Download Achievements in genetic engineering and their influence on

Rev. sci. tech. Off. int. Epiz., 1983, 2 (3), 629-653.
Achievements in genetic engineering
and their influence on the control
and prevention of animal diseases*
H.L. BACHRACH*, J.J. CALLIS**,
F. BROWN*** and K. STROHMAIER****
Summary : Genetic engineering consists of manipulations of cellular or
viral genomes whereby desired gene products (proteins) are made for
use by the host organism or animal. Included in this definition would
be genetic recombination, DNA cloning, DNA injection or transfection, cell fusions, and induction or repression of genome expression.
This technology is already providing effective measures for control of
infectious diseases. A new generation of vaccines is emerging which will
be safe, stable and inexpensive. Such vaccines consist of surface pro­
teins, or segments thereof, of infectious agents produced artificially by
molecular cloning or organic synthesis.
One of the first to be studied is a cloned subunit vaccine for foot
and mouth disease. When administered in two doses, it elicits neutraliz­
ing antibody in cattle and swine and protects against exposure to the
virus. Similarly, an organically-synthesized peptide 20 amino acids long
coupled to a carrier and formulated into a vaccine protected guinea
pigs against the virus and produced antibodies in cattle.
Genetic engineering technology is providing other novel disease con­
trol measures. Monoclonal antibodies derived by the fusion of antibodyproducing cells with myeloma cells are improving the diagnosis, treat­
ment and epitope delineation in infectious and oncogenic diseases.
Interferons, a heterogeneous group of proteins divided into three
classes, alpha, beta and gamma, modulate several immune functions.
Recently, interferon has been produced by genetic engineering in E.
coli in amounts sufficient for testing.
These and other products are being produced by genetic enginee­
ring, and this technology offers to provide within this decade a new
generation of vaccines and therapeutic agents for infectious diseases
and growth hormones for use in several species.
* Report presented at the 51st General Session of the O.I.E., Paris, 23-27 May 1983
(Technical Item I).
** Plum Island Animal Disease Center, U . S . Department of Agriculture, Greenport,
New York, U . S . A .
*** Wellcome F M D V Laboratory, Pirbright, Surrey, U.K.
**** Bundesforschungsanstalt für Viruskrankheiten der Tiere, Tubingen, West Germany.
— 630 —
INTRODUCTION
Genetic engineering consists of manipulations of cellular or viral genomes
whereby desired gene products (i.e. proteins) are made for use either by a
host organism or animal or for treatment of disease or other abnormality in
other hosts. Included in these manipulations are genetic recombination, DNA
cloning, DNA injection or transfection, cell fusions, and induction or repres­
sion of genome expression. This technology is already providing effective
measures for achieving greater control of infectious diseases, and work is
progressing toward introducing and enhancing desirable characteristics in
both animals and humans. A new generation of vaccines, markedly different
from conventional whole-agent (viruses and bacteria) vaccines, is one of the
novel achievements. Although conventional living-attenuated or killed wholeagent vaccines are highly effective for many diseases, they sometimes pro­
duce allergic side reactions and acute or slowly progressive disease. Also, they
can lose potency at ambient temperatures and during extended storage under
refrigeration. In addition, no whole-virus vaccines have been developed for
diseases caused by hepatitis B, retro and many herpes viruses.
A real need exists, therefore, for safe, stable and effective vaccines produ­
ced inexpensively in large quantities covering a broader range of diseases than
is now amenable to whole-agent prophylaxis.
Fortunately, a new generation of vaccines is emerging that promises to
possess these attributes. The vaccines are the surface proteins, or segments
thereof, of infectious agents produced artificially by molecular cloning or
organic synthesis. These two procedures have been used to produce vaccines
for viral and bacterial diseases, and their application to oncogenic diseases
and to certain parasitic diseases, such as trypanosomiasis affecting humans,
can be foreseen. A cloned subunit vaccine for foot and mouth disease (FMD),
the first effective vaccine of its kind, administered in two doses has been
shown to elicit neutralizing antibody and protect cattle and swine against
exposure to the virus (1). Conjugated to a carrier protein, one dose of a
synthetic peptide 20 amino acids long corresponding to residues 141 through
160 of the surface protein of F M D virus induced neutralizing antibody in
rabbits and guinea pigs and protected rabbits against F M D (2); and 3 doses
of a 16mer have elicited neutralizing antibody in rabbits (3).
Similarly, the surface protein of hepatitis B virus produced by cloning (4)
as well as segments produced synthetically (5, 6, 7) have been shown to be
immunogenic. The report that glycoprotein D of herpes simplex virus 1 clo­
ned in E. coli elicits neutralizing antibody against both herpes viruses 1 and 2
presages the development of vaccines against many herpes diseases that
afflict humans and animals (8). In addition, surface proteins have now been
cloned for fowl plague (9), influenza (10, 11), vesicular stomatitis (12) and
rabies (13) viruses as well as for enterotoxigenic E. coli (14) and N. gonor­
rhoeae (15, 16). Cloning is in progress for the surface proteins of canine parvogastroenteritis (W.E. Hahn, personal communication), Rift Valley fever
— 631 —
(U.S. Army Medical Research C o m m a n d and Development Contract, 1982),
arena (17) and paramyxoviruses (P. Choppin, personal communication). The
potency of the subunit vaccines is being enhanced with adjuvants, carrier
proteins and antigen presentation systems (e.g., micelle formation and lipo­
somal entrapment).
Genetic engineering technology is providing other novel disease control
measures distinct from the new protein vaccines. Monoclonal antibodies deri­
ved by the fusion of antibody-producing cells with myeloma cells are impro­
ving the diagnosis, treatment and epitope delineation in infectious and onco­
genic diseases. Monoclonal antibodies also make more realistic the develop­
ment of anti-idiotype antibody vaccines. In addition, alpha, beta and gamma
interferons and modifications thereof made by recombinant D N A procedures
for humans should become inexpensive enough for use in special circumstan­
ces in veterinary medicine, for example, for prize breeding stock, race horses
and valuable show and companion animals.
