Vaccine Provides Protection Against Avian Flu Virus In Animal

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Vaccine Provides 100 Percent Protection Against Avian Flu Virus In

Animal Study

University of Pittsburgh researchers announced they have genetically

engineered an avian flu vaccine from the critical components of the

deadly H5N1 virus that completely protected mice and chickens from

infection. Avian flu has devastated bird populations in Southeast

Asia and Europe and so far has killed more than 80 people.

Because this vaccine contains a live virus, it may be more immune-

activating than avian flu vaccines prepared by traditional methods,

say the researchers. Furthermore, because it is grown in cells, it

can be produced much more quickly than traditional vaccines, making

it an extremely attractive candidate for preventing the spread of the

virus in domestic livestock populations and, potentially, in humans,

according to the study, published in the Feb 15 issue of the Journal

of Virology and made available early online.

" The results of this animal trial are very promising, not only

because our vaccine completely protected animals that otherwise would

have died, but also because we found that one form of the vaccine

stimulates several lines of immunity against H5N1, " said

Gambotto, M.D., assistant professor in the departments of surgery and

molecular genetics and biochemistry, University of Pittsburgh School

of Medicine, and lead author of the study.

Dr. Gambotto and his colleagues constructed the vaccine by

genetically engineering a common cold virus, called adenovirus, to

express either all or parts of an avian influenza protein called

hemagglutinin (HA) on its surface. Found on the surface of all

influenza viruses, HA allows the virus to attach to the cell that is

being infected and is, therefore, critical to the influenza virus'

ability to cause illness and death.

Since the late 1990s, a number of outbreaks of the avian influenza

H5N1 in poultry have occurred in Cambodia, China, Indonesia, Japan,

Laos, South Korea, Thailand and Vietnam. Outbreaks recently have been

reported in Turkey and Romania. To date, H5N1 has caused the most

large-scale and widespread bird deaths in known history--an estimated

150 to 200 million birds have either died in the outbreaks or been

killed as part of infection control actions in the last eight years.

The H5N1 virus does not usually infect humans. However, in 1997, the

first case of spread from a bird to a human occurred in Hong Kong

during an outbreak of bird flu in poultry. The virus caused severe

respiratory illness in 18 people, six of whom died. Since that time,

more than 170 cases of known H5N1 infection have occurred among

humans worldwide, approximately half of whom died.

Based on the published sequence of the Vietnam strain of the H5N1

avian influenza virus, members of the University of Pittsburgh Vector

Core Facility, led by Wentao Gao, Ph.D., research instructor in the

School of Medicine's department of surgery, constructed several

adenovirus " vectors " --viruses that have been modified to serve as a

vector, or delivery vehicle, for foreign genes or DNA--containing

either the full genetic sequence of the HA protein or sequences for

only parts, or subunits, of HA. They also constructed a vector

containing sequences for a portion of the HA protein from the H5N1

Hong Kong strain.

Collaborating with investigators Xiuhua Lu, Ph.D., Doan C. Nguyen,

M.D., Yumi Matsuoka, Ph.D., O. Donis, Ph.D., and Jaquelin M.

Katz, Ph.D., of the Influenza Branch of the Centers for Disease

Control and Prevention, Dr. Gambotto's team tested the ability of

their slightly different vaccines to protect mice from infection by

wild-type H5N1 by comparing its performance to an adenovirus vector

containing no H5N1 genes, or an " empty vector. " The investigators

then observed the H5NI-exposed mice for any signs of illness,

including weight loss and death, and also checked their blood for

anti-viral antibodies and other markers of H5N1-specific immunity.

All of the mice immunized with the empty vector vaccine experienced

substantial weight loss beginning about three days after exposure to

wild-type H5N1, and all were dead within six to nine days of avian

flu exposure. In sharp contrast, most of the mice immunized with the

adenovirus containing either the whole or part of the HA protein

showed only mild and short-lived weight loss and survived H5N1

infection.

