Proc. Nat!. Acad. Sci. USAVol. 88, pp. 3190-3194, April 1991Genetics

Assembly and analysis of a functional vaccinia virus "amplicon"containing the C-repeat region from the M protein ofStreptococcus pyogenes

(homologous recombination/epitope duplication/antlgenlc variation)

DENNIS E. HRUBY*, OLAF SCHNEEWIND , ELIZABETH M. WILSON*, AND VINCENT A. FISCHETTIt*Center for Gene Research and Biotechnology, Department of Microbiology, Oregon State University, Corvallis, OR 97331-3804; and tThe RockefellerUniversity, 1230 York Avenue, New York, NY 10021-6339

Communicated by K. E. van Holde, December 20, 1990

ABSTRACT Previous studies have shown that when inoc-ulated intranasally into nmice, vaccinia virus(W) recombinantsexpressing the carboxyl half of the Streptococcus pyegenes Mprotein [which contains the C-repeat region (CRR)] could elicita protective immune response against subsequent challenge byboth homologous and heterologous serotypes of pathogenicgroupA streptococci. In the present study, an insertion plasmidwas constructed that contained three tandem in-frame repeatsof a 310-base-pair DNA sequence encoding the CRR fromstreptococcal M6 protein under control of a constitutive viralpromoter. The plasmid was used to introduce the bacterialsequences into the W genome by homologous recombination.Surprisingly, the recombinant W:CRR3X virus that wasisolated appeared to represent not an individual recombinantvirus but a complex mixture of variants that contained from 1to >20 tandem copies of the CRR region at the insertion site.This genomic complexity was mirrored at the transcriptionallevel in that a nested set of coterminal transcripts was detectedin W:CRR3X-infected cells, which increased in size from 1400to 6600 bases by increments of ==300 bases. All transcriptscontaining two or more CRR inserts appeared functional, asWestern (immuno) blot analyses of W:CRR3X-infected cellextracts revealed a family of CRR-related proteins with ap-parent molecular masses that increased from 30 kDa upwardin increments of 10 kDa. All data are consistent with thehypothesis that variation in the W:CRR3X recombinants isfrom random crossover events that occur within the CRRregion during viral DNA replication. These results suggest thatthe genomic diversity generated by the "recombinogenic"properties of vaccinia recombinants containing tandem foreigninserts could be used to facilitate induction of a broadlyprotective immune response against antigenically diversepathogenic agents.

Human diseases from infection by Streptococcus pyogenes(group A) remain a significant health problem. In the UnitedStates alone, 25 to 35 million cases of group A streptococcalinfections, which primarily afflict school-age children, arereported annually (1). The high incidence and potentialseverity of streptococcal infections provide impetus for de-velopment of an effective and safe vaccine to prevent strep-tococcal-related diseases.Although the surface of the group-A streptococcus repre-

sents a complex antigenic mosaic, the ability of these orga-nisms to cause infection is attributed to the M protein, acoiled-coil fibrillar molecule on the cell-wall surface thatgives the organism the ability to resist phagocytosis. Thetranslated nucleotide sequence of the M molecules reveals

that >70%6 of the protein is composed of separate blocks oftandem-sequence repeats (2).Development of a vaccine to prevent group A streptococ-

cal infections, particularly pharyngitis, has been hamperedby the facts that opsonic antibodies are type specific and that>80 serotypes have thus far been identified. Type-specificantibodies bind to the hypervariable amino-terminal end ofthe molecule distal to the cell surface; however, the carboxyl-terminal C-repeat region (CRR) located proximal to the cellsurface is highly conserved among streptococci of manydistinct serotypes (2). To determine whether antibodies to theconserved exposed epitopes of M protein influence thecourse of nasopharyngeal colonization by group A strepto-cocci, peptides corresponding to these regions were used asimmunogens in a mouse model (3). It was found that miceimmunized intranasally with a cholera toxin-peptide com-plex showed a significant reduction in colonization comparedwith mice immunized with cholera toxin alone, and theprotection was not type specific (4).As an alternative approach, genetically engineered recom-

