D8271.PDF

Rev. sci. tech. Off. int. Epiz., 1990, 9 (3), 779-794
Biotechnology and veterinary science:
production of veterinary vaccines
C. WRAY and M.J. WOODWARD *
Summary: The fundamental principles of genetic manipulation are explained,
as are the methods ofproduction of vaccines of veterinary importance. Specific
attenuation of micro-organisms may result from
genetically-engineered
mutations which produce metabolic blocks, from the development of effective
bacterial toxoids and from viral gene segment reassortment. Recombinant DNA
techniques have been used to produce multi-component vaccines by the insertion
of genes into carrier organisms, such as pox viruses and attenuated salmonella,
and also to produce subunit vaccines. The use of synthetic peptide vaccines and
anti-idiotype vaccines, together with problems of parasite vaccines, are described.
Biotechnology would appear to have a major role to play in the future prevention
of many animal diseases.
KEYWORDS: Attenuation - Bacteria - Biotechnology - Genetic manipulation Parasites - Recombinant DNA - Subunit vaccines - Virus.
INTRODUCTION
Biotechnology m a y be defined as the application of scientific and engineering
principles t o the processing of materials by biological agents to produce goods and
services. There is nothing new a b o u t biotechnology: m a n has used these techniques
for thousands of years to manufacture products such as beer, wine and bread. W h a t
is new, however, is the increased capabilities of biotechnology since the development
of genetic engineering, more correctly termed recombinant D N A technology, and
monoclonal antibody techniques.
Although vaccines against m a n y animal diseases have been available for m a n y
years, their efficacy has varied. Some of the available bacterial vaccines, such as
clostridial toxoids, have been very successful; others, such as those prepared against
Gram-negative organisms, have been less successful because of their poor
immunogenicity and the occurrence of fatal adverse reactions. Likewise, some viral
vaccines, such as t h a t against rinderpest, have been very successful, whereas others
have shown poor attenuation and stability. Some viral vaccines have been inadvertently
contaminated with other agents, and m a n y of the recent E u r o p e a n outbreaks of foot
and m o u t h disease resulted from inadequately inactivated vaccine.
* Ministry of Agriculture, Fisheries and Food, Central Veterinary Laboratory, New Haw, Weybridge,
Surrey KT 15 3NB, United Kingdom.
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The purpose of this article is t o show briefly how the techniques of biotechnology
m a y be used to produce vaccines. It is not our intention to compile an exhaustive
list of all k n o w n veterinary vaccines. Biotechnology also provides methods that can
lead to a greater understanding of microbial virulence and the nature of protective
immunogens, b o t h of which are important for vaccine development.
GENETIC MANIPULATION
The information needed for cell growth and metabolism is encoded by genes which
are made from a simple chemical alphabet within long sequences of deoxyribonucleic
acid (DNA). T h e D N A consists of two strands each with a backbone of alternate
sugar (deoxyribose) and phosphate groups. Linking the two strands in the form of
a double helix are pairs of the bases adenine (A), guanine (G), cytosine (C) and thymine
(T). E a c h base is attached to a sugar molecule in one strand, and the two strands
of the double helix are linked together by complementary base pairing (that is,
A with T and C with G). The sequence of these bases along the D N A strand is the
code from which specific proteins and enzymes are synthesised, each of the numerous
amino acids that m a k e u p a protein molecule being encoded by a sequence of three
bases.
During reproduction, genetic information from b o t h parents may be reassorted.
Bacteria, however, reproduce by binary fission resulting in clonal populations. Gene
transfer in bacteria m a y also occur by the following mechanisms:
a) Transformation: the entry of naked D N A into bacteria. This usually requires
some pretreatment of bacterial cells, for example, exposure to high concentrations
of calcium ions.
b) Transduction: the transfer of genetic information by bacterial viruses
(bacteriophages) which pick u p host D N A by accident and transfer it to the host cell.
c) Conjugation: a sexual process involving union between two cells, during which
the d o n o r passes D N A to the recipient. Plasmid transfer occurs in this manner.
These mechanisms usually operate only between bacteria of the same or related
species, but following a series of fundamental discoveries in the 1960's it has become
possible to transfer genetic material across these boundaries by the technique known
as "genetic engineering".
