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A Plasmid Family Containing Two Different Expression Cassettes Suitable for Immunomodulation and Genetic Immunization

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A Plasmid Family Containing Two Different Expression Cassettes Suitable for Immunomodulation and Genetic Immunization
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  PLASMID  40,  84–89 (1998) ARTICLE NO . PL981339 SHORT COMMUNICATIONA Plasmid Family Containing Two Different Expression CassettesSuitable for Immunomodulation and Genetic Immunization 1 Silvia A. Ciafre`,* , † Monica Rinaldi,* , ‡ Isabella Vespignani,* Paola Parrella,* , §Davide Seripa,‡ Emanuela Signori,* , ‡ Francesco Ria,§Maria Giulia Farace,* , † and Vito M. Fazio‡ ,  ,2 *  Istituto Medicina Sperimentale, CNR, Rome, Italy;  †  Dipartimento Medicina Sperimentale e Scienze Biochimiche,‘‘Tor Vergata’’ University, Rome, Italy;  ‡ U.R. Oncologia Molecolare e Terapia Genica, IRCCS H.‘‘Casa Sollievo della Sofferenza,’’ San Giovanni Rotondo (FG), Italy;  §  Istituto PatologiaGenerale, Universita` Cattolica (SC), Rome, Italy; and     Department of Molecular Pathology, ‘‘Campus Bio Medico’’ Medical School, Rome, Italy Received June 30, 1997; revised January 21, 1998We have developed an improved eukaryotic expression vector that consists of two distinct,complete, and differentially regulated transcription units. The peculiarities of this prototypevector, named pRC110, are represented by two different strong promoter/enhancer sequences,cytomegalovirus and Rous sarcoma virus, that independently drive transcription of two recombi-nant cDNAs, whichmay be easilyclonedintospecific rare restrictionsites. Moreover, we describea simple way to introduce an optimal translational start site context 5   to any peptide to becloned in our vectors, thus allowing the correct and efficient expression of even a single part of a larger gene or a short synthetic peptide lacking its own AUG and neighboring regions. Wedemonstrate the  in vivo  expression efficacy of pRC110 for use in genetic vaccination throughdirect intramuscular gene transfer: specific antibodies are raised against one of the encodedpeptides 3 weeks after muscle injection, and efficient transcription of the other syngeneic cDNA,mouse interleukin-2, is shown. The development of a ‘‘family’’ of vectors directly deriving frompRC110 is also described, with the common property that one of the encoded proteins maymodulate the effects of the other. We recommend the use of pRC110 for genetic immunizationand immunological response studies, when the concomitant local production of an immunogenicpeptide and of a syngeneic immunomodulating cytokine is required.    1998 Academic Press Two peculiar features of direct intramuscu- the cell’s major histocompatibility complex(MHC) 3 class I pathway (Ulmer  et al.,  1993),lar gene transfer, i.e., the possibility of elic-iting strong immune responses against coded causing activation of cytolytic CD8 / T cells,thus eliciting cell-mediated immunity (Pardollforeign antigens (McDonnell and Askari,1996; Ertl and Xiang, 1996) and the chronic and Beckerleg, 1995). Subsequently, geneticimmunization induces T helper cells and Bsystemic delivery of pharmacologically activeproteins (Rinaldi  et al.,  1995), may be com-bined to exploit a more potent result. An im- 3 Abbreviations used: MHC, major histocompatibility portant advantage of genetic vaccination is complex; CMV, cytomegalovirus; RSV, Rous sarcoma that proteins expressed by this method enter virus; SV40, simian virus 40; mIL2, mouse interleukin-2;hIL2, human interleukin-2; TGF b 1, type b 1 transforming 1 A more detailed description of the experimental pro- growth factor; p/e, promoter/enhancer; r b -glob, rabbit  b -globin; intr, intron; polyA, polyadenylation site; LTR,cedure is available through the Editor of   Plasmid   forreference by interested parties. long terminal repeat; MCS, multiple cloning site; FACS,fluorescence-activated cell sorter; PCR, polymerase chain 2 To whom correspondence should be addressed. Fax: / 39 6 4993 42 57. reaction; RT, reverse transcription.840147-619X/98 $25.00 Copyright    1998 by Academic PressAll rights of reproduction in any form reserved.  85 SHORT COMMUNICATION cells as well, which in turn confer long-lasting and for an enhanced stability of the yieldedtranscripts are contained in a relatively shortimmunological memory and protection (Ertland Xiang, 1996) (for a short review, see plasmid ( Ç 7 kb): Two very efficient promot-ers/enhancers, CMV and RSV, drive the tran-Fazio, 1997). By contrast, standard vaccineantigens are taken up into cells by phagocyto- scription of the two cistrons. A correct andefficient transcription termination is achievedsis or endocytosis and are processed throughthe MHC class II system, which primarily through  b -globin and SV40 polyadenylationsites, respectively, while the stability of thestimulates antibody responses. Moreover, ge-netic vaccines lack an immunological re- yielded transcripts is enhanced through thepresence of