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1. Seed management
2. Method of manufacture
3. In-process control
4. Batch control
5. Tests on the final product
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The control of FMD is usually a national responsibility
and, in many countries, the vaccine may be used only when authorised.
Routine vaccination against FMD is used in many countries where the disease
is endemic. In contrast, a number of disease-free countries have never
vaccinated their livestock but have preferred the use of strict movement
controls and slaughter of infected and contact animals when outbreaks
have occurred. Nevertheless, many disease-free countries maintain the
option to vaccinate and have their own strategic reserves of highly concentrated
inactivated virus preparations. Such antigen reserves offer the potential
of supplying formulated vaccine in an ‘emergency’ at short
notice (16).
FMD vaccines are chemically inactivated cell-culture-derived preparations
of the virus that have been blended with a suitable adjuvant. In the case
of vaccines destined for use in swine, oil adjuvants are preferred.
Because of the presence of multiple serotypes of the virus, many FMD vaccines
are multivalent and it is common practice to prepare vaccines from two
or more different virus strains. In areas where the disease is maintained
by free-living buffalo, it is necessary to include more than one virus
per serotype to ensure broad antigenic coverage against prevailing viruses.
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1.
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Seed management
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a)
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Characteristics of the seed
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Selection of seed viruses should ideally be based on their ease
of growth in cell culture, virus yield, stability and broad antigenic
spectrum (32). The production strains should be characterised and
distributed by the official control laboratories; they should be
selected in accordance with the epidemiological importance of each
variant.
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b)
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Method of culture
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Many manufacturers of FMD vaccines derive their vaccine strains
from local field isolates and, for those grown in cell culture,
adapt them for growth in suspension or monolayer cells by serial
passage. The number of passages in cell culture should be kept to
a minimum as there is evidence of antigenic ‘drift’
of FMD virus during this procedure.
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c)
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Validation as a vaccine
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Seed viruses must be antigenically characterised and proven to
be free from contaminating microorganisms, to establish homology
to the original candidate isolates, purity and effectiveness against
the circulating strains for which they were developed. This often
encompasses a number of methods, but to establish applicability
to field strains a virus neutralisation is often used. Seed viruses
may be stored at -20°C if glycerinated or at a lower temperature
(e.g. -70°C) if not glycerinated. Working seed viruses may be expanded
in one or a few more passages from the mother seed stock and used
to infect the final cell culture at an approximate rate of 1 PFU
(plaque-forming unit) per 100 cells.
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2.
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Method of manufacture
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FMD virus is usually produced in either large-scale monolayer or
suspension cell systems under aseptic conditions. It is essential
that all pipework and vessels be thoroughly sterilised ensuring
that no areas in the system harbour microorganisms. In addition
to general considerations of sterility, it is important to note
that the virus is vulnerable to attack by proteolytic enzymes, such
as those produced by microorganisms (12). Control of pH and temperature
are also critical because of the acid and temperature lability of
the virus (11). Optimum temperature for cell, virus growth and inactivation,
normally around 37°C, should be precisely controlled. During other
stages of manufacture, the temperature should be reduced to 4-6°C.
Virus should be maintained at approximately pH 7.6 and should never
be below pH 7.0.
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A suitable strain of the virus is used to infect a suspension or
monolayer of a transformed cell line, such as BHK. Such cell cultures
should be free from contaminating microorganisms. It is common practice
to keep stocks of BHK cells over liquid nitrogen and revive as necessary.
On revival, they are expanded in nutrient medium to a volume and
cell density appropriate to seeding the main culture. As an approximation,
the main culture is seeded to give an initial density of 0.2-0.5
x 106 cells/ml, which is allowed to multiply to 2-3 x
106 cells/ml before being infected with virus.
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When the virus has reached its maximum titre, which is variously
determined by infectivity, CF or other tests, the culture is clarified
and filtered, often with centrifugation. The virus is subsequently
inactivated by addition of ethyleneimine (EI), usually in the form
of binary ethyleneimine (BEI). This is usually prepared by dissolving,
to a concentration of 0.1 M, 2-bromoethylamine hydrobromide in 0.2
N sodium hydroxide solution, and incubating at 37°C for 1 hour (4).