Several stages are involved. D N A is first carefully extracted from the source tissue
or organism, then cleaved by one of a group of bacterial enzymes called restriction
endonucleases. These enzymes, the natural role of which is to destroy foreign DNA
entering a bacterial cell, cut D N A in a predictable manner by recognising a specific
sequence of bases in the double helix. D N A fragments are thereby produced. For
example, the enzyme EcoRI recognises the sequence:
5' - G
AA TT
3' - C
TT AA
C - 3'
G - 5'
i
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and cuts the D N A strands at the points indicated by the arrows. More than 500 restriction
enzymes have been discovered, which cut at different target sites (Table I). D N A cut
by these enzymes may have short, overhanging single-stranded D N A ends called cohesive
termini or 'sticky ends'. This means that the projection incorporating the four terminal
bases (AATT in the above example) can be joined in vitro, by the enzyme D N A ligase,
to another D N A molecule from another completely different source, which has also
been cut by the same restriction enzyme (in this case EcoRI). Thus a hybrid D N A
molecule incorporating a 'foreign' sequence of base pairs can be produced.
TABLE I
Some examples of restriction
enzymes
showing their cleavage sites
Micro-organism
producing the enzyme
Name of
enzyme
Recognition
sequence
Escherichia coli
EcoRI
Haemophilus
Haelll
GAATTC
CTTAAG
GGCC
CCGG
TGGCCA
ACCGGT
GTCGAC
CAGCTG
AAGCTT
TTCGAA
aegyptius
Brevibacterium albidium
Ball
Streptococcus albus
Sal I
Haemophilus
influenzae
Hind III
Sequences after
cleavage
G
CTTAA
GG
CC
TGG
ACC
G
CAGCT
A
TTCGA
AATTC
G
CC
GG
CCA
GGT
TCGAC
G
AGCTT
A
D N A from bacterial pathogens can be extracted from cells fairly easily and purified
without significant change. A different approach is necessary, however, for cloning
materials from some viruses and eukaryotic pathogens, which possess nuclear
membranes. M a n y viruses d o not use D N A to encode genetic information but use
a similar long-chain molecule, ribonucleic acid (RNA). R N A differs from D N A in
that it is composed of a different sugar, ribose, and one different base, uracil, instead
of thymine. Since R N A itself cannot be cloned, it must first be converted t o D N A
using the retrovirus enzyme, reverse transcriptase, which synthesises complementary
DNA (cDNA) from an R N A template. Once single-stranded c D N A is synthesised,
the R N A can be removed by alkaline hydrolysis and a complementary second strand
of D N A can be produced by means of another enzyme, D N A polymerase; the second
strand becomes a D N A version of the original R N A sequence. After the addition
of short synthetic linker D N A molecules containing restriction enzyme sites, the
double-stranded D N A product can then be ligated to a plasmid or other suitable vector.
Cloning of eukaryotic cells demands a slightly different approach, because the genes
are frequently interrupted by D N A sequences called introns that are transcribed into
messenger R N A (mRNA) during gene expression and then edited from the m R N A
molecule before translation into proteins. Since bacterial recipients such as Escherichia
coli cannot excise introns from m R N A , edited m R N A must be isolated and reversetranscribed t o obtain uninterrupted D N A copies of eukaryotic genes which can be
attached to bacterial regulatory elements for expression. It is also possible chemically
to synthesise chains of nucleotides, or even entire genes, in the laboratory.
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Having obtained a hybrid D N A molecule in vitro, the next step is to transfer the
D N A into a bacterium or other suitable host cell and persuade it to produce the gene
p r o d u c t . In bacteria, this can be achieved by the use of bacterial viruses or plasmids
(Table II). T h e simplest self-replicating unit is the plasmid, which is a small circle
of D N A encoding very few genes. The circle can be opened by restriction enzyme
digestion a n d a new fragment inserted by ligation to complete the circle once more.
It is possible to tailor the insert so that it is placed under the control of a promoter,
a gene switch, which can t u r n on the gene, resulting in its expression and in the
production of the protein for which it codes.
TABLE II
Characterisation
of vectors for cloning recombinant
DNA
Class of vector
Size of vector DNA (kb)
Maximum size of
foreign DNA insert (kb)
Plasmid
Phage
Cosmid
3-15
about 50
25
15
25
50
A n alternative vector is a bacterial virus (phage), whose D N A is larger than plasmid
D N A ; consequently, it is m o r e difficult to manipulate. However, phages will
a c c o m m o d a t e a larger D N A insert and the recombinant D N A will replicate many
hundred-fold in a single bacterial cell, emerging with a complete virus particle which
will reinfect surrounding bacteria and so repeat the cycle of amplification.
A third class of vector is a synthetic hybrid of plasmid and phage k n o w n as a
cosmid. It comprises the D N A insert to be cloned plus two plasmid elements, an
antibiotic resistance gene and a short sequence required to start the process of DNA
copying in the bacterial host. The cohesive end sequences are phage-derived and they
are necessary for circularising the D N A in the bacterial host, itself a prerequisite for
replication, and for packaging the D N A in a viral capsid. These vectors are ideal
for preparing gene libraries because they may carry inserts as large as 50 kb (that
is 50,000 base pairs of double helix D N A ) . Other viruses and D N A viruses such as
adenovirus and SV40 which have been modified, e.g. small R N A retroviruses, are
used to infect animal cells and viral tissue cultures.
