Postexposure Treatment of Marburg Virus Infection

Rhesus monkeys are protected from disease when a recombinant vesicular stomatitis virus–based vaccine is administered 20–30 min after infection with Marburg virus. We protected 5/6 monkeys when this vaccine was given 24 h after challenge; 2/6 animals were protected when the vaccine was administered 48 h postinfection

Pathogenesis of lassa fever in cynomolgus macaques

<p>Abstract</p> <p>Background</p> <p>Lassa virus (LASV) infection causes an acute and sometimes fatal hemorrhagic disease in humans and nonhuman primates; however, little is known about the development of Lassa fever. Here, we performed a pilot study to begin to understand the progression of LASV infection in nonhuman primates.</p> <p>Methods</p> <p>Six cynomolgus monkeys were experimentally infected with LASV. Tissues from three animals were examined at an early- to mid-stage of disease and compared with tissues from three animals collected at terminal stages of disease.</p> <p>Results</p> <p>Dendritic cells were identified as a prominent target of LASV infection in a variety of tissues in all animals at day 7 while Kupffer cells, hepatocytes, adrenal cortical cells, and endothelial cells were more frequently infected with LASV in tissues of terminal animals (days 13.5-17). Meningoencephalitis and neuronal necrosis were noteworthy findings in terminal animals. Evidence of coagulopathy was noted; however, the degree of fibrin deposition in tissues was less prominent than has been reported in other viral hemorrhagic fevers.</p> <p>Conclusion</p> <p>The sequence of pathogenic events identified in this study begins to shed light on the development of disease processes during Lassa fever and also may provide new targets for rational prophylactic and chemotherapeutic interventions.</p

Neutralizing Antibody Fails to Impact the Course of Ebola Virus Infection in Monkeys

Prophylaxis with high doses of neutralizing antibody typically offers protection against challenge with viruses producing acute infections. In this study, we have investigated the ability of the neutralizing human monoclonal antibody, KZ52, to protect against Ebola virus in rhesus macaques. This antibody was previously shown to fully protect guinea pigs from infection. Four rhesus macaques were given 50 mg/kg of neutralizing human monoclonal antibody KZ52 intravenously 1 d before challenge with 1,000 plaque-forming units of Ebola virus, followed by a second dose of 50 mg/kg antibody 4 d after challenge. A control animal was exposed to virus in the absence of antibody treatment. Passive transfer of the neutralizing human monoclonal antibody not only failed to protect macaques against challenge with Ebola virus but also had a minimal effect on the explosive viral replication following infection. We show that the inability of antibody to impact infection was not due to neutralization escape. It appears that Ebola virus has a mechanism of infection propagation in vivo in macaques that is uniquely insensitive even to high concentrations of neutralizing antibody

The broad-spectrum antiviral favipiravir protects guinea pigs from lethal Lassa virus infection post-disease onset

With up to 500,000 infections annually, Lassa virus (LASV), the cause of Lassa fever, is one of the most prevalent etiological agents of viral hemorrhagic fever (VHF) in humans. LASV is endemic in several West African countries with sporadic cases and prolonged outbreaks observed most commonly in Sierra Leone, Liberia, Guinea and Nigeria. Additionally several cases of Lassa fever have been imported into North America, Europe and Asia making LASV a global threat to public health. Despite this, currently no approved therapeutic or vaccine exists to treat or prevent LASV infections. Here, using a passaged strain of LASV that is uniformly lethal in Hartley guinea pigs, we demonstrate that favipiravir, a broad-spectrum antiviral agent and leading treatment option for influenza, has potent activity against LASV infection. In this model, once daily treatment with favipiravir significantly reduced viral titers in tissue samples and reduced mortality rates when compared with animals receiving vehicle-only or ribavirin, the current standard of care for Lassa fever. Favipiravir remained highly effective against lethal LASV infection when treatments were initiated nine days post-infection, a time when animals were demonstrating advanced signs of disease. These results support the further preclinical evaluation of favipiravir for Lassa fever and other VHFs

Preclinical development of filovirus and flavivirus vaccines based on recombinant insect cell expressed subunits