SUBUNIT VACCINES
During the 1970's, it was conclusively demonstrated that proteins isolated
from the surfaces of viruses and some bacteria can induce the production of
neutralizing antibody and protect animals against challenge with homologous
agents. Short segments cleaved from the surface proteins of certain viruses
(e.g., FMD virus) were also shown to be effective immunogens (18, 19, 20).
These findings were compatible with Atassi's finding that antigenic (epitopes)
of proteins (e.g., myoglobin and lysozyme) consist of surface amino acid
sequences 6 to 7 residues long linked continuously or, in part discontinuously, between distant segments of the protein chain brought into contact
by tertiary foldings and stabilization by disulfide bonding (21). A priori rea­
soning based on the intrinsic shortness of the antibody binding domain also
indicated that individual epitopes would be very short.
Results with natural subunit vaccines and the evidence for short epitope
lengths stimulated work in many laboratories on the artificial production of
the surface proteins and active segments therefrom by molecular cloning and
organic synthesis, and the testing of such products for their activity in indu­
cing neutralizing antibody and as protective vaccines. Organic synthesis of
polypeptides and proteins is a well-established manual and automated proce­
dure in organic chemistry, whereas the production of proteins by molecular
cloning has developed only since 1973.
Cloning consists of splicing a segment of D N A (i.e., a gene) coding for
the desired protein into a double-stranded (ds) D N A vector (e.g., bacterial
plasmid or viral DNA) and subsequent transfer to a single-celled host (pro- or
eu-karyote) or animal for replication of the guest gene and its expression as
protein.
— 632
W h i l e most of the cloning procedures have become routine in many labo­
ratories, the preparation of the encoding D N A often proceeds with difficulty.
Thus, if natural D N A does not exist for a protein, as for the proteins of
many R N A viruses, double-stranded complementary D N A (ds cDNA) must
be made enzymatically by treating isolated viral genome R N A or messenger
R N A (mRNA) with reverse transcriptase.
Also, if the R N A is polycistronic (i.e., coding for several proteins), amino
acid sequences of the desired protein have to be determined in order to iden­
tify the desired matching segments of the ds c D N A that are sequenced, in
turn, most often by the method of M a x a m and Gilbert. For a protein enco­
ded by monocistronic m R N A , the required D N A can sometimes be prepared
without first sequencing protein or nucleic acids. Briefly, the nascent protein
still attached to its m R N A (in a cell extract) is precipitated by highly specific
antibody. The m R N A is separated from the protein and transcribed as above
into ds cDNA for insertion into an appropriate vector.
Protein vaccines, cloned or synthetic, have several advantages not posses­
sed by whole-agent vaccines. Being proteins or polypeptides not derived from
virus particles, they are non-infectious, stable to temperature variation and
are much less apt to invoke the adverse side effects that sometimes follow
inoculations with whole-agent vaccines. A disadvantage for now is that the
artificial protein vaccines are still in the experimental stage of development.
It will be some time yet before sufficient data are developed in field trials to
bring them into general use. Subunit vaccines have potential application for
viral, bacterial, parasitic and oncogenic diseases. However, since most work
has been carried out on protein vaccines for viral diseases, progress on it will
be described now in some detail, whereas that for the other diseases will be
taken up to a lesser extent later on.
SUBUNIT VACCINES F O R V I R A L DISEASES
Viruses are grouped into families (Viridae) the members of which possess
distinctive architecture, similar genomic composition and, consequently, very
similar kinds and numbers of proteins in their cores and surfaces. Most impor­
tant to the development of immunity in viral diseases is the immunological res­
ponse mounted by the host animal to the proteins on the surface of the virus.
Table I, modified from Bachrach (22), lists viruses according to familial
membership. The third column identifies those surface proteins which after
being isolated from the virus retain one or more of the following immunoge­
nic activities :
(i) reaction with neutralizing or viral-precipitating antibodies,
(ii) induction of neutralizing or viral-precipitating antibodies and,
(iii) protection of animals against viral challenge.
Also shown (columns 4 and 5) is the present status of the production of
these immunogens, or active segments thereof, by cloning and organic
synthesis.
Yes
QQ
ZZ
Rubivirus
Yes
cj
>H
Split virus E1-3
Glycoprotein V3
Yes
CO CO
Semliki Forest virus
Tick-borne encephalitiss virus
Hog cholera virus, bo\ r'rne
viral diarrhea virus
Rubella virus
Cloned
U
>H
Caliciviridae
Toeaviridae
Alphavirus
Flavivirus
Pestivirus
Glycoprotein
Glycoproteins
Glycoproteins gC and gD
Soluble antigens and hemagglutinins from
intra- and extra-cellular viruses
Soluble antigens
Capsid proteins
Capsid proteins
o 1,CT3 and X 2 proteins
Protein p2
Protein p26 and glycoprotein gp34
VP7.2
24-kd surface protein VP,**
VP,
Protein p61
T (large tumor)
Hexons, pentons fibers
Two-cell membrane turkey herpesvin
proteins
Isolated from virus
Immunogens
53 Q Q Q
Z I * Z Bs Z Z
Picornaviridae
Reoviridae
Orbiviridae
Rotaviridae
Vaccinia virus
H-l virus
Canine parvovirus
Reovirus
Bluetongue virus
Simian virus II
Calf rotavirus
Foot and mouth disease virus
Poliovirus
Vesicular exanthema viirus
Orthopoxviridae
Parvoviridae
Infectious bovine
rhinotracheitis virus
Pseudorabies virus
Herpes simplex virus
Rabbitpox virus
Adenoviridae
Herpetoviridae
Virus
Human papilloma viru; > (wart)
Simian virus 40
Adenovirus
Marek's disease virus
Papovaviridae
Family
Q
QN
CO
GN
1
1
QN
TABLE I
PU
QN
Candidates for subunit vaccines for viral diseases
Yes
Yes
Synthetic
633
QQ
ZZ
WW
CO CO
u u
•> •>
3 3
'S,'S
•t-» _•»-»
00 00
T
Hepatitis B surface anti igen
i
Yes
Yes
Yes
Yes
Synthetic
* Modified from (22).
