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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.) * ** REFERENCES 1. KLEID D . G . , YANSURA D., SMALL B . , DOWBENKO D., MOORE D . M . , GRUBMAN M . J . , MCKERCHER P.D., MORGAN D.O., ROBERTSON B . H . and BACHRACH H . L . (1981). — Cloned viral protein vaccine for foot and mouth disease : Responses in cattle and swine. Science, 214, 1125-1129. 2. BITTLE J . L . , HOUGHTEN R.A., ALEXANDER H . , SHINNICK T . M . , SUTCLIFFE J . G . , LERNER R.A., ROWLANDS D . J . and BROWN F . (1982). — Protection against foot and mouth disease by immunization with a chemically synthesized peptide predic ted from the viral nucleotide sequence. Nature, 298, 30-33. 3. PFAFF E . , MUSSGAY M . , BOHM H . O . , SCHULZ G . E . and SCHALLER H . ( 1 9 8 2 ) . — Antibodies against a preselected peptide recognize and neutralize foot and mouth disease virus. EMBO J., 1, 869-874. 4. EDMAN J . C . , HALLEWELL R.A., VALENZUELA P., GOODMAN H . M . and RUTTER W.J. (1981). — Synthesis of hepatitis B surface and core antigens in E. coli. Nature, 291, 5 0 3 - 5 0 6 . 5. HOPP T . P . and WOODS K.R. (1981). — Prediction of protein antigenic determi nants from amino acid sequences. Proc. Natl. Acad. Sci. USA, 78, 3824-3828. 6. LERNER R.A., GREEN N . . ALEXANDER H . , LIU F . T . , SUTCLIFFE J . G . and SHIN- NICK T . M . (1981). — Chemically synthesized peptides predicted from the nucleo tide sequence of the hepatitis B virus genome elicit antibodies reactive with the envelope protein of Dane particles. Proc. Natl. Acad. Sci. USA, 78, 3403-3407. 7. DREESMAN G . R . , SANCHEZ Y . , IONESCU-MATIU I . , SPARROW J . T . , Six H . R . , PETERSON D . L . , HOLLINGER F . B . and MELNICK J . L . ( 1 9 8 2 ) . — Antibody to hepa- — 648 — titis B surface antigen after a single inoculation of uncoupled synthetic HBsAg peptides. Nature, 295, 158-160. 8. WATSON R.J., WEIS J.H., SALSTROM J . S . and ENQUIST L.W. (1982). — Herpes simplex virus type-1 glycoprotein D gene : nucleotide sequence and expression in Escherichia coli. Science, 218, 381-384. 9. EMTAGE J . S . , TACON W.C.A., CATLIN G.H., JENKINS B., PERTER A.G. and COREY N . H . (1980). — Influenza antigenic determinants are expressed from hemagglutinin genes cloned in Escherichia coli. Nature, 283, 171-174. 10. HEILAND I. and GETHING M . J . (1981). — Cloned copy of the hemagglutinin genes codes for human influenza antigenic determinants in E. coli. Nature, 292, 851-852. 11. DAVIS A.R., NAYAK D.P., UEDA M., H m A . L . , DOWBENKO D. and KLEID D.G. (1981). — Expression of antigenic determinants of the hemagglutination gene of human influenza virus in Escherichia coli. Proc. Natl. Acad. Sci. USA, 78, 53765380. 12. ROSE J . K . and SHAFFERMAN A. (1981). — Conditional expression of the vesicular stomatitis virus glycoprotein gene in Escherichia coli. Proc. Natl. Acad. Sci. USA, 78, 6670-6674. 13. YELVERTON E., NORTON S., OBIJESKI J . F . and GOEDDEL D.V. (1983). — Rabies glycoprotein analogs : biosynthesis in Escherichia coli. Science, 219, 614-620. 14. TREVIS J . and BERTELSEN A. (1982). — Genetic engineering : Promise for agricul tural industries. Feedstuffs, 54 (5), 32-37. 15. Research Report (1982). — Recombinant DNA isolation of the genes specifying an attachment organelle (pili) of Neisseria gonorrhoeae. Walter Reed Army Inst. Res., Div. Communicable Dis. and Immunol., 3-4, 1-2. 