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Antigen-presenting cells (APCs) are critical targets of Ebola virus (EBOV) infection in vivo . However, the susceptibility of monocytes to infection is controversial. Studies indicate productive monocyte infection, and yet monocytes are also reported to be resistant to EBOV GP-mediated entry. In contrast, monocyte-derived macrophages and dendritic cells are permissive for both EBOV entry and replication. Here, freshly isolated monocytes are demonstrated to indeed be refractory to EBOV entry. However, EBOV binds monocytes, and delayed entry occurs during monocyte differentiation. Cultured monocytes spontaneously downregulate the expression of viral entry restriction factors such as interferon-inducible transmembrane proteins, while upregulating the expression of critical EBOV entry factors cathepsin B and NPC1. Moreover, these processes are accelerated by EBOV infection. Finally, ectopic expression of NPC1 is sufficient to rescue entry into an undifferentiated, normally nonpermissive monocytic cell line. These results define the molecular basis for infection of APCs and suggest means to limit APC infection.

As of 13 November 2015, 1618 laboratory-confirmed human cases of Middle East respiratory syndrome coronavirus (MERS-CoV) infection, including 579 deaths, had been reported to the World Health Organization. No specific preventive or therapeutic agent of proven value against MERS-CoV is currently available. Public Health England and the International Severe Acute Respiratory and Emerging Infection Consortium identified passive immunotherapy with neutralizing antibodies as a treatment approach that warrants priority study. Two experimental MERS-CoV vaccines were used to vaccinate two groups of transchromosomic (Tc) bovines that were genetically modified to produce large quantities of fully human polyclonal immunoglobulin G (IgG) antibodies. Vaccination with a clade A γ-irradiated whole killed virion vaccine (Jordan strain) or a clade B spike protein nanoparticle vaccine (Al-Hasa strain) resulted in Tc bovine sera with high enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody titers in vitro. Two purified Tc bovine human IgG immunoglobulins (Tc hIgG), SAB-300 (produced after Jordan strain vaccination) and SAB-301 (produced after Al-Hasa strain vaccination), also had high ELISA and neutralizing antibody titers without antibody-dependent enhancement in vitro. SAB-301 was selected for in vivo and preclinical studies. Administration of single doses of SAB-301 12 hours before or 24 and 48 hours after MERS-CoV infection (Erasmus Medical Center 2012 strain) of Ad5-hDPP4 receptor-transduced mice rapidly resulted in viral lung titers near or below the limit of detection. Tc bovines, combined with the ability to quickly produce Tc hIgG and develop in vitro assays and animal model(s), potentially offer a platform to rapidly produce a therapeutic to prevent and/or treat MERS-CoV infection and/or other emerging infectious diseases.

In the fall of 2014, an international news agency reported that patients suffering from Ebola virus disease (EVD) in Liberia were treated successfully with lamivudine, an antiviral drug used to treat human immunodeficiency virus-1 and hepatitis B virus infections. According to the report, 13 out of 15 patients treated with lamivudine survived and were declared free from Ebola virus disease. In this study, the anti-Ebola virus (EBOV) activity of lamivudine and another antiretroviral, zidovudine, were evaluated in a diverse set of cell lines against two variants of wild-type EBOV. Variable assay parameters were assessed to include different multiplicities of infection, lengths of inoculation times, and durations of dosing. At a multiplicity of infection of 1, lamivudine and zidovudine had no effect on EBOV propagation in Vero E6, Hep G2, or HeLa cells, or in primary human monocyte-derived macrophages. At a multiplicity of infection of 0.1, zidovudine demonstrated limited anti-EBOV activity in Huh 7 cells. Under certain conditions, lamivudine had low anti-EBOV activity at the maximum concentration tested (320 μM). However, lamivudine never achieved greater than 30% viral inhibition, and the activity was not consistently reproducible. Combination of lamivudine and zidovudine showed no synergistic antiviral activity. Independently, a set of in vitro experiments testing lamivudine and zidovudine for antiviral activity against an Ebola-enhanced green fluorescent protein reporter virus was performed at the Centers for Disease Control and Prevention. No antiviral activity was observed for either compound. A study evaluating the efficacy of lamivudine in a guinea pig model of EVD found no survival benefit. This lack of benefit was observed despite plasma lamivudine concentrations in guinea pig of about 4 μg/ml obtained in a separately conducted pharmacokinetics study. These studies found no evidence to support the therapeutic use of lamivudine for the treatment of EVD.

Viruses of the family Filoviridae represent significant health risks as emerging infectious diseases as well as potentially engineered biothreats. While many research efforts have been published offering possibilities toward the mitigation of filoviral infection, there remain no sanctioned therapeutic or vaccine strategies. Current progress in the development of filovirus therapeutics and vaccines is outlined herein with respect to their current level of testing, evaluation, and proximity toward human implementation, specifically with regard to human clinical trials, nonhuman primate studies, small animal studies, and in vitro development. Contemporary methods of supportive care and previous treatment approaches for human patients are also discussed.

Vaccine-induced immunity to Ebola virus infection in nonhuman primates (NHPs) is marked by potent antigen-specific cellular and humoral immune responses; however, the immune mechanism of protection remains unknown. Here we define the immune basis of protection conferred by a highly protective recombinant adenovirus virus serotype 5 (rAd5) encoding Ebola virus glycoprotein (GP) in NHPs. Passive transfer of high-titer polyclonal antibodies from vaccinated Ebola virus-immune cynomolgus macaques to naive macaques failed to confer protection against disease, suggesting a limited role of humoral immunity. In contrast, depletion of CD3(+) T cells in vivo after vaccination and immediately before challenge eliminated immunity in two vaccinated macaques, indicating a crucial requirement for T cells in this setting. The protective effect was mediated largely by CD8(+) cells, as depletion of CD8(+) cells in vivo using the cM-T807 monoclonal antibody (mAb), which does not affect CD4(+) T cell or humoral immune responses, abrogated protection in four out of five subjects. These findings indicate that CD8(+) cells have a major role in rAd5-GP-induced immune protection against Ebola virus infection in NHPs. Understanding the immunologic mechanism of Ebola virus protection will facilitate the development of vaccines for Ebola and related hemorrhagic fever viruses in humans.

Infection with many emerging viruses, such as the hemorrhagic fever disease caused by the filoviruses, Marburg (MARV), and Ebola virus (EBOV), leaves the host with a short timeframe in which to mouse a protective immune response. In lethal cases, uncontrolled viral replication and virus-induced immune dysregulation are too severe to overcome, and mortality is generally associated with a lack of notable immune responses. Vaccination studies in animals have demonstrated an association of IgG and neutralizing antibody responses against the protective glycoprotein antigen with survival from lethal challenge. More recently, studies in animal models of filovirus hemorrhagic fever have established that induction of a strong filovirus-specific cytotoxic T lymphocyte (CTL) response can facilitate complete viral clearance. In this review, we describe assays used to discover CTL responses after vaccination or live filovirus infection in both animal models and human clinical trials. Unfortunately, little data regarding CTL responses have been collected from infected human survivors, primarily due to the low frequency of disease and the inability to perform these studies in the field. Advancements in assays and technologies may allow these studies to occur during future outbreaks.

