November 15, 2014 (Vol. 34, No. 20)
Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications
Guiding Principle Is Global Information Accessibility
On October 12, the Ebola crisis hit home in a new way, as the first case of person-to-person transmission of the virus was reported in Texas. A nurse who helped treat the Liberian man who died from the virus has tested positive for the disease, despite wearing a gown, gloves, mask, and other protective gear while in contact with the victim.
Ebola (EBOV), a single-stranded RNA filovirus, causes infections characterized by immune suppression and a systemic inflammatory response. This results in impairment of the vascular, coagulation, and immune systems, leading to multiorgan failure and shock (in some ways resembling septic shock).
But while overwhelming challenges in controlling and treating this disease remain, the availability of genomic and proteomic data accumulated and shared by researchers since the virus’ discovery in 1976 has already translated into invaluable knowledge about the deadly RNA virus, pinpointing potential targets for diagnostics, vaccines, and therapeutics.
To date, studies have revealed that the EBOV genome consists of seven genes in the order 3′ leader, nucleoprotein, virion protein (VP) 35, VP40, glycoprotein (GP), VP30, VP24, RNA-dependent RNA polymerase (L)—5′ trailer. The glycoprotein is the virus’ only transmembrane surface protein and forms trimeric spikes consisting of glycoprotein 1 and glycoprotein 2—two disulphide-linked furin-cleavage fragments.
The EBOV GP is critical for attachment to host cells and catalysis of membrane fusion. Hence, the EBOV GP gene is a key component of candidate vaccines as well as a target of neutralizing antibodies and inhibitors of attachment and fusion.
Based on contributions from scientists around the world, the University of California Santa Cruz Genomics Institute released in September a new EBOV genome browser—the UCSC Ebola Genome Portal—to assist global efforts to develop a vaccine and antiserum to help stop the spread of the virus. The browser aligns five strains of EBOV with two strains of the related Marburg virus. Within these strains, members of the UC Santa Cruz Genome Browser team have aligned 148 individual viral genomes, including 102 from the current West Africa outbreak.
Researchers from the Broad Institute and Harvard University, with collaborators from the Sierra Leone Ministry of Health and Sanitation and other institutions around the globe, have sequenced and analyzed more than 99 EBOV genomes collected from 78 patients diagnosed with EBOV in Sierra Leone during the first 24 days of the recent outbreak. A portion of the patients contributed samples more than once, allowing, the researchers said, a clearer view into how the virus can change in a single individual over the course of infection.
The team found more than 300 genetic changes that make the 2014 EBOV genomes distinct from the viral genomes tied to previous EBOV outbreaks. They also found sequence variations indicating that, from the samples sequenced, the EBOV outbreak started from a single introduction into humans, subsequently spreading from person to person over many months.
The research team increased the amount of genomic data available on the EBOV four-fold using deep sequencing on all available samples. They sequenced at a depth of 2,000 times on average for each EBOV genome to provide, they said, an “extremely close-up view” of the virus genomes from 78 patients. This high-resolution view allowed the team to detect multiple mutations that alter protein sequences—potential targets for future diagnostics, vaccines, and therapies.
This West African variant likely diverged from central African lineages around 2004, crossed from Guinea to Sierra Leone in May 2014, and has exhibited sustained human-to-human transmission subsequently, with no evidence of additional zoonotic sources.
“We’ve uncovered more than 300 genetic clues about what sets this outbreak apart from previous outbreaks,” said Stephen Gire, Ph.D., a research scientist at the Broad Institute and Harvard. “Although we don’t know whether these differences are related to the severity of the current outbreak, by sharing data with the research community, we hope to speed up our understanding of this epidemic and support global efforts to contain it.” Their findings, they say, could have important implications for rapid field diagnostic tests.
To accelerate response efforts, the research team released the full-length sequences on the National Center for Biotechnology Information’s DNA sequence database in advance of formal scientific publication, making the data available to the global scientific community. The hazardous nature of collecting, storing, and transporting EBOV patient samples is illustrated by the researchers’ tribute to five colleagues and coauthors who succumbed to the disease prior to the publication of their paper on the origin and transmission of EBOV in Science in August.