Efficient expression of cloned genes requires both genetic and biochemical
compatibility within the recipient cell. While the genetic code is the same for all
organisms, the DNA-encoded signals that control gene transcription and translation
vary from one organism to the next and these signals must be recognised by the
host cell if protein synthesis is to proceed. Consequently, much ingenuity has gone
into the development of suitable cloning vectors, which now include multiple
restriction endonuclease sites located adjacent to promoters, and genes for antibiotic
resistance which can be used to select for the presence of recombinant regulatory
sequences which influence the expression of the inserted D N A fragment within the
host cell.
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Invertebrate virus expression vectors have been developed for the expression of
foreign genes in insect cells (18). Autographa californica nuclear polyhedrosis virus
(AcNPV), the prototype baculovirus, has a wide host range a n d infects m o r e t h a n
thirty species of Lepidoptera. The baculovirus utilises the polyhedrin promoter, which
has been modified so that the insertion of prokaryotic and eukaryotic genes produces
recombinant proteins. O n e of the major advantages of this vector over bacteria a n d
yeast expression systems is an a b u n d a n t expression of recombinant proteins, which
are in many cases antigenically and functionally similar to their authentic counterpart.
Many proteins are naturally glycosylated. This system is particularly useful because
the expression of foreign genes in the insect cells often results in near-native
glycosylation.
Host cells for gene expression must be found which are suitable for expression
of the recombinant protein. While E. coli is probably the most commonly used host,
other prokaryotes such as Bacillus subtilis, and simple eukaryotes such as the yeast
Saccharomyces cerevisiae, have also been used. T h e gene encoding the hepatitis B
surface antigen was cloned and expressed in E. coli, but expression of the highly
immunogenic 22 n m particulate form of this antigen was obtained only when the
gene had been inserted into recipient yeast cells (30).
VACCINE PRODUCTION
A number of techniques can be used for the development of new vaccines. In
some cases recombinant D N A technology provides greater specificity t h a n the
empirical approaches of years past, and in other cases the concept is completely novel
(Table III).
TABLE III
Biotechnology
for vaccine
production
Specific attenuation
Bacterial vaccines
a) Mutations - metabolic blocks
b) Toxoid vaccines
Viral vaccines
a) Recombinations and gene segment reassortments
b) Mutations
Recombinant DNA techniques
a) Subunit vaccines
b) Subunit vaccines in heterologous carriers
Viral carriers
Bacterial carriers
Synthetic peptides
Anti-idiotype vaccines
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SPECIFIC A T T E N U A T I O N
It has been known for m a n y years that immunisation with live attenuated
organisms can be very effective for producing immunity. Attenuation has been
achieved by selecting strains of reduced virulence (in the case of bacteria, for example,
rough or semi-rough variants), by repeated passage in the laboratory or by growing
the organisms under growth-limiting conditions. Live vaccine strains selected in such
a m a n n e r have always h a d the disadvantage that they tend to revert to virulence.
BACTERIAL VACCINES
Mutations -
metabolic blocks
Cell wall deficient mutants of Salmonella dublin and Salmonella gallinarum which
are partially rough strains, have been shown to be effective vaccines in calves (24)
and poultry (25) for preventing salmonellosis. However, the S. dublin vaccine causes
swelling at the site of inoculation and the occasional fatal allergic reaction. More
recently, galE m u t a n t s of salmonella with genetic defects created by chemical
mutagenesis have been evaluated for their utility as vaccines. These organisms have
a lesion which affects galactose metabolism, such that they grow normally in the
absence of galactose, but the presence of galactose in the animal leads to their lysis
(11). However, they survive long enough t o produce strong immunity.
Auxotrophic m u t a n t s , also obtained by chemical mutagenesis, have defects which
render them dependent on metabolites not present in the vaccinated animal. Examples
of such m u t a n t s are the purine-requiring (pur) salmonella strains which are
commercially available in the G e r m a n Democratic Republic (19). These strains also
possess a second attenuation marker, usually histidine auxotrophy (his), and a
detection marker to distinguish them from wild-type strains.
A n alternative approach to the production of attenuated auxotrophs has been the
construction of aroA m u t a n t s (14). The aroA gene is one of several genes encoding
enzymes which form the pre-chorismate biosynthetic pathway, the only route by which
aromatic compounds can be synthesised in bacteria. This pathway is absent in animal
cells. The use of aro A mutants as vaccines for farm animals has given good results (26).
