Allen Grolla

Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses

Vaccines and therapies are urgently needed to address public health needs stemming from emerging pathogens and biological threat agents such as the filoviruses Ebola virus (EBOV) and Marburg virus (MARV). Here, we developed replication-competent vaccines against EBOV and MARV based on attenuated recombinant vesicular stomatitis virus vectors expressing either the EBOV glycoprotein or MARV glycoprotein. A single intramuscular injection of the EBOV or MARV vaccine elicited completely protective immune responses in nonhuman primates against lethal EBOV or MARV challenges. Notably, vaccine vector shedding was not detectable in the monkeys and none of the animals developed fever or other symptoms of illness associated with vaccination. The EBOV vaccine induced humoral and apparent cellular immune responses in all vaccinated monkeys, whereas the MARV vaccine induced a stronger humoral than cellular immune response. No evidence of EBOV or MARV replication was detected in any of the protected animals after challenge. Our data suggest that these vaccine candidates are safe and highly efficacious in a relevant animal model.

Panmicrobial Oligonucleotide Array for Diagnosis of Infectious Diseases

To facilitate rapid, unbiased, differential diagnosis of infectious diseases, we designed GreeneChipPm, a panmicrobial microarray comprising 29,455 sixty-mer oligonucleotide probes for vertebrate viruses, bacteria, fungi, and parasites. Methods for nucleic acid preparation, random primed PCR amplification, and labeling were optimized to allow the sensitivity required for application with nucleic acid extracted from clinical materials and cultured isolates. Analysis of nasopharyngeal aspirates, blood, urine, and tissue from persons with various infectious diseases confirmed the presence of viruses and bacteria identified by other methods, and implicated Plasmodium falciparum in an unexplained fatal case of hemorrhagic feverlike disease during the Marburg hemorrhagic fever outbreak in Angola in 2004–2005.

Person-to-person transmission of Nipah virus in a Bangladeshi community.

Transmission of this virus highlights the need for infection control strategies for resource-poor settings.

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.

Vesicular stomatitis virus-based vaccines protect nonhuman primates against aerosol challenge with Ebola and Marburg viruses

Considerable progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against Ebola and Marburg viruses. A vaccine based on recombinant vesicular stomatitis virus (VSV) seems to be particularly robust as it can also confer protection when administered as a postexposure treatment. While filoviruses are not thought to be transmitted by aerosol in nature the inhalation route is among the most likely portals of entry in the setting of a bioterrorist event. At present, all candidate filoviral vaccines have been evaluated against parenteral challenges but none have been tested against an aerosol exposure. Here, we evaluated our recombinant VSV-based Zaire ebolavirus (ZEBOV) and Marburg virus (MARV) vaccines against aerosol challenge in cynomolgus macaques. All monkeys vaccinated with a VSV vector expressing the glycoprotein of ZEBOV were completely protected against an aerosol exposure of ZEBOV. Likewise, all monkeys vaccinated with a VSV vector expressing the glycoprotein of MARV were completely protected against an aerosol exposure of MARV. All control animals challenged by the aerosol route with either ZEBOV or MARV succumbed. Interestingly, disease in control animals appeared to progress slower than previously seen in macaques exposed to comparable doses by intramuscular injection.

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.

Postexposure protection against Marburg haemorrhagic fever with recombinant vesicular stomatitis virus vectors in non-human primates: an efficacy assessment

BACKGROUND Effective countermeasures are urgently needed to prevent and treat infections caused by highly pathogenic and biological threat agents such as Marburg virus (MARV). We aimed to test the efficacy of a replication-competent vaccine based on attenuated recombinant vesicular stomatitis virus (rVSV), as a postexposure treatment for MARV haemorrhagic fever. METHODS We used a rhesus macaque model of MARV haemorrhagic fever that produced 100% lethality. We administered rVSV vectors expressing the MARV Musoke strain glycoprotein to five macaques 20-30 min after a high-dose lethal injection of homologous MARV. Three animals were MARV-positive controls and received non-specific rVSV vectors. We tested for viraemia, undertook analyses for haematology and serum biochemistry, and measured humoral and cellular immune responses. FINDINGS All five rhesus monkeys that were treated with the rVSV MARV vectors as a postexposure treatment survived a high-dose lethal challenge of MARV for at least 80 days. None of these five animals developed clinical symptoms consistent with MARV haemorrhagic fever. All the control animals developed fulminant disease and succumbed to the MARV challenge by day 12. MARV disease in the controls was indicated by: high titres of MARV (10(3)-10(5) plaque-forming units per mL); development of leucocytosis with concurrent neutrophilia at end-stage disease; and possible damage to the liver, kidney, and pancreas. INTERPRETATION Postexposure protection against MARV in non-human primates provides a paradigm for the treatment of MARV haemorrhagic fever. Indeed, these data suggest that rVSV-based filoviral vaccines might not only have potential as preventive vaccines, but also could be equally useful for postexposure treatment of filoviral infections.

