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Vaccinia: Virus, Vector, Vaccine
ADVANCES IN VIRUS RESEARCH, VOL.34
VACCINIA: VIRUS, VECTOR, VACCINE Antonia Piccini and Enzo Paoletti Laboratory of Immunology Wadsworth Center for Laboratories and Research New York State Department of Health Albany, New York 12201
I. Introduction Historical Perspective of Vaccinia Virus 11. The Viral Genome A. Gene Mapping B. DNA Sequence Data 111. Vaccinia Virus as an Expression Vector A. Foreign Genes Expressed by Vaccinia Virus B. General Protocol for the Insertion of Foreign Genes into the Vaccinia Virus Genome C. Analysis of Vaccinia Recombinants IV. Prospectus References
I. INTRODUCTION Historical Perspective of Vaccinia Virus Recently there has been a renaissance in poxvirus research hallmarked by using vaccinia virus as a eukaryotic cloning and expression vector in a variety of biological and clinical applications. This resurgence comes almost 200 years after vaccinia virus was introduced by Edward Jenner as a vaccine against smallpox. Its usefulness as a vaccine was marked by the similar antigenic properties of vaccinia and the smallpox virus, and by the ability of vaccinia to be readily grown in the laboratory, its stability as freeze-dried preparations, and its ease of administration. Vaccination has not been required for the general population since the World Health Organization declared the world free from smallpox in 1980. Vaccinia virus is the prototypic member of a large group of complex animal viruses known as the poxviruses. Detailed discussions on poxviruses can be found in several reviews (Moss, 1985; Dales and Pogo, 1981; Fenner, 1985). An electron micrograph of vaccinia virus is shown in Fig. 1. Enclosed within a brick-shaped outer lipid envelope lies the double-stranded DNA genome of approximately 187,000 base 43
Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ANTONIA PICCINI AND ENZO PAOLETTI
FIG.1. Electron micrograph of negatively stained (phosphotungstic acid) particles of vaccinia virus. Photo generously provided by W. Samsonoff (New York State Department of Health).
pairs (bp). The DNA termini are characteristic in two ways: (1) they are covalently cross-linked so that DNA denaturation results in the formation of a single-stranded circle, and (2) they contain a defined set of direct tandem repetitions which display transcriptional activity and are prone to deletions and rearrangements. The genome is complexed with proteins and enzymes comprising the vaccinia transcriptional machinery. Some of the 200 or so virally encoded gene products include the virion structural polypeptides and an arsenal of enzymes, some of which include RNA polymerase and enzymes that cap and methylate RNA. Additionally, there are a number of nonstructural polypeptides induced on infection, including the DNA polymerase and thymidine kinase. Upon infection, the virus localizes itself to specific regions in the cytoplasm, termed factories, where it undergoes a temporally orchestrated series of developmental stages, including early
VACCINIA: VIRUS, VECTOR, VACCINE
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transcription, DNA replication, late transcription, and virus maturation. Vaccinia virus does not circulate in nature and its exact origin remains obscure. Vaccinia is essentially a laboratory virus. It is highly amenable to in uitro culture techniques and, as an experimentally infectious agent, has a broad host range. Thus, it can infect a variety of animals and is able to replicate in a variety of tissue culture cells. When experimenting with this virus, two unique viral features must be taken into account: (1)the virus replicates independently within the cytoplasm of the infected cell, and (2) the naked DNA of vaccinia is not infectious, unlike the DNA of many other animal viruses, such as herpesviruses, adenoviruses, and papovaviruses. These features, combined with the large DNA size, disallow it from being used as a cloning vector via the “cut and paste” approach commonly used for other viral genomes and plasmids. Instead, the ability to introduce genetic elements into the vaccinia genome is accomplished by marker rescue techniques (Section 111,B). In this review, we will discuss the genetic characterization of vaccinia, a general protocol for the insertion of foreign genes into vaccinia virus, and analysis of the recombinant viruses. It is our intention to show that vaccinia, a virus used historically as a live vaccine for the immunoprophylaxis of smallpox, can now be genetically engineered for the construction of live recombinant viruses and directed against heterologous infectious agents. These recombinants can be used as expression vectors with biological applications ranging from cloning and expression vehicles to live vaccines directed against infectious diseases of both human and veterinary concern. 11. THE VIRALGENOME
A. Gene Mapping Within recent years, the viral genome has been the focus of intense study involving both gene mapping and DNA sequencing. These studies have contributed toward an understanding of the molecular organization and genetic regulation of vaccinia biogenesis, thus defining the molecular characteristics necessary for designing vaccinia as an efficient and versatile expression vector. The mapping and identification of vaccinia genes is a complex endeavor made so by the large genome size, temporal transcription constraints, transcript overlap, and late RNA size heterogeneity. Nevertheless, there has been considerable progress which includes (1)
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ANTONIA PICCINI AND ENZO PAOLE'M'I
mapping genes with assigned in uitro products, (2) mapping genes with assigned in uiuo products and function, and (3) mapping genes with selectable phenotypes. One of the strategies used to map genes involves marker rescue, which is the recovery of genetic characteristics from inactive genomes or genomic fragments. Marker rescue strategies have been used in various bacteriophage and animal virus systems to map genetic loci. The genomic structure of the virus and the infectious nature of the isolated viral DNA are the determining factors for the rescue process. Since purified vaccinia DNA is not infectious, transfected vaccinia DNA could only be rescued by infectious vaccinia virus by in uiuo recombination. Sam and Dumbell (1981) demonstrated this technique using thermosensitive markers and Nakano et al. (1982) demonstrated this approach by reinserting unique vaccinia DNA sequences into vaccinia deletion mutants. Phenotypes such as a-amanitin resistance (Villarreal and Hruby, 1986), temperature sensitivities (Condit et al., 1983; Ensinger and Ravinsky, 1983; Drillien and Spehner, 1983; Thompson and Condit, 1986), and host range (Gillard et al., 1985)have been assigned to DNA segments via marker rescue. A phenotypic property which we have been particularly interested in is vaccinia's sensitivity to the drug rifampicin. Using DNA fragments from a rifampicin-resistant mutant (Rip) in marker rescue experiments, Tartaglia and Paoletti (1985) demonstrated that rifampicin resistance is due to a single-base change. Characterization of the rifampicin locus was continued by demonstrating that this locus is transcriptionally active and that a Rif-specific mRNA complementary to this locus can be translated in uitro into a 63-kDa polypeptide (Tartaglia et al., 1986). In addition to these mapping studies, marker rescue forms the basis for using vaccinia as a eukaryotic cloning vector. As will be discussed later we and others have used modifications of the marker rescue protocol to insert foreign genes into vaccinia (Sections II1,A and 111,B). Additional approaches for gene mapping may employ message selection, which involves isolating specific viral RNAs by hybridization to genomic fragments. The selected RNA can be analyzed directly or translated in vitro to yield products which can either be assayed for enzyme activity or analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A large portion of the vaccinia genome has been analyzed in this way, yielding information pertaining to gene location, RNA polarity, and polypeptide size. If antiserum to an in uiuo product is available, the sera can be used to immunoprecipitate the in uitro translation product to correlate with a viral component. This type of analysis has been used to map viral structural polypeptides, to deter-
VACCINIA: VIRUS, VECTOR, VACCINE
47
mine enzymatic activities, and in some cases for confirmation of expression of a gene after marker rescue. Using message selection, Belle Isle et al. (1981) performed an extensive survey using cloned vaccinia Hind111 fragments to select RNAs, and by in vitro translation identified approximately 75 early and 40 late polypeptides. Although these products were not assigned functions, their identification was indeed useful in establishing the variety of temporally expressed genes and their general location on the vaccinia genome. Moss et al. (1981) and Bedard (1983)used message selection to assign a number of cell-free translation products to the region of the vaccinia genome that distinguishes the two major vaccinia variants (L, large; S, small) of the WR strain. Concentrating on the genomic termini, Wittek et al. (1981) and Cooper et al. (1981) performed transcriptional analyses and in vitro translation which confirmed that the transcripts in these regions encode both early and late gene functions, do not appear to be spliced, have 5' capped ends, can be transcribed in either direction, and are overlapping. These studies established the transcriptional assignments for at least 21 kilobases (kb) of the left end of the vaccinia genome. A considerable amount of mapping has been done within the central region of the genome. For example, Golini et al. (1984) have focused on portions of the Hind F region, Morgan and Roberts (1984) on the Hind NMK regions, and Mahr and Roberts (1984a,b) on the Hind J and H regions. For the most part these investigators presented the general conclusions (1)of the existence of clustered families of RNAs which can be overlapping, sharing 5'-proximal sequences; (2) of transcription occurring bidirectionally and with no particular direction preferred by early or late RNAs; and (3)of late RNAs that are highly heterogeneous at their 3 ' end, a feature that was suspected using hybridization analyses. These studies yielded a great deal of transcriptional information; however, one of the main goals of gene mapping is the identification of a specific function for a particular vaccinia gene product. In this way structure and function mesh t o give a broader picture of the molecular economy of vaccinia gene expression. Vaccinia virus morphogenesis is complex and interdependent such that each event appears to be dependent on the proper completion of the previous one. An understanding of the biological complexity of vaccinia depends upon the elucidation of the interactions of specific components during viral morphogenesis. Therefore, great emphasis has been placed on mapping genes which code for viral components. These include genes encoding some of the vaccinia structural proteins (Wittek et al., 1984a,b; Rose1 and Moss, 1985; Weir and Moss, 1985), an envelope glycoprotein antigen (Hirt et
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ANTONIA PICCINI AND ENZO PAOLETTI
al., 19861, hemagglutinin protein (Shida, 1986), thymidine kinase (Weir et al., 1982; Vassef et al., 1983), DNA polymerase (Jones and Moss, 1984; Traktman et al., 1984), and subunits of the RNA polymerase (Morrison et al., 1985; Broyles and Moss, 1986). A vaccinia core protein was initially identified using an expression vector system. Weir and Moss (1985) used a bacterial expression vector to synthesize a vaccinia product which was then used to generate antiserum for immunoprecipitation of viral components. A vaccinia growth factor was identified (Reisner, 1985; Brown et al., 1985; Bloomquist et al., 1984; Twardzik et al., 1985)using computer technology which allowed the comparison of vaccinia DNA sequences with heterologous DNA sequences in data banks.