A n o t h e r approach to selecting attenuated strains with previously characterised
mutations is to introduce r a n d o m mutations into the genome by means of transposons
and to screen the resulting bacteria for attenuation. The transposon TnphoA contains
a coding region for bacterial alkaline phosphatase. This transposon is constructed
in such a way that if it inserts into a gene for a secreted protein, active alkaline
phosphatase is secreted; a simple chromagenic substrate in the plating medium
differentiates between strains producing active and inactive alkaline phosphatase (28).
T h u s , TnphoA can be used t o identify genes encoding secreted proteins. Deletion
mutations, usually induced by UV-irradiation, are very stable and m a y be used
preferentially.
Toxoid vaccines
In bacteria, small deletions within toxin genes m a y be used to produce toxoid
vaccines which react immunologically the same way as toxin, but lack the active site
of toxicity. Enterotoxigenic E. coli (ETEC) are important causes of diarrhoea and
785
possess two m a i n virulence determinants - an adhesin, which allows the organism
to adhere to the mucosa, and enterotoxins, which are responsible for fluid loss. T h e
enterotoxins m a y b e of two types, a heat-stable toxin (ST), which is a small peptide
and poorly immunogenic, and a heat-labile toxin (LT), which is highly immunogenic.
LT consists of two subunits, the enzymically active A subunit, which is responsible
for the toxic activity, and an immunogenic B subunit, which is responsible for binding
the A subunit to the epithelial cells of the intestine. The toxin is not readily purified
by conventional means and cannot be converted to a toxoid. The L T genes were
cloned, the coding sequences within the A subunit gene deleted and a powerful E. coli
promoter introduced in front of the B subunit gene. This recombinant D N A was
introduced by transfection into a non-pathogenic laboratory strain of E. coli by using
a high copy n u m b e r vector. High levels of B subunit were produced in the form of
a toxoid which, when used as a vaccine, conferred protective immunity against
LT-producing E. coli (7).
Likewise, tetanus toxoid has been produced by cloning portions of the tetanus
toxin gene into E. coli and determining the entire D N A sequence of the gene. Using
E. coli expression vectors, E. coli strains were produced which expressed portions
of the tetanus polypeptide containing the non-toxic but highly immunogenic
C-fragment. These portions protected mice against challenge with tetanus toxin
(8).
VIRAL VACCINES
Recombinations and gene segment reassortments
Viruses with segmented genomes, such as influenza and rotavirus, have the natural
capability to undergo reassortment of segments following viral co-infection. Highly
attenuated temperature-sensitive m u t a n t s of influenza virus or avian strains of the
virus, which do not replicate efficiently in m a m m a l s , have been used with virulent
virus strains to co-infect cell cultures. Some reassortments can be isolated which
contain the six internal genes from the avirulent parent and the two genes for the
surface proteins H A and N A of the wild-type virus. Such reassortments are also being
developed for equine influenza virus (21).
T h e genes of rotaviruses can also undergo reassortment during mixed infections.
The reassortment of the major surface antigen (VP7) of h u m a n rotavirus into bovine
or rhesus rotavirus has been used t o produce a strain having reduced growth
characteristics, which is being evaluated as a vaccine (12).
Mutations
Deletion has also been used t o produce attenuated strains of the herpes
group of viruses. Missense m u t a n t s of influenza and respiratory syncytial viruses
have been produced; temperature-sensitive strains of these viruses, although
attenuated for growth at high temperatures, were found to be genetically unstable
(5).
Another approach has been to select attenuated virus with monoclonal antibodies.
Rotavirus mutants selected in this way contain single amino-acid substitutions although
other viral m u t a n t s (e.g. rabies), selected in a similar m a n n e r , were unstable and
reverted t o virulence (9).
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RECOMBINANT D N A TECHNIQUES
Subunit vaccines
Genes from many infectious agents have been cloned and expressed in prokaryotic
and eukaryotic cells by using recombinant D N A techniques. Hepatitis B virus (HBV)
vaccine is perhaps the most successful vaccine produced so far with these techniques.
The current licensed HBV vaccine which contains purified 22 run H B surface antigen,
is m a d e from plasma of h u m a n carriers and is costly to prepare. Although hepatitis
B surface antigen (HBsAG) could be synthesised in E. coli, the yields in this host
were p o o r . However, when the gene was transferred into yeast
(Saccharomyces
cerevisiae), immunogenic particulate forms of H B s A G , which closely resembled those
found naturally, were produced and readily extracted (30). Evaluation of the utility
of this preparation as a vaccine is underway.