The split influenza virus and hepatitis B surface antigen (isolated from blood)vaccinesin use.
ND = not done to our knowledge.
IP = in progress.
SYN = chemical synthesis of fragments.
.
.
.
Of the cloned immunogens, only the 24-kd surface protein of the fool and mouth disease virus has been reported to protect animals against viral challenge
exposure.
** Also termed VP or VP by some authors.
3
Hemagglutinin and neui •aminiidase
CO
CD
Unclassified
Glycoproteins
>> zz z
CO
CD
Retro viridae
Bs
Protein F
Hemagglutinin and F pi rotein./hemol sin
Glycoprotein G
Glycoprotein G
Glycoprotein gp71
Soluble tumor cell antigen
Bs
Rhabdoviridae
Cloned
P..
Glycoproteins G1-2
Glycoprotein
Glycoprotein 190 kiloda Itons
Split virus, hemagglutini
Hemagglutinin
c
Paramyxoviridae
Arenaviridae
Transmissible gastroentei
virus
Human Coronavirus
Influenza virus
Fowl plague virus
Recombinant viruses,
Rift Valley fever virus
Lymphocytic choriomeni ngitis
virus; others
Sendai virus, Newcastle
disease virus
Simian virus 5,
parainfluenza-3 virus
Measles virus
Vesicular stomatitis viru:
Rabies virus
Friend virus
Feline leukemia virus
Maloney leukemia virus
Hepatitis B
Isolated from virus
Immunogens
W3 CO
CD CD
Bunyaviridae
Orthomyxoviridae
Coronaviridae
Virus
Q Q
ZZ
GN
Family
TABLE I (cont'd)
634 —
— 635 —
Because many hundreds of animal viruses have now been identified, all of
them cannot be included in the Table I. However, an important principle is
that once the isolation, cloning a n d / o r synthesis of a surface immunogen has
been achieved for one member of a viral family, similar procedures should be
successful for the other members of the family. Consequently, we shall dis­
cuss the Table principally with regard to protein immunogens of those mem­
bers of a family for which most progress has been reported to date.
Subunit vaccines for papova, adeno and herpes viruses will be discussed
together owing to similarities in their replication pathways and the ability to
initiate infection from their isolated ds D N A s . For papovaviruses, the clo­
ning of human wart virus D N A has been reported to be in progress for possi­
ble vaccine development (Univ. Minnesota news publication, 1981); and for
simian virus 40, two polypeptides have been synthesized that possess some of
the immunogenicity of the large t u m o r antigen coded for by the virus (23).
For adenoviruses, hexon and penton fibers are reported to elicit neutralizing
antibodies (24), and a library of adenovirus D N A segments has been prepa­
red by molecular cloning and restriction endonuclease cleavages (H.S. Gins­
berg, personal communication). However, no report of the expression of
either of the above surface proteins has as yet been seen by us.
For herpesviruses, two vaccinations with two proteins from the plasma
membranes of cells infected with turkey herpesvirus are reported to protect
chickens against Marek's disease (25), and a crude subunit vaccine prepared
from infectious bovine rhinotracheitis (IBR) virus protects animals against
challenge (26). A glycoprotein purified from IBR virus elicits neutralizing
antibodies in rabbits and cattle (D. Reed, personal communication). Simi­
larly, two vaccinations with a glycoprotein from pseudorabies virus has elici­
ted neutralizing antibody and protected swine against intranasal challenge
(K.B. Platt, personal communication). However, the greatest progress with
subunit vaccines for herpesviruses has been with herpes simplex virus 1
(HSV-1) (8). Subsequent to a report that herpesvirus glycoproteins gC and
gD induce neutralizing antibodies (27), HSV-1 D N A specific for the latter
moiety was cloned in E. coli resulting in the expression of a gD-related poly­
peptide that elicits neutralizing antibodies to both HSV-1 and HSV-2 (8).
This work is significant not only for the potential preparation of protein vac­
cines against venereal herpes, but also for analogous vaccines for the nume­
rous herpesvirus diseases of animals and humans.
There are as many as five important surface antigens on orthopoxviruses
(28). Soluble antigens from intracellular rabbitpox or vaccinia viruses are
reported to elicit neutralizing antibody to the homologous virus but not to
extracellular virus (29, 30). It appears that immunological responses to a late
hemagglutinin in the envelope of extracellular virus and membrane of the
infected cell are important to the protective process. Although effective subu­
nit vaccines have not been described for orthopoxviruses, vaccinia virus can
be bioengineered to act as a cloning and expression vector for carrying
foreign genes into eukaryotes including animals (31).
— 636 —
Thus, cells infected with both vaccinia virus and a plasmid containing the
gene for herpes virus thymidine kinase give rise to recombinant vaccinia virus
that permits the cell to produce the same thymidine kinase. Analogous results
have been obtained for recombinant vaccinia viruses programmed with genes
for the surface proteins of unrelated animal viruses, including that of hepati­
tis B virus (B. Moss, personal communication). Consequently, the apparent
success in eradicating small pox with the help of vaccinia virus presages the
possibility of using its recombinants as live vaccines for the prevention of
other important diseases of animals and h u m a n s .
Parvoviruses represent the class of viruses that contain separately encapsidated positive or negative single-stranded D N A genomes. The isolated sur­
face proteins from both H - l rat and canine parvoviruses are reported to elicit
neutralizing antibodies in guinea pigs (S. Rhode III, personal communica­
tion). In addition, the canine virus surface protein has been cloned and used
to induce neutralizing antibody in mice (W.E. H a h n , personal communica­
tion).