The "World Vaccine Congress 2009" held in Washington D.C. (April 20-23, 2009) sponsored several sessions focused on the vaccine market targeting biodefense. On day one of the congress, a panel discussion outlined the federal progress in medical countermeasure preparedness that included emerging infections, influenza, and biodefense focuses. The second day, a session focused on the biodefense vaccine market with both government and industry members discussing the opportunities and challenges associated with the budding market.

Current methods for inactivating filoviruses are limited to high doses of irradiation or formalin treatment, which may cause structural perturbations that are reflected by poor immunogenicity. In this report, we describe a novel inactivation technique for Zaire Ebola virus (ZEBOV) that uses the photoinduced alkylating probe 1,5-iodonaphthylazide (INA). INA is incorporated into lipid bilayers and, when activated by ultraviolet irradiation, alkylates the proteins therein. INA treatment of ZEBOV resulted in the complete loss of infectivity in cells. Results of electron microscopy and virus-capture assays suggested the preservation of conformational surface epitopes. Challenge with 50,000 pfu of INA-inactivated, mouse-adapted ZEBOV did not cause disease or death in mice. A single vaccination with INA-inactivated ZEBOV (equivalent to 5 x 10(4) pfu) protected mice against lethal challenge with 1000 pfu of ZEBOV. INA-inactivated virus induced a protective response in 100% of mice when administered 3 days before challenge. Thus, INA may have significant potential for the development of vaccines and immunotherapeutics for filoviruses and other enveloped viruses.

With the unprecedented scale of the 2014-2016 West Africa outbreak, the clinical and scientific community scrambled to identify potential therapeutics for Ebola virus disease (EVD). Passive administration of antibodies has a long successful history for prophylaxis and therapy of a variety of infectious diseases, but the importance of antibodies in EVD has been unclear and is the subject of some debate. Recent studies in non-human primates have renewed interest in the potential of antibodies to impact EVD. Currently ongoing clinical evaluation of polyclonal and monoclonal antibody therapy in EVD patients in West Africa may finally offer a definitive answer to this debate.

Brincidofovir (BCV) is the 3-hexadecyloxy-1-propanol (HDP) lipid conjugate of the acyclic nucleoside phosphonate cidofovir (CDV). BCV has established broad-spectrum activity against double-stranded DNA (dsDNA) viruses; however, its activity against RNA viruses has been less thoroughly evaluated. Here, we report that BCV inhibited infection of Ebola virus in multiple human cell lines. Unlike the mechanism of action for BCV against cytomegalovirus and other dsDNA viruses, phosphorylation of CDV to the diphosphate form appeared unnecessary. Instead, antiviral activity required the lipid moiety and in vitro activity against EBOV was observed for several HDP-nucleotide conjugates.

Recently, global public health organizations such as Doctors without Borders (MSF), the World Health Organization (WHO), Public Health Canada, National Institutes of Health (NIH), and the U.S. government developed and deployed Field Decontamination Kits (FDKs), a novel, lightweight, compact, reusable decontamination technology to sterilize Ebola-contaminated medical devices at remote clinical sites lacking infra-structure in crisis-stricken regions of West Africa (medical waste materials are placed in bags and burned). The basis for effectuating sterilization with FDKs is chlorine dioxide (ClO2) produced from a patented invention developed by researchers at the US Army Natick Soldier RD&E Center (NSRDEC) and commercialized as a dry mixed-chemical for bacterial spore decontamination. In fact, the NSRDEC research scientists developed an ensemble of ClO2 technologies designed for different applications in decontaminating fresh produce; food contact and handling surfaces; personal protective equipment; textiles used in clothing, uniforms, tents, and shelters; graywater recycling; airplanes; surgical instruments; and hard surfaces in latrines, laundries, and deployable medical facilities. These examples demonstrate the far-reaching impact, adaptability, and versatility of these innovative technologies. We present herein the unique attributes of NSRDEC’s novel decontamination technologies and a Case Study of the development of FDKs that were deployed in West Africa by international public health organizations to sterilize Ebola-contaminated medical equipment. FDKs use bacterial spores as indicators of sterility. We review the properties and structures of spores and the mechanisms of bacterial spore inactivation by ClO2. We also review mechanisms of bacterial spore inactivation by novel, emerging, and established non-thermal technologies for food preservation, such as high pressure processing, irradiation, cold plasma, and chemical sanitizers, using an array of Bacillus subtilis mutants to probe mechanisms of spore germination and inactivation. We employ techniques of high-resolution atomic force microscopy and phase contrast microscopy to examine the effects of γ-irradiation on bacterial spores of Bacillus anthracis, Bacillus thuringiensis, and Bacillus atrophaeus spp. and of ClO2 on B. subtilis spores, and present in detail assays using spore bio-indicators to ensure sterility when decontaminating with ClO2.

The emergence of Middle East respiratory syndrome coronavirus (MERS-CoV) as a cause of severe respiratory disease highlights the need for effective approaches to CoV vaccine development. Efforts focused solely on the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein may not optimize neutralizing antibody (NAb) responses. Here we show that immunogens based on full-length S DNA and S1 subunit protein elicit robust serum-neutralizing activity against several MERS-CoV strains in mice and non-human primates. Serological analysis and isolation of murine monoclonal antibodies revealed that immunization elicits NAbs to RBD and, non-RBD portions of S1 and S2 subunit. Multiple neutralization mechanisms were demonstrated by solving the atomic structure of a NAb-RBD complex, through sequencing of neutralization escape viruses and by constructing MERS-CoV S variants for serological assays. Immunization of rhesus macaques confers protection against MERS-CoV-induced radiographic pneumonia, as assessed using computerized tomography, supporting this strategy as a promising approach for MERS-CoV vaccine development., Unmet need exists for a vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV). Here the authors report the establishment and evaluation, in mice and primates, of a series of MERS-CoV immunogens and show that they can serve as promising leads for vaccine development.

Filoviruses, consisting of Ebola virus (EBOV) and Marburg virus (MARV), are among the most lethal infectious threats to mankind. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with high mortality rates. Since there is currently no vaccine or antiviral therapy approved for humans, there is an urgent need to develop prophylactic and therapeutic options for use during filoviral outbreaks and bioterrorist attacks. One of the ideal targets against filoviral infection and diseases is at the entry step, which is mediated by the filoviral glycoprotein (GP). In this report, we screened a chemical library of small molecules and identified numerous inhibitors, which are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs, including histamine receptors, 5-HT (serotonin) receptors, muscarinic acetylcholine receptor, and adrenergic receptor. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. The time-of-addition experiment and microscopic studies suggest that GPCR antagonists block filoviral entry at a step following the initial attachment but prior to viral/cell membrane fusion. These results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy. IMPORTANCE Infection of Ebola virus and Marburg virus can cause severe illness in humans with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The 2013-2015 epidemic in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we have identified numerous inhibitors that are known G protein-coupled receptor (GPCR) antagonists targeting different GPCRs. These inhibitors can effectively block replication of both infectious EBOV and MARV, indicating a broad antiviral activity of the GPCR antagonists. Our results strongly suggest that GPCRs play a critical role in filoviral entry and GPCR antagonists can be developed as an effective anti-EBOV/MARV therapy.