Several vaccine candidates have emerged from genomic and proteomic data. All use GP 1 and 2 either alone or in combination with other viral proteins as antigens. A Phase I trial recently began to test a vaccine co-developed by NIAID and GlaxoSmithKline (GSK) that will evaluate its safety and ability to generate an immune system response in healthy adults. Testing is taking place at the NIH Clinical Center in Bethesda, MD.
To protect against the Zaire strain, a preclinical study demonstrated that one injection of the vaccine made from two EBOV gene segments incorporated into a chimpanzee cold virus vector (chimp adenovirus type 3 or ChAd3) protected all four macaque monkeys exposed to high levels of Ebola virus five weeks after inoculation.
While the protective effects of the single inoculation diminished with time, two out of four inoculated animals were protected when challenged with EBOV 10 months after vaccination. To extend immunity, four macaques were first inoculated with the ChAd3 Ebola vaccine; eight weeks later they received a booster vaccine containing EBOV gene segments incorporated into a different vector (a poxvirus). Ten months after the initial inoculation, four out of four animals that received both shots were fully protected from infection with high doses of EBOV, demonstrating that the prime-boost regimen resulted in durable protection.
The investigational vaccine now entering Phase I trials was designed by Nancy J. Sullivan, Ph.D., chief of the Biodefense Research Section in NIAID’s Vaccine Research Center (VRC). She worked in collaboration with researchers at the VRC, the U.S. Army Medical Research Institute of Infectious Diseases, and Okairos, a Swiss-Italian biotechnology company acquired by GSK in 2013.
The study is the first of several Phase I clinical trials that will examine the vaccine and an experimental Ebola vaccine developed by the Public Health Agency of Canada and licensed to NewLink Genetics. NIH is also supporting Crucell in its development of an Ebola/Marburg vaccine as well as Profectus Biosciences, which is working on an EBOV vaccine. Additionally, NIH and Thomas Jefferson University are collaborating to develop a candidate EBOV vaccine based on the established rabies vaccine.
The CDC says it intends to identify the natural reservoir(s) for the known EBOVs and discover previously undetected filoviruses using a next-generation sequencing approach to screen thousands of bat specimens from areas of Africa known to have had filovirus activity. Because known filoviruses are genetically diverse (up to 50% at the nucleotide level), currently available sequence-based tests such as real-time PCR could miss a new virus. For example, Bundibugyo virus (species Bundibugyo ebolavirus), which remained undetected until 2007, caused two large outbreaks in Africa over the last decade. For these reasons the CDC points out that it is important to identify and characterize filovirus diversity in nature.
Drug companies are also looking to target several viral proteins. Tekmira is developing its RNAi-based therapeutic TKM-Ebola, an siRNA delivered through lipid nanoparticles, to combat the Zaire species of Ebola virus (ZEBOV). TKM-Ebola is being developed under a $140 million contract with the U.S. Department of Defense’s Medical Countermeasure Systems BioDefense Therapeutics Joint Product Management Office, which also supports multiple vaccine development efforts.
Preclinical studies were published in the Lancet and demonstrated that when siRNA targeting EBOV and delivered by Tekmira’s LNP technology were used to treat previously infected nonhuman primates, the result was 100% protection from an otherwise lethal dose of ZEBOV. In March, Tekmira was granted a Fast-Track designation from the FDA for its drug. However, the agency is allowing for compassionate use of the medication.
BioCryst is developing the compound BCX4430, a novel synthetic adenosine analog, as an antiviral drug to target EBOV numerous other infections viruses. Phase I testing is expected to begin before the end of the year. Preclinical results, which were published in Nature in April, demonstrated that BCX-4430 given after a viral challenge could stop EBOV and related Marburg infections from taking hold in rodents. Most notably, BCX-4430 given 48 hours after Marburg virus infection conferred complete protection to cynmologous monkeys.
The World Health Organization reports that it will work with all relevant stakeholders to accelerate their new drug and vaccine development efforts and safe use in affected countries. If proven safe, the organization said, a vaccine “could be available in November 2014 for priority use in healthcare workers.”
If this Phase I trial is completed successfully, the vaccine will be fast tracked for use in West Africa. In preparation for this, GSK is preparing a stockpile of 10,000 doses.