An introduction to the Marburg virus vaccine consortium, MARVAC.

The emergence of Marburg virus (MARV) in Guinea and Ghana triggered the assembly of the MARV vaccine "MARVAC" consortium representing leaders in the field of vaccine research and development aiming to facilitate a rapid response to this infectious disease threat. Here, we discuss current progress, challenges, and future directions for MARV vaccines.

Investigational New Drug Enabling Nonclinical Safety Pharmacology Assessment of the Iminosugar UV-4, a Broad-Spectrum Host-Targeted Antiviral Agent.

UV-4 (N-(9-methoxynonyl)-1-deoxynojirimycin) is a broad-spectrum antiviral drug candidate with demonstrated activity in vitro and in vivo against multiple, diverse viruses. Nonclinical safety pharmacology studies were conducted to support the filing of an Investigational New Drug (IND) application. Preliminary in vitro pharmacology testing evaluating potential for binding to "off-target" receptors and enzymes indicated no significant liability for advanced development of UV-4. The safety pharmacology of UV-4 was evaluated in the in vitro human ether-à-go-go-related gene (hERG) assay, in a central nervous system (CNS) study in the mouse (modified Irwin test), in a respiratory safety study in conscious mice using whole body plethysmography, and in a cardiovascular safety study in conscious, radiotelemetry-instrumented beagle dogs. There were no observed adverse treatment-related effects following administration of UV-4 as the hydrochloride salt in the hERG potassium channel assay, on respiratory function, in the CNS study, or in the cardiovascular assessment. Treatment-related cardiovascular effect of decreased arterial pulse pressure after 50 or 200 mg of UV-4/kg was the only change outside the normal range, and all hemodynamic parameters returned to control levels by the end of the telemetry recording period. These nonclinical safety pharmacology assessments support the evaluation of this host-targeted broad-spectrum antiviral drug candidate in clinical studies.

Investigational New Drug Enabling Nonclinical Safety Assessment of the Iminosugar UV-4, a Broad-Spectrum Host-Targeted Antiviral Agent.

The iminosugar UV-4 is a broad-spectrum antiviral drug candidate with activity in vitro and in vivo against multiple, diverse viruses. The toxicological profile of UV-4, dosed as the hydrochloride salt, was evaluated in single-dose and repeat-dose oral toxicity studies in mice, rats, dogs, and non-human primates (NHP). No moribundity or deaths were associated with the drug up to the maximum tolerated dose. No treatment-related adverse effects were observed following single oral doses in dogs, rats, and mice up to 250, 400, 1000 mg/kg, respectively, and in NHP up to 180 mg/kg administered three times daily for 10 days. UV-4-related findings were generally seen at higher doses after 7- or 14-day exposure. The most common clinical pathology findings (increase in aspartate aminotransferase and decreased platelet count) were consistently found across species and each appeared dose related. The kidney, mesenteric lymph nodes, stomach including gastrointestinal tract, and thymus were identified as target organs in mice, rats, and dogs. In 14-day repeat-dose toxicology studies in mice and dogs conducted in compliance with Good Laboratory Practice regulations, the dog was considered to be the most sensitive species to UV-4 exposure based on the treatment-related adverse effects noted in the identified target organs. The results of these studies demonstrate the safety profile of UV-4 hydrochloride and supported the selection of starting and maximal doses for a single ascending dose first-in-human clinical study.

Chikungunya Virus Strains from Each Genetic Clade Bind Sulfated Glycosaminoglycans as Attachment Factors.