Other viral genes which have been cloned and expressed include those encoding
the VP1 protein of foot and m o u t h disease virus (FMDV), influenza A virus
haemagglutinin ( H A ) , rabies virus glycoprotein and herpes simplex glycoprotein. In
the case of F M D V , the sequence coding for V P 1 , one of the viral proteins against
which neutralising antibody is directed, was cloned in E. coli and expressed as a fused
polypeptide. However, the polypeptide prepared in this way had low efficacy as a
vaccine, apparently because the VP1 polypeptide obtained by cloning is folded
differently from the V P 1 polypeptide in its native configuration in the viral coat,
and the immunogenicity is thereby altered (16). Likewise, although a number of
different viral proteins have been expressed successfully, the level of immunity they
induce has been low and much more work is necessary to produce stable immunogenic
products.
Enterotoxigenic E. coli possess somatic pili composed of 14 t o 22 k D a protein
molecules that enable the organism to adhere to the host's mucosa. Immunologically
distinct pili have been isolated from E T E C , such as K88 and 987P from swine and
K99 from cattle, sheep and swine. Vaccines based on purified preparations of these
fimbriae have been used in veterinary medicine for a number of years. In producing
the first commercial recombinant D N A (rDNA) vaccine, a high copy number plasmid
was utilised to increase the yield of the antigen (K88). However, the use of D N A
technology to construct pilus vaccines is complicated by the fact that several genes
are necessary for the transport and processing of the pilus subunit to its final site.
Bacteroides nodosus, the cause of ovine footrot, possesses fimbriae which consist
of a single repeated structural subunit of about 18 kDa. When these genes were cloned
into E. coli, the subunits were not assembled into mature fimbriae but remained
embedded in the cell membrane. However, by cloning into Pseudomonas
aeruginosa,
which possesses compatible type 4 fimbriae, high levels of fimbriae were produced.
The haemagglutinin of fowl plague virus was expressed in larvae of Heliothis
viruscens by using recombinant baculovirus, Autographa
californica
nuclear
polyhedrosis virus (AcNPV), as a vector. The larvae were infected with the
recombinant virus either by parenteral injection or by feeding. T h e a m o u n t of
haemagglutinin synthesised in larvae after parenteral infection was 0 . 3 % of the total
protein as compared with 5 % obtained in cultured insect cells. Likewise, the
haemagglutinin from larvae was found to be only half as active as that from vertebrate
or insect cell culture. However, chickens immunised with larval tissues containing
haemagglutinin, when challenged with the virus, were protected from infection (17).
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Subunit vaccines in heterologous carriers
Virus
carriers
Vaccinia virus, adenovirus and herpes virus have been used as carriers, but in
general m a n y viruses are unsuitable because of a restricted capacity for foreign D N A
and limited growth capacity. Vaccinia virus and related pox viruses, however, retain
infectivity even after a c c o m m o d a t i o n of u p t o 25,000 base pairs of foreign D N A
(equivalent to 20 average genes) in the viral genome. T h e principle of construction
is that foreign D N A flanked by vaccinia D N A sequences within a plasmid vector can
be introduced by transfection into virus-infected cells. Recombination between plasmid
and virus creates recombinant vaccinia virus which express antigens from other viruses.
Also, these recombinant viruses tend to be less virulent t h a n the parental vaccinia
virus in experimental animals and have the potential to produce b o t h cellular a n d
humoral immunity. Vaccinia virus recombinants carrying hepatitis B surface antigen
gene have been produced which express high levels of surface antigen in the
immunologically active, 22 n m particle form (23).
A recombinant vaccinia virus expressing rabies G glycoprotein has been developed
(15). This virus produced high levels of virus neutralising antibodies when fed to foxes,
raccoons and skunks, and conferred long-term protection (4). A hybrid vaccinia virus
expressing the antigens of influenza virus, hepatitis B virus and herpes hominis virus
type 1 elicited antibodies against all three when inoculated into rabbits. Whilst vacciniabased vaccines have given good results in experimental animals, the use of such
vaccines in h u m a n s is controversial because vaccinia itself m a y produce adverse
reactions and complications at a frequency of 1:1000. Likewise, repeated inoculation
with vaccinia virus cannot be used because this will induce immunity to the vaccinia
vector. Consequently, other carriers are being examined.
At present fowl-pox virus appears to be the candidate of choice because its host
range is limited and consequently it is unable to multiply in non-avian hosts. Rabies
glycoprotein genes and avian influenza genes incorporated into the fowl pox genome
are correctly expressed in avian and non-avian cells and, in the latter, poxviral particles
are not produced (27).
