Date: Mon 7 Mar 2005
From: David Swayne <dswayne@seprl.usda.gov>


Vaccines and vaccination to control avian influenza
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The following is provided as background information on the use of vaccines
for control of avian influenza (AI) in poultry.

1. Vaccination should be viewed and used only as a single tool in a
comprehensive control strategy that includes: 1) biosecurity, 2) education,
3) diagnostics and surveillance, and 4) elimination of AI virus infected
poultry. One or more of these components are used to develop AI control
strategies to achieve one of 3 goals or outcomes (Swayne, 2004): 1)
Prevention - preventing introduction of AI; 2) Management - reducing losses
by minimizing negative economic impact through management practices; or 3)
Eradication - total elimination of AI.

2. Protection against avian influenza is the result of immune response
against the hemagglutinin protein (HA), of which there are 15 different HA
subtypes, and to a lesser extent against the neuraminidase protein (NA), of
which there are 9 different NA subtypes (Suarez & Schultz, 2000; Swayne &
Halvorson, 2003). Immune responses to the internal proteins, such as
nucleoprotein or matrix protein, are insufficient to provide field
protection. Therefore, there is no one universal AI vaccine. Practically,
protection is provided against the individual hemagglutinin subtype(s)
included in the vaccine.

3. Experimental and field studies have shown that properly used vaccines
will accomplish several goals: 1) protect against clinical signs and death,
2) reduced shedding of field virus if vaccinated poultry become infected,
3) prevent contact transmission of the field virus, 4) provide at least 20
weeks protection following a single vaccination for chickens (this may
require 2 or more injections in turkeys or longer-lived chickens), 5)
protect against challenges by low to high doses of field virus, 6) protect
against a changing virus and 7) increase a bird´s resistance to avian
influenza virus infection (Swayne, 2003; Capua et al., 2004). These
positive qualities are essential in contributing to AI control strategies.
Most AI vaccine studies and field use have focused on chickens and turkeys
because of their high death rates and the high concentrations of Highly
Pathogenic Avian Influenza (HPAI) virus excreted into the environment by
these species. However, with the changing epidemiology of the H5N1 HPAI
virus in Asia, the infection of domestic ducks and geese has become a very
important contributor to the maintenance and spread of the H5N1 HPAI virus.
Experimentally, vaccines have been shown to significantly reduce AI virus
replication and shedding in domestic ducks and geese and thus decrease
environmental contamination (especially in ponds, lakes and rivers) and
prevent contact transmission. Proper vaccination of domestic ducks and
geese will have a positive impact on control of H5N1 HPAI in Asia.

4. A wide variety of vaccines have been developed and examined in the
laboratory for potential use in the field. However, only vaccines from 2
technologies are licensed and used in poultry: inactivated whole avian
influenza virus vaccines and a recombinant fowlpox virus vectored vaccine
with an H5 AI gene insert (from AI virus A/turkey/Ireland/83 [H5N8]). These
2 vaccine technologies have been shown to produce safe, pure and potent
vaccines. Both vaccine technologies require handling and injection of
individual birds.

5. The quantity of AI vaccine used around the world in poultry is not well
documented, but reliable information suggests the largest single use has
been 2 billion doses of inactivated H5N2 avian influenza vaccine in China
(December 2003 - present). Indonesia also uses H5 inactivated AI vaccine.
In Mexico, an AI vaccination program has been used since January 1995, with
over 1.3 billion doses of inactivated vaccine and 850 million doses of
recombinant fowlpox have been used. H5N2 HPAI has been eradicated (last
isolate was in June 1995), but H5N2 LPAI [low pathogenic avian
influenza]  still circulates in central Mexico. Pakistan began using H7
inactivated AI vaccine in 1995, with use in 3 regions following epizootics
of H7N3 HPAI (1995, 2001 and 2004). By contrast, with low pathogenicity
(LP) AI, H9N2 inactivated vaccines have been and are used in many countries
within Asia, the Middle East and Eastern Europe, but the number of doses is
unknown. Vaccines for control of LPAI have been used sporadically.
Recently, H7 inactivated vaccine is being used in a high risk area of
Northern Italy and in one chicken layer company in the USA to control LPAI.