The BEI formed is then added to a virus suspension held at 20-37°C,
to give a final concentration of 0.001 M. Inactivation is usually
continued for 24 hours, followed by a second dose of EI for
a further 24 hours. After inactivation any residual BEI in the harvest
can be neutralised by adding sodium thiosulphate solution to a final
concentration of 2%. To decrease the likelihood of live virus failing
to contact the EI at the second application, it is essential to
transfer the vessel contents immediately to a second sterile vessel
where inactivation is allowed to go to completion at 48 hours.
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The inactivated virus may be concentrated by ultrafiltration, polyethylene
glycol precipition or polyethylene oxide adsorption (1, 38). These
concentrated antigens can be kept at -70°C or lower temperatures
for many years, if necessary, and made into vaccine when required
by dilution in a suitable buffer and addition of adjuvants (14).
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Conventional FMD vaccines are usually formulated in one of two
ways. The vaccine most commonly used for cattle is prepared by adsorbing
the virus on to aluminium hydroxide gel, one of the adjuvant constituents
of the final vaccine blend. Other components of the final blend
include antifoam, phenol red dye (if permitted by the country requiring
vaccine), lactalbumin hydrolysate, tryptose phosphate broth, antibiotics,
amino acids, vitamins and buffer salts. A second adjuvant, saponin,
derived from the South American tree Quillaja saponaria mollina,
is also incorporated, as well as merthiolate/chloroform as a preservative.
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An alternative formulation uses mineral oils, such as Marcol and
Drakeol, as adjuvants. These preparations offer a number of advantages
over the standard aluminium hydroxide/saponin vaccine, not least
of which is their efficacy in pigs. They are widely used for vaccinating
cattle in South America because of the longer duration of immunity
obtained. The mineral oil is usually premixed with an emulsifying
agent, such as mannide monooleate, before the addition of an equal
volume of the aqueous phase of the vaccine, and emulsified by use
of a colloid mill or continuous mechanical or flow ultrasonic emulsifier.
More complex double emulsions (water/oil/water) may be produced
by emulsifying once more in an aqueous phase containing a small
amount of Tween 80 (25).
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Significant advances made in recent years have seen the introduction
of alternative ‘ready-to-use’ oil adjuvants. Oils containing
esters of octadecenoic acid and 2,5 anhydro-d-mannitol, for example,
readily form double or mixed emulsions (water/oil/water), that are
both stable and of low viscosity, without the requirement of sophisticated
emulsification equipment (5, 16).
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3.
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In-process control
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In general, virus titres reach optimum levels within about 24 hours
of the cell culture being infected. The time chosen to harvest the
culture may be based on a number of assays; for instance cell death.
Virus concentration may be assessed by plaque assay, sucrose density
gradient (13) or serological techniques. It is preferable to use
a method for measuring antigenic mass, such as sucrose density gradient
analysis, as well as one that measures infectivity, as the two properties
do not necessarily coincide and the different methods may complement
one another.
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During inactivation of the virus, timed samples should be taken
at regular intervals for the purpose of monitoring the rate and
linearity of the inactivation process. Virus titres in the samples
are determined by inoculation of cell cultures proven to be highly
susceptible to FMD virus, e.g. BHK or bovine thyroid cells. Such
cultures permit the testing of statistically meaningful samples
under reproducible conditions. The log10 infectivities
of the timed samples are plotted against time, and the inactivation
procedure is not considered to be satisfactory unless at least the
latter part of the slope of the line is linear and extrapolation
indicates that there would be less than one infectious particle
per 104 litres of liquid preparation at the end of the
inactivation period.
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4.
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Batch control
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a)
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Sterility
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The bulk inactivated antigen, the adjuvants, the dilution buffers
and the final formulated product should all undergo sterility testing.