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that causes debilitating musculoskeletal disease. CHIKV displays broad cell, tissue, and species tropism, which may correlate with the attachment factors and entry receptors used by the virus. Cell surface glycosaminoglycans (GAGs) have been identified as CHIKV attachment factors. However, the specific types of GAGs and potentially other glycans to which CHIKV binds and whether there are strain-specific differences in GAG binding are not fully understood. To identify the types of glycans bound by CHIKV, we conducted glycan microarray analyses and discovered that CHIKV preferentially binds GAGs. Microarray results also indicate that sulfate groups on GAGs are essential for CHIKV binding and that CHIKV binds most strongly to longer GAG chains of heparin and heparan sulfate. To determine whether GAG binding capacity varies among CHIKV strains, a representative strain from each genetic clade was tested. While all strains directly bound to heparin and chondroitin sulfate in enzyme-linked immunosorbent assays (ELISAs) and depended on heparan sulfate for efficient cell binding and infection, we observed some variation by strain. Enzymatic removal of cell surface GAGs and genetic ablation that diminishes GAG expression reduced CHIKV binding and infectivity of all strains. Collectively, these data demonstrate that GAGs are the preferred glycan bound by CHIKV, enhance our understanding of the specific GAG moieties required for CHIKV binding, define strain differences in GAG engagement, and provide further evidence for a critical function of GAGs in CHIKV cell attachment and infection.IMPORTANCE Alphavirus infections are a global health threat, contributing to outbreaks of disease in many parts of the world. Recent epidemics caused by CHIKV, an arthritogenic alphavirus, resulted in more than 8.5 million cases as the virus has spread into new geographic regions, including the Western Hemisphere. CHIKV causes disease in the majority of people infected, leading to severe and debilitating arthritis. Despite the severity of CHIKV disease, there are no licensed therapeutics. Since attachment factors and receptors are determinants of viral tropism and pathogenesis, understanding these virus-host interactions can enhance our knowledge of CHIKV infection. We analyzed over 670 glycans and identified GAGs as the main glycan bound by CHIKV. We defined specific GAG components required for CHIKV binding and assessed strain-specific differences in GAG binding capacity. These studies provide insight about cell surface molecules that CHIKV binds, which could facilitate the development of antiviral therapeutics targeting the CHIKV attachment step.

GC-072: A Novel Therapeutic Candidate for Oral Treatment of Melioidosis and Infections Caused by Select Biothreat Pathogens.

Burkholderia pseudomallei (B. pseudomallei), the etiological agent of melioidosis, is a Gram-negative bacterium with additional concern as a biothreat pathogen. The mortality rate from B. pseudomallei varies depending on the type of infection and extent of available health care, but in the case of septicemia left untreated it can range from 50 - 90%. Current therapy for melioidosis is biphasic, consisting of parenteral acute-phase treatment for two weeks or longer, followed by oral eradication-phase treatment lasting several months. An effective oral therapeutic for outpatient treatment of acute-phase melioidosis is needed. GC-072 is a potent, 4-oxoquinolizine antibiotic with selective inhibitory activity against bacterial topoisomerases. GC-072 has demonstrated in vitro potency against susceptible and drug-resistant strains of B. pseudomallei and is also active against Burkholderia mallei, Bacillus anthracis, Yersinia pestis, and Francisella tularensis GC-072 is bactericidal both extra- and intracellularly, with rapid killing noted within a few hours and reduced development of resistance compared to ceftazidime. GC-072, delivered intragastrically to mimic oral administration, promoted dose-dependent survival in mice using lethal inhalational models of B. pseudomallei infection following exposure to a 24 or 339 LD50 challenge with B. pseudomallei strain 1026b. Overall, GC-072 appears to be a strong candidate for first-line, oral treatment of melioidosis.

Role of Antibodies in Protection Against Ebola Virus in Nonhuman Primates Immunized With Three Vaccine Platforms.