When the investigators looked for evidence of a specific immune

response to H5N1, they found similar results. Although they were able

to isolate high levels of infectious H5N1 from multiple organs in the

mice vaccinated with the empty vector, and to various degrees in

animals vaccinated with the vectors containing the HA subunits, they

isolated only very small amounts of H5N1 from the mice immunized with

the full-length HA vaccine three days after infection. Six days after

infection, they could not detect any infectious H5N1 in the organs of

mice immunized with the full-length HA vaccine.

Moreover, when they looked at the cellular immune response to

vaccination, they found that all of the animals immunized with full-

length HA or the subunit vaccines developed strong cellular immune

responses. However, only the full-length HA-immunized mice developed

strong T-cell responses to both of the HA subunits. According to

Simon Barratt-Boyes, B.V.Sc., Ph.D., associate professor, department

of infectious diseases and microbiology, University of Pittsburgh

Graduate School of Public Health, and one of the co-authors of the

study, the ability of this particular recombinant vaccine--a vaccine

carrying only the important immune-stimulating proteins--to induce

both antibody- and T cell-directed immunity is extremely encouraging.

" This means that this recombinant vaccine can stimulate several lines

of defense against the H5N1 virus, giving it greater therapeutic

value. More importantly, it suggests that even if H5N1 mutates, the

vaccine is still likely to be effective against it. How effective, we

are not sure, " Dr. Barratt-Boyes cautioned. " We won't know until that

occurs. "

Based on the superior degree of protection that they found in mice

vaccinated with full-length HA vaccine, Dr. Gambotto's group, working

with E. Swayne, D.V.M., Ph.D., at the U.S. Department of

Agriculture, tested its effectiveness in chickens, which have almost

a 100 percent mortality rate to H5N1 exposure. In all, the

researchers inoculated four groups of chickens either through their

noses (intranasally) or with subcutaneous injections of either the HA-

containing vaccine or the empty vector vaccine. The chickens were

then challenged with a dose of whole H5N1 virus 10,000 times greater

than the dose given to the mice and significantly greater than the

dose farm chickens are likely to be exposed to during a natural

outbreak.

Interestingly, all of the chickens that were immunized subcutaneously

survived exposure to H5N1, developed strong HA-specific antibody

responses and showed no clinical signs of disease. In contrast, half

of the chickens immunized intranasally died and half survived. All of

the chickens immunized with the empty vector (intranasally and

subcutaneously) died within two days of H5N1 exposure. The

researchers are still not yet sure why the subcutaneous delivery is

more effective than the intranasal delivery of the vaccine, but they

suggested it may be because the adenovirus vector they used has

limited infectivity via the nose and respiratory tract.

Dr. Gambotto and his colleagues suggest that rather than replacing

traditional inactivated influenza vaccines, their adenovirus-based

vaccine could be a critically important complement to them. Because

it appears to be so successful in immunizing chickens against H5N1,

widespread inoculation of susceptible poultry populations could

provide a significant barrier to the spread of the virus via that

route in this country and other countries that have so far been

spared from avian flu. Also, if there were a disruption in the

traditional vaccine production pipeline, a recombinant vaccine could

be an attractive alternative for human immunization as well, they

said.

Indeed, according to Dr. Gambotto, there are several major advantages

to this type of vaccine development approach over traditional

approaches. Flu vaccines currently are prepared in fertilized chicken

eggs, a process developed more than 50 years ago that requires

millions of fertilized eggs that would be in short supply if a

pandemic--a widespread, global outbreak--were to occur. The

recombinant vaccine approach grows the vaccine in cell cultures,

which are unlimited in supply. Another major advantage of this

approach is its speed.

" It takes a little over a month for us to develop a recombinant

vector vaccine compared to a minimum of several months via

traditional methods, " he explained. " This capacity will be

particularly invaluable if the virus begins to mutate rapidly, a

phenomenon that often limits the ability of traditional vaccines to

contain outbreaks of mutant strains. " Dr. Gambotto added that his

group is planning a small clinical trial of the vaccine in humans in

the very near future.

###

The research was supported by internal University of Pittsburgh

funds. Others involved in this study include D. Robins, Ph.D.

and Montecalvo, Ph.D., University of Pittsburgh School of

Medicine; and Adam C. Soloff, B.S., University of Pittsburgh Graduate

School of Public Health.

http://www.sciencedaily.com/releases/2006/01/060126185223.htm