B . DNA Sequence Data Presently one of the directions of vaccinia research is toward assessing the DNA sequences responsible for controlling the temporal expression of vaccinia genes. Sequences upstream of mapped RNAs and open reading frames obtained by DNA sequence analysis are being compared to distinguish between early and late transcriptional control signals and/or promotors. Furthermore, since the viral-encoded RNA polymerase is unable to efficiently recognize heterologous promotors, the identification and isolation of vaccinia promotor sequences would facilitate the use of vaccinia as an expression vector. One of the ways in which vaccinia promotor regions have been identified is by mapping RNAs by 5' S, analyses (Berk and Sharp, 1977; Weaver and Weissmann, 1979). Portions of the DNA sequences upstream from the RNA cap site can be tested for promotor activity by using the promotor to direct the expression of a foreign gene in a recombinant vaccinia. An alternative method involves a transient expression system which utilizes the prokaryotic gene encoding chloramphenicol acetyltransferase (CAT). By linking this assayable marker t o a vaccinia sequence and inserting it into a plasmid, CAT activity can be measured as an indication of promotor strength (Cochran et al., 1985a,b). In another promotor search Vassef et al. (1985) used a plasmid rescue technique which allowed the isolation of randomly generated vaccinia DNA fragments which were capable of directing the expression of herpes simplex virus thymidine kinase (HSV-TK) when inserted into a TK- vaccinia virus recombinant. Promotor regions have also been indirectly recognized by sequencing large portions of the vaccinia genome. By converting the DNA sequence to amino acid sequences, open reading frames (ORFs) and their 5' upstream sequences can be established. Furthermore, these ORFs can be corre-
VACCINIA: VIRUS, VECTOR, VACCINE
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lated to in vitro translation products by message selection. In this way Plucienniczak et al. (1985) and Weinrich and Hruby (1986) analyzed 7.6 and 5.1 kb of different portions of the vaccinia genome, respectively. The 5' regions of individual early and late genes have also been sequenced. Some of these include the vaccinia thymidine kinase early gene (Weir and Moss, 1983), the late gene encoding the major core polypeptide p4b (Rose1and Moss, 1985), and a 28-kDa late gene (Weir and Moss, 1985). Attempts to modify vaccinia promotors by in uitro mutagenesis have been used for establishing essential sequences or attenuating expression levels. Examples can be found for a gene encoding the late structural ll-kDa polypeptide (Hanggi et al., 1986) and for a constituitively expressed gene encoding a 7.5-kDa polypeptide (Cochran et al., 1985a). A universal doctrine concerning vaccinia promotors remains to be established. However, the studies mentioned above, as well as others, have described some general properties: 1. Although vaccinia regulatory sequences are unlike their eukaryotic counterparts they are similar in being rich in A-T sequences. 2. The consensus sequences TATA and AATAA, separated by about 25 bp preceding the first ATG codon, were found for a number of early and late genes (Plucienniczak et al., 1985). 3. The 5' end of late transcripts was found to be within five nucleotides of the first ATG.
Some early transcripts have also been shown to have short untranslated regions. For some late promotors, cis-acting regulatory signals were found to reside very close (30-100 bp) to the 5' end of late transcripts. With the ll-kDa structural protein it was found that once a minimum promotor size was established, increasing the promotor size did not seem to affect promotor strength (Bertholet et al., 1985). The study by Hanggi et al. (1986) revealed promotor activity of the ll-kDa polypeptide to be dependent on a TAAAT sequence which overlaps the site of transcription initiation. In addition to searching for vaccinia promotors, DNA sequencing analyses have served to characterize the genomic organization of the vaccinia genome. By determining ORFs it has been shown that vaccinia genes can be tightly clustered, in agreement with RNA mapping studies. For example, Niles et al. (1986) sequenced the 16-kb vaccinia virus Hind111 D fragment. They were able to distinguish 22 ORFs that, in several instances, were so tightly packed that they overlap. They have also assigned previously known temperature-sensitive mutations to specific ORFs within this region.
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ANTONIA PICCINI AND ENZO PAOLE'M'I
111. VACCINIA VIRUSAS
AN
EXPRESSION VECTOR
A . Foreign Genes Expressed by Vaccinia Virus The methodology to insert heterologous genes into vaccinia virus and the demonstration that vaccinia virus can faithfully express these genes at the RNA and protein levels are biologically interesting and exciting. Once constructed, the recombinant virus can be used for a variety of purposes: (1) to understand the mechanisms involved in vaccinia gene expression; (2) to perform the transcriptional, translational, and posttranslational analyses of foreign gene products in a background free from native influences; (3) to synthesize and isolate specific and biologically significant gene products; (4) to perform immunological analyses of defined antigens; ( 5 ) to produce live recombinant vaccines; and (6) to explore the potential of viruses to be used as vehicles for gene replacement therapy. The interest in inserting genes into vaccinia virus has grown tremendously since the first demonstration utilizing the herpes simplex virus thymidine kinase gene (Panicali and Paoletti, 1982; Mackett et al., 1982). Since then a myriad of genes have been inserted into vaccinia virus. The expression of these foreign genes in viable vaccinia recombinants as reported to date is presented in Table I. The list includes genes encoding biochemical markers such as chloramphenicol acetyltransferase, f3-galactosidase, and neomycin resistance. These inserts have enabled vaccinia to be used as a cloning vector with the advantage of assayable markers which can be used for selection, detection, or quantitative procedures. A number of inserts encode human genes, including human factor IX and human preproenkephalin. The majority of the foreign genes that have been inserted into the vaccinia genome encode antigens from a variety of infectious agents. These antigens are responsible for the production of immunity to a specific pathogen, be it viral or parasitic. Upon immunization with the recombinant virion, an immune response ensues which is targeted toward both vaccinia and the foreign antigens. We have focused on this aspect of using vaccinia as an expression vector for the production of live recombinant vaccines, and the remainder of this discussion will address these issues.
B . General Protocol for the Insertion of Foreign Genes into the Vaccinia Virus Genome The general protocol for the insertion of foreign genes into vaccinia virus is diagrammed in Fig. 2. The first step involves isolating the
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TABLE I IN RECOMBINANT VACCINIA VIRUSES FOREIGNGENESEXPRESSED
Reference Herpes simplex virus thymidine kinase Influenza virus hemagglutinin Hepatitis B virus surface antigen Herpes simplex virus glycoprotein D Plasmodium knowlesi sporozoite antigen Chloramphenicol acetyltransferase Rabies virus glycoprotein Transmissible gastroenteritis virus gp195 Vesicular stomatitis virus G protein Vesicular stomatitis virus N protein Influenza virus nucleoprotein Human factor IX (Christmas factor) Neomycin-resistance gene Sindbis virus structural proteins P-Galactosidase Epstein-Barr membrane antigen gp340 Sindbis virus structural proteins Respiratory syncytial virus glycoprotein G Tobacco etch virus proteins Friend murine leukemia virus gp70/p15E HTLV-I11 envelope gene LAV envelope gene Human preproenkephalin
Panicali and Paoletti (1982); Mackett et al. (1982) Panicali et al. (1983);Smith et al. (1983a) Smith et al. (1983b);Paoletti et al. (1984) Paoletti et al. (1984);Cremer et al. (1985) Smith et al. (1984) Mackett et al. (1984) Kieny et al. (1984); Wiktor et al. (1984) Hu et al. (1984) Mackett et al. (1985) Mackett et al. (1985) Yewdell et al. (1985) de la Salle et al. (1985) Franke et al. (1985) Rice et al. (1985) Chakrabarti et al. (1985) Mackett and Arrand (1985) Franke and Hruby (1985) Ball et al. (1986) Dougherty et al. (1986) Stephens et al. (1986) Chakrabarti et al. (1986) Hu et al. (1986) Thomas et al. (1986)
foreign gene of interest. Once obtained, the gene is cloned into a convenient cloning vehicle such as pBR322. In addition to the foreign gene, specific vaccinia sequences are cloned into the pBR322 such that they flank the foreign gene. The vaccinia sequences, which can range from approximately 100 to 10,000 bp on either side of the insert, serve a vital function. They will “direct” the foreign gene to the homologous sequences on the vaccinia genome. The recombinant plasmid containing the chimeric insert is introduced into tissue culture cells via standard transfection procedures utilizing a calcium-orthophosphate-precipitated preparation of plasmid DNA. Concomitantly, the cell is infected with infectious vaccinia virus. This rescuing virus will enter the cytoplasm, where it will proceed through its biogenesis. After uncoating and during DNA replication, a vaccinia genome can come into close proximity to the DNA sequences of the recombinant plasmid. The homologous vaccinia sequences on the plasmid and on the viral genome can pair up by the process of in uiuo recombination, which occurs
52
ANTONIA PICCINI AND ENZO PAOLEWI RECOMBINANT D N A
j?a322 CClNlA SEQUENCES
MBlNATlON
REPLICATION OF RECOMBINED D N A
DNA REPLICATION
FIG.2. General scheme for the insertion of foreign genes into vaccinia virus (see the text for details).
with a frequency of approximately 0.1%.Once this is accomplishedthe virus genome can rescue the foreign gene by incorporating it into its own DNA.Once this has occurred, replication of the recombined DNA molecule can continue followed by maturation of the novel recombinant vaccinia virus.
VACCINIA: VIRUS, VECTOR, VACCINE
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As mentioned earlier, the vaccinia RNA polymerase poorly recognizes heterologous promotor sequences. Therefore, the flanking vaccinia sequences upstream of the transcriptional direction of the foreign gene preferably contain a vaccinia promotor. Genetic manipulations of the chimeric plasmid can be designed so that after in v i m recombination a foreign gene can (1)be inserted in situ together with a vaccinia promotor, (2) replace vaccinia sequences downstream from a vaccinia promotor, or (3)be translocated along with a flanking vaccinia promotor to another part of the genome. All these manipulations require that when the foreign gene is recombined into the vaccinia genome it does not interrupt the flow of essential genetic information. This requirement has led us to search for nonessential sites within the vaccinia genome. Nonessential genes such as the thymidine kinase or DNA sequences deleted from the virus provide obvious targets for the insertion of foreign genetic elements. Perkus et al. (1986)have identified additional nonessential loci and their location is illustrated in Fig. 3.At least a dozen nonessential loci reside within the leftmost 30 kb of the vaccinia genome. In addition to giving us the advantage of using more than one target site for gene insertion, these sites can also be used as loci from which viable deletion mutants of vaccinia virus can be generated. These deletion mutants extend the inherent ability of vaccinia to hold considerable quantities of foreign genetic material (Panicali et al., 1983;Smith and Moss, 1983). In addition to the spontaneously occurring deletion of 9.8 kb toward the left end of the S-variant vaccinia genome (Panicali et al., 1981),other deletion mutants have been generated in the L-variant vaccinia genome and are shown in Fig. 3. Since these areas of deleted DNA are not essential for viral replication, foreign genes can be readily inserted anywhere within these regions. The availability of these viable vaccinia deletion mutants provides additional space for packaging foreign DNA and allows the simultaneous expression of multiple foreign genes, which can result in immunization against multiple pathogens (polyvalent vaccines). Additionally, these multirecombinants can be used for the coordinated expression of a family of related gene products or for the production of a variety of biologically active molecules from a single vaccinia infection. As an example of the versatility of vaccinia virus to express a variety of antigens and its potential use as a polyvalent vaccine, we have constructed a vaccinia recombinant which contains three foreign genes (Perkus et al., 1985). This triple recombinant contains genes coding for three pertinent antigens: the influenza virus hemagglutinin (InfHA), the hepatitis B virus surface antigen (HBsAg), and the herpes virus type 1 glycoprotein D (HSVgD).