Because of the striking structural and functional similarities, progress in
developing subunit vaccines for the Reo-, Orbi- and Rotaviridae will be dis­
cussed together. The similarities are so great that these viruses could readily
be designated as belonging to a single family, with subdivisions as necessary.
For reoviruses, surface protein σ1 is reported to elicit neutralizing antibody,
and for some serotypes σ3 and A 2 proteins also possess this activity (32).
Although most of the Λ2 protein resides in the viral core, a segment of it
extends through to the viral surface.
For orbiviruses, a protein designated p2 appears to be a candidate subunit
vaccine, because of high correlation between protein p2 precipitating and
neutralizing antibody titers in bluetongue virus antisera (33) as well as the
observation that p2-specific monoclonal antibody passively immunizes sheep
(34). For rotaviruses, the isolated unreduced protein p26 and glycoprotein
gp34 of simian SA II virus induces type-specific neutralizing antibodies (35).
Using monospecific polyclonal antiserums, the major neutralization-specific
epitopes of calf rotavirus appear to be present in a minor protein VP 7.2 of
the virion outer shell (36).
Subunit vaccine development for viruses containing an infective singlestranded R N A genome will be considered separately for the four constituent
families (Picorna-, Calici-, Toga- and Coronaviridae) because of their dis­
tinctively different architectures, genomes strategies and protein composi­
tions.
For FMD virus, a member of the Picornaviridae, two vaccinations with a
24 kilodalton (kd) surface protein (generally migrating as V P in high and as
VP1 in low urea concentrations during electrophoresis in polyacrylamide gels;
or designated alternatively as VPT because of its N-terminal threonine) isola­
ted from virus types O and A elicit high levels of neutralizing antibody in
swine (37, 38), and the latter protects swine and cattle against challenge with
3
— 637 —
homologous virus (38). Also, peptides cleaved from the surface protein of
type 0 virus induce neutralizing antibodies in guinea pigs and partial protec­
tion against the generalization of primary lesions when challenged with virus
(18).
And a 13-kd cyanogen bromide fragment of type A surface protein con­
taining amino acids 55 through 179 of the surface protein protects both swine
and cattle against challenge with homologous virus (19, 20). Similar patterns
of neutralizing antibody and protection in swine and cattle have been obtai­
ned by two vaccinations with recombinant type A surface protein produced
in E. coli (1). In addition, two of three goats inoculated repeatedly with
recombinant type O surface protein, developed neutralizing antibodies and
no or very low viremia and milder fevers than two control goats when chal­
lenged with virus (39).
One dose of a synthetic peptide 20 amino acids long corresponding to
residues 141 through 160 of the same surface protein conjugated to a carrier
protein induced high levels of neutralizing antibody in rabbits and protected
these animals against challenge with F M D virus (2). Corresponding synthetic
20mers for two subtype A surface proteins are also reported to raise neutrali­
zing antibodies in rabbits (2). Also, three doses of a type O 16mer (residues
144 through 159) given with carrier protein induced neutralizing antibody in
rabbits (3).
Some of the results using rabbits indicate that synthetic peptides could
protect natural hosts such as cattle and swine against F M D . The conclusion
of a recent discussion paper is very optimistic as to the prospects for the deve­
lopment of practical recombinant and synthetic protein vaccines for F M D
(40).
Except for F M D and Coxsackie (41) viruses, the isolated surface proteins
of other picornaviruses were not reported until recently to possess immuno­
genic activity. Two reports have now appeared that conclusively demonstrate
such activity in VP1 isolated from poliovirus (42, 43).
It will be informative to correlate variability in amino acid sequences of
variants of the immunogenic surface protein of F M D virus with biological
activities such as serotype, immunotype, virulence, pathogenicity, cellreceptor specificity and with the evolution of virus types and subtypes.
Figure 1 shows sequences for a total of eight subtypes of virus types A , O
and C. It is quite apparent that sequence hypervariability in the region 131160 correlates with the reported immunogenic activity in rabbits of synthetic
peptides corresponding to portions of this region (2, 3). Considerable variabi­
lity is also present in the region 43-60, but no biological activity has been
attributed as yet to this domain other than that the right third of this region is
in the 13-kd cyanogen bromide fragment from type A surface protein that
protects livestock against viral challenge (19, 20). The sequences of the two
variants of type O1 surface protein are highly conserved, differing by only
I 2
— 638 —
A5
10
12
24
0 1
1
C 1
3
WW
61
119
Cruz
BFS
K
Obb
Inda
60
50
70
40
30
1
10
20
TTAVGESADPVTTTVENYGGDTQTQRRYYMDVGFIMDRFVKINSLSPTHVIDLMQTHQHGLVGALLRAAT
V
HHT
K I
TT
AT
E V
HHT S
K N
XXXXXXXXXXX
E I
HHT I
Q
T
SA
E I
QHT S
VTPQNQINIL
VPS T
S
QHT S
VTPQNQINIL
IPS T
S
SA
E I
HHT A VL
VTVSGNQ TL V A KDNI
TT
E V
HHT A VL
VHVSGNQ TL V V K S I
TT
E I
A5
10
12
24
0 1
1
C 1
3
Uw
61
119
Cruz
BFS
K
Obb
Inda
130
110
120
140
100
71
80
90
YYFSDLEIVVRHDGNLTWVPNGAPEAALSNTSNPTAYNKVPFTRLALPYTAPHRVLATVYNGTNKYS—T
A
D
—A
G
A
~A
E
S L
A
P
S A—V
A K ERD
K D T
H AL
ECR RNA
A KE D
K D T
H AL
ECR NRNA
A T T K
VS D T
H GL
T TT T—A
A T T K
VS D A
H GL
T T TA T—A
A5
10
12
24
0 1
1
C 1
3
Uw
61
119
Cruz
BFS
K
Obb
Inda
200
170
180
190
216
141
150
160
GGP—RRGDMGSAAARAAKQLPASFNYGAIRAITIHELLVRMKRAELYCPRPLLAIEVSSQDRHKQKIIAPARQLL
SDS— S L I
VT
KT
Y•
K
Q QA
S S-GV
F L PV R
KE
GK S-G
L VV
K OA
K
VPNL- - LQVL QKV RT T
K TRVT
Y
T
HPT-EA
V VK T
VPNL- - LQVL QKV RT T
K TRVT
Y
T
HPT-EA
V VK T
S-T—
LAHLT TR GH T
'- VK E T
T
I P QPT-G
PLV
K
S-A-LAHL AH RH T
- VK E T
T
V PVQPT-G
PL
K
FIG.
l
Amino acid sequences of eight variants of the immunogenic 24-kd surface protein
of foot and mouth disease virus types A, O and C.