6. Historically, AI virus strains selected for manufacturing of inactivated
vaccines have been based on LPAI viruses obtained from field outbreaks that
have homologous hemagglutinin protein; i.e. H5 vaccine virus obtained from
an H5 LPAI outbreak. Rarely, HPAI strains have been used to manufacture
inactivated vaccines, because, to be done properly, such production
requires specialized high biocontainment manufacturing facilities which are
uncommon in the world. Contrary to rumor, HPAI strains do replicate to
sufficient titer in embryonating eggs to be used in inactivated AI
vaccines, but their use is discouraged because of biosecurity and biosafety
manufacturing concerns. Furthermore, LPAI strains, with fewer biosecurity
and biosafety concerns for manufacturing, protect against HPAI viruses of
the same hemagglutinin subtype.

7. Vaccine strains have been shown to provide protection against diverse
field viruses (88-100 percent similarity to the challenge virus
hemagglutinin) isolated over a 38 year period (Swayne et al., 2000b).
Recently, both North American and Eurasian lineages of AI vaccine viruses
from 1968-1986 have been shown to be protective against the most recent
2003-2004 Asian H5N1 HPAI viruses (Swayne, 2004). This broad and
longer-term protection efficacy of poultry AI vaccines, as compared to the
need for frequent change of human influenza vaccine strains, is potentially
the result of the following: 1) poultry vaccines use proprietary
oil-emulsion-adjuvant technology which elicits more intense and
longer-lived immune response in poultry than alum-adjuvant influenza
vaccines, 2) the AI virus immune response in poultry appears to be broader
than in humans, 3) the immunity in the domestic poultry population is more
consistent because of greater host genetic homogeneity than is present in
the human population, and 4) vaccine use in poultry is targeted to a
relatively young, healthy population as compared to humans, in whom the
vaccine is optimized for groups with the highest risk of severe illness and
death.

8. The protection efficacy of individual poultry AI vaccines should be
evaluated every 2-3 years to assure they are still protective against
circulating virus strains. For example, a recent study demonstrated the
1994 Mexican H5N2 vaccine strain is no longer protective against
circulating H5N2 LPAI viruses in Central America, and a change in vaccine
strains is needed (Lee et al., 2004a). With the H5N1 AI virus in Asia
circulating as only an HP strain, future vaccines may require the use of
reverse genetics (Liu et al., 2003; Lee et al., 2004b) to generate new LPAI
vaccine strains, or, other molecular techniques to produce vectored vaccine
products, such as new recombinant fowlpox virus vaccines. Some of these
products use patented technologies and will require legal clarification
before use in the field.

9. "Sterilizing immunity" is not feasible in the field. Some experiments
have reported "sterilizing immunity," but closer examination indicated such
studies used very few experimental birds without statistical evaluation,
used a very low virus challenge, or used low sensitive virus
isolation/detection methods. In the field, vaccines will reduce replication
of challenge virus in respiratory and GI tracts and thus reduce the
environmental load of virus and virus transmission. However, the protection
in conventional poultry in the field will always be less than that seen in
specific-pathogen-free poultry under laboratory conditions because of other
factors, such as improper vaccination technique, reduced vaccine dose,
immunosuppressive viruses and improper storage & handling of vaccines. The
other 4 components of a control strategy, as presented in item 1, are
essential, because vaccines and their use are not perfect.

10. Economics and animal health control drive the use of vaccines in
poultry. Vaccines are used in geographic areas of highest risk and in the
agricultural sector affected, or at greatest risk to be affected. In the
USA, inactivated AI vaccines cost on average USD 0.05/dose and another USD
0.05-0.07 for labor and equipment to administer. In examining new vaccine
technologies, such adoption will only occur when protection is as good as
or better than existing technologies, and the product is cost effective
(Swayne, 2004). If the cost is prohibitively high, the farmer or company
will not be able to use the vaccine.

11. When deciding to use AI vaccine in poultry, a simple animal health
algorithm, in decreasing order of application, should be used: 1) high risk
situations - e.g. as suppressor vaccine in the outbreak zone or as ring
vaccination outside the outbreak zone; 2) rare captive birds, such as those
in zoological collections; 3) valuable genetic poultry stock, such as pure
lines or grandparent stocks whose individual value is high; 4) long-lived
poultry, such as egg layers or parent breeders; and, lastly, 5) meat
production poultry.