This may be carried out directly with components of the vaccine
or the final product, but the preferred method is to collect any
contaminating microorganisms by membrane filtration of the material
to be examined and to detect them by incubation of the membranes
with culture media. The latter procedure allows the removal of preservatives,
etc., which may inhibit the detection of microorganisms. Guidelines
on techniques and culture media, which allow the detection of a
wide range of organisms, are described in the European Pharmacopoeia
1997 (ref. 19; also refer to Chapter I.1.4.).
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b)
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Safety
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Following inactivation, a sample of each batch of inactivated antigen
representing at least 200 doses should be tested for freedom from
infectious virus by inoculation of sensitive monolayer cell cultures,
preferably of the same origin as those used for the production of
antigen. It may be preferable to concentrate the antigen to do this,
in which case it must be shown that the concentrated material does
not interfere with the sensitivity or reading of the assay. The
cell sheets are examined daily over a period of 3 days, after which
the spent medium is transferred to fresh monolayers and the original
monolayers replenished with fresh medium. Using this method, traces
of live virus can be amplified by the passage procedure and detected
on the basis of CPE observed. Two to three passages of the original
virus preparation are commonly used. A variant on this method is
to freeze/thaw the old monolayers to release intracellular virus,
which can be detected by further passage.
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c)
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Potency
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Potency is only examined on the final formulated product (see Section
B.5.b.).
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d)
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Duration of immunity
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In order to establish a satisfactory level of immunity it is usual
to give a primary course of two inoculations, 2-4 weeks apart, followed
by revaccination every 4-12 months. The frequency of revaccination
will depend on the epidemiological situation and the type and quality
of vaccine used. Where access to the animals is difficult, it is
preferable to use oil adjuvanted vaccine at 4 months and 1 year
of age, followed by annual revaccination.
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For calves born of vaccinated dams, the first vaccination should
be delayed as long as possible to allow decline of maternal antibody,
but not beyond 4 months, as at that time a high proportion can be
expected to respond effectively to vaccination. For calves born
of nonvaccinated dams, the first vaccination may be at 1 week
of age (3).
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e)
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Stability
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The shelf life of conventional FMD vaccines is usually 1-2 years
at 4°C, but they are temperature labile and should neither be frozen
nor stored above 4°C.
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f)
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Preservatives
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The most commonly used preservatives are chloroform and merthiolate.
The latter is used at a final concentration of 1/30,000 (w/v).
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g)
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Precautions (hazards)
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Current FMD vaccines are innocuous and present no toxic hazard
to the user. Care must be taken to avoid self-injection with oil-emulsion
vaccines.
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5.
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Tests on the final product
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a)
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Safety
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It is necessary to test FMD vaccines to ensure that the final product
is noninfectious and is not unduly toxic. Some laboratories determine
noninfectivity by eluting the virus from the vaccine, but this is
not universally applicable to all formulations. For example, saponin
influences greatly the elution of FMD from aluminium hydroxide/saponin
vaccines (15). If the elution procedure is appropriate to a particular
formulation, then it may be validated by seeding parallel samples
of vaccine with trace amounts of live virus (6).
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Toxicity and noninfectivity may be assessed simultaneously in an
in-vivo test in cattle (18). Each of three healthy seronegative
cattle are inoculated intradermally on the dorsal surface of the
tongue with 0.1 ml of vaccine at 20 sites (four rows with five
inoculation sites each). The animals are observed for no fewer than
4 days, after which three full bovine doses of vaccine are
administered by the manufacturer’s recommended route to each
animal. The animals are observed for a further 6 days. Should any
of the animals develop signs of FMD, the vaccine will fail the safety
test. Equally, any undue toxicity attributable to the vaccine should
be assessed and may prevent its acceptance. Ideally, vaccines prepared
for species other than cattle should be safety tested in the species
for which they are intended, administering a double dose of vaccine
according to the manufacturer’s recommended route and dose
volume. The animals should be examined daily for a minimum of 7
days for evidence of toxicity or signs of FMD.