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FIG.3. Insertions and deletions of vaccinia virus. Line B illustrates the physical map of the L v a r i a n t vaccinia genome as defined by HindIII restriction enzyme fragments designated A through 0. The location of viable insertion mutants generated by site-specific insertion of modified herpes simplex virus thymidine kinase-coding sequences is indicated by numbers 1 through 12 in line A. Viable deletion mutants derived from the L-variant genome are shown in lines C through F. The deletion mutants are indicated as Vdl and assigned map coordinates based on kilobases of deleted DNA referenced to the left terminus of the L-variant genome. The enzyme sites indicated are as follows: B, BanHI; Bg, BglII; H, HindIII (from Perkus et al., 1986).
VACCINIA: VIRUS, VECTOR, VACCINE
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C . Analysis of Vaccinia Recombinants After a recombinant vaccinia virus is produced in tissue culture cells, the newly aquired foreign DNA can be detected by standard DNA hybridization techniques (Panicali and Paoletti, 1982). Furthermore, genetic manipulations used in recombinant design can provide additional detection/selection of the novel virus by enzyme assays which rely on thymidine kinase activity (Mackett et al., 1982) and pgalactosidase activity (Chakbrabarti et al., 1985) or by drug resistance using the neomycin gene (Franke et al., 1985). Once a recombinant is identified, it can be isolated, purified, and grown to high titers to be used for subsequent analyses. For example, restriction enzyme analysis of the viral DNA can verify the genomic organization of the construct and detect any rearrangements of the vaccinia genome. With respect to foreign gene expression, reports listed in Table I demonstrate the successful ability of vaccinia virus to express foreign genes in a manner similar to, if not identical with native synthesis. The products can be processed correctly and exhibit authentic secondary modifications such as glycosylation. The antigens encoded by the foreign genes are properly localized in the vaccinia virus recombinantinfected cell and are immunologically presented as they are in the natural infection. For example, the hepatitis B virus surface antigen is secreted from infected cells as a morphologically distinct 22-nm particle. In contrast, the influenza hemagglutinin and the herpes simplex glycoprotein D become associated with the membrane of the infected cell. These properties are essential if one wants to use vaccinia virus as an expression vector that exhibits biological fidelity. The heterologous transcripts synthesized from recombinant vaccinia-infected cells can be isolated by standard purification methods and the mRNAs can be detected by Northern blot analysis as shown in Fig. 4. The results shown in lane E verify the synthesis of HSV RNAs in cells infected with the vaccinia recombinant vP60, which expresses the HSV-1 glycoprotein D gene (Paoletti et al., 1984). The major RNA species migrates at approximately 2500 bp, which is similar in size to the dominant RNA synthesized in HSV-l-infected cells as shown in lane F. The minor HSV-1 RNA bands also show similar migration patterns. HSV-lgD RNA is detected in neither host HeLa cells (lane C) nor HeLa cells infected with wild-type vaccinia (lane D).This type of analysis can also be used to characterize transcriptional alterations of the recombinant vaccinia genome. In lane A is shown endogenous RNA from wild-type vaccinia which is complementary to the 3’ region of the genome where the HSV-lgD insertion was made in the recombinant vP60. When RNA from vP60 is hybridized to the vaccinia DNA
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ANTONIA PICCINI AND ENZO PAOLETTI
A
B
C
D
E
F
G 3579 2297 I543
703
FIG.4. Northern blot of RNA isolated from a vaccinia recombinant expressing the herpes simplex virus glycoprotein D gene. Viral RNA was extracted from cells infected at a multiplicity of 10 PFU/cell. Cells were lysed by Dounce homogenization and the RNA was purified by a guanidine-HC1 method (Barth et al., 1982). Early RNA was isolated 3 hours after infection from cells treated with 40 pg/ml of cytosine arabinoside. Control host RNA was extracted from uninfected HeLa cells. The RNAs (20 pg) were separated on 1.5% agarose gels, blotted onto nitrocellulose paper, and hybridized with either a 32P-labeled HSV-lgD clone (lanes C-F) or a Hind111 F subclone (lanes A-B). Hybridization was detected by autoradiography with Kodak XAR-5 film at -70°C for 20 hours. The size markers in lane G were generated by a mixed enzyme digestion of pBR322 and the sizes (bases) are indicated to the right of the bands.
sequences from this region, the endogenous 350-bp RNA is not detected (lane B), but instead a larger RNA is identified. This RNA, which is complementary to the DNA probe at the insertion site, is identical in size to the RNA in the vP60-infected cell that was detected with HSV-lgD sequences. Thus, the endogenous RNA was displaced and it appears that the HSV-lgD RNA contains a small number of vaccinia sequences at its 3' end. Another type of RNA analysis involves determining the steady-state levels of foreign gene RNAs regulated by different vaccinia promotors. A Northern blot, shown in Fig. 5, illustrates differences in RNA expression. Lanes B, F, and J indicate absence of detectable hybridization to RNA isolated from cells infected with wild-type vaccinia virus. Lanes C and D show different HBsAg mRNA levels in cells infected with the vaccinia recombinants vP59 and vP139, respectively. Lanes G
VACCINIA: VIRUS,VECTOR, VACCINE
A B
C
D
E
F
G
H
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I
J
K
FIG.5. Northern blot of RNA isolated from a variety of vaccinia recombinants using different early promotors. Early RNA was isolated from vaccinia recombinants and analyzed as described in Fig. 4. The probes used correspondto the specific genes present in the recombinants as described in the text. Lanes A, E, and I are pBR322 size markers, as described in Fig. 4.
and H show different InfHA mRNA levels in cells infected with vP59 and vP142, respectively. It should be noted that vP142 (lane H) is a double vaccinia recombinant containing InfHA and HSVgD genes under the regulation of two different promotors. HSVgD RNA expression from vP142 is shown in lane K. Various assays are available to measure expression of the foreign gene at the protein level. For example, functional herpes simplex virus thymidine kinase can be quantitated by monitoring the phosphorylation of [1251]iododeoxycytidine(Panicali and Paoletti, 19821, a property not exhibited by eukaryotic or vaccinia thymidine kinases. Assays for the expression of a number of genes in recombinant vaccinia virus have relied on immunological assays. These require that the protein product be structurally correct such that it will react with antiserum generated against the native product. Perkus et al. (1985) demonstrated the presence of InfHA and HSV-lgD gene products at the surface of live cells by using antisera generated against the native product followed by 1251-labeledprotein A. The cell monolayer was lifted onto a filter and the radioactive signal was detected by autoradiography. For HBsAg detection, a commercially available radioimmunoassay kit, AUSRIA I1 (Abbott Laboratories), was used, which utilizes radiolabeled hepatitis antibodies to measure HBsAg in the culture media. These assays gave a qualitative measure of protein expression and showed us that the products can react against antibodies generated against the native antigens. Furthermore, they al-
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ANTONIA PICCINI AND ENZO PAOLETTI
lowed us to follow the cellular localization of the products. For a more quantitative measure of protein expression we have disrupted infected cell monolayers and applied the cell extract onto nitrocellulose filters. The amount of extract can be accurately adjusted based on virus output titer. The filters are treated with the test antiserum followed by 1251-labeledprotein A treatment. After autoradiography, signal intensity is proportional to protein levels. We are using this type of analysis to test the results of fine genetic manipulations at the DNA level, where vaccinia promoters of different strengths are used to direct the synthesis of foreign genes. This is an important consideration since often a sufficient amount of antigenic mass must be produced in order to elicit an immune response adequate for vaccine development. Immunoprecipitation of the radiolabeled product can also be informative with regard to protein level while giving us an analysis of the structural forms of a particular product. For example, Fig. 6 is an autoradiogram of an SDS-polyacrylamide gel showing the [36S]methionine-labeled polypeptides expressed by vaccinia recombinant vP218 (lane E), which expresses the hepatitis B small pre-sAg or pre-S(2) gene (Milich et al., 1986), and by wild-type virus (lane F). The similarities in the observed polypeptide profiles suggest that no dramatic metabolic effects are produced by infection of cells with vP218 as compared with wild-type virus. The polypeptides in lane E and F were immunoprecipitated with antihepatitis antiserum and the results are shown in lanes C and D, respectively. The antiserum did not react with any specific polypeptides produced by wild-type virus (lane D).However, the antiserum did react with specific hepatitis polypeptides produced by vP218-infected cells (lane C). These polypeptides exhibited sizes of 19.9, 23.2, and 30.4 kDa. These sizes and the apparent band heterogeneity exhibited by the largest band correspond to the surface antigen proteins expressed by hepatitis B virus (Heerman et al., 1984) and are similar to those expressed by mammalian cells expressing cloned HBsAg genes (Michel et al., 1984). Our laboratory has been directly involved in designing vaccinia as a live recombinant vaccine. Much of our work has concentrated on assessing the immunogenicity of the foreign gene product using laboratory animals. The test animals are inoculated with a particular vaccinia recombinant and the immune sera are tested for the presence of antibodies. We have detected InfHA antibody levels using a hemagglutinin inhibition assay in which authentic InfHA that normally agglutinate either guinea pig or chicken erythrocytes is prevented from doing so if antibodies are present in the assay. Antibodies against HBsAg can be detected using a commercially available AUSAB radioimmunoassay kit (Abbott Laboratories). Antibodies directed against
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VACCINIA: VIRUS, VECTOR, VACCINE
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14.3K
14.3K
FIG.6. Immunoprecipitation of hepatitis B surface antigen (HBsAg) proteins synthesized by a vaccinia recombinant. As described in the text, an aliquot of the [3~S]methionine-labeledinfected cell lysate was analyzed directly (lane E, vP218; lane F, wild-type virus) and immunoprecipitated with hepatitisB antisera (lane C, vP218; lane D, wild-type virus). The vP218-infected cell lysates were immunoprecipitated with normal serum (lane B) or with immune serum (lane C). Lanes A and G are molecular-weight size markers. After fractionation, the gel was treated with ENSHANCE (New England Nuclear, Boston, Massachusetts) and was analyzed by autoradiography.