Sequences derived from nucleotide sequences determined for the corresponding complementary
D N A s ( 1 , 85, 86, 88, 89). Only differences to the A Ww sequence are specified. Dashes are dele­
tions used to optimize alignment of sequences. The number of amino acid residues in the variant
proteins are : 209 in C1 Obb; 210 in C Indaial; 212 in A Ww, A 61 and A 119; 213 in A
Cruzeiro, O1 BFS and O1 Kaufbeuren. All variants have N-terminal threonine (T) and Cterminal leucine (L). The other abbreviations are : A, alanine; C, cysteine; D , aspartic acid; E,
glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; M, methionine;
N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; V, valine; W, tryptophan; Y,
tyrosine; X, not reported.
5
3
5
1 0
1 2
2 4
— 639 —
three substitutions two of which are in the variable regions just described. It
is also apparent that the type O and C sequences are much more closely rela­
ted than they are to any of the type A sequences. Work is now in progress to
map the number and specificities of the antigenic determinants on these pro­
teins using poly- and monoclonal antibodies, overlapping and immunogenic
synthetic peptides, 3-dimensional structure determinations and related techni­
ques. It would be somewhat premature to speculate further on possible
epitope-activity relationships. Experimental proof will be required, particu­
larly since a single amino acid substitution can greatly alter the structure and
activity of a protein as, for example, in S-hemoglobin of sickle-cell anemia.
For caliciviruses, the single major protein of vesicular exanthema virus of
swine may be a candidate subunit vaccine because two vaccinations of swine
with the isolated protein appear to elicit neutralizing antibodies ( A . H . Dardiri, H.L. Bachrach, unpublished observations).
Togaviruses also have surface protein with subunit vaccine potential. For
the Alpha genus, good protection is obtained against Semliki Forest virus
when its glycoprotein is presented in the form of a multimeric micelle (44).
For the Flavi genus, polymeric complexes of glycoprotein V isolated with
detergent from tick-borne encephalitis virus induced hemagglutinationinhibiting and neutralizing antibodies, and, compared with whole-virus vacci­
nes, they were ten times more active in competition radioimmunoassays and
were equally protective in mice against challenge of immunity (45). For the
Pesti genus, repeated vaccinations with glycoproteins presumed to be E1 and
E from detergent-split hog cholera (HC) virus or infected cells induced neu­
tralizing antibody and protected swine against exposure to H C virus (46).
Detergent-split bovine diarrhea virus (the equivalent of H C virus for bovines)
was less effective as a subunit vaccine against H C than the H C virus glyco­
proteins. For the Rubi genus, results with detergent-split (47) rubella virus
appear to be more promising as a subunit vaccine than hemagglutinin rosette
or virosome preparations (48).
3
2
The coronaviruses, the longest plus-sense stranded R N A viruses (ca.
24,000 nucleotides), also have surface proteins that are candidates for subu­
nit vaccines. Use of antisera specific t o each of three proteins of h u m a n coronavirus indicate that the slower migrating protein (13 kd) induces neutralizing
antibody a g a i n s t o n e v i r u s s t r a i n a n d b o t h n e u t r a l i z i n g a n d
hemagglutination-inhibiting antibodies against another strain (49). Also, two
vaccinations with glycoproteins isolated from transmissible gastroenteritis
virus induce neutralizing antibodies and protect neonatal pigs against direct
and contact exposure to virus (P. Gough, personal communication).
Viruses with segmented negative-stranded R N A genomes /Ortho-, Bunyaand Arenaviridae) have surface glycoproteins that are candidate subunit vac­
cines. For example, a commercial split-virus influenza vaccine effects sero­
conversion and produces fewer side effects than whole-virus vaccine, but two
doses are required for unprimed recipients (50). The gene for the principal
— 640
surface immunogen (hemagglutinin, H A ) of fowl plague virus has been clo­
ned and expressed in E. coli (9) and that for wild-type h u m a n influenza virus
in both E. coli (10, 11) and eukaryotic cell culture (51). Each cell is reported
to produce 10 cell-associated H A molecules that adsorb specifically to eryth­
rocytes and viral antibodies. Use of the gene for an anchor-minus mutant of
the H A permits the cloned H A to be expressed and efficiently secreted into
the extracellular fluid (52). The cloned H A molecules would appear to have
potential for use in a subunit vaccine. However, of twenty overlapping pepti­
des synthesized to 75 percent of the H A 1 portion of the wild-type molecule,
none were reactive with antibody to authentic H A 1 (53).
8
Synthetic peptides corresponding to the 11 amino acids at the N-terminus
of the cell fusion region of the H A 2 part of the H A of influenza viruses A
and B were found to bind to h u m a n antibodies and anti-HA monoclonal
antibodies (54). Although anti-peptide antibodies would bind to the homolo­
gous H A and virus, they failed to neutralize viral activity. Consequently,
more research must be done on the development of cloned and synthetic
influenza protein vaccines, perhaps including the neuraminidase (NA) of the
viral envelope. One such subunit vaccine containing both the H A and NA
moieties of influenza virus shows considerable promise in mouse tests (55).