Direct delivery of aerosolized vaccines to the respiratory mucosa elicits both systemic and mucosal responses. This vaccine strategy has not been tested for Ebola virus (EBOV) or other hemorrhagic fever viruses. Here, we examined the immunogenicity and protective efficacy of an aerosolized human parainfluenza virus type 3-vectored vaccine that expresses the glycoprotein (GP) of EBOV (HPIV3/EboGP) delivered to the respiratory tract. Rhesus macaques were vaccinated with aerosolized HPIV3/EboGP, liquid HPIV3/EboGP, or an unrelated, intramuscular, Venezuelan equine encephalitis replicon vaccine expressing EBOV GP. Serum and mucosal samples from aerosolized HPIV3/EboGP recipients exhibited high EBOV-specific IgG, IgA, and neutralizing antibody titers, which exceeded or equaled titers observed in liquid recipients. The HPIV3/EboGP vaccine induced an EBOV-specific cellular response that was greatest in the lungs and yielded polyfunctional CD8+ T cells, including a subset that expressed CD103 (αE integrin), and CD4+ T helper cells that were predominately type 1. The magnitude of the CD4+ T cell response was greater in aerosol vaccinees. The HPIV3/EboGP vaccine produced a more robust cell-mediated and humoral immune response than the systemic replicon vaccine. Moreover, 1 aerosol HPIV3/EboGP dose conferred 100% protection to macaques exposed to EBOV. Aerosol vaccination represents a useful and feasible vaccination mode that can be implemented with ease in a filovirus disease outbreak situation.

Currently, no approved therapeutics exist to treat or prevent infections induced by Ebola viruses, and recent events have demonstrated an urgent need for rapid discovery of new treatments. Repurposing approved drugs for emerging infections remains a critical resource for potential antiviral therapies. We tested ~2600 approved drugs and molecular probes in an in vitro infection assay using the type species, Zaire ebolavirus. Selective antiviral activity was found for 80 U.S. Food and Drug Administration–approved drugs spanning multiple mechanistic classes, including selective estrogen receptor modulators, antihistamines, calcium channel blockers, and antidepressants. Results using an in vivo murine Ebola virus infection model confirmed the protective ability of several drugs, such as bepridil and sertraline. Viral entry assays indicated that most of these antiviral drugs block a late stage of viral entry. By nature of their approved status, these drugs have the potential to be rapidly advanced to clinical settings and used as therapeutic countermeasures for Ebola virus infections.

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., Author Summary The filoviruses have caused multiple outbreaks among humans this decade, including a 90% lethal outbreak of Marburg virus in Angola and a significant, sustained outbreak of Ebola virus in West Africa. The viral surface glycoprotein (GP), which enables filoviruses to infect host cells, is the primary target of the immune system. Antibodies that target filovirus GP have been shown to provide life-saving therapy in nonhuman primates. However, the majority of known antibodies are only reactive against Ebola virus and not other emerging filoviruses. In this study, we present ten antibodies against Marburg virus, elicited by immunization of mice using engineered forms of its GP. Surprisingly, two antibodies exhibit some cross-reactivity to ebolaviruses (including species Ebola, Sudan, Bundibugyo, Reston). Other antibodies in this panel recognize a novel “wing” feature on a portion of GP that is unique to Marburg and does not exist in ebolaviruses, and protect 90%-100% of mice from lethal exposure. These antibodies, and their structural and functional analysis presented here, illuminate directions forward for therapeutics against Marburg virus.

The Filoviruses Marburg virus and Ebola virus are among the deadliest of human pathogens, causing fulminant hemorrhagic fevers typified by overmatched specific immune responses and profuse inflammatory responses. Keys to both vaccination and treatment may reside, first, in the understanding of immune dysfunctions that parallel Filoviral disease and, second, in devising ways to redirect and restore normal immune function as well as to mitigate inflammation. Here, we describe how Filoviral infections may subvert innate immune responses through perturbances of dendritic cells and neutrophils, with particular emphasis on the downstream effects on adaptive immunity and inflammation. We suggest that pivotal events may be subject to therapeutic intervention as Filoviruses encounter immune processes.

Phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) is a lipid kinase involved in endosome maturation that emerged from a haploid genetic screen as being required for Ebola virus (EBOV) infection. Here we analyzed the effects of apilimod, a PIKfyve inhibitor that was reported to be well tolerated in humans in phase 2 clinical trials, for its effects on entry and infection of EBOV and Marburg virus (MARV). We first found that apilimod blocks infections by EBOV and MARV in Huh 7, Vero E6 and primary human macrophage cells, with notable potency in the macrophages (IC50, 10 nM). We next observed that similar doses of apilimod block EBOV-glycoprotein-virus like particle (VLP) entry and transcription-replication competent VLP infection, suggesting that the primary mode of action of apilimod is as an entry inhibitor, preventing release of the viral genome into the cytoplasm to initiate replication. After providing evidence that the anti-EBOV action of apilimod is via PIKfyve, we showed that it blocks trafficking of EBOV VLPs to endolysosomes containing Niemann-Pick C1 (NPC1), the intracellular receptor for EBOV. Concurrently apilimod caused VLPs to accumulate in early endosome antigen 1-positive endosomes. We did not detect any effects of apilimod on bulk endosome acidification, on the activity of cathepsins B and L, or on cholesterol export from endolysosomes. Hence by antagonizing PIKfyve, apilimod appears to block EBOV trafficking to its site of fusion and entry into the cytoplasm. Given the drug’s observed anti-filoviral activity, relatively unexplored mechanism of entry inhibition, and reported tolerability in humans, we propose that apilimod be further explored as part of a therapeutic regimen to treat filoviral infections., Author summary The recent outbreak of Ebola virus (EBOV) disease in Western Africa highlights the urgent need to develop therapeutics to help quell this devastating hemorrhagic fever virus, especially in resource-limited areas around the globe. Here we show that apilimod, an investigational drug that was well-tolerated in phase 2 clinical trials for rheumatoid arthritis, Crohn’s disease, and psoriasis, is a strong inhibitor of both EBOV and Marburgvirus infections in multiple cell types. Further work shows that apilimod blocks the entry of EBOV particles into the host cell cytoplasm and that it does so by blocking the particles from reaching their normal portal of entry, in Niemann-Pick C1-positive endolysosomes. Our findings are consistent with the identity of phosphatidylinositol-3-phosphate 5-kinase as the molecular target of apilimod, as the kinase and its product phosphatidylinositol 3,5-bisphosphate are required for the proper maturation of late endocytic organelles. Hence we propose that apilimod be further explored for repositioning as part of a therapeutic regimen to help ameliorate the sequelae of filoviral infections.

