined with oseltamivir at 50 mg/kg (−12 h, twice daily for 7 d) in C57BL/6 mice infected with A/Shanghai 2/2013 (H7N9) resulted in significant decreased lung viral load (P = 0.002) and decreased lung cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains.

Safety and Upper Respiratory Pharmacokinetics of the Hemagglutinin Stalk-Binding Antibody VIS410 Support Treatment and Prophylaxis Based on Population Modeling of Seasonal Influenza A Outbreaks

Background Seasonal influenza is a major public health concern in vulnerable populations. Here we investigated the safety, tolerability, and pharmacokinetics of a broadly neutralizing monoclonal antibody (VIS410) against Influenza A in a Phase 1 clinical trial. Based on these results and preclinical data, we implemented a mathematical modeling approach to investigate whether VIS410 could be used prophylactically to lessen the burden of a seasonal influenza epidemic and to protect at-risk groups from associated complications. Methods Using a single-ascending dose study (n = 41) at dose levels from 2 mg/kg–50 mg/kg we evaluated the safety as well as the serum and upper respiratory pharmacokinetics of a broadly-neutralizing antibody (VIS410) against influenza A (ClinicalTrials.gov identifier NCT02045472). Our primary endpoints were safety and tolerability of VIS410 compared to placebo. We developed an epidemic microsimulation model testing the ability of VIS410 to mitigate attack rates and severe disease in at risk-populations. Findings VIS410 was found to be generally safe and well-tolerated at all dose levels, from 2–50 mg/kg. Overall, 27 of 41 subjects (65.9%) reported a total of 67 treatment emergent adverse events (TEAEs). TEAEs were reported by 20 of 30 subjects (66.7%) who received VIS410 and by 7 of 11 subjects (63.6%) who received placebo. 14 of 16 TEAEs related to study drug were considered mild (Grade 1) and 2 were moderate (Grade 2). Two subjects (1 subject who received 30 mg/kg VIS410 and 1 subject who received placebo) experienced serious AEs (Grade 3 or 4 TEAEs) that were not related to study drug. VIS410 exposure was approximately dose-proportional with a mean half-life of 12.9 days. Mean VIS410 Cmax levels in the upper respiratory tract were 20.0 and 25.3 μg/ml at the 30 mg/kg and 50 mg/kg doses, respectively, with corresponding serum Cmax levels of 980.5 and 1316 μg/mL. Using these pharmacokinetic data, a microsimulation model showed that median attack rate reductions ranged from 8.6% (interquartile range (IQR): 4.7%–11.0%) for 2% coverage to 22.6% (IQR: 12.7–30.0%) for 6% coverage. The overall benefits to the elderly, a vulnerable subgroup, are largest when VIS410 is distributed exclusively to elderly individuals, resulting in reductions in hospitalization rates between 11.4% (IQR: 8.2%–13.3%) for 2% coverage and 30.9% (IQR: 24.8%–35.1%) for 6% coverage among those more than 65 years of age. Interpretation VIS410 was generally safe and well tolerated and had good relative exposure in both serum and upper respiratory tract, supporting its use as either a single-dose therapeutic or prophylactic for influenza A. Including VIS410 prophylaxis among the public health interventions for seasonal influenza has the potential to lower attack rates and substantially reduce hospitalizations in individuals over the age of 65. Funding Visterra, Inc.

The Hemagglutinin Stem-Binding Monoclonal Antibody VIS410 Controls Influenza Virus-Induced Acute Respiratory Distress Syndrome.