binant vaccinia virus (VV) strains expressing all or part ofthestreptococcal M protein have been constructed for testing aspotential live-virus vaccines (5, 6). In initial studies, se-quences encoding the entire open reading frame of the Mprotein from serotype 6 S. pyogenes (M6) were inserted byrecombination into the VV genome in a transcriptionallyactive configuration. The derived VV recombinant (VV:M6)was capable of expressing high levels of full-length antigen-ically authentic M protein in either infected tissue culturecells or experimental animals (5).Because the VV:M6 recombinant expressed the entire M

protein, including the immunodominant epitopes and hyper-variable regions found within the amino-terminal half of theM6 molecule (7), this formulation was not a suitable vaccinecandidate. A second-generation VV recombinant (VV:M6')was, therefore, constructed that expressed only the regioncorresponding to sequences found within the conservedcarboxyl-terminal half of the M6 protein. In animal trials,pharyngeal colonization by streptococci after intranasal chal-lenge with these organisms was significantly reduced in miceimmunized intranasally with the VV:M6' virus. M-protein-specific serum IgG was markedly elevated in vaccinatedanimals and absent from controls. Most significantly, theprotective immunity induced by the VV:M6' was also pro-tective against challenge by a heterologous M14 streptococ-cal serotype (6).Because the VV:M6' recombinant virus exhibits obvious

potential as an effective anti-streptococcal immunizationvehicle, further development of this vector system to en-hance antigenicity of the protective M protein epitopesseemed in order. In this communication we report on the

Abbreviations: VV, vaccinia virus; CRR, M protein C-repeat region.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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construction and molecular genetic analysis of a vacciniarecombinant that presents multiple copies of the CRR of theM6 molecule (VV:CRR3X). In examining the characteristicsof the VV:CRR3X virus for use in our animal model we foundthat the virus exhibited some surprising and unusual "re-combinogenic" properties that may enhance the effective-ness of the VV as a vector for similarly constructed antigens.

MATERIALS AND METHODSCells and Virus. VV (WR strain) was grown and titered on

BSC-40 cells as described (8).Construction of pW3:CRR3X. Plasmid pVV3:M6 (5) was

digested with HincII/Pvu II and the 992-base-pair (bp) frag-ment of the emm6.1 gene that contained the CRR wassubcloned into the Sma I site of the pMac5-8 vector (providedby H.-J. Fritz and K. Fiedrich, Max-Planck Institute forBiochemistry, Martinsried, F.R.G.). We used this templateand two oligonucleotides (GAAACTTTGTTGAATTCA-TCTTTTTTAGC and CTTCAAGTTTGAATTCTAGCT-CAGCT) in concert with the site-directed mutagenesis (9) tomutagenize a 310-bp fragment encoding the CRR region(amino acid residues 224-335) of the M6 protein flanked bytwo EcoRI sites. The 310-bp DNA fragment was sequenced,purified, and subjected to partial-ligation conditions. Multi-mers of the CRR were separated by preparative agarose gelelectrophoresis. The 930-bp DNA fragment was ligated to theEcoRI site of expression vector pINIIIompA2 and trans-formed into E. coli DH1. Recombination of the codingsequences was confirmed by restriction analyses and bySDS/PAGE analysis of the expressed polypeptide followedby immunoblotting. The CRR trimer insert was released fromthe vector with Xba I/BamHI cuts and ligated into theBamHI site ofpVV3 (10) after filling all overlapping ends withKlenow polymerase to yield pVV3:CRR3X. This designedprotein was expressed from the VV 7.5-kDa constitutivepromotor imbedded within the coding sequences of the VVthymidine kinase gene (tk). The ribosome-binding site, startcodon, and outer membrane protein A (ompA) signal se-quence come from the expression vector pINIIIompA2. Thepolypeptide ends with a 12-amino acid tail of the pVV3polylinker region.Marker Transfer. The 7.5-kDa:CRR3X chimeric construc-

tion was introduced into the VV by homologous recombina-tion, essentially as described (10) by using calcium phos-phate-mediated transfection procedures (11) in concert withconditional-lethal VV mutants (12, 13). Potential recombi-nants were selected using 5-bromodeoxyuridine (14) andplaque-hybridization procedures (15).