16. MEYER T.F. (1982). — Pilus expression in Neisseria gonorrohoeae involves chro mosomal rearrangement. Cell., 30, 45-52. 17. RAMSINGH A. and LEUNG W.C. (1982). — Molecular cloning and gene mapping of the Pichinde viral genome. IV Intl. Conf. on Comparative Virol., Banff Alberta Canada, Abs. 54-11, 105. 18. KAADEN O . R . , ADAM K . H . and STROHMAIER K . (1977). — Induction of neutrali zing antibodies and immunity in vaccinated guinea pigs by cyanogen bromidepeptides of VP3 of foot and mouth disease virus. J. gen. Virol., 34, 397-400. 19. BACHRACH H.L., MORGAN D . O . and MORE D.M. (1979). — Foot and mouth disease immunogenic capsid protein VPT : N-terminal sequences and immunoge nic peptides obtained by CNBr and tryptic cleavages. Intervirology, 12, 65-72. 20. BACHRACH H.L., MORGAN D . O . , MCKERCHER P.D., MOORE D.M. and ROBERT SON B.H. (1982). — Foot and mouth disease virus : Immunogenicity and struc ture of fragments derived from capsid protein VP3 and of virus containing clea ved VP3. Vet. Microbiol., 7, 85-96. 21. ATASSI M.Z. (1980). — Precise determination of protein antigenic structures has unravelled the molecular immune recognition of proteins and provided a prototype for synthetic mimicking of other binding sites. Mol. Cellular Biochem., 32, 21-44. — 649 — 22. BACHRACH H . L . (1982). — Recombinant DNA technology for the preparation of subunit vaccines. J. Am. Vet. Med. Assoc., 181, 992-999. 23. WALTER G . , SCHEIDTMANN K . H . , CARBONE A., LAUDANO A.P. and DOOLITTLE R.F. (1980). — Antibodies specific for the carboxy- and amino-terminals regions of simian virus 40 large tumor antigen. Proc. Natl. Acad. Sci. USA, 77, 5197-5200. 24. GINSBERG H . S . (1975). — Subunit viral vaccines. In : Notkins A.L. (ed.) : "Viral Immunology and Immunopathology". New York, Academic Press, Inc., 317326. 25. KAADEN O . R . and DIETZSCHOLD B . (1974). — Alterations of the immunological specificity of plasma membranes from cells infected with Marek's disease and turkey herpesvirus. J. gen. Virol., 25, 1-10. 26. LUPTON H . W . and REED D.E. (1980). — Evaluation of experimental subunit vaccines for infectious bovine rhinotracheitis. Am. J. vet. Res., 41, 383-390. 27. Dix R.D., PEREIRA L . and BARINGER J.R. (1981). — Use of monoclonal antibody directed against herpes simplex virus glycoproteins to protect mice against acute virus-induced neurological disease. Infect. Immun., 34, 192-199. 28. BAXBY D. (1982). — The surface antigens of orthopoxviruses detected by crossneutralization tests on cross-absorbed antisera. J. gen. Virol., 58, 251-262. 29. APPLEYARD C . and ANDREWS C . (1974). — Neutralizing activities of antisera to poxvirus soluble antigens. J. gen. Virol., 23, 197-200. 30. BALACHANDRAN N., SETH P. and MOHAPATRA L . N . (1980). Immune response in rabbits to surface components of extracellular and intracellular forms of vacinia virus. Infect. Immun., 29, 846-852. 31. MACKETT M., SMITH G . L . and Moss B . (1982). — Vaccinia virus : A selectable eukaryotic cloning and expression vector. Proc. Natl. Acad. Sci. USA, 79, 74157419. 32. JOKLIK W . K . (1981). — Structure and function of the reovirus genome. Microbiol. Rev., 45, 483-501. 33. HUISMANS H . and ERASMUS B . J . (1981). — Identification of the serotype-specific and group-specific antigens of bluetongue virus. Onderstepoort J. vet. Res., 48, 51-58. 