Ebola virus (EBOV) and related filoviruses cause severe hemorrhagic fever, with up to 90% lethality, and no treatments are approved for human use. Multiple recent outbreaks of EBOV and the likelihood of future human exposure highlight the need for pre- and postexposure treatments. Monoclonal antibody (mAb) cocktails are particularly attractive candidates due to their proven postexposure efficacy in nonhuman primate models of EBOV infection. Two candidate cocktails, MB-003 and ZMAb, have been extensively evaluated in both in vitro and in vivo studies. Recently, these two therapeutics have been combined into a new cocktail named ZMapp, which showed increased efficacy and has been given compassionately to some human patients. Epitope information and mechanism of action are currently unknown for most of the component mAbs. Here we provide single-particle EM reconstructions of every mAb in the ZMapp cocktail, as well as additional antibodies from MB-003 and ZMAb. Our results illuminate key and recurring sites of vulnerability on the EBOV glycoprotein and provide a structural rationale for the efficacy of ZMapp. Interestingly, two of its components recognize overlapping epitopes and compete with each other for binding. Going forward, this work now provides a basis for strategic selection of next-generation antibody cocktails against Ebola and related viruses and a model for predicting the impact of ZMapp on potential escape mutations in ongoing or future Ebola outbreaks.

The cyanobacterial lectin scytovirin (SVN) binds with high affinity to mannose-rich oligosaccharides on the envelope glycoprotein (GP) of a number of viruses, blocking entry into target cells. In this study, we assessed the ability of SVN to bind to the envelope GP of Zaire Ebola virus (ZEBOV) and inhibit its replication. SVN interacted specifically with the protein’s mucin-rich domain. In cell culture, it inhibited ZEBOV replication with a 50% virus-inhibitory concentration (EC50) of 50 nM, and was also active against the Angola strain of the related Marburg virus (MARV), with a similar EC50. Injected subcutaneously in mice, SVN reached a peak plasma level of 100 nm in 45 minutes, but was cleared within 4 hours. When ZEBOV-infected mice were given 30 mg/kg/day of SVN by subcutaneous injection every 6 hours, beginning the day before virus challenge, 9 of 10 animals survived the infection, while all infected, untreated mice died. When treatment was begun one hour or one day after challenge, 70-90% of mice survived. Quantitation of infectious virus and viral RNA in samples of serum, liver and spleen collected on days 2 and 5 postinfection showed a trend toward lower titers in treated than control mice, with a significant decrease in liver titers on day 2. Our findings provide further evidence of the potential of natural lectins as therapeutic agents for viral infections.

Objective: The aim of this study was to determine the relationship of bacterial vaginosis and bacterial vaginosis-associated microorganisms with an HIV-inducing factor (HIF) found in cervicovaginal lavage. Design: A total of 26 cervicovaginal lavage specimens collected from 17 women were used in this study to determine if HIF was significantly associated with features consistent with bacterial vaginosis. Methods: Patients were evaluated for various clinical features including age, HIV status and stage, CD4 cell counts, clinical diagnosis of gynecological infections, vaginal pH, Gram stains of vaginal fluid, phase of menstruation, and presence of cervical dysplasia. Cervicovaginal lavage specimens were analyzed for the presence of HIF by U1 bioassay. The presence of Gardnerella vaginalis, and general Mycoplasmataceae, and specifically Mycoplasma hominis, Ureaplasma urealyticum, M. fermentans, M. genitalium in cervicovaginal lavage were determined by semiquantitative PCR. Results: Eleven cervicovaginal lavage samples from seven women were HIF-positive and 15 cervicovaginal lavage samples from 11 women were HIF-negative (patient No. 8 had two HIF-negative cervicovaginal lavage and one HIF-positive cervicovaginal lavage). The following parameters were significantly associated with HIF: abnormal vaginal fluid pH (> 4.5) (P = 0.006), Gram stains indicative of bacterial vaginosis (P = 0.007), normal menstrual cycle (P = 0.0007) and PCR detection and relative quantity of M. hominis (P = 0.0003, P = 0.002). Conclusions: This study indicates that HIF is closely associated with features of bacterial vaginosis.

The Middle East respiratory syndrome coronavirus (MERS-CoV) presents a novel emerging threat to public health worldwide. Several treatments for infected individuals have been suggested including IFN, ribavirin and passive immunotherapy with convalescent plasma. Administration of IFN-α2b and ribavirin has improved outcomes of MERS-CoV infection in rhesus macaques when administered within 8 h post-challenge. However, detailed and systematic evidence on the activity of other clinically available drugs is limited. Here we compared the susceptibility of MERS-CoV with different IFN products (IFN-α2b, IFN-γ, IFN-universal, IFN-α2a and IFN-β), as well as with two antivirals, ribavirin and mycophenolic acid (MPA), against MERS-CoV (Hu/Jordan-N3/2012) in vitro. Of all the IFNs tested, IFN-β showed the strongst inhibition of MERS-CoV in vitro, with an IC50 of 1.37 U ml−1, 41 times lower than the previously reported IC50 (56.08 U ml−1) of IFN-α2b. IFN-β inhibition was confirmed in the virus yield reduction assay, with an IC90 of 38.8 U ml−1. Ribavirin did not inhibit viral replication in vitro at a dose that would be applicable to current treatment protocols in humans. In contrast, MPA showed strong inhibition, with an IC50 of 2.87 µM. This drug has not been previously tested against MERS-CoV and may provide an alternative to ribavirin for treatment of MERS-CoV. In conclusion, IFN-β, MPA or a combination of the two may be beneficial in the treatment of MERS-CoV or as a post-exposure intervention in high-risk patients with known exposures to MERS-CoV.

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, < virus name > (< strain >/)< isolation host-suffix >/< country of sampling >/< year of sampling >/< genetic variant designation >-< isolate designation >, 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.

Ebola viruses remain a substantial threat to both civilian and military populations as bioweapons, during sporadic outbreaks, and from the possibility of accidental importation from endemic regions by infected individuals. Currently, no approved therapeutics exist to treat or prevent infection by Ebola viruses. Therefore, we performed an in vitro screen of Food and Drug Administration (FDA)– and ex–US-approved drugs and selected molecular probes to identify drugs with antiviral activity against the type species Zaire ebolavirus (EBOV). From this screen, we identified a set of selective estrogen receptor modulators (SERMs), including clomiphene and toremifene, which act as potent inhibitors of EBOV infection. Anti-EBOV activity was confirmed for both of these SERMs in an in vivo mouse infection model. This anti-EBOV activity occurred even in the absence of detectable estrogen receptor expression, and both SERMs inhibited virus entry after internalization, suggesting that clomiphene and toremifene are not working through classical pathways associated with the estrogen receptor. Instead, the response appeared to be an off-target effect where the compounds interfere with a step late in viral entry and likely affect the triggering of fusion. These data support the screening of readily available approved drugs to identify therapeutics for the Ebola viruses and other infectious diseases. The SERM compounds described in this report are an immediately actionable class of approved drugs that can be repurposed for treatment of filovirus infections.