Analysis of Recombinant Virus. Viral DNA was isolatedfrom wild-type or VV:CRR3X-infected cells as described(10), digested with the appropriate restriction endonucleases,and subjected to Southern blot hybridization procedures (16)by using a CRR-specific DNA fragment that had been labeledby the random-primed labeling procedure (17). After expo-sure to film, the blot was stripped and rehybridized with aradioactive probe fragment prepared similarly that was spe-cific for the 5' half of the VV tk gene.

Total cytoplasmic RNA was isolated from cells infectedwith either wild-type or VV:CRR3X virus in the presence ofcycloheximide at 100 ,g/ml to amplify the expression of viralearly mRNA species (18). The RNA was isolated frominfected cells and purified by pelleting through CsCI gradientscontaining 1% N-lauroylsarkosine (19). The different RNAspecies were separated according to size by denaturingagarose/formaldehyde gel electrophoresis (20). After elec-trophoresis, the RNA was transferred to nitrocellulose andsubjected to dual-probe hybridization, as described above.Monolayers of cells were infected with either wild-type or

recombinant VV:CRR3X VV at a multiplicity of 20 plaque-

forming units per cell and incubated at 370C for 18 hr. Theinfected cells were then harvested, washed twice with phos-phate-buffered saline, and lysed in 150 /.l of SDS/gel loadingbuffer. The DNA was sheared by passing the extract througha 22-gauge needle five times. Ten microliters of each samplewas separated by SDS/PAGE on mini-slab gels (21). Afterelectrophoresis the proteins were electroblotted onto nitro-cellulose and immunoblotted, as described (22) by using amonoclonal antibody directed against the CRR region of theM6 protein (23).

RESULTS AND DISCUSSIONConstruction of Recombinant W:CRR3X. From synthetic

peptide studies (4) in conjunction with the VV:M6' results (6),the region responsible for cross-protection against strepto-coccal pharyngeal colonization was localized to the CRR ofthe M protein, which is located within the carboxyl-terminalhalf ofthe molecule. The amino acid sequence correspondingto the CRR is shown in Fig. lA. This 102-amino acid sequenceis derived from residues 234-335 of the native M6 protein.Molecular cloning and site-directed mutagenesis-based pro-cedures were used to construct an artificial gene in which anATG codon in an appropriate mammalian translational con-text abutted an insert consisting of a 310-bp DNA fragmentencoding the CRR, which had been triplicated in-frame,followed by a stop codon. This gene was inserted into the VVgenome by DNA-mediated recombination using standardmarker transfer techniques to produce the VV:CRR3X re-combinant (Fig. 1B). Plaques arising from the VV:CRR3Xrecombinants were detected by plaque hybridization using aCRR-specific probe. The virus was plaque purified, and theviral DNA was extracted and analyzed by Southern blottingprocedures.Genomic Structure of W:CRR3X Recombinant. Fig. 1B