34. LETCHWORTH G . J . and APPLETON J.A. (1982). — Abstract 1st Annual Cong. Hybridoma Res., Feb. 1982. 35. BASTARDO J . W . , MCKIMM-BRESCHKIN J . L . , SONZA S., MERCER L . D . and HOL- MES I.H. (1981). — Preparation and characterization of antisera to electrophoretically purified SA11 virus polypeptides. Infect. Immun., 34, 641-647. 36. KILLEN H . M . and DIMMOCK N.J. (1982). — Identification of a neutralisationspecific antigen of a calf rotavirus. J. gen. Virol., 62, 641-647. 37. LAPORTE J., GROSCLAUDE J., WANTYGHEM J., BERNARD S. and ROUZÉ P. (1973). — Neutralisation en culture cellulaire du pouvoir infectieux du virus de la fièvre aphteuse par des sérums provenant de porcs immunisés à l'aide d'une protéine virale purifiée. C.R. Acad. Sci. Paris, 276, 3399-3401. — 650 — 38. BACHRACH H.L., MOORE D.M., MCKERCHER P.D. and POLATNICK J. (1975). — Immune and antibody responses to an isolated capsid protein of foot and mouth disease virus. J. Immun., 115, 1636-1641. 39. HOFSHNEIDER P.H., BURGELT E., KAUZMANN М., MUSSGAY М., FRANZE R., AHL R., BoHM H., STROHMAIER K., KuPPER H. and OTTO B. (1981). — Studies on the antigenicity and immunogenicity of the foot and mouth disease viral pro tein VP1 expressed in E. coli. In : Bachmann P.A. (ed.) : Munich Symp. on Microbial Biological Products for Viral Diseases. London, Taylor & Francis, Ltd., 105-113. 40. HENDERSON W.M. (1982). — Advances in vaccines against FMD : prospects for the use of vaccines prepared by genetic engineering techniques. O.I.E. Foot and mouth Disease. 16th Conference of the FMD Commission, 299-307. 41. BACHRACH H.L., MOORE D.M., McKERCHER P.D. and POLATNICK J. (1978). — An experimental protein vaccine for foot and mouth disease. In : Pollard M. (ed.) : "Perspectives in Virology", X. New York, Raven Press, 147-159. 42. BLONDEL В., CRAINIC R. and HORODNICEANU F. (1982). — Le polypeptide struc tural VP1 du poliovirus type 1 induit des anticorps neutralisants. C.R. Acad. Sci. Paris, 294, 91-94. 43. CHOW M. and BALTIMORE D. (1982). — Isolated poliovirus capsid protein VP1 induces a neutralizing response in rats. Proc. Natl. Acad. Sci. USA, 79, 75187521. 44. SIMONS K., HELENIUS A., MOREIN В., BALCOROVA J. and SHARP M. (1980). — Development of effective subunit vaccines against enveloped viruses, Mizrahi A., Hertman I., Klingberg M.A., et al. (ed.) : In New Developments with Human and Veterinary Vaccines, New York, Alan R., Liss Inc., pp 217-228. 45. HEINZ F.X., TUMA W. and KUNZ С. (1981). — Antigenic and immunogenic pro perties of defined physical forms of tick-borne encephalitis virus structural pro teins. Infect. Immun., 33, 250-277. 46. DALSGAARD K. and OVERBY E. (1976). — Vaccination of pigs against hog cholera (classical swine fever) with a detergent split vaccine. Acta Vet. Scand., 17, 465-474. 47. CAPPEL R. and DECUYPER F. (1976). — Efficacy and immune response to rubella subunit vaccines. Arch. Virol., 50, 207-213. 48. TRUDEL M. and PAYMENT P . (1982). — Antibody response to rubella virus pro teins in different physical forms. IV Intl. Conf. on Comparative Virol., Banff Alberta Canada, Abs. S3-6, 88. 49. SCHMIDT O.W. and KENNEY G.E. (1982). — Polypeptides and functions of anti gens from human coronaviruses 229E and OC43. Infect. Immun., 35, 515-522. 50. United States Department of Health, Education and Welfare (1976). —Morbidity and Mortality Weekly Report, 25, 357-369. 