12. Several issues which must be resolved before deciding to use AI
vaccines: 1) the vaccine strain must be of the same hemagglutinin subtype
and be shown in animal studies to be protective against the circulating
field virus, 2) standardized manufacturing of vaccines must be followed to
produce consistent and efficacious vaccines, 3) policies must be
established for proper storage, distribution and administration of the
vaccine; 4) adequate serological or virological surveillance must be done
to determine whether the field virus is circulating in vaccinated flocks;
and 5) an exit strategy must be developed to prevent permanent use of
vaccine. In addition, for inactivated whole AI vaccines, the following
should be addressed: 1) the need for adequate AI viral antigen content to
elicit a protective immune response, either by establishing a minimum
hemagglutinin protein content in the vaccine (e.g. minimum of 1-5
micrograms/dose if using generic adjuvant system, less antigen is needed if
a proprietary systems gives higher titers) or by demonstration of a high
level of protection as measured by _in vivo_ challenge studies or the
presence of a minimal hemagglutination inhibition (HI) antibody titer in
vaccinated birds (e.g. minimum of 1:32-1:40 HI test); 2) the need for a
good oil emulsion adjuvant system; and 3) the establishment of a high level
of biosecurity practice for vaccination crews that enter farms to prevent
accidental spreading of field virus. If using recombinant fowlpox vaccine,
the vaccine should only be administered to one day-old chickens in
hatchery, which will give good protection, improved biosecurity and a high
degree of quality control. Before new vaccine technologies should be used
in the field, assessment of safety in target species, environmental impact
to non-target species, purity and efficacy must be demonstrated.

13. Surveillance must be conducted on vaccinated flocks to determine
whether the field virus is circulating and the control strategy is working.
This should be done by both serological and virological surveillance of
vaccinated and non-vaccinated flocks. For serological surveillance, several
methods can be used to identify infections by field virus in vaccinated
populations: 1) placement of unvaccinated sentinel birds and looking for
antibodies against AI viruses, such as in ducks, 2) if using inactivated
vaccine, looking for specific antibodies against the neuraminidase of the
circulating field virus in vaccinated birds (if using an inactivated
vaccine strain with a different neuraminidase subtype than the circulating
field virus (Capua et al., 2003)) or looking for antibodies against the
non-structural protein (Tumpey et al., 2005), or 3) if using recombinant
fowlpox vaccine, looking for antibodies to nucleoprotein/matrix protein.
For virological surveillance, examination for specific AI viral nucleic
acids or proteins, or isolation of the virus, could be used to determine
whether the field virus is circulating. This is best done on sentinel birds
that are showing clinical signs or who die. Alternatively, examination of
dead poultry from vaccinated populations will give an indication of whether
the field virus is circulating.

14. Recently, 2 new vaccines for use in China have been reported (ProMED
20050207.0415 and 20050210.0456): 1) recombinant fowlpox-H5N1 AI vaccine,
and 2) a reverse genetic produced influenza A inactivated vaccine. The new
recombinant fowlpox vaccine is a live, injectable vaccine for chickens and
uses the same technology as the previously licensed
recombinant-fowlpox-virus-AI-H5 vaccine (cDNA copy of the AI hemagglutinin
gene from A/turkey/Ireland/83 [H5N8]), but includes inserted cDNA copies of
AI hemagglutinin (H5) and neuraminidase (N1) genes (both from
A/goose/Guangdong/3/96 [H5N1]) (Qiao et al., 2003). This type of vaccine
can only be used in one-day-old chickens and not in older birds in which
immunity to fowlpox virus will inhibit replication of the vaccine virus and
prevent development of effective immunity (Swayne et al., 2000a). The other
new vaccine is a traditional inactivated oil emulsion AI vaccine, but
unlike current inactivated AI vaccines, the new vaccine virus is not an H5
LP or HPAI field virus. The vaccine virus was produced by reverse genetics
using the 6 internal genes from a human influenza vaccine strain (PR8) and
the hemagglutinin and neuraminidase genes from A/goose/Guangdong/3/96
(H5N1) AI virus. The use of PR8 internal genes imparts the characteristic
of growth to high virus content in embryonating chicken eggs used in the
manufacturing process and thus produces a high concentration of the
protective hemagglutinin protein in the vaccine. Another change in the
vaccine virus: the portion of the gene that codes the hemagglutinin
proteolytic cleavage site has been changed from a sequence of an HP to an
LPAI virus, thus, the vaccine virus is a LPAI virus and can be manufactured
at a lower level of biosafety. Both vaccines require handling and injection
of individual birds. Data published or presented at scientific meetings
indicate that these new vaccines are as efficacious as the existing
licensed vaccines, but no data have been presented to demonstrate they
provide superior protection.