The International Committee on Taxonomy of Viruses (ICTV) organizes the classification of viruses into taxa, but is not responsible for the nomenclature for taxa members. International experts groups, such as the ICTV Study Groups, recommend the classification and naming of viruses and their strains, variants, and isolates. The ICTV Filoviridae Study Group has recently introduced an updated classification and nomenclature for filoviruses. Subsequently, and together with numerous other filovirus experts, a consistent nomenclature for their natural genetic variants and isolates was developed that aims at simplifying the retrieval of sequence data from electronic databases. This is a first important step toward a viral genome annotation standard as sought by the US National Center for Biotechnology Information (NCBI). Here, this work is extended to include filoviruses obtained in the laboratory by artificial selection through passage in laboratory hosts. The previously developed template for natural filovirus genetic variant naming (< virus name > < isolation host-suffix >/< country of sampling >/< year of sampling >/< genetic variant designation >-< isolate designation >) is retained, but it is proposed to adapt the type of information added to each field for laboratory animal-adapted variants. For instance, the full-length designation of an Ebola virus Mayinga variant adapted at the State Research Center for Virology and Biotechnology "Vector" to cause disease in guinea pigs after seven passages would be akin to "Ebola virus VECTOR/C.porcellus-lab/COD/1976/Mayinga-GPA-P7". As was proposed for the names of natural filovirus variants, we suggest using the full-length designation in databases, as well as in the method section of publications. Shortened designations (such as "EBOV VECTOR/C.por/COD/76/May-GPA-P7") and abbreviations (such as "EBOV/May-GPA-P7") 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.

Rift Valley fever virus (RVFV) is an important mosquito-borne veterinary and human pathogen that can cause severe disease including acute-onset hepatitis, delayed-onset encephalitis, retinitis and blindness, or a hemorrhagic syndrome. Currently, no licensed vaccine or therapeutics exist to treat this potentially deadly disease. Detailed studies describing the pathogenesis of RVFV following aerosol exposure have not been completed and candidate therapeutics have not been evaluated following an aerosol exposure. These studies are important because while mosquito transmission is the primary means for human infection, it can also be transmitted by aerosol or through mucosal contact. Therefore, we directly compared the pathogenesis of RVFV following aerosol exposure to a subcutaneous (SC) exposure in the murine model by analyzing survival, clinical observations, blood chemistry, hematology, immunohistochemistry, and virus titration of tissues. Additionally, we evaluated the effectiveness of the nucleoside analog ribavirin administered prophylactically to treat mice exposed by aerosol and SC. The route of exposure did not significantly affect the survival, chemistry or hematology results of the mice. Acute hepatitis occurred despite the route of exposure. However, the development of neuropathology occurred much earlier and was more severe in mice exposed by aerosol compared to SC exposed mice. Mice treated with ribavirin and exposed SC were partially protected, whereas treated mice exposed by aerosol were not protected. Early and aggressive viral invasion of brain tissues following aerosol exposure likely played an important role in ribavirin's failure to prevent mortality among these animals. Our results highlight the need for more candidate antivirals to treat RVFV infection, especially in the case of a potential aerosol exposure. Additionally, our study provides an account of the key pathogenetic differences in RVF disease following two potential exposure routes and provides important insights into the development and evaluation of potential vaccines and therapeutics to treat RVFV infection., Author Summary Rift Valley fever (RVF) is an important neglected tropical disease that has caused severe epidemics and epizootics throughout Africa and the Arabian Peninsula. Severe outbreaks have involved tens of thousands of both human and livestock cases for which no effective, commercially available human vaccines or antiviral drugs are available. Mosquito transmission is the primary means for human infection although it can also be transmitted by aerosol or through mucosal contact. In this study, we directly compared the pathogenesis of RVF virus (RVFV) following aerosol exposure to a peripheral exposure in the mouse model. Additionally, we evaluated the effectiveness of ribavirin, a known antiviral compound, administered prior to virus exposure. The virus affected the liver despite the route of exposure. However, viral invasion of brain tissues occurred much earlier and was more severe in mice exposed by aerosol compared to peripherally exposed mice. Mice exposed peripherally and treated with ribavirin were partially protected whereas treated mice exposed by aerosol were not protected. Our results have important implications for understanding the pathogenesis of RVFV and for the development and evaluation of potential vaccines and therapeutics.

The task of international expert groups is to recommend the classification and naming of viruses. The International Committee on Taxonomy of Viruses Filoviridae Study Group and other experts have recently established an almost consistent classification and nomenclature for filoviruses. Here, further guidelines are suggested to include their natural genetic variants. First, this term is defined. Second, a template for full-length virus names (such as “Ebola virus H.sapiens-tc/COD/1995/Kikwit-9510621”) is proposed. These names contain information on the identity of the virus (e.g., Ebola virus), isolation host (e.g., members of the species Homo sapiens), sampling location (e.g., Democratic Republic of the Congo (COD)), sampling year, genetic variant (e.g., Kikwit), and isolate (e.g., 9510621). Suffixes are proposed for individual names that clarify whether a given genetic variant has been characterized based on passage zero material (-wt), has been passaged in tissue/cell culture (-tc), is known from consensus sequence fragments only (-frag), or does (most likely) not exist anymore (-hist). We suggest that these comprehensive names are to be used specifically in the methods section of publications. Suitable abbreviations, also proposed here, could then be used throughout the text, while the full names could be used again in phylograms, tables, or figures if the contained information aids the interpretation of presented data. The proposed system is very similar to the well-known influenzavirus nomenclature and the nomenclature recently proposed for rotaviruses. If applied consistently, it would considerably simplify retrieval of sequence data from electronic databases and be a first important step toward a viral genome annotation standard as sought by the National Center for Biotechnology Information (NCBI). Furthermore, adoption of this nomenclature would increase the general understanding of filovirus-related publications and presentations and improve figures such as phylograms, alignments, and diagrams. Most importantly, it would counter the increasing confusion in genetic variant naming due to the identification of ever more sequences through technological breakthroughs in high-throughput sequencing and environmental sampling.

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes.