Background: Several vaccine platforms have been successfully evaluated for prevention of Ebola virus (EBOV) disease (EVD) in nonhuman primates and humans. Despite remarkable efficacy by multiple vaccines, the immunological correlates of protection against EVD are incompletely understood. Methods: We systematically evaluated the antibody response to various EBOV proteins in 79 nonhuman primates vaccinated with various EBOV vaccine platforms. We evaluated the serum immunoglobulin (Ig)G titers against EBOV glycoprotein (GP), the ability of the vaccine-induced antibodies to bind GP at acidic pH or to displace ZMapp, and virus neutralization titers. The correlation of these outcomes with survival from EVD was evaluated by appropriate statistical methods. Results: Irrespective of the vaccine platform, protection from EVD strongly correlated with anti-GP IgG titers. The GP-directed antibody levels required for protection in animals vaccinated with virus-like particles (VLPs) lacking nucleoprotein (NP) was significantly higher than animals immunized with NP-containing VLPs or adenovirus-expressed GP, platforms that induce strong T-cell responses. Furthermore, protective immune responses correlated with anti-GP antibody binding strength at acidic pH, neutralization of GP-expressing pseudovirions, and the ability to displace ZMapp components from GP. Conclusions: These findings suggest key quantitative and qualitative attributes of antibody response to EVD vaccines as potential correlates of protection.

AVI-7288 for Marburg Virus in Nonhuman Primates and Humans.

BACKGROUND: AVI-7288 is a phosphorodiamidate morpholino oligomer with positive charges that targets the viral messenger RNA that encodes Marburg virus (MARV) nucleoprotein. Its safety in humans is undetermined. METHODS: We assessed the efficacy of AVI-7288 in a series of studies involving a lethal challenge with MARV in nonhuman primates. The safety of AVI-7288 was evaluated in a randomized, multiple-ascending-dose study in which 40 healthy humans (8 humans per dose group) received 14 once-daily infusions of AVI-7288 (1 mg, 4 mg, 8 mg, 12 mg, or 16 mg per kilogram of body weight) or placebo, in a 3:1 ratio. We estimated the protective dose in humans by comparing pharmacokinetic variables in infected nonhuman primates, uninfected nonhuman primates, and uninfected humans. RESULTS: Survival in infected nonhuman primates was dose-dependent, with survival rates of 0%, 30%, 59%, 87%, 100%, and 100% among monkeys treated with 0 mg, 3.75 mg, 7.5 mg, 15 mg, 20 mg, and 30 mg of AVI-7288 per kilogram, respectively (P<0.001 with the use of the log-rank test for the comparison of survival across groups). No safety concern was identified at doses up to 16 mg per kilogram per day in humans. No serious adverse events were reported. Drug exposure (the area under the curve) was dose-dependent in both nonhuman primates and humans; drug clearance was independent of dose but was higher in nonhuman primates than in humans. The protective dose in humans was initially estimated, on the basis of exposure, to be 9.6 mg per kilogram per day (95% confidence interval, 6.6 to 12.5) for 14 days. Monte Carlo simulations supported a dose of 11 mg per kilogram per day to match the geometric mean protective exposure in nonhuman primates. CONCLUSIONS: This study shows that, on the basis of efficacy in nonhuman primates and pharmacokinetic data in humans, AVI-7288 has potential as postexposure prophylaxis for MARV infection in humans. (Funded by the Department of Defense; ClinicalTrials.gov number, NCT01566877.).

Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein.

UNLABELLED: Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCE: Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus. Since the nature of future outbreaks cannot be predicted, there is an urgent need for therapeutics with broad protective efficacy against multiple filoviruses. Here we describe a set of monoclonal antibodies cross-reactive with multiple filovirus species. These antibodies target novel conserved epitopes within the envelope glycoprotein and exhibit protective efficacy in mice. We further present novel concepts for combination of cross-reactive antibodies against multiple epitopes that show enhanced efficacy compared to monotherapy and provide complete protection in mice. These findings set the stage for further evaluation of these antibodies in nonhuman primates and development of effective pan-filovirus immunotherapeutics for use in future outbreaks.

Pan-ebolavirus and Pan-filovirus Mouse Monoclonal Antibodies: Protection against Ebola and Sudan Viruses.