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vaccinia, InfHA, and HSVgD can also be assayed by testing the ability of the immune sera to affect neutralization of viral infectivity in in uitro plaque reduction assays. Using these assays we demonstrated that animals are able t o make immunoreactive antibodies against a number of foreign antigens expressed in a single vaccinia recombinant (Perkus et al., 1985). Other animal studies have addressed questions regarding the feasibility of immunization regimens with vaccinia recombinants (Perkus et al., 1985).This involved immunizing a rabbit with a HBsAg vaccinia recombinant which resulted in eliciting high titers of HBsAg antibodies for at least 1 year. The animal was then revaccinated with a vaccinia recombinant expressing the same foreign gene. The revaccination resulted in increased levels of antibodies directed against HBsAg. Thus, a booster effect was established for HBsAg that was expressed under vaccinia regulation. To determine whether one could achieve successful revaccination with a different recombinant we immunized a rabbit with a HBsAg vaccinia recombinant, waited approximately 1 year, and then immunized with a InfHA vaccinia recombinant. The initial HBsAg antibody titers remained high and were unaffected by the second immunization, which in itself produced antibodies against InfHA. Taken together, these studies were successful in showing that recombinant vaccinia virus can be used in a revaccination protocol either to give a booster effect or to elicit an immunological response to a different foreign antigen upon revaccination. The success of using recombinant vaccinia vaccines has been demonstrated by protection studies. These studies, showing the protection of vaccinia recombinant-immunized animals against subsequent challenge with the pathogen, can be found in a number of reports (Smith et al., 1983b; Wiktor et al., 1984; Paoletti et al., 1984,1985; Mackett et al., 1985). IV. PROSPECTUS Vaccinia displays a number of characteristics which make it an ideal expression vector. Some of these are (1)the ability to incorporate large amounts of exogenous DNA; (2) the faithful transcription of the heterologous genes producing RNAs, which are translated into protein resembling the native product in structure, function, and localization; and (3) the cytoplasmic site of vaccinia replication, which allows gene expression to proceed without interference by the host genome. These properties are useful for using vaccinia as a cloning vector and for
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vaccine development. However, in the latter case, safety is a critical issue. Although past vaccinations with vaccinia virus were relatively safe, there were rare but occasionally fatal complications involving the skin and central nervous system. However, the use of vaccinia as a vaccine for smallpox protection has been greatly beneficial to the general population. As a prospectus, we can envision vaccinia virus being genetically manipulated to such a degree that we could expect to generate a virus containing only the genetic material for replication and lacking those parts of the genome responsible for pathogenesis. Future research efforts will focus on the vaccinia genome with respect t o defining the genetic parameters which control the expression of both endogenous and exogenous genes. For example, further identification of nonessential vaccinia DNA sequences will establish the minimum amount of genetic material necessary for virus replication; studies aimed at deleting large portions of the vaccinia genome would determine the minimum amount of DNA necessary for vaccinia biogenesis, while making room for the insertion of additional foreign genetic material. This would result in the ability to insert multiple genes for the production of polyvalent vaccines. Thus, the benefit-to-risk ratio would be skewed toward the benefit achieved by a single or low number of vaccinations resulting in immunity to multiple diseases represented by a dozen or so foreign genes expressed by a single vaccinia virus. A polyvalent vaccine would be especially useful for those diseases in which the pathogen may exhibit a number of surface proteins, all of which are immunogenic, as with herpes infection, or in more complicated systems such as the malaria parasite, which expresses different antigens during its life cycle. The ability of the immune system to recognize and react to a variety of antigens remains to be established. However, the diversity should not be significantly greater than that presented by vaccinia alone. In these cases, attenuation of antigen levels may be of influence. With the identification of more viral genome products, molecular dissections of the vaccinia genome will be aimed at defining both host range and virulence. Analysis of regulatory control signals for vaccinia transcription will continue toward defining the properties of a vaccinia promoter. As this data base accumulates, consensus sequences will be generated to determine the subtle differences in the temporal and quantitative control of vaccinia transcription. Additionally, the translation of exogenous gene transcripts encoding foreign proteins will be analyzed in terms of RNA utilization and host RNA competition.
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With all these efforts combined, the complexity of vaccinia biogenesis is sure to be unraveled, revealing a virus with the diversity to be used as an expression vector and to be used once again as a vaccine.
REFERENCES Ball, L. A., Young, K. K. Y., Anderson, K., Collins, P. L., and Wertz, G. W. (1986).Proc. Natl. Acad. Sci. U S A . 83,246-250. Barth, R. K.,Gross, K. W., Gremke, L. C., and Hastie, N. D. (1982).Proc. Natl. Acad. Sci. U S A . 79, 500-504. Bedard, D. L. (1983).J. Virol. 36, 656-660. Belle Isle, H., Venkatesan, S., and Moss, B. (1981).Virology 112,306-317. Berk, A. J., and Sharp, P. A. (1977).Cell 12, 721-732. Bertholet, C., Drillien, R., and Wittek, R. (1985).Proc. Natl. Acad. Sci. U S A . 82,20962100. Bloomquist, M. C., Hunt, L. T., and Barker, W. C. (1984).Proc. Natl. Acad. Sci. U S A . 81,7363-7367. Brown, J. P., Twardzik, D. R., Margquardt, H., andTodaro, G. J. (1985).NaturefLondon) 313,491-492. Broyles, S.S., and Moss, B. (1986).Proc. Natl. Acad. Sci. U S A . 83, 3141-3145. Chakrabarti, S., Brechling, K., and Moss, B. (1985).Mol. Cell. Biol. 5, 3403-3409. Chakrabarti, S., Robert-Guroff, M., WongStaal, F., Gallo, R. C., and Moss, B. (1986). Nature (London) 320, 535-537. Cochran, M.A., Mackett, M., and Moss, B. (1985a).Proc. Natl. Acad. Sci. U S A . 82,1923. Cochran, M.A., Puckett, C., and Moss, B. (198513).J. Virol. 54,30-37. Condit, R. C., Motyczka, A., and Spizz, G. (1983).Virology 128, 429-443. Cooper, J. A., Wittek, R., and Moss, B. (1981).J. Virol. 39, 733-745. Cremer, K. J., Mackett, M., Wohlenberg, C., Notkins, A. L.,and Moss, B. (1985).Science 228, 731-740. Dales, S., and Pogo, B. G. T. (1981).In “Biology of Poxviruses. Virology Monographs; 18” (D. W. Kingsbury and H. Zur Hausen, eds.). Springer-Verlag, New York. de la Salle, H., Altenberger, W., Elkaim, R., Dott, K., Dieterle, A., Drillien, R., Cazenave, J.-P., Tolstoshev, P., and Lecocq, J.-P. (1985).Nature (London) 316, 268270. Dougherty, W. G., Franke, C. A., and Hruby, D. E. (1986).Virology 149, 107-113. Drillien, R., and Spehner, D. (1983).Virology 131, 385-393. Ensinger, M. J., and Rovinsky, M. (1983).J. Virol. 48,419-428. Fenner, F. (1985).In “Virology” (B. N. Fields, ed.), pp. 661-684. Raven, New York. Franke, C. A., and Hruby, D. E.(1985).J . Gen. Virol. 66,2761-2765. Franke, C. A., Rice, C. M., Strauss, J. H., and Hruby, D. E. (1985).Mol. Cell. Biol. 5, 1918-1924. Gillard, S., Spehner, D., and Drillien, R. (1985).J. Virol. 53, 316-318. Golini, F., and Kates, J. R. (1984).J. Virol. 49. 459-470. Hanggi, M., Bannwarth, W., and Stunnenberg, H. G. (1986).EMBO J. 5, 1071-1076. Heermann, K. H.,Goldmann, U.,Schwartz, W., Seyffarth, T., Baumgarten, H., and Gerlich, W. H. (1984).J. Virol. 52,396-402. Hirt, P., Hiller, G., and Wittek, R. (1986).J . Virol. 58, 757-764. Hu, S.,Bruszewski, J., Boone, T., and Souza, L. (1984).In “Modem Approaches to Vaccines: Molecular and Chemical Bases of Virus Virulence and Immunogenecity”
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(R.M. Chanock and R. A. Lerner, eds.), pp. 219-223. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Hu, S., Kosowski, S. G., and Dalrymple, J. H. (1986).Nature (London) 320, 537-540. Jones, E. V., and Moss, B. (1984).J. Virol. 49, 72-77. Kieny, M. P., Lathe, R.,Drillien, R., Spehner, D., Skory, S., Schmitt, D., Wiktor, T., Koprowski, H., and t c o c q , J. P. (1984).Nature (London)312, 163-166. Mackett, M., and Arrand, J. R. (1985).EMBO J . 4,3229-3234. Mackett, M., Smith, G. L., and Moss, B. (1982).Proc. Natl. Acad. Sci. U S A . 79,74157419. Mackett, M., Smith, G. L., and Moss, B. (1984).J. Virol. 49,857-864. Mackett, M., Yilma, T., Rose, J. K., and Moss, B. (1985).Science 227,433-435. Mahr, A., and Roberts, B. E. (1984a).J. Virol. 49,497-509. Mahr, A.,and Roberts, B. E. (1984b).J. Virol. 49,510-520. Michel, M. L., Pontisso, P., Sobczak, E., Malpiece, Y.,Streeck, R. E., and Tiollais, P. (1984).Proc. Natl. Acad. Sci. U S A . 81, 7708-7712. Milich, D. R., MeLachlan, A., Chisari, F. V.,Kent, S. B. H., and Thornton, G. B. (1986).J. Immunol. 137. Morgan, J. R., and Roberts, B. E. (1984).J. Virol. 51, 283-297. Morrison, D. K., Carter, d. K., and Moyer, R. W. (1985).J. Virol. 55, 670-680. Moss, B. (1985).In “Virology” (B. N. Fields, ed.), pp. 685-703.Raven, New York. Moss, B., Winters, E., and Cooper, J. A. (1981).J. Virol.40,387-395. Nakano, E., Panicali, D., and Paoletti, E. (1982).Proc. Natl. Acad. Sci.U S A . 79,15931596. Niles, E. G., Condit, R. C., Caro, P., Davidson, K., Matusick, L., and Seto, J. (1986). Virology 15, 96-112. Panicali, D., and Paoletti, E. (1982).Proc. Natl. Acad. Sci. U S A . 79,4927-4931. Panicali, D., Davis, S. W., Mercer, S. R., and Paoletti, E. (1981).J. Virol.37,1000-1010. Panicali, D.,Davis, S. W., Weinberg, R. L., and Paoletti, E. (1983).Proc.Natl. Acad. Sci. U S A . 80,5364-5368. Paoletti, E., Lipinskas, B. R., Samsonoff, C., Mercer, S., and Panicali, D. (1984).Proc. Natl. Acad. Sci. U S A . 81, 193-197. Paoletti, E., Perkus, M., Piccini, A,, Wos, S., and Lipinskas, B. R. (1985).In “Medical Virology IV” (L. M. de la Maza and E. M. Peterson, eds.), pp. 409-430. Erlbaum, Hillsdale, New Jersey. Perkus, M. E., Piccini, A., Lipinskas, B. R., and Paoletti, E. (1985).Science 229,981-984. Perkus, M., Panicali, D., Mercer, S., and Paoletti, E. (1986).Virology 152, 285-297. Plucienniczak, A.,Schroeder, E., Zettlmeissl, G., and Streeck, R. E. (1985).Nucleic Acids Res. 13, 985-998. Reisner, A. H. (1985).Nature (London) 313,801-803. Rice, C. M., Franke, C. A., Strauss, J. H., and Hru 6 . E. (1985).J . Virol.60,227-239. Rosel, J., and Moss, B. (1985).J. Virol. 56, 830-8 Sam, C. K., and Dumbell, K. R. (1981).Ann. Virol. (Inst. Pasteur) 132E, 135-150. Shida, H.(1986).Virology 150,451-462. Smith, G. L.,and Moss, B. (1983).Gene 25,21-28. Smith, G. L.,Mackett, M., and Moss, B. (1983a).Nature (London) 302,490-495. Smith, G. L., Murphy, B. R., and Moss, B. (1983b).Proc. Natl. Acad. Sci. U S A . 80, 7155-7 159. Smith, G. L., Godson, G. N., Nussenzweig, V., Nussenzweig, R. S., Barnwell, J., and Moss, B. (1984).Science 224, 397-399. Stephens, E. B., Compans, R. W., Earl, P., and Moss, B. (1986).EMBO J. 5, 237-245. Tartaglia, J.,and Paoletti, E. (1985).Virology 147, 394-404.