Bacterial
carriers
The attenuated salmonella strains mentioned previously are also being investigated
as to their suitability as carriers of heterologous genes. Depending o n the strain of
salmonella, a variety of i m m u n e responses in the host, ranging from local to cellmediated, can be produced. Because salmonella and E. coli belong to the same
bacterial family, foreign genes expressed in E. coli can be propagated in salmonella
vaccine strains, usually by means of bacteriophage. Antigens from bacteria (3, 6),
viruses (29) a n d parasites (22) have all been expressed in salmonella. T h u s , a plasmid
from Shigella sonnei has been introduced into the galE typhoid vaccine (Ty21a)
and shown to be safe in volunteers. T h e gene coding for the E. coli heat labile
B subunit toxin has been introduced in salmonella aroA m u t a n t s and shown to
induce an anti-LT-B secretory i m m u n e response and also the production of serum
antibodies. The mice immunised with these constructs are also i m m u n e to
subsequent challenge with virulent salmonella. Likewise, the nucleoprotein of
influenza virus has been introduced into S. typhimurium, where it is expressed at
a high level.
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SYNTHETIC PEPTIDES
Only a small portion of an antigen is necessary to stimulate an immune response
as long as it is immunogenic or has been m a d e immunogenic by being linked to a
carrier. Individual antibodies recognise the small regions of the antigen known as
epitopes, and if the structure of the epitope is known, it is possible to synthesise it
biochemically. Synthetic peptides whose sequences correspond to epitopes recognised
by antibodies, or sequences which are essential for the function of a particular protein,
have been manufactured and used to immunise animals.
The first peptide immunogen, corresponding to the VP1 polypeptide of FMDV,
stimulated the development of neutralising antibodies in guinea pigs. It was, however,
necessary to couple the peptide t o a suitable carrier (keyhole limpet haemocyanin)
and t o use an adjuvant (2). Even so, the immunogenicity was only 1-10% of that
of the whole inactivated virus.
However, recent studies have found that the immunological non-responsiveness
to F M D V peptide can be overcome by addition of 'foreign' helper T-cell determinants
from ovalbumin or sperm-whale myoglobin (10). It was found, however, that the
virus-neutralising activity of the antibody raised against peptide was dependent on
the specific peptide sequence used. T h u s , F M D V peptides with the added sequence
132-148 from sperm-whale myoglobin elicited a high degree of virus neutralising
activity in mice, whereas sera from mice which received the peptides with the added
sequence 105-121 from sperm-whale myoglobin did not neutralise the virus although
they had high levels of anti-peptide activity. The results indicated that the T-cell help
given by foreign epitopes is B-cell clone specific.
Synthetic peptides from other viruses have been less successful. Peptides
representing various regions of the influenza A virus haemagglutinin elicited
antibodies, but these were neither protective nor capable of neutralising virus (13).
Another synthetic peptide corresponding to an influenza A antigen was conjugated
to tetanus toxoid and, when administered with Freund's complete adjuvant, stimulated
the production of antibodies and gave partial protection to mice against challenge
(20). Chemical synthesis of peptide vaccines for bacterial diseases has been
reported. A synthetic peptide, corresponding t o an amino-acid sequence of the
A chain of the diphtheria toxin, when conjugated to a protein carrier elicited
antibodies in guinea pigs that neutralised the dermonecrotic and lethal effects of the
toxin (1).
T w o antibodies are k n o w n t o protect against cholera, one being directed against
Vibrio cholerae lipopolysaccharide and the other against cholera toxin. Antibodies
t o the B subunit of the toxin prevent the binding of toxin molecules t o mucosal
receptors. Consequently, several peptides corresponding to regions of the amino-acid
chain of the B subunit have been synthesised and linked to either tetanus toxoid or
multi-chain poly D-L alanine; these are currently being evaluated (1).
Although synthetic peptides are a promising development and have a number of
important potential applications, their use for vaccines remains uncertain for a number
of reasons. To produce a good immune reponse, Freund's adjuvant is often necessary.
Furthermore, m a n y synthetic peptides lack T-cell recognition sites, which leads to
insufficient co-operation between T and B cells during the early immune response.
Finally, many important epitopes are discontinuous and their antigenicity is therefore
dependent on the conformation of the viral protein.
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ANTI-IDIOTYPE VACCINES
The region of an antibody that interacts with an antigen is called the paratope.
This consists of six hypervariable loops, each of which is made up of a number of
determinants called idiotypes. A portion of the idiotype is the antigen-binding site,
and thus antibodies directed against this site, anti-idiotypes, m a y mimic the
conformation of the antigen and serve as surrogate antigens. The advent of monoclonal
antibodies and r D N A techniques has made the development of anti-idiotype vaccines
a possibility. So far a number of anti-idiotype have been prepared that stimulate
immunity, e.g. injection of mice with HBsAg anti-idiotype primes an anamnestic
response to subsequent injection with H B s A G . The theoretical possibilities of antiidiotype vaccines are numerous. Such vaccines could be used to prime the immune
system, since their immunity is generally specific and of short duration. They could
also be used to overcome the immune restriction to complex carbohydrates that exists
during the neonatal period. Also, they may be able to eliminate the problems associated
with defective configuration of antigens produced by gene cloning of synthetic peptides.