There is limited knowledge of the pathogenesis of human ebolavirus infections and no reported human cases acquired by the aerosol route. There is a threat of ebolavirus as an aerosolized biological weapon, and this study evaluated the pathogenesis of aerosol infection in 18 rhesus macaques. Important and unique findings include early infection of the respiratory lymphoid tissues, early fibrin deposition in the splenic white pulp, and perivasculitis and vasculitis in superficial dermal blood vessels of haired skin with rash. Initial infection occurred in the respiratory lymphoid tissues, fibroblastic reticular cells, dendritic cells, alveolar macrophages, and blood monocytes. Virus spread to regional lymph nodes, where significant viral replication occurred. Virus secondarily infected many additional blood monocytes and spread from the respiratory tissues to multiple organs, including the liver and spleen. Viremia, increased temperature, lymphocytopenia, neutrophilia, thrombocytopenia, and increased alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transpeptidase, total bilirubin, serum urea nitrogen, creatinine, and hypoalbuminemia were measurable mid to late infection. Infection progressed rapidly with whole-body destruction of lymphoid tissues, hepatic necrosis, vasculitis, hemorrhage, and extravascular fibrin accumulation. Hypothermia and thrombocytopenia were noted in late stages with the development of disseminated intravascular coagulation and shock. This study provides unprecedented insight into pathogenesis of human aerosol Zaire ebolavirus infection and suggests development of a medical countermeasure to aerosol infection will be a great challenge due to massive early infection of respiratory lymphoid tissues. Rhesus macaques may be used as a model of aerosol infection that will allow the development of lifesaving medical countermeasures under the Food and Drug Administration’s animal rule.

There is a clear need for novel, effective therapeutic approaches to hemorrhagic fever due to filoviruses. Ebola virus hemorrhagic fever is associated with robust interferon (IFN)–α production, with plasma concentrations of IFN-α that greatly (60- to 100-fold) exceed those seen in other viral infections, but little IFN-β production. While all of the type I IFNs signal through the same receptor complex, both quantitative and qualitative differences in biological activity are observed after stimulation of the receptor complex with different type I IFNs. Taken together, this suggested potential for IFN-β therapy in filovirus infection. Here we show that early postexposure treatment with IFN-β significantly increased survival time of rhesus macaques infected with a lethal dose of Ebola virus, although it failed to alter mortality. Early treatment with IFN-β also significantly increased survival time after Marburg virus infection. IFN-β may have promise as an adjunctive postexposure therapy in filovirus infection.

Two common methods of quantifying filovirus infectivity, a plaque assay and 50% cell culture infectious dose (TCID50) endpoint dilution assay, were compared. The two assays were performed side by side using the same virus stock sample to determine the correlation between the results of the two assays. The TCID50 assay appeared to be more sensitive but slightly more variable, and there was a tenfold difference in the numerical results of these methods of enumeration. The advantages and disadvantages of both assays are discussed. Both methods are useful and practicable in filovirus research, and this comparison will be hugely beneficial to the filovirus research community as it seeks to become more united. Further work in this area should be performed to ensure consistency in filovirus research.

The filovirus plaque assay serves as the assay of choice to measure infectious virus in a cell culture, blood, or homogenized tissue sample. It has been in use for more than 30 years and is the generally accepted assay used to titrate virus in samples from animals treated with a potential antiviral therapeutic or vaccine. As these animal studies are required for the development of vaccines and therapeutics under the FDA Animal Rule, it is essential to have a standardized assay to compare their efficacies against the various filoviruses. Here, we present an evaluation of the conditions under which the filovirus plaque assay performs best for the Ebola virus Kikwit variant and the Angola variant of Marburg virus. The indicator cell type and source, inoculum volumes, length of incubation and general features of filovirus biology as visualized in the assay are addressed in terms of the impact on the sample viral titer calculations. These optimization studies have resulted in a plaque assay protocol which can be used for preclinical studies, and as a standardized protocol for use across institutions, to aid in data comparison. This protocol will be validated for use in GLP studies supporting advanced development of filovirus therapeutics and vaccines.

No countermeasures currently exist for the prevention or treatment of the severe sequelae of Filovirus (such as Ebola virus; EBOV) infection. To overcome this limitation in our biodefense preparedness, we have designed monoclonal antibodies (mAbs) which could be used in humans as immunoprotectants for EBOV, starting with a murine mAb (13F6) that recognizes the heavily glycosylated mucin-like domain of the virion-attached glycoprotein (GP). Point mutations were introduced into the variable region of the murine mAb to remove predicted human T-cell epitopes, and the variable regions joined to human constant regions to generate a mAb (h-13F6) appropriate for development for human use. We have evaluated the efficacy of three variants of h-13F6 carrying different glycosylation patterns in a lethal mouse EBOV challenge model. The pattern of glycosylation of the various mAbs was found to correlate to level of protection, with aglycosylated h-13F6 providing the least potent efficacy (ED 50 = 33 μg). A version with typical heterogenous mammalian glycoforms (ED 50 = 11 μg) had similar potency to the original murine mAb. However, h-13F6 carrying complex N -glycosylation lacking core fucose exhibited superior potency (ED 50 = 3 μg). Binding studies using Fcγ receptors revealed enhanced binding of nonfucosylated h-13F6 to mouse and human FcγRIII. Together the results indicate the presence of Fc N -glycans enhances the protective efficacy of h-13F6, and that mAbs manufactured with uniform glycosylation and a higher potency glycoform offer promise as biodefense therapeutics.

Nine rhesus macaques were implanted with multisensor telemetry devices and internal jugular vein catheters before being infected with Zaire ebolavirus. All animals developed viremia, fever, a hemorrhagic rash, and typical changes of Ebola hemorrhagic fever in clinical laboratory tests. Three macaques unexpectedly survived this usually lethal disease, making it possible to compare physiological parameters in lethally challenged animals and survivors. After the onset of fever, lethal illness was characterized by a decline in mean arterial blood pressure, an increase in pulse and respiratory rate, lactic acidosis, and renal failure. Survivors showed less pronounced change in these parameters. Four macaques were randomized to receive supplemental volumes of intravenous normal saline when they became hypotensive. Although those animals had less severe renal compromise, no apparent survival benefit was observed. This is the first report of continuous physiologic monitoring in filovirus-infected nonhuman primates and the first to attempt cardiovascular support with intravenous fluids.

Metal ion complexes have the potential to form novel types of antiviral compounds, due to their ability to form octahedral and square-planar molecular geometries and their intrinsic charge density. Previously it has been shown that hexamminecobalt(III) chloride (Cohex) has antiviral properties against Sindbis virus (SINV) and adenovirus. Here, we report that Cohex also exhibits activities against two isolates of human immunodeficiency virus (HIV) and the Zaire Ebola (ZEBOV) strain expressing green fluorescent protein (GFP). The therapeutic indices for antiviral activity against the HIV isolates were similar to that found for SINV. Cohex was also effective in decreasing the host translation of viral GFP in four different cell-lines infected with ZEBOV. Toxicity studies in mice found no deleterious effects at up to 8 mg/kg. The 8 mg/kg concentration also prolonged survival of mice infected with ZEBOV. These results point to the potential of Cohex as a new type of broad-spectrum antibiotic compounds.