UNLABELLED: The unprecedented 2014-2015 Ebola virus disease (EVD) outbreak in West Africa has highlighted the need for effective therapeutics against filoviruses. Monoclonal antibody (MAb) cocktails have shown great potential as EVD therapeutics; however, the existing protective MAbs are virus species specific. Here we report the development of pan-ebolavirus and pan-filovirus antibodies generated by repeated immunization of mice with filovirus glycoproteins engineered to drive the B cell responses toward conserved epitopes. Multiple pan-ebolavirus antibodies were identified that react to the Ebola, Sudan, Bundibugyo, and Reston viruses. A pan-filovirus antibody that was reactive to the receptor binding regions of all filovirus glycoproteins was also identified. Significant postexposure efficacy of several MAbs, including a novel antibody cocktail, was demonstrated. For the first time, we report cross-neutralization and in vivo protection against two highly divergent filovirus species, i.e., Ebola virus and Sudan virus, with a single antibody. Competition studies indicate that this antibody targets a previously unrecognized conserved neutralizing epitope that involves the glycan cap. Mechanistic studies indicated that, besides neutralization, innate immune cell effector functions may play a role in the antiviral activity of the antibodies. Our findings further suggest critical novel epitopes that can be utilized to design effective cocktails for broad protection against multiple filovirus species. IMPORTANCE: Filoviruses represent a major public health threat in Africa and an emerging global concern. Largely driven by the U.S. biodefense funding programs and reinforced by the 2014 outbreaks, current immunotherapeutics are primarily focused on a single filovirus species called Ebola virus (EBOV) (formerly Zaire Ebola virus). However, other filoviruses including Sudan, Bundibugyo, and Marburg viruses have caused human outbreaks with mortality rates as high as 90%. Thus, cross-protective immunotherapeutics are urgently needed. Here, we describe monoclonal antibodies with cross-reactivity to several filoviruses, including the first report of a cross-neutralizing antibody that exhibits protection against Ebola virus and Sudan virus in mice. Our results further describe a novel combination of antibodies with enhanced protective efficacy. These results form a basis for further development of effective immunotherapeutics against filoviruses for human use. Understanding the cross-protective epitopes are also important for rational design of pan-ebolavirus and pan-filovirus vaccines.

Protective mAbs and Cross-Reactive mAbs Raised by Immunization with Engineered Marburg Virus GPs.

The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel "wing" feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.

Iminosugars: Promising therapeutics for influenza infection.

Influenza virus causes three to five million severe respiratory infections per year in seasonal epidemics, and sporadic pandemics, three of which occurred in the twentieth century and are a continuing global threat. Currently licensed antivirals exclusively target the viral neuraminidase or M2 ion channel, and emerging drug resistance necessitates the development of novel therapeutics. It is believed that a host-targeted strategy may combat the development of antiviral drug resistance. To this end, a class of molecules known as iminosugars, hydroxylated carbohydrate mimics with the endocyclic oxygen atom replaced by a nitrogen atom, are being investigated for their broad-spectrum antiviral potential. The influenza virus glycoproteins, hemagglutinin and neuraminidase, are susceptible to inhibition of endoplasmic reticulum α-glucosidases by certain iminosugars, leading to reduced virion production or infectivity, demonstrated by in vitro and in vivo studies. In some experiments, viral strain-specific effects are observed. Iminosugars may also inhibit other host and virus targets with antiviral consequences. While investigations of anti-influenza iminosugar activities have been conducted since the 1980s, recent successes of nojirimycin derivatives have re-invigorated investigation of the therapeutic potential of iminosugars as orally available, low cytotoxicity, effective anti-influenza drugs.

Affinity maturation of a broadly neutralizing human monoclonal antibody that prevents acute hepatitis C virus infection in mice.

