A Starting Line-Up: (left to right) Maribeth Eiden, chief, Section on Directed Gene Transfer, Laboratory of Cellular and Molecular Regulation, NIMH; Peter Collins, senior investigator, Laboratory of Infectious Diseases, NIAID; Nidia Oliveira, visiting research fellow, NIMH; Brian Murphy, co-chief, Laboratory of Infectious Diseases, and chief, Respiratory Viruses Section, NIAID; Carolyn Wilson, Center for Biologics Evaluation and Research, FDA; and Kanta Subbarao, senior investigator, Laboratory of Infectious Diseases, NIAID

Maribeth Eiden itemized some of the factors facilitating the spread of infectious diseases in the 21st century: increased global travel, climate change, poverty, enlarged populations, encroachment of domestic livestock, and inadequate surveillance policies.

She then introduced a panel of investigators from NIAID, FDA/CBER, and her own NIMH lab who are working to control or respond to that spread with sophisticated vaccine development and basic explorations into the mechanisms that enable viruses to jump across species.

NIAID-MedImmune CRADA
Aimed at Vanquishing Avian Flu

The global toll of the H5N1 avian flu virus from late 2003 to October 18, 2005, when Kanta Subbarrao delivered her talk on vaccines against potential pandemic flu strains, was 117 laboratory-confirmed human cases and 60 deaths — against the backdrop of 150 million infected birds and transcontinental spread.

The cases have all occurred in the context of a poultry outbreak, direct human contact with infected poultry, and person-to-person transmission of genetically identical virus within the family of an infected person.

There is no rapid human-to-human spread, Subarrao observed, but there are reasons for concern:

n Poultry outbreaks are increasing.

n There is evidence of genetic drift, with new H5N1 genotypes having arisen since its first appearance in 1997.

n Ducks, tigers, and leopards have died of H5N1 avian flu and ferrets and cats can be infected in the laboratory.

n H5N1 viruses can infect people and cause severe disease, and people have no immunity against it. Were it to become transmissible person-to-person, it could cause a pandemic.

 The "H"  in H5N1 stands for hemagglutinin, a key viral surface protein, of which there are 16 subtypes that have been isolated from birds. There are currently vaccines to prtect humans against H1 and H3.

Subbarao, a senior investigator in the Laboratory of Infectious Diseases, NIAID, and Brian Murphy, laboratory co-chief, are heading a team of NIAID scientists that will work its way through all the rest — H2 and H4 through 16 — under a CRADA agreement with MedImmune, Inc., of Gaithersburg, Md.

Each of these avian flu vaccines will be administered intranasally; the live attenuated bird virus will contain the existing backbone of the influenza A component of MedImmune's FluMist® flu vaccine, a live virus that has undergone attenuating mutations.

The project may take up to 10 years, Subbarao said in an interview with The NIH Catalyst. She estimated that it would take about two years to develop vaccines against each subtype and gather initial data from the ensuing phase 1 clinical trial, which will involve healthy adults. The clinical trials will be conducted in an inpatient setting during summers to limit the risk of the expected small amount of shed vaccine virus combining with any circulating wild-type influenza A viruses.

Closing In
On Dengue and West Nile

Murphy and his colleagues in the NIAID Laboratory of Infectious Diseases have been working on live attenuated vaccine constructs against two flaviviruses — dengue and West Nile.

Murphy is aiming to create a tetravalent  vaccine aganst the four dengue serotypes that are responsible for 50 million infections a year.

Meanwhile, the team has introduced a deletion mutation (delta 30) into each of the four wild-type serotypes to achieve attenuation; thus far, good results have been seen with two (DEN1 and DEN4) of the constructs.

The attenuated recombinant DEN4 construct, for instance, "induced good antibody titers" in tests with 80 human volunteers, Murphy said, adding that mild neutropenia and rash were the notable but not dangerous side effects and systemic symptoms were rare.

A second approach, which involves making antigenic chimeric viruses between the DEN4 delta30 virus and the DEN2 or DEN3 wild-type virus, has generated good vaccine candidates for  these two serotypes. A tetravalent vaccine with these four DEN viruses looks good in monkeys, Murphy said.

Preliminary results in monkeys suggest that a boost at day 30 does not elicit a secondary antibody response; however, a four-month interval, he said,  was effective at boosting the antibody response against each of the four serotypes.

Especially significant in low-income countries, Murphy added, is that the equivalent of "one flask could immunize 100,000 inexpensively." He anticipates initiating clinical studies of a tetravalent dengue vaccine in 2006–2007.

The team, led by Alexander Pletnev, is taking a similar approach in developing a vaccine for West Nile virus, which causes "a couple of thousand" infections a year in the United States. Safety studies in mice are encouraging, and one chimeric construct in early human testing was found to raise protective antibodies in seven of nine volunteers in the absence of any symptoms.