51. GETHING M.J. and SAMBROOK J. (1981). — Cell-surfaced expression of influenza haemagglutinin from a cloned DNA copy of the RNA gene. Nature, 293, 620-625. — 651 — 52. GETHING M . J . and SAMBROOK J. (1982). — Construction of influenza haemagglutinin genes that code for intracellular and secreted forms of the protein. Nature, 300, 598-603. 53. GREEN N., ALEXANDER H . , OLSEN A., ALEXANDER S., SHIMMICK T . M . , SUT- CLIFFE J . G . and LERNER R.A. (1982). — Immunogenic structure of the influenza virus haemagglutinin. Cell, 28, 477-487. 54. ATASSI M . Z . and WEBSTER R.G. (1983). — Localization, synthesis and activity of an antigenic site on influenza virus haemagglutinin. Proc. Natl. Acad. Sci. USA, in press. 55. MOREIN B., SUNDQUIST B. and HOGLUND S. (1982). — A new subunit complex including virus membrane proteins as effective vaccines. IV Intl. Conf. on Com parative Virol., Banff Alberta Canada, Abs. 53-4, 87. 56. GENTSCH J.R., ROZHON E.J., KLIMAS R.A., E L SAID L . M . , SHOPE R.E. and BIS HOP D.H.L. (1980). — Evidence from recombinant bunyavirus studies that the M RNA gene products elicit neutralizing antibodies. Virology, 102, 190-204. 57. BISHOP D . H . L . , BEATY B.J. and SHOPE R.E. (1980). — Recombination and gene coding assignments of bunyaviruses and arenaviruses. Ann. N. Y. Acad. Sci., 254, 84-106. 58. BELLINI W . J . , MCFARLIN D.E., SILVER G . D . , MINGIOLI E.S. and MCFARLAND H.F. (1981). — Immune reactivity of the purified hemagglutinin of measles virus. Infect. Immun., 32, 1051-1057. 59. MERZ D.C., SCHEID A. and CHOPPIN P . W . (1980). — Importance of antibodies to the fusion glycoprotein and paramyxoviruses in the prevention of spread of infection. J. Exp. Med., 151, 275-283. 60. HOSAKA Y. (1980). — Separation of Sendai virus glycoproteins by using glutaraldehyde-treated erythrocytes and preparation of monospecific antisera against the glycoproteins. Infect. Immun., 30, 212-218. 61. ROZENBLATT S., GESANG C , LAVIE V. and NEUMANN F.S. (1982). — Cloning and characterization of DNA complementary to the measles virus mRNA enco ding hemagglutinin and matrix protein. J. Virol., 42, 790-797. 62. Cox J . H . , DIETZSCHOLD B., WEILAND F. and SCHNEIDER L . G . (1980). — Prepa ration and characterization of rabies virus hemagglutinin. Infect. Immun., 30, 572-577. 63. ROSE J.K. and BERGMANN J.E. (1982). — Expression from cloned cDNA of cellsurface secreted forms of the glycoprotein of vesicular stomatitis virus in eucaryotic cells. Cell, 30, 753-762. 64. IHLE J.N., LEE J.C., COLLINS J.J., FISCHINGER P.J., PAZMINO N . H . , MOENNING V., SCHAFER W . , HAMIA Jr., M . G . and BOLOGNESI D.P. (1976). — Characteriza tion of the immune response to the major glycoprotein (gp 71) of Friend leukemia virus II. Response in C57BL/6 mice. Virology, 75, 88-101. 65. LEWIS M . G . , MATHES L.E. and OLSEN R.G. (1981). — Protection against feline leukemia by vaccination with subunit vaccine. Infect. Immun., 34, 888-894. — 652 — 66. SUTCLIFF J . G . , SHINNICK T . M . , GREEN N., LIU F . T . , NIMAN H.L. and LERNER R . A . (1980). — Chemical synthesis of polypeptide predicted from nucleotide sequence allows detection of a new retroviral gene product. Nature, 287, 801-805. 67. DREESMAN G . R . , HOLLINGER F . B . , SANCHEZ Y., OEFINGER P. and MELNICK J.L. (1981). — Immunization of chimpanzees with hepatitis B virus-derived poly peptides. Infect. Immun., 32, 62-67. 