PARASITES
Parasite vaccines have been considered separately because of the multicellular
nature of parasites and their wide biological spectrum, which ranges from intracellular
organisms to complex endo- and ectoparasites. During parasite infections, numerous
antigens may be processed by the host immune system and the presentation of multiple
stages has rendered the isolation of the protective antigen(s) difficult.
A number of existing parasite vaccines are available commercially and these range
from the classic lungworm vaccine, which consists of irradiated larvae, to attenuated
Coccidia, Anaplasma and Babesia. It has been shown that non-viable coccidial
sporozoite antigens could induce protective immunity in young chickens and that this
immunity was cross-species. Published evidence suggests that recombinant molecules
have also been successful in conferring immunity. A major surface protein of
Anaplasma marginale, M S P - 1 , has been used t o immunise cattle and reduce postchallenge parasitaemia and clinical disease. D N A from A. marginale was cloned and
expressed in E. coli and M S P - 1 was obtained.
Protective immunity against malaria can be induced by vaccination with
irradiation-attenuated sporozoites. The immunity is mediated by neutralising
antibodies and T-cell effector mechanisms which recognise epitopes in the
circumsporozoite. Thus, passive transfer of cytotoxic T-cell clones into mice provided
protection against challenge. Likewise, salmonella vaccine strains have been engineered
to express the circumsporozoite protein; vaccination with these bacteria provides
protection against murine malaria in the absence of antibodies.
Trichinella spiralis is an important public health problem in many countries.
Infection of an animal host results in solid immunity. It has been shown that vaccination
with a combination of adult and larval antigens confers nearly complete immunity.
A number of studies have identified protective antigens, but the yields of antigen from
the intact parasite have been low; consequently, in vitro translation of T. spiralis m R N A
has been used to prepare a number of polypeptides for protection studies.
790
In the case of ticks and Haemonchus it has been found that parasite gut antigens
can induce protection in the host animal, and it may be possible to produce the antigen
by molecular techniques.
ADJUVANTS
Adjuvants are necessary to augment the immunogenicity of m a n y vaccines. Since
m a n y synthetic antigens are water-soluble, they tend to be less immunogenic than
particulate antigens and adjuvants are often necessary to confer protection. The
adjuvants currently in use are aluminium gels and F r e u n d ' s complete adjuvant (FA),
which is a water-in-oil emulsion with killed mycobacteria. Whilst F A has been used
in laboratory animals, it m a y cause severe local reactions and as a consequence safer
adjuvants are being sought.
O n e candidate is muramyl dipeptide ( M D P ) , which is a synthetic analogue of
monosaccharide tripeptide, the active mycobacterial component of F A . M D P in an
aqueous solution is non-pyrogenic and active even when administered orally. Synthetic
peptide antigens from diphtheria toxin have been covalently linked to M D P and a
carrier; when injected into mice, these induced an effective anti-toxin response. M D P
has also been added to surfactant polymer, squalene-pluronic block emulsion, which
has the ability to bind proteins to the surface of oil drops in the emulsion and possibly
produces good antigen conformation.
A n o t h e r promising adjuvant is immunostimulating complex (ISCOM), which is
made from the adjuvant Quil A (active component of saponin). I S C O M forms a
complex with amphipathic peptides which are presented on the surface of the matrix.
N o t all antigens can be incorporated in ISCOMs but they seem to be particularly
suited to detergent-soluble m e m b r a n e proteins. ISCOMs are very immunogenic and
stimulate b o t h T- and B-cell response. A n equine influenza I S C O M is commercially
available and membrane proteins from parainfluenza-3 (PI3), rabies and measles have
been combined with I S C O M s .
Liposomes, which are spheres of phospholipid bilayers separated by aqueous
compartments, have also been used to entrap antigens; under certain conditions,
the liposome/antigen complex produces higher titres t h a n when the antigen is used
alone.
CONCLUSION
Biotechnology is being used increasingly to clarify the pathogenic mechanisms
and immune response to microbial disease. This will lead to the production of more
effective vaccines in the future. During the last decade, many candidate vaccines have
been produced by these techniques, but few are yet in commercial production.
Problems of strain stability and poor immunogenicity have occurred and production
costs m a y be higher for synthetic vaccines t h a n for conventional vaccines. Licensing
of live genetically-manipulated vaccines may also present additional problems because
791
the use of such vaccines m a y be construed as the release of genetically-manipulated
organisms into the environment. However, given the progress of recent years,
biotechnology has a major role to play in the prevention of m a n y animal diseases.