shows that a HindlII site is contained within each CRR unit,such that digestion with this enzyme should release the310-bp CRR insert plus the chimeric 5' and 3'-flanking DNAfragments that would be expected to be 1.35- and 0.1-kilobasepairs (kbp), respectively. In contrast, Pst I endonucleaseshould cut external to the entire chimeric transcriptional unitand release a single 1.3-kbp fragment that can be detected byusing a CRR-specific probe. Fig. 2 shows the results ofdigesting the starting pVV:CRR3X plasmid DNA, wild-typeVV DNA, and VV:CRR3X DNA with either HindIII or PstI restriction endonucleases. The agarose gel stained withethidium bromide shows the DNA-fragment pattern (Fig. 2Left). The 4.8-kbp HindIII J DNA fragment, which containsthe target recombination site for the CRR:3X insert, is absentin the DNA from the VV:CRR3X recombinant. When this gelwas transferred to nitrocellulose and analyzed with a tk-specific probe, DNA fragments of the predicted sizes (Fig. 1)were detected in all digests with HindIII and Pst I enzymes(Fig. 2 Center). In contrast, although the pattern offragmentsdetected in the VV:CRR3X HindIII digest was as predictedwhen using a CRR-specific probe, the pattern seen in theVV:CRR3X Pst I digest was highly aberrant (Fig. 2 Right).Instead of liberating an insert with an expected size of 1.3kbp, a series of inserts that increased upward from 700 bp, in300-bp increments, was apparent. In darker exposures, frag-ments as large as 6.6 kbp could be seen (data not shown). Thehigher-molecular mass bands seen are incomplete digestionproducts that disappear upon prolonged incubation (data notshown). Assuming the size variation is from recombinationwithin the CRR unit, this variation would correspond to arange of 1-21 inserts. Thus, the VV:CRR3X recombinant isapparently not a single entity but rather represents a familyof recombinants with variable numbers of CRR inserts. Theapparent structure ofthe VV:CRR3X recombinant genome isshown at the bottom of Fig. 1B. Although the sequence ofevents responsible for generating the CRR insert heteroge-

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A XKKTAIAIAVALAGFATVAQ AA

EFNKVSEASRRGLRRDLDASRBAKKQVEKDLANLTAELDKVKBBKQISDASRQGLRRDLDASREAKKQVEKALEEANSKLAALEKLNKELEESKLTESKEKAL

EFNKVSEASRKGLRRDLDASREAKKQVEKDLANLTAELDKVKBEEQISDASRQGLRRDLDASREAKKQVEKALHEANSKLAALBKLNKELEESKKLTEKEKAEL

EFNKVSEABRKQLRRDLDASREAKKQVEKDLANLTAELDKVKEEKQISDASRQGLRRDLDASRZAKKQVEKALBEANSKLAALEKLNKELEESKKLTEKEKAEL

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FIG. 1. Predicted genome structure of recombinant VV:CRR3X in the region containing the M protein CRR insert. (A) Translated aminoacid sequence (in one-letter code) of the artificial gene from VV:CRR3X. The outer membrane protein A (ompA) signal sequence-cleavage siteis indicated by the arrow. Each 104-residue M6 protein CRR segment is separately grouped and starts with the amino acid residues EF derivedfrom the EcoRI linker. The protein ends with a 12-residue tail coded by the polylinker region. (B) Predicted structure of HindIll J region ofrecombinant VV:CRR3X viral genome. Letters above line indicate locations ofHindIll (H) and Pst I (P) restriction endonuclease-cleavage sites.Numbers below line indicate rightward distance in base pairs from the leftmost HindIll-cleavage site, which corresponds to thejunction betweenthe HindIII L and J fragments on the viral genome. Bold line on left and bold arrow at right indicate positions of 5' and 3' halves, respectively,ofthe viral tk gene serving as the genomic insertion site for CRR repeats. Position and orientation of the VV 7.5-kDa promoter element is shownby open box enclosing an arrow. CRR repeats are indicated by black boxes. Grey regions between CRR repeats correspond to EcoRI linkersthat provide in-frame ligations. Hatched box represents a short region derived from the parental CRR plasmid that provides an in-frame ATGcodon. Positions of the early (cross-hatched circle) and late (black circle) viral transcriptional start sites are shown as well as the transcriptionalstop signal used during the early phase of infection (o). Positions of the initiator methionine (*) and translational stop codon (O) are similarlyindicated. The genome below indicates structure ofthe VV:CRR3X recombinant genome (where n equals 1-20) as derived from molecular geneticanalyses.

neity is unknown, the mechanism is probably similar to the Expression of CRR Sequences. To determine whether eachunequal crossover mechanism proposed to account for the member of the VV:CRR3X recombinant family was tran-size diversity generated with the VV terminal repeats during scriptionally active, RNA-blot hybridization analyses werenormal viral replication (24). done on cytoplasmic RNA isolated from recombinant or