the rabbit  b -globin intron se-sponse to vector components, may expressspecific parts of a protein to exhibit only the quence. Moreover, a simple modification of aPCR cloning strategy allows us to clone anyantigenic determinants, or, on the contrary,show minimum constraints regarding size of peptide, even lacking its own appropriateATG, in a translationally favorable context,inserted coding sequences, as compared withother gene transfer vectors, and, finally, they providing an optimal translational start sitecontext, according to the ‘‘Kozak’’ consensusrequire ease of preparation and characteriza-tion with established manufacturing technol- sequence (Kozak, 1987), 5   from an artificialATG. Finally, the possibility of a one-step ori-ogy. The concomitant local production of animmunogenic peptide and of an immunomo- ented cloning between two rare restrictionsites renders this plasmid a useful tool for thedulating cytokine might represent a specificway to further improve immune response insertion of any cDNA.The multistep construction strategy was ac-against insidious agents and/or antigens, espe-cially cancer and lethal or severely impairing complished with standard molecular biologytechniques(Sambrook   et al.,  1989) as outlinedviral diseases (Xiang and Ertl, 1995; Steven-son  et al.,  1995; Chow  et al.,  1997). The best in Fig. 1. Briefly, a complete eukaryotic tran-scription unit coding for the mouse interleu-way of ensuring the coordinate expression of a specific antigen and the immunomodulatory kin-2 (mIL2) cDNA under the control of thestrong CMV major intermediate early pro-cytokine would be to insert both cDNAs intothe same plasmid, thus overcoming the trou- moter/enhancer was inserted on a pUC19(Biolabs) backbone. The stability of the re-bles possibly deriving from the coinjection of different DNA molecules (e.g., targeting of sulting mIL2 RNA is enhanced by the inser-tion of a rabbit  b -globin intron and a rabbitdifferent cells in different amounts, need forand introduction of larger amounts of foreign  b -globin polyadenylation site, respectively, 5  and 3   of the cDNA. The cDNA is cloned atDNA into recipient cells) and allowing a highlocal production of the cytokine, together with a unique  Xho I site from which it can easilybe excised and into which different cytokinelow level, nonrisky, systemic expression.More importantly, the linkage of the expres- coding sequences may be inserted. This kindof mIL2-producing vector, called pUC-CMV-sion of the cytokine to the expression of theforeign antigen in the same cells would ensure mIL2, was used as a recipient plasmid for thecloning of the second eukaryotic transcriptiontheproductionof theimmunomodulatingmol-ecule only when and where needed, that is, as unit, driven by the RSV long-terminal-repeatpromoter/enhancer, and containing a SV40long as the foreign antigen itself is produced.In this paper we report the construction of polyadenylation site preceded by a rabbit  b -globin intron sequence. Two very rare cloninga ‘‘family’’ of plasmids designed for direct  invivo  ‘‘naked’’ DNA transfer, which can be sites,  Nhe I and  Not  I, are present between thepromoter/enhancer and the intron sequences,used for the coexpression of two cDNAs, oneof them encoding for a cytokine and the other thus allowing a directional cloning of anycDNA sequence. The resulting plasmid wasfor the antigenic determinant(s). All the fea-tures needed for a high level of expression called pRC110 and represents the basic plas-  86  SHORT COMMUNICATION  87 SHORT COMMUNICATION mid from which many others were generated (underlined), commonly recognized as astrong translational enhancer (Kozak, 1986;or may be produced in the future for manydiverse applications. Iida and Masuda, 1996), and a  Nhe I site (ital-ics). By this strategy, we were able to cloneThe first, and most trivial, modificationstarting from pRC110 was the replacement of and correctly express the coding sequence fora peptide, Ig H-CDR3, naturally representingmIL2 cDNA with the corresponding humanIL2 (hIL2) cDNA: this simple variation origi- the idiotype-specific part of a larger molecule,and thus lacking its own ATG and neigh-nated a new vector, named pRC120. On theother hand, gene therapy protocols aimed at boring regions. The transcription of thepRC111-encoded mIL2 mRNA in Swiss micedelivering biologically active proteins mightalso benefit from a local silencing of the im- injected muscles was demonstrated either at 2days or at 1 week from DNA injection (Fig.mune response. For this purpose, we modifiedpRC110 by replacing the mIL2 cDNA se- 2A). Furthermore, the functional antigenic ef-fectiveness of the Ig H-CDR3 peptide clonedquence with type b 1 transforming growth fac-tor (TGF b 1) cDNA, thus yielding the plasmid in pRC110 (pRC111) was demonstrated bythe generation of specific antibodies startingcalled pRC130.All the plasmids we generated were tested assoonas3weeksafterplasmidintramuscularinjection: FACS analysis of human Bby in vitro  transfection of CHO cells(Lipofec-tin, Gibco-BRL). mRNA expression analysis