Direct-acting antivirals (DAAs) have led to a high cure rate in treated patients with chronic hepatitis C virus (HCV) infection, but this still leaves a large number of treatment failures secondary to the emergence of resistance-associated variants (RAVs). To increase the barrier to resistance, a complementary strategy is to use neutralizing human monoclonal antibodies (HMAbs) to prevent acute infection. However, earlier efforts with the selected antibodies led to RAVs in animal and clinical studies. Therefore, we identified an HMAb that is less likely to elicit RAVs for affinity maturation to increase potency and, more important, breadth of protection. Selected matured antibodies show improved affinity and neutralization against a panel of diverse HCV isolates. Structural and modeling studies reveal that the affinity-matured HMAb mediates virus neutralization, in part, by inducing conformational change to the targeted epitope, and that the maturated light chain is responsible for the improved affinity and breadth of protection. A matured HMAb protected humanized mice when challenged with an infectious HCV human serum inoculum for a prolonged period. However, a single mouse experienced breakthrough infection after 63 days when the serum HMAb concentration dropped by several logs; sequence analysis revealed no viral escape mutation. CONCLUSION: The findings suggest that a single broadly neutralizing antibody can prevent acute HCV infection without inducing RAVs and may complement DAAs to reduce the emergence of RAVs. (Hepatology 2016;64:1922-1933).

Vaccinating captive chimpanzees to save wild chimpanzees.

Infectious disease has only recently been recognized as a major threat to the survival of Endangered chimpanzees and Critically Endangered gorillas in the wild. One potentially powerful tool, vaccination, has not been deployed in fighting this disease threat, in good part because of fears about vaccine safety. Here we report on what is, to our knowledge, the first trial in which captive chimpanzees were used to test a vaccine intended for use on wild apes rather than humans. We tested a virus-like particle vaccine against Ebola virus, a leading source of death in wild gorillas and chimpanzees. The vaccine was safe and immunogenic. Captive trials of other vaccines and of methods for vaccine delivery hold great potential as weapons in the fight against wild ape extinction.

Dengue Virus Evolution under a Host-Targeted Antiviral.

UNLABELLED: The host-targeted antiviral drug UV-4B reduces viral replication and promotes survival in a mouse model of experimental dengue virus (DENV) infection. UV-4B is an iminosugar that inhibits the α-glucosidase family of enzymes and subsequently the folding of glycosylated proteins, both viral and host. Here, we utilized next-generation sequencing to investigate evolution of a flavivirus under selective pressure by a host-targeted antiviral in vivo. In viral populations recovered from UV-4B-treated mice, there was a significant increase in the number of single-nucleotide polymorphisms (SNPs) and the ratio of nonsynonymous to synonymous SNPs compared to findings in viral populations from vehicle-treated mice. The strongest evidence of positive selection was in the glycosylated membrane protein, thereby providing in vivo validation of the mechanism of action of an iminosugar. In addition, mutations in glycosylated proteins were present only in drug-treated mice after a single passage. However, the bulk of the other mutations were present in both populations, indicating nonspecific selective pressure. Together with the continued control of viremia by UV-4B, these findings are consistent with the previously predicted high genetic barrier to escape mutations in host-targeted antivirals. IMPORTANCE: Although hundreds of millions of people are infected with DENV every year, there is currently no approved vaccine or antiviral therapy. UV-4B has demonstrated antiviral activity against DENV and is expected to enter clinical trials soon. Therefore, it is important to understand the mechanisms of DENV resistance to UV-4B. Host-targeted antivirals are thought to have a higher genetic barrier to escape mutants than directly acting antivirals, yet there are very few published studies of viral evolution under host-targeted antivirals. No study to date has described flavivirus evolution in vivo under selective pressure by a host-based antiviral drug. We present the first in vivo study of the sequential progression of viral evolution under selective pressure by a host-targeted antiviral compound. This study bolsters support for the clinical development of UV-4B as an antiviral drug against DENV, and it provides a framework to compare how treatment with other host-targeted antiflaviviral drugs in humans and different animal models influence viral genetic diversity.

The bioactive lipid 4-hydroxyphenyl retinamide inhibits flavivirus replication.