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Provided that laboratory rodents have been shown to be a satisfactory
alternative to the target species for toxicity testing of FMD vaccines,
these may be used to assess toxicity (17). The test involves the
subcutaneous inoculation of two guinea-pigs and five mice with 2 ml
each and 0.5 ml each of vaccine, respectively. The animals
are observed for 7 days and the test is considered to be satisfactory
if none of the animals dies or shows significant local or systemic
reaction.
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b)
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Potency
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Cattle of at least 6 months of age, obtained from areas free from
FMD, that have not previously been vaccinated against FMD and are
free from antibodies neutralising the different types of FMD virus
should be used. Three groups of no fewer than five cattle per group
should be vaccinated by the route recommended by the manufacturer.
The vaccine should be administered at different doses per group
by injecting different volumes of the vaccine. For example, if the
label states that the injection of 2 ml corresponds to the
administration of 1 dose of vaccine, a 1/4 dose of vaccine would
be obtained by injecting 0.5 ml, and a 1/10 dose would be obtained
by injecting 0.2 ml. These animals and a control group of two
nonvaccinated animals are challenged 3 weeks after vaccination with
a suspension of bovine virus that is fully virulent and appropriate
to the virus types in the vaccine under test by inoculating 10,000
ID50 intradermally into two sites on the upper surface
of the tongue (0.1 ml per site). Animals are observed for 8-10
days. Unprotected animals show lesions at sites other than the tongue.
Control animals must develop lesions on at least three feet. From
the number of animals protected in each group, the PD50
(50% protective dose) content of the vaccine is calculated. The
vaccine should contain at least 3 PD50 per dose for cattle,
when employed for routine prophylactic use, or 6 PD50
per dose when used for emergency (‘ring’) vaccination.
In some cases, vaccine of high potency will prevent the development
of local tongue lesions at the site of challenge. In South American
countries a variation of the potency test is performed, the PGP
test (percentage of protection against generalised foot infection).
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Potency tests in other target species, such as sheep, goats or
buffalo are not common, as a successful test in cattle is considered
sufficient to endorse its use in other species. Under circumstances
where a vaccine is produced for use primarily in one particular
species, it may be more appropriate to potency test the vaccine
in that same species. However, in respect to the limited data for
African buffalo or Asiatic buffalo (Bubalus bubalis) and
sheep, and the often inapparent nature of the disease in these species,
potency results from a cattle test should be a good indicator of
the vaccines applicability in these other species.
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A similar protocol to the cattle test can be adopted for potency
testing FMD vaccines in pigs. Using three groups of five pigs, one
group is vaccinated with the full pig dose recommended by the manufacturer,
one group receives a 1/4 dose, and a third group receives a 1/16
dose of vaccine. Traditionally, the response to oil vaccine is allowed
longer to develop, and not until day 28 after vaccination are the
three groups, plus two unvaccinated control pigs challenged. Challenge
is by intradermal injection into the heel bulbs of one foot with
10,000 TCID50 (0.2 ml) of a virulent challenge virus
homologous to a strain used in the vaccine. The animals are observed
daily for 10 days after challenge for clinical signs of FMD, but
animals are removed as soon as they develop generalised FMD to avoid
excessive challenge to those remaining. Both control animals should
develop clinical signs on more than one foot. Probit tables are
used to determine the potency of the vaccine under test. The vaccine
should contain at least 3 PD50 per dose for pigs.
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Other tests, including measurement following vaccination of virus
neutralising antibodies in cell culture, or ELISA antibodies, or
serum-protecting antibodies in suckling mice, may be used to assess
the potency of a vaccine provided that a statistical evaluation
has established a satisfactory correlation between the results obtained
by the test on the relevant vaccine serotype and the potency test
in cattle (37). For example, the expected percentage of protection
is used to analyse the sera of a group of at least 16 vaccinated
cattle and to express the probability of an animal being protected
by measuring neutralising, ELISA or protecting antibodies. In a
single group of animals given a half dose of vaccine, the expected
percentage protection should be equal to or greater than 80%.
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The presence of more than one serotype in a vaccine does not interfere
with the induction of antibodies against another serotype or the
correlation of antibody titre with protection.
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