lymphoma cells challenged with sera of pRC111 injected mice revealed a significantshowed high levels of expression for bothtranscription units (data not shown). More- specific reactivity (Fig. 2B). Rising specificantibodies were demonstrated at least up to 3over, positive results were produced when weanalyzed the efficiency of pRC110 for use in months from injection, while in mice injectedwith the same plasmid lacking IL2 cDNA,DNA-mediated immunization through directintramuscular plasmid DNA injection in mice. anti-Ig H-CDR3 immune response was sig-nificantly delayed and much less efficientIn particular, pRC110 was used as a vectorfor the  in vivo  expression of a human-specific (Fazio  et al.,  manuscript in preparation).One of the main characteristics of our vec-antigenic determinant (Ig H-CDR3), with theaim of eliciting an anti-idiotype-specific im- tors is the possibility of their modificationthrough the easy insertion of virtually infinitemune response (Stevenson, 1995) possiblysupported by the concomitant production of combinations of cDNAs: substitution of mIL2cDNA in the original plasmid (pRC110) isIL2. In a preliminary trial, a specific antigenicsequence, Ig H-CDR3, was directly amplified easily achievable through an  Xho I or bluntligation, while the cloning site following RSVfrom a human B-cell lymphoma cell line andclonedintopRC110(generatingpRC111). For promoter/enhancer allows a directional clon-ing between two very rare restriction sites,this specific amplification, we designed an up-stream primer (eFW 3 : 5  -TTT GCTAGCC  AC-  Nhe I and  Not  I. We suggest, of course, thatany cDNA, even if lacking  Nhe I and/or  Not  ICATGCACACGGC(C,T)(G,C)TGTATTA-3  ) harboring, besides an artificially inserted ends,canbeclonedintopRC110; infact,thesespecific ends can be added to any sequenceATG in frame with the following coding se-quence, a ‘‘Kozak’’ consensus sequence by amplifying it in the presence of primers FIG. 1.  Schematic diagram of pRC110 construction. (a) pUC-CMV-mIL2 was srcinated by the cloningof the  Xba I–  Bam HI fragment from pBCMG-neo-mIL2 into pUC19 (Biolabs); (b) to obtain pREP4-Intr,the  Bam HI–  Not  I rabbit  b -globin intron (r b -glob intr) fragment from pMV38 was inserted into pREP4(Invitrogen); (c) a  Sal I fragment was excised from pREP4-Intr, blunt-ended by Klenow, and cloned into Sma I digested pUC-CMV-mIL2 to yield pRC110. (d) pRC120 and (e) pRC130 were obtained by  Xho Iexcision of mIL2 cDNA from pRC110 and substitution with human IL2 (hIL2) and porcine TGF b 1 cDNAs,respectively. MCS, multiple cloning site; DCS, directional cloning site.  88  SHORT COMMUNICATION FIG. 2.  (A) RT-PCRanalysis of mIL2 transcription in muscles of pRC111-injectedmice. A representativeresult of the RT-PCR analysis is shown using mRNA prepared from four animals sacrificed at 2 dayspostinjection (lanes 1 through 4, first group) and from four animals sacrificed 5 days later (1 week postinjec-tion) (second set of lanes 1 through 4). Positive samples show a mIL2-specific 158-bp amplified fragment.Lanes C represent noninjected contralateral muscles (negative controls), while the second lane shows theresult of mIL2 PCR performed directly on pRC110 plasmid DNA (positive control). First lane: molecularweight marker, pBR322 MspI. (B) Fluorescence-activated cell sorter (FACS) analysis. Serum from apRC111-immunized mouse was collected 3 weeks after DNA injection and its reactivity against the specificlymphoma cells was tested (shaded area) and compared with that of the preimmune serum (white area).structs. We also thank Giuseppe Saglio and Vania Cilli encompassing them as flushing ends (Ciafre` for patients’ B-cell lymphoma CDR3 H  idiotypic sequence et al.,  1995). Simply by the use of specific amplification. This research was partially supported by primers, weintroducedasequencedesignedto grants from the Italian Ministero della Sanita`, Ricerca act on the translation efficiency of the mRNA  Finalizzata 1995–1998, and from the Italian Ministerodell’Universita` e Ricerca Scientifica, Projects 60% and cloned in pRC110, in view of the fact that 40%. We are also grateful to the IRCCS H. Casa Sollievo these kinds of modifications appear to repre- della Sofferenza for financial support (Ricerca Libera sent the ultimate improvements that might 1996). possibly enhance the power of plasmid ex-pression vectors, as they affect a step of the REFERENCES expression process, translation, which ishighly regulatable but yet poorly exploited Chow, Y. H., Huang, W. L., Chi, W. K., Chu, Y. D., and (Hartikka  et al.,  1996). Tao, M. H. (1997). Improvement of hepatitis B virusDNA vaccines by plasmids coexpressing hepatitis Bsurface antigen and interleukin-2.  J. Virol.  71,  169–178. ACKNOWLEDGMENTS Ciafre`, S. A., Rinaldi, M., Gasparini, P., Seripa, D., Bis-ceglia, L., Zelante, L., Farace, M. G., and Fazio, V. M.We are grateful to Stefano Martinotti, Alberto Gulino,and Franco Pandolfi for kindly providing plasmid con- (1995). Stability and functional effectiveness of phos-
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