HindIIl PstI HindlIl Pstl Hindill PstiP V R P V R P V R P V R P V R P V R

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FIG. 2. Southern blot analyses of genomic DNA from wild-type and recombinant VV:CRR3X VV. The parental insertion plasmidpVV3:CRR3X (P), as well as viral DNA isolated from either purified wild-type VV (V) or the recombinant VV:CRR:3X (R) were digested withHindI11 or Pst I; the resulting DNA fragments were resolved by agarose gel electrophoresis. After being visualized by staining with ethidiumbromide, the DNA fragments were transferred to a nylon membrane and sequentially hybridized with 32P-labeled probes corresponding to aninternal portion of the VV tk gene or theM protein CRR. Sizes in kbp and relative migration ofbacteriophage A HindI11 DNA fragments includedas markers are indicated.

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wild-type VV-infected cells (Fig. 3). The filter was sequen-tially hybridized with radioactive probes specific for eitherVV tk or streptococcal CRR sequences to detect both thenative viral tk gene transcript as well as any chimeric CRR:tktranscripts. Results shown in Fig. 3 Left indicate that, asexpected (25), the tk-specific probe detected a 700-basetranscript present in the RNA isolated from VV-infectedcells. In contrast, when the RNA isolated from VV:CRR3X-infected cells was analyzed, a collection of transcripts wasdetected that began at 300 bases in length and increasedupward in size by increments of -300 bases. As shown in Fig.3 Right, a CRR-specific probe detected the same pattern oftranscripts in recombinant-infected cells but did not hybrid-ize to any sequences in wild-type VV-infected cells. Theseresults substantiate the conclusions reached above regardingstructure of the VV:CRR3X recombinants and suggest thatmost, if not all, recombinants are transcriptionally active.

Ability ofthe chimeric CRR:3X transcripts detected in Fig.3 to be translated in vivo into protein was determined byimmunoblotting infected cell extracts with a monoclonalantibody directed against the CRR. Fig. 4 indicates that noCRR-related proteins were detected in extracts from eithermock-infected or wild-type VV-infected cells, whereas, asreported (6), the VV:M6' recombinant expresses a 29-kDaprotein corresponding to the carboxyl half of the M6 proteincontaining the CRR. Proteins containing CRR epitopes areevident; these had apparent molecular masses of 30, 40, 50,60, 70, and 80 kDa in the extracts from VV:CRR3X-infectedcells. Sizes of these protein products are consistent withthose predicted for VV:CRR3X recombinants containing twoor more CRR inserts. By using longer periods of electro-transfer and/or lower dilutions of monoclonal antibody,CRR-related proteins with molecular masses >80 kDa couldbe detected (data not shown). These results suggest that eachof the nested sets ofCRR:3X transcripts has resulted from an

V R

t k

R v

- 9.5 -

- 7.5 -

- 4.4 -

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- 1.77 -- 1.52 -

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- 0.78 -

- 0.53 -- 0.40 -

- 0.24 -

CRR

FIG. 3. Northern (RNA) blot analysis of the CRR insert-derivedtranscripts. Total cytoplasmic RNA was isolated from BSC-40 cellsinfected with either wild-type VV (V) or recombinant VV:CRR3X(R) virus with cycloheximide to amplify the concentration of earlyviral mRNA species. RNAs were separated according to size bydenaturing agarose/formaldehyde gel electrophoresis. RNA wasthen transferred to a nylon membrane and sequentially hybridizedwith 32P-labeled probes corresponding to an internal portion of theVV tk gene or the M protein CRR. Numbers indicate sizes in kb andrelative migration ofRNA standards included in analysis as markers.