P
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Tartaglia, J., Piccini, A., and Paoletti, E. (1986).Virology 150,45-54. Thomas, G.,Herbert, E., and Hruby, D. E. (1986).Science 232, 1641-1643. Thompson, C. L.,and Condit, R. C. (1986).Virology 150, 10-20. Traktman, P.,Sridhar, P., Condit, R. C., and Roberts, B. E. (1984).J . Virol. 49,125-131. Twardzik, D.R., Brown, J. P., Ranchalis, J. E., Todaro, G. J., and Moss, B. (1985).Proc. Natl. Acad. Sci. U S A . 82, 5300-5304. Vassef, A., Ben-Hamida, F., and Beaud, G. (1983).Anal. Virol. (Inst. Pasteurl 134E, 375-385. Vassef, A.,Mars, M., Dru, A,, Plucienniczak, A., Streeck, R. E., and Beaud, G. (1985).J. Virol. 55, 163-172. Villarreal, E.C., and Hruby, D. E. (1986).J . Virol. 67, 65-70. Weaver, R. F., and Weissman, C. (1979).Nucleic Acids Res. 7, 1175-1193. Weinrich, S.L.,and Hruby, D. E. (1986).Nucleic Acids Res. 14, 3003-3016. Weinrich, S.L.,Niles, E. G., and Hruby, D. E. (1985).J. Virol. 55,450-457. Weir, J. R., and Moss, B. (1983).J. Virol. 46,530-537. Weir, J. P., and Moss, B. (1985).J. Virol. 56, 534-540. Weir, J.P., Bajszar, G., and Moss, B. (1982).Proc. Natl. Acad. Sci. U.S.A.79,1210-1214. Wiktor, T. J.,MacFarlan, R. I., Reagan, K. J., Kietzschold, B., Curtis, P. J., Wunner, W. H., Kieny, M.-P., Lathe, R., Lecocq, J.-P., Mackett, M., Moss, B., and Koprowski, H. (1984).Proc. Natl. Acad. Sci. U.S.A.81, 7194-7198. Wittek, R., Cooper, J. A., and Moss, B. (1981).J. Virol. 39, 722-732. Wittek, R., Hanggi, M., and Hiller, G. (1984a).J. Virol. 49,371-378. Wittek, R., Richner, B., and Hiller, G. (1984b).Nucleic Acids Res. 12, 4835-4848. Yewdell, J. W., Bennink, J. R., Smith, G. L., and Moss, B. (1985).Proc. Natl. Acad. Sci. U.S.A. 82, 1785-1789.
VACCINIA: VIRUS, VECTOR, VACCINE Antonia Piccini and Enzo Paoletti Laboratory of Immunology Wadsworth Center for Laboratories and Research New York State Department of Health Albany, New York 12201
I. Introduction Historical Perspective of Vaccinia Virus 11. The Viral Genome A. Gene Mapping B. DNA Sequence Data 111. Vaccinia Virus as an Expression Vector A. Foreign Genes Expressed by Vaccinia Virus B. General Protocol for the Insertion of Foreign Genes into the Vaccinia Virus Genome C. Analysis of Vaccinia Recombinants IV. Prospectus References
I. INTRODUCTION Historical Perspective of Vaccinia Virus Recently there has been a renaissance in poxvirus research hallmarked by using vaccinia virus as a eukaryotic cloning and expression vector in a variety of biological and clinical applications. This resurgence comes almost 200 years after vaccinia virus was introduced by Edward Jenner as a vaccine against smallpox. Its usefulness as a vaccine was marked by the similar antigenic properties of vaccinia and the smallpox virus, and by the ability of vaccinia to be readily grown in the laboratory, its stability as freeze-dried preparations, and its ease of administration. Vaccination has not been required for the general population since the World Health Organization declared the world free from smallpox in 1980. Vaccinia virus is the prototypic member of a large group of complex animal viruses known as the poxviruses. Detailed discussions on poxviruses can be found in several reviews (Moss, 1985; Dales and Pogo, 1981; Fenner, 1985). An electron micrograph of vaccinia virus is shown in Fig. 1. Enclosed within a brick-shaped outer lipid envelope lies the double-stranded DNA genome of approximately 187,000 base 43
Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG.1. Electron micrograph of negatively stained (phosphotungstic acid) particles of vaccinia virus. Photo generously provided by W. Samsonoff (New York State Department of Health).