Selected References

1. Capua, I., Terregino, C., Cattoli, G., Mutinelli, F., Rodriguez, J.F.,
2003. Development of a DIVA (Differentiating Infected from Vaccinated
Animals) strategy using a vaccine containing a heterologous neuraminidase
for the control of avian influenza. Avian Pathol. 32 (1), 47-55.

2. Capua, I., Terregino, C., Cattoli, G., Toffan, A., 2004. Increased
resistance of vaccinated turkeys to experimental infection with an H7N3
low-pathogenicity avian influenza virus. Avian Pathol. 33 (2), 158-163.

3. Lee, C.W., Senne, D.A., Suarez, D.L., 2004a. Effect of vaccine use in
the evolution of Mexican lineage H5N2 avian influenza virus. J.Virol. 78
(15), 8372-8381.

4. Lee, C.W., Senne, D.A., Suarez, D.L., 2004b. Generation of reassortant
influenza vaccines by reverse genetics that allows utilization of a DIVA
(Differentiating Infected from. Vaccine 22 (23-24), 3175-3181.

5. Liu, M., Wood, J.M., Ellis, T., Krauss, S., Seiler, P., Johnson, C.,
Hoffmann, E., Humberd, J., Hulse, D., Zhang, Y., Webster, R.G., Perez,
D.R., 2003. Preparation of a standardized, efficacious agricultural H5N3
vaccine by reverse genetics. Virology 314 (2), 580-590.

6. Qiao, C.L., Yu, K.Z., Jiang, Y.P., Jia, Y.Q., Tian, G.B., Liu, M., Deng,
G.H., Wang, X.R., Meng, Q.W., Tang, X.Y., 2003. Protection of chickens
against highly lethal H5N1 and H7N1 avian influenza viruses with a
recombinant fowlpox virus co-expressing H5 haemagglutinin and N1
neuraminidase genes. Avian Pathol. 32 (1), 25-32.

7. Suarez, D.L., Schultz, C.S., 2000. Immunology of avian influenza virus:
a review. Dev.Comp.Immunol. 24 (2-3), 269-283.

8. Swayne, D.E., 2003. Vaccines for list A poultry diseases: emphasis on
avian influenza. Developments in Biologics (Basel) 114, 201-212.

9. Swayne, D.E., 2004. Application of new vaccine technologies for the
control of transboundary diseases. Developments in Biologics (Basel) 119,
219-228.

10. Swayne, D.E., Beck, J.R., Kinney, N., 2000a. Failure of a recombinant
fowl poxvirus vaccine containing an avian influenza hemagglutinin gene to
provide consistent protection against influenza in chickens preimmunized
with a fowl pox vaccine. Avian Dis. 44 (1), 132-137.

11. Swayne, D.E., Halvorson, D.A., 2003. Influenza. In: Saif, Y.M., Barnes,
H.J., Fadly, A.M., Glisson, J.R., McDougald, L.R., Swayne, D.E. (Eds.),
Diseases of Poultry, 11th edn. Iowa State University Press, Ames, IA, pp.
135-160.

12. Swayne, D.E., Perdue, M.L., Beck, J.R., Garcia, M., Suarez, D.L.,
2000b. Vaccines protect chickens against H5 highly pathogenic avian
influenza in the face of genetic changes in field viruses over multiple
years. Vet.Microbiol. 74 (1/2), 165-172.

13. Tumpey, T.M., Alvarez, R., Swayne, D.E., Suarez, D.L., 2005. A
diagnostic aid for differentiating infected from vaccinated poultry based
on antibodies to the nonstructural (NS1) protein of influenza A virus.
J.Clin.Microbiol. 43 (2), 676-683.

--
David E. Swayne, DVM, PhD
Laboratory Director
Southeast Poultry Research Laboratory
USDA/ARS