ABSTRACT Most cases of severe influenza are associated with pulmonary complications, such as acute respiratory distress syndrome (ARDS), and no antiviral drugs of proven value for treating such complications are currently available. The use of monoclonal antibodies targeting the stem of the influenza virus surface hemagglutinin (HA) is a rapidly developing strategy for the control of viruses of multiple HA subtypes. However, the mechanisms of action of these antibodies are not fully understood, and their ability to mitigate severe complications of influenza has been poorly studied. We evaluated the effect of treatment with VIS410, a human monoclonal antibody targeting the HA stem region, on the development of ARDS in BALB/c mice after infection with influenza A(H7N9) viruses. Prophylactic administration of VIS410 resulted in the complete protection of mice against lethal A(H7N9) virus challenge. A single therapeutic dose of VIS410 given 24 h after virus inoculation resulted in dose-dependent protection of up to 100% of mice inoculated with neuraminidase inhibitor-susceptible or -resistant A(H7N9) viruses. Compared to the outcomes in mock-treated controls, a single administration of VIS410 improved viral clearance from the lungs, reduced virus spread in lungs in a dose-dependent manner, resulting in a lower lung injury score, reduced the extent of the alteration in lung vascular permeability and protein accumulation in bronchoalveolar lavage fluid, and improved lung physiologic function. Thus, antibodies targeting the HA stem can reduce the severity of ARDS and show promise as agents for controlling pulmonary complications in influenza.

Identification of a Human Monoclonal Antibody To Replace Equine Diphtheria Antitoxin for Treatment of Diphtheria Intoxication

ABSTRACT Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 μg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.

G glycoprotein amino acid residues required for human monoclonal antibody RAB1 neutralization are conserved in rabies virus street isolates

Replacement of polyclonal anti-rabies immunoglobulin (RIG) used in rabies post-exposure prophylaxis (PEP) with a monoclonal antibody will eliminate cost and availability constraints that currently exist using RIG in the developing world. The human monoclonal antibody RAB1 has been shown to neutralize all rabies street isolates tested; however for the laboratory-adapted fixed strain, CVS-11, mutation in the G glycoprotein of amino acid 336 from asparagine (N) to aspartic acid (D) resulted in resistance to neutralization. Interestingly, this same mutation in the G glycoprotein of a second laboratory-adapted fixed strain (ERA) did not confer resistance to RAB1 neutralization. Using cell surface staining and lentivirus pseudotyped with rabies virus G glycoprotein (RABVpp), we identified an amino acid alteration in CVS-11 (K346), not present in ERA (R346), which was required in combination with D336 to confer resistance to RAB1. A complete analysis of G glycoprotein sequences from GenBank demonstrated that no identified rabies isolates contain the necessary combination of G glycoprotein mutations for resistance to RAB1 neutralization, consistent with the broad neutralization of RAB1 observed in direct viral neutralization experiments with street isolates. All combinations of amino acids 336 and 346 reported in the sequence database were engineered into the ERA G glycoprotein and RAB1 was able to neutralize RABVpp bearing ERA G glycoprotein containing all known combinations at these critical residues. These data demonstrate that RAB1 has the capacity to neutralize all identified rabies isolates and a minimum of two distinct mutations in the G glycoprotein are required for abrogation of RAB1 neutralization.

Regulation of hindbrain Pyy expression by acute food deprivation, prolonged caloric restriction, and weight loss surgery in mice

PYY is a gut-derived putative satiety signal released in response to nutrient ingestion and is implicated in the regulation of energy homeostasis. Pyy-expressing neurons have been identified in the hindbrain of river lamprey, rodents, and primates. Despite this high evolutionary conservation, little is known about central PYY neurons. Using in situ hybridization, PYY-Cre;ROSA-EYFP mice, and immunohistochemistry, we identified PYY cell bodies in the gigantocellular reticular nucleus region of the hindbrain. PYY projections were present in the dorsal vagal complex and hypoglossal nucleus. In the hindbrain, Pyy mRNA was present at E9.5, and expression peaked at P2 and then decreased significantly by 70% at adulthood. We found that, in contrast to the circulation, PYY-(1-36) is the predominant isoform in mouse brainstem extracts in the ad libitum-fed state. However, following a 24-h fast, the relative amounts of PYY-(1-36) and PYY-(3-36) isoforms were similar. Interestingly, central Pyy expression showed nutritional regulation and decreased significantly by acute starvation, prolonged caloric restriction, and bariatric surgery (enterogastroanastomosis). Central Pyy expression correlated with body weight loss and circulating leptin and PYY concentrations. Central regulation of energy metabolism is not limited to the hypothalamus but also includes the midbrain and the brainstem. Our findings suggest a role for hindbrain PYY in the regulation of energy homeostasis and provide a starting point for further research on gigantocellular reticular nucleus PYY neurons, which will increase our understanding of the brain stem pathways in the integrated control of appetite and energy metabolism.