*
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BIOTECHNOLOGIE ET SCIENCE VÉTÉRINAIRE : PRODUCTION DE VACCINS
VÉTÉRINAIRES. - C. Wray et M.J. Woodward.
Résumé: Les auteurs expliquent les principes fondamentaux des manipulations
génétiques ainsi que les méthodes de production des vaccins vétérinaires les plus
importants. L'atténuation spécifique des microorganismes peut résulter soit des
mutations introduites par génie génétique et qui produisent des blocages
métaboliques, soit du développement de toxoïdes bactériens efficaces, mais
également du réarrangement d'un fragment de gène viral. Les techniques d'ADN
recombinant ont été utilisées pour produire des vaccins sous-unitaires et pour
concevoir des vaccins multivalents grâce à l'insertion de gènes dans des
organismes porteurs tels que poxvirus et salmonelles atténuées. Cet article fait
état de l'utilisation de vaccins à base de peptides synthétiques et de vaccins antiidiotypes, ainsi que des problèmes posés par les vaccins antiparasitaires. La
biotechnologie devrait apparaître dans le futur comme jouant un rôle majeur
dans la prévention de nombreuses maladies animales.
MOTS-CLÉS : ADN Recombinant - Atténuation - Bactéries - Biotechnologie Manipulation génétique - Parasites - Vaccins sous-unitaires - Virus.
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BIOTECNOLOGÍA Y CIENCIA VETERINARIA: PRODUCCIÓN DE VACUNAS
VETERINARIAS. - C. Wray y M.J. Woodward.
Resumen: Los autores explican los principios fundamentales de las manipulaciones
genéticas, así como las técnicas de producción de las vacunas veterinarias más
importantes. La atenuación especifica de los microorganismos puede resultar de
las mutaciones producidas por ingeniería genética, que producen bloqueos
metabólicos, del desarrollo de toxoides bacterianos eficaces, o bien del
reordenamiento de un segmento de gene vírico. Se han utilizado técnicas de ADN
recombinante para producir vacunas de subunidades y también vacunas
multivalentes mediante la inserción de genes en organismos portadores, como
poxvirus y salmonelas atenuadas. Este artículo comenta el uso de vacunas a base
de péptidos sintéticos y vacunas antiidiotipos, así como los problemas planteados
por las vacunas antiparasitarias. En el futuro, la biotecnología desempeñará sin
duda un papel esencial en la prevención de numerosas enfermedades animales.
PALABRAS CLAVE: ADN recombinante - Atenuación - Bacterias Biotecnología - Manipulación genética - Parásitos - Vacunas de subunidades Virus.
*
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792
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For further reading:
Books
ARNON R. (1987). - Synthetic Vaccines, Vols. 1 and 2 . CRC Press, Inc., Boca Raton, Florida,
USA.
CHANOCK R.M. & LERNER R.A. (1984). - Modern approaches to vaccines: molecular and
chemical basis of virus virulence and immunogenicity. Cold Spring Harbor Laboratory, USA.
794
GERMANIER R. (ed.) (1984). - Bacterial Vaccines. Academic Press, London.
KOHLER H. & LOVERDE P.T. (1988). - Vaccines: new concepts and developments. In
Proceedings of the 10th International Convention on Immunology, Buffalo, New York, July
1986. Harlow Longmans Scientific & Technical, New York, 14-17.
PEARSON T.W. (ed.) (1986). - Parasite antigens: toward new strategies for vaccines. Marcel
Dekker Inc., New York.
Reviews
ARNON R., SHAPIRA M. & JACOB C.O. (1983). - Synthetic Vaccines. J. Immunol. Meth.,
6 1 , 261-273.
BACHRACH H.L. (1985). - New approaches to vaccines. Academic Press Inc., New York.
Adv. vet. Sci. comp. Med., 3 0 , 1-38.
FIELD B.N. & CHANOCK R.M. (1989). - What biotechnology has to offer vaccine
development. Rev. infect. Dis., II. Supplement 3, S519-S523.
GAMBLE H.R. & ZARLENGA D.S. (1986). - Biotechnology in the development of vaccines
for animal parasites. Vet. Parasit., 2 0 , 237-250.
LIEW F . J . (1985). — New aspects of vaccine development. Clin. exp. Immunol., 6 2 , 225-241.
LUCKOW V.A. & SUMMERS M.D. (1988). - Trends in the development of Baculovirus
expression vectors. Biotechnology, 6 , 47-55.
MURRAY P . K . (1989). - Molecular vaccines against animal parasites. Vaccine, 7, 291-299.