pairs (bp). The DNA termini are characteristic in two ways: (1) they are covalently cross-linked so that DNA denaturation results in the formation of a single-stranded circle, and (2) they contain a defined set of direct tandem repetitions which display transcriptional activity and are prone to deletions and rearrangements. The genome is complexed with proteins and enzymes comprising the vaccinia transcriptional machinery. Some of the 200 or so virally encoded gene products include the virion structural polypeptides and an arsenal of enzymes, some of which include RNA polymerase and enzymes that cap and methylate RNA. Additionally, there are a number of nonstructural polypeptides induced on infection, including the DNA polymerase and thymidine kinase. Upon infection, the virus localizes itself to specific regions in the cytoplasm, termed factories, where it undergoes a temporally orchestrated series of developmental stages, including early
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transcription, DNA replication, late transcription, and virus maturation. Vaccinia virus does not circulate in nature and its exact origin remains obscure. Vaccinia is essentially a laboratory virus. It is highly amenable to in uitro culture techniques and, as an experimentally infectious agent, has a broad host range. Thus, it can infect a variety of animals and is able to replicate in a variety of tissue culture cells. When experimenting with this virus, two unique viral features must be taken into account: (1)the virus replicates independently within the cytoplasm of the infected cell, and (2) the naked DNA of vaccinia is not infectious, unlike the DNA of many other animal viruses, such as herpesviruses, adenoviruses, and papovaviruses. These features, combined with the large DNA size, disallow it from being used as a cloning vector via the “cut and paste” approach commonly used for other viral genomes and plasmids. Instead, the ability to introduce genetic elements into the vaccinia genome is accomplished by marker rescue techniques (Section 111,B). In this review, we will discuss the genetic characterization of vaccinia, a general protocol for the insertion of foreign genes into vaccinia virus, and analysis of the recombinant viruses. It is our intention to show that vaccinia, a virus used historically as a live vaccine for the immunoprophylaxis of smallpox, can now be genetically engineered for the construction of live recombinant viruses and directed against heterologous infectious agents. These recombinants can be used as expression vectors with biological applications ranging from cloning and expression vehicles to live vaccines directed against infectious diseases of both human and veterinary concern. 11. THE VIRALGENOME
A. Gene Mapping Within recent years, the viral genome has been the focus of intense study involving both gene mapping and DNA sequencing. These studies have contributed toward an understanding of the molecular organization and genetic regulation of vaccinia biogenesis, thus defining the molecular characteristics necessary for designing vaccinia as an efficient and versatile expression vector. The mapping and identification of vaccinia genes is a complex endeavor made so by the large genome size, temporal transcription constraints, transcript overlap, and late RNA size heterogeneity. Nevertheless, there has been considerable progress which includes (1)
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mapping genes with assigned in uitro products, (2) mapping genes with assigned in uiuo products and function, and (3) mapping genes with selectable phenotypes. One of the strategies used to map genes involves marker rescue, which is the recovery of genetic characteristics from inactive genomes or genomic fragments. Marker rescue strategies have been used in various bacteriophage and animal virus systems to map genetic loci. The genomic structure of the virus and the infectious nature of the isolated viral DNA are the determining factors for the rescue process. Since purified vaccinia DNA is not infectious, transfected vaccinia DNA could only be rescued by infectious vaccinia virus by in uiuo recombination. Sam and Dumbell (1981) demonstrated this technique using thermosensitive markers and Nakano et al. (1982) demonstrated this approach by reinserting unique vaccinia DNA sequences into vaccinia deletion mutants. Phenotypes such as a-amanitin resistance (Villarreal and Hruby, 1986), temperature sensitivities (Condit et al., 1983; Ensinger and Ravinsky, 1983; Drillien and Spehner, 1983; Thompson and Condit, 1986), and host range (Gillard et al., 1985)have been assigned to DNA segments via marker rescue. A phenotypic property which we have been particularly interested in is vaccinia's sensitivity to the drug rifampicin. Using DNA fragments from a rifampicin-resistant mutant (Rip) in marker rescue experiments, Tartaglia and Paoletti (1985) demonstrated that rifampicin resistance is due to a single-base change. Characterization of the rifampicin locus was continued by demonstrating that this locus is transcriptionally active and that a Rif-specific mRNA complementary to this locus can be translated in uitro into a 63-kDa polypeptide (Tartaglia et al., 1986). In addition to these mapping studies, marker rescue forms the basis for using vaccinia as a eukaryotic cloning vector. As will be discussed later we and others have used modifications of the marker rescue protocol to insert foreign genes into vaccinia (Sections II1,A and 111,B). Additional approaches for gene mapping may employ message selection, which involves isolating specific viral RNAs by hybridization to genomic fragments. The selected RNA can be analyzed directly or translated in vitro to yield products which can either be assayed for enzyme activity or analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A large portion of the vaccinia genome has been analyzed in this way, yielding information pertaining to gene location, RNA polarity, and polypeptide size. If antiserum to an in uiuo product is available, the sera can be used to immunoprecipitate the in uitro translation product to correlate with a viral component. This type of analysis has been used to map viral structural polypeptides, to deter-
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mine enzymatic activities, and in some cases for confirmation of expression of a gene after marker rescue. Using message selection, Belle Isle et al. (1981) performed an extensive survey using cloned vaccinia Hind111 fragments to select RNAs, and by in vitro translation identified approximately 75 early and 40 late polypeptides. Although these products were not assigned functions, their identification was indeed useful in establishing the variety of temporally expressed genes and their general location on the vaccinia genome. Moss et al. (1981) and Bedard (1983)used message selection to assign a number of cell-free translation products to the region of the vaccinia genome that distinguishes the two major vaccinia variants (L, large; S, small) of the WR strain. Concentrating on the genomic termini, Wittek et al. (1981) and Cooper et al. (1981) performed transcriptional analyses and in vitro translation which confirmed that the transcripts in these regions encode both early and late gene functions, do not appear to be spliced, have 5' capped ends, can be transcribed in either direction, and are overlapping. These studies established the transcriptional assignments for at least 21 kilobases (kb) of the left end of the vaccinia genome. A considerable amount of mapping has been done within the central region of the genome. For example, Golini et al. (1984) have focused on portions of the Hind F region, Morgan and Roberts (1984) on the Hind NMK regions, and Mahr and Roberts (1984a,b) on the Hind J and H regions. For the most part these investigators presented the general conclusions (1)of the existence of clustered families of RNAs which can be overlapping, sharing 5'-proximal sequences; (2) of transcription occurring bidirectionally and with no particular direction preferred by early or late RNAs; and (3)of late RNAs that are highly heterogeneous at their 3 ' end, a feature that was suspected using hybridization analyses. These studies yielded a great deal of transcriptional information; however, one of the main goals of gene mapping is the identification of a specific function for a particular vaccinia gene product. In this way structure and function mesh t o give a broader picture of the molecular economy of vaccinia gene expression. Vaccinia virus morphogenesis is complex and interdependent such that each event appears to be dependent on the proper completion of the previous one. An understanding of the biological complexity of vaccinia depends upon the elucidation of the interactions of specific components during viral morphogenesis. Therefore, great emphasis has been placed on mapping genes which code for viral components. These include genes encoding some of the vaccinia structural proteins (Wittek et al., 1984a,b; Rose1 and Moss, 1985; Weir and Moss, 1985), an envelope glycoprotein antigen (Hirt et
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al., 19861, hemagglutinin protein (Shida, 1986), thymidine kinase (Weir et al., 1982; Vassef et al., 1983), DNA polymerase (Jones and Moss, 1984; Traktman et al., 1984), and subunits of the RNA polymerase (Morrison et al., 1985; Broyles and Moss, 1986). A vaccinia core protein was initially identified using an expression vector system. Weir and Moss (1985) used a bacterial expression vector to synthesize a vaccinia product which was then used to generate antiserum for immunoprecipitation of viral components. A vaccinia growth factor was identified (Reisner, 1985; Brown et al., 1985; Bloomquist et al., 1984; Twardzik et al., 1985)using computer technology which allowed the comparison of vaccinia DNA sequences with heterologous DNA sequences in data banks.
B . DNA Sequence Data Presently one of the directions of vaccinia research is toward assessing the DNA sequences responsible for controlling the temporal expression of vaccinia genes. Sequences upstream of mapped RNAs and open reading frames obtained by DNA sequence analysis are being compared to distinguish between early and late transcriptional control signals and/or promotors. Furthermore, since the viral-encoded RNA polymerase is unable to efficiently recognize heterologous promotors, the identification and isolation of vaccinia promotor sequences would facilitate the use of vaccinia as an expression vector. One of the ways in which vaccinia promotor regions have been identified is by mapping RNAs by 5' S, analyses (Berk and Sharp, 1977; Weaver and Weissmann, 1979). Portions of the DNA sequences upstream from the RNA cap site can be tested for promotor activity by using the promotor to direct the expression of a foreign gene in a recombinant vaccinia. An alternative method involves a transient expression system which utilizes the prokaryotic gene encoding chloramphenicol acetyltransferase (CAT). By linking this assayable marker t o a vaccinia sequence and inserting it into a plasmid, CAT activity can be measured as an indication of promotor strength (Cochran et al., 1985a,b). In another promotor search Vassef et al. (1985) used a plasmid rescue technique which allowed the isolation of randomly generated vaccinia DNA fragments which were capable of directing the expression of herpes simplex virus thymidine kinase (HSV-TK) when inserted into a TK- vaccinia virus recombinant. Promotor regions have also been indirectly recognized by sequencing large portions of the vaccinia genome. By converting the DNA sequence to amino acid sequences, open reading frames (ORFs) and their 5' upstream sequences can be established. Furthermore, these ORFs can be corre-
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lated to in vitro translation products by message selection. In this way Plucienniczak et al. (1985) and Weinrich and Hruby (1986) analyzed 7.6 and 5.1 kb of different portions of the vaccinia genome, respectively. The 5' regions of individual early and late genes have also been sequenced. Some of these include the vaccinia thymidine kinase early gene (Weir and Moss, 1983), the late gene encoding the major core polypeptide p4b (Rose1and Moss, 1985), and a 28-kDa late gene (Weir and Moss, 1985). Attempts to modify vaccinia promotors by in uitro mutagenesis have been used for establishing essential sequences or attenuating expression levels. Examples can be found for a gene encoding the late structural ll-kDa polypeptide (Hanggi et al., 1986) and for a constituitively expressed gene encoding a 7.5-kDa polypeptide (Cochran et al., 1985a). A universal doctrine concerning vaccinia promotors remains to be established. However, the studies mentioned above, as well as others, have described some general properties: 1. Although vaccinia regulatory sequences are unlike their eukaryotic counterparts they are similar in being rich in A-T sequences. 2. The consensus sequences TATA and AATAA, separated by about 25 bp preceding the first ATG codon, were found for a number of early and late genes (Plucienniczak et al., 1985). 3. The 5' end of late transcripts was found to be within five nucleotides of the first ATG.
Some early transcripts have also been shown to have short untranslated regions. For some late promotors, cis-acting regulatory signals were found to reside very close (30-100 bp) to the 5' end of late transcripts. With the ll-kDa structural protein it was found that once a minimum promotor size was established, increasing the promotor size did not seem to affect promotor strength (Bertholet et al., 1985). The study by Hanggi et al. (1986) revealed promotor activity of the ll-kDa polypeptide to be dependent on a TAAAT sequence which overlaps the site of transcription initiation. In addition to searching for vaccinia promotors, DNA sequencing analyses have served to characterize the genomic organization of the vaccinia genome. By determining ORFs it has been shown that vaccinia genes can be tightly clustered, in agreement with RNA mapping studies. For example, Niles et al. (1986) sequenced the 16-kb vaccinia virus Hind111 D fragment. They were able to distinguish 22 ORFs that, in several instances, were so tightly packed that they overlap. They have also assigned previously known temperature-sensitive mutations to specific ORFs within this region.
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111. VACCINIA VIRUSAS
AN
EXPRESSION VECTOR
A . Foreign Genes Expressed by Vaccinia Virus The methodology to insert heterologous genes into vaccinia virus and the demonstration that vaccinia virus can faithfully express these genes at the RNA and protein levels are biologically interesting and exciting. Once constructed, the recombinant virus can be used for a variety of purposes: (1) to understand the mechanisms involved in vaccinia gene expression; (2) to perform the transcriptional, translational, and posttranslational analyses of foreign gene products in a background free from native influences; (3) to synthesize and isolate specific and biologically significant gene products; (4) to perform immunological analyses of defined antigens; ( 5 ) to produce live recombinant vaccines; and (6) to explore the potential of viruses to be used as vehicles for gene replacement therapy. The interest in inserting genes into vaccinia virus has grown tremendously since the first demonstration utilizing the herpes simplex virus thymidine kinase gene (Panicali and Paoletti, 1982; Mackett et al., 1982). Since then a myriad of genes have been inserted into vaccinia virus. The expression of these foreign genes in viable vaccinia recombinants as reported to date is presented in Table I. The list includes genes encoding biochemical markers such as chloramphenicol acetyltransferase, f3-galactosidase, and neomycin resistance. These inserts have enabled vaccinia to be used as a cloning vector with the advantage of assayable markers which can be used for selection, detection, or quantitative procedures. A number of inserts encode human genes, including human factor IX and human preproenkephalin. The majority of the foreign genes that have been inserted into the vaccinia genome encode antigens from a variety of infectious agents. These antigens are responsible for the production of immunity to a specific pathogen, be it viral or parasitic. Upon immunization with the recombinant virion, an immune response ensues which is targeted toward both vaccinia and the foreign antigens. We have focused on this aspect of using vaccinia as an expression vector for the production of live recombinant vaccines, and the remainder of this discussion will address these issues.