Dengue virus (DENV), a member of the Flaviviridae family, is a mosquito-borne pathogen and the cause of dengue fever. The increasing prevalence of DENV worldwide heightens the need for an effective vaccine and specific antivirals. Due to the dependence of DENV upon the lipid biosynthetic machinery of the host cell, lipid signaling and metabolism present unique opportunities for inhibiting viral replication. We screened a library of bioactive lipids and modulators of lipid metabolism and identified 4-hydroxyphenyl retinamide (4-HPR) (fenretinide) as an inhibitor of DENV in cell culture. 4-HPR inhibits the steady-state accumulation of viral genomic RNA and reduces viremia when orally administered in a murine model of DENV infection. The molecular target responsible for this antiviral activity is distinct from other known inhibitors of DENV but appears to affect other members of the Flaviviridae, including the West Nile, Modoc, and hepatitis C viruses. Although long-chain ceramides have been implicated in DENV replication, we demonstrate that DENV is insensitive to the perturbation of long-chain ceramides in mammalian cell culture and that the effect of 4-HPR on dihydroceramide homeostasis is separable from its antiviral activity. Likewise, the induction of reactive oxygen species by 4-HPR is not required for the inhibition of DENV. The inhibition of DENV in vivo by 4-HPR, combined with its well-established safety and tolerability in humans, suggests that it may be repurposed as a pan-Flaviviridae antiviral agent. This work also illustrates the utility of bioactive lipid screens for identifying critical interactions of DENV and other viral pathogens with host lipid biosynthesis, metabolism, and signal transduction.

Seasonal pulses of Marburg virus circulation in juvenile Rousettus aegyptiacus bats coincide with periods of increased risk of human infection.

Marburg virus (family Filoviridae) causes sporadic outbreaks of severe hemorrhagic disease in sub-Saharan Africa. Bats have been implicated as likely natural reservoir hosts based most recently on an investigation of cases among miners infected in 2007 at the Kitaka mine, Uganda, which contained a large population of Marburg virus-infected Rousettus aegyptiacus fruit bats. Described here is an ecologic investigation of Python Cave, Uganda, where an American and a Dutch tourist acquired Marburg virus infection in December 2007 and July 2008. More than 40,000 R. aegyptiacus were found in the cave and were the sole bat species present. Between August 2008 and November 2009, 1,622 bats were captured and tested for Marburg virus. Q-RT-PCR analysis of bat liver/spleen tissues indicated ~2.5% of the bats were actively infected, seven of which yielded Marburg virus isolates. Moreover, Q-RT-PCR-positive lung, kidney, colon and reproductive tissues were found, consistent with potential for oral, urine, fecal or sexual transmission. The combined data for R. aegyptiacus tested from Python Cave and Kitaka mine indicate low level horizontal transmission throughout the year. However, Q-RT-PCR data show distinct pulses of virus infection in older juvenile bats (~six months of age) that temporarily coincide with the peak twice-yearly birthing seasons. Retrospective analysis of historical human infections suspected to have been the result of discrete spillover events directly from nature found 83% (54/65) events occurred during these seasonal pulses in virus circulation, perhaps demonstrating periods of increased risk of human infection. The discovery of two tags at Python Cave from bats marked at Kitaka mine, together with the close genetic linkages evident between viruses detected in geographically distant locations, are consistent with R. aegyptiacus bats existing as a large meta-population with associated virus circulation over broad geographic ranges. These findings provide a basis for developing Marburg hemorrhagic fever risk reduction strategies.

Virus nomenclature below the species level: a standardized nomenclature for filovirus strains and variants rescued from cDNA.