68. HILLEMAN M . R . , BUYNAK E . B . , MCALEER W . J . , MCLEAN A . A . , PROVOST P.J. and TYTELL A . A . (1981). — Newer developments with human hepatitis vaccines. In : Pollard M . (ed.) : "Perspectives in Virology", XI. New York, Allan R . Liss, Inc., 219-247. 69. SZMUNESS W . , STEVENS C.E., HARLEY E . J . , ZANG E . A . , ALTER H.J., TAYLOR P . E . , DEVERA A . , CHEN G . T . S . and KELLNER A . (1982). — Dialysis vaccine trial study group : Hepatitis B vaccine in medical staff of hemodialysis units : efficacy and subtype cross-protection. New Engl. J. Med., 307, 1481-1486. 70. ISSACSON R . E . (1981). — Pili of enterotoxigenic Escherichia coli. Proc. Int. Symp. Neonatal Diarrhea, 3, 213-236. 71. Research Report : Experimental African Trypanosomiasis (1982). — Studies on animals that undergo self-cure. Walter Reed Army Inst. Res., Div. Biochem., 3-4, 4-5. 72. KOHLER G . and MILSTEIN C (1975). — Continuous cultures of fused cells secre ting antibody of predefined specificity. Nature, 256, 495-497. 73. OXFORD J. (1982). — The use of monoclonal antibodies in virology. J. Hyg., Camb., 88, 361-368. 74. DALCHAU R . and FABRE J . W . (1982). — The purification of antigens and other studies with monoclonal antibody affinity columns : the complementary new dimension of monoclonal antibodies. In : McMichael A . J . , Fabre J . W . (eds.) : "Monoclonal Antibodies in Clinical Medicine". Academic Press, 519-556. 75. DIETSZCHOLD B., WUNNER W . H . , WIKTOR T . J . , LOPES A . W . , LAFON M., SMITH C.L. and KOPROWSKI H. (1983). — Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus. Proc. Natl. Acad. Sci. USA, 80, 70-74. 76. ANTCZAK D . F . (1982). — Monoclonal antibodies : technology and potential use. J. Am. Vet. Med. Assoc., 181, 1005-1010. 77. SHER A. and SNARY D . (1982). — Specific inhibition of the morpho-genesis of Trypanosoma cruzi by a monoclonal antibody. Nature, 300, 639-640. 78. THORPE P.E., EDWARDS D.C., DAVIES A.J.S. and Ross W.C.J. (1982). — Monoclonal antibody-toxin conjugates : aiming the magic bullet. In : McMichael A.J., Fabre J.W. (eds.) : "Monoclonal Antibodies in Clinical Medicine". Acade mic Press, 167-201. 79. REESE K.M. (1983). — Editorial entitled "An antivenom for the brown recluse spider", in Chemical and Engineering News, p. 78. 80. ISSACS A. and LINDENMANN J. (1957). — Virus interference. I . The interferon. Proc. Royal Soc. London (Biol.), 147, 258-267. — 653 — 81. HOOKS J . J . , DETRICK-HOOKS B . and LEVINSON A.I. (1982). — Interferons and immune reactivity. J. Am. Vet. Med. Assoc., 181, 1111-1114. 82. SAKAGUCHI A.Y., STEVENSON D . and GORDON I. ( 1 9 8 2 ) . — Species specificity of interferon action : A functioning homospecific nucleus is required for induction of antiviral activity in heterokaryons. Virology, 116, 441-453. 83. WECKS P . K . , RINDERKNECHT E . , ESTELL D . A . and STEBBING N . ( 1 9 8 2 ) . — Anti viral activity of bacteria-derived human alpha interferons against encephalomyocarditis virus infection of mice. Infect. Immun., 35, 660-665. 84. HEREMANS H . , BILLIAU A., COLUMBATTI A., HILGERS J . and DESOMER P . (1978). — Interferon treatment of N Z B mice : accelerated progression of autoim mune disease. Infect. Immun., 21, 925-930. ERRATUM Page 631, third paragraph, line 6 : Instead of : "antigenic (epitopes)", read : "antigenic determinants (epitopes)".