B . General Protocol for the Insertion of Foreign Genes into the Vaccinia Virus Genome The general protocol for the insertion of foreign genes into vaccinia virus is diagrammed in Fig. 2. The first step involves isolating the
VACCINIA: VIRUS,VECTOR, VACCINE
51
TABLE I IN RECOMBINANT VACCINIA VIRUSES FOREIGNGENESEXPRESSED
Reference Herpes simplex virus thymidine kinase Influenza virus hemagglutinin Hepatitis B virus surface antigen Herpes simplex virus glycoprotein D Plasmodium knowlesi sporozoite antigen Chloramphenicol acetyltransferase Rabies virus glycoprotein Transmissible gastroenteritis virus gp195 Vesicular stomatitis virus G protein Vesicular stomatitis virus N protein Influenza virus nucleoprotein Human factor IX (Christmas factor) Neomycin-resistance gene Sindbis virus structural proteins P-Galactosidase Epstein-Barr membrane antigen gp340 Sindbis virus structural proteins Respiratory syncytial virus glycoprotein G Tobacco etch virus proteins Friend murine leukemia virus gp70/p15E HTLV-I11 envelope gene LAV envelope gene Human preproenkephalin
Panicali and Paoletti (1982); Mackett et al. (1982) Panicali et al. (1983);Smith et al. (1983a) Smith et al. (1983b);Paoletti et al. (1984) Paoletti et al. (1984);Cremer et al. (1985) Smith et al. (1984) Mackett et al. (1984) Kieny et al. (1984); Wiktor et al. (1984) Hu et al. (1984) Mackett et al. (1985) Mackett et al. (1985) Yewdell et al. (1985) de la Salle et al. (1985) Franke et al. (1985) Rice et al. (1985) Chakrabarti et al. (1985) Mackett and Arrand (1985) Franke and Hruby (1985) Ball et al. (1986) Dougherty et al. (1986) Stephens et al. (1986) Chakrabarti et al. (1986) Hu et al. (1986) Thomas et al. (1986)
foreign gene of interest. Once obtained, the gene is cloned into a convenient cloning vehicle such as pBR322. In addition to the foreign gene, specific vaccinia sequences are cloned into the pBR322 such that they flank the foreign gene. The vaccinia sequences, which can range from approximately 100 to 10,000 bp on either side of the insert, serve a vital function. They will “direct” the foreign gene to the homologous sequences on the vaccinia genome. The recombinant plasmid containing the chimeric insert is introduced into tissue culture cells via standard transfection procedures utilizing a calcium-orthophosphate-precipitated preparation of plasmid DNA. Concomitantly, the cell is infected with infectious vaccinia virus. This rescuing virus will enter the cytoplasm, where it will proceed through its biogenesis. After uncoating and during DNA replication, a vaccinia genome can come into close proximity to the DNA sequences of the recombinant plasmid. The homologous vaccinia sequences on the plasmid and on the viral genome can pair up by the process of in uiuo recombination, which occurs
52
ANTONIA PICCINI AND ENZO PAOLEWI RECOMBINANT D N A
j?a322 CClNlA SEQUENCES
MBlNATlON
REPLICATION OF RECOMBINED D N A
DNA REPLICATION
FIG.2. General scheme for the insertion of foreign genes into vaccinia virus (see the text for details).
with a frequency of approximately 0.1%.Once this is accomplishedthe virus genome can rescue the foreign gene by incorporating it into its own DNA.Once this has occurred, replication of the recombined DNA molecule can continue followed by maturation of the novel recombinant vaccinia virus.
VACCINIA: VIRUS, VECTOR, VACCINE
53
As mentioned earlier, the vaccinia RNA polymerase poorly recognizes heterologous promotor sequences. Therefore, the flanking vaccinia sequences upstream of the transcriptional direction of the foreign gene preferably contain a vaccinia promotor. Genetic manipulations of the chimeric plasmid can be designed so that after in v i m recombination a foreign gene can (1)be inserted in situ together with a vaccinia promotor, (2) replace vaccinia sequences downstream from a vaccinia promotor, or (3)be translocated along with a flanking vaccinia promotor to another part of the genome. All these manipulations require that when the foreign gene is recombined into the vaccinia genome it does not interrupt the flow of essential genetic information. This requirement has led us to search for nonessential sites within the vaccinia genome. Nonessential genes such as the thymidine kinase or DNA sequences deleted from the virus provide obvious targets for the insertion of foreign genetic elements. Perkus et al. (1986)have identified additional nonessential loci and their location is illustrated in Fig. 3.At least a dozen nonessential loci reside within the leftmost 30 kb of the vaccinia genome. In addition to giving us the advantage of using more than one target site for gene insertion, these sites can also be used as loci from which viable deletion mutants of vaccinia virus can be generated. These deletion mutants extend the inherent ability of vaccinia to hold considerable quantities of foreign genetic material (Panicali et al., 1983;Smith and Moss, 1983). In addition to the spontaneously occurring deletion of 9.8 kb toward the left end of the S-variant vaccinia genome (Panicali et al., 1981),other deletion mutants have been generated in the L-variant vaccinia genome and are shown in Fig. 3. Since these areas of deleted DNA are not essential for viral replication, foreign genes can be readily inserted anywhere within these regions. The availability of these viable vaccinia deletion mutants provides additional space for packaging foreign DNA and allows the simultaneous expression of multiple foreign genes, which can result in immunization against multiple pathogens (polyvalent vaccines). Additionally, these multirecombinants can be used for the coordinated expression of a family of related gene products or for the production of a variety of biologically active molecules from a single vaccinia infection. As an example of the versatility of vaccinia virus to express a variety of antigens and its potential use as a polyvalent vaccine, we have constructed a vaccinia recombinant which contains three foreign genes (Perkus et al., 1985). This triple recombinant contains genes coding for three pertinent antigens: the influenza virus hemagglutinin (InfHA), the hepatitis B virus surface antigen (HBsAg), and the herpes virus type 1 glycoprotein D (HSVgD).
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FIG.3. Insertions and deletions of vaccinia virus. Line B illustrates the physical map of the L v a r i a n t vaccinia genome as defined by HindIII restriction enzyme fragments designated A through 0. The location of viable insertion mutants generated by site-specific insertion of modified herpes simplex virus thymidine kinase-coding sequences is indicated by numbers 1 through 12 in line A. Viable deletion mutants derived from the L-variant genome are shown in lines C through F. The deletion mutants are indicated as Vdl and assigned map coordinates based on kilobases of deleted DNA referenced to the left terminus of the L-variant genome. The enzyme sites indicated are as follows: B, BanHI; Bg, BglII; H, HindIII (from Perkus et al., 1986).
VACCINIA: VIRUS, VECTOR, VACCINE
55
C . Analysis of Vaccinia Recombinants After a recombinant vaccinia virus is produced in tissue culture cells, the newly aquired foreign DNA can be detected by standard DNA hybridization techniques (Panicali and Paoletti, 1982). Furthermore, genetic manipulations used in recombinant design can provide additional detection/selection of the novel virus by enzyme assays which rely on thymidine kinase activity (Mackett et al., 1982) and pgalactosidase activity (Chakbrabarti et al., 1985) or by drug resistance using the neomycin gene (Franke et al., 1985). Once a recombinant is identified, it can be isolated, purified, and grown to high titers to be used for subsequent analyses. For example, restriction enzyme analysis of the viral DNA can verify the genomic organization of the construct and detect any rearrangements of the vaccinia genome. With respect to foreign gene expression, reports listed in Table I demonstrate the successful ability of vaccinia virus to express foreign genes in a manner similar to, if not identical with native synthesis. The products can be processed correctly and exhibit authentic secondary modifications such as glycosylation. The antigens encoded by the foreign genes are properly localized in the vaccinia virus recombinantinfected cell and are immunologically presented as they are in the natural infection. For example, the hepatitis B virus surface antigen is secreted from infected cells as a morphologically distinct 22-nm particle. In contrast, the influenza hemagglutinin and the herpes simplex glycoprotein D become associated with the membrane of the infected cell. These properties are essential if one wants to use vaccinia virus as an expression vector that exhibits biological fidelity. The heterologous transcripts synthesized from recombinant vaccinia-infected cells can be isolated by standard purification methods and the mRNAs can be detected by Northern blot analysis as shown in Fig. 4. The results shown in lane E verify the synthesis of HSV RNAs in cells infected with the vaccinia recombinant vP60, which expresses the HSV-1 glycoprotein D gene (Paoletti et al., 1984). The major RNA species migrates at approximately 2500 bp, which is similar in size to the dominant RNA synthesized in HSV-l-infected cells as shown in lane F. The minor HSV-1 RNA bands also show similar migration patterns. HSV-lgD RNA is detected in neither host HeLa cells (lane C) nor HeLa cells infected with wild-type vaccinia (lane D).This type of analysis can also be used to characterize transcriptional alterations of the recombinant vaccinia genome. In lane A is shown endogenous RNA from wild-type vaccinia which is complementary to the 3’ region of the genome where the HSV-lgD insertion was made in the recombinant vP60. When RNA from vP60 is hybridized to the vaccinia DNA
56
ANTONIA PICCINI AND ENZO PAOLETTI
A
B
C
D
E
F
G 3579 2297 I543
703
FIG.4. Northern blot of RNA isolated from a vaccinia recombinant expressing the herpes simplex virus glycoprotein D gene. Viral RNA was extracted from cells infected at a multiplicity of 10 PFU/cell. Cells were lysed by Dounce homogenization and the RNA was purified by a guanidine-HC1 method (Barth et al., 1982). Early RNA was isolated 3 hours after infection from cells treated with 40 pg/ml of cytosine arabinoside. Control host RNA was extracted from uninfected HeLa cells. The RNAs (20 pg) were separated on 1.5% agarose gels, blotted onto nitrocellulose paper, and hybridized with either a 32P-labeled HSV-lgD clone (lanes C-F) or a Hind111 F subclone (lanes A-B). Hybridization was detected by autoradiography with Kodak XAR-5 film at -70°C for 20 hours. The size markers in lane G were generated by a mixed enzyme digestion of pBR322 and the sizes (bases) are indicated to the right of the bands.