Vesicular Stomatitis Virus-Based Ebola Vaccine Is Well-Tolerated and Protects Immunocompromised Nonhuman Primates

Ebola virus (EBOV) is a significant human pathogen that presents a public health concern as an emerging/re-emerging virus and as a potential biological weapon. Substantial progress has been made over the last decade in developing candidate preventive vaccines that can protect nonhuman primates against EBOV. Among these prospects, a vaccine based on recombinant vesicular stomatitis virus (VSV) is particularly robust, as it can also confer protection when administered as a postexposure treatment. A concern that has been raised regarding the replication-competent VSV vectors that express EBOV glycoproteins is how these vectors would be tolerated by individuals with altered or compromised immune systems such as patients infected with HIV. This is especially important as all EBOV outbreaks to date have occurred in areas of Central and Western Africa with high HIV incidence rates in the population. In order to address this concern, we evaluated the safety of the recombinant VSV vector expressing the Zaire ebolavirus glycoprotein (VSVΔG/ZEBOVGP) in six rhesus macaques infected with simian-human immunodeficiency virus (SHIV). All six animals showed no evidence of illness associated with the VSVΔG/ZEBOVGP vaccine, suggesting that this vaccine may be safe in immunocompromised populations. While one goal of the study was to evaluate the safety of the candidate vaccine platform, it was also of interest to determine if altered immune status would affect vaccine efficacy. The vaccine protected 4 of 6 SHIV-infected macaques from death following ZEBOV challenge. Evaluation of CD4+ T cells in all animals showed that the animals that succumbed to lethal ZEBOV challenge had the lowest CD4+ counts, suggesting that CD4+ T cells may play a role in mediating protection against ZEBOV.

Immunization with Modified Vaccinia Virus Ankara-Based Recombinant Vaccine against Severe Acute Respiratory Syndrome Is Associated with Enhanced Hepatitis in Ferrets

ABSTRACT Severe acute respiratory syndrome (SARS) caused by a newly identified coronavirus (SARS-CoV) is a serious emerging human infectious disease. In this report, we immunized ferrets (Mustela putorius furo) with recombinant modified vaccinia virus Ankara (rMVA) expressing the SARS-CoV spike (S) protein. Immunized ferrets developed a more rapid and vigorous neutralizing antibody response than control animals after challenge with SARS-CoV; however, they also exhibited strong inflammatory responses in liver tissue. Inflammation in control animals exposed to SARS-CoV was relatively mild. Thus, our data suggest that vaccination with rMVA expressing SARS-CoV S protein is associated with enhanced hepatitis.

Detection of Ebola Virus in Oral Fluid Specimens during Outbreaks of Ebola Virus Hemorrhagic Fever in the Republic of Congo

BACKGROUND Patients who have refused to provide blood samples has meant that there have been significant delays in confirming outbreaks of Ebola virus hemorrhagic fever (EVHF). During the 2 EVHF outbreaks in the Republic of Congo in 2003, we assessed the use of oral fluid specimens versus serum samples for laboratory confirmation of cases of EVHF. METHODS Serum and oral fluid specimens were obtained from 24 patients with suspected Ebola and 10 healthy control subjects. Specimens were analyzed for immunoglobulin G antibodies by enzyme-linked immunosorbent assay (ELISA) and for Ebola virus by antigen detection ELISA and reverse-transcriptase polymerase chain reaction (RT-PCR). Oral fluid specimens were collected with a commercially available collection device. RESULTS We failed to detect antibodies against Ebola in the oral fluid specimens obtained from patients whose serum samples were seropositive. All patients with positive serum RT-PCR results also had positive results for their oral fluid specimens. CONCLUSIONS This study demonstrates the usefulness of oral fluid samples for the investigation of Ebola outbreaks, but further development in antibodies and antigen detection in oral fluid specimens is needed before these samples are used for filovirus surveillance activities in Africa.