Ebola Virus Disease (EVD) is the most prominent example of filovirus disease but despite being characterized as a Category A Priority Pathogen by NIH/NIAID over a decade ago, it lacked public and private research resources due to the absence of a commercial market. Transmission from wild animals into the human population typically causes outbreaks of limited scale in endemic areas located in the forested regions of Central Africa and the Philippines (for Reston ebolavirus), therefore other public health threats garnered more attention. This changed when a Zaire Ebolavirus (EBOV) outbreak of increasing size in several West African countries started to reveal the true epidemic potential that filovirus infections can have when entering an urban setting in a highly mobile society. Despite significant progress with the clinical development of several EBOV vaccine candidates during and after the West African outbreak, no EBOV specific therapeutics and vaccines have yet received regulatory approval. Additional research is needed in particular on understanding the mechanism of protection and defining immune correlates of protection for Ebola and other filovirus vaccines. For our multivalent filovirus vaccine candidate, we have produced soluble recombinant filovirus glycoproteins (GP) from EBOV, Marburg marburgvirus (MARV) and Sudan ebolavirus (SUDV) using the Drosophila S2 cell expression system. The immunogenicity of highly purified recombinant subunits and admixtures formulated with or without clinically relevant adjuvants was evaluated in mice, guinea pigs and macaques. Strong antigen-specific IgG titers as well as virus neutralizing titers were observed after administering two or three doses of adjuvanted formulations. In mice and non-human primates subunit proteins were also shown to elicit cell mediated immune responses. Analysis of secreted cytokines in batch-cultured, antigen-stimulated splenocytes or PBMC’s demonstrated antigen-induced Th1 and Th2 type responses. Recombinant vaccine candidates were tested successfully for protection in the mouse model of EBOV. Further studies allowed us to demonstrate that both humoral and cell-mediated immunity are elicited and can mediate protection. Additional immunogenicity and efficacy studies in guinea pigs were focused on optimized antigen dosing, antigenic balance and adjuvantation. Multiple formulations consistently produced strong antibody responses and demonstrated 100% protective efficacy in the EBOV guinea pig model. Results from studies in two species of non-human primates demonstrate that vaccination with formulations of recombinant EBOV subunits and an emulsion-based adjuvant consistently produces high anti-EBOV IgG and virus neutralizing titers. Such vaccination prevents viremia subsequent to live virus challenge and protects animals from terminal EBOV disease. These studies suggest that we have defined a viable Ebola virus vaccine candidate based on non-replicating viral subunits. In addition to updates on efficacy testing against EBOV and MARV, we will discuss current formulation optimization efforts in our laboratory including thermostabilization of recombinant subunits as well as defining correlates of protection. These are prerequisites to enable efficient clinical development of a monovalent vaccine candidate for protection against EVD and a multivalent, recombinant subunit vaccine with protective efficacy against EBOV, SUDV and MARV infection. Recently we have also demonstrated the applicability of our vaccine platform for the rapid development of vaccines against emerging diseases with a focus on Zika virus (ZIKV), a flavivirus, where we were able to demonstrate efficacy in mice and cynomolgus macaques within approximately 13 months from designing the synthetic gene for antigen expression. While a completely different disease from EVD, the recent outbreak of ZIKV in the Americas provided a similar challenge as no vaccine development efforts have been conducted prior to 2016 and an increasing body of evidence suggests that rather than causing a typical, mild form of disease as previously reported, ZIKV infections can cause neurological sequelae as well as fetal and infant malformations. These results demonstrate that the insect cell expression system can be used to rapidly and efficiently produce recombinant viral subunits from a variety of pathogenic viruses that are highly immunogenic in multiple animal species and are capable of providing effective vaccine protection against live virus challenge

Effective Post-Exposure Treatment of Ebola Infection

Ebola viruses are highly lethal human pathogens that have received considerable attention in recent years due to an increasing re-emergence in Central Africa and a potential for use as a biological weapon. There is no vaccine or treatment licensed for human use. In the past, however, important advances have been made in developing preventive vaccines that are protective in animal models. In this regard, we showed that a single injection of a live-attenuated recombinant vesicular stomatitis virus vector expressing the Ebola virus glycoprotein completely protected rodents and nonhuman primates from lethal Ebola challenge. In contrast, progress in developing therapeutic interventions against Ebola virus infections has been much slower and there is clearly an urgent need to develop effective post-exposure strategies to respond to future outbreaks and acts of bioterrorism, as well as to treat laboratory exposures. Here we tested the efficacy of the vesicular stomatitis virus-based Ebola vaccine vector in post-exposure treatment in three relevant animal models. In the guinea pig and mouse models it was possible to protect 50% and 100% of the animals, respectively, following treatment as late as 24 h after lethal challenge. More important, four out of eight rhesus macaques were protected if treated 20 to 30 min following an otherwise uniformly lethal infection. Currently, this approach provides the most effective post-exposure treatment strategy for Ebola infections and is particularly suited for use in accidentally exposed individuals and in the control of secondary transmission during naturally occurring outbreaks or deliberate release