The principal immunogens of the Bunyaviridae are two glycoproteins G1
and G coded for by the mid-sized viral R N A segment (56). The cloning and
expression underway for these moieties of Rift Valley fever virus may pro­
duce a subunit vaccine for use in the prevention of this dangerous zoonotic
disease. The arenaviruses also contain one or two surface glycoproteins and
although not fully characterized for immunogenic activity (57), glycoprotein
p G P - C encoded by the small R N A of Pichinde virus is considered to be a
candidate subunit vaccine worth cloning (17).
2
The surface glycoproteins of the nonsegmented negative-stranded RNA
viruses (Paramyxo- and Rhabdoviridae) have also been examined for their
immunogenicity. Paramyxoviruses have two surface glycoproteins, one of
them having hemagglutinin (H) activity or hemagglutinin-neuraminidase
(HN) activities and the other cell fusion and hemolytic activities (F protein).
H protein isolated from measles virus (58) or H N protein from Sendai, New­
castle disease, simian-5 and parainfluenza-3 viruses all induce neutralizing
antibody (59, 60). The F proteins induce antibodies that prevent the cell-tocell spread of virus (59). Experiments are in progress in several laboratories
to clone the H , H N , F and matrix proteins of the paramyxoviruses (61, P.
Choppin, personal communication). Rhabdoviruses possess the immunogenic
glycoprotein G (hemagglutinin). Rabies virus subunits or isolated glycopro­
tein G induce neutralizing antibody and immunity in animals (62). Glycopro­
tein G genes of rabies (13) and vesicular stomatitis (VS) (12) viruses have
been cloned in E. coli. The vesicular stomatitis G gene cloned product is pre­
cipitated by antibody to virus, indicating that it contains G epitopes. Also, a
cloned anchorless form of the G protein of VS virus is slowly secreted'from
eukaryotic cells (63). These cloned G proteins are expected to be useful
— 641 —
immunogens especially for rabies, because whole-virus rabies vaccines can
produce severe neurological reactions.
Retroviruses (RNA viruses that replicate by a D N A pathway) have enve­
lope glycoproteins with immunogenic activity. Glycoprotein gp71 isolated
from Friend murine leukemia virus immunizes mice against challenge with
virus (64). Also, a vaccine containing feline leukemia virus subunits and solu­
ble tumor cell antigens is about 80 percent effective in providing protection
against viral challenge (65). Similar results for retrovirus diseases like bovine
leukemia, equine infectious anemia and h u m a n T-cell leukemia/lymphoma
would represent a major step in cancer prevention. Synthetic antigens may
also be of use because a pentadecapeptide encoding a nucleotide sequence at
the 3' end of Maloney leukemia virus D N A has been shown to raise antibody
to envelope proteins of the virus (66).
The hepatitis B surface antigen (HBsAg) from hepatitis B virus (a still
unclassified virus) was shown to protect chimpanzees against challenge with
virus (67), and this antigen isolated from h u m a n sera is now in use as a vac­
cine for humans (68). It appears to show cross-type neutralizing antibodies to
all hepatitis B viruses that possess the a surface antigen (69). The gene for
HBsAg has been cloned by several laboratories, and the products shown to
absorb to antibody to authentic HBsAg (4). Cloning in yeast produces 22 nm
particles of HBsAg similar to the multimeric structure of the natural antigen
(W.J. Rutter, report Intl. Cong. Virol., 1981). In addition, a synthetic hydrophilic peptide simulating residues 138 to 149 of HBsAg reacts with authentic
antibody (5), and other synthetic peptides corresponding to this and other
regions of HBsAg induce antibodies reactive with the native virus envelope
protein (6, 7).
SUBUNIT V A C C I N E S F O R B A C T E R I A L DISEASES
Genetic engineering is being successfully applied to the preparation of
protein vaccines against bacterial diseases. Much of the early work has focu­
sed on the somatic pili of enterotoxigenic E. coli (ETEC) that cause diarrheal
disease in neonatal livestock and h u m a n s . The somatic pili of E T E C are nonflagellar appendages composed of 14 to 22 kd protein molecules called pilin
that promote adhesion to and colonization of the mucosal surface of the
small intestines (70). Immunologically distinctive strains of pili have been iso­
lated from E T E C : K88 and 897P from swine; K99 from cattle, sheep and
swine; CFA 1 and 2 from h u m a n s ; and type I and other common pili. Isola­
ted pili have been used as vaccines against ETEC-induced diarrhea in both
humans and animals. Some pilin genes appear to be coded for on chromoso­
mes (type I and probably 897P) and others on plasmids (K88, K99 and C F A 1
and 2) (70). The production by cloning of some of the pili that are coded for
on plasmids has been achieved and a cloned vaccine is reported to be availa­
ble for use in animals in one or more European countries (14).
A similar pilus vaccine is being engineered for use in humans against
penicillin-resistant strains of Neisseria gonorrhoeae (15, 16). A plasmid vec-
— 642 —
tor containing a gonococcal D N A segment that encodes pili has been construc­
ted and used to transform E. coli. The reprogrammed E. coli have been detec­
ted with pili-specific monoclonal antibody, indicating the expression of protein
carrying epitopes of gonococcal pili. Additional refinements of this work could
produce an effective bioengineered subunit vaccine against gonorrhea.
P R O S P E C T S F O R SUBUNIT V A C C I N E S F O R P A R A S I T I C DISEASES
Not being researchers on parasitic diseases, the present authors will specu­
late only briefly on the extension of genetic engineering principles to the con­
trol of parasitic diseases, using Trypanosoma rhodesiense as an example (71).