Currently, there are no Food and Drug Administration (FDA)-approved antiviral drugs or therapeutics for many of the biosafety level three (BSL-3) and four (BSL-4) pathogens. Many of these high-consequence pathogens, including Venezuelan equine encephalitis virus (VEEV), Ebola virus (EBOV), Marburg virus (MARV), and Lassa virus (LASV), are classified as biothreat agents and the development of therapeutic treatments for these diseases is an important area of research. In recent years, high-throughput screening (HTS) assays have become an effective and robust tool used for drug and therapeutic discovery. There are several types of HTS methods available, including targeted screening, diversity and high-content screening, and RNA interference (RNAi). These screens have been used effectively with a number of BSL-2 pathogens, but present unique challenges for the BSL-3/4 pathogens due to the requirement for higher level biocontainment facilities as well as biosurety requirements. Addressing and overcoming these challenges is essential for the proper adaptation of HTS into higher biocontainment facilities. In this article, we will discuss the advantages and disadvantages of each of the aforementioned HTS methods in the context of BSL-3/4 containment.

Ebola viruses constitute a newly emerging public threat because they cause rapidly fatal hemorrhagic fevers for which no treatment exists, and they can be manipulated as bioweapons. We targeted conserved N-glycosylated carbohydrate ligands on viral envelope surfaces using novel immune therapies. Mannose-binding lectin (MBL) and L-ficolin (L-FCN) were selected because they function as opsonins and activate complement. Given that MBL has a complex quaternary structure unsuitable for large scale cost-effective production, we sought to develop a less complex chimeric fusion protein with similar ligand recognition and enhanced effector functions. We tested recombinant human MBL and three L-FCN/MBL variants that contained the MBL carbohydrate recognition domain and varying lengths of the L-FCN collagenous domain. Non-reduced chimeric proteins formed predominantly nona- and dodecameric oligomers, whereas recombinant human MBL formed octadecameric and larger oligomers. Surface plasmon resonance revealed that L-FCN/MBL76 had the highest binding affinities for N-acetylglucosamine-bovine serum albumin and mannan. The same chimeric protein displayed superior complement C4 cleavage and binding to calreticulin (cC1qR), a putative receptor for MBL. L-FCN/MBL76 reduced infection by wild type Ebola virus Zaire significantly greater than the other molecules. Tapping mode atomic force microscopy revealed that L-FCN/MBL76 was significantly less tall than the other molecules despite similar polypeptide lengths. We propose that alterations in the quaternary structure of L-FCN/MBL76 resulted in greater flexibility in the collagenous or neck region. Similarly, a more pliable molecule might enhance cooperativity between the carbohydrate recognition domains and their cognate ligands, complement activation, and calreticulin binding dynamics. L-FCN/MBL chimeric proteins should be considered as potential novel therapeutics.

Ebola virus (EBOV), a negative-sense RNA virus in the family Filoviridae, is known to cause severe hemorrhagic fever in humans and other primates. Infection with EBOV causes a high mortality rate and currently there is no FDA-licensed vaccine or therapeutic treatment available. Recently, heat-shock protein 90 (Hsp90), a molecular chaperone, was shown to be an important host factor for the replication of several negative-strand viruses. We tested the effect of several different Hsp90 inhibitors including geldanamycin, radicicol, and 17-allylamino-17-demethoxygeldanamycin (17-AAG; a geldanamycin analog) on the replication of Zaire EBOV. Our results showed that inhibition of Hsp90 significantly reduced the replication of EBOV. Classic Hsp90 inhibitors reduced viral replication with an effective concentration at 50% (EC(50)) in the high nanomolar to low micromolar range, while drugs from a new class of Hsp90 inhibitors showed markedly more potent inhibition. These compounds blocked EBOV replication with an EC(50) in the low nanomolar range and showed significant potency in blocking replication in primary human monocytes. These results validated that Hsp90 is an important host factor for the replication of filoviruses and suggest that Hsp90 inhibitors may be therapeutically effective in treating EBOV infection.

Cathepsins B and L contribute to Ebola virus (EBOV) entry into Vero cells and mouse embryonic fibroblasts. However, the role of cathepsins in EBOV-infection of human dendritic cells (DCs), important targets of infection in vivo, remains undefined. Here, EBOV-like particles containing a beta-lactamase-VP40 fusion reporter and Ebola virus were used to demonstrate the cathepsin dependence of EBOV entry into human monocyte-derived DCs. However, while DC infection is blocked by cathepsin B inhibitor, it is insensitive to cathepsin L inhibitor. Furthermore, DCs pre-treated for 48 h with TNFalpha were generally less susceptible to entry and infection by EBOV. This decrease in infection was associated with a decrease in cathepsin B activity. Thus, cathepsin L plays a minimal, if any, role in EBOV infection in human DCs. The inflammatory cytokine TNFalpha modulates cathepsin B activity and affects EBOV entry into and infection of human DCs.

BackgroundMarburg virus (MARV) causes acute hemorrhagic fever that is often lethal, and no licensed vaccines are available for preventing this deadly viral infection. The immune mechanisms for protection against MARV are poorly understood, but previous studies suggest that both antibodies and T cells are required. In our study, we infected BALB/c mice with plaque-purified, nonlethal MARV and used overlapping peptides to map H2d-restricted CD8+ T-cell epitopes.MethodsSplenocytes from mice infected with nonlethal MARV were harvested and stimulated with multiple overlapping 15-mer peptide pools, and reactive CD8+ T cells were evaluated for antigen specificity by measuring upregulation of CD44 and interferon-γ expression. After confirming positive reactivity to specific 15-mer peptides, we used extrapolated 9-mer epitopes to evaluate the induction of cytotoxic T-cell responses and protection from lethal MARV challenge in BALB/c mice.ResultsWe discovered a CD8+ T-cell epitope within both the MARV glycoprotein (GP) and nucleoprotein (NP) that triggered cytotoxic T-cell responses. These responses were also protective when epitope-specific splenocytes were transferred into naïve animals.ConclusionEpitope mapping of MARV GP, NP, and VP40 provides the first evidence that specific MARV-epitope induction of cellular immune responses is sufficient to combat infection. Establishment of CD8+ T-cell epitopes that are reactive to MARV proteins provides an important research tool for dissecting the significance of cellular immune responses in BALB/c mice infected with MARV.

Background. Virus-like particles (VLPs) of Ebola virus (EBOV) and Marburg virus (MARV) produced in human 293T embryonic kidney cells have been shown to be effective vaccines against filoviral infection. In this study, we explored alternative strategies for production of filovirus-like particle-based vaccines, to accelerate the development process. The goal of this work was to increase the yield of VLPs, while retaining their immunogenic properties. Methods. Ebola and Marburg VLPs (eVLPs and mVLPs, respectively) were generated by use of recombinant baculovirus constructs expressing glycoprotein, VP40 matrix protein, and nucleoprotein from coinfected insect cells. The baculovirus-derived eVLPs and mVLPs were characterized biochemically, and then the immune responses produced by the eVLPs in insect cells were studied further. Results. The baculovirus-derived eVLPs elicited maturation of human myeloid dendritic cells (DCs), indicating their immunogenic properties. Mice vaccinated with insect cell-derived eVLPs generated antibody and cellular responses equivalent to those vaccinated with mammalian 293T cell-derived eVLPs and were protected from EBOV challenge in a dose-dependent manner. Conclusion. Together, these data suggest that filovirus-like particles produced by baculovirus expression systems, which are amenable to large-scale production, are highly immunogenic and are suitable as safe and effective vaccines for the prevention of filoviral infection.