Getting the Drop
On Respiratory Viruses

Peter Collins, a senior investigator working with Murphy and other NIAID researchers, discussed his work developing an intranasal vaccine vector for highly pathogenic viruses such as SARS, Ebola virus, and avian flu virus.

Initial studies have used live attenuated human parainfluenza virus (PIV3), an important cause of respiratory tract disease in infants and children, as a vector.

PIV3 has a track record: First, the NIAID team used recombinant DNA techniques to develop live attenuated PIV3 strains; a PIV3 intranasal vaccine is currently in clinical trials. The researchers developed the further strategy of using attenuated PIV3 as a vector to express the protective antigens of additional pediatric viral pathogens, such as human respiratory syncytial virus (RSV). This bivalent PIV3/RSV vaccine is also now in clinical trials sponsored by MedImmune.

Collins described how attenuated PIV3 can also be used to express protective antigens of highly pathogenic agents such as SARS and Ebola virus.

The intranasal route, by drops or nasal spray, has the advantage of directly stimulating local respiratory tract immunity, as well as systemic immunity — a clear plus because the respiratory tract frequently is the portal of entry and egress for viruses; and for respiratory pathogens, it is the major site of viral replication and disease.

Using the SARS-S glycoprotein alone, an intranasal PIV3 vector provided protection against the SARS coronavirus in tests with rodents and nonhuman primate. In addition, an intranasal PIV3 vector expressing the Ebola GP glycoprotein protected guinea pigs against an otherwise lethal dose of Ebola virus. 

Although these vectored vaccines have promise for use in infants and young children, they probably will not be effective in adults, who have naturally acquired immunity that will restrict the replication of the vector, Collins observed.

Therefore, the investigators are developing nonhuman PIV viruses as vectors, such as low-virulence strains of Newcastle disease virus (NDV). In initial studies, NDV expressing a test antigen proved to be both highly attenuated and highly immunogenic as an intranasal vaccine in nonhuman primates.

Unearthing Ebola's
Conserved Domains

The world first became aware of the Ebola virus in 1976; in the past 10 years, there have been 15 Ebola virus outbreaks — notably in Congo (now Zaire), the Sudan, and Côte d'Ivoire.

A natural reservoir has not been identified; incubation is five to seven days; symptoms start with generalized ague and progress to disseminated intravascular coagulation, with death in 50–90 percent of cases.

"But survivors develop antibodies against the virus, and this is the basis for a vaccine strategy," said Carolyn Wilson, of the FDA Center for Biologics Research and Evaluation. (She noted that Nancy Sullivan and her colleagues at the Vaccine Research Center have developed a DNA-prime/adenovirus vector–boost Ebola vaccine currently in clinical trials.)

Working with "the Zaire virus," Wilson's lab has been focusing on an Ebola vaccine strategy that targets conserved domains. They've been mining the glycoprotein section — the heavily glycosylated GP1, which mediates receptor binding, and GP2, which mediates fusion. They used site-directed mutagenesis to functionally screen 15 amino acids from the GP domain and found two that are critical for viral entry and "are conserved across all filovirus strains, including Ebola and Marburg," Wilson said.

Further, Wilson showed that a peptide derived from one of these conserved domains was able to block infection by strains of Ebola virus. Now armed with "proof of concept," the team is continuing its investigations with these two conserved amino acids — F88 and F159.

How Much Can a Koala Bear?

Nidia Oliveira, a visiting research fellow at the NIMH Laboratory of Cellular and Molecular Regulation, traced the meanderings and permutations of a retrovirus that jumped from feral mice in southern Asia to Old World primates to a New World pet primate in San Francisco — and more recently  has been identified in koalas in Australia, where it has gained its greatest lethality.

About 60 percent of koala bear mortality is attributable to neoplasia. Infected koala bears have a neoplastic disease rate that correlates with viral load. The KoRV retrovirus, Oliveira said, has been isolated in 100 percent of Queensland koalas — "it's in their genomes," she said, noting that the rate is about 30 percent elsewhere in southeastern mainland Australia.

Asian rodents are the reservoir for the GALV-like virus that emerged to infect gibbon apes in Thailand and became WMV in woolly monkeys and KoRV in marsupials.

Sequence comparisons establish the lineage, Oliveira said, noting that GALV, WMV, and KoRV use the same receptor — PIT1 — in gaining entry, but that KoRV also uses orthologs that GALV and WMV are unable to use. The KoRV envelope, she said, has an extremely broad host range, and the challenge now is to discern what part of the envelope accounts for that range and for the virus' species-jumping ability.    n

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