People infected with this parasite develop African sleeping sickness. Protec­
tive antibodies are produced that react with the organism's surface glycopro­
tein, thereby initiating the killing of most of the parasitic population. Howe­
ver, resistant parasites develop which have one or another of 20 or more
variant surface glycoproteins that can be coded for and expressed by T. rho­
desiense. This process of immunity and reinfection repeats itself several times
until the host succumbs. The prospect of cloning the many variant surface
glycoproteins that can be presented by T. rhodesiense is a formidable but not
impossible task that will no doubt await work on agents having less surface
protein complexity. Meanwhile, the recent finding of two types of antibodies
against phospholipid regions of the T. rhodesiense membrane that may not
be able to undergo variation presents a new avenue for investigation and a
possible explanation of how rabbits experimentally infected with the parasite
can undergo a self-curing process.
MONOCLONAL ANTIBODY
Monoclonal antibody technology is a form of genetic engineering that has
initiated a revolution in research, diagnosis and control of animal and human
diseases.
Like recombinant D N A technology, monoclonal antibody is a recent
innovation, having been first reported by Kohler and Milstein in 1975 (72)
under the revealing title « Continuous cultures of fused cells secreting anti­
body of predefined specificity ». Such antibody, now known as monoclonal
antibody because it is a homogeneous population of identical molecules, is
produced by a hybridoma resulting from the fusion of an antibodyproducing cell with a neoplastic myeloma cell. The laboratory manipulations
required to produce a hybridoma are lengthy and quite laborious, but once
achieved the hybridoma, through its replication can theoretically produce in
perpetuity a given homogeneous antibody in amounts ranging from up to
100 µg/ml of culture fluid to 10 m g / m l of ascitic fluid from a hybridomabearing mouse (73).
Hybridomas can be frozen in liquid nitrogen for regrowth later on. Whe­
reas monoclonal antibodies are more specific than polyclonal antibodies,
— 643 —
they do have certain limitations. They cannot always form the networks
required to precipitate antigen, and some monoclonal antibodies do not fix
complement. They may also not be very useful for initial screening of a
disease, because their high specificity may recognize only a particular variant
or serotype of the disease agent.
Also, monoclonal antibodies are not strictly monospecific, because they
may bind with considerable avidity to certain viral or cellular antigens that
have structural similarity to the homologous epitope. Nevertheless, the
important uses of monoclonal antibodies far outweigh their few limitations.
The applications of monoclonal antibody are already numerous in both
the investigation and control of animal and h u m a n diseases. Categories of
work include :
(i) purification of antigens,
(ii) elucidation of the complexities of the immune network,
(Hi) analysis of the epitope composition of antigens ranging from single
protein molecules to complex organisms,
(iv) diagnosis of diseases,
(v) treatment of diseases,
(vi) enhancement of progress toward the development of anti-idiotype
vaccines.
Only a few representative examples of the applications of monoclonal
antibodies can be presented here. Monoclonal antibodies covalently coupled
to affinity gel columns are being exploited for their ability to obtain pure
antigens from complex mixtures including valuable products from genetically
engineered hosts (74). F o r this purpose, hybridoma and recombinant D N A
technologies have converged to be highly complementary to each other. With
regard to the complexities of the immune network, monoclonal antibodies
are revealing differences in T-cell populations and are characterizing the
major histocompatibility complex of antigens more precisely than was possi­
ble with polyclonal antibodies.
In virology, in particular, monoclonal antibodies are being used effecti­
vely to construct antigenic maps of the surface proteins and to establish the
loci of the epitopes involved in phenotypically recognizable mutations.
Influenza, herpes, rabies, adeno, mouse m a m m a r y tumor, polio, foot and
mouth, rubella, canine parvogastroenteritis, feline panleukopenia and bluetongue viruses are among those being analyzed with monoclonal antibodies.
In addition to their diagnostic potential, monoclonal antibodies are useful in
identifying epitopes that are involved in generating a protective immune res­
ponse, and consequently of importance in designing effective molecularly
cloned and synthetic vaccines.
They are also useful for the identification of pathogenic sites in viruses
(75). Thus, growth of fixed rabies virus pathogenic for mice in tissue culture
in the presence of anti-glycoprotein monoclonal antibody selects for nonpa-
— 644 —
thogenic variants having isoleucine or glutamine substituted for arginine at
position 333 of the viral surface glycoprotein G, indicating that arginine at
this position is critical for pathogenicity in the mouse.
Antigenic analysis of parasites using monoclonal antibodies is especially
important because of the large number of epitopes involved and their varia­
tion during developmental cycles and exposure to immunological defenses.
Monoclonal antibodies have been produced to several parasites including
trypanosomes, Theileria parva (East Coast fever) and Plasmodium
berghei
(76). Monoclonal antibody has been shown to inhibit a step in the morphoge­
nesis of Trypanosoma cruzi, the agent of Chaga's disease (77).
Because of their high titers especially in ascites fluids, monoclonal antibo­
dies are being used in passive immunotherapy. It has been suggested that
shipping fever in cattle could be controlled by the administration of monoclo­
nal antibody to parainfluenza-3 virus (76).
There are great expectations for monoclonal antibodies in the early dia­
gnosis and treatment of oncogenic diseases. Experimental studies and clinical
trials show promise in causing monoclonal antibodies to couple to tumorspecific antigens on cells with little or no coupling to normal cellular antigens
(8). The monoclonal antibody can carry radio-labels to the tumor cells for
diagnostic purposes and cytotoxic drugs, toxins or cell-lysing enzymes for
treatment of the disease.
Monoclonal antibodies also make the development of anti-idiotype anti­
body vaccines a more realistic goal, particularly of anti-paratope antibody
vaccines (C. Bona, personal communication). The paratope is the binding
site in the crevice of the F portion of antibody and is the molecular comple­
ment of a specific epitope of the inciting antigen. Being foreign to the ani­
mal, paratopes raise antibody having sites that mimick epitopes of the origi­
nal antigen. Consequently, anti-paratope antibodies have potential as vacci­
nes, particularly after amplification in nybridomas or cloning and expression
in single-celled hosts of ds c D N A made on anti-paratope antibody mRNA.
a b
Monoclonal antibodies also hold promise as replacements for antivenoms
produced in rabbits and horses. Monoclonal antivenoms are being developed
aimed specifically at the lipase-like enzyme component in brown recluse spi­
der venom which is more deadly than rattlesnake venom (79). Indications are
that the monoclonal antivenom is superior to the natural antivenom produ­
ced by rabbits.