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FIG. 4. Western (immuno) blot analysis of CRR-related proteinsexpressed in VV:CRR3X-infected cells. Total detergent-soluble cy-toplasmic extracts were prepared from mock-infected BSC-40 cells(MI) or cells infected with either wild-type VV (WT VV), a VVrecombinant expressing the carboxyl halfofthe M protein (VV:M6'),or the VV:CRR3X recombinant (VV:CRR3X). Extracts were sepa-rated by SDS/PAGE and then transferred by electroblotting onto anitrocellulose filter membrane. To detect any CRR-related proteinsthe filter was immunoblotted by using a monoclonal antibody di-rected against the CRR region of the M protein as the primaryantibody, and then horseradish peroxidase-conjugated anti-mouseserum was used as the secondary antibody. Numbers at left indicatesizes and relative migration of protein standards.

in-frame-crossover event that produces a functional messagethat is translated within the infected cell into a polyepitopeprotein containing immunoreactive CRR sequences. Inter-estingly, this hypothesis explains the previously observedsize heterogeneity in the proteins expressed by VV recom-binants expressing a plasmodial S-antigen containing tandemrepetitive sequences (26).

Stability of W:CRR3X Recombinant. Given that theVV:CRR3X isolate represents a complex mixture of CRR-containing recombinants, whether the recombinant popula-tion was stable or in dynamic equilibrium was an interestingquestion. To address this question, individual VV:CRR3Xplaques, which should have arisen from a single infectiousviral particle containing a single DNA molecule with a fixednumber of CRR inserts, were picked and grown underconditions that should allow only a single round of viralreplication to amplify the viral DNA. The genomic DNA fromnine individual VV:CRR3X plaques amplified in this mannerwas extracted and analyzed by Southern blot hybridizationwith a CRR-specific probe (Fig. 5). Relative to the parentalVV:CRR3X DNA shown at right, a spectrum of differentinsert banding patterns was evident in the plaque isolates,ranging from the VV:CRR3X-G isolate, which had predom-inantly a monomer insert, to the VV:CRR3X-B isolate, whichdisplayed a full range ofCRR inserts from 1 to >20, with sixrepeats being the most prevalent. These results confirm that,although the CRR insert content of individual VV:CRR3Xplaques differs, the recombination process continues to occurduring each round of replication to expand the pool of CRRgenomic diversity.

This communication summarizes the construction andanalysis of a VV recombinant (VVCRR:3X) that expressesmultiple copies ofthe CRR from the streptococcal M protein.Although the virus displays unusual recombinogenic proper-

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Plaque IsolatesA B C D E F G H I R

I 23.1

40

-9.4

K,;

- 4.4_ 67

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FIG. 5. Genomic comparisons of individual VV:CRR3X plaqueisolates. An appropriate dilution ofVV:CRR3X was inoculated ontoa BSC-40 cell monolayer under agar. After 48-hr incubation, progenyof individual plaques were picked and inoculated onto cells of a24-well tissue-culture dish. Infections were allowed to proceed for 12hr. Cytoplasmic DNA was then prepared and analyzed by Southernblot hybridization. Lanes A-I correspond to DNA derived from ninedifferent plaque isolates. DNA from the parental VV:CRR3X re-combinant (R) was included as reference. Sizes and relative migra-tion of bacteriophage A HindIII DNA fragments included in theanalysis as size markers are at right.

ties, this ability does not affect its ability to express immu-noreactive CRR-containing proteins in infected cells. There-fore, this recombinant virus should prove useful in addressingthe questions of (t) how the CRR contributes to the inductionof cross-protective immunity and (ii) whether increasingepitope dosage augments or possibly inhibits the develop-ment of protective antibodies. In addition, these VV:CRR3Xresults may have more general implications for the use ofVVas a recombinant vaccine. Our hypothesis is that polyepitopeprotein expression will simply require introduction oftandemcopies of the desired coding sequence into the VV genome,after which the recombinogenic properties ofthe virus shouldoperate on the "amplicon" to generate the level of diversityseen in our experiments. An alternative hypothesis, is thatunder certain conditions, the recombinant virus will deletethe extra copies of the insert. In any case, high levels ofrecombination may be anticipated to introduce random mu-tations into the target sequence at a reasonable frequency.Although some of these mutations will undoubtedly be silentor introduce stop codons, others will introduce missensemutations. As such, the VV recombinant population shouldgenerate antigenic diversity, mirroring the process that the