Background. Currently, there are no licensed vaccines or therapeutics for the prevention or treatment of infection by the highly lethal filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), in humans. We previously had demonstrated the protective efficacy of virus-like particle (VLP)-based vaccines against EBOV and MARV infection in rodents. Methods. To determine the efficacy of vaccination with Ebola VLPs (eVLPs) in nonhuman primates, we vaccinated cynomolgus macaques with eVLPs containing EBOV glycoprotein (GP), nucleoprotein (NP), and VP40 matrix protein and challenged the macaques with 1000 pfu of EBOV. Results. Serum samples from the eVLP-vaccinated nonhuman primates demonstrated EBOV-specific antibody titers, as measured by enzyme-linked immunosorbent assay, complement-mediated lysis assay, and antibody-dependent cell-mediated cytotoxicity assay. CD44 + T cells from eVLP-vaccinated macaques but not from a naive macaque responded with vigorous production of tumor necrosis factor-a after EBOV-peptide stimulation. All 5 eVLP-vaccinated monkeys survived challenge without clinical or laboratory signs of EBOV infection, whereas the control animal died of infection. Conclusion. On the basis of safety and efficacy, eVLPs represent a promising filovirus vaccine for use in humans.

There is an error in the 18th author's name; the middle initial is missing. The correct name is Jody D. Berry. This author's affiliation is also incorrect. The correct affiliation is: Cangene Corp, now a subsidiary of Emergent Biosolutions. Winnipeg, Manitoba, Canada. This author's current address is: Proteos, Inc., Kalamazoo, MI

Mannose-binding lectin (MBL), a serum lectin that mediates innate immune functions including activation of the lectin complement pathway, binds to carbohydrates expressed on some viral glycoproteins. In this study, the ability of MBL to bind to virus particles pseudotyped with Ebola and Marburg envelope glycoproteins was evaluated. Virus particles bearing either Ebola (Zaire strain) or Marburg (Musoke strain) envelope glycoproteins bound at significantly higher levels to immobilized MBL compared with virus particles pseudotyped with vesicular stomatitis virus glycoprotein or with no virus glycoprotein. As observed in previous studies, Ebola-pseudotyped virus bound to cells expressing the lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin). However, pre-incubation of virus with MBL blocked DC-SIGN-mediated binding to cells, suggesting that the two lectins bind at the same or overlapping sites on the Ebola glycoprotein. Neutralization experiments showed that virus pseudotyped with Ebola or Marburg (Musoke) glycoprotein was neutralized by complement, while the Marburg (Ravn strain) glycoprotein-pseudotyped virus was less sensitive to neutralization. Neutralization was partially mediated through the lectin complement pathway, since a complement source deficient in MBL was significantly less effective at neutralizing viruses pseudotyped with filovirus glycoproteins and addition of purified MBL to the MBL-deficient complement increased neutralization. These experiments demonstrated that MBL binds to filovirus envelope glycoproteins resulting in important biological effects and suggest that MBL can interact with filoviruses during infection in humans.

Ebola virus (EBOV)-like particles (eVLP), composed of the EBOV glycoprotein and matrix viral protein (VP)40 with a lipid membrane, are a highly efficacious method of immunization against EBOV infection. The exact requirements for immunity against EBOV infection are poorly defined at this time. The goal of this work was to determine the requirements for EBOV immunity following eVLP vaccination. Vaccination of BALB/c or C57BL/6 mice with eVLPs in conjunction with QS-21 adjuvant resulted in mixed IgG subclass responses, a Th1-like memory cytokine response, and protection from lethal EBOV challenge. Further, this vaccination schedule led to the generation of both CD4+ and CD8+ IFN-γ+ T cells recognizing specific peptides within glycoprotein and VP40. The transfer of both serum and splenocytes, but not serum or splenocytes alone, from eVLP-vaccinated mice conferred protection against lethal EBOV infection in these studies. B cells were required for eVLP-mediated immunity to EBOV because B cell-deficient mice vaccinated with eVLPs were not protected from lethal EBOV challenge. We also found that CD8+, but not CD4+, T cells are absolutely required for eVLP-mediated protection against EBOV infection. Further, eVLP-induced protective mechanisms were perforin-independent, but IFN-γ-dependent. Taken together, both EBOV-specific humoral and cytotoxic CD8+ T cell responses are critical to mediate protection against filoviruses following eVLP vaccination.

The goal of this study was to determine the importance of cellular factors for binding of HIV to cells. HIV primary isolates (PIs) produced in peripheral blood mononuclear cells (PBMCs) bound at relatively high levels to PBMCs but at low levels to cell lines, whereas T cell line-adapted (TCLA) virus produced in the H9 T cell line bound at high levels to both cell lines and PBMCs. Expression of CD4 in CD4-negative cells or blocking CD4 with antibody on CD4-positive cells did not affect virus binding. Blocking of gp120/gp41 with antibodies or a lack of expression of gp120/gp41 in virus particles also did not affect virus binding. However, the cell type from which virus was produced did affect virus binding. Thus, the binding pattern of TCLA virus shifted to that of a PI virus when produced in PBMCs. A PI binding pattern also occurred when a cloned TCLA virus (NL4-3) was produced in PBMCs, indicating that the virus-producing cell type has more of an effect on virus binding than the virus strain. These experiments show that both the virus-producing cell and the target cell have a major influence on HIV binding and suggest that host cell factors incorporated into virions are important for virus binding.

Cellular proteins, including major histocompatibility complex (MHC) class I and class II antigens, are incorporated into the membrane of HIV-1 when virions bud from infected cells. Experiments were performed to determine whether human sera that contained MHC class I and/or class II antibodies would lyse or neutralize a primary isolate of HIV. These results demonstrate that in the presence of complement, sera from some alloimmunized persons mediated significant anti-viral activity against an HIV primary isolate. Both lysis and neutralization of virus were observed. The antiviral effects were complement dependent because heat inactivation eliminated most anti-viral effects. Antiviral activity mediated by sera containing MHC alloantibodies in the presence of complement was > or = activity due to sera from HIV-infected persons as reported in this and a previous study. High levels of antibodies to both MHC class I and class II were present in sera that mediated the highest levels of anti-viral activity. Absorption of serum with platelets (which express class I but not class II antigens) substantially reduced their lytic activity. These studies suggest that MHC antibodies mediate potent anti-viral effects on primary isolates of HIV and support the possibility that deliberately alloimmunizing humans might protect against HIV infection.