sequences from this region, the endogenous 350-bp RNA is not detected (lane B), but instead a larger RNA is identified. This RNA, which is complementary to the DNA probe at the insertion site, is identical in size to the RNA in the vP60-infected cell that was detected with HSV-lgD sequences. Thus, the endogenous RNA was displaced and it appears that the HSV-lgD RNA contains a small number of vaccinia sequences at its 3' end. Another type of RNA analysis involves determining the steady-state levels of foreign gene RNAs regulated by different vaccinia promotors. A Northern blot, shown in Fig. 5, illustrates differences in RNA expression. Lanes B, F, and J indicate absence of detectable hybridization to RNA isolated from cells infected with wild-type vaccinia virus. Lanes C and D show different HBsAg mRNA levels in cells infected with the vaccinia recombinants vP59 and vP139, respectively. Lanes G
VACCINIA: VIRUS,VECTOR, VACCINE
A B
C
D
E
F
G
H
57
I
J
K
FIG.5. Northern blot of RNA isolated from a variety of vaccinia recombinants using different early promotors. Early RNA was isolated from vaccinia recombinants and analyzed as described in Fig. 4. The probes used correspondto the specific genes present in the recombinants as described in the text. Lanes A, E, and I are pBR322 size markers, as described in Fig. 4.
and H show different InfHA mRNA levels in cells infected with vP59 and vP142, respectively. It should be noted that vP142 (lane H) is a double vaccinia recombinant containing InfHA and HSVgD genes under the regulation of two different promotors. HSVgD RNA expression from vP142 is shown in lane K. Various assays are available to measure expression of the foreign gene at the protein level. For example, functional herpes simplex virus thymidine kinase can be quantitated by monitoring the phosphorylation of [1251]iododeoxycytidine(Panicali and Paoletti, 19821, a property not exhibited by eukaryotic or vaccinia thymidine kinases. Assays for the expression of a number of genes in recombinant vaccinia virus have relied on immunological assays. These require that the protein product be structurally correct such that it will react with antiserum generated against the native product. Perkus et al. (1985) demonstrated the presence of InfHA and HSV-lgD gene products at the surface of live cells by using antisera generated against the native product followed by 1251-labeledprotein A. The cell monolayer was lifted onto a filter and the radioactive signal was detected by autoradiography. For HBsAg detection, a commercially available radioimmunoassay kit, AUSRIA I1 (Abbott Laboratories), was used, which utilizes radiolabeled hepatitis antibodies to measure HBsAg in the culture media. These assays gave a qualitative measure of protein expression and showed us that the products can react against antibodies generated against the native antigens. Furthermore, they al-
58
ANTONIA PICCINI AND ENZO PAOLETTI
lowed us to follow the cellular localization of the products. For a more quantitative measure of protein expression we have disrupted infected cell monolayers and applied the cell extract onto nitrocellulose filters. The amount of extract can be accurately adjusted based on virus output titer. The filters are treated with the test antiserum followed by 1251-labeledprotein A treatment. After autoradiography, signal intensity is proportional to protein levels. We are using this type of analysis to test the results of fine genetic manipulations at the DNA level, where vaccinia promoters of different strengths are used to direct the synthesis of foreign genes. This is an important consideration since often a sufficient amount of antigenic mass must be produced in order to elicit an immune response adequate for vaccine development. Immunoprecipitation of the radiolabeled product can also be informative with regard to protein level while giving us an analysis of the structural forms of a particular product. For example, Fig. 6 is an autoradiogram of an SDS-polyacrylamide gel showing the [36S]methionine-labeled polypeptides expressed by vaccinia recombinant vP218 (lane E), which expresses the hepatitis B small pre-sAg or pre-S(2) gene (Milich et al., 1986), and by wild-type virus (lane F). The similarities in the observed polypeptide profiles suggest that no dramatic metabolic effects are produced by infection of cells with vP218 as compared with wild-type virus. The polypeptides in lane E and F were immunoprecipitated with antihepatitis antiserum and the results are shown in lanes C and D, respectively. The antiserum did not react with any specific polypeptides produced by wild-type virus (lane D).However, the antiserum did react with specific hepatitis polypeptides produced by vP218-infected cells (lane C). These polypeptides exhibited sizes of 19.9, 23.2, and 30.4 kDa. These sizes and the apparent band heterogeneity exhibited by the largest band correspond to the surface antigen proteins expressed by hepatitis B virus (Heerman et al., 1984) and are similar to those expressed by mammalian cells expressing cloned HBsAg genes (Michel et al., 1984). Our laboratory has been directly involved in designing vaccinia as a live recombinant vaccine. Much of our work has concentrated on assessing the immunogenicity of the foreign gene product using laboratory animals. The test animals are inoculated with a particular vaccinia recombinant and the immune sera are tested for the presence of antibodies. We have detected InfHA antibody levels using a hemagglutinin inhibition assay in which authentic InfHA that normally agglutinate either guinea pig or chicken erythrocytes is prevented from doing so if antibodies are present in the assay. Antibodies against HBsAg can be detected using a commercially available AUSAB radioimmunoassay kit (Abbott Laboratories). Antibodies directed against
59
VACCINIA: VIRUS, VECTOR, VACCINE
97 K
97K 68K
68 K
43K
43 K
25.7 K
25.7K
18.4K
18.4K
14.3K
14.3K
FIG.6. Immunoprecipitation of hepatitis B surface antigen (HBsAg) proteins synthesized by a vaccinia recombinant. As described in the text, an aliquot of the [3~S]methionine-labeledinfected cell lysate was analyzed directly (lane E, vP218; lane F, wild-type virus) and immunoprecipitated with hepatitisB antisera (lane C, vP218; lane D, wild-type virus). The vP218-infected cell lysates were immunoprecipitated with normal serum (lane B) or with immune serum (lane C). Lanes A and G are molecular-weight size markers. After fractionation, the gel was treated with ENSHANCE (New England Nuclear, Boston, Massachusetts) and was analyzed by autoradiography.
60
ANTONIA PICCINI AND ENZO PAOLETTI
vaccinia, InfHA, and HSVgD can also be assayed by testing the ability of the immune sera to affect neutralization of viral infectivity in in uitro plaque reduction assays. Using these assays we demonstrated that animals are able t o make immunoreactive antibodies against a number of foreign antigens expressed in a single vaccinia recombinant (Perkus et al., 1985). Other animal studies have addressed questions regarding the feasibility of immunization regimens with vaccinia recombinants (Perkus et al., 1985).This involved immunizing a rabbit with a HBsAg vaccinia recombinant which resulted in eliciting high titers of HBsAg antibodies for at least 1 year. The animal was then revaccinated with a vaccinia recombinant expressing the same foreign gene. The revaccination resulted in increased levels of antibodies directed against HBsAg. Thus, a booster effect was established for HBsAg that was expressed under vaccinia regulation. To determine whether one could achieve successful revaccination with a different recombinant we immunized a rabbit with a HBsAg vaccinia recombinant, waited approximately 1 year, and then immunized with a InfHA vaccinia recombinant. The initial HBsAg antibody titers remained high and were unaffected by the second immunization, which in itself produced antibodies against InfHA. Taken together, these studies were successful in showing that recombinant vaccinia virus can be used in a revaccination protocol either to give a booster effect or to elicit an immunological response to a different foreign antigen upon revaccination. The success of using recombinant vaccinia vaccines has been demonstrated by protection studies. These studies, showing the protection of vaccinia recombinant-immunized animals against subsequent challenge with the pathogen, can be found in a number of reports (Smith et al., 1983b; Wiktor et al., 1984; Paoletti et al., 1984,1985; Mackett et al., 1985). IV. PROSPECTUS Vaccinia displays a number of characteristics which make it an ideal expression vector. Some of these are (1)the ability to incorporate large amounts of exogenous DNA; (2) the faithful transcription of the heterologous genes producing RNAs, which are translated into protein resembling the native product in structure, function, and localization; and (3) the cytoplasmic site of vaccinia replication, which allows gene expression to proceed without interference by the host genome. These properties are useful for using vaccinia as a cloning vector and for
VACCINIA: VIRUS, VECTOR, VACCINE
61
vaccine development. However, in the latter case, safety is a critical issue. Although past vaccinations with vaccinia virus were relatively safe, there were rare but occasionally fatal complications involving the skin and central nervous system. However, the use of vaccinia as a vaccine for smallpox protection has been greatly beneficial to the general population. As a prospectus, we can envision vaccinia virus being genetically manipulated to such a degree that we could expect to generate a virus containing only the genetic material for replication and lacking those parts of the genome responsible for pathogenesis. Future research efforts will focus on the vaccinia genome with respect t o defining the genetic parameters which control the expression of both endogenous and exogenous genes. For example, further identification of nonessential vaccinia DNA sequences will establish the minimum amount of genetic material necessary for virus replication; studies aimed at deleting large portions of the vaccinia genome would determine the minimum amount of DNA necessary for vaccinia biogenesis, while making room for the insertion of additional foreign genetic material. This would result in the ability to insert multiple genes for the production of polyvalent vaccines. Thus, the benefit-to-risk ratio would be skewed toward the benefit achieved by a single or low number of vaccinations resulting in immunity to multiple diseases represented by a dozen or so foreign genes expressed by a single vaccinia virus. A polyvalent vaccine would be especially useful for those diseases in which the pathogen may exhibit a number of surface proteins, all of which are immunogenic, as with herpes infection, or in more complicated systems such as the malaria parasite, which expresses different antigens during its life cycle. The ability of the immune system to recognize and react to a variety of antigens remains to be established. However, the diversity should not be significantly greater than that presented by vaccinia alone. In these cases, attenuation of antigen levels may be of influence. With the identification of more viral genome products, molecular dissections of the vaccinia genome will be aimed at defining both host range and virulence. Analysis of regulatory control signals for vaccinia transcription will continue toward defining the properties of a vaccinia promoter. As this data base accumulates, consensus sequences will be generated to determine the subtle differences in the temporal and quantitative control of vaccinia transcription. Additionally, the translation of exogenous gene transcripts encoding foreign proteins will be analyzed in terms of RNA utilization and host RNA competition.
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ANTONIA PICCINI AND ENZO PAOLETTI
With all these efforts combined, the complexity of vaccinia biogenesis is sure to be unraveled, revealing a virus with the diversity to be used as an expression vector and to be used once again as a vaccine.
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