Framework for Leadership and Training of Biosafety Level 4 Laboratory Workers

One-sentence summary for table of contents: Training should include theoretical consideration of biocontainment principles, practical hands-on training, and mentored on-the-job experience

Recombinant Vesicular Stomatitis Virus Vaccine Vectors Expressing Filovirus Glycoproteins Lack Neurovirulence in Nonhuman Primates

The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses an individual filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). The main concern with all replication-competent vaccines, including the rVSV filovirus GP vectors, is their safety. To address this concern, we performed a neurovirulence study using 21 cynomolgus macaques where the vaccines were administered intrathalamically. Seven animals received a rVSV vector expressing the Zaire ebolavirus (ZEBOV) GP; seven animals received a rVSV vector expressing the Lake Victoria marburgvirus (MARV) GP; three animals received rVSV-wild type (wt) vector, and four animals received vehicle control. Two of three animals given rVSV-wt showed severe neurological symptoms whereas animals receiving vehicle control, rVSV-ZEBOV-GP, or rVSV-MARV-GP did not develop these symptoms. Histological analysis revealed major lesions in neural tissues of all three rVSV-wt animals; however, no significant lesions were observed in any animals from the filovirus vaccine or vehicle control groups. These data strongly suggest that rVSV filovirus GP vaccine vectors lack the neurovirulence properties associated with the rVSV-wt parent vector and support their further development as a vaccine platform for human use

Development of a New Vaccine for the Prevention of Lassa Fever

BACKGROUND: Recent importation of Lassa fever into Germany, the Netherlands, the United Kingdom, and the United States by travelers on commercial airlines from Africa underscores the public health challenge of emerging viruses. Currently, there are no licensed vaccines for Lassa fever, and no experimental vaccine has completely protected nonhuman primates against a lethal challenge. METHODS AND FINDINGS: We developed a replication-competent vaccine against Lassa virus based on attenuated recombinant vesicular stomatitis virus vectors expressing the Lassa viral glycoprotein. A single intramuscular vaccination of the Lassa vaccine elicited a protective immune response in nonhuman primates against a lethal Lassa virus challenge. Vaccine shedding was not detected in the monkeys, and none of the animals developed fever or other symptoms of illness associated with vaccination. The Lassa vaccine induced strong humoral and cellular immune responses in the four vaccinated and challenged monkeys. Despite a transient Lassa viremia in vaccinated animals 7 d after challenge, the vaccinated animals showed no evidence of clinical disease. In contrast, the two control animals developed severe symptoms including rashes, facial edema, and elevated liver enzymes, and ultimately succumbed to the Lassa infection. CONCLUSION: Our data suggest that the Lassa vaccine candidate based on recombinant vesicular stomatitis virus is safe and highly efficacious in a relevant animal model that faithfully reproduces human disease

Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus

A previously undescribed coronavirus (CoV) is the etiologic agent responsible for severe acute respiratory syndrome (SARS). Using a panel of contiguous cDNAs that span the entire genome, we have assembled a full-length cDNA of the SARS-CoV Urbani strain, and have rescued molecularly cloned SARS viruses (infectious clone SARS-CoV) that contained the expected marker mutations inserted into the component clones. Recombinant viruses replicated as efficiently as WT virus and both were inhibited by treatment with the cysteine proteinase inhibitor (2S,3S)-transepoxysuccinyl-l-leucylamido-3-methylbutane ethyl ester. In addition, subgenomic transcripts were initiated from the consensus sequence ACGAAC in both the WT and infectious clone SARS-CoV. Availability of a SARS-CoV full-length cDNA provides a template for manipulation of the viral genome, allowing for the rapid and rational development and testing of candidate vaccines and therapeutics against this important human pathogen