Specific alterations (mutations, deletions, insertions) of virus genomes are crucial for the functional characterization of their regulatory elements and their expression products, as well as a prerequisite for the creation of attenuated viruses that could serve as vaccine candidates. Virus genome tailoring can be performed either by using traditionally cloned genomes as starting materials, followed by site-directed mutagenesis, or by de novo synthesis of modified virus genomes or parts thereof. A systematic nomenclature for such recombinant viruses is necessary to set them apart from wild-type and laboratory-adapted viruses, and to improve communication and collaborations among researchers who may want to use recombinant viruses or create novel viruses based on them. A large group of filovirus experts has recently proposed nomenclatures for natural and laboratory animal-adapted filoviruses that aim to simplify the retrieval of sequence data from electronic databases. Here, this work is extended to include nomenclature for filoviruses obtained in the laboratory via reverse genetics systems. The previously developed template for natural filovirus genetic variant naming, (/)///-, is retained, but we propose to adapt the type of information added to each field for cDNA clone-derived filoviruses. For instance, the full-length designation of an Ebola virus Kikwit variant rescued from a plasmid developed at the US Centers for Disease Control and Prevention could be akin to "Ebola virus H.sapiens-rec/COD/1995/Kikwit-abc1" (with the suffix "rec" identifying the recombinant nature of the virus and "abc1" being a placeholder for any meaningful isolate designator). Such a full-length designation should be used in databases and the methods section of publications. Shortened designations (such as "EBOV H.sap/COD/95/Kik-abc1") and abbreviations (such as "EBOV/Kik-abc1") could be used in the remainder of the text, depending on how critical it is to convey information contained in the full-length name. "EBOV" would suffice if only one EBOV strain/variant/isolate is addressed.

Making pain visible: an audit and review of documentation to improve the use of pain assessment by implementing pain as the fifth vital sign.

Pain has been promoted as the fifth vital sign for a decade, but there is little empirical evidence to suggest that doing so has affected the care of individuals suffering pain. This was a three-stage audit of pain assessment in one large teaching hospital in the Northwest of England. Stage one measured the baseline pain assessment activity on surgical and medical wards and identified that the pain assessment tool was not visible to nurses. Stage two redesigned the patient observation charts held at the end of the bed and piloted two versions for clinical utility. Version 2 which had pain assessment alongside the early warning score was adopted and introduced throughout the hospital. Stage three audited pain assessment and management 8 months after the introduction of the new charts. Pain was assessed more regularly at the stage three audit than at the baseline audit. On average, pain was assessed alongside other routine observations 70% of the time across surgical and medical wards. Medical wards appeared to improve their pain assessment using the philosophy of pain being the fifth vital sign better than surgical wards, because they assessed pain alongside routine observations in >90% of cases. Stage three identified that where a high pain score was recorded, analgesia was delivered in the majority of cases (88%). Introducing the philosophy of pain as the fifth vital sign and making pain assessment more visible on the patient observation chart improved the uptake of pain assessment. Pain management strategies were stimulated when high pain scores were identified.

Synthetic human monoclonal antibodies toward staphylococcal enterotoxin B (SEB) protective against toxic shock syndrome.

Staphylococcal enterotoxin B (SEB) is a potent toxin that can cause toxic shock syndrome and act as a lethal and incapacitating agent when used as a bioweapon. There are currently no vaccines or immunotherapeutics available against this toxin. Using phage display technology, human antigen-binding fragments (Fabs) were selected against SEB, and proteins were produced in Escherichia coli cells and characterized for their binding affinity and their toxin neutralizing activity in vitro and in vivo. Highly protective Fabs were converted into full-length IgGs and produced in mammalian cells. Additionally, the production of anti-SEB antibodies was explored in the Nicotiana benthamiana plant expression system. Affinity maturation was performed to produce optimized lead anti-SEB antibody candidates with subnanomolar affinities. IgGs produced in N. benthamiana showed characteristics comparable with those of counterparts produced in mammalian cells. IgGs were tested for their therapeutic efficacy in the mouse toxic shock model using different challenge doses of SEB and a treatment with 200 µg of IgGs 1 h after SEB challenge. The lead candidates displayed full protection from lethal challenge over a wide range of SEB challenge doses. Furthermore, mice that were treated with anti-SEB IgG had significantly lower IFNγ and IL-2 levels in serum compared with mock-treated mice. In summary, these anti-SEB monoclonal antibodies represent excellent therapeutic candidates for further preclinical and clinical development.