native molecule undergoes in vivo, although without selectivepressure. Thus, VV recombinants such as VV:CRR3X mayoffer an opportunity to present the host with an antigenicmosaic for the induction ofa broad array ofantibodies againsta variable epitope of a pathogenic agent and, hence, providemore complete protection. Use of the recombinogenic prop-erties of VV vectors may be of particular relevance withregard to developing effective vaccination strategies againstinfection by serotypically diverse pathogens such as humanimmunodeficiency virus.

This research was supported by Grants Al-00666 (D.E.H.) andAI-11822 (V.A.F.) from the National Institutes of Health and a grantfrom the Mallinckrodt Foundation to V.A.F.

1. Wannamaker, L. W. (1973) Circulation 48, 9-18.2. Fischetti, V. (1989) Clin. Microbiol. Rev. 2, 285-314.3. Bessen, D. & Fischetti, V. (1988) Infect. Immun. 56, 2666-

2672.4. Bessen, D. & Fischetti, V. (1990) J. Immunol. 145, 1251-1256.5. Hruby, D. E., Hodges, W. M., Wilson, E. M., Franke, C. A.

& Fischetti, V. A. (1988) Proc. Natl. Acad. Sci. USA 85,5714-5717.

6. Fischetti, V. A., Hodges, W. M. & Hruby, D. E. (1989) Sci-ence 244, 1487-1490.

7. Fischetti, V. A. & Windels, M. (1988) J. Immunol. 141, 3592-3599.

8. Hruby, D. E., Guarino, L. A. & Kates, J. R. (1979)J. Virol. 29,705-715.

9. Kramer, W., Drutsa, V., Jansen, H.-W., Kramer, B.,Pflugfelder, M. & Fritz, H.-J. (1984) Nucleic Acids Res. 12,9441-9456.

10. Rice, C. M., Franke, C. A., Strauss, J. H. & Hruby, D. E.(1985) J. Virol. 56, 227-239.

11. Graham, F. L. & van der Eb, A. J. (1973) J. Virol. 54, 536-539.12. Condit, R. & Motyczka, A. (1981) Virology 113, 224-241.13. Fahti, Z., Sridhar, P., Pacha, R. F. & Condit, R. C. (1986)

Virology 155, 97-105.14. Mackett, M., Smith, G. & Moss, B. (1984)J. Virol. 49, 857-864.15. Villarreal, L. P. & Berg, P. (1977) Science 196, 183-186.16. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517.17. Feinberg, A. P. & Vogelstein, B. (1983) Anal. Biochem. 132,

6-13.18. Hruby, D. E. & Ball, L. A. (1981) J. Virol. 40, 456-464.19. Glisin, V., Crkvenjakov, R. & Byus, C. (1974) Biochemistry 13,

2633-2637.20. Lehrach, H., Diamond, D., Wozney, J. M. & Boedtker, H.

(1977) Biochemistry 16, 4743-4751.21. Studier, F. W. (1973) J. Mol. Biol. 79, 237-248.22. Miner, J. N. & Hruby, D. E. (1989) Virology 170, 227-237.23. Jones, K. F., Manjula, B. N., Johnston, K. H., Hollingshead,

S. K., Scott, J. R. & Fischetti, V. A. (1985) J. Exp. Med. 161,623-628.

24. Baroudy, B. M. & Moss, B. (1982) Nucleic Acids Res. 10,5673-5679.

25. Hruby, D. E., Maki, R. A., Miller, D. B. & Ball, L. A. (1983)Proc. NatI. Acad. Sci. USA 80, 3411-3415.

26. Langford, C. J., Edwards, S. J., Smith, G. L., Mitchell, G. F.,Moss, B., Kemp, J. & Anders, R. F. (1986) Mol. Cell. Biol. 6,3191-3199.

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