INTERFERONS FOR ANIMALS
Interferons, described in 1957 (80) and investigated extensively since then,
consist of a somewhat heterogeneous group of proteins divided into classes
called
, α,βand y. Interferons modulate several immunological activities
including antibody production, immediate and delayed hypersensitivities as
well as cytotoxic and macrophage functions (81).
— 645 —
The α,β and 7 classes of interferons are produced, respectively, in leu­
kocytes, fibrocytes/epitheliocytes and lymphocytes in response to a variety of
inducers such as viruses, polynucleotides and bacterial products for αand β
interferons, and antigens, antigen-antibody complexes, antilymphocyte
serum and mitogens for 7 interferons.
Interpretations of the induction and functions of interferons have often
been inconclusive because of the large number of variables involved and the
small amount and impurity of the interferon being studied. During the last
three years, however, better preparations of interferons have become availa­
ble from production-scale cell cultures. Very recently, virtually pure recombi­
nant interferon has been produced from E. coli in amounts sufficient for clini­
cal trials against immunological disorders, infectious diseases and neoplasms.
Interferons originally appeared to be animal-species specific, that is inter­
feron produced in calf cells would protect calf cells but not chicken cells
against viral infection, and vice versa (82).
Now, however, it is known that this characteristic is variable depending to
some extent on the class of interferon. T h u s , some h u m a n αinterferons pro­
tect cells of other species, whereas h u m a n βinterferons will not do so. In this
regard, human recombinant αinterferons protect mice against lethal doses of
encephalomyocarditis virus (83). Moreover, recombinant D N A technology
has produced hybrid interferons with relatively high activities on human,
bovine and murine cell lines.
In several autoimmune diseases, the frequency of occurrence and concen­
tration of interferons appears to be generally higher in diseased patients than
in normal individuals. Whereas the precise role of interferon in these diseases
is obscure, the administration of interferon to New Zealand black mice acce­
lerates systemic lupus erythematosus (SLE) in these animals (84). In accor­
dance with this finding, herpes zoster infections appear to occur less fre­
quently in patients with active SLE t h a n in those with latent disease, presu­
mably due to the higher levels of interferon in the former group. In contrast,
certain diseases such as lymphoid malignancies and some autoimmune, viral
and immunodeficiency disorders correlate with defects in 7 interferon pro­
duction (81).
Much remains to be clarified of the specific m o d e of action and therapeu­
tic effectiveness of interferons, but the availability now of recombinant inter­
ferons provide material for use in clinical trials against viral and oncogenic
diseases of humans. The results of this work will eventually benefit veterinary
medicine, for example, in the treatment of valuable breeding stock, race hor­
ses and show and companion animals. Since interferon generally has to be
inoculated at least once daily for several days, timed-release injection
methods would be beneficial.
*
* *
— 646
Appendix
51st G E N E R A L SESSION O F T H E O . I . E .
R E S O L U T I O N N o . II
GENETIC ENGINEERING
INFLUENCE ON CONTROL A N D PREVENTION OF ANIMAL DISEASES
CONSIDERING
that genetic engineering is one of our newest technologies and research is
underway in many laboratories attempting to apply this technology to
improve the quality, quantity, safety and cost of animal vaccines,
that this technology involves manipulation of cellular or viral genomes
whereby desired gene products such as proteins are made by a host organism
or animal for treatment of disease,
that large quantities of immunogens of organisms are being produced by
gene cloning and that the availability of these products in such quantities has
been responsible for more rapid sequencing t h a n would otherwise have been
feasible,
that examination of these sequences enables selection of major antigenic
sites usually much shorter that the entire immunogens, and
that knowledge of these short antigenic sites has made organic synthesis
of peptides feasible and that small linear amino acid sequences synthesised by
different organic chemical methods and coupled to a carrier protein act as
immunogens like natural antigens
THE COMMITTEE
RECOMMENDS
1. That research efforts should continue to explore genetic engineering to
develop through this technology safer, more potent and less costly vaccines
and other products to control animal diseases.
2. That words of caution are also in order, not to stop research and use of
existing vaccines many of which have been effective in controlling diseases.
3. That exploration of monoclonal antibodies is to be encouraged to
increase the basic information and evaluate usefulness of such products for
diagnosis, delivery mechanisms for drugs and study of molecular properties
of microbes.
4. That though much remains to be learned about interferons, especially
their mode of action and therapeutic effectiveness, the availability of recom­
binant interferon provides abundant material for study in clinical trials
against infections. Interferons should benefit veterinary medicine by provi­
ding a means of therapy for infections in valuable breeding stock, race horses
and companion animals and should continue to be researched.
— 647 —
5. That since viruses such as vaccinia a n d SV 40 are also being used as
vectors for immunogens of other viruses, the veterinary and public health
hazard of using such products must be considered. O n the other hand, all
possibilities of t h e new techniques should be explored to develop vaccines
against diseases for which none exist.
6. That the O . I . E . consider promoting through other international agen­
cies like F . A . O . , W . H . O . / P . A . H . O . , I . I . C . A . proposals to develop field
assays and t o coordinate research in genetic engineering. Such actions may
assist in orderly transfer of this technology.
7. That staff in public and private laboratories be cautioned about the
possible hazards of moving microbial genetic material from one country t o
another and, where such material does not now exist, such movements should
be regulated by appropriate control authorities.
8. That the O . I . E . develop guidelines for the international movement of
microbial genetic material.
(Adopted by the International
Committee
of the O.I.E. on 25 May 1983.)
*
**
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ERRATUM
Page 631, third paragraph, line 6 :
Instead of : "antigenic (epitopes)",
read : "antigenic determinants (epitopes)".